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OBJECTIVES: Phenytoin is an anti-epileptic drug that has a narrow therapeutic index, and therefore requires therapeutic drug monitoring. Only the free fraction is pharmacologically active, and in some cases, accurate determination of the free phenytoin concentration may be essential to prevent phenytoin toxicity. Although it is possible to measure free phenytoin concentration, often only the total concentration is measured, with equations used to estimate the free fraction. Several equations are quoted in the literature with no overall consensus with regard to accuracy. This study aimed to assess the correlation between total and free phenytoin in a mixed patient population, and to compare the accuracy of several different equations used to estimate the free phenytoin concentration. METHODS: Fifty-one serum samples were analysed for total phenytoin, free phenytoin and albumin. The measured free phenytoin concentrations were compared against those estimated using five selected equations, identified through a literature search. RESULTS: This study showed poor correlation between the total and measured free phenytoin concentrations, and between the estimated and measured free concentrations. The overall correlation was concentration-dependent, but a correction factor could not be applied to improve the accuracy consistently. The equations assessed showed wide variability between the estimated and measured free phenytoin concentrations, with several showing a clinically significant negative bias when compared with the measured free fraction. DISCUSSION: This study highlights the disparity of the free phenytoin concentrations generated by the equations. Underestimation of free phenytoin concentrations using these equations may result in phenytoin toxicity, bringing into question the safety of using calculated values for patient management in place of physical measurement of free phenytoin concentration by ultra-performance liquid chromatography tandem mass spectrometry.
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Background Liquid chromatography-tandem mass spectrometry (LC-MS/MS) offers advantages over immunoassay due to its increased specificity and ability to multiplex metabolites within a single run. Wide scale adoption of LC-MS/MS in routine clinical laboratories is restricted in part due to the high level of technical expertise required. The Thermo Scientific™ Cascadion™ SM Clinical Analyzer is the first fully automated, random access clinical analyser that utilises LC-MS/MS technology. We report an analytical validation of the 25-hydroxy vitamin D2 and D3 assays on the Cascadion Analyzer and an assessment of its performance within a routine clinical laboratory. Methods Analyser usability was assessed by staff with no previous experience of LC-MS/MS. An analytical validation included analysis of 154 patient samples on two different Cascadion Analyzers and a four-way method comparison of 146 patient samples on Roche and Siemens immunoassays and an in-house LC-MS/MS method. Accuracy was assessed using external quality assurance and reference materials. Seven third party IQC materials were tested on Cascadion. Results Cascadion proved easy to use by scientific and non-scientific staff. The assay passed all validation criteria. Excellent agreement was demonstrated between two different Cascadions (y = 0.97x + 3.9 nmol/L, r2 > 0.99). A method comparison demonstrated no significant difference (p > 0.05) between the Cascadion and the Roche immunoassay. A significant difference (p < 0.0001) was observed between the Cascadion and an LC-MS/MS and Siemens methods. Results obtained from EQA and reference material showed a mean bias of +3.09% and all samples were within ±10% of assigned concentrations. All third party IQC samples tested were compatible for use with Cascadion. Conclusions The Cascadion Analyzer is a fully automated LC-MS/MS system that requires no prior LC-MS/MS expertise. The vitamin D assays demonstrated excellent performance with high levels of accuracy.
