Development of simultaneous analysis method for 11 respiratory drug substances including theobromine and analysis of commercial products using UPLC-PDA

Development of simultaneous analysis method for 11 respiratory drug substances including theobromine and analysis of commercial products using UPLC-PDA | 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 Development of simultaneous analysis method for 11 respiratory drug substances including theobromine and analysis of commercial products using UPLC-PDA Mi Jin Kim, Hyunil Shin, Hwan Seong Choi, Nam Sook Kim, Ji Hyun Lee, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3871996/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Recently, the number of people suffering from respiratory symptoms has continued to increase owing to the effects of COVID-19 and particulate matter, and accordingly, the demand for respiratory-related dietary supplements is increasing. Over the past few years, dietary supplements adulterated with pharmaceutical drug substances have been appearing continuously; thus, preventive measures are needed. In this study, we developed a simultaneous analysis method for 11 respiratory-related pharmaceutical compounds using ultra-performance liquid chromatography with photodiode array (UPLC-PDA). The sample preparation method was optimized by comparing three clean-up methods including the QuEChERS for hard capsule, tablet, powder, and liquid formulations, and the analysis conditions were established by comparing various analysis parameters. In method validation, the limit of detection, limit of quantitation, specificity, linearity, recovery, precision, accuracy, matrix effect, and stability all met the standards of the ICH guidelines. Subsequently, 52 types of dietary supplements promoting relief of respiratory-related symptoms were purchased online and analyzed using UPLC-PDA. Theobromine was detected in one product, and the content was calculated to be 247.7 mg/g (117.1 mg/capsule). This study can contribute to preventively blocking dietary supplements adulterated with respiratory-related substances and can be used as a reference for future research. respiratory dietary supplement adulterated COVID-19 UPLC-PDA Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Recently, the number of people suffering from respiratory symptoms has been increasing owing to the COVID-19 pandemic and the increase in fine dust due to air pollution. Most people infected with the COVID-19 virus experience respiratory illnesses, such as cough and viral pneumonia [ 1 , 2 ]. In addition, particulate matter—floating dust with a diameter of ≤ 10 µm—can accumulate in the alveoli when inhaled by humans. Particulate matter can cause direct toxic damage, increasing lung inflammation and worsening respiratory symptoms [ 3 , 4 ]. Because of the increase in various factors that can worsen respiratory symptoms, interest in respiratory-related dietary supplements is increasing, and demand for some types of dietary supplements increased during the pandemic [ 5 ]. Meanwhile, as general interest in dietary supplements increases, various pharmaceutical compounds have been illegally mixed to achieve medicinal effects [ 6 ]. If pharmaceutical compounds are taken in excessive amounts, there is a risk of experiencing various side effects. Theophylline, which is used to treat respiratory diseases, was found to be contained in dietary supplements for weight control, and the FDA issued a warning against the use of these products [ 7 ]. In Korea, dietary supplements containing theophylline were added to the list of hazardous foods in 2017 [ 8 ]. To prevent this threat in advance, we developed a simultaneous analysis method for 11 respiratory drug substances used to treat respiratory symptoms (Fig. 1). A literature search revealed HPLC methods for caffeine analogues in food samples (coffee) such as theophylline and theobromine[ 9 ], analysis and method validation studies for noscapine in syrup form using HPLC[ 10 ], and HPLC analysis method studies for oxymetazoline in nasal spray formulations[ 11 ]. However, there have been no studies on a simultaneous analysis method for pharmaceutical compounds effective for respiratory symptoms in various dietary supplement formulations. Thus, for quickly analyzing samples of various formulations and reducing the amounts of time and manpower required, research to develop a simultaneous analysis method for 11 respiratory-related drug substances was conducted. In this study, the optimal sample preparation method was established by comparing the recovery rates of 11 compounds for three clean-up methods, and a simultaneous analysis method was developed and validated by comparing various UPLC analysis conditions (Fig. 2). Additionally, we analyzed 52 respiratory-related dietary supplement of various formulations, such as hard capsules, powder, tablets, and liquid. Materials and methods Chemicals and reagents The following standard materials were used in this study: theobromine, theophylline, dropropizine, noscapine, and dexamethasone were purchased from Toronto Research Chemicals (TRC, Toronto, ON, Canada); guaifenesin, azelastine, and fexofenadine were purchased from United States Pharmacopeia (USP, Rockville, MD, USA); tramazoline was purchased from European Pharmacopoeia (Strasbourg, France); olopatadine was purchased from Sigma–Aldrich (St. Louis, MO, USA); and oxymetazoline was purchased from Clearsynth (Mumbai, India). Sodium 1-hexanesulfonate, phosphoric acid (reagent grade), and formic acid (LC-MS grade) were purchased from Sigma–Aldrich (St. Louis, MO, USA), deionized water was prepared using a Milli-Q system (Millipore, Bedford, MA, USA), and acetonitrile and methanol (HPLC grade) were purchased from Merck (Darmstadt, Germany). All the solvents used in this study were filtered through a polytetrafluoroethylene (PTFE) filter (0.2 µm, Millipore) before analysis. Except for theobromine, standard stock solutions were prepared at a concentration of 1000 µg mL − 1 through dissolution in methanol. Theobromine was prepared at the same concentration through dissolution in an ACN:DW (60:40) solution. The stock solutions were stored in a refrigerator at approximately 4 ℃. For calibration curve and method validation, the stock solutions were diluted to the appropriate concentration using HPLC-grade methanol on the same day. Instrumental conditions The analysis method development and validation were conducted using the Waters Acquity UPLC System (Waters, Milford, CT, USA) equipped with a binary pump and photodiode array detector. An Acquity UPLC BEH C 18 Column (2.1 × 150 mm, 1.7 µm) was used for separating the analytes. The column oven temperature was set as 35 ℃. Ultraviolet (UV) absorption was measured at a wavelength of 205 nm, and the temperature of the autosampler was 8 ℃. The flow rate of the mobile phase and injection volume of analytes were 0.2 mL/min and 1.0 µL, respectively. The following mobile phases were selected: (A) 0.5 mM sodium 1-hexanesulfonate buffer in deionized water containing 0.1% phosphoric acid and (B) acetonitrile:water (90:10). The gradient elution was set as follows: 0.0–1.0 min (A: 95%, B: 5%), 8.0–9.5 min (A: 55%, B: 45%), 11.0–13.0 min (A: 40%, B: 60%), 15.5–18.5 min (A: 0%, B: 100%), 19.0–22.0 min (A: 95%, B: 5%). Comparison of clean-up methods To optimize the sample preparation method, various clean-up methods were compared to minimize the impact of sample matrix components. The removal of polar matrix components, fatty acids, fats, and sterols was considered. [ 12 , 13 ] A 0.2-µm PTFE filter and two types of dispersive SPE (d-SPE, commonly referred to as QuEChERS) kits were selected for comparison. For the comparison of the clean-up methods, testing samples were prepared at the same concentration: (a) filter only, (b) QuEChERS kit 1 (Thermo Fisher, Waltham, MA, USA, hereinafter referred as QuE 1), and (c) QuEChERS kit 2 (Waters, Rockwood, TN, USA, hereinafter referred as QuE 2) methods were compared to optimize the clean-up method (Fig. 3). The detailed comparison procedures are as follows. Sample extraction Sample types were categorized into solid and liquid, and the solid samples included hard capsules, tablets, and powder. For the solid samples, hard capsules and tablets were ground using a grinder (IKA Tube mill, Germany), including the outer shell. 1 g of each homogenized sample was weighed into a 50-mL volumetric flask, and 30 mL of 70% aqueous methanol was added. The mixture was extracted in an ultrasonic bath for 30 min and cooled at room temperature, and then 70% methanol was added to obtain a total volume of 50 mL. The extracts were centrifuged for 5 min at 4000 rpm at 8 ℃, and the supernatant was used as a sample extract. (a) Filter only The sample extract (2 mL, prepared as described in “Sample extraction” section) was filtered with a 0.2-µm PTFE syringe filter and then injected into the UPLC-PDA. (b, c) QuEChERS Kits 1 and 2 The sample extract (5 mL) was added to QuE 1 (900 mg MgSO4, 150 mg PSA) and QuE 2 (900 mg MgSO4, 150 mg PSA, 150 mg C 18 ) clean-up kits. It was strongly shaken for 1 min and centrifuged for 10 min at 4000 rpm at 8 ℃. The supernatant was filtered with a 0.2-µm PTFE syringe filter and then injected into the UPLC-PDA. Method validation To validate the developed UPLC-PDA method for analyzing 11 respiratory drug substances, the specificity, limit of detection (LOD), limit of quantitation (LOQ), linearity, accuracy, precision, recovery, matrix effect, and stability were tested. The validation protocol was implemented with reference to the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use [ 14 ]. The specificity was determined by comparing blank matrix and spiked sample chromatograms of solid and liquid samples, respectively. The LOD and LOQ were evaluated at signal-to-noise (S/N) ratios of ≥ 3 and ≥ 10. The linearity was evaluated by plotting the calibration curve using six concentrations, and the correlation coefficient (R 2 ) was ≥ 0.999. The recovery was evaluated by calculating the ratio of the peak areas of the standard solution and the spiked sample at the same concentration. The precision and accuracy were measured by calculating the relative standard deviation (RSD) and recovery of each compound. Matrix effects were calculated by comparing the slope of the calibration curve for the standard solution with that of the spiked sample. The stability of the standard solution was confirmed through analysis after it was stored at room temperature for 6 h, and we checked whether the stability was maintained after 24 and 48 h. The intra-day and inter-day precision, accuracy, recovery, matrix effect, and stability were validated at three concentrations (low: LOQ, medium: 20 LOQ, high: 40 LOQ), and the entire analysis was performed three times. Results and discussion Sample preparation method comparison results To determine the ideal clean-up method for the sample preparation, three clean-up methods were compared by spiking 11 respiratory-related pharmaceutical compounds into solid and liquid blank samples. When samples were extracted and analyzed using 70% methanol, the recovery rates for 11 compounds ranged from 80–120%; thus, this was selected as the extraction solvent(Supplementary Table 1) [ 15 ]. As shown in Table 1 , when the solid sample was cleaned up using QuE 1, the recovery rates of olopatadine and fexofenadine were 126.00% and 121.09%, respectively. When QuE 2 was used, the recovery rate of azelastine was 66.13%. QuE 1 and QuE 2 were not selected, because recoveries were below or above the suitable range (80–120%) of the ICH guideline. Lastly, when a 0.2-µm PTFE syringe filter was used, the recovery of all 11 compounds was 95.75–103.20%; thus, this was selected as a clean-up method for solid samples. Table 1 Recoveries of three clean-up methods (0.2 µm PTFE filter, QuE 1, QuE 2) NO Analytes 0.2 µm Filter QuEChERS (900 mg MgSO 4 + 150 mg PSA) QuEChERS (900 mg MgSO 4 + 150 mg PSA + C 18 ) Solid Liquid Solid Liquid Solid Liquid 1 Theobromine 97.17 ± 0.68 97.40 ± 0.86 111.08 ± 0.61 112.71 ± 0.65 97.41 ± 0.43 99.19 ± 1.40 2 Theophylline 95.75 ± 0.52 96.04 ± 1.12 109.82 ± 0.47 111.81 ± 0.64 99.73 ± 1.45 100.05 ± 2.00 3 Dropropizine 98.52 ± 0.74 98.49 ± 1.78 111.02 ± 1.03 112.16 ± 0.88 96.60 ± 0.56 97.54 ± 1.97 4 Guaifenesin 98.38 ± 0.68 97.50 ± 1.04 112.24 ± 0.33 113.39 ± 1.08 97.47 ± 0.48 98.30 ± 0.22 5 Tramazoline 97.19 ± 2.05 103.81 ± 0.93 114.39 ± 1.86 116.52 ± 1.66 95.17 ± 1.68 97.17 ± 2.38 6 Noscapine 96.35 ± 0.75 95.56 ± 0.88 110.65 ± 0.69 111.46 ± 0.72 85.70 ± 0.31 84.69 ± 0.79 7 Oxymetazoline 96.56 ± 0.98 100.42 ± 1.10 112.90 ± 0.76 113.79 ± 0.81 92.88 ± 0.41 92.84 ± 1.16 8 Olopatadine 97.72 ± 0.70 97.75 ± 0.90 126.00 ± 0.64 129.50 ± 0.95 112.73 ± 0.48 111.82 ± 1.07 9 Azelastine 103.20 ± 0.89 108.31 ± 1.09 112.18 ± 0.56 113.50 ± 0.73 66.13 ± 0.17 62.32 ± 0.59 10 Dexamethasone 98.07 ± 0.81 98.79 ± 0.90 112.04 ± 0.65 114.45 ± 0.64 92.26 ± 1.65 96.17 ± 1.34 11 Fexofenadine 98.72 ± 0.76 98.26 ± 1.09 121.09 ± 0.35 122.35 ± 0.93 98.05 ± 0.39 93.41 ± 0.90 Next, when the liquid sample was cleaned up using QuE 1, the recovery rates of olopatadine and fexofenadine were 129.50% and 122.35%, respectively. When QuE 2 was used, the recovery rate of azelastine was 62.32%. The recoveries of QuE 1 and QuE 2 for liquid samples were also below or above the suitable ranges. Lastly, when a 0.2-µm PTFE syringe filter was used, the recovery of all 11 compounds was 95.56–108.31%; thus, this was selected as a clean-up method for liquid samples. Development of UPLC-PDA condition To determine the optimal Waters ACQUITY UPLC System conditions, two columns, two ratios of the mobile phase, two column temperatures, three flow rates, and three wavelengths were compared. First, in the column comparison, BEH C 18 (2.1 × 150 mm, 1.7 µm) and HSS C 18 (2.1 × 150 mm, 1.7 µm) columns were compared (Supplementary Fig. 1). For the BEH C 18 column, the peaks of all 11 analytes exhibited a resolution of ≥ 1.5, without peak overlap. For the HSS C 18 column, the peaks of the two analytes overlapped. To optimize mobile phase A, the phosphoric acid ratio of 0.5mM sodium 1-hexanesulfonate buffer in deionized water was varied, but there was no difference in the chromatogram; thus, a relatively small phosphoric acid ratio was selected. For mobile phase B, 100% acetonitrile was compared with acetonitrile:water (90:10) (Supplementary Fig. 2). According to the analysis results, the resolution of 11 analytes was improved when acetonitrile:water (90:10) was used; thus, it was selected as mobile phase B. Additionally, a comparative analysis of flow rates of 0.15, 0.2, and 0.3 mL/ min was performed, and 0.2 mL/min was selected, as it exhibited the best separated chromatogram (Supplementary Fig. 3). The UV wavelength was selected as 205 nm after comparing different wavelengths in the range of 200–210 nm (Supplementary Fig. 4). Column temperatures of 35 and 40 ℃ were compared and analyzed, and 35 ℃ was determined as the optimal temperature (Supplementary Fig. 5). Method validation The 11 compounds spiked in the solid and liquid blank samples were all properly separated with a resolution of ≥ 1.5. For all 11 compounds, it was confirmed that there was no interference from blank samples or interference between analytes (Supplementary Fig. 6). The LOD was calculated as 3 times the S/N ratio, and the LOQ was calculated as 10 times the S/N ratio. As shown in Table 2 , for both solid and liquid samples, the LOD concentration range was 0.30–3.00 µg/mL, and the LOQ was 0.90–9.00 µg/mL (Supplementary Table 2). The linearity was evaluated by calculating the correlation coefficient (R 2 ) of the calibration curve using six concentrations (0.90–360 µg/mL; from LOQ to 40 LOQ). The test was performed three times on solid and liquid samples, and R 2 was ≥ 0.999 for all 11 compounds (Supplementary Table 3). The recovery was assessed in three repeated experiments at three concentrations: LOQ, 20 LOQ, and 40 LOQ. The recovery was confirmed to be 88.82–101.30% in solid samples and 86.18–99.74% in liquid samples. The standard deviation (SD) of the solid samples was 0.21–3.54%, and that of the liquid samples was 0.51–2.23%. Thus, the recoveries of solid and liquid samples satisfied the acceptable limits, and reliability was confirmed from three repeated experiments (Supplementary Table 4). As shown in Table 2 , the intra- and inter-day precision and accuracy values satisfied the standards. For the precision, it was 0.04–9.26% (intra-day) and 0.59–8.58% (inter-day) for solid blank samples, and it was 0.19–6.27% (intra-day) and 0.30–2.58% (inter-day) for liquid blank samples. For the accuracy, it was 91.53–107.21% (intra-day) and 99.83–109.51% (inter-day) for solid blank samples, and it was 93.32–104.43% (intra-day) and 92.04–102.04% (inter-day) for liquid blank samples (Supplementary Table 5). The matrix effect was found to be 97.04–100.99% in the solid phase and 96.05–100.10% in the liquid phase, and these results indicated that all 11 substances were not significantly affected by the matrix (Supplementary Table 6). The stability was calculated as the RSD of the peak area of each compound over time (6, 24, and 48 h). The range was 0.14–4.97%, indicating that the mixed standard solution of the 11 compounds was stable for at least 48 h (Supplementary Table 7). Table 2 Summary of validation results Validation Parameters Validation Results Solid Liquid • LOD 0.30–3.00 µg/mL 0.30–3.00 µg/mL • LOQ 0.90–9.00 µg/mL 0.90–9.00 µg/mL • Linearity - Concentration 0.90–360.00 µg/mL 0.90–360.00 µg/mL - Relative coefficient(R 2 ) R 2 ≥ 0.999 R 2 ≥ 0.999 • Accuracy - Intra-day 91.53–107.21% 93.32–104.43% - Inter-day 99.83–109.51% 92.04–102.04% • Precision - Intra-day ≤ 9.26% ≤ 6.27% - Inter-day ≤ 8.58% ≤ 2.58% • Stability ≤ 4.97% • Recovery 88.82–101.30% 86.18–99.74% • Matrix effect 97.04–100.99% 97.21–100.10% Analysis results for commercial products To examine the established analysis method, 52 dietary supplements related to the improvement of respiratory symptoms were purchased online. The 52 samples were classified into hard capsules (20), powders (4), tablets (8), and liquids (20) according to the most commonly sold formulation. Through the analysis, one capsule-type sample was confirmed to have the same RT as theobromine, and a spectrum comparison confirmed that its spectrum was identical to that of theobromine. Subsequently, a quantitative analysis was performed, and 247.7 mg/g (117.1 mg/capsule) theobromine was detected (Fig. 4). The reliability of the established analysis method for 11 types of respiratory drug substances was confirmed through analysis of the 52 commercial products, including one product in which theobromine was detected. Conclusion To develop a simultaneous analysis method for 11 respiratory-related pharmaceutical compounds, we optimized the sample preparation methods for four types of formulations (hard capsules, powders, tablets, liquids) by comparing three clean-up methods, including the QuEChERS kit. Subsequently, instrumental analysis condition development and validation were performed using UPLC-PDA. Through the validation of the developed analysis method, it was confirmed that the specificity, LOD, LOQ, linearity, recovery, precision, accuracy, matrix effect, and stability all met the standards of the ICH guidelines. Using the developed analysis method, 52 dietary supplement products were analyzed, and theobromine was detected in one product. This commercial product analysis confirmed that the developed analysis method can be applied to various dietary supplement formulations. Through qualitative and quantitative analysis for a product containing theobromine, the retention time and spectrum were confirmed to be the same, and the content was calculated to be 247.7 mg/g (117.1 mg/capsule). Consuming excessive amounts of dietary supplements mixed with pharmaceutical compounds can lead to various side effects. In particular, theobromine, which was detected through commercial product analysis, may cause side effects such as nausea and loss of appetite if consumed in excess [ 16 ]. Accordingly, the optimized simultaneous analysis method for 11 respiratory-related drug substances is expected to contribute to improving public health by preventing the distribution of adulterated dietary supplements. Additionally, as interest in respiratory diseases increases, this study can serve as a reference for necessary related research. Declarations Author Contribution Mi Jin Kim & Hyunil Shin conducted overall experiments and equally wrote the manuscript for this article.Hwan Seong Choi supported optimizing the sample preparation method and establishing instrument analysis conditions.Nam Sook Kim was involved in writing and reviewing the introduction.Ji Hyun Lee was involved in writing and reviewing results & discussion and conclusion.Jang Duck Choi was involved in writing and reviewing the entire paper.Lastly, all authors reviewed the manuscript. Disclosure statement No potential conflict of interest was reported by the author(s). Funding This study was supported by a grant [Number:21201MFDS306] from the Ministry of Food and Drug Safety of Korea in 2021 Data availability statement Data supporting the findings of this study are available in the supplementary material of this article. References Çalıca Utku AC, Budak G, Karabay O, Güçlü E. Okan HD, Vatan A (2020) Main symptoms in patients presenting in the COVID-19 period. Scott Med J. https://doi.org/10.1177/0036933020949253 Ferkol T, Schraufnagel D. (2014) The global burden of respiratory disease Ann Am Thorac Soc. https://doi.org/10.1513/AnnalsATS.201311-405PS Kyung SY, Jeong SH (2020) Particulate-matter related respiratory diseases. Tuberc Respir Dis. https://doi.org/10.4046/trd.2019.0025 Kim B, Yoon EJ, Kim S, Lee DK (2020) The effect of risk perceptions related to particulate matter on outdoor activity satisfaction in South Korea. 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Food Addit Contam Part A Chem. https://doi.org/10.1080/19440049.2021.1906954 International Council for Harmonisation, Harmonised guideline (2022) Validation of analytical procedures vol Q2 ( p R2). https://database.ich.org/sites/default/files/ICH_Q2-R2_Document_Step2_Guideline_2022_0324.pdf Kim NS, Kim J, Lim NY, Lee JH, Park S, Kang H (2020) Simultaneous determination of illegal drug substances in dietary supplements for gout and osteoporosis using ultra-performance liquid chromatography and liquid chromatography-quadrupole-time-of-flight mass spectrometry. J Pharm Biomed Anal. https://doi.org/10.1016/j.jpba.2019.113003 Koleva I, van Beek TA, Soffers E, Dusemund B, Rietjens M (2012) Alkaloids in the human food chain – Natural occurrence and possible adverse effects. Mol Nutr Food Res. https://doi.org/10.1002/mnfr.201100165 Additional Declarations No competing interests reported. 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13:22:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1321193,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version.\u003c/p\u003e","description":"","filename":"fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-3871996/v1/3727540b55fc1bf78116d945.png"},{"id":49970396,"identity":"a4bef8ae-17b7-4d2c-834a-21edc2d627a5","added_by":"auto","created_at":"2024-01-22 13:22:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":112240,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version.\u003c/p\u003e","description":"","filename":"fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-3871996/v1/545b78af55e4127e51db6de0.png"},{"id":49970401,"identity":"81df4a1e-24a1-49fd-8fab-5d7bd7a318e7","added_by":"auto","created_at":"2024-01-22 13:22:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":692166,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version.\u003c/p\u003e","description":"","filename":"fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-3871996/v1/8f717d15d518c0e76b659a9a.png"},{"id":50039737,"identity":"5d4caafb-4c29-4ad3-b49d-74418caddb2b","added_by":"auto","created_at":"2024-01-23 15:13:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":896588,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3871996/v1/562628f6-8423-483d-9846-ec657188d715.pdf"},{"id":49970402,"identity":"f4ad9f2b-5337-47a5-b86c-d004e01f7610","added_by":"auto","created_at":"2024-01-22 13:22:31","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1837958,"visible":true,"origin":"","legend":"","description":"","filename":"SupplemantaryMaterials240117.docx","url":"https://assets-eu.researchsquare.com/files/rs-3871996/v1/a519b3537bed57285d80fa44.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development of simultaneous analysis method for 11 respiratory drug substances including theobromine and analysis of commercial products using UPLC-PDA","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eRecently, the number of people suffering from respiratory symptoms has been increasing owing to the COVID-19 pandemic and the increase in fine dust due to air pollution. Most people infected with the COVID-19 virus experience respiratory illnesses, such as cough and viral pneumonia [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In addition, particulate matter\u0026mdash;floating dust with a diameter of \u0026le;\u0026thinsp;10 \u0026micro;m\u0026mdash;can accumulate in the alveoli when inhaled by humans. Particulate matter can cause direct toxic damage, increasing lung inflammation and worsening respiratory symptoms [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBecause of the increase in various factors that can worsen respiratory symptoms, interest in respiratory-related dietary supplements is increasing, and demand for some types of dietary supplements increased during the pandemic [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Meanwhile, as general interest in dietary supplements increases, various pharmaceutical compounds have been illegally mixed to achieve medicinal effects [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. If pharmaceutical compounds are taken in excessive amounts, there is a risk of experiencing various side effects. Theophylline, which is used to treat respiratory diseases, was found to be contained in dietary supplements for weight control, and the FDA issued a warning against the use of these products [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In Korea, dietary supplements containing theophylline were added to the list of hazardous foods in 2017 [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo prevent this threat in advance, we developed a simultaneous analysis method for 11 respiratory drug substances used to treat respiratory symptoms (Fig.\u0026nbsp;1). A literature search revealed HPLC methods for caffeine analogues in food samples (coffee) such as theophylline and theobromine[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], analysis and method validation studies for noscapine in syrup form using HPLC[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], and HPLC analysis method studies for oxymetazoline in nasal spray formulations[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, there have been no studies on a simultaneous analysis method for pharmaceutical compounds effective for respiratory symptoms in various dietary supplement formulations.\u003c/p\u003e \u003cp\u003eThus, for quickly analyzing samples of various formulations and reducing the amounts of time and manpower required, research to develop a simultaneous analysis method for 11 respiratory-related drug substances was conducted. In this study, the optimal sample preparation method was established by comparing the recovery rates of 11 compounds for three clean-up methods, and a simultaneous analysis method was developed and validated by comparing various UPLC analysis conditions (Fig.\u0026nbsp;2). Additionally, we analyzed 52 respiratory-related dietary supplement of various formulations, such as hard capsules, powder, tablets, and liquid.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemicals and reagents\u003c/h2\u003e \u003cp\u003eThe following standard materials were used in this study: theobromine, theophylline, dropropizine, noscapine, and dexamethasone were purchased from Toronto Research Chemicals (TRC, Toronto, ON, Canada); guaifenesin, azelastine, and fexofenadine were purchased from United States Pharmacopeia (USP, Rockville, MD, USA); tramazoline was purchased from European Pharmacopoeia (Strasbourg, France); olopatadine was purchased from Sigma\u0026ndash;Aldrich (St. Louis, MO, USA); and oxymetazoline was purchased from Clearsynth (Mumbai, India). Sodium 1-hexanesulfonate, phosphoric acid (reagent grade), and formic acid (LC-MS grade) were purchased from Sigma\u0026ndash;Aldrich (St. Louis, MO, USA), deionized water was prepared using a Milli-Q system (Millipore, Bedford, MA, USA), and acetonitrile and methanol (HPLC grade) were purchased from Merck (Darmstadt, Germany). All the solvents used in this study were filtered through a polytetrafluoroethylene (PTFE) filter (0.2 \u0026micro;m, Millipore) before analysis. Except for theobromine, standard stock solutions were prepared at a concentration of 1000 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e through dissolution in methanol. Theobromine was prepared at the same concentration through dissolution in an ACN:DW (60:40) solution. The stock solutions were stored in a refrigerator at approximately 4 ℃. For calibration curve and method validation, the stock solutions were diluted to the appropriate concentration using HPLC-grade methanol on the same day.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eInstrumental conditions\u003c/h2\u003e \u003cp\u003eThe analysis method development and validation were conducted using the Waters Acquity UPLC System (Waters, Milford, CT, USA) equipped with a binary pump and photodiode array detector. An Acquity UPLC BEH C\u003csub\u003e18\u003c/sub\u003e Column (2.1 \u0026times; 150 mm, 1.7 \u0026micro;m) was used for separating the analytes. The column oven temperature was set as 35 ℃. Ultraviolet (UV) absorption was measured at a wavelength of 205 nm, and the temperature of the autosampler was 8 ℃. The flow rate of the mobile phase and injection volume of analytes were 0.2 mL/min and 1.0 \u0026micro;L, respectively. The following mobile phases were selected: (A) 0.5 mM sodium 1-hexanesulfonate buffer in deionized water containing 0.1% phosphoric acid and (B) acetonitrile:water (90:10). The gradient elution was set as follows: 0.0\u0026ndash;1.0 min (A: 95%, B: 5%), 8.0\u0026ndash;9.5 min (A: 55%, B: 45%), 11.0\u0026ndash;13.0 min (A: 40%, B: 60%), 15.5\u0026ndash;18.5 min (A: 0%, B: 100%), 19.0\u0026ndash;22.0 min (A: 95%, B: 5%).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eComparison of clean-up methods\u003c/h2\u003e \u003cp\u003eTo optimize the sample preparation method, various clean-up methods were compared to minimize the impact of sample matrix components. The removal of polar matrix components, fatty acids, fats, and sterols was considered. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] A 0.2-\u0026micro;m PTFE filter and two types of dispersive SPE (d-SPE, commonly referred to as QuEChERS) kits were selected for comparison. For the comparison of the clean-up methods, testing samples were prepared at the same concentration: (a) filter only, (b) QuEChERS kit 1 (Thermo Fisher, Waltham, MA, USA, hereinafter referred as QuE 1), and (c) QuEChERS kit 2 (Waters, Rockwood, TN, USA, hereinafter referred as QuE 2) methods were compared to optimize the clean-up method (Fig.\u0026nbsp;3). The detailed comparison procedures are as follows.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eSample extraction\u003c/h2\u003e \u003cp\u003eSample types were categorized into solid and liquid, and the solid samples included hard capsules, tablets, and powder. For the solid samples, hard capsules and tablets were ground using a grinder (IKA Tube mill, Germany), including the outer shell. 1 g of each homogenized sample was weighed into a 50-mL volumetric flask, and 30 mL of 70% aqueous methanol was added. The mixture was extracted in an ultrasonic bath for 30 min and cooled at room temperature, and then 70% methanol was added to obtain a total volume of 50 mL. The extracts were centrifuged for 5 min at 4000 rpm at 8 ℃, and the supernatant was used as a sample extract.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e(a) Filter only\u003c/h2\u003e \u003cp\u003eThe sample extract (2 mL, prepared as described in \u0026ldquo;Sample extraction\u0026rdquo; section) was filtered with a 0.2-\u0026micro;m PTFE syringe filter and then injected into the UPLC-PDA.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e(b, c) QuEChERS Kits 1 and 2\u003c/h3\u003e\n\u003cp\u003eThe sample extract (5 mL) was added to QuE 1 (900 mg MgSO4, 150 mg PSA) and QuE 2 (900 mg MgSO4, 150 mg PSA, 150 mg C\u003csub\u003e18\u003c/sub\u003e) clean-up kits. It was strongly shaken for 1 min and centrifuged for 10 min at 4000 rpm at 8 ℃. The supernatant was filtered with a 0.2-\u0026micro;m PTFE syringe filter and then injected into the UPLC-PDA.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eMethod validation\u003c/h2\u003e \u003cp\u003eTo validate the developed UPLC-PDA method for analyzing 11 respiratory drug substances, the specificity, limit of detection (LOD), limit of quantitation (LOQ), linearity, accuracy, precision, recovery, matrix effect, and stability were tested. The validation protocol was implemented with reference to the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe specificity was determined by comparing blank matrix and spiked sample chromatograms of solid and liquid samples, respectively. The LOD and LOQ were evaluated at signal-to-noise (S/N) ratios of \u0026ge;\u0026thinsp;3 and \u0026ge;\u0026thinsp;10. The linearity was evaluated by plotting the calibration curve using six concentrations, and the correlation coefficient (R\u003csup\u003e2\u003c/sup\u003e) was \u0026ge;\u0026thinsp;0.999. The recovery was evaluated by calculating the ratio of the peak areas of the standard solution and the spiked sample at the same concentration. The precision and accuracy were measured by calculating the relative standard deviation (RSD) and recovery of each compound. Matrix effects were calculated by comparing the slope of the calibration curve for the standard solution with that of the spiked sample. The stability of the standard solution was confirmed through analysis after it was stored at room temperature for 6 h, and we checked whether the stability was maintained after 24 and 48 h. The intra-day and inter-day precision, accuracy, recovery, matrix effect, and stability were validated at three concentrations (low: LOQ, medium: 20 LOQ, high: 40 LOQ), and the entire analysis was performed three times.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSample preparation method comparison results\u003c/h2\u003e \u003cp\u003eTo determine the ideal clean-up method for the sample preparation, three clean-up methods were compared by spiking 11 respiratory-related pharmaceutical compounds into solid and liquid blank samples. When samples were extracted and analyzed using 70% methanol, the recovery rates for 11 compounds ranged from 80\u0026ndash;120%; thus, this was selected as the extraction solvent(Supplementary Table\u0026nbsp;1) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, when the solid sample was cleaned up using QuE 1, the recovery rates of olopatadine and fexofenadine were 126.00% and 121.09%, respectively. When QuE 2 was used, the recovery rate of azelastine was 66.13%. QuE 1 and QuE 2 were not selected, because recoveries were below or above the suitable range (80\u0026ndash;120%) of the ICH guideline. Lastly, when a 0.2-\u0026micro;m PTFE syringe filter was used, the recovery of all 11 compounds was 95.75\u0026ndash;103.20%; thus, this was selected as a clean-up method for solid samples.\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\u003eRecoveries of three clean-up methods (0.2 \u0026micro;m PTFE filter, QuE 1, QuE 2)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\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=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAnalytes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e0.2 \u0026micro;m Filter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eQuEChERS\u003c/p\u003e \u003cp\u003e(900 mg MgSO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;150 mg PSA)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003eQuEChERS\u003c/p\u003e \u003cp\u003e(900 mg MgSO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;150 mg PSA\u0026thinsp;+\u0026thinsp;C\u003csub\u003e18\u003c/sub\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSolid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLiquid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSolid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLiquid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSolid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eLiquid\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTheobromine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e97.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e97.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e111.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e112.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e97.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e99.19\u0026thinsp;\u0026plusmn;\u0026thinsp;1.40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTheophylline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e95.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e96.04\u0026thinsp;\u0026plusmn;\u0026thinsp;1.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e109.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e111.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e99.73\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e100.05\u0026thinsp;\u0026plusmn;\u0026thinsp;2.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDropropizine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e98.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e98.49\u0026thinsp;\u0026plusmn;\u0026thinsp;1.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e111.02\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e112.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e96.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e97.54\u0026thinsp;\u0026plusmn;\u0026thinsp;1.97\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGuaifenesin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e98.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e97.50\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e112.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e113.39\u0026thinsp;\u0026plusmn;\u0026thinsp;1.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e97.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e98.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTramazoline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e97.19\u0026thinsp;\u0026plusmn;\u0026thinsp;2.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e103.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e114.39\u0026thinsp;\u0026plusmn;\u0026thinsp;1.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e116.52\u0026thinsp;\u0026plusmn;\u0026thinsp;1.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e95.17\u0026thinsp;\u0026plusmn;\u0026thinsp;1.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e97.17\u0026thinsp;\u0026plusmn;\u0026thinsp;2.38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNoscapine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e96.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e95.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e110.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e111.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e85.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e84.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOxymetazoline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e96.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e100.42\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e112.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e113.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e92.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e92.84\u0026thinsp;\u0026plusmn;\u0026thinsp;1.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOlopatadine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e97.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e97.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e126.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e129.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e112.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e111.82\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAzelastine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e103.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e108.31\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e112.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e113.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e66.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e62.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDexamethasone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e98.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e98.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e112.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e114.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e92.26\u0026thinsp;\u0026plusmn;\u0026thinsp;1.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e96.17\u0026thinsp;\u0026plusmn;\u0026thinsp;1.34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFexofenadine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e98.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e98.26\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e121.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e122.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e98.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e93.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.90\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\u003eNext, when the liquid sample was cleaned up using QuE 1, the recovery rates of olopatadine and fexofenadine were 129.50% and 122.35%, respectively. When QuE 2 was used, the recovery rate of azelastine was 62.32%. The recoveries of QuE 1 and QuE 2 for liquid samples were also below or above the suitable ranges. Lastly, when a 0.2-\u0026micro;m PTFE syringe filter was used, the recovery of all 11 compounds was 95.56\u0026ndash;108.31%; thus, this was selected as a clean-up method for liquid samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDevelopment of UPLC-PDA condition\u003c/h2\u003e \u003cp\u003eTo determine the optimal Waters ACQUITY UPLC System conditions, two columns, two ratios of the mobile phase, two column temperatures, three flow rates, and three wavelengths were compared. First, in the column comparison, BEH C\u003csub\u003e18\u003c/sub\u003e (2.1 \u0026times; 150 mm, 1.7 \u0026micro;m) and HSS C\u003csub\u003e18\u003c/sub\u003e (2.1 \u0026times; 150 mm, 1.7 \u0026micro;m) columns were compared (Supplementary Fig.\u0026nbsp;1). For the BEH C\u003csub\u003e18\u003c/sub\u003e column, the peaks of all 11 analytes exhibited a resolution of \u0026ge;\u0026thinsp;1.5, without peak overlap. For the HSS C\u003csub\u003e18\u003c/sub\u003e column, the peaks of the two analytes overlapped. To optimize mobile phase A, the phosphoric acid ratio of 0.5mM sodium 1-hexanesulfonate buffer in deionized water was varied, but there was no difference in the chromatogram; thus, a relatively small phosphoric acid ratio was selected. For mobile phase B, 100% acetonitrile was compared with acetonitrile:water (90:10) (Supplementary Fig.\u0026nbsp;2). According to the analysis results, the resolution of 11 analytes was improved when acetonitrile:water (90:10) was used; thus, it was selected as mobile phase B. Additionally, a comparative analysis of flow rates of 0.15, 0.2, and 0.3 mL/ min was performed, and 0.2 mL/min was selected, as it exhibited the best separated chromatogram (Supplementary Fig.\u0026nbsp;3). The UV wavelength was selected as 205 nm after comparing different wavelengths in the range of 200\u0026ndash;210 nm (Supplementary Fig.\u0026nbsp;4). Column temperatures of 35 and 40 ℃ were compared and analyzed, and 35 ℃ was determined as the optimal temperature (Supplementary Fig.\u0026nbsp;5).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMethod validation\u003c/h2\u003e \u003cp\u003eThe 11 compounds spiked in the solid and liquid blank samples were all properly separated with a resolution of \u0026ge;\u0026thinsp;1.5. For all 11 compounds, it was confirmed that there was no interference from blank samples or interference between analytes (Supplementary Fig.\u0026nbsp;6). The LOD was calculated as 3 times the S/N ratio, and the LOQ was calculated as 10 times the S/N ratio.\u003c/p\u003e \u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, for both solid and liquid samples, the LOD concentration range was 0.30\u0026ndash;3.00 \u0026micro;g/mL, and the LOQ was 0.90\u0026ndash;9.00 \u0026micro;g/mL (Supplementary Table\u0026nbsp;2). The linearity was evaluated by calculating the correlation coefficient (R\u003csup\u003e2\u003c/sup\u003e) of the calibration curve using six concentrations (0.90\u0026ndash;360 \u0026micro;g/mL; from LOQ to 40 LOQ). The test was performed three times on solid and liquid samples, and R\u003csup\u003e2\u003c/sup\u003e was \u0026ge;\u0026thinsp;0.999 for all 11 compounds (Supplementary Table\u0026nbsp;3). The recovery was assessed in three repeated experiments at three concentrations: LOQ, 20 LOQ, and 40 LOQ. The recovery was confirmed to be 88.82\u0026ndash;101.30% in solid samples and 86.18\u0026ndash;99.74% in liquid samples. The standard deviation (SD) of the solid samples was 0.21\u0026ndash;3.54%, and that of the liquid samples was 0.51\u0026ndash;2.23%. Thus, the recoveries of solid and liquid samples satisfied the acceptable limits, and reliability was confirmed from three repeated experiments (Supplementary Table\u0026nbsp;4). As shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the intra- and inter-day precision and accuracy values satisfied the standards. For the precision, it was 0.04\u0026ndash;9.26% (intra-day) and 0.59\u0026ndash;8.58% (inter-day) for solid blank samples, and it was 0.19\u0026ndash;6.27% (intra-day) and 0.30\u0026ndash;2.58% (inter-day) for liquid blank samples. For the accuracy, it was 91.53\u0026ndash;107.21% (intra-day) and 99.83\u0026ndash;109.51% (inter-day) for solid blank samples, and it was 93.32\u0026ndash;104.43% (intra-day) and 92.04\u0026ndash;102.04% (inter-day) for liquid blank samples (Supplementary Table\u0026nbsp;5). The matrix effect was found to be 97.04\u0026ndash;100.99% in the solid phase and 96.05\u0026ndash;100.10% in the liquid phase, and these results indicated that all 11 substances were not significantly affected by the matrix (Supplementary Table\u0026nbsp;6). The stability was calculated as the RSD of the peak area of each compound over time (6, 24, and 48 h). The range was 0.14\u0026ndash;4.97%, indicating that the mixed standard solution of the 11 compounds was stable for at least 48 h (Supplementary Table\u0026nbsp;7).\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\u003eSummary of validation results\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eValidation\u003c/p\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eValidation Results\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSolid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLiquid\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026bull; LOD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.30\u0026ndash;3.00 \u0026micro;g/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.30\u0026ndash;3.00 \u0026micro;g/mL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026bull; LOQ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.90\u0026ndash;9.00 \u0026micro;g/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.90\u0026ndash;9.00 \u0026micro;g/mL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026bull; Linearity\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 \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e- Concentration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.90\u0026ndash;360.00 \u0026micro;g/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.90\u0026ndash;360.00 \u0026micro;g/mL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e- Relative coefficient(R\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;\u0026ge;\u0026thinsp;0.999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;\u0026ge;\u0026thinsp;0.999\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026bull; Accuracy\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 \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e- Intra-day\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e91.53\u0026ndash;107.21%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e93.32\u0026ndash;104.43%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e- Inter-day\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e99.83\u0026ndash;109.51%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e92.04\u0026ndash;102.04%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026bull; Precision\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 \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e- Intra-day\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;9.26%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;6.27%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e- Inter-day\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;8.58%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;2.58%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026bull; Stability\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;4.97%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026bull; Recovery\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e88.82\u0026ndash;101.30%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e86.18\u0026ndash;99.74%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026bull; Matrix effect\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e97.04\u0026ndash;100.99%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e97.21\u0026ndash;100.10%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis results for commercial products\u003c/h2\u003e \u003cp\u003eTo examine the established analysis method, 52 dietary supplements related to the improvement of respiratory symptoms were purchased online. The 52 samples were classified into hard capsules (20), powders (4), tablets (8), and liquids (20) according to the most commonly sold formulation.\u003c/p\u003e \u003cp\u003eThrough the analysis, one capsule-type sample was confirmed to have the same RT as theobromine, and a spectrum comparison confirmed that its spectrum was identical to that of theobromine. Subsequently, a quantitative analysis was performed, and 247.7 mg/g (117.1 mg/capsule) theobromine was detected (Fig.\u0026nbsp;4). The reliability of the established analysis method for 11 types of respiratory drug substances was confirmed through analysis of the 52 commercial products, including one product in which theobromine was detected.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eTo develop a simultaneous analysis method for 11 respiratory-related pharmaceutical compounds, we optimized the sample preparation methods for four types of formulations (hard capsules, powders, tablets, liquids) by comparing three clean-up methods, including the QuEChERS kit. Subsequently, instrumental analysis condition development and validation were performed using UPLC-PDA.\u003c/p\u003e \u003cp\u003eThrough the validation of the developed analysis method, it was confirmed that the specificity, LOD, LOQ, linearity, recovery, precision, accuracy, matrix effect, and stability all met the standards of the ICH guidelines. Using the developed analysis method, 52 dietary supplement products were analyzed, and theobromine was detected in one product. This commercial product analysis confirmed that the developed analysis method can be applied to various dietary supplement formulations. Through qualitative and quantitative analysis for a product containing theobromine, the retention time and spectrum were confirmed to be the same, and the content was calculated to be 247.7 mg/g (117.1 mg/capsule).\u003c/p\u003e \u003cp\u003eConsuming excessive amounts of dietary supplements mixed with pharmaceutical compounds can lead to various side effects. In particular, theobromine, which was detected through commercial product analysis, may cause side effects such as nausea and loss of appetite if consumed in excess [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Accordingly, the optimized simultaneous analysis method for 11 respiratory-related drug substances is expected to contribute to improving public health by preventing the distribution of adulterated dietary supplements. Additionally, as interest in respiratory diseases increases, this study can serve as a reference for necessary related research.\u003c/p\u003e "},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eMi Jin Kim \u0026amp; Hyunil Shin conducted overall experiments and equally wrote the manuscript for this article.Hwan Seong Choi supported optimizing the sample preparation method and establishing instrument analysis conditions.Nam Sook Kim was involved in writing and reviewing the introduction.Ji Hyun Lee was involved in writing and reviewing results \u0026amp; discussion and conclusion.Jang Duck Choi was involved in writing and reviewing the entire paper.Lastly, all authors reviewed the manuscript.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eDisclosure statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo potential conflict of interest was reported by the author(s).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by a grant [Number:21201MFDS306] from the Ministry of Food and Drug Safety of Korea in 2021\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData supporting the findings of this study are available in the supplementary material of this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e\u0026Ccedil;alıca Utku AC, Budak G, Karabay O, G\u0026uuml;\u0026ccedil;l\u0026uuml; E. 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Mol Nutr Food Res. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/mnfr.201100165\u003c/span\u003e\u003cspan address=\"10.1002/mnfr.201100165\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"respiratory; dietary supplement, adulterated, COVID-19, UPLC-PDA","lastPublishedDoi":"10.21203/rs.3.rs-3871996/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3871996/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRecently, the number of people suffering from respiratory symptoms has continued to increase owing to the effects of COVID-19 and particulate matter, and accordingly, the demand for respiratory-related dietary supplements is increasing. Over the past few years, dietary supplements adulterated with pharmaceutical drug substances have been appearing continuously; thus, preventive measures are needed. In this study, we developed a simultaneous analysis method for 11 respiratory-related pharmaceutical compounds using ultra-performance liquid chromatography with photodiode array (UPLC-PDA). The sample preparation method was optimized by comparing three clean-up methods including the QuEChERS for hard capsule, tablet, powder, and liquid formulations, and the analysis conditions were established by comparing various analysis parameters. In method validation, the limit of detection, limit of quantitation, specificity, linearity, recovery, precision, accuracy, matrix effect, and stability all met the standards of the ICH guidelines. Subsequently, 52 types of dietary supplements promoting relief of respiratory-related symptoms were purchased online and analyzed using UPLC-PDA. Theobromine was detected in one product, and the content was calculated to be 247.7 mg/g (117.1 mg/capsule). This study can contribute to preventively blocking dietary supplements adulterated with respiratory-related substances and can be used as a reference for future research.\u003c/p\u003e","manuscriptTitle":"Development of simultaneous analysis method for 11 respiratory drug substances including theobromine and analysis of commercial products using UPLC-PDA","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-22 13:22:26","doi":"10.21203/rs.3.rs-3871996/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"526a9779-df15-4ead-b708-0a70ce1d9fde","owner":[],"postedDate":"January 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-01-23T15:05:12+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-22 13:22:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3871996","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3871996","identity":"rs-3871996","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}