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We have compared the diagnostic ability of different concentrations of 0.125% and 0.0625% dilute pilocarpine for detecting denervation supersensitivity in unilateral Adie's tonic pupil. This retrospective, observational, case-control study involved 117 subjects, consisting of 56 patients with unilateral Adie's tonic pupil and 61 controls with other causes of unilateral dilated pupils. Subjects underwent the dilute pilocarpine test with one of the two concentrations, 0.125% or 0.0625%. Pupillary light reflex was recorded with a dynamic pupillometer at baseline and at 30-40 min after instilling one of the two concentrations of dilute pilocarpine. Diagnostic accuracy of two different concentrations of the dilute pilocarpine test, 0.125% group versus 0.0625% group, were compared by area under the receiver operating characteristic curve (AUC). Diagnostic ability of the dilute pilocarpine test for detecting denervation supersensitivity in unilateral Adie's tonic pupil was significantly better in the 0.0625% group than in the 0.125% group (AUC = 0.954 vs. 0.840, respectively, P = 0.047). In the 0.0625% group, the change in maximal pupil diameter of ≥ 0.5 mm after topical pilocarpine instillation showed 100% sensitivity and 82.8% specificity for detecting Adie's tonic pupil. This study confirmed that pupillary constriction with 0.0625% pilocarpine is better than 0.125% pilocarpine for detecting denervation supersensitivity in Adie's tonic pupil. Digital pupillometry is a reliable method for assessing denervation supersensitivity in Adie's tonic pupil.

Mass spectrometry (MS) imaging is a useful tool for direct and simultaneous visualization of specific molecules. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is used to evaluate the abundance of molecules in tissues using sample homogenates. To date, however, LC-MS/MS has not been utilized as an imaging tool because spatial information is lost during sample preparation. Here we report a new approach for LC-MS/MS imaging using a thermal film-based laser microdissection (LMD) technique. To isolate tissue spots, our LMD system uses a 808-nm near infrared laser, the diameter of which can be freely changed from 2.7 to 500 μm; for imaging purposes in this study, the diameter was fixed at 40 μm, allowing acquisition of LC-MS/MS images at a 40-μm resolution. The isolated spots are arranged on a thermal film at 4.5-mm intervals, corresponding to the well spacing on a 384-well plate. Each tissue spot is handled on the film in such a manner as to maintain its spatial information, allowing it to be extracted separately in its individual well. Using analytical LC-MS/MS in combination with the spatial information of each sample, we can reconstruct LC-MS/MS images. With this imaging technique, we successfully obtained the distributions of pilocarpine, glutamate, γ-aminobutyric acid, acetylcholine, and choline in a cross-section of mouse hippocampus. The protocol we established in this study is applicable to revealing the neurochemistry of pilocarpine model of epilepsy. Our system has a wide range of uses in fields such as biology, pharmacology, pathology, and neuroscience. Graphical abstract Schematic Indication of LMD-LC-MS/MS imaging.

Pilocarpus microphyllus Stapf ex Wardleworth (jaborandi, Rutaceae) is one of the most important Brazilian medicinal species owing to its content of pilocarpine (PIL), an alkaloid used for treating glaucoma and xerostomia. This species contains another alkaloid, epiisopiloturine (EPI), which has demonstrated effectiveness against schistosomiasis. The aim of this work was to assess seasonal changes of PIL and EPI in three populations of cultivated P. microphyllus from northeastern Brazil over one year, including the dry and rainy seasons. Alkaloid profiles were correlated to phenotypic and genetic patterns in the morphological and molecular characterizations. PIL was the primary alkaloid and its levels differed among populations in all months except September. The S01 population (green line) showed an especially high PIL content compared to populations S02 and S03 (traditional line), which had similar alkaloid contents. PIL content gradually decreased in the three populations in the rainy season.EPI content was significantly different between the green line (S01) and the traditional line (S02 and S03).S01 had a significantly lower EPI content in all months, demonstrating that it was not the best source for EPI extraction. Inter simple sequence repeat (ISSR) markers and morphological analyses clearly separated S01 from S02 and S03, in agreement with the alkaloid results. This study shows the first correlation between the chemical, morphological, and molecular markers of P. microphyllus and highlights the potential benefits of a multidisciplinary research approach aimed at supporting both industry and conservation of natural resources., Competing Interests: The authors have declared that no competing interests exist.

Objectives: A common experimental model of status epilepticus (SE) utilizes intraperitoneal administration of the cholinergic agonist pilocarpine preceded by methyl-scopolamine treatment. Currently, activation of cholinergic neurons is recognized as the only factor triggering pilocarpine SE. However, cholinergic receptors are also widely distributed systemically and pretreatment with methyl-scopolamine may not be sufficient to counteract the effects of systemically injected pilocarpine. The extent of such peripheral events and the contribution to SE are unknown and the possibility that pilocarpine also induces SE by peripheral actions is yet untested., Methods: We measured in vivo at onset of SE: brain and blood pilocarpine levels, blood-brain barrier (BBB) permeability, T-lymphocyte activation and serum levels of IL-1beta and TNF-alpha. The effects of pilocarpine on neuronal excitability was assessed in vitro on hippocampal slices or whole guinea pig brain preparations in presence of physiologic or elevated [K+](out)., Results: Pilocarpine blood and brain levels at SE were 1400 +/- 200 microM and 200 +/- 80 microM, respectively. In vivo, after pilocarpine injection, increased serum IL-1beta, decreased CD4:CD8 T-lymphocyte ratios and focal BBB leakage were observed. In vitro, pilocarpine failed to exert significant synchronized epileptiform activity when applied at concentrations identical or higher to levels measured in vivo. Intense electrographic seizure-like events occurred only in the copresence of levels of K+ (6 mM) mimicking BBB leakage., Conclusions: Early systemic events increasing BBB permeability may promote entry of cofactors (e. g. K+) into the brain leading to pilocarpine-induced SE. Disturbance of brain homeostasis represents an etiological factor contributing to pilocarpine seizures.

Methods developed on conventional particle-packed C18 columns for pilocarpine, propranolol, glibenclamide, glimepiride, insulin and their respective degradation products or related compounds were transferred from the conventional Superspher 100RP-18e column to Chromolith Performance RP-18e columns. All transfers were successful applying the same chromatographic conditions, except for insulin where the acetonitrile content of the mobile phase was reduced by 0.5%. The intraday and interday precisions for both retention time and peak area were evaluated over a wide concentration range. Results were found to be equal, or slightly better on Chromolith Performance with RSD%<1.1% in all cases. Monolithic batch to batch repeatability of both retention time and peak area, compared for monolithic columns from different batches gave an RSD% of less than 1.3%. The separation of each drug and its related products was investigated on monolithic columns at flow rates from 1 to 9 ml/min, and superior resolution was always obtained using monolithic over conventional columns at the same flow rate. A total of seven monolithic columns from four different batches were used in this study.

Background/aim: In glaucoma therapy beta-blocator, timolol-maleate, is the first-line medicine of selection, whereas a miotic, pilocarpine chloride, is one of the oldest medicines used for the treatment of this illness. They are applied as eye drops. In order to achieve a better therapeutic effect and improve life quality of the ill, we produced and tested eye drops formulations based on combining pilocarpinechloride and timolol maleate in buffers of diferent pH values., Methods: Following the general pharmacopoeial eye drops preparation regulation, we prepared formulations, of solution type, of pilocarpine chloride and timolol maleate combination, of pilocarpine choloride alone and of timolol maleate alone. A modified phosphate buffer according to Sorensen at 7.4, 7.7 and 8.0 pH values was used as a solvent. The quality of the produced formulations was examined using physical and physico-chemical methods and biological tests. Following pharmacopoeial regulations, we examined clarity, pH value and sterilty. Pilocarpine chloride level was determined by means of ion-par (High Performance Liguid Chromatography) RP-HPLC method, whereas UV/VIS absorption spectrophotometry was used for determining the level of both timolol maleate, and timolol., Results: Results showed that monocomponent and combined preparations complied with the regulation demands. With the increase in the pH value of the solution pilocarpine chloride level decreased in relation to its initial content, whereas timolol level showed a tendency of moderate increase., Conclusion: Magistrally prepared pilocarpine chloride eye drops with timolol maleate have satisfied all the required conditions for an ophtalmological preparation. A modified phosphate buffer according to Sorensen at 7.4 pH value proved to be the most optimal solvent.

Monolithic Performance C18 HPLC columns (Chromolith Performance RP-18e, Merck) were applied for the determination of pilocarpine hydrochloride in the presence of its degradation products isopilocarpine, pilocarpic acid and isopilocarpic acid. The method was validated using a set of six monolithic columns and compared to a conventional C18 column. The separation of pilocarpine from its degradation products was investigated on monolithic columns at different flow rates from 1 to 9 ml/min. Superior resolution was obtained using monolithic columns over the conventional C18 column at the same flow rate of 1 ml/min with a total run time of 9 min compared to 13.5 min for the conventional C18 column. Comparable resolution to that obtained in the C18 column (but with better peak symmetry) was obtained at a flow rate of 4 ml/min, although the total run time was reduced to 3.5 min. The precision for both retention time and peak area was investigated over a wide concentration range and found to be equal, or slightly better on Chromolith Performance compared to the conventional column. The overall RSDs% ranged from 0.5% to 1.16% for the conventional column, while for monolithic columns ranged from 0.38% to 0.87% at a flow rate of 1 ml/min and from 0.38% to 0.89% at a flow rate of 4 ml/min. Monolithic column to column reproducibility (n = 6) was measured. The RSDs% ranged from 0.34% to 0.68% for retention time and from 0.3% to 0.94% for peak areas. The detection and quantitation limits on monolithic columns at both flow rates (1 and 4 ml/min) were found to be 0.17 microg/ml and 0.5 microg/ml, compared to 0.31 microg/ml and 1 microg/ml on the conventional column. Monolithic silica rods have also shown the advantage of reducing the time to wash and to re-equilibrate the column. The method showed good linearity and recovery and was found to be suitable for the analysis of pilocarpine hydrochloride formulations.

Amblyomma americanum (Linneaus) (Acari: Ixodidae), an important tick vector of human and animal disease, is not a competent vector of the bacterial agent of Lyme disease, Borrelia burgdorferi, although its range overlaps the geographical distribution of Lyme disease within the United States. A possible mechanism that could prevent acquisition of B. burgdorferi spirochetes from infected hosts is the toxic effect of A. americanum saliva on B. burgdorferi. The data presented here indicate that after 24 and 48 h of exposure to A. americanum saliva, significantly fewer B. burgdorferi were alive compared to treatment controls as assessed by spirochete motility under dark-field microscopy and resistance to the dead stain, propidium iodide. After 48 h, fewer than 13% of saliva-exposed B. burgdorferi were alive. In contrast, significantly more B. burgdorferi exposed to Ixodes scapularis (Acari: Ixodidae) saliva survived after 24 or 48 h compared to A. americanum saliva or treatment controls.

Pilocarpine is often applied or injected into ticks to induce salivation, and the resulting saliva used to test for various pharmacological, biochemical and immunological activities. To measure the amount of pilocarpine in pilocarpine-induced tick saliva, an HPLC-MS/MS method, based on capillary strong cation exchange chromatography online with an ion trap mass spectrometer, was used to measure pilocarpine in the pg to ng range. Results indicate large concentrations of pilocarpine in Ixodes scapularis Say and Amblyomma americanum (Linnaeus) (Acari: Ixodidae) saliva, ranging from 3 to 50 mm. Due to the known effects of pilocarpine on smooth muscle and immune cells, appropriate controls are proposed and discussed for proper interpretation of results using this saliva preparation.

Objective: To develop an RP-HPLC method for assay of pilocarpine in rabbit ocular aqueous humor., Methods: The RP-HPLC method was performed on a column of ODS-C(18) with the mobile phase consisting of 0.5% of triethylamine (TEA) of phosphate solutions (10 mmol/L, pH 2.5) and acetonitrile (98/2,v/v). The detection wavelength was 215 nm and flow rate was 1.0 ml/min. Ninety albino rabbits were divided into 3 groups (30 in each):group 1 received 50 microl of eye drops containing 1% generic pilocarpine, group 21% mixture pilocarpine solution consisting of aqueous sample and liposome and group 31% liposome pilocarpine, respectively. The aqueous humor was withdrawn at 5, 10, 30, 40, 60, 90, 120, 180, 240 and 360 min. Pilocarpine was extracted from aqueous humor with dichloromethane., Result: The linear calibration curve was obtained in the concentration range of 0.1 - 20 microg/ml. The average recovery was (68.1+/-2.7)% (n=9). Inter-day and intra-day RSD were 4.33% and 2.87%, respectively. In three formations 1% liposome pilocarpine was the best for the areas under curve and measurable amounts., Conclusion: The RP-HPLC method is simple and reliable for pilocarpine measurement in ocular aqueous. Liposome formulation can significantly increase the bioavailability of pilocarpine in ocular aqueous.

The influence of artificial tear fluid (AT) on ionic and nonionic ophthalmic polymer excipients was rheologically established. In usual concentrations polyvinylalcohol, polyvinylpyrrolidone, dextran, hydroxypropylmethylcellulose, hydroxyethylcellulose and methylcellulose did not show any changes. In contrast, solutions of polyacrylic acid, sodium hyaluronate (S-Hya), sodium alginate (S-Alg) and chitosan decrease the apparent viscosity in contact with AT, while gellan solution increases the viscosity and shows thixotropy. The adhesion of selected polymers (polysaccharides) on mucin was evaluated using a rheological method and resulted in the order S-Hya > Gellan > S-Alg > dextran. Miosis testing of Gellan containing pilocarpine HCl formulations in rabbits shows a possible reduction of drug concentration from 2% to 0.5% obtaining the same bioavailability.

Gelatin particles containing pilocarpine HCl were prepared using a desolvation method. The influence of the gelatin type and the preparation pH on particle properties was studied. The drug entrapment amounted 44 to 57%, zetapotential was slightly negative. Particle size varied from 312 nm to 500 nm depending on preparation pH. Franz diffusion cell experiments showed a release approaching zero-order kinetics and a sustained release compared to an aqueous pilocarpine HCl solution.

A method is described for the determination of pilocarpine and its degradation products isopilocarpine, pilocarpic acid and isopilocarpic acid in human plasma and urine. The method is based on a simple sample preparation step -- ultrafiltration for plasma and dilution for urine samples -- followed by a reversed-phase liquid chromatographic separation of the analytes and detection by means of tandem mass spectrometry. Parameters affecting the performance of these steps are discussed. The high sensitivity and selectivity of the method allow low ng/ml concentrations to be determined for all compounds in plasma and undiluted urine, which enables the investigation of the metabolic fate and elimination of pilocarpine after oral administration to humans.

The separation of pilocarpine and its degradation products by micellar electrokinetic capillary chromatography (MECC) has been optimized by using fractional factorial design of the experiments. Critical parameters were identified in a screening design, and an optimization design was used to optimize the separation. The optimal separation method was based on a borate buffer with sodium dodecyl sulfate (SDS). It is concluded that by using fractional factorial design it is possible to improve the separation of pilocarpine, its trans epimer, isopilocarpine and their hydrolysis products, pilocarpic acid and isopilocarpic acid.

The amounts of 2 principal neurotransmitters, acetylcholine (ACh) and norepinephrine (NE) in the 3 major salivary glands, and pilocarpine-, isoproterenol- and phenylephrine-induced salivation in male mice fed a powdered diet for 16 weeks were compared with those in mice fed a standard pellet diet (as control). There were no significant differences in the final body weights of mice fed the powdered diet and the control diet. The only salivary gland in the powdered diet fed mice to increase significantly in weight was the sublingual gland. Mice fed the powdered diet had significantly increased ACh concentrations and contents, but had decreased amounts of NE, in the submandibular and sublingual, but not the parotid glands. The salivation stimulated by pilocarpine was markedly decreased in mice fed the powdered diet, whereas the salivation stimulated by phenylephrine or isoproterenol was not. These findings indicate that reduced mastication affects not only the secretory function but also the amounts of these neurotransmitters in the salivary glands of mice.

A sensitive assay for pilocarpine in biological fluids has been developed involving HPLC of a fluorescent derivative of 4-bromomethyl-7-methoxycoumarin. Pilosine as internal standard was added before the derivatisation step. The fluorescent derivatives were well resolved and separated from excess reagent and endogenous compounds on a cyanopropyl silica column. The detection limit of pilocarpine in biological fluids was 1.0 ng/ml and the assay was linear up to a concentration of 150 ng/ml. The assay was applied to a preliminary study of pilocarpine disposition in man after a single oral dose. This is the first report of pilocarpine excretion into saliva.

A new method has been developed for rapid analysis and determination of pilocarpine in aqueous humour using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry. The chromatography was carried out on a reversed-phase phenyl column with 0.1% acetic acid-acetonitrile (95:5, v/v). Pilocarpine and its analogues, isopilocarpine, pilocarpic acid and isopilocarpic acid, were separated. An aqueous humour sample was deproteinized with methanol. After evaporation, the residue was dissolved in the mobile phase. The method was applied to the analysis of the metabolite in aqueous humour after the topical application of 2% pilocarpine (w/v) eye-drops. The main metabolite, pilocarpic acid, was easily identified. The protonated molecular ion of pilocarpine was used for the determination. The calibration curve had a good linearity within the concentration range investigated (2 ng to 10 micrograms/ml). The limit of determination was estimated to be an aqueous humour concentration of ca. 2 ng/ml. The method was applied to the determination of unchanged pilocarpine after the topical application of 2% pilocarpine (w/v) eye-drops.

Capillary zone electrophoresis was used for the separation of pilocarpine from its epimer, isopilocarpine, using coated fused-silica capillaries of 20 cm x 25 microm I.D., 8 kV running voltage, migration buffer of 0.1 M sodium dihydrogenphosphate pH 8, detection at 217 nm and injection by electromigration. Injections of aqueous, acid and basic solutions were compared. Linearity of the signal for pilocarpine hydrochloride up to 200 microg ml(-1) in 0.05 M hydrochloric acid was obtained, using naphazoline nitrate as internal standard. Optimization of migration buffer pH using coated silica capillaries of 50 cm x 50 microm I.D. showed that at pH 6.9 pilocarpine can be separated from ++isopilocarpine. Inclusion of beta-cyclodextrin in the buffer allows full baseline separation of both epimers. The method was applied to the analysis of a commercial ophthalmic pilocarpine solution.

A high-performance liquid chromatographic (HPLC) method for the determination of pilocarpine, isopilocarpine, pilocarpic acid and isopilocarpic acid was developed. A beta-cyclodextrin column achieved the separation in less than 10 min. Baseline resolution of all four compounds permitted the determination of each degradation product in the presence of pilocarpine. The calibration graphs for each compound were linear, and pilocarpine degradation products could be determined without a correction for the ultraviolet detector response using a pilocarpine standard. A comparison of beta-cyclodextrin separation with the USP HPLC method demonstrated similar results for pilocarpine contents in several commercial ophthalmic formulations.

A rapid high-resolution high pressure liquid chromatographic method was developed for assaying pilocarpine. Pilocarpine in ophthalmic solutions decomposes fairly rapidly to give isopilocarpine, pilocarpic acid and isopilocarpic acid. The quality of an ophthalmic solution can be assessed by assaying these decomposition products. Existing high pressure liquid chromatographic methods suffer from long analysis times and poor resolution. The new method uses as the mobile phase 6 ml/l of triethylamine in water (pH 2.3, adjusted with 85% phosphoric acid) at a flow of 1.5 ml/min and as the stationary phase a C18-silica 125 x 4.6 mm column. 2-Amino-1-phenyl-1,3-propanediol is used as an internal standard. Complete separation was obtained within 8 min. Pilocarpine eye drops were stored under different conditions and then analysed for decomposition products. During heat treatment, decomposition to isopilocarpine predominated over decomposition to pilocarpic or isopilocarpic acid. However, when stored at room temperature or in a refrigerator, formation of pilocarpic acid clearly prevailed. Thus, from assessment of decomposition products, the cause of decomposition can be established.

The properties of pilocarpine as a ligand toward the halides of cobalt(II), nickel(II), copper(II), and zinc(II) were investigated. Pilocarpine behaved as a monodentate ligand, giving rise to compounds with the general formula methyl(pilocarpine)2X2. The coordinating geometry at the metal ion was pseudotetrahedral in every case. PMR studies showed that the pyridine-type nitrogen of the imidazole ring of pilocarpine was the donor atom of the ligand.

Previous studies of saliva from engorged female cattle ticks revealed a component which induced contration of some isolated smooth muscles. Fractionation and further characterisation have shown that this substance is of the "slow-reacting" type, but that it is neither a bradykinin nor slow-reacting substance of anaphylaxis. The substance is deactivated by incubation with 15-hydroxprostaglandin dehydrogenase and its pharmacological properties also support its classification as a prostaglandin. A second pharmacologically-active component has now been found in the saliva but has not yet been characterised.

A high-performance liquid chromatographic method is described which allows the simultaneous estimation of pilocarpine, isopilocarpine, pilocarpic acid and isopilocarpic acid. The method offers advantages over existing chromatographic procedures in that pilocarpine and the degradation products are eluted within 15 min making the procedure suitable for routine quality control. Pilocarpine eye-drop preparations available on the Australian market were determined to contain between 0.4 and 1.9% isopilocarpine and between 2.2 and 6.3% total pilocarpic acids. These findings are comparable to those reported by Weber in a survey of pilocarpine containing products available in the U.S.A.

A method for separating and determining pilocarpine and 2 degradation products was developed and subjected to collaborative study. Pilocarpine, isopilocarpine, and pilocarpic acid were isolated on a reverse phase liquid chromatographic phenyl bonded column and detected by UV spectrophotometry at 220 nm. Nine collaborators received commercial samples labeled to contain 2, 1, and 0.5% pilocarpine and a 2% practice sample. The collaborative results for pilocarpine were excellent; coefficients of variation ranged from 3.20 to 4.10%. The method was adopted official first action for determination of the active component, pilocarpine, in the presence of isopilocarpine and pilocarpic acid. Although quantitative results for the degradation products were not as good, the method is suitable as a limits test for these substances.

A derivatization procedure and a high-performance liquid chromatographic (HPLC) method of analysis for pilocarpine are described. The method is based on the quaternization of the 3'-tertiary amino group of the methylimidazole ring of pilocarpine with p-nitrobenzyl bromide. The HPLC system employs and RP-ODS column with a methanol-water mobile phase containing octanesulfonate as an ion-pairing agent. The sensitivity of the method permits the analysis of pilocarpine in biological fluids such as aqueous humor. The method is selective for pilocarpine in the presence of isopilocarpine. Its applicability to the analysis of aromatic heterocyclic and alkyl tertiary amines is demonstrated.

The comparative effect on pilocarpine flux across rabbit cornea induced by two hydrogel polymer lenses containing equal doses was quantitated in a transport chamber. This closed system featured continuous flow of a tear analog but excluded variables of the internal eye influencing concentration. Flux induced by both lenses increased linearly with time. At 240 minutes total flux was a whole order greater than that induced by the same pilocarpine dose in free fluid. Analysis of pilocarpine in tear analog effluent showed the flux to be independent of the available dose retained in the hydrogel polymer lens, suggesting that corneal transport of pilocarpine to the aqueous may involve mediation by a carrier system.

Pilocarpine undergoes 2 important reactions in solutions, epimerization to isopilocarpine and hydrolysis to pilocarpic acid. There is no official USP limit for isopilocarpine or pilocarpic acid in pilocarpine hydrochloride ophthalmic solutions. An existing high-pressure liquid chromatographic (HPLC) method was modified by changing the buffer system to permit its use with constant-pressure as well as constant-volume instruments. The procedure separates all 3 components on a cation exchange resin; pilocarpine and isopilocarpine are determined directly and pilocarpic acid indirectly. Ophthalmic solutions and drug substances were obtained from substantially all the United States marketers of pilocarpine opthalmic solutions and were analyzed by the modified HPLC method. Results of the survey show that isopilocarpine is present to the extent of 0.4-3% and pilocarpic acid at levels of 0.6-7% of total alkaloid.

A rapid and convenient reversed-phase high-performance liquid chromatographic procedure for the quantitative determination of pilocarpine and its degradation products was used to analyze 10 clinical ophthalmic pilocarpine formulations.

A quick method for the determination of pilocarpine in eye drops in the presence of decomposition products is described. The method involves complexation of the alkaloid with bromocresol purple at pH 6. After treatment with 0.1N NaOH, the liberated dye is measured at 580 nm. The method has a relative standard deviation of 1.99%, and has been successfully applied to the analysis of 2 batches of pilocarpine eye drops. The recommended method was also used to monitor the stability of a pilocarpine nitrate solution in 0.05N NaOH at 65 degrees C. The BPC method failed to detect any significant decomposition after 2 h incubation, but the recommended method revealed 87.5% decomposition.

A method for the determination of pilocarpine was developed in which the imidazole ring of pilocarpine was acylated with heptafluorobutyric anhydride, using triethylamine as a catalyst. After cleanup, the pilocarpine derivative was analyzed using GLC with electron-capture detection. The limit of sensitivity was 25-50 pg of pilocarpine, which had been subjected to the derivatization and cleanup procedures. The method was specific for pilocarpine, with the isopilocarpine derivative eluting prior to the pilocarpine derivative.

A high-performance liquid chromatographic assay is described for the simultaneous assay of pilocarpine and physostigmine in the presence of their decomposition products. This assay method was used to quantitatively determine these medicaments in an eye drop formulation which had been sterilized by autoclaving at 115 degrees C for 30 min and at 121 degrees C for 15 min. There was no significant loss of pilocarpine whereas physostigmine was degraded. The major decomposition product is shown to be eseroline.

We used 20-day-old rabbits as a model to show that the ocular bioavailability of topically applied pilocarpine nitrate increased as the instilled volume of the drug was decreased. Decreasing the instilled volume from 25 to 5 microliter permitted a dosage reduction of greater than 2.5 times without sacrificing overall drug concentrations in the eye. Since only a small fraction of topically applied doses to the eye actually reached the interior of the eye, the remainder of the dose was lost and available for systemic absorption. The reduction in dosage permitted by this approach resulted in less drug appearing in the general circulation, as shown by comparative plasma level-time profiles. The advantages of reducing drop size are improved ocular bioavailability permitting the use of smaller doses; and less systemic drug loss, thus reducing the potential for systemic side effects. These advantages could be especially significant in the pediatric and geriatric age groups.

Effect of methylcellulose and polyethylene glycol (PEG1 and PEG2) gel formulations on in-vitro release of combination of 0.25% w/w physostigmine salicylate and 1% w/w pilocarpine hydrochloride was investigated in comparison with a fatty base containing the same drugs by using a stationary dialysis technique. The results showed that the release of both drugs was very slow from the fatty base while a maximum release was attained from methylcellulose gel formulation followed by PEG1 and then PEG2 gel formulations as indicated by the calculated area under curves (AUC) for the amounts of drugs released from different formulations.