Development of a High-Performance Liquid Chromatographic Method for Analysis of Glibenclamide from Dissolution Studies

Abstract

A HPLC method for the detection and quantification of glibenclamide, from dissolution studies of glibenclamide tablets (5 mg), was developed. The dissolution test employed was the basket method, operating at 100 rpm, using 1000ml phosphate buffer pH 7.4 as the dissolution medium. Elution was performed on LC-18 reverse phase, SupelcosilTM ODS column (4.6mm x 25cm, 5mm) using a mobile phase consisting of 0.02M monobasic ammonium phosphate in 60%v/v acetonitrile in water at a flow rate of 2ml/min, using phenacetin as the internal standard. The eluent was monitored at 254nm with an UV detector. Retention times of the glibenclamide and phenacetin peaks were 3.61 minutes and 1.8 minutes respectively.

Introduction

Glibenclamide is the most extensively used sulphonylurea in many parts of the world for the management of non-insulin-dependent diabetes mellitus (NIDDM) [1] A search of the registry of drugs approved for marketing in Malaysia, kept at the Drug Evaluation and Safety Division of the National Pharmaceutical Control Bureau, revealed a total of 32 glibenclamide preparations registered as at July 1999. These included the innovator products, namely DaonilÒ and EugluconÒ, as well as 30 generic preparations, of which 14 were imported.

Glibenclamide is documented to possess low aqueous solubility [2]. Large inter- and intra-individual responses following administration of glibenclamide preparations have also been reported [3][4][5]. Such variations are undesirable and may expose susceptible patients to the danger of hypoglycaemia or other hazards when changing a patient’s therapy from one preparation to another.

Since 1970, dissolution requirements have been added to tablet and capsule monographs, in general, in response to concerns for bioavailability. Of equal significance is the recognition of the immense value of dissolution testing as a tool for quality control. Thus, equivalence in dissolution behaviour was sought in light of both bioavailability and quality control considerations [2]. The United States Pharmacopoeia (USP) 1995, however, does not require glibenclamide tablets to comply to the dissolution test [2]. Nonetheless, dissolution profiles are often used by the industry to ascertain the release rates of glibenclamide from tablet formulations as a quality assurance tool.

Signoretti et al (1983) and El-Sayed et al (1989) conducted studies to evaluate the physico-chemical characteristics, including the dissolution profiles, of various glibenclamide preparations which might contribute to the unpredictable behaviour of the drug products [4][6]. In their studies, Signoretti et al (1983) used a method based on ultraviolet spectrophotometry to analyze glibenclamide from dissolution samples.

However, in terms of sensitivity, precision and specificity, a high-performance liquid chromatographic (HPLC) method may offer additional advantages (5,7-9). The USP (1995) documents a HPLC method for the assay of glibenclamide tablets using progesterone as the internal standard [2]. The use of an internal standard is required for evaluating system suitability and is not necessary for assays, which have been proven to be accurate, precise, sensitive and specific. However, the authors felt that the incorporation of a suitable internal standard provides an added value to a HPLC technique, as an additional calibration tool, to accommodate any changes in the system and to improve retention reproducibility throughout the analytical period [10].

Aim

This study aims to develop a HPLC method, with the incorporation of an internal standard, for the detection and quantification of glibenclamide from dissolution studies.

Materials and Methods

Materials

Two of the 5mg glibenclamide tablet preparations available in the Malaysian market namely, Brand A (expiry date: August 2002) and Brand B (expiry date: April 2002) were used in the dissolution studies. Glibenclamide RS, progesterone RS and phenacetin RS were obtained from the Reference Standard Unit, National Pharmaceutical Control Bureau (NPCB). HPLC-grade acetonitrile and methanol as well as AR-grade monobasic ammonium phosphate and phosphoric acid were used in preparing the mobile phase.

Apparatus

In vitro dissolution studies were carried out in a Erweka® DT 70 dissolution apparatus using the basket method, operated at 100 rpm. The HPLC system consisted of a dual-pump Waters® solvent delivery system (Model 600E) a Rheodyne® (7725 i) variable-volume, syringe-loading sample injector, a Waters® UV detector (Model 486) set at 254 nm and Millennium® 2010 chromatography Manager, version 2.1 data system as the integrator. A stainless steel Supelcosil™ LC-18 ODS (4.6 mm x 25 cm, 5 mm) column was used as the stationary phase.

Assay procedures and validation

Stock solutions of 0.05%w/v of glibenclamide RS in methanol:phosphate buffer pH7.4 (2:98%v/v), 0.001%w/v progesterone RS in acetonitrile and 0.001%w/v of phenacetin RS in phosphate buffer were prepared separately. Standard solutions of varying concentrations of glibenclamide (0.05, 0.1, 0.2, 0.5, 0.75, 0.8, 1, 1.5, 2 and 5mg/ml) were prepared. This range was selected based on 5mg/ml being the maximum concentration of glibenclamide in the dissolution medium, upon complete dissolution of the tablet.

To each 1ml aliquot of the standard solutions, 0.5ml of the internal standard solution (0.001%w/v progesterone or 0.001%w/v phenacetin) was added. 10ml aliquots of the mixture were then injected into the HPLC (minimum of n=5 for each mixture). For the mixtures incorporating progesterone as the internal standard, the following mobile phases were used:

1. 0.02M monobasic ammonium phosphate in acetonitrile:water (55:45%v/v) b) 0.02M monobasic ammonium phosphate in acetonitrile:water (60:40%v/v) For each of the mobile phases, the pH was adjusted to 5.25 ± 0.10, using phosphoric acid, and it was delivered isocratically at 2ml/min. Mobile phase
2. as also used for aliquots of mixtures containing phenacetin as the internal standard.

For the assessment of intra-day precision, 10ml aliquots of the complete set of glibenclamide standard and the internal standard (phenacetin) mixtures were injected (n=3) at 4 different times over an 18-hour period, namely in the early morning, noon, mid-evening and night. This was repeated over 3 days to measure inter-day precision.

To further validate the accuracy of the assay technique, the phosphate buffer pH 7.4 used in the dissolution experiments was spiked with 3 different known concentrations of the glibenclamide standard (0.65, 1.30 and 1.95 mg/ml) and assayed. Phenacetin was used as the internal standard for the assay of the spiked samples.

Apart from measuring the retention times of the analyte peaks, calibration curves of peak area ratios (PAR) of glibenclamide:internal standard versus the known glibenclamide concentrations were also constructed.

Dissolution experiments

1000 ml of phosphate buffer pH 7.4 @ 37oC was used as the dissolution medium. Dissolution of the tablets was carried simultaneously in 6 vessels, using the basket method, operating at 100 rpm. 2ml samples were drawn at 5, 10, 15, 30, 60, 90 and 120 minutes from the onset of the dissolution studies. Equal volumes of phosphate buffer pH 7.4 preheated to 37oC, was added into each vessel to replace the withdrawn volumes. The samples were filtered through a 0.45mm (millipore) membrane filter . To each 1ml aliquot of the samples, 0.5ml of 0.01mg/ml phenacetin in phosphate buffer pH7.4 was added as the internal standard. 10ml aliquots of the sample and internal standard mixture were then analysed by HPLC (n=3).

Results and Discussion

Table 1. Analysis data of phosphate buffer pH 7.4 spiked with known concentrations of glibenclamide.

Known concentration of glibenclamide added to buffer solutions (mg/ml) [a]	Quantitated mean concentration of glibenclamide from HPLC assay (mg/ml) [b]	Difference between known concentration and detected mean concentration (mg/ml) [a]-[b]	Percentage of difference from known concentration (%) [a]-[b] / [a]x 100
0.65	0.65	0	0
1.30	1.28	0.02	1.54
1.95	1.93	0.02	1.03

A mobile phase composing of 0.02M monobasic ammonium phosphate in 55%v/v acetonitrile in water was initially used, with progesterone as the internal standard, as recommended by the United States Pharmacopoeia (1995). The retention times for the glibenclamide and progesterone peaks were 4.5 minutes and 7.9 minutes respectively. The prolonged retention time of progesterone coupled with its extremely poor aqueous solubility rendered it unsuitable as an internal standard for this assay. It was subsequently substituted with phenacetin. To reduce the retention time of the glibenclamide peak, the composition of the acetonitrile in the mobile phase was increased to 60%v/v. The retention time of phenacetin was found to be 1.8 minutes (Figure 1) while the mean retention time for the glibenclamide peaks was 3.61 minutes with Relative Standard Deviation (RSD) values between 0.08% and 1.6% (n=12). The maximum RSD at 1.6% showed that the precision of this method was acceptable.

The calibration curve for glibenclamide was linear in the concentration range 0.05 to 5 mg/ml (R2 = 0.997; y = 0.1384x + 0.0138). The intercept was not significantly different from zero. However, for the dissolution experiments, preliminary studies revealed that there was incomplete dissolution of the glibenclamide tablets from both Brands A and B. Less than 2mg/ml of glibenclamide was detected in the dissolution medium at 120 minutes from the onset of the dissolution studies. As such, the concentration range for the calibration curve utilized for the dissolution studies was narrowed down to 0.05-2 mg/ml. The linearity (R2 = 0.9908) was found to be acceptable for this range as displayed in Figure 2.

Using peak area ratios of glibenclamide:phenacetin (internal standard), the coefficients of variation for both intra- and inter-day analyses were shown to range from 0.91% to 5.91% and 0.39% to 6.26% respectively for the complete range of glibenclamide standards. As such the intra- and inter- day precision of the assay were found to be acceptable.

Table 1 compares the results of the assayed concentrations ( calculated from the standard curve) of the spiked samples of phosphate buffer pH 7.4 to the known concentrations of glibenclamide added. The differences between the known concentration values and the values quantitated from the assay method were not significant as reflected by the very low values (<2%) of the percentage of the [difference ¸ known concentration]. This further validated the accuracy of the assay method.

Figures 3 and 4 display the dissolution profiles from the studies conducted on the two commercial glibenclamide preparations, Brands A and B, using the HPLC method developed. It was found that the HPLC method developed was suitable to measure the low levels of glibenclamide released into the dissolution medium.

For both brands, dissolution of the tablets were not complete, even at 120 minutes. USP (1995) generally requires that, for an immediate release tablet, at least 75% of its active ingredient is dissolved within 45 minutes [2]. However, the pharmacopoeia does not specify dissolution testing in the glibenclamide tablet monograph and as such these tablets need not comply to the general requirement. Nonetheless, due to its poor aqueous solubility, glibenclamide tablet formulations may potentially face bioavailability problems if its dissolution profile is found to be relatively poor. Thus, the industry does utilize dissolution studies as a quality assurance tool.

Conclusion

A HPLC method for the detection and quantification of glibenclamide from dissolution studies had been successfully developed, with acceptable retention times of the drug and internal standard peaks, of less than 4 minutes per assay. The HPLC method is able to detect glibenclamide concentrations as low as 0.05mg/ml with a Relative Standard Deviation ranging between 0.08% and 1.6%. Apart from the greater precision and sensitivity attained using this HPLC method, the specificity offered is undoubtedly another advantage compared to the UV method of analysis.

Acknowledgement

The authors wish to thank University of Malaya R&D Unit for its financial support and Mr. Mohd Nasir of the Reference Standard Unit, National Pharmaceutical Control Bureau, Ministry of Health Malaysia for his prompt supply of the reference standards.

References

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