Class identity assignment for amphetamines using neural networks and GC-FTIR data.

An exploratory analysis was performed in order to evaluate the feasibility of building of neural network (NN) systems automating the identification of amphetamines necessary in the investigation of drugs of abuse for epidemiological, clinical and forensic purposes. A first neural network system was built to distinguish between amphetamines and nonamphetamines. A second, more refined system, aimed to the recognition of amphetamines according to their toxicological activity (stimulant amphetamines, hallucinogenic amphetamines, nonamphetamines). Both systems proved that discrimination between amphetamines and nonamphetamines, as well as between stimulants, hallucinogens and nonamphetamines is possible (83.44% and 85.71% correct classification rate, respectively). The spectroscopic interpretation of the 40 most important input variables (GC-FTIR absorption intensities) shows that the modeling power of an input variable seems to be correlated with the stability and not with the intensity of the spectral interaction. Thus, discarding variables only because they correspond to spectral windows with weak absorptions does not seem be not advisable.

Validation of 5 routine assays for serum free testosterone with a candidate reference measurement procedure based on ultrafiltration and isotope dilution-gas chromatography-mass spectrometry.

BACKGROUND/METHOD: The analytical validity of free testosterone (FTe) analog immunoassays is subject to much controversy. We revisited the validation of 4 analog assays and 1 FTe calculation procedure with a metrologically traceable reference measurement procedure (RMP) based on ultrafiltration and isotope dilution-mass spectrometry for direct measurement of Te in the ultrafiltrate. To this end, we performed split-sample measurements of 40 male sera. RESULTS: Deming regression showed that 3 of the immunoassays had moderate to good correlation (0.8474 < or = r < or = 0.9241) with the RMP; however, the slope was markedly below 1. The FTe calculation procedure was in good agreement with this result. The Sy/x values for all assays were higher than the combined imprecision values, which indicate their susceptibility to matrix-related effects. CONCLUSIONS: The study demonstrated substantial differences in analytical quality of FTe assays; however, the results suggested that after extending the validation with a larger variety of samples, recalibration of some analog assays might be worth further investigation.

The production in high yield of N'-(4-[11C]methyl)-imipramine.

A method for the routine production in high yield of N'-(4-[11C]methyl)-imipramine is presented. The label is incorporated by reaction of C-11 methyl iodide (11CH3I) upon desipramine in dimethylsulfoxide. Quaternization of the tertiary amine by 11CH3I is minimized by using an excess of desipramine. The reaction proceeds at room temperature for 10 min and the product is isolated by means of high-performance liquid chromatography (HPLC). The entire production takes only 40 min and results in a radiochemical yield of 60%. About 60 mCi of labeled product are available for medical application; the specific activity, at the time of use, is 50 mCi/mumole. The product was characterized by chromatographic and spectrometric methods.

Novel perfluoroacyl derivatives of corticosteroids and related substances for potential use in quantitative gas chromatography mass spectrometry.

We investigated the potential of perfluoroacylation for gas chromatographic mass spectrometric determination of corticosteroids and related substances. Structure elucidation of the reaction products was performed with high and low resolution mass spectrometry and with proton and carbon nuclear magnetic resonance spectrometry. Besides the well known 3-enol ester formation, 17ß-methyl-18-nor-13(14)-ene steroids were formed via loss of the 17-α hydroxyl group followed by a Wagner-Meerwein rearrangement. Compounds that bear an 11ß-hydroxy group formed additionally a 9(11) double bond when acetone was used as solvent, whereas acetonitrile or cyclohexane led to formation of 11ß-perfluoroacyl esters. In particular, perfluoroacylation of cortisol led to a clearly defined product instead of complex mixtures observed before, which thus makes it a valuable alternative to methoxime formation-silylation of cortisol for quantitative gas chromatographic mass spectrometric analyses.

Longitudinal study on the prevalence of benzodiazepine (mis)use in a prison: importance of the analytical strategy.

AIMS: This study evaluates the suitability of gas chromatographic-mass spectrometric (GC-MS) analysis to follow-up the extent of benzodiazepine (mis)use in a Belgian prison population and compares it to other analytical strategies (e.g. screening followed by confirmation of the positive samples). DESIGN AND PARTICIPANTS: From February to August 1998, 598 persons were jailed of which 188 (31.4% of the incoming detainees) volunteered to be screened. Urine samples (530 in total) were collected on the day of arrival and after 14, 30 and 90 days of imprisonment. MEASUREMENTS: All samples were screened by EMIT(R) for benzodiazepines and analysed subsequently by GC-MS. FINDINGS: EMIT(R) screening yielded 117 (22.1%) positive samples, a number which increased to 174 (32.8%) after GC-MS analysis. Of these 174 GC-MS positive samples, 119 (68.4%) contained one benzodiazepine while for the remaining samples multiple benzodiazepine (mis)use could be demonstrated. A significant increase in benzodiazepine (mis)use was indicated only from day 0 to day 14 based on the GC-MS results but not on the immunoassay results, even when the latter were complemented with GC-MS analysis of the positively screened samples. The GC-MS data also demonstrated that benzodiazepines are mainly (mis)used by subjects on benzodiazepine prescription as almost 50% of these subjects took additional non-prescribed benzodiazepines. During GC-MS analysis other drugs were co-extracted unintentionally and chromatographed and 23.9% of the volunteers were positive for illegal drugs on the day of arrival. CONCLUSION: Immunoassay results yield an underestimation of the problem of benzodiazepine (mis)use in prison due to the high false negative rate. GC-MS analysis of all samples therefore is the recommended strategy for this type of longitudinal study as it yields more correct and detailed information than the immunoassay results.

Quality control of azure B preparations by liquid chromatography and standardization with azure B tetrafluoroborate.

A liquid chromatographic procedure for the quantitative determination of the thiazine dye azure B, the principal constituent of Romanowsky stains, is presented. Unlike previous methods relying on peak area normalization, the present approach involves real quantitation through calibration with the reference standard azure B tetrafluoroborate. The method has been used for the quality control of commercial azure B preparations and to study their stability in stock and staining solutions, either or not in the presence of eosin Y. Results suggest that highly pure azure B perchlorate meets the requirements of a reference material, useful for standardization of Romanowsky-Giemsa staining in haematology.

External quality assessment of laboratory performance in haematology in Belgium: analysis of two and a half years' experience.

In Belgium, external quality assessment of health laboratories is organised according to the directives of the government. The programme in the discipline haematology, as conceived by our laboratory, has now been carried out for about two and a half years. The results obtained in the different surveys are reported. A few general conclusions on the state of the art of laboratory performance in routine haematological tests are drawn from these experimental data.

Liquid chromatographic estimation of doxycycline in human tissues.

A rapid, sensitive and specific liquid chromatographic method is described for the determination of doxycycline in human tissues. The procedure involves mechanical homogenization of tissue samples in hydrochloric acid followed by extraction of the drug and an internal standard into ethyl acetate. Chromatographic separation is performed on a reversed phase column and allows quantitation of tissue levels as low as 0.68 nmol/g using a 200-400 mg sample. Application of the assay to tissue samples obtained from 36 patients confirmed the excellent penetration of doxycycline in organs. The method supersedes the classical microbiological assays in specificity and speed.

Comparison of a cytosol radioreceptor assay with a radioimmunoassay for 1,25-dihydroxyvitamin D in serum or plasma.

Human plasma and serum levels for 1 alpha,25-dihydroxyvitamin D were determined by a cytosol radioreceptor assay (RRA) and a radioimmunoassay (RIA). For both assays, 1.5 ml of human serum or plasma is used. Prior to RRA or RIA, extraction with benzene is performed followed by 'high-performance' liquid chromatography (HPLC) on a silica column (25 X 0.46 cm) with hexane/isopropanol (9/1 by vol), to isolate 1 alpha,25-dihydroxyvitamin D from the other vitamin D metabolites. The cytosol receptor was isolated from the intestine of healthy chickens. The antisera were raised in rabbits to 1 alpha,25-dihydroxyvitamin D3-3-hemisuccinate coupled to bovine serum albumin. The standard curves for RRA and RIA are prepared with 1 alpha,25-dihydroxyvitamin D3. 1 alpha,25-dihydroxy[3H]vitamin D3 of high spec act (158 kCi/mol) is used as tracer. The reactants are incubated for 16 h at 4 degrees C. Then, bound and free ligand are separated after the addition of dextran-coated charcoal. Both assays have a sensitivity of 2 pg/tube. The cytosol receptor and the antibodies have about the same absolute affinity for 1 alpha,25-dihydroxyvitamin D3 but the cytosol receptor has a higher relative affinity for 1 alpha,25-dihydroxyvitamin D3 (compared with other vitamin D metabolites). Reproducibility and precision are better for the RIA. The between- and within-assay CVs are 16.0% (mean = 58.7 ng/l, n = 16) and 11.2% (mean = 52.1 ng/l, n = 15), respectively, for RRA and 12.6% (mean = 61.8 ng/l, n = 27) and 7.4% (mean = 61.8 ng/l, n = 15), respectively using RIA. Reference values obtained by both assays on healthy males and healthy premenopausal females are the same for both sexes; 53.9 +/- 31.0 ng/l (n = 46) using RRA and 51.8 +/- 30.2 ng/l (n = 91) for RIA (mean +/- 2 SD).

A modified statistical approach for the detection of outlying values in external quality control: comparison with other techniques.

Laboratory results submitted to External Quality Assessment Schemes (EQAS) are evaluated against the arithmetic mean (m) and standard deviation (SD) of the result from other participants assuming a normal distribution of individual results. The sample statistics m and SD can only be reliable estimates of the theoretical mean (mu) and standard deviation (sigma), if they are not distorted by outlying values. Outliers are commonly detected by defining a confidence interval m +/- k SD, typically with k-values fixed at 3.0 or 3.2. The objective of our study was to prove the deficiency of the use of these fixed limits in large-scale EQAS, because it results in an unacceptable increase of outlier rate and hence in a serious distortion of the estimate of sigma. We have proposed a modified approach using a variable k-value which is dependent upon sample size and keeping the significance level constant at 5%. The influence of different decision limits on the number of outliers and on the sample statistics m and SD was compared using data from the Belgian National EQAS obtained in 6 consecutive surveys.

A method for the statistical evaluation of results in external quality control surveys.

A method for the evaluation of results in external quality control surveys is developed on purely statistical grounds. This system allows to establish accuracy and precision of assay methods by comparing statistical data from subsequent surveys. However, for a particular survey the overall accuracy, precision and quality of each laboratory are defined in statistical terms and visually displayed. Results for each laboratory are summarized on a two-page computer printout. Per component assayed and combining all components determined in the particular survey, the laboratory is assigned one of the four possible qualifications i.e. "very good", "moderate" or "bad". A few general conclusions drawn from experimental data of previous surveys are reported.

External quality control of clinical chemistry laboratories in Belgium.

A description is given of one of the existing National External Quality Assessment Schemes as organised in Belgium. The results of the six surveys held in 1981 in the disciplines Clinical Chemistry and Hormonology are reported. A few general conclusions are drawn from experimental data.

Increased concentrations of endogenous 13-cis- and all-trans-retinoic acids in diffuse idiopathic skeletal hyperostosis, as demonstrated by HPLC.

Endogenous 13-cis- and all-trans-retinoic acids have been quantitated in human serum using a solvent extraction procedure followed by isocratic reversed phase high performance liquid chromatography and UV detection. In healthy adults, after an overnight fasting period, the concentrations of 13-cis- and all-trans-retinoic acids yielded 5.3 +/- 2.43 nmol/l and 11.8 +/- 3.3 nmol/l, respectively (mean +/- SD). The method has been successfully applied to the analysis of both isomers in serum from patients with idiopathic skeletal hyperostosis in whom, the 13-cis- as well as all-trans-retinoic acid levels were raised as compared to the control group.

Fatty acid composition and kinetic behaviour of liver retinyl esters in vitamin A sufficient and deficient rats.

Weanling rats were fed vitamin A deficient diets (-A) or diets supplemented with vitamin A (+A) (4.4 mg retinol equivalents/kg diet) for a period of 7 or 6 wk, respectively. In liver tissues of these two groups of animals both the subcellular localization as well as the fatty acid composition of the retinyl esters was studied. During vitamin A supplementation or deprivation, the kinetics of the different ester forms were investigated. Results indicate that the subcellular localization of all retinyl esters is similar and dependent on age. Two pools exist, ie one consisting of the nuclear/cell debris and mitochondrial-lysosomal fractions and the other containing the microsomal and cytosol fractions. HPLC analysis showed retinyl palmitate as the predominating (80%) form of the various retinyl esters. By supplementation clearly two kinetic behaviours can be demonstrated: one being a relatively stable storage of the palmitate and stearate, increasing with time and the second one being a more labile pattern for the ester forms with other saturated and unsaturated fatty acids. By vitamin A depletion all retinyl esters are affected indicating that the ester forms other than palmitate and stearate are also storage forms of vitamin A.

Production, purification and characterization of antibodies to 1,25-dihydroxyvitamin D raised in chicken egg yolk.

For this sensitive RIA for 1 alpha,25-dihydroxyvitamin D, we used antibodies to 1 alpha,25-dihydroxycholecalciferol-3-hemisuccinate conjugated to bovine serum albumin, raised in eggs by immunization of chickens. We describe an efficient method for purification of IgG from egg yolk. We characterized these antibodies with immunoelectrophoresis and by radioimmunoassay. These antibodies show a high affinity for 1 alpha,25-dihydroxyvitamin D3 but cross react with other vitamin D metabolites as well. Extraction and liquid chromatography are necessary to isolate the 1 alpha,25-dihydroxyvitamin D from human serum or plasma before determination by RIA. The sensitivity of the assay is estimated at 5 pg/tube.

Plasma vitamin A in haemodialysis patients.

Different high performance liquid chromatographic systems were applied to the investigation of vitamin A metabolism in subjects undergoing haemodialysis. Plasma levels of retinol, retinyl esters and retinoic acid were measured. There was a significant elevation of plasma retinol and dialysis failed to normalise this level. No correlation with plasma concentrations of creatinine or urea was found. No differences in retinyl ester and retinoic acid levels were observed between healthy subjects and haemodialysis patients. These results suggest that retinol accumulation is not caused by a deficiency in its oxidative metabolism.

Evaluation of [11C]thymidine for measurement of cell proliferation in fast dividing tissues.

The purpose of this study was to investigate the possibility of using [11C]thymidine as a tumor marker for positron emission tomography studies. Biodistribution studies were set up to investigate the in vivo behavior of [11C]thymidine. Simultaneously, the DNA incorporation in fast dividing tissues and catabolism was studied. Our results confirm that [11C]thymidine can be used for detection of cell proliferation by positron emission tomography. As such, it can produce supplementary information in cancer research.

Chemometric detection of thermally degraded samples in the analysis of drugs of abuse with gas chromatography-Fourier-transform infrared spectroscopy.

We present a chemometric procedure for the identification of the reference standard chromatographic peak in cases where the GC-FTIR analysis of commercial standards results in the appearance of more than one peak in the GC chromatogram. The procedure has been designed for phenethylamines, which represent the class with the largest number of individual molecules on the illicit drug market, and which are abused for their stimulant and/or hallucinogenic effects. The similarity between their vapor-phase FTIR spectra was modeled using principal component analysis (PCA), and class identity was assigned on the basis of soft independent modeling of class analogy (SIMCA). Additional peaks could be assigned to impurities in the standards, but most often they were artifacts formed during the GC-FTIR analysis of thermolabile or chemically unstable compounds. The latter case is illustrated by the identification of the reference standard chromatographic peak and FTIR spectrum of the potent psychotropic amphetamine derivative N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine (MBDB), and by the elucidation of the chemical changes that occur in the molecule of MBDB due to thermal degradation.

Quantitative analysis of urinary C-peptide by liquid chromatography-tandem mass spectrometry with a stable isotopically labelled internal standard.

We describe the first results of a quantitative LC-tandem mass spectrometry method for urinary C-peptide with the use of [2H14]C-peptide as internal standard. LC was based on gradient elution of a Hypersil PEP C18 column. Mass spectrometry was performed in the negative electrospray ionization mode and by monitoring of the transitions at m/z 1514/1334 ([2H14]C-peptide) and 1507/1320 (C-peptide). For sample preparation, we applied ultrafiltration. The analytical performance of the method in terms of measurement precision gave an RSD of <2% (n=10). The overall imprecision was investigated from independent analysis of two urine samples in six-fold and resulted in an RSD<5%. The limit of detection, expressed as signal-to-noise ratio 3, was approximately 0.15 ng C-peptide injected. Analysis of 10 random urine samples from laboratory volunteers showed interference-free ion chromatograms at a signal-to-noise ratio of approximately 75 on average. The C-peptide concentrations calculated from quantification by the bracketing calibration technique ranged from 32 to 165 ng/ml.

Comparison of phenyl-type columns in the development of a fast liquid chromatographic system for eighteen opiates commonly found in forensic toxicology.

We report a precise and reliable method for the detection of 18 of the most commonly found opiates on the Belgian legal and illicit market, by ion-exchange, reversed-phase high-performance liquid chromatography, using a conventional phenyl-type analytical column (150x4.6 mm I.D., particle size 5 microm) and diode-array detection. We also describe a performance (efficiency and sensitivity) comparison of this column to a recently developed "high-speed" column (53x7.0 mm I.D., particle size 3 microm) packed with the same stationary phase, and used under slightly adjusted flow and gradient conditions. The final method, using the "high-speed" column, showed a significant reduction (55%) in analysis time without loss of resolution and sensitivity.