An Ultraviolet-Spectrophotometric Method for the Determination of Glimepiride in Solid Dosage Forms

Abstract

Background:

Considering the cost of acquiring a liquid chromatographic instrument in underdeveloped economies, the rising incidence of diabetes mellitus, the need to evaluate the quality performance of glimepiride generics, and the need for less toxic processes, this research is an imperative.

Methods:

The method was validated for linearity, recovery accuracy, intra- and inter-day precision, specificity in the presence of excipients, and inter-day stability under laboratory conditions. Student's t test at the 95% confidence limit was used for statistics.

Results:

Using 96% ethanol as solvent, a less toxic and cost-effective spectrophotometric method for the determination of glimepiride in solid dosage forms was developed and validated. The results of the validated parameters showed a λ_max of 231 nm, linearity range of 0.5–22 μg/mL, precision with relative SD of <1.0%, recovery accuracy of 100.8%, regression equation of y = 45.741x + 0.0202, R ² = 0.999, limit of detection of 0.35 μg/mL, and negligible interference from common excipients and colorants. The method was found to be accurate at the 95% confidence limit compared with the standard liquid chromatographic method with comparable reproducibility when used to assay the formulated products Amaryl^® (sanofi-aventis, Paris, France) and Mepyril^® (May & Baker Nigeria PLC, Ikeja, Nigeria). The results obtained for the validated parameters were within allowable limits.

Conclusion:

This method is recommended for routine quality control analysis.

Introduction

G limepiride is a medium-to-long-acting sulfonylurea antidiabetes drug. It is the first third-generation sulfonylurea and is very potent.^1,2 Structurally, it is 3-ethyl-4-methyl-N-(4-[N-((1r,4r)-4-methylcyclohexylcarbamoyl)sulfamoyl]phenethyl)-2-oxo-2,5-dihydro-1H-pyrrole-1-carboxamide.

Clinically, the desired goal of any drug is to achieve a therapeutic effect on administration. It is therefore imperative that the quality of the drug or drug product must be assured.

The incidence of diabetes among the productive age group in Nigeria has been on the rise. To complement existing hypoglycemic medications like glibenclamide, metformin, and pioglitazone, increasing numbers of glimepiride generics have been introduced into the Nigerian market recently. Ascertaining the quality of the several formulations of glimepiride has therefore become an imperative.

Several high-performance liquid chromatographic (LC) methods have been described for quantitative determination of glimepiride in human plasma and biological samples.^3

–6 The British Pharmacopoeia recommends an LC method for its determination in raw material and in dosage forms.⁷ However, high-perfomance LC techniques for routine analysis are often time consuming and expensive. For most developing nations, acquisition of sophisticated LC instruments for use in their laboratories is a capital-intensive venture. Compared with chromatographic methods, spectrophotometric methods are suitable for routine analysis because they are economical, simple, rapid, and of low maintenance cost. They also show comparable accuracy and precision with high-performance LC methods, especially where analysis does not involve studies of stability or degradation products.⁸ Literature describing simple spectrophotometric assay of glimepiride is scarce. Moreover, the spectrophotometric methods described for determination of glimepiride require the use of highly toxic solvents such as methanol⁹ and dimethylformamide.¹⁰ Methanol is associated with the toxicity of wood fuel and is also linked to blindness.¹¹

As an alternative to the existing methods, the aim of this study was to develop, validate, and apply a less toxic, inexpensive, useful, and simple ultraviolet spectrophotometric method for quantitative determination of glimepiride in commercial pharmaceutical tablet preparations. This will not only help in checking the influx of fake, substandard, or adulterated drugs into the market and their attendant clinical problems, but also reduce the cost of quality control testing requirements, especially in a third world society like Nigeria.

Experimental Procedures

Materials

Pure glimepiride (99.98%) was kindly received as a gift from May & Baker Nigeria PLC (Ikeja, Lagos, Nigeria). Ethanol (96%) (analytical grade) was purchased from Sigma Aldrich (St. Louis, MO). A Cecil Instruments (Cambridge, UK) spectrophotometer (model CE7200) was used for the analysis. Excipients such as sodium starch glycolate, lactose, maize starch, and Avicel (Gujarat Microwax Ltd., Ahmedabad, India) of manufacturing grade were obtained from May & Baker Nigeria PLC.

Method development and validation

Determination of wavelength of maximum absorption

Pure glimepiride (0.01 g) was dissolved in 100 mL of 96% ethanol using a 100-mL volumetric flask to give a 100 μg/mL glimepiride stock solution (R_o). Ten milliters of R_o was further diluted to 100 mL with distilled water to give a 10 μg/mL glimepiride solution, which was scanned in the region 190–800 nm to determine the wavelength of maximum absorption (λ_max) using 96% ethanol as the reagent blank.

Linearity study

The 100 μg/mL glimepiride solution (R_o) used for the λ_max determination was used as the stock solution for the linearity study. Aliquots in the range of 50–2,200 μL of this solution were taken and diluted to 10 mL with 96% ethanol to obtain different concentrations within the range 0.5–22 μg/mL and used for the linearity calibration plot.

Intra-day precision study

Aliquots (600, 800, and 1,200 μL) of the 100 μg/mL glimepiride stock solution were taken and diluted to 10 mL with 96% ethanol to obtain three concentrations of 6.0, 8.0, and 12.0 μg/mL, respectively. Triplicate absorbance measurements of each were taken, and the mean, SD, and relative SD (RSD) were calculated.

Inter-day precision study

The selected concentrations for the intra-day precision study were again analyzed the following day after refrigeration for 24 h at −4°C, and the mean, SD, and RSD were calculated.

Recovery accuracy study using the new method

This study was carried out using preformulated granules containing 1.1764% and 2.3529% (wt/wt) pure glimepiride and common excipients such as sodium starch glycolate, maize starch, lactose, and Avicel. The granulation (0.170 g) was then transferred into a 50-mL volumetric flask. Ethanol (96%) (25 mL) was then added, shaken for 15 min using a vortex-mixer, and diluted to the 50 mL mark with the same solvent. The mixture was filtered to obtain the sample stock solution (P_o). This stock solution (20 mL) was further diluted to 100 mL with 96% ethanol and then assayed for the content of glimepiride using the proposed method with a solution containing 8.0 μg/mL pure glimepiride as the standard for comparison. All analyses were carried out in triplicate at 231 nm using 96% ethanol as the blank.

Limit of detection and limit of quantitation

This was determined by using the expressions limit of detection = 3(SD/a) and limit of quantitation = 10(SD/a), where SD is the SD of the intercept and a is the average slope obtained from the calibration plot.

Recovery accuracy study using the Pharmacopoeia LC method

This was done using the standard British Pharmacopoeia method.⁷

Specificity in the presence of excipients

This test was carried out using common excipients such as sodium starch glycolate, lactose, maize starch, colorants, and Avicel. Dummy granules devoid of pure glimepiride were prepared as in the recovery study above, and their absorbance reading at 231 nm was measured and compared with both that of the blank (96% ethanol) and that obtained for the recovery study.

Assay of content of glimepiride in selected marketed brands

This was carried out using the developed and validated method as follows.

Sample preparation

Accurately weighed tablet powder, equivalent to 2 mg of glimepiride, was transferred into a 50-mL volumetric flask. Twenty-five milliliters of 96% ethanol was added, shaken for 15 min using a vortex-mixer, and diluted to the 50 mL mark with same solvent. It was then filtered to obtain sample stock solution (P_o). Twenty milliliters of the filtrate (P_o) was further diluted to 100 mL with 96% ethanol and then assayed for content of glimepiride using the proposed method with a solution containing 8.0 μg/mL pure glimepiride as the standard for comparison. All analyses were carried out in triplicate.

Reference standard preparation

Pure glimepiride (10.0 mg) was accurately weighed and dissolved in 100 mL of 96% ethanol. From this solution, 8 mL was further diluted to the 100 mL mark with 96% ethanol to give an 8.0 μg/mL glimepiride standard solution. The absorbances of the sample preparation and reference standard solution were measured using 96% ethanol as the blank. The content of anhydrous glimepiride in the marketed formulations was determined using Eqs. 1, 2a, and 2b: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}L_{ \rm H} ( \% ) = 20 ( A_{ \rm p} \times W_{ \rm s} ) /( A_{ \rm s} \times W_{ \rm p} ) \tag{1} \end{align*} \end{document} \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}\% \ & \hbox{\rm stated dose of anhydrous glimepiride for} \ 2.0 { -} \hbox{\rm mg} \\ & \rm{dose \ formulation} = ( L_{\rm H} \times W_{20} ) / 40 \tag{2{\rm a}} \end{align*} \end{document} \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}\% \ & \hbox{\rm stated dose of anhydrous glimepride for} \ 4 {-} \hbox{\rm mg} \\ & \rm{dose \ formulation} = ( L_{\rm H} \times W_{20} ) / 80 \tag{2{\rm b}} \end{align*} \end{document}

where L _H is the content of glimepiride (% [wt/wt]), A _p is the absorbance of generic sample solution, A _s is the absorbance of reference glimepiride standard solution, W _s is the weight of reference glimepiride powder, and W _p is the weight of generic powder sample, with the factor 20 due to dilution factors in Eq. 1. In Eq. 2a and 2b, W ₂₀ is the weight (g) of 20 tablets of generic sample, and the factor 40 and 80 are due to the labeled claim of 2.0 and 4.0 mg of glimepiride per tablet, respectively.

Statistical analysis

Where applicable, results were expressed as mean ± SD values and analyzed statistically using Student's t test. They were considered significant at the 95% confidence limit.

Results

The λ_max was observed to be 231 nm. The linearity parameter (Table 1) and the corresponding regression data indicated excellent linear relationship (r ² = 0.999) over the working concentration range (0.50–22.00 μg/mL).

Table 1.

Linearity Parameter Data

Parameter	Result
λ_max (nm)	231
Beer's law linearity range	0.50–22.0 μg/mL
Regression equation: y = bx + a	y = 045.741x + 0.0202
Intercept (a)	0.0202
Slope (b)	45.741
Correlation coefficient (r)	R ² = 0.999
LOD	0.35 μg/mL
LOQ	1.15 μg/mL
N	16

LOD, limit of detection; LOQ, limit of quantitation.

Table 2 presents the intra- and inter-day precision of the new method, confirming adequate sample stability and method reliability over a 24-h period. This is because for the three selected concentrations within the linearity range, the observed RSDs were all <1%. The mean analyte recovery of 102.37 ± 2.0365% (n = 3) with an RSD of 1.9894% was not statistically significant from that obtained using the Pharmacopoeia LC method (99.48 ± 3.2085%, n = 2) as the calculated value of t (2.01886) was substantially less than the tabulated value of 2.3534 (n = 3) at the 95% confidence level. This showed that the proposed method could satisfactorily be used for the assay of glimepiride in dosage forms.

Table 2.

Intra- and Inter-Day Precision Data

	Mean ± SD absorbance		RSD (%)
Concentration (μg/mL)	Day 1	Day 2	Day 1	Day 2	Calculated value of t
6.0	0.309 ± 0.00158	0.306 ± 0.00265	0.51136	0.8660	2.8203^*
8.0	0.372 ± 0.0020	0.376 ± 0.00301	0.5376	0.9601	2.6619^*
12.0	0.575 ± 0.00458	0.570 ± 0.00354	0.7865	0.6211	3.4412^*

n = 3, P = 0.05.

No significant difference between day 1 and day 2 mean values at 95% confidence level considering tabulated t value of 4.3000 at four degrees of freedom.

RSD, relative SD.

This was confirmed when the new method was used to determine the percentage absolute drug content of two glimepiride generics in Nigeria; the tradename Amaryl^® (sanofi-aventis, Paris, France) contained the indicated claims (percentage of label) of 99.42–101.40% as shown in Table 3.

Table 3.

Assay of Selected Formulations Using the New Method

Formulation	Labeled claim	New method % of labeled claim assay	SD	RSD (%)
A (Mepyril)	2 mg	99.42	0.9636	0.9692
B (Mepyril)	4 mg	100.34	0.6768	0.6745
C (Amaryl)	4 mg	101.40	1.8420	1.8166

RSD, relative SD.

Discussion

Compared with LC instruments, the cost of acquisition and maintenance of spectrophotometers is low, and methods based on spectrometry show comparable accuracy and precision with LC methods. The new method has been validated as per International Conference on Harmonization guidelines,^12,13 and the results of the validation parameters were within acceptable limits. The observed linearity range where Beer–Lambert's law was obeyed, the corresponding regression data (r = 0.999), and the limit of detection of 0.05 μg/mL showed a high degree of sensitivity. Using Student's t test, recovery accuracy was statistically insignificant from the expected value as the calculated t value was substantially less than the theoretical value at the 95% confidence level. Intra- and inter-day precision data also indicate good precision with RSD <1%, which is within the allowable limit of ≤15%.^14,15 This also shows that sample solutions are appreciably stable under refrigeration. When the new method was applied to determine the content of glimepiride in two marketed formulations, the method showed excellent reproducibility of the recovery accuracy and precision validation results in addition to their assay data being well within Pharmacopoeia limits for tablets.⁷ Although the method was not applied to liquid preparations like injections and co-formulated solid dosage forms, the observed recovery accuracy and precision are likely to be reproduced where noninterfering species and excipients at the λ_max of 231 nm are used in their formulation.

Conclusion

Considering the observed excellent validation results, the method could be used for routine quality control assay of glimepiride where noninterfering species and/or excipients at the λ_max of 231 nm are used in their formulation.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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