Plasma or serum 5-hydroxyindoleacetic acid can be used interchangeably in patients with neuroendocrine tumours.

5-hydroxyindole acetic acid, a metabolite of serotonin, is used in the diagnosis and monitoring of patients with neuroendocrine tumours, in particular patients with small intestinal neuroendocrine tumours associated with the carcinoid syndrome. Analysis of 5-hydroxyindole acetic acid was commonly performed in urine, but blood-based assays are now becoming available. The objective of this study was to assess how 5-hydroxyindole acetic acid compares in plasma and serum as a biochemical marker of neuroendocrine tumours. Twenty-four-hour urine, plasma and serum samples were obtained from 80 patients with neuroendocrine tumours and 30 healthy volunteers. We developed a liquid chromatography tandem mass spectrometry assay for plasma and serum 5-hydroxyindole acetic acid. Comparison was made between them, and their cut-off was determined using a receiver-operating characteristic curve. A close correlation was shown between plasma and serum 5-hydroxyindole acetic acid. At a cut-off of 135 nmol/l, a sensitivity of 91.2% with a specificity of 61.9% was obtained for both compared to the urinary assay. A statistically significant agreement was shown when plasma and serum 5-hydroxyindole acetic acid were compared with the currently used urine assay in patients with neuroendocrine tumours; κ = 0.675 (95% CI 0.49 to 0.86), p < 0.001 and healthy volunteers; 0.967 (95% CI 0.828 to 0.999), p = <0.001. In conclusion, 5-hydroxyindole acetic acid in plasma and serum were comparable, hence either sample type can be used interchangeably.

Ultra-fast LC-MS/MS in therapeutic drug monitoring: Quantification of clozapine and norclozapine in human plasma.

A novel approach to high-throughput, targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis has been developed. A single chromatographic system can be used for the analysis of a range of 20 drugs and metabolites with a total analysis time of 36 s (one 96-well plate of prepared samples per hour). To demonstrate the applicability of this approach to quantitative analysis, a method has been validated for the therapeutic drug monitoring of clozapine and norclozapine following automated extraction from human plasma. Chromatographic retention times were 11.4 and 12.4 s for norclozapine and clozapine, respectively (for both analytes the chromatographic peak width was less than 1 s). Comparison with a conventional LC-MS/MS method (5 min analysis time) showed excellent agreement. This new approach offers analysis times more akin to flow-injection analysis, but is likely to be more widely applicable because of chromatographic resolution from residual matrix components and isobaric interferences.

Automated Analysis of Clozapine and Norclozapine in Human Plasma Using Novel Extraction Plate Technology and Flow-Injection Tandem Mass Spectrometry.

BACKGROUND: Analysis of plasma clozapine and N-desmethylclozapine (norclozapine) for therapeutic drug monitoring purposes is well established. To minimize analysis times and facilitate rapid reporting of results, we have fully automated sample preparation using novel AC Extraction Plates and a Tecan Freedom EVO 100 liquid handling platform, and minimized extract analysis times using flow-injection tandem mass spectrometry (FIA-MS/MS). METHODS: Analytes and deuterium-labeled internal standards were extracted from plasma (100 µL) at pH 10.6 and extracts analyzed directly using tandem mass spectrometry [20 µL injection, 0.7 mL/min methanol carrier flow, analysis time (injection-to-injection) approximately 60 seconds]. RESULTS: Validation data showed excellent intraplate and interplate accuracy (95%-104% nominal concentrations). Interbatch precision (% RSD) at the limit of quantitation (0.01 mg/L) was 3.5% and 5.5% for clozapine and norclozapine, respectively. Matrix effects were observed for both clozapine and norclozapine, but were compensated for by the internal standards. Overall process efficiency was 56%-70% and 66%-77% for clozapine and norclozapine, respectively. Mean relative process efficiency was 98% and 99% for clozapine and norclozapine, respectively. Comparison of results from patient samples (n = 81) analyzed using (1) manual liquid-liquid extraction with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and (2) automated extraction with FIA-MS/MS gave y = 1.01x - 0.002, R(2) = 0.9943 and y = 1.01x + 0.009, R(2) = 0.9957 for clozapine and norclozapine, respectively. Bland-Altman plots revealed a [mean (95% limits of agreement) bias of 0.0074 (-0.04 to 0.06) mg/L and of 0.015 (-0.02 to 0.05) mg/L for clozapine and norclozapine, respectively]. CONCLUSIONS: FIA-MS/MS used with automated extraction offers a rapid, simple, cost-effective alternative to manual liquid-liquid extraction and conventional LC analysis for clozapine therapeutic drug monitoring.