| During the past two decades, liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has proven to be one of the most effective tools in biomedical research, particularly for the analysis of trace level compounds in complex biological matrices because of its excellent specificity, speed, and sensitivity. However, certain chemical substances are not readily ionizable under the currently available atmospheric pressure ionization (API) techniques, making them difficult to be detected by the wide-spread LC-MS/MS method. This paper describes the development of efficient derivatization approaches to facilitate mass spectrometric analysis of poor-ionizable and/or chemically reactive compounds and the application of chemical derivatization combined with LC-MS/MS in drug metabolism research.1. Phase I clinical and pharmacokinetic study of houttuyninHouttuynin (Decanoyl acetaldehyde), a beta-dicarbonyl compound, is the major antibacterial constituent in the volatile oil of Houttuynina cordata Thunb. In the present work, the detection of houttuynin in human plasma based on the chemical derivatization with 2,4-dinitrophenylhydrazine (DNPH) coupled with liquid chromatography/tandem mass spectrometry was described. The primary reaction products between the beta-dicarbonyl compound and DNPH in aqueous phase were identified as heterocyclic structures, of which the mass spectrometric ionization and fragmentation behavior were characterized with the aid of high resolution multi-stage mass spectral analysis. For quantification, houttuynin and internal standard (IS, benzophenone) in plasma were firstly converted to their DNPH derivatives without sample purification, then extracted from human plasma with n-hexane and detected by liquid chromatography tandem mass spectrometry performed in selected reaction monitoring mode. This method allowed for a lower limit of quantification of 1.0 ng/ml using 100μL plasma. The validation results showed high accuracy (%bias<2.1) and precision (%CV<7.2) at broad linear dynamic range (1.0-5000 ng·mL-1). The simple and quantitative derivatization coupled with tandem mass spectrometric analysis facilitates a sensitive and robust method for determination of plasma houttuynin in pharmacokinetic studies. 2. Pharmacokinetic study of zofenopril and its active metabolite zofenoprilatZofenopril is a prodrug designed to undergo metabolic hydrolysis yielding the active free sulfhydryl compound zofenoprilat, which is an angiotensin converting enzyme (ACE) inhibitor, endowed also with a marked cardioprotective activity. In this study, a simple and specific derivatization in human plasma based on the sulfhydryl alkylation with 2, 4'-dibromoacetophenone (p-BPB) was employed and optimized to prevent the oxidative degradation of zofenoprilat. At the beginning of the reaction, captopril was added to the reaction system to generate its derivative as the internal standard. The reactions took place at 25℃by mixing the freshly prepared drug-containing plasma of 1 mL with 100μL of p-BPB (2 mg·mL-1) at pH 8 and maintained for 20 min. On the basis of the derivatization above, a highly sensitive and specific LC/MS/MS method was established and validated for the simultaneous determination of zofenopril and zofenoprilat in human plasma. For quantification, zofenopril, p-BPB attached zofenoprilat and p-BPB attached captopril were extracted from 200μL of the derivatized plasma with 3 mL of ether. Separation was achieved using an ODS column and isocratic elution. Excellent linearity was obtained for the entire calibration range from 0.10 to 400 ng·mL-1 for zofenopril and from 0.20 to 2000 ng·mL-1 for zofenoprilat. The intra-day and inter-day relative standard deviations were less than 14.4%. The proposed method was successfully applied to the pharmacokinetic studies of zofenopril calcium tablets in healthy subjects.3. Quantitative analysis of five major cytochrome P450 isoenzymesThe development of a novel method for absolute quantification of five most clinically relevant CYP450 isoenzymes is described based on chemical derivatization of cysteine residues. The sulfhydryl reactive reagents, 2-bromo-4'-chloroacetophenone (p-CPB) and 2-bromo-4'-bromoacetophenone (p-BPB), are proposed for use in quantitative proteomics. After reducing and denaturing, the P450s are derivatized with p-CPB for sulfhydryl alkylation then subjected to trypsin digestion. The resulted p-CPB attached peptides are enriched with phenyl-resin solid phase cartridge, then separated on a Zorbax 300SB reversed-phase column, and detected under positive electrospray ionization source in the multiple reaction monitoring mode. Quantification is achieved using p-BPB modified peptides as internal standards. Validation results demonstrated that this method showed good linearity between the concentration range of 10 fmol·μg-1 to 5 pmol·μg-1 for selected six peptides in complex matrix (rat liver microsomal protein). Intra-day and inter-day precision, expressed by relative standard deviation, were all less than 18%. Assay accuracy was within±20% as terms of relative error. The quantitative derivatization approach was proved to be reproducible, cost-effective and readily suitable for high-throughput assays. The reliability of this method for quantification of intact P450s was demonstrated through comparing with the well-applied ICAT method.
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