Journal of Chromatography B (v.1005, #C)

A novel UHPLC method for the rapid and simultaneous determination of daidzein, genistein and equol in human urine by Begoña Redruello; Lucía Guadamuro; Isabel Cuesta; Jorge R. Álvarez-Buylla; Baltasar Mayo; Susana Delgado (1-8).
This work reports on a novel method involving reverse-phased ultra-high performance liquid chromatography (UHPLC) plus a spectrophotometric photodiode array/fluorescence (FLR) detection system for determining the concentration of equol and major soy isoflavones (daidzein and genistein) in human urine. The proposed method was validated in terms of its linearity, sensitivity, accuracy (recovery) and precision (intra- and inter-day repeatability). The isoflavone profiles of urine samples from a group of menopausal women following oral soy isoflavone supplementation were determined and compared. Screening for equol-producer status was accomplished with high sensitivity (detection limit of the FLR detector 2.93 nM). The method involves a short chromatographic run time compared to conventional HPLC methods while allowing for the simultaneous and reliable quantification of daidzein, genistein and equol in human urine. It also allows for the rapid screening of multiple urine samples when testing for equol production status and checking patient adherence to isoflavone treatment regimens.
Keywords: UHPLC; Solid phase extraction; Human urine; Soy isoflavones; Equol; Menopausal women;

Gypenoside LVI and gypenoside XLVI are the major bioactive dammarane saponins from Gynostemma pentaphyllum. Gypenoside LVI, gypenoside XLVI, and their metabolite 2α-OH-protopanaxadiol (2α-OH-PPD) possess potent non-small cell lung carcinoma A549 cell inhibitory activity. A sensitive liquid chromatography tandem mass spectrometry method was developed and validated to study the pharmacokinetics of gypenoside LVI and XLVI, 2α-OH-PPD, metabolite 1 (M1), and metabolite 2 (M2) after administration of gypenosides or 2α-OH-PPD. Plasma samples from rats were protein precipitated with methanol. Analytes were detected by triple quadrupole MS/MS with an electrospray ionization source in the positive multiple reaction monitoring mode. The transition m/z 441.4 → 109.2 was selected to quantify gypenoside LVI and XLVI, and 2α-OH-PPD, because of the extensive conversion of the gypenosides to aglycone in the ionization source. M1 and M2 are isomers that shared the transition m/z 493.4 → 143.1. To avoid interference, the baseline separation of each analyte was performed on a SunFire C18 column with a gradient of acetonitrile (0.1% formic acid, v/v) and water (0.1% formic acid, v/v). The chromatographic run time was 10 min. The linearity was validated over a plasma concentration range from 2.00 to 2000 ng/mL for M1 and M2, and from 10.0 to 2000 for gypenosides LVI and XLVI, and 2α-OH-protopanaxadiol. The lower limits of quantification were 10.0, 10.0, 10.0, 2.00, and 2.00 ng/mL for gypenoside LVI, gypenoside XLVI, 2α-OH-PPD, M1, and M2, respectively, with acceptable intra-/inter-day precision and accuracy. The extraction recovery rates were >86.9% for each compound. No apparent matrix effect or instability was observed during each step of the bioanalysis. After full validation, this method was proved to be simple, fast, and efficient in analyzing large batches of plasma samples for the analytes.
Keywords: Gypenoside; 2α-OH-Protopanaxadiol; Gynostemma pentaphyllum; Validation; Pharmacokinetic;

Soxhlet-assisted matrix solid phase dispersion to extract flavonoids from rape (Brassica campestris) bee pollen by Shuangqin Ma; Xijuan Tu; Jiangtao Dong; Peng Long; Wenchao Yang; Xiaoqing Miao; Wenbin Chen; Zhenhong Wu (17-22).
Soxhlet-assisted matrix solid phase dispersion (SA-MSPD) method was developed to extract flavonoids from rape (Brassica campestris) bee pollen. Extraction parameters including the extraction solvent, the extraction time, and the solid support conditions were investigated and optimized. The best extraction yields were obtained using ethanol as the extraction solvent, silica gel as the solid support with 1:2 samples to solid support ratio, and the extraction time of one hour. Comparing with the conventional solvent extraction and Soxhlet method, our results show that SA-MSPD method is a more effective technique with clean-up ability. In the test of six different samples of rape bee pollen, the extracted content of flavonoids was close to 10 mg/g. The present work provided a simple and effective method for extracting flavonoids from rape bee pollen, and it could be applied in the studies of other kinds of bee pollen.
Keywords: Matrix solid phase dispersion; Continuous extraction; Soxhlet extraction; Bee pollen; Flavonoids;

Irisolidone, a major isoflavone found in Pueraria lobata flowers, exhibits a wide spectrum of bioactivities, while its metabolic pathways and the pharmacokinetics of its metabolites in vivo have not been investigated yet. In the present study, an ultra performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF MS) method was employed to investigate the metabolic pathways of irisolidone and the pharmacokinetics of its main metabolites in rats, after a single 100 mg/kg oral dose of irisolidone. Protein precipitation method was used to prepare plasma samples. A total of 15 metabolites included irisolidone were detected and tentatively identified based on the mass spectral fragmentation patterns, elution order or confirmed using available reference standards. The pharmacokinetics of the main metabolites included three glucuronide metabolites tectorigenin-7-O-glucuronide (Te-7G), 6-hydroxybiochanin A-6-O-glucuronide (6-OH-BiA-6G), irisolidone-7-O-glucuronide (Ir-7G), and three sulfate metabolite tectorigenin-7-O-sulfate-4′-O-sulfate (Te-7S-4′S), tectorigenin-7-O-sulfate (Te-7S) and irisolidone-7-O-sulfate (Ir-7S), and aglycone tectorigenin (Te), and irisolidone (Ir) were evaluated. The plasma concentrations reached maximal values of 0.297 μmol/L at 10.3 h for Te-7S-4′S, 0.199 μmol/L at 21.7 h for Te-7G, 0.154 μmol/L at 8.00 h for Te-7S, 4.10 μmol/L at 15.3 h for 6-OH-BiA-6G, 10.7 μmol/L at 9.71 h for Ir-7G, 0.918 μmol/L at 11.3 h for Te, 0.150 μmol/L at 8.67 h for Ir-7S, and 0.843 μmol/L at 9.67 h for Ir, respectively. Since the total plasma concentrations of conjugated metabolites were much higher than that of the irisolidone aglycone, an extensive phase II metabolism plays an important role in the pharmacokinetics of irisolidone in vivo.
Keywords: Irisolidone; Plasma pharmacokinetics; Metabolites; Rat; UHPLC/Q-TOF MS;

Quantitative analysis of unconjugated and total bisphenol A in human urine using solid-phase extraction and UPLC–MS/MS: Method implementation, method qualification and troubleshooting by Brigitte Buscher; Dick van de Lagemaat; Wolfgang Gries; Dieter Beyer; Dan A. Markham; Robert A. Budinsky; Stephen S. Dimond; Rajesh V. Nath; Stephanie A. Snyder; Steven G. Hentges (30-38).
The aim of the presented investigation was to document challenges encountered during implementation and qualification of a method for bisphenol A (BPA) analysis and to develop and discuss precautions taken to avoid and to monitor contamination with BPA during sample handling and analysis. Previously developed and published HPLC–MS/MS methods for the determination of unconjugated BPA (Markham et al. Journal of Analytical Toxicology, 34 (2010) 293–303) [17] and total BPA (Markham et al. Journal of Analytical Toxicology, 38 (2014) 194–203) [20] in human urine were combined and transferred into another laboratory. The initial method for unconjugated BPA was developed and evaluated in two independent laboratories simultaneously. The second method for total BPA was developed and evaluated in one of these laboratories to conserve resources. Accurate analysis of BPA at sub-ppb levels is a challenging task as BPA is a widely used material and is ubiquitous in the environment at trace concentrations. Propensity for contamination of biological samples with BPA is reported in the literature during sample collection, storage, and/or analysis. Contamination by trace levels of BPA is so pervasive that even with extraordinary care, it is difficult to completely exclude the introduction of BPA into biological samples and, consequently, contamination might have an impact on BPA biomonitoring data. The applied UPLC–MS/MS method was calibrated from 0.05 to 25 ng/ml. The limit of quantification was 0.1 ng/ml for unconjugated BPA and 0.2 ng/ml for total BPA, respectively, in human urine. Finally, the method was applied to urine samples derived from 20 volunteers. Overall, BPA can be analyzed in human urine with acceptable recovery and repeatability if sufficient measures are taken to avoid contamination throughout the procedure from sample collection until UPLC–MS/MS analysis.

Crystallization-induced dynamic resolution R-epimer from 25-OCH3-PPD epimeric mixture by Sainan Zhang; Yun Tang; Jiaqing Cao; Chen Zhao; Yuqing Zhao (39-46).
25-OCH3-PPD is a promising antitumor dammarane sapogenin isolated from the total saponin-hydrolyzed extract of Panax ginseng berry and Panax notoginseng leaves. 20(R)-25-OCH3-PPD was more potent as an anti-cancer agent than 20(S)-25-OCH3-PPD and epimeric mixture of 25-OCH3-PPD. This paper describes the rapid separation process of the R-epimer of 25-OCH3-PPD from its epimeric mixture by crystallization-induced dynamic resolution (CIDR). The optimized CIDR process was based on single factor analysis and nine well-planned orthogonal design experiments (OA9 matrix). A rapid and sensitive reverse phase high-performance liquid chromatographic (HPLC) method with evaporative light-scattering detector (ELSD) was developed and validated for the quantitation of 25-OCH3-PPD epimeric mixture and crystalline product. Separation and quantitation were achieved with a silica column using a mobile phase consisting of methanol and water (87:13, v/v) at a flow rate of 1.0 mL/min. The ELSD detection was performed at 50 °C and 3 L/min. Under conditions involving 3 mL of 95% ethanol, 8% HCl, and a hermetically sealed environment for 72 h, the maximum production of 25(R)-OCH3-PPD was achieved with a chemical purity of 97% and a total yield of 87% through the CIDR process. The 25(R)-OCH3-PPD was nearly completely separated from the 220 mg 25-OCH3-PPD epimeric mixture. Overall, a simple and steady small-batch purification process for the large-scale production of 25(R)-OCH3-PPD from 25-OCH3-PPD epimeric mixture was developed.
Keywords: 25-OCH3-PPD epimeric mixture; Crystallization-induced dynamic resolution; Anticancer activity; HPLC-ELSD; Separation and quantitation;

To understand the role of l-arginine depletion in impaired nitric oxide synthesis in disease, it is important to simultaneously quantify arginine, citrulline, and ornithine in the plasma. Because the three amino acids are endogenous analytes, true blank matrix for them is not available. It is necessary and valuable to compare the performance of different approaches due to lack of regulatory clarity for validation. A two-step sample preparation method using methanol as protein precipitation reagent was developed in this study is used for sample preparation. Because true blank matrix for endogenous analytes is not available, water as blank matrix, 1% BSA in PBS as blank matrix, surrogate analyte, and background subtraction were designed to establish successful quantification methods. Four methods to simultaneously quantify arginine, citrulline, and ornithine in human plasma using hydrophilic interaction liquid chromatography and electrospray tandem mass spectrometry were developed, validated, and compared. The developed two-step sample preparation method using methanol as protein precipitation reagent in this study needs less time and provides higher recovery comparing with other approaches. Three of the four methods, water as blank matrix, 1% BSA in PBS as blank matrix, and surrogate analyte, have been successful in fulfilling all the criteria, while background subtraction has failed. Results of the measured concentrations in 97 human plasma samples using the three methods show that the difference between any two methods or among the three methods presents 100% of samples with less than 20% for all the three amino acids and majority of them are under 10%. The developed two-step sample preparation method using methanol as protein precipitation reagent is simple and convenient. Three of the four methods are fully validated and the validation is successful. The BSA functioned effectively as a blank matrix for these three amino acids, considering cost, data quality, matrix similarity, and practicality.
Keywords: Arginine; Citrulline; Ornithine; HILIC-MS/MS; Plasma; Validation;

Leek, leaf lettuce and garland chrysanthemum are troublesome vegetables containing large amount of pigments which may bring serious matrix interferences in mass spectrometry analysis. Multi-walled carbon nanotubes (MWCNTs) have a good effect for the cleanup of troublesome matrix. So the study was designed to develop a multi-residue method for the determination of 70 pesticide residues in leek, leaf lettuce and garland chrysanthemum based on a modified QuEChERS procedure using MWCNTs as reversed-dispersive solid phase extraction (r-DSPE) materials to remove the interferences of pigments. PSA and GCB were used as comparison. LC–MS/MS was used to identify and quantify the residue levels of multi-pesticides. The clean-up performance of MWCNTs was demonstrated to be obviously superior to GCB and PSA. This method was validated on leek, leaf lettuce and garland chrysanthemum spiked at the concentration of 10, 50 and 100 μg kg−1 with five replicates. The recoveries of 70 pesticides ranged from 74% to 119%, with relative standard deviations (RSDs) lower than 14.2%. Good linearity (R 2  ≥ 0.9903) was obtained at the range of 10–1000 μg/L for all pesticides in the selected matrices. The limit of quantification (LOQs) and limit of detection (LODs) of the 70 pesticides for the selected matrices ranged from 0.3 to 7.9 μg kg−1 and from 0.1 to 2.4 μg kg−1 respectively. The method was successfully applied to the routine monitoring of pesticide residues in market samples.
Keywords: MWCNTs; QuEChERS; r-DSPE; LC–MS/MS; Pesticide residues;