Automated and Fully Validated High-Throughput LC-MS/MS Assay for Analyzing Multiple Drugs of Abuse in Oral Fluids Using Novel Features in Sample Preparation and Chromatographic Conditions

Abstract

Oral fluid sampling offers advantages over other biological matrices, mainly due to its noninvasive procedure avoiding privacy intrusion. The fully automated sample preparation procedure is based on salting-out assisted liquid-liquid extraction (SALLE) combined with high-efficiency LC-MS/MS methods for both screening and confirmation of 37 drugs and incorporates novel features enabling direct injection of acetonitrile extracts into an innovative chromatographic system. The methods' drug panel includes opioids, benzodiazepines, benzodiazepine-like drugs, cannabinoids, and stimulants. A full method validation was performed using OF/buffer from Greiner Bio-ONE International and Quantisal saliva collection devices. The validation included assessments of linearity, sensitivity, precision, accuracy, extraction recovery, matrix effects, process efficiency, stability, and carryover. All compounds demonstrated linearity across the concentration range 1-25 ng/mL, with R² ≥ 0.99. Both methods' limit of detection ranged between 0.001 and 0.03 ng/mL, and the limit of quantification ranged between 0.02 and 0.09 ng/mL. Precision was ≤ 14.8% for screening and ≤ 8.5% for the confirmation method. Accuracy was ± 13.6% for screening and ± 8.7% (except at 0.5 and 1 ng/mL, where it was ± 25.3% and ± 17.6%, respectively) for the confirmation method. Extraction recoveries ranged from 40.0% to 95.1%, except for hydromorphone (27.4%) and morphine (34.4%). Although matrix effects were observed for a large number of compounds to varying degrees, they were largely compensated for by the use of deuterium- and ¹³C-labeled internal standards (IS). IS-corrected overall process efficiency ranged from 100.7% to 119.1% with precision (CV%) ≤ 10.8% for all compounds. Spiked calibrators and QC samples in OF were stable in autosampler for up to 72 h and in the freezer for 3 days. Methanol working solutions were stable for 6 months. No significant carryover was observed. The methods have been successfully implemented in the routine analysis of approximately > 1000 samples per month since March 2024.

Keywords: Greiner Bio‐One; LC‐MS/MS; Quantisal; SALLE; drugs of abuse; liquid chromatography tandem mass spectrometry; oral fluid; saliva; salting‐out assisted liquid–liquid extraction.

FIGURE 1

The EIC of the Q1 product ions in the confirmation method for the 1 ng/mL calibrator.

FIGURE 2

The chromatogram showing peaks of mostly polar compounds when saturated AcN extract of OF injected onto the RP C18 column.

FIGURE 3

The chromatogram showing peaks of polar compounds when saturated AcN extract of OF injected onto the RP PS C18 column.

FIGURE 4

The chromatogram showing peaks of mostly polar compounds when saturated AcN extract of OF prepared calibrator 1 ng/mL after addition of 30‐μL ammonium bicarbonate injected onto the RP PS C18 column.

FIGURE 5

(a, b) The MS response for a selected number of compounds chosen with different polarities using the following salting‐out reagents: 10‐M AmAc, 10‐ and 12‐M AmF, 6.2‐M NaCl, and 2.9‐M MgSO₄. The displayed area represents the average across three replicates.

FIGURE 6

The structure of Tramadol, Venlafaxine, and its metabolites.

FIGURE 7

MRM for drugs of abuse in saliva showing the detection of tramadol in the real patient sample containing ODMV and tramadol 25 ng/mL

FIGURE 8

(a, b) The distribution of positive findings screened by LC‐MS/MS positives during a period of 3 months.

FIGURE 9

Number of positive findings per sample screened positive by LC‐MS/MS.