Selection and application of internal standard for LC-MS in biological analysis


biological analysis

When processing and analyzing biological samples, the loss of analyte and signal fluctuation often occur. These situations may occur in the process of sample transfer, adsorption, evaporation and injection, especially in the process of mass spectrometry detection. Matrix effects can cause changes in the detection signal of the analyte. By adding equal amounts of internal standards with similar physicochemical properties to the analyte to all samples in the same analytical batch, and using the ratio of the response values ​​of the analyte to the internal standard to quantify According to calculations, the signal fluctuations and quantitative losses of most analytes can be corrected, so the use of appropriate internal standards can greatly improve the accuracy and precision of analytical results and the reliability of the method.

1. How to choose a suitable internal standard and what are the requirements?
Internal standards are generally divided into two types, structural analog internal standards and isotope-labeled internal standards.

A. Structural analog internal standard
① The internal standard of structural analog preferably has the same key chemical structure and functional group (such as -COOH.-SO2.-NH2, etc.) as the analyte. The difference is limited to the C-H part. If the key chemical structure and functional group are different, it will lead to ionization efficiency. and recovery rate difference.
② The internal standard of the analog should not be the same as any metabolite of the analyte in the body to avoid affecting the test results.

B. Isotope Labeled Internal Standard (SIL-IS)
① 13C and 15N-labeled internal standards are better than deuterium (2H/D)-labeled internal standards.
② When selecting a deuterated internal standard, pay attention to the position of deuterium atom substitution, and should choose a position to replace a stable functional group structure (such as -CH2-CD3), rather than a position with more active chemical properties (such as -CD2-(COOH)2 ) to ensure that no deuterium-hydrogen exchange occurs during sample preparation.
③ In order to reduce the influence of isotope interference, the isotope internal standard is preferably at least 4-5 mass units higher than the unlabeled analyte.

Under the premise that the experiment requires the precision, accuracy and stability of the method, the isotope-labeled internal standard should be used as much as possible, and compared with the analog internal standard, SIL-IS can broaden the linear range of the method.

2. What should be paid attention to when using deuterated isotope internal standard?
Although SIL-IS has many advantages and good performance compared to analog internal standards, it does not solve all the problems faced in all biological analysis. When you encounter the following situations, you need to pay attention.
For the deuterated internal standard, due to the differences in physicochemical properties between the deuterated compounds substituted by some groups and the compounds, there will be slight differences in peak time, differences in recovery in the same extraction step, and The exchange reaction between hydrogen atoms, especially the substitution of deuterium atoms similar to the adjacent ketone group (-CD2-CHO), will lead to a large loss of deuterium atoms due to enol interconversion, which will lead to systematic errors in the detection results.

3. How to determine the concentration of the internal standard?
The first factor to consider is the size of the cross signal between the internal standard and the analyte, which is mainly due to chemical impurities or isotopic interference in the standard. When the analyte has a cross contribution to the signal of the internal standard, the As the internal standard concentration decreases, the standard curve will become more and more non-linear, and inappropriate internal standard concentration can lead to significant systematic errors during analysis. When the internal standard concentration is 5% of the analyte’s upper limit of quantification, The curve is curved.

According to regulatory guidelines and acceptance criteria, the usual acceptance criteria are that the signal contribution of the internal standard to the analyte does not exceed 20% of the lower limit of quantitation response, and the signal response of the analyte to the internal standard does not exceed 5% of the internal standard response.
Therefore, in general, for the internal standard and the analyte without interference and cross-signal, the internal standard concentration is recommended to be above 40% ULOQ. For the interference between the internal standard and the analyte within the acceptable range, the The standard concentration is recommended to be above 70% ULOQ concentration.

The second factor is the detection sensitivity of mass spectrometry to the analyte and internal standard. When the sensitivity of the internal standard is high, its concentration can be reduced. Otherwise, a high concentration of internal standard should be used to ensure a sufficient signal-to-noise ratio (S/N) to Reduces the effect of random noise in the internal standard response.

The third factor is the influence of the matrix effect. Because of the matrix effect, the closer the chromatographic response of the internal standard and the analyte is, the more the internal standard can simulate the actual situation that the analyte is affected by the matrix effect, so as to better offset the effect of the matrix effect. influences. Generally speaking, the concentration range of 1/3 to 2/3 of a qualified standard curve is very important, which can basically cover the actual sample concentration to be detected. Therefore, when selecting the internal standard concentration, it is necessary to refer to the The ratio is close to the compound concentration of the actual sample to be analyzed.

For some special experimental projects, when the analyte signal will be inhibited by the co-eluting internal standard, a low concentration internal standard should be used to maintain the detection sensitivity of the analyte, and when the internal standard is inhibited by the analyte, a high concentration should be selected. concentration of the internal standard for good reproducibility.