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PubMed2006

Combining desorption electrospray ionization mass spectrometry and nuclear magnetic resonance for differential metabolomics without sample preparation.

Desorption electrospray ionization mass spectrometry (DESI-MS) and nuclear magnetic resonance (NMR) spectroscopy are used to provide data on urine examined without sample preparation to allow differentiation between diseased (lung cancer) and healthy mice. Principal component analysis (PCA) is used to shortlist compounds with potential for biomarker screening which are responsible for significant differences between control urine samples and samples from diseased animals. Similar PCA score plots have been achieved by DESI-MS and NMR, using a subset of common detected metabolites. The common compounds detected by DESI and NMR have the same changes in sign of their concentrations thereby indicating the usefulness of corroborative analytical methods. The effects of different solvents and surfaces on the DESI mass spectra are also evaluated and optimized. Over 80 different metabolites were successfully identified by DESI-MS and tandem mass spectrometry experiments, with no prior sample preparation.

Animals↗
PubMed1998

Urinary organic acid screening by solid-phase microextraction of the methyl esters.

We developed a new sample preparation method for profiling organic acids in urine by GC or GC-MS. The method includes derivatisation of the organic acids directly in the aqueous urine using trimethyloxonium tetrafluoroborate as a methylating agent, extraction of the organic acid methyl esters from the urine by solid-phase microextraction, using a polyacrylate fiber with a thickness of 85 microm and transfer of the methyl esters into the GC or the GC-MS instrument. Desorption of the analytes takes place in the heated injection port. The proposed sample preparation is very simple. There is no need for any evaporation step and for the use of an organic solvent. The risk of contamination and the loss of analytes are minimized. The total sample preparation time prior to GC or GC-MS analysis is about 40 min, and therefore more rapid than other sample preparation procedures. The urinary organic acids are well separated by GC and 29 substances are identified by GC-MS.

Borates↗
PubMed2003

Determination of polydimethylsiloxanes by 1H-NMR in wine and edible oils.

Fourier transform (1)H-nuclear magnetic resonance (NMR) spectroscopy was suitable for the quantitative determination of polydimethylsiloxanes (PDMS) in wine and edible oil samples. This approach offers highly specific qualitative and quantitative analysis due to silicone-specific location of proton signals linked to carbon atoms located directly next to silicon atoms (0-0.5 ppm), as well as a different location of signals in the range for different organosilicon structures. The method can be used for the control of PDMS at regulatory limits in foodstuffs (10 mg kg(-1)) using hexamethyldisiloxane (HDMS) as an internal standard. Samples were prepared by extraction under suitable conditions to separate the analyte, and with analyte enrichment before (1)H-NMR analysis. Analytical procedures were developed to permit the determination of PDMS at 0.06 mg kg(-1) in wine and at 6 mg kg(-1) in edible oils samples using readily available NMR instrumentation. It was, however, possible to lower the limit of detection to 6 microg kg(-1) for wine and to 60 microg kg(-1) for edible oils using a higher field instrument (500 MHz). Relative standard deviations (S(r)) were obtained for wine (0.028) and for oil samples (0.043), which when compared with values obtained for samples spiked with PDMS (0.021) indicated that the sample preparation was the main factor determining the precision of the method. The average recovery rates for PDMS were 97 and 95% for wine and edible oils, respectively. PDMS was detected in four brands of Italian wine, with Chianti-Rafaello containing the highest concentration (0.35 mg kg(-1)), and in four types of edible oils, highest concentration (11.9 mg kg(-1)) being found in Italian corn oil. None of the levels of PDMS found in the food samples exceeded the permissible standards laid down by the Codex Alimentarius Commission (10 mg kg(-1)), with the exception of the one corn oil sample.

Dimethylpolysiloxanes↗
PubMed2000

Development of a method for rapid quantitation of amino acids by liquid chromatography-tandem mass spectrometry (LC-MSMS) in plasma.

A new analytical method has been developed and is proposed for the rapid determination of eighteen common amino acids, including tryptophan, in plasma and dried blood spots, by liquid chromatography coupled with ionspray tandem mass spectrometry. Potentially the method can include other amino acids and can be used for the diagnosis of metabolic disease. The use of the ionspray tandem mass spectrometry approach permits extremely rapid chromatographic separation of all amino acids requiring less than four minutes for the analysis of each sample, after a simple sample preparation procedure. The chromatographic separation of the analytes was achieved using a CN normal phase column and a water/acetonitrile/trifluoroacetic acid mobile phase at flow rate of 1 ml/min. The mass spectrometer was operated in multiple reaction monitoring mode, where each analyte had its own unique precursor and product ion setting. The quantitative analysis of amino acids was achieved using as internal standards just two representative isotopically labeled amino acids: D4-Ala and D5-Phe. Calibration is made externally by using aqueous solutions with the same labelled amino acids as internal standards. The high specificity of tandem mass spectrometry coupled with a fast chromatographic process is suitable for the rapid and reliable assay of metabolically significant amino acids. The liquid chromatography-tandem mass spectrometry method is more effective than other published tandem mass spectrometry methods at distinguishing isobaric amino acids like Leu, Ile and HO-Pro and certainly far more rapid than HPLC or ion-exchange chromatographic methods.

Amino Acids↗
PubMed2004

Determination of Abacavir in maternal plasma, amniotic fluid, fetal and placental tissues by a polarity switching liquid chromatography/tandem mass spectrometry method.

A rapid and efficient high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) method for the determination of concentrations of the carbocyclic nucleoside antiviral Abacavir in maternal rat plasma, amniotic fluid, placental and fetal tissue samples has been developed and validated. All tissue samples were homogenized in water prior to analysis and all samples were prepared by acetonitrile precipitation followed by dilution with HPLC-grade water. Separation of the analyte and internal standard from the matrices was achieved on a C(8) analytical column (2.1 x 150 mm, 5 microm particle size). The mobile phase consisted of 10 mM ammonium acetate/acetonitrile using a gradient method at a flow rate of 0.25 mL/min for all matrices. The method yields retention times of approximately 3.4 and 5.1 min for the internal standard (Azidouridine) and Abacavir, respectively. For all matrices the limit of detection was approximately 1 ng/ml. Recoveries from the different matrices ranged from 53-87% for Abacavir and from 69-84% for Azidouridine. Within- and between-run precision (%RSD) and accuracy (%Error) were under 15% for all matrices.

Amniotic Fluid↗
PubMed2006

[AAS method for determination of manganese in fluorspar].

An AAS method for the determination of manganese in fluorspar was established for the inspection of export product. The fluorspar sample was treated with HF, then fumed with HClO4. After cooling, the residue was dissolved by adding HCl and boric acid with gentle heating. The series of standards for calibration were prepared by adding the same amount of Ca for matrix matching. The linear range of the determination of manganese is 0.09-2.5 microg x mL(-1). The determination limit of the method for Mn in fluorspar was 0.001 0%. Analytical results of real samples with various levels of content by the proposed method were in good agreement with those by the ISO method of spectrophotometry. The method features good precision, easy operation, and convenience for application.

English Abstract↗
PubMed1985

Immunochemical methods for measurement of transferrin in serum: effects of analytical errors and inappropriate reference intervals on diagnostic utility.

We estimated analytical errors of the Calbiochem, Kallestad, Hyland, Meloy, Helena, and Beckman immunochemical methods for serum transferrin. Intermethod biases were determined by analysis of the "Reference Preparation for Serum Proteins" of the College of American Pathologists and by analysis of 106 patients' serum samples. We judged the acceptability of errors by comparing confidence limits for total errors with 1/4 of the normal reference intervals. The transferrin status of each patient's sample was interpreted by comparing the result of each method with the normal reference interval claimed by the corresponding manufacturer. We found that the combined effects of medically unacceptable analytical errors and inappropriate normal intervals caused results of the tested methods not to be interchangeable. The Calbiochem method identified 61 serum samples (57%) as having abnormally high transferrin concentrations. In contrast, for the same specimen, with the Meloy method we found abnormally high transferrin concentrations for only two samples (1.8%).

Analysis of Variance↗
PubMedDate not supplied

Direct determination of free methionine in soy-based infant formula.

A method was developed for the direct determination of free methionine in soy-based infant formula, with analyte separation and quantitation by reversed-phase liquid chromatography (LC), and UV absorbance at 214 nm, respectively. Sample preparation required only dilution with mobile phase and syringe filtration. Using a 0.02M KH2PO4 mobile phase (pH adjusted to 2.9 with 85% o-phosphoric acid) and 0.7 mL/min flow rate, methionine eluted at approximately 8 min, and total run time was 14 min after column regeneration with acetonitrile-water. System linearity was demonstrated as peak area versus analyte concentration, ranging from 80 to 120% of the formula specification for free methionine (r > 0.999, and all residuals < 0.45%). Intermediate precision relative standard deviation values were < 1.5% for ready-to-feed and reconstituted powder samples, and recoveries ranged from 98.0 to 103.5% for inter-method comparison with an amino acid analyzer method. The limit of quantitation was 3 mg methionine/L in the "as fed" infant formula. Despite the relatively weak UV absorptivity of methionine, the 214 nm signal was sufficiently intense in the 30-65 mg/L (201-436 microM) range to afford quantitation by peak area proportionation versus a 2-point external standard calibration. This direct UV detection after reversed-phase LC separation provides a simple and accurate method for determining free methionine without derivatization.

Chromatography, Liquid↗
PubMed2000

Determination of morphine 3-esters in rabbit plasma by high-performance liquid chromatography with ultraviolet detection.

A sensitive high-performance liquid chromatographic (HPLC) method for the quantitation of the morphine 3-esters 1[3-(2, 2-dimethylvaleroyl)-morphine (A), 3-(2-phenylbenzoyl)-morphine (B) and 3-(2,2-diphenylpropionyl)-morphine (C)] in rabbit plasma is described. Sample preparation was based on reversed-phase solid-phase extraction. The compounds were separated on C(18) reversed-phase analytical columns and then determined by ultraviolet detection. The recovery from plasma was 78.7 +/- 7.4%, 69.1 +/- 6.9% and 75 +/- 7.2% (mean +/- SD) for A, B, and C, respectively. The present method enabled the detection limit of 0.2, 0.2 and 0.1 ng and quantification limit of 20, 10 and 10 ng/ml for A, B and C, respectively. The developed method was used for determination of the plasmakinetics of these morphine 3-esters in rabbits.

Animals↗
PubMed1984

A comparison of methods for the estimation of the proportion of microbial nitrogen in duodenal digesta, and of correction for microbial contamination in nylon bags incubated in the rumen of sheep.

Four sheep, each fitted with cannulas in the rumen and proximal duodenum, were given two diets (1390 g dry matter (DM)/d) consisting of lucerne (Medicago sativa) pellets (24.2 g nitrogen/kg DM) plus pelleted reed canary grass (Phalaris arundinacea; 14.1 g N/kg DM) or chopped hay (11.8 g N/kg DM) at intervals of 2 h. Flow of duodenal digesta measured by reference to the markers 51Cr-EDTA and 103Ru-phenanthroline indicated a net gain of 5.8-7.5 g non-ammonia-N (NAN) between mouth and duodenum. The proportion of microbial N in duodenal digesta N was estimated using 15N and 35S incorporation into bacteria and digesta. Two methods of analysis for 35S content, the Bird & Fountain (1970; B&F method) and the Mathers & Miller (1980; M&M method), were used. (15NH4)2SO4 and Na2(35)SO4 were infused into the rumen for 3.5 d before and 4.0 d during sampling. A bacterial fraction was prepared from the fluid phases of sampled duodenal digesta and rumen contents by differential centrifugation. In addition, samples of ground canary grass and of lucerne were incubated in nylon bags in the rumen for 3-48 h during the infusion. Each of the 35S analytical methods yielded similar values of 35S content of isolated rumen or duodenal bacteria, but there was more (P less than 0.05) incorporation of 15N into rumen than into duodenal bacteria. Relative to values obtained using the M&M method and 15N incorporation, the B&F method for S analysis yielded higher (P less than 0.05) estimates of microbial content of duodenal digesta from sheep given chopped reed canary grass. 35S activity associated with washed nylon-bag residues increased rapidly with time-period of incubation and was substantially greater (P less than 0.05) when analysed by the B&F method compared with the M&M method. The 35S content (/g DM) of adherent bacteria removed from nylon-bag residues by homogenization in a second experiment varied from 0.65 to 1.88 that of free-living bacteria isolated from rumen fluid by differential centrifugation. The difference in 35S content in digesta and nylon-bag residues as measured using the two analytical methods was considered in relation to 35S-labelled extracellular material postulated to be produced by bacteria adherent to plant residues. Estimates of disappearance of dietary N from nylon bags after correction for microbial contamination indicated a disparity with estimates based on in vivo information.

Animal Feed↗
PubMed2000

Simultaneous determination of bosentan and its three major metabolites in various biological matrices and species using narrow bore liquid chromatography with ion spray tandem mass spectrometric detection.

An analytical method was developed for the determination of bosentan and its three main metabolites in various matrices and species with focus on robustness. The drug assay involved protein precipitation, followed by liquid-liquid extraction and column switching in combination with narrow bore HPLC-MS-MS. Deuterated analogues of the analytes were used as internal standards. The sample preparation procedure was optimised with respect to minimise the suppression effects from different matrices. The drug and its metabolites could be analysed in plasma, serum, bile, and liver samples from man, dog, and rat with a run cycle time of 10 min. The method used always calibration samples made up in human plasma, whereas quality control samples were prepared in human plasma as well as in the identical matrix as the unknown samples. Calibration graphs for the drug and for the metabolites were linear in the range from 1 or 2 to 2000 or 10,000 ng/ml using a sample volume of 0.25 ml. Mean inter-assay precision and accuracy were 3.0% and 98.4%, respectively. Two additional methods were derived from the main method for the analysis of plasma samples only with focus on reduced manual effort and instrumental run cycle time. The modified methods showed a mean inter-assay precision and accuracy of 5.0% and 99.9% for the method using column-switching, and 3.5% and 98.8% for the method using off-line SPE, respectively. All methods proved to be robust, sensitive, and selective during the analysis of several thousand samples.

Animals↗
PubMed2005

Determination of promethazine hydrochloride in pharmaceuticals by capillary isotachophoresis.

Capillary isotachophoresis (ITP) in cationic regime of the separation with conductometric detection has been used for the separation and determination of promethazine hydrochloride (PRO) in commercial mass-produced pharmaceutical preparations. Several electrolyte systems of different compositions and pH were examined and the optimized ITP electrolyte system consisted of 10 mmol/l of potassium acetate adjusted to pH 4.8 with acetic acid as the leading electrolyte with electroosmotic flow (EOF) suppressing additive, 0.2% (w/v) methylhydroxyethylcellulose (m-HEC), and 5 mmol/l of beta-alanine as the terminating electrolyte. The proposed electrophoretic method was successfully validated. It was convenient for the sensitive, simple, rapid, and highly reproducible assay of promethazine. The calibration graph relating the ITP zone length to concentration of the analyte was rectilinear in the range of 40-200 mg/l of the drug standard, with a coefficient of determination r(2)=0.9992. The relative standard deviation (RSD) was 1.12% (n=6) when determining 100 mg/l of PRO in standard sample. Good quantitation was obtained in short analysis time (a single analysis took 6 min). The recoveries of drug from samples were found to be 97.22% (tablets), 99.72% (injections), and 99.14% (syrup). The minimal sample pretreatment and low running cost make the proposed ITP method a good alternative to commonly used analytical methods.

Electrophoresis, Capillary↗
PubMed2005

Simultaneous determination of phenytoin and dextromethorphan in urine by solid-phase extraction and HPLC-DAD.

A rapid and simple high-performance liquid chromatographic method with photodiode array detection was developed for the separation and the simultaneous determination of phenytoin and dextromethorphan in human urine. Analysis was performed in less than 4.5 min in isocratic mode on a reversed-phase C18 column (5 microm; 150 x 4.6 mm) using a mobile phase composed of acetonitrile-buffer phosphate 0.01 M (60:40, v/v) adjusted to pH 6.0, at 1 mL/min flow rate and UV absorbance at 210 nm. The elution order of analytes was dextromethorphan (DXM), Internal Standard (IS), and phenytoin (PHT). Calibration curves were linear in the 7.5-25 microg/mL range for PHT and in the 10-30 microg/mL range for DXM. Spike recoveries for urine samples prepared at three spiking levels ranged from 97.8 to 102.3% for PHT and from 94.8 to 100.4% for DXM. The detection limit (LOD) values ranged from 0.08 microg/mL for PHT to 0.5 microg/mL for DXM. The quantitation limit (LOQ) values ranged from 0.3 microg/mL for PHT to 1.6 microg/mL for DXM. The sample preparation method involves a rapid and simple procedure based on solid-phase extraction using a C18 reversed-phase column. Validation of the optimised method was carried out according to the ICH guidelines. The method developed in this study allows the reliable simultaneous analysis of PHT and DXM, drugs that were never quantified together in previously reported analytical methods. The described method has the advantage of being rapid and easy and it could be applied in therapeutic monitoring of these drugs in human urine of epileptic patients.

Anticonvulsants↗
PubMed2004

Comparison of the analysis of corticosteroids using different techniques.

In this work we have optimized the analysis of 18 human corticosteroids, some endogenous (tetrahydrocortisol, tetrahydrocortisone, cortisol, and cortisone) and others synthetic (betamethasone, budesonide, cortisone acetate, desonide, dexamethasone, dexamethasone acetate, flunisolide, fluocinolone acetonide, halcinonide, methylprednisolone, prednisolone, prednisone, triamcinolone, and triamcinolone acetonide). Three analytical techniques were developed: ELISA, gas chromatography coupled with mass spectrometry (GC-MS), and liquid chromatography coupled with mass spectrometry (LC-MS). Several sample-preparation methods were optimized for each technique and enabled compounds of interest to be extracted from small urine samples (several mL). The results enabled us to assess the possibilities and the sensitivity of each technique for application to doping tests.

Adrenal Cortex Hormones↗
PubMed2000

Ceramide analysis utilizing gas chromatography-mass spectrometry.

Suitable analytical methods are a prerequisite of a detailed investigation of ceramides. Therefore, a new gas chromatograph-mass spectrometry method with electron impact ionization was developed. Samples have been prepared for gas chromatography by the formation of volatile trimethylsilyl derivatives. The method provides high separation efficiency, sensitivity and specificity. Mass spectra facilitate the structural characterization of each species, because certain fragments indicate the fatty acid as well as the sphingoid base moiety. In a 30-mm run even very similar ceramides are baseline separated. The method is compared to a recently published assay for liquid chromatography-mass spectrometry.

Animals↗
PubMed1999

Trace analysis of pesticides by gas chromatography.

The analysis of pesticides is relevant to both food quality and the environment. Many laboratories are occupied with the analysis of pesticides in food, water or soil. Capillary gas chromatography is the technique most widely used in pesticide analysis. In present laboratory practice it serves as a screening method for over 300 pesticides. In this review we describe the role of gas chromatography as an analytical tool in combination with currently used or recently developed sample preparation techniques.

Chromatography, Gas↗
PubMed1982

Gas chromatographic-mass spectrometric analysis of amine metabolites in urine.

An analytical method for the separation and identification of amine metabolites in human urine has been developed using a gas chromatograph-mass spectrometer-computer system. The sample preparation consists of organic solvent extraction, adsorption on a cation-exchange resin, evaporation, and acylation. N-3-Hydroxypropyl-1, 4-diaminobutane was identified for the first time in urine. Methylguanidine, guanidine, putrescine, cadaverine, p-tyramine, 3-methoxytyramine, spermidine, and spermine were detected in normal urine with good gas chromatographic separation.

Amines↗
PubMed2004

Chemometric determination of amiloride hydrochloride, atenolol, hydrochlorothiazide and timolol maleate in synthetic mixtures and pharmaceutical formulations.

Different chemometric methods such as classical least squares (CLS), principal components regression (PCR) and partial least squares with one dependent variable (PLS-1) applied on UV spectral data (0 D) and on their first derivatives (1 D) were evaluated for the simultaneous quantification of samples containing mixtures of amiloride hydrochloride, atenolol, hydrochlorothiazide and timolol maleate. Their performances were compared by means of ANOVA tests, which evidenced that 0 D-PCR, 0D-PLS-1, 1D-PCR, 1D-PLS-1, were reproducible and gave statistically similar results, while 0 D-CLS and 1D-CLS displayed higher variances than the former and failed to comply with the Levene's variance homogeneity test at different stages of the method comparison and validation process. The four statistically equivalent procedures were successfully applied to the analysis of synthetic samples with two to four analytes and to commercial tablet preparations containing amiloride hydrochloride and hydrochlorothiazide alone or in association with atenolol or timolol maleate.

Amiloride↗