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PubMed2001

Determination of free amino acids in infant food by capillary zone electrophoresis with mass spectrometric detection.

A method for the quantitative determination of free amino acids in infant food samples using capillary electrophoresis (CE) with electrospray-mass spectrometric (ESI-MS) detection is presented. According to the zwitterionic nature of the analytes, two different modes of separation as well as detection were tested; highly acidic carrier electrolytes combined with MS detection in the positive ion mode proved to be the optimum solution. Sensitivity as well as linearity of the method were sufficient to allow the analysis of all solutes of interest in a single run. In this way, free amino acids could be analyzed in a variety of infant food preparations without any sample pretreatment or derivatization step.

Amino Acids↗
PubMed1979

Isotachophoresis of CSF proteins in gel tubes especially gammaglobulins. An analytical and preparative technique for high-separation of CSF proteins.

An isotachophoretic method using polyacrylamide gel (PAG-ITP) in a simple disc electrophoretic equipment with plastic tubes containing the gels, was elaborated and especially designed for studying the gammaglobulins in CSF and serum from control subjects and patients with neurological disorders, especially known or probable MS. The device and the ITP system used, including leading and terminating electrolytes and spacer substances, dividing the gammaglobulins in a reproducible way, are described. No cooling of the gel tubes was needed. The sample volumes varied between 5--500 microliters, and the separation time was 1.5--3.0 h. CSF from patients with verified or probable MS revealed characteristic, increased low-mobility gammaglobulin fractions. Using other ITP systems, such as other spacer compositions, the anodic proteins can also be studied in more detail. PAG-ITP in gel tubes is a simple and inexpensive technique which can be used for both analytical and preparative procedures for biological material such as CSF, serum and extractions from nervous tissues.

Cerebrospinal Fluid Proteins↗
PubMed2007

Electroelution of intact proteins from SDS-PAGE gels and their subsequent MALDI-TOF MS analysis.

A method has been developed for the extraction of intact proteins from SDS-PAGE gels and for subsequent MALDI-TOF MS analysis to determine precise molecular mass. The method consists of an electroelution step and a subsequent low-temperature matrix-analyte cocrystallization step prior to MALDI-TOF MS. Proteins were first extracted from polyacrylamide gels using electroelution (ProteoPLUS). Following electroelution, salts, SDS, and dye were removed by dialysis using the same electroelution tubes. The proteins were then concentrated in a vacuum centrifuge. Using bovine serum albumin (BSA) and phosphorylase B (Phos B) as standards, it was found that electroelution was more effective than passive elution in extracting intact proteins from gels. Optimized protein recoveries were determined to be 89% for BSA and 58% for Phos B, supporting the hypothesis of an inverse relationship between recovery and protein size. The traditional practice of "fixing gels" using methanol and acetic acid was found to reduce protein recoveries significantly and should be avoided if possible. A subsequent sample preparation method involving the low-temperature cocrystallization of whole proteins with MALDI matrices (trans-3,5-dimethoxy-4-hydroxycinnamic acid, sinapinic acid, 10 mg/mL) was also developed to remove residual impurities further. Proteins and MALDI matrices were mixed, sealed, and stored at 4 degrees C to allow for overnight cocrystallization of matrix and protein. The supernatant was removed and protein-matrix crystals redissolved using the same matrix solution. MALDI-TOF MS signal intensities were found to increase three- to fourfold using the low-temperature cocrystallization method relative to the more common method of immediate analysis following matrix-analyte mixing and "dried droplet" deposition. The reported method is illustrated using two beta-1,3-endoglucanase isoforms from wheat apoplast extracts.

Cellulase↗
PubMed2002

Direct-injection high performance liquid chromatography ion trap mass spectrometry for the quantitative determination of olanzapine, clozapine and N-desmethylclozapine in human plasma.

A specific and sensitive direct-injection high performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) method has been developed for the rapid identification and quantitative determination of olanzapine, clozapine, and N-desmethylclozapine in human plasma. After the addition of the internal standard dibenzepin and dilution with 0.1% formic acid, plasma samples were injected into the LC/MS/MS system. Proteins and other large biomolecules were removed during an online sample cleanup using an extraction column (1 x 50 mm i.d., 30 microm) with a 100% aqueous mobile phase at a flow rate of 4 mL/min. The extraction column was subsequently brought inline with the analytical column by automatic valve switching. Analytes were separated on a 5 microm Symmetry C18 (Waters) analytical column (3.0 x 150 mm) with a mobile phase of acetonitrile/0.1% formic acid (20:80, v/v) at a flow rate of 0.5 mL/min. The total analysis time was 6 min per sample. The inter- and intra-assay coefficients of variation for all compounds were <11%. By eliminating the need for extensive sample preparation, the proposed method offers very large savings in total analysis time.

Antipsychotic Agents↗
PubMed2001

Analysis of metabolites of glucose pathways in human erythrocytes by analytical isotachophoresis.

The aim of this study was to establish an isotachophoretic (ITP) method for the determination of the main compounds of glycolysis in human erythrocytes in order to analyze the influence of different glucose concentrations (mimicking the situation in diabetes mellitus) on this pathway. Samples for ITP were prepared by isolation of erythrocytes, lysis of the cells by heating in double-distilled water and subsequent ultrafiltration (Mr cut-off: 5000). All the main compounds of glycolysis were characterized by ITP. The influence of different glucose concentrations on the main compounds of the energy metabolism (ATP, ADP, lactate, pyruvate) and 2,3-diphosphoglycerate were analyzed in short- and long-time incubations.

Adenosine Triphosphate↗
PubMed2004

Enzyme-linked immunosorbent assay for the detection of yessotoxin and its analogues.

Polyclonal antibodies were produced for the development of competitive enzyme-linked immunoassays for use in quantifying yessotoxins in shellfish, algal cells, and culture supernatants. Immunizing and plate coating antigens were prepared by derivatization of yessotoxin either by ozonolysis or bromination and conjugation to proteins. Two assays that were the most sensitive for yessotoxin were optimized and characterized. Cross-reactivity studies indicated that the antibodies raised have broad specificity and that binding to analogues was strongly affected by changes to the A-ring and, to a lesser extent, the K-ring regions of the toxin molecule. ELISA provides a sensitive and rapid analytical method that is suitable for screening large numbers of samples and detects all the yessotoxin analogues that the European Commission currently requires shellfish to be tested for. The assay limit of quantitation for yessotoxin in whole shellfish flesh is 75 microg/kg; therefore, assay sensitivity is sufficient to measure toxin levels well below the maximum permitted level set by the European Commission. The antibodies produced can be used in additional applications such as the immunolocalization of yessotoxins in shellfish and preparation of immunoaffinity columns.

Antibody Specificity↗
PubMed2006

Enantiospecific separation and quantitation of mephenytoin and its metabolites nirvanol and 4'-hydroxymephenytoin in human plasma and urine by liquid chromatography/tandem mass spectrometry.

A sensitive method using enantiospecific liquid chromatography/tandem mass spectrometry detection for the quantitation of S- and R-mephenytoin as well as its metabolites S- and R-nirvanol and S- and R-4'-hydroxymephenytoin in plasma and urine has been developed and validated. Plasma samples were prepared by protein precipitation with acetonitrile, while urine samples were diluted twice with the mobile phase before injection. The analytes were then separated on a chiral alpha(1)-acid glycoprotein (AGP) column and thereafter detected, using electrospray ionization tandem mass spectrometry. In plasma, the lower limit of quantification (LLOQ) was 1 ng/mL for S- and R-4'-hydroxymephenytoin and S-nirvanol and 3 ng/mL for R-nirvanol and S- and R-mephenytoin. In urine, the LLOQ was 3 ng/mL for all compounds. Resulting plasma and urine intra-day precision values (CV) were <12.4% and <6.4%, respectively, while plasma and urine accuracy values were 87.2-108.3% and 98.9-104.8% of the nominal values, respectively. The method was validated for plasma in the concentration ranges 1-500 ng/mL for S- and R-4'-hydroxymephenytoin, 1-1000 ng/mL for S-nirvanol, and 3-1500 ng/mL for R-nirvanol and S- and R-mephenytoin. The validated concentration range in urine was 3-5000 ng/mL for all compounds. By using this method, the metabolic activities of two human drug-metabolizing enzymes, cytochrome P450 (CYP) 2C19 and CYP2B6, were simultaneously characterized.

Chromatography, Liquid↗
PubMed2006

Routine analysis of off-flavor compounds in water at sub-part-per-trillion level by large-volume injection GC/MS with programmable temperature vaporizing inlet.

"Earthy-musty" off-flavor problem in water samples are due to organic compounds present at the sub-part-per-trillion level. Most of the developments in the analysis of tastes and odorous compounds focus on the extraction pre-concentration technique, with detection at picogram per liter level of the earthy-musty off-flavor compounds difficult to be achieved. The objective of this study is to develop a new method involving the large-volume injection (LVI) GC/MS via programmable temperature vaporizing (PTV) inlet and continuous liquid-liquid extraction, to attain analytical sensitivity equal to or better than olfactory sensitivity. Six "earthy-musty" organic compounds, 2-methylisoborneol (MIB), geosmin, 2,4,6-trichloroanisole, 2,3,6-trichloroanisole, 2,3,4-trichloroanisole and 2,4,6-tribromoanisole, were used as probes for this study. It was found that LVI via PTV could greatly improve system sensitivity towards off-flavor compound detection. Variable instrument conditions and sample preparation conditions were studied in detail. This reliable and efficient method has been optimized and validated. The method detection limit were found to be from 35 to 70 pg/L for haloanisoles, 50 pg/L for geosmin and 0.34 ng/L for MIB, with relative standard deviation of calibration curve ranging from 4.7% to 15.1% and recovery ranging from 58% to 96%. This method has been successfully applied to test off-flavor compounds in different types of water samples with satisfactory results.

Calibration↗
PubMed1985

Enzymatic correction of interference in the kinetic Jaffé reaction for determining creatinine in plasma.

We evaluated a simple, specific method for measuring plasma creatinine concentration, involving enzymatic degradation of creatinine before analysis by a kinetic Jaffé reaction. The enzymes--prepared in a dry, stable form--are convenient to use and eliminate the need to correct for sample dilution. As little as 50 microL of plasma is required, making the procedure ideal for use with samples from children. Between-day precision studies for determination of plasma creatinine at concentrations of 70 and 470 mumol/L yielded CVs of 7.8 and 4.3%, respectively. Analytical recovery of creatinine added to plasma samples ranged from 94 to 113%. Linear regression analysis demonstrated excellent agreement between results by this method and those by a "high performance" liquid-chromatographic technique.

Amidohydrolases↗
PubMed1999

Capillary zone electrophoresis determination of galanthamine in biological fluids and pharmaceutical preparatives: experimental design and artificial neural network optimization.

Galanthamine is a third-generation cholinesterase inhibitor used against Alzheimer's disease. New analytical methods for the determination of galanthamine in pharmaceutical preparatives and biological fluids, such as urine and serum, were developed. An experimental design and artificial neural network approach were used for method optimization. Thirty-five ppb of galanthamine were determined in serum samples (with addition of 10 mM magnesium chloride and using solid-phase preconcentration).

Alzheimer Disease↗
PubMed2006

Chromatographic analysis of penicillins in pharmaceutical formulations and biological fluids.

Natural penicillin (benzylpenicillin) is the oldest antibiotic observed by Alexander Fleming in 1928. To broaden its spectrum of activity, natural penicillin was modified, giving rise to a group of antibiotics under the name 'penicillins'. Although an increasing number of bacteria appear to be resistant to them, penicillins are used to treat a variety of bacterial infections including Gram-positive, Gram-negative aerobic and anaerobic bacteria. Consequently, they are widely used in human and veterinary medicine to prevent and treat diseases. This review covers the analytical methodologies, mainly chromatographic, employed to the penicillins determination in pharmaceutical formulations, biological fluids and in production-scale fermentations reported in the literature. Results of published assays are comparatively presented focusing on sample preparation regarding isolation and purification, chromatographic conditions and method validation. Information on chemical structure, spectrum of activity and action mechanism of common penicillins has also been given.

Chromatography, High Pressure Liquid↗
PubMed2004

Comparative study of the continuous wavelet transform, derivative and partial least squares methods applied to the overlapping spectra for the simultaneous quantitative resolution of ascorbic acid and acetylsalicylic acid in effervescent tablets.

The simultaneous spectrophotometric determination of ascorbic acid (AA) and acetylsalicylic acid (ASA) in effervescent tablets in the presence of the overlapping spectra was accomplished by the continuous wavelet transform (CWT), derivative spectrophotometry (DS) and partial least squares (PLS) approaches without using any chemical pre-treatment. CWT and DS calibration equations for AA and ASA were obtained by measuring the CWT and DS amplitudes corresponding to zero-crossing points of spectra obtained by plotting continuous wavelet coefficients and first-derivative absorbance values versus the wavelengths, respectively. The PLS calibration was constructed by using the concentration set and its full absorbance data consisting of 850 points from 220 to 305 nm in the range of 210-310 nm. These three methods were tested by analyzing the synthetic mixtures of the above drugs and they were applied to the real samples containing two commercial pharmaceutical preparations of subjected drugs. A comparative study was carried out by using the experimental results obtained from three analytical methodologies and precise and accurate results were obtained.

Ascorbic Acid↗
PubMed1997

Development of an assay for the simultaneous determination of sildenafil (Viagra) and its metabolite (UK-103,320) using automated sequential trace enrichment of dialysates and high-performance liquid chromatography.

The development of the ASTED (automated sequential trace enrichment of dialysates) system to prepare plasma samples prior to high-performance liquid chromatography (HPLC) of sildenafil and its demethylated metabolite (UK-103,320) is described. Investigations to elucidate potential pitfalls of the ASTED on-line sample preparation system prior to separation of the analytes by HPLC are presented. The procedure is shown to be selective for sildenafil and UK-103,320, and linear over the range 1.00-250 ng/ml. The intra-batch imprecision (C.V.) of the method at plasma analyte concentrations of 1.00, 5.00, 50.0 and 200 ng/ml was 11.2, 3.10, 1.50, and 1.30%, respectively, and the corresponding inter-batch imprecision was estimated to be 13.5, 7.09, 3.69, and 5.43%. At these plasma analyte concentrations the overall inaccuracy (% bias) of the procedure ranged from 3.6 to 8.4%. The method showed similar assay performance for the estimation of the metabolite, UK-103,320. The application of the assay to a pharmacokinetic investigation during a clinical study is presented.

Chromatography, High Pressure Liquid↗
PubMed1998

Determination of methazolamide concentrations in human biological fluids using high performance liquid chromatography.

Methazolamide is a carbonic anhydrase inhibitor used to treat glaucoma. In vivo, methazolamide readily distributes into red blood cells. Therefore, both blood and plasma concentration data are needed in order to characterize the pharmacokinetics of methazolamide. In the present study, an analytical method using high performance liquid chromatography was validated for determination of methazolamide concentrations in several biological fluids. Through slight modification of a previously reported method for acetazolamide, another carbonic anhydrase inhibitor, methazolamide was readily quantitated in whole blood, plasma and urine. Sample preparation involved liquid-liquid extraction with ethyl acetate followed by a washing step using phosphate buffer (pH 8.0). After back extraction into glycine buffer (pH 10.0), samples were then washed with ether and injected onto the chromatograph. Chromatography was performed using a C-18, 5 microns reverse-phase column with UV detection at a wavelength of 285 nm. Mobile phase consisted of 0.05 M sodium acetate (pH 4.0) and acetonitrile (20%). The assay was validated over two standard concentration ranges from 1 to 100 micrograms ml-1, concentrations reflective of those expected in vivo, Calibration curves were linear for all biological fluids and coefficients of variation for interday and intraday reproducibility studies were less than 8% (range 3.1-7.9%). The method was used to measure methazolamide concentrations in blood, plasma and urine following oral administration to five human subjects.

Acetazolamide↗
PubMed2001

A facile spectrophotometric method for the determination of selenium.

A rapid and sensitive spectrophotometric method is described for the determination of trace amounts of selenium using Variamine Blue (VB) as a chromogenic reagent. The proposed method is based on the reaction of selenium with potassium iodide in an acidic medium to liberate iodine, which oxidizes Variamine Blue to form a violet-colored species having an absorption maximum at 546 nm. Beer's law is obeyed in the range 2-20 g of selenium in a final volume of 10 ml. The molar absorptivity and Sandell's sensitivity were found to be 2.6 x 10(4) l mol-1 cm-1 and 0.003 microgram cm-2, respectively. The optimum reaction conditions and other analytical parameters were evaluated. The effect of interfering ions on the determination is described. The proposed method has been successfully applied to the determination of selenium in real samples of water, soil, plant materials, human hair, and synthetic samples of cosmetics and pharmaceutical preparations.

Brassicaceae↗
PubMed1996

Simultaneous determination of coumarin, 7-hydroxycoumarin and 7-hydroxycoumarin glucuronide in human serum and plasma by high-performance liquid chromatography.

A HPLC method was developed for the determination of the metabolites of coumarin and 7-hydroxycoumarin in plasma and serum. Separation was based on gradient elution of 7-hydroxycoumarin glucuronide, 7-hydroxycoumarin, coumarin and finally 4-hydroxycoumarin (which is used as an internal standard). Standards, prepared in plasma or serum, and samples were treated with trichloroacetic acid, mixed and centrifuged. The supernatant was removed and analyzed by reversed-phase high-performance liquid chromatography on a C18 column. The limit of detection was 50 ng/ml for 7-hydroxycoumarin and 200 ng/ml for coumarin and 7-hydroxycoumarin glucuronide. The linear range was 0.5-100 micrograms/ml for each of the analytes. The percentage relative standard deviation about the mean measured concentrations were all below 10%. There was no statistical difference between the standard curves prepared in plasma or serum. The method developed was applied to the determination of each of the three compounds in serum, after the administration of 7-hydroxycoumarin, and in plasma after the administration of coumarin. The concentrations of total 7-hydroxycoumarin in the serum samples were also determined by another HPLC method and the results were compared. There was no statistical difference between the results determined.

Chromatography, High Pressure Liquid↗
PubMed1997

An isotope-dilution gas chromatography-mass spectrometry method for trace analysis of xylene metabolites in tissues.

A gas chromatography-mass spectrometry (GC-MS) method using isotope dilution was developed to measure trace levels of xylene metabolites in brain tissues. The primary metabolites of xylene are dimethylphenol (DMP), methylbenzyl alcohol (MBA), toluic acid (TA), and methylhippuric acid (MHA). The internal standard was a mixture of deuterated DMP-d3, TA-d7, and MHA-d7. DMP-d3 was commercially available and was used as the internal standard for both DMP and MBA. TA-d7 and MHA-d7 were biosynthesized by administering xylene-d10 to rats and collecting their urine. Based on the noise peaks in 10 blank samples, the on-column limits of quantitation (mean +10 SD of noise peaks) were approximately 305, 1220, 545, and 386 pg for DMP, MBA, TA, and MHA, respectively. Analyte detection and recovery tests from brain tissues of control rats were conducted by spiking the tissues with 32 nmol/g of each analyte, together with the deuterated metabolites. The tissues were homogenized, extracted with ethyl acetate, and derivatized by trimethylsilylation. One microliter of the sample was injected into the GC-MS. The recoveries of the analytes were 104 +/- 8%, 80 +/- 9%, 93 +/- 10%, and 92 +/- 11% (mean +/- SD, n = 7) for DMP, MBA, TA, and MHA, respectively. The tissue preparation efficiency, which was indicated by absolute recoveries of internal standards, was approximately 33% for DMP, MBA, and TA and approximately 80% for MHA. No metabolites were detected in untreated control tissues. This simple and sensitive method to simultaneously detect major xylene metabolites in brain tissues could also be used for the analysis of blood and urine samples from workers to monitor p-xylene exposure.

Animals↗
PubMed2005

Separation of proteins with a molecular mass difference of 2 kDa utilizing preparative double-inverted gradient polyacrylamide gel electrophoresis under nonreducing conditions: application to the isolation of 24 kDa human growth hormone.

A method for separating proteins with a molecular mass difference of 2 kDa using SDS-PAGE under nonreducing conditions is presented. A sample mixture containing several human growth hormone (hGH) isoforms was initially separated on a weak anion-exchange column. Fractions rich in 24 kDa hGH as determined by analytical SDS-PAGE were pooled and further separated by cation-exchange chromatography. The fractions pooled from the cation-exchange chromatography contained two hGH isoforms with a 2 kDa molecular mass difference according to SDS-PAGE analysis, 22 and 24 kDa hGH. The 22 and 24 kDa hGH were separated using continuous-elution preparative double-inverted gradient PAGE (PDG-PAGE) under nonreducing conditions. The preparative electrophoresis gel was composed of three stacked tubular polyacrylamide matrices, a 4% stacking gel, a 13-18% linear gradient gel, and a 15-10% linear inverted gradient gel. Fractions containing purified 24 kDa hGH were pooled and Western blot analysis displayed immunoreactivity to antihGH antibodies. PDG-PAGE provides researchers with an electrophoretic technique to preparatively purify proteins under nonreducing conditions with molecular mass differences of 2 kDa.

Chromatography↗