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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.

This study applied electrospray ionization-isotope dilution-liquid chromatography-tandem mass spectrometry for the evaluation of five urinary C-peptide immunoassays via split-sample measurements. The immunoassays measured in duplicate in the same run, the comparison method in triplicate over different runs. From the data, the within-run imprecision and the method comparison total RSDs were calculated. Regression analysis revealed on the one hand systematic differences, on the other, an excellent correlation between the test and comparison methods. From the spread of the data around the regression line in comparison with the 95% prediction intervals from the total RSD, sample-related effects and/or specificity problems were apparent and investigated.

A method using liquid chromatography coupled to tandem mass spectrometry is described for the determination of drugs of abuse in oral fluid. The method is able to simultaneously quantify amphetamines (amphetamine, methamphetamine, MDA, MDMA and MDEA), opiates (morphine and codeine), cocaine and benzoylecgonine. Only 200 micro of oral fluid is spent for analysis. The sample preparation is easy and consists of mixed mode phase solid-phase extraction. Reversed-phase chromatography is carried out on a narrow bore phenyl type column at a flow-rate of 0.2 ml/min. A gradient is applied ranging from 6 to 67.6% methanol with ammonium formate (10 mM, pH 5.0) added to the mobile phase. The column effluent was directed into a quadrupole-time-of-flight instrument by electrospray ionization, without the use of a splitter. A validation study was carried out. Recovery ranged from 52.3 to 98.8%, within-day and between-day precision expressed by relative standard deviation were less than 11.9 and 16.8%, respectively, and inaccuracy did not exceed 11.6%. The limit of quantification was 2 ng/ml (0.66 x 10(-5)-1.48 x 10(-5) M) for all compounds. Internal standards were used to generate quadratic calibration curves (r(2)>0.999). The method was applied to real samples obtained from suspected drug users. An interference was observed from the device used to sample the oral fluid, consequently this was excluded from the method which was validated on oral fluid obtained by spitting in a test-tube.

Background: In clinical chemistry, traceability of measurements is of high priority., Methods: In this literature review, current recommendations on the process of establishing traceability (or standardization) are critically discussed., Results: Traceability is to be established to the highest international standards by a comprehensive reference measurement system. Elementary to this system are a metrological basis, a measurement unit system, i.e., the Système International d'Unités (SI), its embodiment by a material standard and a calibration hierarchy for transfer of accuracy/trueness to the manufacturer's product calibrators and routine methods. However, for analytes lacking an unequivocally recognized entity, the International Unit (IU) and International Standard (IS) concept have been developed. On this basis, the review distinguishes between traceability of SI- and IU-analytes., Conclusions: SI-traceability, exemplified by cortisol, is straightforward. However, special attention is needed for "free analytes" and "analyte families". For traceability of IU-analytes, exemplified by hCG, the standardization process passes different phases in function of the history of the analyte (discovery, ISs, measurement by bioassays/immunoassays, complete structure elucidation). However, perspectives to the development of an SI-based reference measurement system are realistic. Last but not least, for successful global implementation of the standardization process, consensus of all major players in the field will be required.

A fast liquid chromatographic method with tandem diode array-fluorescence detection for the simultaneous determination of in total 17 opium alkaloids and opioids is presented. Blank blood and urine samples (1 ml) were spiked with different concentrations of a standard mixture, as well as with the internal standard, butorphanol (2000 ng/ml). After solid-phase extraction, based on weak cation exchange (Bond Elut CBA SPE columns), the extracts were examined by HPLC-DAD-FL. By using a "high-speed" phenyl column (53 x 7.0 mm I.D., particle size 3 microm) eluted with a gradient system (A: water-methanol (90:10, v/v), B: methanol, both containing 25 mM triethylammoniumformate (pH(A) = 4.5)) all compounds could be baseline separated within 12 min. The method was validated and its applicability was demonstrated by the analysis of real-time forensic cases.

A gas chromatographic-mass spectrometric method was developed for the simultaneous analysis of 15 low-dosed benzodiazepines, both parent compounds and their corresponding metabolites, in human urine. The target compounds are alprazolam, alpha-hydroxyalprazolam, 4-hydroxyalprazolam, flunitrazepam, 7-aminoflunitrazepam, desmethylflunitrazepam, flurazepam, hydroxyethylflurazepam, nitrogen-desalkylflurazepam, ketazolam, oxazepam, lormetazepam, lorazepam, triazolam and alpha-hydroxytriazolam. Nitrogen-methylclonazepam is used as the internal standard. The urine sample preparation involves enzymatic hydrolysis of the conjugated metabolites with Helix pomatia beta-glucuronidase for 1 h at 56 degrees C followed by solid-phase extraction on a phenyl-type column. The extracted benzodiazepines are subsequently analyzed on a polydimethylsiloxane column using on-column injection to enhance sensitivity. The extraction efficiency exceeded 80% for all compounds except for oxazepam, lorazepam and 4-hydroxyalprazolam which had recoveries of about 60%. The LODs ranged from 13 to 30 ng/ml in the scan mode and from 1.0 to 1.7 ng/ml in the selected ion monitoring (SIM) mode. Linear calibration curves were obtained in the concentration ranges from 50 to 1000 ng/ml in the scan mode and from 5 to 100 ng/ml in the SIM mode. The within-day and day-to-day relative standard deviations at three different concentrations never exceeded 15%.

In an in vitro study, less compound A was formed when a KOH-free carbon dioxide absorbent was used. To confirm this observation we used a lung model in which carbon dioxide was fed in at 160 ml min(-1) and sampling gas was taken out for analysis at 200 ml min(-1); ventilation aimed for a PE'CO2 of 5.4 kPa. The soda lime canister temperatures in the inflow and outflow ports (Tin and Tout) were recorded. In six runs of 240 min each, a standard soda lime, Sodasorb (Grace, Epernon, France) was used and in eight runs KOH-free Sofnolime (Molecular Products, Thaxted, UK) was used. Liquid sevoflurane was injected using a syringe pump to obtain 2.1% E'. Compound A was measured by capillary gas chromatography combined with mass spectrometry. Median (range) compound Ainsp increased to a maximum of 22.7 (7.9) ppm for Sodasorb and 33.1 (20) for Sofnolime at 60 min and decreased thereafter; the difference between groups was significant (P<0.05) at each time of analysis up to 240 min. The canister temperatures were similar in both groups and increased to approximately 40 degrees C at 240 min. Contrary to expectation, compound A concentrations were greater with the KOH-free absorbent despite similar canister temperatures with both absorbents.

GC-MS screening conditions were developed for 15 low-dosed benzodiazepines, covering alprazolam, flunitrazepam, flurazepam, ketazolam, lorazepam and triazolam, and the corresponding metabolites alpha-hydroxyalprazolam, 4-hydroxyalprazolam; 7-aminoflunitrazepam, desmethylflunitrazepam, 7-aminodesmethylflunitrazepam; hydroxyethylflurazepam, N-desalkylflurazepam; oxazepam and alpha-hydroxytriazolam, respectively. Benzodiazepines are analyzed on a polydimethylsiloxane column in both the scan and the multiple ion monitoring modes using on-column injection to attain maximal sensitivity. The reactive compounds are acetylated with pyridine and acetic anhydride for 20 min. The derivatives are stable for at least 4 days. The relative standard deviation observed with standard compounds at the low nanogram-level ranged from 1.13 to 4.87% within-day and from 1.12 to 4.94% between-day. Unequivocal identification potential, high chromatographic resolution and sensitivity are combined with minimal thermal degradation. The presented screening conditions provide the basis for a unique routine screening method for low-dosed benzodiazepines with a broad polarity range.

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.

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.

In aquaculture, alpha-tocopheryl acetate (alpha-TA) is the main source of vitamin E used to fortify fish feed. alpha-TA in fish is often determined indirectly, i.e., by alkaline hydrolysis, followed by quantitation of "total alpha-tocopherol" (alpha-T) and subtraction of the natively present alpha-T. The aim of this study was to develop an HPLC method for the simultaneous quantitative determination of alpha-TA and free tocopherols in aquatic organisms and fish feed. The assay consists of a simple extraction with methanol containing butylhydroxytoluene (BHT) as an antioxidant, followed by reversed-phase chromatography with consecutive UV and fluorescence detection of alpha-TA and tocopherols, respectively. The peak of the internal standard tocol in the fluorescence trace was used for quantitation. Linearity was achieved over the range of 0.2 to 4.2 micrograms alpha-TA per ml extract of Artemia nauplii, which would correspond to 30.7 to 614.4 micrograms/g dry mass. The within-run coefficient of variation was 1.9% at a level of 310 micrograms/g dry mass. The recovery of alpha-TA ranged from 97.7 to 100.8% (concentration = 2.1 and 20.5 micrograms/ml, n = 6). The detection limit was about 7 ng and the quantification limit on spiked samples was 0.2 microgram/ml. This method was routinely applied to determine alpha-TA and alpha-, gamma- and delta-tocopherol (alpha-T, gamma-T, delta-T) simultaneously in Artemia, fish feed, shrimp eggs and various other aquatic organisms.

We present carbon-13 nuclear magnetic resonance (13C-NMR) and mass spectral data for several androstanes and estranes having a 17,17-dialkyl-18-nor-13(14)-ene substructure. These compounds are formed by a Wagner-Meerwein rearrangement of steroids bearing a tertiary 17-hydroxy group during the derivatization reaction with heptafluorobutyric anhydride. The 13C-NMR assignments are compared with those of natural products having a similar substructure. The mass spectra show characteristic fragment ions for which a fragmentation mechanism is proposed.

The potentials and limitations of high-performance liquid chromatography-photodiode array detection are highlighted in respect to its use in the analysis of different biological matrices followed by the identification of unknowns. The logical analytical approach used in clinical and forensic toxicology, vital for the identification of one or more toxic substances as a cause of intoxication, is largely based on both simple and fast "general unknown screening" methods which cover most relevant drugs and potentially hazardous chemicals. In this field of systematic toxicological analysis, a literature overview shows that HPLC can play a substantial role. Both column packing material and eluent composition have their impact on intra- and interlaboratory reproducibility. In view of the sometimes different retention characteristics of various HPLC columns, several possibilities are addressed to enhance the discriminating power of primary retention parameters. The advantages of photodiode array detection as compared to UV detection have been of paramount importance to the success of HPLC in toxicological analysis. Dedicated libraries with spectral information and searching software are powerful tools in the process of identification of an unknown substance. In the present paper, these aspects are also verified in a number of real cases, i.e., trazodone and dothiepin, azide, chloroquine and cocaine, in which we illustrate from our own experience the potentials of HPLC-photodiode array detection in systematic toxicological analysis.

A liquid chromatographic (HPLC) method has been developed for the quantitative determination of different forms of vitamin E (alpha-, gamma-, and delta-tocopherol) in aquatic organisms. The assay consists of a simple extraction with methanol containing butylhydroxytoluene (BHT) as an antioxidant, followed by reversed-phase chromatography with fluorescence detection. The efficiency of the extraction method was equivalent or superior to that of more complex approaches for the isolation of tocopherols. Linearity has been achieved over the range of 0.02 to 3 micrograms/ml for alpha-tocopherol and within-run and between-run coefficients of variation at three levels were 0.7-2.9 and 1.2-3.7%, respectively. The recovery at three concentrations ranged from 73.8 to 96.6% and the minimal quantity that could be detected was 0.6 ng. Comparable figures were obtained for gamma- and delta-tocopherol. This method has been routinely applied to determine vitamin E in Artemia, rotifers, turbot and sea bass larvae, and shrimp postlarvae.

We present a new candidate reference method for determining cortisol in serum. The method is based on isotope dilution-gas chromatography/mass spectrometry and makes use of derivatization with heptafluorobutyric anhydride and selected ion monitoring at m/z 489 and 491. A detection limit of 0.08 pmol (30 pg) was achieved. Twenty-four structurally related steroids were tested for interference and found negative. Verification of the analytical quality specifications was done from measurement in two laboratories of the certified reference materials 192 and 193 of the Bureau Communautaire de Référence and a number of commercial quality control materials. The maximum systematic error was estimated to be 1.0% and the mean imprecision was 1.0%. The total error was lower than 2.05%. The method was applied for target-setting in external quality assessment and internal accuracy control and for measurement of patient samples.

As part of ongoing studies in the biosynthesis of the lantibiotic nisin, we investigated the proteolytic cleavage of a synthetic Fmoc-labeled decapeptide mimicking a key amino acid sequence of the precursor prenisin in extracts of a Lactococcus lactis subsp. lactis strain. Reverse-phase high-performance liquid chromatography with photodiode array detection was used to trace and purify potential enzymatic conversion products. Of the three newly appearing chromatographic peaks, one was identified by means of electrospray mass spectrometry, amino acid analysis, and amino acid sequencing as an Fmoc-labeled hexapeptide derived from cleavage at the Arg-1-Ile+1 bond. This assay will be useful to monitor the purification of the endoproteinase that reportedly cleaves prenisin at the same Arg-1-Ile+1 site present in the model substrate.

We evaluated two internal standards for HPLC determination of creatinine in human serum after ultrafiltration: 2-iminoimidazolidin-4-one for normal-phase HPLC on aluminium oxide, and thymine for C18 reversed-phase HPLC. Detection of 2-iminoimidazolidin-4-one was done at the same wavelength as that used for creatinine, i.e. 240 nm. For thymine, the wavelength was switched to 280 nm. The suitability of the selected compounds to serve as an internal standard in the described measurement procedures, including ultrafiltration of serum, was evaluated from the precision and accuracy obtained. The method based on normal-phase HPLC with 2-iminoimidazolidin-4-one showed an imprecision expressed as R.S.D. ranging from 0.8 to 3.4% (mean: 2.1%) and an inaccuracy, calculated from the deviations from target values determined by isotope-dilution gas chromatography-mass spectrometry, ranging from -1.3 to +1.8% (mean: +0.4%). For the reversed-phase HPLC procedure with thymine, the imprecision ranged from 0.3 to 1.3% (mean: 1.0%) and the inaccuracy from +0.1 to +3.9% (mean: +1.7%). The occasional observation of interferences with 2-iminoimidazolidin-4-one limited the application of the normal-phase method to a certain extent.

Embutramide is a general anesthetic having a strong narcotic effect on the central nervous system where it paralyzes the brain center that controls respiration. It is a constituent of T61, a veterinary euthanasia drug. This paper describes a gas chromatographic procedure using nitrogen-phosphorus detection for the determination of embutramide in biological matrices. The drug and the internal standard (ambucetamide) are extracted with dichloromethane under alkaline conditions. The method is linear from 100 to 3000 ng/ml. The within-day and day-to-day coefficients of variation range from 5.1 to 5.7% and from 9.1 to 10.0%, respectively. The recovery is above 80% while the minimum detectable level under the conditions described is 40 ng/ml analyzing a 1-ml or a 1-g aliquot of a sample (blood or tissue). The method is also applied to different samples from dogs euthanized with T61.

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.

A liquid chromatographic method for the determination of oxytetracycline in bovine plasma was developed. Contrary to most current tetracycline assays involving solid-phase extraction, oxytetracycline and the internal standard tetracycline were isolated from buffered plasma by double-phase extraction with ethyl acetate-isopropyl alcohol. The latter component was found to be essential to ensure reproducible partitioning of both tetracyclines into the organic layer. Chromatography was conducted on a Lichrosorb RP8 column, compounds were eluted with 0.01 M oxalic acid-methanol-acetonitrile, and detection was at 357 nm. Linearity was observed over the 0-8.5-micrograms/mL range, and the detection limit approximated 5 ng/mL. The extraction yield was 80.4 +/- 3.4% (n = 12), and the coefficients of variation for repetitive within-run and between-run analyses at two concentration levels (0.25 and 2 micrograms/mL) were 1.0-4.3 and 2.4-7.2%, respectively. The method was applied to a pharmacokinetic study of oxytetracycline in three nonlactating cows after a single intramuscular administration of a long-acting preparation.

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.

A reliable analytical method has been developed for measurement of 13-cis- and all-trans-acitretin (Neotigason) in human plasma by normal-phase high-performance liquid chromatography, with ultraviolet detection. Human plasma was obtained after centrifugation of whole blood samples and deproteinized by ethanolic denaturation. After liquid-liquid extraction with water-n-hexane, and aliquot ws chromatographed on a silica column using isocratic elution with n-hexane-methylsalicylate-acetic acid (200:18:0.6, v/v). The wavelength was set at 360 nm, and for plasma samples a limit of quantification of 3-4 ng/ml was obtained. All manipulations were carried out under dim light conditions to prevent photoisomerization.

Quality control of albumin solutions for human use is of utmost importance because the presence of impurities can provoke adverse reactions in treated patients. Three different techniques were used to detect the presence of albumin oligomers and polymers as well as foreign proteins in commercial solutions. The relative concentrations of the former two were estimated using size-exclusion high-performance liquid chromatography. Isoelectric focusing and two-dimensional immunoelectrophoresis allowed detection of other contaminants of proteinaceous nature. The application of this combination of techniques supersedes the traditional approaches (gel filtration on polydextran gels, electrophoresis) in specificity and speed. Analysis of 34 lots of commercial albumin solutions from 22 manufacturers revealed the superior quality of preparations of placental rather than plasmatic origin.

A high-performance liquid chromatographic method on a polymeric column was developed for the analysis of xanthene dyes. The rigid polystyrene-divinylbenzene column was connected to a photodiode array detector to verify the identity and the purity of the dyes. For eosin Y a within-day precision of 1-2% was obtained, and on a day-to-day basis the coefficient of variation was 4.2%. The purity of commercial xanthene dyes was investigated, and the results show the divergence between the actual dye contents and the dye contents indicated on the label.

A high-performance liquid chromatographic procedure is described for the simultaneous determination of etretinate (Tigason), all-trans-acitretin (Neotigason) and 13-cis-acitretin in human plasma. The compounds are extracted from the plasma with n-hexane under acidic conditions. Quantification is performed on a normal-phase column (CP-Spher Si, 5 microns), followed by UV detection at 350 nm. The limit of quantification is 3 ng/ml. The day-to-day precision was 6.7, 13.6 and 9.1% for etretinate (means = 53 ng/ml), all-trans-acitretin (means = 95 ng/ml) and 13-cis-acitretin (means = 149 ng/ml), respectively (n = 13 for each compound). The within-day precision of nine determinations was 3.2, 11.7 and 6.5%, respectively, with mean concentrations of 128.53 and 261 ng/ml, respectively. The method was also applied to the study of the long-term pharmacokinetic behaviour of etretinate in psoriatic patients previously treated with etretinate but now on therapy with all-trans-acitretin.

The polar molecule, fluorouracil, is a monoanion at pH 10 and may be quantitatively extracted from aqueous solutions with quaternary alkylammonium ions into an organic solvent such as dichloromethane as the ion-pair. Extraction constants of fluorouracil with the tetrapentylammonium ion in dichloromethane or dichloromethane-1-butanol (9:1) and with the tetrahexylammonium ion in dichloromethane were determined. Slope analysis demonstrated that association of the ion components in the aqueous phase occurred as the side reaction. A column ion-pair extraction technique, using tetrapentylammonium as the counterion and dichloromethane as the eluting phase, was developed and allowed quantitative transfer of fluorouracil to the organ solvent. The applicability of this method was shown by determining plasma levels of fluorouracil in cancer patients to whom 1 g of active substance was administered intravenously.

The reference interval for serum vitamin K1(20) levels was assayed in healthy fasting adults by a method based on high-performance liquid chromatography (HPLC). The isolation procedure involves a solvent extraction of the plasma lipids followed by two chromatographic steps, consisting of a purification of the extract on a semipreparative adsorption column and a final quantitation on a reverse-phase column. Vitamin K1(20) and vitamin K1(25), the internal standard, are monitored by fluorescence detection after postcolumn reduction with a methanolic solution of tetramethylammonium octahydridotriborate. This reaction is performed in an open tubular reaction coil at elevated temperature. The median plasma concentration in 50 healthy fasting adults was 247 pg/ml. The levels showed a skewed distribution with a range of 62 to 980 pg/ml [log x +/- 2 SD (log x)]. The method is linear over the entire physiological range and has a within-run precision of 3.6% (n = 5, mean = 311 pg/ml). The minimum detectable amount in serum is 50 pg/ml. Other extraction procedures resulted in lower recoveries or in interferences in the final measurement. The vitamin K1(20) levels as reported by other research groups are also discussed.

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.

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.

Various procedures for converting 5-fluorouracil into its methyl, butyl and hexyl derivatives are described. Structures were established as the N,N'-dialkyl derivatives using mass spectrometry or combined gas-liquid chromatography-mass spectrometry. The reaction conditions, i.e., the amount of derivatization reagents and reaction time, were optimized. Gas-liquid chromatographic characteristics of the derivatives were investigated on different stationary liquid phases, and 2% or 3% SP-2250, 5% XE-60 and 5% OV-1 were found to be superior. With 5-chlorouracil as the internal standard a linear response for the various derivatives was observed in the microgram range. The applicability of the different dialkyl derivatives in the measurement of 5-fluorouracil in biological materials is discussed.

A sensitive and specific high-performance liquid chromatographic (HPLC) method for the determination of cortisol in only 200 microliters of serum is described. Cortisol and two internal standards, 19-nortestosterone (IS1) and 6 alpha-methylprednisolone (IS2) are extracted with dichloromethane and analyzed on a C18 reversed-phase column eluted with a mobile phase of methanol:water at a flow rate of 0.75 ml/min. Ultraviolet absorption at 254 nm is used for detection and quantitation is performed by peak height ratio measurement. Using 200 microliters of serum, the lower limit of detection for cortisol is 10 ng/ml, the analytical recovery is 104 +/- 3.6% (n = 8), and the day-to-day precision was 1.69% at a level of 90 ng/ml (n = 16). Cortisol values obtained by this method were generally lower than those obtained by radioimmunoassay or by fluorometry. A serum pool was analyzed both by HPLC and by isotope dilution/mass spectrometry (ID/MS). A mean value of 90.1 ng/ml was obtained by HPLC (n = 16, CV = 1.7%), whereas ID/MS yielded a mean of 90.8 ng/ml (n = 28, CV = 0.4%). These results clearly demonstrate the high specificity and the accuracy of the HPLC procedure. The use of two internal standards not only compensates for losses during the sample manipulation but also prevents erroneous results in case of medication by either of these two products.

A review is presented of current gas and liquid chromatographic methods for the determination of the fat-soluble vitamins A, D, E and K and the provitamin A beta-carotene in biological samples of human origin. For each vitamin, the discussion successively focuses on procedures for sample preparation, gas and liquid chromatographic systems and principles of detection. The emphasis is on liquid chromatography, which is gradually becoming a standard technique in fat-soluble vitamin assays. New trends in the liquid chromatography of these compounds include the use of smaller particles and shorter columns, to improve speed, and the advance of electrochemical detection as an alternative to absorbance and fluorescence detection. Bonded phases, both normal and reversed phase, tend to be preferred over underivatized silica as column supports. Gas chromatography remains of particular value in combination with mass spectrometry, a technique which may form the basis of reference methods. In general, despite the availability of well established analytical methods for fat-soluble vitamins, the wealth of recent literature in this area indicates that there continues to be a need for new assays with enhanced speed, specificity and sensitivity.

A rapid and specific flame-ionization GLC method was developed for the determination of plasma fluorouracil. The chloro analog is used as the internal standard. The method involves the isolation of both the drug and the internal standard from plasma on a strong anion-exchange column at pH 10. Elution is performed with acetic acid in methanol. The evaporated eluate is dissolved in tetrahexylammonium hydroxide. An aliquot of the resulting solution is introduced directly into the gas chromatograph, where conversion to the bishexyl derivatives and subsequent separation take place. The extraction recovery from blank plasma, to which fluorouracil was added, was 96.8 +/- 2.4% (SD). Linearity was proven in the range from 0 to 25 micrograms/ml, whereas the detection limit of the method was estimated at about 2 micrograms/ml of plasma. The within-run precision was determined at three different fluorouracil levels. To demonstrate method applicability, plasma samples obtained from cancer patients to whom 1 g of fluorouracil had been administered intravenously were analyzed.

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.

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.

Seven solvent systems for the thin-layer chromatographic separation of hydrazidic monoamine oxidase inhibitor drugs have been developed. Detection of compounds at the microgram level was performed with the aid of thirteen spray reagents.

A rapid, specific, and sensitive liquid chromatographic assay for minocycline in human serum is described. The drug and an internal standard (oxytetracycline) were extracted into ethyl acetate from 0.5 ml of buffered serum (pH 6.5). Chromatographic separation was achieved on a 10-micrometer Lichrospher 100 CH 8 column with acetonitrile--citric acid (0.1 M) as the eluent. The column effluent was monitored at 352 nm. The assay was linear up to 3 micrograms/ml with a mean coefficient of variation of 3.3% (n = 6). An extraction recovery of 89.4 +/- 3.2% (mean +/- SD, n = 17) was obtained over the 0.5--2.6 micrograms/ml range. The detection limit averaged 50 ng/ml. A serum concentration-time profile in humans after oral intake is presented.

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.

Reversed-phase liquid chromatography is used for the separation of 5-fluorouracil, its deoxyribo- and ribonucleosides and nucleotides. The bases and nucleosides are easily separated from their naturally occurring analogues on an octadecyl silica column eluted with 2.10(-2) M KH2PO4 (pH 5.0) containing 5% (v/v) of methanol. This system can be applied to the measurement of 5-fluoro-2'-deoxyuridine serum levels down to 0.1 microgram/ml. Addition of small amounts (10(-3) M) of tetrabutylammonium phosphate to the eluent results in a large retention increase for the nucleotides, while the capacity ratios of the bases and nucleosides remain unchanged. The influence of the tetrabutylammonium phosphate and ammonium phosphate concentrations and pH of the eluent on the various k' values was investigated. Evidence is presented indicating that the quaternary ammonium compound is adsorbed onto the octadecyl silica surface: nucleotides are probably retained as adsorbed tetrabutylammonium ion-pairs.

A sensitive combined adsorption and reversed-phase high-performance liquid chromatographic procedure is described that permits the determination of endogenous vitamin K1 levels in serum. The separation of vitamin K1 (cis- + trans-isomers), vitamin K1 epoxide and the menaquinones MK-2, MK-4 and MK-9 by adsorption, reversed-phase and cyano-bonded phase chromatography is discussed. The methodology was further developed for the quantitative measurement of vitamin K1 in human serum. Concentrations as low as 500 pg/ml in serum could be detected. The identity of the vitamin K1 peak was confirmed by UV absorption spectrophotometry and re-chromatography. The method has been applied to the determination of serum levels in normal healthy individuals and patients treated with vitamin K1.

A GLC method for the specific identification and quantitation of iproiazid, a potent monamine oxidase inhibitor drug, is described. The free drug isolated from urine samples by two-phase extraction reinforced with salting out. Identification and quantitative determination at the microgram level are done on aliquots of the chloroform extract by GLC, using the 2-butyl analog as an internal standard. Iproniazid and potentially interfering compounds present in the extracts are also identified by GLC-mass spectrometry and TLC for supporting evidence of the GLC method's specificity.

A reversed-phase high-performance liquid chromatographic method for quantitative doxycycline determination in human serum and urine is described. The drug was extracted from buffered (pH 6.1) serum or urine into ethyl acetate. A structural analog, demeclocycline, was added as the internal standard. A 10-cm X 2-mm i.d., 5-micrometers Lichrosorb RP8 column with acetonitrile-0.1 M citric acid as the eluent was used. The effluent was monitored at 350 nm. The extraction recovery from spiked serum was 87-8 +/- 4.3% (mean +/- SD, n = 11); for urine, a value of 92.2 +/-2.0% (mean +/- SD, n = 10) was found. Within-run and within-day relative standard deviations averaged (x = 2.5 micrograms/ml, n = 10) and 4.75% (x = 2.6 micrograms/ml, n = 9), respectively. The detection limit was estimated at 50 ng/ml of serum. No significant extra peaks were observed in chromatograms obtained on serum or urine extracts, suggesting the probable absence of metabolic processes in vivo.

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.

A quantitative determination of physiological levels of all-trans-retinoic acid (vitamin A acid) in human serum has been developed. A double-phase extraction of 3.5 ml of serum followed by a specific and sensitive high performance liquid chromatography procedure allowed measurement of levels down to 1 ng/ml. Serum concentrations in 37 fasting volunteers ranged from 2.7 to 4.2 ng/ml and fitted a normal-Gaussian distributional shape with a mean value of 3.5 ng/ml and SD of 0.4 ng/ml, as demonstrated by the Kolmogorov-Smirnov test.

A sensitive and rapid method for the gas chromatographic (with electron-capture detection) confirmation of derivable sympathomimetic amines is described. Extractive derivatization with pentafluorobenzoyl chloride is performed on 2-ml urine or plasma samples. Especially for primary amines, the method appears to be very sensitive. Mass spectral data allowed confirmation of the monobenzoylation of all congeners.

A specific GLC method was developed for the determination of floxuridine in plasma using the thermionic nitrogen detector. The method involves the isolation of the compound and internal standard from plasma on a strong anion-exchange column at pH 10, followed by elution with 0.3 M acetic acid in methanol. The eluate is evaporated to dryness, and the residue is dissolved in dimethyl sulfoxide and permethylated with potassium tert-butoxide and methyl iodide. The permethylated compounds are reextracted from the reaction mixture with cyclohexane-methylene dichloride (9:1). The organic solution is evaporated to dryness, the residue is dissolved in ethyl acetate, and an aliquot is analyzed by GLC on a 3% OV-17 column. The extraction recovery from spiked plasma was 93.2 +/- 2.1% (SD), whereas linearity for the overall procedure was in the 0-1-microgram/ml range. The detection limit of the thermionic nitrogen detector was 50 ng/ml. The within-run and within days precision (CV) were 4.0 and 6.2%, respectively, at 300 ng/ml.