INTRODUCTION:

Repaglinide (RPG), chemically is (S) -2-Ethoxy-4-2-[[methyl-1-[2-(1-piperidinyl) phenyl]butyl]amino]-2-oxoethyl] benzoic acid[1], is an oral antidiabetic drug(Figure 1). It’s an oral postprandial hypoglycemic agent for type 2 diabetes mellitus. It reduces fasting sugar in type 2 diabetes patients and helps control sugar by the amount of insulin released by the pancreas. It induces a rapid onset short acting insulin release[2,3]. Stability indicating methods have become an important aspect of any analytical method validation and a part of US FDA requirements. The stability indicating method is essential when the manufacturing of the drug is done as the accuracy and precision of the method gets established [4].

Liquid chromatography –tandem mass spectrometry (LC/MS/MS) and normal chiral HPLC methods for determining of RPG are reported [5,6,7]. Literature survey also reveals that simple HPLC with UV [8] or spectroflurometric [9] have been reported. These methods are costly. Comparatively, the HPTLC provides a cheaper solution for the same. The drug stability test guidelines Q1A (R2) issued by International Conference on Harmonization (ICH) requires that analytical test procedures for stability samples should be fully validated and the assay should be stability indicating.

The purpose of this work was to develop and validate simple, specific, sensitive, accurate, precise, rapid and cost effective HPTLC method for the estimation of Repaglinide in bulk drug as per ICH guidelines.[10,11]

Figure1: Chemical structure of Repaglinide

MATERIALS AND METHODS:

Working standard:

Working standard of Repaglinide was provided by USV Ltd. Daman and was used as such without further purification. Methanol, toluene, conc. HCl, NaOH and H₂O₂ (all from S. D. fine Chem. Laboratories Pvt. Ltd., Mumbai, India) used were of analytical reagent grade.

Instruments:

Aluminium plates precoated with silica gel 60 F₂₅₄, purchased from E-Merck, Germany were used. For method development, Camag HPTLC system consisting of Linomat-V applicator with a100ml syringe (Hamilton Switzerland) was used.. The linear ascending development was carried out using the twin trough chamber at room temperature. A Camag TLC Scanner 3 with a radiation source of Deuterium Lamp (Range 400-200nm) and WinCATS software V 1. 4. 3were used. For photo-degradation studies, Photo-stability Chamber was used (Make-Newtronic). All the weighing was done on Shimadzu balance (ModelAY-120).

Chromatographic Conditions:

Samples were applied on the 10 cm x 10 cm plate as band with a width of 4 mm and slit dimensions were kept as 3.00 x 0.45 mm. 25 μl volume of each sample was applied on TLC plate. Chamber saturation time was 15 minutes and the migration distance was 80 mm.

METHOD:

Wavelength selection:

After chromatographic development, the bands were scanned over the range of 200-400 nm. It was observed that drug showed considerable absorbance at 242 nm. So, 242nm was selected as the wavelength for detection (fig.-2).

Figure 2: UV spectrum of Repaglinide

Preparation of Standard Stock Solution:

The standard was prepared by dissolving 25mg RPG in 25ml methanol to get 1000mg/ml. This solution was used for further evaluation.

STRESS DEGRADATION STUDIES:

Degradation under alkali catalyzed hydrolytic conditions:

5ml of the working standard was mixed with 1ml 0.1N NaOH and volume was made upto 25ml with methanol. The solution was kept for 15hrs. A spot of 10ml was applied on the TLC plate.

Degradation under acid catalysed conditions:

5ml of the working standard was mixed with 1ml 0.1N HCl and volume was made upto 25ml with methanol. The solution was kept 15hrs. A spot of 10ml was applied on the TLC plate.

Degradation under oxidative conditions:

5ml of the working standard was mixed with 1ml of 3% Hydrogen peroxide and volume was made upto 25ml with methanol. The solution was kept 15hrs. A spot of 10ml was applied on the TLC plate.

Degradation under dry heat:

Dry heat studies were performed by keeping the samples in the oven at 50 °C for 4 days. Sample were withdrawn at appropriate time, and dissolved in methanol to get a final concentration of 100mg/ml. A spot of 15ml was applied to the TLC plate.

Photo- degradation:

The study was carried out by exposing the sample to UV (200 watts hour/square metre) and cool white fluorescent light (1.2 million lux hrs). Samples were collected at, weighed accurately and diluted to get 100mg/ml as final concentration. Then 15ml of each was applied on TLC plate.

Since the peak purity of RPG peak was within limits even for stress degradation samples, by the developed method, it was taken up for validation study.

METHOD VALIDATION:

Linearity and range:

The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample. It was studied by analyzing five samples of the drug and process was repeated five times. It was done over a range of 500- 2500ng/band.

Precision:

Precision of the system was analyzed by evaluating six independent sample preparations and % RSD value obtained was calculated to determine any intra-day variation. These studies were also repeated on different days to determine inter day variation.

Accuracy:

For accuracy of the method, recovery studies were carried out by addition of standard drug solution to pre analyzed sample solution at three different levels 80, 100 and 120 %.

Limit of detection and Quantitation:

The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample, which can be detected but not necessarily quantitated as an exact value. Based on the Standard Deviation of the Response and the Slope, detection limit (LOD) may be expressed as:

LOD = 3.3s/ S

The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample, which can be quantitatively determined with suitable precision and accuracy. Based on the Standard Deviation of the Response and the Slope, The quantitation limit (LOQ) may be expressed as:

LOQ = 10s/S

Where,

s = the standard deviation of the response for the lowest conc. in the range

S = the slope of the calibration curve.

Specificity:

The specificity of the method was determined by peak purity profiling studies. Purity of the drug peak was found by analyzing the spectrum at peak start, middle and at peak end. The peak purity was determined on WinCATS software V 1. 4. 3.

RESULTS:

Development of optimum mobile phase:

Chromatographic separation studies were carried out on the working standard solution of Repaglinide (100mg/ml). Initially varying solvents were used with methanol and toluene. Later varying proportions of methanol and toluene were used. After several trials the mobile phase methanol: toluene (2:8 v/v) as the mobile phase, for HPTLC, which resulted in good resolution and acceptable peak parameters. Rf was found to be 0.36 ± 0.02 (Figure-3).

Figure3: Representative Densitogram of Repaglinide (1500ng/band)

Degradation behavior (table-1):

In the evaluation for Alkaline conditions the degradation was observed. Though no separate peaks for degradation were observed the peak area was reduced. There was 20% degradation.

In acid degradation the peak area was reduced and no separate peaks for the degraded product were obtained. The degradation was observed to be 30%.

For oxidative degradation the peak area showed no signs of degradation product but degradation of drug was observed to be 10%.

After dry heat degradation there was no degradation observed.

On exposure to cool white fluorescent light no degradation was observed.

After the UV exposure degradation of about 10% was observed.

Table-1. The degradation summary

Sr. No	Stress Condition	Peak (Front)	Peak (Tail)	% Recovered
1	Alkaline (0.1N for 15 hrs)	0.99946	0.99887	80
2	Acidic (0.1N for 15 hrs)	0.999934	0.998502	70
3	Oxidation (3% Hydrogen Peroxide)	0.999657	0.995222	90
4	UV	0.99924	0.99742	90
5	Cool white fluorescent light	0.99956	0.99684	100
6	Thermal 50°C (72 hrs)	0.99989	0.99838	100

Validation of the Developed Stability Indicating Method

Linearity:

The data obtained in the linearity experiment was subjected to linear-regression analysis. A linear relationship between peak areas and concentrations was obtained in the range of 500- 2500 ng/ band. The correlation coefficient was 0.9988, with mean slope of 2.3971 and a mean -intercept of 596.39 with r² = 0.9988 .

Limit of detection:

The limit of detection as calculated by standard formula as given in ICH guidelines was found to be 8.04 ng/ band

Limit of Quantitation:

The limit of quantitation as calculated by standard formula as given in ICH guidelines was found to be 24.36 ng/band

Precision:

Table-2. The precision table

Theoretical

Observed

(mean ng/band± SD)

Precision (% RSD)

Intra-day

1000ng/band

1500ng/band

2000ng/band

Average

1003.186± 4.578

1510.68 ± 7.20599

2060.67±13.74094

0.456

0.477

0.668

0.534 ± 0.116

Inter-day

1000ng/band

1500ng/band

2000ng/band

Average

1005.466±5.6974

1507.659±4.5697

2044.3±14.06541

0.565

0.303

0.688

0.518 ± 0.196

% R.S.D. = SD/mean x 100, accuracy = observed/theoretical x 100

The developed method was found to be precise as the % RSD value for repeatability studies was less than 2%, where as the %RSD for inter-day precision was 0.518 ± 0.196 (table-2) .

Accuracy:

Excellent recoveries were obtained at each level of added concentration. The result obtained (n = 3 for each level) indicated the mean recovery between 99% to 100% for RPG (table-3) (fig.4).

Table-3. The accuracy table

Concentration (Theoretical) ng	Concentration (practical)	% Recovery
1000+800	1782	99.033
1000+1000	1987.3	99.36
1000+1200	2208.1	100.34

Figure4: Densitogram of Repaglinide accuracy study

Specificity:

The specificity of the method was ascertained by peak purity profiling studies. The peak purity values were found to be > 0.995, indicating no interference in the detection of Repaglinide.

DISCUSSION:

The optimized HPTLC method for determination of Repaglinide involves only a binary mixture of constituent solvents, thus very simple to prepare leading to reproducible results. The drug was found to be sensitive to alkaline, acidic, oxidative and UV degradation. Though separate peak for the products of degradation were not obtained, the peak purity of the drug peak was confirmed by peak purity studies using WinCats software. This method may be used for monitoring the stability and purity of Repaglinide.

ACKNOWLEDGEMENT:

The authors are thankful to USV Ltd., Daman for providing a working standard of Repaglinide. The authors are also thankful to the Management, AISSMS College of pharmacy for providing necessary facilities and constant encouragement.

REFERENCES:

1) The United States Pharmacopoeia NF, United States Pharmacopoeia Commission, CD ROM version, 25 (30) :2007: 3110

2) Cutler SJ. and Cocolas GH.: Cardiovascular Agents. In Wilson and Gisvold’s Textbook of Organic medicinal and Pharmaceutical Chemistry Edited by Block HJ, Beale Jr. JM. Lippincot Williams and Wilkins , Philadelphia, 10th ed;1998: 671.

3) Tripathi KD: Essentials of Medial Pharmacology, Jaypee, New Delhi. 6th ed;2009:269

4) Hong DD, Shah M, Cartensen JT. and Rhodes CT.: Drug Stability Principles and Practices, Marcel and Dekker Inc. New York. 3rd ed;2005:20, 146-148

5) Patel DR, Patel LJ andPatel MM, Development and Validation of stability indicating method for the determination of Repaglinide in Pharmaceutical dosage form using High Performance Liquid Chromatography. International Journal of ChemTech Research . 3 (2) ;2011 : 539-546.

6) Sharma MC and Sharma S, Stability Indicating RP-HPLC Method for Determination and Validation of Repaglinide in Pharmaceutical Dosage Form. International Journal of ChemTech Research . 3(1) ;2011: 210-216.

7) Rane VP, Shinde DB. A Validated Chiral LC for the Enantiomeric Separation of Repaglinide on Amylose based Stationary Phase. Chromatographia.66 (7-8); 2007:583-587.

8) Gandhimati M, Ravi TK and Susan KR. Determination of Repaglinide in Pharmaceutical Formulations by HPLC with UV Detection. Analytical Sciences .19; 2003: 1675 -1677.

9) Kaushal N, Jain S and Tiwary AK. Development of Spectrofluorimetric and HPLC Methods for In-vitro Analysis of Repaglinide. Indian Journal of Pharmaceutical Sciences. 72(2) ;2010: 240-244.

10) ICH Q1A (R2): Stability Testing of new Drug Substances And Products, International Conference on Harmonization, Geneva 2003

11) ICH Q2 (B): Validation of Analytical Procedures: Text and Methodology. International Conference on Harmonization,

Geneva 2005

Received on 06.12.2012 Modified on 18.12.2012

Research J. Pharm. and Tech. 6(2): Feb. 2013; Page 158-161