Current Pharmaceutical Analysis - Volume 15, Issue 1, 2019

Background: Synthetic glucocorticoids like Betamethasone (BE) are used in the treatment of inflammatory diseases due to its effective and potent effect. BE is available in three different esters: dipropionate (BD), sodium phosphate (BP), and acetate (BA). BD/BP combined in suspension has antiinflammatory, anti-allergic, and anti-rheumatic effects. The aim of this study was to describe the bioavailability of a single-dose of the injectable formulation of BP/BD in healthy Mexican subjects. Methods: This was a randomized, open-label, longitudinal, not therapeutic, single-dose trial of an intramuscular administration of BD/BP (5 mg/2 mg), in healthy Mexican subjects under fasting condition. Twenty-six healthy Mexicans volunteers of both genders who were between the ages of 18 and 45 were enrolled in the study. Results: From non-compartmental estimation of data, it was observed that the BE highest mean concentration was 15.70 ± 3.93 ng/mL reaching it in 2.83 ± 1.35 h. The values of elimination half-life were 10.89 ± 2.02 h. No clinically significant adverse effects were presented during the study. Conclusion: The reported PK parameters for BE suggest that the BD/BP suspension has a similar release velocity in Mexican healthy subjects compared with previous studies.

Background: In order to realize current aminoglycosides supervision in food and environment, our team improved the sensitivity and separation efficiency of the portable ITO detector, based on the technology of microchip capillary electrophoresis and contactless conductivity detection. Experiment: Parameters (the separation voltage, buffer concentration, electrodes gap, elicitation frequency, elicitation voltage) were optimized for the detection of three aminoglycosides, gentamicin, kanamycin and streptomycin and the separation of their mixture in background electrolyte consists of 2-(N-Morpholino) ethanesulfonic acid (MES) and L-Histidine (His). The enhanced method was also applied to other types of aminoglycosides. Results: Under optimal conditions, the monitoring of three types of aminoglycosides obtained such a sensitive response that the limits of detection of gentamicin sulfate, kanamycin sulfate and streptomycin sulfate were calculated as 3.1 μg/ml, 0.89 μg/ml and 0.96 μg/ml, at signal-to-noise ratio 3, respectively. In addition they got separated completely from each other only in 40 s. The results of other varieties of aminoglycosides including tobramycin sulfate and amikacin sulfate also met the standard. Conclusion: We successfully proposed here an unprecedentedly portable, miniaturized and rapid microchip capillary electrophoresis contactless conductivity detection system to realize current aminoglycosides supervision in food and environment.

Background: Pantoprazole (PTZ) and Levosulpiride (LS) were proven as effective agents for the treatment of Gastro-Esophageal Reflux Disease (GERD). It is a complex motor disorder that results in regurgitation of the gastric contents into the lower esophagus with consequent symptoms such as heart burn, retrosternal pain, dysphagia and belching. Methods: A rapid, sensitive, selective and specific liquid chromatography- electro spray ionization tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous quantification of Pantoprazole (PTZ) and Levosulpiride (LS) in spiked Human Plasma. The method utilized SPE as sample preparation technique and the analysis was carried out on a HPLC system utilizing electro spray ionization as interface and triple quadrupole mass analyzer for quantification in MRM possitive mode. Iloperidone was used as internal standard (IS). Chromatographic separation was performed on a Phenomenex C-18 Column (4.6 mm x 50 mm, 5μ) with an isocratic elution mode utilizing a mobile phase composition of Solution containing a mixture of 70 volumes of acetonitrile: 30 volumes of methanol and 10mM ammonium formate (pH 4.0) at the ratio of 80:20 % v/v. The flow rate was maintained at 0.3 mL/min. Results: PTZ, LS and IS were detected and quantified with proton adducts at m/z 383.37→200.00, m/z 341.42→112.15 and 426.48→261.00 respectively. The linearity and range was established by fortifying blank plasma samples in the concentration range of 3.5-2000 ng/mL for PTZ and 3.0-2400 ng/mL for LS. The correlation coefficient (r2) was found to be ≥ 0.993 for PTZ and (r2) ≥ 0.990 for LS. The lower limit of quantification for PTZ was 3.5 ng/mL and LS was 3.0 ng/mL. The intra and inter day precision and accuracy for PTZ and LS were within the limits fulfilling the international acceptance criteria. PTZ and LS were found to be stable throughout three freeze-thaw cycles, bench top and short term stability studies. Conclusion: The proposed validated LC-MS/MS method offers a sensitive quantification of PTZ and LS in spiked human plasma and can be utilized for the quantification of PTZ and LS in real-time samples.

Background: Ephedrine, an alpha/beta-adrenergic agonist, is one of the most common doping agents not only among athletes but also the ordinary people, therefore its detection at low trace levels with a sensitive and cost effective method has become a priority to investigate many analytical methods. Objective: In this work, solvent bar microextraction followed by high-performance liquid chromatography (HPLC-UV) was used for extraction and determination of ephedrine at low trace levels from urine samples at optimum condition. Methods: In this study, a designed experiment was carried out using solvent bar microextraction technique, which has been proved to be a green method. This method requires three phases consisting of a donor phase with an alkaline pH, an acceptor phase with an acidic pH, and organic solvent to impregnate the pores of the hollow fiber. The obtained results were used for estimating the optimum ranges for each parameter, analyzing the effect of different parameters, simultaneously. Results: Under optimized circumstances, the preconcentration factor was 129. The calibration curves represented good linearity for urine sample with coefficient estimations higher than 0.9991. The limit of detection and quantitation for ephedrine were 16.7 μg L-1 and 50 μg L-1, respectively. The relative standard deviations of analysis were 3.5% within a day (n=3) and 4.1% between days (n=9). Conclusion: According to the results and previous studies, it can be concluded that the preconcentration factor for ephedrine was the best result ever reported considering selectivity and cost-effectiveness.

Introduction: This paper presents the development and validation of a novel, fast, sensitive and accurate high performance liquid chromatography (HPLC) method for the simultaneous quantitative determination of dibucaine HCl, fluocortolone pivalate and fluocortolone caproate in pharmaceutical preparations. Experiment: Development of the chromatographic method was based on an experimental design approach. A five-level-three-factor central composite design requiring 20 experiments in this optimization study was performed in order to evaluate the effects of three independent variances including mobile phase ratio, flow rate and amount of acid in the mobile phase. Conclusion: The optimum composition for mobile phase was found as a methanol:water:acetic acid mixture at 71.6 : 26.4 : 2 (v/v/v) ratio and optimum separation was acquired by isocratic elution with a flow rate of 1.3 mL/min. The analytes were detected using a UV detector at 240 nm. The developed method was validated in terms of linearity, precision, accuracy, limit of detection/quantitation and solution stability and successfully applied to the determination of dibucaine HCl, fluocortolone pivalate and fluocortolone caproate in pharmaceutical topical formulations such as suppositories and ointments.

Introduction: Quantitative NMR spectroscopy (qNMR) is a rapid, simple and efficient method for the assay of sulfasalazine (SSZ) in commercial tablet formulation. Materials and Methods: The qNMR method was demonstrated using maleic acid as an internal standard and DMSO-d6 as a solvent. The characteristic signals of SSZ at δ 8.36 ppm and maleic acid at δ 6.28 ppm were quantified. The reliability of the quantification method had been implemented successfully in validated experiments including specificity and selectivity, linearity, recovery, precision concentration rang, limit of detection (LOD), limit of quantification (LOQ), stability and robustness. Conclusion: The method was found to be liner (R2 = 0.9991) from 8.62 to 20.14 mg/0.6 mL DMSO-d6 in the drug concentration range. The maximum relative standard deviation (RSD) of recovery and precision were tested to be 0.59% and 0.65%, respectively. The LOD and LOQ were determined to be 0.02, 0.07 mg/mL, respectively. The RSD of stability was 0.05%. The robustness was demonstrated by changing four different parameters with the maximum difference less than 0.9%. In addition, the result of qNMR showed in good agreement with the HPLC and UV methods. Based on the experiments, the developed method was successfully applied to the determination of SSZ in commercial tablet.

Background: Piroxicam is a non-steroidal anti-inflammatory drug. The prevailing clinical use and investigation of piroxicam necessitate a rapid and sensitive method for its determination. A carbon ionic liquid electrode, fabricated using graphite and the ionic liquid 1-octylpyridinium hexafluorophosphate (OPFP) was used as an electrochemical sensor for piroxicam determination. Methods: The surface of the proposed electrode was characterized by scanning electron microscopy. Cyclic voltammetry (CV) was applied to study the oxidation of piroxicam and to acquire information about the reaction mechanism. Differential pulse voltammetry was also used as an analytical technique for quantification of the sub-micromolar concentration of piroxicam. Results: One oxidation peak at 0.55V was observed at CILE. The oxidation peak at the CPE was weak, while the response was notably increased at the CILE. The proposed electrode exhibited interesting sensitivity towards the determination of piroxicam and the anodic peak current versus piroxicam concentration was linear in the ranges of 0.2-60 μM. The detection limit of 40 nM was achieved. Conclusion: The electroxidation process was irreversible and revealed adsorption controlled behavior. The method was successfully applied for the determination of piroxicam content in pharmaceutical samples.

Background: Flunixin is a Non-Steroidal Anti-Inflammatory Drug (NSAID), because it can effectively alleviate the organism of pyrexia, inflammation and pain, it has been widely used in veterinary practice. In order to better study flunixin in the body's absorbed, distributed, metabolized and excreted, our team developed a UPLC-MS/MS method for determination of flunixin in swine plasma, urine and feces. Methods: Flunixin was extracted from the sample by liquid-liquid extraction and cleaned-up using a mixed-mode Oasis MCX solid-phase extraction column. Analysis was performed on UPLC-MS/MS operating in Multiple Reaction Monitoring (MRM) mode. Internal standard was used for quantitation of target drug. Results: Recoveries of fortified samples ranged from 90.2% to 101.4%, with Relative Standard Deviations (RSD) lower than 17.0%. The Limits Of Detection (LODs) and Quantification (LOQs) in plasma were 0.25 and 0.5 μg L-1, in urine were 0.25 and 0.5 μg L-1, and in feces were 0.5 and 1 μg kg-1, respectively. This validated method was successfully applied to the determination of flunixin in real samples. The half-life of flunixin after the last dose in pigs was 7.37±1.60 h after intramuscular administration of 2.2 mg/kg of flunixin, and approximately 6.8% and 1.9% of the administered dose was excreted as parent compound in urine and feces respectively. Conclusion: The developed UPLC-MS/MS method for determination of flunixin in swine plasma, urine and feces was validated and successfully applied to monitor flunixin from real samples.

Background: Capecitabine is an orally available prodrug of 5-flurouracil used in the treatment of breast cancer, metastatic colorectal cancer and stage III colorectal cancer. Various studies have reported the HPLC, HPLC-MS, MS/MS methods for estimation of capecitabine. However, till date HPTLC method for estimation of capecitabine and its validation is not reported in tablet dosage form. Introduction: Presented study deals with the development and validation of stability indicating high performance thin layer chromatography method for the determination of Capecitabine in tablet dosage form. Methods: The method was developed using precoated HPTLC plates with silica gel 60 F254 as stationary phase and toluene-methanol the ratio of 7.5:2.5 v/v as the mobile phase. Capecitabine (RF 0.48 ± 0.03) and its degradation products were well resolved. The wavelength selected for study was 240 nm. The method was linear in the concentration range 50–550 ng/band with a correlation coefficient of 0.994. The repeatability for six samples was 1.25% RSD. The intraday and interday precisions were 1.46-1.71%RSD and 1.31-1.67% RSD, respectively. The accuracy (recovery) was found to be in the range of 99.10-101.23% with LOD and LOQ were found to be 0.650 and 1.765 mg/band. The mean content of drug in tablet dosage form was found to be 101.51% with a % RSD of 1.20. The drug was subjected to stress conditions such as hydrolysis, oxidation, photolysis, and heat. Results: Degradation products produced as a result of the stress conditions did not interfere with the detection of Capecitabine; therefore, the proposed technique can be considered stability-indicating. Capecitabine did not degrade under thermal and photolytic conditions but showed degradation under acidic and alkaline conditions with 15 and 11% decompositions respectively. Conclusion: The developed method was found to be facile, simple, specific, precise, and stabilityindicating. It can be employed for the routine analysis of capecitabine in tablet dosage form.

Background: Ferruginol (FRGN) exhibits a broad range of pharmacological properties which make it a promising candidate for chemoprevention. However, little is known about its absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties. Methods: A rapid, sensitive and specific HPLC-DAD method was established to quantify FRGN in the plasma and tissues of Wistar rats. After extraction of FRGN with ethyl acetate (EtOAc), chromatographic separation was performed on a YMC ODS C18 column (250 4.6 mm I.D., 5 μm) with a mobile phase consisting of methanol-water (92:8, v/v) at a flow rate of 0.9 mL/min. Detection was conducted with a wavelength of 273 nm at 25 °C. Results: The calibration curves for FRGN were linear in the concentration range of 0.5-20 μg/mL for plasma, 0.5-10 μg/mL for heart, liver, spleen, lung, kidney, stomach, intestine, brain and muscle. After three cycles of freezing and thawing, the concentration variations were within ± 7% of nominal concentrations, indicating no significant substance loss during repeated thawing and freezing. The assay was applied to pharmacokinetic and tissue distribution study in rats. Results suggested that lung, heart, liver, spleen and kidney were the major distribution tissues of FRGN in rats, and FRGN could permeate the blood-brain barrier to distribute in the brain of rats. Conclusion: The information provided by this research is very useful for gaining knowledge of the pharmacokinetic process and tissue distribution of FRGN.

Introduction: A rapid, sensitive and convenient ultra-performance liquid chromatography with tandem mass spectrometric detection (UPLC-MS/MS) method has been validated and applied to the simultaneous determination of kirenol, rosmarinic acid and caffeic acid after oral administration of the extract of Manxingshizhen preparation in rat plasma. Materials and Methods: Puerarin was selected as the internal standard (IS). The plasma sample preparation was pretreated by liquid-liquid extraction of the mixture with ethyl acetate. All analytes were simultaneously detected in multiple reaction monitoring (MRM) mode via both the positive electrospray ionization (ESI+) and negative electrospray ionization (ESI-). In the experiment, all calibration curves revealed good linearity (r > 0.999). The LLOQ were between 0.80-2.00 ng/mL, respectively. Besides, the intra-day and inter-day precision ranged from 6.4 to 13.8%, respectively. Moreover, the accuracy was within - 11.4% and 12.8% for all the QC levels of all analytes. The extraction recoveries of the analytes and IS in plasma at three concentration levels ranged from 88.5 to 103.2%, moreover, the matrix effects of all the analytes and the IS were found to be satisfied with the acceptable range of 89.8%-101.7%. Meanwhile, the RSD values of stability met the requirement of not more than 15%. Furthermore, the pharmacokinetic parameters of three compounds were analyzed using concentrationtime profiles. Conclusion and Results: Plasma concentrations of the three compounds were determined up to 24 h after oral administration, and their pharmacokinetic parameters were in agreement with previous studies. The validated method was successfully applied in a pharmacokinetic study in rat plasma after oral administration of Manxingshizhen preparation.

Introduction: Tenofovir Disoproxil Fumarate/Lamivudine/Efavirenz Tablets are a pharmaceutical dosage form indicated for the treatment of Human Immunodeficiency Virus (HIV) infection. Methods: A simple, time efficient and stability indicating Reverse Phase Ultra Performance Liquid Chromatography (RP – UPLC) method was developed for the simultaneous determination of the three drugs present in the tablets. Mobile phase-A is 0.1% Trifluoro Acetic acid in water and mobile phase-B is acetonitrile in gradient elution mode. Flow rate at 0.4 mL/min was fixed in the method. Acquity BEH Phenyl (100 mm 2.1 mm), 1.7 μ column was used for the separation of analytes at 260 nm wavelength. The developed method has been validated as specified in International Conference on Harmonization (ICH) guideline. Forced degradation study was conducted for Tenofovir Disoproxil fumarate (TDF), Lamivudine (LAM) and Efavirenz (EFA) pharmaceutical tablets to identify the degradation behaviour of individual drugs under stress study. Results: The method was observed to be linear from 6-90 μg/mL concentration for TDF & LAM and 12-180 μg/mL for EFA with correlation coefficient values of more than 0.999. The method was found to be precise with RSD (Relative standard deviation) values of below 2% for replicate measurements. Recovery results were found to be between 99.0 and 100.8%. LOQ values for TDF, LAM & EFA were 0.041 μg/mL, 0.034 μg/mL & 0.053 μg/mL, respectively. Specificity of the analytical method was checked by injecting the stressed samples and evaluating the homogeneity of peaks using Photo Diode Array (PDA) detector. Peak purity results indicated that no interference was observed from degradation impurities for the quantification of TDF, LAM and EFA in the dosage form. The method was found to be robust under the deliberately modified chromatographic parameters. Conclusion: The developed UPLC method is stability indicating, successfully validated and suitable for quantification purpose.

Background: Therapeutic drug monitoring is recommended for patients taking vancomycin and teicoplanin to ensure pharmaceutical efficacy and prevent toxicity. Only few studies were reported regarding the simultaneous determination of vancomycin and teicoplanin in human plasma. Objective: The study aimed at developing and validating a Liquid Chromatography-Mass Spectrometry (LC-MS) method for simultaneous determination and therapeutic drug monitoring of vancomycin and teicoplanin in patients with severe infection. Method: Plasma was processed by protein precipitation extraction. The analytes were separated on a C18 column by gradient elution with 0.1% formic acid and acetonitrile as mobile phase and measured by electrospray ionization source in positive selective ion monitoring mode at m/z 724.7 (vancomycin), 940.7 (teicoplanin) and 329.0 (bergenin). The plasma samples (104) were obtained from patients who were taking vancomycin or teicoplanin for further analysis. Results: The calibration curves were linear within the range of 0.25–40 μg/mL for vancomycin, and 0.5-40 μg/mL for teicoplanin. Either inter- or intra-day precision was less than 10.01 %. The extraction recoveries ranged from 89.99 to 94.29% for vancomycin and from 39.83 to 40.16 % for teicoplanin. Vancomycin and teicoplanin in plasma were stable at various storage conditions. The measured mean trough concentrations were 12.313 μg/mL for vancomycin and 8.765 μg/mL for teicoplanin. Conclusion: This method was successfully applied to therapeutic drug monitoring of vancomycin and teicoplanin in patients. It is with great clinic value for monitoring and predicting the individual response of patients under treatment.