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PubMed2006

An intercalibration study on organobromine compounds: Results on polybrominated diphenylethers and related dioxin-like compounds.

An intercalibration study on organobromine compounds has been conducted to evaluate the accuracy and reliability in the analysis of brominated flame retardants (BFRs) such as polybrominated diphenyl ethers (PBDEs) and their related compounds like polybrominated and monobromo-polychlorinated dibenzodioxins and -furans (PBDDs/DFs, MoBPCDDs/DFs). This paper reports the results for these compounds in 'Mixed Standard Solutions' and 'Air-Dried Sediment'. The relative standard deviations (RSDs) for PBDEs, PBDDs/DFs and MoBPCDDs/DFs in mixed standard solutions ranged from 9% to 24%, 4% to 20% and 8% to 27%, respectively. The results of this study are better than those reported in earlier international interlaboratory studies due to general improvement in analytical methods and an increasing number of available authentic standards, particularly for PBDEs. In the analysis for higher brominated compounds such as octabromodibenzodioxin, the participants were advised to optimize the calibration curves befitting the range of concentrations found in samples because variation of relative response factors was noted. The results for 'Air-Dried Sediment' were also reasonable with RSDs from 10% to 38% for PBDEs, 8% to 38% for PBDDs/DFs and 17% to 36% for MoBPCDDs/DFs. In the sediment sample, possible errors in the sample pretreatment and/or interference by other compounds/impurities were indicated. The concentrations of pentabromodiphenylether, BDE100, varied more than the other PBDE isomers due to its poor separation in some chromatographic conditions. In addition, interference by PBDEs was observed in the analysis for PBDFs. Potential degradation/secondary formulation of PBDEs and PBDFs during the Soxhlet extraction were suggested when copper powder was added into the sediment samples. Inspite of these observations, the results in this study are better than those reported in other interlaboratory studies due to the advice given to participants for improving the results. Compared with the results for PCDDs/DFs (5-23% RSDs) and Co-PCBs (6-24% RSDs), the RSDs for most of the organobromine compounds were high, indicating an immature QA/QC approach for the analysis of PBDEs and related compounds in comparison to common chlorinated dioxins.

Calibration↗
PubMed2005

Solving liquid chromatography mass spectrometry coelution problems in the analysis of environmental samples by multivariate curve resolution.

Multivariate curve resolution-alternating least squares (MCR-ALS) is shown to be a powerful tool to resolve coelution problems in liquid chromatograpy-mass spectrometry (LC-MS) in scan mode. This investigation was performed using two types of LC columns, one traditional LC column of 25 cm length with a slow gradient and a shorter LC column of 7.5 cm with a rapid gradient which allowed much faster analysis and save of reagents and solvents. Mixtures of multiple biocide compounds were simultaneously analyzed in standard mixtures and in environmental samples (sediment and wastewater samples) with little sample pretreatment. Using the more traditional LC 25 cm column, all biocide compounds were properly resolved by MCR-ALS and quantitatively analyzed with estimated errors always below 20%. When fast chromatography (LC column of 7.5 cm) was used, MCR-ALS resolution of the more strongly coeluted compounds was also achieved but limitations were found in their simultaneous quantitative determination, specially for environmental samples.

Acetamides↗
PubMed1994

Rapid determination of the antimycotic drug flucytosine in human serum by micellar electrokinetic capillary chromatography with direct sample injection.

The rapid determination of flucytosine (5-FC) in human serum by micellar electrokinetic capillary chromatography (MECC) with direct sample injection is discussed. Minute (nanoliter) quantities of patient sera are applied to the beginning of a fused silica capillary filled with a phosphate/borate buffer (pH 9.2) containing 75 mM sodium dodecyl sulfate. Upon application of an electric field along the capillary, endogenous and drug substances are transported toward the cathode and separated into distinct zones that are detected by on-column ultraviolet absorption. Depending on the capillary and instrument used, 5-FC is shown to elute within 2-3.5 min of current application and free of endogenous interferences. 5-FC becomes also well separated from another often coadministered antimycotic drug, amphotericin B. Thus, quantitation of 5-FC is accomplished without any sample pretreatment. MECC data of 60 patient sera produced on two different automated instruments and 5-FC serum levels obtained by an agar-based bioassay are shown to agree well. For MECC, intraday and interday reproducibility data (80 micrograms/ml level) are shown to be < or = 4.5 and < 7%, respectively. Due to short run times and short capillary equilibration time intervals between runs, a sample throughput of 15/h is feasible. Thus, this technology is suitable for rapid determination of 5-FC serum levels, the time requirement for running a complete calibration, two control sera, and several patient samples being approximately 1 h only.

Calibration↗
PubMedDate not supplied

Measures to overcome HAMA interferences in immunoassays.

The incidence of human anti-mouse antibodies (HAMA) in different patient sera panels may be debatable, the interference of HAMA, if present, in immunochemically based assays is, however, a fact. This interference can lead to falsely elevated or depressed results depending on the nature of the HAMAs involved and the particular assay format chosen. For several reasons, assays for serum tumor markers may be especially prone to HAMA interference. Consequently, in the development of such assays, special attention should be given to the HAMA issue. In our experience, the degree of HAMA interference varies greatly from one assay to another. Nevertheless, the HAMA issue has to be addressed. Several methods have been described to remove HAMA (and other interfering substances) via sample pretreatment. Alternatively, there are also some options to counterbalance the potential effect of HAMA by using assay reagents optimized for minimal HAMA susceptibility, e.g. inclusion of an excess of non-relevant mouse antibodies. However, there is no guarantee that any given assay will not be affected by HAMA. This is especially true if a portion of the HAMA in a patient's sample is comprised of anti-idiotypic "internal image" antibodies.

Animals↗
PubMed1995

High-performance liquid chromatographic determination of the antifungal drug fluconazole in plasma and saliva of human immunodeficiency virus-infected patients.

A high-performance liquid chromatographic (HPLC) assay has been developed for the determination of the antifungal drug fluconazole in saliva and plasma of patients infected with the human immunodeficiency virus (HIV). Samples can be heated at 60 degrees C for 30 min to inactivate the virus without loss of the analyte. The sample pretreatment involves a liquid-liquid extraction with chloroform-1-propanol (4:1, v/v). The chromatographic analysis is performed on a Lichrosorb RP-18 (5 microns) column by isocratic elution with a mobile phase of 0.01 M acetate buffer (pH 5.0)-methanol (70:30, v/v) and ultraviolet (UV) detection at 261 nm. The lower limit of is 100 ng/ml in plasma (using 500-microliters samples) and 1 microgram/ml in saliva (using 250-microliters samples) and the method is linear up to 100 micrograms/ml in plasma and saliva. At a concentration of 5 micrograms/ml the within-day and between-day precision in plasma are 7.1 and 5.7%, respectively. In saliva the within-day and between-day precision is 10.8% (at 5 micrograms/ml). The methodology is now being used in pharmacokinetic studies in HIV-infected patients in our hospital.

Calibration↗
PubMed1990

Pyrolytic methylation-gas chromatography of whole bacterial cells for rapid profiling of cellular Fatty acids.

A novel, on-line derivatization technique has been developed which enables generation of fatty acid methyl ester (FAME) profiles from microorganisms by gas chromatography-mass spectrometry without the need for laborious and time-consuming sample preparation. Microgram amounts of bacterial cells are directly applied to a thin ferromagnetic filament and covered with a single drop of methanolic solution of tetramethylammonium hydroxide. After air drying, the filament is inserted into a special gas chromatograph inlet equipped with a high-frequency coil, thus enabling rapid inductive heating of the ferromagnetic filament. This so-called Curie-point heating technique is shown to produce patterns of bacterial FAMEs which are qualitatively and quantitatively nearly identical to those obtained from extracts of methylated lipids prepared by conventional sample pretreatment methods. Relatively minor differences involve the loss of hydroxy-substituted fatty acids by the pyrolytic approach as well as strongly enhanced signals of FAMEs derived from mycolic acids. This type of pyrolysis enables on-line derivatization and thermal extraction of volatile derivatives for analysis, whereas the residual components remain on a disposable probe (ferromagnetic wire) of a pyrolytic device. The reduced sample size (micrograms instead of milligrams) and the lack of sample preparation requirements open up the possibility of rapid microbiological identification of single colonies (thus overcoming the need for time-consuming subculturing) as well as analysis of FAME profiles directly from complex environmental samples.

Journal Article↗
PubMed2002

Simultaneous quantitative analysis of sphingoid base 1-phosphates in biological samples by o-phthalaldehyde precolumn derivatization after dephosphorylation with alkaline phosphatase.

This paper describes a simultaneous analytical method for the measurement of sphingoid base 1-phosphates and sphingoid bases from a variety of biological samples. This method consists of two steps of sample pretreatment: the enzymatic dephosphorylation of sphingoid base 1-phosphates by alkaline phosphatase (APase) and the subsequent analysis of o-phthalaldehyde (OPA) derivatives of the liberated sphingoid bases by HPLC. By introducing C17-sphingosine 1-phosphate and C17-sphingosine as internal standards, not only phytosphingosine 1-phosphate, sphingosine 1-phosphate, and sphinganine 1-phosphate but also phytosphingosine, sphingosine, and sphinganine present in a sample could be quantified in 12 min on a C18 reversed-phase column with a simple mobile phase of acetonitrile:deionized distilled water (90:10, v/v). With this HPLC method, we could reproducibly analyze the levels of sphingoid base 1-phosphates over a broad range of concentrations from 0.5 to 100.0 pmol from various biological samples including serum, cultured cells, and rat tissue homogenates. The conversion of sphingoid base 1-phosphates into sphingoid bases increased the stability of the OPA adducts. Thus, this indirect measurement of sphingoid base 1-phosphates increased the sensitivity and reproducibility of the method. This HPLC method was also used to measure the changes in the levels of sphingoid base 1-phosphates in cultured cells after treatment with 1,25-(OH)2D3, a sphingosine kinase activator, or with fumonisin B1, a sphinganine N-acyltransferase inhibitor.

Alkaline Phosphatase↗
PubMed2001

Molecular epidemiology of malaria in Yaounde, Cameroon. VIII. Multiple Plasmodium falciparum infections in symptomatic patients.

The extent of genetically distinct parasite populations coinfecting individual human hosts (i.e., multiplicity) was studied by polymerase chain reaction amplification of 3 polymorphic genetic markers, circumsporozoite protein and merozoite surface antigens (MSA) 1 and 2, in symptomatic children and adults and analyzed in relation with age and initial parasitemia. Of the total of 177 DNA samples analyzed (of which 115 were paired pre- and posttreatment samples), 101 (57%) were composed of multiclonal infections, with up to 7 distinguishable parasite populations. Among the 3 polymorphic markers, msa-2 yielded the highest proportion of clinical isolates with multiclonal populations. Patients with multiclonal infections before treatment had, on average, 2.9 genetically distinct parasite populations. The extent of multiplicity decreased significantly (P < 0.05) in recrudescent parasites, but not with reinfections, as compared with the pretreatment samples. Neither age (5-60 years) nor initial parasitemia was correlated with multiplicity. Further studies in different epidemiological settings are required to understand the role of multiclonal Plasmodium falciparum infections in influencing malaria transmission.

Adolescent↗
PubMed1994

The effect of methylprednisolone on monocyte eicosanoid production in patients with multiple sclerosis.

The in vitro effect of methylprednisolone on prostaglandin E2 (PGE2), leukotriene B4 (LTB4) and thromboxane B2 (TXB2) synthesis by adherent monocytes was examined using samples of peripheral blood from 15 patients with multiple sclerosis and 18 normal controls. Eicosanoid production by monocytes was reduced in patients compared with controls and there was a dose-dependent inhibitory effect of methylprednisolone on eicosanoid production in both groups. In vitro production of PGE2 and TXB2 but not LTB4 was reduced in patients with multiple sclerosis following intravenous treatment with methylprednisolone compared with pretreatment samples. In a separate cohort of 20 patients with multiple sclerosis and 15 controls, the in vitro inhibition of PGE2 release by methylprednisolone was not associated with reduced pokeweed-mitogen-stimulated immunoglobulin G synthesis by peripheral blood mononuclear cells. These results suggest that methylprednisolone inhibits monocyte-macrophage function, but this effect is not specific to patients with multiple sclerosis.

Adult↗
PubMed1992

Determination of stavudine, a new antiretroviral agent, in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection.

A sensitive high-performance liquid chromatographic assay has been developed to determine the levels of a new antiretroviral agent, stavudine (2',3'-didehydro-3'-deoxythymidine, d4T), in human plasma. Didanosine (2',3'-dideoxyinosine, ddI) was used as the internal standard. The very selective sample pretreatment involved solid-phase extraction using silica gel columns. Chromatography was carried out on a mu Bondapak phenyl column, using a mobile phase of 0.005 M phosphate buffer (pH 6.8)-methanol (90:10, v/v) and ultraviolet detection at 265 nm. The method has been validated, and stability tests under various conditions have been performed. The detection limit is 10 ng/ml (using 500-microliters human plasma samples). The bioanalytical assay has been used in a single pharmacokinetic experiment in a rat to investigate the applicability of the method in vivo.

Animals↗
PubMed1979

Clindamycin-associated enterocolitis in guinea pigs: evidence for a bacterial toxin.

Experimental enterocolitis was induced in guinea pigs by intraperitoneal injection of clindamycin. Specimens of feces were collected daily in phosphate-buffered saline (pH 7.0) and pooled every second day. The pooled samples were centrifuged to remove solids, and the supernatant was sterilized by membrane filtration. The sterile fecal supernatants were then dialyzed for 48 h against two 15-liter changes of phosphate-buffered saline and subsequently tested for toxicity in cultured monolayers of mouse adrenal cells. A filterable toxin(s) was found in the fecal supernatants on days 2, 4, and 6 postchallenge and not in pretreatment samples. The toxin(s) caused enterocolitis when administered orogastrically to healthy animals and altered the morphology of cultured mouse adrenal cells. The alteration of adrenal cell morphology was neutralized by specific antitoxin to Clostridium histolyticum.

Adrenal Glands↗
PubMed2006

Determination of ethyl sulfate--a marker for recent ethanol consumption--in human urine by CE with indirect UV detection.

A CE method for the determination of the ethanol consumption marker ethyl sulfate (EtS) in human urine was developed. Analysis was performed in negative polarity mode with a background electrolyte composed of 15 mM maleic acid, 1 mM phthalic acid, and 0.05 mM cetyltrimethylammonium bromide (CTAB) at pH 2.5 and indirect UV detection at 220 nm (300 nm reference wavelength). This buffer system provided selective separation conditions for EtS and vinylsulfonic acid, employed as internal standard, from urine matrix components. Sample pretreatment of urine was minimized to a 1:5 dilution with water. The optimized CE method was validated in the range of 5-700 mg/L using seven lots of urine. Intra- and inter-day precision and accuracy values, determined at 5, 60, and 700 mg/L with each lot of urine, fulfilled the requirements according to common guidelines for bioanalytical method validation. The application to forensic urine samples collected at autopsies as well as a successful cross-validation with a LC-MS/MS-based method confirmed the overall validity and real-world suitability of the developed expeditious CE assay (sample throughput 130 per day).

Adult↗
PubMed2002

Determination of bromate in drinking water by zone electrophoresis-isotachophoresis on a column-coupling chip with conductivity detection.

The use of capillary zone electrophoresis (CZE) on-line coupled with isotachophoresis (ITP) sample pretreatment (ITP-CZE) on a poly(methylmethacrylate) chip, provided with two separation channels in the column-coupling (CC) arrangement and on-column conductivity detection sensors, to the determination of bromate in drinking water was investigated. Hydrodynamic and electroosmotic flows of the solution in the separation compartment of the chip were suppressed and electrophoresis was a dominant transport process in the ITP-CZE separations. A high sample load capacity, linked with the use of ITP in this combination, made possible loading of the samples by a 9.2 microL sample injection channel of the chip. In addition, bromate was concentrated by a factor of 10(3) or more in the ITP stage of the separation and, therefore, its transfer to the CZE stage characterized negligible injection dispersion. This, along with a favorable electric conductivity of the carrier electrolyte solution, contributed to a 20 nmol/L (2.5 ppb) limit of detection for bromate in the CZE stage. Sample cleanup, integrated into the ITP stage, effectively complemented such a detection sensitivity and bromate could be quantified in drinking water matrices when its concentration was 80 nmol/L (10 ppb) or slightly less while the concentrations of anionic macroconstituent (chloride, sulfate, nitrate) in the loaded sample corresponding to a 2 mmol/L (70 ppm) concentration of chloride were still tolerable. The samples containing macroconstituents at higher concentrations required appropriate dilutions and, consequently, bromate in these samples could be directly determined only at proportionally higher concentrations.

Bromates↗
PubMed2004

Quantitative analysis of the P-glycoprotein inhibitor Elacridar (GF120918) in human and dog plasma using liquid chromatography with tandem mass spectrometric detection.

A liquid chromatographic/tandem mass spectrometric (LC/MS/MS) method for the determination of the P-glycoprotein and breast cancer resistance protein inhibitor Elacridar in human and dog plasma is described. The internal standard was stable isotopically labelled Elacridar. Sample pretreatment involved liquid-liquid extraction with tert-butyl methyl ether. Analysis of Elacridar and internal standard was performed by reversed-phase LC on a basic stable minibore analytical column with an eluent consisting of acetonitrile and aqueous ammonia. An API-2000 triple-quadrupole mass spectrometer with an electrospray ion source was used in the positive-ion multiple reaction monitoring mode. The run time per sample was only 6 min. The method is sensitive and specific, with a dynamic range from 1 to 500 ng ml(-1) from 100 microl of human or dog plasma. The accuracy of the method was within 15% bias and the precision was lower than 15% for all tested concentration levels and in both matrices. The method is simple and the liquid-liquid extraction produces clean samples. This method was successfully applied to support the pharmacokinetics of a clinical trial in which orally applied Elacridar was used as a bioavailability enhancer.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Rapid quantification of delavirdine, a novel non-nucleoside reverse transcriptase inhibitor, in human plasma using isocratic reversed-phase high-performance liquid chromatography with fluorescence detection.

Delavirdine is a novel non-nucleoside reverse transcriptase inhibitor for the treatment of HIV-1-infected patients. A simple and rapid high-performance liquid chromatographic method for the quantification of delavirdine in human plasma suitable for drug monitoring in patients is described. Sample pretreatment consists of protein precipitation with acetonitrile and subsequent evaporation of the extract to concentrate the analyte. The drug is separated from endogenous compounds by isocratic reversed-phase, high-performance liquid chromatography coupled with fluorescence detection. The optimal excitation and emission wavelengths are 300 and 425 nm, respectively. The method has been validated over the range of 50-50 000 ng/ml using only 200 microl of plasma samples. The assay is linear over this concentration range as indicated by the F-test for lack of fit. Within- and between-day precisions are less than 4.4% for all quality control samples. The lower limit of quanititation is 50 ng/ml. Recovery of delavirdine from human plasma is 93.8%. Delavirdine is stable under various conditions, for example 1 h at 60 degrees C and one week at 4 degrees C. This validated assay is suited for use in pharmacokinetic studies with delavirdine and can readily be implemented in the setting of a hospital laboratory for the monitoring of delavirdine concentrations.

Anti-HIV Agents↗
PubMed2004

Derivative-differential UV spectrophotometry and compensation technique for the simultaneous determination of zidovudine and lamivudine in human serum.

Three new simple, precise, rapid and selective determination methods are described for zidovudine (ZID) and lamivudine (LAM) in human serum and in pharmaceutical formulations. The first method, based on the compensation technique is presented for the derivative spectrophotometric determination of binary mixtures with overlapping spectra. Using ratios of the derivative maxima or the derivative minimum, the exact compensation of either component in the binary mixture and human serum can be achieved, followed by its determination. The second method, differential derivative spectrophotometry, comprised of measurement of the difference absorptivities derivatized in the first order (deltaD1) of a tablet extract in 0.1 N NaOH relative to that of an equimolar solution in methanol at wavelengths of 246 nm and 263 nm, respectively. Neither sample pretreatment nor separation were required. The third method is based on HPLC on a reversed-phase column using a mobile phase of 0.01M sodium dihydrogen phosphate : methanol : acetonitrile (4 : 2 : 3 v/v/v), with detection at 285 nm. Repeatability and reproducibility studies for each compound showed no significant differences at 95% confidence level. The proposed methods were used for the simultaneous determination of the drugs in human serum samples and binary mixtures with good recoveries.

Adult↗
PubMed2005

Upstream sample processing facilitates PCR detection of Listeria monocytogenes in mayonnaise-based ready-to-eat (RTE) salads.

Sample pretreatment to reduce volume and concentrate cells of the target organism(s) prior to molecular detection offers a useful supplement or alternative to cultural enrichment. The purpose of this study was to develop an upstream processing method to facilitate the detection of Listeria monocytogenes in ready-to-eat (RTE) salads by PCR. Potato salad, a model RTE commodity, was seeded with L. monocytogenes and processed by two alternative upstream sample processing methods (designated one-step and two-step centrifugation), followed by DNA extraction, PCR amplification, and Southern hybridization. The two-step method resulted in 1,000-fold improvements in the PCR detection limit, from 10(6) Cfu/g (no sample processing) to 10(3) Cfu/g. The two-step method was applied for upstream sample processing of four representative deli salad items artificially inoculated with L. monocytogenes at levels ranging from 10(1)-10(6) Cfu/g. Following DNA extraction, PCR amplification, and Southern hybridization, detection was achieved at input levels of 10(5) Cfu/g for chicken salad, 10(4) Cfu/g for macaroni salad, and 10(3) Cfu/g for potato and seafood salads. The two-step method reported here facilitates the production of a final sample concentrate of reduced volume and improved purity which was compatible with PCR amplification. This approach offers further progress in our efforts to reduce or eliminate cultural enrichment in an effort to speed time to results when applying molecular methods to the detection of pathogens in foods.

Blotting, Southern↗
PubMed2005

Ultrasound-assisted analyte extraction for the determination of sulfate and elemental sulfur in zinc sulfide by different liquid chromatography techniques.

The speciation and determination of sulfate (SO4(2-)) and elemental sulfur (S degree) in zinc sulfide (ZnS) using ion-chromatography (IC) and reversed-phase liquid chromatography (RPLC) respectively is described. Three sample pretreatment approaches were employed with the aim of determining sulfate: (i) conventional water extraction of the analyte; (ii) solid-liquid aqueous extraction with an ultrasonic probe; and (iii) elimination of the zinc sulfide matrix via ion-exchange dissolution (IED). The separation of sulfate was carried out by an anion-exchange column (IonPac AS17), followed by suppressed conductivity detection. Elemental sulfur was extracted ultrasonically from the acid treated sample solution into chloroform and separated on a reversed phase HPLC column equipped with a diode array detector (DAD) at 264 nm. The achievable solid detection limits for sulfate and sulfur were 35 and 10 microg g(-1) respectively.

Journal Article↗