Imir Metushi (Presenter)
UC San Diego Health
Bio: Imir finished his BS in Chemistry from York University (Toronto, Canada) specializing in analytical chemistry. He then went to pursue a Ph.D in Pharmacology at the University of Toronto where he developed an interest in biomarker development for adverse drug reactions (ADRs). He did a one year post doc at the La Jolla Institute for Allergy and Immunology where he worked on the development of immunoassays used to pre screen for drugs that could have an HLA-linked ADR. He has 11 first author publications in high quality journals and has given several oral presentations in meetings such as: Society of Toxicology and Gordon Conference on Drug Metabolism. His interests are in biomarker discovery as well as performing mass spectrometry applications utilized in the toxicology laboratory. He is currently a Clinical Chemistry Fellow at UCSD.
Authorship: Imir G. Metushi (1), Robert Fitzgerald (1)
(1) UC San Diego Health, Department of Pathology, San Diego CA
Urine drug screening is among the most widely used procedures in clinical toxicology laboratories. Recently, high resolution mass spectrometry (HRMS) such as time of flight-mass spectrometry (TOF-MS) has been proposed as an alternative for non-targeted drug screening. This session will discuss the applications of time-of-flight high resolution mass spectrometry (TOF-HRMS) in toxicology during drug testing and illustrates the approach that our institution has taken in validating a TOF-HRMS method for urine drug screening.
Urine drug screening is among the most widely used procedures in clinical toxicology laboratories. Immunoassay is the primary method used for drug screening in many laboratories. Positive immunoassay drug screens are often confirmated with liquid chromatography coupled to unit resolution tandem mass spectrometry (LC-MS/MS). LC-MS/MS isolates the protonated analyte [(M+H)+] of interest followed by fragmentation in the collision cell and monitoring of one or more product ions. However, this method is limited by: 1) co-eluting compounds of similar structure 2) fragmentation that does not always produce product ions, and 3) difficulties performing non-targeted screening. Recently, high resolution mass spectrometry (HRMS) such as time of flight-mass spectrometry (TOF-MS) has been proposed as an alternative for non-targeted drug screening.
TOF-MS offers several advantages to the traditional low-resolution tandem MS, some of which include: greater specificity and the ability to retrospectively analyze data without the need to have a reference standard. Therefore, HRMS has the ability to perform broad spectrum screening with superior specificity than immunoassay without the need for additional confirmation by LC-MS/MS. We validated a UPLC-TOF HRMS method for drug screening by assessing reproducibility (within run and between run precision), patient comparison studies and carry over effects.
Sixty one drugs were divided into six groups at three different concentrations (100 ng/mL, 1000 ng/mL and 5000 ng/mL) and within run (five injections), between run (twenty injections over 20 days) and patient comparison studies were performed. Identification criteria for a positive result involved accurate mass (within 5 ppm), one fragment (within 10 ppm) and retention time match (within 0.2 min). Chromatographic separation was achieved using Waters UPLC BEH C18 guard column (1.7 μm, 5 mm) and analytical column (1.7 μm, 2.1 × 150 mm) with flow rate of 0.4 mL/min at 50°C. Mobile phase A and B were 5 mmol/L ammonium formate (pH 3) and 0.1% formic acid in acetonitrile, respectively. Five percent B was maintained for initial 0.5 min, increased linearly to 50% B at 6 min, then to 95% B at 7 min, where it was held for 2 min. Reconditioning of the column was achieved by dropping the gradient to 5% B at 9.05 min and maintaining it for 2 min. We used a TOF-MS (Xevo G2 TOF; Waters Corp) with a resolution of 20,000; cone voltage of 20V; capillary voltage of 0.8 kV; source block temperature of 130 °C; and desolvation gas flow of 1000 L/H at 450 °C. Data were acquired in profile mode with MassLynx software version 4.1, SCN 862 (Waters Corp). UNIFI (v 1.6.1) was used as the software for processing data. A MRM-based LC-MS/MS (UPLC-Xevo TQ-S) was used to confirm positive results and served as the reference method.
Within run studies revealed that out of 5 injections, norpropoxyphene, EDDP and tramadol were not identified at a low concentration of 100 ng/mL; however, detection greatly improved at the higher concentrations of 1000 ng/mL and 5000 ng/mL. Increasing the mass error threshold limit from 5 ppm to 10 ppm increased the positive identification rate for EDDP but it did not make a difference for norpropoxyphene and tramadol. The remaining drugs were all identified 5/5 times at all three concentrations. Between run precision studies revealed that, as in the case of within run precision, norpropoxyphene and tramadol were not identified at 100 ng/mL. In addition, other drugs such as morphine, norbuprenorphine, propoxyphene, EDDP and MDA were identified 17/20, 14/20, 17/20, 6/20 and 18/20 times respectively at a concentration of 100 ng/mL. Upon increasing the concentration of these drugs to 1000 ng/mL and 5000 ng/mL the detection rate improved significantly. Changing the mass error detection window for the parent ion from 5 ppm to 3 ppm greatly reduced the number of drugs identified. Initial patient comparison studies for 23/61 drugs in the panel were performed. The LC-MS/MS method identified a total of 244 compounds while the TOF-HRMS method identified 190 and 188 compounds respectively when samples were run in duplicate. The drugs with the highest false positive rate of identification were oxycodone, EDDP, hydrocodone, lorazepam and methamphetamine. Drugs with the highest false negative rate identification were: fentanyl, morphine, norfentanyl, hydroxyalprazolam, amphetamine, norfentanyl, methamphetamine and nordiazepam. TOF-HRMS was able to identify a number of drugs that were not part of the normal confirmation panel through LC-MS/MS.
Our UPLC-TOF HRMS method for drug screening offers the advantage of performing non-targeted drug screening (compared to MRM based LC-MS/MS) and has the potential for higher sensitivity and specificity than conventional immunoassays. Current validation studies show that at low drug concentrations, some of the drugs such as norpropoxyphene and tramadol were missed, mainly because due to the lack of fragment identification. However, upon increasing the drug concentrations the identification status improved significantly. Patient comparison studies indicate that some drugs such as oxycodone and methamphetamine show a higher rate of false positive identification when using TOF HRMS and results should be interpreted with caution or in conjunction with another confirmatory method.
References & Acknowledgements:
IP Royalty: no
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