Analysis of cyclosporin A, tacrolimus, sirolimus, and everolimus in dried blood spot samples using liquid chromatography tandem mass spectrometry.

In the last few years, an increasing number of dried blood spot (DBS) sampling assays have been developed. With this increase, more insight is gained in the factors that possibly influence the performance of DBS assays. We have developed an assay for four commonly used immunosuppressants; some of them are possibly concomitantly prescribed: cyclosporin A (CsA), tacrolimus (TcR), sirolimus (SiR), and everolimus (EvE). Chromatographic separation from possible ion suppression was obtained within the total runtime of 4.2 min. Trifluoroacetic acid and ammonium acetate were used as mobile phase additives. The linearity ranged from 23.6 to 787, 1.14 to 30.3, 1.34 to 35.8, and 1.26 to 33.7 µg/L, for CsA, TcR, SiR, and EvE, respectively. Between- and within-run accuracy and precision were all within 15 % and extensive validation for DBS samples, such as hematocrit, blood spot volume, and spot punch location was performed. None of these factors were found to be of influence on the performance of the DBS assay.

Limited stability of thiopurine metabolites in blood samples: relevant in research and clinical practise.

BACKGROUND: Monitoring of thiopurine metabolites 6-thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine (6-MMP) is used to assess compliance and explain adverse reactions in IBD-patients. Correlations between dosage, metabolite concentrations and therapeutic efficacy or toxicity are contradictive. Research is complicated by analytical problems as matrices analyzed and analytical procedures vary widely. Moreover, stability of thiopurine metabolites is not well documented, yet pivotal for interpretation of analytical outcomes. Therefore, we prospectively investigated metabolite stability in blood samples under standard storage conditions. METHODS: Stability at room temperature and refrigeration (22 degrees C, 4 degrees C) was investigated during 1 week and frozen samples (-20 degrees C, -80 degrees C) were analyzed during 6 months storage. Ten patient samples were analyzed for each study period. RESULTS: Median 6-TGN concentrations on day 7 decreased significantly to 53% and 90% during storage at ambient temperature or refrigeration. Median 6-MMP concentrations on day 7 decreased significantly to 55% and 86%, respectively. Samples stored at -20 degrees C also showed significant decreases in both 6-TGN and 6-MMP in comparison with baseline values. At -80 degrees C, only 6-MMP showed a significant decrease in values compared to baseline. CONCLUSION: The stability of thiopurine metabolites is clearly a limiting factor in studies investigating utilisation of TDM and correlations with therapeutic outcome in IBD-patients. This has to be accounted for in clinical practice and (multi-center) trials investigating thiopurine drugs.

Analysis of mycophenolic acid in dried blood spots using reversed phase high performance liquid chromatography.

At our laboratory a reversed phase high performance liquid chromatography assay was developed for analysis of mycophenolic acid in dried blood spot samples. The assay was validated in the range of 0.74-23.4mg/L and proved to be accurate and precise. The developed sample pretreatment procedure was consistent and has a recovery of 95.2% for mycophenolic acid. Hematocrit showed to have influence on the physics of the blood spots and thus on the concentration of mycophenolic acid, therefore standard and control samples should be made with a standardized hematocrit.

Simultaneous quantification of erlotinib, gefitinib, and imatinib in human plasma by liquid chromatography tandem mass spectrometry.

A quantitative liquid chromatography (LC)-mass spectrometry (MS)/MS method in human plasma was developed and validated for the tyrosine kinase inhibitors erlotinib, gefitinib, and imatinib in human plasma. Pre-treatment of the samples was achieved by using liquid-liquid extraction using D-8 imatinib as internal standard. Separation was performed on a Waters Alliance 2795 LC system using an XBridge RP18 column. The mass spectrometer Micromass was equipped with an electro spray ionization probe, operating in the positive mode. The calibration curves in plasma were linear for erlotinib, gefitinib, and imatinib over the concentration range of 5 to 3,000; 5 to 3,000, and 5 to 5,000 ng/mL, respectively. The intraday and interday accuracy ranged from 90% to 110% and the intraday and interday precision of the method was within 5%. The reported method provided the necessary linearity, precision, and accuracy to determine tyrosine kinase inhibitors in clinical research and for therapeutic drug monitoring.

Analysis of cyclosporin A in dried blood spots using liquid chromatography tandem mass spectrometry.

Dried blood spot sampling is a promising and patient friendly alternative for venous sampling. A liquid chromatography tandem mass spectrometer assay was developed for analyzing cyclosporin A in dried blood spots. Linearity ranged from 25 to 1440 microg/L. Within and between run accuracy and precision were within limits. The developed assay has a negligible matrix-effect and a recovery of 97%. The dried blood spots were stable during a period of at least 17 days in the refrigerator. The developed assay is suitable for analyzing cyclosporin A in dried blood spots.

Toxicokinetics of dothiepin: 2 case reports.

In this article, 2 cases of intentional dothiepin intoxication are presented. Both patients survived after receiving appropriate supportive care. The dothiepin and metabolite levels were measured at different times after ingestion. The initial levels of dothiepin were 1900 microg/L in case 1 and 5500 microg/L in case 2. In case 1, a toxicokinetic model was fitted, suggesting a higher peak level, corresponding with the QRS duration and clinical symptoms. In case 2, a combined dothiepin-ethanol intoxication was seen, complicating the interpretation of clinical parameters, such as the QRS duration. In conclusion, the severity of dothiepin intoxication is poorly predicted by plasma levels alone, as time of ingestion can be critical for interpretation. However, especially in combined intoxications, interpretation of the QRS duration may also fail. Therefore, it is important to evaluate the whole range of clinical parameters, QRS duration, clinical symptoms, dothiepin concentration(s), and other possible intoxications.