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.

Development of a novel ion-pairing UPLC method with cation-exchange solid-phase extraction for determination of free timolol in human plasma.

A novel UPLC-UV method was developed for analysis of timolol in human plasma using a simple, fast, and cost effective ion-exchange SPE procedure, followed by separation on a C18 UPLC column with a mobile phase consisting of acetonitrile, phosphate buffer, and sodium 1-octane sulfonate as an ion pairing agent. The method was fully validated according to US-FDA guidelines, and was found to be sufficiently accurate and precise for analysis of timolol in human plasma for clinical pharmacokinetic studies. The application of ion-exchange SPE cartridges for purification of timolol in plasma produced excellent percent recoveries and good sample clean-up, while the ion-pairing separation described here allowed quantitation of timolol without interference from endogenous sample components. The method lower limit of detection was 1.7 ng/mL and the lower limit of quantitation was 5.0 ng/mL, allowing for analysis of therapeutic concentrations of timolol in plasma.

Pharmacokinetics, Efficacy, and Safety of the Preservative-free Fixed Combination of Tafluprost 0.0015% and Timolol 0.5% in Healthy Volunteers: A Phase I Comparison vs. the Corresponding Preservative-free Monotherapies.

PURPOSE: Plasma concentrations of tafluprost acid and timolol were compared after single (Day 1) and repeated (Day 8) instillations of once-daily tafluprost 0.0015%-timolol 0.5% preservative-free (PF) fixed-dose combination (FDC), once-daily PF tafluprost 0.0015%, and twice-daily PF timolol 0.5%. PATIENTS AND METHODS: Fifteen healthy volunteers were randomized to this double-masked, single-center, three-period cross-over study. A wash-out interval of at least 4 weeks separated each three 8-day dosing period. Blood samples were drawn on the first and last day of each dosing period, prior to the morning dose, as well as 5, 10, 15, 30, and 45 min, and 1, 1.5, 2, 4, 8, and 12 h post-dosing. Sample plasma concentrations of tafluprost acid and/or timolol were determined and maximum concentration (C max), area under the concentration-over-time curve from time zero to the last time point with a quantifiable measurement (AUC0-last), and time to maximum concentration were calculated. Intraocular pressure (IOP), adverse events, and ocular/systemic safety variables were also evaluated. RESULTS: Plasma concentrations of tafluprost acid were low, with similar levels measured subsequent to either single or repeated dosing of PF FDC and PF tafluprost. On both sampling days, concentrations peaked at 10 min after the dose, and were cleared from the blood circulation by 30 min; average C max ranged from 17 to 24 pg/mL, and AUC0-last from 3 to 5 pg*h/mL. Plasma concentrations of timolol were comparable after the first dose of PF FDC or PF timolol. Concentrations peaked at 15 min post-dose and diminished in a similar manner after 2 h; average C max was 800 pg/mL and AUC0-last 3900 pg*h/mL. As expected, PF timolol produced a higher Day 8 pre-dose timolol concentration than PF FDC (235 vs. 37 pg/mL; p < 0.001, respectively). The Day 8 post-dose changes in timolol concentrations were relative to this pre-dose difference. All study treatments were well tolerated and safe. PF FDC seemed to provide the best IOP reduction. CONCLUSIONS: PF FDC demonstrated good IOP-lowering efficacy and displayed similar pharmacokinetic characteristics to the monotherapy agents. Exposure to timolol was reduced via the halved dosing.

A prospective self-controlled study of topical timolol 0.5% cream for large superficial infantile hemangiomas.

Timolol has recently been reported to be an effective and safe treatment for small and superficial infantile hemangiomas (IH). However, it is controversial to choose it as an alternative to oral propranolol for large superficial IH. In this study, we generated a new modified timolol agent as the base of an ointment. To evaluate the efficacy and safety of this new agent, we recruited 20 patients with large superficial IH. The average age was 4 months old. The average area of the IH was 28.8 cm(2) . The treatment was continued for 2-6 months. Three assessors were asked to judge the changes in both the treated and untreated parts separately by comparing photographs. After an average of 3.25 months of treatment, the average visual analog scale scores were 5.5 and 4.3 for those with and without the medication, respectively. The treated parts regressed significantly more than the untreated parts (P < 0.05). There were no side-effects observed during treatment. High performance liquid chromatography was used to detect the serum concentration of timolol, and no timolol was detected in any of the blood samples (<0.02 µg/mL). Our new modified timolol agent is proven to be an effective therapy option for IH. Prospective studies with high-precision serum timolol concentrations, with heart rate or blood pressure monitoring during treatment, are needed to evaluate potential systemic absorption when using timolol on large IH.

Two-dimensional transport analysis of transdermal drug absorption with a non-perfect sink boundary condition at the skin-capillary interface.

A transient percutaneous drug absorption model was solved in two dimensions. Clearance of the topically-applied pharmaceutical occured at the skin-capillary boundary. Timolol penetration profiles in the dermal tissue were produced revealing concentration gradients in the directions normal and parallel to the skin surface. Ninety-eight percent of the steady-state flux was reached after 85 h or four time constants. The analytical solution procedure agreed with published results. As the clearance rate increased relative to diffusion, the delivery rate and amount of drug absorbed into the bloodstream increased while the time to reach the equilibrium flux decreased. Researchers can apply the closed-form expressions to simulate the process, estimate key parameters and design devices that meet specific performance requirements.

Plasma levels and systemic safety of 0.1% unpreserved timolol maleate gel, 0.5% timolol aqueous solution and 0.5% timolol maleate gel.

PURPOSE: To compare systemic absorption of three formulations of timolol eye drops: 0.1% timolol maleate gel, 0.5% timolol aqueous solution, and 0.5% timolol maleate gel. METHODS: This was a double cross-over phase I study. Cross-over 1: two weeks of 0.1% timolol gel once daily, followed by a 3-week wash-out period and then two weeks of 0.5% timolol aqueous solution twice a day (group 1) or the reverse (group 2). Cross-over 2: two weeks of 0.1% timolol gel once daily, followed by a 3-week wash-out period, and then two weeks of 0.5% timolol gel once daily (group 3) or the reverse (group 4). Subjects underwent tonometry, blood sampling, and heart rate and blood pressure assessments (during bicycle exercise and head-up tilt tests) before and after instillation at the beginning and end of each treatment period. RESULTS: Forty-three healthy volunteers were randomized: 11 subjects in groups 1, 2, and 3, and 10 subjects in group 4. Areas under the concentration-time curve (AUC) values after administration of timolol 0.5% formulations were 15- to 38-fold higher than those seen after administration of timolol 0.1% gel. Maximum timolol concentrations after instillation of 0.1% gel are reduced by almost 90% compared to concentrations obtained after both 0.5% aqueous solution and 0.5% gel instillation. The AUC between 0 and 12 h post-administration were also reduced by up to 93 to 98%. CONCLUSIONS: After treatment with a timolol 0.1% gel formulation, systemic concentrations found were considerably lower than after administration of timolol 0.5% gel or in aqueous solution.

Simultaneous determination of timolol maleate, rosuvastatin calcium and diclofenac sodium in pharmaceuticals and physiological fluids using HPLC-UV.

A novel HPLC-UV method was developed for the simultaneous determination of timolol (TM), rosuvastatin (RST), and diclofenac sodium (DS) in pharmaceuticals, human plasma and aqueous humor using naproxen sodium as internal standard (IS). The target compounds were analyzed on Hypersil BDS C(18) column (250 mm × 4.6 mm, 5 µm), applying 0.2% triethylamine (TEA) and acetonitrile (ACN) (40:60, v/v), in isocratic mode as mobile phase, pH 2.75 adjusted with 85% phosphoric acid at a flow rate of 1 ml/min. The column oven temperature was kept at 45°C and the peak response was monitored at 284 nm after injecting a 50 µl sample into HPLC system. The direct liquid-liquid extraction procedure was applied to human plasma and bovine aqueous humor samples using mobile phase as an extraction solvent after deproteination with methanol. The different HPLC experimental parameters were optimized and the method was validated according to standard guidelines. The recoveries of the suggested method in human plasma were 98.72, 96.04, and 95.14%, for TM, RST, and DS, while in aqueous humor were 94.99, and 98.23%, for TM, and DS, respectively. The LOD values were found to be 0.800, 0.500, and 0.250 ng/ml, for TM, RST, and DS, respectively, while their respective LOQ values were 2.00, 1.50, and 1.00 ng/ml. The co-efficient of variation (CV) were in the range of 0.1492-1.1729% and 1.0516-4.0104%, for intra-day and inter-day studies, respectively. The method was found accurate in human plasma and bovine aqueous humor and will be applied for the quantification of these compounds in plasma, and aqueous humor samples using animal models and in pharmaceuticals.

Influence of plasma protein binding on pharmacodynamics: Estimation of in vivo receptor affinities of beta blockers using a new mechanism-based PK-PD modelling approach.

The objective of this investigation was to examine in a systematic manner the influence of plasma protein binding on in vivo pharmacodynamics. Comparative pharmacokinetic-pharmacodynamic studies with four beta blockers were performed in conscious rats, using heart rate under isoprenaline-induced tachycardia as a pharmacodynamic endpoint. A recently proposed mechanism-based agonist-antagonist interaction model was used to obtain in vivo estimates of receptor affinities (K(B,vivo)). These values were compared with in vitro affinities (K(B,vitro)) on the basis of both total and free drug concentrations. For the total drug concentrations, the K(B,vivo) estimates were 26, 13, 6.5 and 0.89 nM for S(-)-atenolol, S(-)-propranolol, S(-)-metoprolol and timolol. The K(B,vivo) estimates on the basis of the free concentrations were 25, 2.0, 5.2 and 0.56 nM, respectively. The K(B,vivo)-K(B,vitro) correlation for total drug concentrations clearly deviated from the line of identity, especially for the most highly bound drug S(-)-propranolol (ratio K(B,vivo)/K(B,vitro) approximately 6.8). For the free drug, the correlation approximated the line of identity. Using this model, for beta-blockers the free plasma concentration appears to be the best predictor of in vivo pharmacodynamics.

Ophthalmic timolol: plasma concentration and systemic cardiopulmonary effects.

Timolol maleate is a non-selective beta-adrenoceptor antagonist currently used mainly as an ocular preparation for the treatment of glaucoma and ocular hypertension. Despite the topical administration, ophthalmic timolol causes systemic adrenergic beta-blocking because of absorption from the eye into the systemic circulation. Gel formulations of ophthalmic timolol have been developed to reduce systemic absorption and adverse effects in comparison with conventional aqueous solution formulations. Timolol is metabolized by the polymorphic cytochrome P450 2D6 enzyme (CYP2D6). The changes in heart rate (HR) are the most striking effects of the systematically absorbed fraction of ophthalmic timolol, with 0.5 % aqueous formulations presenting larger effects than 0.1 % hydrogel formulations, especially during exercise. Plasma levels of ophthalmic timolol correlate with the changes in HR. Neither 0.5 % aqueous nor 0.1 % hydrogel formulations of timolol have exerted noteworthy effects on systolic (SAP) or diastolic (DAP) arterial pressures, probably because of a compensatory increase in systemic vascular resistance due to the attenuation of HR. Ophthalmic timolol does not exert remarkable effects on pulmonary parameter peak expiratory flow (PEF) and forced expiratory volume in 1 s (FEV1) in non-asthmatic patients. CYP2D6 activity is clearly associated with the pharmacokinetic parameters, particularly when 0.5 % aqueous solution of timolol is used: peak plasma concentration, elimination half-life and area-under-the-curve are highest in CYP2D6 poor metabolizers. Finally, since there is a correlation between the plasma level of timolol and several haemodynamic effects - especially HR in the state of elevated beta-adrenergic tonus - the CYP2D6 poor metabolizers may be more prone to bradycardia during treatment with (aqueous) ophthalmic timolol.

Comparison of a non-preserved 0.1% T-Gel eye gel (single dose unit) with a preserved 0.1% T-Gel eye gel (multidose) in ocular hypertension and glaucomatous patients.

AIM: This comparative, open design, phase III study was to assess the non-inferiority of the non-preserved T-Gel 0.1% single dose unit (SDU) versus its preserved multidose (MD) reference. METHODS: 175 patients with bilateral POAG or OHT were randomised: 87 patients were to receive one drop daily of T-Gel 0.1% MD and 88 patients were to receive one drop daily of T-Gel 0.1% SDU, for a treatment period of 12 weeks. The primary efficacy variable was the change in intraocular pressure (IOP) in the worse eye between the baseline and the last assessment. Subjective and objective ocular signs as well as adverse events were recorded for safety. Global tolerance was assessed by the investigator and by the patient. RESULTS: The mean percentage reduction from baseline IOP was 24% for both treatments groups, which was consistent with previous studies. The safety results were comparable in both treatment groups. Because of gel formulation, mild short lasting episodes of blurred vision occurred for about 20% of patients. The global tolerance assessment reported that both treatments were well tolerated. CONCLUSION: The overall study results demonstrated that T-Gel 0.1% SDU is not inferior to T-Gel 0.1% MD.

Polymorphisms of genes CYP2D6, ADRB1 and GNAS1 in pharmacokinetics and systemic effects of ophthalmic timolol. A pilot study.

OBJECTIVE: To test the hypotheses that (1) CYP2D6 genotype is associated with pharmacokinetics of ophthalmic timolol and (2) variation in genotypes of ADRB1 (beta(1)-adrenoceptor) and GNAS1 (alpha-subunit of G-protein) modulate heart rate (HR), and systolic (SAP) and diastolic (DAP) arterial pressure responses to timolol. METHODS: Nineteen glaucoma patients and eighteen healthy volunteers were treated with 0.5% aqueous and 0.1% hydrogel formulations of ophthalmic timolol using a randomised cross-over design. The participants conducted head-up tilt and maximum exercise test at four visits. Plasma concentration of timolol was measured twice for glaucoma patients and ten times for healthy volunteers on each visit. Also, the genotypes for CYP2D6, ADRB1 and GNAS1 were determined. RESULTS: Among healthy volunteers using aqueous timolol, poor metabolisers (PMs, n=2) of CYP2D6 had higher maximum plasma concentrations (C(max), values 2.63 and 2.94 ng/ml), longer elimination half-lives ( T(1/2), 5.49 and 6.75 h), and higher area-under-curve (AUC, 19.54 and 23.25 ng.h/ml) than intermediate [IMs, n=6, mean+/-SD 1.73+/-0.59 ng/ml (not significant), 3.30+/-0.48 h, 11.32+/-3.72 ng.h/ml], extensive (EMs, n=8, 1.60+/-0.72 ng/ml, 3.24+/-1.24 h, 8.52+/-6.12 ng.h/ml) and ultra-rapid (UMs, n=2, values 1.23 and 1.67 ng/ml, 2.22 and 2.52 h, 6.16 and 6.94 ng.h/ml) metabolisers. The IMs, EMs and UMs did not differ from each other for any of the kinetic variables. Also, the elevation of HR from rest to maximum level tended to differ between PMs and IMs, and between PMs and UMs. The pharmacokinetics and pharmacodynamics between the CYP2D6 groups did not differ with statistical significance when hydrogel timolol was used. Upon head-up tilt, the Ser49 homozygotes (n=26) had higher SAP (P=0.03) and DAP (P<0.01) than the Gly carriers (n=11). The change in DAP from rest to maximum during exercise was lower (P<0.01) in subjects with CC alleles of GNAS1 (n=13) than those with at least one T allele (n=24). CONCLUSION: The CYP2D6 poor metabolisers may be more prone to systemic adverse events with aqueous timolol than extensive metabolisers. Since CYP2D6 genotyping is not routine clinical practice, using 0.1% timolol hydrogel instead of 0.5% aqueous preparation will increase patient safety.

Application of substrate depletion assay for early prediction of nonlinear pharmacokinetics in drug discovery: assessment of nonlinearity of metoprolol, timolol, and propranolol.

The purpose of this study was to investigate the advantages of the substrate depletion assay for evaluating linearity of pharmacokinetics compared with the metabolite formation assay. For propranolol, metoprolol, and nisoldipine with multiple and/or sequential metabolisms, the Michaelis constant (Km) and maximum metabolic intrinsic clearance obtained from the depletion assay using rat and human liver microsomes showed a good correlation with relevant parameters with the formation assay. In vitro kinetics and in vivo pharmacokinetic profiles after oral administration of timolol, metoprolol, and propranolol, were investigated in rats using the depletion assay. The same rank order was found between nonlinearities based on dose-normalized areas under the plasma concentration curve (AUC/Dose) and Km values. Using the kinetic parameters of these compounds, AUC was predicted based on a physiological based pharmacokinetic model incorporated saturable metabolism. The AUCs predicted for propranolol and metoprolol had a good relationship with those observed in the in vivo studies, implying that the depletion assay could be useful for assessing linearity of pharmacokinetics.

Association between low plasma levels of ophthalmic timolol and haemodynamics in glaucoma patients.

OBJECTIVES: The aims of the study were to assess the correlation between the plasma concentration of ophthalmic timolol and cardiovascular parameters, and the influence of timolol on advanced haemodynamic variables, such as stroke (SI), cardiac (CI) and systemic vascular resistance (SVRI) indices and arterial pulse wave velocity (PWV). METHODS: Twenty-five glaucoma or ocular hypertensive patients were treated with 0.5% aqueous and 0.1% hydrogel formulations of timolol using a randomised, double-masked, crossover, multicentre design. All the patients were subjected to passive head-up tilt, electrocardiography, exercise test and measurement of plasma concentration of timolol. In the analysis, the data on the two treatments were combined, and the Spearman correlation coefficients between the plasma level of timolol and physiological effects were calculated. RESULTS: During the head-up tilt test before rising the bed up, the resting heart rate (HR; R=-0.52, P=0.001) and PWV (R=-0.34, P=0.04) were inversely correlated with timolol level. In the upright position, ophthalmic timolol effectively suppressed the rise in HR (R=-0.36, P=0.03). The SI did not change with timolol concentration, while CI diminished as timolol concentration rose (R=-0.39, P=0.02). The SVRI correlated with timolol concentration (R=0.38, P=0.02). In the exercise test, correlation between HR and plasma level of timolol steadily grew stronger as the load increased, reaching R=-0.60 (P<0.0001) at the maximum load. Systolic and diastolic arterial pressures were not associated with the timolol concentration. CONCLUSION: The plasma concentration of ophthalmic timolol correlates with several haemodynamic effects. As HR decreases, SVRI increases and blood pressure is kept unchanged.

Cardiovascular effects of ophthalmic 0.5% timolol aqueous solution and 0.1% timolol hydrogel.

The objective of this randomized, double-masked, cross-over study was to compare the cardiovascular effects of two glaucoma formulations, ophthalmic 0.5% timolol aqueous solution and 0.1% timolol hydrogel. Twenty-four young healthy subjects received for 2 weeks either twice daily 0.5% timolol solution or once daily 0.1% timolol hydrogel. Heart rate (HR), blood pressure, atrio-ventricular conduction (PR interval), corrected QT time (QTc) and heart rate variability (HRV) were measured in supine position and during head-up tilted position. The mean peak concentrations of timolol in plasma were significantly higher after administration of 0.5% aqueous solution than after 0.1% hydrogel. A 0.5% timolol aqueous solution decreased HR on average by 3 bpm in supine position and by 7 bpm in head-up tilted position while no significant effects were observed with 0.1% timolol hydrogel. During tilt test HR was significantly lower after administration of timolol aqueous solution than after timolol hydrogel (mean +/- SD, 77 +/- 11 bpm versus 86 +/- 13 bpm, P < 0.05). Timolol aqueous solution slightly decreased QTc during tilt (5.9 +/- 5.6 ms, P < 0.01). During tilt tests, timolol aqueous solution slightly increased atrio-ventricular conduction (7.2 ms, P = 0.02). No significant differences were found in HRV. These results indicate that in healthy volunteers, ophthalmic 0.5% timolol aqueous solution produces more pronounced cardiac beta-blocking effects than 0.1% timolol hydrogel.

Water-activated, pH-controlled patch in transdermal administration of timolol. II. Drug absorption and skin irritation.

The feasibility of the water-activated, pH-controlled silicone reservoir devices for transdermal administration was investigated using timolol maleate as a model drug. Timolol patches were applied to the arm of 12 volunteers for 81 h, two patches per subject. Timolol absorption from patches was compared to that from a peroral timolol tablet formulation (Blocanol((R)) 10 mg). Furthermore, in vivo plasma levels of timolol were compared with those predicted by kinetic simulations. Skin irritation induced by timolol patches was assessed by visual scoring and color reflectance measurements. With water-activated, pH-controlled patches both steady-state concentrations of timolol in plasma and its duration could be controlled. However, a considerable, inter-individual variability in the transdermal absorption of timolol was observed. This is due to the high fractional skin control in timolol delivery. Timolol patches were well tolerated by subjects. Skin irritation induced by the combination of timolol with long-term occlusion was mild, and after removal of the patches, skin changes were practically reversed in 24 h. Simulation model was useful in prediction of timolol levels in plasma after transdermal administration.

Determination of low plasma timolol concentrations following topical application of timolol eye drops in humans by high-performance liquid chromatography with electrochemical detection.

A simple and sensitive high-performance liquid chromatographic assay was developed for determination of timolol in human plasma following administration of two drops of a 5% timolol ophthalmic solution. A 4% butyl alcohol-hexane extract of an alkalized sample of plasma was chromatographed on a reversed-phase column and the components in the column effluent were monitored by coulometric detection. The extraction efficiency of timolol was 69.02 +/- 4.16% (mean +/- S.D.) and its detection limit was 107.2 pg/ml. The effect of mobile phase pH, buffer concentration and the working potential of the detector on column performance and the electrochemical response are described.

A model to predict aqueous humor and plasma pharmacokinetics of ocularly applied drugs.

PURPOSE: To develop methods for constructing a pharmacokinetic model to predict the time course of aqueous humor and plasma drug concentrations after topical dosage in rabbits using the simulation program iThink (formerly STELLA; High Performance Systems, Lyme, NH). METHOD: The model was constructed in experimentally verifiable segments using previously published data on intravenous, nasal, and ocular dosage, and was used to describe the influence of prolonging precorneal retention and varying drug release rate on the ratio of drug absorbed locally to drug absorbed systemically in rabbits. RESULTS: The model developed is comprehensive; it includes precorneal kinetics, nasal absorption kinetics, and plasma kinetics. CONCLUSIONS: Such a model may be useful in designing drug delivery strategies to improve the safety of topical eye medications through minimization of systemic absorption and maximization of drug delivery to ocular tissues.

Determination of the beta-adrenergic blocker timolol in plasma by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry.

A method based on LC-MS-MS has been developed for the determination of timolol in plasma using the (CD3)3-labelled species as the internal standard. Timolol is isolated from plasma by a simple solid-phase extraction and converted to its oxazolidin-2-one prior to analysis on a 50 x 4.6 mm reversed-phase high-performance liquid chromatography column packed with SynChropak, C18, 5 microns. The column eluate is passed by means of a heated nebulizer interface into a corona discharge atmospheric pressure chemical ionization source where the analyte and its internal standard are detected using multiple reaction monitoring (MRM). The very high specificity of this technique permits chromatographic run times of less than 2 min. The method has a lower quantifiable limit of 0.5 ng ml-1, with intra- and inter-day relative standard deviations less than 10%, and enables the determination of timolol in plasma after ocular administration to volunteers.

Assay of timolol in human plasma using gas chromatography with electron-capture detection.

A simple and sensitive gas chromatographic method has been developed for the determination of timolol in plasma using electron-capture detection and propranolol as internal standard. Timolol was extracted using butyl chloride and derivatized using trifluoroacetic anhydride in butyl acetate. The lower detection limit for the assay was found to be 1 ng/ml from 1 ml of plasma. Extracted standards gave within-day precision of 12.55, 9.68 and 3.78% for 1, 20 and 100 ng/ml plasma samples, respectively. A recovery of at least 80% of timolol was found using the extraction method described. The assay was used in a randomized cross-over bioequivalence trial using an oral administration of 20 mg of timolol. Pharmacokinetic parameters compare favourably with other literature values.