Anal. Chem. 1992,64,2233-2237
2233
Rates of Peptide Proteolysis Measured Using Liquid Chromatography and Continuous-Flow Fast Atom Bombardment Mass Spectrometry Richard B. van Breemen' and Roderick G. Davis Department of Chemistry, Box 8204, North Carolina State University, Raleigh, North Carolina 27695-8204
An lmmoblllzeddlgestlve enzyme assay, whlch has been used to determlne whether orally admlnlstered peptlde drugs are hydrolyzed by the dlgestlve system, was applled to the measurement of rates of proteolyslsof blologlcallyactlve p e p tldes. I n thls study, the rates of hydrolysls by trypsln and chymotrypdn of the pressor agent anglotensln II, the peptlde hormone [Arg']vasopresdn, and the peptlde drug [deamlnoCysl,bArg']vasopressIn were measured. Enzyme knmoblllzatlon prevented autolytic proteolysls and provided a stable enzyme preparatlon durlng the assays. For rate determlnatlons, the dlsappearance of substrate was measured over time by using elther flow Injectlon continuous-flow fast atom bombardment (FAB) mass spectrometry wHh selected Ion monltorlng or revenredghase hlgh-performance llquld chromatography(HPLC) wlth UV absorbancedetocth. Compared to the HPLC method, c o n t l m k w FAB was faster, provlded more confldent Identlflcatlon of the analyte because molecular welght data was obtained, and could be used for all enzymatic reactlons Instead of only those In which complete chromatographlc resolutlon of substrate from proteolytic fragments was obtained, The In vltro proteolytlc rates measured for the vasopresslns were compared to data from rat bioassays and conflrmed that the llmltlng factor In the oral bloavallablllty of [Arg8]vasoprdn was rapld hydrolysls by trypdn In the lntestlnal lumen. The more bloactlve compound, [deamlno-Cysl,bArg']vasopressin, was more stable to chymotryptlc dlgestlon and completely resistant to trypslnlzatlon.
INTRODUCTION Many drugs under development including antibiotics and antihypertensive agents contain peptide bonds or are peptide analogs. Examples of peptide drugs already in clinical use include vancomycin,l cyclosporin A,* and ~aptopril.~ For oral administration, peptide drugs should resist hydrolysis by the digestive system, otherwise they must be administered by more invasive means such as by injection as in the case of in~ulin.~ Recently, we reported an in vitro method using immobilized digestive enzymes to measure whether compounds that are structurally similar to peptides are hydrolyzed by digestive enzymes in the stomach, in the intestinal lumen, or by mucosal peptidases at the intestinal brush border.5 In that study, continuous-flow fast atom bombardment liquid chromatography-mass spectrometry was used to rapidly identify the hydrolysis p r ~ d u c t s . ~
* Corresponding author.
(1)Pfeiffer, R. R. Rev. Infect. Dis. 1981,3,5205-209. (2)Petcher, T. J.; Weber, H.-P.; Ruegger, A. Helu. Chim. Acta 1976, 59,1480-1488. (3)Ondetti, M. A,;Rubin, B.; Cushman, D. W. Science 1977,196,441444. (4)Saffran, M.;Kumar, G.S.; Savariar, C.;Burnaham, J. C.; Williams, F.; Neckers, D.C. Science 1986,233,1081-1084. (5)van Breemen, R. B.; Bartlett, M. G.; Tsou, Y.; Culver, C.; Swaisgood, H.;Unger, S. E. Drug Metab. Dispos. 1991,19,683-690. 0003-2700/92/0364-2233903.0010
Since its introduction by Barber and co-workers? fast atom bombardment (FAB) mass spectrometry has become a standard method for the analysis of underivatized, polar, nonvolatile, and thermally labile compounds. FAB ionization has been combined with a liquid sample introduction system called continuous-flow FAB mass Spectrometry? which has been used for peptide mapping: monitoring of enzymatic hydrolysis of peptide mixtures? measurement of trypsin kinetic parameters,lO and peptide quantitation.11 The advantages of continuous-flow FAB over standard FAB mass spectrometry include reduced chemical noise from the FAB matrix? increased signal-to-noise? less suppressionof sample ions when mixtures of samples are analyzed,l*and the fact that samples can be analyzed much more rapidly and reprod~cibly.~ In an effort to extend our in vitro studies on the susceptibility of orally administered peptide drugs to include measurements of their rates of hydrolysis, the disappearance of angiotensin I1and two vasopressin substrates were followed during incubations with either trypsin or chymotrypsin. Vasopressin is an antidiuretic agent produced in the hypothalamus, and the oral bioavailabilityof vasopressin and synthetic analogs is under investigation for the treatment of diabetes insipidus.13 Angiotensin I1 was selected for study because of the clinical importance of angiotensin converting enzyme inhibitors14 and because it is hydrolyzed by every enzyme used in our digestive enzyme assay.5 Previously, CapriolilO used flow injection continuous-flow FAB selected ion monitoring mass spectrometry to measure reaction rates and other kinetic parameters for the hydrolysis of a tetrapeptide by trypsin. In our investigation, Caprioli's continuous-flow FAB selected ion monitoring method was applied to the measurement of rates of proteolysis of orally administered peptide drugs by digestive enzymes. The method of Caprioli'o was extended to include the addition of an internal standard for increased precision and the use of immobilized instead of soluble enzymes so that enzyme autolysis was prevented. Rate measurements were carried out using either gradient reversed-phase high-performance liquid chromatography (HPLC) separation followed by UV absorbance detection or, because HPLC could not be used for some analyses, flow (6) Barber, M.; Bordoli, R. S.; Sedgwick, R. D.; Tyler, A.N. J.Chem. SOC.,Chem. Commun. 1981,325-327. (7) Caprioli, R. M.; Fan, T.; Cottrell, J. D. Anal. Chem. 1986,58,29492954. (8) Whaley, B.; Caprioli, R. M. Biol. Mass Spectrom. 1991,20,210214. (9)Caprioli, R. M. Anal. Chem. 1990,62,477A-485A. (10)Caprioli, R. M. Biochemistry 1988,27,513-621. (11)Lieek, C. A.;Bailey, J. E.; Benson, L. M.; Yaksh, T. L.; Jardine, I. Rapid Commun. Mass Spectrom. 1989,3, 43-46. (12)Caprioli, R. M.; Moore, W. T.; Fan, T. Rapid Commun. Mass Spectrom. 1987,I , 15-18. (13)Saffran, M.;Bedra, C.; Kumar, G. S.; Neckers, D. C . J. Pharm. Sci. 1988. 33-38. .... -. - - , 77. -(14)Roark, W. H.; Tinney, F. J.; Cohen, D.; Eseenburg, A.D.; Kaplan, H. R. J. Med. Chem. 1985,28,1291-1295. I
@3 I9S2 Amerlcan Chemlcal Society
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 19, OCTOBER 1, 1992
1 hour
-
f
Chymotrypsin-.
N
0
5
10
15
I/I
amidinopheny1)methanesulfonylfluoride (PMSF)to irreversibly inhibit residual trypsin activity.16 Briefly, 2 mL of 1mM aqueous PMSF was added to 1 mL of beads and stirred for 1 h at room temperature. The beads were rinsed with lOmL of water followed by 10 mL of 0.1 M ammonium acetate, pH 7.5.
6 hours I
20
25
5
0
10
I
1/ l 2 hours A
Retention Time (mtn)
15
20
Enzymatic Reactions. The autolysis of soluble proteases was investigated by following the disappearance of chymotrypsin (1 mg/mL) in 0.1 M ammonium acetate at pH 7.5 and 37 OC. Every 3 h, 50-pL aliquota were removed from the incubation and analyzed by using reversed-phase HPLC as described in the next section. In all subsequent studies, immobilized enzymes were used. Substrate solutions containing 87 pM angiotensin 11, vasopressin (AVP), or [deamino-Cysl,D-Arga]vasopressin (DDAVP) were individually incubated in 0.1 M aqueous ammonium acetate, pH 7.5, at 37 "C with 80 pL of beads containing immobilized trypsin. The total reaction volume was 2.00 mL. Incubationswith immobilizedchymotrypsinwere identical except that they contained 125 pM substrate. All incubations were carried out a minimum of three times. The substrate-to-enzyme ratio in these reactions was 200:l and 5 0 1 for chymotrypsin and trypsin, respectively. Micropipetting techniques were practiced until the error in measuringeach aliquot of beads was determined to be approximately 5 % (RSD). After allowing 4 min for equilibration of each incubation mixture, aliquota of 10 pL each were withdrawn at regular intervalsthroughout the incubation period, lyophilizedto dryness, and stored at -20 "C. Incubation times ranged from 3 min for rapidly hydrolyzed substrates to 60 h for stable or slowly hydrolyzed peptides. The sum of all aliquota removed from a single incubation mixture was lees than 5 % of the initial reaction volume. During selected analyses, angiotensin I (10 pL of a 100 pM solution) was added to each aliquot as an internal standard. Lyophybd sampleswere reconstituted in 10pL of the incubation buffer for analysis using HPLC, or the continuous-flow FAB carrier solvent (see below) for mass spectrometric analysis. Chromatography. Reversed-phase HPLC analyses were carried out using a Waters Associates (Bedford, MA) gradient HPLC system consisting of two Model 501 pumps, Model 740 integrator/recorder, Model 680 gradient controller,Biorad (Richmond, CA) gradient mixer, Rheodyne (Cotati, CA) Model 7125 injector, Vydac (Hesperia, CA) 5 pm, 330 A pore size Cl8-silica HPLC column (4.6 X 250 mm), and Applied Biosyetems (Foster City, CA) Model 757 variable-wavelength absorbance detector set at 214 nm. A 7-pL portion of each 10-pLaliquot was injected on to the HPLC column every 45 min.
25
'
[Are]-
11
Retention Time (min)
Flgurr 1. Autolysis of soluble chymotrypsin (1 mg/mL) In 0.1 M ammonium acetate at pH 7.5 and 37 O C . The reaction was followed by analyzlng 50-pL aiiquots using reversed-phase HPLC as described in the Experimental Section.
injection continuous-flow FAB mass spectrometry using selected ion monitoring (SIM). The mass spectrometric technique of SIM has a lower detection limit and is more suitable for quantitation than standard scanning mass spectrometric methods because only signals from the analytes of interest are recorded. The advantages and shortcomings of both the HPLC and continuous-flow FAB mass spectrometric techniques were evaluated.
EXPERIMENTAL SECTION Reagents. All solventswere HPLC-grade and were purchased from Fisher Scientific (Springfield, NJ). The enzymes chymotrypsin and TPCK-treated trypsin, substrates angiotensin 11, [Ar$]vasopressin, and [deamino-Cysl,~-Ar$]vasopressin,and controlled-pore glass beads (120/200 mesh, 200-nm mean pore diameter) were purchased from Sigma Chemical Co. (St. Louis, MO). The procedures for trypsin and chymotrypsin immobilization and determination of enzyme activity have been previously reporteda6 The enzyme activites were 101and 121 pmol min-l g1(beads), 30.6 and 14.4 pmol mi& mgl (protein), and 31and 35 pmol min-l mL-l (beads)for trypsin and chymotrypsin, respectively. Immobilized chymotrypsin was treated with (4-
Table I. Structures of Peptide Substrates and Initial Tryptic and Chymotryptic Peptides amino acid sequence angiotensinI1 Asp-Arg-Val-Tyr-ne-His-Pro-Phe tryptic products Aw-Arg Val-Tyr-ne-His-Pro-Phe chymotrypticproducts Asp-Arg-Val-Tyr ne-His-Pro-Phe [Ar$]vasopressin (AVP) Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH,
chymotryptic products
1047 290
775 552 513 1086
I
I
tryptic producta
[M+ HI+
Gly-NHz Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg
75a
1030 1103
Phe-Gln-Asn-Cys-PreArg-Gly-NHp I
Cys-Tyr Gln-Asn-Cys-Pro-Arg-GIy-NH2
1103
I
Cys-Tyr-Phe
[deamino-Cys1,~Ar$]vasopressin(DDAVP)
deaminoCys-Tyr-Phe-Gln-Asn-Cys-Pro-D-Arg-Gly-NH2 I
tryptic products chymotrypticproducts
none expected nor observed Phe-Gln-Asn-$ys-PreD-Arg-Gly-NH2
10Ea
dearninok ys-Ty r Gln-Asn-Cys-PreD-Arg-Gly-NH2
1088
I
deaminocys-Tyr-Phe
a
Not detected during FAE3 mass spectrometry.
1070
I
ANALYTICAL CHEMISTRY, VOL. 64, NO. 19, OCTOBER 1, 1902 0 min.
Table 11. Initial Hydrolysis Rates
enzymes“ substrates chymotrypsin trypsin angiotensin I1 4380 (f360) 20 (f2) 5050 (f480)* A W 24 (f2) 7200 (f1080)* DDAW 9 (fl) 0 0
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5 10 15 Retention Time (min)
20
Flgurr 2. Reversed-phase HPLC chromatograms showlng the progresslve hydrolysis of anglotensin I Iby immobllized chymotrypsinover time. The band at approximately 2.5 min corresponds to the solvent front. For analysis of tryptic digests,intact substrate was separated from hydrolysis products using a solvent gradient from 9 o : l O O . l to O1oO:O.l water/acetonitrile/trifluoroaceticacid (v/v/v) over 20 min. The purity of each HPLC band was determined by using static positive ion FAB mass spectrometry. For the analysis of chymotryptic digests of the vasopressins, the peptide mixture could be resolved more rapidly using a 15-min gradient from 9 o : l O O . l to 55450.1 water/acetonitrile/trifluoroaceticacid (v/ v/v). Enzymatic reaction rates were determined by measuring the initial rate of disappearance of substrate using reversed-phase HPLC. The area under the curve of the HPLC band correspondingto substrate was integratedand background subtracted. Standard curves were prepared and used to calculate the concentration of substrate contained in each incubation aliquot. Hydrolysis rates were determined from the slope of the best straight line of a plot of substrate concentration (rM) versus incubation time as calculated using linear least squaresregreeaion analysis. Mass Spectrometry. Positive ion continuous-flowFAB mass spectra and selected ion monitoring chromatogramswere obtained using a JEOL (Tokyo, Japan) HXllOHF double-focusing mass spectrometer equipped with a frit-FAB version of a continuousflow FAB interface and DA5000 data system. Xenon fast atoms at 6 kV were used for FAB ionization. The resolving power was approximately 1000. The mobile phase for continuous-flowFAB mass spectrometry consisted of water/acetonitrile/trifluoroacetic acid/glycerol(80:2O0.1:0.5; v/v/v/w). Glycerol functioned as the FAB matrix. The flow rate of the mobile phase into the continuous-flowFAB interfacewas 5 rL/min and was controlled by an Applied Biosystems (Foster City, CA) Model 140A dualsyringe pump.. Every 6 mm, 1-pL aliquots of each reaction mixture were injected using a Rheodyne (Cotati,CA) Model 8125 injector and analyzed using continuous-flow FAB SIM mass spectrometry without a chromatographic column. Except for the initial continuous-flowFAB experiments, angiotensin I was used as an internal standard. During continuous-flow FAB analysis, SIM was used to measure the abundance of the protonated molecule of the peptide substrate and, if present, the internal standard. The sampling rate for SIM was 100 Hz. Standard curves were constructed for each set of analyses in which the area under the curve of the signal corresponding to protonated substrate was plotted versus the concentration bM) of injected substrate. The concentration of substrate in each incubation aliquot was determined using the appropriate standard curve. These concentrations were plotted versus incubation time, and the rate of hydrolysis of each substrate was determined from the slope of the best straight line through these data points as calculated using linear least squares regression analysis.
RESULTS AND DISCUSSION To facilitate the measurement of hydrolysis rates of bioactive peptides using digestive proteases, the enzymes had to (15) Laura,R.;Robison, D. J.; Bing,D. H. Biochemistry 1980,19,4859.
approx dose (pmol/rat) for 50% reduction in urine flow oral 1v poten@ potencyd 3500
20
5 1
a The units for all rate measurements are pmol pL-l h-l mgl (enzyme).b Determined by using continuous-flow FAB mass spectrometry with SIM. All other measurementswere made using HPLC with UV absorbance detection. From Saffran et al. (see ref 13). From Manning et al. (see ref 18).
[M+H]+ 1086
m/z
Flguro 3. Poslthre lon FAB mass spectrum of the HPLC band eluting at 22.5 mln in the reversed-phase HPLC chromatogram of a tryptic dtgest of AVP. The HPLC band consisted of an unresolved mixture of the substrate AVP and Its tryptic hydrolysis product, AVP-QIyNHs, detected at mlz 1086 and 1030, respectlvely.
be stable during the course of the reaction. When using soluble enzymes, the primary cause of loss of soluble enzyme activity was autolytic proteolysis, which is illustrated in Figure 1for soluble chymotrypsin. Autolytic proteolysis was prevented by using immobilized enzymes. Immobilized trypsin and chymotrypsin have been shown to maintain greater than 80 5% of their original activity after being used in 35 assays of 20 h each.16 Another advantage of using immobilized proteases in this study was the absence of enzyme or enzymatic autolysis products in aliquots removed from incubations for analysis. Furthermore, proteolysis ended immediately in aliquots as they were removed from the incubation medium containing the immobilized enzyme. The structures of the three substrates used in this investigation and their tryptic and chymotryptic hydrolysis produde are shown in Table I. Because trypsin hydrolyzes peptides on the carboxylic acid side of lysine and arginine residues, angiotensin I1 and AVP were each cleaved into two peptides. DDAW was not hydrolyzed by trypsin because the D-arginine enantiomer was not recognized by this enzyme. Chymotrypsin, which cleaves peptides on the carboxylic acid side of aromatic amino acid residues, hydrolyzed angiotensin I1 into two tetrapeptides and formed ring opening products from AVP and DDAVP. Whether ring opening occurred a t the tyrosine or phenylalanine residues was not determined. During the time course of the reactions, no further hydrolysis to release phenylalanine from either vasopressin was detected. The molecular weights of the tryptic and chymotryptic peptides were confirmed by measuring the (16) Chung, S.-Y.; Swaisgood, H. E.; Catignani, H.E. J. Agric. Food Chem. 1986,34, 579-584.
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 19, OCTOBER 1, 1992
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400 R =0.955
r
-
/
A]
0
Angiotensin II (pM) '
5-
R I 0.997 RSDS e 2.6%
10 20 Reaction Time (min)
0
B Slop = 4.157 RSDS = 14.4%
70
1
Angiotensin II (pM)
FWr 4. Standardcurvesforthe quantitatknof angiotensin I I obtalned by using continuous-flow FAB SIM mass spectrometry (A) w h u t an internal standard or (B) with angiotensin I as the internal standard. R = correlation coefficient; RSDS = relative standard deviation of the slope; n = 1.
m/z values of the corresponding protonated molecules using positive ion FAB mass spectrometry (see Table I). Using HPLC with UV absorbance detection, standard curves were generated for peptide quantitation that showed high precision and accuracy. Correlation coefficients for the linear regression analysis of these data ranged from 0.997 to 1.OOO. Next, the rates of disappearance of peptide substrates during enzymatic digestions were measured. For example, a series of HPLC analyses of aliquots from an incubation of angiotensin I1 with chymotrypsin is shown in Figure 2. The peak area corresponding to intact angiotensin I1 decreased as a function of time, and two new bands appeared at shorter retention times. Using positive ion FAB mass spectrometry, the protonated molecules of the bands corresponding to angiotensin I1 and the two hydrolysis products were confirmed to be 1047,552,and 513,respectively (see Table I). No peptide or protein contaminants such as autolysis products or enzyme released from the glass beads were detected. Following HPLC analysis, the substrate concentrations in each incubation aliquot were calculated using the straight line equation for the standard curve. Next, the concentration values were plotted versus incubation time, and the best fit straight line through these data points was determined using linear regression analysis. The correlation coefficients for these plots ranged from 0.985 to 0.998. The slopes of the lines are reported as the initial hydrolysis rates in Table 11. The standard deviation in each slope was used to estimate the error for the rate measurement. The fastest hydrolysis rate measured during the chymotrypsin incubations, 4380 (f360)pmol pL-l h-l mgl, corresponded to the hydrolysis of angiotensin 11. Rates for chymotryptic digestion of AVP and DDAVP were 24 (f2) and 9 (fl) pmol pL-l h-' m g l , respectively. Because hydrolysis of the vasopressins by chymotrypsin required ring opening, chymotrypsin hydrolyzed these molecules at a much slower rate than the open-chain angiotensin 11. Cyclic regions of peptides might not bind to proteases in the most favorable conformation for hydrolysis. The presence of unusual groups such as deaminocysteine and/or D-argininein DDAVP further reduced its susceptibility to hydrolysis by chymotrypsin compared to AVP.
40
0
40 80 Reaction Time (sec)
120
Figurr 5. Rate curves for the hydrolysis of (A) angiotensin I1 by chymotrypsin and (8) AVP by trypsin. The disappearance of each peptide substrate was measured by analyzing incubation aiiquots using flow injection positive ion continuous-flow FAB SIM mass spectrometry with angiotensin I as an internalstandard. The standard devlation for each data point (n = 3) is indicated by the error bars.
Next, the rates of tryptic hydrolysis of the three peptide substrates was investigated using HPLC. As expected, DDAVP was not hydrolyzed by trypsin because the D-arginine enantiomer was not a substrate recognizedby the enzyme active site. Although angiotensin I1 was hydrolyzed slowly (see Table 11), the rate of trypsinization of AVP could not be determined by using reversed-phase HPLC because one of the hydrolysisproducts was not chromatographicallyresolved from AVP. Attempts to resolve these two peptides using different solvent gradients failed. The presence of intact AVP and one its hydrolysis products was confirmed by collecting the peptide band eluting at 22.5 min in the HPLC chromatogram and analyzing it by using positive ion FAB mass spectrometry. The protonated molecule of AVP was detected at mlz 1086,and the [M + HI+ of the tryptic peptide formed by release of the terminal glycine residue by trypsin was observed at mlz 1030 (see Figure 3). In order to measure the rate of trypsinization of AVP, continuous-flow FAB mass spectrometry with selected ion monitoring was used with flow injection. Also, the rate of hydrolysis of angiotensin I1 by chymotrypsin was measured using continuous-flow FAB and compared to that obtained using HPLC. Because no HPLC column was used during these analyses, samples could be injected every 6 min with no sample carry over instead of every 45 min as was necessary during HPLC. The area under the peak recorded in each selected ion chromatogram was used for quantitation. Without an internal standard, the standard curve for angiotensin I1had a correlation coefficient of 0.955with a relative standard deviation (RSD) in the slope of 15.6% (Figure 4). The correlation coefficient increased to 0.997with an RSD in the slope of 2.6% when angiotensin I was used as an internal standard (Figure 4). Therefore, internal standards were used for all subsequent analyses. The rate of hydrolysis of angiotensin I1 by chymotrypsin was measured using continuous-flowFAB mass spectrometry with S I M and found to be consistent (within experimental error) with the rate measured using the HPLC method (see Table 11). The graph depicting the rate of hydrolysis of angiotensin I1 measured by using continuous-flow FAB SIM
ANALYTICAL CHEMISTRY, VOL. 64, NO. 19, OCTOBER 1, 1992
0 sec
I 20sec
40 sec
i 70 sec
100 sec
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. . \.-,=
min
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130 sec
Flgure 6. Ion chromatograms obtained using positive ion continuous-flow FAB SIM with flow injection showing the disappearance of AVP (protonated molecule at mlz 1086) over time durlng a single incubation with immobilized trypsin. Angiotensin I at m/z 1298 was used as the internal standard and is shown in the inset for each AVP trace.
mass spectrometry is shown in Figure 5. Next, mass spectrometry was used to measure the rate of hydrolysis of AVP by trypsin, which is shown in Figure 5 and Table 11. A series of selected ion chromatograms showing the disappearance of AVP over time during a single incubation with immobilized trypsin is shown in Figure 6. Whether obtained using HPLC or mass spectrometry, the RSD for all measurements was approximately 109%. Rates were measured over the range 9-7200 pmol pL-l h-' mgl, and no upper limit to the reaction rates that could be determined was observed. During the analysis of peptide mixtures using static FAB mass spectrometry, peptides with low surface excess concentration (typically the more hydrophilic compounds) tend to produce less abundant protonated molecules in positive ion FAB mass spectra than the more hydrophobicand surface active peptides in the mixture.17 The extent of such peptide ion 'suppression" during FAB mass spectrometry is reduced during continuous-flowFAB, presumably because of the rapid and continuous mixing of the matrix and analyte, the thin f i i of matrix present at the probe surface, and the continuous replenishment of the sampleand matrix at the surface exposed to the FAB beam.12 During the rate measurements described above usingcontinuous-flowFAB, the initial rates of substrate disappearancewere measured and showed no enzyme product feed-back inhibition nor any ion suppression effects since the concentration of product peptides remained low during the analyses. The addition of a peptide internal standard to the samples analyzed during continuous-flow FAB mass spectrometry might produce more significantion suppression effects. However, these effects would not affect the results because they would be incorporated into the standard curve, which was generated using a constant concentration of internal standard and a varying concentration of analyte. Saffran et al.13compared the biological activities in rats of orally administered AVP or DDAVP. In order to achieve a (17) Clench, M. R.: Garner, G. V.: Gordon, D. B. Biomed. Mass Spectrom. 1985, 12, 355-357. (18) Manning, M.; Gzronka, Z.; Sawyer, W. B. In The Pituitary; Beardwell, C., Robinson, G., Eds.; Butterworths: Kent, UK, 1981; pp 265-296.
50% reduction in urine flow, 3500 pmol of AVP and only 20
pmol of DDAVP had to be admini~tered.'~When administered intravenously, 5 pmol of AVP and 1pmol of DDAVP elicitedthe same effectla(seeTable 11). The ratea of hydrolysis shown in Table I1 indicate that the most likely cause of low oral bioavailability of AVP is rapid hydrolysis in the intestinal lumen by trypsin. The synthetic analogue of AVP, DDAVP was shown here to be completely resistant to trypsinization because of the incorporation of D-arginine. Furthermore, the effect of this replacement and the incorporation of deaminocysteine into DDAVP resulted in a nearly 3-fold reduction in its rate of hydrolysis by chymotrypsin.
CONCLUSIONS Because small differences in hydrolysis rates of peptide drugs by digestive enzymes can be measured using this digestive enzyme assay, this approach could be used to quantitatively demonstrate the effect of small changes in drug structure on proteolysis. Similarly, a series of structurally similar drugs under development could be assayed so that those compounds that are the most resistant to proteolysis in the digestive tract could be selected for further study and development as drugs for oral administration. Although HPLC with UV detection is more widely available and much less expensive than mass spectrometry, continuous-flowFAB mass spectrometry with SIM was shown to be much faster and independent of chromatographic resolution.
ACKNOWLEDGMENT This work was carried out at the Mass Spectrometry Laboratory for BiotechnologyResearch,which was established by a grant from the North Carolina Biotechnology Center. Preliminary results were presented at the 39th ASMS Conference on Mass spectrometry and Allied Topics, Nashville, TN, May 19-24,1991. RECEIVED for review March 23, 1992. Accepted June 24, 1992.
