Journal of Chromatography B (v.973, #C)

A validated LC–MS/MS method for the simultaneous determination of BMS-791325, a hepatitis C virus NS5B RNA polymerase inhibitor, and its metabolite in plasma by Long Yuan; Hao Jiang; Naiyu Zheng; Yuan-Qing Xia; Zheng Ouyang; Jianing Zeng; Billy Akinsanya; Jack L. Valentine; Jeffrey D. Moehlenkamp; Yuzhong Deng; Anne-Françoise Aubry; Mark E. Arnold (1-8).
BMS-791325 is a hepatitis C virus (HCV) non-structural protein 5B (NS5B) RNA polymerase inhibitor that is being developed for the treatment of HCV infection. A rugged and accurate LC–MS/MS method was developed and validated for the quantitation of BMS-791325 and its metabolite, BMS-794712, in rat and dog plasma. This method utilized stable-isotope labeled [D6]-BMS-791325 and [13CD3]-BMS-794712 as internal standards. The samples were extracted using liquid–liquid extraction with n-butyl-chloride. Chromatographic separation was achieved with gradient elution on a Waters Atlantis dC18 analytical column (2.1 mm × 50 mm, 3.0 μm). Analytes and their stable isotope labeled internal standards were detected by positive ion electrospray tandem mass spectrometry. The standard curves, which ranged from 5.00 to 2000 ng/mL for BMS-791325 and from 1.00 to 400 ng/mL for BMS-794712, were fitted to a 1/x 2 weighted linear regression model. For both species, the intra-assay precision was within ±4.3% CV, the inter-assay precision was within ±6.2% CV, and the assay accuracy was within ±10.8% of the nominal values for BMS-791325 and BMS-794712. The validated method was successfully applied to support pre-clinical toxicokinetic studies.
Keywords: HCV; BMS-791325; Metabolite; Quantitation; LC–MS/MS;

Isolation and identification of cyclic lipopeptides from Paenibacillus ehimensis, strain IB-X-b by Gleb Aktuganov; Jouni Jokela; Henri Kivelä; Elvira Khalikova; Alexander Melentjev; Nailia Galimzianova; Lyudmila Kuzmina; Petri Kouvonen; Juha-Pekka Himanen; Petri Susi; Timo Korpela (9-16).
Antifungal lipopeptides produced by an antagonistic bacterium, Paenibacillus ehimensis strain IB-X-b, were purified and analyzed. The acetone extract of the culture supernatant contained an antifungal amphiphilic fraction stainable with ninhydrin on thin layer chromatography. The fraction was further purified with water–methanol extraction followed by a chromatography on a C18-support. The analysis with LC–MS showed presence of two main series of homologous compounds, family of depsipeptides containing a hydroxy fatty acid, three 2,4-diaminobutyric acid (Dab) residues, five hydrophobic amino acids and one Ser/Thr residue, and cyclic lipopeptides of bacillomycin L and fengycin/plipastatin/agrastatin families. The prevailing compounds in this group are bacillomycin L-C15, fengycin/plipastatin A-C16 together with their homologues responsible for the majority of fungal growth inhibition by P. ehimensis IB-X-b.
Keywords: Antifungal compounds; Microbial antagonism; Paenibacillus ehimensis; Biocontrol; Cyclic lipopeptides; Purification; LC–MS;

Fast analysis of polyphenols in royal jelly products using automated TurboFlow™-liquid chromatography–Orbitrap high resolution mass spectrometry by Noelia López-Gutiérrez; María del Mar Aguilera-Luiz; Roberto Romero-González; José Luis Martínez Vidal; Antonia Garrido Frenich (17-28).
This study describes the development of a novel, simple and fast analytical method for the detection and quantification of polyphenols in royal jelly products, using an in-house database containing more than 50 compounds. The extraction method consisted of sample dilution, followed by a fast on-line system composed of turbulent flow chromatography (TurboFlow™) coupled to liquid chromatography (LC)–Exactive-Orbitrap analyzer. The total run time was 18 min, including automated extraction, analytical chromatography and re-equilibration. The method was validated obtaining limits of quantification (LOQ) ranging from 10 to 150 μg/kg. The linearity range was up to 2000 μg/L and determination coefficients (R 2) were higher than 0.994. Adequate recoveries were obtained at three concentration levels (500, 1000 and 2000 μg/kg). This method was applied to the analysis of nine samples and the concentration of polyphenols ranged from 14 (apigenin) to 18,936 μg/kg (ferulic acid).
Keywords: Royal jelly; Polyphenol; TurboFlow; UHPLC–Orbitrap;

In this work, an on-line preconcentration method of field-enhanced sample injection (FESI) was implemented in the determination of four β2-agoinsts terbutaline (TER), procaterol (PRO), formoterol (FOR) and bambuterol (BAM) by capillary electrophoresis coupled with capacitively coupled contactless conductivity detection (CE-C4D). Under optimized conditions the background electrolyte (BGE) was 5 mM Tris(hydroxymethyl)aminomethane (Tris) and 10 mM citric acid (Cit) at a pH of 3.2 while the sample dilution solution was obtained by methanol. The detection limits (defined as S/N = 3) of this method were 0.02 mg/L for TER, PRO, FOR, BAM, which were much lower than that of the conventional CE-C4D method without preconcentration procedure, the enhancement factors were greatly improved to be 30–40-fold. The linearity ranges of four β2-agoinsts were 0.1–15 mg/L, with good linear correlation coefficients (r 2  > 0.9900). In order to evaluate the application potential of the developed method, real sample from pig feed was analyzed with recoveries of 91.4–106.2%.
Keywords: Field-enhanced sample injection (FESI); β2-Agonists; Capillary electrophoresis; Contactless conductivity detection;

A rapid and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the quantification of miltirone concentration in rat plasma. The cytotoxic activity of miltirone was firstly evaluated by the MTT assay and compared with other tanshinones. Quantification was carried out on an Agilent triple quadrupole LC–MS system using multiple reaction monitoring (MRM) mode in positive mode. After simple protein precipitation with acetonitrile, the chromatographic separation of miltirone was achieved by using a Waters Symmetry C18 analytical column (2.1 mm × 100 mm, 3.5 μm) with a mobile phase of acetonitrile (A)–water (B) (75:25, v/v) containing 0.5% formic acid. The monitored transitions were set at m/z 283.1 → 223.1 and m/z 361.0 → 232.9 for miltirone and IS, respectively. The calibration curve was linear over the concentration range of 0.5–200 ng/mL with lower limit of quantification of 0.5 ng/mL. The intra- and inter-day accuracy and precision of miltirone were both within acceptable limits. The developed method was successfully applied to a pharmacokinetic study of following oral administration of 20, 40, 60 mg/kg and an intravenous administration of 0.5 mg/kg to rats. The results indicated that miltirone had linear pharmacokinetic properties within the tested dosage range and was poorly absorbed with an absolute bioavailability of approximately 3.4%.
Keywords: Miltirone; LC–MS/MS; Rat plasma; Pharmacokinetics; Bioavailability;

A fast, sensitive and specific hydrophilic interaction chromatography combined with tandem mass spectrometry (HILIC-MS/MS) method was developed for the determination of tobramycin in human plasma. With sisomycin as internal standard, the analysis was carried out on a hilic column (150 mm × 2.1 mm, 3.5 μm) using a mobile phase consisting of acetonitrile: 5 mM ammonium acetate and 0.1% formic acid (60:40, v/v). The detection was performed by tandem spectrometry via electrospray ionization (ESI). Linear calibration curves were obtained in the concentration range of 10.51–1051 ng/mL for tobramycin, with a lower limit of quantification of 10.51 ng/mL. The intra- and inter-day precision (RSD) values were below 15% and accuracy (RE) was 1.3–5.7% at all QC levels. The method was applicable to the clinical study of the pharmacokinetics of tobramycin in healthy volunteers.
Keywords: Tobramycin; HILIC-MS/MS; Human plasma; Tandem mass spectrometry; Pharmacokinetics;

Metabolomics research on the hepatoprotective effect of Angelica sinensis polysaccharides through gas chromatography–mass spectrometry by Peng Ji; Yanming Wei; Hongguo Sun; Wenxin Xue; Yongli Hua; Pengling Li; Wenquan Zhang; Ling Zhang; Haifu Zhao; Jinxia Li (45-54).
Angelica sinensis polysaccharides (ASP) have an established hepatoprotective effect, but the mechanism for this effect remains unclear. A novel approach using biochemical parameters coupled with metabolomics based on gas chromatography–mass spectrometry (GC–MS) and chemometrics was established in this study to explain the hepatoprotective effect mechanism of ASP. The superoxide dismutase activity, malonaldehyde content, alanine aminotransferase, aspartate aminotransferase, and γ-glutamyl transpeptidase in plasma were measured. Pathological changes in the liver were observed. Plasma and liver homogenate obtained from mice were analyzed using GC–MS. Distinct changes in metabolite patterns in the plasma and liver homogenate after being induced by carbon tetrachloride and drug intervention were observed using principal component analysis (PCA) and partial least squares-discriminate analysis (PLS-DA). Potential biomarkers were found using PLS-DA and T-test. The results of the pathological changes observed in the liver, the biochemical parameters in plasma, and the metabolomics of the plasma and liver homogenate all showed that liver injury was successfully reproduced, ASP exhibited hepatoprotective effect, and the medium dose of ASP exhibited the best. Nine endogenous metabolites in the liver homogenate and ten endogenous metabolites in the plasma were all considered as potential biomarkers. They were considered to be in response to hepatoprotective effects of ASP involved in the amino acids metabolism, energy metabolism, and lipids metabolism. Therefore metabolomics is a valuable tool in measuring the efficacy and mechanisms of action of traditional Chinese medicines.
Keywords: Metabolomics; Angelica sinensis polysaccharide; Hepatoprotective effect; Gas chromatography–mass spectrometry; Carbon tetrachloride;

Cyclooxygenase-2 (COX-2) inhibitors may be used to efficiently treat inflammation or cancer diseases. In the present study, we established a new screening assay based on magnetic Fe3O4@SiO2–COX-2 ligand fishing combination with high-performance liquid chromatography–diode array detector–mass spectrometry (HPLC–DAD–MSn) to screen and identify COX-2 inhibitors from green tea. Optimized conditions (pH at 7.4, temperature at 30 °C, and incubation time for 30 min) for fishing out COX-2 inhibitors were achieved by testing positive control, celecoxib, with active and inactive COX-2. Notably, immobilized COX-2 showed high stability (remained 94.7% after ten consecutive cycles), reproducibility (RSD < 10% for batch-to-batch evaluation). Finally, eight catechins with COX-2 binding activity were screened in green tea, and their structures were characterized by ultraviolet (UV), accurate molecular weight, diagnostic fragment ions and nuclear magnetic resonance (NMR). Particularly, the COX-2 inhibitory activities of two rare catechins, [(−)-epigallocatechin-3-(3″-O-methyl)-gallate (3″-O-methyl-EGCG, IC50  = 0.17 ± 0.03 μM 0.16 ± 0.01), (−)-epicatechin-3-(3″-O-methyl)-gallate (3″-O-methyl-ECG, IC50  = 0.16 ± 0.02 μM)], were reported for the first time. The results indicated that the proposed method was a simple, robust and reproducible approach for the discovery of COX-2 inhibitors from complex matrix.
Keywords: Cyclooxygenase-2; Magnetic ligand fishing; HPLC–DAD–MSn; Green tea; Catechin;

Determination of 5-hydroxythiabendazole in human urine as a biomarker of exposure to thiabendazole using LC/MS/MS by Eva Ekman; Moosa H. Faniband; Margareta Littorin; Margareta Maxe; Bo A.G. Jönsson; Christian H. Lindh (61-67).
Thiabendazole (TBZ) is widely used as a pre-planting and post-harvest agricultural fungicide and as an anthelminthic in humans and animals. TBZ is of toxicological concern, since adverse effects including nephrogenic, hepatogenic, teratogenic and neurological effects have been reported in mammals. Occupational exposure can occur among agricultural workers and the general public may be environmentally exposed to TBZ through the diet. The metabolite 5-hydroxythiabendazole (5-OH-TBZ) was chosen as biomarker of exposure to TBZ and a LC/MS/MS method for the quantification of 5-OH-TBZ in human urine was developed. The method includes enzyme hydrolysis, as 5-OH-TBZ is conjugated to glucuronide and sulphate in urine. Sample through put was optimised using 96-well plates for sample handling as well as for solid phase extraction (SPE). The method has excellent, within-run, between-run and between-batch precision between 4 and 9%. The limit of detection (LOD) of 0.05 and a limit of quantification (LOQ) of 0.13 ng 5-OH-TBZ/mL urine enable detection in environmentally exposed populations. When applying the method in a general Swedish population, 52% had levels above LOD. The method was also applied in one oral and one dermal human experimental exposure study in two individuals. After oral exposure, the excretion of 5-OH-TBZ in urine was described by a two-compartment model and both the first rapid and the second slower elimination phase followed first-order kinetics, with estimated elimination half-life of 2 h and 9–12 h. The recoveries in urine were between 21 and 24% of the dose. Dermal exposure was described by a one compartment model and followed first order kinetics, with estimated elimination half-life of 9–18 h. The recovery in urine was 1% of the administrated dose of TBZ. Although these studies are limited to two individuals, the data provide new basic information regarding the toxicokinetics of TBZ after oral and dermal exposure.
Keywords: Biomarker; Dermal; LC/MS/MS; Oral; Pesticide; Thiabendazole;

Evaluation of the effect of apatinib (YN968D1) on cytochrome P450 enzymes with cocktail probe drugs in rats by UPLC–MS/MS by Yunfang Zhou; Shuanghu Wang; Ting Ding; Mengchun Chen; Li Wang; Mingdong Wu; Guoxin Hu; Xianghong Lu (68-75).
An accurate and validated liquid chromatography method and a triple quadrupole mass spectrometry method were developed and validated to simultaneously evaluate the cytochrome P450 (CYP) enzymes in vivo using the co-administration of these probes. Phenacetin, losartan, metoprolol and midazolam were used as the probe substrates for rat CYP1A2, CYP2C11, CYP2D4 and CYP3A1 enzymes, respectively. The purpose of the study was to investigate the effect of apatinib on these cytochrome P450 enzymes in vivo with co-administration of these probes. Plasma samples were prepared by precipitating protein with acetonitrile. The analytes were separated using a reversed-phase BEH C18 column (2.1 mm × 100 mm, 1.7 μm, Waters, USA) maintained at 40 °C. The mobile phase consisted of acetonitrile and water (containing 0.1% formic acid) with a gradient elution pumped at a flow rate of 0.4 mL/min. The analytes were detected with positive electrospray ionization in multiple reaction monitoring (MRM) mode for target fragment ions m/z 180.05 → 109.94 for phenacetin, m/z 423.1 → 207.2 for losartan, m/z 268.12 → 115.8 for metoprolol, m/z 326.02 → 290.99 for midazolam and m/z 285.1 → 193.1 for diazepam (IS). Good linearity was achieved to quantify the concentration ranges of 10–2000 ng/mL for phenacetin, 10–1000 ng/mL for losartan, 10–1000 ng/mL for metoprolol and 1–100 ng/mL for midazolam in rat plasma. The mean recoveries of phenacetin, losartan, midazolam and metoprolol from the plasma exceeded 77.07%. The intra-run and inter-run assay precisions were both less than 8.9%. This method was successfully applied to evaluate the effects of apatinib on the cytochrome P450 enzymes in rats.
Keywords: Apatinib; Cytochrome P450; Drug–drug interactions; Cocktail; UPLC–ESI-MS/MS;

The purpose of this study was to explore the plasma protein binding (PPB), pharmacokinetics profiles and tissue distribution of chrysophanol in rats. Biological samples were extracted by ethyl ether, separated and determined by a novel, sensitive and specific GC–MS method under selected ion monitoring (SIM) mode for the first time. This method was validated for the quantification of chrysophanol, in the range of 0.005–1.50 μg mL−1 in rat plasma and 0.04–12 μg mL−1 in tissue homogenates, respectively. The limit of detection (LOD) was 0.4 ng mL−1 or 5.0 ng g−1, which obtained excellent sensitivity for the pharmacokinetics analysis in rats. The intra- and inter-day assay of precisions in plasma and tissues were less than 10% and the intra- and inter-day accuracies were 82.1–92.9%. The method recoveries of all samples were more than 90%, except for liver samples (>80%), which indicated that chrysophanol may be partly metabolized in liver homogenates. The PPB rates in rat plasma, human plasma and bovine serum albumin were 83 ± 2, 88 ± 3 and 58 ± 3%, respectively. The main pharmacokinetic parameters were the time of peak concentration (T max) = (0.665 ± 0.034) h, the peak concentration (C max) = (929.8 ± 102.7) ng mL−1, the area under the curve (AUC) = (1197.6 ± 258.5) ng h mL−1 and the clearance (CL) = (0.013 ± 0.0028) mL mg−1  h−1. The tissue distribution of chrysophanol in rats after the oral administration showed a decreasing tendency in different tissues (heart > kidney > spleen > liver > lung > cerebrum).
Keywords: Chrysophanol; GC/MS; Pharmacokinetics; Tissue distribution; Plasma protein binding;

Recovery of casein-derived peptides with in vitro inhibitory activity of angiotensin converting enzyme (ACE) using aqueous two-phase systems by Evaldo Cardozo de Souza; Jane Sélia dos Reis Coimbra; Eduardo Basílio de Oliveira; Renata Cristina Ferreira Bonomo (84-88).
Peptides inhibiting the activity of angiotensin converting enzyme (ACE) were obtained by trypsin-catalyzed hydrolysis of bovine milk casein, performed at 37 °C, during 1, 2, 5, 8 and 24 h. Results of in vitro inhibitory activity ranged between 13.4% and 78.5%. The highest ACE inhibitory activity was evidenced for hydrolysates obtained after 2 h of reaction. Aqueous two-phase systems (ATPS) formed by polyethylene glycol of 1500 g mol−1 (PEG 1500) + sodium phosphate or potassium phosphates were produced and evaluated, in terms of partition coefficients (K) and extraction yields (y), to recovery the casein hydrolysates at room temperature. In ATPS containing sodium phosphate, the peptides showed a slightly greater affinity toward the bottom salt-rich phase (0.1 ≤  K  ≤ 0.9; 5.7% ≤ y ≤ 47%). In the case of ATPS containing potassium phosphates, these molecules showed substantially greater affinity toward the top polymer-rich phase (137 ≤  K  ≤ 266; y ≥ 99%). These results point out extraction using PEG 1500/potassium phosphate ATPS is an efficient technique to recover casein hydrolysates containing ACE inhibitors peptides. Outlined data will be helpful in integrating such unit operation to larger scale processes.
Keywords: Bioseparation; Bovine milk; Phase equilibrium; Protein; Trypsin; Antihypertensive;

Simultaneous determination of resorcylic acid lactones, β and α trenbolone and stilbenes in bovine urine by UHPLC/MS/MS by L. Fernández-Arauzo; D. Pimentel-Trapero; M. Hernández-Carrasquilla (89-96).
Treatment of cattle with α-zearalanol (zeranol, α-ZAL), a resorcylic acid lactone (RAL), is illegal in European Union countries. Zearalenone, a common contaminant of cattle feed, is also a RAL and there is evidence that it, or its metabolites, can be converted in vivo to α-ZAL (or to β-zearalanol, β-ZAL). To determine whether an animal has been treated with α-ZAL it is necessary to quantify separately all the RALs. This work presents the simultaneous determination in urine of RALs, β-trenbolone (β-TB) and its metabolite α-trenbolone (α-TB) and the stilbenes diethylstilbestrol (DES), dienestrol (DEN) and hexestrol (HEX) using Ultra High Performance Liquid Chromatography/Mass Spectrometry (UHPLC/MS/MS). Several chromatographic UHPLC columns were tested in order to achieve chromatographic separation of the analytes and the results are shown. Baseline separation of all compounds was not possible, so that careful consideration of the MRM transitions was necessary. The separation chosen for the validation work used a 100 mm × 2.1 mm × 1.7 μm Phenyl column eluting with a gradient of acetonitrile/methanol/water. The method validation according to EU Decision 657/2002 included linearity, within laboratory reproducibility and trueness, decision limit (CCα) and detection capability (CCβ). For all compounds the method was linear in the range 2–12 μg/l (1 and 6 μg/l for DES) with determination coefficients greater than 0.97 and linear residuals below 20%. Within laboratory reproducibility was lower than 25% and trueness less than 11% for all compounds and concentration levels. CCα ranged from 0.6 μg/l (DES) to 1.6 (α-TB) and CCβ was 0.8 μg/l (α-zearalenol) to 1.9 μg/l (α-TB).
Keywords: Resorcylic acid lactones; Trenbolone; Stilbenes; urine; UHPLC/MS/MS;

A new, sensitive and efficient ultra fast liquid chromatography–tandem mass spectrometry (UFLC–MS/MS) method has been developed and validated for simultaneous quantification of cefazedone and etimicin in beagle dog plasma. After addition of the internal standard (IS) metronidazole, plasma samples were treated by protein precipitation procedure, and then separated on a Venusil MP C18 column (100 mm × 2.1 mm, 3.0 μm) (Venusil, China) using gradient elution with the mobile phase consisting of 0.01% heptafluorobutyric acid (HFBA) in acetonitrile and 0.01% HFBA in water at a flow rate of 0.4 mL min−1. The detection of the analytes was performed on 4000Q UFLC-MS/MS system with turbo ion spray source in the positive ion and multiple reaction-monitoring (MRM) mode. The linear range was 1.0–200 μg mL−1 for cefazedone and 0.5–100 μg mL−1 for etimicin, with lower limits of quantification of 1.0 and 0.5 μg mL−1, respectively. Intra-day and inter-day precisions were within 7.2% and 4.3%, respectively for both analytes, and the accuracy (relative error, RE, %) was less than 10.7% and 12.7%, respectively. The mean absolute extraction recoveries of analytes and IS from beagle dog plasma were all more than 73.22%. The validated method was successfully applied to the pharmacokinetic study of cefazedone and etimicin in beagle dog after intravenous administration of cefazedone injection combined with etimicin injection and the two single injections alone, respectively. The results indicated there were not obvious differences between the pharmacokinetic behaviors between the combined group and either of the single groups.
Keywords: Cefazedone; Etimicin; Ultra fast liquid chromatography–tandem mass spectrometry; Pharmacokinetics;

A simple, rapid and sensitive ultra high performance liquid chromatography with fluorescence detection (UHPLC-FLD) method was developed and validated for quantification of parishin and its metabolites in rats. Plasma samples were prepared by protein precipitation and then analyzed using UHPLC-FLD system. Repeated optimization showed that parishin and its metabolites, including gastrodin, p-hydroxybenzyl alcohol, parishin B and parishin C, could be sensitively detected based on the autofluorescence when excitation and emission wavelengths were set at 225 nm and 295 nm, respectively. The limit of detections (LODs) of GAS, HBA, PB, PC and PA reached 0.6, 0.8, 1, 1 and 1 ng/mL, respectively. The linearity for all targets was within the range 2.5–5000 ng/mL and the correlation coefficient (r 2) was larger than 0.999. Importantly, our method was almost free from matrix effects and the recoveries were higher than 80%. Additionally, our method also had high precision and accuracy for all analytes, presenting RSDs and REs within ±6% and ±14%, respectively. Finally, the validated UHPLC-FLD method was successfully applied for studying the pharmacokinetics of parishin following intragastrically administration in rats.
Keywords: Parishin; UHPLC-FLD; Metabolites; Pharmacokinetics;

A simple, rapid and specific high performance liquid chromatography–electrospray ionization tandem mass spectrometry method was developed for simultaneous determination of dabigatran etexilate (BIBR 1048 MS), the intermediate metabolite (BIBR 1087 SE) and dabigatran (BIBR 953 ZW). In this method, a stacked protein precipitation with methanol was performed in Sirocco 96-well filtration plates to extract analytes using only 50 μL plasma. The analysis was performed on an Ultimate TM XB-C18 (4.6 × 50 mm, 5 μm) column using gradient elution with a mobile phase composed of methanol containing 0.01% formic acid and pure water at a flow rate of 0.3 mL/min. The gradient was set to 90% methanol containing 0.01% formic acid for the first 1.0 min, after which it dropped to 10%, and then was kept at 10% for the next 5 min followed by an additional 1.0 min at the initial composition of 90% methanol containing 0.01% formic acid for equilibration. Detection was performed on a triple-quadrupole mass spectrometer electrospray ionization interface in positive ion mode. Linear calibration curves were obtained over the concentration ranges of 1–500 ng/mL for all analytes. The validated LC–MS/MS method for its selectivity, sensitivity, linearity, precision, accuracy, recovery, matrix effect and stability had been successfully applied to a pharmacokinetic study of analytes in rat plasma following a single oral administration of 15 mg/kg dabigatran etexilate.
Keywords: Dabigatran; Sirocco 96-well filtration plates; LC–MS/MS; Method validation; Pharmacokinetic study;

A simple, sensitive and rapid ultra high performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) method has been developed and validated for the quantification of liquiritigenin, a promising anti-tumor agent. Liquiritigenin and the internal standard were separated on an Agilent Extend C18 column and eluted with a gradient mobile phase system of acetonitrile and water. The analysis was performed on a negative ionization electrospray mass spectrometer via multiple reaction monitoring (MRM). Transitions of m/z 255.0 → 119.0 for liquiritigenin and m/z 269.0 → 117.0 for the IS were monitored. One-step protein precipitation with acetonitrile was used to remove impurities and extract the analytes from plasma. The method had a chromatographic run time of 4.5 min and a good linearity in the range of 1–1000 ng/mL. The precision (R.S.D.) of intra-day and inter-day ranged from 4.54 to 10.65% and 5.94 to 13.81%, respectively; while the accuracy of intra-day and inter-day ranged from 104.06 to 109.28% and 94.98 to 112.05%. The recovery and stability were also within the acceptable limits. The validated method was applied to a linear pharmacokinetic study of liquiritigenin in rat plasma for the first time.
Keywords: Liquiritigenin; UHPLC–MS/MS; Intravenous administration; Linear pharmacokinetic study;

Development of an intermediate chromatography step in an insulin purification process. The use of a High Throughput Process Development approach based on selectivity parameters by Eva Heldin; Sara Grönlund; Jamil Shanagar; Elisabeth Hallgren; Kjell Eriksson; Mariza Xavier; Heloisa Tunes; Luciano Vilela (126-132).
Recent innovations in designing purification processes for biopharmaceutical production have enabled initial screening (optimization) of chromatographic conditions for binding to be performed in miniaturized batch format. The present report demonstrates the possibility of using this format to screen for selectivity and illustrates the need for careful adjustment of protocols when highly abundant, tightly-binding impurities are present in the sample. This batch format approach was used to choose a chromatography medium (resin) from a selection of available resins for the purification of recombinant insulin expressed in E. coli and to screen binding and elution conditions.Subsequent optimization was performed in small packed columns using a Design of Experiments (DoE) approach with statistical modeling before scaling up to a small pilot scale experiment. In this study insulin was effectively purified from the more tightly-binding C-peptide, and a reduction in insulin variants was also noted using the optimized conditions.
Keywords: Microtiter filter plates; Batch adsorption; Insulin; Purification; Multimodal chromatography; HTPD; Screening of elution conditions;

A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the determination and quantification of four predominantly used analgosedatives in the intensive care unit: ketamine, lorazepam, midazolam and sufentanil in human serum. The extraction procedure consisted of protein precipitation of serum samples with acetonitrile and subsequent centrifugation. D5-fentanyl and D4-midazolam served as internal standards (ISTD). Separation of analytes was performed with a Hypersil C18 column and a mobile phase with acetonitrile and 0.1% formic acid (60/40, v/v) under isocratic conditions at a flow rate of 280 μl/min. Analytes were simultaneously detected with a triple-stage quadrupole mass spectrometer (LC–MS/MS) in a selected reaction monitoring (SRM) mode with positive heated electrospray ionization (HESI) within a single 2-min run. Calibration curves were linear over a range of 50–2000 for ketamine, 10–1000 for lorazepam, 5–500 for midazolam and 1–100 for sufentanil (ng/ml). The limit of detection and the lower limit of quantification were 0.01 and 10.00 for ketamine, 0.005 and 10.00 for lorazepam, 0.018 and 5.00 for midazolam and 0.068 and 0.25 for sufentanil (ng/ml). Intra- and inter-day accuracies and precisions of all analytes were less than 15%. Bench stability with spiked serum samples was ensured after 12, 24 and 48 h at room temperature, freeze- and thaw-stability after 3 cycles of thawing and freezing. The method was successfully established according to International Conference on Harmonization (ICH) guideline Q2 (R1) “Validation of Analytical Procedures” and applied in critically ill adult patients in the intensive care unit. We suggest its suitability for parallel quantification of the sedative analgesics ketamine, lorazepam, midazolam and sufentanil. The method serves as an instrumental tool for therapeutic drug monitoring (TDM) and pharmacokinetic studies [1].
Keywords: Liquid chromatography–tandem mass spectrometry; Ketamine; Lorazepam; Midazolam; Sufentanil; Intensive care unit; Therapeutic drug monitoring (TDM);

Hollow fiber-solid phase micro-extraction (HF-SPME) technique containing sol–gel-derived Fe3O4/SiO2/TiO2 core-double shell nanocomposite as a novel high efficiency sorbent, coupled with high performance liquid chromatography was used to extraction and determination of six non-steroidal anti-inflammatory drugs; acetylsalicylic acid, naproxen, piroxicam, diclofenac, indomethacin and mefenamic acid, in hair samples. First, magnetite nanoparticles (Fe3O4-NPs) were synthesized by chemical co-precipitation of Fe(II) and Fe(III) ions (where the ratio of Fe(II) to Fe(III) is 1:2 and a non-oxidizing environment), in alkaline medium to produce magnetite particles. Subsequently, surface of Fe3O4-NPs was modified with SiO2 and TiO2 using layer-by-layer chemical technique. A core–shell structure of Fe3O4/SiO2/TiO2 composite was prepared by coating magnetite core particles with silica and titania layers. In the proposed method, NSAIDs were extracted by the synthesized nanocomposite and analyzed by HPLC. The parameters affecting the efficiency of magnetic nanoparticle (MNPs) assisted HF-SPME were investigated and optimized. The method validation was included and satisfying results with high pre-concentration factors (405 up to 2450) were obtained. It owes large surface area and porosity of the nano-adsorbent. Under the optimal conditions, the method detection limits (S/N = 3) were in the range of 0.01–0.10 μg ml−1 and the limits of quantification (S/N = 10) between 0.04 and 0.30 μg ml−1. Relative standard deviations were 3.09–6.61%. Eventually, the method was successfully applied to human hair after administration of NSAIDs.
Keywords: Hollow-fiber solid-phase micro-extraction (HF-SPME); Fe3O4/SiO2/TiO2 nano composite; Sol–gel; Non-steroidal anti-inflammatory drugs; Hair sample;