Journal of Chromatography B (v.1011, #C)

Magnetic molecularly imprinted polymer nanoparticles for selective solid phase extraction and pre-concentration of Tizanidine in human urine by Golaleh Sheykhaghaei; Moayad Hossainisadr; Salah Khanahmadzadeh; Mirabdollah Seyedsajadi; Awat Alipouramjad (1-5).
In this work, the magnetic molecularly imprinted polymer nanoparticles (MMIP-NPs) for the selective pre-concentration of Tizanidine have been described. The polymer nanoparticles were synthesized by the polymerization of methacrylic acid as a functional monomer, ethylene glycol dimethacrylate as a cross-linker, 2,2-azobisisobutyronitrile as an initiator and Tizanidine as a template molecule. The MMIP-NPs were characterized by scanning electron microscopy (SEM) and thermogravimeteric analysis (TGA). Imprinted Tizanidine molecules were removed from the polymeric structure using acetic acid in methanol (10:90 V/V%), as the eluent solvent. The limits of detection (L.O.D) for Tizanidine were 1.13 × 10−6  M and 1.68 × 10−6  M in ultrapure water and urine, respectively. Also, the relative standard deviations (R.S.D) in ultrapure water and urine were 2.21% and 2.58%, respectively. The method was applied to the determination of Tizanidine in the human urine samples.
Keywords: Magnetic nanoparticles; Molecularly imprinted polymer; Magnetic solid phase extraction (MSPE); Ttizanidine; Human urine;

Preparative separation of gallocatechin gallate from Camellia ptilophylla using macroporous resins followed by sephadex LH-20 column chromatography by Kaikai Li; Xuelin Zhou; Cheuk-lun Liu; Xiaorong Yang; Xiaoqiang Han; Xianggang Shi; Xiaohong Song; Chuangxing Ye; Chun-hay Ko (6-13).
Gallocatechin gallate (GCG) possesses multiple potential biological activities. However, the content of GCG in traditional green tea is too low which limits its in-depth pharmacological research and application. In the present study, a simple, efficient and environment-friendly chromatographic separation method was developed for preparative enrichment and separation of GCG from cocoa tea (Camellia ptilophylla) which contains high content of GCG. In the first step, the adsorption properties of selected resins were evaluated, and XAD-7HP resin was chosen by its adsorption and desorption properties for GCG. In order to maximize column efficiency for GCG collection, the operating parameters (e.g., flow rate, ethanol concentration, and bed height) were optimized. We found that the best combination was the feed concentration at 20 mg/mL, flow rate at 0.75 BV/h and the ratio of diameter to bed heights as 1:12. Under these conditions, the purity of GCG was 45% with a recovery of 89%. In order to obtain pure target, a second step was established using column chromatography with sephadex LH-20 gel and 55% ethanol–water solution as eluent. After this step, the purity of the GCG was 91% with a recovery of 68% finally.
Keywords: Macroporous resins; Sephadex LH-20; Gallocatechin gallate; Camellia ptilophylla; Preparative enrichment and separation;

A sensitive and efficient method of high performance liquid chromatography using 1-(2-naphthyl)-3-methyl-5-pyrazolone (NMP) as pre-column derivatization reagent coupled with UV detection (HPLC-UV) and online mass spectrometry identification was established for determination of the most common N-Acetylhexosamines (N-acetyl-d-glucosamine (GlcNAc) and N-acetyl-d-galactosamine (GalNAc)) and N-acetylneuraminic acid (Neu5Ac). In order to obtain the highest liberation level of the three monosaccharides without destruction of Neu5Ac or conversion of GlcNAc/GalNAc to GlcN/GalN in the hydrolysis procedure, the pivotal parameters affecting the liberation of N-acetylhexosamines/Neu5Ac from sample were investigated with response surface methodology (RSM). Under the optimized condition, maximum yield was obtained. The effects of key parameters on derivatization, separation and detection were also investigated. At optimized conditions, three monosaccharides were labeled fast and entirely, and all derivatives exhibited a good baseline resolution and high detection sensitivity. The developed method was linear over the calibration range 0.25–12 μM, with R 2  > 0.9991. The detection limits of the method were between 0.48 and 2.01 pmol. Intra- and inter-day precisions for the three monosaccharides (GlcNAc, GalNAc and Neu5Ac) were found to be in the range of 3.07–4.02% and 3.69–4.67%, respectively. Individual monosaccharide recovery from spiked milk was in the range of 81%–97%. The sensitivity of the method, the facility of the derivatization procedure and the reliability of the hydrolysis conditions suggest the proposed method has a high potential for utilization in routine trace N-acetylhexosamines and Neu5Ac analysis in biological samples.
Keywords: N-acetylhexosamines; N-acetylneuraminic acid; Pre-column derivatization; 1-(2-naphthyl)-3-methyl-5-pyrazolone; HPLC-UV-MS/MS; Milk-based products;

Intact mass analysis of monoclonal antibodies by capillary electrophoresis—Mass spectrometry by Mei Han; Brooke M. Rock; Josh T. Pearson; Dan A. Rock (24-32).
Characterization of monoclonal antibody (mAb) therapeutics by intact mass analysis provides important information on sequence integrity and post-translational modifications. In order to obtain domain specific information, monoclonal antibodies are reduced to heavy and light chain components or enzymatically digested into smaller portions or peptides. Liquid chromatography (LC) is widely used for separation of the antibody fragments in line with mass spectrometry (MS) for characterization. Capillary electrophoresis (CE) is an analytical technique with high separation efficiency, high sensitivity, and minimal inter-run sample carryover. Combining the resolving power of CE with electrospray ionization (ESI) MS has great potential in regards to accurate mass characterization of protein therapeutics and has been a long sought-after approach. However, the intrinsic technical difficulty in coupling CE to MS has hindered the broad application of CE-MS across the biopharmaceutical industry. Recently, a CE-MS interface has been developed [1] and commercialized. Herein, we report implementation of this technology for coupling CE to an Agilent time-of-flight (TOF) mass spectrometer. CE-MS provides an attractive complement to LC–MS for separation and intact mass determination of mAbs and antibody-based therapeutics.
Keywords: Capillary electrophoresis; CE–MS; Intact mass analysis; Heavy chain; Light chain; IdeS; PTMs; Oxidation; Deamidation;

The purpose of this paper is to introduce a solid-phase extraction method of separating and determining of phthalic acid esters (PAEs) by PPy-coated Fe3O4 magnetic nanoparticles (Fe3O4@PPy MNPs). In the process, nanoparticles were served as sorbents and the optimal conditions of the extraction have been explored. The composite was synthesized through the chemical oxidation method, combining pyrrole monomer with Fe3O4 magnetic ball in the form of aggregation state and the coated nanoparticles possessed core–shell construction. The PPy-coated Fe3O4 magnetic microspheres have been extensively characterized by transmission electron microscopy, Fourier transform infrared spectroscopy and vibrating sample magnetometry. The optimum conditions were investigated by orthogonal experimental design, which was used to testing the influence of main factors and the interactions among them. The orthoplan is famous for the merit that it can study the whole experiments comprehensively through minimal tests. Under the optimal extraction conditions: 20 mg of modified magnetite nanoparticles, eluting with acetic ester of 2 mL, 40 min of MSPE, eluting in 1 h and 20 mL of sample volume, good linearity (r 2  > 0.9912) of all calibration curves was obtained in validation experiments. And the limits of detection (LODs) were from 0.006 to 0.021 ng/mL. The recoveries in different sample matrices were in the range from 80.4% to 108.2% with relative standard deviations less than 12.8%. The present work demonstrates the applicability of the developed method for the determination of PAEs in water sample and the results justified that it can be applied successfully to the selective isolation and enrichment of PAEs in real water samples.
Keywords: Polypyrrole coated Fe3O4 magnetic microsphere; Magnetic solid-phase extraction; Orthogonal experimental design; Phthalic acid esters; Gas chromatography–mass spectrometry;

A chiral UFLC-MS/MS method was established and validated for quantifying d-threo-methylphenidate (d-threo-MPH), l-threo-methylphenidate (l-threo-MPH), d-threo-ethylphenidate (d-threo-EPH), l-threo-ethylphenidate (l-threo-EPH) and d,l-threo-ritalinic acid (d,l-threo-RA) in rat plasma over the linearity range of 1–500 ng/mL. Chiral separation was performed on an Astec Chirobiotic V2 column (5 μm, 250 × 2.1 mm) with isocratic elution using methanol containing 0.003% ammonium acetate (w/v) and 0.003% trifluoroacetic acid (v/v) at a flow of 0.3 mL/min. All analytes and IS were extracted from rat plasma by a one-step liquid–liquid extraction (LLE) method. The intra- and inter-run accuracies were within 85–115%, and the intra- and inter-run precision were <10% for all analytes. Extraction recoveries were 55–62% for d-threo-MPH, 54–60% for l-threo-MPH, 55–60% for d-threo-EPH, 53–57% for l-threo-EPH and 25–30% for d,l-threo-RA. The validated UFLC-MS/MS method successfully applied to the pharmacokinetic interaction study of oral d-threo-MPH and l-threo-MPH (alone or in combination) in female Sprague Dawley rats. The EPH was not detected in rat plasma following oral administrated MPH without EtOH. As far as it is known to the authors, this study is the first one step liquid–liquid extraction method to extract and UFLC-MS/MS method to quantify d-threo-MPH, l-threo-MPH, d-threo-EPH, l-threo-EPH and d,l-threo-RA simultaneously.
Keywords: Methyphenidate; Ethylphenidate; Chiral separation; UFLC-MS/MS; Pharmacokinetics; Drug interaction;

Enantioselective analysis of 4-hydroxycyclophosphamide in human plasma with application to a clinical pharmacokinetic study by Francine Attié de Castro; Gabriel dos Santos Scatena; Otávio Pelegrino Rocha; Maria Paula Marques; Quézia Bezerra Cass; Belinda Pinto Simões; Vera Lucia Lanchote (53-61).
Cyclophosphamide (CY) is one of the most common immunosuppressive agents used in autologous hematopoietic stem cell transplantation. CY is a prodrug and is metabolized to active 4-hydroxycyclophosphamide (HCY). Many authors have suggested an association between enantioselectivity in CY metabolism and treatment efficacy and/or complications. This study describes the development and validation of an analytical method of HCY enantiomers in human plasma by high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) that can be applied to pharmacokinetic studies, filling this gap in the literature. HCY enantiomers previously derivatized with phenylhydrazine were extracted from 200-μL plasma aliquots spiked with antipyrine as internal standard and a mixture of hexane and dichloromethane (80:20, v/v) was used as the extraction solvent. The derivatized HCY enantiomers were resolved on a Chiracel® OD-R column using water:acetonitrile:formic acid (55:45:0.2, v/v) as the mobile phase. No matrix effect was observed and the analysis of HCY enantiomers was linear for plasma concentrations of 5–5000 ng of each enantiomer/mL plasma. The coefficients of variation and inaccuracy calculated in precision and accuracy assessments were less than 15%. HCY was stable in human plasma after three successive freeze/thaw cycles, during 3 h at room temperature, and in the autosampler at 4 °C for 24 h after processing, with deviation values less than 15%. The method was applied to evaluate the kinetic disposition of HCY in a patient with multiple sclerosis who was pretreated with intravenous racemic CY for stem cell transplantation. The clinical study showed enantioselectivity in the pharmacokinetics of HCY.
Keywords: 4-hydroxycyclophosphamide; Enantiomers; LC–MS/MS; Metabolism; Pharmacokinetics;

A sensitive LC–MS/MS method to quantify methylergonovine in human plasma and its application to a pharmacokinetic study by Yanhui Gao; Qichao Sun; Dongming Liu; Bowen Ma; Hengli Zhao; Zengjun Fang; Haisheng Wang; Hongxiang Lou (62-68).
Methylergonovine (ME) is a semisynthetic ergot alkaloid that is used for the treatment and prophylaxis of postpartum hemorrhage. In recent years, methylergonovine has been effective in the control of refractory headaches and is likely to be employed as chemosensitizers for cancer. However, this alkaloid sometimes causes elevated blood pressure. Therefore, a sensitive and accurate method for the quantification of this drug in biological matrices is necessary. In this study, ME was extracted from 500 μL plasma samples by a liquid–liquid extraction under alkaline conditions and detected using positive multi-reaction-monitoring mode (+MRM) mass spectrometry. The method was validated according to US FDA guidelines and covered a working range from 0.025 to 10 ng/mL with a lower limit of quantification (LLOQ) of 0.025 ng/mL. In conclusion, a rapid, sensitive, selective and accurate quantification by an LC–MS/MS method was developed and successfully applied to a clinical pharmacokinetics study in female volunteers after a single intramuscular injection or oral administration of a 0.2 mg dose of ME maleate. It is suitable for both preclinical and clinical studies on ME.
Keywords: Methylergonovine; HPLC–MS/MS; Pharmacokinetics; Human plasma;

A high-performance liquid chromatography tandem mass spectrometry (LC–MS/MS) assay for the quantitative determination of NAD+ in human whole blood using a surrogate analyte approach was developed and validated. Human whole blood was acidified using 0.5 N perchloric acid at a ratio of 1:3 (v:v, blood:perchloric acid) during sample collection. 25 μL of acidified blood was extracted using a protein precipitation method and the resulting extracts were analyzed using reverse-phase chromatography and positive electrospray ionization mass spectrometry. 13C5-NAD+ was used as the surrogate analyte for authentic analyte, NAD+. The standard curve ranging from 0.250 to 25.0 μg/mL in acidified human blood for 13C5-NAD+ was fitted to a 1/x 2 weighted linear regression model. The LC–MS/MS response between surrogate analyte and authentic analyte at the same concentration was obtained before and after the batch run. This response factor was not applied when determining the NAD+ concentration from the 13C5-NAD+ standard curve since the percent difference was less than 5%. The precision and accuracy of the LC–MS/MS assay based on the five analytical QC levels were well within the acceptance criteria from both FDA and EMA guidance for bioanalytical method validation. Average extraction recovery of 13C5-NAD+ was 94.6% across the curve range. Matrix factor was 0.99 for both high and low QC indicating minimal ion suppression or enhancement. The validated assay was used to measure the baseline level of NAD+ in 29 male and 21 female human subjects. This assay was also used to study the circadian effect of endogenous level of NAD+ in 10 human subjects.
Keywords: LC–MS; Surrogate analyte; Human blood; NAD+; Baseline level; Circadian effect;

Microfluidic immunomagnetic cell separation from whole blood by Sajay Bhuvanendran Nair Gourikutty; Chia-Pin Chang; Poenar Daniel Puiu (77-88).
Immunomagnetic-based separation has become a viable technique for the separation of cells and biomolecules. Here we report on the design and analysis of a simple and efficient microfluidic device for high throughput and high efficiency capture of cells tagged with magnetic particles. This is made possible by using a microfluidic chip integrated with customized arrays of permanent magnets capable of creating large magnetic field gradients, which determine the effective capturing of the tagged cells. This method is based on manipulating the cells which are under the influence of a combination of magnetic and fluid dynamic forces in a fluid under laminar flow through a microfluidic chip. A finite element analysis (FEA) model is developed to analyze the cell separation process and predict its behavior, which is validated subsequently by the experimental results. The magnetic field gradients created by various arrangements of magnetic arrays have been simulated using FEA and the influence of these field gradients on cell separation has been studied with the design of our microfluidic chip. The proof-of-concept for the proposed technique is demonstrated by capturing white blood cells (WBCs) from whole human blood. CD45-conjugated magnetic particles were added into whole blood samples to label WBCs and the mixture was flown through our microfluidic device to separate the labeled cells. After the separation process, the remaining WBCs in the elute were counted to determine the capture efficiency, and it was found that more than 99.9% WBCs have been successfully separated from whole blood. The proposed design can be used for positive selection as well as for negative enrichment of rare cells.
Keywords: Magnetophoresis; Magnetic separation; Magnetic force; Magnetic particles; Trapping; Negative enrichment; Cell separation;

Determination of residual dextran sulfate in protein products by SEC–HPLC by Loubna M. Tazi; Shiranthi Jayawickreme (89-93).
Dextran sulfate is a polyanionic derivative of dextran, produced by esterification of dextran with chlorosulphonic acid. Dextran sulfate with an average molecular weight of 8000 Da can be added to the cell culture to inhibit binding of proteins to cells, increasing cellular growth and productivity. Residual dextran sulfate levels must be monitored during the purification process development to insure clearance. A size-exclusion chromatography based HPLC assay has been developed for the separation and quantitation of dextran sulfate in a highly concentrated purified protein drug substance sample. Trichloroacetic acid (TCA) was used to precipitate the protein and separate the dextran sulfate. Detection and quantitation of dextran sulfate was achieved by post column reaction with dimethylene blue to form a metachromatic complex that absorbs visible light at 530 nm. The quantitation limit (LOQ) was determined to be 1.5 μg/mL dextran sulfate in high concentration protein samples.
Keywords: Dextran sulfate; Protein products; SEC–HPLC;

A sensitive and rapid ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed to determine tedizolid and linezolid in rat plasma simultaneously. Chromatographic separation was carried out on an Acquity UPLC BEH C18 column and mass spectrometric analysis was performed using a XEVO TQD triple quadruple mass spectrometer coupled with an electrospray ionization (ESI) source in the positive ion mode. Multiple reaction monitoring (MRM) mode was used for quantification using target fragment ions m/z 371.4 → 343.2 for tedizolid, and m/z 338.3 → 56.1 for linezolid. This assay method has been fully validated in terms of selectivity, linearity, recovery and matrix effect, accuracy, precision and stability. The linearity of this method was found to be within the concentration range of 5–5000 ng/mL for tedizolid, and 10–10,000 ng/mL for linezolid in rat plasma, respectively. Only 3.0 min was needed for an analytical run. This assay was used to support a preclinical study where multiple oral doses were administered to rats to investigate the pharmacokinetics of tedizolid and linezolid.
Keywords: Tedizolid; Linezolid; UPLC-MS/MS; Rat plasma;

Molecular distillation residue (MD-R) from ginger had the most total phenol content of 247.6 mg gallic acid equivalents per gram (GAE/g) among the ginger oils. High-speed counter-current chromatography (HSCCC) technique in semi-preparative scale was successfully performed in separation and purification of 6-gingerol from MD-R by using a two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water (10:2:5:7, v/v/v/v). The target compound was isolated, collected, purified by HSCCC in the head–tail mode, and then analyzed by HPLC. A total of 90.38 ± 0.53 mg 6-gingerol was obtained from 600 mg MD-R, with purity of 99.6%. In addition, the structural identification of 6-gingerol was performed by EI/MS, 1H NMR and 13C NMR. Moreover, the orders of antioxidant activity were vitamin E (VE) > supercritical fluid extraction oleoresin (SFE-O) = MD-R = 6-gingerol > molecular distillation essential oil (MD-EO) and butylated hydroxytoluene (BHT) = VE > 6-gingerol > MD-R = SFE-O > MD-EO, respectively in 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) scavenging and β-Carotene bleaching.
Keywords: High-speed counter-current chromatography (HSCCC); Molecular distillation residue; 6-Gingerol; Antioxidant activity;

Preparative separation of sesamin and sesamolin from defatted sesame meal via centrifugal partition chromatography with consecutive sample injection by Je-Seung Jeon; Chae Lee Park; Ahmed Shah Syed; Young-Mi Kim; Il Je Cho; Chul Young Kim (108-113).
A preparative separation method using consecutive sample injection centrifugal partition chromatography (CPC) was developed to obtain sesamin and sesamolin from defatted sesame meal extracts. A two-phase solvent system consisting of n-hexane–ethyl acetate–methanol–water (8:2:8:2, v/v) was applied in reversed-phase mode (descending mode). Preliminary experiments with an SCPC-100 (column volume: 100 mL) were performed to select the appropriate two-phase solvent system and sample injection times; these parameters were then used with an SCPC-1000 (column volume: 1000 mL) in a 10-fold scale-up preparative run. A sample containing 3 g of crude extract was consecutively injected four times onto the SCPC-1000, which yielded 328 mg of sesamin and 168 mg of sesamolin. These compounds were analyzed by high-performance liquid chromatography and determined to have purities of 95.6% and 93.9%, respectively. Sesamin and sesamolin (30 μM) increased antioxidant response element (ARE) luciferase activity 2.6-fold and 1.9-fold, respectively.
Keywords: Centrifugal partition chromatography; Consecutive sample injection; Sesamin; Sesamolin; Antioxidant response element;

Capillary electrophoresis of RNA in hydroxyethylcellulose polymer with various molecular weights by Zhenqing Li; Chenchen Liu; Dawei Zhang; Shaopeng Luo; Yoshinori Yamaguchi (114-120).
Recent research demonstrates that large numbers of long noncoding RNAs (lncRNAs) in mammals exhibit indices of functionality, and thus analysis of longer RNAs is of great significance. In the present work, we investigated the effect of molecular weight on the separation performance of long RNA by capillary electrophoresis (CE). Results demonstrate that (1) low molecular weight of hydroxyethylcellulose (HEC) (90k) favors the separation of short RNA (<1000 nt). The resolution for short RNA was improved and the migration time was linearly extended with the increase of polymer concentration. (2) In the longer chain HEC (250k, 720k and 1300k), the resolution for the small RNA fragment (<1000 nt) became better as the polymer concentration increased, whereas the resolution for the large ones (>3000 nt) deteriorated. (3) Based on logarithmic plot, there exist two migration regimes for RNA in short chain HEC (90k), three regimes in moderate chain HEC (250k and 720k), and four regimes in the long chain HEC (1300k). Such a systematic investigation of long RNAs may be useful for research on lncRNAs in the length range of 100–10,000 nt.
Keywords: RNA; Capillary electrophoresis; Hydroxyethylcellulose;

A simple, sensitive, rapid, and reproducible analytical method of manassantin B in rat plasma by high performance liquid chromatography with fluorescence detection (HPLC-FL) was developed for its application to pharmacokinetic study in rats. Valsartan (VST) was used as an internal standard (IS) in this quantitative analytical method. Manassantin B and VST were extracted by simple and efficient protein precipitation method. Manassantin B was detected at 282/322 nm (excitation/emission) wavelengths using FL detector. The chromatographic separation was obtained with reverse phase C18 column and the mobile phase composed of potassium phosphate buffer containing 0.025% trifluoroacetic acid (pH 2.5; 5 mM) and acetonitrile including 0.025% trifluoroacetic acid (20:80, v/v) at 1.0 mL/min flow rate. The linearity was established at 25.0–10000 ng/mL and the lower limit of detection (LLOD) was 7 ng/mL. The intra- and inter-day accuracy and precision values of manassantin B were within ± 15% of the theroretical values and <9% from the nominal concentrations, respectively. Accuracy and precision values of manassantin B after stability tests were also within the acceptable ranges. Developed assay was also successfully applied to pharmacokinetic study after intravenous administration of manassantin B in rats.
Keywords: Manassantin B; Validation; HPLC; Fluorescence detection; Pharmacokinetics;

Simultaneous quantification of 5 main components of Psoralea corylifolia L. in rats’ plasma by utilizing ultra high pressure liquid chromatography tandem mass spectrometry by Qianqian Gao; Zisheng Xu; Genhua Zhao; Heng Wang; Zebin Weng; Ke Pei; Li Wu; Baochang Cai; Zhipeng Chen; Weidong Li (128-135).
Psoralea corylifolia L. has long been used in traditional Chinese medicine for treating and preventing many diseases. A group of flavonoid components are regarded as the active principals within the seeds. In this research, a rapid, accurate and sensitive ultra high pressure liquid chromatography tandem mass spectrometry (UHPLC/MS/MS) method has been established for simultaneous quantification of its 5 main components, namely, neobavaisoflavone, bavachin, isobavachalcone, bavachinin and corylifol A in rats’ plasma after the rats were orally administrated with Buguzhi extract. Negative ion electrospray mode was applied in the detection process. Multiple reactions monitoring (MRM) mode was utilized for simultaneous quantitative analyzing of neobavaisoflavone (m/z 321.1 →  m/z 265.1), bavachin (m/z 323.1 →  m/z 119.0), isobavachalcone (m/z 323.2 →  m/z 119.0), bavachinin (m/z 337.2 →  m/z 119.0), corylifol A (m/z 389.2 →  m/z 277.0) and liquiritigenin (Internal Standard, m/z 255.1 →  m/z 119.0). Chromatographic separation of the above mentioned components was conducted on a Waters BEH-C18 column (100 mm × 2.1 mm, 1.7 μm) with gradient elution system at flow rate of 0.3 mL/min. The mobile phase was composed of acetonitrile and 0.1% formic acid solution. The lower limit of quantification (LLOQ) for each of the above analytes was 0.5 ng/mL. Each of the analytes exhibited good linearity within the concentration range of 0.5–100 ng/mL. The method was fully validated for its selectivity, accuracy, precision, stability, matrix effect and extraction recovery. The validated method has been successfully applied for simultaneous determination of the 5 flavonoids in rat plasma for the first time.
Keywords: Psoralea corylifolia L.; Flavonoids; UHPLC–MS/MS; Pharmacokinetics;

Display OmittedA novel pretreatment method involving microwave-assisted extraction and solid-phase purification combined with dispersive liquid–liquid microextraction (MAE-SPP-DLLME) followed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS) was established for the simultaneous determination of six antibacterial pharmaceuticals including metronidazole, tinidazole, chloramphenicol, thiamphenicol, malachite green and crystal violet. The conditions of MAE were optimized using an orthogonal design and the optimal conditions were found to be 8 mL for acetonitrile, 50 °C for 5 min. Then, neutral alumina column was employed in the solid-phase purification. Finally, the critical parameters affecting DLLME, including selection of extraction and dispersive solvent, adjustment of pH, salt concentration, extraction time, were investigated by single factor study. Under optimum conditions, good linearities (r  > 0.9991) and satisfied recoveries (Recoveries > 87.0%, relative standard deviation (RSD) < 6.3%) were observed for all of the target analytes. The limits of detection and quantification were 4.54–101.3 pg kg−1 and 18.02–349.1 pg kg−1, respectively. Intra-day and inter-day RSDs were all lower than 3.6%. An obvious reduction in matrix effect was observed by this method compared with microwave assisted extraction followed by purification. The established method was sensitive, rapid, accurate and employable to simultaneously determine target analytes in farmed fish, river fish and marine fish.
Keywords: Microwave-assisted extraction; Dispersive liquid–liquid microextraction; UHPLC–MS/MS; Veterinary pharmaceuticals residues; Fish;

This paper describes a method to reveal the illegal use of chloramphenicol (CAP) in animals intended for human consumption based on the detection of free CAP and chloramphenicol-glucuronide (CAP-glu) in urine. It details the different steps of the method, including hydrolysis of CAP-glu, extraction and cleanup with molecularly imprinted polymers and detection by LC–MS/MS, as well as the validation design. The efficiency of chloramphenicol release during the hydrolysis step and the stability of CAP-glu in urine samples stored at −20 °C were also investigated. These verifications were important to ensure the method’s suitability for checking CAP misuse in veterinary medicine. Validation results were fully compliant with the qualitative and quantitative criteria required by European regulations. Intraday relative standard deviations were all below 7.5%, while interday relative standard deviations were below 6.9%. Recoveries lay between 93.3 and 104.6%. Purification appears very effective since no matrix effect was demonstrated. CAP-glu was found to be stable for at least 3 months, and the mean recovery following deconjugation was assessed to be 79.4%. The decision limits (CCa) were all found to be lower than 0.1 μg/kg.
Keywords: Chloramphenicol-glucuronide; Urine; MIP; LC–MS/MS; Validation; Stability;

A method based on a simplified sample extraction by matrix solid phase dispersion (MSPD) followed by HPLC determination is validated for analysis of five lignans in Schisandra chinensis. The MSPD parameters that affect the extraction efficiency of lignans from S. chinensis were examined and optimized. The optimal extraction conditions were determined to be that silica gel was used as dispersing sorbent, the ratio of silica gel to sample mass was selected to be 2:1, and 4 mL of methanol was used as elution solvent. The method recoveries were determined to be from 92.25 to 101.17% and the RSDs from 1.3 to 4.9%. The extraction yields of five lignans obtained by the MSPD were higher than those of traditional reflux and sonication extraction with reduced requirements on sample, solvent and time. In addition, the optimized method was applied for analyzing five real S. chinensis samples obtained from different cultivated areas.
Keywords: Schisandra chinensis; Lignans; Matrix solid phase dispersion; HPLC;

Peucedanum praeruptorum Dunn (BaiHuaQianHu in Chinese) is a traditional Chinese medicine that has a long history of use in China. In this study, HEK 293 α1A adrenergic cell membrane chromatography was coupled with UHPLC-ESI-MS/MS and successfully used to identify active components from Peucedanum praeruptorum Dunn. Paeruptorin A, paeruptorin B, and paeruptorin C were identified with α1A adrenergic receptor activity. Pharmacological assays showed that tamsulosin hydrochloride, paeruptorin A, paeruptorin B, and paeruptorin C in concentrations of 1 × 10−8 to 1 × 10−4  mol/mL could relax prostate strips pre-contracted with adrenalin in a concentration dependent manner. Therefore, the HEK293 α1A cell membrane chromatography coupled UHPLC–ESI–MS/MS system may be a potentially useful drug discovery method for screening for medicinal herbal components with α1A adrenergic receptor inhibitory activity.
Keywords: Cell membrane chromatography; UHPLC–ESI–MS/MS; Peucedanum praeruptorum Dunn; α1A adrenergic receptor;

A stability-indicating HPLC method for simultaneous determination of morphine and naltrexone by Milad Jafari-Nodoushan; Jalal Barzin; Hamid Mobedi (163-170).
This study developed a stability-indicating reversed-phase HPLC method for the simultaneous determination of morphine sulfate and naltrexone hydrochloride content in bulk, Solid dosage forms and in-vitro dissolution samples to support product development and quality control efforts. Chromatographic separation of the pharmaceutical compound was achieved on a perfectSil™ MZ C18 column (250 × 4.6 mm, 5 μm) with an isocratic mobile phase composed of a mixture of acetate buffer (10 mM, pH 4, containing 0.1% of 1-heptanesulfonic acid sodium salt) and acetonitrile with 80/20 at a flow rate of 1.5 ml min−1. Both analytes were quantified using a photodiode array detector set at a wavelength of 280 nm and column temperature was set to 30 °C. naltrexone, morphine and a mixture of the two were subjected to thermal, peroxide, acid, base and photolytic degradation and their peak homogeneity was obtained using a photodiode array detector, demonstrating the specificity of method. These pharmaceuticals were spiked in biological fluid to examine method selectivity. The method was validated for system suitability, linearity, accuracy, precision, detection and quantification limits and robustness and was found it is acceptable in range of 2–250 μg ml−1 for morphine and 4–100 μg ml−1 for naltrexone.
Keywords: Simultaneous determination; Morphine; Naltrexone; Stability-indicating; HPLC;

Detoxification of lithocholic acid (LCA) to lithocholic acid sulfate (LCA-S) is catalyzed by sulfotransferases, mainly SULT2A1. We developed and validated an ultra-high performance liquid chromatography–tandem mass spectrometric (UPLC–MS/MS) method to quantify human liver cytosolic-dependent LCA sulfation. Chromatographic separation was achieved on an UPLC C18 column (2.1 × 50 mm, 1.7 μm) and a gradient elution of 0.1% formic acid in water and acetonitrile. Negative electrospray ionization with multiple reaction monitoring (MRM) mode was used to quantify LCA-S (455.3 → 97.0) and cholic acid (407.2 → 343.3; internal standard). The retention time was 3.51 min for LCA-S and 3.08 min for cholic acid. The lower limit of quantification of LCA-S was 0.5 nM (or 0.23 ng/ml in 400 μl total volume) and the assay was linear from 0.2 to 200 pmol. Intra-day and inter-day accuracy and precision were <14%. The quality control samples were stable at room temperature for 4 h, 4 °C for 24 h, −20 °C for 14 days, and after three freeze–thaw cycles. The matrix (20–100 μg cytosolic protein) did not affect LCA-S quantification. This is the first UPLC–MS/MS method applied to optimization of the human liver cytosolic LCA sulfation assay. The optimal levels of MgCl2 and 3′-phosphoadenosine 5′-phosphosulfate (PAPS) cofactor were 2.5 mM and 20 μM, respectively. Addition of reducing agents (2-mercaptoethanol and DL-dithiothreitol) did not affect LCA-S formation. Human liver cytosolic LCA sulfation was linear with 20–100 μg of cytosolic protein and 5–30 min incubation time. This UPLC–MS/MS approach offers a specific, sensitive, fast, and direct approach for quantifying human liver cytosolic LCA sulfation.
Keywords: Human liver cytosol; Lithocholic acid; Lithocholic acid sulfate; Sulfotransferase; Sulfotransferase 2A1; Ultra-high performance liquid chromatography–tandem mass spectrometry;

Comparative UPLC-QTOF-MS-based metabolomics and bioactivities analyses of Garcinia oblongifolia by Ping Li; Harini AnandhiSenthilkumar; Shi-biao Wu; Bo Liu; Zhi-yong Guo; Jimmie E. Fata; Edward J. Kennelly; Chun-lin Long (179-195).
Display Omitted Garcinia oblongifolia Champ. ex Benth. (Clusiaceae) is a well-known medicinal plant from southern China, with edible fruits. However, the phytochemistry and bioactivity of the different plant parts of G. oblongifolia have not been studied extensively. Comparative metabolic profiling and bioactivities of the leaf, branch, and fruit of G. oblongifolia were investigated. A total of 40 compounds such as biflavonoids, xanthones, and benzophenones were identified using UPLC-QTOF-MS and MSE, including 15 compounds reported for the first time from this species. Heatmap analyses found that benzophenones, xanthones, and biflavonoids were predominately found in branches, with benzophenones present in relatively high concentrations in all three plant parts. Xanthones were found to have limited distribution in fruit while biflavonoids were present at only low levels in leaves. In addition, the cytotoxic (MCF-7 breast cancer cell line) and antioxidant (ABTS and DPPH chemical tests) activities of the crude extracts of G. oblongifolia indicate that the branch extract exhibits greater bioactivity than either the leaf or the fruit extracts. Orthogonal partial least squares discriminate analysis was used to find 12 marker compounds, mainly xanthones, from the branches, including well-known antioxidants and cytotoxic agents. These G. oblongifolia results revealed that the variation in metabolite profiles can be correlated to the differences in bioactivity of the three plant parts investigated. This UPLC-QTOF-MS strategy can be useful to identify bioactive constituents expressed differentially in the various plant parts of a single species.
Keywords: Garcinia oblongifolia; UPLC-QTOF-MS; PCA; Cytotoxicity; Antioxidants; Xanthones; Benzophenones; Biflavonones;

Determination of metabolites of di(2-ethylhexyl) terephthalate (DEHTP) in human urine by HPLC-MS/MS with on-line clean-up by Frederik Lessmann; André Schütze; Tobias Weiss; Thomas Brüning; Holger M. Koch (196-203).
Di(2-ethylhexyl) terephthalate (DEHTP) is used as a substitute for ortho-phthalate based plasticizers like di(2-ethylhexyl) phthalate (DEHP) which are discussed and regulated due to their reproductive toxicity. We developed a fast and rugged method to quantify side chain oxidized monoesters of DEHTP in human urine, namely 5OH-MEHTP, 5oxo-MEHTP, 2cx-MMHTP and 5cx-MEPTP. Sample preparation was kept simple with enzymatic deconjugation and a two column assembly for on-line sample clean up. Metabolites were identified with authentic standards and quantified via isotope dilution LC–MS/MS. The limit of quantification was 0.2 μg/L for 5cx-MEPTP and 5oxo-MEHTP, 0.3 μg/L for 5OH-MEHTP and 0.4 μg/L for 2cx-MMHTP. Accuracy (relative recovery: 95.8–111%) and precision (relative standard deviation: <7%) were highly acceptable. In a pilot biomonitoring study with 34 volunteers (aged 25–61 (median 42), 20 female and 14 male) not known to be occupationally exposed to DEHTP, we could detect 5cx-MEPTP above the limit of quantification in 94% of the samples (median: 0.9 μg/L, maximum: 38.7 μg/L). The other metabolites investigated were detected at a lower rate and at lower concentration levels (5oxo-MEHTP: 21%, maximum: 1.8 μg/L; 5OH-MEHTP: 18%, maximum: 3.4 μg/L; 2cx-MMHTP: 9%, maximum: 0.9 μg/L). All target analytes can be regarded as promising and specific urinary biomarkers for DEHTP exposure. With this method we provide a basis for quantitatively investigating the human metabolism of DEHTP and for performing exposure and risk assessments in the general population and the working environment.
Keywords: Di(2-ethylhexyl) terephthalate; DEHTP; Plasticizer; Exposure assessment; Urinary metabolite; Human biomonitoring;

An ultra-high performance liquid chromatography/tandem mass spectrometry (UPLC–MS/MS) method for the simultaneous determination of (4S,5R)-5-[3,5-bis (trifluoromethyl)phenyl]-3-{[4′-fluoro-5′-isopropyl-2′-methoxy-4-(trifluoromethyl)biphenyl-2-yl] methyl}-4-methyl-1,3-oxazolidin-2-one (anacetrapib, I) and [13C5 15N]-anacetrapib, II in human plasma has been developed to support a clinical study to determine the absolute bioavailability of I. The analytes and the stable-isotope labeled internal standard ([13C7 15N2H7]-anacetrapib, III) were extracted from 100 μL of human plasma by liquid–liquid extraction using 20/80 isopropyl alcohol/hexane (v/v). The chromatographic separation of the analytes was achieved using Waters BEH Shield RP 18 (50 × 2.1 mm × 1.7 μm) column and mobile phase gradient of 0.1% formic acid in water (Solvent A) and 0.1% formic acid in acetonitrile (Solvent B) at 0.6 mL/min flow rate. The MS/MS detection was performed on AB Sciex 5000 or AB 5500 in positive electrospray ionization mode, operated in selected reaction monitoring mode. The assay was validated in the concentration range 1–2000 ng/mL for I; and a lower curve range, 0.025–50 ng/mL for II.In addition to the absolute bioavailability determination, it was desired to better elucidate the pharmacokinetic behavior of several hydroxylated metabolites of I. Toward this end, two exploratory assays for the hydroxy metabolites of I were qualified in the concentration range 0.5–500 ng/mL. All metabolites were separated on a Supelco Ascentis Express Phenyl-Hexyl (50 × 2.1 mm, 2.7 μm) column. Metabolite M4 was analyzed in the negative mode with a mobile phase consisting of a gradient mixture of water (A) and acetonitrile (B). The other three metabolites, M1–M3 were analyzed in the positive mode using a mobile phase gradient of water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B).The assays were utilized to support a clinical study in which a microdosing approach was used to determine the pharmacokinetics of anacetrapib and its metabolites.
Keywords: Anacetrapib; Microdosing; UPLC–MS/MS; Bioavailability; Metabolites;

Simultaneous determination of gefitinib and its major metabolites in mouse plasma by HPLC–MS/MS and its application to a pharmacokinetics study by Nan Zheng; Can Zhao; Xi-Ran He; Shan-Tong Jiang; Shu-Yan Han; Guo-Bing Xu; Ping-Ping Li (215-222).
Gefitinib (Iressa) is the first oral EGFR tyrosine kinase inhibitor and it brings benefits to non-small cell lung cancer patients with EGFR mutation. In this study, a simple, rapid and credible high performance liquid chromatography–tandem mass spectrometry method was established and validated for the simultaneous quantification of gefitinib and its main metabolites M523595, M537194, M387783 and M608236 in NSCLC tumor-bearing mouse plasma. Sample extraction was done by protein precipitation using acetonitrile containing dasatinib as the internal standard. The chromatography run time was 6 min using an Agilent RRHD SB-C18 column with a gradient of acetonitrile and water (0.1% formic acid, v/v). The mass analysis was performed by a triple quadrupole mass spectrometry in positive multiple reaction monitoring mode. The calibration range was 0.5–100 ng/mL for M608236 and 1–200 ng/mL for other analytes with the correlation coefficients (r 2) ≥ 0.99. For quality control samples, inter- and intra-assay precision was less than 15% and accuracies ranged from 92.6% to 107.58% for all analytes. The extraction recoveries were in the range of 86–105% and no significant matrix effect was observed. This simple and reproducible high-throughput method was successfully applied to the pharmacokinetic study of gefitinib and its major metabolites in mouse.
Keywords: Gefitinib; Metabolites; LC–MS/MS; Mouse plasma; Pharmacokinetics;

Chemical characterization and antioxidant activities comparison in fresh, dried, stir-frying and carbonized ginger by Yuxin Li; Yan Hong; Yanquan Han; Yongzhong Wang; Lunzhu Xia (223-232).
Ginger (Zingiber officinale Rosc.) is a common dietary adjunct that contributes to the taste and flavor of foods, and is also an important Traditional Chinese medicine (TCM). Different processing methods can produce different processed gingers with dissimilar chemical constituents and pharmacological activities. In this study, an ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC/QTOF-MS) was applied to identify the complicated components from fresh, dried, stir-frying and carbonized ginger extracts. All of the 27 compounds were identified from four kinds of ginger samples (fresh, dried, stir-frying and carbonized ginger). Five main constituents (zingerone, 6-gingerol, 8-gingerol, 6-shogaol and 10-gingerol) in these four kinds of ginger sample extracts were simultaneously determined by UPLC-PDA. Meanwhile, the antioxidant effect of fresh, dried, stir-frying and carbonized gingers were evaluated by three assays (2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid) diammonium salt (ABTS), and ferric reducing antioxidant power (FRAP)). The results demonstrated that antioxidant activity of dried ginger was the highest, for its phenolic contents are 5.2-, 1.1- and 2.4-fold higher than that of fresh, stir-frying and carbonized ginger, respectively, the antioxidant activities’ results indicated a similar tendency with phenolic contents: dried ginger > stir-frying ginger > fresh ginger > carbonized ginger. The processing contributed to the decreased concentration of gingerols and the increased levels of shogaols, which reducing the antioxidant effects in pace with processing. This study elucidated the relationship of the heating process with the constituents and antioxidant activity, and provided a guide for choosing different kinds of ginger samples on clinical application.
Keywords: Ginger; Processing; UPLC/QTOF-MS; Chemical characterization; Antioxidant activity;

The chemical fingerprint and metabolic profile of traditional Chinese medicine is very complicated and has been a great challenge. In the present study, chemical fingerprint of ethyl acetate fraction of Gastrodia elata (EtAcGE) and metabolic profile of rat plasma sample after intragastric administration of EtAcGE (2.5 g/kg) were investigated using ultra-high performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry (UPLC/Q-TOF MS). A total of 38 chemical constituents of EtAcGE were identified by comparing their retention time, accurate molecular mass and characteristic fragment ions with those of references, or tentatively characterized by comparing molecular formula, fragment ions with that of known compound or information available in literature. And 40 compounds were detected in dosed rat plasma sample, including 16 prototypes and 24 metabolites underwent metabolic process of glucuronidation, glucosylation, sulfation, methylation, hydroxylation, dehydrogenation or mixed modes. The metabolic “soft spots” was hydroxyl or carboxy group. This is the first research for chemical fingerprint and metabolic profile of EtAcGE, which lay a foundation for the further investigation of EtAcGE.
Keywords: Chemical fingerprint; Metabolic profile; EtAcGE; UPLC/Q-TOF MS;