Journal of Chromatography B (v.844, #2)
Editorial Board (CO1).
Review of methodology for the determination of macrocyclic lactone residues in biological matrices by Martin Danaher; Laurence C. Howells; Steven R.H. Crooks; Vesna Cerkvenik-Flajs; Michael O’Keeffe (175-203).
The macrocyclic lactones (MLs) are probably the anti-parasitic agents most widely used in the treatment of food producing animals, poultry, aquaculture and crops. Ivermectin was the first macrocyclic lactone product to be licensed for use about 20 years ago. A number of alternative products such abamectin, doramectin, emamectin, eprinomectin, moxidectin, milbemycin and selamectin, have been marketed since. Because of the increase in the number of ML drugs, there has been a steady increase in the number of published analytical methods for determination of their residues. In this paper, the structure and properties of the different ML drugs available on the market are described. The occurrence and persistence of ML residues in food is discussed in relation to marker residues and current maximum residue limits (MRLs) as defined in the European Union (EU). Methodologies for determination of ML residues in biological matrices are described in terms of extraction and clean-up methods used for different matrices. Detection systems for determination of ML residues are discussed with a particular emphasis placed on new developments in screening technologies and liquid chromatography with fluorescence or mass spectrometry.
Keywords: Avermectins; Milbemycins; Extraction; SPE; Derivatisation; HPLC Fluorescence; LC–MS/MS;
Evaluation of stationary phases for 2-dimensional HPLC of proteins by E. Reh; B. Hahn; S. Lamotte (204-212).
RP-separation with TFA-based water/acetonitrile eluents is widely used for peptides and small proteins but is well known difficult for large or membrane proteins. Especially in proteomics or other complex biological matrices reliable elution patterns are difficult to achieve. New commercial stationary phases are validated regarding long term stability, protein recovery, carry over, symmetry and selectivity using 10 different proteins with different molar weights, isoelectric points and glycosylation. It could be demonstrated that some stationary phases had poor protein elution performances. They did not elute a protein at all or with minor recovery, peak symmetries. Sometimes bad and formidable carry over effects for peak areas in the following run were observed. Selectivity in separation of different isomers or glycosylated proteins is also different. The results suggest that neither surface area nor pore diameter play an important role in the application of reversed phases for HPLC of proteins. The investigations leads one to suppose that the bonding chemistry seems to be an important aspect. Most critical fact is that some RP-phases did not elute a protein at all others only 20% of the injected protein mass, which makes the objective of an RP-chromatogram highly questionable.
Keywords: HPLC; Proteins; Reversed phase; Stability; Recovery; Carry over; Symmetry; Selectivity;
A hemagglutinin with mitogenic activity from dark red kidney beans by Lixin Xia; T.B. Ng (213-216).
A 67-kDa hemagglutinin composed of two identical subunits was purified from Phaseolus vulgaris cv. ‘Dark Red Kidney Bean’. It was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel. The hemagglutinin was highly purified after the two aforementioned chromatographic steps as revealed by a single peak in gel filtration on Superdex 75 and a single band in SDS-PAGE. The hemagglutinating activity was stable between 25 °C and 70 °C, and between pH 4 and pH 11, and in the presence of a variety of divalent metal chlorides at 500 mM concentration. The activity was reduced by 50% at 80 °C, and also when the pH was lowered to 3 or elevated to 12. The activity was reduced by 75% in the presence of 250 mM KCl or NaCl. A variety of sugars tested failed to inhibit the hemagglutinating activity of the hemagglutinin. Although the hemagglutinin exhibited mitogenic activity toward murine splenocytes, it had no effect on the activity of HIV-1 reverse transcriptase or mycelial growth in the fungi Botrytis cinerea, Fusarium oxysporum and Mycosphaerella arachidicola. It exerted an antiproliferative activity on leukemia L1210 cells.
Keywords: Dark red kidney bean; Isolation; Hemagglutinin;
Aqueous–aqueous two-phase systems composed of low molecular weight of polyethylene glycols and dextrans for counter-current chromatographic purification of proteins by Yoichi Shibusawa; Naoko Takeuchi; Kazusa Sugawara; Akio Yanagida; Heisaburo Shindo; Yoichiro Ito (217-222).
New aqueous–aqueous two-phase systems composed of relatively low molecular weight polymers such as polyethylene glycol (PEG) (Mr: 1000–4000) and dextran (Mr: 10,000 and 40,000) were evaluated for purification of proteins by counter-current chromatography (CCC). The compositions of aqueous two-phase systems were optimized by measuring parameters such as viscosity and volume ratio between the two phases. CCC purification of a glucosyltransferase (GTF) from Streptococcus mutans (SM) cell-lysate was successfully demonstrated with a 7.5% PEG 3350–10% dextran T40 system containing 10 mM potassium phosphate buffer at pH 9.0. After CCC purification, both PEG and dextran contained in the CCC fractions were easily removed by ultrafiltration in a short period of time. The fractionated column contents containing GTF were analyzed by enzymatic activity as well as sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The recovery of the enzyme from CCC fraction was over 95% as estimated by enzymatic activities.
Keywords: Aqueous–aqueous two-phase systems; Counter-current chromatography; Proteins; Glucosyltransferase;
Quantitative measurement of sulforaphane, iberin and their mercapturic acid pathway metabolites in human plasma and urine using liquid chromatography–tandem electrospray ionisation mass spectrometry by Ahmed A. Al Janobi; Richard F. Mithen; Amy V. Gasper; P. Nicholas Shaw; Richard J. Middleton; Catharine A. Ortori; David A. Barrett (223-234).
A quantitative liquid chromatography positive ion electrospray tandem mass spectrometric method for the simultaneous determination of sulforaphane, iberin and their metabolites in human urine and plasma is described. The stability of the metabolites was determined in aqueous solution and in human plasma. Gradient liquid chromatographic separation was performed on a Zorbax SB-Aq 3.5 μm (100 × 2.1 mm) column, using a mobile phase (flow rate 0.25 mL/min) consisting of ammonium acetate buffer at pH 4 and acetonitrile. Butyl thiocarbamoyl l-cysteine was used as internal standard. The assay was linear (r 2 > 0.99) over the range of 0.03–300 μM in urine and 0.03–15 μM in plasma with intra- and inter-day assay precision (<10% CV) and accuracy (<20%). The lower limits of quantitation were in the range of 10–150 nmol/L. The method has been used to report, for the first time, individual quantitative measurement of each of the mercapturic acid pathway metabolites of sulforaphane and iberin in both human plasma and urine following a dietary study of broccoli consumption.
Keywords: Isothiocynates; Glucosinolates; Sulforaphane; Iberin; Electrospray ionisation mass spectrometry; Liquid chromatography; Metabolism; Mercapturic acid pathway; Plasma; Urine;
Quantification of a cytochrome P450 3A4 substrate, buspirone, in human plasma by liquid chromatography–tandem mass spectrometry by Wade M. Chew; Min-Jian Xu; Catherine A. Cordova; H-H. Sherry Chow (235-239).
A sensitive HPLC-tandem mass spectrometry method was developed for determination of buspirone levels in human plasma. After solid phase extraction and reversed phase HPLC separation, detection of buspirone and the internal standard (prazosin) was performed using eletrospray ionization and selected reaction monitoring in the positive ion mode. Linear calibration curves were established over a concentration range of 0.025–2.5 ng/ml when 0.5 ml aliquots of plasma were used. Satisfactory results of within-day precision (RSD of 1.9–7.7%) and accuracy (% difference of 0.5–6.6%) and between-day precision (RSD of 3.7–11.1%) and accuracy (% difference of 2.2–6.8%) were obtained. The assay has been successfully applied to the analysis of buspirone levels in more than 500 human plasma samples collected from a drug interaction study.
Keywords: Buspirone; Human plasma; Liquid chromatography; Tandem mass spectrometry; Electrospray ionization;
Determination of specific activities and kinetic constants of biotinidase and lipoamidase in LEW rat and Lactobacillus casei (Shirota) by Kou Hayakawa; Lei Guo; Elena A. Terentyeva; Xiao-Kang Li; Hiromitsu Kimura; Masahiko Hirano; Kazuyuki Yoshikawa; Takeaki Nagamine; Noriyuki Katsumata; Tsutomu Ogata; Toshiaki Tanaka (240-250).
Enzyme kinetic parameters, such as K m, V max (or V), k cat/K m, and K i (by biotin or lipoic acid) for biotinidase and lipoamidase were determined in Lewis (LEW) rat and Lactobacillus casei (Shirota) using fluorimetric high-performance liquid chromatography (HPLC). It was found that the final protein concentration below 0.1 mg/ml is sufficient to obtain linear hydrolytic reaction and to determine the Michaelis–Menten type kinetic parameters (K m, V, K i ). We applied this HPLC enzyme assay method onto the rat and some bacteria. The highest specific activities (Vs) for biotinidase were found in Lactobacillus casei (Shirota) and rat kidney. It was also found that the largest K i by product for biotinidase and lipoamidase were present in the Lactobacillus casei (Shirota). There has been found specie (between rat and mouse) differences and tissue (organ) differences, together with tissue region differences and sex differences in some tissues. Summary of the distributions of both enzymes in LEW rat was also presented. Therefore, this HPLC determination method for the enzyme kinetic parameters in tissues is expected to be an indispensable tool for the investigation of the various diseases in humans.
Keywords: Enzyme kinetics; Biotinidase; Lipoamidase (lipoyl-X hydrolase); LEW rat; Lactobacillus casei (Shirota); Tissue homogenate; HPLC-fluorimetric enzyme assay; SEC protein assay;
Performances of a multidimensional on-line SPE-LC-ECD method for the determination of three major catecholamines in native human urine: Validation, risk and uncertainty assessments by E. Rozet; R. Morello; F. Lecomte; G.B. Martin; P. Chiap; J. Crommen; K.S. Boos; Ph. Hubert (251-260).
A novel, multidimensional on-line SPE-LC method with electrochemical detection is described for the fully automated and direct analysis of the catecholamines norepinephrine, epinephrine and dopamine in urine. The integrated extractive clean-up of the raw biofluid is based on a SPE-column packed with restricted access material (RAM) which is modified with the affinity ligand nitrophenylboronic acid. The method was fully validated according to a recent approach based on an accuracy profile. The acceptance limits were set at ±15% of the nominal concentration values. The method was found accurate over a concentration range from 15 to 500 μg/l for norepinephrine, from 5 to 500 μg/l for epinephrine and from 50 to 500 μg/l for dopamine. The relative risk for the use of the validated method in routine analysis was also assessed based on this validation strategy. It was found that at most 3.5% of future sample measurements will fall outside the acceptance limits. This demonstrates the high reliability of the analytical method described. Moreover, the measurements uncertainties were deduced from the validation experiments without any additional effort.
Keywords: Catecholamines; Norepinephrine; Epinephrine; Dopamine; Urine; On-line SPE; Restricted access materials; RAM; Multidimensional; ECD; Column-switching; Validation; Accuracy profile; Risk analysis; Measurement uncertainty;
Liquid chromatography with tandem mass spectrometry for the simultaneous determination of baicalein, baicalin, oroxylin A and wogonin in rat plasma by Young Hoon Kim; Dong Won Jeong; In Bok Paek; Hye Young Ji; Youn-Chul Kim; Dong Hwan Sohn; Hye Suk Lee (261-267).
A rapid, sensitive and selective liquid chromatography–tandem mass spectrometric (LC–MS/MS) method for the determination of baicalein, baicalin, oroxylin A and wogonin, Scutellaria baicalensis active components in rat plasma was developed. After liquid–liquid extraction with 2-(3,4-dimethoxy-phenyl)-5,7-dihydroxy-chromen-4-one as internal standard, baicalein, baicalin, oroxylin A and wogonin were eluted from an Atlantis C18 column within 7 min with isocratic mobile phase consisting of methanol and 0.1% formic acid (60:40, v/v). The analytes were detected using an electrospray ionization tandem mass spectrometry in the multiple reaction monitoring (MRM) mode. The standard curves were linear (r = 1.000) over the concentration ranges of 5–500 ng/ml for baicalein, wogonin and oroxylin A and 5–5000 ng/ml for baicalin. The coefficients of variation and relative errors of baicalein, wogonin, oroxylin A and baicalin for intra- and inter-assay at three or four quality control (QC) levels were 0.8–6.1% and −4.0 to 5.8%, respectively. The lower limits of quantification for baicalein, wogonin, oroxylin A and baicalin were 5 ng/ml using 50 μl of plasma sample. This method was successfully applied to the pharmacokinetic study of baicalein, baicalin, wogonin and oroxylin A after an intravenous administration of Scutellariae radix extract to male Sprague–Dawley rats.
Keywords: LC–MS/MS; Baicalein; Baicalin; Oroxylin A; Wogonin; Rat plasma;
Development and validation of a selective and robust LC–MS/MS method for high-throughput quantifying rizatriptan in small plasma samples: Application to a clinical pharmacokinetic study by Yi Chen; Haijun Miao; Mei Lin; Guorong Fan; Zhanying Hong; Huiling Wu; Yutian Wu (268-277).
An analytical method based on liquid chromatography with positive ion electrospray ionization (ESI) coupled to tandem mass spectrometry detection (LC–MS/MS) was developed for the determination of a potent 5-HT1B/1D receptor agonist, rizatriptan in human plasma using granisetron as the internal standard. The analyte and internal standard were isolated from 100 μL plasma samples by liquid–liquid extraction (LLE) and chromatographed on a Lichrospher C18 column (4.6 mm × 50 mm, 5 μm) with a mobile phase consisting of acetonitrile–10 mM aqueous ammonium acetate–acetic acid (50:50:0.5, v/v/v) pumped at 1.0 mL/min. The method had a chromatographic total run time of 2 min. A Varian 1200 L electrospray tandem mass spectrometer equipped with an electrospray ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/z 270 → 201 (rizatriptan) and 313.4 → 138 (granisetron) used for quantitation. The assay was validated over the concentration range of 0.05–50 ng/mL and was found to have acceptable accuracy, precision, linearity, and selectivity. The mean extraction recovery from spiked plasma samples was above 98%. The intra-day accuracy of the assay was within 12% of nominal and intra-day precision was better than 13% C.V. Following a 10 mg dose of the compound administered to human subjects, mean concentrations of rizatriptan ranged from 0.2 to 70.6 ng/mL in plasma samples collected up to 24 h after dosing. Inter-day accuracy and precision results for quality control samples run over a 5-day period alongside clinical samples showed mean accuracies of within 12% of nominal and precision better than 9.5% C.V.
Keywords: Rizatriptan; 5-HT1B/1D receptor agonist; LC–MS/MS; Pharmacokinetics;
Determination of d-serine and related neuroactive amino acids in human plasma by high-performance liquid chromatography with fluorimetric detection by Suzanne L. Grant; Yanina Shulman; Philip Tibbo; David R. Hampson; Glen B. Baker (278-282).
A simple and versatile methodology using high-performance liquid chromatography (HPLC) with fluorimetric detection was developed to simultaneously determine d-serine along with other metabolically related neuroactive amino acids in the glutamatergic system: l-serine, l-glutamate, l-glutamine, and glycine. On-column sensitivity was in the lower picomole range. Of two chiral thiol reagents investigated, amino acid derivatives of o-phthaldialdehyde (OPA) in combination with N-isobutyryl-l-cysteine were found to have consistently higher responses than their corresponding N-tert-butyloxycarbonyl-l-cysteine derivatives. This methodology was applied to the quantitative detection of amino acids in human plasma and lays the foundation for further investigations of the role of neuroactive amino acids in the pathophysiology and treatment of neurological and psychiatric disorders.
Keywords: NMDA receptor; Serine racemase; Chiral thiol; Brain; Neurochemistry; Schizophrenia; Alzheimer's disease; Epilepsy;
Simultaneous determination of selegiline and desmethylselegiline in human body fluids by headspace solid-phase microextraction and gas chromatography–mass spectrometry by Ayako Kuriki; Takeshi Kumazawa; Xiao-Pen Lee; Chika Hasegawa; Mitsuru Kawamura; Osamu Suzuki; Keizo Sato (283-291).
A method for the simultaneous determination of selegiline and its metabolite, desmethylselegiline, in human whole blood and urine is presented. The method, which combines a fiber-based headspace solid-phase microextraction (SPME) technique with gas chromatography–mass spectrometry (GC–MS), required optimization of various parameters (e.g., salt additives, extraction temperatures, extraction times and the extraction properties of the SPME fiber coatings). Pargyline was used as the internal standard. Extraction efficiencies for both selegiline and desmethylselegiline were 2.0–3.4% for whole blood, and 8.0–13.2% for urine. The regression equations for selegiline and desmethylselegiline extracted from whole blood were linear (r 2 = 0.996 and 0.995) within the concentration ranges 0.1–10 and 0.2–20 ng/ml, respectively. For urine, the regression equations for selegiline and desmethylselegiline were linear (r 2 = 0.999 and 0.998) within the concentration ranges 0.05–5.0 and 0.1–10 ng/ml, respectively. The limit of detection for selegiline and desmethylselegiline was 0.01–0.05 ng/ml for both samples. The lower and upper limits of quantification for each compound were 0.05–0.2 and 5–20 ng/ml, respectively. Intra- and inter-day coefficients of variation for selegiline and desmethylselegiline in both samples were not greater than 8.7 and 11.7%, respectively. The determination of selegiline and desmethylselegiline concentrations in Parkinson's disease patients undergoing continuous selegiline treatment is presented and is shown to validate the present methodology.
Keywords: Selegiline; Desmethylselegiline; Solid-phase microextraction (SPME);
Characterization of metabolites and cytochrome P450 isoforms involved in the microsomal metabolism of aconitine by Yuguang Wang; Shengqi Wang; Yongxue Liu; Liangping Yan; Guifang Dou; Yue Gao (292-300).
Aconitine, a major Aconitum alkaloid, is well known for its high toxicity that induces severe arrhythmias leading to death. The current study investigated the metabolism of aconitine and the effects of selective cytochrome P450 (CYP) inhibitors on the metabolism of aconitine in rat liver microsomes. The metabolites were separated and assayed by liquid chromatography-ion trap mass spectrometry (LC/MS n ) and further identified by comparison of their mass spectra and chromatographic behaviors with reference substances. Various selective inhibitors of CYP were used to identify the isoforms of CYP, that involved in the metabolism of aconitine. A total of at least six metabolites were found and characterized in rat liver microsomal incubations. Result showed that the inhibitor of CYP 3A had an inhibitory effect on aconitine metabolism in a concentration-dependant manner, the inhibitor of CYP1A1/2 had a modest inhibitory effect, whereas inhibitors of CYP2B1/2, 2D and 2E1 had no obvious inhibitory effects on aconitine metabolism. Aconitine might be metabolized by CYP 3A and CYP1A1/2 isoforms in rat liver microsome.
Keywords: Aconitine; Cytochrome P450; Metabolites; Metabolism; Microsome;
Chemical fingerprint analysis of rhizomes of Gymnadenia conopsea by HPLC–DAD–MS n by Min Cai; Yan Zhou; Suolang Gesang; Ciren Bianba; Li-Sheng Ding (301-307).
A high-performance liquid chromatography–diode array detection–tandem mass spectrometry (HPLC–DAD–MS n ) method has been firstly developed for chemical fingerprint analysis of rhizomes of Gymnadenia conopsea R. Br. and rapid identification of major compounds in the fingerprints. Comparing the UV and MS spectra with those of reference compounds, seven main peaks in the fingerprints were identified as adenosine (1), 4-hydroxybenzyl alcohol (2), 4-hydroxybenzyl aldehyde (3), dactylorhin B (4), loroglossin (5), dactylorhin A (6) and militarine (7). Compounds 4–7 were succinate derivative esters and firstly discovered from this species. The Computer Aided Similarity Evaluation System (CASES) for chromatographic fingerprint of traditional Chinese medicine was employed to evaluate the similarities of 10 samples of the rhizomes of G. conopsea collected from Sichuan, Qinghai and Hebei provinces, Tibet autonomous region of China, and Nepal. These samples from different sources had similar chemical fingerprints. This method is specific and may serve for quality identification and comprehensive evaluation of this traditional Tibetan remedy.
Keywords: Chemical fingerprint; HPLC–DAD–MS n ; Identification; Succinate derivative ester; Gymnadenia conopsea;
Determination of echinacoside in rat serum by reversed-phase high-performance liquid chromatography with ultraviolet detection and its application to pharmacokinetics and bioavailability by Cunqin Jia; Haiming Shi; Xiangmei Wu; Yinzeng Li; Jingjing Chen; Pengfei Tu (308-313).
A rapid and simple high-performance liquid chromatographic (HPLC) method has been developed and validated for the determination of echinacoside (ECH) in rat serum. After protein precipitation of serum sample with trichloroacetic acid, the supernatant was directly injected and analyzed on a C18 CapcellACR analytical column (150 mm × 4.6 mm I.D. 5 μm) with a mobile phase consisting of acetonitrile–0.5% acetic acid (15.5:84.5, v/v). The UV detector was set at 330 nm. The lower limit of detection and quantification were 9 and 29.2 ng/mL, respectively, and the calibration curves were linear over the concentration range of 29.2–18250 ng/mL. The assay method was successfully applied to the study of the pharmacokinetics and bioavailability of ECH in rat.
Keywords: Echinacoside; Pharmacokinetics; Bioavailability; HPLC; Phenylethanoid glycoside;
A simple and sensitive bioanalytical assay for simultaneous determination of omeprazole and its three major metabolites in human blood plasma using RP-HPLC after a simple liquid–liquid extraction procedure by Naser L. Rezk; Kevin C. Brown; Angela D.M. Kashuba (314-321).
A simple, sensitive and specific reverse-phase high-performance liquid chromatography (HPLC) assay for the simultaneous quantitative determination of omeprazole and its three metabolites in human plasma was developed and validated. This method provides excellent chromatographic resolution and peak shape for the four components and the internal standard within a 17 min run time. The simple extraction method results in a clean base line and relatively high extraction efficiency. The method was validated over the range of 2–2000 ng/mL, with 2.0 ng/mL as the lower limit of quantification. Within- and between-day accuracies for five different concentrations ranged from 95 to 102%, and 95 to 114%, respectively. Within- and between-day precision ranged from 1.1 to 6.3% and 0.5 to 6.2%, respectively. Simplicity and high throughput make this method suitable for clinical pharmacokinetic studies.
Keywords: Chromatography; HPLC; Omeprazole; 5-Hydroxyomeprazole; Omeprazole sulfone; Omeprazole sulfide;
Simple, rapid and sensitive assay method for simultaneous quantification of urinary nicotine and cotinine using gas chromatography–mass spectrometry by Che Nin Man; Lay-Harn Gam; Syazwani Ismail; Razak Lajis; Rahmat Awang (322-327).
Nicotine is a major addictive compound in cigarette. Its smoke is rapidly and extensively metabolized to several metabolites in human. Cotinine as a major metabolite of nicotine is commonly used as a biomarker to determine active and passive smokers. Cotinine has a longer half-life (∼20 h) compared to nicotine (∼2 h). A simple, sensitive, rapid and high throughput GC–MS method was developed for simultaneous quantification of urinary nicotine and cotinine in passive and active smokers. In the sample preparation method, the analytes and internal standard were first basified and followed by liquid–liquid extraction. Upon completion, anhydrous sodium sulphate was added to the solvent mixture to trap moistures. The clear extract obtained was directly injected into GC–MS, operating under selective ion monitoring (SIM) mode. Calibration curves in the range of 0.5–5000 ng/mL of the analytes in urine matrix were established with linear correlation coefficients (r 2) greater than 0.997. The limit of detection for both nicotine and cotinine were 0.20 ng/mL. The mean recoveries for nicotine and cotinine were 93.0 and 100.4%, respectively. The within- and between-assay accuracies were between 2.1 and 7.9% for nicotine and between 0.7 and 11.1% for cotinine. Within- and between-assay precisions of 3.3–9.5% for nicotine and 3.4–9.8% for cotinine were also achieved. The method can be used in routine assessment and monitoring of active smoking and exposure to environmental tobacco smoke. The applicability of the assay was demonstrated in a small-scale comparison study between smokers and non-smokers.
Keywords: Nicotine; Cotinine; Urine; GC–MS; Environmental tobacco smoke (ETS);
The effect of the hexahistidine-tag in the oligomerization of HSC70 constructs by Mouna Amor-Mahjoub; Jean-Philippe Suppini; Nathalie Gomez-Vrielyunck; Moncef Ladjimi (328-334).
The hexahistidine is a fusion tag used for the isolation of proteins via an immobilized metal-ion affinity chromatography (IMAC). In the present study, we have purified and analyzed two constructs of the heat shock protein HSC70 in the presence or the absence of the His-tag (C30WT-His+/C30WT and C30ΔL-His+/C30ΔL). The oligomerization properties of the constructs were analyzed by size exclusion chromatography (SEC) and analytical ultracentrifugation (AU). Results from SEC analysis indicated that the His-tag promotes the dimerization of C30ΔL-His+ but has no effect on the elution profile of C30WT-His+, compared to their respective untagged forms C30ΔL and C30WT. These observations were also confirmed by AU analysis which indicates that C30ΔL is stabilized in the dimeric form in the presence of the His-tag. These results emphasize the need to remove the His-tag before structural characterization of some recombinant proteins.
Keywords: Immobilized metal affinity chromatography (IMAC); HSC70; Mutagenesis; Histidine-tag; Oligomerization;
High-performance liquid chromatographic method for the determination of histamine and 1-methylhistamine in biological buffers by Vivica von Vietinghoff; Gotthold Gäbel; Jörg R. Aschenbach (335-339).
A method for the determination of histamine and its catabolite 1-methylhistamine (1-MH) was developed, using HPLC with fluorescence detection. Derivatization of both compounds occurred on-column with o-phthaldialdehyde dissolved in an alkaline borate buffer, followed by separation on a reversed phase C18 column. Histamine and 1-MH could be detected with comparable sensitivity (limit of quantification, 50 nM). The method was proven suitable to investigate catabolism of histamine by epithelia of pig colon. The method should be useful in research on histamine metabolism.
Keywords: High-performance liquid chromatography; Histamine; Histamine absorption; Histamine metabolism; Histamine release; Intestine; 1-Methylhistamine; Ussing chamber;
Author Index Vol. 844 (340-342).
Keyword Index Vol. 844 (343-350).