Analysis of propantheline bromide and related quaternary ammonium drugs by reversed-phase,ion-pair liquid chromatography with two counterions in the eluent

Summary The addition of appropriate concentrations of an organic amine and an alkylsulphonate to the mobile phase in reversed-phase, ion-pair liquid chromatography can introduce unique selectivity in to the chromatographic system allowing separation of complex mixtures of basic, acidic and neutral compounds. As an example, the methodology for a specific stability-indicating determination of propantheline bromide, a quaternary ammonium anticholinergic agent, on several reversedphase stationary phases, was developed. The retention mechanism was studied and it was concluded that both ion-interaction processes and ion-exchange processes were involved in the separation technique developed with two counterions of opposite charge in the eluent.     

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.     

Determination of phosphonoformate (foscarnet) in biological fluids by ion-pair reversed-phase liquid chromatography

Bioanalytical liquid chromatographic methods for the determination of phosphonoformate (foscarnet) have been developed. Biological fluids, after simple pre-treatment (ultrafiltration and/or treatment with charcoal), were injected into a reversed-phase liquid chromatographic system with electrochemical detection. Foscarnet was retained as an ion pair with tetrahexylammonium; addition of pyrophosphate was necessary in order to obtain an acceptable peak. This additive could also be used for the fine regulation of the retention to achieve the necessary selectivity.     

反相离子对色谱法测定间苯二磺酸钠

运用反相高效离子对色谱法测定了间苯二磺酸钠.采用C18色谱柱(250 mm×4.6 mm i.d.,5 μm),流动相为V(乙腈):V(50 mmol/L NaH2PO4)＝7:93(含0.05%四丁基氢氧化铵),流速为1.0 mL/min,检测波长210 nm,柱温:30 ℃.间苯二磺酸钠在0.32×10-3～81.92×10-3 mg/mL范围内线性关系良好,r＝0.9999,加样回收率为98.8%～101.1%,RSD＝0.73%(n＝7).本法为工业生产间苯二磺酸钠提供了分析方法.     

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.     

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.     

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.     

Determination of S-carboxymethylcysteine in serum by reversed-phase ion-pair liquid chromatography with column switching following pre-column derivatization with o-phthalaldehyde

A method is described for the determination of S-(carboxymethyl)-L-cysteine in serum. After addition of S-(carboxyethyl)-L-cysteine as internal standard, both compounds are extracted into methanol, converted into fluorescent derivatives with o-phthalaldehyde and quantitatively determined by reversed-phase high-performance liquid chromatography. Chromatography of unwanted amino acid derivatives is avoided by column switching, thereby shortening analysis time and increasing column lifetime. The technique was applied in a study of the bioavailability of S-(carboxymethyl)-L-cysteine after oral administration to humans. The concentration-response curve was linear from 2 to 16 micrograms/ml; mean serum concentrations are reported.     

Characterization of small interfering RNA by non-denaturing ion-pair reversed-phase liquid chromatography

Small interfering RNAs (siRNA) are emerging as a novel therapeutic modality for the specific inhibition of target gene expression. siRNA are typically formed by annealing of two complementary single stranded oligoribonucleotides. Compared to purity determination of non-hybridized single strands by denaturing chromatographic methods, characterization of the hybridized duplex is challenging. Here we are reporting a non-denaturing ion pairing-reversed phase (IP-RP) chromatography method capable of separating optimal duplex (full-length single strands only) from non-optimal duplex variants (containing shortmers, longmers and 2',5'-isomers) using ultraviolet- and mass spectrometric detection. The impact of different annealing conditions on siRNA composition was investigated. Optimized annealing conditions lead to a significant increase in optimal duplex, while total duplex content remained constant. The non-denaturing method reported herein showed high mass spectrometric sensitivity and superior separation efficiencies compared to other IP-RP buffer systems. The method is useful for in-process control and release testing of therapeutic double stranded nucleic acids such as siRNA.     

Speciation of arsenical species by anion-exchange and ion-pair reversed-phase liquid chromatography

Summary Speciation and quantitative analysis of arsenical compounds are performed by using high-performance liquid chromatography (HPLC) with direct UV detection. Ion chromatography has been used to separate mixtures of arsenical compounds (arsenite, MMA, DMA, arsenate) on an anion-exchange column using phosphate buffer (1 mmol/l, pH=5.3) as eluent. Ion -pair reversed-phase chromatography has been investigated to resolve mixtures of arsenite, arsenate, MMA, DMA, arsenobetaine and arsenocholine on an octadecyl-bonded silica column using water as mobile phase (pH=7.3) and tetrabutylammonium cation as ion-pairing reagent. The influence of several parameters (pH, the ion-pairing reagent concentration or the amount of methanol in the mobile phase) has been studied to determine the best chromatographic conditions.     

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.     

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.     

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.     

Determination of propionylpromazine hydrochloride in formulation matrixes using reversed-phase ion-pair small bore liquid chromatography

Propionylpromazine hydrochloride (PPZHCl) has been investigated for use with leghold traps to reduce the amount of self-inflicted trauma experienced by animals restrained by these traps. Three types of PPZHCl formulations made with Karo dark syrup, K-Y Jelly, and Vaseline were used in 2 types of tranquilizer trap devices (TTDs). A reversed-phase ion-pair liquid chromatography (LC) method using a small bore C18 column was used to: (1) determine the purity of the PPZHCl material used in these formulations, and (2) to determine the resulting PPZHCl content of each formulation. Analyte quantitation was done using UV absorption at 280 nm. Regression analysis of calibration standard solutions indicated a linear and directly proportional relationship between analyte response and PPZHCl concentration over the range evaluated. Recovery data from: (1) Vaseline formulations containing 38.8, 16.2, and 8.78% PPZHCl were 104, 92.9, and 90.2%, respectively, (2) Karo dark syrup formulations containing 26.5, 18.1, and 10.3% PPZHCl were 97.7, 99.3, and 106%, respectively, and (3) K-Y Jelly formulations containing 33.0, 23.5, and 13.4% PPZHCl were 100, 99.4, and 88.7%, respectively. The relative standard deviation (RSD) values from triplicate analysis of these formulations ranged from 0.7 to 6.7%. The PPZHCl content from 9 manufactured TTDs, 3 for each formulation type, were analyzed in triplicate and produced RSD values ranging from 0.7-6.8%. These results indicate that the formulation extraction presented could be used to evaluate the PPZHCl content in TTDs prior to field use. The use of a small bore LC column reduced the amount of solvents consumed and hazardous waste generated, compared to sample analysis that uses a more conventional analytical LC column.     

反相离子对色谱法测定脱氧精胍菌素药物中的主成分

建立了反相离子对色谱法分离测定脱氧精胍菌素药物中主成分的方法。分别考察了色谱柱类型、离子对试剂种类及浓度、缓冲盐浓度和流动相pH值等参数对实验结果的影响。确定了分离脱氧精胍菌素的最佳条件: C18色谱柱(250 mm×4.6 mm, 5 μm);以5 mmol/L磷酸氢二钾水溶液(含5 mmol/L戊烷磺酸钠,pH 3.6±0.3)-乙腈(90:10, v/v)为流动相,流速为1.0 mL/min;检测波长为210 nm;柱温为30 ℃;进样量为20 μL。该方法实现了对脱氧精胍菌素的良好分离,且线性关系良好,检出限可达0.5 mg/L。