Comparison of sample preparation techniques on twenty-three drugs in human whole blood and urine
McGowan, Courtney K.
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In forensic toxicology, analysis of drugs and metabolites in biological fluids is performed to determine cause of death, suspected drug use, drug facilitated sexual assaults, or whether someone was driving under the influence. Analyte identification and concentration determination can be determined in a variety of matrices (e.g., blood, urine, or oral fluid) and can be complex. It is therefore necessary to have optimal sample preparation and instrumental conditions that work for all matrices of interests. Determining the best approach can be challenging due to the amount of time and resources to perform expansive evaluations of sample preparation, stationary/mobile phases, liquid chromatography (LC) conditions and mass spectrometry (MS) operating parameters. In this study three different sample preparation methods were validated for blood and urine. The three sample preparation methods were solid-phase extraction (SPE), supported liquid extraction (SLE), and liquid-liquid extraction (LLE). Six different drug groups were used as the analytes being tested by the methods. These drug groups were amphetamines, local anesthetics, opioids, hallucinogens, antidepressants, and novel psychoactive substances (NPS). A total of twenty-three drugs were used: amphetamine, methamphetamine, (3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxy-N-ethylamphetamine (MDEA), and 3,4-methylenedioxymethamphetamine (MDMA), benzoylecgonine (BZE), cocaine, lidocaine, codeine, methadone, morphine, 6-monoacetylmorphine (6-MAM), fentanyl, oxycodone, lysergic acid diethylamide (LSD), phencyclidine (PCP), amitriptyline, citalopram, fluoxetine, trazodone, ethylone, α-pyrrolidinopentiophenone (α-PVP), and 25I-NBOMe. The methods were validated according to guidelines set forth by the Scientific Working Group for Forensic Toxicology (SWGTOX) Standard Practices for Method Validation in Forensic Toxicology and the American Academy of Forensic Science (AAFS) Standards Board (ASB) draft of Standard Practices for Method Validation in Forensic Toxicology. Parameters of calibration model, bias, precision, limit of detection (LOD), limit of quantitation (LOQ), dilution integrity, ion suppression/enhancement, interference studies, and stability were evaluated. Recovery was also assessed to determine the efficiency of the extraction. Calibration models met the 0.98 R2 minimum requirement. For all sample preparations the compounds evaluated in each were found to be stable for at least 72 hours. Interferences were found to be similar across all three sample preparation methods. Parameters of bias, precision, and dilution integrity were largely comparable between all three methods. Overall for LOD, SLE resulted in lower values for blood and urine ranging for 0.1 to 5 ng/mL. Overall for LOQ, SLE resulted in lower values for blood and LLE resulted in lower values for urine in the range of 0.5-10 ng/mL. SLE resulted in the highest recovery for all twenty-three analytes, due to LLE failing to extract consistently or completely for benzoylecgonine, morphine, and 6-monoacetylmorphine. Overall, SLE resulted in the lowest percent values for ion suppression and enhancement for both blood and urine. Overall, blood resulted in high ion suppression (exceeding -20%) for SPE and LLE. Final determination overall was that SLE was the best sample preparation method for all twenty-three analytes. This was determined based on the evaluation of recovery, ion suppression/enhancement, and LOD, as well as sample preparation time. Sample preparation time for SLE was approximately 1 hour, while SPE took 2.5 hours and LLE 2 hours.