Journal of Chromatography B (v.1059, #C)

Development and validation of an UPLC-UV method for determination of a novel triazine coccidiostat ethanamizuril and its metabolite M3 in chicken tissues by Xiao Zhao; Youfa Xu; Lifang Zhang; Chunmei Wang; Chunna Guo; Chenzhong Fei; Keyu Zhang; Xiaoyang Wang; Yingchun Liu; Mi Wang; Wenli Zheng; Feiqun Xue (1-6).
Ethanamizuril (EZL) is a novel anti-coccidial triazine synthesized by the Shanghai Veterinary Research Institute of the Chinese Academy of Agricultural Sciences. As a novel coccidiostat, it is essential to study its residue in food animal tissues. In this study, a reliable UPLC method for the determination of ethanamizuril (EZL) and one of its metabolites (M3) in chicken muscle, skin and fat, liver, and kidney samples was developed. Analytes were extracted using acetonitrile and 10% sodium carbonate solution, defatted by n-hexane, and further purified by using an Oasis MCX cartridge. The calibration curves for both test compounds were linear in all tissue matrices we tested: muscle, skin and fat, liver and kidney. The mean relative recoveries of EZL and M3 were from 87.3% to 103.2% between all tissue samples. The inter-day and intra-day relative standard deviations were within 15% and 20%, respectively. This method is accurate, reproducible and is ready to be field-tested.
Keywords: Novel triazine coccidiostat; UPLC; SPE; Residues; Chicken tissues;

An isotope-dilution UPLC–MS/MS technique for the human biomonitoring of the internal exposure to glycidol via a valine adduct at the N-terminus of hemoglobin by Jan Hielscher; Bernhard H. Monien; Klaus Abraham; Sönke Jessel; Albrecht Seidel; Alfonso Lampen (7-13).
Fatty acid esters of glycidol (glycidyl esters) are processing contaminants generated as a byproduct of the industrial deodorization of vegetable oils and fats. Oral intake of glycidyl esters leads to the release of glycidol in the gastrointestinal tract. Glycidol is carcinogenic, genotoxic and teratogenic in rodents. It is rated as probably carcinogenic to humans (IARC group 2A). The determination of internal exposure of glycidol may support the assessment of the possible human health risks related to glycidyl ester intake. For this purpose, hemoglobin adducts of glycidol may be suitable biomarkers reflecting the cumulative exposure of up to four months. We applied a modified Edman degradation to assess the glycidol adduct at the N-terminal valine, N-(2,3-dihydroxypropyl)-valine (2,3-diHOPr-Val), of hemoglobin. The modified valine was cleaved with fluorescein-5-isothiocyanate (FITC), resulting in the formation of N-(2,3-dihydroxypropyl)-valine fluorescein thiohydantoin (DHP-Val-FTH). An isotope-dilution technique was developed for the quantification of the thiohydantoin analyte by ultra performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS) and DHP-Val-d7 -FTH as reference standard. The limit of detection was 4 fmol DHP-Val-FTH per injection corresponding to 0.7 pmol 2,3-diHOPr-Val/g hemoglobin. The adduct levels in blood samples of 12 non-smoking participants were in the range of 2.2–4.9 pmol 2,3-diHOPr-Val/g hemoglobin. The current work presents the first isotope-dilution technique using UPLC–MS/MS for the quantification of 2,3-diHOPr-Val at the N-terminus of hemoglobin as a sensitive and convenient alternative to earlier GC–MS methods.
Keywords: Glycidol; Edman degradation; Protein adduct; Biomarker; UPLC–MS/MS;

Comparative pharmacokinetic study of baicalin and its metabolites after oral administration of baicalin and Chaiqin Qingning capsule in normal and febrile rats by Xiao-Kui Huo; Bo Wang; Lu Zheng; Hai-Jian Cong; Ting Xiang; Shu-mei Wang; Cheng-peng Sun; Chao Wang; Lin Zhang; Sa Deng; Bin Wu; Xiao-Chi Ma (14-20).
An accurate, precise, selective, and sensitive high performance liquid chromatography coupled to tandem mass spectrometry (HPLC–MS/MS) method was developed for the simultaneous determination of baicalin and its metabolite, baicalein 6-O-glucopyranuronoside, in normal and febrile rats plasma. Two analytes, along with hesperidin as an internal standard, were determined by multiple reactions monitoring (MRM) operated in the positive electrospray ionization (ESI) mode. Chromatographic separation was performed on an Agilent ZORBAX Extend-C18 column (100 mm × 2.10 mm, 3.5 μm) with a mobile phase of 0.1% formic acid solution and acetonitrile at a flow rate of 0.6 mL/min. The calibration curves showed good linearity (r ≥ 0.9974) with the concentration ranges of 2.000–2000 ng mL−1 for baicalin and baicalein 6-O-glucopyranuronoside. The inter- and intra-day accuracies (relative error, RE%) were between −6.62% and 6.75%, and the precisions (relative standard deviation, RSD%) were less than 9.09% for quality control samples (QCs). The method also possessed good selectivity, recovery and stability, and was successfully applied to a comparative pharmacokinetic study of baicalin and baicalein 6-O-glucopyranuronoside in normal and febrile rats after oral administration of baicalin and Chaiqin Qingning capsule.
Keywords: Pharmacokinetic; Baicalin; Chaiqin Qingning capsule; Febrile rats;

Dynamic changes of flavonoids in Abelmoschus manihot different organs at different growth periods by UPLC–MS/MS by Xinxin Pan; Leyue Du; Jinhua Tao; Shu Jiang; Dawei Qian; Jinao Duan (21-26).
Abelmoschus manihot (Linn.) Medicus has been clinically used to treat chronic kidney disease, oral ulcers, burns, and dysmenorrhea in China for many centuries. The major pharmacologically-active components of A. manihot are flavonoids. In this study, a rapid and highly sensitive UPLC–MS/MS analysis method was established and successfully applied to the simultaneous determination of five major flavonoids (rutin, hyperoside, isoquercitrin, quercetin, and myricetin) in different parts of A. manihot harvested at ten growth periods. Under the optimized chromatographic conditions, good separation for five target components was obtained on an Acquity UPLC BEH C18 column within 18 min. The total contents of the five investigated flavonoids in A. manihot roots, stems, leaves and flowers ranged from 2.86 to 123.7 μg/g, 46.39 to 141.0 μg/g, 929.4 to 3096 μg/g, and 10,150 to 19,390 μg/g, respectively, indicating that the total flavonoids in the four parts could be mainly arranged in a decreasing order as flower > leaf > stem > root. The peak of total flavonoids in flowers and leaves appeared at G8 and G9, respectively. These results will be helpful for the determination of the suitable harvest time of A. manihot and the improvement of the utility value of the disused parts.
Keywords: Abelmoschus manihot; Flavonoids; Growth period; UPLC–MS/MS;

A novel magnetic solid-phase extraction (MSPE) based method was established for aromatic amino acids (AAAs) selective determination. Central to the method was the adopted extractant. An explored composite of metal organic frameworks (MOFs) and magnetic carbon nanotubes (CNTs) was employed for that, which exhibited superior adsorption affinity and selectivity to AAAs as compared to other amino acids with the mechanisms attributed to multiple hydrogen bonding and π-π electron-donor-acceptor (EDA) interactions. The morphology, structure and magnetic behavior of the composite were characterized and related MSPE procedure was established. Critical extraction conditions including pH, extraction time, temperature and salt addition were investigated and optimized. Subsequently the concentrations of three AAAs tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe) in Lanzhou lily were determined by the composite based MSPE procedure coupled with high performance liquid chromatography-ultraviolet detection (HPLC-UV). The composite provided a superior sample clean-up function and many of the matrix interferences were eliminated, thus ensured AAAs were accurately and efficiently determined. The results showed that the method had good linearities with the linear coefficients above 0.99, desirable recoveries ranged from 88.0% to 96.8% with the RSD less than 5.1%, satisfactory precision and low limits of detection (LODs) which were respectively 0.04, 0.11, and 0.87 ng/g for Trp, Tyr and Phe. The composite based MSPE-HPLC-UV method has great potentials for the AAAs selective determination from complex matrix samples.
Keywords: Magnetic solid-phase extraction; Fe3O4-MWCNTs-OH@poly-ZIF67; Aromatic amino acid; High performance liquid chromatography; Lanzhou lily;

Pharmacokinetics study of Erhuang decoction extracts in rats by HPLC–MS/MS by Jinglong Wang; Dandan Zheng; Yingzi Wang; Chao Zhang; Xiumei Sun (35-42).
To study the pharmacokinetics of Erhuang decoction extracts, a high performance liquid chromatography-tandem mass spectrometry (HPLC–MS/MS) method was established for the determination of effective substances in rat plasma. The extracts prepared by water extraction (WE) method were given to rats by oral administration. After collected from the orbital venous plexus, plasma was treated by protein precipitation method. Then, the concentration of index components, including baicalin, liquiritin, berberine, palmatine and glycyrrhetinic acid, were determined by HPLC–MS/MS. Gradient elution mode was used to the chromatographic separation with an Inertsil ODS-SP column (100 mm × 2.1 mm, 5 μm), with acetonitrile and 0.1% formic acid containing 10 mmol L−1 ammonium acetate as the mobile phase. MS analysis was conducted by multiple reactions monitoring (MRM) with Electrospray Ionization (ESI). The extraction recoveries of the five active ingredients from plasma were greater than 86.04%, and the intra- and inter-day precisions were less than 16.57%. Results indicated that active ingredients in plasma of rats with oral administration of extracts showed certain difference in the pharmacokinetic parameters, which proved that the active ingredients were effectively absorbed. The established HPLC–MS/MS analytical method was sensitive and accurate, suitable for the pharmacokinetic study of active ingredients in Erhuang decoction.
Keywords: Pharmacokinetics; Erhuang decoction; Active ingredients; HPLC–MS/MS;

A UPLC–MS/MS method for the quantitation of Ulipristal acetate in human serum by Renu Nandakumar; Piyapa Praditpan; Carolyn L. Westhoff; Serge Cremers (43-48).
The progesterone receptor modulator, Ulipristal acetate (UPA) has proven to be an effective emergency contraceptive. Conflicting data has been reported that suggests different efficacy of the drug in different populations, which may be explained by the systemic exposure to the drug. A UPLC–MS/MS method was developed and validated for the accurate and sensitive measurement of UPA in human serum to address this matter. UPA was extracted from human serum using liquid-liquid extraction with a combination of hexane and dichloromethane. An analytical platform comprising reverse-phase chromatographic separation followed by mass spectrometric detection by positive electrospray ionization in multiple reaction monitoring was used for quantitation of UPA within 7 min. The method was linear from 0.1 to 250 ng/mL. The matrix effect was minimal and intra- and inter-assay precision and accuracy were all within the acceptable limits. UPA was found to be stable at all processing and storage conditions. The method was used to investigate the pharmacokinetics of UPA in a clinical trial designed to explore the effect of obesity on its bioavailability.
Keywords: Ulipristal acetate; LC–MS/MS; Validation; Pharmacokinetics;

Quantification of cystatin-C in human serum by stable isotope dilution liquid chromatography electrospray ionization tandem mass spectrometry by Huoyan Ji; Jianxin Wang; Shaoqin Ju; Hui Cong; Xudong Wang; Jianyou Su; Huimin Wang (49-55).
Cystatin C (CysC)-based estimated glomerular filtration rate is an excellent marker for early diagnosis of Chronic Kidney Disease (CKD). Particle-enhanced nephelometric immunoassay (PENIA) and particle-enhanced turbidimetric immunoassay (PETIA) are commonly used methods for CysC quantification in clinical testing. However, both of them lack of specificity which mass spectrometry offers. In this paper, an isotope dilution liquid chromatography tandem mass spectrometry (ID-LC–MS/MS) method was established for quantification of CysC in human serum. After CysC denaturation, reduction and alkylation of cysteine residues, trypsin was added for proteolytic digestion of CysC. Specific peptide ALDFAVGEYNK was selected as surrogates for intact CysC protein, then quantified CysC by stable isotope-labelled internal standard peptide A[13C615N]LDFAVGEYNK based on calibration curve method. The calibration curves were y  = 0.1565x  − 1.6715 (R 2  = 0.988) and y  = 1.8785x  − 2.2497 (R 2  = 0.991) for y 9 and y 6, respectively. The linear range was 0.1–10 mg/L and 0.5–15 mg/L for y 9 and y 6, respectively. The limit of quantification (LOQ) was 0.052 mg/L. At different concentrations of CysC, the recoveries were in the range of 80.5%–93.7%, the intraday precisions were in the range of 0.3%–2.2%, and the inter-day precisions were in the range of 0.2%–2.8%. The results show that ID-LC–MS/MS and PETIA methods have higher consistency (y  = 0.8021x  + 0.6611, p  = 0.14), and the mean difference of the two methods was −0.29 mg/L, and 95% of the individual difference values were in the range of −0.91 mg/L–0.33 mg/L.
Keywords: Cystatin C; ID-LC–MS/MS; Proteolytic digestion; Calibration curve;

Study on pharmacokinetics and bioequivalence of Vonoprazan pyroglutamate in rats by liquid chromatography with tandem mass spectrometry by Yuan Qiao; Jun Zhao; Xuanfeng Yue; Yang Zhang; Ruitao Zhang; Yuan Xu; Xiuling Tang; Xueying Liu; Qingwei Wang (56-65).
Vonoprazan Fumarate (TAK-438 F) is a new and effective drug approved in Japan in 2014 for treatment and prevention of acid-related diseases (ARDs), which exhibits many advantages compared with traditional proton-pump inhibitors (PPIs). However, the clinical applications of TAK0-438 F suffers limitation due to the lack of injection dosage form. Efforts to overcome this limitation lead to the systhesis of Vonoprazan pyroglutamate (TAK-438 P) for its high water solubility and more potent antisecretory effect. This was the first report to establish and validate a reliable and sensitive LC–MS/MS method for the quantification of TAK-438 P in rat plasma and tissues (heart, liver, spleen, liver, kidney, rain, stomach and small intestine). All the features of the developed method suggested it was within bioanalytical criteria recommended by regulatory authorities. Furthermore, the developed method was applied to the exploration of the bioequivalence between TAK-438 P and TAK-438 F, as well as the pharmacokinetics and tissue distribution of TAK-438 P. The results showed that there was no significant differences between TAK-438 P and TAK-438 F after oral administration of the same dose. Besides, TAK-438 P was rapidly absorbed and eliminated in rat plasma. And it was widely distributed and there was no long-term accumulation in most tissues. Notably, more than 2000 ng/mL was observed in stomach 12 h after oral administration. The high accumulation revealed that stomach was likely to be the target organs of TAK-438 P.
Keywords: Vonoprazan pyroglutamate (TAK-438 P); Water solubility; Pharmacokinetics; Tissue distribution; LC–MS/MS;