Journal of Chromatography B (v.1006, #C)

Analysis of stereochemistry and biosynthesis of epicatechin in tea plants by chiral phase high performance liquid chromatography by Yumei Qian; Xianqian Zhao; Lei Zhao; Lilan Cui; Li Liu; Xiaolan Jiang; Yajun Liu; Liping Gao; Tao Xia (1-7).
Tea (Camellia sinensis) is rich in flavan-3-ols (catechins), especially epicatechin (EC), which is the predominant extension unit of polymeric proanthocyanidins (PAs). However, studies assessing EC’s stereochemistry are scarce. Here, a high performance liquid chromatography column using amylose tris-(3, 5-dimethylphenylcarbamate) immobilized on silica-gel as chiral stationary phases (CSPs) was applied to explore its stereochemistry and biosynthetic pathway in tea plants. The results revealed (−)-epicatechin [(−)-EC] was the predominant di-hyroxy-non-galloylated-catechins, while (+)-epicatechin [(+)-EC] was not detected. Interestingly, (−)-EC was the only product obtained from cyanidin using the partially purified native C. sinensis anthocyanidin reductase (CsANR) in the presence of reduction nicotinamide adenine dinucleotide phosphate (NADPH); meanwhile, (+)-EC was the main product using recombinant CsANR in the same conditions. In addition, (−)-EC could be obtained from (+)-catechin [(+)-C] using recombinant CsANR, which displayed C3-epimerase activity in the presence of oxidation nicotinamide adenine dinucleotide phosphate (NADP+). But the partially purified native CsANR did not possess this function. Finally, (−)-EC could result from the de-gallate acid reaction of epicatechin gallate (ECG) catalyzed by a novel partially purified native galloylated catechins hydrolase (GCH) from tea leaves. In summary, (−)-EC is likely the product of native protein from the tea plants, and (+)-EC is only produced in a reaction catalyzed by recombinant CsANR in vitro.
Keywords: High performance liquid chromatography; Chiral phase; Stereochemistry; Biosynthesis; Epicatechin; Tea;

A rapid and sensitive liquid chromatography–tandem mass spectrometric (LC–MS/MS) assay method was developed and validated for simultaneous quantification of simvastatin (SV), its metabolite simvastatin hydroxy acid (SVA) and berberine (BBR) in rat plasma. Separation was performed on Poroshell 120 EC-C18 column (4.6 × 50 mm, 2.7 μm) using gradient elution by mobile phase containing acetonitrile and 10 mM ammonium acetate (pH 4.5). Polarity switch (positive–negative–positive ionization mode) was performed in a total run time of 4.0 min. The lower limits of quantification (LLOQ) for SV, SVA and BBR were 0.10, 0.20 and 0.10 ng/mL, respectively. The response function was established for concentration range of 0.10–100 ng/mL for SV and BBR and 0.20–3000 ng/mL for SVA, with a coefficient of correlation of >0.99 for all the compounds. The proposed method was applied to the drug–drug pharmacokinetic interaction study of SV combined with BBR after oral administration in rats.
Keywords: Simvastatin; Berberine; LC–MS–MS; Determination; Pharmacokinetics; Drug–drug interaction;

Role of Bai-Shao towards the antidepressant effect of Chaihu-Shu-Gan-San using metabonomics integrated with chemical fingerprinting by Xing Chang; Hongmei Jia; Chao Zhou; Hongwu Zhang; Meng Yu; Junshan Yang; Zhongmei Zou (16-29).
This study provides insight into the antidepressant active constituents of Bai-Shao and the metabolic regulation of Bai-Shao contributed to the therapeutic effect of CSGS.Display OmittedChaihu-Shu-Gan-San (CSGS) is a classical traditional Chinese medicine formula for the treatment of depression. As one of the single herbs in CSGS, Bai-Shao displayed antidepressant effect. In order to explore the role of Bai-Shao towards the antidepressant effect of CSGS, the metabolic regulation and chemical profiles of CSGS with and without Bai-Shao (QBS) were investigated using metabonomics integrated with chemical fingerprinting. At first, partial least squares regression (PLSR) analysis was applied to characterize the potential biomarkers associated with chronic unpredictable mild stress (CUMS)-induced depression. Among 46 differential metabolites found in the ultra-performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-Q-TOF/MS) and 1H NMR-based urinary metabonomics, 20 were significantly correlated with the preferred sucrose consumption observed in behavior experiments and were considered as biomarkers to evaluate the antidepressant effect of CSGS. Based on differential regulation on CUMS-induced metabolic disturbances with CSGS and QBS treatments, we concluded that Bai-Shao made crucial contribution to CSGS in the improvement of the metabolic deviations of six biomarkers (i.e., glutamate, acetoacetic acid, creatinine, xanthurenic acid, kynurenic acid, and N-acetylserotonin) disturbed in CUMS-induced depression. While the chemical constituents of Bai-Shao contributed to CSGS were paeoniflorin, albiflorin, isomaltopaeoniflorin, and benzoylpaeoniflorin based on the multivariate analysis of the UPLC-Q-TOF/MS chemical profiles from CSGS and QBS extracts. These findings suggested that Bai-Shao played an indispensable role in the antidepressant effect of CSGS.
Keywords: Bai-Shao; Chaihu-Shu-Gan-San; Active constituents; Integrated strategy; Urinary metabonomics; Chemical fingerprinting;

An ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC–MS/MS) approach was developed and validated for the determination of ginkgolide L (GL) in rat plasma and tissues using diazepam as internal standard (IS). Detection was performed on a triple quadrupole MS system using multiple reaction monitoring (MRM) mode in positive mode. Sample preparation was carried out through a liquid–liquid extraction with ethyl acetate. The chromatographic separation was achieved by using an Agilent ZORBAX SB-Aq column with a mobile phase of 0.5% aqueous formic acid (A) and methanol (B). The monitored transitions were set at m/z 391.14 → 271.10 for GL and m/z 285.08 → 193.10 for IS, respectively. The validated method was successfully applied to the pharmacokinetic and tissue distribution study of GL in rats after intravenous administration. Good linearity was found between 2.5–2000 ng/mL (r  > 0.996) for plasma samples, and calibration curves were also linear for other tissue samples over a wide range. The results indicated that GL has linear pharmacokinetic properties after intravenous administration at three doses. GL could distribute to tissues quickly and the major distribution tissue of GL in rats was liver. This was the first report of pharmacokinetic and tissue distribution data for GL.
Keywords: Ginkgolide L; Ginkgo; Pharmacokinetics; Tissue distribution; UHPLC-MS/MS;

Solvent modulation strategy for superior antibody monomer/aggregate separation in cation exchange chromatography by Simon Kluters; Christian Frech; Thomas von Hirschheydt; Andreas Schaubmar; Sebastian Neumann (37-46).
Cation exchange chromatography (CEX) is an integral part of many downstream processes for monoclonal antibodies (mAbs). However, in some cases CEX methods with standard mobile phase conditions do not lead to a sufficient removal of soluble antibody aggregates. The addition of neutral polymers such as polyethylene glycol (PEG) to the mobile phase can improve the separation of proteins in IEC remarkably. The applicability of this solvent modulation technique is limited by protein precipitation at higher PEG concentrations. To overcome this limitation solubility enhancers like polyols and amino acids can be added to the mobile phase. These additives are known to inhibit PEG-induced protein precipitation in solution.This new solvent modulation strategy was tested with three different mAbs on two different CEX resins in the presence of PEG in combination with various solubility enhancers. In order to assess the general applicability of this method, mAbs were selected that show major differences with respect to their sensitivity to PEG-induced precipitation and monomer/aggregate resolution performance that is achieved by CEX under standard conditions. For all three mAbs precipitation could be prevented without elimination of the positive PEG-effect. The addition of solubility enhancers gives access to improved separation at elevated PEG concentrations and high protein loadings without running into precipitation issues. Our data indicate that this method is generically applicable and leads to a superior antibody monomer/aggregate separation.
Keywords: Monoclonal antibodies (mAbs); Aggregate removal; Mobile phase additives; Solvent modulation; Polyethylene glycol (PEG); Solubility enhancer;

Affinity analysis and application of dipeptides derived from l-tyrosine in plasmid purification by Soraia Ferreira; Josué Carvalho; Joana F.A. Valente; Marta C. Corvo; Eurico J. Cabrita; Fani Sousa; João A. Queiroz; Carla Cruz (47-58).
The developments in the use of plasmid DNA (pDNA) in gene therapy and vaccines have motivated the search and improvement of optimized purification processes. In this context, dipeptides l-tyrosine-l-tyrosine and l-tyrosine-l-arginine are synthetized to explore their application as affinity ligands for supercoiled (sc) plasmid DNA (pDNA) purification. The synthesis is based on the protection of N-Boc-l-tyrosine, followed by condensation with l-tyrosine or l-arginine methyl esters in the presence of dicyclohexylcarbodiimide (DCC), which after hydrolysis and acidification give the afforded dipeptides. The supports are then obtained by coupling l-tyrosine, l-tyrosine-l-tyrosine and l-tyrosine-l-arginine to epoxy-activated Sepharose and are characterized by high resolution magic angle spinning (HR-MAS) NMR and Fourier transform infrared spectroscopy (FTIR). Surface plasmon resonance (SPR) biosensor is used to establish the promising ligand to be used in the chromatographic experiments and ascertain experimental conditions. Sc isoform showed the highest affinity to the dipeptides, followed by linear (ln) pDNA, being the open circular (oc) the one that promoted the lowest affinity to l-tyrosine-l-arginine. Saturation transfer difference (STD)-NMR experiments show that the interaction is mainly hydrophobic with the majority of the 5′-mononucleotides, except for 5′-GMP with l-tyrosine-l-arginine Sepharose that is mainly electrostatic. The support l-tyrosine Sepharose used in chromatographic experiments promotes the separation of native pVAX1-LacZ and pcDNA3-FLAG-p53 samples (oc+sc) by decreasing the salt concentration. The results suggest that it is possible to purify different plasmids with the l-tyrosine Sepharose, with slight adjustments in the gradient conditions.
Keywords: l-tyrosine dipeptides; Plasmid DNA; Surface plasmon resonance; Nuclear magnetic resonance; Chromatography;

Reduction of urinary uric acid excretion in patients with proteinuria by Huiqing Zou; Mingfeng Xiang; Xinming Ye; Yuanzhen xiong; Baogang Xie; Jianghua Shao (59-64).
Serum uric acid (UA) concentration is positively associated with proteinuria. However, the relationship between proteinuria and urinary metabolites of purine metabolism remains unknown. This study developed a hydrophilic interaction chromatography (HILIC)-based HPLC method with ultraviolet detection (UV) to quantify creatinine (Cr), UA, xanthine, and hypoxanthine in human urine simultaneously. The urinary concentrations of UA and Cr obtained by our method are consistent with those measured by an autoanalyzer. The HPLC-HILIC-UV method was validated as selective and robust with simple sample preparation for measuring UA, xanthine, hypoxanthine and Cr, which is suitable for large clinical studies. The UA/Cr ratios in random urine samples were 5.5 times lower in proteinuria patients (0.077 ± 0.008) than in healthy individuals (0.424 ± 0.037). Moreover, the UA/hypoxanthine ratio in proteinuria patients was approximately 10 times lower than that in healthy individuals. Our findings revealed a reduced urinary UA excretion, which is one of the factors leading to increased serum UA in proteinuria patients.
Keywords: Proteinuria; HPLC-HILIC-UV; Uric acid; Xanthine; Hypoxanthine and creatinine;

In recent years, the study of chiral compounds in vivo has received much attention. In this study, a novel method based on high performance liquid chromatography (HPLC) coupled with chemiluminescence (CL) detection was developed for the separation of tryptophan (Trp) enantiomers. o-Phthalaldehyde and N-acetyl-l-cysteine were used as chiral derivatization reagents for Trp before it can be detected by HPLC-CL method. The separation was carried out on an ODS column using a mobile phase composed of methanol—0.01 mol/L phosphate buffer (40/60, v/v). Under the optimum conditions, satisfactory results were obtained, including complete separation, good relative standard deviations and low detection limits. The applicability of the proposed method has been validated by determining Trp in biological samples. Linear responses (r  > 0.9990) were observed over the range of 2.5 × 10−7 to 1.2 × 10−5  g/mL of Trp enantiomers, with quantitation limit of 2.5 × 10−7  g/mL. The assay method shows good specificity to Trp enantiomers, and thus it will have great potential application in clinical diagnosis. The mean extraction efficiency of Trp enantiomers in mice plasma samples were 98.48% and 97.40%, respectively. The mean relative standard deviation (RSD) of Trp enantiomers were <3%.
Keywords: HPLC-CL; Tryptophan; Indirect chiral separation; Indirect CL;

Size-exclusion HPLC as a sensitive and calibrationless method for complex peptide mixtures quantification by Alice Bodin; Xavier Framboisier; Dominique Alonso; Ivan Marc; Romain Kapel (71-79).
This work describes an original methodology to quantify complex peptide mixtures by size-exclusion high-performance liquid chromatography (SE-HPLC). The methodology was first tested on simulated elutions of peptide mixtures. For this set of experiments, a good estimation of the total peptide concentration was observed (error less than 10 %). Then 30 fractions obtained by ultrafiltration of hydrolysates from two different sources were titrated by Kjeldahl or BCA analysis and analysed by SE-HPLC for an experimental validation of the methodology. Very good matchs between methods were obtained. The linear working range depends on the hydrolysate but is generally between 0.2 and 4 g L−1 (i.e. between 10 and 200 μg). Moreover, the presence of organic solvents or salts in samples does not impact the accuracy of the methodology contrary to common quantification methods. Hence, the findings of this study show that total concentration of complex peptide mixture can be efficiently determinate by the proposed methodology using simple SE-HPLC analysis.
Keywords: Protein hydrolysates; Quantification; Size-exclusion HPLC;

Oridonin (ORI) is an active natural ent-kaurane diterpenoid ingredient originating from well-known traditional Chinese herb medicine and is expected to be pursued as a new anticancer agent. In the present study, a novel and efficient approach was developed for in vivo screening and identification of ORI metabolites using ultra high performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UPLC-Triple-TOF-MS/MS). This analytical strategy was as follows: an effective on-line data acquisition method multiple mass defect filter (MMDF) combined with dynamic background subtraction (DBS), was developed to trace all of potential metabolites of ORI. The MMDF and DBS method could trigger an information dependent acquisition scan, which could give the information of low-level metabolites masked by background noise and endogenous components in complex matrix. Moreover, the sensitive and specific multiple data-mining techniques including extracted ion chromatography, mass defect filtering, product ion filtering and neutral loss filtering were employed to identify the metabolites of ORI. Then, structures for the metabolites were successfully assigned based on accurate masses, the mass fragmentation of ORI and metabolic knowledge. Finally, an important parameter Clog P was used to estimate the retention time of isomers. Based on the proposed strategy, 16 phase I and 2 phase II metabolites were detected in rats after oral administration of ORI. The main biotransformation route of ORI was identified as reduction, oxidation, dehydroxylation and glucuronic acid conjugation. This is the first study of ORI metabolism in vivo. This study not only proposed a practical strategy for rapidly screening and identifying metabolites, but also provided useful information for further study of the pharmacology and mechanism of ORI in vivo. At the same time this methodology can be widely applied for the structural characterization of the metabolites of other ent-kaurane diterpenoid.
Keywords: Oridonin; Metabolite; UPLC-Triple-TOF-MS; MMDF; Multiple data processing; ent-Kaurane diterpenoid;

Since serotonin, homocysteine and oxytocin are known to fluctuate during mammalian gestation, we screened amines altered in pregnant-associated hypertensive (PAH) mice by tagging their amino groups with 6-aminoquinoline carbamoyl (AQC) group in concert with ultra high-performance liquid chromatography (UPLC). Interestingly, a candidate amine significantly increased in PAH mice was recovered to the basal level, when treated with antihypertensive drugs. Mass spectrometric analyses indicated that the molecular mass of this amine was 61.2, which was identified as ethanolamine.
Keywords: Amines; Ethanolamine; Pregnancy-associated hypertension; Anti-hypertensive drugs; UPLC; MALDI-TOF/MS;

Serum metabolomics in rats models of ketamine abuse by gas chromatography–mass spectrometry by Meiling Zhang; Congcong Wen; Yuan Zhang; Fa Sun; Shuanghu Wang; Jianshe Ma; Kezhi Lin; Xianqin Wang; Guanyang Lin; Lufeng Hu (99-103).
This study aims to evaluate the effect of ketamine on rats by examining the differences in serum metabolites between ketamine abuse group (Ket-group) and control group (Con-group). Compared to the Con-group, the level of phosphate, propanoic acid, ribitol and d-fructose of the Ket-group increased after continuous intraperitoneal administration of ketamine for 7 days, while the level of alanine, glycine, butanoic acid, valine, l-serine, l-proline, mannonic acid, octadecanoic acid and cholesterol decreased. After 14 days’ administration, the level of alanine, butanoic acid, valine, l-leucine, phosphate, l-serine, l-threonine, propanetricarboxylic acid, hexadecanoic acid and oleic acid of the ketamine group increased while the level of mannonic acid, octadecanoic acid and cholesterol decreased. After stopping ketamine administration for 2 days, the level of butanoic acid, phosphate, aminomalonic acid, gluconic acid, hexadecanoic acid, oleic acid and arachidonic acid of Ket-group increased, while the level of glycine, l-lysine and cholesterol decreased. This study can provide invaluable information for the metabolites changes due to ketamine abuse.
Keywords: Serum; Metabolomics; GC/MS; Ketamine; Abuse;

Liquid chromatography-tandem mass spectrometry (LC–MS/MS) has been widely utilized for the analysis of compounds in biological matrices due to its selectivity and sensitivity. This study describes the application of an LC–MS/MS-based approach toward the analysis of cobinamide in Yorkshire pig plasma. The selectivity, accuracy, precision, recovery, linearity, range, carryover, sensitivity, matrix effect, interference, stability, reproducibility, and ruggedness of the method were investigated in pig plasma. The accuracy and precision of the method was determined to be within 10% over three different days over a range of concentrations (25–10,000 ng/mL) that spanned more than two orders of magnitude. The lower limit of quantitation (LLOQ) for dicyanocobinamide was determined to be 25 ng/mL in pig plasma. Carryover was acceptable, as the area response of the carryover blanks were ≤15% of the area response of the nearest LLOQ standard for the analyte, while it was nonexistent for the internal standard. Specificity was ensured using six different lots of pig plasma. While the matrix effects of dicyanocobinamide in plasma were enhanced, ginsenoside Rb1 experienced signal suppression under the described conditions. The absolute recovery results for both compounds were consistent, precise, and reproducibly lower than expected at ∼60% for dicyanocobinamide and ∼22% for ginsenoside Rb1, confirming that a matrix standard curve was required for accurate quantitation. Cobinamide was shown to be very stable in matrix at various storage conditions including room temperature, refrigerated, and frozen at time intervals of 20 h, 4 days, and 60 days respectively. This method was demonstrated to be sensitive, reproducible, stable, and rugged, and it should be applicable to the analysis of cobinamide in other biological matrices and species.
Keywords: Cobinamide; Dicyanocobinamide; Nitrocobinamide; Potassium cyanide; Yorkshire pig plasma; Liquid chromatography–tandem mass spectrometry;

A UPLC–MSMS method for the analysis of olanzapine in serum—with particular emphasis on drug stability testing by Trine Naalsund Andreassen; Berit Margrethe Hasle Falch; Olav Spigset (112-120).
A method including a rapid and automated extraction of olanzapine from serum followed by ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) has been developed and validated. Serum aliquots (100 μL) and internal standard (olanzapine-d3, 25 μL) were pipetted onto an OstroTM 96-well filtration plate and protein precipitated with acidic acetonitrile (300 μL) before removal of endogenous phospholipids by filtration followed by analysis. Chromatography was achieved using an HSST 3 (2.1 × 100 mm, 1.8 μm) column and gradient elution with acidic water in combination with methanol at a flow rate of 0.5 mL/min. The runtime was 1.5 min. The mass spectrometer was monitored in positive mode and multiple reaction monitoring (MRM). The m/z 313.1 > 256.1 and 313.1 > 198.0 transitions were monitored for olanzapine (m/z 316.1 > 256.1 for olanzapine-d3). The quadratic calibration curves ranged from 5 to 500 nM (R 2  ≥ 0.999). Limit of quantification was 0.5 nM (CV 9.6%, accuracy 110%). Within-assay and between-assay inaccuracies were 2.6–11.9% (CV ≤ 4.8%). Recovery was 84–95% (CV ≤ 1.4%) and matrix effects ranged from 100 to 103% (CV ≤ 2.6%). Extensive stability testing showed that at ambient temperature, olanzapine in patient serum samples were stable for at least seven hours on the laboratory bench and for at least 48 h in darkness. When exposed to 3000 lux, however, significant degradation had occurred after 48 h. Notably, olanzapine in spiked serum was unstable already after four hours when exposed to 3000  lux. At 4–8 °C and exposure to 550 lux, both patient serum and spiked serum were stable for more than 48 h but less than a week, whereas in darkness, the samples were stable for at least 14 days. The cumulative light exposure causing significant degradation of olanzapine in patient serum was 50,000–100,000 lux-h. In some individual samples, however, the effect of light exposure was more pronounced. Therefore, it seems pertinent to recommend protecting all samples from light, although we found no indication that a few hours of exposure to standard indoor illumination will affect the olanzapine concentration to any significant degree.
Keywords: UPLC–MSMS; Olanzapine; Serum; Simple sample preparation; Drug stability;

Sodium cromoglicate (SCG), antihistaminic agent, and tetryzoline hydrochloride (TZH), a sympathomimetic agent, are formulated together as an ophthalmic preparation. An ultra-performance liquid chromatographic method with UV detection (UPLC–UV) was developed and validated for the quantitative determination of SCG and TZH in rabbit aqueous humor. Due to the instability of both SCG and TZH under alkaline conditions, the UPLC method was applied for their determination in the presence of their possible degradation impurities. The separation was performed using C18 column (1.7 μm particle size) and isocratic elution system with methanol: 1% o-phosphoric acid (65: 35, v/v).The optimum flow rate was 0.5 ml/min and the detection was done at 230 nm. The suggested method was validated in compliance with the ICH guidelines and was successfully applied for determination of sodium cromoglicate (SCG) and tetryzoline HCl (TZH) as prepared synthetically in laboratory mixtures, and in the presence of their alkali-induced degradation impurities. The suggested method was effectively applied the determination of spiked rabbit aqueous humor samples as well as commercial pharmaceutical formulation.
Keywords: Sodium cromoglicate; Tetryzoline HCl; UPLC; Aqueous humor; Degradation;

Comparative characterization of nucleotides, nucleosides and nucleobases in Abelmoschus manihot roots, stems, leaves and flowers during different growth periods by UPLC-TQ-MS/MS by Le-yue Du; Da-wei Qian; Shu Jiang; Er-xin Shang; Jian-ming Guo; Pei Liu; Shu-lan Su; Jin-ao Duan; Min Zhao (130-137).
Nucleotides, nucleosides and nucleobases have been proven as important bioactive compounds related to many physiological processes. Abelmoschus manihot (L.) Medicus from the family of Malvaceae is an annual herbal plant of folk medicine widely distributed in Oceania and Asia. However, up to now, no detailed information could be available for the types and contents of nucleotides, nucleosides and nucleobases contained in A. manihot roots, stems, leaves as well as the flowers. In the present study, an UPLC-TQ-MS/MS method was established for detection of the twelve nucleotides, nucleosides and nucleobases. The validated method was successfully applied to identify the 12 analytes in different parts of A. manihot harvested at ten growth periods. 2′-deoxyinosine was not detected in all of the A. manihot samples. The data demonstrated that the distribution and concentration of the 12 compounds in A. manihot four parts were arranged in a decreasing order as leaf > flower > stem > root. Based on the results, the leaves and flowers of A. manihot could be developed as health products possessed nutraceutical and bioactive properties in the future. This method might also be utilized for the quality control of the A. manihot leaves and other herbal medicines being rich in nucleotides, nucleosides and nulecobases.
Keywords: Abelmoschus manihot; UPLC-TQ-MS/MS; Nucleotides; Nucleosides; Nucleobases; Growth periods;

Separation and purification of two new and two known alkaloids from leaves of Nitraria sibirica by pH-zone-refining counter-current chromatography by Mahinur Bakri; Qibin Chen; Qingling Ma; Yi Yang; Abdumijit Abdukadir; Haji Akber Aisa (138-145).
The total alkaloids from Nitraria sibirica leaves have been confirmed to exhibit significant protective effects against inflammatory renal injury, hypertension and albuminuria in angiotensin II-salt hypertension. In the present study, a separation method of pH-zone-refining counter-current chromatography was established for separation of the alkaloids from N. sibirica. The separation was performed with a solvent system of MtBE-n-BuOH-H2O (2:2:5, v/v) at a flow rate of 2.0 mL/min. And 15 mM triethylamine (TEA) was added to the upper organic phase, while 10 mM hydrochloric acid was added to the lower aqueous phase. As a result, a new alkaloid, schobemine (5.6 mg), and a known alkaloid, nitraramine (5.0 mg), together with fractions A and B were obtained from the total alkaloids of N. sibirica. The fractions A and B were further purified by means of pH-zone-refining counter-current chromatography with solvent systems of n-hexane-n-BuOH-H2O (1.5:3.5:5, v/v) and (2:3:5, v/v), respectively. TEA (10 mM) was added to the upper phase, and 10 mM of HCl was added to the lower phase in above two solvent systems, respectively. As a result, a known alkaloid, schoberidine (5.0 mg), and a new alkaloid, schoberimine (3.0 mg) were obtained from fractions A and B, respectively. The purities of the compounds were measured by HPLC–ELSD, and their structures were identified by ESI–MS, 1D and 2D NMR.
Keywords: pH-zone-refining counter-current chromatography; Separation and purification; Alkaloids; Novel compounds;

A simple, sensitive and selective method for determination of urapidil hydrochloride was developed using capillary electrophoresis with electrochemiluminescence (CE-ECL) technique for the first time. Under the optimized experimental conditions, the ECL intensity was linear with the concentration of urapidil hydrochloride in the range from 0.050 to 50.0 ng/mL and the detection limit was 0.014 ng/mL (S/N = 3). The proposed method was used for studying pharmacokinetics of urapidil hydrochloride in rat plasma and the main pharmacokinetic parameters of the peak concentration (C max), half life time (T 1/2) and peak concentration time (T max) were 240.45 ± 21.15 ng/mL, 0.58 ± 0.16 h and 1.08 ± 0.13 h, respectively. The recoveries of urapidil hydrochloride in the diluted extracts of rat plasma samples ranged from 96.68 to 98.82%. The RSD was lower than 3%.
Keywords: Capillary electrophoresis; Electrochemiluminescence; Urapidil hydrochloride; Pharmacokinetics; Rat plasma;

A simple and sensitive derivatization method using toluene-3,4-dithiol as a derivatization reagent for the simultaneous analysis of seven arsenic compounds (roxarsone, nitarsone, p-arsanilic acid, o-arsanilic acid, phenylarsonic acid, phenylarsine oxide, and mono-methylarsonic acid) in chicken muscle was developed and validated by ultra-performance liquid chromatography coupled with ultraviolet detection (UPLC-UV). The structure of the derivatized arsenic compounds was confirmed by liquid chromatography-ion trap mass spectrometry or gas chromatography-mass spectrometry. Optimization of the derivatization reaction conditions was carried out by investigating the influence of reagent concentration, buffer or additive acids, temperature, and time. The optimized conditions were a derivatization reagent concentration of 20 mg/mL with 0.05 mol/L HCl as an additive acid at 60 °C for 15 min. In this study, baseline separation of arsenic compounds could be achieved within 13 min, except for phenylarsonic acid and phenylarsine oxide whose derivatized products are equal. The developed method was successfully validated and applied to 12 chicken muscle samples from Korean districts and other countries.
Keywords: Arsenic compounds; Derivatization; Toluene-3,4-dithiol; Ultra-performance liquid chromatography-ultraviolet detection; Chicken muscle;

Investigation of cell culture volatilomes using solid phase micro extraction: Options and pitfalls exemplified with adenocarcinoma cell lines by Kristin Schallschmidt; Roland Becker; Christian Jung; Jana Rolff; Iduna Fichtner; Irene Nehls (158-166).
Three strategies to sample volatile organic compounds (VOC) from lung cancer cell lines cultured in vitro were compared. Headspace solid phase microextraction was applied in situ to culture flasks and alternatively to subsamples of headspace gas or to nutrient solution subsamples followed by gas chromatography–mass spectrometry. The direct quantification of 55 VOC in the headspace of cell cultures was validated and is discussed with respect to reproducibility and system-related interferences. The role of the VOC background from culture media and usually employed polystyrene culture vessels is examined and was seen to invoke potentially misleading conclusions. The commercial A549 and two further adenocarcinoma cell lines displayed largely similar VOC profiles with distinct differences regarding certain individual substances. There is evidence for the inappropriateness of the standard cell culturing methods in the search for volatile cancer markers.
Keywords: A549; Lung cancer cell culture; GC–MS; SPME; VOC; Volatilome;

Simultaneous, quantitative determination of intracellular nucleoside triphosphates and other polar metabolites using liquid chromatography with electrospray ionization tandem mass spectrometry (LC-MS/MS) represents a bioanalytic challenge because of charged, highly hydrophilic analytes presented at a large concentration range in a complex matrix. In this study, an ion pair LC-MS/MS method using triethylamine (TEA)—hexafluoroisopropanol (HFIP) ion-pair mobile phase was optimized and validated for simultaneous and unambiguous determination of 8 nucleoside triphosphates (including ATP, CTP, GTP, UTP, dATP, dCTP, dGTP, and dTTP) in cellular samples. Compared to the the less volatile ion-pair reagent, triethylammonium acetate (100 mM, pH 7.0), the combination of HFIP (100 mM) and TEA (8.6 mM) increased the MS signal intensity by about 50-fold, while retaining comparable chromatographic resolution. The isotope-labeled internal standard method was used for the quantitation. Lower limits of quantitation were determined at 0.5 nM for CTP, UTP, dATP, dCTP, and dTTP, at 1 nM for ATP, and at 5 nM for GTP and dGTP. The intra- and inter-day precision and accuracy were within the generally accepted criteria for bioanalytical method validation (<15%). While the present method was validated for the quantitation of intracellular nucleoside triphosphates, it had a broad application potential for quantitative profiling of nucleoside mono- and bi-phosphates as well as other polar, ionic metabolic intermediates (including carbohydrate derivatives, carboxylic acid derivatives, co-acyl A derivatives, fatty acyls, and others) in biological samples.
Keywords: Ion pair chromatography; Ribonucleoside triphosphate (NTP); Deoxyribonucleoside triphosphate (dNTP); LC-MS/MS; Metabolomics;

The manuscript presents the development of a new method for the quantification of 16 sulfonamides in beeswax. Different sample preparation techniques were tested and modified to maximise the recovery of the target analytes and minimise the amount of coeluted impurities under conditions that provide reproducible results. The proposed method consisted of melting and dilution of beeswax in a mixture of n-hexane and isopropanol followed by extraction with 2% acetic acid. The extract was cleaned up by solid-phase extraction using strong cation exchange phase. Determination of the sulfonamides was achieved by liquid chromatography coupled to tandem mass spectrometry with the use of a pentafluorophenyl analytical column and applying a gradient elution with acetonitrile and 0.01% acetic acid as mobile phases. The limits of detection and limits of quantification ranged from 1 to 2 μg/kg and from 2 to 5 μg/kg, respectively. The recoveries varied between 65.2% and 117.8% while coefficient of variation of the method was less than 24.2% under intermediate precision conditions. Finally, the method was applied to the analysis of real samples of beeswax from beekeepers and commercial foundations manufacturers.
Keywords: Sulfonamides; Beeswax; Liquid chromatography; Mass spectrometry; Optimization;

The elution of certain protein affinity tags with millimolar concentrations of diclofenac by Martina Baliova; Anna Juhasova; Frantisek Jursky (187-193).
Diclofenac (2-[(2, 6-dichlorophenyl)amino] benzeneacetic acid) is a sparingly soluble, nonsteroidal anti-inflammatory drug therapeutically acting at low micromolar concentrations. In pH range from 8 to 11, its aqueous solubility can be increased up to 200 times by the presence of counter ions such as sodium. Our protein interaction studies revealed that a millimolar concentration of sodium diclofenac is able to elute glutathione S-transferase (GST), cellulose binding protein (CBD), and maltose binding protein (MBP) but not histidine-tagged or PDZ-tagged proteins from their affinity resins. The elution efficiency of diclofenac is comparable with the eluting agents normally used at similar concentrations. Native gel electrophoresis of sodium diclofenac-treated proteins showed that the interaction is non-covalent and non-denaturing. These results suggest that sodium diclofenac, in addition to its pharmaceutical applications, can also be exploited as a lead for the development of new proteomics reagents.
Keywords: Diclofenac; Glutathione S-transferase; GST; Maltose binding protein; MBP; Cellulose binding protein; CBD; PDZ (PSD95/Discs large/ZO-1);

Pharmacokinetics and tissue distribution study of PA-824 in rats by LC–MS/MS by Libin Wang; Yetao Ma; Hongtao Duan; Jiahui Yao; Li Liang; Ruitao Zhang; Xuejiao Zhou; Xueying Liu; Qingwei Wang; Shengyong Zhang (194-200).
A simple, sensitive and rapid LC–MS/MS method has been developed and validated for determination of PA-824 in rat biological samples using darunavir as internal standard. Chromatographic separation was achieved on an Inertsil®ODS3 C18 column (150 mm × 4.6 mm, 5 μm) using gradient elution of methanol-0.1% ammonia in water (90:10, v/v) with fast gradient elution at a flow rate of 0.6 mL/min and run time of 5 min. The mass spectrometer was run in positive electrospray ionization (ESI) mode using multiple reaction monitoring (MRM) to monitor the mass transitions. The optimized ion transition pairs for quantitation were m/z  360.1 →  m/z  175.0 for PA-824, m/z  548.5 →  m/z  504.2 for IS. The method was validated for accuracy, precision, linearity, range, selectivity, lower limit of quantification (LLOQ), recovery, matrix effect and robustness. All validation parameters met the acceptance criteria according to regulatory guidelines. The LLOQ was 0.05 μg/mL. The calibration curves showed a good linearity over the concentration range of 0.05–50 μg/mL. The calibration curves for all biological samples showed good linearity (r 2  > 0.9978) over the concentration ranges tested. The recoveries obtained for PA-824 were ≥88.8%. The developed method was successfully applied to investigate the pharmacokinetics and tissue distribution of PA-824 in rats following oral administration. It was also the first study to investigate the tissue distribution of PA-824 in rats following oral administration.
Keywords: PA-824; Pharmacokinetics; Tissue distribution;

Multi-allele genotyping platform for the simultaneous detection of mutations in the Wilson disease related ATP7B gene by Maria Amvrosiadou; Margarita Petropoulou; Myrto Poulou; Maria Tzetis; Emmanuel Kanavakis; Theodore K. Christopoulos; Penelope C. Ioannou (201-208).
Wilson’s disease is an inherited disorder of copper transport in the hepatocytes with a wide range of genotype and phenotype characteristics. Mutations in the ATP7B gene are responsible for the disease. Approximately, over 500 mutations in the ATP7B gene have been described to date. We report a method for the simultaneous detection of the ten most common ATP7B gene mutations in Greek patients. The method comprises 3 simple steps: (i) multiplex PCR amplification of fragments in the ATP7B gene flanking the mutations (ii) multiplex primer extension reaction of the unpurified amplification products using allele-specific primers and (iii) visual detection of the primer extension reaction products within minutes by means of dry-reagent multi-allele dipstick assay using anti-biotin conjugated gold nanoparticles. Optimization studies on the efficiency and specificity of the PEXT reaction were performed. The method was evaluated by genotyping 46 DNA samples of known genotype and 34 blind samples. The results were fully concordant with those obtained by reference methods. The method is simple, rapid, cost-effective and it does not require specialized instrumentation or highly qualified personnel.
Keywords: Wilson disease; ATP7B gene mutations; Genotyping; Primer extension reaction; Visual detection; Multi-allele DNA biosensor;