Validation of a high-performance liquid chromatographic method for determination of isoflavonoids in soybeans. Study of the extraction procedure by experimental design

Summary An improved analytical method, reversed-phase high-performance liquid chromatography on a narrow-bore C₁₈ column, has been developed for the simultaneous determination of genistein, daidzein, formononetin, and biochanin A. The method was validated in terms of detection limits, quantitation limits (LOQ), linearity, and precision.LOQ in the 0.04–0.1 μg mL⁻¹ range were calculated, enabling determination of these compounds of nutritional concern at trace levels. Good linearity was demonstrated over three orders of magnitude of concentration for each analyte (r ²=0.998–1.000). The intra-day repeatability was evaluated in terms ofRSD (%) at two concentration levels for each analyte (RSD (%) <1.8%). Good inter-day reproducibility of data was proved by performing homoscedasticity and ANOVA tests (P>0.05 at the 95% confidence level). The method was applied to the determination of genistein and daidzein in yellow soybeans, after optimization of the method for extraction of isoflavonoid aglycones from soybeans by experimental design, i.e. central composite design. Extraction recoveries up to 87±4% were obtained when the corresponding glycosidic forms (genistin and daidzin) were added to soybean samples.     

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.     

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%.     

Use of experimental design for the purge-and-trap-gas chromatography-mass spectrometry determination of methyl tert.-butyl ether,tert.-butyl alcohol and BTEX in groundwater at trace level

An efficient method for the simultaneous determination of methyl tert.-butyl ether, tert.-butyl alcohol, benzene, toluene, ethylbenzene and xylene isomers in groundwater by purge-and-trap-gas chromatography-mass spectrometry was developed and validated. Experimental design was used to investigate the effects of temperature of extraction, time of extraction and percentage of salt added to the water samples. Regression models and desirability functions were applied to find the experimental conditions providing the highest global extraction yield. Validation was carried out in terms of limits of detection (LOD), limits of quantitation (LOQ), linearity and precision. LOD values ranging from 2.6 to 23 ng l(-1) were achieved, whereas linearity was statistically verified over two orders of magnitude for each compound. Precision was evaluated testing two concentration levels. Good results were obtained both in terms of intra-day repeatability and intermediate precision: RSD% lower than 4.5% at the highest concentration and lower than 13% at the lowest one were calculated for intra-day repeatability. A groundwater sample suspected of contamination by leaking underground petroleum storage tanks was analysed and some of the analytes were detected and quantitated.     

Liquid chromatography-UV determination and liquid chromatography-atmospheric pressure chemical ionization mass spectrometric characterization of sitosterol and stigmasterol in soybean oil.

A narrow-bore HPLC-UV method was developed for the analysis of two of the more abundant naturally occurring phytosterols in vegetable oils: sitosterol and stigmasterol. The method enabled detection of the compounds at a concentration of 0.42 microg/ml and quantitation at concentrations of 0.52 and 0.54 microg/ml for sitosterol and stigmasterol, respectively. An excellent linearity was determined over two orders of concentration magnitude (r2 0.999-1.000) and verified by applying the Mandel fitting test (p>0.099) and the lack-of-fit test (p>0.057) performed at the 95% confidence level. A good intra-day precision ranging from 0.15 to 1.16% was calculated at two concentration levels (2 and 100 microg/ml). The inter-day reproducibility was verified on 3 different days by performing an homoscedasticity test and analysis of variance. A solid-phase extraction method was developed on silica cartridges for the isolation of phytosterols from soybean oil providing recovery values of 101+/-9 and 106+/-7% for sitosterol and stigmasterol, respectively. Good accuracy of the method was statistically demonstrated since no matrix effect was found for both the analytes. The developed method was applied to the quantitative assay of phytosterols in a soybean oil sample (61+/-5 mg/100 g of stigmasterol and 118+/-4 mg/100 g sitosterol). The HPLC-atmospheric pressure chemical ionization MS technique enabled the identification of stigmasterol, sitosterol and campesterol in the oil sample.     

气相色谱-负化学源-质谱法检测水中10种全氟羧酸化合物

建立了气相色谱-负化学源-质谱（GC-NCI-MS）检测水中10种全氟羧酸化合物的分析方法。使用硅烷衍生化试剂N-甲基-N-三甲基硅基三氟乙酰胺（MSTFA）对全氟羧酸化合物进行衍生化，水样经弱阴离子交换固相萃取柱净化富集后进样。实验优化了样品前处理、衍生化和仪器条件。结果表明，10种全氟羧酸化合物在0.1~10 mg/L范围内线性关系良好，相关系数为0.9956~0.9993；方法的检测限（LOD）和定量限（LOQ）分别为0.5~1.5 μg/L和1.5~4.5 μg/L。在空白水样中进行了3个添加水平的加标回收试验，10种全氟羧酸化合物的平均回收率为70.2%~112.6%，相对标准偏差（RSD）为2.1%~14.5%（n=6）。该法原理简单，灵敏度高，准确、精密，可实现水体中10种全氟羧酸化合物同时检测的要求。     

Simultaneous determination of nine intense sweeteners in foodstuffs by high performance liquid chromatography and evaporative light scattering detection--development and single-laboratory validation

A high performance liquid chromatographic method with evaporative light scattering detection (HPLC-ELSD) has been developed for the simultaneous determination of multiple sweeteners, i.e., acesulfame-K, alitame, aspartame, cyclamic acid, dulcin, neotame, neohesperidine dihydrochalcone, saccharin and sucralose in carbonated and non-carbonated soft drinks, canned or bottled fruits and yoghurt. The procedure involves an extraction of the nine sweeteners with a buffer solution, sample clean-up using solid-phase extraction cartridges followed by an HPLC-ELSD analysis. The trueness of the method was satisfactory with recoveries ranging from 93 to 109% for concentration levels around the maximum usable dosages for authorised sweeteners and from 100 to 112% for unauthorised compounds at concentration levels close to the limit of quantification (LOQs). Precision measures showed mean repeatability values of <4% (expressed as relative standard deviation) for highly concentrated samples and <5% at concentration levels close to the LOQs. Intermediate precision was in most cases <8%. The limits of detection (LODs) were below 15 microg g(-1) and the LOQs below 30 microg g(-1) in all three matrices. Only dulcin showed slightly higher values, i.e., LODs around 30 microg g(-1) and LOQs around 50 microg g(-1)     

Comparison of solid-phase extraction sorbents for the fractionation and determination of important free and glycosidically-bound varietal aroma compounds in wines by gas chromatography-mass spectrometry

A critical comparison was made of seven solid-phase extraction (SPE) sorbents for the fractionation and isolation of 21 important free and glycosidically-bound varietal volatile aroma compounds. The sample was subjected to SPE and the free aromatics were eluted with dichloromethane followed by elution of the glucoconjugates with methanol; after fractionation, the free fraction was analyzed directly by GC-MS while the sugar-bound fraction was enzymatically hydrolyzed to liberate the free compounds before analysis by GC-MS. The extraction efficiency for the free compounds was evaluated based on the analytes’ signal recovery and for the glycosidically-bound compounds in terms of the relative peak areas. The best results for both the free and bound fractions were obtained with the Isolute ENV+ resin. Following selection of the most efficient SPE material, a GC-MS method was validated (in terms of selectivity, linearity, limits of detection (LODs) and limits of quantification (LOQs), recovery, repeatability, within-laboratory reproducibility and uncertainty) for the quantitative determination of the free primary volatiles in white wines. Validation results are presented at 4 fortification levels (10, 50, 200 and 500 μL^?1). Regarding linearity, the correlation coefficient of the matrix-matched calibration plots was ≥0.99 for all the compounds. The LOQs were in the range 0.6–17.5 μg L^?1. Recoveries ranged from 61% to 120% while the% relative standard deviation of the within-laboratory reproducibility was in the range 1.3% to 17.7%. Finally, the% expanded uncertainty ranged from 3.1% to 40.3%. The method has been successfully applied to the analysis of 20 white wine samples.      

The experimental design approach to the optimization of a simple quick easy cheap effective rugged and safe method for the analysis of phytoestrogens in complex soy‐based food

The present paper deals with the multivariate optimization of an extraction‐purification strategy for the determination of phytoestrogens (daidzein, genistein, coumestrol, formononetin, and biochanin A) in soy‐based meat substitutes by high performance liquid chromatography with tandem mass spectrometry. For a reliable quantitation of these new concerning compounds in such a complex matrix, recovery and matrix effect must be carefully evaluated. Therefore, two sequential experimental designs were used to optimize the sample‐pretreatment of soy‐based burgers: the chosen technique was the quick, easy, cheap, effective, rugged and safe methodology, which does not require any particular facility or instrumentation. Thanks to the first screening design (Plackett‐Burman), the significant factors influencing the studied responses were identified and further investigated through a response surface design (Box‐Behnken). The optimal values of the variables (volume of extraction solvent mix/sample mass ratio and two clean‐up sorbents) led to quantitative recoveries (97–104%) and low ion suppression (matrix effect 60–93%) for all analytes. This optimized method was characterized by low detection limits (0.2–1.5 ng/g) and excellent intraday precision (RSD 2–4%). It was applied to the determination of the considered compounds in several soy‐burgers from the Italian market, detecting low ng/g levels (up to 40 ng/g) of coumestrol, formononetin, and biochanin A, and high concentrations (7.9–78 µg/g) of genistein and daidzein.     

高效液相色谱-柱后衍生法检测养殖水体及沉积物中11种磺胺药物残留

建立了养殖水体及沉积物中11种磺胺化合物的高效液相色谱-柱后衍生分析方法。养殖水体过滤后采用HLB固相萃取柱进行净化、富集;沉积物采用甲醇/EDTA-Mcllvaine缓冲液(1:1, v/v)提取,HLB固相萃取柱净化富集。经高效液相色谱分离,用荧光胺衍生试剂进行柱后衍生,荧光检测器检测。对柱后衍生系统参数进行了优化,确定了荧光胺溶液的浓度、流速和反应温度分别为0.2 g/L、0.15 mL/min和50 ℃,磺胺化合物在0.01~1.0 mg/L范围内线性显著,其相关系数r²值大于0.99995。11种磺胺类药物在养殖水体和沉积物中的加标回收率分别为79.3%~100.7%和74.6%~95.3%,相对标准偏差为2.2%~11.0%和2.6%~10.3%,检出限(LOD, S/N=3)为0.9~5.5 ng/L和0.3~1.3 μg/kg,定量限(LOQ, S/N=10)为3.0~18.1 ng/L和1.0~4.4 μg/kg。该法可应用于养殖环境中磺胺类药物的定性定量检测,具有较好的实用性。     

GC-MS法快速测定铅笔涂层中18种光引发剂

建立了快速测定铅笔涂层中18种光引发剂的气相色谱-质谱联用法(GC/MS)。样品以二氯甲烷为提取溶剂,在30℃温度下,超声提取30 min后,萃取液用100 mg N-丙基乙二胺(PSA)分散固相萃取净化,以气相色谱-质谱法(GC-MS)测定,外标法定量。18种光引发剂在0.2~4.0 mg/L(或0.1~4.0 mg/L)范围内线性良好,相关系数R²均大于0.997,方法检出限(LODs)为0.4~1.0 mg/kg,定量限(LQDs)为1.0~2.0 mg/kg。3个加标水平(2.0或4.0、10.0和40.0 mg/kg)的回收率在75.2%~115.2%之间,相对标准偏差(RSDs)为1.0%~9.3%(n=6)。该方法可用于铅笔涂层中18种光引发剂含量的高通量检测。     

Determination of the isoflavone genistein in soybeans by high-performance liquid chromatography following cloud point extraction

A simple and sensitive method has been developed for preconcentration and determination of genistein in soybeans. This method is based on cloud point extraction (CPE) of genistein from soybeans using ethylene glycol monoalkyl ether (Genapol X-080) as a nonionic surfactant. The concentration of extracted genistein was determined by HPLC with a UV detector. Optimum experimental conditions were established. With 5% Genapol X-080 (v/v), a liquid/solid ratio of 25:1 mL/g, and ultrasonic-assisted extraction at 40 degrees C for 45 min, the extraction percentage of genistein reached its highest value. The preconcentration factor for genistein was about 16.5. The RSD for seven replicate measurements and the LOD were +/- 4.45% and 15.0 ng/mL, respectively. CPE is simple, inexpensive, and suitable for extraction of genistein from soybean. It uses environmentally friendly surfactants and offers a convenient alternative to more conventional extraction systems.     

Development and validation of a solid phase micro-extraction-gas chromatography-mass spectrometry method for the determination of furan in baby-food

An efficient and simple method for the determination of furan in baby-food (vegetables and fruits) by solid phase micro-extraction-gas chromatography-mass spectrometry (SPME-GC-MS) was developed and validated. Experimental design was used to investigate the effects of temperature and time of extraction. The calculated regression model was used to find the experimental conditions providing the optimal SPME extraction yield. Validation was carried out in terms of limit of detection (LOD), limit of quantitation (LOQ), linearity, precision and trueness. LOD and LOQ values in the low ngkg(-1) were achieved, whereas linearity was established over two order of magnitude. Good precision was obtained both in terms of intra-day repeatability and between-day precision on two concentration levels (RSD% lower than 3.6%). Recovery values of 91.5+/-6.2% and of 96.1+/-1.3% (n=3) were calculated at 75 ngkg(-1) and 75 microgkg(-1) level. Finally, the applicability of the method to the determination of furan in a number of commercial and home-made baby-food samples was demonstrated.     

高效液相色谱-串联质谱法测定蜂蜜中9种农药残留

建立了同时测定蜂蜜中9种苯并咪唑类和新烟碱类农药的全自动固相萃取-高效液相色谱-串联质谱检测方法。蜂蜜样品用磷酸盐缓冲液（pH=7.8）溶解，超声提取，经亲水亲脂平衡（hydrophilic-lipophilic balance，HLB）固相萃取小柱净化，氮吹浓缩，定容，过滤膜后进行高效液相色谱-串联质谱分析，采用多反应监测（MRM）模式测定，以内标法定量。结果表明，在0.002~0.05 mg/L范围内9种农药呈现出较好的线性关系（相关系数r² ≥ 0.99），检出限和定量限分别为0.1~1.0 μg/kg和0.3~2.0 μg/kg。对阴性蜂蜜，在5.0、10.0、20.0 μg/kg 3个水平下分别进行加标回收试验，测出9种农药的平均回收率在78.2%~101.2%之间，相对标准偏差为1.3%~14.3%（n=6）。该方法可适用于大批量蜂蜜样品的快速准确测定。     

气相色谱-质谱法同时检测尿液中8种卡西酮类新精神活性物质

卡西酮类新精神活性物质是在诸多国家滥用较为广泛的一类人工合成的新精神活性物质。该文采用气相色谱-质谱法建立了尿液中8种卡西酮类新精神活性物质同时定性、定量的分析方法。取尿液试样适量,加入内标N,N-二甲基苯胺,经环己烷溶剂萃取,离心取上清液进样分析。结果表明,尿液中8种目标物在各自浓度范围内线性关系良好,检出限(LOD)为0.002~0.01μg/mL,定量下限(LOQ)为0.005~0.02μg/mL,平均回收率为92.4%~99.4%,日内精密度不高于7.8%,日间精密度不高于8.8%。所建立的方法具有快速、灵敏、高效、准确等特点,不仅能为涉毒案件相关物证的鉴定、案件侦破与诉讼等提供一定的科学理论与技术支持,而且能为打击毒品违法犯罪提供一定帮助。     

超声提取正相高效液相色谱法测定大豆及大豆油中的磷酸甘油酯

建立了超声提取-固相萃取纯化/正相高效液相色谱测定大豆及大豆油中磷脂酰胆碱(PC)、磷脂酰乙醇胺(PE)、磷脂酰肌醇(PI)的方法。考察了提取溶剂、超声功率、超声时间、提取温度及净化方式的影响,并研究了不同色谱固定相对磷酸甘油酯分离效果的影响。优化的实验条件为:以氯仿-甲醇(2∶1,体积比)为提取溶剂,1 500 W功率超声提取30 min;氨基固相萃取柱为纯化小柱;正己烷-异丙醇-1%HAc(8∶8∶1,体积比)为流动相。在该条件下,PC、PE、PI的线性范围分别为0.08～8.00、0.15～15.00、0.30～20.00 g.L-1,定量下限分别为0.021、0.050、0.060 g.L-1,检出限在8～23 mg.L-1之间,其在大豆和大豆油中的回收率为85%～108%。日内与日间精密度分别不大于4.7%和8.6%。     

Compositional changes induced by UV-B radiation treatment of common bean and soybean seedlings monitored by capillary electrophoresis with diode array detection

In this work, a new CE method with diode array detection (DAD) was developed for the monitoring and quantitation of flavonoids in different beans treated and untreated with UV-B radiation. Flavonoid concentration was monitored in UV-B-treated and untreated sprouts of three common beans (Zolfino ecotype, cv. Verdone, cv. Lingua di Fuoco) and one soybean (cv. Pacific). After acid hydrolysis of extracts, the CE-DAD method provides reproducible quantitative determinations of daidzein, glycitein, genistein, and kaempferol at ppm level in these natural matrices within a relatively short time (less than 16 min). Total flavonoid content determined by CE-DAD was 159 +/- 8, 26 +/- 2, 13 +/- 1, and 1.3 +/- 0.3 microg/g fresh weight for untreated sprouts of Pacific soybean, Verdone bean, Zolfino bean, and Lingua di Fuoco bean, respectively. UV-B treatment caused no significant quantitative effect on Pacific soybean sprouts, whereas it enhanced the total isoflavone content by 1.5, 1.8, and 3.2-fold in Verdone, Zolfino, and Lingua di Fuoco beans, respectively. The proposed method shows (i) the potentialities of bean sprouts as a natural source of bioactive compounds (antioxidants); (ii) the technological role of UV-B treatment for sprout isoflavone enrichment; and (iii) the good capabilities of CE-DAD to monitor this process.     

Headspace Single-Drop Microextraction with Gas Chromatography for Determination of Volatile Halocarbons in Water Samples

A simple, rapid and inexpensive procedure for extraction and analysis of volatile halocarbons in water samples was presented using the headspace single-drop microextraction (HS-SDME) technique and gas chromatography with microcell electron capture detector (GC-μECD). Operation parameters. such as extraction solvent. headspace volume. organic drop volume. salt concentration. temperature and sampling time, were studied and optimized. Extraction of 10 volatile halocarbon compounds was achieved using the optimized method. Calibration curves of 10 target compounds yielded good linearity in the respective range of concentration (R ² ≥ 0.9968, chlorodibromomethane in the concentration range of 0.05–50 μg/L). The limits of detection were found between 0.002 (tetrachloroethene) and 0.374μg/L (1,1,2-trichloroethane). and relative standard deviations (RSD%) ranged between 4.3 (chloroform) and 9.7% (1,1,2,2-tetrachloroethane). Spiked recoveries of tap water and ground water agreed well with the known values between 118.97 (20.0μg/L of 1,1,2-trichloroethane) and 82.61% (10.0μg/L of tetrachloroethene), demonstrating that the HS-SDME combined GC-μECD was a useful and reliable technique for the rapid determination of volatile halocarbon compounds in water samples.     

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.     

凝固漂浮有机液滴-分散液液微萃取-高效液相色谱-串联质谱法测定海水中苯并三唑类紫外线过滤剂

建立了海水中7种苯并三唑类紫外线过滤剂的凝固漂浮有机液滴-分散液液微萃取-高效液相色谱-串联质谱分析方法。优化后的萃取实验条件：20 μL十二醇为萃取溶剂,400 μL甲醇为分散溶剂,NaCl质量分数为8%,pH小于6,涡旋振荡时间2 min。目标化合物经Hypersil GOLD色谱柱（150 mm×2.1 mm,5 μm）结合甲醇-水梯度洗脱分离后,用正离子多反应监测模式进行质谱分析。在较宽的线性范围内,7种化合物的线性相关系数（r²）均大于0.99;基质加标回收率为68.3%~127.5%,相对标准偏差为0.9%~15.2%,方法的检出限为0.001~0.090 μg/L,定量限为0.003~0.300 μg/L。将建立的方法用于大连8个海滨浴场海水中苯并三唑类紫外线过滤剂的测定,部分浴场海水有不同程度的检出。该方法简便、快速、环境友好、灵敏度高,可用于海水中苯并三唑类紫外线过滤剂的分析检测。