Analysis of Glimepiride in Human Blood and Urine by Thin-Layer Chromatography and UV-Spectrophotometry

Objective: An increasing numbers of cases of poisonings by glimepiride, either attempted suicide or accidental, combined with the absence of reliable methods for the detection and quantitation of glimepiride in biological matrices is the basis for the need for the development of new analytical techniques for forensic analysis. Materials and Methods: Analyses were performed using drugfree biological fluids (whole blood and urine). Specimens were spiked with chromatographically pure glimepiride. After hydrolysis with diluted hydrochloric acid at 50-60 0C for 15-20 min and a double extraction into chloroform, glimepiride was identified by thin-layer chromatography. Standard solution of glimepiride (1 mg/mL) and Sorbfi l chromatographic plates were used for thinlayer chromatography. The thin-layer chromatography studies showed that the best mobile phase was chloroform:acetone (9:1), Rf value of glimepiride in five examinations was 0.37±0.02. Visualization of glimepiride was achieved byspraying with Dragendorff”s, Bushard's, or diphenylcarbazone-chloroform solution followed by mercuric sulphate. The limit of detection of pure glimepiride by thin-layer chromatography was 0.5 μ/mL, 1.5 μ g/mL in whole blood and 1.0 μ g/mL in urine. For spectrophotometric determinations of glimepiride, a UV/VIS spectrophotometer with 1 cm matches quartz cell was used. Standard solutions of glimepiride in ethanol were prepared at concentrations of 1-50 μ g/mL and scanned in full-scan mode between 200-400 nm. Results and Conclusion: The wavelength maxima for glimepiride was found to be 227 nm with molar absorptivity of 3.2685×10 4 l/mol/cm. Beer's law was obeyed in the concentration range of 2-40 μ g/mL. The limit of detection and limit of quantification were found to be 0.97 μ g/mL and 2.70 μ g/mL, respectively. The results have been successfully applied in blood of patients after oral administration and on postmortem blood in an overdose death.


M e t h o d s s u c h a s h i g h p e r f o r m a n c e l i q u i d chromatography, gas chromatography and high performance
thin-layer chromatography etc. are precise with good reproducibility, but the cost of analysis is quite high owing to expensive instrumentation, reagents and need for expertise (12)(13)(14)(15).Hence it is worthwhile to develop simpler and cost effective method for qualitative and quantitative analysis.
In forensic chemical laboratories of Uzbekistan thin-layer chromatography and spectrophotometric methods are widely used for routine analysis.
The aim of the present investigation was to use thin-layer chromatographic and UV-spectrophotometric methods for the determination of GLM in biological fluids.

1.Chemicals and Reagents
Glimepiride (purity 99.30%) was obtained as a gift sample from the Main Directorate of the Quality Control of Medicines and Medical Equipment of the Ministry of Health of the Uzbekistan Republic.
All the reagents were of analytical grade.Glass double distilled water was used throughout the experiment.

Instrumentation
For the spectrophotometric determination of GLM a U V / V I S s p e c t r o p h o t o m e t e r ( M o d e l 8 4 5 3 , A g i l e n t Technologies, USA) with 1 cm matches quartz cell was used.

3.Preparation of standard stock solution of glimepiride
An accurately weighed 10 mg sample of glimepiride was dissolved in 2 mL of ethanol in a 5 ml volumetric flask and then brought to volume with additional ethanol to obtain a stock solution of 2 mg/mL (for TLC determinations).
An accurately weighed 5 mg sample of glimepiride was dissolved in 10 ml of ethanol in a 50 ml volumetric flask and then brought to volume with additional ethanol to obtain a stock solution of 100 μg/mL (for UV-spectrophotometric estimation).
All solutions were freshly prepared on the day of analysis.

TLC Analysis
Standard solutions of GLM (2 mg/mL) and Sorbfil chromatographic plates were used for TLC.The solvent mixture was optimiazed by studying the chromatographic mobility of the GLM.Different solvents such as methanol, ethanol, acetone, acetonitrile, dimethyl formamide, chloroform, diethyl ether, ammonia, cyclohexane, toluene, d i e t h y l w e r e a t t e m p t e d i n v a r i o u s p r o p o r t i o n s .
Chromatograms were visualized with Dragendorff"s, Bushard's, and diphenylcarbazone-chloroform solution followed with mercuric sulphate.10 µl of standard solution of GLM and sample extracts were applied to the chromatographic plate using glass capillaries.

UV-spectrophotometric Analysis
Aliquots of 0.1 to 5.0 mL of stock solution were transferred to a series of 10 ml volumetric flasks and were adjusted to 10 ml with distilled water to obtain a concentration of range of 1-50 μg/mL.Spectral scans of each sample of standard against ethanol were recorded in the range 200 to 400 nm where maxima was observed at 227 nm (figure 2).

Linearity
To establish linearity of the proposed method, six separate series of solutions of the drug (2-40 μg/mL) in ethanol were prepared from the stock solutions and analyzed.A six point calibration curve was constructed and found to be linear (fig 3).
Least square regression analysis was used to obtain data (table 2).

Precision
The precision of an analytical method is the degree of agreement among individual test results when the method is applied repeatedly to multiple samplings of a homogenous sample (United States Pharmacopeia, 2000).In order to determine the precision of the method, solutions containing known amounts of pure drug were prepared and analyzed in three replicates and the absorbances were recorded in six replicates to get the mean.The low %RSD values obtained from the analyses of pure standards of GLM indicated that the method has high precision.The analytical results obtained from these investigations for the methods are showed in table 2.

Limit of detection and quantitation
The detection limit (LOD) may be expressed as: The above procedure was repeated three times.Mean absorbance values along with the statistical data for the method are shown in table 1. Tab. 2 represents the optical characteristics and validation parameters of the proposed method for estimation of glimepiride.

UV-spectrophotometric method validation
The proposed method was validated in the light of FDA Guidelines for linearity, precision, sensitivity and selectivity, stability and recovery (16).LOQ may be expressed as:

LOD S  
Where σ is the standard deviation of the response and S is the slope of the calibration curve.The slope, S, may be estimated from the calibration curve of the analyte.A specific calibration curve should be studied using samples, containing an analyte in the range of LOQ.The residual standard deviation of regression lines or the standard deviation of y-intercepts of regression lines were used as the standard deviation.The limit of quantitation of glimepiride was 2.70 μg/mL.

Application of the method in human biological fluids
Analyses were performed using drug-free biological fluids: 5 mL of whole blood and 10 mL of urine.Blood samples were diluted with distilled water (1:1).Specimens were spiked with

TLC Analysis
The TLC studies showed that the best mobile phase was chloroform:acetone (9:1), The Rf value of GLM in five examinations was 0.37±0.02.In the examination of visualization reagents for GLM, the spraying of Dragendorff"s test solution produced a pale yellow-red spot, Bushard's test solution-a brown spot, diphenylcarbazone chloroform solution followed by spraying mercury sulphate -a blue spot (remains after drying the plate) The limit of detection (LOD) of pure GLM by TLC was 0.5 μg/mL, 1.5 μg/mL in whole blood and 1.0 μg/mL in urine.

UV-spectrophotometric Analysis
Glimepiride in ethanol yields a characteristic curve when scanned in the ultraviolet wavelength range between 200 and 400 nm.The scan (figure 2) shows absorption maxima at 227 and 275 nm (table 2).The British Pharmacopoeia uses275 nm figure-1) used in the treatment of non-insulin dependent diabetes mellitus (8-10).Today glimepiride (GLM) is widely used in the treatment of type 2 diabetes mellitus in Uzbekistan.The number of registered patients in Uzbekistan on January 1, 2011 amounted

4 .
Preparation of samples from biological fluids Analysis was performed using drug-free biological fluids: 5 mL of whole blood and 10 mL of urine.Blood samples were diluted with distilled water (1:1).Specimens were spiked with chromatographically pure GLM.After hydrolysis with diluted hydrochloric acid at 50-60 ⁰C for 15-20 min and a double extraction into chloroform, the serum and urine extracts were passed through coarse filter paper and evaporated to dryness in disposable plastic 25-mL conical beakers.After evaporation to dryness, 3 mL of ethanol were added in conical beakers for dissolution of the dried extracts.The absorbance of sample solution was measured and amount of glimepiride was determined by referring to the calibration curve.
pure GLM.After hydrolysis with diluted hydrochloric acid at 50-60 ⁰C for 15-20 min and double extraction into chloroform, the serum and urine extracts were passed through coarse filter paper and evaporated to dryness in disposable plastic 25-mL conical beakers.After evaporation to dryness, 3 mL of ethanol were added in conical beakers for dissolution of dried extracts.The absorbance of sample solution was measured at 227 nm and amount of glimepiride was determined by referring to the calibration curve.The above procedure was carried out in triplicate and absorbance readings were recorded three times to get the mean.Results of these determinations are included in tables 3-4.

Table 1 .
Mean absorbance values and statistical data of the calibration curve for the estimation of glimepiride.
a Mean of three values.

Table 2 .
Optical characteristics and validation parameters for standard glimepiride.

Table 3 .
Results of the proposed method for estimation of GLM in whole blood a Confidence limits at P=0.95

Table 4 .
Results of the proposed method for estimation of GLM in urine a Confidence limits at P=0.95