Journal of Chromatography B (v.936, #C)

A novel core shell beaded chromatographic materials was prepared by grafting of glycidyl methacrylate (GMA) on to the surface of poly(hydoxypropyl methacrylate/ethyleneglycol dimethacrylate), p(HPMA/EGDMA) beads via surface-initiated atom transfer radical polymerization (SI-ATRP). For grafting GMA, p(HPMA/EGDMA) beads were first modified with an ATRP initiator. A reaction with 2-bromo-2-methylpropionyl bromide of the hydroxyl groups of the beads led to ATRP initiator-covered surfaces. The grafted p(GMA) fibrous chains on the beads were decorated with two different ligands (i.e., Protein L and l-histidine) for separation of Immunoglobulin's (Igs) from aqueous solution in batch system. The maximum Igs adsorptions on the p(HPMA/EGDMA)-g-p(GMA)-Protein L and p(HPMA/EGDMA)-g-p(GMA)-l-histidine affinity beads were found to be 81.8 and 112.3 mg/g at pH 7.5 and 5.5, respectively. The purity of Igs from human serum was analyzed by HPLC. The Protein L immobilized affinity beads provided purity about 98%. The novel core shell polymeric beads decorated with Protein L showed a good selectivity for Igs molecules from diluted human serum. Adsorption studies of Igs onto Protein L and l-histidine immobilized affinity beads were also carried out in a continuous system.
Keywords: Affinity beads; Protein L; l-Histidine; Adsorption; Separation; Immunoglobulins;

A simple multiresidue method was developed for detecting and quantifying twenty analytes from 5 classes of prohibited veterinary drugs (β-agonists (9), anabolic hormones (4), quinoxalines (4), tranquilizers (1), cyproheptadine, and clonidine in animal feeds using a QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) approach. Feed samples were extracted by ultrasonic-assisted extraction with a mixture of methanol–acetonitrile (50:50, v/v), followed by a cleanup using a dispersive solid-phase extraction with PSA (primary secondary amine). Target compounds were separated and determined by a liquid chromatography tandem quadrupole mass spectrometer operating in positive electrospray ionization mode, using multiple reaction monitoring (MRM). The recoveries of these compounds were between 56.7% and 103% at three spiked levels. The repeatability was lower than 10%, whereas reproducibility was no more than 15% except for nandrolone (17% at 10 μg kg−1) and diazepam (19% at 10 μg kg−1). Decision limits (CCαs) and detection capabilities (CCβs) ranged from 0.42 to 5.74 μg kg−1 and 5.70–9.81 μg kg−1, respectively. The method was successfully applied to screening of real samples obtained from local feed markets and confirmation of the suspected target analytes.
Keywords: Multiclass drugs; Liquid chromatography–tandem mass spectrometry; QuEChERS; Feed;

P-glycoprotein (P-gp) is the most frequently proposed factor for multi-drug resistance. It is traditionally measured using antibody-based methods. While these techniques can provide relative quantification values for P-gp levels, the important information that is usually missing is its amount in the biological system. In this study, a novel and advanced liquid chromatography–tandem mass spectrometry (LC/MS/MS)-based targeted proteomics assay was developed and validated for the determination of P-gp in the breast cancer drug sensitive cell line MCF-7/WT and the drug resistant cell line MCF-7/ADR. Three tryptic peptides (434STTVQLMQR442, 674GSQAQDR680 and 368IIDNKPSIDSYSK380) can specifically represent P-gp. Among these peptides, 434STTVQLMQR442 was selected as the surrogate analyte for quantification, and a stable isotope-labeled synthetic peptide with the same sequence was used as an internal standard. The calibration range was validated from 10 to 1000 ng/mL. The intra- and inter-day precisions were within 5.9% and 3.7%, respectively. The accuracy for the quality control (QC) samples was within 8.0%. Using this assay, the amounts of P-gp were accurately quantified as 3.53 fg/cell (∼2.08 × 10−2  amol/cell) in the MCF-7/WT cells and 34.5 fg/cell (∼2.02 × 10−1  amol/cell) in the MCF-7/ADR cells. This outcome was then compared with those obtained by conventional analytical methods including confocal microscopy, western blotting and flow cytometry. The comparative results show that not only is the LC/MS/MS-based targeted proteomics assay able to monitor the protein levels in a more accurate manner, but the large discrepancy observed between the other methods was most likely due to the lack of specificity and the semi-quantitative nature of the conventional assays
Keywords: P-glycoprotein; Liquid chromatography–tandem mass spectrometry; Targeted proteomics; Stable isotope labeling; Methods comparison;

Abundant evidence suggests a prominent role for eicosanoids and metabolites in the pathogenesis and prognosis of inflammatory diseases. A sensitive and high-throughput SPE UPLC–MS/MS method was developed to quantitatively interrogate the levels of 18 eicosanoids in human and monkey plasma samples. A limit of quantitation of 0.25 ng/mL was achieved for all 18 investigated compounds with linear ranges spanning four orders of magnitude. Bioanalytical performance of this assay was fully characterized including SPE extraction efficiency, matrix effect, autosampler stability, benchtop stability and freeze–thaw cycle variability. Endogenous levels of the eicosanoids and analogs within a set of monkey plasma samples challenged with lipopolysaccharide and human plasma samples were quantified by this ultra performance liquid chromatography–mass spectrometry (UPLC–MS/MS) assay. Quantitative eicosanoid profiles of the human samples were further analyzed by a non-supervised cluster analysis, which revealed a set of potential positive and negative lipid biomarkers to distinguish the following three groups: healthy individuals, hypertensive patients and severe atherosclerosis patients. The components of the negative biomarker cluster (8-HETE, LTB4, 9-HODE and 13-HODE) are putative ligands of peroxisome proliferator-activated receptors (PPARs), a family of master genes controlling the resolution of inflammatory signaling.
Keywords: UPLC–MS/MS; Eicosanoid; Atherosclerosis; Lipid biomarkers; Cluster analysis;

Evaluation of mixed-mode chromatographic resins for separating IgG from serum albumin containing feedstock by Rong-Zhu Wang; Dong-Qiang Lin; Hong-Fei Tong; Hui-Li Lu; Shan-Jing Yao (33-41).
Mixed-mode chromatography has been focused as a cost-effective new technique for antibody purification. In this study, four mixed-mode resins with N-benzyl-N-methyl ethanol amine, 2-benzamido-4-mercaptobutanoic acide, 4-mercapto-ethyl-pyridine and phenylpropylamine as the ligands were tested and the multi-functional interactions between ligand and protein were discussed. Immunoglobulin G (IgG), bovine serum albumin (BSA) and the binary mixture of BSA and IgG were used as the model feedstock to compare the separation behaviors by pH gradient elution. The comparison analysis showed mixed-mode resin with N-benzyl-N-methyl ethanol amine as the ligand had the best ability to separate IgG and BSA. The results indicated that for four resins tested ionic interaction might play the dominant role in the separation of IgG and BSA while the hydrophobic interactions and hydrogen bonding have some subsidiary effects. The pH stepwise elution and sample loading were optimized to improve the IgG purification from serum albumin containing feedstock. High purity (92.3%) and high recovery (95.6%) of IgG were obtained. The results indicated that mixed-mode chromatography would be a potential option for antibody purification with the control of loading and elution conditions.
Keywords: Mixed-mode chromatography; Immunoglobulin G; Serum albumin; Gradient elution; Step elution; Purification;

Development and validation of a new UPLC-PDA method to quantify linezolid in plasma and in dried plasma spots by Lorena Baietto; Antonio D’Avolio; Alessandra Ariaudo; Silvia Corcione; Marco Simiele; Jessica Cusato; Rosario Urbino; Giovanni Di Perri; V. Marco Ranieri; Francesco Giuseppe De Rosa (42-47).
Linezolid is an oxazolidinone antibiotic used for the treatment of pneumonia and uncomplicated and complicated skin and soft tissues infections caused by Gram positive bacteria. It is also used as second line agent in multi-drug resistant tuberculosis. Therapeutic drug monitoring (TDM) of linezolid represents a valid tool in clinical practice to optimize therapy, especially in critically ill patients. Spreading of TDM is mainly limited by high costs shipment and lack of laboratories that offer a TDM service. To overcome these problems, the use of dried plasma spots or dried blood spots is increasing. The aim of this work was to develop and validate a new chromatographic method to analyze linezolid in plasma and in dried plasma spots and to evaluate the correlation between the two extraction methods. Linezolid extraction from plasma and from dried plasma spots was obtained using acetonitrile. Quinoxaline was used as internal standard. Analysis was performed by an ultra performance liquid chromatography (UPLC) system coupled with photo diode array (PDA) detector, at 254 nm. Both analytical methods were linear (r 2  > 0.999) over the calibration range of 30–0.117 mg/L. Limit of quantification and limit of detection were 0.117 mg/L and 0.058 mg/L, respectively. Intra and inter-day precision (R.S.D.%) and accuracy (%) were <15%. Long term stability of linezolid in dried plasma spots showed absence of degradation at room temperature (20–25 °C) and at 4 °C, for at least one month. Linear regression analysis confirmed that the two methods of extraction have good correlation. Thus they are suited for TDM of linezolid and for pharmacokinetic studies.
Keywords: Linezolid; Dried plasma spots; HPLC; UPLC; Therapeutic drug monitoring TDM; Human plasma; Stability;

Boldine belongs to the group of aporphine alkaloids isolated from Boldo tree. In contrast with numerous reports on the pharmacological effects of boldine, the data about its pharmacokinetics and biotransformation are scarce. No validated bioanalytical method of sufficient sensitivity has so far been described in the literature which could be used for quantification of boldine in various body fluids collected in pharmacokinetic studies. This work presents, for the first time, the assay for boldine in the plasma, bile and urine of rats. It includes liquid–liquid extraction/back-extraction of boldine, its chromatographic separation and sensitive fluorescence detection. Separation was carried out on a pentafluorophenyl core–shell column (Kinetex PFP, 150 × 3 mm, 2.6 μm) in gradient elution mode with solvent system consisting of an acetonitrile–ammonium formate buffer (5 mM, pH = 3.8). Fluorimetric detection (λ EX  = 320 nm, λ EM  = 370 nm) was used for quantitative work. Validation according to the EMEA guideline proved the assay LLOQ (0.1 μmol L−1), linearity over a broad range of 0.1–50 μmol L−1, precision (intra- and inter-day CVs less than 4.5% and 6.1%, respectively) and accuracy (relative errors between −5.8% and 4.8%). In a pilot pharmacokinetic experiment, the concentration–time profiles were described for boldine (single i.v. bolus 50 mg kg−1) in plasma and bile and cumulative excretion in urine was investigated. The major metabolites identified by means of LC–MS n were boldine-O-glucuronide, boldine-O-sulphate and disulphate, boldine-O-glucuronide-O-sulphate and N-demethyl-boldine-O-sulphate.
Keywords: Boldine; Metabolites; Pharmacokinetics; LC/MS/MS; Core–shell column; Fluorescence;

Four iridoid glucosides, shanzhiside methyl ester, phloyoside II, chlorotuberside, and penstemonoside, were isolated and purified from an herbal medicinal plant for the first time by high-speed counter-current chromatography (HSCCC) using a two-phase solvent system composed of ethyl acetate–n-butanol–water (5:14:12, v/v/v). A total of 37 mg of shanzhiside methyl ester, 29 mg of phloyoside II, 27 mg of chlorotuberside, and 21 mg of penstemonoside with the purity of 99.2%, 98.5%, 97.3%, and 99.3%, respectively, were obtained in one-step separation within 4 h from 150 mg of crude extract. To the best of our knowledge, this is the first report of separation and purification of iridoid glucosides from natural sources by HSCCC. The chemical structures of all the four compounds were identified by ESI-MS, 1H NMR, and 13C NMR.
Keywords: Lamiophlomis rotata (Benth.) Kudo; Shanzhiside methyl ester; Phloyoside II; Chlorotuberside; Penstemonoside;

A novel ion pairing LC/MS metabolomics protocol for study of a variety of biologically relevant polar metabolites by Jose M. Knee; Teresa Z. Rzezniczak; Aiko Barsch; Kevin Z. Guo; Thomas J.S. Merritt (63-73).
We report a method of ion-pairing liquid chromatography coupled to mass spectrometry (IP-LC–MS) that we have developed for the sensitive detection and quantification of a variety of biologically relevant polar molecules. We use the ion-pairing agent diamyl ammonium to improve chromatographic resolution of polar compounds, such as nucleotide cofactors, sugar phosphates, and organic acids, that are generally poorly retained by conventional reverse phase chromatographic methods. This method showed good linearity (average R value of 0.996) and reproducibility (generally RSD values <10%). We demonstrate the utility of this method by investigating the metabolomic signature of three distinct biological systems: the metabolic response to lack of superoxide dismutase activity and to paraquat induced oxidative stress, and the metabolic profiles of four different Drosophila species.
Keywords: Ion pairing chromatography; IP-LC–MS; Diamyl ammonium (DAA); Drosophila metabolomics; Polar metabolites;

Competitive immunoassay of progesterone by microchip electrophoresis with chemiluminescence detection by Fanggui Ye; Jinwen Liu; Yong Huang; Shutin Li; Shulin Zhao (74-79).
A sensitive and rapid homogeneous immunoassay method based on microchip electrophoresis-chemiluminescence detection (MCE-CL) using luminol-hydrogen peroxide as chemiluminescence system catalyzed by horseradish peroxidase (HRP) was developed for the determination of progesterone (P). The assay was based on the competitive immunoreactions between HRP-labeled P antigen (HRP-P) and P with a limited amount of anti-P mouse monoclonal antibody (Ab), and MCE separation of free HRP-P and HRP-P-Ab immunocomplex followed by CL detection. The effect of various factors such as conditions for the CL reaction, MCE and incubation time for the immunoreactions were examined and optimized. Under optimal assay conditions, the MCE separation was accomplished within 80 s. The linear range of detection for P was 8–800 nM with a detection limit of 3.8 nM (signal/noise ratio = 3). This present method has been applied to determine P in human serum samples from normal and pregnant women. The result indicates that the proposed MCE-CL based homogeneous immunoassay method can serve as an alternative tool for clinical assay of P.
Keywords: Chemiluminescence detection; Competitive immunoassay; Human serum; Microchip electrophoresis; Progesterone;

UV-filters are widely used in many personal care products and cosmetics. Recent studies indicate that some organic UV-filters can accumulate in biota and act as endocrine disruptors, but there are few studies on the occurrence and fate of these compounds in humans. In the present work, a new liquid chromatography–tandem mass spectrometry (LC–MS/MS) method to assess the presence of six UV-filters in current use (benzyl salicylate, phenyl salicylate, octyl salicylate, homosalate, 3-(4-methylbenzylidene) camphor, and 3-benzylidene camphor) in human placental tissue is proposed. The method involves the extraction of the analytes from the samples using ethyl acetate, followed by a clean-up step using centrifugation prior to their quantification by LC–MS/MS using an atmospheric pressure chemical ionization (APCI) interface. Bisphenol A-d16 was used as surrogate for the determination of benzyl salicylate, phenyl salicylate, octyl salicylate and homosalate in negative mode and benzophenone-d10, was used as surrogate for the determination of 3-(4-methylbenzylidene) camphor and 3-benzylidene camphor in positive mode. The found limits of detection ranged from 0.4 to 0.6 ng g−1 and the limits of quantification ranged from 1.3 to 2.0 ng g−1, while variability was under 13.7%. Recovery rates for spiked samples ranged from 97% to 104%. Moreover, the interactions of these compounds with the human estrogen receptor alpha (hERα) and androgen receptor (hAR), using two in vitro bioassays based on reporter gene expression and cell proliferation assessment, were also investigated. All tested compounds, except benzyl salicylate and octyl salicylate, showed estrogenic activity in the E-Screen bioassay whereas only homosalate and 3-(4-methylbenzylidene) camphor were potent hAR antagonists. Although free salicylate derivatives and free camphor derivatives were not detected in the human placenta samples analyzed, the observed estrogenic and anti-androgenic activities of some of these compounds support the analysis of their occurrence and their role as endocrine disrupters in humans.
Keywords: UV-filters; Placental tissue; LC–MS/MS; E-Screen; Gene expression;

In this study, we fabricate a monolithic poly(methylmethacrylate) (PMMA) microdevice on which solid phase-based DNA preparation and flow-through polymerase chain reaction (PCR) units were functionally integrated for one-step sample preparation and amplification operated by pressure. Chelex resin, which is used as a solid support for DNA preparation, can capture denatured proteins but releases DNA, and the purified DNA can then be used as a template in a subsequent amplification process. Using the PMMA microdevices, DNA was successfully purified from both Escherichia coli and human hair sample, and the plasmid vector inserted in E. coli and the D1S80 locus in human genomic DNA were successfully amplified from on-chip purified E. coli and human hair samples. Furthermore, the integration potential of the proposed sample preparation and flow-through PCR units was successfully demonstrate on a monolithic PMMA microdevice with a seamless flow, which could pave the way for a pressure-driven, simple one-step sample preparation and amplification with greatly decreased manufacture cost and enhanced device disposability.
Keywords: Sample preparation; Solid phase; Chelex resin; Flow-through polymerase chain reaction (PCR); Poly(methylmethacrylate) (PMMA); Integrated microdevice;