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Development and validation of the liquid chromatography-tandem mass spectrometry method for quantitative estimation of candesartan from human plasma

Development and validation of the liquid chromatography-tandem mass spectrometry method for... Original Article Development and validation of the liquid chromatography-tandem mass spectrometry method for quantitative estimation of candesartan from human plasma Introduction: A simple and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for estimation of candesartan in human plasma using the protein precipitation technique. Materials and Methods: The chromatographic separation was performed on reverse phase using a Betasil C (100 x 2.1 mm) 5-µm column, mobile phase of methanol:ammonium tri-floro acetate buffer with formic acid (60:40 v/v) and flow rate of 0.45 ml/min. The protonated analyte was quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. The mass transitions m/z 441.2 → 263.2 and 260.2 → 116.1 were used to measure candesartan by using propranolol as an internal standard. Results: The linearity of the developed method was achieved in the range of 1.2–1030 ng/ml (r ≥ 0.9996) for candesartan. Conclusion: The developed method is simple, rapid, accurate, cost-effective and specific; hence, it can be applied for routine analysis in pharmaceutical industries. Key words: Candesaminan, LC-MS/MS method, propranolol Shailesh T. Prajapati, Pratik K. Patel, Marmik INTRODUCTION Patel , Vijendra B. Chauhan, Chhaganbhai Candesartan is used in the management of hypertension, and has been [1,2] N. Patel investigated in heart failure. Candesartan is an anti-hypertensive drug from a category of angiotensin-II receptor antagonists. Angiotensin II is formed from Department of Quality Assurance, Shri Sarvajanik Pharmacy angiotensin I in a reaction catalyzed by angiotensin-converting enzyme (ACE, College, Mehsana, Zydus Cadila kinase II). Angiotensin II is the principal pressor agent of the renin–angiotensin Healthcare Limited, Ahmedabad, Gujarat, India system, with effects that include vasoconstriction, stimulation of synthesis and release of aldosterone, cardiac stimulation and renal reabsorption of sodium. Address for correspondence: Candesartan blocks the vasoconstrictor and aldosterone-secreting effects of Dr. Shailesh T. Prajapati, angiotensin II by selectively blocking the binding of angiotensin II to the AT1 Department Quality Assurance, Shri Sarvajanik Pharmacy receptor in many tissues, such as vascular smooth muscle and adrenal gland. Its College, Mehsana - 384 001, action is, therefore, independent of the pathways for angiotensin II synthesis. Gujarat, India. Email: stprajapati@gmail.com The chemical name of candesartan is {(±)-1-Hydroxyethyl 2-ethoxy-1-[ p-(o-1H- [2] tetrazol-5-ylphenyl) benzyl]-7-benzimidazole carboxylate}. The structure of Access this article online candesartan is shown in Figure 1. On a detailed literature survey, it was found Website: www.phmethods.org that there was only one liquid chromatography-tandem mass spectrometry DOI: 10.4103/2229-4708.84460 (LC-MS/MS) gradient method reported for the estimation of candesartan from [3] Quick response code human plasma, and one method was reported to estimate candesartan in rat [4] plasma by LC-MS/MS. Some methods were reported to estimate candesartan from human plasma and solid dosage forms by the high-performance liquid [5-9] [10] chromatography (HPLC) and UV spectrophotometric methods, which were found to be time-consuming and costly. Hence, the objective of the present work was to develop a simple bioanalytical method to estimate candasartan from human plasma with due consideration of accuracy, sensitivity, rapidity, economy, Pharmaceutical Methods | April-June 2011 | Vol 2 | Issue 2 Abstract Prajapati, et al.: Quantitative estimation of candesartan from human plasma by LC-MS/MS dissolved in 5 ml of methanol and the volume up made up to the mark with methanol to prepare a 1 mg/ml solution. The final concentration of 0.1 mg/ml (100000 ng/ml) was carried out by dilution of 1 ml of the above 1 mg/ml solution up to 10 ml with methanol. The working solutions of candesartan were prepared using the diluent. The final concentration was made up to 58.270, 116.939, 708.723, 2531.155, 10124.620, Figure 1: Structure of candesartan 25960.563, 37086.519, 46353.149 and 51509.054 ng/ ml. Similarly, the lower quality control (LQC) selectivity and stability indicating according to the concentration (162.254 ng/ml), middle quality control US-FDA guidelines. (MQC) concentration (16225.352 ng/ml), higher quality control (HQC) concentration (36056.338 ng/ MATERIALS AND METHODS ml) and lower limit of quantification (LLOQ; 64.902 ng/ml) samples were prepared. Required numbers of samples of concentration of candesartan ranging Chemical and reagents from 1 to 1000 ng /ml were prepared by making up the The working standard or Candesartan and Propranolol volume with drug-free plasma and labelling them as as internal standard were gifted by Zydus Cadila STD-1 to STD-9, which are 1.169, 2.339, 14.174, 50.623, Healthcare Limited, Ahmedabad, India. Human 202.492, 519.211, 741.730, 927.163 and 1030.181 ng/ml, plasma samples were procured from Prathama Blood respectively. Bank, Ahmedabad, India. Methanol (HPLC grade) and ammonium trifloroacetate (GR grade) were purchased from Spectrochem, Hyderabad, India. Formic acid Sample preparation supra pure grade was purchased from Merck, Mumbai 0.10 ml of sample into was accurately pipetted into (India) and Milli-Q water was procured from Zydus prelabeled vials and 500 µl of propranolol (internal Cadila Healthcare Limited. standard) was added and mixed for 2 min (for blank sample, 500 µl of methanol solution was added instead of the internal standard solution). Methanol Instrumentation in propranolol solution was used for protein An HPLC (Shimadzu Corporation, Kyoto, Japan) precipitation. Samples were centrifuged at 4800 rpm coupled to an API 4000 mass spectrometer (Thermo at less than 10°C for 15 min. Then, 0.4 ml supernatent Finnigan Ltd., Stafford Ho, UK) was employed for the was transferred into the prelabeled vial containing 0.4 analysis. A pH meter (Thermo Orion, Asheville, NC , ml diluent and mixed properly. 0.5 µl of this mixture USA , Model 420) and sonicator (Oscar Ultra Sonics, was then injected into an HPLC system using an auto Andheri (E), Mumbai , India OU-72 SPL) were used sampler. for this work. The chromatographic conditions were as follows: The concentration of candesartan and propranolol was calculated from the area ratio v/s spiked plasma Column: Betasil C (100 x 2.1 mm), 5 µm; injection concentration regression equations, with reciprocate volume: 5 µL; flow rate: 0.45 ml/min; column oven of the drug concentration as a weighting factor (1/ temperature: 40ºC; mobile phase: methanol:buffer 2 2 [concentration] , i.e. 1/X ): y = mx + c (60:40); 2 ml of formic acid in 1000 ml mobile phase; diluent: methanol:water (50:50) + 2 ml of formic where, y = peak area ratio of candesartan to Propranolol, acid in 1000 ml of diluents; retention time: 2.1 min m = slope of the calibration curve, x = concentration for candesartan (analyte); 1.0 min for propranolol of candesartan, c = y-axis intercept of the calibration (internal standard); run time: 3.3 min; extraction curve technique: protein precipitation. Method validation Preparation of standards for calibration and The specified LC-MS/MS method was validated to quality control estimate candesartan in human plasma as per the [11] Accurately transferred about 10 mg of the candesartan US-FDA guidelines. Various validation parameters, working standard into a 10 ml volumetric a fl sk. It was such as linearity, precision, accuracy, specificity, Pharmaceutical Methods | April-June 2011 | Vol 2 | Issue 2 131 Prajapati, et al.: Quantitative estimation of candesartan from human plasma by LC-MS/MS stability study and matrix effect, were carried out to preparation. For intrabatch and interbatch accuracy, prove the capability of the proposed method. %nominal concentration of the back-calculated value for LLOQ, LQC, MQC and HQC, analyzed in a single analytical batch and thee different batches, were Linearity calculated respectively as per formula. %nominal A calibration curve comprising of a “blank matrix” concentration was found to be within the criteria of (matrix processed without analyte and internal 85–115%. standard), a “zero standard” (blank matrix processed only with internal standard) and nine calibration For intrabatch and interbatch precision, standard standards covering the expected range were processed deviation and %coefficient of variation for LLOQ, and analyzed. The linearity of the developed LQC, MQC and HQC samples, analyzed on one method was achieved in the range of 1.2–1030 ng/ batch and five different batches, were calculated, ml (r = 0.9996). The present method was capable of respectively, which were found to be within criteria quantifying the lower concentration of candesartan ≤15, except LLOQ (≤20). Results of the interbatch accurately [Figure 2]. %nominal values for all the precision and accuracy study are described in Table 1. standards were within the limits of 85–115%, except for STD-1, which was between 80 and 120%, as per [11] Recovery the US-FDA guidelines. Recovery for analyte and internal standard was performed by comparing the area of the extracted Accuracy and precision samples at three different concentrations (LQC, Calibration standards and six replicates each of LLOQ, MQC and HQC) with unextracted standards area LQC, MQC and HQC samples were processed and that represents 100% recovery. %recovery of an analyzed as per the procedure described in sample analyte(s) at LQC, MQC and HQC samples and an internal standard were calculated, which were found to be 101.9% for candesartan and 87% for the internal standard (propranolol), as depicted in Table 2. Specificity and selectivity Plasma matrix including four normal plasma lots with the anticoagulant, one lipemic plasma and one hemolyzed plasma lot were processed and analyzed. One sample each of the six plasma lots at blank and LLOQ level were processed and analyzed as per the procedure described in sample preparation. Area response at the RT of candesartan in the blank was less than 20% of the LLOQ area response and the area response at the RT of propranolol (internal standard) in the blank plasma was less than 5% of the internal standard area response as per the limit. Sensitivity Calibration standards, zero standard (matrix spiked only with internal standard) and six sets of matrix sample spiked at LLOQ concentration using blank matrix lot were processed and analyzed as per the procedure described in sample preparation. Response of candesartan at the LLOQ level was greater than five-times that of the blank plasma. %coefficient of variation (CV) and %nominal concentration were found to be 10.2% and 94.8%, respectively, which passes the limit of %CV (≤20) and %nominal Figure 2: Chromatogram of candesartan in the lower limit of quantification sample concentration (80–120%). Pharmaceutical Methods | April-June 2011 | Vol 2 | Issue 2 132 Prajapati, et al.: Quantitative estimation of candesartan from human plasma by LC-MS/MS Table 1: Results of interday and intraday precision P and A Interday Intraday LLOQ LQC (3.245 MQC HQC LLOQ LQC MQC HQC (1.298 ng/ml) (324.51 (721.13 (1.298 (3.245 (324.51 (721.13 ng/ml) ng/ml) ng/ml) ng/ml) ng/ml) ng/ml) ng/ml) Mean concentration* ± SD 1.34 ± 11.3 3.437 ± 7.3 324.10 ± 2.7 739.05 ± 2.1 1.44 ± 7.9 3.39 ± 6.7 324.74 ± 3.6 736.96 ± 2.4 Nominal (%) 103.4 100.4 99.9 102.5 111.0 104.5 100.1 102.2 *Average of six determinations, SD = Standard deviation Table 2: Results of accuracy study Sample Candesartan Propranolol (internal standard) (50.0 ng/ml) Mean area* ± SD % recovery Mean area ± SD %recovery Extracted LQC (3.245 ng/ml) 13720 ± 10.9 101.9 354722.2 ± 8.2 87.0 Unextracted 13466.7 ± 6.0 407833.3 ± 1.5 Extracted MQC (324.51 ng/ml) 1607500 ± 7.0 102.8 Unextracted 1563333.3 ± 2.2 Extracted HQC (721.13 ng/ml) 3590000 ± 1.6 110.6 Unextracted 3245000.0 ± 1.4 *SD = Standard deviation Table 3: Results of the stability study Type of stability study %CV Mean %change Long-term stock solution stability 0.8 0.6 LQC (n = 6) HQC (n = 6) LQC (n = 6) HQC (n = 6) Freeze and thaw stability study 3.2 0.9 0.5 2.5 Process stability in auto aampler 6.0 1.1 12.4 4.6 Bench-top stability study 6.4 2.0 1.2 1.1 Dilution integrity after three freeze and thaw cycles at LQC and HQC Analyte spiking stock solution was spiked in blank levels. Mean %changes were 0.5% and 2.5% for LQC plasma to get a concentration equivalent to three-times and HQC, respectively. This fulfilled the criteria of of the upper limit of quantification and diluted with mean %change (within 15% as shown in Table 3). blank plasma to get 1/5 and 1/10 concentrations of Process stability of candesartan at 6C in an auto the spiked sample or as per requirement. Calibration sampler for 24 h standards and six aliquots each of the diluted samples Process stability of the analyte is determined at LQC (1/5 and 1/10 dilutions) were processed and analyzed and HQC levels. Mean %changes for LQC and HQC as per the procedure described in sample preparation. were calculated to be 12.4% and 4.6%, respectively %nominal concentration was found to be 111.97% and [Table 3]. 108.3% for both the dilutions, which passed the limit of 85–115%. Bench-top stability of candesartan at room temperature for 6 h Matrix effect LQC and HQC samples were spiked in human Calibration standards, in the same matrix which was plasma and kept at room temperature for 6 h and to be used during validation experiment, and three were analyzed along with freshly prepared LQC and replicates from three different plasma matrices at HQC samples. Mean %changes during the stability LQC and HQC levels were processed and analyzed period were found to be 1.2% and 1.1% for the LQC as described in sample preparation. %nominal and HQC, respectively [Table 3]. concentration of LQC and HQC were found to be 100.4% and 106.4%, respectively, which fulfilled the Long-term stock solution stability of candesartan at criteria of %nominal concentration (85–115%). 2–8°C for 6 days The main stock solution of candesartan was freshly prepared and an aliquot of the stock was kept at 2–8°C Stability study for 6 days (stability sample). Aqueous equivalent Freeze and thaw stability of candesartan Freeze and thaw stability of the analyte was determined highest calibration standard of candesartan was Pharmaceutical Methods | April-June 2011 | Vol 2 | Issue 2 133 Prajapati, et al.: Quantitative estimation of candesartan from human plasma by LC-MS/MS prepared from the stability samples and analyzed. CONCLUSIONS Areas of stability samples and freshly prepared The proposed isocratic method is able to estimate a very samples were compared to determine the %mean low concentration of candesartan in human plasma change and %CV. %mean change and %CV were at less-retention time with high recovery compared found to be 0.6 and 0.8, respectively [Table 3]. with the reported method. Therefore, the developed method is simple, rapid, specific, selective, precise and RESULTS AND DISCUSSION accurate. The protein precipitaion technique used for the extraction purpose made this method time saving. Methanol and ammonium trifluoroacetate buffer were used for preparation of the mobile phase after taking various trials. Buffer concentration was optimized to ACKNOWLEDGMENTS 1 M after using various concentrations, and formic The authors are thankful to Zydus Cadila Healthcare acid was used to acidify the buffer. The ratio of the Limited, Ahmedabad, India, for providing reference buffer was increased to allow for better peak shape standards and all facilities to complete this research work. and resolution in plasma. Best results were obtained by using the ratio: methanol:buffer (60:40 v/v). The REFERENCES Betasil C8 column was selected to reduce the run 1. FDA.gov [drug information] Office of clinical pharmacology review time instead of the C columns. Low flow rate was of Candesartan. Available from: http://www.fda.gov/ downloads/ selected to 0.45 ml/min to increase the efficiency of the drugs/ developmentapprovalprocess/ development resources/ column and to reduce the usage of the mobile phase. ucm189128.pdf. [Last cited on 2008 May 21]. 2. Fenton C, Scott L. Candesartan Cilexetil: A review of its use in the No interference from endogenous substance was management of chronic heart failure. Drugs 2005;65:537-58. observed in the selectivity exercise at the retention time 3. Ferreiros N, Dresen S, Alonso RM, Weinmann W. Validated of candesartan. This is explicit from the chromatogram quantitation of angiotensin II receptor antagonists (ARA-II) in human plasma by liquid-chromatography-tandem mass spectrometry using of the sample [Figure 2] LOQ plasma samples spiked minimum sample clean-up and investigation of ion suppression. with internal standard. The R-square was consistently Ther Drug Monit 2007;29:824-34. 0.99 or greater during the course of the validation. 4. Kondo T, Yoshida K, Yoshimura Y, Motohashi M, Tanayama S. Characterization of conjugated metabolites of a new angiotensin The best fit for calibration curve of chromatographic II receptor antagonist, candesartan cilexetil, in rats by liquid response versus concentration was determined by chromatography/electrospray tandem mass spectrometry following the weighted least square regression analysis, with chemical derivatization. J Mass Spectrom 1996;31:873-8. 5. Nevin E. Simultaneous analysis of candesartan Cilexetil and weighting factor of 1/concentration.2 The data of intra- hydrochlorothiazide in human plasma and dosage forms using and interday precision and accuracy for candesartan HPLC with a photo diode array detector. J Liq Chromatogr Relat from QC samples are summarized in Tables 1 and 2, Technol 2003;26:2581-91. 6. Gonzalez L, Lopez JA, Alonso RM, Jimenez RM. Fast screening respectively. The precision and accuracy of this method method for the determination of Angiotensin II receptor antagonists conform to the FDA guidance document, which states in human plasma by high-performance liquid chromatography with that the accuracy determined at each concentration fluorimetric detection. J Chromatogr A 2002;949:49-60. 7. Subba R, Radhakrishnanand R, Suryanarayana M, Himabindu level must not exceed 15% (20% for LOQ) and V. A stability-indicating LC method for Candesartan cilexetil. precision must be within 15% (20% for LOQ) of the Chromatographia 2007;66:7-8. nominal value. The extraction recoveries from the QC 8. Yu Z, Wang Q, Wang F. Determination of the content of Candesartan Cilexetil by HPLC. J Shenyang Pharm Univ 2002;02:11-8. samples at low, middle and high concentrations were 9. Xiu HW, Yu-kai H, Xinhua Z, Luo J. HPLC determination of 101.9%, 102.8% and 110.6% for candesartan, whereas candesartan cilexetil and its related substances, Central South it was 87% for the internal standard. Recoveries were Pharmacy. 2004;02:12-7. 10. Mehta BH, Morge SB. HPTLC-densitometric analysis of candesartan good, and it was consistent, precise and reproducible cilexetil and hydrochlorothiazide in tablets. J Planar Chrom. Modern with this proposed extraction method [Table 2]. The TLC. 2008;21:173-6. results from the stability test are presented in Table 3, 11. FDA.gov. [Guidance for Industry: Bioanalytical Method Validation] US Department of Health and Human Services, which demonstrated a good stability of candesartan Food and Drug Administration, CDER, Rockville, USA. under the conditions evaluated. Bench-top stability Available from: http:// www.fda.gov/ downloads/ Drugs/ in matrix (7 h), post-preparative (in injector) stability Guidanc eComplianc eRegulatoryInform ation/Guidances/ ucm070107.pdf. [Last cited on 2001 May 24]. at 5 ± 1°C for 24 h and freeze–thaw stability were determined at six replicates of the low and high QC How to cite this article: Prajapati ST, Patel PK, Patel M, concentration at -70°C for 24 h, and were found to be Chauhan VB, Patel CN. Development and validation of the liquid acceptable. There was negligible or null matrix factor chromatography-tandem mass spectrometry method for quantitative and matrix effect. Values for all method validation estimation of candesartan from human plasma. Pharm Methods 2011;2:130-4. parameters indicate that the methods can be applied Source of Support: Nil, Conflict of Interest: None declared. for routine bioanalysis. Pharmaceutical Methods | April-June 2011 | Vol 2 | Issue 2 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Pharmaceutical Methods Pubmed Central

Development and validation of the liquid chromatography-tandem mass spectrometry method for quantitative estimation of candesartan from human plasma

Pharmaceutical Methods , Volume 2 (2) – Jul 1, 168

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Pubmed Central
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2229-4708
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10.4103/2229-4708.84460
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Abstract

Original Article Development and validation of the liquid chromatography-tandem mass spectrometry method for quantitative estimation of candesartan from human plasma Introduction: A simple and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for estimation of candesartan in human plasma using the protein precipitation technique. Materials and Methods: The chromatographic separation was performed on reverse phase using a Betasil C (100 x 2.1 mm) 5-µm column, mobile phase of methanol:ammonium tri-floro acetate buffer with formic acid (60:40 v/v) and flow rate of 0.45 ml/min. The protonated analyte was quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. The mass transitions m/z 441.2 → 263.2 and 260.2 → 116.1 were used to measure candesartan by using propranolol as an internal standard. Results: The linearity of the developed method was achieved in the range of 1.2–1030 ng/ml (r ≥ 0.9996) for candesartan. Conclusion: The developed method is simple, rapid, accurate, cost-effective and specific; hence, it can be applied for routine analysis in pharmaceutical industries. Key words: Candesaminan, LC-MS/MS method, propranolol Shailesh T. Prajapati, Pratik K. Patel, Marmik INTRODUCTION Patel , Vijendra B. Chauhan, Chhaganbhai Candesartan is used in the management of hypertension, and has been [1,2] N. Patel investigated in heart failure. Candesartan is an anti-hypertensive drug from a category of angiotensin-II receptor antagonists. Angiotensin II is formed from Department of Quality Assurance, Shri Sarvajanik Pharmacy angiotensin I in a reaction catalyzed by angiotensin-converting enzyme (ACE, College, Mehsana, Zydus Cadila kinase II). Angiotensin II is the principal pressor agent of the renin–angiotensin Healthcare Limited, Ahmedabad, Gujarat, India system, with effects that include vasoconstriction, stimulation of synthesis and release of aldosterone, cardiac stimulation and renal reabsorption of sodium. Address for correspondence: Candesartan blocks the vasoconstrictor and aldosterone-secreting effects of Dr. Shailesh T. Prajapati, angiotensin II by selectively blocking the binding of angiotensin II to the AT1 Department Quality Assurance, Shri Sarvajanik Pharmacy receptor in many tissues, such as vascular smooth muscle and adrenal gland. Its College, Mehsana - 384 001, action is, therefore, independent of the pathways for angiotensin II synthesis. Gujarat, India. Email: stprajapati@gmail.com The chemical name of candesartan is {(±)-1-Hydroxyethyl 2-ethoxy-1-[ p-(o-1H- [2] tetrazol-5-ylphenyl) benzyl]-7-benzimidazole carboxylate}. The structure of Access this article online candesartan is shown in Figure 1. On a detailed literature survey, it was found Website: www.phmethods.org that there was only one liquid chromatography-tandem mass spectrometry DOI: 10.4103/2229-4708.84460 (LC-MS/MS) gradient method reported for the estimation of candesartan from [3] Quick response code human plasma, and one method was reported to estimate candesartan in rat [4] plasma by LC-MS/MS. Some methods were reported to estimate candesartan from human plasma and solid dosage forms by the high-performance liquid [5-9] [10] chromatography (HPLC) and UV spectrophotometric methods, which were found to be time-consuming and costly. Hence, the objective of the present work was to develop a simple bioanalytical method to estimate candasartan from human plasma with due consideration of accuracy, sensitivity, rapidity, economy, Pharmaceutical Methods | April-June 2011 | Vol 2 | Issue 2 Abstract Prajapati, et al.: Quantitative estimation of candesartan from human plasma by LC-MS/MS dissolved in 5 ml of methanol and the volume up made up to the mark with methanol to prepare a 1 mg/ml solution. The final concentration of 0.1 mg/ml (100000 ng/ml) was carried out by dilution of 1 ml of the above 1 mg/ml solution up to 10 ml with methanol. The working solutions of candesartan were prepared using the diluent. The final concentration was made up to 58.270, 116.939, 708.723, 2531.155, 10124.620, Figure 1: Structure of candesartan 25960.563, 37086.519, 46353.149 and 51509.054 ng/ ml. Similarly, the lower quality control (LQC) selectivity and stability indicating according to the concentration (162.254 ng/ml), middle quality control US-FDA guidelines. (MQC) concentration (16225.352 ng/ml), higher quality control (HQC) concentration (36056.338 ng/ MATERIALS AND METHODS ml) and lower limit of quantification (LLOQ; 64.902 ng/ml) samples were prepared. Required numbers of samples of concentration of candesartan ranging Chemical and reagents from 1 to 1000 ng /ml were prepared by making up the The working standard or Candesartan and Propranolol volume with drug-free plasma and labelling them as as internal standard were gifted by Zydus Cadila STD-1 to STD-9, which are 1.169, 2.339, 14.174, 50.623, Healthcare Limited, Ahmedabad, India. Human 202.492, 519.211, 741.730, 927.163 and 1030.181 ng/ml, plasma samples were procured from Prathama Blood respectively. Bank, Ahmedabad, India. Methanol (HPLC grade) and ammonium trifloroacetate (GR grade) were purchased from Spectrochem, Hyderabad, India. Formic acid Sample preparation supra pure grade was purchased from Merck, Mumbai 0.10 ml of sample into was accurately pipetted into (India) and Milli-Q water was procured from Zydus prelabeled vials and 500 µl of propranolol (internal Cadila Healthcare Limited. standard) was added and mixed for 2 min (for blank sample, 500 µl of methanol solution was added instead of the internal standard solution). Methanol Instrumentation in propranolol solution was used for protein An HPLC (Shimadzu Corporation, Kyoto, Japan) precipitation. Samples were centrifuged at 4800 rpm coupled to an API 4000 mass spectrometer (Thermo at less than 10°C for 15 min. Then, 0.4 ml supernatent Finnigan Ltd., Stafford Ho, UK) was employed for the was transferred into the prelabeled vial containing 0.4 analysis. A pH meter (Thermo Orion, Asheville, NC , ml diluent and mixed properly. 0.5 µl of this mixture USA , Model 420) and sonicator (Oscar Ultra Sonics, was then injected into an HPLC system using an auto Andheri (E), Mumbai , India OU-72 SPL) were used sampler. for this work. The chromatographic conditions were as follows: The concentration of candesartan and propranolol was calculated from the area ratio v/s spiked plasma Column: Betasil C (100 x 2.1 mm), 5 µm; injection concentration regression equations, with reciprocate volume: 5 µL; flow rate: 0.45 ml/min; column oven of the drug concentration as a weighting factor (1/ temperature: 40ºC; mobile phase: methanol:buffer 2 2 [concentration] , i.e. 1/X ): y = mx + c (60:40); 2 ml of formic acid in 1000 ml mobile phase; diluent: methanol:water (50:50) + 2 ml of formic where, y = peak area ratio of candesartan to Propranolol, acid in 1000 ml of diluents; retention time: 2.1 min m = slope of the calibration curve, x = concentration for candesartan (analyte); 1.0 min for propranolol of candesartan, c = y-axis intercept of the calibration (internal standard); run time: 3.3 min; extraction curve technique: protein precipitation. Method validation Preparation of standards for calibration and The specified LC-MS/MS method was validated to quality control estimate candesartan in human plasma as per the [11] Accurately transferred about 10 mg of the candesartan US-FDA guidelines. Various validation parameters, working standard into a 10 ml volumetric a fl sk. It was such as linearity, precision, accuracy, specificity, Pharmaceutical Methods | April-June 2011 | Vol 2 | Issue 2 131 Prajapati, et al.: Quantitative estimation of candesartan from human plasma by LC-MS/MS stability study and matrix effect, were carried out to preparation. For intrabatch and interbatch accuracy, prove the capability of the proposed method. %nominal concentration of the back-calculated value for LLOQ, LQC, MQC and HQC, analyzed in a single analytical batch and thee different batches, were Linearity calculated respectively as per formula. %nominal A calibration curve comprising of a “blank matrix” concentration was found to be within the criteria of (matrix processed without analyte and internal 85–115%. standard), a “zero standard” (blank matrix processed only with internal standard) and nine calibration For intrabatch and interbatch precision, standard standards covering the expected range were processed deviation and %coefficient of variation for LLOQ, and analyzed. The linearity of the developed LQC, MQC and HQC samples, analyzed on one method was achieved in the range of 1.2–1030 ng/ batch and five different batches, were calculated, ml (r = 0.9996). The present method was capable of respectively, which were found to be within criteria quantifying the lower concentration of candesartan ≤15, except LLOQ (≤20). Results of the interbatch accurately [Figure 2]. %nominal values for all the precision and accuracy study are described in Table 1. standards were within the limits of 85–115%, except for STD-1, which was between 80 and 120%, as per [11] Recovery the US-FDA guidelines. Recovery for analyte and internal standard was performed by comparing the area of the extracted Accuracy and precision samples at three different concentrations (LQC, Calibration standards and six replicates each of LLOQ, MQC and HQC) with unextracted standards area LQC, MQC and HQC samples were processed and that represents 100% recovery. %recovery of an analyzed as per the procedure described in sample analyte(s) at LQC, MQC and HQC samples and an internal standard were calculated, which were found to be 101.9% for candesartan and 87% for the internal standard (propranolol), as depicted in Table 2. Specificity and selectivity Plasma matrix including four normal plasma lots with the anticoagulant, one lipemic plasma and one hemolyzed plasma lot were processed and analyzed. One sample each of the six plasma lots at blank and LLOQ level were processed and analyzed as per the procedure described in sample preparation. Area response at the RT of candesartan in the blank was less than 20% of the LLOQ area response and the area response at the RT of propranolol (internal standard) in the blank plasma was less than 5% of the internal standard area response as per the limit. Sensitivity Calibration standards, zero standard (matrix spiked only with internal standard) and six sets of matrix sample spiked at LLOQ concentration using blank matrix lot were processed and analyzed as per the procedure described in sample preparation. Response of candesartan at the LLOQ level was greater than five-times that of the blank plasma. %coefficient of variation (CV) and %nominal concentration were found to be 10.2% and 94.8%, respectively, which passes the limit of %CV (≤20) and %nominal Figure 2: Chromatogram of candesartan in the lower limit of quantification sample concentration (80–120%). Pharmaceutical Methods | April-June 2011 | Vol 2 | Issue 2 132 Prajapati, et al.: Quantitative estimation of candesartan from human plasma by LC-MS/MS Table 1: Results of interday and intraday precision P and A Interday Intraday LLOQ LQC (3.245 MQC HQC LLOQ LQC MQC HQC (1.298 ng/ml) (324.51 (721.13 (1.298 (3.245 (324.51 (721.13 ng/ml) ng/ml) ng/ml) ng/ml) ng/ml) ng/ml) ng/ml) Mean concentration* ± SD 1.34 ± 11.3 3.437 ± 7.3 324.10 ± 2.7 739.05 ± 2.1 1.44 ± 7.9 3.39 ± 6.7 324.74 ± 3.6 736.96 ± 2.4 Nominal (%) 103.4 100.4 99.9 102.5 111.0 104.5 100.1 102.2 *Average of six determinations, SD = Standard deviation Table 2: Results of accuracy study Sample Candesartan Propranolol (internal standard) (50.0 ng/ml) Mean area* ± SD % recovery Mean area ± SD %recovery Extracted LQC (3.245 ng/ml) 13720 ± 10.9 101.9 354722.2 ± 8.2 87.0 Unextracted 13466.7 ± 6.0 407833.3 ± 1.5 Extracted MQC (324.51 ng/ml) 1607500 ± 7.0 102.8 Unextracted 1563333.3 ± 2.2 Extracted HQC (721.13 ng/ml) 3590000 ± 1.6 110.6 Unextracted 3245000.0 ± 1.4 *SD = Standard deviation Table 3: Results of the stability study Type of stability study %CV Mean %change Long-term stock solution stability 0.8 0.6 LQC (n = 6) HQC (n = 6) LQC (n = 6) HQC (n = 6) Freeze and thaw stability study 3.2 0.9 0.5 2.5 Process stability in auto aampler 6.0 1.1 12.4 4.6 Bench-top stability study 6.4 2.0 1.2 1.1 Dilution integrity after three freeze and thaw cycles at LQC and HQC Analyte spiking stock solution was spiked in blank levels. Mean %changes were 0.5% and 2.5% for LQC plasma to get a concentration equivalent to three-times and HQC, respectively. This fulfilled the criteria of of the upper limit of quantification and diluted with mean %change (within 15% as shown in Table 3). blank plasma to get 1/5 and 1/10 concentrations of Process stability of candesartan at 6C in an auto the spiked sample or as per requirement. Calibration sampler for 24 h standards and six aliquots each of the diluted samples Process stability of the analyte is determined at LQC (1/5 and 1/10 dilutions) were processed and analyzed and HQC levels. Mean %changes for LQC and HQC as per the procedure described in sample preparation. were calculated to be 12.4% and 4.6%, respectively %nominal concentration was found to be 111.97% and [Table 3]. 108.3% for both the dilutions, which passed the limit of 85–115%. Bench-top stability of candesartan at room temperature for 6 h Matrix effect LQC and HQC samples were spiked in human Calibration standards, in the same matrix which was plasma and kept at room temperature for 6 h and to be used during validation experiment, and three were analyzed along with freshly prepared LQC and replicates from three different plasma matrices at HQC samples. Mean %changes during the stability LQC and HQC levels were processed and analyzed period were found to be 1.2% and 1.1% for the LQC as described in sample preparation. %nominal and HQC, respectively [Table 3]. concentration of LQC and HQC were found to be 100.4% and 106.4%, respectively, which fulfilled the Long-term stock solution stability of candesartan at criteria of %nominal concentration (85–115%). 2–8°C for 6 days The main stock solution of candesartan was freshly prepared and an aliquot of the stock was kept at 2–8°C Stability study for 6 days (stability sample). Aqueous equivalent Freeze and thaw stability of candesartan Freeze and thaw stability of the analyte was determined highest calibration standard of candesartan was Pharmaceutical Methods | April-June 2011 | Vol 2 | Issue 2 133 Prajapati, et al.: Quantitative estimation of candesartan from human plasma by LC-MS/MS prepared from the stability samples and analyzed. CONCLUSIONS Areas of stability samples and freshly prepared The proposed isocratic method is able to estimate a very samples were compared to determine the %mean low concentration of candesartan in human plasma change and %CV. %mean change and %CV were at less-retention time with high recovery compared found to be 0.6 and 0.8, respectively [Table 3]. with the reported method. Therefore, the developed method is simple, rapid, specific, selective, precise and RESULTS AND DISCUSSION accurate. The protein precipitaion technique used for the extraction purpose made this method time saving. Methanol and ammonium trifluoroacetate buffer were used for preparation of the mobile phase after taking various trials. Buffer concentration was optimized to ACKNOWLEDGMENTS 1 M after using various concentrations, and formic The authors are thankful to Zydus Cadila Healthcare acid was used to acidify the buffer. The ratio of the Limited, Ahmedabad, India, for providing reference buffer was increased to allow for better peak shape standards and all facilities to complete this research work. and resolution in plasma. Best results were obtained by using the ratio: methanol:buffer (60:40 v/v). The REFERENCES Betasil C8 column was selected to reduce the run 1. FDA.gov [drug information] Office of clinical pharmacology review time instead of the C columns. Low flow rate was of Candesartan. 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[Last cited on 2001 May 24]. at 5 ± 1°C for 24 h and freeze–thaw stability were determined at six replicates of the low and high QC How to cite this article: Prajapati ST, Patel PK, Patel M, concentration at -70°C for 24 h, and were found to be Chauhan VB, Patel CN. Development and validation of the liquid acceptable. There was negligible or null matrix factor chromatography-tandem mass spectrometry method for quantitative and matrix effect. Values for all method validation estimation of candesartan from human plasma. Pharm Methods 2011;2:130-4. parameters indicate that the methods can be applied Source of Support: Nil, Conflict of Interest: None declared. for routine bioanalysis. Pharmaceutical Methods | April-June 2011 | Vol 2 | Issue 2

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