Ludmila Matysová

Advantages of application of UPLC in pharmaceutical analysis

Ultra Performance Liquid Chromatography (UPLC) is a relatively new technique giving new possibilities in liquid chromatography, especially concerning decrease of time and solvent consumption. UPLC chromatographic system is designed in a special way to withstand high system back-pressures. Special analytical columns UPLC Acquity UPLC BEH C(18) packed with 1.7 microm particles are used in connection with this system. The quality control analyses of four pharmaceutical formulations were transferred from HPLC to UPLC system. The results are compared for Triamcinolon cream containing trimacinolone acetonide, methylparaben, propylparaben and triamcinolone as degradation product, for Hydrocortison cream (hydrocortisone acetate, methylparaben, propylparaben and hydrocortisone degradation product), for Indomethacin gel (indomethacin and its degradation products 4-chlorobenzoic acid and 5-methoxy-2-methylindoleacetic acid) and for Estrogel gel (estradiol, methylparaben, propylparaben and estrone as degradation product). The UPLC system allows shortening analysis time up to nine times comparing to the conventional system using 5 microm particle packed analytical columns. In comparison with 3 microm particle packed analytical columns analysis should be shortened about three times. The negative effect of particle decrease is back-pressure increase about nine times (versus 5 microm) or three times (versus 3 microm), respectively. The separation on UPLC is performed under very high pressures (up to 100MPa is possible in UPLC system), but it has no negative influence on analytical column or other components of chromatographic system. Separation efficiency remains maintained or is even improved. Differences and SST parameters, advantages and disadvantages of UPLC are discussed.

Development and validation of HPLC method for determination of clotrimazole and its two degradation products in spray formulation

A novel simple isocratic HPLC method with UV detection for the determination of three compounds in spray solution (active component clotrimazole and two degradation products imidazole and (2-chlorophenyl)diphenylmethanol) using ibuprofen as an internal standard was developed and validated. The complications with different acido-basic properties of the analysed compounds in HPLC separation - while clotrimazole has pK(a) 4.7, imidazole has pK(a) 6.9 compared to relatively more acidic (2-chlorophenyl)diphenylmethanol - were finally overcome using a 3.5mum Zorbax((R)) SB-Phenyl column (75mmx4.6mm i.d., Agilent Technologies). The optimal mobile phase for separation of clotrimazole, degradation products imidazole and (2-chlorophenyl)diphenylmethanol and ibuprofen as internal standard consists of a mixture of acetonitrile and water (65:35, v/v) with pH* conditioned by phosphoric acid to 3.5. At a flow rate of 0.5mlmin(-1) and detection at 210nm, the total time of analysis was less than 6min. The method was applied for routine analysis (batch analysis and stability tests) in commercial spray solution.

Separation and determination of terbinafine and its four impurities of similar structure using simple RP-HPLC method.

A novel reversed-phase HPLC method for the simultaneous determination of active component terbinafine, its one impurity 1-methylaminomethylnaphtalene and three degradation products, beta-terbinafine, Z-terbinafine and 4-methyl-terbinafine occurring in pharmaceutical formulations after long-term stability tests, was developed and validated using propylparaben as an internal standard. The chromatographic separation was performed on a NUCLEOSIL 100-5-CN column, mobile phase for separation of all compounds consisted of a mixture of tetrahydrofurane, acetonitrile and citrate buffer pH 4.50 (10:20:70,v/v/v). The analysis time was less than 32 min at flow-rate of 0.8 ml min(-1). UV detection was performed at 226 nm. The method was validated and system suitability parameters were investigated. Method robustness and short-term standard solution stability were verified. Limits of detection for terbinafine degradation products/impurity were from 0.023 to 0.098 microg ml(-1), limits of quantitation were from 0.078 to 0.327 microg ml(-1). The method was applicable for routine determination of terbinafine and all its found impurities of similar structure with sufficient selectivity, precision and accuracy.

Application of monolithic columns in pharmaceutical analysis. Determination of indomethacin and its degradation products.

Comparison of HPLC methods using conventional particle-based and monolithic columns for determination of indomethacin and its two degradation products, viz. 5-methoxy-2-methylindoleacetic acid and 4-chlorobenzoic acid, was carried out. Ketoprofen was used as an internal standard for data evaluation throughout the study. Conventional separation was based on analytical column Zorbax SB-Phenyl (75x4.6 mm; 3.5 microm particles) used with a mobile phase composition of acetonitrile and phosphoric acid 0.2% (50:50, v/v) and isocratic flow at 0.6 mL/min. Three different lengths of Chromolith columns RP-18e (25x4.6 mm, 50x4.6 mm, and 100x3 mm) were tested with respect to the validation parameters peak asymmetry, resolution, height equivalent to a theoretical plate, repeatability, and after optimisation compared to values obtained using a conventional Zorbax column. The developed methods were used to determine all three compounds in a pharmaceutical formulation--Indobene gel. Chromatographic parameters were comparable to those of a conventional particle-based column. The analysis time was shortened as expected (retention times were lowered by a factor of two). Moreover, the repeatability of peak areas and retention times obtained with a 50 mm monolithic column was greatly improved (RSD values were lower than 0.40%).

Advantages of pentafluorophenylpropyl stationary phase over conventional C18 stationary phase--application to analysis of triamcinolone acetonide.

A pentafluorophenylpropyl (PFPP) stationary phase was for the first time tested for the simultaneous determination of triamcinolone acetonide, its degradation product triamcinolone and two preservatives, methylparaben, and propylparaben. A new simple isocratic reversed phase HPLC method with UV detection, using estradiol hemihydrate as an internal standard, has been developed and validated. Chromatography was performed on a Discovery HS F5 column (150 mm x 4.6 mm, 5 microm) using a binary mobile phase composed of acetonitrile and water 45:55 (v:v). The flow-rate was 0.6 mL/min, the column temperature 25 degrees C and the UV detection was accomplished at 240 nm. The chromatography results using PFPP stationary phase were compared with those obtained using conventional C18 columns.

Optimisation of an HPLC method for the simultaneous determination of pyrantel pamoate, praziquantel, fenbendazole, oxfendazole and butylhydroxyanisole using a phenyl stationary phase

A novel and simple isocratic reversed phase HPLC method with UV and fluorimetric detection for the simultaneous determination of the active anthelmintic components pyrantel pamoate, praziquantel, fenbendazole, its degradation product oxfendazole, and the antioxidant butylhydroxyanisole (BHA) has been developed and validated. The chromatography was performed using a Phenomenex Luna 3 μm phenyl–hexyl column (150 × 3.0 mm), and the mobile phase was composed of 0.5% triethylamine at pH 9.0 and acetonitrile 55 : 45 (v/v) at a flow rate of 1.0 ml min−1. The UV detection was performed at 290 nm for pyrantel, oxfendazole and fenbendazole and at 220 nm for praziquantel. For BHA, fluorescence detection was used with excitation and emission wavelengths of 287 nm and 322 nm, respectively. The method was validated and was applied for the determination of active compounds in various dosage forms of veterinary formulations.

Application of HILIC stationary phase to determination of dimethindene maleate in topical gel

A novel high performance liquid chromatography method for the determination of dimethindene maleate in pharmaceutical gel using hydrophilic interaction liquid chromatography (HILIC) with UV detection was developed and validated. Following optimal conditions for the analysis of dimethindene maleate were used: analytical column SeQuant ZIC-HILIC (50mmx2.1mm, 5microm), and mobile phase consisted of a mixture of acetonitrile and aqueous solution of acetic acid (25mM) and ammonium acetate (2.5mM) (87.5:12.5, v:v). The analysis time was less than 3min at a flow rate of 0.3mlmin(-1). UV detection was performed at 258nm. The method was validated and system suitability parameters were evaluated. The method is suitable for application for routine determination of dimethindene maleate in topical pharmaceutical preparation.

Development of a Gradient HPLC Method for the Simultaneous Determination of Sotalol and Sorbate in Oral Liquid Preparations Using Solid Core Stationary Phase

A selective and sensitive gradient HPLC-UV method for quantification of sotalol hydrochloride and potassium sorbate in five types of oral liquid preparations was developed and fully validated. The separation of an active substance sotalol hydrochloride, potassium sorbate (antimicrobial agent), and other substances (for taste and smell correction, etc.) was performed using an Ascentis Express C18 (100 × 4.6 mm, particles 2.7 μm) solid core HPLC column. Linear gradient elution mode with a flow rate of 1.3 mL min−1 was used, and the injection volume was 5 µL. The UV/Vis absorbance detector was set to a wavelength of 237 nm, and the column oven was conditioned at 25°C. A sodium dihydrogen phosphate dihydrate solution (pH 2.5; 17.7 mM) was used as the mobile phase buffer. The total analysis time was 4.5 min (+2.5 min for reequilibration). The method was successfully employed in a stability evaluation of the developed formulations, which are now already being used in the therapy of arrhythmias in pediatric patients; the method is also suitable for general quality control, that is, not only just for extemporaneous preparations containing the mentioned substances.

Chromatographic determination of active compounds in topical formulations

The aim of this study was to develop and validate new methods for the determination of active compounds in topical gels. The method for the determination of nonoxinol-9 and trimecaine hydrochloride in lubricant gel by HPLC with UV detection was described. Separation was carried out using a Discovery ZR-PBD analytical column (150 × 4.6 mm, 5 μm), and a mobile phase composed of a mixture of triethylammonium acetate buffer (pH 9; 50 mM) and methanol (22 : 78, v/v). Determination of terpinen-4-ol in lubricant gel was achieved by GC with a flame ionization detector (FID). An Alltech-AT-624 (0.32 mm ID × 30 m, 1.8 μm film thickness) fused-silicacapillary column and helium as the carrier gas, at a flow rate of 28 cm s−1, were employed. The analysis time was less than 7.5 min for HPLC and 5 min for GC analyses. Both methods were successfully applied to the analysis of pharmaceuticals.

Furosemide ethanol-free oral solutions for paediatric use: formulation, HPLC method and stability study

Background Oral liquid solutions of the diuretic active ingredient furosemide (FUR) marketed across Europe do not comply with recent requirements for paediatric preparation owing to their ethanol content and, moreover, in some countries only tablet or injection dosage forms of furosemide are available. Objectives To formulate extemporaneous paediatric ethanol-free solutions of FUR (2 mg/mL) with suitable solubility in the aqueous vehicle and an acceptable taste and to evaluate their stability under two different storage conditions during a 9-month study period. Methods Our work presents two developed formulations of FUR ethanol-free paediatric oral solutions 2 mg/mL for easy extemporaneous compounding in a pharmacy. FUR solubility avoiding the use of ethanol was achieved using sodium hydroxide (formulation F1) or disodium hydrogen phosphate dodecahydrate (formulation F2). The preparations were stored at 25°C±3°C or at 40°C±0.5°C and protected from light. For FUR and preservative, methylparaben (MP), a stability assay was conducted by a high-performance liquid chromatography validated method and determination of pH stability. Results The remaining FUR concentration was >90% of the initial concentration after 270 days in both formulations at both storage conditions, 25°C and 40°C. The concentration of MP decreased significantly in the formulation F2 stored at 40°C. Conclusions Both formulations were stable when stored at room temperature for up to 9 months; formulation F1 was stable even at 40°C. MP used as an antimicrobial agent fully satisfied the recommended criteria for preservative efficacy in oral preparations according to the European Pharmacopoeia 9.0 (5.1.3).