Journal of Chromatography B (v.861, #1)

Quantification of cidofovir in human serum by LC–MS/MS for children by André Breddemann; Linda Hsien; Edith Tot; Stephanie Läer (1-9).
A new method for the quantification of cidofovir (CDV), an acyclic nucleotide analogue of cytosine with antiviral activity against a broad-spectrum of DNA viruses, in human serum, using high-performance liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) has been developed. A strong anion exchange (SAX) solid-phase extraction procedure was applied for the sample preparation. The tandem mass spectrometer was tuned in the multiple reaction monitoring mode to monitor the m/z 278.1 → 234.9 and the m/z 288.1 → 133.1 transitions for CDV and the internal standard 9-(2-phosphonylmethoxyethyl)guanine (PMEG), respectively, using negative electrospray ionization. The MS/MS response was linear over the concentration range from 78.125 ng/ml to 10,000 ng/ml, with a lower limit of quantification of 78.125 ng/ml. The intra- and inter-day precisions (relative standard deviation (%)) for CDV were less than 7.8% and the accuracies (% of deviation from nominal level) were within ±12.1% for quality controls. The novel LC–MS/MS method allowed a specific, sensitive and reliable determination of CDV in human serum and was applied to investigate the yet unknown pharmacokinetic properties of CDV in a paediatric cancer patient.
Keywords: Cidofovir; PMEG; LC–MS/MS; Pharmacokinetic study; Children;

Development and validation of a liquid chromatographic/electrospray ionization mass spectrometric method for the determination of salidroside in rat plasma: Application to the pharmacokinetics study by Sen Yu; Li Liu; Tao Wen; Yuchun Liu; Dianlei Wang; Yuxian He; Yan Liang; Xiaodong Liu; Lin Xie; Guangji Wang; Wenzhi Wei (10-15).
A sensitive and specific liquid chromatography–electrospray ionization-mass spectrometry (LC–ESI-MS) method has been developed and validated for the identification and quantification of salidroside, a major active constituent from Rhodiola rosea L., in rat plasma using helicid as an internal standard. The method involves a simple single-step liquid–liquid extraction with n-butanol. The analytes were separated by isocratic gradient elution on a Shim-pack ODS (4.6 μm, 250 mm × 2.0 mm i.d.) column and analyzed in selected ion monitoring (SIM) mode with a negative electrospray ionization (ESI) interface using the respective [M  + Cl] ions, m/z 335 for salidroside, m/z 319 for internal standard. The method was validated over the concentration range of 5–2000 ng/mL for salidroside. Within- and between-batch precision (R.S.D.%) were all within 6% and accuracy ranged from 96 to 112%. The lower limits of quantification was 5 ng/mL. The extraction recovery was on average 86.6% for salidroside. The validated method was used to study the pharmacokinetic profile of salidroside in rat plasma after intravenous and oral administration of salidroside. The bioavailability of salidroside in rats is 32.1%.
Keywords: Liquid chromatography/mass spectrometry; Salidroside; Rat plasma; Pharmacokinetics; Bioavailability;

A new analytical method using gas chromatography with mass spectrometry (GC–MS) for the quantitative determination of lufenuron, a benzoylphenylurea (BPU) class of insecticide, from wheat flour has been developed and applied for time-dependant residue monitoring in treated wheat flour. The analyte was extracted from wheat flour by a single step solid–liquid extraction by using ethyl acetate and subsequently cleaned up using the Primary Secondary Amine as a sorbent prior to GC–MS analysis. The present method provides sufficient sensitivity as reflected by the values of limit of detection (LOD) and limit of quantification (LOQ), 5 ng/mL (S/N ∼3) and 50 ng/mL (the lowest validation point on the calibration curve), respectively. The calibration curve showed an excellent linearity in the concentration range of 50–1000 ng/mL (r 2  = 0.998). The average recovery for spiked samples at three concentrations (150, 300, and 450 ng/g) was 98.23 ± 2.52% R.S.D. The method was applied for the determination of lufenuron residues in treated wheat flour samples. Simultaneous determination of bio-efficacy of lufenuron residues was also carried out against the red flour beetle, Tribolium castaneum to correlate the actual residual effect of lufenuron as detected by the analytical method, over a period of 3 months. The findings revealed that the residual concentration of lufenuron were neither uniform nor in descending order over a period of 3 months in wheat flour, possibly because of an uneven dispersal in the treated wheat which was subsequently milled into flour, as confirmed by GC–MS analysis. However, the residues of lufenuron were sufficient to produce 100% mortality of T. castaneum larvae up to 3 months. The results have been discussed in view of the potential of lufenuron as a candidate molecule for the control of stored product pests.
Keywords: Lufenuron; GC–MS; Method development; Residue analysis; Tribolium castaneum; Wheat; Bio-efficacy;

A method based on direct injection of diluted urine for the identification and quantification of amphetamine, methamphetamine, 3,4-methylenedioxymetamphetamine and 3,4-methylenedioxyamphetamine in human urine by electrospray ionisation liquid chromatography-tandem mass spectrometry was validated for use as a confirmation procedure in urine drug testing. Two deuterium labelled analogues, amphetamine-D5 and 3,4-methylenedioxymetamphetamine-D5, were used as internal standards. Twenty microliter aliquots of urine were mixed with 80 μL internal standard solution in autosampler vials and 10 μL was injected. The chromatographic system consisted of a 2.0 mm × 100 mm C18 column and the gradient elution buffers used acetonitrile and 25 mmol/L formic acid. Two product ions produced from the protonated molecules were monitored in the selected reaction monitoring mode. The intra- and inter-assay variability (coefficient of variation) was between 5 and 16% for all analytes at 200 and 6000 ng/mL levels. Ion suppression occurred early after injection but did not affect the identification and quantification of the analytes in authentic urine samples. The method was further validated by comparison with a reference gas chromatographic–mass spectrometric method using 479 authentic urine samples. The two methods agreed almost completely (99.8%) regarding identified analytes when applying a 150 ng/mL reporting limit. Four deviating results were observed for 3,4-methylenedioxymethamphetamine and this was due to uncertainty in quantification around the reporting limit. For the quantitative results the slope of the regression lines were between 0.9769 and 1.0146, with correlation coefficients >0.9339. We conclude that the presented liquid chromatographic-tandem mass spectrometric method is robust and reliable, and suitable for use as a confirmation method in urine drug testing for amphetamines.
Keywords: Urine drug testing; Amphetamine; Methamphetamine; 3,4-Methylenedioxymethamphetamine; 3,4-Methylenedioxyamphetamine; LC–MS/MS;

Cross species applicability of abundant protein depletion columns for ribulose-1,5-bisphosphate carboxylase/oxygenase by Nicholas A. Cellar; Krishnamoorthy Kuppannan; Marsha L. Langhorst; Weiting Ni; Ping Xu; Scott A. Young (29-39).
In plants, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is an important enzyme in the Calvin cycle, catalyzing the first step of carbon fixation. Because of its critical role in photosynthesis, RuBisCO comprises 30–60% of the total protein content in green leaf tissue and represents a major protein which can interfere with determination of lower abundance proteins in plant proteomics. A potential solution to aid in the determination of low level proteins in plant proteomics are RuBisCO immunodepletion columns. Two formats, spin and LC, of Seppro™ IgY RuBisCO depletion columns were evaluated for cross species applicability. The spin and LC columns were found to deplete arabidopsis RuBisCO by greater than 90 and 98%, respectively, and automation could be achieved with the LC format. Canola RuBisCO was depleted to a similar extent, and there was evidence suggesting that corn and tobacco RuBisCO were also highly depleted in flow through fractions. Model proteins were spiked into samples to provide insight into the degree of non-specific binding. Finally, improved detection and identification of lower abundance proteins was demonstrated after depletion.
Keywords: RuBisCO; Ribulose-1,5-bisphosphate carboxylase/oxygenase; Protein; Immunodepletion; Arabidopsis; Tobacco; Corn; Canola; Proteomics; Peptide mass fingerprint; Abundant protein depletion; Depletion;

Chemopreventive and antioxidant action of betalain pigments can differ in dependence on their stereoselective properties, therefore, it is necessary to use relevant methods for monitoring of their possible stereoisomers. Chromatographic characterisation of a group of new isomers of various 6′-O-acylated betacyanins and decarboxylated betacyanins which were generated at low concentration by intramolecular pH-dependent acyl migration was studied in aqueous solutions by HPLC separation with diode-array and mass spectrometric detection. Under alkaline conditions (pH 10.5) the rate of migration was dramatically accelerated, however, always favouring the 6′-O-position and it was much less prominent at lower pH (under 7.0). The possible products of the partial rearrangement were tentatively identified as the 3′-O- and 4′-O-acylated forms and their relative retention times were provided. In malonylated betacyanins and 17-decarboxy-betacyanins the 4′-O-forms were characterised in RP-HPLC by higher retention than the 6′-O forms, whereas the 3′-O-forms were always the most polar. In contrast, the isomerisation of hylocerenin and 17-decarboxy-hylocerenin resulted in different chromatographic profiles of the migration products. In 2-decarboxy- and 2,17-bidecarboxy-betacyanins the 3′-O- and 4′-O-acylated forms eluted always before the 6′-O-acylated betacyanins. The investigations on acyl migration in isolated 4′-O-malonyl-betanin confirmed the strong tendency of reverse acyl migration (4′ → 6′) and also partial 4′ → 3′ rearrangement which were leading to the final monoester regioisomeric distribution (%) close to 87:7:6 (6′-O-, 4′-O-, 3′-O-).
Keywords: Acyl migration; HPLC–ESI-MS; HPLC-DAD; Betanin; Phyllocactin; Hylocerenin; Mammillarinin; Apiosyl; Decarboxy-betacyanins;

Isoprostanes are formed after peroxidation of arachidonic acid and are promising biomarkers for reactive oxygen species. A LC–MS/MS based method was developed for the quantitation of two isoprostanes (iPF-III and 8,12-iso-iPF-VI) in hepatocytes, tissue and urine samples of rats. A column switching method was used to reduce sample preparation to a minimum. Precision was 9.4% and accuracy was between 96 and 114% for free iPF-III in tissue at concentrations from 1.9 to 6.1 ng/g. Treatment of rats with CCl4 to induce oxidative stress resulted in a dose-dependent increase (two- to three-fold) of iPF-III and 8,12-iso-iPF-VI in liver and kidney. For both isoprostanes an increase of four- to five-fold was observed in CCl4 treated hepatocytes and six- to eight-fold in CCl4 treated and glutathione depleted hepatocytes. In conclusion, the presented method is sensitive, specific and precise to be applied for the quantitation of iPF-III and 8,12-iso-iPF-VI which are shown to increase by CCl4 treatment in vitro and in vivo.
Keywords: Isoprostanes; 8-iso PGF; iPF-III; 8,12-iso-iPF-VI; Oxidative stress; Carbon tetrachloride; LC–MS/MS; Column switching; Electrospray;

Dextromethorphan (DEM) is a widely used probe drug for human cytochrome P450 2D6 isozyme activity assessment by measuring the ratio between DEM and its N-demethylated metabolite dextrorphan (DOR). DOR is excreted in urine mainly conjugated to glucuronic acid. Prior to quantification, DOR must be deconjugated to avoid variability caused by the polymorphic glucuronosyltransferase enzyme. A chemical hydrolysis method was optimized using a chemometric approach. Three factors (acid concentration, hydrolysis time and temperature) were selected and simultaneously varied to study their effect on conjugated DOR hydrolysis. Hydrolysis conditions that maximize DOR release without significant degradation of both DEM and DOR were chosen and results were compared to those obtained by enzymatic method using β-glucuronidase. An HPLC method with fluorescence detection was developed for the simultaneous quantitation of DEM, DOR and levallorphan, used as an internal standard. Separation was performed on a phenyl analytical column (150 mm × 4.6 mm i.d., 5 μm) with a mobile phase consisting of 18% acetonitrile and 50 mM phosphoric acid (pH 3). The overall analytical procedure was validated and showed good performances in terms of selectivity, linearity, sensitivity, precision and accuracy. Finally, this assay was used to determine DEM/DOR molar ratios in fibromyalgia patients for the purpose of determining phenotype status for the CYP2D6.
Keywords: Chemical hydrolysis; Experimental design; CYP2D6; Phenotype; Dextromethorphan;

Effect of IDA and TREN chelating agents and buffer systems on the purification of human IgG with immobilized nickel affinity membranes by Mariana Borsoi Ribeiro; Mookambesvaran Vijayalakshmi; Daniele Todorova-Balvay; Sonia Maria Alves Bueno (64-73).
The purification of IgG from human plasma was studied by comparing two affinity membranes complexed with Ni(II), prepared by coupling iminodiacetic acid (IDA) and Tris(2-aminoethyl)amine (TREN) to poly(ethylenevinyl alcohol), PEVA, hollow fiber membranes. The Ni(II)-TREN-PEVA hollow fiber membrane had lower capacity for human IgG than the complex Ni(II)-IDA-PEVA, but with similar selectivity. The IgG in peak fractions eluted from the Ni(II)-IDA-PEVA with a stepwise concentration gradient of Tris–HCl pH 7.0 (100–700 mM) reached a purity of 98% (based on IgG, IgM, IgA, albumin, and transferrin nephelometric analysis). Adsorption IgG data at different temperatures (4–37 °C) were analyzed using Langmuir model resulting in a calculated maximum capacity at 25 °C of 204.6 mg of IgG/g of dry membrane. Decrease in K d with increasing temperature (1.7 × 10−5 to 5.3 × 10−6  M) indicated an increase in affinity with increased temperature. The positive value of enthalpy change (26.2 kJ/mol) indicated that the adsorption of IgG in affinity membrane is endothermic. Therefore, lower temperature induces adsorption as verified experimentally.
Keywords: Affinity membrane; Purification; Human IgG; Adsorption; IMAC;

The aim of this study is to develop magnetically loaded nanosorbents carrying specific monoclonal antibodies (namely CD105 and CD73) for separation of mesenchymal stem cells from cell suspensions. Super-paramagnetic magnetite (Fe3O4) nanoparticles were produced and then coated with a polymer layer containing carboxylic acid functional groups (average diameter: 153 nm and polydispersity index: 0.229). In order to obtain the nanosorbents, the monoclonal antibodies were immobilized via these functional groups with quite high coupling efficiencies up to 80%. These nanosorbents and also a commercially available one (i.e., microbeads carrying CD105 antibodies from Miltenyi Biotec., Germany) were used for separation of CD105+ and CD73+ mesenchymal stem cells from model cell suspension composed of peripheral blood (97.6%), human bone marrow cells (1.2%) and fibroblastic cells (1.2%). The initial concentrations of the CD105+ and CD73+ cells in this suspension were measured as 5.86% and 6.56%, respectively. A flow-through separation system and a very simple homemade batch separator unit were used. We were able to increase the concentration of CD105+ cells up to about 86% in the flow-through separation system with the nanosorbents produced in this study, which was even significantly better than the commercial one. The separation efficiencies were also very high, especially for the CD73+ cells (reached to about 64%) with the very simple and inexpensive homemade batch unit.
Keywords: Mesenchymal stem cell; Isolation; Flow-through and batch systems; Magnetic polymeric nanosorbents; CD105 and CD73 antibodies;

Recently there has been some debate regarding the presence and associated health risk of low molecular weight carrageenan in foodstuffs. Unfortunately measurement of the low molecular weight tail (LMT) of food-grade carrageenans (defined here as the carrageenan having relative molecular mass (Mr) below 50,000) is not trivial, largely due to its low abundance. So far methods employing light scattering have been unsuccessful in producing reproducible results, probably due to the poor detector response at low masses. In this work a method based on high performance size exclusion chromatography coupled to a refractive index detector (HPSEC-RI) has been used for the measurement of the LMT in food-grade carrageenan ingredients and in a carrageenan-containing finished product (a jelly). Over the course of half a year, 19 measurements were made on a reference carrageenan; the results demonstrated that the method had excellent reproducibility. Applied to a number of different carrageenan ingredients, it was found that, in general, the LMT represents less than 8% of the total carrageenan in ingredients, and under the correct conditions increases little during food processing. The data also indicated that pH appears to be a critical factor during food processing and pH levels below 4.0 should be avoided.
Keywords: HPSEC; Poligeenan; Low molecular weight carrageenan; κ-Carrageenan; ι-Carrageenan; Food;

A fast method for the quantification of methylamine in fermentation broths by gas chromatography by Valérie Jérôme; Markus Hermann; Frank Hilbrig; Ruth Freitag (88-94).
The objective of this study was to develop a method for the quantitative analysis of the methylamine concentration in fermentation broths of Hyphomicrobium zavarzinii ZV 580 cultures. For this purpose an established method for the quantification of free amino acids in such matrices was adapted and validated. The detection limit was 10 μM, the calibration curve showed good linearity (R 2  = 0.9998) in the concentration range between 0.1 and 8 mM. The standard deviation of the injection-to-injection reproducibility (n  = 10) of the retention coefficient was <1%, that of the peak area <5%. In case of the sample-to-sample reproducibility (n  = 8), the standard deviation was <5% for the retention coefficient and <10% for the peak area. The validated method was successfully applied for monitoring a fed-batch bioprocess (starting volume: 8 L, initial methylamine hydrochloride concentration: 10 mM) producing a dye-linked formaldehyde dehydrogenase in H. zavarzinii ZV 580.
Keywords: Hyphomicrobium zavarzinii; Methylamine; Gas chromatography; Fermentation;

A highly sensitive assay for ritodrine in human serum by hydrophilic interaction chromatography-tandem mass spectrometry by Tomofumi Ohmori; Mitsuhiro Nakamura; Shin Tada; Tadashi Sugiyama; Yoshinori Itoh; Yasuhiro Udagawa; Kazuyuki Hirano (95-100).
We developed a sensitive assay for ritodrine (RTD), a β2-adrenergic agonist, in human serum. This method was based upon the selective and sensitive technique by a tandem mass spectrometry (MS/MS) using a hydrophilic interaction chromatography (HILIC) technique. This method involved a mixed-mode cation-exchange solid-phase extraction of RTD and isoxsuprine, the internal standard (IS), from serum with Waters Oasis® MCX cartridges. The detection was made using a Micromass Quattromicro™ API LC–MS/MS system with electrospray ionization source in positive ion mode. The separation of the analytes was achieved within 4 min on a silica column with a mobile phase of ammonium acetate (10 mM, pH 4.5) and acetonitrile (10:90, v/v). Multiple reaction monitoring was utilized by monitoring 288.2 → 121.1 for RTD, 302.2 → 107.0 for IS. The calibration curve for RTD was linear over a range of 0.5–1000 ng/mL. When 50 μL serum was used for extraction, the lower limit of quantification was 0.39 ng/mL (97.5 fg on-column). The percent coefficient of validation for accuracy and precision (inter- and intra-day) was less than 9.8% and the recovery was ranged from 83.5 to 94.7% for RTD. This method enabled us to successfully determine RTD in maternal and fetal sera.
Keywords: Ritodrine; Tandem mass spectrometry; Hydrophilic interaction chromatography; Solid-phase extraction;

Development of accurate classification method based on the analysis of volatile organic compounds from human exhaled air by J.J.B.N. Van Berkel; J.W. Dallinga; G.M. Möller; R.W.L. Godschalk; E. Moonen; E.F.M. Wouters; F.J. Van Schooten (101-107).
Analysis of exhaled air leads to the development of fast accurate and non-invasive diagnostics. A comprehensive analysis of the entire range of volatile organic compounds (VOCs) in exhaled air samples will enable the identification of VOCs unique for certain patient groups. This study demonstrates proof of principle of our developed method tested on a smoking/non-smoking study population. Thermal desorption and gas chromatography coupled to time-of-flight mass spectrometry were used to analyse exhaled air samples. The VOC profiles obtained from each individual were combined into one final database based on similarity of mass spectra and retention indexes (RI), which offers the possibility for a reliable selection of compounds of interest. As proof of principle we correctly classified all subjects from population of smoking (N  = 11) and non-smoking (N  = 11) based on the VOC profiles available in their exhaled air. Support vector machine (SVM) analysis identified 4 VOCs as biomarkers of recent exposure to cigarette smoke: 2,5-dimethyl hexane, dodecane, 2,5-dimethylfuran and 2-methylfuran. This approach contributes to future development of fast, accurate and non-invasive diagnostics of inflammatory diseases including pulmonary diseases.
Keywords: VOC; Exhaled air; Breath; Classification; GC/MS; Smokers;

A fast method for the quantitative determination of amoxicillin (AMO), amoxicilloic acid (AMA) and amoxicillin diketopiperazine-2′,5′-dione (DIKETO) in pig edible tissues (kidney, liver, fat and muscle) with liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS) is presented. The method uses a simple liquid–liquid extraction of the tissue matrix with a 10 mM potassium dihydrogen phosphate buffer (pH 4.5) as extraction solvent. After deproteinisation by ultrafiltration, the tissue extract was directly injected onto the LC column. Chromatographic separation of the components was performed on a PLRP-S polymeric column using 0.1% of formic acid in water and acetonitrile. The mass spectrometer was operated in the positive electrospray MS/MS mode. The method was fully validated according to EU requirements (linearity, precision, trueness, quantification limit, detection limit and specificity). The stability of the components was evaluated over the pH range from 1.2 to 8.0. Biological samples of pigs medicated with AMO and AMO/clavulanic acid were analyzed using the developed method.
Keywords: Amoxicillin; Amoxicilloic acid; Amoxicillin diketopiperazine-2′,5′-dione; Liquid chromatography/electrospray ionization-tandem mass spectrometry; Stability; Pig tissues;

Fast and direct quantification of adrenal steroids by tandem mass spectrometry in serum and dried blood spots by Nils Janzen; Stefanie Sander; Michael Terhardt; Michael Peter; Johannes Sander (117-122).
We present a fast and reproducible method for steroid analysis (corticosterone, deoxycorticosterone, progesterone, 17α-hydroxyprogesterone, 11-deoxycortisol, 21-deoxycortisol, androstenedione, testosterone, dihydrotestosterone and cortisol) in small volumes of serum and in dried blood spot samples by LC-MS/MS. No derivatisation was needed. LC separation was achieved by using an Atlantis® C18 column and water–methanol–formic acid gradient as a mobile phase and a flow rate of 250 μL/min over a run time of 6 min. Steroids were measured in MRM mode with electrospray interface (positive ion mode). Validation showed excellent precision, sensitivity, recovery and linearity with coefficients of determination r 2  > 0.992.
Keywords: Steroid profile; Tandem mass spectrometry; Adrenal cortex disorders;

An HPLC-ICP-MS technique for determination of cadmium–phytochelatins in genetically modified Arabidopsis thaliana by Baki B.M. Sadi; Anne P. Vonderheide; Ji-Ming Gong; Julian I. Schroeder; Jodi R. Shann; Joseph A. Caruso (123-129).
A reversed-phase high-performance liquid chromatographic technique was developed to separate cadmium–phytochelatin complexes (Cd-PC2, Cd-PC3, and Cd-PC4) of interest in the plant Arapidopsis thaliana. High-performance liquid chromatography (HPLC) was coupled to an inductively coupled plasma mass spectrometric (ICP-MS) system with some modification to the interface. This was done in order to sustain the plasma with optimum sensitivity for cadmium detection in the presence of the high methanol loads used in the gradient elution of the reversed-phase separation. The detection limits were found to be 91.8 ng l−1, 77.2 ng l−1 and 49.2 ng l−1 for Cd-PC2, Cd-PC3, and Cd-PC4 respectively. The regression coefficients (r 2) for Cd-PC2 to Cd-PC4 detection ranged from 0.998 to 0.999. The method was then used to investigate the occurrence and effect of cadmium–phytochelatin complexes in wild-type Arabidopsis and a phytochelatin-deficient mutant cad1-3 that had been genetically modified to ectopically express the wheat TaPCS1 phytochelatin synthase enzyme. The primary complex found in both wild-type and transgenic plants was Cd-PC2. In both lines, higher levels of Cd-PC2 were found in shoots than in roots, showing that phytochelatin synthases contribute to the accumulation of cadmium in shoots, in the Cd-PC2 form. Genetic modification did, however, impact the overall accumulation of Cd. Transgenic plants contained almost two times more cadmium in the form of Cd-PC2 in their roots than did the corresponding wild-type plants. Similarly, the shoot samples of the modified species also contained more (by 1.6 times) cadmium in the form of Cd-PC2 than the wild type. The enhanced role of PC2 in the transgenic Arabidopsis correlates with data showing long-distance transport of Cd in transgenic plants. Targeted transgenic expression of non-native phytochelatin synthases may contribute to improving the efficiency of plants for phytoremediation.
Keywords: Phytoremediation; HPLC-ICP-MS; Arabidopsis thaliana; Genetic modification; Cadmium;

A HPLC method with UV detection was developed and validated for the determination of thiorphan in human plasma. Nevirapine was used as the internal standard. Separation was performed by a Waters sunfire C18 reversed-phase column maintained at 35 °C. The mobile phase was a mixture of 0.05 M phosphate buffer with the pH adjusted to 2.6 and acetonitrile (74:26, v/v) at a flow rate of 1.0 mL/min. The UV detector was set at 210 nm. An original pre-treatment of plasma samples was developed, based on solid-phase extraction (SPE) with solid-phase extraction cartridges (Oasis HLB 3 mL, 60 mg). The extraction recovery for plasma samples of thiorphan at 0.1, 0.4 and 2.0 μg/mL was 93.5%, 98.2% and 97.8%, respectively. The calibration curve was linear with the correlation coefficient (r) above 0.9998. Linearity was verified over the range of 0.05–4 μg/mL thiorphan in plasma. The limit of quantification (LOQ) is 0.05 μg/mL. The mean accuracy was 92.7–99.6%. The coefficient of variation (precision) in the within- and between-batch was 2.2–8.4% and 4.1–8.1%, respectively. This method is simple, economical and specific, and has been used successfully in a pharmacokinetic study of thiorphan.
Keywords: Racecadotril; Thiorphan; Human plasma; High performance liquid chromatography; Solid-phase extraction;

The suitability of micellar electrokinetic capillary chromatography (MEKC) with laser-induced fluorescence (LIF) detection for the determination of pheomelanin in biological materials has been investigated. 5-Carboxyfluorescein succinimidyl ester was chosen as the labeling reagent to precapillary derivatize the two marker aminohydroxyphenylalanine (AHP) isomers produced after reductive hydrolysis of pheomelanin with hydriodic acid (HI). Various parameters affecting derivatization and separation were systematically studied. Under optimal conditions, the analytes could be separated within 18 min, and the relative standard deviations (R.S.D.) of migration time and corrected peak areas were less than 5.5%. Compared with the conventional high-performance liquid chromatography (HPLC) method with electrochemical detection, the 100-fold improvements in sensitivity were achieved by applying LIF detection. As a preliminary application, this method has been successfully applied to the determination of pheomelanin in two human melanoma cell cultures, black hair, melanoma tissue and urine samples of human melanoma patients with the spiked recoveries in the range of 88–96%.
Keywords: Pheomelanin; Aminohydroxyphenylalanine; Laser-induced fluorescence; Micellar electrokinetic capillary chromatography; Derivatization;

Combination of HSCCC and Sephadex LH-20 methods by Changjun Yang; Daxiang Li; Xiaochun Wan (140-144).
In order to separate the main individual theaflavin monomers from black tea, high-speed countercurrent chromatography (HSCCC) and Sephadex LH-20 column chromatography were applied. The results showed that theaflavin (TF1), theaflavin-3-gallate (TF2A), theaflavin-3′-gallate (TF2B) and theaflavin-3,3′-digallate (TF3) can be obtained by HSCCC using a solvent system composed of n-hexane–ethyl acetate–methanol–water (1:3:1:6, v/v/v/v), but the TF1 was containing epicatechin-3-gallate (ECG). Similarly, Sephadex LH-20 can also effectively separate TF2A(B) and TF3, but epigallocatechin-3-gallate (EGCG) contaminated TF1, too. Combination of HSCCC and Sephadex LH-20, the preferably purified TF1, TF2A(B) and TF3 were obtained than single separation technique. In addition, ECG and EGCG were also suggested to be able to be comprehensively separated by combination of the two techniques.
Keywords: High-speed countercurrent chromatography; Sephadex LH-20; Theaflavin; Theaflavin-3-gallate; Theaflavin-3′-gallate; Theaflavin-3,3′-digallate; Black tea;

A comparison of the analytical performance of atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) for the quantitative determination of six urinary phytoestrogens (daidzein, O-desmethylangolensin, equol, enterodiol, enterolactone and genistein) by high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) is presented here. Both APCI and ESI were suitable for the analysis of these compounds; however, ESI did improve measurement imprecision and sensitivity in certain cases. Method imprecision (between-run coefficients of variation [CVs] from duplicate analysis of three quality control [QC] urine pools across 20 runs) was 5.6–12% for ESI, as opposed to 5.3–30% for APCI. At low concentrations (3–60 ng/mL, analyte dependent) imprecision was lower with ESI, whereas both techniques were generally commensurate at high concentrations (200–1000 ng/mL, analyte dependent). Method accuracy (spiked analyte recovery from the QC pools) was comparable between techniques: 86–114% for ESI; 95–105% for APCI. Limits of detection (LODs) were equivalent or better with ESI compared to APCI, with the most significant LOD improvement observed for equol (ESI: 0.3 ng/mL; APCI: 2.7 ng/mL). This translated into a substantial increase in equol detection frequency (% of sample results above LOD) within a random patient sample subset (98% for ESI, compared to 81% for APCI, n  = 378). Correlation (Pearson) and agreement (Deming regression, Bland-Altman bias) between ESI and APCI results in the patient subset was better in cases where imprecision and sensitivity was similar for both techniques (daidzein, enterolactone, genistein: r  = 0.993–0.998; slope = 0.98–1.03; bias = −4.2 to −0.8%); correlation and/or agreement was poorer for analytes, where APCI imprecision and sensitivity were inferior (equol, O-desmethylangolensin, enterodiol). Baring significant factors arising from differences in ionization source design, these observations suggest that ESI is more appropriate for urinary biomonitoring of these compounds by LC–MS/MS.
Keywords: Phytoestrogens; High-performance liquid chromatography; Tandem mass spectrometry; Electrospray ionization; Atmospheric pressure chemical ionization;

Determination of tegaserod by LC–ESI-MS/MS and its application to a pharmacokinetic study in healthy Chinese volunteers by Jian-Jun Zou; Xiao-Jie Bian; Li Ding; Yu-Bin Zhu; Hong-Wei Fan; Da-Wei Xiao (151-157).
A simple, rapid and sensitive high performance liquid chromatography–electrospray ionization-tandem mass spectrometry (HPLC–ESI-MS/MS) assay for determination of tegaserod in human plasma using diazepam as internal standard (IS) was established. After adjustment to a basic pH with sodium hydroxide, plasma was extracted by ethyl acetate and separated by high performance liquid chromatography (HPLC) on a reversed-phase C18 column with a mobile phase of methanol: 5 mM ammonium acetate (75:25, v/v, adjusting the pH to 3.5 with glacial acetic acid). The quantification of target compounds was obtained by using multiple reaction monitoring (MRM) transitions; m/z 302.5, 173.2 and 285.4, 193.2 were measured in positive mode for tegaserod and internal standard (diazepam), respectively. The lower limit of quantification (LLOQ) was 0.05 ng/ml. The calibration curves were linear over the range 0.05–8.0 ng/ml (r  = 0.9996) for tegaserod. The mean absolute recovery of tegaserod was more than 85.56%. Intra- and inter-day variability values were less than 9.21% and 10.02%, respectively. The samples were stable for 8 h under room temperature (25 °C, three freeze–thaw cycles in 30 days and for 30 days under −70 °C). After administration of a single dose of tegaserod maleate 4 mg, 6 mg and 12 mg, respectively, the area under the plasma concentration versus time curve from time 0 h to 12 h (AUC0–12) were (2.89 ± 0.88), (5.32 ± 1.21) and (9.38 ± 3.42) ng h/ml, respectively; peak plasma concentration (C max) were (1.25 ± 0.53), (2.21 ± 0.52) and (4.34 ± 1.66) ng/ml, respectively; apparent volume of distribution (V d /F) were (6630.5 ± 2057.8), (7615.2 ± 2242.8) and (7163.7 ± 2057.2) l, respectively; clearance rate (CL/F) were (1851.4 ± 496.9), (1596.2 ± 378.5) and (1894.2 ± 459.3) l/h, respectively; time to C max (T max) were (1.00 ± 0.21), (1.05 ± 0.28) and (1.04 ± 0.16) h, respectively; and elimination half-life (t 1/2) were (3.11 ± 0.78), (3.93 ± 0.92) and (3.47 ± 0.53) h, respectively; MRT were (3.74 ± 0.85), (4.04 ± 0.56) and (3.28 ± 0.66) h, respectively. The essential pharmacokinetic parameters after oral multiple doses (6 mg, b.i.d) were as follows: C ssmax, (2.72 ± 0.61) ng/ml; T max, (1.10 ± 0.25) h; C ssmin, (0.085 ± 0.01) ng/ml; C av, (0.54 ± 0.12) ng/ml; DF, (4.84 ± 0.86); AUCss, (6.53 ± 1.5) ng h/ml. This developed and validated assay method had been successfully applied to a pharmacokinetic study after oral administration of tegaserod maleate in healthy Chinese volunteers at a single dose of 4 mg, 6 mg and 12 mg, respectively. The pharmacokinetic parameters can provide some information for clinical medication.
Keywords: Tegaserod; Pharmacokinetics; LC–ESI-MS/MS;