Paul H. Gamache

Analysis of phytoestrogens and polyphenols in plasma, tissue, and urine using HPLC with coulometric array detection

Abstract The study of phytoestrogens in food sources and their metabolism, effects, and mechanism of action in animals requires very selective and often sensitive analytical techniques. We have applied coulometric array detection, which uses a series of flow-through electrochemical sensors each providing 100% electrolytic efficiency, for measurement of a variety of phytochemicals in complex matrices. Recent work has involved the resolution of coumestrol (COM), daidzein (DE), daidzin (DI), diethylstil-bestrol (DES), enterodiol (ED), enterolactone (EL), equol (EQ), estradiol (E2), estriol (E3), estrone (E), genistein (GE), and quercetin (QE). Binary gradient reversed-phase (C18) chromatography was used with a sodium acetate buffer (pH 4.8)-methanol-acetonitrile solvent system. Eight coulometric sensors were set at 260, 320, 380, 440, 500, 560, 620, and 680 mV (vs Pd reference). Compounds were resolved in 30 min via both their oxidation/reduction characteristics and chromatographic behavior. Respective maximal oxidation potentials (mV) were: COM = 380; DE = 500; DI = 620; DES = 440; ED = 620; EL = 620; EQ = 560; E2 = 560; E3 = 560; E1 = 560; GE = 500; and QE = 260 with limits of detection of 5–50 pg. Uterine tissue homogenates (30 mg/ml in Tris-EDTA) and plasma from Sprague-Dawley rats sacrificed 1 hr after sc injection with either vehicle, dimethylsulfoxide, 10 μg DES, or 1.0 mg EQ were analyzed before and after enzymatic hydrolysis with β-glucuronidase/sulfatase. Urine samples from humans receiving a Boston-area diet with or without soy protein isolate supplements were also analyzed. Ethanol extracts were evaporated and reconstituted in 20% methanol before HPLC analysis. DE, ED, EL, EQ, and GE were determined in urine with less than 5% (R.S.D.) intraassay imprecision and 85%-102% recovery. Levels (ng/ml) of GE (1.8), QE (11.2), and EQ (1.7) were found in control plasma before hydrolysis and GE (293), QE (183), and EQ (22) after hydrolysis. Higher concentrations, corresponding to sc injection, in free and total EQ were found in both tissue and plasma.

Simultaneous Measurement of Monoamine, Amino Acid, and Drug Levels, Using High Performance Liquid Chromatography and Coulometric Array Technology: Application to In Vivo Microdialysis Perfusate Analysis

Abstract An automated HPLC coulometric array-ECD method is described for the simultaneous analysis of monoamines, their metabolites, derivatized amino acids, and pharmacological agents. This method has been used with in vivo microdialysis in urethane-anesthetized animals to examine extracellular fluid levels of endogenous and exogenous analytes after the peripheral administration of drugs. An aliquot of dialysate was initially analyzed for the monoamines, their metabolites and drugs by isocratic elution and detection on eight serial coulometric electrodes (0 to 490 mV; 70 mV increment). The remaining sample was then derivatized, pre-column, with OPA/sME and, after column switching, was analyzed on a parallel isocratic system with detection on four electrodes (set at 250, 450, 550 and 650 mV respectively). Compounds were identified by their retention time and electrochemical profile across the arrays. This method had a limit of detection of 0.125 pg/μl for the monoamines and 0.75 pg/μl for amino acids (bot...

Determination of tropane alkaloids by heart cutting reversed phase – Strong cation exchange two dimensional liquid chromatography

Current Chinese Pharmacopoeia (ChP) standards apply liquid extraction combined with one dimensional liquid chromatography (1DLC) method for determining alkaloids in herbal medicines. The complex pretreatments lead to a low analytical efficiency and possible component loss. In this study, a heart cutting reversed phase - strong cation exchange two dimensional liquid chromatography (RP - SCX 2DLC) approach was optimized for simultaneously quantifying tropane alkaloids (anisodine, scopolamine and hyoscyamine) in herbal medicines and herbal medicine tablets without further treatment of the filtered extract. The chromatographic conditions were systematically optimized in terms of column type, mobile phase composition and flow rate. To improve peak capacity and obtain symmetric peak shape of alkaloids, a polar group embedded C18 column combined with chaotropic salts was used in the first dimension. To remove the disturbance of non-alkaloids, achieve unique selectivity and acquire symmetric peak shape of alkaloids, an SCX column combined with phosphate buffer was used in the second dimension. Method validation was performed in terms of linearity, precision (0.54-0.82%), recovery (94.1-105.2%), limit of detection (LOD) and limit of quantification (LOQ) of the three analytes varied between 0.067-0.115mgL-1 and 0.195-0.268mgL-1, respectively. The method demonstrated superiority over 1DLC method in respect of resolution (less alkaloid co-eluted), sample preparation (no pretreatment procedure) and transfer rate (minimum component loss). The optimized RP - SCX 2DLC approach was subsequently applied to quantify target alkaloids in five herbal medicines and herbal medicine tablets from three different manufactures. The results demonstrated that the developed heart cutting RP - SCX 2DLC approach represented a new, strategically significant methodology for the quality evaluation of tropane alkaloid in related herbal medicines that involve complex chemical matrix.

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).