Reversed-phase Liquid Chromatographic Procedure Research Articles

Robustness assessment in computer-assisted liquid chromatography procedures based on desirability functions

A novel approach based on the use of desirability functions is presented for the robustness assessment of liquid chromatographic separations as derived from computer-assisted methods development processes. The approach is based on generally accepted hypothesis that a robust separation procedure will be inert to small random variations of the operational variables, typically encountered in the day-to-day routine analytical practice. This means that peak positions along the chromatograms must keep standstill or move insignificantly when operational variables are not intentionally changed. Thus, the degree of peak positions variation as evaluated from mathematical retention models can be used to assess the robustness of the developed procedures before testing the actual performance experimentally. In the approach proposed, this assessment is obtained by fixing a bilateral partial desirability window around each peak in the simulated chromatogram. The whole chromatogram robustness is characterized by an overall desirability value calculated as the geometric mean of the partial desirability windows evaluation. An added advantage of this approach is that the robustness value calculated is normalized between zero and one and thus, easy to interpret. Thus, when chromatograms are simulated and small random variations are introduced into the operational factors of the model, values for the overall desirability close to one means that the procedure performs robustly. On the contrary, low values for the overall desirability clearly indicated a serious lack of robustness. When used in conjunction with the Pareto optimality approach, as shown here, this robustness assessment strategy allows testing several Pareto front solutions before the final experimental testing which is always needed. In this way, a dramatical reduction of the experimental effort is obtained. Although the approach is theoretically applicable to any chromatographic separation, examples of reversed phase liquid chromatographic procedures are used to show the performance of the proposed methodology.

Development and Validation of Stability Indicating Assay Method for Simultaneous Estimation of Glibenclamide and Metformin

A high performance reverse phase liquid chromatographic procedure is developed for simultaneous estimation of Metformin and Glibenclamide in combined tablet dosage form. The method was carried out on Agilent Hypersil ODS (4.6 x 250 mm) column with a mobile phase used consisting of Methanol: Water (0.1 % OPA) OPA= Ortho Phosphoric acid (80:20) and the pH of buffer was adjusted to 3 using 2M Orthophosphoric acid. The detection of the combined dosage form was carried out at 228 nm and a flow rate employed was 1 ml/min and column oven temperature at 300C. The retention times of Metformin andGlibenclamide were 3.4667 and 7.3500 minutes respectively. The developed method was validated in terms of accuracy, precision, linearity, limit of detection, limit of quantification as per ICH norms. The proposed method can be used for the estimation of these combined drugs.

Methylation on RNA: A Potential Mechanism Related to Immune Priming within But Not across Generations.

Invertebrate immune priming is a growing field in immunology. This phenomenon refers to the ability of invertebrates to generate a more vigorous immune response to a second encounter with a specific pathogen and can occur within and across generations. Although the precise mechanism has not been elucidated, it has been suggested that methylation of DNA is a cornerstone for this phenomenon. Here, using a novel method of analytical chemistry (a reversed-phase liquid chromatography procedure) and the beetle Tenebrio molitor as a model system, we did not find evidence to support this hypothesis taking into account the percentage of methylated cytosine entities in DNA (5mdC) within or across generations. However, we found a lower percentage of methylated cytosine entities in RNA (5mC) within but not across generations in immune priming experiments with adults against the bacteria Micrococcus lysodeikticus and larvae against the fungus Metarhizium anisopliae. To our knowledge, this is the first report suggesting a role of differential methylation on RNA during immune priming within generations.

Simultaneous estimation of metformin hydrochloride and pioglitazone hydrochloride by RPHPLC method from combined tablet dosage form

A high performance reverse phase liquid chromatographic procedure is developed for simultaneous estimation of metformin hydrochloride and pioglitazone hydrochloride in combined tablet dosage form. The mobile phase used was a combination of acetonitrile:water:acetic acid (60:40:0.3) and the pH was adjusted to 5.5 by adding triethylamine. The detection of the combined dosage form was carried out at 230 nm and a flow rate employed was 1 ml/min. Linearity was obtained in the concentration range of 0.015 to 0.120 μg/ml of pioglitazone hydrochloride and 0.5 to 4.0 μg/ml of metformin hydrochloride with a correlation coefficient of 0.9992 and 0.9975. The results of the analysis were validated statistically and recovery studies confirmed the accuracy and precision of the proposed method.

Micellar liquid chromatography in clinical chemistry: application to the monitorization of B6 vitamins

Background: A micellar reversed-phase liquid chromatographic procedure was developed for the determination of B6 group vitamins, i.e. pyridoxine, pyridoxal and pyridoxamine, in human serum. Methods: Chromatographic conditions used were a C18 column, isocratic mode, flow-rate of 1 ml/min and UV-detection at 290 nm. Optimization of the composition of the mobile phase was performed using an interpretative strategy. Results: After modeling, the composition of the selected mobile phase was 150 mM sodium dodecyl sulphate (SDS)–2% (v/v) pentanol–dihydrogenphosphate buffer 10 mM at pH 3. In this mobile phase, serum samples were injected without pretreatment and analysis time was below 14 min. Calibrations for the three vitamins were linear, with coefficient regression better than 0.999, and intra- and inter-day precision, achieved according to ICH, offering values below 4.3% and 3.2%, respectively. The method was applied to the determination of the B6 vitamins in spiked serum samples, with recoveries around 100%, and in the pharmacokinetic determination of pyridoxine half-life in serum, which was found to be 47.5±3.2 min ( n=5). The procedure was also applied for the analysis of pyridoxine in human serum spiked with several pharmaceutical preparations that contain other drugs which do not produce any kind of interference. Finally, solutions of B6 vitamins kept at −201 °C are stable for up to 3 months. Conclusions: Using the method proposed here, with an SDS–pentanol mobile phase, it is possible to carry out the fast sensitive determination of B6 vitamins in serum following direct injection, without sample pretreatment.

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), B 1 (thiamine), B 2 (riboflavin), B 6 (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 C 18 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 °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.

Quantitation of Antihistamines in Pharmaceutical Preparations by Liquid Chromatography with a Micellar Mobile Phase of Sodium Dodecyl Sulfate and Pentanol

A reversed-phase liquid chromatographic procedure with a micellar mobile phase of sodium dodecyl sulfate (SDS), containing a small amount of pentanol, was developed for the control of 7 antihistamines of diverse action in pharmaceutical preparations (tablets, capsules, powders, solutions, and syrups): azatadine, carbinoxamine, cyclizine, cyproheptadine, diphenhydramine, doxylamine, and tripelennamine. The retention times of the drugs were <9 min with a mobile phase of 0.15M SDS-6% (v/v) pentanol. The recoveries with respect to the declared compositions were in the range of 93-110%, and the intra- and interday repeatabilities and interday reproducibility were <1.2%. The results were similar to those obtained with a conventional 60 + 40 (v/v) methanol-water mixture, with the advantage of reduced toxicity, flammability, environmental impact, and cost of the micellar-pentanol solutions. The lower risk of evaporation of the organic solvent dissolved in the micellar solutions also increased the stability of the mobile phase.

HYDRO-ORGANIC AND MICELLAR-ORGANIC REVERSED-PHASE LIQUID CHROMATOGRAPHIC PROCEDURES FOR THE EVALUATION OF SULPHONAMIDES IN PHARMACEUTICALS

Two reversed-phase liquid chromatographic procedures were developed for the analysis of ten pharmaceutical formulations used in medicine and veterinary, which contained one of the following sulphonamides: sulphacetamide, sulphadiazine, sulphaguanidine, sulphamethazine, sulphamethizole, sulphamethoxazole, or sulphathiazole. In both chromatographic modes, the same C18 column was utilized and the mobile phase contained a low amount of acetonitrile and citric buffer at pH 3.0. In one of the procedures, the surfactant sodium dodecyl sul phate was added to the mobile phase. The presence of micelles allowed a decrease in the amount of organic solvent from 11% to 6%, and facilitated sample dissolution. The surfactant also gave rise to changes in the elution order and selectivity of the drugs. The analyses of the formulations were usually in good agreement with the claimed levels, and the results showed good precision. The procedures were simple and fast, the chromatographic runs took less than 15 min and the samples were injected directly into the chromatograph after dilution and filtration.

Simultaneous Determination of Zopiclone and Its Two Major Metabolites (N-Oxide and N-Desmethyl) in Human Biological Fluids by Column Liquid Chromatography After Solid-Phase Extraction

Abstract A reverse phase liquid chromatographic procedure with fluorescence detection for the simultaneous determination of zopiclone and its main metabolites, N-desmethylzopiclone and zopiclone-N-oxide, in serum, blood and urine is described. An aliquot (0.5–1 mL) of the sample after the addition of 0.25 mL of 250 ng/mL solution of harmane in 0.2 M NaH2PO4 as the internal standard is passed through a 1-mL BondElut C18 silica extraction column. The column is selectively washed with water and acetonitrile to remove polar, neutral and acidic compounds. The desired compounds are eluted with a 0.25 mL aliquot of a mixture of methanol + 35% perchloric acid (100:1 v/v). A 10–25 μL aliquot of the eluate is injected onto a 150 × 4.6 mm I.D. column packed with 5-μm C18 silica particles which is eluted at ambient temperature with a mobile phase of acetonitrile - 0.1% tetramethylammonium perchlorate (17:83 v/v) adjusted to pH 3.8 with 10% perchloric acid at a flow rate of 1.8 mL/min. The peaks are detected with a fl...

Study of the influence of free dissolved amino acids on copper(II) adsorption/remobilization from inorganic fractions of marine sediments using a reversed-phase liquid chromatographic procedure

A reversed-phase liquid chromatographic procedure for the determination of amino acids and copper(II)-amino acid complexes was improved and applied to studies dealing with adsorption and remobilization of copper from sediments in the presence of free and complexed amino acids. In particular, the adsorption by inorganic fractions of a marine sediment was investigated by means of batch equilibration experiments. The results indicate that two adsorption mechanisms, one due to electrostatic interaction and the other one based on hydrophobic characteristics of the considered species, are simultaneously possible. Scanning electron microscopy with electron probe microanalysis was utilized to provide further information.

Liquid chromatography—electrochemistry procedure for the determination of chlorophenolic compounds in pulp mill effluents and receiving waters

An isocratic, reversed-phase liquid chromatography procedure is described for the separation and determination of chlorophenolic compounds, including several priority pollutant chlorophenols. The separation was experimentally optimised, and selective and sensitive detection was obtained by dual electrode amperometry. The most suitable combination was a silica-based column with phenyl functional groups, and a mobile phase of acetonitrile-acetate buffer (40:60, pH 5). Dual glassy-carbon electrodes were used in a parallel configuration in a thin-layer flow cell, with differential current measurement (+0.90 V relative to +0.60 V). A solid-phase extraction scheme using a polymeric sorbent (Chromosorb 102) was devised for the extraction and preconcentration of selected chlorophenols, chloroguaiacols, and a chlorosyringol from aqueous samples. Use of tribromophenol as an internal standard was also investigated. The total procedure —preconcentration and chromatography— was applied to the analysis of effluents discharged by a paper mill, and a pulp mill.

Rapid chromatographic separation of the AVP-like locust peptides from synthetic and biological mixtures. Quest for these peptides in some insect, batrachian and mammalian nervous systems

The AVP-like insect diuretic hormone, an antiparallel dimer and its monomeric form are present together in the locust ventral nerve cord. Classical reversed-phase liquid chromatographic procedures were developed to rapidly separate dimer and monomer from their synthetic and native mixtures. These procedures were then used to look for AVP-like peptides in cockroach, stick-insect, bumble-bee, frog and rat nervous tissues. Cockroach corpora cardiaca as well as a stick-insect and bumble-bee ventral nerve cords contain AVP-like peptides whose chromatographic properties are similar to those of the locust AVP-like peptides.

Micro Method for Liquid Chromatographic Determination of Cyclosporin A in Whole Blood with Use of a Rapid Extraction Procedure

In this precise, reversed-phase liquid chromatographic procedure, Cyclosporin A (Cy A) is extracted from 300 microL of whole blood with cyclosporin D as internal standard by liquid-liquid extraction. A 20-microL aliquot of the extract, injected onto a Nucleosil octyl analytical column heated at 72 degrees C, is eluted with a mixture of acetonitrile and 0.01 M phosphate buffer, pH 5.5 at a flow rate of 1 mL/min. Detection is set at 210 nm. The chromatography is complete within 10 min. The detection limit is 25 micrograms/L. Between-run CVs range from 2.3 to 6.2% and recovery is 92.1 +/- 6.3%. The major advantages of our extraction procedure are the rapid clean-up method and the small volume required. This procedure is especially suitable for cyclosporine determination in pediatric transplantations.

Determination of Fluoxetine and Norfluoxetine in Serum by Liquid Chromatography with Fluorescence Detection

Abstract A reversed phase liquid chromatographic procedure with fluorescence detection for the simultaneous determination of fluoxetine and its active metabolite, norfluoxetine in human serum is described. A 0.5-mL aliquot of the sample after the addition of protriptyline as the internal standard is passed through a 1-mL BondElut C18 silica extraction column. The column is selectively washed to remove polar, neutral, acidic and weakly basic compounds. The desired compounds are eluted with a 0.25-mL aliquot of a mixture of 0.1 N perchloric acid + acetonitrile (1:3). A 20-uL aliquot of the eluate is injected onto a 15 cm × 4.6 mm (i.d.) column packed with 5-um C8-silica particles, which is eluted at ambient temperature with a mobile phase containing tetramethyl-ammonium perchlorate. The peaks are detected with a fluorescence detector (ex = 235 nm; em = 310 nm). In the resulting chromatogram, there are only few extraneous peaks, fluoxetines give sharp peaks which are well resolved from peaks for solvent and ...

Formation of hemoglobin-benzo[ a]pyrene adducts in human erythrocytes incubated with benzo[a]pyrene and hamster embryo cells

Evidence is accumulating that the levels of covalent carcinogen-macromolecule adducts, including adducts with hemoglobin, reflect biologically effective levels of carcinogen exposure. The purposes of the present study were (a) to establish a cellular system for obtaining adducts between intracellular human hemoglobin and metabolites of polycyclic aromatic hydrocarbons (PAH), and (b) to evaluate techniques for chromatographic characterization of the adducts. We showed that hemoglobin-benzo[ a]pyrene adducts were formed when human erythrocytes were treated with [ 3 H] benzo[a] pyrene (BP) in the presence of hamster embryo fibroblasts, which are known to be effective for BP metabolism. After lysis of the erythrocytes, noncovalently bound BP and its metabolites were effectively removed from hemoglobin under mild conditions by using hydrophobic interaction and size-exclusion liquid chromatography. Three to five distinct adducts were resolved by reversed-phase and ion-exchange liquid chromatography. As determined by a two-step, reversed-phase liquid chromatographic procedure, trypsin treatment of globin from the cellular system yielded at least three of the four 7,8,9,10-tetrahydro-7,8,9,10-tetrahydroxy BP tetrols known to arise from mammalian metabolism of BP. This observation is consistent with both (a) the recently described formation of labile carboxyl esters via reaction of BP-7,8-dihydrodiol-9,10-epoxide (BPDE) with hemoglobin and (b) the known formation of both anti- and syn-BPDE in hamster embryo fibroblasts. In addition, high-performance liquid chromatographic analysis demonstrated the presence of other products presumed to be BP-peptide adducts because of their susceptibility to thermolysin treatment.

Elimination of caffeine interference in HPLC determination of urinary nicotine and cotinine.

We report an analytical reversed-phase liquid-chromatographic procedure for quantifying nicotine and cotinine in urine, taking into account the presence of interfering caffeine frequently encountered in such specimens. These analytes are extracted from the alkalinized urine with chloroform. After evaporation of the chloroform, the residue is dissolved in methanol and injected into a chromatographic C18 column. Extraction recoveries averaged 80% to 97%. Chromatographic conditions were investigated to obviate caffeine interference. The proposed eluent mobile phase is a polar mixture of water, acetonitrile, methanol, and a pH 4 acetoacetate buffer (65/2/29/4 by vol) adjusted to pH 4.30 +/- 0.02 with triethylamine. High resolution and linearity were obtained for each analyte up to a concentration of 200 mg/L. The minimum detectable amount of each compound was 20 ng per injection, corresponding to 10 micrograms per liter of urine. Correlation with results of gas-liquid chromatography was excellent (r = 0.99). This simple, rapid procedure allows routine screening of tobacco exposure with acceptable precision: within- and between-run coefficients of variation were less than 2% and less than 5%, respectively.

Phenylalanine and tyrosine in serum and eluates from dried blood spots as determined by reversed-phase liquid chromatography.

We have developed a reversed-phase liquid-chromatographic procedure for simultaneously determining phenylalanine and tyrosine in serum and eluates of dried blood spots. Batch derivatization with phenylisothiocyanate and a 10-min linear gradient chromatographic assay with ultraviolet absorbance detection provide rapid sample throughput. Interrun precision (CV) is less than 12%; analytical recovery (from blood spot samples) exceeds 85%. Results for patients' samples correlate well with those from an amino acid analyzer and we encountered no apparent interferences. The speed and specificity of this assay facilitate the rapid diagnosis and monitoring of patients with phenylketonuria.

Analysis of Pharmaceutical Dosage Forms for Oxfendazole: II. Simultaneous Liquid Chromatographic Determination of Oxfendazole and Trichlorfon in Equine Paste

A reverse phase liquid chromatographic procedure is described for the simultaneous determination of oxfendazole [2-(methoxycarbonylamino)-5-phenylsulfinylbenzimidazole] and trichlorfon [(2,2,2-trichloro-1-hydroxyethyl)phosphonic acid dimethyl ester] in equine paste. The sample is extracted by sonication in methanol. Insoluble excipients are removed by centrifugation and an aliquot plus internal standard are diluted with dilution solvent (water-acetonitrile-phosphoric acid, 80 + 20 + 1). The samples are filtered and injected onto a Partisil-5 ODS-3 column with acetonitrile-0.01 M phosphate buffer pH 6.0 (20 + 80) as mobile phase. Method specificity is confirmed using an absorbance rationing technique. The method yields mean recoveries of 100.9 and 100.0% for trichlorfon and oxfendazole, respectively. Dependence of chromatographic performance characteristics on mobile phase organic content, pH, and buffer concentration is also reported.

Combined enzyme immunoassay-LCEC method for the identification, confirmation, and quantitation of opiates in biological fluids.

An enzyme multiplied immunoassay technique (EMIT) was used to test for opiates (morphine, hydromorphone, and codeine) in extracts of blood, bile, and tissue homogenates. All immunoassay opiate positive specimens were then tested by a reversed-phase liquid chromatographic procedure using electrochemical detection (LCEC). Blood specimens were then quantitated by LCEC. The sensitivity of the immunoassay (as morphine) was 0.020 mg/L, 0.200 mg/L, and 0.100 mg/kg for blood, bile, and tissue homogenates, respectively, with 2% intrarun and 7% interrun precision. The LCEC method was linear from 0.005 to 0.300 mg/L for morphine, hydromorphone, and codeine (nalorphine internal standard) with detection limits of 0.005 mg/L for each analyte. Intrarun and interrun precision varied from 1 to 2% and 6 to 11%, respectively. Recoveries, using a double extraction technique, ranged from 70 to 95%. These two methods, applied to 495 post mortem cases, demonstrated a 6% incidence of opiates, with no false positives.

Improved liquid-chromatographic determination of mexiletine, an antiarrhythmic drug, in plasma.

In this improved reversed-phase liquid-chromatographic procedure for determination of mexiletine in plasma, mexiletine and an internal standard, chlorodisopyramide, are extracted with methylene chloride from 0.5 mL of serum or plasma; the extract is then concentrated and injected onto a C18 chromatographic column. Mexiletine in the column effluent is detected by monitoring absorbance at 210 nm. It is quantified by use of mexiletine-internal standard peak-height ratios. The relation between this ratio and mexiletine concentration is linear from 0.1 to 5.0 mg/L. The lower limit of detection is about 50 micrograms/L. At a mexiletine concentration of 2.0 mg/L in serum, intrarun precision (CV) is 2.9% and inter-run precision is 5.9%; at 0.5 mg/L, these CVs are 5.7% and 9.6%, respectively. Analytical recovery of added mexiletine in serum is 68-88%. Therapeutic concentrations of some commonly administered drugs in patients' specimens did not interfere. In serum from 38 patients receiving mexiletine for cardiac arrhythmia, concentrations measured by this method correlated with therapeutic efficacy.