A non-derivative method for the quantitative analysis of isosteroidal alkaloids from Fritillaria by high performance liquid chromatography combined with charged aerosol detection.

A non-derivative method was developed for the qualitative and quantitative analysis of isosteroidal alkaloids from Fritillaria thunbergii. During method development the performance of two universal detectors, the charged aerosol detector (CAD) and evaporative light scattering detector (ELSD), were evaluated. The CAD was found to be 30 to 55 times more sensitive than ELSD enabling the measurement of low levels of reference compound impurities that could not be detected by ELSD. The peak area percent of the reference compound, peimisine, obtained by CAD was 50.10%, but 91.66% by ELSD showing that CAD is suitable to estimate the presence of impurities. The CAD showed good reproducibility with overall intra- and inter-day peak area RSD values of less than 1.8% and 2.7%, respectively and had a linear dynamic range of up to 4 orders of magnitude (0.06-44mg/L) for peimine and peiminine. The optimized method was used for the quantitative analysis of peimine and peiminine from F. thunbergii.

Determination of oxidized and reduced CoQ10 and CoQ9 in human plasma/serum using HPLC-ECD.

This chapter describes the use of reversed-phase HPLC with multichannel coulometric electrochemical detection for the routine, sensitive, and simultaneous measurement of oxidized and reduced CoQ10 and CoQ9 in human plasma and serum. Analytes are first resolved chromatographically prior to electrochemical detection using three serially placed flow-through coulometric sensors set for oxidation-reduction-re-oxidation. Such electrochemical manipulation of analytes not only improves selectivity and specificity (decreasing the likelihood of co-elution), but also leads to improved sensitivity and decreased noise. The method is completed in ,18 min, shows excellent linearity, good intra-day (% RSD = 1.2-2.3) and inter-day (% RSD 2.2-3.9) precision, and has a limit of detection to low pg levels (on column). This approach was used to measure oxidized and reduced CoQ10 and CoQ9 in 30 human plasma samples, and oxidized and reduced CoQ10 in 10 human serum samples (NIST Micronutrients Measurement Quality Assurance Program for fat-soluble vitamins).

Metabolomic applications of electrochemistry/mass spectrometry.

Analytical techniques used for multivariate analysis of endogenous metabolites in biological systems (e.g., metabolomics, metabonomics) must be capable of accurately and selectively monitoring many known and unknown molecules that span a diverse chemical spectrum and over extremely large dynamic concentration ranges. Mass spectrometric (MS) and electrochemical array (EC-Array) detection have been widely used for multi-component analysis with applicability to low-level (fmol) metabolites. Described here are practical considerations and results obtained with the combined use of EC-Array and MS for HPLC-based multivariate metabolomic analysis. Data presented include the study of changes in rat urinary metabolite profiles associated with xenobiotic toxin exposure analyzed by HPLC using water:acetonitrile binary gradient conditions and post-column flow splitting between EC-Array and MS detectors. Results show complementary quantitative and qualitative analysis and the ability to differentiate sample groups consistent with xenobiotic-induced histopathological changes. The potential applicability of this hyphenated technique for biomarker elucidation through measurement of redox active compounds that are commonly associated with disease pathology and xenobiotic toxicity is discussed. The use of EC reactor cells in series with MS is also presented as a means of producing likely metabolites to facilitate structural elucidation and confirmation.