Journal of Mass Spectrometry and Advances in the Clinical Lab最新文献

Background

Methods

Results

Conclusion

Introduction

Objective

Methods

Results

Conclusion

Background

Methods

Results

Conclusions

Mass spectrometry (MS) is a versatile analytical tool used in various fields such as biochemistry, pharmacology, omics, and clinical analysis for determining and quantifying compounds based on their molecular mass and structure through the mass-to-charge ratio. While MS offers high specificity and selectivity, it encounters challenges including matrix effects, in-source fragmentation, and other interferences caused by natural isotopic abundance, as well as isomeric and isobaric compounds. These challenges can impede accurate qualitative and quantitative analysis. Visual aids such as graphical illustrations can help elucidate the chemical differences and similarities among isotopes, isomers, and isobaric compounds.

Objective

The rapid detection and differentiation of strains of the BA.2.86 lineage including the new sub-variant JN.1 (BA.2.86.1.1) is demonstrated employing selected ion monitoring (SIM) and high resolution mass spectrometry.

Methods

A study of a preliminary set of BA.2.86 lineage positive specimens, identified BA.2.86 and BA.2.86.1.1 peptide markers in 62.5 % and 29.1 % of samples.

Results

Peptide-specific markers in the surface spike protein associated with the L455S mutation are confidently detected with high sensitivity in protein and virus digests.

The virus was thus confidently assigned in over 91 % of positive specimens.

Conclusions

A rise in the global prevalence of the JN.1 (BA.2.86.1.1) immune evasive sub-variant, that emerged in late 2023, requires that new strategies and protocols to detect such strains in human specimens are accelerated and implemented.

Background

Reference measurement procedures are an essential element in the standardization and comparability of analytical measurement results in laboratory medicine. No LC-MS/MS-based reference measurement procedure for cefepime in serum has been published previously.

Materials and methods

An isotope-dilution based two-dimensional LC-MS/MS reference measurement procedure for cefepime concentrations in human serum was developed and tested. The value assignment of unknown samples is based on a defined measurement series validation. Six unknown samples can be measured per series. Pass criteria for the run and the samples were determined empirically based on a performance evaluation. For this purpose, a between-run determination of five runs of the defined measurement series with six cefepime samples was carried out and evaluated. The goal was to define rigorous, realistic target limits and minimize measurement uncertainty. The final defined target limits are used for series-based validation and value assignment. The results for the six unknown samples are provided with the associated measurement uncertainty for this series.

Results

The developed and extensively studied measurement procedure for the quantification of cefepime in serum was found to be practicable and fit for its purpose. The between-run mean imprecision of the six cefepime samples was ≤ 2.0 %, for the QCs it was ≤ 2.3 % and the between-run mean inaccuracy of the QCs was within ± 1.1 %.

Conclusion

The novel isotope-dilution-LC-MS/MS measurement procedure in accordance to ISO 15193 can be recommended as candidate reference measurement procedure for the value assignment of cefepime concentrations in human serum.

Introduction

Internal standards correct for measurement variation due to sample loss. Isotope labeled analytes are ideal internal standards for the measurement of fatty acids in human plasma but are not always readily available. For this reason, quantification of multiple analytes at once is most often done using only a single or few internal standards. The magnitude of the impact this has on method accuracy and precision is not well studied for gas chromatography-mass spectrometry systems.

Objective

This study aims to estimate bias and changes in uncertainty associated with using alternative fatty acid isotopologue internal standards for the estimation of similar or dissimilar long chain fatty acids.

Method

Using a previously reported method for the quantification of 27 fatty acids in human plasma using 18 internal standards we obtained estimates of bias and uncertainty at up to three levels of fatty acid concentration.

Results

With some notable exceptions, method accuracy remained relatively stable when using an alternative internal standard (Median Relative Absolute Percent Bias: 1.76%, Median Spike-Recovery Absolute Percent Bias: 8.82%), with larger changes in method precision (Median Increase in Variance: 141%). Additionally, the degree of difference between analyte and internal standard structure was related to the magnitude of bias and uncertainty of the measurement.

Conclusion

The data presented here show that the choice of internal standard used to estimate fatty acid concentration can affect the accuracy and reliability of measurement results and, therefore, needs to be assessed carefully when developing analytical methods for the measurement of fatty acid profiles.

Disclaimer: The findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the Centers for Disease Control and Prevention/the Agency for Toxic Substances and Disease Registry. Use of trade names is for identification only and does not imply endorsement by the Centers for Disease Control and Prevention, the Public Health Service, and the US Department of Health and Human Services.

When quantifying therapeutic drugs using LC-MS/MS instrumentation in clinical laboratories, batch-mode analysis with a calibration curve consisting of 6–10 concentrations for each analyte is the most widely used approach. However, this is an inefficient use of this technology since it increases cost, delays result availability and precludes random instrument access. Various alternative methods to reduce the calibrator use and improve efficiency without compromising analytical quality have been investigated, and a single-point calibration has been reported to be the simplest, least expensive and the quickest approach.

This study compares a single and a multi-point calibration method using LC-MS/MS with 5-fluorouracil (5-FU) as a model drug. The method was validated for quantitative analysis of 5-FU over a concentration range of 0.05–50 mg/L. Patients undergoing cancer treatment with intravenous 5-FU had plasma 5-FU concentrations measured, and their dose adjusted in real time based on the calculated area under the time-concentration curve (AUC). Subsequently, a single point calibration method using a concentration at 0.5 mg/L was compared to the multi-point calibration method in terms of accuracy and precision. A Bland-Altman bias plot and a Passing-Bablok regression analysis showed a good agreement between the two methods (mean difference = −1.87 %, slope = 1.002, respectively) when comparing patient plasma 5-FU concentrations. The calibration method did not impact the AUC results nor the decision on 5-FU dose adjustments. Our study demonstrated that a single point calibration method produced analytically and clinically comparable results to those produced by a multi-point method when quantifying 5-FU and is feasible to be used clinically.

Introduction

Benzodiazepines are frequently prescribed and misused therefore urine drug screening (UDS) is performed in many patient populations. Most current benzodiazepine immunoassays have poor sensitivity, particularly for detecting the metabolites of newer benzodiazepines such as lorazepam in urine.

Objectives

We aimed to verify the clinical performance of the new qualitative Roche Benzodiazepines II (BNZ2) immunoassay, as well as compare its performance to the Roche Benzodiazepines Plus (BENZ) assay in two patient populations: UDS in the emergency department (ED) and compliance monitoring.

Methods

An initial verification study was performed, selecting for samples containing clonazepam and lorazepam metabolites. Performance of the BNZ2 and BENZ assays was compared to liquid chromatography-tandem mass spectrometry (LC-MS/MS) as the reference method. Sensitivity, specificity, false positive rate (FPR) and false negative rate (FNR) were determined.

Results

We verified the performance claims in the initial verification and demonstrated similar precision, with coefficient of variations (CVs) of 12.8% and 7.7% for negative and positive controls, respectively. Furthermore, we observed higher clinical sensitivity and lower FNR with the BNZ2 assay in both the ED and compliance monitoring populations due to improved cross-reactivity for lorazepam and clonazepam metabolites. Despite these improvements, the BNZ2 assay was unable to detect 27% of specimens positive by LC-MS/MS, including specimens from patients using benzodiazepines without prescription.

Discussion

Due to its improved performance and rapid turnaround time, the BNZ2 assay should be implemented for UDS in the ED. However, the assay should not replace LC-MS/MS testing for compliance monitoring, as unsuspected benzodiazepine use may go undetected.