SAMHSA drug panel screening in oral fluid: Development of screening method at 8 seconds per sample using LDTD-MS/MS

Abstract

In 2019, the US Department of Health and Human Services (via the SAMHSA agency) has established scientific and technical guidelines for federal workplace drug testing programs in oral fluids (Federal Register/Vol. 84, No. 207, 2019). Screening various drug classes requires several different immunoassay reagents or an LC-MS/MS method with a longer analysis time per sample. LDTD-MS/MS technology combines the speed and the analysis of different drug classes with a single method. The goal of this presentation is to use an automated sample preparation method for LDTD-MS/MS screening of all compounds in a single operation. Quantisal collection devices were used in this method. Drug-free oral fluids of different volunteers were collected using the Quantisal device. After the collection of the oral fluid, the pad is transferred into a tube containing an extraction buffer. During this process, oral fluids are diluted by a factor of 3. The SAMHSA drug panel for an Oral fluid screen was spiked in extracts at a concentration around the decision point cut-off (Amphetamine: 50 ng/mL, Methamphetamine: 50 ng/mL, MDA: 50 ng/mL, MDMA: 50 ng/mL, PCP: 100 ng/mL, Morphine: 30 ng/mL, Hydromorphone: 30 ng/mL, Codeine: 30 ng/mL, Hydrocodone: 30 ng/mL, Cocaine: 30 ng/mL, Oxymorphone: 30 ng/mL, Oxycodone: 30 ng/mL, 6-AM: 4 ng/mL and THC: 4 ng/mL). Samples were extracted using the Azeo automated extraction system. The robot scans the barcode of the sample, generates a batch file for the LDTD-MS/MS system, extracts the sample and spots the LazWell plate. Salt assisted liquid-liquid extraction (SALLE) was used as the sample preparation method. LDTD (Phytronix Luxon) uses a carrier gas (air) flow rate of 6 L/min with water infusion at 20 uL/sec. Samples were desorbed from the surface of the plate using a laser ramp of 6 seconds to 65% power and 2 seconds hold at 65% power. Mass spectrometer (Shimadzu 8060) operates in positive MRM mode with a specific transition for the following drugs: Amphetamine, Methamphetamine, MDA, MDMA, PCP, Morphine, Hydromorphone, Codeine, Hydrocodone, Cocaine, Oxymorphone, Oxycodone, 6-AM and THC. Data acquisition systems of mass spectrometers were not designed to deal with signals of only a few seconds per sample. The synchronization sequence adds 6 to more than 15 seconds between each sample. To bypass this, all samples are acquired in a single file. To allow the analysis of such data, Cascade™ software is designed to detect, split, and integrate every sample peak acquired in a single file. Spiked samples around the decision point and blank solutions are used to validate the precision of the method. Each concentration must not exceed 20% CV and the mean concentration ± 2 times the standard deviation must not overlap with other concentrations at the decision point. The peak area against the IS ratio was used to normalize the signal. Replicate extractions are deposited on a LazWell plate and dried before analysis. No overlapping at the decision point is observed for all curves and the CV% was below 15% for within-run experiments. A dry and wet stability of sample extracts were performed. Precision criteria were applied. All results are within the acceptable criteria range. Oral fluids are collected from eight different volunteers. Samples are screened to verify the presence of each analyte (all samples were negative). Drugs are spiked at 50% cut-off (QC-L) and 200% cut-off (QC-H) and screened as unknown to verify the method sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy. A percentage of 100% was obtained for all validation parameters of all drugs. Luxon Ion Source® combined to a Shimadzu 8060 mass spectrometer system allows ultra-fast (8 seconds per sample) screening of a SAMHSA drug panel in oral fluid using a simple and automated sample preparation method.