Current Pharmaceutical Analysis - Volume 17, Issue 8, 2021

Pharmaceutical analysis plays an important role in all steps of drug development processes. Analysis of active pharmaceutical ingredients in biological samples needs sample preparation techniques to prevent the signal of the analyte from interferences coming from matrix components. Ultrafiltration is a well-known technique used in the food and pharmaceutical industry. Commercial ultrafiltration devices have been frequently used on proteomics and metabolomics studies for sample preparation. In pharmaceutical analysis, these devices have been employed to analyze the free concentration of drugs in biological fluids after filtration. However, they have been rarely used to determine the total concentration of targeted compounds when it is compared with some other common sample preparation techniques. Ultrafiltration-based sample preparation might be used to clean-up the sample easily from matrix components especially on bioanalysis performed with high-performance liquid chromatography (HPLC). In the case of using protein precipitation agents on filtration procedure, the quantitative recovery of this non-selective unique technique is competitive with solid-phase extraction.

Background: Metallic impurities are the traces of metals that can be found in finished drug products. Description: These metallic impurities in pharmaceutical preparations can enter from formulation ingredients, catalysts, and process equipment, containers and closures. They are not completely removed from the product by practical manufacturing techniques and should be evaluated relative to safetybased limits. They can affect drug efficacy or produce direct toxic effect on the patient. Methods: In this paper, an attempt has been made to review these metallic impurities including potential sources and analytical procedures to quantify these impurities. ICH guideline on these impurities and measures to control impurities has also been discussed in the paper. Results: The implementation of ICH Q3D guideline with the quality risk assessment approach is an important milestone to harmonize control of elements worldwide. Conclusion: This approach allows manufacturers to provide vital information about the contribution of impurities in the drug product.

Every "entity" or compound has physical and chemical properties as references for the synthesis and determination of the entity's structure. Thermodynamically, solid-state is the most stable matter in the universe and to be the ideal form in structure elucidation of pharmaceutical. The dry treatments, such as mechanochemistry, microwave heating, and the using of deep eutectic agent are becoming popular. These techniques are viewed as futuristic methods for reducing environmental damage, in line with "green pharmacy" concept. On the other hand, solid-state analysis methods from the simplest to the most sophisticated one have been used in the long decades, but most are for qualitative purposes. Recently many reports have proven that solid-state analysis instruments are reliable and prospective for implementing in the quantitative measurement. Infrared spectroscopy, powder x-ray diffraction, and differential scanning calorimetry have been employed in various kinetics and content determination studies. A revolutionary method developed for structural elucidation is single-crystal diffraction, which is capable of rapidly and accurately determining a three-dimensional chemical structure. Hereby it is shown that the accurate, precise, economic, ease, rapid-speed, and reliability of solidstate analysis methods are eco-benefits by reducing the reagent, catalyst, and organic solvent.

Background: Rifampicin (RIF), also known as rifampin, a bactericidal antibiotic having broad antibacterial activity against various gram-positive and gram-negative bacteria acts by inhibiting DNA dependent RNA polymerase. RIF has been administered in different dosage forms like tablets, capsules, injections, oral suspension, powder, etc. for the treatment of several types of bacterial infections, including tuberculosis, Mycobacterium avium complex, leprosy and Legionnaires’ disease. Introduction: To ensure the quality, efficacy, safety and effectiveness of RIF drug product, effective and reliable analytical methods are of utmost importance. To quantify RIF for quality control or pharmacokinetic purposes, alternative analytical methods have been developed along with the official compendial methods. Methods: In this review paper, an extensive literature survey was conducted to gather information on various analytical instrumental methods used so far for RIF. Results: These methods were high-performance liquid chromatography (42%), hyphenated techniques (18%), spectroscopy (15%), high-performance thin-layer chromatography or thin-layer chromatography (7%) and miscellaneous (18%). Conclusion: All these methods were selective and specific for the RIF analysis.

Background: Udenafil, a recently discovered drug used for erectile dysfunction treatment, has been widely prescribed and its effect on human systems has been extensively studied. However, there is little research on the human metabolites of udenafil. Three metabolites have been identified in rats. Objective: Herein, highly sensitive and accurate liquid chromatography–quadrupole time-of-flight tandem mass spectrometry (LC-Q-TOF-MS/MS) was conducted to identify new udenafil metabolites. Methods: Human liver microsomes were incubated with udenafil for in vitro samples, and rat urine and faeces samples were collected from udenafil-administered rats for in vivo samples. Each sample was deproteinated with acetonitrile and extracted by solid phase extraction. The purified samples were separated and analyzed by LC-Q-TOF-MS, and some metabolite candidates were reanalyzed for further structural analysis using LC-Q-TOF-MS/MS. Results: Eleven and three metabolites were identified in the in vitro and in vivo samples, respectively, and were found to be hydrolyzed, oxidized, or demethylated forms of udenafil or its metabolites. The error of the metabolic analysis was −8.7 to 7.6 ppm, indicating the high accuracy of the method. Conclusion: These metabolic results could be useful for further investigation of udenafil and new phosphodiesterase-5 inhibitors.

Background: The serum levels of Docosahexaenoic Acid (DHA), Eicosapentaenoic Acid (EPA) and Arachidonic Acid (AA) under the state of Pressure Ulcers (PUs) are still unclear. Introduction: In order to investigate serum levels of DHA, EPA, and AA in PUs rats, an ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/ MS) method was developed and validated. Methods: Chromatographic separation of DHA, EPA, AA was carried out on a BEH C18 column and gradient elute consisted of 5 mM ammonium acetate-0.1% formic acid and acetonitrile. Subsequently, fifty rats were divided into five groups (n=10), four PU groups (A-D) underwent various pressure and release time protocols, with group E as the control. The concentrations of DHA, EPA, AA from five groups were determined by using a validated method. Results: The results showed there was good linearity for DHA (327.3/283.4), EPA (301.2/257.0), and AA (303.1/258.9) within 0.05-6.4 μg/mL. In control group, the levels of DHA, AA and EPA were 1.16±0.68, 0.59±0.19 and 0.78±0.21 μg/mL. At the end of modeling, concentrations of DHA, EPA and AA were increased after long and persistent pressure (>8 h). Especially, the level of DHA was significantly higher (P<0.01) than that of control group. Conclusion: A stable, reliable and accurate UPLC-MS/MS for determination of DHA, EPA, AA in blood was developed. Serum concentrations of DHA, EPA and AA were altered differently after long and persistent pressure (>8 h), and DHA is a remarkable one.

Background: Cuscutae Semen (CS) is reported to show a hepatoprotective effect. Chlorogenic acid, hyperoside and astragalin are three major biologically active components from CS. Objective: A sensitive method based on ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed and validated to quantify the three components in rat plasma and was successfully used to study pharmacokineticsin liver injury rats. Methods: Plasma samples were prepared with protein precipitation by acetonitrile. Chromatographic separation was achieved on ACQUITY-XBridge BEH C18 column with gradient elution using the mobile phase containing 0.05% formic acid in water (A) and acetonitrile (B). The three components were quantified using Electrospray Ionization (ESI) source in the negative multiple Reaction Monitoring (MRM) mode. Results: Calibration curves of each analyte showed good linearity with correlation coefficients over 0.99. Accuracies (RE%) and precisions (RSD%) were within 15%. The method was stable. Recovery of the target compounds in plasma samples ranged from 87.00% to 102.29%. No matrix effect was found to influence the quantitative method. Conclusion: The UPLC-MS/MS method met the acceptance criteria and was successfully applied to the simultaneous determination of chlorogenic acid, hyperoside and astragalin in rat plasma for the first time. It is suitable for pharmacokinetic application in liver injury rats. It provides the foundation for further development and utilization of the hepatoprotective effect of cuscutae semen.

Background: D-amino acids are closely related to the development and progression of Alzheimer's disease (AD) and are expected as the novel biomarkers for AD diagnosis. Objective: The aim was to investigate the potential clinical value of D-amino acids for Alzheimer's disease. Methods: A simple and sensitive HPLC/MS-MS method was developed for the simultaneous determination of D-alanine, D-glutamine, D-proline and D-serine in rat urine. The samples were firstly pretreated by methanol, then derivatized by 7-chloro-4-nitrobenzoxadiazole with Fudosteine as internal standard, enantioseparated on Sumichiral OA-2500S column, using a mobile phase composed of acetonitrile- methanol (50:50, v/v) containing 0.5% formic acid, and detected with 4000 Qtrap MS/MS in electrospray-ionization source by negative ion mode. Results: The established method was successfully applied to determine the D-amino acid levels in rat urine from 20 Alzheimer's disease rats and 20 age-matched normal controls. The mean levels of Damino acids in the urine of Alzheimer's disease rats were all significantly lower than those in normal controls. Based on the contents of D-amino acids, the distinction model between Alzheimer's disease rats and normal controls was established by the Bayesian discriminant analysis. Conclusion: The relationship between Alzheimer's disease and D-amino acids revealed that D-amino acids would be potential biomarkers for Alzheimer’s disease.

Introduction: This study introduces an effective strategy, which combines high performance liquid chromatography coupled with diode array detection (HPLC-DAD) with multivariate calibration methods for the simultaneous determination of paracetamol (PAR), pseudoephedrine HCl (PSE), dextromethorphan HBr (DEX) and doxylamine succinate (DOX) along with sweetener saccharin (SAC) in syrup formulation. Methods: PLS-2 and PCR calibration algorithms were selected for data processing. Based on the strategy, all target analytes were rapidly quantified within 5.3 min under the simple isocratic elution (water: methanol, 20/80, v/v) without a complete separation. The performances of the proposed methods were confirmed by analyzing a series of synthetic solutions including different concentrations of analytes. Results: The average recovery values were in the range of 100.33 to 103.70%, and the REP (relative error of prediction) values ranged from 1.96 to 4.36% showed that these methods could provide satisfactory predictions. Conclusion: Novel HPLC methods coupled with PLS and PCR algorithm enable a simple, fast and low-cost analysis of similar pharmaceutical products for simultaneous determination of the target compounds.

Background: N’,N’-diethyl-m-toluamide (DEET) is the most widely used repellent substance worldwide. It is formulated as aerosol, solution, lotion, gel and patches. However, the official compendia report monographs to analyze only DEET drug substance and solution. Objective: In this study, an isocratic HPLC method was validated to assay DEET in lotion, gel and solution, under the same analytical conditions. Methods: The method was validated according to ICH requirements and DEET detection was achieved at around 11 min, using a C-18 column, a mobile phase composed by methanol, acetonitrile and water pH 4.5 (45:10:45), flow rate at 1 mL min^-1 and detection at 270 nm. Results: A linear relationship was observed in the range of 2.5 to 100 μg mL^-1, the method was precise (relative standard deviation < 2%) and accuracy was demonstrated by DEET recovery values ranging from 99.5 to 100.2%. The specificity was studied by a forced degradation test, where degradation products were observed after alkaline degradation and ultraviolet radiation. Appropriate resolution between DEET, degradation products and excipient peaks indicated the method specificity. Robustness was evaluated by a full factorial design, and no effect on DEET assay was observed under simultaneous variation in analytical parameters. The method was applied to assay nine marketed formulations, demonstrating its good applicability. Conclusion: The validated HPLC method was successfully applied to the quantitative analysis of DEET in lotion, gel and solution, contributing to improve the quality control and the efficacy of these formulations.

Background: The development of sound bioanalytical LC-MS (liquid chromatography-mass spectroscopy) method(s) is of paramount importance during the process of drug discovery, development and culminating in a marketing approval. The use of oral antidiabetic agents has been increased significantly from the last decades and till now no bioanalytical method is available for quantitation of sitagliptin (SG) and ertugliflozin (EG) in biological matrix which can be applied to pharmacokinetic studies using LC-MS/MS. Objective: To develop a new, rapid and sensitive LC-MS/MS method for the simultaneous estimation of sitagliptin (SG) and ertugliflozin (EG) in rat plasma by Liquid-Liquid Extraction method (LLE) using deutereated sitagliptin (SGd6) and ertugliflozin (EGd6). Methods: Chromatographic separation was carried out on a reverse phase Waters, Xetrra C18 (150mm x 4.6mm, 2μm) column using a mixture of acetonitrile and OPA buffer (50:50v/v) at a flow rate of 1ml/min in isocratic mode. Quantification was achieved using an electrospray ion interface operating in positive mode, under Multiple Reaction Monitoring (MRM) conditions. Results: The method showed excellent linearity over the concentration range of 5.00- 75.00pg/mL for sitagliptin and 0.75- 11.35pg/mL ertugliflozin. The intra-batch and inter batch precision (%CV) was ≤ 4.3% and matrix effect (%CV) was 0.02% and 0.12% for sitagliptin at HQC and LQC, respectively. Matrix effect (%CV) was 0.08% and 0.33% for ertugliflozin at HQC and LQC, respectively. Conclusion: The simplicity of the method allows for application in laboratories, presents a valuable tool for pharmacokinetic studies. The particular assay has been proficiently put on pharmacokinetic study in rats subjects.

Background: An enantiomeric separation of the stability-indicating high-performance liquid chromatographic method was developed and validated for the analysis of Meclizine enantiomers. The degradation behavior of Meclizine Hydrochloride was investigated under different stress conditions recommended by the International Conference on Harmonization (ICH). Methods: Enantiomeric resolution of the drug and complete separation from its degradation products were successfully achieved on a Phenomenex® lux cellulose 1 C18 (250 mm × 4.6 mm i.d, 5 μm particle size) column, using UV detector at a wavelength of 230 nm, with a mobile phase consisting of acetonitrile, 20mM ammonium bicarbonate at the ratio of 75:25 (v/v), and a flow rate of 1 mL/min. The drug was subjected to alkaline, acidic, neutral, oxidative and photolytic conditions in order to mimic stress conditions. Results: The degradation products were well resolved from the main peak, proving the stabilityindicating power of the method. The developed method provided linear responses within the concentration range of 1-5 μg/mL, and regression analysis showed a correlation coefficient value (r2) of 0.999. The HPLC method was validated as per ICH guidelines with respect to specificity, precision, linearity and robustness. Limit of Detection (LOD) and Limit of Quantification (LOQ) were found to be 0.25 μg/mL and 1.00 μg/mL, respectively. Conclusion: The method provides good sensitivity and excellent precision and reproducibility. The method was highly selective, in which degradation products and co-formulated compounds did not interfere. The proposed method was successfully applied in pharmaceutical preparations.

Background: In case of meningitis, the meninges are inflamed and the blood brain barrier is distorted, therefore, there is no hindrance to drug penetration. The problem arises when the disease is at the verge of cure, the meninges become uninflamed and the permeability of the drug is reduced to such extend that it becomes nearly impossible to maintain the minimum inhibitory concentration of drug at the site of infection. This problem was overcome by formulating ceftriaxone loaded NLCs and administrating it through intraperitoneal route to Wistar Albino rat. For quantitative assessment of drugs in rat serum and cerebrospinal spinal fluid, a new RP-UFLC (reverse-phase ultra-fast liquid chromatography) method has been developed and validated. Objective: Development and validation of RP-UFLC (using ion-pairing reagent) method for accurate estimation of ceftriaxone in rat serum and CSF. Methods: Method validation is done according to the ICH Guidelines (Q2) for the estimation of ceftriaxone in rat serum and its CSF (cerebrospinal fluid) by the RP-UFLC method. The blood was collected from the rat tail vein and CSF was collected carefully from cisterna magna of the rats by a 23G syringe. The mobile phase was used in a ratio of 70:30%v/v of phosphate buffer with ion-pairing reagent and acetonitrile with a pH of 8.0 Results: Limit of detection and limit of quantification of ceftriaxone in rat serum was 1.08 μg/ml and 3.84 μg/ml, respectively. Similarly, the limit of detection and limit of quantification of ceftriaxone in CSF of rats was 0.94 μg/ml and 2.84 μg/ml, respectively. In both cases, the R² value was more than 0.99 and showed 99% accuracy. Conclusion: The experimental results suggest that the new RP-UFLC method developed and validated can be effectively used to assess ceftriaxone in preclinical studies in rats.

Background: Alfuzosin is recently co-formulated with solifenacin for relieving two coincident urological diseases, namely; benign prostate hyperplasia and overactive bladder. Objective: Herein, green, simple and rapid spectrophotometric methods were firstly developed for simultaneous determination of the two cited drugs in their co-formulated pharmaceutical capsule. Methods: Alfuzosin, which is the major component in the dosage form, was directly assayed at its extended wavelength at 330.0 nm. The challenging spectrum of the minor component, solifenacin, was resolved by five spectrophotometric methods, namely; Dual Wavelength (DW) at 210.0 & 230.0 nm, first derivative (¹D) at 222.0 nm, Ratio Difference (RD) at 217.0 - 271.0 nm , derivative ratio (¹DD) at 223.0 and mean centering of ratio spectra (MC) at 217.0 nm. Results: The proposed methods were successfully validated as per ICH guidelines. Alfuzosin showed linearity over the range of 4.0 - 70.0 μg/mL, while that of solifenacin were 4.0 - 50.0 μg/mL for DW, 2.0 - 70.0 μg/mL for ¹D and RD methods, 1.0 - 70.0 μg/mL for ¹DD and 4.0 - 70.0 μg/mL for MC method. Statistical comparison with their official ones showed no noticeable differences. The methods showed good applicability for assaying drugs in their newly combination. Besides the eco-scale, the greenness profile of the methods was assessed and compared with the reported spectrophotometric one via the newest metric tool; Green Analytical Procedure Index (GAPI). Conclusion: The proposed methods are superior in not only being smart, accurate, selective, robust and time-saving, but also in using distilled water as an eco-friendly and cheap solvent.

Background: PARTIAL Least Squares (PLS) and Principal Component Regression (PCR) are two well-known chemometric methods based on dimension reduction techniques. They can be very practical analyzing a large data set of multiple correlated predictor variables. Objective: In the presented work, the resolving power of spectrophotometric assisted mathematical techniques was implemented for the simultaneous determination of two active ingredients; Ephedrine Hydrochloride (EPH) and naphazoline nitrate (NAPH), in a matrix of excipients. Methods: To build the PLS and PCR models, a calibration set was prepared where the two drugs, in combination with the interfering parabens, were modeled by multilevel multifactor design. The proposed models successfully predicted the concentrations of both drugs in validation samples with low Root Mean Squared Error of Prediction (RMSEP). Results: The results revealed the ability of the mentioned multivariate calibration models to analyze EPH and NAPH in the presence of the interfering parabens with high selectivity in the concentration ranges of 4.00-20.00 μg mL^-1 and 1.00-9.00 μg mL^-1, respectively. Conclusion: A commercially available nasal spray was successfully analyzed using the developed methods without interfering with other dosage form additives.