DETERMINATION OF CARISOPRODOL IN HUMAN PLASMA BY LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY

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1 WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES Narapusetti et al. SJIF Impact Factor Volume 7, Issue 1, Research Article ISSN DETERMINATION OF CARISOPRODOL IN HUMAN PLASMA BY LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY Anjaneyulu Narapusetti 1 *, Alla Teja Sri 2, Kalyan Chakravarthy Janjanam 3 and Repaka Naga Kishore 4 1 Department of Pharmaceutical Analysis, Geethanjali College of Pharmacy, Cheeryal , India. 2 Asst.Professor, School of Pharmacy, Anurag Group of Institutions, Venkatapur, Ghatkesar, India. 3 University College of Pharmaceutical Sciences, Jawaharlal Nehru Technological University, Kukatpally, Hyderabad , India. 4 Department of Pharmacology, Geethanjali College of Pharmacy, Cheeryal , India. Article Received on 14 Nov. 2017, Revised on 04 Dec. 2017, Accepted on 25 Dec DOI: /wjpps *Corresponding Author Dr. Anjaneyulu Narapusetti Department of Pharmaceutical Analysis, Geethanjali College of Pharmacy, Cheeryal , India. ABSTRACT A liquid chromatographic tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination of carisoprodol in human plasma. Carisoprodol d3 was used as an internal standard (IS). The plasma samples were extracted by simple liquid liquid extraction method. These samples were then chromatographed on a Zorbax XDB phenyl column by using a mixture of 0.1% formic acid in water and acetonitrile (20:80, v/v) as the mobile phase. The method was validated in the range of ng/ml with r The intra day and inter day precision and accuracy results in four validation batches across five concentration levels were well within the acceptance limits. KEYWORDS: Carisoprodol; Human Plasma; Liquid-liquid Extraction (LLE); LC MS/MS. INTRODUCTION Chemically, carisoprodol is a 2-{[(aminocarbonyl)oxy]methyl}-2-methylpentyl isopropyl carbomate and its chemical formula is C12H24N2O4. Carisoprodol is a centrally acting skeletal muscle relaxant indicated for the relief of discomfort associated with acute Vol 7, Issue 1,

2 musculoskeletal spam in adults. [1] Acute musculoskeletal spasm of the back is a common disorder that causes localized pain, reduced mobility, impaired activities of daily living and sleep disturbances. Carisoprodol was approved for use in the United States in Carisoprodol has poor solubility and its permeability numbers can be obtained from altered permeability assays. [2] However, carisoprodol has a rapid, 30-minute onset of action. It is metabolized in the liver via the cytochrome P450 oxidase isozyme CYP2C19, excreted by the kidneys and has about an eight-hour half-life. A considerable proportion of carisoprodol is metabolized to meprobamate, which is a known drug of abuse and dependence; this could account for the abuse potential of carisoprodol. Several LC-MS/MS methods have been reported for the determination of carisoprodol along with its metabolite(s) and/or other drugs [3-6] in various biological matrices. However, these methods are not suitable for high throughput analysis of carisoprodol in human plasma. An efficient high-throughput bioanalytical method should be simple, sensitive, have shorter [7, 8] runtime, low sample volume and reproducible extraction procedure. Hence, authors developed a simple LC-MS/MS method with simple and economical high throughput bioanalytical method for the determination of carisoprodol in human plasma. The developed method was validated as per the current FDA guidance. The use of deuterated internal standard makes the method more reliable and can be easily adopted in pharmaceutical industry to support the bioequivalence or pharmacokinetic study of carisoprodol in human volunteers. EXPERIMENTAL Standards and reagents Carisoprodol (98.9% pure) and carisoprodol methyl d3 sodium salt (98% pure) reference standards were obtained from Clearsynth Limited (Mumbai, India). Water used for the LC- MS/MS analysis was prepared by using Milli Q water purification system procured from Millipore (Bangalore, India). Acetonitrile, methanol (HPLC grade), Ethyl acetate and n- Hexane were purchased from J.T. Baker (Phillipsburg, USA). Extra pure formic acid and ammonium formate were purchased from Fluka (Stinheim, Germany). Oasis HLB cartridges were obtained from Waters (Massachusetts, U.S.A). The blank human plasma was purchased from Deccan s Pathological Lab s (Hyderabad, India). Vol 7, Issue 1,

3 LC MS/MS instrument and conditions An HPLC system (Shimadzu, Kyoto, Japan) consisting of a Zorbax XDB phenyl 75 X 4.6 mm 3.5 µm; Agilent Technologies, Santa Clara, CA, USA), a binary LC-20AD prominence pump, an auto sampler (SIL-HTc) and a solvent degasser (DGU-20A3) were used for the study. Aliquots of the processed samples (10 µl) were injected into the column, which was kept at ambient temperature. An isocratic mobile phase consisting of a 80:20 (v/v) mixture of acetonitrile and 0.1% formic acid was used to separate the analytes and delivered at a flow rate of 1.0 ml/min into the electrospray ionization chamber of the mass spectrometer. Quantification was achieved with MS-MS detection in positive ion mode using an MDS Sciex API-3000 mass spectrometer (Foster City, CA, USA) equipped with a Turboionspray interface at 550 ºC. The ion spray voltage was set at 5500 V. The source parameters viz. the nebulizer gas, curtain gas and collision gas were set at 8, 8 and 6 psi, respectively. The compound parameters viz. the declustering potential (DP), collision energy (CE) and collision cell exit potential (CXP) were 40, 12 and 9 for carisoprodol and 50, 12 and 9 V for internal standard. Detection of the ions was carried out in the multiple-reaction monitoring mode (MRM), by monitoring the transition pairs of m/z for carisoprodol (Fig. 1 A) and the m/z for internal standard (Fig. 2 B). The analysis data obtained were processed by Analyst software (version 1.4.2). Sample Dilutions and Spiking Stock solutions of carisoprodol and internal standard were dissolved in methanol at a concentration of 1 mg/ml. From these stock solutions, appropriate dilutions were made to produce working standard solutions using a 60:40 (v/v) mixture of methanol and water as a diluent. Calibration curve (CC) standard solutions of carisoprodol in blank plasma were prepared by spiking with an appropriate volume of the working solutions, giving final concentrations of 25.0, 50.0, 150.9, 301.9, 603.9, , , and ng/ml for carisoprodol. The CC samples were analyzed along with the quality control (QC) samples for each batch of plasma samples. The QC samples were prepared at four different concentration levels of 25.2 (LLOQ), 75.3 (LQC), (MQC1), (MQC2) and (HQC) ng/ml in blank plasma. All the prepared plasma samples were stored at 80 C. Vol 7, Issue 1,

4 Sample processing The samples were thawed at room temperature and vortexed to ensure complete mixing of the contents. 100 µl of the plasma sample was pipetted 15 ml glass stoppered tubes, 10 µl of ng/ml Carisoprodol Methyl D3 dilution was added to it and vortexed, except in blank plasma samples where 10 µl diluent was added and vortexed. To this, 100 μl of 10mM ammonium formate was added and vortexed. 4 ml of extraction solvent was added and shaken for 20 min on reciprocating shaker at 200 rpm. Samples were centrifuged at 4000 rpm for 10 minutes at 4 C. Then supernatant organic layer (3.0 ml) was transferred to prelabelled glass dry test tubes and evaporated to dryness under gentle stream of nitrogen at 40 C. The samples were reconstituted in 500 µl of mobile phase. Aliquot of 10 μl of the extract was injected into the LC-MS/MS system. Method validation parameters The present method was validated for matrix effect, sensitivity, linearity, precision and accuracy, recovery, dilution integrity and stability as per the recent US FDA guidelines. [9] RESULTS AND DISCUSSION Method development Mass parameters were tuned in both positive and negative ionization modes for the analyte and internal standard. Good response was found in positive ionization mode. Data in the multiple reaction monitoring (MRM) mode was considered, which showed better selectivity. The compound and source dependent parameters were suitably altered to get most intense signals and reproducible response. Data in the multiple reaction monitoring (MRM) mode was considered, which showed better selectivity. [10,11] Chromatographic conditions, especially the composition of the mobile phase, were optimized through several trials to achieve good resolution, desired intensity of the signals of the analyte and short run time. The presence of a small amount of formic acid in the mobile phase improved the detection of the analyte. It was found that a mixture of acetonitrile and 0.1% formic acid (80:20, v/v) could achieve this purpose and was finally adopted as the mobile phase. Zorbax XDS Phenyl (75 mm ˣ 4.6 mm, 3.5 µm) column gave good peak shapes and response even at lowest concentration level. The mobile phase was operated at a flow rate of 1.0 ml/min. The retention time of carisoprodol and the IS were low enough (0.74 and 0.74 min) allowing a small run time of 2 min. A simple and efficient liquid liquid extraction (LLE) technique was employed for the sample preparation in this work and provides high recoveries of the drugs. Vol 7, Issue 1,

5 Selectivity and chromatography The degree of interference by endogenous plasma constituents with the analyte and the IS was assessed by inspection of chromatograms derived from processed blank plasma sample. As shown in Fig. 2, no significant direct interference in the blank plasma traces was observed from endogenous substances in drug-free plasma at the retention time of the analyte. Sensitivity The lowest limit of reliable quantification for the carisoprodol was set at the concentration of the LLOQ. Representative chromatogram of LLOQ and HQC shown in Fig.3. The precision and accuracy at LLOQ concentration were found to be 7.44% and %. Matrix effect No significant matrix effect was observed in all the six batches of human plasma for the raltegravir at low and high quality control concentrations. The precision at LQC and HQC concentrations were found to be 6.12% and Linearity The nine-point calibration curve was found to be linear over the concentration range of ng/ml. After comparing the two weighting models (1/x and 1/x2), a regression equation with a weighting factor of 1/x2 of the drug to the IS concentration was found to produce the best fit for the concentration-detector response relationship in human plasma. The mean correlation coefficient of the weighted calibration curves generated during the validation was Precision and accuracy As shown in Table 1, the precision and accuracy of carisoprodol in the intra-day and interday runs were within ± 15% at LQC, MQC1, MQC2 and HQC concentrations and within ± 20% at LLOQ QCs. Extraction efficiency Six replicates at low, medium-2 and high quality control concentration were prepared for recovery determination. The recoveries of analyte and IS were good and reproducible. The mean overall recoveries (with the precision range) of carisoprodol and IS were % and % respectively. Vol 7, Issue 1,

6 Stability studies In the different stability experiments carried out viz. bench top stability (10 h), auto-sampler stability (40 h), repeated freeze-thaw cycles (4 cycles), wet extract stability (33 h at 2-8 C) and long term stability at 80 C for 82 days, the mean % nominal values of the analytes were found to be within 15% of the predicted concentrations for the analytes at their LQC and HQC levels (Table 2). Thus, the results were found to be within the acceptable limits during the entire validation. Table 1: Precision and accuracy data for carisoprodol. Quality Concentration found Accuracy Run Precision (%) control Mean±SD (ng/ml) (%) Intra day variations (n=6 at each concentration) LLOQ LQC MQC MQC HQC Inter day variations (n=30 at each concentration) LLOQ LQC MQC MQC HQC Nominal concentrations of LLOQ, LQC, MQC1, MQC2 and HQC are 25.23, 75.30, , and ng/ml, respectively Table 2: Stability data for carisoprodol in plasma (n=6). Stability test QC (spiked concentration (ng/ml)) Mean SD (ng/ml) Precision (%) Accuracy/ Stability (%) Process a Process b Bench top c FT d Long term e a after 40 h in autosampler at 10 C; b after 33 h at 2 8 C; c after 10 h at room temperature; d after 4 freeze and thaw cycles; e at 80 C for 82 days Vol 7, Issue 1,

7 Figure 1: Mass spectra of carisoprodol (a) and internal standard (b). Figure 2: Typical MRM chromatograms of carisoprodol (upper panel) and IS (lower panel) in human blank plasma. Vol 7, Issue 1,

8 Figure 3: Typical MRM chromatograms of carisoprodol a) LLOQ; b) HQC. CONCLUSIONS The LC-MS/MS assay method described in this paper is rapid, simple, specific and more sensitive for quantification of carisoprodol in human plasma and is fully validated as per the FDA guidelines. The proposed method described the simple and economical liquid-liquid extraction method with consistent and reproducible recoveries for the analyte and internal standard from plasma. The method provided good linearity range which can be applied to pharmacokinetic or bioequivalence study. A sample turnover rate of less than 2.0 min and simple extraction method makes it an attractive procedure in high-throughput bioanalysis of carisoprodol. ACKNOWLEDGEMENTS The authors gratefully acknowledge PCR Laboratories (Hyderabad, India) for providing necessary facilities to carry out this work. REFERENCES 1. Simon S, Andrea CD, Wheeler WJ and Sacks H. Bioavailability of oral carisoprodol 250 and 350 mg and metabolism to meprobamate: a single-dose crossover study. Current Therapeutic Research, 2010; 71: Devang S, Sundeep P, Muralikrishna M, Gajendra C, Murali S, Ajay S, Matthew GS, John H, Roy H, Punit M, Sandhya M. A systematic evaluation of solubility enhancing Vol 7, Issue 1,

9 excipients to enable the generation of permeability data for poorly soluble compounds in Caco-2 model. Drug metabolism letter, 2014; 8(2): Skinner W, McKemie D and Stanley S. Quantitative determination of carisoprodol and its metabolites in equine urine and serum by liquid chromatography tandem mass spectrometry. Chromatographia, 2004; 59: S61 S Matsumoto T, Sano T, Matsuoka T, Maeno Y and Nagao M. Simultaneous determination of carisoprodol and acetaminophen in an attempted suicide by liquid chromatography mass spectrometry with positive electrospray ionization. Journal of Analytical Toxicology, 2003; 27: Nirogi R, Kandikere V, Mudigonda K, Ajjala D, Suraneni R and Thoddi P. Simultaneous extraction of acetylsalicylic acid and salicylic acid fromhuman plasma and simultaneous estimation by liquid chromatography and atmospheric pressure chemical ionization/tandem mass spectrometry detection. Application to a pharmacokinetic study. Arzneimittel-Forschung, 2011; 61: Vudagandla S, Mullangi R, Inamadugu JK, Ravi VB, Nageswara RP and Abburi K. Simultaneous determination of carisoprodol and asprin in human plasm using liquid chromatography-tandem mass spectrometry in polarity switch mode: application to a human pharmacokinetic study. Journal of biomedical chromatography, 2012; 27: Matta MK, Pilli NR, J V L N SR. A validated liquid chromatography and tandem mass spectrometric method for simultaneous quantitation of tenofovir, emtricitabine and efavirenz in human plasma and its pharmacokinetic application. Acta Chromatographica, 2015; 27: Matta MK, Burugula L, Pilli NR, Inamadugu JK, J V L N SR. A novel LC-MS/MS method for simultaneous quantification of tenofovir and lamivudine in human plasma and its application to a pharmacokinetic study. Biomedical Chromatography, 2012; 26: US DHHS, FDA and CDER. Guidance for Industry: Bioanalytical Method Validation. US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research and Center for Veterinary Medicine, Matta MK, Pilli NR, Inamadugu JK, Burugula L, J V L N SR. Simultaneous quantification of lamivudine, zidovudine and nevirapine in human plasma by liquid chromatography tandem mass spectrometry and its application to a pharmacokinetic study. Acta Pharm Sin B, 2012; 2: Vol 7, Issue 1,

10 11. Putluru SP, Matta MK, Ahire D, Subramanian M, Sinz M, Mandlekar S. A novel liquid chromatography tandem mass spectrometry method for the estimation of bilirubin glucuronides and its application to in vitro enzyme assays. Drug Metab Lett, 2016; 10: Vol 7, Issue 1,