Using mass spectrometry to overcome the longstanding inaccuracy of a commercially-available clinical testosterone immunoassay.

Accurate measurement of testosterone is important for the diagnosis of gonadal disorders in men, women, and children. Testosterone measurement has limited accuracy at low concentrations by most commercially available immunoassays. We aimed to develop an LC-MS/MS assay to address the inaccuracy of the in-house immunoassay observed over the past decade and to replace it with the new assay. Testosterone in serum/plasma was extracted with commercial supported liquid extraction plates. Method validation was performed following the CLSI C62-A guideline. A total of 126 samples were used for method comparison between the Beckman UniCel DxI immunoassay and LC-MS/MS. Results by immunoassay were 20% lower compared with LC-MS/MS and had minimal correlation (R2 = 0.403) with LC-MS/MS below 100 ng/dL. When comparing specimens from the Accuracy-Based Survey from the College of American Pathologists, the newly developed assay agreed well with the CDC reference measurement procedure. In summary, immunoassay measurement of testosterone can be significantly inaccurate, especially at low concentrations. The newly developed LC-MS/MS assay provides accurate results across the entire measurable range.

Calibrating from Within: Multipoint Internal Calibration of a Quantitative Mass Spectrometric Assay of Serum Methotrexate.

BACKGROUND: Clinical LC-MS/MS assays traditionally require that samples be run in batches with calibration curves in each batch. This approach is inefficient and presents a barrier to random access analysis. We developed an alternative approach called multipoint internal calibration (MPIC) that eliminated the need for batch-mode analysis. METHODS: The new approach used 4 variants of 13C-labeled methotrexate (0.026-10.3 µM) as an internal calibration curve within each sample. One site carried out a comprehensive validation, which included an evaluation of interferences and matrix effects, lower limit of quantification (LLOQ), and 20-day precision. Three sites evaluated assay precision and linearity. MPIC was also compared with traditional LC-MS/MS and an immunoassay. RESULTS: Recovery of spiked analyte was 93%-102%. The LLOQ was validated to be 0.017 µM. Total variability, determined in a 20-day experiment, was 11.5%CV. In a 5-day variability study performed at each site, total imprecision was 3.4 to 16.8%CV. Linearity was validated throughout the calibrator range (r2 > 0.995, slopes = 0.996-1.01). In comparing 40 samples run in each laboratory, the median interlaboratory imprecision was 6.55%CV. MPIC quantification was comparable to both traditional LC-MS/MS and immunoassay (r2 = 0.96-0.98, slopes = 1.04-1.06). Bland-Altman analysis of all comparisons showed biases rarely exceeding 20% when MTX concentrations were >0.4 µM. CONCLUSION: The MPIC method for serum methotrexate quantification was validated in a multisite proof-of-concept study and represents a big step toward random-access LC-MS/MS analysis, which could change the paradigm of mass spectrometry in the clinical laboratory.

Quantification of Methotrexate in Human Serum and Plasma by Liquid Chromatography Tandem Mass Spectrometry.

Mass spectrometry (MS) is a highly specific and sensitive technique that is used for the detection of many different analytes with diverse chemical characteristics. It has been adopted by clinical laboratories for the quantification of small molecules and, by extension, has been widely used for therapeutic drug monitoring. It is an attractive alternative to immunoassay methods, because it is not subject to the same interferences. A limitation of MS (relative to immunoassays) is the turnaround time. However, this can be addressed by workflow parallelization with other assays. Herein we describe a tandem LC-MS/MS method for the detection and quantification of methotrexate in human plasma with a lower limit of quantification of 0.01 µM and within-assay and between-assay coefficients of variation of less than 15%. This method lacks interference from high-abundance metabolites and utilizes kindred chromatography to improve turnaround time in the therapeutic drug monitoring laboratory.

Design and implementation of software for automated quality control and data analysis for a complex LC/MS/MS assay for urine opiates and metabolites.

BACKGROUND: Mass spectrometry provides a powerful platform for performing quantitative, multiplexed assays in the clinical laboratory, but at the cost of increased complexity of analysis and quality assurance calculations compared to other methodologies. METHODS: Here we describe the design and implementation of a software application that performs quality control calculations for a complex, multiplexed, mass spectrometric analysis of opioids and opioid metabolites. RESULTS: The development and implementation of this application improved our data analysis and quality assurance processes in several ways. First, use of the software significantly improved the procedural consistency for performing quality control calculations. Second, it reduced the amount of time technologists spent preparing and reviewing the data, saving on average over four hours per run, and in some cases improving turnaround time by a day. Third, it provides a mechanism for coupling procedural and software changes with the results of each analysis. We describe several key details of the implementation including the use of version control software and automated unit tests. CONCLUSIONS: These generally useful software engineering principles should be considered for any software development project in the clinical lab.