Journal of Chromatography B (v.1020, #C)

Solanesol extraction from tobacco leaves by Flash chromatography based on molecularly imprinted polymers by Xiaoqin Ma; Zihui Meng; Lili Qiu; Jing Chen; Yushu Guo; Da Yi; Tiantian Ji; Hua Jia; Min Xue (1-5).
A novel solanesol extraction method based on molecularly imprinted polymer (MIP) as the Flash chromatography stationary phase was established and evaluated. Spherical MIP particles in a size range of 250–350 μm (d (0.5) = 320 μm) for solanesol were synthesized by suspension polymerization, with imprinting factor of 3.9. The MIP particles (5.5 g) were packed in common Teflon column as the stationary phase while the sample solution and elution solvent were confirmed as methanol and methanol/acetic acid solution (80/20, v/v), loading at 4 ml/min and eluting 8 ml/min, respectively. Under the optimal chromatographic conditions, the adsorption capacity of the MIP-Flash column was determined as 107.3 μmol/g, and in each process, 370.8 mg purified solanesol (98.4%) could be obtained from the extract (20 mM, 40 ml) of tobacco leaves (14.7 g), and the yield of solanesol was 2.5% of the dry weight of tobacco leaves. The results reported here confirm the feasibility to extract highly purified active ingredients directly from natural products on a large scale by MIP-Flash chromatography.
Keywords: Solanesol; Tobacco leaves; Flash chromatography; Molecularly imprinted polymers;

p-Coumaric acid, a novel and effective biomarker for quantifying hypoxic stress by HILIC-ESI-MS by Yuliya E. Silina; Claudia Fink-Straube; Rainer G. Hanselmann; Henrike Peuschel; Dietrich A. Volmer (6-13).
Display OmittedIn this study, we report p-coumaric acid as novel and effective response marker for indirectly measuring the levels of hypoxia in normal primary bronchial epithelial cells. We developed a simple and rapid technique based on hydrophilic interaction chromatography-electrospray ionization-mass spectrometry (HILIC-ESI–MS). During 168 h of hypoxia without induction of reactive oxygen species (ROS), an almost linear increase of p-coumaric acid levels was observed. We interpret the increasing p-coumaric acid concentrations during hypoxia as a result of cell damage, triggered by reduced co-enzyme Q10 levels, because the oxidative cascade was not able to supply sufficient energy. The HILIC-ESI–MS assay within p-coumaric acid exhibited a linear dynamic range from 60 to 610 ng/μL with correlation coefficient of 0.9998. The precision of the assay was ≤15% RSD and method accuracies between 97 and 108%.
Keywords: Cellular hypoxia; ROS; p-Coumaric acid; Co-enzyme Q10; HILIC-ESI–MS;

Determination of “new psychoactive substances” in postmortem matrices using microwave derivatization and gas chromatography–mass spectrometry by Cláudia Margalho; Alice Castanheira; Francisco Corte Real; Eugenia Gallardo; Manuel López-Rivadulla (14-23).
Despite worldwide efforts aiming to ban the marketing and subsequent abuse of psychoactive substances such as synthetic cathinones and phenethylamines, there has been an alarming growth of both in recent years. Different compounds similar to those already existing are continuously appearing in the market in order to circumvent the legislation.An analytical methodology has been validated for qualitative and quantitative determinations of d-cathine (d-norpseudoehedrine), ephedrine, methcathinone, 1-(4-methoxyphenyl)-propan-2-amine (PMA), mephedrone, methedrone, 2,5-dimethoxy-4-methylamphetamine (DOM), 4-bromo-2,5-dimethoxyamphetamine (DOB), 2,5-dimethoxyphenethylamine (2C-H), 4-bromo-2,5-dimethoxyphenethylamine (2C-B), 4-iodo-2,5-dimethoxyphenethylamine (2C-I), 2-[2,5-dimethoxy-4-(ethylthio)phenyl]ethanamine (2C-T-2), 2,5-dimethoxy-4-isopropylthiophenethylamine (2C-T-4) and 2-[2,5-dimethoxy-4-(propylthio)phenyl]ethanamine (2C-T-7), in low volumes of vitreous humor (100 μL), pericardial fluid (250 μL) and whole blood (250 μL), using deutered amphetamine, ephedrine and mephedrone as internal standards. The validation parameters included selectivity, linearity and limits of detection and quantification, intra- and interday precision and trueness, recovery and stability. The method included mixed-mode solid phase extraction, followed by microwave fast derivatization and analysis by gas chromatography-mass spectrometry operated in selected ion monitoring mode. The procedure was linear between 5 and 600 ng/mL, with determination coefficients higher than 0.99 for all analytes. Intra- and interday precision ranged from 0.1 to 13.6%, while accuracy variability was within 80–120% interval from the nominal concentration at all studied levels. The extraction efficiencies ranged from 76.6 to 112.8%. Stability was considered acceptable for all compounds in the studied matrices. The developed assay was applied to authentic samples of the Laboratory of Chemistry and Forensic Toxicology, Centre Branch, of the National Institute of Legal Medicine and Forensic Sciences, Portugal.
Keywords: Cathinones; Phenethylamines; Alternative biological matrices; Microwave fast derivatization; Gas chromatography–mass spectrometry;

LVV-h7 (LVVYPWTQFR) is a bioactive peptide that can be obtained from blood as waste of food industry, more precisely from hemoglobin hydrolysis by pepsin. This opioid peptide belongs to the hemorphins family and have strong physiological effects that bring its use in pharmaceutics and various therapeutic treatments attractive, in particular for substituting its costly chemically synthetized analogous. Hemoglobin hydrolysis by pepsin generates a huge variety of peptides among whose LVV-h7 can be purified by liquid–liquid extraction (LLE). Herein, selective preparation of this peptide is proposed by a microfluidic-based continuous reaction-separation process. Hemoglobin hydrolysis in microreactor was firstly coupled to LVV-h7 LLE in octan-1-ol and then coupled to LVV-h7 back LLE in acidic water. This continuous process allowed to prepare pure LVV-h7, as confirmed by liquid chromatography and mass spectrometry. The microfluidic circuit also allowed octan-1-ol recycling in a closed loop, making this method more sustainable than similar biphasic batch process.
Keywords: Enzymatic hydrolysis; Bioactive peptide; Liquid–liquid extraction; Microfluidics; Solvent recycling;

An ultrasensitive label-free antibody-free detection method for malachite green and crystal violet is presented using nonlinear laser wave-mixing spectroscopy and capillary zone electrophoresis. Wave-mixing spectroscopy provides a sensitive absorption-based detection method for trace analytes. This is accomplished by forming dynamic gratings within a sample cell, which diffracts light to create a coherent laser-like signal beam with high optical efficiency and high signal-to-noise ratio. A cubic dependence on laser power and square dependence on analyte concentration make wave mixing sensitive enough to detect molecules in their native form without the use of fluorescent labels for signal enhancement. A 532 nm laser and a 635 nm laser were used for malachite green and crystal violet sample excitation. The use of two lasers of different wavelengths allows the method to simultaneously detect both analytes. Selectivity is obtained through the capillary zone electrophoresis separation, which results in characteristic migration times. Measurement in capillary zone electrophoresis resulted in a limit of detection of 6.9 × 10−10  M (2.5 × 10−19  mol) for crystal violet and 8.3 × 10−11  M (3.0 × 10−20  mol) for malachite green at S/N of 2.
Keywords: Malachite green; Crystal violet; Gentian violet; Wave mixing; Capillary; Electrophoresis;

A rapid and sensitive assay based on supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS) has been developed and validated for the determination of oxcarbazepine (OXC) and its chiral metabolite licarbazine (Lic) in beagle dog plasma using carbamazepine as internal standard. Chiral analysis in a run time of only 3 min was performed on an ACQUITY UPC2 ™ Trefoil™ CEL2 column (3.0 × 150 mm, 2.5 μm) at 50 °C by isocratic elution with a mobile phase of supercritical carbon dioxide (purity ≥ 99.99%) and methanol (60:40, v/v) at a flow rate of 2.3 mL/min. The assay was linear over the concentration ranges 5–1000 ng/mL for OXC and 0.5–100 ng/mL for the enantiomers of Lic with corresponding lower limits of quantitation of 5 ng/mL and 0.5 ng/mL. Intra- and inter-day precisions were in the range 0.78–14.14% with accuracies in the range −10.80% to 0.42%. The method was successfully applied to a pharmacokinetic study involving a single oral administration of 16 mg/kg OXC as Trileptal@ tablets to beagle dogs.
Keywords: SFC-MS/MS; UPC2; Oxcarbazepine; Chiral analysis; S-Licarbazepine; R-Licarbazepine;

A bioanalytical HPLC method for coumestrol quantification in skin permeation tests followed by UPLC-QTOF/HDMS stability-indicating method for identification of degradation products by Sara E. Bianchi; Helder F. Teixeira; Samuel Kaiser; George G. Ortega; Paulo Henrique Schneider; Valquiria L. Bassani (43-52).
Coumestrol is present in several species of the Fabaceae family widely distributed in plants. The estrogenic and antioxidant activities of this molecule show its potential as skin anti-aging agent. These characteristics reveal the interest in developing analytical methodology for permeation studies, as well as to know the stability of coumestrol identifying the major degradation products. Thus, the present study was designed, first, to develop and validate a versatile liquid chromatography (HPLC) method to quantify coumestrol in a hydrogel formulation in different porcine skin layers (stratum corneum, epidermis, and dermis) in permeation tests. In the stability-indicating test coumestrol samples were exposed to stress conditions: temperature, UVC light, oxidative, acid and alkaline media. The degradation products, as well as the constituents extracted from the hydrogel, adhesive tape or skin were not eluted in the retention time of the coumestrol. Hence, the HPLC method showed to be versatile, specific, accurate, precise and robust showing excellent performance for quantifying coumestrol in complex matrices involving skin permeation studies. Coumestrol recovery from porcine ear skin was found to be in the range of 97.07–107.28 μg/mL; the intra-day precision (repeatability) and intermediate precision (inter-day precision), respectively lower than 4.71% and 2.09%. The analysis using ultra-performance liquid chromatography coupled to a quadrupole time-of-flight high definition mass spectrometry detector (UPLC-QTOF/HDMS) suggest the MS fragmentation patterns and the chemical structure of the main degradation products. These results represent new and relevant findings for the development of coumestrol pharmaceutical and cosmetic products.
Keywords: Bioanalytical method; Coumestrol; Stability-indicating method; Skin permeation; Validation; UPLC-QTOF/HDMS;

Acute hydrodynamic damage induced by SPLITT fractionation and centrifugation in red blood cells by Adriana Urbina; Ruben Godoy-Silva; Mauricio Hoyos; Marcela Camacho (53-61).
Though blood bank processing traditionally employs centrifugation, new separation techniques may be appealing for large scale processes. Split-flow fractionation (SPLITT) is a family of techniques that separates in absence of labelling and uses very low flow rates and force fields, and is therefore expected to minimize cell damage. However, the hydrodynamic stress and possible consequent damaging effects of SPLITT fractionation have not been yet examined. The aim of this study was to investigate the hydrodynamic damage of SPLITT fractionation to human red blood cells, and to compare these effects with those induced by centrifugation. Peripheral whole blood samples were collected from healthy volunteers. Samples were diluted in a buffered saline solution, and were exposed to SPLITT fractionation (flow rates 1–10 ml/min) or centrifugation (100–1500  g) for 10 min. Cell viability, shape, diameter, mean corpuscular hemoglobin, and membrane potential were measured. Under the operating conditions employed, both SPLITT and centrifugation maintained cell viability above 98%, but resulted in significant sublethal damage, including echinocyte formation, decreased cell diameter, decreased mean corpuscular hemoglobin, and membrane hyperpolarization which was inhibited by EGTA. Wall shear stress and maximum energy dissipation rate showed significant correlation with lethal and sublethal damage. Our data do not support the assumption that SPLITT fractionation induces very low shear stress and is innocuous to cell function. Some changes in SPLITT channel design are suggested to minimize cell damage. Measurement of membrane potential and cell diameter could provide a new, reliable and convenient basis for evaluation of hydrodynamic effects on different cell models, allowing identification of optimal operating conditions on different scales.
Keywords: SPLITT fractionation; Centrifugation; Red blood cells; Hydrodynamic damage; Energy dissipation rate;

A rapid, sensitive and specific ultra performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS) method for the quantification of shanzhiside methyl ester, 8-O-acetylshanzhiside methyl ester and luteolin-7-O-β-d-glucopyranoside of Lamiophlomis rotata Pill in rat plasma was developed and validated. After liquid–liquid extraction with n-butyl alcohol/ethyl acetate (70:30, v/v), analytes and paeoniflorin (internal standard, IS) were separated on an Acquity BEH UPLC C18 column (100 × 2.1 mm, 1.7 μm) with gradient elution at a flow rate of 0.2 mL/min. All calibration curves had good linearity (r > 0.9929) over the concentration ranges of 1–1000 ng/mL for shanzhiside methyl ester and 8-O-acetylshanzhiside methyl ester, 0.3–150 ng/mL for luteolin-7-O-β-d-glucopyranoside. The intra- and inter-day precisions were all within 11.1% and the accuracy (relative error, RE%) all ranged from −13.6% to 5.3%. The method also guaranteed an acceptable selectivity, recovery and stability, which was successfully applied to a pharmacokinetic study of the three analytes in rats after oral administration of Lamiophlomis rotata Pill.
Keywords: Lamiophlomis rotata pill; Pharmacokinetics; UPLC–MS/MS; Rat plasma;

A sensitive and simple liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for simultaneous determination of nicotine and its main metabolite cotinine in human serum samples. Liquid–liquid extraction using ethyl acetate was employed for serum sample extractions. Chromatographic separation was achieved on Phenomenex Luna® HILIC column (150 mm x 3.0 mm, 5 μm). Isocratic elution was performed using acetonitrile:100 mM ammonium formate buffer (pH = 3.2) (90:10, v/v) as the mobile phase, at a flow rate of 0.4 mL/min. Tandem mass spectrometric detection was employed at positive electrospray ionization in MRM mode for the determination of both nicotine and cotinine and their stable isotope labeled internal standards. Analysis was carried out in 8 min over a concentration range of 0.26–52.5 ng/mL and 7.0–1500 ng/mL for nicotine and cotinine, respectively. The assay was validated according to FDA guidelines for bioanalytical method validation and satisfactory results were obtained; the accuracy ranged between 93.39% and 105.73% for nicotine and between 93.04% and 107.26% for cotinine. No significant matrix effect was observed. Stability assays indicated both nicotine and cotinine were stable during sample storage, preparation and analytical procedures. The method was successfully applied to biological samples obtained from a pharmacokinetic study conducted in adult smokers to investigate heat effect on nicotine and cotinine serum levels after nicotine transdermal delivery system (TDS) application.
Keywords: Nicotine; Cotinine; LC–MS/MS; HILIC; TDS;

Identification of in vivo and in vitro metabolites of 4,5-dimethoxycanthin-6-one by HPLC–Q-TOF-MS/MS by Xiaolei Miao; Junjun Wang; Liang Chen; Zhihong Peng; Yong Chen (78-84).
4,5-Dimthexycanthin-6-one and 5-hydroxy-4-methoxycanthin-6-one are the main active ingredients of Picrasma quassioides, which is a widely used herbal medicine for the treatment of gastroenteritis, snakebite, infection and hypertension in China. In the present study, the in vitro metabolites of 4,5-dimethoxycanthin-6-one in rat, mouse, dog and human liver microsomes, as well as the in vivo metabolites in rat plasma and urine following a single oral dose of 4,5-dimethoxycanthin-6-one, were identified by high-performance liquid chromatography combined with triple TOF mass spectrometry (HPLC–TOF/MS/MS). The metabolites were elucidated based on an accurate mass measurement, the MS/MS fragmentation patterns, the retention times of the parent drug and its metabolites, and the relevant drug biotransformation rules. After incubation in liver mcrosomes for 50 min, 4,5-dimethoxycanthin-6-one produced 8 phase I metabolites including 2 mono-demethylated metabolites (M1, M2), 3 mono-hydroxylated metabolites (M3-M5), and 3 mono-demethylated and mono-hydroxylated metabolites (M6-M8) in rat and mouse liver microsomes, 7 phase I metabolites (without M7) in dog and human liver microsomes. After a single oral administration of 4,5-dimethoxycanthin-6-one to rats, there were 3 phase I metabolites (M1, M2 and M5) detected in rat plasma and 5 phase I metabolites (M1–M5) in rat urine. Phase II metabolites were not detected in rat plasma and urine. Among these metabolites, mono-demethylated metabolites (M1 and M2) were the major metabolites of 4,5-dimethoxycanthin-6-one, mono-hydroxylated metabolites (M3–M5) were the minor metabolites of 4,5-dimethoxycanthin-6-one.
Keywords: 4,5-Dimethoxycanthin-6-one; Metabolite identification; In vitro and in vivo; HPLC–TOF/MS/MS;

An HPLC–MS method for the quantification of new acetylcholinesterase inhibitor PC 48 (7-MEOTA-donepezil like compound) in rat plasma: Application to a pharmacokinetic study by Martin Mzik; Jan Korabecny; Eugenie Nepovimova; Viktor Voříšek; Vladimir Palička; Kamil Kuca; Jana Zdarova Karasova (85-89).
A simple, rapid and sensitive method based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been developed and validated for the quantitative determination in rat plasma of a new candidate for AD treatment, namely PC 48 (a 7-MEOTA-donepezil like compound) in rat plasma. Sample preparation involved pH adjustment with sodium hydroxide followed by solvent extraction with ethyl acetate:dichloromethane (80:20, v/v). The chromatographic separation was achieved on an Ascentis Express RP-Amide column (75 mm × 2.1 mm, 2.7 μm) with a gradient mobile phase consisting of 0.05 M aqueous formic acid and acetonitrile. Detection was carried out using positive-ion electrospray tandem mass spectrometry on an LTQ XL system using the MS/MS CID (collision-induced dissociation) mode. The method was linear in the range 0.1–1000 ng/ml (r2  = 0.999) with a lower limit of quantitation of 0.1 ng/mL. Extraction recovery was in the range 63.5–72.1% for PC 48 and 70.5% for reserpine (internal standard, IS). Intra- and inter-day precisions measured as relative standard deviation were below 10.8% and accuracy was from −7.2% to 7.4%. The method was successfully applied to a pharmacokinetic study involving intramuscular application of 3.86 mg/kg PC 48 to rats for the first time. Pharmacokinetic parameters for PC 48 include Cmax 39.09 ± 4.45 ng/mL, Tmax 5.00 ± 3.08 min, AUC0 − t 23374 ± 4045 min ng/mL and t1/2 1065 ± 246 min.
Keywords: Acetylcholinesterase; Donepezil; 7-MEOTA; PC 48; Alzheimer disease; HPLC–MS;

Screening for toxic phorbol esters in jerky pet treat products using LC–MS by Upul Nishshanka; Hiranthi Jayasuriya; Chaitali Chattopadhaya; Philip J. Kijak; Pak-Sin Chu; Renate Reimschuessel; Andriy Tkachenko; Olgica Ceric; Hemakanthi G. De Alwis (90-95).
Since 2007, the U.S. FDA’s Center for Veterinary Medicine (CVM) has been investigating reports of pets becoming ill after consuming jerky pet treats. Jerky used in pet treats contains glycerin, which can be made from vegetable oil or as a byproduct of biodiesel production. Because some biodiesel is produced using oil from Jatropha curcas, a plant that contains toxic compounds including phorbol esters, CVM developed a liquid chromatography-mass spectrometry (LC–MS) screening method to evaluate investigational jerky samples for the presence of these toxins. Results indicated that the samples analyzed with the new method did not contain Jatropha toxins at or above the lowest concentration tested.
Keywords: Jatropha curcas; Phorbol esters; Jerky pet treats; High resolution; Accurate mass; LC–MS method;

Semen Oroxyli, a Traditional Chinese Medicine, has many significant pharmacological activities such as analgesic, apoptotic, anti-inflammatory, anticancer, and immunostimulant activities. A sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the simultaneous quantitation of four flavonoids (oroxin A, oroxin B, baicalin, and chrysin) of Semen Oroxyli in rat plasma. After the addition of internal standard, plasma samples were pretreated with acetonitrile via a single-step protein precipitation. Chromatographic separation was performed on a Capcell Pak C18 column (100 mm × 2.0 mm, 5 μm particles) with isocratic elution using a mobile phase of methanol and 2 mM ammonium acetate buffer solution (75:25, v/v) at a flow rate of 0.45 mL/min. The analytes were detected without interference in the selection reaction monitoring mode with negative electrospray ionization. The validated method exhibited good linearity over a wide concentration range (r ≥ 0.9958), and the lower limits of quantification were 1.0–5.5 ng/mL for all the analytes. The mean extraction recoveries of the analytes from rat plasma exceeded 80.6%. The intra- and inter-day precisions at three QC levels were both less than 11.5%, and the accuracies ranged from −6.2% to 10.3%. The LC–MS/MS method was successfully applied to a pharmacokinetic study of the four flavonoids in rat plasma after oral administration of Semen Oroxyli extract.
Keywords: Semen oroxyli; Liquid chromatography–tandem mass spectrometry; Rat plasma; Pharmacokinetics;

Analysis of prasugrel active metabolite R-138727 in human plasma: a sensitive, highly selective and fast LC–MS/MS method by Sreekanth Kakarla; Peda Varma Datla; Geetha kodali; Ganapaty Seru (103-110).
A cost effective, sensitive, simple, and rapid high-performance liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of the prasugrel metabolite in human plasma. Following solid phase extraction, the analyte (prasugrel active metabolite; R-138727) and internal standard (emtricitabine) were separated using a mobile phase in an isocratic elution mode on a reverse phase C18 column and were analyzed by an LC–MS/MS in the multiple reaction monitoring mode using the respective [M + H]+ ions, m/z 498.3–206.0 for R-138727 and m/z 248.2–130.1 for the internal standard. The assay exhibited a linear dynamic range of 0.2–120 ng/mL. The lower limit of quantification was 0.2 ng/mL with a relative standard deviation of 5.0%. This LC–MS/MS method was validated with intra-batch and inter-batch precision and accuracy. Results for precision and accuracy are in range of 3.9–9.6% and 95.2–102.2% respectively. This validated method is simple and repeatable to use in bioequivalence/pharmacokinetic studies.
Keywords: Prasugrel metabolite (R-138727); Liquid chromatography-tandem mass spectrometry; Human plasma;

Simultaneous determination of itraconazole, hydroxy itraconazole, keto itraconazole and N-desalkyl itraconazole concentration in human plasma using liquid chromatography with tandem mass spectrometry by Xiaorong Liang; Michael Van Parys; Xiao Ding; Ning Zeng; Luke Bi; Drew Dorshort; Janine McKnight; Dennis Milanowski; Jialin Mao; Yuan Chen; Joseph A Ware; Brian Dean; Cornelis E.C.A. Hop; Yuzhong Deng (111-119).
A high-performance liquid chromatography tandem mass spectrometry (LC–MS/MS) assay was developed and validated for simultaneous determination of itraconazole (ITZ), hydroxy-itraconazole (OH-ITZ), keto-itraconazole (keto-ITZ) and N-desalkyl itraconazole (ND-ITZ) concentration in human plasma. One hundred and fifty microliters of human plasma were extracted using a solid-supported liquid extraction (SLE) method and the final extracts were analyzed using reverse-phase chromatography and positive electrospray ionization mass spectrometry. The standard curve range is 5–2500 ng/mL for ITZ and OH-ITZ and 0.4–200 ng/mL for keto-ITZ and ND-ITZ. The curve was fitted to a 1/x 2 weighted linear regression model for all analytes. The precision and accuracy of the LC–MS/MS assay based on the five analytical quality control (QC) levels were well within the acceptance criteria from both FDA and EMA guidance for bioanalytical method validation. Average extraction recovery was 97.4% for ITZ, 112.9% for OH-ITZ, 103.4% for keto-ITZ, and 102.3% for ND-ITZ across their respective curve range. Matrix factor was close to 1.0 at both high and low QC levels of all 4 analytes, which indicates minimal ion suppression or enhancement in our validated assay. Itraconazole and all three metabolites are stable in human plasma for 145 days stored at −70 °C freezers. The validated assay was successfully applied to a clinical study, which has a drug-drug interaction (DDI) arm using ITZ as a cytochrome P450, family 3, subfamily A (CYP3A) inhibitor.
Keywords: LC–MS; Human plasma; Itraconazole; Hydroxy itraconazole; Keto itraconazole; N-desalkyl itraconazole; Drug-drug interaction;

Esculin, a coumarin derivative found in Fraxinus rhynchophylla, has been reported to possess multiple biological activities. This present study is designed to investigate the metabolic profile of esculin in vivo based on ultra high performance liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (UHPLC-FT-ICR-MS) for the first time. After oral administration of esculin (100 mg/kg) for rats, plasma, urine, feces and bile samples were collected to screen metabolites. As a result, a total of 19 metabolites (10 phase I metabolites and 9 phase II metabolites) were found and identified. Results showed that metabolic pathways of esculin included hydrolysis, dehydrogenation, hydroxylation, methylation, dehydrogenation, glucuronidation, sulfation, and glycine conjugation. It was also found that after oral administration of esculin, the esculin could be metabolized to esculetin in vivo via deglycosylation, and esculetin was found in all biological samples. This study also laid solid basis for in-depth development of esculin and provided important information for clarifying the biotransformation process of esculin in vivo.
Keywords: Esculin; Identification; Metabolism; Rats; FT-ICR-MS;

An experimental model of affinity filtration for the isolation of egg white Lysozyme using Cibacron Blue immobilized to yeast cells. by María del Pilar Ferraris; Ulises A. Gonzalez; Carlos F. Aguilar; Jorge A. Rodríguez (129-133).
An experimental model of affinity filtration process was designed using a macroligand composed by Cibacron Blue F3GA immobilized to yeast cells. Its performance was evaluated, at bench scale, through the recovery of egg white Lysozyme. The selective and reversible binding between the Cibacron ligand molecule and the enzyme is described. The separation of Lysozyme from the protein mixture included the application of stages such as affinity adsorption, concentration, diafiltration and elution. A tangential microfiltration system with an inorganic membrane was designed. The main finding was the development of the diafiltration operation, key stage in the enzyme isolation. The macroligand particle kept its integrity along the whole process and the degree of purity of the isolated Lysozyme was significant.
Keywords: Affinity filtration; Macroligand; Cibacron Blue; Lysozyme isolation; Hen egg white;

A comparative study of various physicochemically modified capillaries used in CE technique for the three distinct analytical purposes by Paweł Mateusz Nowak; Michał Woźniakiewicz; Magdalena Garnysz; Paweł Kościelniak (134-141).
The five different commercially offered capillaries, bare silica, one dynamically and three permanently coated, have been tested with respect to the three distinct bioanalytical issues: (i) achiral and chiral separation of small mass molecules, warfarin and its six isomeric hydroxy-derivatives; (ii) ultraselective separation of transferrin and albumin including differentiation between the diferric, two monoferric and iron-free forms of transferrin; and (iii) pioneering identification of albumin-induced shifts of acid dissociation constant by using CE instrumentation, revealed for warfarin and six hydroxywarfarins. As a result all of these pharmacologically and biochemically-relevant purposes have been reached, but using different capillaries. The dynamically coated silica capillary has allowed for the first time to separate warfarin from its six main hydroxy-metabolites by CE with high resolution. The bare silica capillary has performed well in cyclodextrin-assisted enantioseparation of these compounds. The neutral capillary has provided excellent resolution and performance in separation of proteins. The amine capillary, in turn, has occurred to be the best choice for identification of albumin-induced pK a shifts occurring in vivo, omitting capillary clogging and providing better sensitivity than the neutral capillary. This work reveals specific predispositions and shows that there is no universal capillary which may be applied to all issues.
Keywords: Acid dissociation constant; Capillary electrophoresis; Capillary coating; Transferrin; Warfarin;

There is a relationship between circadian rhythm and metabolic disorders. The active agent, SR8278, could competitively bind to and inhibit the nuclear receptor, Rev-erb (a major modulator of mammalian circadian clock system), to regulate the metabolism in organisms. However, we had limited knowledge of the pharmacokinetic (PK) characteristics of SR8278. Here, we describe a sensitive and reproducible ultra-performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method to quantify SR8278 in vivo. The linearity range and the limit of quantification (LOQ) for SR8278 were 30–3000 ng/mL and 6 ng/mL, respectively. The inter-day and intra-day variability were within 10%. This UPLC-MS/MS method was successfully used to characterize the PK behaviors of SR8278 in normal and diabetic rats after intravenous (i.v.) injection at a dosage of 2 mg/kg. No significant differences were observed in the PK parameters of SR8278 in normal and diabetic rats. Specifically, the values of areas under plasma concentration time curves (AUC), initial plasma concentrations (C0), elimination half-lives (t1/2), and clearances (CL) were 608.33 ± 295.25 vs. 598.59 ± 276.92 ng·h/mL, 2410.25 ± 202.36 vs. 3742.11 ± 1300.21 ng/mL, 0.17 ± 0.08 vs. 0.11 ± 0.04 h, 3330.83 ± 1609.48 vs. 3364.81 ± 1111.38 mL/kg·h for SR8278 in normal rats vs. diabetic rats, respectively. In conclusion, a UPLC-MS/MS method was successfully developed and validated for the first time, with a wide linearity range, low LOQ, small sample volume (10 μL), rapid analysis (4 min) and excellent recoveries (>80%). It was also used to clarify the PK characteristics of SR8378 in rats. The same PK behaviors of SR8278 in normal and diabetic rats showed that diabetes may have little or no effect on the disposition, metabolism and/or elimination in vivo, which may be of great importance for future clinical studies.
Keywords: UPLC-MS/MS; SR8278; Pharmacokinetics; Rev-erb; Diabetes;

The charge variations of therapeutic monoclonal antibody reveal important information of the post-translational modifications that may potentially impact the potency and safety of pharmaceutical products, especially during the evaluation of biosimilarity of therapeutic proteins. In this work, a novel SpeB-based proteolysis strategy coupling with imaged capillary isoelectric focusing was developed for the determination of domain-specific charge heterogeneities of innovator and generic Rituximab drug products from United States, European and Indian markets. It was observed that innovator Rituximab from the United States and Europe share highly similar peak distributions and charge heterogeneities with 26.2–26.6% Fc/2, 28.9–29.3% LC and 44.4–44.5% Fd peak areas detected, respectively, while multiple basic variations of Fc/2 and less acidic LC and Fd species were found from generic Rituximab from India with 20.9% Fc/2, 32.3% LC and 46.9% Fd peak areas detected. It was also demonstrated that structural changes caused by Carboxypeptidase B treatment and deamidation study at pH extremes could be sensitively captured with the established method, with the results further indicating that the generic product’s basic variations of Fc/2 were un-cleaved Lysine residues, while the lack of certain acidic peaks on LC and Fd probably was due to the lower level of deamidation. This new strategy could become a useful tool to reveal domain-specific charge heterogeneities profiles of a variety of therapeutic monoclonal antibodies in regulated environments.
Keywords: Rituximab; Biosimilar; SpeB; Proteolysis; Imaged capillary isoelectric focusing; CIEF; Deamidation; Monoclonal antibody; mAb;

We report the improvement of a method for the detection of ochratoxin A (OTA) and its thermal degradation product 2′R-ochratoxin A in dried blood spots (DBS) by high performance liquid chromatographic (HPLC) tandem mass spectrometry (MS/MS). The DBS technique was advanced for the analysis of these two compounds in DBS with unknown amounts of blood as well as varying hematocrit values. Furthermore the comparability of venous vs. capillary blood was investigated.Human whole blood samples were spotted, dried, and extracted with a solvent consisting of acetone, acetonitrile and water for analysis by HPLC-MS/MS. Quantification was carried out by stable isotope labelled internal standards. Blood samples of volunteers (n = 50) were used to further optimize and simplify the procedure. Ochratoxin A and 2′R-ochratoxin A concentrations found in the entire spots (approx. 100 μL blood) were compared with punched DBS discs of 8.8 mm size containing approximately 20 μL blood. As a result the amounts of both toxins in a punched 8.8 mm disc correlate well with the entire DBS. Also the use of capillary blood from finger-pricks versus venous blood was evaluated. The analyte levels correlate as well indicating that the less invasive finger-prick sampling gives also reliable results. The influence of hematocrit was investigated in a range of 25–55% according to the hematocrit in the used real blood samples (34–46% hematocrit). However no significant hematocrit effect was observed for the utilized real blood samples. Moreover different blood volumes were spotted and punched as a minimal spot size is usually recommended for accurate analysis. In this experiment finger-prick samples typically consist of about 90 μL blood. Therefore spots of 75, 100 and 125 μL blood were prepared and analyzed. Similar to the hematocrit effect, no considerable influence was observed.
Keywords: Biomonitoring; Dried blood spot; Hematocrit; Metabolism; Mycotoxin; Ochratoxin;