Journal of Chromatography B (v.960, #C)

Application of optimized dispersive liquid–liquid microextraction for determination of melatonin by HPLC–UV in plasma samples by M.S. Talebianpoor; S. Khodadoust; A. Rozbehi; M. Akbartabar Toori; M. Zoladl; M. Ghaedi; R. Mohammadi; A.S. Hosseinzadeh (1-7).
Melatonin (N-acetyl-3-(2-aminoethyl)-5-methoxyindole) is biologically active as a neurohormone and antioxidant agent. The optimized dispersive liquid–liquid microextraction (DLLME) followed by high-performance liquid chromatography–ultra violet detection (HPLC–UV) was used for the analysis of melatonin in human plasma. Influence variables such as volume of extracting (carbon tetrachloride: CCl4) and dispersing solvents (acetonitrile: ACN), pH and ionic strength, extraction time and centrifugation time were screened in a 26−2 fractional factorial design (FFD) and then the significant variables were optimized by using a central composite design (CCD). At optimum conditions values of variables set as pH 6.0, 1.5 mL ACN, 140 μL CCl4, 1.0 min extraction time and 3.0 min centrifugation at 4500 rpm. At optimum conditions method has linear response over 2.0–500.0 ng mL−1 with detection limit of 0.5 ng mL−1 with relative standard deviations (RSDs) less than 5.0%. The values of intra-day and inter-day RSD were 4.3% and 8.5%, respectively. The method was applied successfully for the analysis of melatonin in plasma sample.
Keywords: Central composite design; Dispersive liquid–liquid microextraction; Fractional factorial design; HPLC; Melatonin; Plasma;

Microwave-assisted derivatization: Application to steroid profiling by gas chromatography/mass spectrometry by Gregori Casals; Josep Marcos; Oscar J. Pozo; José Alcaraz; María Jesús Martínez de Osaba; Wladimiro Jiménez (8-13).
Gas chromatography–mass spectrometry (GC–MS) remains as the gold-standard technique for the study of the steroid metabolome. A main limitation is the need of performing a derivatization step since incubation with strong silylations agents for long periods of time (usually 16 h) is required for the derivatization of hindered hydroxyls present in some steroids of interest. In the present work, a rapid, simple and reproducible microwave-assisted derivatization method was developed. In the method, 36 steroids already treated with methoxyamine (2% in pyridine) were silylated with 50 μl of N-trimethylsilylimidazole by using microwave irradiation, and the formed methyloxime-trimethylsilyl derivatives were analyzed by GC–MS. Microwave power and derivatization time silylation conditions were optimized being the optimum conditions 600 W and 3 min respectively. In order to evaluate the usefulness of this technique, the urine steroid profiles for 20 healthy individuals were analyzed. The results of a comparison of microwave irradiation with the classical heating protocol showed similar derivatization yields, thus suggesting that microwave-assisted silylation is a valid tool for the rapid steroid metabolome study.
Keywords: Gas chromatography/mass spectrometry; Human urine; Steroids; Metabolome; Microwave assisted derivatization;

Determination and pharmacokinetic study of the novel anti-tumor candidate drug DG-7 in rat plasma by liquid chromatography–tandem mass spectrometry by Juan Li; Dan-Dan Liu; Yu Ke; Fei Guo; Xiao-Ming Ding; Shuai-Liang Wang; Yu Chen; Hong-Min Liu; Ying-Qian Su; Yan-Yang Nan (14-18).
DG-7 (11,14-dihydroxy-7,20-epoxy-20-O-derivative of ent-kaurene diterpenoid) is a novel anti-tumor candidate drug. A sensitive and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the quantification of DG-7 in rat plasma. An aliquot of 50 μL plasma sample was prepared by liquid–liquid extraction with ethyl acetate. Chromatographic separation was accomplished on a Waters XTerra C18 column (2.1 mm × 150 mm, 5 μm) with an isocratic elution system consisting of methanol and water. Detection was performed by multiple reaction monitoring (MRM) mode using electrospray ionization in the positive ion mode. The optimized fragmentation transitions for MRM were m/z 590.1 →  m/z 260.0 for DG-7 and m/z 180.3 →  m/z 110.1 for phenacetin (internal standard). The method was linear over the concentration range of 5–2500 ng/mL. The intra- and inter-day precisions were less than 7.9% and the accuracy was within ±9.0%. The mean recovery of DG-7 ranged from 76.8% to 79.2%. The validated method has been successfully applied to a pharmacokinetic study in rats after intravenous administration of DG-7.
Keywords: Pharmacokinetics; DG-7; LC–MS/MS; Anti-tumor; Rat plasma;

(−)-Securinine (SE) is a major alkaloid found in plant Securinega suffruticosa, which has a wide range of pharmacological activities including anticancer, anti-parasitic and central nervous system stimulating effects, etc. To aid the pharmacological study of SE, we developed an LC–MS/MS method for quantitative determination of SE in mouse plasma. In this method, plasma samples were first prepared with salting-out assisted liquid–liquid extraction using cold acetonitrile (−20 °C) and 2.00 M ammonium acetate. Separation of SE and the internal standard (IS) from sample matrix was achieved on a Gemini Nx C18 column using 40% acetonitrile and 60% 10.0 mM ammonium acetate at a flow rate of 0.200 mL min−1. Quantification of SE was accomplished with positive electrospray ionization tandem mass spectrometry using mass transitions m/z 218.1 → 84.1 for SE, and m/z 204.1 → 70.2 for the IS. This method has a lower limit of quantitation (LLOQ) of 0.600 ng mL−1 and a linear calibration range up to 600 ng mL−1 in mouse plasma. The intra- and inter-run accuracy (%RE) and precision (%CV) were ≤±6% and 6%, respectively. The IS normalized matrix factors from six lots of plasma matrices ranged 0.92–1.07, and the recoveries of plasma SE were 99–109%. The validated method has been applied to the measurement of SE in plasma samples of a mouse study.
Keywords: Securinine; Norsecurinine; LC–MS/MS; Salting-out assisted liquid–liquid extraction; Myeloid differentiation inducing agent; Method validation;

Development, validation and application of an HPLC–MS/MS method for the determination of fentanyl and nor-fentanyl in human plasma and saliva by Sudeep R. Bista; Michael Lobb; Alison Haywood; Janet Hardy; Angela Tapuni; Ross Norris (27-33).
Monitoring fentanyl concentration in saliva and plasma may be useful in pharmacokinetic/pharmacodynamic studies. Salivettes® have been used widely for collecting saliva samples. However due to its lipophilicity, fentanyl adsorbs to the cotton dental bud (CDB) used in this device. Furthermore, due to dry mouth being a common adverse effect seen in patients treated with opioids, obtaining enough saliva for analysis is often a challenge. Hence, a simple simultaneous method to quantify fentanyl and its metabolite in both human plasma and saliva was developed and validated. A novel extraction method was also developed and validated to recover fentanyl in saliva directly from the CDB. This extraction method utilises acetonitrile to recover the fentanyl directly from the CDB rather than recovery by centrifugation, which is not always possible. Reverse phase chromatographic separation was performed on a Shimadzu LC 20A HPLC system using gradient elution. The electrospray ion source (ESI) was operated in positive ion mode using an Applied Biosystems API 3200 LC/MS/MS as detector. Deuterated fentanyl (D5) and nor-fentanyl (D5) were used as internal standards (IS). The retention times for fentanyl and nor-fentanyl were 3.70 min and 3.20 min respectively. The lower limit of quantitation (LLOQ) was determined to be 0.030 μg/L in plasma and 0.045 in saliva for fentanyl and nor-fentanyl. Acceptable linearity for fentanyl and nor-fentanyl in both plasma and saliva was demonstrated from 0.02 to 10 μg/L (R 2 0.9988–0.9994). Accuracy for fentanyl and nor-fentanyl in both plasma and saliva samples was between 96% and 108%. Total imprecision expressed as the co-efficient of variation was between 1.0 and 15.5% for both analytes in both matrices. The validated method was applied successfully in 11 paired plasma and saliva samples obtained from patients with cancer pain receiving transdermal fentanyl (Duragesic®) at doses from 25 μg to 100 μg.
Keywords: Fentanyl; HPLC–MS/MS; Plasma; Saliva; Salivette®; Adsorption;

A downstream process allowing the efficient isolation of a recombinant amphiphilic protein from tobacco leaves by Elisa Gecchele; Stefan Schillberg; Matilde Merlin; Mario Pezzotti; Linda Avesani (34-42).
The 65-kDa isoform of human glutamic acid decarboxylase (hGAD65) is a major autoantigen in autoimmune diabetes. The heterologous production of hGAD65 for diagnostic and therapeutic applications is hampered by low upstream productivity and the absence of a robust and efficient downstream process for product isolation. A tobacco-based platform has been developed for the production of an enzymatically-inactive form of the protein (hGAD65mut), but standard downstream processing strategies for plant-derived recombinant proteins cannot be used in this case because the product is amphiphilic. We therefore evaluated different extraction buffers and an aqueous micellar two-phase system (AMTPS) to optimize the isolation and purification of hGAD65mut from plants. We identified the extraction conditions offering the greatest selectivity for hGAD65mut over native tobacco proteins using a complex experimental design approach. Under our optimized conditions, the most efficient initial extraction and partial purification strategy achieved an overall hGAD65mut yield of 92.5% with a purification factor of 12.3 and a concentration factor of 23.8. The process also removed a significant quantity of phenols, which are major contaminants present in tobacco tissue. This is the first report describing the use of AMTPS for the partial purification of an amphiphilic recombinant protein from plant tissues and our findings could also provide a working model for the initial recovery and partial purification of hydrophobic recombinant proteins from transgenic tobacco plants.
Keywords: AMTPS; Autoimmune diabetes; hGAD65mut; Purification; Transgenic plant;

Component analysis and structure identification of active substances for anti-gastric ulcer effects in Radix Astragali by liquid chromatography and tandem mass spectrometry by Xiao-hua Liu; Liang-gong Zhao; Jing Liang; Long Guo; Ying-lai Yang; Fang Hu; Rui-juan Zhu; Shi-lan Feng (43-51).
This study provided a comprehensive component analysis and structure identification of active substances for the anti-gastric ulcer effects of Radix Astragali. The data were generated by organically combining the results from in vivo pharmacodynamic experiments, a cell growth-promoting assay, structure identification, content determination, fingerprinting, and correlation analyses. The fingerprints from high-performance liquid chromatography coupled with a diode array detector (HPLC–DAD) and from HPLC coupled with evaporative light scattering detectors (ELSD) from 95% ethanol extracts of Radix Astragali (ERA) were determined using HPLC–DAD–ELSD. The structures of 16 compounds were identified using ultra-pressure liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS). The contents of these 16 compounds were simultaneously determined in a single run using HPLC–DAD–ELSD. The strength of the anti-ulceration effect of each of the 16 compounds was correlated to its content in the HPLC spectrum using gray relation statistics. The sequence of the contribution from each of the 16 compounds to the anti-gastric ulcer effect was determined. The results showed that ononin, astragalosideIII, and astragalosideIV contributed most to the observed anti-gastric ulcer effects and that these three compounds also exhibited strong growth-promoting effects in cultured GES-1 cells. The results of this study can be used to evaluate the quality of Radix Astragali and to provide a theoretical foundation for its further study.
Keywords: Radix Astragali; Fingerprint; Pharmacodynamics; Spectrum–effect relationship; GES-1; LC–ESI–MS;

Comparison of a novel TiO2/diatomite composite and pure TiO2 for the purification of phosvitin phosphopeptides by Yang Zhang; Junhua Li; Fuge Niu; Jun Sun; Yuan Dou; Yuntao Liu; Yujie Su; Bei Zhou; Qinqin Xu; Yanjun Yang (52-58).
A novel TiO2/diatomite composite (TD) was prepared and then characterized by scanning electron microscope (SEM) and Fourier Transform Infrared (FTIR). The results of SEM showed that after modification, the porous surface of diatomite was covered with TiO2. Both diatomite and TD had clear disc-shaped structures with average grain diameters of around 25 μm. Then TD and pure TiO2 were applied in the purification of phosvitin phosphopeptides (PPPs) from the digest of egg yolk protein, and a comparative study of adsorption properties of PPPs on TD and TiO2 was performed. In the study of adsorption kinetics, the adsorption equilibrium of PPPs on TD and TiO2 fitted well with the Langmuir model, and the time needed to reach adsorption equilibrium were both around 10 min. The maximum dynamic adsorption capacity of TD (8.15 mg/g) was higher than that of TiO2 (4.96 mg/g). The results of repeated use showed that TD and TiO2 were very stable after being subjected to ten repeated adsorption–elution cycles, and TD could easily be separated from aqueous solution by filtration. On the other hand, the present synthetic technology of TD was very simple, cost-effective, organic solvent-free and available for large-scale preparation. Thus, this separation method not only brings great advantages in the purification of PPPs from egg yolk protein but also provides a promising purification material for the enrichment of phosphopeptides in proteomic researches.
Keywords: TiO2; TiO2/diatomite composite; Phosvitin phosphopeptides; Purification; Yolk;

Corynoline and acetycorynoline are active compounds of Corydalis bungeana Turcz. with various pharmacological effects such as sedation, anti-leptospira and liver injury protection effects. A specific, simple and sensitive UHPLC–ESI–MS/MS method was developed and validated for the pharmacokinetic study of corynoline and acetycorynoline in rat plasma. Corynoline and acetycorynoline were extracted from plasma samples by liquid–liquid extraction. The analysis was carried out on an Agilent SB-C18 column (1.8 μm, 50 mm × 2.1 mm) with the mobile phase of acetonitrile–0.1% formic acid (80:20, v/v). The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source in positive mode. The current UHPLC–ESI–MS/MS assay was validated for linearity, intra-day and inter-day precisions, accuracy, extraction recovery and stability. The lower limits of quantification were 0.10 ng/mL for corynoline and 0.353 ng/mL for acetycorynoline. Intra-day and inter-day precision were less than 12.3% and accuracy ranged from 0.18% to 14.9%. The mean extraction recoveries of analytes and internal standard (IS) from rat plasma were all more than 76.2%. This validated method was successfully applied to pharmacokinetic study in rats after oral administration of corynoline, acetycorynoline and the extract of Corydalis bungeana Turcz.
Keywords: Corydalis bungeana Turcz.; UHPLC–ESI–MS/MS; Corynoline; Acetycorynoline; Pharmacokinetics;

A sensitive and selective ultra high performance liquid chromatography–tandem mass spectrometric (UHPLC–MS/MS) method for the determination of phlorizin and phloretin in human plasma has been firstly developed. Samples were prepared after protein precipitation and analyzed on a C18 column interfaced with a triple quadrupole tandem mass spectrometer. Negative electrospray ionization was employed as the ionization source. The mobile phase consisted of acetonitrile–water (0.02% formic acid), using a gradient procedure. The analytes and internal standard dihydroquercetin were both detected by use of multiple reaction monitoring mode. The method was linear in the concentration range of 2.5–1000.0 ng/mL. The lower limit of quantification (LLOQ) was 2.5 ng/mL. The intra- and inter-day relative standard deviation across three validation runs over the entire concentration range was less than 9.2%. The accuracy determined at three concentrations was within ±7.3% in terms of relative error. The total run time was 12.0 min. This assay offers advantages in terms of expediency, and suitability for the analysis of phlorizin and phloretin in various biological fluids.
Keywords: Phlorizin; Phloretin; UHPLC–MS/MS; Pharmacokinetics;

Water-soluble vitamins are an important class of compounds that require quantification from food sources to monitor nutritional value. In this study we have analysed six water-soluble B vitamins ([thiamine (B1), riboflavin (B2), nicotinic acid (B3, NAc), nicotinamide (B3, NAm), pyridoxal (B6), folic acid (B9)], and ascorbic acid (vit C) with hydrophilic interaction liquid chromatography (HILIC), and compared UV, fluorescent (FLD) and coulometric detection to optimise a method to quantitate the vitamins from food sources.Employing UV/diode array (DAD) and fluorimetric detection, six B vitamins were detected in a single run using gradient elution from 100% to 60% solvent B [10 mM ammonium acetate, pH 5.0, in acetonitrile and water 95:5 (v:v)] over 18 min. UV detection was performed at 268 nm for B1, 260 nm for both B3 species and 284 nm for B9. FLD was employed for B2 at excitation wavelength of 268 nm, emission of 513 nm, and 284 nm/317 nm for B6. Coulometric detection can be used to detect B6 and B9, and vit C, and was performed isocratically at 75% and 85% of solvent B, respectively. B6 was analysed at a potential of 720 mV, while B9 was analysed at 600 mV, and vit C at 30 mV. Retention times (0.96 to 11.81 min), intra-day repeatability (CV 1.6 to 3.6), inter-day variability (CV 1.8 to 11.1), and linearity (R 0.9877 to 0.9995) remained good under these conditions with limits of detection varying from 6.6 to 164.6 ng mL−1, limits of quantification between 16.8 and 548.7 ng mL−1. The method was successfully applied for quantification of six B vitamins from a fortified food product and is, to our knowledge, the first to simultaneously determine multiple water-soluble vitamins extracted from a food matrix using HILIC.
Keywords: Vitamins; HPLC; Detection; Analysis;

Bone distribution study of anti leprotic drug clofazimine in rat bone marrow cells by a sensitive reverse phase liquid chromatography method by Cheruvu Hanumanth Srikanth; Pankaj Joshi; Anil Kumar Bikkasani; Konica Porwal; Jiaur R. Gayen (82-86).
The aim of the present study was to investigate the distribution of clofazimine (CLF) in rat bone marrow cells by a validated reverse phase high performance liquid chromatography. CLF and chlorzoxazone (I.S) were extracted by liquid–liquid extraction from plasma and rat bone marrow cells. The chromatographic separation was performed in isocratic mode by the mobile phase consisting of 10 mM ammonium formate (pH 3.0 with formic acid) and acetonitrile in a ratio of 50:50 (v/v). The method was accurate and precise in the linear range of 15.6–2000.0 ng/mL with a correlation coefficient (r 2) of 0.996 and 0.995 in rat plasma and bone marrow cells, respectively. After single oral dose of 20 mg/kg, the maximum concentration of CLF in plasma and bone marrow cells were obtained at 12 h with the concentrations of 593.2 and 915.4 ng/mL, respectively. The AUC0−t and mean elimination half life (t 1/2) of CLF in bone marrow cells were 54339.02 ng*h/mL and 52.46 h, respectively, which signified the low body clearance and high distribution of CLF in bone marrow cells. The single oral dose pharmacokinetic investigation was confirmed the CLF endure for a long period in rat due to high distribution in various tissues. The developed method was successfully applied to the estimation of the pharmacokinetic parameters of CLF in plasma and bone marrow cells after administration of single oral dose of 20 mg/kg to rats.
Keywords: Clofazimine; Clastogenic effect; Bone marrow distribution; Rat bone marrow cells; Rat plasma; Liquid–liquid extraction; RP-HPLC;

Simultaneous determination of 16 synthetic colorants in hotpot condiment by high performance liquid chromatography by Bobin Tang; Cunxian Xi; Yun Zou; Guomin Wang; Xianliang Li; Lei Zhang; Dongdong Chen; Jinzhong Zhang (87-91).
A simultaneous determination method for 16 synthetic colorants in hotpot condiment was developed by high performance liquid chromatography. The samples were successively extracted with 2 mol/L carbamide solution containing 5% ammonia (dissolved in methanol) and methanol–acetone solution, and then the target analytes could be divided into two groups named as lipid-soluble and water-soluble colorants by ethyl acetate-cyclohexane with liquid–liquid extraction. The lipid-soluble and water-soluble colorants were purified by gel permeation chromatography and solid phase extraction column packed with polyamide resin, respectively. The obtained two eluates were combined, concentrated, and separated by C18 column and determined by diode array detector. Good linear relationships between peak areas and the concentrations of the synthetic colorants were obtained in the range of 0.01–50.0 mg/L with correlation coefficients above 0.999 (n  = 10). The limits of detection and quantitation were 1–3 and 10 μg/kg for 16 synthetic colorants, respectively. The average recoveries at the spiked levels of 5, 10, 20 and 50 μg/kg were in the range of 63.2–97.1% with relative standard deviations (n  = 6) around 1.5–10.6%. This method is sensitive and reliable, and can be used to simultaneously determine 8 lipid-soluble and 8 water-soluble colorants in hotpot condiment.
Keywords: Hotpot condiment; Synthetic colorant; Simultaneous determination; HPLC;

A liquid chromatography–tandem mass spectrometry method to quantify carvedilol enantiomers in human plasma was developed and validated as a measure of compliance in clinical research. Carvedilol enantiomers were extracted from human serum (0.5 mL) via liquid–liquid extraction with methyl tert-butyl ether (2.5 mL). Carvedilol-related compound C served as the internal standard. The analyte and internal standard were separated on a Sino-Chiral AD column (150 × 4.6 mm, 5 μm, amylose tris-3,5-dimethylphenylcarbamate coated on silica-gel) using isocratic elution with mobile phases of methanol, water and diethylamine (94:6:0.01, v/v). The total run-time was 10.5 min. Carvedilol enantiomers were quantified using a triple quadrupole mass spectrometer operated in multiple-reaction-monitoring mode using positive electrospray ionisation. The mass transitions monitored for quantitation were carvedilol (m/z 407 → 222) and carvedilol-related compound C (m/z 497 → 222). The limits of quantification for the S- and R-carvedilol enantiomers in plasma were both 0.08 ng/mL. The method was validated in the linear range of 0.08–50 ng/mL with acceptable inter- and intra-assay precision and accuracy and stability suitable for routine laboratory practice. The method was successfully applied to samples taken from research volunteers treated with carvedilol sustained-release tablet 18 mg. C max and AUClast were 9.1 ± 5.1 ng/mL and 59.4 ± 39.6 ng h/mL for R-carvedilol, 4.0 ± 2.3 ng/mL and 24.7 ± 15.0 ng h/mL for S-carvedilol, respectively. t max and t 1/2 were 4.6 ± 1.9 h and 9.6 ± 4.5 h for R-carvedilol, and 4.7 ± 1.0 h and 10.7 ± 5.7 h, respectively.
Keywords: Carvedilol; Enantiomer; Chiral column; LC–MS/MS; Human plasma; Method validation;

A simple, sensitive and cost-effective assay based on reversed phase high performance liquid chromatography (RP-HPLC) with isocratic mode for simultaneous determination of bendamustine (BM) and its active metabolite, gamma-hydroxy-bendamustine (γ-OH-BM) in human plasma and urine was developed and validated. Sample preparation involved protein precipitation by 10% perchloric acid-methanol solution. The peaks were recorded by using fluorescence detector (excitation wavelength 328 nm and emission wavelength 420 nm). The calibration curves were linear over concentration ranges of 8.192–10,000 ng mL−1 and 5–1000 ng mL−1 for BM in human plasma and urine as well as 10–1000 ng mL−1 and 5–1000 ng mL−1 for γ-OH-BM in human plasma and urine, respectively. Intra- and inter-run precisions of BM and γ-OH-BM were less than 15% and the bias were within ±15% for both plasma and urine. This validated method was successfully applied to a pharmacokinetic study enrolling 10 Chinese patients with indolent B-cell non-Hodgkin lymphoma and chronic lymphocytic leukemia administered a single intravenous infusion of 100 mg m2 bendamustine hydrochloride.
Keywords: Bendamustine; Gamma-hydroxy-bendamustine; HPLC-fluorescence detector; Pharmacokinetics; Human plasma;

Determination of nitroimidazole residues in aquaculture tissue using ultra high performance liquid chromatography coupled to tandem mass spectrometry by Anna Gadaj; Valentina di Lullo; Helen Cantwell; Martin McCormack; Ambrose Furey; Martin Danaher (105-115).
An UHPLC-MS/MS method was developed for the quantitative confirmatory analysis of residues of nitroimidazole drugs (dimetridazole, ipronidazole, metronidazole, ornidazole and ronidazole) and their corresponding hydroxy metabolites (HMMNI, ipronidazile-OH and metronidazole-OH) in aquaculture tissue. Samples were extracted by shaking in acetonitrile, water, MgSO4 and NaCl before being defatted with n-hexane pre-saturated with acetonitrile and concentrated under nitrogen. Nitroimidazole residues were determined by UHPLC-MS/MS operating in positive electrospray ionisation mode using a reversed phase BEH C18 column. The method was validated according to the EU Commission Decision 2002/657/EC guidelines. The following performance studies were carried out: specificity, selectivity, linearity, within laboratory repeatability (WLr)/reproducibility (WLR), accuracy, precision, decision limit (CCα), detection capability (CCβ), absolute recovery and stability. The analytical range of the method is 0.1–20 μg kg−1. Accuracy and precision of the method, under within-laboratory reproducibility conditions, ranged from 83 to 105% and 2.3 to 14.0%, respectively. CCα were 0.07–1.0 μg kg−1 depending on analyte and matrix. A total of 50 samples can be analysed in a single day using the assay. The method has been extensively evaluated through application to real test samples.
Keywords: Nitroimidazole residues; UHPLC-MS/MS; Aquaculture; Prawn; Shrimp; Salmon; Trout; Sea bass; Tilapia;

A selective, sensitive and reliable ultra fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) method has been developed for the simultaneous determination of two iridoid glycosides (geniposide and genipin gentiobioside), two anthraquinones (rhein and emodin) and four flavonoid glycosides (isonaringin, naringin, hesperidin and neohesperidin), the major active ingredients of Zhi-Zi-Da-Huang decoction (ZZDHD), in rat plasma using paeoniflorin as internal standard (IS). After liquid–liquid extraction with ethyl acetate-isopropanol (1:1, v/v), separation was achieved on a Shim-pack XR-ODS C18 column (75 mm × 3.0 mm, 2.2 μm) using gradient elution with a mobile phase consisting of water (containing 0.1% formic acid) and acetonitrile at a flow rate of 0.4 mL/min. Detection was performed on 4000 QTRAP mass spectrometry equipped with turbo ion spray source in the negative ionization and multiple reaction monitoring (MRM) mode. The intra- and inter-day precisions (as relative standard deviation) were less than 11.4%, and accuracy (as relative error) was within ±10.0%. The lower limits of quantification (LLOQ) were 4.0, 0.5, 2.0, 0.1, 1.0, 2.0, 1.0, 2.0 ng/mL for geniposide, genipin gentiobioside, rhein, emodin, isonaringin, naringin, hesperidin and neohesperidin, respectively. The extraction recoveries of the analytes and IS from rat plasma were all more than 86.0%. The method was fully validated and applied to compare the pharmacokinetic profiles of the analytes in normal and cholestatic liver injury (CLI) rats after oral administration of ZZDHD. Results showed that there were remarkable differences in pharmacokinetic properties of the analytes between normal and CLI group.
Keywords: Zhi-Zi-Da-Huang decoction; UFLC-MS/MS; Pharmacokinetics; Rat plasma; Cholestatic liver injury;

Development of simple and effective methods for high-throughput, high-fidelity screening and identification of cyclooxygenase-1 (COX-1) inhibitors from natural products are important for drug discovery to treat inflammation and carcinogenesis. Here, we developed a new screening assay based on cyclooxygenase-1 (COX-1) functionalized magnetic nanoparticles (i.e. Fe3O4@SiO2–COX-1) for solid phase ligand fishing, and then mass spectrometry (MS) was applied for structural identification. Incubation conditions were optimized. High specificity for isolating COX-1 inhibitors was achieved by testing positive control, indomethacin, with active and inactive COX-1. Moreover, high stability of immobilized COX-1 (remained 95.3% after ten consecutive cycles) allows the analysis reproducible. When applied to turmeric extract, four curcuminoids (i.e. curcumin, demethoxycurcumin, bisdemethoxycurcumin, and 1-(4-hydroxy-3,5-dimethoxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)-(1E,6E)-1,6-heptadiene-3,5-dione), difficult to be distinguished from original MS spectrum of turmeric extract, were isolated as main COX-1 inhibitors. Their structures were characterized based on their accurate molecular weight and diagnostic fragment ions. The results indicated that the proposed method was a simple, robust and reproducible approach for the discovery of COX-1 inhibitors from complex matrixes.
Keywords: Cyclooxygenase-1; Ligand fishing; MS; Turmeric; Curcuminoid;

Corrigendum to “Rapid and sensitive LC–MS/MS assay for the quantitation of 20(S)-protopanaxadiol in human plasma” [J. Chromatogr. B 877 (5–6) (2009) 581–585] by Dan Zhang; Yingwu Wang; Jiangbin Han; Weisong Yu; Lili Deng; J. Paul Fawcett; Zeyuan Liu; Jingkai Gu (133).

Simultaneous determination of seven β-lactam antibiotics in human plasma for therapeutic drug monitoring and pharmacokinetic studies by Fekade Bruck Sime; Michael S. Roberts; Jason A. Roberts; Thomas A. Robertson (134-144).
There is strong evidence in literature supporting the benefit of monitoring plasma concentrations of β-lactam antibiotics in the critically ill to ensure appropriateness of dosing. The objective of this work was to develop a method for the simultaneous determination of total concentrations piperacillin, benzylpenicillin, flucloxacillin, meropenem, ertapenem, cephazolin and ceftazidime in human plasma. Sample preparation involved protein precipitation with acetonitrile containing 0.1% formic acid and subsequent dilution of supernatant with 0.1% formic acid in water. Chromatographic separation was achieved on a reversed phase column (C18, 2.6 μm, 2.1 × 50 mm) via gradient elution using water and acetonitrile, each containing 0.1% formic acid, as mobile phase. Tandem mass spectrometry (MSMS) analysis was performed, after electrospray ionization in the positive mode, with multiple reaction monitoring (MRM). The method is accurate with the inter-day and intra-day accuracies of quality control samples (QCs) ranging from 95 to 107% and 95 to 108%, respectively. It is also precise with intra-day and inter-day coefficient of variations ranging from 4 to 12% and 5 to 14%, respectively. The lower limit of quantification was 0.1 μg/mL for each antibiotic except flucloxacillin (0.25 μg/mL). Recovery was greater than 96% for all analytes except for ertapenem (78%). Coefficients of variation for the matrix effect were less than 10% over the six batches of plasma. Analytes were stable over three freeze-thaw cycles, and for reasonable hours on the bench top as well as post-preparation. This novel liquid chromatography tandem mass spectrometry method proved accurate, precise and applicable for therapeutic drug monitoring and pharmacokinetic studies of the selected β-lactam antibiotics.
Keywords: β-lactam; Antibiotics; LC-MS/MS; Therapeutic drug monitoring; Pharmacokinetics;

Microwave assisted extraction (MAE) followed by microplate solid phase extraction (MPSPE) coupled with ultra high performance liquid chromatography (UHPLC) for the semi-quantitative determination of colchicine, 3-demethyl colchicine and 2-demethyl colchicine in postmortem rat bone is described. Rats (n  = 4) received 50 mg/kg colchicine (i.p), and euthanized by CO2 asphyxiation. Remains decomposed to skeleton outdoors and vertebral bones were collected cleaned, and ground to a fine powder. Powdered bone underwent MAE using methanol in a closed microwave system, followed by MPSPE and analysis using UHPLC–PDA. MAE analyte stability was assessed and found to be stable for at least 60 min irradiation time. The majority (>95%) of each analyte was recovered after 15 min. The MPSPE–UHPLC method was linear between 10 and 2000 ng/mL, with coefficients of variation <20% in triplicate analysis, with a limit of detection of 10 ng/mL for each of the three analytes. Following MAE for 30 min (80 °C, 1200 W), MPSPE–UHPLC analysis of vertebral bone of colchicine-exposed rats detected colchicine (1.8–4.1 μg/g), 3-demethyl colchicine (0.77–1.8 μg/g) and 2-demethyl colchicine (0.43–0.80 μg/g) in all samples assayed.
Keywords: UHPLC; Colchicine; Metabolites; Forensic toxicology; Bone; Microwave assisted extraction;

An LC-ESI–MS/MS method using high-throughput solid-phase extraction (SPE) was developed and validated to measure crizotinib in human and mouse plasma to support ongoing clinical and preclinical pharmacokinetic studies. Chromatographic separation of mouse or human plasma extracts was performed on a Supelco Discovery c18 column (50 mm × 2.1 mm, 5.0 μ) with gradient elution using a combination of acidified aqueous and methanol (MeOH) mobile phases. The mass-to-charge transition monitored for detection and quantitation of crizotinib was m/z 450.2 > 260.2 while the stable label internal standard (ISTD) was monitored at m/z 457.2 > 267.3. The validation studies demonstrated that the assay is both precise and accurate with %CV < 9% and accuracies within 8% of nominal target concentration across all concentrations tested for both the human and mouse plasma matrices. Sample volumes required for analysis were 50 and 25 μL for human plasma and mouse plasma, respectively. Calibration curves were linear over a range of 5–5000 ng/mL for human plasma and 2–2000 ng/mL for mouse plasma. The use of a 96-well plate format enabled rapid extraction as well as compatibility with automated workflows. The method was successfully applied to analyze crizotinib concentrations in plasma samples collected from children enrolled on a phase I pediatric study investigating the use of crizotinib for treatment of pediatric brain tumors.
Keywords: Crizotinib; Human plasma; Solid phase extraction (SPE); Liquid chromatography-electrospray ionization–tandem mass spectrometry (LC-ESI–MS/MS); Lower limit of quantitation (LLOQ); Pharmacokinetic studies;

Determination of deltamethrin in rat plasma and brain using gas chromatography–negative chemical ionization mass spectrometry by Darren Gullick; Andrew Popovici; Holly C. Young; James V. Bruckner; Brian S. Cummings; Pei Li; Michael G. Bartlett (158-165).
Quantification of the pyrethroid deltamethrin (DLM) in small (100 μL) biological samples from rodents is essential for toxicokinetic studies of trace levels of the insecticide in foods. Such empirical kinetic data are necessary for construction of valid physiologically-based toxicokinetic models. There are no validated methods in the literature for determining deltamethrin in 100 μL plasma and brain samples. Plasma and brain samples were stabilized using sodium fluoride as an esterase inhibitor, and the DLM was extracted by protein precipitation using acetonitrile and phosphoric acid. The samples were vortexed, centrifuged, evaporated to dryness, and reconstituted in toluene prior to injection into a gas chromatograph equipped with a quadrupole mass analyzer. Samples were ionized via electron capture in the negative ion mode using methane, and the molecular ion and fragment ions of DLM were monitored using Selected-Ion Monitoring (SIM) for quantitation and verification of the analyte. Cis-permethrin was used as the internal standard for the method, which was validated according to current US FDA guidelines. Linearity was determined between 0.3 and 1000 ng/mL, with a limit of detection of 150 pg/mL. The intra- and inter-batch variation for precision (as % relative standard deviation, RSD) and accuracy (as % bias) of the method were better than 20% at the limit of quantitation and better than 15% across the remaining linear range (n  = 18), with recoveries of 113% and 68% for plasma and brain respectively. Benchtop stability, autosampler stability, and freeze/thaw stability studies of the method (over a 3-day freeze/thaw cycle) were found to be within the acceptance criteria of 20% RSD and bias. This optimized method was applied to the quantitation of DLM in plasma and brain homogenate samples obtained up to 12 h after oral dosing of Sprague-Dawley rats with 1 mg DLM/kg body weight.
Keywords: Deltamethrin; Pyrethroids; Neurotoxicity; Plasma; Brain homogenate; Bioanalytical method validation;

α-Amylase inhibitors play an important role in management of diabetes and obesity. In order to rapidly discover potent α-amylase inhibitors from medicinal plants, a ligands-screening method based on enzyme-immobilized magnetic nanoparticles integrated with HPLC was developed. Amine-terminated magnetic nanoparticles were prepared for the immobilization of α-amylase. Based on the affinity theory, the α-amylase-coated magnetic nanoparticles were employed to fish out the ligands from the extracts of Garcinia xanthochymus, and the elutes were examined by HPLC. As a result, three ligands were screened out. Isolation and identification were carried out subsequently. By analyzing the UV, MS and NMR spectra, they were identified as three biflavonoids including GB2a glucoside (2), GB2a (3) and fukugetin (4). The IC50 values of the three compounds were also determined. The results suggest the proposed approach is efficient and accurate, and has great potential in rapid discovery of drug candidates from medical plants.
Keywords: Ligand fishing; Magnetic nanoparticles; Screening; α-Amylase inhibitors; Garcinia xanthochymus;

Aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine and benzoylhypaconine are six main Aconitum alkaloids from traditional Chinese medicine, Aconiti kusnezoffii radix, which possess highly bioactive as well as highly toxic character for medicinal use. In the present study, for the purpose of better utilizing the toxic herbal material, the performance characteristics of NKA-II, D101, X-5, AB-8, S-8, HPD722 and HPD750 macroporous resins for the enrichment and purification of these six Aconitum alkaloids were critically evaluated. Results showed that NKA-II offered the best adsorption and desorption capacities for six Aconitum alkaloids among the seven macroporous resins tested, which were affected significantly by the pH value. Subsequently, dynamic adsorption and desorption experiments had been carried out with the column packed by NKA-II resin to optimize the separation process of six Aconitum alkaloids. After one run treatment with NKA-II resin, the content of total six Aconitum alkaloids were increased from 5.87% to 60.3%, the recovery was 75.8%. Meanwhile, a validated HPLC–MS method had been developed to qualitative and quantitative these six Aconitum alkaloids. This method would provide scientific references to the large-scale production of six Aconitum alkaloids from Aconiti kusnezoffii radix or other plants and might also expand the secure application of these highly toxic components for pharmacy.
Keywords: Enrichment; Purification; Aconitum; HPLC–MS; Macroporous resins;

A semi-automated LC–MS/MS method for the determination of LCI699, a steroid 11β-hydroxylase inhibitor, in human plasma by Wenkui Li; Suyi Luo; Sam Rebello; Jimmy Flarakos; Francis L.S. Tse (182-193).
A novel liquid chromatographic method with tandem mass spectrometric detection (LC–MS/MS) for the determination of LCI699 was developed and validated with dynamic ranges of 0.0500–50.0 ng/mL and 1.00–1000 ng/mL using 0.0500 mL and 0.100 mL, respectively, of human plasma. LCI699 and the internal standard, [M + 6]LCI699, were extracted from fortified human plasma via protein precipitation. After transfer or dilution of the supernatant followed by solvent evaporation and/or reconstitution, the extract was injected onto the LC–MS/MS system. Optimal chromatographic separation was achieved on an ACE C18 (50 mm × 4.6 mm, 3 μm) column with 30% aqueous methanol (containing 0.5% acetic acid and 0.05% TFA) as the mobile phase run in isocratic at a flow rate of 1.0 mL/min. The total analysis cycle time is approximately 3.5 min per injection. The addition of an ion-pair reagent, TFA (0.05%, v/v), to the mobile phases significantly improved the chromatographic retention and resolution of the analyte on silica based reversed-phase column. Although addition of TFA to the mobile phase suppresses the ESI signals of the analyte due to its ion-pairing characteristics in the gas phase of MS source, this negative impact was effectively alleviated by adding 0.5% acetic acid to the mobile phase. The current method was validated for sensitivity, selectivity, linearity, reproducibility, stability and recovery. For the low curve range (0.0500–50.0 ng/mL), the accuracy and precision for the LLOQs (0.0500 ng/mL) were −13.0 to 2.0% bias and 3.4–19.2% CV, respectively. For other QC samples (0.100, 6.00, 20.0 and 40.0 ng/mL), the precision ranged from 1.2 to 9.0% and from 3.8 to 8.8% CV, respectively, in the intra-day and inter-day evaluations. The accuracy ranged from −11.3 to 8.0% and −7.2 to 1.6% bias, respectively, in the intra-day and inter-day batches. For the high curve range (1.00–1000 ng/mL), the accuracy and precision for the LLOQs (1.00 ng/mL) were 1.0–15.0% bias and 7.4–9.2% CV, respectively. For the other QC samples (3.00, 20.0, 200 and 750 ng/mL), the precision ranged from 0.8 to 7.0% and from 1.9 to 5.2% CV, respectively, in the intra-day and inter-day evaluations. The accuracy ranged from −2.5 to 4.0% and 0.7–1.0% bias, respectively, in the intra-day and inter-day batches. Additional assessments of incurred sample stability (ISS) and incurred sample reanalysis (ISR) were conducted to demonstrate the ruggedness and robustness of the assay method. The absence of adverse matrix effect and carryover was also demonstrated. The validated method was successfully used to support rapid turnaround human pharmacokinetic studies.
Keywords: LCI699; Steroid 11β-hydroxylase inhibitor; LC–MS/MS; Plasma;

Simultaneous determination of fludarabine and clofarabine in human plasma by LC–MS/MS by Liusheng Huang; Patricia Lizak; Christopher C. Dvorak; Francesca Aweeka; Janel Long-Boyle (194-199).
A method for quantification of fludarabine (FDB) and clofarabine (CFB) in human plasma was developed with an API5000 LC–MS/MS system. FDB and CFB were extracted from EDTA plasma samples by protein precipitation with trichloroacetic acid. Briefly, 50 μL plasma sample was mixed with 25 μL internal standard (50 ng/mL aqueous 2-Cl-adensosine) and 25 μL 20% trichloroacetic acid, centrifuged at 25,000 ×  g (20,000 rpm) for 3 min, and then transfered to an autosampler vial. The extracted sample was injected onto an Eclipse extend C18 column (2.1 mm × 150 mm, 5 μm) and eluted with 1 mM NH4OH (pH 9.6) – acetonitrile in a gradient mode. Electrospray ionization in positive mode (ESI+) and multiple reaction monitoring (MRM) were used, and ion pairs 286/134 for FDB, 304/170 for CFB and 302/134 for the internal standard were selected for quantification. The retention times were typically 3.72 min for FDB, 4.34 min for the internal standard, 4.79 min for CFB. Total run time was 10 min per sample. Calibration range was 0.5–80 ng/mL for CFB and 2–800 ng/mL for FDB. The method was applied to a clinical pharmacokinetic study in pediatric patients.
Keywords: Fludarabine; Clofarabine; LC–MS/MS; Plasma;

A simple and sensitive method based on the magnetic solid phase extraction with modified magnetic nanoparticles followed by high performance liquid chromatography with fluorescence detection has been developed for extraction and determination of aflatoxins B1 (AFB1) and B2 (AFB2) in cereal products. Magnetic nanoparticle coated with 3-(trimethoxysilyl)-1-propanthiol (TMSPT) and modified with 2-amino-5-mercapto-1,3,4-thiadiazole (AMT) was used as an antibody-free adsorbent. Under the optimal conditions, the calibration curves for AFB1 and AFB2 were linear in the ranges of 0.2–15 μg L−1 and 0.04–3 μg L−1, respectively. Detection limit was 0.041 μg L−1 for AFB1 and 0.013 μg L−1 for AFB2. The proposed method was successfully applied to the determination of AFB1 and AFB2 in spiked corn and rice samples with an average recovery of 93.5%. The results demonstrated that the developed method is simple, rapid, inexpensive, accurate and remarkably free from interference effects.
Keywords: Aflatoxins B1 and B2; Modified magnetic nanoparticles; Magnetic solid phase extraction; Cereals; HPLC-FD;

A study on the nature of interactions of mixed-mode ligands HEA and PPA HyperCel using phenylglyoxal modified lysozyme by J. Pezzini; C. Cabanne; J.-W. Dupuy; R. Gantier; X. Santarelli (209-213).
Mixed mode chromatography, or multimodal chromatography, involves the exploitation of combinations of several interactions in a controlled manner, to facilitate the rapid capture of proteins. Mixed-mode ligands like HEA and PPA HyperCel™ facilitate different kinds of interactions (hydrophobic, ionic, etc.) under different conditions. In order to better characterize the nature of this multi-modal interaction, we sought to study a protein, lysozyme, which is normally not retained by these mixed mode resins under normal binding conditions. Lysozyme was modified specifically at Arginine residues by the action of phenylglyoxal, and was extensively studied in this work to better characterize the mixed-mode interactions of HEA HyperCel™ and PPA HyperCel™ chromatographic supports. We show here that the adsorption behaviour of lysozyme on HEA and PPA HyperCel™ mixed mode sorbents varies depending on the degree of charge modification at the surface of the protein. Experiments using conventional cation exchange and hydrophobic interaction chromatography confirm that both charge and hydrophobicity modification occurs at the surface of the protein after lysozyme reaction with phenylglyoxal. The results emanating from this work using HEA and PPA HyperCel sorbents strongly suggest that mixed mode chromatography can efficiently separate closely related proteins of only minor surface charge and/or hydrophobicity differences.
Keywords: Mixed mode chromatography; HEA HyperCel; PPA HyperCel; Lysozyme; Phenylglyoxal; Purification;

Hydrophilic interaction liquid chromatography-solid phase extraction directly combined with protein precipitation for the determination of triptorelin in plasma by Jixia Wang; Song Kong; Jingyu Yan; Gaowa Jin; Zhimou Guo; Aijin Shen; Junyan Xu; Xiuli Zhang; Lijuan Zou; Xinmiao Liang (214-221).
Peptide drugs play a critical role in therapeutic treatment. However, as the complexity of plasma, determination of peptide drugs using liquid chromatography–tandem mass spectrometry (LC–MS/MS) is a daunting task. To solve this problem, hydrophilic interaction liquid chromatography-solid phase extraction (HILIC-SPE) directly combined with protein precipitation (PPT) was developed for the selective extraction of triptorelin from plasma. The extracts were analyzed by reversed-phase liquid chromatography (RPLC). Proteins, phospholipids and highly polar interferences could be removed from plasma by the efficient combination of PPT, HILIC-SPE and RPLC–MS/MS. This method was evaluated by matrix effect, recovery and process efficiency at different concentration levels (50, 500 and 5000 ng/mL) of triptorelin. Furthermore, the performance of HILIC-SPE was compared with that of reversed-phase C18 SPE and hydrophilic lipophilic balance (Oasis HLB) SPE. Among them, HILIC-SPE provided the minimum matrix effect (ranging from 96.02% to 103.41%), the maximum recovery (ranging from 80.68% to 90.54%) and the satisfactory process efficiency (ranging from 82.83% to 92.95%). The validated method was successfully applied to determine triptorelin in rat plasma.
Keywords: HILIC-SPE; PPT; RPLC–MS/MS; Triptorelin; Plasma;

A rapid and precise method for quantification of fatty acids in human serum cholesteryl esters by liquid chromatography and tandem mass spectrometry by Songlin Yu; Jun Dong; Weiyan Zhou; Ruiyue Yang; Hongxia Li; Haijian Zhao; Tianjiao Zhang; Hanbang Guo; Shu Wang; Chuanbao Zhang; Wenxiang Chen (222-229).
We described a rapid and precise method for simultaneous quantification of eleven fatty acids in human serum cholesteryl esters (CEFAs) by liquid chromatography and tandem mass spectrometry (LC–MS/MS). After extraction of serum lipids with isopropanol, CEFAs were separated on reversed phase liquid chromatography and detected by mass spectrometry in positive ion mode with multiple reaction monitor. Individual CEFA was quantified by peak area normalization method and expressed as molar percent of total CEFAs. The run time was less than 5 min and detection limits were from 0.31 to 14.50 × 10−5  mmol/L. Recoveries of the CEFAs ranged from 91.85% to 104.83% with a mean of 99.12%. The intra and total CVs for the measurement of CEFAs were 0.87–7.70% and 1.02–7.65%, respectively. This LC–MS/MS method required no internal standards, eliminated natural isotope interferences, and provided reproducible and reliable results for 11 major CEFAs in human serum. This method can be used in monitoring and evaluating dietary fatty acid intake. Additional studies are needed to evaluate the associations between serum CEFAs and cardiovascular disease risk factors.
Keywords: Fatty acids; Cholesteryl ester; Cardiovascular diseases; Liquid chromatography and tandem mass spectrometry;

Size-exclusion chromatography (SEC) is commonly used to monitor low molecular weight fragments and aggregates present in recombinant monoclonal antibody (mAb) biopharmaceuticals. It has been previously demonstrated that SEC could be coupled with mass spectrometry (MS) to directly measure the molecular weights of these protein species to aid in their identification. However, the use of certain mobile phase modifiers led to compromised sensitivity in MS detection. In this work, we demonstrate that the use of m-nitrobenzyl alcohol (m-NBA) as a post-column additive in an SEC-MS method is able to improve the ionization of antibody light chain and heavy chain approximately 7-fold and 2-fold, respectively, and thus allows the MS detection of low-abundance size variants present in mAb biopharmaceuticals. Application of the 15-min reducing SEC-UV/MS method enabled the direct identification of size variants present in an IgG1 mAb sample. One high molecular weight species observed under reducing conditions was identified to be a thioether-linked heterodimer of light chain and heavy chain. Multiple lower molecular weight species were found to result from cleavage of the heavy chain at a number of sites throughout the conserved sequence. The reducing SEC-UV/MS method provides a straightforward approach for identification of size variants present in mAb and may be applicable generally to all types of mAb biopharmaceuticals.
Keywords: Size-exclusion chromatography; Liquid chromatography; Mass spectrometry; Antibody fragment; Aggregate;

Protein imprinted ionic liquid polymer on the surface of multiwall carbon nanotubes with high binding capacity for lysozyme by Shifang Yuan; Qiliang Deng; Guozhen Fang; Jianhua Wu; Wangwang Li; Shuo Wang (239-246).
In this research, ionic liquid as functional monomer to prepare molecularly imprinted polymers for protein recognition was for the first time demonstrated, in which, 1-vinyl-3-butylimidazolium chloride was selected as functional monomer, acrylamide as co-functional monomer and lysozyme (Lyz) as template protein to synthesize imprinted polymers on the surface of multiwall carbon nanotubes in aqueous medium. The results indicated that ionic liquid was helpful to improve binding capacity of imprinted polymers, which had a maximum binding capacity of 763.36 mg/g in the optimum adsorption conditions. The prepared imprinted polymers had a fast adsorption rate and a much higher adsorption capacity than the corresponding non-imprinted polymers, with the difference in adsorption capacity up to 258.31 mg/g. The obtained polymer was evaluated by Lyz, bovine serum albumin (BSA), bovine hemoglobin (BHb), equine myoglobin (MB) and cytochrome c (Cyt c). The selectivity factor (β) for Lyz/BSA, Lyz/Mb, Lyz/BHb, and Lyz/Cyt c were 7.17, 2.12, 1.76 and 1.57, respectively, indicating the imprinted polymers had a good selectivity. Easy preparation of the imprinted polymers as well as high ability and selectivity to adsorb template proteins makes this polymer attractive and broadly applicable in biomacromolecular separation, biotechnology and sensors.
Keywords: Ionic liquids; Multiwall carbon nanotubes; Molecularly imprinted polymers; Protein recognition; Lysozyme;

The rapid, sensitive, and selective liquid chromatography–electrospray ionization-tandem mass spectrometry method (LC–ESI-MS/MS) for the simultaneous estimation and pharmacokinetic investigation of glimepiride and pioglitazone in human plasma has been developed and fully validated. Glimepiride and pioglitazone, compounds which exert synergistic effects on blood glucose control, were investigated in human plasma using deuterium-labeled analogs as internal standards (IS). Liquid–liquid extraction was carried out on 0.2 mL of human plasma using ethyl acetate, and chromatographic separation was performed on an Agilent Eclipse plus C18 column (4.6 mm × 100 mm, 3.5 μm) using a mobile phase consisting of methanol–water–formic acid (95:5:0.1, v/v/v, plus 5 mM ammonium acetate) at a flow rate of 0.8 mL/min. To quantify glimepiride, pioglitazone and their IS, multiple reaction monitoring (MRM) transitions of m/z 491.2→ 352.2, m/z 496.2 → 357.2, m/z 357.2 → 134.2 and m/z 361.2 → 138.2 were performed in positive mode. The total run time was 3.0 min and the elution time was about 2.4 min. The method exhibited good separation of analytes, without interference from endogenous substances. The linear calibration curves were 0.2–250 ng/mL for glimepiride and 0.2–1250 ng/mL for pioglitazone; the lower limit of quantification (LLOQ) was 0.2 ng/mL for both analytes. Intra- and inter-day reproducibility was less than 10% for glimepiride and less than 5% for pioglitazone, with relative errors ranging from −8.00% to 2.80% at the three concentrations of analytes used for quality control (QC). The matrix effect was negligible and recoveries were similar for each analyte and its IS. Glimepiride and pioglitazone were found to be stable under the assay conditions and the method was successfully applied to the evaluation of pharmacokinetic studies of glimepiride and pioglitazone, following oral doses of 2 mg glimepiride tablets and 15 mg pioglitazone tablets to 16 healthy volunteers.
Keywords: LC–MS/MS; Glimepiride; Pioglitazone; Pharmacokinetic;

A critical evaluation of a recent attempt to measure inositol hexakisphosphate (IP6) in mammalian plasma by mass spectroscopy leads to the conclusion that as yet there is no unambiguous evidence that plasma contains any IP6.
Keywords: Inositol; Inositol phosphates; Inositol hexakisphosphate; Phytate;

Phytate levels in biological fluids of mammals by Joan Perelló; Felix Grases (255-257).
Keywords: Phytate levels; Myo-inositol hexaphosphate; Biological fluids;