Journal of Chromatography B (v.990, #C)

Polybrominated diphenyl ethers (PBDEs) have become ubiquitous environmental contaminants due to their incorporation into many consumer products. Their ability to bioaccumulate to alarming levels in fat-rich matrices such as fish demands fast and efficient methods to monitor these contaminants. We present an analytical method for selective-pressurised liquid extraction (S-PLE) of PBDEs from fish tissue. Fat removal performance of different mixtures of Florisil, silica gel and sulphuric acid-impregnated silica gel were evaluated using a response surface experimental design approach for determining the optimal fat-retaining mixture for S-PLE. Acid-silica gel had the greatest individual effect on fat retention; with a two-thirds acid-silica one-third Florisil mixture found to be the most efficient (>97%). Method validation was performed using recovery experiments at three spiked concentration levels (0.05, 0.5 and 5 ng g−1 ww). Mean recoveries of target analytes in spiked samples ranged from 70 to 124%, with relative standard deviations <27%. The S-PLE lipid removal efficiency combined with the sensitivity of triple quadrupole mass spectrometers provides a fast and comparatively inexpensive analytical method for analysis of PBDEs in fish samples.
Keywords: Pressurised liquid extraction; S-PLE; Accelerated solvent extraction; In-cell cleanup; Polybrominated diphenyl ethers; Fish;

Purity determination of amphotericin B, colistin sulfate and tobramycin sulfate in a hydrophilic suspension by HPLC by Corina Pfeifer; Georg Fassauer; Hagen Gerecke; Thomas Jira; Yvonne Remane; Roberto Frontini; Jonathan Byrne; Robert Reinhardt (7-14).
A suspension comprising of the three antibiotic substances amphotericin B, colistin sulfate and tobramycin sulfate is often used in clinical practice for the selective decontamination of the digestive tract of patients in intensive care. Since no detailed procedures, specifications or stability data are available for manufacturing this suspension, there may be discrepancies regarding formulation and stability of suspensions prepared in different pharmacies. The aim of this work is to develop a standardized formulation and to determine its stability under defined storage conditions. This would help guarantee that all patients receive the same preparation, therefore ensuring similar efficacy and improved safety. The first step in this process is to develop the required analytical tools to measure the content and purity of the drug substances in this complex mixture. In this paper, the development and validation of these tools as well as the development of the drug suspension formulation is described. The formulation comprises of Ampho-Moronal®-Suspension (Dermapharm) and a buffered, preservated aqueous solution of colistin sulfate and tobramycin sulfate. Two simple, well established high-performance liquid chromatography (HPLC) methods in the European Pharmacopoeia (EP) for impurity profiling of the two active ingredients amphotericin B and colistin sulfate were combined with a newly developed sample extraction procedure for the suspension. Sufficient selectivity and stability-indicating power have been demonstrated. Additionally, a new robust routine method was developed to determine possible degradation products of tobramycin sulfate in the investigated suspension. The specificity, precision, accuracy and linearity of the analytical procedures were demonstrated. The recovery rate was in the range of 90–110%. The precision results for the calculated impurities showed variation coefficients of <10%. The calibration curves were found to be linear with correlation of greater than 0.9994 for all components. The results show the suitability of the methods for the quality control analysis of the suspension.
Keywords: Amphotericin B; Colistin sulfate; Tobramycin sulfate; Suspension; HPLC; ELSD;

Column chromatography has been widely used as a scalable purification strategy for recombinant adeno-associated virus (rAAV) vectors. The rAAV1, 2, 4, 5, 6, 8 and 9 serotypes could be separated using affinity resins, ion exchange resins or other types of resins. Apatite resin has displayed outstanding performance in protein purification in the past 10 years, and ceramic hydroxyapatite (CHT) chromatography resin with a polyethylene glycol (PEG) modulation has recently been used for rAAV1 and rAAV9 vectors. This study reports the use of CHT chromatography modulated by calcium ions instead of PEG for rAAV9 purification. Calcium-ion-containing buffers effectively improve the inclusion of CHT as a capture resin, the resin-binding capacity and the yield. The optimum calcium ion concentration is 30 ppm, and the optimum pH is 7.0. A frontal analysis indicated that the binding capacity of CHT at 2 ml/min reaches 65.1 mg total protein per ml of resin. A previously developed purification strategy consists of CHT followed by ANX anion exchange chromatography. The vector yield of this approach is approximately 70%, and a software analysis indicated a vector purity exceeding 98%. The residual host cell (HEK293) protein contents are 24.75 ± 2.32 ng and 67.21 ± 2.10 ng, and the Benzonase residue contents are 1.55 ± 0.10 pg and 1.95 ± 0.16 ng per 1013 vector genome copies (G.C.) separated by CHT/ANX and CsCl. In addition, CHT/ANX yields 798.44 ± 50.10 pg of plasmid DNA and 2.17 ± 0.11 ng of HEK293 DNA, while CsCl purification yields 840.27 ± 76.14 pg of plasmid DNA and 2.43 ± 0.19 of HEK293 DNA. The two methods produce vectors with similar in vitro and in vivo potencies. The results indicated that the CHT/ANX method is suitable for the scalable purification of the rAAV9 vector.
Keywords: Calcium ion; Ceramic hydroxyapatite resin; Scalable purification; rAAV9;

Development of a SPE-HPLC–MS/MS method for the determination of most prescribed pharmaceuticals and related metabolites in urban sewage samples by Robert Gurke; Julia Rossmann; Sara Schubert; Tobias Sandmann; Martin Rößler; Reinhard Oertel; Joachim Fauler (23-30).
Based on regional prescription data several pharmaceuticals with variable amounts of prescription and corresponding metabolites were selected and analyzed in influent and effluent samples of the sewage treatment plant (STP) in Dresden, Germany. Pharmaceuticals of the following most prescribed therapeutic groups were chosen: antibiotics, antifungals, anticonvulsants, antipsychotics, antidepressants, and cardiovascular active compounds like beta blockers and angiotensin-converting enzyme inhibitors. To analyze the selected compounds, a multi-target method was developed and applied to 24-h composite wastewater samples for three single days in May and June 2014. The method was based on a cleanup of a sample with a volume of 1 mL using solid phase extraction followed by a high performance liquid chromatography coupled to a tandem mass spectrometer. Analytes were separated in a 15 min chromatographic separation and quantified using 23 Internal Standards and a calibration curve in 40-fold diluted blank urine. The limit of quantification varied between 50 and 200 ng/L and for all analytes good accuracy and precision as well as linearity for the calibration curve with the correlation coefficient R 2 higher than 0.99 was reached. A total of 41 and 40 of the selected 55 analytes were detected and quantified in the influent and effluent samples of the studied STP, respectively. Valsartan was the compound with the highest maximum concentration in influent (27.1 μg/L) and effluent (15.7 μg/L). Furthermore, analytes like bezafibrate, candesartan, carbamazepine, gabapentin, metoprolol, levetiracetam, pregabalin and telmisartan as well as the metabolite O-desmethyl venlafaxine were detectable in influent and effluent samples, respectively, with a concentration higher than 1 μg/L.
Keywords: Pharmaceutical; Metabolites; Sewage; SPE-HPLC–MS/MS; Multi-target; Regional prescription data;

A sensitive and selective liquid chromatography tandem mass spectrometric method was developed and validated for the simultaneous determination of five pyridine alkaloids contained in tripterygium glycosides tablets (triptolide, wilforine, wilforgine, wilfording and wilfortrine) in dog plasma. The analysis was carried out on a Sepax GP-Phenyl column using a mixture of methanol and 10 mmol/L ammonium formate buffer solution containing 0.1% formic acid (75:25, v/v) as the mobile phase pumped at a flow-rate of 1.0 mL/min. All MS data were obtained in the positive ESI mode with selective multiple reaction monitoring of ion transitions. The method was fully validated to be accurate and precise with a linear range of 0.2–1000 ng/mL for triptolide and 0.05–1000 ng/mL for the other four pyridine alkaloids. The intra-day and inter-day precisions (relative standard deviation, RSD, %) were within 10.6% and 14.0%, respectively, and the relative error (RE, %) were all less than 13.1%. The method was successfully applied to multi-components pharmacokinetic study of the five pyridine alkaloids in beagle dogs after a single oral administration of 3 mg/kg and 30 mg/kg tripterygium glycosides tablets, respectively, and a multiple oral administration of 30 mg/kg for 6 consecutive days.
Keywords: Triptolide; Wilforine; Wilforgine; Wilfording; Wilfortrine; LC–MS/MS;

Determination of cyproheptadine in feeds using molecularly imprinted solid-phase extraction coupled with HPLC by Jianwen Yang; Zongnan Wang; Tong Zhou; Xuqin Song; Qingyong Liu; Yuman Zhang; Limin He (39-44).
A novel method was developed for the determination of cyproheptadine in feeds using molecularly imprinted solid-phase extraction coupled with high-performance liquid chromatography. The polymers were prepared using cyproheptadine as a template molecule, methacrylic acid as a functional monomer, ethylene glycol dimethacrylate as a cross-linking agent, and dichloromethane as a solvent by bulk polymerization. Under the optimum solid-phase extraction conditions, the molecular imprinting cartridge can selectively extract and enrich cyproheptadine from a variety of feeds. Mean recoveries of cyproheptadine from four kinds of feeds spiked at 0.1, 1.0 and 10 mg kg−1 ranged from 85.5% to 96.2%, with intra-day and inter-day relative standard deviation less than 10%. The calibration curve of cyproheptadine was good linear relationship (r  > 0.9993) within the range of 0.1–50 μg mL−1. The limit of detection (LOD) and the limit of quantification (LOQ) were 0.04 and 0.1 mg kg−1, respectively.
Keywords: Molecularly imprinted polymers; Cyproheptadine; High performance liquid chromatography; Feeds;

Triacylglycerols (TAGs) are a large class of neutral lipids that naturally occur in both plant and animal oils and fats. Their analyses in Non-Aqueous Reversed Phase Liquid Chromatography (NARP) require a mixture of weak solvent (mostly acetonitrile) and strong solvent. In the present work, we have established eluotropic solvent strength scale of several binary mobile phases on C18 bonded silica at different temperatures (acetonitrile/methylene chloride, acetonitrile/acetone, acetonitrile/ethyl acetate, acetonitrile/propan-2-ol, and acetonitrile/butan-1-ol at 25 °C, 43 °C, 63 °C and 85 °C); it is based on the methylene selectivity and the use of homologous series. We show that this scale is well suited to the TAGs analysis. The analysis of nine seed oils (Aleurites fordii, Calophyllum inophyllum, Glycina max, Olea europea, Orbignya olifeira, Pinus koraiensis, Pistacia lentiscus, Punica granatum and Ribes nigrum) in iso-eluotropic conditions leads to propose unambiguously the couple MeCN/BuOH at 25 °C as the best system to separate TAGs. The use of butanol, as strong solvent, provides very good TAGs congeners separations and avoids the use of chlorinated solvents which gave to this day the best separations.
Keywords: Non-aqueous reversed phase; Triacylglycerols; Eluotropic solvent strength scale; Iso-eluotropic mobile phases; Butanol; Temperature effect;

Lipidomic analysis of plasma, erythrocytes and lipoprotein fractions of cardiovascular disease patients using UHPLC/MS, MALDI-MS and multivariate data analysis by Michal Holčapek; Blanka Červená; Eva Cífková; Miroslav Lísa; Vitaliy Chagovets; Jitka Vostálová; Martina Bancířová; Jan Galuszka; Martin Hill (52-63).
Differences among lipidomic profiles of healthy volunteers, obese people and three groups of cardiovascular disease (CVD) patients are investigated with the goal to differentiate individual groups based on the multivariate data analysis (MDA) of lipidomic data from plasma, erythrocytes and lipoprotein fractions of more than 50 subjects. Hydrophilic interaction liquid chromatography on ultrahigh-performance liquid chromatography (HILIC-UHPLC) column coupled with electrospray ionization mass spectrometry (ESI-MS) is used for the quantitation of four classes of polar lipids (phosphatidylethanolamines, phosphatidylcholines, sphingomyelins and lysophosphatidylcholines), normal-phase UHPLC—atmospheric pressure chemical ionization MS (NP-UHPLC/APCI-MS) is applied for the quantitation of five classes of nonpolar lipids (cholesteryl esters, triacylglycerols, sterols, 1,3-diacylglycerols and 1,2-diacylglycerols) and the potential of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is tested for the fast screening of all lipids without a chromatographic separation. Obtained results are processed by unsupervised (principal component analysis) and supervised (orthogonal partial least squares) MDA approaches to highlight the largest differences among individual groups and to identify lipid molecules with the highest impact on the group differentiation.
Keywords: Lipids; Lipidomics; Cardiovascular diseases; Lipoprotein fractions; UHPLC/MS; Multivariate data analysis;

Epimedii herba is a traditional Chinese medicine for the treatment of osteoporosis. Epimedin A, B and C and icariin are the primary effective ingredients of this medicine. In this study, a simple and low-cost method based on pipette tip solid-phase extraction, high-performance liquid chromatography separation, and diode array detection has been developed for the simultaneous analysis of four flavonoids (epimedin A, B and C and icariin) from Epimedii herba in rat serum samples. In this novel extraction configuration, the sorbents were placed between a filter (hollow fiber) and the pipette tip. Pipette tip solid-phase extraction has several advantages compared to conventional extraction methods: faster extraction time (6.0 min); lower sample volume (100 μL); lower solvent volume (100 μL); and less solvent waste. Under the optimum extraction conditions, the method showed good linearity (0.05–10.0 μg mL−1), acceptable intra- and inter precision (RSD < 6%), low limits of quantification (0.027–0.045 μg mL−1) and satisfactory relative recoveries (98.63–103.18%). This method was successfully applied to investigate the pharmacokinetics of the major flavonoids in Epimedii herba extract after oral administration to rats (10 g kg−1 body weight). The primary pharmacokinetic parameters for rats were determined as follows: C max, 0.45–4.11 μg mL−1; T max, 0.21–0.26 h; t 1/2α, 0.06–0.12 h; t 1/2β, 2.02–3.48 h; AUC0–∞: 0.50–2.58 μg h mL−1; CL, 19.53–44.72 L kg−1  h−1; and MRT0–∞, 2.25–3.77 h. The developed method has the potential to promulgate the pharmacokinetics and provide more information for clinical applications.
Keywords: Pipette tip extraction; Flavonoids; Epimedii herba; Serum; Pharmacokinetics;

Affinity extraction based on the interaction between a target molecule and a specific affinity ligand offers a novel separation system for biomolecules in an aqueous two-phase system, however, most of affinity ligands are expensive. In the present study, metal affinity extraction of histidine (His) derivatives using a complex between Cu(II) and a commercially available chelating ligand was studied in a poly(ethylene glycol) (PEG)/Li2SO4 ATPS. Alizarin complexone (3-[N,N-bis(carboxymethyl)amino methyl]-1,2-dihydroxy anthraquinone, AC) was selected as the chelating ligand because of the good extractability of Cu(II) into the upper PEG-rich phase. On the basis of coordinate bonding with Cu(II), the extraction of His in the presence of the Cu(II)–AC complex under neutral condition was 73%, which was much higher than that under Cu(II) free condition (13%). Among a series of divalent transition metal ions (Cu(II), Ni(II), Co(II), and Zn(II)), Cu(II) was the most effective for the extraction of His. Derivatives of His were selectively extracted in the presence of many other amino acids because of the specificity of the interaction between Cu(II) and imidazole group of His. Extracted His was quantitatively stripped from the Cu(II)–AC complex using competitive complexation with agents such as iminodiacetic acid and imidazole.
Keywords: Aqueous two-phase system; Extraction; Immobilized metal affinity chromatography; Histidine; Alizarin complexone;

A validated LC–MS/MS method for determination of periplogenin in rat plasma and its application in pharmacokinetic study by Fang Bo; Ting Dou; Xingrui Wang; Paul Owusu Donkor; Huizi Ouyang; Yanxu Chang; Yaru Tu; Xiumei Gao; Jun He (80-83).
A method coupling high performance liquid chromatography with tandem mass spectrometry has been developed and validated for quantifying periplogenin in rat plasma using psoralen as an internal standard (IS). Plasma samples were pretreated using a simple liquid–liquid extraction with ethyl acetate and the chromatographic separation of periplogenin and psoralen was achieved on a Waters XBridge™ BEH C18 column with 0.1% formic acid and acetonitrile as mobile phase at a flow rate of 0.4 mL/min. The detection was performed on a positive ion mode with electrospray ionization (ESI) source. The optimized ion transition pairs for quantitation were m/z 391.3 →  m/z 337.2 for periplogenin and m/z 187.0 →  m/z 131.0 for IS. The total run time was 9.0 min. The calibration curve was linear over the range of 0.2–250 ng/mL (r  > 0.99) with the lower limit of quantitation (LLOQ) at 0.2 ng/mL. The intra- and inter-day precision were below 9.85% and the mean accuracy were from −10.03% to 10.26%. The average recoveries of periplogenin in plasma ranged from 85.1% to 95.6%. The proposed method was successfully applied in evaluating the pharmacokinetics of periplogenin after an oral dose of 30 mg/kg Cortex Periplocae extract in rats.
Keywords: Periplogenin; LC–MS/MS; Pharmacokinetics; Cortex Periplocae;

Lysozyme is an important polypetide used in medical and food applications. We report a novel magnetic strong cation exchange beads for efficient purification of lysozyme from chicken egg white. Magnetic chitosan (MCHT) beads were synthesized via phase inversion method, and then grafted with poly(glycidyl methacrylate) (p(GMA)) via the surface-initiated atom transfer radical polymerization (SI-ATRP). Epoxy groups of the grafted polymer, were modified into strong cation-exchange groups (i.e., sulfonate groups) in the presence of sodium sulfite. The MCTH and MCTH-g-p(GMA)-SO3H beads were characterized by ATR-FTIR, SEM, and VSM. The sulphonate groups content of the modified MCTH-g-p(GMA)-4 beads was found to be 0.53 mmol g−1 of beads by the potentiometric titration method. The MCTH-g-p(GMA)-SO3H beads were first used as an ion-exchange support for adsorption of lysozyme from aqueous solution. The influence of different experimental parameters such as pH, contact time, and temperature on the adsorption process was evaluated. The maximum adsorption capacity was found to be 208.7 mg g−1 beads. Adsorption of lysozyme on the MCTH-g-p(GMA)-SO3H beads fitted to Langmuir isotherm model and followed the pseudo second-order kinetic. More than 93% of the adsorbed lysozyme was desorbed using Na2CO3 solution (pH 11.0). The purity of the lysozyme was checked by HPLC and SDS gel electrophoresis. In addition, the MCTH-g-p(GMA)-SO3H beads prepared in this work showed promising potential for separation of various anionic molecules.
Keywords: Chitosan; Magnetic beads; Ion-exchange beads; Adsorption; Purification; Lysozyme;

Fabrication of a polystyrene microfluidic chip coupled to electrospray ionization mass spectrometry for protein analysis by Xianqiao Hu; Yuanyuan Dong; Qiaohong He; Hengwu Chen; Zhiwei Zhu (96-103).
A highly integrated polystyrene (PS) microfluidic chip coupled to electrospray ionization mass spectrometry for on-chip protein digestion and online analysis was developed. The immobilized enzymatic microreactor for on-chip protein digestion was integrated onto microchip via the novel method of region-selective UV-modification combined with glutaraldehyde-based immobilization. The micro film electric contact for applying high voltage was prepared on chips by using UV-directed electroless plating technique. A micro-tip was machined at the end of main channel, serving as the interface between microchip and mass spectrometric detector. On-chip digestion and online detection of protein was carried out by coupling the microchip with mass spectrometry (MS). The influences of methanol flow rate in side channel on the stability of spray and intensity of signals were investigated systematically. Also the influence of sample flow rate on the performance of immobilized enzymatic reactor were investigated. Stable spray was obtained at the spray voltage of 2.8–3.0 kV and the methanol flow rate of 500–700 nL min−1 with the relative standard deviation (RSD) of total ion current (TIC) less than 10%. The influence of sample flow rate on the performance of immobilized enzymatic reactor was also studied. The sequence coverage of protein identification decreased with the increase of flow rate of the sample solution. A sequence coverage of 96% was obtained with immobilized enzymatic reactor at the sample flow rate of 100 nL min−1 with the reaction time of 8.4 min. It could detect cytochrome c as low as 10 μg mL−1 with the developed system. No obvious decrease in protein digestion efficiency was observed after the chip continuously performed for 4 h and stored for 15 d.
Keywords: Microfluidic chip; Electrospray ionization–mass spectrometry; Polystyrene; Immobilized enzymatic reactor; Gold film electric contact; Interface;

Isolation of C-glycosylflavonoids with α-glucosidase inhibitory activity from Passiflora bogotensis Benth by gradient high-speed counter-current chromatography by Geison Modesti Costa; Paola Andrea Cárdenas; Andressa Córneo Gazola; Diana Marcela Aragón; Leonardo Castellanos; Flávio Henrique Reginatto; Freddy Alejandro Ramos; Eloir Paulo Schenkel (104-110).
In this study, we applied a gradient High-Speed Counter-Current Chromatography (HSCCC) method that allowed, by direct injection of an aqueous crude extract of the leaves of Passiflora bogotensis, the successful isolation of six flavonoids in a single run, with purity of each compound higher than 81%. This separation enabled the isolation of two new flavonoid glycosides, apigenin-6-C-α-l-rhamnopyranosyl-(1 → 2)-(6″-O-acetyl)-β-d-glucopyranoside (2) and luteolin-6-C-α-l-rhamnopyranosyl-(1 → 2)-(6″-O-acetyl)-β-d-glucopyranoside (4), and four known ones, isovitexin (1), isoorientin (3), isovitexin-2″-O-rhamnoside (5) and isoorientin-2″-O-rhamnoside (6). The structures of the isolated compounds were identified by HPLC-DAD, LC-MS, 1H and 13C NMR and comparison with literature data. The inhibitory activities of all of these compounds were evaluated in vitro on α-glucosidase from S. cerevisiae, and the IC50 was determinate. This is the first study concerning the chemical composition and biological activity of Passiflora bogotensis.
Keywords: High-speed counter-current chromatography; Passiflora bogotensis; C-glycosylflavonoids;

Urolithins were separated from the intestinal metabolites of pomegranate ellagitannins by high-speed counter current chromatography in two steps using two solvent systems composed of n-hexane-ethyl acetate-methanol-acetic acid-water (2.5:2:0.25:5, v/v/v/v/v) and n-hexane-ethyl acetate-methanol-acetic acid-water (2.5:0. 8:0.25:5, v/v/v/v/v) for the first time. Each injection of 100 mg extract yielded 21 mg of pure urolithin A and 10 mg of pure urolithin B. High-performance liquid chromatography analyses revealed that the purity of urolithin A and urolihtin B was over 98.5%. The structures of urolithin A and urolitihn B were identified by high resolution-MS, NMR and single crystal x-ray analysis. Urolithins reduced the oxidative stress status in colon cancer by decreasing the intracellular ROS and malondialdehyde levels, and increasing SOD activity in H2O2 treated Caco-2 cells.
Keywords: Pomegranate husk; Urolithins; High-speed counter-current chromatography (HSCCC); Intestinal metabolites; Anti-oxidant;

Determination of N-methylcytisine in rat plasma by UPLC-MS/MS and its application to pharmacokinetic study by Shuanghu Wang; Haiya Wu; Xueli Huang; Peiwu Geng; Congcong Wen; Jianshe Ma; Yunfang Zhou; Xianqin Wang (118-124).
In this work, a sensitive and selective UPLC-MS/MS method for determination of N-methylcytisine in rat plasma is developed. After addition of hordenine as an internal standard (IS), protein precipitation by acetonitrile-methanol (9:1, v/v) was used to prepare samples. Chromatographic separation was achieved on a UPLC BEH HILIC (2.1 mm × 100 mm, 1.7 μm) with acetonitrile (containing 10 mM ammonium formate) and water (containing 0.1% formic acid and 10 mM ammonium formate) as the mobile phase with gradient elution. An electrospray ionization source was applied and operated in positive ion mode; multiple reaction monitoring (MRM) mode was used for quantification using target fragment ions m/z 205.1→58.0 for N-methylcytisine, and m/z 166.1→121.0 for IS. Calibration plots were linear throughout the range 2-2000 ng/mL for N-methylcytisine in rat plasma. Mean recoveries of N-methylcytisine in rat plasma ranged from 86.1% to 94.8%. RSD of intra-day and inter-day precision were both < 13%. The accuracy of the method was between 94.5% and 109.4%. The method was successfully applied to pharmacokinetic study of N-methylcytisine after either oral or intravenous administration. For the first time, the absolute bioavailability of N-methylcytisine was reported as high as 55.5%.
Keywords: N-methylcytisine; UPLC-MS/MS; pharmacokinetics; rat plasma;

In this study, a sensitive and rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated to simultaneously determinate andrographolide (AP), dehydroandrographolide (DP), and neoandrographolide (NP) in plasma of beagle dogs after oral administration of Andrographis paniculata tablet (A. paniculata). The analytes and bilobalide (internal standard) were separated on an Agilent ZORBAX XDB-C18 column (50 mm × 2.1 mm, 3.5 μm) by using gradient elution consisting of methanol and water at a flow rate of 0.50 mL/min in 7 min. Multiple reaction monitoring (MRM) mode was performed to quantify data under monitoring precursor-product ion transitions of m/z 348.8 → 286.9, 330.9 → 107.9, 479.1 → 160.8 and 325.0 → 163.0 for AP, DP, NP and internal standard (IS) at negative ion mode, respectively. This method was developed at linearity ranging from 0.50 to 250 ng/mL for AP, 1.00 to 500 ng/mL for DP and 0.20 to 100 ng/mL for NP. The accuracy of each analyte ranged between 94.8% and 107.1% and the precision was within 14.6%. No significant matrix effect was observed. AP, DP and NP were stable during sample storage, preparation and analytic procedures. Furthermore, this method was successfully applied in the investigation of the pharmacokinetic profile of AP, DP and NP in beagle dogs after oral administration of A. paniculata tablet (49.5 mg for AP, 7.0 mg for DP, 22.0 mg for NP). Biological half-life (t 1/2) was 2.08 ± 0.99, 3.13 ± 1.19 and 1.07 ± 0.38 h for AP, DP and NP, respectively. The areas under curves (AUC0–t ) of AP, DP and NP was 494.50 ± 150.64, 26.01 ± 8.72 and 78.78 ± 18.29 ng h/mL, respectively.
Keywords: Andrographis paniculata tablet; Andrographolide; LC–MS/MS; Beagle dog plasma; Pharmacokinetics;

The original analytical method for the simultaneous determination and confirmation of neonicotinoids insecticides (imidacloprid, clothianidin, acetamiprid, thiametoxam, thiacloprid, nitenpyram, dinotefuran) and some of their metabolites (imidacloprid guanidine, imidacloprid olefin, imidacloprid urea, desnitro-imidacloprid hydrochloride, thiacloprid-amid and acetamiprid-N-desmethyl) in honey bee and honey was developed. Preparation of honey bee samples involves the extraction with mixture of acetonitrile and ethyl acetate followed by cleaned up using the Sep-Pak Alumina N Plus Long cartridges. Honey samples were dissolved in 1% mixture of acetonitrile and ethyl acetate with addition of TEA, then extracts were cleaned up with Strata X-CW cartridges. The identity of analytes was confirmed using liquid chromatography tandem mass spectrometry. All compounds were separated on a Luna C18 column with gradient elution. The whole procedure was validated according to the requirements of SANCO 12571/2013. The average recoveries of the analytes ranged from 85.3% to 112.0%, repeatabilities were in the range of 2.8–11.2%, within-laboratory reproducibility was in the range of 3.3–14.6%, the limits of quantitation were in the range of 0.1–0.5 μg kg−1, depending of analyte and matrices. The validated method was successfully applied for the determination of clothianidin, imidacloprid and imidacloprid urea in real incurred honey bee samples and clothianidin in honey.
Keywords: Neonicotinoids; Metabolites; Honey bee; Honey; LC-MS/MS;

Fosinopril is an angiotensin-converting enzyme inhibitor containing a phosphate ester group which undergoes esterase hydrolysis to its active metabolite, fosinoprilat. EDTA was utilized as an anticoagulant to inhibit the hydrolysis of fosinopril in whole blood during blood collection and processing. To prevent the ex vivo conversion to fosinoprilat, formic acid was added to rat plasma to effectively stabilize fosinopril. A sensitive, rapid and robust ultra-fast liquid chromatography–tandem mass spectrometry (UFLC-MS/MS) method was developed and validated for simultaneous determination of fosinopril and fosinoprilat in rat plasma. Protein precipitation was employed for plasma sample clean-up. Chromatographic separation was achieved on a Welch Ultimate XB-C18 column using gradient elution with a total run time of 5 min. Analytes and their stable isotope labeled internal standards were detected by positive ion electrospray tandem mass spectrometric assay. The assay involves quantitation of both analytes in small-volume (50 μL) plasma, with the lower limit of quantification of 0.1 and 1 ng/mL for fosinopril and fosinoprilat, respectively. The method was fully validated in linear calibration ranges of 0.1–150 ng/mL for fosinopril and 1–1500 ng/mL for fosinoprilat with acceptable accuracy and precision. Assay recoveries were high (>95% for fosinopril and >91% for fosinoprilat) and matrix effect was negligible. Both analytes were found to be stable in stabilized rat plasma for 6 h at room temperature, 30 days at −80 °C, and following three freeze–thaw cycles and were also stable in processed samples for 36 h at 4 °C. The validated method was successfully applied to sample analyses for pharmacokinetic study of fosinopril and can be extended to the measurement of fosinopril in other biological samples.
Keywords: Fosinopril; Fosinoprilat; UFLC-MS/MS; Esterase inhibitor; Formic acid;

Evaluation of four derivatization methods for the analysis of fatty acids from green leafy vegetables by gas chromatography by Anna Topolewska; Karolina Czarnowska; Łukasz P. Haliński; Piotr Stepnowski (150-157).
Green leafy vegetables are valuable secondary sources of nutrients, including lipids, commonly consumed in developing countries. However, method development for the analysis of fatty acids is usually focused on the animal lipid samples, rarely including natural plant extracts. Hence, the usefulness of four derivatization methods for the gas chromatographic analysis of plant lipids was studied. Methylation using 10% solution of BF3 in methanol and 2.0 M solution of (trimethylsilyl)diazomethane (TMSD) in hexane, trimethylsilylation and tert-butyldimethylsilylation were compared using lipid standards and extracts from the leaves of Solanum macrocarpon and S. melongena after saponification. While silylation was found effective and precise using lipid standards, it initially did not perform well in the analysis of plant lipids due to the presence of transesterification products in samples. Optimization of the hydrolysis conditions resulted in an effective analysis of these derivatives, but poor separation of FA(18:0) from unsaturated FA(18:X) compounds and the presence of larger amounts of interferences disqualified the use silylation for the analysis of plant fatty acids in applied analytical conditions. Methylation using TMSD gave more precise quantitative results when compared to BF3/MeOH method. Also, it produced a significantly lower amount of interferences when applied to plant lipid samples. Additionally, the TMSD-based method is simple, safe and less time-consuming when compared to other procedures. Thus, we suggest using TMSD-based methylation as a method of choice in the GC analysis of plant-derived fatty acids.
Keywords: Fatty acids; Plant lipids; Green leafy vegetables; Derivatization; Gas chromatography; (Trimethylsilyl)diazomethane;

Liquid chromatography–mass spectrometry method development for monitoring stress-related corticosteroids levels in pig saliva by Ledicia Rey-Salgueiro; Elena Martínez-Carballo; Jesús Simal-Gándara (158-163).
Biochemical response stressors results in an increase of adrenocortical activity. Before knowing the corticosteroid levels in saliva in a stressful situation, baselines salivary levels should be established. A method for simultaneous determination of five corticosteroids was developed, validated and applied to pig saliva at farms. The method employs solid-phase extraction (SPE) coupled with clean-up extraction step using silica cartridge in the same step followed by liquid chromatography/tandem mass spectrometry (LC–MS/MS), using electrospray ionization (ESI) in positive mode. The overall method quantification limits range from 0.050 to 0.30 μg/L for the enrichment of 1.0 mL saliva samples and analyte recoveries are between 60 and 90% (RSD < 11%). Some factors studied were: pig sex, breeds, and time at farm. The analytical method clearly shows that CRL and CRS levels of, respectively, 3.0 and 4.0 μg/L in saliva can be indicative of maxima non-stress levels in different pig breeds at farm.
Keywords: Solid-phase extraction; Liquid chromatography/tandem mass spectrometry; Pig breeds; Saliva; Stress hormones; Corticosteroids;

Fosfomycin is a small, hydrophilic antibiotic drug with activity against Gram-positive as well as Gram-negative pathogens. It is in increasing use in intensive care units as a last line antibiotic since it shares no cross-resistance with other antibiotics. It is not metabolized and plasma levels are dependent on renal excretion rate and renal replacement therapy such as hemofiltration or hemodialysis. Measurement of fosfomycin plasma concentrations is therefore highly desirable in order to optimize dosing. We have developed a method for the quantification of fosfomycin in human plasma using HILIC chromatography on a silica stationary phase and tandem mass spectrometric detection. Sample preparation consisted only of protein precipitation without derivatization. Propylphosphonic acid was used as internal standard. Two calibration ranges from 15 to 150 μg/ml and 100 to 750 μg/ml were necessary to cover the whole range of plasma concentrations expected from intensive care patients. Intraday precision ranged from 4.0% to 6.4%, depending on the concentration level, with accuracies ranging from −1.1% to 11.5%. The corresponding interday precisions and accuracies were 2.0–11.0% and 0.6–7.8%, respectively. Fosfomycin was stable in human plasma under all storing conditions relevant for clinical samples. First experiences with this method in clinical routine use confirmed the applicability and ruggedness of the analytical procedure.
Keywords: Fosfomycin; Human plasma; HILIC; LC–MS/MS;

Development of ESI-MS-based continuous enzymatic assay for real-time monitoring of enzymatic reactions of acetylcholinesterase by Qiang Fu; Jun Tang; Meng Cui; Zhong Zheng; Zhiqiang Liu; Shuying Liu (169-173).
The continuous enzymatic assay based on ESI-MS was developed to real-time monitoring of enzymatic reactions of acetylcholinesterase (AChE). The changes of product concentrations were continuously measured. Calibration curves were established for quantitative calculation. By this method, the Michaelis constant (Km) of acetylcholinesterase was determined to be 70.60 ± 0.93 μM and Huperzine A as an effective inhibitor of acetylcholinesterase displayed a mixed inhibition with competitive and noncompetitive inhibition behaviors. The half maximal inhibitory concentration (IC50) and inhibition constant (Ki) value of Huperzine A were also calculated as 48.51 ± 1.16 nM and 26.73 ± 0.27 nM, respectively. This method provides the rapid and accurate ways to monitor enzyme reactions.
Keywords: Continuous enzymatic assay; ESI-MS; Michaelis constant (Km); Half maximal inhibitory concentration (IC50); Inhibition constant (Ki);

In this study, we present an on-line measurement of enzyme activity and inhibition of Glucose-6-phosphate dehydrogenase (G6PDH) enzyme using capillary electrophoresis based immobilized enzyme micro-reactor (CE-based IMER). The IMER was prepared using a two-step protocol based on electrostatic assembly. The micro-reactor exhibited good stability and reproducibility for on-line assay of G6PDH enzyme. Both the activity as well as the inhibition of the G6PDH enzyme by six inhibitors, including three metals (Cu2+, Pb2+, Cd2+), vancomycin, urea and KMnO4, were investigated using on-line assay of the CE-based IMERs. The enzyme activity and inhibition kinetic constants were measured using the IMERs which were found to be consistent with those using traditional off-line enzyme assays. The kinetic mechanism of each inhibitor was also determined. The present study demonstrates the feasibility of using CE-based IMERs for rapid and efficient on-line assay of G6PDH, an important enzyme in the pentosephosphate pathway of human metabolism.
Keywords: Glucose-6-phosphate dehydrogenase; Enzyme micro-reactor; Enzyme assay; Inhibition kinetics; Capillary electrophoresis;

Determination of trifolirhizin in rat plasma by UPLC: Application to a pharmacokinetic study by Kong-hai Ni; Zheng-de Wen; Xin-ce Huang; Chen-xi Wang; Tian-tian Ye; Guo-xin Hu; Meng-tao Zhou (181-184).
In this study, a simple, sensitive, and robust analytical method based on ultra-performance liquid chromatography (UPLC) has been developed for the determination of trifolirhizin in rat plasma using pirfenidone as internal standard (IS). After sample preparation by a simple liquid–liquid extraction, chromatography was performed on an Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm particle size) and ultraviolet detection set at a wavelength of 366 nm. The method was linear over the concentration range 25–1000 ng/mL with a lower limit of quantification (LLOQ) of 25 ng/mL. Inter- and intra-day precision (RSD%) were all within 10.2% and the accuracy (RE%) was equal or lower than 9.3%. The recovery was in the range of 78.5–86.4% for trifolirhizin and 87.4% for IS. Stability studies showed that trifolirhizin was stable under a variety of storage conditions. The method was successfully applied to a pharmacokinetic study involving oral administration of trifolirhizin to rats.
Keywords: Trifolirhizin; Rat plasma; Pharmacokinetic; UPLC;

Simultaneous determination of azilsartan and chlorthalidone in rat and human plasma by liquid chromatography-electrospray tandem mass spectrometry by Rachumallu Ramakrishna; Santosh kumar Puttrevu; Manisha Bhateria; Veenu Bala; Vishnu L. Sharma; Rabi Sankar Bhatta (185-197).
Azilsartan medoxomil (AZM), an ester prodrug of azilsartan (AZ), and chlorthalidone (CLT) have recently been approved as a combination therapy for the management of hypertension. This is the first report which described a selective and sensitive method for the simultaneous quantification of AZ and CLT in rat and human plasma using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). AZ and CLT were extracted from plasma by liquid-liquid extraction technique and separated on a C18 reverse phase column using ammonium acetate (10 mM, pH 4)-mixture of methanol and acetonitrile (8:92, v/v) as a mobile phase at a flow rate of 0.7 mL/min. Detection was performed by electrospray ionization (ESI) operated in negative multiple reaction monitoring (MRM) mode. The lower limit of quantitation (LLOQ) of this method was 1 ng/mL and the calibration curves were linear (r 2  ≥ 0.995) over the concentration range of 1–4000 ng/mL for both the analytes. The intra- and inter-day precision and accuracy were well within the acceptable limits. The mean extraction recoveries were found to be about 80% and no matrix effect was observed. AZ and CLT were found to be stable under all relevant storage conditions. The method was successfully applied to the oral pharmacokinetic study of AZM and CLT in rats. Further, the sensitivity of the method enabled the determination of protein binding of AZ and CLT in human plasma.
Keywords: Azilsartan medoxomil; Azilsartan; Chlorthalidone; LC-MS/MS; Pharmacokinetics; Protein binding;

Chromatographic properties of two columns was compared: commercial IAM.PC.DD2 that imitates the cell membrane and home-made Amino-P-C18 (N,O-dialkylphosphoramidate C18). The comparison has been done by correlation of retention (log  k w parameters) of a series of solutes: hydrophobic (alkyl benzene derivatives and PAHs) and polar, with both acidic (flavonoids) and basic (nucleosides and nucleic bases) character. The slope of correlation plots for hydrophobic compounds and polar basic was very close to 1.0 that confirms the chromatographic similarity. Only for flavonoids the slope of correlation plot was 1.5. For hydrophobic compound retention parameters log  k w were also correlated with hydrophobic parameter log  P with very good determination coefficients.
Keywords: Liquid chromatography; Retention mechanism; Mimics of cell membrane;