- (2013) Volume 5, Issue 2
Nishant Paliwal1 , Peeyush Jain1 , Naveen Dubey1 , Sandeep Sharma1 , Siddharth Khurana1 , Rishabh Mishra1 and Sarvesh Kumar Paliwal2
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| Corresponding Author: Peeyush Jain , E-mail: peeyush_jain108@yahoo.com |
| Received:18 December 2012 Accepted: 23 December 2012 |
| Citation: Nishant Paliwal, Peeyush Jain, Naveen Dubey, Sandeep Sharma, Siddharth Khurana, Rishabh Mishra and Sarvesh Kumar Paliwal “Liquid Chromatography Tandem Mass Spectrometry Method for Quantification of Solifenacin in Human Plasma and its Application to Bioequivalence Study” Int. J. Drug Dev. & Res., April-June 2013, 5(2):91-101. doi: doi number |
| Copyright: © 2013 IJDDR, Peeyush Jain et al. This is an open access paper distributed under the copyright agreement with Serials Publication, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
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A hasty, specific and robust assay based on liquid-liquid extraction and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI MS-MS) has been developed and validated for the quantitative analysis of Solifenacin ( a drug used for urinary incontinence ) in human plasma using Solifenacin D5 as internal standard (ISTD). The precursor to product ion transitions of m/z 363.20/110.10 and m/z 368.14/110.20 were used to measure the analyte and the ISTD, respectively. The method was validated in terms of selectivity, matrix effect, sensitivity, linearity, precision and accuracy, various stabilities (standard stock solution stability in refrigerator and at room temperature, stock dilution stability at refrigerator and room temperature, auto sampler stability, freeze thaw stability, long term stability- 65 o C ± 10o C & long term stability- 22 o C ± 5°C, reagent stability, bench top stability, dry extract stability, wet extract stability in refrigerator, effect of potentially interfering drugs, dilution integrity, recovery, lon suppression through infusion, and blood Stability. The mean percentage recovery of Solifenacin and the internal standard was 65.39 ± 3.646% and 66.24 ± 2.209% respectively. The assay exhibited a linear dynamic range of 0.200 to 30.361 ng mL-1. The RSD % of intra-day and inter-day assay was ≤15%. The application of this assay was demonstrated in a bioequivalence study and will be ideal for clinical pharmacokinetic studies in study population with as lower as 0.200 ng mL-1 analytical sensitivity and as little as 300 μL plasma sample.
Keywords |
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| Solifenacin; electrospray ionization; tandem mass spectrometry; human plasma; bioequivalence study | ||||||
Introduction |
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| Urinary incontinence associated with overactive bladder (OAB) is often considered as a reason for hygiene related embarrassment caused among the pregnant ladies, old age persons and patients gone through prolonged antidepressant or diuretic treatment. Behavioral modification techniques and pharmacotherapy are the two different ways suggested for the treatment.(1-2) Behavioral modification techniques as pelvic exercises and bladder retraining drills are proven to be cumbersome for patients with impaired mobility. Until 2004 antimuscaranics as oxybutynin, flavoxate and trospium were considered as first line of pharmacotherapy for controlling the urinary urgency. Treatment with these drugs has shown inconsistent efficacy due nonselective nature towards the muscaranic receptors. Solifenacin has emerged as the future drug to treat the urinary incontinence and bring better quality of life among the patients. Solifenacin has reduced central nervous system penetration and have better selectivity for the M3 subclass of muscaranic receptors which brings about improved tolerability. Solifenacin is the first antimuscarinic that has shown improvements in all the key symptoms of OAB – frequency, urgency, incontinence and nocturia. Solefanacin acts by blocking the activation of muscarinic receptor present on the bladder wall. | ||||||
| Solifenacin is approximately 96% bound to human plasma proteins, extensively metabolized in liver and bears long elimination half life . [3] Thus clinical pharmacokinetic studies progression requires rapid, selective, sensitive and robust bioanalytical methods.Very few bioanalytical procedures have been reported for the determination of Solifenacin that include HPLC and LC/MS-MS based methods. Recently, Jan Macek et al. had reported a liquid chromatography–electrospray tandem mass spectrometry method to quantitate Solifenacin in human plasma [4]. The assay was based on protein precipitation which compromise the applicability of the method for a longer bioequivalence study as poor sample clean up associated with precipitation techniques can clog the ion source, whereas the matrix components supplemented with the analysed fraction can bring down the sensitivity. Hiren N. Mistri et al had reported a liquid-liquid extraction LC MSMS based method for the simultaneous quantification of Alfuzosin and Solifenacin in human plasma using propranolol as internal standard [5]. The method bears limitation for its use in pharmacokinetic and bioequivalence studies of Solifenacin alone as it requires an additional demonstration of selectivity of Solifenacin in the presensce of Alfuzosin by spiking it in calibration curve standards and quality control samples. Further the sensitivity of the method was also high. Takamitsu Yanagihara et al had reported a liquid-liquid extraction HPLC based method for the simultaneous quantification of Solifenacin and its metabolite in rat plasma [6]. The method bears limitation as it requires a large plasma volume for analysis with a poor sensitivity. The purpose of the current study was to develop and validate a sensitive, robust and a rapid LC–ESIMS/ MS method for determination of Solifenacin in human plasma over a wide range which could make it applicable for use in a bioequivalence study. The method was validated according to published USFDA guidelines [7]. | ||||||
EXPERIMENTAL |
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Materials and Method |
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| Solifenacin (> 99.97% w/w ) and Solifenacin D5 (> 94.21% w/w ), were obtained from Clearsynth lab Pvt Ltd, India (Fig. 1). HPLC-grade methanol and acetic acid were purchased from SD Fine Chem. Ltd (Mumbai, India). HPLC-grade acetonitrile was purchased from JT Baker (Phillipsburg, USA). Ammonia was purchased from Fluka (Fluka Chemie, GmbH, Germany). Tertiary Butyl Methyl Ether was purchased from Rankem ,India. Milli-Q water 18.2m (milliohm) and TOC 50 ppb (parts per billion)] from Milli-Q system (Millipore SAS, Molsheim, France) was used. Hypurity Advance C18 column (4.6 x 50 mm,5μ) was purchased from Thermo Scientific, Germany. All other reagents and chemicals used for these studies were of HPLC grade unless specified. | ||||||
| Instrumentation | ||||||
| LC MS-MS analysis was performed using API 3000 triple quadrupole instrument (Applied Biosystems MDS SCIEX, Toronto, Canada) coupled with Shimadzu SIL HTC system, USA) in multiple reaction monitoring (MRM) mode. A turbo electrospray interface in positive ionization mode was used for ionization. Data processing was performed on Analyst software version 1.4.2 (Applied Biosystems MDS SCIEX, Toronto, Canada). | ||||||
| Standard and Quality Control Sample Preparation | ||||||
| Primary stock solution of Solifenacin (1.0 mg mL-1) and Solifenacin D5 (0.1 mg mL-1) were prepared in methanol .Aqueous dilution for spiking were prepared by serially diluting the primary stock solution of Solifenacin in water:methanol (50:50, v/v). Spiking of aqueous dilutions in human plasma was done to give eight-point calibration curve (0.200-30.361 ng mL-1) of Solifenacin. Separate spiking standards were prepared for making quality control samples at four concentration levels viz. 0.200 ng mL-1 as lower limit of quantitation (LOQQC), 0.546 ng mL-1 as low quality control (LQC), 16.531 ng mL-1 as middle quality control (MQC) and 24.797 ng mL-1 as high quality control (HQC) samples. ISTD working solution of 60 ng mL-1 was prepared by diluting the primary stock with methanol:water (50:50 v/v). Primary stock solutions were kept at 2-8°C when not in use. Spiked calibration standards and QC samples were stored below –65°C. | ||||||
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Sample Preparation |
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| Sample preparation involved a simple liquid-liquid extraction method .An aliquot of 300μL of plasma was admixed with 50 μL of internal standards working solution in ria vial. These samples were vortexed and 2.0 mL of tertiary butyl methyl ether was added to them for extracting the analyte. Subsequently the the plasma organic solvent mixture was vortex, centrifuged and allowed to flash freeze. The plasma matrix got freezed at the bottom and the analyte got extracted in the supernatant organic solvent. The supernatant was then poured in separate tubes and dried to eliminate any traces of matrix that remain associated with analyte. The dried samples were reconstituted in mobile phase comprising of 85% acetonitrile and 15% of 0.05% ammonia in water. | ||||||
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Chromatographic and Mass Spectrometric Conditions |
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| The analytes were chromatographically separated using reversed-phase high-performance liquid chromatography (HPLC) with isocratic elution. The mobile phase consisted of acetonitrile: 0.05% ammonia in water (85:15 v/v) at a flow rate of 0.700 mL min-1 through the Hypurity Advance (4.6 x 50 mm,5μ) C18 column. For all analyses 15 μL of sample was injected. The total run time was 4.0 min. The mass spectrometer was operated in the electro spray ionization mode with positive ion detection to monitor the ions with m/z 363.20/110.10 a.m.u. for Solifenacin and m/z 368.14/110.20 a.m.u. for Solifenacin D5. For Solifenacin and Solifenacin D5 the optimized source parameters were nebulizer gas (NEB): 12 psi, ion spray voltage (IS): 3000 V, source temperature (TEM): 400°C, collision gas (CAD): 5 psi and curtain gas (CUR): 8 psi. The optimized compound parameters for Solifenacin and Solifenacin D5 were declustering potential (DP): 66 V , enterance potential (EP):10 V ,collision energy (CE): 37 and 41 V ,focusing potential (FP):180 and 160 V and cell exit potential (CXP):8 and 10 V. | ||||||
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Data processing and Regression |
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| The MRM chromatographic peaks were integrated using Analyst software version 1.4.2 after which peak area ratios of Solifenacin to Solifenacin D5 were plotted versus concentration and a linear curve fit, weighted by 1/x2 (where x = concentration) was used to produce the regression line. | ||||||
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Bioanalytical Method Validation |
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| As a part of method validation stock solution and stock dilution stability, In-injector, bench-top, freezethaw and long-term stability of Solifenacin was evaluated. The stock solution and stock dilution stability was evaluated at room temperature and at 2- 8°C by comparing with freshly prepared stock solution. In order to determine the autosampler stability ,against a freshy prepared calibration standards a set of freshy prepared and extracted quality control samples were compared with quality control samples that were kept in auto sampler at 5°C for 63 hrs and 20 min. The stability of analyte in human plasma stored at room temperature (benchtop stability) was evaluated similarly for 7 hrs and 6 min by comparing with freshly prepared and extracted samples. The freeze-thaw stability was conducted by comparing the stability samples that had been frozen and thawed five times with freshly prepared quality control samples by analyzing against freshly prepared calibration standards . The long-term stability was conducted by analyzing low and high quality control samples stored below -65°C and -22°C along with freshly prepared quality control samples for 41 days with freshly prepared calibration standards. | ||||||
RESULTS AND DISCUSSION |
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Method Development and Optimization |
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| The optimization of mass spectrometric parameters for the analyte and internal standard was carried out by direct infusion and adjustment of the compound dependent parameters as declustering potential (DP), focusing potential (FP) and entrance potential (EP) leading to production of abundant protonated molecular ions (MH)+ at m/z 363.20 (Solifenacin) and 368.14 (ISTD) under positive ion polarity. Negative ion polarity was not tried as the structure of the compound rendered it as proton acceptor. Parameters such as DP, FP and EP were ramped to provide best signal to noise level. Subsequently scanning was done for the product ions. The optimization of parameters as collision energy, cell exit potential was carried out by trying different combinations of the volatages and comparing against the ramped values. Results showed that collision energy of 37 V for Solifenacin and 41 V for ISTD along with the cell exit potential of 8 V for Solifenacin and 10 V for ISTD gives the more intense product ion. Source dependent parameters as nebulizer gas, curtain gas and collision associated dissociation energy were optimized by flow injection analysis using a union in place of column. Afterwards chromatographic conditions were optimized to account for higher sensitivity, better peak shape and shorter chromatographic run time. The selection of mobile phase was done by keeping symmetric peak shapes, cost effective organic components and shorter run time in view. Results derived from several combinations showed that 85% acetonitrile and 15% of 0.05% ammonia in water serves the desired purpose with utmost effectiveness. Presence of high percentage of organic content in the mobile phase results in proper spraying condition within the ionization source in comparison to aqueous part that takes more time to nebulise. Acetonitrile has lower density in comparison to methanol, hence it was found as more suitable organic component as it lays lower back pressure on the column and prolongs column life on a longer bioequivalence study. Solifenacin is reported to have pKa on slightly basic side . Addition of small percentage of ammoniated solution as the aqueous fraction of the mobile phase helps in keeping the pH of the mobile phase within the range of pKa of Solifenacin. The choice of the appropriate column for a particular application is always a daunting task. Within the broad range of bonded phases offering available, the Hypurity Advance (4.6 x 50 mm,5μ) C18 column was found optimal as it meet most separation needs with better LC/MS compatibility and highly selective behavior at higher pH. The use of proper internal standard was necessary as it eliminates the quantitative bias caused by matrix effect and instrumental variation.Solifenacin D5 was found to be the most suitable compound for use as an internal standard due to its deuterated nature as it contain enough mass increase to show a signal outside the natural mass distribution of the analyte. Solifenacin D5 has shown the same extraction recovery, ionization response in ESI mass spectrometry. Solifenacin D5 has shown same chromatographic retention time as the analyte and it use to co-elute with the compound to be quantified. Fig 1a & 1b | ||||||
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ASSAY PERFORMANCE AND VALIDATION |
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Specificity and Sensitivity |
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| Selectivity is the ability of the analytical method to differentiate and quantify the analyte in the presence of other expected components in the sample. Selectivity of the analyte and internal standard was demonstrated in normal,haemolysed and lipemic plasma lots. The rapid and simple liquid liquid extraction based method of extraction was found to be highly selective for the analysis of Solifenacin and ISTD in human plasma. The selectivity was carried out in eight different lots of blank normal human plasma, four haemolysed and four lipemic plasma lots. No interference of endogenous matrix/impurities was found at the retention time of the analyte and internal standard. Representative chromatograms of extracted blank human plasma (Fig. 2a) and blank human plasma fortified with ISTD (Fig. 2b), demonstrated the selectivity and sensitivity of the method. Sensitivity was determined by analyzing six replicates of blank human plasma spiked with the analyte at the lowest level of the calibration curve (0.200 ng mL-1) against a calibration curve. The representative chromatogram for the LLOQ (0.200 ng mL-1) showing sensitivity is depicted in Fig. 2c. | ||||||
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Linearity |
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| Calibration curves were linear over the concentration range 0.200-30.361ng mL-1. Each batch of spiked plasma samples evaluated for linearity, precision and accuracy included one complete set of calibration curve standards and six replicates of quality control samples at LOQQC, LQC, MQC and HQC levels. The best linear fit and least square residuals for the calibration curve were achieved with a 1/x2 weighing factor, giving a mean linear equation for the calibration curve of y = (0.10 ± 0.0034) x + (0.0017± 0.0005) where y is the peak area ratio of analyte to the ISTD and x is the concentration of analyte. The correlation coefficient (r) for Solifenacin was found above 0.999. | ||||||
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Precision and Accuracy |
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| The linearity, precision and accuracy evaluations were executed on three batches of spiked plasma samples. The intra day and inter day precision and accuracy was demonstrated by running three precision and accuracy batches (each including one set of calibration curve standards and six sets of quality control samples at every level). Not more than two acceptable precision and accuracy batches were run on a single day. The intra day and inter day accuracy was determined by calculating percentage bias of quality control sample from the theoretical concentration. The intra day and inter day precision was determined in terms of relative standard deviation (% RSD). Precision of the assay was measured by the percent coefficient of variation over different concentration levels. The acceptance criteria for within and between batch precision were 20% or better for LOQQC and 15% or better for other nonzero concentrations. The accuracy of the assay was defined as the ratio of absolute value of the calculated mean values of the quality control sample to their respective nominal values, expressed as percentage and the criteria for accuracy was 100 ± 20% or better for LOQQC and 100 ± 15% or better for other concentrations. For intra day experiments, the precision ranged from 1.00% to 3.74% and the accuracy ranged from 98.00% to 98.75%. For the interday, the precision ranged from 1.65% to 3.65% and the accuracy ranged from 98.41% to 101.56%. The intra and inter day precision and accuracy data is presented in Table 1. | ||||||
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Recovery |
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| Recovery relates to the extraction efficiency of an analytical method within the limits of variability. Recovery of Solifenacin from the extraction procedure was determined by a comparison of the peak area response of Solifenacin and Solifenacin D5 in spiked plasma samples (six replicates of low, medium and high quality control samples) with the peak area response of aqueous mixture including Solifenacin and Solifenacin D5 at concentrations representing 100% extraction of quality control samples at low, middle and high concentration (nonextracted samples).The % CV of mean percentage recovery for analyte(s) and internal standard across the different quality control samples should be <15%.The absolute recovery of Solifenacin at LQC, MQC and HQC levels was found to be 63.31, 69.60 and 63.26% respectively. The absolute recovery of Solifenacin D5 at LQC, MQC and HQC levels was found to be 67.54 ,63.69 and 67.49%. The mean percentage recovery of Solifenacin and Solifenacin D5 was 65.39 ± 3.646% and 66.24 ± 2.209%, respectively. | ||||||
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Stability study |
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| Stability studies were performed to evaluate the Solifenacin and Solifenacin D5 stability in stock solution ,stock dilution and in matrix samples under different conditions (bench top stability, auto sampler stability, freeze thaw stability, long term stability - 65 °C ± 10°C & long term stability - 22 °C ± 5°C, dry extract stability, wet extract stability in refrigerator and at bench top, blood stability). Stock solutions of Solefanacin and Solefanacin D5 were stable at room temperature for 26 hrs and 50 min and in refrigerator at 2-8°C for 9 days. Stock dilution of Solifenacin and Solifenacin D5 were stable at room temperature for 26 hrs and 50 min Spiked Solifenacin was stable in human plasma (bench top stability) at room temperature for 8hrs and 17 min. Solifenacin in the final extract was found to be stable in the autosampler up to 63 hrs and 20 min. Solifenacin was found to be stable for at least five freeze and thaw cycles. The Solifenacin spiked plasma samples stored below -65°C and -22°C for long- term stability experiment and were found to be stable for 41 days. The processed long term stability - 65 °C ± 10°C and -22 °C ± 5°C quality control samples and freshly spiked quality control samples were quantified against the freshly spiked calibration curve standards. Dry Extract Stability of Solifenacin was demonstrated for 71 hrs, 13 min. Wet Extract Stability of Solifenacin was demonstrated for 72 hrs. Blood stability for spiked samples is carried out to assess the stability of the analyte(s) in blood. Blood stability was performed by the preparation of six sets of quality control samples Medium Quality Control (MQC) and High Quality Control samples (HQC) by spiking 2 % of MQC and HQC aqueous dilution in blood which were kept on the bench at room temperature for approximately 1 hour. The samples were processed with freshly spiked MQC and HQC after centrifuged at 4000 rpm at 4°C for 15 minutes to separate the plasma. Blood stability is expressed as the percentage change between the fresh and the stability samples. The % change for Solifenacin were - 3.05 % (MQC) and -1.13 % (HQC) respectively. Additionally reagent stability was carried out to confirm the stability of the reagents during the period of their use in analysis. For estimating reagent Stability, six sets of quality control samples (LQC and HQC) were processed in aged solutions that were kept on bench at room temperature for the stability duration and they were quantified against a calibration curve standards, which was processed using fresh solutions. The precision and accuracy of the quality control samples is used as an estimate of the reagent stability. Reagents used in analysis were found stable for duration of 7 days The stability data of the analyte in matrix and aqueous solution is presented in Table 2(a) and 2(b). | ||||||
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Effect of potentially interfering drug |
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| Effect of potentially interfering drug was evaluated to demonstrate the effect of potential drugs that could affect the analysis (by creating any interference at the retention levels of analyte and internal standard) because of their administration to the study volunteers during the clinical phase of the study. Potentially co-administered drugs prepared at therapeutic concentrations are tested to verify their interfering potential at the RT of an analyte and the IS. PID LLOQ samples were prepared by the spiking of 2 % of the respective PIDs at their expected Cmax concentration and 2 % of Lower Limit of Quantification (LLOQ) in blank matrix. These samples were analysed against a calibration curve. The effect of potentially interfering drugs (PID) (Ibuprofen, Caffeine, Acetaminophen and Acetyl salicylic acid) on Solifenacin analysis was performed. The back calculated concentrations of QC sample spiked with PID were found to be within ± 15 % of the actual concentration of the QC sample for Solefanacin. Hence, the above-mentioned PIDs have no effect on the analysis of Solefanacin. | ||||||
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Dilution Integrity |
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| Dilution integrity was carried out by using the drug free matrix for the required number of dilution (1.50 to 1.75 times of ULOQ concentration) .Six quality control samples for dilution integrity were prepared by spiking approximately 2 times (51.134 ng mL-1) of ULOQ concentration of Solifenacin. Six dilution integrity quality control samples were analyzed with the appropriate dilution factor 1/2 and 1/5 against a calibration curve standards. The precision (% CV) and accuracy (mean % nominal) ranged upto 2.59 % and 102.20 % with a dilution factor of 1/2 whereas the precision (% CV) and accuracy (mean % nominal) ranged upto 5.28 % and 104.74 % with a dilution factor of 1/5. | ||||||
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Ion Suppression through Infusion |
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| The ions suppression through infusion was done at the retention time of Solifenacin and Solifenacin D5. There was no suppression observed at the retention time of the compounds. | ||||||
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Matrix effect assessment |
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| Matrix effect is the direct or indirect interference in response of analyte and internal standard due to the presence of interfering substances in the sample. Matrix effect evaluation signifies the absence of interference from endogenous components in plasma which could affects the measurement of the analytes. The quantitative measurement of the matrix effect was carried out at low and high quality control samples level in normal,haemolysed and lipemic plasma by the determination of matrix factor ( the ratio of the analyte peak response in the presence of matrix ions to the analyte peak response in the absence of matrix ions). The % Matrix Effect expressed as % CV of the matrix factor should be ≤15%. The variability of matrix factor (reported as %CV of matrix factor) was found to be 2.53 % (HQC) and 6.97 % (LQC) for Solifenacin. The variability of matrix factor (reported as %CV of matrix factor) was 6.08% (HQC) and 12.03% (LQC) for Solifenacin D5. The variability of matrix factor of haemolysed plasma (reported as %CV of matrix factor) was 4.43 % (HQC) and 7. 79 % (LQC) for Solifenacin.The variability of matrix factor of Haemolysed Plasma (reported as % CV of matrix factor) was 4.97 % (HQC) and 6.98 % (LQC) for Solifenacin D5, The variability of matrix factor of Lipemic Plasma (reported as %CV of matrix factor) was 0.28 % (HQC) and 3.02 % (LQC) for Solifenacin. The variability of matrix factor of Lipemic Plasma (reported as %CV of matrix factor) was 0.95 % (HQC) and 3.02 % (LQC) for Solifenacin D5 | ||||||
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Application of the method |
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| The validated method was successfully applied for the determination of plasma concentration during bioequivalence study comparing test product (Solifenacin Succinate tablets 10 mg of Jubilant Life Sciences Limited, India) with reference Product (Vesicare® [solifenacin succinate] tablets 10 mg of Astellas Pharma Canada, Inc.) in healthy Adult, human Subjects, under Fasting Conditions. | ||||||
Conclusion |
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| A highly selective and rapid LC-ESI MS-MS method for the determination of Solifenacin in human plasma has been developed and validated with a range of 0.200-30.361 ng mL-1. The underlying advantage of this validated method is that it uses only 300 μL of human plasma thus minimizing untoward matrix effect, also the injection volume was restricted to 15μL, i.e. only 0.455 ng was injected onto the column at ULOQ which eliminate any chances of response saturation. Further the added advantage of simple and inexpensive liquid-liquid extraction procedure and shorter (4.0 min) analytical run time ensures high throughput and renders it suitable for sample analysis of routine volunteers. Method sensitivity of 0.200 ng mL-1 was found suitable for bioequivalence study purposes | ||||||
Conflict of Interest |
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| NIL | ||||||
Source of Support |
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| NONE | ||||||
Tables at a glance |
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Figures at a glance |
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