TEMPLATE DESIGN © 2008 www.PosterPresentations.com Quantification of metformin in human plasma with sold phase extraction using sensitive ultra flow liquid chromatography –tandem mass spectrometric Detection Raghunadha Reddy Seelam a,* , Nageswara Rao Pilli b , Ravindra Reddy S c , Seelam Sai Satyanaraya Reddy c a Department of Pharmaceutical Science, School of Pharmacy, University of Maryland, Pine Street, Baltimore, Maryland 21201, USA. b Piramal clinical research, Hyderabad,Telangana, India. c Vardhaman College of Engineering, Hyderabad, Telangana, India. . Abstract Literature Cited Conclusion Acknowledgements A selective and sensitive method have been developed for quantification of metformin in human plasma by using UFLC–MS/MS method. Phenformin was used as an internal standard(IS). The extraction of the metformin from human plasma was performed using solid phase extraction. Inertsil ODS-3 (250x4.6 mm,5µm) reverse phase column was employed for chromatographic separation of metformin and phenformin(IS) for MS/MS detection at 1 ml/min flow using 90:10 (v/v) methanol: 10 mM ammonium acetate. Detection was performed at transitions of m/z 130.000→71.000 for metformin and m/z 206.100→ 105.000 for phenformin by positive electro-spray ionization (ESI+) in multiple reaction monitoring (MRM) mode using tandem mass spectrometry. Analysis was carryout within 3.0 min. The calibration curves were linear over a concentration range of 25.0 ng/mL to 3500.0 ng/mL for quantification of Metformin with the correlation coefficients demonstrating good linearity (0.994-0.999). The lower limits of quantification were 25.0 ng/mL for metformin. The developed method was compared in the terms of validation parameters including specificity,linearity, sensitivity, precision, accuracy and stability. The method was shown rugged when new batch was performed with a different column, different analyst and with fresh solutions. No effect was observed in presence of hemolysed or lipemic content in plasma sample and in presence of potentially interfering drugs. Matrix based samples were stable at room temperature for >16 hrs, processed samples were stable at least for >46 hrs and also stable at four freeze-thaw cycles. This validated method was successfully applied for quantification of metformin in human plasma for bioequivalence study. Materials and Methods Chemicals and reagents : Metformin HCl was supplied by Shreeji Pharma International. Phenformin HCl was commercially procured from Selleck USA. All the solvents used were of HPLC grade. Acetonitrile and methanol were of HPLC grade and obtained from J.T.Bakers. Acetic acid and Ammonium Acetate were obtained from Merck. Double distilled water is obtained from Sartorius apparatus. Standard solutions preparation Stock solution preparation Approximately 5 mg of Metformin / 2mg of Phenformin (IS) working standard was weighed and transferred to 10.0 mL volumetric flask, to this 5.0 mL of Methanol was added and sonicated to aid dissolution and the final volume was made up with Methanol. Preparation of internal standard dilution The Phenformin internal standard (ISTD) dilution of about 100 ng/mL from the ISTD stock solution (IS stock) using (80:20methanol: water) as the diluent was prepared. Preparation of calibration curve (CC) standards and quality control (QC) samples Appropriate dilutions of the stock solutions with diluent were made subsequently in order to prepare the working standard solution in the range of 1.268 µg /mL to 177.400 µg/mL. All the solutions were stored in a refrigerator between 2°C and 8°C. Calibration standards and quality control samples, in the range of 25.0 ng/mL to 3500.0 ng/mL were prepared for calibration. Accuracy and precision, quality control and stability assessment was done by spiking 0.5mL of drug free plasma with appropriate volume of working solution. Solutions used: 50%v/v Methanol in Water: Prepared the diluent by mixing methanol and water in 1:1 ratio. 10 mM Ammonium Acetate: Weighed accurately about 0.385g of ammonium acetate into 500 mL volumetric flask and make up to the mark with HPLC Grade water, mixed well and transferred it into 500 mL reagent bottle. Mobile phase Preparation: [MeOH : 10mM Ammonium Acetate (90:10)]:Taken 900 mL of methanol into 1000mL reagent bottle and added 100mL of 10 mM ammonium acetate mixed well and sonicated for about 1 minute and filter through 0.45µm nylon membrane filter. Rinsing Solvent for Auto Injector [MeOH : Milli – Q water (80:20)]: Taken 800mL of Methanol in a 1000mL reagent bottle, added 200mL of water, mixed well, sonicated for 1 minute. Total preparation volume was adjusted according to the requirement, by keeping the ratios constant. 5% ammonia solution: Taken 5 ml of ammonia solution in to 100mL volumetric flask containing 30mL Milli-Q water, mixed well and make up to the mark with Milli-Q Water. Transfered it into a reagent bottle. Results The method was applied successfully to the analysis of plasma samples obtained for pharmacokinetic, bioavailability or bioequivalence study after therapeutic doses of Metformin. The established LC-MS/MS method is sensitive and suitable for the study of Metformin in human plasma. Because of the relative short chromatographic runtime (3.0 min), the method is easy to follow and can be adopted for clinical drug monitoring. The authors would like to thank S.Venu Gopal, Y.K.Naidu, V. Ravi Kiran, D.Koteswara Rao and S.Sandhya Rani for their technical assistance. 1. Koteswara Rao.Divi, I. Sarath Chandiran, K.N. Jayaveera, Y.K. Naidu, M.P. Kalyan Reddy. Development and validation of high-throughput liquid chromatography–tandem mass spectrometric method for simultaneous quantification of Clopidogrel and its metabolite in human plasma, Journal of Chromatography B, Elsevier Publications, February- 2010, volume-878, Issue:3-4, pages:502–508, Impact factor-2.977, ISSN: 1570-0232. 2. Y.K.N, Raghunadha Reddy Seelam, Koteswara Rao Divi, Penchala Kalyan Reddy Mule, Sarath Chandiran I and Jayaveera K N. Simultaneous Quantification of Alverine And Its Metabolite P-Hydroxy Alverine In Human Plasma With Robotic Liquid-Liquid Extraction By Using Fully Validated LC-MS/MS and Its Application To A Bioequivalence Study, Journal Of Pharmacy Research, 2010, Volume-3, Issue-6, Page-1407-1411, Impact Factor-1.5, ISSN: 0974-6943. 3. Raghunadha Reddy.S, Koteswara Rao.Divi, I.Sarath chandiran and K.N. Jayaveera. Quantification of Artemether in human plasma with liquid- liquid extraction by using fully validated high performance liquid Chromatography–Tandem mass spectrometric method, Journal of Pharmacy Research, August-2010, Voumel-3, Issue-8, Impact Factor-1.5, ISSN: 0974-6943. 4. I.Sarath Chandiran, K. N. Jayaveera and Raghunadha Reddy. S., High-Throughput Liquid Chromatography–Tandem Mass Spectrometric Method for Simultaneous Quantification of Carvedilol and Its Metabolite 4-Hydroxyphenyl Carvedilol in Human Plasma and Its Application to Bioequivalence Study, Journal of Chemical and Pharmaceutical Research, , 2011, 3[2]:341-353, ISSN No: 0975-7384, CODEN[USA]: JCPRC5. 5. I.Sarath Chandiran, K. N. Jayaveera and Ragunadha Reddy. S. Development and Validation of High-Throughput Liquid Chromatography-Tandem Mass Spectrometric Method for Quantification of Itraconazole and its Metabolite in Human Plasma. Scholars Research Library, Der Pharmacia Lettre, 2011, 3[2]: 316-328. 6. .Sarath Chandiran K.N. Jayaveera and Raghunadha Reddy.S, Pharmacokinetic and Bioequivalence Comparison Between Extended Release Capsules of Venlafaxine Hydrochloride 150mg: An Open Label, Balanced, Randomized-Sequence, Single-Dose, Two-Period Crossover Study In Healthy Indian Male Volunteers, International Research Journal Of Pharmacy [IRJP], 2[3], 2011,262-269, ISSN 2230-8407. 7. RaghunadhaR.S, I. Sarath Chandiran, K. N. Jayaveera and Koteswara Rao. Divi. Quantification of Ursodeoxy Cholic acid in human plasma by using High performance liquid chromatography–tandem mass spectrometric method and its applications in pharmacokinetics, Journal of Chemical and Pharmaceutical Research, 2010, volume-2, issue-3, Pages:59-69 and ISSN No: 0975-7384. 8. Sarath Chandiran I, Koteswara Rao Divi, Jayaveera K. N., Development and Validation of High Performance Liquid Chromatography-Tandem Mass Spectrometric Method for Simultaneous Quantification of Telmisartan in Human Plasma, International journal of pharmaceutical sciences and Drug Research, 2010, Volume-2, Issue-3, Pages: 188-192 and ISSN No: 0975-248X. 9. .Sarath chandiran, K.N. Jayaveera and Koteswara Rao.Divi. Quantification of ibuprofen in human plasma by using high throughput liquid chromatography–tandem mass spectrometric method and its applications in pharmacokinetics, scholars research library, archives of applied research, 2010, volume-2, issue-3, pages-101-111, ISSN- 0975-508x,coden[USA] AASRC9. 10. RRS, Koteswara Rao.Divi, Y.K.Naidu, I.Sarath Chandiran, k.N. Jayaveera and M.P.Kalyan Reddy. Development and validation of high-performance liquid chromatography tandem mass spectrometric method for quantification of Clonidine in human plasma, journal of chemical and pharmaceutical sciences, April - June’2010, volume-3, issue-2. Sample preparation Retrieved the frozen CC, QC and subject samples from the deep freezer and thawed in water bath maintained at room temperature, vortexed to mix. Removed the caps from the polypropylene tubes. An aliquot of 100 μL of CC, QC and subject samples in a ria vial tube was spiked with 50 μL of the IS solution (100 ng/mL) and vortex mixed for 30secs. Then 100 µL of 5 %v/v ammonia solution was added and vortex-mixed for 30 secs. SPE cartridges (strata-X) were conditioned with 1mL methanol, equilibrated with 1mL water and loaded the sample into SPE cartridges(strata-X). The SPE cartridges were washed twice with 1mL of Methanol and 1mL of Water (Milli-Q/HPLC grade). The samples were eluted with 1mL methanol into pre labeled Ria vials. Then evaporated the samples to dryness under the nitrogen pressure with 50°C of temperature. The residual was reconstituted in 100 µL of a mobile phase and centrifuged at 4,000rpm for 5 min. Then, 10 μL aliquot was injected on to the LC-MS/MS system. For optimal stability, the auto-sampler temperature was set at 5 ˚C. Data processing : Chromatograms were acquired on a API 3200 of MDS Sciex tandem mass spectrometry equipped with Electrospray ionization (ESI) and connected to a PC runs with the standard software Analyst -1.5.1. Mass spectroscopic detection was performed on a Triple quadrapole instrument (API 3200 of MDS Sciex). The calibration curve is constructed by weighted 1/x 2 least-square linear regression analysis of the peak area ratio (drug/ISTD) vs. the concentration of drug. Chromatographic and mass spectrometric conditions UFLC separation was carried out on a Inertsil ODS-3 (250x4.6 mm,5µm) with mobile phase A-methanol and B-10mM Ammonium Acetate (A:B=90:10) at a flow rate of 1 mL/min and the column temperature was maintained at 35˚C. The sample injection volume was 10 μL and the analytical run time was 3.0 min. The eluent from the analytical column was introduced directly to the MS/MS system using ESI source in the positive ion mode. Source specific and compound specific mass spectrometric parameters are given in Table-1. Method Validation: Specificity and selectivity Six human plasma samples from six individual healthy donors receiving no medication were extracted and analyzed for the assessment of potential interferences with endogenous substances. The apparent response at the retention time of drug and internal standard were compared to the response at the lower limit of quantification (LLOQ) for drug to the response at the working concentration for internal standard. Observed retention times were about 0.79 min (metformin) and 2.08 min (phenformin) respectively. No additional peak due to endogenous substances that could have interfered with the detection of the compounds of interest was observed. Representative chromatograms of extracted human blank plasma and extracted human blank plasma spiked with Drug and IS are shown in Fig.1.A and 1.B. Linearity The mean accuracy and precisions for back calculated concentrations of each standard calculated from calibration curves were tabulated as Table 2. Recovery The recoveries of metformin and phenformin were evaluated with 6 replicates at 3 different concentration levels. In our method we got 95.6% and 93.2% recovery for metformin and phenformin, which are within the acceptance criteria. Precision and accuracy The accuracy and precision for inter day and intra day was tabulated for drug in Table 3. Stability In our study quality control plasma samples were used subject to bench top (>16h), Auto injector (10–84 h), freeze–thaw (-80 to +20 °C) cycles, wet extract (28 h) at room temperature, wet extract at 2-8°C (>46h) and long term ( 90 days) at deep freezer (at -80 °C) tests are performed. The values obtained for present stability studies are tabulated (Table 4), which are within the acceptance criteria. Application of the method The present method was applied for a randomized cross-over bioequivalence study of two different metformin preparations in 16 healthy male volunteers. After single oral administration of the drug blood samples were collected at a suitable time intervals. This method was successfully used to measure the plasma concentrations of metformin. Parameter Metformin Phenformin Q1 130.00 206.10 Q3 71.00 105.00 Dwell Time(msec) 200.00 200.00 Declustering Potential (DP) 30.00 25.00 Entrance Potential (EP) 10.00 10.00 Collision Cell Entrance Potential (CEP) 13.00 15.51 Collision Energy (CE) 30.00 35.00 Collision Cell Exit Potential (CXP) 10.00 10.00 Scan Type MRM Polarity Positive Curtain Gas (CUR) 25.00 Collision Associated Dissociation (CAD) 6.00 Ion Spray Voltage (ISV) 4500 Heater Temperature (TEM) 475.00°C Nebulizer Gas (GS1) 45.00 Heater Gas (GS2) 35.00 ihe ON Table-1:Source specific and compound specific mass spectrometric parameters Fig.1.A and 1.B. Representative chromatograms of extracted human blank plasma and extracted human blank plasma spiked with Drug and IS Table 2: Back calculated concentrations from calibration curves Nominal concentration (ng/mL) Mean Accuracy (%) Precision (% RSD) 3500.0 95.6 1.6 3015.8 94.4 3.7 2292.0 93.3 1.2 1558.6 91.7 4.6 623.4 100.8 2.5 155.6 97.2 6.7 50.7 96.8 8.3 25.0 92.4 1.7 Nominal Conc. (ng/mL) 25.5 68.8 1720.8 2868.0 Intra-day accuracy(%)(day1) 95.8 104.2 104.3 98.2 Intra-day precision(%)(day1) 2.7 5.2 4.2 6.7 Intra-day accuracy(%)(day2) 98.1 94.5 94.5 92.5 Intra-day precision(%)(day2) 6.5 4.3 2.1 3.9 Intra-day accuracy(%)(day3) 98.4 94.6 98.5 96.2 Intra-day precision(%)(day3) 2.9 5.4 4.7 1.4 Overall accuracy (%) 92.4 91.9 93.2 92.4 Overall Precision (%) 2.6 5.4 4.7 7.1 Number of determinations 18 18 18 18 Table 3.Assessment of Accuracy and precision of the method Nominal Conc. (ng/mL) 2868.0 25.5 Precision Accuracy Precision Accuracy Freeze thaw stability 2.1 91.6 5.8 94.8 Bench top stability 3.4 101.4 5.3 102.5 Wet extract stability at room temperature 1.2 104.9 4.1 95.3 Wet extract stability at 2-8ºC 9.6 94.6 8.4 104.5 Auto sampler stability 3.5 104.5 5.2 107.2 Long term stability 4.5 108.2 4.2 91.3 Interim storage stability at -25 °C 3.7 102.5 4.7 100.4 Table 4.Stability results