Measurement of Urinary Free Cortisol and Cortisone by LC-MS/MS.

Monitoring urinary free cortisol (UFC) excretion helps assess adrenal function and is used to screen for endogenous Cushing's syndrome caused by an adrenal or pituitary tumor. While serum cortisol levels fluctuate in response to time of day, stress, and concentrations of cortisol-binding globulin (CBG), a 24-h urine collection measures the cortisol produced over the entire day and does not suffer from as much variability as a serum measurement.We describe here a method of measurement of urinary free cortisol (UFC) and cortisone using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Urine samples, combined with stable isotope-labeled internal standards, are extracted by liquid-liquid extraction using ethyl acetate and hexane. An API 5500 mass spectrometer operated in positive atmospheric pressure chemical ionization (APCI) mode is used for detection.

Iatrogenic Cushing syndrome in 24-hour urine free cortisol measurement.

Cushing syndrome (CS) is caused by an excess of glucocorticoids that results in a variety of symptoms such as central obesity, moon facies, hirsutism, and reddish-purple stretch marks. Cortisol is the most potent endogenous glucocorticoid, and measuring the total amount excreted in the urine over a 24-hour period is useful to screen for CS caused by a tumor. However, most cases of CS are believed to be caused by exogenous glucocorticoids, such as prednisone and prednisolone, which are administered for anti-inflammatory and immunosuppressive treatments. This is often referred to as iatrogenic (drug-related or exogenous) CS. We modified an LC-MS/MS method for urine free cortisol to detect the presence of prednisone and prednisolone in patient samples. We wanted to understand the potential prevalence of exogenous CS in our patient population.

Detection and characterization of estradiol (E2) and unconjugated estriol (uE3) immunoassay interference due to anti-bovine alkaline phosphatase (ALP) antibodies.

OBJECTIVES: Competitive immunoenyzmatic assays for estradiol (E2) and unconjugated estriol (uE3) on UniCel DxI 800 Access immunoassay systems (Beckman Coulter) utilize bovine alkaline phosphatase (ALP) for amplification. In these assays, rare 'IND' error flags indicate that a relative light unit (RLU) raw result is past the high or low end of the calibration curve but cannot be differentiated from an instrument error or analytical interference. The present studies were conducted to establish a protocol to identify analytical interference and to characterize its mechanism when present. DESIGN AND METHODS: Matrix and recovery studies were conducted to establish a protocol for interference identification. Spiking experiments with inactivated calf intestinal ALP were performed to determine whether interference could be blocked. Commercial anti-ALP antibodies (Abs) were spiked into human serum to model assay interference. Three E2 immunoassays which do not include ALP as a reagent component (cobas e602, Roche; Centaur XP, Siemens; ARCHITECT i2000SR, Abbott) were tested for comparative purposes. RESULTS: 1:2 dilution of specimen into Access Sample Diluent A (Beckman) differentiated IND error flags due to true low results (e.g. less than the analytical measurement range; AMR) from those due to assay interference. Interferences were reduced by pre-incubation with inactivated ALP and could be replicated by spiking with commercial anti-ALP Abs. CONCLUSIONS: Patient anti-bovine ALP Abs can cause interference on DxI 800 E2 and uE3 assays. This model can be used to investigate interference risk with other ALP-dependent assays.

An exploratory study Evaluating the impact of opioid and non-opioid pain medications on serum/plasma free testosterone and free estradiol concentrations.

Chronic use of opioid medications has been reported to cause altered sexual function. It is not known if non-opioid pain medications have similar effects. Assessment of this effect through the measurement of concentrations of free hormones is limited. Positivity of opioid medications (hydrocodone, oxycodone, morphine, methadone, tramadol and fentanyl) and non-opioid pain medications (gabapentin or pregabalin) in human serum and plasma samples from adult men and women were evaluated for association with concentrations of free testosterone (fTe) and free estradiol (fE2) measured using equilibrium dialysis-liquid chromatography-tandem mass spectrometry methods. Lower concentrations of fTe (p = 0.0253) were observed in samples positive for the hydrocodone, oxycodone and morphine group compared to age matched controls. The presence of methadone, tramadol, fentanyl and pregabalin had no effect on fTe. When compared with age-matched controls, women between 48-55 years of age showed reduced fE2 concentrations in samples positive for tramadol, fentanyl and gabapentin (p = 0.0243, 0.0045 and 0.0050, respectively). Particular opioid medications such as methadone, tramadol or fentanyl and non-opioid medications such as pregabalin or gabapentin may offer advantages over opioid medications for treating pain with fewer endocrinologic side effects. Measurement of free hormones in pain medication users could be important in determining their association with sexual function. Copyright © 2017 John Wiley & Sons, Ltd.

Identification of a novel pathogenic Borrelia species causing Lyme borreliosis with unusually high spirochaetaemia: a descriptive study.

BACKGROUND: Lyme borreliosis is the most common tick-borne disease in the northern hemisphere. It is a multisystem disease caused by Borrelia burgdorferi sensu lato genospecies and characterised by tissue localisation and low spirochaetaemia. In this study we aimed to describe a novel Borrelia species causing Lyme borreliosis in the USA. METHODS: At the Mayo clinic, from 2003 to 2014, we tested routine clinical diagnostic specimens from patients in the USA with PCR targeting the oppA1 gene of B burgdorferi sensu lato. We identified positive specimens with an atypical PCR result (melting temperature outside of the expected range) by sequencing, microscopy, or culture. We collected Ixodes scapularis ticks from regions of suspected patient tick exposure and tested them by oppA1 PCR. FINDINGS: 100 545 specimens were submitted by physicians for routine PCR from Jan 1, 2003 to Sept 30, 2014. From these samples, six clinical specimens (five blood, one synovial fluid) yielded an atypical oppA1 PCR product, but no atypical results were detected before 2012. Five of the six patients with atypical PCR results had presented with fever, four had diffuse or focal rash, three had symptoms suggestive of neurological inclusion, and two were admitted to hospital. The sixth patient presented with knee pain and swelling. Motile spirochaetes were seen in blood samples from one patient and cultured from blood samples from two patients. Among the five blood specimens, the median oppA1 copy number was 180 times higher than that in 13 specimens that tested positive for B burgdorferi sensu stricto during the same time period. Multigene sequencing identified the spirochaete as a novel B burgdorferi sensu lato genospecies. This same genospecies was detected in ticks collected at a probable patient exposure site. INTERPRETATION: We describe a new pathogenic Borrelia burgdorferi sensu lato genospecies (candidatus Borrelia mayonii) in the upper midwestern USA, which causes Lyme borreliosis with unusually high spirochaetaemia. Clinicians should be aware of this new B burgdorferi sensu lato genospecies, its distinct clinical features, and the usefulness of oppA1 PCR for diagnosis. FUNDING: US Centers for Disease Control and Prevention Epidemiology and Laboratory Capacity for Infectious Diseases (ELC) Cooperative Agreement and Mayo Clinic Small Grant programme.

Effectiveness of Residential Acaricides to Prevent Lyme and Other Tick-borne Diseases in Humans.

BACKGROUND: In the northeastern United States, tick-borne diseases are a major public health concern. In controlled studies, a single springtime application of acaricide has been shown to kill 68%-100% of ticks. Although public health authorities recommend use of acaricides to control tick populations in yards, the effectiveness of these pesticides to prevent tick bites or human tick-borne diseases is unknown. METHODS: We conducted a 2-year, randomized, double-blinded, placebo-controlled trial among 2727 households in 3 northeastern states. Households received a single springtime barrier application of bifenthrin or water according to recommended practices. Tick drags were conducted 3-4 weeks after treatment on 10% of properties. Information on human-tick encounters and tick-borne diseases was collected through monthly surveys; reports of illness were validated by medical record review. RESULTS: Although the abundance of questing ticks was significantly lower (63%) on acaricide-treated properties, there was no difference between treatment groups in human-tick encounters, self-reported tick-borne diseases, or medical-record-validated tick-borne diseases. CONCLUSIONS: Used as recommended, acaricide barrier sprays do not significantly reduce the household risk of tick exposure or incidence of tick-borne disease. Measures for preventing tick-borne diseases should be evaluated against human outcomes to confirm effectiveness.

Direct Measurement of Free Estradiol in Human Serum and Plasma by Equilibrium Dialysis-Liquid Chromatography-Tandem Mass Spectrometry.

We describe a direct method of measurement of free estradiol using equilibrium dialysis followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Serum aliquots and internal standards are extracted by liquid-liquid extraction using methyl-tert-butyl ether (MTBE) followed by derivatization with dansyl chloride. An API 5500 mass spectrometer operated in positive electrospray mode is used for detection.

Human Infection with Ehrlichia muris-like Pathogen, United States, 2007-2013(1).

An Ehrlichia muris-like (EML) pathogen was detected among 4 patients in Minnesota and Wisconsin during 2009. We characterized additional cases clinically and epidemiologically. During 2004-2013, blood samples from 75,077 patients from all 50 United States were tested by PCR from the groEL gene for Ehrlichia spp. and Anaplasma phagocytophilum. During 2007-2013, samples from 69 (0.1%) patients were positive for the EML pathogen; patients were from 5 states: Indiana (1), Michigan (1), Minnesota (33), North Dakota (3), and Wisconsin (31). Most (64%) patients were male; median age was 63 (range 15-94) years; and all 69 patients reported likely tick exposure in Minnesota or Wisconsin. Fever, malaise, thrombocytopenia, and lymphopenia were the most common symptoms. Sixteen (23%) patients were hospitalized (median 4 days); all recovered, and 96% received doxycycline. Infection with the EML pathogen should be considered for persons reporting tick exposure in Minnesota or Wisconsin.

Performance enhancement in the measurement of 5 endogenous steroids by LC-MS/MS combined with differential ion mobility spectrometry.

BACKGROUND: Challenges for steroid analysis by LC-MS/MS include low ionization efficiency, endogenous isobars with similar fragmentation patterns and chromatographic retention. Differential ion mobility spectrometry (DMS) provides an additional degree of separation prior to MS/MS detection, and shows promise in improving specificity of analysis. We developed a sensitive and specific method for measurement of corticosterone, 11-deoxycortisol, 11-deoxycorticosterone, 17-hydroxyprogesterone and progesterone in human serum and plasma using an ABSciex 5500 mass spectrometer equipped with a differential ion mobility interface. METHODS: 250µL aliquots of serum were spiked with deuterated internal standards and extracted with MTBE. The samples were analyzed using positive mode electrospray LC-DMS-MS/MS. The method was validated and compared with immunoassays and LC-MS/MS methods of reference laboratories. RESULTS: Inter and intra assay imprecision was <10%. Limits of quantification and detection in nmol/L were 0.18, 0.09 for corticosterone and 17-hydroxyprogesterone, 0.30, 0.16 for 11-deoxycortisol, 0.12, 0.06 for progesterone and 0.06, 0.03 for 11-deoxycorticosterone. Comparison for progesterone and 17-hydroxyprogesterone with immunoassay showed slopes of 0.97 and 1.0, intercepts of 0.16 and 0.10 and coefficients of determination (r(2)) of 0.92 and 0.97, respectively. Progesterone by immunoassay showed positive bias in samples measuring <3.18nmol/L. Reference intervals for progesterone and 11-deoxycorticosterone in post-menopausal women were found to be <2.88 and <0.28nmol/L respectively. CONCLUSIONS: We developed and validated an LC-DMS-MS/MS method for analysis of five endogenous steroids suitable for routine measurements in clinical diagnostic laboratories. Specificity gained with DMS allows reducing the complexity of sample preparation, decreasing LC run times and increasing speed of the analysis.

Enhancement of specificity of aldosterone measurement in human serum and plasma using 2D-LC-MS/MS and comparison with commercial immunoassays.

BACKGROUND: Accurate measurement of aldosterone is important for the standardized testing of primary hyperaldosteronism. Commercial immunoassays show substantial between-method variations resulting in significant clinical consequences. We developed a specific two dimensional (2D)-LC-MS/MS method for measuring aldosterone in human serum and plasma and compared it with three commercial immunoassays and an LC-MS/MS method. METHODS: 250 µL samples, controls and calibrators spiked with d4-aldosterone were subjected to liquid-liquid extraction. The samples were analyzed using negative mode electrospray and 2D-LC followed by MS detection using an ABSciex 5500 mass spectrometer and compared with immunoassays of Siemens (Coat-A-Count), DiaSorin (CLIA-LIAISON), and IBL (ELISA). Data was acquired using multiple reaction-monitoring mode. RESULTS: LOQ and LOD of the method were 0.04 and 0.02 nmol/L respectively. The assay was linear up to 166 nmol/L. Inter and intra-assay imprecision at 0.13, 1.38 and 8.30 nmol/L were <10%. Interferences were absent and no differences were observed between serum and plasma matrices. Method recovery ranged from 95% to 113%. Ion suppression was not observed. Evaluated immunoassays showed positive biases ranging between 22% and 37% when compared with the developed method. CONCLUSIONS: We developed and validated an accurate method for measurement of aldosterone in human serum and plasma using 2D-LC-MS/MS which is suitable for clinical purposes. The method is faster than previously published LC-MS/MS methods, uses less sample, has adequate sensitivity while being able to preserve high specificity in a cost effective manner. Linearity of the assay makes it promising for urine and adrenal venous samples. Comparison with three commercial immunoassays demonstrates the advantages of the developed method.

Direct measurement of free estradiol in human serum by equilibrium dialysis-liquid chromatography-tandem mass spectrometry and reference intervals of free estradiol in women.

BACKGROUND: Measurement of free estradiol offers a better representation of the bioactive fraction of the hormone. We describe a direct equilibrium dialysis-liquid chromatography-tandem mass spectrometry (ED-LC-MS/MS) method for serum free estradiol. METHODS: Two hundred fifty microliter aliquots of serum were dialyzed for 22h followed by liquid-liquid extraction and derivatization with dansyl chloride. Free estradiol was measured using LC-MS/MS with an AB SCIEX 5500 mass spectrometer in positive ion and multiple reaction monitoring (MRM) mode. RESULTS: The limits of detection and quantification for free estradiol were 0.25 and 0.5pg/ml (0.9 and 1.8pmol/l) respectively. Total imprecision was less than 10%. Results of method comparison showed 3 times overestimation using indirect methods of measurement. Reference intervals in pre-menopausal women in follicular, mid-cycle, and luteal phases of cycle were <2.4, <3.1 and <2.6pg/ml (8.8, 11.4, 9.5pmol/l) respectively; in post menopausal women the concentrations were ≤0.5pg/ml (1.8pmol/l). CONCLUSIONS: ED-LC-MS/MS is a direct method for accurately measuring free estradiol, independent of total estradiol or sex hormone binding globulin concentrations. Imprecision and sensitivity of the method are adequate for clinical diagnostic applications. The degree of variation observed in the method comparison reinforces the relevance of method specific reference ranges.

Analysis of cortisol, cortisone and dexamethasone in human serum using liquid chromatography tandem mass spectrometry and assessment of cortisol: cortisone ratios in patients with impaired kidney function.

BACKGROUND: We developed a high sensitivity method for simultaneous measurement of cortisol, cortisone and dexamethasone. Using this method, we compared concentrations of cortisol, cortisone and their ratios in samples from intensive care unit (ICU) and non-ICU patients, and cortisol and dexamethasone concentrations in patients with Cushing's and suspected Cushing's syndrome. METHODS: Two hundred microliters of human serum aliquots were extracted using solid phase extraction and analyzed using liquid chromatography-tandem mass spectrometry. Primary mass transitions monitored for cortisol, cortisone and dexamethasone were m/z 363/121, 361/163 and 393/373 respectively. RESULTS: The limits of quantification for cortisol and cortisone were 0.3 µg/l (0.8 nmol/l) and for dexamethasone it was 0.5 µg/l (1.2 nmol/l). Total imprecision was <10.9%. Median cortisol to cortisone ratio of ICU patient samples was found to be 2 times higher than non-ICU samples. 54.2% of patients after 1mg dose of overnight dexamethasone could be categorized as consistent with Cushing's syndrome. CONCLUSIONS: The method has high sensitivity and specificity. High cortisol to cortisone ratios in samples from ICU patients suggest change in activity of 11ß-hydroxysteroid dehydrogenase in modulation of systemically available cortisol. Simultaneous measurement of dexamethasone and cortisol can be used to differentially diagnose diseases causing increased concentrations of cortisol.

Rapid analysis of 25-hydroxyvitamin D(2) and D(3) by liquid chromatography-tandem mass spectrometry and association of vitamin D and parathyroid hormone concentrations in healthy adults.

Measurement of 25-hydroxyvitamin D (25OH-vitD) is used to assess vitamin D status. We developed a high-sensitivity measurement method for 25OH-vitD and assessed the relationship between 25OH-vitD and parathyroid hormone (PTH) in healthy adults. Aliquots (100 microL) of serum were spiked with internal standard, proteins were precipitated, and samples were analyzed by liquid chromatography-tandem mass spectrometry using 2-dimensional chromatographic separation. Total imprecision was less than 10%, and the limit of quantitation was 1.0 ng/mL. We determined the distribution of concentrations of 25OH-vitD(2) and 25OH-vitD(3) in healthy adults using samples collected during winter and summer and evaluated the association between 25OH-vitD and PTH. The difference between median concentrations of 25OH-vitD in samples collected during winter and summer was 11 ng/mL (27 nmol/L). Statistically significant differences in concentrations of PTH were observed between groups of samples with 25OH-vitD less than 11 (27 nmol/L) and 11 to 15 ng/mL (27-37 nmol/L) and between groups with 25 to 30 (62-75 nmol/L) and more than 40 ng/mL (100 nmol/L). Among the advantages of this method are its high sensitivity and specificity.