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Accurate, sensitive and green HPTLC chromatographic method was proposed for simultaneous determination of metformin, glipizide and sitagliptin in the presence of metformin potential toxic impurities melamine and cyanoguanidine. The separation was completed on silica gel HPTLC F254 plates using a mixture of ethyl acetate: methanol: ammonia: formic acid (7: 2: 0.2: 0.2, by volume) as a developing system with UV scanning for the developed bands at 235 nm. The Rf values for metformin, glipizide, sitagliptin, melamine and cyanoguanidine were 0.17, 0.84, 0.67, 0.47 and 0.75, respectively. Linear responses were observed in the ranges of 0.2-3, 0.07-1.5, 1.5-5, 0.8-4 and 0.4-2 μg/band with correlation coefficients of 0.9999, 0.9998, 0.9997, 0.9996 and 0.9998 for metformin, glipizide, sitagliptin, melamine and cyanoguanidine, respectively. The proposed method was validated as per ICH criteria with respect to linearity, accuracy, precision, specificity and robustness. The validated method was successfully applied for determination of the studied drugs in Janumet® and Engilor® tablets; also, the results were statistically compared to those obtained by the reported spectrophotometric method and no significant difference was found between them. This method permitted the accurate simultaneous determination of the studied drugs, indicating its ability to be used for routine quality control assays of these drugs., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background and Objectives: Rheumatoid factor (RF) and anti-cyclic citrullinated protein (anti-CCP) have been used to improve the diagnosis and prognosis of rheumatoid arthritis (RA). However, their association with RA disease phenotypes, individually and in combination, is not well studied. The aim of the study was to compare patients' and disease characteristics, activity and severity in double seronegative (DNRA), single seropositive RF, single seropositive anti-CCP and double seropositive (DPRA) patients., Methods: Adults subjects with RA from Egyptian College of Rheumatology (ECR) database who had RF and anti-CCP results available were included. Demographic, clinical features, disease activity score 28 (DAS28), Health Assessment Questionnaire (HAQ) and laboratory data were collected and compared among different RA groups., Results: 5268 RA patients with mean age of 44.9±11.6 years, and 4477 (85%) were females. 2900 (55%) had DPRA, 892 (16.9%) had single positive RF, 597 (11.3%) had single positive anti-CCP while 879 (16.7%) had DNRA. Patients with DPRA had significantly high percentage of metabolic syndrome (19.3%, P < 0.001), and functional impairment using HAQ ( P = 0.01). Older age (RRR [relative risk ratio]: 1.03, 95%CI: 1.0, 1.0, P = 0.029), greater DAS28 (RRR: 1.51, 95%CI: 1.2, 1.9, P < 0.001), higher steroid use (RRR: 2.4, 95%CI: 1.36, 4.25, P = 0.002) were at higher risk of DPRA while longer disease duration (RRR: 1.08, 95%CI: 1.01, 1.16, P = 0.017) and fibromyalgia syndrome (RRR: 2.54, 95%CI: 1.10, 5.88, P = 0.028) were associated with higher odds of single positive RF status., Conclusion: Dual antibody-positive status has higher disease activity and severity, and higher chance of development of metabolic syndrome; highlighting the implicated role of inflammation, atherogenesis and cardiovascular disease risk in RA., Competing Interests: Conflict of Interest The authors declare no conflict of interest., (© 2024 Nevin Hammam, Passant N. El-Husseiny, Suzan S. Al-Adle, Nermeen Samy, Nora Y. Elsaid, Dina F. El-Essawi, Eman F. Mohamed, Samar M. Fawzy, Samah A. El Bakry, Maha Nassr, Samah I. Nasef, Hanan M. El-Saadany, Shereen Elwan, Nada M. Gamal, Abdelhfeez Moshrif, Osman Hammam, Rawhya R. El Shereef, Faten Ismail, Samar Tharwat, Doaa Mosad Mosa, Mervat I Abd Elazeem, Enas A. Abdelaleem, Tamer A. Gheita; on behalf of The Egyptian College of Rheumatology (ECR) Rheumatoid Arthritis (ECR-RA) Study Group, published by De Gruyter on behalf of NCRC-DID.)

Bromhexine (BR), guaiafenesin (GUF) and salbutamol (SAL) are formulated as Ventocough syrup® (with and without sugar), labeled to contain propyl paraben and sodium benzoate as inactive ingredients. They are used to make coughing more productive and easier. A crucial element and a major issue in the pharmaceutical industry is the control of organic related impurities to obtain safe and effective treatment. Guaiacol (GUL) is reported to be GUF related impurity that was proved to be extremely toxic (toxic rating class 5), and its use should be banned. In this work, In-Silico study and ADMET estimation were conducted to predict GUL pharmacokinetic properties and its toxicity profile. Additionally, two chromatographic methods were conducted to analyze the studied components along with GUF impurity in the presence of the labeled dosage form excipients. The In-Silico study assured that GUL has oral rat acute toxicity and it is considered to be skin sensitizer. On the other hand, the developed TLC- densitometeric method depended on using a mobile phase mixture of hexane: methylene chloride: triethylamine (5.0:6.0:0.3, by volume) as a developing system. UV-Scanning was performed immediately at 275 nm for SAL, GUF and GUL, while scanning at 310 nm was used for scanning BR. Linearity was established in the ranges of 0.25-4.0, 0.25-4.0, 0.5-8.0 and 0.1-1.6 µg/band for BR, SAL, GUF and GUL, respectively. In the developed HPLC method, separation was performed on X-Bridge® C 18 column (250 × 4.6 mm, 5 μm) using a solvent mixture of 0.05M disodium hydrogen phosphate pH 3 with aqueous phosphoric acid: methanol (containing 0.3%, v/v triethylamine) (40:60, v/v). Detection was done at 225 nm and separation was achieved within 10 min. Linearity was proved in the range of 2-50 µg/mL for the proposed drugs. Validation of the developed methods was done and all the calculated parameters were within the acceptable limits recommended by ICH guidelines. After that, methods were used to examine the potency of the selected marketed dosage forms and concentrations of all drugs were within the acceptable limits. Additionally, complete separation between the studied drugs and the additives were observed. The developed methods can be used during routine quality control analysis of the proposed drugs when the required issues concern on sensitivity, selectivity and analysis time., (© 2024. The Author(s).)

The common antihypertensive drugs are B-blockers and diuretics. For the determination of beta-blocker medicines (bisoprolol fumarate and carvedilol) and diuretic drug (Furosemide), new and accurate chromatographic method has been developed. The separation was achieved using a developing system that includes chloroform:methanol:ethyl acetate:ammonia (6:2:2:0.2 by volume) as a mobile phase and the bands were detected at 240 nm. The concentration ranges were 5-25, 1-7, and 1-3.5 μg/band for bisoprolol fumarate, carvedilol, and furosemide, respectively. This chromatographic approach is the first methodology for simultaneously determining bisoprolol fumarate, carvedilol, and furosemide in their pure forms and in their pharmaceutical dosage forms. The advantages of using known analytical procedures are their simplicity, speed, cost effectiveness, lack of laboriousness, and ability to save time as the three tablets are determined in one step and can be used for routine analysis of the investigated combinations in quality control laboratories. According to International Conference of Harmonization guidelines, the established procedures have been validated, and the results were statistically compared to those obtained by the reported reversed-phase-high-performance liquid chromatography methods using Student's t-test and F-test, with no significant difference between them, indicating that the proposed methods can be used for routine drug quality control analysis., (© 2023 Wiley-VCH GmbH.)

Objective: To study the prevalence and impact of comorbidities among a cohort of patients with systemic lupus erythematosus (SLE)., Methods: This study is retrospective, multicenter including 902 Egyptian patients with SLE. Medical records were reviewed for demographic data, clinical characteristics, routine laboratory findings, immunological profile, and medications. Moreover, SLE Disease Activity Index (SLEDAI), and the Systemic Lupus International Collaborating Clinics/American College Rheumatology Damage Index scores were calculated., Results: Comorbidities were found in 75.5% of the studied group with hypertension and dyslipidemia as the most frequent comorbidities (43.1% and 40.1%, respectively), followed by sicca features, avascular necrosis, diabetes, osteoporosis and renal failure (11.5%,9%, 9%,8.9%, and 7.1%, respectively). Multivariate regression model showed statistically significant relation between the presence of comorbid condition and each of age ( P  = 0.006), disease duration ( P  = 0.041), SLEDAI at onset ( P  < 0.001), cyclophosphamide intake ( P  = 0.001), and cumulative pulse intravenous methylprednisone ( P  < 0.001). Also, when adjusted to age and sex, those with multiple comorbid conditions had 18.5 increased odds of mortality compared to those without comorbidities (odds ratio (OR), 95% confidence interval (CI) = 18.5 (6.65-51.69)]., Conclusion: Patients with SLE suffer from several comorbidities, with an increasing risk with age, longer disease duration, higher SLEDAI at onset, cyclophosphamide intake and cumulative pulse intravenous methylprednisone. Risk of mortality is exponentiated with multiple comorbidities., Competing Interests: Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Green, selective and accurate high-performance liquid chromatography (HPLC) chromatographic method is presented for simultaneous separation and quantitation of the co-prescribed drugs in chemotherapy omeprazole, ondansetron and deflazacort in spiked human plasma. An isocratic HPLC separation was performed on X Bridge C18 (4.6 × 250 mm) column with 5 μm particle size using mobile phase consisting of methanol: ammonium acetate buffer pH 4 adjusted by acetic acid (60: 40, v/v). The injection volume was 20μL with UV detection wavelength at 237 nm at room temperature. Flow rate of the mobile phase was adjusted to be 2.0 ml/min. Dexamethasone was used as internal standard to correct the variation during sample pretreatment. FDA guidelines were followed to validate the developed method. Successful application of the developed method was revealed by simultaneous determination of omeprazole, ondansetron and deflazacort in spiked human plasma in ranges of 1-20, 0.1-8 and 0.2-8 μg mL-1 for omeprazole, ondansetron and deflazacort, respectively. Four greenness assessment tools were used to evaluate the greenness of the developed method and the results were accepted. This method permitted the accurate simultaneous determination of the studied drugs, thus it can be used during therapeutic drug monitoring in daily clinical practice., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Rheumatoid arthritis (RA) is a chronic auto-inflammatory disease of connective tissue with progressive joint damage and systemic disorders. RA is considered a multifactorial disease triggered by a genetic predisposition and environmental factors. Polymorphisms have been identified in the Xq28 locus as risk loci for RA. The aim of study was to assess the association between two polymorphisms in the Xq28 region containing Transmembrane Protein 187 (TMEM187) gene (rs13397) and interleukin1 receptor associated kinase (IRAK1) gene (rs1059703) with the disease susceptibility and activity in Egyptian RA patients. This study was conducted on 100 RA patients and 100 age and sex matched normal controls, together were recruited from the Rheumatology Department, Beni-Suef University Hospital. We detected TMEM187 (rs13397) and IRAK1 (rs1059703) gene polymorphisms using the real time PCR TaqMan allelic discrimination assays. We found that the frequency of the major genotypes (GG) of TMEM 187 gene was higher in RA group (54%) compared to controls (50%); while the minor genotypes (AA) was higher in the control group (22%) compared to the diseased one (18%), but such differences did not reach statistical significance (p=0.599). Regarding the IRAK1 gene, we revealed that the frequency of the major genotypes (AA) of the rs1059703 was slightly higher in RA group (48%) compared to controls (46%); while the minor genotypes (GG) was the same in both groups (26%). However, there was higher incidence of minor genotype in the TMEM187 and IRAK1 genes in males; with a statistical significance (p=0.004 and 0.015, respectively). We concluded that the major allele G of TMEM187(rs 13397) could be considered as a risk genetic allele for RA in Egyptian populations., (Copyright© by the Egyptian Association of Immunologists.)

Secondary osteoporosis is commonly caused by long-term intake of glucocorticoids (GCs), such as dexamethasone (DEX). Diosmin, a natural substance with potent antioxidant and anti-inflammatory properties, is clinically used for treating some vascular disorders. The current work targeted exploring the protective properties of diosmin to counteract DEX-induced osteoporosis in vivo. Rats were administered DEX (7 mg/kg) once weekly for 5 weeks, and in the second week, vehicle or diosmin (50 or 100 mg/kg/day) for the next four weeks. Femur bone tissues were collected and processed for histological and biochemical examinations. The study findings showed that diosmin alleviated the histological bone impairments caused by DEX. In addition, diosmin upregulated the expression of Runt-related transcription factor 2 (Runx2) and phosphorylated protein kinase B (p-AKT) and the mRNA transcripts of Wingless (Wnt) and osteocalcin. Furthermore, diosmin counteracted the rise in the mRNA levels of receptor activator of nuclear factor-kB ligand (RANKL) and the reduction in osteoprotegerin (OPG), both were induced by DEX. Diosmin restored the oxidant/antioxidant equilibrium and exerted significant antiapoptotic activity. The aforementioned effects were more pronounced at the dose level of 100 mg/kg. Collectively, diosmin has proven to protect rats against DEX-induced osteoporosis by augmenting osteoblast and bone development while hindering osteoclast and bone resorption. Our findings could be used as a stand for recommending supplementation of diosmin for patients chronically using GCs., Competing Interests: Conflict of Interest Statement The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

To depict the spectrum of rheumatoid arthritis (RA) in Egypt in relation to other universal studies to provide broad-based characteristics to this particular population. This work included 10,364 adult RA patients from 26 specialized Egyptian rheumatology centers representing 22 major cities all over the country. The demographic and clinical features as well as therapeutic data were assessed. The mean age of the patients was 44.8 ± 11.7 years, disease duration 6.4 ± 6 years, and age at onset 38.4 ± 11.6 years; 209 (2%) were juvenile-onset. They were 8750 females and 1614 males (F:M 5.4:1). 8% were diabetic and 11.5% hypertensive. Their disease activity score (DAS28) was 4.4 ± 1.4 and health assessment questionnaire (HAQ) 0.95 ± 0.64. The rheumatoid factor (RF) and anti-cyclic citrullinated peptide (anti-CCP) were positive in 73.7% and 66.7% respectively. Methotrexate was the most used treatment (78%) followed by hydroxychloroquine (73.7%) and steroids (71.3%). Biologic therapy was received by 11.6% with a significantly higher frequency by males vs females (15.7% vs 10.9%, p = 0.001). The least age at onset, F:M, RF and anti-CCP positivity were present in Upper Egypt (p < 0.0001), while the highest DAS28 was reported in Canal cities and Sinai (p < 0.0001). The HAQ was significantly increased in Upper Egypt with the least disability in Canal cities and Sinai (p = 0.001). Biologic therapy intake was higher in Lower Egypt followed by the Capital (p < 0.0001). The spectrum of RA phenotype in Egypt is variable across the country with an increasing shift in the F:M ratio. The age at onset was lower than in other countries., (© 2023. The Author(s).)

Objectives: Leptin and adiponectin are adipose-derived immune modulators (adipokines) that may contribute to SLE pathology and symptoms. This study aimed to evaluate the associations of serum adiponectin and leptin with clinical manifestations and disease activity in SLE patients. Methods: This is a case control study, where 70 SLE patients and 50 age- and sex-matched healthy controls were enrolled from the Rheumatology and Rehabilitation Department of Beni-Suef University Hospital from June 2020 till April 2022. The SLE disease activity index (SLEDAI) and Systemic Lupus International Collaborative clinics/America Collage of Rheumatology damage index were used to assess disease severity. Laboratory parameters including erythrocyte sedimentation rate (ESR) and serum concentrations of antinuclear antibody (ANA), anti-double stranded DNA, complement 3 and 4, lipids, and C-reactive protein (CRP) were measured and compared between SLE and control groups. Serum adiponectin and leptin were also measured by enzyme-linked immunosorbent assays (ELISA). Results: Compared to healthy controls, SLE patients exhibited significantly greater serum leptin (21.1 vs 3.9 ng/mL, p < 0.001) and adiponectin (18.1 vs 4.8 ng/mL, p < 0.001), and both values were positively correlated with SLEDAI scores ( p = 0.048 and 0.042). Higher serum leptin was significantly associated with lupus nephritis (LN) ( p = 0.048) as well as greater body mass index ( p = 0.010), ESR ( p = 0.002), serum CRP ( p = 0.003), total cholesterol ( p = 0.013), and uric acid ( p = 0.002), while higher adiponectin was significantly associated with LN ( p = 0.046). Conclusion: Serum leptin and adiponectin levels are associated with the clinical and pathological manifestations of SLE, suggesting direct involvement in disease progression and utility as diagnostic biomarkers and therapeutic targets.

Objectives: The aim of this work was to explore the expression of miR-320a level in fibromyalgia patients in comparison to healthy controls, and to clarify its impact on the severity of symptoms and the cerebral processing of pain assessed by middle latency somatosensory evoked potentials (SSEPs)., Design: Case-control study., Setting: Rheumatology and Neurology outpatient clinics., Subjects: Seventy-four fibromyalgia patients and seventy-four normal healthy controls., Methods: The included patients were subjected to detailed history taking, assessment of severity of fibromyalgia symptoms using the Fibromyalgia Impact Questionnaire Revised (FIQR), assessment of pain intensity using the Neuropathic Pain Symptom Inventory (NPSI), measurement of the serum level of miR-320a in addition to of measurement peak latencies and amplitudes of middle latency SSEPs., Results: Fibromyalgia patients had significantly higher micro-RNA-320a levels (0.907 ± 0.022) in comparison to controls (0.874 ± 0.015) (P-value < .001). The mean values of micro-RNA-320a levels were significantly higher in fibromyalgia patients with insomnia, chronic fatigue syndrome, persistent depressive disorder, and primary headache disorder than those without (P-value = .024, <.001, .006, .036 respectively). There were statistically significant positive correlations between micro-RNA-320a levels, and disease duration, FIQR, and NPSI total scores (P-value <0.001, 0.003, 0.002 respectively). There were no statistically significant correlations between micro-RNA-320a levels and middle latency SSEPs., Discussion: Micro-RNA-320a level is significantly upregulated in fibromyalgia patient. It has a crucial impact on the severity of symptoms but not related to the cerebral processing of pain., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Academy of Pain Medicine. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Tetracyclines are frequently employed in animal husbandry. Bromhexine is a mucolytic drug that improves the efficacy of tetracyclines. It has been reported that residues of tetracyclines in milk may have negative effects on humans. Two versatile and accurate methods were developed for concurrent analysis of oxytetracycline (OTC) and bromhexine (BR) residues in spiked milk samples. Sample preparation was carefully considered for extraction and cleanup using the ecofriendly chemicals acetic acid, 0.1 N EDTA and ethanol or methanol. The first method was a TLC-densitometric method in which TLC plates previously treated with 10% EDTA (pH 9 with 40% sodium hydroxide) were used as a stationary phase. A solvent mixture of methanol : methylene chloride : 2% aqueous acetic acid (8 : 2 : 0.5, by volume) was the developing system, and detection was carried out at 254 nm. Metformin was used as the internal standard, and linearity was achieved in the ranges of 0.2-10 and 0.04-2 μg per band for OTC and BR, respectively. The second method was a RP-HPLC method; separation was performed on a C18 column using an isocratic mixture of ethanol : 7.5% aqueous acetic acid (70 : 30, v/v). Separation was achieved within 10 minutes, and linearity was proven in the ranges of 0.05-50 and 0.05-30 μg mL -1 for OTC and BR, respectively. Diclofenac sodium was used as an internal standard. The proposed methods were validated in accordance with the FDA Center for Veterinary Medicine guidelines. Moreover, the performance and health and environmental impacts of the methods were evaluated using several greenness metrics, namely, the National Environmental Methods Index (NEMI), modified NEMI, Green Analytical Procedure Index (GAPI), Analytical Eco-Scale and Analytical GREEnness (AGREE) metric approaches. All the obtained results proved the validity of the developed methods concerning its performance and ecological effects. The methods can be used to investigate the presence of OTC residues in various marketed milk samples to maintain public health.

Favipiravir and Meropenem have been concurrently used as directly acting antiviral and antibiotic agents for the treatment of coronavirus disease in human plasma. Accurate and specific reversed phase ultra-performance liquid chromatographic and high-performance thin-layer chromatographic methods were developed and validated for the first time analysis of this combination in spiked human plasma using Cefepime as an internal standard. In the developed ultra-high-performance liquid chromatography method, separation was performed on a BEH C18 column with a mixture of ACN and 0.05 M potassium dihydrogen orthophosphate (pH = 3) in a ratio of 10:90 (v/v) as an eluate. Scanning of the separated peaks was at 298 nm. The developed method showed high sensitivity, and the drugs showed linearity in the range of 5-70 μg/ml for Favipiravir and 2-50 μg/ml for Meropenem. The proposed high-performance thin-layer chromatographic method included the separation using a mixture of ethyl acetate:methanol:deionized water:formic acid (5:4:1.5:0.3, by volume), then spots detection at 300 nm. Methods were investigated for greenness using the eco-scale and national environmental method index tools and were validated according to food and drug administration guidelines. Methods can be applied for bio-analysis and therapeutic drug monitoring studies., (© 2022 Wiley-VCH GmbH.)

Innovative therapeutic protocols to the rapidly spreading coronavirus disease (COVID19) epidemic is highly required all across the world. As demonstrated by clinical studies, Favipiravir (FVP) and Remdesivir (REM) are new antiviral medicines that are effective against COVID-19. REM is the first FDA approved antiviral medicine against COVID-19. In addition to antivirals, corticosteroids such as dexamethasone (DEX), and anticoagulants such as apixaban (PX) are used in multidrug combinations protocols. This work develops and validates simple and selective screening of the four medicines of COVID -19 therapeutic protocol. FVP, REM, DEX, and PX as internal standard in human plasma using UPLC method by C18 column and methanol, acetonitrile, and water acidified by orthophosphate (pH = 4) in a ratio of (15: 35: 50, by volume) as an eluate flowing at 0.3 mL/min. The eluent was detected at 240 nm. The method was linear over (0.1-10 μg/mL) for each of FVP, REM, and DEX. The validation of the UPLC method was assessed in accordance with FDA guidelines. The method can detect as low as down to 0.1 μg/mL for all. The recoveries of the drugs in spiked human plasma ranged from 97.67 to 102.98 percent. Method accuracy and precision were assessed and the drugs showed good stability. The method was proven to be green to the environment after greenness checking by greenness profile and Eco-Scale tool., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 Elsevier B.V. All rights reserved.)

One of the main aims of green analytical chemistry (GAC) is the reduction of solvents and chemicals consumed. Recycling the mobile phase in chromatographic techniques provides an efficient way to implement GAC principles. However, this is not an easy job, particularly in the case of the gradient mode. Analysis of multi-pharmaceuticals for the same manufacturer using one mobile phase system dramatically reduces consumed solvents, time, and cost for pharmaceuticals analysis in quality control laboratories. This work is an attempt to reduce time, cost and effort needed for quality control analysis of several dosage forms produced by the same manufacturer. Our novel and green RP-HPLC method is able to separate and quantify a tertiary mixture of piracetam, ketoprofen and omeprazole produced by the same manufacturers. The analyst can easily quantify the three drugs in the three dosage forms in one run using the gradient elution mode of methanol and water (from 50% methanol to 85% methanol in ten minutes) with a flow rate 1.5 mL min -1 on a non-polar C 18 column. Suitable dilutions were done for the working solution of the mixed pharmaceutical formulations prior to chromatographic analysis. This procedure will dramatically reduce the consumed solvents and save time and money during pharmaceutical analysis. The calibration ranges are (5-25), (5-25) and (3-20) μg mL -1 for the three studied drugs. The International Council for Harmonization (ICH) procedures were followed in the validation process and the results were evaluated in comparison with official HPLC methods, where no noteworthy differences were found. The green profile of the method and pictograms of AGREE and Green Analytical Procedure Index (GAPI) approaches proved the eco-friendly character for the studied drugs. The simultaneous quantitative analysis for Stimulan® and Hyposec® capsules, and Ketolgin® tablets from the Amoun Pharmaceutical Company, Egypt, can be accomplished via the novel method. Also, Memoral® ampoules, Topfam® tablets, and Gastroloc® capsules from Sigma Pharmaceutical Industries, Egypt, could be analyzed simultaneously. Omez® capsules and Ketogesic® tablets from the Pharaonia Pharmaceuticals, Egypt, could be determined simultaneously too. Applying this RP-HPLC method, a significant reduction of the total cost is assured as the required amount of solvent is noticeably decreased when performing multi-analyses in comparison to single component analysis., Competing Interests: No conflict exists regarding this study., (This journal is © The Royal Society of Chemistry.)

Background: The presented quadruple divisor spectrophotometric method was able to resolve and analyze a complex quintuple drug matrix with severe overlapped spectra without previous separation or extraction steps or need of complicated apparatus like chromatographic methods and had the advantage of being green as the solvent used was water., Method: A simple, sensitive, and precise quadruple devisor spectrophotometric method was developed for simultaneous determination of metformin, glipizide, and sitagliptin in the presence of metformin potential impurities melamine and cyanoguanidine. The proposed method was applied for analysis of metformin, glipizide, and sitagliptin in pure form and pharmaceutical formulation (tablets). The developed method was validated and met the requirements for ICH guidelines with respect to linearity, accuracy, precision, specificity, and robustness., Results: A linear response was observed in the range of 2-27, 2-20, 1-20, 0.5-10, and 1-10 μg/mL for metformin, glipizide, sitagliptin, melamine, and cyanoguanidine, respectively, with a correlation coefficient of 0.9996, 0.9998, 0.9997, 0.9997, and 0.9996 for metformin, glipizide, sitagliptin, melamine, and cyanoguanidine, respectively., Conclusions: The validated method was successfully applied for determination of the studied drugs in Janumet® and Engilor® tablets; moreover, the results were statistically compared to those obtained by the reported RP-HPLC method and no significant difference was found between them, indicating the ability of the proposed method to be used for routine quality-control analysis of these drugs., Highlights: Novel application of quadruple divisor spectrophotometric technique is introduced for resolving mixtures containing five components with severely overlapping spectra. A simple and sensitive spectrophotometric method was developed for simultaneous determination of metformin, glipizide, and sitagliptin in the presence of metformin potential impurities melamine and cyanoguanidine. The validity of the proposed method was revealed as per ICH guidelines., (© The Author(s) 2021. Published by Oxford University Press on behalf of AOAC INTERNATIONAL. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

We indented to evaluate of the role miRNA-146a-5p expression level as a possible biomarker associated with the incidence of rheumatoid arthritis and the detection of its relevance with different disease parameters. The study included 60 clinically diagnosed RA patients fulfilling 2010 American College of Rheumatology / European League Against Rheumatism (ACR/EULAR) classification criteria for Rheumatoid Arthritis and 45 age and sex matched control subjects. Disease activity was assessed by Disease Activity Score 28 (DAS28). The expression levels of miR-146a in whole blood was measured using reverse transcriptase quantitative real time polymerase chain reaction (RT-PCR). There median expression level of miRNA-146a-5p was significantly higher in RA patients compared with the control group (P = 0.036). MiR-146a expression level was positively correlated with individual activity markers, including erythrocyte sedimentation rate (ESR) (P = 0.04), visual analogue scale (VAS) (P = 0.047) and Modified Health Assessment Questionnaire (MHAQ) (P = 0.050). A lower expression level of miRNA-146a-5p was detected in RA patients on antimalarial drugs as compared to patients whose treatment protocol did not include antimalarial drugs (P = 0.016). In conclusion, data of this study suggested a possible association between miRNA 146a-5p expression level and the incidence of RA in Egyptian patients., (Copyright© by the Egyptian Association of Immunologists.)

Novel, accurate, selective, and rapid high-performance liquid chromatography mass spectrometry method was developed for simultaneous analysis of amoxicillin trihydrate, dicloxacillin sodium, and their official impurity 6-aminopenicillanic acid. The chromatographic separation was carried out by applying the mixture on a C 18 column (3.5  µ m ps, 100 mm × 4.6 mm id) using acetonitrile:water (65 : 35 by volume) as a mobile phase within only 4 min. The quantitative analysis was executed using single quadrupole mass spectrometer in which electrospray ionization, selected ion monitoring, and negative mode were operated. The retention times were 1.61, 2.54, and 3.50 mins for amoxicillin, 6-aminopenicillanic acid, and dicloxacillin, respectively. The method was validated in linear ranges of 2-28  µ g mL -1 , 2-35  µ g mL -1 , and 1-10  µ g mL -1 for amoxicillin, dicloxacillin, and 6-aminopenicillanic acid, respectively. The results obtained from the suggested HPLC/MS were statistically compared with those obtained from the reported HPLC method, where no significant difference appeared respecting accuracy and precision. According to the analytical eco-scale assessment method, the proposed method was proved to be greener than the reported one, where the analysis time and the amount of the wasted effluent decreased., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2021 Atiah H. Almalki et al.)

Innovative and specific double dual wavelength, dual ratio subtraction spectrophotometric methods were carried out along with a successive ratio subtraction spectrophotometric method for determination of dacarbazine and its related impurities including toxic and hazardous ones. For determination of dacarbazine by the double dual wavelength method, the absorbance differences between 323 and 350 nm of the zero order absorption spectra of dacarbazine were used. The values of absorbance difference between 267.2 and 286.2 nm of the zero order spectra of 5-amino-imidazole-4 carboxamide were used for its determination by the dual ratio subtraction method. The zero order absorption spectrum of 2-azahypoxanthine at 235 nm was used for its determination after applying the successive ratio subtraction method. ICH guidelines were followed for validation of the developed methods, where linear relationships were obtained in the range of 4-20, 1-16, and 2-20 μg mL -1 for dacarbazine, 5-amino imidazole-4-carboxamide and 2-azahypoxanthine, respectively. Accurate, precise, and specific results were obtained upon applying the proposed methods according to ICH guidelines. Furthermore, the developed methods were successfully applied for determination of dacarbazine in its pharmaceutical formulation. Comparing the results of the developed methods with those of the official USP spectrophotometric method statistically showed no significant difference. The developed methods don't need any sophisticated techniques so they are considered cost effective methods. Moreover, the introduced methods have the advantages of being green where water was used as a solvent. The methods proved to be more economic, fast and simple than other reported HPLC methods., Competing Interests: There are no conflicts of interests to declare., (This journal is © The Royal Society of Chemistry.)

Ulcerative colitis is an autoimmune inflammatory disorder with a negative impact on the life quality of patients. Cinnamaldehyde and hesperetin were chosen due to their antioxidants and anti-inflammatory effects. This study explored the protective effects of cinnamaldehyde (40 and 90 mg/kg, po) and hesperetin (50 and 100 mg/kg, po) on 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced ulcerative colitis in rats. Cinnamaldehyde and hesperetin significantly improved macroscopic and histopathological examinations with a significant reduction in myeloperoxidase and intracellular adhesion molecule-1 expression. They significantly reduced colon oxidative stress by a significant elevation in both reduced glutathione content and superoxide dismutase activity with a significant reduction of NO content. Furthermore, cinnamaldehyde and hesperetin alleviated the inflammatory injury by a significant reduction in interleukin-6 along with suppression of nuclear factor-κB, receptor for advanced glycation end products, and tumor necrosis factor-α expression. Moreover, cinnamaldehyde and hesperetin significantly decreased p-JAK2 and p-STAT3 while significantly increased suppressors of cytokine signaling 3 (SOCS3) protein expression. In conclusion, cinnamaldehyde and hesperetin counteracted TNBS-induced ulcerative colitis through antioxidant, anti-inflammatory properties as well as modulation of the JAk2/STAT3/SOCS3 pathway., (© 2021 Wiley Periodicals LLC.)

A simple and reliable HPLC method is developed for isocratic separation of a ternary mixture of Salbutamol Sulfate (SAL), Guaifenesin (GUI) and its impurity Guaiacol (GUA) either in pure powder or in pharmaceutical formulation. Chromatographic separation was applied on a Hypersil GOLD CN column with a mobile phase consisting of 0.05 M KH2PO4 (containing 0.1% triethylamine, pH adjusted to 3.7 by phosphoric acid): methanol (60: 40 by volume) using 0.6 mL min-1 flow rate and detection of peaks at 275 nm at 25°C with run time around 6 min. The calibration plots were linear over the concentration ranges of 0.5-20, 0.5-30 and 0.1-10 μg mL-1 for SAL, GUI and GUA, respectively. ICH guidelines were used for the validation of presented method. The obtained results were compared with the results obtained from the reported HPLC method and no significant difference was found regarding accuracy and precision., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The aim of this work is to develop and validate a simple, rapid, highly sensitive and selective spectrofluorometric method for determination of Metformin HCl (MFH) and Glibenclamide (GLB) in their binary mixture without prior separation. The proposed method based on measuring the native fluorescence intensity of GLB at λ emission  = 348 nm, after excitation at λ excitation  = 226 nm and measuring the fluorescence intensity of the fluorescent product produced from the derivatization of MFH using 9,10-phenanthraquinone in alkaline media at λ emission  = 416 nm after excitation at λ excitation  = 240 nm. The proposed spectrofluorometric method allowed sensitive determination of the studied drugs with a limit of quantitation of 0.04 and 0.01 μg mL -1 for MFH and GLB, respectively, providing greater sensitivity than the reported one. Validation of the proposed method was carried out according to ICH guidelines with respect to linearity, accuracy, precision, and selectivity. The developed method was successfully applied for the determination of MFH and GLB in laboratory prepared mixtures and pharmaceutical formulation and the results obtained were statistically compared to those of the reported HPLC method with no significant difference between them., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Two green, simple, and accurate chromatographic methods were developed and validated for the simultaneous determination of omeprazole and aspirin mixture in the presence of salicylic acid, a major impurity of aspirin. Method A is a reversed-phase ultra-high-performance liquid chromatography; the separation was performed on a C18 column, with a mobile phase composed of ethanol:0.1% aqueous solution of triethylamine acidified with orthophosphoric acid (pH 3) (30:70, v/v) at 0.15 mL/min flow rate and 230 nm. Omeprazole, aspirin, and aspirin impurity retention times were 7.47, 4.40, and 5.13 min, respectively. Good linearity was achieved in the concentration ranges of 5-80, 5-85, and 3-50 μg/mL for the three mentioned components, respectively. Method B is thin-layer chromatography (TLC) where silica gel TLC F254 plates were utilized to achieve separation using ethanol:ethyl acetate (2:8, v/v) as a developing system at 240 nm. The resulted R f values were 0.83, 0.65, and 0.23 for omeprazole, aspirin, and impurity, respectively. The concentration ranges of 0.1-3 μg/band for the three drugs showed good linearity. The proposed methods are eco-friendly and greener when compared to the already reported method (Microchemical Journal, 152, 104350). This is the first use of TLC method for the determination of the three drugs. International Council for Harmonization (ICH) guidelines were followed to ensure the validity of developed methods., (© 2020 John Wiley & Sons, Ltd.)

This work presents a sensitive, accurate and selective RP-HPLC method for simultaneous determination of cyproheptadine HCl (CPH), its impurity B (dibenzosuberone) and CPH oxidative degradation product (10,11-dihydroxy-dibenzosuberone) in bulk powder and in pharmaceutical formulation. The RP-HPLC method depends on isocratic elution using C8 column and mobile phase consisting of 0.05 M KH2PO4 buffer:methanol (35:65, v/v, pH = 4.5) at a flow rate of 2 mL/min, and the eluant was monitored at 245 nm. Good resolution was obtained with tR values of 3.05, 7.54 and 6.17 min for CPH, impurity and oxidative degradate, respectively. The proposed method has been validated as per ICH guidelines using pure forms of CPH, its impurity and degradation product in pharmaceutical formulation with an accuracy of 100.48, 100.16 and 100.11, respectively. Additional spiking experiments yielded an accuracy of 100 ± 1.6%. Repeatability and intermediate precision results indicated acceptable low <2% RSD values. Moreover, the developed method's statistical results were favorably compared to the previously reported method results regarding both accuracy and precision. The developed method can be applied for analysis of the three components in quality control laboratories., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The aim of the presented work is to compare two popular chemometric methods which are partial least squares regression (PLSR) and support vector regression (SVR). The comparison shows their characteristics via application of the suggested methods to analysis of Norfloxacin (NF) and Tinidazole (TZ) with the presence of a potential impurity of Tinidazole; 2-Methyl-5-nitro-1H-imidazole (MNZ). For appropriate analysis, a 3 factor 4 level experimental design was constructed, which results in a training set composed of 16 mixtures which contains different concentrations of the three components; achieving symmetry, rotatability and orthogonality in mixture space. In order to validate the prediction ability of the suggested models, an independent test set consisting of 8 in-space and 8 out-of-space mixtures was used. The presented results show high specificity and accuracy of the mentioned multivariate calibration models for analysis of in-space samples of NF and TZ in presence of (MNZ) using UV spectral data. Statistical comparisons of predictive abilities of proposed models against classical least squares CLS model and against each other was performed; whether for analysis of test set mixtures or dosage form. CLS model showed lower predictive ability compared to other models. Results obtained by SVR model are as accurate as PLSR model, however, optimization and implementation of PLSR is faster and easier, hence PLSR could be of choice for this given case study. The developed chemometric models were validated as directed by ICH strategies. The validated methods were efficiently used for estimation of NF and TZ in pure powders and pharmaceuticals which indicates their suitability for application in quality control examination of both of the drugs., Competing Interests: Declaration of competing interest The researchers declare that no conflicts of interest in this research article., (Copyright © 2020 Elsevier B.V. All rights reserved.)

This manuscript reports on the development of a capacitive sensor for the detection of imidacloprid (IMD) in water samples based on molecularly imprinted polymers (MIPs). MIPs used as recognition elements were synthesized via a photo-initiated emulsion polymerization. The particles were carefully washed using a methanol (MeOH) /acetic acid mixture to ensure complete template removal and were then dried. The average size of the obtained particles was less than 1 µm. The imprinting factor (IF) for IMD was 6 and the selectivity factor (α) for acetamiprid, clothianidin, thiacloprid and thiamethoxam were 14.8, 6.8, 7.1 and 8.2, respectively. The particles were immobilized on the surface of a gold electrode by electropolymerization. The immobilized electrode could be spontaneously regenerated using a mixture of MeOH/10 mM of phosphate buffer (pH = 7.2)/triethylamine before each measurement and could be reused for 32 times. This is the first-time that automated regeneration was introduced as part of a sensing platform for IMD detection. The developed sensor was validated by the analysis of artificially spiked water samples. Under the optimal conditions, the linearity was in the range of 5-100 µM, with a limit of detection (LOD) of 4.61 µM.

Development and validation of accurate and selective spectrophotometric methods were carried out for determination of a new combination of Mebendazole and Quinfamide in bulk powder and pharmaceutical formulation. Extended ratio subtraction coupled with isoabsorptive point methods were used for determination of the binary mixture. A comparative study was carried out between the newly developed methods; simultaneous ratio subtraction, absorbance subtraction, and dual wavelength spectrophotometric methods were applied as well. The developed methods were validated in accordance with the ICH guidelines. The developed methods were successfully carried out for determination of Quinfamide and Mebendazole in Vermox Plus® tablets. Upon statistical comparison of the results obtained by the developed methods with those obtained by the reported simultaneous equation spectrophotometric method, no significant difference was shown among them. Moreover, the introduced methods have the advantages of being more sensitive and have wider linearity ranges than other reported spectrophotometric methods., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Felodipine is a calcium channel blocker used together with metoprolol succinate for treatment of hypertension., Objective: Two chromatographic methods were developed for simultaneous determination of felodipine (FEL) and metoprolol succinate (MET), and their major metabolites, dehydrofelodipine and metoprolol acid, respectively., Methods: The first method was RP-HPLC which comprised separation of the studied components by gradient elution using a Phenomenex C8 column and a mobile phase composed of water (adjusted to pH 3.5 with o-phosphoric acid)-acetonitrile - methanol (45:40:15, by volume) for the first 6 min and (30:60:10, by volume) for the next 4 min at a flow rate of 1 mL/min followed by UV detection of the eluted peaks at 225 nm. The second method was an HPTLC method where separation was achieved using a mobile phase consisting of toluene-ethyl acetate-methanol-ammonia-formic acid (10:5:2.5:0.3:0.1, by volume) and scanning of the separated bands at 225 nm., Results: Validation of the developed methods was done according to ICH guidelines. Successful application of the developed methods was carried out for determination of the studied drugs in human spiked plasma and in Logimax® tablets., Conclusions: The developed RP-HPLC and HPTLC methods can be further applied for quality control testing of the studied drugs., Highlights: RP-HPLC and HPTLC methods for determination of FEL, MET and their major metabolites. The developed methods were successfully applied for determination of FEL and MET in Logimax® tablets., (© AOAC INTERNATIONAL 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Amiloride hydrochloride (AM) is a potassium sparing diuretic, while hydrochlorothiazide (HCZ) is the protype of thiazide diuretics. The combining of the studied drugs exhibits a synergistic effect. Moreover, HCZ prevents the potassium depletion side effect caused by AM., Objective: Two accurate and precise simultaneous chromatographic separation methods were promoted and investigated to quantify AM, HCZ, official impurities of HCZ (cholorothiazide and salamide), and the official impurities of AM (methyl 3, 5-diamino-6-chloropyrazine-2-carboxylate)., Methods: The components of the quintuple mixture were quantified by two methods. The first method was high-performance thin layer chromatography (HPTLC), where exemplary separation was achieved on silica gel HPTLC F254 plates at the stationary phase using ethyl acetate-ethanol-ammonia solution (8 + 2 + 0.2, v/v) as a developing system. Scanning of bands at 273 nm was done. The second method was a reversed-phase chromatography (RP-HPLC) method using C18 (4.6 × 100 mm) column and mobile phase comprising 0.1% phosphoric acid solution-acetonitrile (90 + 10, v/v) with UV determination at 273 nm. Adjustment of the flow rate at 1 mL/min and pH at 3.6 was performed., Results: Regarding RP-HPLC, optimum separation of the quintuple mixture was achieved within just five minutes. According to HPTLC, symmetric and sharp peaks were separated on the resulted chromatogram. Validity of the introduced methods was investigated by applying international conference on harmonization (ICH) guidelines., Conclusions: The methods were successfully applied for assays of the studied drugs in their pure and tablet forms. No significant difference was revealed through application of statistical comparison between results of the suggested methods and those of the reported method regarding both accuracy and precision., (© AOAC INTERNATIONAL 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Two selective and sensitive chromatographic methods were developed for simultaneous determination of new combination of quinfamide and mebendazole in bulk powder and in pharmaceutical formulation. The first method is HPTLC by which separation was obtained using silica gel HPTLC F254 plates and a simple mobile phase consisting of methanol:toluene (2:6, v/v) and the separated bands were scanned at 254 nm. The second method RP-HPLC that comprised isocratic separation of both drugs on a Phenomenex C18 column using a green mobile phase consisting of double distilled water:methanol (30:70, v/v) at a flow rate of 0.8 mL/min and UV detection at 254 nm. The developed methods were validated and proved to meet ICH guidelines. Successful application of the developed methods was carried out for determination of quinfamide and mebendazole in Vermox Plus® tablets. Statistical comparison between the developed chromatographic methods and the reported simultaneous equation spectrophotometric method showed that there was no significant difference between them, proving the ability of applying the proposed methods in quality control testing of the studied drugs. The developed methods are considered the first chromatographic methods for simultaneous determination of quinfamide and mebendazole; moreover, they offered sensitive and selective eco-friendly methods for analysis of the studied drugs., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

A comparative study using novel quadruple divisor and mean centering of ratio spectra spectrophotometric methods was developed for resolution of five- component mixture of Tolnaftate, β-naphthol (Tolnaftate alkaline degradation product and its toxic impurity), methyl(m-tolyl)carbamic acid (Tolnaftate alkaline degradation product), N-methyl-m-toluidine (Tolnaftate toxic impurity) and methyl paraben (as a preservative). For the novel quadruple divisor method, each component in the quinary mixture was determined by dividing the quinary mixture spectrum by a sum of standard spectrum of equal concentration of the other four components as a quadruple divisor. First derivative of each ratio spectra was then obtained which allowed selective determination of each component without interference from other components in the mixture. The second method was mean centering of ratio spectra that depended on utilizing the mean centered ratio spectra in four successive steps leading to enhancement of the signal to noise ratio. The absorption spectra of the five studied components were recorded in the wavelength range of 210-350 nm. The mean centered fourth ratio spectra amplitudes for each component were used for its determination. The developed methods were successfully applied for determination of laboratory prepared quinary mixtures to ensure method's specificity, then, were further applied on Tinea Cure® cream where no interference from excipients. For the first time, Tolnaftate was determined along with its toxic impurity; β-naphthol, that could be absorbed by the skin, causing systemic toxic effects, unlike Tolnaftate that poorly absorbed, indicating the significance of this work. The proposed methods were statistically compared with each other and with the reference method. Furthermore, ICH guidelines were followed for their validation., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Tolnaftate, a thionoester anti-fungal drug, was subjected to alkaline hydrolysis to produce methyl(m-tolyl)carbamic acid and β-naphthol (tolnaftate impurity A). N-Methyl-m-toluidine, tolnaftate impurity D, was synthesized and structurally elucidated along with tolnaftate alkaline degradation products using IR, H 1 NMR and MS. Two stability-indicating HPTLC and RP-HPLC methods were developed and validated, for the first time, for determination of tolnaftate, its alkaline degradation products and toxic impurities in the presence of methyl paraben, as a preservative in Tinea Cure ® cream. The proposed HPTLC method depended on separation of the studied components on TLC silica gel F 254 plates using hexane-glacial acetic acid (8:2, v/v) as a developing system and scanning wavelength of 230 nm. The proposed RP-HPLC method was based on separation of the five components on an Eclipse plus C 18 column. The mobile phase used was acetonitrile-water containing 1% ammonium formate (40:60, v/v), with a flow rate of 1 mL/min and detection wavelength of 230 nm. The proposed methods allowed the assay of tolnaftate toxic impurities, β-naphthol and N-methyl-m-toluidine, down to 2%, allowing tracing of β-naphthol that could be absorbed by the skin causing systemic toxic effects, unlike tolnaftate, indicating the high significance of such determination. International Conference on Harmonization guidelines were followed for validation., (© 2019 John Wiley & Sons, Ltd.)

A green, accurate and specific high-performance thin-layer chromatographic (HPTLC) method was developed and validated for simultaneous quantitative determination of pyridostigmine bromide (PR), impurity B (IMP B);3-hydroxy-N-methylpyridinium bromide and impurity A (IMP A); pyridin-3-yl-dimethylcarbamate. The two pharmacopeial impurities are also its main inactive metabolites. Furthermore, IMP B is known to be its alkaline-induced degradation product. Achievable separation of the studied components required silica gel HPTLC F254 plates as a stationary phase and acetone: acetic acid (80:20, v/v) as a developing system. Scanning of the separated bands was done at 260 nm. According to green solvent selection guidelines, acetone and acetic acid are eco-friendly solvents. Validation of the developed method was insured by its acquiesce to international conference on harmonization (ICH) guidelines. The introduced method was successfully achieved for the quantitative determination of PR, IMP B and IMP A in the range of 0.4-10, 2-11 and 0.4-3.5 μg/band, respectively. Successful application of the developed method was done for determination of PR in human plasma in the range of 0.6-10 μg/band, so the proposed HPTLC can be applied in the pharmacokinetic studies. The studied drug was also analyzed in Mestinon® tablets using the developed method., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Resolution and quantitative determination of ternary mixture with severely overlapped spectra without any preliminary separation steps represents a big challenge for any analyst. Smart and novel spectrophotometric methods are continuously innovated for achieving the above mentioned target. Novel applications of ratio difference spectrophotometric technique utilizing ratio spectra and derivative ratio spectra are applied in presented work. The proposed methods included derivative ratio difference (DRD) and ratio subtraction ratio difference (RSRD) methods. Comparative study was achieved between the proposed methods and the recently developed induced ratio difference (IRD) method. The developed methods were assessed through the analysis of ternary mixtures with different ratios of Pyridostigmine bromide (PR) and its related substances; impurity a (IMP A) and impurity b (IMP B). Analysis of PR in a pharmaceutical dosage form without any interference from other inactive ingredients was also a successful application of the proposed methods. As per ICH guidelines, the proposed methods were validated ensuring their accuracy, precision and specificity. Statistical comparison between the developed methods and the reference method was done, where calculated F and t values were less than the theoretical ones in regards to accuracy and precision., (Copyright © 2018. Published by Elsevier B.V.)

Ascorbic acid (ASC), paracetamol (PAR) and guaifenesin (GUF) are co-formulated together as analgesic and expectorant effervescent powder. This work describes a selective stability indicating HPTLC-densitometric method which is the first developed stability indicating one for chromatographic separation of the three studied drugs in presence of paracetamol toxic impurity [4-aminophenol (4-AP)] and guaifenesin related substance [impurity, (guaiacol) (GUC)]. The chromatographic separation was carried out on HPTLC 60F254 plates using chloroform-acetone-trifluoroacetic acid (6.5:3.5:0.30, by volume) as a developing system with UV detection at 254 nm. Factors affecting chromatographic separation have been optimized and good separation was obtained between the studied components where (ASC Rf = 0.05, 4-AP Rf = 0.12, GUF Rf = 0.24, PAR Rf = 0.41 and GUC Rf = 0.56). Method validation has been performed according to ICH guidelines and linearity was achieved in the range of 0.4-2.4, 0.4-2.8 and 4-15 μg/band for ASC, PAR and GUF, respectively. In order to evaluate the developed method it was applied for determination of the cited drugs in G.C.Mol® effervescent powder and the obtained results were statistically compared with those obtained by applying the reported HPLC method using Student's t- and F-tests where no significant difference was observed. The developed method has the advantages over the reported HPLC one of being more sensitive and selective which permit its application in quality control analysis of the cited their in presence of drug impurities.

Aim of the Study: The present case control study was conducted to assess the association of LTA 252 A>G, TNF-α 308 G>A, and TNF-α 1031 T>C gene polymorphisms with rheumatoid arthritis (RA), and their involvement in disease activity and severity., Material and Methods: A total of 70 Egyptians, including 35 RA patients and 35 healthy control individuals, were included in the study. The RA patients comprised 34 females and one male. Cases with RA were diagnosed by a rheumatologist and fulfilled the 2010 ACR/EULAR criteria. Modified disease activity score (DAS28) was used to assess disease activity. Van Der Heijde-modified Sharp score (vdHSS) was used to assess radiological changes for assessment of disease severity. PCR-RFLP was used to detect the association of LTA 252 A>G, TNF-α 308 G>A, and TNF-α 1031 T>C gene polymorphisms with RA., Results: TNF-α 308 G allele and TNF-α 308 GG genotype were significantly higher in RA patients compared to healthy control subjects (p = 0.04 and p = 0.001, respectively). TNF-α 308 G allele and GG genotype were significantly higher in the RA non-remission group compared to the remission group (p = 0.008, p < 0.001). Patients with the TNF-α 308 AG genotype had higher mean of Sharp score compared to the patients with the GG and AA genotypes (p = 0.007). There was no significant association between LTA 252 A>G and TNF-α 1031 T>C gene polymorphisms and RA., Conclusions: Our results suggest that TNF-α 308 G/A gene polymorphism is genetically associated with RA and involved in disease activity and severity in Egyptian patients., Competing Interests: The authors declare no conflict of interest., (Copyright: © 2019 Polish Society of Experimental and Clinical Immunology.)

Sensitive, selective and accurate high-performance thin layer chromatographic HPTLC method for quantitative determination of Norfloxacin (NF), Tinidazole (TZ) and 2-Methyl-5-nitroimidazole (MNZ) as potential impurity of Tinidazole is developed and validated in the presented work. Calibration curves were linear over the concentration ranges of 0.4-2.4, 0.4-1.6, 0.2-1.2 μg/band for NF, TZ and MNZ, respectively. The method depends on separation and quantitation of NF, TZ and MNZ on aluminium plates pre-coated with silica gel HPTLC 60F254 as stationary-phase using chloroform: methanol: formic acid (7.5:1: 0.3, by volume) as developing system followed by densitometric measurement of bands at 298 nm. The developed method was validated and proved to meet the requirements delineated by ICH guidelines with respect to linearity, accuracy, precision, specificity and robustness. The validated method was successfully applied for determination of studied drugs in bulk powders and in their pharmaceutical formulation indicating the ability of proposed method to be used for routine quality control analysis of these drugs.

Toxic solvents are widely used in chemical laboratories, which are dangerous on health, safety of workers, and environment. Green chemistry established different principles to keep safety of environment, one of these goals is to replace toxic solvents by greener alternatives or by minimizing the used volumes. Paracetamol (PAR), Guaifenesin (GUF), Oxomemazine (OX), and Sodium benzoate (SB) combination is a widely used cough preparation. 4- aminophenol (4-AP) is PAR poisonous impurity and related substance. Guiacol (GUC) is GUF impurity and related substance; its presence may lead to rejection of GUF sample. An eco-friendly HPTLC method was developed to quantify the studied drugs and their impurities. Chromatographic separation was achieved on HPTLC 60F254 plates using ethylacetate: methanol: 0.05 M ammonium chloride buffer (100: 2: 5, by volume) as a mobile phase and scanning at 225 nm. The linear ranges were 0.25-3.50, 0.50-8.00, 0.25-4.00, 0.20-8.00, 0.05-4.00, and 0.25-4.00 µg/band for PAR, GUF, OX and SB, 4-AP, and GUC. Method was successfully applied to available syrup and suppositories. It compared well with the reported method. It can be considered as an alternative green method for previously developed TLC method. Greenness profile of the method proved that it is greener than the reported methods being time and solvents saving.

Two selective and accurate chromatographic methods are presented for simultaneous quantitation of spironolactone (SP) and furosemide (FR) and canrenone (CN), the main degradation product and the main active metabolite of SP. Method A was HPTLC, where separation was completed on silica gel HPTLC F 254 plates using ethyl acetate-triethylamine-acetic acid (9:0.7:0.5, by volume) as a developing system and UV detection at 254 nm. Method B was a green isocratic RP-HPLC utilizing a C 18 (4.6 × 100 mm) column, the mobile phase consisting of ethanol-deionized water (45: 55, v/v) and UV estimation at 254 nm. Adjustment of flow rate at 1 mL/min and pH at 3.5 with glacial acetic acid was done. Regarding the greenness profile, the proposed RP-HPLC method is greener than the reported one. ICH guidelines were followed to validate the developed methods. Successful applications of the developed methods were revealed by simultaneous determination of FR, SP and CN in pure forms and plasma samples in the ranges of 0.2-2, 0.05-2.6 and 0.05-2 μg/band for method A and 5-60, 2-60 and 2-60 μg/mL for method B for FR, SP and CN, respectively., (© 2018 John Wiley & Sons, Ltd.)

Two simple, rapid chromatographic methods were developed for the simultaneous determination of Theophylline (THP), Guaifenesin (GUI) and Guaifenesin impurity namely Guaiacol (GUA). The first method is an isocratic reversed-phase high-performance liquid chromatography (RP-HPLC) method, where separation of THP, GUI and GUA was achieved on C18 column using methanol:water (containing 0.1% triethylamine):acetonitrile (30:60:10, by volume) as a mobile phase with a flow rate of 0.7 mL /min and UV detection at 275 nm. The separation was achieved at retention times (Rt 3.76, 6.76 and 8.79 for THP, GUI and GUA, respectively). The calibration plots were linear over the concentration range of 2-25, 2-37 and 0.5-10 μg /mL for THP, GUI and GUA, respectively. The second method is high pressure thin layer liquid chromatography method, which was developed using silica gel plates 60F254 as a stationary phase with ethyl acetate:hexane:methanol:ammonia (65:35:10:2, by volume) as a developing system. The densitometric measurements were performed at 275 nm with good Rf values (0.13, 0.35 and 0.8) for THP, GUI and GUA, respectively. The calibration plots showed good correlation over the range (0.4-2 μg/band) for both THP and GUI, and (0.4-1.2 μg/band) for GUA. The two proposed methods were validated according to ICH guidelines. The results for the two methods were statistically compared to those obtained by a reported high-performance liquid chromatography method and no significant difference was found regarding accuracy and precision.

Furosemide and spironolactone are commonly prescribed antihypertensive drugs. Canrenone is the main degradation product and main metabolite of spironolactone. Ratio subtraction and extended ratio subtraction spectrophotometric methods were previously applied for quantitation of only binary mixtures. An extension of the above mentioned methods; successive ratio subtraction, is introduced in the presented work for quantitative determination of ternary mixtures exemplified by furosemide, spironolactone and canrenone. Manipulating the ratio spectra of the ternary mixture allowed their determination at 273.6nm, 285nm and 240nm and in the concentration ranges of (2-16μgmL -1 ), (4-32μgmL -1 ) and (1-18μgmL -1 ) for furosemide, spironolactone and canrenone, respectively. Method specificity was ensured by the application to laboratory prepared mixtures. The introduced method was ensured to be accurate and precise. Validation of the developed method was done with respect to ICH guidelines and its validity was further ensured by the application to the pharmaceutical formulation. Statistical comparison between the obtained results and those obtained from the reported HPLC method was achieved concerning student's t-test and F ratio test where no significant difference was observed., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Two multivariate chemometric models, namely, partial least-squares regression (PLSR) and linear support vector regression (SVR), are presented for the analysis of amoxicillin trihydrate and dicloxacillin sodium in the presence of their common impurity (6-aminopenicillanic acid) in raw materials and in pharmaceutical dosage form via handling UV spectral data and making a modest comparison between the two models, highlighting the advantages and limitations of each. For optimum analysis, a three-factor, four-level experimental design was established, resulting in a training set of 16 mixtures containing different ratios of interfering species. To validate the prediction ability of the suggested models, an independent test set consisting of eight mixtures was used. The presented results show the ability of the two proposed models to determine the two drugs simultaneously in the presence of small levels of the common impurity with high accuracy and selectivity. The analysis results of the dosage form were statistically compared to a reported HPLC method, with no significant difference regarding accuracy and precision, indicating the ability of the suggested multivariate calibration models to be reliable and suitable for routine analysis of the drug product. Compared to the PLSR model, the SVR model gives more accurate results with a lower prediction error, as well as high generalization ability; however, the PLSR model is easy to handle and fast to optimize.

A sensitive, accurate and selective high performance thin layer chromatography (HPTLC) method was developed and validated for the simultaneous determination of paracetamol (PAR), its toxic impurity 4-aminophenol (4-AP), pseudoephedrine HCl (PSH) and loratidine (LOR). The proposed chromatographic method has been developed using HPTLC aluminum plates precoated with silica gel 60 F254 using acetone-hexane-ammonia (4:5:0.1, by volume) as a developing system followed by densitometric measurement at 254 nm for PAR, 4-AP and LOR, while PSH was scanned at 208 nm. System suitability testing parameters were calculated to ascertain the quality performance of the developed chromatographic method. The method was validated with respect to USP guidelines regarding accuracy, precision and specificity. The method was successfully applied for the determination of PAR, PSH and LOR in ATSHI(®) tablets. The three drugs were also determined in plasma by applying the proposed method in the ranges of 0.5-6 µg/band, 1.6-12 µg/band and 0.4-2 µg/band for PAR, PSH and LOR, respectively. The results obtained by the proposed method were compared with those obtained by a reported HPLC method, and there was no significance difference between both methods regarding accuracy and precision., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Validated sensitive and highly selective methods were developed for the quantitative determination of cefoperazone sodium (CEF) in the presence of its reported impurities; 7-aminocephalosporanic acid (7-ACA) and 5-mercapto-1-methyl-tetrazole (5-MER). Method A is high-performance liquid chromatography (HPLC), where the mixture of CEF and the reported impurities; 7-ACA and 5-MER were separated on a C8 column (5 µm ps, 250 mm × 4.6 i.d.) using methanol:0.05 M KH2PO4 buffer (22.5:77.5 v/v, pH 7.5) as a mobile phase. The three components were detected at 254 nm with a concentration range of 10-90 µg mL(-1) and the mean percentage recovery 99.67% (SD 1.465). Method B is high-performance thin layer chromatography (HPTLC), where the mixture of CEF and the reported impurities were separated on silica gel HPTLC F254 plates using (acetone:methanol:ethyl acetate:2% sodium lauryl sulfate:glacial acetic acid) (3:2:3:0.8:0.2, by volume) as a developing system and scanning at 254 nm over a concentration range of 1-10 µg per band with the mean percentage recovery 99.95% (SD 1.335). The proposed methods were statistically compared with a reported HPLC method with no significant difference regarding accuracy and precision; indicating the ability of the proposed methods to be reliable and suitable for routine analysis of drug product. The proposed HPTLC method proved to be more sensitive, while the HPLC gave more reproducible results besides saving time., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Two chromatgraphic methods were developed for determination of Paracetamol (PCM) and Pamabrom (PAM) in presence of P-aminophenol (PAP) and Theophylline (THEO) as potential impurities of both drugs respectively. First method is HPTLC which depends on separation and quantitation of the studied drugs on aluminum plates pre-coated with silica gel 60 F₂₅₄ as a stationary phase using chloroform:methanol:ethyl acetate:glacial acetic acid (8:0.8:0.6:0.2, v/v/v/v) as mobile phase followed by densitometric measurement of the bands at 254 nm. Second method is RP-HPLC which comprises separation of the studied drugs on a Phenomenex C8 column by gradient elution using mobile phase consisting of sodium dihydrogen phosphate buffer (0.05 M): methanol:acetonitrile (85:10:5, v/v/v) at a flow rate of 1 mL/min for first 7.5 min and (70:20:10, v/v/v) at a flow rate of 1.5 mL/min for the next 5 min. The proposed methods were successfully applied for determination of the potential impurities of PCM and PAM after resolving them from the pure drugs. The developed methods have been validated and proved to meet the requirements delineated by ICH guidelines with respect to linearity, accuracy, precision, specificity and robustness. The validated methods were successfully applied for determination of the studied drugs in their pharmaceutical formulation. The results were statistically compared to those obtained by the reported RP-HPLC method where no significant difference was found; indicating the ability of proposed methods to be used for routine quality control analysis of these drugs., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A comparison between partial least squares regression and support vector regression chemometric models is introduced in this study. The two models are implemented to analyze cefoperazone sodium in presence of its reported impurities, 7-aminocephalosporanic acid and 5-mercapto-1-methyl-tetrazole, in pure powders and in pharmaceutical formulations through processing UV spectroscopic data. For best results, a 3-factor 4-level experimental design was used, resulting in a training set of 16 mixtures containing different ratios of interfering moieties. For method validation, an independent test set consisting of 9 mixtures was used to test predictive ability of established models. The introduced results show the capability of the two proposed models to analyze cefoperazone in presence of its impurities 7-aminocephalosporanic acid and 5-mercapto-1-methyl-tetrazole with high trueness and selectivity (101.87 ± 0.708 and 101.43 ± 0.536 for PLSR and linear SVR, resp.). Analysis results of drug products were statistically compared to a reported HPLC method showing no significant difference in trueness and precision, indicating the capability of the suggested multivariate calibration models to be reliable and adequate for routine quality control analysis of drug product. SVR offers more accurate results with lower prediction error compared to PLSR model; however, PLSR is easy to handle and fast to optimize.

Simple, selective and sensitive high-performance thin layer chromatographic (HPTLC) method has been developed and validated for the simultaneous determination of hydrochlorothiazide (HCZ) in the presence of its impurities (chlorothiazide (CT) and salamide (DSA)), in two quaternary mixtures with benazepril hydrochloride (BZ) or amiloride hydrochloride (AM). The separation was carried out on HPTLC silica gel 60 F254 using ethyl acetate-methanol-glacial acetic acid (85:2:0.3 v/v/v) followed by densitometric measurement of bands at 240 nm for the first mixture containing HCZ, CT, DSA, BZ and by using ethyl acetate-methanol-water-ammonia (90:10:5:3 v/v/v) followed by densitometric measurement at 278 nm for the second mixture containing HCZ, CT, DSA, AM. Calibration curves were constructed in the range of (0.2-1.8 µg/band) and (0.4-2.2 µg/band) with good accuracy for HCZ and BZ, respectively, for the first mixture and in the range of (0.6-1.8 µg/band) and (0.4-2.4 µg/band) with good accuracy for HCZ and AM, respectively, for the second mixture. The developed method was validated according to ICH guidelines and demonstrated good accuracy and precision. Moreover, the methods were successfully applied for the determination of HCZ and BZ and AM in pure form and pharmaceutical dosage forms. The results were statically compared with the reported methods with no significant difference, indicating the ability of the proposed method to be used for routine analysis of drug product., (© The Author [2014]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Partial least squares regression (PLSR) and support vector regression (SVR) are two popular chemometric models that are being subjected to a comparative study in the presented work. The comparison shows their characteristics via applying them to analyze Hydrochlorothiazide (HCZ) and Benazepril hydrochloride (BZ) in presence of HCZ impurities; Chlorothiazide (CT) and Salamide (DSA) as a case study. The analysis results prove to be valid for analysis of the two active ingredients in raw materials and pharmaceutical dosage form through handling UV spectral data in range (220-350 nm). For proper analysis a 4 factor 4 level experimental design was established resulting in a training set consisting of 16 mixtures containing different ratios of interfering species. An independent test set consisting of 8 mixtures was used to validate the prediction ability of the suggested models. The results presented indicate the ability of mentioned multivariate calibration models to analyze HCZ and BZ in presence of HCZ impurities CT and DSA with high selectivity and accuracy of mean percentage recoveries of (101.01±0.80) and (100.01±0.87) for HCZ and BZ respectively using PLSR model and of (99.78±0.80) and (99.85±1.08) for HCZ and BZ respectively using SVR model. The analysis results of the dosage form were statistically compared to the reference HPLC method with no significant differences regarding accuracy and precision. SVR model gives more accurate results compared to PLSR model and show high generalization ability, however, PLSR still keeps the advantage of being fast to optimize and implement., (Copyright © 2014 Elsevier B.V. All rights reserved.)