Determination of some phytoestrogens in soybeans and their processed products with HPLC and coulometric electrode array detection

A reliable and sensitive method for quantification of daidzein and genistein by HPLC with coulometric electrode array detection is presented using bisphenol A as internal standard. Acid hydrolysis during extraction of foods allows the quantitative determination of total phytoestrogens as aglycones. The substances are separated on a reversed phase column (Hypersil^? Elite C-18), eluted with methanol/acetonitrile/50 mM sodium acetate pH 4.8 (40/ 20/40, v/v/v) and detected in a coulometric electrode array detector using an oxidative detection mode (+390 to +810 mV in increments of 60 mV, vs palladium reference electrode). Phytoestrogen levels from several food items were determined. High levels of daidzein (819 mg/kg) and genistein (960 mg/kg) were found in soy products whereas biochanin A could not be detected in any of these food samples. The recovery depends on the kind of food and was found to be between 72 and 94% for daidzein and genistein. Received: 29 September 1998 / Revised: 30 November 1998 / Accepted: 4 December 1998     

Analysis of flavonoids in honey by HPLC coupled with coulometric electrode array detection and electrospray ionization mass spectrometry

The analysis of flavonoids in unifloral honeys by high-performance liquid chromatography (HPLC) coupled with coulometric electrode array detection (CEAD) is described. The compounds were extracted by a nonionic polymeric resin (Amberlite XAD-2) and then separated on a reversed phase column using gradient elution. Quercetin, naringenin, hesperetin, luteolin, kaempferol, isorhamnetin, and galangin were detected in a coulometric electrode array detection system between +300 and +800 mV against palladium reference electrodes, and their presence was additionally confirmed by HPLC coupled with electrospray ionization mass spectrometry. The method was applied to analysis of 19 honeys of different varieties and origin. The limits of detection and quantitation ranged between 1.6 and 8.3 μg/kg and 3.9 and 27.4 μg/kg, respectively. The recoveries were above 96% in fluid and above 89% in creamy honeys. Some of these honeys (melon, pumpkin, cherry blossom, dandelion, maple, and pine tree honey) were investigated for their flavonoid content and profile for the first time. Differences between honeys were observed both in flavonoid concentrations and in the flavonoid profiles. The flavonoid concentrations ranged from 0.015 to 3.4 mg/kg honey. Galangin, kaempferol, quercetin, isorhamnetin, and luteolin were detected in all investigated honeys, whereas hesperetin occurred only in lemon and orange honeys and naringenin in lemon, orange, rhododendron, rosemary, and cherry blossom honeys.     

Determination of salbutamol in human plasma and urine by high-performance liquid chromatography with a coulometric electrode array system

A method is developed to determine salbutamol in human plasma and urine using high-performance liquid chromatography (HPLC) with a coulometric electrode array system, based on the electrochemical behavior of salbutamol at graphite electrode. The mobile phase component A is 30 mM sodium dihydroxy phosphate-30 mM triethylamine and is adjusted to pH 6.0 with 20% phosphate acid. The mobile phase component B is methanol. The optimized mobile phase composition was A and B in the proportion of 90:10 (v/v). Paracetamol is selected as the external standard. The human plasma and urine samples are pretreated using solid-phase extraction cartridges (Sep-Pak Silica), and the eluting solution is monitored by the coulometric electrode array system. The electrode potentials are set at 300, 400, 550, and 650 mV, respectively. Calibration curves show good linearity, and the recovery of salbutamol proves to be constant and unaffected by the concentration of the drug. This method, developed using HPLC-electrochemical detection, is reproducible and sensitive enough for the determination of salbutamol in human plasma and urine.     

Determination of pipecuronium bromide and its impurities in pharmaceutical preparation by high-performance liquid chromatography with coulometric electrode array detection

A sensitive and selective HPLC method with coulometric electrode array detection for the determination of pipecuronium bromide and its four impurities has been developed. The coulometric electrode array detection at increasing potentials from +300 to +900mV of the porous graphite electrode versus the palladium reference electrode was used. The limit of detection and quantitation for pipecuronium bromide was 8 and 25ngml(-1), respectively. This elaborate method for the simultaneous analysis of pipecuronium bromide and its impurities proved to be fast, precise, accurate, sensitive, and could be applied to analysis in substances and in pharmaceutical preparations.     

Determination of the levels of isoflavonoids in soybeans and soy-derived foods and estimation of isoflavonoids in the Japanese daily intake

The levels of 6 kinds of isoflavonoids found in 11 domestic and imported soybeans, and 12 kinds of soybean-based processed foods in Japan were systematically analyzed, and the Japanese daily intake of isoflavonoids from those foods was estimated. The total isoflavonoids (daidzein, glycitein, and genistein) were analyzed with acid hydrolysis and the intact isoflavonoids (daidzein, glycitein, genistein, daidzin, glycitin, and genistin) were analyzed without hydrolysis. This was followed by cleanup with an ODS cartridge column and determined by liquid chromatography with a diode array detector. The highest content of isoflavonoids was found in kinako (a roasted soybean powder) and the lowest was found in soy sauce. The contents and composition of the isoflavonoids in the 11 soybeans varied by species and country of origin. The level of isoflavonoids found in the processed foods varied by manufacturing method or ingredients. The percentage of aglycone tended to be higher in miso (fermented soybean paste) and soy sauce, which are heated and fermented during the manufacturing process. Japanese daily intake of isoflavonoids from soybeans and soybean-based processed foods was estimated as 27.80 mg per day (daidzein 12.02 mg, glycitein 2.30 mg, and genistein 13.48 mg).     

Amperometric Detection of Histamine with a Pyrroloquinoline‐Quinone Modified Electrode

A modified glassy carbon (GC) electrode was developed for the amperometric detection of biogenic amines, particularly histamine. The electrode was modified with the co‐enzyme pyrroloquinoline quinone (PQQ) by entrapment during electropolymerziation of pyrrole to form polypyrrole (PPy). This method formed a thin film on the electrode surface possessing very good stability with a shelf‐life exceeding one month without loss of signal. Optimal conditions for the PQQ/PPy electrode were determined and a linear response was found for histamine in phosphate buffer (pH 6) at +550 mV from 40 to 170 mg L^?1 with a limit of detection (S/N≥3) of 38 mg L^?1. The practical linear range offered by this method suggests ideal use for spoilage detection in fermented foods.     

Analysis of isoflavones in soybeans employing direct analysis in real-time ionization-high-resolution mass spectrometry

A direct analysis in real‐time (DART) ion source coupled to a high‐resolution orbitrap mass spectrometer was used for the quantitative analysis of isoflavones isolated from soybeans. For the isolation of genistein, daidzein, glycitein, and their respective acetyl, malonyl, and glucoside forms, an extraction employing 80% aqueous MeOH enhanced by sonication was used. As far as the total isoflavones (expressed as aglycones) were to be determined, an acid hydrolysis with 80% aqueous EtOH and refluxing had to be employed, while in the latter case a good agreement of the results with the data generated by the UHPLC‐orbitrap MS method was achieved, in the case of the analysis of non‐hydrolyzed extracts, some overestimation of the results as compared with those generated by UHPLC‐orbitrap MS was observed. A careful investigation of this phenomenon showed that the free aglycones originated from the conjugated forms of isoflavones in the DART ion source, thus contributing significantly to the “free” genistein/daidzein/glycitein signals during the DART analysis. Good recoveries (95–102%) and repeatabilities (RSD: 7–15%) were obtained at the spiking levels of 0.5, 1, and 0.05 g/kg, for daidzein, genistein, and glycitein, respectively. The limits of detection estimated for the respective analytes were 5 mg/kg.     

Simultaneous determination of 11 fluorescent whitening agents in food‐contact paper and board by ion‐pairing high‐performance liquid chromatography with fluorescence detection

4,4′‐Diaminostilbene‐2,2′‐disulfonic acid based fluorescent whitening agents (DSD‐FWAs) are prohibited in food‐contact paper and board in many countries. In this work, a reliable high‐performance liquid chromatography method was developed for the simultaneous determination of 11 common DSD‐FWAs in paper material. Sample preparation and extraction as well as chromatographic separation of multicomponent DSD‐FWAs were successfully optimized. DSD‐FWAs in prepared samples were ultrasonically extracted with acetonitrile/water/triethylamine (40:60:1, v/v/v), separated on the C₁₈ column with the mobile phase containing tetrabutylammonium bromide, and then detected by a fluorescence detector. The limits of detection were 0.12–0.24 mg/kg, and the calibration curves showed the linear correlation (R² ≥ 0.9994) within the range of 8.0–100 ng/mL, which was equivalent to the range of 0.80–10 mg/kg in the sample. The average recoveries and the RSDs were 81–106% and 2–9% at two fortification levels (1.0 and 5.0 mg/kg) in paper bowls, respectively. The successful determination of 11 DSD‐FWAs in food‐contact paper and board obtained from local markets indicated that the newly developed method was rapid, accurate, and highly selective.     

Optimization of a rapid microwave assisted extraction method for the liquid chromatography-electrospray-tandem mass spectrometry determination of isoflavonoid aglycones in soybeans

A very fast chromatographic separation of isoflavonoids genistein, daidzein, formononetin and biochanin A was developed on a C18 high-speed column under isocratic conditions. The method was validated in terms of detection limits, quantitation limits (LOQs), linearity and precision. LOQs in 0.04-0.2 microg/g range were calculated, making feasible the determination of these compounds of nutritional concern at trace levels. Good linearity was demonstrated over three concentration orders of magnitude for each analyte (r2 0.990-1.000). The intra-day and inter-day repeatability was evaluated in terms of relative standard deviation (RSD%) at two concentration levels for each analyte (RSD% <9%). An optimization strategy was adopted to find the best conditions for the extraction of isoflavonoid aglycones from yellow soybeans using microwave-assisted extraction. The most relevant parameters resulted to be the microwave power, the extraction time and the acid concentration, optimal values being 600 W, 1 min and 12 M, respectively. When performing sample treatment on a fortified soybean sample, high recovery percentage was obtained for both compounds (94+/-8% for daidzein and 97+/-5% (n = 4) for genistein). The concentration level at which daidzein and genistein were found in the soybean sample were 1.21+/-0.15 mg/g and 2.38+/-0.09 mg/g (n=4), respectively.     

Determination of daidzein and genistein in soybean foods by automated on-line in-tube solid-phase microextraction coupled to high-performance liquid chromatography

An automated on-line method for the determination of the isoflavones, daidzein and genistein, was developed using in-tube solid-phase microextraction coupled to high-performance liquid chromatography (in-tube SPME-HPLC). In-tube SPME is a new extraction technique for organic compounds in aqueous samples, in which analytes are extracted from the sample directly into an open tubular capillary by repeated draw/eject cycles of sample solution. Daidzein, genistein and their glucosides tested in this study were clearly separated within 8 min by HPLC using an XDB-C8 column with diode array detection. In order to optimize the extraction of these compounds, several in-tube SPME parameters were examined. The glucosides daidzin and genistin were analyzed as aglycones after hydrolysis because the glucosides were not concentrated by in-tube SPME. The optimum extraction conditions for daidzein and genistein were obtained with 20 draw/eject cycles of 40 microl of sample using a Supel-Q porous layer open tubular capillary column. The extracted compounds were easily desorbed from the capillary by mobile phase flow, and carryover was not observed. Using the in-tube SPME-HPLC method, the calibration curves of these compounds were linear in the range 5-200 ng/ml, with a correlation coefficient above 0.9999 (n = 18), and the detection limits (S/N = 3) were 0.4-0.5 ng/ml. This method was successfully applied to the analysis of soybean foods without interference peaks. The recoveries of aglycones and glucosides spiked into food samples were above 97%.     

Method for the determination of gamma-L-glutamyl-L-dihydroxyphenylalanine and its major metabolites L-dihydroxyphenylalanine, dopamine and 3,4-dihydroxyphenylacetic acid by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic method for the analysis of gamma-L-glutamyl-L-dihydroxyphenylalanine (gludopa) and its major metabolites L-dihydroxyphenylalanine (L-DOPA), dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) is described. High sensitivity is achieved with a multi-cell coulometric detector utilising the specific electrochemical properties of gludopa (limit of detection 10 pg on-column). The retention time of gludopa was both pH-dependent and sensitive to negatively charged ion-pairing agents. An alumina-based solid-phase sample preparation technique with dihydroxybenzylamine as internal standard is described for plasma and urine (limit of detection 40 pg/ml) and an ultrafiltration technique is described for tissues (limit of detection 1-10 ng/g). After treatment with 50 mg/kg gludopa, in excess of twenty separate catecholic metabolic peaks can be detected in rat urine, whereas in humans after 9 mg/kg the only catechols detected were L-DOPA, dopamine and DOPAC.     

基质固相分散-高效液相色谱法测定鲫鱼肌肉中残留的辛硫磷

建立了鲫鱼肌肉中残留的辛硫磷的基质固相分散-高效液相色谱-二极管阵列检测(MSPD-HPLC-DAD)的分析方法。通过优化样品处理条件,确定选取0.50 g鲫鱼肌肉样品与1.5 g弗罗里硅土、0.5 g无水硫酸钠混合研磨,并采用丙酮-正己烷溶液(体积比为40:60)为洗脱剂,洗脱剂用量为25 mL。优选的最佳色谱条件为：ODS色谱柱(250 mm×4.6 mm,5 μm),流动相为甲醇-水(体积比为50:50),流速0.6 mL/min,检测波长270 nm,进样量为20 μL。在上述条件下,辛硫磷质量浓度在0.01～10 mg/L范围内与响应信号呈良好的线性关系(r20.9994),检出限为3.3 μg/kg;相对标准偏差为1.1%～6.3%(n7);3个添加水平(0.05,0.1,1 mg/kg)下得到的回收率为88%～112%。该方法操作简单,耗时少,精密度高,符合农残分析的要求。     

Trace determination of bisphenol A and phytoestrogens in infant formula powders by gas chromatography-mass spectrometry

This investigation describes a reliable and sensitive method for simultaneously determining bisphenol A (BPA) and two major phytoestrogens, daidzein and genistein, in powdered milks and infant formulas by gas chromatography-mass spectrometric analysis after trimethylsilylation. To reduce the matrix interference associated with the constituents of the formulas, the dissolved formula solutions were firstly ultra-centrifuged and the analytes in the supernatant were then extracted using a C18 solid-phase extraction column. The accuracy and precision of the method were determined and the technique was successfully employed to measure trace concentrations of BPA, daidzein and genistein in powdered formulas. The results show that BPA, daidzein and genistein were detected in all the testing samples (n = 6) at concentrations from 45 to 113 ng/g (except one infant formula), 20 to 2050 ng/g and 21 to 6510 ng/g, respectively. The highest concentrations of daidzein and genistein (i.e., 2050 and 6510 ng/g) were detected in a soy-based powdered infant formula. The quantitation limits were 1.0 ng/g for BPA, and 10 ng/g for daidzein and genistein using 0.5 g powdered milk samples.     

Determination of naltrexone and 6 beta-naltrexol in plasma by high-performance liquid chromatography with coulometric detection

A simple and reliable reversed-phase high-performance liquid chromatographic method with coulometric detection is described for the quantitation of naltrexone and its metabolite, 6 beta-naltrexol, in plasma samples of healthy volunteers who received orally 50 mg of naltrexone. The analytes and the internal standard, naloxone, are extracted with an octadecyl solid-phase extraction column before chromatography. The mobile phase is 0.01 M potassium phosphate (pH 3)-acetonitrile (85:15, v/v) and it is pumped at 0.8 ml/min. The coulometric detector is formed by two electrodes set at +0.20 V and +0.70 V, with a palladium reference electrode. The limit of quantitation observed was 5 ng/ml for both naltrexone and 6 beta-naltrexol. This method can be used to investigate pharmacokinetic parameters of different pharmaceutical preparations of this opioid antagonist.     

The presence of three isoflavonoid compounds in Psoralea corylifolia.

The optimization of a high-performance liquid chromatographic method to determine three isoflavonoids (daidzein, genistein, and biochanin A) in the fruit of Psoralea corylifolia is developed and validated. Dried psoralea fruit powder is extracted with aqueous methanol followed by the hydrolysis of the analytes' conjugated glycosides with hydrochloric acid. The HPLC assay is performed on a reverse-phase C18 column with gradient elution using acetonitrile and 10% acetic acid as the mobile phase at a flow rate of 0.8 mL/min. Flavone is used as the internal standard and the substances are detected at 260 nm. Calibrations are linear (correlation coefficient > or = 0.995) for all three analytes. The limits of detection are 0.01 microg/mL for daidzein and genistein and 0.1 microg/mL for biochanin A. The overall intra- and interassay precision range from 2.5% to 4.9% and from 0.5% to 4.7%, respectively. The method proved to be sensitive, specific, accurate, and precise for the determination of daidzein, genistein, and biochanin A in Psoralea corylifolia.     

Quantitative determination of isoflavones and coumestrol in soybean by column liquid chromatography

Four different stationary phases and a variety of solvents in varying proportions were examined in this study. Daidzein, genistein, formononetin, biochanin A and coumestrol were separated within 24 min on a phenyl column with acetonitrile-water (33:67, v/v) as eluent. The proposed method showed an acceptable repeatability with a RSD of quantitation <6%. The mean recoveries of daidzein, genistein, formononetin, biochanin A and coumestrol from soybean ranged from 89 to 104%. The identity of the individual analytes was confirmed by LC-MS-MS. The four isoflavones and coumestrol were isolated from soybean by hydrolysis with acid and heat. Neutralization of the soybean samples prior to identification did not alter the concentration of daidzein and genistein in soybean.     

超高效液相色谱法测定水果和饮料中残留的氟嘧菌酯和嘧螨酯

建立了多种水果和饮料中氟嘧菌酯和嘧螨酯残留的超高效液相色谱测定方法。样品用乙酸乙酯-环己烷(体积比为1∶1)超声波萃取,凝胶渗透色谱法净化,超高效液相色谱-二极管阵列检测器检测,外标法定量。采用BEH C18色谱柱(50 mm×2.1 mm,1.7 μm),流动相为水-乙腈(体积比为3∶7),流速为0.3 mL/min,柱温为40 ℃,紫外检测波长为251 nm。实验结果表明：氟嘧菌酯和嘧螨酯在0.05～2 mg/L范围内线性关系良好(r>0.999),在不同的基质中添加3个浓度水平(0.01,0.05,0.1 mg/kg)的氟嘧菌酯和嘧螨酯,两者的回收率均在82.60%～101.11%之间,相对标准偏差为5.4%～15.3%;检出限不大于6 μg/kg,定量限不大于20 μg/kg。     

Determination of tetracyclines in ovine milk by high-performance liquid chromatography with a coulometric electrode array system

A method has been developed to analyze residual tetracyclines (TCs) (oxytetracycline (OTC), tetracycline (TC), chlortetracycline (CTC), methacycline (MTC), doxycycline (DC)) in ovine milk, using high-performance liquid chromatography (HPLC) with a coulometric electrode array system. The samples were pretreated, using liquid-liquid extraction based on hexane. The chromatography was performed, using a C18 column (150 mm x 4 mm i.d. and 5 microm) with a mobile phase: sodium phosphate monobasic dihydrate (pH 2.2, 0.05 M)-acetonitrile (78:22, v/v). The flow rate of mobile phase was kept constantly at 1ml/min. The residues were monitored by an ESA electrochemical detector. Potentials of four electrodes in series were set at 400, 660, 680 and 700 mV, respectively. The first electrode was set to remove those interfering substances that may co-elute with TCs and the other three electrodes were used for quantification. The maximal potential of our detection was 700 mV. Calibration curve showed good linearity and the detection limit of TCs was 12.5, 20, 25, 10 and 25 ng/ml, respectively. Optimization of the pH of the mobile phase, the proportion of acetonitrile and the pH of the pretreatment were also performed. Recoveries of TCs from spiked samples were more than 88% and the relative standard deviations were less than 4.3%. This method was reliable, sensitive, economical and suited for routine monitoring of TC residues in ovine dairy milk.     

Determination of puerarin and daidzein in Puerariae radix and its medicinal preparations by micellar electrokinetic capillary chromatography with electrochemical detection

The use of micellar electrokinetic capillary chromatography (MECC) with electrochemical detection is described for the determination of puerarin and daidzein in Puerariae radix and its medicinal preparations. Operated in a wall-jet configuration, a 300 μm diameter carbon-disk electrode was used as the working electrode, which exhibits good responses at +900 mV (versus SCE) for the two analytes. Under the optimum conditions, the analytes were base-line separated within 11 min in a sodium dodecyl sulphate—borax (pH 7.8) running buffer, and excellent linearity was obtained in the concentration range from 5.0×10⁻⁴ to 5.0×10⁻⁶ mol/l. The detection limit (S/N=3) was 6×10⁻⁷ and 1.1×10⁻⁶ mol/l for puerarin and daidzein, respectively. This work provides a useful method for the analysis of traditional Chinese medicines.     

高效液相色谱-紫外检测法同时测定食品接触材料中7种苯多酸及其衍生物的特定总迁移量

建立了高效液相色谱-紫外检测(HPLC-UV)同时测定5种食品模拟物(10%(v/v)乙醇、20%(v/v)乙醇、50%(v/v)乙醇、3%(w/v)乙酸和橄榄油)中偏苯三甲酸、偏苯三甲酸酐、间苯二甲酰氯、间苯二甲酸、对苯二甲酰氯、邻苯二甲酸、对苯二甲酸的特定总迁移量(SML(T))的方法。用食品模拟物浸泡待测样品,冷却至室温并混匀,水基食品模拟物经亲水性聚四氟乙酸针头过滤器过滤后进样;橄榄油用0.1%(w/v)乙酸铵水溶液提取后,下层清液用亲水性聚四氟乙烯针头过滤器过滤后进样。用Synergi Polar-RP色谱柱(250 mm×4.6 mm, 4 μm)分离,梯度洗脱,检测波长为232 nm。5种食品模拟物中的定量限为0.1~0.2 mg/kg;水基食品模拟物在0.5~12 mg/L、橄榄油食品模拟物在0.5~12 mg/kg范围内线性关系良好(r² > 0.99991); 1.25、2.5、6.25 mg/kg水平的加标回收率为94.3%~105%,相对标准偏差为0.1%~2.3%。结果表明,该方法的色谱分离和线性关系较好,回收率和准确度高,完全满足欧盟(EU)No 10/2011法规附表2中7种苯多酸及其衍生物的SML(T)的限量要求,并已应用于实际样品的检测。