Per- and polyfluoroalkyl substances (PFASs) include persistent organic pollutants whose spread is still ubiquitous. Efforts to substitute substances of high concern with fluorinated alternatives, such as HFPO-DA (GenX), DONA (ADONA), and cC6O4, have been made. The aim of this work was to develop and validate an isotopic dilution liquid chromatography-tandem mass spectrometry (LC–MS/MS) method suitable to quantify 30 PFASs in human plasma. Analytes included legacy PFASs (PFOA, PFOS, and PFHxS), fluorinated alternatives (PFBA, PFBS, 6:2 FTSA, HFPO-DA, DONA, and cC6O4), and newly identified compounds (F-53B and PFECHS). The sample preparation was rapid and consisted of simple protein precipitation and centrifugation. Calibration standards and quality control solutions were prepared with a human pooled plasma containing relatively low background levels of the considered analytes. A complete validation was carried out: the lower limits of quantitation (LLOQs) ranged from 0.009 to 0.245 µg/L; suitable linearity (determination coefficients,R²0.989–0.999), precision (2.0–19.5%, relative standard deviation), and accuracy (87.9–113.1% of theoretical) were obtained for considered concentration ranges. No significant variations of analyte responses were recorded under investigated storage conditions and during matrix effect tests. The external verification confirmed the accuracy of the method, although limited to 12 analytes. The method was also applied to 38 human plasma samples to confirm its applicability. The developed assay is suitable for large-scale analyses of a wide range of legacy and emerging PFASs in human plasma. To our knowledge, this is the first published method including cC6O4 for human biomonitoring.Graphical abstract

Objective The use of late-night salivary cortisol (LNSalC) for diagnosing subclinical hypercortisolism (SH) is debated. No data are available regarding the role of LNSalC as measured by liquid chromatography–tandem mass spectrometry (LC–MS/MS) in SH diagnosis. The aim of this study was to evaluate the diagnostic accuracy of LNSalC measured by LC–MS/MS in SH. Design Cross-sectional prospective study of outpatients. Methods In 70 consecutive patients with adrenal incidentalomas (AI), without signs and symptoms of hypercortisolism, we diagnosed SH in the presence of at least two of the following: cortisol after 1 mg overnight dexamethasone suppression test (1 mg DST) >83 nmol/l, 24-h urinary free cortisol (UFC) >193 nmol/24 h, and morning ACTH <2.2 pmol/l. The LNSalC levels by LC–MS/MS at 2300 h (normal values <2.8 nmol/l) and the presence of hypertension, type 2 diabetes mellitus (T2DM), and osteoporosis (OP) were assessed. Results The increased LNSalC levels (>2.8 nmol/l) had an 83.3% specificity (SP) and a 31.3% sensitivity (SN) for predicting the biochemical diagnosis of SH. The increased LNSalC had an 85.2% SP and a 55.6% SN for predicting the presence of hypertension, T2DM, and OP, while the combination of LNSalC >1.4 nmol/l (cutoff with 100% SN) plus 1 mg DST >50 nmol/l had an 88.9% SN and an 85.2% SP (similar to SH criterion at enrollment). Conclusions In AI patients, LNSalC measured by LC–MS/MS appears to be useful in combination with 1 mg DST for diagnosing SH, while it is not useful as a single criterion.

Purpose: The current cut-offs for the diagnosis of adrenal insufficiency (AI) have been established using outdated immunoassays. We compared the cortisol concentrations measured with Roche Cortisol I (R1), the newly available Roche Cortisol II (R2), and liquid chromatography tandem mass spectrometry (LC-MS/MS), the gold standard procedure to measure steroids in patients undergoing the corticotropin (ACTH) test. Methods: We enrolled 30 patients (age 47 ± 21 years) referred to undergo the ACTH test (1 or 250 μg). Cortisol was measured at 0, 30, and 60 min after stimulation with R1, R2, and LC-MS/MS. AI was diagnosed for R1-stimulated peak cortisol concentrations < 500 nmol/L. Results: Mean cortisol concentrations measured with R2 and LC-MS/MS were comparable, while mean cortisol concentrations measured by R1 were higher than those of both R2 and LC-MS/MS (respectively, basal 411 ± 177, 287 ± 119, and 295 ± 119 nmol/L; at 30 min, 704 ± 204, 480 ± 132, and 500 ± 132 nmol/L; at 60 min, 737 ± 301, 502 ± 196, and 519 ± 201 nmol/L, p ≤ 0.01 for R1 vs. both R2 and LC-MS/MS at each point). Considering the 500 nmol/L cortisol peak cut-off, AI was diagnosed in 5/30 patients using R1 and in 12/30 using R2 (+ 140%). Based on the correlation between R1 and R2, the threshold of 500 nmol/L became 351 nmol/L (12.7 μg/dL) when cortisol was measured with R2, and 368 nmol/L (13.3 μg/dL) with LC-MS/MS. Conclusions: The use of more specific cortisol assays results in lower cortisol concentrations. This could lead to misdiagnosis and overtreatment when assessing AI with the ACTH test if a different cut-off for cortisol peak is not adopted.