Evaluation of drugs of abuse extraction from oral fluid using supported liquid extraction (SLE) prior to UPLC-MS/MS analysis

Objectives Drug screening using oral fluid has gained popularity over recent years due to its simple, non-invasive collection means. Screening drugs of abuse can be complicated due to the wide variation of functional groups associated with different analyte classes. Most extraction techniques cannot extract all analytes using a single procedure without using non-optimal extraction protocols resulting in compromised extract cleanliness. Supported liquid extraction allows for the simultaneous analysis of cross functional analytes in a single extraction protocol without forfeiting extract cleanliness. The aim was to develop a UPLC-MS/MS assay for the determination of a range of drugs of abuse from oral fluid using supported liquid extraction (SLE), after collecting specimens from a variety of collection devices. The devices evaluated were Intercept from Orasure and Quantisal from Immunalysis. Methods Negative oral fluid samples for method development purposes were obtained using the Immunalysis Quantisal and Orasure Intercept collection devices. To ensure maximum oral fluid extraction on SLE+, samples from Quantisal and Intercept collection devices were modified in pH terms using 10–15 μL of concentrated NH 4 OH. The target for elevated pH was between 8.2–8.5 to provide a balance for the extraction of the basic drugs but also to avoid any potential hydrolysis of 6-MAM to morphine. Final pH control used 15 μL of neat NH 4 OH and 10 μL 0.5% NH 4 OH resulting in loading pHs of 8.3 and 8.5 for the Quantisal and Intercept devices, respectively. Extraction evaluation was performed loading 300 μL on ISOLUTE SLE+ 400 μL capacity columns, followed by elution with 2 × 1 mL of either MTBE, DCM, 95/5 DCM/IPA or EtOAc. Benzodiazepines, z drugs, amphetamines, cathinones, opiates, cocaine, buprenorphine, THC-COOH, fentanyl and ketamine spiked oral fluid extracts were analyzed using a Waters UPLC-MS/MS system (Waters). Positive ions were acquired using electrospray ionization operated in multiple reaction monitoring mode. Results The DOA multisuite showed optimum extraction at pH condition between 8–8.5. No degradation of 6-MAM was observed in this pH environment. The use of 95/5 DCM/IPA suffered from non-optimal extract cleanliness associated with co-extraction of buffer additives and was discontinued for both devices. Optimization of loading volume provided the optimal balance of extract cleanliness and recoveries when using DCM for Quantisal and EtOAc for Intercept. Recoveries were greater than 80% for the vast majority of analytes for both collection devices. BZE recovery was low in EtOAc, however a sub-nanogram LLOQ was achievable in spite of this. Calibration curves were constructed from 1–500 ng/mL and good linearity was universally achieved, demonstrating coefficients of determination > 0.99 for both collection devices. When utilizing optimum pH control and extraction solvent for each oral fluid device, it was possible to extract a wide range of drugs of abuse with varying logP and pKa values, demonstrating the possibility of a single extraction. Conclusion This poster demonstrates the extraction of a range of drugs of abuse prior to UPLC-MS/MS analysis. The analyte list includes benzodiazepines, z drugs, amphetamines, cathinones, opiates, cocaine, buprenorphine, THC-COOH, fentanyl and ketamine.