The sample preparation for the method described here attempted to reduce time, extraction solvent, sample volume, and cost. Twenty pediatric serum samples, aged 2–15 years, with mixed gender and race (not plasma matched) were analyzed to produce the results shown in the SI Table 6. In addition, looking at the percent difference among the three samples from each individual donor, the average between serum and plasma, or between plasma LH and plasma K2EDTA was ∼ 1%, ranging from 0.04 to 11.6%. On each day of analysis, we added the two concentration levels of NIST 971 as an extra quality control from a different source. First, the method was calibrated with 40 Phase 1 samples from the program and two NIST 971 SRMs.
High levels may point to precocious (early) puberty. Free testosterone is easier for your body to use. Your healthcare provider may recommend this test if you have symptoms that could point to high or low testosterone. A testosterone level that’s too low or high can cause health problems regardless of your sex. Your body functions best when your testosterone is in a certain range.
Measurement values obtained with this method are in high agreement with established reference methods and meets performance criteria for accuracy used by the CDC Hormone Standardization Program . Some methods described in literature use derivatization procedures to enable analysis by GC-MS or to achieve higher sensitivity of LC-MS measurements 47–49. Interferences from components in serum separator tubes (SST) have been reported but were not found with our method using BD Vacutainer® SST™ (Franklin Lakes, NJ) probably because of the use of different chromatographic conditions.
To investigate this we infused a 340 pmol/L solution of D5 testosterone in 40% (v/v) methanol/water directly into the mass spectrometer to give a constant background signal. The ratios of analyte peak area to internal standard peak area were plotted against testosterone concentration in pmol/L. About 15 mL of venous blood was drawn from an antecubital vein to give 3 x 2.5mL aliquots of serum (in cryovials for storage at -80°C).
While devices are available to ensure controlled collection of blood spots, these devices can be costly and require strict patient compliance, which diminishes the simplicity and cost‐effectiveness of the DBS method. A major challenge in DBS sampling is the ‘HCT effect’ which occurs when the blood's HCT level influences the spreading of the blood on the filter paper, leading to uneven distribution and potential assay bias . Accurate and reliable measurement of testosterone levels is essential in clinical settings, particularly for diagnosing conditions such as hypogonadism, polycystic ovary syndrome, and other endocrine disorders 1, 3. Testosterone, a key androgen hormone, plays a crucial role in regulating numerous physiological functions in both men and women, including reproductive health, muscle mass, bone density, and overall well‐being 1, 2. The testosterone was extracted from DBS using a liquid‐liquid method and analyzed with LC‐MS/MS.

Gender: Female

Social links