Journal of Chromatography B (v.889-890, #C)

Karel Macek (1928–2011) by Jaroslav Janák (1).

Purification, identification and profiling of serum amyloid A proteins from sera of advanced-stage cancer patients by Jing Li; Zhensheng Xie; Linan Shi; Zhiqiang Zhao; Junjie Hou; Xiulan Chen; Ziyou Cui; Peng Xue; Tanxi Cai; Peng Wu; Sutang Guo; Fuquan Yang (3-9).
► We established a workflow for the purification, identification and profiling of the targeted serum proteins found by SELDI-TOF-MS. ► Ten proteins were purified from sera of advanced-stage cancer patients and identified as SAA proteins. ► Our results suggest that maybe SAA should not be used alone as a biomarker for any specific cancer type.Surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) is a powerful tool for screening potential biomarkers of various pathological conditions. However, low resolution and mass accuracy of SELDI-TOF-MS remain a major obstacle for determination of biological identities of potential protein biomarkers. We report here a refined workflow that combines ZipTip desalting, acetonitrile precipitation, high-performance liquid chromatography (HPLC) separation and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) analysis for the profiling, purification and identification of the targeted serum proteins found by SELDI-TOF-MS. By using this workflow, we purified ten targeted proteins from the sera of patients with various types of advanced stage (stage III–IV) cancers. These proteins were identified as isoforms of the human serum amyloid protein A (SAA) family with or without truncations at their N-terminals. This was confirmed by Western blot analysis. Different SAA expression patterns were observed by MALDI-TOF-MS profiling. SAA has long been reported as a biomarker for various cancer types such as lung cancer, ovarian cancer, and breast cancer. However, in this study we found increased SAA expression in the sera of advanced-stage cancer patients with different cancer types. Our results suggest that maybe SAA should not be used alone as a biomarker for any specific cancer type.
Keywords: SELDI-TOF-MS; MALDI-TOF-MS; Purification; Targeted protein; SAA;

Development and validation of a sensitive LC–MS/MS assay for simultaneous quantitation of ranolazine and its three metabolites in human plasma by Yuan Wang; Xiaoyan Chen; Zuoming Sun; Yong Yang; Ying Zhang; Wanhui Liu; Dafang Zhong (10-16).
► A rapid and sensitive LC–MS/MS assay has been developed and validated. ► Ranolazine and three metabolites were simultaneously determined in human plasma. ► The LLOQ was 4 ng/mL for ranolazine, CVT-2514 and CVT-2738, and 8 ng/mL for CVT-4786. ► The method was applied to a pharmacokinetic study in humans.A rapid, sensitive and reliable LC–MS/MS method was developed and validated for the simultaneous determination of ranolazine and its three metabolites, CVT-2514, CVT-2738, and CVT-4786, in human plasma. The plasma samples were prepared by protein precipitation. Chromatographic separation was achieved on a Gemini C18 column (50 mm × 2.0 mm, 5 μm) using methanol: 5 mM ammonium acetate as the mobile phase with gradient elution. Mass detection was carried out by electrospray ionization in both positive and negative ion multiple reaction monitoring (MRM) modes. The calibration curves were linear over a concentration range of 4–2000 ng/mL for ranolazine, 4–1000 ng/mL for CVT-2514 and CVT-2738 and 8–1000 ng/mL for CVT-4786. The intra-day and inter-day accuracy and precision were within the acceptable limits of ±15% at all concentrations. The method was successfully applied for the simultaneous estimation of ranolazine and its three metabolites in human plasma from a clinical pharmacokinetics study.
Keywords: Liquid chromatography–tandem mass spectrometry; Ranolazine; Metabolites; Pharmacokinetics;

► The use of RAM column for extraction and quantification. ► No methods have been described for direct injection of human milk. ► Drug and active metabolite found in milk in high concentration.This work reports the use of a liquid chromatography ion trap tandem mass spectrometry (LC–IT-MS/MS) system for quantification in human milk samples of both carbamazepine (CBZ) and its active metabolite, carbamazepine 10,11-epoxide (CBZE). An octadecyl restricted-access media bovine serum albumin column (RAM-BSA C18) was used in single-column mode. Selectivity, extraction efficiency, accuracy and precision were achieved employing 100 μL of the sample, without preparation, with detection limits of 20.0 ng/mL for CBZ and 40.0 ng/mL for CBZE. The matrix effect was investigated for the compounds by post-column infusion (qualitative) and by on-line extraction (quantitative). It was observed suppression effect for CBZ and CBZE by post-column infusion, ion suppression of 0.80 for CBZ, and enhancement of 1.28 for CBZE by on-line extraction. The developed method was validated and applied to analyze breast milk samples from one nursing mother. CBZ and CBZE were quantified in the concentrations of 2.26 μg/mL and 1.54 μg/mL, respectively. To our knowledge, this is the first report on the simultaneous determination of CBZ and its active metabolite by direct injection of human milk serum.
Keywords: Human milk; Carbamazepine; Carbamazepine 10,11-epoxide; Liquid chromatography ion trap mass spectrometry; Restricted-access media; Single mode;

► So far no environmental transformation studies of antipsychotic drug Chlorpromazine (CPR). ► Investigation of aerobic/anaerobic biodegradability (OECD/ISO) and abiotic photolysis of CPR. ► LC–MS n and special software found transformation products with low intensity in difficult matrix. ► CPR not readily/inherently biodegradable; 3 aerobic and 1 anaerobic product; bacterial toxicity. ► CPR photolysis revealed 57 photoproducts; 3 main photoproducts were structurally elucidated.The search for environmental transformation products of organic pollutants (like drugs) is a difficult task and usually only few compounds are detected. This might be due to effective degradation but could also be a result of analytical deficits dealing with complex matrices. Especially transformation products of very low concentrations in sludge were difficult to identify so far. Additionally, the use of standard separation techniques might lead to the loss of isomeric compounds, which possess identical spectroscopic and spectrometric properties. To date no complete study investigating the environmental fate of any tricyclic antipsychotic drug has been reported. Therefore, this study investigated the popular neuroleptic drug chlorpromazine and its potential transformation by all main environmental pathways: aerobic and anaerobic biodegradation as well as abiotic photolytic degradation by sunlight. Analysis of test samples by high performance liquid chromatography coupled to multiple stage mass-spectrometry (HPLC–MS n ) allowed the detection of numerous compounds. Further, the use of a special software allowed distinguishing between transformation products of small intensities and background “noise” caused by sludge or matrix. Three aerobic tests of different bacterial density (the Closed Bottle test, OECD 301D; the Manometric Respiratory test, OECD 301F; the modified ZahnWellens test, 302B; one anaerobic test (a modified anaerobic degradation test according to ISO 11734) as well as a photodegradation test were performed in the present study. According to the individual test guidelines, chlorpromazine had to be classified as not biodegradable in all of the biodegradation tests. However, a special chromatographic column and gradient along with mass spectrometric fragmentation experiments of higher order uncovered the presence of a total of 61 abiotic and biotic transformation products which where formed during the course of the tests. The structures of three aerobic and one anaerobic biotransformation products were elucidated by HPLC-UV-Flourescence–MS n . Photodegradation showed almost complete elimination of chlorpromazine after 4 h of irradiation with a xenon arc lamp. 57 photoproducts were found and for 28 of them LC–MS n fragmentation experiments (n  = 4) were performed. The molecular structures of the three main photolysis products were elucidated. The identified transformation products are expected to be found in the aquatic environment, yet nothing is known about their ecotoxicological properties. As some of the performed tests showed toxic effects of chlorpromazine or its transformation products on bacteria, further risk assessment upon this drug and its fate is strongly recommended.
Keywords: Biodegradation; Photodegradation; Photolysis; Anaerobic; Toxicity; Risk;

Determination of sinomenine sustained-release capsules in healthy Chinese volunteers by liquid chromatography–tandem mass spectrometry by Meng-Xiang Su; Min Song; De-Zhu Sun; Hua Zhao; Xiao Gu; Ling Zhu; Xiao-Le Zhan; Zhong-Nan Xu; Ai-Dong Wen; Tai-Jun Hang (39-43).
► LC–MS/MS quantification of sinomenon in human plasma was developed. ► This method achieved a LLOQ as low as 0.5 ng/mL with a simple pretreatment procedure. ► Clinical pharmacokinetic of sinomenine sustained-release capsules was characterized. ► The accumulation of sinomenine in body was observed after repeated dosing.A sensitive and selective liquid chromatographic tandem mass spectrometric method was developed and validated for the determination of sinomenine in human plasma. Plasma samples were precipitated using methanol with metronidazole as internal standard. Separation was carried out on an Inertsil ODS-3 column using a mixture of 0.2% ammonium acetate solution (A) and methanol (B) as the mobile phase with linear gradient elution as follows: 0 min (50%B) → 1.5 min (80%B) → 4.5 min (80%B) → 4.6 min (50%B) → 6.0 min (50%B). All mass data were obtained in the positive ion mode, and the fragmentation transitions for the selective multiple reaction monitoring were m/z 330 → 181 and 172 → 128 for sinomenine and metronidazole, respectively. The method was fully validated to be accurate and precise with a linear range of 0.5–500 ng/mL and applied to a single- and multiple-dose pharmacokinetics study of sustained-release capsules of sinomenine hydrochloride in 20 healthy Chinese volunteers. After oral administration of a single 60-mg dose, the T max, C max, AUC0–96 and t 1/2 were 7.9 ± 2.0 h, 123 ± 22 ng/mL, 3032 ± 682 ng h/mL and 13.4 ± 1.6 h, respectively. After oral administration of the 60 mg capsules twice-daily for 7 consecutive days, these parameters were 4.4 ± 3.6 h, 279 ± 69 ng/mL, 7333 ± 2096 ng h/mL and 15.1 ± 1.3 h, respectively. The AUC and C max values after multiple-dose treatment were significantly higher than those after a single-dose treatment (P  < 0.01), with an accumulation factor of 2.49 ± 0.77.
Keywords: Sinomenine; Sustained-release capsules; Pharmacokinetics; LC/MS/MS;

► Theophylline and its metabolites were validated simultaneously in rat plasma. ► Protein precipitation was applied in an assay using LC–MS/MS. ► The combination of LC separation and the mass parameters shorten HPLC run times. ► The assay demonstrated a high degree of suitable precision and accuracy. ► It makes the method practical for cost-effective, high-throughput sample analyses.A rapid, specific, and reliable LC–MS/MS-based bioanalytical method was developed and validated in rat plasma for the simultaneous quantitation of theophylline and its four metabolites: 1,3-dimethyluric acid (1,3-DMU), 3-methylxanthine (3-MX), 1-methylxanthine (1-MX), and 1-methyluric acid (1-MU). Chromatographic separation of these analytes was achieved on a Gemini C18 column (50 mm × 4.60 mm, 5 μm) using reversed phase chromatography. The analytes were monitored by electrospray ionization in negative ion multiple reaction monitoring mode. Modification of collision energies was performed in parallel with chromatographic separation to further eliminate interference peaks. The method was validated from 0.05 to 30 μg/mL for 1-MX, 1,3-DMU, 1-MU, and theophylline and from 0.1 to 30 μg/mL for 3-MX using 0.2 mL of plasma sample. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels exhibited relative standard deviations (RSD) of less than 13% and with relative error (RE) values of −8.8% to 9.7%. The method was successfully applied for the quantitation of theophylline and its metabolite in rat plasma samples.
Keywords: Theophylline; 1-Methyl xanthine; 3-Methyl xanthine; 1,3-Dimethyl uric acid; 1-Methyl uric acid; LC–MS/MS;

Purification and characterization of catalase from sprouted black gram (Vigna mungo) seeds by Sai Srikar Kandukuri; Ayesha Noor; S. Shiva Ranjini; M.A. Vijayalakshmi (50-54).
► Catalase content was estimated in black gram sprouts, seedlings and leafs. ► Day 4 sprouts showed the maximum catalase activity. ► A single step purification of catalase was achieved using Seph 4B-IDA-Zn(II). ► Purity of catalase was confirmed qualitatively by SDS–PAGE and Zymogram analysis. ► Kinetic and stability studies were performed with purified catalase.Black gram (Vigna mungo) is a legume which belongs to Fabaceae family. It is a rich source of protein. It has been known to have interesting small molecule antioxidant activity. However, its enzymatic antioxidant properties have not been explored much. In the present work we studied catalase, a principal antioxidant enzyme from black gram seeds. Day four sprouted black gram seeds were found to have a significant catalase content approximately of 15,240 U/g seeds. IMAC (Seph 4B-IDA-Zn(II)) was used for purifying this catalase, a purification fold of 106 and a high specific activity of 25,704 U/mg was obtained. The K m and V max of the purified catalase were found to be 16.2 mM and 2.5 μmol/min. The effect of inhibitors like Sodium azide (NaN3) and EDTA and different metal ions on catalase activity were studied. NaN3, Fe3+and Cu2+ were found to have profound inhibitory effects on the enzyme activity. Other metal ions like Ni2+, Ca2+, Mg2+ and Mn2+ had both enhancing and inhibitory effects. The enzyme showed optimal activity at a temperature of 40 °C and pH 7.0. It was stable over a broad range of pH 6.0–10.0 and had a half life of 7 h 30 min at 50 °C.
Keywords: Enzyme purification; Catalase; IMAC;

► A porous poly(N-isopropylacrylamide-co-ethyleneglycol dimethacrylate) [poly(NIPAAm-co-EDMA)] monolithic column was prepared by in situ free-radical polymerization. ► The prepared poly(NIPAAm-co-EDMA) monolithic column showed excellent permeability and high selectivity, which was suitable for SPE pre-column. ► The prepared poly(NIPAAm-co-EDMA) monolithic column was used as SPE sorbent to simultaneously clean up of protein and enrich of nifedipine, nitrendipine and nisoldipine in plasma and urine. ► The sample pretreatment step was embedded into the LC chromatographic system and manual intervention was minimized. ► The established method was also applied in clinical plasma studies.A porous poly(N-isopropylacrylamide-co-ethyleneglycol dimethacrylate) [poly(NIPAAm-co-EDMA)] monolithic column was prepared by in situ free-radical polymerization. The morphology of monolithic column and pressure drop across the columns were characterized. The results showed excellent permeability and high selectivity. Nifedipine, nitrendipine and nisoldipine were simultaneously selected to validate the extraction efficiency of the prepared monolith both in plasma and urine. The extracted nifedipine, nitrendipine and nisoldipine from plasma and urine samples have been on-line tested quantitatively by using the prepared monolith connected with RP-C18 column. The total analytical run time was 38 min. For all analytes, linear calibration curves were obtained over a range of 2–500 ng/mL with coefficient of correlation > 0.997. Precision for inter- and intra-day assay showed acceptable results for quantitative assay with relative standard deviation (RSD) less than 12%. The accuracy and recovery was found to be in the range of 89–109% and 88–106%. The results indicated that the prepared monolith was feasible to be used as an on-line SPE sorbent material and the method was especially appropriate for multi-analytes monitoring in plasma and urine samples. Finally, the proposed method was successfully applied to simultaneously screen nifedipine, nitrendipine and nisoldipine in plasma.
Keywords: P(NIPAAm-EDMA) monolithic column; On-line solid-phase extraction; Three dipine series; Human plasma; Human urine;

► A single step purification of a novel alkaline protease from salt tolerant alkaliphilic actinomycetes, Nocardiopsis alba strain OK-5 has been described. ► Since the halophilic enzymes are difficult to purify, development of a simple purification procedure with good yield would be quite useful for purification of other extremozymes. ► A detailed characterization of protease from actinomycetes has not been conducted and to the best of our knowledge there are no reports on the analysis of the thermodynamic and kinetic parameters of protease from salt tolerant alkaliphilic actinomycetes. Therefore, biochemical, thermodynamic and kinetic properties of extracellular protease would add significantly to the biocatalysis from this group of microbes. ► The enzyme had high activity and significant stability at higher salt, temperature, pH and a range of metal ions. The enzyme also displayed extreme resistance against urea denaturation, oxidizing and reducing agents and surfactants, a finding which is rather unique and restricted to only few proteins. ► The results would be significant on macromolecular stability and to explore biotechnological potential of enzymes from less attended haloalkaliphilic actinomycetes.An alkaline protease from salt tolerant alkaliphilic actinomycetes, Nocardiopsis alba strain OK-5 was purified to homogeneity by 27 and 13 fold with a yield of 35 and 13% using two-steps and one-step method, respectively. The purification methods involved hydrophobic interaction on phenyl sapharose matrix. The apparent molecular mass was 20 kDa. The temperature optimum shifted from 70 to 80 °C in 4 M NaCl and 30% Na-glutamate, with significant stability at 60–80 °C in Na-glutamate. Deactivation rate constant (K d) increased and half life (t 1/2) decreased with the increasing temperatures from 37 to 80 °C. The order of stability was: 30% Na-glutamate > 4 M NaCl > 2 M NaCl > 0 M NaCl. The enzyme was stable even at 80 °C in 30% Na-glutamate with K d 4.11 and t 1/2 168.64 min. The activation energies (E), enthalpy (ΔH*) and entropy (ΔS*) for protease deactivation in with Na-glutamate were 31.97 kJ/mole, 29.23 kJ/mole and −211.83 J/mole, respectively. The change in free energy (ΔG*) for protease deactivation at 60 °C in 30% Na-glutamate was 101.70 kJ/mole. Protease had the highest activity and stability at pH 10–11. While the enzyme was highly resistant against chemical denaturation, it had varied responses to metal ions. Complete inhibition by PMSF confirmed serine nature of the protease. Na-glutamate, H2O2, β-mercaptoethanol and different surfactants enhanced the activity.
Keywords: Salt-tolerant alkaliphilic actinomycetes; Thermostability; Alkaline protease; Enzyme purification; Enzyme kinetics; Thermodynamics; Denaturation constant;

► Presentation of a human biomonitoring method for epichlorohydrin and 2-chloroprene. ► Simultaneous determination of six mercapturic acids in human urine. ► Validation of the method proved good sensitivity and accuracy. ► The method enables for the first time human biomonitoring studies on 2-chloroprene.We developed and validated an analytical method for the simultaneous determination of several chlorine and non-chlorine containing mercapturic acids in urine as specific metabolites of the hazardous chemicals 2-chloroprene and epichlorohydrin. The method involves an online column switching arrangement for online solid phase extraction of the analytes with subsequent analytical separation and detection using LC–MS/MS. The developed method enables for the first time the determination of Cl-MA-I (4-chloro-3-oxobutyl mercapturic acid), Cl-MA-II (4-chloro-3-hydroxybutyl mercapturic acid), Cl-MA-III (3-chloro-2-hydroxy-3-butenyl mercapturic acid) and HOBMA (4-hydroxy-3-oxobutyl mercapturic acid) as potential biomarkers of 2-chloroprene in urine. Additionally, CHPMA (3-chloro-2-hydroxypropyl mercapturic acid) as a specific metabolite of epichlorohydrin in urine and DHBMA (3,4-dihydroxybutyl mercapturic acid) can be determined. The analytical method proved to be both sensitive and reliable with detection limits ranging from 1.4 μg/L (for Cl-MA-III) to 4.2 μg/L (for HOBMA). Intra- and interday imprecision was determined to range from 4.7 to 11.8%. Due to the good accuracy and precision and the low limits of detection the developed method is well suited for application in biomonitoring studies in order to determine occupational exposure to 2-chloroprene and epichlorohydrin.
Keywords: Biomonitoring; Mercapturic acid; 2-Chloroprene; Epichlorohydrin; Alkylating agents; Occupational medicine;

Liquid chromatography and tandem mass spectrometry method for the quantitative determination of saxagliptin and its major pharmacologically active 5-monohydroxy metabolite in human plasma: Method validation and overcoming specific and non-specific binding at low concentrations by Xiaohui (Sophia) Xu; Roger Demers; Huidong Gu; Lisa J. Christopher; Hong Su; Laura Cojocaru; David W. Boulton; Mark Kirby; Bruce Stouffer; William G. Humphreys; Mark E. Arnold (77-86).
► A liquid chromatography and tandem mass spectrometry (LC–MS/MS) method was developed and validated to simultaneously determine the concentrations of saxagliptin and its major active metabolite, 5-hydroxy saxagliptin. ► The sample pre-treatment process was carefully controlled to disrupt DPP4-specific binding and non-specific binding observed at lower concentrations. ► Under these chromatographic conditions, the isomers of saxagliptin and 5-hydroxy saxagliptin were chromatographically separated from saxagliptin and 5-hydroxy saxagliptin. ► The assay has been used to support multiple clinical studies and regulatory approvals.A liquid chromatography and tandem mass spectrometry (LC–MS/MS) method was developed and validated to simultaneously determine the concentrations of saxagliptin (Onglyza™, BMS-477118) and its major active metabolite, 5-hydroxy saxagliptin to support pharmacokinetic analyses in clinical studies. The dynamic range of the assay was 0.1–50 ng/mL for saxagliptin and 0.2–100 ng/mL for 5-hydroxy saxagliptin. Protein precipitation (PPT) with acetonitrile was used to extract the analytes from plasma matrix before injecting on an Atlantis® dC18 column (50 mm × 2.1 mm, 5 μm) for LC–MS/MS analysis. The sample pre-treatment process was carefully controlled to disrupt DPP4-specific binding and non-specific binding observed at lower concentrations. The recoveries for both analytes were >90%. The assay was selective, rugged and reproducible; storage stability of at least 401 days at −20 °C was demonstrated. Under these chromatographic conditions, the isomers of saxagliptin and 5-hydroxy saxagliptin were chromatographically separated from saxagliptin and 5-hydroxy saxagliptin. The assay has been used to support multiple clinical studies and regulatory approvals.
Keywords: Antidiabetic agent; Onglyza™; Saxagliptin; BMS-477118; 5-Hydroxy saxagliptin metabolite; Dipeptide-peptidase; DPP4; LC–MS/MS; Liquid chromatography–tandem mass spectrometry; Diastereomer; Protein precipitation; Human plasma; Cross-validation;

Evaluation of enantioselective binding of propanocaine to human serum albumin by ultrafiltration and electrokinetic chromatography under intermediate precision conditions by María Amparo Martínez-Gómez; Laura Escuder-Gilabert; Rosa María Villanueva-Camañas; Salvador Sagrado; María José Medina-Hernández (87-94).
► First evidence on enantioselective binding of propanocaine to human serum albumin. ► Approximate in vivo conditions allows in vitro data unapproachable from in vivo assays. ► Optimised experimental design reduces methodological errors. ► Direct equations enable statistical advantages and robust results. ► Data from two days and two processed fractions provide reliable uncertainty.Stereoselectivity in protein binding can have a significant effect on the pharmacokinetic and pharmacodynamic properties of chiral drugs. In this paper, the enantioselective binding of propanocaine (PRO) enantiomers to human serum albumin (HSA), the most relevant plasmatic protein in view of stereoselectivity, has been evaluated by incubation and ultrafiltration of racemic PRO–HSA mixtures and chiral analysis of the bound and unbound fractions by electrokinetic chromatography using HSA as chiral selector. Experimental conditions for the separation of PRO enantiomers using HSA as chiral selector and electrokinetic chromatography have been optimised. Affinity constants and protein binding in percentage (PB) were obtained for both enantiomers of PRO, as well as the enantioselectivity (ES) to HSA. Data were obtained in two independent working sessions (days). The influence of the session and fraction processed factors were examined. A univariate direct-estimation approach was used facilitating outliers’ identification and statistical comparison. Non-linear fitting of data was used to verify the stoichiometry and affinity estimations obtained by the direct approach. Robust statistics were applied to obtain reliable estimations of uncertainty, accounting for the factors (day and processed fraction), thus representing intermediate precision conditions. Mimicking in vivo experimental conditions, information unapproachable by in vivo experiments was obtained for PRO enantiomers interacting with HSA. For the first (E1) and the second (E2) eluted PRO enantiomers the results were: 1:1 stoichiometry, medium affinity constants, log  K E1  = 3.20 ± 0.16 and log K E2  = 3.40 ± 0.14, medium protein binding percentage, PB = 48.7 and 60.1% for E1 and E2, respectively, and moderate but significant enantioselectivity, ES =  K E2/K E1  = 1.5 ± 0.3.
Keywords: Propanocaine; Electrokinetic chromatography; Enantioselective binding; Human serum albumin; Intermediate precision;

Molecular imprinting based composite cryogel membranes for purification of anti-hepatitis B surface antibody by fast protein liquid chromatography by Sevgi Asliyuce; Lokman Uzun; Abbas Yousefi Rad; Serhat Unal; Ridvan Say; Adil Denizli (95-102).
Display Omitted► Molecular imprinted cryogel membranes (MI-CMs) for anti-HBs purification by FPLC. ► The MI-CMs: a novel candidate for specific anti-HBs purification from human plasma. ► Competitive adsorption of anti-HBs, total anti-HAV and total IgE were carried out. ► The MI-CMs can be used many times without any significant decrease in the capacity. ► The chromatographic purification performances of the MI-CMs were evaluated.In the present study, we have focused our attention to prepare molecular imprinted composite cryogel membranes for purification of hepatitis B surface antibody (anti-HBs) by fast protein liquid chromatography. Before the preparation of the molecular imprinted composite cryogel membranes (MI-CMs) by free radical polymerization at sub-zero temperature, we have synthesized and characterized the anti-HBs imprinted particles. Then, the cryogel membranes (CMs) were characterized by swelling test, scanning electron microscopy and Fourier transform infrared spectroscopy. Prior to chromatographic purification studies, the effective parameters on the anti-HBs adsorption process were evaluated by investigating the dependency of the adsorption capacity on flow-rate, anti-HBs concentration, contact time and ionic strength. The maximum anti-HBs adsorption capacity was calculated as 701.4 mIU/g CM. The selectivity of the MI-CMs was shown by competitive adsorption of anti-HBs, total anti-hepatitis A antibody (anti-HAV) and total immunoglobulin E (IgE) adsorption studies. The MI-CMs have relative selectivity coefficients as 5.45 for anti-HBs/total anti-HAV and 9.05 for anti-HBs/total IgE, respectively. The phosphate buffer solution (pH 7.4) containing 1.0 M NaCl was used for elution, almost completely, of adsorbed anti-HBs molecules. The MI-CMs could be used many times without any significant decrease in the adsorption capacity. The chromatographic purification performances of the MI-CMs were also investigated. The chromatographic parameters such as capacity and separation factors, the theoretical plate number and resolution of the MI-CMs were calculated as 5.48, 6.02, 1153.9, and 1.72 for anti-HBs molecules, respectively. As a conclusion, we can say that the MI-CMs could be used for specific purification of anti-HBs from anti-HBs positive human plasma.
Keywords: Molecular imprinted polymers; Anti-hepatitis B surface antibody; Composite cryogel membrane; Fast protein liquid chromatography; Affinity purification;

► The first LC–MS/MS method for quantitation of Y101 was established. ► The method provided good sensitivity (1 ng/mL for Y101). ► A simple one-step protein precipitation was chosen to pretreat samples. ► The method has been successfully applied to a pharmacokinetic study for the first time in rats.A simple, accurate and sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed and validated for quantitation of bentysrepinine (Y101) in rat plasma. After the addition of diphenhydramine (internal standard, IS), plasma samples were pretreated by protein precipitation. Chromatographic separation was carried out on an Atlantis® analytical column (4.6 mm × 100 mm, 5 μm, Waters) with methanol: 20 mM ammonium formate consisting of 1.0% formic acid (65:35, v/v) as the mobile phase at an isocratic flow rate of 0.4 mL/min for 7.5 min. The multiple reaction monitoring (MRM) transitions were performed at m/z 490.2 → 339.5 for Y101and m/z 256.0 → 167.0 for IS on a SCIEX API 4000 mass spectrometer in the positive ion mode with electrospray ionization (ESI) source. Good linearity was achieved over the concentration range of 1–2500 ng/mL. The intra- and inter-day precisions were less than 8.3%, and the accuracy ranged from −4.0% to 2.8%. Y101 was stable during the analysis and the storage period. The pharmacokinetic profiles of Y101 at three dose levels were successfully studied for the first time in rats by this method. After single intra-gastric administration of Y101 at the doses of 25, 50 and 100 mg/kg, C max and AUC0–t were proportional to the doses given.
Keywords: Bentysrepinine (Y101); LC–MS/MS; Pharmacokinetics;

A sensitive HPLC-based method to quantify adenine nucleotides in primary astrocyte cell cultures by Dhaval P. Bhatt; Xuesong Chen; Jonathan D. Geiger; Thad A. Rosenberger (110-115).
► We describe a sensitive HPLC method to quantify adenine nucleotides in cell culture. ► Optimized ion-pair reagent provides reproducible separation of adenine nucleotides. ► Optimized derivatization to maximum yield and limit hydrolysis. ► Surpasses conventional methods used to quantify AMP and ADP in cell cultures. ► Used to accurately measure adenylate energy charge in an astrocyte cell culture.In mono-layered primary cell cultures baseline AMP and ADP levels are found nominally in the mid to low picomolar range and are thus difficult to measure with conventional HPLC methods that often require the pooling of samples or require indirect detection methods using radiotracers or enzyme coupled assays. To address this issue, we developed a highly sensitive and selective ion-pairing HPLC method with fluorescence detection to quantify adenine nucleotides and the adenylate energy charge in primary astrocyte cell cultures. To accomplish this, we optimized the fluorescence derivatization conditions and the HPLC parameters to achieve baseline separation and quantification of all adenine nucleotides. Nucleotides were converted to their respective 1, N6-etheno derivatives by incubating with chloroacetaldehyde at pH 4.5 and 60 °C for 60 min. Under these conditions, the loss of the adenine nucleotides due to hydrolysis was minimized with a derivatization yield of 94.1% for 1, N6-ethenoadenosine. The optimal concentration of tetrabutylammonium phosphate, the ion-pairing reagent, required to achieve a reproducible separation of the adenine nucleotides was found to be 0.8 mM. Calibration curves of nucleotide standards were linear within the range of 0.16–10.4 pmol for adenosine, 0.16–20.6 pmol for AMP, 0.15–19.2 pmol for ADP, and 0.15–19.5 pmol for ATP. The limits of detection and quantification for all adenine nucleotides were approximately 0.08 and 0.16 pmol, respectively. The intra- and inter-day variability for this method was less than 5.1 and 3.4%, respectively. This method was successfully used to measure all adenine nucleotides and an adenylate energy charge of 0.92 ± 0.02 in primary astrocyte cell cultures.
Keywords: Adenylate energy charge; AMP; Chloroacetaldehyde; Etheno-adenine nucleotides; Fluorescence; Primary astrocyte cultures;

Determination of biomarkers of tobacco smoke exposure in oral fluid using solid-phase extraction and gas chromatography–tandem mass spectrometry by B.M. da Fonseca; I.E.D. Moreno; A.R. Magalhães; M. Barroso; J.A. Queiroz; S. Ravara; J. Calheiros; E. Gallardo (116-122).
► First GC–MS/MS method for the determination of nicotine and metabolites in oral fluid samples. ► Excellent quantitation limits using only 0.2 mL of sample. ► Absolute recoveries higher than 85% for all compounds. ► Low limits allow monitoring tobacco smoke exposure in non-smoking individuals.A new, simple and sensitive method was described for the simultaneous determination of nicotine, cotinine and trans-3′-hydroxycotinine in oral fluid samples using solid-phase extraction and gas chromatography/tandem mass spectrometry (GC–MS/MS). This technique was developed using only 0.2 mL of sample, and deuterated analogues were used as internal standards. The method was found to be linear between 0.5 and 1000 ng/mL, with determination coefficients higher than 0.996 for all analytes. Intra- and interday precision and accuracy were in conformity with the criteria normally accepted in bioanalytical method validation. All analytes were stable in the samples for at least 24 h at room temperature, for at least 72 h at 25 °C in processed samples and for at least three freeze/thaw cycles. Absolute recoveries ranged from 89 to 92% for all analytes. GC–MS/MS has demonstrated to be a powerful tool for the simultaneous quantitation of the analytes, providing adequate selectivity and sensitivity. In addition, its performance characteristics allow its routine use in the analysis of biomarkers of tobacco smoke exposure, extending the window of analyte detection in nicotine cessation programs, using a sample amount as low as 0.2 mL of human oral fluid.
Keywords: Biomarkers of tobacco smoke exposure; GC–MS/MS; Oral fluid;

Determination of antimalarial compound, ARB-89 (7β-hydroxy-artemisinin carbamate) in rat serum by UPLC/MS/MS and its application in pharmacokinetics by Deepthi Pabbisetty; Anuradha Illendula; K.M. Muraleedharan; Amar G. Chittiboyina; John S. Williamson; Mitchell A. Avery; Bonnie A. Avery (123-129).
► Highly active 7β-hydroxy derivative of artemisinin is selected as a lead compound. ► High through put UPLC/MS/MS method is developed and validated for quantitation of the derivative in rat serum. ► This method can be used to explore the therapeutic potential of this derivative in the future. ► Oral bioavailability was improved than some of the existing artemisinin analogs.Among all the antimalarial agents, artemisinin and its semi synthetic family of analogs are the most potent antimalarials available for the treatment of Plasmodium falciparum infections. But these analogs have a few issues such as shorter half-lives and low oral bioavailability values. In order to overcome these inherent problems, novel artemisinin analogs were synthesized from 7β-hydroxy artemisinin by the Department of Medicinal Chemistry, University of Mississippi using a new synthesis mechanism. Out of all the 7β-hydroxy artemisinin analogs synthesized, 7β-hydroxy artemisinin carbamate (ARB-89) was chosen as a lead compound because of its high in vitro and in vivo activity. In this manuscript, a sensitive and rapid ultra-performance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) method was developed and validated for the quantification of ARB-89 in rat serum. The analysis was carried out on an Acquity™ UPLC BEH C18 column (1.7 μm, 2.1 mm × 50 mm) with a flow rate of 0.3 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer in positive electrospray ionization (ESI) mode. The selected mass-to-charge (m/z) ratio transitions used in the multiple reaction monitoring (MRM) for ARB-89 and artemisinin (internal standard) were m/z 778.4 > 253.4 and m/z 283.4 > 151.1 respectively. The calibration curve was linear from 1.00 ng/mL to 10.0 μg/mL (r 2  = 0.999). A simple protein precipitation method was used for extraction. Moreover, the inter-day and intra-day precision values were found to be less than 15%. The recoveries of the method ranged from 94.0% to 96.7% at three concentrations. ARB-89 in rat serum was found to be stable at room temperature for 12 h. This method was successfully used to quantitate the novel antimalarial compound ARB-89 after intravenous and oral administration to rats.
Keywords: Artemisinin analogs; UPLC/MS/MS; Pharmacokinetics; Antimalarial; Rat serum; Bioavailability;

► We develop and validate an LC–MS/MS method for glycopyrrolate in horse urine. ► We construct tolerance intervals for glycopyrrolate in urine and plasma for threshold limits. ► Urine to plasma concentration ratios of glycopyrrolate can enhance the regulatory control of glycopyrrolate. ► Renal clearance of glycopyrrolate in the horse can be estimated without volumetric urine collections.We describe a validated, rapid, sensitive, and specific UHPLC–MS/MS method to detect and quantify glycopyrrolate in 0.5 mL of horse urine. Further, we investigated the elimination of glycopyrrolate in urine after both intravenous and oral administration of clinically relevant doses to Thoroughbred horses. Quantification was performed by weighted, linear regression analysis using a deuterated analogue of glycopyrrolate as internal standard (IS). The method was characterized by a linear range of 5–2500 pg/mL, a lower limit of quantification of 5 pg/mL and a limit of detection of 1 pg/mL. The intra and inter-batch imprecisions were <10% RSD and accuracy of the method ranged between 94 and 104%. Glycopyrrolate remained detectable in urine samples collected through 168 h after intravenous administration and through 24 h after oral administration. Analytical method validation requirements for linearity, specificity, precision, accuracy, stability, dilution integrity, matrix effect, and ruggedness have been fulfilled. The urine method described in this report is simple and efficient and is the first reported method with sufficient sensitivity, accuracy, and precision to regulate the use of glycopyrrolate in urine samples collected more than one day after dosing of horses. Urine to plasma glycopyrrolate concentration ratios were calculated and were approximately 100:1 in samples collected from 24 h through the end of sample collection.
Keywords: Glycopyrrolate; Horse; LC/MS/MS; Threshold; Urine;

► Different C18 particle sizes have been compared to analyse aflatoxins. ► Solid core particles allowed the reduction of the analysis time. ► Reductions of analysis costs were achieved by reducing time and solvents use. ► The suitability to analyse aflatoxin from different matrixes was demonstrated.In this work we compared the performance of chromatography columns with particles of 5 and 3 μm with the new 2.7 μm solid core particles for the analysis of aflatoxins B1, G1, B2, and G2 using trifluoroacetic acid pre-column derivatization. Three different columns have been used and chromatographic parameters as retention time, resolution, limit of detection (LOD), limit of quantification (LOQ) were obtained from all of them and compared. The results show that comparing with the traditional columns, shorter columns (100 mm × 4.6 mm) with the new solid core particles are suitable for the analysis of these mycotoxins and allowed the reduction of the analysis time by 45.5% and 33.3% with respect to columns with particle size 5 μm (150 mm × 4.6 mm) and 3 μm (150 mm × 4.6 mm) respectively, without any detrimental effect on performance. This leads to the reduction of the analysis costs by saving on organic solvents and increasing the total number of analyses per day. The capability of these columns for analyzing samples, in different culture media, was assessed by analyzing different samples from: yeasts extract sucrose medium, corn meal agar medium and fresh hazelnut media.
Keywords: Aflatoxin; Particle size; HPLC; Solid core;

Liquid chromatography–tandem mass spectrometric assay for the mutated BRAF inhibitor vemurafenib in human and mouse plasma by Rolf W. Sparidans; Selvi Durmus; Alfred H. Schinkel; Jan H.M. Schellens; Jos H. Beijnen (144-147).
► The first bioanalytical assay for vemurafenib has been reported. ► The assay is simple and fast, precise and accurate. ► Vemurafenib is stable under all conditions relevant for the assay. ► The assay was successfully validated in the 0.1–100 μg/ml calibration range.A bioanalytical assay for the mutated BRAF inhibitor vemurafenib was developed and validated. For the quantitative assay, human plasma samples were pre-treated using protein precipitation with water-acetonitrile (1/3, v/v) containing sorafenib as internal standard. The extract was directly injected into the chromatographic system. This system consisted of a sub-2 μm particle, trifunctional bonded octadecyl silica column with isocratic elution using 0.01% (v/v) of formic acid in a mixture of water and methanol. The eluate was transferred into the electrospray interface with positive ionization and the analyte was detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The assay was validated in a 0.1–100 μg/ml calibration range. Within day precisions were 1.6–3.2%, between day precisions 2.7% and 8.2% and accuracies were between 99% and 106% for the whole calibration range. The drug was stable under all relevant conditions. Finally, the assay was successfully used to assess drug levels in a pharmacokinetic mouse study.
Keywords: Vemurafenib; Mutated BRAF inhibitor; LC–MS/MS; Plasma;