Journal of Chromatography B (v.879, #28)

Self-association of long-acting insulin analogues studied by size exclusion chromatography coupled to multi-angle light scattering by Malene H. Jensen; Per-Olof Wahlund; Jes K. Jacobsen; Bente Vestergaard; Marco van de Weert; Svend Havelund (2945-2951).
Two structurally very different insulin analogues analysed here, belong to a class of analogues of which two have been reported to have a protracted action through self-assembly to high molar mass in subcutis. The process of self-association of insulin analogues LysB29 (Nɛω-carboxyheptadecanoyl) des(B30) human insulin and LysB29 (Nɛ-lithocholyl) des(B30) human insulin was investigated using size exclusion chromatography (SEC) in connection with multi-angle light-scattering. Self-assembly to high molar mass was obtained by exchanging the formulation containing phenolic preservatives with an isotonic eluent during SEC. It was shown that increasing amounts of zinc in the formulations of the two analogues increased the size of the self assemblies formed during gel filtration. The addition of 0.2 mM phenol to the elution buffer slowed down the self-association process of zinc containing formulations and shed light on the initial association process. The results indicated that a dihexamer is a possible building block during self-association of LysB29 (Nɛω-carboxyheptadecanoyl) des(B30) human insulin. Surprisingly, in the absence of zinc the two analogues behaved very differently. LysB29 (Nɛω-carboxyheptadecanoyl) des(B30) human insulin was in equilibrium between oligomers smaller than a hexamer, whereas LysB29 (Nɛ-lithocholyl) des(B30) human insulin self-associated and formed even larger complexes than in the presence of zinc.
Keywords: Long-acting insulin analogue; Acylated insulin analogue; Soluble basal insulin; Self-association of insulin analogues; Depot formation; Size exclusion chromatography coupled with multi-angle light-scattering;

Hb Riesa or β93 (F9) Cys → Ser, a new electrophoretically silent haemoglobin variant interfering with haemoglobin A1c measurement by Emmanuel Bissé; Agnès Hovasse; Sabine Preisler-Adams; Thomas Epting; Oswald Wagner; Gabriele Kögel; Alain Van Dorsselaer; Christine Schaeffer-Reiss (2952-2956).
A new β variant was found in a German diabetic patient whose blood samples appeared to contain 45% Hb A1c using Bio-Rad Variant V-II A1c-analyzer but 7.6% on boronate affinity chromatography. Structural studies using, HPLC, mass spectrometry, and the genomic DNA analysis revealed a new substitution in which the cysteine residue at position β93 was replaced by serine. The variant was named Hb Riesa or β93 (F9) Cys → Ser and accounted for 54.3% of the total haemoglobin. This suggests that the protein-synthesis processes for the mutant could be slightly more promoted than those of the wild-type. Hb Riesa is clinically and electrophoretically silent.
Keywords: Hb variant; Hb A1C; Diabetes; HPLC; TOF-ESI-MS; LC–MS/MS;

Preparation and application of a partially degradable gel in mass spectrometry-based proteomic analysis by Jian Zhou; Yongbo Hu; Yong Lin; Haipeng Liu; Peng Xie (2957-2962).
In-gel digestion is an attractive route in mass spectrometry-based proteomic analysis, which, however, often suffers from a certain amount of sample loss mainly due to insufficient protein digestion and peptide extraction. To address this, herein we establish a partially degradable gel-assisted protein digestion and peptide recovery method by means of a simple replacement of bis-acrylamide (BA) with bis-acrylylcystamine (BAC). Concretely, the protein sample solubilized using high concentrations of sodium dodecyl sulfate (SDS) and urea were directly entrapped and immobilized into BAC-crosslinked gel by vacuum-dried gel absorption followed by fixation treatment. After removal of SDS and urea by repeated washing, the proteins were subjected to in-gel digestion and the gel was reductively treated. The tryptic peptides were recovered from the partial degradation of the gel and analyzed afterwards by capillary liquid chromatography coupled with tandem mass spectrometry (CapLC–MS/MS). Compared with conventional BA-crosslinked gel method, this new method increased the numbers of identified proteins and unique peptides by 20.2% and 20.4%, respectively. The further statistical analysis demonstrated that the method improved the recovery of tryptic peptides particularly larger and/or hydrophobic peptides, thereby significantly facilitating protein identification. Thus, the newly developed method is a promising alternative for BA-crosslinked gel-based shotgun workflows and has potential application in the related fields of protein chemistry and proteomics.
Keywords: Bis-acrylylcystamine; In-gel digestion; Mass spectrometry; Peptide recovery; Proteomics;

Monitoring cellular accumulation of 3′-deoxy-3′-fluorothymidine (FLT) and its monophosphate metabolite (FLT-MP) by LC–MS/MS as a measure of cell proliferation in vitro by Wenlin Li; Marcela Araya; Mark Elliott; Xiaolin Kang; Phillip M. Gerk; Matthew S. Halquist; H. Thomas Karnes; Cathy Zhang; Peter J. O’Brien (2963-2970).
Accurate measurement of in vitro cell growth is critical for oncology drug development, but cell counting and the most accurate indirect proliferation assays are impractical. Here, we describe a robust alternative method that monitors proliferating cell thymidine kinase 1 (TK1) activity via LC–MS/MS quantification of 3′-deoxy-3′-fluorothymidine (FLT) and its monophosphate metabolite FLT-MP. LNCaP prostate cancer cells were cultured at four densities (20,000; 10,000; 5000; and 500 cells/well) and incubated with 2000 ng/mL FLT in multi-well plates. Internal standards were FLT-d3 for FLT and d4-thymidine for FLT-MP. In culture medium, peak area ratios of FLT to FLT-d3 and FLT-MP to d4-thymidine were linear over the range 0.25–100 ng/mL (r 2  ≥ 0.998). Accuracy for quality controls was between −7.3% and 6.3% for FLT, and from −3.3% to 1.7% for FLT-MP. Quality control precision was from 2.4% to 5.7% for FLT and 3.2% to 7.5% for FLT-MP. The limit of quantification was 0.25 ng/mL, with good control results (precision of 9.6% for FLT and 14.8% for FLT-MP). FLT-MP formation was linearly proportional to cell number from 500 to 20,000 cells/well 1 h after FLT addition. FLT-MP and ATP generation were comparable in LNCaP cells exposed to cell cycle inhibitor drugs (Spearman r  = 0.925, p  < 0.0001), demonstrating assay suitability for drug screening. This fit for purpose method is amenable to analysis of tumor tissue extracts, and should enable direct assessment of in vitroin vivo relationships in animal models of cancer.
Keywords: Cell proliferation assay; Detection of tumor growth; 3′-Deoxy-3′-fluorothymidine (FLT); 3′-Deoxy-3′-fluorothymidine monophosphate (FLT-MP); Microplate format; LC–MS/MS;

► A conjoint chromatography column with SEC and recDsbA Sepharose FF was constituted. ► Less loss of biological activity of DsbA was achieved with the protection of SEC. ► The conjoint chromatography offers high productivity in protein refolding in vitro. ► The conjoint chromatography refolding system shows excellent column reusability.DsbA (disulfide bond formation protein A) located in the periplasm of Escherichia coli is a disulfide isomerase, which is vital to disulfide bonds formation directly affecting the nascent peptides folding to the correct conformation. In this paper, recombinant DsbA was firstly immobilized onto NHS-activated Sepharose Fast Flow gel. Then Sephadex G-100 gel was sequentially packed on the top of recDsbA Sepharose Fast Flow, and a so-called conjoint chromatography column composed of SEC and immobilized recombinant DsbA was constructed. Denatured lysozyme was applied on the conjoint column. The effect of SEC volume, flow rate, loading amount and volume, pre-equilibrium mode and KCl concentration in the buffer on lysozyme refolding were investigated in detail and the stability of DsbA immobilization was evaluated. Finally the reusability of the conjoint refolding column was also tested. When loading 2.4 mg denatured lysozyme in 0.5 ml solution, the activity recovery reached 92.7% at optimized experimental conditions, and the conjoint column renaturation capacity decreased only 7.7% after six run reuse due to the use of SEC section in the chromatographic refolding process. The conjoint chromatography offers an efficient strategy to refold proteins in vitro with high productivity and column reusability.
Keywords: Conjoint column; DsbA; Immobilization; Lysozyme; Chromatographic refolding; Protein refolding in vitro;

► Liquid–liquid microextraction-high performance liquid chromatography method for opium alkaloids. ► Determination of morphine, codeine, papaverine, noscapine and thebaine in urine samples. ► Study of optimum experimental conditions for extraction process. ► Linear calibration over 0.50–500 μg L−1 and limit of detections in the ranges 0.2–10 μg L−1.A simple, rapid and sensitive method based on dispersive liquid–liquid microextraction (DLLME) combined with high-performance liquid chromatography–ultraviolet detection (HPLC-UV) was used to determine opium alkaloids in urine samples. Some effective parameters on extraction were studied and optimized. Under the optimum conditions, enrichment factors and recoveries for different opiates are in the range of 63.0–104.5 and 31.5–52.2%, respectively. The calibration graphs are linear in the range of 0.50–500 μg L−1 and limit of detections (LODs) are in the range of 0.2–10 μg L−1. The relative standard deviations (RSDs) for 200 μg L−1 of morphine, codeine and thebaine, 5.0 μg L−1 of papaverine and 10.0 μg L−1 of noscapine in diluted urine sample are in the range of 2.8–6.1% (n  = 7). The relative recoveries of urine samples spiked with alkaloids are 84.3–106.0%. The obtained results show that DLLME combined with HPLC-UV is a fast and simple method for the determination of opium alkaloids in urine samples.
Keywords: Dispersive liquid–liquid microextraction; Opium alkaloids; High-performance liquid chromatography; Urine analysis;

A sensitive combined assay for the quantification of paclitaxel, docetaxel and ritonavir in human plasma using liquid chromatography coupled with tandem mass spectrometry by Jeroen J.M.A. Hendrikx; Michel J.X. Hillebrand; Bas Thijssen; Hilde Rosing; Alfred H. Schinkel; Jan H.M. Schellens; Jos H. Beijnen (2984-2990).
► A validated LC–MS/MS method is described for the simultaneously quantification of paclitaxel, docetaxel and ritonavir. ► Drugs with diverse individual chemical properties, concentration ranges and ionisation responses are quantified. ► An alkaline mobile phase increases the sensitivity for docetaxel and paclitaxel. ► Application of the method to study orally administered taxanes combined with ritonavir is described.A combined assay for the determination of paclitaxel, docetaxel and ritonavir in human plasma is described. The drugs were extracted from 200 μL human plasma using liquid–liquid extraction with tertiar-butylmethylether, followed by high performance liquid chromatography analysis using 10 mM ammonium hydroxide pH 10:methanol (3:7, v/v) as mobile phase. Chromatographic separation was obtained using a Zorbax Extend C18 column. Labelled analogues of the analytes are used as internal standards. For detection, positive ionization electrospray tandem mass spectrometry was used. Method development including optimisation of the mass transitions and response, mobile phase optimisation and column selection are discussed. The method was validated according to FDA guidelines and the principles of Good Laboratory Practice (GLP). The validated range was 0.5–500 ng/mL for paclitaxel and docetaxel and 2–2000 ng/mL for ritonavir. For quantification, quadratic calibration curves were used (r 2  > 0.99). The total runtime of the method is 9 min and the assay combines analytes with differences in ionisation and desired concentration range. Inter-assay accuracy and precision were tested at four concentration levels and were within 10% and less than 10%, respectively, for all analytes. Carry-over was less than 6% and endogenous interferences or interferences between analytes and internal standards were less than 20% of the response at the lower limit of quantification level. The matrix factor and recovery were determined at low, mid and high concentration levels. The matrix factor was around 1 for all analytes and total recovery between 77.5 and 104%. Stability was investigated in stock solutions, human plasma, dry extracts, final extracts and during 3 freeze/thaw cycles. The described method was successfully applied in clinical studies with oral administration of docetaxel or paclitaxel in combination with ritonavir.
Keywords: Paclitaxel; Docetaxel; Ritonavir; Liquid chromatography/tandem mass spectrometry; Acid/alkaline mobile phases;

Immobilized metal affinity chromatography (IMAC) and metal oxide type affinity chromatography (MOAC) techniques have been widely used for mass spectrometry-based phosphorylation analysis. Unlike MOAC techniques, IMAC requires rather complete removals of buffering reagents, salts and high concentrations of denaturant prior to sample loading in order for the successful enrichment of phosphopeptides. In this study, a simple off-line capillary column-based IMAC phosphopeptide enrichment method can shorten sample preparation time by eliminating the speed-vac step from the desalting process. Tryptic digest peptide samples containing 2 M urea can be directly processed and the entire IMAC procedure can be completed within 6 h. When tryptic digest peptide samples prepared from mouse whole brain tissues were analyzed using our method, an average of 249 phosphoproteins and 463 unique phosphopeptides were identified from single 2-h RPLC–MS/MS analysis (∼88% specificity). An additional advantage of this method is the significantly improved reproducibility of the phosphopeptide enrichment results. When four independent phosphopeptide enrichment experiments were carried out, the peak areas of phosphopeptides identified among four enrichment experiments were relatively similar (less than 16.2% relative standard dev.). Because of this increased reproducibility, relative phosphorylation quantification analysis of major phosphoproteins appears to be feasible without the need for stable isotope labeling techniques.
Keywords: IMAC; Label-free; Phosphopeptide enrichment; Relative quantification; RPLC–MS/MS;

► A novel analytical method employing SPE and LC–MS/MS was developed. ► The method can analyze 31 EDCs in surface water simultaneously. ► The first study to investigate 31 EDCs in surface water of Shanghai.A novel analytical method employing MCX (mixed-mode cationic exchange) based solid phase extraction (SPE) coupled with liquid chromatography tandem mass spectrometry (LC–MS/MS) was developed to detect 31 endocrine-disrupting compounds (EDCs) in surface water samples simultaneously. The target EDCs belong to five classes, including seven estrogens, eight androgens, six progesterones, five adrenocortical hormones and five industrial compounds. In order to simultaneously concentrate the target EDCs and eliminate matrix interferences in the water samples, MCX SPE cartridges were employed for SPE, and then followed by a simple and highly efficient three-step sequential elution procedure. Two electrospray ionization (ESI) detection modes, positive (ESI+) and (ESI−), were optimized for HPLC–MS/MS analysis to obtain the highest sensitivity for all the EDCs. The limits of detection (LODs) were 0.02–1.9 ng L−1, which are lower than or comparable to these reported in references. Wide linear ranges (LOD-100 ng L−1 for ESI+ mode, and LOD-200 ng L−1 for ESI− mode) were obtained with determination coefficients (R 2) higher than 0.99 for all the compounds. With five internal standards, good recoveries (84.4–103.0%) of all the target compounds were obtained in selected surface water samples. The developed method was successfully applied to investigate the EDCs occurrence in the surface water of Shanghai by analyzing surface water samples from 11 sites. The results showed that nearly all the target compounds (30 in 31) were present in the surface water samples of Shanghai, of which three industrial compounds (4-t-OP, BPA, and BPF) showed the highest concentrations (median concentrations were 11.88–23.50 ng L−1), suggesting that industrial compounds were the dominating EDCs in the surface water of Shanghai, and much more attention should be paid on these compounds. Our present research demonstrated that SPE with MCX cartridges combined with HPLC–MS/MS was convenient, efficient and reliable for multiclass analysis of EDCs in surface water.
Keywords: Endocrine-disrupting compounds; Solid phase extraction; LC–MS/MS; Surface water;

► Comparative metabolism of sildenafil in liver microsomes from 5 species was studied. ► Liquid chromatography–mass spectrometry and multivariate statistical analysis were used. ► Peak area ratio for 12 metabolites was used as variables for multivariate analyses. ► Mice showed the most similar pattern with humans regarding sildenafil metabolism.Sildenafil metabolism in liver microsomes obtained from different species was studied in vitro and compared using liquid chromatography–mass spectrometry (LC/MS) and multivariate statistical analysis. Sildenafil (1, 5, and 25 μM) was incubated with rat, mouse, dog, monkey, and human liver microsomes along with NADPH, and the reaction mixtures were analyzed by LC/MS to obtain species-specific metabolic profiles of sildenafil. A total of 12 metabolites were detected and their peak area ratio values were used as variables for multivariate analyses to evaluate the interspecies differences in sildenafil metabolism. Principal components analysis of the metabolic profiles showed that the mouse samples were generally clustered closer to the human samples on the principal component score plot. Similarity index (SI) indicated that sildenafil metabolism in mice, compared to the other animals, was highly analogous (SI = 0.764 at 25 μM) to that in humans. These results suggest that LC/MS-based multivariate analytical approaches are useful for the evaluation of interspecies differences in the metabolism of xenobiotics.
Keywords: Sildenafil; Comparative metabolism; Multivariate analysis; LC/MS;

This paper presents a study of the synthesis of a polymer monolith column and its application to the analysis of PAHs in smoked meat products. A poly(butyl methacrylate-co-ethylene glycol dimethacrylate) monolith capillary has been successfully prepared with in situ polymerization method. The polymer monolith microextraction combined with HPLC determinations is employed for the analysis of naphthalene, biphenyl, phenanthrene, and anthracene. Various parameters affecting the extraction efficiency have been investigated and optimized. Under the optimum experimental conditions, the method provides an acceptable linearity (2–10,000 μg/L), low limits of detection (1.4–2.0 μg/L), and good precision (intraday relative standard deviations < 4.1%, interday relative standard deviations < 5.7%). When applied to the determination of the four PAHs in smoked meat samples, recoveries are obtained in the range of 86.6–101.5%.
Keywords: Poly(butyl methacrylate-co-ethylene glycol dimethacrylate) monolith microextraction; Polycyclic aromatic hydrocarbons; High performance liquid chromatography; Smoked meat products;

The objective of this study was to adapt and improve an environmentally friendly and fast routine method for the analysis of ferulic and p-coumaric acids released from grass cell-walls by alkaline hydrolysis. This methodological development was performed on maize samples selected for their contrasted contents in ferulic and p-coumaric acids as a consequence of their different maturity stages (from stage of 7th leaf with visible ligule to stage of silage harvest). We demonstrate that the Carrez method is an efficient substitute to the common solvent-consuming extraction by ethyl acetate for the preparation of samples suitable for HPLC–ESI-MS analysis. We prove that it is possible to replace methanol by ethanol in the Carrez step and at last we propose a scale reduction of this procedure that offer a first step towards high throughput determinations. The new method leads to a solvent consumption reduced by a factor 100 and only requires ethanol as organic solvent.
Keywords: Ferulic acid; p-Coumaric acid; Maize cell wall; Environmentally friendly purification method; Carrez reagents; HPLC–ESI-MS;

► MAE was applied for the extraction of xanthones from Garcinia mangostana. ► Two xanthones was isolated by HSCCC in one-step separation from MAE extraction. ► MAE coupled with HSCCC is an efficient method for the separation of xanthones.A microwave-assisted extraction (MAE) method is presented for the extraction of xanthones, α-mangostin and γ-mangostin from Garcinia mangostana. The MAE conditions including extraction temperature, liquid/solid ratio, extraction time and concentration of ethanol were optimized with an orthogonal test, and 5 g sample was extracted with the optimized conditions. The crude extraction of MAE was successfully isolated and purified by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of petroleum ether–ethyl acetate–methanol–water (0.8:0.8:1:0.6, v/v) in one-step separation. The separation yielded 75 mg of α-mangostin at 98.5% purity, and 16 mg of γ-mangostin at 98.1% purity from 360 mg crude extract of G. mangostana in less than 7 h. The purity of the two xanthones was determined by HPLC. Their structures were further identified by ESI-MS, 1H NMR and 13C NMR.
Keywords: Garcinia mangostana; Microwave-assisted extraction; High-speed counter-current chromatography; Xanthones;

► A new strategy to improve the yield and efficiency of on-column exopeptidase cleavage is described. ► Maximum on-column cleavage yield in excess of 99% was achieved in 3 h of incubation at 300 mM imidazole. ► The effect of protease concentration and incubation time on the new strategy is significant. ► Comparisons are made for different tag removal strategies.This paper describes a new strategy, which aims to make on-column poly-histidine tag removal more useful in the production of recombinant proteins by improving the yield and efficiency of on-column exopeptidase cleavage. This involves improvement of the on-column cleavage condition by using imidazole concentrations in the range of 100–500 mM in the cleavage buffer. At 300 mM imidazole, maximum on-column cleavage yield (in excess of 99%) was achieved in 3 h of incubation. However, as a result of the increased imidazole concentration, this new strategy of on-column cleavage results in some residual uncleaved poly-histidine tagged proteins (∼0.1%) and the production of cleaved dipeptides, both of which need to be further removed in a subsequent step. A method involving the recirculation of recovered proteins and peptides through the immobilized metal affinity chromatography (IMAC) column (same-column recirculation) was found to be superior to subtractive IMAC for the purpose of contaminant clearance. Recovery of the detagged target proteins was achieved using 10 column volumes of recovery buffer, which had the effect of diluting the imidazole concentration to a suitably low level for contaminant removal by same-column recirculation. This strategy was also applicable at a higher adsorbent loading of 10 mg protein/mL adsorbent with an optimal ratio of 200 mU of DAPase per mg of adsorbed tagged maltose binding protein (MBP), giving a cleavage yield of 99.1% in 3 h. Finally, on-column cleavage conditions including the effect of protease concentration and incubation time on the new strategy have been investigated and comparisons are made for different tag removal strategies.
Keywords: Maltose binding protein; Purification; Immobilized metal affinity chromatography; Poly-histidine fusion tag cleavage; Exopeptidase; Tag clearance;

The new pre-concentration technique, hollow fiber-solid phase microextraction based on carbon nanotube reinforced sol–gel and liquid chromatography–photodiode array detection was applied to determination of aflatoxins B1, B2 (AFB1, AFB2) in rice, peanut and wheat samples. This research provides an overview of trends related to synthesis of solid phase microextraction (SPME) sorbnents that improves the assay of aflatoxins as the semi-polar compounds in several real samples. It mainly includes summary and a list of the results for a simple carbon nanotube reinforced sol–gel in-fiber device. This device was used for extraction, pre-concentration and determination of aflatoxins B1, B2 in real samples. In this technique carbon nanotube reinforced sol was prepared by the sol–gel method via the reaction of phenyl trimethoxysilane (PTMS) with a basic catalyst (tris hydroxymethyl aminomethan). The influences of microextraction parameters such as pH, ageing time, carbon nanotube contents, desorption conditions, desorption solvent and agitation speed were investigated. Optimal HPLC conditions were: C18 reversed phase column for separation, water–acetonitril–methanol (35:10:55) as the mobile phase and maximum wavelength for detection was 370 nm. The method was evaluated statistically and under optimized conditions, the detection limits for the analytes were 0.074 and 0.061 ng/mL for B1 and B2 respectively. Limit of quantification for B1 and B2 was 0.1 ng/mL too (n  = 7). The precisions were in the range of 2.829–2.976% (n  = 3), and linear ranges were within 0.1 and 400 ng/mL. The method was successfully applied to the analysis of cereals (peanut, wheat, rice) with the relative recoveries from 47.43% to 106.83%.
Keywords: Solid phase microextraction; Multi-walled carbon nanotube; Hollow fiber; AFB1; AFB2; Sol–gel;

To evaluate the penetration of the blood–brain barrier by 9-fluoropropyl-(+)-dihydrotetrabenazine (AV-133), microdialysis probes were implanted simultaneously into rat blood and brain, and a liquid chromatography–tandem mass spectrometric method was developed and validated to monitor the AV-133 concentration in the microdialysates. The chromatographic separation was performed on an XTerra C18 column (150 mm × 2.1 mm i.d., 5 μm particles) with gradient elution. The mass spectrometer was operated in positive mode using electrospray ionization. The analytes were measured using the multiple-reaction-monitoring mode. The calibration curves were linear over the range of 5.00–1000 ng/mL AV-133, with a coefficient of determination >0.995. The accuracies ranged from 99.5% to 105.0% and the precisions were <10% for AV-133. This method was used to determine the concentrations of AV-133 and its pharmacokinetics in the brains and blood of rats. The blood and brain concentration–time profiles for AV-133 were obtained, and the blood–brain barrier penetration was evaluated.
Keywords: 9-Fluoropropyl-(+)-dihydrotetrabenazine; Pharmacokinetics; Liquid chromatography–tandem mass spectrometry; Microdialysis;

Simple and sensitive liquid chromatography–tandem mass spectrometry methods for quantification of paraquat in plasma and urine: Application to experimental and clinical toxicological studies by Klintean Wunnapuk; Gregory A. Medley; Xin Liu; Jeffrey E. Grice; Sudheera Jayasinghe; Indika Gawarammana; Nicholas A. Buckley; Michael S. Roberts (3047-3052).
► Simple sample preparation with a one-step protein precipitation. ► Analytical separation was achieved by using the HILIC silica column and ion-pair reagents are not required for the method. ► The LLOQ of the method was 10 ng/mL in both plasma and urine samples with acceptable precision and accuracy. ► Method was successfully applied in clinical and animal studies.Simple, sensitive and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) methods have been developed and validated for quantification of paraquat (PQ) in plasma and urine. Plasma and urine sample preparation were carried out by one-step protein precipitation using cold acetonitrile (−20 to −10 °C). After centrifugation, an aliquot of 10 μL of supernatant was injected into a Kinetex™ hydrophilic interaction chromatography (HILIC) column with a KrudKatcher™ Ultra in-line filter. The chromatographic separation was achieved using the mobile phase mixture of 250 mM ammonium formate (with 0.8% aqueous formic acid) in water and acetonitrile at a flow rate of 0.3 mL/min. Detection was performed using an API2000 triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode via an electrospray ionization (ESI) source. The calibration curve was linear over the concentration range of 10–5000 ng/mL, with an LLOQ of 10 ng/mL. The inter- and intra-day precision (% R.S.D.) were <8.5% and 6.4% for plasma and urine, respectively with the accuracies (%) within the range of 95.1–102.8%. PQ in plasma and urine samples was stable when stored at −70 °C for three freeze–thaw cycles. The methods were successfully applied to determine PQ concentration in rat and human samples.
Keywords: Paraquat; Herbicide; LC–MS/MS; HILIC column;

► Conformational behavior of lysozyme during the process of adsorption/desorption. ► On the surface of Fe3O4 (PEG + CM-CTS) NPs, the lysozyme would adopt a more compact conformation state. ► The alterations of lysozyme secondary structure on desorption from nanoparticles were confirmed. ► Fe3O4 (PEG + CM-CTS) NPs is capable of preserving the structure and bioactivity of lysozyme.A fundamental understanding of the conformational behaviors of lysozyme during the process of adsorption and desorption has been studied using spectrophotometric techniques, and interpreted in terms of the secondary structures in this work. FTIR data show an increase in α-helix and β-sheet content when lysozyme interaction with magnetite nanoparticles (Fe3O4 (PEG + CM-CTS) NPs) which indicates that the lysozyme would adopt a more compact conformation state. The mechanism of fluorescence quenching of lysozyme by magnetite nanoparticles is due to the formation of lysozyme–nanoparticles complex. High desorption of lysozyme from Fe3O4 (PEG + CM-CTS) NPs were achieved using phosphate buffer solution (PBS) (20 mM, pH 5.0, 0.2 M NaCl), PBS (20 mM, pH 5.0, 0.5 M NaCl) and acetic acid (0.2 M, pH 4.0) as eluents. The alterations of lysozyme secondary structure on desorption from nanoparticles were confirmed by circular dichroism and fluorescence spectroscopy. Lysozymes desorbed by PBS (20 mM, pH 5.0, 0.2 M NaCl) and PBS (20 mM, pH 5.0, 0.5 M NaCl) retain high fraction of its native structure with negligible effect on its activity, and about 92.4% and 89.5% activity were retained upon desorption from nanoparticles, however, lysozyme desorbed by acetic acid (0.2 M, pH 4.0) solution showed significant conformational changes. The stability of NPs-conjugated protein and retention of higher activity may find useful applications in biotechnology ranging from enzyme immobilization to protein purification.
Keywords: Lysozyme; Conformational change; Desorption; Bioactivity; Fluorescence spectroscopy; Circular dichroism;

► Tetrahydrocannabinolic acid A is the biogenetic precursor of THC in the Cannabis plant. ► We present an isolation procedure for THCA with two flash chromatography systems. ► System 1 runs on normal phase, system 2 on reversed phase columns. ► Purity was tested with HPLC-DAD and GC–MS analysis, identity confirmed with NMR. ► The methods are rapid, do not use harmful solvents and yield purity grades >98.8%.Two isolation procedures for Δ9-tetrahydrocannabinolic acid A (THCA), the biogenetic precursor in the biosynthesis of the psychoactive Δ9-tetrahydrocannabinol (THC) in the cannabis plant, are presented. Two flash chromatography systems that can be used independently from each other were developed to separate THCA from other compounds of a crude cannabis extract. In both systems UV absorption at 209 and 270 nm was monitored. Purity was finally determined by HPLC-DAD, NMR and GC–MS analysis with a focus on the impurity THC. System 1 consisted of a normal phase silica column (120 g) as well as cyclohexane and acetone – both spiked with the modifier pyridine – as mobile phases. Gradient elution was performed over 15 min. After the chromatographic run the fractions containing THCA fractions were pooled, extracted with hydrochloric acid to eliminate pyridine and evaporated to dryness. Loading 1800 mg cannabis extract yielded 623 mg THCA with a purity of 99.8% and a THC concentration of 0.09%. System 2 was based on a reversed-phase C18 column (150 g) combined with 0.55% formic acid and methanol as mobile phases. A very flat gradient was set over 20 minutes. After pooling the THCA-containing fractions methanol was removed in a rotary evaporator. THCA was re-extracted from the remaining aqueous phase with methyl tert-butyl ether. The organic phase was finally evaporated under high vacuum conditions. Loading 300 mg cannabis extract yielded 51 mg THCA with a purity of 98.8% and a THC concentration of 0.67%.
Keywords: Δ9-Tetrahydrocannabinolic acid A; Isolation; Flash chromatography;

► A method based on liquid-phase microextraction and ion mobility spectroscopy is reported. ► Amantadine was determined in human urine and plasma by the method. ► The method shows relatively low detection limit, good precision, high recovery and efficient sample clean-up. ► Compared with other method, the present method consumes less sample and organic solvent and does not need sample pretreatment steps.A method based on liquid–liquid–liquid microextraction combined with corona discharge ion mobility spectrometry was developed for the analysis of amantadine in human urine and plasma samples. Amantadine was extracted from alkaline aqueous sample as donor phase through a thin phase of organic solvent (n-dodecane) filling the pores of the hollow fiber wall and then back extracted into the organic acceptor phase (methanol) located in the lumen of the hollow fiber. All variables affecting the extraction of analyte including acceptor organic solvent type, concentration of NaOH in donor phase, ionic strength of the sample and extraction time were studied. The linear range was 20–1000 and 5–250 ng/mL for plasma and urine, respectively (r 2  ≥ 0.990). The limits of detection were calculated to be 7.2 and 1.6 ng/mL for plasma and urine, respectively. The relative standard deviation was lower than 8.2% for both urine and plasma samples. The enrichment factors were between 45 and 54. The method was successfully applied for the analysis of amantadine in urine and plasma samples.
Keywords: Hollow fiber-based liquid–liquid–liquid microextraction; Corona discharge ion mobility spectrometry; Amantadine; Biological samples;

► An LC-MS/MS method was validated to determine dronedarone and debutyldronedarone in human plasma. ► The method has an LLOQ of 0.200 ng/mL for both analytes. ► Sample preparation was simple and quick with acetonitrile protein precipitation. ► The method was applied to a pharmacokinetic study of dronedarone in humans.Dronedarone is a derivative of amiodarone – a popular antiarrhythmic drug. It was developed to overcome the limiting iodine-associated toxicities of amiodarone. Debutyldronedarone is a major circulating active metabolite of dronedarone in humans. To investigate the pharmacokinetics of dronedarone, a rapid, simple, and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated to simultaneously determine dronedarone and debutyldronedarone in human plasma using amiodarone as internal standard (IS). Acetonitrile with IS was used to precipitate proteins from a 50-μL aliquot of plasma. Effective chromatographic separation was performed on a CAPCELL PAK C18 MG (100 mm × 4.6 mm, 5 μm) column with gradient elution (5 mmol/L ammonium acetate–acetonitrile, with each phase containing 0.2% acetic acid) at a flow rate of 0.7 mL/min. Complete separation was achieved within 5.5 min. Detection was carried out on an tandem mass spectrometer in multiple reaction monitoring mode using a positive atmospheric pressure chemical ionization interface. A lower limit of quantification of 0.200 ng/mL was achieved for both dronedarone and debutyldronedarone, with acceptable precision and accuracy. The linear range of the method was from 0.200 to 200 ng/mL for each analyte. Intra- and inter-day precisions were lower than 7.2% in relation to relative standard deviation, while accuracy was within ±5.1% in terms of relative error for analytes. Our findings demonstrate the successful application of the validated LC–MS/MS method to a pharmacokinetic study after a single oral administration of 400 mg dronedarone to six healthy volunteers.
Keywords: Liquid chromatography–tandem mass spectrometry; Dronedarone; Debutyldronedarone; Pharmacokinetics;