Anal. Chem. 1995, 67,4431-4436
Structural Identification of the Degradation Products of the Antitumor Peptide Antagonist [Arg6,~-Trp7~a,MePhe8]Substance P (6- II) J. LOon E. Reubsaet,* Jos H. BeUnen, Auke Bult, Ed Hop,Raymond Vennaas, Yunus Kellekule, J. Jantien Kettenesvan den Bosch, and Willy J. M. Underberg Faculty of Pharmacy, Univetsiteit Utrechf, P.O. Box 80082, 3508 TB Utrecht, The Nethedands
The basic hexapeptide antagonist [Aqf,~-Trp~~~,MePhe~lsubstance P (6-11) was degraded in acid and alkaline media. In acid solution, only one degradation product is found whereas in allcaline solution at least six products are formed. These compounds were analytically characterized and strucAwallyidentified by reversed-phasehighperformance liquid chromatography, capillary electroFigure 1. Structure of antagonist [Arg6,~-Trp7~g,MePhe8]substance phoresis, liquid ch”atography/mass spectrometry,fast P (6-1 1) (antagonist G). atom bombardment tandem mass spectrometry, optical rotation analysis, and chiral gas chromatography. The yielding a sulfoxide or a ~ulfone.~ Other reactions are hydrolysis product formed in acidic solution is the terminally deamidated antagonist ~ , ~ - T r p ~ ~ ~ , M e P h e ~ l s u bPs t a n cofethe peptide bonds and racemization of the amino acid residues in alkaline solutions.5 Another potential degradation reaction is (6- 11); this product was also found in alkaline degradadeamidation at the C-terminus of methionine, yielding the free tion mixtures. Other important degradation products carboxylic acid analog, comparable with the deamidation of originate from racemization of the amino acid residue L-Met, formation of ornithine from Arg, and the oxidation asparagine and g l ~ t a m i n e . ~ , ~ Cummings et al.’oJl investigated the in vitro stability and of Met to its sulfoxide form. metabolism of antagonist G in plasma using reversed-phasehighliquid chromatography (RP-HPLC) with electrcThe basic hexapeptide antagonist [ A r ~ , ~ - T r p ~ s ~ , M e P h e ~performance lchemical detection. A study of the degradation of antagonist G substance P (6-11) (antagonist G, Figure 1) is a neuropeptide in aqueous solution with a stability-indicatingRF-HPLC systemI2 antagonist that inhibits the growth of small cell lung cancer showed that in the pH range 0-14 at least seven degradation (SCLC) Antagonist G is structurally related to a part of products appear.I3 bombesin, which is an autocrine growth factor in SCLC. This Since antagonist G, which is in phase I trials as an antitumor peptide antagonist owes its cytotoxic properties to, competitively, drug, is pharmaceutical formulated as an infusion liquid, informainhibiting the receptor binding of bombesin by blocking the tion on the degradation of the drug in solution is appropriate. It receptor intermediating in the action of bombesin, which leads is of clinical importance, in order to optimize the pharmaceutical to the rapid mobilization of calcium and the arrest of DNA formulation, to clarify the nature of the degradation products. In synthesis.’ this paper, the analytical and structural characterization of Degradation of peptides and proteins proceeds via a number antagonist G degradation products formed in acidic and alkaline of well-defined pathways and can be divided in chemical and solutions will be described. physical degradation reaction^.^,^ In a small peptide like antagonist G, physical degradation reactions such as aggregation and MATERIALS AND METHODS precipitation are unlikely to occur, in contrast to chemical Chemicals. Antagonist G was obtained through the New degradation. Considering the chemical structure of antagonist Drug Development Office of the European Organization for G, several reactions are possible. Arginiine can be hydrolyzed into ornithine or citrulline5whereas sidechain oxidation of tryptophan (7) Brot, N.; Weissbach, H. Arch. Biochem. Biophys. 1983,223,271-81. results in a ringqpened residue! Methionine also can be oxidized, (8) L ~ u D. , T.-Y. TIBTECH 1992,10, 364-9. (1) Penella, J. W.; Rozengurt, E. Cancer Res. 1990,50,3968-73.
(2) Cuttita, F.; Carney, D. N.; Mulshine, J.; Moody, T. W.; Fedorko, J.; Fischler, A; Minna, J. D. Nafure 1985,316, 823-6. (3) Manning, M. C.; Patel, K; Borchardt, R T. P h a n . Res. 1989,6,903-18. (4) Oliyai, C.; Schoneich, C.; Wilson, G. S.; Borchardt, R T. In Topics in Pharmaceutical Sciences; Crommelin, D. J. A, Midha, K IC,Eds.; Medpharm Scientific Publishers: Stuttgart, 1991; pp 23-46. (5) Whitaker, J. R; Feeney, R E. CRC Crif. Rev. Food Sci. Nufr. 1977,19 (3). 173-212. (6) Means. G. E.; Feeney, R E. Chemical Mod$icution OfProteins, 1st ed.; HoldenDay, Inc.: San Francisco, 1971; Chapter 8. 0003-2700/95/0367-4431$9.00/0 0 1995 American Chemical Society
(9) Capasso, S.; Mazzarella, L.; Sica, F.; Zagari, A Pepf. Res. 1989,2, 195200. (10) Cummings, J.; Maclellan, A; Langdon, S. P.; Rozengurt, E.; Smyth, J. F. ]. chroma tog^ B 1994,653, 195-203. (11) Cummings, J.; Maclellan, A; Langdon, S. P.; Smyth, J. F.]. Pharm. Biomed. Anal. 1994,12,811-9. (12) Reubsaet, J. L E.; Beijnen, J. H.; Bult, A; Teeuwsen, J.; Koster. E. H. M.; Waterval, J. C. M.; Underberg, W. J. M. Anal. Biochem. 1994,220,98102. (13) Reubsaet, J. L E.; Beijnen, J. H.; Bult, A; Van der Houwen, 0. A G. J.; Teeuwsen, J.; Koster, E. H. M.; Underberg, W. J. M. Anal. Biochem., in press.
Analytical Chemistry, Vol. 67, No. 23, December 7, 7995 4431
Research and Treatment of Cancer (NDDO-EORTC, Amsterdam, The Netherlands). All other chemicals used were of analytical grade, and deionized water was used throughout the study. RP-HPLC. The development of a stability-indicatingisocratic W-HPLC system was described earlier.12 In addition, a gradient RP-HPLC system was used consisting of a Gynkotek highprecision pump Model 300 CS and a Gynkotek high-precision pump Model 480 gradient controller (Separations, Hendrik-IdoAmbacht, The Netherlands), a homepacked Hypersil ODS 5pm column (12.5 x 0.39 cm), and an Applied Biosystems 785A programmable absorbance detector (Separations). Mobile phase A consisted of 0.1%trifluoroacetic acid (TI?A), 10 mM ammonium acetate, and 5% (w/w) acetonitrile. Mobile phase B consisted of 0.1%TFA, 10 mM ammonium acetate, and 90% (w/w) acetonitrile. The separation was carried out with a linear gradient in 20 min from 40%to 70%mobile phase B. The injection volume was 200 pL and the flow 1.0 mL/min. W detection was performed at 214 nm. Capillary Electrophoresis. ?jqTo capillary electrophoretic systems used, free zone capillary electrophoresis (FZCE) at pH 9.0 and dynamically coated capillary electrophoresis @C-CE) at pH 6.5, were described earlier.'* A FZCE system at pH 12.7 was also applied. The running buffer at pH 12.7 was a 50 mM borate buffer. The applied voltage was 15 kV.
Liquid Chromatography/Mass Spectrometry (LC/MS). The LC system used was the same as described under gradient W-HPLC; the flow was reduced to 0.1 mL/min prior to component elution. MS detection was performed using a VG Platform Benchtop LC/MS (Fisons Instruments, Altricham, UK). An electrospray interface was used to ionize the molecules (positive ion mode). The nebulizing gas had a flow of 25 L/h; the drying gas had a flow of 300 L/h. The applied voltage to the capillary was 3.4 kV; a low cone voltage (22.0 V) was applied to prevent fragmentation. The MS was calibrated from 166 to 1060 Da with a mixture of horse heart myoglobin (multiply charged) and low molecular peptides with molecular masses ranging from 166 to 754 Da. Fast Atom Bombardment Tandem Mass Spectrometry (Fab-MS/MS). Fab-MS/MS was carried out with a JMSSX/SX 102 A mass spectrometer (BEBE JEOL, Tokyo, Japan). Operating at an accelerating voltage of 10 kV, a xenon beam with an energy of 6 keV was applied to obtain fast atom bombardment spectra. In all experiments, glycerol was used as the matrix. MS/ MS spectra were recorded at 50%precursor ion reduction with air as the collision gas. Optical Rotation, Racemization of antagonist G was studied with a Jasco Model DIP-4 micropolarimeter (Charles Go&, Maastricht, The Netherlands). The wavelength used was 589 nm, and the cuvet length was 10 cm. Chiral Gas Chromatography(Chiral GC). The racemization of amino acids in antagonist G was investigated by chiral GC with a Chrasil-L-Valfused-silica column (25 m x 0.25 mm) (Chrompack, Bergen op Zoom, The Netherlands). The carrier gas was hydrogen. The temperature program started at 75-200 "C (5 "C/ min) after which the temperature was held at 200 "C for 10 min. Both the injector and detector temperatures were set at 250 "C. Detection was carried out with a nitrogen/phosphorous detector (N/PD), and the injection volume was 1 pL. 4432 Analytical Chemistry, Vol. 67, No. 23, December 1 , 1995
Antagonist G and degraded samples were hydrolyzed to single amino acids as described by Creighton14with 6 M HCl for 24 h at 110 "C under nitrogen in a sealed vial. After hydrolysis, the samples were evaporated to dryness under nitrogen at 50 "C. Hydrolyzed products and L- and amino acids were derivatized with 2 M HCl in 2-propanol at 110 "C (carboxyl esterification) and with Muoracetic acid anhydride/ethyl acetate in a 4:l ratio at 110 "C (amino acylation) under nitrogen. Samples were injected directly. Degradation Conditions. For RP-HPLC,FZCE, and DC-CE analysis, degradations were carried out at pH/Ho 0.3 or pH/H13; antagonist G concentrations were 0.2 mg/mL according to Reubsaet et Since in the pH range 0-3 the nature of the reaction mechanism does not change, pH/Ho 0.3 was choosen to have rapid degradation. The same goes for the pH range 5- 14. At pH/H- 13, the degradation reaction is much faster than at physiological conditions although the same degradation reactions occur. Looking at the degradation mechanism in the pH range 3-5 it can be seen that it is a transition between proton- and hydroxyl-mediated degradation. Variation of temperature, ionic strength, buffer components, and antagonist G concentration only affects the degradation reaction rate.13 By inter- and extrapolation of the results, predictions can be made for pharmaceutically important matrices. For LC/MS, Fab-MS/MS, and optical rotation tests, the same conditions were used but with an antagonist G concentration of 0.8 mg/mL. RESULTS
RP-HPIX and CE. The degradation products formed in both alkaline and acidic media elute prior to the parent compound in RP-HPLC. The proton-mediated degradation yielded one product, whereas at least six products appear in alkaline degradation mixtures (Figure 2). The results from FZCE analysis of the degraded antagonist G samples are partly contradictory to the RP-HPLC data. A sample degraded in acidic media showed again one product when analyzed with FZCE at pH 9.0 and pH 12.7. However, alkaline degradation mixtures also showed only one product in FZ-CE (Figure 3B), whereas six products were observed in RP-HPLC. Analysis of fractions, isolated after RP-HPLC separation of an acidic degraded sample, with FZCE at pH 9.0 shows that the parent compound and the degradation product migrate at different times corresponding to the analysis of a sample which was directly analyzed with FZCE at pH 9.0. From the products formed by alkaline degradation, all but one comigrated with the parent compound. Peak 7 in RP-HPLC ( F i r e 2B) comigrated with peak 2 in FZCE (Figure 3B) and eluted after the parent compound. Since antagonist G has two basic but no acidic groups (Figure 1) it has no isoelectric point and no negative charge and cannot migrate with negatively charged molecules in FZ-CE. Analysis of degraded samples showed that, even at pH 12.7, antagonist G still migrates before the uncharged molecules and that the only detectable degradation product migrates after the uncharged molecules (Figure 4). DC-CE analysis of degraded samples was in better agreement with the W-HPLC data.12 The acidic degradation again yielded one degradation product, which migrated ahead of the parent compound. In the alkaline-degraded (14)Creighton. T. E. Proteins, Stncctures and Molecular Properties, 2nd ed.; Freeman and Co.: New York, 1993; Chapter 1.
40.0
105 ,
A
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I
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."n
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/ 0
I
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time (minutes) Figure 3. FZ-CE electropherogram at pH 9.0, of antagonist G degradation at f112. (A) pH/Ho 0.3. (6)pH/H- 13. peak 1, antagonist G; peak 2,degradation product(s).
7.5
0.0
0
i
2
3
d
5
6
7
8
9
10
time (minutes) Figure 2. RP-HPLC chromatograms of antagonist G degradation at f112. (A) pH/Ho 0.3: peak 1, antagonist G; peak 2, degradation product. (6)pHIH- 13: peak 1, antagonist G; peaks 2-7,degradation products.
sample, at least seven peaks were detected, all migrating prior to the parent compound. Due to the high detection limit in DC-CE, analysis of RF-HPLC fractions was not possible. LC/MS and Fab-MS/MS. The degradation product formed in acidic media and the parent compound were analyzed by LC/ MS. The parent compound shows the MH+ ion at mas&-charge ratio (m/z) 951.5 (the calculated mass 951.495), the degradation product at m/z of 952.6. FabMS/MS at the m/z 952.6 compound, after isolation with RP-HPLC, showed that the mass change occurs in the C-terminal amino acid of antagonist G. LC/MS analysis of alkaline-degraded samples showed that peaks 1-7 (Figure 2B) predominantly possess the molecular ions at m/z 951.5 and 952.5. The peak of the parent compound only showed a pseudomolecular ion at m/z 951.5, peak 7 only at m/z 952.5. Peaks 3-7 showed MH+ ions at m/z 951.5,952.5, and 909.5. A MH+ at m/z 967.4 was only found in peak 3/4 (coeluting in LC/MS). Due to its low intensity, no MS data could be obtained from peak 2.
FabMS/MS of m/z 952.5 in the alkaline degradation showed the same fragmentation pattern as the degradation product in acidic media. FabMS/MS of the compound with MH+ at m/z 909.5 showed that the antagonist G molecule was modfied at the N-terminus in the Arg guanidino group of the peptide. Product m/z 967.4 had a fragmentation pattern showing that in this product the C-terminal Met residue of antagonist G was mowed. Fab MS/MS of products formed in the alkaline degradation was performed on a desalted degradation mixture. Optical Rotation. LC/MS data showed that some degradation products which appear after hydroxyl-mediated degradation have G molecular ions at the same m/z as the parent antagonist, which may be due to racemization reactions. In neutral solution, the specific optical rotation of antagonist G is -42.0'. During degradation in acidic media, the optical rotation did not change, demonstrated by the fact that a reference sample (concentration 0.8 mg/mL) antagonist G with a rotation a of -0.033' did not change in rotation during 3 half-lives. After alkaline degradation of antagonist G, the optical rotation disappeared. After 3 half-lives, the a value was -0.003'. Gas Chromatography. No racemization occurred after degradation of antagonist G in acidic media. Figure 5A shows the chromatogram of a degraded sample after hydrolysis in acidic media. Comparing chromatograms of the D/L-amino acids with the chromatogram of the degraded antagonist G, it is evident that no detectable amounts of D-MePhe, D - ~ u D-Arg, , and ~-Trp have been formed during protoncatalyzed degradation. The peak for D-Met found in this chromatogram is also found, in equal amounts, in the chromatogram of the hydrolyzed parent compound. Analytical Chemistry, Vol. 67, No. 23, December 1, 7995
4433
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Figure 4. FZ-CE electropherogram at pH 12.7, of antagonist G degradation at t112, pH/H- 13: peak 1, antagonist G; peak 2, uncharged molecules; peak 3, degradation product(s).
0
1 I
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l5
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Figure 5. Chiral GC chromatograms of hydrolyzed antagonist G samples after (A) degradation in acidic media or (B) degradation in alkaline media: peak 1, L-Leu; peak 2A, D-Met; peak 2B, L-Met; peak 3, L-MePhe; peak 4, L-Arg; peak 5, D-Trp.
In alkaline solutions racemization did occur, as expected from the data obtained from LC/MS and the optical rotation experiments. Only Met racemized, showing both D and L forms (Figure 5B), while ~-Trp,DMePhe, D-L~u, and DArg were not detectable. DISCUSSION AND CONCLUSION The product formed in acidic media is less lipophilic than the parent compound and also possesses a different charge at pH 12.7, pH 9.0, and pH 6.5. At pH 12.7 the product migrated with the negatively charged molecules. At pH 9.0 the product combgated with the uncharged molecules; at pH 6.5 the product was positively charged but had less net positive charge than antagonist G. These findings suggest that an extra negative charge has been introduced into the molecule. Possibly this is due to the deamidation of the amide terminal of antagonist G (PI -10.8). LC/MS and Fab-MS/MS data confirmed that the product formed was the deamidated antagonist G. In this reaction, NH2 is eliminated from methionine and is replaced by an OH group (Figure 6A). Formation of the acid results in an increase in mass of 1 atomic mass unit (amu) which is in accordance with the results of LC/ MS, the m/z of antagonist G being 951.5, and that of the degradation product 952.6. Amino acid sequencing of the product with FabMS/MS according to Biemann15by selecting MHf m/z 952.6 showed that the amino acid Met was modified and that there were no alterations in the other amino acids Arg, Trp, MePhe, Trp, and Leu. Fragments A2-A5 (nomenclature according to Roepstorff'G) ) were found. In Table 1 the calculated and the experimental values for m/z and the assigned amino acid sequences of the (15) Biemann, K. In Protein Sequencing: a practical approach; Findlay, J. B. C., Giesow, M. J., Eds.; IRL Press: Oxford, U.K., 1989 pp 99-118. (16) Roepstorff, P.; Fohlman, J. Biomed. Mass Specfrom. 1984, 11, 601.
4434
Analytical Chemistry, Vol. 67, No. 23, December 1, 7995
A
Figure 6. (A) Deamidation product of antagonist G. (B) Oxidation product of antagonist G.
fragments found are given. Also, some D-fragments @I and DqD6) were detected. Table 1 shows the theoretical and experimental values for m/z of the fragments as well as their composition. This result also indicates that alterations did not take place in either the amino acids Arg, Trp, MePhe, Trp, and Leu or the backbone of fragment &. The only Gterminal fragments found are Y6, 26, 5,V,, and Ws. Fragments Y.5, v6, and ws correspond with the m/z of the free acid. In Table 1, the theoretical and experimental values for m/z of the Z fragments found and their amino acid sequences are listed. These results prove that the degradation product of the proton-mediated degradation of antagonist G is the free acid instead of the amide form of the peptide.
Table 1. Fragments and Mass-tomchargeRatios from Fab-MSIMS of Selected Mass 952.6 (Formed in Acid Degradation)
fragment,, A2
A3
A4 A5
DI D4 D5 D6 26
25
mass (MH+) calcd exptl 315.2 476.3 662.4 775.4 44.0 547.3 733.4 846.5 935.5 779.4
315.2 476.4 662.5 775.6 44.1 547.3 733.6 846.9 935.9 779.6
fragment sequence Arg-Trp Arg-TrpMePhe Arg-TrpMePheTrp Arg-TrpMePheTrpLeu fragment A ~ C H ~ N Z + fragment &-C&N fragment &-C3H6 fragment A&zH~S Arg-TrpMePheTrpLeu-Met-COOH TrpMePhe-TrpLeu-Met-COOH
Figure 7. Ornithine derivative of antagonist G. Table 2. Fragments and Mass-to-Charge Ratios from Fab-MSIMS of Selected Mass 909.5 (Formed in Alkaline Degradation)
fragment,,
A3 As expected from literature data,3-5 racemization in acidic media did not occur. Optical rotation data and chiral gas chromatography show that the original configurations of the amino acids remain the same. As in the parent compound, the only amino acids detected are L-Arg, ~-Trp,L-MePhe, L-L~u, and L-Met (Figure 5 4 . The products formed in alkaline media appeared to be less lipophilic than the parent compound. The discrepancy between the RP-HPLC and E - C E analyses in detecting degradation products can be explained by the fact that of the products detected in RP-HPLC all (peaks 2-6) but one (peak 7) comigrated with the parent compound in FZ-CE. This suggests that the values for m/z of products 2-6 are equal to that of antagonist G ( F i r e 2B). Product 7 (Figure 2B) migrated in FZCE at pH 9.0 (peak 2, Figure 3B) with the uncharged molecules and in FZCE at pH 12.7 as a negatively charged molecule (peak 3, Figure 4). Product 7 in Figure 2B is the same as product 2 in Figure 2A. This means that in both media deamidation of antagonist G takes place. Products with molecular ions at m/z 952.5, 909.5, and 967.4 were sequenced with FabMS/MS. The mlz 952.5 product showed the fragmentation pattern of the free acid instead of the amide form, identical with the pattern of product 2 obtained from acidic degradation. The carboxyl group was introduced at the C-terminus of Met due to deamidation of the amide. Products with m/z at 909.5 were found in peaks 3-7 (Figure 2B) with LC/MS but in far lower concentration than mlz 951.5 and 952.5. Fragmentation with FabMS/MS showed that the modfication took place in the Arg residue. N-terminalfragments &, B3, B6, and C5 were detected, all differing by 42 amu from the corresponding fragments in antagonist G. C-terminal fragments Y4, Y5, Z5, and Z6 were detected. Only fragment Z6 appeared to have a mass 42 lower than fragment & in antagonist G. All other C-terminal fragments had the same mass as those in antagonist G. Fragments D4, D5, Iz, 13, and L showed no difference from those in antagonist G. These results indicate that no alterations take place in the amino acids Trp, MePhe, Leu, and Met. A decrease in mass of 42 in the Arg residue could be a consequence of the formation of ornithine from Arg: the guanidino side chain of arginine can be hydrolyzed in alkaline solutions yielding an aliphatic amine5J4 (Figure 7). In Table 2, the calculated and experimentalvalues for m/z and the amino acid sequences of the fragments are shown. The product with m/z at 967.4 was only found in very low amounts in peak 3/4 (Figure 2B). An increase of 16 in the mass suggests oxidation. In the MS/MS spectrum only the N-terminal
B3 B6 c5 Y4
Y5 25
26 D4 D5 Iz 13 14
mass (MH+) calcd exptl 434.3 462.3 892.5 778.4 609.3 795.4 778.4 892.5 505.3 691.4 159.1 134.1 159.1
434.1 462.2 892.3 778.4 609.3 795.4 778.4 892.3 505.3 691.4 159.1 134.1 159.1
fragment sequence Om-TrpMePhe Om-TrpMePhe Om-TrpMePheTrpLeu-Met OmTrpMePhe-TrpLeu MePhe-TrpLeu-Met TrpMePheTrpLeu-Met TrpMePheTrpLeu-Met Om-TrpMePheTrpLeu-Met fragment &-C8HeN fragment &-C3H6
Table 3. Fragments and Mass-to-Charge Ratios from Fab-MSIMS of Selected Mass 967.4 (Formed in Alkaline Degradation)
fragment,, A2
A3
A4 As Bz B6
Dz
mass (MH+) calcd exptl 315.2 476.3 662.4 775.4 343.2 950.5 200.2
315.1 476.2 662.3 775.3 342.9 950.4 200.1 903.4
fragment sequence ArgTrp Arg-TrpMePhe Arg-TrpMePheTrp Arg-TrpMePheTrpLeu Arg-T$ Arg-TrpMePhe-TrpLeu-Met,x fragment & & H a MH+-SOCH4+
fragments &-&, Bz, and B6 were detected. Fragments &-&, and Bz have values for m/z comparable to the Corresponding fragments in antagonist G. Fragment Bs has a m/z at 950.5 which is 16 higher than fragment B6 in antagonist G. These results show that no alterations took place in Arg, Trp, MePhe, and Leu. The only residue left for the oxidation is Met, where the methylthiol group can be oxidized to a sulfoxide (Figure 6B). The presence of a fragment m / z 903.4 is probably the loss of a SOCH4+group (difference of 64 amu with the m/z 967.4). In Table 3 the calculated and the experimental values for m/z and the amino acid sequence of the fragments is shown. As stated before, peaks 3-6 ( F i r e 2B) mainly consist of products with their m/z at 951.5 and 952.5. Optical rotation of samples after alkaline degradation virtually disappears. Chiral GC analysis of the degraded sample showed that the amino acid L-Met had racemized. Detected amino acids were L-Arg, L-L~u, D/L-Met, ~-Trp, and L-MePhe (Figure 5B). This finding suggests that all detected masses would be detected twice, as racemized and nonracekzed products. However, due to the similar elution behavior, many products coelute. Therefore, for products with molecular ions at m/z 909.5, 910.5, 967.4, and 968.4, it is almost Analytical Chemistry, Vol. 67, No. 23, December 1, 1995
4435
impossible to determine whether there are one or two products with the same value for m/z. Racemized products with a m/z at 952.5 are probably the result of secundaryreactions. This can also be said of the small fractions with molecular ions at m/z 968.4 and 910.5, respectively. These products are probably the result of the combination of deamidation with racemization, oxidation, or ornithine formation. In summary, it can be said that in acidic media deamidation is the only degradation reaction whereas in alkaline media besides
4436 Analytical Chemistry, Vol. 67, No. 23, December 1, 1995
deamidation racemization, ornithineformation, and oxidation also takes place. ACKNOWLEDGMENT
The authors thank Mr C. Versluis for his assistance with the FabMS/MS analysis. Received for review June 26, 1995. Accepted September 15, 1995.@ AC950633+ @Abstractpublished in Advance ACS Abstracts, November 1, 1995.
