Development and validation of a highly sensitive GC/MS method for the determination of buprenorphine and nor-buprenorphine in blood.

A sensitive and specific GC/MS method for the determination of buprenorphine (BPN) and its main metabolite nor-buprenorphine (nor-BPN) in blood has been developed, optimized and validated. Sample preparation includes solid-phase extraction of both analytes and their derivatization with acetic anhydride in pyridine. BPN-d4 was used as internal standard for the determination of both analytes. Limits of detection and quantification for BPN and nor-BPN were 0.02 and 0.05 µg/L, respectively. The calibration curves were linear within the dynamic range of each analyte (0.05-30.0 µg/L) with a correlation coefficient higher than 0.996. Absolute recovery ranged from 90.2 to 97.6% for both analytes and their internal standard. Intra- and inter-day accuracy was found to be between -5.40 to 1.73% and -2.45 to 2.80%, respectively, while intra- and inter-day precision were less than 5.8 and 4.7%, for both analytes. The method was applied to real blood samples obtained from patients that follow BPN maintenance program. The developed method can be used in routine every day analysis by clinical and forensic laboratories, for pharmacokinetic studies, for therapeutic drug level monitoring in order to adjust BPN dosage of BPN maintained patients or for the investigation of forensic cases.

Development and validation of an EI-GC-MS method for the determination of benzodiazepine drugs and their metabolites in blood: applications in clinical and forensic toxicology.

Benzodiazepines are used widely in daily clinical practice, due to their multiple pharmacological actions. The frequent problems associated with the wide use of benzodiazepines, as well as the multiple incidents of poisonings, led to the necessity for the development of a precise, sensitive and rapid method for the simultaneous determination of the 23 most commonly used benzodiazepines (diazepam, nordiazepam, oxazepam, bromazepam, alprazolam, lorazepam, medazepam, flurazepam, fludiazepam, tetrazepam, chlordiazepoxide, clobazam, midazolam, flunitrazepam, 7-amino-flunitrazepam, triazolam, prazepam, nimetazepam, nitrazepam, temazepam, lormetazepam, clonazepam, camazepam) in blood. A gas chromatographic method combined with mass spectrometric detection was developed, optimized and validated for the determination of the above substances. This method includes liquid-liquid extraction with chloroform at pH 9 and two stages of derivatization using tetramethylammonium hydroxide and propyliodide (propylation), as well as a mixture of triethylamine:propionic anhydride (propionylation). The recoveries were higher than 74% for all the benzodiazepines. The calibration curves were linear within the dynamic range of each benzodiazepine with a correlation coefficient higher than 0.9981. The limits of detection and quantification for each analyte were statistically calculated from the relative calibration curves. Accuracy and precision were also calculated and were found to be less than 8.5% and 11.1%, respectively. The developed method was successfully applied for the investigation of both forensic and clinical toxicological cases of accidental and suicidal poisoning.

Development and validation of an EI-GC-MS method for the determination of methadone and its major metabolites (EDDP and EMDP) in human breast milk.

Methadone is used extensively for the maintenance of opioid-addicted pregnant women. Because methadone and the two major metabolites, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenylpyrroline (EMDP), are excreted into breast milk, a sensitive and specific gas chromatographic-mass spectrometric method has been developed, optimized, and validated for their quantitative determination in human breast milk. The procedure combined protein precipitation with acetonitrile and solid-phase extraction, using Isolute Confirm HCX mixed-mode SPE columns, with minimal matrix effect. The optimum extraction conditions for all three analytes were evaluated using spiked human breast milk, and the recovery exceeded 93.0%. This assay uses methadone-d(9) as internal standard for the determination of methadone and EMDP, and EDDP-d(3) for the determination of EDDP. Calibration curves were linear within the range of 2.00-1000 microg/L for methadone (R(2) > 0.995) and 1.00-500 microg/L for EDDP (R(2) >0.997) and EMDP (R(2) > 0.991). Intra- and interday accuracy and precision were within the range of 0.8-5.7% and 1.3-5.2%, respectively, for all analytes. The stability study was assessed by fortifying human breast milk with methadone and its metabolites at two different concentrations and keeping the samples at different temperature conditions. The analytes were found to be stable in breast milk at room temperature for at least 4 h and at -20 degrees C for at least one month. The method was used for the determination of methadone and its major metabolites in human breast milk samples obtained from women in the postpartum period participating in a methadone maintenance program.