A micellar liquid chromatographic method for quality control of pharmaceutical preparations containing tricyclic antidepressants

Summary Micellar liquid chromatography methods for quality control of pharmaceutical preparations (capsules, pills, tablets, injections) containing the tricyclic antidepressants amineptine, amitriptiline, clomipramine, doxepin, imipramine, melitracen and nortriptyline alone or together with other CNS drugs like diazepam, medazepam and perphenazine are described. The methods using micellar solutions of cetyltrimethylammonium bromide as mobile phases and UV detection are rapid and reproducible. Due to the versatility of interactions in micellar liquid chromatography, it is possible determine highly hydrophobic compounds such as TCAs in a short time using mobile phases containing low organic solvent concentrations and usual flow rates, in contrast with the RP-HPLC methods proposed for these compounds. Samples preparation only requires solution and adequate dilution with the mobile phase before injection into the chromatographic system.     

Determination of catecholamines as aminochromes by micellar liquid chromatography with thermal lens spectrophotometric detection

Summary The determination of catecholamines (CAs) using micellar liquid chromatography with thermal lens spectrophotometric detection has been studied. CAs are oxidized with hexacyanoferrate(III) to aminochromes which are separated with a mobile phase of 0.05 M sodium dodecyl sulphate, 7% propanol and 0.03 M citrate buffer, pH 4.8, on a partially endcapped C₁₈ column. The aminochrome-micelles and aminochrome-stationary phase association constants are evaluated. Using the 488 nm line of an Ar⁺ laser with 250 mW pump power the limits of detection are about 4 ng mL^–1. The technique is applied to the determination of unconjugated CAs in urine using isoproterenol as internal standard.     

Micellar electrokinetic capillary chromatography as an alternative method for determination of hydrocortisone and its most important associated compounds in local pharmaceutical preparations

Summary A new, simple, and accurate micellar electrokinetic chromatographic (MEKC) method is described for quantification of hydrocortisone, hydrocortisone hemisuccinate, hydrocortisone acetate, mystatin, oxytetracycline, Zn-bacitracin, polymyxin B, and lidocaine in ocular and cutaneous pharmaceutical products. The separation was performed at 25°C and 25 kV, with 15mm phosphate +15mm borate buffer, pH 8.2, and 60mm sodium dodecylsulfate (SDS) in 10∶1 (%,v/v) methanol-water as background electrolyte. Under these conditions the analysis time is approximately 23 min. The method has been used for quantification of these compounds in different commercial pharmaceutical products and gave good results when compared with reference spectrophotometric and HPLC methods.     

Determination of piribedil in pharmaceutical formulations by micellar electrokinetic capillary chromatography

A fast and simple micellar electrokinetic capillary chromatographic method was developed for the analysis of piribedil in pharmaceutical formulations. The effects of buffer concentration, buffer pH, sodium dodecyl sulphate (SDS) concentration, organic modifier, applied voltage and injection time were investigated. Optimum results were obtained with a 50 mM borate buffer at pH 8.0 containing 50 mM SDS by using a fused silica capillary (50 m internal diameter, 72 cm effective length). The sample was injected hydrodynamically for 4 s at 50 mbar pressure and the applied voltage was +30 kV. The detection wavelength was set at 205 nm. Diflunisal was used as an internal standard. The analysis was performed at 25 °C and the total run time was 14 min. The method was suitably validated with respect to linearity range, limit of detection and quantification, precision, accuracy, specificity and robustness. The linear calibration range was 5–100 g mL^–1 and the limit of detection was determined as 1 g mL^–1. The method developed was successfully applied to the determination of piribedil in pharmaceutical formulations. The results were compared with a spectrophotometric method reported in the literature and no significant difference was found statistically.     

Determination of vitamin A in pharmaceutical preparations by high-performance liquid chromatography with diode-array detection

Summary A very simple high-performance liquid chromatographic method for determination of Vitamin A in pharmaceutical preparations without the need for saponification was developed. A reversed-phase (Nova-Pack C₁₈, 4 m) column was used with a mobile phase of acetonitrile-tetrahydrofuran-water (55378) and a flowrate of 1.5 ml/min. Sample treatment only consisted of the extraction of retinol acetate content from capsules or tablets with methanol. Total extraction was achieved by shaking vigorously with the aid of magnetic stirring for three hours at room temperature. No change of solvent is necessary to introduce the sample in the chromatographic system. This method is suitable for routine quantification of Vitamin A.     

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 > 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.     

Determination of water-soluble vitamins in pharmaceutical preparations by reversed-phase high-performance liquid chromatography with a mobile phase containing sodium dodecylsulphate andn-propanol

Summary A reversed-phase liquid chromatographic method with sodium dodecylsulphate-n-propanolwater as mobile phase has been used to separate and determine six water-soluble vitamins in twelve minutes. The analytical characteristics linear range, sensitivity, detection limits, and precision were evaluated. The lowest detection limits were those of nicotinic acid (not usually present in pharmaceutical products), 0.7 mgL⁻¹, nicotinamide, 1.3 mg L⁻¹, and pyridoxine, 1.4 mg L⁻¹. When the method was applied to the determination of the vitamins in pharmaceutical samples the values found agreed with those on the labels.     

Determination of rofecoxib,in the presence of its photodegradation product,in pharmaceutical preparations by micellar electrokinetic capillary chromatography

A simple and rapid micellar electrokinetic capillary chromatographic (MEKC) method for analysis of rofecoxib (ROF) and its photodegradation product (PDP) in pharmaceutical preparations has been developed and validated. Analyses were conducted in a fused silica capillary (72 cm effective length, 50 m i.d.) with a background electrolyte consisting of 25 mmol L^–1 borate buffer at pH 7.0 containing 15 mmol L^–1 sodium dodecyl sulfate (SDS) and 10% acetonitrile (ACN). The separation was performed by voltage-controlled system, applying 30 kV at 30 °C, detecting at 225 nm; injection was hydrodynamic at 50 mbar for 2 s. Nifedipine was used as internal standard (IS). Under the optimum conditions ROF, PDP, and IS were well separated with in 10 min. The method was validated with regard to linearity, limit of detection and quantitation, precision, accuracy, specificity, and robustness. The detection limit of the method was low, 0.8 g mL^–1, and the linearity range was wide, 2.5 to 125 g mL^–1. The method was highly efficient—5×10⁵ plates m^–1 for ROF. The method was applied to the tablet form of ROF-containing pharmaceutical preparations. The data were compared with those from the voltammetric method described in literature. No statistically significant difference was found.     

Determination of preservatives in cosmetics and food samples by micellar liquid chromatography

An analytical procedure has been developed for the analysis of benzoic acid, p-hydroxybenzoic acid, methyl-, ethyl-, propyl-, isopropyl-, and butyl esters of p-hydroxybenzoic acid by micellar liquid chromatography. After dilution in n-propanol the sample was directly injected onto a Lichrosorb ODS, 5 microm (250 x 4.6 mm ID) column and eluted with aqueous 2% Brij-35 adjusted to pH 3.0 with phosphoric acid:propanol (80:20 v/v) at a flow rate of 1 mL min(-1) and UV detection at 254 nm. A linear calibration curve was obtained simultaneously for each component in the range of 50-500 microg mL(-1) for benzoic acid and 5-150 microg mL(-1) for the other components; detection limits were within 25-250 ng mL(-1) corresponding to 125-1250 pg per injection (5 microL). The reproducibility in terms of average peak area and average retention time was obtained with coefficients of variation (CV) of 1.2% and 0.5%. The method was applied to analysis of these compounds in cosmetics (shampoos, hand lotions, creams, and bath foam) and food samples.     

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.     

Ionic liquids as surfactants in micellar liquid chromatography

This paper is devoted to application of ionic liquids as surfactants in LC of organic compounds, derivatives of 1,4‐thiosemicarbazides. According to HPLC requirements the most advantageous conditions such as transparency for ultraviolet light, low CMC, additional inorganic salt additives, and appropriate organic solvent were established. The CMC was determined using conductivity measurements. Suitability of two different stationary phases: RP‐C18 and cyanopropyl bonded phase was examined under micellar conditions. Chosen ionic liquid surfactant was compared to common traditional amphiphilic reagent – SDS. Elaborated on chromatographic micellar conditions were tested as a pilot technique for prediction of distribution coefficients of organic analytes in ionic liquid‐based aqueous two‐phase system.     

Causes and remediation of reduced efficiency in micellar liquid chromatography

Broad peaks are obtained when purely aqueous micellar phases are used in micellar liquid chromatography (MLC). The causes of reduced efficiency in MLC are investigated. Slow solute mass-transfer kinetics between micelles, the aqueous phase and the surfactant covered stationary phase are the origins of the efficiency loss. Knox plots show that the reduced efficiency comes from A term increase and, for lipophilic solutes, A and C terms increases. Surfactant adsorption reduces the pore volume and surface area of the stationary phase changing the flow anisotropy (A term). The surfactant adsorbed layer slows down the mass transfer (C term). Three ways for efficiency loss remediation are known: flow-rate reduction, temperature increase and alcohol addition. Alcohols are known to change the micelle structure and to increase the kinetics of micelle formation-destruction. It is shown that the ratio of the alcohol chain length to surfactant alkyl chain length, C_{n, OH}/C_{nm surf}, should be equal or higher than 1/3 to produce the best efficiency enhancements in MLC. Also, the volume of alcohol to be added is not absolute but relative to the surfactant concentration. The alcohol to surfactant concentration ratio should be kept constant. Temperature increases and especially alcohol additions reduce the retention factors. Thermodynamic and kinetics of the micellar exchanges in MLC cannot be dissociated.     

Simultaneous isocratic separation of phenolic acids and flavonoids using micellar liquid chromatography

The simultaneous isocratic separation of a mixture of five phenolic acids and four flavonoids (two important groups of natural polyphenolic compounds with very different polarities) was investigated in three different RPLC modes using a hydro‐organic mobile phase, and mobile phases containing SDS at concentrations below and above the critical micellar concentration (submicellar LC and micellar LC (MLC), respectively). In the hydro‐organic mode, methanol and acetonitrile; in the submicellar mode methanol; and in the micellar mode, methanol and 1‐propanol were examined individually as organic modifiers. Regarding the other modes, MLC provided more appropriate resolutions and analysis time and was preferred for the separation of the selected compounds. Optimization of separation in MLC was performed using an interpretative approach for each alcohol. In this way, the retention of phenolic acids and flavonoids were modeled using the retention factors obtained from five different mobile phases, then the Pareto optimality method was applied to find the best compatibility between analysis time and quality of separation. The results of this study showed some promising advantages of MLC for the simultaneous separation of phenolic acids and flavonoids, including low consumption of organic solvent, good resolution, short analysis time, and no requirement of gradient elution.     

Determination of sulfonamides in milk after precolumn derivatisation by micellar liquid chromatography

A simple method to identify and determine six sulfonamides (sodium sulfacetamide, sulfamethizole, sulfaguanidine, sulfamerazine, sulfathiazole and sulfamethoxazole) in milk by micellar liquid chromatography (MLC) is reported. The assay makes use of a precolumn diazotisation-coupling derivatisation including the formation of an azo dye that can be detected at 490 nm. Furthermore, the use of MLC as an analytical tool allows the direct injection of non-purified samples. The separation was performed with an 80 mM SDS-8.5% propanol eluent at pH 7. Analysis times are below 16 min with a complete resolution. Linearities (r > 0.9999), as well as intra- and inter-day precision (below 2.7%), were studied in the validation of the method. The limits of detection and quantification ranged from approximately 0.72 to 0.94 and 2.4 to 3.1 ng mL⁻¹, respectively. The detection limit was below the maximum residue limit established by the European Community. Finally, recoveries in spiked milk samples were in the 83-103% range.     

Evaluation of distribution coefficients in micellar liquid chromatography

The possibilities of micellar liquid chromatography for evaluating distribution coefficients are discussed. Determination of solute-micelle association constants and distribution coefficients of solutes between stationary-aqueous, stationary-micellar and aqueous-micellar phases is described. Application of the calculation of distribution coefficients to the study of the retention mechanism of solutes in the chromatographic system and prediction of separation selectivity is also presented.     

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.     

Determination of procaine and tetracaine in plasma samples by micellar liquid chromatography and direct injection of sample

Summary A liquid chromatographic procedure is proposed for the determination of procaine and tetracaine in plasma samples with direct injection. The method uses a Spherisorb octadecylsilane ODS-2 C₁₈ analytical column and a micellar mobile phase containing 0.15 M sodium dodecyl sulphate, 0.5% triethylamine at pH 2.5 and 10% propanol. The UV detection was carried out at 300 nm. Plasma sample preparation required only adequate dilution with the mobile phase before injection into the chromatographic system. The proposed method allows the determination of procaine and tetracaine in plasma at therapeutic levels.     

Modelling of retention behaviour of solutes in micellar liquid chromatography

In micellar liquid chromatography (MLC), the resolution for a given multi-component mixture can be optimized by changing several variables, such as the concentrations of surfactant and organic modifier, the pH and temperature. However, this advantage can only be fully exploited with the development of mathematical models that describe the retention and the separation mechanisms. Several reports have appeared recently on the possibilities of accurately predicting the solute retention in MLC. Although the retention and selectivity may strongly change with varying concentrations of surfactant, organic modifier and/or pH, the observed changes are very regular, and are well described by simple models. This characteristic enables a successful prediction of retention times and compensates the negative effect of the broad and tailed chromatographic peaks obtained for some solutes when micellar eluents are used. An overview of the models proposed in the literature to describe the retention behaviour in pure micellar eluents and micellar eluents containing an organic modifier, at a fixed pH or at varying pH, is given. The equations derived permit the evaluation of the strength of micelle-solute and stationary phase-solute interactions. The prediction of the retention based on molecular properties and the use of neural networks, together with the factors affecting the prediction capability of the models (linearization of the equations, dead time, critical micellar concentration, ionic strength and temperature) are commented on. The strategies used for the optimization of resolution are also given.     

Determination of synthetic antioxidants in dairy products and dietetic supplements by micellar liquid chromatography with direct sample injection

Summary A simple and rapid HPLC method for the determination of synthetic antioxidants (propyl gallate, tert-butylhydroquinone, 2,4,5-trihydroxybutyrophenone, nordihydroguaiaretic acid, octyl gallate, 3-tert-butyl-4-hydroxyanisole and dodecyl gallate) in powdered and liquid milk, cream of milk and dietetic supplements is described. The samples are diluted or solved in a micellar solution, filtered and directly injected. The retention behavior of the antioxidants on a C18 column, with micellar mobile phases containing SDS (0.05–0.15 M), n-propanol (1–9%, v/v) and 10 mM phosphate at pH 3, has been studied by using mathematical models. Retention is predicted with errors below 3%. To optimize the mobile phase composition, a procedure which takes into account the position and shape of the peaks is applied. The optimized mobile phase, which contains 0.090 M SDS and 6.6% n-propanol, allows the separation of six antioxidants in less than 13 min. Calibration curves are linear (r>0.9998) and the limits of detection range from 0.05 to 0.3 ng antioxidant, which correspond to concentrations well below the levels allowed in foods. Repeatabilities for standards containing 5 μg mL⁻¹ ranged from 0.2 to 1.6%.