Quantification of isoflavones and lignans in serum using isotope dilution liquid chromatography/tandem mass spectrometry

Phytoestrogens (isoflavones and lignans) are receiving increasing attention due to a potential protective effect against a number of complex diseases. However, in order to investigate these associations, it is necessary to accurately quantify the levels of phytoestrogens in foods and biological fluids. We report an assay for three isoflavones (daidzein, genistein, and glycitein), two metabolites of daidzein (O-desmethylangolensin and equol), and two lignans (enterodiol and enterolactone) in human serum using electrospray ionisation liquid chromatography/mass spectrometry (LC/MS) with selective reaction monitoring. A simple, highly automated sample preparation procedure requires only 200 microL of sample and utilises one solid-phase extraction stage. Limits of detection are in the region of 10 pg/mL for all analytes except equol, which had a limit of detection of approximately 100 pg/mL. The method developed is suitable for measuring the concentrations of phytoestrogens in blood samples collected from large epidemiological studies. The results of the analysis of serum samples from 300 men and women living in the UK are reported.     

The synthesis of daidzein sulfates

The first syntheses of the urinary isoflavone metabolites, daidzein 4′-sulfate, daidzein 7-sulfate and daidzein 4′,7-disulfate are described. These syntheses employ a key protecting group strategy, allowing regiospecific sulfation.     

An accurate and reproducible method for the quantitative analysis of isoflavones and their metabolites in rat plasma using liquid chromatography/mass spectrometry combined with photodiode array detection

To study the safety and potential health benefits of soy isoflavones, a rapid and simple method based on liquid chromatography combined with mass spectrometry (LC/MS) and photodiode array detector (PDA) was developed for the determination of isoflavones in rat plasma. The analytes included daidzein, genistein, glycitein, equol, 4-ethyl phenol, and biochanin A over a concentration range of 1.0-4320.0 nM using 75 microL of rat plasma. Rat plasma samples were hydrolyzed by adding an enzyme mixture from Helix pomatia containing glucuronidase and sulfatase to convert the isoflavone beta-glycosides daidzin, genistin, and glycitin to their active aglycone forms. A liquid-liquid extraction method using ethyl acetate as the extraction solvent was used to extract aglycones and the internal standards (phenolphthalein beta-D glucuronide, 4-methylumbelliferyl sulfate, and apigenin) from digested plasma samples. The extract was evaporated to dryness under a nitrogen stream, reconstituted with 0.1% formic acid in water-acetonitrile (85 + 15), and injected into a Zorbax SB-CN reversed-phase column (4.6 x 75 mm, 3.5 microm particle size). The Micromass ZQ detector was operated in the positive ion selected-ion monitoring mode. The flow rate for LC was 1.0 mL/min, with a split where 25% of the effluent was introduced into the electrospray ionization probe of the MS instrument and 75% into the PDA. The chromatographic run time was 16.0 min, with delay of 10 min/injection. The interday precision and accuracy of the standard samples were <2.6% relative standard deviation and <10% relative error, respectively. Recovery of the reported isoflavones with this method varied from 86 to 100%.     

Protective effect of the isoflavonoid equol against hairless mouse skin carcinogenesis induced by UV radiation alone or with a chemical cocarcinogen

Isoflavones derived from many edible plants, such as genistein from the soybean, have well-documented antioxidant and estrogenic activity but may also be anticarcinogenic. In this study, we examined the potential of the isoflavone equol [(S)-4',7-dihydroxyisoflavane] to protect from skin carcinogenesis in the hairless mouse. Daily topical applications of equol lotions significantly protected against skin carcinogenesis induced by chronic exposure to solar-simulated UV radiation (SSUV) or by topical treatment with the chemical carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) or by the combined cocarcinogenic treatment of DMBA followed by chronic SSUV. Monitoring of tumor development for 40 weeks showed significantly delayed tumor appearance and reduced tumor multiplicity in all equol-treated groups. In mice treated with either SSUV or DMBA + SSUV, equol significantly reduced the proportion of tumors progressing from benign papillomas to malignant squamous cell carcinoma (SCC) by 33-58% and reduced the average diameter of SCC by 71-82%. In a short-term study, equol dose dependently inhibited the SSUV induction of the tumor promotion biomarker enzyme, ornithine decarboxylase, in the skin, suggesting the anticarcinogenic activity of equol may be attributed to its inhibition of the tumor promotion phase of carcinogenesis.     

Determination of isoflavones in ready-to-feed soy-based infant formula

An alkaline hydrolysis/liquid chromatography (LC) method was developed for determination of isoflavones in ready-to-feed soy-based infant formula. The method consists of a 15 min methanol extraction, 10 min alkaline hydrolysis, HCl neutralization, gravity filtration, aqueous dilution, and 50 min LC analysis with UV detection at 262 and 250 nm to quantify 6 isoflavone analytes: daidzin, glycitin, genistin, daidzein, glycitein, and genistein. The concentration averages for 10 commercial batches (microg aglycone/g formula) were daidzein, 6.12 +/- 1.23; glycitein, 1.19 +/- 0.16; genistein, 12.8 +/- 2.35; and total, 20.1 +/- 3.61. Validation experiments demonstrated extraction completion and analyte stability to alkaline hydrolysis. Spike recoveries ranged from 97.6 to 104.1%, and a series of accuracy assessments showed that isoflavone concentration determined by the method was within 5% of the true value. The relative standard deviation values for repeatability ranged from 0.4 to 2.2% (n = 10), and from 0.3 to 2.7% (n = 4) for intermediate precision. Isoflavone peak purity was verified by comparing sample and standard peak area ratios (262/250 nm). The limits of detection and quantitation (microg/ formula) ranged from 0.02 to 0.05 and 0.08 to 0.18 microg/g, respectively. The difference between our concentrations and those reported by others in 1995-1998 is attributable to the well-established seasonal variation in soybean isoflavone levels. Although the method was applied exclusively to ready-to-feed formula in the present study, it is equally suitable for powder and concentrated liquid infant formulas.     

Biotin-avidin amplified enzyme-linked immunosorbent assay for determination of isoflavone daidzein

A biotin-avidin amplified enzyme-linked immunosorbent assay (BA-ELISA) method was developed and optimized for the determination of a weakly estrogenic isoflavone daidzein in serum, urine and Puerariae radix. Specific polyclonal antibody was produced against daidzein by immunization of rabbits with a conjugate of 7-O-(carboxymethyl)-daidzein and bovine serum albumin (BSA). The polyclonal antibody showed specific recognition of daidzein, while cross-reactivities to coumarin, 4-hydroxycoumarin, phenol, and other isoflavones such as puerarin and rutin were all lower than 1%. The linear range of daidzein calibration curve was 0.1-1000 ng mL⁻¹. The detection limit was found to be 0.04 ng mL⁻¹, and the intra-assay and inter-assay coefficients of variation were 7 and 16%, respectively. Human serum and urine samples were spiked with known amounts of daidzein and measured by the established BA-ELISA. Recoveries were between 91 and 107%. Daidzein in P. radix was determined by the BA-ELISA method and HPLC method, and the content of daidzein was determined to be 0.0219 and 0.0194%, respectively. The results indicated that there was a good agreement between the two methods. The established method is very useful for monitoring daidzein in biological samples and traditional Chinese medicine.     

Determination of Daidzein in Rat Plasma by LC

Daidzein (4′,7-dihydroxylisoflavone), a water-insoluble isoflavone, is mainly present in leguminous plants. This study was aimed at investigating the absorption characteristics of daidzein across the intestinal membranes. Borneol has been used to enhance the intestinal absorption and bioavailability of daidzein. We chose an in vitro diffusion chamber system using isolated rat intestinal membranes as a model. A sensitive, accurate and reliable LC method was developed and validated to determine daidzein in rat plasma. A linear relationship for daidzein was found in the range of 20–800 ng mL^?1. The mean absolute recoveries of daidzein determined over three concentrations were 89.8 ± 4.8, 91.3 ± 2.8 and 95.4 ± 1.7%. The coefficients of variation for inter-day and intra-day assay were found to be less than 6.1%. The plasma concentration of daidzin following oral administration was significantly increased by coadministration of borneol in rats, and a 1.5-fold enhancement of relative bioavailability of daidzein was observed as compared to daidzein administered alone.     

Synthesis of haginin E, equol, daidzein, and formononetin from resorcinol via an isoflavene intermediate

New syntheses of haginin E, equol, daidzein, and formononetin are described in this Letter. Through a sequence of a Wittig reaction, O-alkylation, and another Wittig reaction, 4-benzyloxysalicylaldehyde, which was prepared from resorcinol in two steps, was converted into the desired diene in one pot. Subsequently, the prepared diene was subjected to ring-closing metathesis using Grubbs’ catalyst (II) to construct the desired isoflavene intermediate. Using the prepared isoflavene, certain isoflavonoids such as haginin E, equol, daidzein, formononetin, and other related compounds were derived smoothly and in good overall yields.     

Characterization and quantification of soy isoflavone metabolites in serum of renal transplanted patients by high-performance liquid chromatography/electrospray ionization mass spectrometry

During a dietary intervention study on 16 renal transplanted patients, in which 25 g/day of animal proteins were replaced with 25 g of soy proteins, the metabolic profile of soy isoflavones in serum was characterized. This paper describes a reliable and fast liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) method, in negative ion mode, allowing the characterization and simultaneous quantification of several soy isoflavone metabolites. Six metabolites were identified and quantified: daidzein ([M-H](-) at m/z 252.8), dihydrodaidzein (DHD, [M-H](-) at m/z 254.8), equol ([M-H](-) at m/z 240.9), O-desmethylangolensin (O-DMA, [M-H](-) at m/z 256.8), genistein ([M-H](-) at m/z 268.8), and dihydrogenistein (DHG, ([M-H(+)](-) at m/z 270.8). Quantification was assessed using two deuterated internal standards, D(3)-daidzein and D(4)-genistein. This method permitted a limit of quantification (LOQ, S/N = 10) and a limit of detection (LOD, S/N = 3) of 0.05 microM and 0.005 microM for all analytes, except for genistein, where the LOQ and LOD were 0.005 microM and 0.001 microM, respectively. The linearity ranges were from 0.005 to 1.5 microM for genistein, from 0.05 to 1.5 microM for DHG, and from 0.05 to 0.7 microM for the other metabolites. The relative standard deviations (RSDs) were between 0.19% and 13.9% at the LOQ concentration for all metabolites, and between 0.6% and 4.8% at the maximum concentration. On the basis of the results obtained in the dietary intervention study, it was possible to split the patients into five groups characterized by different metabolic pathways.     

反相高效液相色谱法制备纯化大豆异黄酮糖苷

利用制备高效液相色谱法从大豆总异黄酮提取物中制备出了3种大豆异黄酮糖苷。在Nova-Pak HR C18色谱柱(100 mm×25 mm i.d.,6 μm)上,以甲醇-体积分数为0.1%的乙酸水溶液(体积比为23∶77)为流动相,流速为20 mL/min,采用 等度洗脱方式,制备了3种大豆异黄酮糖苷,经质谱分析,确认它们分别为大豆苷、黄豆苷和染料木苷。高效液相色谱分析 表明,所制备的3种化合物的纯度均达到了99%以上。     

Supercritical fluid extraction of isoflavones from biological samples with ultra-fast high-performance liquid chromatography/mass spectrometry

An efficient method of modifier addition for supercritical fluid extraction (SFE) of polar isoflavones was developed and yielded extraordinarily high recoveries. To find the optimal extraction conditions, a temperature and pressure optimization and modifier impact study was performed in naturally contaminated and spiked samples. Ultra-fast high-performance liquid chromatography/mass spectrometry (HPLC/MS) was used for the determination of isoflavones on an Atlantis dC18 high-speed reversed phase chromatographic column (20 x 2.1 mm, 3 microm particle size). A newly elaborated supercritical fluid extraction (SFE) procedure allowed more accurate (< 5%) and precise (< 4-7%) determination of isoflavones in biological materials. The HPLC/MS method significantly reduced analysis time with simultaneous improvement of sensitivity and detection limits. The on-column limits of detection LOD (S/N = 3) for isoflavone glycosides (daidzin, genistin, glycitin, ononin, and sissotrin) were 1.3-3.6 fmol and 0.2-1.0 fmol for aglycones (daidzein, glycitein, genistein, formononetin, and biochanin A), respectively.     

Quantification of tamoxifen and metabolites and soy isoflavones in human plasma using liquid chromatography with electrospray ionization tandem mass spectrometry

Tamoxifen is an important estrogen receptor antagonist used successfully for the treatment and prevention of breast cancer. The use of complementary and alternative medicines is an increasingly popular means for patients to participate in their own health care, and soy products, which contain phytoestrogens, have been widely promoted for possible beneficial effects on menopausal symptoms. The possibility that soy isoflavones could reduce tamoxifen efficacy has been demonstrated in animal models of post-menopausal breast cancer, but the occurrence of such an effect in women has not been explored. This paper describes the development and validation of a sensitive method using solid-phase extraction and isotope dilution liquid chromatography/tandem mass spectrometry with multiple reaction monitoring for the concurrent analysis of the major soy isoflavones (genistein and daidzein), an important metabolite of daidzein (equol), tamoxifen, and its important metabolites (4-hydroxytamoxifen, N-desmethyltamoxifen, and 4-hydroxy-N-desmethyltamoxifen or "endoxifen") in the serum of rats and women. The limits of quantification achieved are sufficient to determine accurately and precisely the concentrations of all of these analytes in women consuming soy foods and/or therapeutic doses of tamoxifen at levels consistent with modulation of estrogen receptor-mediated functions. These procedures enable future investigations of the possible impact of diet on the outcome of breast cancer therapy.     

大豆苷元的前药修饰研究进展

大豆异黄酮大豆苷元具有抗氧化、心血管保护、抗癌、抗炎、抗溃疡及雌激素等多种药理功能.本文综述了大豆苷元的化学修饰研究进展,为充分利用大豆资源和发展以大豆苷元为先导物的化学活性修饰物提供研究信息.     

Determination of isoflavones in red clover and related species by high-performance liquid chromatography combined with ultraviolet and mass spectrometric detection

High-performance liquid chromatography-UV-electrospray ionization-mass spectrometric detector (HPLC-UV-ESI-MSD) method for determination of isoflavones in red clover (Trifolium pratense L.) and related species has been developed. The separated isoflavones including aglycones, glycosides and glycoside malonates, were individually analyzed and identified by their molecular ions and characteristic fragment ion peaks using LC-MSD under MS and MS-MS mode, and in comparison with the standard isoflavones. A total of 31 isoflavones were detected in red clover. Several isoflavones were also identified for the first time in related species, T. repense L. (white clover), T. hybridum L. (alsike clover) and T. campestre Schreber (hop trefoil). Based on reversed phase HPLC, all 10 isoflavone aglycones, daidzein, formononetin, genistein, pseudobaptigenin, glycitein, calycosin, prunetin, biochanin A, irilone and pratensein in acidic hydrolyzed extracts were successfully separated within 40 min and quantified individually by UV and MS detectors. For the 10 target compounds, the investigated concentrations ranged from approximately 24 to approximately 12500 ng/ml for UV detection and approximately 6 to approximately 3125 ng/ml for MS detection, and good linearities (r2 > 0.999 for UV and r2 > 0.99 for MS) for standard curves were achieved for each isoflavone. The accuracy and repeatability (n = 10) were within 15% for these 10 compounds. This is the first method reported that enables the simultaneous quantitation of all 10 isoflavone aglycones in red clover and related species.     

Automated online and off-line solid-phase extraction methods for measuring isoflavones and lignans in urine

Automated online and off-line solid-phase extraction (SPE) methods coupled to isotope dilution-high-performance liquid chromatography-tandem mass spectrometry for measuring four isoflavones (daidzein, genistein, equol, and O-desmethylangolensin) and two lignans (enterolactone and enterodiol) in urine are developed. The SPE recoveries for the online SPE method are excellent for most analytes (83-94%) and somewhat lower for enterolactone (61%). The recoveries for all analytes with the off-line SPE method are also very good (65-80%). The limit of detection is lower for the online method (0.1-0.7 ng/mL) than for the off-line method (0.4-3.3 ng/mL). Similarly, the reproducibility is generally better for the online method [coefficient of variation (CV) of 4-12%) than for the off-line method, except for enterolactone, which has a higher CV (18-19%) that is consistent with its lower online SPE recovery. Both methods are adequate for analyzing a large number of samples for epidemiological studies to assess the prevalence of human exposure to isoflavones and lignans.     

Quantitative analysis of isoflavone aglycones in human serum by solid phase extraction and liquid chromatography-tandem mass spectrometry

This paper describes a liquid chromatography-electrospray-tandem mass spectrometry (LC-ESI-MS/MS) for the qualitative and quantitative analysis of three isoflavone aglycones (glycitein, daidzein and genistein) in human serum. Positive ion mode was used for the detection of these compounds and selective reaction monitoring (SRM) was employed for quantitative measurement. The SRM transitions monitored were as 285.0 → 242.0, 270.0 for glycitein, 255.0 → 137.0, 153.0, 181.0, 199.0 for daidzein and 271.0 → 153.0, 215.0 for genistein. d₃-Daidzein was used as an internal standard for quantitative measurement. The linearity was good from 0.5 to 500 ng/ml. The detection limit based on a signal-to-noise ratio of three was 0.27, 0.38 and 0.29 ng/ml for glycitein, daidzein and genistein, respectively. A newly developed solid phase extraction (SPE) procedure was developed for sample pre-treatment. Good recovery, 92.3-103.2%, for three isoflavone aglycones were obtained. This newly developed method was successfully applied to evaluate isoflavone pharmacokinetic in human serum after oral administration.     

Simultaneous analysis of nine active components in Gegen Qinlian preparations by high-performance liquid chromatography with diode array detection

HPLC with diode array detection (HPLC/DAD) was employed to determine the quantities of puerarin, daidzin, daidzein, berberine, palmatine, coptisine, baicalin, baicalein, and glycyrrhizin in Gegen Qinlian preparations of three different pharmaceutical forms including decoction, dispensing granule and pill. The calibration curves for the nine bioactive components were linear in the given concentration ranges. The precision of the method was in the range of 0.2 - 5.0% (RSD), and the recoveries of this method were between 96.5 and 104.1%. The proposed method was applicable to analyze Gegen Qinlian preparations.     

Analysis and retention behavior of isoflavone glycosides and aglycones in Radix Astragali by HPLC with hydroxypropyl‐β‐cyclodextrin as a mobile phase additive

In order to determine isoflavone glycosides (calycosin‐7‐O‐β‐d ‐glucoside and formononetin‐7‐O‐β‐d ‐glucoside) and aglycones (calycosin and formononetin), a simple HPLC method with isocratic elution employing hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) as a mobile phase additive was developed. Various factors affecting the retention of isoflavone glycosides and aglycones in the C₁₈ reversed‐phase column, such as the nature of cyclodextrins, HP‐β‐CD concentration, and methanol concentration, were systematically studied. The results show that HP‐β‐CD, as a very effective mobile phase additive, can markedly reduce the retention of isoflavone glycosides and aglycones, and the decrease magnitudes of isoflavone aglycones are more than those of their glycosides. The role of HP‐β‐CD in the developed HPLC method is attributed to the formation of the inclusion complexes between isoflavone glycosides (or aglycones) and HP‐β‐CD. So, the apparent formation constants of the isoflavone glycosides (or aglycones)/HP‐β‐CD inclusion complexes also were investigated. Isoflavone glycosides (and aglycones) form the 1:1 inclusion complexes with HP‐β‐CD, and the isoflavone aglycones/HP‐β‐CD complexes are more stable than the isoflavone glycosides/HP‐β‐CD complexes. Finally, the optimized method was successfully applied for the determination of isoflavone glycosides and aglycones in Radix Astragali samples.     

Ultrasound‐assisted solid‐phase extraction coupled with photodiode‐array and fluorescence detection for chemotaxonomy of isoflavone phytoestrogens in Trifolium L. (Clover) species

Detailed chemotaxonomic studies were undertaken to establish the qualitative profile and real amounts of the pharmacologically active isoflavone aglycones genistein, daidzein, formononetin, and biochanin A in aerial parts of thirteen Trifolium L. (clover) species, native to Poland. A newly elaborated micropreparative technique – SPE – on BakerBond octadecyl, cyclohexyl, and phenyl cartridges was used in combination with ultrasound‐assisted extraction for isolation of isoflavone aglycones from hydrolyzed samples. The effectiveness of all three SPE sorbents in the purification of plant extracts was compared and very high recoveries (>96%) were documented for four isoflavones. Classical photodiode‐array and very sensitive fluorescence detection, coupled with reversed‐phase high‐performance liquid chromatography (RP‐HPLC), were employed to obtain the most reliable qualitative and quantitative results. Chemotaxonomic differences combined with flower color variability were demonstrated within thirteen clover species. Concentration levels of particular isoflavones in ten Trifolium species possessing flowers with white, pink, or purple‐red corolla ranged from ∼︁3 to ∼︁3300 μg/g dry weight, while in three yellow flowering clovers (T. aureum, T. dubium, and T. campestre) isoflavone compounds have not been detected at all. RSD values, determined for intra‐ and inter‐day precision of the quantitative results, were not higher than 6.2% and 7.1%, respectively.     

Protection against photoaging in the hairless mouse by the isoflavone equol

Topical application of the isoflavone equol immediately following solar-simulated UV (SSUV) radiation exposure has previously been demonstrated to have significant photoprotective effects. Equol reduced both the inflammatory edema and the systemic suppression of the contact hypersensitivity reaction in hairless mice. Furthermore, daily topical equol application immediately following irradiation during a 10-week chronic SSUV exposure regime also reduced photocarcinogenesis severity in the mouse. This study examines the potential for topical equol to prevent photoaging in response to chronic SSUV irradiation for up to 30 weeks. We did not find consistent expression of the characteristic markers of photoaging until 30 weeks, although moderate epidermal hyperplasia and a transient increase in dermal mast cell numbers were evident after 1 week. Daily application of 10 muM equol lotion significantly reduced these early changes. However after 30 weeks of SSUV exposure, photoaging was well developed, as shown histologically by markedly increased epidermal hyperplasia, increased dermal mast cell number, pronounced focal elastotic deposits, degraded dermal collagen and deposition of glycosaminoglycans in the lower dermis. Topical equol treatment protected significantly from each of these impairments, as demonstrated histologically and quantitatively. Additionally, equol was found to have strong antioxidant action against acute UVA (320-400 nm)-induced lipid peroxidation of mouse skin, this property accounting for its antiphotoaging mechanism. The evidence for equol's antiphotoaging activity, taken together with its anti-inflammatory, immunoprotective and anticarcinogenic efficacy against SSUV irradiation in the mouse, suggests that equol could be developed as a helpful topical photoprotective agent for daily use by humans.