Optical separation of racemic 5-(p-hydroxyphenyl)-5-phenylhydantoin by reversed phase high-performance liquid chromatography using eluents containing beta-cyclodextrin.

Both S-(-)- and R-(+)-enantiomers of 5-(p-hydroxyphenyl)-5-phenylhydantoin (p-HPPH), a main oxidative metabolite of the achiral antiepileptic drug phenytoin, could be determined simply, sensitively and accurately using reversed phase high-performance liquid chromatography by using a methanol-monopotassium phosphate eluent containing beta-cyclodextrin. Using this assay procedure, it was determined that an S-(-)-enantiomer was formed predominantly by the oxidation of phenytoin in isolated rat hepatocytes.     

5-(对-羟基苯基):-5-苯基乙内酰脲的反相高效液相手性分离测定法

以β－ＣＤ为手性流动相添加剂、苯巴比妥为内标,于ＦＬＣ－Ｃ８反相柱上建立了鼠肝微粒体中５－（对－羟基苯基）－５－苯基乙内酰脲（ｐ－ＨＰＰＨ）外消旋体的拆分方法。测得ｐ－ＨＰＰＨ对映体的线性范围为０．５～１１０ｍｇ／Ｌ（ｒ＝０．９９９６）;最低检出量为５ｎｇ（Ｓ／Ｎ＝３）;Ｓ－ｐ－ＨＰＰＨ的回收率为９３．６％±２．８％,Ｒ－ｐ－ＨＰＰＨ的回收率为９４．７％±１．８％;日内和日间精密度ＲＳＤ值均小于２％。所建立的方法具有结果准确、操作方便等特点。     

Simultaneous determination of acetylsalicylic acid and salicylic acid by gas-liquid chromatography using a new methylation technique

Summary A GLC method of simultaneous determination of acetylsalicylic acid and salicylic acid after a simple and elegant methylation is presented. Acetonic solution of these acids is refluxed with methyl iodide and potassium carbonate for 30 minutes and then gas chromatographed with a column of 3% OV 17 on Varaport 30, 70–80 mesh, AW/DMCS. The methylation is quantitative in 0.001–0.1 M solutions. The detection limit is about 25 ng.     

Simultaneous determination of N-acetylglucosamine, N-acetylgalactosamine, N-acetylglucosaminitol and N-acetylgalactosaminitol by gas-liquid chromatography

A gas-liquid chromatographic procedure is described which will concomitantly separate and quantitate N-acetylglucosamine, N-acetylgalactosamine, N-acetylglucosaminitol and N-acetylgalactosaminitol in a single analytical run. The hexosamines, as their O-methyloximes, and the hexosaminitols can be separated as either their per-O-acetylated or per-O-trimethylsilylated derivatives. This method is particularly useful with samples that possess both N-acetylhexosaminitols and N-acetylhexosamines as are seen with N-linked oligosaccharides that are cleaved from glycoproteins by alkaline borohydride treatment. This procedure demonstrates a range of acceptable linearity of 1-1000 nmoles for each type of amino sugar.     

Determination of indomethacin in serum and urine by electron-capture gas-liquid chromatography.

A specific and sensitive method for the quantitative determination of indomethacin in serum and urine is described. The drug is extracted at pH 5.0 with 1,2-dichloroethane and a portion of the organic extract is concentrated and made to react with diazoethane in diethyl ether. The ethyl ester derivative is analyzed by electron-capture gas-liquid chromatography, quantitation being achieved by comparison of peak areas for samples and standards, which are prepared in serum or urine and treated in the same manner as the samples. The limit of sensitivity is 50 ng/ml and the relative standard derivation for repeat determinations on the same sample is about 3%.     

Simultaneous determination of amiodarone and its major metabolite desethylamiodarone in plasma, urine and tissues by high-performance liquid chromatography

A simple and sensitive high-performance liquid chromatographic method for the simultaneous assay of amiodarone and desethylamiodarone in plasma, urine and tissues has been developed. The method for plasma samples and tissue samples after homogenizing with 50% ethanol, involves deproteinization with acetonitrile containing the internal standard followed by centrifugation and direct injection of the supernatant into the liquid chromatograph. The method for urine specimens includes extraction with a diisopropyl ether-acetonitrile (95:5, v/v) mixture at pH 7.0 using disposable Clin-Elut 1003 columns, followed by evaporation of the eluate, reconstitution of the residue in methanol-acetonitrile (1:2, v/v) mixture and injection into the chromatograph. Separation was obtained using a Radial-Pak C18 column operating in combination with a radial compression separation unit and a methanol-25% ammonia (99.3:0.7, v/v) mobile phase. A wavelength of 242 nm was used to monitor amiodarone, desethylamiodarone and the internal standard. The influence of the ammonia concentration in the mobile phase on the capacity factors of amiodarone, desethylamiodarone and two other potential metabolites, monoiodoamiodarone (L6355) and desiodoamiodarone (L3937) were investigated. Endogenous substances or a variety of drugs concomitantly used in amiodarone therapy did not interfere with the assay. The limit of sensitivity of the assay was 0.025 micrograms/ml with a precision of +/- 17%. The inter- and intra-day coefficient of variation for replicate analyses of spiked plasma samples was less than 6%. This method has been demonstrated to be suitable for pharmacokinetic and metabolism studies of amiodarone in man.     

High-performance liquid chromatographic method for direct separation of 5-(p-hydroxyphenyl)-5-phenylhydantoin enantiomers using a chiral tris(4-methylbenzoate) column.

After simple purification of the incubation mixture of phenytoin in isolated rat hepatocytes, 5-(p-hydroxyphenyl)-5-phenylhydantoin (p-HPPH), which formed as a major metabolite, was readily resolved to each enantiomer by direct high-performance liquid chromatography on a cellulose tris(4-methylbenzoate) column, with a mobile phase of ethanol-water. It was also observed that the formation of S-(-)-p-HPPH was dominant, and the S/R ratio was 11.5.     

Simultaneous determination of neopynamine and piperonyl butoxide by capillary column gas-liquid chromatography

A method is developed for the simultaneous determination of neopynamine and piperonyl butoxide by chromatography on a quartz capillary column using flame ionization detection. Benzyl mandelate is used as an internal standard. Calibration graphs are linear down to at least 3.75 mg% and 3 mg% for neopynamine and piperonyl butoxide, respectively.     

Simultaneous determination of some active ingredients in cough-cold syrups by gas-liquid chromatography

A simple, efficient and accurate gas-liquid chromatography (GLC) method for the simultaneous determination of eight active ingredients in cough-cold syrups has been developed. The active ingredients under study were bromhexine, chlorpheniramine, codeine, dextromethorphan, diphenhydramine, ephedrine/pseudoephedrine, guaiphenesin and papaverine. Before injection, the active ingredients were first separated, from the excipients present in the cough-cold syrups, with chloroform, from alkaline medium. They were then separated by GLC on a glass column (5 ft x 2 mm i.d.) packed with 3% of OV-25 supported on Supelcoport (80-100 mesh). The column temperature was maintained at 170 degrees C for 1 min, then programmed to 265 degrees C at a rate of 10 degrees C min-1, and maintained at this temperature for 10 and 1 min, respectively, for samples with and without papaverine. The flow-rate of the nitrogen carrier gas was 30 ml min-1. A flame-ionisation detector was used for detection, and clomipramine hydrochloride was used as the internal standard. The recoveries of the drugs ranged from 96.0 to 99.7%, and the relative standard deviations for ten replicate determinations ranged from 0.49 to 4.7%. Results are reported for nine commercially available cough-cold syrups.     

Mass fragmentographic determination of diphenylhydantoin and its main metabolite, 5-(4-hydroxyphenyl)-5-phenylhydantoin, in human plasma.

A method is described for the mass fragmentographic determination of diphenylhydantoin and its main metabolite, 5-(-4-hydroxyphenyl)-5-phenylhydantoin (4-OH-DPH), in human plasma as their dimethyl and trimethyl derivatives, respectively. The derivatives are formed by using the recently described extractive alkylation technique. Pentadeuterated 4-OH-DPH is used as the internal standard. Following acidic hydrolysis of the plasma sample, conjugated 4-OH-DPH and, indirectly, the dihydrodiol metabolite, 5-(3,4-dihydroxy-1,5-cyclohexadien-1-yl)-5-phenylhydantoin, are measured. Using 100-mul plasma samples, the lower limit of detection is about 10 ng/ml 00.03 nmole/ml).     

Simultaneous determination of the sulphonylurea glimepiride and its metabolites in human serum and urine by high-performance liquid chromatography after pre-column derivatization

A sensitive and selective high-performance liquid chromatographic method has been developed for a new sulphonylurea, glimepiride, and its metabolites. The assay involves extraction with diethyl ether, thermolysis of the sulphonylureas at 100 degrees C and trapping of the resulting amines with 2,4-dinitrofluorobenzene. The derivatives were quantitated on a reversed-phase column by absorbance at 350 nm using a step gradient for the three compounds in serum and an isocratic run for the metabolites in urine. Analogous compounds were used as internal standards. The detection limit was 5 ng/ml for glimepiride and metabolite II and 10 ng/ml for metabolite I using 1 ml of serum. The method has been applied to the analysis of serum and urine samples from pharmacokinetic studies in humans.     

Simultaneous determination of phenolphthalein and phenolphthalein glucuronide from dog serum, urine and bile by high-performance liquid chromatography.

A procedure is described to simultaneously quantitate phenolphthalein and its glucuronide metabolite from dog serum, urine and bile using high-performance liquid chromatography. The major advantages of this over pre-existing methods include direct analysis of the parent compound and glucuronide metabolite without enzymatic hydrolysis, increased sensitivity and the potential for automation of a large number of samples. Analytes were extracted from serum and urine using a combination of liquid- and solid-phase extraction methodology. Bile samples were analyzed directly after a twenty-fold dilution with mobile phase. The components plus internal standard were separated by reversed-phase high-performance liquid chromatography using step gradient elution and quantitated by the absorbance of ultraviolet light at 230 nm. Limits of detection from 1 ml of serum, 0.1 ml of urine and 0.05 ml of bile were 0.1, 0.5 and 10 microgram/ml for phenolphthalein and 0.1, 10 and 50 microgram/ml for phenolphthalein glucuronide, respectively.     

Fluorescence determination of 5-fluorouracil and 1-(tetrahydro-2-furanyl)-5-fluorouracil in blood serum by high-performance liquid chromatography

Fluorescence derivatization of 5-fluorouracil (5-FU) and 1-(tetrahydro-2-furanyl)-5-fluorouracil (ftorafur, FT) with 4-bromomethyl-7-methoxycoumarin using 18-crown-6 as a catalyst is studied with aim of developing a sensitive and selective liquid chromatographic method. 5-FU and FT form virtually substituted derivatives which possess maxima in their fluorescence emission spectra near 400 nm. These derivatives are separated by thin-layer chromatography and high-performance liquid chromatography to confirm the completion of reaction. For the determination of 5-FU and FT in serum, the reversed-phase high-performance liquid chromatographic separation of the derivatives is studied with a C13 column. This chromatography is of importance for the accurate determination of 5-FU and FT, which are, respectively, an important antitumour agent for the treatment of solid tumours in clinical medicine and a masked form of 5-FU generated in vivo.