Therapeutic drug monitoring of plasma clozapine and of its principal plasma metabolite N-desmethylclozapine (norclozapine) (predose or "trough" sample) can help in monitoring adherence, in dose adjustment, and in minimizing the risk of toxicity. To obtain data to assist in the interpretation of analytical results, the results from a clozapine therapeutic drug monitoring service, 1993-2007, have been audited. There were 104,127 samples from 26,796 patients [18,750 (70%) men aged at time of first sample (median, range) 34 (10-89) years, and 7763 (30%) female aged 38 (12-90) years]. Clozapine was not detected (plasma concentration <0.01 mg/L) in 1.5% of samples (prescribed clozapine dose up to 900 mg/d). Plasma clozapine was either below 0.35 mg/L or greater than 0.60 mg/L in 42.5% and 28.4% of samples, respectively; in 0.4% samples plasma clozapine was 2.0 mg/L or more. Although plasma clozapine was broadly related to prescribed dose, there was much variation: 1.2% of samples had plasma clozapine >1.0 mg/L at prescribed clozapine doses up to 150 mg/d (76.2% < 0.35 mg/L), whereas 23.3% of samples had plasma clozapine < 0.35 mg/L at doses of 850 mg/d and over (18.0% > 1.0 mg/L). The highest plasma clozapine and norclozapine concentrations encountered were 4.95 and 2.45 mg/L, respectively. Although the median plasma clozapine:norclozapine ratio was 1.25 at plasma clozapine concentrations < 0.35 mg/L, the median ratio was 2.08 at plasma clozapine concentrations > 1.0 mg/L. Data (median, 10th-90th percentile) for both clozapine and norclozapine by prescribed clozapine dose band are useful in assessing partial adherence. Analysis of the plasma clozapine:norclozapine ratio by clozapine concentration provides clear evidence that clozapine N-demethylation becomes saturated at higher plasma clozapine concentrations and adds urgency to the requirement for dose adjustment should smoking habit change. A clozapine:norclozapine ratio greater then 2 suggests either a nontrough sample, or that clozapine N-demethylation has become saturated.
Objectives
The aim of this study was to develop high-throughput, quantitative and highly selective mass spectrometric, targeted immunoassays for clinically important proteins in human plasma or serum.
Design and methods
The described method coupled mass spectrometric immunoassay (MSIA), a previously developed technique for immunoenrichment on a monolithic microcolumn activated with an anti-protein antibody and fixed in a pipette tip, to selected reaction monitoring (SRM) detection and accurate quantification of targeted peptides, including clinically relevant sequence or truncated variants.
Results
In this report, we demonstrate the rapid development of MSIA-SRM assays for sixteen different target proteins spanning seven different clinically important areas (including neurological, Alzheimer's, cardiovascular, endocrine function, cancer and other diseases) and ranging in concentration from pg/mL to mg/mL. The reported MSIA-SRM assays demonstrated high sensitivity (within published clinical ranges), precision, robustness and high-throughput as well as specific detection of clinically relevant isoforms for many of the target proteins. Most of the assays were tested with bona-fide clinical samples.
In addition, positive correlations, (R2 0.67–0.87, depending on the target peptide), were demonstrated for MSIA-SRM assay data with clinical analyzer measurements of parathyroid hormone (PTH) and insulin growth factor 1 (IGF1) in clinical sample cohorts.
Conclusions
We have presented a practical and scalable method for rapid development and deployment of MS-based SRM assays for clinically relevant proteins and measured levels of the target analytes in bona fide clinical samples. The method permits the specific quantification of individual protein isoforms and addresses the difficult problem of protein heterogeneity in clinical proteomics applications.
Serum steroid paneling by LC-MS/MS is useful for diagnosing ACC by combining the measurement of steroid hormones and their precursors in a single analysis.
The method is suitable for high-throughput therapeutic drug monitoring of DOACs. The acquisition of full scan data allows for the retrospective identification of metabolites. The method can be used to identify a particular DOAC if information on the drug taken is lacking.
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