Development of Quantitative HPTLC–Densitometry Methods for The

Original Research Paper

Development of Quantitative HPTLC–Densitometry Methods for the Analysis of Amiodarone HCl, Carvedilol, Doxylamine Succinate, Magnesium Salicylate, Metoprolol Succinate, Nebivolol HCl, and Salicylamide Using a Model Process Developed Earlier for the Transfer of TLC Screening Methods

K. Nguyen and J. Sherma* Department of Chemistry, Lafayette College, Easton, PA, USA

Received: 27 August 2017; accepted: 01 September 2017

A model process, previously developed in a series of studies, allows for the transfer of thin-layer chromatography (TLC) methods for qualitative screening of counterfeit drug products published in the Global Pharma Health Fund (GPHF) Minilab manual and US Food and Drug Administration (FDA) Compendium of Unofficial Methods for Screening of Pharmaceuticals by TLC to quantitative high-performance TLC (HPTLC)–densitometry methods. This article describes HPTLC–densitometry methods developed and validated according to this model process for pharmaceutical products of amiodarone HCl, carvedilol, doxylamine succinate, magnesium salicylate, metoprolol succinate, nebivolol HCl, and salicylamide, for which qualitative screening methods have not been published in the Minilab manual or FDA Compendium. These methods use relatively inexpensive and nontoxic “green solvents” for sample and standard solution and mobile phase preparation, Merck Premium Purity silica gel 60 F254 plates, automated standard and sample solution bandwise application, and automated densitometry for the assessment of peak purity and identity and quantification. Corresponding to the quantitative HPTLC–densitometry methods, qualitative TLC screening methods for these drug products were developed and posted online with open access as supplements to the FDA Compendium. Keywords: Amiodarone HCl, carvedilol, doxylamine succinate, magnesium salicylate, metoprolol succinate, nebivolol HCl, salicylamide, thin-layer chromatography, densitometry, drug analysis

Introduction No. 18917-89-0) and salicylamide (CAS No. 65-45-2). Qualita- tive TLC screening methods corresponding to these new The model process developed earlier [1–3] has allowed for the HPTLC–densitometry methods were subsequently developed transfer of visual, qualitative TLC drug screening methods, and published online with open access in a supplement to the which are published in the Global Pharma Health Fund (GPHF) FDA Compendium on the Website of Dr. Thomas Layloff. Minilab manual [4] and US Food and Drug Administration The model process includes standard and sample solution (FDA) Compendium of Unofficial Methods for Screening of preparation, establishment of linear and second order polynomial Pharmaceuticals by TLC [5], to quantitative HPTLC–densitome- regression calibration curves by applying standards representing try methods that can be used in support of regulatory sanctions. 70–130% of the product's label value, assay of three individual Use of this model process has been reported for the development tablets or capsules in triplicate for comparison of the label value, and validation of HPTLC–densitometry methods for analyzing a peak purity and identity tests, and validation of the method by variety of drugs in pharmaceutical products [1–14]. In addition standard addition analysis of 50%, 100%, and 150% spike levels. to the methods that were transferred from the Minilab manual or Only the “green” solvents and reagents acetone, concentrated am- FDA Compendium method, the model process has been used to monium hydroxide, ethanol, ethyl acetate, glacial acetic acid, develop and validate quantitative methods for amitriptyline HCl hydrochloric acid, methanol, sulfuric acid, and toluene specified [2]; acyclovir [7]; naproxen sodium, loperamide HCl, and lorati- in the Minilab manual were considered for use in the develop- dine [12]; caffeine, fluoxetine HCl, and gabapentin [14]; and ment of these methods. This eliminates the use of many common desloratadine, etodolac, famotidine, omeprazole, oxaprozin, and TLC solvents and reagents for sample and standard solutions and phenazopyridine HCl [15], for which no TLC screening methods mobile phases, such as hexane, butanol, chloroform, dioxane, exist in these sources. This article details the development and methylene chloride, acetonitrile, formic acid, and triethylamine validation of HPTLC–densitometry methods for the following additional pharmaceutical products for which no Minilab manual or FDA Compendium methods have been published: the anti- Experimental arrhythmic agent amiodarone HCl (CAS No. 19774-82-4); the β blockers carvedilol (CAS No. 72956-09-3), metoprolol succi- Standard and Sample Preparation. Preparation of standard nate (CAS No. 98418-47-4), and nebivolol HCl (CAS No. and sample solutions followed the procedures described 152520-56-4); the antihistamine doxylamine succinate (CAS previously [1–3], unless otherwise noted. Standards and No. 562-10-7); and the analgesics magnesium salicylate (CAS tablets ground by mortar and pestle were dissolved in their respective solvents by 10 min of magnetic stirring followed by 10 min of sonication. Metoprolol succinate sample * Author for correspondence: [email protected] solutions were sonicated for 15 min to obtain sufficient

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Unauthenticated | Downloaded 09/26/21 02:06 AM UTC K. Nguyen and J. Sherma dissolution. All sample solutions, before further dilution or 4 mm; distance from the left edge of the plate, 17 mm; and direct application onto the plates, were syringe-filtered to distance from the bottom of the plate, 1 cm. Development was remove excipients. Nebivolol HCl sample solutions, at a carried out for a distance of 7 cm beyond the bottom of the plate concentration of 10.9 mg/mL, were allowed to stand for in a mobile phase vapor saturated CAMAG twin-trough chamber; 15 min prior to filtration to allow excipients to settle to the the mobile phases and their respective Rf values are listed in bottom of the vial. This allowed for less difficult filtration and Table 2. A CAMAG Scanner 3 controlled by winCATS software more plentiful filtrate of a relatively concentrated solution. If was used for automated HPTLC–densitometry with 4.00 × necessary to the preparation of a working solution, volumetric 0.45 mm microslit dimensions and a 20 mm/s scan rate. All drugs flasks, measuring pipets, and volumetric pipets of appropriate for which the analytical methods are detailed in this article volume designation were used. Solutions were refrigerated in quenched the fluorescence of the phosphor in the layer and were Parafilm-sealed glass vials. Table 1 describes the source of the scanned with 254 nm ultraviolet (UV) radiation. The winCATS pharmaceutical products as well as the methods employed to scanner software generated two calibration curves (linear and prepare the 100% standard and sample solutions for each drug second-order polynomial regressions) for each sample, by analysis. determining the relationship between the scan areas and the HPTLC. Premium Purity silica gel 60 F254 plates (20 × 10 cm; weights of standards applied. Sample weights were interpolated Merck KGaA, Darmstadt, Germany; Catalog No. 1.05648.0001) from calibration curves based on the bracketed scan areas of were used without prewashing. Calibration curves were generated samples. Spectral comparison was used to test peak purity and by applying 7.00, 9.00, 11.0, and 13.0 μL of the 100% sample identity. Validation of the developed methods was performed using solution, representing 70–130% of the label value of the active standard addition with spiking at 50%, 100%, and 150% levels, as pharmaceutical ingredient. Assays were carried out by applying described by Popovic and Sherma [3]. 10.0 μL of each sample solution in triplicate. A CAMAG (Wilmington, NC, USA) Linomat 4 equipped with a 100 μL Results syringe was used for semi-automated, bandwise, zone application. An application rate of 4 s/μL was used for all solutions. The band Calibration curve r values for the assays and validations were length was 6 mm; table speed, 10 mm/s; distance between bands, all greater than 0.99, all pharmaceutical products were assayed

Table 1. Preparation of 100% standard and 100% sample solutions Pharmaceutical product 100% Standard solution 100% Sample solutiona Amiodarone HCl 0.500 μg 10.0 μL−1: Dissolve 25.0 mg standard 0.500 μg 10.0 μL−1: Dissolve a tablet in (200 mg; Zydus Pharmaceuticals, (Sigma-Aldrich, St. Louis, MO, USA, No. A8423) 100 mL of ethanol, then dilute 1.00 mL with Pennington, NJ, USA) in 50.0 mL of ethanol and then dilute 1.00 mL 3.00 mL ethanol for a total of 4.00 mL, and dilute with 9.00 mL of ethanol, for a total volume of 10.0 mL. 1.00 mL further with an additional 9.00 mL of ethanol for a total volume of 10.0 mL. Carvedilol (25.0 mg; 10.0 μg 10.0 μL−1: Dissolve 25.0 mg standard 10.0 μg 10.0 μL−1: Dissolve a tablet in 50.0 mL Aurobindo Pharma (Sigma-Aldrich, No. PHR1265) in 50.0 mL of methanol of methanol and then dilute 1.00 mL with Limited, Hyderabad, India) and then dilute 1.00 mL with 4.00 mL of methanol 4.00 mL of methanol for a total volume of 5.00 mL. for a total volume of 5.00 mL. Doxylamine Succinate 2.50 μg 10.0 μL−1: Dissolve 25.0 mg standard 2.50 μg 10.0 μL−1: Dissolve a tablet in (25.0 mg; Perrigo, (Sigma-Aldrich, No. PHR1420) in 100 mL of ethanol. 100 mL of ethanol. Allegan, MI, USA) Magnesium Salicylate 4.67 μg 10.0 μL−1: Dissolve 43.2 mg salicylic acid standard 4.67 μg 10.0 μL−1: Dissolve a tablet in 100 mL (467.2 mgb; Ciba (Sigma-Aldrich, No. S-6271) in 100 mL of ethanol. of ethanol and then dilute 1.00 mL with 9.00 mL Self-Medication, Inc., 4.32 μg salicylic acid 10.0 μL−1 is equivalent to 4.67 μg of ethanol for a total volume of 10.0 mL. Woodbridge, NJ, USA) magnesium salicylate 10.0 μL−1. 9.50 μg 10.0 μL−1: Dissolve 50.0 mg metoprolol tartrate 9.50 μg 10.0 μL−1: Dissolve a tablet in Metoprolol Succinate (47.5 mg; standard (Sigma-Aldrich, No. PHR1076) in 50.0 mL of 50.0 mL of methanol. Mylan Pharmaceuticals Inc., − methanol. 10.0 μg metoprolol tartrate 10.0 μL 1 is Morgantown, WV, USA) − equivalent to 9.50 μg metoprolol succinate 10.0 μL 1. Nebivolol HCl (5.45 mg; 10.9 μg 10.0 μL−1: Dissolve 10.9 mg of standard 10.9 μg 10.0 μL−1: Dissolve a tablet in Forest Pharmaceuticals, Inc. (Sigma-Aldrich, No. N1915) in 10.0 mL of methanol. 5.00 mL of methanol. Subsidiary of Forest Laboratories, LLC, Cinncinnati, OH, USA) 4.56 μg 10.0 μL−1: Dissolve 76.0 mg of standard 4.56 μg 10.0 μL: Dissolve a tablet in 100 mL Salicylamide (152 mgc; (Sigma-Aldrich, No. S-0750) in 50.0 mL of ethanol of ethanol and then dilute 3.00 mL with First Aid Only Inc., and then dilute 3.00 mL with 7.00 mL of ethanol 7.00 mL of ethanol for a total volume of 10.0 mL. Vancouver, WA 98682) for a total volume of 10.0 mL. aConcentrations indicated for all 100% sample solutions are theoretical concentrations based on label values. b580 mg of magnesium salicylate tetrahydrate equivalent to 467.2 mg anhydrous magnesium salicylate. cCoformulated with 162 mg aspirin, 110 mg acetaminophen, and 32.4 mg caffeine.

Table 2. Mobile phases used for the analysis of pharmaceutical products containing amiodarone HCl, carvedilol, doxylamine succinate, magnesium salicylate, metoprolol succinate, nebivolol HCl, and salicylamide a Pharmaceutical product Mobile phase Rf Amiodarone HCl Acetone–toluene–glacial acetic acid (10:10:1) 0.24 Carvedilol Ethyl acetate–toluene–methanol–concentrated ammonium hydroxide (50:30:15:5) 0.62 Doxylamine Succinate Ethyl acetate–methanol–concentrated ammonium hydroxide (24:3:1) 0.54 Magnesium Salicylate Ethyl acetate–glacial acetic acid (95:5) 0.58 Metoprolol Succinate Ethyl acetate–toluene–methanol–concentrated ammonium hydroxide (50:30:15:5) 0.50 Nebivolol HCl Ethyl acetate–methanol–concentrated ammonium hydroxide (8.5:1:0.5) 0.42 Salicylamide Ethyl acetate–acetone–glacial acetic acid (18:4:0.1) 0.60 aAll solutions are shown in volume proportions.

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Unauthenticated | Downloaded 09/26/21 02:06 AM UTC Quantitative HPTLC–Densitometry Methods within 85–115% of the label value as is required by the United In the development of the method for analyzing carvedilol, the States Pharmacopeia (USP) for single tablet or capsule analysis use of methanol as the solvent for sample and standard solution (Table 3), all standard addition recoveries in the method valida- preparation was transferred from the methods of Abdel-Gawad tions were within ± 5% (Table 4), peak purity and identity r et al. [17] and Patel et al. [18] for assay of pharmaceutical formu- values were greater than 0.99, and all relative standard deviation lations of carvedilol. However, the mobile phases used in these (RSD) values were no higher than 3% in accordance with the re- methods, acetone–toluene–ethanol–ammonia (45:45:10:1) [17] quirements of the model process. The preferred regression mode and ethyl acetate–toluene–methanol (1:4:3.5) [18], both gave was determined based on whether polynomial or linear regression broad, streaked zones that could not be successfully scanned. gave better r values for the calibration curve, assay and standard The mobile phase described by Ramadan et al. [19] for determi- addition recovery values closer to 100%, and lower RSD values nation of metoprolol phosphate and hydrochlorthiazide mixed for the triplicate analyses. pharmaceutical product was successfully used to develop our method for carvedilol (Table 2). Ethanol as the solvent for the 100% standard and sample solu- Discussion tions for doxylamine succinate product analysis was suggested The model process for directly transferring Minilab manual or by DiGregorio and Sherma [20]. Their mobile phase, ethyl Compendium TLC methods to HPTLC–densitometry methods acetate–methanol–concentrated ammonium hydroxide (85:10:5), involves use of the same solvents for sample and standard solu- gave an Rf value of 0.66 and acceptable calibration curve r tion preparation, weight of sample and standard applied (in value, but zones were found to be unequally spaced after devel- 10.0 μL for the densitometry methods, instead of 2.00 μLor opment on the Premium Purity plates and could not be scanned 3.00 μL as in the Minilab manual or Compendium, respectively), accurately. The mobile phase from our previously published mobile phase, and detection method. As no Minilab manual or HPTLC–densitometry method for loperamide HCl [12] (Table 2) Compendium method had been published for the pharmaceutical was found to give excellent results for the doxylamine succinate products included in this article, our extensive previous experi- determination according to the model process. ence in drug product analysis and exhaustive literature searches Based on DiGregorio and Sherma's study [21], the magnesium through SciFinderW (Chemical Abstracts), ISI Web of Science, salicylate quantification method used ethanol as the solvent for and Google Scholar were employed to guide our method devel- sample and standard solution preparation, ethyl acetate–glacial opment and validation research. acetic acid (95:5) as the mobile phase, and 100% sample and For the amiodarone quantification method, the mobile phase standard solutions with a concentration of 4.67 μg10.0μL−1. ethyl acetate–methanol–concentrated ammonium hydroxide An additional pharmaceutical product, DiurexW Water Pills, con- (85:10:15) used for this drug in a study of the identification of tains magnesium salicylate (162.5 mg) and caffeine (50 mg). It drugs using chemometrics [16] was tested first, but this ternary was verified that this formulation could be successfully analyzed solution was cloudy. The solvent proportions were adjusted to by the developed method because the two active ingredients are (34:6:6) to achieve the necessary clear solution, but this mobile well separated by the mobile phase used (Table 2) with Rf values phase produced an Rf value that was too high (0.87) to give sym- of 0.58 and 0.18, respectively. metrical peak scans on a flat baseline and a calibration curve with For the metoprolol succinate quantification method, the mobile an adequately high r value. Additional mobile phases from our phase ethyl acetate–toluene–methanol–concentrated ammonium previously published drug product analysis articles were tested, hydroxide (50:30:15:5) and methanol as the solvent for standard and acetone–toluene–glacial acetic acid (10:10:1) [13] gave ideal and sample solution preparation were used as reported earlier results satisfying all model process guidelines. The published arti- [19]. A method for a 23.75-mg formulation of metoprolol succi- cle [16] did not specify the amount of drug applied to the plates nate (equivalent to 25.0 mg of metoprolol tartrate, Dr. Reddy's for TLC, and a trial and error process was necessary to determine Laboratories Limited, Bachupally, 500090, India) was also devel- that 100% standard and sample solutions with concentrations of oped and validated successfully according to the model process 0.500 μg 10.0 μL−1 met the model process guidelines. (data not given). The tablet is dissolved in 25.0 mL of methanol

Table 3. Assay results for pharmaceutical products containing amiodarone HCl, carvedilol, doxylamine succinate, magnesium salicylate, metoprolol succinate, nebivolol HCl, and salicylamide Pharmaceutical product Regression mode Tablet 1 Tablet 2 Tablet 3 Assay (%) RSD (%) Assay (%) RSD (%) Assay (%) RSD (%) Amiodarone HCl Polynomial 106 1.12 107 0.568 108 0.913 Carvedilol Polynomial 109 0.318 110 0.456 108 0.694 Doxylamine succinate Polynomial 94.4 0.488 103 1.35 101 1.23 Magnesium salicylate Linear 97.9 1.14 102 1.01 101 1.05 Metoprolol succinate Linear 101 1.02 100 1.05 103 0.872 Nebivolol HCl Linear 92.2 2.34 91.9 1.38 94.4 0.786 Salicylamide Linear 100 0.678 102 1.27 106 1.07

Table 4. Validation results for pharmaceutical products containing desloratadine, etodolac, famotidine, omeprazole, oxaprozin, and phenazopyridine HCl Pharmaceutical product Regression mode 50% Spike 100% Spike 150% Spike Rec.a (%) RSD (%) Rec. (%) RSD (%) Rec. (%) RSD (%) Amiodarone HCl Polynomial 103 0.525 101 0.642 97.8 2.21 Carvedilol Polynomial 103 0.595 103 1.15 101 0.316 Doxylamine succinate Polynomial 102 0.445 100 1.09 96.9 0.965 Magnesium salicylate Linear 95.2 1.78 103 1.16 101 1.88 Metoprolol succinate Linear 100 1.00 105 0.201 102 0.581 Nebivolol HCl Linear 100 0.592 100 0.856 96.9 1.61 Salicylamide Linear 102 2.33 99.0 0.618 96.5 1.96 aRec. = recovery.

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Unauthenticated | Downloaded 09/26/21 02:06 AM UTC K. Nguyen and J. Sherma to give a 100% sample solution concentration of 9.50 μg 10.0 μL−1, with all other method parameters identical to those of the metoprolol succinate 47.5 mg tablet quantification method described above. Both metoprolol succinate products gave two well-resolved zones on the plate after development (Figure 1): a faint satellite zone (Rf = 0.27) and the primary zone (Rf = 0.50) used for quantification. In the development of the nebivolol HCl 5.45 mg tablet method, the mobile phase ethyl acetate–methanol–concentrated ammonium hydroxide (8.5:1:0.5) and methanol as the solvent for the sample and standard solutions were used as recom- mended by Bhat et al. [22]. Nebivolol HCl is also available as a 10.9-mg tablet that could be analyzed using the method described above except for dissolving the tablet in 10.0 mL of methanol to give a 100% sample solution concentration of 10.9 μg 10.0 μL−1. The use of ethanol as the solvent for salicylamide 100% sam- μ ple and standard solution preparation was based on the method Figure 2. Densitogram of 10.0 L of the 100% sample solution of salicylamide, showing the salicylamide peak (Rf = 0.60), acetaminophen/ of Sullivan and Sherma [23]; their employment of a calibration aspirin peak (R = 0.44), and caffeine peak (R =0.21) curve centered at 5.00 μg influenced the decision to prepare f f these solutions at 4.56 μg10.0μL−1, which was a convenient concentration given the 152 mg tablet label value. The mobile Conclusion phase of the earlier method [23], dichloromethane–acetone (4:1), HPTLC–densitometry methods for the determination of could not be employed because dichloromethane is not on the amiodarone, carvedilol, doxylamine succinate, magnesium sa- list of solvents approved for the model process. The mobile licylate, metoprolol succinate, nebivolol, and salicylamide in phase used for the developed method (Table 2), which gave pharmaceutical preparations were developed and validated good separation of salicylamide from the three coformulants in according to the previously described model procedure for the analyzed product (32.4 mg caffeine, 162 mg aspirin, and transfer of qualitative TLC screening methods. The methods 110 mg acetaminophen; see Figure 2), was taken from a previ- should be fully validated according to the International Con- ously published method for the analysis of artesunate pharma- ference on Harmonization (ICH) guidelines [24] or by interla- ceutical products [7]. boratory studies [25] if future applications require. Since Qualitative TLC screening methods based on the format of the neither the Minilab manual nor the FDA Compendium contain FDA Compendium [5] were developed corresponding to the new qualitative TLC screening methods for these drugs that are HPTLC–densitometry methods. The qualitative methods gener- able to used in the field, these were subsequently developed ally employed the same solvents used in sample and standard so- and posted online with open access as supplements to the lution preparation, weights of analytes spotted on the plate (in FDA Compendium on Dr. Thomas Layloff's website [26]. 3.00 μL for the qualitative method, instead of the 10.0 μLused in the quantitative method), mobile phases, and methods of detection. If necessary, parameters of the qualitative methods (mo- Acknowledgments. J.S. thanks Dr. Thomas Layloff, bile phase or weights spotted) were adjusted to improve visual Senior Quality Assurance Advisor, Supply Chain Management differences among 85%, 100%, 115% of the drug product, rela- Systems (SCMS), Arlington, VA, USA, for his help in tive Rf of coformulants, if present, and spot shape. These supple- devising the model transfer process and its application to mental FDA Compendium methods, which are available online, pharmaceutical products. The authors also thank Dr. Gerd open access [26], can be easily converted to Minilab manual Battermann, Head of Instrumental Analytics Franchise, Merck methods by spotting the same drug weights in the 2.00 μL appli- KGaA, Darmstadt, Germany, for providing the Premium cation volume specified in the Minilab manual and substitution Purity HPTLC plates used in this research. Kaitlin Nguyen of the Sigma-Aldrich standards we used with authentic drug was financially supported by a Camille and Henry Dreyfus products available to the GPHF. Foundation Senior Scientist Mentor Program award to J.S. and by the Lafayette College EXCEL Scholars Program.

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