scholarly journals Measurement of Free Testosterone in Serum Using Equilibrium DialysisCoupled With ID-UHPLC-MS/MS: Comparison Between Equilibrium Devices

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A760-A760
Author(s):  
Hui Zhou ◽  
Ashley Ribera ◽  
Amonae Dabbs-Brown ◽  
Uliana Danilenko ◽  
Hubert W Vesper
Keyword(s):  
High Throughput ◽  
High Performance ◽  
Equilibrium Dialysis ◽  
Internal Standard ◽  
Free Testosterone ◽  
Performance Comparison ◽  
Total Testosterone ◽  
Method Performance ◽  
Endocrine Society ◽  
Clinical Patient

Abstract Free testosterone (FT) has been used as a biomarker in clinical patient care and public health research to assess and manage patients with androgenic abnormalities. The latest Endocrine Society clinical practice guideline for testosterone therapy in men with hypogonadism recommends measuring FT for those with borderline and low total testosterone concentrations, or those who have conditions that change SHBG concentrations, such as some metabolic or hormonal diseases, certain medication use, or SHBG genetic polymorphisms. Measuring FT is technically challenging and shows high variability. The CDC clinical standardization program is developing a high throughput method using the gold-standard equilibrium dialysis (ED) procedure with isotope dilution ultra-high-performance liquid chromatography tandem mass spectrometry (ID-UHPLC-MS/MS). A serum sample was dialyzed against a protein-free HEPES buffer (pH 7.4) at 37 °C until equilibrium. After isolating endogenous FT from protein-bound testosterone by ED, isotope-labeled internal standard (13C3-testosterone) was added to the dialysate for quantification. Certified pure primary reference material (National Measurement Institute M914) was used to prepare calibrators, enabling traceable quantitation and ensuring measurement trueness. FT was further isolated from the dialysate matrix using supported liquid extraction and a chromatographic separation from interfering compounds and quantitation by tandem MS. The dialysis step requires maintaining the endogenous free hormone equilibrium so that results in dialysate reflect FT concentrations in the blood without influence from dilution, temperature, or pH. The dialyzer system has a 1:1 sample-to-buffer volume and has been used in reference measurement procedures for free hormone measurements, serving as the standard for method performance comparison. Four commercially available devices designed for high throughput in a multiple well-plate format, requiring respective sample-to-buffer ratios, were evaluated for their recovery, speed, ease of automation by a liquid handling system, repeatability, and robustness. Preliminary results showed that a device with 1:1 sample-to-buffer volume had the most comparable results to those obtained from the standard dialyzer, with the mean bias less than 15%. The device with the highest sample-to-buffer ratio showed bias as high as 50%. These data suggest that controlling sample-to-buffer ratio is a critical step in ED FT method.

scholarly journals SUN-066 Prenatal Sex Steroid Serum Concentrations in Relation to Sex-Typical Play Behavior at 4 Years of Age

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Swathi Sethuram ◽  
Shanna Swan ◽  
Christina C L Wang ◽  
Emily Barrett ◽  
Ruby Nguyen ◽  
...  
Keyword(s):  
Sex Steroids ◽  
Infant Development ◽  
Parental Attitudes ◽  
Equilibrium Dialysis ◽  
Objective Measure ◽  
Free Testosterone ◽  
Small Sample ◽  
Play Behavior ◽  
Total Testosterone ◽  
Quadratic Term

Abstract INTRODUCTION: Sex differences in play behavior have been seen in rodents, primates and humans with literature showing some associations with maternal sex steroids. The PreSchool Activities Inventory (PSAI) is a validated tool to assess sexually-dimorphic play behavior. We assessed the associations between prenatal maternal estradiol (E2), free testosterone (fT) and total testosterone (T) and children’s PSAI scores at age 4. METHODS: Data were collected in The Infant Development and the Environment Study (TIDES), a US-based multi-center pregnancy cohort. The analysis included 399 pregnant women with complete data on sex steroids, PSAI and covariates. Early pregnancy E2 and T were analyzed by liquid chromatography tandem mass spectrometry and fT by equilibrium dialysis using labeled testosterone. PSAI scores (masculine, feminine and composite) were calculated from questionnaires completed by mothers when child was approximately 4 years old. Covariates included: maternal age and education, child age at questionnaire completion, number of older brothers and sisters, infant race and parental attitudes towards sex-atypical play behavior. After demonstrating nonlinear associations between PSAI scores and sex steroids, we used multivariable sex-stratified quadratic regression to model these relationships. RESULTS: The analysis included 192 boys and 207 girls (mean age: 4.5 years). Sex steroids in serum collected at 6-20 weeks’ gestation (mean: 11 weeks) were similar in mothers of boys and girls. In girls, E2 showed a quadratic relationship with the masculine score (beta (E2) = 0.003, p = 0.002, beta (E22) = -7.3E-7, p <0.001). Masculine scores were lower among girls whose mothers had E2 above the 75th percentile (2160 pg/ml). In boys, we observed a positive linear association of borderline significance (beta (fT) = 9.11, p = 0.06) and a significant negative association for the quadratic term (beta (fT2) = -7.44, p = 0.04). PSAI measures were not associated with E2 in mothers of boys, fT in mothers of girls, or T in either sex. DISCUSSION: This study did not replicate previously reported associations between T and masculine scores in girls. However, we found that, while most maternal E2 levels were not associated with play behavior in either sex, levels above the 75th percentile were associated with reduced masculine scores in girls at age 4. Our data also suggest a somewhat reduced feminine scores in boys born to mothers with fT above the 75th percentile. These results, based on a small sample using a maternal assessment of play behavior, should be examined in a larger sample using an objective measure of play.

In women, no significant variances of calculated free testosterone (cFT) are observed when a fixed value of albumin (Alb: 4.3 g/dL) is used instead of measured albumin values

2014 ◽  
Vol 20 (3) ◽  
Author(s):  
Dinamarie Garcia-Banigan ◽  
Andre Guay ◽  
Abdul Traish ◽  
Gheorghe Doros ◽  
John Gawoski
Keyword(s):  
Equilibrium Dialysis ◽  
Free Testosterone ◽  
Sex Hormone Binding Globulin ◽  
Total Testosterone ◽  
Hormone Binding ◽  
R Software ◽  
Significant Variance ◽  
Clinical Evaluations ◽  
Data Points ◽  
Two Measures

AbstractCalculated free testosterone (cFT) is determined from the values of total testosterone (TT), sex hormone binding globulin (SHBG), and albumin (Alb) using mathematical formulae. We evaluated any potential cFT variance when determined with fixed Alb (4.3 g/dL) compared to measured Alb, and the point at which low SHBG and Alb combinations produced significant cFT variance.We analyzed 2050 data points in 1222 women. cFT values with fixed vs. the actual measured Alb values were evaluated and contrasted. cFT levels were determined theoretically for all possible combinations of TT, SHBG, and Alb.Agreement between the two measures was assessed with Lin’s concordance coefficient. Statistical analyses were performed using R software version 2.12.1.Mean Alb was 4.05±0.30 g/dL. Mean SHBG 73.0±53.3 nmol/L. A fixed Alb of 4.3 g/dL produced no significant variance for most evaluations of cFT. The accuracy decreased with Alb ≤3.5 g/dL in combination with SHBG ≤30 nmol/L and exists in 1.0% of the samples.A fixed Alb of 4.3 g/dL is acceptable for most clinical evaluations. If Alb is ≤3.5 g/dL, along with SHBG ≤30 nmol/L, the variance increases and a free testosterone (FT) measurement by equilibrium dialysis is warranted for better accuracy.

An improved ultrafiltration method for determining free testosterone in serum.

1982 ◽  
Vol 28 (11) ◽  
pp. 2286-2291 ◽  
Author(s):  
I Vlahos ◽  
W MacMahon ◽  
D Sgoutas ◽  
W Bowers ◽  
J Thompson ◽  
...  
Keyword(s):  
Pregnant Women ◽  
Equilibrium Dialysis ◽  
Free Testosterone ◽  
Total Testosterone ◽  
Unbound Fraction ◽  
Men And Women ◽  
Healthy Men ◽  
Centrifugal Ultrafiltration ◽  
Dialyzed Serum

Abstract In this method, we use the Amicon MPS-1 centrifugal ultrafiltration device and the YMB membrane in measuring free testosterone in serum. Two independent assays are combined: total testosterone and the ultrafiltrable fraction of added [3H]testosterone. The unbound fraction is determined in 0.15-0.5 mL ultrafiltrates of 0.6 to 1 mL of variably diluted serum that has been equilibrated with [3H]testosterone at 37 degrees C. The assay is rapid (less than 1 h), practicable (requires 0.6 mL of serum), and reproducible (CV 3.2% within assay, 3.9% between assays). Accuracy was evaluated as the fraction of free testosterone in the ultrafiltrate of dialyzed serum vs that in a prior dialysate; they were the same confirming the validity of the free testosterone measurement. Samples from ostensibly healthy men and women and from hirsute and pregnant women gave results that agreed with those obtained by equilibrium dialysis. Total testosterone concentrations for normal and hirsute women showed considerable overlap, but data on free testosterone concentrations in these populations were better resolved.

scholarly journals Validity of free testosterone calculation in pregnant women

2019 ◽  
Vol 8 (6) ◽  
pp. 672-679
Author(s):  
M P Schuijt ◽  
C G J Sweep ◽  
R van der Steen ◽  
A J Olthaar ◽  
N M M L Stikkelbroeck ◽  
...  
Keyword(s):  
Pregnant Women ◽  
Equilibrium Dialysis ◽  
Free Testosterone ◽  
Sex Hormone Binding Globulin ◽  
Total Testosterone ◽  
Testosterone Concentration ◽  
Testosterone Levels ◽  
Liquid Chromatography Tandem Mass ◽  
Highly Correlated ◽  
Design And Methods

Objective Increased maternal testosterone concentration during pregnancy may affect the fetus. Therefore it is clinically relevant to have a quick and reliable method to determine free testosterone levels. Current calculators for free testosterone are suspected to perform poorly during pregnancy due to suggested competition between high levels of estradiol and free (bio-active) testosterone for sex hormone-binding globulin (SHBG) binding. Therefore, it is claimed that reliable calculation of free testosterone concentration is not possible. However, recent evidence on SHBG-binding sites questions the estradiol effect on the testosterone-SHBG binding during pregnancy. In this study, we investigated whether the free testosterone concentration can be calculated in pregnant women. Design and methods Free testosterone was measured with a specially developed equilibrium dialysis method combined with liquid chromatography tandem mass spectrometry (LC-MS/MS). Free testosterone was also calculated with the formulas of Vermeulen et al. and Ross et al. Results Total and free testosterone measured in healthy men and women were in good agreement with earlier reports. In pregnant women, total testosterone values were higher than in non-pregnant women, whereas free testosterone values were comparable. Calculated free testosterone levels in pregnant women were highly correlated, but marginally higher, compared to measured free testosterone levels. Conclusions We developed an equilibrium dialysis–LC-MS/MS method for the measurement of free testosterone in the low range of pregnant and non-pregnant women. Although during pregnancy total testosterone is increased, this is not the case for free testosterone. The free testosterone formulas perform well in pregnant women.

scholarly journals Analog-Based Free Testosterone Test Results Linked to Total Testosterone Concentrations, Not Free Testosterone Concentrations

2008 ◽  
Vol 54 (3) ◽  
pp. 512-516 ◽  
Author(s):  
Kristofer S Fritz ◽  
Alastair J S McKean ◽  
Jerald C Nelson ◽  
R Bruce Wilcox
Keyword(s):  
Serum Proteins ◽  
Equilibrium Dialysis ◽  
Serum Testosterone ◽  
Free Testosterone ◽  
Sex Hormone Binding Globulin ◽  
Normal Male ◽  
Total Testosterone ◽  
Test Results ◽  
Experimental Conditions ◽  
Serum Free

Abstract Background: Analog-based free testosterone test results, sex hormone binding globulin (SHBG) concentrations, and total testosterone concentrations are somehow related. This study used new experiments to clarify these relationships. Methods: An analog-based free testosterone immunoassay and a total testosterone immunoassay were applied to well-defined fractions of serum testosterone. First, they were applied to the 2 fractions (retentate and dialysate) of normal male serum obtained by equilibrium dialysis. Second, they were applied to covaried concentrations of SHBG and total testosterone. Third, they were applied to decreasing concentrations of SHBG and protein-bound testosterone, offset by increasing concentrations of protein-free testosterone, while total testosterone was held constant. Results: The analog-based free testosterone assay and the total testosterone assay detected and reported serum testosterone test results from serum retentate, whereas neither assay detected the free testosterone in serum dialysate. Test results reported by the analog-based free testosterone assay followed varied concentrations of SHBG and total testosterone. When total testosterone was held constant, however, analog-based free testosterone test results did not follow varied concentrations of serum proteins or of free testosterone. Conclusion: An analog-based free testosterone immunoassay reported free testosterone test results that were related to total testosterone concentrations under varied experimental conditions. This alleged free testosterone assay did not detect serum free testosterone (the test results it reported were nonspecific) and should not be used for this purpose.

Development and validation of a LC-MS/MS method for quantification of mobocertinib (TAK-788) in plasma and its application to pharmacokinetic study in rats

2020 ◽  
Vol 23 ◽  
Author(s):  
Bo Li ◽  
Jin Wang ◽  
Xinyao Dou ◽  
Xinjie Zhang ◽  
Xianbei Xue ◽  
...  
Keyword(s):  
Oral Administration ◽  
Pharmacokinetic Study ◽  
High Performance ◽  
Kinase Inhibitor ◽  
Internal Standard ◽  
Selected Reaction Monitoring ◽  
Precipitation Method ◽  
Plasma Samples ◽  
Bioanalytical Method Validation ◽  
Extraction Recovery

Aim and Objective:: An analytical method for the determination of mobocertinib, an investigational tyrosine kinase inhibitor, was developed and optimized by high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) in rat plasma. Materials and Methods:: Plasma samples were pretreated by the protein precipitation method with a methanol solution of osimertinib as the internal standard (IS). Chromatographic separation was performed using an Inertsil ODS-3 column (50 mm × 4.6 mm, I.D. 5 μm) column with the temperature maintained at 40 °C. The mobile phase consisted of water (containing 0.1% formic acid) and methanol in a gradient mode at a flow rate of 0.5 mL/min. Mass spectrometric detection was carried out in the selected reaction monitoring (SRM) mode with positive electrospray ionization, and the mass transitions of mobocertinib and osimertinib were m/z 587.01 → 71.88 and m/z 499.80 → 71.94, respectively. The method was validated in terms of selectivity, linearity, accuracy and precision, extraction recovery and matrix effect, stability and carryover as per the guidelines for bioanalytical method validation (FDA, 2018). The method was applied to the pharmacokinetic study of mobocertinib in rats by oral gavage at the doses of 2, 6, and 18 mg/kg. A total of 216 plasma samples from 18 rats were analyzed. Results:: It showed good linearity over the range of 1-1000 ng/mL (R2 = 0.9957). The intra-batch accuracy was within 94.65-102.59% and the precision was within 5.49-10.46%. The inter-batch accuracy was within 97.08-102.25% with a precision of 7.54-10.13%. The extraction recovery and matrix factor were acceptable for the bioanalysis of mobocertinib. Additionally, mobocertinib was found to be stable under the detected conditions. Mobocertinib showed linear pharmacokinetic characteristics following oral administration to rats at 2.0-18.0 mg/kg. Conclusion:: The developed and validated method was successfully employed in the pharmacokinetic study in rats following oral administration of mobocertinib at the doses of 2, 6, and 18 mg/kg.

Prevalence of and Risk Factors for Low Free Testosterone Levels in Japanese Men with Well-controlled Human Immunodeficiency Virus Infection

2020 ◽  
Vol 18 (5) ◽  
pp. 381-386
Author(s):  
Yusuke Yoshino ◽  
Ichiro Koga ◽  
Yoshitaka Wakabayashi ◽  
Takatoshi Kitazawa ◽  
Yasuo Ota
Keyword(s):  
Risk Factors ◽  
Human Immunodeficiency Virus ◽  
Standard Deviation ◽  
Hiv Infection ◽  
Free Testosterone ◽  
Serum Levels ◽  
University Hospital ◽  
Rapid Decline ◽  
Total Testosterone ◽  
Immunodeficiency Virus

Background: The change in the prevalence of hypogonadism with age in men with human immunodeficiency virus (HIV) infection is subject to debate. Objective: To address this issue, we diagnosed hypogonadism based on serum levels of free testosterone (fTST) rather than total testosterone which is thought to be an inaccurate indicator. We also determined the relationship between age and fTST levels and identified risk factors for hypogonadism in men with HIV infection. Method: We retrospectively reviewed fTST levels and associated clinical factors in 71 wellcontrolled HIV-infected men who were treated at Teikyo University Hospital between April 2015 and March 2016 and who had data available on serum fTST levels, measured >6 months after starting antiretroviral therapy. fTST was measured using radioimmunoassay on blood samples collected in the morning. Risk factors for hypogonadism were identified using Welch’s t-test and multiple regression analysis. Results: The men had a mean (± standard deviation) age of 47.4 ± 13.6 years, and mean (± standard deviation) serum fTST level of 13.0 ± 6.1 pg/mL. Fifteen (21.1%) men had hypogonadism based on a fTST <8.5 pg/mL. Serum fTST levels significantly decreased with age (−0.216 pg/mL/year). Older age and low hemoglobin levels were identified as risk factors for hypogonadism. Conclusion: The men in the study experienced a more rapid decline in fTST levels with age than men in the general population (−0.161 pg/mL/year). Serum fTST levels in men with HIV infection should be monitored, especially in older men and those with low hemoglobin levels.

Revealing changes in curcumin bioavailability using vitamin C as an enhancer by HPLC-MS/MS

2019 ◽  
Vol 16 ◽  
Author(s):  
Xufen Dai ◽  
Jiaxue Hao ◽  
Ying Feng ◽  
Jing Wang ◽  
Qiannan Li ◽  
...  
Keyword(s):  
Vitamin C ◽  
High Performance ◽  
Internal Standard ◽  
Fold Increase ◽  
Rat Plasma ◽  
Pharmacokinetic Parameters ◽  
Solution Ph ◽  
Esi Ms ◽  
Simple Strategy ◽  
Isolated Compound

Background: Curcumin (CUR), a natural isolated compound from turmeric, has been the promising star in fighting many diseases but the broad application of curcumin has been limited ascribed to low bioavailability. Objective: The aim of this study is to pursue the enhancement of curcumin bioavailability through co-administration of vitamin C. Methods: Such purpose was achieved through the analysis of curcumin pharmacokinetics by high performance liquid chromatography coupled with electrospray ionization - tandem mass spectrometry (HPLC - ESI - MS/MS). The plasma was separated on a C18 reverse phase column using acetonitrile and ammonium formate solution (pH 6.5; 2.0 mM) at 0.8 mL/min. MS/MS detection was carried out in negative mode using mass patterns of m/z 367.0 > 216.7 for curcumin and m/z 265.2 > 223.9 for internal standard (honokiol). Results: Successful application of the proposed method in the pharmacokinetic study presented clear changes in key pharmacokinetic parameters including the growth of AUC (0-t) up to 2.4 times, 2.2-fold increase of Cmax, 2.2-fold loss of CL, and 1.5-fold diminishment of t1/2. Conclusion: We developed an HPLC-ESI-MS/MS method for determination of curcumin in rat plasma and validated the improvement of bioavailability of curcumin through co-administration of vitamin C. We reasoned these changes to the inhibition of lipid peroxidation induced by the use of vitamin C. Such a simple strategy is possible to become an alternative for enhancing curcumin efficiency in practice.

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