Microbore reversed-phase chromatography of proteins with conventional gradient equipment for high-performance liquid chromatography

Reversed-phase high-performance liquid chromatography with microbore columns (50 X 1.0 mm) was used effectively for the separation and analysis of proteins down to 1 ng at flow-rates of 0.1-0.2 ml/min. With the use of standard low-pressure gradient HPLC equipment, the peak volumes were five times smaller when compared with a conventional column at equal chromatographic efficiencies and analysis time. The sensitivity of detection was further increased by a reduction in solvent peaks, resulting in a 20-fold overall increase.     

Micellar liquid chromatography determination of B vitamins with direct injection and ultraviolet absorbance detection

A micellar reversed-phase liquid chromatographic procedure was developed for the control of five water-soluble vitamins, B (nicotinamide), B1 (thiamine), B2 (riboflavin), B6 (pyridoxine and pyridoxamine), in multivitamin pharmaceutical formulations (capsules, pills and syrups). Optimization procedure includes studies about the composition of the mobile phase (sodium dodecyl sulphate and the modifiers propanol, butanol or pentanol), flow-rate and temperature. Chromatographic analysis of all vitamins was carried out using a single mobile phase of 0.1 M SDS-4% (v/v) pentanol at pH 3, in a C18 column in isocratic mode, and UV-detection at 270, 290 and 325 nm. The flow-rates selected were 1.0 ml/min in the interval 0 to 6 min, and 2.0 ml/min until the end of the chromatogram and temperature was 45 degrees C. In the micellar liquid chromatographic system, the samples were injected without pretreatment, and the analysis time was below 12 min. Repeatabilities and intermediate precision were achieved according to ICH, and were below 5%. When the method is applied to real samples, the amount found with respect to the declared compositions were within the 91-105% range. These results were similar to those obtained with a conventional 60:40 (v/v) methanol-water mixture for some of the vitamins, but with the advantage of use a single mobile phase for the analyses of the five vitamins, with direct injection of the samples and reduced toxicity, flammability, environmental impact and cost of the micellar-pentanol solutions.     

Ultra-short columns and ballistic gradients: considerations for ultra-fast chromatographic liquid chromatographic-tandem mass spectrometric analysis

Ultra-fast chromatographic separations has enabled fast chromatographic method development and rapid analysis for sample quantification. Decreasing over-all analytical time has become a factor of major importance for all aspects of drug discovery. However, merely decreasing chromatographic analysis time by decreasing k' can lead to inconsistent quantitative or qualitative results due to ineffective separations in complex matrices. We have found that by changing column length and gradient slope we can maintain chromatographic integrity of chemically diverse analytes and achieve the analytical speed required for bioanalytical drug discovery quantitative analysis. We have optimized method development strategy by performing separations on 2x20 mm HPLC columns at flow-rates of 1.5 ml/min to 2 ml/min with full linear gradients achieved in 1 min for the quantification of pharmaceuticals and their metabolites from biological matrices. This method development strategy can be readily adapted to other matrices. This paper will discuss the effects of column length and gradient time in ultra-fast chromatographic resolution.     

Control of voltammetric experiments by means of the Commodore 64 microcomputer

An electronic interface for the Commodore 64 microcomputer suitable for generation of voltammetric waveforms and for data acquisition has been built. Used together with analogue voltammetric instruments the interface makes updating of the measurement techniques possible. Also, fast A/D-conversion and a floppy disk drive make the system useful as a universal data-acquisition unit in the laboratory. The system has been tested together with an amperometric detector in the square-wave voltammetric determination of paracetamol and iodide.     

Rapid reversed-phase separation of proteins and peptides using optimized 'moulded' monolithic poly(styrene-co-divinylbenzene) columns

Monolithic macroporous poly(styrene-co-divinylbenzene) stationary phases have been prepared by free radical polymerization within the confines of 4.6-mm I.D. chromatographic columns. The optimized porous properties allow the mobile phase to flow through these columns at flow-rates of up to 10 ml/min. As opposed to the simultaneously tested columns packed with either silica or synthetic polymer beads, the monoliths exhibit only modest back pressure. The monolithic columns were able to separate mixtures of peptides and proteins in a very short time. Under the optimized conditions, the separation of five proteins can be easily achieved in less than 20 s.     

On-line photo-oxidation for the determination of organoarsenic compounds by atomic-absorption spectrometry with continuous arsine generation

A method has been developed for on-line conversion of organoarsenicals into arsenate, which is readily detected by atomic-absorption spectrometry with continuous arsine generation. The photoreactor consists of a mercury lamp wrapped with 5 m of PTFE tubing (0.5 mm i.d.). The photo-oxidation conditions were optimized, with a flow-injection analysis procedure, for arsenobetaine, monomethylarsonic acid, dimethylarsinic acid, o-arsanilic acid, and phenylarsonic acid, all organoarsenicals of biological and environmental importance. Solutions were continuously pumped at a flow-rate of 2.0 ml/min and combined with a stream of potassium persulfate (flow-rate 0.6 ml/min) before entering the photo-reactor. With reactor dwell times of 36 sec, conversion efficiencies for these compounds were above 95% under these conditions. Interfacing of this flow-through conversion and detection system with a chromatographic inlet permits real-time analysis of mixtures of these organoarsenic compounds, in a manner suitable for environmental or biomedical samples.     

Separation of oligonucleotides on novel monolithic columns with ion-exchange functional surfaces.

Porous monolithic columns have been prepared by the direct free radical copolymerization of glycidyl methacrylate and ethylene dimethacrylate within the confines of a 50x8 mm I.D. chromatographic column in the presence of porogens. The epoxide groups of these monoliths were modified to different extents by reaction with diethylamine to afford 1-N,N-diethylamino-2-hydroxypropyl functionalities useful for ion-exchange chromatography. Following characterization of the monoliths, the columns were tested in the chromatographic separation of a homologous series of oligodeoxyadenylic [pd(A)(12-18)] and oligothymidylic acids [d(pT)(12-24)] at different flow-rates. Very good separations of the oligonucleotides were achieved even at the high flow-rate of 4 ml/min.     

HPLC Determination of 6-Thiouric Acid and 6-Mercaptopurine in Organ Transplant Patient Serum

Abstract

A sensitive high performance liquid chromatographic method for the simultaneous determination of 6-thiouric acid and 6-mercaptopurine in serum is described. Our intent was to develop a procedure that could be used for pharmacokinetic studies and therapeutic drug monitoring in organ transplant patients taking azathioprine. Serum samples were precipitated with acetonitrile containing 6-n-propyl-2-thiouracil as the internal standard. The chromatographic separation was performed with an octadecylsilane column and gradient solvent system consisting of acetonitrile and 0.01 M sodium dihydrogen phosphate, pH 6.1. An initial acetonitrile concentration of 1% was used to elute 6-thiouric acid but was increased to 16% to recover the 6-mercaptopurine and internal standard. The flow rate was increased from 1.3 ml/min to 1.5 ml/min during the analysis. The column effluent was monitored at 353 nm and 323 nm for detection of 6-thiouric acid and 6-mercaptopurine, respectively. Statistical analysis of standard curve data showed good intra- and inter-day accuracy, precision and reproducibility throughout a concentration range of 10–2500 ng for 6-thiouric acid and 10–500 ng for 6-mercaptopurine/ml of serum. The method has been applied to the quantification of 6-thiouric acid and 6-mercaptopurine in serum from two kidney allograft recipients.     

Comparison of a jet separator and an open splitter as an interface between a multi-capillary gas chromatographic column and a time-of-flight mass spectrometer

A gas chromatographic/time-of-flight mass spectrometric (GC/TOFMS) interface is being developed for fast on-line analysis utilizing multi-capillary column technology. A variable gap-distance jet separator has been constructed and its performance compared with that of a commercially supplied post-column open splitter recommended for use between the multi-capillary column and a mass spectrometer. Both interfaces were found to be compatible with the GC/TOFMS system at high carrier gas flow-rates, facilitating high-speed and high-resolution separations. The systems were investigated and tested with a mixture of volatile organic compounds (VOCs) with molecular masses from 85 to 166: dichloromethane, toluene, m-dichlorobenzene, o-dichlorobenzene and tetrachloroethylene. The optimum tip-to-tip gap distance corresponding to the highest efficiency of the jet separator was found to be 0.030 mm for each compound at carrier gas flow-rates of 20, 40 and 60 ml min(-1) giving, in the ion source housing, ion gauge pressure readings of 1.6 x 10(-6), 5.0 x 10(-6) and 5.8 x 10(-6) mbar, respectively. The efficiency of the jet separator (10-30% yields) was significantly higher than that of the open splitter (6-9% yields). The observation that the open splitter did not provide a constant flow-rate to the ion source was not in agreement with the manufacturer's specifications. A method for measuring the gas flow-rates in all parts of the equipment is described. The correlation between yield in the jet separator and molecular mass for the heterogeneous set of compounds studied was found to be less linear than usually reported for homologous series of compounds in jet separator studies. The result suggests that the pressure conditions in the jet may be sufficient for the separation process to be partly controlled by diffusion rather than predominately by effusion. Copyright 2000 John Wiley & Sons, Ltd.     

Selection of optimal pH and solvent composition for the separation of organic acids using three-dimensional diagrams and a computer program

Summary The retention behaviour of different types of organic acids was investigated on an ODS-Hypersil column using eluents of various pH and of various methanol-water ratios. Equations introduced by Horvath and coworkers were fitted onto the experimental data, and the obtained constants were used to calculate the three-dimensional k′-surfaces for the compounds in the investigated pH and solvent composition range. Using the data of the individual surface diagrams the optimal pH and solventg composition was computed for the separation of six selected compounds. To verify the applicability of the above procedure the chromatographic separation of the six compounds was carried out with the predicted conditions, and the chromatogram obtained is presented.     

High-performance liquid chromatographic determination of mitoxantrone in plasma utilizing non-bonded silica gel for solid-phase isolation to reduce adsorptive losses on glass during sample preparation

Mitoxantrone, a highly active antineoplastic agent, was found to bind strongly to non-bonded silica gel and glassware. When a Hamilton syringe was used to load and inject a mitoxantrone solution (0.4 microgram/ml in water) on to a high-performance liquid chromatographic (HPLC) system, about 95% of the loaded compound was found to bind to the glass surface of the syringe barrel and could not be removed by rinsing with water. It could, however, be removed slowly with an acidic solution and thus a small peak of mitoxantrone was present on the chromatogram whenever a blank acidic solution was injected with the syringe. The bound mitoxantrone could be removed effectively from the syringe surface with a solution of tetramethylammonium chloride, citric acid, methanol and water (elution solvent). This binding introduces a large error in assay results and might be one of the major factors responsible for contradictory pharmacokinetic data that have been reported. A new plasma preparation scheme and an HPLC method for mitoxantrone were developed to address this binding problem. Mitoxantrone was extracted directly from plasma samples with a plastic mini-column packed with non-bonded silica gel and eluted with the above elution solvent. The eluent was analysed by HPLC on an ODS column with an absorbance detector at 658 nm. The mobile phase was 0.1 M triethylamine phosphate (pH 3.0) in water-tetrahydrofuran-methanol (69:1:30) containing 0.02 M tetramethylammonium chloride. Methylene blue was added as an internal standard. Preliminary results showed that mitoxantrone levels in human plasma followed a triphasic decay curve after an intravenous bolus injection. The terminal elimination half-lives measured in three patients (mean t1/2 gamma = 25 min) were all shorter than the published values which ranged from 56 min to 9 days.     

Fast screening method for the determination of angiotensin II receptor antagonists in human plasma by high-performance liquid chromatography with fluorimetric detection

A selective, accurate and precise high-performance liquid chromatographic assay coupled to fluorescence detection was developed for the detection of some angiotensin II receptor antagonists (ARA II): Losartan, Irbesartan, Valsartan, Candesartan cilexetil and its metabolite Candesartan MI. The analytes and the internal standard (bumetanide, a high-ceiling diuretic) were extracted from plasma under acidic conditions by means of solid-phase extraction using C8 cartridges. This procedure allowed recoveries close to 80% for all these drugs excluding Candesartan cilexetil (70%) which presented adsorption processes on glass and plastic walls. The analytes and potential interferences were separated on a reversed-phase column, muBondapak C18, at room temperature. A gradient elution mode was used to carry out the separation, the optimal mobile phase being composed of acetonitrile-5 mM acetate buffer, pH 4, at variable flow-rates (from 1.0 to 1.2 ml/min). Fluorescence detector was set at an excitation wavelength of 250 nm and an emission wavelength of 375 nm. Intra- and inter-day relative standard deviations for all the compounds were lower than 8% except for Losartan (12%) and the method assesses a quite good accuracy (percentage of relative error approximately 6% in most of the cases). The limit of quantitation for these compounds was 3 ng/ml for Candesartan cilexetil and M1, 16 ng/ml for Losartan and 50 ng/ml for Irbesartan and Valsartan, which allows their determination at expected plasma concentration levels. This assay method has been successfully applied to plasma samples obtained from hypertensive patients under clinical studies after oral administration of a therapeutic dose of some of these ARA II compounds.     

Spiral coils for counter-current chromatography using aqueous polymer two-phase systems

Retention properties of polyethylene glycol-phosphate aqueous two-phase systems in a spiral coil (5 mm I.D.) on Type-J synchronous counter-current chromatographic devices have been compared for the elution mode where the lower phase is the mobile phase and flows from the inside head terminal. This was achieved with the aid of digital imaging under stroboscopic illumination, an image analysis and measurement of the displaced volume of the stationary phase. For the spiral coil, high and stable stationary phase retention at mobile phase flow rates up to 64 ml/min has been obtained. Wave-like disturbance of the interface near the proximal point was observed and analyses have been made for possible use in protein separation.     

Improved N-P thermionic-GLC resolution using the SGE-solids injector

Summary The improvement of N/P-GLC chromatogram resolution is described. The use of a SGE-solids injector eliminates off-scale tailing solvent peaks, eliminates loss of information during desensitization of the detector and permits injection from any solvent.     

气相色谱质谱联用仪与微机的数据传输和处理

报道了一种用于气相色谱质谱联用仪和微机之间实现数据传输和处理的方法。方法可更有效地利用质谱仪采集的数据，解决了工作站处理数据的局限性。经数据格式转换，原始数据可以在微机上实现色谱峰再现，从而为色谱条件的优化和定量数据处理创造了条件。     

Rapid high-pressure liquid chromatographic analysis of verapamil in blood and plasma

A high-pressure liquid chromatographic assay procedure has been developed for verapamil in blood or plasma. A paired-ion solvent system with a reversed-phase column is employed. The procedure is specific for verapamil and the retention times of the major metabolites are identified. This procedure is sensitive to a lower blood concentration of 1 ng/ml and standard curves were found to be linear up to the highest concentration tested, 500 ng/ml. Several drugs were tested for interference with the assay, but none were found to cause any problems. The procedure is simple, rapid and permits the analysis of up to 25 samples per day.     

Quantitative inorganic paper chromatography direct determination of cobalt by a scanning method

Cobalt can be determined in the presence of other elements by chromatography followed by photometric scanning. The chromatographic solvent used is a mixture of acetone, n-butanol, hydrochloric acid and acetylacetone, and the cobalt spot is made visible by spraying with a solution of rubeanic acid. The filter paper is cut in strips which are scanned along their length either in a Beckman DU spectrophotometer or a Photovolt densitometer Mod. 525 at 422 mμ. The absorbance curves are plotted and the Co concentration is calculated from the area under the peaks. Chromatograms with 8 spots, each containing 0.4 — 2 μg Co, of which 4 are standards, are convenient. The coefficient of variation for the mean of a single chromatogram is 6 %. Alloys containing as little as 0.2 % Co can be analysed with an accuracy of 5 % or better.     

Hydrophilic interaction liquid chromatographic tandem mass spectrometric determination of atenolol in human plasma

A hydrophilic interaction liquid chromatographic method with tandem mass spectrometry for the determination of atenolol, a beta-blocking agent, in human plasma has been developed and validated over the curve range of 10--2000 ng/mL. The assay was based on protein precipitation followed by evaporation of the extraction solvent, reconstitution with acetonitrile, and chromatography on an Hypersil silica column (50 x 4.6 mm) using a low aqueous--high organic mobile phase. The mobile phase consists of 85% acetonitrile, 15% water, 0.5% acetic acid and 0.04% trifluoroacetic acid and runs isocratically at a flow rate of 2.0 mL/min. The column ef fluent was split so that 50% of it was transferred into the LC-MS/MS interface operated in positive electrospray ionization mode. The chromatographic run time was 2.0 min per injection. Atenolol and the internal standard, atenolol-d(7), showed a retention time of 1.0 min. The inter-day and intra-day precision and accuracy of the quality control samples were <5.3% relative standard deviation and <8.0% relative error, respectively. To explore the application of the current method for the analysis of other beta-blocking agents, propranolol and metoprolol were tested under the same chromatographic conditions with retention times of 0.68 and 0.75 min, respectively. The present method could be used for therapeutic drug monitoring, pharmacokinetic and drug--drug interaction studies of beta-blocking agents.     

Convection/diffusion- and diffusion-controlled rapid-scan voltammetry in liquid chromatographic systems with a large-volume wall-jet detector

On-line voltammetric measurements under liquid chromatographic (LC) conditions yield voltammograms which are either purely diffusion- or purely convection/diffusion-controlled, or a combination of both. The dependence of this behaviour on flow-rate, scan-rate and electrode diameter is investigated for a large-volume wall-jet detector. It is shown that for 4 V s^?1 scan-rates at 1 ml min^?1 flow-rates (conventional LC conditions), S-shaped (convection/diffusion-controlled) voltammograms can be obtained with macro-electrodes (? 1 mm diameter). Distortion of voltammogram shape by the cell time constant is discussed for macro-electrodes. The behaviour of the cell in microbore and micro-LC applications is demonstrated. The advantage of being able to change from convection/diffusion-controlled to diffusion-controlled behaviour is discussed.     

溶剂浮选-HPLC法测定茵陈中滨蒿内酯的研究

摘要：建立茵陈中滨蒿内酯的溶剂浮选分离富集方法。方法 考察了浮选溶剂、氮气流速、试液 pH、浮选时间及电解质 NaCl 等因素对浮选效果的影响,优选出最佳浮选条件,并由高效液相色谱测定其含量。结果 对最佳条件下的浮选效果进行了评价。结论 加标回收率92.31 ~ 99.97 %; RSD = 3.20 %.溶剂浮选分离富集方法可行。