Column classification and selection for the determination of antibiotics by micellar liquid chromatography

Seven commercially available: Zorbax C18, Kromasil C18, C8, cyano, phenyl, monolithic and amino stationary phases columns, have been characterized and classified into broadly similar types to simplify column choice. The results were evaluated employing cluster analysis, which shows several interesting groups based on distances (Minkowski and Euclidean) in agreement with the manufacturer’s claims: chain density of the stationary phase used, and the presence or not a silica base. Finally, results showed that C18 columns offer the best chromatographic characteristics for separation and quantification of antibiotics in micellar liquid chromatography

Simultaneous separation and determination of quinolones in pharmaceuticals by micellar liquid chromatography

A rapid and simple liquid chromatographic procedure using micellar mobile phases is reported for the separation and determination of four quinolones (pipemidic acid, levofloxacin, norfloxacin and moxifloxacin) in pharmaceuticals. This purpose was achieved without any previous pretreatment step in a C18 column using a micellar mobile phase of 0.15 M sodium dodecyl sulphate, 2.5% propanol and 0.5% triethylamine at pH 3, with retention times below 12 min. For detection, the diode-array UV-Vis set at 276 nm was used. The limits of detection and quantification were between 8-51 and 28-171 ng/mL, respectively. This method was validated in terms of intra-day and inter-day precision and accuracy, and robustness. Calibration curves over the concentration range of 0.1-50 μg/mL were linear (r2 > 0.9997) and. Good claim percentages (96–106 %) were obtained in the analysis of pharmaceutical formulations. The results show that the procedure is suitable for the routine analysis of drugs

Monitoring of HAART regime antiretrovirals in serum of acquired immunodeficiency syndrome patients by micellar liquid chromatography

A methodology based on micellar liquid chromatography to monitor five antiretroviral drugs (lamivudine, stavudine, tenofovir, zidovudine and efavirenz) was proposed. Antiretrovirals were studied in sets of three, corresponding to each highly active antiretroviral therapy (HAART) regime, prescribed to acquired immunodeficiency syndrome (AIDS)-infected patients. Four aqueous micellar mobile phases buffered at pH 7 were optimized to separate these compounds, using sodium dodecyl sulfate as the tensioactive, and 1-propanol or 1-pentanol as the organic modifier. The composition of each mobile phase was optimized for each antiretroviral. The common separation conditions were: C18 apolar column (125 4.6 mm, 5 mm particle size), UV detection set at 214 nm, and mobile phase running at 1 mL min␣1 without controlling the temperature. The finally suggested method was validated for five analysed antiretroviral drugs following the US Food and Drug Administration guidelines in terms of: linearity between 0.5 and 50 ppm (r2 > 0.9995), sensitivity (LOD lower than 0.25 ppm), intra- and inter-day precision (<7.1 and <5.2%, respectively) and accuracy (recovery 88.5– 105.3% and 93.5–101.3%, respectively), as well as robustness (<6.5%). The proposed method was used to monitor the level of antiretrovirals in the serum of AIDS patients. The suggested methodology was found to be useful in the routine analysis of antiretrovirals in serum samples

Use of micellar mobile phases for the chromatographic determination of melamine in dietetic supplements

Melamine is a nitrogen-rich industrial chemical which is occasionally used to increase the apparent protein content of different products destined for human and animal consumption. In this work, a liquid chromatographic procedure that uses micellar mobile phases of sodium dodecyl sulfate (SDS) buffered at pH 3, a C18 column and UV detection is reported for the determination of melamine in dietetic supplements. Samples were reconstituted with a SDS solution and were directly injected, thus avoiding long extraction and experimental procedures. Melamine was eluted in less than 10 min with no interference by other compounds of the matrices. The optimum mobile phase composition was taken by a chemometrical approach that considers the retention factor, efficiency and peak shape. Validation was performed following the indications of the European Commission (Decision 2002/657/EC). The following parameters were considered: linearity (0.02-100 μg mL(-1); R(2) = 0.9996), intra- and inter-day precisions (<12.4%), accuracy (90.0-101.3%), and robustness (less than 9.8% and 5.1%, for retention time and peak area, respectively). The limits of detection and quantification were 9 and 20 ng mL(-1), respectively. Recoveries for several spiked samples were in the 85.8-114.3% range. These results indicate that the proposed methodology is useful for routine analysis of control quality of infant formula and adult dietetic supplement

Application of a liquid chromatographic procedure for the analysis of penicillin antibiotics in biological fluids and pharmaceutical formulations using sodium dodecyl sulphate/propanol mobile phases a.

A direct injection liquid chromatography procedure was developed for the simultaneous determination of four penicillin antibiotics (amoxicillin, ampicillin, cloxacillin and dicloxacillin) in pharmaceutical formulations and physiological fluids (urine) using hybrid micellar mobile phases. These antimicrobials are used to treat gastrointestinal and systemic infections. The four penicillins were analysed using a Zorbax C18 reversed-phase column and detected at 210 nm. These antibiotics were separated by an interpretive optimisation procedure based on the accurate description of the retention and shape of the chromatographic peaks. Antibiotics were eluted in less than 16 min with no interference by the urine protein band or endogenous compounds using the mobile phase 0.11 M sodium dodecyl sulphate–6% propanol–0.01 M NaH2PO4 buffered at pH 3. The method was validated according to the Food and Drug Administration guideline, including analytical parameters such as linearity (R2 > 0.993), intra- and inter-day precisions (RSD, %: 0.1–4.4 and 1.2–5.9, respectively), and robustness for the four compounds. This method is sensitive enough for the routine analysis of penicillins at therapeutic urine levels, with limits of detection in the 1.5–15 ng mL−1 range and limits of quantification of 50 ng mL−1. Recoveries in a micellar medium and a spiked urine matrix were in the 92.4–108.2% and 96–110% ranges, respectively. Finally, the method was successfully applied to determine these antibiotics in urine samples and pharmaceutical formulations

Development and validation of a method to determine amoxicillin in physiological fluids using micellar liquid chromatography

A simple and robust method was developed for the routine identification and quantification of amoxicillin by micellar liquid chromatography. Amoxicillin, a β-lactamase inhibitor, is one of the most commonly prescribed drugs in the treatment of urine and skin structure infections. In this work, amoxicillin was determined in urine samples without any pretreatment step in a phenyl column using a micellar mobile phase of 0.10 M sodium dodecyl sulfate and 4% butanol at pH 3. A UV detection set at 210 nm was used. Amoxicillin is eluted at 5.1 min with no interference by the protein band or endogenous compounds. Linearities (r > 0.9998), intra- and inter-day precisions were determined (RSD (%) 0.4-2.7% and 0.3-5%, respectively in micellar media, and 0.14-2.6% and 0.13-6%, respectively in urine), and robustness was studied in the validation of the method. LOD and LOQ were 0.06 and 0.4 μg/mL in micellar media and 0.11 and 0.4 μg/mL in urine, respectively. Recoveries in the urine matrix were in the range of 95-110%. The validated method proved to be reliable and sensitive for the determination of amoxicillin in urine sample

Analysis of omeprazole and its main metabolites by liquid chromatography using hybrid micellar mobile phases

Omeprazole is a selective inhibitor of gastric acid secretion and is one of the most widely prescribed drugs internationally. A chromatographic procedure that uses micellar mobile phases of sodium dodecyl sulphate and propanol buffered at pH 7 and a C18 column is reported for the determination of omeprazole and its principal metabolites (omeprazole sulphone and hydroxyomeprazole) in urine and serum samples. In this work, direct injection and UV detection set at 305 nm was used. Omeprazole and its metabolites were eluted in less than 11 min with no interference by the protein band or endogenous compounds. Adequate resolution was obtained with a chemometric approach, in which the retention factor and shape of the chromatographic peaks were taken into account. The analytical parameters including linearity (r &gt; 0.9998), intra- and inter-day precision (RSD, %: 0.6-7.9 and 0.14-4.7, respectively) and robustness were studied in the validation of the method for the three compounds. The limits of detection and quantification were less than 6 and 25 ng mL-1, respectively. Recoveries in micellar medium, plasma and urine matrices were in the 98-102% range. Finally, the method was successfully applied to the determination of omeprazole and its metabolites in physiological samples. Omeprazole was also analysed in pharmaceutical formulations. © 2008 Elsevier B.V. All rights reserved

Is it really necessary to validate an analytical method or not? That is the question

Method validation is an important requirement in the practice of chemical analysis. However, awareness of its importance, why it should be done and when, and exactly what needs to be done, seems to be poor amongst analytical chemists. Much advice related to method validation already exists in the literature, especially related to particular methods, but more often than not is underused. Some analysts see method validation as something that can only be done by collaborating with other laboratories and therefore do not go about it. In addition, analysts’ understanding of method validation is inhibited by the fact that many of the technical terms used in the processes for evaluating methods vary in different sectors of analytical measurement, both in terms of their meaning and the way they are determined. Validation applies to a defined protocol, for the determination of a specified analyte and range of concentrations in a particular type of test material, used for a specified purpose. In general, validation should check that the method performs adequately for the purpose throughout the range of analyte concentrations and test materials to which it is applied. It follows that these features, together with a statement of any fitness-for-purpose criteria, should be completely specified before any validation takes place

Validation of a MLC method with fluorescence detection for the determination of quinolones in urine samples by direct injection

A sensitive and robust method was developed and validated for the routine identification and quantification of five quinolones in urine samples directly injected into a micellar liquid chromatographic system without any pre-treatment step. Since the simultaneous elution of the five compounds was not resolved, two mobile phases have been proposed: (a) for ciprofloxacin and levofloxacin 0.15 M sodium dodecyl sulphate, 12.5% propanol and 0.5% triethylamine at pH 3.0 as the mobile phase and the detector at excitation wavelength 285 nm and emission wavelength 465 nm; and (b) for lomefloxacin, ofloxacin and moxifloxacin 0.05 M sodium dodecyl sulphate, 12.5% propanol and 0.5% triethylamine at pH 3.0 as the mobile phase and the detector at excitation wavelength 295 nm and emission wavelength 485 nm. Using these conditions, and in accordance with the food and drug analysis (FDA) guideline, the limit of quantification was 1 ng/mL, and the relative standard deviation and accuracy of the inter-day assay were 1.0-8.4% and 0.11-1.5%, respectively. Detection of the urinary excretion of four quinolones was followed up at 12 h after the healthy volunteers had taken the drug. No potential interference from metabolites was observed. This procedure permits the rapid and reproducible measurement of low levels of quinolones in a small amount of urine. © 2009 Elsevier B.V. All rights reserved

Quinolones control in milk and eggs samples by liquid chromatography using a surfactant-mediated mobile phase

Four quinolones (danofloxacin, difloxacin, flumequine and marbofloxacin) were determined in milk and egg samples by a simplified high-performance liquid chromatographic procedure using a micellar mobile phase. No extraction was needed to precipitate the proteins from the matrices since they were solubilised in micelles. The only pretreatment steps required were homogenisation, dilution and filtration before injecting the sample into the chromatographic system. An adequate resolution of the quinolones was achieved by a chemometrics approach where retention was modelled as a first step using the retention factors in only five mobile phases. Afterwards, an optimisation criterion was applied to consider the position and shape of the chromatographic peaks. Analytical separation involved a C18 reversed-phase column, a hybrid micellar mobile phase of 0.05 M sodium dodecyl sulphate, 10% (v/v) butanol and 0.5% (v/v) triethylamine buffered at pH 3 and fluorimetric detection. Quinolones were eluted in less than 15 min without the protein band or other endogenous compounds from the food matrices interfering. The calculated relevant validation parameters, e.g., decision limit (CCα), detection capability (CCβ), repeatability, within-laboratory reproducibility, recoveries and robustness, were acceptable and complied with European Commission Decision 2002/657/EC. Finally, the proposed method was successfully employed in quantifying the four quinolones in spiked egg and milk samples