LC–MS for Detecting 37 Drugs in Oral Fluids

Researchers from the Karolinska University Hospital in Stockholm, Sweden developed a new liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for analyzing drugs of abuse in oral fluids. Their findings were published in the Journal of Mass Spectrometry (1).

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Most methods for drug analysis have relied on techniques such as immunoassays and spectrophotometry, which lack specificity and sensitivity, the researchers wrote. However, in recent decades, mass spectrometry (MS) has become a powerful tool in toxicological laboratories, pushing various advancements in drug analysis.

MS is widely used in nearly all areas of clinical diagnostics, notably with drugs of abuse (DoA). It offers various advantages in measuring small molecules compared to immunoassays, which stem from its enhanced specificity, superior sensitivity, and adaptability to a wide variety of applications. The most abused drugs include opioids, cocaine, cannabis, amphetamines, and new psychoactive substances (NPSs), with most drug use disorders associated with cannabis and opioids. More sensitive methods need to be developed for detecting legal and illicit drugs in the fields of toxicology, roadside testing, workplace drug testing, and rehabilitation programs.

In this study, the scientists developed a fully automated sample preparation procedure 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, including benzodiazepines, cannabinoids, and stimulants. This method also incorporates novel features that enable direct injection of acetonitrile extracts into a new chromatographic system. According to the scientists, the method is capable of injecting saturated organic solvent directly into LC–MS/MS without sample extract evaporation, concentration or reconstitution, representing a novel chromatographic feature. The method was validated using oral fluid (OF)/buffer from different saliva collection devices, with the procedures involved including assessments of linearity, sensitivity, precision, accuracy, extraction recovery, matrix effects, process efficiency, stability, and carryover.

All compounds demonstrated linearity across the concentration range of 1–25 ng/mL, with R² ≥ 0.99. Both methods’ limits of detection (LODs) ranged between 0.001 and 0.003 ng/mL, with the limits of quantification ranging 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%). The newly created methods displayed various other advantages over their counterparts, including easier and faster sample processing, a wide range of analytes covered, and high analytical sensitivity, as an organic extract of the diluted OF samples, is directly injected.

The developed LC–MS/MS screening has gained popularity compared to immunoassay screening, as it covers a wide panel of drugs and, due to its high sensitivity and selectivity, does not require confirmation in clinical routines. According to the scientists, their methods have become the preferred choice for DoA testing in OF among clinical services. In the future, this sample preparation technique could be used for the simultaneous detection of a wide range of compounds with variable physicochemical properties.

References

(1) Tan, J.; Rylski, A.; Bergqvist, A.; Stephanson, N. N. 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. J. Mass Spectrom. 2025, 60 (5), e5132. DOI: 10.1002/jms.5132