Current Pharmaceutical Analysis - Volume 14, Issue 1, 2018

Objective: To establish a particular, sensitive and accurate High Performance Liquid Chromatography (HPLC) technique for the specific and effective analysis of 5-Fluorouracil (5-FU) in mobile phase and in plasma. Method: Analytical methods for 5-FU in mobile phase and plasma were developed. An assay consisted of isocratic elution of 5-FU in prepacked column discovery R-HS C-18, 5micrometer, Catalog # 568523-U, column dimensions : 25cm x 4.6mm using mobile phase composed of 5 mmol/L potassium dihydrogen phosphate (pH 6.0) and methanol at a ratio of 96:4 v/v respectively. 5-FU was detected at 254 nm with the flow rate of 1 ml/min in both mobile phase and plasma. Results: Retention time was found to be 3.457 minutes in mobile phase and 2.543 minutes in plasma. System suitability test showed no robustness among the inter-day, intra-day analysis, mobile phase and column. Accuracy of the developed method proved reliability of the purposed procedure. %RSD analysis was found to be less than 2% and coefficient of correlation was found to be 1. Conclusion: Valuable precision, recovery and accuracy were observed indicating method reliability. Method can also be applicable for bioequivalence study.

Background: Polymorphism in drugs is a common phenomenon which can lead to compromised quality due to changes in their physicochemical properties and may impact therapeutic efficacy. Objective: The main objective of the present study is to investigate the polymorphic behaviour of Pioglitazone Hydrochloride (PGH) under different preparative conditions and to evaluate the solid forms. Method: The solid-state forms of PGH were developed under different preparative conditions like solvent addition, neat grinding, solvent assisted grinding, cooling, slurrying etc. Which were characterized by Fourier Transform Infrared Spectroscopy (FTIR), X-Ray Powder Diffraction (XRPD), Scanning Electron Microscopy (SEM) and Differential Scanning Calorimetry (DSC). Results: Solid forms D and E, crystallized from cooling and solvent evaporation methods exists in Form I of PGH, while solid form A obtained from antisolvent method underwent solid-solid transition from Form I to Form II. Form G obtained by slurrying in dimethylformamide contains Form II with trace amount of Form I. Grinding did not show any effect on polymorphic nature of PGH. The dissolution behaviour of these solid Forms reveals that solid Form A containing Form II showed improved dissolution (92.6 %). No significant difference in anti-diabetic activity was observed between Form I and Form II of PGH. Conclusion: PGH readily undergoes polymorphic transitions under certain preparative and processing conditions. However, no significant difference in anti-diabetic activity was observed between Form I and Form II of solid form of PGH.

Background: Sulfonamide antibiotics are the second most frequent cause of allergic drug reactions (after the β-lactams). Activity and side effects of sulfonamide may be attributed to their metabolites. To the best of our knowledge there is no in vitro metabolism study for sulfamerazine (SMR) available in the literature. The main objectives of this research are to develop an analytic method for screening, identifying and quantifying SMR and its active metabolites and for in vitro metabolism study. Methods: An ultra performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometric (UPLC-QTOF-MS) method was used for establishing the accurate mass databases of SMR and N-ASMR as well as non-target analytes and for in vitro screening, identification, and quantification of SMR and its metabolites N4-acetylsulfamerazine (N-ASMR) in mice liver homogenates. Result: The limit of detection (LOD) and limit of quantification (LOQ) were 1.0 and 5.0 μg L-1 for SMR and 12.0 and 20 μgL-1 for N4-acetylsulfamerazine (N-ASMR), respectively. Peak area intra-day RSD (n=6) and inter-day RSD (n=9) were 5.0 and 8.2 % for SMR, and 4.3 and 7.2% for N-ASMR, respectively. The identification of targeted analytes (SMR and N-ASMR) was conducted based on the established accurate mass database (both retention time and accurate mass). SMR could be metabolized via acetylating by acetoacetyl coenzyme A, and two isomers of ASMR were confirmed. Methylation of SMR was found, and four isomers of a metabolite of SMR were confirmed. Conclusion: The developed UPLC-QTOF-MS method is rapid and effective for screening, identification, and quantification of SMR and its metabolites. The metabolism mechanism and accurate mass databases for target and non-target metabolites of SMR in mice liver are valuable for metabolism study, with high accurate mass.

Background: Vitamin has an important function in biochemical processes and plays an important role in the main process of the human body. Vitamin products are used to maintain health, restore vitality, control weight and help in disease prevention. To comply with regulatory requirements and to establish nutrient intake in some populations, the quality of vitamins in food and nutritional supplements needs to be ensured. Methods: This study aims to obtain a simultaneous method for the quantification of thiamine hydrochloride, riboflavin, nicotinamide, dexpanthenol, pyridoxine hydrochloride, cyanocobalamin and ascorbic acid in pediatric syrup by Ultra Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS/MS). ACQUITY UPLC® BEH Shield C8 column (2.1 mm x 50 mm, 1.7 m), mobile phase is a mixture of 10 mM ammonium formate in 0.2% formic acid pH 2.8 with methanol by gradient elution at a flow rate of 0.4 mL/min achieved in 6.5 minutes was used for separation. Ionization method was positive electrospray ionization (ESI +) with Multiple Reaction Monitoring (MRM). Result: Confirmation result showed 7 water-soluble vitamin parent ions and daughter ion of thiamine HCl at m/z 265.15> 122; riboflavin at m/z 377.20> 243.10; nicotinamide at m/z 123> 80.10; pyridoxine HCl at m/z 170.10> 152.00; cyanocobalamin at m/z 678.45> 147.05; dexpanthenol at m/z 206.05> 76.00 and ascorbic acid at m/z 177.00> 94.90. The results of method validation showed that the method fulfills the acceptance criteria of precision, recovery and linearity Conclusion: The method can be used for the routine analysis of determination of seven water soluble vitamins in pediatric syrup.

Background: Fifteen pairs of geometric isomers of new sulfonamide derivatives, exhibiting antitumor activities were analyzed with the use of quantitative structure-retention relationships method. The set of studied compounds bears thiohydrazone moiety, which leads to in-solution formation of an equilibrium between cis and trans isomer. The semi-empirical level of in silico molecular modeling was performed for calculations of molecular descriptors. Methods: During the study, OPLS and MLR were compared, as two types of chemometric approach. QSRR model was proposed applying partial least squares regression method based on chosen descriptors. Results: The relationship between the isomers geometry and chromatographic retention properties derived from in silico model was able to describe that observed pairs of peaks are most likely cis and trans isomers. Conclusion: The applied chemometric techniques also revealed the most influential features of molecules responsible for chromatographic behavior. In silico applied approach seems to be a good supportive tool helpful in identifying geometric isomers with the use of chromatographic retention data.

Objective: This study aims to establish an efficient, sensitive and specific analytical method (in vitro and in vivo) for syringic acid. Method: Syringic acid loaded liposome was prepared and characterized. In vitro and in vivo chromatographic conditions for the determination of syringic acid were investigated, with the application verified by pharmacokinetics and biodistribution studies. Results: The developed liposomal formulation produced homogeneous regular, spherical and multilamellar- shaped liposome. Efficient HPLC method for the analysis of syringic acid was established and validated. The chromatographic separation was performed on a Waters symmetry C18 column (4.6 150 mm, 5 μm) at 25 °C by using mobile phases of 30 % methanol and 70 % acetic acid solution (0.1 %) (in vitro) and 28 % methanol and 72 % acetic acid solution (0.1 %) (in vivo) at a flow rate of 0.8 mL/min. The wavelength was set at 272 nm. Vanillin was used as internal standard for the in vivo analysis. The established linearity ranges of SA were 1 - 100 μg/mL (R2 = 0.9997) in vitro and 0.125 – 64 μg/mL (R2 = 0.9991) in vivo. In vitro accuracy of the method was > 99%, and in vivo accuracies were between 91.23 % - 109.54 %, while the precisions met the acceptance criterion. In vivo HPLC method was also confirmed by pharmacokinetics and biodistribution studies. Conclusion: Established HPLC method exhibited high linearity, precision, accuracy and specificity, could be used for quantitative determination of SA in vitro and in vivo.

Background: The combination of cyclophosphamide and doxorubicin is commonly used in the adjuvant treatment of breast cancer in women with a high risk of recurrent disease. It is essential to devise dosing strategies for such drugs in order to minimise their in-use toxicity and to maximise their efficacy. Data have shown that concentration directed rather than dose targeted therapy provides better clinical outcomes. Therefore, analytical methods that permit the simple, sensitive and accurate determination of such compounds in human plasma will assist in drug dosing strategies Methods: We report herein a method for the LC-MS/MS determination of cyclophosphamide and doxorubicin concentrations in patient plasma, following intravenous chemotherapy using protein precipitation without time-consuming supernatant drying and reconstitution steps, at the same time using a HILIC pre-column to further remove residual phospholipids. Results: The method has a less than 15% RE and less than 5% RSD both intra- and inter- day. It has following principal advantages over recently published LC-MS/MS methods for cyclophosphamide and doxorubicin in human plasma: 1) the sample preparation method is very simple and rapid; 2) the method has a very low LLOQ (cyclophosphamide 0.5 ng/mL and doxorubicin 1.0 ng/mL) at the same time the precision and accuracy meeting the primary requirements established by the FDA; and 3) the procedure only requires a very small plasma volume (30 μL). Conclusion: Such a methodology, when applied in close temporal and geographical proximity to patients will permit a short turn-around time and thereby facilitate the clinical interpretation and feedback to improve patient outcomes.

Background: Simultaneous determination of binary mixtures with simple and cost effective analysis is always of interest. Progressive advancement in chemometrics enables spectral resolution of drugs in the presence of their degradation products or impurities. Methods and Result: Three stability indicating chemometric methods are applied for the simultaneous determination of Dantrolene sodium (DNT) and Paracetamol (PAR). Partial Least Squares (PLS), Concentration Residuals Augmented Classical Least Squares (CRACLS) and Multivariate Curve Resolution- Alternating Least Squares (MCR-ALS) were selected for that purpose. DNT and PAR were determined in the linearity range of (2 – 10 μg mL-1) and (12 – 28 μg mL-1), respectively, in the presence of their degradation products. The presented methods were compared for their qualitative and quantitative analyses and validated according to the ICH guidelines. Furthermore, statistical comparison between the results obtained by the proposed methods and the reported chromatographic method showed no significant differences. Conclusion: The proposed multivariate calibrations were accurate and specific for quantitative analysis of the studied components. Furthermore, CRACLS and MCR-ALS methods succeeded in both quantitative and qualitative of the studied components and their degradation products.

Introduction: Chromatographic methods for determination of betamethasone or dexamethasone in their pharmaceutical preparations by Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry were developed. Method: Analytes were eluted from Acquity BEH C18 (50 mm.1 mm, 1.7 μm) column using stepwise gradient with mobile phase consisted of acetonitrile and 0.1% (v/v) acetic acid in water. Quantitation of dexamethasone and betamethasone was performed using 451>361 m/z on MRM mode. Results: Methods were linear over the concentration range from 10 to 1500 ng mL-1 for both analyte. Limits of detection for BTM or DXM were 1 ng mL-1. The highest relative error and relative standard deviation values were not over 2%. Conclusion: Presented methods were specific, rugged and robust, successfully applied to the analysis of their pharmaceutical preparations.

Background: The field of research in pharmacy is rapidly shifting towards nanocarrier based drug delivery systems. There is a need to develop accurate and specific analytical techniques for the analysis of the drugs in nanocarriers. The performance of such system will be based on how accurately these are analyzed during their development stage. Aim and Objective: The present study deals with the development, validation and application of simple, precise and accurate HPLC method for the determination of colchicine in pharmaceutical formulations and solid lipid nanoparticles. Methods: The analytical conditions were optimized on Phenomenex Luna C18 Column (150 x 4.6 mm, 5 μm) at room temperature. The mobile phase consists of acetonitrile:0.1 % orthophosporic acid in 35:65 v/v ratio. Injection volume was 10 μL. The flow rate was maintained at 1.0 ml/min and analysis was carried out at 245nm. Results and Discussion: The method was found to be linear in the concentration range 2-12 μg/mL with regression coefficient (r2) of 0.999. The method was found to be precise with % relative standard deviation below 2%. The limit of detection and limit of quantification were found to be 0.6121μg/ml and 1.854 μg/ml respectively. The percent recovery of the developed method is 99.273 %. The applicability of the method for the quantification of drug in novel carriers like solid lipid nanoparticles (SLNs) was assessed by determining entrapment efficiency and in-vitro drug release profile. The entrapment efficiency of prepared SLNs was found to be 37.35 % ± 0.54. The in vitro release data was subjected to kinetic model fitting which shows Korsmeyer- peppas as a best fit model for COL release from SLNs. Conclusion: A fast, simple, precise, accurate and robust HPLC method for the determination of entrapment efficiency and in vitro drug release of colchicine from solid lipid nanoparticles has been developed and validated in accordance with ICH guidelines.