| Objects Bio-samples, charactered as trace detected compound in complex mass bio-matrices, are more difficult to analyse when compared to the samples of crude drugs and preparations. Moreover, detections are usually interfered by endogenous components present in the same sample, so it requires a higher sensitivity and selectivity in process of bio-analysis. For this reason, liquid chromatography /mass spectrometry (LC/MS) technique has been widely used in the field of bio-analysis. However, when it is used to detect compounds, there are still several problems. For example, some poorly- or non-ionizable compounds lead to low sensitivity. Bio-matrices could suppress the ionization of the analyte which is called matrix effect. In addition, some compounds can not retain on analytical columns, so they are easily interfered by endogenous material. An appropriate derivatization could change the structure of the analyte and therefore change its physical and chemical properties, resulting in improved performances of LC/MS, increased detection sensitivity or a detectable compound for LC/MS.Methods (1) Some HPLC derivatization reagents commonly used were chosen to react with compounds containing reactive functional group in solution, such as phenols, ketones, aldehydes and amines. Then the samples were detected with LC/MS to investigate whether the derivatization reaction happened, the detectability of derivates for LC/MS and the derivatization conditions, such as pH of the buffer, the concentration of derivatization reagent, reaction temperature. (2) To evaluate the role of the derivatization reagent in the LC/MS detection of compounds in biological samples, a comparison of the performances of chromatography and mass spectrometry of the compound before and after derivatization was made. (3) The methods of LC/MS with derivatization for the determination of ferulic acid, ethinylestradiol and 4-DMAP in biological samples were established. According to work requirements, the pharmacokinetic study of 4-DMAP in rat and Beagle dog were carried on.Results (1) The investigation of detection of ketones and aldehyde by LC/MS derivatized with toluene sulfonyl hydrazine(TSH)and dansyl hydrazine(DNSH), detection of amines by LC/MS derivatized with 4-Chloro-7-nitrobenzofurazan(NBD-Cl)and 9-fluorenylmethyl chloroformate(FMOC-Cl), and detection of phenols by LC/MS derivatized with dansyl chloride(DNS-Cl) showed that only that DNS-Cl reacted with single phenolic hydroxyl compounds in solution got successful. (2) The common derivatization conditions of single phenolic hydroxyl compounds in biological samples derivatized with dansyl chloride was that pH of the buffer was about 10, the concentration of derivatization reagent was 1 mg·mL-1, reaction temperature was 60℃, reaction time was 10 min. In LC performance, the retention time of the compounds became longer after derivatization. In MS performance, the molecular weight of the compounds increased of 233 which could avoid the interference of endogenous substances. Regardless of ESI (+) detection mode or ESI (-) detection model of compounds before derivatization, all derivatives were ESI (+) detection mode after derivatization, and sensitivity increased in different degree. (3) Typical compounds ferulic acid and ethinylestradiol were chosen to establish methods of LC/MS to determine them in biological samples. The results showed that the developed methods met the requirements for bio-analysis, and the sensitivity got enhanced in some degree. (4) According to PK requirements, the methods of LC/MS with derivatization for the determination of 4-DMAP in biological samples were established, and the pharmacokinetic study of 4-DMAP in rat and Beagle dog was carried on. The study showed that the great individual differences of pharmacokinetics of 4-DMAP were observed both in rats and dogs. After a single im (15 mg·kg-1) of 4-DMAP in rat, the T1 / 2 was 8.9±1.2min; MRT was 14.7±1.2min, Tmax was about 7.0 min. After a single iv (3.25 mg·kg-1) of 4-DMAP in Beagle dog, the T1 / 2 was 5.9±0.8min, MRT was 5.9±2.9min. After a single im (3.25 mg·kg-1)of 4-DMAP in Beagle dog, the Tmax was 8.4±3.5min, T1 / 2 was13.9±6.4min, MRT was 14.5±4.2min. Absolute bioavalability was 92.2±39.0% in Beagle dog.Conclusion (1) Dansyl chloride is a good derivatization reagent for single phenolic hydroxyl compounds in LC/MS detection, because the derivatization reaction is easy to operate, well repeatable, and the derivatives are stable, and the detection signal gets a greater enhancement. (2) The developed methods established by LC/MS with derivatization in biological samples could improve the LC performances, and increase the MS detection sensitivity. (3) The pharmacokinetic study of 4-DMAP using methods established by LC/MS with derivatization provides some data for new drug declaration.
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