Use of micellar liquid chromatography to analyze oxolinic acid, flumequine, marbofloxacin and enrofloxacin in honey and validation according to the 2002/657/EC decision.

A micellar liquid chromatographic method was developed for the analysis of oxolinic acid, flumequine, marbofloxacin and enrofloxacin in honey. These quinolines are unethically used in beekeeping, and a zero-tolerance policy to antibiotic residues in honey has been stated by the European Union. The sample pretreatment was a 1:1 dilution with a 0.05M SDS at pH 3 solution, filtration and direct injection, thus avoiding extraction steps. The quinolones were eluted without interferences using mobile phase of 0.05M SDS/12.5% 1-propanol/0.5% triethylamine at pH 3, running at 1mL/min under isocratic room through a C18 column. The analytes were detected by fluorescence. The method was successfully validated according to the requirements of the European Union Decision 2002/657/EC in terms of: specificity, linearity (r(2)>0.995), limit of detection and decision limit (0.008-0.070mg/kg), lower limit of quantification (0.02-0.2mg/kg), detection capability (0.010-0.10mg/kg), recovery (82.1-110.0%), precision (<9.4%), matrix effects, robustness (<10.4%), and stability. The procedure was applied to several commercial honey supplied by a local supermarket, and the studied antibiotics were not detected. Therefore, the method was rapid, simple, safe, eco friendly, reliable and useful for the routine analysis of honey samples.

Validation of micellar LC-based methods applied to analyze foodstuffs.

The validation of several micellar LC-based analytical methodologies was described. These methods were able to quantify quinolones in fish from fisheries, hydroxytyrosol in olive extracts and biogenic amines in anchovy sauce. The validation was performed following the requirements of official guides to provide more reliability. Two guides suggested by renowned institution are described: US FDA Guidance for Industry and EU Regulation 2002/657/EC Decision. The appropriate guide was used for each method, depending of the analyte, the matrix and the scope of sample. The calculated validation parameters were those proposed by the guide: selectivity, calibration range, linearity, LOD and LOQ, inter- and intra-day accuracy and precision, limit of decision, detection capability, robustness, recovery and stability. The methodologies were successfully validated by the selected guideline, indicating their suitability to be applied to analysis of real samples, proven to be useful to its intended purpose.

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 µm 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 (r(2) > 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.

Validation of an analytical methodology to quantify melamine in body fluids using micellar liquid chromatography.

Measurement of urine and plasma melamine-concentration is helpful in confirming melamine-associated renal diseases. A chromatographic procedure using a C18 column and a micellar mobile phase of sodium dodecyl sulphate (0.2M), buffered at pH 3 and detection set at 210 nm, was reported for the resolution and quantification of melamine in plasma and urine. In this work, direct injection was used, thus avoiding long extraction and experimental procedures. Melamine was eluted in nearly 6.3 min without overlapping the protein band or other endogenous compounds. The optimal mobile phase composition was taken by studying the influence of each chromatographic parameter. Validation was satisfactorily performed following the US Food and Drug Administration (FDA), in terms of: linearity (0.25-25 ppm; r2>0.9995 in both cases), sensitivity, limit of detection (50 ppb), limit of quantification (250 ppb), intra- and inter-day precision (R.S.D. 0.7-10.2% and 1.0-9.1%, respectively) and recovery, calculated as accuracy (85.7-103.8% and 94.8-103.6%, respectively) and robustness (R.S.D.<7.1%). The suggested methodology has been applied to the analysis of real samples of volunteers, and no melamine was found in any of them.

Analytical determination of hydroxytyrosol in olive extract samples by micellar liquid chromatography.

Hydroxytyrosol is a well-known natural phenolic component obtained from olive extract samples with antioxidant effects. A micellar liquid chromatography method to detect and quantify hydroxytyrosol in olive extract samples is described. Matrix resolution was performed using a Kromasil C18 column and a micellar mobile phase of sodium dodecyl sulphate (SDS) 0.05M and 4% methanol buffered at pH 7. Detection was set by absorbance at 280nm. Samples were diluted with 0.05M SDS at pH 3 and directly injected, thus avoiding long tedious extractions. Hydroxytyrosol was eluted in 3.5min without overlapping other matrix compounds. Validation was performed following the US FDA guideline. The main analytical parameters studied were: linearity (0.03-250µgmL-1; r2=0.999), limit of detection and quantification (3 and 30ngmL-1, respectively), intra- and inter-day precision (RSD, % <1.4 and <8.2, respectively), and robustness (RSD, %<6.6). Recoveries were in the 88.5-98.9% range.

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 (CC(α)), detection capability (CC(ß)), 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.

Simultaneous determination of tyramine and tryptamine and their precursor amino acids by micellar liquid chromatography and pulsed amperometric detection in wines.

Two biogenic amines, tryptamine and tyramine, and their precursors, tryptophan and tyrosine, were determined by a liquid chromatographic procedure. A hybrid micellar mobile phase of sodium dodecyl sulphate (SDS) and 1-propanol, a C18 column and electrochemical detection were used. A pH study in the range of 3-9 was performed and pH 3 was finally selected in accordance with resolution and analysis time. Oxidation potential was also checked in the range 0.6-0.9V: the maximum area obtained in all those potentials was at 0.8V, which was selected to carry out the analysis using a sequence of pulsed amperometric detection waveform. The four compounds were resolved using a mobile phase of 0.15M SDS-5% 1-propanol with an analysis time of 16 min. Repeatabilities and intermediate precision were evaluated at three different concentrations for each compound with RSD values lower than 2.6 and 4.8%, respectively. Limits of detection and quantification were also obtained within the 10-40 and 33-135 ng/ml ranges, respectively. Finally, the applicability of the procedure was tested in several types of wine and no matrix effect was observed. The possibility of direct sample introduction simplifies and greatly expedites the treatments with reduced cost, improving the accuracy of the procedures.

Determination of trazodone in urine and pharmaceuticals using micellar liquid chromatography with fluorescence detection.

A simple and reliable liquid chromatographic procedure is described for the determination of trazodone in pharmaceutical formulations and urine samples. The optimized procedure uses fluorimetric detection, a C18 column and a micellar mobile phase of sodium dodecyl sulfate (SDS) and 1-butanol. The mobile phase selected for use was 0.2M SDS and 8% 1-butanol fixed at pH 3 with phosphate buffer. The total analysis time was 10 min. For the analysis of urine samples, one great advantage of the method is that no extraction step is required. The quantification limit was 9.5 ng mL(-1), ensuring the analysis of the drug in biological fluids. The procedure shows good accuracy, repeatability and selectivity. Repeatability and intermediate precision were tested for several concentrations of the drug. Good claim percentages were obtained in the analysis of pharmaceutical formulations. Calibration repeatability in urine matrix was also studied in the 0.06-22.4 microg mL(-1) range. Good recoveries were obtained from spiked urine samples. No interferences from common additives frequently administered with trazodone or from endogenous compounds in urine samples were found. The results show that the procedure is suitable for routine analysis of the drug.

Micellar liquid chromatographic determination of five antianginals in pharmaceuticals.

A procedure was developed for the determination of five antianginals (diltiazem, nadolol, nifedipine, propranolol and verapamil), using hybrid micellar mobile phases of sodium dodecyl sulphate (SDS) and pentanol, a C18 column and UV detection. All possible combinations of antianginals were resolved and determined using a mobile phase of 0.05 M SDS-5% pentanol with an analysis time of 9 min. Repeatabilities and intermediate precision were evaluated at four different drug concentrations in the 2-20 microg/ml (n=5) range. Limits of detection were in the range 0.028 microg/ml for diltiazem and 0.130 microg/ml for verapamil. The range of the limit of quantitation was from 0.092 to 0.431 microg/ml for the same compounds. Antianginal drugs were studied in pharmaceuticals with no interference from related compounds. The results of the analyses of pharmaceuticals formulations were in agreement with the declared compositions.

Direct determination of verapamil in urine and serum samples by micellar liquid chromatography and fluorescence detection.

Verapamil, a calcium channel antagonist, is one of the most commonly prescribed drugs in the treatment of hypertension. In this work, it was determined in serum and urine samples by a sensitive and precise chromatographic procedure without any pre-treatment step in a C18 column using a micellar mobile phase of 0.15M sodium dodecyl sulfate and 5% pentanol at pH 7. Fluorescence detection set at 230 nm (excitation) and 312 nm (emission) was used. Verapamil is eluted at 12.5 min with no interference by the protein band or endogenous compounds. Linearities (r > 0.998), as well as intra- and inter-day precision, were studied in the validation of the method. LODs were also calculated to be 11.0, 18.5 and 20.2 ng/mL in micellar solution, serum and urine, respectively. Recoveries in the biological matrices were in the 97-99% range. Drug excretion in urine was studied in a volunteer receiving treatment for hypertension, and verapamil, as an unchanged drug, was separated from other metabolites. The procedure developed can be useful in the field of toxicology and clinical analysis.

Photostability studies for micellar liquid chromatographic determination of nifedipine in serum and urine samples.

Nifedipine is a photosensitive compound that is converted into its 4-(2-nitrophenyl) pyridine and 4-(2-nitrosophenyl) pyridine homologue. In order to obtain the most adequate conditions for handling nifedipine solutions in the analytical laboratory, a number of studies on the decomposition of this compound were performed. A simple micellar liquid chromatographic procedure was described to determine nifedipine in different biological matrices such as serum and urine, and to control its decomposition. To perform the analysis, nifedipine was dissolved in 0.1 m SDS at pH 3 and chromatographed using a mobile phase containing 0.125 m SDS-3% pentanol, pH 3 on a C18 column and UV detection at 235 nm. The chromatographic analysis time was 8 min. The response of the drug for both biological matrices was linear in the 1-100 microg/mL range, with r2>0.997 at all times. Repeatability, intermediate precision (CV, %) and limits of quantification and detection (ng/mL) were 0.19, 4.3, 104 and 31 in serum and 0.81, 2.1, 136 and 41 in urine. The method developed here does not show interferences or matrix effects produced by endogenous compounds. Micellar media and mobile phases have the advantage of stabilising the compounds, thus preventing photodegradation and allowing the direct injection of biological samples.

Rapid determination of acetaminophen in physiological fluids by liquid chromatography using SDS mobile phase and ED detection.

Acetaminophen is determined in serum and urine samples by a rapid, sensitive, and precise chromatographic method without any pretreatment step in a C18 column using a pure micellar mobile phase of 0.02M sodium dodecyl sulfate at pH 7. Acetaminophen is eluted in less than 5 min with no interference of the protein band. The use of electrochemical and UV detection is compared. Linearities (r > 0.999), as well as intra- and interday precision, are studied in the validation of the method. Limits of detection (LOD) are also calculated to be 0.56, 0.83, and 0.74 ng/mL in micellar solution, serum, and urine using electrochemical detection. The developed micellar liquid chromatographic method is useful for the quantitation of acetaminophen in serum and urine. Recoveries in the biological matrices are in the 98-107% range and results are compared with those obtained using a reference method. Drug excretion (in urine) and serum distribution are studied in several healthy volunteers, and no interference from metabolites is found. The developed procedure can be applied in routine analyses, toxicology, and therapeutic monitoring.

Micellar liquid chromatography determination of some biogenic amines with electrochemical detection.

A simple and reliable liquid chromatographic procedure is successfully applied to the simultaneous determination of the biogenic amines, dopamine, serotonin, their metabolites (homovalinic acid (HVA) and hydroxyindoleacetic acid (HIAA)) as well as tyramine in serum samples. After an optimization procedure using a C18 column, the mobile phase selected was 0.15 M sodium dodecyl sulfate buffered at pH 3, in which the serum samples were directly injected and the analysis time for the five substances was less than 12 min. The use of electrochemical (ED) and ultraviolet (UV) detection was compared. The limits of detection of the biogenic amines studied were drastically improved using ED detection. Repeatability and intermediate precision were tested at three different concentrations and the relative standard deviations were below 1.5% for most assays. Finally, the method was successfully applied to the determination of biogenic amines in serum samples.

Determination of furosemide in urine samples by direct injection in a micellar liquid chromatographic system.

A sensitive, selective and efficient micellar liquid chromatographic (MLC) procedure was developed for the determination of furosemide (4-chloro-N-furfuryl-5-sulfamoylanthranilic acid) in urine samples by direct injection and UV detection. The procedure makes use of a C18 reversed-phase column and a micellar mobile phase of 0.05 mol l(-1) sodium dodecyl sulfate-6% v/v propanol and phosphate buffer at pH 3 to resolve furosemide from its photochemical degradation products. The importance of protecting the standards and urine samples to be analysed from light in the assay of furosemide, avoiding its degradation, was verified. The limit of quantification was 0.15 microg ml(-1) and the relative standard deviation of the inter-day assay was 0.8-0.04% in the 6-82 microg ml(-1) range. Detection of urinary excretion of furosemide was followed up to 12 h after ingestion of the drug by a healthy volunteer. No potential interference from the major metabolite (furosemide acylglucuronide) and its hydrolytic product (4-chloro-5-sulfamoylanthranilic acid) was observed. Commonly administered drugs also did not interfere. The proposed MLC procedure permits the rapid and reproducible measurement of low levels of furosemide in a small amount of urine.

Chromatographic analysis of phenethylamine-antihistamine combinations using C8, C18 or cyano columns and micellar sodium dodecyl sulfate-pentanol mixtures.

The chromatographic behaviour of binary and ternary mixtures of several phenethylamines (phenylephrine, phenylpropanolamine, ephedrine, pseudoephedrine and methoxyphenamine) and antihistamines (pheniramine, carbinoxamine, doxylamine, chlorpheniramine, dexchlorpheniramine, dexbrompheniramine, diphenhydramine, tripolidine, azatadine and phenyltoloxamine), found in cough-cold pharmaceutical preparations, was studied using C8, C18 and cyano columns, micellar mobile phases of sodium dodecyl sulfate (SDS) and pentanol and UV detection. Using a C8 column and mobile phases of 0.05 mol l-1 SDS-6% v/v pentanol or 0.15 mol l-1 SDS-2% v/v pentanol at pH 7, more than 30 different phenethylamine-antihistamine combinations can be resolved in < 15 min. Intra- and inter-day repeatabilities and reproducibilities evaluated at three different drug concentrations (0.5, 5 and 25 micrograms ml-1, n = 10) were below 1.6, 2.5 and 2.4%, respectively. The drug amounts found in 18 formulations agreed with those declared by the manufacturers within the tolerance limits, and with those obtained using a mobile phase of 55% v/v methanol at pH 7. No interference was observed from other accompanying drugs such as acetylsalicylic acid, ascorbic acid, betamethasone, bromhexine, caffeine, codeine, dextromethorphan, paracetamol, prednisolone, salicylamide and tartrazine. The proposed procedure has the advantage over the conventional aqueous-organic procedure of using a small amount of organic solvent, which is highly retained in the SDS solution. The efficiencies are also greater. On the other hand, in the micellar system, the retentions of phenethylamines and antihistamines are similar, although the compounds can be easily resolved. In contrast, using the methanol-water mobile phase, the phenethylamines are weakly retained, whereas the antihistamines usually show a high retention.

Determination of active ingredients in cough-cold preparations by micellar liquid chromatography.

The chromatographic behaviour of some active ingredients in cough-cold pharmaceutical preparations, the antihistamine chlorpheniramine (or the dextro enantiomer dexchlorpheniramine), and the phenethylamines phenylephrine, phenylpropanolamine and pseudoephedrine, has been studied using a C(18) column, micellar mobile phases of sodium dodecyl sulphate (SDS) and pentanol, and with UV detection. All possible combinations of chlorpheniramine/phenethylamine were resolved and determined using a mobile phase of 0.15 M SDS-6% (v/v) pentanol at pH 7, with analysis time below 7 min. Repeatabilities and within laboratory precisions were evaluated at four different drug concentrations in the range 0.5-25 mug ml(-1) (n=5), resulting RSDs below 1.6%. The drug amounts found in the analysis of 14 commercialised preparations agreed with those declared by the manufacturers within the tolerance limits, and with those obtained using an aqueous 60% (v/v) methanol reference mobile phase. No interference was observed from other accompanying drugs such as acetylsalicylic acid, ascorbic acid, betamethasone, caffeine, codeine phosphate, diphenhydramine, lactose, paracetamol, and prednisolone. The studied combinations required a rather high amount of methanol in conventional RPLC to be eluted from the column. In contrast, the proposed procedure used a much lower amount of organic solvent (pentanol), which is highly retained in the SDS solution, being also less toxic than methanol.

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.

Use of a three-factor interpretive optimisation strategy in the development of an isocratic chromatographic procedure for the screening of diuretics in urine samples using micellar mobile phases.

Screening of diuretics in urine is feasible through direct injection of the samples into the chromatographic system and isocratic reversed-phase liquid chromatography (RPLC) with micellar-organic mobile phases of sodium dodecyl sulfate (SDS) and 1-propanol. The surfactant coverage of the chromatographic column makes the addition of organic competing amines less necessary than in conventional aqueous-organic RPLC to achieve well-shaped peaks. Also, the range of elution strengths of micellar mobile phases required to elute mixtures of hydrophobic and hydrophilic diuretics is smaller. This allows the isocratic separation of the diuretics within adequate analysis times. An interpretive methodology is applied to optimise the resolution of a mixture of 15 diuretics of diverse polarity and acid-base behaviour (althiazide, amiloride, bendroflumethiazide, benzthiazide, bumetanide, canrenoic acid, chlorthalidone, ethacrynic acid, furosemide, piretanide, probenecid, torasemide, triamterene, trichloromethiazide and xipamide), using pH and concentrations of surfactant and organic modifier in the mobile phase as separation factors. Twelve diuretics were resolved in 25 min using 0.055 M SDS-6.0% 1-propanol at pH 3.0. The mixture of 15 diuretics was also resolved with two mobile phases showing complementary behaviour: 0.05 M SDS-5.6% 1-propanol at pH 5.4 and 0.11 M SDS-5.4% 1-propanol at pH 4.2. The results were applied to the analysis of urine samples with limits of detection similar to those usually reported for aqueous-organic RPLC, taking into account that the samples were injected without any previous treatment to separate or preconcentrate the analytes.

Furosemide assay in pharmaceuticals by Micellar liquid chromatography: study of the stability of the drug.

A simplified high-performance liquid chromatographic procedure is described for the determination of furosemide (4-chloro-N-furfuryl-5-sulphamoylanthranillic acid), which makes use of UV detection, a C18, reversed-phase column, and micellar mobile phases of sodium dodecyl sulphate (SDS) and 1-propanol at pH 3 buffered with phosphate system. The most adequate experimental conditions to handle furosemide solutions in the analytical laboratory are studied. The mixture of furosemide and its degradation products which are formed upon light exposition was resolved with a mobile phase of 0.04 M SDS-2% propanol. Separation of furosemide from its common impurities and the hydrolytic product, 4-chloro-5-sulphamoylanthranillic acid, was also possible. A mobile phase of larger elution strength, such as 0.06 M SDS-8%, propanol was preferred to assay furosemide in several dosage forms (tablets, capsules, injectables and drops). The validity of the procedure was checked by analysing 27 pharmaceuticals commercialised in several countries. The label claim percentages and coefficients of variation were in the 95-102% and 0.05-1.3% ranges, respectively. The results showed that the procedure is suitable for routine analysis of the diuretic.

Comparison of the performance of butanol and pentanol as modifiers in the micellar chromatographic determination of some phenethylamines.

A procedure was developed for the determination of several phenethylamines (amphetamine, arterenol, ephedrine, phenylephrine, phenylpropanolamine, mephentermine, methoxyphenamine, pseudoephedrine and tyramine), using micellar mobile phases of sodium dodecyl sulfate (SDS), a C18 column and UV detection. The drugs were eluted at short retention times with conventional acetonitrile-water or methanol-water mobile phases. In contrast, in the micellar system, they were strongly retained due to association with the surfactant adsorbed on the stationary phase, and needed the addition of butanol or pentanol to be eluted from the column. These modifiers allowed a simple way of controlling the retention. The chromatographic efficiencies obtained with the hybrid mobile phases of SDS-butanol and SDS-pentanol were also very high, mostly in the N=3000-7000 range, significantly greater than those achieved with a conventional acetonitrile-methanol-water mobile phase. Butanol and pentanol yielded similar selectivities, but the latter modifier permitted significantly shorter retention times than butanol, and was preferred to expedite the analysis of the pharmaceuticals. Most binary combinations of the nine phenethylamines can be resolved with these mobile phases. A mobile phase of 0.15 M SDS-5% pentanol was used to assay five of the phenethylamines (amphetamine, ephedrine, phenylephrine, phenylpropanolamine and pseudoephedrine) in 22 pharmaceutical preparations, which contained diverse accompanying compounds. The results agreed with the declared compositions and with those obtained with a mobile phase of methanol-acetonitrile-0.05 M phosphate buffer (pH 3) 10:5:85, with no interferences and relative errors usually below 2%. However, with the aqueous-organic mobile phase, the retention time for phenylephrine was too low and could not be usually evaluated.