An alternative approach for assessment of liquid chromatography-mass spectrometry matrix effects using auto-sampler programmed co-injection.

We report the use of auto-sampler programmable functions to co-inject analyte standard solution and matrix extract to assess ion enhancement and suppression (matrix effects) in LC-MS. This is effectively an automated post-extraction addition (APEA) procedure, emulating the manual post-extraction addition (PEA) approach widely adopted for assessment of matrix effects. To verify that APEA was comparable to the conventional PEA approach, matrix effects were determined using both methods for a selection of 31 illicit and pharmaceutical drugs in 10 different human urine extracts. Matrix effects measured using APEA were statistically indistinguishable from manual PEA methodology for 27 of the 31 drugs. Of the four drugs that showed significant differences using the two methods, three differed by less than 2 %, which is within the expected accuracy limits required for matrix effect determinations. The remaining analyte, trimeprazine, was found to degrade in the spiked PEA matrix extract, accounting for the difference between matrix effects measured by the PEA and APEA approaches. APEA enables a single matrix extract to be assessed at multiple analyte concentrations, resulting in a considerable reduction in sample preparation time. In addition, APEA can reduce the quantity of analyte-free sample matrix required for matrix effect assessment, which is an important consideration in certain analytical and bioanalytical fields. This work shows that APEA may be considered as an acceptable alternative to PEA for the assessment of matrix effects in LC-MS method validation and may be applicable to a variety of matrices such as environmental samples.

Bacterial degradation of risperidone and paliperidone in decomposing blood.

The stability of two benzisoxazole antipsychotics was determined in vitro in decomposing porcine blood inoculated with bacteria, utilizing a high-performance liquid chromatography with ultraviolet and fluorescence detection method for drug quantitation. Stability experiments for risperidone and paliperidone were conducted at 7, 20 and 37°C for 4 days using sterile and bacterially inoculated porcine blood. The drugs were stable in sterile blood at each temperature and in inoculated blood at 7°C, but degraded significantly in inoculated blood at 20 and 37°C. Complete loss occurred within 2 days when incubated at 37°C. The benzisoxazole-cleaved degradation products for both drugs were identified as 2-hydroxybenzoyl-risperidone and 2-hydroxybenzoyl-paliperidone utilizing liquid chromatography quadrupole-time-of-flight mass spectrometry and accurate mass measurements. The degradation products have been found in postmortem case studies, including one case where risperidone and paliperidone were not detected, indicating complete conversion can occur in situ.

Stability of serotonin-selective antidepressants in sterile and decomposing liver tissue.

It is well established that bacteria are capable of degrading selected drugs during decomposition. The aim of this study was to investigate the stability of several serotonin-selective reuptake inhibitor antidepressants and venlafaxine during putrefaction in porcine liver macerate inoculated with porcine cecal contents rich in bacteria. Blank liver matrices, sterile liver macerates, and sterile aqueous controls were included with the experiment performed for 57 days at 20°C under anaerobic conditions. A liquid chromatography/mass spectrometry method was developed for quantitative determination of the drugs investigated in both sterile and decomposed liver matrices. The method was found to encounter matrix effects not detected during the validation stage. Citalopram, paroxetine, sertraline, venlafaxine, and fluoxetine were found to be stable under the experimental conditions; however, fluvoxamine was found to be decreased by c. 50% over 57 days in bacterially inoculated liver macerate. This study suggests that fluvoxamine concentrations in cases with evidence of decomposition/putrefaction should be interpreted with extra caution.

Vomiting, diarrhea, and sudden death with recent southeast asian travel : Fatal colchicine toxicity.

A 41-year-old male was referred for autopsy from a hospital with a diagnosis of sepsis of uncertain etiology. As he had recently been attached to a military base in Southeast Asia, and had only just returned home, there was considerable concern that an unknown infectious agent may have been involved, which would necessitate screening of coworkers and contacts, with possible quarantine of the facilities where he had been working. His clinical history included a day of vomiting and diarrhea. Despite rehydration and antibiotic therapy, he died within hours of hospitalization. His only past medical history was of gout, for which he was prescribed allopurinol. At autopsy there was evidence of multiorgan failure but no focal sepsis. Postmortem microbiological tests including blood cultures, lung swab, colonic fecal culture, and a small intestinal swab were negative. Histological examination of small intestinal mucosa demonstrated numerous mitotic figures, which, in concert with the presentation, raised the possibility of colchicine toxicity. Subsequent reinterview of family members confirmed that the deceased had ingested an unknown quantity of colchicine on the day prior to his illness and toxicological evaluation demonstrated a toxic/potentially lethal level of 0.05 mg/L of colchicine in the blood. Death was therefore attributed to colchicine toxicity and not to occult sepsis. This case clearly demonstrates that causes of gastrointestinal illness other than sepsis need to be considered when patients have presented with vomiting and diarrhea. There may also be considerable public health implications if a death is incorrectly attributed to sepsis and then a specific infectious agent is not identified. Maintaining a broad approach to diagnostic possibilities is essential if forensic practitioners are to maintain a useful role in the investigation of unexpected deaths.

Application of liquid chromatography-tandem mass spectrometry to the analysis of benzodiazepines in blood.

A sensitive and selective high-performance liquid chromatography/atmospheric pressure chemical ionisation tandem mass spectrometry (HPLC/APCI-MS/MS) method for the simultaneous detection of 18 benzodiazepines and metabolites in human blood is described. The procedure utilises butyl chloride extraction at alkaline pH followed by reversed-phase liquid chromatography. The technique is suitable for screening analyses and confirmation of identity of the benzodiazepines at their lowest reported therapeutic concentrations using 500 microL of blood. The method has been successfully applied in forensic cases involving low concentrations of benzodiazepines.