Development and Validation of Facile RP-HPLC Method for Simultaneous Determination of Timolol Maleate, Moxifloxacin Hydrochloride, Diclofenac Sodium and Dexamethasone in Plasma, Aqueous Humor and Pharmaceutical Products.

The present study aimed to develop a validated RP-HPLC method for the simultaneous determination of timolol maleate (TM), moxifloxacin hydrochloride (MOXI), diclofenac sodium (DS) and dexamethasone (DEXA) in human plasma, bovine aqueous humor and pharmaceutical preparations. The chromatographic separation was studied using the C18 column. The chromatographic conditions, such as composition, pH, the flow rate of mobile phase, the temperature of column, wavelength of absorption and injection volume of the sample, were studied. The method was validated to confirm specificity, linearity and accuracy in accordance with an International Conference on Harmonization guidelines. The optimum conditions for separation included mobile phase 0.05 M monobasic phosphate buffer: acetonitrile (65:35 v/v), pH of buffer adjusted to 6.2 and the flow rate of 1 mL/minute. The optimum temperature of the column was found to be 35°C, absorption wavelength 265 nm and injection volume 50 µL. The baseline separation of all four drugs with good sensitivity, resolution, and a less than 15 min run time was achieved. The retention time of TM, MOXI, DS and DEXA were 4.3,5.7,9.9 and 13.5 minutes respectively. The limit of detection (LOD) values were 6.2, 4.8, 0.8 and 1.2 ng/mL for TM, MOXI, DS and DEXA, respectively, whereas their respective limit of quantification (LOQ) values was: were 42.6, 26.8, 5.6 and 6.2 ng/mL. The coefficient of variation for intra-day and inter-day were in the range of 0.32-1.57 and 1.29-3.07%, respectively. The method was found to be sensitive, precise and accurate in human plasma and bovine aqueous humor and can be applied for the quantification of these compounds in plasma, aqueous humor and pharmaceuticals.

Full Factorial Design for Development and Validation of a Stability-Indicating RP-HPLC Method for the Estimation of Timolol Maleate in Surfactant-Based Elastic Nano-Vesicular Systems.

A novel isocratic stability-indicating chromatographic method was developed, optimized and validated using Design-Expert® following ICH guidelines for the quantification of Timolol maleate (TM). The intrinsic stability of TM was assessed by force degradation studies, which concluded no extensive degradation except under alkaline and oxidative conditions. TM was quantified accurately in the surfactant-based elastic vesicular system by separating it on Hypersil BDS C8 column using triethylamine in H2O (0.15%v/v; pH 3.0) and acetonitrile (ACN; 65:35%v/v). The influence of variable factors like mobile phase pH, injection volume (µL), flow rate (mL/min) and ACN content (%) on method responses were assessed using a full factorial design. The method was linear between 0.05 and 10 µg/mL with an R2 value of 0.9993. Limit of detection and limit of quantification were found to be 0.90 and 27.2 ng/mL. The method was specific, with recovery in plain drug solution of 89-92% and elastic nanovesicles of 90-93%. The experimental model was significant (P < 0.0001) as indicated by deliberate changes in the method analyzed through analysis of variance. The total drug content in elastic nanovesicles was estimated to be 9.53 ± 0.01 mg/20-mL dispersion and entrapment efficiency was 44.52 ± 0.73%. The developed method was rapid, economic and precise for the quantification of TM in bulk and vesicular system.

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.

Simultaneous spectrofluorometric analysis of tablets containing hydrochlorothiazide combined with timolol maleate or amiloride hydrochloride.

Green and sensitive spectrofluorometric methods have been developed and validated for the determination of timolol maleate (TML)/hydrochlorothiazide (HCT) and amiloride hydrochloride (AMH)/hydrochlorothiazide in tablets. The proposed spectrofluorometric procedures were found to be linear in the range of 4-12, 5-35 and 0.025-0.2 mg L-1 for HCT, TML and AMH, resp. The excitation and emission wavelengths for HCT, TML and AMH at room temperature were 270 and 375, 295 and 435, 330 and 415 nm, resp. The methods were validated with respect to ICH guidelines. The AMH showed higher sensitivity with lower values of LOD and LOQ values compared to HCT and TML. The proposed methods were applied to two pharmaceutical formulations; the method for HCT and AMH has proven as reliable assaying method, whereas the method for TML, when combined with HCT, is applicable to screening semi-quantitative analyses.

Analytical quality by design development of an ecologically acceptable enantioselective HPLC method for timolol maleate enantiomeric purity testing on ovomucoid chiral stationary phase.

Official method in Ph. Eur. for evaluation of timolol enantiomeric purity is normal-phase high performance liquid chromatography (NP-HPLC) method. Compared to other HPLC modes, NP is depicted as quite expensive with high consumption of organic solvents which leads to chronic exposure of analysts to toxic and carcinogenic effects. In order to overcome above-mentioned drawbacks, the aim of this study was to develop new method with better eco-friendly features. This was enabled by using protein type Chiral Stationary Phase (CSP) in reversed-phase mode that required up to 10 % (v/v) of organic solvent. Therefore, an enantioselective HPLC method was developed and validated for quantification of (S)-timolol and its chiral impurity, (R)-isomer. Optimized separation conditions on ovomucoid column were set using Analytical Quality by Design (AQbD) approach in method development. Optimization step was performed following the Box-Behnken experimental plan and the influence of three critical method parameters (CMPs) towards enantioseparation of the above-mentioned peak pair was examined. CMPs included variation of acetonitrile content in the mobile phase (5-10 %, v/v), pH value of the aqueous phase (6.0-7.0) and ammonium chloride concentration in the aqueous part of the mobile phase (10-30 mmol L-1). The most relevant critical method attributes (CMAs) in this case were the separation criterion between studied critical pair and retention factor of the second eluting peak, (S)-timolol. Qualitative Design Space (DS) was defined by Monte Carlo simulations providing adequate assurance of method's qualitative robustness (π = 95 %). The selected working point situated in the middle of the DS was characterized by following combination of CMPs: acetonitrile content in the mobile phase 7 % (v/v), pH value of the aqueous phase 6.8 and concentration of ammonium chloride in aqueous phase 14 mmol L-1. In the next step, the quantitative robustness was tested by Plackett-Burman experimental design. The validation studies confirmed adequacy of the proposed method for its intended purpose. Finally, Analytical Eco-Scale metric tool was applied to confirm that developed method represents excellent green analytical method compared to the official one.

A versatile HPLC method with an isocratic single mobile phase system for simultaneous determination of anti-glaucoma formulations containing timolol.

Timolol is a non-cardioselective beta blocker and has different combined ophthalmic dosage forms for treatment of glaucoma. This research introduce an HPLC method for the separation of three drugs used in combination with timolol simultaneously by applying isocratic mobile phase system in a single run and the same detection wavelength with short time. The drugs included in the separation procedures are; dorzolamide, brinzolamide, and brimonidine. The HPLC method was carried out through a single mobile phase system, which contains acetonitrile: 0.05M phosphate buffer at the ratio of 30:70, respectively at pH 3.5 and wavelength of 220nm. The method, regarding its simplicity allows determination of the studied drugs simultaneously using single run in about 8minutes. The method was rectilinear in the ranges of concentration: 1.25-25µg/mL for timolol, 4-80µg/mL for dorzolamide, 5-50µg/mL for brinzolamide and 2-20µg/mL for brimonidine. Different factors affecting the separation are thoroughly studied. The developed method was validated based on the official guidelines and the results were compared statistically with previously published methods and showed non-significant difference.

Electrochemical determination of antihypertensive drugs by employing costless and portable unmodified screen-printed electrodes.

Hypertension increases the risk of heart disease and stroke, is commonly known as a silent killer disease and considered as one of the key risk factor for premature death and disability over the world. Herein, we report for the first time a sensitive, costless and reproducible voltammetric method for individual determination of five antihypertensive drugs namely, propranolol (PRO), timolol (TIM), amlodipine (AML), amiloride (AMI) and triamterene (TRI) using differential pulse voltammetry at bare/unmodified screen-printed carbon electrodes (SPEs) in presence of sodium dodecyl sulfate (SDS). Each drug exhibits an electrochemical signal in aqueous media which is significantly enhanced in presence of optimized concentration of SDS due to accumulation of the protonated drug molecules and electrostatically interaction with negatively charged micellar structures. As a result, the spherical micellar orientation of SDS onto the graphitic surface of SPEs offered the analytically sensitive determination of the target drugs over a wide linear concentration range with nano-molar detection limits possible negating the need for any complicated surface modifications. Finally, the proposed voltammetric method was successfully utilized in the individual determination of the target antihypertensive drugs in pharmaceutical formulations and human urine samples.

Validated HPLC-DAD Method for the Simultaneous Determination of Six Selected Drugs Used in the Treatment of Glaucoma.

This work presents a simple, sensitive, and generic HPLC-diode-array detection method for the simultaneous determination of six drugs prescribed for the treatment of open-angle glaucoma and ocular hypertension. The investigated drugs include brimonidine tartarate (BMN), acetazolamide (AZA), brinzomaide (BZA), dorzolamide HCl (DZA), levobunolol HCl (LVB), and timolol maleate (TIM). Efficient chromatographic separation was achieved using a Thermo Hypersil BDS C18 column (4.6 × 250 mm, 5 µm) with a mobile phase consisting of phosphate buffer pH 5 and acetonitrile in a ratio of 78 + 22. The flow rate was 1 mL/min, and quantification was based on measuring peak areas at 298 nm for TIM and 254 nm for the other drugs. Peaks were perfectly resolved, with retention times at 3.06, 3.87, 4.53, 5.78, 7.31, and 10.78 min for BMN, AZA, DZA, TIM, LVB, and BZA respectively. The developed method was validated according to International Conference on Harmonization guidelines with respect to system suitability, linearity, ranges, accuracy, precision, robustness, and LODs and LOQs. The proposed method showed good linearity in the ranges of 2-80, 2.5-100, 2.5-100, 5-200, 3.75-150, and 1.75-70 µg/mL for BMN, AZA, DZA, TIM, LVB, and BZA, respectively. LODs were 0.20-1.01 µg/mL for the analyzed compounds. Applicability of the proposed method to real-life situations was assessed through the analysis of five different pharmaceutical formulations, and satisfactory results were obtained.

The application of artificial neural networks and support vector regression for simultaneous spectrophotometric determination of commercial eye drop contents.

In the present study, artificial neural networks (ANNs) and support vector regression (SVR) as intelligent methods coupled with UV spectroscopy for simultaneous quantitative determination of Dorzolamide (DOR) and Timolol (TIM) in eye drop. Several synthetic mixtures were analyzed for validating the proposed methods. At first, neural network time series, which one type of network from the artificial neural network was employed and its efficiency was evaluated. Afterwards, the radial basis network was applied as another neural network. Results showed that the performance of this method is suitable for predicting. Finally, support vector regression was proposed to construct the Zilomole prediction model. Also, root mean square error (RMSE) and mean recovery (%) were calculated for SVR method. Moreover, the proposed methods were compared to the high-performance liquid chromatography (HPLC) as a reference method. One way analysis of variance (ANOVA) test at the 95% confidence level applied to the comparison results of suggested and reference methods that there were no significant differences between them. Also, the effect of interferences was investigated in spike solutions.

A New UPLC Method with Chemometric Design-Optimization Approach for the Simultaneous Quantitation of Brimonidine Tartrate and Timolol Maleate in an Eye Drop Preparation.

A new ultra-performance liquid chromatography (UPLC) with photodiode array was proposed for the quantitation of Brimonidine Tartrate (BRI) and Timolol Maleate (TIM) in eye drop using experimental design and optimization methodology. A 33 full factorial design was applied to uncover the effects of the selected factors and their interactions on the chromatographic response function for the optimization of experimental conditions in the development of a new UPLC method. As a result, the optimal chromatographic conditions giving a better separation and short analysis time were found to be 49.2°C for column temperature; 0.38 mL/min for flow rate and 56.7 % (v/v) for 0.1 M CH3COOH used in mobile phase. The elution of BRI and TIM was reported as 0.508 and 0.652 min within a short runtime of 1.5 min, respectively. Calibration graphs for BRI and TIM were obtained by the regression of the concentration on the peak area, which was detected at 246 and 298 nm, respectively. The method validation was performed by the analysis of the synthetic mixtures, intra-day and inter-day samples and standard addition samples. This study shows that the optimized and validated UPLC method is very promising and available for the quantification of BRI and TIM in an eye drop formulation.

Effects of timolol maleate, levobunolol and apraclonidine on intraocular pressure, pupil size, blood pressure and heart rate in beagles / Efeitos do maleato de timolol, do levobunolol e da apraclonidina sobre a pressão intraocular, o diâmetro pupilar, a pressão sanguínea e a frequência cardíaca, em cães da raça Beagle

The aim of this study was to evaluate changes in intraocular pressure (IOP), pupil size (PS), blood pressure (BP), heart rate (HR), and ECG variables (Pms wave PmV, PR interval, QRS complex, RMV wave and QT intervals) over time during the instillation of 0.5% timolol, 0.5% levobunolol and 0.5% apraclonidine in clinically normal dogs. Ten adult beagles were used. Baseline values were measured at 8a.m., 2p.m. and 8p.m., for three consecutive days. A waiting period of 10 days between the administrations of each drug was established. For 15 consecutive days, the drug being tested was instilled in one eye of each dog twice a day (7a.m. and 7p.m.). The parameters were evaluated at the aforementioned times on days 3, 6, 9, 12 and 15. Data were statistically compared using the Bonferroni test and one-way repeated measures analysis of variance (P<0.05). The Pearson test was used to evaluate any correlation between QT interval, HR and BP. The tested drugs did not find a decrease in IOP. A significant decreased in PS was observed in almost all dogs following levobunolol administration, relative to the control eye. A significant decrease in HR was observed on day 3 following levobunolol treatment, while apraclonidine induced an increase on day 15. Blood pressure was reduced in all measurement time points following apraclonidine treatment. A negative correlation between QT interval and HR was only observed in dogs treated with timolol. In conclusion, levobunolol was the only drug that induced significant alterations in PS. Apraclonidine was the only drug that induced systemic hypotension. Timolol was the only drug to that induced a negative correlation between QT and HR.(AU)

O objetivo deste estudo foi avaliar as mudanças na pressão intraocular (PIO), no diâmetro pupilar (DP), na pressão sanguínea (PS), na frequência cardíaca (FC) e nas variáveis eletrocardiográficas (onda Pms, PmV, intervalo PR, complexo QRS, onda RmV e intervalo QT), ao longo do tempo da instilação do timolol 0,5%, do levobunolol 0,5% e da apraclonidina 0,5% em cães clinicamente normais. Dez Beagles adultos compuseram o estudo. Valores basais foram mensurados às oito,, 14 e 20 horas, durante três dias consecutivos. Foi instituído um período de espera de 10 dias entre a administração de cada fármaco. Durante 15 dias consecutivos, um olho de cada animal recebeu uma gota de cada um deles, a intervalos de 12 horas (às sete e às 19 horas). Os parâmetros foram avaliados nos momentos acima referidos, nos dias três, seis, nove, 12 e 15. Os dados foram comparados estatisticamente empregando-se o teste de Bonferroni após análise de variância para medidas repetidas (P<0,05). Teste de Pearson foi utilizado para correlação entre o intervalo QT com a FC e a PS. Não se encontrou diminuição da PIO. Observou-se redução significativa do DP na quase totalidade dos animais que receberam levobunol, relativamente ao olho controle. Diminuição significativa da FC foi vista ao terceiro dia após a administração do levobunolol, enquanto apraclonidina induziu aumento no 15º dia. A pressão arterial foi reduzida em todos os momentos com a apraclonidina. Observou-se correlação negativa entre o intervalo QT e a FC apenas nos indivíduos tratados com o timolol. Em conclusão, levobunolol foi o único fármaco que induziu alterações significativas no DP. A apraclonidina foi o único fármaco que induziu hipotensão sistêmica significativa. O timolol foi o único a ensejar correlação negativa entre o intervalo QT e a FC.(AU)

Simultaneous determination of timolol maleate in combination with some other anti-glaucoma drugs in rabbit aqueous humor by high performance liquid chromatography-tandem mass spectroscopy.

In this work, a sensitive, selective, accurate and precise LC-MS/MS method has been developed for the simultaneous determination of an anti-glaucoma ß-blocker, timolol maleate (TIM) with other co-administered anti-glaucoma drugs of different classes, namely; dorzolamide hydrochloride (DOR), brinzolamide (BRZ) and brimonidine tartrate (BRM) in rabbit aqueous humor (AH) using eslicarbazepine as an internal standard (IS). Liquid-liquid extraction was used for the purification and pre-concentration of analytes from rabbit AH matrix. The chromatographic separation was achieved using a mobile phase consisting of 10mM ammonium formate pH=7: methanol: acetonitrile (5: 50: 45, v/v/v) in isocratic mode of elution at a flow rate of 0.8mL/min on an INERTSIL(®) C18 ODS-3 column (150mm×4.6mm, 3.5µm). The method was operated using electrospray ionization source in the positive ionization mode prior to detection by multiple reaction monitoring (MRM) at the following transitions: m/z 317.2→261.0 for TIM, m/z 325.1→199.0 for DOR, m/z 384.2→281.0 for BRZ, m/z 292.1→212.0 for BRM and m/z 255.0→237.0 for IS. The separation was done in only 3min and the lower limit of quantitation (LLOQ) was (50ng/ml) for all cited drugs. A detailed validation of the bio-analytical method was performed as mentioned in US-FDA and EMA guidelines and the standard calibration curves were found to be linear in the range (50-5000ng/ml) for all drugs with good mean regression coefficient for all drugs.

Deformability properties of timolol-loaded transfersomes based on the extrusion mechanism. Statistical optimization of the process.

The purpose of this work was to analyze the deformability properties of different timolol maleate (TM)-loaded transfersomes by extrusion. This was performed because elastic liposomes may contribute to the elevation of amount and rate of drug permeation through the corneal membrane. This paper describes the optimization of a transfersome formulation by use of Taguchi orthogonal experimental design and two different statistical analysis approaches were utilized. The amount of cholesterol (F1), the amount of edge-activator (F2), the distribution of the drug into the vesicle (F3), the addition of stearylamine (F4) and the type of edge-activator (F5) were selected as causal factors. The deformability index, the phosphorous recovery, the vesicle size, the polydispersity index, the zeta potential and percentage of drug entrapped were fixed as the dependent variables and these responses were evaluated for each formulation. Two different statistical analysis approaches were applied. The better statistical approach was determined by comparing their prediction errors, where regression analysis provided better optimized responses than marginal means. From the study, an optimized formulation of TM-loaded transfersomes was prepared and obtained for the proposed ophthalmic delivery for the treatment of open angle glaucoma. It was found that the lipid to surfactant ratio and type of surfactant are the main key factors for determining the flexibility of the bilayer of transfersomes. From in vitro permeation studies, we can conclude that TM-loaded transfersomes may enhance the corneal transmittance and improve the bioavailability of conventional TM delivery.

Determination of phosphate concentration in glaucoma eye drops commercially available in Spain. / Determinación de la concentración de fosfatos en los colirios antiglaucomatosos comercializados en España.

OBJECTIVES: To identify and analyze the phosphate concentration in glaucoma eye drops available in Spain. MATERIAL AND METHODS: Glaucoma medications containing phosphates were identified according to the 2013 Vademecum and the website of the Spanish Agency for Medicines and Medical Devices. Phosphate concentration was determined in these eye drops using ultraviolet molecular absorption spectrophotometry, and pH was determined using scan image analysis algorithms of pH strips. RESULTS: A total of 37 phosphate containing glaucoma eye drops were identified. The mean phosphate concentration was 97.72±75.52mM. The group with higher concentration of active substance was timolol (204.85±42.38mM) followed by brimonidine/timolol (200.9mM). No statistically significant difference was found between brand name (95.65±71.11mM) and generic eye drops (99.14±80mM, P=.892). Although no statistically significant difference was found between products containing preservatives (99.24±76.78mM) and those without preservatives (85.17±72.86mM) (P=.730), a lower phosphate concentration was observed in the preservative-free Timolol and Latanoprost. Single dose samples showed a lower phosphate concentration than multi-dose ones (102.04±75.39 vs. 22.24±2.98mM, P<.001). The mean pH was 7.13±0.63. No statistical correlation was found between phosphate concentration and pH (r: 0.07). CONCLUSION: The phosphate concentration in glaucoma eye drops exceeded the tear film physiological level (1.45mM). No difference was observed between brand names and generic eye drops. Lower phosphate concentration was observed in preservative-free single dose eye drops.

Development and validation of a stability-indicating micellar liquid chromatographic method for the determination of timolol maleate in the presence of its degradation products.

A stability-indicating micellar liquid chromatographic (MLC) method was developed and validated for the quantitative determination of timolol maleate (TM) in the presence of its degradation products resulting from accelerated degradation in a run time not more than 8 min. TM was subjected to stress conditions of hydrolysis (including alkaline, acidic and thermal hydrolysis) and oxidation. An isocratic, rapid and mobile phase saving the micellar LC method was developed with a BioBasic phenyl column (150 × 1.0 mm, 5 µm particle size) and a micellar mobile phase composed of 0.1 M sodium dodecyl sulfate, 10% of 1-propanol and 0.1% of triethylamine in 0.035 M ortho-phosphoric acid. The flow rate of the mobile phase was 0.1 mL/min. UV detection was adjusted at 298 nm and performed at room temperature. The method has been validated according to the International Conference on Harmonisation guidelines. The method is successfully applied for the determination of TM in bulk powder and pharmaceutical dosage form.

Simultaneous determination of dorzolomide and timolol in aqueous humor: a novel salting out liquid-liquid microextraction combined with HPLC.

A Snovel method for the simultaneous separation and determination of two antiglaucoma drugs namely, dorzolamide hydrochloride (DOR) and timolol maleate (TIM) in aqueous humor samples (AH) was developed by using salting-out assisted liquid-liquid microextraction (SALLME) combined with HPLC-UV method. Box-Behnken experimental design and response surface methodology were employed to assist the optimization of SALLME conditions, including salt concentration, the pH of sample solution and vortex time as variable factors. The optimal extraction conditions were as follows: to 50 µL of AH sample, 100 µL of phosphate buffer (100 mmol L(-1), pH 11.9), 90 µL of acetonitrile (ACN) and 0.11 g of (NH4)2SO4 salt were added into an Eppendorf vial (1 mL) then vortexed for 1.1 min. As an effort to miniaturize SALLE system, a 1 mL syringe adapted with a capillary tube was employed as the phase separation device. Once the phase separation occurred, the upper layer could be narrowed into the capillary tube by pushing the plunger; thus, the collection of the upper layer solvent was simple and convenient. By miniaturization, the consumption of the organic solvent was decreased as low as possible. The chromatographic separation was achieved on Gemini C18 column using a mobile phase of ACN: 30 mmol L(-1) potassium dihydrogen phosphate buffer containing 0.1% triethylamine, pH 3.5 (20:80, v/v) at a flow rate of 1 mL min(-1) and UV detection at 254 and 295 nm for DOR and TIM, respectively. Mepivacaine hydrochloride was used as an internal standard. The described method showed better separation with enhanced sensitivities than the previously reported methods with limits of quantitation of 8.75 and 10.32 ng mL(-1) in aqueous solution and 15.97 and 23.53 ng mL(-1) in AH for DOR and TIM, respectively. The simple, rapid and eco-friendly SALLME-HPLC method has been successfully applied for the simultaneous pharmacokinetic studies of DOR and TIM in rabbit AH.

[Determination of enantiomeric impurity of timolol maleate in bulk substances and eye drops].

OBJECTIVE: To determine the enantiomeric impurity contents of domestic timolol maleate in bulk drugs and eye drops. METHODS: Enantiomer impurity of timolol was assayed by chiral high performance liquid chromatography. The chromatographic conditions were as follows:chiralcel OD chiral column (4.6 mm ×150 mm, 5µm), detection wavelength:297 nm, mobile phase:hexane-isopropanol-diethylamine (480:20:1), column temperature:25 ℃, flow rate:1.0 ml/min, sample injection volume:5 µl. RESULTS: The resolution between R- and S-timolol was more than 4. The enantiomeric impurity contents were less than 0.67% on average in two batches of timolol maleate bulk drugs, and 0.31% on average in three batches of timolol maleate eye drops. CONCLUSION: Enantiomeric impurity contents in each batch of products all meet European Pharmacopoeia criteria, which can be used as references in Chinese Pharmacopoeia criteria.

Novel spectrophotometric methods for simultaneous determination of timolol and dorzolamide in their binary mixture.

Two smart and novel spectrophotometric methods namely; absorbance subtraction (AS) and amplitude modulation (AM) were developed and validated for the determination of a binary mixture of timolol maleate (TIM) and dorzolamide hydrochloride (DOR) in presence of benzalkonium chloride without prior separation, using unified regression equation. Additionally, simple, specific, accurate and precise spectrophotometric methods manipulating ratio spectra were developed and validated for simultaneous determination of the binary mixture namely; simultaneous ratio subtraction (SRS), ratio difference (RD), ratio subtraction (RS) coupled with extended ratio subtraction (EXRS), constant multiplication method (CM) and mean centering of ratio spectra (MCR). The proposed spectrophotometric procedures do not require any separation steps. Accuracy, precision and linearity ranges of the proposed methods were determined and the specificity was assessed by analyzing synthetic mixtures of both drugs. They were applied to their pharmaceutical formulation and the results obtained were statistically compared to that of a reported spectrophotometric method. The statistical comparison showed that there is no significant difference between the proposed methods and the reported one regarding both accuracy and precision.

Determination of (R)-timolol in (S)-timolol maleate active pharmaceutical ingredient: validation of a new supercritical fluid chromatography method with an established normal phase liquid chromatography method.

An enantioselective supercritical fluid chromatography (SFC) method was developed and validated to meet the current European Pharmacopoeia requirements of a limit test for the determination of S-timolol maleate enantiomeric purity in timolol maleate drug substance. The developed method is presented as an alternative to the current normal phase high performance liquid chromatography (NP-HPLC) method described in the European Pharmacopoeia (Timolol Maleate Monograph). Using a 4.6mm×250mm Chiralcel OD-H (dp: 5µm) column and a mobile phase of (93:7) CO2/0.1% (v/v) TEA in MeOH delivered at 4.0mLmin(-1) resolution of 2.0 was achieved within 5min, representing a 3-fold reduction in run-time and an 11-fold reduction in solvent consumption relative to the NP-HPLC method. Method robustness was examined by the variation of flow rate (±0.5mLmin(-1)), column temperature (±5°C) and column back-pressure (±10bar) and resolution was maintained at ≥1.9 in all cases. R-timolol was resolved from all potential impurities and the limit of detection was improved by increasing the sample concentration threefold compared to the NP-HPLC method such that the method could detect the R-timolol enantiomer at 0.5% (w/w) with respect to S-timolol maleate. Additional validation parameters demonstrated that the potential of the method to be used for routine release testing of timolol maleate raw material for drug product manufacturing in which the quantitation of R-timolol impurity in S-timolol maleate drug substance would be a requirement.

Comparative study of in vitro ocular surface cytotoxicity of a fixed combination of 0.5% timolol/1% dorzolamide eyedrop and its components with 0.005% benzalkonium chloride.

We evaluated the cytotoxicity of antiglaucoma ophthalmic solutions preserved with the same concentration of benzalkonium chloride (BAK) in four cultured corneal and conjunctival cell lines. The viability of cell cultures was determined following the exposure of cells to timolol maleate, dorzolamide, and their fixed combination, Kosoputo(®) (MSD, a Japanese formulation of Cosopt(®) (Merck)), and two commercially available eyedrop solutions, 0.5% Timpotol(®) (containing 0.5% timolol maleate, MSD) and 1% Trusopt(®) (containing 1% dorzolamide, MSD) for varying exposure times and at various dilutions using the MTT and neutral red assays. All the three commercially available eyedrop solutions tested in this study were preserved with 0.005% BAK. The toxicity of each solution was compared using the % cell viability score (CVS) . Cell viability was also subjected to statistical analysis using ANOVA, Dunnett's multiple comparison tests and a chi-square test. %CVS50/%CVS40/80s for the tested solutions were 53/-13 for 0.5% Timoptol(®), 100/88 for preservative-free 0.5% timolol maleate, 50/ -10 for 1% Trusopt(®), 72/100 for preservative-free 1% dorzolamide, and 44/-17 for Kosoputo(®). The results of statistical analysis were consistent to them. In conclusion, Kosoputo(®) had greater cytotoxicity than each component; however, in actual use it may have the advantages of reduced toxicity (side effect) due to reduced instillation frequency, and better patient adherence to the treatment regimen as well as a comparable pressure reduction effect.