Detection of the isoflavone aglycones genistein and daidzein in urine using solid-phase microextraction–high-performance liquid chromatography–electrospray ionization mass spectrometry

An improved method of detection of the isoflavone aglycones, genistein and daidzein, is reported using solid-phase microextraction–high-performance liquid chromatography–electrospray ionization mass spectrometry (SPME–HPLC–ESI-MS). Extraction of the isoflavonoids from urine using SPME with a Carbowax–templated resin fiber coating allows rapid preconcentration of the analytes without the usual sample preparation required by other methods. Detection of the analytes is accomplished by HPLC–ESI-MS. Analysis of spiked samples of urine resulted in a linear range of 0.25 to 250 ng/ml for daidzein and 0.27 to 27.0 ng/ml for genistein. Limits of detection of daidzein and genistein were measured at 25.4 pg/ml for daidzein and 2.70 pg/ml for genistein. Daidzein and genistein were detected in urine following consumption of a soy drink.     

A comparison of thermospray and direct liquid introduction high-performance liquid chromatography/mass spectrometry for the analysis of candidate antimalarials

The analysis of antimalarials by high-performance liquid chromatography (HPLC)/mass spectrometry demonstrates a new dimension in specificity along with increased sensitivity compared to conventional HPLC detection methods. Both direct liquid introduction and thermospray HPLC/mass spectrometry interfaces provided molecular weight information as well as characteristic fragment ions for antimalarials not normally amenable to direct probe or gas chromatographic/mass spectrometric techniques. The direct liquid introduction interface, which incorporated a 1/100 split, showed a detection limit of 30 ng using selected ion monitoring. The thermospray technique showed less than 1 ng detection limits using selected ion monitoring.     

Measurement of intact sulfate and glucuronide phytoestrogen conjugates in human urine using isotope dilution liquid chromatography-tandem mass spectrometry with [13C(3)]isoflavone internal standards

A method has been developed for the analysis of phytoestrogens and their conjugates in human urine using liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Stable isotopically labeled [13C(3)]daidzein and [13C(3)]genistein were synthesized and used as internal standards for isotope dilution mass spectrometry. Free aglycons and intact glucuronide, sulfate, diglucuronide, disulfate, and mixed sulfoglucuronide conjugates of isoflavones and lignans were observed in naturally incurred urine samples. Sample pretreatment was not necessary, other than addition of internal standards and pH adjustment. Urine was injected directly onto the analytical column. The limits of detection were generally <50ng/ml, precision was generally <10% CV for conjugates. Total hydrolyzed daidzein and genistein were measured against quality assurance urine sample and were accurate to within 12%. The accuracy of conjugate measurement can not be ascertained, as no reference samples are available. The mean sum of daidzein and its conjugates was within 20% of the hydrolyzed value. Concentrations of the free aglycons of up to 22% of genistein and 18% of daidzein were observed. The average pattern was ca. 54% 7-glucuronide, 25% 4(')-glucuronide, 13% monosulfates, 7% free daidzein, 0.9% sulfoglucuronides, 0.4% diglucuronide, and <0.1% disulfate. Selective enzymatic deconjugation with glucuronidase and mixed glucuronidase/sulfatase were used to validate the accuracy of the quantitation of the intact daidzein conjugates. There were no apparent sex differences, or conditioning effects on the conjugation profile of isoflavones after chronic dosing.     

Analysis of lignans from Phyllanthus niruri L. in plasma using a simple HPLC method with fluorescence detection and its application in a pharmacokinetic study

A simple analytical method using HPLC with fluorescence detection was developed for the simultaneous determination of four lignans, phyllanthin (1), hypophyllanthin (2), phyltetralin (3) and niranthin (4) from Phyllanthus niruri L. in plasma. The method recorded limits of detection for 1, 2, 3 and 4 as 1.22, 6.02, 0.61 and 1.22 ng/ml, respectively, at a signal-to-noise ratio of 5:1 whereas their limits of quantification were 4.88, 24.41, 4.88 and 9.76 ng/ml, respectively, at a signal-to-noise ratio of 12:1. These values were comparable to those of other sensitive methods such as gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography-MS (HPLC-MS) and HPLC-electrochemical detection (HPLC-ECD) for the analysis of plasma lignans. A further advantage over known methods was its simple protocol for sample preparation. The within-day and between-day accuracies for the analysis of the four lignans were between 87.69 and 110.07% with precision values below 10.51%. Their mean recoveries from extraction were between 91.39 and 114.67%. The method was successfully applied in the pharmacokinetic study of lignans in rats. Following intravenous administration, the lignans were eliminated slowly from the body with a mean clearance of 0.04, 0.01, 0.03 and 0.02 l/kg h and a mean half-life of 3.56, 3.87, 3.35 and 4.40 h for 1, 2, 3 and 4, respectively. Their peak plasma concentration upon oral administration was 0.18, 0.56, 0.12 and 0.62 microg/ml, respectively, after 1h. However, their absorption was incomplete with a calculated absolute oral bioavailability of 0.62, 1.52, 4.01 and 2.66% for 1, 2, 3 and 4, respectively.     

Atmospheric pressure chemical ionization-mass spectrometry method to improve the determination of dansylated polyamines

Determination of polyamine pools is still a step impossible to circumvent in studies aimed at determining the pathophysiological role of natural polyamines. In addition, polyamine measurement in biological fluids and tissues may have clinical relevance, especially in cancer patients. Among the wide panel of analytical methods developed for the quantification of polyamines, high-performance liquid chromatographic (HPLC) separation of polyamines after derivatization with dansyl chloride remains the most commonly used method. In this work, we show that atmospheric pressure chemical ionization-mass spectrometry (MS) can be used to detect and quantify biologically relevant polyamines after dansylation, without chromatographic separation. Positive-ion mass spectra for each dansylated polyamine were generated after optimization by flow injection analysis (FIA). FIA coupled with MS detection by selected ion monitoring greatly increased the sensitivity of the polyamine detection. The method is linear over a wide range of polyamine concentrations and allows detection of quantities as low as 5 fmol. The FIA/MS method is about 50-fold more sensitive than the conventional HPLC/fluorimetry procedure. A good correlation (r>0.98) between these two methods was observed. The FIA/MS method notably reduces the time of analysis per sample to 1.5 min and turns out to be rapid, efficient, cost saving, reproducible, and sufficiently simple to allow its routine application.     

High-performance liquid chromatographic methods for the determination of topoisomerase II inhibitors

Various methods for separating eleven different types of topoisomerase II (TOPO-2) inhibitors, including epipodophyllotoxins, anthracyclines, anthracenediones, anthrapyrazoles, anthracenebishydrazones, indole derivatives, aminoacridines, benzisoquinolinediones, isoflavones, bisdioxopiperazines and thiobarbituric acids, are summarized. Proper sample preparation and storage is critical to the successful analysis of some TOPO-2 inhibitors due to difficulties associated with adsorption, instability and complex biological components. Solid-phase and liquid–liquid extractions are widely used to separate TOPO-2 inhibitors from biological samples, although simple deproteinization followed by direct analysis of the supernatant is preferable to extraction based on its speed and simplicity. High-performance liquid chromatography (HPLC) is the favored method for the bioanalysis of TOPO-2 inhibitors. UV or diode array detection is generally employed for early pharmacokinetic studies, while fluorescence or electrochemical detection is used more frequently for analytes with fluorescent or oxidative–reductive properties. For analyses requiring highly sensitive and/or specific detection, electrospray mass spectrometry (ESI-MS or ESI-MS–MS) provides a suitable alternative. A comprehensive compilation of the HPLC techniques currently used to separate TOPO-2 inhibitors will aid the future development of analytical methods for new TOPO-2 inhibitors.     

Sensitive, quantitative, and fast modifications for Coomassie Blue staining of polyacrylamide gels

In spite of the high sensitivity of silver staining and the wide dynamic range of various fluorescent detection methods, Coomassie Brilliant Blue staining is still the most widely used protein detection technique for proteins separated by polyacrylamide gel electrophoresis. There are several reasons: Low price, Visible with the eye, Desk top scanners can be employed for image acquisition, Better for quantitative analysis than silver staining, Possible modifications for fast or highly sensitive staining, Mass spectrometry compatible.     

Commentary the Measurement of Oxidative Damage to DNA by HPLC and GC/MS Techniques

Oxidative damage to DNA has been measured by quantitating 8-hydroxy-2′-deoxyguanosine (8-OHdGuo) after enzymic digestion of DNA, followed by HPLC separation and electrochemical detection. Alternatively, 8-hydroxyguanine (and a wide range of other base-derived products of free radical attack) may be measured after acidic hydrolysis of DNA or chromatin, followed by derivatization and gas-chromatography/mass spectrometry. Both techniques have comparable sensitivity, but GC/MS enables determination of a wide variety of chemical changes to all four DNA bases and it can be applied to DNA-protein complexes. However, the two techniques do not always give similar results. Potential reasons for this are discussed. Greater attention to methodological questions is required before using measurement of 8-OHdGuo as a “routine” marker of oxidative DNA damage in vivo.     

Development and validation of a RP-HPLC method for quantification of isoflavone aglycones in hydrolyzed soy dry extracts

Isoflavones are widely used as an alternative treatment to hormone replacement therapy and also for prevention of several chronic diseases, including cancers. Genistein, daidzein and glycitein are the most abundant isoflavone aglycones found in soy extracts, where they also occur as glycosides. This paper describes the development and validation of an isocratic reversed-phase HPLC (RP-HPLC) method for the analysis of isoflavone aglycones, released after acid hydrolysis of soy dry extracts, used as pharmaceutical raw material. The quantification was carried out in a C(18) endcapped column, using a mobile phase composed of 0.1% acetic acid and methanol (52:48), at a flow rate of 1.0 ml/min and diode array detection (DAD) at 254 nm. The method showed to be linear (r(2)>0.99), precise (R.S.D.<2%), accurate (recovery of 98.88% for daidzein and 98.12% for genistein), robust and specific.     

Simultaneous determination of cytotoxic bufadienolides in the Chinese medicine ChanSu by high-performance liquid chromatography coupled with photodiode array and mass spectrometry detections

ChanSu (toad venom) is a traditional Chinese medicine for the treatment of serious liver and gastric cancers. The major cytotoxic compounds in ChanSu are bufadienolides. In this paper, a strategy combining qualitative LC/MS analysis and quantitative HPLC determination of major bufadienolides was used for global quality control of ChanSu crude drug. Majority of the bufadienolides in methanol extract of ChanSu were unambiguously characterized by high-performance liquid chromatography coupled with atmospheric pressure chemical ionization tandem mass spectrometry (HPLC/APCI-MS/MS), and by comparing with pure compounds. In addition, eight major bufadienolides were simultaneously determined in one single HPLC run within 30 min with photodiode array detection (DAD). All compounds showed good linearity in a wide concentration range, and their limits of detection (LOD) were around 1 ng. Thus, > 95% of the bufadienolides in ChanSu could be characterized, and > 90% of them were quantitated. The established method is rapid, simple and sensitive, and could be used for the routine analysis of ChanSu crude drug and its preparations. This research sets a good example for the comprehensive quality control of traditional medicine.     

The electroanalytical approach to lipid peroxide determinations

An electroanalytical method for the determination of lipid peroxides in physiological material is described. The technique is based on electrochemical detection for HPLC as the means for enhancing sensitivity. Samples containing organic peroxides, including lipid peroxides, can be analyzed directly using a modified polarographic detector (Lloyd, J.B.F.; Optimization of the operational parameters of the supported mercury drop electrode detector in high performance liquid chromatography. Anal. Chim. Acta 154:121-131; 1983.) for reversed phase HPLC determinations. Detection limits for fatty acid hydroperoxides were found to be in the low nanogram range.     

A novel, sensitive method for determining benzo[a]pyrene-diones using high-performance liquid chromatography with post-column zinc reduction

A novel and sensitive high-performance liquid chromatography (HPLC) method was developed to analyze dione metabolites of benzo[a]pyrene (BaP). Because BaP-diones do not fluoresce, detection of low concentrations is difficult to achieve when analyzing these chemicals with a simple HPLC system. We developed a method to increase the detection sensitivities for BaP-diones using reduction by zinc after the chromatographic separation. A post-column zinc reducer was used to convert BaP-diones, in-line, to their corresponding fluorescent BaP-hydroquinones, which can be measured by fluorescence detection with high sensitivity. With 20-muL injections, the limits of detection for the BaP-diones tested (BaP-1,6-dione, BaP-3,6-dione, and BaP-6,12-dione) were all below 1.0 nM. In addition to the high detection sensitivity, this HPLC method provides a wide linear dynamic range for BaP-dione detection (1.0-220 nM). We also studied the extraction recovery of BaP-diones from recombinant human cytochrome P450 and epoxide hydrolase. To demonstrate the application of this method, the kinetics of BaP-dione formation was studied by incubating BaP with these recombinant enzymes. The present method enhances the detection sensitivity for BaP-diones by more than two orders of magnitude compared with traditional ultraviolet detection. Moreover, the method avoids the time-consuming derivatization or reduction steps required by other methods.     

Analysis of mutagenic imidazo[4,5-f]quinolines and -quinoxalines (IQ compounds). Comparison of electrochemical and ultraviolet detection

Two synthetic imidazoquinolin-2-amines (IQ and MeIQ) and two imidazoquinoxalin-2-amines (MeIQx and 4,8-DiMeIQx), all known potent mutagens, have been separated by reversed-phase HPLC and detected by two methods - UV detection and electrochemical (EC) detection. The limits of detection were found to be 2.5 pmoles for UV detection and 0.5-1.5 pmoles for electrochemical detection.     

Depletion of multiple high-abundance proteins improves protein profiling capacities of human serum and plasma

Systematic detection of low-abundance proteins in human blood that may be putative disease biomarkers is complicated by an extremely wide range of protein abundances. Hence, depletion of major proteins is one potential strategy for enhancing detection sensitivity in serum or plasma. This study compared a recently commercialized HPLC column containing antibodies to six of the most abundant blood proteins ("Top-6 depletion") with either older Cibacron blue/Protein A or G depletion methods or no depletion. In addition, a prototype spin column version of the HPLC column and an alternative prototype two antibody spin column were evaluated. The HPLC polyclonal antibody column and its spin column version are very promising methods for substantially simplifying human serum or plasma samples. These columns show the lowest nonspecific binding of the depletion methods tested. In contrast other affinity methods, particularly dye-based resins, yielded many proteins in the bound fractions in addition to the targeted proteins. Depletion of six abundant proteins removed about 85% of the total protein from human serum or plasma, and this enabled 10- to 20-fold higher amounts of depleted serum or plasma samples to be applied to 2-D gels or alternative protein profiling methods such as protein array pixelation. However, the number of new spots detected on 2-D gels was modest, and most newly visualized spots were minor forms of relatively abundant proteins. The inability to detect low-abundance proteins near expected 2-D staining limits was probably due to both the highly heterogeneous nature of most plasma or serum proteins and masking of many low-abundance proteins by the next series of most abundant proteins. Hence, non2-D methods such as protein array pixelation are more promising strategies for detecting lower abundance proteins after depleting the six abundant proteins.     

High throughput quantification of phytoestrogens in human urine and serum using liquid chromatography/tandem mass spectrometry (LC-MS/MS)

Phytoestrogens are currently the subject of intense study owing to their potential protective effects against a number of complex diseases. However, in order to investigate the interactions between phytoestrogens and disease state effectively, it is necessary to have analytical methods which are sensitive, reproducible, and require low sample volumes. We report an assay for three isoflavones (daidzein, genistein, and glycitein), two metabolites of daidzein (equol and O-desmethylangolensin), three lignans (secoisolariciresinol, enterodiol, and enterolactone), and one flavanone (naringenin) in human urine and serum. A high throughput of samples has been achieved via the use of 96-well plate sample extraction and liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis incorporating column switching, thus making the assay suitable for use on large sample numbers, such as those found in epidemiological studies. The robustness of the assay was proven via the comparison of data generated on two different LC-MS/MS systems, with and without column switching.     

Separation methods used in the determination of choline and acetylcholine

Cholinergic neurotransmission has been the subject of intensive investigations in recent years due to increasing recognition of the importance of its roles in physiology, pathology and pharmacology. The fact that the disposition of a neurotransmitter may reflect its functional status has made the measurement of acetylcholine and/or its precursors and metabolites in biological fluids an integral part of cholinergic research. With evolving complexity in experimental approaches and designs, and correspondingly increasing demand on sensitivity, specificity and accuracy matching advancements in sophistication in analytical methods have been made. The present review attempts to survey the array of analytical techniques that have been adopted for the measurement of acetylcholine or its main precursor/metabolite choline ranging from simple bioassays, radioenzymatic assays, gas chromatography (GC) with flame ionization detection, GC with mass spectrometry (GC–MS) detection, high-performance liquid chromatography (HPLC) with electrochemical detection (ED), HPLC with MS (HPLC–MS) to the sophisticated combination of micro-immobilized enzymatic reactor, microbore HPLC and modified electrode technology for the detection of ultra-low levels with particular emphasis on the state of the art techniques.     

Advances in techniques for ecdysteroid analysis

Summary

Recent improvements in chromatographic methods for ecdysteroids are reviewed. Minor improvements in liquid chromatography have increased the separation and range of compounds which can be accomodated, some useful techniques have been introduced in thin layer chromatography, and with new radioactivity scanners and direct mass spectrometry from TLC, this technique retains an important place among the tools of the ecdysonist. Supercritical fluid chromatography has been suggested as a rapid and more sensitive alternative to liquid chromatography. In mass spectrometry, fast atom bombardment has proved most useful, though coupling HPLC through thermospray and ptasmaspray methods has been attempted, but as yet, these techniques lack sensitivity. New solid phase immunoassay methods using colorimetric rather than radioactivity measurements promise to give a better means of analysis of small amounts of ecdysteroids, capable of use in the presence of radiolabeled compounds. A solid phase separation of C-20, 22 diol ecdysteroids with immobilized phenylboronic acid has been reported.     

HPLC study of oxidation products of hydroethidine in chemical and biological systems: ramifications in superoxide measurements

Methods for the detection and quantitation of hydroethidine (HE) and its oxidation products by HPLC analysis are described. Synthetic methods for preparation of authentic standards (2-hydroxyethidium and diethidium) are provided. Potential applications of the HPLC methods to chemical and biological systems are discussed. Specific examples of chromatograms obtained using UV–Vis absorption, fluorescence, electrochemical, and mass spectrometry detectors are provided. The development of a dual electrochemical and fluorescence detection methodology and its applications are described. The HPLC-based method enables analyses of HE and its oxidation products such as ethidium and the dimeric products of HE. The ramifications of HPLC measurement of HE and its oxidation products in the detection and quantitation of 2-hydroxyethidium, the diagnostic marker product of superoxide and HE, in the intracellular milieu are discussed. Similarly, mitochondria-targeted HE conjugated to a triphenylphosphonium group (Mito-HE or Mito-SOX) also forms oxidation products (dimers of Mito-HE and Mito-E⁺) that can affect the detection and quantitation of 2-hydroxy-mito-ethidium, the diagnostic marker product of Mito-HE and superoxide in mitochondria.     

Simple and rapid measurement of acetylcholine and choline by HPLC and enzymatic-electrochemical detection

A simple, rapid and sensitive method for the detection of acetylcholine and choline in tissue extracts is reported. Acetylcholine and choline are first separated by HPLC then react in a mini-column with acetylcholinesterase and choline oxidase immobilized on Sepharose. The resulting H₂O₂ produced by choline oxidase is then detected electrochemically. The assay is more sensitive than existing methods. We believe that the principle involved in this method namely the combination of immobilized enzymes and the high sensitivity of electrochemical detection may be applied to other substances that can be converted by immobilized enzymes into an electrochemically detectable compound.     

Quantitative determination of intact glucosinolates in broccoli, broccoli sprouts, Brussels sprouts, and cauliflower by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry

Many methods have been proposed to analyze glucosinolates, a class of phytochemicals whose breakdown products are thought to be responsible for an improvement in health; however, few are quantitative and many are time consuming. A selective and sensitive quantitative method for direct determination of intact glucosinolates was developed using liquid chromatography coupled to electrospray ionization tandem mass spectrometry with selected reaction monitoring detection. Detection limits for glucoiberin, sinigrin, progoitrin, glucoerucin, and glucotropaeolin were 1.75, 1.38, 1.36, 0.6, and 0.63 pmol, respectively. Intraassay precision of the method was within 10% for each compound. The method was successfully applied to quantify 10 individual glucosinolates in broccoli, broccoli sprouts, Brussels sprouts, and cauliflower. The advantage of the proposed method includes analysis of individual intact glucosinolates rather than the conversion to desulfoglucosinolates, an increased selectivity through the use of mass spectrometry, and a 10-fold improvement in detection sensitivity over conventionally used HPLC techniques.