Development and validation of a quantitative assay based on tandem mass spectrometry

Abstract

Many routine hospital and clinical research assays have relied upon immunoassay procedures to achieve sensitive measurements of a range of important analytes. Some of the methods have been developed in-house but increasingly commercial kits and automated analysers have become commonplace. The accuracies of these methods are under question in health care. Mass spectrometry (MS) is potentially a more accurate technique with the ability to demonstrate specificity. An introduction of the basic analytical aspects of liquid chromatography (LC) -MS/MS leads on to the validation of the method before general use. LC coupled with MS and tandem mass spectrometry (MS n ) is being adopted in a number of hospital laboratories for the quantitative analysis of a number of analytes from physiological matrices, but standards for development and validation of such assays are not easily available. Most assays can be regarded as in-house methods and herein may lay the failure so far for mass spectrometric methods to improve quality of results between laboratories for an analyte using the same technology. Manufacturers are taking on board the experience of clinical laboratories with kits containing all or most of the disposable items and reagents. A number of documents and guidelines have been consulted. These documents are expensive to purchase, are often very long and not easy to read. This review highlights the specific requirements for introduction of a tandem mass spectrometric test for small molecules into a routine hospital laboratory. A number of experiments need to be planned and executed in order to describe a new quantitative method in terms of selectivity, accuracy, imprecision, sensitivity and stability. The introduction of a quantitative method based on tandem MS requires careful validation. This review has distilled out important points from a number of key documents in order to provide a working validation guideline for clinical laboratories. In a supplementary file a working document for assembling the assay validation is proposed.

Has the era of individualised medicine arrived for antifungals? A review of antifungal pharmacogenomics

“…Assay is fully validated 190,191 196 In salvage therapy for invasive aspergillosis, response rate increased with increasing serum levels and in patients with average levels above 1.25 mg/L response rate was 75% 197 Table 6 summarises therapeutic ranges for itraconazole, voriconazole and posaconazole, and Table 7 lists the suggested indications for TDM. Use of TDM is becoming more widespread and data from reference laboratories indicate that therapeutic levels are not achieved in a significant proportion of routine samples.…”

Multiple Reaction Monitoring with Multistage Fragmentation (MRM3) Detection Enhances Selectivity for LC-MS/MS Analysis of Plasma Free Metanephrines

“…Liquid chromatography coupled to tandem quadrupole mass spectrometry (LC-MS/MS) is widely used as a powerful tool for quantifying target analytes in clinical chemistry laboratories, owing to the selectivity provided by multiple reaction monitoring (MRM) (1)(2)(3). 3 However, for a number of assays, interferences exist that can cause falsely increased values when using MRM detection (4 -7 ).…”

“…3 However, for a number of assays, interferences exist that can cause falsely increased values when using MRM detection (4 -7 ).…”

“…Enhanced selectivity can be achieved by the use of quadrupole-ion trap tandem mass spectrometers that allow multistage fragmentation (MRM 3 ), in which primary fragment ions, created by collision-induced dissociation, can be isolated and then further fragmented into secondary fragment ions for quantification. Early MRM 3 studies with quadrupole linear ion trap (Q/LIT) instruments focused on characterization and identification in proteomics (8,9 ) and genomics (10 ).…”

“…Early MRM 3 studies with quadrupole linear ion trap (Q/LIT) instruments focused on characterization and identification in proteomics (8,9 ) and genomics (10 ). Newer-generation Q/LIT instruments have demonstrated greater quantitative sensitivity along with reduced cycle times owing to decreased excitation times for MRM 3 experiments (11,12 ). These technological advances have made MRM 3 detection more compatible with both the fast liquid chromatography used by modern LC-MS platforms and the sensitivity requirements of quantitative clinical analysis.…”