In Acworth

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 monoamines, metabolites and amino acids in brain tissue and microdialysis perfusates.

A high-performance liquid chromatographic method with coulometric array electrochemical detection is described for the simultaneous analysis of monoamines, their metabolites and o-phthalaldehyde (OPA)-derivatized amino acids. This method has been used to examine metabolite levels in both striatal tissue homogenates and striatal microdialysis perfusates. An aliquot of sample was initially analyzed for monoamines and metabolites by isocratic elution and electrochemical detection on a serial electrode array of eight coulometric flow-through graphite electrodes (0 to 490 mV; 70-mV increment). The remaining sample was derivatized pre-column with OPA-beta-mercaptoethanol and after column switching was analyzed for amino acids on a second isocratic system with electrochemical detection on four electrodes. Metabolites were then identified based on their retention time as well as electrochemical behavior across the arrays. The analysis, derivatization procedure, column switching, data reduction and peak identification were fully automated. The limit of detection for striatal tissue homogenates was approximately 1.38 ng/g wet weight for the monoamines and 8.25 ng/g wet weight for amino acids. The limit of detection for striatal perfusates was approximately 2.5 pg per 20-microliters sample for the monoamines and 15 pg per 20-microliters sample for the amino acids with analysis completed within 25 min making it ideal for microdialysis samples.

Improved method for the estimation of hydroxyl free radical levels in vivo based on liquid chromatography with electrochemical detection

Free radical damage to proteins, lipids, DNA and RNA has been thought to play an important role in many diseases as well as the aging process. One free radical, the hydroxyl free radical (HFR), is extremely reactive and is difficult to measure directly. HFRs were quantified by measuring the hydroxylation products 2,3- and 2,5-dihydroxybenzoic acids (DHBAs) formed as a result of the reaction between HFR and systemically administered salicylate (SAL). DHBAs and SAL concentrations were determined using RP-HPLC with dual coulometric electrode detection. The method has limits of detection of 1 pg for the DHBAs and 100 pg for SAL (signal-to-noise ratio 3:1). A detailed interference study as well as analyte stability and linearity studies were performed. This method was used to determine basal ratios of DHBA/SAL in a variety of tissues and to study the effects of glutamatergic and dopaminergic drugs on DHBA/SAL ratios in brain region homogenates.

Improved method for the routine analysis of acetylcholine release in vivo : quantitation in the presence and absence of esterase inhibitor

An improved high-performance liquid chromatographic (HPLC) method using electrochemical detection (ED) is described capable of routinely measuring the low levels of acetylcholine (ACh) typically found in rat brain microdialysis samples. Microdialysis was performed in the striatum of the urethane anesthetized rat using a 4-mm membrane length, high recovery (40% at 1.0 microliters/min; ambient conditions), loop-design probe perfused with an artificial cerebrospinal fluid (aCSF) solution containing physiologically normal calcium levels (1.2 mM). The HPLC method utilizes a polymeric stationary phase to resolve choline (Ch) from ACh. These analytes are then converted to hydrogen peroxide (H2O2) by a solid-phase reactor (containing immobilized choline oxidase and acetylcholinesterase enzymes). The H2O2 is detected amperometrically and quantitated on a platinum (Pt) working electrode (+300 mV; with a unique analytical cell featuring a solid-state palladium reference electrode). Two designs of the Pt working electrode were examined, differing only in the support material used (Kel-F or PEEK). The Kel-F/Pt electrode had a limit of detection (LOD) for both analytes of < 30 fmol per 10 microliters with a signal-to-noise ratio of 3:1. Striatal microdialysis perfusates were monitored for ACh and Ch over a 0-1000 nM range of neostigmine (NEO) in the CSF perfusion medium. Using the 4-mm probe, basal ACh and Ch levels were detected with a NEO level as low as 10 nM and were found to be 37 +/- 3 fmol and 22 +/- 1 pmol per 10 microliters (mean +/- S.E.M., n = 6 replicates) respectively. In similar experiments using 3-mm concentric probes comparable (lower) levels of ACh were found with the 50 and 1000 nM NEO doses (n = 4-21 animals). ACh could not be reliably quantitated when animals were perfused with the 10 nM dose of NEO (n = 4). The PEEK/Pt electrode had an improved LOD of < 20 fmol per 10 microliters due to a two- to three-fold decrease in the background noise component. Basal striatal levels of ACh in the absence of NEO approached the LOD and were found to be 15 +/- 2 fmol per 10 microliters; Ch was 5 +/- 1 pmol per 10 microliters (n = 2, mean of five basal samples). The analytical system requires very little maintenance; a simple electrochemical electrode cleaning step eliminates the need for routine polishing of the Pt electrode and the mobile phase is stable for up to one week.(ABSTRACT TRUNCATED AT 400 WORDS)

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...

Direct determination of tissue aminothiol, disulfide, and thioether levels using HPLC-ECD with a novel stable boron-doped diamond working electrode.

This chapter describes two different methods using reversed-phase HPLC with electrochemical detection on a boron-doped diamond (BDD) working electrode for the direct, routine, sensitive and simultaneous measurement of a number of aminothiols, disulfides, and thioethers, in either plasma or tissue homogenates.

Method for the Detection of Nucleosides, Bases, and Hydroxylated Adducts Using Gradient HPLC with Coulometric Array and Ultraviolet Detection

Free radicals are compounds with an unpaired electron capable of independent existence. These highly reactive species have been implicated in many disease states and can react with cell membranes, lipids, proteins, and DNA. When an oxygen radical reacts with DNA, base damage, cross-linking (e.g., DNA-DNA or DNA-protein), or DNA backbone damage (e.g., single- or double-strand breaks) can occur and often result in cell death. The field of oxidative metabolism as it relates to DNA damage has grown tremendously, with more DNA adducts being identified as biomarkers. These biomarkers are indicative of DNA damage. Measurement of these biomarkers has proved to be a challenge because of their relatively low occurrence (1 per 10(5)-10(6) bases). Methodologies for the measurement of DNA damage include thin-layer chromatography, enzyme-linked immunosorbent assay, gas chromatography-mass spectrometry, DNA sequencing, high-performance liquid chromatography (HPLC)-ultraviolet, and HPLC-ECD. HPLC-ECD (electrochemical detection) is a powerful technique that is both sensitive and selective. However, HPLC-ECD is generally not amenable to gradient analyses, so its utility is restricted. In addition, many of the bases and nucleosides are not electrochemically active. Gradient HPLC separation coupled to both a coulometric electrochemical array detector and an ultraviolet detector overcomes these limitations. Presented here is a gradient HPLC method that measures a wide variety of nucleosides, bases, and hydroxylated adducts using the inherent stability, sensitivity, and wide dynamic range of a coulometric electrochemical array detector and the universal detection qualities of an ultraviolet detector. Linear ranges, limits of detection, and detailed methods development are presented.

Novel, Universal Approach for the Measurement of Natural Products in a Variety of Botanicals and Supplements

Introduction Botanicals contain a great diversity of compounds that can show extreme variation in their physicochemical properties. Analysis of potentially active components can be challenging as not all contain a chromophore or can be ionized, thereby limiting the use of UV absorbance and mass spectrometry, respectively. HPLC with charged aerosol detection is a sensitive, universal (nonselective) approach that can measure any nonvolatile and many semi-volatile compounds. This mass-based detector is especially well-suited for the determination of any nonvolatile analyte, independent of chemical characteristics. As shown in Figure 1, the detector uses nebulization to create an aerosol. The mobile phase evaporates in the drying tube, leaving analyte particles that become charged in the mixing chamber. The charge is then measured by a highly sensitive electrometer, providing reproducible, nanogram-level sensitivity. This technology has greater sensitivity and precision than evaporative light scattering (ELS) or refractive index (RI) detection, and it is simpler to operate than a mass spectrometer (MS).

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).