Validation and comparison of three sample preparation techniques for quantitation of amobarbital, butalbital and phenobarbital in blood and urine using UFLC-MS/MS
Chan, Chi Hin
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This research study successfully completed three objectives: 1) validate liquid-liquid, supported-liquid, and solid-phase extractions for the quantitation of three barbiturates (amobarbital, butalbital, and phenobarbital) in blood and urine using liquid chromatography-tandem mass spectrometry; 2) to compare the efficiency and effectiveness among methods in accomplishing extraction of barbiturates under the laboratory setting at Boston University School of Medicine; and 3) to report all the analytical data to RTI International for interlaboratory comparison. For the validation study, a six-point linear calibration model (20-2000 ng/mL) with inversely weighted concentration (1/x) was reproducible in all three sample preparation methods for both blood and urine with r2 greater than or equal to 0.994. Bias and precision evaluated from three controls throughout the range of the curve were within ±20% and ±20%CV, respectively. Neither carryover nor interference was observed. Detection limits were evaluated down to 5 ng/mL depending on the extraction procedure. Samples were able to be diluted up to 50 times prior to instrumental analysis. Samples were stable on autosampler at room temperature up to 72 hours after their initial analysis. Recovery of barbiturates from blood and urine all ranged from 45% to 86%. The effect of ionization suppression or enhancement was found to have minimal impact on the validation. For choosing the most suitable method quantifying barbiturates, efficiency and effectiveness were studied. Efficiency evaluates the time and ease of sample preparation required to prepare a sample for analysis. Supported-liquid extraction was found to be the most efficient method for extracting barbiturates as it required the least amount of time to perform and could be easily automated with minimal training. Effectiveness is an assessment of one’s ability to selectively recover target analyte at a reasonably low concentration. By considering a method’s recovery, extract cleanliness, detection limits, and reproducibility, liquid-liquid extraction was the best at quantifying barbiturates in blood and supported-liquid extraction was the most suitable method for extracting barbiturates from urine. For interlaboratory comparison, all the data collected has been reported to RTI International. These findings can be used for examining the overall reliability and reproducibility of the validated methods. Results obtained can also be used to explore the possibility for streamlining sample preparation in the forensic laboratory, and hence reducing the case backlog.