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), B1 (thiamine), B2 (riboflavin), B6 (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 C18 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 degrees 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.     

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.     

Heat capacities,volumes and solubilities of pentanol in aqueous surfactant solutions

Apparent molar heat capacities and volumes of pentanol (PentOH) 0.05m in dodecyltrimethylammonium chloride (DTAC), dodecyldimethylammonium chloride (DDAC) and dodecylamine hydrochloride (DAC) micellar solutions were measured at 25°C. They were assumed to approach the standard infinite dilution values and rationalized by means of previously reported equations. The distribution constant between the aqueous and the micellar phase and heat capacity and volume of pentanol in both phases were thus derived. The results show that the presence of methyl groups on the surfactant head group does not appreciably influence the apparent molar volume and heat capacity of pentanol in micellar phase and the free energy of transfer of pentanol from the aqueous to the micellar phase. Also, the apparent molar heat capacities of pentanol in micellar solutions as a function of surfactant concentration show evidence of two maxima for DAC and of one maximum for DTAC whereas no maxima were detected for DDAC. According to the literature data for alkyltrimethylammonium bromides these maxima can be ascribed to the presence of structural post-micellar transitions. It is shown that the C_,PentOH vs. surfactant molality curve for DAC lies between that for hexadecyltrimethylammonium bromide and that for tetradecyltrimethylammonium bromide. This evidence, which is similar to that found for solubilities, agrees with the previously reported idea that the removal of a CH₃ group from the head group of surfactant is equivalent to the introduction of a CH₂ group in its hydrophobic moiety. By comparing data for DTAC with those for the corresponding bromide, the role of the nature of the counterion in the thermodynamics of solubilization of pentanol in micellar solutions is derived.     

Comparison of C18 silica bonded phases selectivity in micellar liquid chromatography

The paper describes a new test designed in micellar LC (MLC) to compare the commercial C18 stationary phase properties. This test provides the total hydrophobicity, hydrophilicity, steric selectivity, hydrogen bonding, and ion‐exchange capacity properties calculation of the ODS stationary phases. Both the test compounds and chromatographic separation conditions choice for column characterization in MLC are detailed. The chromatographic performance of several stationary phases that are used in MLC was evaluated with specific chromatographic test comprising nine test compounds, possessing different physico‐chemical properties, which were injected on different supports with two micellar mobile phases: one at pH 7.0 (0.075 mol/L SDS and 1.5% v/v 1‐pentanol), and other at pH 2.7 (0.075 mol/L SDS and 1.5% v/v 1‐pentanol adjusted to pH by TFA). Fundamental column chromatographic properties were obtained under these conditions and were treated by hierarchical cluster analysis. From the results of cluster analysis, two closely related groups of columns are distinguished, and it was shown that the chosen column characteristic parameters allow characterizing both sorbent and micellar chromatographic system properties. Eleven columns were analyzed by this test, which allows a comparison of columns with the aim of the selection of suitable and analogous column for the analysis with MLC.     

Use of micellar mobile phases for the chromatographic determination of clorazepate, diazepam, and diltiazem in pharmaceuticals

An ODS-2 column, a micellar mobile phase of high elution strength containing 0.1M sodium dodecyl sulfate and 3% (v/v) butanol, and ultraviolet detection at 230 nm are used for the determination of either of two benzodiazepines (clorazepate and diazepam) and a benzothiazepine (diltiazem) in pharmaceuticals. The procedure is shown to be competitive against conventional chromatography with methanol-water mobile phases, especially for diltiazem. The composition of the micellar mobile phase is selected using a predictive strategy based on an accurate retention model and assisted by computer simulation. Calibration graphs are linear at least in the 2.5 to 20 microg/mL, 4 to 20 microg/mL, and 5 to 40 microg/mL ranges for clorazepate, diazepam, and diltiazem, respectively. The intra- and interday repeatabilities (%) are clorazepate (1.7, 5.2), diazepam (0.43, 3.7), and diltiazem (0.36, 3.1). Limits of detection are well below the concentrations of the drugs found in the commercial pharmaceutical preparations analyzed. The drug contents evaluated with the proposed procedure are compared with the declared contents given by the manufacturers. The achieved percentages of label claim are usually between 95 and 104%.     

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.     

Simultaneous determination of three opiates in serum by micellar liquid chromatography using direct injection

A simple and reliable micellar liquid chromatographic method was developed for the simultaneous determination of 3 opiates (codeine, morphine, and thebaine) in serum, using direct injection and ultraviolet detection. The separation of the drugs was optimized on a C18 column, thermostatically controlled at 25 degrees C, by evaluating mobile phases containing sodium dodecyl sulfate (SDS) and various modifiers (propanol, butanol, or pentanol). Adequate resolution of the opiates was obtained with a chemometrics approach, in which retention was modeled as a first step by using the retention factors for several mobile phases. Next, an optimization criterion that takes into account the position and shape of the chromatographic peaks was applied. The 3 opiates were totally resolved and determined in 12 min with the mobile phase 0.15M SDS-7% (v/v) butanol buffered at pH 7. The limits of detection for codeine and morphine were greatly improved by using fluorimetric detection. Repeatability and intermediate precision were tested for 3 different concentrations of the drugs, and the relative standard deviations were <0.8% for most of the assays. Finally, the method was successfully applied to the determination of morphine and codeine in serum samples.     

Heat capacities,volumes and solubilities of pentanol in aqueous alkyltrimethylammonium bromides

Apparent molar heat capacities and volumes of pentanol, 0.05m in decyl-, tetradecyl- and hexadecyltrimethylammonium bromides micellar solutions, were measured at 25°C. They were assumed to approach the standard infinite dilution values and rationalized by means of previously reported equations following which the distribution constant between the aqueous and the micellar phase, heat capacity, and volume of pentanol in both phases are simultaneously derived. The present results show that the volume of the micellar core does not seem to have a significant effect on the apparent molar volume and heat capacity of pentanol in the micellar phase and on the free energy of transfer of pentanol from the aqueous to the micellar phase. We report an equation correlating the free energy of transfer of alcohols in alkyltrimethylammonium bromides as a function of the number of carbon atoms in the alcohol and surfactant alkyl chain. Also, the apparent molar heat capacities of pentanol in micellar solutions as a function of surfactant concentration show evidence of two maxima, which, by increasing the alkyl chain length of surfactant display an opposite dependence on concentration. The second maximum can be attributed to a sphere to rod transition. The second transition was also found in the case of butoxyethanol in hexadecyltrimethylammonium bromide. It is more difficult to explain the nature of the first maximum although an attempt is made.     

Monitoring bronchodilators with direct injection

A procedure was developed for the determination of caffeine and theophylline using a C18 column (5 microm, 250 mm x 4.6 mm) and micellar liquid chromatography using hybrid mobile phases containing sodium dodecyl sulfate (SDS) and propanol, butanol or pentanol as modifiers. Detection was performed with a variable wavelength UV-vis detector at 272 nm. After the application of an interpretative strategy for the selection of the optimimum mobile phase, caffeine and theophylline can be resolved and determined in serum samples by direct injection, using a mobile phase made up of 50 mM SDS-2.5% (v/v) propanol-10 mM KH2PO4, pH 7, with an analysis time below 5 min. Calibration was linear in the range 0.05 to 50 microg mL(-1) with r > 0.999. The statistical evaluation of the method was examined by performing intra-day (n = 6) and inter-day calibration (n = 7) and was found to be satisfactory, with highly accurate and precise results. The proposed method was suitably validated and applied to the determination of caffeine and theophylline in serum samples of patients treated with bronchodilators.     

Determination of some banned stimulants in sports by micellar liquid chromatography

Summary A procedure has been developed for the determination, in <12 min, of several stimulants (amphetamine, ephedrine, methoxyphenamine, phenylephrine and phenylpropanolamine) in spiked urine samples after direct injection, using a hybrid micellar mobile phase of 0.15 M sodium dodecyl sulfate and 3% pentanol at pH 7, on a C₁₈ column with UV detection. Recoveries were 94–102% and limits of detection 4.5 ng·mL⁻¹ for methoxyphenamine and 0.39 μg·mL⁻¹ for amphetamine, similar to those obtained for aqueous solutions. Linearity reached 0.99 and intermediate precision was <8.4 and 5.3, for the two different concentrations tested.     

High-performance liquid chromatographic determination of diuretics in urine by micellar liquid chromatography.

The use of micellar liquid chromatography for the determination of diuretics in urine by direct injection of the sample into the chromatographic system is discussed. The retention of the urine matrix at the beginning of the chromatograms was observed for different sodium dodecyl sulphate (SDS) mobile phases. The eluent strengths of a hybrid SDS-methanol micellar mobile phase for several diuretics were compared and related to the stationary phase/water partition coefficient with a purely micellar mobile phase. The urine band was appreciably narrower with a mobile phase of 0.05 M SDS-5% methanol (v/v) at 50 degrees C (pH 6.9). With this mobile phase the determination of bendroflumethiazide and chlorthalidone was adequate. Acetazolamide, ethacrynic acid, furosemide, hydrochlorothiazide and probenecid were overlapped by the urine matrix, and the retention of amiloride and triamterene was too long.     

Therapeutic monitoring of imipramine and desipramine by micellar liquid chromatography with direct injection and electrochemical detection

A micellar liquid chromatographic (MLC) procedure was developed for the clinical monitoring of imipramine and its active metabolite, desipramine. The determination of these highly hydrophobic substances was carried out after direct injection of the serum samples using a mobile phase composed of 0.15 m SDS--6% (v/v) pentanol buffered at pH 7, pumped at 1.5 mL/min into a C(18) column (250 x 4.6 mm), and electrochemical detection at 650 mV. Using this MLC method, calibration was linear (r > 0.995) and the limits of detection (ng/mL) were 0.34 and 0.24 for imipramine and desipramine, respectively. Repeatabilities and intermediate precision were tested at three different concentrations in the calibration range and a CV (%) below 2.2 was obtained. In this MLC procedure, the serum is determined without treatment, thus allowing repeated serial injections without changes in retention factors, and reducing the time and consumables required to carry out the pretreatment process. The assay method can be applied to the routine determination of serum imipramine and its metabolite in therapeutic drug monitoring.     

Biological assay and liquid chromatographic method for analysis of moxifloxacin in tablets

A microbiological assay and a liquid chromatographic method were validated for quantitation of moxifloxacin in tablets. The microbiological method consisted of a cylinder-plate agar diffusion assay using Micrococcus luteus ATCC 9341 as the test microorganism and phosphate buffer (0.1M, pH 8.0) as the diluent solution. The response graphs for standard and sample solutions were linear (r = 0.9479), and no parallelism deviations were detected in the tested levels of concentration (4.0, 8.0, and 16.0 microg/mL). The interday precision was 2.73%. Recovery values were between 96.25 and 100.5%. The chromatographic analyses were performed using a Shim-pack CLC-ODS column (250 x 4.6 mm, 5 microm) with a mobile phase consisting of (A) a mixture of phosphoric acid (0.17%, v/v) with tetramethylammonium hydroxide (0.05M) and acetonitrile (95 + 5, v/v) and (B) methanol (55 + 45, v/v) adjusted to pH 3.0. The flow rate was 1.0 mL/min, and detection was made at 294 nm. The method was linear in a range from 12.0 to 42 microg/mL (r = 0.9999), and the interday precision was 1.39%. Recovery ranged between 101.9 and 103.81%. Both validated methods were used to quantify the moxifloxacin content in tablets exposed to ultraviolet radiation, and similar results were obtained.     

Improved efficiency in micellar liquid chromatography using triethylamine and 1-butanol as mobile phase additives to reduce surfactant adsorption

The effect of triethylamine as a mobile phase modifier on chromatographic efficiency in micellar liquid chromatography (MLC) is reported for nine different columns with various bonded stationary phases and silica pore sizes, including large-pore short alkyl chain, non-porous, and perfluorinated. Reduced plate height (h) versus reduced velocity (nu) plots were constructed for each column and the A' and C' terms calculated using a simplified Van Deemter equation introduced in our previous work. To further explore the practicality of using triethylamine in the micellar mobile phase, the efficiency of nine polar and non-polar substituted benzenes was studied on seven columns. Surfactant adsorption isotherms were measured for five columns with three micellar mobile phases to understand the relationship between adsorbed surfactant, mobile phase additive, and column efficiency. Clear improvements in efficiency were observed with the addition of 2% (v/v) triethylamine to a 1-butanol modified aqueous micellar mobile phase. This finding is supported by the lower amount of surfactant adsorbed onto the stationary phase when TEA is present in the mobile phase compared to an SDS only or a 1-butanol modified SDS mobile phase.     

Screening and monitoring antiretrovirals and antivirals in the serum of acquired immunodeficiency syndrome patients by micellar liquid chromatography

Thirteen different antiretrovirals are commonly used in hospital protocols for suppressing the activity of the human immunodeficiency virus (HIV) and associated opportunistic diseases in patients with acquired immunodeficiency syndrome (AIDS). In this work, three micellar mobile phases are recommended for screening these substances, using UV detection, and the process can be performed in less than 18 min. The first mobile phase (sodium dodecyl sulphate or SDS 50 mM) is used for the group consisting of acyclovir, didanosine, ganciclovir, stavudine and zidovudine. The second mobile phase (SDS 120 mM/4.5% propanol) is used for the group containing abacavir, lamivudine, nevirapine, valaciclovir and zalcitabine, whereas the third mobile phase (SDS 150 mM/5% pentanol) is used for efavirenz, indinavir and ritonavir. The use of micellar liquid chromatography (MLC) as an analytical tool allows serum samples to be injected directly. The method was validated over the range of 0–10 μg mL⁻¹. The limits of detection (signal-to-noise ratio of 3), which ranged from 6 to 30 ng mL⁻¹, were adequate for monitoring these substances. Intra- and inter-day relative standard deviations of the assay were below 3% for all compounds. The recoveries in spiked serum samples were in the 89.5–104.4% range. The method can be applied to the screening, monitoring and control of patients’ treatment with antiretrovirals and antivirals.     

Monitoring disopyramide, lidocaine, and quinidine by micellar liquid chromatography

A micellar liquid chromatography (MLC) method using a C18 column was developed to determine three antiarrhythmic drugs--disopyramide, lidocaine, and quinidine--that are most usually monitored in serum samples. After the application of an interpretative strategy for optimization of sodium dodecyl sulfate (SDS) and modifier concentrations in order to ensure the minimum analysis time, maximum sensitivity, and good resolution, the optimum chromatographic conditions for the determination of the three antiarrhythmics were flow rate, 1 mL/min; injection volume, 20 microL; separation temperature, 25 degrees C; mobile phase, 150 mmol/L SDS-7% (v/v) butanol-phosphate buffer, 10 mmol/L, pH 7-0.9% (w/v) NaCl; and detection at 214 nm. The calibration curves for the drugs were linear (r2 > 0.999). The intraday and interday precisions were lower than 3.9% (CV). Recoveries were 100 +/- 0.6% when the method was applied to both serum samples spiked with the antiarrhythmics (n = 10) and real serum samples. In all cases, the results were similar to those obtained using the reference method (fluorescence polarization immunoassay) usually used in the Spanish hospital. The proposed method is useful for hospital monitoring of the antiarrhythmics by direct injection into the chromatograph.     

Development and Validation of Stability-Indicating HPLC Methods for Quantitative Determination of Pravastatin,Fluvastatin, Atorvastatin,and Rosuvastatin in Pharmaceuticals

Abstract

High-performance liquid-chromatographic (HPLC) methods were validated for determination of pravastatin sodium (PS), fluvastatin sodium (FVS), atorvastatin calcium (ATC), and rosuvastatin calcium (RC) in pharmaceuticals. Two stability-indicating HPLC methods were developed with a small change (10%) in the composition of the organic modifier in the mobile phase. The HPLC method for each statin was validated using isocratic elution. An RP-18 column was used with mobile phases consisting of methanol–water (60:40, v/v, for PS and RC and 70:30, v/v, for FVS and ATC). The pH of each mobile phase was adjusted to 3.0 with orthophosphoric acid, and the flow rate was 1.0 mL/min. Calibration plots showed correlation coefficients (r) > 0.999, which were calculated by the least square method. The detection limit (DL) and quantitation limit (QL) were 1.22 and 3.08 µg/mL for PS, 2.02 and 6.12 µg/mL for FVS, 0.44 and 1.34 µg/mL for ATC, and 1.55 and 4.70 µg/mL for RC. Intraday and interday relative standard deviations (RSDs) were <2.0%. The methods were applied successfully for quantitative determination of statins in pharmaceuticals.     

Quantitative determination of seven chemical constituents and chemo‐type differentiation of chamomiles using high‐performance thin‐layer chromatography

A simple and rapid high‐performance thin‐layer chromatographic method was developed for the separation and determination of six flavonoids (rutin, luteolin‐7‐O‐β‐glucoside, chamaemeloside, apigenin‐7‐O‐β‐glucoside, luteolin, apigenin) and one coumarin, umbelliferone from chamomile plant samples and dietary supplements. The separation was achieved on amino silica stationary phase using dichloromethane/acetonitrile/ethyl formate/glacial acetic acid/formic acid (11:2.5:3:1.25:1.25 v/v/v/v/v) as the mobile phase. The quantitation of each compound was carried out using densitometric reflection/absorption mode at their respective absorbance maxima after postchromatographic derivatization using natural products reagent (1% w/v methanolic solution of diphenylboric acid‐β‐ethylamino ester). The method was validated for specificity, limits of detection and quantification, precision (intra‐ and interday) and accuracy. The limits of detection and quantification were found to be in the range from 6–18 and 16–55 ng/band for six flavonoids and one coumarin, respectively. The intra‐ and interday precision was found to be <5% RSD and recovery of all the compounds was >90%. The data acquired from high‐performance thin‐layer chromatography was processed by principal component analysis using XLSTAT statistical software. Application of principal component analysis and agglomerative hierarchial clustering was successfully able to differentiate two chamomiles (German and Roman) and Chrysanthemum.     

Separation optimization of quercetin,hesperetin and chrysin in honey by micellar liquid chromatography and experimental design

The chemometrics approach was applied for the separation optimization of flavonoid markers (quercetin, hesperetin and chrysin) in honey using micellar liquid chromatography (MLC). The investigated method combines SPE of flavonoids from honey using C₁₈ cartridge and their separation and quantification by micellar liquid chromatography. A two level full factorial design was carried out to evaluate the effect of four experimental factors including concentration of SDS, alkyl chain length of the alcohol used as the organic modifier (N), volume percentage of the organic modifier (V_m) and volume percentage of acetic acid (AcOH) in mobile phase on analytes retention times. Experiments for analytes retention times modeling and optimization of separation were performed according to central composite design. Multiple linear regression method was used for the construction of the best model based on experimental retention times. Pareto optimal method was used to find suitable compatibility between resolution and analysis time of analytes in honey. The optimum mobile phase composition for separation and determination of analytes in honey were [SDS]=0.124 mol/L; 7.8% v/v ethanol and 5.0% v/v AcOH. Limits of detection and linear range of flavonoid markers were 0.0079–0.0126, 0.05–50.0 mg/L, respectively.