Journal of Chromatography B (v.811, #2)
News Section (N1).
Ion-exchange chromatography method for the purification of genomic DNA fraction from Mycobacterium bovis Bacillus Calmette-Guérin by Wang Jinga ; Sun Shuhanb ; Hu Zhenlinb ; Zhou Fengjuanb ; Ling Yilinga (103-107).
The goal of this study was to provide practical strategies for purifying genomic DNA fraction from Mycobacterium bovis Bacillus Calmette-Guérin (BCG-DNA) by ion-exchange chromatography. A multistep process was developed to purify BCG-DNA. The process consisted of sonication, heating, trypsin digestion, ion-exchange chromatography, gel-filter chromatography, and lyophilization. After ion-exchange chromatography, BCG-DNA was highly purified and possessed potent biological effects. The methods described were efficient and had good reproducibility. Further, this was the first reported chromatography method to purify BCG-DNA.
Keywords: Purification; DNA;
Determination of a neuroprotective agent (S)-(+)-BMS-204352 in human, rat and dog plasma by enantioselective liquid chromatography-tandem mass spectrometry by Jianing Zeng; Holly Palme; Nuggehally Srinivas; Mark Arnold (109-117).
A sensitive liquid chromatography-electrospray ionization tandem mass spectrometry method (LC/ESI/MS/MS) for the enantioselective determination of (S)-(+)-BMS-204352, a potent and specific maxi-K channel opener, in human, rat and dog plasma was developed. (S)-(+)-BMS-204352, its enantiomer (R)-(−)-BMS-204353 and the internal standard (13C-deuterated racemate of (S)-(+)-BMS-204352) were extracted from plasma using toluene. Chromatographic separation for the enantiomers was achieved on a Chiralcel OD-H analytical column with a run time of 8 min. An aqueous mobile phase modifier was added post column to enhance the mass spectrometer sensitivity. ESI mass spectra were acquired in the negative mode with selected reaction monitoring. The limit of quantitation (LLOQ) is 0.10 ng/mL for human plasma assay. Samples from a clinical study and two animal studies were processed using these procedures. Based on the in vivo data, lack of inversion of (S)-(+)-BMS-204352 to (R)-(−)-BMS-204353 was demonstrated in human, rat and dog after administration of the drug. A sensitive non-enantioselective LC/ESI/MS/MS assay has also been developed for (S)-(+)-BMS-204352 which uses a similar extraction procedure with a C18 column with a limit of quantitation at 0.05 ng/mL. Human study samples were analyzed by both methods and the correlation coefficient between both sets of data is greater than 0.99.
Keywords: (S)-(+)-BMS-204352; Enantioselective liquid chromatography; Tandem mass spectrometry;
Molecular recognition based cadmium removal from human plasma by Müge Andaç; Ridvan Say; Adil Denizli (119-126).
Molecularly imprinted polymers (MIPs) are easy to prepare, stable, inexpensive and capable of molecular recognition. MIPs can be considered as affinity separation media. Cadmium is a carcinogenic and mutagenic element. There is no specific treatment available for acute or chronic metal poisoning. Besides supportive therapy and hemodialysis, metal poisoning is often treated with commercially available chelating agents including EDTA and dimercaprol. However, there is histopathological evidence for increased toxicity in animals when these agents are utilized. The aim of this study is to prepare ion-imprinted polymers, which can be used for the selective removal of Cd2+ ions from Cd2+-overdosed human plasma. N-Methacryloly-(l)-cysteinemethylester (MAC) was chosen as the complexing monomer. In the first step, Cd2+ was complexed with MAC and the Cd2+-imprinted p(HEMA-MAC) beads were synthesized by suspension polymerization. After that, the template (i.e., Cd2+ ions) were removed using 0.1 M thiourea solution. The specific surface area of the Cd2+-imprinted poly(HEMA-MAC) beads was found to be 19.4 m2/g with a size range of 63–140 μm in diameter and the swelling ratio was 78%. According to the elemental analysis results, the beads contained 42.1 μmol MAC/g polymer. The maximum adsorption capacity was 32.5 μmol Cd2+/g beads. The relative selectivity coefficients of imprinted beads for Cd2+/Pb2+ and Cd2+/Zn2+ were 7.8 and 1683 times greater than non-imprinted matrix, respectively. The Cd2+-imprinted poly(HEMA-MAC) beads could be used many times without decreasing their adsorption capacities significantly.
Keywords: Ion-imprinting; Molecular recognition; Cadmium removal; Metal detoxification; Affinity binding;
The bioactivity of 2,5-dimethoxy-4-ethylthiophenethylamine (2C-T-2) and its detection in rat urine by capillary electrophoresis combined with an on-line sample concentration technique by Yu-Chih Chiu; Shiu-Huey Chou; Ju-Tsung Liu; Cheng-Huang Lin (127-133).
The bioactivity of 2,5-dimethoxy-4-ethylthiophenethylamine (2C-T-2) on nitric oxide (NO) production and the proliferation of spleen and thymus lymphocytes to mitogen stimulation in mice are reported for the first time. NO production by T and B lymphocytes in spleen and T cells in the thymus of mice decreased after the oral administration of 2C-T-2. This indicates that 2C-T-2 intake may perturb both neural and immune activity since a decrease in NO production is indicative of a weakened defense function. 2C-T-2 (the parent drug) in rat urine samples was detected by means of capillary electrophoresis/UV absorbance combined with an on-line sample concentration technique. When the CZE and MEKC modes were employed, the detection limit was found to be 4.5 and 5.0 μg/mL (at a 92.1% confidence level); whereas when on-line sample concentration methods, including stacking and sweeping-micellar electrokinetic chromatography were used, the detection limits were improved to 19.2 and 9.1 ng/mL, respectively. In an analysis of some actual samples from animal experiments, three male rats were administered 20 μg/g of body weight of 2C-T-2 by intra-peritoneal injection. The first- and second-day urine fractions were collected after the administration, for use in the analysis. As a result, 2.9 μg/mL and 0.25 μg/mL of 2C-T-2, respectively, were detected after ingestion of the doses.
Keywords: 2,5-Dimethoxy-4-ethylthiophenethylamine (2C-T-2); Nitric oxide (NO); Lymphocytes; Capillary electrophoresis; Urine;
Determination of CC-5013, an analogue of thalidomide, in human plasma by liquid chromatography–mass spectrometry by Tanyifor M. Tohnya; Kyunghwa Hwang; Erin R. Lepper; Howard A. Fine; William L. Dahut; Jürgen Venitz; Alex Sparreboom; William D. Figg (135-141).
A high-performance liquid chromatographic assay with MS detection has been developed for the quantitative determination of the anti-angiogenic agent CC-5013 in human plasma. Sample pretreatment involved liquid–liquid extraction with acetonitrile/1-chlorobutane (4:1, v/v) solution containing the internal standard, umbelliferone. Separation of the compounds of interest was achieved on a column packed with Waters C18 Nova-Pak material (4 μm particle size; 300 mm × 3.9 mm internal diameter) using acetonitrile, de-ionized water, and glacial acetic acid in ratios of 20:80:0.1 (v/v/v) (pH 3.5) delivered at an isocratic flow rate of 1.00 ml/min. Simultaneous MS detection was performed at m/z 260.3 (CC-5013) and m/z 163.1 (umbelliferone). The calibration curve was fit to a linear response–concentration data over a range of 5–1000 ng/ml using a weighting factor of 1/x. Values for accuracy and precision, obtained from four quality controls analyzed on three different days in replicates of five, ranged from 98 to 106% and from 5.5 to 15.5%, respectively. The method was successfully applied to study the pharmacokinetics of CC-5013 in a cancer patient receiving the drug as single daily dose.
Keywords: CC-5013; LC–MS; Pharmacokinetics; Angiogenesis inhibitor;
Metabolism of the designer drug 4-bromo-2,5-dimethoxyphenethylamine (2C-B) in mice, after acute administration by Helena Carmo; Douwe de Boer; Fernando Remião; Félix Carvalho; Lesseps A. dos Reys; Maria de Lourdes Bastos (143-152).
4-Bromo-2,5-dimethoxyphenethylamine (2C-B) is a psychoactive drug of abuse often sold under the general street name “Ecstasy”. Recent reports on the abuse of 2C-B and analogues denote the lack of knowledge on this drug metabolism. In the present study, we investigated the metabolic profile of 2C-B in the mouse and found unchanged 2C-B and several metabolites, which could be identified by GC/MS in the mice urine. The identification of 2C-B metabolites may give important clues for the biological and toxicological effects of this drug of abuse and provides new important data for forensic analysis on samples taken from 2C-B abusers.
Keywords: 2C-B; Designer drugs of abuse; Metabolism; Mice;
Sensitive determination of midazolam and 1′-hydroxymidazolam in plasma by liquid–liquid extraction and column-switching liquid chromatography with ultraviolet absorbance detection and its application for measuring CYP3A activity by Norio Yasui-Furukori; Yoshimasa Inoue; Tomonori Tateishi (153-157).
This manuscript described a new sensitive determination of midazolam and its metabolite 1′-hydroxymidazolam by automated column-switching high-performance liquid chromatography. The test compounds were extracted from 2 ml of plasma using chloroform-hexane (30:70, v/v) and the extract was injected into a column I (TSK-PW precolumn, 10 μm, 35 mm × 4.6 mm i.d.) for clean-up and column II (C18 STR ODS-II analytical column (5 μm, 150 mm × 4.6 mm i.d.) for separation. The mobile phase for separation consisted of phosphate buffer (0.02 M, pH 4.6), perchloric acid (60%) and acetonitrile (57.9:0.1:42, v/v/v) and was delivered at a flow-rate of 0.6 ml/min. The peak was detected using a UV detector set at 254 nm. The method was validated for the concentration range 0.3–100 ng/ml, and good linearity (r > 0.998) was confirmed. Intra- and inter-day coefficient variations for midazolam and 1′-hydroxymidazolam were less than 8.5 and 6.1%, respectively, at the concentrations of 0.5, 5 and 50 ng/ml for the test compounds. Relative errors ranged from −14 to 6% and mean recoveries were 78–85%. The limit of quantification was 0.5 ng/ml for each compound. This method was sensitive enough for pharmacokinetic studies measuring CYP3A activity in human volunteers following an intravenous (1 mg) and a single-oral administration (2 mg) of a subtherapeutic midazolam dose.
Keywords: Midazolam; 1′-Hydroxymidazolam; HPLC; CYP3A4;
Liquid chromatographic determination of the plasma concentrations of cefotaxime and desacetylcefotaxime in plasma of critically ill patients by M.T. Rosseel; K.H. Vandewoude (159-163).
A method for the simultaneous determination of cefotaxime (CTX) and desacetylcefotaxime (DES) in plasma was developed, using acetonitrile protein precipitation and high-performance liquid chromatography (HPLC) with UV-detection at 285 nm. Desacetylcefotaxime was also analysed after conversion in highly acidic medium to its lactone form (DES-lactone). The chromatographic separation was performed on a C18 Aqua column. The lower limit of quantitation was 1 μg/ml for CTX and 0.5 μg/ml for DES and DES-lactone, using 25 μl of plasma samples. The linearity of the calibration curves was satisfactory as indicated by correlation coefficients of ≥0.990. The within-day and between-day precisions were <12% (n = 18) for the two products and the accuracy was between 88 and 101%. The developed HPLC method was applied for CTX and DES determination in plasma samples of critically ill patients after continuous intravenous infusion of CTX.
Keywords: Cefotaxime; Desacetylcefotaxime; HPLC;
High-sensitivity capillary electrophoresis determination of inorganic anions in serum and urine using on-line preconcentration by transient isotachophoresis by Takeshi Hirokawa; Masato Yoshioka; Hikaru Okamoto; Andrei R. Timerbaev; Gottfried Blaschke (165-170).
Concentrations of inorganic anions, both as individual species and biotransformation products, in physiological fluids are of strong concern in clinical studies. To date, analytical methodologies have either required different analytical procedures to determine these analytes in plasma and urine, or extensive sample preparation, or unconventional and often expensive detection schemes, or both. A simple and sensitive capillary electrophoresis (CE) method with direct UV detection was developed for the simultaneous determination of iodide, bromide and nitrate in human plasma and urine, with a special focus on reliable quantification of the trace serum iodide. With the latter objective, the method incorporates a transient isotachophoresis (tITP) procedure enabling an efficient on-line preconcentration of iodide (limit of detection, 1.4 μg l−1) as well as other moderately mobile analytes that fall into the tITP range. The analyses of both types of biofluids were performed using an acidic electrolyte system composed of 0.25 mol l−1 sodium chloride and 7.5 mmol l−1 cetyltrimethylammonium chloride at pH 2.2 and 0.5 mol l−1 2-(N-morpholino)ethanesulfonate (pH 6.0) as terminating electrolyte. Relative standard deviations (R.S.D.) below 3.0% and 9.2% were obtained for within-day and between-day precision, respectively. Resolution and quantification of oxalic acid was also feasible under optimized tITP-CE conditions. Sample preparation required only ultrafiltration (serum) and dilution (urine). A number of plasma and urine samples were evaluated with this assay and the iodide, bromide and nitrate concentrations were in the expected clinical concentration ranges.
Keywords: Inorganic ion analysis; Human serum; Urine; Capillary electrophoresis; Transient isotachophoresis;
Investigation of matrix effects of urine on a molecularly imprinted solid-phase extraction by Kristina Möller; Ulrika Nilsson; Carlo Crescenzi (171-176).
This study investigates matrix effects on a molecularly imprinted solid-phase extraction (MISPE) method developed for the clean-up of diphenyl phosphate (a hydrolysis product of the commonly used flame retardant and plasticizer, triphenyl phosphate) in urine samples. The influence of potentially interfering compounds that naturally occur in urine was examined with respect to extraction recovery, repeatability and selectivity. The components tested were NaCl, urea, creatinine and hippuric acid. The imprinted polymer was prepared using 2-vinylpyridine as the functional monomer, ethylene glycol dimethacrylate as crosslinker and a structural analogue of the analyte as the template molecule. The recovery of diphenyl phosphate from water standards was over 90% using MISPE, compared to less than 25% using a non-imprinted SPE (NISPE) counterpart. The selectivity of MISPE compared to NISPE was achieved in a wash step with a basic modifier in methanol. The recovery and repeatability of the MISPE method were affected most by NaCl in the tested concentrations, while urea, creatinine and hippuric acid had no significant influence. NaCl most likely weakens the binding during the loading of the sample. This effect could be suppressed by diluting the sample with a citrate buffer at pH 4.0.
Keywords: Molecularly imprinted polymers; Solid-phase extraction; Matrix effects; Urine samples;
Determination of polyethylene glycols of different molecular weight in the stratum corneum by I. Jakasa; F. Calkoen; S. Kezic (177-182).
We developed a sensitive method for determination of polyethylene glycols (PEGs) of different molecular weight (MW) in the human stratum corneum (SC) obtained by tape stripping. The analysis is based on derivatization with pentafluoropropionic anhydride (PFPA) and gas chromatography–electron capture detection (GC–ECD). The identification and quantification of PEGs was done using individual oligomers. The method showed to be suitable for studying permeability in normal and impaired skin with respect to MW in the range of 150–600 Da.
Keywords: Polyethylene glycol (PEG); Tape stripping; Percutaneous penetration; Stratum corneum;
Determination of 2′,3′-dideoxycytidine in maternal plasma, amniotic fluid, placental and fetal tissues by high-performance liquid chromatography by Yan Ding; Leah N. Williamson; Catherine A. White; Michael G. Bartlett (183-189).
2′,3′-Dideoxycytidine (DDC) is a nucleoside reverse transcriptase inhibitor that has been shown to inhibit the human immunodeficiency virus (HIV). DDC is a candidate for treatment of pregnant women to prevent prenatal transmission of HIV/AIDS to their unborn children. A quick and simple high-performance liquid chromatography (HPLC) method has been developed and validated for the determination of DDC concentrations in samples collected from a pregnant rat model (maternal plasma, amniotic fluid, placental and fetal tissues). Extraction of DDC and its internal standard 2′,3′-dideoxy-3′-thiacytidine (3TC) in plasma and amniotic fluid was carried out by protein precipitation. Extraction from placental and fetal homogenates was achieved by solid phase extraction using Waters Oasis HLB solid phase extraction cartridges. Chromatographic separation was achieved on a Waters Spherisorb S3W silica column (4.6 mm × 100 mm) equipped with a Phenomenex guard column. The mobile phase used was 10% methanol in water with 22 mM formic acid. The flow rate was 0.5 ml/min, and the detection wavelength was optimized at 275 nm. Under these chromatographic conditions, DDC eluted around 12 min, and 3TC eluted around 10 min. The calibration curves for each day of validation and analysis showed good linear response through the range of 0.15–75.0 μg/ml in each of the four matrices. The relative recovery for DDC in each of the matrices ranged from 87.8% to 103.0%. Acceptable intra- and inter-day assay precision (<15% R.S.D.) and accuracy (<15% error) were observed over 0.15–75.0 μg/ml for all four matrices.
Use of molecularly imprinted polymers from a mixture of tetracycline and its degradation products to produce affinity membranes for the removal of tetracycline from water by Roongnapa Suedee; Teerapol Srichana; Thitima Chuchome; Urairut Kongmark (191-200).
The possibility of introducing multiple recognition in artificial receptors by imprinting polymers, using a mixture of tetracycline (TC) and its degradation products as templates, has been examined. The recognition ability of the resulting molecularly imprinted polymer (MIP), as evaluated by batch rebinding assay, was found to be group-specific to tetracyclines, while the single tetracycline imprinted polymer (MIP-2) prepared using TC free from degradation products as the print molecule showed considerably high selectivity for doxycycline (DC) and modest selectivity for TC and other TC derivatives, oxytetracycline (OTC) and chlortetracycline (CTC). Based on the recognition property of the multiple tetracycline imprinted polymer (MIP-1), the polymer was applied in affinity membrane extraction as a class-selective adsorption phase to remove tetracyclines residues from water. For this purpose, the ground MIP was incorporated in a plasticized poly(vinyl chloride)-membrane by casting method. Affinity separation of the obtained membrane was evaluated for the extraction of tetracycline and its analogs (CTC, OTC or DC) in aqueous solutions by a dialysis method. The membrane exhibited significantly stronger extraction ability towards tetracycline and structurally related compounds than a “blank” membrane having a non-printed polymer (NIP) as the adsorption phase. The result of these membrane extraction studies also indicates that the drug saturating at the receptor sites of MIP (deposited in membrane) faster will also be released into the receptor chamber faster. These affinity membranes were able to extract tetracyclines from water at all pHs, the highest selectivity being shown at pH 7 of the feed solution, which gives the lowest flux of the drug. Moreover, presence of salt in the feed solution increases the release of tetracycline bound in membrane. The results of the present study show that imprinting simultaneous with TC and TC degradation products formed in situ as a mixture template generates the group selectivity towards tetracyclines for the polymeric material. High affinity to a class of tetracycline of the membrane fabricated with this receptor, together with its fast and simple MIP fabrication, provides good possibilities for its application in separation processes of tetracycline antibiotics, which often contaminate the aqueous environment.
Keywords: Molecularly imprinted polymers; Affinity membrane; Tetracyclines; Poly(vinyl chloride);
Investigation of the silylation of ephedrines using N-methyl-N-trimethylsilyl-trifluoroacetamide by Guro Forsdahl; Günter Gmeiner (201-208).
This paper describes a gas chromatography (GC)–mass spectrometry (MS) method for the simultaneous quantification of ephedrine, pseudoephedrine, cathine, norephedrine and methylephedrine in urine. The sample preparation step includes solid phase extraction and derivatisation with N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA). An evaluation of various silylation conditions compatible with screening methods in doping control analysis is presented. The method was found to be well suited for quantification of ephedrines in doping control.
Keywords: Ephedrine; Pseudoephedrine; Cathine; Norephedrine; Methylephedrine; Derivatisation; Doping; Silylation; N-Methyl-N-timethylsilyltrifluoroacetamide;
Liquid chromatography–electrospray ionisation mass spectrometry method for the determination of escitalopram in human plasma and its application in bioequivalence study by Sonu Sundd Singh; Hiten Shah; Sapna Gupta; Manish Jain; Kuldeep Sharma; Purav Thakkar; Ruchy Shah (209-215).
A novel liquid chromatographic–electrospray ionisation mass spectrometric (LC–ESI-MS) method has been developed for the determination of escitalopram, an antidepressant in human plasma using paroxetine as internal standard. The method involved liquid–liquid extraction of the analyte from human plasma with a mixture of diethyl ether and dichloromethane (70:30, v/v). The chromatographic separation was achieved within 7.0 min by using 2.0 mM ammonium acetate (pH 5.0)–acetonitrile (54:46, v/v) as mobile phase and a ODS YMC™ AQ 150 mm × 4.6 mm analytical column; the flow-rate was 1.0 ml/min. Ion signals m/z 325.0 and 330.0 for escitalopram and internal standard, were measured in the positive mode. A detailed validation of the method was performed as per USFDA guidelines and the standard curves were found to be linear in the range of 1.0–200 ng/ml with a mean correlation coefficient more than 0.99. The absolute recovery was more than 75% for both escitalopram and internal standard. The method was simple, sensitive, precise, accurate and was successfully applied to the bioequivalence study of escitalopram in healthy, male, human subjects.
Keywords: Escitalopram; Validation; Bioequivalence;
A three-step purification strategy for isolation of hamster TIG2 from CHO cells: characterization of two processed endogenous forms by Annette Busmann; Michael Walden; Martin Wendland; Christian Kutzleb; Wolf-Georg Forssmann; Harald John (217-223).
We have recently isolated a bioactive, circulating protein of human tazarotene-induced gene-2 (TIG2) as the natural ligand of the orphan receptor ChemR23. Here we describe a simplified method for the isolation of hamster TIG2 protein from Chinese hamster ovary (CHO) cell supernatant. Using a heparin-affinity column followed by two reversed phase chromatography steps resulted in the isolation of pure biologically active material. Two processed bioactive forms of Chinese hamster TIG2 were identified by Edman sequencing and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) mass fingerprint analysis, representing the amino acid residues T20 to F156, and T20 to A155 of the 163 amino acid propeptide. Comparison with the predicted aa-sequence indicates a mutation or modification within the C-terminal end of the peptide.
Keywords: TIG2; GPCR; Heparin-affinity chromatography; FLIPR; CHO cells;
Quantitation of tigecycline, a novel glycyclcycline, by liquid chromatography by Chonghua Li; Christina A. Sutherland; Charles H. Nightingale; David P. Nicolau (225-229).
An ion-paired HPLC assay was developed to determine tigecycline (GAR-936) concentrations in Hank's balanced salts solution, tigecycline intra-cellular concentrations in human polymorphonuclear neutrophils (PMNs) and tigecycline concentrations in human serum. Minocycline was used as internal standard, 5% trichloroacetic acid was added to lyse PMNs and also precipitate proteins in PMNs and serum. The top aqueous layer was aspirated for HPLC assay. The chromatograms were performed with a reversed-phase C18 column with UV detector. The mobile phase consisted of acetonitrile, phosphate buffer (pH 3) and 1-octanesulfonic acid at a flow rate of 1 ml/min. Good linearity and recovery were achieved over the range of standard curves. The relative standard deviations of three quality controls for intra- and inter-day precision were less than 6.4%, and the relative errors of the intra- and inter-day accuracy were less than 7.0%. Tigecycline in Hank's buffer, PMNs and serum was stable under different test conditions. This new liquid chromatography assay is a simple, accurate and reproducible method for determining tigecycline in different matrix.
Keywords: Tigecycline; HPLC;
Development of a Micellar electrokinetic capillary chromatography method for the determination of three drugs employed in the erectile dysfunction therapy by J. Rodríguez Flores; J.J. Berzas Nevado; G. Castañeda Peñalvo; N. Mora Diez (231-236).
A Micellar electrokinetic capillary chromatography method is proposed for the determination of sildenafil, vardenafil and tadalafil, which are employed in oral therapy for erectile dysfunction. Optimum conditions for the separation were investigated. A background electrolyte solution consisting of 10 mM phosphate buffer adjusted to pH 12.0, sodium dodecyl sulfate (SDS) 25 mM, hydrodynamic injection, and 25 kV as separation voltage were used. Relative standard deviations (R.S.D.s) were 1.0, 1.0, 0.4% and 2.9, 2.9, 1.9% for migration time and corrected peak area (CPA) (n = 9) for sildenafil, vardenafil and tadalafil, respectively. Detection limits obtained for the three drugs ranged from 0.19 to 0.61 mg L−1. A linear concentration range between 1 and 20 mg L−1 was obtained. A ruggedness test of this method was checked using the fractional factorial model of Plackett–Burman, in which the influence of six factors at three different levels was tested on different electrophoretic results: resolution and corrected peak area. The statistical evaluation of the electrophoretic results was achieved by Youden and Steiner method. The described method is rapid, sensitive and rugged and it was tested in the pharmaceutical formulations analysis obtaining recoveries between 98 and 107% respect to the nominal content
Keywords: Micellar electrokinetic capillary chromatography; Electrophoresis; Sildenafil; Vardenafil; Tadalafil; Erectile dysfunction; Ruggedness; Pharmaceutical formulations;
A sensitive internal standard method for the determination of melatonin in mammals using precolumn oxidation reversed-phase high-performance liquid chromatography by Kenji Hamase; Junzo Hirano; Yuki Kosai; Tatsunosuke Tomita; Kiyoshi Zaitsu (237-241).
A sensitive and accurate internal standard method to determine melatonin in mammalian tissues and physiological fluids has been described. This method includes the oxidation of melatonin to a highly fluorescent compound, N-[(6-methoxy-4-oxo-1,4-dihydroquinolin-3-yl)methyl]acetamide (6-MOQMA), and the determination of 6-MOQMA by a reversed-phase HPLC system. For the accurate and reliable determination, several melatonin analogs were designed and utilized as the internal standards, and ethyl and isopropyl analogs (having the corresponding alkyl group via the amide bond of melatonin instead of the methyl group) were found to be promising. Using these internal standards, highly accurate and sensitive determination could be accomplished using rat pineal gland samples, and the clear circadian rhythms are demonstrated. This method was also successfully applied to the determination of melatonin in a small amount (20 μL) of human saliva.
Keywords: Melatonin; Fluorescence detection; Precolumn oxidation; RP-HPLC;
Preparative high-performance liquid chromatographic separation and analysis of the Maltacine complex – a family of cyclic peptide antibiotics from Bacillus subtilis by Gunnar Hagelin; Inger Oulie; Åse Raknes; Kjell Undheim; Ole G. Clausen (243-251).
Purification of secondary metabolites from fermentation broths can be a challenging task both due to the complexity of the medium, inherently unstable molecular structures or by the action of enzymes present in the fermentation broth leading to poor isolation yield and loss of antibiotic activity. A combination of different purification techniques has usually been used to arrive at acceptable purities for characterisation of the target molecules. Due to rapid decay of antimicrobial activity a rapid preparative high-performance liquid chromatography (HPLC) method was developed that provided separation and resolution of a family of 18 closely related cyclic peptides within 110 min with minimal loss of activity. Characterisation of the peptides with LC–MS, UV/IR spectroscopy and amino acid analysis disclosed 20 different peptides with cyclic structures (lactones) with molecular weights between 1447.7 and 1519.8 Da. No peptide antibiotics with identical molecular weights have previously been reported in the literature, which lead us to conclude that this peptide complex has not been discovered before. We have named them Maltacines.
Keywords: Preparative HPLC; LC–MS; Cyclic peptide antibiotics; Bacillus subtilis; Maltacines;
Application of high-performance capillary electrophoresis to the purification process of Escherichia coli K4 polysaccharide by Nicola Volpi (253-256).
The high-performance capillary electrophoresis (HPCE) (electrokinetic chromatography with sodium dodecyl sulphate) technique was applied to the extraction and purification process of the K4 polysaccharide from cultured bacteria in several stages. HPCE proved to be a technique with high resolution and sensitivity in analyzing K4 polysaccharide during its purification, in particular by using a strong anion-exchange resin. This is of paramount importance to monitor the product during the extraction and purification process or to test the purity of the final product. Furthermore, HPCE is able to verify that the extraction and purification process adopted is not carried out under drastic conditions capable of inducing fructose removal from the polysaccharide backbone.
Keywords: Escherichia coli; Capillary electrophoresis; K4 polysaccharide; Glycosaminoglycans; Chondroitin;
Development of a high performance liquid chromatography/mass spectroscopy method for the determination of strychnine concentrations in insects used to assess potential risks to insectivores by Randal S. Stahl; Wendy M. Arjo; Kim K. Wagner; Carol Furcolow; Dale L. Nolte; John J. Johnston (257-262).
A high performance liquid chromatography/mass spectrometry (HPLC/MS) method was developed in support of a study to assess potential tertiary risks posed to insectivores by strychnine baited pocket gophers (Thomomys sp.). Necropholous insects are primary consumers of pocket gopher carcasses. A field study was conducted to collect insects from strychnine-baited and control pocket gopher carcasses. The majority of the insects collected were from the orders Diptera (flies, assayed separately as adults and larvae), Coleoptera (beetles), and Hymenoptera (ants and wasps, assayed separately). Samples (0.5 g) were extracted in acetic acid (2%) and analyzed with the mass spectrometer configured for tandem mass spectrometry. For most of the samples the strychnine concentrations were less than the method limit of detection. However, strychnine concentrations as high as 0.338, 0.341, 0.698, and 0.034 μg/g were detected in ants, fly adults, fly larvae, and beetles, respectively. This information collected with the HPLC/MS method is critical for assessing potential non-target hazards for insectivores.
Keywords: Strychnine; Mass spectrometry; Tertiary risk;
Identification and separation of PCR products based on their GC content by denaturing high-performance liquid chromatography by Eugeni Belda; Vicente Sentandreu; Francisco J. Silva (263-268).
We show that denaturing high-performance liquid chromatography is a suitable method for the separation of DNA molecules of similar sizes but with different GC contents. A mixture of homologous molecules coming from different bacterial species may be obtained when PCR with degenerate primers is used for the amplification of a specific gene from an environmental sample. We have observed that, by selecting an appropriate temperature for the partial denaturation of the molecules, we are able to separate them according to the GC content of each DNA product. This allows us to determine if one or several types of molecules are amplified in the course of a PCR reaction. In the latter case it is possible, even with minority products, to isolate them by collecting the eluted volumes, followed by cloning, sequencing or reamplifying them by PCR, depending on the DNA concentration. We have applied this analysis to the amplification of a fragment of the ribA gene in the bacterial endosymbionts of insects, obtaining a high correlation coefficient (0.978) between retention time and the GC content of the molecules.
Keywords: Denaturing high-performance liquid chromatography; GC content; Bacterial endosymbionts; Buchnera aphidicola;
Author Index to Vol. 811 (269-271).
Keyword Index to Vol. 811 (273-277).