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

Introduction

Oxidized LDL (oxLDL) is formed by the spontaneous reaction between aldehyde byproducts of lipid peroxidation and lysine residues of apolipoprotein B within LDL. Clinically, oxLDL is used as a marker of coronary artery disease and predictor of metabolic syndrome risk. Despite its popularity as a clinical marker, no systematic studies of oxLDL stability, in which serum or plasma has been pre-analytically exposed to an array of different time and temperature conditions, have been carried out.

Objective

To systematically evaluate the stability of oxLDL in human serum samples exposed to thawed conditions (> −30 °C) for varying periods of time while monitoring a second protein/small molecule redox system as a positive control for non-enzymatic biomolecular activity.

Methods

OxLDL was measured in serum samples, from 24 different humans, that had been pre-exposed to three different time courses at 23 °C, 4 °C and −20 °C using ELISA kits from Mercodia that employ the 4E6 mouse monoclonal antibody. A liquid chromatography/mass spectrometry-based marker of serum exposure to thawed conditions known as ΔS-Cys-Albumin was employed as a positive control.

Results

OxLDL was stable in serum exposed to 23 °C for up to 48 h, 4 °C for 21 days, or −20 °C for 65 days. ΔS-Cys-Albumin changed dramatically during these time courses (p < 0.001).

Conclusions

OxLDL is remarkably stable ex vivo in human serum samples exposed to thawed conditions.

Background

Steroids play a key role in numerous physiological processes. Steroid determination is a useful tool to explore various endocrine diseases. Because of its specificity, mass spectrometry is considered to be a reference method for the determination of steroids in serum compared to radioimmunoassay. This technology could progress towards more automation for the optimal organization of clinical laboratories and ultimately for the benefit of patients.

Methods

A fully automated ultra-high-performance liquid chromatography-tandem mass spectrometry method was developed and fully validated to determine five steroids in serum. Sample preparation was based on protein precipitation with filtration followed by online solid phase extraction. Chromatographic separation was performed using a biphenyl stationary phase.

Results

The method was successfully validated according to European Medicine Agency guidelines. Coefficients of variation did not exceed, respectively, 8.4% and 8.1% for intra- and inter-assay precision. Method comparison with radioimmunoassay showed a proportional bias for all compounds, except for testosterone in men. Comparison with another LC-MS/MS method demonstrated acceptable concordance for all steroids, although a small bias was observed for androstenedione.

Conclusion

The novelty of this method is that it has been fully automated. Automation provides benefits in traceability and allows significant savings in cost and time.

Objectives

Highly selective and sensitive multi-analyte methods for the analysis of steroids are attractive for the diagnosis of endocrine diseases. Commercially available kits are increasingly used for this purpose. These methods involve laborious solid phase extraction, and the respective panels of target analytes are incomplete. We wanted to investigate whether an improvement of kit solutions is possible by introducing automated on-line solid phase extraction (SPE) and combining originally separate analyte panels.

Methods

Sample preparation was performed using automated on-line SPE on a high-pressure stable extraction column. Chromatographic separation, including isobaric compounds, was achieved using a 0.25 mM ammonium fluoride-methanol gradient on a small particle size biphenyl column. Standard compounds and internal standard mixtures of two panels of a commercially available kit were combined to achieve an optimized and straightforward detection of 15 endogenous steroids. Validation was performed according to the European Medicines Agency (EMA) guidelines with slight modifications.

Results

Validation was successfully performed for all steroids over a clinically relevant calibration range. Deviations of intra- and inter-assay accuracy and precision results passed the criteria and no relevant matrix effects were detected due to highly effective sample preparation. External quality assessment samples showed the applicability as a routine diagnostic method, which was affirmed by the analyses of anonymized clinical samples.

Conclusions

It was found possible to complement a commercially available kit for quantitative serum steroid profiling based on isotope dilution LC-MS/MS by implementing automated on-line SPE, thereby improving the practicality and robustness of the measurement procedure.

The need for high-throughput analysis of multiple analytes for inborn errors of metabolism in newborn screening (NBS) has led to the introduction of tandem mass spectrometry (MS/MS) into the NBS laboratory. In a flow-injection analysis (FIA), the predominant MS/MS method utilized for NBS, samples are introduced directly into the mass spectrometer without chromatographic separation. When a high-throughput FIA-based MS/MS method is implemented on newer generations of mass spectrometers with increased sensitivity, the risk of carryover and contamination increases. In the present study, we report the carryover of ornithine identified during the implementation of the NeoBase™ 2 (PerkinElmer) non-derivatized kits on the Xevo-TQD platform (Waters Corporation) and describe the source of the carryover, which was traced to the stainless-steel frit-type inline filter. Furthermore, a possible compound-dependent interaction with the stainless-steel frit is suggested based on the structure of ornithine and its effect on separation techniques. Investigation and mitigation of carryover can be a time and resource consuming process, and to this end, our report on identification of a stainless-steel frit as the source of delayed elution and carryover of ornithine should be recognized as a rare, albeit possible source of carryover in FIA-MS/MS methods adopted for NST.

Background

Analytical methods to measure trace and toxic elements are essential to evaluate exposure and nutritional status. A ten-element panel was developed and validated for clinical testing in whole blood. Retrospective data analysis was conducted on patient samples performed at ARUP Laboratories.

Methods

A method was developed and validated to quantify ten elements in whole blood by ICP-MS. Fifty microliters of sample were extracted with 950 μL of diluent containing 1 % ammonium hydroxide, 0.1 % Triton X-100, 1.75 % EDTA along with spiked internal standards. Four calibrators were used for each element and prepared in goat blood to match the patient specimen matrix. Samples were analyzed with an Agilent 7700 ICP-MS with a Cetac MVX 7100 μL Workstation autosampler.

Results

The assay was linear for all elements with inter- and intra-assay imprecision less than or equal to 11% CV at the low end of the analytical measurement range (AMR) and less than or equal to 4% CV at the upper end of the AMR for all elements. Accuracy was checked with a minimum of 40 repeat patient samples, proficiency testing samples, and matrix-matched spikes. The linear slopes for the ten elements ranged from 0.94 to 1.03 with intercepts below the AMR and R² ranging from 0.97 to 1.00.

Conclusions

The multi-element panel was developed to analyze ten elements in whole blood to unify the sample preparation and increase batch run efficiency. The improved analytical method utilized matrix-matched calibrators for accurate quantification to meet regulatory requirements. The assay was validated according to guidelines for CLIA-certified clinical laboratories and was suitable for clinical testing to assess nutritional status and toxic exposure.

Introduction

The sample matrix composition, which is greatly affected by the type of blood collection tube used during phlebotomy, is of major importance in laboratory testing as it can influence test results. We developed an LC-MRM-MS test to molecularly characterize antithrombin in citrate plasma. The test principle differs greatly from traditional laboratory tests and the influence of varying plasma sample matrices is largely unknown.

Objectives

To identify whether variations in sample matrix affect the LC-MRM-MS test for antithrombin and assess whether sample pre-processing by immunocapture reduces matrix-specific effects.

Methods

Samples (n = 45) originating from four different blood collection tubes (sodium citrate, lithium heparin, K₂-EDTA and K₂-EDTA with protease inhibitors) were processed directly or after immunocapture. Antithrombin was digested into proteotypic peptides, which were monitored by LC-MRM-MS. Results from lithium heparin and the K₂-EDTA matrices were compared to the standard sample matrix, sodium citrate, using Deming regression analysis and repeated measures one-way ANOVA.

Results

Deming regression analysis of directly processed samples revealed slopes deviating >5% from the line of identity for at least six out of 22 peptides in all matrices. Significant differences between all matrices were found upon analysis by ANOVA for at least 10 peptides. Pre-processing by immunocapture led to slopes within 5% of the line of identity for nearly all peptides of the matrices. Furthermore, significant differences between matrices after immunocapture were only observed for four peptides.

Conclusion

Variations in the sample matrix affect the measurement of antithrombin by LC-MRM-MS, but observed effects are greatly reduced upon pre-processing by immunocapture.

Introduction

Adherence to medication is an important determinant of outcomes in chronic diseases like heart failure. Drug assays provide objective adherence biomarkers. Dried blood spots (DBS) are appealing samples for drug assays due to less demanding transportation and storage requirements.

Objectives

To analytically validate a LC-MS/MS method for the simultaneous quantification of carvedilol, enalaprilat, and perindoprilat in DBS and evaluate the feasibility of using the method as an adherence determining assay. To validate the assay further clinically by establishing correlation and agreement between plasma and DBS samples from a pharmacokinetic pilot study.

Methods

The method was validated over a concentration range of 1.00–200 ng/mL according to FDA guidelines. Adherence tracking ability of the assay was evaluated using a pharmacokinetic pilot study. Correlation and agreement were evaluated through Deming regression and Bland-Altman analysis, respectively.

Results

Accuracy, precision, selectivity, and sensitivity were proven with complete and reproducible extraction recovery at all concentrations tested. Stability of the analytes in the matrix and throughout sample processing was proven. The full range of concentrations of the pharmacokinetic pilot study could be quantified for enalaprilat, but not for carvedilol and perindoprilat. The difference between the observed and calculated plasma concentrations was less than 20 % of their mean for >67 % of samples for all analytes.

Conclusions

The assay is suitable as a screening tool for carvedilol and perindoprilat, while suitable as an adherence determining assay for enalaprilat. Equivalence between observed and predicted plasma concentrations proves DBS and plasma concentrations can be used interchangeably.