Rabbit Aqueous Humor Research Articles

Selective spectrofluorimetric determination of two corticosteroids along with their co-formulated drugs and degradation products in ophthalmic solution and aqueous humour.

Prednisolone acetate (PNO) and fluorometholone (FRT) are corticosteroids, co-formulated with moxifloxacin HCl (MFX) and cromolyn sodium (CML), respectively. PNO has a negligible quantum yield and its hydrolytic degradation products have enhanced fluorescence, which is 250-fold greater. FRT is a nonfluorescent drug, but its hydrolytic degradation products show reasonable fluorescence; MFX and CML have native fluorescence. Two methods were proposed based on the determination of PNO and FRT via their hydrolytic degradation products in the presence of other degradation products. Method (A) was developed for simultaneous determination of PNO and MFX in the presence of PNO degradation products by measuring peak amplitudes of the first derivative (1 D) of its enhanced fluorescence; PNO and MFX were measured at 345 and 473nm, respectively. Method (B) is a synchronous fluorescence spectroscopic method for simultaneous determination of FRT and its co-formulated drug CML in the presence of its degradation products. Fluorescence intensities were measured at λem 283 and 347nm for FRT and CML, respectively, using Δλ=99.20nm. Validation of the proposed methods was conducted as per International Council for Harmonisation (ICH) guidelines. The proposed methods were successfully applied for the determination of the proposed drugs in bulk powder, ophthalmic solution, and rabbit's aqueous humour.

UPLC-MS/MS assay of Tedizolid in rabbit aqueous humor: Application to ocular pharmacokinetic study

Tedizolid phosphate (TZP) a prodrug of Tedizolid (TDZ) is a novel oxazolidinone antibiotic, used for the treatment of acute bacterial skin, skin structure infections and other serious gram positive and MRSA infections. In the present study, a sensitive UPLC-MS/MS analytical method was developed and validated for the quantification of TDZ in rabbit’s aqueous humor (AqH) by using linezolid as internal standard (IS). Both TDZ and IS were separated on an Acquity™ HILIC column using an isocratic mobile phase comprising of acetonitrile: 20 mM ammonium acetate (85:15, v/v), eluted at 0.3mLmin−1 flow rate with total run time of 3 min. The AqH samples were processed by protein precipitation method by using acetonitrile as precipitating agent. TDZ and IS were detected in positive mode using electrospray ionization source. The precursor to product ion transitions at m/z 371.15 to 343.17 for TDZ and m/z 338.18 to 296.22 for IS were used for the quantification in multiple reaction monitoring mode. The calibration curve was linear in the concentration range of 4.98–1000ngmL−1 and the lower limit of detection was 1.97ngmL−1 only. The method was validated following US-FDA-guidelines and the results of validation parameter were found within the set limits. The developed UPLC-MS/MS method was fast, sensitive and reliable for the quantification of TDZ in the rabbit AqH and was successfully employed for ocular pharmacokinetic study of TDZ in AqH after topical ocular application of TZP-containing formulations in rabbit eyes.

Determination of naphazoline HCl, pheniramine maleate and their official impurities in eye drops and biological fluid rabbit aqueous humor by a validated LC-DAD method.

A simple RP-HPLC-DAD method was developed and validated, as per the ICH guidelines, for simultaneous determination of naphazoline HCl (NPZ) & pheniramine maleate (PHN) along with three of their official impurities. Chromatographic separation was performed on a hypersil ODS column (5 mm, 250–4.6 mm i.d.) with isocratic elution using phosphate buffer pH 6.0: acetonitrile (70 : 30, v/v) as mobile phase, at a flow rate of 1.0 mL min−1 and UV detection at 260.0 nm. The developed method was found to be linear over the concentration ranges of 5.00–45.00 μg mL−1 for NPZ and NPZ impurity B and 10.00–110.00 μg mL−1, 10–70 μg mL−1 and 10–120 μg mL−1 for PHN, and PHN impurity A and B, respectively, with correlation coefficient values <0.999 for the five cited compounds. The method was confirmed to be accurate, robust and precise with RSD >2.0%. LOD and LOQ values for the five cited compounds were calculated. Moreover, the method was also validated in rabbit aqueous humor as per the US food and drug administration (FDA) bioanalytical validation guidelines. Finally, the proposed method was applied for the analysis of the two drugs along with their impurities in dosage form and spiked aqueous humor samples.

Comprehensive Ocular and Systemic Pharmacokinetics of Brinzolamide in Rabbits After Intracameral, Topical, and Intravenous Administration

Brinzolamide is a topical carbonic anhydrase inhibitor which reduces the production of aqueous humor in the ciliary body, thereby reducing intra-ocular pressure. It is formulated as an ophthalmic suspension. The pharmacokinetics of ocular suspensions is not well understood. The objective of this study was to characterize the pharmacokinetics of brinzolamide in rabbit aqueous humor, iris-ciliary body, plasma, and whole blood. New Zealand White rabbits were dosed via intracameral, topical and intravenous administration. After intracameral administration (4.5 μg) of solubilized brinzolamide, aqueous humor concentrations were described with a two-compartment model, the estimated clearance was 4.12 μL/min, apparent volume of distribution at steady-state 673 μL, and terminal half-life 3.4 h. After topical administration of 1% brinzolamide suspension (500 μg), absolute bioavailability based on aqueous humor AUC0-∞ was 0.10%. After intravenous administration of brinzolamide solution (0.75 mg/kg) elimination half-life in plasma and whole blood appeared to be over two weeks. The ratios of the measured concentrations of irisciliary body to whole blood, to plasma, and to aqueous humor concentrations enabled direct comparisons, and helped identify the significant contribution of the conjunctival-scleral pathways of absorption to the ciliary body. This study shows for the first-time the absolute bioavailability in aqueous humor and provides comprehensive pharmacokinetic parameters following administration of a topical suspension.

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 (150mm×2.1mm, 1.6μm) 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 0.2mL/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.

DEVELOPMENT OF NANOEMULSION TO IMPROVE THE OCULAR BIOAVAILABILITY AND PATIENT COMPLIANCE IN POSTOPERATIVE TREATMENT USING INDOMETHACIN

Objective: To develop a new cationic nanoemulsion (NE) for ophthalmic delivery of indomethacin (IND) to improve the permeability and retention time of formulations, thereby improving the drug's ocular bioavailability.&#x0D; Methods: Based on the solubility profile of indomethacin in various solvents, captex 8000 was selected as oil phase, span 20 as a surfactant and tween 20 as co-surfactant to construct pseudo ternary phase diagrams and nanoemulsion region was recognized. Sonication was used as the method of NE preparation. Optimization was done using 32 factorial designs by considering the oil and the ratio of surfactant to co-surfactant (Smix) quantities as independent variables and evaluated for different physicochemical properties. Ex vivo transcorneal permeability was studied using bovine cornea, the In vivo drug pharmacokinetics of optimized NE and marketed formulation were assessed in rabbit aqueous humor and also in plasma.&#x0D; Results: The mean globule size, zeta potential, viscosity, refractive index, pH, surface tension and the osmolarity values for the prepared indomethacin nanoemulsions (IND-NEs) were found between 129.8±1.1 to 191.4±1.6 nm,+13.20±4.6 to+23.45±4.82, 15.3±0.1 to 32.7±0.0 mPas, 1.346±0.007 to 1.386±0.005, 5.5±0.4 to 6.9±0.9, 32.0±2.6 to 52.3±3.4 mN/m and 303-395 mOsm/l respectively and all these values found to be falling under the recommended values for ophthalmic use. From the In vitro release studies, it was found that the IND-NEs exhibited sustained drug release with 67.91±2.01 to 95.90±1.93 % drug release at 24h when compared to the drug solution which showed 99.81±5.21 % drug release within 2h. The Ex vivo drug permeation through the corneal membrane at 4h from the optimized NE and drug solution was found to be 524±1.5 µg/cm2 and 175±2.6 µg/cm2 respectively. Further, the optimized NE was found to be nonirritant with the lowest ocular irritation potential (Iirr) of 1 towards the rabbit's eyes. The area under the drug concentration vs. time curve for 24h (AUC (0–24h)) for optimized NE and the marketed formulation was found to be 1514.99 ng/ml/h and 974.14 ng/ml/h in aqueous humour; 2266.83 ng/ml/h and 778.15 ng/ml/h in plasma respectively.&#x0D; Conclusion: Due to its improved corneal absorption and prolonged drug release along with less systemic absorption, the optimized NE offers an effective postoperative treatment with increased ocular bioavailability and improved patient compliance with a decrease in the number of installations per day and a decrease or disappearance of systemic side effects of IND.

Development and optimization of ocular in situ gels loaded with ciprofloxacin cubic liquid crystalline nanoparticles

Ciprofloxacin (Cf) is antibiotic used oculary for treatment of conjunctivitis and corneal ulcers. The barriers for its use are: high dosage frequency and poor solubility lead to ocular irritation. This study sought to develop ciprofloxacin-cubosomal (Cf-Cub) in situ gel in order to improve eye permeation, prolong the ocular retention time, and enhance its antimicrobial activity. Box-Behnken design was used to prepare cubosomes. Three independent variables were; Phytantriol amount, Lutrol amount, and hydration media pH, while particle size, encapsulation, and steady state flux were evaluated as dependent variables. The optimised cubosomes were evaluated for antimicrobial activity, and in vivo estimation of Cf in the aqueous humour of rabbits’ eyes. The results indicated that, the optimised formula consisted of 60 mg of Cf, 100 mg phytantriol, 25 mg Lutrol, and hydration media pH equal 5.8 produced cubosomes with 121 nm size, 75% entrapment, and 2.54 times antimicrobial activity compared with commercially eye drops. The estimation of Cf in aqueous humour indicated that once daily administration of the optimised formula maintain the drug concentration above MIC compared with commercial drops which require four times daily administration. This confirmed that optimised formula improved eye permeation, prolonged the ocular retention time, and enhanced the antimicrobial activity.

Development and validation of a novel UPLC-MS/MS method for quantification of delafloxacin in plasma and aqueous humour for pharmacokinetic analyses

Acute bacterial skin and skin structure infections are one of the most frequent infectious disease requiring hospitalization for treatment. Delafloxacin is a clinically approved fluoroquinolone antibiotic for the treatment of ABSSSIs. In spite of being marketed since 2017, there is no published analytical method for quantification of delafloxacin in biological samples. Herein, a selective and sensitive UPLC-MS/MS method was developed and validated for quantitative analysis of delafloxacin in rat plasma and rabbit aqueous humour samples. The liquid liquid extraction (using ethyl acetate) was used for analyte extraction form rat plasma, whereas protein precipitation (acetonitrile) was used for aqueous humour samples preparations. An Acquity UPLC BEH C18 column was used for chromatographic separation of delafloxacin and internal standard (rivaroxaban). The mobile phase composition of acetonitrile (containing 0.1% formic acid) and 10 mM ammonium acetate in ratio of 60:40 were used for sample elution at 300 µL/min flow rate. The electrospray ionization operated in positive mode was used for sample ionization and detection of analyte and internal standard were performed by multiple reaction monitoring (MRM) mode. The MRM transitions were set to 441.14 > 379.09 and 436.89 > 144.87 for delafloxacin and internal standard, respectively. The method was validated as per USFDA guideline for bioanalytical method and all the evaluated parameters were within the acceptable ranges. The developed method in plasma was successfully used to analyze samples in pharmacokinetic study of newly developed stearic acid-chitosan solid lipid nanoparticles formulation of delafloxacin in rat.

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 254nm. The second method was HPLC using a mobile phase of acetonitrile: methanol: deionized water, pH2.8 (25.0: 35.0: 40.0, v/v/v), at 254nm. 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 0.20-50.00μg band-1, for PDN and MXF by HPTLC, while for HPLC, it was 0.02-50.00 and 0.10-50.00μgmL-1 for both drugs, in order.

An electrochemical sensing platform to determine tetrahydrozoline HCl in pure form, pharmaceutical formulation, and rabbit aqueous humor.

In the pharmaceutical industry, finding cost-effective and real-time analyzers that provide valid data is a good aim. The purpose of this work was to propose a link between the pharmaceutical industry and the recent innovations in solid-contact ion-selective electrodes (SC-ISEs) for the utilization of these electrodes as real-time analyzers to evaluate the concentration of tetrahydrozoline HCl in different matrices. The backbone of these new potentiometric sensors is the conjunction of calix[6]arene and (2-hydroxypropyl)-β-cyclodextrin as molecular recognition elements and a network of multi-walled carbon nanotubes as a solid transducer material between an ionophore-doped PVC membrane and microfabricated Cu electrodes. The proposed sensors were optimized to determine tetrahydrozoline, and their performances were assessed according to the IUPAC recommendations. The proposed solid-contact sensors were compared with liquid contact sensors, and the former sensors were found to be better than the latter sensors in terms of durability, handling, and easier adaptation to industry with comparable sensitivity. The measurements were implemented using phosphate buffer (pH: 6). The best obtained linearity range was 1 × 10-2 to 1 × 10-7 M, and the best LOD was 1 × 10-8 M. The sensors with the best performance were successfully applied to determine tetrahydrozoline in a pharmaceutical eye preparation and rabbit tears. The obtained results were statistically compared to those obtained by the official method of analysis, and no significant difference was obtained. The eco-score of the method was assessed using the eco-scale tool and also compared with that of the official method. The proposed approach was validated according to the International Council for Harmonisation (ICH) guidelines.

Quantitative proteomic analysis of aqueous humor after rabbit lensectomy reveals differences in coagulation and immunomodulatory proteins.

Compared to adults, children experience increased postoperative scarring and inflammation following intraocular surgery. While the underlying causes of the exaggerated immune response in children are not understood, proteins play key roles in postoperative scarring and wound healing processes. To identify and quantify proteins associated with the robust postoperative immune response, this study applied quantitative proteomics approaches to a juvenile rabbit model of lensectomy with intraocular lens (IOL) insertion. Twenty-six 6-7 week-old New Zealand white rabbits underwent unilateral portions of lensectomy with IOL insertion including: anterior chamber paracentesis, corneal incision with wound suture, lensectomy only, and lensectomy with IOL insertion. Aqueous humor was collected immediately prior and three days after each procedure. Semi-quantitative protein discovery was achieved by label-free quantitation using data dependent and data independent acquisition modes. Based on the discovery results, targeted quantitation by parallel reaction monitoring of 3 proteins of interest, fibrinogen-beta chain, transforming growth factor beta-2, and retinol binding protein 3, was used to confirm the observed quantitative trends. Total protein concentration levels increased with each progressive surgical step of lensectomy with IOL insertion. Proteins related to the complement and coagulation cascades were found to increase in relative abundance, while proteins related to ocular immunosuppression decreased in abundance following surgery. These data provide insights into the postoperative response by providing the first surgical step-wise views of the AH proteome before and after surgery. Overall, this work provides the foundation for future investigations targeting specific proteins for therapeutic interventions aimed at minimizing postoperative complications after pediatric intraocular surgery.

Nanoemulsion Based Vehicle for Effective Ocular Delivery of Moxifloxacin Using Experimental Design and Pharmacokinetic Study in Rabbits.

Nanoemulsion is one of the potential drug delivery strategies used in topical ocular therapy. The purpose of this study was to design and optimize a nanoemulsion-based system to improve therapeutic efficacy of moxifloxacin in ophthalmic delivery. Moxifloxacin nanoemulsions were prepared by testing their solubility in oil, surfactants, and cosurfactants. A pseudoternary phase diagram was constructed by titration technique and nanoemulsions were obtained with four component mixtures of Tween 80, Soluphor® P, ethyl oleate and water. An experiment with simplex lattice design was conducted to assess the influence of formulation parameters in seven nanoemulsion formulations (MM1–MM7) containing moxifloxacin. Physicochemical characteristics and in vitro release of MM1–MM7 were examined and optimized formulation (MM3) was further evaluated for ex vivo permeation, antimicrobial activity, ocular irritation and stability. Drug pharmacokinetics in rabbit aqueous humor was assessed for MM3 and compared with conventional commercial eye drop formulation (control). MM3 exhibited complete drug release in 3 h by Higuchi diffusion controlled mechanism. Corneal steady state flux of MM3 (~32.01 µg/cm2/h) and control (~31.53 µg/cm2/h) were comparable. Ocular irritation study indicated good tolerance of MM3 and its safety for ophthalmic use. No significant changes were observed in the physicochemical properties of MM3 when stored in the refrigerator for 3 months. The greater aqueous humor concentration (Cmax; 555.73 ± 133.34 ng/mL) and delayed Tmax value (2 h) observed in MM3 suggest a reduced dosing frequency and increased therapeutic efficacy relative to control. The area under the aqueous humor concentration versus time curve (AUC0–8 h) of MM3 (1859.76 ± 424.51 ng·h/mL) was ~2 fold higher (p < 0.0005) than the control, suggesting a significant improvement in aqueous humor bioavailability. Our findings suggest that optimized nanoemulsion (MM3) enhanced the therapeutic effect of moxifloxacin and can therefore be used as a safe and effective delivery vehicle for ophthalmic therapy.

HPLC-MS/MS-based analysis of everolimus in rabbit aqueous humor: pharmacokinetics of in situ gel eye drops of suspension.

Aim: Everolimus is an inhibitor of mTOR which is derived from rapamycin. Its high corneal permeability and bioavailability makes it an effective treatment for ocular disorders. To study the pharmacokinetics of in situ gel eye drops of everolimus suspension after ocular administration in rabbit aqueous humor, we developed a rapid and reliable HPLC-MS/MS method. Methodology/results: Ascomycin was selected as the internal standard.HPLC-MS/MS method was used to investigate the concentration-time relationship of everolimus in rabbit aqueous humor through protein precipitation technique. The validated results showed good linearity, accuracy and precision. Conclusion: The method developed in this study is rapid, sensitive and reliable for the determination of the concentration of everolimus in rabbit aqueous humor.

Pharmacokinetic study of sirolimus ophthalmic formulations by consecutive sampling and liquid chromatography–tandem mass spectrometry

Sirolimus is regarded as one of the most effective immunosuppressants receiving extensive attention over the years, for which the ocular application needs further development in clinical keratoplasty. In order to study the transcorneal absorption effect of ophthalmic administration, there was a need to study the pharmacokinetics of drugs in aqueous humor. In this work, a validated and reliable HPLC-ESI-MS/MS method was established to study the pharmacokinetics of sirolimus nanoformulations in rabbit aqueous humor. The analysis conditions were as follows. Ascomycin was chosen as internal standard. After a simple precipitation extraction procedure, the aqueous humor samples were separated on a XBridge C18 column (4.6 mm × 150 mm, 3.5 μm, Waters Co., USA) with a mobile phase comprised of water (0.1% formic acid and 5 mM ammonium formate) and methanol (0.1% formic acid) at the ratio of 10:90 (v/v). The mass analysis was achieved by positive ionization with multiple reaction monitoring (MRM) mode. The highest response ion pairs m/z at 931.5→864.5 were chosen for sirolimus. The validated results showed that the calibration range was 0.3–100.6 ng/mL with r = 0.9997 (n = 6). The R.S.D. values of the intra- and inter-day precision were less than 11% and the average accuracy values were between 94.73%–100.20%. Besides, for reducing the consumption of rabbits and the variation of the data, we designed a consecutive sampling method in pharmacokinetic study, with only seven rabbits consumed for each formulation. In conclusion, the developed analysis method was more reliable and practical than previously reported experiments. Meanwhile, the validated method was successfully applied to study the pharmacokinetics of sirolimus micelle and sirolimus nanosuspension after ophthalmic administration.

Validated potentiometric method for the determination of sulfacetamide sodium; application to its pharmaceutical formulations and spiked rabbit aqueous humor

Specific, accurate and precise electrochemical method was developed and validated for the determination of sulfacetamide sodium in presence of its co-formulated drug (prednisolone acetate) and its pharmacopoeial impurities. The method was based on fabrication of membrane sensor. The characteristics of electrochemical response were estimated, and the proposed sensor displayed excellent characteristics for the determination of sulfacetamide sodium in bulk powder, laboratory prepared mixtures, dosage forms and in spiked biological fluid (Rabbit aqueous humor). The sensor was constructed through the use of tetradodecylammonium bromide (TDB) as an anion exchanger and 2-nitrophenyl octyl ether (NPOE) as a plasticizer in polyvinyl chloride (PVC) matrix. The performance characteristics, sensitivity and selectivity were evaluated according to IUPAC guidelines. Linearity was achieved over the concentration range of 1 × 10−4.5–1 × 10−2 M with Nernstian slope of 51.086 mV/decade over the pH range of 5–7. The sensor showed a rapid response (10–15 s) and good stability (up to 4 weeks). The obtained results were statistically compared with the official methods and no significant difference was found regarding accuracy and precision.

Potentiometric ion-selective electrodes for determination of cyclopentolate hydrochloride and phenylephrine hydrochloride in their challenging ophthalmic formulation

Introduction of potentiometric ion-selective electrodes (ISEs) opened a new bright area in pharmaceutical analysis acknowledged as being an eco-friendly, simple, and energy-saving technique that is well-suited with microfabrication. In this contribution, potentiometric ISEs were employed as an alternative green analytical tool with the crucial goal of expanding the effective application of the potentiometric sensors in different disciplines of drug-stability studies and quality-control investigations. Four novel cyclopentolate hydrochloride and phenylephrine hydrochloride selective membrane sensors were constructed and evaluated. Sensors’ fabrication was achieved using potassium tetrakis (4-chlorophenyl) borate, a cationic exchanger, in a polyvinyl chloride polymeric matrix plasticized with 2-nitrophenyl octyl ether and using 2-hydroxy propyl-β-cyclodextrin as an ionophore. A comparative potentiometric study was implemented using two designs of ISEs; a conventional liquid inner contact and a glassy carbon solid-contact one. Using solid-contact ISEs, detection limit was substantially decreased and the discriminative ability in the presence of the most interfering substances was enhanced. This permits simultaneous estimation of both drugs, in spite of their similar ionic characteristics, abolishing the need for any pretreatment or separation steps in their challenging combined ophthalmic formulation as well as in rabbit aqueous humor and in the presence of their degradation products.

Validated HPTLC and HPLC methods for determination of fluorometholone and sodium cromoglycate in presence of their impurities and degradation products; application to kinetic study and on rabbit aqueous humor

ABSTRACTFluorometholone (FLM) and Sodium Cromoglycate (CMG) are co-formulated in ophthalmic preparation and showed marked instability under different conditions. Two specific, sensitive and precise stability-indicating chromatographic methods have been developed and validated for their determination in the presence of their degradation products and FLM impurity. Ten components were efficiently separated by them. The first method was HPTLC-spectrodensitometry, where the separation was achieved using silica gel 60 F254 HPTLC plates and developing system of ethyl acetate: methanol (9:1, v/v). The second method was a reversed phase HPLC associated with kinetic study of the degradation process and was successfully applied for determination of the studied compounds in spiked rabbit aqueous humor. The mobile phase was acetonitrile: methanol: 0.05 M potassium dihydrogenphosphate (0.1% trimethylamine); pH 2.5, adjusted with orthophosphoric acid (20: 30: 50, by volume). In both methods, the separated components were detected at 240 nm and system suitability was checked. Good correlation was obtained in the range of 0.10–24.00 and 0.20–48.00 µg band−1, for FLM and CMG by HPTLC. While for HPLC, the linearity ranges from 0.01–50.00 and 0.05–50.00 µg mL−1 for both drugs. The methods were applied in pharmaceutical formulation, where they were compared to the reported method with no significant difference.

Development and Validation of a New Ultra-fast HPLC Method for Quantification of Levofloxacin in Rabbit Aqueous Humour: Application to a Pharmacokinetic Study

A specific, sensitive, reliable, low-cost, isocratic, ultra-fast HPLC-DAD for Levofloxacin quantification in rabbit aqueous humor was developed, validated and applied to a pharmacokinetic study, conducted on commercial formulation of Levofloxacin. Separations were obtained on a XB-C18 column (100A, 100 mm × 4.60 mm, 2.6 μm, Phenomenex) with an isocratic mobile phase consisting of 18% acetonitrile and 82% triethylamine 0.5% in water (pH adjusted to 2.5 with H3PO4) at a flow rate of 0.5 ml/min. Detection of levofloxacin was done at 292 nm, and the column temperature was 40°C. The total analysis run-time was 5 min per sample, and no interfering peaks from aqueous humor were detected. Method was found to be selective, linear (R2=0.99984), accurate (intra-day recovery, 98.24%-100.04%) and precise (RSD, ≤ 5.50%). Pharmacokinetic parameters in rabbit aqueous humors were calculated by PKSolver add in program and are in agreement with literature data. The outlined method can be efficiently applied for monitoring levofloxacin in aqueous humor in pharmacokinetics studies.

EXPERIMENTAL EVALUATION OF AQUEOUS HUMOUR LOW-MOLECULAR PROTEINS DEGRADATION DUE TO COMBINED CORNEAL, IRIDIAL AND LENS PENETRATING EYE INJURY: THE INFLUENCE OF PROSTAGLANDINS AND THEIR SYNTHESIS INHIBITORS

The task of the study was to evaluate the effect of prostaglandins and their synthesis inhibitors on proteolytic activity of rabbit aqueous humour due to combined corneal, iridial and lens penetrating eye injury. 40 eyes of 40 Shinshilla rabbits were treated. The intensity of low-molecular proteins lysis due to penetrating ciliary body injury does not change, while collagen activity of aqueous humour increases throughout all period of the experiment. PGЕ1 and PG F2α are determined to reduce degradation degree of low-molecular proteins, PG Е2 increases the intensity of asocasein lysis, therefore decreasing intensity of proteolytic collagen disintegration (cleavage). Paracetamol, diclofenac and dexamethasone decrease the proteolylysis of low-molecular proteins, but essentially raise collagenalytic activity of anterior chamber aqueous humour of rabbit eyes due to combined corneal, iris and lens penetrating injury. Differential choice of used drugs prevents excessive fibrosis of the ocular tissues.

Stability indicating HPLC-DAD method for analysis of Ketorolac binary and ternary mixtures in eye drops: Quantitative analysis in rabbit aqueous humor.

Ketorolac tromethamine (KTC) with phenylephrine hydrochloride (PHE) binary mixture (mixture 1) and their ternary mixture with chlorpheniramine maleate (CPM) (mixture 2) were analyzed using a validated HPLC-DAD method. The developed method was suitable for the in vitro as well as quantitative analysis of the targeted mixtures in rabbit aqueous humor. The analysis in dosage form (eye drops) was a stability indicating one at which drugs were separated from possible degradation products arising from different stress conditions (in vitro analysis). For analysis in aqueous humor, Guaifenesin (GUF) was used as internal standard and the method was validated according to FDA regulation for analysis in biological fluids. Agilent 5 HC-C18(2) 150×4.6mm was used as stationary phase with a gradient eluting solvent of 20mM phosphate buffer pH 4.6 containing 0.2% triethylamine and acetonitrile. The drugs were resolved with retention times of 2.41, 5.26, 7.92 and 9.64min for PHE, GUF, KTC and CPM, respectively. The method was sensitive and selective to analyze simultaneously the three drugs in presence of possible forced degradation products and dosage form excipients (in vitro analysis) and also with the internal standard, in presence of aqueous humor interferences (analysis in biological fluid), at a single wavelength (261nm). No extraction procedure was required for analysis in aqueous humor. The simplicity of the method emphasizes its capability to analyze the drugs in vivo (in rabbit aqueous humor) and in vitro (in pharmaceutical formulations).