Current Pharmaceutical Analysis - Volume 15, Issue 6, 2019

Introduction: The purpose of this study is the development and validation of assay test for Tenofovir Disoproxil Fumarate (TDF), Emtricitabine (FTC) and Efavirenz (EFV) in combined tablet dosage form by Reverse Phase (RP) HPLC. Materials and Methods: The assay method by HPLC was found to be linear in the concentration range of 15-150 μg/mL, 10-100 μg/mL and 30-300 μg/mL for TDF, FTC, and EFV, respectively. Successful separation of combined drugs was achieved by isocratic elution on a Phenomenex® C8 column (250 mm x 4.6 mm, 5μm). The mobile phase was composed of buffer pH: 7.0 ± 0.05 potassium dihydrogen phosphate, acetonitrile and methanol (40:40:20 v/v) at the flow rate of 1 mL/min using UV detection at 262 nm, column oven temperature 25ºC, and injection volume 20 μL. Results: The analytical results were validated by recovery studies. All the parameters of validation were in the acceptance range. This developed method was successfully applied for simultaneous estimation the amount of TDF, FTC and EFV in the bulk and marketed dosage forms.

Background: Signal transducer and activator of transcription 3 (STAT3), an oncogenic protein found constitutively active in many types of human malignancies, is considered to be a promising target for cancer therapy. Objective: In this study for the first time, a simple and accurate method has been developed for the determination of a STAT3 dimerization inhibitor called stattic in aqueous and plasma samples. Methods: A reverse-phase high-performance liquid chromatography (RP-HPLC) composed of C18 column as stationary phase, and the mixture of acetonitrile (60%) and water (40%) as mobile phase with a UV detection at 215 nm were applied for quantification of stattic. The developed method was validated by Food and Drug Administration (FDA) guideline. Results: The method provided a linear range between 1-40 and 2.5-40 μg mL-1 for aqueous and plasma samples, respectively, with a correlation coefficient of 0.999. The accuracy (as recovery) of the developed method was found to be between 95-105% for aqueous medium and 85-115% for plasma samples. The precision (as relative standard deviation) for aqueous and plasma samples was less than 6% and 15%, respectively. The sensitivity of the developed method based on FDA guideline was 1 μg mL-1 for aqueous and 2.5 μg mL-1 for plasma samples. Conclusion: These results show that the established method is a fast and accurate quantification for stattic in aqueous and plasma samples.

Objective: This study was aimed at conducting a pharmacokinetic evaluation of metformin in rabbit plasma samples using rapid and sensitive HPLC method and UV detection. Methods: Acetonitrile was used for protein precipitation in the preparation of plasma samples. Reverse phase chromatography technique with silica gel column (250 mm x 4.6 mm, 5 μm) at 30°was used for the separation purpose. Methanol and phosphate buffer (pH 3.2) mixture was used as a mobile phase with flow rate 0.8 ml/min. The wavelength of UV detector was adjusted at 240 nm. Results: The calibration curve was linear in a range of 0.1-1 μg/ml with R² = 0.9982. The precision (RSD, %) values were less than 2%, whereas, accuracy of method was higher than 92.37 %. The percentage recovery values ranged between 90.14 % and 94.97 %. LOD and LOQ values were 25 ng/ml and 60 ng/ml, respectively. Cmax and AUC0-t values were found to be 1154.67 ± 243.37 ng/ml and 7281.83 ± 210.84 ng/ml.h, respectively after treating rabbits with a formulation containing 250 mg metformin. Conclusion: Based on the above findings, it can be concluded that present method is simple, precise, rapid, accurate and specific and thus, can be efficiently used for the pharmacokinetic study of metformin.

Introduction: The root of Millettia speciosa Champ. (Leguminosae) is one of the wellknown traditional Chinese medicines abundant in phenolic compounds and plays important roles in the treatment of pain or numbness of the joints, blood deficiency sallow, chronic bronchitis and chronic hepatitis. Objective: An ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS) based chemical profiling approach was established for the separation and characterization of isoflavonoids in the roots of Millettia speciosa Champ. Methods: The roots of Millettia speciosa Champ. were prepared by ethanol extraction followed by further extraction with chloroform. The extracts were analyzed by UPLC-Q-TOF-MS in positive and negative electrospray ion modes at different Collision Energy (CE) values. Results: A total of 48 components were simultaneously detected, of which 38 components, including 21 isoflavones, 4 phenolic acids, 4 isoflavanones, 2 phenolic aldehydes, 2 flavanones, 1 aliphatic acid, 1 chalcone, 1 flavonol, 1 isoflavane and 1 pterocarpan were unambiguously identified or tentatively assigned based on the retention time, UV spectra, characteristic molecular ions, MS/MS fragmentation data and reference standards. The isomers of isoflavonoids were distinguished using accurate mass, the diagnostic fragmentations of C-ring and UV spectra. The major diagnostic fragment ions of isoflavonoids were observed and the corresponding fragmentation pathways were proposed. Conclusion: This investigation provides important analysis data for further quality control, pharmacological and toxicological research on Millettia speciosa Champ.

Background: A successful attempt has been done to develop and validate a simple stability indicating HPTLC method for the estimation of Mometasone furoate (MF) and its degradation product in the presence of Salicylic acid (SA). The degradation product was isolated, characterized and tested for cytotoxicity. Introduction: Mometasone furoate (MF) is chemically 9,21-Dichloro-17α-[(2-furanylcarbonyl) oxy]- 11β-hydroxy-16-α-methylpregna-1,4-diene-3,20-dione, a high potency glucocorticoid. Salicylic acid (SA) has antiseptic, antifungal and keratolytic properties. Combination of MF and SA is available in the market as an ointment and is used for the treatment of skin inflammation, skin diseases, acne, skin redness and other conditions. Till now, there is no scientific documentation on HPTLC method for simultaneous estimation of MF and SA in the topical formulation; stress testing of drugs and determination of degradation products. Methods: Combination of Toluene: Ethyl Acetate: Methanol: Ammonia (6.4:1.5:2.0:0.1) was selected as the mobile phase. Detection was done by UV absorbance mode at wavelength 250 nm. Topical formulation containing MF and SA was analyzed by the developed method. The developed method was validated as per ICH guidelines. The standard drugs were subjected to stress testing like hydrolysis, oxidative, thermal and photolytic degradation. Results: Good separation with Rf values 0.61 ± 0.02 (MF) and 0.21 ± 0.02 (SA) was achieved by optimized chromatographic conditions. The % drug content was found to be 97.41±1.15 and 99.43 ± 0.73 for MF and SA, respectively in a topical formulation. From the results of validation parameters, the developed method was found to be specific, accurate, precise, sensitive and robust. After stress testing, SA was found to be stable under different stress conditions. Whereas, MF was found to be base sensitive and single degradation product was observed and isolated by preparative TLC. It was characterized by LC-MS and LC-MS/MS studies. Isolated degradation product was subjected to cytotoxicity testing on A549 and SiHa cell lines. Conclusion: A simple stability indicating HPTLC method was developed and validated for the estimation of MF and its degradation product in presence of SA. Probable structure of degradation product of MF and probable pathway of degradation was interpreted. Results of cytotoxicity testing showed that the degradation product was more cytotoxic as compared to MF against both the cell lines.

Introduction: Engeletin is the main active component in the engelhardia leaf that promotes circulation and removes stasis, and has hypoglycemic, hypolipidemic, and anti-inflammatory actions. The aim of this study was to develop an ultra-performance liquid chromatography- tandem mass spectrometry method to detect engeletin in plasma and tissues and investigate its absorption, distribution, and mechanism in mice, which could provide very useful information for its pharmacological effect in vivo. Materials and Methods: Twenty-five mice were intraperitoneally injected with 20 mg/kg engeletin, and five mice were sacrificed using 4% chloral hydrate 0.25, 0.5, 2, 4, and 6 h later. The tissues (brain, kidney, heart, liver, spleen, and lung) and blood were collected. Acetonitrile precipitation was applied to remove protein and further process the mouse plasma and tissue homogenate samples. Multiple reactions monitoring mode in negative mode was used to quantify the engeletin. Results and Conclusion: Linearity of engeletin in plasma and tissues was good (R2 > 0.995), within the range of 2-2,000 ng/mL in plasma and 2-2,000 ng/g in tissues, and the lower limit of quantitation was 2 ng/mL in plasma and 2 ng/g in tissues. Inter-day precision of engeletin in plasma or tissues (brain, kidney, heart, liver, spleen, and lung) was < 14%, and intra-day precision was < 15%. After the mice were intraperitoneally injected with engeletin (20 mg/kg), the distribution in kidney and liver was the highest, followed by blood, spleen, lung, heart, and brain. Engeletin concentration in the brain was low, suggesting that engeletin can penetrate through the blood brain barrier, which could also help with engeletin investigations of the brain.

Background: Analytical Quality by design (AQbD) is one of the upcoming regulatory requirements in the field of pharmaceutical analysis for the development of reliable and robust analytical methods. Montelukast sodium and theophylline available in tablet dosage forms are used widely for the treatment of airway disorders. Liquid chromatographic methods for estimation of the drugs are reported but those chromatographic methods developed by the AQbD approach are not available. Methods: A systematic design of experiments has been employed to develop a liquid chromatographic method for simultaneous estimation of montelukast sodium and theophylline. Critical process parameters are defined and optimization of methanol content in mobile phase, the concentration of trifluoroacetic acid and flow rate was employed within the framework of the full factorial design. The statistical analysis of the experimental data for critical quality attributes namely tailing factor of both the drugs and resolution was performed. Results: A multi-dimensional design space for the critical process parameters was obtained. The optimal chromatographic separation of the two API was achieved using C18 (250mmx4.6mm, 5μm) column as stationary phase and mobile phase composed of acetonitrile, trifluoroacetic acid (TFA) (0.2%v/v in water) and methanol in the ratio of 25:15:60 (v/v/v) at flow rate of 1.0 ml min-1. The influence of the CPP was evaluated by various data tables for example, analysis of variance and lack of fit. Interpretation of various plots obtained from the experimental data such as predicted vs. actual, predicted vs. run, pareto chart, perturbation plot and contour plot provided the information on the manner in which each factor affects the critical quality attributes. Conclusion: A design space generated by design of experiments is an evidence for the robustness of the method. The assay results show the applicability of the method for analysis of commercially available tablets.

Background: Etamsylate (ETS), a haemostatic drug, is formulated with mefenamic acid (MFA) for pain relief. Objective: The aim of this work was to develop chromatographic methods for the estimation of ETS and MFA in the presence of their main impurities. These methods could be used in the routine analysis in quality control laboratories. Methods: The first method was RP-HPLC method, the separation was carried out on an Inertsil® ODS- 3V C18 column using a mobile phase composed of acetonitrile: potassium dihydrogen phosphate buffer adjusted to pH 7 with 0.1 N NaOH (55: 45, v/v) at a flow rate of 1 ml/ min. The detection was carried out at 220 nm. The second method was a TLC-densitometric method where the studied components were separated using a developing system composed of dichloromethane: ethyl acetate: methanol: triethylamine (6: 2: 2: 0.5, v/v/v/v) on TLC silica gel 60 F254 plates, followed by densitometric scanning at 300 nm. Results: In RP-HPLC method, the peaks were sharp and well separated, good retention times were obtained. Linearity was obtained over the concentration range 20-90 μg/ml, for both ETS and MFA. In the TLC-densitometric method, well separation of drug spots and linear relationship were achieved over the concentration range of 0.4-2.8 μg/spot, for both ETS and MFA. Method validation was conducted according to ICH guidelines. Conclusion: The developed methods were applied for the determination of the cited drugs in laboratory prepared mixtures and in tablets containing the two drugs. The methods are simple and precise and can be used for routine analysis of the drugs in combined dosage forms in quality control laboratories.

Background: Monitoring of plasma concentrations is a necessity for narrow therapeutic index potent drugs. Development of non-invasive methods can save the patients from the trauma of needles and hence is considered as a research priority. Introduction: Gabapentin, an anti-epileptic drug requires therapeutic monitoring because of its narrow therapeutic index. The objective of the study was to develop a suitable method for the non-invasive extraction of gabapentin for the same. Methods: Transdermal reverse iontophoresis was performed using pig ear skin as a barrier membrane. Three compartment iontophoretic cells were used for the extraction study. Extractions were carried out under low intensity electric field (current intensity- 0.5 mA/cm2, electrical field approximately 5 V). The donor compartment was charged with aqueous gabapentin (10 μg/ml in phosphate buffer of pH 7.4). For studying the effect of receiving vehicle (pH, ionic strength, and enhancer) on the extraction efficiency of gabapentin, the two receiver chambers were charged with media having varying concentration of these factors. Drug content was determined by HPLC. Results: Compared to other pHs, cumulative extraction of gabapentin at pH 5 was significantly higher at both anode and cathode (p<0.001). At low ionic strength, extraction of gabapentin increased linearly with the increase in concentration of ions up to a certain value but at very high ionic strength the pattern reversed. Similar results were obtained with enhancer (polyethylene glycol 400). Extraction increased with increase in polyethylene glycol 400 up to 3% and then decreased. Conclusion: Extraction flux can be optimized by manipulation of the receiver media.

Background: Of two main alkaloids extracted from Gelsemium, koumine was shown to be a promising analgesic, while gelsemine proved to be deleterious. Many patients suspected to be poisoned by Gelsemium cannot be timely diagnosed due to the lack of UPLC-MS/MS. Additionally, the concentration of alkaloids in humans has never been reported. The aim of this study was to establish a more economical and accessible method using HPLC-UV for diagnosis and quantitative analysis of Gelsemium poisoning. Methods: Plasma spiked with an internal standard, oxcarbazepine, was prepared with solid-phase extraction. Koumine and gelsemine were separated on a C18 column using a mobile phase consisting of methanol, water, and di-n-butylamine (58:42:0.01) pumped at a flow rate of 1.00 mL/min. The detection wavelength was set at 263 nm. Plasma concentrations of two different times were determined for the patients. Results: The calibration curves for both monomers possessed good linearity from 0.05-50 mg/L (r=0.9997 and 0.9999, respectively). The extraction recoveries were greater than 88.5 %. Variation for intraday and interday assays of koumine and gelsemine were less than 8.3% and 7.7%, respectively. The concentrations of the two alkaloids were identified in 5 patients with Gelsemium poisoning by using the established method. Conclusion: The established method by using HPLC-UV is applicable for diagnosis and quantitative analysis of Gelsemium poisoning in such cases. TDM of koumine and gelsemine in patients with Gelsemium poisoning may provide additional information for the clinic to improve rescue strategy.

Background: Glucosamine sulfate sodium chloride (glucosamine-SP) is mainly used for the treatment of osteoarthritis. During quality control of glucosamine-SP capsules, an unknown impurity was detected. Another unknown degradation product was generated together with above-mentioned impurity in heat condition. Objective: The study aimed to characterize an unknown impurity in glucosamine-SP capsules. Methods: A new volatile HPLC method compatible with mass spectrometry detection was set up. An amino column at 35 °C with a mobile phase consisting of water and acetonitrile (20: 80, v/v) was used at a flow rate of 1.5 ml/min at 297 nm. High-performance liquid chromatography quadrupole time-offlight mass spectrometry (HPLC-Q-TOF MS) was used to identify the impurity with the electrospray ionization (ESI) source in the positive ionization mode. Results: The results of HPLC-Q-TOF MS analysis indicated that the protonated molecule ions [M + H]+ of the unknown impurity and the novel degradation product were both at m/z 287. Preparative LC method was put into practice with a Prep-C18 column with a mobile phase consisting of water and acetonitrile (99: 1, v/v) at a flow rate of 20.0 ml/min at 297 nm. The assignment of the 1D and 2D NMR signals was performed for the unknown impurity. In addition, the formation of impurities was also studied. Conclusion: An unknown impurity and a degradation product in glucosamine-SP capsules were characterized. They were assigned as (1R, 2S, 3R)-1-(5-((S, E)-3, 4-dihydroxybut-1-en-1-yl) pyrazin-2-yl) butane-1, 2, 3, 4-tetraol and (1R, 2S, 3R)-1-(5-((S, Z)-3, 4-dihydroxybut-1-en-1-yl) pyrazin-2-yl) butane- 1, 2, 3, 4-tetraol.

Background: The poor oral bioavailability of newly discovered chemical entities and marketed formulations are usually related to poor aqueous solubility or poor permeability, leading to drug failure in the development phases or therapeutic failure in a clinical setting. However, advancement in drug formulations and delivery technologies have enabled scientists to improve the bioavailability of formulations by enhancing solubility or permeability. Objective: This study reports the enhancement of the oral bioavailability of ibrutinib (IBR), a poorly soluble anticancer drug in Wistar albino rats. Methods: IBR loaded nanoparticles were formulated through the nanoprecipitation method by utilizing poly lactide-co-glycolide (PLGA) as a safe, biodegradable and biocompatible polymer, and poloxamer or pluronic 127 as a stabilizer. Animals were administered with a dose of 10 mg/kg of IBR suspension or an equivalent amount of IBR loaded nanoparticles. Plasma samples were extracted and analyzed by state of the art UPLC-MS/MS technique. Pharmacokinetic (PK) parameters and bioavailability were calculated by non-compartmental analysis. Results: There was an approximately 4.2-fold enhancement in the oral bioavailability of IBR-loaded nanoparticles, as compared to the pure IBR suspension. The maximum plasma concentration (Cmax; 574.31 ± 56.20 Vs 146.34 ± 5.37 ng/mL) and exposure (AUC; 2291.65 ± 263.83 vs 544.75 ± 48.33 ng* h/mL) of IBR loaded nanoparticles were significantly higher than those exhibited through pure IBR suspension. Conclusion: The outcomes of the present study suggested the potential of PLGA nanoparticles in the enhancement of bioavailability and the therapeutic efficacy of IBR.