Determination of Glimepiride in Rat Serum by Rp-Hplc Method

doi:10.4236/ajac.2011.22017

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American Journal of Anal yt ical Chemistry, 2011, 2, 152-157

doi:10.4236/ajac.2011.22017 Published Online May 2011 (http://www.SciRP.org/journal/ajac)

Determination of Glimepiride in Rat Serum by RP-HPLC

Method

Sujatha Samala, Sandhya Rani Tatipamula, Ciddi Veeresham

University College of Pharmaceutical Sciences, Kakatiya University, Warangal, India

E-mail: [email protected]

Received August 27, 2010; revised November 12, 2010; accepted November 17, 2010

Abstract

A simple and sensitive reverse phase high performance liquid chromatography (RP-HPLC) method was de-

veloped for the determination of glimepiride in rat serum. The assay involves one step liquid-liquid extrac-

tion with methanol. Gliclazide was used as an internal standard. Chromatographic separation was performed

on a C18 column using a mobile phase of methanol: 10 mM phosphate buffer (80:20 v/v) adjusted to pH 3.0

with orthophosphoric acid, at a flow rate of 1.0 ml/min and UV detection at 230 nm. The retention time of

glimepiride and gliclazide was found to be 5.5 and 4.0 min and separation was complete in less than 10 min.

The method was validated for linearity, accuracy and precision were found to be acceptable over the range of

0.5 - 500 µg/ml for glimepiride. The method was found suitable to analyse rat serum samples for application

in pharmacokinetic, pharmacodynamic, bioavailability/bioequivalence studies.

Keywords: Glimepiride, Gliclazide, RP-HPLC, Rat Serum

1. Introduction

The sulfonylurea glimepiride (1-[[p-[2-(3-ethyl-4-methy

l-2-oxo-3-pyrroline-1-carboxamido)ethyl]phenyl]sulfonyl]-

3-(trans-4-methylcyclohexyl) urea) used widely in the

treatment of non-insulin dependent Type II diabetes mel-

litus [1,2]. It acts by stimulating insulin secretions from

the beta cells of pancreas and is also known to increase

peripheral insulin sensitivity thereby decreasing insulin

resistance. It can be used in combination with metformin,

thiazolidinediones, alpha-glucosidase inhibitors and in-

sulin [3,4]. After oral administration, it is completely

absorbed from the gastrointestinal tract. Peak plasma

concentration is reached 2 - 3 h after dosing. Its

bioavailability changes a little with food and glimepiride

(99.5%) are bound to proteins. Glimepiride is completely

metabolised in liver [5]. The structures of glimepiride and

gliclazide (internal standard IS) are shown in Figure 1.

To date there is no method was available for determi-

nation of glimepiride in rat serum, so an RP-HPLC

method was developed for determination of glimepiride

in rat serum. Several different methods have been re-

ported for qualitative and quantitative analysis of glime-

piride in human plasma and biological samples; these

include micellar electrokinetic capillary chromatography

(MECC) with diode-array detection (DAD) or ultraviolet

(UV) detection [6], high performance liquid chromatog-

raphy (HPLC) with DAD [7] and UV detection [8] and

derivative UV spectrophotometric detection [9], liquid

chromatography-electrospray ionization mass spectrome-

try (LC-ESI/MS) [10-13], an HPLC method for the

quantification of glimepiride in tablets [14], the determi-

nation of related substances in glimepiride [15], the

quantification of cis-isomer of glimepiride by normal

phase chromatography [16] and the quantification of

cis-isomer of glimepiride in a bulk drug substance by

(a)

(b)

Figure 1. Chemical structures of (a) Gliclazide and (b) Gli-

mepiride.

S. SAMALA. ET AL.153

reverse-phase chromatography [17] have been reported.

However, these methods are not ideal for pharmacoki-

netics work, because they are time consuming owing to

derivatization, arduous sample preparation and long

chromatographic run times.

The main objective of this work was to develop a sim-

ple, rapid and sensitive RP-HPLC method for the deter-

mination of glimepiride in rat serum by liquid-liquid

extraction. The developed and validated method is rapid,

reproducible with simple mobile phase, sample prepara-

tion steps, improved sensitivity and a short chroma-

tographic run time. The usability of this method can be

explored further by the possibility of its application to a

human pharmacokinetic study using similar chroma-

tographic condition. The technique was validated, suc-

cessfully applied to the pharmacokinetic study of glime-

piride in rats after oral administration.

2. Experimental

2.1. Chemicals

Glimepiride and gliclazide (IS) were obtained from Dr.

Reddy’s laboratories (Hyderabad, India). Methanol of

HPLC grade, Potassium dihydrogen orthophosphate and

orthophosphoric acid of AR grade (99.5%) were pro-

cured from Merck and Milli-Q water was used.

2.2. Instrumentation and Chromatographic

Conditions

The HPLC system consisted of a Shimadzu LC-10AT

pump, a Rheodyne 7725i sample injector with a 20 µl

loop and a Shimadzu SPD-M10Avp diode array detector.

The data acquisition was performed by processing soft-

ware “LC Solution” (Shimadzu Corp.). The method was

developed on a LiChrosphere 100 RP 18 e (125 × 4.0 mm

i.d, 5 µm) column maintained at ambient temperature.

The mobile phase was 80:20 (v/v) mixture of metha-

nol and 10 mM Potassium dihydrogen orthophosphate

(pH adjusted to 3.0 with orthophosphoric acid) delivered

at a flow rate of 1ml/min. The column was maintained at

30˚C and the detection was carried out at a wavelength

of 230 nm. The injection volume was 20 µL.

2.3. Preparation of the Standard Solutions

2.3.1. Stock and Working Standard Solutions

The stock solution of glimepiride (1000 µg/ml) was

prepared by dissolving 25 mg in 25 ml methanol and

further dilutions were prepared in methanol to obtain

working standards in a concentration range of 0.1 -

500 µg/ml.

2.3.2. Internal Standard (IS)

For IS stock solution 10mg of gliclazide was weighed

and dissolved in 10 ml of methanol. The stock solution

was again diluted with methanol to working solution of

gliclazide which was at 25µg/ml. All solutions were

stored at –20˚C.

2.3.3. Sample Preparation

Serum samples were stored at –20˚C and allowed to thaw

at room temperature before processing. In brief, to 100 µL

serum, 100 µL aliquot of working standard solution of

glimepiride was added in a polypropylene centrifuge

tubes; 100 µL aliquot of gliclazide solution (25 µg/ml)

was added as an IS and the tube was shaken for 1 min.

To this, 100 µL of methanol was added for precipitation

and the tubes were vortexed each for 1min. Then all the

tubes were centrifuged for 20 min at 3000 rpm. Clear

supernatant was collected in another centrifuge tubes and a

20 µL aliquot was injected into the analytical column.

2.3.4. Appl ic a ti on of the Assay

The method described above was applied to the pharma-

cokinetic studies of glimepiride in rats. Sprague-Da-

wley rats (200 - 250 g) were housed with free access to

food and water. The rats were fasted overnight with free

access to water before administration of drug. After a

single oral administration of 1 mg/kg of glimepiride,

0.5 ml of blood samples were collected from retro orbital

plexus sinus at 0.5, 1, 2, 4, 6, 8, 12 and 24 h time-points.

Serum was separated by centrifugation and stored at

–20˚C until analysis. Aliquots of 0.1 ml serum samples

were processed and analyzed for glimepiride concentra-

tions.

The pharmacokinetic parameters were calculated with

a Non-Compartmental model using Kinetica TM Soft-

ware (version 4.4.1 Thermo Electron Corporation, U.S.A).

Each value is expressed as Mean ± SD.

3. Results and Discussion

3.1. Method Validation

3.1.1. Selectivity and Specificity

Blank serum was tested for endogenous interference. A

representative chromatogram of the serum blank is

shown in Figure 2(a). No additional peaks of endoge-

nous substances were observed. Figure 2(b) shows the

chromatograms of calibration standard containing 30 µg/ml

of glimepiride and 25 µg/ml of gliclazide in serum.

Typical chromatogram of serum samples which were

collected 1 h after oral administration of 1 mg/kg of

glimepiride to a rat is shown in Figure 2(c). The ob-

served retention times are 4.0 and 5.5 min for gliclazide

and glimepiride, respectively.

S. SAMALA. ET AL.

154

(a)

(b)

Retention factor (k’) = 1.2, Separation factor (α) = 2, and Resolution factor (RS) = 2.5

(c)

Figure 2. HPLC trace of glimepride and gliclazide (IS) using Ultraviolet detection at 230 nm. (a) Blank serum sample; (b)

Quality control standard (30 µg/ml) and IS (25 µg/ml); (c) Serum sample 1 hr post administration of 1 mg/kg glimepiride.

S. SAMALA. ET AL.

155

3.1.2. Linearity and Limit of Quantitation

Linear calibration curves with correlation coefficients

greater than 0.9999 were obtained over the concentration

range 0.5 - 100 µg/ml for glimepiride in rat serum. The

typical equation of the calibration curve is as follows;

Y = 0.0686x + 0.0192, r = 0.9999. The results shown that

within the concentration range indicated there was an

excellent correlation between peak area ratio and each

concentration of glimepiride.

The limit of quantitation, defined as the lowest con-

centration analyzed with an accuracy of ±15% and a co-

efficient of variation <15%, was 0.5 µg/ml for the deter-

mination of glimepiride in serum.

3.1.3. Precision and Accuracy

Precision and accuracy of the assay were determined,

using replicate analyses (n = 6) of quality control samples

at three concentrations, by performing the complete ana-

lytical runs on the same day and also on three consecu-

tive days. The data obtained for glimepiride were within

the acceptable limits to meet the guidelines for bioana-

lytical methods. The results are depicted in Table 1.

3.1.4. Extraction Recovery

Extraction recovery of glimepiride was determined by

comparing peak areas obtained from extracted serum

samples with those found by extracting blank matrices

through the extraction procedure and spiking with a

known amount of glimepiride. The results showed that

the mean extraction recoveries of glimepiride were >85%

at concentrations of 1.0, 5.0, and 10.0 µg/ml respectively

(Table 2). Different organic extraction solvents were

evaluated in the experiment, including Methanol, Ethy-

lacetate, Acetonitrile, Dichloromethane, Chloroform and

Diethylether. Methanol proved to be the most efficient in

extracting glimepiride from rat serum and had a small

variation in extraction recoveries over the concentration

range.

3.1.5. Stability

The amount of glimepiride recovered over a period of 30

days in serum samples stored at –20˚C did not differ

from the initial concentrations, which were shown in

Table 3.

3.2. Application of the Analytical Method in

Pharmacokinetic Studies

The described method was applied to a pharmacokinetic

study in rats. After a single oral administration of glime-

piride (1 mg/kg) to rats, serum concentrations were de-

termined over a period of 24 h after administration. The

mean serum concentration-time curve after an oral dose

of 1 mg/kg glimepiride is shown in Figure 3 and the

main pharmacokinetic parameters are summarized in

Table 4. The Cmax of glimepiride detected in the rats was

17.56 µg/ml, and the Tmax was 4 hrs.

Table 1. Inter-day and Intra-day accuracy and precision for the analysis of Glimepiride in rat serum.

Inter-day Intra-day

Nominal concentration

(µg/ml) Calculated concentration

Mean (µg/ml)a ± SD

Accuracy

(%)b

Precision

(% RSD)c

Calculated concentration

Mean (µg/ml)a ± SD

Accuracy

(%)b

Precision

(% RSD)c

1 1.045 ± 0.02 104.5 1.91 1.02 ± 0.1 102.1 9.79

5 5.01 ± 0.1 100.2 2.00 4.92 ± 0.25 98.4 5.08

10 10.08 ± 0.52 100.8 5.16 10.01 ± 1.05 100.1 10.5

a: Averaged for six measurements at each concentration level (n = 6); b: Accuracy = (mean observed concentration) (spiked concentration)−1 × 100; c: Preci-

sion (% RSD) = (SD × 100) (mean observed concentration)−1.

Table 2. The percent extraction recovery of measurement of

glimepiride in rat serum.

Nominal concentration

(µg/ml) % Recoverya,b

1 91.2

5 86.4

10 89.7

a: Averaged for six measurements at each concentration level (n = 6);

b: % recovery = (response of extracted spike) (response of post-extracted

spike)–1 × 100.

Table 3. Glimepiride stability results: Blank rat serum was

spiked with 3 different concentrations of glimepiride and

stored at –20˚C over a period of 30 days.

Concentration obtained (µg/ml)

Concentration added

(µg/ml) Day 1 Day 14 Day 21Day 30

1 1.02 0.98 1.05 1.002

5 4.92 5.01 4.98 5.05

10 10.01 10.12 9.98 9.99

S. SAMALA. ET AL.

156

Figure 3. Mean serum concentration-time profile of glimepiride after oral administration of 1 mg/kg glimepiride in rats.

Table 4. The main pharmacokinetic parameters of mean drug serum concentration time curve (mean ± SD, n = 6) of glime-

piride in rats after single oral administration at 1 mg/kg.

max (µg/ml) Tmax (hrs)AUCo-n (µg.h/ml)AUCtot (µg.h/ml)t½ (hrs) MRT (hrs)

Glimepiride 17.56 ± 0.3 4.0 ± 1.255.6 ± 1.4 57.5 ± 5.3 1.05 ± 0.8 4.14 ± 2.6

4. Conclusion

A sensitive and selective reverse phase HPLC method

was developed for the pharmacokinetic study of glime-

piride in rats. This analytical procedure is inexpensive

and simple because it requires fewer preparation steps, is

less time consuming than methods using pre-column

derivatization, and is particularly suitable when tandem

mass spectrometric detection is not available. The assay

has been validated and the results have shown that the

method is sensitive, accurate and reproducible. As a re-

sult, the proposed method is found to be appropriate and

suitable for the determination of glimepiride in serum

samples for pharmacokinetic, bioavailability or bio-

equivalence studies.

5. Acknowledgements

The authors are thankful to Dr. Reddy’s laboratories,

Hyderabad, India for the gift samples of glimepiride and

gliclazide.

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