The application of chemical derivatization technology in biological analysis |

In general, compounds suitable for LC-MS analysis need to have the following characteristics: good chemical stability, easy ionization (ESI, APCI), high abundance, good selectivity of product ions, and moderate polarity, with suitable retention. Biological analysis often encounters some poor chemical stability, containing unstable groups such as sulfhydryl (-SH), large polarity, poor chromatographic retention, low ionization efficiency, poor instrument sensitivity, serious residues, or difficult to separate interference. In this case, it may be difficult to use conventional liquid-mass combination, so chemical derivatization can be tried.

Chemical derivatization is a technology that converts a compound into a substance of similar chemical structure by using the basic organic functional groups in the chemical structure. Chemical derivatization technology has the following advantages: (1) For difficult compounds, it can improve the sensitivity and accuracy of the instrument; (2) For compounds that are easily vaporized and thermally unstable, their volatility can be reduced and their thermal stability improved; (3) For mixtures that are difficult to separate, one of the components can be transformed through derivatization technology, while keeping other components unchanged, so that the component can be smoothly separated and the separation performance can be improved.

In the chemical derivatization method, the selection of derivatization reagent is the core problem, which needs to consider the following factors: (1) Derivatization reagent needs to contain the reaction functional group with obvious characteristics or introduce the easily ionized group, so as to improve the mass spectrum response and improve the sensitivity; (2) The derivated reagent must be chemically stable, not easily decomposed, and can react exclusively with the specific functional group of the substance to be tested; (3) The derivatization reagent itself and the generated by-products have little interference with the instrument; (4) The derivatization reaction conditions are mild and the reproducibility is good; (5) derivatization reagents should be non-toxic and harmless, and will not cause pollution to the environment.

At present, we have several projects applying derivatization techniques to bioanalysis. For the detection of 25-hydroxyvitamin D3 in plasma, Cookson reagent is used to introduce a large number of high proton affinity elements (O, N) to improve the ionization efficiency and improve the separation degree of endogenous interfering substances from the tested substance, so as to achieve accurate detection; By introducing easily ionized quaternary ammonium groups with Girard T reagent, the sensitivity can be increased by nearly 10 times, and the chromatographic separation can be improved by the reaction of characteristic groups. The stability of compounds containing sulfhydryl groups can be improved by masking their unstable groups.

The key factors for the success of derivatization are as follows: (1) Conduct sufficient preliminary evaluation according to the structural characteristics, metabolic conditions and biological matrix types of metabolites; (2) Select the appropriate derivatization reagent; (3) Conduct scientific experimental design: fully evaluate the reaction system, temperature, duration, etc.; (4) Select appropriate internal standard substances, extraction conditions and separation conditions, and establish a robust, reproducible and easy to operate method.