Validation of an isotope dilution mass spectrometry (IDMS) measurement procedure for the reliable quantification of steroid hormones in waters.

Reliable data are compulsory to efficiently monitor pollutants in aquatic environments, particularly steroid hormones that can exert harmful effects at challenging analytical levels below the ng L-1. An isotope dilution two-step solid-phase extraction followed by an ultra-performance liquid chromatography separation coupled to tandem mass spectrometry (UPLC-MS/MS) detection method was validated for the quantification of 21 steroid hormones (androgens, estrogens, glucocorticoids, and progestogens) in whole waters. To achieve a realistic and robust assessment of the performances of this method, the validation procedure was conducted using several water samples representative of its intended application. These samples were characterized in terms of concentration of ionic constituents, suspended particulate matter (SPM), and dissolved organic carbon contents (DOC). For estrogens that are part of the European Water Framework Directive Watchlist (17beta-estradiol and estrone), the performances met the European requirements (decision 2015/495/EU) in terms of limit of quantification (LQ) and measurement uncertainty. For 17alpha-ethinylestradiol, the challenging LQ of 0.035 ng L-1 was reached. More generally, for 15 compounds out of 21, the accuracy, evaluated in intermediate precision conditions at concentrations ranging between 0.1 and 10 ng L-1, was found to be within a 35% tolerance. The evaluation of the measurement uncertainty was realized following the Guide to the expression of Uncertainty in Measurement. Finally, a water monitoring survey demonstrated the suitability of the method and pointed out the contamination of Belgium rivers by five estrogens (17alpha-ethinylestradiol, estriol, 17alpha-estradiol, 17beta-estradiol, and estrone) and three glucocorticoids (betamethasone, cortisol, and cortisone) which have been up to now poorly documented in European rivers.

Overcoming matrix effects in quantitative liquid chromatography-mass spectrometry analysis of steroid hormones in surface waters.

RATIONALE: Accurate and reliable measurements are mandatory in the field of environmental monitoring. Matrix effects are often depicted as the Achilles' heel of liquid chromatography-mass spectrometry analysis since they may be prejudicial for analytical performances such as detection capability and accuracy, if not documented or compensated. Here a methodology for the evaluation and compensation of matrix effects is described. METHODS: Natural and synthetic representative water samples were used for the evaluation of matrix effects with the post-extraction addition technique. Samples were analysed using ultra-performance liquid chromatography separation coupled to tandem mass spectrometry and electrospray ionization. Isotopic dilution was investigated as a way to allow compensation of signal alteration and therefore satisfactory quantification. When this approach was not possible, a methodology was conducted for choosing the most appropriate internal standard. RESULTS: The matrix effects were dependent on both matrix composition and nature of analyte. They ranged from total signal suppression to signal enhancement of +27% but were independent of compound concentration. The correction of matrix effects by internal standards was satisfactory, particularly for compounds benefiting from isotope dilution leading to acceptable quantification performances. CONCLUSIONS: Even if no exhaustive or agreed criteria exist for the final interpretation of matrix effects, this study highlights the interest in isotope dilution for reducing their inherent prejudicial effects in quantification and the need to conduct this type of study for representative matrices. Moreover, a methodological approach is proposed for choosing the most appropriate available internal standard when isotope dilution is not possible.

Harmonization status of procalcitonin measurements: what do comparison studies and EQA schemes tell us?

Sepsis represents a global health priority because of its high mortality and morbidity. The key to improving prognosis remains an early diagnosis to initiate appropriate antibiotic treatment. Procalcitonin (PCT) is a recognized biomarker for the early indication of bacterial infections and a valuable tool to guide and individualize antibiotic treatment. To meet the increasing demand for PCT testing, numerous PCT immunoassays have been developed and commercialized, but results have been questioned. Many comparison studies have been carried out to evaluate analytical performance and comparability of results provided by the different commercially available immunoassays for PCT, but results are conflicting. External Quality Assessment Schemes (EQAS) for PCT constitute another way to evaluate results comparability. However, when making this comparison, it must be taken into account that the variety of EQA materials consist of different matrices, the commutability of which has not yet been investigated. The present study gathers results from all published comparison studies and results from 137 EQAS surveys to describe the current state-of-the-art harmonization of PCT results. Comparison studies globally highlight a significant variability of measurement results that nonetheless seem to have a moderate impact on medical decision-making. For their part, EQAS for PCT provides highly discrepant estimates of the interlaboratory CV. Due to differences in commutability of the EQA materials, the results from different peer groups could not be compared. To improve the informative value of the EQA data, the existing limitations such as non-harmonized conditions and suboptimal and/or unknown commutability of the EQA materials have to be overcome. The study highlights the need for commutable reference materials that could be used to properly evaluate result comparability and possibly standardize calibration, if necessary. Such an initiative would further improve the safe use of PCT in clinical routine.

Development of an antibody-free ID-LC MS method for the quantification of procalcitonin in human serum at sub-microgram per liter level using a peptide-based calibration.

The quantification of low abundant proteins in complex matrices by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) remains challenging. A measurement procedure based on optimized antibody-free sample preparation and isotope dilution coupled to LC-MS/MS was developed to quantify procalcitonin (PCT) in human serum at sub-microgram per liter level. A combination of sodium deoxycholate-assisted protein precipitation with acetonitrile, solid-phase extraction, and trypsin digestion assisted with Tween-20 enhanced the method sensitivity. Linearity was established through peptide-based calibration curves in the serum matrix (0.092-5.222 µg/L of PCT) with a good linear fit (R2 ≥ 0.999). Quality control materials spiked with known amounts of protein-based standards were used to evaluate the method's accuracy. The bias ranged from -2.6 to +4.3%, and the intra-day and inter-day coefficients of variations (CVs) were below 2.2% for peptide-based quality controls. A well-characterized correction factor was determined and applied to compensate for digestion incompleteness and material loss before the internal standards spike. Results with metrological traceability to the SI units were established using standard peptide of well-characterized purity determined by peptide impurity corrected amino acid analysis. The validated method enables accurate quantification of PCT in human serum at a limit of quantification down to 0.245 µg/L (bias -1.9%, precision 9.1%). The method was successfully applied to serum samples obtained from patients with sepsis. Interestingly, the PCT concentration reported implementing the isotope dilution LC-MS/MS method was twofold lower than the concentration provided by an immunoassay.

Evaluation of the necessity and the feasibility of the standardization of procalcitonin measurements: Activities of IFCC WG-PCT with involvement of all stakeholders.

Procalcitonin (PCT) is an important biomarker for sepsis diagnosis and management. To date, there is no higher-order reference measurement procedure (RMP) and certified reference material to achieve global standardization of results and results traceability to the SI units. Although efforts have been made to harmonize PCT results, a number of comparison studies and external quality assessment (EQA) schemes show conflicting results regarding results comparability and to date, equivalence of PCT results across the assays remains questionable in absence of studies relying on commutable EQA materials. In this context, the IFCC initiated activities to fill these gaps through the creation of the working group on standardization of PCT assays that gathers experts from National Metrology Institutes, calibration laboratories, clinicians, biologists, EQA providers and assay manufacturers. Among the activities, a higher order RMP and commutable reference materials are under development to build a robust reference measurement system (RMS). A commutability study is being organized to identify EQA materials that are fit for purpose to reliably estimate the current comparability of PCT results. This work will make it possible to evaluate the necessity and the feasibility for establishing and maintaining a new RMS for PCT assays, if deemed necessary.

Heterogeneity of Borrelia burgdorferi Sensu Stricto Population and Its Involvement in Borrelia Pathogenicity: Study on Murine Model with Specific Emphasis on the Skin Interface.

Lyme disease is a multisystemic disorder caused by B. burgdorferi sl. The molecular basis for specific organ involvement is poorly understood. The skin plays a central role in the development of Lyme disease as the entry site of B. burgdorferi in which specific clones are selected before dissemination. We compared the skin inflammatory response (antimicrobial peptides, cytokines and chemokines) elicited by spirochete populations recovered from patients presenting different clinical manifestations. Remarkably, these spirochete populations induced different inflammatory profiles in the skin of C3H/HeN mice. As spirochete population transmitted into the host skin is heterogeneous, we isolated one bacterial clone from a population recovered from a patient with neuroborreliosis and compared its virulence to the parental population. This clone elicited a strong cutaneous inflammatory response characterized by MCP-1, IL-6 and antimicrobial peptides induction. Mass spectrometry of this clone revealed 110 overexpressed proteins when compared with the parental population. We further focused on the expression of nine bacterial surface proteins. bb0347 coding for a protein that interacts with host fibronectin, allowing bacterial adhesion to vascular endothelium and extracellular matrix, was found to be induced in host skin with another gene bb0213 coding for a hypothetical protein. These findings demonstrate the heterogeneity of the B. burgdorferi ss population and the complexity of the interaction involved early in the skin.

Effects of controlled ingestion of kaolinite (5%) on food intake, gut morphology and in vitro motility in rats.

Geophagia is found in various animal species and in humans. We have previously shown that spontaneously ingested kaolinite interacts with the intestinal mucosa modifies nutrient absorption and slows down gastric emptying and intestinal transit in rats in vivo. However, the precise mechanisms involved are not elucidated. The aim of this work was to investigate the effects of controlled kaolinite ingestion on food intake, gut morphology and in vitro motility in rats. Male Wistar rats were fed with 5% kaolinite in standard food pellets during 7, 14 and 28 days. Body mass and food consumption were measured daily. Intestinal morphological and proteomic analyses were conducted. The length of mucosal lacteals was evaluated. Plasmatic levels of leptin and adiponectin were determined. Finally, organ bath studies were conducted to evaluate smooth muscle contractility. Food consumption was significantly increased during the first two weeks of kaolinite ingestion without any mass gain compared to controls. Kaolinite induced weak variations in proteins that are involved in various biological processes. Compared to control animals, the length of intestinal lacteals was significantly reduced in kaolinite group whatever the duration of the experiment. Leptin and adiponectin plasmatic levels were significantly increased after 14 days of kaolinite consumption. Changes in spontaneous motility and responses to electrical nerve stimulation of the jejunum and proximal colon were observed at day 14. Altogether, the present data give evidence for a modulation by kaolinite-controlled ingestion on satiety and anorexigenic signals as well as on intestinal and colonic motility.