Summary of Analytical Method for Quantitative Estimation of Fingolimod and Fingolimod Phosphate from Human Whole Blood Samples

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Fingolimod Whole Blood Analysis Summary of Analytical Method for Quantitative Estimation of Fingolimod and Fingolimod Phosphate from Human Whole Blood Samples Study Detail: - Determination of

1 Fingolimod Whole Blood Analysis Summary of Analytical Method for Quantitative Estimation of Fingolimod and Fingolimod Phosphate from Human Whole Blood Samples Study Detail: - Determination of Fingolimod and Finglomod Phosphate from human Whole Blood samples after administration of x. mg Capsules of Fingolimod to healthy human subjects Bioanalytical method for quantitative estimation of Fingolimod and Finglomod Phosphate from human whole blood samples was developed and validated as per EMEA/US FDA guidelines for Bioanalytical method validation using LC-MS/MS (API-) Matrix: Human whole blood Matrix Volume: µl Fingolimod Calibration range.pg/ml to. pg/ml Sample ID minal Concentration (pg/ml) Accurac y (n=) CS.. QC samples.,. and. pg/ml Calibration curve plot MV_RUN.rdb (Sapropterin): "Linear" Regression (" / (x * x)" weighting): y =. x + -. (r =.) CS.. CS.. CS.. Analyte Area / IS Area CS CS.... Analyte Conc. / IS Conc. CS.. CS.. Slope=., Intercept=-., Regression =., Curve Type= Linear Regression, Weighting factor = /x Method is Precise and Accurate over the range of.pg/ml to.pg/ml, The information in this document is provided by Clinical Research Services of Reliance Life Sciences Pvt. Ltd. ( CRS ) and the contents, including all figures, tables, and drawings, are the intellectual property of CRS. Forwarding or reproduction of this document or parts thereof, regardless of reason or form, is not Page of

2 Fingolimod Method Validation Summary Selectivity interference observed in out of different lots of human plasma. Specificity (Cross reactivity) significant interference was observed at the RT of both Analyte and IS. carry over is observed for analyte and Injector Carryover Test internal standard. carry over is observed for analyte and internal standard for different instrument. Within-run accuracy QC % minal value. to. Within run precision QC Coefficient of variation. to.% Extended run accuracy QC % minal value. to.% Extended run precision QC Coefficient of variation. to.% Between run accuracy QC % minal value. to. Between run precision QC Coefficient of variation. to.% Ruggedness- Different analyst: accuracy QC % minal value. to. Ruggedness- Different analyst: precision QC Coefficient of variation. to.% Ruggedness- Different column: accuracy QC % minal value. to. Ruggedness- Different column: precision QC Coefficient of variation. to.% Recovery of analyte. to.% Recovery of Internal standard.% Dilution Integrity accuracy QC % minal value. to. Dilution Integrity precision QC Coefficient of variation. to.% Re-injection Reproducibility Analyte A response was reproducible. Anticoagulant effect accuracy QC % minal value. to. Anticoagulant effect precision QC Coefficient of variation. to.% Matrix effect precision (Internal Standard normalized MF for LQC) Coefficient of variation.% Matrix effect precision (Internal Standard normalized MF for HQC) Coefficient of variation.% Short term stability of analyte in stock solution on bench Mean % Change after hours minutes is -. stock solution on bench Mean % Change after hours minutes is -. Short term stability of analyte in working Mean % Change after hours Page of

3 Sample Name: "MB_RP" Sample ID: "" File: "MV_RUN RES_.wiff" Peak Name: "Fingolimod" Mass(es): "./. amu". Concentration:. pg/ml Acq. Time: :: PM Sample Name: "MB_RP" Sample ID: "" File: "MV_RUN RES_.wiff" Peak Name: "Fingolimod D(IS)" Mass(es): "./. amu" Concentration:. pg/ml. Acq. Time: :: PM solution on bench minutes is -. working solution on bench Mean % Change after hours minutes is -. Freeze and thaw stability at -± C after cycles QC Mean % Change -. to. Coolant stability of analyte in matrix in dry ice QC Mean % Change after hours minutes is. to. Autosampler stability at C QC Mean % Change after hours minutes is. to. Bench Top stability of analyte in matrix at room temperature QC Mean % Change after hours minutes is. to. Long Term stability of analyte in stock solution at - C Mean % Change is -. after days stock solution at - C Mean % Change is -. after days Long Term stability of analyte in working solution at - C Mean % change is -. after days working solution at - C Mean % Change is -. after days Fingolimod Figure Blank: Human whole blood.. Page of

4 Sample Name: "CS_RP" Sample ID: "" File: "MV_RUN RES_.wiff" Peak Name: "Fingolimod" Mass(es): "./. amu" Concentration:. pg/ml Calculated Conc:. pg/ml Acq. Time: :: PM ise Threshold:. cps Area Threshold:. cps Sep. Width:. Exp. Adj. Ratio:. Exp. Val. Ratio:. Use Relative RT: Retention Time:. min Start Time:. min End Time:. min Sample Name: "CS_RP" Sample ID: "" File: "MV_RUN RES_.wiff" Peak Name: "Fingolimod D(IS)" Mass(es): "./. amu" Concentration:. pg/ml Acq. Time: :: PM ise Threshold:. cps Area Threshold:. cps Sep. Width:. Exp. Adj. Ratio:. Exp. Val. Ratio:. Use Relative RT: Retention Time:. min Start Time:. min End Time:. min.. LLOQ:.pg/ml Proc. Algorithm: Analyst Classic Sep.. Exp. Peak Ratio:. RT Window:. sec Expected RT:. min Area: counts.e+ cps Proc. Algorithm: Analyst Classic Sep.. Exp. Peak Ratio:. RT Window:. sec Expected RT:. min Area: counts.e+ cps Fingolimod Phospahte: Calibration range.pg/ml to.pg/ml Sample ID minal Concentration (pg/ml) Accuracy (n=) CS.. QC samples.,. and. pg/ml Calibration curve plot MV_RUN.rdb (Sapropterin): "Linear" Regression (" / (x * x)" weighting): y =. x + -. (r =.) CS..... CS.. CS.. Analyte Area / IS Area CS CS CS.. Analyte Conc. / IS Conc. CS.. Slope=., Intercept=- -., Regression =., Curve Type= Linear Regression, Weighting factor = /x Method is Precise and Accurate over the range of.pg/ml to.pg/ml, Page of

5 Fingolimod Phospahte Method Validation Summary Selectivity interference observed in out of different lots of human plasma. Specificity (Cross reactivity) significant interference was observed at the RT of both Analyte and IS. carry over is observed for analyte and Injector Carryover Test internal standard. carry over is observed for analyte and internal standard for different instrument. Within-run accuracy QC % minal value. to. Within run precision QC Coefficient of variation. to.% Extended run accuracy QC % minal value. to. Extended run precision QC Coefficient of variation. to.% Between run accuracy QC % minal value. to. Between run precision QC Coefficient of variation. to.% Ruggedness- Different analyst: accuracy QC % minal value. to. Ruggedness- Different analyst: precision QC Coefficient of variation. to.% Ruggedness- Different column: accuracy QC % minal value. to. Ruggedness- Different column: precision QC Coefficient of variation. to.% Recovery of analyte.to.% Recovery of Internal standard.% Dilution Integrity accuracy QC % minal value. to. Dilution Integrity precision QC Coefficient of variation. to.% Re-injection Reproducibility Analyte B response was reproducible. Anticoagulant effect accuracy QC % minal value. to. Anticoagulant effect precision QC Coefficient of variation. to.% Matrix effect precision (Internal Standard Coefficient of variation.% normalized MF for LQC) Matrix effect precision (Internal Standard Coefficient of variation.% normalized MF for HQC) Short term stability of analyte in stock solution on bench Mean % Change after hours minutes is -. stock solution on bench Mean % Change after hours minutes is -. Short term stability of analyte in working solution on bench Mean % Change after hours minutes is -. working solution on bench Mean % Change after hours minutes is -. Freeze and thaw stability at -± C after QC Mean % Change -. to. Page of

6 Sample Name: "MB_RP" Sample ID: "" File: "MV_RUN RES_.wiff" Peak Name: "Fingolimod phosphate" Mass(es): "./. amu" Concentration:. pg/ml Acq. Time: :: PM Sample Name: "MB_RP" Sample ID: "" File: "MV_RUN RES_.wiff" Peak Name: "Fingolimod phosphate D(IS)" Mass(es): "./. amu". Concentration:. pg/ml Acq. Time: :: PM cycles Coolant stability of analyte in matrix in dry ice Autosampler stability at C Bench Top stability of analyte in matrix at room temperature Long Term stability of analyte in stock solution at - C stock solution at - C Long Term stability of analyte in working solution at - C working solution at - C QC Mean % Change after hours minutes is -. to. QC Mean % Change after hours minutes is. to. QC Mean % Change after hours minutes is -. to. Mean % Change is -. after days Mean % Change is -. after days Mean % Change is -. after days Mean % Change is -. after days Fingolimod phosphate figure: Blank: Human whole blood LLOQ:.pg/ml Page of

7 Sample Name: "CS_RP" Sample ID: "" File: "MV_RUN RES_.wiff" Peak Name: "Fingolimod phosphate" Mass(es): "./. amu" Concentration:. pg/ml Calculated Conc:. pg/ml Acq. Time: :: PM Proc. Algorithm: Analyst Classic ise Threshold:. cps Area Threshold:. cps Sep. Width:. Sep.. Exp. Peak Ratio:. Exp. Adj. Ratio:. Exp. Val. Ratio:. RT Window:. sec Expected RT:. min Use Relative RT: Retention Time:. min Area: counts.e+ cps Start Time:. min End Time:. min. Sample Name: "CS_RP" Sample ID: "" File: "MV_RUN RES_.wiff" Peak Name: "Fingolimod phosphate D(IS)" Mass(es): "./. amu" Concentration:. pg/ml Acq. Time: :: PM Proc. Algorithm: Analyst Classic ise Threshold:. cps Area Threshold:. cps Sep. Width:. Sep.. Exp. Peak Ratio:. Exp. Adj. Ratio:. Exp. Val. Ratio:. RT Window:. sec Expected RT:. min Use Relative RT: Retention Time:. min Area: counts.e+ cps Start Time:. min End Time:. min.... Page of

Pharmacologyonline 1: (2010) Newsletter Mohan et al.

Pharmacologyonline 1: (2010) Newsletter Mohan et al. SIMPLE AND ROBUST METHOD FOR THE QUANTIFICATION OF LEVONORGESTREL IN PLASMA BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY ELECTROSPRAY IONIZATION MASS SPECTROMETRY Reddy C.Mohan 1, Gajjar A.K 2 and Khan A.Hussian