Journal of Chromatography B (v.959, #C)

Since 2008 a significant amount of work has focused on the development of methods to analyze contaminated heparin. This work focuses on utilizing heparin's ability to serve as a chiral selector as a means for determining contamination. Specifically, the effect of contamination on the separation of pheniramine and chloroquine enantiomers was explored. Separations were conducted using heparin contaminated with chondroitin sulfate at varying levels. For each pair of enantiomers, electrophoretic mobility and resolution were calculated. For pheniramine enantiomers, an increase in contamination leads to a decrease in the electrophoretic mobility and resolution. A linear relationship between contamination level and electrophoretic mobility of the pheniramine enantiomers was observed for the entire contamination range. A linear relationship was also found between contamination level and resolution of the enantiomers between 0 and 70 percent contamination. For the separation of chloroquine enantiomers, it was found that at low levels of contamination, the resolution of enantiomers was increased due to the secondary interaction between the chloroquine enantiomers and the chondroitin sulfate. Results of this study illustrate the potential of using chiral recognition as a means to determine heparin contamination as well as the improvement of the chiral resolution of chloroquine with the additional of low levels of chondroitin sulfate A.
Keywords: Capillary electrophoresis; Heparin; Chiral;

We present a robust clinical assay for the measurement of red blood cell uridine diphosphate galactose-4-epimerase enzyme activity for the diagnostic confirmation of patients positive for a newborn screen for inherited galactosemia in whom galactose-1-phosphate uridyltransferase activity is normal. Previous assays required the use of ion-pairing reagents and frequent need for system maintenance that was not appropriate for heavy clinical use where patient results should be quickly available. We have designed a two-step enzyme assay which converts stable-isotope-labeled UDP-galactose to isotope-labeled-UDP-glucose which is converted in the second reaction to the final product of [13C6]-UDP-glucuronic acid. Measurement conditions t remove potential interference from endogenous UDP-glucose and UDP-galactose. We also report a significant ion suppression effect of the red cell preparation for which we have optimized assay sample volume to minimize this effect.
Keywords: Galactosemia; Epimerase enzyme assay; UPLC–tandem mass spectrometry;

Development of LC–MS determination method and back-propagation ANN pharmacokinetic model of corynoxeine in rat by Jianshe Ma; Jinzhang Cai; Guanyang Lin; Huilin Chen; Xianqin Wang; Xianchuan Wang; Lufeng Hu (10-15).
Corynoxeine(CX), isolated from the extract of Uncaria rhynchophylla, is a useful and prospective compound in the prevention and treatment for vascular diseases. A simple and selective liquid chromatography mass spectrometry (LC–MS) method was developed to determine the concentration of CX in rat plasma. The chromatographic separation was achieved on a Zorbax SB-C18 (2.1 mm × 150 mm, 5 μm) column with acetonitrile–0.1% formic acid in water as mobile phase. Selective ion monitoring (SIM) mode was used for quantification using target ions m/z 383 for CX and m/z 237 for the carbamazepine (IS). After the LC–MS method was validated, it was applied to a back-propagation artificial neural network (BP-ANN) pharmacokinetic model study of CX in rats. The results showed that after intravenous administration of CX, it was mainly distributed in blood and eliminated quickly, t 1/2 was less than 1 h. The predicted concentrations generated by BP-ANN model had a high correlation coefficient (R  > 0.99) with experimental values. The developed BP-ANN pharmacokinetic model can be used to predict the concentration of CX in rats
Keywords: Corynoxeine; LC–MS; Pharmacokinetics; BP-ANN;

A liquid chromatography–electrospray ionization tandem mass spectrometry method was established and validated for the determination of bevantolol in human plasma using propranolol as the internal standard. The optimal chromatographic behavior of bevantolol and propranolol was achieved on a Welch Ultimate XB-C18 column (5 μm, 150 mm × 2.1 mm, Maryland, USA) with a mobile phase of acetonitrile–water (40:60, v/v) containing 10 mM ammonium acetate and 0.1% formic acid. The mass spectrometer was operated in selected reaction monitoring mode using the transition m/z 346.1 > 165.1 for bevantolol and m/z 260.3 > 116.1 for propranolol. Sample preparation was carried out through protein precipitation with acetonitrile. The calibration curves were linear over the range of 5.00–1000 ng/ml. The intra- and inter-day precisions were less than 6.7% and 6.6%, respectively. This method was successfully applied to the bioequivalence study of two kinds of bevantolol hydrochloride tablets in 24 Chinese male volunteers in fasting and postprandial experiment.
Keywords: Bevantolol; LC–MS/MS; Bioequivalence;

Two high-performance liquid chromatography (HPLC) coupled with electrospray ionization (ESI) mass spectrometry methods were developed and validated for the simultaneous determination of asperosaponin VI (A-VI) and hederagenin (HG) in rats’ various tissues. Biological samples were processed with methanol extraction, and glycyrrhetinic acid was chosen as the internal standard (IS). The analytes were separated on a C18 column with two gradient elution programs for different rat tissue samples. The MS/MS detection was carried out by monitoring the transitions of m/z [M―H] 927.5 → 603.4 for A-VI, m/z [M―H] 471.3 → 471.3 for HG and m/z [M―H] 469.4 → 425.4 for the IS, respectively. The lower limits of quantification (LLOQ) for the two analytes in different rat tissues were 2–6 ng/mL, respectively. The methods were successfully applied to a tissue distribution study of A-VI and its active metabolite HG in rats. The results of the tissue distribution study showed that the highest concentration of A-VI was in the gastrointestinal (GI) tract. Besides, A-VI was mainly distributed in lung, liver, fat and ovary. HG was almost undetectable in most tissues except for the GI tract.
Keywords: Asperosaponin VI; Hederagenin; LC–MS/MS; Tissue distribution;

Development of a dynamic multiple reaction monitoring method for determination of digoxin and six active components of Ginkgo biloba leaf extract in rat plasma by Zhi Rao; Hongyan Qin; Yuhui Wei; Yan Zhou; Guoqiang Zhang; Fan Zhang; Yunyun Shao; Jing Huang; Xin’an Wu (27-35).
A new liquid chromatography–tandem mass spectrometry (LC–MS/MS) method by using dynamic multiple reaction monitoring (DMRM) has been developed and validated for the simultaneous determination of digoxin (DGX) and six main components of Ginkgo biloba leaf extract (GBE) in rat plasma. Comparing with the conventional multiple reaction monitoring (MRM), DMRM dramatically decreases the number of concurrent MRM transitions, and significantly extended the dwell time, which provided much higher sensitivity and reproducibility than MRM when complex multi-component samples were quantified. The plasma samples were protein precipitated with methanol, the detection was accomplished with electro-spray ionization (ESI) as the ion source operating in the negative ionization mode, with methanol and water as mobile phase, and with an Agilent Zorbax eclipse plus C18 column (4.6 × 100 mm, 3.5 μm) as the analytical column. The total run time was 12.0 min. The validation of the method was implemented including specificity, linearity, accuracy, precision, recovery, matrix effect and stability. This method was successfully applied to the herb–drug pharmacokinetic interaction study of DGX combined with GBE after oral administration to rats. The result indicated that co-administration of GBE and DGX significantly influenced the pharmacokinetics of DGX when compared to that of single DGX-treated rats.
Keywords: LC–MS/MS; Dynamic multiple reaction monitoring; Digoxin; Ginkgo biloba leaf extract; Pharmacokinetics; Herb–drug interaction;

A valproic acid (VPA) LC-MS/MS analytical method using analyte adduct formation was developed and validated in human serum. The fragmentation of the sodium acetate adduct (mass transition: 225/143) and acetic acid adduct (mass transition: 203/143) were used as the target and qualifier mass transition, respectively. A Luna 5 μm C18 (2) 100 A, 150 mm × 2 mm analytical column and a mobile phase consisting of A (H2O/methanol = 95/5, v/v) and B (H2O/methanol = 3/97, v/v), both with 10 mM ammonium acetate and 0.1% acetic acid (pH = 3.2) were used. A binary flow pumping mode with a total flow rate of 0.4 mL/min was applied. Protein precipitation with 1 mL of the mobile phase B was used as sample preparation. The calculated limit of detection/quantification was 0.45/1.0 μg/mL and the inter-/intra-day precision was <6%. The application of a deuterated internal standard resulted in a good adduct formation reproducibility. The strategy applied made the VPA LC-MS/MS analysis in human serum on the basis of two mass transitions possible. Therefore, it is an interesting alternative for the VPA pseudo multiple reaction monitoring methods (mass transition 143/143) and a proof that the developed strategy is also useful for the analysis of compounds which do not produce any stable ion fragments detectable by tandem mass spectrometry.
Keywords: Valproic acid; Adduct formation; Liquid chromatography; Mass spectrometry;

In this work, poly(HEMA-co-GMA) cryogel was synthesized by using cryopolymerization technique and this cryogel was functionalized with Cibacron Blue F3GA dye. Prepared dye attached cryogel was used for the reversible immobilization of alcohol dehydrogenase from its aqueous solution. Cibacron Blue F3GA attached poly(HEMA-co-GMA) cryogel was characterized by environmental scanning electron microscopy (ESEM), energy dispersive X-ray (EDX) analysis and swelling studies. Surface morphology of the cryogel was considerably porous and the pore size was found to be around 30–50 μm. Effects of medium pH, initial alcohol dehydrogenase concentration, medium temperature and ionic strength on the alcohol dehydrogenase adsorption were also investigated and maximum alcohol dehydrogenase adsorption onto the dye-attached cryogel was found to be 8.55 mg/g cryogel. Adsorbed alcohol dehydrogenase was desorbed from the cryogel by using 10 mL of NaCl solution (1.0 M; in pH 4.0 acetate buffer). Synthesized cryogel was able to reuse for 25 sequential cycles and it was found that, there was no negligible decrease in the adsorption capacity of the dye-attached cryogel.
Keywords: Cryogel; Alcohol dehydrogenase; Cibacron Blue F3GA; Reversible immobilization;

Metabonomic analysis of the toxic effects of TM208 in rat urine by HPLC-ESI-IT-TOF/MS by Haisong Yang; Wensi Lin; Jianmei Zhang; Weiwei Lin; Peng Xu; Jing Li; Xiaomei Ling (49-54).
4-Methylpiperazine-1-carbodithiocacid-3-cyano-3,3-diphenylpropyl ester hydrochloride (TM208) was a potential antitumor new drug with many preliminary studies in pharmacokinetics and pharmacodynamics. This study aims to determine whether TM208 elicits toxic effects by metabonomics for the first time. Sprague Dawley (SD) rats were exposured to TM208 at a single therapeutic dose (100 mg/kg/d) for 5 days, metabolites of urine samples from both control and TM208-treated groups were analyzed using high performance liquid chromatography-electrospray ionization source in combination with hybrid ion trap and high-resolution time-of-flight mass spectrometry (HPLC-ESI-IT-TOF/MS). Metabolites such as aminoadipic acid, creatine, gluconic acid, cis-aconitic acid, succinic acid and pipecolic acid which changed significantly, were identified as potential biomarkers. These results suggest that the changes in urinary metabolites of rats after exposure to TM208 were mainly related to energy metabolism and amino acid metabolism, which may be helpful to further understand the mechanism of TM208 toxicity in rats and a new drug development.
Keywords: Metabonomics; HPLC-ESI-IT-TOF/MS; TM208; Toxicity; TCA cycle;

This paper described a novel application of PEGylated magnetic carbon nanotubes as solid-phase extraction nanosorbents for the determination of puerarin in rat plasma by high performance liquid chromatography (HPLC). A solvothermal method was employed for the synthesis of monodisperse magnetites anchored onto multi-walled carbon nanotubes (MWCNTs@Fe3O4). In order to enhance the water solubility of MWCNTs@Fe3O4 that ensured sufficient contact between nanosorbents and analytes in the sampling procedure, the obtained nanomaterials were further noncovalently functionalized using a phospholipids–polyethylene glycol (DSPE-PEG). The PEGylated MWCNTs@Fe3O4 nanomaterials had an extremely large surface area and exhibit a strong interaction capability for puerarin with π–π stacking interactions. The captured puerarin/nanosorbents were easily isolated from the plasma by placing a magnet, and desorbed by acetonitrile. The experimental variables affecting the extraction efficiency were investigated. The calibration curve of puerarin was linear from 0.01 to 20 μg/ml, and the limit of detection was 0.005 μg/ml. The precisions ranged from 2.7% to 3.5% for within-day measurement, and for between-day variation was in the range of 3.1–5.9%. The method recoveries were acquired from 95.2% to 98.0%. Moreover, the analytical performance obtained by PEGylated magnetic MWCNTs was also compared with that of magnetic MWCNTs. All results showed that our proposed method was an excellent alternative for the analysis of puerarin in rat plasma.
Keywords: PEGylated magnetic carbon nanotubes; Puerarin; Rat plasma; High performance liquid chromatography;

Emission rates of selected volatile organic compounds from skin of healthy volunteers by Paweł Mochalski; Julian King; Karl Unterkofler; Hartmann Hinterhuber; Anton Amann (62-70).
Gas chromatography with mass spectrometric detection (GC–MS) coupled with solid phase micro-extraction as pre-concentration method (SPME) was applied to identify and quantify volatile organic compounds (VOCs) emitted by human skin. A total of 64 C4-C10 compounds were quantified in skin emanation of 31 healthy volunteers. Amongst them aldehydes and hydrocarbons were the predominant chemical families with eighteen and seventeen species, respectively. Apart from these, there were eight ketones, six heterocyclic compounds, six terpenes, four esters, two alcohols, two volatile sulphur compounds, and one nitrile. The observed median emission rates ranged from 0.55 to 4790 fmol cm−2  min−1. Within this set of analytes three volatiles; acetone, 6-methyl-5-hepten-2-one, and acetaldehyde exhibited especially high emission rates exceeding 100 fmol cm−2  min−1. Thirty-three volatiles were highly present in skin emanation with incidence rates over 80%. These species can be considered as potential markers of human presence, which could be used for early location of entrapped victims during Urban Search and Rescue Operations (USaR).
Keywords: Volatile organic compounds; Human skin emanation; Human odor; Entrapped victims; Emission rate; SPME-GCMS;

LC–MS/MS and GC–MS/MS measurement of plasma and urine di-paracetamol and 3-nitro-paracetamol: Proof-of-concept studies on a novel human model of oxidative stress based on oral paracetamol administration by Arne Trettin; Darko Modun; Sanja Madunic; Jonatan Vukovic; Maja Radman; Sandor Batkai; Thomas Thum; Jens Jordan; Dimitrios Tsikas (71-81).
Paracetamol (acetaminophen) is a widely used safe analgesic drug when administered at therapeutic doses. Given the chemical reactivity of its phenolic group towards electrophilic species, we assumed that detection of paracetamol metabolites distinctly different from its known phase I metabolite N-acetyl-p-benzoquinone imine (NAPQI) and the phase II glucuronic, sulfuric and mercapturic acids in biological samples upon oral administration of paracetamol (e.g., a 500-mg tablet) may represent a novel model of oxidative stress in humans. Such potential paracetamol metabolites are di-paracetamol and 3-nitro-paracetamol, in analogy to the well-investigated endogenous biomarkers di-tyrosine and 3-nitro-tyrosine. Di-paracetamol and 3-nitro-paracetamol are known to be formed both by enzymatic and non-enzymatic routes. In the present work we report on mouse and human pilot studies on the formation and appearance of di-paracetamol and 3-nitro-paracetamol in blood of mice intraperitoneally administered paracetamol, as well as in plasma and urine samples of healthy subjects who received a 500-mg paracetamol tablet or placebo. For the analysis of di-paracetamol and 3-nitro-paracetamol in plasma and urine samples, analytes were extracted by solvent extraction with ethyl acetate and subsequently analyzed by LC–MS/MS without and with derivatization with pentafluorobenzyl bromide. GC–MS/MS was used to detect 3-nitro-paracetamol and quantify paracetamol as pentafluorobenzyl derivatives. Our studies indicate that di-paracetamol and 3-nitro-paracetamol appear in plasma and urine when paracetamol is given orally to healthy humans at the therapeutic dosage of 5–7 mg/kg. The molar ratio of di-paracetamol to paracetamol in urine was determined to be 1:535 in the paracetamol group and 1:6844 in the placebo group; the molar ratio of 3-nitro-paracetamol to paracetamol in urine was determined to be 1:199 in the paracetamol group and 1:8657 in the placebo group. Our studies suggest that a fraction of circulating and excretory di-paracetamol and 3-nitro-paracetamol may be formed artefactually during sample workup including derivatization. Further studies based on the quantitative determination of di-paracetamol and 3-nitro-paracetamol in biological samples by LC–MS/MS and/or GC–MS/MS using stable-isotope labeled analogues as internal standards are warranted to test the utility of paracetamol as a probe of oxidative stress in animals and in humans in health and disease.
Keywords: Clinical trial; Dimerization; Human model; Nitration; Oxidative stress; Tandem mass spectrometry;