Current Pharmaceutical Analysis - Volume 13, Issue 6, 2017

Objective: Green pharmaceutical and bioanalytical methods for Voriconazole and Tadalafil were developed and validated using an environment friendly organic modifier without any extraction step. Replacing toxic solvent in bioanalytical HPLC is the first greening approach in chromatographic drug analysis in plasma. Method: Analytical C18 columns and mobile phases containing conventional solvents or propylene carbonate with/without ethanol and/or buffer were used in optimization study. Results: Optimized chromatographic conditions were Shim-Pack XR-ODS (100 × 2.0 mm, 2.2 µm) column and the mobile phase consisted of propylene carbonate/(70% phosphate buffer (50 mM, pH=3.0) + 30% ethanol) (10:90, v/v). Limit of detection and limit of quantification in tablet analysis were respectively 0.1 µg mL-1 and 0.5 µg mL-1 for both Voriconazole and Tadalafil. Lower limit of quantification in plasma analysis was found to be 0.005 µg mL-1 for both analyte. Conclusion: Green HPLC methods presented here were accurate, precise, specific, sensitive, rugged, robust, reliable in long-term use and successfully applied to pharmaceutical and plasma analysis.

Background: Ketoconazole is an antifungal drug, available in different pharmaceutical dosage forms. The current published methods for the ketoconazole determination confirmed the primacy of the HPLC method, but high toxic solvents were used, also. Introduction: A new simple, precise, accurate, and cost-effective RP-HPLC method for the determination of ketoconazole in tablets, topical cream, and shampoo has been developed and validated. Method: The chromatographic separation was conducted on a LiChrospher®100 C-18 column (150 mm length x 4.6 mm i.d., 5 µm particle size) using a mixture of methanol and water (90:10 v/v) adjusted to pH 8.90 with a phosphate buffer. The HPLC analysis was carried out at 25 °C column temperature with 1.0 mL/min isocratic flow rate of the mobile phase. The robustness of the method was evaluated changing the flow rate of the mobile phase (0.9 mL/min and 1.0 mL/min) and the column temperature (23 °C and 25 °C). Result: The mean recovery data for the ketoconazole pharmaceutical dosage forms were in the range of 92.67–105.23%. Conclusion: The developed and validated method was successfully used for the quantitative analysis of ketoconazole pharmaceutical dosage forms; tablets, topical cream, and shampoo.

Intrduction: The present study reports validated stability indicating high performance thin layer chromatographic (HPTLC) method of analysis for estimation of an immunosuppressant drug; mycophenolate sodium in bulk drug and formulations. Method: Silica gel pre-coated aluminium plates 60F254 were used as stationary phase. Various mobile phases were tried, out of which chloroform: methanol and glacial acetic acid (9:0.3:0.1; v/v/v) provided best separation at 254 nm (Rf 0.66±0.03) by densitometric evaluation. Results: The linearity range was 100-2500 ng with R2 value 0.9977 while, % RSD of intra-day and inter- day precision was found to be less than 1.5% indicating that the method is precise. Limit of detection (LOD) and limit of quantification (LOQ) of mycophenolate sodium were found to be 30ng/spot and 90 ng/spot respectively. The recovery of mycophenolate sodium was found to be 100.2±0.011%. Inherent stability of mycophenolate sodium was studied by exposing it to various stress conditions as per ICH guidelines viz. acid, base, dry heat, oxidation, photolysis (sunlight, UV) and neutral hydrolysis. The peaks of the degradation products and pure drug exhibited different Rf values indicating significant resolution. Degradation studies revealed 11-20% degradation of mycophenolate sodium in 1-3 h by acidic (1 N HCl), alkaline (1N NaOH), dry heat, neutral hydrolysis and oxidative degradation by 3% H2O2. However it remained stable to photo-degradation. Conclusion: Proposed method was found to be more sensitive, precise, economic and less time consuming than the reported stability indicating HPLC method. Moreover, it is simple, accurate, and reproducible. It could be applied for analysis of bulk drug/formulation, dissolution study and stress degradation kinetics.

Introduction: Rifaximin is an oral antimicrobial, intestine-selective and non systemic with adverse effects compared to placebo. Materials and Methods: Rifaximin, until then, does not have standardized analytical methods in most official compendia. Fast, economic, environmentally friendly and selective method indicative of stability by thin chromatographic (TC) for the determination of rifaxmin and its degradation products was designed. The TC method used silica gel as stationary phase and ethyl acetate: ethyl alcohol, 90:10 (v/v), as mobile phase to achieve spots for rifaximin (Rf=0.62) and its degradation products basic (Rf=0.33, 0.49, 0.56), acidic (Rf=0.47, 0.54), oxidative (Rf=0.28) and neutral (Rf=0.32). The plates were visualized in chamber UV at 254 nm. Simultaneously, analysis of high performance liquid chromatography (HPLC) was performed using Eclipse Plus C18 (150 mm x 4.6 mm) column and purified water + 0.1 % glacial acetic acid and ethanol in the ratio 52:48 (v/v) as mobile phase at 290 nm. The objective was to verify the interchangeability of TLC and HPLC methods in obtaining the degradation products of rifaximin tablets. In HPLC, as in TC was observed the rifaximin (RT=5.5) and its degradation products acidic, basic (RT =4.2 and 4.7), oxidative (RT =3.6 min) and neutral (RT =4.7 min). Conclusion: These techniques can be used as methods indicative of stability because they identify the rifaximin and its degradation products. Therefore, they can be effectively applied in quality control of rifaximin in tablets. The study of alternative methods should be encouraged by reducing costs, allowing the use of environmentally friendly solvents, optimize analysts and equipment while providing quality analyses.

Introduction: An accurate and precise spectrophotometric method has been developed for the determination of the antiallergenic drug, promethazine with ruthenium (III). Methods: The proposed method involves complexation of promethazine with Ru (III) (PM.Ru(III)) at 60ºC and λmax = 510 nm, which is applicable in pharmaceutical tablets and analytical data. The values for Beer's law, molar absorptivity, Sandell's sensitivity, and mean correlation coefficient were determined to be 0.2-28 µg mL-1, 1.54 x 103 L mol-1cm-1, 0.021 and 0.9996, respectively. Conclusion: The complex of promethazine with Ru(III) was characterised using FTIR, 1H-NMR, ESR, magnetic susceptibility, thermal (TG-DTA), and PXRD. The most plausible structure for the complex is reported with molecular formula [C37H52ClN4O2RuS2]·3Cl. The proposed method could be implemented for an easy and effective analysis for promethazine.

Introduction: Rifaximin is an antibiotic with a wide spectrum of antibacterial activity. Due to its non-absorbable nature and mechanism of action, it is commonly used for treating travelers’ diarrhea. It still does not have standardized methods in most official compendiums. This paper aimed to validate an environmentally friendly spectrophotometric method in the visible region for quantification of rifaximin in tablets. Method: Validation was performed in terms of International Conference on Harmonization guidelines, examining linearity, precision, selectivity, specificity, accuracy and robustness. The method considered rifaximin solutions in purified water and ethyl alcohol (5:1, v/v) at a wavelength of 477 nm. Conclusion: The method showed great linearity in the concentration range between 15 and 50 µg L-1 with correlation coefficient higher than 0.9998. Selectivity was determined by submitting the rifaximin in tablets to the forced degradation in ultraviolet light and acidic, basic, oxidative, neutral solution. The interday and intraday precision presented deviations of less than 1 %. The accuracy was 99.12 %. The method was robust to the variation in wavelength and source of purified water. The validated method proved to be fast, cost effective and suitable for routine quality control of rifaximin in tablets, avoiding the use of polluting reagents.

Objective: An analytical assay with focus on UV spectrophotometric method was proposed as alternative for the analysis of the antiplatelet ticagrelor. Method: Method performance was continuously evaluated, establishing limits for reported parameters that denote reproducibility during routine analysis. By selecting solvent and extractive system, the validation parameters were well executed based on experimental protocols that were exhaustively tested. Results: Preliminary tests indicated an adequate UV profile using methanol and applying ultrasonic bath as mechanical support to extract ticagrelor from tablets matrix. The extractive solution was compared to placebo solution following the same sample preparation, in order to evaluate eventual interference from matrix excipients and affirming the method's specificity. Linearity assay at 255 nm showed a good capacity on drug estimation in the range of 7.5 - 30.0 µg/mL (r = 0.9996). In parallel, quantitation was applied with focus on precision and accuracy, whose results indicated good response on data reproducibility (RSD intraday=1.60-1.84%; RSD interday=0.39) and potential to determine the drug in spiked matrix (% of standard recovery=101.90; RSD< 2.0%). The known robustness profile for spectrophotometric assay was confirmed, demonstrating reliability on drug quantitation after small variations in solvent brand, equipment and λ (nm) (% drug content=100.78-104.44%). Conclusion: By statistical comparison to HPLC, the validated spectrophotometric assay can be used alternatively for ticagrelor determination on routine analysis.

Introduction: The modern analytical demand requires fast, inexpensive and easy convey methods, therefore several sensor and biosensor methods have been developed. Methods: In this context two different enzyme electrodes for the determination of ethanol were assembled by the authors, immobilizing alcohol oxidase or catalase in a k-Carrageenan gel layer overlapping an amperometric gaseous diffusion Clark type oxygen electrode. The two enzyme sensors used different formats, as the alcohol oxidase biosensor was a conventional direct enzyme sensor, while the catalase biosensor was based on the antagonism of a different substrate for the same enzyme. The response of the two biosensors toward standard solution of ethanol was recorded, compared and discussed. Conclusion: Finally, by using the fabricated biosensors, ethanol concentrations of several pharmaceutical formulations were determined and the results, obtained with two enzyme electrodes, were compared. The precision of the two methods on pharmaceutical samples was comparable and recovery tests on real matrices were satisfactory for both the biosensors, however in the applications to pharmaceutical formulations the alcohol oxidase biosensor seems to be more reliable.

Backround: A new spectrophotometric method based on the oxidation reaction between iodide and iodate in the presence of the carboxylic acid form of losartan potassium (LP) was developed for the estimation of LP in pharmaceutical products. Method and Results: HPLC/MS, kinetic studies and central composite design were used for the formation reactions, equilibration, and determination of stability duration and experimental conditions, respectively. The developed spectrophotometric method was validated. As such, the equilibration period was calculated to be 10 min and 9.7 mmol/L KI and 0.59 mmol/L KIO3 were utilized. The optimal temperature was 32.3 °C. The method was determined to be linear between 4-30 µg/mL with a correlation coefficient of 0.9996. The limit of detection and limit of quantification were calculated to be 0.55 and 1.85 µg/mL, respectively. Pharmaceutical preparations were analyzed by the developed kinetic spectrophotometric method. The obtained results ranged between 94.9 % and 100.9 %. Conclusion: According to our results the developed method can be used as an alternative for the simple and routine determination of LP in industrial quality control laboratories. Moreover, this proposed method use experimental design which the optimized reaction conditions to be determined with a minimal number of experiments.

Introduction: A suitable liquid chromatography tandem mass spectrometry (LC-MS/MS) method to determine mazindol in human plasma is needed for bioequivalence and pharmacokinetic studies of celecoxib preparations. The present study describes a simple, rapid, reproducible, and reliable LC-MS/MS method to determine mazindol concentrations in human plasma. Method: After one-step liquid-liquid extraction (LLE) using ethyl acetate : hexane = 8 : 2, mazindol and fluoxetine (internal standard, IS) were eluted on a Kinetex HILLIC column (50.0 × 2.1mm i.d. 2.6 µm) with an isocratic mobile phase, which consisted of 20 mM ammonium formate in water: acetonitrile (20:80, v/v) at a flow rate of 0.2 mL/min. The calibration curve was linear (correlation coefficient were > 0.99) over the concentration range (0.05-10 ng/mL). Results: The intraday and interday precisions ranged 0.74 - 8.78% and 4.49 - 7.49%, respectively, and its accuracies ranged 92.00 - 108.48% and 97.73 - 105.20%, respectively. The stability of mazindol was evaluated by the addition of buffer to the plasma. Conclusion: The devised method was successfully applied to bioequivalence study of two formulations of mazindol, Sanorex tablet® and Mazanor tablet® in 50 healthy Korean male volunteers following single oral administration.

Background: The control of structural variations of therapeutic proteins is a big challenge requiring sophistic and accurate analytical tools for the analysis of the conformational stability of the final formulations. Methods: This paper reports the biochemical, structural and biological tests used to evaluate the identity and purity of a recombinant human erythropoietin alfa, used in the formulation of the first Tunisian approved biosimilar (Epomax 2000 I.U/mL) and the comparative assessment between this final product and an equivalent commercialized biosimilar in Tunisia with respect to the protein content, physiochemical integrity, immunogenicity and biological activity. Results: Our results demonstrated that Epomax showed high and similar consistence in total protein content (2.605 mg/mL for Bradford assay), isotonicity (271 mOsm/kg for Osmolarity measurement), purity (a single band at 37.40 kDa for western blot analysis), and potency (1724.00 I.U./mL for in vivo bioassay) when compared to the commercialized biosimilar product (2.969 mg/mL for Bradford assay, 291 mOsm/kg for Osmolarity measurement, a single band at 36.29 kDa and 1690.79 I.U./mL for in vivo bioassay). Conclusion: The analyzed erythropoietin raw material is highly pure and in compliance with the specifications of the European Pharmacopeia. As well as, the final product Epomax has shown highly comparability with the commercialized reference product.

Background: The availability of a low-cost bioanalytical method, easy to transfer and to set up, represents an advantage in therapeutic drug monitoring and industrial research. Objective: A simple, sensitive and rapid high performance liquid chromatographic (HPLC) method is developed and validated for quantitative determination of milnacipran hydrochloride in rabbit plasma. Method: Simple and effective solid phase extraction technique was performed for sample treatment and it has resulted in consistent and high recoveries (93–95%) at all concentrations studied. Efficient chromatographic separation has been performed on a LiChrospher® C18 column using a mobile phase consisting of a mixture of phosphate buffer, acetonitrile and methanol (65:25:10; v/v/v) at a flow rate of 0.8 mL/min. Results: The method has demonstrated linearity from 25 ng to 2000 ng/mL with a regression coefficient of 0.9998. The accuracy was found to be very high (99.31 to 101.44 %). %RSD values for inter-day and intra-day variation were not more than 3.58. The method has demonstrated high sensitivity with a lower limit of quantification of 25 ng/mL and excellent stability of milnacipran in rabbit plasma. The method was applied for pharmacokinetic investigation of immediate release and controlled release milnacipran tablets. The in-vivo study results indicated that developed HPLC method was sensitive to accurately quantify the concentration of milnacipran in plasma, which is an essential requirement for determination of pharmacokinetic parameters for drug formulation studies as well as future bioavailability or bioequivalence studies. Conclusion: A simple and sensitive bioanalytical HPLC method was developed, validated and applied for pharmacokinetic studies.