Journal of Chromatography B (v.891-892, #C)

► A selective, rapid and sensitive UPLC/MS method was developed and validated to quantitate a highly selective mixed-affinity sigma receptor ligand. ► The validated analytical method resulted in a run time of 4 min. The calibration curve exhibited excellent linearity over a concentration range of 5–4000 ng/mL (LLOQ, 5 ng/mL). The intra- and inter-day precision values were below 15% and accuracy ranged from −6.5% to 5.0%. ► The validated method was successfully applied to an intravenous pharmacokinetic study of CM156 in rats.A selective, rapid and sensitive ultra performance liquid chromatography mass spectrometry (UPLC/MS) method was developed and validated to quantitate a highly selective mixed-affinity sigma receptor ligand, CM156 (3-(4-(4-cyclohexylpiperazin-1-yl)butyl)benzo[d] thiazole-2(3H)-thione), in rat plasma. CM156 and the internal standard (aripiprazole) were extracted from plasma samples by a single step liquid–liquid extraction using chloroform. The analysis was carried out on an ACQUITY UPLC™ BEH HILIC column (1.7 μm, 2.1 mm × 50 mm) with isocratic elution at flow rate of 0.2 mL/min using 10 mM ammonium formate in 0.1% formic acid and acetonitrile (10:90) as the mobile phase. The detection of the analyte was performed on a mass spectrometer operated in selected ion recording (SIR) mode with positive electrospray ionization (ESI). The validated analytical method resulted in a run time of 4 min and the retention times observed were 2.6 ± 0.1 and 2.1 ± 0.1 min for CM156 and the IS, respectively. The calibration curve exhibited excellent linearity over a concentration range of 5–4000 ng/mL with the lower limit of quantification of 5 ng/mL. The intra- and inter-day precision values were below 15% and accuracy ranged from −6.5% to 5.0%. The mean recovery of CM156 from plasma was 96.8%. The validated method was applied to a pilot intravenous pharmacokinetic study in rats.
Keywords: Sigma receptor ligand; UPLC/MS; Method validation; Pharmacokinetics;

Determination of landiolol, an ultra-short-acting β1-receptor antagonist, in human plasma by liquid chromatography–tandem mass spectrometry by Qun He; Meiyun Shi; Xidong Liu; Yantong Sun; Lianghai Hu; Yan Yang; J. Paul Fawcett; Jingkai Gu; Limei Zhao (7-11).
► A novel method for determination of landiolol in plasma by LC–MS/MS was developed. ► Landiolol in blood/plasma samples is stabilized by pyridostigmine bromide. ► The plasma samples were prepared by liquid–liquid extraction. ► The LLOQ of the method is 0.5 ng/ml which is the lowest reported so far. ► The method was successfully applied to a clinical pharmacokinetic study of landiolol.A method for the determination of landiolol, an ultra-short-acting β1-adrenoreceptor antagonist, in human plasma has been developed and validated. With the addition of pyridostigmine bromide to stabilize landiolol in the blood/plasma samples, and bisoprolol as internal standard, plasma samples were subjected to liquid–liquid extraction with diethyl ether:dicholoromethane (60:40, v/v) prior to assay by liquid chromatography–tandem mass spectrometry. Separation was performed on a TC-C18 column (150 mm × 4.6 mm, 5 μm) using a mobile phase of methanol:10 mM ammonium acetate containing 1% formic acid (65:35, v/v) in a run time of 3.5 min. Detection involved electrospray ionization in the positive ion mode followed by multiple reaction monitoring of the precursor-to-product ion transitions of landiolol at m/z 510.1 → 157.2 and bisoprolol at m/z 326.3 → 116.1. The method was linear over the concentration range 0.5–500 ng/ml with a lower limit of quantitation of 0.5 ng/ml. Intra- and inter-day precisions (as relative standard deviation, RSD) were <4.4% and <10.0%, respectively, with accuracy (as relative error, RE) <10.0%. The method was successfully applied to a clinical pharmacokinetic study involving a continuous infusion of landiolol hydrochloride to healthy Chinese volunteers.
Keywords: Landiolol; β1-Receptor antagonist; LC–MS/MS; Determination; Plasma;

► A new approach for the GC–MS determination of 22 pharmacologically active substances in urine and blood samples is presented. ► Miniaturization of the sample treatment system using a continuous solid-phase extraction unit. ► Simplification of the derivatizing reaction through the use of a household microwave oven that reduces the reaction time to ca. 3 min. ► Successful analysis of human and animal (lamb, veal and pig) blood and urine samples.A sensitive method based on gas chromatography–mass spectrometry was used to determine 22 pharmacologically active substances (frequently used in the treatment of human and animal's diseases) including analgesics, antibacterials, anti-epileptics, antiseptics, β-blockers, hormones, lipid regulators and non-steroidal anti-inflammatories in blood and urine samples. Samples were subjected to continuous solid-phase extraction in a sorbent column (Oasis HLB), and then the target analytes were eluted with ethyl acetate and derivatized in a household microwave oven at 350 W for 3 min. Finally, these products were determined in a gas chromatograph–mass spectrometer equipped with a DB-5 fused silica capillary column. The analyte detection limits thus obtained ranged from 0.2 to 1.3 ng L−1 for urine samples and 0.8–5.6 ng L−1 for blood samples. Recoveries from both blood and urine ranged from 85 to 102%, and within-day and between-day relative standard deviations were all less than 7.5%. The proposed method offers advantages in reduction of the exposure danger to toxic solvents used in conventional sample pretreatment, simplicity of the extraction processes, rapidity, and sensitivity enhancement. The method was successfully used to quantify pharmacologically active substances in human and animal (lamb, veal and pig) blood and urine. The hormones estrone and 17β-estradiol were detected in virtually all samples, and so were other analytes such as acetylsalicylic acid, ibuprofen, ketoprofen and triclosan in human samples, and florfenicol, pyrimethamine and phenylbutazone in animal samples.
Keywords: Pharmacologically active substances; Biological fluids; Continuous solid-phase extraction; Microwave-assisted derivatization; Gas chromatography–mass spectrometry;

► First LC–MS/MS method to quantify gadoxetate in human serum and cell lysates. ► Substantially increased sensitivity compared to previously published methods. ► Monitoring of the intact Gd-complex (compared to AES methods). ► Comprehensive method validation; application to analyze in vitro/in vivo studies. ► Identification of gadoxetate to be a substrate of the uptake transporter OATP1B1.Gadoxetate (Gd-EOB-DTPA, Primovist®) is a frequently used liver-specific magnetic resonance imaging (MRI) contrast agent which disposition is so far not fully understood in humans. Here, we describe the development and validation of a selective and sensitive quantification method to measure cellular in vitro concentrations as well as human serum concentrations of gadoxetate. The drug was measured after protein precipitation with acetonitrile and ethyl acetate-mediated sample concentration using amoxicillin as internal standard and liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) for detection. Hydrophilic interaction chromatography (HILIC) was performed by using the column Atlantis® HILIC Silica (2.1 mm × 100 mm), a step-elution gradient with acetonitrile and ammonium acetate (5 mM, pH 3.8) as mobile phases and a flow rate of 200 μl/min. The MS/MS detection was done in the negative multiple reaction monitoring (MRM) mode by monitoring the m/z transitions 681.3/635.2 for gadotrexate and 363.8/222.7 for the internal standard. The method was validated between 5 and 4000 ng/ml in serum and between 1.25 and 500 ng/ml in cell lysates. The method was shown to possess sufficient specificity, accuracy, precision and stability without any matrix effects, thereby fulfilling current bioanalytical guidelines. The developed assay was successfully applied to quantify gadoxetate in cellular uptake studies in OATP1B1-transfected cell lines and to monitor serum concentrations-time profiles from a clinical pilot study performed in healthy volunteers carrying the wild-type or the functionally relevant variants T521C (*5) and A388G (*1b) of the hepatic uptake transporter OATP1B1.
Keywords: Gadoxetate; Gd-EOB-DTPA; LC–MS/MS; OATP1B1;

► A magnetic porous polymer of Fe3O4/SiO2/poly(AMPS-co-EGDMA) was prepared. ► The magnetic polymer was firstly used as MSPE medium in CKs analysis. ► A MSPE–HILIC–MS/MS method for determination of CKs in plant tissues was established. ► The MSPE–HILIC–MS/MS method was proved to be a high sensitive and rapid method.A 2-acrylamido-2-methyl-1-propanesulfonic acid-co-ethylene glycol dimethacrylate (Fe3O4/SiO2/P(AMPS-co-EGDMA)) copolymer was prepared and used as a magnetic solid phase extraction (MSPE) medium for recovery of endogenous cytokinins (CKs) from plant extracts. This magnetic porous polymer was characterized by electron microscopy, nitrogen sorption experiments, elemental analysis and Fourier-transformed infrared spectroscopy. It was demonstrated to have high extraction capacity toward CKs in plants due to its specificity, surface area and porous structure. Coupled with hydrophilic interaction chromatography–tandem mass spectrometry (HILIC–MS/MS), a rapid, simple, and effective MSPE–HILIC–MS/MS analytical method for the quantitative analysis of endogenous CKs in Oryza sativa (O. sativa) roots was successfully established. Good linearities were obtained for all CKs investigated with correlation coefficients (R 2) > 0.9975. The results showed that LODs (S/N = 3) were ranged from 0.18 to 3.65 pg mL−1. Reproducibility of the method was obtained with intra-day and inter-day relative standard deviations (RSDs) less than 16.1% and the recoveries in plant samples ranged from 72.8% to 115.5%. Finally, the MSPE–HILIC–MS/MS method was applied to several plant samples, and the amounts of endogenous CKs in O. sativa roots, leaves and Arabidopsis thaliana (A. thaliana) were successfully determined.
Keywords: Magnetic solid-phase extraction; Fe3O4/SiO2/P(AMPS-co-EGDMA); Cytokinins; Hydrophilic interaction chromatography; Mass spectrometry;

Measurements of polybrominated diphenyl ethers and polychlorinated biphenyls in a single drop of blood by Dasheng Lu; Dongli Wang; Ho Sai Simon Ip; Frank Barley; Robert Ramage; Jianwen She (36-43).
► A method was developed and well validated to determine PCBs and PBDEs in a small volume (50 μL) of blood samples and in dried blood spots. ► Measurements of PBDE and PCBs in dried blood spots have not been reported in the previous studies. ► PCBs and PBDEs were measured in filter paper blanks from different manufacturing years (1987–2009). ► Levels of PBDE/PCB in dried blood spot samples did not show any changes during a period of 30 days.A quantitative method that requires only a small volume (50 μL) of blood has been developed for the determination of polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs). Target analytes in both plasma sample (DBSV) and dried blood spot (DBS) were analyzed by a gas chromatography/high resolution mass spectrometer (GC/HRMS). Measurements of standard reference materials by the developed method were in agreement with those certified values. Linear correlation coefficients were found to be 0.9984 and 0.9965 for DBS and DBSV analysis, respectively. Other analytical criteria, such as limits of detection, recoveries, precision, accuracy and linearity of the proposed method are also reported. From recovery studies, the addition of formic acid to the extraction solvent was found to be effective in extracting PBDEs and PCBs from filter paper. The PBDE and PCB levels in spiked DBS were monitored at room temperature for up to 30 days and the variations of target analytes were found to be insignificant. Our results suggest that DBS sampling technique is feasible for PBDE and PCBs biomonitoring in human population.
Keywords: PBDEs; PCBs; Dried blood spots; DBS; POPs; Small volume of blood;

► Metabolite identification has been facilitated in high resolution LC–MS/MS. ► The method was successfully applied to study pharmacokinetics of almotriptan. ► Simple protein precipitation method was used for rat plasma in sample preparation. ► Good separation and resolution were obtained on Lichrospher RP-18 column.A highly sensitive and specific liquid chromatography–electrospray ionization tandem mass spectrometric (LC–ESI-MS/MS) method for investigating the in vivo metabolites of almotriptan in rat plasma, feces and urine was developed. Chromatographic separation was achieved on a Lichrospher RP-18 column (250 mm × 4.6 mm, 5 μm), using 20 mM ammonium acetate (pH 3.5) and acetonitrile (60:40, v/v) as a mobile phase at 25 °C. MS/MS detection was performed by positive ion electrospray ionization using target ions at m/z 336 [M+H]+, m/z 368 and m/z 282 [M+H]+ for almotriptan and its two metabolites, respectively. Two metabolites viz., γ-aminobutyric acid and sulfonamide were detected in plasma as well as feces after 24 h of oral administration of almotriptan, while only γ-aminobutyric acid was found in urine. The method was sensitive with a lower limit of quantification of 1.43 ng/mL and linear over the range of 1.43–5000 ng/mL in plasma. The method was validated and successfully applied to a pharmacokinetic study of almotriptan in rat plasma using sumatriptan as an internal standard. The peak plasma concentration (C max) after 0.3 h of 5 mg/kg oral dose of almotriptan was determined to be 69.85 ng/mL.
Keywords: Antimigraine; Almotriptan; In vivo metabolites; LC–ESI-MS/MS; Rat plasma; Pharmacokinetics;

► An effective method for the detection of trace levels of acetaldehyde in peritoneal dialysis fluids (PDFs). ► Analysis of acetaldehyde via 2,4-dinitrophenylhydrazone (DNPH). ► Directly suspended droplet microextraction technique (DSDME) coupled with HPLC.The aim of present study was to develop and validate a rapid, sensitive, inexpensive and reliable method for the detection of trace levels of acetaldehyde in peritoneal dialysis fluids (PDFs) by 2,4-dinitrophenylhydrazine (DNPH) derivatization and extraction. Separation and analysis of acetaldehyde via 2,4-dinitrophenylhydrazone (DNPH) was by reverse phase high performance liquid chromatography (HPLC). In order to remove co-eluting interferences and to pre-concentrate acetaldehyde, the extraction and clean-up of the sample has been performed using a liquid phase microextraction technique. In this research directly suspended droplet microextraction technique (DSDME) coupled with HPLC was used to determine acetaldehyde in PDFs. In DSDME method a free suspended droplet of an organic solvent (1-octanol) used as extraction phase. Important factors such as organic solvent, extraction time, droplet volume, sample and reagent solution volumes and rate of stirring were optimized. After extraction under optimal conditions the samples were analyzed by HPLC with UV detection at 360 nm. The linearity ranged from 0.01 to 100 mg L−1 with a relative standard deviation (RSD%; n  = 3) 5.6 Enrichment factor and limit of detection (LOD; n  = 5) were 54 and 1.12 μg L−1, respectively.
Keywords: Directly suspended droplet microextraction technique (DSDME); Acetaldehyde; 2,4-Dinitrophenylhydrazine; Peritoneal dialysis fluids; HPLC;

► Development of a HPLC–MS/MS assay for the determination of oseltamivir and oseltamivir carboxylate in plasma. ► Assessment of the ex vivo stability of oseltamivir in whole blood and plasma. ► Validation of the assay according to FDA guidelines.Oseltamivir, the ethyl ester prodrug of the neuramidase inhibitor oseltamivir carboxylate, is licensed for the treatment of patients with influenza virus infection. Here we describe the development and validation of an assay for the simultaneous quantification of oseltamivir and oseltamivir carboxylate in human fluoride EDTA plasma including the ex vivo stability using liquid chromatography coupled to tandem mass spectrometry. Sample pretreatment consisted of protein precipitation with 8% (v/v) trichloroacetic acid in water using only 50 μL plasma. Chromatographic separation was performed on a reversed phase C18 column (150 mm × 2.0 mm ID, particle size 4 μm) with a stepwise gradient using 0.1% formic acid and methanol at a flow rate of 250 μL/min. A triple quadrupole mass spectrometer operating in the positive ionization mode was used for detection and drug quantification. The method was validated over a range of 3–300 ng/mL for oseltamivir and 10–10,000 ng/mL for oseltamivir carboxylate. Deuterated oseltamivir and oseltamivir carboxylate were used as internal standards. The intra-assay accuracies and precisions for oseltamivir were between −8.8 and 16.3% at the LLOQ level, whereas for all other concentration levels this was −8.6 and 14.5%. For oseltamivir carboxylate the intra-assay accuracies and precisions were between −10.9 and 10.7% at all levels. Furthermore, oseltamivir was stable in plasma and whole blood ex vivo in commercially available fluoride EDTA tubes for at least 24 h at 2–8 °C. This method is now applied for the determination of both compounds in specific patient populations to evaluate current dosing guidelines.
Keywords: Oseltamivir; Oseltamivir carboxylate; Plasma; Stability; Bioanalysis; Liquid chromatography; HPLC–MS/MS;

► A LC–MS/MS assay was developed and validated for simultaneous determination of FAU and its active metabolite FMAU in human plasma. ► FAU and FMAU were extracted from plasma samples using solid-phase extraction. ► The method has been successfully employed to study plasma pharmacokinetics of FAU and FMAU in cancer patients.A liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) assay was developed and validated for simultaneous determination of 1-(2′-deoxy-2′-fluoro-β-d-arabinofuranosyl) uracil (FAU) and its active metabolite 1-(2′-deoxy-2′-fluoro-β-d-arabinofuranosyl) 5-methyluracil (FMAU) in human plasma. FAU and FMAU were extracted from plasma samples using solid-phase extraction with Waters Sep-Pak® Vac C18 cartridge. Chromatographic separation was achieved on a Waters Atlantis T3 C18 column with a gradient mobile phase consisting of methanol and water with 0.45% formic acid (v/v) running at a flow rate of 0.2 ml/min. The analytes were monitored by triple quadrupole mass spectrometer under positive ionization mode. The lower limit of quantitation (LLOQ) was 10 and 2 ng/ml for FAU and FMAU in plasma, respectively. Calibration curves were linear over FAU and FMAU plasma concentration range of 10–2000 and 2–1000 ng/ml, respectively. The intra-day and inter-day accuracy and precision were within the generally accepted criteria for bioanalytical method (<15%). The method has been successfully employed to characterize the plasma pharmacokinetics of FAU and FMAU in cancer patients receiving 1-h intravenous infusion of FAU 50 mg/m2.
Keywords: FAU; FMAU; High performance liquid chromatography; Mass spectrometry; LC–MS/MS; Pharmacokinetics;

► We evaluated matrix effect and recovery by supported liquid extraction. ► Over 75% recovery was achieved and the matrix effect was considerably reduced. ► Methyl tert-butyl ether and DCM eluted less phospholipids than ethylacetate. ► ACN and methanol in loading matrix increased the phospholipids’ recovery. ► Major suppression zones overlap the elution of phospholipids.In past a few years, there has been a large increase in the application of supported liquid extraction (SLE) for LC–MS/MS based bioanalysis due to its distinct practical advantage in reduced time cost, ease of operation and the feasibility for automation. The main purpose of this study was to systematically evaluate supported liquid extraction in reducing matrix effect and improving extraction efficiency/recovery under various extraction conditions with 10 model pharmaceutical compounds in liquid chromatography coupled to electrospray tandem mass spectrometry (LC–ESI-MS/MS) analysis. Selected compounds have diverse physicochemical properties where log  P ranges from 0.1 to 6.24 and pK a ranges from 4.0 to 11.1. The factors that may have the impact on the recovery of analytes and phospholipids (PL) were assessed. Over 75% recovery was achieved for every analyte under its respectively optimized extraction conditions where the selection of the polarity of extraction solvent and buffered pH can be critical for efficient recovery. Furthermore, the matrix effect was assessed by postextraction spike and postcolumn infusion method. The matrix effect was considerably reduced for all analytes under most extraction conditions evaluated for SLE, compared with protein precipitation (PPT) method. The correlation between matrix effect and residual phospholipids in sample extract was clearly shown. Although analyte-dependent matrix effect was observed prominently in sample extract prepared by PPT, it was minimized by SLE sample preparation process that effectively removes the majority of phospholipids. Sample extracted by ethyl acetate contained more phospholipids and demonstrated stronger matrix effect than by other organic solvents. Water-miscible organic content, such as methanol and acetonitrile in samples prior to loading has significant impact on PL recovery when eluting with methyl tert-butyl ether. However, isopropanol does not enhance the recovery of PL when adding to dichloromethane for elution. In addition, the compromise between improved extraction efficiency by SLE and reduced matrix effect is sometimes necessary to yield clean extract with acceptable recovery. The effective removal of phospholipids and reduction of matrix effect, while achieving good recovery for all pharmaceutical compounds with diverse physicochemical properties, demonstrated that SLE is a valuable alternative technique to liquid–liquid extraction (LLE) in high throughput LC–MS/MS based bioanalysis.
Keywords: Supported liquid extraction; Phospholipids; Matrix effects; LC–MS/MS; Quantitative analysis; Sample preparation; Recovery efficiency;

Plasma persistence of 2-aminothiazoline-4-carboxylic acid in rat system determined by liquid chromatography tandem mass spectrometry by Ilona Petrikovics; Jorn C.C. Yu; David E. Thompson; Prashanth Jayanna; Brian A. Logue; Jessica Nasr; Raj K. Bhandari; Steven I. Baskin; Gary Rockwood (81-84).
► ATCA persistence was determined after injecting it directly to the blood stream. ► ATCA concentration in blood decreased to half within 2.5 h in the rat system. ► After the initial loss, ATCA level stayed constant (700 ng/ml) over 48 h. ► Endogenous ATCA level in blood was found 141 ng/ml. ► The role of ATCA as a biomarker for CN exposure needs more future investigations.2-Aminothiazoline-4-carboxylic acid (ATCA) was intravenously injected to rats in order to investigate its plasma distribution. ATCA was extracted from plasma samples by solid phase extraction (SPE) and molecularly imprinted polymer stir bar sorption extraction (MIP-SBSE). Detection and quantification of ATCA were achieved by using liquid chromatography–tandem mass spectrometry (LC–MS/MS). It was found that the intravenously injected ATCA concentration quickly decreased to half within 2.5 h in the rat system. However, after 2.5 h, the concentration of ATCA in plasma stayed constant at least 5 folds above the endogenous ATCA level for more then 48 h. This finding can be used for evaluating ATCA's diagnostic and forensic value as a biomarker for cyanide exposure.
Keywords: Diagnostic biomarker; Forensic biomarker; Cyanide exposure; 2-Aminothiazoline-4-carboxylic acid (ATCA); LC–MS/MS;

► We developed a method for detecting allantoin using stable isotope HILIC–MS/MS. ► This resulted in a rapid method for quantifying allantoin in biological tissues. ► The method is significantly more sensitive than other published methods. ► The method was shown to have good precision and accuracy.Allantoin is the major oxidation product of urate in humans and is a potential biomarker of oxidative stress. Several methods are used to measure allantoin in biological samples but they have inherent issues that can include lack of specificity and sensitivity, difficulty in sample preparation, or artefactual generation of allantoin. We have developed a method for measuring allantoin using hydrophilic liquid chromatography with stable isotope dilution tandem mass spectrometry (HILIC–MS/MS). It was validated for measuring allantoin in plasma, synovial fluid and urine from human subjects. The limit of quantification was determined to be 10 fmol and the assay displayed excellent linearity for the wide range of concentrations found in clinical samples. Relative standard deviations were <5% for between-day and <7% for within-day variation. Accuracy was between 100% and 104%. Concentrations of allantoin in plasma of healthy controls (2.0 μM; interquartile range 1.4–3.6 μM, n  = 35) was significantly lower (p  < 0.001) than that in plasma from patients with rheumatoid arthritis (3.7 μM; IQR 3.0–5.6 μM, n  = 43) and in synovial fluid of patients with gout (3.3 μM; IQR 2.8–5.8 μM, n  = 10). This newer HILIC–MS/MS method is a simple and highly sensitive assay for detection of allantoin. It can be used to assess the level of oxidative stress in human pathologies.
Keywords: Allantoin; Urate; Biomarker; Oxidative stress; HILIC; Mass spectrometry;

Recovery of active anti TNF-α ScFv through matrix-assisted refolding of bacterial inclusion bodies using CIM monolithic support by Krishnan Sushma; Chuvappumkal Joseph Bilgimol; Mookambeswaran A. Vijayalakshmi; Padikara Kutty Satheeshkumar (90-93).
► Simultaneous refolding and purification of inclusion bodies of anti TNF-α ScFv. ► Methacrylate-CIM support was used for the first time in matrix assisted refolding. ► The yield and activity of the protein were comparable to the soft gel purification. ► Protein refolding scale up systems could be more efficient with this technology.Anti TNF-α molecules are important as therapeutic agents for many of the autoimmune diseases in chronic stage. Here we report the expression and purification of a recombinant single chain variable fragment (ScFv) specific to TNF-α from inclusion bodies. In contrast to the conventional on column refolding using the soft gel supports, an efficient methodology using monolithic matrix has been employed. Nickel (II) coupled to convective interaction media (CIM) support was utilized for this purpose with 6 M guanidine hydrochloride (GuHCl) as the chaotropic agent. The protein purified after solubilization and refolding proved to be biologically active with an IC50 value of 15 μg. To the best of our knowledge, this is the first report showing the application of methacrylate based chromatographic supports for matrix-assisted refolding and purification of Escherichia coli inclusion bodies. The results are promising to elaborate the methodology further to exploit the potential positive features of monoliths in protein refolding science.
Keywords: Tumor necrosis factor-α (TNF-α); Single chain variable fragment (ScFv); Monolithic matrix support; Convective interaction media (CIM);

► Nucleotides and their constituents were separated by spiral CCC. ► A series of polar solvent systems was used to optimize the partition coefficients. ► Ultra polar samples of ATP, ADP, AMP, and adenosine were well resolved.A set of nucleic acid constituents were separated with ultra polar two-phase solvent systems by a spiral multilayer coil mounted on the rotary frame of a type-J coil planet centrifuge. These two-phase systems were composed of 1-butanol/ethanol/50% saturated aqueous ammonium sulfate at various volume ratios. Nucleobases including adenine, cytosine, uracil, and thymine; nucleosides including adenosine, guanosine, cytidine, and uridine; and nucleotides including, AMP, GMP, CMP, UMP, and TMP are partitioned in each group with suitable solvent ratios. Adenine derivatives such as adenosine, AMP, ADP, and ATP were well resolved in the most polar solvent system composed of ethanol/50% saturated aqueous ammonium sulfate at a volume ratio of 1:2. It was found that cytosine and cytidine peaks showed some irregular two peaks probably due to their keto and enol isomers, while the separation of AMP forms two peaks especially when TMP was added in the sample solution, the mechanism of which is now under investigation in our laboratory.

► A novel chiral LC–MS/MS method simultaneously quantifies R- and S-TJ0711. ► A UPLC-MRM-IDA-EPI method supports metabolite identification of TJ0711 enantiomers. ► Racemic TJ0711 HCl undergoes enantioselective metabolism in vitro. ► Demethylation and hydroxylation are the principle metabolism pathways for both enantiomers.A novel liquid chromatography–tandem mass spectrometry (LC–MS/MS) method employing chiral analytical techniques was developed and validated for in vitro enantioselective metabolic stability study of racemic 1-[4-(2-methoxyethyl) phenoxy]-3-[[2-(2-methoxyphenoxy) ethyl]amino]-2-propanol hydrochloride (TJ0711 HCl), a newly developed vasodilatory β-blocker. Robust enantiomeric separations were achieved on a chiral SUMICHIRAL OA-2500 column using ethanol and hexane (40:60, v/v) as a mobile phase. Metabolic stability results demonstrated that both TJ0711 enantiomers underwent a rapid phase I metabolism, but preferential metabolism of R-TJ0711 was observed. Our previously reported ultra-performance liquid chromatography-multiple reaction monitoring-information dependent acquisition-enhanced product ion (UPLC-MRM-IDA-EPI) method was finally chosen for metabolite profiling study of TJ0711 enantiomers, because the newly developed HPLC-based method resulted in compromised chromatographic separation, particularly for TJ0711 metabolites. A number of metabolic products were detected and the structures of formed metabolites were predicted. Similar to racemic TJ0711 HCl, demethylation and hydroxylation were proposed to be the principle metabolism pathways during in vitro incubations of each enantiomer with human liver microsomes.
Keywords: LC–MS/MS; Enantioselective metabolism; Metabolite profiling; TJ0711 hydrochloride;

► The simultaneous determination of pimpinellin, isopimpinellin and phellopterin. ► Simple and rapid one-step liquid–liquid extraction. ► Short analytical time (4.5 min). ► It was applied to the pharmacokinetic study.A rapid and selective ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was developed for simultaneous determination of three bioactive coumarins of Toddalia asiatica extract including pimpinellin, isopimpinellin and phellopterin in rat plasma for the first time. Phenacetin was used as the internal standard (IS). Plasma samples were extracted by liquid–liquid extraction with methyl tert-butyl ether. The chromatographic separation was carried out on an ACQUITY UPLC™ BEH C18 column with an isocratic mobile phase consisting of methanol-5 mmol/L ammonium acetate (65:35, v/v). The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) via electrospray ionization (ESI) source with positive ionization mode. The method was linear for all analytes over investigated range with all correlation coefficients greater than 0.9942. The lower limits of quantification (LLOQ) were 25.0 ng/mL for pimpinellin, 10.0 ng/mL for isopimpinellin and 5.00 ng/mL for phellopterin. The intra- and inter-day precision (RSD%) was within 12% and the accuracy (RE%) ranged from −2.3% to 5.5%. The rapid and sensitive method was fully validated and successfully applied to the pharmacokinetic study of pimpinellin, isopimpinellin and phellopterin in rats following oral administration of Toddalia asiatica extract.
Keywords: Coumarins; UPLC–MS/MS; Rat plasma; Pharmacokinetics; Toddalia asiatica;

Comparison of IMAC and MOAC for phosphopeptide enrichment by column chromatography by Luc Negroni; Stephane Claverol; Jean Rosenbaum; Eric Chevet; Marc Bonneu; Jean-Marie Schmitter (109-112).
► IMAC-Fe3+- and TiO2-column chromatography were compared. ► Specificity of IMAC-Fe3+ increases when 0.1 M TFA is used instead of 0.1 M acetic acid. ► Fe3+ is released for loading/washing step when 0.1 M TFA is used as loading buffer. ► Fe3+ precipitates for elution of phosphopeptides with NH4OH. ► IMAC-Fe3+ is not a convenient media for repetitive use on the contrary to TiO2.Automated phosphopeptide enrichment prior to MS analysis by means of Immobilized Metal Affinity Chromatography (IMAC) and Metal Oxide Affinity Chromatography (MOAC) has been probed with packed columns. We compared POROS-Fe3+ and TiO2 (respectively IMAC and MOAC media), using a simple mixture of peptides from casein–albumin and a complex mixture of peptides isolated from mouse liver. With theses samples, selectivity of POROS-Fe3+ and TiO2 were pH dependant. In the case of liver extract, selectivity increased from 12–18% to 58–60% when loading buffer contained 0.1 M acetic acid or 0.1 M trifluoroacetic acid, respectively. However, with POROS-Fe3+ column, the number of identifications decreased from 356 phosphopeptides with 0.1 M acetic acid to 119 phosphopeptides with 0.1 M TFA. This decrease of binding capacity of POROS-Fe3+ was associated with strong Fe3+ leaching. Furthermore, repetitive use of IMAC-Fe3+ with the 0.5 M NH4OH solution required for phosphopeptide elution induced Fe2O3 accumulation in the column. By comparison, MOAC columns packed with TiO2 support do not present any problem of stability in the same conditions and provide a reliable solution for packed column phosphopeptide enrichment.
Keywords: Phosphopeptide enrichment; IMAC; TiO2; LC–MS/MS;