Determination of endogenous steroid hormones in urine by liquid chromatography-tandem mass spectrometry

Summary

This assay is mainly used for routine analysis of endogenous steroid hormone stimulants in body fluids.

Operation method

Liquid chromatography-tandem mass spectrometry

Principle

The urine samples were digested by glucuronidase and then extracted with liquid-liquid extraction. The separation was performed on a Cosmosil C18 column using methanol-0.1% formic acid buffer (containing 0.02 mol/L ammonium acetate) (68∶32, v/v) as the mobile phase, and the determination of five hormones, namely, dehydroepiandrosterone (DHEA), testosterone, epididymis, androstenedione, and phenylcholanolone, and phenylchondroitolone, was performed by a triple quadrupole mass spectrometer (Triple QMMS) with multi-reaction monitoring scanning mode. androstenedione, testosterone, epitestosterone, androstenedione and phenylcholanolone in urine samples.

Materials and Instruments

Urine Sample
Testosterone Epitestosterone Androsterone Phenylcholanolone DHEA Internal Standard Methyltestosterone Standard Methanol Formic Acid Ammonium Acetate Phosphate Buffer β-Glucuronidase Ether
Triple quadrupole tandem mass spectrometer Liquid chromatography system Constant temperature water bath

Move

1.Sample collection

There were 6 healthy volunteers, including 3 males and 3 females. Blank urine was collected before the volunteers were subjected to the test; after 120 mg DHEA tablets were orally administered, the urine was collected from 0 to 24 h and stored at -20℃.

2. Sample processing

Take 210 mL of urine in a test tube, add 5 ng of methyltestosterone (internal standard) and 210 mL of phosphate buffer pH 6.8, 150 μL of β-glucuronidase (3 000 U), incubate in a thermostatic water bath at 80 ℃ for 3 h. Remove the sample and cool it to room temperature. Add 3.5 mL of ether, mix and spin, centrifuge, remove the organic phase and evaporate in a 60 ℃ water bath, and add 100 μL of mobile phase to the residue for LC-MS/MS analysis.

3. Chromatographic and mass spectrometric conditions

Liquid chromatography: A Cosmosil C18 column (150 mm×2 mm, 5 μm) and a Phenom enex C18 pre-protected column were used. The mobile phase was methanol-0.1% formic acid buffer (containing 0.02 mol/L ammonium acetate) (68∶32, v/v) at a flow rate of 200 μL/min, and the injection volume was 5 μL.

Tandem mass spectrometry (TMS): an electrospray ionization source ( ES I ) was used and scanned in multiple reaction monitoring (MRM) positive ion mode. The pressure of collision gas (CAD ) was 48 263 Pa; the pressure of curtain gas (CUR) was 68 948 Pa; the pressure of nebulizing gas (GS1) was 241 317 Pa; and the pressure of auxiliary heating gas (GS2) was 275 790 Pa. The spray voltage was 5 500 V; and the ionization temperature ( TEM ) was 500 ℃.

Two groups of characteristic parent/daughter ion pairs of each target were selected, and their optimal collision voltages (CE) and de-clustering voltages (D P ) were optimized, respectively. The more abundant parent/daughter ion pair was used for quantitative analysis.

Caveat

1. The optimization of liquid chromatographic separation conditions is the key to this study. Testosterone and epitestosterone, androstenedione and phenylcholanolone are isomers, with the same parent ion and daughter ion, which must be separated by chromatographic methods.

2. Three kinds of samples with low, medium and high concentrations were selected for each target, and the recoveries were obtained by comparing the results of the three kinds of samples with those of the corresponding standard solutions, and the precision and accuracy of the method were investigated.

Common Problems

Source: Determination of endogenous steroid hormones in urine by liquid chromatography-tandem mass spectrometry.

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