Analysis of vitamin K1 and major K2 variants in rat/human serum and lipoprotein fractions by a rapid, simple, and sensitive UHPLC-MS/MS method.

Determination of the various forms of vitamin K, which are involved in coagulation and other physiological processes in humans, is challenging and no standardized method is yet available. Therefore, a reliable and practical method was developed to quantify vitamin K levels in serum and additionally in lipoprotein fractions to clarify its distribution. The LC-MS/MS method for the determination of vitamin K1 and the three main isoforms of vitamin K2 (MK-4, MK-7, MK-9) was combined with a gradient ultracentrifugation technique to allow the separation of lipoprotein fractions. The chromatographic separation was carried out on a Kinetex™ C18 column using a mobile phase consisting mainly of methanol. The target analytes were detected by electrospray ionization mass spectrometry. The separation of all four substances was achieved after a simple sample preparation technique based on miniaturized liquid-liquid extraction. Our method of only 8.5 min revealed the levels of the major forms of vitamin K in 59 human and 12 rat sera and confirmed our hypothesis that vitamin K is primarily (about 50 %) found in the high-density lipoprotein fraction. The median concentrations of vitamin K1, MK-4, MK-7, and MK-9 were found to be 1.19, 2.98, 0.43, and < 0.71 nmol/L in human serum and 1.74, 6.75, less than 0.2, and less than 0.5 nmol/L in rat serum, respectively.

Comparison of the liquisolid technique and co-milling for loading of a poorly soluble drug in inorganic porous excipients.

Drug loading into mesoporous carriers may help to improve the dissolution of poorly aqueous-soluble drugs. However, both preparation method and carrier properties influence loading efficiency and drug release. Accordingly, this study aimed to compare two preparation methods: formulation into liquisolid systems (LSS) and co-milling for their efficiency in loading the poorly soluble model drug cyclosporine A (CyA) into mesoporous magnesium aluminometasilicate Neusilin® US2 (NEU) or functionalized calcium carbonate (FCC). Scanning electron microscopy was used to visualize the morphology of the samples and evaluate the changes that occurred during the drug loading process. The solid-state characteristics and physical stability of the formulations, prepared at different drug concentrations, were determined using X-ray powder diffraction. In vitro release of the drug was evaluated in biorelevant media simulating intestinal fluid. The obtained results revealed improved drug release profiles of the formulations when compared to the milled (amorphous) CyA alone. The dissolution of CyA from LSS was faster in comparison to the co-milled formulations. Higher drug release was achieved from NEU than FCC formulations presumably due to the higher pore volume and larger surface area of NEU.

Comparative study of drug release from electrospun nanofibers loaded with clotrimazole via two different approaches using a fully automated sequential injection system.

The development of new drug delivery platforms including the use of nanotechnology has been found of great interest in recent years. Two different loading approaches of the model antimycotic drug clotrimazole into the nanofibrous polycaprolactone and polydioxanone structures including electrospinning of a drug-polymer blend and impregnation of nanofibers with drug have been tested. The final amount of clotrimazole in the nanofibrous materials was determined by HPLC analysis and Raman spectroscopy. The electrospinning of blend approach allowed the adsorption of clotrimazole in a quantity of up to 30 % using mixtures with polymer/clotrimazole ratios from 2:1 to 8:1 (w/w). Ethanolic clotrimazole solutions with concentrations from 2.5 to 3.5 mg L-1 were used for adsorbing clotrimazole in blank nanofibers for 1-3 h with final clotrimazole content ranging from 3.0 to 5.7 %. Furthermore, a comparative liberation study including comparison with commercially available creams was carried out in low pressure flow system. The results obtained confirmed well controlled release of clotrimazole from both types of nanofibers. Compared to commercial pharmaceutical formulations containing 1 % clotrimazole where first-order release kinetics was observed, nanofibrous materials provided linear controlled release (zero-order kinetics) in the tested 3 h period.

Analysis of terbinafine in PLGA-based drug delivery systems by a fast and sensitive UHPLC-DAD method.

A novel ultra-high performance chromatography method with multichannel detection that allows fast, sensitive, and robust analysis of an antifungal drug terbinafine and its three main impurities ß-terbinafine, (Z)-terbinafine, and 4-methylterbinafine in just 5.0 min has been developed. Analysis of terbinafine is important in pharmaceutical analysis since it enables the detection of its impurities at very low concentrations. In this study, we focused on the development, optimization, and validation of the UHPLC method as well as its subsequent application in the evaluation of terbinafine and its three main impurities in the dissolution medium to reveal the incorporation of terbinafine in two poly(lactic-co-glycolic acid) (PLGA) carriers and testing of the drug release at pH 5.5. PLGA based drug delivery systems such as solid dispersions, thin films, microparticles, and nanoparticles are new favorable ways of terbinafine administration. PLGA features excellent tissue compatibility, biodegradation, and adjustable drug release profile. Our pre-formulation study indicates that poly(acrylic acid) branched PLGA polyester has more suitable properties than tripentaerythritol branched PLGA polyester. Therefore, the former is likely to enable design of a new drug delivery system for topically applied terbinafine that could facilitate its administration and increase patient compliance.

Terbinafine-loaded branched PLGA-based cationic nanoparticles with modifiable properties.

Although the systemic administration of terbinafine is quite well tolerated, topical treatment of the local infections is often preferred. New formulation strategies in topical antifungal therapy represent the polymeric nanoparticles (NPs). We successfully employed the originally synthesized PLGA derivatives of branched architectures of various molar masses, branching ratio, and high number of terminal hydroxyl or carboxyl groups for compounding of terbinafine loaded nanoparticles by nanoprecipitation method. Employing the polymers with tailored properties allowed us to formulate the NPs with desired particle size, loading capacity for drug, mucoadhesive properties, and drug release profile. The hydrophobicity and the polyester concentration revealed the main impact on the NPs size ranging from 100 to 600 nm. The stability of the nanosuspension is demonstrated by zeta potential >25 mV, and polydispersity index values <0.2. We used terbinafine in its less dissolved form of the base to increase the drug loading and delay the release. Cationic surfactant as stabilizer give the NPs high positive surface charge enhancing the adhesion to the mucosal surfaces. All formulations provided prolonged sustained release of terbinafine for several days. Antimicrobial potential has been proven by agar-well diffusion method.

Determination of urinary retinol and creatinine as an early sensitive marker of renal dysfunction.

Determination of urinary retinol, which is a new promising early biomarker of renal damage typically expressed in the clinical environment as retinol/creatinine ratio, is currently difficult to accomplish. We have developed and validated the new ultra-high-performance liquid chromatography method with UV and mass spectrometry detection for the separation and quantification of retinol and creatinine in human urine in a single run. The separation of these two substances with completely different physicochemical properties was achieved using a column packed with fluorinated stationary phase and acetonitrile and aqueous ammonium formate buffer as the mobile phases. The separation was completed within 4 min. Our new method involves very fast and simple sample preparation requiring small amount of sample matrix and solvents. Deuterium labeled internal standard was used for the more precise quantification. The method was tested with real-life samples using urine collected from patients suffering from breast, colorectal, head, and neck cancer.

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).

Extemporaneous sotalol hydrochloride oral solutions for use in paediatric cardiology: formulation and stability study.

OBJECTIVES: Sotalol hydrochloride (SOT) is an antiarrhythmic ß-blocker which is highly effective for the treatment of supraventricular tachycardia in children. However, a licensed paediatric dosage form with sotalol is not currently available in Europe. The aim of this work was to formulate paediatric oral solutions with SOT 5 mg/mL for extemporaneous preparation in a hospital pharmacy with the lowest possible amount of excipients and to determine their stability. METHODS: Three aqueous solutions were formulated. One preparation without any additives for neonates and two preparations for children from 1 month of age were compounded using citric acid to stabilise the pH value, potassium sorbate 0.1% w/v as a preservative, and simple syrup or sodium saccharin as a sweetener. The samples were stored at room temperature and in a refrigerator, respectively, and the content of SOT and potassium sorbate was determined simultaneously using a validated high performance liquid chromatography method at different time points over 180 days. RESULTS: At least 95% of the initial sotalol concentration remained throughout the 180-day study period in all three preparations at both temperatures. The content of potassium sorbate decreased by 17% with sodium saccharin stored at room temperature. CONCLUSIONS: The three proposed oral aqueous solutions of SOT for neonates and infants were stable for 180 days. Storage in a refrigerator is preferred, particularly with sodium saccharin. The additive-free solution of SOT can be autoclaved to ensure microbiological stability and used particularly for neonates and in emergency situations.

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.

Pediatric oral solutions with propranolol hydrochloride for extemporaneous compounding: the formulation and stability study.

UNLABELLED: The aim of this study is to formulate an extemporaneous pediatric oral solution of propranolol hydrochloride (PRO) 2 mg/ml for the therapy of infantile haemangioma or hypertension in a target age group of 1 month to school children and to evaluate its stability. A citric acid solution and/or a citrate-phosphate buffer solution, respectively, were used as the vehicles to achieve pH value of about 3, optimal for the stability of PRO. In order to mask the bitter taste of PRO, simple syrup was used as the sweetener. All solutions were stored in tightly closed brown glass bottles at 5 ± 3 °C and/or 25 ± 3 °C, respectively. The validated HPLC method was used to evaluate the concentration of PRO and a preservative, sodium benzoate, at time intervals of 0-180 days. All preparations were stable at both storage temperatures with pH values in the range of 2.8-3.2. According to pharmacopoeial requirements, the efficacy of sodium benzoate 0.05 % w/v was proved (Ph.Eur., 5.1.3). The preparation formulated with the citrate-phosphate buffer, in our experience, had better palatability than that formulated with the citric acid solution. KEYWORDS: propranolol hydrochloride pediatric preparation extemporaneous preparation solution stability testing HPLC.

High-resolution monolithic columns--a new tool for effective and quick separation.

This work describes a comparison of three types of commercial high-performance liquid chromatography silica monolithic columns with different inner diameters and generations of monolithic sorbent: a "classic" monolithic column, the first generation (Onyx™ monolithic C(18), 100 mm × 4.6 mm, Phenomenex); a "narrow" monolithic column for fast separation at lower flow rates (Chromolith® Performance RP-18e, 100 mm × 3 mm, Merck); and a recently introduced "high-resolution" monolithic column, the next generation (Chromolith® HighResolution RP-18e, 100 mm × 4.6 mm, Merck). Separation efficiency (number of theoretical plates, height equivalent to a theoretical plate and van Deemter curves), working pressure, the symmetry factor and resolution were critical aspects of the comparison in the case of the separation of ascorbic acid, paracetamol and caffeine. The separations were performed under isocratic conditions with a mobile phase consisting of 10:90 (v/v) acetonitrile-phosphoric acid (pH 2.80). Detailed comparison of the newest-generation monolithic column (Chromolith® HighResolution) with the previously introduced monolithic sorbents was performed and proved the advantages of the Chromolith® HighResolution column.

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.

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.

Determination of estradiol and its degradation products by liquid chromatography.

A novel HPLC method for simultaneous determination of estradiol and its seven degradation products in topical gel was developed. Zorbax SB-CN (150 mm x 4.6 mm, 5 microm) analytical column and mobile phase composed of acetonitrile, phosphoric acid 0.085%, and tetrahydrofurane (27:63:10, v/v/v) at flow-rate 1.0 ml min(-1) were used for the chromatographic separation using UV detection at 225 nm. The active substance estradiol was separated from all its known degradation products successfully. Two degradation products estrone and Delta(9(11))-estrone were not separated sufficiently, their peaks were evaluated as a sum of two components. The method was validated according to ICH guideline recommendations and thereafter it was successfully applied for stability tests of topical cream Estrogel HBF in the quality control laboratory. Limits of detection for degradation products ranged from 1.03 x 10(-5) to 1.14 x 10(-4) mg ml(-1), limits of quantitation for degradation products were in the range 3.43 x 10(-5) to 3.81 x 10(-4) mg ml(-1). The developed method is selective, precise, accurate and sensitive enough for determination of estradiol and its known degradation products.

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.

Comparison of performance of C18 monolithic rod columns and conventional C18 particle-packed columns in liquid chromatographic determination of Estrogel and Ketoprofen gel.

The performance of monolithic HPLC columns Chromolith (made by Merck, Germany) and conventional C18 columns Discovery (Supelco, Sigma-Aldrich, Prague, Czech Republic) was tested and the comparison for two topical preparations Ketoprofen gel and Estrogel gel was made. The composition of mobile phases - for Ketoprofen analysis a mixture of acetonitrile, water and phosphate buffer adjusted to pH 3.5 (40:58:2) and for Estrogel analysis a mixture of acetonitrile, methanol, water (23:24:53) - was usually not optimal for analyses at all types of columns. Thus an adjustment of components ratio was necessary for sufficient resolution of the compounds analysed. Various flow rates (1.0-5.0 ml/min) and mobile phases (usually increasing ratio of water content) were applied. Determination of active substances, preservatives and impurities and comparison of retention times and system suitability test parameters was accomplished. For Estrogel gel, following chromatographic conditions were found: using Chromolith Flash RP-18e monolith column, mobile phase was acetonitrile, methanol, water (13:24:63, v/v/v) and flow-rate 3.0 ml/min. Using monolith column ChromolithSpeedROD RP-18e, the mobile phase was acetonitrile, methanol, water (18:24:58, v/v/v) and flow-rate 4.0 ml/min. For the monolith column Chromolith Performance RP-18e, the mobile phase was acetonitrile, methanol, water (23:24:53, v/v/v), flow-rate 3.0ml/min. Analysis of Ketoprofen gel gave the best results using following analytical conditions: for monolith column Chromolith Flash RP-18e, mobile phase as a mixture of acetonitrile, water, phosphate buffer pH 3.5 (30:68:2, v/v/v) was used, at flow-rate 2.0 ml/min. For ChromolithSpeedROD RP-18e monolith column, acetonitrile, water, phosphate buffer pH 3.5 (35:63:2, v/v/v) was used as a mobile phase at flow-rate 3.0 ml/min. Chromolith Performance RP-18e gave the best results using mobile phase acetonitrile, water, phosphate buffer pH 3.5 (30:68:2, v/v/v) at the flow-rate 5.0 ml/min. It was proved that monolith columns, due to their porosity and low back-pressure, can save analysis time by about a factor of three with sufficient separation efficiency. Thus, for example 11 min long analysis can be performed in 4 min with comparable results.

HPLC determination of estradiol, its degradation product, and preservatives in new topical formulation Estrogel HBF.

This paper deals with the development of a novel method for simultaneous determination of estradiol, its degradation product estrone, and two preservatives, methylparaben and propylparaben, in the topical preparation Estradiol HBF. After optimization of the analytical conditions the method was validated and applied in studies of the stability of the topical preparation Estrogel HBF. Separation of all these compounds was performed on a Supelco Discovery C18 (250 mm x 3.0 mm, 5 microm) analytical column. A mixture of acetonitrile, methanol, and water (23:24:53 v/ v) was chosen as mobile phase. UV absorbance at 225 nm was used for detection and quantitation of analytes. The total analysis time was less than 12 min at a flow rate of 0.9 mL min(-1). All the compounds were isolated from the topical gel by simple extraction with an acetonitrile solution of hydrocortisone, as internal standard, and using sonication and centrifugation thereafter. The supernatant was injected directly on to the analytical column. The recovery of the procedure was from 96.9 to 100.4%. Validation of method according international guidelines was successfully performed.