Exquisite integration of quality-by-design and green analytical approaches for simultaneous determination of xylometazoline and antazoline in eye drops and rabbit aqueous humor, application to stability study.

This work implements a stability indicating HPLC method developed to simultaneously determine xylometazoline (XYLO) and antazoline (ANT) in their binary mixture, rabbit aqueous humor and cited drug's degradates by applying analytical quality-by-design (AQbD) combined with green analytical chemistry (GAC) experiment for the first time. This integration was designed to maximize efficiency and minimize environmental impacts, as well as energy and solvent consumption. Analytical quality-by-design was applied to achieve our aim starting with evaluation of quality risk and scouting analysis, tracked via five parameters chromatographic screening using Placket-Burman design namely: pH, temperature, organic solvent percentage, flow rate, and wavelength detection. Recognizing the critical method parameters was done followed by optimization employing central composite design and Derringer's desirability toward assess optimum conditions that attained best resolution with satisfactory peak symmetry with short run time. Optimal chromatographic separation was attained by means of an XBridge® C18 (4.6 × 250 mm, 5 µm) column through isocratic elution using a mobile phase consists of phosphate buffer (pH 3.0): ethanol (60:40, by volume) at a 1.6 mL/min flow rate and 230.0 nm UV detection. Linearity acquired over a concentration range of 1.0-100.0 µg/mL and 0.5-100.0 µg/mL for XYLO and ANT, respectively. Furthermore, imperiling cited drugs' stock solutions to stress various conditions and satisfactory peaks of degradation products were obtained indicating that cited drugs are vulnerable to oxidative degradation and basic hydrolysis. Degradates' structures were elucidated using mass spectrometry. Applying various assessment tools; namely: analytical greenness (AGREE), green analytical procedure index (GAPI), analytical eco-scale, and national environmental method index (NEMI), Greenness method's evaluation was applied and proved to be green. In fact, the developed method is established to be perceptive, accurate, and selective to assess cited drugs for routine analysis.

[Effect of Shelf Life on the Physical Stability of Suspended Ophthalmic Solutions].

Quality changes associated with physical changes in suspended eye drops are difficult to predict. In this study, we attempted to evaluate the aggregation and redispersability in commercially available suspended eye drops (fluorometholone ophthalmic solutions). The 0.1% fluorometholone ophthalmic solutions (the original product and 4 generic products) were gently mixed by hand after short-term (4 months) or long-term (40 months) storage, and the drug concentration in the first drop and physical stability (redispersability and particle size) were measured. All eye drops produced a cloudy precipitate on the bottom surface of the container, and the amount of precipitate decreased with mixing time. The drug concentration per drop in the original product was approximately 70% of the labeled value after mixing 10 times, and the drug particle size was approximately 4 µm. After mixing the generic products stored short-term 10 times, the concentration ranged from less than 50% to almost 100%. In addition, some generic products after long-term storage had a reduced redispersion ability and labeled concentration. These results suggested that at least 10 mixing were required before the using of fluorometholone original product. In addition, some generic products may not provide sufficient drug exposure even when mixed in the same manner as the original products.

Imprinted ß-ketoenamine-linked covalent organic frameworks as dispersive sorbents for the fluorometric determination of timolol.

Timolol accompanied the formation of fluorescent ß-ketoenamine-linked covalent organic frameworks (COFs) via the Sc(Tof)3-catalyzed condensation of derivated carbaldehyde and hydrazide in a 1,4-dioxane/mesitylene porogen to construct timolol-imprinted COFs (TICOFs). With high imprinting factors, the synthesis-optimized TICOFs were characterized by fluorescence, UV-Vis spectrometry, X-ray diffraction, N2 adsorption/desorption analyses, scanning electron microscopy, and FTIR spectrometry. The TICOF fluorescence measured at 390 nm/510 nm is dynamically quenched by timolol and was thus utilized to quantify timolol in a linear range of 25-500 nM with a LOD of 8 nM. The TICOF recovered 99.4% of 0.5% timolol maleate in a commercial eye drop (RSD = 1.1%, n = 5). In addition, TICOF was used as a dispersive sorbent to recover 95% of 2.0 nM timolol from 20 mg of TICOF in 25 mL phosphate buffer. Dilution factors of 25 and 75 were the maximum tolerated proportions of the urine and serum matrix spiked with 2.0 nM timolol to reach recoveries of 92.4% and 90.3%, respectively.

Chromatographic bioanalysis of antiglaucoma drugs in ocular tissues.

Glaucoma is a heterogeneous group of multifactorial optic neuropathies and the leading cause of irreversible blindness and visual impairment. Epidemiological data has estimated that in 2020 there will be more than 80 million individuals affected by the disease worldwide. Nowadays, intraocular pressure (IOP) lowering is carried out mainly by pharmacotherapy, with different drugs. The study of ocular pharmacokinetics of antiglaucoma drugs, crucial for better understanding of ocular distribution, bioavailability, and pharmacodynamic parameters, can benefit the development of antiglaucoma drugs or formulations. Bioanalysis of drugs in ocular matrices is still underestimated, since it is challenging and rarely performed. Therefore, this review summarized the chromatographic methods employed for the quantification of several antiglaucoma drugs in different ocular matrices, discussing bioanalytical steps, such as sample preparation, separation, and detection. Animals and matrices as well as the challenges faced in ocular bioanalysis were also discussed. Ocular bioanalysis has been performed mainly in rabbits, the most adequate animal model for ocular studies. The matrix most used is aqueous humor, because it is cleaner and easier to sample. Sample preparation was carried out primarily employing classic techniques, such as liquid-liquid extraction, protein precipitation, and solid-phase extraction, with conventional solvents and sorbents. Chromatographic separation was achieved predominantly by reversed-phase liquid chromatography. Ultraviolet spectrophotometry and tandem mass spectrometry prevailed for detection, although other techniques, such as fluorimetry, have also been used. It was evidenced that more efforts must be directed towards miniaturized, eco-friendly, and non-terminal sampling for sample preparation. In its turn, ultra high-performance liquid chromatography and mass spectrometry should gain prominence in ocular bioanalysis for separation and detection, respectively, since it combines high separation capacity with selectivity and sensitivity, in addition to being an environmental friendly approach.

Latanoprost Quantification in Ocular Implants and Tissues: HPLC-Fluorescence vs HPLC-UV.

Anti-glaucoma latanoprost-loaded ocular implants provide prolonged delivery and enhanced bioavailability relative to the conventional eye drops. This study aims at the development and validation of a reversed-phase high-performance liquid chromatography method for quantitative analysis of nanogram levels of latanoprost in the eye, and for the first time, compares the use of fluorescence vs ultraviolet (UV) detectors in latanoprost quantification. The mobile phase was composed of acetonitrile:0.1% v/v formic acid (60:40, v/v) with a flow rate of 1 mL/min and separation was done using a C18 column at temperature 40°C. The fluorescence excitation and emission wavelengths were set at 265 and 285 nm, respectively, while the UV absorption was measured at 200 nm. The latanoprost concentration-peak area relationship maintained its linearity (R2 = 0.9999) over concentration ranges of 0.063-10 µg/mL and 0.212-10 µg/mL for the fluorescence and UV detectors, respectively. The UV detector showed better precision, while the fluorescence detector exhibited higher robustness and greater sensitivity, with a detection limit of 0.021 µg/mL. The fluorescence detector was selected for quantification of latanoprost released from ocular implants in vitro and in porcine ocular tissues. The developed method is a robust, rapid and cost-effective alternative to liquid chromatography-mass spectrometry for routine analysis of latanoprost released from ocular implants.

Efficacy of Sub-Tenon Micro-Perfusion of Cyclophosphamide in Rabbits with Severe Ocular Inflammation.

PURPOSE: To explore the feasibility of cyclophosphamide (CP) via a sub-Tenon micro-perfusion system (SMS) in rabbits, and assess its therapeutic efficacy in severe ocular inflammation. MATERIALS AND METHODS: Distribution and pharmacokinetics of CP were evaluated in vivo, and the concentrations of CP in plasma, vitreous humor, and retina/choroid were quantitated by ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) at different time points. After induction of severe experimental uveitis, rabbits were divided into three groups (n=8 in each): the SMS group, subconjunctival injection (SI) group, and control group. Clinical inflammatory score was assessed in rabbits. Electroretinography and histopathology were performed on post-treatment day 8. Statistical analyses were performed using Mann-Whitney and Kruskal-Wallis tests. P-value less than 0.05 was considered significant. RESULTS: The concentrations of CP in vitreous humor and retina/choroid in the SMS group were significantly higher than that of the SI group at 3, 6, 10, and 24 hours (P<0.01), while plasmatic CP concentrations were comparable at all time points in the SMS group and SI group (P>0.05). The SMS group showed significantly less inflammation compared to the control group and SI group. Furthermore, the restoration of retinal structure and function were more obvious in the SMS group compared with conventional SI application. CONCLUSION: Sub-Tenon micro-perfusion of CP exhibited satisfied therapeutic efficacy in rabbits with severe ocular inflammation and may provide a promising alternative for controlling ocular inflammatory disease and immune-mediated ocular diseases.

Validated Liquid Chromatography-Tandem Mass Spectroscopic Method for Simultaneous Determination of Certain Drugs Used in Cataract Surgery in Rabbit Aqueous Humor.

A sensitive, selective, accurate and precise ultra-high performance liquid chromatography-Tandem mass spectrometry (MS/MS) method was developed and validated for the simultaneous determination of drugs used as eye drops in cataract surgery in aqueous humor. Cataract surgery requires a powerful mydriatic eye drops combination such as cyclopentolate hydrochloride and phenylephrine hydrochloride to dilate the pupil and facilitate eye lens replacement and also requires strong fluoroquinolone antibiotic such as lomefloxacin hydrochloride. The method was performed with positive ion electrospray ionization and the analytes were quantified and monitored on a triple quadrupole mass spectrometer using multiple reaction monitoring scanning mode. Liquid-liquid extraction was used for the purification and preconcentration of analytes from rabbit aqueous humor matrix. Chromatographic elution was performed using an Phenomenex Luna® C18 (150 mm × 2.1 mm, 1.6 µm) column and moxifloxacin hydrochloride as internal standard with a mobile phase consisting of methanol:water:formic acid (70:29:1, by volume) at flow rate of 0.2 mL/min. Satisfactory results regarding linearity, recovery, stability, accuracy and precision of the analytes were obtained. Full validation of the procedure was performed according to the US Food and Drug Administration guidance for industry: bioanalytical method validation and European Medicines Agency (EMA) guideline on bioanalytical method validation.

Measurement of Free Iodine in Different Formulations of Povidone-Iodine Eye Drops 5.

There are multiple studies in the literature that support the use of povidone iodine in the preparation of the surgical field of cataract as the most effective means to reduce the bacteria present in the ocular surface and the risk of infection. The concentration of free iodine is related to the antiseptic activity of these compounds, being, therefore, a good indicator of its effectiveness. The objective of this study was to determine the amount of free iodine and the evolution of it in different formulations of povidone iodine eye drops. The povidone iodine 5% eye drops were prepared starting from Betadine 10% dermal or the active principle and using a solvent, phosphate-citrate buffer solution, and sodium chloride 0.9% or sterile water for injection. Aliquots of 5 mL were packed in low-absorption absorption eye drops, topaz glass vials, and polyethylene syringe. The determination of free iodine was made by volumetric titration. Titration was performed with 0.1 M sodium thiosulfate using a starch solution as an indicator. Of the 0.1 M sodium thiosulfate, 1 mL is equivalent to 12.69 mg of available iodine, and it is expressed as a percentage of free iodine in the iodized povidone (% free iodine). Eyewash titrations were performed by replacing the substance with 5 mL of eye drops and following the remaining steps. Valuations were made on days 0, 7, and 14, as well as the measurement of pH and osmolarity. The results show that there are no differences between the average results at the three measurements taken on days 0, 7, and 14. We conclude that the free iodine remains stable during the stability period of 14 days. Regarding the pH and osmolarity data, the authors believe that the best tolerated formula will be that elaborated with povidone iodine and a phosphate- citrate buffer solution.

Microbial safety implications of in-use topical diagnostic ophthalmic medications in eye clinics in Ghana.

PURPOSE: To determine the microbial contaminants and its clinical importance in topical diagnostic ophthalmic medications (cycloplegics/mydriatics and miotics) in eye clinics in Ghana. METHOD: A cross-section of eye clinics was sampled for the diagnostic agents (Atropine, Phenylephrine, Tropicamide and Cyclopentolate, Pilocarpine). Standard laboratory procedures and protocols were observed in culturing the samples on different Agars. Microscopy and various biochemical tests were performed to identify microbial species. Antimicrobial susceptibility testing was also performed to ascertain the clinical importance of the isolated microbes. RESULTS: A total of 113 samples were obtained, from which 334 bacteria were isolated which included Bacilli spp. 91(27.25%), Coagulase Negative Staphylococci spp. 59(17.66%), Moraxella spp. 47(14.07%), Staphylococcus aureus 41(12.27%), Streptococcus spp. 21(6.29%), Klebsiella spp. 20(5.99%), Pseudomonas spp. 13(3.89%), Proteus spp. 12(3.59%), Escherichia coli. 12 (3.59%), Serratia spp. 10(2.99%), Shigella spp. 7(2.09%), Salmonella spp. 1(0.3%). There were 96 isolated fungal contaminants mainly Penicillium spp. 41(42.71%), Cephalosporium spp. 19(19.79%), Cladosporium spp. 15(15.63%), Aspergillus spp. 13(13.54%), Cercospora spp. 8(8.33%). The diagnostic agent with the most bacteria contamination was Phenylephrine 90 (26.95%) and the least being Pilocarpine 49 (14.67%). Also, the diagnostic agent with the most fungal contamination was Cyclopentolate 29 (30.2%) and the least was Tropicamide and Pilocarpine with 15 (15.63%) each. Gentamicin and Ciprofloxacin were the only antibiotics that showed 100% activity against all the bacterial isolates. Fungal contaminants were more susceptible to Ketoconazole as compared to Fluconazole. CONCLUSION: Topical diagnostic ophthalmic preparations used in clinical settings in Ghana are contaminated with clinically important bacteria and fungi.

HPLC-UV method for simultaneous determination of sparfloxacin and dexamethasone sodium phosphate in eye drops.

A simple, sensitive liquid chromatographic method was developed and validated for the simultaneous estimation of sparfloxacin and dexamethasone sodium phosphate in bulk and pharmaceutical formulations. Optimum separation was achieved in less than 10 min using a C18 column (250 mmx4.6 mm i.d, 5µ particle size) by isocratic elution. The mobile phase consisting of a mixture of mixed phosphate buffer (pH 6.8) and acetonitrile (50:50, v/v) was used. Column effluents were monitored at 224nm at a flow rate of 1ml/min. Retention times of sparfloxacin and dexamethasone sodium phosphate were 3.01 and 6.47 min respectively. The linearity of sparfloxacin and dexamethasone sodium phosphate was in the range of 3-18µg/ml and 1-6µg/ml respectively. Developed method was economical because, the time taken and amount of solvent consumed for each analysis was less. The method was validated and was applied to the simultaneous determination of sparfloxacin and dexamethasone sodium phosphate in bulk and pharmaceutical formulations.

A validated size exclusion chromatography method coupled with fluorescence detection for rapid quantification of bevacizumab in ophthalmic formulations.

Bevacizumab is a full-length human monoclonal antibody used to treat various neovascular diseases such as wet age-related macular degeneration (AMD), diabetic eye disease and other problems of the retina. Monthly intravitreal injections of bevacizumab (Avastin®) are effective in the treatment of wet AMD. However, there is a growing demand in the development of sustained release ophthalmic formulations. Therefore, this study aims, for the first time, to develop a rapid, simple, and sensitive method using size exclusion chromatography coupled with fluorescence detection for routine quantification of bevacizumab in ophthalmic formulations and during in vitro release studies. The selected chromatographic conditions included an aqueous mobile phase composed of 35 mM sodium phosphate buffer and 300 mM sodium chloride (pH 6.8), a flow rate of 0.5 mL/min, and the fluorescence detector was operated at excitation and emission wavelengths of 280 and 340 nm, respectively. The peak area-concentration relationship maintained its linearity over concentration range of 0.1-20 µg/mL (R2 = 0.9993), and the quantitation limit was 100 ng/mL. The method was validated for specificity, accuracy, precision, and robustness. The developed method had a run time of 6 min at temperature 25 °C, making it a unique validated method for rapid and cost-effective quantification of bevacizumab.

Validación galénica del colirio de plasma rico en factores de crecimiento / Galenic validation of plasma rich in growth factors eye drops

Objetivo: Evaluación galénica del proceso de obtención y almacenamiento del colirio de plasma rico en factores de crecimiento PRGF-Endoret(R). Método: Para evaluar la asepsia en la obtención del colirio de PRGFEndoret(R) se realizó un ensayo de esterilidad siguiendo las normas descritas en la Farmacopea Europea y se analizó la estanqueidad de los dispensadores de colirio de PRGF-Endoret(R). Asimismo, se estudiaron las propiedades químicas y biológicas del colirio tras su proceso de obtención y almacenamiento. Se incluyeron ensayos de filtración del colirio, de un ciclo de congelación a -20 ºC y descongelación, así como de estabilidad durante tres y seis meses almacenados a -20 ºC. Resultados: Los ensayos de esterilidad mostraron que no hubo crecimiento microbiano en ninguno de los dispensadores analizados y se observó que el 100% de los monodosis analizados y el 98,4% de los tapones mantenían el hermetismo. Todos los factores de crecimiento analizados permanecieron constantes tras el filtrado del colirio de PRGF-Endoret(R). Además, todos los estudios de estabilidad llevados a cabo con el colirio de PRGF-Endoret(R) en el presente estudio mostraron que no se produjeron cambios significativos en los niveles de factores de crecimiento, en la actividad proliferativa celular ni en las características químicas analizadas. Conclusiones: El presente trabajo muestra que el proceso de elaboración del colirio de PRGF-Endoret(R) se lleva a cabo de forma controlada, aséptica y segura, siguiendo las normas descritas en la Farmacopea Europea. Además, el colirio de PRGF-Endoret(R) obtenido mantiene sus propiedades físico-químicas y biológicas tras someterlo a diferentes tiempos y temperaturas de almacenamiento

Objective: Galenic evaluation of the process for obtaining and storing the platelet rich in growth factors PRGF-Endoret(R) eye drops. Method: To assess whether the PRGF-Endoret(R) eye drops process is aseptically obtained, a sterility test was carried out on the eye drops; the tightness of the PRGF-Endoret(R) eye drops containers was also analyzed. Likewise, the chemical and biological properties of the PRGF-Endoret(R) eye drops were evaluated after the obtaining process and storage. Eye drop filtration tests, one cycle of freezing at -20 °C and thawing, and eye drop stability for three and six months stored at -20 °C were included. Results: The results obtained in the sterility test showed no microbial contamination in any of the analyzed eyedropper; tightness test showed that 100% of the eyedrop containers and the 98.4% of the plugs analyzed remained hermetic. On the other hand, all the growth factors measured remained constant after filtering the PRGF-Endoret(R) eye drops. Furthermore, the different eye drop stability tests carried out in this study showed no significant changes in the growth factors levels, cell proliferative activity or in the chemical characteristics analyzed. Conclusions: The PRGF-Endoret(R) eye drops are obtained in a safety and aseptic manner following the guidelines issued by the Spanish Agency for Drugs and Health Products and the Ministry of Health to obtain medicines for human use. The PRGF-Endoret(R) eye drops maintain their physical-chemical and biological properties after being subjected to different storage times and temperatures

Galenic validation of plasma rich in growth factors eye drops.

OBJECTIVE: Galenic evaluation of the process for obtaining and storing the platelet rich in growth factors PRGF-Endoret® eye drops. METHOD: To assess whether the PRGF-Endoret® eye drops process is aseptically obtained, a sterility test was carried out on the eye drops; the tightness of the PRGF-Endoret® eye drops containers was also analyzed. Likewise, the chemical and biological properties of the PRGF- Endoret® eye drops were evaluated after the obtaining process and storage.  Eye drop filtration tests, one cycle of freezing at -20 °C and thawing, and  eye drop stability for three and six months stored at -20 °C were included. RESULTS: The results obtained in the sterility test showed no microbial  contamination in any of the analyzed eyedropper; tightness test showed that  100% of the eyedrop containers and the 98.4% of the plugs analyzed  remained hermetic. On the other hand, all the growth factors measured  remained constant after filtering the PRGF-Endoret® eye drops.  Furthermore, the different eye drop stability tests carried out in this study  showed no significant changes in the growth factors levels, cell proliferative  activity or in the chemical characteristics analyzed. Conclusions: The PRGF-Endoret® eye drops are obtained in a safety and aseptic manner following the guidelines issued by the Spanish Agency  for Drugs and Health Products and the Ministry of Health to obtain medicines for human use. The PRGF-Endoret® eye drops maintain their physical-chemical and biological properties after being subjected to different  storage times and temperatures.

Objetivo: Evaluación galénica del proceso de obtención y almacenamiento del colirio de plasma rico en factores de crecimiento PRGF- Endoret®. Método: Para evaluar la asepsia en la obtención del colirio de PRGFEndoret ® se realizó un ensayo de esterilidad siguiendo las normas  descritas en la Farmacopea Europea y se analizó la estanqueidad de los dispensadores de colirio de PRGF-Endoret®. Asimismo, se estudiaron las propiedades químicas y biológicas del colirio tras su proceso de obtención y almacenamiento. Se incluyeron ensayos de filtración del colirio,  de un ciclo de congelación a ­20 ºC y descongelación, así como de  estabilidad durante tres y seis meses almacenados a ­20 ºC.Resultados: Los ensayos de esterilidad mostraron que no hubo crecimiento microbiano en ninguno de los dispensadores analizados y se  observó que el 100% de los monodosis analizados y el 98,4% de los tapones mantenían el hermetismo. Todos los factores de crecimiento  analizados permanecieron constantes tras el filtrado del colirio de PRGF- Endoret®. Además, todos los estudios de estabilidad llevados a cabo con el  colirio de PRGF-Endoret® en el presente estudio mostraron que no se  produjeron cambios significativos en los niveles de factores de crecimiento,  en la actividad proliferativa celular ni en las características químicas  analizadas.Conclusiones: El presente trabajo muestra que el proceso de elaboración del colirio de PRGF-Endoret® se lleva a cabo de forma  controlada, aséptica y segura, siguiendo las normas descritas en la  farmacopea Europea. Además, el colirio de PRGF-Endoret® obtenido  mantiene sus propiedades físico-químicas y biológicas tras someterlo a  diferentes tiempos y temperaturas de almacenamiento.

Respuesta de los autores: Comentarios al artículo:La atropina superdiluida al 0.01% frena el aumento de la miopía en niños españoles. Un estudio a largo plazo 5 años de evolución: seguridad y eficacia / Author's reply: Comments on: Superdiluted atropine at 0.01% reduces progression of myopia in children and adolescents. A 5-year study of safety and effectiveness

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Analytical Eco-Scale for Assessing the Greenness of a Developed Potentiometric Method for Lomefloxacin Hydrochloride Determination in its Different Dosage Forms, Plasma, and Dissolution Medium.

Background: Traditional methods for Lomefloxacin hydrochloride (LOM) determination involve pretreatment steps, which extend analysis time and use hazardous chemicals. Objective: The ability to provide a rapid route without sample pretreatment for quantitative determination of compounds via a low-cost instrument is a challenging task. In this work, a simple potentiometric method was developed to determine the antibacterial LOM via in-house fabricated ion selective electrodes. Methods: Different sensors were fabricated using a poly vinyl chloride-based membrane, potassium tetrakis(4-chlorophenyl) borate as a cation exchanger, and 2-Nitrophenyl octyl ether as a plasticizer (sensor 1). To increase the selectivity of sensor 1, a selective molecular recognition component 2-hydroxypropyl-ß-cyclodextrin was used as ionophore (sensor 2). Results: The proposed method was validated according to International Union of Pure and Applied Chemistry recommendations, in which the proposed sensors show a linear dynamic range from 1 × 10-5 to 1 × 10-2 mol/L, with Nernstian slopes of 55.829 and 58.229 mV/decade for sensors 1 and 2, respectively. It was applied to determine LOM in bulk powder, in different dosage forms, and in plasma with no sample pretreatment. Also, the suggested method can be used as a green, in-line bench top real-time analyzer for in-process monitoring of LOM release from its tablets, under U.S. Food and Drug Administration dissolution regulations, with clear discrimination from common excipients. Results obtained by the proposed potentiometric method were compared with those obtained by a reported HPLC method. Conclusions: The proposed method is considered as a perfect alternative to traditional reported methods for LOM determination.

Selective and Sensitive Chromatographic Methods for Determination of a Co-Formulated Binary Mixture in Antibacterial Eye Drops and Aqueous Humor in the Presence of Their Degradation Products and Potential Impurities.

Prednisolone acetate (PDN) is a corticosteroid anti-inflammatory liable to degradation under different conditions and used with antibiotics in eye drops. Two selective stability-indicating separation techniques were developed for simultaneous determination of PDN and moxifloxacin HCl (MXF) binary mixture in pure forms, ophthalmic formulation, in the presence of PDN impurities and in the presence of their degradation products. The first method was based on HPTLC separation using silica gel 60 F254 HPTLC plates, and a developing system of toluene: ethyl acetate: methanol: ammonia (5.0: 6: 2.0: 0.05, v/v/v/v) is used with detection at 254 nm. The second method was HPLC using a mobile phase of acetonitrile: methanol: deionized water, pH 2.8 (25.0: 35.0: 40.0, v/v/v), at 254 nm. A kinetic study utilizing the developed HPLC method for PDN degradation under different stress conditions was performed. Furthermore, the method was applied for determination in rabbit aqueous humor. Validation was conducted as per ICH guidelines, and system suitability was ascertained. The calibration curves were constructed in the range 0.10-25.00 and 0.20-50.00 µg band-1, for PDN and MXF by HPTLC, while for HPLC, it was 0.02-50.00 and 0.10-50.00 µg mL-1 for both drugs, in order.

Chemical and toxicological studies on different brands of Asmad (Antimony sulphide) available in Pakistan and Saudi Arabia.

Eye is the most beautiful, important and sensitive organ of human body. It is not only linked with visionary complex optical system also has the ability to differentiate among the millions of colors. The apparent human personality is also associated with it. Asmad/Antimony Sulfide/Kohl/Surma powder is one of the eye preparation has been used since ancient time. There are several aesthetic and ophthalmic preparations available for human eye and they have closed association between the aesthetic and medicinal significance such as cleansing, soothing, strengthening and anti-infectious actions along with beautifying purpose of eye. The main objective of present research is to provide scientific findings regarding beneficial and toxic effects of Asmad products available in market for the frequent users. The chemical and toxicological investigations on ten selected famous brands of Pakistan samples (PHS1, PHS2, PLS, PMS and PSS) and Saudi Arabia samples (SBS, SAS, SHS, SMS and STS) were carried out through advanced and sophisticated technique Scanning Electron Microscope (SEM) linked with Energy Dispersive X-ray Spectroscopy (EDS) which is used to determine the presence different percentages of organic and inorganic elements in all the brands of Pakistani and Saudi Arabian samples. The safety and toxicity depends on the Na, Mg, Ca, K, Al, Cu, Zn, Fe, Bi, Si, O, C, S and Pb percentages respectively of the Asmad products.

Contaminação microbiana em colírios de pacientes em tratamento de glaucoma / Microbial contamination in eye drops of patients in glaucoma treatment

Resumo Objetivos: Avaliar o grau de contaminação por fungos e bactérias e o modo de conservação destes colírios hipotensores por parte dos pacientes no ambulatório de Glaucoma da Santa Casa de Ribeirão Preto. Métodos: Foram selecionados aleatoriamente cinquenta e cinco pacientes, em seguimento no ambulatório, e após consentimento dos mesmos os colírios eram coletados e enviados via correio para análise por microbiologista e patologista em até 72 horas. Foi analisado 0,5ml aproximadamente das medicações e os pacientes respondiam a um questionário simples sobre o método de conservação e se consideravam estes adequados. Resultados: Dos 55 colírios analisados, cinco (9,01%) estavam com seu conteúdo líquido contaminado. Entre os microrganismos isolados haviam 4 bactérias Gram negativas, sendo 1 (1,8%) por Serratia marcescens, 1 (1,8%) Pseudomonas aeruginosa e 2 (3,6%) Stenotrophomas maltophilia. Um colírio estava contaminado pelo fungo Cândida ssp Todos pacientes do estudo julgam seus métodos de armazenamento e instilação adequados. Os pacientes que tiveram os colírios positivados eram convocados para exame clínico e passavam por novo questionário pelo investigador. Conclusão: O tempo de abertura dos frascos e os métodos de conservação influenciam na contaminação dos medicamentos, todos os colírios com crescimento de microrganismos no presente estudo estavam abertos entre 30 e 90 dias. O fato de que a maioria dos pacientes levam seus colírios em tarefas cotidianas, aumenta a exposição dos frascos e podem ser um fator relevante para determinar a contaminação destas medicações.

Abstract Objetives: To assess the degree of fungal and bacterial contamination of hypotensive eye drops and the way these are preserved by the patients at the Glaucoma outpatient clinic of Santa Casa Hospital in Ribeirão Preto. Methods: Fifty-five patients were randomly assigned to follow-up in the outpatient clinic and, after their consent, an eye drop was collected per patient and later sent by mail for analysis by microbiologist and pathologist in up to 72 hours. Approximately 0.5ml of the medications were analyzed and the patients were asked to answer a simple questionnaire on the method of drug conservation and whether they considered it adequate. Results: Of the 55 analysed eye drops, five (9.01%) had their liquid contents contaminated. Among the microorganisms isolated there were 4 Gram negative bacteria, 1 (1.8%) by Serratia marcenses, 1 (1.8%) Pseudomonas aeruginosa and 2 (3.6%) Stenotrophomas maltophilia. An eye drop was contaminated by the fungus Candida ssp. All the patients in the study judged their methods of storage and instillation appropriate. The patients who had the positive coliria were summoned for clinical examination and passed through a new questionnaire by the investigator. Conclusion: The time and methods of preservation influence the contamination of medicinal products. All the eye drops that presented growth of microorganisms in the present study were open between 30 and 90 days. The fact that most patients take their eye drops on daily tasks increases the exposure of the bottles and can be a relevant fact to determine the contamination of these medications.

Investigation of the spectrofluorimetric behavior of azelastine and nepafenac: Determination in ophthalmic dosage forms.

The first spectrofluorimetric report investigating the fluorimetric behavior of the antihistaminic drug, azelastine (AZEL), and the non-steroidal anti-inflammatory drug, nepafenac (NEP), either in bulk or in their dosage forms, eye drops and ophthalmic suspension. After a full investigation of the factors that may influence their spectrofluorimetric behavior: pH, different organized media and organic solvents, the optimum factors were set in order to enable the analysis of each drug with maximum sensitivity. The AZEL spectrofluorimetric analysis was set at 286/364 (λex/λem) in distilled water while for NEP, the analysis was set at 228/303 (λex/λem) in methanol. The linearity range for AZEL was from 0.1 to 1.5 µg/mL while that of NEP was from 0.2 to 1.5 µg/mL. The linearity yielded good regression parameters with low LOD (0.022 and 0.032 µg/mL for AZEL and NEP, respectively) and LOQ (0.073 and 1.08 µg/mL for AZEL and NEP, respectively) when compared with those obtained from many previous spectroscopic and chromatographic reports in literature. The method was ICH validated and was applied to the analysis of AZEL and NEP with good selectivity regarding the inactive ingredients.