The comparison of serum T and free-T with Sal-T (LC-MS/MS) for both males and females is shown in Figure 4. Two outliers were excluded from the analysis because there was insufficient sample to repeat the test. Sal T analysisThe LC-MS/MS method for measuring Sal-T in males has previously been shown to have good analytical performance (13).
Measured testosterone concentrations of 20 matched serum/plasma pediatric samples and the corresponding correlation plot. While the %CV of female samples was slightly higher than that of male samples due to a much lower testosterone concentration range, it remained within the acceptable value of ≤ 5.3% for both sample preparation methods. Pearson correlation between CDC reference and SCIEX concentration values using 40 serum samples from CDC testosterone HoSt program (phase 1). Method comparison (mean difference plot) between CDC reference and SCIEX concentration values using 40 serum samples from CDC HoSt program for testosterone (Phase 1). The reference concentrations of these 42 samples were compared to those determined by our method using Pearson correlation and percent bias calculation. In pediatric samples, total testosterone levels are the lowest in females and males aged 2–10 years and can be , . To develop an accurate and robust total testosterone ESI-LC-MS/MS quantification method with a simple sample preparation workflow and sufficient sensitivity for serum or plasma samples of all gender and age groups, via ketone functional group derivatization (using Amplifex™ Keto Reagent).
Reliable measurement of total testosterone is essential for the diagnosis, treatment and prevention of a number of hormone-related diseases affecting adults and children. Laboratories may have different reference ranges for normal testosterone levels. The amount of deep sleep varies between nights and individuals, but on average, adults should spend 15-20% of their total sleep time in deep sleep. It ideally makes up about 20-25% of your total sleep time but tends to decrease with age. It’s important to realize that the total time you spent in bed does not necessarily equate with the amount of total sleep. The Oura ring app shows your average HRV (from 5-minute samples measured during the whole night), and your nightly HRV curve.
Sal-T measured by LC-MS/MS correlated significantly with serum-T across the full adult range of concentrations in males as well as females. As with serum measurements, therefore, saliva samples for T measurement should be collected in the morning, preferably before 10am. Monthly samples of paired serum and saliva were obtained over four consecutive months to assess within-individual variations in T. Three paired serum and saliva samples obtained 30 minutes apart in the morning from 11 men and 12 women were analysed to assess the repeatability or test-retest reliability. The sample size was sufficient to detect a statistically significant (5% significance level) correlation between salivary and blood testosterone if the underlying correlation is 0.7 with a power of more than 0.99. Lastly, to investigate month-to-month variation, saliva and blood samples were collected (before 10.00h), once a month over four months.
These findings suggest that this method is either minimally affected by interfering substances or that it is not at all affected. The highest QI/CI ratio observed in an individual sample was 1.24, which is well-below the suggested maximum differences of 20% difference 30,38,39. Measurement specificity has been another concern with testosterone measurements. In addition, we do not use protein precipitation, which is another common sample preparation approach . In the second step, polar lipids such as phospholipids are largely removed from the sample extract as evidenced in reduction in SRM (m/z 184–184) and precursor-ion scanning (pre m/z 184) signals that are indicative for these types of lipids 43,44.
7A and B illustrate the individual sample bias throughout the cycle year for each of the sample preparation methods. Method performance after the first cycle year of HoSt Phase 2 program for total testosterone. Representative calibration curve comprised of 6 concentration levels, 1.8–1843 ng/dL. Based on repeated serial dilutions of NIST 971 female sample and from the S/N ratio of the lowest calibrator level over different days, the LLOQ was estimated to be 1 ng/dL (10 pg/mL). SCIEX Analyst® MD 1.6.2 and MultiQuant MD 3.0.2 programs were used for LC-MS/MS method optimization, data acquisition, data processing and quantification.
Within-individual variations in Sal-T and serum-T were explored in paired samples of saliva and blood in 11 men and 12 women. 2) determine whether Sal-T concentration accurately reflect serum-T concentrations in both men and women and 3) investigate practical aspects of salivary sample collection, including stability in transport and storage. 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. This described method provides a consistently high accurate and sensitive LC-MS/MS assay for testosterone measurements in serum at all concentration levels typically observed in men as well as in women and children.

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