URINARY AND SALIVARY CORTISOL IN LIQUID CHROMATOGRAPHY–TANDEM MASS SPECTROMETRY: METHOD VALIDATION AND EXPECTED VALUES DETERMINATION

BACKGROUND: Cortisol measurement is useful in evaluation of Cushing syndrome, adrenal insufficiency, mineralocorticoid excess and congenital adrenal hyperplasia. We developed a liquid chromatography–tandem mass spectrometry (LCMS/MS) method for salivary and urinary cortisol and we determined the 95th percentile (p95) for the urinary and salivary cortisol. We compared them to the Mayo Clinic expected values. METHODS: Saliva at 8 am and 11 pm and 24h urine were obtained from 32 healthy (22 female, 34.3±9.3 yo) volunteers. We performed validation with the enoval software (Arlenda, Belgium). For the validation, we used water or urine with spiked known amounts of cortisol for the CORS and CTU respectively. For the CORS, samples were centrifuged, deuterium labelled cortisol was added as internal standard and the protein precipated by acetonitril. The supernatant was evaporated, dissolved in methanol acidified with acetic acid and analyzed by LCMS/MS. For CTU, samples were centrifuged, deuterium labelled cortisol was added as internal standard and diluted by the ammonium acetate and analyzed by LCMS/MS. At the Mayo Clinic, the expected values were 1-7.5 μg/L (7 a.m-9 a.m) and <1 μg/L (11-12 p.m) for CORS and 3.5-45 μg/24h (<18yo) for CTU. RESULTS: For the CTU, the with-in run did not exceed 3% (0.4-3%) and the between-run did not exceed 3.1% (0.4-3.1%) for 1.5-750 μg/L. The limit of quantification was 1.5 μg/L. The linearity was good between 1.5 and 750 μg/L. The recovery is 97.9±2.2% (95%CI for the mean: 92.4-101.1%). For the CORS, the with-in run and between run did not exceed 8% (1.9-8%) for 1.15-8.65 μg/L. The limit of quantification was 1.15 μg/L. The analyse presents a good linearity between 1.15 and 8.65 μg/L. The recovery is 99.9±2.9% (95%CI for the mean: 94.2-108.7%). The p95 for the CTU according to the CLSI C28-A3 was 33 μg/24h, and for the CORS was 5.42 μg/L at 8 am and 0.7 μg/L at 12 pm. CONCLUSIONS: Our developed method in liquid chromatography tandem mass spectrometry was validated for the measurement of urinary and salivary cortisol. Our findings indicate that the proposed analytical methods were suitable for routine purposes and useful in many pathological conditions.The expected values confirm these defined by the Mayo Clinic

Establishment of reference values for 6 six steroids in serum by LC-MS/MS

peer reviewe

Le Cortisol et la DHEA comme marqueurs de la placentite chez la jument gestante : une étude préliminaire

peer reviewedPlacentitis in mares is a subclinical cause of abortion, high- lighting the importance of early detection for successful treatment. Sexual steroids could serve as biomarkers, but cross-reactivity and a limited detection range of immunoassays may hinder ac- curate measures. This study evaluated some steroids as poten- tial markers of placentitis using liquid chromatography coupled to mass spectrometry (LC-MS), known to improve detection and quantification of steroids with good selectivity, specificity, and allowing multiplexing capabilities (Conley et al., Reproduction. 2019;158:197-208; Ledeck et al., Theriogenology. 2022;189:86- 91). From the 7th month of pregnancy, mares were transrectally scanned to measure the combined thickness of the uterus and placenta (CTUP), and blood samples were collected at a fixed time of day. Ten healthy mares (HM) and nine mares diagnosed with non-experimentally-induced placentitis (PM) between 8 and 10 months were enrolled, based on ultrasonographic placentitis signs: heterogeneous echogenicity and/or thickened CTUP. Post- partum allantochorion examination confirmed the diagnosis. Sub- sequently, PM were further excluded from the study as they re- ceived treatments for placentitis. Serum concentrations of pro- gesterone, 17α-hydroxyprogesterone (17αOHP), dehydroepiandros- terone (DHEA), and cortisol were assayed using LC-MS. Non- normally distributed data were presented as medians, and groups were compared at the same month of pregnancy using the Mann- Whitney test. There were no significant differences in 17αOHP and progesterone concentrations between groups at any month. DHEA concentrations were significantly higher (p=0.0297) at 8 months in PM (3.552μg/L) compared to HM (2.240μg/L). A sim- ilar trend was observed at 9 months, with DHEA concentra- tions of 2.126μg/L and 1.445μg/L in PM and HM, respectively (p=0.0604). In contrast, HM had significantly higher (p=0.022) cortisol concentrations at 7 months (71.75μg/L) compared to PM (38.62μg/L). This difference tended (p=0.0529) to be observed at 8 months with cortisol concentrations of 52.25μg/L (HM) and 37.28μg/L (PM). The DHEA/cortisol ratio was significantly higher in PM than in HM at 9 months (p=0.013) and tended to be in- creased at 7 and 8 months (respectively, p=0.0659 and p=0.0529). Placentitis has been shown to increase pregnenolone production (Ousey et al., Theriogenology. 2005;63:1844-1856). In this prelim- inary study, PM were observed to metabolize pregnenolone into DHEA rather than cortisol, without changes in 17αOHP concen- trations. Reduced cortisol concentrations at 7 months could be an early but non-specific biomarker of placentitis. Conversely, elevated DHEA Ievels only appear together with ultrasonographic signs at 8 months but could become a more specific biomarker, which dif- fers from previous findings in mares with experimentally-induced placentitis (Canisso et al., Equine Veterinary Journal. 2017;49:244- 249). These changes result in a higher DHEA/cortisol ratio in PM at 9 months. However, further research should explore mod- ifications of steroids’ pathways in PM to confirm the value of DHEA and cortisol for early diagnosis of placentitis in mares.Study of equine placental sexual steroids’ metabolism using Mass Spectrometry (LC-MS/MS) and in vitro model to develop early diagnosis methods of placental pathologie

Serum 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D in dogs with sinonasal aspergillosis

peer reviewedSinonasal aspergillosis (SNA) is a common cause of chronic nasal disease with a still poorly understood pathophysiology and which remains a challenge to treat. There is increasing evidence that vitamin D plays a role in both innate and adaptative immunity. A preliminary retrospective study showed that serum 25-hydroxyvitamin D concentration, measured by high performance liquid chromatography (HPLC), was significantly lower in dogs affected with SNA compared to healthy dogs. Objectives of this prospective study were 1) to compare serum 25(OH)D and 24,25(OH)2D concentrations in dogs with SNA to healthy control dogs and dogs with lymphoplasmacytic rhinitis (LPR) or nasal neoplasia; and 2) to determine if serum 25(OH)D and 24,25(OH)2D concentrations in dogs with SNA change from the time of diagnosis to when a cure is achieved. Twenty dogs with a novel diagnosis of SNA, 12 healthy control dogs, 9 dogs with LPR, 10 dogs with nasal neoplasia were included. Nine dogs with SNA were available for follow up until cure. Serum vitamin D concentrations were measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) and compared: 1) among the different groups using a One-way ANOVA and 2) from diagnosis to cure with a paired t-test (significant p-value <0,05 for both tests). The vitamin D metabolite ratio (VMR) was calculated by dividing the 25(OH)D by the 24,25(OH)2D concentration. Serum 25(OH)D and 24,25(OH)2D were lower in dogs with SNA at the time of diagnosis (mean ± standard deviation = 23.5 ± 7,1 ng/ml – 10,5 ± 4,2 ng/ml, respectively) than in healthy dogs (34,1 ± 7,5 ng/ml; p=0,017 - 18,2 ± 5.4 ng/ml; p = 0,005) while there was no difference between healthy and dogs with tumor (27,8 ± 10,9 ng/ml – 15,4 ± 6,5 ng/ml) or LPR (27,4 ± 13,7 ng/ml – 14,3 ± 8,7 ng/ml). There was no significant difference in serum 25(OH)D and 24,25(OH)2D between dogs with SNA at the time of diagnosis and dogs achieving cure. The VMR was higher in SNA dogs (2,4 ± 0,7) than in control dogs (1,9 ± 0,3; p=0,031 t-test), indicating a decreased catabolic clearance of vitamin D in SNA dogs. These results further support the rationale that vitamin D could play a role in dogs with SNA as it does in human with aspergillosis. Whether hypovitaminosis D could contribute to the development of SNA or if oral supplementation could be a beneficial adjunctive therapy in affected dogs is unknown and warrants future investigations