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Data to support a simultaneous testosterone and estradiol assay in serum by LC-MS/MS.

ABSTRACT: A very sensitive LC-MS/MS assay was developed implementing a liquid-liquid extraction step followed by mass spectrometry which was operated in both positive and negative ion modes. The assay was calibrated with readily available commercial calibrators and compared with international reference standards. This data is also presented in "Sensitive Simultaneous Quantitation of Testosterone and Estradiol in Serum by LC-MS/MS without Derivatization and Comparison with the CDC HoSt Program" (Schofield et al., 2017). This article includes the comparison of the LC-MS/MS assay with a commonly available chemiluminescencent immunoassay for the quantitation of both estradiol and testosterone. In addition we show baseline separation of estradiol and testosterone from other structurally related and/or isobaric compounds that could potentially interfere with the assay. In addition, various calibrator materials were tested and compared with internationally-recognized reference materials.

SUBMITTER: Schofield RC

PROVIDER: S-EPMC6141700 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Data to support a simultaneous testosterone and estradiol assay in serum by LC-MS/MS.

Schofield Ryan C RC Ramanathan Lakshmi V LV Pessin Melissa S MS Carlow Dean C DC

Data in brief 20180830

A very sensitive LC-MS/MS assay was developed implementing a liquid-liquid extraction step followed by mass spectrometry which was operated in both positive and negative ion modes. The assay was calibrated with readily available commercial calibrators and compared with international reference standards. This data is also presented in "Sensitive Simultaneous Quantitation of Testosterone and Estradiol in Serum by LC-MS/MS without Derivatization and Comparison with the CDC HoSt Program" (Schofield ...[more]

PMID: 30246107

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Project description:BackgroundHighly 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.ObjectiveTo 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).MethodA 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.ResultsUsing 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%.ConclusionsFeasibility 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).

Dataset Information

Data to support a simultaneous testosterone and estradiol assay in serum by LC-MS/MS.

Publications

Data to support a simultaneous testosterone and estradiol assay in serum by LC-MS/MS.

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