Current Pharmaceutical Analysis - Volume 15, Issue 2, 2019

Background: Lamivudine was approved by Food and Drug Administration of the United States for the treatment of both HIV and HBV infection, which has been widely used as monotherapy or a component of combination therapy in clinics in many countries and nationalities. Methods: In this paper, the recent chromatographic and mass spectrometry analytical methods for the determination of lamivudine individually or combination with other drugs simultaneously were presented. These methods were widely applied in pharmacokinetics studies, bioequivalence studies, therapeutic drug monitoring studies, cell and animal experiments. Conclusion: The review paper might provide references for determining lamivudine in biological fluids, the intracorporal process of lamivudine, and the clinical practice by monitoring plasma concentration of lamivudine in the future.

Microanalytical systems have aroused great interest because they can analyze extremely small sample volumes, improve the rate and throughput of chemical and biochemical analysis in a way that reduces costs. Microchip Electrophoresis (ME) represents an effective separation technique to perform quick analytical separations of complex samples. It offers high resolution and significant peak capacity. ME is used in many areas, including biology, chemistry, engineering, and medicine. It is established the same working principles as Capillary Electrophoresis (CE). It is possible to perform electrophoresis in a more direct and convenient way in a microchip. Since the electric field is the driving force of the electrodes, there is no need for high pressure as in chromatography. The amount of the voltage that is applied in some electrophoresis modes, e.g. Micelle Electrokinetic Chromatography (MEKC) and Capillary Zone Electrophoresis (CZE), mainly determines separation efficiency. Therefore, it is possible to apply a higher electric field along a considerably shorter separation channel, hence it is possible to carry out ME much quicker.

Background: Imatinib (IM) is a chemotherapy medication metabolized by CYP3A4 to Ndesmethyl imatinib (NDI), which shows similar pharmacologic activity to the parent drug. Although methods for determination of IM and/or NDI have been developed extensively, only few observations have been addressed to simultaneously determine IM and NDI in biological tissues such as liver, kidney, heart, brain and bone marrow. Methods: A validated LC-MS/MS method was developed for the quantitative determination of imatinib (IM) and N-desmethyl imatinib (NDI) from rat plasma, bone marrow, brain, heart, liver and kidney. The plasma samples were prepared by protein precipitation, and then the separation of the analytes was achieved using an Agilent Zorbax Eclipse Plus C18 column (4.6 100 mm, 3.5 μm) with gradient elution running water (A) and methanol (B). Mass spectrometric detection was achieved by a triplequadrupole mass spectrometer equipped with an electrospray source interface in positive ionization mode. Results: This method was used to investigate the pharmacokinetics and the tissue distributions in rats following oral administration of 25 mg/kg of IM. The pharmacokinetic profiles suggested that IM and NDI are disappeared faster in rats than human, and the tissue distribution results showed that IM and NDI had good tissue penetration and distribution, except for the brain. This is the first report about the large penetrations of IM and NDI in rat bone marrow. Conclusion: The method demonstrated good sensitivity, accuracy, precision and recovery in assays of IM and NDI in rats. The described assay was successfully applied for the evaluation of pharmacokinetics and distribution in the brain, heart, liver, kidney and bone marrow of IM and NDI after a single oral administration of IM to rats.

Background: Jingzhiguanxin (JZGX) tablet, a traditional Chinese prescription, is commonly used for treating coronary heart disease and angina pectoris in the clinic. There are six active components (Danshensu (DSS), Protocatechuic aldehyde (PD), Paeoniflorin (PF), Ferulic acid (FA), Salvianolic acid B (Sal B) and Tanshinone IIA (TA)) in JZGX tablet. Objective: In this paper, a simple and reliable method was used for simultaneous determining the six active components by high-performance liquid chromatography coupled with diode array detector (HPLC-DAD). Methods: These six active components were separated on an Agilent Zorbax Eclipse XDB-C18 column (150 mmx4.6 mm, 5 μm) at 30 °C. Acetonitrile (A), methanol (B) and 0.5% H3PO4 aqueous solution (C) were used as mobile phase for gradient elution. The flow rate was 1 mL/min and the detection wavelengths were set at 280 nm for DSS, PD and Sal B, 230 nm for PF, 320 nm for FA and 270 nm for TA, respectively. Results: All of the six components showed good linearity regressions (r2≥0.9997) in the detected concentration range. The recovery rates and coefficient of variation (CV) for all analytes were 98.66%- 100.18% and 0.75%-1.89%, respectively. This method was successfully applied to simultaneously determine the six components in JZGX tablet from different batches and manufacturers. Conclusion: The validated method can be used in routine quality control analysis of JZGX tablet without any interference.

Background: The carbamide peroxide is the most commonly active ingredient used for home dental whitening products, its quantification in pharmaceutical products is of extreme importance due to the relation with the products potency and the previously related low carbamide peroxide stability. Once, there is only one official carbamide peroxide determination based on iodometric titration, this method is time-consuming and generates a lot of residues. The aim of this study was to carry out development and validation of a simple and fast ultraviolet spectrophotometer assay to quantify an innovative dental whitening gel. Methods: The proposed method was validated according international conference on harmonization guideline. Procedure is based on the iodide/iodine redox chemistry; iodine released through the action of hydrogen peroxide of carbamide peroxide with ultraviolet detection at 350 nm. Results: The procedure was linear in the concentration range of 1.0-4.0 μg/mL, specific to the excipients, robust for the evaluated parameters (variation of wavelength (± 5 nm); reagent addition (± 10%)), showing the results of RSD 1.88 and 0.39% respectively. Repeatability precision was RSD = 1.42%, with accurate RSD = 2.15% by adding reference solution. The assay used only water as solvent for sample preparation. In comparison to the pharmacopeial method, the latter is more time-consuming, as it generates a lot of residues, and it could not quantify small CP dosages. Conclusion: Thus, the proposed method was proved to be suitable to determine carbamide peroxide during the development and characterization of nanoparticle formulations in the present study.

Background: Lipid-Core Nanocapsules (LNC) containing co-encapsulated ρ-coumaric acid and coumarin are under development. However, there is a lack of analytical methods to assay these bioactives in nanoformulations. Objective: The aim of this study was to validate an LC analytical method for the simultaneous determination of ρ-coumaric acid and coumarin in lipid-core nanocapsules. Methods: The mobile phase was composed of acetonitrile:water (40:60 v/v) adjusted to pH 4 and a C- 18 reversed-phase column was used. Both bioactives were detected at 275 nm. Specificity, linearity, range, precision and accuracy of the method were assessed, according to the official requirements. Results: Nanocapsules containing ρ-coumaric and coumarin had monomodal particle size distribution, spherical-shape and Z-average size of 207 ± 2 nm. LC method was specific, linear (5 to 30 μg.mL-1), precise (RSD < 5%) and accurate (97 - 103%). It was applied to assay the content and encapsulation efficiency of the bioactive substances in LNC, which were close to 0.5 mg.mL-1 and 72%, respectively. Conclusion: The proposed analytical method is reliable for the simultaneous assay of ρ-coumaric acid and coumarin in nanocapsules and can be further used in their development.

Background: Quantification of preservatives in herbal formulation, simultaneously by high performance liquid chromatography analysis is very complex and involves series of steps including sample preparation, selection of suitable mobile phase and its validation for routine applications. Introduction: Application of Quality by Design (QbD) in the development of novel, simple, accurate and precise RP-HPLC method for concurrent quantification of quaternary preservatives in herbal formulation, focusses on development of robust method. Methods: Isocratic analysis was carried out using C18 column at 231 nm. Risk assessment studies were executed to determine the critical method parameters which were defined as acetonitrile volume in the mobile phase, volume of injection and orthophosphoric acid concentration in the mobile phase. The effect of the critical method parameters on critical method attributes, i.e. retention time, resolution and chromatographic optimization function was further evaluated by means of central composite design and the optimal conditions were determined through derringer’s desirability approach of multi-criteria decision making technique. Results: The method was statistically validated according to ICH guidelines having good resolution using optimized mobile phase, acetonitrile: 0.11% orthophosphoric acid in water (12.30: 87.70 % v/v) giving acceptable retention time i.e. 3.7128 ± 0.0138 of bronopol, 4.5106 ± 0.00542 of sodium propyl paraben, 10.7228 ± 0.029 of sodium benzoate and 12.252 ± 0.027 of sodium methyl paraben. Conclusion: Hence, the QbD based method development assisted in generating a design space with knowledge of all method performance characteristics leading to a better understanding of the method, and achieving desirable method quality.

Background: A gas chromatography and mass spectrometry (GC/MS) procedure was developed and validated for the evaluation and quantification of pregabalin (PGN) in pharmaceutical preparations. Introduction: Pregabalin is a γ-amino-n-butyric acid derivative used as an antiepileptic drug for the management of fibromyalgia, and has analgesic, anxiolytic, and anticonvulsant activities. Few studies have been reported on the determination of PGN content in pharmaceutical preparations involving gas chromatography - mass spectroscopy. Methods: Pregabalin was extracted with MSTFA/NH4F/β- mercapto-ethanol at 60°C for 30 min. The acquired derived molecule of pregabalin was identified by specific ion monitoring mode applying the analytical ions m/z 232 and 331. Propranolol was used as Internal Standard (IS). The following validation parameters were taken into consideration: precision, linearity, accuracy, stability, specificity, robustness, ruggedness, Limit Of Detection (LOD) and Limit Of Quantitation (LOQ). Results: The method was selective, precise, sensitive, linear and specific. The linearity of the method was between 3.5 and 300 ng/ml. The precise values were ≤ 3.62% of both intra- and interday validation. The LOD accurate values for Intraday and interday validation were in the range of -0. 25 -2.05%. While LOQ accurate values for intraday and interday were 1.5x10-6 and 3.5 x10-6mg/ml, respectively. Conclusion: Therefore, the developed GC-MS method was effectively implemented to identify PGN in a pharmacological preparation.

Background: In this study, we have developed a novel, rapid enzymatic hydrolysis method for conversion of omega lipids (omega fatty acid triglycerides, phospholipids, omega conjugates) in to free fatty acids at room temperature using lipase and esterase enzymes. Objective: To develop simple enzymatic hydrolysis and rapid sample extraction method for quantification of free (un-esterified) and conjugated (esterified) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) to provide the total EPA and DHA lipids present in human plasma. Quantification of total EPA/DHA was performed using liquid chromatography and tandem mass spectrometer instrument. Methods: The plasma sample is digested with lipase and esterase enzymes and extracted by using combined precipitation and liquid-liquid techniques. The LC-MS/MS method was optimized using EPA-D5 and DHA-D5 as labeled internal standards for EPA/DHA respectively. The analytical method is validated, utilized for simultaneous quantification of total EPA and DHA lipids in plasma collected from healthy human volunteers clinical study. Results: The reproducibility of the established enzymatic hydrolysis method was demonstrated by incurred sample reanalysis and the results for total EPA and DHA lipid were 93.33% and 96.67% respectively. The pharmacokinetic and statistical analysis was performed using baseline corrected concentration of total EPA and DHA lipids. Conclusion: The enzymatic hydrolysis method for conversion of omega fatty acid triglycerides, phospholipids, omega conjugates in to free fatty acid was reported first time for the quantitative application. The shorter time for sample workup procedure, simple enzymatic hydrolysis at room temperature and 3 minutes chromatography run time are well suitable for bioavailability/ bioequivalence studies.

Introduction: Deltaline, an aconitine-type alkaloid, was detected in mouse blood using an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method, and the pharmacokinetics of deltaline following intravenous administration in mice was studied. Materials and Methods: The gelsenicine was used as the internal standard (IS). Deltaline and IS were eluted at a flow rate of 0.4 ml/min and separated on a UPLC BEH C18 column by gradient elution using acetonitrile and 10 mmol/L ammonium acetate (0.1% formic acid) as a mobile phase. The following transitions were obtained at m/z 508.2→75.0 for deltaline and m/z 327.1→107.8 for gelsenicine in multiple reactions monitoring mode. Acetonitrile was used to precipitate protein. Six mice after intravenous administration of a single dose of deltaline (1 mg/kg), 20-μL blood samples from each mouse were collected from the tail vein. Results: The UPLC-MS/MS method was sensitive and linear (r>0.995) with a lower limit of quantitation (LLOQ) of 0.1 ng/mL over the range of 0.1-500 ng/mL. Intra- and inter-day precisions were below 13%, the accuracy range was between 88.0% and 108.2%, the recovery was higher than 90.1%, and the matrix effect was between 102.9% and 108.1%. Conclusion: The method was sensitive, fast, specific, and has been successfully applied to a pharmacokinetic study of deltaline after intravenous administration.

Background: Cinacalcet is one of the second-generation calcimimetics which consists of a chiral center. The pharmacological effect of R-cinacalcet is 1000 times greater than that of the Scinacalcet. As mentioned in many literatures, 1-(1-naphthyl)ethylamine is used as the starting material for the synthesis of cinacalcet. The absolute structure of cinacalcet is influenced by the starting materials. Methods: We present the chiral separation of cinacalcet and its starting material, 1-(1-naphthyl) ethylamine along with one of its intermediates, N-(1-(naphthalen-1-yl) ethyl)-3- (3-(trifluoromethyl) phenyl) propanamide by high-performance liquid chromatography with chiral stationary phase and chiral mobile phase additives. Results: On vancomycin and cellulose tri 3,5-dimethylphenylcarbamate) chiral stationary phase, cinacalcet and 1-(1-naphthyl)ethylamine achieved enantioseparation under normal phase with addition of triethylamine additives, respectively. Meanwhile, 1-(1-naphthyl)ethylamine and N-(1-(naphthalen-1- yl)ethyl)-3-(3-(trifluoromethyl) phenyl) propanamide achieved enantioseparation on 1-napthalene vancomycin chiral stationary phase using D-tartaric acid, diethyl L-tartrate and diethyl D-tartrate as chiral mobile phase additives. Conclusion: The chiral recognition in our experiment was based on the hydrogen-bonding, dipoledipole and π-π interactions among the solutes, chiral stationary phases and chiral mobile phase additives. In addition, the space adaptability of chiral stationary phases also affected the separation efficacy.