Application of bioanalysis in understanding traumatic brain injury and opiate pharmacology

The research is primarily on the design, development and validation of chromatographic-based bioanalytical methods for identification and quantification of trace small molecules in biological samples. Two major projects are included: (1) identification and quantification of endogenous bioactive eicosanoids in brain samples in support of traumatic brain injury study; and (2) identification and quantification of centrally-acting analgesic drug, buprenorphine, and its major metabolite, norbuprenorphine, in brain and plasma samples in support of opiate pharmacological study.; In traumatic brain injury project, trace analytical approaches were developed to identify and quantify arachidonic acid (AA), and it's metabolites, prostaglandins (PGs), hydroxyeicosatetraenoic acids (HETEs), dihydroxyeicosatrienoic acids (DiHETrEs), and epoxyeicosatrienoic acids (EETs), in brain samples. A sensitive fluorescence HPLC method was developed initially. In this method, a selective reversed-phase solid phase extraction (SPE) procedure was developed for brain tissues; the eicosanoids were derivatized with 2-(2,3-naphthalimino)ethyl-trifluoromethanesulphonate, followed by separation at high sensitivity using reversed-phase HPLC with fluorescence detection, and further identified via LC/MS. To improve the specificity and throughput, a more sensitive, specific, robust, and rapid LC/MS method was developed and validated, which allows simultaneous analysis of AA, PGF 2alpha, PGE2, PGD2, PGJ2, 14,15-DiHETrE, 11,12-DiHETrE, 8,9-DiHETrE, 5,6-DiHETrE, 20-HETE, 15-HETE, 12-HETE, 9-HETE, 8-HETE, 5-HETE, 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET in rat brain tissues. PGF2alpha-d4, PGD2-d4, 15-HETE-d 8, 14,15-EET-d8, 11,12-EET-d8, 8,9-EET-d 8, and AA-d8 were used as internal standards. A gradient LC/MS method using a C18 column and electrospray ionization with negative ion mode was optimized for the best sensitivity and separation within 35 min. The method validation, including LC/MS instrument qualification, specificity, calibration model, accuracy, precision, and extraction efficiency was performed at pg levels for metabolites, and ng levels for AA respectively.; In opiate pharmacology project, a specific and reproducible LC/MS method was developed and validated to measure buprenorphine and norbuprenorphine in rat brain and plasma samples. d4-Buprenorphine and d3-norbuprenorphine were used as internal standards. Cation-exchange reversed-phase 96-well plate SPE procedures were developed. An isocratic method using a C8 column and electrospray ionization with positive ion mode was optimized. The matrix-based method validation, including specificity, calibration model, accuracy, precision, extraction efficiency, and matrix effect was performed for buprenorphine and norbuprenorphine.