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ANALYTICAL CHEMISTRY, VOL. 57. NO. 1, JANUARY 1985
In today's regulatory environment, the drug development process is a costly and time-consuming endeavor. Pharmaceutical companies are attempting to implement programs that will accelerate this process if possible or at least prevent avoidable delays. An important contribution to this ef. fort is achieved via preformulation testing, which is conducted during the early stages of the development process. Preformulation testing encompasses those studies that define the physical and chemical properties of a new drug substance that are critical to the development of stable, efficacious dosage forms. One of the most challenging activi. ties of the preformulation scientist is the identification of degradation pathways, i.e., oxidation, hydrolysis, etc., and the structure elucidation of degradation products of new drug entities. This information is vital both for developing dosage forms possessing optimum stability characteristics and for estahlishing analytical methodology capable of assaying both the intact drug and its degradation products. To accomplish these objectives, a deliberate attempt is made to degrade the drug substance, either partially or totally, by subjecting the compound to a variety of stress conditions such as elevated temperature, high relative humidity, intense light, a hostile chemical environment, or a comhination of these conditions. The resulting mixture is analyzed for evidence of decomposition and, if positive, an effort is made to isolate and identify the degradation product(s). Generally, the 0003-2700/84/0351-068A$O 1.50/0
@ 1984 American Chemical Society
Michael J. Frank Hoechst-Roussel Pharmaceuticals Inc. Somerville, N.J. 08876
DegmdationStudies inthehy
preformulation analyst need apply only routine analytical techniques such as thin-layer chromatography (TLC) or ultraviolet-visible (UV-VIS) spectrophotometry to obtain confirmation that decomposition has occurred. The subsequent identification of the degradation product(s) may he a simple or formidable task depending on the complexity of the parent compound as well as the information available in the literature. In all prohability, sophisticated instrumental techniques including nuclear magnetic resonance spectrometry (NMR) and mass spectrometry (MS) will be required. The.compound of interest-clohazam (I), 7-chloro-1-methyl-5-phenyllH-1,5-henzodiazepine-2,4(3H,5H)dione, a tranquilizer (see Figure 1)was submitted for a stability investigation. The drug, which was synthesized in 1969 ( I ) , had been investigated extensively, but a detailed study of the stability of the compound when subjected to acid and base hydrolysis
T o determine the acid and hase hydrolysis products of clobazam (I),solutions of the compound in 0.1 N HCI and 0.1 N NaOH were refluxed for 1 h to facilitate the decomposition process. Because of the Door auueous soluhility of the drug, the acidand hase solutions were actually hydroalcoholic mixtures prepared by mixing equal volumes of water and methanol. After reflux, a substantial amount of precipitate was present in the hase sample, hut the acid solution remained clear.
had not been reported in the literaThe analytical approach ture. The fate of a comDound of simiThe nniilytiral iipproach selected t u liir structure, 1.5-dimethyl-]H-1.5resche the problem at hand involved hen~[rdia2euine-;'..Ir3H,SH,-dioni,( I I J , the following sequence: after boiling in dilute sulfuric acid had Verification of decomposition upon been studied previously (21, and the reflux via TLC and UV spectrophohydrolysis product was identified as 1,2,3-trimethyl-2-hydroxy-2,3-dihy- tometry. Separation, purification, and identidrohenzimidazole (1111,in which N [(2-methyl-amino)-phenyl]-N-methyl- fication of solid residues using routine analytical techniques as well as MS acetamide (IV) was postulated as an and NMR. intermediate which cyclized to give Separation of soluble decomposition the final product (111).A subsequent products from the parent compound investigation on a series of o-phenylby appropriate extraction techniques enediamine derivatives (31, including and confirmation of the separation by (IV), led some authors ( 4 , 5 )to conhigh-pressure liquid chromatography clude that the actual product of the (HPLC) and UV spectrophotometry. hydrolysis of (11) was indeed comIdentification of the isolated deuound (IV). Based on the chemical cumls~sitionproducts by comparing structure ~ r (IJ, i n theoretical eslimatheir sulution c~hemiitr?.and 1IV and tiun of its putcntiol for hydrulytic degmass spectral prarpvrtics with tnuw ut rad;it:on h3d heen propusrd 16,. hut irnicturally related ccimpinmd~. concrete experimental data were lackThe iaci that decumpusitiim upun ing. I n summary. the literature iugrrtlux does indeed occur \!ai verified gestcd that acid hydrdysii should hy (I\' spertropholumt.try ,Figurrs 2 yield an o-phenylenediaminespecies mid ;II. A cornparism d'the sprrtra in and hase hydrolysis a compound ronANALYTICAL CHEMISTRY, VOL. 5 7 , b 0 1, JANUARY 1985
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69A
8 0.1
m
8
/Ocl---. cHa
C1
6
i
0.2
0.:
250
V
3w
350
Wavelength (nm) Figure 2. Absorption spectrum of clobazam in 0.1 N hydroalcoholic NaOH (a) Initial: (b) lh reflux-supernatant: flux-precipitate
I Ja
CI
0
M
X C%
I
c1'
0
(c) 14 re-
I
250
iH,
350
Wavelength (nm)
xu
XI
303
Figure 3. Absorption spectrum of CID bazam in 0.1 N hydroalcoholic HCI
Figure 1. Structures of pertinent compounds
(1)IniIUl;
each figure clearly indicates that clobazam (I) degrades in both basic and acidic media as evidenced by the dramatic change in the UV absorption svectra of the solutions before and &ter reflux. The twofold task of determinine the number of components present in-the base refluxed supernatant and of identifying the corresponding precipitate formed during reflux was performed first. Preliminary experiments with TLC indicated that the supernatant contained only two species, Le.,
magq spectrum obtained hy both techniques indicated a molecular weight of 274 and a molecular formula of C16Hl5C1N*0. Analysis of the fragmentation pattern as well as a knowledge of possible degradation products suggested that the decomposition product could be one of two compounds-N-[(4-chloro-2-phenylamino)phenyl]4"methylacetamide (V) or N-[(5-chloro-2-methylamino)phenyl]-N-phenyl-acetamide(VI). Since MS failed to differentiate conclusively between the two possible
70A
clohazam and a compound that corresponds to the precipitate. HPLC confirmed the results obtained by TLC that the only basic decomposition product of clohazam (I) isthe precipitate formed on reflux. In addition, the precipitate was found to he pure by hoth chromatographic techniques. T o determine the molecular weight of the precipitate, the mass spectrum of the material was obtained by elec tron impact mass spectrometry (EIMS) as well as chemical ionization mass spectrometry (CI-MS). The
ANALYTICAL CKMISTRY. VOL. 57, NO. 1, JANUARY 1985
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Table 1. Proton NMR data for clobazam base hydrolysis species 6
6
1.9
2.1
1.92
3.2
2.7
3.22
5.4
3.3
0
11 +-CH3
I -N--CHJ
I -N--H
(very weak) Aromatic
7.0-7.5
3:3:1:8
structures, the sample was subjected to NMR. Using proton magnetic resonance data for structurally similar compounds, it is possible to predict the characteristics of the NMR spectrum of compounds (V) and (VI) and by comparing the predicted data with the experimental results to arrive at a structure for the base decomposition product of clobazam (I).The NMR data are summarized in Table I. The data show that the NMR spectrum of the precipitate and that predicted for compound (VI are nearly identical, thus verifying the structure of the base hydrolysis product. Finally, UV spectrophotometry provides additional evidence that the base hydrolysis product conforms to compound (V). From the literature, compounds similar to (V) exhibit ahsorhance maxima in the 280-290-nm region of the spectrum, whereas com-
I .I
0.1
0.:
250
300
350
Wavelength (nm) owe 4. Absorption spectrum of the :id hydrolysis product of clobazam in 1 N hydroalcoholic HCl 72 A
7.0-7.5
pounds similar to (VI) absorb in the 235-245-nm region of the spectrum. The large difference in the position of the absorbance maxima of the two compounds renders UV spectrophotometry capable of distinguishing between the two structural entities and again points to compound (V) as the correct structure for the base hydrolysis product of clohazam (I). In summary, the evidence derived from MS, proton NMR data, and UV spectrophotometry allows us unequivocally to assign structure (V) to the base hydrolysis product of clobazam (I),which, ironically, is the product predicted in the literature for acid hydrolysis. The identification of the species present after reflux of clobazam (I) in acidic medium followed a more circuitous path than that taken for the study in basic medium. Although UV spectrophotometry provided undeniable evidence of decomposition (Figure 3), TLC failed to separate the compounds and yielded only a single hand. HPLC demonstrated the presence of clohazam (I)and an unknown compound and suggested that the polarities of the two compounds were substantially different. The pronounced difference in the polarities of the two components implied that separation of the two species could he accomplished by partitioning between an aqueous and an organic phase. Accordingly, equal volumes of the refluxed solution and water were combined with an equal volume of chloroform and shaken vigorously. The chloroform and aqueous phases were separated, an additional volume of chloroform was added to the aqueous phase, and the extraction procedure was repeated. The aqueous phase and chloroform phases were analyzed hy HPLC and UV spectrophotometry. The results showed that the organic phase contains only the parent compound, clohazam (I), that one extraction with chloroform is sufficient
ANALYTICAL CHEMISTRY, vcn. 57, NO. I, JANUARY 1985
to remove the clobazam from the aqueous phase, and that the aqueous phase also contains a single species whose UV spectrum is depicted in Figure 4. The benzimidazoles possess characteristic absorption spectra in the UV region, which may he of use for their identification. Since the literature data suggested that base hydrolysis of clohazam (I) should result in the formation of a compound containing the benzimidazole moiety, the UV spectrum of benzimidazole (VII) and benzimidazolone (VIII) had been recorded during preliminary experiments and are presented in Figure 5. Benzimidazolone (VIII) can exist as 2hydroxy-benzimidazole (IX)because of the possibility of keto-enol tautomerism. A comparison of the spectra in Figures 4 and 5 demonstrates that the spectrum of the acid hydrolysis product of clohazam (I) is similar to that of benzimidazole (VII). In addition, henzimidazolone (VIII) is essentially in the keto form since its spectrum hears no resemblance to that of henzimidazole. In the course of the study, it had been observed that when a solution of the acid hydrolysis product is made alkaline the UV spectrum rapidly changes to that of the base hydrolysis product (V); however, under the same conditions the absorption spectrum of benzimidazole (VU) and henzimidazolone (VIII) remains unchanged. When the base hydrolysis product (V) is dissolved in 0.1 N hydroalcoholic HCI, the absorption spectrum gradually reverts to that of the acid hydrolysis product over a 1-h period. The spec-
1.c
0.8
0.2
300350 Wavelength (nm)
250
Figure 5. Absorption spectrum of (a) benzimidazoloneand (b) benzimidazole in 0.1 N hydroalcoholic HCI
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 1, JANUARY 1985
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