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PubMed1998

Constitutive expression of cellular retinoic acid binding protein II and lack of correlation with sensitivity to all-trans retinoic acid in acute promyelocytic leukemia cells.

The up-regulation of cellular retinoic acid binding protein-II (CRABP-II) has been invoked as an important mechanism of clinically acquired resistance to all-trans retinoic acid (RA) therapy in acute promyelocytic leukemia (APL). To test this hypothesis, we used quantitative reverse transcription-PCR and fast performance liquid chromatography procedures to examine the levels of CRABP-II mRNA and RA binding activity in APL patient samples. We found that CRABP-II mRNA in APL cells from pretreatment patients (n = 36) was constitutively expressed at relatively high levels (median, 0.92; range, 0.16-4.13) relative to the level in CRABP-H protein-expressing NB4 cells (arbitrarily set at 1.0 unit). Consistent with this finding, the RA binding activity of CRABP in APL cells from three pretreatment cases (range, 27.2-53.2 fmol/mg protein) was similar to that of NB4 cells (22.6 +/- 5.4 fmol/mg protein). Furthermore, in the pretreatment samples, there was no association between CRABP-H mRNA expression level and APL cellular sensitivity to RA-induced differentiation in vitro. After 45 days of remission induction therapy on Eastern Cooperative Oncology Group protocol E2491, CRABP-II mRNA was modestly increased from day 0 values in patients treated with either RA (median increase, 0.41) or chemotherapy (median increase, 0.56), and there was no significant difference between the two treatment groups (P = 0.91). In patients studied after relapse from RA therapy (n = 7), there was a significant decline in APL cell sensitivity to RA-induced differentiation in vitro compared with patients after relapse from chemotherapy (n = 5; P = 0.015-0.055 at three RA concentrations tested), but in the RA relapse cases, there was no change from pretreatment levels of CRABP-II mRNA (median, 0.98) or, in three relapse cases studied, of RA protein binding activity (range, 22.1-70.7 fmol/mg protein). Taken together, our data strongly imply that variations in CRABP-II expression and RA binding activity are not causally related to the development of clinically acquired APL cellular RA resistance, but rather, they suggest that constitutive expression of CRABP-II could have a facilitative role in the response of APL cells to RA.

Cell Differentiation↗

PubMed2006

Determination of polycyclic aromatic hydrocarbons in olive oil by a completely automated headspace technique coupled to gas chromatography-mass spectrometry.

A new and completely automated method for the determination of ten relevant polycyclic aromatic hydrocarbons (PAHs) in olive oil is proposed using an extraction by the headspace (HS) technique. Quantification and confirmation steps are carried out by gas chromatography-mass spectrometry (GC-MS) combining simultaneously selected-ion monitoring (SIM) and tandem mass spectrometry (MS/MS). This combination offers on one hand an increased sensitivity and on the other hand, selective and reliable qualitative information. Sample pretreatment or clean-up are not necessary because the olive oil sample is put directly into an HS vial, automatically processed by HS and introduced into the GC-MS instrument for analysis. Because of its high selectivity and sensitivity, a triple-quadrupole (QqQ) detector coupled with the gas chromatograph allows us to limit handling. Each sample is completely processed in approximately 63 min (45 min for HS isolation and 18 min for GC-MS determination), a reduced time compared with previously published methods. The chemical and instrumental variables were preliminarily optimized using uncontaminated olive oil samples spiked with 25 microg kg(-1) of each target compound. The final method was validated to ensure the quality of the results. The precision was satisfactory, with relative standard deviation (RSD) values in the range 3-9%. Recovery rates ranged from 96 to 99%. Limits of detection (LOD) were calculated as 0.02-0.06 microg kg(-1) and the limits of quantification (LOQ) were obtained as 0.07-0.26 microg kg(-1). It must be mentioned that the LOD and LOQ are much lower than the maximum levels established by the European Union (EU) in oils and fats intended for direct human consumption or for use as an ingredient in foods, which are set at 2 microg kg(-1). All the figures of merit are completely in accordance with the latest EU legislation. This fact makes it possible to consider the proposed method as a useful tool for the control of PAHs in olive oils.

Food Analysis↗

PubMed1979

[Determination of carbamate pesticides in fruits and vegetables by means of high pressure liquid chromatography (author's transl)].

It is shown that carbamate pesticides can be determined at the ppm level in different kinds of fruits and vegetables using high pressure liquid chromatography. The lower detection limit corresponding to a signal to noise ratio of three is between 0,025 and 0,25 ppm depending on the type of carbamate and plant material. In all cases it is below the maximum permissible value for pesticide residues. Due to the high separating power of high pressure liquid chromatography a simple sample pretreatment procedure can be used. The carbamates are extracted from the biological sample by dichloromethane and injected directly into the liquid chromatograph after replacing the extraction solvent by the mobile phase. In many cases a definite identification and quantitation of the carbamate is possible with a single chromatographic column. Some plants with a more complex matrix require a two-column operation in which the effluent fraction of the first column containing the pesticide is transferred by column switching to a second column in order to achieve a complete separation. The improved resolution in the two-column operation is caused by the relative enrichment effect of the fractionation and the increased column length.

Carbamates↗

PubMed2003

Direct determination of four fluoroquinolones, enoxacin, norfloxacin, ofloxacin, and ciprofloxacin, in pharmaceuticals and blood serum by HPLC.

A rapid, accurate and sensitive method has been developed for the quantitative determination of four fluoroquinolone antimicrobial agents, enoxacin, norfloxacin, ofloxacin and ciprofloxacin, with high in-vitro activity against a wide range of Gram-negative and Gram-positive organisms.A Kromasil 100 C(8) 250 mm x 4 mm, 5 microm analytical column was used with an eluting system consisting of a mixture of CH(3)CN-CH(3)OH-citric acid 0.4 mol L(-1) (7:15:78 %, v/v). Detection was performed with a variable wavelength UV-visible detector at 275 nm resulting in limits of detection: 0.02 ng per 20 microL injection for enoxacin and 0.01 ng for ofloxacin, norfloxacin and ciprofloxacin. Hydrochlorothiazide (HCT) was used as internal standard at a concentration of 2 ng microL(-1). A rectilinear relationship was observed up to 2 ng microL(-1) for enoxacin, 12 ng microL(-1) for ofloxacin, 3 ng microL(-1) for norfloxacin, and 5 ng microL(-1) for ciprofloxacin. Separation was achieved within 10 min. The statistical evaluation of the method was examined by performing intra-day (n=8) and inter-day precision assays (n=8) and was found to be satisfactory with high accuracy and precision. The method was applied to the direct determination of the four fluoroquinolones in human blood serum. Sample pretreatment involved only protein precipitation with acetonitrile. Recovery of analytes in spiked samples was 97+/-6% over the range 0.1-0.5 ng microL(-1).

Anti-Infective Agents↗

PubMed1982

Sensitivity of herpes virus isolates from acyclovir clinical trials.

Clinical isolates of herpes simplex virus and varicella zoster virus have been examined for changes in drug sensitivity after treatment of patients with acyclovir. Some preliminary sensitivity results determined by plaque reduction assays in Vero cells for herpes simplex virus and MRC-5 cells for varicella zoster virus are presented. Where both pretreatment and posttreatment virus isolates were available, no significant changes in sensitivity to acyclovir were observed. Similarly, isolates taken after treatment was begun, without a pretreatment sample, gave IC50 values no greater than expected when compared with other pretreatment isolates.

Acyclovir↗

PubMed1992

Biodetermination of N-(deacetyl-O-4-vinblastoyl-23)-L-tryptophan, a metabolite of vintriptol, by high-performance liquid chromatography with fluorescence detection.

The determination of N-(deacetyl-O-4-vinblastoyl-23)-L-tryptophan (vintriptol acid, VtrpA), a metabolite of the investigational semi-synthetic vinca alkaloid vintriptol [N-(deacetyl-O-4-vinblastoyl-23)-L-ethyltryptophan, VtrpE], in plasma and urine samples is described. Sample pretreatment included liquid-liquid extraction of the buffered (pH 5.0) biological samples with chloroform-2-propanol (95:5, v/v). The analyses were performed by ion-exchange high-performance liquid chromatography on normal-phase silica with fluorescence detection. The assay was applied to the analysis of samples from cancer patients who had been treated with VtrpE in a phase I clinical study. VtrpA was found to be a principal metabolite of VtrpE with up to 1.2% of the administered dose excreted in the urine.

Antineoplastic Agents↗

PubMed1994

Simultaneous determination of tryptophan, 5-hydroxytryptophan, 5-hydroxytryptamine, 5-hydroxyindoleacetic acid, 4-hydroxy-3-methoxyphenylacetic acid and 3-methoxy-4-hydroxyphenylglycol in human cerebrospinal fluid.

Serotonin (5-hydroxytryptamine) metabolism may be influenced by its precursor tryptophan. A method utilizing reversed-phase high-performance liquid chromatography and electrochemical and ultraviolet detection with a mobile phase composed of acetate buffer and methanol has been developed for determination of tryptophan, its metabolites 5-hydroxytryptophan, serotonin, 5-hydroxyindoleacetic acid, as well as 4-hydroxy-3-methoxyphenylacetic acid (homovanillic acid) and 3-methoxy-4-hydroxyphenylglycol in human cerebrospinal fluid (CSF). The electrochemical potential is set at 0.6 V in order to reduce the background current. Since tryptophan is not electroactive at this potential, it is detected by ultraviolet absorbance. The present method is simple, rapid, specific and accurate as compared with a previously reported method. No sample pretreatment is necessary and it takes ca. 20 min to run a sample. The concentrations of the compounds measured in CSF are similar to those obtained by HPLC in previous reports, although there are still arguments about the true level of serotonin in CSF.

5-Hydroxytryptophan↗

PubMed2000

Determination of soluble anions and cations from waters of pulp and paper mills with on-line coupled capillary electrophoresis.

When the degree of closure of the paper machine wet end waters increases, wet end problems also become more difficult to control without specific and selective on-line measurements. The need to measure the concentrations of individual compounds in order to explain wet end phenomena is growing. This study was performed to set up a CE system to a paper machine and to determine soluble inorganic and organic ions in different locations of pulp and paper process waters with real time analyses by two on-line CE methods. A reconstructed commercial CE apparatus was connected to a papermaking machine via an apparatus, which was a combined sampling and sample pretreatment instrument, the role of which was to filter and dilute the samples before on-line determination by CE. The on-line procedures were optimized for simultaneous determination of anions as chloride, sulfate, oxalate, formate and acetate and for determination of cations as potassium, calcium, sodium, magnesium and traces of aluminium. The quantification was performed with external standard methods using the programs available in the commercial CE instrument. The concentrations of the ions were transferred by using a computerized transfer algorithm exporting the results from the analysis instrument to the process control unit. The developed on-line procedures were tested three times in paper and paperboard mills for 1 month at a time. Correlations were observed between the CE results and changes in the processes.

Anions↗

PubMedDate not supplied

Screening of clostebol and its metabolites in bovine urine with ELISA and comparison with GC-MS results in an interlaboratory study.

An extraction procedure for clostebol metabolites in urine is developed including enzymatic hydrolysis of conjugated metabolites with Helix pomatia juice (SHP) and solid-phase extraction (SPE) with further cleanup of sample extracts. For the enzymatic deconjugation step, variables such as buffer pH, amount of enzyme, incubation time, and temperature are examined. For the SPE step, different wash solutions and combinations with subsequent liquid-liquid extractions are examined. Incurred bovine urine samples, obtained through oral and intramuscular administration of clostebol acetate to animals, are used to test the performance of the developed method. In addition to the optimization of the sample pretreatment procedure, an interlaboratory study for the analysis of the incurred urine samples with ELISA and GC-MS is performed and good agreements are observed.

Administration, Oral↗

PubMed1998

Interference from digitoxin-like immunoreactive factors reduced in a new monoclonal chemiluminescent digitoxin assay.

Endogenous digoxin-like immunoreactive factors (DLIF) can interfere with some digoxin immunoassays. We looked for similar interference, called digitoxin-like immunoreactive factors (DTLIF) in two digitoxin immunoassays: A new chemiluminescent assay (CLIA), processed on the automated random access immunoassay system ACS:180, and a fluorescent polarization assay (FPIA), processed on the semiautomated TDx batch analyzer. One hundred thirty-seven samples of sera were tested from nondigitalized pregnant women, patients with liver or kidney diseases, and cord blood. The CLIA digitoxin assay uses a murine monoclonal antibody and requires no sample pretreatment; the FPIA digitoxin assay uses a polyclonal rabbit antibody and requires sample precipitation. Both assays have a similar dynamic range and sensitivity and give comparable results with commercial controls and external quality control survey samples. Although the CLIA detected no digitoxin in any sample tested, the FPIA showed apparent digitoxin concentrations of more than 2.0 ng/ml for 100% and 44% among cord blood and liver disease specimens, respectively. The highest DTLIF concentration was found in serum from a patient with liver disease (18.1 ng/ml). When spiked with 32 ng/ml digitoxin, six of the samples containing DTLIF generated FPIA digitoxin values of 6% to 27.5% more than the expected digitoxin levels. Two specimens with no detectable DTLIF activity were run as controls, and when spiked with digitoxin, showed target digitoxin concentrations in the FPIA. The CLIA recovered near the target digitoxin values (32 ng/ml) in all spiked samples. It was concluded that the polyclonal FPIA digitoxin assay may give discordant digitoxin concentrations in some patient groups because of interference from digitoxin-like immunoreactive factors. The CLIA digitoxin assay is not affected by DTLIF interference.

Antibodies, Monoclonal↗

PubMed2005

Validation of nitrite and nitrate measurements in exhaled breath condensate.

BACKGROUND: Inflammatory markers in exhaled breath condensate (EBC) are investigated as a non-invasive approach to monitoring of inflammation in the respiratory tract. EBC concentrations of nitrite and nitrate, the stable end products of oxidative metabolism of nitric oxide, are increased in patients with asthma, especially during acute exacerbations. OBJECTIVES: To examine methodological aspects of nitrite and nitrate measurements in EBC such as sample collection, storage and analysis. METHODS: In a randomized study, EBC was collected twice within 1 h (with and without a nose clip) in 20 healthy adults and 20 patients with well-controlled asthma and no symptoms of allergic rhinitis. Nitrite and nitrate were assayed by ionex chromatography and fluorimetrically after derivatization with diaminonaphthalene. RESULTS: The geometric mean [exp (mean +/- SD)] EBC levels of nitrite and nitrate in healthy subjects [4.3 (3.0-6.1) and 11.0 (5.3-22.7) micromol/l] and patients [4.6 (2.6-7.3) and 8.7 (3.2-23.8) micromol/l] did not differ (p = 0.13). Wearing a nose clip (p = 0.3) did not influence nitrite and nitrate concentrations. The mean intra-subject %CVs of EBC concentrations of nitrite were 26 and 21% in healthy subjects and patients, while those of nitrate achieved 49 and 88%, respectively. CONCLUSIONS: Ionex chromatography of nitrite and nitrate requires no sample pretreatment and provides comparable results as a more laborious diaminonaphthalene method. EBC samples should be kept cold (8 degrees C) and analyzed for nitrite and nitrate within 24 h of collection or stored in the freezer and thawed preferably only once. Wearing a nose clip during EBC collection has no influence on nitrite and nitrate concentrations. Short-term repeatability of nitrite and nitrate measurements was worse compared to published data on exhaled nitric oxide.

Adult↗

PubMed1996

Chromium, copper, iron, manganese, selenium and zinc levels in dairy products: in vitro study of absorbable fractions.

Because milk and dairy products are some of the most widespread foods in the human diet, they contribute a large fraction of mineral intake. We determined levels of chromium, copper, iron, manganese, selenium and zinc in 60 samples of 10 widely consumed dairy products. Graphite furnace atomic absorption spectrometry was used to analyze samples processed with a slurry procedure to minimize sample pretreatment. The accuracy and precision of our method were verified. In analyzed samples, mean values ranged from not detectable to 0.950 microgram/g for Cr, from 0.020 to 2.800 micrograms/g for Cu, from 0.750 to 20.0 micrograms/g for Fe, from 0.010 to 0.900 microgram/g for Mn, from not detectable to 0.140 microgram/g for Se, and from 0.250 to 4.500 micrograms/g for Zn. The highest levels of Cr, Cu, Fe, Mn and Zn were detected in children's milk. Increased concentrations of Cr, Cu, Fe and Mn were detected in products packaged in glazed ceramic containers. We also studied the absorbable fractions of these elements using in vitro techniques which simulate human gastric and intestinal digestion.

Absorption↗

PubMed1987

Erythrocytic glucose-6-phosphate dehydrogenase measured by a differential pH technique.

We propose a new quantitative electrochemical method for determining glucose-6-phosphate dehydrogenase (EC 1.1.1.49) activity in purified erythrocytes or in whole blood, based on measurement of the pH change caused by oxidation of glucose 6-phosphate to 6-phosphogluconic acid, with simultaneous reduction of NADP+ to NADPH + H+. No sample pretreatment (e.g., preparation of hemolysate) is needed, and the automatic correction for sample blanks obviates interference from 6-phosphogluconate dehydrogenase (EC 1.1.1.44). The method is simple and fast, and the standard curve is linear to at least 2200 U/L at 37 degrees C. The within-day CV was 3.9% for activities in healthy individuals (mean value 1204 U/L), and 10% for deficient ones (classified as belonging to class II, mean value 407 U/L). Results (y) correlated well with those obtained with the WHO-recommended method (x): y = 1.13x + 0.02 (r = 0.971) for purified erythrocytes. Normal reference intervals are reported for the enzyme in purified erythrocytes and in whole blood.

Erythrocytes↗

PubMed2004

Determination of MS-275, a novel histone deacetylase inhibitor, in human plasma by liquid chromatography-electrospray mass spectrometry.

A rapid method was developed for the quantitative determination of the novel histone deacetylase inhibitor, MS-275, in human plasma. Calibration curves were constructed in the range of 1-100 ng/ml, and were analyzed using a weight factor proportional to the nominal concentration. Sample pretreatment involved a one-step protein precipitation with acetonitrile of 0.1 ml samples. The analysis was performed on a column (75 mm x 4.6 mm i.d.) packed with 3.5 microm Phenyl-SB material, using methanol-10mM ammonium formate (55:45 (v/v)) as the mobile phase. The column effluent was monitored by mass spectrometry with positive electrospray ionization. The values for precision and accuracy were always < or =5.58 and <11.4% relative error, respectively. The method was successfully applied to examine the pharmacokinetics of MS-275 in a cancer patient.

Administration, Oral↗

PubMed2005

Determination of the heat shock protein 90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin in plasma by liquid chromatography-electrospray mass spectrometry.

A rapid method was developed for the quantitative determination of the novel heat shock protein 90 inhibitor, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG; NSC707545), in human plasma. Calibration curves were constructed, and were analyzed using a weight factor proportional to the nominal concentration. Sample pretreatment involved a one-step extraction with ethyl acetate of 0.5-ml samples. The analysis was performed in the range of 1-100 ng/ml on a column (75 mm x 2.1 mm internal diameter with 3.5 microm C18 particle size), using 55% methanol in water containing formic acid as the mobile phase. The column effluent was monitored by mass spectrometry with positive electrospray ionization. The values for precision and accuracy were always <8% and <10% relative error, respectively. The method was successfully applied to examine the pharmacokinetics of 17-DMAG in a cancer patient.

Benzoquinones↗

PubMed2000

Numerical chromosomal changes in metastatic prostate cancer following anti-androgen therapy: fluorescence in situ hybridization analysis of 5 Japanese cases.

We used fluorescence in situ hybridization with centromere-specific probes for chromosomes 7, 8, 10, and Y to determine the copy number of these chromosomes in metastatic prostate cancers of five Japanese cases. Freshly prepared samples were obtained from prostate needle biopsies at different phases of clinical treatment; pretreatment, 1 week, 4 weeks, 12 weeks, 24 weeks post-treatment (PT), and clinical relapse. Gain of chromosomes 7 and 8, as noted in pretreatment samples; however, in post-treatment specimens (four of five cases), a remarkable reduction in the number of cells with extra copies of these chromosomes was detected. This decrease in the number of cells with additional chromosome 7 and 8 signals was correlated with the clinicohistopathological findings until 4 weeks PT. Chromosomes Y and 10 did not show numerical aberrations before treatment or changes in cells with aneusomy after treatment in all five cases. Our results suggest that gains of chromosomes 7 and 8 correlate with high grade and stage, and that changes in the cell number with aneusomy of chromosomes 7 and 8 reflect the clinical effects of anti-androgen therapy at an early phase, which may also indicate the androgen dependency of prostate cancer cells.

Aged↗

PubMed2002

Determination of ciprofloxacin, enrofloxacin and flumequine in pig plasma samples by capillary isotachophoresis--capillary zone electrophoresis.

Quinolones are a group of synthetic antibiotics that are widely used in veterinary medicine. Their residues may remain in tissues, milk, etc. intended for human consumption. The European Union fixes the maximum residue limits (MRLs) of veterinary medicinal products in foodstuffs of animal origin. Analytical methods are therefore needed to determine them in biological samples. In this study, we describe capillary isotachophoresis-capillary zone electrophoresis (ITP-CZE) to analyze three quinolones, enrofloxacin (ENR), ciprofloxacin (CPR) and flumequine (FLU), in pig plasma samples. We used solid-phase extraction with Oasis HLB cartridges as a sample pretreatment clean-up step. Capillary zone electrophoresis (CZE) requires low amounts of sample and is not as sensitive as one would wish. ITP-CZE is an easy way to increase the sample loadability and sensitivity. With this system sensitivity increases 40-fold. The detection limits for CPR, ENR and FLU were 70, 85 and 50 microg l(-1), respectively, which were lower than their MRLs in different kinds of samples. This method is simple and sensitive, and is therefore an alternative tool to the existing HPLC methods for analyzing the residuals of these quinolones in biological samples.

Animals↗

PubMed2001

Measurement of pyruvate in blood by high-performance liquid chromatography with fluorescence detection.

Accurate measurement of pyruvate is particularly important in the detection of mitochondrial enzyme disorders. describe a simple high-performance liquid chromatography assay for pyruvate with derivatization and fluorescence detection. Between-run variation is below 4.5% and the method is linear between I and 1,200 micromol/L. The mean recovery is 101%. After sample pretreatment, the calculated pyruvate concentration proved to be stable for 15 days when samples were kept at -20 degrees C.

Calibration↗