Semester

Spring

Date of Graduation

2009

Document Type

Dissertation

Degree Type

PhD

College

Eberly College of Arts and Sciences

Department

Chemistry

Committee Chair

Suzanne Bell

Abstract

Development and validation of ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) methodologies for the purpose of detecting and quantifying common drugs of abuse in human meconium specimens is described. Meconium is the first stool passed by a newborn infant. Meconium formation occurs over several months of gestation and subsequent toxicological analysis of the specimens can be useful for identifying drugs and other xenobiotics indicative of prenatal drug exposure.;Ultra performance liquid chromatography (UPLC) is an emerging analytical technique which draws upon the principles of chromatography to run separations at higher flow rates for increased speed, while simultaneously achieving superior resolution and sensitivity. Tandem mass spectrometry experiments were performed using a triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source operating in positive ion mode. Methodologies were developed and validated to detect and quantify amphetamine, methamphetamine, cocaine, benzoylecgonine, morphine, codeine, hydromorphone, and 6-monoacetylmorphine (6-MAM) in authentic meconium specimens.;Analytes were extracted from the meconium matrix using either a mixed mode solid phase extraction (SPE), or a supported-liquid extraction (SLE) employing columns containing a modified form of diatomaceous earth. Data acquisition was performed using multiple reaction monitoring (MRM) and quantitation of each analyte was performed using a working standard calibration curve. The analytical methodologies were fully validated for the meconium matrix, where linearity, matrix equivalence, selectivity, accuracy, precision, sensitivity, stability, and recovery were evaluated.;Equivalence studies indicated that in all cases, certified drug free whole blood was a suitable matrix for the preparation of working calibration curves. Blank meconium specimens containing analytes at the LOQ, were spiked with various exogenous interferences and analyzed to assess the selectivity of the analytical method. All methodologies were deemed highly selective for the analyte of interest even in the presence of exogenous compounds commonly encountered in forensic specimens. Accuracy, precision, stability, and recovery were assessed at three different analyte concentrations corresponding to the LOQ, the ULOQ, and a concentration point midway between the two. Mean accuracies ranged from 94.6% to 99.6% over the three concentrations for the cocaine/benzoylecgonine methodology while the amphetamine and opiate assays also exhibited high accuracies with mean ranges of 93.6% to 98.4% and 93% to 99.6%, respectively. Inter and intra batch precision data indicated enhanced method precision and reproducibility relative to existing techniques. Intra-batch CV values ranged from 1.6 to 11.8% for the cocaine/benzoylecgonine methodology while inter-batch CV values ranged from 3.9% to 6.2%. Intra-batch CV values ranged from 0.7% to 8.5% for the amphetamine/methamphetamine methodology while inter-batch CV values ranged from 1.9% to 6.2%. The opiate methodology was also highly precise with intra-batch CV values ranging from 1.2% to 10.7% while interbatch CV values ranged from 0.5% to 6.1% across the four analytes. While mean analyte recoveries ranged from 9.3% for benzoylecgonine to 76.3% for 6-monoacetylmorphine, the consistency and reproducibility of the extraction was acceptable for all analytes. Stability studies indicated that all analytes are stable in the meconium matrix when stored at 4°C and subjected to multiple freeze-thaw cycles over a 72 hour period. Limits of detection ranged from 250 pg/mL for methamphetamine, to 2.5 ng/mL for all four opiate analytes. Linear calibration for the cocaine/benzoylecgonine, amphetamine/methamphetamine, and opiate methodologies was achieved over the range of 10--250 ng/mL, 5--500 ng/mL, and 10--500 ng/mL, respectively.;Recent technological advances made in the field of particle chemistry mean that liquid chromatographic separations can be performed at higher flow rates for increased speed without sacrificing resolution or sensitivity. The increased speed, resolution, sensitivity, and separation efficiency afforded by UPLC combined with the inherent selectivity and sensitivity of the tandem mass spectrometer allowed for the accurate quantitation of all 8 analytes in the meconium matrix in a time and cost effective manner. Development and validation of such analytical methodologies will prove beneficial for the identification of prenatal substance abuse which is an ongoing concern across socioeconomic lines.

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