Determination of steroid hormones by high performance liquid chromatography-tandem mass spectrometry

Summary

High performance liquid chromatography-tandem mass spectrometry (LC-MS) is a means of qualitative and quantitative analysis of samples using high performance liquid chromatography and mass spectrometry mass analysis in tandem. It can be used to (1) analyze the composition of non-volatile or polar compounds; (2) analyze and determine biological macromolecules (including proteins, peptides, polymers, etc.); and (3) accurately quantify components with extremely low levels of content down to the pico level.

Operation method

High performance liquid chromatography-tandem mass spectrometry

Principle

High Performance Liquid Chromatography - Tandem Mass Spectrometry (HPLC-MS/MS), i.e., HPLC as the means of separation and MS/MS as the means of identification and determination, are connected to form a complete instrument by means of an appropriate interface. The sample is fed through the liquid chromatography system, separated by the column, and then enters the interface. In the interface, the sample is converted from ions or molecules in the liquid phase to ions in the gas phase, which are then focused in a mass analyzer and separated according to the mass-to-charge ratio. Finally, the ion signal is converted to an electrical signal, which is detected by an electron multiplier, amplified and transmitted to a computerized data processing system.

Materials and Instruments

Food of Animal Origin
Standard Testosterone Methyltestosterone Progesterone Trenbolone
TSQ Quantum Triple Quadrupole Tandem Mass Spectrometer Electrospray Ionization Source Surveyor AS Autosampler

Move

1. Sampling: weigh about 500 g of animal muscle, liver, completely chopped, homogenized and spare; take 10 fresh eggs, shelled, homogenized and spare; take 500 mL of fresh milk, mixed and spare.

2. Extraction: 5 g of sample, placed in a 50 mL centrifuge tube, add 3 mL of 10 % sodium carbonate solution and 10 mL of ethyl acetate, homogenized for 30 s, shaking for 10 min (4 ℃), centrifuged at 6000 r/min for 10 min, and transferred the upper layer of the organic phase to a pear-shaped bottle. The extraction was repeated once more with 10 mL of ethyl acetate, and the upper organic phases were combined. It was rotary evaporated to dryness at 40 °C. The residue was dissolved with 1 mL of 50 % aqueous acetonitrile solution, the solution was frozen for 30 min, centrifuged at 16000 r/min for 5 min, and an appropriate amount of the solution was passed through a 0.22 μm filter membrane for LC/MS/MS determination.

3. Sample analysis

Caveat

1. The ion source, mass analyzer and detector of a mass spectrometer must all operate in a high vacuum to reduce background interference and avoid unwanted ion-molecule reactions. Insufficient vacuum results in insufficient sensitivity.

2. The LC-MS interface avoids non-volatile buffers, buffers containing phosphorus and chlorine, and components containing which and potassium must be ＜1mmol/1. Too high a salt content will inhibit the signal of the ion source and clog the spray needle and contaminate the instrument.

3. Choose the right voltage: when the DP voltage (de-clustering voltage) is high, the sample is decomposed or fragmented within the source, which also makes the ratio of multi-charged ions low and the polymers reduced.

Common Problems

1. The choice of ion source is crucial for the qualitative and quantitative analysis and detection of the compounds to be tested. Generally speaking, electrospray ion source is preferred for medium or large polar compounds, while atmospheric pressure chemical ionization is preferred for small polar compounds and small molecule compounds; electrospray is beneficial for the analysis of biomacromolecules or other compounds with large molecular weights.

2. Positive and negative ion mode selection: the general principle is that positive ions are suitable for alkaline samples, which can be used to acidify the sample with acetic acid or formic acid, and the positive ion mode can be preferred when the sample contains secondary ammonia or tertiary ammonia. Negative ions are suitable for acidic samples, and can be used to alkalize the sample with ammonia.

3. From Drug Chromatography, edited by Ding Li.

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