Bioanalytical Method Development and Validation for the Simultaneous Determination of Vildagliptin and Telmisartan in Rabbit Plasma Using RP-HPLC

A simple, reproducible bioanalytical method of liquid chromatography and PDA detector was developed and validated for the simultaneous Determination of Vildagliptin and Telmisartan in Rabbit Plasma using liquid-liquid extraction technique. K2 EDTA was used as anti-coagulant. Analytes were extracted by Methyl-tert-Butyl Ether (MTBE) and subsequent separation on a Kromasil C18 column (5 μ, 100 × 4.6 mm) using Acetonitrile : Methanol 75:25 v/v as mobile phase at a flow rate of 1 mL/min and (40±1) ° C column oven temperature. Analytes were monitored with PDA detector at an isosbestic point of 225 nm for both Vildagliptin and Telmisartan. Retention times of Vildagliptin and Telmisartan were found to be at 2.545 mins and 6.633 mins respectively. Original Research Article The method was validated over a linear (r μg/ml for Vildagliptin and 1.011precisions were found to be less than 15 The developed HPLC-PDA method was fully validated for all the other parameters as per FDA guidelines like selectivity, matrix effect, recovery and stability as well. Due to the high degree of sensitivity, very less time consuming, easy extraction procedure and low requirement of sample volume, the method will be applicable for therapeutic drug monitoring.


INTRODUCTION
Vildagliptin (VIL), S-1-[N-(3 -hydroxyl adamantyl) glycyl] pyrrolidine -2 ( Fig. 1), is an oral hypoglycemic drug of the dipeptidylpeptidase-4 (DPP-4) inhibitor class [1,2]. DPP-4 inhibitors represent a new therapeutic approach to the treatment of type 2 diabetes [3]. Telmisartan (TEL) is 2-6-(1-methylbenzimidazol propylbenzimidazol-1-yl]-methyl]phenyl] benzoic acid (Fig. 2), is a synthetic analogue of angiotensin II receptor blocker, used for the treatment of hypertension. TEL is a poorly water soluble drug which displays a dissolution rate limited absorption pattern in humans and animals. Hence, it can be used as a model drug to assess the influence of various physicochemical, physiological, and dosage form factors on the absorption kinetics and bioavailability of hydrophobic drugs [4 Chemical structures of VIL and TEL were shown in Figs. 1 and 2 respectively.

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The method was validated over a linear (r 2 = 0.9979) concentration range of 24.979 -202.559 µg/ml for Telmisartan. The inter-day and intra precisions were found to be less than 15% and the accuracy was all within ±15% (at LLOQ ±20%).
PDA method was fully validated for all the other parameters as per FDA guidelines like selectivity, matrix effect, recovery and stability as well. Due to the high degree of ty, very less time consuming, easy extraction procedure and low requirement of sample volume, the method will be applicable for therapeutic drug monitoring. liquid chromatography; vildagliptin; telmisartan, rabbit plasma.
hydroxyl -1 -2 -carbonitrile 1), is an oral hypoglycemic drug of the 4) inhibitor class 4 inhibitors represent a new therapeutic approach to the treatment of type 2 -[4-[[4-methylmethylbenzimidazol-2-yl)-2 methyl]phenyl] benzoic 2), is a synthetic analogue of angiotensin II receptor blocker, used for the treatment of hypertension. TEL is a poorly watersoluble drug which displays a dissolution ratettern in humans and animals. Hence, it can be used as a model drug to assess the influence of various physicochemical, physiological, and dosage form factors on the absorption kinetics and bioavailability of hydrophobic drugs [4][5][6][7].
VIL and TEL were shown

Telmisartan
Literature survey reveals that vildagliptin can be estimated by UV spectroscopic method [8], RP HPLC method, which is a time method being the retention time is more than 10 min [9], RP-LC/MS method, requires mass spectroscopy detection [10]. Few other methods for the quantitative estimation of VIL separately and in combination with other drugs were reported [11][12][13][14]. A variety of methods have been developed for determination of TEL individually or with combination with some other antihypertensive agents in biological samples [15][16][17]. This includes, HPLC coupled with mass spectrometric (HPLC-MS) and spectrofluorimetric detection. [18,19] In general, spectrofluorimetric method lack sensitivity and cannot distinguish degradation products from the parent compound. Although HPLC provide excellent sensitivity, they are not available in all laboratories because of their special requirements and economic considerations. Moreover, spectrofluorimetric method utilized either a column switching system or an expensive solid phase extraction cartridges.
With respect to these, all reported methods for the determination of VIL and TEL limitations: time-consuming sample clean laborious extraction steps and long chromatographic elution time. Moreover, to the best of author's knowledge no bioanalytical method was reported for simultaneous estimation of VIL and TEL in rabbit plasma. Thus, the present study has been undertaken to develop and validate a simple, sensitive, accurate, precise and reproducible bioanalytical HPLC PDA method for estimation of the simultaneous estimation of VIL and TEL in rabbit plasma.
The bioanalytical methods used for the quantification of drugs in biological samples play a very important role in the evaluation and interpretation of bioavailability and bioequivalence data. Therefore, complete validation of the analytical method was ; Article no.JPRI.64733 = 0.9979) concentration range of 24.979 -5003.808 day and intra-day % and the accuracy was all within ±15% (at LLOQ ±20%). PDA method was fully validated for all the other parameters as per FDA guidelines like selectivity, matrix effect, recovery and stability as well. Due to the high degree of ty, very less time consuming, easy extraction procedure and low requirement of sample Literature survey reveals that vildagliptin can be estimated by UV spectroscopic method [8], RP-HPLC method, which is a time consuming method being the retention time is more than 10 LC/MS method, requires mass spectroscopy detection [10]. Few other methods for the quantitative estimation of VIL separately and in combination with other drugs were variety of methods have been developed for determination of TEL individually or with combination with some other antihypertensive agents in biological samples 17]. This includes, HPLC coupled with mass MS) and 19] In general, spectrofluorimetric method lack sensitivity and cannot distinguish degradation products from the parent compound. Although HPLC-MS methods provide excellent sensitivity, they are not available in all laboratories because of their quirements and economic considerations. Moreover, spectrofluorimetric method utilized either a column switching system or an expensive solid phase extraction With respect to these, all reported methods for the determination of VIL and TEL have various consuming sample clean-up, laborious extraction steps and long chromatographic elution time. Moreover, to the best of author's knowledge no bioanalytical method was reported for simultaneous estimation t plasma. Thus, the present study has been undertaken to develop and validate a simple, sensitive, accurate, precise and reproducible bioanalytical HPLC-PDA method for estimation of the simultaneous estimation of VIL and TEL in rabbit plasma. tical methods used for the quantification of drugs in biological samples play a very important role in the evaluation and interpretation of bioavailability and bioequivalence data. Therefore, complete validation of the analytical method was performed in accordance with USFDA guidelines [20] to obtain reliable results that could be interpreted satisfactorily.

HPLC-PDA Instrumentation and Chromatographic Conditions
The HPLC system was an C Waters (Waters, Milford, MA, USA) consisting of quaternary gradient system (600 Controller), in line degasser (Waters, model AF), photodiode array detector (Water, 2998 model) and auto sampler (Waters, model 717 plus). Data was processed using Empower Pro software (Waters, Milford, MA, USA). Chromatographic separation assay was performed with a Kromasil C18 column (5µ, 100 × 4.6 mm) maintained at ambient temperature. The mobile phase consists of Acetonitrile: Methanol 75:25 v/v. The mobile phase was pumped at a flow rate of 1.0 mL/min. The detection wavelength was 225 nm. Mobile phase was used as diluent for the preparation of working standards of VIL and TEL. Injection volume was 10μl. The run time was 9 mins and the retention time of VIL and TEL was found to be 2.545 min and 6.633 mins respectively.

Chemicals and Reagents
The reference sample of VIL (99.99%) and TEL (99.98%) was gifted by Cadila Health Care Ltd., Ahmadabad, India. HPLC type II water from Millipore's Milli-Q system was used throughout the analysis. HPLC grade Methanol, Acetonitrile and Methyl-tert-Butyl Ether (MTBE) were purchased from Merck, Mumbai, India. All other chemicals were of analytical grade purchased from SD Fine Chem, Mumbai, India.

Animals and Ethical Committee
This study was conducted on four groups of animals, each group contains six healthy white male albino rabbits which were purchased from animal house of Adita Biosys Private Limited (1868/PO/BT/S/16/CPCSEA), Bangalore, with average body weight of 1250 ± 50 g. Rabbits were chosen due to larger volume of blood was required to perform Pharmacokinetic study and other parameters of study. Animals were kept under prerequisite temperature of (23 ± 2) 0 C with humidity 50-60% in a light-dark cycle of 12 h each. Both control and tested animals were kept in same environment and provided with the standard food. Steel bottom cages were used to keep each rabbit separately with free access of food and water.

Blood Samples Collections
The blood samples were collected from the marginal ear vein of normal healthy rabbits at the time intervals of 0, 1, 2, 4, 6, 8, 10, 12 and 14 hours (H) respectively after dosing.

Preparation of Stock and Working Solutions
The

Sample Preparation
A volume of 200 μl of plasma was transferred into a vial, then 50 μl of VIL (25 µg / ml) and 50 μl of TEL (36 µg / ml) were added and 100 μl of 2% formic acid were added and stirred for 10 minutes. 2.5 ml of methyl tert-butyl ether were added and centrifuged at 2500 RPM for 10 minutes, the organic layer was transferred to a new vial and evaporated to dryness under a light stream of gaseous nitrogen at 45°C. The residue was reconstituted with 500 μl of diluent and 10 μL aliquots were injected into the HPLC system.

Quantification
VIL and TEL were determinate quantitatively using an eight-point calibration curve which was established in plasma using peak area ratio of analytes. The unknown concentration of samples and QC's were calculated by interpolation from the calibration curve.

Method Validation
Method validation protocol was prepared based on the guidelines of the United States Food and Drug Administration (USFDA) [20].

Calibration curve and lower limit of quantitation (LLOQ)
A series of eight calibration curve standards, were prepared to assess the linearity of the method. During the course of validation, each calibration curve was analyzed by using a 1/x 2 weighted least square regression analysis of standard plots associated with an eight-point standard curve. The standard curve was chosen to cover the range of clinically significant concentrations of patients. The curve was found to be validated; at least 6 of 8 calibration standards should be less than 15% of the coefficient of variation (CV). A Correlation of more than 0.99 shall be desirable for all the calibration curves. The lowest concentration on the calibration curve was considered as the Lower limit of quantitation. The error of accuracy and CV should be less than 15% for all calibration standards and less than 20% for LLOQ. The response of LLOQ of the analyte should be at least 5 times higher than the response of in blank.

Precision and accuracy
To measure reproducibility precision and repeatability are significant factors. Precision and accuracy of the method were developed by analyzing six replicate samples of each LLOQ, low, mid (1 &2) and high quality control samples.
To determine intraday precision and accuracy, the plasma samples were analyzed on the same day. The interday precision and accuracy were assessed by performing precision and accuracy batches on different days. Accuracy was measured as the percentage difference between the theoretical and the measured value according to the equation:

Accuracy (%) = ((Measured Concentration -Theoretical Concentation) / (Theoretical Concentation )) X100%
The % deviation of each concentration level from the nominal concentration in the accuracy and precision must be less than 15% for all except for LLOQ, for which it should not be more than 20%.

Recovery from plasma
The extraction efficiency (recovery) of VIL and TEL were measured as the ratio between the mean analyte concentrations in plasma following LLE of QC samples of High quality control

Optimization of Sample Preparation and Chromatographic Conditions
It is very essentials to adjust or tune the UV and chromatographic conditions as well, for the optimum detection and quantification of the analytes (VIL and TEL) in rabbit plasma VIL and TEL both the analytes showed good response and prominent peaks. Sample preparation is one of the key fundamental steps in the development of the bioanalytical method. Sample preparation must be fast, simple and easy to proceed and get the maximum recovery of analytes with a minimum amount of reagents and solvents. Literature review revealed the use of solid phase extraction (SPE) technique for the extraction of VIL. However, SPE is time consuming and expensive technique when compared to LLE method. Hence we used the LLE method for the sample preparation to cut the cost and shorten the processing time and acquire desired recoveries of the analyte. Some methods were reported to use higher plasma volumes for sample preparation and injection volume for the chromatographic development. Whereas the current method was developed with less plasma volume and injection volume, leads to better acceptability of the method. In order to get maximum recovery, a wide variety of extraction solvents and buffers were used to extract analyte from human plasma such as Diethyl Ether, Ethyl acetate, Methyl-tert-Butyl Ether: n-Hexane (80:20), Methyl-tert-Butyl Ether, etc. better recovery and response were obtained with Methyl-tert-Butyl Ether. There was no interference from any exogenous and endogenous plasma matrix.
In order to achieve good sensitivity, peak shape, and symmetry as well as short chromatographic run time for both analyte all chromatographic conditions were adjusted and optimized. In this study thermo, Gemini, symmetry, waters and luna columns with various mobile phases such as acetonitrile, methanol, formic acid, ammonium formate, ammonium acetate, phosphate buffers, and aqueous ammonia, etc were tired. Finally, Kromasil C18 column (5µ, 100 × 4.6 mm) using Acetonitrile : Methanol (75:25 v/v) as mobile phase at a flow rate of 1 mL/min and 40 ± 1 o C column oven temperature were selected because of better separation and detection. Due to low injection volume of 10 µL reduced overloading of the column with analytes, thereby ensuring more number of analyses on the same column. Finally, VIL and TEL were eluted at 2.545 min and 6.633 mins respectively. Figs. 3-5 represents the chromatograms of HQC (Fig. 3), MQC (Fig. 4) and LQC (Fig. 5).

Selectivity
There was no interference peaks observed due to endogenous or exogenous components at a retention time of the sample of VIL and TEL, extracted from rabbit plasma as represented in Figs. 3-5. The response of drug in blank plasma was less 2%. Typical retention times of VLD and TEL was 2.545 min and 6.633 mins respectively. µg / ml. The coefficient of correlation was found to be better than 0.99 for all the six calibration curves analyzed. The present bioanalytical method provided a lower limit of quantitation and a good range of linearity were shown in Table 1 exhibits the mean concentrations obtained for the calibration curve.

Precision and Accuracy
The intraday accuracy of the method was between 88.41 to 97.90 % with a precision of 0.41 -12.23 % for VIL. The inter-day accuracy was between 93.01 -107.55 % with a %CV of 2.87 -6.63% for TEL. The data indicate that the method possessed adequate repeatability and reproducibility. Table 2 shows the data on precision and accuracy.

Recovery
The percentage recovery was determined by comparing the average area of the peak in samples taken with fresh non-extracted samples prepared in three concentrations. The % of average recoveries was determined by measuring the concentrations of the plasma quality control samples extracted in HQC, MQC1, MQC2 and LQC compared to the quality control samples extracted in HQC, MQC1, MQC2 and LQC. Results of recovery of VIL and TEL were shown in Tables 3 and 4 respectively. It has been documented that recovery% must be 80% in analytical methods. While the development of the bioanalytical method, the purpose of recovery, is not considered a problem as long as the method produces sensitivity, Accuracy and precision.

Application to a Pharmacokinetic Study
Overlay graphs of mean concentration versus time of the two drugs are shown in Fig. 6. The area under the curve from 0 to 12 h was determined with the help of the linear trapezoidal rule. The extrapolation to infinity that is necessary for AUC 0-∞ was calculated using a linear regression model from the last three data points in the elimination phase that has been logtransformed. Maximum concentration achieved (C max ) was obtained directly from measured concentration without interpolation. The parametric point estimates for the mean of test medication were found within the commonly accepted bioequivalence range of 0.8-1.25. Therefore, the results indicate that the proposed method is suitable for pharmacokinetic studies to determine the concentration of Vildagliptin and Telmisartan in rabbit plasma. The study was conducted strictly in accordance with guidelines laid down by the International Conference on Harmonization (ICH) and USFDA. The pharmacokinetic data are tabulated in Table  5.

CONCLUSION
The proposed HPLC-PDA method is rapid, sensitive and reproducible for the quantification of VIL and TEL in rabbit plasma with a wide linear dynamic range. It was validated and met all the requirements according to the USFDA standards guidelines with a high degree of accuracy and precision. Absence of matrix effects was adequately demonstrated. In addition, the stability study indicated that analytes were stable in plasma during the sample preparation process and other storage conditions. The lower LLOQ, smaller plasma volume and shorter run time make our new method particularly suitable and applicable to characterize the clinical pharmacokinetics and bioequivalence assay studies of VIL and TEL in rabbit plasma.

CONSENT
It is not applicable.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.