Quantitative Screening of 46 Illicit Drugs in Urine using Exactive Ultrahigh Resolution and Accurate Mass system Kevin Mchale Thermo Fisher Scientific, San Jose CA
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
Presentation overview Exactive system introduction Principle of operation Method overview and salient features Urine analysis results Conclusions Appendix 3
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 5-4 4 Polarity switching One positive and one negative scan < 1 second (25K Resolution)
Orbitrap Principle of Operation r z φ Hyper-logarithmic potential distribution: ideal Kingdon trap k 2 2 2 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, 1156-1162.
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
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
Results - XIC@ ± 5 ppm at LOQs in urine R T :.5-4.5 5 2 2 4 2 5 5 4 2 2 S M : 7G AA : 15 961 AA : 187 81 A AA : 11 446 5 A A: 7 511 8 B C D AA : 37 32 A A: 95 964 A A: 16 41 61 A A: 5 295 8 E A A: 2 498 AA : 36 98 F AA : 636 32 A A: 1 452 7 AA : 65 25 AA: 231 75 AA : 15 46 1 G A A: 1 17 77 H 1 2 3 4 Tim e (m in) R T : 4. - 5.5 2 1 4 2 2 5 6 4 2 2 1 6 4 2 S M : 7G AA : 68 965 I A A: 15 19 4 J A A: 95 14 3 K AA: 2 13 492 L A A: 19 327 5 M AA : 57 388 N A A: 1 798 96 O 4. 4.5 5. 5.5 T im e (m in ) R T : 4. - 7. 2 1 5 2 1 2 2 1 1 5 2 4 2 S M : 7G AA: 7 37 68 P AA: 245 518 Q A A: 7 5 R A A: 8 96 9 S A A: 6 219 5 AA: 6 264 A A: 92 81 9 T AA: 344 575 U AA : 1 573 7 V A A: 18 74 8 W 4 5 6 7 T im e (m in) 8 Note: Drug names and LOQs on slide 9 & 1
Results - XIC @± 5ppm at LOQs in urine. 9 RT: 6.25-7.75 5 5 4 2 2 2 2 1 1 5 1 S M: 7G AA : 21 467 5 AA A A: 1 837 64 BB A A: 1 13 82 CC A A: 8 31 5 DD A A: 8 413 EE A A: 49 967 FF AA: 371 1 6.5 7. 7.5 Time (min) GG A A: 3 517 4 HH RT: 7.25-8.25 S M: 7G A A: 4 384 4 1 5 2 1 2 4 2 1 5 2 1 6 4 2 Note: Drug names and LOQs on slide 9 & 1 AA : 46 52 JJ II AA : 764 58 KK A A: 1 644 73 LL MM A A: 3 491 7 A A: 6 548 2 NN AA: 158 753 OO AA: 9 34 9 7.4 7.6 7.8 8. 8.2 Time (min) RT: 7.75-9. S M: 7G AA : 17 777 78 4 2 5 2 4 2 1 5 2 1 5 1 5 PP A A: 1 726 25 A A: 79 129 QQ RR AA: 135 478 SS AA: 4 64 1 TT AA : 8 28 UU AA : 37 469 8 VV AA: 3 588 98 WW 8. 8.5 9. Time (min)
List of drugs and mass errors @ LOQ 1
List of drugs monitored and mass errors @ LOQ 11
Need for 5, resolution Nicotine mass spectra at experimental (Exp) and different simulated (Sim) resolutions (FWHM) 12 1 5 1 5 1 5 1 5 163.1229 C 1 H 15 N 2 163.123 C 1 H 15 N 2 163.1 222 C 1 H 15 N 2 163.1326 C 8 H 19 O 3 163.1329 C 8 H 19 O 3 163.1328 C 8 H 19 O 3 163.129 6 C 4 H 15 O N 6 Exp: 5, Sim: 5, 163.1 163.15 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 Res.Pwr. @FWHM 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 Res.Pwr. @FWHM 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 Res.Pwr. @FWHM Note: Experimental Nicotine at RT 1.4 min; exact mass = 163.123
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
Appendix 14
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