Screening and quantitation of drugs in oral fluids using Q Exactive system

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1 Screening and quantitation of drugs in oral fluids using Q Exactive system Marta Kozak November 28,2011

2 Goal To develop a robust and sensitive quantitative LC-MS/MS analysis method for drugs of abuse in oral fluid using Q Exactive. The data to be developed to demonstrate quant performance for a forensic lab in USA. 2

3 Samples info provided to TMO 3

4 Samples Orals fluid SPE processed samples prepared by Forensic Labs 0.1 ml of oral fluid was processed with SPE, evaporated and reconstituted in 200 ul of 20% ACN Calibrators and QC 5 levels calibrators: 1, 10, 100, 1000 and pg/ml Low QC concentration pg/ml 4

5 19 analytes and 14 internal standards 5

6 Instrumentation QExactive mass spectrometer with HESI source 600 Accela pump, CTC autosampler 6

7 LC method Column: 50x2.1 mm, 2.6 um Acucore PFP column Mobile Phase A: 0.2% FA in DIW B: 0.1% FA in MeOH Gradient: 4.42 min Injection volume: 50 ul 4.42 minutes LC gradient 7

8 MS method- Targeted SIM multiplexed 9 8

9 MS method - Inclusion list 9

10 Chromatogram of 10 pg/ml standard C:\Documents and Settings\...\10pg 12/2/2011 3:58:49 PM RT: SM: 3B Relative Abundance Nortriptyline RT: 1.97 MA: SN: 190RMS Time (min) RT: 2.32 MA: SN: INF RT: 1.77 MA: SN: 77RMS RT: 2.08 Doxepin MA: SN: INF RT: 2.22 MA: SN: INF NL: 2.43E5 O-DM Venlafaxine Venlafaxine DM Doxepin m/z= F: FTMS + p ESI SIM msx ms [ , , , , , , , , ] MS 10pg NL: 1.76E6 m/z= F: FTMS + p ESI SIM msx ms [ , , , , , , , , ] MS 10pg NL: 2.58E6 m/z= F: FTMS + p ESI SIM msx ms [ , , , , , , , , ] MS 10pg NL: 2.73E5 m/z= F: FTMS + p ESI SIM msx ms [ , , , , , , , , ] MS 10pg NL: 3.60E5 m/z= F: FTMS + p ESI SIM msx ms [ , , , , , , , , ] MS 10pg RT: Relative Abundance SM: 3B Maprotiline Norfluoxetine RT: 2.03 MA: SN: INF Citalopram Time (min) RT: 2.26 MA: SN: INF RT: 2.26 MA: SN: INF NL: 1.68E5 m/z= F: FTMS + p ESI SIM msx ms [ , , , , , , , , ] MS 10pg NL: 1.18E5 m/z= F: FTMS + p ESI SIM msx ms [ , , , , , , , , ] MS 10pg NL: 6.61E6 m/z= F: FTMS + p ESI SIM msx ms [ , , , , , , , , ] MS 10pg DM Citalopram RT: 2.16 NL: 3.77E5 MA: m/z= F: FTMS + p ESI SIM SN: INF msx ms [ , , , , , , , , ] MS 10pg 10

11 Chromatogram of 10 pg/ml standard C:\Documents and Settings\...\10pg 12/2/2011 3:58:49 PM RT: SM: 3B RT: 2.24 MA: SN: INF Desipramine NL: 2.50E5 m/z= F: FTMS + p ESI SIM msx ms [ , , , , , , , , ] MS 10pg RT: SM: 5B RT: 2.55 MA: SN: INF Sertraline NL: 3.21E4 m/z= F: FTMS + p ESI SIM msx ms [ , , , , , , , , ] MS 10pg RT: 2.14 MA: SN: INF Paroxetine NL: 1.51E5 m/z= F: FTMS + p ESI SIM msx ms [ , , , , , , , , ] MS 10pg RT: 2.28 MA: SN: INF Norsertraline NL: 1.19E4 m/z= F: FTMS + p ESI SIM msx ms [ , , , , , , , , ] MS 10pg Relative Abundance RT: 2.49 MA: SN: INF RT: 2.46 MA: SN: INF Fluoxetine Imipramine NL: 5.11E6 m/z= F: FTMS + p ESI SIM msx ms [ , , , , , , , , ] MS 10pg NL: 3.12E5 m/z= F: FTMS + p ESI SIM msx ms [ , , , , , , , , ] MS 10pg Relative Abundance Clomipramine RT: 2.90 MA: SN: INF 2.79 RT: 2.51 MA: SN: INF NL: 1.05E5 m/z= F: FTMS + p ESI SIM msx ms [ , , , , , , , , ] MS 10pg NL: 6.00E5 m/z= F: FTMS + p ESI SIM msx ms [ , , , , , , , , ] MS 10pg DM Clomipramine Amitriptyline Time (min) RT: 3.49 MA: SN: INF 3.71 NL: 1.05E6 m/z= F: FTMS + p ESI SIM msx ms [ , , , , , , , , ] MS 10pg RT: 2.68 MA: SN: INF Time (min) NL: 3.55E4 m/z= F: FTMS + p ESI SIM msx ms [ , , , , , , , , ] MS 10pg Trimipramine 11

12 LLOQ and linearity Analyte LOQ (pg/ml) Linear range R2 Amitriptyline Citalopram DM Citalopram (two data points) Clomipramine DM Clomipramine Desipramine Doxepin DM Doxepin Fluoxetine Norfluoxetine Imipramine Maprotiline Sertraline Norsertraline Nortriptyline Paroxentine Trimipramine Venlafaxine CDM Venlafaxine (quadratic) 12

13 QC sample (1 injection) Analyte Obtained concentration (pg/ml) Amitriptyline Citalopram DM Citalopram % Difference from expected mean Expected concentration: pg/ml Clomipramine DM Clomipramine Desipramine Doxepin DM Doxepin Fluoxetine Norfluoxetine Imipramine Maprotiline Sertraline Norsertraline Nortriptyline Paroxentine Trimipramine Venlafaxine CDM Venlafaxine

14 Conclusion All compounds can be detected in samples with concentration of 10 pg/ml. 9 out of 19 compounds can be quantified with LOQ of 10 pg/ml. 9 compounds can be quantified with LOQ of 100 pg/ml and 1 compound can be quantified with LOQ of 1000 pg/ml. Lower internal standards concentrations are recommended for methods quantifying in range of 10 pg/ml since contribution of analyte from deuterated internal standard is detected in blanks spiked with internal standards 14

15 Appendix 15

16 Amitriptyline 16

17 Citalopram 17

18 DM Citalopram 18

19 Clomipramine 19

20 DM Clomipramine 20

21 Desipramine 21

22 Doxepin 22

23 DM Doxepin 23

24 Fluoxetine 24

25 Norfluoxetine 25

26 Imipramine 26

27 Maprotiline 27

28 Sertraline 28

29 Norsertraline 29

30 Nortriptyline 30

31 Paroxentine 31

32 Trimipramine 32

33 Venlafaxine 33

34 O-DM Venlafaxine 34

35 Amitriptyline-D3 N=8 35

36 Citalopram-D6 36

37 Clomipramine-D3 37

38 Desipramine-D3 38

39 Doxepin-D3 39

40 Fluoxetine-D6 40

41 Imipramine-D3 41

42 Norfluoxetine-D6 42

43 Nortriptyline-D3 43

44 O-DM Venlafaxine-D6 44

45 Paroxetine-D6 45

46 Sertraline-D3 46

47 Trimipramine-D3 47

48 Venlafaxine-D6 48

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