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Abstract INTRODUCTION:
The “gold standard” biological matrices for toxicological testing are typically blood and urine, however, the collection of these two matrices is considered invasive. The analysis of drugs of abuse(DoA) in oral fluids is a solution that is gaining popularity due to its ease of collection compared to that of blood or urine. However, when performing the oral fluid analysis, it can be difficult to remove all of the buffer surfactants present in the oral fluid collection device’s buffer solution. These surfactants can cause matrix effects and poor column lifetime. 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. In this work, a dilute-and-shoot sample preparation was compared against supported liquid extraction (SLE) and salt-assisted liquid-liquid extraction (SALLE).
OBJECTIVE:
The primary objective of this work is to compare the recovery for DoA and novel psychoactive substances (NPS) in oral fluids using three sample preparation techniques: dilute-and-shoot, SLE, and SALLE.
METHODS:
An LC-MS/MS method was developed using a Raptor Biphenyl 50 x 2.1 mm, 2.7 µm analytical column equipped with a Raptor Biphenyl EXP 5 x 2.1 mm, 2.7 µm guard column. A total of 68 analytes were separated using gradient conditions, consisting of water (MPA) and methanol (MPB), both containing 0.1% formic acid, for a total cycle time of 10 minutes. Samples were prepared in oral fluid and combined with QuantisalTM buffer. Aliquots from the buffer underwent each of the sample preparation techniques, dilute-and-shoot, SALLE, and SLE. Samples were dried down and reconstituted in 90:10 MPA: MPB and moved to the instrument for analysis.
RESULTS:
The biphenyl stationary phase resolved all 68 analytes, including sets of isobaric analytes, such as methamphetamine and phentermine, isotonitazene and protonitazene, and eutylone and pentylone. All sets of isobars achieved a resolution of 1.5 or greater, providing accurate quantitation of the analytes. Performance metrics such as recovery, linearity, matrix effects, and accuracy and precision were evaluated. All analytes passed both intra and inter-day accuracy and precision requirements, without matrix interferences or effects being observed. Linearity was demonstrated using 1/x weighted linear regression and with an r2 of ≥ 0.991. An evaluation was completed, comparing analyte recovery when using dilute-and-shoot, to SLE and SALLE approaches. Dilute-and-shoot failed to remove the buffer surfactants compared to SLE and SALLE. Both SALLE and SLE returned increased sensitivity compared to dilute-and-shoot for all analytes (excluding pregabalin, gabapentin, THC, and CBD for the SLE workflow). SALLE yielded higher sensitivity for a broader range of analytes, while SLE achieved increased sensitivity for most of the opiates.
DISCUSSION:
This work demonstrates an accurate and robust solution for the analysis of these analytes. Additionally, this workflow demonstrates quick and efficient sample clean up procedures that remove buffer surfactants without the need for SPE or other tedious extraction techniques. All analytes showed an improvement in recovery when using the SALLE technique as compared to dilute-and-shoot. Most analytes showed an improvement in recovery when using SLE as compared to dilute-and-shoot aside from a few (THC, CBD, pregabalin, and gabapentin). These sample preparations can be catered based on analyte list and LOD requirements to ensure the best recovery for all analytes of interest. Utilizing quick and efficient sample preparation techniques leads to faster processing of samples in high through-put laboratories.
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= Discovery stage. (53.14%, 2025)
= Translation stage. (22.33%, 2025)
= Clinically available. (24.53%, 2025)
