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PubMedDate not supplied

Identification of hydrolyzed inulin syrup and high-fructose corn syrup in apple juice by capillary gas chromatography: PVM 4:1999.

A peer-verified, gas chromatographic (GC) method is presented for the identification of hydrolyzed inulin syrup (HIS) and high-fructose corn syrup (HFCS) in apple juice. The procedure involves determining the Brix value of the apple juice or apple juice concentrate and preparing a dilution of the test sample to 5.5 degrees Brix. A 100 microL aliquot of the 5.5 degrees Brix test solution is then freeze-dried in a GC autosampler vial. The sugars in the freeze-dried residue are converted to trimethylsilyl derivatives, by the addition of an appropriate silylation reagent, and the vial is heated at 75 degrees C for 30 min. After derivatization, the solution is introduced into a gas chromatograph where the analytes are separated on a 30 m, 0.25 mm id DB-5 column. The method can use hydrogen, helium, or nitrogen as the carrier gas. The analytes and marker compounds are measured by use of a flame ionization detecone of the 2 syrups at 2 levels. Dilution was ascertained by the presence of retrograde sugar markers found in the 2 sugar syrups. All 3 laboratories involved in the study were able to identify the correct diluent in the blind, randomly coded, apple juice test portions. The levels of dilution in the test portions were 0, 6.9% (HIS), 16.0% (HIS), 8.1% (HFCS), and 17.0% (HFCS). No false positive results were reported. Quantitative conclusions can be drawn when the same syrup is used for dilution and as a reference standard.

Beverages↗
PubMed1999

Determination of ABT-089 in human plasma by high performance liquid chromatography using in situ precolumn derivatization with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole.

ABT-089 is a potent, selective neuronal cholinergic channel modulator with cognition enhancing activity in several animal paradigms. A simple and sensitive chromatographic method for the specific determination of ABT-089 in human plasma has been developed and validated. The method utilizes in situ precolumn fluorescence derivatization of the sample with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) prior to liquid-liquid extraction followed by reverse phase HPLC and fluorescence detection (lambda(ex) 495 nm, lambda(em) 533 nm). The described method significantly simplifies sample preparation. The derivatized product was separated from interference using a YMC ODS-AQ, 5 microm, 250x4.6 mm i.d. column using a mobile phase consisting of 30:5:65 (v:v:v) acetonitrile/methanol/aqueous buffer at a flow rate of 0.75 ml min(-1). The aqueous buffer consisted of 0.01 M tetramethylammonium perchlorate, 0.1% (v:v) trifluoroacetic acid, pH 3.0. An Alltech Absorbosphere CN, 5 microm, cartridge guard column was also used before the analytical column. Plasma samples were alkalinized with 0.1 M NaHCO3, 300 microl of a 1 mg ml(-1) ethanolic solution of NBD-F was added and the samples were heated in a water bath for 10 min at 50 degrees C. The samples were then extracted with tert-butylmethylether, evaporated to dryness and then reconstituted in mobile phase. For 1 ml of plasma, a limit of quantitation (LOQ) of 1.6 ng ml(-1) was obtained. The method was linear from 1.6 to 836 ng ml(-1). Inter and intra-day assay RSD (n = 6) were less than 9%. Accuracy determinations showed the quality control samples to range between 88-114% of the theoretical concentration.

4-Chloro-7-nitrobenzofurazan↗
PubMed1992

Spectrofluorimetric flow injection method for the individual and successive determination of L-cysteine and L-cystine in pharmaceutical and urine samples.

A flow injection configuration is proposed for the determination of L-cysteine and L-cystine individually and for mixtures of both analytes. The procedure is based on the rapid oxidation of L-cysteine by thallium(III) with concomitant formation of fluorescent thallium(I). The inclusion of a selecting valve and of a copper-coated cadmium column in the configuration allows the successive determination of two analytes. Linear calibration graphs were obtained between 5 x 10(-6) and 5 x 10(-5) mol dm-3 of L-cysteine and between 2 x 10(-6) and 2 x 10(-5) mol dm-3 of L-cystine. The applicability of the method to the determination of L-cysteine and L-cystine in pharmaceutical preparations was demonstrated by investigating the effect of potential interferents and by the analysis of commercial preparations. The method was successfully applied to the determination of L-cysteine and L-cystine in urine samples.

Cysteine↗
PubMed2002

Rapid determination of underivatized pyroglutamic acid, glutamic acid, glutamine and other relevant amino acids in fermentation media by LC-MS-MS.

Determination of amino acids in a complex matrix without derivatization is advantageous, however, difficulties are found in both the detection and the separation of those compounds. In this study, a rapid and reliable LC-MS-MS method for the quantitation of underivatized amino acids in exocellular media was established. Injections were made directly after centrifugation of the samples, without further preparation. The separation of seven underivatized amino acids was achieved on a reversed-phase C18 column with pentadecafluorooctanoic acid as a volatile ion-pair reagent, and the specific detection of most amino acids was achieved by MS-MS of the specific transitions [M + H]+-->[M + H - 46]+. The calibration curves of all analytes were linear over the range of 1.0-1000 microg ml(-1) and the detection limits ranged from 0.1 to 5 ng ml(-1), with an injection volume of 20 microl. The inter-day and intra-day precisions ranged from 2.6 to 5.7% and 4.8 to 8.2%, respectively; the mean recoveries of the seven analytes were 81-104%, 91-107% and 93-101% respectively at the spiked level of 10, 40 and 200 microg ml(-1). A large number of fermentation samples were analysed using this method. The technique is simple, rapid, selective and sensitive, and shows potential for the high-throughput quantitation of amino acids from other biological matrices.

Amino Acids↗
PubMed2001

Critical assessment of the performance of electronic moisture analyzers for small amounts of environmental samples and biological reference materials.

Two electronic moisture analyzers were critically evaluated with regard to their suitability for determining moisture in small amounts (< or = 200 mg) of various environmental matrices such as leaves, needles, soil, peat, sediments, and sewage sludge, as well as various biological reference materials. To this end, several homogeneous bulk materials were prepared which were subsequently employed for the development and optimization of all analytical procedures. The key features of the moisture analyzers included a halogen or ceramic heater and an integrated balance with a resolution of 0.1 mg, which is an essential prerequisite for obtaining precise results. Oven drying of the bulk materials in a conventional oven at 105 degrees C until constant mass served as reference method. A heating temperature of 65degrees C was found to provide accurate and precise results for almost all matrices investigated. To further improve the accuracy and precision, other critical parameters such as handling of sample pans, standby temperature, and measurement delay were optimized. Because of its ponderous heating behavior, the performance of the ceramic radiator was inferior to that of the halogen heater, which produced moisture results comparable to those obtained by oven drying. The developed drying procedures were successfully applied to the fast moisture analysis (1.4-6.3 min) of certified biological reference materials of similar provenance to the investigated the bulk materials. Moisture results for 200 mg aliquots ranged from 1.4 to 7.8% and good agreement was obtained between the recommended drying procedure for the reference materials and the electronic moisture analyzers with absolute uncertainties amounting to 0.1% and 0.2-0.3%, respectively.

Adsorption↗
PubMedDate not supplied

Proficiency testing as a tool to assess the performance of visual TLC quantitation estimates.

The use of rapid and inexpensive nonlaboratory-based screening tests for drug quality assessments is recommended as a component of a drug quality assurance program in poor resource settings. We have established routine Minilab test procedures to screen product quality and a proficiency testing program to determine the competency of the inspectors and reliability of results. Samples for the proficiency testing were prepared by pulverizing a standard reference tablet of the appropriate drug and making serial dilutions with starch to obtain concentrations of 0, 40, and 100%. The samples, which were labeled only with the drug name and an identifying letter, were given to inspectors for quality screening using Minilab procedures. In round 1 of the proficiency test, only 3 of 28 substandard samples were correctly identified. Round 2 of the proficiency test, which was administered after a performance qualification test for the analytical method, showed much improvement: 19 of 27 substandard drugs were correctly identified, while 5 out of 9 inspectors made the correct inference on the quality of 45 samples. However, in both rounds, 2 inspectors failed to identify substandard samples, indicating that their technical competencies need to be improved for the reliability of the results. Although the thin-layer chromatography screening methods provide a rapid means for drug quality assessment, they need to be put in the hands of competent users. The inclusion of a proficiency test in the screening program provides a measure of determining competency of the personnel and reliability of the results.

Chromatography, Thin Layer↗
PubMed2000

Comparison of extraction techniques for the recovery of veterinary drug residues from animal tissues.

Four different techniques for the extraction of veterinary drugs were compared. The use of a high speed mixer/emulsifier, an ultrasonic bath, a Stomacher and an end-over-end mixer were used to extract both incurred and fortified residues of chlortetracycline, sulphadiazine and flumequine from chicken muscle. For each drug, similar analytical recoveries from fortified muscle samples were obtained using each of the extraction techniques. However, for each analyte the highest drug concentration detected in incurred samples was obtained following preparation using a mixer/emulsifier extraction. Residue concentrations obtained using sonication, Stomacher or end-over-end mixer procedures were as low as 20% of those obtained using the mixer/emulsifier. This highlights the need to measure both incurred and spiked samples during method validation. A survey of published methods indicated that 75% of laboratories use some form of high-speed homogenization for the extraction of drug residues from tissue. However only 52% reported detection of incurred residues. More than two-thirds of methods that used other forms of extraction did not measure incurred residues. The use of such methods has implications for the statutory detection of veterinary drug residues.

4-Quinolones↗
PubMedDate not supplied

Rapid confirmation/quantitation of ecgonine methyl ester, benzoylecgonine, and cocaine in urine using on-line extraction coupled with fast HPLC and tandem mass spectrometry.

A rapid, rugged, and highly specific assay for the quantitation of cocaine (COC) and especially its primary metabolites benzoylecgonine (BZE) and ecgonine methyl ester (EME) in human urine has been established. Here, we investigated the use of on-line sample extraction coupled to rapid chromatography systems for tandem mass analysis of COC, EME, and BZE in human urine. Using this method, sample preparation consisted of a sole centrifugation step. Combined extraction and chromatographic run times were < 3.5 min. The lower limits of detection were 0.5 ng/mL, 2.0 ng/mL, and 0.5 ng/mL for EME, BZE, and COC, respectively. Linear calibration curves ranging from 7.5 ng/mL to 1000 ng/mL were produced for the test analytes. Within-day and between-day precision and accuracy of the assay were determined using human urine quality-control specimens at 5, 10, or 15; 150; and 1000 ng/mL. The analyses were performed over the course of five days, rendering %CVs < 10% for EME, BZE, and COC. Percent mean accuracy for the three analytes of 97 to 113% were obtained. Our data suggest that on-line sample extraction coupled with rapid high-performance liquid chromatography-tandem mass spectrometry may be a viable alternative for EME, BZE, and COC analyses in human urine.

Chromatography, High Pressure Liquid↗
PubMedDate not supplied

Evaluation of a preclinical blood test for scrapie in sheep using immunocapillary electrophoresis.

An analytical method is described for detection of endogenous disease-associated prion protein in the buffy coat fraction from the blood of sheep infected with scrapie. The method has been improved and evaluated for its performance in the preclinical diagnosis of ovine transmissible spongiform encephalopathies. The test system uses a protocol for sample preparation that includes extraction and concentration and a test method that uses a liquid-phase competitive immunoassay for prion protein. Antibodies directed to a peptide sequence at the C-terminus of the prion protein (PrP) and a fluorescein-labeled peptide conjugate are used in the assay. Free zone capillary electrophoresis with laser-induced fluorescence for detection is used to separate the antibody-bound fluorescently labeled peptide and free labeled peptide. In this assay, the PrP competes with the fluorescently labeled peptide for limited antibody binding sites, which results in a reduction of the peak representing the immunocomplex of the antibody bound to the fluorescently labeled peptide. When blood samples from scrapie-infected sheep aged 7-12 months and of the scrapie-susceptible PrP genotypes VRQ/VRQ and VRQ/ARQ were analyzed, the abnormal PrP was found in blood samples. These results correlated with the post-mortem diagnosis of scrapie. The sheep were preclinical and appeared normal at the time of testing but later died with clinical disease approximately 12 months after testing. In older animals, and those with clinical signs, a smaller percentage of animals tested positive. This study has demonstrated that this technology can be used as a sensitive, rapid preclinical test to detect the disease-associated PrP in the blood of scrapie-infected sheep. Improvements in the extraction protocol and capillary electrophoresis conditions will enhance the robustness of this test.

Animals↗
PubMed1990

[A rapid and simple approach for radioimmunochemical determination of aflatoxin B1 in cereals and feed using a commercial analysis kit].

In order to facilitate a practical large-scale introduction of the radioimmunochemical control of aflatoxin B1 contamination in cereals and feeds a new method of the preparation of sample extracts was verified. In comparison with the procedure used until now when water-nonmiscible chloroform must be evaporated from a chloroform extract and the solids must be dissolved in acetone, while using the tested method the sample is extracted by methyl alcohol or acetone and the extract is diluted by a buffer before the radioimmunoanalysis. This new method of sample extract preparation displays aflatoxin B1 recovery comparable to that of the older method (83-90% for concentrations of 2-20 micrograms per kg), the worse theoretical detection limit (1 microgram per kg vs. 0.2 microgram per kg) but it prevents the result distortion by interfering substances and it makes the whole analytical procedure simpler and expeditious. Radioimmunochemically aflatoxin was quantified by means of commercially produced analytical kits developed in one institute at Kosice.

Aflatoxin B1↗
PubMed1993

Herbal mixtures with claimed slimming activity: determination by TLC and HPLC of illegally added drugs.

A method for the detection and quantitation of several undeclared drugs in herbal preparations with slimming activity is proposed. Samples containing various anorexics, hypoglycemics and antidepressants were prepared by addition of the drugs to a synthetic mixture containing the most commonly used plant powders for those preparations. Each sample was subjected to a treatment that permitted, after a simple ethanolic extraction, the identification of the drugs by TLC using three different solvent systems. A further purification of the ethanolic solution through a polyamide column allowed for quantitative analysis of the drugs by a RP-HPLC method. The analytical recovery was good (88-97%); the calibration curves were linear over a wide range of drug concentrations (30-500 micrograms/ml) (r > 0.9995); the precision was high (CV% = 0.4-2.8) as well as the accuracy (96-102%).

Appetite Depressants↗
PubMed1998

[Review of bioanalytic methods in pharmacokinetics].

The analytical methods used in pharmacokinetic practice are reviewed. The system of special pharmacokinetic demands on bioanalytical methods for carrying out the different pharmacokinetic, metabolite kinetic, bioequivalence and food interaction studies is demonstrated in details (high sensitivity; the parallel measurability of the parent compound and its metabolites; species and biological matrices dependent methods; anticoagulation; sample preparation; derivatisation; special calibration system; method selectivity; reproducibility; validation; economicalness). The different immunological methods and separation techniques used in pharmacokinetic practice and the sensitivity of the detectors have been compared and characterized. Without the requirement of completeness, the author has tried to summarize the most important bioanalytical techniques in the view of general and specific feature; the possibilities of application, characteristics of operation and limitations.

Animals↗
PubMed1985

[Chemical analysis of veterinary drugs in food. 1. General methods and gas chromatographic procedures].

A review is presented that in the first section describes current techniques for preparation, extraction and clean up of food samples for the determination of veterinary drug residues by chemical methods. The second section gives an overview of gas chromatographic methods published up to 1983 for the analysis of meat, liver, kidney, egg and milk for residues of antibiotics and chemotherapeutic agents. Discussed are stability of the compounds, derivatization, detection and particular aspects of their metabolism. The structural formula is given for all drugs mentioned. The procedures for determination are summarized in a table, together with the most important analytical parameters.

Adrenergic beta-Antagonists↗
PubMed2003

[Study on preparation process and analytical methods of ESAC from Ganoderma lucidum].

OBJECTIVE: To develop the procedure for separating the ethanol-soluble and acidic components (ESAC) from Ganoderma lucidum, and to establish a method for quantifying ESAC in G. lucidum. METHOD: The ethanol extract of G. lucidum was extracted with a saturated NaHCO3 solution, acidified and re-extracted by chloroform to obtain ESAC. The quantitative analysis of ESAC was based on the characteristic color reaction between ESAC and H2SO4. RESULT: The optimal conditions for separating ESAC on a 10 g scale are as follows: ratio of material and ethanol (mL), 1:15; immersing time, 24 h; volume of saturated NaHCO3 and chloroform, 1300 mL; extract 3 times. The condition for measuring ESAC is as follows: sample weight, 1 g; solution volume, 1.5 mL; immuersing time, 0.5 h; detecting reagent, 50% H2SO4 in ethanol; heating time in 100 degrees C water bathe, 3 min; measuring wavelength, 490 nm. CONCLUSION: The procedure for ESAC preparation is simple and well-designed, and the established method for ESAC can be used for the qualitative analysis of the G. lucidum related products.

Drugs, Chinese Herbal↗
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↗
PubMed2000

The use of nuclear magnetic resonance spectroscopy in the detection of drug intoxication.

The use of nuclear magnetic resonance (NMR) spectroscopy as a method for drug analysis has the advantages of reduced pre-analytical preparation time and the potential to detect and quantitate drug conjugates and metabolites simultaneously. NMR was investigated as a method to screen for organic substances (and metabolites) in 25 patients who presented to the Emergency Department with clinical indications of a drug overdose. Urine specimens were examined by 1H NMR spectroscopy at 300 MHz and the results compared with gas chromatography-mass spectrometry (GC-MS) results. There was a 56% concordance (14 of 25 samples) between NMR and GC-MS. NMR identified acetaminophen, ibuprofen, aspirin, valproate, carbamazepine, and pseudoephedrine as parent compounds or metabolites. For a patient for whom GC-MS results were negative, NMR strongly suggested the presence of erythromycin. NMR was most successful in identifying analgesics and antiepileptic drugs (sensitivity 83-100%). In 10 patients, signals from 1,2-propanediol, a common vehicle for some pediatric medications, were observed by NMR spectroscopy. NMR had 0% sensitivity in identifying tricyclic antidepressants and antipsychotic drugs. In these samples, GC-MS detected a variety of compounds, including tricyclic antidepressants and their metabolites and chlorpromazine. In addition, other substances that had not been disclosed as having been ingested, such as caffeine, diphenhydramine, and nicotine, were detected by GC-MS. NMR spectroscopy represents an emerging supplementary analytical technique that is applicable to a wide range of possible intoxicants and to the evaluation of the intoxicated patient, particularly when larger amounts of the intoxicant (> 200 mg) are ingested.

Adolescent↗
PubMed1999

Chromatographic separation of carotenoids.

The carotenoids are extremely reactive and consequently unstable due to their long system of conjugated double bonds. Several precautions, such as protection against light and oxygen, use of low temperature and antioxidants, analysis in the shortest possible time, should be taken during isolation and chromatography. The food samples, preferably fresh, are homogenized and immediately extracted with a suitable organic solvent. Saponification has been employed in order to hydrolyze the carotenoid esters, remove fatty material and destroy chlorophyll. This optional step facilitates subsequent carotenoid separation, identification and quantification. The separation of carotenoids is usually carried out by column chromatography, thin layer chromatography and high performance liquid chromatography, in analytical or preparative scale, on many stationary phases such as silica-gel, alumina, MgO, Ca(OH)2 and reversed-phase material (C18 and C30). The choice of the most suitable chromatographic method depends on the amount of sample, carotenoid composition, resolution, speed and purity required. Examples of carotenoid separation in different stationary phases will be shown and discussed.

Carotenoids↗
PubMed2005

Fluorimetric liquid chromatographic analysis of amantadine in urine and pharmaceutical formulation.

A simple and sensitive liquid chromatographic method is described for the analysis of amantadine and memantine. The method is based on the derivatization of amantadine and memantine extracted from alkalified samples with (2-naphthoxy)acetyl chloride at mild conditions. The resulting derivatives were analyzed by isocratic HPLC with a fluorimetric detector (lambdaex, 227 nm; lambdaem, 348 nm). The linear range for the determination of amantadine or memantine spiked in urine (1.0 ml) was 1.0-10.0 nmol with a detection limit of about 0.2 nmol (S/N = 3; injected sample 20 microl). Only amantadine preparations are available on our local market, and application of the method to the analysis of amantadine in formulation and in the urine of a dosed subject was demonstrated and proved feasible. Quantitation of AT in tablets or capsules is capable in the linear range of 2.0-50.0 microM. Toluene was used as the solvent for extracting amantadine or memantine in samples and the resulting toluene extract was directly subjected to subsequent derivatization without solvent replacement leading to a simpler analytical procedure.

Amantadine↗