Development of Quantitative HPTLC-Densitometry Methods

Journal of Liquid Chromatography & Related Technologies

ISSN: 1082-6076 (Print) 1520-572X (Online) Journal homepage: http://www.tandfonline.com/loi/ljlc20

Development of quantitative HPTLC-densitometry methods following a model approach for transfer of TLC screening methods for pharmaceutical products of atenolol, chloramphenicol, furosemide, glibenclamide, penicillin V potassium, and praziquantel

Bingsong Zeng, Kaitlin Nguyen & Joseph Sherma

To cite this article: Bingsong Zeng, Kaitlin Nguyen & Joseph Sherma (2018) Development of quantitative HPTLC-densitometry methods following a model approach for transfer of TLC screening methods for pharmaceutical products of atenolol, chloramphenicol, furosemide, glibenclamide, penicillin V potassium, and praziquantel, Journal of Liquid Chromatography & Related Technologies, 41:6, 324-328, DOI: 10.1080/10826076.2018.1448689 To link to this article: https://doi.org/10.1080/10826076.2018.1448689

Published online: 24 May 2018.

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Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ljlc20 JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES 2018, VOL. 41, NO. 6, 324–328 https://doi.org/10.1080/10826076.2018.1448689

none defined Development of quantitative HPTLC-densitometry methods following a model approach for transfer of TLC screening methods for pharmaceutical products of atenolol, chloramphenicol, furosemide, glibenclamide, penicillin V potassium, and praziquantel Bingsong Zeng, Kaitlin Nguyen, and Joseph Sherma Department of Chemistry, Lafayette College, Easton, PA, USA

ABSTRACT KEYWORDS Transfer of six thin-layer chromatography (TLC) Global Pharma Health Fund E.V. Minilab manual protocols Amitriptyline HCL; for detecting fake drugs in pharmaceutical products to quantitative high-performance TLC (HPTLC)- amodiaquine; atenolol; densitometry methods was performed following a previously published model process. The developed chloramphenicol; and validated methods for tablets or capsules containing atenolol, chloramphenicol, furosemide, diphenhydramine HCL; furosemide; glibenclamide; glibenclamide, penicillin V potassium, and praziquantel involved use of a limited list of inexpensive, mebendazole; model relatively nontoxic, readily available solvents and other reagents; silica gel 60 F254 plates; automated process for tlc to bandwise sample and standard solution application; ascending mobile phase development of plates in a quantitative hptlc transfer; chamber; and automated slit scanning densitometry for detection, identification, and quantification. penicillin V potassium; Validation data for methods developed in an early version of the transfer model process that did not praziquantel; thin-layer include standard addition validation are reported for pharmaceutical products containing amitriptyline chromatography HCl, amodiaquine, diphenhydramine HCl, and mebendazole.

GRAPHICAL ABSTRACT

Introduction HCl, mebendazole, diphenhydramine HCl, and amodiaquine + artesunate;[2] diazepam and amodiaquine;[3] lumefantrine + Development of a model process for transfer of thin-layer artemether;[4] albendazole and amodiaquine + artesunate;[5] chromatography (TLC) screening methods for pharmaceutical pyrazinamide + ethambutol + isoniazid + rifampicin;[6] quin- products with quality defects to quantitative high-performance ine sulfate, mefloquine, and dihydroartemisinin + TLC (HPTLC)-densitometry methods was described in three piperaquine;[7] azithromycin, imipramine HCl, and papers[1–3] and applied to pharmaceutical products containing sulfadoxine + pyrimethamine;[8] cefixime, cefuroxime axetil, paracetamol (acetaminophen), acetylsalicylic acid (aspirin), cephalexin • H O, ciprofloxacin HCl, levofloxacin, and ibuprofen, and chlorpheniramine maleate;[1] amitriptyline 2

CONTACT Joseph Sherma, [email protected] Department of Chemistry, 326 Hugel Science Center, Lafayette College, 701 Sullivan Road, Easton, PA 18042, USA. Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/ljlc. © 2018 Taylor & Francis JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES 325

Table 1. Preparation of 100% standard and 100% sample solutions for the newly developed drug product analysis methods. Pharmaceutical product 100% standard solution 100% sample solutiona Atenolol (50 mg; Denk Pharma 10.0 µg/10.0 µL; dissolve 100 mg standard (Sigma-Aldrich, 10.0 µg/10.0 µL; dissolve a tablet in 50.0 mL methanol GmbH & Co. KG, Munich, Germany) St. Louis, MO, USA, No. A7655-1 G) in 100 mL methanol Chloramphenicol (250 mg; S Kant 1.67 µg/10.0 µL; dissolve 250 mg standard (Sigma-Aldrich, 1.67 µg/10.0 µL; dissolve a capsule in 50.0 mL methanol, Healthcare Ltd., Gujarat, India) No. C0378) in 50.0 mL methanol, then dilute 1.00 mL then dilute 1.00 mL with 9.00 mL methanol, then with 9.00 mL methanol, then dilute 1.00 mL with dilute 1.00 mL with 2.00 mL methanol 2.00 mL methanol Furosemide (40 mg; Sandoz, Inc., 0.800 µg/10.0 µL; dissolve 20.0 mg standard (Sigma- 0.800 µg/10.0 µL; dissolve a tablet in 100 mL acetone, Princeton, NJ, USA) Aldrich, No. PHR1057-1 G) in 50.0 mL acetone, then then dilute 1.00 mL with 4.00 mL acetone dilute 1.00 mL with 4.00 mL acetone Glibenclamide (5 mg; Remedica Ltd., 2.00 µg/10.0 µL; dissolve 100 mg standard (Sigma-Aldrich, 2.00 µg/10.0 µL; dissolve a tablet in 25.0 mL methanol- Limassol, Cyprus) No. G0639-5 G) in 50.0 mL methanol-ethyl acetate (1:1), ethyl acetate (1:1) Glibenclamide (2.5 mg; Tianjin Pacific then dilute 5.00 mL with 45.0 mL methanol-ethyl 2.00 µg/10.0 µL; dissolve a tablet in 12.5 mL methanol- Ocean Pharmacy Ltd., Tianjin, acetate (1:1) ethyl acetate (1:1) China) Penicillin V potassium (262 mgb; Jilin 12.5 µg/10.0 µL; dissolve 125 mg standard (Sigma-Aldrich, 13.1 µg/10.0 µL; dissolve a tablet in 100 mL deionized Wantong Pharmacy Group Co., No. 46616-250MG) in 100 mL deionized water water, then dilute 1.00 mL with 1.00 mL of deionized Jilin, China) water Praziquantel (600 mg; Medochemie 30.0 µg/10.0 µL; dissolve 150 mg standard (Sigma-Aldrich, 30.0 µg/10.0 µL; dissolve a tablet in 50.0 mL methanol, Ltd., Limassol, Cyprus) No. PHR1391-1 G) in 50.0 mL methanol then dilute 1.00 mL with 3.00 mL methanol aConcentrations indicated for 100% sample solutions are based on label values. bThe sample is labeled as a tablet of 236 mg penicillin V. A conversion factor of 1.1087 was applied to calculate the weight of penicillin V potassium, which was 262 mg. metronidazole;[9] clarithromycin, azithromycin, and Darmstadt, Germany; Part No. 1.05648.0001) were used aminodiaquine + artesunate;[10] and potassium clavulanate without prewashing. Application of 7.00, 9.00, 11.0, and and metformin.[11] In this paper, we report the use of the 13.0 μL aliquots of the 100% standard solution of each drug model process to transfer six TLC screening methods for (representing 70-130% of the active pharmaceutical ingredient pharmaceutical products in the Global Pharma Health Fund content based on the label value) and 10.0 μL of 100% sample (GPHF) E.V. Minilab manual,[12] including atenolol (CAS solution were applied to the plate origin using a spray-on No. 29122-68-7, beta blocker), chloramphenicol (CAS No. CAMAG (Wilmington, NC, USA) Linomat 4 applicator with 56-75-7, antibiotic), furosemide (CAS No. 54-31-9, diuretic), the following settings: band length 6 mm, application rate glibenclamide (CAS No. 10238-21-8, anti-diabetic), penicillin 15 s/μL for penicillin V potassium with its aqueous solvent V potassium (CAS No. 132-98-9, antibiotic), and praziquantel and 4 s/μL for other drugs with organic solvents, table speed (CAS No. 55268-74-1, anti-parasite). In an early version of the 10 mm/s, distance between bands 4 mm, distance from the left transfer model,[2] validation was not yet included. Therefore, edge of the plate 17 mm, and distance from the bottom of the we also report here data on validation for four of the five plate 1 cm. HPTLC-densitometry in the absorption-reflectance developed methods in that paper: amitriptyline HCl, meben- mode was performed using a CAMAG Scanner 3 densitometer dazole, diphenhydramine, and amodiaquine; the validation with 4.00 � 0.45 mm micro slit dimensions and 20 mm/s scan for artesunate was not performed because a modified method rate. Six different mobile phases were used for the eight for artesunate without the need of a detection reagent was pharmaceutical products, respectively, as shown in Table 2. subsequently developed and validated.[10] The fluorescence quenching zones were scanned under 254 nm ultraviolet (UV) light from the deuterium light source. The densitometer winCATS software automatically created Standard and sample preparation calibration curves (linear or second order polynomial) based General preparation procedures were carried out as described on scan areas versus standard weights applied, interpolated in detail in previous papers[1–3] unless otherwise specified. All weights of drugs in bracketed samples based on scan areas, standards and ground (by mortar and pestle) tablets or capsule and tested peak purity and identity of the sample by spectral contents were dissolved with the aid of 10 min each of comparison. Accuracy of the developed methods was validated magnetic stirring and sonication before syringe filtration to remove undissolved excipients prior to further dilution or direct application. Dilutions were made using appropriate Table 2. Mobile phases used for the development of plates for atenolol tablet, chloramphenicol capsule, furosemide tablet, glibenclamide tablet (5 mg), volumetric flasks and transfer and measuring pipets. Solutions glibenclamide tablet (2.5 mg), penicillin V potassium tablet, and praziquantel were stored in sealed vials wrapped in parafilm in a refriger- tablet. a ator. Detailed procedures for standard and sample preparation Pharmaceutical product Mobile phase Rf of each analyzed drug product are shown in Table 1. Atenolol Methanol-ammonia (40:0.4) 0.35 Chloramphenicol Ethyl acetate-toluene-methanol (30:10:6) 0.52 Furosemide Toluene-ethyl acetate-acetic acid (17:13:1) 0.30 Glibenclamide (5 mg) Ethyl acetate-methanol-toluene-ammonia 0.61 HPTLC Glibenclamide (2.5 mg) (22:14:2:2) 0.63 Penicillin V potassium Ethyl acetate-methanol-acetic acid 0.48 Detailed HPTLC-densitometry methods and instruments were (30:15:1.5) [1–3] described in earlier papers. Silica gel 60 F254 premium Praziquantel Acetone-toluene (28:14) 0.65 purity HPTLC glass plates (20 � 10 cm; Merck KGaA, aAll solutions are shown in volume proportions. 326 B. ZENG ET AL. by using standard addition analysis with a 70-130% calibration Discussion curve as described earlier.[3] For glibenclamide, only one of the According to the previously published process, direct transfer two sample formulations was used for validation. of TLC screening methods to HPTLC-densitometry methods involves use of the same solvent in sample and standard Results solution preparation, application of the same weight in 10.0 µL of 100% standard and sample solutions as in 2.00 μL Assay and validation results for transfer of the eight pharma- in the Minilab manual method, and use of the same mobile ceutical preparations are listed in Tables 3 and 4, respectively. phase and detection mode. Methods for glibenclamide The optimal regression mode for assays and validation of each (Minilab manual Volume II, Method 6.17, pp. 96-99), pharmaceutical product was chosen based on the best results praziquantel (Minilab manual Volume II, Method 6.30, obtained in terms of calibration curve r-values, assay values pp. 148-151), and atenolol (Volume II, Supplement 2015, closer to the label value, accuracy of validations, and smaller Method 6.80, pp. 4-7) were directly transferred to HPTLC- RSDs. The guidelines of the model process require that cali- densitometry methods. The Minilab method for atenolol states bration curves have r-values of at least 0.99, assays are within that this drug “performs weak” when observed under 254 nm 85-115% specification limits of the label values as specified by light, but we found detection of fluorescence quenching was the United States Pharmacopeia (USP) for individual tablets, sensitive enough to perform a direct transfer with the same validation recoveries are 95-105%, and RSDs are no higher than applied weights. 3%. These guideline values were met in all cases except for the The chloramphenicol Minilab method (Volume II, Method 150% validation spike level RSD of penicillin V potassium 6.9, pp. 64-67) could not be directly transferred because the (6.38%). This high precision value was due to one anomalous weight of drug applied in the 100% standard solution of the result out of three recovery analyses, which could be rejected Minilab manual method (5.00 µg/2.00 µL) resulted in scan by applying the statistical Q-test for three observations leaving areas that were too high to give a satisfactory calibration curve an average recovery value of 98.0% with a percent difference of for densitometry The applied weight was lowered to one only 0.204% for the two remaining results. third of the weight specified in the Minilab method (1.67 µg/ In the second published version of the model process,[2] 10.0 µL) to prepare the calibration curve. validation was not yet included. Data for validation of the The furosemide Minilab manual method (Volume II, developed amitriptyline HCl, mebendazole, diphenhydramine, Method 6.16, pp. 92-95) required changes in the applied and amodiaquine methods are given in Table 5. Validation for weight and the mobile phase for successful transfer. The these pharmaceutical products met the same aforementioned 4.00 µg/2.00 µL 100% standard solution was lowered to model process requirements.

Table 3. Assay results for the newly developed drug product analysis methods. Tablet 1 Tablet 2 Tablet 3 Pharmaceutical product Regression mode Assay (%) RSD (%) Assay (%) RSD (%) Assay (%) RSD (%) Atenolol Polynomial 106 2.16 113 1.08 105 1.64 Chloramphenicol Polynomial 93.7 1.35 110 0.950 102 2.21 Furosemide Linear 103 0.522 100 0.0464 108 0.898 Glibenclamide (5 mg) Polynomial 98.8 0.558 98.0 1.27 98.6 1.23 Glibenclamide (2.5 mg) Polynomial 96.5 0.581 99.8 2.91 99.4 0.423 Penicillin V potassium Polynomial 98.6 2.47 104 0.282 102 2.02 Praziquantel Polynomial 96.2 0.420 98.9 1.34 98.3 2.88

Table 4. Validation results for the newly developed drug product analysis methods. 50% spike 100% spike 150% spike Pharmaceutical product Regression mode Rec.a (%) RSD (%) Rec. (%) RSD (%) Rec. (%) RSD (%) Atenolol Polynomial 102 1.21 103 2.11 104 2.03 Chloramphenicol Polynomial 101 0.716 100 1.11 102 0.871 Furosemide Linear 95.7 1.34 100 1.84 102 0.304 Glibenclamide (5 mg) Polynomial 102 2.03 104 1.19 105 1.17 Penicillin V potassium Polynomial 102 0.565 101 1.11 102 6.38 Praziquantel Polynomial 104 1.03 98.2 1.12 102 0.470 aRec. ¼ recovery.

Table 5. Validation results for previously developed drug product analysis methods. 50% spike 100% spike 150% spike Pharmaceutical product Regression mode Rec. (%) RSD (%) Rec. (%) RSD (%) Rec. (%) RSD (%) Amitriptyline HCl Linear 104 1.14 105 1.52 103 2.17 Amodiaquine Polynomial 103 1.05 101 0.783 96.1 2.75 Diphenhydramine HCl Polynomial 101 0.467 98.9 0.626 96.0 0.971 Mebendazole Polynomial 105 0.326 104 0.322 100 0.374 JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES 327

Figure 1. Densitogram of 10.0 μL of penicillin V potassium 100% sample solution, representing 13.1 µg of penicillin V potassium when interpolated from the calibration curve based on its area.

0.800 µg/10.0 µL to achieve a good calibration curve, and the as accuracy, precision (repeatability and intermediate mobile phase was changed from methanol-acetone toluene precision), specificity, linearity, range, and robustness under (21:2:2), which gave irregularly shaped densitometer scans, relevant guidelines such as those described by the Inter- to toluene-ethyl acetate-glacial acetic acid (17:13:1). national Conference on Harmonization[15] or subjected to an The Minilab manual method for penicillin V potassium interlaboratory study[16] to prove that they are suitable for (Volume II, Method 6.29, pp. 144-147) specified use of ethyl their intended purpose by users. acetate-glacial acetic acid-water (15:5:5) mobile phase and iodine staining to detect a brown spot at Rf 0.85. Other chromo- genic detection reagents for penicillin V were suggested in the Acknowledgements literature such as ferric chloride-potassium ferricyanide or JS thanks Thomas Layloff, Senior Quality Assurance Advisor, Supply sulfuric acid-charring,[13] but no quantitative TLC-densitome- Chain Management System (SCMS), Arlington, VA, USA, for his help try method has been reported. The mobile phase was changed in designing the model transfer process and its application to the analysis to the one listed in Table 2 and the drug migrated in a tight, of pharmaceutical products, and for arranging delivery of drug product samples supplied by Dar es Saleem, Tanzania, Office of SCMS. We thank symmetrical band near the optimal middle area of the plate Dr. Gerd Battermann, Head of Instrumental Analytics Franchise, Merck and naturally quenched fluorescence under 254 nm UV light, KGaA, Darmstadt, Germany, for providing the HPTLC plates used in which gave good scans (Figure 1) that allowed development our experiments. We also thank Danhui Zhang for performing the and validation of the HPTLC-densitometry method described validation of the mebendazole method. Bingsong Zeng and Katlin above. No previous publication reports the natural fluorescence Nguyen were financially supported by a Camille and Henry Dreyfus Foundation Senior Scientist Mentor Program award to JS and by the quenching of penicillin V potassium zones on silica gel plates Lafayette College EXCEL Scholars Program. to eliminate the need of a detection reagent. In addition to the HPTLC-densitometry method based on fluorescence quenching detection mentioned above, a modified Minilab screening References method that does not require the use of iodine vapor detection [1] O’Sullivan, C.; Sherma, J. A Model Procedure for the Transfer of was developed for another penicillin V potassium formulation TLC Pharmaceutical Product Screening Methods Designed for use and published online open access on the website of Dr. Thomas in Developing Countries o Quantitative HPTLC-Densitometry Layloff.[14] In addition to the change in detection method, the Methods. Acta Chromatogr. 2012, 24, 241–252. DOI: 10.1556/ applied weight of penicillin V potassium was increased to appro- achrom.24.2012.2.7. [2] Lianza, K.; Sherma, J. Application of an Expanded Model Procedure ximately 240% of the weight specified in the Minilab manual. for Transfer of TLC Screening Methods for Substandard and Fake The validation data shown in Table 5 for methods Drugs Designed for use in Developing Countries to Quantitative transferred in an earlier version of the model process[2] were HPTLC-Densitometry Methods. J. Liq. Chromatogr. Relat. Technol. obtained using exactly the same methods except that Merck 2013, 36, 2446–2462. premium purity plates were substituted for regular Merck [3] Popovic, N.; Sherma, J. Transfer of TLC Screening Methods Designed for use in Developing Countries to Quantitative silica gel 60 F254 HPTLC plates. HPTLC-Densitometry Methods for Diazepam and Amodiaquine Depending on the applications of the methods described in Tablets. Acta Chromatogr. 2014, 26, 615–623. DOI: 10.1556/ this paper, they should be fully validated for parameters such achrom.26.2014.4.4. 328 B. ZENG ET AL.

[4] Nguyen, M.; Sherma, J. Use of a Model Procedure for Transfer of HCl, Levofloxacin, and Metronidazole. Acta Chromatogr. 2017, 29, Minilab Qualitative Screening TLC Methods for Lumefantrine and 484–486. In press. DOI: 10.1556/1326.2016.29409. Artemether in a Combined Tablet Formulation to Individual and [10] Armour, E.; Sherma, J. Transfer of Silica Gel TLC Screening Simultaneous Quantitative HPTLC-Densitometry Methods. Trends Methods for Clarithromycin, Azithromycin, and Amodiaquine + Chromatogr. 2013, 8, 131–135. Artesunate to HPTLC-Densitometry with Detection by Reagentless [5] Nguyen, M.; Sherma, J. Development of Quantitative HPTLC- Thermochemical Activation of Fluorescence Quenching. J. Liq. Densitometry Methods for Analysis of Fake and Substandard Chromatogr. Relat. Technol. 2017, 40, 282–286. DOI: 10.1080/ Pharmaceutical Products Following a Mode Approach for Transfer 10826076.2017.1298029. of TLC Screening Methods: Albendazole, Amodiaquine + [11] Nguyen, K.; Zhang, D.; Sherma, J. Development of Quantitative Artesunate, Aciclovir, and Amoxicillin. J. Liq. Chromatogr. HPTLC-Densitometry Methods Following a Model Approach for Relat. Technol. 2014, 37, 2956–2970. DOI: 10.1080/10739149.2014. Transfer of TLC Screening Methods for Pharmaceutical Products 906988. of Metformin HCl, Potassium Clavulanate, Caffeine, Fluoxetine [6] Strock, J.; Nguyen, M.; Sherma, J. Transfer of Minilab TLC HCl, and Gabapentin. Studia UBB Chemia 2017, 62, 9–18. DOI: Screening Methods to Quantitative HPTLC-Densitometry for 10.24193/subbchem.2017.2.01. Pyrazinamide, Ethambutol, Isoniazid, and Rifampicin in a [12] Global Pharma Health Fund (GPHF) E.V. https://www.gphf.org/ Combination Tablet. J. Liq. Chromatogr. Relat. Technol. 2015, 38, (accessed Aug 9, 2017). 1126–1130. DOI: 10.1080/10826076.2015.1028292. [13] McGilveray, I.; Strickland, R. Detection and Separation of Penicillins [7] Strock, J.; Nguyen, M.; Sherma, J. Use of a Model Process for by Thin-Layer Chromatography. J. Pharm. Sci. 1967, 56, 77–79. Transfer of Minilab TLC Screening Methods for Quinine Sulfate, DOI: 10.1002/jps.2600560116. Mefloquine, and Dihydroartemisinin + Piperaquine Phosphate [14] Zeng, B.; Armour, E.; Sherma, J. Supplement to a Compendium of Tablets to Quantitative HPTLC-Densitometry Methods. Acta Unofficial Methods for Rapid Screening of Pharmaceuticals by Thin Chromatogr. 2016, 28, 363–372. DOI: 10.1556/1326.2016.28.3.6. Layer Chromatography. 2017 http://www.layloff.net. [8] Zhang, D.; Strock, J.; Sherma, J. Transfer of TLC Screening Methods [15] Ferenczi-Fodor, K.; Vegh, Z.; Nagy-Turak, A.; Renger, B.; Zeller, M. for Azithromycin, Imipramine HCl, and Sulfadoxine + Validation and Quality Assurance of Planar Chromatography Pyrimethamine Tablets to Quantitative HPTLC-Densitometry Procedures in Pharmaceutical Analysis. J. AOAC Int. 2001, 84, Methods. J. Liq. Chromatogr. Relat. Technol. 2016, 39, 277–280. 1265–1276. DOI: 10.1080/10826076.2016.1163465. [16] Kaale, E.; Risha, P.; Reich, E.; Layloff, T. P. An Interlaboratory [9] Zhang, D.; Armour, E.; Sherma, J. Development of Quantitative Investigation on the Use of High Performance Thin Layer HPTLC-Densitometry Methods Following a Model Approach for Chromatography to Perform Assays of Lamivudine-Zidovudine, Transfer of LC Screening Methods for Pharmaceutical Products of Metronidazole, Nevirapine, and Quine Composite Samples. Cefixime, Cefuroxime Axetil, Cephalexin • H2O, Ciprofloxacin J. AOAC Int. 2010, 93, 1836–1843. 本文献由“学霸图书馆-文献云下载”收集自网络，仅供学习交流使用。

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