Identification of the Degradation Products of Gonadorelin and Three

In acidic solution, gonadorelin and triptorelin are deamidated, whereas buserelin and goserelin undergo debutylation. In the pH range 5−6, the pepti...
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Anal. Chem. 1997, 69, 4972-4978

Identification of the Degradation Products of Gonadorelin and Three Analogues in Aqueous Solution Marnix A. Hoitink,* Jos H. Beijnen, Marcel U. S. Boschma, Auke Bult, Ed Hop,† Jack Nijholt, Cees Versluis,‡ Gerard Wiese, and Willy J. M. Underberg

Department of Pharmaceutical Analysis, Faculty of Pharmacy, Utrecht University, Sorbonnelaan 16, NL-3584 CA Utrecht, The Netherlands

Degradation products of the peptides gonadorelin and its analogues buserelin, goserelin, and triptorelin were characterized with LC/MS, chiral amino acid analysis, and FAB-MS(-MS). Differences in chemical structures of gonadorelin, its analogues, and their respective decomposition products were evaluated in relation to the putative degradation mechanisms. In acidic solution, gonadorelin and triptorelin are deamidated, whereas buserelin and goserelin undergo debutylation. In the pH range 5-6, the peptide backbone of all four analogues is hydrolyzed at the N-terminal side of the 4serine residue. The hydroxyl moiety of the 4serine residue catalyzes this hydrolysis. Goserelin possesses an azaglycinamide residue, which is also subject to degradation in the neutral pH region. At pH > 7, 4serine epimerization is the main pathway of degradation of all four peptides. Parallel to the epimersation, hydrolysis of gonadorelin, goserelin, and triptorelin occurs.

have not been evaluated in relation with the degradation mechanisms. Consequently, their relevancy remains unknown. In our laboratory, the chemical stabilities of gonadorelin and three of its analogues, buserelin, goserelin, and triptorelin (Figure 1), are currently under investigation, both qualitatively and quantitatively. Structural differences are limited to substitution of the sixth amino acid residue and differences in the C-terminal group. The degradation products of these four compounds have now been characterized and the degradation mechanisms evaluated. These results might contribute to the possibility to predict the reactivity of other peptides with similar structural elements.

* To whom correspondence should be addressed. Tel: 31 30 2536917. Fax: 31 30 2535180. E-mail: [email protected]. † Permanent address: Synthon BV, Microweg 22, NL-6545 CM Nijmegen, The Netherlands. ‡ Permanent address: Bijvoet Center for Biomolecular Research, Utrecht University, P.O. Box 80083, NL-3508 TB Utrecht, The Netherlands. (1) Motto, M. G.; Hamburg, P. F.; Graden, D. A.; Shaw, C. J.; Cotter, M. L. J. Pharm. Sci. 1991, 80, 419-23. (2) Okada, J.; Seo, T.; Kasahara, F.; Takeda, K.; Kondo, S. J. Pharm. Sci. 1991, 80, 167-70. (3) Oyler, A. R.; Naldi, R. E.; Lloyd, J. R.; Graden, D. A.; Shaw, C. J.; Cotter, M. L. J. Pharm. Sci. 1991, 80, 271-5. (4) Strickley, R. G.; Brandl, M.; Chan, K. W.; Straub, K.; Gu, L. Pharm. Res. 1990, 7, 530-6.

EXPERIMENTAL SECTION Chemicals. Buserelin acetate is obtained from Hoechst Holland NV (Amsterdam, The Netherlands), LH-RH acetate and R-chymotrypsin (type 1-s) from Sigma Chemical Co. (St. Louis, MO), goserelin acetate from Zeneca Pharma BV (Ridderkerk, The Netherlands), and triptorelin acetate from Ferring AB (Malmo¨, Sweden). Handling of the dry compounds is performed in a downflow laminar air flow cabinet to prevent inhalation of the substances. All chemicals used are of analytical grade, and deionized water is applied throughout the study. Preparation of the Degradation Samples. Degradation products are generated by heating sealed glass ampules with solutions of the analogues in water baths. Samples at pH 2, 5, and 9 are prepared using 0.01 M perchloric acid, 25 mM acetate buffer, and 25 mM borate buffer, respectively. Initial peptide concentrations are 500 µg/mL at pH 2 and 9 and 1000 µg/mL at pH 5. The pH 2 and 9 samples are heated at 70 °C for 24-48 h. At pH 5, the goserelin-containing solutions are heated at 70 °C for 360 h; all other solutions are heated at 80 °C for 720 h. RP-HPLC. The RP-HPLC device for analysis of the gonadorelin analogues consists of a Gynkotech Model 300 precision pump (Separations, H.I. Ambacht, The Netherlands) used at 1 mL/min and connected to an ISS 100 sampling system (PerkinElmer Corp., Norwalk, CT). The mobile phase contains acetonitrile, water, and 0.1% trifluoroacetic acid (v/w). The mobile phase varies in acetonitrile concentration: buserelin, gonadorelin, goserelin, and triptorelin are analyzed with 23, 16, 22, and 22% (w/w), respectively. The column is a Lichrospher 100 RP-18 (5 µm), 125 mm × 4 mm i.d. (Merck, Darmstadt, Germany). The Applied Biosystems 785A programmable absorbance detector (Separations) is operated at 214 nm, and data acquisition is

4972 Analytical Chemistry, Vol. 69, No. 24, December 15, 1997

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Peptides and proteins are subject to an array of degradation processes. The chemical stability of specific structural elements in proteins can best be studied in small peptides of 2-10 amino acid residues, with the major advantage that no denaturation takes place. The gonadorelin analogues (luteinizing hormone-releasing hormone (LH-RH) analogues), peptides of 9-11 residues, are extremely useful for investigating structure-reactivity relationships because a relatively large number of compounds are available with typical structural differences. Although structure-activity relationships of gonadorelin analogues have been throroughly investigated, the structure-stability relationships have not been established so far. The identities of degradation products of gonadorelin,1 fertirelin,2 histrelin,3 and antagonist 263064 have been established; however, except for the latter, these results are not correlated to structural elements and

© 1997 American Chemical Society

Figure 1. Chemical structures of buserelin, gonadorelin, goserelin, and triptorelin.

executed on a DP 700 data processor (Carlo Erba Instruments, Milan, Italy). RP-HPLC for chiral amino acid analysis is executed with a Model 510 pump (Waters, Milford, MA) operating at 1 mL/min. Serine and histidine are analyzed with different mobile phases. The mobile phase for analysis of L/D-serine contains 3 mL of acetic acid, 1.8 mL of ethylenediamine, 51 g of methanol, and 935 g of water. The mobile phase for L/D-histidine contains 4.1 g of sodium acetate, 32 g of methanol, and 960 g of water. Derivatization, as described in the section Sample Pretreatment for Chiral Amino Acid Analysis, and injection of the samples is performed in a Model 231 sample injector, equipped with a Model 401 dilutor (both from Gilson, Villiers, France). The injection volume is set at 20 µL. The column is a Lichrospher 100 RP-8 (5 µm), 125 mm × 4 mm i.d. (Merck), and detection is achieved with a 821-FP fluorescence detector (Jasco Ltd., Hachioji City, Japan) at excitation and emission wavelengths of 335 and 432 nm, respectively. All data collected are edited with Gynkosoft software version 5.3 (Separations). The RP-HPLC system for collection of fractions for FAB-MSMS analysis consists of two Model 510 pumps (Waters) controlled by an automated gradient controller (Waters) and operating at a flow of 1 mL/min. Mobile phase A contains a buffer, consisting of 1.14 mL of acetic acid and 0.75 mL of 25% ammonia in 1000 g of water; mobile phase B contains the same buffer components in a mixture of 500 g of water and 395 g of methanol. The gradient procedure is as follows: 0-5 min, 75% A isocratic; 5-10 min, linear gradient to 40% A; 10-15 min, 40% A isocratic; 15-16 min, linear gradient to 75% A. An U6K injector (Waters) is used to inject 100-200 µL of the samples. The column is a Lichrospher 100 RP-18 (5 µm), 125 mm × 4 mm i.d. (Merck), and detection is realized using an Applied Biosystems 785A programmable absorbance detector (Separations), operating at 214 nm.

Synthesis and Degradation of Product S3. One milligram of triptorelin was dissolved in 1 mL of 25 mM phospate buffer, pH 7.5 . After addition of 42 µg of R-chymotrypsin, the solution was allowed to react for 17 h at room temperature. Isolation was performed with the RP-HPLC for analysis of the gonadorelin analogues under gonadorelin analysis conditions. The acetonitrile in the collected fraction was removed under a nitrogen stream. The remaining solution was diluted with 5 mL of 25 mM acetate buffer pH 5.3. This mixture was divided over 10 ampules, and degradation of these ampules was performed at 80 °C at increasing time intervals and over a total period of 14 days. Analysis was performed with the RP-HPLC for analysis of the gonadorelin analogues with a mobile phase containing 12% (w/w) acetonitrile. Sample Pretreatment for Chiral Amino Acid Analysis. Fractions are collected using the RP-HPLC for analysis of the gonadorelin analogues with a trifluoroacetic acid concentration of 0.2% (v/v) and methanol instead of acetonitrile. The methanol concentrations for buserelin, gonadorelin, goserelin, and triptorelin analysis are 41, 30, 41, and 35% w/w, respectively. Evaporation of the fractions is executed under a nitrogen stream. This has to be performed thoroughly, because small amounts of methanol can cause ampules to explode during heating. The fractions are dissolved in 6 M hydrochloric acid and brought into glass ampules, which are subsequently sealed. The sealed ampules are kept for 16 h at 110 °C in an air-heated oven. After that, the ampules are cooled to -20 °C in a freezer to prevent explosion during opening. After opening of the ampules, the 6 M hydrochloric acid is removed under a nitrogen stream. Samples are dissolved in 75 mM carbonate buffer, pH 11, followed by derivatization of 40 µL of sample with 40 µL of reagent at room temperature. After 2 min, the reaction is terminated by adding 40 µL of 0.2 M acetic acid, and the derivatized sample is injected into the RP-HPLC for chiral amino acid analysis. The reagent is prepared as follows: a stock solution is made by dissolving 8 mg of o-phthalaldehyde and 10 mg of N-acetyl-Lcysteine in 1 mL of methanol.5 This stock solution is diluted 10 times with water before use. Liquid Chromatography/Mass Spectrometry (LC/MS). The liquid chromatograph consists of a LC-10 AD liquid chromatograph (Shimadzu, Tokio, Japan) and a 7125 injector (Rheodyne, Cotati, CA). The column used is a Superspher 100 RP-18 (5 µm), 119 mm × 2 mm i.d. ( Merck). The mobile phase contains acetonitrile and water with 0.1 % trifluoroacetic acid (v/w). For analysis of gonadorelin-containing samples, 16% acetonitrile (w/ w) is used; all other samples are analyzed with 22% acetonitrile (w/w). The flow is set at 0.1 ml/min. This LC system is directly coupled to a VG Platform II mass spectrometer (Fisons, Altrincham, UK), operated in the positive ion mode. Calibration was performed with an aqueous 4 mg/mL sodium iodide solution. The scanned mass range is 100-1500 m/z, scan time is set at 3 s, and low- and high-mass resolution are 12.5 and 15 (instrumental units), respectively. The cone voltage was 25 V, and the probe temperature was kept at 120 °C. Data acquisition and calculation of the m/z values are done with MassLynx version 2.1 (Fisons). Fast Atom Bombardment Tandem Mass Spectrometry (FAB-MS-MS). All FAB-MS(-MS) analysis is performed on a JMS-SX/SX102 A BEBE mass spectrometer (JEOL, Tokyo, Japan) operated at 10 kV accelerating voltage. A beam of 6 keV xenon (5) Nimura, N.; Kinoshita, T. J. Chromatogr. 1986, 352, 169-77.

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Table 1. Abundant Fragment Ions in FAB-MS(-MS) Spectra of Gonadorelin Analogues

Figure 2. Chromatograms of degraded solutions of triptorelin at pH 2 (A), 9 (B), and 5 (C). P parent compound; S1-S3, degradation products occurring in all analogues; T1-T7, products originating from triptorelin.

atoms is produced by a MS-FAB 10 D FAB gun (JEOL). The MS scan range is 0-1500 m/z at a speed of 30 s/scan. Nitrogen gas is used as collision gas, and the flow rate is adjusted to obtain a 50% reduction of the main beam. As standard matrix, glycerol is used. Samples are dissolved in 20 µL of water, and 1 µL is brought into the matrix. Prior to this, fractions are collected using the RP-HPLC for collection of fractions for FAB-MS-MS, a gradient RP-HPLC with a volatile buffer. Excess methanol in the fractions is removed under a nitrogen gas stream. The remaining solutions are then freeze-dried over a period of 16 h. RESULTS AND DISCUSSION The degradation mechanisms of gonadorelin and its analogues can be divided into three types: proton-, solvent- and hydroxylcatalyzed mechanisms.6 Therefore, degradation products emerging at pH 2, 5, and 9, respectively, are studied. The names given to the products consist of the first character of the analogue from which it originates, followed by a unique number. For gonadorelin, the letter L of LH-RH is used. Three products of the solvent-catalyzed degradation are identical for all analogues, and their names start with the character S. Typical degradation product patterns are illustrated by the chromatograms of degraded triptorelin samples in Figure 2. Proton-catalyzed degradation of all four analogues yields one main product for each analogue. From the literature,1,6 it is known that gonadorelin is deamidated in acidic medium. The reactivity at pH 5 is more complex since a lot of products are involved. Buserelin, gonadorelin, and triptorelin appear to react similarly, but goserelin tends to degrade differently. (6) Hoitink, M. A.; Beijnen, J. H.; Bult, A.; Van der Houwen, O. A. G. J.; Nijholt, J.; Underberg, W. J. M. J. Pharm. Sci. 1996, 85, 1053-9.

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analogue

peptide backbone fragments

side-chain fragments

buserelin gonadorelin goserelin triptorelin

A2,8-9, B2-4,8, X2, Y′′3,5, Y5 A2,8-9, B2,4,6,8, Y′′6 A2,8-9, B2-4,8, Y′′4-5, Y5 A2,8-9, B2,6-8, X4, Y′′6

V4-7, W3-4,6 V6-8, W4,7 V4-7, W3-4 V5,7-8, W4,7

In the chromatograms at pH 9, one main product is observed. Epimerization of serine is considered the most important degradation reaction for hydroxyl-catalyzed degradation of gonadorelin analogues.1,3,7 Analytical Procedures. Mass spectrometry is very important in identifying the degradation products of peptides. As RP-HPLC conditions are already determined for the parent compound, LC/ MS is a logical choice. Therefore, initial characterization of the degradation products is performed using LC/MS. In the RPHPLC for analysis of the gonadorelin analogues, trifluoroacetic acid is used in the mobile phase as ion pairing agent and to maintain a low pH value. This RP-HPLC system can be coupled directly to an electrospray mass spectrometer if the flow is reduced from 1 to less than 0.2 mL/min. With a 2 mm i.d. column and a flow of 0.1 mL/min, an acceptable compromise between analysis time and sensitivity is obtained. Columns with a diameter less than 2 mm tend to behave differently with respect to resolution, which makes the direct comparison with chromatographic data from kinetic experiments analyzed on a 4 mm i.d. column difficult. Buserelin, gonadorelin, goserelin, and triptorelin behave very similarly in the electrospray MS. Parent compounds and most products give intense, doubly charged ions with singly charged ions at approximately 10% of their intensity. This preference for two charges can be explained by the presence of two strong basic residues, histidine and arginine. Confirmation of a number of degradation products was performed with FAB-MS-MS after isolation and freeze-drying. The nomenclature of the fragment ions is according to Roepstorff and Fohlman.8 The azaglycinamido residue in goserelin is treated not as a residue but as a C-terminal derivatization. Fragmentation of the gonadorelin analogues occurs both at the peptide backbone ions (A, B, X, Y′′) and in the side chain (V, W).9 In Table 1, the measured abundant fragment ions of gonadorelin and its three analogues are summarized. The more C-terminally located arginine residue, present in the analogues, directs the fragmentation pathway. The proton affinity of this residue is relatively high, which causes the vast majority of the [M + H]+ ions to be protonated at this site. Due to the presence of the arginine residue and its location near the C-terminus, V and W ions are abundant, as a result of so called “charge remote” fragmentation.10 For the same reason, A8, B8, and A9 fragment ions are observed in all spectra. Another basic residue, histidine, is located at the N-terminus. The basicity of histidine is lower than that of arginine, and its influence on the fragmentation pattern is, therefore, limited to a relatively strong abundance of A2 and B2-6 ions. (7) Nishi, K.; Ito, H.; Shinagawa, S.; Hatanaka, C.; Fujino, M.; Hattori, M. Pept. Chem. 1979, 175-80. (8) Roepstorff, P.; Fohlman, J. Biomed. Mass Spectrom. 1984, 11, 601. (9) Papayannopoulos, I. A. Mass Spectrom. Rev. 1995, 14, 49-73. (10) Johnson, R. S.; Martin, S. A.; Biemann, K. Int. J. Mass Spectrom. Ion Processes 1988, 86, 137-54.

Table 2. Mass Balance at 214 nm after More than One Halflife in the Degradation of Gonadorelin Analogues percentage of initial peak area (no. of products) analogue

pH 2

pH 5

pH 9

buserelin gonadorelin goserelin triptorelin

89 (2) 86 (2) 89 (2) 75 (2)

79 (5) 75 (9) 61 (6) 77 (9)

94 (2) 86 (4) 79 (2) 97 (4)

Because mass spectrometry cannot discriminate between Land D-isomers of the amino acid residues, epimerization was studied with chiral amino acid analysis. RP-HPLC with a derivatization step was preferred over GC because of the higher sensitivity. The o-phthalaldehyde reaction with a reaction time of 2 min at room temperature, developed by Nimura and Kinoshita5 was selected. The derivatization and analysis procedures were automated using a programmable injector, making it more reliable and reproducible. Mass Balance. The areas of the peaks found in the UV chromatograms at 214 nm were summed to establish a mass balance of the reactions. The results are displayed in Table 2. Acceptable percentage were found at pH 2 and 9, since it must be kept in mind that most degradations involve hydrolysis of peptide bonds, resulting in losses in UV absorbance. A large number of degradation reactions is possible, and this might lead to a large number of products in very low quantities. Because of the threshold in the integrator, these products will not be detected; hence, no mass balance is obtained. This is especially the case at pH 5, since some proton- and hydroxyl-catalyzed degradation take place of both the parent and the products of the solventcatalyzed degradation, resulting in a large number of products in low quantities. The reactions given in this paper should, therefore, be considered as main pathways of degradation at certain conditions instead of the only pathway of degradation. Products of the Proton-Catalyzed Degradation. The results of the LC/MS characterization are shown in Table 3. Protoncatalyzed degradation of gonadorelin and triptorelin involves mainly deamidation, resulting in products L1 and T1, determined through a mass increment of 1. The ethylamide moiety of buserelin was found not to degrade: primary amides are apparently more sensitive to hydrolysis. The stability of peptides with C-terminal primary amido groups might, therefore ,be enhanced by alkylating these amide groups. The main degradation of goserelin and buserelin is accompanied by a mass loss of 56, yielding products B1 and G1. After isolation of B1 and G1, FAB-MS spectra of these two

products were recorded. Identification was performed using Y′′3,4 and W4. Y′′3 and W4 are indentical compared to the parent compound, while Y′′4 is 56 mass units lower. This corresponds to elimination of the tert-butyl moiety of the O-tert-butyl-D-serine residue. Two less important acidic degradation reactions, similar for buserelin, gonadorelin, and triptorelin, provide a mass difference of +18 and -111. Both reactions are likely to occur at the pyroglutamyl group, respectively hydrolysis of the ring and the earlier reported total elimination of the residue.1,3 In the goserelin samples, only the +18 product can be found. Since these two reactions contribute to a minor extent to the total proton-catalyzed degradation, it was not possible to isolate sufficient quantities to perform FAB-MS-MS analysis. Products of the Solvent-Catalyzed Degradation. LC/MS characterization of the solvent-catalyzed degradation products is presented in Table 4. Products S1-S3 are formed from all four analogues. To confirm that all gonadorelin analogues yield the same products, the capacity factors of S1-S3 were determined for every analogue and turned out to be identical. S1 is, according to the literature,1,3 cyclo-(L-His-L-Trp), a so-called diketopiperazine (DKP). S1 was subjected to FAB-MS-MS analysis, and the resulting spectrum is shown in Figure 3. Fragments 296.1 and 307 are likely to arise through opening of the ring, followed by CO and NH3 elimination, respectively, according to a review published by Eckhardt.11 The resulting fragment ions are of the A and Z types, respectively.8 The immonium ions of histidine at m/z 110.0 and tryptophan at m/z 159.0 can also be found in the spectrum. Beside peptide bond fragmentation, sidechain fragmentation of tryptophan takes place, resulting in fragments at m/z 194.0 (C9H8N elimination) and 195.0 (C9H7N radical elimination). The most abundant ion is the tryptophan side chain itself at m/z 130.0, typically for tryptophan, as is m/z 82 for histidine.12 The molecular ion and the fragmentation patterns clearly point t0 cyclo(His-Trp). Formation of DKPs has been described previously.13-15 DKP formation is especially known to occur from di- and tripeptides, and it was found that, during degradation of some gonadorelin analogues in the neutral pH region cylco-(His-Trp) was formed.1-3 S2 has the same mass as S1, indicating epimerization. Chiral amino acid analyses revealed that S2 is the D-histidine epimer (Table 4). The clearly distinct L- and D-histidine percentages may deviate from the theoretical values as a result of two factors. First, separation of S1 and S2 is not complete. Second, the signal of histidine was low compared to, for example, that of serine, probably due to degradation of histidine during hydrolysis in 6 M hydrochloric acid. However, the difference in L-histidine

Table 3. Molecular Ions of the Products of the Proton-Catalyzed Degradation of Gonadorelin Analogues m/z

a

H]+

product

[M +

buserelin B1 B2 B3 gonadorelin L1 L2 L3

1240.0 1183.9 nda nd 1182.8 1183.7 nd nd

m/z [M+2H]2+

product

[M +

620.7 592.4 629.7 565.1 592.0 592.2 600.8 536.5

goserelin G1 G2 triptorelin T1 T2 T3

1269.9 1213.8 nd 1311.8 1312.9 nd nd

H]+

[M+2H]2+ 635.7 607.4 644.5 656.6 657.1 665.7 601.1

nd, not detected.

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Table 4. Molecular Ions and the L-Histidine Content of the Products Formed in the Solvent-Catalyzed Degradation of Gonadorelin Analogues m/z

m/z

product

[M + H]+

[M+2H]2+

buserelin gonadorelin goserelin triptorelin S1 S2 S3 B4 B5

1240.0 1182.8 1269.9 1312.0 324.3 ( 0.0 324.5 ( 0.3 453.3 ( 0.2 805.8 805.6

620.7 591.9 635.7 656.8 nda nd nd 403.3 403.5

a

L-histidine

content (%)

85 39

product

[M + H]+

[M+2H]2+

L4 L5 G3 G4 G5 T4 T5

748.3 748.7 835.8 1227.0 1213.0 877.7 877.7

374.8 374.6 418.3 614.2 607.1 439.6 439.4

L-histidine

content (%)

nd, not detected.

Figure 3. FAB-MS-MS spectrum of product S1.

percentages between S1 and S2 strongly indicates the epimerization of histidine. DKPs were found to racemize relatively easily,16 which explains the formation of cyclo-(D-His-L-Trp). Racemization of L-histidine residue in S1 is, therefore, likely to occur because of the properties of DKPs and not of the histidine residue; otherwise, D-histidine epimers of S3 and the parent compound should have been found. Contrarily to earlier reports,1,3,7 histidine racemization in the other compounds could not be observed. According to FAB-MS-MS analysis, product S3 is very likely pGlu-His-Trp and results from peptide backbone hydrolysis. Identification was performed with abundant A2 and B2 ions and less abundant C′′2, Y′′2, and M3 (side-chain elimination) ions. The presence of tryptophan and histidine was proven by fragment ions m/z 159 and 130, respectively m/z 110 and 82. This route of degradation was found for other gonadorelin analogues with identical first four amino acid residues1-3 but also for antagonist RS-26306, in which the first three amino acid residues are different, while the fourth residue is identical, namely serine.4 Strickley et al.4 show a serine-catalyzed degradation mechanism for this reaction. The serine hydroxyl group was also found to catalyze peptide bond hydrolysis in di- and tripeptides.17 The formation of product S1 involves the cleavage of two peptide bonds, namely the bond between the 1pyroglutamyl and the 2histidine residues and the bond between the 3tryptophan and 4serine residues. As this is unlikely to occur in one (degradation) step, product S1 might very well be a secondary degradation 4976

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product, the result of degradation of another product. The primary product S3 can give rise to S1 by the cleavage of only one bond. To find out if S3 is the precursor of S1, degradation experiments on isolated S3 were performed. S3 was enzymatically generated from triptorelin with chymotrypsin, isolated, and subjected to degradation. The degradation products S1 and S2 found in the S3 samples degraded during 14 days are small in concentration, 2% or less of compound S3. A lot of products with retention times smaller than that of S3 can be seen, unlike what is found in the chromatograms of degraded gonadorelin analogues (Figure 2C). S3 is, therefore, not the precursor of S1. Another precursor might be the depyroglutamyl gonadorelin analogues found at pH 2 (Table 3). Although these products are not found at pH 5, fast conversion into S1 might be responsible for their absence. Analogue-specific products are the hexa- and heptapeptides resulting from the cleavage of the peptide bond on the N-terminal side of the L-serine residue: B4, L4, T4, and G3 and their epimers B5, L5, and T5. B4, L4, and T4 were isolated and subjected to FAB-MS-MS analysis. In the FAB-MS-MS spectra of B4, L4, and T4 and in the spectra of the parent compounds, identical W and V ions are found but totally different high A and B ions. The identity of the products could clearly be verified. The amount of G3, a minor product in the goserelin degradation, was too small for FAB-MS-MS analysis. The products B5, L5, and T5 are possibly the D-serine epimers, degradation products of B4, L4, and T4, respectively. Goserelin degrades mainly through a totally different mechanism, resulting in products G4 and G5. The C-terminal azaglycinamide residue appears to be responsible for this degradation process, as the mass changes in G4 and G5 correspond to partial hydrolysis and elimination of the semicarabazide moiety, respectively (Figure 4). The degradation rate constant is more than 5 times higher than that of the serine-catalyzed backbone hydrolysis. FAB-MS-MS spectra of G4 and G5 were recorded to confirm the proposed structures. Fragment ions A8-9 and B8-9 of G4 were identical to those of the parent, proving that the mass loss of 57 (11) Eckhart, K. Mass Spectrom. Rev. 1994, 13, 23-55. (12) Heerma, W.; Kulik, W. Biomed. Environ. Mass. Spectrom. 1988, 16, 1559. (13) Steinberg S. M.; Bada J. L. Science 1981, 213, 544-5. (14) Steinberg, S. M.; Bada, J. L. J. Org. Chem. 1983, 48, 2295-8. (15) Smith, G. G.; Evans, R. C.; Baum, R. J. Am. Chem. Soc. 1986, 108, 732732. (16) Smith, G. G.; Baum, R. J. Org. Chem. 1987, 52, 2248-55. (17) Noll, B. W.; Jarboe, C. J.; Hass, C. F. Biochemistry 1974, 13, 5164-9.

Table 5. Molecular Ions and L-Serine Content of the Products of the Hydroxyl-Catalyzed Degradation of Gonadorelin Analogues m/z

m/z

product

[M + H]+

[M+2H]2+

buserelin B6 gonadorelin L6 goserelin G6 triptorelin T6

1240.0 1239.9 1182.8 1182.8 1269.9 1269.8 1312.0 1312.0

620.7 620.7 591.9 592.0 635.5 635.4 656.6 656.5

L-histidine

content (%)

55 4 98 6 61 13 98 12

Figure 4. Hydrolysis of the azaglycinamide moiety of goserelin and reaction products of G5 with glycerol and nitrobenzyl alcohol (NBA) during FAB-MS-MS analysis.

is caused by elimination of the azaglycinamide moiety. The [M + H]+ ion of G5 disappeared rapidly from the MS spectra during scanning, making FAB-MS-MS analysis impossible. Another ion, at 72 mass units higher, was formed, indicating that product G5 reacts in the mass spectrometer. When nitrobenzyl alcohol was used as matrix instead of glycerol, a reaction product was formed at 133 mass units higher. The mass increments of 72 and 133 are most likely caused by addition of glycerol and nitrobenzyl alcohol, respectively, accompanied by elimination of 20 Da, and the process is, therefore, not a simple condensation. Fragment ions A8-9 and B8 prove that all nine amino acid residue are unchanged. In the FAB-MS-MS spectrum of the glycerol product, the C′′9 ion is abundant, which indicates that the acylhydrazin moiety is cleaved at the bond between the nitrogen atoms. An identical C′′9 was present in the spectra of goserelin, so it can be concluded that the first nine amino acid residues are unchanged. Since glyceraldehyde can be obtained from glycerol through mild oxidation,18 it is possible that in the glycerol, but also in the nitrobenzyl alcohol, traces of aldehydes are present that can react with G5 under elimination of a water molecule. Proposed structures are depicted in Figure 4. Further research on model peptides with the glycinamido moiety should cast more light on this matter. (18) Witzemann, E. J. J. Am. Chem. Soc. 1914, 36, 2227.

product

[M + H]+

[M+2H]2+

L1 G4 G7 G8 G9 T1 T7

1183.8 1227.0 1227.0 1252.2 1251.8 1312.9 1313.0

592.5 613.7 614.1 626.6 626.5 657.1 657.1

L-histidine

content (%)

Figure 5. Chiral amino acid analysis of (A) buserelin and (B) product B6. Peaks 1-4 represent L-glutamine, D-glutamine, L-serine, and D-serine, respectively.

Products of the Hydroxyl-Catalyzed Degradation. Main products of the hydroxyl-catalyzed degradation are B6, G6, L6, and T6, having m/z values identical with those of their parent compounds (Table 5). Serine epimerization can be expected,1,3,7 and the RP-HPLC for chiral amino acid analysis was optimized for L/D-serine resolution. Both epimer and parent were isolated, followed by amino acid hydrolysis, according to Sample Pretreatment for Chiral Amino Acid Analysis. As an example, the chiral analysis of buserelin and B6 is shown in Figure 5. The results, indeed, show that epimerization is located at the serine residue (Table 5). Gonadorelin and triptorelin have only one serine residue at the fourth position, and this residue is in the Dconfiguration in products L6 and T6. Buserelin and goserelin contain two serine residues, the “normal” L-4serine residue and an O-tert-butyl-D-6serine residue. The percentages of L-serine vary from 4 to 12% in products B6 and G6 and from 55 to 61% in the parent compounds. From these results, it can be concluded that the tert-butyl-D-serine residue is not epimerized; otherwise, the L-serine percentages in the products B6 and G6 would have been higher. Epimerization of amino acid residues is known to occur Analytical Chemistry, Vol. 69, No. 24, December 15, 1997

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elimination. Possibly it is an intermediate for the formation of G4 and G7, but only for the hydroxyl-catalyzed mechanism, since it was absent in the degraded goserelin samples at pH 5. Again, stability research on smaller model peptides might result in elucidation of the reaction mechanism.

Figure 6. Epimerization mechanism of serine.

through a carbanion intermediate.19 A relatively stable sixmembered intermediate with a hydrogen bridge is proposed for the serine racemization, which explains the relative high rate of racemization of the L-serine residue compared to other amino acid residues (Figure 6). The hydrogen interaction was, for example, found in the study of the 3D structure of a serine dipeptide by Dey et al.20 The tert-butylated D-serine cannot form the sixmembered intermediate, because the hydrogen bridge is not possible. Epimerization will, therefore, not take place. Less important degradation reactions are deamidation of gonadorelin and triptorelin and hydrolysis of the azaglycinamide residue of goserelin. In Figure 2B, the peaks of triptorelin and its three products, T1, T6, and T7, are shown. T1 is deamidated triptorelin, T6 is epimerized triptorelin, and T7 is epimerized, deamidated triptorelin. For gonadorelin, the same products were expected; however, only L1 and L6 were found. The m/z values of the deamidated, epimerized product could not be determined due to its low intensity and its overlap with the abundant LH-RH parent peak. Hydrolysis of goserelin results in the products G4, G7, G8, and G9. G4 is a product with an N-terminal hydrazide (see Solvent-Catalyzed Degradation), and G7 is probably its D-4serine epimer. The structure of G8 and its epimer G9 are unknown, but the mass decrement corresponds to a water molecule (19) Neuberger, A. Adv. Protein Chem. 1948, 4, 297. (20) Dey, S.; Kaur, P.; Singh, T. P. Int. J. Pept. Protein Res. 1996, 48, 299-303.

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CONCLUSIONS From this study of the stability of gonadorelin analogues, it can be concluded that accelerated degradation reactions occur at a very limited number of residues. Serine is involved in both hydroxyl-catalyzed epimerization and solvent-catalyzed backbone hydrolysis. Deamidation of C-terminal primary amides occurs both in acidic and alkaline solution, while secondary amides are considerably less sensitive to degradation. The introduction of a tert-butyl ether, however, increases the proton-catalyzed degradation by a factor 2-3. The azaglycinamide moiety in goserelin is relatively unstable in neutral and alkaline solutions. Mass spectrometry is an important technique in the stability research. LC/MS is a powerful technique in the screening for degradation products, and, because of the high sensitivity, minor products can also be characterized. FAB-MS-MS analysis of peptides of 2-10 amino acids generally gives unambiguous results, especially when structure-fragmentation relationships are considered, instead of just correlating computer-generated m/z values with found peaks. Because mass spectra do not contain configurational data, chiral amino acid analysis is necessary to detect epimerization. RP-HPLC methods are less complex than GC methods, and the selected o-phthalaldehyde/N-acetylcysteine method is fast, easy to automate, and very cost-effective. ACKNOWLEDGMENT We thank Hoechst-Marion-Roussel BV (Hoevelaken, The Netherlands), Zeneca Pharma BV (Ridderkerk, The Netherlands), and Ferring (Hoofddorp, The Netherlands) for their kind donation of buserelin acetate, goserelin acetate, and triptorelin acetate, respectively. Received for review June 18, 1997. Accepted September 25, 1997.X AC970634X X

Abstract published in Advance ACS Abstracts, November 1, 1997.