Simultaneous Quantification of Citicoline and Tyrosine by Green HPTLC-Densitometry: Application to Pharmaceutical Formulations and Biological Fluids

Simultaneous Quantification of Citicoline and Tyrosine by Green HPTLC-Densitometry: Application to Pharmaceutical Formulations and Biological Fluids | 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 Simultaneous Quantification of Citicoline and Tyrosine by Green HPTLC-Densitometry: Application to Pharmaceutical Formulations and Biological Fluids Amira M. Hegazy, Hesham Salem, Maha M. Abdelrahman, Dina Z. Mazen, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7835319/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 11 You are reading this latest preprint version Abstract Citicoline (CTN) and Tyrosine (TSN) are co-formulated in fixed-dose therapy, leveraging their synergistic effects to enhance cognitive function and mental clarity. This study presents the development of a high-performance thin-layer chromatography (HPTLC) method for the simultaneous determination of Citicoline (CTN) and Tyrosine (TSN) in a pharmaceutical capsules, plasma, and urine. A green HPTLC system composed of phosphate buffer pH = 4 and methanol (70:30 v/v) was optimized and applied on silica gel TLC plates (G60 F254). (CTN) and (TSN) were quantified within linear ranges of 0.05‒1.25 µg/band and 0.12‒2.5 µg/band, respectively, using UV detection at 256 nm. The proposed HPTLC method was statistically validated according to ICH in terms of linearity, precision, accuracy, specificity, robustness, detection limits, with LOQ values of 0.043 µg/band for (CTN) and 0.107 µg/band for (TSN). Application of this method extended to pharmaceutical formulations, spiked human plasma, and urine, confirming peak identity and purity. A statistical comparison with published analytical techniques revealed no significant differences, highlighting the reliability of this approach. To assess environmental impact of the advised method, the eco-scale, Green Analytical Procedure Index (GAPI),AGREE metric and Blue Analytical Greenness Index (BAGI). tools were employed, demonstrating the sustainability of this method. High recovery rates further validate its efficacy, positioning it as a green, accurate, and reproducible alternative for routine analysis of (CTN) and (TSN). Citicoline Tyrosine HPTLC Greenness evaluation Figures Figure 1 Figure 2 Figure 3 1. Introduction Citicoline (CTN), also known as cytidine 5'-diphosphocholine, is an intermediate in the biosynthesis of phosphatidylcholine, a key component of cell membranes. Structurally, it is 5′-trihydrogen diphosphate p′-[2-(trimethylammonio)ethyl] ester inner salt [ 1 ].(Fig. 1 A). CTN is naturally occurring compound in human and animal tissues, particularly in the organs. CTN is widely used in the pharmacotherapy of neurological disorders, including stroke, brain trauma, Parkinson’s disease, and cognitive impairment, due to its ability to stimulate phospholipid biosynthesis, preserve neuronal energy reserves, inhibit apoptosis, and enhance acetylcholine production [ 2 ]. A review of published literature reveals that CTN has been analyzed using various techniques, including chromatographic methods [ 3 – 9 ], spectrophotometry [ 10 ], and spectrofluorometry [ 11 ], either as a single component or in combination. Tyrosine (TSN), chemically known as 4-hydroxyphenylalanine[ 1 ], is an aromatic amino acid essential for protein synthesis. It serves as a precursor for the neurotransmitter's norepinephrine and dopamine, contributing to moodelevation, memory enhancement, and mental alertness. Additionally, TSN plays a critical role in melanin production and thyroxin (thyroid hormone) synthesis. Several analytical techniques—including HPTLC [ 12 ], LC [ 13 ], HPLC [ 14 – 16 ], spectrophotometry [ 17 – 21 ], and spectrofluorometry [ 22 – 26 ].have been reported for the determination of TSN, both individually and in combination. In Egypt, CTN and TSN are formulated together in the pharmaceutical product Cervitolka Plus® (BioMed Pharmaceutical Company), where they work synergistically to support cognitive function and mental alertness. A review of the available literature reveals no existing analytical method for the simultaneous determination of CTN and TSN in their binary mixture. To address this, we present, for the first time, developed HPTLC method the employs green organic solvents. This approach not only reduces environmental hazards but also eliminates the need for toxic solvents [ 27 , 28 ] In the simultaneous estimation of these compounds. The green analytical profile was further evaluated using Eco-Scale [ 29 ], Green Analytical Procedure Index (GAPI) [ 30 ], AGREE [ 31 ] metrics and Blue Analytical Greenness Index (BAGI)[ 32 ]. to assess its sustainability compared to previously published. The successful implementation of this method to spiked human plasma and urine samples demonstrates its potential for adaptation to pharmacokinetic studies and clinical applications. 2. Experimental 2.1. Instrumentation The CAMAG® TLC scanner 3, which was operated by vision CATS software (version 3.1.21109.3, Muttenz, Switzerland), was used for the analysis. Using a 100 µL Hamilton syringe (Bonaduz, Switzerland) and nitrogen gas for solvent evaporation, samples are placed as bands on TLC plates using the CAMAG® Linomat 5 sample application equipment. Deuterium and tungsten lamps were used for the scanning, which had a 5 × 0.2 mm slit and a scanning speed of 20 mm/s. The stationary phase consisted of aluminum sheets (20 × 20 cm, Merck, Darmstadt, Germany) covered with TLC silica gel 60 F254. Additionally, the sample was processed using a Centurion low-speed digital centrifuge (4000 rpm, United Kingdom). Other instruments included an analytical balance (Switzerland) and a Jenway 3510 pH meter (England), which ensured accurate measurements throughout the analysis. 2.2. Active pharmaceutical ingredients and chemical reagents The Pyramid Office in Cairo, Egypt, provided the pure CTN and TSN. Tyrosine (purity: 99.17%) and citicoline (purity: 99.5%) were employed in this investigation. Using statistical analysis based on the mean values from three independent experiments for each compound, The accuracy of the authentic samples was verified HPLC-grade methanol was supplied by Sigma–Aldrich Chemie GmbH, Germany, while sodium dihydrogen phosphate was provided by El-Nasr Pharmaceutical Chemicals Co. (Cairo, Egypt). To prepare a phosphate buffer (pH = 4), dissolve 3.2 g of sodium dihydrogen phosphate in a 25 mL volumetric flask and fill with deionized water (source: Otsuka Pharmaceuticals, Cairo, Egypt). Plasma samples were provided by the blood bank at Minia University Hospital (Minia, Egypt), and urine samples were collected from a healthy volunteer. The silica gel 60 F254 (20 × 20 cm) pre-coated TLC aluminum sheet was used. Pharmaceutical formulation:Cervitolka Plus® capsule (batch No. 230858) were manufactured and supplied by BioMed Pharmaceuticals (Cairo, Egypt). Each CAPULE is claimed to contain 500 mg of CTN and 50 mg of TSN. 2.3. Standard solutions To formulate standard stock solutions, 100 mg of CTN and TSN were dissolved in 100 milliliters of water. .to have stock solutions of (1mg/mL) Appropriate dilutions were made using water to obtain standard working solutions at concentration of 500 µg/mL of each CTN and TSN . A 5 µL injection volume was applied onto aluminum-backed TLC silica gel 60 F256 plates. 2.4. Cervitolka plus ® capsule sample The content of one Cervitolka plus® capsule were weighed and transferred into clean 100mL volumetric flask. Approximately 15 mL of distilled water was added, and the mixture was sonicated for 5 minutes to ensure complete dissolution of citicoline and tyrosine. After cooling to room temperature, the solution was filtered through Whatman filter paper to remove any undissolved particles. The filtrate was then diluted to volume with distilled water to obtain a stock solution of 5 mg/mL for citicoline and 0.5 mg/mL for tyrosine. 2.5. Spiked biological samples Healthy participants' blood samples were collected carefully toward heparinized tubes and centrifuged at 4000 rpm for 20 minutes to obtain plasma, which was then transferred to a sterile new tube and stored at -20°C. Additionally, urine samples from healthy individuals were collected and maintained at 4°C until analysis. In sterile centrifuge tubes, 1.0 mL of plasma was spiked with 1.0 mL of each medication at a stock concentration of 1000 µg/mL in order to prepare the plasma sample. To denature proteins, two milliliters of acetonitrile were added, and the mixture was stirred for ten minutes. After diluting the resultant solution with 10 milliliters of pure water, it was centrifuged for 30 minutes at 3500 rpm. At a concentration of 100 µg/mL, the clear supernatant was separated for further dilution. To prepare the urine sample, 1.0 mL of each medication (1000 µg/mL) was combined with 1.0 mL of urine. After adding two milliliters of methanol, the liquid was stirred for ten minutes. After that, the solution was centrifuged for 30 minutes at 3500 rpm after being diluted with 10 mL of distilled water. As necessary, the supernatant was further diluted. To prepare blank samples (plasma and urine), the same procedures were followed without the drug solution. 2.6 Optimization of chromatographic conditions 5 µL of sample solutions and standard dilutions were placed as bands 10 mm from the bottom of TLC plates (20 cm × 9 cm). Phosphate buffer (pH = 4) and methanol in a 70:30: v/v ratio made up the mobile phase. The plate was dried with a cold stream of air until it dried all the way to the top after 20 minutes of saturation. After removal, the plate was allowed to dry before being scanned in reflectance/absorbance mode at 256 nm. 3. Results and discussion 3.1. Method optimization Citicoline, is a polar molecule and classified as a water-soluble compound. While Tyrosine exhibits both polar and nonpolar characteristics due to its molecular structure. While its hydroxyl (-OH) group confers polarity through hydrogen bonding with water, the presence of an aromatic ring introduces hydrophobic properties. This dual nature sometimes makes its classification ambiguous. Under physiological pH conditions, tyrosine generally remains in a neutral, uncharged state. The polar nature of the analytes necessitates the use of polar solvents to achieve proper migration on TLC plates without significant retention. The mobile phase composition was determined using trial-based analysis, taking into account the polarity differences between CTN and TSN. Initially, a mobile phase consisting of phosphate buffer and water 70:30 (v/v) was used and gave satisfactory results; further trials were necessary to refine the separation and increase resolution. Among various tested systems, a 60:40 (V/V) ethanol-water mobile phase was tested, but poor separation was obtained due to differences in polarity—TSN, being less polar. migrated near the solvent front due to its weak interaction with the stationary phase, while the highly polar CTN remained near the baseline. When the same solvent system was tested in an acidic medium, CTN exhibited even stronger retention at the baseline. Protonation of its phosphate group increased its polarity, enhancing interaction with the stationary phase and further restricting its movement, while TSN continued to migrate freely. Even after adjusting the mobile phase composition to 30:70 (v/v), separation was not achieved. An alternative mobile phase system using triethylamine-water (10:90 v/v) was employed but failed to produce adequate separation between CTN and TSN. The spots were either overlapped or insufficiently resolved, indicating that this solvent system lacked the necessary selectivity. Consequently, phosphate buffer was explored at various pH levels, resulting in successful separation of the two compounds. The optimal developing system was identified as phosphate buffer (pH 4) with methanol (70:30 v/v), providing well-resolved bands with suitable Rf values and minimal tailing. Figure 2 displays the densitogram produced by this mobile phase system. To find the ideal period, trials were also carried out with different saturation times (10 to 30 minutes). It was found that 20 minutes provided the best separation between the two bands, yielding the most reliable Rf values. The UV absorption spectra of the two active medicinal substances under examination showed maxima for TSN and CTN at 280 nm and 272 nm, respectively. 256 nm was determined to be the optimum compromise after a number of wavelengths were investigated for the appropriate detection of the medications under study (Figs. 2 & 3 ). 3.2. Method validation The proposed approach was confirmed to adhere to the International Council for Harmonization's (ICH) guidelines. [ 33 ]. 3.2.1. Linearity, limit of quantification (LOQ), and detection (LOD) The linearity ranges of CTN and TSN were (0.05‒1.25 µg/band) and (0.12–2.5 µg/band), respectively, with correlation coefficient from 0.9999 for the cited drugs. Based on statistical estimations, the formulae LOD = 3.3 x SD of intercept/slope and LOQ = 10 × SD of intercept/slope were developed. The statistical results show that the method's sensitivity for detecting the medications in consideration is acceptable. The important quantitative features are displayed in Table 1 . The linear regression model performed well for the medications under study, according to the lack-of-fit test. 3.2.2. Accuracy The correctness of a technique is determined by the degree of agreement between the values found and the values accepted as acceptable reference values or conventional true values. Table 2 demonstrates the accuracy of the suggested techniques with a low percentage RSD and good recovery. Table 2 Accuracy and precision of the proposed approach Drug Amount (µg/band) Accuracy Recovery ± RSD a Precision Intra-day Recovery ± RSD (%) Precision Inter-day Recovery b ± RSD (%) CTN 0.3 99.16 ± 1.66 99.75 ± 1.15 99.41 ± 1.10 0.6 99.37 ± 1.80 99.53 ± 0.66 100.34 ± 1.91 0.9 100.55 ± 0.92 100.31 ± 0.99 100.71 ± 1.03 TSN 0.3 99.72 ± 0.72 98.77 ± 1.01 100.02 ± 0.89 0.6 99.58 ± 1.25 100.14 ± 0.35 99.18 ± 0.47 0.9 100.73 ± 0.49 100.10 ± 0.65 98.93 ± 0.88 a, b Three determination were averaged c Nine determinations were averaged in 3 days Three replicates of one by one of the three distinct concentrations of CTN and TSN (0.3, 0.6, and 0.9 µg/band) were used to test the accuracy of the suggested approach. 3.2.3. Precision Precision experiments conducted intra- and inter-day demonstrated the accuracy of the suggested analytical method. medications that were mentioned. Three copies of each of the three distinct amounts—0.3, 0.6, and 0.9 µg/band per CTN and TSN, respectively—were used in order to attain intra-day accuracy. For three consecutive days, equal doses of the medicines under study (0.3, 0.6, and 0.9 µg/band) were used separately to assess inter-day precision. The same three doses of the aforementioned medications—0.3, 0.6, and 0.9 µg/band—were utilized to test inter-day precision for three consecutive days. The %RSD value was computed in order to determine intra-day and inter-day variability. To identify both intra- and inter-day fluctuations, the percentage RSD was calculated as shown in Table 2 . The results of the research show that the %RSD is less than 2%, demonstrating the high level of accuracy of the suggested procedures. 3.2.4. Selectivity and specificity The capability of an analytical approach for assessing analytes without affecting other matrix components is known as selectivity. Researchers were able to assess the method's selectivity by analyzing the CTN and TSN combination that had been tampered with using plasma and urine samples. Figure 3 shows that the examined drug peaks did not have any overlapping peaks of plasma or urine components. The drug's RF values in plasma and urine were identical to those in the reference solutions. The system suitability parameters [ 34 ]. for the CTN and TSN separation utilizing the discussed approach are displayed in Table 3 . Table 3 System suitability parameters for the separation of CTN and TSN using the developed method parameter CTN TSN Reference value [ 34 ] Tailing factor (T) 0.87 1.01 ≥ 1 Capacity factor(k´) 0.75 0.19 0–10 Selectivity factor (α) 3.95 > 1 Resolution (RS) 2.12 > 1.5 Table 4 Comparison study of the proposed TLC method and the reported TLC- method (n = 6,3) [ 3 ],[ 12 ] c Items Proposed method Reported method Dosage form Cervitolka plus® capsule % Recovery ± %RSD a CTN TSN CTN [ 3 ] TSN [ 12 ] 100.06 ± 1.04 100.66 ± 0.88 99.91 ± 1.07 100.31 ± 0.67 Standard addition technique 30 µg/mL 100.14 ± 0.63 99.97 ± 1.05 ------------- ----------- 40 µg/mL 99.43 ± 0.87 100.03 ± 0.97 ------------- ------------ t test b ---------------- --------------- 0.06 (2.306) 0.28 (2.571) F test b ---------------- --------------- 1.05 (4.76) 1.71 (9.28) a Average of three determinations b At a 95% confidence level, the theoretical values for the t (2.306, 2.571) and F tests were (4.76,9.28) for CTN and TSN respectively. c [ 3 ] TLC—densitometry for determination of citicoline sodium in prescence of its alkaline degradation using ammonia—ethyl acetate—triethylamine (6:3.5:0.5, v / v ) as the mobile phase to give compact spots for citicoline sodium ( R F = 0.35) and its degradation product ( R F = 0.1); the chromatogram was scanned at 272 nm. [ 12 ] HPTLC method for determination of tyrosine and tryptophan using acetonitrile, ethyl acetate, and Britton-Ribonson buffer with a pH of 9 (50:20:20 v/v/v) mobile phase with Rf = 0.35 for tryptophane and (Rf = 0.23) for tyrosine at dual wavelengths of 280 and 225 nm for absorption and fluorescence modes . 3.2.5. Robustness Robustness is a measure of a procedure's capacity to continue unaltered with minor but intentional modifications to its parameters. As indicated in Table 1 , analyses were performed on the slight variations in the ratios of the used mobile phase (phosphate buffer (pH = 4): methanol) and saturation time (20 ± 3min). 3.3. Applications of the suggested approach 3.3.1. Pharmaceutical formulations Oral capsules of Cervitolka plus® were evaluated following the technique outlined in "2.4. Procedure for capsule." Acceptable percentage recoveries and standard deviations that were statistically equivalent to the outcomes obtained using the reported approach were obtained using the tabulated t-test and F-test at a 95% confidence level [ 3 ] and [ 12 ]. There is no appreciable difference in precision and accuracy between the reported and suggested methods 3.3.2. Spiked human plasma and urine Table 5 contains the information obtained by spiking human urine and plasma. Low RSD values and good recovery percentages were discovered after the plasma samples were analyzed using three different quantity ranges (Table 5 ). These results demonstrated the selectivity of the approach by demonstrating a high degree of drug extraction capacity from plasma without interacting with plasma and urine components. The outcomes demonstrated that actual plasma could be treated using the recommended method. Table 5 Results of the proposed method in spiked human plasma and urine spiked human plasma Urine samples Drug Amount taken (µg/band) Amount found (µg/band) % Amount found (mean ± RSD) Amount taken (µg/band) Amount found (µg/band) % Amount found (mean ± RSD) CTN 0.25 0.24 94.50 ± 0.40 0.25 0.23 90.75 ± 0.25 0.5 0.46 92.50 ± 0.64 0.5 0.46 91.25 ± 0.25 1 0.94 93.87 ± 0.24 1 0.91 91.00 ± 0.50 TSN 0.25 0.32 93.00 ± 0.54 0.25 0.23 90. 50 ± 0.28 0.5 0.46 91.62 ± 0.95 0.5 0.46 92.75 ± 0.39 1 0.90 90.25 ± 0.42 1 0.92 91.50 ± 0.69 3.4. Greenness evaluation of the eluted TLC technique Table 6 contains the green assessment results of the proposed TLC method and comparison of the proposed method with the previously reported methods. The findings of evaluating and comparing the proposed procedures with the reported chromatographic methods using the three assessment tools (GAPI,AGREE, and BAGI) showed that the suggested chromatographic method was comparatively ecologically begin and had few danger. On the other hand, it has the highest sensitivity to estimate the studied drugs in spiked human plasma and urine. Green analytical procedure index (GAPI) As illustrated in Table 6 , the GAPI pictograms clearly differentiate the greenness profiles of the compared chromatographic methods. The developed TLC-UV method achieved the most favorable profile, comprising eight green, five yellow, and two red sections. This distribution reflects the use of less hazardous components in the mobile phase which composed of (phosphate buffer-methanol.70:30 (V/V). In contrast, the reported TLC method [ 3 ] exhibited six green, four yellow, and five red sections, indicating a higher environmental burden primarily due to the use of ammonia- ethyl acetate-triethyleamin as the mobile phase. The reported HPTLC method [ 12 ] demonstrated a moderate profile with seven green, five yellow, and three red sections. Analytical greenness calculator(AGREE) AGREE software assessment confirmed the superior greenness of the developed TLC-UV method, which achieved the highest score (0.86),followed by HPTLC method [ 12 ] (0.72), whereas the reported TLC method [ 3 ] showed the score (0.59). these results demonstrate that the developed method adheres more closely to the 12 principles of green analytical chemistry, making it more environmentally benign compared to the previously published methods. Blue Analytical Greenness Index (BAGI ) In addition to GAPI and AGREE, the Blue Analytical Greenness Index (BAGI) was also applied for a more comprehensive evaluation of the environmental impact developed TLC-UV method exhibited the highest BAGI score (77.5),confirming its superior greenness profile and alignment with sustainable practices. In contrast, the reported TLC [ 3 ] method gave the lowest BAGI score (35.0),indicating significant environmental drawbacks associated with the solvents and conditions employed. The HPTLC [ 12 ] method showed an intermediate BAGI value (40.0),which, although better than the reported TLC,still lagged behind the developed TLC-UV method. To complete these findings, the Eco-scale tool was employed, offering another quantitative perspective on the enviromemental impact of the developed method as in Table 7 . Table 7 Eco-scale penalty points for the developed TLC method for determining CTN and TSN simultaneously. Reagents Penalty points Solvents Methanol 6 water 0 phosphate 1 Instruments Energy used (UV Scanner) 1 (< 1.5 kWh per sample) 1 Occupational hazard 3 Waste 3 Total penalty points 14 Analytical eco-scale score 86 Waste, the volume of the mobile phase/no of bands per aluminum-backed TLC silica gel 60 F 254 plate 4. Conclusion A sensitive, selective, and eco-friendly TLC approach was developed in conjunction with UV detection to accurately and precisely separate and identify the CTN and TSN in their synthetic combination, administered form, and (spiked plasma and urine) samples. Every experimental parameter was adjusted to achieve high sensitivity.Numerous benefits of the suggested method include low solvent consumption, a big sample capacity per run, and a fast analysis time. The unique TLC technique is environmentally safe and green, according to the test greenness results. Declarations Credit authorship contribution statement Declaration of competing interest Ethics approval and consent to participate, the research ethical commission of the Deraya University University—Faculty of Pharmacy (New Minia, Egypt) has accepted this work, with serial number DCSR-010-024-29. Every participant gave their informed consent. The authors declare no conflict of interest . Funding The work has not received fund from any particular organization. Data availability All data will be available upon request. Ethics approval and consent to participate The current study was carried out in accordance with relevant guidelines and regulations Credit authorship contribution statement Amira M. Hegazy: Investigation and Conceptualization, Hesham Salem: Supervision, Maha M. Abdelrahman: Supervision , Dina Z. Mazen: Resources and Software, Maimana A. Magdy: Investigation and Conceptualization References O'Neil, M.J., The Merck index: an encyclopedia of chemicals, drugs, and biologicals. 2013: RSC Publishing. Brayfield, A., Martindale: the complete drug reference. 2017. 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Capello, C., Fischer, U. and Hungerbühler, K.J.G.C., What is a green solvent? A comprehensive framework for the environmental assessment of solvents.Green Chemistry, 2007. 9.927-934. DOI: 10.1039/B617536H Gałuszka, A., Migaszewski, Z.M., Konieczka, P. and Namieśnik, J.J.T.T.i.A.C., Analytical Eco-Scale for assessing the greenness of analytical procedures. 2012. 37.61-72. https://doi.org/10.1016/j.trac.2012.03.013 Płotka-Wasylka, J. and Wojnowski, W., Complementary green analytical procedure index (ComplexGAPI) and software.Green Chemistry. 2021. 23.8657-8665. DOI: 10.1039/D1GC02318G Pena-Pereira, F., Wojnowski, W. and Tobiszewski, M., AGREE—Analytical GREEnness metric approach and software.Analytical chemistry.2020.92.10076-10082. https://pubs.acs.org/doi/10.1021/acs.analchem.0c01887. Manousi a,N. Wojciech Wojnowski bc. PłoWasylka J and Samanidou, v"Blue applicability grade index (BAGI) and software: a new tool for the evaluation of method practicality." Green chemistry 25(19): 760410.1039/D3GC02347H Guideline, I.H.T. Validation of Analytical Procedures: Text and Methodology, Q2 (R1) Geneva, 2005. in The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use.[Google Scholar]. 2013. https://doi.org/10.1556/1006.2015.28.3.8 The United States Pharmacopeia, Formulary N. USP–NF standards. Secun derabad: United States Pharmacopeia Convention Inc; 2023. Table 1 and 6 Table 1 and 6 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1and6.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 31 Oct, 2025 Reviews received at journal 24 Oct, 2025 Reviews received at journal 19 Oct, 2025 Reviewers agreed at journal 16 Oct, 2025 Reviewers agreed at journal 16 Oct, 2025 Reviewers agreed at journal 15 Oct, 2025 Reviewers invited by journal 15 Oct, 2025 Editor invited by journal 15 Oct, 2025 Editor assigned by journal 15 Oct, 2025 Submission checks completed at journal 15 Oct, 2025 First submitted to journal 11 Oct, 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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1","display":"","copyAsset":false,"role":"figure","size":101523,"visible":true,"origin":"","legend":"\u003cp\u003eChemical structure of Citicoline (a) \u0026nbsp;and tyrosine (b).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7835319/v1/e1ce2024d45dc3c5c866e099.png"},{"id":94673105,"identity":"ad455acd-685f-4482-a961-7a5b3ec25333","added_by":"auto","created_at":"2025-10-29 13:41:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":172890,"visible":true,"origin":"","legend":"\u003cp\u003eTLC densitogram of a mixture of CTN and TSN at 256 nm \u0026nbsp;by phosphate buffer (PH=4): methanol (70:30, v/v).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7835319/v1/c801d5b67b18dc9841989455.png"},{"id":94673381,"identity":"959bf98c-692c-4324-a4b2-ab131f382340","added_by":"auto","created_at":"2025-10-29 13:41:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":473529,"visible":true,"origin":"","legend":"\u003cp\u003eTLC densitogram of spiked human plasma (a) and urine (b) samples\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7835319/v1/1b148cb788a2e1bd013cb2aa.png"},{"id":94731069,"identity":"362717ab-9acc-4b5a-9500-0be24d5c93d4","added_by":"auto","created_at":"2025-10-30 07:07:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2084203,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7835319/v1/5867cda6-2b5f-4540-a346-337d688955e0.pdf"},{"id":94670634,"identity":"53725607-86a7-49ac-8168-79ef98b33cf8","added_by":"auto","created_at":"2025-10-29 13:12:36","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":351815,"visible":true,"origin":"","legend":"","description":"","filename":"Table1and6.docx","url":"https://assets-eu.researchsquare.com/files/rs-7835319/v1/0edbf50ebd0c329709de446e.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eSimultaneous Quantification of Citicoline and Tyrosine by Green HPTLC-Densitometry: Application to Pharmaceutical Formulations and Biological Fluids\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCiticoline (CTN), also known as cytidine 5'-diphosphocholine, is an intermediate in the biosynthesis of phosphatidylcholine, a key component of cell membranes. Structurally, it is 5\u0026prime;-trihydrogen diphosphate p\u0026prime;-[2-(trimethylammonio)ethyl] ester inner salt [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). CTN is naturally occurring compound in human and animal tissues, particularly in the organs. CTN is widely used in the pharmacotherapy of neurological disorders, including stroke, brain trauma, Parkinson\u0026rsquo;s disease, and cognitive impairment, due to its ability to stimulate phospholipid biosynthesis, preserve neuronal energy reserves, inhibit apoptosis, and enhance acetylcholine production [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eA review of published literature reveals that CTN has been analyzed using various techniques, including chromatographic methods [\u003cspan additionalcitationids=\"CR4 CR5 CR6 CR7 CR8\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], spectrophotometry [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], and spectrofluorometry [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], either as a single component or in combination.\u003c/p\u003e\u003cp\u003eTyrosine (TSN), chemically known as 4-hydroxyphenylalanine[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], is an aromatic amino acid essential for protein synthesis. It serves as a precursor for the neurotransmitter's norepinephrine and dopamine, contributing to moodelevation, memory enhancement, and mental alertness. Additionally, TSN plays a critical role in melanin production and thyroxin (thyroid hormone) synthesis. Several analytical techniques\u0026mdash;including HPTLC [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], LC [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], HPLC [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR15\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e16\u003c/span\u003e], spectrophotometry [\u003cspan additionalcitationids=\"CR18 CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e21\u003c/span\u003e], and spectrofluorometry [\u003cspan additionalcitationids=\"CR23 CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e26\u003c/span\u003e].have been reported for the determination of TSN, both individually and in combination.\u003c/p\u003e\u003cp\u003eIn Egypt, CTN and TSN are formulated together in the pharmaceutical product Cervitolka Plus\u0026reg; (BioMed Pharmaceutical Company), where they work synergistically to support cognitive function and mental alertness.\u003c/p\u003e\u003cp\u003eA review of the available literature reveals no existing analytical method for the simultaneous determination of CTN and TSN in their binary mixture. To address this, we present, for the first time, developed HPTLC method the employs green organic solvents. This approach not only reduces environmental hazards but also eliminates the need for toxic solvents [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eIn the simultaneous estimation of these compounds.\u003c/p\u003e\u003cp\u003eThe green analytical profile was further evaluated using Eco-Scale [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e29\u003c/span\u003e], Green Analytical Procedure Index (GAPI) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e30\u003c/span\u003e], AGREE [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e31\u003c/span\u003e] metrics and Blue Analytical Greenness Index (BAGI)[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. to assess its sustainability compared to previously published.\u003c/p\u003e\u003cp\u003eThe successful implementation of this method to spiked human plasma and urine samples demonstrates its potential for adaptation to pharmacokinetic studies and clinical applications.\u003c/p\u003e"},{"header":"2. Experimental","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Instrumentation\u003c/h2\u003e\u003cp\u003eThe CAMAG\u0026reg; TLC scanner 3, which was operated by vision CATS software (version 3.1.21109.3, Muttenz, Switzerland), was used for the analysis. Using a 100 \u0026micro;L Hamilton syringe (Bonaduz, Switzerland) and nitrogen gas for solvent evaporation, samples are placed as bands on TLC plates using the CAMAG\u0026reg; Linomat 5 sample application equipment.\u003c/p\u003e\u003cp\u003eDeuterium and tungsten lamps were used for the scanning, which had a 5 \u0026times; 0.2 mm slit and a scanning speed of 20 mm/s.\u003c/p\u003e\u003cp\u003eThe stationary phase consisted of aluminum sheets (20 \u0026times; 20 cm, Merck, Darmstadt, Germany) covered with TLC silica gel 60 F254. Additionally, the sample was processed using a Centurion low-speed digital centrifuge (4000 rpm, United Kingdom).\u003c/p\u003e\u003cp\u003eOther instruments included an analytical balance (Switzerland) and a Jenway 3510 pH meter (England), which ensured accurate measurements throughout the analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Active pharmaceutical ingredients and chemical reagents\u003c/h2\u003e\u003cp\u003eThe Pyramid Office in Cairo, Egypt, provided the pure CTN and TSN. Tyrosine (purity: 99.17%) and citicoline (purity: 99.5%) were employed in this investigation. Using statistical analysis based on the mean values from three independent experiments for each compound, The accuracy of the authentic samples was verified\u003c/p\u003e\u003cp\u003eHPLC-grade methanol was supplied by Sigma\u0026ndash;Aldrich Chemie GmbH, Germany, while sodium dihydrogen phosphate was provided by El-Nasr Pharmaceutical Chemicals Co. (Cairo, Egypt).\u003c/p\u003e\u003cp\u003eTo prepare a phosphate buffer (pH\u0026thinsp;=\u0026thinsp;4), dissolve 3.2 g of sodium dihydrogen phosphate in a 25 mL volumetric flask and fill with deionized water (source: Otsuka Pharmaceuticals, Cairo, Egypt). Plasma samples were provided by the blood bank at Minia University Hospital (Minia, Egypt), and urine samples were collected from a healthy volunteer. The silica gel 60 F254 (20 \u0026times; 20 cm) pre-coated TLC aluminum sheet was used.\u003c/p\u003e\u003cp\u003ePharmaceutical formulation:Cervitolka Plus\u0026reg; capsule (batch No. 230858) were manufactured and supplied by BioMed Pharmaceuticals (Cairo, Egypt). Each CAPULE is claimed to contain 500 mg of CTN and 50 mg of TSN.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Standard solutions\u003c/h2\u003e\u003cp\u003eTo formulate standard stock solutions, 100 mg of CTN and TSN were dissolved in 100 milliliters of water.\u003c/p\u003e\u003cp\u003e.to have stock solutions of (1mg/mL) Appropriate dilutions were made using water to obtain standard working solutions at concentration of 500 \u0026micro;g/mL of each CTN and TSN .\u003c/p\u003e\u003cp\u003eA 5 \u0026micro;L injection volume was applied onto aluminum-backed TLC silica gel 60 F256 plates.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Cervitolka \u003csup\u003eplus\u003c/sup\u003e\u0026reg; capsule sample\u003c/h2\u003e\u003cp\u003eThe content of one Cervitolka plus\u0026reg; capsule were weighed and transferred into clean 100mL volumetric flask. Approximately 15 mL of distilled water was added, and the mixture was sonicated for 5 minutes to ensure complete dissolution of citicoline and tyrosine.\u003c/p\u003e\u003cp\u003eAfter cooling to room temperature, the solution was filtered through Whatman filter paper to remove any undissolved particles. The filtrate was then diluted to volume with distilled water to obtain a stock solution of 5 mg/mL for citicoline and 0.5 mg/mL for tyrosine.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Spiked biological samples\u003c/h2\u003e\u003cp\u003eHealthy participants' blood samples were collected carefully toward heparinized tubes and centrifuged at 4000 rpm for 20 minutes to obtain plasma, which was then transferred to a sterile new tube and stored at -20\u0026deg;C. Additionally, urine samples from healthy individuals were collected and maintained at 4\u0026deg;C until analysis.\u003c/p\u003e\u003cp\u003eIn sterile centrifuge tubes, 1.0 mL of plasma was spiked with 1.0 mL of each medication at a stock concentration of 1000 \u0026micro;g/mL in order to prepare the plasma sample. To denature proteins, two milliliters of acetonitrile were added, and the mixture was stirred for ten minutes. After diluting the resultant solution with 10 milliliters of pure water, it was centrifuged for 30 minutes at 3500 rpm. At a concentration of 100 \u0026micro;g/mL, the clear supernatant was separated for further dilution.\u003c/p\u003e\u003cp\u003eTo prepare the urine sample, 1.0 mL of each medication (1000 \u0026micro;g/mL) was combined with 1.0 mL of urine. After adding two milliliters of methanol, the liquid was stirred for ten minutes. After that, the solution was centrifuged for 30 minutes at 3500 rpm after being diluted with 10 mL of distilled water. As necessary, the supernatant was further diluted.\u003c/p\u003e\u003cp\u003eTo prepare blank samples (plasma and urine), the same procedures were followed without the drug solution.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Optimization of chromatographic conditions\u003c/h2\u003e\u003cp\u003e5 \u0026micro;L of sample solutions and standard dilutions were placed as bands 10 mm from the bottom of TLC plates (20 cm \u0026times; 9 cm). Phosphate buffer (pH\u0026thinsp;=\u0026thinsp;4) and methanol in a 70:30: v/v ratio made up the mobile phase.\u003c/p\u003e\u003cp\u003eThe plate was dried with a cold stream of air until it dried all the way to the top after 20 minutes of saturation. After removal, the plate was allowed to dry before being scanned in reflectance/absorbance mode at 256 nm.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Method optimization\u003c/h2\u003e\u003cp\u003eCiticoline, is a polar molecule and classified as a water-soluble compound. While Tyrosine exhibits both polar and nonpolar characteristics due to its molecular structure. While its hydroxyl (-OH) group confers polarity through hydrogen bonding with water, the presence of an aromatic ring introduces hydrophobic properties. This dual nature sometimes makes its classification ambiguous. Under physiological pH conditions, tyrosine generally remains in a neutral, uncharged state. The polar nature of the analytes necessitates the use of polar solvents to achieve proper migration on TLC plates without significant retention.\u003c/p\u003e\u003cp\u003eThe mobile phase composition was determined using trial-based analysis, taking into account the polarity differences between CTN and TSN. Initially, a mobile phase consisting of phosphate buffer and water 70:30 (v/v) was used and gave satisfactory results; further trials were necessary to refine the separation and increase resolution.\u003c/p\u003e\u003cp\u003eAmong various tested systems, a 60:40 (V/V) ethanol-water mobile phase was tested, but poor separation was obtained due to differences in polarity\u0026mdash;TSN, being less polar. migrated near the solvent front due to its weak interaction with the stationary phase, while the highly polar CTN remained near the baseline.\u003c/p\u003e\u003cp\u003eWhen the same solvent system was tested in an acidic medium, CTN exhibited even stronger retention at the baseline. Protonation of its phosphate group increased its polarity, enhancing interaction with the stationary phase and further restricting its movement, while TSN continued to migrate freely. Even after adjusting the mobile phase composition to 30:70 (v/v), separation was not achieved.\u003c/p\u003e\u003cp\u003eAn alternative mobile phase system using triethylamine-water (10:90 v/v) was employed but failed to produce adequate separation between CTN and TSN. The spots were either overlapped or insufficiently resolved, indicating that this solvent system lacked the necessary selectivity.\u003c/p\u003e\u003cp\u003eConsequently, phosphate buffer was explored at various pH levels, resulting in successful separation of the two compounds. The optimal developing system was identified as phosphate buffer (pH 4) with methanol (70:30 v/v), providing well-resolved bands with suitable Rf values and minimal tailing.\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e displays the densitogram produced by this mobile phase system. To find the ideal period, trials were also carried out with different saturation times (10 to 30 minutes).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIt was found that 20 minutes provided the best separation between the two bands, yielding the most reliable Rf values. The UV absorption spectra of the two active medicinal substances under examination showed maxima for TSN and CTN at 280 nm and 272 nm, respectively. 256 nm was determined to be the optimum compromise after a number of wavelengths were investigated for the appropriate detection of the medications under study (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026amp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Method validation\u003c/h2\u003e\u003cp\u003e The proposed approach was confirmed to adhere to the International Council for Harmonization's (ICH) guidelines.\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e3.2.1. Linearity, limit of quantification (LOQ), and detection (LOD)\u003c/h2\u003e\u003cp\u003eThe linearity ranges of CTN and TSN were (0.05‒1.25 \u0026micro;g/band) and (0.12\u0026ndash;2.5 \u0026micro;g/band), respectively, with correlation coefficient from 0.9999 for the cited drugs. Based on statistical estimations, the formulae LOD\u0026thinsp;=\u0026thinsp;3.3 x SD of intercept/slope and LOQ\u0026thinsp;=\u0026thinsp;10 \u0026times; SD of intercept/slope were developed. The statistical results show that the method's sensitivity for detecting the medications in consideration is acceptable. The important quantitative features are displayed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The linear regression model performed well for the medications under study, according to the lack-of-fit test.\u003c/p\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e3.2.2. Accuracy\u003c/h2\u003e\u003cp\u003eThe correctness of a technique is determined by the degree of agreement between the values found and the values accepted as acceptable reference values or conventional true values. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e demonstrates the accuracy of the suggested techniques with a low percentage RSD and good recovery.\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\u003eAccuracy and precision of the proposed approach\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDrug\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAmount\u003c/p\u003e\u003cp\u003e(\u0026micro;g/band)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAccuracy\u003c/p\u003e\u003cp\u003eRecovery\u0026thinsp;\u0026plusmn;\u0026thinsp;RSD\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePrecision\u003c/p\u003e\u003cp\u003eIntra-day\u003c/p\u003e\u003cp\u003eRecovery\u0026thinsp;\u0026plusmn;\u0026thinsp;RSD (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePrecision\u003c/p\u003e\u003cp\u003eInter-day\u003c/p\u003e\u003cp\u003eRecovery\u003csup\u003eb\u003c/sup\u003e \u0026plusmn; RSD (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCTN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e99.16\u0026thinsp;\u0026plusmn;\u0026thinsp;1.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e99.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e99.41\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e99.37\u0026thinsp;\u0026plusmn;\u0026thinsp;1.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e99.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e100.34\u0026thinsp;\u0026plusmn;\u0026thinsp;1.91\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e100.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e100.71\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTSN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e99.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e98.77\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e100.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e99.58\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e99.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e100.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e98.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003ea, b\u003c/sup\u003e Three determination were averaged\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003ec\u003c/sup\u003e Nine determinations were averaged in 3 days\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThree replicates of one by one of the three distinct concentrations of CTN and TSN (0.3, 0.6, and 0.9 \u0026micro;g/band) were used to test the accuracy of the suggested approach.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e3.2.3. Precision\u003c/h2\u003e\u003cp\u003ePrecision experiments conducted intra- and inter-day demonstrated the accuracy of the suggested analytical method. medications that were mentioned. Three copies of each of the three distinct amounts\u0026mdash;0.3, 0.6, and 0.9 \u0026micro;g/band per CTN and TSN, respectively\u0026mdash;were used in order to attain intra-day accuracy. For three consecutive days, equal doses of the medicines under study (0.3, 0.6, and 0.9 \u0026micro;g/band) were used separately to assess inter-day precision. The same three doses of the aforementioned medications\u0026mdash;0.3, 0.6, and 0.9 \u0026micro;g/band\u0026mdash;were utilized to test inter-day precision for three consecutive days.\u003c/p\u003e\u003cp\u003eThe %RSD value was computed in order to determine intra-day and inter-day variability. To identify both intra- and inter-day fluctuations, the percentage RSD was calculated as shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The results of the research show that the %RSD is less than 2%, demonstrating the high level of accuracy of the suggested procedures.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003e3.2.4. Selectivity and specificity\u003c/h2\u003e\u003cp\u003eThe capability of an analytical approach for assessing analytes without affecting other matrix components is known as selectivity.\u003c/p\u003e\u003cp\u003eResearchers were able to assess the method's selectivity by analyzing the CTN and TSN combination that had been tampered with using plasma and urine samples. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows that the examined drug peaks did not have any overlapping peaks of plasma or urine components. The drug's RF values in plasma and urine were identical to those in the reference solutions. The system suitability parameters [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. for the CTN and TSN separation utilizing the discussed approach are displayed in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\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\u003eSystem suitability parameters for the separation of CTN and TSN using the developed method\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\u003eparameter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCTN\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTSN\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eReference value [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTailing factor (T)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCapacity factor(k\u0026acute;)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u0026ndash;10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSelectivity factor (α)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e3.95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eResolution (RS)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e2.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;1.5\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\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\u003eComparison study of the proposed TLC method and the reported TLC- method (n\u0026thinsp;=\u0026thinsp;6,3) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e],[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eItems\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eProposed method\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003eReported method\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e\u003cp\u003eDosage form\u003c/p\u003e\u003cp\u003eCervitolka plus\u0026reg; capsule\u003c/p\u003e\u003cp\u003e% Recovery \u0026plusmn; %RSD\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCTN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTSN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCTN [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eTSN [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e100.06\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e99.91\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e100.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eStandard addition technique\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e30 \u0026micro;g/mL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e100.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e99.97\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-------------\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-----------\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e40 \u0026micro;g/mL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e99.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-------------\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e------------\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cem\u003et\u003c/em\u003e test\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e----------------\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e---------------\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.06\u003c/p\u003e\u003cp\u003e(2.306)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.28\u003c/p\u003e\u003cp\u003e(2.571)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cem\u003eF\u003c/em\u003e test\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e----------------\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e---------------\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.05\u003c/p\u003e\u003cp\u003e(4.76)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.71\u003c/p\u003e\u003cp\u003e(9.28)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003ea\u003c/sup\u003e Average of three determinations\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003eb\u003c/sup\u003e At a 95% confidence level, the theoretical values for the \u003cem\u003et\u003c/em\u003e (2.306, 2.571) and \u003cem\u003eF\u003c/em\u003e tests were (4.76,9.28) for CTN and TSN respectively.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003ec\u003c/sup\u003e [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] TLC\u0026mdash;densitometry for determination of citicoline sodium in prescence of its alkaline degradation using ammonia\u0026mdash;ethyl acetate\u0026mdash;triethylamine (6:3.5:0.5, \u003cem\u003ev\u003c/em\u003e/\u003cem\u003ev\u003c/em\u003e) as the mobile phase to give compact spots for citicoline sodium (\u003cem\u003eR\u003c/em\u003e\u003csub\u003eF\u003c/sub\u003e = 0.35) and its degradation product (\u003cem\u003eR\u003c/em\u003e\u003csub\u003eF\u003c/sub\u003e = 0.1); the chromatogram was scanned at 272 nm.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] HPTLC method for determination of tyrosine and tryptophan using acetonitrile, ethyl acetate, and Britton-Ribonson buffer with a pH of 9 (50:20:20 v/v/v) mobile phase with Rf\u0026thinsp;=\u0026thinsp;0.35 for tryptophane and (Rf\u0026thinsp;=\u0026thinsp;0.23) for tyrosine at dual wavelengths of 280 and 225 nm for absorption and fluorescence modes .\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003e3.2.5. Robustness\u003c/h2\u003e\u003cp\u003eRobustness is a measure of a procedure's capacity to continue unaltered with minor but intentional modifications to its parameters. As indicated in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, analyses were performed on the slight variations in the ratios of the used mobile phase (phosphate buffer (pH\u0026thinsp;=\u0026thinsp;4): methanol) and saturation time (20\u0026thinsp;\u0026plusmn;\u0026thinsp;3min).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Applications of the suggested approach\u003c/h2\u003e\u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\u003ch2\u003e3.3.1. Pharmaceutical formulations\u003c/h2\u003e\u003cp\u003eOral capsules of Cervitolka plus\u0026reg; were evaluated following the technique outlined in \"2.4. Procedure for capsule.\" Acceptable percentage recoveries and standard deviations that were statistically equivalent to the outcomes obtained using the reported approach were obtained using the tabulated t-test and F-test at a 95% confidence level [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] and [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. There is no appreciable difference in precision and accuracy between the reported and suggested methods\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\u003ch2\u003e3.3.2. Spiked human plasma and urine\u003c/h2\u003e\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e contains the information obtained by spiking human urine and plasma. Low RSD values and good recovery percentages were discovered after the plasma samples were analyzed using three different quantity ranges (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These results demonstrated the selectivity of the approach by demonstrating a high degree of drug extraction capacity from plasma without interacting with plasma and urine components. The outcomes demonstrated that actual plasma could be treated using the recommended method.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eResults of the proposed method in spiked human plasma and urine\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\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003espiked human plasma\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e\u003cp\u003eUrine samples\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDrug\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAmount taken\u003c/p\u003e\u003cp\u003e(\u0026micro;g/band)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAmount found (\u0026micro;g/band)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e% Amount found\u003c/p\u003e\u003cp\u003e(mean\u0026thinsp;\u0026plusmn;\u0026thinsp;RSD)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAmount taken\u003c/p\u003e\u003cp\u003e(\u0026micro;g/band)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAmount found (\u0026micro;g/band)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e% Amount found\u003c/p\u003e\u003cp\u003e(mean\u0026thinsp;\u0026plusmn;\u0026thinsp;RSD)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eCTN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e94.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e90.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e92.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e91.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e93.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e91.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eTSN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e93.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e90. 50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e91.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e92.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e90.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e91.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69\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\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Greenness evaluation of the eluted TLC technique\u003c/h2\u003e\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e contains the green assessment results of the proposed TLC method and comparison of the proposed method with the previously reported methods.\u003c/p\u003e\u003cp\u003e The findings of evaluating and comparing the proposed procedures with the reported chromatographic methods using the three assessment tools (GAPI,AGREE, and BAGI) showed that the suggested chromatographic method was comparatively ecologically begin and had few danger. On the other hand, it has the highest sensitivity to estimate the studied drugs in spiked human plasma and urine.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGreen analytical procedure index (GAPI)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs illustrated in Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, the GAPI pictograms clearly differentiate the greenness profiles of the compared chromatographic methods. The developed TLC-UV method achieved the most favorable profile, comprising eight green, five yellow, and two red sections. This distribution reflects the use of less hazardous components in the mobile phase which composed of (phosphate buffer-methanol.70:30 (V/V). In contrast, the reported TLC method [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] exhibited six green, four yellow, and five red sections, indicating a higher environmental burden primarily due to the use of ammonia- ethyl acetate-triethyleamin as the mobile phase. The reported HPTLC method [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] demonstrated a moderate profile with seven green, five yellow, and three red sections.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnalytical greenness calculator(AGREE)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAGREE software assessment confirmed the superior greenness of the developed TLC-UV method, which achieved the highest score (0.86),followed by HPTLC method [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] (0.72), whereas the reported TLC method [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] showed the score (0.59). these results demonstrate that the developed method adheres more closely to the 12 principles of green analytical chemistry, making it more environmentally benign compared to the previously published methods.\u003c/p\u003e\u003cp\u003e\u003cb\u003eBlue Analytical Greenness Index (BAGI\u003c/b\u003e)\u003c/p\u003e\u003cp\u003eIn addition to GAPI and AGREE, the Blue Analytical Greenness Index (BAGI) was also applied for a more comprehensive evaluation of the environmental impact developed TLC-UV method exhibited the highest BAGI score (77.5),confirming its superior greenness profile and alignment with sustainable practices. In contrast, the reported TLC [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] method gave the lowest BAGI score (35.0),indicating significant environmental drawbacks associated with the solvents and conditions employed. The HPTLC [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] method showed an intermediate BAGI value (40.0),which, although better than the reported TLC,still lagged behind the developed TLC-UV method.\u003c/p\u003e\u003cp\u003eTo complete these findings, the Eco-scale tool was employed, offering another quantitative perspective on the enviromemental impact of the developed method as in Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEco-scale penalty points for the developed TLC method for determining CTN and TSN simultaneously.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eReagents\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePenalty points\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eSolvents\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMethanol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ewater\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ephosphate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eInstruments\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEnergy used (UV Scanner) 1 (\u0026lt;\u0026thinsp;1.5 kWh per sample)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOccupational hazard\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWaste\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal penalty points\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnalytical eco-scale score\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e86\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"2\"\u003eWaste, the volume of the mobile phase/no of bands per aluminum-backed TLC silica gel 60 F\u003csub\u003e254\u003c/sub\u003e plate\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eA sensitive, selective, and eco-friendly TLC approach was developed in conjunction with UV detection to accurately and precisely separate and identify the CTN and TSN in their synthetic combination, administered form, and (spiked plasma and urine) samples.\u003c/p\u003e\u003cp\u003eEvery experimental parameter was adjusted to achieve high sensitivity.Numerous benefits of the suggested method include low solvent consumption, a big sample capacity per run, and a fast analysis time. The unique TLC technique is environmentally safe and green, according to the test greenness results.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCredit authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate, the research ethical commission of the Deraya University University—Faculty of Pharmacy (New Minia, Egypt) has accepted this work, with serial number DCSR-010-024-29. Every participant gave their informed consent.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work has not received fund from any particular organization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data will be available upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe current study was carried out in accordance with relevant guidelines and regulations\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCredit authorship contribution statement\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAmira M. Hegazy:\u003c/strong\u003e Investigation and Conceptualization,\u003cstrong\u003e\u0026nbsp;Hesham Salem:\u0026nbsp;\u003c/strong\u003eSupervision, \u003cstrong\u003eMaha M. Abdelrahman:\u0026nbsp;\u003c/strong\u003eSupervision\u003cstrong\u003e, Dina Z. Mazen:\u0026nbsp;\u003c/strong\u003eResources and\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eSoftware,\u003cstrong\u003e\u0026nbsp; Maimana A. Magdy:\u0026nbsp;\u003c/strong\u003eInvestigation and Conceptualization\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eO\u0026apos;Neil, M.J., The Merck index: an encyclopedia of chemicals, drugs, and biologicals. 2013: RSC Publishing.\u003c/li\u003e\n\u003cli\u003eBrayfield, A., Martindale: the complete drug reference. 2017.\u003c/li\u003e\n\u003cli\u003eMahmoud, O.A., Hegazy, M.A., Salem, H. and Moustafa, A.A., Comparative study of reversed-phase high-performance liquid chromatography versus thin-layer chromatography\u0026mdash;densitometry for determination of citicoline sodium in presence of its alkaline degradation products.Journal of Planar Chromatography\u0026ndash;Modern TLC, 2015. 28.241-247. https://doi.org/10.1556/1006.2015.28.3.8\u003c/li\u003e\n\u003cli\u003eAbdelrahman, M.M., Ahmed, A.B., Omar, M.A., Derayea, S.M. and Abdelwahab, N.S., Development and validation of stability indicating chromatographic methods for simultaneous determination of citicoline and piracetam. 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A comprehensive framework for the environmental assessment of solvents.Green Chemistry, 2007. 9.927-934. DOI: 10.1039/B617536H \u003c/li\u003e\n\u003cli\u003eGałuszka, A., Migaszewski, Z.M., Konieczka, P. and Namieśnik, J.J.T.T.i.A.C., Analytical Eco-Scale for assessing the greenness of analytical procedures. 2012. 37.61-72. https://doi.org/10.1016/j.trac.2012.03.013 \u003c/li\u003e\n\u003cli\u003ePłotka-Wasylka, J. and Wojnowski, W., Complementary green analytical procedure index (ComplexGAPI) and software.Green Chemistry. 2021. 23.8657-8665. DOI: 10.1039/D1GC02318G\u003c/li\u003e\n\u003cli\u003ePena-Pereira, F., Wojnowski, W. and Tobiszewski, M., AGREE\u0026mdash;Analytical GREEnness metric approach and software.Analytical chemistry.2020.92.10076-10082. https://pubs.acs.org/doi/10.1021/acs.analchem.0c01887.\u003c/li\u003e\n\u003cli\u003eManousi a,N. Wojciech Wojnowski bc. PłoWasylka J and Samanidou, v\u0026quot;Blue applicability grade index (BAGI) and software: a new tool for the evaluation of method practicality.\u0026quot; Green chemistry 25(19): 760410.1039/D3GC02347H\u003c/li\u003e\n\u003cli\u003eGuideline, I.H.T. Validation of Analytical Procedures: Text and Methodology, Q2 (R1) Geneva, 2005. in The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use.[Google Scholar]. 2013. https://doi.org/10.1556/1006.2015.28.3.8\u003c/li\u003e\n\u003cli\u003eThe United States Pharmacopeia, Formulary N. USP\u0026ndash;NF standards. Secun derabad: United States Pharmacopeia Convention Inc; 2023.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1 and 6","content":"\u003cp\u003eTable 1 and 6 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"bmc-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccjo","sideBox":"Learn more about [BMC Chemistry](https://bmcchem.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ccjo/default.aspx","title":"BMC Chemistry","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Citicoline, Tyrosine, HPTLC, Greenness evaluation","lastPublishedDoi":"10.21203/rs.3.rs-7835319/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7835319/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCiticoline (CTN) and Tyrosine (TSN) are co-formulated in fixed-dose therapy, leveraging their synergistic effects to enhance cognitive function and mental clarity. This study presents the development of a high-performance thin-layer chromatography (HPTLC) method for the simultaneous determination of Citicoline (CTN) and Tyrosine (TSN) in a pharmaceutical capsules, plasma, and urine. A green HPTLC system composed of phosphate buffer pH\u0026thinsp;=\u0026thinsp;4 and methanol (70:30 v/v) was optimized and applied on silica gel TLC plates (G60 F254). (CTN) and (TSN) were quantified within linear ranges of 0.05‒1.25 \u0026micro;g/band and 0.12‒2.5 \u0026micro;g/band, respectively, using UV detection at 256 nm. The proposed HPTLC method was statistically validated according to ICH in terms of linearity, precision, accuracy, specificity, robustness, detection limits, with LOQ values of 0.043 \u0026micro;g/band for (CTN) and 0.107 \u0026micro;g/band for (TSN). Application of this method extended to pharmaceutical formulations, spiked human plasma, and urine, confirming peak identity and purity. A statistical comparison with published analytical techniques revealed no significant differences, highlighting the reliability of this approach.\u003c/p\u003e\u003cp\u003eTo assess environmental impact of the advised method, the eco-scale, Green Analytical Procedure Index (GAPI),AGREE metric and Blue Analytical Greenness Index (BAGI).\u003c/p\u003e\u003cp\u003etools were employed, demonstrating the sustainability of this method. High recovery rates further validate its efficacy, positioning it as a green, accurate, and reproducible alternative for routine analysis of (CTN) and (TSN).\u003c/p\u003e","manuscriptTitle":"Simultaneous Quantification of Citicoline and Tyrosine by Green HPTLC-Densitometry: Application to Pharmaceutical Formulations and Biological Fluids","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-29 13:12:32","doi":"10.21203/rs.3.rs-7835319/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-31T10:36:53+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-24T19:07:42+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-19T15:22:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"324197851638640361451182164232907456751","date":"2025-10-16T09:39:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"316624068221698018724740165826049976585","date":"2025-10-16T04:26:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"50780777843099690350411129477530851445","date":"2025-10-15T23:23:56+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-15T18:22:49+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-10-15T10:08:39+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-15T05:54:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-15T05:53:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Chemistry","date":"2025-10-11T12:44:04+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccjo","sideBox":"Learn more about [BMC Chemistry](https://bmcchem.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ccjo/default.aspx","title":"BMC Chemistry","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e2981494-fb39-4e90-a655-ad093e375a87","owner":[],"postedDate":"October 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-04-03T15:40:02+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-29 13:12:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7835319","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7835319","identity":"rs-7835319","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}