Reference ranges of late-night salivary cortisol and cortisone measured by LC-MS/MS and accuracy for the diagnosis of Cushing's syndrome.

PURPOSE: International guidelines recommend salivary cortisol for the diagnosis of Cushing's syndrome. Despite mass spectrometry-based assays are considered the analytical gold-standard, there is still the need to define reference intervals and diagnostic accuracy of such methodology. METHODS: 100 healthy volunteers and 50 consecutive patients were enrolled to compare LC-MS/MS and electrochemiluminescence assay for the determination of late-night salivary cortisol and cortisone. Moreover, we aimed to determine reference intervals of salivary steroids in a population of healthy individuals and diagnostic accuracy in patients with suspected hypercortisolism and in a population including also healthy individuals. RESULTS: Method comparison highlighted a positive bias (51.8%) of immunoassay over LC-MS/MS. Reference intervals of salivary cortisol (0.17-0.97 µg/L), cortisone (0.84-4.85 µg/L) and ratio (0.08-0.30) were obtained. The most accurate thresholds of salivary cortisol for the diagnosis of hypercortisolism were 1.15 µg/L in the population with suspected hypercortisolism (AUC 1) and 1.30 µg/L in the population including also healthy individuals (AUC 1). Cut-off values of salivary cortisone (7.23 µg/L; Se 92.9%, Sp 97.2%, AUC 0.960 and Se 92.9%, Sp 99.1%, AUC 0.985 in suspected hypercortisolism and in overall population, respectively) and cortisol-to-cortisone ratio (0.20; Se 85.7%, Sp 80.6%, AUC 0.820 and Se 85.7%, Sp 85.5%, AUC 0.855 in suspected hypercortisolism and in overall population, respectively) were accurate and similar in both populations. CONCLUSION: LC-MS/MS is the most accurate analytical platform for measuring salivary steroids. Obtained reference intervals are coherent with previously published data and diagnostic accuracy for diagnosis of overt hypercortisolism proved highly satisfactory.

Effective validation of chromatographic analytical methods: The illustrative case of androgenic steroids.

The increasing need to develop quantitative chromatographic methods with upgradable multi-targeted approach, allowing flexible and reliable application on large daily workload makes the implementation of an efficient strategy of method's validation and maintenance crucial for the quality assurance policy. The expounding case of a gas chromatographic-mass spectrometric method for the urinary endogenous steroid profiling is presented to illustrate a validation strategy that combines rigorous estimation of validation parameters with highly efficient use of the collected data. The analysis of blank urine samples fortified at six concentration levels with 18 targeted steroids was replicated nine times in three working sessions along twelve days. This dataset of 54 analysis formed the groundwork on which the statistical evaluation of several validation parameters was founded, including calibration, intra- and inter-day accuracy and precision, limit of detection (LOD), limit of quantification, ion abundance ratio repeatability, selectivity, specificity, and carry-over. The preliminary comparison of the response variances at different concentration levels provided the evaluation for heteroscedasticity. Then, the most appropriate calibration model was determined for each steroid, in terms of order (linear vs. quadratic) and weighting, allowing to complete their quantitation in each solution. Intra- and inter-day accuracy and precision were calculated therefrom. LOD values were computed with the Hubaux-Vos method from the weighted linear segment of the calibration curves. Only the assessment of recovery and ionization suppression/enhancement required the execution of further independent experiments. The case study demonstrated that the application of adequate statistical testing typically produced non-homogeneous models of calibration curves, mostly arising from heteroscedastic and quadratic distribution of datasets, unlike what is reported in overly simplified approaches. The misleading information obtained from the regression coefficient R2 to evaluate linearity was evidenced. The strong dependence of calculated LOD and accuracy from the selected calibration parameters was highlighted, making the implementation of an adequate calibration maintenance policy highly advisable.

Toward the Interpretation of Positive Testing for Fentanyl and Its Analogs in Real Hair Samples: Preliminary Considerations.

The detection of new psychoactive substances (NPS) in hair has become extensively researched in recent years. Although most NPS fall into the classes of synthetic cannabinoids and designer cathinones, novel synthetic opioids (NSO) have appeared with increasing frequency in the illicit drug supply. While the detection of NSO in hair is now well documented, interpretation of results presents several controversial issues, as is quite common in hair analysis. In this study, an ultra-high-performance liquid chromatography-tandem mass spectrometry method able to detect 13 synthetic opioids (including fentanyl analogs) and metabolites in hair was applied to 293 real samples. Samples were collected in the USA between November 2016 and August 2018 from subjects who had reported heroin use in the past year or had already tested positive to hair testing for common opiates. The range, mean and median concentrations were calculated for each analyte, in order to draw a preliminary direction for a possible cut-off to discriminate between exposure to either low or high quantities of the drug. Over two-thirds (68%) of samples tested positive for fentanyl at concentrations between LOQ and 8600 pg/mg. The mean value was 382 pg/mg and the median was 95 pg/mg. The metabolites norfentanyl and 4-ANPP were also quantified and were found between LOQ and 320 pg/mg and between LOQ and 1400 pg/mg, respectively. The concentration ratios norfentanyl/fentanyl, 4-ANPP/fentanyl and norfentanyl/4-ANPP were also tested as potential markers of active use and to discriminate the intake of fentanyl from other analogs. The common occurrence of samples positive for multiple drugs may suggest that use is equally prevalent among consumers, which is not the case, as correlations based on quantitative results demonstrated. We believe this set of experimental observations provides a useful starting point for a wide discussion aimed to better understand positive hair testing for fentanyl and its analogs in hair samples.

Use of Cold Plasma To Inactivate Escherichia coli and Physicochemical Evaluation in Pumpkin Puree.

This work aimed to study the pumpkin puree processing by cold plasma corona discharge as an alternative to heat treatment to reduce Escherichia coli contamination and evaluate physicochemical alterations, using argon (Ar) as the process gas. The treatment time to verify E. coli inactivation was between 2 and 20 min, while physicochemical alterations were analyzed at 5, 10, 15, and 20 min. Cold plasma corona discharge technology to inactivate E. coli proved to be promising, reaching 3.62 log cycles of reduction at 20 min of treatment. The inactivation kinectics showed a tendency of higher decrease with time. Physicochemical characteristics indicate that plasma induces a decrease of pH; however, there is an indication that process gases have an important role and react with the environment and procedure reactive species. This technology may reduce the total carotenoid content of pumpkin puree and in color, mainly the a* parameter, which showed great reduction.

Biodegradation and ecotoxicological assessment of pectin production wastewater.

The chemical composition of pectin production wastewater and its toxicity during biological treatment were investigated. Samples of wastewater from different steps of a pectin production wastewater biological treatment plant were investigated including the influent of the treatment (1), after denitrification tank (2), after anaerobic treatment (3) and final effluent (4). The conventional physicochemical characteristics of samples did not indicate wastewater toxicity. However, toxicity assessments carried out on Vibrio fischeri and Scenedesmus subspicatus indicated low EC50 values. The fractionation of the samples using an XAD resin showed that the toxicity was associated with the organic matter. Wastewater apparent molecular mass distributions were 14.3, 25.0, 24.4 and 29.6 kDa for samples 1-4, respectively. Finally, characteristics of the sample by pyrolisis-gas chromatography-mass spectrometry (Py-CG-MS) demonstrated its polyphenolic nature and a 23% increase in the levels of such compounds after the first biological treatment step.

Cassava starch fermentation wastewater: characterization and preliminary toxicological studies.

Cassava starch fermentation wastewater is an industrial residue composed mainly of lactic acid bacteria with predominance of the genera Lactobacillus, and organic acids. To evaluate the safety of this residue for possible production of probiotic beverages, acute in mice and sub-chronic (28-day repeated dose) toxicity studies in rats were carried. The administration of a single dose of 5 g/kg/body weight did not produce mortality in mice. Rats treated with water containing 0 (control), 25%, 50%, and 100% of the residue for 28 days, did not present alterations in behaviour or in food and water consumption. There were no treatment-related changes of toxicological significance in the relative weight of the organs neither in the haematological nor in the biochemical parameters. Histopathologic alterations observed in the small intestine did not seem to be associated with the treatment.