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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