Quantitative Screening of 46 Illicit Drugs in Urine using Exactive Ultrahigh Resolution and Accurate Mass system

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1 Quantitative Screening of 46 Illicit Drugs in Urine using Exactive Ultrahigh Resolution and Accurate Mass system Kevin Mchale Thermo Fisher Scientific, San Jose CA

2 Presentation Overview Anabolic androgenic steroids landscape Triple quadrupole MS/MS characteristics and performance AAS behavior in ESI-MS Sample preparation methods Orbitrap high resolution mass spectrometry Quantitative analysis of AAS in OF using Q Exactive Testosterone in serum using TurboFlow and Q Exactive Summary 2

3 Presentation overview Exactive system introduction Principle of operation Method overview and salient features Urine analysis results Conclusions Appendix 3

4 Exactive Benchtop LC-MS Resolution 1, at 1 scan per second 1, at 1 scans per second Mass accuracy Sub ppm Sensitivity 5 fg Buspirone with S/N >1:1 Dynamic range >1, within a spectrum Scan speed Up to 1 scans per second Mass range m/z Polarity switching One positive and one negative scan < 1 second (25K Resolution)

5 Orbitrap Principle of Operation r z φ Hyper-logarithmic potential distribution: ideal Kingdon trap k U ( r, z) = { z r / 2 + Rm ln( r / R m )} 2 Characteristic frequencies: Frequency of rotation ω φ Frequency of radial oscillations ω r Frequency of axial oscillations ω z ω z = k m / q ω z R m 2 R m ω ϕ = 1 ω r = ω z 2 2 R R 2 5 Makarov A. Anal. Chem. 2, 72,

6 Method overview and salient features Negative standard urine diluted with 25% ACN spiked with 46 drugs at different concentrations. Isotopically labeled internal standards at constant 5 ng/ml. No sample preparation. A 1 ul aliquot directly injected onto reverse phase column. Exactive operated at a resolution of 5, FWHH External calibration mass accuracy better than 5 ppm. Analysis time <1 minutes Note: LC/MS method details in appendix 6

7 Method overview and salient features Negative urine sample Dilute with 25% ACN Spike 46 drugs at different concentrations Spike isotopically labeled int standards at 5 ng/ml Inject 1 ul Hypersil GOLD PFP 2.1 x 1 mm, 3 µm Note: LC/MS method details in appendix Operated at res. 5, w/ Ext. Calib. Mass accuracy better than 5 ppm 7

8 Results - XIC@ ± 5 ppm at LOQs in urine R T : S M : 7G AA : AA : A AA : A A: B C D AA : A A: A A: A A: E A A: AA : F AA : A A: AA : AA: AA : G A A: H Tim e (m in) R T : S M : 7G AA : I A A: J A A: K AA: L A A: M AA : N A A: O T im e (m in ) R T : S M : 7G AA: P AA: Q A A: 7 5 R A A: S A A: AA: A A: T AA: U AA : V A A: W T im e (m in) 8 Note: Drug names and LOQs on slide 9 & 1

9 Results - 5ppm at LOQs in urine. 9 RT: S M: 7G AA : AA A A: BB A A: CC A A: DD A A: EE A A: FF AA: Time (min) GG A A: HH RT: S M: 7G A A: Note: Drug names and LOQs on slide 9 & 1 AA : JJ II AA : KK A A: LL MM A A: A A: NN AA: OO AA: Time (min) RT: S M: 7G AA : PP A A: A A: QQ RR AA: SS AA: TT AA : 8 28 UU AA : VV AA: WW Time (min)

10 List of drugs and mass LOQ 1

11 List of drugs monitored and mass LOQ 11

12 Need for 5, resolution Nicotine mass spectra at experimental (Exp) and different simulated (Sim) resolutions (FWHM) C 1 H 15 N C 1 H 15 N C 1 H 15 N C 8 H 19 O C 8 H 19 O C 8 H 19 O C 4 H 15 O N 6 Exp: 5, Sim: 5, m/z Sim: 2, Sim: 1, NL: 1.5 3E5 drugs_abuse_urine_5k_313#18 1 RT: 1.4 AV: 1 T: FTMS {,} + p ESI Full ms [11.-8.] NL: 5.6 7E4 C 8 H 18 O 3 *1. + C 1 H 14 N 2 *.5 +H: p (gss, s /p:15) Chrg 1 R: 5 NL: 5.7 4E4 C 8 H 18 O 3 *1. + C 1 H 14 N 2 *.5 +H: p (gss, s /p:15) Chrg 1 R: 2 NL: 6.9 1E4 C 8 H 18 O 3 *1. + C 1 H 14 N 2 *.5 +H: p (gss, s /p:15) Chrg 1 R: 1 Note: Experimental Nicotine at RT 1.4 min; exact mass =

13 Conclusions The method is capable of easy confirmatory and quantitative analysis of 46 illicit drugs at LOQs of.5 5 ng/ml in urine. Ultrahigh mass resolution of 5, FWHM provided the necessary selectivity to identify 46 drugs in urine in 9 minutes with minimal sample preparation Analyte confirmation is achieved by utilizing better than 5 ppm mass accuracy with external mass calibration. Good quantitative accuracy achieved by isotopically-labeled standard. Nicotine example demonstrates the need for 5, resolution in addition to exact masses. 13

14 Appendix 14

15 LC/MS Method HPLC system Accela UHPLC Pump, PAL autosampler (CTC Analytics) LC Column 2.1 x 1 mm, 3 µm Hypersil GOLD PFP Injection Volume = 1 µl Mobile Phase (A) 5 mm NH4OOCH +.1% HCOOH, (B) ACN +.1% HCOOH Flow Rate.35 ml/min MS system Exactive (Thermo Scientific) Heated ESI; Polarity: Positive; Scan Range: m/z 11 8 Resolution: 5, FWHM; External mass calibration 15

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