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  • 1 Lafayette College, Easton, PA, USA
  • 2 Lafayette College, Easton, PA, USA
  • 3 Lafayette College, Easton, PA, USA
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Transfer of six thin-layer chromatography (TLC) Global Pharma Health Fund Minilab kit protocols for detecting fake or markedly substandard drugs in pharmaceutical products in the field to quantitative high-performance TLC (HPTLC)–densitometry methods was carried out using a model process published earlier. The developed and validated methods for tablets or capsules containing cefixime, cefuroxime axetil, cephalexin·H2O, ciprofloxacin HCl, levofloxacin, and metronidazole involved use of EMD Millipore Premium Purity silica gel 60 F254 plates, automated sample and standard solution application with a CAMAG Linomat 4, and automated densitometry with a CAMAG Scanner 3 for detection, identification, and quantification.

Abstract

Transfer of six thin-layer chromatography (TLC) Global Pharma Health Fund Minilab kit protocols for detecting fake or markedly substandard drugs in pharmaceutical products in the field to quantitative high-performance TLC (HPTLC)–densitometry methods was carried out using a model process published earlier. The developed and validated methods for tablets or capsules containing cefixime, cefuroxime axetil, cephalexin·H2O, ciprofloxacin HCl, levofloxacin, and metronidazole involved use of EMD Millipore Premium Purity silica gel 60 F254 plates, automated sample and standard solution application with a CAMAG Linomat 4, and automated densitometry with a CAMAG Scanner 3 for detection, identification, and quantification.

Introduction

A model process was previously described [18] for transfer of qualitative–semiquantitative thin-layer chromatography (TLC) screening methods for pharmaceutical products with quality defects contained in the Global Pharma Health Fund EV (GPHF) Minilab manual [9] or US Food and Drug Administration Compendium of Unofficial Methods for Rapid Screening of Pharmaceuticals by Thin Layer Chromatography [10] to quantitative high-performance TLC (HPTLC)–densitometry methods. The model process was applied earlier to formulations containing acetylsalicylic acid, acetaminophen, ibuprofen, and chlorpheniramine maleate [1]; mebendazole, diphenhydramine HCl, amodiaquine, and artesunate [2]; amodiaquine and diazepam [3]; lumefantrine + artemether [4]; albendazole, amodiaquine + artesunate [5]; pyrazinamide + ethambutol + isoniazid + rifampicin [6]; quinine sulfate, mefloquine, and dihydroartemisinin + piperaquine phosphate [7]; and azithromycin, imipramine HCl, and sulfadoxine + pyrimethamine [8]. In this article, we report the use of the model process to transfer methods for pharmaceutical products included in the Minilab manual containing cefixime (CAS No. 79350-37-1), cefuroxime axetil (CAS No. 64544-07-6), cephalexin·H2O (CAS No. 23325-78-2), ciprofloxacin HCl (CAS No. 86393-32-0), levofloxacin (CAS No. 100986-85-4), and metronidazole (CAS No. 443-48-1). All of these compounds are antimicrobial pharmaceuticals. In addition, ciprofloxacin HCl and levofloxacin are antituberculosis pharmaceuticals.

Standard and Sample Preparation

General preparation procedures were carried out as described in detail [13] unless otherwise specified. All standards and ground (by mortar and pestle) tablets or capsule contents were dissolved with the aid of 10 min each of magnetic stirring and sonication before syringe filtration to remove undissolved excipients prior to further dilution or direct application. Dilutions were made using appropriate volumetric flasks and transfer and measuring pipets. Solutions were stored in sealed vials wrapped in parafilm in a refrigerator. Detailed procedures for standard and sample preparation are shown in Table 1.

Table 1.

Preparation of 100% standard and 100% sample solutions

Pharmaceutical product100% Standard solution100% Sample solution
Cefixime (100 mg; Henan Zhongjie Pharmaceutical Co., Ltd. Henan, China)2.00 μg 10.0 μL−1: dissolve 50.0 mg standard (Sigma-Aldrich, St. Louis, MO, Catalog No. 1097658) in 50.0 mL methanol, then dilute 1.00 mL with 4.00 mL methanol2.00 μg 10.0 μL−1,a: dissolve a tablet in 50.0 mL methanol, then dilute 1.00 mL with 9.00 mL methanol
Cefixime (200 mg; Torrent Pharmaceuticals, Ltd, Ahmedabad, India)2.00 μg 10.0 μL−1: dissolve a tablet in 100 mL methanol, then dilute 1.00 mL with 9.00 mL methanol
Cefuroxime axetil (125 mg; Shenzhen Zhijun Pharmaceutical Co., Ltd., Shenzhen, China)2.50 μg 10.0 μL−1: dissolve 25.0 mg standard (Sigma-Aldrich, No. 1098220) in 100 mL methanol2.50 μg 10.0 μL−1: dissolve a capsule in 50.0 mL methanol, then dilute 1.00 mL with 9.00 mL methanol
Cephalexin·H2O (125 mg; Huabei Pharmaceutical Group Co., Ltd., Hebei, China)2.50 μg 10.0 μL−1: dissolve 25.3 mg standard (Sigma-Aldrich, No. C4895) in 100 mL methanolb2.50 μg 10.0 μL−1: dissolve a capsule in 50.0 mL methanol, then dissolve 1.00 mL with 9.00 mL methanol
Ciprofloxacin HCl (250 mg; Henan Zhongjie Pharmaceutical Co., Ltd., Henan, China)1.25 μg 10.0 μL−1: dissolve 100 mg standard (Sigma-Aldrich, No. 1134335) in 50.0 mL methanol, then dilute 1.00 mL with 15.0 mL methanol1.25 μg 10.0 μL−1: dissolve a capsule with 100 mL methanol, then dilute 1.00 mL with 19.0 mL methanol
Levofloxacin [100 mg; Yangtze River Pharmaceutical (Group) Co., Ltd, Jiangsu, China]1.25 μg 10.0 μL−1: dissolve 100 mg standard (Sigma-Aldrich, No. 28266) in 50.0 mL glacial acetic acid-water (1:49, v/v), then dilute 1.00 mL with 15.0 mL methanol1.25 μg 10.0 μL−1: dissolve a capsule in 50.0 mL glacial acidic-water (1:49, v/v), then dilute 1.00 mL with 15.0 mL methanol
Metronidazole (200 mg; Jiangsu Pingguang Pharmaceutical Co., Ltd., Jiangsu, China)10.3 μg 10.0 μL−1: dissolve 25.7 mg standard (Sigma-Aldrich, No. M3761) in 25.0 mL methanol10.0 μg 10.0 μL−1: dissolve a tablet in 25.0 mL methanol, then dilute 1.00 mL with 7.00 mL methanol

Concentrations indicated for 100% sample solutions are theoretical concentrations.

The concentration indicated here, 2.50 μg 10.0 μL−1, is with respect to cephalexin·H2O. However, the standard acquired was cephalexin·xH2O, where x was calculated to be 1.22 since it was given that the standard contains 5.94% water. Adjusted by the molecular weight ratio of cephalexin·xH2O and cephalexin·H2O, which is 369/365 = 1.01, the concentration of the standard solution was adjusted to 2.50 μg/10.0 μL * 1.01 = 2.53 μg/10.0 μL, and thus, 25.3 mg of standard was used.

HPTLC

Detailed HPTLC–densitometry methods and instruments were described earlier [15]. Silica gel 60 F254 Premium Purity HPTLC glass plates (20 × 10 cm; EMD Millipore Corp., Billerica, MA, a division of Merck KGaA, Darmstadt, Germany; Part No. 1.05648.0001) were used as received. Application of 7.00, 9.00, 11.0, and 13.0 μL aliquots of the 100% standard solution of each drug (representing 70–130% of the active pharmaceutical ingredient [API] content based on label value) and triplicate 10.0 μL aliquots of 100% sample solution were applied using a CAMAG (Wilmington, NC, USA) Linomat 4 spray on applicator (band length, 6 mm; application rate, 4 s μL−1; table speed, 10 mm s−1; distance between bands, 4 mm; distance from the left edge of the plate, 17 mm; and distance from the bottom of the plate, 1 cm). HPTLC–densitometry in the absorption–reflectance mode was performed using a CAMAG Scanner 3 (4.00 × 0.45 mm micro-slit dimensions, 20 mm s−1 scan rate). Six different mobile phases were used for the six pharmaceutical products respectively, as shown in Table 2. The fluorescent levofloxacin zones were scanned with 366 nm ultraviolet (UV) light from the high-pressure mercury light source, while the fluorescence-quenching zones of all other drugs were scanned with 254 nm UV light from the deuterium light source. The Scanner 3 winCATS software automatically created calibration curves (linear or second order polynomial) based on scan areas versus standard weights applied, interpolated weights of drugs in bracketed samples based on scan areas, and tested peak purity and identity of the sample based on spectral comparison [3]. Accuracy of the developed methods was validated by using standard addition with a 70–130% calibration curve as described earlier [3].

Table 2.

Mobile phases selected in our methods for pharmaceutical products containing cefixime, cefuroxime axetil, cephalexin·H2O, ciprofloxacin HCl, levofloxacin, and metronidazole, respectively

Pharmaceutical productMobile phasea
CefiximeMethanol–water–acetic acid (70:20:1)
Cefuroxime axetilAcetone–toluene–glacial acetic acid (10:10:1)
Cephalexin·H2OEthyl acetate–acetone–glacial acetic acid–water (12.5:5:5:2.5)
Ciprofloxacin HClMethanol–acetone–toluene–concentrated ammonia (10:5:2.5:5)
LevofloxacinMethanol–concentrated ammonia–water (7:2:1)
MetronidazoleEthyl acetate–methanol–concentrated ammonia (15:5:0.5)

All solutions are shown in volume proportions.

Results

Results of the transfer for the six pharmaceutical preparations are displayed in Table 3. The optimal regression mode for assays and validation of each pharmaceutical product was chosen based on the best results in terms of calibration curve r-values, assay values closer to the label value, accuracy of the standard addition validation analyses, and lower relative standard deviation (RSD) values. R-values of all calibration curves in our assay and validation experiments were at least 0.99, all validation recoveries were within ±5%, RSDs for triplicate assays and validation analyses at 50, 100, and 150% spike levels were within 3%, and peak purity and identity r-values were 0.99 consistent with the model requirements. All assays were within 85–115% specification limits of the label value as specified by the United States Pharmacopeia (USP) for individual tablets except for the second tablet of cefuroxime axetil, which was quantified to be 124% of the label declaration. However, good validation data for this drug confirm the accuracy of this high value.

Table 3.

Assay and validation results for pharmaceutical products containing cefixime, cefuroxime axetil, cephalexin H2O, ciprofloxacin HCl, levofloxacin, and metronidazole, respectively

Pharmaceutical productRegression modeValidation analyses
Tablet 1
Tablet 2
Tablet 3
50% Spike
100% Spike
150% Spike
Assay (%)RSD (%)Assay (%)RSD (%)Assay (%)RSD (%)Rec.a (%)RSD (%)Rec. (%)RSD (%)Rec. (%)RSD (%)
Cefixime (100 mg)Polynomial95.72.2894.60.71097.30.5491031.041000.6111010.594
Cefixime (200 mg)Polynomial96.00.29694.31.2388.92.541040.8691030.6111040.594
Cefuroxime axetilLinear1071.161241.361041.121050.7841040.1151010.975
Cephalexin·H2OLinear96.41.471070.4551100.1671050.3951050.9951012.31
Ciprofloxacin HClLinear1111.151091.421061.151001.0796.81.311040.170
LevofloxacinLinear1040.7871122.031042.3799.21.251032.0797.40.759
MetronidazolePolynomial96.51.881080.6471080.6001051.951022.071020.929

Rec. indicates recovery.

Discussion

Direct transfer to a HPTLC–densitometry method according to the earlier published process involves use of the same solvents in preparing the sample and standard solutions, application of the same weights in 10.0 μL as in 2.00 μL in the Minilab method, and use of the same mobile phase and detection mode. Minilab methods for cefuroxime axetil (Volume II, Supplement 2010, Method 6.45, pp. 16–19), cephalexin (Volume II, Method 6.8, pp. 60–63), ciprofloxacin (Volume II, Method 6.11, pp. 72–75 and Volume II, Supplement 2013, Method 6.67, pp. 12–15), and metronidazole (Volume II, Method 6.25, pp. 128–131) were directly transferred.

The cefixime HPTLC quantitative method was not able to be directly transferred from the available Minilab TLC screening method (Volume II, Supplement 2010, Method 6.44, pp. 12–15). The modified mobile phase methanol–water–acetic acid (70:20:1) was used to give more compact bands instead of ethyl acetate–acetone–glacial acetic acid–water (12.5:5:5:2.5) mobile phase specified in the Minilab manual. A modified Minilab screening method showing better drug spots on a silica gel layer with this new mobile phase was published on the website of Dr. Thomas Layloff [11].

In the transfer of the Minilab TLC screening method for levofloxacin (Volume II, Supplement 2010, Method 6.47, pp. 24–27), the only change was that half of the concentration was used for the standard and sample solutions as compared to those of a direct transfer to the HPTLC–densitometry method in order to obtain less intense bands and higher r-value for linear regression calibration curve. Levofloxacin was found to both fluoresce and quench fluorescence, as specified in the Minilab manual. As shown in Figure 1, scanning at 366 nm excitation/>400 nm emission filter with the CAMAG Scanner 3 provided more symmetrical peaks for levofloxacin that are approximately two times as sensitive as the peaks obtained when levofloxacin bands were scanned using the 254 nm UV lamp. Therefore, 366 nm UV was chosen to scan levofloxacin in our method specified in order to optimize the analytical results.

Figure 1.
Figure 1.

Densitogram of 10.0 μL of levofloxacin 100% sample solution, representing 1.29 μg of levofloxacin when interpolated from the calibration curve based on its area, developed on Premium Purity silica gel plate with methanol–concentrated ammonia–water (7:2:1, v/v/v) mobile phase

Citation: Acta Chromatographica Acta Chromatographica 29, 4; 10.1556/1326.2016.29409

Depending on the applications of the methods described in this paper, they should be fully validated for parameters such as accuracy, precision (repeatability and intermediate precision), specificity, linearity, range, and robustness under relevant guidelines such as those described by the International Conference on Harmonization [12] or subjected to an interlaboratory study [13] to prove that they are suitable for their intended purpose by users.

Conclusion

The previously developed model process for transfer of manual, visual TLC screening methods to quantitative, instrumental, HPTLC–densitometry methods using only a limited list of readily available, relatively inexpensive, and relatively nontoxic solvents and other chemicals allowed for use with the Minilab was successfully applied to formulations of six drugs. The process involves preparation of sample and standard solutions; choice of a mobile phase for development of Merck Premium Purity silica gel plates; construction of a calibration curve spanning 70–130% of the label value; assay of three samples for comparison to the label value; checking of peak identity and peak purity with the CAMAG Scanner 3 WinCATS software; and validation of the method by standard addition analysis at 50%, 100%, and 150% fortication levels. The new methods overcome deficiencies in technology of the screening methods and can be used for stability studies of the drugs, forensic analyses, or in support of regulatory actions after more complete validation if required depending on the specific application.

Acknowledgments

The authors thank Thomas Layloff, Senior Quality Assurance Advisor, Supply Chain Management System (SCMS), Arlington, VA, USA for his support of this research and review of the article prior to its submission for publication. We also thank EMD Millipore Corp. for providing the Premium Purity glass HPTLC plates used in our experiments. Danhui Zhang and Ellen Armour were supported by a Camille and Henry Dreyfus Foundation Senior Scientist Mentor Program awarded to Professor Joseph Sherma.

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