The analysis of drugs of abuse (DoA) and Novel Psychoactive Substances (NPS) in oral fluids by LC-MS/MS

Abstract

Aim

The primary objective of this work is to demonstrate the analysis of drugs of abuse and (DoA) and NPS compounds in oral fluids by LC-MS/MS using a salt-assisted liquid-liquid extraction (SALLE).

Testing for drugs in biological matrices is an important part of toxicology and workplace drug testing. The “gold standard” matrices that have been used for decades are typically blood and urine, however, the collection of these two matrices is invasive. The analysis of drugs of abuse in oral fluids is a solution that is gaining popularity due to its ease of collection compared to blood or urine collection. However, there are some issues with the buffer used in the collection devices when performing the oral fluid analysis. It can be difficult to remove all of the surfactants and preservatives present in the oral fluid collection device's buffer solution which can cause matrix effects and poor column lifetime. It can also be challenging to get full recovery of all of the analytes due to different techniques of emptying the sponge on the collection device. Often times solid phase extraction (SPE), or lengthy extraction techniques are utilized. Finding a workflow that uses a simple sample preparation paired with accurate and robust quantitation of the analytes is important for laboratories running these tests.

Method

A LC-MS/MS method was developed using a Raptor Biphenyl 50 × 2.1 mm, 2.7 μm analytical column equipped with a Raptor Biphenyl EXP 5 × 2.1 mm, 2.7 μm guard column. Mobile phase A consisted of 0.1% formic acid in water and mobile phase B consisted of 0.1% formic acid in methanol. A total of 65 analytes were separated under gradient conditions, with a total cycle time of 10 minutes. This method utilized both positive and negative ESI modes. Samples were prepared in oral fluid and combined with QuantisalTM buffer. An aliquot from the buffer underwent sample preparation using a salt-assisted liquid-liquid extraction (SALLE). This technique used a saturated sodium chloride solution along with acetonitrile to extract the analytes from the matrix and buffer. The samples were dried down and reconstituted in 90:10 MPA: MPB.

Results

The biphenyl stationary phase offered superior selectivity and was able to resolve all 65 analytes, including all sets of isobaric analytes including, methamphetamine and phentermine, isotonitazene and protonitazene, and eutylone and pentylone among others. All sets of isobars had a resolution of 1.5 or greater, offering accurate quantitation of the analytes. Performance metrics such as recovery, linearity, matrix effects, and accuracy and precision were evaluated. All analytes included in this method passed both intra and inter-day accuracy and precision requirements, and no matrix interferences or effects were observed. Linearity was demonstrated using 1/ × weighted linear regression and all analytes showed a r2 of 0.99 or greater. An evaluation was completed, comparing dilute-and-shoot, which involved minimal clean-up, to the SALLE approach. Dilute-and-shoot failed to remove all of the buffer surfactants compared to SALLE. It was also difficult to achieve certain limits of detection using the dilute and shoot method for analytes such as buprenorphine. A recovery study on the collection device will also be discussed.

Conclusion

A panel of 65 DoA and NPS were analyzed in oral fluid using a SALLE sample preparation technique and LC-MS/MS. This method demonstrates an accurate and robust solution for the analysis of these analytes. This method also offers a quick and efficient sample preparation procedure that cleans up with sample and removes buffer surfactants, without the need for SPE or other tedious extraction techniques, leading to faster processing of samples in high throughput laboratories.