A sensitive LC-MS/MS methotrexate assay capable of assessing adherence to methotrexate therapy in rheumatoid arthritis.

OBJECTIVES: To develop a sensitive liquid chromatography-tandem mass spectrometry method capable of measuring serum methotrexate in patients with rheumatoid arthritis to assess adherence to drug treatment. METHODS: Isotopically labelled internal standard and deionised water were added to sample prior to solid phase extraction using a Waters Oasis Max ion-exchange 96-well plate. Following extraction, samples were analysed by LC-MS/MS on a TQS-micro mass spectrometer. RESULTS: Mean recovery was 107 % for four different concentrations of methotrexate spiked into seven patient samples, whilst post extraction spiking gave a mean recovery of 100 %. Between-batch and within-batch CVs were ≤6 % at three different concentrations of methotrexate in fresh frozen plasma. Mean bias was <5 % for between-batch and within batch analysis at three different weighed in concentrations of methotrexate certified reference material. The lower limit of quantification of the assay was 0.1 nmol/L with linearity up to approximately 100 nmol/L. Dilution linearity studies were used to validate the dilution of patient samples prior to analysis. There was no significant interference in the method from lipaemia, haemolysis or icterus. CONCLUSIONS: A sensitive LC-MS/MS assay for methotrexate has been developed and validated. The method has been used to measure methotrexate adherence in patient samples from clinical trials and could be used in future research to assess the ability of the assay as a biofeedback intervention to improve adherence to methotrexate therapy.

Development and validation of a novel 7α-hydroxy-4-cholesten-3-one (C4) liquid chromatography tandem mass spectrometry method and its utility to assess pre-analytical stability.

OBJECTIVES: 7α-Hydroxy-4-cholesten-3-one (C4) is the common intermediary of both primary bile acids. C4 is recommended by the British Society of Gastroenterology for the investigation of bile acid diarrhoea (BAD) in patients with chronic diarrhoea. This project aimed to develop and validate an assay to quantitate C4 in serum and assess the stability of C4 in unseparated blood. METHODS: Accuracy was underpinned by calibrating to quantitative nuclear magnetic resonance analysis. C4 was analysed in a 96-well plate format with a deuterated C4 internal standard and liquid-liquid extraction. Validation followed the 2018 Food and Drug Administration guidelines. To assess C4 stability, healthy volunteers (n=12) donated 8 fasted samples each. Samples were incubated at 20 °C for up to 72 h and retrieved, centrifuged, aliquoted and frozen for storage at different time points prior to C4 analysis. RESULTS: The C4 method demonstrated excellent analytical performance and passed all validation criteria. The method was found to be accurate, precise, free from matrix effects and interference. After 72 h of delayed sample separation, C4 concentration gradually declined by up to 14 % from baseline. However, the change was not significant for up to 12 h. CONCLUSIONS: We present a robust method of analysing serum C4, offering a convenient alternative to 75SeHCAT for BAD investigation. C4 was found to decline in unseparated blood over time; however, after 12 h the mean change was <5 % from baseline. Our results suggest C4 is suitable for collection from both primary and secondary care prior to gastroenterology referral.

Report from the HarmoSter study: different LC-MS/MS androstenedione, DHEAS and testosterone methods compare well; however, unifying calibration is a double-edged sword.

OBJECTIVES: Current liquid chromatography-tandem mass spectrometry (LC-MS/MS) applications for circulating androgen measurements are technically diverse. Previously, variable results have been reported for testosterone. Data are scarce for androstenedione and absent for dehydroepiandrosterone sulfate (DHEAS). We assessed the agreement of androstenedione, DHEAS and testosterone LC-MS/MS measurements among nine European centers and explored benefits of calibration system unification. METHODS: Androgens were measured twice by laboratory-specific procedures in 78 patient samples and in EQA materials. Results were obtained by in-house and external calibration. Intra- and inter-laboratory performances were valued. RESULTS: Intra-laboratory CVs ranged between 4.2-13.2 % for androstenedione, 1.6-10.8 % for DHEAS, and 4.3-8.7 % and 2.6-7.1 % for female and male testosterone, respectively. Bias and trueness in EQA materials were within ±20 %. Median inter-laboratory CV with in-house vs. external calibration were 12.0 vs. 9.6 % for androstenedione (p<0.001), 7.2 vs. 4.9 % for DHEAS (p<0.001), 6.4 vs. 7.6 % for female testosterone (p<0.001) and 6.8 and 7.4 % for male testosterone (p=0.111). Median bias vs. all laboratory median with in-house and external calibration were -13.3 to 20.5 % and -4.9 to 18.7 % for androstenedione, -10.9 to 4.8 % and -3.4 to 3.5 % for DHEAS, -2.7 to 6.5 % and -11.3 to 6.6 % for testosterone in females, and -7.0 to 8.5 % and -7.5 to 11.8 % for testosterone in males, respectively. CONCLUSIONS: Methods showed high intra-laboratory precision but variable bias and trueness. Inter-laboratory agreement was remarkably good. Calibration system unification improved agreement in androstenedione and DHEAS, but not in testosterone measurements. Multiple components, such as commutability of calibrators and EQA materials and internal standard choices, likely contribute to inter-laboratory variability.

Discriminatory Value of Steroid Hormones on Polycystic Ovary Syndrome and Clustering of Hyperandrogenism and Metabolic Factors.

OBJECTIVE: We determined (1) if 11-oxygenated androgens better identify polycystic ovary syndrome (PCOS) diagnosis in women with obesity compared to total or free testosterone (T) and free androgen index; (2) how biochemical hyperandrogenism and metabolic factors cluster in a cohort of women with infertility and obesity. METHODS: Women with obesity and PCOS comprised the study group (N = 132). Ovulatory women with obesity and idiopathic, tubal or male factor infertility were the control group (N = 83). Steroid hormones were measured by means of liquid chromatography tandem mass spectrometry. Receiver operating characteristic curves and principal component analysis were used. RESULTS: Women with obesity and PCOS had higher 11-ketotestosterone (11 KT) (1.22 nmol/L [0.84; 1.65] vs 1.05 [0.78; 1.35], P = .04) compared to controls, but not 11ß-hydroxyandrostenedione 4.30 [2.87; 5.92] vs 4.06 [3.22; 5.73], P = .44). 11-ketotestosterone (area under the curve: 0.59) did not better discriminate PCOS in women with obesity compared to: total T (0.84), free T (0.91), and free androgen index (0.85). We identified 4 principal components (PCs) in the PCOS group (72.1% explained variance): (1) insulin resistance status; (2) blood pressure; (3) obesity; (4) androgen status and 4 PCs in the control group (68.7% explained variance) with variables representing metabolism being dispersed in component 2, 3, and 4. CONCLUSIONS: Eleven-oxygenated androgens do not aid in the diagnosis of PCOS in women with obesity. Insulin resistance is the strongest PC in the PCOS group. There is no major dominant characteristic that defines obese non-PCOS women.

Diurnal 11-ketotestosterone and 17-hydroxyprogesterone saliva profiles in paediatric classical congenital adrenal hyperplasia.

OBJECTIVES: The most suitable biochemical markers for therapy adjustment in patients with congenital adrenal hyperplasia are controversial. 11-Oxygenated androgens are a promising new approach. The objective of this study was to investigate the diurnal rhythm of 11-ketotestosterone in children and adolescents in saliva and to correlate it with salivary 17-hydroxyprogesterone. METHODS: Fifty-one samples of steroid day-profiles from 17 patients were additionally analysed for 11-ketotestosterone, retrospectively. All patients were treated in our university outpatient clinic for paediatric endocrinology between 2020 and 2022. Steroid day-profiles of 17 patients could be examined. The cohort showed a balanced sex ratio. The median age was 13 years. The measurements for 17-hydroxyprogesterone were carried out during routine care by immunoassay. The measurements of 11-ketotestosterone were performed from frozen saliva samples using an implemented in-house protocol for liquid chromatography-tandem mass spectrometry (LC-MS/MS). The most important outcome were the absolute values for 11-ketotestosterone, their diurnal rhythmicity and the correlation with 17-hydroxyprogesterone. RESULTS: Both steroids show a circadian diurnal rhythm. 17-hydroxyprogesterone and 11-ketotestosterone correlate significantly. 11-Ketotestosterone showed a positive correlation with BMI at all times of the day. CONCLUSIONS: 11-Ketotestosterone shows circadian rhythmicity in our cohort and correlates with 17-hydroxyprogesterone. These findings serve as an important basis for prospective research into 11-oxygenated androgens as therapeutic markers in paediatrics. However, 11-ketotestosterone appears to be very dependent on BMI.

Home Waking Salivary Cortisone to Screen for Adrenal Insufficiency.

BACKGROUND: Worldwide, adults and children are at risk of adrenal insufficiency as a result of adrenal suppression from use of anti-inflammatory glucocorticoids and opiates, as well as infectious diseases. The adrenocorticotropin (ACTH) stimulation test is the reference standard for diagnosis of adrenal insufficiency but requires clinic attendance and venesection. Salivary cortisone reflects free serum cortisol, and samples can be collected at home and posted to a laboratory. We tested whether home waking salivary cortisone level could be used to screen for adrenal insufficiency. METHODS: A prospective, diagnostic accuracy study was performed in patients at high risk of adrenal insufficiency. Patients collected a home salivary sample on waking and then attended the clinical facility for an ACTH stimulation test. Salivary cortisone was measured by liquid chromatography­tandem mass spectrometry. Receiver-operating characteristic curves were computed, and positive and negative predictive values were calculated. RESULTS: Two hundred twenty patients were recruited. As measured by an ACTH stimulation test, the prevalence of adrenal insufficiency was 44%. The area under the receiver-operating characteristic curve for waking salivary cortisone as a predictor of adrenal insufficiency was 0.95 (95% confidence interval [CI], 0.92 to 0.97). Cutoffs to ensure a minimum of 95% sensitivity and specificity gave a negative predictive value of 96% (95% CI, 90 to 99) and a positive predictive value of 95% (95% CI, 87 to 99) to exclude and confirm adrenal insufficiency, respectively. Waking salivary cortisone data provided information similar to that of an ACTH stimulation test in 70% of participants. Eighty-three percent of patients preferred home salivary collection to clinic attendance. CONCLUSIONS: Home waking salivary cortisone sampling has accuracy for the diagnosis of adrenal insufficiency similar to that of a standard ACTH stimulation test. Patients found the at-home test to be more convenient than the hospital-based test. (Funded by the National Institute for Health Research.)