Current Pharmaceutical Analysis - Volume 17, Issue 6, 2021

Aim/Background: In this study, a stability-indicating method of the anticancer agent cabazitaxel was developed and validated. This method will be used to determine the chemical stability of commercially available concentrate-solvent mixture cabazitaxel (Jevtana®) to examine the possibility of multi-dosing from the same product vial after storage. The impossibility to re-use leftovers today is contributing to unnecessary and significant financial waste. Methods: A forced degradation study of cabazitaxel was performed under different conditions to produce degradation products. Acidic, basic, oxidation, heat, and Ultraviolet (UV) light conditions were tested. The method to determine the stability was developed so that potential degradation products would be shown in the UV spectra after separation from cabazitaxel with a C18 column in a High- Performance Liquid Chromatography (HPLC) system. The only degradation product occurring during storage in room temperature and ambient light was identified by accurate mass Orbitrap Mass Spectrometry. Results: A stability-indicating method for cabazitaxel (Jevtana®) concentrate-solvent mixture has been developed. We demonstrated that this method can be applied to stability studies with the purpose of multi-dosing cabazitaxel from a chemical/physical stability perspective within the tested period of time and conditions. Conclusion: As an addition, the only naturally occurring degradation product found has been identified and a degradation reaction has been suggested.

Background: Xuezhikang capsule, which contains cholesterol synthase inhibitors and a large number of natural statins, is put in the clinical application of lipid-lowering and so on. However, the specific use of dose, lipid-lowering effect and the relationship between metabolites are to be further studied. Introduction: Metabonomics is the study of the relationship between the change of quantity and physiological changes from metabolites. At present, metabolomics has been widely used in drug development and testing. In this study, we developed a metabolomic method based on gas chromatographymass spectrometry (GC-MS) to find out hyperlipemia-related substances, and study the lipid-lowering mechanism of Xuezhikang. Methods: Fifty SD rats (220 ± 20 g) were given a high-fat diet. After four-weeks modeling, they were randomly divided into semi-control groups, high fat group, simvastatin intervention group and Xuezhikang intervention group (0.23, 0.69, 1.15 mg/kg, low, medium, high), each dosage in eight rats. The control group (rest eight rats) were given a normal diet, and no specific treatment. The rats were sacrificed at the end of the experiment. Results: The biochemical and body weight indexes of the normal control group and the high fat group were significantly different (P <0.05), which indicated that the model of hyperlipidemia was established success. There was a significant difference (P <0.05) between the Xuezhikang intervention group and high-fat control group (P <0.05), and hyperlipemia metabolomics related markers, oxalic acid, butyric acid, mannitol, glucose, glucuronic acid were found. Glucuronic acid and non-binding bilirubin combined with bilirubin, combined with some of the liver harmful substances, play a detoxification effect. Conclusion: The results of metabonomics showed that the high-fat group and the control group have significant differences. Mannose, glucose content is relatively stable, lipid metabolism in high-fat group stearic acid, palmitic acid levels decreased, suggesting that high-fat diet disorders rat body lipid metabolism. It is worth mentioning that the experimental evaluation of rats, such as biochemical indicators and pathological results are prompted to model success, Xuezhikang intervention effect is more significant, consistent with the expected.

Background: Fu Zheng Fang (FZF) is an important Chinese medicine prescription for tumor treatment in hospitals, which has two different types, traditional Chinese medicine (TCM) decoction pieces and TCM formula granules. Objective: This study aimed to determine the effective composition of the drug FZF. Methods: In this research, FZF decoction pieces and FZF formula granules were collected and their composition, determined by HPLC-Q-Exactive Orbitrap/MS, and multivariate statistical analysis, was applied to distinguish differential metabolite patterns between two groups. Results: A clear cut difference in the composition of the two groups was observed. 124 differential chemical compositions could be identified in the positive mode, while 59 differential chemical compositions could be identified in the negative mode. The differential chemical compositions were mainly concentrated in flavonoids, organic acids, fatty acids, amino acid compounds, and presenting different change rules, mainly involved in two metabolic pathways; the flavonoid biosynthesis, and flavone and flavonol biosynthesis. Conclusion: This study provides basic information that may be of use in the formulation of the drug in different dosages and in the examination of their efficacy.

Background: To control the potential presence of heavy metals in pharmaceuticals, the United States Pharmacopeia (USP) and International Conference on Harmonization (ICH) have put forth new requirements and guidelines. USP <232> and ICH Q3D specify 24 elemental impurities and their concentration limits in consideration of the permitted daily exposure (PDE) of different drug categories (oral, parenteral and inhalation). while USP <233> describes more information about sample preparation and method validation procedure. Objective: To establish and verify an ICP-MS method for the determination of 24 elemental impurities (Cd, Pb, As, Hg, Co, V, Ni, Tl, Au, Pd, Ir, Os, Ph, Ru, Se, Ag, Pt, Li, Sb, Ba, Mo, Cu, Sn, Cr) in ubenimex APIs according to USP/ICH guidelines. Methods: Samples were analyzed by ICP-MS after direct dissolution in diluted acid solution. All elements were detected in He/HEHe mode (except for Li, which was in No gas mode). Results: The spiked recoveries were within 80-120% except Hg (79.4% at 0.5J level in HEHe mode) and Cd (121.9% at 0.5J level in HE mode). The RSD of repeatability (N = 6) for all elements were < 7.0% and intermediate precision (N = 12) were < 9.0%. The correlation coefficients of linear (R) for 24 elements were all > 0.998. The Limits of Detection (LOD) were < 1 ng/mL except that Ni was 1.23 ng/mL in HEHe mode. The contents of 24 elements in 3 batches of samples were significantly lower than the actual target limit of ICH, while the highest content of Pd did not exceed 10 μg/g. Conclusion: The established method was proved to be simple, sensitive and accurate. It successfully applied to the elemental impurity determination in 3 batches of ubenimex APIs from different manufactories. This method also provided technical guidance for the determination of multiple elements in pharmaceutical products.

Background: Pedunculoside (PE) is a triterpene saponin from the barks of Ilex rotunda, a Traditional Chinese Medicine called Jiubiying, which is used for the treatment of cold and fever, tonsillitis, sore throat, acute and chronic hepatitis. Previous studies have confirmed that crude extract orally has a significant therapeutic effect on myocardial infarction. Methods: A simple, sensitive, and specific method by using UPLC-MS/MS to study the pharmacokinetics of PE in rats was developed and validated, with ilexsaponin A as an internal standard. Methanol was used as a protein precipitation reagent for blood sample extraction. A Waters Acquity C18 column (2.1 mm × 50 mm, 1.7 μm) was used for chromatographic separation with a gradient elution of CH3CN: 0.1% formic acid (0.3 mL·min^-1). Negative ion electrospray ionization was used for detection in multiple reaction monitoring mode. Results: PE was linear within the concentration range of 0.14-1118.00 ng/mL. The LLOQ was 0.14 ng/mL for the plasma samples. The intra-day and inter-day precision were ranged from 1.18% to 10.48%, while the accuracy ranged from -1.32% to 1.68%, indicating satisfactory precision and accuracy of the assay. The extraction recoveries for PE and IS were ranged from 81.40% to 86.65%, with no significant variation among the three concentrations, respectively. PE remained stable at room temperature (25°C) for 3 h, in auto-sampler (4°C) for 24 h, after three freeze-thaw cycles, and in long-term storage at 128;’20°C for 30 days. The PK results of PE indicated its poor oral bioavailability (3.37%). Conclusion: Non-compartmental pharmacokinetics parameters indicated that PE was rapidly distributed to the tissues and metabolized. The pharmacokinetic data of this paper highlighted the first-time report of PE oral bioavailability with two different administration manners, which will help to better understand how PE metabolized in rats and exerted its pharmacological effect in vivo.

Background: Prodrugs principle is widely used to improve the pharmacological and pharmacokinetic properties of some active drugs. Much effort was made to develop metronidazole prodrugs to enhance antibacterial activity and/or to improve pharmacokinetic properties of the molecule or to lower the adverse effects of metronidazole. Objective: In this work, the pharmacokinetic properties of some of monoterpenes and eugenol pro metronidazole molecules that were developed earlier were evaluated in vitro. The kinetic hydrolysis rate constants and half-life time estimation of the new metronidazole derivatives were calculated using the validated RP-HPLC method. Methods: Chromatographic analysis was performed using Zorbbax Eclipse eXtra Dense Bonding (XDB)-C18 column of dimensions (250 mm, 4.6 mm, 5 μm), at ambient column temperature. The mobile phase was a mixture of sodium dihydrogen phosphate buffer of pH 4.5 and methanol in gradient elution, at 1ml/min flow rate. The method was fully validated according to the International Council for Harmonization (ICH) guidelines. The hydrolysis process was carried out in an acidic buffer pH 1.2 and in an alkaline buffer pH 7.4 in a thermostatic bath at 37 ºC. Results: The results followed pseudo-first-order kinetics. All metronidazole prodrugs were stable in the acidic pH, while they were hydrolysed in the alkaline buffer within a few hours (6-8 hr). The rate constant and half-life values were calculated, and their values were found to be 0.082- 0.117 hr^-1 and 5.9- 8.5 hr., respectively. Conclusion: The developed method was accurate, sensitive, and selective for the prodrugs. For most of the prodrugs, the hydrolysis followed pseudo-first-order kinetics; the method might be utilised to conduct an in vivo study for the metronidazole derivatives with monoterpenes and eugenol.

Background: Apranax Plus® is a new bilayer tablet dosage form, which combines two active pharmaceutical ingredients: naproxen sodium and codeine phosphate. Objective: The purpose of this work was to develop an Ultra-High-Performance Liquid Chromatography (UHPLC) method for the separation and determination of codeine phosphate hemihydrate impurities in a bilayer tablet dosage form. Methods: The separation and determination of codeine phosphate hemihydrate and its impurities, methylcodeine, morphine, codeine dimer, 10-hydroxycodeine, 14-hydroxycodeine, thebaine and codeinone were achieved by using reversed-phase liquid chromatography with TUV (Tunable UV Detector) and PDA (Photodiode Array Detector) detection by UHPLC. The new proposed method utilized by the Waters Acquity UHPLC® TUV and PDA systems using a UHPLC column Waters Acquity, BEH, C18, 2.1x100 mm, 1.7 μm particle size with a mixture of component A and acetonitrile in a gradient mode at a flow rate of 0.3mL/min, at 25°C with a load of 5μL. The detection for all eluted compounds was carried out at 245nm. Results: The codeine phosphate hemihydrate and peaks of its impurities were adequately obtained, thus proving the stability-indicating power of the method. The developed method was validated as per the ICH guidelines with respect to parameters such as precision, accuracy, linearity, Limit of Detection (LOD), Limit of Quantification (LOQ) and robustness. Conclusion: It was verified as being adequate for all the mentioned impurities of codeine phosphate hemihydrate. The described method was found to be useful for routine purity testing and was also found suitable for the analysis of the stability samples of the drug product.

Background: Currently, ovulation stimulation with clomiphene citrate is the most commonly used treatment for infertility. It stimulates the secretion of gonadotropins necessary for the release of an egg. Objective: In this study, microextraction method of liquid phase was utilized using hollow fiber with high-performance liquid chromatography and ultraviolet (HPLC-UV) detector for pre-concentration and measuring clomiphene citrate in plasma. Methods: Clomiphene citrate was extracted from a 15 ml sample solution with pH=11 in an organic solvent (Octanol), which was located in the vent wall of the fiber. Subsequently, the drug was introduced into an internal organic solvent in an acidic pH=2 receptor that was located inside the fiber. In the following, effective factors in microextraction including the pH of the donor phases and acceptor phases, the type of organic solvent, ion phase strength, extraction time and stirring speed were investigated and optimized. Results: After extracting the drug with an optimal pre-concentration factor of 212, extraction recovery was 91%, the detection limit was 1.50 ng/ml, limiting the linearity of 5-1000 ng/mL and %RSD=6.98. Conclusion: The proposed method was successfully implemented and optimized for trace detection of clomiphene citrate in plasma, therefore the clinical and investigational application of this method is facilitated by this study.

Background: The illegal virtual market for food supplements facilitates fraud and adulteration. Worldwide concern for consumer safety is growing on the part regulatory agencies, healthcare professionals and consumers. Objective: This work aimed to evaluate the presence of sexual stimulants and anabolic steroids commonly used in the adulteration of dietary supplements through the development of a high performance liquid chromatography with the photodiode array detection (HPLC-DAD) method for the identification and quantification of these compounds. Methods: The mobile phase composed of an ammonium acetate solution, acetonitrile and methanol led to the efficient separation of vardenafil, testosterone base, testosterone propionate, tadalafil, sildenafil and yohimbine. Results: The assay was linear (r²> 0.999), precise (RSD% <0.5), accurate (99.1 to 105.2%), and the limits of detection and quantification were less than 0.05 and 0.15 μg/mL, respectively. Four samples of dietary supplements contained testosterone (n=1), tadalafil (n=2) and yohimbine (n=1) as adulterants. The adulterants found were in subtherapeutic doses, probably to reduce possible adverse effects and the action expected to appear natural. Since about 80% of adverse drug reactions are dose dependent, unpredictable adverse drug reactions are dose independent and based on idiosyncratic or allergic mechanisms or intolerance. Conclusion: The developed method is convenient and easily applicable for adulteration detection of the analyzed drugs in the multicomponent supplements.

Objective: A particular, easy, and precise reverse phase liquid chromatographic technique was developed for the determination of Zaltoprofen in human plasma. Methods: The ODS C18 (250mm x 4.6mm, 5μm) column was utilized for determination. The mobile phase contains buffer and acetonitrile (55:45 v/v). The rate of flow was 1.0 ml/min. The volume of infused sample was 10 μl. The column was kept at a temperature of ~30°C and equilibrated for no less than 30 min, prior to injection of the solutions. The detection wavelength was set at 331 nm. Results: The linearity experiment was performed for Zaltoprofen in the range of 0.15 to 20 μg/ml. The observed recovery of Zaltoprofen was 98.32 %. Conclusion: The suggested technique has been validated and has proved to be very helpful for zaltoprofen determination in human plasma.

Background: A simple and sensitive quantitation analytical technique by liquid chromatography– tandem mass spectrometry (LC-MS/MS) is essential for fedratinib in biological media with kinetic study in healthy rabbits. Objective: The main objectives of the present research work are the development of the LC-MS/MS method and to validate a procedure for the quantitation of fedratinib and its application to kinetic study in rabbits. Methods: Separation of processed samples was done on zorbax SB C18 column (50mm×4.6 mm) 3.5μm with a movable phase of methanol, acetonitrile and 0.1% formic acid in the ratio of 30:60:10. The movable phase was monitored through column at 0.8 ml/min flow rate. The drug and ibrutinib internal standard (IS) were evaluated by monitoring the transitions of m/z -525.260/57.07 and 441.2/55.01 for fedratinib and IS, respectively in multiple reaction monitoring mode. Results: The linear equation and coefficient of correlation (R²) results were y =0.00348x+0.00245 and 0.9984, respectively. Intra and inter-day precision RSD findings of the developed technique were found in the range of 2.4 - 5.3% for the quality control (QC)-samples (252.56, 1804.0 and 2706 ng/ml). The proposed method was subjected to pharmacokinetic study in healthy rabbits and the kinetic study, fedratinib showed mean AUClast 13190±18.1 hr*ng/ml and Cmax was found to be 3550±4.31 ng/ml in healthy rabbits. Conclusion: The validated method can be applicable for the pharmacokinetic and toxicokinetic studies in the clinical and forensic analysis of fedratinib in different kinds of biological matrices successfully.

Background: A tablet dosage form widely used in the treatment of cough and cold, containing phenylephrine hydrochloride, paracetamol, caffeine, and diphenhydramine hydrochloride as active pharmaceutical ingredient was selected for the development of a novel, rapid, simultaneous isocratic reversed phase-high performance liquid chromatography (RP-HPLC) method. Objective: The objective of this paper was to develop and validate a novel, rapid, simple, precise, accurate and reproducible RP-HPLC method for simultaneous estimation of phenylephrine hydrochloride, paracetamol, caffeine, diphenhydramine hydrochloride in bulk and pharmaceutical dosage form. Method: Optimized chromatographic conditions were an isocratic elution with prontosil C18-column (250×4.6 mm, 5 μ), methanol and 20mM phosphate buffer (55:45 v/v, pH 3 ) as mobile phase, flow rate 1.0 mL/min and UV detector set at λ max 220 nm. The method was validated for specificity, precision, linearity, accuracy, sensitivity, and robustness as per the International Council for Harmonization guidelines. Result: The retention times of phenylephrine hydrochloride, paracetamol, caffeine, diphenhydramine hydrochloride were found to be 2.8 min, 3.3 min, and 4.0 min and 7.3 min, respectively. This novel method was found to be rapid, simple, linear (R²> 0.99), preciserelative standard deviation < 2.0 %), accurate (recovery 98-102%), sensitive and robust. Conclusion: The proposed novel isocratic RP-HPLC method is rapid (short run time below 10min), highly selective, precise, accurate, sensitive and robust. The method was successfully applied for the simultaneous analysis of phenylephrine hydrochloride, paracetamol, caffeine, diphenhydramine hydrochloride in a pharmaceutical dosage form.

Background: Acefylline (Acef) is a derivative of theophylline that has bronchodilator effects. Two binary mixtures were marketed for Acef: Acefylline piperazine/ Phenobarbitone (Acef- P/Phen) and Acefylline heptaminol/ Cinnarizine (Acef-H/ Cinn). To our knowledge, none of the reported methods had the capacity to determine Acef in its binary mixture in the presence of its degradation products and potential impurity theophylline (Theo). Methods: Two validated RP-LC methods were established for the determination of Acef-P/Phen and Acef-H/ Cinn, in the presence of Acef degradation products and its’ potential impurity Theo. A complete study of the forced acidic, alkaline, and oxidative degradation of Acef was presented. The methods were based on LC separation on RP C18 columns using isocratic and gradient elution for Acef-P /Phen and Acef-H /Cinn mixtures, respectively. Different chromatographic conditions were examined and optimized. Results: Linear responses were attained over concentration ranges of 75-500/15-1000 μg/mL and 100- 1000 /50-500 μg/mL with mean percentage recoveries of (100.72±1.23)%/ (99.29±1.12)% and (100.44±1.27)%/ (99.01±0.97)% for Acef-P/Phen and Acef-H /Cinn, respectively. ICH guidelines were used for methods validation and all the parameters were found to be acceptable. Conclusion: The methods showed to be accurate, precise, and specific for the analysis of Acef-P/Phen and Acef-H /Cinn in drug substance, drug product, and in laboratory prepared mixtures in the presence of Theo and up to 50% of degradation products. The structures of the main degradation products and the expected degradation pathway were suggested using the MS data.

Purpose: Hydrazones are a class of azomethines with a wide spectrum of pharmacological properties that are influenced by the pH of the media. The purpose of this study was to investigate acidbase properties of five 4-nitrobenzaldehyde-4-substitutedphenyl-1-carbonylhydrazones in sodium hydroxide media (14>pH>7). Methods: The dissociation process was followed by UV-Vis spectroscopy, in ethanol-water (V/V, 1:1) solutions, at room temperature. Semiempirical methods AM1 and PM3 were applied for determination of the deprotonation enthalpies. Results: The changes in the UV-Vis spectra, as well as the deprotonation enthalpies, suggested that the dissociation process for four investigated hydrazones with an amide group took place in one step. The exception with two dissociation steps was hydrazone with amide and hydroxyl group. The pH region of dissociation was from pH 10.8 to pH 11.6 for the first step and between pH 11.7 and pH 12.1 for the second step of dissociation. The influence of the ethanol on the UV-Vis spectra was eliminated by the method of Characteristic Vector Analyses (CVA). The stoichiometric dissociation constants were determined numerically (pKHA = n·pH + logI) and graphically (intercept of the dependence of logI on pH) from the absorbance data using experimental and reconstructed UV-Vis spectra, at three different ionic strengths. Thermodynamic dissociation constants were estimated graphically as an intercept of dependence of dissociation constant on the square root of the ionic strength. Conclusion: The obtained results demonstrated that the influence of the substituents on pKHA values was not significant, except for hydrazone with amide and hydroxyl group. Namely, the dissociation of the amide group of this hydrazone was retarded due to the influence of the phenolic group.

Introduction: Clarithromycin is a macrolide antibiotic that is active against a variety of microorganisms. It is widely used in the local and international market in different pharmaceutical dosage forms. However, its chemical structure lacks a chromophore and hence it has a low absorption and this makes it more difficult to be detected at low concentrations. In this research project, we proposed an easy and feasible chemical derivatization of clarithromycin to introduce a chromophore in order to increase its absorptivity at low concentration using a simple reverse phase HPLC analytical method. Methodology: Chemical derivatization of clarithromycin involved an introduction of benzoyl groups as a chromophore through esterification reaction. A reverse phase analytical HPLC method was developed to quantify clarithromycin at a very low concentration compared to the standard official pharmacopeia. Results: Clarithromycin was successfully derivatized and a hyperchromic and bathochromic shift to UV absorption lambda max (λmax) was achieved (λmax = 245nm.) A successful chromatographic separation was obtained using reverse phase HPLC chromatography. The developed method was capable of detecting and quantifying clarithromycin at very low concentration. The Limit of Quantification (LOD) and Limit of Quantification (LOQ) was found to be 2*10^-8 mg/ml and 2*10^-6 mg/ml respectively. Conclusion: Clarithromycin was successfully derivatized to a chromophore containing molecule. The developed reverse phase HPLC method is capable to detect and quantify clarithromycin at a very low concentration. The method can successfully quantify clarithromycine when present in low concentration such as in biological and enviromental samples.