Validating Bioavailable Testosterone by LC-MS/MS: A Clinically Accurate Alternative to Free Testosterone

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* Clinicians often prefer bioavailable testosterone as it more closely resembles the total bioactive testosterone in circulation when compared to free testosterone. Validation to ensure clinical accuracy of our bioavailable testosterone method modification involved a comparison of the reference interval flags between this method and verified free and total testosterone flags.


* Recently, our bioavailable testosterone assay underwent a method modification to enhance calibrator storage and quality, which required subsequent validation to confirm adequate clinical performance. It has been shown that a strong correlation exists between bioavailable and free testosterone. As such clinical performance was validated by investigating the concordance between the reference interval (RI) flags (low, normal, or high) obtained using the modified BAT method and the RI flags from validated LC-MS/MS total and free testosterone assays.







MSACL 2025 Abstract


To Free or Not to Free? An LC-MS/MS-Based Method Modification to Validate Clinical Performance of Bioavailable Testosterone Measurement
Chelsea B. Swartchick, Jennifer V. Kemp, Stefan K. Grebe, Irina Bancos, and Anthony D. Maus
Mayo Clinic, Rochester, MN


Abstract

INTRODUCTION

Measurement of total testosterone is an invaluable method for assessing an individual’s androgen status. This assay is often performed in conjunction with free testosterone measurement, which should encompass approximately 1 to 2% of the total testosterone concentration. However, measurement of free testosterone is influenced by abnormalities in both the concentration and/or function of sex hormone binding globulin (SHBG), the primary transport and regulatory protein for testosterone, along with albumin, the most abundant plasma protein
. Therefore, analysis of bioavailable testosterone, which accounts for both free and testosterone loosely bound to albumin, is a more reliable representation of an individual’s true testosterone status. Recently, our bioavailable testosterone assay underwent a method modification to enhance calibrator storage and quality, which required subsequent validation to confirm adequate clinical performance. It has been shown that a strong correlation exists between bioavailable and free testosterone. As such clinical performance was validated by investigating the concordance between the reference interval (RI) flags (low, normal, or high) obtained using the modified BAT method and the RI flags from validated LC-MS/MS total and free testosterone assays.


METHODS


Residual serum samples (n = 65) from patients with recent (< 2 weeks) total and free testosterone measurements were used, where 23 were from women and 42 were obtained from men. Reference interval flags were categorized as high, normal, or low as determined being above, within, or below established reference intervals, respectively. There were 14 specimens classified as high, 36 as normal, and 15 as low, by both their total and free testosterone measurements. Bioavailable testosterone sample preparation involved the differential precipitation of testosterone-bound SHBG with ammonium sulfate. All non-SHBG bound testosterone remained in the supernatant, which subsequently was spiked with internal standard, carbon-13 labeled testosterone. Liquid-liquid extraction was performed, and the resulting isolate was derivatized before being quantified by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). All 65 bioavailable testosterone results were then categorized as either high, normal, or low according to the RI dependent on the patient’s sex and age. Flags from the bioavailable testosterone assay and flags from the free/testosterone measurements were evaluated for agreement. Serum SHBG was quantified using a chemiluminescent sandwich immunoassay. Serum albumin concentration was determined using a colorimetric method using the absorbance at 570 nm.


RESULTS

59 of the 65 (90.7%) samples were correctly categorized with a RI flag as either low, normal, or high by both the free/total testosterone assays and the modified bioavailable testosterone method. The 6 samples that had discrepant flags required further investigation in which serum SHBG and albumin concentrations were evaluated. Of the discordant samples, 4 had elevated free and total testosterone, but normal bioavailable testosterone. Follow-up of this group demonstrated high SHBG concentrations. Elevated SHBG would expectedly bind more testosterone and decrease the bioavailable testosterone concentration, which provides a rationale of the normal categorization in this population. In a similar manner, one sample had a slightly elevated free and total testosterone, but normal bioavailable testosterone. The SHBG concentration of this sample was nearing the upper limit of normal, which follows the same principle as above where the bioavailable testosterone would be flagged as normal. The remaining discrepant sample had normal free and total testosterone, but elevated bioavailable testosterone. This specimen had an SHBG at the lower end of normal and an albumin at the higher end of normal. As there is less SHBG to bind testosterone and more albumin, an elevated bioavailable testosterone is appropriate.


CONCLUSION

Clinicians often prefer bioavailable testosterone as it more closely resembles the total bioactive testosterone in circulation when compared to free testosterone. Validation to ensure clinical accuracy of our bioavailable testosterone method modification involved a comparison of the reference interval flags between this method and verified free and total testosterone flags. In theory, samples that flag normal by both free and total testosterone assays should flag similarly with the bioavailable assay. We observed concordance in 90.7% of our samples, while the remaining 9.3% specimens had abnormal SHBG and/or albumin levels that were reflected appropriately in bioavailable testosterone RI flagging. In summary, our method modification performs clinically and can reflect the true testosterone status within a patient.
 


BIOAVAILABLE TESTOSTERONE

Ammonium Sulfate Precipitation of Sex Hormone-Binding Globulin-Bound Testosterone


As a technique used to measure bioavailable testosterone, ammonium sulfate precipitation involves the mixing of tracer-labeled testosterone with serum followed by the precipitation of SHBG via the addition of ammonium sulfate (Table 3).Protocols often utilize saturated ammonium sulfate solution in a 1:1 ratio with the sample specimen. The remaining tracer-labeled testosterone is then multiplied by total testosterone to yield an estimation of bioavailable testosterone in serum. Although this technique correlates well with equilibrium dialysis, it has several disadvantages such as its reliability on the accuracy of the total testosterone assay and the potential impact of tracer impurities on results.19

An alternative to precipitation of SHBG with ammonium sulfate has been proposed with the use of concanavalin A separation.34 Early evidence suggests that this method may have increased specificity over ammonium sulfate.35
Giton and colleagues compared the results of 131 samples assessed for bioavailable testosterone using both ammonium sulfate precipitation and concanavalin A separation methods. They found similar results from both methods.


Concanavalin A has the benefit of eliminating errors associated with nonspecific albumin precipitation that may occur in poorly controlled assay conditions.35 Further evaluation of this relatively novel method should precede its adoption into clinical practice.




Table 3 A select summary of assays available to measure bioavailable testosterone
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Scientific Reference

Lakshman KM, Kaplan B, Travison TG, Basaria S, Knapp PE, Singh AB, LaValley MP, Mazer NA, Bhasin S. The effects of injected testosterone dose and age on the conversion of testosterone to estradiol and dihydrotestosterone in young and older men. J Clin Endocrinol Metab. 2010 Aug;95(8):3955-64.

DOI: 10.1210/jc.2010-0102 | PMID: 20534765 | PMCID: PMC2913038

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