How to Quantify Desired Substances Using Internal Standard Method in Liquid Chromatography-Mass Spectrometry Without Standard Samples?
In liquid chromatography-mass spectrometry (LC-MS) analysis, if there are no standards for the target substance, quantitative analysis can be performed using the internal standard method. The principle of the internal standard method is to add a compound (internal standard) to the sample that has similar chemical properties to the target substance but slightly different structure or isotopic composition, and use the response ratio between the internal standard and the target substance for quantitative analysis. The general steps for quantitative analysis using the internal standard method without standards are as follows:
1. Selection of an appropriate internal standard:
First, it is necessary to select an internal standard that has similar chemical properties to the target substance and is stable under experimental conditions. Typically, the internal standard should have a similar molecular structure, polarity, and mass spectrometric response to the target substance. The ideal internal standard is an isotopically labeled form of the target substance, such as a 13C or 15N labeled form of a known compound. If isotopically labeled internal standards are unavailable, structurally similar compounds can be chosen as internal standards.
2. Addition of the internal standard:
Before conducting the experiment, a known amount of internal standard must be added to the sample. The concentration of the internal standard should be within a similar range to that of the target substance. Additionally, ensure that the internal standard remains stable throughout the experiment to minimize errors.
3. Establishment of an internal standard calibration curve:
Due to the absence of standards for the target substance, an external calibration curve cannot be directly established. However, an internal standard calibration curve can be used for quantification. First, prepare a series of solutions with known concentrations of the internal standard. Then, perform LC-MS analysis on these solutions alongside the samples, recording the peak area or height for both the internal standard and the target substance. Finally, establish the internal standard calibration curve based on the response ratio between the internal standard and the target substance.
4. Sample analysis:
Perform LC-MS analysis on the samples treated with the internal standard, recording the mass spectrometric signals of both the internal standard and the target substance. Then, calculate the concentration of the target substance using the internal standard calibration curve.
It should be noted that the accuracy of the internal standard method is affected by the choice of internal standard, the stability of the internal standard, and experimental conditions. Therefore, in practice, an appropriate internal standard should be selected, and experimental conditions should be optimized to improve the accuracy of quantitative analysis. Additionally, to verify the reliability of the internal standard method, it is advisable to include quality control samples in the experiment or use other independent methods for cross-validation.
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