Capillary Electrophoresis-Mass Spectrometry at Trial by Metabo-Ring: Effective Electrophoretic Mobility for Reproducible and Robust Compound Annotation.

Capillary zone electrophoresis-mass spectrometry (CE-MS) is a mature analytical tool for the efficient profiling of (highly) polar and ionizable compounds. However, the use of CE-MS in comparison to other separation techniques remains underrepresented in metabolomics, as this analytical approach is still perceived as technically challenging and less reproducible, notably for migration time. The latter is key for a reliable comparison of metabolic profiles and for unknown biomarker identification that is complementary to high resolution MS/MS. In this work, we present the results of a Metabo-ring trial involving 16 CE-MS platforms among 13 different laboratories spanning two continents. The goal was to assess the reproducibility and identification capability of CE-MS by employing effective electrophoretic mobility (µeff) as the key parameter in comparison to the relative migration time (RMT) approach. For this purpose, a representative cationic metabolite mixture in water, pretreated human plasma, and urine samples spiked with the same metabolite mixture were used and distributed for analysis by all laboratories. The µeff was determined for all metabolites spiked into each sample. The background electrolyte (BGE) was prepared and employed by each participating lab following the same protocol. All other parameters (capillary, interface, injection volume, voltage ramp, temperature, capillary conditioning, and rinsing procedure, etc.) were left to the discretion of the contributing laboratories. The results revealed that the reproducibility of the µeff for 20 out of the 21 model compounds was below 3.1% vs 10.9% for RMT, regardless of the huge heterogeneity in experimental conditions and platforms across the 13 laboratories. Overall, this Metabo-ring trial demonstrated that CE-MS is a viable and reproducible approach for metabolomics.

Highly sensitive and selective separation of intact parathyroid hormone and variants by sheathless CE-ESI-MS/MS.

Parathyroid hormone (PTH) is a common clinical marker whose quantification relies on immunoassays, giving variable results as batch, brand, or target epitope changes. Sheathless CE-ESI-MS, combining CE resolution power and low-flow ESI sensitivity, was applied to the analysis of PTH in its native conformation in the presence of related forms. Fused silica and neutral-coated capillaries were investigated, as well as preconcentration methods such as transient isotachophoresis, field-amplified sample injection (FASI), and electrokinetic supercharging (EKS). The method for the separation of PTH and its variants was first developed using fused-silica capillary with UV detection. An acidic BGE was used to separate 1-84 PTH (full length), 7-84 PTH, and 1-34 PTH. Acetonitrile was added to the BGE to reduce peptide adsorption onto the capillary wall and transient isotachophoresis was used as analyte preconcentration method. The method was then transferred to a sheathless CE-ESI-MS instrument. When using a fused silica capillary, CE-MS was limited to µg/mL levels. The use of a neutral coating combined with FASI or EKS allowed a significant increase in sensitivity. Under these conditions, 1-84 PTH, 7-84 PTH, and 1-34 PTH were detected at concentrations in the low ng/mL (FASI) or pg/mL (EKS) range.

Qualitative and quantitative comparison of different commercially available 1-84 parathyroid hormone proteins to the WHO international standard 95/646 using orthogonal methods.

Relative quantitation methods rely on the use of reference substances to determine the content of samples. The aim of this study was to compare 1-84 parathyroid hormone (PTH) standards from different manufacturers to the WHO international standard 95/646. CE and LC with UV detection were investigated as quick and inexpensive quantitation methods, with an emphasis on selectivity between intact 1-84 PTH and its oxidized forms. Both methods were fully validated according to ICH Q2R1. Moreover, method performance was also evaluated according to guidelines defining the maximum allowable measurement uncertainty (MU) of a biological parameter from its intraindividual variation (CVI), as well as the proportion of that MU devoted to the reference material. This study highlighted the fact that some 1-84 PTH standards have a content that is actually twice as high as the one stated on the label, which was confirmed by an amino acid analysis investigation. Our approach offers a quick and inexpensive way to estimate the content of 1-84 PTH standards.

Development and validation of a fast and reliable method for the quantification of glucagon by liquid chromatography and tandem mass spectrometry.

INTRODUCTION: The quantitation of glucagon remains a challenging immunoassay, mainly due to cross-reactivity. A sensitive, rapid and specific intact glucagon method is therefore necessary for quality routine analysis. A tandem mass spectrometry method to fulfill this objective is described in this work. METHODS: Glucagon was extracted from plasma employing a mixed-mode anion exchange solid-phase extraction. Sample stability was assessed in K2-EDTA and P800 tubes at different temperatures. We compared our method to two different immunoassays. FDA and EMA guidelines were followed for validation. An external quality control program served for comparison with other laboratories. RESULTS: Assay imprecision was below 4%. Recoveries were within 95-103%. LoQ was 8.75 pg/mL. Total analytical CV was 2.91%. Samples were found stable at 4 °C for less than 4 h. Diasource® RIA disagreed with our method. Mercodia® ELISA provided a closer agreement, also proven by external quality control samples. CONCLUSIONS: A rapid and specific LC-MS/MS method for glucagon quantitation has been developed, validated and is suitable to routine care. The simplicity and the good performances in terms of time and specificity, could open the possibility to establish a standardized method for glucagon.

A simple LC-MS method for the determination of iohexol and iothalamate in serum, using ioversol as an internal standard.

BACKGROUND: Chronic kidney disease (CKD) is diagnosed and explored through the determination of the glomerular filtration rate (GFR). Our goal was to develop a simple LC-MS method for the determination in serum of 2 popular GFR markers, contrast agents iohexol and iothalamate, for routine use and comparison studies between the two markers. A similar contrast agent, ioversol, was used as an internal standard and the method underwent a rigorous validation protocol based on ß-expectation tolerance intervals. METHODS: We adapted the HPLC-UV method from Cavalier et al. to our LC-MS system. Data treatment for the validation was performed using Multiquant 3.0 (Sciex, Framingham, MA, USA) and e.noval 3.0 software (Arlenda, Liège, Belgium). RESULTS: According to the validation results our method will give accurate and reliable results for concentrations ranging from 6.8 to 250µg/ml for iohexol and 6.15µg/ml to 250µg/ml for iothalamate. In our practice these intervals are sufficient to determine both compounds in most patient samples. Samples with higher detected concentrations can always be diluted into range. CONCLUSION: With its internal standard and extensive validation, our method is now ready for routine and clinical research use.

Problems with the PTH assays.

Even if the first assay for parathyroid hormone (PTH) was published in the early 1960s, its determination remains a challenge even today. Indeed, in the circulation, PTH is present in its active form (PTH 1-84), but many PTH fragments can also be present. These fragments accumulate when renal function declines and are recognized, at different extents, by the 2nd generation ("intact") PTH assays that are widely used in the clinical laboratories. Some assays, called "3rd generation PTH" do not recognize these fragments, but are not available everywhere. Hence, different problems are also linked with PTH determination. Among them, one can cite the lack of a reference method, the lack of standardization of the assays and, sometimes, the lack of consistent reference range. We can also point out stability problems and a large intra-individual variation. A workgroup is working on these problems under the auspices of the IFCC and we hope that some of these problems will be resolved in the next years. In this article, we will discuss all the possible issues of PTH determination.