Development of a CDC-certified total testosterone assay for adult and pediatric samples using LC-MS/MS.

BACKGROUND: Highly accurate and sensitive method to measure testosterone in hypogonadal male, female and children is vital for proper diagnosis of hormone-related conditions and their treatment. OBJECTIVE: 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). METHOD: A simple sample preparation method to accommodate both low and high numbers of samples was developed using simultaneous protein precipitation and derivatization with Amplifex™ Keto reagent, followed by centrifugation and direct injection of supernatant into an LC-MS/MS system (SCIEX Topaz™ IVD LC-MS/MS, in which MS is equivalent to a SCIEX 4500MD Mass Spectrometer). Total testosterone in human serum or plasma samples was quantified using an external calibration curve generated by calibrators spanning a broad concentration range of ∼1-2000 ng/dL (10-20,000 pg/mL), traceable to NIST 971 SRM. 13C3-enriched testosterone was used as an internal standard to correct for both analyte loss during sample preparation and matrix effect during analysis (Supplementary Information: SI Fig. 4C). Two methods, one using a 96-well filter plate and another using Eppendorf tubes, were developed. Both methods were certified by the Centers for Disease Control (CDC) hormone standardization (HoSt) program for total serum testosterone. The feasibility of implementing the method for plasma and serum samples was tested via a small-scale method comparison study between matched pediatric serum and plasma samples derived from the same donor. In addition, plasma samples originating from the same donor collected in two different anticoagulant tube types (Li-heparin and K2EDTA) were compared. RESULTS: Using in-house formulated NIST 971-traceable calibrators, the method was linear (r2 > 0.999) between 1 and 2000 ng/dL (10 and 20,000 pg/mL) with a limit of detection of approximately 1 ng/dL (10 pg/mL). The testosterone concentration bias against 40 reference samples from the HoSt certification program was absolute <3% with an average %CV of ∼3-4%. More than 78% of samples passed the CDC bias criterion of ±6.4%. Comparison between pediatric matched serum and plasma samples resulted in high correlation (r2 = 0.997) and bias of <5%. The calculated % difference between matched adult serum and plasma samples was ∼1%. CONCLUSIONS: Feasibility for an accurate and streamlined method suitable for measuring total testosterone in all human samples was demonstrated with a choice of sample preparation workflow to suit low or high number of samples. The method can potentially be used for plasma matrix from different blood collection tubes (Li-Heparin and K2EDTA).

A blood test for cerebrotendinous xanthomatosis with potential for disease detection in newborns.

Cerebrotendinous xanthomatosis (CTX) is a rare, difficult-to-diagnose genetic disorder of bile acid (BA) synthesis that can cause progressive neurological damage and premature death. Detection of CTX in the newborn period would be beneficial because an effective oral therapy for CTX is available to prevent disease progression. There is no suitable test to screen newborn dried bloodspots (DBS) for CTX. Blood screening for CTX is currently performed by GC-MS measurement of elevated 5α-cholestanol. We present here LC-ESI/MS/MS methodology utilizing keto derivatization with (O-(3-trimethylammonium-propyl) hydroxylamine) reagent to enable sensitive detection of ketosterol BA precursors that accumulate in CTX. The availability of isotopically enriched derivatization reagent allowed ready tagging of ketosterols to generate internal standards for isotope dilution quantification. Ketosterols were quantified and their utility as markers for CTX was compared with 5α-cholestanol. 7α,12α-Dihydroxy-4-cholesten-3-one provided the best discrimination between CTX and unaffected samples. In two CTX, newborn DBS concentrations of this ketosterol (120-214 ng/ml) were ∼10-fold higher than in unaffected newborn DBS (16.4 ± 6.0 ng/ml), such that quantification of this ketosterol provides a test with potential to screen newborn DBS for CTX. Early detection and intervention through newborn screening would greatly benefit those affected with CTX by preventing morbidity and mortality.

A sensitive and selective LC-differential mobility-mass spectrometric analysis of allopregnanolone and pregnanolone in human plasma.

A sensitive and selective method was developed to quantitate allopregnanolone and its 5ß isomer pregnanolone in human plasma using liquid chromatography-differential mobility separation combined with MS/MS detection. The method employed a simple liquid-liquid extraction of 100 µL plasma with hexane/ethyl acetate. After extraction, the sample was derivatized using a quaternary aminooxy reagent. Separation of allopregnanolone, pregnanolone, and their 3ß epimers (epiallopregnanolone and epipregnanolone) was achieved using a Phenomenex Kinetex C18 2.1 × 100-mm 2.6-µm column. A linear calibration curve was obtained over the concentration range from 10 to 25,000 pg/mL, and the inter- and intra-day accuracy of the quality control samples were between 90 and 110 % with the inter- and intra-day precision less than 10 %. The lower limit of quantitation is 50 fg (157 amol) on column for both allopregnanolone and pregnanolone which is 100-fold less than the underivatized compounds. The recovery is above 95 %, and the extracted samples are stable for at least 6 days when stored at 4 °C. Plasma samples from normal, pregnant, and postpartum women were analyzed using this method.

LC-ESI-MS/MS analysis of testosterone at sub-picogram levels using a novel derivatization reagent.

Testosterone analysis by LC-MS/MS is becoming the analytical method of choice over immunoassays due to its specificity and accuracy. However, neutral steroid hormones possess poor ionization efficiency in MS/MS, resulting in insufficient sensitivity for analyzing samples with trace concentrations of the hormones. The method presented here utilizes a derivatization step involving a novel, permanently charged, quaternary aminooxy (QAO) reagent or MS-tag that reacts to the ketone functionality of testosterone and significantly enhances its ESI-MS/MS sensitivity. This derivatization method enabled quantitation of total testosterone in human serum (200 µL) with a lower limit of quantitation (LLOQ) of 1 pg/mL (3.47 pmol/L), total testosterone in dried blood spots (8-10 µL) with a LLOQ of 40 pg/mL, and free testosterone in serum ultrafiltrate (400 µL) with a LLOQ of 0.5 pg/mL. The linearity of each of the high sensitivity applications was maintained over a broad dynamic range of 1-5000 pg/mL for the serum samples and 40-10,000 pg/mL for the dried blood spots (DBS) with R(2) >0.998. The %CV at the LLOQ was <15 for all applications. The QAO derivatization and sample preparation workflows are quick, simple, and robust. Comparison of the derivatization method with an LC-ESI-MS/MS nonderivatization method yielded high correlation and agreement. The derivatization reagent is universal and reacts with other compounds containing ketone or aldehyde functionality.