LC-MS/MS imaging with thermal film-based laser microdissection.

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.

Dilute pilocarpine test for diagnosis of Adie's tonic pupil.

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.

Evaluation of monolithic HPLC columns for various pharmaceutical separations: method transfer from conventional phases and batch to batch repeatability.

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.

Seasonal change in main alkaloids of jaborandi (Pilocarpus microphyllus Stapf ex Wardleworth), an economically important species from the Brazilian flora.

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.

Evaluation of monolithic C18 HPLC columns for the fast analysis of pilocarpine hydrochloride in the presence of its degradation products.

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.

Corneal metabolism of pilocarpine in pigmented rabbits.

Cornea, aqueous humor, and iris-ciliary body levels of pilocarpine and its metabolite pilocarpic acid were determined in mixed-breed rabbits following topical dosing with 25 microliters of 1 X 10(-2) M pilocarpine. From the time-drug concentration profile it is clear that extensive metabolism of pilocarpine occurs in the cornea of pigmented rabbits. This finding contrasts sharply with similar studies in albino rabbits where relatively low levels of pilocarpine acid were observed. It is estimated that the first-order metabolism rate constant in albino rabbits is approximately two orders of magnitude smaller than in pigmented animals. A significant observation from this finding is the possibility that the reported greater dose requirement for heavily pigmented individuals may not be due to drug-pigment binding alone but also to extensive corneal drug metabolism.

Ocular bioavailability and systemic loss of topically applied ophthalmic drugs.

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.

Simultaneous determination of pilocarpine and isopilocarpine in pharmaceutical preparations by liquid chromatography.

A liquid chromatographic assay method is described for the simultaneous determination of pilocarpine and isopilocarpine in pharmaceutical preparations. The method involves separation of the isomers on a high-resolution ion-exchange column, followed by detection of pilocarpine and isopilocarpine by UV absorption in the 217-nm region. The specificity of the method is such that pilocarpine and isopilocarpine can be assayed separately in the presence of one another and in the presence of the excipients commonly found in commercial pilocarpine solutions. Its sensitivity for isopilocarpine is at least 25 ppm (independent of the concentration of pilocarpine). In contrast, USP methods lack both specificity and sensitivity. It is concluded that this method is applicable to the routine analysis of commercial pilocarpine preparations and is an improvement over the official methods.

Analysis of pilocarpine and isopilocarpine in ophthalmic solutions by UV spectrophotometry-polarimetry.

An improved analytical method was developed that simultaneously quantitates pilocarpine and isopilocarpine in the presence of each other and pilocarpic acid. Pilocarpine and isopilocarpine are first separated from any pilocarpic acid present in the sample by eluting with water-washed chloroform through a column packed with acid-washed diatomaceous earth. The concentrations of pilocarpine and isopilocarpine then are determined by a combination of UV spectrophotometric and polarimetric measurements. UV absorbance is measured at the absorption maximum (215 nm), and optical rotation is measured at the 254-nm line of mercury. Standard curve and standard recovery data are presented. The method is applicable to several commercially available ophthalmic solutions of pilocarpine and is compared to both the USP colorimetric method and a high-performance liquid chromatographic method.

Measurement of pilocarpine and its degradation products by high-performance liquid chromatography.

An assay method for pilocarpine using reversed-phase high-performance liquid chromatography is presented. This method also measures isopilocarpine, the stereoisomer of pilocarpine, and the degradation products pilocarpic acid and isopilocarpic acid. Maximum sensitivity was obtained with optical absorbance at 216 nm.

High-speed liquid chromatographic determination of pilocarpine in pharmaceutical dosage forms.

A specific method for the direct determination of pilocarpine in aqueous pharmaceuticals in the presence of decomposition products, methylcellulose, and other ingredients usually present in pharmaceuticals is described. The method involves separation by high-speed liquid chromatography using, in series, octadecylsilane bonded to silica and cyanopropylsilane bonded to silica columns and a tetrahydrofuran-pH 9.2 borate buffer (3:7) eluant. Quantitation is achieved by monitoring the absorbance of the effluent at 254 nm and using a pyridine internal standard and a calibration curve prepared from known concentrations of pilocarpine nitrate. The reproducibility of the retention time and peak area was better than 2.0%.

Quantitative determination of pilocarpine, isopilocarpine, pilocarpic acid, and isopilocarpic acid in clinical ophthalmic pilocarpine formulations by reversed-phase liquid chromatography.

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.

Subnanogram assay for pilocarpine in biological fluids.

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.

Complexes between pilocarpine and cobalt(II), nickel(II), copper(II), and zinc(II) ions.

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.

Quantitative analysis of degradation products in pilocarpine hydrochloride ophthalmic formulations.

The presence of isopilocarpine, an epimer of pilocarpine, and of pilocarpinic acid, a hydrolytic degradation product of pilocarpine, was established and all three substances were assayed in various commercial ophthalmic formulations of pilocarpine hydrochloride by 13C-Fourier transform spectroscopy. Assay was based upon integrated intensities of selected resonances from any formulation calibrated against the intensity of tetramethylammonium bromide, used as a common external reference. The normalized intensities were then related to those of a reference solution of pilocarpine hydrochloride, thereby eliminating any factor arising from variability of 13C-relaxation times. The 13C-resonance for the N-methyl group, being common to all products, provides a convenient basis for the assay of the total alkaloid content whereas the C-8 resonances are best suited for assaying residual pilocarpine and its degradation products. This procedure, estimated as accurate to +/- 5%, constitutes the first comprehensive analytical method to differentiate between pilocarpine and its degradation products.

Analysis of pilocarpine and isopilocarpine in ophthalmic solutions by normal-phase high-performance liquid chromatography.

A normal-phase high-performance liquid chromatographic separation for pilocarpine and isopilocarpine was developed which is suitable for the routine analysis of ophthalmic preparations. The method utilizes a column packed with 5-micron silica with a mobile phase of hexane-2% ammonium hydroxide in 2-propanol (70:30). Peak detection is by UV at 220 nm. It is suggested that a partition separation mechanism is involved rather than adsorption. A separation factor (alpha) of 1.17 was obtained with a relative separation (Rs) of 2.13. Column lifetimes were typically 6-8 months with daily use. Several standard pilocarpine-isopilocarpine mixtures and five commercially available ophthalmic solutions from different manufacturers were analyzed. The method was specific for pilocarpine and isopilocarpine in the presence of each other and pilocarpine acid and is a significant improvement over the nonspecific colorimetric methods of analysis.

Pilocarpine prodrugs I. Synthesis, physicochemical properties and kinetics of lactonization of pilocarpic acid esters.

Various alkyl and aralkyl esters of pilocarpic acid were synthesized and evaluated as prodrug forms for pilocarpine with the purpose of improving the ocular bioavailability of pilocarpine through increased corneal membrane permeability. The esters were found to undergo a quantitative cyclization to pilocarpine in aqueous solution of pH 3.5-10, the rate of cyclization being a function of the polar and steric effects within the alcohol portion of the esters. The rates of lactonization increased proportionally with the hydroxide ion activity over the pH range studied which is in accord with a reaction mechanism involving intramolecular nucleophilic attack of alkoxide ion on the ester carbonyl moiety. At pH 7.4 and 37 degrees C, half-times of lactonization ranging from 30 min (p-chlorobenzyl ester) to 1105 min (n-hexyl ester) were observed for the various esters. The esters are markedly more lipophilic than pilocarpine. The results suggested that the pilocarpic acid esters may be potentially useful prodrugs, especially when further derivatized to give in vitro stable pilocarpic acid diesters.

High-performance liquid chromatographic determination of pilocarpine in aqueous humor: derivatization by quaternization of methylimidazole tertiary amine group.

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.

HPLC method for the simultaneous determination of pilocarpine, isopilocarpine, pilocarpic acid and isopilocarpic acid.

A high-performance liquid chromatographic method suitable for the determination of pilocarpine, isopilocarpine, pilocarpic acid and isopilocarpic acid is described. The column used is a Spherisorb-CN bonded phase at 25 degrees C. An aqueous solution of triethylamine (0.1%, v/v) at pH 2.5 is used as mobile phase. Peak detection is by UV absorption at 220 nm. The elution orders are found to be pilocarpic-isopilocarpic acids with a resolution of 9 and K' of 5 and 6, respectively, isopilocarpine-pilocarpine with a resolution of 1.8 and K' of 13 and 13.5, respectively, the peaks being symmetrical.

The effects of a long-term powdered diet on the amounts of two principal neurotransmitters in the major salivary glands and on stimulated salivary secretion in mice.

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.