Acta Chromatographica, No. 7, 1997

ANALYSIS OF TABLETS AND CAPLETS CONTAINING KETOPROFEN BY NORMAL-

AND REVERSED-PHASE HPTLC WITH ULTRAVIOLET ABSORPTION DENSITOMETRY

ON PREADSORBENT PLATES

J. Sherma and C. D. Incarvito

Department of Chemistry, Lafayette College, Easton, PA 18042, USA

SUMMARY

An HPTLC method has been developed for the quantitative determination of ketoprofen in analgesic tablets and caplets using preadsorbent normal phase silica gel and reversed phase C-18 chemically bonded silica gel plates with fluorescent indicator and scanning densitometry of fluorescence-quenched zones of samples and standards. Pharmaceutical tablets and caplets were analyzed and the results compared to the label declarations. Precision was evaluated by performing replicate analyses and accuracy validated by recovery analysis of a sample fortified with a known concentration of ketoprofen.

INTRODUCTION

Quantitative thin layer chromatography methods were described earlier for the determination of aspirin, phenacetin, and caffeine [1], aspirin, acetaminophen, and caffeine [2], and naproxen and ibuoprofen [3] in analgesic tablets. In this paper, the quantitative TLC methodology is extended to the determination of the newest analgesic product, ketoprofen.

Methods available in the literature for quantitatively determining ketoprofen and metabolites in pharmaceutical dosage forms involve high performance liquid chromatography (HPLC) with UV detection [4, 5] and gas chromatography [6], and qualitative TLC screening procedures have been reported [7, 8]. This paper describes a simple, sensitive, and fast quantitative HPTLC method for determination of ketoprofen employing direct densitometry of fluorescence-quenched sample and standard zones on high performance preadsorbent silica gel and C-18 chemically bonded silica gel plates containing fluorescent indicator. The method was applied

- 124 -

to the two dosage forms available in the United States market, 12.5 mg tablets and caplets.

EXPERIMENTAL

Preparation of standard solutions

Ketoprofen (2-[3-benzoylphenyl]propionic acid; Sigma, St. Louis, MO, USA) stock standard solution was prepared at a concentration of 1.00 mg/mL in methanol. The TLC standard solution was prepared by 1:10 dilution with methanol to a concentration of 0.100 g/L A 6.25 mg/mL spiking solution in methanol was also prepared.

Preparation of sample solutions

A tablet or caplet with a label values of 12.5 mg of ketoprofen was placed in a 100 mL volumetric flask and crushed into numerous pieces with a flattened glass rod. Methanol was added to the line, and the solution was magnetically stirred for 15 minutes to completely dissolve the active ingredient. Some excipients remained undissolved and were removed by filtering through glass wool packed in a small disposable pipet in order to prepare a clear solution prior to spotting onto the TLC plate.

Thin layer chromatographic analysis

Analyses were performed on Whatman (Clifton, NJ, USA) LHPKDF 10 x 20 cm high performance silica gel layers with 19 lanes, preadsorbent spotting area, and fluorescent phosphor, and on Whatman LKC18F 20 x 20 cm C-18 chemically bonded silica gel preadsorbent plates with fluorescent phosphor. Plates were prewashed by development with dichloromethane-methanol (1 + 1). For analysis of the samples, 2.50, 5.00, and 7.50 l aliquots of the TLC standard solution (0.250-0.750 g) and duplicate 4.00 l aliquots of the sample solution (containing 0.500 g of ketoprofen based on the label value) were spotted in adjacent lanes using a 10 L Drummond (Broomall, PA, USA) digital microdispenser. Silica gel plates were developed for a distance of 6.5 cm beyond the preadsorbent-silica gel interface with ethyl acetate-glacial acetic acid (95 + 5) mobile phase in a vapor-equilibrated, paper-lined Camag (Wilmington, NC, USA) HPTLC twin-trough chamber. The development time was ca. 11-14 minutes. C-18 plates were developed in a twin-trough TLC chamber for a

- 125 -

distance of 13 cm (ca. 120 min) with methanol-0.5 M NaCl (55 + 45). The plates were air dried and inspected under 254 nm UV light in a viewing box, and standard and sample zones scanned at 264 nm with a Shimadzu CS-930 densitometer in the single-beam, UV reflectance mode. This wavelength was found to provide maximum absorption by measurement of the in situ spectrum of a ketoprofen standard zone on a silica gel plate using the spectral mode of the densitometer. A calibration equation relating standard weights and scan areas was determined by use of a linear regression program on a personal computer, and the weight of ketoprofen in the sample was calculated from the calibration equation using the average area of the duplicate sample aliquots. The experimental weight was compared to the theoretical label value.

The method was validated by the following recovery experiment. A ketoprofen tablet test solution was prepared as described above, and the theoretical 0.125 mg/mL concentration of a 50.0 mL portion was doubled by addition of 6.25 mg of ketoprofen as 1.00 ml of a 6.25 mg/mL methanol stock solution, followed by dilution with 49.0 mL of methanol. Duplicate 4.00 L aliquots of the unspiked and diluted spiked samples were analyzed against a calibration curve prepared from three TLC standard aliquots spotted on the same plate. Percentage recovery of the spike was calculated using the following equation: [(g found in spiked sample x 2 - g found in unspiked sample)/(theoretical amount of added spike)] x 100. The factor of two corrects for the dilution of the spiked sample prior to spotting, and the denominator is 0.500 g.

RESULTS AND DISCUSSION

Development on HPTLC silica gel and C-18 layers containing fluorescent indicator using the mobile phases described above produced dark, flat compact zones on a bright green background with respective R f

values of 0.72 and 0.25 when viewed under 254 nm UV light. Detection of ketoprofen by fluorescence quenching was about equally sensitive on the two layers, allowing 0.25 g amounts to be visually detected and reproducibly scanned.

Two individual ketoprofen tablets were analyzed by the proposed TLC method on silica gel, and the results were 98.4% and 99.4% relative to the 12 mg label value. Triplicate analysis of a third tablet on silica gel gave 97.3+/-2.2% (mean+/-standard deviation) relative to the label value. Two ketoprofen caplets were analyzed on C-18 layers, and the results were

- 126 -

100.6% and 101.1% relative to the 12 mg label value. Triplicate analysis of a third caplet gave 99.1%+/-0.88% relative to the label value.

Calibration curves for ketoprofen in the 0.25-0.75 g range calculated for each plate on which samples were analyzed typically had linear correlation coefficient (r) values of 0.990-0.999 during these analyses. As another measure of reproducibility, the percent difference between the scan areas of the duplicate sample aliquots was in the range of 0.9-6% with an average of ca. 3.5%. No additional, interfering zones appeared in any of the sample lanes for tablets or caplets on either layer.

Analysis of the diluted, spiked sample aliquots on a silica gel plate gave an average value of 0.496 g ketoprofen while the unspiked sample gave an average value of 0.504 g. These weights represent a recovery of 97.6% of the added 0.500 g spike, which demonstrates the accuracy of the proposed method.

The analyses of all tablets and caplets were within the 95-105% range generally specified in the U.S. Pharmacopeia for active ingredients of pharmaceutical dosage forms. The recoveries as a percentage of label value, recovery of the spiked sample, and standard deviations are typical of those obtained for established pharmaceutical TLC analyses [9] and satisfactory for routine use of the method in a pharmaceutical company analytical laboratory. The TLC method involves very low solvent usage on a per sample basis compared to HPLC, leading to savings in purchase and disposal costs, and the ability to analyze multiple samples on a single plate containing only three standard zones permits very high sample throughput. Use of both NP- and RP-TLC provides excellent confirmation of qualitative and quantitative results.

- 127 -

REFERENCES

[1] J. Sherma, S. Stellmacher, and T.J. White, J. Liq. Chromatogr., 8, 2961 (1985)

[2] J. Sherma and C.D. Rolfe, J. Planar Chromatogr.-Mod. TLC, 5, 197 (1992)

[3] M. Lippstone and J. Sherma, J. Planar Chromatogr.-Mod. TLC, 8, 427 (1995)

[4] C.Y. Wong, M.K. Yeh, and D.P. Wang, J. Liq. Chromatogr., 15, 1215, (1992)

[5] R.T. Sane, V.J. Banavalikar, M.D. Joshi, and V. Nayak, Indian Drugs, 27, 54 (1989)

[6] E. Benoit, P. Jaussaund, P. Besse, B. Videmann, D. Courtot, and P. Delatour, J. Chromatogr. Biomed. Appl., 583, 167 (1992)

[7] P.X. Iten, Contrib. Forensic Toxicol., Proc. Int. Meet. Int. Assoc. Forensic Toxicol., 31st, R.K. Mueller (Ed), 1994; 299-303.

[8] H. Schuetz, A. Pielmeyer, and G. Weiler, Aerztl. Lab., 36, 113 (1990)

- 128 -

[9]

- 129 -