Journal of Chromatography B (v.1049-1050, #C)

Rapid and selective extraction of target compounds from complex matrices was significantly important for their quantification. Here, hollow porous molecular imprinted polymers (HPMIPs) were prepared using cinnamic acid (CMA) as template, 4-vinylpyridine (4-VP) as functional monomer, and MCM-48 as sacrificial support. Hollow porous structure with large surface area (534.1 m2/g) made most recognition sites locate on the surface of HPMIPs, resulting in high binding capacity (14.84 mg/g) and fast kinetic binding (40 min). In comparison, binding capacity and binding equilibrium time for solid MIPs were 4.01 mg/g and 16 h. Equilibrium data fitted well to Freundlich equation, and adsorption process with multi-diffusion mechanisms could be described by pseudo-second order model. In addition, HPMIPs exhibited favorable selectivity. Finally, HPMIPs were successfully used as a molecular sorbent to efficiently and selectively extract CMA from apple and orange juices. Coupling with high-performance liquid chromatography and UV detection (HPLC–UV), the limit of detection for CMA was 0.006 mg/L, and the recoveries (89.3–103.5%) with RSD values ranged from 4.3% to 8.0% were relatively satisfactory. Results indicated the superiority of HPMIPs in the selective extraction field.
Keywords: Molecularly imprinted polymers; Hollow porous structure; Selective analysis; Cinnamic acid; Juice;

Gas chromatography-mass spectrometry profiles of urinary organic acids in healthy captive cheetahs (Acinonyx jubatus) by Adrian Stephen Wolferstan Tordiffe; Mari van Reenen; Fred Reyers; Lodewyk Jacobus Mienie (8-15).
In captivity, cheetahs (Acinonyx jubatus) frequently suffer from several unusual chronic diseases that rarely occur in their free-ranging counterparts. In order to develop a better understanding of their metabolism and health we documented the urine organic acids of 41 apparently healthy captive cheetahs, in an untargeted metabolomic study, using gas chromatography–mass spectrometry. A total of 339 organic acids were detected and annotated. Phenolic compounds, thought to be produced by the anaerobic fermentation of aromatic amino acids in the distal colon, as well as their corresponding glycine conjugates, were present in high concentrations. The most abundant organic acids in the cheetahs' urine were an as yet unidentified compound and a novel cadaverine metabolite, tentatively identified as N 1,N 5-dimethylpentane-1,5-diamine. Pantothenic acid and citramalic acid concentrations correlated negatively with age, while glutaric acid concentrations correlated positively with age, suggesting possible dysregulation of coenzyme A metabolism in older cheetahs. This study provides a baseline of urine organic acid reference values in captive cheetahs and suggests important avenues for future research in this species.
Keywords: Cheetahs; Acinonyx jubatus; Urine organic acids; Metabolomics; GC–MS;

Purification of foot-and-mouth disease virus by heparin as ligand for certain strains by Ping Du; Shiqi Sun; Jinjie Dong; Xiaoying Zhi; Yanyan Chang; Zhidong Teng; Huichen Guo; Zaixin Liu (16-23).
The goal of this project was to develop an easily operable and scalable process for the recovery and purification of foot-and-mouth disease virus (FMDV) from cell culture. Heparin resins HipTrap Heparin HP and AF-Heparin HC-650 were utilized to purify FMDV O/HN/CHA/93. Results showed that the purity of AF-Heparin HC-650 was ideal. Then, the O/HN/CHA/93, O/Tibet/CHA/99, Asia I/HN/06, and A/CHA/HB/2009 strains were purified by AF-Heparin HC-650. Their affinity/virus recoveries were approximately 51.2%/45.8%, 71.5%/70.9%, 96.4%/73.5, and 59.5%/42.1%, respectively. During a stepwise elution strategy, the viral particles were mainly eluted at 300 mM ionic strength peaks. The heparin affinity chromatography process removed more than 94% of cellular and medium proteins. Anion exchange resin Capto Q captured four FMD virus particles; 40% of binding proteins and 80%–90% of viral particles were eluted at 450 mM NaCl. Moreover, ionic strength varied from 30 to 450 mM had no effect on the immunity to FMDV. The results revealed that heparin sulfate may be the main receptor for CHA/99 strain attachment-susceptible cells. Heparin affinity chromatography can reach perfect results, especially when used as a ligand of the virus. Anion exchange is useful only as previous step for further purification.
Keywords: Foot-and-mouth disease virus; Heparin affinity chromatography; Anion exchange; Virus purification;

Development of high throughput LC/MS/MS method for analysis of perfluorooctanoic acid from serum, suitable for large-scale human biomonitoring by Eduard Rogatsky; Colleen O’Hehir; James Daly; Amelie Tedesco; Richard Jenny; Kenneth Aldous (24-29).
A simple method for determination of perfluorooctanoic acid (PFOA) from human serum by liquid chromatography/tandem mass spectrometry (LC/MS/MS) and utilizing high-throughput sample preparation was developed by New York State Public Health Emergency Preparedness Laboratory and used for biomonitoring studies. 50 μl of serum was mixed with a 0.4 ml of acetonitrile containing isotopically labeled internal standard, followed by phospholipid removal/protein precipitation. The extract was partially dried and analyzed by LC/MS/MS. The linear range of PFOA analysis was 0.5–100 ng/ml. LLOQ was selected as 0.5 ng/ml. The method was validated following APHL guidelines for LRN-C laboratories and about 6000 specimens were successfully prepared and analyzed using this simple LC/MS method. Due to sample preparation simplicity, the presented method can be used in large-scale clinical testing, such as public health surveillance studies.
Keywords: PFOA; Sample preparation; High throughput; LC/MS;

Green tea (GT) is one of the most consumed beverages worldwide. Tyrosine kinase inhibitors (TKIs) belong to the oral targeted therapy that gained much interest in oncology practice, among which are erlotinib (ERL) and lapatinib (LAP). Since green tea polyphenols (GTP) are known to be inhibitors of receptor tyrosine kinases, GTE could likely potentiate the anticancer effect of TKIs, but with a possibility of pharmacokinetic (PK) interaction with co-administered TKIs. In this study, the effect of GTE on the PK of ERL/LAP in rats was studied. UPLC–ESI–MS/MS method has been developed and validated for the quantification of ERL and LAP in rat plasma, using gefitinib (GEF) as the internal standard. Plasma samples were treated extensively by protein precipitation (PPT) followed by solid phase extraction (SPE) using octadecyl C 18/14% cartridges. Chromatographic analysis was carried out on Acquity UPLC BEH™ C18 column with a mobile phase consisting of water: acetonitrile (20: 80, v/v), each with 0.15% formic acid. Quantification was performed in the positive electrospray ionization (ESI + ) mode with multiple reaction monitoring (MRM) of the transitions m/z 394.29 → 278.19 (ERL), m/z 581.07 → 365.13 (LAP), and m/z 447.08 → 128.21 (GEF). The method was fully validated as per the FDA guidelines showing linearity over the range of 0.4–1000 (ERL) and 0.6–1000 (LAP) ng/mL with very low lower limit of quantification (LLOQ) of 0.4 and 0.6 ng/mL for ERL and LAP, respectively. The applicability of the method was extended to perform a comparative study of the PK of ERL/LAP following short-term and long-term administration of GTE, compared with their single oral administration. The results revealed that a significant reduction in the oral bioavailability was recorded with both ERL and LAP following the ingestion of GTE particularly for short-term administration. A reduction in Cmax (AUC) by 67.60% (69.50%) and 70.20% (73.96%), was recorded with short-term administration of GTE, compared with only 16.03% (21.09%) and 13.53% (22.12%) reduction for ERL and LAP, respectively, with long-term administration. Thus patients taking TKIs should preferably avoid drinking GT or ingesting GTE capsules during the period of treatment with TKIs.
Keywords: Green tea extract; Erlotinib; Lapatinib; UPLC–ESI–MS/MS; Pharmacokinetic interaction; Rat plasma;

An analytical approach including fingerprint, quantitative analysis and rapid screening of anti-oxidative components was established and successfully applied for the comprehensive quality control of Rhizoma Smilacis Glabrae (RSG), a well-known Traditional Chinese Medicine with the homology of medicine and food. Thirteen components were tentatively identified based on their retention behavior, UV absorption and MS fragmentation patterns. Chemometric analysis based on coulmetric array data was performed to evaluate the similarity and variation between fifteen batches. Eight discriminating components were quantified using single-compound calibration. The unit responses of those components in coulmetric array detection were calculated and compared with those of several compounds reported to possess antioxidant activity, and four of them were tentatively identified as main contributors to the total anti-oxidative activity. The main advantage of the proposed approach was that it realized simultaneous fingerprint, quantitative analysis and screening of anti-oxidative components, providing comprehensive information for quality assessment of RSG.
Keywords: Rhizoma Smilacis Glabrae; Accelerated solvent extraction; Mass spectrometry; Charged aerosol detection; Coulometric array detection;

Determination of catecholamines and endogenous related compounds in rat brain tissue exploring their native fluorescence and liquid chromatography by Beatriz M. Fonseca; Márcio Rodrigues; Ana C. Cristóvão; Daniela Gonçalves; Ana Fortuna; Liliana Bernardino; Amílcar Falcão; Gilberto Alves (51-59).
The profiling analysis of catecholamines and their metabolites in brain tissue offers a crucial key to understand their functions in the body and the opportunity to follow up neural diseases. A rapid and simple liquid chromatography-fluorescence detection (LC-FLD) method was developed and validated for simultaneously measuring several catecholamines and endogenous related compounds in the rat brain tissue samples. The target analytes measured in this bioanalytical assay were levodopa (L-DOPA), dopamine (DA), norepinephrine (NE), epinephrine (E), 3-O-methyldopa (3-O-MD), and homovanillic acid (HVA), being the 3,4-dihydroxybenzylamine (DHBA) used as internal standard (IS). The six analytes (L-DOPA, DA, NE, E, 3-O-MD and HVA) can be determined in a single chromatographic run of less than 12 min, and all the compounds (analytes and IS) were detected using their native fluorescence and monitored at excitation/emission wavelengths of 279 nm/320 nm, respectively. The chromatographic and detection conditions were experimentally optimized and then several validation parameters (linearity, limits of quantification and detection, precision and accuracy, recovery, stability and selectivity) were examined. In accordance with the international guidelines of the Food and Drug Administration and European Medicines Agency the method described herein exhibited limits of quantification in the range of 2–25 ng mL−1, linearity in wide concentration ranges (r 2  ≥ 0.994), and acceptable precision (coefficient variation ≤8.76%) and accuracy (bias ±14.65%) levels. Since the bioanalytical procedure does not involve pre-purification or derivatization of the sample, the absolute recovery was found to be around 100%. Moreover, the developed LC-FLD method was successfully applied for the determination of the compounds of interest in tissue samples of different rat brain regions (cerebellum, amygdala, cortex, hippocampus, striatum, mesencephalon, medulla oblongata, substantia nigra and ventral tegmental area). Hence, this assay represents a valuable bioanalytical tool to support several pre(non)clinical studies in the broad field of neurosciences, requiring the quantitative analysis of these bioamines and their metabolites.
Keywords: Catecholamines; Native fluorescence; Brain tissue; Bioanalytical method validation;