Journal of Chromatography B (v.1035, #C)

We discuss the use of a laterally-fed membrane chromatography (or LFMC) device for single-step purification of mono-PEGylated lysozyme. Recent studies have shown such LFMC devices to be suitable for high-resolution, multi-component separation of proteins in the bind-and-elute mode. The device used in this study contained a stack of rectangular cation-exchange membranes having 9.25 mL bed volume. PEGylation of lysozyme was carried out in batch mode using 5 kDa methoxy-polyethyleneglycol propionaldehyde (or m-PEG propionaldehyde) in the presence of sodium cyanoborohydride as reducing agent. Membrane chromatographic separation was carried out at 1.62 membrane bed volumes per minute flow rate, in the bind-and-elute mode. When a salt gradient was applied, the higher PEGylated forms of lysozyme (i.e. the byproducts) eluted earlier than mono-PEGylated lysozyme (the target product), while lysozyme eluted last. Under elution conditions optimized for resolution and speed, the separation could be carried out in less than 15 membrane bed volumes. High purity and recovery of mono-PEGylated lysozyme was obtained. The resolution of separation of mono-PEGylated lysozyme obtained under the above condition was comparable to that reported in the literature for equivalent cation-exchange resin columns while the flow rate expressed in bed volumes/min was 21.7 times higher. Also, the number of theoretical plates per meter was significantly higher with the LFMC device. Therefore the LFMC based purification process discussed in this paper combined high-productivity with high-resolution.
Keywords: Membrane chromatography; Device; PEGylated protein; Laterally-fed membrane chromatography; Protein purification;

UPLC–MS/MS determination of florfenicol and florfenicol amine antimicrobial residues in tilapia muscle by Eduardo Adilson Orlando; Aline Gabriela Costa Roque; Marcos Eliseu Losekann; Ana Valéria Colnaghi Simionato (8-15).
Despite the benefits to fish farmers, the use of antimicrobials in aquaculture has concerned consumers and competent authorities. The indiscriminate use of such substances promotes the emergence of resistant microorganisms, decreases the effectiveness of treatments, and causes possible toxic effects in humans. In Brazil, florfenicol is the only antimicrobial registered for use in aquaculture and is often used in tilapia in cage creation. Thus, this study aimed to develop a method for determination of florfenicol residues and its metabolite florfenicol amine in tilapia fillet by UPLC–MS/MS. Analytes were extracted with ethyl acetate, followed by liquid-liquid partition clean-up with hexane and SPE. The sorbents C18, phenyl and HLB-Oasis were evaluated by SPE. Phenyl sorbent showed the best results, and the extraction conditions were optimized in the sample matrix with fractional factorial design 24−1. The analytes were separated on a C18 chromatographic column (50 × 2.1 mm × 1.7 μm) using water (A) and acetonitrile (B) as mobile phase at a flow rate of 0.3 mL min−1 with a linear gradient (in% B): 0–2.0 min: 20%; 2.0–2.5 min: increase to 90%; 2.5–3.5 min: 90%; 3.0–3.5 min: decrease to 20%; 4.0–5.0 min: 20%. The analytes were monitored in a MS/MS triple quadrupole system by MRM mode with transitions at m/z 356.1 > 336.1 (florfenicol) and m/z 248.1 > 130.1 (florfenicol amine). The optimized method was validated obtaining LOQ values of 3 and 25 ng g−1 for florfenicol and florfenicol amine, respectively, precision between 20 and 36%, absolute extraction efficiency between 38 and 80%, and adequate linearity. The method was applied to samples intended for human consumption, and within the 15 evaluated samples, only one showed florfenicol residue at 30 ng g−1, which is below the maximum residue limit established in Brazil.
Keywords: Florfenicol; Florfenicol amine; Residue; Fish; Tilapia; SPE;

Preparation of soluble isotopically labeled human growth hormone produced in Escherichia coli by Jin-Hee Lee; Ji-Seon Jeong; Sook-Kyung Kim; Jimyeong Song; Ji Youn Lee; Soyun Baek; Jun-Hyuk Choi (16-24).
Isotopically labeled proteins have been used as internal standards for mass spectrometry (MS)-based absolute protein quantification. Although this approach can provide highly accurate analyses of proteins of interest within a complex mixture, one of the major limitations of this method is the difficulty in preparing uniformly labeled standards. Human growth hormone (hGH) is one of the most important hormones that circulate throughout the body, and its measurement is primarily of interest in the diagnosis and treatment of growth disorders. In order to provide a useful internal standard for MS-based hGH measurement, we describe an efficient strategy to produce a potentially valuable, stable isotope-labeled hGH with high purity and yield. The strategy involves the following steps: solubilization of hGH under labeling conditions, detection of stable isotope incorporation, large-scale purification, analysis of the labeled protein, and assessment of the labeling efficiency. We show that the yield of soluble hGH under selective isotopic labeling conditions can be greatly increased by optimizing protein expression and extraction. Our efficient method for generating isotopically labeled hGH does not influence the structural integrity of hGH. Finally, we assessed the efficiency of stable isotope labeling at the intact protein level, and the result was further verified by amino acid analysis. These results clearly indicate that our labeling approach allows an almost complete incorporation of 13C6 15N4-arginine into the hGH expressed in E.coli without detectable isotope scrambling.
Keywords: Recombinant protein; Stable isotope; Purification; Human growth hormone; Escherichia coli;

Biomimetic ELISA detection of malachite green based on magnetic molecularly imprinted polymers by Lu Li; Zheng-zhong Lin; Ai-hong Peng; Hui-ping Zhong; Xiao-mei Chen; Zhi-yong Huang (25-30).
A direct competitive enzyme-linked immunosorbent assay (ELISA) method was used for the detection of malachite green (MG) with a high sensitivity and selectivity using magnetic molecularly imprinted polymers (MMIPs) as a bionic antibody. MMIPs were prepared through emulsion polymerization using Fe3O4 nanoparticles as magnetic nuclei, MG as a template, methacrylic acid (MAA) as a functional monomer, ethylene glycol dimethacrylate (EGDMA) as a crosslinking agent and span-80/tween-80 as mixed emulsifiers. The MMIPs were characterized by scanning electron micrographs (SEM), thermal-gravimetric analyzer (TGA), Fourier transform infrared spectrometer (FT-IR) and vibrating sample magnetometer (VSM), respectively. A high magnetic saturation value of 54.1 emu g−1 was obtained, resulting in rapid magnetic separation of MMIPs with an external magnet. The IC 50 of the established ELISA method was 20.1 μg L−1 and the detection limit (based on IC 85) was 0.1 μg L−1. The MMIPs exhibited high selective binding capacity for MG with cross-reactivities less than 3.9% for MG structural analogues. The MG spiking recoveries were 85.0%–106% with the relative standard deviations less than 4.7%. The results showed that the biomimetic ELISA method by using MMIPs as bionic antibody could be used to detect MG rapidly in fish samples with a high sensitivity and accuracy.
Keywords: Magnetic molecularly imprinted polymer; ELISA; Malachite green; Bionic antibody; Fish;

A sensitive and high-throughput method was established and validated for the simultaneous determination of 22 mycotoxins in Pheretima aspergillum (E.Perrier) and Pheretima guillelmi (Michaelsen). A modified Quick Easy Cheap Effective Rugged and Safe (QuEChERS) method was used for sample preparation with recoveries ranging from 73% to 105% with relative standard deviations (RSDs) <8.0% for all target analytes. Ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC–MS/MS) was applied for separation and detection in ESI (+) and ESI (−) modes with the limits of detection (LOD) in the range of 0.05–10 μg kg−1. The 22 compounds could be accurately quantified in the 0.5–1000 μg kg−1 concentration range with correlation coefficients >0.99. In all cases, the intra- and inter-day precisions were lower than 6% and 10%, respectively. Matrix-matched calibration was utilized for quantification purposes to compensate for the matrix effects. Furthermore, the established method was successfully applied in 17 batches of normal real samples collected from different areas of China and 2 batches of moldy samples due to improper storage, only mold-contaminated samples were confirmed to have fumonisin B1 (FB1) and fumonisin B2 (FB2) contamination at 2.54–3.78 μg kg−1. The constructed method could serve as a practical application of the UHPLC–MS/MS method for the trace analysis of multiple mycotoxins in complex matrixes, especially for those with high lipid contents.
Keywords: Pheretima; Mycotoxins; QuEChERS; UHPLC–MS/MS;

Trimethylamine-N-oxide (TMAO) is a metabolite generated from choline, betaine and carnitine in a gut microbiota-dependent way. This molecule is associated with development of atherosclerosis and cardiovascular events. A sensitive liquid chromatographic electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) has been developed and validated for the simultaneous determination of TMAO related molecules including TMAO, betaine, choline, and carnitine in mouse plasma. Analytes are extracted after protein precipitation by methanol and subjected to LC-ESI-MS/MS without preliminary derivatization. Separation of analytes was achieved on an amide column with acetonitrile-water as the mobile phase. This method has been fully validated in this study in terms of selectivity, linearity, sensitivity, precision, accuracy, and carryover effect, and the stability of the analyte under various conditions has been confirmed. This developed method has successfully been applied to plasma samples of our mouse model.
Keywords: HPLC-ESI-MS/MS; TMAO; Choline; Betaine; Carnitine;

Monitoring of proteins involved in cellular life and death processes is of high scientific interest since it permits the elucidation of functional changes in a variety of diseases. In this study, we have developed a nanoLC–MS/MS assay for the simultaneous detection and quantification of 24 selected proteins that are known to be important for cellular homeostasis. The Selected Reaction Monitoring (SRM) assay applies heavy-labeled peptide analogues for the relative quantification of proteins with central functions in cellular stress and metabolism, including many mitochondrial proteins. The assay includes proteins involved in the quality control of mitochondrial proteins, oxidative stress, respiratory chain, and fatty acid degradation, as well as the cytosolic glyceraldehyde 3-phosphate dehydrogenase, lactate dehydrogenase and ribosomal proteins. The assay can thus quantitate the balance between mitochondrial and cytosolic pathways, which is relevant in many disease states, and can be studied by comparing patient and control samples. The measured validation parameters showed satisfactory results for the proteins included in the analysis. The linear range of the monitored proteins was 0.01–20 nM, with a median precision of less than 10%. The assay performed well in monitoring proteins in both cultured human skin fibroblast cells as well as in isolated peripheral blood mononuclear cells. We therefore believe that this assay is applicable for the study of cellular stress response in various types of cell defects and disease states.
Keywords: Mitochondrion; Proteomics; Metabolic disease; Biomarker; Chaperone; PBMC; Apoptosis;

Determination of personal care products benzophenones and parabens in human menstrual blood by I. Jiménez-Díaz; L.M. Iribarne-Durán; O. Ocón; E. Salamanca; M.F. Fernández; N. Olea; E. Barranco (57-66).
Benzophenones and parabens are synthetic chemicals used in many personal care products, foods and pharmaceuticals. Benzophenones are used to protect the skin and materials from the adverse effects of UV-radiation, and parabens are used as preservatives. Despite their widespread occurrence and proven endocrine disrupting activity, relatively little is known about human exposure to these compounds. In the present work, an analytical method based on sample treatment using dispersive liquid-liquid microextraction (DLLME) for the extraction of six benzophenones (benzophenone-1, −2, −3, −6, −8 and 4-hydroxybenzophenone) and four parabens (methyl-, ethyl-, propyl- and butyl- paraben) from human menstrual blood samples, followed by ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS) analysis, is proposed and validated. The method was validated using matrix-matched standard calibration followed by a recovery assay with spiked samples. The limits of detection ranged from 0.1 to 0.3 ng mL−1, with recoveries of 93.8% to 108.9%, and precision (evaluated as relative standard deviation) lower than 14% for all selected compounds. This method was successfully applied for the determination of the target compounds in 25 samples of human menstrual blood. Methylparaben and benzophenone-3 were the most frequently detected compounds (96%).
Keywords: Personal care products; Benzophenones; Parabens; Human menstrual blood; DLLME; UHPLC–MS/MS;

A simple and rapid high-performance liquid chromatography method with diode-array detection (HPLC-DAD) using microextraction by packed sorbent (MEPS) during the sample preparation step was developed and validated to quantify lamotrigine (LTG) in rat plasma and brain samples. MEPS variables such as pH, number of draw-eject cycles, and washing and desorption conditions were optimized. The chromatographic resolution of LTG and chloramphenicol, used as internal standard (IS), was accomplished in less than 5 min on a C18 column, at 35 °C, using an isocratic elution with acetonitrile (13%), methanol (13%) and water-triethylamine (99.7:0.3, v/v; pH 6.0) pumped at a flow rate of 1 mL/min. Detection was performed at 215 nm. Calibration curves were linear over the range of 0.1–20 μg/mL (r2  ≥ 0.9947) for LTG in both rat plasma and brain homogenate samples. The intra and interday imprecision did not exceed 8.6% and the intra and interday inaccuracy ranged from −8.1 to 13.5%. LTG was extracted from rat plasma and brain homogenate samples with an average absolute recovery ranging from 68.0 to 86.7%, and its stability was demonstrated in the assayed conditions. No interferences were observed at the retention times of the analyte (LTG) and IS. To the best of our knowledge, this is the first bioanalytical assay that uses MEPS procedure for the determination of LTG not only in rat plasma but also in tissue (brain) samples. This novel method was successfully applied to a preliminary pharmacokinetic study in rats and it seems to be a cost-effective tool to support non-clinical pharmacokinetic-based studies involving LTG treatment.
Keywords: Lamotrigine; Bioanalytical method validation; High-performance liquid chromatography; Microextraction by packed sorbent; Rat plasma and brain;

Urinary hippuric acid (HA) and phenylacetylglycine (PAG) are biomarker candidates for drug-induced phospholipidosis (PLD). To confirm their utility in preclinical and clinical settings, it is essential to develop and validate their quantification method in advance. In this study, we have applied liquid chromatography-tandem mass spectrometry (LC/MS/MS) for simultaneous quantification of HA and PAG in rat urine, and matrix based ion suppression was assessed by post-column infusion assay. Effective sample dilution reduced matrix effect of urine to be negligible level and calibration curves showed good correlation between those in urine diluent and buffer alone. Reliability of this assay was confirmed by the assessments for intra- and inter-day precisions and accuracies of quality control samples. The method was applied to rat urine after multiple oral administrations of PLD-inducing drugs, and the changes in HA and PAG concentrations and their ratio were successfully detected as rat plasma in previous report. This is the first report to quantify HA and PAG easily and accurately as potential biomarkers to monitor PLD status. This assay would be useful tool for monitoring PLD in toxicological studies by non-invasive sampling.
Keywords: Drug-induced phospholipidosis; Hippuric acid; Phenylacetylglycine; LC/MS/MS; Analytical validation;

Antibiotic industrial dregs, generated from the production of antibiotics by fermentation, are banned in China as animal feed additives. Official monitoring programs require the analysis of feeds for possible illegal use of the dregs. A rapid and sensitive method was developed for the simultaneous determination of streptomycin and streptidine as markers for streptomycin industrial dregs in pig and poultry compound feeds. After extraction with 20% aqueous trichloroacetic acid and pH adjustment, sample cleanup was performed by weak cation-exchange solid-phase extraction. UPLC–ESI–MS/MS was carried out using a hydrophilic interaction chromatography(HILIC)column to achieve separation. Quantification required matrix-matched calibrations in a linear range of 50–1000 μg kg−1; the calibration curves were linear in this range with coefficients of determination of 0.991 and 0.994 for streptomycin and streptidine, respectively. The method validity parameters—LODs (20 μg kg−1) and LOQs (50 μg kg−1), recoveries (71–78% and 75–84%, respectively), and relative reproducibility (5.4–9.6%)—satisfy the requirements of routine analysis.
Keywords: Solid-phase extraction; UPLC–MS/MS; Streptomycin; Streptidine; Streptomycin industrial dregs; Compound feed;

Synthetic cathinones continue to present a formidable challenge to forensic toxicology laboratories despite the fact that they are often encountered in impaired driving and death investigations. Due to limitations in immunoassay-based screening technologies, many forensic toxicology laboratories must rely on more labor intensive chromatographic-based screening approaches in order to detect these drugs in biological evidence. Solid phase extraction (SPE) and liquid chromatography-quadrupole/time of flight (LC-Q/TOF) mass spectrometry were used to identify twenty-two synthetic cathinones in urine and blood. Target drugs included methcathinone, ethcathinone, pentedrone, buphedrone, 3-fluoromethcathinone (3-FMC), 4-fluoromethcathinone (4-FMC), 4-methylethcathinone (4-MEC), 4-ethylmethcathinone (4-EMC), mephedrone, methedrone, 3,4-dimethylmethcathinone (3,4-DMMC), ethylone, butylone, pentylone, eutylone, methylone, methylenedioxypyrovalerone (MDPV), 4-methylpyrrolidinobutiophenone (MPBP), 3,4-methylenedioxypyrrolidinobutiophenone (MDPBP), α-pyrrolidinopentiphenone (α-PVP), pyrovalerone, and naphyrone. A total of nine deuterated internal standards were employed. Using traditional reversed phase chromatography all positional isomers, including 3-FMC and 4-FMC, were separated in 12 min. The procedure was validated in accordance with the Scientific Working Group for Forensic Toxicology (SWGTOX) Standard Practices for Method Validation. Extraction efficiencies were 84–104% and 81–93% in urine and blood, respectively. Limits of quantitation in both matrices were 0.25–5 ng/mL. Precision, bias and matrix effect were all within acceptable thresholds and the assay was free from more than fifty interferences. The validated method was used to identify cathinones in authentic urine case samples (n = 20) and these results highlight important considerations for cathinone stability and the subsequent interpretation of results.
Keywords: Cathinone; Designer drug; Liquid chromatography mass spectrometry; High resolution mass spectrometry; Forensic toxicology;

New psychoactive substances (NPS) have become an increasing drug problem in the past decade. For detection of NPS, new analytical methods have to be developed, and the methods also have to be updated regularly. This study aimed at developing a multi-component LC–MS/MS method for detection and quantification of 11 NPS of the benzodiazepine sub-class (“designer benzodiazepines”) in urine specimens. The method involves dilution of urine with internal standard and hydrolysis of any glucuronide conjugated forms. Separation of the compounds was achieved on a BEH Phenyl column, followed by MS/MS detection in positive electrospray mode. Method validation was performed following the EMA guideline. The method was applied to study the occurrence of designer benzodiazepines in Sweden in 2014–2015, by analysis of 390 samples retrieved from a routine drug testing laboratory. In 40% of these samples, selected based on a positive immunoassay benzodiazepine screening but a negative MS confirmation for the standard set of prescription benzodiazepines, intake of designer benzodiazepines was revealed. These results stress the importance of using and updating confirmation methods to include the increasing number of designer benzodiazepines appearing on the NPS market.
Keywords: Designer benzodiazepines; LC–MS/MS; NPS; Urine; Drug testing;