Journal of Chromatography B (v.784, #1)
News Section (N1-N2).
Editorial Board (iii).
Publisher`s Note (vii).
Determination of trenbolone and its metabolite in bovine fluids by liquid chromatography–tandem mass spectrometry by F. Buiarelli; G.P. Cartoni; F. Coccioli; A. De Rossi; B. Neri (1-15).
A liquid chromatographic–tandem mass spectrometric (LC–MS–MS) method has been developed for the determination of trenbolone in bovine urine and serum. The aim was a control of the misuse of trenbolone in food-producing animals. The procedure involved, in both cases, a preliminary solid-phase clean-up followed by a liquid–liquid extraction for urine samples after a preliminary enzymatic hydrolysis. The extracts have been directly analysed by reversed-phase LC–MS–MS in selected reaction monitoring (SRM), acquiring two diagnostic product ions from the chosen precursor [M+H]+. The procedures were validated across the concentration range of 1–1500 ng/ml. The linearity, the inter- and intra-day accuracy and precision have been determined. The procedure was specific and the accuracy values were better than 20% at the limit of quantitation of spiked samples. The limit of quantification (LOQ) and the limit of detection (LOD) were, respectively, 1 ng/ml and 350 pg/ml for urine and serum. According to the draft, SANCO/1805/2000, we determined the decision limit CCα and the detection capability CCβ. The recovery values for urine ranged from 87 to 128%, and for plasma the recovery was 70±4%. The procedure proved to be simple and suitable for routine and confirmatory purposes such as those developed for residue studies.
Keywords: Trenbolone; Steroids;
Determination of cefaclor in human plasma by a sensitive and specific liquid chromatographic–tandem mass spectrometric method by Xiaoyan Chen; Dafang Zhong; Bin Huang; Jian Cui (17-24).
A sensitive and specific liquid chromatographic–tandem mass spectrometric method is described for the determination of cefaclor in human plasma. The plasma samples were treated by two sample preparation procedures, i.e. protein precipitation (PPT) and solid-phase extraction (SPE). The pretreated samples were analyzed on a C18 HPLC column interfaced with a triple quadrupole tandem mass spectrometer. Positive electrospray ionization (ESI) was employed as the ionization source. The analyte and internal standard ampicillin (for PPT) or cefetamet (for SPE) were detected by use of selected reaction monitoring (SRM) mode. The lower limit of quantitation obtained as a result of the PPT procedure was 100 ng/ml. The intra- and inter-run precision, calculated from quality control (QC) samples was less than 12% for cefaclor. The accuracy as determined from QC samples was within ±3% for the analyte. The SPE procedure could provide the lower limit of quantitation of 2 ng/ml. The precision and accuracy were measured to be below 7.1% and between −3.6% and 1.1%, respectively, for all QC samples. The method was applied for the evaluation of the pharmacokinetic profiles of cefaclor sustained-release formulation.
Simple and sensitive determination of the new antitumor drug CKD-602 in human plasma by liquid chromatography by Joo-Youn Cho; Hyo-Bum Seo; Kyung-Sang Yu; Kyun-Seop Bae; So-Young Yi; In-Jin Jang; Sang-Goo Shin (25-31).
A simple and sensitive high-performance liquid chromatographic with fluorescence detection method has been developed and validated for the determination of the new antitumor agent CKD-602 in human plasma. Plasma proteins were precipitated with methanol and the samples were acidified with 7% (v/v) perchloric acid. The supernatants were analyzed by HPLC using a Capcell Pak C18 UG120 column and a mixture of methanol–0.1 M hexane-1-sulfonic acid in methanol–0.01 M TEMED in water at pH 6.0 (40:1:59, v/v) as the mobile phase. The lower limit of quantification was 0.2 ng/ml using 200 μl plasma samples. Mean within-run precision and between-run precision at six tested concentrations (0.2–400 ng/ml) were ≤10% and mean accuracy was ≤15%. Stability studies showed that CKD-602 is stable in both plasma and methanol extracts for at least 3 months at −30 °C. The described method was used for the pharmacokinetic analysis of CKD-602 during clinical phase I studies, in patients with solid tumors.
One step purification of alpha1-acid glycoprotein from human plasma by Federica Azzimonti; Daniel H Atchley; Catherine Anne Morrison; Seetal Dodd; David W Boulton; C.Lindsay DeVane; Philippe Arnaud (33-38).
Alpha1-acid glycoprotein is a plasma protein that exhibits both microheterogeneity and polymorphism. Its purification from human plasma is usually performed using a sequence of different fractionation steps. Here we report a one-step isolation technique of this protein based upon pseudo-ligand affinity chromatography on immobilized Cibacron Blue F3GA at acidic pH. In addition, the use of two narrow pH elution buffers allows us to separate the two genetic products of this protein, which differ from each other by 21 amino acid substitutions. This technique will facilitate the study of the structural, biological and pharmacokinetic properties of each individual allele product.
Keywords: α1-Acid glycoprotein; Polymorphic allele products;
Determination of prednisolone and the most important associated compounds in ocular and cutaneous pharmaceutical preparations by micellar electrokinetic capillary chromatography by J.M Lemus Gallego; J Pérez Arroyo (39-47).
A micellar electrokinetic capillary chromatographic method to separate prednisolone, prednisolone acetate, naphazoline, Zn–bacitracin, sulfacetamide and phenylefrine is described. The separation was carried out by using a fused-silica capillary (57 cm×75 μm I.D.) at 25 °C and 30 kV, using a 5 mM phosphate–5 mM borate buffer adjusted to pH 8.2, 50 mM sodium dodecyl sulfate (SDS) and 10% methanol–water (v/v) as background electrolyte. Under these conditions, the run time was 8 min and the limits of quantification were about 1.0 mg/l for every component. The method was applied to pharmaceutical preparations and the results provided recoveries close to 100% and the method gave good results when compared with a reference multivariate calibration spectrophotometric method.
Keywords: Prednisolone; Naphazoline; Bacitracin, Zn; Sulfacetamide; Phenylefrine;
Determination of testosterone metabolites in human hepatocytes by Gerhard Friedrich; Thorsten Rose; Klaus Rissler (49-61).
A rapid and sensitive RP–HPLC assay for determination of 6β-hydroxytestosterone in human hepatocytes with corticosterone as the internal standard is described. The procedure employs on-line sample enrichment using a BioTrap 500 MS™ (20×4 mm I.D.) extraction pre-column and subsequent gradient separation on a Prontosil 60-5 C18-H (250×2 mm I.D., 5 μm particle size) analytical column in the back-flush mode using a ternary eluent system composed of methanol, tetrahydrofuran and water. Signal monitoring was done by measurement of the responses from liquid chromatography coupled to mass spectroscopy (LC–MS/MS) using an atmospheric pressure chemical ionization (APCI) source conducted in the selected reaction monitoring (SRM) mode. Mean recoveries of 6β-hydroxytestosterone from an estimate of the biological matrix, i.e., Dulbecco’s modified Eagle medium “High Glucose”, ranged from 101.8–104.4% for samples containing the target analyte at the 250, 500 and 1000 ng/ml level. The limit of quantitation (LOQ) was 20 ng/ml at an injection volume of 100 μl determined in the same matrix. Linearity of signal responses versus concentration for all three analytes was accomplished in the range of 100–4000 ng/ml. Mean values of the coefficients of variation (C.V.) for the target analyte obtained for the concentrations 250, 500 and 1000 ng/ml at 5 different days in quintuplicate ranged from 1.5–7.7% (within-day) and 4.8–7.3% (between-day). The corresponding values for the accuracy ranged from 87.7–106.1% for the within-day and from 98.8–102.5% for the between-day measurements. The target analyte was sufficiently stable at both storage and sample preparation conditions because no substantial deviations between analyte concentrations measured before and after subsequently performed freeze and thaw cycles were observed.
Keywords: Testosterone; 6β-Hydroxytestosterone;
Determination of cortisol in human saliva using liquid chromatography–electrospray tandem mass spectrometry by Bo A.G. Jönsson; Birgitta Malmberg; Åsa Amilon; Anne Helene Garde; Palle Ørbæk (63-68).
The aim of this work was to develop a method for determination of cortisol in saliva by liquid chromatography–tandem mass spectrometry (LC–MS–MS). Saliva was sampled on Salivette tubes. These were centrifuged, deuterium-labeled cortisol was added as internal standard and the proteins precipitated by acetonitrile. The supernatant was evaporated, dissolved in methanol acidified with acetic acid and analyzed by LC–MS–MS. The with-in run precision, tested by pooling saliva samples from volunteers and then analyzing these in a single run, was found to be 7% at 0.7 μg l−1. The between-run precision was tested by analysis of the same samples at different days and found to be 11% at 2.5 μg l−1. The limit of quantification was 0.5 μg l−1. The method was applied for analysis of saliva samples from three volunteers during their last week before vacation and the first and second week on vacation. In addition, the method was compared to analysis by an immunological method. The values from the immunological method were 2.7 times higher than the LC–MS–MS results.
Synthesis of [18O2]valproic acid and its use as an internal standard for the quantitative measurement by gas chromatography–electron ionization mass spectrometry by Hans Jörg Leis; Werner Windischhofer; Gerald N. Rechberger; Günter Fauler (69-75).
A specific method for the quantitative determination of valproic acid in human plasma is presented. Valproate was extracted from acidified plasma by hexane extraction and converted to its trimethylsilyl derivative without sample concentration. The derivatives were analyzed without any further purification. Using gas chromatography–electron ionization mass spectrometry, diagnostic useful fragment ions at m/z 201 and 205 were obtained for valproic acid and [18O2]valproic acid internal standard, respectively. [18O2]Valproic acid was synthesized from unlabeled valproate by acid-catalyzed exchange reaction in H2 18O. The method was validated in the expected concentration range of a pharmacokinetic study. Thus, calibration graphs were linear within a range of 0.47–120 μg/ml plasma. Intra-day precision was 2.29% (0.47 μg/ml), 2.93% (4 μg/ml), 3.22% (20 μg/ml) and 4.40% (80 μg/ml), inter-day variability was found to be 1.49% (0.47 μg/ml), 3.79% (20 μg/ml), 2.74% (40 μg/ml) and 3.03% (80 μg/ml). Inter-day accuracy showed deviations of 1.94% (0.47 μg/ml), 0.53% (4 μg/ml), −0.32% (20 μg/ml) and 0.06% (80 μg/ml). The method is rugged and robust and has been applied to the batch analysis of valproate during pharmacokinetic profiling of the drug.
Keywords: [18O2]Valproic acid;
Accurate quantification of basal plasma levels of 3-nitrotyrosine and 3-nitrotyrosinoalbumin by gas chromatography–tandem mass spectrometry by Dimitrios Tsikas; Edzard Schwedhelm; Friedericke K Stutzer; Frank-Mathias Gutzki; Iris Rode; Christina Mehls; Jürgen C Frölich (77-90).
Measurement of 3-nitro-l-tyrosine (NO2Tyr) and protein-related 3-nitro-l-tyrosine in human plasma is associated with numerous methodological problems which result in highly divergent basal plasma levels often ranging within two orders of magnitude. Recently, we have described an interference-free GC–tandem MS-based method for NO2Tyr which yielded the lowest basal plasma NO2Tyr levels reported thus far. This method was extended to quantify protein-associated 3-nitrotyrosine and in particular 3-nitrotyrosinated albumin (NO2TyrALB) in human plasma. NO2TyrALB and albumin (ALB) were extracted from plasma by affinity column extraction and digested enzymatically at neutral pH. 3-Nitro- l-[2H3]tyrosine was used as internal standard. In plasma of 18 healthy young volunteers the molar ratio of NO2TyrALB to albumin-derived tyrosine (TyrALB), i.e. NO2TyrALB/TyrALB, was determined to be 1.55±0.54×1:106 (mean±SD). The plasma concentration of NO2TyrALB was estimated as 24±4 nM. The NO2Tyr plasma levels in these volunteers were determined to be 0.73±0.53 nM. In the same volunteers, NO2TyrALB/TyrALB, NO2TyrALB and NO2Tyr were measured 15 days later and the corresponding values were determined to be 1.25±0.58×1:106, 25±6 nM and 0.69±0.16 nM. For comparison, NO2Tyr and NO2TyrALB were measured in six plasma samples from healthy volunteers by GC–MS and GC–tandem MS. Different values were found for NO2Tyr, i.e. 5.4±2.8 versus 2.7±1.5 nM, and comparable values for NO2TyrALB/TyrALB, i.e. 0.5±0.2×1:106 versus 0.4±0.1×1:106, by these methods. The ratio of the values measured by GC–MS to those measured by GC–tandem MS were 2.9±3.1 for NO2Tyr and 1.2±0.2 for NO2TyrALB/TyrALB. The present GC–tandem MS method provides accurate values of NO2Tyr and NO2TyrALB in human plasma.
Keywords: 3-Nitrotyrosine; 3-Nitrotyrosinoalbumin;
Development and validation of a bioanalytical method for the determination of the cholecystokinin type-1 (CCK1) receptor antagonist dexloxiglumide in human plasma by R Brodie; A Peard; A Roth; S Persiani; F Makovec; M D’Amato (91-100).
A sensitive bioanalytical method for the measurement of dexloxiglumide, a new selective and potent cholecystokinin type-1 (CCK1) receptor antagonist, in plasma, is reported. The method is based on reversed-phase liquid chromatography with ultraviolet absorption detection. Samples are extracted under acidic conditions into an organic solvent, and following evaporation, reconstitution and centrifugation stages, the supernatant is injected on to an ODS column with detection at 244 nm. The method has been validated over the concentration range 0.2–20 μg/ml, 0.2 μg/ml being the lower limit of quantification. The overall precision and accuracy (expressed as relative error) of the method was less than 6.1 and 2.3%, respectively. Dexloxigulmide was shown to be stable in plasma when stored at −20 °C for at least 200 days. The method is suitable for studying the pharmacokinetics of dexloxiglumide in man.
Keywords: Cholecystokinin type-1; Dexloxiglumide; Receptor antagonist;
Analysis of intracellular nucleoside triphosphate levels in normal and tumor cell lines by high-performance liquid chromatography by Dan Huang; Yazhuo Zhang; Xiaoguang Chen (101-109).
A reversed-phase ion-pair high-performance liquid chromatographic method for the direct and simultaneous determination of ribonucleoside triphosphates and their corresponding deoxyribonucleoside triphosphates in trichloroacetic acid cell extracts is presented. Using this system, high resolution of nine acid-soluble compounds, including ADP, CTP, dCTP, GTP, UTP, dGTP, dTTP, ATP and dATP in 16 normal or tumor cell lines, is obtained. The method is based on an extraction of nucleotides from cells with a solution of 6% trichloroacetic acid followed by neutralization with the addition of 5 M K2CO3 just prior to HPLC analysis. Chromatographic separations were performed using a Symmetry C18 3.5 μm (150×4.6 mm) column (Waters) equipped with a NovaPak C18 Sentry guard column with UV detection at 254 nm. The HPLC columns were kept at 27 °C. The mobile phase was delivered at a flow-rate of 1.0 ml/min, with the following stepwise gradient elution program: A–B (60:40) at 0 min→(40:60) at 30 min→(40:60) at 60 min. Solvent A contained 10 mM tetrabutylammonium hydroxide, 10 mM KH2PO4 and 0.25% MeOH, and was adjusted to pH 6.9 with 1 M HCl. Solvent B consisted of 5.6 mM tetrabutylammonium hydroxide, 50 mM KH2PO4 and 30% MeOH, and was neutralized to pH 7.0 with 1 M NaOH. The calibration curves (r>0.99) of the components in cell extracts were established with their aqueous standards. The average within-day precision for the nine compounds was 0.9%, and the average day-to-day precision was 5.0%. The detection limits (pmol) of the nine reagents were 1.39 (ADP), 4.32 (CTP), 15.5 (dCTP), 2.38 (GTP), 4.42 (UTP), 9.45 (dGTP), 14.6 (dTTP), 2.44 (ATP) and 11.8 (dATP). The recovery of this method for the standards ranged from 82.4 to 120.5%. The results for the detection of nucleotide pools in 16 normal and tumor cell lines were presented. In conclusion, this simplified analytical method enables the simultaneous quantitation of NTP and dNTP in cell or tissue extracts and may represent a valuable tool for the detection of minute alterations of intracellular NTP/dNTP pools induced by anticancer/antiviral drugs and diseases.
Keywords: Nucleoside triphosphates;
Determination of 6-hydroxychlorzoxazone and chlorzoxazone in porcine microsome samples by Sherry K. Cox; Tina Hamner; Joe Bartges (111-116).
A simple, accurate and sensitive HPLC method for the in vitro determination of 6-hydroxychlorzoxazone and chlorzoxazone in porcine microsome samples is described. Chromatography was performed on a YMC-Pack ODS-AQ column using a mobile phase of 0.05% phosphoric acid pH 3–methanol (60:40, v/v). UV detection was carried out at 287 nm. The detector response was linear over the concentration range 25–2000 ng/ml. This assay produced quick, accurate, and repeatable results.
Keywords: Hydroxychlorzoxazone; Chlorzoxazone;
Use of large particles support for fast analysis of methadone and its primary metabolite in human plasma by liquid chromatography–mass spectrometry by S Souverain; S Rudaz; D Ortelli; E Varesio; J.-L Veuthey (117-123).
A bioanalytical method was developed for the quantitation of methadone (MTD) and its primary metabolite, (EDDP) in plasma. The extraction step was performed within a capillary column packed with large particles (35×0.3 mm I.D.; d p 30 μm) at high flow-rate conditions (450 μl/min). The separation was performed on a microbore analytical column (55×2 mm I.D.; d p 3 μm) coupled to a mass spectrometer (MS). This procedure was based on a column-switching unit. Analytes of interest were retained on the precolumn by hydrophobic interactions and backflushed from the precolumn to the analytical column. The detection was carried out with a MS single quadrupole equipped with an electrospray interface. The total analysis time was 6 min. The limits of quantification were evaluated at 10 and 25 ng/ml for MTD and EDDP, respectively. At this level, good accuracies were obtained for both analytes with repeatability values less than 18%.
Keywords: Methadone; 2-Ethylidene-1,5-dimethyl-1,3-diphenylpyrrolidine;
Determination of diaminopimelic acid in rat feces by high-performance liquid chromatography using the Pico Tag method by Luis A Rubio (125-129).
The purpose of the study was to develop a method for the determination of diaminopimelic acid (DAPA) concentrations in rat feces by reversed-phase high-performance liquid chromatography (HPLC) using the Pico Tag method. Precolumn derivatization with phenylisothyocyanate (PITC) and UV (254 nm filter) detection were used. Samples were hydrolysed in 6 M HCl at 110 °C for 24 h. Hydrolysates were then diluted, dried and derivatized, and samples (10 μl injected onto a 300×3.9 mm NovaPak C18 (Waters) HPLC column. Under the conditions used, DAPA eluted as one single peak between those of tyrosine and valine. On-column DAPA concentrations in standards were 41.5–83 pmol, which were in the range of the amounts present in fecal samples of rats fed semisynthetic diets. Amounts of DAPA determined in fecal samples of rats fed broad bean- or chickpea-based diets were, respectively, 2.56 and 2.98 mg g−1. The advantages of the method and the relevance of the results for nutritional studies in monogastric animals are discussed.
Keywords: Diaminopimelic acid; Phenylisothiocyanate;
Effect of different sample pretreatment methods on the concentrations of excitatory amino acids in cerebrospinal fluid determined by high-performance liquid chromatography by H. Zhang; S.D. Zhai; Y.M. Li; L.R. Chen (131-135).
Using high-performance liquid chromatography (HPLC), the effect of different sample pretreatment methods on the concentrations of excitatory amino acids (EAAs, glutamate and aspartate) measured in cerebrospinal fluid (CSF) was investigated. The results showed that the measured values of glutamate and aspartate were constant when the samples were stored at −80 °C and then methanol was used for CSF deproteinization before assay; the values of glutamate (Glu) increased when 0.3 M perchloric acid was used for CSF deproteinization with the CSF subsequently being stored at −20 °C; the values of Glu changed when the samples were stored at −20 °C over 8 weeks with methanol subsequently being used for CSF deproteinization before assay. This reference data suggested that the CSF sample would be better stored at −80 °C. If the sample is stored at −20 °C over 8 weeks, the Glu values change with the storage time. If strong acidic reagents are used for precipitation of protein in the CSF sample and then stored at −20 °C, Glu values are abnormally increased. From this study, an accurate and sensitive reversed-phase high-performance liquid chromatographic method has been developed for anti-excitotoxicity therapy and thorough study of EAAs in a clinical setting.
Keywords: Glutamate; Aspartate;
Quantitation of soy-derived phytoestrogens in human breast tissue and biological fluids by high-performance liquid chromatography by Julie Maubach; Marc E. Bracke; Arne Heyerick; Herman T. Depypere; Rudolphe F. Serreyn; Marc M. Mareel; Denis De Keukeleire (137-144).
A new and reliable HPLC method for the quantitation of daidzein, equol, and genistein in human breast tissue has been developed. The method was applied to biopsies from women undergoing breast reductions, who, prior to surgery, had ingested either a soy isoflavone preparation or a placebo tablet. The results were compared with data collected for urine and serum of the same subjects using standard methods. The limits of detection in the breast tissue homogenate were 24.7 nmol/l for daidzein, 148.0 nmol/l for equol, and 28.4 nmol/l for genistein (S/N of 3). The chromatographic limits of quantitation were 62.5 nmol/l for daidzein and genistein, and 125.0 nmol/l for equol, for which the accuracies were 86.0%, 83.6%, and 81.8%, respectively. The coefficients of variation of these measurements were all below 20% (11.1% for daidzein, 16.4% for genistein, and 13.2% for equol). The sample preparation comprised a concentration step and the absolute limits of quantitation were, therefore, 4.7 nmol/l, 18.8 nmol/l, and 0.94 nmol/l for daidzein and genistein, and 9.4 nmol/l, 37.5 nmol/l, and 1.9 nmol/l for equol in urine, serum, and breast tissue homogenate, respectively. Recoveries were between 70% (±5.6%) in breast tissue homogenate and 100% (±14.1%) in urine and serum for all three compounds. Equol (less than 1 μmol/l homogenate) was found to be the predominant phytoestrogen in breast tissue and its concentrations exceeded those in serum. The concentrations of phytoestrogens were at least 100-fold higher in urine than in serum and breast tissue.
Keywords: Phytoestrogens; Daidzein; Equol; Genistein;
Gas chromatographic method for detection of urinary sucralose: application to the assessment of intestinal permeability by Ashkan Farhadi; Ali Keshavarzian; Earle W Holmes; Jeremy Fields; Lei Zhang; Ali Banan (145-154).
We developed a capillary column gas chromatography (CCGC) method for the measurement of urinary sucralose (S) and three other sugar probes including, sucrose, lactulose (L) and mannitol (M) for use in in vivo studies of intestinal permeability. We compared the capillary method with a packed column gas chromatography (PCGC) method. We also investigated a possible role for sucralose as a probe for the measurement of whole gut permeability. Sample preparation was rapid and simple. The above four sugars were detected precisely, without interference. We measured intestinal permeability using 5- and 24-h urine collections in 14 healthy volunteers. The metabolism of sugars was evaluated by incubating the intestinal bacteria with an iso-osmolar mixture of mannitol, lactulose and sucralose at 37 °C for 19 h. Sugar concentrations and the pH of the mixture were monitored. The use of the CCGC method improved the detection of sucralose as compared to PCGC. The average coefficient of variation decreased from 15% to 4%. It also increased the sensitivity of detection by 200–2000-fold. The GC assay was linear between sucralose concentrations of 0.2 and 40 g/l (r=1.000). Intestinal bacteria metabolized lactulose and acidified the media but did not metabolize sucralose or mannitol. The new method for the measurement of urinary sucralose permits the simultaneous quantitation of sucrose, mannitol and lactulose, and is rapid, simple, sensitive, accurate and reproducible. Because neither S nor M is metabolized by intestinal bacteria, and because only a tiny fraction of either sugar is absorbed, this pair of sugar probes appears to be available for absorption throughout the GI tract. Thus, the 24-h urinary concentrations of S and M, or the urinary S/M ratio following an oral dose of a sugar mixture, might be good markers for whole gut permeability.
Keywords: Sucralose; Mannitol; Lactulose; Sucrose;
Isolation and identification of two [3H]norharman- ([3H]β-carboline)-binding proteins from rat liver by Alexa Greube; Hans Rommelspacher (155-168).
Norharman (9H-pyrido-[3,4-b]indol) represents a member of the mammalian alkaloids with the group name β-carbolines. In mammals, it exhibits psychotropic and co-mutagenic actions. Highly specific [3H]norharman binding sites have been detected in the liver of rats (B max: 11 pmol mg−1 protein; K D: lower nanomolar range). Two [3H]norharman binding proteins with apparent molecular masses of 60 and 80 kDa (SDS–PAGE) were isolated from rat liver crude membrane fraction and identified as the enzyme carboxylesterase (EC 184.108.40.206; 60 kDa) and the stress protein glucose-regulated protein 78 (GRP78; 78 kDa). Possible functional consequences of the interaction of norharman with these two proteins are discussed.
Keywords: Norharman; β-Carboline; Proteins;
On-line clean-up by multidimensional liquid chromatography–electrospray ionization tandem mass spectrometry for high throughput quantification of primary and secondary phthalate metabolites in human urine by Holger M Koch; Luis M Gonzalez-Reche; Jürgen Angerer (169-182).
We developed a new and fast multidimensional on-line HPLC-method for the quantitative determination of the secondary, chain oxidized monoester metabolites of diethylhexylphthalate (DEHP), 5-hydroxy-mono-(2-ethylhexyl)-phthalate (5OH-MEHP) and 5-oxo-mono-(2-ethylhexyl)-phthalate (5oxo-MEHP) in urine samples from the general population. Also included in the method were the simple monoester metabolites of DEHP, dioctylphthalate (DOP), dibutylphthalate (DBP), butylbenzylphthalate (BBzP) and diethylphthalate (DEP). Except for enzymatic hydrolysis for deconjugation of the metabolites no further sample pre-treatment step is necessary. The phthalate metabolites are stripped from urinary matrix by on-line extraction on a restricted access material (LiChrospher® ADS-8) precolumn, transferred in backflush-mode and chromatographically resolved by reversed-phase HPLC. Eluting metabolites are detected by ESI-tandem mass spectrometry in negative ionization mode and quantified by isotope dilution. Within a total run time of 25 min we can selectively and sensitively quantify seven urinary metabolites of six commonly occurring phthalate diesters including the controversial di(2-ethylhexyl)phthalate (DEHP). The detection limits for all analytes are in the low ppb range (0.5–2.0 μg/l urine). First results on a small non-exposed group (n=8) ranged for 5OH-MEHP from 0.59 to 124 μg/l, for 5oxo-MEHP from <LOQ to 73.0 μg/l, and for MEHP from <LOQ to 41.1 μg/l. The other short chain monoester metabolites were detectable in every sample with mean concentrations for MnBuP of 36.5 μg/l, for MBzP of 7.19 μg/l and MEP of 1.0 mg/l. With this rapid and economic method we can determine the internal exposure of the general population to DEHP and other phthalates as well as the body burden of occupationally and medically exposed subjects. The results can help to rank the risks of phthalates in the areas of carcinogenesis, peroxisome proliferation and endocrine disruption. Since secondary, functionalized metabolites of DEHP are included in the method an enduring problem of the past is excluded: sample contamination in the pre-analytical and analytical phase by both di- and monoesters.
Expanded bed adsorption processing of mammalian cell culture fluid: comparison with packed bed affinity chromatography by Y González; N Ibarra; H Gómez; M González; L Dorta; S Padilla; R Valdés (183-187).
A comparison between expanded bed adsorption and conventional packed bed Protein A Fast Flow to purify the anti-rHBsAg mAbs from feedstock is presented in this work. Direct capture by STREAMLINE expanded bed adsorption chromatography resulted in 92% product recovery and sevenfold more concentrated product with similar purity levels compared to that obtained by the standard packed method. The process time and buffer consumption were reduced in the expanded bed adsorption method not only with the binding-elution conditions but also with the use of NaOH during the cleaning-in-place step. The latter is the most widely accepted agent in downstream processing, being a cost effective technique that provides not only efficient cleaning but also sanitizes complete column systems and destroys pirogens.
Keywords: Monoclonal antibodies;
Liquid chromatography method for the analysis of adenosine compounds by Gesine Haink; Andreas Deussen (189-193).
A newly available chromatography column packing material that employs hybrid particle technology was used to improve the analysis of adenosine compounds. Using a TBAS buffer/acetonitrile gradient this material permits separation of etheno-adenosine compounds in less than 4 min with excellent resolution and sensitivity (50 fmol). Variability of compound quantification is small (coefficients of variation 0.23±0.14% for 50 pmol and 1.70±0.53% for 0.5 pmol). The new method is well suited for the analysis of adenosine compounds in small biological samples and permits a high sample throughput in autosampler setups with high precision and reproducibility.
Keywords: Adenosine compounds;
Simultaneous determination of irbesartan and hydrochlorothiazide in human plasma by liquid chromatography by Nevin Erk (195-201).
A simple, selective, sensitive and precise high-performance liquid chromatographic plasma assay for the antihypertensive drugs, irbesartan and hydrochlorothiazide is described. Good chromatographic separation was achieved using a Supelcocil C18 (5 μm, 15 cm×4.6 mm) column and a mobile phase consisting of 10 mM potassium dihydrogen phosphate:methanol:acetonitrile (5:80:15 v/v/v) (pH:2.5) while at a flow-rate of 1.0 ml min−1. Irbesartan and hydrochlorothiazide were detected at 275 nm and were eluted 5.8 and 7.8 min, respectively, after injection. No endogenous substances were found to interfere. The method utilizes protein precipitation with acetonitrile as the only sample preparation involved prior to reversed-phase high-performance liquid chromatography. No internal standard was required. Linearity range for irbesartan and hydrochlorothiazide was 10.0–60.0 μg ml−1 and 4.0–20.0 μg ml−1, respectively. The determination of intra- and inter-day precision (RSD) was less than 2.5 and 3.5%, at all concentration levels, while the inter- and intra-day accuracy (% difference) was less than 4.9–6.2%. This method is being used in a therapeutic drug monitoring service to quantitate these therapeutic agents in patients for pharmacokinetic studies.
Keywords: Irbesartan; Hydrochlorothiazide;
Improved lectin-mediated immobilization of human red blood cells in superporous agarose beads by Ingo Gottschalk; Per-Erik Gustavsson; Bo Ersson; Per Lundahl (203-208).
A new type of agarose bead, superporous agarose, was used as a gel support for immobilization of human red blood cells (RBCs) mediated by wheat germ lectin. The number of immobilized cells was similar to that obtained with commercial wheat germ lectin–agarose but the cell stability appeared to be superior. This allowed improved frontal affinity chromatographic analyses of cytochalasin B (CB)-binding to the glucose transporter GLUT1 which established a ratio of one CB-binding site per GLUT1 dimer for both plain RBCs or those treated with different poly amino acids. The measured dissociation constants, 70±14 nM for CB and 12±3 mM for glucose binding to GLUT1, are similar to those reported earlier.
Keywords: Wheat germ agglutinin; Cytochalasin B; GLUT1;
Instructions to Authors (209-217).