ABSTRACT
AIM: Serum 7α-hydroxy-cholesten-3-one (C4) has been reported as a biomarker to assess CYP7A1 enzyme activity and bile acid synthesis. To support a clinical program, a sensitive and reliable assay without derivatization was required for the analysis of C4 in human serum. Methodology & results: A systematic approach was used to optimize mass spectrometry, LC and sample extraction conditions, therefore, significantly improved assay sensitivity, and achieved the required quantification limit without derivatization. A surrogate matrix approach was used to overcome the interference from endogenous C4. A stable isotope-labeled C4 was used as internal standard. The samples were extracted using a simple protein precipitation method with 2% formic acid in acetonitrile. CONCLUSION: A simple, fast, sensitive and robust UHPLC-MS/MS method for the quantification of 0.50 ng/ml C4 in 100 µl human serum was developed and fit for purpose validated. The method was successfully applied to the bioanalysis of C4 in a clinical study.
Subject(s)
Biomarkers/blood , Blood Chemical Analysis/methods , Cholestenones/blood , Chromatography, High Pressure Liquid , Tandem Mass Spectrometry , Blood Chemical Analysis/instrumentation , Calcifediol/chemistry , Cholestenones/standards , Cholesterol 7-alpha-Hydroxylase/metabolism , Chromatography, High Pressure Liquid/standards , Humans , Isotope Labeling , Quality Control , Reference Standards , Tandem Mass Spectrometry/standardsABSTRACT
OBJECTIVE: To establish chromatographic fingerprint of Sichuan native medicinal plant Alisma plantago-aquatica by RP-HPLC for the quality control. METHOD: The gradient elution mode was applied in chromatographic separation, and data were analyzed by "Computer Aided Similarity Evaluation" software to compare the quality of A. plantago-aquatica samples from different habitats. RESULT: The HPLC chromatographic fingerprinting of A. plantago-aquatica with 26 characteristic peaks was established from 17 lots of A. plantago-aquatica samples, peak 16 and 22 were identified as 24-acetyl alisol A and 23-acetyl alisol B, respectively. CONCLUSION: The chromatographic fingerprinting of A. plantago-aquatica with high specificity can be used to control its quality and assure lot to lot consistency. The RP-HPLC fingerprint method is repeatable, feasible in analysis of A. plantago-aquatica.
Subject(s)
Alisma/chemistry , Cholestenones/analysis , Chromatography, High Pressure Liquid/methods , Plants, Medicinal/chemistry , China , Cholestenones/standards , Drugs, Chinese Herbal/analysis , Drugs, Chinese Herbal/standards , Plant Roots/chemistry , Quality Control , Reproducibility of ResultsABSTRACT
The quality of Alismatis Rhizoma was evaluated by reversed-phase high performance liquid chromatographic method. Alisol B 23-acetate was used as a standard marker for evaluation. This component was fully separated from the other components in the plant extracts on a ODS column. Identification of alisol B 23-acetate was carried out by comparing the LC/MS spectrum of separated peak from the extract with that of standard. Alisol B 23-acetate contents in Alismatis Rhizoma obtained from several herbal markets were varied from 0.15% to 0.56%.
