Current Pharmaceutical Analysis - Volume 17, Issue 5, 2021

Introduction: Epilepsy is one of the most common neurological diseases in clinical practice. The combined application of metabolomics technology plays a great advantage in the screening of biomarkers. Methods: In this study, Wistar rats were used as experimental subjects to model intractable epilepsy and to detect the metabolic changes of small molecules in plasma. UPLC-MS/MS was used to determine the small molecules in rat plasma. UPLC HSS C18 (2.1mmx100mm, 1.7 μm) column was used for separation, column temperature of 40°C. The initial mobile phase was acetonitrile -0.3% formic acid with gradient elution, the flow rate was 0.3 mL/min, total running time 4.0 min. Quantitative analysis was performed with multi-response monitoring (MRM). Results: Compared to the control group, the L-Alanine and L-Arginine decreased in the Epilepsy group (p<0.05); while Cytosine, Adenosine, L-Tyrosine, Citric acid, Fructose increased (p<0.05). Conclusion: In the screening of epilepsy biomarkers using metabolomics, various amino acids that lead to increased energy production and neurotransmitter imbalance play an important role in epileptic seizures.

Background: Up to date, generic linezolid injections produced by Chinese manufacturers were not widely used in clinics in China. Quality evaluation of linezolid injections produced in China is a prerequisite, which has rarely been performed. Objective: This study aimed to evaluate the quality of branded and generic injections from different manufacturers and to provide a basis for quality control. Methods: In this study, the content of linezolid, related impurities and enantiomer of linezolid were determined by high-performance liquid chromatography. The content of glucose was determined by the iodine method. The insoluble particles and visible and sub-visible particles were determined by light blockage and lamp test, respectively. Osmotic pressure was determined by the freezing point depression method. The standard solution control method was used to check the color of the injection. Linezolid injections from different manufacturers were evaluated uniformly. Results: No significant difference was found in the content of linezolid, glucose, related impurities, visible particles, insoluble particles, pH value, and solution color between branded and generic drugs from different manufacturers in China. Conclusion: The quality of samples from different manufacturers is consistent. Although the physicochemical similarity does not guarantee the bioequivalence of studied branded and generic linezolid injections, the results provide references for further bioequivalence study. Generic injections offer more affordable treatment options for patients with infections than expensive branded drugs.

Objective: Apatinib, a novel small-molecule Tyrosine Kinase Inhibitor (TKI), is under development to treat advanced gastric cancer. For the pharmacokinetic evaluation and routine drug monitoring of apatinib, a quantitative ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) method in rat plasma was developed with tinidazole used as an internal standard (IS). Methods: Protein precipitation (PPT) was selected as a sample pre-treatment method to extract apatinib. Then, chromatography was performed on a Kinetex C8 column (2.1x100 mm, 2.6 μm) using a constant mobile phase including 0.2% formic acid and 10 mM ammonium acetate in water and methanol (30:70, v/v) with a gradient flow rate from 0.2 mL/min to 0.4 mL/min. Chromatographic analysis was performed in only 4.5 min. Mass spectrometric detection was carried on positive electrospray ionization (ESI+) mode with Multiple-Reaction Monitoring (MRM). Results: The standard calibration curve showed good linearity in 2-1000 ng/mL with the correlation coefficient (R²) > 0.99. The Lower Limit of Quantitation (LLOQ) was 2 ng/mL. The precision, accuracy, extraction recovery, matrix effect, stability and carryover were all within the acceptable range. Conclusion: This method was simple, accurate, selective and successfully used for a pharmacokinetic study following seven rats orally administrated a single of 60 mg/kg apatinib.

Introduction: Spectrophotometry was investigated for the determination of epsilon aminocaproic acid (EACA) with p-nitrophenol (PNP). The method was based on Charge Transfer (CT) complexation of this drug as n-electron donor with π-acceptor PNP. Methods: The experiment indicated that CT complexation was carried out at room temperature for 10 minutes in dimethyl sulfoxide solvent. The spectrum obtained for EACA/PNP system showed the maximum absorption band at a wavelength of 425 nm. The stoichiometry of the CT complex was found to be a 1:1 ratio by Job’s method between the donor and the acceptor. Different variables affecting the complexation were carefully studied and optimized. At the optimum reaction conditions, Beer’s law was obeyed in a concentration limit of 1~6 μg mL^-1. The relative standard deviation was less than 2.9%. The apparent molar absorptivity was determined to be 1.86x104 L mol^-1cm^-1 at 425 nm. The CT complexation was also confirmed by both FTIR and 1H NMR measurements. Results: The thermodynamic properties and reaction mechanism of the CT complexation have been discussed. Conclusion: The developed method could be applied successfully for the determination of the studied compound in its pharmaceutical dosage forms with good precision and accuracy compared to the official method comprising t- and F-tests.

Background: Polyethylene glycol 400 (PEG400), as a good traditional Chinese medicine solvent, diluent and solubilizer, is widely used as a main pharmaceutical excipient in traditional Chinese medicine compound preparations containing active ingredient baicalin. PEG400 could increase the solubility and release of baicalin in vivo, but it was unknown that PEG400 affected the absorption and distribution of baicalin or not. Objective: At present, the effects of PEG400 on the pharmacokinetic characteristics and tissue distribution behaviors of the main flavonoid metabolites baicalin, baicalein 6-O-β-D-glucopyranoside (B6G) and baicalein after oral administration of baicalin were investigated by a rapid, efficient and sensitive ultra- high performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) method. Moreover, we respectively studied the effects of PEG400 on the activities and protein expressions of two subtypes UDP-glucuronyltransferase 1 A8/A9 (UGT1A8 and UGT1A9) of UDP-glucuronosyltransferases (UGTs) in vitro and in vivo experiments to determine the partial mechanisms by which PEG400 altered the pharmacokinetics and tissue distribution behaviors of the three flavonoid metabolites. Methods: A rapid, efficient and sensitive ultra-high performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) method and ELISA and so on. Results: The results showed that PEG400 significantly increased the Cmax and AUC0-t values (P < 0.05 or P < 0.01) of baicalin and B6G while baicalein could not be quantified due to its extremely low concentration (lower the LLOQ) in plasma. Baicalin, B6G and baicalein were mainly distributed in the stomach, small intestine, kidney and liver. PEG400 changed the distribution of three flavonoid metabolites in various tissues and also increased the activities and expressions of UGT1A8 and UGT1A9. Conclusion: In conclusion, PEG400 significantly altered the pharmacokinetic characteristics and tissue distribution behaviors of three flavonoid metabolites may partly result from PEG400 upregulated the activities and expressions of the drug biphasic metabolic enzymes UGT1A8 and UGT1A9, which provided a material basis and useful information to reveal the mechanism of action and clinical application of PEG400.

Background: Levocetirizine is chemically know as (R)-(-)-2-[4-(4-chlorobenzhydryl)-1- piperazinyl]-ethoxy acetic acid dihydrochloride. Many publications have reported the synthetic routes of levocetirizine. Several related substances have been detected in levocetirizine hydrochloride drug substances. In our study, a pharmaceutical impurity, at the level of >0.1% w/w, was isolated, purified ed and identified. It is not included in the European Pharmacopoeia (EP). Objective: Identification, isolation and characterization of a new pharmaceutical impurity in levocetirizine hydrochloride. Methods: The impurity was enriched by normal phase silica gel, and was further purified by semipreparative HPLC. It was separated from the crystallization mother liquor of levocetirizine hydrochloride for the first time. Mass spectrometry and nuclear magnetic resonance spectroscopy are the ultimate tools in structure elucidation. Results: The structure was identified as levocetirizine quaternary ammonium. The formation mechanisms of the impurity are also presented. The method was applied to the determination of the impurity in levocetirizine hydrochloride in real samples. Conclusion: The method was applied to the determination the impurity of levocetirizine hydrochloride in real samples.

Background: Danyikangtai powder, a Traditional Chinese Medicine (TCM) formula, shows promise to become a novel drug candidate for the simultaneous treatment of chronic cholecystitis and chronic pancreatitis. However, the pharmacokinetic behavior of Danyikangtai powder remains unclear. Objective: We investigated the comparative pharmacokinetics of four flavonoids in rats after oral administration of Danyikangtai powder and three compatibilities. Materials and Methods: The comparative pharmacokinetics was studied by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS). Chromatographic separation was performed on a Universil XB-C18 column with a gradient mobile phase containing 0.1% (v/v) aqueous formic acid and acetonitrile. All analytes and internal standard were quantitated in the multiple reaction monitoring modes with a positive electrospray ionization interface. Results and Discussion: Danyikangtai powder and Scutellariae radix have similar pharmacokinetic behaviors in rats after oral administration. However, the elimination of four flavonoids in rats after oral administration of Danyikangtai powder was accelerated, which was possibly related to the reduction of the potential hepatotoxicity of Scutellariae radix. The varying degrees of change in pharmacokinetic parameters after oral administration of different herb combinations suggested that herb–herb interactions occurred in vivo. Conclusion: This study will be helpful to reveal the safety, rational and mechanism of Danyikangtai powder formula compatibility, thereby providing pre-clinical research data for its new drug development and guidance for its rational clinical application.

Aims: Characterization of antimicrobial metabolites of novel Streptomyces sp. UK-238. Background: Novel antimicrobial drug discovery is urgently needed due to emerging multi antimicrobial drug resistance among pathogens. Since many years, natural products have provided the basic skeletons for many therapeutic compounds including antibiotics. Bioprospection of un/under explored habitats and focussing on selective isolation of actinobacteria as a major reservoir of bio and chemodiversity has yielded good results. Objective: The main objectives of the study were the identification of UK-238 by 16S rDNA sequencing and antimicrobial metabolite fingerprinting of culture extracts. Methods: In the present study, a promising isolate, UK-238, has been screened for antimicrobial activity and metabolite fingerprinting from the Himalayan Thano Reserve forest. It was identified by 16S rDNA sequencing. Ethyl acetate extract was partially purified by column chromatography. The pooled active fractions were fingerprinted by GC-MS and compounds were tentatively identified by collated data analysis based on Similarity Index, observed Retention Index from Databases and calculated Retention Index. Results: UK-238 was identified as Streptomyces sp. with 98.4% similarity to S. niveiscabiei. It exhibited broad-spectrum antibacterial and antifungal activity. The GC-MS analysis of active fractions of ethyl acetate extract showed the presence of eighteen novel compounds, some with antimicrobial activity, belonging to four major categories- alcohols, alkaloids, esters and peptide. Conclusion: The study confirms that bioprospection of underexplored habitats can elaborate novel bio and chemodiversity.

Background: The determination of trace drugs in aquatic environments is important. For this purpose, many methods such as High Performance Liquid Chromatography (HPLC) and gas chromatography mass spectrometry (GC/MS) have been used. Objective: This study introduces a simple, sensitive, and rapid colorimetric method for the spectrophotometric determination of sitagliptin (STG) in drinking water, tablet, human plasma, and human urine using gold nanoparticles (AuNPs). Methods: The Surface Plasmon Resonance (SPR) property of AuNPs and the interaction between STG and AuNPs are the base of the colorimetric method. The addition of STG into AuNPs led to the aggregation of AuNPs. Transmission Electron Microscopy (TEM) proved the aggregation of AuNPs in the presence of STG. Also, the size of the nanoparticles distribution was evaluated by Dynamic Light Scattering (DLS). In addition, Fourier-Transform Infrared Spectrophotometer (FTIR) was used to study the chemical structure of AuNPs, STG, and AuNPs in the presence of STG. Results: The parameters that affect the absorbance such as pH, type and volume of buffer, AuNPs concentration, interaction time, ionic strength, and interfering ions were investigated and optimized. Under the optimum conditions, the determination of STG was performed via this method over the range of 50-300 μgL^-1 (R²=0.9941) with the Limit of Detection (LOD) and Limit of Quantification (LOQ) of 1.23 and 1.39 μgL^-1, respectively. Conclusion: Eventually, the results showed that the proposed method has a high potential for simple, rapid, sensitive, and accurate determination of STG.

Background: Haloperidol (HP) and Risperidone (RIS) are antipsychotic drugs and the simultaneous determination of these drugs is important. Estimation of HP and RIS alone or in combination with other drugs has been performed in a variety of ways. Objective: The aim of this paper was to propose a rapid, simple, accurate, and robust method for the simultaneous determination of HP and RIS using Artificial Neural Networks (ANNs), Partial Least Squares (PLS), and Principal Component Regression (PCR) methods along with spectrophotometry technique. Methods: The simultaneous spectrophotometric determination of HP and RIS in synthetic mixtures and biological fluid was performed by applying ANNs Containing Feed-forward Backpropagation (FFBP) and Radial Basis Function (RBF) networks as intelligent methods, as well as PLS, and principal component regression PCR as multivariate calibration methods. The Levenberg- Marquardt (LM), Scaled Conjugate Gradient (SCG), and Resilient Back-propagation (RP) algorithms with different layers and neurons were used in FFBP network and obtained results were compared with each other. Results: Among various algorithms of the FFBP network, the LM algorithm was selected as the best model with a lower Mean Square Error (MSE). MSE of the RBF model was 1.46x10^-25 and 1.62x10^-23 for HP and RIS, respectively. On the other hand, the mean recovery of PLS and PCR was 99.91%, 100.01% and 98.60%, 101.90% for HP and RIS, respectively. Conclusion: The proposed models and High-Performance Liquid Chromatography (HPLC) as a reference method were compared with each other by one-way Analysis of Variance (ANOVA) test at the 95 % confidence level for the urine sample. It was observed that the developed methods presented comparable results for the simultaneous determination of HP and RIS.

Background: Sample preparation is one of the most challenging phases in pharmaceutical analysis, especially in biological matrices, affecting the whole analytical methodology. Objective: In this study, a new Ca(II)/Cu(II)/alginate/CuO Nanoparticles Hydrogel Fiber (CCACHF) was synthesized through a simple, green procedure and applied for fiber micro solid-phase extraction (FMSPE) of diazepam (DIZ) and oxazepam (OXZ) as model drugs prior to high-performance liquid chromatography-UV detection (HPLC-UV). Methods: Composition and morphology of the prepared fiber were characterized and the effect of main parameters on the fiber fabrication and extraction efficiency have been studied and optimized. Results: In optimal conditions, calibration curves were linear, ranging between 0.1–500 μg L⁻¹ with regression coefficients of 0.9938 and 0.9968. Limit of Detection (LOD) (S/N=3) and Limit of Quantification (LOQ) (S/N=10) of the technique for DIZ and OXZ were 0.03 to 0.1 μg L⁻¹. Within-day and between-day Relative Standard Deviations (RSDs) for DIZ and OXZ were 6.0–12.5% and 3.3–9.4%, respectively. Conclusion: The fabricated adsorbent has been substantially employed to the extraction of selected benzo-diazepines (BZDs) from human serum real specimens and the obtained recoveries were also satisfactory (82.1-109.7%).

Introduction: A reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the simultaneous analysis of two drugs, levamisole hydrochloride (LH) and oxyclozanide (OX), in co-formulation for veterinary use. Materials and Methods: The new HPLC method was validated as per the ICH and other guidelines. A C18 column was used with a gradient program; eluent A was an equal mixture of methanol and acetonitrile, and eluent B included a 25 mM phosphate buffer at pH 7.0 containing 30 mM sodium decanesulfonate and triethylamine (50:50:1 v/v) with pH adjusted to 7.0 using H3PO4 [51:49 v/v] .The detection wavelength was set at 220 nm. For the final gradient program, the retention times were 8.2(for LH) and 13.6(for OX) minutes, respectively, at a flow rate of 1 ml/min over 20 minutes run time. Results: The method was precise, specific and robust. The correlation coefficient, R² was 0.9998 and 0.9999 for LH and OX, respectively in the range of 5 – 280 μg / mL. The percent y-intercepts and percent residual standard deviations were 1.6%/0.4% and 1.4%/1.0% for LH and OX, respectively. The LOD and LOQ of the method were 0.21 μg / mL and 0.62 μg / mL for LH and 0.06 μg / mL and 0.18 μg / mL for OX. The method has an average accuracy of 100.5% for LH and 101.1% for OX when tested on veterinary bolus formulations, and the samples could be stored under typical lab conditions for about 7 days without significant degradation. Conclusion: This HPLC method is suitable for assaying levamisole hydrochloride and oxyclozanide simultaneously from veterinary formulations.