[Live] Efficiency and Quality in LC/MS Urine Drug Testing

Wednesday, January 30, 2019 – 11:00am EDT / 8:00am PDT / 3:00pm GMT
Duration: 1 hour(s)
Speaker: Dr. Jan Palaty
Sponsored by Kura Biotec and Agilent Technologies

Broad spectrum LC/MS is a powerful tool to expand the scope and reliability of urine drug forensic analysis. Methods suitable to the mid-size lab for optimizing workflow and adding extensive quality control measures will be discussed.

Broad spectrum (e.g. 30-50 compounds or more) LC/MS is now widely used by forensic laboratories of all sizes to monitor drugs in urine. In this webinar, I will discuss experience gained from two distinct implementations in our mid-sized laboratories which handle several hundred samples daily. Both used automated mixed-mode SPE of hydrolyzed urine samples (Kura BG100® for former, Kura BGTURBO® for latter) but the latter used a liquid handler for sample pipetting followed by incubation of the entire SPE plate with a high-activity recombinant enzyme. The first implementation used qualitative analysis for 150 drugs/metabolites whereas the second used a 6- or 7-point polynomial calibration curve for 65 compounds. Analyses were performed using biphenyl columns, with or without alternating column regeneration to increase throughput. Carryover proved problematic for compounds such as fentanyl and methadone. The use of in-house spreadsheets to track inventory and perform calibrator pipetting calculations was invaluable for standards management, optimizing calibration mixtures, updating the test menu and providing a clear audit trail for calibrator preparation. Method validation was guided by the SWGTOX guidelines: data from both sites will be presented.

As the manual review of data from such methods is potentially time-intensive and subjective, an extensive Microsoft Excel-based program (‘Griffin’) was constructed to deal with both concerns. This program uses exception-based reporting to track both pre-analytical (e.g. sample dilution, adulteration) and analytical issues such as low internal standard areas, carryover, cross-talk (e.g.between wells of SPE plate) and efficiency of hydrolysis. Compounds are identified by Griffin in a graded approach which exploits the fact that urine usually contains both parent and metabolite of a given drug. Accordingly, every compound is identified by two MRMs based on retention time and ion ratio using CLSI-suggested tolerances: these two MRMs may belong to parent and metabolite (one each) or to the target compound exclusively. The grading system assigns a suffix to compounds not meeting the full identification criteria which helps guide the operator in determining whether or not the detected peak truly corresponds to the target compound. Nevertheless, a small number of early-eluting compounds (e.g. morphine, hydromorphone, gabapentin) did not give reliable results, necessitating their individual manual review. After all tentative identifications have been accepted or rejected by the user, Griffin constructs a text string report for each sample in the batch which is then uploaded as a single comma-delimited file to the laboratory information system. A network drive is used to save the batch raw data and reports as a single Excel workbook, a format which greatly facilitates any subsequent inquiries or data analysis compared with that of a typical MS instrument file.

Given that a large fraction of the drugs in a typical LC/MS panel is not detected in any sample, the calibration/QC review process was streamlined for those compounds by reducing the acceptance criteria from a quantitative method to those of a qualitative method only (i.e. lower required r2 for calibrators, fewer required levels and wider QC tolerance). All of these steps have allowed a diverse group of users to consistently report a typical batch of 60-80 samples in well under an hour.

Detailed Learning Objectives include:

1. Configuration of a mid-size (65 compound) LC/MS method.
2. Detection of analytical and pre-analytical errors.
3. Using Excel middleware for data reduction and quality control.

*For Forensic Use.

Price: Funding for this course has been provided by Agilent Technologies

Course Type: On-Demand Presentation
Length: Approximately 60 minutes
Audience: Forensic Practitioners
Course Accreditation: Certificate of Attendance