Mobile Phase Delivery Research Articles

Development of a Fast and Highly Sensitive UPLC–MS/MS Technique for Simultaneous Estimation of Artemether and Dihydroartemisinin with its Application to Pharmacokinetic and Biodistribution Studies

Background: Artemether (ART) has been recognized as a potent and rapidly acting antimalarial agent metabolized by cytochrome P450 isoenzyme 3A4 into a more active form dihydroartemisinin (DHA). Hence, it was required to study the pharmacokinetics and biodistribution of ART and DHA for the optimization of dose. Objectives: The current research aims to develop an ultra-performance liquid chromatographytandem mass spectrometry (UPLC-MS/MS) technique with high sensitivity for quantification of ART and DHA simultaneous in the biological fluid. Methods: An isocratic elution mode was applied for delivery of mobile phase (A) ammonium acetate (2mM) in UPLC grade water and (B) acetonitrile (20:80, v/v) comprising 0.1% v/v formic acid in each solvent system with the rate of flow maintained at 400 μL/min. For analyte detection and parent to daughter ion transition tracking, multiple reaction monitoring system was adopted. Artemisinin was selected as the internal standard (IS) for its structural similarity with the analytes. Results: The linearity range of the calibration curve was between 0.5-200 ng/mL. The regression equation indicated r2 = 0.996 for ART and r2 = 0.997 for DHA. Precision and accuracy set in terms of relative error (RE%: ±15) and relative standard deviation (RSD%: ±15) expressed in percentage were within the acceptability range. The % recoveries were within the acceptable limit (90–110%). The detection and quantification limits of the analyte were 0.2 ng/mL and 0.5 ng/mL, respectively. Conclusion: The method demonstrated a useful technique for quantification of ART and DHA for evaluating the pharmacokinetics and biodistribution studies in rat plasma following orally administered nanoformulation.

A Validated RP-HPLC method for related substances of Dabigatran etexilate mesylate

The scope of this research work was to develop a reverse-phase liquid chromatographic method for the quantification of related impurities of dabigatran and to validate the method according to ICH guidelines. Chromatographic conditions were optimised with Poroshell SB C18, 150mm, 4.6mm, 2.7µm particle size column, mixer of Phosphate buffer with phosphoric acid in water and acetonitrile with percentage of 10:90 (v/v) as solvent-A and acetonitrile and buffer with a percentage of 70:30 (v/v) as solvent-B. Gradient compassion mode is employed for mobile phase delivery with a flow rate of 0.8 mL/min. Stationary phase was maintained at 35°C temperature and detection at 230 nm, with 10 µL of sample injection volume. Water and acetonitrile in the percentage 30:70(v/v) were used as a diluent. The developed RP-HPLC method was validated according to ICH guidelines. LOD and LOQ values for dabigatran its impurities were in the range from 33 to 55 ppm and 112 to 168 ppm correspondingly. Method validation results for all the parameters are meeting the ICH guidelines acceptance criteria for the parameters of robustness, ruggedness, linearity, reproducibility and recovery. The proposed method was found to be suitable for the quantitative determination of potential impurities in the bulk samples of Dabigatran etexilate mesylate API.

On the feasibility to conduct gradient liquid chromatography separations in narrow-bore columns at pressures up to 2000 bar

The limits in operating pressures are extended for narrow-bore columns in gradient elution up to 2000bar. As the required pumps for these pressures are incompatible with common chromatographic solvents and are not suitable to apply a mobile phase composition gradient, a mobile phase delivery and injection system is described and experimentally validated which allows to use any possible chromatographic solvent in isocratic and gradient elution. The mobile phase delivery and injection system also allows to perform multiple separations without the need to depressurize the column. This system consists out of 5 dual on/off valves and two large volume loops in which the gradient and equilibration volume of initial mobile phase are loaded by a commercial liquid chromatography pump. The loops are then flushed toward the column at extreme pressures. The mobile phase delivery and injection system is first evaluated in isocratic elution and shows a comparable performance to a state-of-the-art commercial flow-through-needle injector but with twice the pressure rating. Distortion of the loaded gradient by dispersion in the gradient storage loop is studied. The effect of the most important parameters (such as flow rate, pressure and gradient steepness) is experimentally investigated. Different gradient steepnesses and volumes can be applied at different flow rates and operating pressures with a good repeatability. Due to the isobaric operation of the pumps, the gradient is monitored in real-time by a mass flow meter installed at the detector outlet. The chromatograms are then converted from time to volume-base. A separation of a 19-compound sample is performed on a 300×2.1mm column at 1000bar and on a 600×2.1mm column at 2000bar. The peak capacity was found to increase from 141 to 199 and thus scales with L as is predicted by theory. This allows to conclude that the inlet pressure for narrow-bore columns in gradient elution can be increased up to 2000bar without fundamental pressure-induced limitations.

Direct determination of non-steroidal anti-inflammatory drugs by column-switching LC–MS

A method for determination of 16 non-steroidal anti-inflammatory drugs (NSAIDs) in human plasma samples without time-consuming sample pre-treatments was developed. The system consisted of two pumps for mobile phase delivery, a six-port switching valve, a pre-column (Oasis HLB Cartridge Column), and a reversed phase analytical column (COSMOSIL 3C18-MS-II). The analytes were trapped on the precolumn and subsequently separated on the analytical column. The present method allowed on-line sample clean-up and enrichment, leading to improved sensitivity without any tedious sample preparation. The recoveries of NSAIDs from human plasma by column-switching were greater than 72.6%. The total analysis time for a single analytical run was approximately 11 min. The detection limits of NSAIDs were 0.0025 to 0.2 microg/mL using the selected ion monitoring mode.

Study on pressurizing electroosmosis pump for chromatographic separation

A porous core electroosmosis pump was studied and improved in accordance with the electroosmosis theory. Hexamethylene tetraamine (HMTA) was used as the additive of pump carrier solution to improve the flow rate stability and delivery efficiency. The influences of the electric field strength, porous core dimension and acetonitrile concentration of carrier solution on the pump flow rate and output pressure were investigated in detail. The improved electroosmosis pump can provide not only steady flow rate and large flow range, but also moderate output pressure. With the pump carrier solution of 0.5 mmol/L HMTA and the working voltage of 4950 V, the pump output pressure, flow rate and delivery efficiency achieved 1.1 MPa, 1.3 mL/min and 3.2 mL/(min mA), respectively. The pump can be employed for mobile phase delivery in the reversed-phase chromatographic separation of monolithic silica columns.

An electroosmotic pump for packed capillary liquid chromatography

An electroosmotic pump (EOP) capable of generating pressure above 3 MPa and μl/min flow rate with reverse phase mobile phases of HPLC was constructed and evaluated. The pump consisted of three parallel connected fused silica capillary columns (25 cm×320 μm I.D.) packed with 2 μm silica materials, hollow electrodes, a high voltage DC power supply, and a liquid pressure transducer. The EOP was applied in a capillary liquid chromatographic system for mobile phase delivery instead of a mechanical pump. Standard samples containing thiourea, naphthalene, anthracene, phenanthrene and acetonitrile were separated on a 15 cm×320 μm I.D. 5 μm Chromasil C 18 packed capillary column with acetonitrile/water as mobile phase.

Application of a horizontal DS chamber to planar electrochromatography

A commercially available horizontal DS chamber has been applied to the planar electrochromatographic (PEC) separation of a dye mixture and of the enantiomers of dl-phenylalanine on non-polar stationary phases with ethanol-buffer or acetonitrile-buffer as mobile phases. The mobile phase was driven electroosmotically by applying a 50–250 V cm −1 field along a 10-cm RP18 TLC plate. Use of a counter-plate to the chromatographic plate enables elimination of problems associated with excessive delivery of mobile phase to the chromatographic adsorbent layer. Compared with conventional TLC separations the efficiency of electrochromatography was higher and the development time shorter.

Evaluation of Automated Isocratic and Gradient Nano-Liquid Chromatography and Capillary Electrochromatography

An automated liquid nano-separation system has been developed for nano-liquid chromatography (nano-LC) and capillary electrochromatography (CEC) using both isocratic and gradient elution. One fused-silica nanocolumn, typically 75 μm i.d. × 39 cm (25 cm effective packed length), packed with Spherisorb ODS 1, 3 μm particle size, can be used with either technique without having to remove the column upon switching from one mode to the other. The mobile phase is delivered by two reciprocating micro-LC pumps at a flow rate of 30 μL/min to a postinjection splitter that houses the nanocolumn inlet. The splitter is directly connected to a micro-injection valve with a 0.5 μL injection volume. In the CEC mode, pressure is not applied (no restriction on splitter) to the column inlet or outlet and the voltage is continuously applied during sample injection and mobile phase delivery. In the nano-LC mode, the restrictor is coupled to the splitter. Using the same nanocolumn under isocratic conditions, the repeatabilities of retention time and peak area for nano-LC were better than 0.2% and 4%, respectively, and those for CEC were better than 0.6% and 6%, respectively. On average, column efficiency was 57% higher in CEC compared to nano-LC. Gradient elution separations of parabens and polynuclear aromatic hydrocarbons (PAHs) were accomplished by CEC.

Selective stability-indicating high-performance liquid chromatographic assay for recombinant human regular insulin.

This report presents a selective HPLC assay capable of separating recombinant human regular insulin from insulin decomposition and transformation products. The assay utilizes an isocratic delivery of mobile phase, a C18 peptide column, UV detection and is performed at ambient temperature. The standard curve ranges from 0.2 to 2.5 U/ml. The inter-day and intra-day variabilities are less than 7 and 5%, respectively, at the concentrations studied. The accuracy and precision are within 5% over the range of the standard curve.

CAPILLARY SFC WITH DUAL-FLAME PHOTOMETRIC DETECTION USING PRESSURE PROGRAMMING OF A BINARY ON-LINE MIXED MOBILE PHASE

Using a primary mobile phase of supercritical carbon dioxide, the effects of various organic solvents as modifiers are studied. The on-line mixed mobile phase delivery for capillary SFC is served by a system which was modified a Jasco SFC Super-200 by incorporating a double-stage flow splitter. Organosulfur compounds were separated on a capillary column (SB-Phenyl-5, 50μmx10m, 0.25μm film thickness). The mobile phases which various modifiers up to 0.15mL/min were mixed with a constant supercritical carbon dioxide flow (2.0mL/min at pump outlet) were used to separate the test solute mixture. Selective detection of organosulfur compounds has been carried out with a dual-flame photometric detector without interference of organic modifier solvents. Modifier - pressure programming are shown to yield decreased analysis time with increasing modifier concentration. But early eluted compounds (thiophenol and benzo[b]thiophene) were more retained since fluids with modifier were probably not supercritical at an initial SFC condition. Also approximately linear relationships existed between capacity factors (k') and the solubility parameter of organic modifiers.

Pressure-programming of a binary, on-line mixed mobile phase for capillary column SFC using a packed column SFC pumping system

Mixed mobile phase delivery for CSFC is served by a system previously designed for packed column SFC, where a back-pressure regulator controls pressure programming. Piston pumps deliver separate flows of carbon dioxide and modifier (2-propanol). The on-line mixed phase with the injected sample is split to the capillary column. Compared to a commercially available CSFC instrument the system shows no significant differences in resolution. Applications show the advantages of using modifiers in CSFC, such as separations of polar, ionic and high molecular weight compounds, influences on selectivity and shorter retention times.

New principle of sample introduction integrated with mobile phase delivery for micro-column liquid chromatography

The possibilities and limitations of a liquid micro-chromatograph with integrated sample introduction and mobile phase delivery systems are considered. Basic values of chromatographic constants such as capacity ratio and number of theoretical plates are expressed as a function of a defined and limited retention volume. A new type of the liquid micro-chromatograph is described. The sample introduction system is integrated with a mobile phase delivery system in one instrumental section. A gradient of the mobile phase can be generated in the device. The new principle is applicable as a portable, fully automated analyser. Volumes of 0.01–500 μl can be injected. A controlled sample introduction velocity makes it possible to generate extremely small dispersions. A column efficiency of near to theoretical levels can be achieved with an amperometric detector. Both trace analyses and rapid chromatographic separations are demonstrated.