Development and Validation of a Reverse Phase Liquid Chromatographic Method for Quantitative Estimation of Telmisartan in Human Plasma V. Kabra1, V. Agrahari1, P. Trivedi2 1 2
Department of Pharmacy, Shri G. S. Institute of Technology & Science (SGSITS), Indore, India School of Pharmaceutical Sciences, Rajiv Gandhi Technical University (RGTU), Bhopal, India
Abstract — A sensitive, rapid, reverse phase and isocratic high-performance liquid chromatography (HPLC) method has been developed for the estimation of telmisartan in human plasma. Sample detection was carried out at 297 nm using an Ultraviolet (UV)-PDA detector. Plasma sample pretreatment consist of protein precipitation extraction with methanol. The compounds were separated using a mobile phase of sodium dihydrogen phosphate buffer (pH 3.0): acetonitrile (ACN) (42:58, v/v) on a Phenomenex LUNA C18, column (250×4.6 mm i.d., 5m) at a flow rate of 1.2 ml/min. The total run time for the assay was 4.2 min. The method was validated over the range of 40-1600 ng/ml. The lowest limit of quantification and limit of detection was found 40 and 2.8 ng/ml respectively. The recovery for low, medium and high quality control samples were found 81.13, 79.96 & 82.40 % respectively. The method was found to be accurate and precise. Stability data of analytes revealed that they are stable in plasma under different stability conditions. The results observed conclude that the developed method is very appropriate and applicable for antihypertensive research programs of telmisartan.
drawbacks such as use of extensive sample preparation methods, excess quantity of sample requirement, long run time and higher cost of sample analysis due to the use of sensitive detectors which is a tedious and costly job for lab scale analysis of telmisartan. In this paper, we describe a sensitive, isocratic & simple HPLC–UV method for the determination of telmisartan in human plasma which includes short run time & small plasma sample volume. The method was validated for different parameters. On the basis of the results obtained it can be said that the method described here would be suitable for determination of pharmacokinetic profiles, drug monitoring and in bioequivalence studies of telmisartan.
Keywords — High performance liquid chromatography, telmisartan, plasma, reverse phase, stability
Fig. 1 The chemical structure of telmisartan (TLMS)
I. INTRODUCTION
II. EXPERIMENTAL
Bioanalytical method is the qualitative and quantitative analysis of drug substances in biological fluids or tissues. It plays a significant role in the evaluation & interpretation of bioavailability, bioequivalence and pharmacokinetic data. The sensitivity and selectivity of the bioanalytical methods are essential to the success of preclinical and clinical studies during all phases of the drug development process, i.e. from the initial preclinical phase to the final clinical phases. Telmisartan, [1,1-biphenyl]-2-carboxylic acid, 4-[(1, 4 -dimethyl-2-propyl[2,6-bi-1H-benzimidazol]-1-yl)methyl] (Fig. 1) is an angiotensin II receptor antagonist that lowers blood pressure through blockade of the rennin-angiotensinaldosterone system and shows non-competitive inhibition, used in the treatment of hypertension [1]. A few analytical methods for the determination of telmisartan have reported [2-11]. However no methods have been reported for the determination of telmisartan in plasma by HPLC with ultraviolet detection. All these reported methods have some
A. Reagents and Chemicals Telmisartan was kind gift sample from Dr. Reddy’s laboratories ltd., Hyderabad, India. Methanol and Acetonitrile (ACN) were of HPLC grade and purchased from Merck ltd., New Delhi, India. Human plasma was kindly obtained from blood bank department, Bhopal Memorial Hospital & Research Centre (BMHRC), Bhopal, India. All other chemicals were of analytical grade and used without further purification. B. Instrumentation The HPLC system (Shimadzu Corporation, Japan) consisted of a LC-10AT VP pump, SPD-10AT VP photo diode array (PDA) detector. A manual injector attached with a 20 μl sample loop was used for sample loading. Class LC10/M10A work station was used for data collection
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and acquisition. The analytical column was Phenomenex Luna C18 (250mm X 4.6mm, 5m), protected by Phenomenex HPLC guard cartridge system.
limit quality control (LLOQ, 40 ng/ml) samples were selected to perform various validation parameters.
C. Chromatographic conditions
F. Limit of detection (LOD) and lowest limit of quantification (LLOQ)
The chromatographic separations were performed at ambient temperature with an isocratic elution. The mobile phase composed of sodium dihydrogen phosphate buffer (pH 3.0, adjusted with orthophosphoric acid): ACN (42:58 v/v) and was set at a flow rate of 1.2 ml/min. The chromatogram was monitored with UV detection at a wavelength of 297 nm. The mobile phase was prepared daily, filtered & sonicated before use.
LOD was defined as the concentration that yields a signal-to- noise ratio of 3. LLOQ was the smallest analytical concentration which could be measured with defined accuracy and precision under the given experimental conditions. LLOQ was calculated to be the lowest analyte concentration that could be measured with a signal-to-noise ratio of 10. The LLOQ & LOD was found to be 40 ng/ml & 2.8 ng/ml respectively.
D. Sample extraction procedure
G. Linearity
Protein precipitation technique was used for extraction of telmisartan from plasma samples by using methanol as a precipitating agent. The satisfactory values for recovery for each plasma sample with single extraction were observed when 1:4 ratios of plasma & methanol were mixed thoroughly and vortexes at room temperature. It was then centrifuged at 10,000 rpm for 10 min at 4 0C. The decanted clear supernatant liquid was filtered through 0.45 syringe filter and injected into HPLC system by using a small sample volume (20μl).
Calibration plots for the analytes in plasma were prepared by spiking drug-free plasma with standard stock solutions to yield concentrations of 40–1600 ng/ml (40, 80, 160, 320, 640, 960, 1280 and 1600 ng/ml) for telmisartan. Five replicate injections (n =5) of each concentration were performed. The linear regression data analysis was used for the calculation of intercept, slope and correlation coefficient (r2) which were then used to calculate the analytes concentration in each sample. H. Selectivity
E. Preparation of standard stock solution and quality control samples (QC) Standard stock solution of telmisartan (100μg/ml) was prepared in methanol and stored at 4°C. Serial dilution of the standard stock solution with methanol, lead to solution of concentration of 40-1600 ng/ml. For the preparation of calibration samples, 0.8 ml was taken from the standard stock solution and diluted up to 10 ml with blank plasma to give standard spiked plasma stock solution of 8 μg/ml. From the standard spiked plasma stock solution different concentrations were prepared by taking 0.05, 0.1, 0.2, 0.4, 0.8, 1.2, 1.6 & 2.0 ml and the volume of each was made up to 2 ml with blank plasma. 8 ml of precipitating agent (methanol) was added in all to obtain the final concentration of 40, 80, 160, 320, 640, 960, 1280 and 1600 ng/ml of telmisartan in plasma respectively. After that these different concentrations were followed to sample extraction procedure as described above, and the supernatant was filtered, directly injected into HPLC column for each concentrations. QC samples at different levels of high quality control (HQC, 1600 ng/ml), middle (MQC, 640 ng/ml), low quality control (LQC, 80 ng/ml) and lowest
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The ability of an analytical method to differentiate and quantify the analyte in the presence of other components in the sample is termed as selectivity. For selectivity, blank sample of the plasma was prepared from six different sources. I. Accuracy The accuracy of an analytical method describes the closeness of mean test results obtained by the method to the true value of the analyte. The mean value should be within ± 15% of the actual value except for LLOQ, where it should not deviated by more than ± 20%. It is determined by calculating the percent difference (% bias) between the mean concentrations and the corresponding nominal concentrations. The accuracy samples were analyzed for five replicates (n =5) of LLOQ, LQC, MQC and HQC. J. Precision The precision of an analytical method describes the closeness of individual measure of an analyte. Precision of the assay in plasma was determined by assaying all quality
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Development and Validation of a Reverse Phase Liquid Chromatographic Method for Quantitative Estimation of Telmisartan …
control samples including LLOQ in five replicate (n =5). To determine different precision parameters sample was analyzed within the same day (repeatability of precision), on other day (inter day precision) & by other analyst (analyst to analyst precision). Precision was reported in terms of percent relative standard deviation (% RSD). K. Recovery Recovery analysis should be performed by comparing the analytical results of extracted samples with unextracted samples for HQC, MQC & LQC samples which representing 100% recovery. The percentage of the drug recovered from the plasma samples was determined by comparing the peak height ratio after extraction with those of unextracted methanolic solution containing same concentration of telmisartan as in plasma. L. Stability Stability analysis should evaluate the stability of the analytes during sample collection and handling after longterm and short-term (bench top) storage at room temperature and after going through freeze and thaw cycles. The different stability conditions are described are Stock solution stability (SSS), Short term (bench top) storage stability (BTS), Post-Preparative Stability (PPS), Freeze– thaw stability (FTS), Long term storage stability (LTS). III. RESULTS AND DISCUSSION A. Selection of HPLC chromatographic conditions Various combinations of different solvents were tried & investigated to optimize the mobile phase composition to performance and peak shape. The separation of telmisartan and plasma were affected by the composition and pH of the obtain good results for the separation, sensitivity, column mobile phase. Peak shape and run time were improved by adjusting the pH of water to 3.0 with orthophosphoric acid (OPA). Under the chromatographic conditions described above, blank plasma sample did not give any peak at the
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retention time of telmisartan (Fig. 2) i.e. the peaks were well separated. The retention times of plasma and of telmisartan were 1.78 ± 0.3 min and 3.5 ± 0.3 min, respectively (Fig. 3). B. Validation of the optimized method 1. Selectivity No interfering endogenous compound peak was observed at the retention time of blank plasma and telmisartan. Thus the method was selective for analysis of telmisartan in plasma. 2. Linearity Linearity was determined over the concentration range of 40–1600 ng/ml, which display good linear response to the method. The calibration curves were fitted by linear leastsquare regression and the correlation coefficient was found to be 0.9998. 3. Recovery of the method Recovery for telmisartan was evaluated. The overall mean value of recovery was found to be 81.16%. The recovery of the extraction procedure at HQC, MQC and LQC samples of telmisartan were found to be 82.4 %, 79.96 % and 81.13 % respectively (Table 1). 4. Assessment of stability Telmisartan was found stable under different storage and processing conditions. The stability data of telmisartan under various storage conditions was shown in Table 2. 5. Accuracy and precision measurement The accuracy (% N conc.) for telmisartan was found to be 100.93 101.30, 103.99 & 108.05 for HQC, MQC, LQC & LLOQ samples respectively (Table 1). The precision data for HQC, MQC, LQC & LLOQ samples were analyzed in replicates of five (n = 5) and were shown in Table 1. For intra-day it was found 1.31 %, 1.53 %, 4.68 % & 7.40 %, for inter-day 1.50 %, 4.82 %, 3.20 % & 5.71 % and for analyst to analyst precision it was found 2.15 %, 1.89 %, 4.67 % & 5.53 % for HQC, MQC, LQC & LLOQ samples respectively. The results were within the acceptance criteria for precision and accuracy, i.e., deviation values were within ± 15% of the authentic values, except for LLOQ, which could show a ± 20% deviation.
Table 1 Accuracy, precision & recovery of the developed method QC samples r LLOQb LQCc MQCd HQCe a
Accuracy
Precision (% RSD)
Mean Recovery (%)
% N.Conc.a
% Bias
Intra day
Inter day
Analyst-to-analyst
108.05 103.99 101.30 100.93
8.05 3.99 1.30 0.93
7.40 4.68 1.53 1.31
5.71 3.20 4.82 1.50
5.53 4.67 1.89 2.15
% Nominal concentration;
b
lowest limit of quantitation;
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c
low quality control;
d
medium quality control;
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high quality control.
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Table 2 Stability of the developed method at various conditions Stability Conditions
Telmisartan LQC % N. Conc.
% Change
HQC % N. Conc.
% Change
SSSa 093.60 05.28 103.20 3.08 BTSb 102.40 01.81 094.63 8.57 PPSc 098.42 04.73 092.65 6.10 FTSd 103.30 02.70 107.96 4.70 LTSe 110.57 -5.60 112.40 2.96 a stock solution stability; b bench top stability; c post processing stability; d freeze & thaw stability; e long term stability.
ACKNOWLEDGEMENT The authors are grateful to Prof. P. B. Sharma (Ex-Vice Chancellor, RGTU, Bhopal, India) for providing generous access to laboratory facility to perform this study, and to the blood bank department, Bhopal Memorial Hospital & Research Centre, Bhopal, India for providing plasma samples. This work was also supported by a grant from the Ministry for Human Resource Development (MHRD), New Delhi, India.
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Fig. 2 The representative chromatogram of blank plasma
Fig. 3 The extracted chromatogram of telmisartan from plasma IV. CONCLUSION A rapid, sensitive, selective and validated assay for the quantitative determination of telmisartan in human plasma was described. The result observed from this study concludes that the developed method is very appropriate for quantitative analysis and applicable for toxicokinetic, pharmacokinetic, bioavailability, bioequivalence, therapeutic drug monitoring (TDM) and routine plasma monitoring studies of telmisartan in hypertensive patients.
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Wienen, W, Entzeroth M, Stangier, J et al. (2000) A Review on Telmisartan: A Novel, Long-Acting Angiotensin II-Receptor Antagonist. Cardiovasc Drug Rev 18:127-154 2. Hillaert S, Bossche W V D (2002) Optimization and validation of a capillary zone electrophoretic method for the analysis of several angiotensin-II-receptor antagonists. J Chromatogr A 979:323-333 3. Su C A, Stangier J, Fraunhofer A et al. (2000) Pharmacokinetics of acetaminophen and ibuprofen when coadministered with telmisartan in healthy volunteers. J Clin Pharmacol 40:1338-1346 4. Stangier J, Schmid J, T¨urck D et al. (2000) Absorption, metabolism, and excretion of intravenously and orally administered telmisartan in healthy volunteers. J Clin Pharmacol 40:1312-1322 5. Stangier J, Su C A, Van H et al. (2001) Inhibitory Effect of Telmisartan on the Blood Pressure Response to Angiotensin II Challenge. J Cardiovasc Pharmacol 38:672-685 6. Torrealday N, Gonzalez L, Alonso R M et al. (2003) Experimental design approach for the optimisation of a HPLC-fluorimetric method for the quantitation of the angiotensin II receptor antagonist telmisartan in urine. J Pharm Biomed Anal 32:847-857 7. Xu M T, Song J, Li N (2003) Rapid determination of telmisartan in pharmaceuticals and serum by the parallel catalytic hydrogen wave method. Anal Bioanal Chem 377:1184-2650 8. Xu M, Song J, Liang Y (2004) Rapid determination of telmisartan in pharmaceutical preparations and serum by linear sweep polarography. J Pharm Biomed Anal 34:681-687 9. Chen B M, Liang Y Z, Wang Y L et al. (2005) Development and validation of liquid chromatography–mass spectrometry method for the determination of telmisartan in human plasma. Anal Chim Acta 540:367-373 10. Li P, Wang Y W, Wang Y et al. (2005) Determination of telmisartan in human plasma by liquid chromatography–tandem mass spectrometry. J Chromatogr B 828:126-129 11. Hempen C, Schwarz L G, Kunz U et al. (2006) Determination of telmisartan in human blood plasma: Part II: Liquid chromatographytandem mass spectrometry method development, comparison to immunoassay and pharmacokinetic study. Anal Chim Acta 560:41-49
Author: Ms. Vibhuti Kabra Institute: Department of Pharmacy, Shri G. S. Institute of Technology & Science (SGSITS) Street: 23-Park Road City: Indore Country: India Email:
[email protected]
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