Quantitative Analysis of Synthetic Opioids in Urine using Exactive LC-MS System

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1 Quantitative Analysis of Synthetic Opioids in Urine using Exactive LC-MS System Clinical Research Applications Group Thermo Fisher Scientific San Jose, CA

2 Overview Customer workflow Analytes and Internal Standards used Sample Preparation Analytes and Internal Standards M/Z and retention times Linearity and Dynamic Range data Results on QC s Conclusions Appendix 2

3 Customer Workflow Quick Semi Quantitative Screening using Exactive Positive Samples Run On Immunoassay Near future plans to use only Exactive LC-MS No Immunoassay The Workflow Primarily Driven by Cost Effectiveness 3

4 Analytes and Internal Standards No Analyte Ketamine Norketamine Nalbuphine Fentanyl Norfentanyl Alfentanil Sufentanil Butorphanol Internal Standard Ketamine-D4 Norketamine-D4 Fentanyl-D Fentanyl-D Norfentanyl-D Fentanyl-D Sufentanil-D Fentanyl-D 4

5 Sample Preparation Dilute n Shoot X dilution with DI water Standards and QC samples Analytes and internal standards Cerilliant. Calibrators - standard spiked in blank urine Commercial QC samples

6 Analytes and Internal standards Accurate Masses and Retention Times No Analyte Exact mass Ret time No Analyte Exact mass Ret time 1 Ketamine Ketamine-D Norketamine Nalbuphine Fentanyl Norfentanyl Alfentanil Norketamine-D4 Fentanyl-D Norfentanyl-D Sufentanil-D Sufentanil Butorphanol Ketamine

7 Ketamine Good Linearity in the Desired Range Ketamine Y = *X R^2 =.9892 W: 1/X^ ng/ml Area Ratio Calibrator ng/ml % Difference Note: 4 point cal curve. Objective was semi quantitative analysis 7

8 Ketamine Calibrator S/N ng/ml std - m/z= RT:.8 SM: -.8 NL:1.39E4 F: FTMS {,} + p ESI Full ms [ ] Relative Intensity RT: 3. AA: SN: Time (min) 8

9 Norketamine Good Linearity in the Desired Range Norketamine Y = *X R^2 =.9884 W: 1/X^ ng/ml Area Ratio Calibrator ng/ml % Difference Note: 4 point cal curve. Objective was semi quantitative analysis 9

10 Norketamine Calibrator S/N = ng/ml std - m/z= RT:.6 SM: -.6 NL:7.72E3 F: FTMS {,} + p ESI Full ms [ ] Relative Intensity RT: 3.1 AA: 3919 SN: Time (min).18

11 Nalbuphine Good Linearity in the Desired Range Area Ratio Nalbuphine Y = e-*X R^2 =.994 W: 1/X^2. ng/ml Calibrator ng/ml % Difference Note: 4 point cal curve. Objective was semi quantitative analysis 11

12 Nalbuphine S/N = ng/ml std - m/z= RT:.72 SM: -.72 NL:7.3E3 F: FTMS {,} + p ESI Full ms [ ] Relative Intensity RT: 3.22 AA: 2439 SN: Time (min) 12

13 Fentanyl Good Linearity in the Desired Range Fentanyl Y = *X R^2 =.998 W: 1/X^ ng/ml Area Ratio Calibrator ng/ml % Difference Note: 4 point cal curve. Objective was semi quantitative analysis 13

14 Fentanyl Calibrator S/N = ng/ml std - m/z= RT:2.2 SM: NL:7.41E4 F: FTMS {,} + p ESI Full ms [ ] Relative Intensity RT: 4.2 AA: SN: Time (min) 14

15 Norfentanyl Calibrator Good Linearity in the Desired Range Norfentanyl Y = *X R^2 =.9898 W: 1/X^2 Area Ratio ng/ml Calibrator ng/ml % Difference Note: 4 point cal curve. Objective was semi quantitative analysis 1

16 Norfentanyl Calibrator S/N = ng/ml std - m/z= RT:.81 SM: -.81 NL:1.E4 F: FTMS {,} + p ESI Full ms [ ] Relative Intensity RT: 3.31 AA: 6678 SN: Time (min) 16

17 Alfentanil Good Linearity in the Desired Range Area Ratio Alfentanil Y = *X R^2 =.9994 W: 1/X^ ng/ml Calibrator ng/ml % Difference Note: 4 point cal curve. Objective was semi quantitative analysis

18 Alfentanil Calibrator S/N Very ng/ml std - m/z= RT:1.8 SM: -6.8 NL:9.67E4 F: FTMS {,} + p ESI Full ms [ ] Relative Intensity RT: 4.3 AA: SN: INF Time (min) 18

19 Sulfentanil Good Linearity in the Desired Range Sufentanil Y = *X R^2 =.9971 W: 1/X^2 Calibrator ng/ml % Difference Area Ratio ng/ml Note: 4 point cal curve. Objective was semi quantitative analysis 19

20 Sulfentanil Calibrator S/N = ng/ml std - m/z= RT:2.23 SM: -7.2 NL:1.14E F: FTMS {,} + p ESI Full ms [ ] Relative Intensity RT: 4.73 AA: 773 SN: Time (min)

21 Butorphanol Good Linearity in the Desired Range Butorphanol Y = *X R^2 =.9997 W: 1/X.. Calibrator ng/ml % Difference Area Ratio ng/ml Note: 4 point cal curve. Objective was semi quantitative analysis 21

22 Butorphanol Calibrator S/N = ng/ml std - m/z= SM: RT: NL: 4.62E4 F: FTMS {,} + p ESI Full ms [ ] Relative Intensity RT: 4.18 AA: 2173 SN: Time (min) 22

23 Quality Control Samples QC lowest calibrator concn. ( ng/ml) Acceptance Criteria Values </= % of the target value Target Value from the supplier ng/ml 23

24 QC Results Ketamine 7.8 ng/ml qcfent6 - m/z= RT:.79 -SM:.79 NL:1.92E4 F: FTMS {,} + p ESI Full ms [ ] Relative Intensity RT: 3.29 AA: 9244 SN: Time (min) Norketamine 46.2 ng/ml qcfent6 - m/z= RT:.66 -SM:.66 NL:6.34E3 F: FTMS {,} + p ESI Full ms [ ] Relative Intensity RT: 3.16 AA: 3111 SN: Time (min) 24

25 QC Results Nalbuphine 9. ng/ml Fentanyl 8. ng/ml qcfent6 - m/z= RT: NL:9.31E3 SM: F: FTMS {,} + p ESI Full ms [ ] Relative Intensity RT: 3.18 MA: SN: NA Time (min) qcfent6 - m/z= RT: NL:9.3E4 SM: F: FTMS {,} + p ESI Full ms [ ] Relative Intensity RT: 4.1 AA: 4878 SN: Time (min) 2

26 QC Results Norfentanyl 4.3 ng/ml Alfentanil 9. ng/ml qcfent6 - m/z= RT: NL:1.7E4 SM: F: FTMS {,} + p ESI Full ms [ ] Relative Intensity 1.2 RT: 3.31 AA: 7 SN: Time (min) qcfent6 - m/z= RT: NL:1.E SM: F: FTMS {,} + p ESI Full ms [ ] Relative Intensity RT: 4.36 AA: SN: Time (min) 26

27 QC Results Sulfentanil 49.4 ng/ml Butorphanol. ng/ml qcfent6 - m/z= RT:2.2 SM: - 7.2NL:1.9E F: FTMS {,} + p ESI Full ms [ ] Relative Intensity RT: 4.7 AA: SN: Time (min).9 qcfent6 - m/z= RT:1.67 SM: NL:6.18E4 F: FTMS {,} + p ESI Full ms [ ] Relative Intensity RT: 4.17 MA: SN: NA Time (min) 27

28 Conclusion Method met the requirements for a semi quantitative assay It is very cost efficient Full calibration and further validation will replace immunoassay The method is expected to be the only method used in the lab 28

29 Appendix 29

30 LC method Analytical Column: GOLD PFP, x 2.1 mm, um Column temp: room temperature Mobile phases A: mm Ammonium Acetate and.1% FA in DI water B:.1% FA in ACN LC gradient Time %A %B Flow (ul/min)

31 MS method HESI in positive ionization Spray voltage: Heater temp: Capillary temp: 3 Sheet gas: Aux gas: 31

32 Data acquisition method Positive ionization mode Full scan data Mass range: Resolution: Max Injection time: ms 32

33 Data processing method Chromatographic peaks were obtained by reconstructing analytes and internal standard masses with accuracy of ppm. Analyte to internal standard peaks area ratios were used in quantitative calculations 33

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