Return to Cover of Wave to the Future

ABSTRACT. The degree of crosslinking, measured by percentage of amoxicillin released from formaldehyde-stressed hard gelatin capsules (HGCs), was accurately predicted by near-infrared spectrophotometry (NIR). When HGCs were exposed to a 150 ppb atmosphere of formaldehyde for 2.25, 4.60, 9.42, 16.0 and 24.0 hours, the in vitro dissolution of amoxicillin from the capsules at pH 1.2 was found to decrease with increasing time of exposure to the formaldehyde atmosphere. Principal component (PC) regression was employed to analyze the spectra of the intact capsules. The set of principal components was formed by a linear transformation of the absorbance values at each wavelength scanned. A good correlation was established (r²=0.963) when PC values from the NIR spectra of the HGCs were regressed against percentage of amoxicillin dissolved at 45 minutes and several other times of dissolution, at pH 1.2.

KEY WORDS: gelatin; crosslinking; formaldehyde; dissolution; near-infrared spectrophotometry

¹ Division of Medicinal Chemistry and Pharmaceutics, College of Pharmacy, University of Kentucky, Lexington, KY 40536-0082.

² To whom correspondence should be addressed.

INTRODUCTION

Gelatin is valuable to the pharmaceutical industry because of the variety of formulations that can be incorporated into the hard gelatin capsule (HGC). Gelatin capsules are useful due to their strong yet flexible backbone, polished appearance, ability to hold dyes, and their solubility in aqueous solutions.(1)

The susceptibility of gelatin to chemical modification is well known. Of the variety of reagents capable of interacting covalently with gelatin, formaldehyde has been studied most extensively(2). Indeed, crosslinking of gelatin with formaldehyde has been used to produce enteric hard and soft capsules(1-4). However, when gelatin capsules intended for immediate release of their contents are exposed to trace levels of formaldehyde, the effect on in vitro dissolution rates may be adverse (1,5). For example, literature indicates that corn starch, a common pharmaceutical excipient, may incorporate a small amount of hexamethylenetetramine stabilizer (6-9), which upon hydrolysis, forms ammonia and formaldehyde(10).

Current research indicates that reduced in vitro dissolution rates, rather than decreased in vivo bioavailability of drugs, are the principal consequence emanating from the crosslinking of HGCs with low levels of formaldehyde (1,5). This phenomenon has been investigated by several workers (11-14), who utilized carbon-13 nuclear magnetic resonance (¹³C NMR) and ¹³C-enriched formaldehyde to show that gelatin reacts with formaldehyde through initial formation of amine methylols (carbinolamines) on lysine and arginine residues, with subsequent production of methylene bridges (crosslinks) between lysine and arginine (11-14). More importantly, the same studies showed that pancreatin, a proteolytic enzyme present in the gastrointestinal tract, can depolymerize crosslinked gelatin (11).

It has become evident that a nondestructive, noninvasive technique suitable for the detection of HGC crosslinking with low levels of formaldehyde could be of vital importance to the pharmaceutical industry. The present work introduces a novel concept of monitoring the extent of formaldehyde-induced crosslinking in HGCs by near-infrared (NIR) spectrophotometry, pharmaceutical applications of which include the determination of water uptake in tablets (15) and hard gelatin capsules(16), detection of tampering in capsules (17) and tablets (18), and analysis of degradation products (19). In the present study, the percent of amoxicillin dissolved at 45 minutes was used as a reference to correlate the degree of crosslinking with data obtained by NIR, which predicted dissolution of the drug from intact HGCs.

MATERIALS AND METHODS

Materials. The dissolution medium consisted of 0.2% wt/v sodium chloride and 0.7% v/v conc. HCl in distilled water. Gelatin capsules (size 2, clear, Lot No. 026539) were provided by Capsugel, Greenwood, SC. Formaldehyde, 37 wt% solution in water (stabilized with 15% methanol) was purchased from Aldrich, Milwaukee, WI. The formaldehyde reagent consisted of an aqueous solution of 2.0 M ammonium acetate (98%, Aldrich) and 0.020 M 2,4-pentanedione (>99%, Aldrich), adjusted to pH 6.0. The amoxicillin reagent consisted of 464 ml of 0.10 M citric acid and 536 ml of 0.20 M disodium hydrogen phosphate. The pH of this reagent was adjusted to 5.20 with the either pure phosphate or citric acid buffer. Fifteen ml of 0.0158 M copper sulfate pentahydrate (98%, Spectrum, Gardena, CA) was diluted to a volume of 1L with the pH 5.20 citric acid-phosphate mixed buffer. Amoxicillin trihydrate used for filling the stressed HGCs was Amoxil^® (SmithKline Beecham, Philadelphia, PA) brand formulation, while amoxicillin trihydrate (99% pure) utilized for preparation of standards was obtained from Sigma, St. Louis, MO.

Instrumentation. The fluorescence spectrophotometer used was a Hitachi model F-2000 (Hitachi, Tokyo, Japan) with a 150 W xenon short arc lamp. Fluorescence excitation and emission were at 410 and 510 nm, respectively. A Vanderkamp^® 600 (VanKel Industries, Inc., Edison, NJ) six-spindle dissolution tester was used to conduct dissolution tests. Apparatus II was used and the paddles rotated at 50 rpm. The solutions were maintained at 37°C. The NIR spectral data were collected by a Flex^® (Bran + Luebbe, Elmsford, NY) spectrophotometer. Reflectance values were obtained from capsules in a conical reflector over a range from 1445 to 2348 nm (16) using a 10 nm bandpass. The data were collected with an IBM PS/2 model 50 (International Business Machines, Armonk, NY) computer. Data analysis was performed with a Pentium (133 MHz) personal computer with software written in Speakeasy IV Eta (Speakeasy Corporation, Chicago, IL). The UV-visible spectrophotometer used was a Cary 2200 (Varian, Palo Alto, CA) with a 1.0 nm bandwidth and =320 nm.

Procedure. Twelve HGCs were exposed to a formaldehyde atmosphere by placing them on an aluminum wire screen support inside a vacuum desiccator containing no desiccant. The vacuum port was fitted with a rubber septum to allow introduction of formaldehyde and sampling of the air above the capsules. Exactly 1.0 µL of a solution of 37% formaldehyde in water was introduced into the desiccator chamber. Exactly one hour after formaldehyde introduction, 2.00 ml of the desiccator atmosphere was sampled through the vacuum port with a syringe (Hamilton, Reno, NV). The contents of the syringe were immediately introduced into 2.00 ml of 2,4-pentanedione reagent. The resulting solution was incubated at 37°C for 1 h and subsequently cooled to ambient temperature. Fluorescence was determined on the latter solution in a 1.00 cm cuvette. Fluorescence was likewise determined on standard solutions (50 to 400 ng/ml) of formaldehyde in distilled water, with 2.00 ml standard added to an equal volume of reagent prior to incubation. At 2.25 h after introduction of the formaldehyde into the desiccator chamber (1.25 h after atmosphere sampling), the gelatin capsules were removed from the formaldehyde chamber and were allowed to remain at ambient conditions for 72 h. Four additional experiments were conducted whereby HGCs were exposed to formaldehyde vapors for longer times: 4.60, 9.42, 16.0, and 24.0 h. The chamber atmosphere was always sampled 1 h after formaldehyde introduction. Upon removal of the HGCs from the chamber, they were filled with fresh amoxicillin formulation (Amoxil^®). As a control, six fresh, unstressed HGCs which had not been exposed to formaldehyde vapor were also filled with fresh amoxicillin drug blend.

The HGCs were placed in a 90° conical reflector and scanned in the NIR spectrometer. A steel wire support was used to position the capsule vertically within the cone.

The dissolution profile of amoxicillin from the HGCs was determined. Ten mL samples were taken from each dissolution vessel from 0 to 90 min at 15 min intervals, with fresh medium (pH 1.2, 10 mL) replacing the sampled medium. The concentration of amoxicillin in each of the sampled solutions was determined based on the spectrophotometric measurement of a copper-amoxicillin degradation product complex (20). Accordingly, a 0.500 mL aliquot of the dissolution sample was diluted into 5.00 ml of copper sulfate reagent. The resulting 5.50 ml solution was split into two equal portions. One of the two solutions was heated at 75°C for 30 min and, using an ice bath, was cooled to ambient temperature. The optical density of the latter solution was determined spectrophotometrically in a 1.00 cm cuvette, using the unheated solution as reference.

RESULTS AND DISCUSSION

A fluorimetric assay (21) was used to determine formaldehyde concentration inside the desiccator chamber in which HGC were incubated. Figure 1 represents the standard curve (r²=0.992), which included formaldehyde concentrations from 25 to 200 ng/ml (ppb). This represented a range of 50 - 400 ng/ml of formaldehyde standard solution as prepared, since the latter solutions were diluted with an equal volume of formaldehyde reagent. The fluorescence intensities of the solutions containing the 1 h desiccator-sampled atmosphere from each of the five experiment groups (2.25, 4.60, 9.24, 16.0, and 24.0 h exposure of 12 HGCs to formaldehyde vapor) were determined. From the standard curve (Figure 1), concentration values of the five experiment groups were extrapolated, the mean of which was 145.4 ng/ml of formaldehyde present in the desiccator atmosphere (145.4 ppb). This value agreed closely with the theoretical calculation of 151.7 ppb, in which the mass of formaldehyde (400.7 ng) was divided by the volume of the desiccator (2640 ml) into which the formaldehyde was introduced. The determination of formaldehyde in some methods of analysis may be complicated by its existence in both monomeric and polymeric forms. However, the assay utilized in the present work which involves the reaction of one mole ammonia, two moles 2,4-pentanedione, and one mole formaldehyde, requires the latter to be in its monomeric form. Since formaldehyde hydrate (monomer) is consumed in the aforementioned reaction, the equilibrium of formaldehyde in water shifts toward the monomer, as predicted by the law of mass action. Therefore, whether the formaldehyde sampled from the desiccator chamber atmosphere was in polymeric or monomeric form is of little consequence, because the reaction equilibrium assured that all formaldehyde which was present in the aqueous solution of ammonia and 2,4-pentanedione would be available for assay. In Figure 2, the obtained dissolution profiles (pH 1.2 medium) for amoxicillin from HGCs are shown. With increasing exposure time (up to 16 h)of the HGCs to the 150 ppb formaldehyde atmosphere, the percentage of amoxicillin dissolved at any time point decreased
(Figure 2 andFigure 3. However, the dissolution curves of HGCs, exposed to either 16 or 24 h of 150 ppb formaldehyde, were virtually superimposable (Figure 2). The similarity of the two dissolution curves (16 and 24 h, Figure 2) may be the result of the fact that, given ample time (16 or more hours), the formaldehyde (150 ppb) inside the desiccator was the limiting reagent with respect to the reactive sites, -amino and guanidino functionalities of lysine and arginine residues, respectively, within the gelatin polypeptide (11-14). Further, the presence of significant amoxicillin in the 90 minute dissolution samples from the HGCs exposed for 16 and 24 h to 150 ppb formaldehyde (~40% amoxicillin dissolved at 90 minutes, respectively, Figure 2) supports the hypothesis that not all of the reactive sites on the gelatin molecule had been modified by formaldehyde (1,11). In general, reaction of gelatin with excessive quantities of formaldehyde produces crosslinked, hydrophobic peptide chains (11) of increased average molecular weight which are no longer soluble in aqueous media and through which drug is not able to migrate.

Figure 4 is the NIR reflectance spectra of fresh and formaldehyde-stressed HGCs. Because the NIR spectra contained baseline shifts which were both additive and multiplicative, simple calibration methods were not suitable for data analysis. Instead, principal component regression (PCR) was used to analyze the NIR spectra(22). PCR reduces the dimensionality of a data set, in which there are a large number of correlated variables, by linear transformation into a new set of uncorrelated variables called principal components (PCs). The PCs are structured so that the first few retain most of the variation contained in all the original variables. Thus, the first PC contains information from the constituent which contributes most to the total NIR spectral variation of the data. The second PC is orthogonal to the first and weights most heavily the wavelengths which contribute the most variation to the spectra after removal of the first PC. Progressively smaller contributions to the spectral variation are described by additional, orthogonal PCs. Most of the variation in the NIR spectra (96%) was described by six principal components. Examination of the transformation matrix connecting wavelength and PC hyperspace showed that the signal on the first PC was due to water. The latter has NIR absorbances at 1450 and 1940 nm, and these peaks' intensities were found to decrease with the capsules' increasing time spent in the formaldehyde atmosphere (Figure 4). Since water is a product of many crosslinking phenomena, including that which occurs between formaldehyde and gelatin, the exclusion of water from the HGC shell with increasing time of exposure to formaldehyde (NIR spectra, Figure 4) substantiates the idea that covalent crosslinking is indeed occurring. The first six PCs of the capsule spectra were regressed against percentage of amoxicillin dissolved at 45 minutes from the HGCs exposed to 150 ppb formaldehyde (Figure 5). The NIR spectra of the capsules show a strong linear correlation to actual dissolution of amoxicillin from the formaldehyde-stressed HGCs (Figure 5, SEE (standard error of estimate) = 6.23%, SEP (standard error of prediction) = 7.67%). The dissolution experiments to which the NIR spectra were correlated were conducted in acidic medium because of amoxicillin's superior solubility at low pH. Thus, correlation was established not between the inherent solubility of drug and the NIR spectral data, but rather, between the dissolution from the formaldehyde-stressed capsule shell and its respective NIR spectrum. The effect of acid on the methylene crosslink, which covalently links the -amino nitrogen of lysine with the guanidino nitrogen of arginine to form an aminal (1), has not been established definitively. Recently, it has been demonstrated, using ¹³C-NMR and ¹³C-enriched formaldehyde, that crosslinking of aqueous gelatin solutions does not occur as readily at pH 2 as in neutral (pH 7) or even alkaline (pH 13) media (11). This suggests that the methylene crosslink (aminal) in gelatin behaves similar to an acetal and thus, may be susceptible to acid hydrolysis. Correlation of dissolution data with NIR spectra may therefore have been further improved had dissolution occurred in neutral medium, where the formaldehyde-produced methylene crosslink would be less likely to undergo hydrolysis.

After removal of the first principal component, which was determined by matrix transformation to be due to water, the capsule spectra were reconstructed from PCs where crosslinking appears, with wavelength plotted against standard deviation units (Figure 6). The NIR spectra of fresh control HGCs was selected as the zero standard deviation point on the graph in Figure 6. At 1734 and 1782 nm, most of the spectra of the formaldehyde-stressed capsules deviate from spectra of control (unstressed) HGCs in a positive direction (Figure 6). Conversely, the capsule spectra show both positive and negative deviations from spectra of unstressed HGCs at 1760 nm (Figure 6). Infrared spectra (2500-15000 nm) are the result of rhythmical changes in the dipole moment of molecules which absorb vibrational energy. These fundamental molecular vibrations, representing either bond stretching or bending, give rise to harmonic and overtone frequencies which are observed in the near-infrared (1000-2500 nm) region. Thus, simple IR spectra (a few absorption frequencies) often give rise to more complicated (many absorption frequencies) NIR spectra. More information can therefore be obtained about chemical environment from the NIR spectral region than from the IR region. The chemical bonds broken and formed during formaldehyde-induced crosslinking reactions in gelatin absorb vibrational energy and give rise to fundamental IR frequencies as well as harmonics in the NIR region.

Since IR absorption frequencies are well-characterized with respect to functional groups, connection of the NIR spectra of the formaldehyde-stressed HGCs to IR absorption frequencies would permit explanation of gelatin crosslinking mechanism. This approach is currently being investigated in these laboratories.

CONCLUSIONS

The degree of crosslinking, measured by dissolution of amoxicillin from intact hard gelatin capsules exposed to low levels of formaldehyde was shown to be predictable using NIR spectrophotometry. When NIR was coupled to principal component analysis, it was possible to distinguish which wavelength(s) contributed most to overall spectral variation of the gelatin capsules. Water content of the capsules was the largest determinant in the variation between HGC spectra at each exposure time, with NIR absorbances of the capsules at 1450 and 1949 nm decreasing with increasing exposure time to formaldehyde. That there are chemical reactions which contribute to the decreased ability of the formaldehyde-stressed HGCs to imbibe water was supported by NIR spectral reconstruction from principal components where crosslinking occurred. The spectrophotometric method described here permits a rapid and nondestructive analysis of intact capsules with or without drug product inside. Using a relatively small set of standards, capsules may be rapidly categorized according to their (NIR) predicted release rate of drug content, and those which fail to meet predetermined criteria may be singled out. Furthermore, these laboratories are currently investigating the coupling of NIR imaging and laser technologies, which may permit more efficient, on-line prediction of drug release from dosage formulations.

REFERENCES

1. G. A. Digenis, T. B. Gold, and V.P. Shah. Cross-linking of gelatin capsules and its relevance to their in vitro-in vivo performance. J. Pharm. Sci. 83:915-921 (1994).

2. B. E. Jones. History of the hard gelatin capsule, in Hard Capsules-Development and Technology; K. Ridgway, (ed.), The Pharmaceutical Press, London, 1987, p. 11.

3. B. T. Palerno and S. C. McMillion. Method of treating gelatin capsules and product resulting therefrom. U.S. Patent 2 578 943, 1951.

4. E. A. Swinyard and W. Lowenthal in Pharmaceutical necessities in Remington's Pharmaceutical Sciences, 18th edition; A. R. Gennaro, (ed.), Mack, Eaton, PA, 1990, p. 1306.

5. G. A. Digenis and T. B. Gold. Chemistry of gelatin cross-linking. Pharm. Res. 11:S146, PT 6064 (1994).

6. E. Doelker and A. C. Vial-Bernasconi. Interactions contenant-contenu au sein des capsules gelatineuses et evaluation critique de leurs effects sur la disponibilite des principes actifs, S. T. P. Pharma. 4:298-306 (1988).

7. H. Mohamad, R. Renoux, S. Aiache and J.-M. Aiache. Study on the biopharmaceutical stability of medicines: application to tetracycline hydrochloride capsules I. In vitro study. S. T. P. Pharma. 2:531-535 (1986).

8. H. Mohamad, R. Renoux, S. Aiache, J.-M. Aiache and J.-P. Kantelip. Study on the biopharmaceutical stability of medicines: application to tetracycline hydrochloride capsules II. In vivo study. S. T. P. Pharma. 2:630-635 (1986).

9. H. Mohamad, J.-M. Aiache, R. Renoux, P. Mougin and J.-P. Kantelip. Study on the biopharmaceutical stability of medicines: application to tetracycline hydrochloride capsules IV. Complementary in vivo study. S. T. P. Pharma. 3:407-411 (1987).

10. A. Martin, Anti-infective agents in Wilson and Gisvold's Textbook of Organic Medicinal and Pharmaceutical Chemistry, 9th edition, Jaime N. Delgado and W. A. Remers, Eds., J. B. Lippincott Co., NY, p 157 (1991).

11. T. B. Gold, S. L. Smith and G. A. Digenis. Studies on the influence of pH and pancreatin on ¹³C-formaldehyde-induced gelatin cross-links using nuclear magnetic resonance. Pharm. Dev. Tech. 1:21-26 (1996).

12. S. K. Taylor, F. Davidson and D. W. Ovenall. Carbon-13 nuclear magnetic resonance studies on gelatin crosslinking by formaldehyde. Photogr. Sci. Eng. 22:134-138 (1978).

13. K. Albert, B. Peters, E. Bayer, U. Treiber and M. Zwilling. Crosslinking of gelatin with formaldehyde; a ¹³C NMR study. Z. Naturforsch. 41b:351-358 (1986).

14. K. Albert, E. Bayer, A. Worsching and H. Vogele. Investigation of the hardening reaction of gelatin with 13C labeled formaldehyde by solution and solid state 13C NMR spectroscopy. Z. Naturforsch. 46b:385-389 (1991).

15. M. S. Kamat, R. A. Lodder and P. P. DeLuca. Near-infrared spectroscopic determination of residual moisture in lyophilized sucrose through intact glass vials. Pharm. Res. 6:961-965 (1989).

16. R. G. Buice, Jr., T. B. Gold, R. A. Lodder and G. A. Digenis. Determination of moisture in intact gelatin capsules by near-infrared spectrophotometry. Pharm. Res. 12:161-163 (1995).

17. R. A. Lodder, M. Selby and G. M. Hieftje. Detection of capsule tampering by near-infrared reflectance analysis. Anal. Chem. 59:1921-1930 (1987).

18. R. A. Lodder and G. M. Hieftje. Analysis of intact tablets by near-infrared reflectance spectroscopy. Appl. Spectrosc. 42:556-558 (1988).

19. J. K. Drennen and R. A. Lodder. Nondestructive near-infrared analysis of intact tablets for determination of degradation products. J. Pharm. Sci. 79:622-627 (1990).

20. J. W. G. Smith, G. E. de Grey and V. J. Patel. The spectrophotometric deter- mination of ampicillin. Analyst 92:247-252 (1967).

21. S. Belman. The fluorimetric determination of formaldehyde. Anal. Chim. Acta. 29:120-126 (1963).

22. J. E. Jackson. A User's Guide to Principal Components, John Wiley & Sons, Inc., New York, 1991.

23. R.G. Buice, Jr., T.B. Gold, R.A. Lodder and G.A. Digenis. The use of near-infrared imaging for determination of uniformity of hydration and extent of formaldehyde induced cross-linking in intact hard gelatin capsules. Pharm. Res. 12:S36 (1995).

Table 1: NIR wavelengths (in nm) over which the gelatin capsules were scanned.

Figure Captions

Figure 1. Standard curve showing fluorescence intensity vs. concentration (ng/ml) of formaldehyde in water.

Figure 2. Dissolution curves of amoxicillin (pH 1.2 medium) from hard gelatin capsules exposed for various times (0, 2.25, 4.60, 9.42, 16.0, and 24.0 h) to 150 ppb formaldehyde atmosphere.

.Dissolution of amoxicillin from HGCs at 45 minutes vs. time spent in 150 ppb formaldehyde.

Figure 4. NIR spectra of 18 HGCs (three for each of the six exposure times: 0, 2.25, 4.60, 9.42, 16.0, 24.0) exposed to 150 ppb formaldehyde atmosphere.

Figure 5. Correlation of actual dissolution of amoxicillin from HGCs exposed to 150 ppb formaldehyde to dissolution predicted from NIR spectral data. Principal component values from NIR spectra of the HGCs were regressed against percentage of amoxicillin dissolved (pH 1.2 media) from the capsules at 45 minutes (SEE=6.48%, SEP=7.67% from cross validation). The cross validation samples are shown superimposed on calibration line, with error bars representing range (extreme values).

Figure 6. NIR spectra reconstructed from PCs where crosslinking appears. PCs were eliminated which, after examination of transformation matrices connecting PCs and NIR spectral wavelength, were determined to be due to water absorbance. The y-axis is standard deviation of the formaldehyde-exposed capsules from unstressed, control capsules (0 h of exposure to the formaldehyde atmosphere).

Return to Wave of the Future Table of Contents

Return to Cover of Wave to the Future