Title: Liquid chromatography high resolution time of flight analysis: investigation of MSE for broad-spectrum drug screening

Chindarkaret al.

List of supplemental materials

  1. Materials and Methods
  2. Materials
  3. TOF-MS: Lock spray settings
  4. LC-MS/MS conditions
  5. Analytes selection
  6. References
  7. Supplemental Data Table 1.Comparison of overall results of UPLC-TOF-MS analysis with reference method results
  8. Supplemental Data Table 2. List of compounds and their cutoff concentration (ng/mL) included in reference methods compared to UPLC-TOF-MS method. Each sample set was analyzed for listed compounds by respective method. All four sample sets were analyzed for 61 compounds by UPLC-TOF-MS method.

Materials and Methods

Materials

Sixty one reference analytes and five deuterium labeled internal standards used in this report were purchased from Cerilliant (Round Rock TX, USA). Methanol, acetonitrile, and formic acid (all optima LC/MS grade) were purchased from Fisher Scientific (Fair Lawn, NJ). β-Glucuronidase (Helix pomatia) was purchased from Sigma Aldrich (St. Louis, MO, USA). Deionized (DI) water (18.2 MΩ-cm) was produced in-house by Elga, Purelab Ultra system (ELGA LLC, Woodridge, IL, USA). Mass calibration for time of flight-mass spectrometer (TOF) was performed with sodium formate [10% formic acid:0.1 M sodium hydroxide:acetonitrile (1:1:8)]. Leucine enkephalin used for lock mass and detector setup was supplied by Waters Corporation (Milford, MA, USA).

TOF-MS: Lock spray settings

The Xevo G2 time of flight-mass spectrometer (TOF-MS) had two electrospray tips in the ion source isolated by a moving baffle. One electrospray tip was for the LC flow (specimen) and the other was for lock-mass spray. During the data acquisition, 2 ng/µL of Leucine Enkephalin in acetonitrile: 0.1% formic acid (50:50 v/v) was continuously infused through the lock mass spray tip at 5 µL/minute and the lock mass data were acquired every 15 sec for 0.1 sec. For the rest of the time, the baffle in the ion source blocked the entry of lock spray into the MS, allowing only the LC flow (specimen) into MS.

LC-MS/MS conditions

A UPLC-tandem quadrupoleMS (Xevo TQ-S, Waters) was used to confirm additional positivesthat were identified by the UPLC-TOF-MS method but were not detected by the initial reference methods. The conditions employed were cone voltage, 20 V; capillary voltage, 1.0 kV; source block temperature, 150°C; and desolvation gas flow of 1000 L/h at 500°C. Data were acquired with MassLynx (Waters) software and processed with TargetLynx(Waters) software.Confirmation using the quadrupole MS were based on two multiple reaction monitoring (MRM) transitions for each of the 61 compounds of interest. A positive result on the tandem confirmation consisted of a retention time match (± 0.2 min), S/N of greater than 100, analyte response area of greater than theresponse of 10ng/mL concentration (spiked in drug free urine and subjected to DHS), presence of parent or metabolite meeting above criteria and/or ion ratios within ± 30% of the drug standards.

Analytes selection

The target analytes in this broad-spectrum screen were selected based on ‘2011 Annual Report of the American Association of Poison Control Centers National Poison Data System (NPDS): 29th Annual Report’(1), ‘Results from the 2012 National Survey on Drug Use and Health (NSDUH): Detailed Tables’ (2), ‘National Institute on Drug Abuse: Trends and Statistics’ (3), drug profiles provided by The European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) (4), and consultation with the emergency department physicians at UC San Diego Health System.After this review, we decided to focus on basic and neutral analytes because they were involved in more toxicology cases. Omitting acidic compounds allowed us to avoid using negative mode ionization, thus improving MS duty cycle time and maximizing the sensitivity of the assay. A list of these compounds is shown in Table 1 in the main text.

References

1.Bronstein AC, Spyker DA, Cantilena LR, Jr., Rumack BH, Dart RC. 2011 Annual report of the American Association of Poison Control Centers' National Poison Data System (NPDS): 29th Annual Report. Clin Toxicol (Phila) 2012;50:911-1164.

2.U.S. Department of Health and Human Services, Substance Abuse and Mental Health Services Administration, Center for Behavioral Health Statistics and Quality.Results from the 2012 National Survey on Drug Use and Health: August 20, 2013)

3.NIH: National Institute on Drug Abuse (NIDA). Trends & Statistics. (Accessed August 20, 2013)

4.The European Monitoring Centre for Drugs and Drug Addiction (EMCDDA). Drug Profiles; Available from: (Accessed August 20, 2013)

Supplemental Data Table 1.Comparison of overall results of UPLC-TOF-MS analysis with reference method results.

Specimen type / # of
specimen / rt, 20 ppm / rt, 5 ppm / rt, 5 ppm,
fragment / Reference
Positives / NF / NI / Total additional
positives
Benzodiazepines / 10 / 36 / 35 / 34 / 36 / 1 / 1 / 21
Opiates / 11 / 60 / 58 / 50 / 55 / 4 / 1 / 45
Postmortem / 15 / 70 / 67 / 49 / 53 / 4 / 0 / 38
Pain management / 25 / 166 / 146 / 120 / 131 / 8 / 3 / 32
Total / 61 / 332 / 306 / 253 / 275 / 17 / 5 / 136
False positive / - / 57 / 31 / - / - / - / - / -
Note : % false positive = (# positives – reference positives/reference positives ) x 100
NF: no fragment (for identified protonated analyte) NI: (protonated analyte) not identified; rt: retention time

Supplemental Data Table 2.List of compounds and their cutoff concentrations (ng/mL) included in reference methods compared to the UPLC-TOF-MS method. Each sample set was analyzed for listed compounds by respective methods.All four sample sets were analyzed for 61 compounds by the UPLC-TOF-MS method.

UPLC-TOF-MS / Reference method-1 / Reference method-2 / Reference method-3 / Reference method-4
Sample set 1 / Sample set 2 / Sample set 3 / Sample set 4
Opiates / Benzodiazepines / Postmortem / Pain management
α-hydroxyalprazolam / 20 / 20
6-acetylmorphine / 10 / 10 / 10
7-aminoclonazepam / 20 / 20
Amitriptyline / 50
Amphetamine / x / 100
Atenolol / x
Benzoylegonine / x / 50
Buprenorphine / 10
Carisoprodol / 100
Citalopram / x / 25
Codeine / 100 / x / 50
Cyclobenzaprine / 50
Dextromethorphan / 50
Diazepam
Diltiazem
Doxepine
EDDP / 100 / x / 100
Fentanyl / 100 / x / 2
Flunitrazepam
Hydrocodone / 100 / x / 50
Hydromorphone / 100 / 50
Ketamine / x / 50
Lorazepam / 20 / 40
MDA
MDEA
MDMA
MDPV
Meperidine / 50
Mephedrone / 3
Meprobamate / x / 100
Methadone / 100 / x / 100
Methamphetamine / x / 100
Methylone / 3
Methylphenidate / 50
Metoprolol
Morphine / 100 / x / 50
Norbuprenorphine / 20
Nordiazepam / 20 / 40
Norfentanyl / 10 / x / 8
Norhydrocodone / x / 50
Normeperidine / 50
Noroxycodone / 50
Norpropoxyphene / 100
Nortriptyline / 50
Oxazepam / 20 / 40
Oxycodone / 100 / x / 50
Oxymorphone / 100 / x / 50
Phencyclidine / 10
Promethazine / x
Propoxyphene / 100
Propranolol / x
Quetiapine / x
Tapentadol / 50
Temazepam / 20 / 50
Tramadol / x / 100
Trazadone
Venlafaxine / 100
Verapamil
Zaleplon
Zolpidem / 10
Zopiclone
Notes. EDDP: 2-ethylidine-1,5dimethyl-3,3-diphenylpyrrolidine; MDA: 3,4-methylenedioxyamphetamine; MDEA: 3,4-methylenedioxy-N-ethylamphetamine; MDMA: 3,4-methylenedioxy- N-methylamphetamine; MDPV: Methylenedioxypyrovalerone. x: no cut-off value was available

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