Historical analgesic tablets revisited: Development of a versatile RP-HPLC method for the stability study of single- and multi-component pharmaceutical preparations

Historical analgesic tablets revisited: Development of a versatile RP-HPLC method for the stability study of single- and multi-component pharmaceutical preparations | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Historical analgesic tablets revisited: Development of a versatile RP-HPLC method for the stability study of single- and multi-component pharmaceutical preparations Tomáš Lener, Martin Štícha, Markéta Bauerová, Klára Ulbertová, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6801134/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Aug, 2025 Read the published version in Monatshefte für Chemie - Chemical Monthly → Version 1 posted 4 You are reading this latest preprint version Abstract A versatile RP-HPLC method using phenyl-hexyl stationary phase and gradient elution (0.1% aqueous acetic acid and methanol) was developed to investigate the chemical integrity of twelve historical analgesic single- and multi-component preparations (1940s to 1990s). UV/VIS detection, supported by tandem mass spectrometry, was employed for compound identification and potential degradation product screening. Twelve active pharmaceutical ingredients (APIs), including acetylsalicylic acid, bromisoval, caffeine, codeine, narcotine, phenacetine, phenobarbital, salicylic acid, and four different barbiturates. Calibration models were established with limits of quantification as low as 3.0 mg dm⁻³. Coelution of phenobarbital and aminophenazone was effectively resolved through mathematical correction using dual-wavelength detection. Most APIs demonstrated remarkable long-term stability (within ±10% of declared content), with the exception of codeine and acetylsalicylic acid in specific preparation. Effervescent and poorly stored tablets exhibited significant degradation, highlighting the importance of dosage form and storage conditions. In addition, the tandem mass spectrum of bromisoval is presented here for the first time in the literature, and a plausible fragmentation mechanism is proposed. Analgesic Preparation Degradation RP-HPLC Shelf life Stability Tandem Mass Spectrometry Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Analgesics are drugs that induce a painless state, also known as analgesia. While acute pain has a biological function of warning the organism of something potentially harmful, chronic pain merely burdens the organism in many ways. The biological function makes pain an important and inseparable part of life and the most common reason for seeking a suitable medication. Although certain preparations were historically used to soothe pain, the documented history of analgesic drugs does not truly begin until the mid-19th century [ 1 ]. The early discoveries of analgesic drugs depended on geographical conditions and were obtained empirically. Not only were certain plants (poppy, valerian, hops, or mandrake) utilized, but also the appease effect of ethanol was employed to induce analgesia. One of the most potent and historically significant analgesic drugs in history remains opium [ 2 , 3 ]. This dried latex of opium poppy ( Papaver somniferum L.) has been used for thousands of years, and has served as an inspiration for the development of many analgesic drugs, some of which have indeed often been misused for other purposes. The development of science in the 19th century led to the discovery and synthesis of many new compounds including some with analgesic effects. The development of industry further enabled the production of a wide range of different analgesic pharmaceutical preparations which nowadays vary in their active pharmaceutical ingredients (APIs) and excipients. In the case of analgesic preparations, not only single-component preparations but also variable combinations of APIs are often used. These preparations remain a fundamental and indispensable part in the management of both acute and chronic pain conditions in clinical practice. In this study, the development of a versatile RP-HPLC method for the stability study of single- and multi-component analgesic preparations is presented. The developed method was subsequently used for the analysis of a unique set of twelve historical analgesic preparations: six single-component (Fig. 1 ), and six multi-components (Fig. 2 ). These preparations contained in total twelve APIs: acetylsalicylic acid, codeine, caffeine, bromisoval, narcotine, phenacetine, phenobarbital, salicylic acid, and four different barbiturates (see below for the exact composition of specific products). To characterize the individual APIs used in the analyzed preparations, the following can be briefly mentioned. An important and widely known compound with analgesic effects is acetylsalicylic acid (ASA), also often known as aspirin [ 1 ]. In fact, it is a prodrug; the active compound is salicylic acid which is formed in the gastrointestinal tract by hydrolysis. Both acids have analgesic, anti-inflammatory, and antipyretic effects, which have led to their widespread use in medicine. However, the disadvantage of salicylic acid is its unpleasant taste and strong stomach irritation, making the use of acetylsalicylic acid more tolerable. Since it is an inhibitor of cyclooxygenases, it reduces blood clotting, and it is also a proven teratogen, caution is needed when using it. Acetylsalicylic acid was first synthesized from salicylic acid and acetic anhydride by Charles von Gerhart in 1853, and Felix Hoffman later prepared it for the German company Bayer in 1897 [ 4 ]. The inspiration for the discovery and analgesic use of this compound was willow bark decoction, which had been used for its weak analgesic effect since times immemorial, and the Latin name of willow ( Salix ) is the root of the acid’s name. Codeine (methylmorphine) is an opioid used for its antitussive and analgesic effects, often in combination with other non-opioid analgesic substances, such as acetylsalicylic acid or paracetamol. Like other opioids, codeine has the dangerous disadvantage of possible addiction because, similar to morphine, it releases histamine in the body, and its mild euphoric and sedative effects are the reason for its misuse. The name is derived from the Greek word for the capsule of the poppy head (κωδεια). Codeine was discovered by Pierre-Jean Robiquet, who identified it by chance while researching morphine derivatives in 1832 [ 5 ]. A few years earlier, in 1817, the same person discovered another opioid alkaloid, narcotine (noscapine) whose antitussive activity was discovered in 1930 [ 6 ]. In contrast to codeine, narcotine lacks significant analgesic, sedative, and euphoric effects; therefore, there is no problem of addiction. Caffeine is an alkaloid and a strong stimulant of the central nervous system. Although it has no analgesic effect itself, it is often used in analgesic preparations because it can potentiate the effect of other active substances (e.g., paracetamol). Like other xanthine alkaloids, caffeine was used for its diuretic effect until the introduction of thiazides [ 6 ]. Caffeine was first isolated from coffee beans by Ferdinand Friedlieb Runge in the first half of the 19th century. Barbiturates (in this work allo-, apro-, pheno-, and butobarbital) are derivatives of barbituric acid (malonylurea), which was first synthesized by Adolf von Baeyer in 1864 [ 7 ]. Despite barbituric acid itself having no specific effects, its derivatives have diverse therapeutic applications, and more than 2,500 of them have been synthesized to date. However, only about fifty of barbiturates have been used for their sedative and hypnotic properties in treatment. Barbiturates affect the central nervous system and were the first truly effective tools for managing epileptic seizures. Nevertheless, the risk of addiction and death by overdose has led to a decrease in the use of these compounds. Phenobarbital was synthesized by Hörlein in 1911 and was the first barbiturate used for epilepsy treatment. Bromisoval (bromvalerlurea) is a monoureide derivative, synthesized from isopentyl alcohol, and has been used for its mild, short-acting sedative effect since 1908 [ 8 ]. Nowadays, it is largely obsolete and is rarely used due to its high potential for toxicity. The problem with the therapeutic use of bromisoval is its ability to cause excessive sedation and chronic intoxication characterized by confusion, ataxia, disorientation, delirium, hallucinations, and diplopia. In 1887, Carl Hinsberg first prepared phenacetin (acetophenetidin) while seeking a use for an accumulated byproduct of dye works, p nitrophenol [ 9 ]. Phenacetin was used for almost a century for its analgesic and antipyretic effects. When combined with caffeine or administered in higher doses, it has a euphoric effect, which was often the cause of chronic poisoning typically manifested by grayish-blue skin coloration. Moreover, chronic use causes kidney damage; thus, this substance was withdrawn in many countries and replaced by its metabolite, paracetamol, which is used instead[ 10 ]. Aminophenazone (aminopyrine), a derivative of phenazone, was first synthesized by Friedrich Stolz in 1893 [ 9 ]. Both phenazone and aminophenazone have analgesic, antipyretic and anti-inflammatory effects. The high popularity of aminophenazone lasted until the 1960s when its market availability decreased due to its unfavorable side effects (e.g., agranulocytosis). The stability of pharmaceutical preparations is one of the most important criteria that must be controlled, because physical and chemical degradation of the preparations – or of the APIs – can cause changes in therapeutic efficacy or even result in toxic effects [ 11 ]. Therefore, many stability tests are conducted before the preparation can be introduced onto the market and approved for use. The interest in studying the long-term stability and shelf-life of medicinal preparations is illustrated by the Shelf-Life Extension Program, which was initiated in the USA in 1986 [ 12 ]. This program aimed to identify opportunities to extend the expiration date of drugs appropriately stored in government emergency stockpiles, thereby reducing the costs associated with the regular replacement of these stockpiles. In 2019, Zilker et al. [ 13 ]extensively reviewed available data on the stability of finished pharmaceutical products and drug substances beyond their labeled expiry dates, and discussed the pros and cons of possible extending shelf-life beyond the usual five-year limit. More recent studies addressing this issue are reviewed in our recent publication[ 14 ]. The longterm stability of APIs in decades-old pharmaceutical products has been a persistent research focus in our laboratory [ 15 ]. We have focused on the stability of various injectable solutions [ 16 – 19 ], as well as solid dosage forms [ 20 – 22 ]. Results and discussion RP-HPLC method development Drawing on the literature [ 23 – 27 ], the binary mobile phase, based on 0.1% aqueous acetic acid (solvent A) and methanol (solvent B), was selected. Based on the dissociation constants of all twelve analytes considered (Fig. 3 ), the pH of the aqueous component of the mobile phase was set to 5.00 (adjusted by the addition of an aqueous ammonia), at which most of the analytes are present in solution exclusively in either fully protonated or fully deprotonated form. For the optimization of RP-HPLC method, three different types of columns were tested. Traditionally, the C18 stationary phase is the first option for reverse-phase methods. Using XBridge® BEH C18 2.5 µm (150×3.0 mm) column, isocratic elution with methanol (solvent B) concentrations ranging from 40–50%, and a total runtime of 45 min, constituted the initial step in the gradient program development. Subsequently, various gradient programs were tested, with solvent B ranging from 20–80% and different total runtimes. However, two analytes – bromisoval and phenobarbital – both concurrently present in Veralgin and, unfortunately, sharing a similar absorption maximum available for UV detection, coeluted and could not be separated by modification of the gradient. Subsequently, two other stationary phases were tested (i) XBridge® BEH Amide 2.5 µm (150×3.0 mm), and (ii) Kinetex 2.5 µ Phenyl-Hexyl 100Å (100×3.0 mm). The Phenyl-Hexyl stationary phase provided the best separation of most of the analytes. The only remaining issue was the coelution of phenobarbital and aminophenazone, concurrently present in Veralgin , which could not be resolved by alternating the gradient. Fortunately, these two analytes significantly differ in their UV absorption maxima available for detection (phenobarbital at 210 nm and aminophenazone at 260 nm). Therefore, baseline separation was not required for quantification, and the issue was effectively addressed by employing dual-wavelength detection (see below). Moreover, a slight modification of the gradient program shortened the total runtime of analysis from 38 to 30 minutes. Thus, the Phenyl-Hexyl stationary phase was selected as the best option for the analysis (Fig. 4 ). Calibration curves for studied APIs Under optimized conditions for RP-HPLC separation, calibration curves based on standards of the analyzed APIs were measured in the range of 10.00–150.0 mg dm − 3 . UV/VIS detection was used, and the area of the chromatographic peak measured at the absorption maximum of each analyte served as the analytical signal. The obtained and statistically evaluated parameters of the calibration curves are summarized in Table 1 . The resolution calculated from retention parameters, was ≥ 1.3 for all peaks. Table 1 Parameters of the calibration curves for RPHPLC–UV determination of APIs based on peak area (analyte, retention time, detection wavelength, slope, intercept, correlation coefficient, limit of quantification). Measurements were performed on a Kinetex 2.5 µ Phenyl-Hexyl 100Å (100×3.0 mm) column using gradient elution with (A) 0.1% aqueous acetic acid (pH = 5.00) and (B) methanol (for gradient program see Fig. 1 ), flow rate 0.4 cm 3 min − 1 . Compound, CASRN t r /min λ/nm Slope /µAU min mg − 1 dm 3 Intercept /µAU min R 2 LOQ /mg dm − 3 Acetylsalicylic acid, [50-78-2] 1.79 210 8000 – a 0.9887 70 Salicylic acid, [69-72-7] 1.99 210 15700 – a 0.9994 15.9 Codeine, [76-57-3] 2.38 210 8900 – a 0.9996 12 Caffeine, [58-08-2] 3.90 271 7900 – a 0.9999 4.6 Allobarbital, [52-43-7] 8.06 210 7000 – a 0.9987 20 Bromisoval, [496-67-3] 9.53 210 4300 – a 0.9994 15 Phenacetine, [62-44-2] 10.00 245 10700 – a 0.9998 8.44 Aprobarbital, [77-02-1] 10.44 210 6300 – a 0.9997 11 Phenobarbital, [50-06-6] 11.60 210 9100 – a 0.9995 14 Aminophenazone, [58-15-1] 11.60 260 6020 −11000 0.9999 5.4 Butobarbital, [77-28-1] 16.06 210 5620 −7000 1.0000 3 Narcotine, [128-62-1] 24.20 210 19900 – a 0.9997 10.2 a Statistically insignificant. In order to solve the problem of quantifying phenobarbital and aminophenazone due to their co-elution, calibration curves were measured in the concentration range of 10–150 mg dm − 3 for one analyte at a fixed concentration of the second analyte (at levels 0.0, 10.0, 80.0, and 150.0 mg dm − 3 ). For both analytes, the areas of the chromatographic peaks at wavelengths of 210 and 260 nm served as the analytical signal. From the obtained data, it was possible to construct a system of two equations with two unknowns, which allows determination of the concentration of phenobarbital and aminophenazone in the case of co-elution A 210 = 8000 c A + 8600 c p (1) A 260 = 5800 c A + 380 c p (2) where A 210 and A 260 are the peak areas at 210 and 260 nm, respectively [µAU min mg − 1 dm 3 ], c A is the concentration of aminophenazone [mg dm − 3 ], and c P is the concentration of phenobarbital [mg dm − 3 ]. The accuracy of the system was verified using five training sets of solutions with different concentration of analytes. Finally, the potential influence of ultrasound treatment and filtration using a syringe filter during the extraction of the specified APIs from historical analgesic tablets on the quantification of the analyte was evaluated. It was found that neither of these operations affected the quantification. Quantification of APIs in historical analgesic tablets analyzed Twelve historical analgesic preparations were analyzed using the developed RP-HPLC method; three tablets from each preparation were analyzed individually. The results, including statistical evaluation, are summarized in Table 2 . Table 2 Quantification of APIs in historical analgesic tablets analyzed (preparation, date of manufacture; API and declared content; measured API content expressed as a percentage of the declared amount). Preparation, date of manufacture Measured / % of declared content Compound, declared content Tablet #1 Tablet #2 Tablet #3 Median L 1,2 s r /% Acylpyrin , 1986 Acetylsalicylic acid, 500 mg/tbt 90.0 93.6 92.1 92.1 4.6 0.023 Recalculated ASA a 92.4 96.6 95.0 95.0 5.5 0.026 Acylpyrin effervescens , 1982 Acetylsalicylic acid, 500 mg/tbt < LOD 21.0 < LOD 21.0 n.a. n.a. Recalculated ASA a, b 105.6 94.2 105.8 105.6 15.1 0.065 Acidum acetylosalicylicum , 1953 Acetylsalicylic acid, 500 mg/tbt 102.1 86.9 87.2 87.2 19.7 0.103 Recalculated ASA a 104.8 89.9 91.6 91.6 19.3 0.096 Codeinum phosphoricum , 1953 Codeine, 20 mg/tbt 86.2 88.4 88.9 88.4 3.6 0.018 Codein , 1993 Codeine, 15 mg/tbt 75.7 73.2 – c 74.5 –468.5 –0.871 Isoneurin , ca. 1940s Bromisoval, 300 mg/tbt 95.6 95.4 92.5 95.4 4.1 0.019 Algena , 1981 Caffeine, 50 mg/tbt 91.1 90.1 92.7 91.1 3.4 0.017 Aprobarbital, 50 mg/tbt 96.3 90.3 88.8 90.3 9.7 0.049 Phenacetine, 150 mg/tbt 103.5 100.2 96.7 100.2 8.8 0.040 Aminophenazone, 300 mg/tbt 98.7 93.8 86.4 93.8 15.9 0.077 Alnagon , 1983 b Acetylsalicylic acid, 380 mg/tbt 42.6 41.4 41.2 41.4 1.9 0.020 Codeine, 20 mg/tbt 46.8 58.9 59.1 58.9 16.0 0.124 Caffeine, 80 mg/tbt 86.2 93.9 90.6 90.6 10.0 0.050 Phenobarbital, 20 mg/tbt 104.5 97.9 94.4 97.9 13.1 0.061 Recalculated ASA a 105.7 107.3 105.8 105.8 2.0 0.009 Alnagon , 1990 d Acetylsalicylic acid, 380 mg/tbt 46.9 60.7 34.8 46.9 33.7 0.327 Codeine, 20 mg/tbt 53.3 67.9 50.7 53.3 22.4 0.191 Caffeine, 80 mg/tbt 77.0 103.6 98.6 98.6 34.5 0.159 Phenobarbital, 20 mg/tbt 101.6 113.2 87.3 101.6 33.7 0.151 Recalculated ASA a 95.2 117.3 122.1 117.3 34.9 0.135 Veralgin , 1973 Caffeine, 25 mg/tbt 101.0 99.1 86.7 99.1 18.6 0.085 Bromisoval, 250 mg/tbt 108.7 108.7 101.9 108.7 8.8 0.037 Phenobarbital, 15 mg/tbt 107.4 113.2 103.0 107.4 13.2 0.056 Aminophenazone, 200 mg/tbt 99.9 103.0 92.7 99.9 13.4 0.061 Dinyl , 1977 Caffeine, 50 mg/tbt 101.0 91.1 96.3 96.3 12.9 0.061 Allobarbital, 18 mg/tbt 93.2 91.4 94.1 93.2 3.5 0.017 Phenacetin, 200 mg/tbt 106.9 100.5 103.6 103.6 8.3 0.036 Aminophenazone, 200 mg/tbt 91.0 95.0 94.4 94.4 5.2 0.025 Butobarbital, 12 mg/tbt 109.7 109.1 111.5 109.7 3.1 0.013 Gelonida , ca. 1940s Acetylsalicylic acid, 250 mg/tbt 74.2 76.3 88.8 76.3 19.0 0.113 Phenacetine, 250 mg/tbt 103.9 99.2 101.8 101.8 6.1 0.027 Narcotine, 30 mg/tbt 103.8 89.3 81.9 89.3 28.5 0.145 Recalculated ASA a 102.8 99.0 108.0 102.8 11.7 0.052 a Recalculated content of acetylsalicylic acid based on determined amount of salicylic acid, considering the hydrolysis of acetylsalicylic acid to salicylic acid. b Two tablets were homogenized together during measurements 2 and 3. c Only two tablets were available in the package analyzed. d Analysis of brown tablets; discussed in more detail below. Acetylsalicylic acid was a relatively problematic analyte for accurate quantification due to its instability – namely, hydrolysis to salicylic acid – in all commonly used organic solvents for HPLC [ 29 ], as well as its low solubility in water. Therefore, all solutions (historical samples and calibration solutions) containing this analyte were analyzed immediately after preparation in methanol, with a maximum time delay of 15 min. To evaluate the effect of acetylsalicylic acid hydrolysis on the quantification accuracy, a methanolic solution of acetylsalicylic acid at a concentration level in the middle of the calibration range (75 mg dm − 3 ) was repeatedly analyzed over time. The results showed that even after one hour, the error did not exceed 3.5% (Fig. 5 ). Thus, the “Recalculated ASA” parameter is introduced in Table 2 , to represent the presumed amount of acetylsalicylic acid originally present in the analyzed tablet prior to extraction of given tablet by methanol. The recalculated ASA value is the sum of the measured amount of acetylsalicylic acid and the amount of acetylsalicylic acid equivalent to the measured salicylic acid amount, assuming hydrolysis of acetylsalicylic acid to salicylic acid in a 1:1 molar ratio. The difference between “Recalculated ASA” and the directly measured amount of acetylsalicylic acid indicates the extent of its hydrolytic degradation that occurred during long-term storage. Acetylsalicylic acid is the only API in the first three singlecomponent preparations analyzed. In the case of tablets Acylpyrin (manufactured in 1986), 92.1% of acetylsalicylic acid remained after 39 years of their production. Similarly, tablets Acidum acetylosalicylicum (manufactured in 1953) remained 87.2% of declared content even after 72 years of production. This represents remarkable stability, in contrast to Cantrell et al. [ 30 ], who reported more than 90% degradation of acetylsalicylic acid in multicomponent pharmaceutical preparations aged 28–40. On the other hand, different results were obtained from the analysis of effervescent tablets Acylpyrin effervescens (manufactured in 1982). In this package, all 15 tablets present had completely disintegrated into powder, such that their original shape was no longer discernible. The package was filled to the brim with a light greyish to brownish powder, and the odor of the content was also notable. Despite the disintegration, the entire contents of the package were weighed, homogenized, and an aliquot was analyzed. As shown in Table 2 , salicylic acid was identified as the major constituent, and acetylsalicylic acid was detected only in one of the three aliquots. The appearance, along with the results, indicates a high degree of degradation of acetylsalicylic acid in effervescent tablets and suggests lower long-term stability of this dosage form. Surprisingly high instability was observed in the case of codeine, contrary to previously reported data in the literature [ 21 , 30 , 31 ]. Codeine was relatively stable in the single-component tablets Codeinum phosphoricum (manufactured in 1953), with 88.4% of the declared content remaining after 72 years. In contrast, in 40-year-younger single-component tablets Codein (manufactured in 1993), only 74.5% of declared content was found. This lower content in the more recent preparation may be attributed to differences in the storage conditions. However, the percentage of codeine was even lower in both multi-component Alnagon tablets: 58.9% in the 1983 batch and 53.3% in the 1990 batch, respectively. The results were verified using two independent calibration curves, based on two different batches of codeine standards. Furthermore, no degradation products were detected by UV/Vis or mass spectrometry. It can therefore be hypothesized that the source of the low codeine content in both analyzed preparations may be due to a manufacturing error. From the perspective of codeine degradation, the analysis of tablets Alnagon (manufactured in 1990) was particularly informative, as it showed a strong correlation between codeine content and the physical states of tablet degradation. Three distinct types of tablets, differing visibly in their degree of degradation, were observed within the package (Fig. 6 ): (i) one white tablet, solid, with some darker spots; (ii) five brown tablets, solid, with colorless crystals on their surface; and, (iii) two black tablets, soft, with abundant colorless crystals on their surface. As shown in Table 3 , the APIs content differed across these tablet types. The amount of acetylsalicylic acid was 97.4% in the white tablet, 46.9% in the brown tablet, and below the limit of detection in the black tablet. A similar trend was observed in the case of codeine. Caffeine content appeared relatively stable in all tablet types, although in one of three brown tablets analyzed, it dropped to 77.0% (Table 2 ). The results also indicated high stability of phenobarbital across tablet types. Mass spectrometry was employed to verify these findings; however, no degradation products or compound responsible for the discoloration were identified – except salicylic acid, which was confirmed as the degradation product of acetylsalicylic acid. It is likely that colorless crystals are salicylic acid, as their presence correlated with more advanced stages of degradation. Therefore, it seems to be probable that the original codeine content was lower than declared by manufacturer. The influence of different codeine forms (e.g., phosphate, sulfate, hemihydrate, sesquihydrate) was also considered. However, even if a different form of codeine than declared by the manufacturer was used during production, the variation in codeine content would not exceed 5%. The observed differences among the three tablet types most likely resulted from external factors during the storage. The presence of black tablets alongside white and brown ones is unusual, as one would typically expect a gradual transition in colors. Unfortunately, the exact storage conditions are unknown, so the factor(s) responsible for this variation – such as heat, humidity, or light – can only be hypothesized. These findings underscore the importance of storage conditions and their evaluation when analyzing expired pharmaceutical products. On the other hand, undefined conditions often better reflect the real-world storage environment of pharmaceuticals in everyday use [ 32 ]. Table 3 Quantification of APIs in historical analgesic preparation Alnagon , manufactured in 1990 (API and declared content; measured API content expressed as a percentage of the declared value). API, declared content Measured / % of declared content White tablet Brown tablet b Black tablet Acetylsalicylic acid, 380 mg/tbt 97.4 46.9 < LOD ASA recalculated a 115.3 117.3 134.5 Codeine, 20 mg/tbt 77.8 53.3 38.9 Caffeine, 80 mg/tbt 97.9 98.6 96.2 Phenobarbital, 20 mg/tbt 96.3 101.6 102.9 a Recalculated content of acetylsalicylic acid based on determined amount of salicylic acid, considering the hydrolysis of acetylsalicylic acid to salicylic acid. b Median value from three brown tablets analyzed. High stability of bromisoval was revealed by the analysis of the single-component preparation tablets Isoneurin (manufactured in the 1940s), in which 95.4% of the declared content was retained even after approximately 85 years, despite the fragile physical state of the tablets. Similarly, multi-component tablets Veralgin (manufactured in 1973) showed 108.7% of declared amount of bromisoval. This finding is particularly important, as no data on bromisoval stability have been reported in the literature to date. Caffeine, phenacetine, aminophenazone, and all four barbiturates (aprobarbital, phenobarbital, allobarbital, and butobarbital) exhibited high stability in all tested preparations, with deviations within ± 10% of the declared amount, which is consistent with published data [ 13 , 20 , 30 ]. The content of narcotine – present only in tablets Gelonida antineuralgica (manufactured in the 1940s) – was slightly below the ± 10% threshold set by current pharmacopoeial standards. Approximately 85 years after manufacturing, the content of narcotine was found to be 89.3% of the declared value. As no degradation products were detected by mass spectrometry, this minor content may be attributed to manufacturing inaccuracy. High‑resolution tandem mass spectrometry of the studied APIs All historical samples and API standards were also analyzed by RPHPLC coupled with a high-resolution tandem mass spectrometer to confirm their identity in the respective preparations and to detect potential degradation products. The ions of studied APIs, as identified by tandem mass spectrometry, are summarized in Table 4 . A collision energy of 35.0 eV was used for MS 2 fragmentation of the analytes. For barbiturates, only precursor ions were measured, as their MS 2 fragments could not be obtained at this high collision energy. The diagnostic ions of other APIs were consistent with values reported in the literature. Table 4 The m/z ions of studied APIs identified by tandem mass spectrometry, used for compound confirmation (compound, type and m/z of the molecular ion, m/z of diagnostic ions, and reference to literature). Only the ions with a reasonable molecular formula and mass error ≤ 6 ppm were considered. Compound Parent ion Diagnostic ions m/z Ref. Type m/z Exper. Theor. Δ ppm Acetylsalicylic acid a [M + Na] + 203.0316 203.0315 0.59 121.0281 [ 33 ] Allobarbital [M − H] − 207.0772 207.0775 −1.53 164.0718 b [ 34 ] Aminophenazone [M + H] + 232.1441 232.1444 1.60 187.0862; 177.1014; 159.0913; 149.1073; 139.0864; 113.1073; 98.0837 c [ 35 , 36 ] Aprobarbital [M − H] − 209.0930 209.0932 −0.79 166.0871 b [ 34 ] Bromisoval [M + H] + 223.0076 223.0077 −0.26 181.9993; 134.9810; 119.9452; 101.0841; 86.0602; 69.0602; 69.0338; 57.0703 n.a. Butobarbital [M − H] − 211.1090 211.1088 0.87 168.1028 b [ 34 ] Caffeine [M + H] + 195.0875 195.0877 −0.78 138.0659; 123.0426; 110.0711 [ 37 ] Codeine [M + H] + 300.1590 300.1594 −1.40 282.1485; 267.1239; 243.1010; 225.0905; 215.1061; 199.0749; 183.0799; 161.0593 [ 37 , 38 ] Narcotine [M + H] + 414.1553 414.1547 1.38 353.1013; 323.0907; 220.0969; 206.0810; 179.0701 [ 39 ] Phenacetine [M + H] + 180.1017 180.1019 −1.14 138.0915; 110.0602; 93.0336; 82.0654 [ 35 ] Phenobarbital [M − H] − 231.0773 231.0775 −0.93 188.0723 b [ 20 , 35 ] Salicylic acid [M − H] − 137.0246 137.0244 1.33 93.0347; 65.0401 [ 40 ] a Sodium adduct caused by the internal standard (sodium formate). b Ions obtained by MS 2 fragmentation experiments. c Dimer ion. The mass spectra of all APIs studied in this work have already been well described in the literature, with the exception of bromisoval. For bromisoval, only molecular ion m/z values have been reported thus far [ 41 , 42 ]. Therefore, the tandem mass spectrum of bromisoval in the positive mode was studied in detail. The ion at m/z = 223.0076 was selected as the precursor ion, its MS 2 spectrum and proposed fragmentation pathway are shown in Fig. 7 . Conclusions A robust RP-HPLC-UV method was developed and applied for the quantitative analysis of twelve historical analgesic preparations, containing twelve APIs of various structural classes. The method enabled effective separation and quantification, including cases of co-elution addressed via dual-wavelength detection. The results demonstrated that most APIs – including barbiturates, phenacetine, aminophenazone, and caffeine – retained high chemical stability over several decades, with contents within ± 10% of the declared values. Bromisoval showed remarkable stability, despite a complete lack of prior stability data. Conversely, acetylsalicylic acid was stable in solid dosage forms but degraded significantly in effervescent tablets. Codeine showed inconsistent results: high stability in older single-component tablets, but substantially lower content in multi-component formulations, without detectable degradation products – suggesting possible manufacturing variability. Marked differences in degradation were observed among visually distinct tablets from a single Alnagon batch (manufactured in 1990), underlining the impact of storage conditions. High-resolution MS/MS was used to confirm compound identity and investigate degradation. Bromisoval fragmentation pattern was described here for the first time. These findings support the notion that many APIs can remain chemically stable long past their labeled expiration dates, although the effects of formulation type and storage conditions must be carefully considered. Experimental Analyzed samples, standards of APIs, and chemicals All twelve historical analgesic preparations analyzed originate from collection of the Department of Analytical Chemistry, Faculty of Science, Charles University. An overview of all samples is provided in Figs. 1 and 2 . The standards of the analyzed APIs were: acetylsalicylic acid (p.a., Herbacos Recordati, Czech Republic), salicylic acid (p.a., Merck), codeine phosphate hemihydrate (Ph. Eur. 11, Lékárna Fakultní nemocnice Motol, Czech Republic), codeine phosphate (Ph. Eur. 11, Zentiva, Czech Republic), caffeine (extra pure, Fischer Chemical), allobarbital (p.a., Zentiva, Czech Republic), bromisoval (p.a., Zentiva, Czech Republic), phenacetine (for HPLC, Sigma-Aldrich), aprobarbital (p.a., Léčiva Měcholupy, Czech Republic), phenobarbital (p.a., Zentiva, Czech Republic), aminophenazone (p.a., Sigma-Aldrich), butobarbital (p.a., Zentiva, Czech Republic) and narcotine (from the collection of the Department of Analytical Chemistry, Faculty of Science, Charles University [ 2 ]). The other chemicals used were: methanol (LC-MS grade, Fischer Chemical), acetic acid (p.a., Lach-ner, Czech Republic), aqueous ammonia 28% (p.a., VWR Chemicals, France) and deionized water with specific conductivity < 0.05 µS cm − 1 , obtained using a Milli-Q system (Millipore, USA). Samples preparation Each historical sample tablet was weighed and placed in a 100 cm 3 volumetric flask, followed by dissolution in methanol. If necessary, samples were sonicated in an ultrasonic bath for 2 to 10 minutes until fully dissolved. Dilution to match the calibration concentration range was calculated based on the declared content of the respective analytes. The resulting extracts were filtered through a 0.2 µm PVDF centrifuge filter. Instrumentation and procedures For RP-HPLC with UV/VIS detection, a UHPLC Nexera XR System equipped with DAD detection (Shimadzu) was used. The stationary phase was a Kinetex 2.5µ Phenyl-Hexyl 100Å (100×3 mm) column, maintained at 30°C. The binary mobile phase consisted of 0.1% aqueous acetic acid (solvent A) and methanol (solvent B). The mobile phase flow rate was set at 0.4 cm 3 min − 1 . The gradient elution program (total runtime: 30 min) was as follows: 0 − 7 min: 30% B (isocratic); 7 − 15 min: linear gradient to 35% B; 15 − 20 min: linear gradient to 70% B; 20 − 25 min: 70% B (isocratic); 25–26 min: return to 30% B, 26–30 min: 30% B (re-equilibration). The volume of the injected sample was 2 mm 3 . The detection wavelengths were: 271 nm for caffeine, 245 nm for phenacetin, 260 nm for aminophenazone, 210 nm for all other analytes. Data were processed using LabSolutions 2010 (Shimadzu). For HPLC with MS/MS detection, an Agilent 1290 Infinity II LC System with a binary pump model was operated under the same conditions as the HPLC–UV method. High-resolution tandem mass spectrometry was performed using a Bruker QqTOF compact instrument, operated via Compass otof Control 4.0 software (Bruker Daltonics, Germany). Data were processed with Compass DataAnalysis 4.4 (Build 200.55.2969) software (Bruker Daltonics, Germany). ESI-MS 2 data were collected in both positive and negative ion modes, within a mass range of m / z 50–1000. The drying gas temperature was 220°C, with a flow rate 5.0 dm 3 min − 1 . The capillary (cone) voltage was 2,800 V. Fragmentation data were interpreted using Compass Compound Crawler 3.0 (Bruker, Germany). Declarations Acknowledgements The financial support by the project Cooperatio Chemistry of Charles University is gratefully acknowledged. Conflict of interest The authors have no financial or proprietary interests in any material discussed in this article. Data availability statement The experimental data supporting the findings of this study are available from the corresponding author, T.L., upon reasonable request. References Rainsford KD (2004) Aspirin and Related Drugs. CRC Press, Boca Raton Nesměrák K, Kudláček K, Štícha M, Kozlík P, Červený V, Kunešová J (2019) Monatsh Chem 150:1593 Nesměrák K, Štícha M, Belianský M, Červený V, Kozlík P, Kudláček K, Kunešová J (2021) Monatsh Chem 152:1089 Sneader W (2000) Br Med J 321:1591 Rida PCG, LiVecche D, Ogden A, Zhou J, Aneja R (2015) Med Res Rev 35:1072 Sneader W (2005) Drug Discovery: A History. Wiley, New York López-Muñoz F, Ucha-Udabe R, Alamo C (2005) Neuropsychiatr Dis Treat 1:329 Baselt RC (2014) Disposition of Toxic Drugs and Chemicals in Man. 10th Ed. Biomedical Publications, Seal Beach Raviña E (2011) The Evolution of Drug Discovery. Wiley, Weinheim Kar A (2007) Medicinal Chemistry. 4th Ed. New Age International, New Delhi Yoshioka S, Stella VJ (2002) Stability of Drugs and Dosage Forms. Kluwer, New York Courtney B, Easton J, Inglesby TV, SooHoo C (2009) Biosecur Bioterror 7:101 Zilker M, Sörgel F, Holzgrabe U (2019) J Pharm Biomed Anal 166:222 Lener T, Nesměrák K (2024) Monatsh Chem 155:757 Nesměrák K, Kudláček K, Babica J (2017) Monatsh Chem 148:1557 Nesměrák K, Štícha M, Čvančarova M (2010) Anal Lett 43:2572 Kudláček K, Nesměrák K, Štícha M, Kozlík P, Babica J (2017) Monatsh Chem 148:1613 Kudláček K, Nesměrák K, Štícha M, Kozlík P, Babica J (2018) Monatsh Chem 149:1555 Nesměrák K, Kudláček K, Štícha M, Kozlík P, Babica J (2019) Monatsh Chem 150:1611 Nesměrák K, Štícha M, Belianský M, Tomnikova A (2022) Monatsh Chem 153:735 Nesměrák K, Kudláček K, Kozlík P, Šícha M, Vašíčková P, Babica J (2020) Chem Listy 114:52 Nesměrák K, Kudláček K, Hraníček J, La Nasa J, Ribechini E, Colombini MP (2024) Monatsh Chem 155:791 Franeta JT, Agbaba D, Eric S, Pavkov S, Aleksic M, Vladimirov S (2002) Farmaco 57:709 Sun S, Liu G, Wang Y (2006) Chromatographia 64:719 Krieger DJ (1984) J AOAC Int 67:339 Acheampong A, Gyasi WO, Darko G, Apau J, Addai‑Arhin S (2016) SpringerPlus 5:625 Dams R, Benijts T, Lambert WE, De Leenheer AP (2002) J Chromatogr B 773:53 Prankerd RJ (2007) In: Brittain HG (ed) Profiles of Drug Substances, Excipients, and Related Methodology. 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Elsevier, Amsterdam Skibinski R, Komsta L (2016) Curr Issues Pharm Med Sci 29:39 Cantrell L, Suchard JR, Wu A, Gerona RR (2012) Arch Intern Med 172:1685 Lyon RC, Taylor JS, Porter DA, Prasanna HR, Hussain AS (2006) J Pharm Sci 95:1549 Gikonyo D, Gikonyo A, Luvayo D, Ponoth P (2019) Afr Health Sci 19:2737 Fagbohun OF, Joseph JS, Salami OA, Msagati TAM (2021) Biol Trace Elem Res 199:1633 Wachełko O, Tusiewicz K, Zawadzki M, Szpot P (2023) J Pharm Biomed Anal 228:115318 Singh A, Thatikonda T, Kumar A, Wazir P, V V, Nandi U, Singh PP, Singh S, Gupta AP, Tikoo MK, Singh G, Vishwakarma R (2018) J Pharm Biomed Anal 149:387 Guo C, Gong L, Wang W, Leng J, Zhou L, Xing S, Zhao Y, Xian R, Zhang X, Shi F (2020) Int J Mass Spectrom 447:116250 Thörngren JO, Östervall F, Garle M (2008) J Mass Spectrom 43:980 Cristoni S, Cantu M, Bernardi LR, Gerthoux P, Mocarelli P, Brambilla M, Gonella E, Guidugli F (2005) J Mass Spectrom 40:1609 Fang ZZ, Krausz KW, Li F, Cheng J, Tanaka N, Gonzalez FJ (2012) Br J Pharmacol 167:1271 Algethami FK, Jlizi S, Znati M, Elamin MR, Hamadi NB, Jannet HB (2024) Nat Prod Res 38:3881 Kokatsu J, Yomoda R, Suwa T (1992) Chem Pharm Bull (Tokyo) 40:1517 Hori Y, Fujisawa M, Shimada K, Hirose Y, Yoshioka T (2006) Biol Pharm Bull 29:7 Supplementary Files Graphicalabstract.docx Cite Share Download PDF Status: Published Journal Publication published 04 Aug, 2025 Read the published version in Monatshefte für Chemie - Chemical Monthly → Version 1 posted Reviewers agreed at journal 03 Jun, 2025 Reviewers invited by journal 03 Jun, 2025 Editor assigned by journal 03 Jun, 2025 First submitted to journal 02 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6801134","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":465837596,"identity":"730377ff-3b7d-49a1-81e9-b47e4f0f4682","order_by":0,"name":"Tomáš Lener","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIiWNgGAWjYPACCxDB+AAhYIBbLQ+EkgARzAYIpURqYZPAqxQG7Nm7Ez/+YJCQM2fvfVZdUPMncXsD8zPpggIGefkGHLbwnN0szcMgYWzZc9zs9oxjBolzDrCZSc8wYDBsxKVFIneDNNBhiRtupLHd5mEzSJzBwMMmzWPAkMCMyy8SuZt/Ah1Wv+H+M7Zinn9IWthwa9kmAXRYgsENNjZm3jYkLTy4tJw5u82ax0DCcMOZNGZp3j5j4xnMbMZgkRk4tLC3926++aPCRt7g+DHGzzzf5GRnsDc/vM3zxwZniEEASmxAvC2BT/0oGAWjYBSMAgIAADMJRBpDtyHEAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0009-0000-4688-775X","institution":"Charles University Faculty of Science: Univerzita Karlova Prirodovedecka fakulta","correspondingAuthor":true,"prefix":"","firstName":"Tomáš","middleName":"","lastName":"Lener","suffix":""},{"id":465837599,"identity":"c154b415-afa3-4210-84ca-fb9023c94294","order_by":1,"name":"Martin Štícha","email":"","orcid":"","institution":"Charles University Faculty of Science: Univerzita Karlova Prirodovedecka fakulta","correspondingAuthor":false,"prefix":"","firstName":"Martin","middleName":"","lastName":"Štícha","suffix":""},{"id":465837600,"identity":"90d48f28-ee59-4d6a-b340-7a2ca7101263","order_by":2,"name":"Markéta Bauerová","email":"","orcid":"","institution":"Charles University Faculty of Science: Univerzita Karlova Prirodovedecka fakulta","correspondingAuthor":false,"prefix":"","firstName":"Markéta","middleName":"","lastName":"Bauerová","suffix":""},{"id":465837601,"identity":"e629685a-c730-4cf9-a093-015f891aa3c5","order_by":3,"name":"Klára Ulbertová","email":"","orcid":"","institution":"Charles University Faculty of Science: Univerzita Karlova Prirodovedecka fakulta","correspondingAuthor":false,"prefix":"","firstName":"Klára","middleName":"","lastName":"Ulbertová","suffix":""},{"id":465837604,"identity":"196dab67-9a1e-4700-a654-224d1d7bf252","order_by":4,"name":"Karel Nesměrák","email":"","orcid":"","institution":"Charles University Faculty of Science: Univerzita Karlova Prirodovedecka fakulta","correspondingAuthor":false,"prefix":"","firstName":"Karel","middleName":"","lastName":"Nesměrák","suffix":""}],"badges":[],"createdAt":"2025-06-02 10:21:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6801134/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6801134/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00706-025-03350-4","type":"published","date":"2025-08-04T15:58:02+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":84070994,"identity":"7c1e5504-593a-4105-890f-e1e260e38394","added_by":"auto","created_at":"2025-06-06 12:15:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":26779982,"visible":true,"origin":"","legend":"\u003cp\u003ePackaging and tablet appearance of the historical single-component analgesic preparations analyzed: \u003cstrong\u003e(a)\u003c/strong\u003e \u003cem\u003eAcylpyrin\u003c/em\u003e (manufactured 1986),\u003cstrong\u003e (b) \u003c/strong\u003e\u003cem\u003eAcylpyrin effervescens\u003c/em\u003e (manufactured 1982),\u003cstrong\u003e (c)\u003c/strong\u003e \u003cem\u003eAcidum acetylosalicylicum\u003c/em\u003e (manufactured 1953),\u003cstrong\u003e (d)\u003c/strong\u003e \u003cem\u003eCodeinum phosphoricum\u003c/em\u003e (manufactured 1953), \u003cstrong\u003e(e)\u003c/strong\u003e \u003cem\u003eCodein\u003c/em\u003e (manufactured 1993),\u003cstrong\u003e (f)\u003c/strong\u003e \u003cem\u003eIsoneurin\u003c/em\u003e (manufactured in the 1940s)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6801134/v1/dac02377081e36b4bde52505.png"},{"id":84070995,"identity":"6ed555f1-8d0e-4ea1-922d-4188127dafe4","added_by":"auto","created_at":"2025-06-06 12:15:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":23115567,"visible":true,"origin":"","legend":"\u003cp\u003ePackaging and tablet appearance of the historical multiple-component analgesic preparations analyzed:\u003cstrong\u003e (a)\u003c/strong\u003e \u003cem\u003eAlgena\u003c/em\u003e (manufactured 1981), \u003cstrong\u003e(b) \u003c/strong\u003e\u003cem\u003eAlnagon\u003c/em\u003e (manufactured 1983), \u003cstrong\u003e(c)\u003c/strong\u003e \u003cem\u003eAlnagon\u003c/em\u003e (manufactured 1990), \u003cstrong\u003e(d) \u003c/strong\u003e\u003cem\u003eVeralgin\u003c/em\u003e (manufactured 1973), \u003cstrong\u003e(e)\u003c/strong\u003e \u003cem\u003eDinyl\u003c/em\u003e (manufactured 1977), \u003cstrong\u003e(f) \u003c/strong\u003e\u003cem\u003eGelonida antineuralgica\u003c/em\u003e (manufactured in the 1940s).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6801134/v1/f63030eb0889d42c8207a66d.png"},{"id":84070983,"identity":"c2932811-dfc7-40e4-81fd-149353012d79","added_by":"auto","created_at":"2025-06-06 12:15:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":200075,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical representation of the dissociation states of the individual APIs studied as a function of the solution pH (dissociation exponents taken from [28]). The red line indicates the selected pH of the aqueous component of the mobile phase (i.e., solvent A) used for RP‑HPLC separation.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6801134/v1/d72f27e047c108bcca5269a4.png"},{"id":84070989,"identity":"0eabb533-c00b-4147-b232-da9365fc74a2","added_by":"auto","created_at":"2025-06-06 12:15:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":818090,"visible":true,"origin":"","legend":"\u003cp\u003eRP-HPLC-UV chromatogram of a mixture of standards of twelve analyzed APIs: \u003cstrong\u003e1\u003c/strong\u003e\u0026nbsp;acetylsalicylic acid, \u003cstrong\u003e2\u003c/strong\u003e\u0026nbsp;salicylic acid, \u003cstrong\u003e3\u003c/strong\u003e\u0026nbsp;codeine, \u003cstrong\u003e4\u003c/strong\u003e\u0026nbsp;caffeine, \u003cstrong\u003e5\u003c/strong\u003e\u0026nbsp;allobarbital, \u003cstrong\u003e6\u003c/strong\u003e\u0026nbsp;bromisoval, \u003cstrong\u003e7\u003c/strong\u003e\u0026nbsp;phenacetine, \u003cstrong\u003e8\u003c/strong\u003e\u0026nbsp;aprobarbital, \u003cstrong\u003e9\u003c/strong\u003e and \u003cstrong\u003e10\u0026nbsp;\u003c/strong\u003ecoelution of phenobarbital and aminophenazone, \u003cstrong\u003e11\u003c/strong\u003e\u0026nbsp;butobarbital, and \u003cstrong\u003e12\u0026nbsp;\u003c/strong\u003enarcotine. Kinetex 2.5\u0026nbsp;µ Phenyl-Hexyl 100Å (100×3.0 mm) column, gradient elution with (A) 0.1% aqueous acetic acid (pH = 5.00) and (B)\u0026nbsp;methanol, flow rate 0.4 cm\u003csup\u003e3\u003c/sup\u003e\u0026nbsp;min\u003csup\u003e−1\u003c/sup\u003e, detection at 210\u0026nbsp;nm, concentration of each API 40.00\u0026nbsp;mg\u0026nbsp;dm\u003csup\u003e−3\u003c/sup\u003e in methanol\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6801134/v1/53cb21a0edc05edf9c057538.png"},{"id":84071671,"identity":"43105bbd-cefc-45f5-954e-61b5018a72c6","added_by":"auto","created_at":"2025-06-06 12:23:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1418955,"visible":true,"origin":"","legend":"\u003cp\u003eTime-dependent decrease of \u003cstrong\u003e1\u003c/strong\u003e the acetylsalicylic acid peak, and simultaneous increase of \u003cstrong\u003e2\u003c/strong\u003e the salicylic acid peak in RP-HPLC-UV chromatogram of 75\u0026nbsp;mg\u0026nbsp;dm\u003csup\u003e−3\u003c/sup\u003e acetylsalicylic acid solution in methanol measured at: \u003cstrong\u003ea\u003c/strong\u003e\u0026nbsp;5\u0026nbsp;min, \u003cstrong\u003eb\u0026nbsp;\u003c/strong\u003e15\u0026nbsp;min, \u003cstrong\u003ec\u0026nbsp;\u003c/strong\u003e30\u0026nbsp;min, \u003cstrong\u003ed\u0026nbsp;\u003c/strong\u003e45\u0026nbsp;min, and \u003cstrong\u003ee\u0026nbsp;\u003c/strong\u003e60\u0026nbsp;min after solution preparation. Kinetex 2.5 µ Phenyl-Hexyl 100Å (100×3.0 mm) column, gradient elution with (A) 0.1% aqueous acetic acid (pH = 5.00) and (B) methanol, flow rate 0.4\u0026nbsp;cm\u003csup\u003e3\u003c/sup\u003emin\u003csup\u003e1\u003c/sup\u003e, detection at 210 nm\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6801134/v1/9138b31be3d73f49c875d174.png"},{"id":84070998,"identity":"6e0091ff-13cc-4c6a-8a6c-425da2e4a14f","added_by":"auto","created_at":"2025-06-06 12:15:32","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":13545576,"visible":true,"origin":"","legend":"\u003cp\u003eAppearance of the historical analgesic preparation \u003cem\u003eAlnagon\u003c/em\u003e(manufactured in 1990) analyzed \u003cstrong\u003ea\u003c/strong\u003e blister pack, and the details of three different types of tablets \u003cstrong\u003eb\u003c/strong\u003e white,\u003cstrong\u003ec \u003c/strong\u003ebrown, and\u003cstrong\u003e d \u003c/strong\u003eblack with visible crystals.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6801134/v1/fe64acb7c7326d2f23617536.png"},{"id":84071004,"identity":"764c95eb-6d75-4fd8-94bf-0081975bba06","added_by":"auto","created_at":"2025-06-06 12:15:32","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":322056,"visible":true,"origin":"","legend":"\u003cp\u003eAppearance of the historical analgesic preparation \u003cem\u003eAlnagon\u003c/em\u003e(manufactured in 1990) analyzed \u003cstrong\u003ea\u003c/strong\u003e blister pack, and the details of three different types of tablets \u003cstrong\u003eb\u003c/strong\u003e white,\u003cstrong\u003ec \u003c/strong\u003ebrown, and\u003cstrong\u003e d \u003c/strong\u003eblack with visible crystals.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6801134/v1/183d1c2f65a6fd3f86780efc.png"},{"id":88814643,"identity":"54b9e692-82f6-4c7f-9dd7-c5b273f41a68","added_by":"auto","created_at":"2025-08-11 16:09:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":59634718,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6801134/v1/41962fc3-3192-4788-a0ad-9ae6c3a7bddf.pdf"},{"id":84071666,"identity":"9327ab0a-cd42-4b41-83cd-7417f215f665","added_by":"auto","created_at":"2025-06-06 12:23:31","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":131976,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-6801134/v1/08e92fa4a1ae0ace3a280a36.docx"}],"financialInterests":"","formattedTitle":"Historical analgesic tablets revisited: Development of a versatile RP-HPLC method for the stability study of single- and multi-component pharmaceutical preparations","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAnalgesics are drugs that induce a painless state, also known as analgesia. While acute pain has a biological function of warning the organism of something potentially harmful, chronic pain merely burdens the organism in many ways. The biological function makes pain an important and inseparable part of life and the most common reason for seeking a suitable medication. Although certain preparations were historically used to soothe pain, the documented history of analgesic drugs does not truly begin until the mid-19th century [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe early discoveries of analgesic drugs depended on geographical conditions and were obtained empirically. Not only were certain plants (poppy, valerian, hops, or mandrake) utilized, but also the appease effect of ethanol was employed to induce analgesia. One of the most potent and historically significant analgesic drugs in history remains opium [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. This dried latex of opium poppy (\u003cem\u003ePapaver somniferum\u003c/em\u003e L.) has been used for thousands of years, and has served as an inspiration for the development of many analgesic drugs, some of which have indeed often been misused for other purposes.\u003c/p\u003e \u003cp\u003eThe development of science in the 19th century led to the discovery and synthesis of many new compounds including some with analgesic effects. The development of industry further enabled the production of a wide range of different analgesic pharmaceutical preparations which nowadays vary in their active pharmaceutical ingredients (APIs) and excipients. In the case of analgesic preparations, not only single-component preparations but also variable combinations of APIs are often used. These preparations remain a fundamental and indispensable part in the management of both acute and chronic pain conditions in clinical practice.\u003c/p\u003e \u003cp\u003eIn this study, the development of a versatile RP-HPLC method for the stability study of single- and multi-component analgesic preparations is presented. The developed method was subsequently used for the analysis of a unique set of twelve historical analgesic preparations: six single-component (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and six multi-components (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These preparations contained in total twelve APIs: acetylsalicylic acid, codeine, caffeine, bromisoval, narcotine, phenacetine, phenobarbital, salicylic acid, and four different barbiturates (see below for the exact composition of specific products).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo characterize the individual APIs used in the analyzed preparations, the following can be briefly mentioned. An important and widely known compound with analgesic effects is acetylsalicylic acid (ASA), also often known as aspirin [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In fact, it is a prodrug; the active compound is salicylic acid which is formed in the gastrointestinal tract by hydrolysis. Both acids have analgesic, anti-inflammatory, and antipyretic effects, which have led to their widespread use in medicine. However, the disadvantage of salicylic acid is its unpleasant taste and strong stomach irritation, making the use of acetylsalicylic acid more tolerable. Since it is an inhibitor of cyclooxygenases, it reduces blood clotting, and it is also a proven teratogen, caution is needed when using it. Acetylsalicylic acid was first synthesized from salicylic acid and acetic anhydride by Charles von Gerhart in 1853, and Felix Hoffman later prepared it for the German company Bayer in 1897 [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The inspiration for the discovery and analgesic use of this compound was willow bark decoction, which had been used for its weak analgesic effect since times immemorial, and the Latin name of willow (\u003cem\u003eSalix\u003c/em\u003e) is the root of the acid\u0026rsquo;s name.\u003c/p\u003e \u003cp\u003eCodeine (methylmorphine) is an opioid used for its antitussive and analgesic effects, often in combination with other non-opioid analgesic substances, such as acetylsalicylic acid or paracetamol. Like other opioids, codeine has the dangerous disadvantage of possible addiction because, similar to morphine, it releases histamine in the body, and its mild euphoric and sedative effects are the reason for its misuse. The name is derived from the Greek word for the capsule of the poppy head (κωδεια). Codeine was discovered by Pierre-Jean Robiquet, who identified it by chance while researching morphine derivatives in 1832 [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA few years earlier, in 1817, the same person discovered another opioid alkaloid, narcotine (noscapine) whose antitussive activity was discovered in 1930 [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In contrast to codeine, narcotine lacks significant analgesic, sedative, and euphoric effects; therefore, there is no problem of addiction.\u003c/p\u003e \u003cp\u003eCaffeine is an alkaloid and a strong stimulant of the central nervous system. Although it has no analgesic effect itself, it is often used in analgesic preparations because it can potentiate the effect of other active substances (e.g., paracetamol). Like other xanthine alkaloids, caffeine was used for its diuretic effect until the introduction of thiazides [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Caffeine was first isolated from coffee beans by Ferdinand Friedlieb Runge in the first half of the 19th century.\u003c/p\u003e \u003cp\u003eBarbiturates (in this work allo-, apro-, pheno-, and butobarbital) are derivatives of barbituric acid (malonylurea), which was first synthesized by Adolf von Baeyer in 1864 [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Despite barbituric acid itself having no specific effects, its derivatives have diverse therapeutic applications, and more than 2,500 of them have been synthesized to date. However, only about fifty of barbiturates have been used for their sedative and hypnotic properties in treatment. Barbiturates affect the central nervous system and were the first truly effective tools for managing epileptic seizures. Nevertheless, the risk of addiction and death by overdose has led to a decrease in the use of these compounds. Phenobarbital was synthesized by H\u0026ouml;rlein in 1911 and was the first barbiturate used for epilepsy treatment.\u003c/p\u003e \u003cp\u003eBromisoval (bromvalerlurea) is a monoureide derivative, synthesized from isopentyl alcohol, and has been used for its mild, short-acting sedative effect since 1908 [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Nowadays, it is largely obsolete and is rarely used due to its high potential for toxicity. The problem with the therapeutic use of bromisoval is its ability to cause excessive sedation and chronic intoxication characterized by confusion, ataxia, disorientation, delirium, hallucinations, and diplopia.\u003c/p\u003e \u003cp\u003eIn 1887, Carl Hinsberg first prepared phenacetin (acetophenetidin) while seeking a use for an accumulated byproduct of dye works, \u003cem\u003ep\u003c/em\u003enitrophenol [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Phenacetin was used for almost a century for its analgesic and antipyretic effects. When combined with caffeine or administered in higher doses, it has a euphoric effect, which was often the cause of chronic poisoning typically manifested by grayish-blue skin coloration. Moreover, chronic use causes kidney damage; thus, this substance was withdrawn in many countries and replaced by its metabolite, paracetamol, which is used instead[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAminophenazone (aminopyrine), a derivative of phenazone, was first synthesized by Friedrich Stolz in 1893 [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Both phenazone and aminophenazone have analgesic, antipyretic and anti-inflammatory effects. The high popularity of aminophenazone lasted until the 1960s when its market availability decreased due to its unfavorable side effects (e.g., agranulocytosis).\u003c/p\u003e \u003cp\u003eThe stability of pharmaceutical preparations is one of the most important criteria that must be controlled, because physical and chemical degradation of the preparations \u0026ndash; or of the APIs \u0026ndash; can cause changes in therapeutic efficacy or even result in toxic effects [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Therefore, many stability tests are conducted before the preparation can be introduced onto the market and approved for use. The interest in studying the long-term stability and shelf-life of medicinal preparations is illustrated by the Shelf-Life Extension Program, which was initiated in the USA in 1986 [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. This program aimed to identify opportunities to extend the expiration date of drugs appropriately stored in government emergency stockpiles, thereby reducing the costs associated with the regular replacement of these stockpiles. In 2019, Zilker et al. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]extensively reviewed available data on the stability of finished pharmaceutical products and drug substances beyond their labeled expiry dates, and discussed the pros and cons of possible extending shelf-life beyond the usual five-year limit. More recent studies addressing this issue are reviewed in our recent publication[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The longterm stability of APIs in decades-old pharmaceutical products has been a persistent research focus in our laboratory [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. We have focused on the stability of various injectable solutions [\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], as well as solid dosage forms [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003eRP-HPLC method development\u003c/p\u003e \u003cp\u003eDrawing on the literature [\u003cspan additionalcitationids=\"CR24 CR25 CR26\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], the binary mobile phase, based on 0.1% aqueous acetic acid (solvent A) and methanol (solvent B), was selected. Based on the dissociation constants of all twelve analytes considered (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), the pH of the aqueous component of the mobile phase was set to 5.00 (adjusted by the addition of an aqueous ammonia), at which most of the analytes are present in solution exclusively in either fully protonated or fully deprotonated form.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor the optimization of RP-HPLC method, three different types of columns were tested. Traditionally, the C18 stationary phase is the first option for reverse-phase methods. Using XBridge\u0026reg; BEH C18 2.5 \u0026micro;m (150\u0026times;3.0 mm) column, isocratic elution with methanol (solvent B) concentrations ranging from 40\u0026ndash;50%, and a total runtime of 45 min, constituted the initial step in the gradient program development. Subsequently, various gradient programs were tested, with solvent B ranging from 20\u0026ndash;80% and different total runtimes. However, two analytes \u0026ndash; bromisoval and phenobarbital \u0026ndash; both concurrently present in \u003cem\u003eVeralgin\u003c/em\u003e and, unfortunately, sharing a similar absorption maximum available for UV detection, coeluted and could not be separated by modification of the gradient. Subsequently, two other stationary phases were tested (i) XBridge\u0026reg; BEH Amide 2.5 \u0026micro;m (150\u0026times;3.0 mm), and (ii) Kinetex 2.5 \u0026micro; Phenyl-Hexyl 100\u0026Aring; (100\u0026times;3.0 mm). The Phenyl-Hexyl stationary phase provided the best separation of most of the analytes. The only remaining issue was the coelution of phenobarbital and aminophenazone, concurrently present in \u003cem\u003eVeralgin\u003c/em\u003e, which could not be resolved by alternating the gradient. Fortunately, these two analytes significantly differ in their UV absorption maxima available for detection (phenobarbital at 210 nm and aminophenazone at 260 nm). Therefore, baseline separation was not required for quantification, and the issue was effectively addressed by employing dual-wavelength detection (see below). Moreover, a slight modification of the gradient program shortened the total runtime of analysis from 38 to 30 minutes. Thus, the Phenyl-Hexyl stationary phase was selected as the best option for the analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCalibration curves for studied APIs\u003c/p\u003e \u003cp\u003eUnder optimized conditions for RP-HPLC separation, calibration curves based on standards of the analyzed APIs were measured in the range of 10.00\u0026ndash;150.0 mg dm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e. UV/VIS detection was used, and the area of the chromatographic peak measured at the absorption maximum of each analyte served as the analytical signal. The obtained and statistically evaluated parameters of the calibration curves are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The resolution calculated from retention parameters, was \u0026ge;\u0026thinsp;1.3 for all peaks.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eParameters of the calibration curves for RPHPLC\u0026ndash;UV determination of APIs based on peak area (analyte, retention time, detection wavelength, slope, intercept, correlation coefficient, limit of quantification). Measurements were performed on a Kinetex 2.5 \u0026micro; Phenyl-Hexyl 100\u0026Aring; (100\u0026times;3.0 mm) column using gradient elution with (A) 0.1% aqueous acetic acid (pH\u0026thinsp;=\u0026thinsp;5.00) and (B) methanol (for gradient program see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), flow rate 0.4 cm\u003csup\u003e3\u003c/sup\u003e min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompound, CASRN\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003et\u003c/em\u003e\u003csub\u003er\u003c/sub\u003e/min\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eλ/nm\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSlope\u003c/p\u003e \u003cp\u003e/\u0026micro;AU min mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dm\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIntercept\u003c/p\u003e \u003cp\u003e/\u0026micro;AU min\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eR\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eLOQ\u003c/em\u003e/mg dm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcetylsalicylic acid, [50-78-2]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e210\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.9887\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSalicylic acid, [69-72-7]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e210\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e15700\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.9994\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e15.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCodeine, [76-57-3]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e210\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8900\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.9996\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCaffeine, [58-08-2]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e271\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7900\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.9999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAllobarbital, [52-43-7]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e210\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.9987\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBromisoval, [496-67-3]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e210\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.9994\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhenacetine, [62-44-2]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e245\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10700\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.9998\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8.44\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAprobarbital, [77-02-1]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e210\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.9997\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhenobarbital, [50-06-6]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e11.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e210\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.9995\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAminophenazone, [58-15-1]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e11.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e260\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026minus;11000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.9999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eButobarbital, [77-28-1]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e16.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e210\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5620\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026minus;7000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNarcotine, [128-62-1]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e24.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e210\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e19900\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.9997\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e10.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003e \u003cem\u003ea\u003c/em\u003e \u003c/sup\u003e Statistically insignificant.\u003c/p\u003e \u003cp\u003eIn order to solve the problem of quantifying phenobarbital and aminophenazone due to their co-elution, calibration curves were measured in the concentration range of 10\u0026ndash;150 mg dm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e for one analyte at a fixed concentration of the second analyte (at levels 0.0, 10.0, 80.0, and 150.0 mg dm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e). For both analytes, the areas of the chromatographic peaks at wavelengths of 210 and 260 nm served as the analytical signal. From the obtained data, it was possible to construct a system of two equations with two unknowns, which allows determination of the concentration of phenobarbital and aminophenazone in the case of co-elution\u003c/p\u003e \u003cp\u003e \u003cem\u003eA\u003c/em\u003e \u003csub\u003e210\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;8000 \u003cem\u003ec\u003c/em\u003e\u003csub\u003eA\u003c/sub\u003e + 8600 \u003cem\u003ec\u003c/em\u003e\u003csub\u003ep\u003c/sub\u003e (1)\u003c/p\u003e \u003cp\u003e \u003cem\u003eA\u003c/em\u003e \u003csub\u003e260\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;5800 \u003cem\u003ec\u003c/em\u003e\u003csub\u003eA\u003c/sub\u003e + 380 \u003cem\u003ec\u003c/em\u003e\u003csub\u003ep\u003c/sub\u003e (2)\u003c/p\u003e \u003cp\u003ewhere \u003cem\u003eA\u003c/em\u003e\u003csub\u003e210\u003c/sub\u003e and \u003cem\u003eA\u003c/em\u003e\u003csub\u003e260\u003c/sub\u003e are the peak areas at 210 and 260 nm, respectively [\u0026micro;AU min mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dm\u003csup\u003e3\u003c/sup\u003e], \u003cem\u003ec\u003c/em\u003e\u003csub\u003eA\u003c/sub\u003e is the concentration of aminophenazone [mg dm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e], and \u003cem\u003ec\u003c/em\u003e\u003csub\u003eP\u003c/sub\u003e is the concentration of phenobarbital [mg dm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e]. The accuracy of the system was verified using five training sets of solutions with different concentration of analytes.\u003c/p\u003e \u003cp\u003eFinally, the potential influence of ultrasound treatment and filtration using a syringe filter during the extraction of the specified APIs from historical analgesic tablets on the quantification of the analyte was evaluated. It was found that neither of these operations affected the quantification.\u003c/p\u003e \u003cp\u003eQuantification of APIs in historical analgesic tablets analyzed\u003c/p\u003e \u003cp\u003eTwelve historical analgesic preparations were analyzed using the developed RP-HPLC method; three tablets from each preparation were analyzed individually. The results, including statistical evaluation, are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eQuantification of APIs in historical analgesic tablets analyzed (preparation, date of manufacture; API and declared content; measured API content expressed as a percentage of the declared amount).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePreparation, date of manufacture\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"6\" nameend=\"c7\" namest=\"c2\"\u003e \u003cp\u003eMeasured / % of declared content\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompound, declared content\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTablet #1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTablet #2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTablet #3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMedian\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eL\u003c/em\u003e\u003csub\u003e1,2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003es\u003c/em\u003e\u003csub\u003e\u003cem\u003er\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e/%\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAcylpyrin\u003c/b\u003e, \u003cb\u003e1986\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcetylsalicylic acid, 500 mg/tbt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e90.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e93.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e92.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e92.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.023\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRecalculated ASA\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e92.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e96.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e95.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e95.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.026\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAcylpyrin effervescens\u003c/b\u003e, \u003cb\u003e1982\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcetylsalicylic acid, 500 mg/tbt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt; \u003cem\u003eLOD\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt; \u003cem\u003eLOD\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003en.a.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003en.a.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRecalculated ASA\u003csup\u003e\u003cem\u003ea, b\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e105.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e94.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e105.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e105.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.065\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAcidum acetylosalicylicum\u003c/b\u003e, \u003cb\u003e1953\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcetylsalicylic acid, 500 mg/tbt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e102.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e86.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e87.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e87.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e19.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.103\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRecalculated ASA\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e104.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e89.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e91.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e91.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e19.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.096\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCodeinum phosphoricum\u003c/b\u003e, \u003cb\u003e1953\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCodeine, 20 mg/tbt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e86.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e88.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e88.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e88.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.018\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCodein\u003c/b\u003e, \u003cb\u003e1993\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCodeine, 15 mg/tbt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e75.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e73.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;\u003csup\u003e\u003cem\u003ec\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e74.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026ndash;468.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026ndash;0.871\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIsoneurin\u003c/b\u003e, \u003cb\u003eca. 1940s\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBromisoval, 300 mg/tbt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e95.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e95.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e92.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e95.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.019\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAlgena\u003c/b\u003e, \u003cb\u003e1981\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCaffeine, 50 mg/tbt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e91.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e90.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e92.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e91.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.017\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAprobarbital, 50 mg/tbt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e96.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e90.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e88.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e90.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.049\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhenacetine, 150 mg/tbt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e103.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e96.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e100.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.040\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAminophenazone, 300 mg/tbt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e98.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e93.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e86.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e93.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.077\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAlnagon\u003c/b\u003e, \u003cb\u003e1983\u003c/b\u003e\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcetylsalicylic acid, 380 mg/tbt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e41.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.020\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCodeine, 20 mg/tbt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e46.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e58.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e59.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e58.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e16.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.124\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCaffeine, 80 mg/tbt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e86.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e93.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e90.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e90.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.050\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhenobarbital, 20 mg/tbt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e104.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e97.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e94.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e97.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.061\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRecalculated ASA\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e105.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e107.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e105.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e105.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.009\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAlnagon\u003c/b\u003e, \u003cb\u003e1990\u003c/b\u003e\u003csup\u003e\u003cb\u003ed\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcetylsalicylic acid, 380 mg/tbt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e46.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e60.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e46.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e33.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.327\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCodeine, 20 mg/tbt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e53.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e67.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e53.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e22.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.191\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCaffeine, 80 mg/tbt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e77.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e103.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e98.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e98.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e34.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.159\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhenobarbital, 20 mg/tbt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e101.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e113.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e87.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e101.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e33.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.151\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRecalculated ASA\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e95.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e117.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e122.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e117.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e34.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.135\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVeralgin\u003c/b\u003e, \u003cb\u003e1973\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCaffeine, 25 mg/tbt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e101.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e99.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e86.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e99.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e18.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.085\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBromisoval, 250 mg/tbt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e108.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e108.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e101.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e108.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.037\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhenobarbital, 15 mg/tbt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e107.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e113.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e103.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e107.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.056\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAminophenazone, 200 mg/tbt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e99.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e103.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e92.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e99.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.061\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDinyl\u003c/b\u003e, 1977\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCaffeine, 50 mg/tbt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e101.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e91.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e96.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e96.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.061\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAllobarbital, 18 mg/tbt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e93.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e91.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e94.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e93.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.017\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhenacetin, 200 mg/tbt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e106.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e103.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e103.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.036\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAminophenazone, 200 mg/tbt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e91.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e95.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e94.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e94.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.025\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eButobarbital, 12 mg/tbt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e109.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e109.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e111.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e109.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.013\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGelonida\u003c/b\u003e, \u003cb\u003eca. 1940s\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcetylsalicylic acid, 250 mg/tbt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e74.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e76.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e88.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e76.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e19.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.113\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhenacetine, 250 mg/tbt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e103.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e99.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e101.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e101.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.027\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNarcotine, 30 mg/tbt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e103.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e89.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e81.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e89.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e28.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.145\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRecalculated ASA\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e102.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e99.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e108.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e102.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e11.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.052\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003e \u003cem\u003ea\u003c/em\u003e \u003c/sup\u003e Recalculated content of acetylsalicylic acid based on determined amount of salicylic acid, considering the hydrolysis of acetylsalicylic acid to salicylic acid.\u003c/p\u003e \u003cp\u003e \u003csup\u003e \u003cem\u003eb\u003c/em\u003e \u003c/sup\u003e Two tablets were homogenized together during measurements 2 and 3.\u003c/p\u003e \u003cp\u003e \u003csup\u003e \u003cem\u003ec\u003c/em\u003e \u003c/sup\u003e Only two tablets were available in the package analyzed.\u003c/p\u003e \u003cp\u003e \u003csup\u003e \u003cem\u003ed\u003c/em\u003e \u003c/sup\u003e Analysis of brown tablets; discussed in more detail below.\u003c/p\u003e \u003cp\u003eAcetylsalicylic acid was a relatively problematic analyte for accurate quantification due to its instability \u0026ndash; namely, hydrolysis to salicylic acid \u0026ndash; in all commonly used organic solvents for HPLC [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], as well as its low solubility in water. Therefore, all solutions (historical samples and calibration solutions) containing this analyte were analyzed immediately after preparation in methanol, with a maximum time delay of 15 min. To evaluate the effect of acetylsalicylic acid hydrolysis on the quantification accuracy, a methanolic solution of acetylsalicylic acid at a concentration level in the middle of the calibration range (75 mg dm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e) was repeatedly analyzed over time. The results showed that even after one hour, the error did not exceed 3.5% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Thus, the \u0026ldquo;Recalculated ASA\u0026rdquo; parameter is introduced in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, to represent the presumed amount of acetylsalicylic acid originally present in the analyzed tablet prior to extraction of given tablet by methanol. The recalculated ASA value is the sum of the measured amount of acetylsalicylic acid and the amount of acetylsalicylic acid equivalent to the measured salicylic acid amount, assuming hydrolysis of acetylsalicylic acid to salicylic acid in a 1:1 molar ratio. The difference between \u0026ldquo;Recalculated ASA\u0026rdquo; and the directly measured amount of acetylsalicylic acid indicates the extent of its hydrolytic degradation that occurred during long-term storage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAcetylsalicylic acid is the only API in the first three singlecomponent preparations analyzed. In the case of tablets \u003cem\u003eAcylpyrin\u003c/em\u003e (manufactured in 1986), 92.1% of acetylsalicylic acid remained after 39 years of their production. Similarly, tablets \u003cem\u003eAcidum acetylosalicylicum\u003c/em\u003e (manufactured in 1953) remained 87.2% of declared content even after 72 years of production. This represents remarkable stability, in contrast to Cantrell et al. [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], who reported more than 90% degradation of acetylsalicylic acid in multicomponent pharmaceutical preparations aged 28\u0026ndash;40. On the other hand, different results were obtained from the analysis of effervescent tablets \u003cem\u003eAcylpyrin effervescens\u003c/em\u003e (manufactured in 1982). In this package, all 15 tablets present had completely disintegrated into powder, such that their original shape was no longer discernible. The package was filled to the brim with a light greyish to brownish powder, and the odor of the content was also notable. Despite the disintegration, the entire contents of the package were weighed, homogenized, and an aliquot was analyzed. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, salicylic acid was identified as the major constituent, and acetylsalicylic acid was detected only in one of the three aliquots. The appearance, along with the results, indicates a high degree of degradation of acetylsalicylic acid in effervescent tablets and suggests lower long-term stability of this dosage form.\u003c/p\u003e \u003cp\u003eSurprisingly high instability was observed in the case of codeine, contrary to previously reported data in the literature [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Codeine was relatively stable in the single-component tablets \u003cem\u003eCodeinum phosphoricum\u003c/em\u003e (manufactured in 1953), with 88.4% of the declared content remaining after 72 years. In contrast, in 40-year-younger single-component tablets \u003cem\u003eCodein\u003c/em\u003e (manufactured in 1993), only 74.5% of declared content was found. This lower content in the more recent preparation may be attributed to differences in the storage conditions. However, the percentage of codeine was even lower in both multi-component \u003cem\u003eAlnagon\u003c/em\u003e tablets: 58.9% in the 1983 batch and 53.3% in the 1990 batch, respectively. The results were verified using two independent calibration curves, based on two different batches of codeine standards. Furthermore, no degradation products were detected by UV/Vis or mass spectrometry. It can therefore be hypothesized that the source of the low codeine content in both analyzed preparations may be due to a manufacturing error.\u003c/p\u003e \u003cp\u003eFrom the perspective of codeine degradation, the analysis of tablets \u003cem\u003eAlnagon\u003c/em\u003e (manufactured in 1990) was particularly informative, as it showed a strong correlation between codeine content and the physical states of tablet degradation. Three distinct types of tablets, differing visibly in their degree of degradation, were observed within the package (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e): (i) one white tablet, solid, with some darker spots; (ii) five brown tablets, solid, with colorless crystals on their surface; and, (iii) two black tablets, soft, with abundant colorless crystals on their surface. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the APIs content differed across these tablet types. The amount of acetylsalicylic acid was 97.4% in the white tablet, 46.9% in the brown tablet, and below the limit of detection in the black tablet. A similar trend was observed in the case of codeine. Caffeine content appeared relatively stable in all tablet types, although in one of three brown tablets analyzed, it dropped to 77.0% (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The results also indicated high stability of phenobarbital across tablet types. Mass spectrometry was employed to verify these findings; however, no degradation products or compound responsible for the discoloration were identified \u0026ndash; except salicylic acid, which was confirmed as the degradation product of acetylsalicylic acid. It is likely that colorless crystals are salicylic acid, as their presence correlated with more advanced stages of degradation. Therefore, it seems to be probable that the original codeine content was lower than declared by manufacturer. The influence of different codeine forms (e.g., phosphate, sulfate, hemihydrate, sesquihydrate) was also considered. However, even if a different form of codeine than declared by the manufacturer was used during production, the variation in codeine content would not exceed 5%. The observed differences among the three tablet types most likely resulted from external factors during the storage. The presence of black tablets alongside white and brown ones is unusual, as one would typically expect a gradual transition in colors. Unfortunately, the exact storage conditions are unknown, so the factor(s) responsible for this variation \u0026ndash; such as heat, humidity, or light \u0026ndash; can only be hypothesized. These findings underscore the importance of storage conditions and their evaluation when analyzing expired pharmaceutical products. On the other hand, undefined conditions often better reflect the real-world storage environment of pharmaceuticals in everyday use [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eQuantification of APIs in historical analgesic preparation \u003cem\u003eAlnagon\u003c/em\u003e, manufactured in 1990 (API and declared content; measured API content expressed as a percentage of the declared value).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAPI, declared content\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eMeasured / % of declared content\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWhite tablet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBrown tablet\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBlack tablet\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcetylsalicylic acid, 380 mg/tbt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e97.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt; \u003cem\u003eLOD\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eASA recalculated\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e115.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e117.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e134.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCodeine, 20 mg/tbt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e77.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e53.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e38.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCaffeine, 80 mg/tbt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e97.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e98.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e96.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhenobarbital, 20 mg/tbt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e96.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e101.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e102.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003e \u003cem\u003ea\u003c/em\u003e \u003c/sup\u003e Recalculated content of acetylsalicylic acid based on determined amount of salicylic acid, considering the hydrolysis of acetylsalicylic acid to salicylic acid.\u003c/p\u003e \u003cp\u003e \u003csup\u003e \u003cem\u003eb\u003c/em\u003e \u003c/sup\u003e Median value from three brown tablets analyzed.\u003c/p\u003e \u003cp\u003eHigh stability of bromisoval was revealed by the analysis of the single-component preparation tablets \u003cem\u003eIsoneurin\u003c/em\u003e (manufactured in the 1940s), in which 95.4% of the declared content was retained even after approximately 85 years, despite the fragile physical state of the tablets. Similarly, multi-component tablets \u003cem\u003eVeralgin\u003c/em\u003e (manufactured in 1973) showed 108.7% of declared amount of bromisoval. This finding is particularly important, as no data on bromisoval stability have been reported in the literature to date.\u003c/p\u003e \u003cp\u003eCaffeine, phenacetine, aminophenazone, and all four barbiturates (aprobarbital, phenobarbital, allobarbital, and butobarbital) exhibited high stability in all tested preparations, with deviations within \u0026plusmn;\u0026thinsp;10% of the declared amount, which is consistent with published data [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe content of narcotine \u0026ndash; present only in tablets \u003cem\u003eGelonida antineuralgica\u003c/em\u003e (manufactured in the 1940s) \u0026ndash; was slightly below the \u0026plusmn;\u0026thinsp;10% threshold set by current pharmacopoeial standards. Approximately 85 years after manufacturing, the content of narcotine was found to be 89.3% of the declared value. As no degradation products were detected by mass spectrometry, this minor content may be attributed to manufacturing inaccuracy.\u003c/p\u003e \u003cp\u003eHigh‑resolution tandem mass spectrometry of the studied APIs\u003c/p\u003e \u003cp\u003eAll historical samples and API standards were also analyzed by RPHPLC coupled with a high-resolution tandem mass spectrometer to confirm their identity in the respective preparations and to detect potential degradation products. The ions of studied APIs, as identified by tandem mass spectrometry, are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. A collision energy of 35.0 eV was used for MS\u003csup\u003e2\u003c/sup\u003e fragmentation of the analytes. For barbiturates, only precursor ions were measured, as their MS\u003csup\u003e2\u003c/sup\u003e fragments could not be obtained at this high collision energy. The diagnostic ions of other APIs were consistent with values reported in the literature.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe \u003cem\u003em/z\u003c/em\u003e ions of studied APIs identified by tandem mass spectrometry, used for compound confirmation (compound, type and \u003cem\u003em/z\u003c/em\u003e of the molecular ion, \u003cem\u003em/z\u003c/em\u003e of diagnostic ions, and reference to literature). Only the ions with a reasonable molecular formula and mass error\u0026thinsp;\u0026le;\u0026thinsp;6 ppm were considered.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eParent ion\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDiagnostic ions \u003cem\u003em/z\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eRef.\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eType\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003em/z\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eExper.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTheor.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eΔ ppm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcetylsalicylic acid\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e[M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e203.0316\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e203.0315\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e121.0281\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAllobarbital\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e[M\u0026thinsp;\u0026minus;\u0026thinsp;H]\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e207.0772\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e207.0775\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026minus;1.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e164.0718\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAminophenazone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e[M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e232.1441\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e232.1444\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e187.0862; 177.1014; 159.0913; 149.1073; 139.0864; 113.1073; 98.0837\u003csup\u003e\u003cem\u003ec\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAprobarbital\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e[M\u0026thinsp;\u0026minus;\u0026thinsp;H]\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e209.0930\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e209.0932\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026minus;0.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e166.0871\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBromisoval\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e[M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e223.0076\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e223.0077\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026minus;0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e181.9993; 134.9810; 119.9452; 101.0841; 86.0602; 69.0602; 69.0338; 57.0703\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003en.a.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eButobarbital\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e[M\u0026thinsp;\u0026minus;\u0026thinsp;H]\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e211.1090\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e211.1088\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e168.1028\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCaffeine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e[M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e195.0875\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e195.0877\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026minus;0.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e138.0659; 123.0426; 110.0711\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCodeine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e[M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e300.1590\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e300.1594\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026minus;1.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e282.1485; 267.1239; 243.1010; 225.0905; 215.1061; 199.0749; 183.0799; 161.0593\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNarcotine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e[M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e414.1553\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e414.1547\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e353.1013; 323.0907; 220.0969; 206.0810; 179.0701\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhenacetine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e[M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e180.1017\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e180.1019\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026minus;1.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e138.0915; 110.0602; 93.0336; 82.0654\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhenobarbital\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e[M\u0026thinsp;\u0026minus;\u0026thinsp;H]\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e231.0773\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e231.0775\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026minus;0.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e188.0723\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSalicylic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e[M\u0026thinsp;\u0026minus;\u0026thinsp;H]\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e137.0246\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e137.0244\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e93.0347; 65.0401\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003e \u003cem\u003ea\u003c/em\u003e \u003c/sup\u003e Sodium adduct caused by the internal standard (sodium formate).\u003c/p\u003e \u003cp\u003e \u003csup\u003e \u003cem\u003eb\u003c/em\u003e \u003c/sup\u003e Ions obtained by MS\u003csup\u003e2\u003c/sup\u003e fragmentation experiments.\u003c/p\u003e \u003cp\u003e \u003csup\u003e \u003cem\u003ec\u003c/em\u003e \u003c/sup\u003e Dimer ion.\u003c/p\u003e \u003cp\u003eThe mass spectra of all APIs studied in this work have already been well described in the literature, with the exception of bromisoval. For bromisoval, only molecular ion \u003cem\u003em/z\u003c/em\u003e values have been reported thus far [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Therefore, the tandem mass spectrum of bromisoval in the positive mode was studied in detail. The ion at \u003cem\u003em/z\u003c/em\u003e\u0026thinsp;=\u0026thinsp;223.0076 was selected as the precursor ion, its MS\u003csup\u003e2\u003c/sup\u003e spectrum and proposed fragmentation pathway are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eA robust RP-HPLC-UV method was developed and applied for the quantitative analysis of twelve historical analgesic preparations, containing twelve APIs of various structural classes. The method enabled effective separation and quantification, including cases of co-elution addressed via dual-wavelength detection.\u003c/p\u003e \u003cp\u003eThe results demonstrated that most APIs \u0026ndash; including barbiturates, phenacetine, aminophenazone, and caffeine \u0026ndash; retained high chemical stability over several decades, with contents within \u0026plusmn;\u0026thinsp;10% of the declared values. Bromisoval showed remarkable stability, despite a complete lack of prior stability data. Conversely, acetylsalicylic acid was stable in solid dosage forms but degraded significantly in effervescent tablets.\u003c/p\u003e \u003cp\u003eCodeine showed inconsistent results: high stability in older single-component tablets, but substantially lower content in multi-component formulations, without detectable degradation products \u0026ndash; suggesting possible manufacturing variability. Marked differences in degradation were observed among visually distinct tablets from a single \u003cem\u003eAlnagon\u003c/em\u003e batch (manufactured in 1990), underlining the impact of storage conditions.\u003c/p\u003e \u003cp\u003eHigh-resolution MS/MS was used to confirm compound identity and investigate degradation. Bromisoval fragmentation pattern was described here for the first time.\u003c/p\u003e \u003cp\u003eThese findings support the notion that many APIs can remain chemically stable long past their labeled expiration dates, although the effects of formulation type and storage conditions must be carefully considered.\u003c/p\u003e"},{"header":"Experimental","content":"\u003cp\u003eAnalyzed samples, standards of APIs, and chemicals\u003c/p\u003e \u003cp\u003eAll twelve historical analgesic preparations analyzed originate from collection of the Department of Analytical Chemistry, Faculty of Science, Charles University. An overview of all samples is provided in Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThe standards of the analyzed APIs were: acetylsalicylic acid (p.a., Herbacos Recordati, Czech Republic), salicylic acid (p.a., Merck), codeine phosphate hemihydrate (Ph. Eur. 11, L\u0026eacute;k\u0026aacute;rna Fakultn\u0026iacute; nemocnice Motol, Czech Republic), codeine phosphate (Ph. Eur. 11, Zentiva, Czech Republic), caffeine (extra pure, Fischer Chemical), allobarbital (p.a., Zentiva, Czech Republic), bromisoval (p.a., Zentiva, Czech Republic), phenacetine (for HPLC, Sigma-Aldrich), aprobarbital (p.a., L\u0026eacute;čiva Měcholupy, Czech Republic), phenobarbital (p.a., Zentiva, Czech Republic), aminophenazone (p.a., Sigma-Aldrich), butobarbital (p.a., Zentiva, Czech Republic) and narcotine (from the collection of the Department of Analytical Chemistry, Faculty of Science, Charles University [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]).\u003c/p\u003e \u003cp\u003eThe other chemicals used were: methanol (LC-MS grade, Fischer Chemical), acetic acid (p.a., Lach-ner, Czech Republic), aqueous ammonia 28% (p.a., VWR Chemicals, France) and deionized water with specific conductivity\u0026thinsp;\u0026lt;\u0026thinsp;0.05 \u0026micro;S cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, obtained using a Milli-Q system (Millipore, USA).\u003c/p\u003e \u003cp\u003eSamples preparation\u003c/p\u003e \u003cp\u003eEach historical sample tablet was weighed and placed in a 100 cm\u003csup\u003e3\u003c/sup\u003e volumetric flask, followed by dissolution in methanol. If necessary, samples were sonicated in an ultrasonic bath for 2 to 10 minutes until fully dissolved. Dilution to match the calibration concentration range was calculated based on the declared content of the respective analytes. The resulting extracts were filtered through a 0.2 \u0026micro;m PVDF centrifuge filter.\u003c/p\u003e \u003cp\u003eInstrumentation and procedures\u003c/p\u003e \u003cp\u003eFor RP-HPLC with UV/VIS detection, a UHPLC Nexera XR System equipped with DAD detection (Shimadzu) was used. The stationary phase was a Kinetex 2.5\u0026micro; Phenyl-Hexyl 100\u0026Aring; (100\u0026times;3 mm) column, maintained at 30\u0026deg;C. The binary mobile phase consisted of 0.1% aqueous acetic acid (solvent A) and methanol (solvent B). The mobile phase flow rate was set at 0.4 cm\u003csup\u003e3\u003c/sup\u003emin\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The gradient elution program (total runtime: 30 min) was as follows: 0\u0026thinsp;\u0026minus;\u0026thinsp;7 min: 30% B (isocratic); 7\u0026thinsp;\u0026minus;\u0026thinsp;15 min: linear gradient to 35% B; 15\u0026thinsp;\u0026minus;\u0026thinsp;20 min: linear gradient to 70% B; 20\u0026thinsp;\u0026minus;\u0026thinsp;25 min: 70% B (isocratic); 25\u0026ndash;26 min: return to 30% B, 26\u0026ndash;30 min: 30% B (re-equilibration). The volume of the injected sample was 2 mm\u003csup\u003e3\u003c/sup\u003e. The detection wavelengths were: 271 nm for caffeine, 245 nm for phenacetin, 260 nm for aminophenazone, 210 nm for all other analytes. Data were processed using LabSolutions 2010 (Shimadzu).\u003c/p\u003e \u003cp\u003eFor HPLC with MS/MS detection, an Agilent 1290 Infinity II LC System with a binary pump model was operated under the same conditions as the HPLC\u0026ndash;UV method. High-resolution tandem mass spectrometry was performed using a Bruker QqTOF compact instrument, operated via Compass otof Control 4.0 software (Bruker Daltonics, Germany). Data were processed with Compass DataAnalysis 4.4 (Build 200.55.2969) software (Bruker Daltonics, Germany). ESI-MS\u003csup\u003e2\u003c/sup\u003e data were collected in both positive and negative ion modes, within a mass range of \u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e 50\u0026ndash;1000. The drying gas temperature was 220\u0026deg;C, with a flow rate 5.0 dm\u003csup\u003e3\u003c/sup\u003e min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The capillary (cone) voltage was 2,800 V. Fragmentation data were interpreted using Compass Compound Crawler 3.0 (Bruker, Germany).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThe financial support by the project Cooperatio Chemistry of Charles University is gratefully acknowledged.\u003c/p\u003e\n\u003cp\u003eConflict of interest\u003c/p\u003e\n\u003cp\u003eThe authors have no financial or proprietary interests in any material discussed in this article.\u003c/p\u003e\n\u003cp\u003eData availability statement\u003c/p\u003e\n\u003cp\u003eThe experimental data supporting the findings of this study are available from the corresponding author, T.L., upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRainsford KD (2004) Aspirin and Related Drugs. 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Elsevier, Amsterdam\u003c/li\u003e\n\u003cli\u003eSkibinski R, Komsta L (2016) Curr Issues Pharm Med Sci 29:39\u003c/li\u003e\n\u003cli\u003eCantrell L, Suchard JR, Wu A, Gerona RR (2012) Arch Intern Med 172:1685\u003c/li\u003e\n\u003cli\u003eLyon RC, Taylor JS, Porter DA, Prasanna HR, Hussain AS (2006) J Pharm Sci 95:1549\u003c/li\u003e\n\u003cli\u003eGikonyo D, Gikonyo A, Luvayo D, Ponoth P (2019) Afr Health Sci 19:2737\u003c/li\u003e\n\u003cli\u003eFagbohun OF, Joseph JS, Salami OA, Msagati TAM (2021) Biol Trace Elem Res 199:1633\u003c/li\u003e\n\u003cli\u003eWachełko O, Tusiewicz K, Zawadzki M, Szpot P (2023) J Pharm Biomed Anal 228:115318\u003c/li\u003e\n\u003cli\u003eSingh A, Thatikonda T, Kumar A, Wazir P, V V, Nandi U, Singh PP, Singh S, Gupta AP, Tikoo MK, Singh G, Vishwakarma R (2018) J Pharm Biomed Anal 149:387\u003c/li\u003e\n\u003cli\u003eGuo C, Gong L, Wang W, Leng J, Zhou L, Xing S, Zhao Y, Xian R, Zhang X, Shi F (2020) Int J Mass Spectrom 447:116250\u003c/li\u003e\n\u003cli\u003eTh\u0026ouml;rngren JO, \u0026Ouml;stervall F, Garle M (2008) J Mass Spectrom 43:980\u003c/li\u003e\n\u003cli\u003eCristoni S, Cantu M, Bernardi LR, Gerthoux P, Mocarelli P, Brambilla M, Gonella E, Guidugli F (2005) J Mass Spectrom 40:1609\u003c/li\u003e\n\u003cli\u003eFang ZZ, Krausz KW, Li F, Cheng J, Tanaka N, Gonzalez FJ (2012) Br J Pharmacol 167:1271\u003c/li\u003e\n\u003cli\u003eAlgethami FK, Jlizi S, Znati M, Elamin MR, Hamadi NB, Jannet HB (2024) Nat Prod Res 38:3881\u003c/li\u003e\n\u003cli\u003eKokatsu J, Yomoda R, Suwa T (1992) Chem Pharm Bull (Tokyo) 40:1517\u003c/li\u003e\n\u003cli\u003eHori Y, Fujisawa M, Shimada K, Hirose Y, Yoshioka T (2006) Biol Pharm Bull 29:7\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"monatshefte-fur-chemie-chemical-monthly","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mccm","sideBox":"Learn more about [Monatshefte für Chemie - Chemical Monthly](https://www.springer.com/journal/706)","snPcode":"706","submissionUrl":"https://www.editorialmanager.com/mccm/","title":"Monatshefte für Chemie - Chemical Monthly","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Analgesic Preparation, Degradation, RP-HPLC, Shelf life, Stability, Tandem Mass Spectrometry","lastPublishedDoi":"10.21203/rs.3.rs-6801134/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6801134/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA versatile RP-HPLC method using phenyl-hexyl stationary phase and gradient elution (0.1% aqueous acetic acid and methanol) was developed to investigate the chemical integrity of twelve historical analgesic single- and multi-component preparations (1940s to 1990s). UV/VIS detection, supported by tandem mass spectrometry, was employed for compound identification and potential degradation product screening. Twelve active pharmaceutical ingredients (APIs), including acetylsalicylic acid, bromisoval, caffeine, codeine, narcotine, phenacetine, phenobarbital, salicylic acid, and four different barbiturates. Calibration models were established with limits of quantification as low as 3.0 mg dm⁻³. Coelution of phenobarbital and aminophenazone was effectively resolved through mathematical correction using dual-wavelength detection. Most APIs demonstrated remarkable long-term stability (within ±10% of declared content), with the exception of codeine and acetylsalicylic acid in specific preparation. Effervescent and poorly stored tablets exhibited significant degradation, highlighting the importance of dosage form and storage conditions. In addition, the tandem mass spectrum of bromisoval is presented here for the first time in the literature, and a plausible fragmentation mechanism is proposed.\u003c/p\u003e","manuscriptTitle":"Historical analgesic tablets revisited: Development of a versatile RP-HPLC method for the stability study of single- and multi-component pharmaceutical preparations","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-06 12:15:27","doi":"10.21203/rs.3.rs-6801134/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-06-03T16:44:46+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-03T11:52:40+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-03T06:20:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"Monatshefte für Chemie - Chemical Monthly","date":"2025-06-02T06:20:04+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"monatshefte-fur-chemie-chemical-monthly","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mccm","sideBox":"Learn more about [Monatshefte für Chemie - Chemical Monthly](https://www.springer.com/journal/706)","snPcode":"706","submissionUrl":"https://www.editorialmanager.com/mccm/","title":"Monatshefte für Chemie - Chemical Monthly","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9deb4df8-900f-41e3-bd47-f80e2e07514f","owner":[],"postedDate":"June 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-11T16:06:41+00:00","versionOfRecord":{"articleIdentity":"rs-6801134","link":"https://doi.org/10.1007/s00706-025-03350-4","journal":{"identity":"monatshefte-fur-chemie-chemical-monthly","isVorOnly":false,"title":"Monatshefte für Chemie - Chemical Monthly"},"publishedOn":"2025-08-04 15:58:02","publishedOnDateReadable":"August 4th, 2025"},"versionCreatedAt":"2025-06-06 12:15:27","video":"","vorDoi":"10.1007/s00706-025-03350-4","vorDoiUrl":"https://doi.org/10.1007/s00706-025-03350-4","workflowStages":[]},"version":"v1","identity":"rs-6801134","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6801134","identity":"rs-6801134","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}