Spectrodensitometric and ultra-performance liquid chromatographic quantification of dapagliflozin and saxagliptin in their dosage form and human plasma

Purpose: To simultaneously quantify dapagliflozin (DAPA) and saxagliptin (SAX) in a pharmaceutical product and human plasma. Methods: Separation and quantification of DAPA and SAX were performed on pre-coated TLC plates in TLC-densitometric method using a solvent system of chloroform, ethyl acetate and methanol at a volume ratio of 8:1:1 as the mobile phase. The developed spots were scanned at 225 and 210 nm in absorbance mode. Moreover, the studied drugs were concurrently determined in human plasma using ultra-performance liquid chromatography (UPLC). The separation process was carried out in WatersTM Acquity C18 BEH column using a solvent system of 0.02 M KH2PO4 buffer, pH 4; MeOH and acetonitrile (2:1:1, v:v:v) isocratically at a flow speed of 0.5 mL/min. The absorbance of each eluent was read at 220 nm. Results: Concurrent evaluation of DAPA and SAX was carried without separation using TLC-densitometric method, and it was successful in determination of DAPA and SAX in concentration ranges of 10 – 70 μg/band and 5 – 60 μg/band, respectively. In addition, retardation factor (Rf) values for SAX and DAPA were 0.17 and 0.31, respectively. Furthermore, the studied drugs were concurrently determined in human plasma using UPLC, which was sensitive enough to quantify DAPA and SAX in concentration ranges of 100 – 1000 and 20 – 200 ng/mL, respectively. Conclusion: These methods can be utilized for sensitive monitoring of DAPA and SAX in pharmacokinetic and bioequivalence studies

Selective simultaneous ultra-performance liquid chromatographic quantification of some benzodiazepines drug residues in pharmaceutical industrial wastewater

Purpose: To investigate the sensitivity and selectivity of ultra-performance liquid chromatographic (UPLC) quantification of bromazepam (BRZ) and diazepam (DZP) in pharmaceutical industrial wastewater. Methods: Wastewater samples were collected from the effluents of a pharmaceutical industrial plant producing BRZ and DZP in tablet dosage forms. The quantification of BRZ and DZP was done after their solid-phase extraction. The resolution process was performed on WatersTM column as the stationary phase. The mobile phase was acetonitrile: methanol: 0.05 M phosphate buffer (pH 6.5), at a volume ratio of 5:2:3, with a flow rate of 0.7 mL/min. Detection was carried out at 240 nm in a concentration range of 10 – 250 ng/mL. The method was fully validated in line with ICH-Q2B regulations. Results: The UPLC method was validated for the quantification of BRZ and DZP. The relative percentage recoveries were 99.55 ± 0.48 (n = 5) and 101.34 ± 0.86 (n = 5), for BRZ and DZP, respectively, in spiked distilled water, and 99.16 ± 0.77 (n = 5) and 99.32 ± 0.56 (n = 5), in tap water, respectively. The UPLC revealed effluent content ranging from 20.68 – 44.77 mg/mL for BRZ and 22.77 – 41.83 ng/mL for DZP. These values were not significantly different from their reference standards (p > 0.05). Conclusion: A sensitive and selective UPLC-method has been developed for the reproducible determination of BRZ and DZP in industrial wastewater samples. The effective monitoring of the pharmaceutical industrial pollutant will help to conserve the environment and minimize the hazardous effects of these pollutants

Solid-phase extraction and validated spectrofluorimetric quantification of pamidronate in human plasma

Purpose: To design a simple and sensitive quantification procedure for pamidronate disodium (PAM) after its solid phase extraction from plasma.Methods: The procedure was based on derivatization of PAM using a suitable fluorogen, 4-chloro-7-nitro-2,1,3-benzoxazole. The  product was determined spectrofluorometrically at excitation and emission wavelengths of 390 and 535 nm, respectively. The method was optimized for all factors that affect the reaction between PAM and the fluorogen. These factors include diluting solvent, pH of the reaction medium, volume of fluorogen solution, buffer pH, buffer volume, temperature and heating time. The method was fully validated according to US-FDA guidelines with respect to linearity, accuracy, precision, recovery, robustness and stability.Results: PAM was successfully extracted from human plasma with solid-phase extraction technique. A linear response was obtained in the concentration range of 10 – 100 ng/mL, with correlation coefficient of 0.998. Mean maximum plasma concentration of PAM was 9.73.2 ± 3.2 µmol/L, which was within the linear range of the proposed method, thereby confirming its sensitivity for the determination of plasma PAM.Conclusion: The proposed procedure is suitable for the quantification of PAM in human plasma after its solid phase extraction. The method is sensitive enough for use in PAM determination in pharmacokinetic studies. Moreover, it is likely a more cost-effective and simpler alternative method than high performance liquid chromatograph (HPLC) methods. Keywords: Pamidronate disodium, Derivatization, Spectrofluorimetry, Fluorogen, Quantificatio

Ultra-performance liquid chromatography–tandem mass spectrometric determination of ramipril in human plasma

Purpose: To develop a sensitive and accurate ultra-performance liquid chromatography–tandem mass spectrometric (UPLC-MS) method for quantification of ramipril in human plasma.Methods: Ramipril was extracted from biological fluid using equal volumes of n-hexane and propanol (1:1, v/v), and then chromatographed in a suitable C18 column with methanol: 0.1 % HCOOH (4: 1, v/v) as mobile phase. Atorvastatin was used as an internal standard for the  chromatographic separation and quantification. The method was validated according to the United States Food and Drug Administration guidelines for standard indices.Results: Ramipril was determined in the concentration range 0.05 and 1000 ng/mL the validation procedure exhibited a correlation coefficient of 0.9979 + 0.002 (p = 0.05). The studied drug was quantified with lower ceiling of 0.05 ng/mL, and showed an accuracy of 105.00 %.Conclusion: A sensitive UPLC-MS analytical method has been successfully developed for the quantification of ramipril in human plasma. This method can be applied efficiently for the quantification of ramipril in bioavailability and pharmacokinetic studies. Keywords: Liquid chromatography–tandem mass, Ramipril, Stability, Biological fluids, Plasm

Solid phase extraction and LC-MS/MS quantification of ibandronate in human plasma

Purpose: To develop and validate a simple, highly sensitive and accurate method for the quantification of ibandronate (IBN) in human plasma.Methods: Electrospray ionization liquid chromatography tandem mass spectrometry (LC-MS/MS) in positive ion mode was applied to obtain optimum signals. The parent ion was acquired under collisionactivated dissociation conditions, and the abundant fragments used to design multiple reaction monitoring experiments for monitoring two ibandronate transitions (m/z 376 to m/z 114, and m/z 376 to m/z 250). The IBN was isolated from plasma with weak anion exchange solid phase extraction columns with ‘on-cartridge’ derivatization using tri-methylsilyl-diazomethane (TMSDZ) reagent to convert IBN to tetra-methyl derivative.Results: The studied drug was successfully extracted from plasma samples without any interference at a retention time of 3.2 min. The matrix effect averaged 110 %, indicating that endogenous materials had little effect on ionization. The relationship between plasma analyte concentration and IBN signal area was satisfactorily linear, with correlation coefficient (r2) ranging from 0.9817 to 0.9942 in the concentration range of 0.5 – 200 ng/ml. The lower and upper limits of quantification (LLOQ and ULOQ) for IBN were 0.5 and 200 ng/ml, respectively. Relative recovery of IBN from plasma after extraction and derivatization at 3 distinct concentrations was 83.93 to 85.06 %, relative to standard solutions. The ranges of intra- and inter-day accuracies of quantification of quality controls were 89.39 - 106.40 %, and 90.50 - 107.96 %, respectively. Processed plasma IBN extracts were stable in autosampler at 4 0C (91.12 to 103.49%). Long-term stability in plasma after 30 days at -24 0C ranged from 89.52 to 113.18 %.Conclusion: This validated LC-MS/MS method can be successfully applied for determination of IBN in pharmacokinetic studies. It is a sensitive and specific assay for plasma IBN in bioequivalence studies. Keywords: Ibandronate, LC-MS/MS, Validation, Derivatization, Solid-phase extractio

Spermatozoal Fractalkine Signaling Pathway Is Upregulated in Subclinical Varicocele Patients with Normal Seminogram and Low-Level Leucospermia

Background. Fractalkine is produced in seminal plasma in small amounts and correlates with sperm motility. Purpose. To investigate the possible effect of low-level leucospermia on spermatozoa oxidative stress and sDNA fragmentation in patients with subclinical varicocele and apparently normal seminogram, and also to study the role of spermatozoal fractalkine and its receptor (CX3CR1) gene expression as a marker of spermatozoa inflammatory response. Methods. This study included 80 patients with subclinical varicocele (45 fertile and 35 infertile) and 45 age-matched fertile volunteers. In semen samples, fractalkine and CX3CR1 gene expression were investigated by qRT-PCR. Moreover, seminal plasma malondialdehyde (MDA) and total antioxidant capacity (TAC) were measured. Results. There are significant decrease in semen quality and significant increase in seminal leucocytes count in subclinical varicocele. Our results show a significant increase in MDA and TAC levels, DNA fragmentation, and expression levels of fractalkine and its receptor (CX3CR1) in subclinical varicocele groups. Conclusion. Subclinical varicocele induces seminal and spermatozoal subclinical inflammatory response in the form of low-level leucospermia and increased mRNA expression of the fractalkine signaling pathway, leading to increased spermatozoal ROS production, oxidative stress, and DNA fragmentation. These could cooperate in the pathogenesis of delayed fertility in males with subclinical varicocele

HER2/neu expression status of post BCG recurrent non-muscle-invasive bladder urothelial carcinomas in relation to their primary ones

Background: Transurethral resection (TUR) followed by adjuvant therapy is still the treatment of choice of Non-Muscle-Invasive Bladder Urothelial Carcinoma (NMIBUC). However, recurrence is one of the most troublesome features of these lesions. Early second resection and adjuvant BCG therapy has been shown to improve the outcome. Objective: To evaluate the prognostic value of C-erbB-2 (HER2/neu) expression status in Non-Muscle-Invasive Bladder Urothelial Carcinoma cases, before and after intravesical Bacillus Calmette Guerin (BCG immunotherapy). Materials and methods: HER2/neu expression was studied in 120 (Ta-T1) Non-Muscle-Invasive Urothelial Carcinoma cases. The expression was evaluated and compared to the expression after Bacillus Calmette Guerin (BCG) immunotherapy. Results: HER2/neu expression in low and high grade of the Non- Muscle-Invasive Urothelial Carcinoma was (38%) and (83%) respectively. The difference of the expression rates by tumor grade was statistically significant. In recurring lesions post BCG therapy, C-erbB-2 expression was markedly decreased (31.6%) when compared to its expression before therapy (65%). Conclusions: The HER2/neu expression increased as the tumor grade rose. The reduction in expression following BCG treatment in Non-Invasive transitional cell carcinoma cases could reflect a reduction of the potential malignancy of the tumor

Antimicrobial resistance among migrants in Europe: a systematic review and meta-analysis

BACKGROUND: Rates of antimicrobial resistance (AMR) are rising globally and there is concern that increased migration is contributing to the burden of antibiotic resistance in Europe. However, the effect of migration on the burden of AMR in Europe has not yet been comprehensively examined. Therefore, we did a systematic review and meta-analysis to identify and synthesise data for AMR carriage or infection in migrants to Europe to examine differences in patterns of AMR across migrant groups and in different settings. METHODS: For this systematic review and meta-analysis, we searched MEDLINE, Embase, PubMed, and Scopus with no language restrictions from Jan 1, 2000, to Jan 18, 2017, for primary data from observational studies reporting antibacterial resistance in common bacterial pathogens among migrants to 21 European Union-15 and European Economic Area countries. To be eligible for inclusion, studies had to report data on carriage or infection with laboratory-confirmed antibiotic-resistant organisms in migrant populations. We extracted data from eligible studies and assessed quality using piloted, standardised forms. We did not examine drug resistance in tuberculosis and excluded articles solely reporting on this parameter. We also excluded articles in which migrant status was determined by ethnicity, country of birth of participants' parents, or was not defined, and articles in which data were not disaggregated by migrant status. Outcomes were carriage of or infection with antibiotic-resistant organisms. We used random-effects models to calculate the pooled prevalence of each outcome. The study protocol is registered with PROSPERO, number CRD42016043681. FINDINGS: We identified 2274 articles, of which 23 observational studies reporting on antibiotic resistance in 2319 migrants were included. The pooled prevalence of any AMR carriage or AMR infection in migrants was 25·4% (95% CI 19·1-31·8; I2 =98%), including meticillin-resistant Staphylococcus aureus (7·8%, 4·8-10·7; I2 =92%) and antibiotic-resistant Gram-negative bacteria (27·2%, 17·6-36·8; I2 =94%). The pooled prevalence of any AMR carriage or infection was higher in refugees and asylum seekers (33·0%, 18·3-47·6; I2 =98%) than in other migrant groups (6·6%, 1·8-11·3; I2 =92%). The pooled prevalence of antibiotic-resistant organisms was slightly higher in high-migrant community settings (33·1%, 11·1-55·1; I2 =96%) than in migrants in hospitals (24·3%, 16·1-32·6; I2 =98%). We did not find evidence of high rates of transmission of AMR from migrant to host populations. INTERPRETATION: Migrants are exposed to conditions favouring the emergence of drug resistance during transit and in host countries in Europe. Increased antibiotic resistance among refugees and asylum seekers and in high-migrant community settings (such as refugee camps and detention facilities) highlights the need for improved living conditions, access to health care, and initiatives to facilitate detection of and appropriate high-quality treatment for antibiotic-resistant infections during transit and in host countries. Protocols for the prevention and control of infection and for antibiotic surveillance need to be integrated in all aspects of health care, which should be accessible for all migrant groups, and should target determinants of AMR before, during, and after migration. FUNDING: UK National Institute for Health Research Imperial Biomedical Research Centre, Imperial College Healthcare Charity, the Wellcome Trust, and UK National Institute for Health Research Health Protection Research Unit in Healthcare-associated Infections and Antimictobial Resistance at Imperial College London

Surgical site infection after gastrointestinal surgery in high-income, middle-income, and low-income countries: a prospective, international, multicentre cohort study

Background: Surgical site infection (SSI) is one of the most common infections associated with health care, but its importance as a global health priority is not fully understood. We quantified the burden of SSI after gastrointestinal surgery in countries in all parts of the world. Methods: This international, prospective, multicentre cohort study included consecutive patients undergoing elective or emergency gastrointestinal resection within 2-week time periods at any health-care facility in any country. Countries with participating centres were stratified into high-income, middle-income, and low-income groups according to the UN's Human Development Index (HDI). Data variables from the GlobalSurg 1 study and other studies that have been found to affect the likelihood of SSI were entered into risk adjustment models. The primary outcome measure was the 30-day SSI incidence (defined by US Centers for Disease Control and Prevention criteria for superficial and deep incisional SSI). Relationships with explanatory variables were examined using Bayesian multilevel logistic regression models. This trial is registered with ClinicalTrials.gov, number NCT02662231. Findings: Between Jan 4, 2016, and July 31, 2016, 13 265 records were submitted for analysis. 12 539 patients from 343 hospitals in 66 countries were included. 7339 (58·5%) patient were from high-HDI countries (193 hospitals in 30 countries), 3918 (31·2%) patients were from middle-HDI countries (82 hospitals in 18 countries), and 1282 (10·2%) patients were from low-HDI countries (68 hospitals in 18 countries). In total, 1538 (12·3%) patients had SSI within 30 days of surgery. The incidence of SSI varied between countries with high (691 [9·4%] of 7339 patients), middle (549 [14·0%] of 3918 patients), and low (298 [23·2%] of 1282) HDI (p < 0·001). The highest SSI incidence in each HDI group was after dirty surgery (102 [17·8%] of 574 patients in high-HDI countries; 74 [31·4%] of 236 patients in middle-HDI countries; 72 [39·8%] of 181 patients in low-HDI countries). Following risk factor adjustment, patients in low-HDI countries were at greatest risk of SSI (adjusted odds ratio 1·60, 95% credible interval 1·05–2·37; p=0·030). 132 (21·6%) of 610 patients with an SSI and a microbiology culture result had an infection that was resistant to the prophylactic antibiotic used. Resistant infections were detected in 49 (16·6%) of 295 patients in high-HDI countries, in 37 (19·8%) of 187 patients in middle-HDI countries, and in 46 (35·9%) of 128 patients in low-HDI countries (p < 0·001). Interpretation: Countries with a low HDI carry a disproportionately greater burden of SSI than countries with a middle or high HDI and might have higher rates of antibiotic resistance. In view of WHO recommendations on SSI prevention that highlight the absence of high-quality interventional research, urgent, pragmatic, randomised trials based in LMICs are needed to assess measures aiming to reduce this preventable complication

A Comparative Study between Screen-Printed and Solid-Contact Electrodes for the Stability-Indicating Determination of Bromazepam

Stability-indicating methods are awesome tools to ensure the safety and efficacy of active pharmaceutical ingredients (APIs). An accurate comparative study involving the use of potentiometric sensors for the determination of bromazepam (BRZ) in the presence of its main product of degradation and impurity was performed by the fabrication of two membrane electrodes. A screen-printed electrode (SPE) and a solid-contact glassy carbon electrode (SCE) were fabricated and their performance optimized. The fabricated sensors showed a linear electrochemical response in the concentration range 1.0 &times; 10&minus;6 M to 1.0 &times; 10&minus;2 M. The electrodes exhibited Nernstian slopes of 59.70 mV/decade and 58.10 mV/decade for the BRZ-SPE and BRZ-SCE membrane electrodes, respectively. The electrochemical performance was greatly affected by the medium pH. They showed an almost ideal electrochemical performance between pH 3.0 and pH 6.0. The fabricated membranes were applied successfully for the quantification of BRZ in the presence of up to 90% of its degradation product. Moreover, a successful application of the fabricated electrodes was performed for the sensitive and selective quantification of BRZ in its tablet form without any pretreatment procedure