Identification of cosmetic dyes by ion-pair reversed-phase high-performance liquid chromatography

A method based on ion-pair reversed-phase high-performance liquid chromatography with detection at four wavelengths between 400 and 600 nm is reported for the separation and identification of the most common synthetic colour additives in cosmetic products. All the dyes generally employed in the U.S.A. and almost all those in current use in cosmetics in the European Community have been taken into account. The chromatography was performed on a C8 bonded silica packed column, with a 60-min gradient changing from 10 to 95% acetonitrile in water containing 10(-2) M sodium perchlorate (pH 3.0) as mobile phase (flow-rate 2.5 ml/min). Detection limits are in the range 20-100 ng for all dyes investigated. The method has been applied to the analysis of commercial lipsticks.     

Assay of sialyltransferase activity by reversed-phase ion-pair high-performance liquid chromatography.

Sialyltransferases (CMP-N-acetylneuraminic acid:glycoprotein sialyltransferases, EC 2.4.99.1) are involved in the transfer of a sialic acid moiety from CMP-N-acetylneuraminic acid (CMP-NeuAc) to an oligosaccharide side-chain of an acceptor, asialoglycoprotein (AGP), according to the following reaction: CMP-NeuAc + AGP----NeuAc-O-AGP + CMP. This enzyme occurs in elevated levels in the sera of patients with a wide variety of neoplastic diseases and its assay might be useful in monitoring treatment. Radioactive CMP-NeuAc has been used in assays and the radioactive sialylated product separated and counted by liquid scintillation spectrometry. This study shows that a simple, rapid, non-radiochemically based high-performance liquid chromatographic method developed for the analysis of CMP-sialic acid synthetase can be used for the quantitation of sialyltransferase activity by monitoring simultaneously the utilization of CMP-NeuAc and the release of CMP. We describe the application of this method to assay of commercially available sialyltransferase activity and to activities from synovial, ascites and gastric fluids.     

Separation of translationally active mRNAs by reversed-phase ion-pair high-performance liquid chromatography

An ion-pair high-performance liquid chromatographic method on C4 columns was developed for the separation of mRNAs. The addition of methylmercuric hydroxide markedly influenced the separation according to length of these molecules. A method is given to recover minute amounts of translatable mRNA from the organic phase. The resolution of mRNAs improved with increasing pore size of the column support.     

Molecular species analysis of phosphatidylcholine by reversed-phase ion-pair high-performance liquid chromatography

The retention behavior of molecular species of phosphatidylcholine (PC) is studied by reversed-phase (RP) ion-pair high-performance liquid chromatography (HPLC). Mobile phases contain tetraalkyl ammonium phosphates (TAAPs) in methano-acetonitrile-water. The stationary phase is alkyl-bonded silica. Competitive interactions of TAAPs, analyte solutes, and an RP-HPLC column result in reduced retention of PC molecular species. PC molecular species are eluted at longer retention times with a larger size of TAAP in the mobile phase, and an increase in the TAAP concentration invariably causes a decrease in PC molecular species retention times. There is a linear correlation between the logarithmic retention factors (k) of PC molecular species and the total number of carbon atoms of TAAP, and the logarithm of k values of PC molecular species can be approximated as a linear function of the logarithm of the counter-ion concentration. There is found to be no distinct dependence between k values of PC molecular species and the mobile phase pH.     

Determination of nitrophenol derivatives in various crops by reversed-phase ion-pair high-performance liquid chromatography

A method for the determination of nitrophenol derivatives in various crops and soil is described. The sample is extracted with dichloromethane; the extract is evaporated to dryness and the residue is dissolved in an alkaline methanol/water mixture. After filtration this solution is injected. The eluent contains methanol, water, a phosphate buffer and hexadecyltrimethylammonium as the pairing ion. Blank chromatograms for various crops do not show interfering peaks with detection at 365 or 405 nm. The limit of detection is usually around 0.01 mg kg^-1; recoveries generally exceed 80% (coefficient of variation, 5–10%). For 2,4-dinitrophenylthiocyanate and dinocap, recoveries are somewhat lower.     

Detection of non-typical porphyrin isomers in human urines by ion-pair reversed-phase high-performance liquid chromatography

An improved ion-pair reversed-phase high-performance liquid chromatographic system has been developed for the separation of uroporphyrin isomers I, II and III, whereas the isomers III and IV could not be resolved. Application of this method to the analysis of urines from porphyric patients indicated the presence of small amounts of the non-typical uroporphyrin isomer II. The questionable presence of the isomer IV was confirmed by acid-catalyzed decarboxylation to the corresponding coproporphyrin isomers, which were completely separated by a modified ion-pair method at elevated column temperatures. These procedures enabled the detection of small fractions of the atypical isomers II (1-3%) and IV (8-15%) besides the normal isomers I and III in urines of patients suffering from attacks of acute intermittent porphyria. Because such urines contain large amounts of porphobilinogen, the nonenzymatic self-condensation of porphobilinogen to uroporphyrinogens was studied under mild reaction conditions. In these experiments quite similar isomeric compositions were observed as compared to those in urines of patients with acute intermittent porphyria. Thus the non-typical uroporphyrin isomers II and IV present in human urines originate from a simple non-enzymatic condensation of porphobilinogen.     

Measurement of tobramycin by reversed-phase high-performance liquid chromatography with mass spectrometry detection

Analysis of tobramycin faces challenges owing to its significant basicity, hydrophilicity and lack of a UV absorbing chromophore. Chromatographic methods, coupled with derivatization to introduce chromophores for tobramycin analysis, were extensively studied. A direct reversed-phase HPLC method for tobramycin analysis has not been reported. Here, we would like to report a simple LC/MS method for quantitative analysis of tobramycin in pharmaceutical formulations. Reversed-phase HPLC analysis of tobramycin was achieved using a pH stable C18 column with basic (pH 11) aqueous mobile phase (ammonium hydroxide buffer), while direct detection was carried out employing a single quadruple mass detector in negative mode via electrospray ionization. This unique separation-detection combination provided simple and specific determination of tobramycin. This method was found to be linear at a tobramycin concentration range of 0.2-0.8 mg/mL with a correlation coefficient value of 0.999. The quantitation limit and detection limit were calculated as 0.210 and 0.063 μg/mL, respectively, with 99.994% confidence. This method was successfully applied to measure tobramycin content in matrices containing tobramycin and other pharmaceutical formulation ingredients. Recoveries of 101.8, 97.8 and 106.7% were obtained for tobramycin spiked in the pharmaceutical formulation at concentrations of 1.68, 1.0 and 0.35 mg/mL, respectively. The relative standard deviations for six injections of spiked samples ranged from 0.2 to 3.2%, indicating good method repeatability.     

Determination of paraquat in rat brain using ion-pair solid-phase extraction and reversed-phase high-performance liquid chromatography with ultraviolet detection

A simple and sensitive procedure for the measurement of N-methylisoquinolinium ion (NMIQ+), a putative neurotoxin, was devised using high-performance liquid chromatography (HPLC) with fluorescence detection. Separation of NMIQ+ was carried out by gel filtration and reversed-phase HPLC on a column of hydrophilic polymer gels (Asahipak GS-302H). The method was sensitive enough to measure 50 fmol of NMIQ+. Uptake of NMIQ+ into rat striatal slices was confirmed by this method.     

Determination of remoxipride in human plasma and urine by reversed-phase ion-pair high-performance liquid chromatography.

A sensitive method is described for the measurement of remoxipride in human plasma and urine. Remoxipride and its internal standard are extracted from plasma or urine at pH 12 with a mixture of hexane and methyl tert.-butyl ether. After washing the organic phase with base, the compounds are extracted into acid and analyzed on a C18 column with ultraviolet detection at 214 nm. The mobile phase is composed of acetonitrile and aqueous buffer (sodium perchlorate and phosphoric acid, pH 1.7). The limits of reliable quantitation for remoxipride are 12.5 and 50 ng/ml for plasma and urine, respectively. The run times are 6 min for plasma and 3 min for urine. The method has been successfully used to assay remoxipride clinical study samples. This mobile phase has also been successfully applied to the analysis of other basic drugs such as cimetidine, codeine, diltiazem and quinidine with minor modifications.     

Use of an immobilized enzyme reactor for the analysis of residues of 17 alpha-methyltestosterone in trout by high-performance liquid chromatography

We have recently observed in trout that 48 h after ingestion of a single dose of [14C]-17 alpha-methyltestosterone ([14C]-MT), 25% of the radioactivity was still in the carcass, corresponding to metabolites of 17-MT. These compounds have no appreciable chromophore, fluorophore or electrophore, therefore the usual detection systems was not satisfactory for their analysis. Consequently a method was developed for high-performance liquid chromatographic separation and detection of two of the major tissue metabolites of 17-MT: 5 alpha-androstane-17 alpha-methyl-3 alpha,17 beta-diol and 5 beta-androstane-17 alpha-methyl-3 alpha,17 beta-diol. A column of immobilized 3 alpha-hydroxysteroid dehydrogenase was prepared and used for detection. The NADH produced from 3-hydroxysteroids by this immobilized enzyme reactor was monitored fluorimetrically. The detection limit of this method, as obtained from the calibration curve, was at the picomole level; the limit of quantification in muscle was 1 microgram/kg, at a signal-to-noise ratio of 4.     

Determination of ranitidine and its metabolites in human urine by reversed-phase ion-pair high-performance liquid chromatography

A method using ion-pair high-performance liquid chromatography is presented for determining ranitidine, ranitidine N-oxide, ranitidine S-oxide and desmethyl ranitidine in the urine from four volunteers, given on separate occasions an intravenous and oral dose of 100 mg ranitidine. This method has been used to study the metabolism and pharmacokinetics of ranitidine by man. It was found that the elimination half-life of ranitidine ranged from 110-246 min. The mean renal clearance of ranitidine in these four volunteers was 512 ml/min.     

Quantitative analysis of manganese, chromium and molybdenum by ion-pair reversed-phase high-performance liquid chromatography with pre-column derivatization and UV-visible detection.

An ion-pair reversed-phase high-performance liquid chromatographic method with UV-visible spectrophotometric detection is proposed for the simultaneous determination of manganese, chromium and molybdenum. By using a C18-bonded silica column, 4-(2-pyridylazo)resorcinol (PAR) chelates of Mn(II), Cr(VI) and Mo(VI) were successfully separated and accurately determined at 480 nm. Tetrabutylammonium bromide (TBAB) was used as the ion-pair reagent. Effects of pH, the buffer system, the concentration of buffer, the color developing time, the concentration of chelating reagent and the ion-pair reagent on the resolution were investigated. PAR chelates were eluted within 20 min at a flow-rate of 1.0 ml min(-1) with a methanol aqueous mobile phase, CH3OH-water (20:80, v/v), containing 1.0 x 10(-3) mol l(-1) acetate buffer (pH 6.5), 1.8 x 10(-2) mol l(-1) TBAB and 2.0 x 10(-4) mol l(-1) PAR. The feasibility of the proposed method was verified with the standard reference materials of nickel-based alloys. The nickel-based alloys were analyzed chromatographically after ammonium pretreatment. Under the optimum conditions, the detection limits for the chelates of Mn(II), Cr(VI) and Mo(VI) were 0.31, 4.2 and 4.6 ng with 100 microl injection, respectively. The accuracy of the proposed chromatographic method was verified by good agreement between the values obtained by this method and certified values.     

Determination of tetracycline antibiotics by reversed-phase high-performance liquid chromatography with fluorescence detection.

A highly sensitive method for the determination of tetracycline antibiotics (TCs) using reversed-phase high-performance liquid chromatography with fluorescence detection is presented. This method was based on the use of disodium ethylenediaminetetraacetate (EDTA) and calcium chloride as fluorescence-increasing reagents in the mobile phase. The concentrations of each reagent in the mobile phase greatly influenced the fluorescence intensity of TCs. When the concentration of EDTA and calcium chloride were 25 and 35 mM, respectively, and the pH of the mobile phase was 6.5, the maximum fluorescence intensity was obtained. The column temperature hardly influenced the fluorescence intensity. At 3.75 ng of TCs injected, the precision (relative standard deviation) ranged from 1.12 to 2.20%. In the range 0.075-37.5 ng for tetracycline and oxytetracycline and 0.225-37.5 ng for chlortetracycline, a linear response was observed. The detection limits of this method were 49-190 pg for three different TCs. The proposed method was applied to the determination of one of the TCs in pharmaceuticals by the internal standard method using other TCs as internal standards and was also applied to determination of TCs added to fish tissue.     

Analysis of heparins by size-exclusion and reversed-phase high-performance liquid chromatography with photodiode-array detection

High-performance liquid chromatography (HPLC) of heparins was carried out with on-line photodiode-array detection. Average molecular weights and molecular weight distribution were calculated using computer data acquisition and handling; size-exclusion chromatography was performed using different selective microparticulate columns and narrow molecular weight distribution heparin standards for calibration; heparin peak purity was investigated using several methods for evaluation. Additional UV-absorbing compounds present in heparin preparations were characterized and quantitated by reversed-phase HPLC. These methods are useful for the analysis of the molecular weight distribution and peak purity of heparins and for the determination of additional drugs, additives or impurities.