Resumption of ovulation in anovulatory women with PCOS and obesity is associated with reduction of 11ß-hydroxyandrostenedione concentrations.

STUDY QUESTION: Is resumption of ovulation after a 6-month lifestyle intervention in women with PCOS and obesity associated with differential changes in endocrine and metabolic parameters (weight, insulin resistance, anti-Müllerian hormone (AMH), and androgens) compared to women with PCOS who remained anovulatory? SUMMARY ANSWER: Resumption of ovulation after a 6-month lifestyle intervention in women with PCOS and obesity is associated with changes in serum 11ß-hydroxyandrostenedione (11OHA4) concentrations. WHAT IS KNOWN ALREADY: Lifestyle interventions have been shown to reduce clinical and biochemical hyperandrogenism in women with PCOS. Weight loss of 5-10% may reverse anovulatory status, thereby increasing natural conception rates. However, the mechanisms underlying why some women with PCOS remain anovulatory and others resume ovulation after weight loss are unclear. Reproductive characteristics at baseline and a greater degree of change in endocrine and metabolic features with lifestyle intervention may be crucial for ovulatory response. STUDY DESIGN, SIZE, DURATION: We used data and samples originating from an earlier randomized controlled trial (RCT), which examined the efficacy of a 6-month lifestyle intervention prior to infertility treatment compared to prompt infertility treatment on live birth rate in women with obesity. A total of 577 women with obesity (BMI > 29 kg/m2) were randomized between 2009 and 2012. Anovulatory women with PCOS who were allocated to the intervention arm of the original RCT (n = 95) were included in the current analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: We defined women as having resumed ovulation (RO+) based on the following criteria: spontaneous pregnancy; or assignment to expectant management; or IUI in natural cycles as the treatment strategy after lifestyle intervention. Steroid hormones were measured using liquid chromatography tandem mass spectrometry. Generalized estimating equations with adjustment for baseline measures and interaction between group and time was used to examine differences in changes of endocrine and metabolic parameters between RO+ (n = 34) and persistently anovulatory women (RO-, n = 61) at 3 and 6 months after intervention. MAIN RESULTS AND THE ROLE OF CHANCE: At baseline, the mean ± SD age was 27.5 ± 3.6 years in the RO+ group and 27.9 ± 4.1 years in the RO- group (P = 0.65), and the mean ± SD weights were 101.2 ± 9.5 kg and 105.0 ± 14.6 kg, respectively (P = 0.13). Baseline AMH concentrations showed significant differences between RO+ and RO- women (median and interquartile range [IQR] 4.7 [3.2; 8.3] versus 7.2 [5.3; 10.8] ng/ml, respectively). Baseline androgen concentrations did not differ between the two groups. During and after lifestyle intervention, both groups showed weight loss; changes in 11OHA4 were significantly different between the RO+ and RO groups (P-value for interaction = 0.03). There was a similar trend for SHBG (interaction P-value = 0.07), and DHEA-S (interaction P-value = 0.06), with the most pronounced differences observed in the first 3 months. Other parameters, such as AMH and FAI, decreased over time but with no difference between the groups. LIMITATIONS, REASONS FOR CAUTION: No high-resolution transvaginal ultrasonography was used to confirm ovulatory status at the end of the lifestyle program. The small sample size may limit the robustness of the results. WIDER IMPLICATIONS OF THE FINDINGS: Reduction of androgen concentrations during and after lifestyle intervention is associated with recovery of ovulatory cycles. If our results are confirmed in other studies, androgen concentrations could be monitored during lifestyle intervention to provide individualized recommendations on the timing of resumption of ovulation in anovulatory women with PCOS and obesity. STUDY FUNDING/COMPETING INTEREST(S): The study was supported by a grant from ZonMw, the Dutch Organization for Health Research and Development (50-50110-96-518). The Department of Obstetrics and Gynecology of the UMCG received an unrestricted educational grant from Ferring Pharmaceuticals BV, The Netherlands. A.H. reports consultancy for the development and implementation of a lifestyle App MyFertiCoach developed by Ferring Pharmaceutical Company. All other authors have no conflicts to declare. TRIAL REGISTRATION NUMBER: The LIFEstyle RCT was registered at the Dutch trial registry (NTR 1530).

Insulin resistance variability in women with anovulatory and ovulatory polycystic ovary syndrome, and normal controls.

Women with polycystic ovary syndrome (PCOS) were found to have a higher biological variability in insulin resistance (IR) compared to controls, but it is unknown whether this variability in IR differs between PCOS who are anovulatory compared to those who have an ovulatory cycle. The primary aim of this study was to compare and contrast the variability of IR in women with ovulatory and anovulatory PCOS, in comparison to normal subjects. 53 Caucasian women with PCOS and 22 normal ovulating women were recruited. Fasting blood was collected each day on 10 consecutive occasions at 3-4 day intervals for analysis of insulin, glucose, progesterone, and testosterone. Analysis of progesterone levels showed 22 of 53 women with PCOS to have had an ovulatory cycle. Insulin resistance was calculated by HOMA method. Women with anovulatory PCOS had higher mean and variability of IR compared to those having an ovulatory cycle, and both were significantly higher than controls (mean ± SEM; HOMA-IR 4.14 ± 0.14 vs. 3.65 ± 0.15 vs. 2.21 ± 0.16, respectively) after adjustment or BMI. The mean BMI for individual PCOS patients correlated with mean HOMA-IR (p=0.009). Insulin resistance in women with anovulatory PCOS is both higher and more variable than in ovulatory PCOS. Since anovulatory PCOS therefore mimics the IR features of type 2 diabetes more closely, anovulation may be particularly associated with a higher cardiovascular risk compared to PCOS patients who ovulate.

Effect of metformin, orlistat and pioglitazone treatment on mean insulin resistance and its biological variability in polycystic ovary syndrome.

CONTEXT: Mean insulin resistance (IR) is greater and it is also more variable in overweight women with polycystic ovarian syndrome (PCOS) compared to weight matched controls. Whilst treatment will reduce the mean IR, it is not known if the IR variability is also reduced. OBJECTIVE: To compare the change in IR and its variability before and after treatment with insulin sensitization through metformin and pioglitazone, compared to that induced by weight loss with orlistat. DESIGN: Randomized, open labelled parallel study. SETTING: Endocrinology outpatient clinic at a referral centre. PATIENTS: Thirty obese PCOS patients [BMI 36.0 +/- 1.2 kg/m(2) (mean +/- SEM)] participated in the study. INTERVENTION: The change in biological variability (BV) was assessed by measuring IR (homeostasis model assessment method) at 4-day intervals on 10 consecutive occasions before and 12 weeks after randomization to metformin, pioglitazone or orlistat. OUTCOME MEASURED: The primary end point of the study was a change in BV of IR. RESULTS: Treatment with pioglitazone, orlistat and metformin reduced the overall IR by 41.0 +/- 4.1%, 19.7 +/- 6.4% and 16.1 +/- 6.8% (P = 0.005, P = 0.013, P = 0.17, respectively) and IR variability by 28.5 +/- 18.0%, 41.8 +/- 11.6% and 23.7 +/- 17.0 (P = 0.20, P = 0.015 and P = 0.28, respectively). Free androgen index reduced significantly with all treatments. CONCLUSION: Only orlistat reduced both IR and its variability significantly, though all three drugs were effective in reducing hyperandrogenism within the 12-week period of the study.

Prediction of haematocrit in dried blood spots from the measurement of haemoglobin using commercially available sodium lauryl sulphate.

Background When preparing dried blood spots (DBSs), haematocrit (Hct) can affect the ability of the blood to spread through the filter paper, thus resulting in varying quantities of sample being measured when fixed subpunches of the DBSs are taken. It may be important to predict the sample Hct to correct volume differences. Methods Blood (10 µL) was applied to Perkin Elmer 226® paper. The samples ( n = 165) were allowed to dry for 24 h, and the entire blood spots were cut out. Subpunch analysis was also performed on blood spots prepared from 75 µL EDTA blood, taking 6 mm subpunches centrally and peripherally from the spots ( n = 59). The spots were eluted with 100 µL water, and a 10 µL aliquot of lysate was added to sulfolyser reagent (80 µL) in a microtitre plate. Hb was measured at 550 nm using an ELISA plate reader. DBS samples were compared against blood samples measured on a routine Sysmex XN-9000 analyser. Results The Passing and Bablock regression showed Hct (DBS-predicted) = 0.99 Hct (Sysmex) -0.02, R2 = 0.87. Intra-assay imprecision measured at Hct values of 0.27, 0.40 and 0.52, gave CVs of 4.1%, 2.8% and 4.2%, respectively. Inter-assay imprecision showed CVs of 6.2%, 5.2% and 4.2%, respectively. DBS samples were stable for up to two days at 60℃, one month at room temperature and six months at 4℃. Conclusion This method provides a simple and fast estimation of predicted Hct in dried blood spots.

Simultaneous determination of androstenedione and testosterone in human serum by liquid chromatography-tandem mass spectrometry.

BACKGROUND: We aimed to develop a sensitive assay to quantitate serum concentrations of both androstenedione and testosterone within the female range simultaneously, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for use in the routine clinical laboratory and to compare this method with immunoassay. METHOD: Samples (200 microL) were prepared by liquid-liquid extraction using (1 mL) methyl-tert-butyl-ether. Deuterated androstenedione and testosterone were used as internal standards. RESULTS: The standard curve was linear to 50 nmol/L, the lower limit of quantitation was 0.25 nmol/L, and intra- and inter-assay coefficients of variation were < 10% for both androgens over the range 0.3-35 nmol/L. There was a poor relationship between the LC-MS/MS and the radioimmunoassay methods for androstenedione with the LC-MS/MS generally giving lower results. For testosterone, the LC-MS/MS and immunoassay methods compared well at all concentrations. However, when female samples only were examined, the agreement deteriorated. CONCLUSIONS: We have developed a sensitive and precise LC-MS/MS method, which gives more accurate results for all androstenedione measurements and low testosterone concentrations than immunoassay.

Affective alterations in patients with Cushing's syndrome in remission are associated with decreased BDNF and cortisone levels.

OBJECTIVE: Affective alterations and poorer quality of life often persist in patients with Cushing's syndrome (CS) in remission. Brain-derived neurotrophic factor (BDNF) regulates the hypothalamic-pituitary-adrenal axis (HPA) and is highly expressed in brain areas controlling mood and response to stress. Our aims were to assess affective alterations after long-term remission of CS and evaluate whether they are associated with serum BDNF, salivary cortisol (SalF) and/or cortisone (SalE) concentrations. SUBJECTS AND METHODS: Thirty-six CS patients in remission (32 females/4 males; mean age (±s.d.), 48.8 ± 11.8 years; median duration of remission, 72 months) and 36 gender-, age- and BMI-matched controls were included. Beck Depression Inventory-II (BDI-II), Center for Epidemiological Studies Depression Scale (CES-D), Positive Affect Negative Affect Scale (PANAS), State-Trait Anxiety Inventory (STAI), Perceived Stress Scale (PSS) and EuroQoL and CushingQoL questionnaires were completed and measured to evaluate anxiety, depression, stress perception and quality of life (QoL) respectively. Salivary cortisol was measured using liquid chromatography/tandem mass spectrometry (LC/TMS). BDNF was measured in serum using an ELISA. RESULTS: Remitted CS patients showed worse scores in all questionnaires than controls: STAI (P < 0.001), BDI (P < 0.001), CES-D (P < 0.001), PANAS (P < 0.01), PSS (P < 0.01) and EuroQoL (P < 0.01). A decrease in BDNF was observed in CS vs controls (P = 0.038), and low BDNF was associated with more anxiety (r = -0.247, P = 0.037), depression (r = -0.249, P = 0.035), stress (r = -0.277, P = 0.019) and affective balance (r = 0.243, P = 0.04). Morning salivary cortisone was inversely associated with trait anxiety (r = -0.377, P = 0.040) and depressed affect (r = -0.392, P = 0.032) in CS patients. Delay to diagnosis was associated with depressive symptoms (BDI-II: r = 0.398, P = 0.036 and CES-D: r = 0.449, P = 0.017) and CushingQoL scoring (r = -0.460, P < 0.01). CONCLUSIONS: Low BDNF levels are associated with affective alterations in 'cured' CS patients, including depression, anxiety and impaired stress perception. Elevated levels of SalE might also be related to poor affective status in these patients.

Salivary Testosterone Levels and Health Status in Men and Women in the British General Population: Findings from the Third National Survey of Sexual Attitudes and Lifestyles (Natsal-3).

CONTEXT: Salivary T (Sal-T) measurement by liquid chromatography-tandem mass spectroscopy resents the opportunity to examine health correlates of Sal-T in a large-scale population survey. OBJECTIVE: This study sought to examine associations between Sal-T and health-related factors in men and women age 18-74 years. DESIGN AND SETTING: Morning saliva samples were obtained from participants in a cross-sectional probability-sample survey of the general British population (Natsal-3). Self-reported health and lifestyle questions were administered as part of a wider sexual health interview. PARTICIPANTS: Study participants included 1599 men and 2123 women. METHODS: Sal-T was measured using liquid chromatography-tandem mass spectroscopy. Linear regression was used to examine associations between health factors and mean Sal-T. RESULTS: In men, mean Sal-T was associated with a range of health factors after age adjustment, and showed a strong independent negative association with body mass index (BMI) in multivariable analysis. Men reporting cardiovascular disease or currently taking medication for depression had lower age-adjusted Sal-T, although there was no association with cardiovascular disease after adjustment for BMI. The decline in Sal-T with increasing age remained after adjustment for health-related factors. In women, Sal-T declined with increasing age; however, there were no age-independent associations with health-related factors or specific heath conditions with the exception of higher Sal-T in smokers. CONCLUSIONS: Sal-T levels were associated, independently of age, with a range of self-reported health markers, particularly BMI, in men but not women. The findings support the view that there is an age-related decline in Sal-T in men and women, which cannot be explained by an increase in ill health. Our results demonstrate the potential of Sal-T as a convenient measure of tissue androgen exposure for population research.

Prednisolone measurement in human serum using liquid chromatography tandem mass spectrometry.

Prednisolone is a commonly prescribed corticosteroid used in the treatment of many diseases. Despite high doses of prednisolone, some patients appear to have subtherapeutic concentrations of the drug. It would be useful to measure prednisolone in this group to determine if they have poor absorption or compliance. Hence, we have developed a liquid chromatography-tandem mass spectrometry method for the determination of prednisolone in serum. Chromatography was performed using a C18 column, giving a retention time for both prednisolone and deuterated prednisolone (internal standard) of 1.6 min. Two transitions were monitored for both prednisolone and deuterated prednisolone. These were m/z 361.2 > 343.0 and m/z 361.2 > 146.9 for prednisolone, and m/z 367.2 > 349.0 and m/z 367.2 > 149.9 for the internal standard. The intra- and inter-batch imprecision was < 7% in both cases over a concentration range of 62.5-750 microg/L. The imprecision at the lower limit was 8%, the lower limit of quantitation was determined to be 30 microg/L and the method was linear up to 5000 microg/L. The method allows rapid prednisolone analysis because of a simplified sample extraction step, and has a cycle time of 3.5 min.

Development of a method for the measurement of dehydroepiandrosterone sulphate by liquid chromatography-tandem mass spectrometry.

BACKGROUND: Dehydroepiandrosterone sulphate (DHEAS) is a steroid that is increasingly being recognized as a potential drug of abuse in many countries. This is due to its reputation as a hormone that may be able to retard the ageing process. The measurement of DHEAS is useful in the diagnosis of medical conditions such as congenital adrenal hyperplasia and polycystic ovary syndrome. Thus, a liquid chromatography-tandem mass spectrometry method has been developed to determine DHEAS concentrations in human serum. METHOD: The chromatography was performed using a Waters 2795 Alliance HT LC system coupled to a Mercury Fusion-RP column fitted with a SecurityGuard column. RESULTS: DHEAS and the internal standard, deuterated DHEAS, both had a retention time of 1.5 min. The transition determined by the Micromass Quattro tandem mass spectrometer for DHEAS was m/z 367.3>4 96.7 and for the internal standard m/z 369.3>96.6. The method was linear up to 20 micromol/L; the lower limit of detection and the lower limit of quantitation were both 1 micromol/L. The intra- and interassay imprecision were <11% over a concentration range of 1-18 micromol/L for the in-house quality control and <12% for the intra- and interassay imprecision for the Bio-Rad Lyphocheck QC. CONCLUSION: The measurement of DHEAS by liquid chromatography-tandem mass spectrometry is robust and has a simple sample preparation procedure with a rapid cycle time of only 4 min.

Measurement of citrate in urine using liquid chromatography tandem mass spectrometry: comparison with an enzymatic method.

BACKGROUND: Measurement of urine citrate is used to assess the risk of further urinary stone formation and to assess the benefit of treatment in affected individuals. We wanted to develop a simple and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the analysis of urinary citrate and to compare it with our current enzymatic assay. METHODS: For the LC-MS/MS assay, samples were prepared in a deep-well block by adding 10 microL of urine and 20 microL of internal standard to 400 microL of water. After mixing, 3 microL of the diluted sample was injected into the LC-MS/MS system. An LC system was used to isocratically elute a C18 column (50 x 2.1 mm) with 0.4 mL/min water containing 2 mmol/L ammonium acetate and 0.1% (v/v) formic acid. A step gradient of 100% methanol containing 2 mmol/L ammonium acetate and 0.1% (v/v) formic acid was used to wash the column. The retention times were 1.4 min for citrate and 1.4 min for d4-citrate. Cycle time was 4.0 min, injection to injection. The analytes were monitored using a tandem mass spectrometer operated in multiple reaction monitoring mode using the following transitions, citrate m/z 191.0>111.0 and d4-citrate m/z 195.0>113.0. RESULTS: Within and between-batch coefficients of variation were <3% over the range 480-3800 micromol/L. The lower limit of quantification was 24.0 micromol/L. Regression analysis showed LC-MS/MS = 0.8781 (enzymatic assay) + 102.5, r = 0.964, n = 73. CONCLUSIONS: We have developed a simple LC-MS/MS method for urinary citrate measurement that shows acceptable performance.

Biological variation of glycated hemoglobin. Implications for diabetes screening and monitoring.

OBJECTIVE: To assess the inherent potential of glycated hemoglobin as a screening test for type 2 diabetes by determining the biological variation in nondiabetic subjects. RESEARCH DESIGN AND METHODS: HbA1c values were measured by high-performance liquid chromatography (HPLC) in 12 nondiabetic subjects (7 men and 5 women; median age, 40 years [range, 21-55 years]) on 10 fortnightly occasions. The nondiabetic index of individuality (IOI) for HbA1c (i.e., the square root of the ratio of intra- to interindividual variance) was determined. Any test with an IOI of 1.4 has the most potential in disease screening, while one of 0.6 will be of little value. RESULTS: The analytical variance contributed to 9% of the total test variance, intraindividual variance, 6%; and interindividual variance, 85%. The IOI was, therefore, only 0.27. Thus, nondiabetic HbA1c values vary markedly between subjects, while values in the same individual change little with time. As such, to lie outside the assay reference range, the HbA1c values of some nondiabetic subjects must exceed 12 SD from their usual mean value, while in others a change of only 2 SD would be sufficient. CONCLUSIONS: This fundamental characteristic of HbA1c means that even if analytical methods improve, glycated hemoglobin measurements will always be of limited value when screening for type 2 diabetes. If similar interindividual differences also exist in diabetic subjects, then patients with the same glycemic control may vary by at least 1-2%, which has implications in setting glycated hemoglobin targets.

Determinants of raised C-reactive protein concentration in type 1 diabetes.

As a marker of systemic inflammation, raised C-reactive protein (CRP) concentrations which are still within the normal range have been associated with an increased incidence of coronary heart disease (CHD) in non-diabetic subjects. This study aimed to establish potential determinants of raised CRP concentrations in type 1 diabetic patients. We used a sensitive assay to measure 'low-level' CRP concentrations in 167 type 1 patients (93M, 74F, median age 30 years, range 13-67). Stepwise multivariate analysis was used to relate these CRP levels to known cardiovascular risk factors and demographic data. Only six patients had established CHD (median CRP 3.34 mg/l vs. 0.83 mg/l, p=0.032). In subjects without overt CHD, multivariate analysis showed increases in subject age (p=0.0025), BMI (p=0.001) and HbA(1) (p=0.012) to be associated with a higher CRP concentration, as was female sex (p=0.026) and a history of CHD in a first-degree relative (p=0.018, n=57). The duration of diabetes, current smoking status, presence of microvascular complications, lipid status and presence of hypertension were unrelated. This study suggests that some of the risk factors associated with CHD in type 1 patients are also independently predictive of high CRP concentrations. The reasons for this, and whether intervention would prove useful, require further investigation.

Analysis of sweat chloride by reversed phase high-performance liquid chromatography.

A high-performance liquid chromatography (HPLC) method for the determination of sweat chloride was developed and evaluated. This method is capable of measuring chloride ion in sweat eluates with an analytical working range from 0.5 mmol/L to 6 mmol/L (15 to 180 mmol/L in undiluted sweat samples). Precision studies showed that at the levels of 1 mmol/L, 2.5 mmol/L and 3.5 mmol/L, the within batch coefficients of variation were 1.26%, 0.88% and 0.43%, respectively, and the between batch coefficients of variation were 1.31%, 1.75% and 1.07%, respectively. The minimum detection limit was 0.5 mmol/L. This method correlated well with the ion-selective electrode (ISE) method currently in use.

A rapid anion exchange high-performance liquid chromatography method for the measurement of HbA2 in whole blood.

We developed a binary gradient high-performance liquid chromatography (HPLC) method for measuring HbA2 in whole blood samples using a Pharmacia Mono Q column (1 mL) and measurement at 415 nm. The assay requires a simple lysis and centrifugation step before injection onto the column. We found good agreement of results between the HPLC method and the Helena column chromatography method. The within batch precision was 2-6% and between batch precision was 4-6%. We found that using 30 mM Tris buffers (pH 7-8) with a sodium chloride gradient resulted in short analysis times and good chromatographic separation of HbA2, HbS and HbA. We conclude that this is a robust assay for the diagnosis of beta-thalassaemia.

Plasma concentrations of tumour dimeric pyruvate kinase are increased in patients with chronic cardiac failure.

BACKGROUND: The aim of the study was to assess the effect of chronic cardiac failure (CCF) on circulating plasma concentrations of tumour metabolic marker dimeric pyruvate kinase type M2 (M2-PK). METHODS: Fifty patients with clinically stable CCF were studied. Patients with a history of past or ongoing malignancy were excluded. Dimeric M2-PK was measured by enzyme-linked immunosorbent assay in EDTA plasma. RESULTS: Dimeric M2-PK concentration increased significantly (P = 0.005) with increasing clinical severity of CCF as assessed by the New York Heart Association classification grade. CONCLUSIONS: Chronic cardiac failure results in an increased circulating plasma concentration of M2-PK, probably related to increased glycolytic flux. The physiological significance of the results is at present unclear but may relate to maintaining a balance between energy production and the supply of glycolytic intermediates to maintain synthetic processes. The results of this investigation will also have significance in the clinical interpretation of M2-PK concentrations.

Evaluation of a rapid micro-scale assay for tacrolimus by liquid chromatography-tandem mass spectrometry.

BACKGROUND: The immunosuppressive drug tacrolimus has complex and unpredictable pharmacokinetics, therefore regular monitoring is required in patients receiving tacrolimus therapy. We have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring tacrolimus concentrations in whole blood and have compared it with a microparticle enzyme immunoassay. METHODS: For the LC-MS/MS assay, samples were prepared in a 96-deep well microtitre plate by adding 10 micro L of blood to 40 micro L of 0.1 mol/L zinc sulphate solution. Proteins were precipitated by adding 100 micro L acetonitrile containing ascomycin internal standard. After vigorous mixing and centrifugation, 20 micro L of the supernatant was injected into the LC-MS/MS system. A C18 cartridge (3 mm x 4 mm) was eluted with a step gradient of 50% to 100% methanol containing 2 mmol/L ammonium acetate and 0.1% (v/v) formic acid, at 0.6 mL/min. The column was maintained at 55 degrees C. RESULTS: The retention times were 0.98 min for ascomycin and 0.98 min for tacrolimus. Cycle time was 2.5 min, injection to injection. The analytes were monitored using a Quattro micro trade mark tandem mass spectrometer operated in multiple reaction monitoring mode using the following transitions: m/z821 > 768 (tacrolimus) and m/z809 > 756 (ascomycin). The limit of quantitation was 0.5 micro g/L and the assay was linear up to 30 micro g/L. Precision of the method, over the concentration range 2.5-15.0 micro g/L, was < 7% within-batch and < 6% between-batch. Total time to analyse 24 samples including result generation was 90 min. CONCLUSION: We conclude that the LC-MS/MS method is quick, precise and robust and will provide a fast turn around of results for the transplant physician.

A comparison of three procedures for the detection of Bence-Jones proteinuria.

A traditional electrophoretic procedure for detection of Bence-Jones proteinuria, employing Amido black stain on 200-fold concentrated urine, has been compared to two procedures employing highly sensitive protein stains not requiring prior urine concentration. All three procedures were carried out on 80 random urine samples screened for Bence-Jones proteinuria and 10 samples were provided by patients attending a myeloma clinic. A new procedure employing modified Coomassie brilliant blue stain on unconcentrated urine showed comparable sensitivity to the established procedure (82% versus 88%, respectively) and specificity (77% versus 74%, respectively), when assessed against immunofixation as a reference method. However, the new method is considerably quicker and cheaper. A second method, employing Gold stain, showed enhanced sensitivity (94% versus 88% for Amido black) but lower specificity (62% versus 74% for Amido black). However, this method is labour intensive and relatively expensive. Our data suggest that the procedure employing modified Coomassie brilliant blue may be a suitable alternative to the traditional procedure commonly used in many clinical laboratories.

Salivary testosterone measurement by liquid chromatography tandem mass spectrometry in adult males and females.

BACKGROUND: Salivary testosterone (Sal-T) may be a useful surrogate of serum free testosterone. The study aims were to use a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) assay to determine whether Sal-T concentrations accurately reflect Sal-T concentrations in both sexes and to investigate practical aspects of sample collection. METHODS: Saliva and serum samples were collected in 104 male and 91 female subjects. A more sensitive LC-MS/MS assay was developed to enable Sal-T quantitation in the low concentrations found in females. Saliva (200 µL) was extracted with 1 mL of methyl-tert-butyl ether following the addition of D5-testosterone. Quantitation was performed using a Waters TQ-S mass spectrometer. RESULTS: The assay achieved a lower limit of quantification of 5 pmol/L, sufficiently sensitive to measure testosterone in female saliva. Sal-T showed a diurnal variation but samples taken at weekly and monthly intervals showed no significant differences. Sal-T was stable at ambient temperature for up to 5 days, after freeze-thawing and 3 years frozen storage. Reference intervals for Sal-T were 93-378 pmol/L in males and 5-46 pmol/L in females. Sal-T correlated significantly with serum calculated free-T in males (r = 0.71, P < 0.001) and in females (r = 0.39, P < 0.001). CONCLUSIONS: These results confirm that testosterone can be reliably and accurately measured by LC-MS/MS in both adult male and female saliva samples. These results lay the foundation for further exploration of the clinical application of Sal- T as a reliable alternative to serum testosterone in the diagnosis and management of androgen disorders and assessment of androgen status in clinical research.

Is calibration the cause of variation in liquid chromatography tandem mass spectrometry testosterone measurement?

BACKGROUND: Testosterone measurement by liquid chromatography tandem mass spectrometry (LC-MS/MS) is well accepted as the preferred technique for the analysis of testosterone. Variation is seen between assays and this may be due to differences in calibration as commercial calibrators for this assay are not readily available. We investigated the effects calibration in routine clinical LC-MS/MS assays. METHODS: All LC-MS/MS users that were registered with the UKNEQAS external quality assurance scheme for testosterone were invited to take part in the study. A set of seven serum samples and serum-based calibrators were sent to all laboratories that expressed an interest. The laboratories were instructed to analyse all samples using there own calibrators and return the results and a method questionnaire for analysis. RESULTS: Fifteen laboratories took part in the study. There was no consensus on supplier of testosterone or matrix for the preparation of calibrators and all were prepared in-house. Also, a wide variety of mass spectrometers, internal standards, chromatography conditions and sample extractions were used. The variation in results did not improve when the results were corrected with a common calibrator. CONCLUSIONS: The variation in results obtained could not be attributed to variations in calibrators. The differences in methodologies between laboratories must be the reason for this variation.

Testosterone measurement by liquid chromatography tandem mass spectrometry: the importance of internal standard choice.

BACKGROUND: Testosterone measurement by liquid chromatography tandem mass spectrometry (LC-MS/MS) is well accepted as the preferred technique for the analysis of testosterone. Variation is seen between assays and is likely to be due to method differences. One area of inconsistency among assays is the choice of internal standard. We investigated the effects of three internal standards. METHODS: Testosterone with two deuterium (D2), five deuterium (D5) and three carbon 13 enrichment (C13) were separately assessed. Samples were extracted using ether following the addition of 10 µL of internal standard. All aliquots were prepared in triplicate, one for each type of internal standard. After mixing, the ether was transferred to a 96-deep well block, and then evaporated to dryness. Extracts were reconstituted with 50% mobile phases and analysed using a Waters Acquity UPLC and Quattro Premier tandem mass spectrometer. This method had previously been shown to have excellent agreement with a reference method using the D2 internal standard and this was considered the target. RESULTS: Lower results were obtained when using D5 testosterone when compared with D2 testosterone. The C13 internal standard also gave lower results, but was closer to the D2 target than the D5 internal standard. CONCLUSIONS: The choice of internal standard alone can have a significant affect on the results obtained by LC-MS/MS assays for testosterone using this chromatography. The effects of the combination of chromatography and internal standard choice should be investigated during method development.