Current Pharmaceutical Analysis - Volume 16, Issue 6, 2020

Electrode modification is a technique performed with different chemical and physical methods using various materials, such as polymers, nanomaterials and biological agents in order to enhance sensitivity, selectivity, stability and response of sensors. Modification provides the detection of small amounts of analyte in a complex media with very low limit of detection values. Electrochemical methods are well suited for drug analysis, and they are all-purpose techniques widely used in environmental studies, industrial fields, and pharmaceutical and biomedical analyses. In this review, chemically modified electrodes are discussed in terms of modification techniques and agents, and recent studies related to chemically modified electrodes in electrochemical drug analysis are summarized.

Background: In order to organize and give a better understanding of the existing population of protease activity units together with their respective methods of enzymatic activity assessment, there is a need of their clear classification system. Results and Conclusion: The following system has been proposed: Enzyme Centered Units (ECU) equivalent to Enzyme Process Unit notation; Protein Centered Units (PCU) equivalent to Protein Process Unit notation; Legal Authority and Enzyme Centered Units (LAECU) equivalent to Enzyme Centered Units system additionally related to a legal authority or an organization. The suitable ways for the mutual conversion of commonly used units and their conversion into the standard SI units have been included. A convenient gravity/spectrophotometer test of proteolytic activity with the use of three protein types has also been proposed. The test gives high degree of confidence of the experimental determination for a wide spectrum of protease activity in samples of plant origin. The whole paper allows both theoretical and practical orientation in the range of different proteolytic activity units as well as in the methods of their determination.

At present, no one can imagine drug development, marketing and post-marketing without rigorous quality control at each stage. Only modern, selective, accurate and precise analytical methods for determination of active compounds, their degradation products and stability studies are able to assure the appropriate amount and purity of drugs administered every day to millions of patients all over the world. For routine control of drugs simple, economic, rapid and reliable methods are desirable. The major focus of current scrutiny is placed on high-performance thin layer chromatography and derivative spectrophotometry methods, which fulfill routine drug estimation’s expectations [1-4]. The present paper reveals state-of-the-art and possible applications of those methods in pharmaceutical analysis between 2010 and 2018. The review shows advantages of high-performance thin layer chromatography and derivative spectrophotometry, including accuracy and precision comparable to more expensive and time-consuming methods as well as additional fields of possible applications, which contribute to resolving many analytical problems in everyday laboratory practice.

Background: The simultaneous determination of multiple components in a sample is an important factor in the quality control of traditional Chinese medicines and can give an indication of potential clinical applications. Introduction: A rapid and sensitive method has been introduced for the simultaneous quantitative analysis of eight bioactive flavonoid constituents from Scutellariae Radix using ultra-high performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry. Methods: The separation was performed on a Waters Acquity UPLC C18 column (2.1 mmx100 mm, 1.7 μm), under optimized mass spectrometry conditions, with a flow rate of 0.3 mL/min. The column temperature was maintained at 35°C and the injection volume was 3 μL. Result: The method showed a good linear relationship of each component; all R2 values were above 0.9990 in the experiment. The RSDs of the precision test, repeatability test, stability test and recovery test were all not more than 2.86 %. We found that the total percentage amounts of the eight flavonoids were 22.19%, 18.63% and 10.86% in Raw Scutellariae Radix (RSR), Wine Scutellaria Radix (WSR) and Scutellaria Radix Charcoal (SRC) respectively. Conclusion: The method was successfully applied to the simultaneous determination of the eight bioactive flavonoids of Raw Scutellariae Radix, Wine Scutellaria Radix and Scutellaria Radix Charcoal.

Introduction: Fenugreek- the seeds of Trigonella foenum-graecum- are known for their medicinal value since the very early-recorded human history. This study is conducted to explore the effect of environmental conditions on the trigonelline content of the plant seeds. Methods: Trigonelline contents in different samples of fenugreek were estimated by HPTLC. The established method was proved to be accurate, precise and robust according to ICH guidelines. The used mobile phase was a mixture of ethyl acetate: methanol: water: formic acid 7.5: 2.5: 3: 1 (%, v/v/v/v) on 20 x 10 cm glass coated silica gel 60 F254 plates. The plates were scanned and quantified densitometrically at λ = 267 nm after development. Results: A valid linear relationship correlating peak area and amount of trigonelline in the range of 100- 700 ng/spot was obtained. The amount of trigonelline varied according to the country of origin. Conclusion: The Indian sample has the highest amount of trigonelline followed by the Moroccan sample. The two locations represent areas with moderate temperature, low altitude and rainy winter.

Background: The aim of this study was to determine the concentrations of khasianine in mouse whole blood sample and its application for the pharmacokinetics by a rapid, selective and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method. Methods: The blood samples were preprocessed by one-step protein precipitation with acetonitrile. The study was performed on an ACQUITY I-Class UPLC system with a UPLC BEH column. Lannaconitine (internal standard, IS) and khasianine were gradient eluted by a mixture of acetonitrile and water with 0.1% formic acid at a flow rate of 0.4 mL/min. The mass spectrometer was equipped with an Electrospray Ionization (ESI) source in positive mode. The quantitative detection was performed in a multiple reaction monitoring modes at transitions m/z 722.4→70.7 for khasianine and m/z 585.3→119.9 for the corresponding IS. Results: The calibration curve was of good linearity ranging from 0.5 to 1000 ng/mL (r > 0.995). The Lower Limit of Detection (LLOD) and Lower Limit of Quantitation (LLOQ) were 0.2 and 0.5 ng/mL, respectively. The inter-day and intra-day precision (RSD%) were both less than 14%, and the accuracy ranged from 86.6% to 108.3%. The matrix effects were between 98.0% and 103.7%, and the average recovery was better than 67.4%. Conclusion: This assay established a sensitive, rapid, selective UPLC-MS/MS method which was successfully used for the pharmacokinetic study of khasianine in mouse blood, and the absolute availability of khasianine was 0.78% which exhibited a poor oral absorption.

Background: Xiexin Tang (XXT) is a classic Traditional Chinese Medicine (TCM) formula that has been used in herbal clinics for more than 1800 years. Recently, many studies have investigated the pharmacological effects and chemical composition of XXT. However, there is little information about systematic studies on the material basis of its efficacy. In the present study, the serum pharmacochemistry technique and HPLC-DAD-Q-TOF/MS were performed to screen and analyze the multiple absorbed bioactive components and metabolites of orally dosed XXT in rat serum. Methods: Bio-samples and herbal extracts were analyzed and detected by HPLC-DAD-Q-TOF/MS. Upon comparison of the chromatograms of the single-constituent decoctions with that of the XXT formulation, the peak quantity and peak intensity of the formulated decoction showed some variation from those of the single-constituent decoctions. Results: Twenty-one serum-adsorbed constituents were identified after intragastric administration of herbal extracts, of which 8 originated from Rhei Radix et Rhizoma (RRR), 5 from Coptidis Rhizoma (CR), and 8 from Scutellariae Radix (SR). The results showed that the main adsorbed constituents in the serum were anthraquinones, anthrones, chromones, and butyrophenones, alkaloids, and flavonoids. Conclusion: The results demonstrate that an effective and reliable analytical method is set up for screening the bioactive components of Chinese herbal medicine, which provided a meaningful basis for further pharmacology and active mechanism research of XXT.

Background: Urinary or recombinant Follicle-Stimulating Hormones (uFSH and rhFSH) are regularly applied in controlled ovarian stimulation procedure of assisted reproductive technology. Specific activity and purity of these reagents are of great importance since subtle variations in the contents and glycosylation status of FSH may result in differences in clinical efficacy and safety. Objective: The purpose of this study was to comprehensively analyze the FSH contents, glycosylation status and non-specific protein components of the widely used rhFSH Gonal-F and two Chinese marketed FSHs, r-FSH (JSH) and urinary-derived FSH (LSB). Methods: FSH contents, glycosylation status, and other protein contents in these FSH products were assessed with benchtop assays including SDS-PAGE, HPLC and MALDI-MS. Results: HPLC results showed that the purity of the three products was 81.5±0.06% for Gonal-F, 79.6±0.25% for LSB and 76.5±0.36% for JSH, respectively. In addition, MALDI-MS analysis demonstrated that the Gonal-F contained more types of glycosylated isoforms compared to the local rFSHs. The analytical assessment showed that the urinary-derived FSH contained several other protein components. Conclusion: These results suggest that rhFSH Gonal-F is with high purity and potential high activity.

Background: Tigecycline is a known antibiotic in the tetracycline family and a chemical analog of minocycline. It may be used for the treatment against drug-resistant bacteria. Methods: HPLC method was used for related substance analysis. The degraded impurities during the process were isolated and characterized by IR, HRMS (High Resolution Mass Spectrometry) and NMR spectral analysis. Result: Four impurities of tigecycline, a broad spectrum antibacterial agent, were identified, synthesized and characterized. The in vitro biological evaluation of the isolated compounds showed significant antimicrobial and antioxidant properties to that of tigecycline. Apart from these, the tigecycline drug substance showed significant degradation under oxidation conditions. Conclusion: The extensive investigational data confirm the structure of the four impurities. The specification limit for these impurities is applied based on the toxicological data. The antimicrobial activity revealed that the impurity 4 shows excellent activity towards both Gram-positive and Gram-negative bacteria when compared with tigecycline. The results obtained for DPPH (2,2-diphenyl-1- picrylhydrazyl) antioxidant activity concluded that the impurity 2 and the impurity 3 showed good antioxidant properties when compared with tigecycline. There was no activity observed on fungi for both isolated degradants as well as tigecycline.

Background: Vitex negundo (Nagod) is a very useful medicinal plant growing throughout India. The leaves of Nagod are aromatic, tonic and vermifuge. They are useful in the treatment of many ailments. Introduction: Both seasonal and geographical variations have been observed in the active constituents in Vitex negundo. A comprehensive and reproducible HPLC method based on HPLC fingerprint analysis was developed for assessing the quality of Vitex negundo. Methods: Sixteen samples of Nagod collected from different locations and seasons of India were analysed by HPLC and chromatograms were recorded for each of them using PDA detector and 10 peaks were considered for further data analysis. The data were then treated for PCA and cluster analysis using Minitab software. Results: PCA and HCA analysis were used in determining the variability in the leaves of Nagod collected from different places and seasons. Conclusion: The method was useful for discriminating the location of plant within or outside Gujarat but was unable to display any effective seasonal variation in collected samples.

Background: Food and herbal extracts rich in Quercetin (QRT) are often self-medicated by diabetics and can potentially alter the pharmacokinetics (PK) of Metformin HCl (MET) and Canagliflozin (CNG) leading to food or herb-drug interactions and reduced therapeutic efficacy. However, the impact of these flavonoids on the pharmacokinetic behaviour of MET and CNG is mostly unknown. Methods: A simple one-step protein precipitation method was developed for the determination of MET and CNG from rat plasma. The mobile phase chosen was MeOH 65% and 35% water containing 0.1% formic acid at a flow rate of 1mL/min. Results: The retention time of MET, internal standard (Valsartan) and CNG was 1.83, 6.2 and 8.2 min, respectively. The method was found to be linear in the range of 200 - 8000 ng/mL for CNG and 100 = 4000 ng/ml for MET. Precision and accuracy of the method were below 20% at LLOQ and below 15% for LQC, MQC, and HQC. Conclusion: The method was successfully applied for the determination of PK of MET and CNG by using 100 μL of rat plasma. QRT co-administration affects the PK parameters of MET and CNG. This alteration in PK parameters might be of significant use for clinicians and patients.

Background: Terpene lactones are major components of ginkgo biloba extract which are used in cardiovascular and degenerative diseases. To study the involvement of transporters in the transport/disposition of ginkgolides A, B, C, and bilobalide, a bioanalytical assay was developed by LCMS/ MS system for the quantitation of intracellular levels of terpene lactones in cells expressing organic cation transporter 2 (OCT2). Methods: The assay involved an optimized simple sample handling with methyl tert-butyl ether for liquid-liquid extraction and reconstitution in modified dissolution solution. Pretreatment of samples with 50 μM ascorbic acid and the addition of ascorbic acid and formic acid in dissolution solution significantly reduced matrix effect and stabilized the postpreparative samples. Separations were performed by Zobrax RRHD column (extend-C18 1.8μm, 3.0 x 100mm) and acetonitrile gradient elution. The analysis was carried out in the negative ion scan mode using multiple reaction monitoring. Results: The method was validated for linearity (concentration range of 20-5000nM), accuracy (±13.1%), precision (<11.0%), recovery (94.31–105.9%), matrix effect (93.8-111.0%) and stability. Finally, the method was applied in the determination of intracellular concentrations of ginkgolides A, B, C, and bilobalide in Madin-Darby canine kidney (MDCK-mock) and MDCK-OCT2 cells in uptake study. Conclusion: The developed method was successfully validated. Results suggest that OCT2 is involved in the renal disposition of ginkgolide A, B, and bilobalide. This method would foster the study of transport mediated activity via the interaction of ginkgolides and bilobalide with cellular systems.

Background: Ilexsaponin A1, one of the most representative triterpene saponin components in the roots of I. pubescens, showed its effects in anticoagulation and antithrombosis, attenuating ischemia-reperfusion-induced myocardial, angiogenesis and inhibiting phosphodiesterase. Objective: Reveal the key intestinal bacterial strains responsible for ilexsaponin A1 metabolism, and clarify their metabolic behavior. Methods: An accurate and sensitive LC-MS/MS method for the determination of “ilexsaponin A1 in General Anaerobic Medium (GAM) broth” was established and systematically validated. Then it was applied to screen and study the metabolic potential of the intestinal bacterial strains in an anaerobic incubation system. Results: Quantitation of ilexsaponin A1 could be performed within an analytical run time of 14.5 min, in the linear range of 2 - 2000 ng/ml. Enterobacter sakazakii, Bifidobacterium breve, Bifidobacterium adolescentis, Bifidobacterium catenulatum, and Bifidobacterium angulatum were identified to have a potential effect to metabolize ilexsaponin A1 to different extents; and further bacterial metabolic studies were performed to clarify their metabolic capacity and behavior. Conclusion: This paper contributes to a better understanding of the intestinal bacterial metabolism of ilexsaponin A1 and provides scientific evidence for its clinical application. Additionally, the importance of intestinal bacterial strains in the disposition of natural products was also highlighted.

Background: Sodium hyaluronate (NaHA) is generally supplemented in products related to contact lenses for increasing comfort during wearing. The quantity of sodium hyaluronate and the material of lenses affect the retention of sodium hyaluronate on the contact lenses. Methods: We developed a convenient and sensitive but unconventional chromatographic method to quantify sodium hyaluronate and analyze its release behavior from contact lenses. The reverse-phase chromatography eluted sodium hyaluronate with high molecular masses in the shortest time and could separate salt and small compounds from sodium hyaluronate. Results: This method could accurately quantify sodium hyaluronate with diverse molecular sizes. Because sodium hyaluronate was eluted in a narrow time frame, sensitivity was significantly enhanced, and the limit of detection of this method was 0.45 μg/mL. According to this quantitation method, the attached quantity of sodium hyaluronate is related to the water content of the material. Furthermore, a material test indicated that the release efficiency of sodium hyaluronate depends on the material of lenses. Nonionic Polymacon had a longer half-life in the sodium hyaluronate release curve than negative Methafilcon A and silicone hydrogel. Conclusion: This hyaluronate quantification method is a fast, sensitive and accurate method, making it suitable for the in vitro hyaluronate research without further derivatization.

Background: Chromatographic methods for determination of insulin degludec in rabbit plasma by Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry were developed. Methods: Analytes were eluted from Waters ACQUITY UPLC® Peptide BEH C18 (2.1×50mm, 300Å) column with a mobile phase of water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B). Quantitation of insulin degludec was performed using 1222.06 > 641.24 m/z on Multiple- Reaction Monitoring (MRM) mode. Results: Good linearity was observed in the concentration range of 500-50000 ng/mL (r >0.99), and the lower limit of quantification was 500ng/mL. The within-run and between-run precision (expressed as relative standard deviation, RSD) of insulin degludec were ≤ 14.16% and ≤ 13.64% respectively, and the accuracy was within 94.37-96.35%. The recovery and matrix effects were both within acceptable limits. Conclusions: This method was successfully applied for the pharmacokinetic study of insulin degludec in rabbit after subcutaneous administration.