Determination of tropicamide and its major impurity in raw material by the HPLC-DAD analysis and identification of this impurity using the off-line HPLC-FT-IR coupling.

This study presents TLC, HPLC-DAD and HPLC-FT-IR analyses concerning the detection, identification and determination of organic impurities of commercial tropicamide ((R,S)-N-ethyl-3-hydroxy-2-phenyl-N-(4-pyridylmethyl)propanamide) as a medical substance designed for the production of eye drops. In the examined samples from several random production batches, only one impurity (defined by Ph. Eur. 6th Ed.) was discovered in the amount sufficient for the quantitative analysis. On the basis of comparison of retention times, UV and IR spectra of the impurity and its synthesized standard, this impurity was identified as apotropicamide (N-ethyl-2-phenyl-N-(4-pyridylmethyl)prop-2-enamide). For the chemical identification of organic compounds occurring in the tropicamide samples, an off-line coupling of HPLC with FT-IR was used. The structure of a standard of apotropicamide was confirmed by (1)H NMR and (13)C NMR analysis. Validation of the HPLC-DAD method of determination both tropicamide and apotropicamide in the tropicamide medical substance was performed. This method is suitable for use in the quality control of tropicamide during its production.

Determination of tropicamide in pharmaceutical formulations using high-performance liquid chromatography.

An isocratic, reversed-phase liquid chromatographic method was developed for determination of tropicamide using atropine as an internal standard in a pharmaceutical dosage form. Tropicamide and atropine sulfate were separated using a microBondapak ODS (C18) column by isocratic elution of mobile phase with flow rate of 2.0 ml/min. The mobile phase composition was methanol-50 mM phosphate buffer (pH 4; 30:70, v/v). The eluate was monitored at 257 nm with detector range setting fixed at 0.01 AUFS. Under these conditions, the retention times were 4.81 min for atropine and 11.89 min for tropicamide. The standard calibration curve was linear over a sample concentration range from 2 to 300 microg/ml, with limit of detection of 0.15 microg/ml. The assay linearity was good (typically r2 = 0.9992) and the standard curves were linear in the detection range. The precision of the method (expressed by relative standard deviation) and the accuracy (mean error in percent) were <5% for both intra- and inter-day assays. Recovery at 80-120% of labeled claim ranged from 98.4 to 100.7% for tropicamide. The proposed method was satisfactorily applied to the determination of tropicamide in pharmaceutical preparation and stability indicating studies.

Stable isotopic characterization of active pharmaceutical ingredients.

Stable isotopic characterization or "fingerprinting" of active pharmaceutical ingredients (APIs) is a highly-specific means of defining the provenance of these pharmaceutical materials. The isotopic analysts in this study were provided with 20 blind samples of four APIs (tropicamide, hydrocortisone, quinine HCL, and tryptophan) from one-to-five production batch(es) from one-to-five manufacturer(s). Only the chemical identity of the APIs was initially provided to the isotopic analysts. Depending on the API chemical composition, isotopic ratios of either three or four elements (13C/12C, 15N/14N, 18O/16O, and/or D/H) were measured by either elemental analyzer/isotope ratio mass spectrometry (EA/IRMS: carbon (delta13C) and nitrogen (delta15N)) or by thermal conversion-EA/IRMS (TCEA/IRMS; hydrogen (deltaD) and oxygen (delta15N)); in all cases, the isotopic results are reported in the standard delta-notation which represents part-per-thousand () variations from the isotopic ratios of international standards. The stable isotopic analyses of the four suites of APIs spanned broad ranges in absolute value (deltadelta) and in estimated specificity (a product of dynamic ranges (DR, unitless)--note that these are upper limits of specificity because some of these isotope values may be partially interdependent). The five samples of tropicamide from one production batch and one manufacturer demonstrated the narrowest ranges (deltadelta13C=0.13 ; deltadelta15N=0.52 ; deltadelta18O=0.24 ; deltadeltaD=2.8 ) and the smallest specificity of 1:30.9. By contrast, the five samples of tryptophan that came from five separate manufacturers had some of the widest isotopic ranges observed (deltadelta13C=21.32 ; deltadelta15N=5.26 ; deltadelta18O=22.07 ; deltadeltaD=55.3 ) and had the largest specificity of 1:19.6 x 10(6). The isotopic provenance of the four suites of APIs readily emerged from bivariate plots of selected isotope ratios, particularly deltaD versus delta18O.

[Enantioseparation of tropicamide by capillary electrophoresis with square wave amperometric detection].

An enantioseparation method for tropicamide by high performance capillary electrophoresis with square wave amperometric detection (SWAD) was developed. The enantiomers of tropicamide were baseline separated in 16 min with an uncoated fused-silica capillary (75 microm i.d. x 50 cm) under the optimum conditions: 7 mmol/L Tris-10 mmol/L citric acid-2 mmol/L H3BO(3)-15 mmol/L beta-cyclodextrin (beta-CD) (pH 3.0) as background electrolyte, SWAD balance potential (E(b)) + 0.80 V (vs. Ag/AgCl), separation voltage 15 kV, injection height 20 cm, and injection time 10 s. The calibration curve of the enantiomers showed good linearity in the range from 5 micromol/L to 750 micromol/L with detection limit of 2 micromol/L. The average recoveries of added standards were 96% - 103%. The effects of the concentrations of beta-CD and the boric acid, the pH of the background electrolyte on resolutions (Rs) of the enantiomers were discussed in details. The proposed method was applied to the determination of a commercial tropicamide eye-drops sample without pre-treatment, and satisfactory results were obtained.

High-performance liquid chromatographic determination of phenylephrine and tropicamide in human aqueous humor.

A high-performance liquid chromatographic method for the simultaneous determination of phenylephrine and tropicamide in human aqueous humor was developed. After centrifugation, an aliquot of the supernatant was injected onto the column and the eluent was monitored at 280 nm then 254 nm after 5 min. Separation was performed on a CN column with 0.01 M Pic B8 (octane sulfonic acid)-acetonitrile (65:35, v/v) as mobile phase. The standard curves were linear in the detection range. The precision of the method (expressed by relative standard deviation) and the accuracy (mean error in per cent) were <5% for both intra- and interassays.

[Contribution to the stability of tropicamide solutions (author's transl)]. / Beitrag zur Stabilität von Tropicamidlösungen.

The course of the degradation of tropicamide solutions was elucidated. Thus, inter alia, intramolecular elimination of water leads to a compound comparable to apoatropine that the authors designated by atropicamide. Furthermore, methods are described that permit (after thin-layer chromatographic repartition) to detect and to determine quantitatively the active principle in the presence of its degradation products. Tropicamide is stable in aqueous solution; eye-drops prepared from such a solution (Mydrum) are stable for no less than 5 years.