Validated chiral high-performance liquid chromatography method for a novel anti-methicillin-resistant Staphylococcus aureus fluoroquinolone WCK 771

A sensitive, simple, specific, precise, accurate and rugged method for the assay and determination of enantiomeric purity of S-(-)-9-fluoro-6,7-dihydro-8-(4-hydroxypiperidin-1-yl)-5-methyl-1-oxo-1H,5H-benzo[i,j]quinolizine-2-carboxylic acid L-arginine salt tetrahydrate (WCK 771) in bulk drug has been developed. The method is RP-HPLC using endcapped C-18 stationary phase and chiral mobile phase. Chirality to the mobile phase was imparted with addition of beta-cyclodextrin. The UV-vis detector was operated at 290 nm. The flow rate of mobile phase was 2 ml/min. The method offers excellent separation of two enantiomers with resolution more than 2 and tailing factor less than 1.5. The method was validated for the assay of WCK 771 and quantification of R-(+)-enantiomer impurity in bulk drug. The calibration curves showed excellent linearity over the concentration range of 0.05-0.15 mg/ml for WCK 771 and 0.5-7.5 microg/ml for R-(+)-enantiomer. The precision (RSD) of the assay was 0.23%. The limit of detection and limit of quantitation of the method for WCK 771 were 0.015 and 0.06 microg/ml, respectively. The limit of detection and limit of quantitation for R-(+)-enantiomer were 0.025 and 0.09 microg/ml, respectively. The average recovery of the R-(+)-enantiomer was 100.5%. Same method was applied for the assay and determination of enantiomeric purity of WCK 771 in the intravenous formulation.     

Development and validation of a capillary zone electrophoretic method for the determination of bisphosphonate and phosphonate impurities in clodronate

Capillary zone electrophoresis with direct UV detection at low wavelength and reversed polarity was applied for the separation and quantitation of bisphosphonate and phosphonate impurities in clodronate bulk material. Polyacrylamide-coated capillaries were used to reduce the interactions between the analytes and the electric double layer of the capillary, and to minimize electroosmotic flow. Study was made of the major factors affecting the separation, i.e., pH and ionic strength of the electrolyte solution and various instrumental parameters. The developed method provided reproducible separations of clodronate and related impurities (between-day precision of migration times: RSD < 2.3%, 275 runs). Acceptable validation results in the impurity quantitation range of 0.5-7.5 microg ml(-1) (corresponding to 0.1-1.5% of clodronate working concentration) were obtained in specificity, within-day and between-day precision, accuracy and linearity.     

Simultaneous determination of dopa and carbidopa enantiomers by capillary zone electrophoresis

Dopa and carbidopa, components of the dual therapy for Parkinson's disease treatment, are both provided as single enantiomers, since their D-forms are inactive. To ensure the efficiency and safety of the therapy, these D-enantiomers, therefore, should be considered as impurities. In this paper, the enantioseparation power of different types of cyclodextrins, both neutral and charged ones, on dopa and carbidopa enantiomers was tested. Three methods of simultaneous separation of dopa and carbidopa enantiomers were developed, using highly sulfated beta-cyclodextrin and sulfated beta-cyclodextrin as chiral selector, in normal and reversed polarity mode. Two methods among these three were found sensitive enough for the quantitation of 0.1% D-enantiomers in L-forms (impurity level). After the optimization study, the best method was selected, using 16 mM sulfated beta-cyclodextrin in 15 mM sodium phosphate buffer pH 2.45, an uncoated fused-silica capillary (50 num inner diameter, 30 cm total length), and an applied voltage of -12 kV. This method is robust and efficient, with very high resolution for all peaks within a short analysis time of 10 min. Quantitatively, the method offers a limit of detection (LOD) of 0.2 nug/mL and a limit of quantitation (LOQ) of 0.5 nug/mL for both D-dopa and D-carbidopa, which is equivalent to 0.02% and 0.05% against the respective L-enantiomers. A linear relationship was found between the concentration of the analyte and the corresponding peak area in a range of 0.5-2.0 nug/mL.     

Rapid capillary electrophoretic method for the determination of clozapine and desmethylclozapine in human plasma

A rapid and sensitive high-performance capillary electrophoretic method for the determination of clozapine and its main metabolite desmethylclozapine in human plasma was developed. The separation of the two analytes was carried out in an untreated fused-silica capillary [33 cm (8.5 cm effective length) x 50 microm I.D.] filled with a background electrolyte at pH 2.5 containing beta-cyclodextrin. Baseline separation of clozapine and desmethylclozapine was recorded in less than 3 min. An accurate sample pretreatment by means of solid-phase extraction and subsequent concentration allows for reliable quantitation of clozapine in the plasma of schizophrenic patients under treatment with the drug. The method showed good precision (mean RSD = 4.0%) as well as satisfactory extraction yields (approximately 88%) and a good sensitivity (limit of quantitation = 0.075 microg ml(-1), limit of detection = 0.025 microg ml(-1)).     

Rapid and direct analysis of gamma-hydroxybutyric acid in urine by capillary electrophoresis-electrospray ionization ion-trap mass spectrometry

The present work was aimed at the development of a capillary electrophoretic analysis of gamma-hydroxybutyric acid (GHB) using electrospray ion trap mass spectrometry to achieve the direct and unequivocal detection of this analyte in human urine. Optimized capillary electrophoretic conditions were: injection, 20 s at 0.5 psi (1 psi = 6894.76 Pa); buffer electrolyte, 12.5 mM ammonium formate adjusted to pH 8.35 with diethylamine; fused silicacapillary: 100 cm x 50 microm i.d.; separation voltage, 25 kV (forward polarity) + 0.5 psi; room temperature. Electrospray and mass spectrometric conditions were: drying gas and nebulizing gas (nitrogen) at flow rate 3 l/min, temperature 250 degrees C, nebulizer pressure: 10 psi; sheath liquid solution: methanol-water (90:10) containing 0.1% ammonia delivered at 3 microl/min; spray voltage 3.5 kV. Mass spetrometric detection was carried out in the selected ion monitoring mode of negative molecular ions at 103 m/z for GHB and 115 m/z for maleic acid (I.S.). Under these conditions the baseline separation of GHB and the I.S. was obtained. The selectivity of the analysis allowed for direct injection of unextracted urine, previously diluted 1:4 with water. Linearity was assessed in the GHB concentration range from 80 to 1280 microg/ml in urine. Analytical sensitivity (as limit of detection) resulted about 5 microg/ml in water and 20 microg/ml in original urine. Analytical precision was fairly acceptable with R.S.D. values lower than 5% for migration times and 18% for quantitation in real samples, in both intra day and day-to-day experiments. On these grounds, the developed method can be adopted for rapid identification of acute intoxications from GHB in humans.     

Determination of nonsteroidal anti-inflammatory drugs in biological fluids by automatic on-line integration of solid-phase extraction and capillary electrophoresis

A new, automatic method for the clean-up, preconcentration, separation, and quantitation of nonsteroidal anti-inflammatory drugs (NSAIDs) in biological samples (human urine and serum) using solid-phase extraction coupled on-line to capillary electrophoresis is proposed. Automatic pretreatment is carried out by using a continuous flow system operating simultaneously with the capillary electrophoresis equipment, to which it is linked via a laboratory-made mechanical arm. This integrated system is controlled by an electronic interface governed via a program developed in GWBasic. Capillary electrophoresis is conducted by using a separation buffer consisting of 20 mM NaHPO4, 20 mM beta-cyclodextrin and 50 mM SDS at pH 9.0, an applied potential of 20 kV and a temperature of 20 degrees C. The analysis time is 10 min and the detection limits were between 0.88 and 1.71 microg mL(-1). Automatic clean-up and preconcentration is accomplished by using a C-18 minicolumn and 75% methanol as eluent. The limit of detection of NSAIDs can be up to 400-fold improved when using sample clean-up. The extraction efficiency for these compounds is between 71.1 and 109.7 microg mL(-1) (RSD 2.0-7.7%) for urine samples and from 77.2 to 107.1 microg mL(-1) (RSD 3.5-7.1%) for serum samples.     

Capillary zone electrophoresis for the determination of light-absorbing anions in environmental samples.

A rapid and simple method for separation and determination of inorganic anions by capillary zone electrophoresis was described. The detection was carried out directly with a diode array detector. The experimental conditions, such as concentration of carrier electrolyte, capillary length, voltage, and temperature were optimized. In order to improve selectivity, different organic modifiers were also investigated. The baseline separation of 10 light-absorbing anions was accomplished within 3.5 min with a background electrolyte consisting of 50 mM sodium tetraborate containing 5% MeOH. Linear plots were obtained in the concentration range of 0.1-10 microg/ml. With sample stacking injection, the quantitation limits of the anions were found to be in the range of 0.02-0.1 microg/ml. The proposed method was successfully applied to the determination of inorganic anions in environmental samples and in effluents of a power plant.     

Semiquantitative determination of residues of amphetamine in sewage sludge samples

A procedure based on HPLC and mass spectrometric detection has been developed for screening of residues of the illicit drug amphetamine in sewage sludge. Sample pretreatment consisted in extraction by 50 mM formic acid and methanol (80:20 v/v), followed by adjustment of the pH to 10 and preconcentration by SPE at poly(di-vinylbenzene)-N-vinylpyrrolidone. HPLC separation of the extract was done on a C18 RP with a mixture of 50 mM formic acid and methanol (80:20 v/v) as mobile phase. The mass spectrometer was operated in the MS2 and MS3 mode using the transition from m/z 136 to 119 and from m/z 119 to 91. Due to the complex matrix, ionization suppression effects as well as shifts in the sensitivity of the detector within a series of runs could not be fully excluded. Therefore, quantitation was done by standard addition together with external standards, so that semiquantitative results could be obtained down to concentrations of 2 microg/kg sewage sludge. Samples taken from various municipal sewage treatment plants indicate that amphetamine residues are ubiquitous in urban areas.     

Resonance light scattering spectroscopy of beta-cyclodextrin-sodium dodecylsulfate-protein ternary system and its analytical applications.

A simple, highly sensitive and dye-less assay for proteins was reported using a resonance light-scattering (RLS) technique based on the enhanced RLS intensity of beta-cyclodextrin (beta-CD)-sodium dodecylsulfate (SDS)-protein system. Under the optimum conditions, the enhanced RLS intensity is in proportion to the concentration of proteins in the range of 0.01 to 2.3 microg ml(-1) for bovine serum albumin (BSA), 0.01 to 2.0 microg ml(-1) for human serum albumin (HSA), 0.015 to 5.0 microg ml(-1) for gamma-globulin (gamma-G), 0.02 to 3.5 microg ml(-1) for egg albumin (EA), 0.02 to 4.0 microg ml(-1) for pepsin (Pep), and 0.02 to 3.6 microg ml(-1) for alpha-chymotrypsin (Chy). Their detection limits (S/N = 3) are 1.1, 1.6, 2.4, 6.7, 5.4 and 4.2 ng ml(-1), respectively. Synthetic samples and human serum samples were determined satisfactorily, and the results were in reasonable agreement with those obtained by a documented spectrophotometric (Bradford) method.     

Determination of the flavone tricin in human plasma by high-performance liquid chromatography

Tricin is a flavone constituent of brown rice and rice bran, which interferes potently with the survival of human-derived breast and colon cancer cells in vitro. A specific and simple high-performance liquid chromatographic (HPLC) method was developed for the determination of tricin in human plasma with UV-visible detection. HPLC separation on Hypersil-BDS C(18) (4.6 x 250 mm) was carried out with an isocratic mobile phase of 52% methanol in 0.1 m ammonium acetate, pH 5.10, containing 0.27 mm disodium ethylenediamine tetraacetic acid and detection at 355 nm. The retention times of tricin and quercetin (internal standard) were 14.2 and 7.8 min, respectively. The assay was linear in the range 1-100 microg/mL (r(2 ) > or = 0.995). Tricin in plasma was efficiently extracted with 0.1 m acetic acid in acetone, and the recoveries were in the range 92.6-102.8% (n = 6) with relative standard deviation below 10% for three concentrations of tricin, 5, 10 and 100 microg/mL. The lower limit of quantitation (relative standard deviation <20%) was 1 microg/mL.     

Determination of triamcinolone in human plasma by a sensitive HPLC-ESI-MS/MS method: application for a pharmacokinetic study using nasal spray formulation

A liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method for the quantitation of triamcinolone in human plasma after nasal spray application was developed and validated. Betamethasone was used as internal standard (IS). The analytes were extracted by a liquid-liquid procedure and separated on a Zorbax Eclipse XDB C(18) column with a mobile phase composed of 2 mM aqueous ammonium acetate pH 3.2 and acetonitrile (55:45). Selected reaction monitoring was performed using the transitions m/z 435 → 415 and m/z 393 → 373 to quantify triamcinolone acetonide and betamethasone, respectively. Calibration curve was constructed over the range of 20-2000 pg/ml for triamcinolone acetonide. The lower limit of quantitation was 20 pg/ml. The mean RSD values were 4.6% and 5.7% for the intra-run and inter-run precision, respectively. The mean accuracy value was 98.5% and a recovery rate corresponding to 97.5% was achieved. No matrix effect was detected in the samples. The validated method was successfully applied to determine the plasma concentrations of triamcinolone acetonide in healthy volunteers, in a pharmacokinetic study with nasal spray formulation.     

Determination of chamazulene carboxylic acid in serum by high-performance liquid chromatography. Development and validation

A new, simple and sensitive high-performance liquid chromatographic (HPLC) method was developed for the determination of chamazulene carboxylic acid (CCA) in serum. The technique is based on a single liquid-liquid extraction of the substance using ibuprofen as internal standard (I.S.). The separation was achieved on a C(18) reversed-phase column using acetonitrile/water (4:6, pH 3) as mobile phase. The effluent was monitored at 221 and 286 nm. The calibration curves were linear over the concentration range of 0.1-30 microg/ml. The intra- and inter-day RSDs were in all cases less than 15 and 11%, respectively. The limit of quantitation was 0.1 microg/ml. The assay was developed and validated to be applied in a pharmacokinetic study in healthy volunteers.     

Separation of steroids using temperature-dependent inclusion chromatography

The influence of temperature on retention and separation of estrogens, progesterone derivatives and beta-cyclodextrin in reversed-phase high-performance liquid chromatography has been studied. Steroids were detected using direct UV detection at 240 and 280 nm. Detection of beta-cyclodextrin was achieved using a post-column indirect photometric method. Chromatographic experiments were performed using an acetonitrile-water mobile phase (30%, v/v) and a wide range of column temperatures from 0 to 80 degrees C with 20 degrees C steps. Linear Van't Hoff plots were observed for steroids and beta-cyclodextrin when an unmodified binary mobile phase was applied. The retention of steroids was strongly influenced by temperature when the mobile phase was modified with beta-cyclodextrin at a concentration of 12 mM. Particularly, for 17beta-estradiol and 20alpha-hydroxyprogesterone a strong deviation from the linear Van't Hoff plots and a remarkable affinity for beta-cyclodextrin was observed. Polynomial regression calculations were performed to fit the set of experimental data points. Using third-order polynomial equations, minimum separation factor values (alphamin) were calculated for temperatures from -10 to + 100 degrees C with 1 degrees C steps. The best chromatographic conditions for separation of multicomponent samples were chosen. A possible retention mechanism for solutes in the presence of macrocyclic additives is discussed. The results presented describe the role of temperature in high-performance liquid chromatography systems in which the mobile phase is modified with an inclusion agent.     

Gas and liquid chromatography of metal chelates of pentamethylene dithiocarbamate

Capillary GC and HPLC of metal chelates of pentamethylene dithiocarbamate were examined. Copper(II), nickel(II), cobalt(III), iron(III), manganese(II) and chromium(III) chelates formed in slightly acidic media (pH 5) were extracted in methyl isobutyl ketone or chloroform. Capillary GC elution and separation was carried out on methylsilicone DB-1 column (25 m x 0.2 mm I.D.) with film thickness 0.25 microm. Electron-capture detection was used. Elution was carried at initial column temperature 200 degrees C with an increment at a rate of 5 degrees C/min up to 250 degrees C and maximum temperature was maintained for 10 min. Symmetrical peaks with baseline separation were obtained with the metal chelates investigated with linear calibration range between 5 and 25 microg/ml for each metal ion and detection limits in the range of 0.5-6.0 microg/ml corresponding to 27-333 pg of metal ion reaching to the detector. HPLC separation was carried out from LiChrosorb ODS, 5 microm column and complexes eluted with methanol-water-1 mM sodium acetate (70:28:2, v/v) with a flow-rate of 1.2 ml/ml. UV detection was at 260 nm. The detection limits obtained were in the range 2-6 microg/ml. The methods were applied to the determination of metal ions in canal water and coal samples with RSD values within 4.15%. The results when compared with a standard flame atomic absorption spectrophotometric method and revealed no significant difference.     

Development and validation of spectrophotometric, TLC and HPLC methods for the determination of lamotrigine in presence of its impurity

Three reliable, rapid and selective methods have been developed and validated for the determination of lamotrigine in the presence of its impurity, 2,3-dichlorobenzoic acid. The first method is spectrophotometric method using p-chloranilic acid forming a colored product with lambda(max) 519+/-2 nm. All variables affecting the reaction have been investigated and the conditions were optimized. Beer's law was obeyed over a concentration range of 10-200 microg ml(-1) with mean accuracy 100.13+/-0.44%. The molar ratio of the formed ion-association complex is found to be 1 : 1 as deduced by Job's method. The conditional stability constant (K(f)), standard free energy (DeltaG), molar absorptivity(epsilon), and sensitivity index were evaluated. The second method is based on TLC separation of the cited drug (Rf=0.75+/-0.01) from its impurity (Rf=0.23+/-0.01) followed by densitometric measurement of the intact drug spots at 275 nm. The separation was carried on silica gel plates using ethyl acetate : methanol : ammonia 35% (17 : 2 : 1 v/v/v) as a mobile phase. The linearity range was 0.5-10 microg/spot with mean accuracy 99.99+/-1.33%. The third method is accurate and sensitive stability-indicating HPLC method based on separation of lamotrigine from its impurity on a reversed phase C(18) column, using a mobile phase of acetonitrile : methanol : 0.01 M potassium orthophosphate (pH 6.7+/-0.1) (30 : 20 : 50 v/v/v) at ambient temperature 25+/-5 degrees C and UV detection at 275 nm in an overall analysis time of about 6 min., based on peak area. The injection repeatability, intraday and interday repeatability were calculated. The procedure provided a linear response over the concentration range 1-12 microg ml(-1) with mean accuracy of 99.50+/-1.30%. The proposed methods were successfully applied for the determination of lamotrigine in bulk powder, in dosage form and in presence of its impurity. The results obtained were analyzed by ANOVA to assess that no significant difference between each of the three methods and the reported one. The validation was performed according to USP guidelines.     

Rapid enantiomeric separation of polychlorinated biphenyls by electrokinetic chromatography using mixtures of neutral and charged cyclodextrin derivatives

Electrokinetic chromatography with cyclodextrin derivatives (CD-EKC) was used to achieve the rapid enantiomeric separation of chiral polychlorinated biphenyls (PCBs). Thirteen of the 19 chiral PCBs stable at room temperature were individually separated into their two enantiomers by using 2-morpholinoethanesulfonic acid (MES) buffer (pH 6.5) containing carboxymethylated gamma-cyclodextrin (CM-gamma-CD) as pseudostationary phase mixed with beta-cyclodextrin (beta-CD) or permethylated beta-cyclodextrin (PM-beta-CD). Urea was also added to increase the solubility of PCBs and cyclodextrins in the aqueous separation buffer. Several experimental parameters such as the nature, concentration, and pH of the buffer, nature and concentration of the cyclodextrin derivatives used, and the addition of different additives were studied in order to improve the enantiomeric separation. In addition, the effect of some instrumental parameters such as separation temperature and applied voltage was also investigated. PCBs were enantiomerically separated in less than 12 min by using a 50 mM MES buffer (pH 6.5) containing 20 mM CM-gamma-CD, 10 mM beta-CD or 20 mM PM-beta-CD, and 2 M urea at a temperature of 45 degrees C and an applied voltage of 20 kV.     

Analysis of vancomycin and related impurities by micellar electrokinetic capillary chromatography. Method development and validation

A fast and highly selective micellar electrokinetic capillary chromatography (MEKC) method for quantitative analysis of vancomycin and related impurities is described. Among the tested surfactants, cetyltrimethylammonium chloride (CTAC) offered the best selectivity. Another important parameter, which strongly influenced the selectivity, was buffer pH. It was found that the selectivity increased with buffer pH decreasing from 9 to 5. Using Tris-phosphate buffer containing CTAC, satisfactory separation could be obtained in the pH range from 5.0 to 5.5. Excellent repeatability in terms of migration time and peak area could be obtained when the capillary was carefully washed between two runs. In order to obtain optimal conditions and to evaluate the method robustness, a central composite experimental design was carried out. The optimal conditions were: 44 cm length of fused-silica capillary with 50 microm ID, 120 mM Tris-phosphate buffer (pH 5.2) containing 50 mM CTAC, -15 kV applied voltage, UV detection at 210 nm, and a column temperature of 25 degrees C. Under the optimal conditions, more than 20 peaks could be separated within 8 min. The method has a linearity range from 0.004 to 1.2 mg/ml (concentration of vancomycin B, active component). The limit of detection (LOD) and limit of quantitation (LOQ) were 0.4 microg/mL vancomycin, equivalent to 0.3 microg/mL vancomycin B (0.04%) and 1.1 microg/mL vancomycin, equivalent to 0.9 microg/mL vancomycin B (0.1%), respectively.     

Chromatographic behaviour of selected steroids and their inclusion complexes with beta-cyclodextrin on octadecylsilica stationary phases with different carbon loads

Retention and separation studies of selected estrogens, progestogens and their inclusion complexes with beta-cyclodextrin were conducted using two C18 HPLC columns with different carbon loads. The difference in carbon load between investigated octadecylsilica packing materials was about 50%. The mobile phases were composed of a 30% v/v acetonitrile-water mixture without and with addition of beta-cyclodextrin at a concentration of 12 mM. The experimental data revealed that retention of the steroids was significantly reduced on the column with the lower carbon load. Moreover, it was found that this column offers better separation power and shorter analysis time at the temperatures studied. However, the calculated values of the retention factor ratios (k0(mMCD))/k(12mMCD)) of the steroids were similar for both columns investigated. This observation suggests that the stationary phase structure appears to have little effect on the formation of host-guest complexes if the complexation process is localised to the chromatographic mobile phase. From a practical point of view, when the mobile phase is modified with beta-cyclodextrin, the separation of the steroids is strongly influenced by temperature. The best chromatographic conditions were determined for the separation of multicomponent samples on the column with lower carbon load. A possible retention mechanism for components of interest in the presence of macrocyclic additives is discussed.     

Liquid chromatographic-tandem mass spectrometric determination of amprenavir (agenerase) in serum/plasma of human immunodeficiency virus type-1 infected patients receiving combination antiretroviral therapy

A selective assay method for quantitation of amprenavir (agenerase) in human immunodeficiency virus type-1 infected patient serum or plasma using liquid chromatography-tandem mass spectrometry (LC-MS-MS) is described. Amprenavir and an internal standard (reserpine) are extracted by liquid-liquid extraction and chromatographically separated by a reversed-phase C18-analytical column. The triple quadrupole LC-MS-MS system is operated in the positive-ion mode and multiple reaction monitoring is used for drug quantitation. The method has been validated over the range of 0.05-10.0 microg/ml. The RSDs for the intra-day and inter-day determinations ranged from 5.3 to 6.1% and from 4.7 to 6.2%, respectively. The average assay accuracy at two different concentrations ranged from 96.0 to 103.0% and the extraction recovery of amprenavir was 90.8%. The lower limit of quantitation was 0.05 microg/ml. Using a short microbore column, the analysis was completed in less than 5 min.     

Determining eight colorants in milk beverages by capillary electrophoresis

Milk beverages are popular because of their high nutritional value, and milk products that are enhanced with various fruit flavors are especially in high demand in Asia. Colorants are usually added to fruit flavored milk in order to increase its attraction and appearance, therefore, the detection and measurement of colorants in this type of beverage are relatively important for health issue reasons. Carminic acid, a natural colorant, along with tartrazine, Fast green FCF, Brilliant blue FCF, Allura Red AC, Indigo carmine, Sunset yellow FCF, and New coccine, which are seven different synthetic food colorants, are commonly used as food additives, therefore, this study would focus on the development of an analytical method for the detection of these common colorants in milk beverages. A high efficiency capillary electrophoresis separation method was finished by a pH 10.0 running buffer containing 7.0 mM beta-cyclodextrin, and the eight colorants were separated with baseline resolution within 9 min. In order to reduce the matrix interference resulting from the constituents of milk, a suitable polyamide column solid-phase extraction (SPE) was also investigated for milk sample pretreatment. The combination of the simple SPE pretreatment and the fast separation method of capillary electrophoresis, was able to determine successfully without matrix interference the content of these colorant additives in commercial milk beverages. The recoveries of the eight food colorants in milk beverages were better than 85% and the detection limits were also lower than 0.5 microg/ml by the developed method.