Journal of Chromatography B (v.1014, #C)

In conventional ionic liquid-based dispersive liquid-liquid microextraction (IL-DLLME) procedures, most of the IL disperser remains in the aqueous phase resulting in low recovery for moderately and weakly polar analytes due to the “carry-over effect”. Herein, we successfully developed a “NH4PF6-enhanced, non-organic solvent, dual microextraction” method (ANSDM) for pretreatment of phthalate (PAE) metabolites with weak to moderate polarity. This method utilized in situ reaction of NH4PF6 as an ion-exchange reagent and disperser to realize two microextractions after using [C8MIM]PF6 as an extraction solvent and [C4MIM]BF4 as a disperser for conventional DLLME. Single-factor experiments, a two-level full factorial experimental design and central composite design were applied for optimizing operational parameters using 3D response surfaces and contour lines. Under optimized conditions, the newly developed method provided high extraction recoveries (93.8–99.1%) and low LODs (ca. 0.3 μg L−1) for three phthalate metabolites in human urine. The primary advantages of the ANSDM method include: (1) integration of in situ reaction and conventional DLLME techniques to effectively extract both weak and moderately polar pollutants simultaneously; (2) non-organic solvent use in the microextraction procedure making the process safer and more environmental friendly; and (3) a time-saving, simple operation that is fully compatibility with HPLC analysis. To the best of our knowledge, our group is the first to develop the “non-organic solvent, dual microextraction” method and it has great potential as a sample pre-treatment technique for organic pollutants with weak to moderate polarity in biological and environmental matrices.
Keywords: Phthalate metabolites; NH4PF6-enhanced; Non-organic solvent; Dual microextraction; Central composite design; Weak and moderately polar chemicals; Human urine;

A rapid and simple reversed phase liquid chromatographic system has been developed for simultaneous analysis of terpenoid indole alkaloids (TIAs) and their precursors. This method allowed separation of 11 compounds consisting of eight TIAs (ajmalicine, serpentine, catharanthine, vindoline, vindolinine, vincristine, vinblastine, and anhydrovinblastine) and three related precursors i.e., tryptophan, tryptamine and loganin. The system has been applied for screening the TIAs and precursors in Catharanthus roseus plant extracts. In this study, different organs i.e., flowers, leaves, stems, and roots of C. roseus were investigated. The results indicate that TIAs and precursor accumulation varies qualitatively and quantitatively in different organs of C. roseus. The precursors showed much lower levels than TIAs in all organs. Leaves and flowers accumulate higher level of vindoline, catharanthine and anhydrovinblastine while roots have higher level of ajmalicine, vindolinine and serpentine. Moreover, the alkaloid profiles of leaves harvested at different ages and different growth stages were studied. The results show that the levels of monoindole alkaloids decreased while bisindole alkaloids increased with leaf aging and upon plant growth. The HPLC method has been successfully applied to detect TIAs and precursors in different types of C. roseus samples to facilitate further study of the TIA pathway and its regulation in C. roseus plants.
Keywords: HPLC-DAD; Simultaneous; Terpenoid indole alkaloids; Alkaloids precursors; Catharanthus roseus;

Metabolomics study on the antitumor effect of marine natural compound flexibilide in HCT-116 colon cancer cell line by Dan Gao; Yini Wang; Weiyi Xie; Ti Yang; Yuyang Jiang; Yuewei Guo; Jin Guan; Hongxia Liu (17-23).
A marine natural compound flexibilide isolated from the soft coral Sinularia flexibilis has been found to have antitumor activity. However, its pharmacological mechanism on tumor cells has not been studied. Herein, an ultra-performance liquid chromatography coupled to quadrupole time of-flight mass spectrometry (UPLC/Q-TOF MS) based metabolomics approach was established to investigate the antitumor effect of flexibilide on HCT-116 cells and its action mechanism. Q-TOF MS and MS/MS were used to identify significantly different metabolites. Comparing flexibilide-treated HCT-116 cells group with control group (dimethyl sulfoxide), 19 distinct metabolites involved in sphingolipid metabolism, alanine, aspartate and glutamate metabolism, d-glutamine and d-glutamate metabolism, glycerophospholipid metabolism, pyrimidine metabolism and others were discovered and identified. The significant decrease of phosphatidylcholine (PC) and phosphocholine levels and increase of lysophosphatidylcholine (LysoPC) levels in flexibilide treated cells suggested down-regulation of PC biosynthesis pathway. The decrease of sphingolipids reflected the lesions of cell membrane, and the up-regulation of sphingosine-1-phosphate indicated that TRAF2 and caspase-8 were likely to be activated by flexibilide and further caused cell apoptosis. Furthermore, TCA cycle was deemed to be down-regulated after flexibilide treatment, which might lead to an unsustainable of mitochondrial transmembrane potential MMP). The further measured descreased MMP with the increasing concentration of flexibilide treatment indiciated the dysfunction of mitochondrial which might finally lead to apoptosis. The UPLC/Q-TOF MS based metabolomics approach provides new insights into the mechanistic studies of flexibilide on tumor cells, which benefit its further improvement and application.
Keywords: Metabolomics; UPLC/Q-TOF MS; Flexibilide; HCT-116 cells; Action mechanism;

Over the last few years, the use of organophosphate flame retardants (OPFRs) has been on the rise; however, there are knowledge gaps in both the human health effects of OPFRs and levels of human exposure. Currently, human biomonitoring data on the levels of OPFR metabolites in the Canadian population are still non-existent. Herein we describe a novel method to measure nine urinary OPFR metabolites using solid phase extraction and ultra performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). The method detection limits were between 0.08 and 0.25 ng/mL for target metabolites. The newly developed and validated method was applied to the analysis of 24 urine samples collected in 2010–12 from pregnant Canadian women. The most frequently detected OPFR metabolite in urine of study participants (detection frequency: 97%) was diphenyl phosphate (DPHP), with concentrations ranging between <0.13–25.2 ng/mL, followed (75%) by the sum of two metabolites (DoCP: Di-o-cresyl phosphate and DpCP: Di-p- cresyl phosphate) of tricresyl phosphate, with concentrations between <0.13–4.38 ng/mL. With the exception of desbutyl-tris-(2-butoxy-ethyl) phosphate which was not detected in any of the samples, all other OPFR metabolites measured were found among study participants with variable detection frequency, suggesting pregnant Canadian women may be exposed to OPFRs.
Keywords: Organophosphate flame retardants (OPFRs); Urinary metabolites; Pregnant women; UPLC-; MS/MS;

Determination of residual fipronil in chicken egg and muscle by LC–MS/MS by Meiyu Zhang; Kui Bian; Tong Zhou; Xuqin Song; Qingying Liu; Chenying Meng; Limin He (31-36).
A simple, sensitive and reliable method was developed and applied to the residue analysis of fipronil in chicken egg and muscle by liquid chromatography-tandem mass spectrometry (LC–MS/MS). Chicken egg and muscle samples were extracted with acetronitrile, then salting out by dehydration with anhydrous magnesium sulfate and sodium chloride. The extracts were purified by the C18 solid phase extraction cartridge prior to analysis by LC–MS/MS. The matrix-matched calibration curve showed a good linear within the concentration range from 0.01 to 2.00 μg kg−1 (r 2  ≥ 0.999). The average recoveries of fipronil at three spiked levels of 0.01, 0.1 and 1.0 μg kg−1 ranged from 79.7% to 98.0%, and the relative standard deviations were less than 8.8%. The decision limit (CCα) and detection capability (CCβ) of fipronil in chicken egg and muscle matrices were all 0.002 μg kg−1 and 0.01 μg kg−1, respectively. The method has also been successfully applied to monitoring fipronil in the real samples.
Keywords: Fipronil; Liquid chromatography–tandem mass spectrometry; Residue; Chicken; Egg;

Metabolic profile of anhydrosafflor yellow B in rats by ultra-fast liquid chromatography/quadrupole time-of-flight mass spectrometry by Shijun Yue; Liang Wu; Jun Wang; Yuping Tang; Cheng Qu; Xuqin Shi; Pengxuan Zhang; Yahui Ge; Yujie Cao; Hanqing Pang; Chenxiao Shan; Xiaobin Cui; Li Qian; Jin-ao Duan (37-44).
Anhydrosafflor yellow B (AHSYB) is one of the major active water-soluble pigments from Carthamus tinctorius, which has been found to inhibit ADP-induced platelet aggregation and possess significant antioxidant activity. However, the metabolic fate of AHSYB in vivo remains unknown. In order to explore whether AHSYB is extensively metabolized, the metabolites of AHSYB in plasma, urine, bile, and feces samples after intravenous administration to the rats were investigated by ultra-fast liquid chromatography/quadrupole time-of-flight mass spectrometry (UFLC/Qq-TOF–MS/MS) combined with Metabolitepilot™ software. In total, AHSYB and 22 metabolites including both phase I and phase II metabolism processes were found and tentatively identified from the bio-samples. The metabolic pathways were involved in oxidation, reduction, hydroxylation, methylation, dimethylation, O-acetylation, hydrolyzation, sulfation, glucuronidaton, glutathionation and combination with glucose. The results showed that the renal and biliary routes play an important role in the clearance and excretion of AHSYB as well as hepatocyte metabolism. All of these results were reported for the first time and would contribute to a further understanding of the in vivo intermediated processes and metabolic mechanism of AHSYB and its analogs.
Keywords: Carthamus tinctorius; Anhydrosafflor yellow B; AHSYB; Metabolic profile; UFLC; Qq-TOF–MS/MS;

Rutin, hyperoside and hesperidin were effectively extracted from Sorbus tianschanica leaves by an ionic liquid vacuum microwave-assisted method. A series of ionic liquids with various anions and alkyl chain length of the cations were studied and the extraction was performed in [C6mim][BF4] aqueous solution. After optimization by a factorial design and response surface methodology, total extraction yield of 2.37 mg/g with an error of 0.12 mg/g (0.71 ± 0.04 mg/g, 1.18 ± 0.06 mg/g and 0.48 ± 0.02 for rutin, hyperoside and hesperidin, respectively) was achieved under −0.08 MPa for vacuum, 19 min and 420 W for microwave irradiation time and power, and 15 mL/g for liquid–solid ratio. The proposed method here is more efficient and needs a shorter extraction time for rutin, hyperoside and hesperidin from S. tianschanica leaves than reference extraction techniques. In stability studies performed with standard rutin, hyperoside and hesperidin, the target analytes were stable under the optimum conditions. The proposed method had a high reproducibility and precision. In addition, separation of rutin, hyperoside and hesperidin from [C6mim][BF4] extraction solution was completed effectively by AB-8 macroporous resin adsorption and desorption process. Ionic liquid vacuum microwave-assisted extraction is a simple, rapid and efficient sample extraction technique.
Keywords: Ionic liquid; Vacuum microwave-assisted extraction; Macroporous resin; Rutin; Hyperoside; Hesperidin; Sorbus tianschanica;

High throughput LC–MS/MS method for the simultaneous analysis of multiple vitamin D analytes in serum by Carl Jenkinson; Angela E. Taylor; Zaki K. Hassan-Smith; John S. Adams; Paul M. Stewart; Martin Hewison; Brian G. Keevil (56-63).
Recent studies suggest that vitamin D-deficiency is linked to increased risk of common human health problems. To define vitamin D ‘status’ most routine analytical methods quantify one particular vitamin D metabolite, 25-hydroxyvitamin D3 (25OHD3). However, vitamin D is characterized by complex metabolic pathways, and simultaneous measurement of multiple vitamin D metabolites may provide a more accurate interpretation of vitamin D status.To address this we developed a high-throughput liquid chromatography-tandem mass spectrometry (LC–MS/MS) method to analyse multiple vitamin D analytes, with particular emphasis on the separation of epimer metabolites. A supportive liquid-liquid extraction (SLE) and LC–MS/MS method was developed to quantify 10 vitamin D metabolites as well as separation of an interfering 7α-hydroxy-4-cholesten-3-one (7αC4) isobar (precursor of bile acid), and validated by analysis of human serum samples.In a cohort of 116 healthy subjects, circulating concentrations of 25-hydroxyvitamin D3 (25OHD3), 3-epi-25-hydroxyvitamin D3 (3-epi-25OHD3), 24,25-dihydroxyvitamin D3 (24R,25(OH)2D3), 1,25-dihydroxyvitamin D3 (1α,25(OH)2D3), and 25-hydroxyvitamin D2 (25OHD2) were quantifiable using 220 μL of serum, with 25OHD3 and 24R,25(OH)2D3 showing significant seasonal variations.This high-throughput LC–MS/MS method provides a novel strategy for assessing the impact of vitamin D on human health and disease.
Keywords: LC–MS/MS; Vitamin D; Method validation; Chiral separation; Serum analysis;

Herb–drug interaction of Epimedium extract on the pharmacokinetic of dapoxetine in rats by Thomas Y. Hsueh; Jing-Kai Ho; Lie-Chwen Lin; Allen W. Chiu; Chi-Hung Lin; Tung-Hu Tsai (64-69).
The aim of study is to develop a high performance liquid chromatography tandem mass spectrometry (LC–MS/MS) method to investigate the pharmacokinetic interaction of Epimedium extract on the dapoxetine in rats. Experimental rats were divided into the following four parallel groups: (1) dapoxetine alone (10 mg/kg, i.v.); (2) oral administration of Epimedium extract (2 g/kg) for 3 consecutive days and on the fourth day dapoxetine was administered (10 mg/kg, i.v.); (3) dapoxetine alone (10 mg/kg, p.o.); (4) oral administration of Epimedium extract (2 g/kg) for 3 consecutive days and on the fourth day dapoxetine was administered (10 mg/kg, p.o.). The calibration curves of dapoxetine were acquired over a concentration ranges from 1 to 500 ng/mL with the R 2  = 0.999. The mean matrix effects and extraction recoveries of dapoxetine at three different concentrations (1, 10, 500 ng/mL) ranged from 107.3 to 110.9% and from 25.5 to 28.2% respectively. The interday and intraday relative standard deviation were both <6% while the bias were both <14%. The pharmacokinetic results demonstrated that pretreated with/without Epimedium extract for three consecutive days did not significant alter the pharmacokinetics of dapoxetine in rats. The oral bioavailability of dapoxetine was about 75% in rats.
Keywords: Dapoxetine; Epimedium sagittatum extract; Herbal drug interaction; Traditional Chinese medicine; Pharmacokinetics;

Study on the potential application of salivary inorganic anions in clinical diagnosis by capillary electrophoresis coupled with contactless conductivity detection by Lin Guo; Yu Wang; Yiliang Zheng; Zhipeng Huang; Yiyuan Cheng; Jiannong Ye; Qingcui Chu; Dongping Huang (70-74).
A capillary electrophoresis approach with capacitively coupled contactless conductivity detection method has been developed for the determination of inorganic metabolites (thiocyanate, nitrite and nitrate) in human saliva. Field amplified sample injection, as a simple sample stacking technique, was used in conjunction for online preconcentration of above inorganic anions. A selective separation for the target anions from other coexisting constituents present in saliva could be obtained within 14 min in a 10 mmol/L His—90 mmol/L HAc buffer (pH 3.70) at the separation voltage of −18 kV. The limits of detection and limits of quantification of the three analytes were within the range of 3.1–4.9 ng/mL (S/N  = 3) and 10–16 ng/mL (S/N  = 10), respectively. The average recovery data were in the range of 81–108% at three different concentrations. This method provides a simple, rapid and direct approach for metabolite analyses of nitric oxide and cyanide based on noninvasive saliva sample, which presents a potential fast screening tool for clinical test.
Keywords: Saliva; Clinical analysis; Inorganic anions; Field amplified sample injection; Capillary electrophoresis; Capacitively coupled contactless conductivity detection;

Iron is an essential element for higher plants, and its acquisition and transportation is one of the greatest limiting factors for plant growth because of its low solubility in normal soil pHs. Higher plants biosynthesize ferric iron [Fe(III)] chelator (FIC), which solubilizes the iron and transports it to the rhizosphere. A high-performance liquid chromatography (HPLC) post-column method has been developed for the analysis of FICs using the luminol/H2O2 system for chemiluminescence (CL) detection. A size-exclusion column was the most suited in terms of column efficiency and CL detection efficiency. Mixing of the luminol with H2O2 in a post-column reaction was feasible, and a two-pump system was used to separately deliver the luminol and H2O2 solutions. The luminol and H2O2 concentrations were optimized using Fe(III)–EDTA and Fe(III)–citrate (Cit) solutions as analytes. A strong CL intensity was obtained for Fe(III)-Cit when EDTA was added to the luminol solution, probably because of an exchange of Cit with EDTA after separation on the HPLC column; CL efficiency was much higher for Fe(III)–EDTA than for Fe(III)–Cit with the luminol/H2O2 system. The present method can detect minute levels of Fe(III)–FICs; the detection limits of Fe(III)–EDTA, Fe(III)–Cit and Fe(III)–nicotianamine were 0.77, 2.3 and 1.1 pmol, respectively.
Keywords: Chemiluminescence; HPLC; Luminol; Mugineic acids; Nicotianamine; Organic acids; Phytosiderophores;

Simultaneous determination of corynoline and acetylcorynoline in human urine by LC–MS/MS and its application to a urinary excretion study by Ruijuan Liu; Lu Zheng; Minlu Cheng; Yao Wu; Pan Gu; Yujie Liu; Pengcheng Ma; Li Ding (83-89).
Corynoline and acetycorynoline, the major active components derived from Corydalis bungeana Herba, showed multiple pharmacological activities. However, quantification of the two compounds in human urine has not been reported. A simple liquid chromatography with tandem mass spectrometry method for the simultaneous determination of corynoline and acetycorynoline in human urine has been developed and fully validated. The analytes were extracted from urine samples by simple liquid-liquid extraction. Chromatographic separation was achieved on a Hedera ODS-2C18 column with the mobile phase of water (containing 0.5% formic acid) and acetonitrile (28:72, v/v) at a flow rate of 0.4 mL/min. A tandem mass spectrometric detection was conducted using multiple reaction monitoring via an electrospray ionization source in positive mode. The monitored ion transitions were m/z 368.1 → 289.1 for corynoline, m/z 410.2 → 289.2 for acetycorynoline and m/z 380.2 → 243.2 for donepezil (internal standard), respectively. The calibration curves were linear (correlation coefficients > 0.9970) over the concentration ranges of 3.0–3000 pg/mL for corynoline and 3.0–1000 pg/mL for acetycorynoline. The established method was highly sensitive with the lower limit of quantification (LLOQ) of 3.0 pg/mL for both analytes. The intra- and inter-day precision was lower than 10% in terms of relative standard deviation for the low, medium, and high quality control samples, and lower than 16% for the LLOQ samples of the analytes. The accuracy was within ±10% in terms of relative error for both analytes. The method was successfully applied to a urinary excretion study after oral administration of the Chinese medicine formula Shuanghua Baihe tablets in healthy volunteers. The urinary excretion profiles of corynoline and acetycorynoline in human were first reported. The results of this study suggest that renal excretion was not the main excretion pathway of corynoline and acetycorynoline in humans.
Keywords: Corynoline; Acetycorynoline; LC–MS/MS; Human urine; Urinary excretion; Shuanghua Baihe tablets;

Serum albumins are the most abundant carrier proteins in blood plasma and participate in the binding and transportation of various exogenous and endogenous compounds in the body. This work was designed to investigate the recognition and binding of three typical β-lactam antibiotics including penicillin G (Pen G), penicillin V (Pen V) and cefalexin (Cef) with bovine serum albumin (BSA) by frontal affinity chromatography in combination with UV–vis absorption spectra, fluorescence emission spectra, binding site marker competitive experiment and molecular docking under simulated physiological conditions. The results showed that a BSA only bound with one antibiotic molecule in the binding process, and the binding constants for Pen G-BSA, Pen V-BSA and Cef-BSA complexes were 4.22 × 101, 4.86 × 102 and 3.32 × 103 (L/mol), respectively. All the three β-lactam antibiotics were mainly inserted into the subdomain IIA (binding site 1) of BSA by hydrogen bonds and Van der Waals forces. The binding capacity between the antibiotics and BSA was closely related to the functional groups and flexibility of side chains in antibiotics. This study provided an important insight into the molecular recognition and binding interaction of BSA with β-lactam antibiotics, which may be a useful guideline for the innovative clinical medications and new antibiotic designs with effective pharmacological properties.
Keywords: β-lactam antibiotics; Bovine serum albumin; Recognition and binding; Frontal affinity chromatography; Fluorescence spectroscopy; Molecular docking;

Determination of valnemulin in swine and bovine tissues by ultra-high performance liquid chromatography–tandem mass spectrometry by Hui Li; Yingyu Wang; Xiaowei Li; Qin Fu; Yawen Shan; Tianhe Liu; Xi Xia (102-106).
A sensitive and reliable method has been developed and validated for the determination of valnemulin in swine and bovine muscle, liver, and kidney using solid-phase extraction (SPE) and ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS). The tissue samples were extracted with mixture solution of acetonitrile and 0.01 mol/L hydrochloric acid, defatted by n-hexane, and further cleaned up using SPE cartridges with polymeric sorbent. Gradient UHPLC separation was performed using an Acquity BEH C18 column with water and acetonitrile as the mobile phase. Multiple reaction monitoring mode of two precursor-product ion transitions for valnemulin was used. Mean recoveries from fortified samples ranged from 93.4 to 104.3% with 3.3–10.7% relative standard deviation. The limit of detection and quantification was 0.2 and 1 μg/kg for the analyte, respectively.
Keywords: Valnemulin; Ultra-high performance liquid chromatography; Tandem mass spectrometry; Tissues;

Display OmittedQuantification of energy and redox cofactors is of great value to synthetic biologists to infer the balance of energy metabolism in engineered microbial strains and assess each strain's potential for further improvement. Most currently used methods for intracellular cofactor measurement suffer from incomplete coverage, low reproducibility, suboptimal sensitivity or specificity. In this study, we described an SPE–HILIC/MS approach for simultaneous determination of six cofactor targets (ATP, ADP, NAD, NADH, NADP, NADPH) in Escherichia coli cells. Sufficient linearity, precision and metabolite recoveries of this new approach justified its reliability in targeted cofactor quantification. Our approach was then compared with conventional enzymatic assays to demonstrate its superior performance. We applied the SPE–HILIC/MS approach to profile shift of cofactor balances in several engineered E. coli strains with varying isobutanol production. Our cofactor analysis clearly revealed that optimal energy fitness was achieved in the highest-yield strain through combined modulation of a transhydrogenase and a NAD+ kinase. Apart from the targeted cofactors, the SPE enrichment procedure also allowed for confident identification of 39 groups of polar metabolites mainly involved in central carbon metabolism in E. coli cells.
Keywords: SPE; HILIC–MS; Cofactor; Targeted metabolome; Energy metabolism; Isobutanol production;