Simplified method for determination of the tetracyclic antidepressant mianserin in human plasma using gas chromatography with nitrogen detection

A simplified gas chromatographic method for determination of the antidepressant drug mianserin in human plasma is described. Application of a nitrogen-sensitive detector reduces the assay procedure to extraction, concentration and gas chromatographic determination. The method is suitable to determine mianserin in human plasma at the 1 ng/mol level on a routine basis. At the 20 ng/ml level the deviation of the mean from the true value and the relative standard deviation amount to 1.0% and 6.8%, respectively.     

A rapid,sensitive method for accurate determination of the lecithin/sphingomyelin ratio by thin-layer chromatography and reflectance spectrofluorometry

A highly reproducible thin-layer chromatographic procedure has been developed for accurate determination of the lecithin/sphingomyelin ratio. Two interfering compounds, phosphatidyl inositol and phosphatidyl serine, have been investigated and eliminated by adsorption onto DEAE-cellulose. A uniform fluorescence staining procedure employing 2′,7′-dichlorofluorescein has been developed. Accurate quantitation was performed by direct measurement of the reflected fluorescence intensity of the lecithin and sphingomyelin fluorophore spots with a spectrofluorometer equipped with a thin-layer scanning attachment. Stability and reproducibility studies are reported.     

Simple,rapid and micro high-pressure liquid chromatographic method for the simultaneous determination of tolbutamide and carboxy tolbutamide in plasma

A rapid high-pressure liquid chromatographic (HPLC) assay is described for the quantitative analysis of tolbutamide and its major metabolite, carboxy tolbutamide, in plasma. An aliquot (25–100 μl) of plasma was prepared for chromatography by deproteinization as follows. One volume of plasma and 2.5 volumes of acetonitrile were vortex mixed for a few seconds and then centrifuged for approx. 1 min. A 50-μl sample of the clear supernatant was injected into the chromatograph. A μBondapak C₁₂ reversed-phase column was used with a mobile phase of acetonitrile-0.05% phosphoric acid (45:55) at a flow-rate of 1.5 ml/min. The column effluent was monitored by a variable-wavelength UV detector set at 200 nm. Tolbutamide and its metabolite had retention times of 5.75 and 3.25 min, respectively. The procedure yields reproducible results with sensitivity adequate for routine clinical monitoring of plasma levels or for single-dose pharmacokinetic studies. A number of commonly used drugs do not interfere with the method. A single plasma sample can be analyzed in approx. 9 or 10 min.     

Differential gas-liquid chromatography method for determination of uronic acids in carbohydrate mixtures

An integrated gas-liquid chromatography method is described for the quantitation of mixtures containing simple monosaccharides in addition to mannuronic, glucuronic, and/or galacturonic acids. A hydrolyzed sample is divided into two portions. One portion is analyzed by the standard aldononitrile method. Glucuronic, galacturonic, and mannuronic acids are converted into compounds that do not chromatograph in the region of the standard aldononitrile acetates. Thus, this analysis gives an accurate estimation of the neutral monosaccharide content. The second portion is analyzed by a modified alditol acetate procedure. The reduction step is repeated three times to convert mannuronic, galacturonic, and glucuronic acids to their corresponding alditols via their intermediate lactones. The results of this gas-liquid chromatography analysis reflect the sum of the monosaccharides present plus their corresponding uronic acids. The difference between the values obtained by the aldononitrile acetate method and the modified alditol acetate method, therefore, is a measure of the uronic acid(s) present.     

Simple and sensitive procedure for the assay of serotonin and catecholamines in brain by high-performance liquid chromatography using fluorescence detection

A simple, rapid and specific method for the determination of serotonin and catecholamines in brain is described. After tissue homogenisation, catecholamines are isolated by adsorption onto alumina and elution with perchloric acid. Serotonin is isolated by extraction into n-heptanol and back-extraction into acid. High-performance liquid chromatography of the acid extracts is performed with a C₁₈ reversed-phase column and simple mobile phases. Detection is by the intrinsic fluorescence of the amines on excitation at 200 nm. Detection limits are 100 pg for norepinephrine, 300 pg for dopamine and 20 pg for serotonin. The results are found to correlate well with a catechol O-methyl transferase radioenzymatic assay for catecholamines and a ninhydrin derivatisation procedure for serotonin.     

Analysis of fatty acids from human lipids by gas chromatography

A rapid, quantitative method is described for the analysis of fatty acids from human lipids, namely serum lipids and lipids from adipose tissue biopsies. The method includes extraction of serum lipids with chloroform—methanol, hydrolysis with tetramethylammonium hydroxide, methylation with methyl iodide and N,N-dimethylformamide and gas chromatographic analysis on a Supelcoport SP-2330 column. Fat biopsies are analysed without extraction. Optimal hydrolysis conditions have been investigated.     

A sensitive method for determination of 5-fluorouracil and 5-fluoro-2'-deoxyuridine in human plasma by high-pressure liquid chromatography

Polymerization-competent extracts of suspension-cultured HeLa cells and porcine brain tissue were assayed for tubulin content. Five different methods were used to assay identically prepared extracts: two types of sodium dodecyl sulfate-containing acrylamide gels, a DEAE retention assay, a colchicine-binding assay, and a radioimmunoassay. The colchicine-binding and radioimmunoassay results were in close agreement and are therefore considered reliable assays for tubulin content in cell extracts. The DEAE retention assay gave slight overestimates, but the gel methods seriously overestimated tubulin content. Based on data from colchicine binding and radioimmunoassay, the proportion of soluble cell protein which is tubulin is 4.3% for HeLa cell extracts and 12.1% for brain tissue extracts.     

Determination of methenamine in biological samples by gas—liquid chromatography

Methenamine (hexamethylenetetramine), a urinary disinfectant, was determined in human plasma and urine by gas—liquid chromatography with a short (10 m) open-bone glass capillary column (split ratio 1:20) and nitrogen-selective detector. An almost quantitative recovery (92.1%) was achieved by simple dilution of water-containing samples (0.5 ml) with acetone (4.5 ml). After centrifugation and aliquot (2 μl) of the supernatant was injected into the gas chromatograph. Selectivity and sensitivity of the nitrogen detector allowed the quantitation of unchanged methenamine in plasma and urine up to 24 h after a single therapeutic dose of 1 g.Reproducibility of the method was 7.6 and 2.1% (C.V.) in serum and urine, respectively. The time required for the analysis of one sample was approx. 2 min. Due to the simple extraction and short analysis time it was possible to analyze the samples concurrently with sample taking. Absorption of standard tablets and an enterosoluble preparation of methenamine hippurate was compared.     

Tryptophan and kynurenine determination in untreated urine by reversed-phase high-pressure liquid chromatography

A fast and sensitive method for the analysis of tryptophan and some of its metabolites is discussed. A reversed-phase chromatographic system with water mobile phase can separate tryptophan, N-formalkynurenine, kynurenine and 3-hydroxykynurenine in less than 15 min at a flow-rate of 1 ml/min. The application of the method to the analysis of tryptophan and kynurenine in untreated urine of a patient loaded with tryptophan is described. The ease and speed of analysis makes the method very attractive for clinical purposes. Among other things, it was found that tryptophan in untreated urine degrades with time, even if the sample is frozen at ?11°.     

Micro method for the gas chromatographic determination of serum theophylline utilizing an organic nitrogen sensitive detector

A gas chromatographic micro method utilizing an organic nitrogen sentitive detector for the determination of serum theophylline is described. The method incorporates 3-isobutyl-1-methylxanthine as the internal standard and involves extraction and off-column derivatization of theophylline and the internal standard to their pentyl derivatives. Using 50 μl of serum, concentrations of 1 μg/ml in serum can easily be measured. The method is linear up to 50 μg/ml and the precision of the method is 3.4% in the therapeutic range. No interferences from endogenous compounds or from drugs commonly co-administered with theophylline have been encountered.     

Determination of urinary thiamine by high-pressure liquid chromatography utilizing the thiochrome fluorescent method

A sensitive, reproducible, and specific method for the determination of urinary thiamine has been established. Unique to this method is the use of high-pressure liquid chromatography (HPLC) to separate the fluorescent thiamine derivative from interfering fluorescent compounds. Urine samples were passed through a Decalso cation-exchange column, washed with 0.5 M KCl to remove some interfering compounds, and eluted with 3.4 M KCl. The eluted thiamine was converted to the fluorescent derivative, thiochrome, by reaction with alkaline potassium ferricyanide. The reaction mixture was extracted with isobutanol and subjected to HPLC monitored by a fluorescent detector.Within-day and day-to-day coefficients of variation proved to be 2.5% and 1.2%, respectively. Recovery of added thiamine (range 0.04 to 2.0 μg/ml) averaged 99.9 ± 5.3%. The sensitivity of this method was 0.03 μg/ml.