Current Pharmaceutical Analysis - Volume 14, Issue 6, 2018

Background: Analytical method development and validation remain the key prerequisites for the successful development of a product. Methods: An endeavour, therefore, was made for the development of an effortless, swift, sensitive and cost-effective method for estimation of galantamine hydrobromide using the established paradigms of Quality by Design (QbD). Results: Chromatographic separation was accomplished on a reverse phase C18 column employing an optimized mixture of methanol, acetonitrile and ammonium formate buffer, adjusted to pH 9, at a flow rate of 0.7 mL/min with UV detection at 212 nm. At the onset, risk assessment and factor screening studies using Taguchi design facilitated the understanding of the factors that are crucial in varying Critical Analytical Attributes (CAAs). The mobile phase ratio and the flow rate through the column were identified as the Critical Method Parameters (CMPs), and were subsequently employed for systematic method optimization using a Face-Centred Cubic Design (FCCD), employing four Critical Analytical Attributes (CAAs) viz. retention time, peak area, peak tailing and theoretical plates. Statistical modeling was accomplished, followed by response surface analysis leading to complete analytical understanding and comprehending plausible interaction(s) among CMPs, if any. Numerical and graphical optimizations were employed in order to demarcate the design space and eventually search for an optimum solution. Conclusion: The studies successfully reveal the utility of analytical QbD (AQbD) paradigms for developing quite sensitive and precise liquid chromatographic method, for galantamine hydrobromide, with enhanced method performance and improved analytical understanding.

Background and Objectives: To establish a simple and reproducible High Performance Liquid Chromatography (HPLC) method for the determination of mizoribine in human plasma using glipizide as Internal Standard (IS). Methods: The mizoribine in plasma was precipitated with 10% perchloric acid and separated on a Phenomenex Luna NH2 (250 mm 4.6 mm, 5 μm) column with a mobile phase consisted of acetonitrile and 0.3% glacial acetic acid aqueous solution (48:52, V/V) at a flow rate of 1.0 mL/min. The detection wavelength was 280 nm and the column temperature was room temperature. Results: The assay exhibited a linear range of 0.05-10.0 μg/mL (r = 0.9992) and the lower limit of quantification was 0.05 μg/mL. The specificity, linearity, accuracy, precision and stability were in accordance to China Food and Drug Administration (CFDA) guidelines. Conclusion: Therefore, the method was successfully applied in the routine therapeutic mizoribine monitoring in Chinese kidney transplant patients after an oral administration of 100 mg (2 Bredinin® 50 mg tablets) or 150 mg (3 Bredinin® 50 mg tablets) mizoribine.

Background: Teneligliptin is anti diabetic drug acting as dipeptidyl peptidase-4 inhibitor. HPLC methods are reported for the estimation of teneligliptin but till date no HPTLC method has been reported. HPTLC method has advantage of short analysis time, requires less quantity of mobile phase, and accurate and comparable results can be obtained with that of HPLC. Objective: Objective of the present study is to develop sensitive, precise, stability indicating high performance thin layer chromatographic method for the estimation of teneligliptin. Methods: HPTLC analysis was performed using aluminum plates pre-coated with silica gel 60F254 as the stationary phase and 0.25% ammonium sulphate in water: ethyl acetate: methanol (10: 2.5: 2.5, v/v/v) as mobile phase. The Rf value was observed to be 0.33 ± 0.02. The densitometric scanning was carried out in absorbance mode at 243 nm. The method was validated with respect to accuracy, precision, specificity and robustness. Results and Discussion: The developed method was found to be linear in the range of 400 – 1600 ng/band. The recovery of teneligliptin was found in the range of 99.41- 99.97 %. Stability-indicating nature of the HPTLC method was established by performing various forced degradation studies. The results clearly indicated that the degradation products were well separated from the pure drug with significant difference in Rf values. The method was validated as per ICH guideline and can be used for routine analysis of stability samples.

Background: Exhaled breath has recently been identified as an alternative matrix for the detection of drugs and chemical compounds. By keeping in mind that different chemical and biological compounds have already been determined in the exhaled air, the Exhaled Breath Condensate (EBC) was found to be a suitable sample for non-invasive assessment of the respiratory system. Thus, the aim of this work is determination of ammonia and nitrite in EBC samples. Methods: The determination of the analytes is based on the derivatization of ammonia using Berthelot method. The absorbance of colored compound was determined at 655 ± 3 nm. Nitrite ion was determined after its reduction to ammonia by using Zn/HCl reagent. Results: Good calibration curves were obtained for ammonia with linearity in the range of 10– 620 ng mL -1 , and correlation coefficients (r2) of more than 0.999. The Limit of Detection and Quantification (LOD, LOQ) and Lower Limit of Quantification (LLOQ) were found to be 1.5, 4.5 and 10 ng mL -1 , respectively. The recoveries of the method were calculated to be 94 to 102.5% and the intra- and interday precisions were lower than 1.4 and 3.4%, respectively. Conclusion: The results presented here demonstrate that the concentrations of ammonia and nitrite in samples of healthy humans can be determined with good accuracy, high sensitivity, and simple operation. The proposed EBC methodology possessed several advantages like simplicity and low cost, especially if more sophisticated techniques such as HPLC are not available.

Background: Ganoderma lucidum is a well-known edible mushroom and traditional Chinese medicine, but there were no comprehensive reports on the extraction, analysis and activities test of Ganoderma lucidum Amino Acids (GLAA). Objective: The present study was aimed to optimize the extraction process of GLAA and analyze the amino acid composition of GLAA, combined with preliminary activity evaluation. Methods: The hydrochloric acid-hydrolysis method for extraction of GLAA was optimized by Response Surface Methodology (RSM) with Box-Behnken Design (BBD). The amino acid composition of GLAA was analyzed by PITC pre-column derivation with HPLC method and amino acid automatic analyzer. The hypoglycemic and antioxidant activities of GLAA were assayed by inhibitory rate of α- glucosidase and DPPH radical scavenging rate, respectively. Results: The optimal extraction conditions were obtained: liquid-to-solid ratio 20:1 mL/g, extraction time 9.5 h, hydrochloric acid concentration 7.0 mol/L, and the yield of GLAA was 4.78 ± 0.21 %. The result of amino acid automatic analyzer showed that the content of total amino acids in GLAA was 2.94 %, including 18 kinds of amino acids, and the most abundant amino acid was leucine (0.37 %). The results of activity tests in vitro showed that GLAA exhibited high α-glucosidase inhibitory rate with IC50 value of 380.62 μg/mL and a certain scavenging DPPH activity with IC50 of 484.54 μg/mL. Conclusion: The RSM with BBD was successfully implemented for optimization of the extraction parameters of GLAA. The GLAA was rich in 18 kinds of amino acids and possessed strong hypoglycemic and antioxidant activities.

Background: Green electrochemical sensor (an in-situ plated bismuth film glassy carbon electrode) was used for the first time in combination with adsorptive striping voltammetry to elaborate the novel, simple, cheap and sensitive alternative analytical procedure for determination of rutin. Methods and Results: In this paper, the electrochemical procedure for the determination of rutin using a square-wave adsorptive stripping voltammetry and a bismuth film modified glassy carbon electrode was developed. Rutin provided a well-defined, single oxidation peak at +0.45 V (vs. Ag/AgCl) in the solution containing 0.025 mol L-1 of acetate buffer (pH = 4.0 ± 0.1), 5.0 10-7 mol L-1 Bi(NO3)3. Under optimum conditions, the obtained results showed a linear calibration graph of the peak current (Ip) versus rutin concertation in the range of 1.0 10-10 to 1.0 10-8 mol L-1 with a correlation coefficient of 0.9969. The determined detection limit was 2.2 10-11 mol L-1 for the accumulation time of 60 s. The sensor prepared in situ was successfully applied to the determination of rutin content in the tablets without previous separation. Conclusion: In this paper, significant progress has been made in the development of suitable electrochemical procedure for rutin determination in comparison to our previous paper as well as other works described in the literature, due to the application of bismuth film electrode.

Background: Eremurus spectabilis BIEB. (Liliaceae) is an edible and medicinal plant in Turkey. Introduction: This study was designed to isolate and quantify isoorientin in leaves of E. spectabilis and exhibit its effect on SH-SY5Y neuroblastoma cell line. Methods: Purity and identification of isoorientin were evaluated by 1D-NMR, 2D-NMR and Q-TOF were isolated from E. spectabilis via column chromatography. An HPLC method was also developed and validated for isoorientin. Results: Quantitative measurements indicated that contents of isoorientin in E. spectabilis leaves were 81.01 mg/g and MeOH extract were 23.75 mg/gr. All measurements were performed at 350 nm. Anticancer activity was investigated on cell culture. IC50 doses of isoorientin were detected as 250 μM at the 48th hour in SH-SY5Y cells by XTT assay. Real-time PCR analysis in SH-SY5Y cells showed that CCND1, CDK6, casp-9, Bax, ATR, Bcl-2, CHEK1 and CHEK2, expressions significantly reduced in experimental group when compared with the control group. p53, p21, caspase-3, caspase-8, Bcl-2, ATM and ERCC1 expressions increased in the experimental group when compared with the control group (P <0.05). Conclusion: Measurements revealed that E. spectabilis contains high amount of isoorientin and can be used as a new source of this compound. In addition to this, isoorientin affects cell proliferation of neuroblastoma cells by cell cycle control and apoptosis gene expression and it could be used as a therapeutic agent, however, more studies must be performed to clarify its mechanism.

Background: Cuphea glutinosa is a medicinal species extensively distributed in South of Brazil, being known in the folk medicine as diuretic, anti-inflammatory, laxative and anti-hypertensive. In the present work, its majority chemical components present in infusions and macerations obtained from leaves and roots were investigated. The analytical responses were well worked using a UPLC-MS, whose detection was standardized for flavonoids due to their importance for this genus and the biological effects reported. Conclusion: Using a mobile phase composed by acetonitrile:methanol and 0.1% formic acid, eluted by gradient, and a ionization mode by positive ESI, four flavonoids were identified: quercetin-3-glucoside, quercetin-3-arabinoside, quercetin-3-glucuronide and isorhamnetin. Another quercetin derivative was also detected. The extracts were investigated for antifungal activity by broth microdilution method, whose results indicated activity against Candida parapsilosis, Candida tropicalis, and Trichosporon asahii. This antimicrobial potential can be attributed to the chemical profile predominant in flavonoids.

Background: A new UHPLC method was developed and subsequently validated for determination of the anti-hypertensive combination Trandolapril (TAD) and verapamil hydrochloride (VRP) in bulk powder, dosage form (Tarka® 2 mg TAD/ 180 mg VRP), and spiked human plasma. Moreover, the method was utilized for studying the stability of the combination under hydrolytic and thermal conditions. Methods: Separation was achieved by using BEH C18 (1.7 μm, 2.1 x 50 mm) analytical column (Waters ® Acquity UPLC) and a mobile phase composed of 15 mM tris buffer pH 6.5 adjusted with 1 M HCl acetonitrile (20:80 v/v). Forced degradation studied was carried out using Waters® Xevo G2-S QToF coupled with Waters® Acquity UPLC system with binary Solvent Manager (I-Class) via electrospray ionization (ESI) interface. Results: In bulk powder, the retention times were 0.450± 0.004 and 1.290± 0.011 minutes, where the linearity ranges were found to be 0.12 to 240 μg/mL and 0.10 to 360 μg/mL for TAD and VRP, respectively. Moreover, linearity was achieved in spiked human plasma samples by using Ledipasvir as an internal standard and linearity ranges were found to be 55 to 90 μg/mL and 15 to 80 μg/mL for TAD and VRP, respectively. The amount of trandolapril and verapamil hydrochloride present in the dosage form was determined in triplicate to give a concentration of 99.60%±0.48 (1.97 mg) and 99.96%0±0.63 (179.93 mg), respectively. The structure of the degradation products was elucidated by UHPLC-ESI-QToF. Conclusion: The method was able to extract 95.61% of trandolapril and 97.83% of verapamil hydrochloride from human plasma and found to be stable. Furthermore, the combination is stable towards acidic and thermal conditions used.

Objective: A simple, sensitive, robust method for the estimation of zaltoprofen was developed and validated. The forced degradation study was carried out as per FDA guidelines. Methods: The method was developed by using HPLC Binary Gradient System (Model no.: HPLC 3000 Series) of Analytical Technologies Ltd. equipped with the UV-3000-M detector, P-3000-M Reciprocating (40MPa) Pump, Grace C18 (250mm x 4.6ID, Particle size: 5 microns) Column. The mobile phase optimized for the analysis of Zaltoprofen was Methanol and water in the ratio of 90:10 v/v having pH 3.The RT obtained for Zaltoprofen was 4.6 min. The Zaltoprofen was determined at 227nm. Results: The linearity range was found to be 10-50 μg/ml with R2 0.997. The LOD and LOQ were found to be 0.1125μg/ml and 0.3409μg/ml respectively. The results of robustness and ruggedness were within the acceptable limit. The results of recovery were 99.80,99.84,99.74%. Forced degradation of Zaltoprofen was carried out by exposing to 0.1N HCL, 0.1N NaOH at 60°C for 30min., an oxidizing agent (3% H2O2), at 24 hrs., photolytic degradation study for 24 hrs., and thermal degradation at 600 °C for 24 hrs. The results of which were 9.08, 16.78, 6.4, 2.1, 3.46% degradation, respectively. Conclusion: The method is economical and can be implemented for routine analysis of Zaltoprofen in bulk and tablet formulation. Forced degradation study will be useful for the determination of stability indicating properties of zaltoprofen.

Background: Most of the fat-soluble vitamins are not stable and decompose when exposed to light. Therefore, stability testing of the analytes in solutions and biological matrix is required during development and validation of analytical methods devoted to determination of these vitamins in biological fluids. Objective: Determination of the stability of lipophilic vitamins, including retinol, α-tocopherol, β- carotene and 25-hydroxy-metabolites of vitamin D at all stages of the analysis. Methods: Stability studies in methanol solutions and plasma samples were performed, including shortterm and long-term stability, freeze and thaw stability and autosampler test. For determination of vitamins in solutions, spectrophotometric method was applied. Analysis of vitamins in samples obtained following extraction of the analytes from plasma was performed using validated HPLC methods with UV detection. Results: Short-term stability test showed that all studied vitamins were stable up to 4 hours when stored at +4°C and in ambient temperature without protection from light. During long-term stability test performed within 46 days at room temperature with no access to the light, retinol and β-carotene in solutions showed degradation while other analytes proved to be stable. No significant decomposition of vitamins in solutions stored at +4°C was observed during the above storage time. All the vitamins were stable in samples stored for 24 hours in autosampler and during three freeze-thaw cycles of plasma samples stored at -80°C. Conclusion: In solutions, the most stable analyte was α-tocopherol, whereas the less stable was retinol. All vitamins were stable in plasma samples in the studied storage conditions. To avoid decomposition of these vitamins low temperature and protection from the light is recommended.

Background: Synthesis of substituted N'-aryl-N'-adipic acid thiourea (1-7) was, carried out adipic acid chlorides with potassium thiocynate in situ produced isocyanate followed by reflux with substituted anilines in dry acetone till the completion of reaction. Methods: Structure elucidation of all the synthesized compounds was carried out by UV-Vis, FT-IR and 1HNMR analysis. All the synthesized compounds were evaluated for their biological screening by subjecting to antimicrobial and antioxidant activities. Conclusion: In case of antibacterial screening most significant results were obtained by compound 7 having fluoro group at the para position with 16.5±1.0 zone of inhibition against methicilline resistant S. aureus (MRSA 09) while no activity was observed in case of antifungal activities. Compound 7 also showed significant antioxidant activities in DPPH assay with percent scavenging values of 63.51±.8.3.

Background: The purpose of this study is the development and validation of assay test for Dapoxetine Hydrochloride in the pharmaceutical dosage forms by HPLC. The assay method by HPLC was found to be linear in the concentration range of 25 to 150 μg/mL. The mobile phase was composed of methanol and buffer pH: 7.0 ammonium dihydrogen phosphate (80:20 v/v) at the flow rate of 1 mL/min using UV detection at 232 nm. Results and Conclusion: The analytical results were validated by recovery studies. The percentage recovery method was found to be 99.58-100.75%. The LOD and LOQ were found to 0.008 μg/mL and 0.027 μg/mL. All the parameters of validation were in the acceptance range. This developed method was successfully applied to estimate the amount of Dapoxetine Hydrochloride in the tablets.

Background: Non-ionic X-ray contrast agents constitute a very important class of pharmaceutical compounds produced in large quantities. Iohexol is an important example of such compounds. Objective: Three simple and selective stability indicating spectrophotometric methods utilizing ratio spectra were proposed for the determination of the widely used X-ray contrast medium, iohexol in the presence of its acidic degradate and in its pharmaceutical formulation. Methods: The first method is the first derivative of ratio spectra method (DD1), the second is the Ratio Difference Method (RD), and the last one is the Mean Centering method (MC). Results: The three proposed methods showed a good linearity over the concentration range of 4-40 μg.mL-1. The selectivity of the three developed methods was evaluated by analyzing different laboratory- prepared mixtures and satisfactory results were obtained. Conclusion: Iohexol has been successfully determined in its pure form and pharmaceutical formulation (Omnipaque® vials) utilizing the proposed methods with no interference from the present additives. The results obtained by each of the proposed methods were statistically compared to the official United States pharmacopeial method and non-significant difference was obtained regarding accuracy or precision.

Introduction: Curcumin possesses a wide range of biological properties, including antiinflammatory, anti-oxidant, anti-proliferative, and anti-microbial activities. Paraquat (PQ) is a highly toxic herbicide, and many cases of acute poisoning and death have been reported over the past decades in humans and animals. In this study, we developed a tissue (lung, liver, kidney, heart) metabolomics method by Gas Chromatography-Mass Spectrometry (GC-MS) combined with analysis of pathologic changes in tissues to evaluate the effect of curcumin in rats that underwent acute PQ poisoning. Materials and Methods: Thirty-six rats were randomly divided into the acute PQ poisoning group, the curcumin group, the acute PQ poisoning and curcumin intervention group, and the control group, with 9 rats in each group. Partial Least Squares-Discriminate Analysis (PLS-DA) revealed that intragastric administration of curcumin induced metabolic perturbations of acute PQ poisoning. Results: When rats were treated with curcumin, compared to the acute PQ poisoning group, certain biomarkers (L-alanine, L-proline, D-ribose, arachidonic acid, 9,12-octadecadienoic acid, octadecanoic acid, and hexadecanoic acid) changed in tissues (lung, liver, kidney, and heart) of the acute PQ poisoning and curcumin intervention group. The PLS-DA 3D score chart showed that the rats in the acute PQ poisoning group were clearly distinguished from the rats in the control group or acute PQ poisoning with curcumin intervention group, and suggested that there was some improvement in rats that received curcumin intervention for acute PQ poisoning. Pathologic changes in tissues (lung, liver, kidney, and heart) showed that curcumin intervention for PQ poisonings reduced the degree of pulmonary fibrosis. Conclusion: Curcumin intervention for acute PQ poisoning resulted in some improvement. Moreover, the metabolomic method by GC-MS may be useful to elucidate the effects of curcumin in rats that have been acutely poisoned by PQ.