Studies on the Influence of pH and Pancreatin on 13C-Formaldehyde-Induced Gelatin Cross-Links Using Nuclear Magnetic Resonance

Abstract

Carbon-13 nuclear magnetic resonance (13C-NMR) and 13C-enriched formaldehyde (13CH2O) were utilized to observe cross-linking in gelatin. Thus, when a 6% solution of gelatin in water was treated with 2000 ppm 13CH2O at 20 degrees C, the 15 hr 13C-NMR spectrum of the crosslinked gel showed peaks representing carbinolamines (methylols) of arginine and lysine, as well as a peak ascribed to a methylene cross-link between arginine and lysine. Similar results were obtained when these cross-linking reactions were conducted using only 100 ppm 13CH2O. When pancreatin (1% w/v) was added to the solution of 6% gelatin cross-linked with 2000 ppm 13CH2O, the gel began to revert to a clear fluid solution. After incubation for 24 hr at 37 degrees C, the 13C-NMR spectrum of this solution confirmed the presence of the methylols of lysine and arginine, and the lysine-arginine cross-link. When 13CH2O (2000 ppm) was added to a 6% solution of gelatin at pH 13.0, the arginine methylol and the lysine-arginine crosslinks were produced. The 13CH2O-induced crosslinking of gelatin at pH 2.0, however, yielded the lysine methylol as the sole product.

Dissolution of Gelatin Capsules: Evidence and Confirmation of Cross-Linking

“…The peaks at 800, 1020, and 1095 cm -1 were assigned to the out-of-plane bending of the ring-like formation of arginine methylol, the C-O stretch of the lysine methylol, and the C-O stretch of the arginine methylol. These observed spectral changes conform to the reaction intermediates proposed in previous works based on NMR experiments (52,53). Further spectral changes for the capsules exposed to the aldehyde for longer durations were reported to be mostly from unreacted aldehydes absorbed to the gelatin.…”

“…The sensitivity of the 13 C-NMR methodology to determine cross-linking should be assessed carefully. Gold et al correlate the degree of the cross-linking caused by adding 100 ppm formaldehyde to a 6% solution of gelatin in water (53). It is still challenging to determine the level of cross-linking that made a drug product in gelatin capsule just fail the USP Tier-1 dissolution test and provide a confirmative evidence of cross-linking as expected by the USP.…”

“…Gold et al also performed solution 13 C-NMR studies to investigate formaldehyde-induced cross-linking in gelatin, with a focus on the influence of pH and the enzyme pancreatin (53). Under alkaline conditions (i.e., pH 7.2 and 13.0), when a 6% solution of gelatin in water is treated with 2000 ppm or 100 ppm 13 C-enriched formaldehyde, the unprotonated ɛ-amino functional group in lysine and the guanidine functional group in arginine reacted with the formaldehyde resulting in cross-linking.…”

Soft Gelatin Capsules (Softgels)

“…196,197,[206][207][208][209][210][211] Both of these amino acids, lysine and arginine, have long, reactive side chains that can extend farther out from the polypeptide chain than other groups and thus can participate more easily in the inter and intra molecular reactions. Possible mechanism of formation of methylols of lysine and arginine and subsequent cross-links formation in gelatin (adopted from Taylor et al, 207 Albert et al, 209 Gold et al 210 ). Possible mechanism of formation of methylols of lysine and arginine and subsequent cross-links formation in gelatin (adopted from Taylor et al, 207 Albert et al, 209 Gold et al 210 ).…”

“…6). 210 In contrast, since both of these functional groups exist primarily in their unprotonated forms at alkaline pH, they can potentially react with aldehydes, resulting in crosslinking. [207][208][209] As the formation of arginine methylols was identified to coincide with the initiation of gelatin cross-linking reactions, its formation was hypothesized to be the rate limiting step in the cross-linking of gelatin.…”