B-421 Validation of high-resolution mass spectrometry-based targeted drug screening

Abstract

High-resolution mass spectrometry (HRMS) is a rapidly growing technique available in the clinical laboratory that is more sensitive, specific, and comprehensive for quantitative and qualitative drug analysis compared to traditional immunoassay screening. A 66 drug and metabolite qualitative panel, with emphasis on the detection of common drugs of abuse and drugs used to manage patients with chronic pain, was developed and validated for clinical use. This test provides a more comprehensive panel to assist clinicians in a variety of patient settings including pain management and addiction medicine Urine specimen preparation included glucuronidase-mediated hydrolysis and purification by mixed-mode cation exchange. Chromatographic separation was achieved by Vanquish Duo UHPLC System (Thermo Scientific) on a C18 column using a 6.4 min gradient. A combination of a full MS/ddMS2 scan and parallel reaction monitoring (PRM) was used to monitor analytes by Q Exactive Orbitrap Mass Spectrometer (Thermo Scientific). Following the policy of CLIA and guidance of CLSI, this method was validated as a qualitative method including matrix effects, specificity, sensitivity, precision, accuracy, hydrolysis efficiency, carryover, and stability experiments Method precision was evaluated at 50% and 200% of the threshold cutoff. Intra-and inter-day precision (CV) of the 66 analytes ranged from 0.5-13.5% and 0.1-9.6%, respectively. All the samples met the required categorical agreement. Quantitative matrix effects were evaluated with a minimum of 10 lots of negative patient specimens and the average matrix factors for 66 analytes ranged from 0.46-1.58. The coeluted isotopic internal standards showed similar matrix effects, which resulted in the average relative matrix effects between 0.75-1.13. Sensitivity was verified, which is 25 ng/ml for most analytes except for fentanyl and its analogs at 2 ng/ml and buprenorphine and norbuprenorphine at 5 ng/ml. The total error calculated at the threshold was less than 12% for all analytes. The limit of the blank for most analytes is less than 0.1 ng/ml. No significant interference was observed from common endogenous interferences and more than 80 potential exogenous substances. Enzymatic hydrolysis of 11 glucuronides was evaluated and all met categorical agreement. A total of 160 urine specimens were evaluated using the HRMS method and compared to those from the current in-house LC-MS/MS method and national reference labs. More than 95% of the categorical agreement was achieved for all the analytes except for speciociliatine which had no comparative method available. Accuracy spikes were prepared as supplement of method comparison for this analyte and overall agreement was 100%. Carryover and stability were also evaluated for all analytes Overall, HRMS coupled with high performance liquid chromatography (HPLC) method provides a powerful tool for specific and sensitive drug detection. Its performance met the requirement and specification for a laboratory-developed clinical test. This HRMS test provides an expanded panel of analytes to assess compliance and drug exposure