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PubMed1994

Determination of thiamine and its phosphate esters in human erythrocytes by high-performance liquid chromatography with isocratic elution.

A high-performance liquid chromatographic method for the simultaneous determination of thiamine and its phosphate esters in human erythrocytes, using postcolumn derivatization, is presented. The sample preparation and the choice of the analytical column avoid the use of an elution gradient. The four thiamine compounds (thiamine and thiamine monophosphate, diphosphate and triphosphate) are eluted within less than 15 min with a detection limit of ca. 20 fmol. The reproducibility and accuracy of the assay are satisfactory. Normal physiological red blood cell concentrations of the four thiamine compounds are included.

Adult↗
PubMed2000

Analytical techniques for determining biotin.

Biotin is a vitamin of the B-complex, which plays an important biochemical role in every living cell. In the recent years, the interest in this vitamin has been rekindled, mainly due to its association with serious human disorders, such as the inherited syndrome multiple carboxylase deficiency, which can be successfully treated with biotin administration. Diagnosis of biotin deficiency as well as monitoring of biotin levels in biological fluids of patients receiving biotin treatment is crucial. Equally important is the determination of biotin levels in pharmaceutical preparations as well as in food and food supplement products, which constitute the main source of biotin in humans. Several analytical methods for measuring biotin in various samples, e.g. human fluids, pharmaceutical formulations, food material etc., have been reported in the literature. In this review, the most representative of these methods are presented, and their characteristics are evaluated.

Biotin↗
PubMed1975

Background documentation of evaluation of occupational exposure to airborne asbestos.

This review presents background information and literature documentation to supplement the "Recommended Procedures for Sampling and Counting Asbestos Fibers: Procedures for the Evaluation of Occupational Exposure to Airborne Asbestos" prepared by the joint ACGIH-AIHA Aerosol Hazards Evaluation Committee. It reviews the nature of the inhalation hazard associated with asbesots fibers, the sampling and analytic methods which have been used, and a rationale for the selection of the membrane filter sampling-optical phase microscope identification and assay methodology which is recommended.

Air Pollution↗
PubMed1994

Detection of Coxiella burnetii in cow's milk using the polymerase chain reaction (PCR).

A PCR approach (transposon PCR) with primers based on repetitive transposon-like sequences, which--depending on the isolate--were found at a minimum frequency of 19 on the C. burnetii genome, was established for the highly sensitive and specific detection of C. burnetii. This study describes the analytical detection of C. burnetii in milk, which requires a special preparation method prior to PCR. Because of the low level of C. burnetii particles in milk samples, template DNA was concentrated by a factor of 200, using cetyltrimethylammonium bromide as the precipitation reagent. Using this particular preparation method, even a single C. burnetii particle could be detected in 1 ml milk.

Animals↗
PubMed2005

Correction and reporting of potassium results in haemolysed samples.

BACKGROUND: Potassium is usually the most important analyte affected by in vitro haemolysis and the result obtained may falsely indicate or disguise a life-threatening abnormality and so give rise to inappropriate treatment. The purpose of the study was to provide a solution to the problem of reporting potassium on haemolysed samples, taking into account both clinical needs and analytical concerns (inter-individual and inter-sample variability). METHODS: Using a new procedure that mimics the collection process in an actual clinical setting, haemolysed samples were prepared from 41 volunteers with a range of inter-individual factors - haemoglobin 80-173 g/L, red blood cells 2.42-6.77 x 10(12)/L, leucocytes 3.0-306 x 10(9) /L and platelets 31-710 x 10(9)/L - in order to develop a more accurate correction equation using a haemolytic index (HI) corresponding to g Hb/L in plasma. RESULTS: The mean (range) potassium increase was 0.0036 mmol/L (0.0029-0.0053 mmol/L) per unit HI. The following equation was developed to estimate potassium increase per HI, in order to compensate approximately for potassium leakage in haemolysed samples: Corrected K+ = Measured K+ -(HI x 0.004). CONCLUSION: The balanced solution is this: instead of reporting the post-haemolysis corrected potassium result a qualitative comment is given, indicating the likely range of the potassium concentration. If the potassium result is in a critically low or high range, it is communicated promptly to the requesting clinician.

Blood Specimen Collection↗
PubMed2002

Structural identification and biological activity of 7-methyl-10,11-ethylenedioxy-20(S)-camptothecin, a photodegradant of lurtotecan.

An additional chromatographic peak was observed in plasma samples of patients receiving NX 211, a liposomal formulation of the topoisomerase I inhibitor lurtotecan. We have isolated and purified this product by sequential solid-phase extractions, and we report its structure and cytotoxicity relative to lurtotecan and related agents. Nuclear magnetic resonance data indicate that cleavage of the piperazino moiety occurred at the N-C bond of the B-ring, yielding 7-methyl-10,11-ethylenedioxy-20(S)-camptothecin (MEC). Tests of the growth inhibition potential of MEC in seven human tumor cell lines showed that the compound was approximately 2-18-fold more cytotoxic than lurtotecan, topotecan, and 7-ethyl-10-hydroxy-20(S)-camptothecin (SN-38). Subsequently, we found that MEC was the product of rapid photolysis of lurtotecan, with the rate of degradation inversely proportional to NX 211 concentrations, and greatly depends on light intensity. Furthermore, MEC concentrations were found to increase significantly in plasma samples exposed to laboratory light but not in blood. MEC was not produced from NX 211 in the presence of human liver microsomes, suggesting that it is not a product of cytochrome P-450 metabolism. Using a validated analytical method, trace levels of MEC were quantitated in blood samples of two patients. These observations confirm that the precautions for protection from light currently specified for preparation and administration of NX 211 dose solutions are critical. Procedures to minimize formation of MEC, by the use of amber vials for NX 211 and by preparation of dilutions immediately before clinical use in a fashion completely protected from light, are now being routinely implemented.

Adult↗
PubMedDate not supplied

[Urinary excretion of mutagens and cisplatin among the nursing staff at a medical oncology department exposed to cytostatic drugs].

Exposure to cytostatic drugs was assessed in a group of 9 nurses employed in a hospital cancer therapy department by measuring the post-shift levels of urinary mutagens and cis-platinum. A slight but significant increase in urinary mutagenic activity compared to 11 controls was observed in the non-smokers: the mean values of mutagenic activity on the Ta100 strain in the presence of both microsomal and deconjugating enzymes were 4418 +/- 1186 and 2468 +/- 1681 respectively. Conversely, the urinary platinum concentration was below the detection limit of the analytical method (10 micrograms/l) in all samples. The increased urinary mutagenic activity in the exposed group can probably be attributed to the absorption of cyclophosphamide either during preparation and administration of the drug, or due to accidental contact with contaminated biological fluids, in view of the fact that the level of mutagens in urine samples from cyclophosphamide-treated patients is extremely high (up to 319,478 revertants/g creatinine in the case we examined).

Antineoplastic Agents↗
PubMed1983

[A rapid determination of cholesterol in egg yolks using the Bio-La-Test].

A technique of direct one-stage determination of the total cholesterol content in egg yolk is proposed. Yolk samples are treated by dilution into a form suitable for a diagnostic application of the Bio-La-Test Cholesterol (manufacturer Lachema, n.e., Brno, CSSR), designed for the determination of cholesterol in blood serum. The procedure of applying the bio-assay was partly modified. Modification of the Bio-La-Test for egg yolk provides results with a methodical error of 1.5%. The time needed for sample preparation and the analysis itself does not exceed 40 minutes. It was found out by checking the proposed analytical procedure that in the egg yolk of the Hisex laying hybrid the content of cholesterol fluctuated from 1.14 to 1.41 weight percent during the laying period.

Animals↗
PubMed1981

Excretion of an aflatoxin-guanine adduct in the urine of aflatoxin B1-treated rats.

Administration of aflatoxin B1 (AFB1) to rats resulted in the urinary excretion of 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1. This is the major product formed by the interaction in vivo of AFB1 with rat liver nucleic acids. The adduct was isolated from urine by the combined use of preparative and analytical high-pressure liquid chromatography and was quantitated by measurement of absorbance at 365 nm. The method allowed reproducible quantitation of adduct in urine samples from rats treated with AFB1 by i.p. injection at levels as low as 0.125 mg/kg. Application of the method to urine samples from rats given injections of AFB1 (1 mg/kg) revealed the presence of a compound chromatographically identical to authentic 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1. Spectral and chemical analysis of microgram quantities of this compound provided strong evidence that this compound is identical to authentic adduct. Measurement of this adduct in the urine of rats given injections of different doses of AFB1 showed that excretion occurs in a dose-dependent manner. Comparison of the dose-response curve for adduct excretion with that previously observed for adduct formation in rat liver DNA in vivo revealed a high degree of qualitative similarity, with the levels of adduct excreted in urine representing 30 to 40% of the levels seen initially in liver DNA.

Aflatoxin B1↗
PubMedDate not supplied

Gas chromatographic/nitrogen-phosphorus detection method for determination of ethylene thiourea in finished drinking waters: collaborative study.

A joint U.S. Environmental Protection Agency (USEPA)-AOAC interlaboratory method validation study was conducted on USEPA National Pesticide Survey (NPS) Method 6, "Determination of Ethylene Thiourea (ETU) in Finished Drinking Water by Gas Chromatography with a Nitrogen-Phosphorus Detector." The purpose of the study was to determine and compare the mean recoveries and precision for determination of ETU in reagent water and finished drinking waters. The study design was based on Youden's nonreplicate plan for collaborative tests of analytical methods. The waters were spiked with ETU at 6 concentrations levels, prepared as 3 Youden pairs. In the method, the test water is extracted by passing the sample through an absorbent matrix type tube. ETU is recovered from the tube with methylene chloride, the extract is solvent-exchanged to ethyl acetate, and an aliquot of each extract is analyzed by gas chromatography using a nitrogen-phosphorus detector. Twelve laboratories participated in the study. Data were analyzed using a USEPA computer program, which measured recovery and precision for ETU and compared the performance of the method between the 2 water types. Over the concentration range tested, the mean percent recoveries of ETU were 82-92% in reagent water and 85-98% in finished drinking water. The range of the between-laboratory relative standard deviations (RSDR) for the 6 concentrations was 5-24% in reagent water, but was only 4-9% in finished drinking water. The range of the within-laboratory relative standard deviations (RSDr) was 6-14% for reagent water and 6-10% for finished drinking water. Results for the 2 water matrixes showed no statistically significant differences.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1998

Automated simultaneous quantification of the immunosuppressants 40-O-(2-hydroxyethyl) rapamycin and cyclosporine in blood with electrospray-mass spectrometric detection.

A new analytical method to quantify 40-O-(2-hydroxyethyl)rapamycin (SDZ RAD) and cyclosporine (Cs) simultaneously in blood is presented. The combination of an on-line solid-phase extraction step with an HPLC system coupled to an electrospray mass spectrometer gave excellent specificity, sensitivity, and reproducibility. Aliquots of deproteinized blood samples were injected into the HPLC system and extracted on-line, using a conventional C18 guard column. The extract was eluted from the guard column in the backflush mode and injected into the liquid chromatography-mass spectrometry system. The calibration functions for SDZ RAD and Cs extracted from blood with added analyte were linear from 0.15 to 30 microg/L (r2 = 0.999) and from 1.5 to 1000 microg/L (r2 = 0.999), respectively. The CVs of peak areas were 6.2% at 10 microg/L SDZ RAD (n = 6) and 6.2% at 100 microg/L Cs (n = 6). Recovery ranged from 84.3% to 102.3% for SDZ RAD and from 81.7% to 92.2% for Cs. The lower limit of detection for both drugs was 0.05 microg/L. A rate of four samples per hour was maintained during the consecutive analysis of SDZ RAD and Cs in >500 blood samples with one single extraction and analytical column. The method described is a powerful tool for the simultaneous determination of SDZ RAD and Cs in blood. It works without time-consuming sample preparation steps and with excellent reproducibility. Because of the detection performance of electrospray mass spectrometry, this system offers flexibility in the working range, which is essential for therapeutic drug monitoring under different conditions.

Chromatography, High Pressure Liquid↗
PubMedDate not supplied

Confirmation of marijuana, cocaine, morphine, codeine, amphetamine, methamphetamine, phencyclidine by GC/MS in urine following immunoassay screening.

Rapid, reliable, sensitive, qualitative, and quantitative methods using small urine volumes (0.2-0.5 mL) were developed primarily for confirmation of marijuana, cocaine, benzoylecgonine, ecgonine methyl ester, morphine, codeine, amphetamine, methamphetamine, and phencyclidine. Using capillary gas chromatography/mass spectrometry (GC/MS) and selected ion monitoring (SIM), mass spectra were obtained for each analyte. Samples were prepared by hydrolysis where applicable, organic solvent extraction, and derivatization where necessary. Confirmation was achieved by comparing abundance of major ions and retention time of the total ion current (TIC) of an analyte with those of the appropriate analytical standard. Quantitation was achieved and calibration curves derived by obtaining the molecular ion ratios of that analyte/internal standard (IS) over a concentration range of 10-300 ng/mL (0.16-4.0 ng total injected into GC/MS). The overall extraction efficiency for these analytes ranged from 53% to 96%. Statistically significant cut-off values (p less than 0.01) were obtained for each analyte. The slope, y-intercept, and coefficient of determination (r2) were calculated for each analyte. All of the GC/MS methods were extensively tested against urine samples determined positive or negative by immunoassay (IA) and are now used in our laboratory.

Amphetamine↗
PubMedDate not supplied

Synthesis, identification, and acute toxicity of alpha-benzylphenethylamine and alpha-benzyl-N-methylphenethylamine. Contaminants in clandestine preparation of amphetamine and methamphetamine.

Amphetamine samples obtained from clandestine laboratories often contain other by-product amines. The most common of these are the alpha-benzylphenethylamine derivatives. This article reports the independent synthesis of reference samples of these amines, spectrophotometric and chromatographic methods for their identification, as well as acute toxicological studies. The utility of the analytical methods is demonstrated by the analysis of several amphetamine samples containing these alpha-benzylphenethylamines.

Amphetamine↗
PubMed2002

Effect of analytical error on the assessment of cardiac risk by the high-sensitivity C-reactive protein and lipid screening model.

BACKGROUND: Several prospective epidemiologic studies have demonstrated that high-sensitivity C-reactive protein (hsCRP) is an effective serum marker for cardiac risk assessment. When hsCRP is considered in conjunction with traditional lipid screening, its clinical utility is further increased. In this report, hsCRP, HDL-cholesterol (HDLC), and total cholesterol (TC) assay imprecision is evaluated in terms of the impact on the cardiac risk assessment process. METHODS: Cardiac risk assessment events were simulated using software written in Visual Basic for Applications with Microsoft Excel. Representative sets of analyte concentrations were used for true patient cardiac marker values. Monte Carlo simulations about the marker values were run using assay SD estimates based on College of American Pathologists surveys and journal articles. RESULTS: Risk distributions for reasonable assay imprecision showed clinically significant variation. Estimated relative risks of cardiovascular disease using the Ridker-Rifai quintile model varied by as much as 2.5-, 3.5-, and 6-fold for conditions of low, medium, and high laboratory test imprecision, respectively. The true relative risks were underestimated by >25% in 3%, 6.7%, and 10.5% of cases under conditions of low, medium, and high laboratory test imprecision, respectively, and overestimated by >25% in 4.4%, 8.5%, and 11.1% of cases under those conditions. CONCLUSIONS: Use of the Monte Carlo simulation method as a tool to assess the impact of analytical variation on the clinical decision-making process is valuable. From this analysis, it is shown that multiple measurements of HDLC may reduce misclassifications that result from assay imprecision. This is most important when using HDLC assays that include sample preparation with 500 000 molecular weight dextran sulfate. For these assays the total assay method SDs are higher than for other assay methods. In addition, as demonstrated by a propagation of error analysis, HDLC has the largest component of overall error in the relative risk estimate. Under conditions of typical TC and hsCRP assay performance, replication of these assays is less important.

C-Reactive Protein↗
PubMed2000

Trace analysis of pentachlorophenol (PCP) in wood and wood-based products--comparison of sample preparation procedures.

The main problem with routine analyses of pentachlorophenol (PCP) and sodium pentachlorophenolate (Na-PCP) in wood and wood-based products is to determine critical PCP-contents. This task requires a reliable analytical method and statistical testing. An analytical procedure is described, which permits the determination of PCP and Na-PCP with sufficient sensitivity and accuracy. A medium size sieve (4 x 4 mm quadratic mesh) was found suitable for the grinding step. Different extraction techniques and solvents were tested systematically. Extraction by a combination of ultrasonication and shaking in the solvent mixture toluene/sulfuric acid showed best recoveries. The eluted PCP and Na-PCP were derivatized with acetic anhydride and determined by GC/ECD. The limits of detection and determination were 0.14 mg/kg and 0.40 mg/kg, respectively.

Acetic Anhydrides↗
PubMed1994

Chemical synthesis and characterization of peptides and oligomeric proteins designed to form transmembrane ion channels.

A strategy for the synthesis of peptides and oligomeric proteins designed to form transmembrane ion channels is described. A folding motif that exhibits a functional ionic pore encompasses amphipathic alpha-helices organized as a four-helix bundle around a central hydrophilic pore. The channel-forming activity of monomeric amphipathic peptides may be examined after reconstitution in lipid bilayers in which peptides self-assemble into conductive oligomers. The covalent attachment of channel-forming peptides to the lysine epsilon-amino groups of a template molecule (KKKPGKEKG) specifies oligomeric number and facilitates the study of ionic permeation and channel blockade. Here we describe detailed protocols for the total synthesis of peptides and template-assembled four-helix bundle proteins, exemplified with the sequence of M2 delta (EKM-STAISVLLAQAVFLLLTSQR), considered involved in lining the pore of the nicotinic acetylcholine receptor channel. For comparison, the synthesis of a second four-helix bundle, T4CaIVS3 with the sequence of predicted transmembrane segment S3 (DPWNVFDFLIVIGSIIDVILSE) of the fourth repeat of the L-type voltage-gated calcium channel, is included. Peptides and proteins are synthesized step-wise by solid-phase methods, purified by reversed-phase HPLC, and homogeneity ascertained by analytical HPLC, capillary zone electrophoresis, SDS/PAGE, amino acid analysis and sequencing. Optimization of synthetic procedures for hydrophobic molecules include reducing resin substitution to avoid steric hindrance and aggregation of the final product. Protocols for the preparation of the samples prior to HPLC purification as well as the conditions and columns required for successful purification are presented. The methods developed are generally applicable for the chemical synthesis, purification and characterization of amphipathic peptides and template directed helical bundle proteins.

Amino Acid Sequence↗
PubMed2003

Determination of herbicides and a metabolite in human urine by liquid chromatography-electrospray ionization mass spectrometry.

A method was developed to determine simazine, atrazine and their metabolite, 2-chloro-4,6-diamino-1,3,5-triazine, in urine. The presence of these herbicides in urine may reflect possible exposure to pesticides. Sample preparation involved protein precipitation and solid-phase extraction. The samples were analyzed by high-performance liquid chromatography-mass spectrometry. The detection limits were 0.4 microg/l and the analytes have a linear response in the interval 6-800 microg/l. The precision of the method was reflected in the RSD of < 2.4% for the herbicides studied. Based on the detectable herbicide levels from spiked urine samples collected from unexposed volunteers, this method can be used to determine the low levels necessary for establishing reference values of the selected herbicides and the metabolite.

Chromatography, Liquid↗
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↗