Current Pharmaceutical Analysis - Volume 17, Issue 9, 2021

Roxithromycin is one of the most frequently used macrolide antibiotics, a safe group of antimicrobials, that acts against Gram-positive bacteria and some Gram-negative bacteria. It is sold in several countries in different dosage forms (tablets and capsules) and strengths (50, 100, 150 and 300 mg). Several analytical methods have been described to quantify roxithromycin in different matrices, such as biological and environmental samples and food. Identifying the main characteristics of the drugs and selecting appropriate analytical methods for their quantification are of paramount importance for understanding the behavior of drugs, metabolites and impurities. This review presents the physical-chemical and microbiological characteristics, properties as well as methods for quantification of roxithromycin in biological fluids, pharmaceutical preparations, food and environmental samples. HPLC coupled to various detection systems is the most used analytical technique to determine roxithromycin in these matrices. Although many analytical methods have been reported for the analysis of this drug, it is very important to include in this context a prospective view to implement new analytical technologies for the quality control of this antimicrobial that contributes to the preservation of economic and environmental impacts.

Background: Neuropathy is the most common perplexity of diabetes type 1 and 2. About 50% of diabetic patients develop diabetic neuropathic pain (DNP). At the beginning of diabetic neuropathy, there is a loss of sense, especially in the lower limb, accompanied by pain, and difficulty in movement. Glucose regulation effectively prevents the development of diabetic neuropathy in type 1 diabetic patients, but the consequences in type 2 diabetic patients are more drastic. Introduction: No single treatment exists to prevent or renovate the pain caused by diabetic neuropathy. The drugs used for the treatment of DNP come in various formulations and with different storage conditions. To date, a number of analytical methodologies have been reported for the analysis of DNP drugs. Few reports are published describing analytical methods of single DNP drugs. Methods: The main objective of this review is to compile the different analytical methods developed at UV-Vis (Ultraviolet-visible) spectrophotometer, HPLC (High Performance Liquid Chromatography), and LC-MS (Liquid Chromatography-Mass spectrometry) to identify and quantify the drug content in various formulations, which are used to treat or prevent the diabetic neuropathic pain mainly focusing on γ-aminobutyric acid analogues, anti-depressants, Serotonin Noradrenaline Reuptake Inhibitors (SNRIs), aldose reductase inhibitors, opioids, and dietary supplements. Results and Discussion: We have compiled UV-Vis, HPLC, and Liquid Chromatography-Mass spectrometry analytical methods developed to study the pharmacokinetic profile, quantify drug content, and their metabolites in plasma as well as pharmaceutical formulations. Conclusion: The authors believe that the mentioned studies in the report will help audible readers to select a suitable method for analysis of these drugs and also help researchers to develop a more convenient, fast, and sensitive method for these.

Pharmaceuticals are the chemical molecules which were invented to improve the life of living things through diagnosing, curing, treating, or preventing diseases. Pharmaceuticals can do their maximum function or work against disease or prevent from harmful effects of diseases only if they are free from impurities. The pharmaceutical analysis consists of different essential and important procedures to ascertain “identity, strength, quality and purity” of drug molecules. There are three different possibilities to originate impurities, which include (i) at the developmental stages of pharmaceuticals, (ii) at the time of transportation, and (iii) storage conditions. Therefore, the pharmaceuticals must be examined at all stages and the impurities should be discovered and quantified. The pharmaceutical analysis also admits that the analysis of intermediates and raw materials during the synthesis of drug molecules. Instrumentation is required to perform pharmaceutical analysis and methods can be developed to certain required levels. This review affords the basic theory of UV-Visible spectrophotometry and its applications, pharmaceutical method development and validation, and a list of some reported spectrophotometric methods, which are employed in pharmaceutical analysis from 2011 to 2020.

Background: Since propofol is rapidly metabolized and excreted from the body, it is not easy to quantify its intake in blood or urine samples over the time. In this case, the hair sample would be more advantageous to estimate during the abuse period. However, the presence of protein and lipid in the hair sample could interfere with extraction and be problematic during mass spectrometric analysis. Objective: The aim of this study is to develop a simple and less time-consuming method for the extraction of propofol glucuronide by removing hair interferences with the centrifugal filter. Methods: Hair samples were washed and dissolved with sodium hydroxide solution. This dissolved hair solution was applied to the centrifugal filter and centrifuged. The filtrate was extracted with ethyl acetate and evaporated to dryness. The residue was reconstituted with methanol and analyzed by liquid chromatography coupled with tandem mass spectrometry. This developed analytical method was validated by testing linearity, selectivity, accuracy, precision, recovery, matrix effect and stability of propofol glucuronide. Results: The validation results showed good linearity over the concentration range of 0.5~500 pg/mg, with a correlation coefficient of 0.9991. The LOD and LLOQ were 0.2 and 0.5 pg/mg, respectively. The intra-and inter-day precision and accuracy were acceptable within 14.5% for precision and 10.1% for accuracy. Similarly, the developed method revealed high sample recovery (>88%), low hair matrix effect (<10%) and highly-efficient extraction procedure. Conclusion: This well-validated procedure was successfully applied to determine propofol glucuronide in a rat hair sample and can be applicable, with high potential, in the field of forensic toxicology especially with increasing abuse and accidental overdose of propofol.

Background: Morin flavonoid exerts neuroprotective effects with potential interest in neurodegenerative disorders. For this, the use of surface-modified polymeric nanoparticles loaded with morin is an interesting approach. Objective: To develop and validate an HPLC method for the quantification of morin released from a new delivery system consisting of poly lactic-co-glycolic (PLGA) nanoparticles functionalized with the dipeptide phe-phe (phenylalanine-phenylalanine) used to facilitate their access to the CNS. Methods: The HPLC procedure was developed and validated with morin hydrate dissolved either in methanol (method A) or in methanol: 0.1N HCl (50:50, v/v, method B). Two new nanoparticle formulations were developed and characterized: morin-loaded PLGA nanoparticles (formulation F1), and morin-loaded PLGA phe-phe nanoparticles (formulation F2). Results: Method A was linear within the concentration range of 5-30 μg.mL^-1 and, 1-30 μg mL^-1 for method B. LOD and LOQ with method A were 1.23 μg.mL^-1 and 3.90 μg.mL^-1, respectively, and 0.481 μg.mL^-1 and 1.458 μg.mL^-1, respectively for method B. The average amount of phe-phe bound to formulation F2 (50 mg of NPs) was 431.33 μg. The encapsulation efficiency of morin within PLGA nanoparticles was around 80%. After functionalization, this value decreased significantly. Conclusion: Method B showed better sensitivity, accuracy, and precision for the quantification of morin. The procedure used to functionalize the nanoparticles was adequate for linking the dipeptide to their surfaces, but this procedure is not adequate when encapsulating water-soluble compounds.

Background: The activation or overexpression of histone deacetylases (HDACs) are associated with the development of numerous cancers. The importance of HDACs in new anticancer therapeutic strategies has led researchers to develop promising anticancer drugs, design, and synthesis of novel HDACs inhibitors are also in progress. E14 was synthesized and acted as a selective inhibitor of HDACs I. Methods: We develop a sensitive UPLC-QTOF-MS/MS method to discover the new phase I and II metabolites of E14 from complex biological matrices in plasma, feces, and urine of rats. Then a strategy comparing samples after administration with blank samples was applied to distinguish metabolites. The accurate measurements of the product ions obtained by high-resolution mass spectrometry, the structure of the parent drug and its detected metabolites are used to verify its fragmentation pathway and to obtain the proposed structure of the metabolites. Results: Fourteen metabolites were tentatively identified, including twelve metabolites in urine, five in plasma and one metabolite in feces. Hydroxylation, oxidation, methylation, N-demethylation, dehydration, glucuronidation, dehydroxylation and hydrolysis were the main metabolic pathways of E14. Conclusion: E14 is a drug with a variety of metabolites and metabolic pathways. E14 metabolites was excreted most in urine, indicating the main metabolic organ was the kidney.

Background: Current methods for assaying bioactivity of GLP-1 analogues are costly and time consuming, mainly dependent on AlphaScreen luminous beads, based on fluorescent resonance energy transfer technology, and cAMP-GloTM Max assay kit, and on ATP to generate a negative- association curve. Objective: To establish a sensitive, stable, and low-cost method for the determination of biological activity of therapeutic glucagon-like peptide-1(GLP-1) and their analogous drugs in vitro in quality control. Methods: An applicable fluorescent high-content system (fHCS) method was developed in U2OS cell, which was stably transfected with green fluorescent protein (GFP) labeled at the C-terminal of glucagon-like peptide-1 receptor (GLP-1R). HCS was firstly used to measure per unit area of fluorescent intensity changes as a result of drug-stimulated receptor endocytosis. Results: The detection sensitivity improved at least 30 folds than the other reported methods. This method showed a wide detection range with a good linear regression of R2>0.98. In five scgs from 50~150% SCG, the accuracy achieved the standard of the 41st United States Pharmacopoeia with all biases being less than 7.5%. The precision attained the 9.0th European Pharmacopoeia standard with all relative standard deviations (RSDs) below 10%. This assay result can be kept stable over 100 h at room temperature. Furthermore, the U2OS/GLP-1R cells showed a sharp specificity towards GLP-1 analogues during detecting activities in a mixture of different ligands. Conclusion: These results suggested that the GLP-1R/fHCS method can be employed broadly in the bioactivity determination of GLP-1 analogous drugs and as a platform for screening GLP-1 similar active ingredients in natural products.

Objectives: Riptolide (TPL) has been shown to have a good clinical effect on rheumatoid arthritis (RA). We designed TPL microspheres (TPL-MS) and investigated its metabolic behavior in human, dog, rabbit and rat liver microsomes (HLM, DLM, RLM and SDRLM) with UPLC- MS/MS method. Methods: First, a UPLC-MS/MS method was established to measure the concentration of TPL in samples. The sample was separated on a C18 column (2.100 mm, 1.8μm) and eluted with a gradient elution. The precursor ions/product ions were m/z 378.1/361.0 for TPL and 260.0/116.2 for the internal standard. Then T1/2, Vmax and CLint were calculated from the above data. Finally, the metabolites of TPL-MS were identified by high-resolution UPLC-MS/MS. The sample was separated on a C18 column (2.100 mm, 2.2 μm) and eluted with isocratic elution. Mass spectrometric detection was carried out on a thermo Q-exactive mass spectrometer with HESI. The scanning range of precursor ions was from m/z 50 to m/z 750. Results: Through several indicators including standard curve, precision, accuracy, stability, matrix effect and recovery rate, the enzymatic kinetics parameters including T1/2, Vmax and CLint were evaluated. Several metabolites of TPL-MS were identified. Discussion: UPLC-MS/MS method is an accurate and sensitive method for the determination of TPL in liver microsome samples with good precision, accuracy and stability. The variation of parameters indicated that the microspheres can delay the elimination of TPL in liver microsomes. Conclusion: The metabolism of TPL-MS varied among species, but no new metabolites appeared.

Background: Cannabidiol (CBD) is the main, physiologically active, but psycho-inactive constituent of the glandular hairs of Cannabis sativa. CBD exhibits diverse pharmacological activities and it is used as an anticonvulsant, sedative and hypnotic agent. Other biological properties shown by CBD are antipsychotic, anti-inflammatory and neuroprotective. Objective: The aim of this study is to develop, validate and apply high performance liquid chromatography (HPLC/DAD) method with diode array detection for identification and assay of CBD in food supplements obtained by different types of extraction and purification. Method: HPLC method with isocratic elution using column C18 ODS, 250 x 4.6, 100 Å, 5 μm, mobile phase - a mixture of acetonitrile and water in ratio 80:20 v/v, a flow rate of 1.0 ml/min and DAD detection at 220 nm, was used. Results: The method was developed and validated according to European Pharmacopoeia and International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH). For the analytical purposes, parameters like repeatability, linearity, accuracy, LOD, LOQ, specificity and system suitability were studied. The developed method was successfully applied on a series of food supplements containing CBD. Conclusion: The developed method is of practical importance for the determination of purity, identification and assay tests of food supplements containing CBD extract in accordance with EU regulation concerning food authorizations because it is useful for analytical tests for identification, purity determination and assay of products containing CBD extract and could serve as part of the testing process in the pharmaceutical industry.

Aim: EPAS (evaporative precipitation into aqueous solution) was used in the current studies to prepare azithromycin nanosuspensions and investigate the physicochemical characteristics for the nanosuspension batches with the aim of enhancing the dissolution rate of the nanopreparation to improve bioavailability. Methods: EPAS method used in this study for preparing azithromycin nanosuspension was achieved through developing an in-house instrumentation method. Particle size distribution was measured using Zetasizer Nano S without sample dilution. Dissolved azithromycin nanosuspensions were also compared with raw azithromycin powder and commercially available products. The total drug content of nanosuspension batches was measured using an Ultra-Performance Liquid Chromatography (UPLC) system with Photodiode Array (PDA) detector while residual solvent was measured using Gas Chromatography (GC). Results: The average particle size of azithromycin nanosuspension was 447.2 nm and total drug content was measured to be 97.81% upon recovery. Dissolution study data showed a significant increase in the dissolution rate for nanosuspension batch when compared to raw azithromycin and commercial version (microsuspension). The residual solvent found for azithromycin nanosuspension is 0.000098023 mg/mL or 98.023 ppb. Conclusion: EPAS was successfully used to prepare azithromycin nanoparticles that exhibited a significantly enhanced dissolution rate. Further studies are required to scale up the process and determine long term stability of the nanoparticles.

Background: The presence of residual solvents in pharmaceuticals can be a potential risk factor to human health because of its toxicity. Gas chromatography is used for its excellent separation abilities and lower limit of detection. Racecadotril, an antidiarrheal drug, is reported having four residual solvents, namely n-Hexane, isopropyl alcohol, toluene, and dimethylformamide. Estimation of the amount of these solvents in active pharmaceutical ingredients to ensure that they are within the permissible limits as per ICH guidelines is necessary. Objective: To develop and validate a new, simple, and sensitive gas chromatographic method for simultaneous determination of n-Hexane, isopropyl alcohol, toluene, and dimethylformamide in racecadotril. Methods: The residual solvents of racecadotril were estimated using a gas chromatographic method by direct injection using FID as a detector. This method employed a 30-meter long DB-FFAP nitroterephthalic-acid-modified polyethene glycol column with 0.53 mm in inner diameter and 1 μm film thickness. The separation was achieved using nitrogen as the carrier gas at a flow rate of 2.8 mL/min using a split ratio of 1:10. Results: The peak shape for all the residual solvents from racecadotril was symmetric with excellent resolution eluting at reasonable retention time. The limit of detection of n-Hexane, isopropyl alcohol, Toluene, and dimethylformamide was found to be 6, 27, 14, and 42 ppm, respectively. The developed method exhibited excellent linearity for each residual solvents in the range studied. Conclusion: The developed gas chromatographic method is simple, specific, precise, accurate, and sensitive. Hence, the method can be successfully used in in the pharmaceutical companies and research laboratories for simultaneous determination of residual solvents in racecadotril active pharmaceutical ingredients.

Background: Haemodialysis is a process to remove the waste products and excess fluids that build up in the human body when kidneys stop working. Haemodialysis concentrate consists of two parts, acid concentrate (Part A) that contains mineral ions (Na, K, Mg and Ca) and buffering agent acetic acid /citric acid whereas basic concentrate (Part B) comprises sodium bicarbonate alone or in combination with sodium chloride. Aim: Present research aimed to develop and validate a rapid method for the determination of cations and anions in haemodialysis concentrates in solution and powder form by choosing one technique among various suggested in ISO 13958:2014. Methods: The analysis was done by flame emission (flame photometer), atomic absorption spectrometer (AAS), polarimetric and titrimetric techniques. ICH guidelines were followed for method validation by addressing the linearity, accuracy, precision and detection limits (LOD and LOQ). Results and Discussion: The calibration curves were linear for Mg, Ca, K and Na with coefficient of determination (R²) ≥ 0.9978 and regression data indicating the excellent linearity for the selected concentration range for each content. The recovery results were obtained between the ranges 96.9-102.5 % which were good enough to justify the suitability of the proposed method for its intended applications. Statistical analysis indicated that there was no significant difference was observed between the proposed and compendial method at 95 % confidence of interval. Conclusion: The advantage of the proposed method over the compendial method by virtue of its economy and fast with reliable results because more reagents and chemicals are consumed in the compendial method.