Separation of Diethylstilbestrol and Derivatives in Biological Fluids and Tissues by High Pressure Liquid Chromatography

Abstract

A method was developed to identify radiolabeled DES and DES metabolites in biological fluids and tissues. After a rapid initial clean-up step, the biological samples were analyzed with a single-column, reversed-phase, highpressure liquid chromatography system. The parent compound and metabolites were simultaneously resolved and tentatively identified by comparing their retention times to those of known standards. Positive identification of some metabolites was achieved by field-desorption and capillary-column mass-spectrometric analysis. The method was found to be rapid and reproducible, and sample recovery was >80%.     

Analysis of Cimetidine in Biological Fluids by High Performance Liquid Chromatography

Abstract

The clean-up and analysis of cimetidine in human urine and blood is described. Samples were prepared by adsorption on Waters Sep-pak C-18 disposable pre-columns in basic solution followed by elution in 1 ml methanol. For blood samples, the eluate was concentrated under a stream of nitrogen; urine samples required no further concentration. The separation was performed on a reversed phase column using a mixture of methanol-1 mM sodium dodecyl sulphate in a 10 mM phosphate buffer of pH 3.0 (35:65) as mobile phase. Procaine was used as internal standard. Detection was by UV at 228 nm yielding a minimum detectable quantity of 20 ng with linearity over three decades of concentration.     

Analysis of Indenolol In Biological Fluids By High Performance Liquid Chromatography

Abstract

The analysis of indenolol in plasma and urine is described. The method involves extraction of the drug from plasma or urine using chloroform at basic pH. The separation was performed on CN column using methanol and 0.01M potassium dihydrogen phosphate solution 50:50. The efficiency of extraction was 97%. Minimum detectable amount by fluorescence was 20 ng/ml.     

Separation of Biological Pyridines by High Pressure Liquid Chromatography

Abstract

The separation of the six pyridine compounds which comprise the pyridine nucleotide cycle, nicotinamide adenine dinucleotide phosphate and para-aminobenzoic acid, a compound biologically related to these pyridines, can be achieved rapidly utilizing high pressure liquid chromatography. Optimum separation is accomplished using ion-ion pairing in reverse phase chromatography with a C₁₈ stationary phase and an aqueous mobile phase of 5mM pentanesulfonic acid and 25 mM KH₂PO₄. The effect of temperature on the separation is minimal. As little as 10 ng of these compounds is detected via absorption of ultraviolet light at a wavelength of 254 nm.     

Liquid Chromatographic Analysis of Adriamycin and Metabolites in Biological Fluids

Abstract

A definitive approach to the analysis of plasma, bile, and urine levels of the widely used antitumor drug Adriamycin using two complementary high performance liquid chromatographic systems, one normal phase and the other reversed phase, is described. Sensitivity in the 2–10 picomole per sample range is achieved by means of fluorescence detection. The use of the two systems in parallel provides an unequivocal basis for the characterization and determination of Adriamycin and its principal metabolite, adriamycinol, and enables the measurement of anthracycline fluorescent aglycones and conjugates, as well.     

The Determination of Metribuzin and Its Metabolites by High Pressure Liquid Chromatography

Abstract

A method for the determination of metribuzin and its metabolites in plant tissues has been developed using High Pressure Liquid Chromatography (HPLC). The system used involves reversed-phase chromatography on a C-18 HPLC column and a 62:38 methanol/0.05 M acetic acid mobile phase. Under these conditions, metribuzin and the three known metabolites [deaminated metribuzin (DA), deaminated diketometribuzin (DADK) and diketometribuzin (DK)] are completely resolved from each other. Detection is by UV absorbance at 254 nm.     

Determination of Chlordiazepoxide and Its Metabolites in Plasma by High Pressure Liquid Chromatography

Abstract

A rapid, sensitive and specific high pressure liquid chromatographic (HPLC) assay was developed for the determination of chlordiazepoxide and its metabolites from plasma. The assay involves extraction of chlordiazepoxide and its metabolites into diethyl ether from plasma buffered to pH 9. The overall recovery of chlordiazepoxide is 80 ± 5.0% (S.D.) and the sensitivity limit of detection is 50 to 100 ng/ml of plasma, using a 1 ml specimen. The assay was used in the determination of plasma levels of chlordiazepoxide and its metabolites in man following oral administration of chlordiazepoxide. HCl.

The chromatographic behavior of other clinically important benzodiazepines and their major metabolites is also reported.     

Determination of Flurbiprofen in Dosage form and in Biological Fluids by High Performance Liquid Chromatography

Abstract

A high performance liquid chromatograpy method is described for the determination of flurbiprofen in both dosage form and in biological fluids (urine and plasma) using fluorescence detection. The method for dosage forms involves grinding of a 100 mg tablet, suspension in methanol, filtration and adjusting to the appropriate concentration and 10 ul is injected onto the column. In the case of biological fluids a series of standard solutions were prepared in 0.IN sodium hydroxide and a known amount was added to 1 ml of serum or urine which was then acidified, extracted with ethylacetate, evaporated and the residue was then dissolved in a known volume of the mobile phase. Complete separation of the drug was achieved in about 6.5 minutes under the used conditions.     

High Performance Liquid Chromatography of Tryptophan and Serotonin Metabolites

Abstract

Serotonin is a neurotransmitter in cerebral centers and its perturbations can produce humor and behavior disorders. In addition, exploration of tryptophan and serotonin metabolism is extremely important for early detection and supervision of treatment in carcinoïd tumors. High performance liquid chromatography (HPLC) enabled us to separate and titrate different metabolites (5-hydroxyindolylacetic acid, serotonin, tryptophan, 5-hydroxy-tryptophan and N-acetyl tryptophan (NAT) as internal standard) in several types of biological tissues: blood, urine, brain and cerebrospinal fluid. This method is original because it connects HPLC with fluorescence in continuous flow, which allows to change the conditions of pH and buffer. This technique is highly sensitive (below 100 picograms) and very quick (ten minutes).     

体液直接进样固定相的研究进展

评述了生物体液直接进样主效液相色谱固定相的制备、结构性能及应用。其特点可概括为：只允许生物体液中蛋白质等大分子物质与亲水的生物相容的固定相表现接触、使其在柱中无保留地洗脱、而小分子物质可获得保留、达到直接进样分析的目的。     

Liquid Chromatography with Fluorescence Detection in the Analysis of Biological Fluids

ABSTRACT

Liquid chromatography with fluorescence detection is well suited to the analysis of biological fluids, as it combines both selectivity and sensitivity. The determinations are not limited to fluorescent compounds, as non-fluorescent substances can be converted to fluorescent derivatives by appropriate reactions. As a consequence of progress in methodology and of the development of new reagents, a great number of biological substances and drugs can now be successfully analyzed by this technique. Reliable automated procedures using pre-column derivatization are available, in particular for the analysis of amino acids and amines. In addition, systems using short columns, reduced particle size of the stationary phase and ultramicro detector cells represent a promising approach to the analysis of very small volumes of sample.     

Fluorometric Determination of Histamine in Biological Fluids and Tissue by High-Performance Liquid Chromatography

Abstract

High-pressure liquid chromatography was used to separate the fluorescent adduct formed from the reaction of histamine with o-phthalaldehyde (OPT) from other biogenic amines in tissue, cerebrospinal fluid (CSF), sweat and urine samples. Using off-column derivatization and isocratic elution techniques fluorescent OPT adducts can be detected in the low picogram range. Perchloric acid extracts of tissue samples from Aplysia california and urine specimens collected from healthy adult males, including internal standard, were derivatized with OPT buffer, pH 9.5 and extracted with ethylacetate to increase sensitivity and stabilization of the fluorescent adduct prior to chromatography. Sweat and CSF samples were reacted with OPT buffer and aliquots of this mixture injected directly onto the chromatographic column (μBondapak CN) with methanol/0.08 mol/liter acetic acid (52/48 by volume) as the mobile phase. Assay of pooled urine containing added histamine (1 μg/ml) gave a with-in run coefficient of variation of 2.5%. The use of o-phthalaldehyde as an off-column HPLC derivatization agent for fluorometric determination of low-levels of biogenic amines is rapid, sensitive and easily adapted to routine use in a clinical or neurobiological laboratory.     

High Performance Liquid Chromatographic Determination of Phenacemide in Biological Fluids

Abstract

A sensitive and reliable high performance liquid chromatographic procedure has been developed for the quantitation of phenacemide in plasma or urine. After simple extraction of the drug with ethylacetate from alkalinized samples and evaporation to dryness, the reconstituted extract was chromatographed using a C₈ reversed phase analytical column with UV detection at 254 nm. Regression analyses for the calibration plots obtained on 3 different days for the drug concentrations ranging 1–15 mcg/ml indicated excellent linearity (r >0.999) and reproducibility (CV< 4%, p >0.01). The mean recovery of spiked phenacemide in plasma and urine from the lower limit of quantitation (1 mcg/ml) to 15 mcg/ml was 97.9 and 96.3%, respectively and their respective CV was 3.53 and 2.58%. The method was applied to monitor the plasma vs. time profile of the drug following a single bolus IV dose of 12 mg/kg in a dog.     

非衍生化高效液相色谱-串联质谱法快速检测生物体液中草甘膦、草铵膦及代谢物

建立了一种非衍生化高效液相色谱-串联质谱快速检测生物体液中草甘膦、草铵膦及其代谢物等8种极性农药的方法.8 种极性农药经Metrosep A Supp 5 阴离子色谱柱(150 mm x4.0 mm,5 μm)分离,以纯水-200 mmol/L碳酸氢铵溶液(含0.1％氨水)为流动相进行梯度洗脱,负离子多反应监测(MRM...     

Determination Of Chlorpromazine And Its Sulfoxide And 7-Hydroxy Metabolites By Ion-Pair High Pressure Liquid Chromatography

Abstract

An ion-pair HPLC approach with ordinary silica has been applied, with detection by ultraviolet absorption, to the assay of plasma for chlorpromazine and its sulfoxide on the one hand, and for 7-hydroxychlorpromazine (an active metabolite) on the other hand. The respective sample-preparation procedures entail extraction of the plasma with heptane at strongly alkaline pH, or else with diethyl ether at a less alkaline pH and with ensuing back-extraction and re-extraction. For each of the compounds, levels as low as 10 ng. ml^?1 are measurable. The conditions adopted are such that specificity and reproducibility are satisfactory although chlorpromazine and its various metabolites, especially 7-hydroxychlorpromazine, are chemically unstable and, moreover, are readily lost onto glass. With the unorthodox separation system adopted, adsorption rather than partition appears to be the dominant mechanism.     

A High Pressure Liquid Chromatography Procedure for the Separation of Metabolites of 2-Acetylaminofluorene from Cells in Culture

Abstract

A method is presented for the determination of pmole levels of the carcinogenic metabolite N-hydroxy-2-acetylaminofluorene (N-hydroxy-AAF) formed from N-2-acetylaminofluorene (AAF) by cells in culture. The extract from the cells and cell incubation medium was given a preliminary cleanup using thin layer chromatography. N-hydroxy-AAF was then isolated using high-pressure liquid chromatography (HPLC) with a Waters Bondapak C₁₈/Corasil column and an acetonitrile-water elution system. Ring hydroxylation products were eluted with 23% acetonitrile, and AAF and the deacetylation product 2-aminofluorene (AF) were eluted with 33% acetonitrile. Resolution of N-hydroxy-AAF was accomplished using a linear gradient of from 33% to 99% acetonitrile.

When cells in culture were incubated with 22 nmoles/ml of AAF for 6 h, 690 pmoles of N-hydroxy-AAF per 10⁶ cells were formed by hamster hepatocytes compared with 13 and 8 pmoles/10⁶ cells for human embryo and hamster embryo cells respectively. The rate of formation of N-hydroxy-AAF by hamster liver microsomes was 140 nmoles per h of incubation per mg of microsomal protein.     

High Pressure Liquid Chromatography of Selected Sulfur Compounds

Abstract

High pressure liquid chromatography was used to separate and determine quantitatively the following groups of sulfur compounds: thiols, sulfides, disulfides, sulfones, isothiocyanates, thioamides, and thioureas. Amperometric and UV detectors were compared; for thiols, thioureas, isothiocyanates, and thioamides, the former was generally more sensitive. With the exception of alkyl and cycloalkyl sulfides, the liquid chromatographic method can be used for the analysis of the investigated sulfur compounds below the ppm range. The method developed was compared to gas chromatography with flame photometric detection. The latter was found to be superior for the analysis of alkyl and cycloalkyl thiols, sulfides and disulfides of molecular weight below 200, whereas the former was more suitable for the analysis of aromatic thiols, sulfides and disulfides, as well as thioamides, isothiocyanates, and thioureas. Both methods were equivalent for the analysis of aromatic sulfones.     

High Pressure Liquid Chromatography of Thyromimetic Iodoamino Acids

Abstract

The chromatographic properties of 16 thyromimetic iodoamino acids and related compounds on microparticulate non-polar stationary phases have been examined and conditions determined which allow optimised resolution with analysis time ca. 60 minutes. These compounds elute in order of increasing hydrophobicity which correlates with the progressive increase in the number of iodogroups present in the tyrosine or thyronine aromatic nucleus. The reverse isomers, e.g. rT3, have consistently greater k' values than their corresponding analogues, e.g. T3. Conditions for the direct application of the rapid HPLC analyses of the iodoamino acids in biological or pharmaceutical samples have been examined.     

Analysis of Organic Acids in Physiological Fluids by High Performance Liquid Chromatography

Abstract

Most organic acids in physiological fluids are carboxylic acids or their glycine or glucuronide conjugates. Also included are hydroxyl compounds, such as phenols or cresols. By definition, compounds with a primary amino group, detectable by conventional amino acid analysers, are excluded. Organic acids are produced continuously in the body as intermediates in the metabolism of amino acids, carbohydrates and fats, and of drugs and food additives. They do not accumulate in the body, since they are rapidly converted to non-acidic end products or excreted as water soluble metabolites i n urine. However, if they are produced in excess, or if their metabolism is prevented by an inherited enzyme defect, concentrations increase in the tissues, blood and urine. Examples of excessive production are during fasting ketosis, in which acetoacetic and 3-hydroxybutyric acids and a range of dicarboxylic acids derived from breakdown of fatty acids accumulate, and lactic acidosis, secondary to hypoxia, when increases in lactic, pyruvic and 2-hydroxybutyric acids are common.     

Use of High Pressure Liquid Chromatography and Thin Layer Chromatography for the Separation and Detection of Testosterone and its Metabolites from in vitro Incubation Mixtures

Abstract

Testosterone and its 6β-, 7α-, and 16α-hydroxylated metabolites were resolved by high pressure liquid chromatography and thin layer chromatography. Separation by HPLC was achieved in less than 45 min on a microparticulate silica gel column using isocratic elution with isopro-panol:tetrahydrofuran:hexane (5:15:80) as the mobile phase. TLC systems utilizing silica gel on glass and plastic plates, and polysilicic acid on glass fiber sheets are presented. The monohydroxylated metabolites of testosterone formed during incubation of (¹⁴C)-testosterone with liver postmitochondrial preparations from adult male rats pre-treated with phenobarbital or Aroclor 1254 were separated and quantitated by both HPLC and TLC. The results using both techniques are compared with those obtained by paper chromatography.