Validated HPTLC Method for Determination of Repaglinide in Bulk and Tablet Dosage Form

Seema M. Dhole¹*, Pramod B. Khedekar², Nikhil D. Amnerkar³

¹Department of Pharmaceutical Chemistry, J. L. Chaturvedi College of Pharmacy, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur-440016, Maharashtra, India.

²University Department of Pharmaceutical Sciences, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur-440033, Maharashtra, India.

³Department of Pharmaceutical Chemistry, Sharad Pawar College of Pharmacy, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur-441110, Maharashtra, India.

*Corresponding Author E-mail: seemadhole@gmail.com

ABSTRACT:

A simple, rapid, accurate and precise high performance thin layer chromatographic (HPTLC) method has been developed and validated for the estimation of the repaglinide as bulk and in tablet dosage form. The method employed TLC precoated silica gel 60 F₂₅₄ aluminium plates as the stationary phase. The solvent system consisted of methanol: chloroform: toluene: triethylamine (5:3:2:0.1 v/v/v/v). The detection of spots was carried out densitometrically using a UV detector at 241 nm in absorbance mode. This system was found to give compact spot for repaglinide with (Rf value of 0.45 ± 0.08). The method was validated in terms of linearity, accuracy, precision, limit of detection, limit of quantification and specificity as per ICH guidelines. The calibration curve was found to be linear over the concentration range of 100-500 ng/spot for repaglinide with significantly high value of correlation coefficient (r² > 0.99). The limits of detection and quantitation were found to be 20 ng/spot and 90 ng/spot, respectively for repaglinide. The % assay was found to be 99.14 ± 0.34(SD). Accuracy of the method was accessed by percentage recovery and mean % value was found to be 99.51±0.74. The proposed method was found to be accurate, precise, reproducible and specific and can be successfully applicable for the quality control analysis of repaglinide as in bulk drug and pharmaceutical formulations.

KEYWORDS: Repaglinide, High-performance thin layer chromatography (HPTLC), Method Validation.

Today TLC is rapidly becoming a routine analytical technique due to its advantages of low operating costs, high sample throughput and the need for minimum sample preparation. The major advantage of TLC is that several samples can be run simultaneously using a small quantity of mobile phase unlike HPLC thus reducing the analysis time and cost per analysis.

MATERIAL AND METHODS:

Chemicals and Reagents

Pure drug sample of repaglinide was kindly gifted from USV Lab. Pvt. Ltd., Mumbai, India. Eurepa tablets labeled to contain repaglinide 2 mg, manufactured by M/S Torrent Pharmaceutical Ltd., Baddi (H.P.), India, were obtained from commercial sources within their shelf life period. All the reagents and solvents used were of analytical reagent (AR) grade, obtained from Merck Chemicals, Mumbai.

Instrumentation and Chromatographic Conditions

The samples were spotted in the form of bands of width of 6mm with a Camag 100 microlitre sample syringe (Hamilton, Bonaduz, Switzerland) on precoated silica gel aluminum plate 60 F₂₅₄ (20 cm×10cm with 250 μm thickness; E. Merck, Darmstadt, Germany) using a Camag Linomat V sample applicator (Switzerland). The plates were prewashed with methanol and activated at 110^oC for 5 min prior to chromatography. A constant application rate of 0.1µl/s was used was employed, and the space between two bands was 8 mm. The slit dimension was kept 6 mm × 0.45 mm. The mobile phase consisted of methanol: chloroform: toluene: triethylamine (5: 3: 2: 0.1, v/v/v/v) was selected, which gave sharp and symmetrical peak with Rf value 0.45. Linear ascending development was carried out in a 20 cm X 10 cm twin rough glass chamber (Camag, Muttenz, Switzerland) saturated with the mobile phase. The optimized chamber saturation time was 30 min at room temperature (25^◦C ± 2^◦C) and relative humidity 60% ± 5%. The length of chromatogram run was approximately 8 cm. Subsequent to the development; TLC plates were dried with the help of an air dryer in a wooden chamber with adequate ventilation. Densitometric scanning was performed using Camag TLC scanner III equipped with win CATS software at 241 nm. The source of radiation utilized was deuterium lamp emitting a continuous UV spectrum in the range of 200–400 nm. Evaluation was performed using peak area with linear regression.

Preparation of standard solution

An accurately weighed quantity of repaglinide (10 mg) was dissolved in methanol and the volume was made up to 10 ml (1000 μg/ml). The aliquot of standard stock solution was diluted with methanol to produce 100 µg/ml of repaglinide.

Optimization of HPTLC Method

Initially, various mobile phases with different ratios of methanol, chloroform and toluene were tried in attempts to obtain resolved and sharp peak of repaglinide. Finally, the mobile phase containing methanol: chloroform: toluene: triethylamine (5: 3: 2: 0.1, v/v/v/v) gives sharp and symmetrical peak with Rf value of 0.45 at 241nm. The densitogram of repaglinide is shown in Figure 2.

Application of proposed method to formulation

Twenty tablets were weighed and average weight was calculated. Tablets were triturated to fine powder. Approximately a portion of tablet powder equivalent to 10 mg of repaglinide was accurately weighed and dissolved in 20 ml methanol in 50 ml volumetric flask. The contents of the flask were sonicated for 10 min to dissolve repaglinide completely. Then volume was made up to mark with methanol and filtered. An aliquot of this solution was diluted with methanol to produce 100 µg/ml final tablet sample solution. From the above sample solution 3 µl was applied on prewash TLC plate which gave final concentration of 300 ng/spot for repaglinide, followed by chromatographic development with selected mobile phase under the optimized chromatographic conditions. Content of repaglinide was calculated by comparing peak areas of sample with that of the standard.

Method Validation

The validation procedure was followed the ICH guidelines and United State Pharmacopoeia for the analysis of repaglinide by HPTLC method. The performance parameters evalutated for the method were linearity, precision, accuracy, limits of detection and quantitation, specificity and ruggedness.

Linearity

For linearity study, aliquots of 1, 2, 3, 4and 5 μl from standard solution of repaglinide were applied on TLC plate to obtain the concentrations of 100, 200, 300, 400 and 500 ng/spot. TLC plates were developed under the above optimized conditions. Area under peak was recorded and plotted against concentration. The standard calibration graph is shown in Figure 3.

Figure 3. Calibration graph of Repaglinide

Accuracy

The accuracy of the method was determined by calculating recoveries of repaglinide by the standard addition method at different levels to the pre-analyzed sample. For that known amounts of standard solutions of repaglinide (80, 100, and 120 % of test concentration) were added to pre-analyzed sample of tablet dosage form and determining their contents.

Precision

The precision of the method was verified by repeatability studies. Repeatability studies were performed by analysis the sample solutions of the drug six times on the same day. Series of diluted standard solutions were prepared and analyzed by proposed method.

Limit of detection and limit of quantitation

The limit of detection (LOD) and limit of quantitaton (LOQ) were separately determined based on standard deviation of the y-intercept and the slope of the calibration curve by using the equations (1) and (2), respectively.

LOD = 3.3 δ ----- (1)

LOQ =10 δ -----(2)

Where, δ: standard of y-intercept and S: slope of calibration curve.

Ruggedness

Ruggedness of the method was performed by spotting 300 ng of drug between different time intervals, days and by three different analysts maintaining same experimental and environmental conditions.

Specificity

The specificity of the method was determined by analyzing standard drug and test samples. The spot for repaglinide in the formulation sample was confirmed by comparing the Rf and spectrum to the spot with that of a standard. The peak purity of repaglinide was determined by comparing at three different regions of the spot i.e. peak start (S), peak apex (M) and peak end (E).

RESULTS AND DISCUSSION:

An attempt has been made to develop a rapid, sensitive, economic, precise and accurate HPTLC method for estimation of repaglinide in tablet dosage form. The TLC procedure was optimized with a view to develop a method for determination of repaglinide. Optimization of mobile phase with methanol: chloroform: toluene: triethylamine (5 : 3 : 2 : 0.1 v/v/v/v) and stationary phase consisting of aluminium plate precoated with silica gel 60 F₂₅₄ (10 cm × 10 cm) gives satisfactorily resolved, sharp and symmetrical peak having Rf value of 0.45 ± 0.08 (Fig. 2). Pre-saturation of TLC chamber with mobile phase for 30 min assured better reproducibility in migration of repaglinide and better resolution.

The method was found to be linear in the range of 100-500 ng/spot and the regression analysis data are presented in Table 1. The regression coefficients (R²) obtained was higher than 0.99 which attest the linearity of the method.

Table 1. Regression analysis data for repaglinide

Parameters	Repaglinide
Linear Range (ng/spot)	100-500
Regression equation	y = 1.733x + 116.1
Correlation Co-efficient (r²)	0.9979

The precision data obtained for the evaluated method are demonstrated in Table 2. Mean contents of repaglinide in repeatability of measurement for precision analysis (n=6) were very close to labeled claim drug. The %RSD value was lower than 2%, assure the precision of the method.

Table 2. Analysis of formulation using the HPTLC method

Drug	Label claim mg/tablet	Parameter (n=6)	Results
Repaglinide	2.0	Mean (%)	99.14
		Amount found (mg)	1.98
		SD^*	0.3432
		% RSD^*	0.3461

SD: Standard deviation; RSD: Relative standard deviation

Accuracy was investigated by means of recovery studies using the proposed method. The percent recoveries after spiking with additional standard drug afford recovery in the range of 98.92-100.34% which demonstrates an adequate accuracy and the results are listed in Table 3.

The results of analysis of pharmaceutical dosage form by the proposed method (Table 2), expressed as percentage of label claim were in good agreement with the label claims thereby suggesting that there is no interference from any of the excipients which are normally present in tablets. Peak purities higher than 99.0% were obtained for repaglinide in the chromatogram of sample solution and show no interference due to excipients in the same retention time of drugs which shows the specificity of method. In case of ruggedness study, the % RSD values for intra-day, inter-day and different analysts were found to be less than 2% showed ruggedness of the proposed method. The results of ruggedness were presented in Table 4. The validation parameters were studied for proposed HPTLC method and summary of validation parameters is shown in Table 5.

Table 4: Results of ruggedness parameters

Drug	Parameters	Intraday	Interday	Different analysts
Repaglinide	(%) Mean	99.13	99.13	99.61
	±SD	0.6352	0.904	0.6009
	% RSD	0.6408	0.9126	0.6033

Table 5. Validation parameters of evaluated method

Parameters	Repaglinide
Linearity range(ng/spot)	100-500
Precision, n=6 (%RSD)	0.3461
Recovery, n=9 (%±SD)	99.51±0.7414
LOD(ng/spot)	20
LOQ(ng/spot)	90
Ruggedness(n=3) (%RSD)
Intraday	0.6408
Interday	0.9126
Analysts	0.6033
Specificity	Specific

CONCLUSION:

The developed and validated HPTLC method was found to simple, precise, specific and accurate. Statistical analysis proves that the method is suitable for the analysis of repaglinide in pharmaceutical formulation without any interference from the excipients. It was concluded that the developed method offered several advantages such as rapid, cost effective, simple mobile phase and sample preparation steps and improved sensitivity made it specific, reliable and easily reproducible in any quality control analysis providing all the parameters are followed accurately for its intended use.

ACKNOWLEDGEMENTS:

Authors are thankful to the Manager, USV Lab. Pvt. Ltd., Mumbai, India for providing the gift samples and also thankful to Dr. K. P. Bhusari, Principal, Sharad Pawar College of Pharmacy, Nagpur for providing experimental facilities for this work.

REFERENCES:

1. Rang HP, Dale MM, Ritter JM, Moore PK. Pharmacology. Edinburgh:Churchill Livingstone. 2003; 5^th ed: pp. 310.

2. Budavari S. The Merck Index, In: An Encyclopedia of Chemicals, Drugs and Biologicals. Whitehouse Station, NJ: Merck and Co Inc. 2001; 13th ed: pp. 790.

3. Reynolds JEF. Martindale, The Complete Drug Reference. Pharmaceutical Press, London. 2002; 33rd ed: p. 334.

4. Goyal A, Singhvi I. Visible spectrophotometric methods for estimation of repaglinide in tablet formulation. Indian Journal Pharmaceutical Sciences. 5(3); 2006: 79-80.

5. Sharma S, Sharma MC. Simultaneous determination of repaglinide in pharmaceutical dosage form using indigo carmine. American-Eurasian Journal of Scientific Research. 6(1); 2011: 47-51.

6. Patel JR, Suhagia BN, Patel BH. Simultaneous specrophotometric estimation of metformin and Repaglinide in a synthetic mixture. Indian Journal Pharmaceutical Sciences. 69(6); 2007: 844-846.

7. Kaushal N, Jain S, Tiwary AK. Development of spectrofluorimetric and HPLC methods for In vitro analysis of repaglinide. Indian Journal Pharmaceutical Sciences. 72; 2010: 240-244.

8. Khalidi B, Shtaiwi M, Khatib H, Mohammad M, Bustanji Y. A Comparative Study of First-Derivative Spectrophotometry and Column High-Performance Liquid Chromatography Applied to the Determination of Repaglinide in Tablets and for Dissolution Testing. Journal of AOAC International. 91(3); 2008: 530-535.

9. Gandhimathi M, Renu TK. Determination of repaglinide in pharmaceutical formulation by HPLC with UV detection. Analytical Sciences. 19(12); 2003: 1675-1677.

10. Prameela RA, Bala SC, Archana N, Siva TP, Aruna B. Determination of repaglinide in pharmaceutical formulation by HPLC. Journal of Applied Sciences Research. 5(10); 2009: 1500-1504.

11. Sharma MC, 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.

12. Ruzilawati AB, Wahab MSA, Imran A, Ismail Z, Gana SH. Method development and validation of repaglinide in human plasma by HPLC and its application in pharmacokinetic studies. Journal of Pharmaceutical and Biomedical Analysis. 43; 2007: 1831-1835.

13. International Conference on Harmonization Q2B, Validation of Analytical Procedures: Methodology, Geneva, 1996.

14. The United States Pharmacopoeia 24 and National Formulary 19, Asian edition. Rockville (MD): United States Pharmacopoeia convection, 2000: pp. 2149-52.

Received on 15.02.2013 Modified on 01.03.2013

Asian J. Research Chem. 6(3): March 2013; Page 268-271

S. No.	Level (%)	Amount of std drug added (µg/ml)	% Recovery*	±SD	CV
1	80	240	98.92	0.6912	0.6987
2	100	300	99.26	0.5320	0.5359
3	120	360	100.34	0.7416	0.7391

INTRODUCTION: