Spectrophotometric and spectrofluorimetric methods for determination of Racecadotril.

Two accurate and sensitive spectrophotometric and spectrofluorimetric methods were developed for determination of Racecadotril. In the first method reduction of Fe3+ into Fe2+ in presence of o-phenanthroline by Racecadotril to form a stable orange-red ferroin chelate [Fe-(Phen)3]2+ was the basis for its determination. The absorbance at 510 nm was measured and linear correlation was obtained in the concentration range of 2.5-25 µg mL(-1). In the second method the native fluorescence of Racecadotril in acetonitrile solvent at λ=319 nm when excitation was at 252 nm is used for its determination. Linear correlation was obtained in the concentration range of 50 to 500 ng mL(-1). The proposed methods were applied for determination of Racecadotril in bulk powder with mean accuracy of 100.39±1.239 for the spectrophotometric method and 100.09±1.042 for the spectrofluorimetric method. The proposed methods were successfully applied for determination of Racecadotril in its pharmaceutical dosage form.

Stability-indicating methods for the determination of racecadotril in the presence of its degradation products.

Three stability-indicating methods were developed for the determination of racecadotril (RCT) in the presence of its alkaline degradation products. The first was an HPLC method in which efficient chromatographic separation was achieved on a C18 analytical column and a mobile phase of acetonitrile-methanol-water-acetic acid (52:28:20:0.1, v/v/v/v). Linearity was obtained in the range of 4-40 microg/mL with mean accuracy of 99.5 +/- 0.88%. The second method was a densitometric evaluation of thin-layer chromatograms of the drug using a mobile phase of isopropanol-ammonia (33%)-n-hexane (9:0.5:20, v/v/v). The chromatograms were scanned at 232 nm, a wavelength at which RCT can be readily separated from its degradation products and determined in the range of 2-20 microg per spot with mean accuracy of 99.5 +/- 0.56%. The third method is based on the use of first-derivative spectrophotometry (D1) at 240 nm, and the drug was determined in the range of 5-40 microg/mL with mean accuracy of 99.2 +/- 1.02%. The three methods provided satisfactory recovery of the intact drug (100.8 +/- 0.82, 100.4 +/- 0.55, and 99.9 +/- 0.72%, respectively) in the presence of up to 90% of its degradation products. Determination was also successful when analyzing RCT in a formulation in the form of acetorphan packets. Results were statistically analyzed and found to be in accordance with those given by a reported method.

Structural studies of racecadotril and its process impurities by NMR and mass spectroscopy.

Three unknown impurities in racecadotril bulk drug at levels below 0.5% were detected by simple reverse phase isocratic high performance liquid chromatography (HPLC). Structures for these impurities were proposed by molecular ion information and their fragmentation pattern obtained by LC-MS and these impurities were confirmed by NMR spectroscopy. The impurities I, II and III were characterized as benzyl 2-methyl carboximido acetate, benzyl 2-phenyl ethyl carboximido acetate, and benzyl 2-(1-benzyl vinyl carboximido) acetate. These structures were further confirmed by co-injecting of synthetic standards of impurities with racecadotril. The mechanism of the formation of these process related impurities is discussed.

A stability study involving HPLC analysis of aqueous thiorphan solutions in the presence of human serum albumin.

The stability of thiorphan (1.0 mg/ml) in normal saline containing 1% human serum albumin (HSA) was determined in order to find the most appropriate storage conditions. Direct liquid chromatographic analysis of this solution was feasible through the use of a micellar chromatographic system and proved to be stability indicating. During 8 weeks the percentages of the initial thiorphan concentration remaining after storage at 4, 20, 30, and 50 degrees C were determined. An Arrhenius plot was composed using the rate constants of thiorphan degradation at these temperatures. The thiorphan solution was stable for at least 2 months if stored at -20 degrees C. Taking into account the oxidative degradation of about 7% after thawing, we determined that the solution can be kept in a refrigerator for 4 days. Storage at room temperature should be limited to 1 day. By identification of the degradation products it could be concluded that thiorphan is degraded mainly via oxidation forming disulfides. Therefore, it is recommended that the solvent be purged with nitrogen before thiorphan is dissolved.

Stereoselective protection of exogenous and endogenous atrial natriuretic factor by enkephalinase inhibitors in mice and humans.

We compared the relative potencies of sinorphan and retorphan, the S- and R-enantiomers of acetorphan a potent inhibitor of enkephalinase (EC 3.4.34.11), to inhibit membrane metalloendopeptidase in vivo and to protect exogenous and endogenous ANF after oral administration. In mice, sinorphan was 2-3 fold as potent as retorphan in inhibiting the specific in vivo binding of [3H]acetorphan to kidney enkephalinase. The same potency ratio was found for the enhancement of trichloroacetic acid-precipitated radioactivity in kidneys of mice that had received 125I-ANF, which is used as a test for the protection of the hormone against inactivation in vivo. In nine healthy human volunteers who had received a low oral dosage of sinorphan or retorphan in a double-blind, placebo-controlled, randomized trial, sinorphan was also 2-3 fold more potent than retorphan in inhibiting plasma enkephalinase activity. These effects were accompanied by a related rise in plasma ANF immunoreactivity, which also reflected the difference in the effectiveness of the two compounds. Sinorphan was also more potent than retorphan in enhancing urinary cyclic GMP excretion and sodium excretion in five of these subjects. These data indicate that, in humans as in rodents, enkephalinase plays a crucial role in the inactivation of ANF, its partial inhibition in vivo being accompanied by a significant protection of the exogenous or endogenous hormone as well as by typical ANF-like responses. Thus orally administered sinorphan appears to be a promising compound for therapeutic use in cardiovascular and renal diseases in which ANF has been postulated to exert beneficial effects.