Determination of Serum Testosterone Levels with Liquid Chromatography-Isotope Dilution Tandem Mass Spectrometry and Comparision with Other Immunoassays

In adrenal cortex, placenta and gonads, testosterone is produced from the cholesterol precursor. Mitochondria and smooth endoplasmic reticulum have a major function for this production. Testosterone has three main forms in circulation: Tightly bound to sex hormone binding globulin (SHBG), bound loosely to albumin and free fraction. The free fraction is about 1-2% of total testosterone and this free form is active through the target gene expression [1].


Introduction
In adrenal cortex, placenta and gonads, testosterone is produced from the cholesterol precursor. Mitochondria and smooth endoplasmic reticulum have a major function for this production. Testosterone has three main forms in circulation: Tightly bound to sex hormone binding globulin (SHBG), bound loosely to albumin and free fraction. The free fraction is about 1-2% of total testosterone and this free form is active through the target gene expression [1].
Determination of testosterone levels is useful for investigation of androgen-producing tumors, anti-androgen therapy in prostate cancer and testicular functions in men [2]. It is also an informative marker for congenital adrenal hyperplasia and sex assignment in newborns [3]. For women, testosterone concentrations in circulation accounts 10-15% of men. Testosterone has physiological properties like maintaining bone mineral density, mood, libido and measurement of serum testosterone concentrations gives valuable clinical information for clinical conditions like hirsutism, alopecia, acne, oligo-amenorrhea presenting with hyperandrogenism [4]. For determining sex steroids, radioimmunoassay (RIAs) is inexpensive and requires minimum sample preparation. Although RIA kits which have extraction procedures or directly measuring testosterone levels compare well with LC-MS/MS and GC-MS, results obtained from studies performed with these kits do not always compare well with LC-MS/MS due to testosterone binding globulins and structurally similar molecules in serum [5]. Because of less interference, GC-MS and LC-MS are taken into consideration as gold standard method [6].
For excessive production or deficiency, measurement of serum or plasma testosterone is performed using automated immunoassays in most clinical laboratories. Commercial immunoassays are rapid, cost-effective and simple. Because of these properties they become widely used in clinical quantification of steroids. But there is a technical difficulty for measurement of testosterone especially in low concentrations [7]. Mostly used techniques measuring testosterone have some disadvantages like low precision, cross-reactivity, limited linear range, poor correlation. Relatively low circulating testosterone concentrations especially in children and women lead to limited precision, unacceptable cross-reactivity and result with positive bias [8].
For these reasons we described performance characteristics and established LC-MS/MS method to our laboratory for clinical practice.

Study design
Patients: Blood samples were taken among the patient samples sent to the laboratory at Ankara Numune Education and Training Hospital, Ankara, Turkey, and centrifuged at 3000 g for 4 minutes to prepare three different serum pools. The first pool was prepared from female patients(aged between 20-30 years) who had low levels of testosterone aged between 20-30 years, second was from high levels of testosterone from men. Third was from intermediate levels of testosterone from geriatric men patients aged over 65 years. These serum pools divided into 20 aliquots and stored at -20°C until analysis. Serum testosterone levels were performed from prepared pools four times a day (Two times in the morning, two times in the afternoon). These study plan was designed for 20 days. Institutional review board approval was obtained from Ankara Numune Education and Training Hospital, Turkey.
Sample preperation: After centrifugation at 3000 g for 4 minutes, 250 µL sample, standart and control were taken into eppendorf tubes. 3 µL internal standart was added to all samples (calibrator,control samples, patient samples). Testosterone was extracted by adding 1000 µL methyl tertbutyl ether (MTBE) to mixture, vortex-mixed for 2 minutes and centrifuged at 3000 g for 5 minutes. The upper organic layer was evaporated under nitrogen gas at 45°C. Then 500 µL 80% methanolwater and 1000 µL heptane were added,vortex-mixed for 2 minutes and upper layer was transferred to clean glass tube for evaporation under nitrogen gas at 45°C. 100 µL 50% methanol-water was added, vortexmixed for a few seconds then transferred to autosampler vials for LC-MS/MS analysis. 30 µL was used as injection volume.

LC-MS/MS analysis:
Determination of serum testosterone was performed with API 3200 mass spectrometer according to the method previously described by Chen et al. [7] in Ankalab Laboratories (Ankara,Turkey). Briefly, HPLC was conducted using Shimadzu Prominence LC unit in lineer gradient mode at a flow rate 1 mL/min through an Phenomenex Luna C8 column employing a mobile phase consisting of methanol-water (20/80, v/v) for 1 minute, increasing 100 % methanol over 5 minutes and maintained at 100% methanol for 2 minutes then reequilibrate for 2 minutes at methanol-water (20/80, v/v). API 3200 (Applied Biosystems/Sciex, Concord, ON, Canada) mass spectrometer coupled with an atmospheric pressure chemical ionization source was operated in the positive ion mode at 5 kV and 400°C source temperature. Testosterone and d 5 -testosterone were detected at the ion-transitions of m/z 289.2→109.1 and 294.2→113.2, respectively. Analyst software (version 1.4.2) was used to data acquisition, peak area integration and calculation of analyte concentrations from standard curves. Statistical analysis was carried out by EP Evaluator 8 program (RHOADS, USA).
For accuracy and linearity test, "pass" criteria is defined as mentioned in testosterone assay performance goals [9].

Discussion
Reference method for steroid measurement is isotope-dilution GC-MS. Published methods using LC-MS and GC-MS require high sample volumes [10,11]. Our study allows simple, useful analysis of low level of testosterone in women by short analysis time (10 minutes) and less sample volume (250uL).
Ogibene et al. [23] compared three non-isotopic RIA methods for determination of testosterone in adult population. Two methods give different results in the lower concentrations of dynamic range when compared with ID/GC-MS. Consistent method has limitations for measurement of testosterone in children and women.
Also, Jockenho et al. [24] reported commercially available testosterone kits give higher results at lower testosterone concentrations.
All commercial testosteron assays have a good correlation when compared with each other. However, they lack sensitivity and specifity in comparision with ID/GC-MS.
Roche Modular Systems E-170 testosterone assay has the best correlation in comparision with LC-MS/MS. We believe our method have advantages like short analysis time (10 minutes), minimum sample volume (250 uL).
Wang et al reported a similiar sample pretreatment as our study with a higher required sample volume (2 mL vs 250 uL) [7]. This method is useful for testosterone determination especially from women, children serum or late-onset hypogonadism mens' samples in clinical laboratories.
Some automated platform immunoassays are more accurate than others and the RIA is considered a more reliable immunoassay. However, the same holds for different LC-MS/MS methods. Validation between LC-MS/MS methods is necessary, preferably by comparison with a reference method.