Current Pharmaceutical Analysis - Volume 14, Issue 4, 2018

Background: 7, 8-dihydroxyflavone (7, 8-DHF) has been discovered to be a TrkB receptorspecific agonist and demonstrated remarkable therapeutic efficacy in vivo. These promising findings prompted the current pharmacokinetic study, in which we first established a practical LC-MS quantitative method for 7, 8-DHF then applied it in a pilot pharmacokinetic study in rats. Methods: A simple HPLC-MS system was employed for the determination of 7, 8-DHF in rat plasma after the plasma samples were extracted with ethyl acetate by vortex-mixing. The method was thoroughly validated regarding accuracy, precision, sensitivity, linearity, selectivity, recovery, and stability. Results: This method exhibited linearity within the concentration range of 9.024 (LLOQ) and 1128 ng/mL. The accuracy ranged from 87.9 to 105.6% of the actual values. The intra- and inter-day precisions ranged from 1.7 to 5.1% and from 2.2 to 4.4% (RSD, %). In the pharmacokinetic study, 7, 8-DHF was detectable in rat plasma for up to 24 h, with a terminal half-life of 22.8 h. Conclusion: Overall, the results showed that this method was fully validated and successfully applied to a pilot pharmacokinetic study, which will facilitate the determination of the pharmacokinetic profile of 7, 8-DHF in future animal studies.

Background: Fraxetin is mainly an active ingredient of Fraxini Cortex which is an important and traditional Chinese medicine for clinical applications, has heat-clearing, dispelling dampness, antitussive and anti-asthmatic effects. Fraxetin has a variety of pharmacological activities. Method: The chromatographic separation was carried out on a Zorbax Eclipse XDB C18 column. The mobile phase consisted of acetonitrile (0.1 % formic acid) and ammonium acetate (0.1 % formic acid). Samples were prepared with protein precipitation. Result: The method had good linearity within 10 - 2500 ng/mL. Both intra- and inter-assay precisions were acceptable and ≤ 8.5 %. Extraction recovery was 75.9 - 89.5 %, and the lower limit of quantification for fraxetin was 10 ng/mL. This proposed method was successfully used to study the pharmacokinetic and bioavailability of fraxetin after 5 and 25 mg/kg fraxetin were administered to rats via intravenous and oral routes, respectively. The half-life (t1/2) of fraxetin for i.v was (3.86 ± 0.65) h. The t1/2 of fraxetin for p.o was (4.41 ± 1.79) h. The bioavailability of fraxetin is 12.6%. Conclusion: The results provided some useful information for the future development and clinical medication of fraxetin.

Objective: A simple and rapid method for the measurement of paracetamol in oral pharmaceutical preparations by a commercial glucometer was proposed. Methods: The established method was successfully applied to the determination of paracetamol in oral solid preparations, and exhibited the excellent capability of resisting interference from co-existing excipients. The commercial disposable test strips of hand-held glucometer showed high consistency of electrocatalytic activity toward the oxidation of paracetamol between each other. Results: The linear range was from 0.2 mg/ml to 3.5 mg/ml with the detection limit of less than 0.2 mg/ml. The results demonstrated that the test strips can be used as a sensitive, selective and low-cost sensor for amperometric determination of paracetamol in oral solid dosage forms. Conclusion: The unique application of household glucometer presented a convenient home-use tool for the paediatric subministration of the home medicine in common use.

Background: Clorsulon is a drug substance in other words Active Pharmaceutical Ingredient (API). It is listed in the United States Pharmacopoeia (USP). The related substances analysis as per USP is by Thin Layer Chromatography (TLC). It has been extremely difficult for the analyst to take decision on quality as the test was not able to reproduce the result and hence the purity by HPLC was planned. It was observed from the HPLC analysis that there was presence of one unknown impurity at Relative Retention Time (RRT) of about 1.15 minute. The same impurity was observed in several batches and resulted out of specification (OOS). The light is thrown to investigate the impurity and establish the impurity profile. Objective: Any unknown impurities present in the drug substance are toxic to animal safety and affect the drug efficacy. The research work was aimed to identify and characterize the impurity and understand the structure. It helps toxicological evaluation and based on level of toxicity, the limit for the specific impurity is arrived and recommended for monograph. This work helps pharmaceutical manufacturers and regulators to comply with qualification requirements of both USP and VICH (International cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products). Methods: HPLC method was used for related substances analysis. Preparative HPLC was used to isolate the impurity. LC-MS was used to identify the mass and further NMR was used for characterization. Results: The impurity isolated by preparatory HPLC was targeted and confirmed by spike study by regular HPLC method. The purity of the impurity was 96.9%. The molecular formula of the impurity is C9H10Cl3N3O4S2 and Mass is 394.38. The NMR data of both 1H and 13C characterization deduce the structure of the compound as “4-(Methylamino)-6-(trichloroethenyl) benzene-1, 3-disulfonamide”. Conclusion: The extensive investigational data confirm the structure of the impurity. The specification limit for this impurity is applied based on toxicological data.

Background: Tolterodine was quantitatively determined using variable analytical methods including; spectrophotometric and chromatographic methods. Spectrophotometric methods are usually fast, easy to handle, cost effective and very reliable for the routine assay during in process quality control procedures. Eosin Y has been used to determine several basic compounds through the formation of colored complexes. In this research work, spectrophotometric method is developed to determine Tolterodine in bulk and in tablet via ion pair complex with Eosin as complexing agent. Methods: The method developed in this work was based on the formation of a colored binary complex between the tertiary amine group in (TOL) molecule and the hydroxyl group in Eosin Y molecule. The absorbance of the chloroform extractable complex was measured at 530 nm using spectrophotometric technique. Result: The method obeyed Beers Lambert law and the linearity range was 18.6 -58.2 μg mL-1. The calibration curve equation is Y=9.28X + 0.07 with a linear regression of 0.992. The method was applied for the assay of (TOL) in commercially available tablets. Conclusion: Sensitive, specific, accurate and cost effective analytical method was developed in this work. It was found that the method could be used for a quick assay of the Tolterodine tartrate in bulk and in pharmaceutical dosage form for research and quality control purposes.

Background: Protodioscin, a furostanolic saponin type phytocompound, is claimed to have aphrodisiac and anticancer activities. Determination of protodioscin is challenging problem since it lacks chromophoric or florophoric groups. Pharmacokinetic data after administration of protodioscinrich extract prepared from Trigonella foenum-graecum in rabbit was not yet reported. A selective and sensitive LC–MS/MS method with solid phase extraction was developed and validated to quantify the concentration of protodioscin in rabbit plasma. Methods: Chromatographic separation was accomplished by using a Shiseido Spolar C18 column (150.6 mm, 5μ) with a mobile phase consisting of 0.2% formic acid in Milli-Q water-acetonitrile (40:60, v/v) at a flow rate of 0.5 mL min-1. Digoxin was used as an internal standard. Samples were prepared by solid phase extraction using Orochem C18 cartridges. A triple quadrupole tandem mass spectrometer with ESI was used as a detector operated by multiple reaction monitoring in positive ion mode with m/z 1031.3→869.2 and m/z 798.4→651.7 for protodioscin and internal standard, respectively. Results: The calibration curve was linear (r2 ≥ 0.99) over the concentration range of 0.1 to 100 μg/mL. The precision, accuracy and stability experiments results were meeting the acceptance criteria. Conclusion: The method can be applied to study the pharmacokinetic parameters of protodioscin in rabbit.

Purpose: To develop two PVC membrane sensors for determination of ciprofloxacin (CIP). Method: Beta - or γ-cyclodextrin was investigated as electroactive material for the formation of sensor 1 and 2 in the presence of potassium tetrakis (4-chlorophenyl) borate (KTpClPB) as ion additive. Results: The investigated sensors appeared fast, accurate, sensitive and showed near-Nernstian response (50 mV/ decade) with a calibration range for CIP (1 0-3 - 80-6 and 1 10-3 - 1.00-5 mol L-1). The detection limits were 90-6, and 80-6 mol L-1 for β- or γ- cyclodextrin sensor, respectively. The measuring pH of the developed sensors was in 3 - 6 range. Selectivity coefficient of the proposed methods indicates that both sensors showed a high selectivity for CIP in the presence of many ions and different common drug excipients. The proposed mechanism is based on inclusion complex or host-guest interaction. Conclusion: The advantages of the developed sensors are that it is accurate, selective, sensitive and cost less compared with published instrumental methods of analysis. The proposed sensors have been utilized for quantification of CIP in bulk and its formulation with good reproduicibilty compared with British pharmacopeia method.

Background: Penthorum chinense Pursh has a long history as a traditional medicine for the treatment of liver diseases in China. Despite its common use, investigations on the hepatoprotective components of P. chinense have not been carried out clearly. Experiment: Four extraction methods related to reflux, cold-soak, microwave and ultrasonic extraction were compared for optimum condition, combined with in vitro antioxidant activities and in vivo hepatoprotective effects to explore the chemical principles responsible for its liver protective effects. Result: Of four methods, the reflux extraction displayed the highest efficiency, producing the most abundant flavonoid of 82.36 mg/g (as rutin equivalents). Furthermore, among the 10 flavonoid constituents identified with their mass spectrometry data, HPLC analysis showed the contents of six major ones from reflux extraction were significantly higher, namely protocatechuic acid (1), catechin (2), 2,6- dihydroxyacetophenone-4-O-[4',6'-(S)-hexahydroxydiphenoyl]-β-D-glucose (3), pinocembrin-7-O-[3- O-galloyl-4'',6''-hexahydroxydiphenoyl]-β-D-glucose (PGHG) (4), quercetin-3-O-α-L-rhamnoside (5) and thonningianins A (Th A) (6). Compounds 2, 4, 5 and 6 exhibited potent antioxidant activity, while compounds 4, 5 and 6 remarkably prevented the elevation of alanine aminotransferase in an alcoholic liver injury mouse model. Hence, PGHG, quercetin rhamnosides and Th A could be considered as key active biomarkers of P. chinense for the treatment of hepatitis.

Background: In this work, we developed and validated a simple, rapid and accurate reverse- HPLC for the multi-determination of enrofloxacin (EFX), ciprofloxacin (CFX), and oxytetracycline (OXY) in raw materials and veterinary pharmaceutical formulations. Method: Optimum chromatographic separations among these three analytes have been achieved within 3 min employing an Agilent Poroshell 120 EC-C18 column (100 mm 3.0 mm, 2.7 μm) as the stationary phase using a mobile phase composted of water:acetonitrile (87: 13, v/v) plus 0.1% trifluoroacetic acid, flow rate at 3 mL min-1 and detection at 280 nm. Result: The method resulted in excellent separation (Rs > 3) and good efficiency (theoretical plates > 5000) for all of the analytes. The method showed acceptable results in the procedure of validation according with ICH and USP guidelines with respect to selectivity, linearity, limits of detection and quantitation, accuracy, precision and robustness. Finally, the developed method can be applied in the determination of EFX, CFX and OXY in routine quality control of raw materials and veterinary pharmaceutical formulations.

Background: Daptomycin (DPT) is the first lipopeptide antibiotic available for commercialization, approved by FDA in 2003 and not included in any official compendia. Introduction: A simple capillary zone electrophoresis method (CZE) to assay DPT injection was developed according to international guidelines. Methods: The method employed a 15 mmol L-1 pH 8.0 phosphate buffer and acetonitrile (85:15) as background electrolyte, with a voltage of 27 kV, hydrodynamic injection (50 mBar/5 s), and detection at 223 nm. The separation was achieved in a fused silica capillary with 40 cm of effective length, at 22°C, and acetylsalicylic acid was used as internal standard. The specificity was evaluated through a stress test combined with the PDA detector. As a result, the method was specific, even in the presence of degradation products. Detection was assessed around 5.5 min and the method was linear in the range of 20-120 μg mL-1 (r=0.9989). Results: The results also indicated the precision (RSD values of repeatability and intermediate precision < 2%), and accuracy (mean recovery of 101.26%) of the method. By a full factorial design 23, it was observed that none of the single factors or the combination of them affected the DPT assay, confirming the method robust. Conclusion: The method proved to be suitable to determine daptomycin injection, in quality control routine assays or in stability studies, and represents an environmentally friendly method.

Objective: To devise a rapid, simple, selective and sensitive High Performance Liquid Chromatography (HPLC) method for simultaneous estimation of hypertension drug Valsartan and diabetic drug Gliclazide in bulk and dosage forms. Methods: Valsartan and Gliclazide were measured using the HPLC method with UV detector at 234 nm wave length. Drug analytes were separated on 5μm inertsil C18 column (4.6x250mmx5μm) using 55:45 v/v 10mM phosphate buffer pH 4.8, acetonitrile and 100μl of triethanolamine as mobile phase at a flow rate of 1 ml/min. Results: Chromatograms of high resolution were obtained and the retention times of Valsartan and Gliclazide were 2.50 and 3.71 minutes respectively. The method was linear over the concentrations ranging from 2-20μg/ml for Valsartan and 3-15μg/ml for Gliclazide respectively. The percentage assays of Valsartan and Gliclazide in active pharmaceutical ingredient were found to be 99.27 % and 98.55 % respectively. The devised method was validated by determining its accuracy, precision and system suitability. Conclusion: The results showed good reproducibility and are within the acceptable limits. It was demonstrated that the method can be successfully applied for the routine determination of Valsartan and Gliclazide in bulk and in its pharmaceutical dosage forms.

Background: A mixture of aspirin and dipyridamole has been proved to significantly reduce the stroke recurrence. As the use of mixture of aspirin and dipyridamole is increased, the accurate concentration of each component should be determined to avoid side effects and to improve the treatment process in pharmaceutical preparation. Objective: The goal of the present study is the simultaneous determination of aspirin and dipyridamole mixtures in pharmaceuticals using the chemometrics methods. Methods: The simultaneous spectrophotometric determination of aspirin and dipyridamole was carried out using the genetic algorithm as feature selection, coupled with partial least squares for regression analysis. Results: The linearity range of calibration curve was obtained over the range of 30-250 and 1-20 μg·mL-1 for aspirin and dipyridamole, respectively. The results indicated that the developed methods are of high accuracy and can be used as a simple and fast techniques for analyzing of these compounds. Conclusion: The developed techniques are simple and with high precision and accuracy can be used in real samples for pharmaceutical formulation.