Anal. Chem. 1995,67,3871-3878
XPS and TOF=SlMSMicroanalysis of a Peptide/ Polymer Drug Delivery Device C. M. John,t R. W. Odom,*l* L. Sahmti,S A. Annapragada,S and M. Y. Fu Lug Department of Pharmaceutical Chemistty, School of Pharmacy, University of Califomia, San Francisco, Califomia 9414k3-0446,Charles Evans & Associates, Redwood City, Califomia 94063, and Abbott Laboratories,Abbott Park, Illinois 60064-3500
The localization of a peptide drug dispersed in a solid tnatrix of hydroxypropyl cellulose (HPC) was determined at micrometer lateral resolutionusing secondary ion mass spectrometry (SIMS)/ion microscopy. Leuprolide formulated as a sustained release drug delivery device has been selected as a model compound for this investigation. One key facet of this study was to attempt to understand the distribution and ultimate bioavailability of the peptide dispersed in an inert polymer matrix. The results reported in this paper demonstrate that the lateral distribution of leuprolide along the surfaces of cross sections prepared fiom different polymer formulations is different. Ion microscopy directly measures the lateral distribution of protonated molecular ions as well as specific fiagment peaks and provides a direct method of determinii peptide distributions in polymers. Ion images of leupolide dispersed in HPC demonstrate that the peptide distribution is critically dependent on polymer composition. The mass spectrometry results augment quantititative X-ray photoelectron (XPS) measurement of C and N levels in different polymedpeptide formulations. The combination of X P S and TOF-SIMS techniques provides a powerful method for determining the distribution of peptides in polymer matrices. The localization of organic molecules in biological tissues and biopolymers is becoming increasingly important in pharmaceutical research and development.' For example, the ability to localize selected pharmaceutical compounds or metabolites within speciflc regions of a tissue or organ can provide tremendous advantages over existing methodologies for development and evaluation of drug delivery and efficacy.2 Biomaterials utilized as artificial tissue, skin, prosthetic devices, and intraocular lens are typically synthetic polymers with some form of biocompatible surface layer.3 The ultimate acceptance or rejection of the artificial material by an organism depends on the compatibility of the implant surface with the cells with which it makes intimate ~ontact.~ Another important, practical application of biopolymers is their use as solid media for innovative time-release pharmaceutical University of California. Charles Evans & Associates. 3 Abbott Laboratories. (1) Kasedmo, B.; Lausmaa, J. In Sutface Characterization ofBiomaterials; Ratner, B. D., Ed.; Elsevier: New York, 1988; Chapter 1. (2) Prescott. L. F. In Novel Drug Delivey and Its Therapeutic Application; Prescott. L. F., Nimmo, W. S., Eds.; John Wiley & Sons: New York, 1989; Chapter 1. (3) Ratner, B. D. J. Biomed. Mater. Res. 1993,27, 837. +
0003-2700/95/0367-3871$9.00/0 0 1995 American Chemical Society
formulation^.^ For example, the development of protein and peptidic drugs that can regulate a number of physiological processes such as growth, immune responses, blood pressure, blood clotting, and bone calcification has been limited by the necessity of administering these drugs by injection since oral administratioin is generally ineffective? Polymer matrices containing peptidic drugs could provide a highly efficient method for drug administration. However, the polymer matrices must have wellcharacterizeddrug release rates, and of course, the polymers must be biocompatible. The work reported in this paper illustrates one of the first examples of direct molecular microanalysis of a medicinal patch in which the distribution of a peptide is determined using molecular imaging secondary ion mass spectrometry. This study was performed on samples containing the drug leuprolide, dissolved in a matrix of hydroxypropyl cellulose (HPC). Leuprolide is an orally inactive synthetic nonapeptide analog of ovine or porcine gonadotropin-releasing hormone (GnRH), which is used as an antineoplastic agent in the treatment of endometriosis and precocious puberty. The structure of leuprolide is SoxoPro-HisTrpSer-Tyr-D-Leu-Leu-Arg-Pro-NHCzH5. Leuprolide is more potent than GnRH and differs from the naturally occurring hormone by the presence of the D isomer of leucine at position 6 and the ethylamide which replaces the glycine at position 10. A research effort was initiated to design a device that can be placed in direct contact with a biological membrane, and hence, the drug distribution in the polymer matrix is a critical parameter in these experimental devices. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) provided a direct method of determining the leuprolide distribution along cross sections of different peptide/polymer blends. TOFSIMS localized the distribution of protonated molecular ions, (M H)+,and fragment ions of leuprolide at lateral resolutions on the order of 3-5 pm along cross sections 100pm or more thick. The cross-sectional distributions of the drug were found to be strongly dependant on the drug-to-polymer ratio. The addition of 12%oleic acid to the drug/HPC blend significantly changed the axial distribution of leuprolide compared to formulations that contained only leuprolide and HPC. X-ray photoelectron spectroscopy (XPS) was also employed in this study to measure the atomic concentrationsand functional forms of C, 0, and N on the sample surfaces.
+
(4) Kelly, P.; Atkins, T. W. In Microencapsulation of Drugs; Whateley, T. L.,
Ed.; Hanvood Academic Publishers: Chur, Switzerland, 1991;p 33. (5) Aungst, B.J. In Drug Permeation Enhancement: Theory and Applications; Hsieh, D. S., Ed.; Marcel Dekker, Inc.: New York, 1991;p 323.
Analytical Chemisfry, Vol. 67,No. 21,November 1, 1995 3871
Table 1. Composition of Leuprolide/HPC Films
sample
leuprolide (mg)
HPC (mg)
oleic acid (mg)
A1 B1 c1 01
3.0
6.0 6.0 6.0 6.0
0.0 0.0
1.0 0.5
1.0
0.0 1.0
Table 2. Atomic Percent of 0, N, and C Determined by XPS
sample HPC (control) AI
B1
c1 01
EXPERIMENTAL SECTION Table 1 lists the formulations of the leuprolide/HPC films investigated in this study. The films were prepared using 2% by weight HPC in methanol solutions, and all films contained 6 mg of HPC. The films were prepared by pipeting 100 pL of the dispersions onto a clean PTFE substrate and air-drying at room temperature. XPS analyses were performed on both air and substrate surfaces of the cast films. These latter surfaces were exposed by removing the films from the substrates. TOF-SIMS analyses were performed on both the air and substrate surfaces as well as cross sections of the polymer mixtures. Cross-sectional thickness ranged between 100 and 200 pm, and cross sections were prepared by shock freezing the samples in liquid nitrogen followed by freeze fracturing. Frozen sections were warmed to room temperature prior to analysis. Reference data was acquired for both leuprolide and HPC. Leuprolide was dissolved in ethanol and solution cast onto an acidetched Ag substrate. The reference HPC film was cast onto a PTFE substrate using the same procedure described above for the drug patches. XPS spectra were collected on a Perkin-Elmer 5600 XPS/SIMS instrument. XPS scans were acquired using a monochromatic Al X-ray (AlKa = 1486.6 eV) source to minimize radiation damage to the sample and to optimize the instrumental energy resolution in the analysis. A lowenergy electron gun was used to minimize sample charging. XPS data were acquired in both low-resolution survey scans (0-1100 ev) and high-resolution multiplex scans for each of the elements detected on the surface region. TOF-SIMS spectra and ion images were acquired using the Charles Evans & Associates TFS time-of-flight secondary ion mass spectrometer. Secondary ions are produced by a pulsed primary ion beam and are accelerated to a k e d kinetic energy before entering the time-of-flight drift region of the mass spectrometer. TOF-SIMS mass analysis measures the time required for secondary ions to travel the distance between the sample surface and an ion detector! Low primary ion doses were used to minimize chemical alteration of the surface during the analysis. Typical primary ion doses were lo1*ions/cm2, which nominally consume 0.1%of the top monolayer. Lowdose conditions produce elemental, molecular, and/or structurally significantfragment ions from the near-surface region from essentially intact inorganic and organic solids. TOF-SIMS mass spectra and ion images were acquired using an ion microprobe technique in which a pulsed, microfocused primary ion beam is rastered over the sample surface. Secondary ion images are acquired by synchronizing the ion arrival times (ion masses) with the position of the primary ion beam in the raster. This raster imaging technique is similar to those employed in scanning electron microscopy (SEM),’ and TOF-SIMS images _
_
_
_
~
(6) John, C. M.; Chakel, J. A; Odom, R W. In Secondary Ion Moss Specfromety SIMSWIk Benninghoven, A,, Janssen. K T. F., Tumpner, J., Werner, H. W.. Eds.; Wiley & Sons: New York, 1991; p 657.
3872 Analytical Chemisffy, Vol. 67,No. 27, November 7, 7995
O(air) O(sub) 34.8 23.0 28.0 29.9 25.9
29.8 33.8 32.6 31.8 22.9
N(air) N(sub) C(air) C(sub) ndn
ndn
12.6
0.6 1.9 2.2 9.1
7.1 4.9 7.4
65.2 64.1 64.6 65.1 65.6
60.0 62.1 63.9 65.2 66.6
nd, not detected.
contain the distribution of mass-resolved secondary ions within the image field! A liquid metal ion gun W I G ) employing a microfocused ‘j9Ga+beam was used in these microbeam analyses. The ‘j9Ga+beam used in these experiments was focused to a 0.5 pm diameter spot size determined using a copper grid having 25 pm diameter grid bars mounted on an aluminum substrate. Submicrometer image resolutions were validated from both the grid edges and small adventitious particles. The lateral resolution of SIMS ion images depend on the spot size of the primary ion beam and the topography of the sample surface. The image resolutions reported below ranged between 3 and 5 pm and this factor of 6-10 decrease in image resolution with respect to the primary beam spot size is due primarily to sample topography. Mass resolution in these experiments was typically >lo00 measured at mass 41. Typical primary ion impact energies were 21.5 (+ions) and 28.5 keV (-ions). Sample surfaces were flooded with pulses of lowenergy electrons (average impact energies, a few electronvolts) between primary ion pulses which m i n i i e d sample charging during the analysis. All ion images were acquired into 256 x 256 pixel areas at two different image magnifications correspondingto image areas of 150 x 150 pm2 to 250 x 250 pm2. These image fields contain 1.7 and 1.0 pixels per pm, respectively. The secondary ion intensities within each image are the integrated secondary ion counts at each pixel location. Thus, the intensity scale in the images directly represents the number of ion counts in that image. A positive ion fast atom bombardment (FAI3) mass spectrum of leuprolide was recorded on a Finnigan MAT MAT95 mass spectrometer using a glycerol/thioglycerol matrix. RESULTS AND DISCUSSION XPS Results. Table 2 summarizes the atomic surface concentrations measured by XPS, and the two sets of values correspond to the polymer/& interface and polymer/substrate surfaces. The data show large variations in the nitrogen levels of the two surfaces, and since the peptide is the only sign5cant source of N in the mixtures, the data indicate that the two surfaces have different peptide concentrations. In contrast to this behavior, drug/polymer films containing oleic acid show higher N concentrations at the substrate surface. For example, although samples 01 and B1 have similar N concentrations at the air surface; the N levels at the substrate surface of sample 01 is > 4 times the value observed for sample B1. If the drug were uniformly distributed through the HPC matrix, the observed N concentrations would have been 7%for sample 01 and 10%for sample B1. (7) Goldstein, J. I.; Newbury, D. E.; Echlin, P.; Joy, D. C.; Fiori, C.; Lifshin. E. Scanning Electron Microscopy and X-Ray Microanalyis; Plenum Press: New York, 1984. (8) Schueler, B. Microsc. Microanal. Microstruct. 1992,3, 1.
0 50
i'
r
X 40
,600 'OOi
0.Peptide Ions
80
f
601
I ,580
830 800
do
120
I
11210)
A
1iw
1OW
K)
l d
2r
loo; 80
11% 200
250
300
350
400
450
500
Figure 1. Positive ion TOF-SIMS spectrum of the polymerlair surface of pure HPC (control). Prominent Na+ and K+ ion peaks and apparent polymer fragment ion peaks, such as those at mlz59, 155, 21 3, 301, 359, are detected. Some of these fragment ions are likely sodiated species.
The differences between the calculated and measured values suggest that leuprolide is nonuniformly distributed and the oleic acid significantly affects the drug distribution. TOF-SIMS Mass Spectra. A positive ion TOF-SIMS spectrum of a HPC control sample shown in Figure 1 contains a number of peaks characteristic of hydroxypropyl cellulose including peaks at mass-to-charge ratios (mlz) 31 (CH30+) and 45 (CzHsO+) and a peak at m / z 59 corresponding to C3H70+. The spectrum is displayed out to mlz 500, and no peaks were observed above this mass. However, there are a number of low-intensity peaks between m/z 100 and 500 including peaks at 101,113,127, 141, 155,213,271, 301, and 359 Da. Several of the peaks in the mlz 100-200 range are separated by 14 Da, indicating probable loss of a methylene (CH2)fragment. If these ions are fragments of hydroxypropyl cellulose, they are not produced by simple bond cleavages. In addition to these organic peaks, a series of peaks characteristic of siloxanes such as poly(dimethylsi1oxane)are also detected in this spectrum. This siloxane has the general formula [-(CH3)2Si0-ln, and positive ions produced from this organic silicone include peaks at 73, 147,207,221,and 281 Da.9 Siloxanes are commonly observed surface contaminants and are typically introduced by exposing the surface to materials containing silicones or silicone contamination. Figure 2 is a positive ion TOF-SIMS spectrum produced from a solid residue of leuprolide dispersed on an Ag substrate. The protonated molecular ion, (M H)+, is readily apparent at 1210 Da. Leuprolide molecular ions formed by cationization with Ag+ produce peaks at m / z 1316 and 1318 corresponding to the molecular adduct with the two Ag isotopes. In addition, ions corresponding to (M - H + Ag)'+ are detected at m/z 1315 and 1317. The lower mass region of the spectrum also contains a number of peaks characteristic of fragments of leuprolide including peaks at m/z 353 and 466, which are discussed in more detail below.
+
(9) Briggs, D.; Brown, A,; Vickeman, J. C. Handbook ofstatic Secondary Ion Muss Spectrometry; John Wiley & Sons: Chichester, UK, 1989.
1225
1200
1242 1250
1300
1350
Figure 2. Positive TOF-SIMS spectrum of leuprolide on a gold substrate. Protonated molecular ions are apparent at mlz 1210 as are Ag cationized ions at mlz 1316 and 1318.
0= Peptide Ions 80-
60 40 20
300
400
500
600
700
800
11209) I
100
l
60 40-
l
20-
900
1000
1100
1300
1200
Figure 3. Positive ion FAB spectrum of leuprolide in glycerol. Matrix ion peaks can be observed at mlz 369, 461, and 553. A prominent protonated molecular at m/z 1210 and a series of y-ion fragments can be seen. Two w n ion peaks are detected at mlz 353 and 466.
Figure 3 is a FAB spectrum of leuprolide dissolved in a glycerol/thioglycerolmatrix. Molecular and protonated molecular ions detected at m/z 1209 and 1210 are the most intense peaks, and a series of C-terminal y, ions are also detected at 525, 688, 776, 962, and 1098 Da corresponding to the series y4-y~, respectively.lOJ1In addition,peptide molecules form w,,fragments by loss of substituents located on 4, carbons of amino acid residues,11J2and the detection of w, ions provides a direct method of distinguishing leucine and isoleucine residues. Two abundant leuprolide fragments formed as C-terminal wn ions (w3 and w4) are observed at mlz 353 and 466 in this FAB spectrum. The presence of basic amino acid residues such as arginine near the C-terminus favor the formation of C-terminal ions in FAB and liquid secondary ion mass spectrometry (LSIMS) s p e ~ t r a . ' ~ J ~ ~
(10) Roepstorff, P.; Fohlman, J. Biomed.
Muss Spectrow. 1984, 11, 601.
(11) Biemann, K In Methods in Enzymology; McCloskey, J. A., Ed.; Academic Press: San Diego, 1990; Vol. 193, pp 455-479. (12) Johnson, R S.; Martin, S. A; Biemann, IC;Stults, J. T.; Watson, J. T. Anal. Chem. 1987,59, 2621.
Analytical Chemistry, Vol. 67, No. 21, November 1, 1995
3873
2.5
2.0
(M +lip
801 i
207
1
::i 2.
40
221
2.5
1.0
Wt
(M + H).
I
I
(M + Nap
0.5 0.0
Flgure 4. Positive ion TOF-SIMS spectrum of a cross section of a polymer film composed of 6 mg of HPC and 3 mg of leuprolide (sample A l ) . Peaks observed in TOF-SIMS spectra of the pure polymer are detected at m/z 101, 113, 127, 155, 213, 271, 301, and 359. A number of peptide fragment ions can also be observed as indicated along with protonated molecular ion at m/z 1210.
However, C-terminal w, ions such as w3 and w4 are not generally observed in FAB or LSIMS spectra unless the molecular ion undergoes highenergy collision-induced dissociation (CID) .11J2J4-16 Positive ion TOF-SIMS spectra of cross sections through the polymerlair surfaces of films containing leuprolide only (sample Al) and leuprolide oleic acid (sample 01) are illustrated in Figures 4 and 5, respectively. Protonated molecular ions are detected in both spectra along with several leuprolide fragment ions detected in FAB spectra of leuprolide/glycerol mixtures. Peaks for (M H)+ and (M Na)t ions are detected at mlz 1210 and 1232, respectively, in Figure 5. A protonated molecular ion peak can also be observed in Figure 4 along with a higher mass adduct at 1242 Da. The identity of this adduct is not certain, but the ion mass is consistent with an oxidized species (M + 02 H)t. Peaks are also detected in both TOF-SIMS spectra for the w3 and w4 ions observed in the FAB spectrum. The intensities of these two w, ions are approximately equal to the molecular ion intensity. In addition, peaks for sodium adducts of the two w,, fragment ions are detected at mlz 375 and 488, respectively. Two z, ion peaks are also detected in these spectra, zz at 284 Da and 23 at 397 Da. Two peaks for N-terminal ions, the bz and possibly bl ions, are also observed at mlz 249 and 112 in the spectrum of the sample 0 1 . Immonium ions having the general formula (NH2=CHR)+,where R is the amino acid side chainloor immonium-like ionsI7 corresponding to His, Trp,Tyr, and Arg, are labeled with the single letter abbreviation for the amino acid residues. The peak at mlz 112 could be immonium-like ions from
+
+
+
+
(13) Johnson, R S.; Martin, S. A; Biemann, K Int. J Mass Spectrom. Zon Processes 1988,86,137. (14) Martin, S. A.; Biemann, K. Znt. J. Mass Spectrom. Zon Processes 1987,78, 213. (15) Naylor, S.; Moneti, G. Rapid Commun. Mass Spectrom. 1989,3, 405. (16)Stults, J. T. In Biomedical Applications of Mass Spectromety; Suelter, C. J., Watson, J. T., Eds.; John Wiley & Sons: New York, 1990. (17) McCormack, A L.; Somogy, A; Dongr, A. R; Wysocki, V. H. Anal. Chem. 1993,65,2859.
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Analytical Chemistry, Vol. 67, No. 21, November 1, 1995
Flgure 5. Positive ion TOF-SIMS spectrum of a cross section of a polymer film composed of 6 mg of HPC and 1 mg each of oleic acid and leuprolide (sample 01). The peaks associated with the HPC polymer are less intense and the peptide-related peaks are more intense relative to the spectrum of sample A1 shown in Figure 4. The protonated molecular ions of leuprolide are more abundant in the spectrum of sample 0 1 as well.
Arg17 or bl ions containing the N-terminal pyroglutamic acid (or 5-oxoproline) residue. More intense peptide peaks are produced from TOF-SIMS spectra of leuprolide in mixtures of HPC and oleic acid. Figure 6 illustrates the normalized relative intensity, expressed as percent of the total ion intensity in the spectra, for the 12 most abundant peptide peaks produced from these two formulations. Two spectra were averaged for the leuprolide and HPC material while three spectra were averaged for the leuprolide oleic acid sample. The percent relative standard deviation (%RSD) for these data varied between 5 and 30%for most of the peaks. Interestingly, although the average total intensity in sample 0 1 spectra is only 50%higher than the average intensity in sample A1 spectra, the relative intensity of peptide peaks produced from the leuprolide + oleic acid samples is 3 times larger. This larger relative intensity is especially noteworthy since the bulk leuprolide concentration in the 0 1 samples is l/3 the bulk leuprolide concentration in the A1 samples. Thus, the addition of oleic acid to the HPC matrix has increased the ionization efficiency of the peptide molecular and fragment peaks by 1 order of magnitude. If the ion intensity is proportionalto peptide surface Concentration, the addition of oleic acid appears to have significantlyincreased the leuprolide concentration over the cross section of the polymer. The XPS data (Table 2) do not demonstrate an increase in leuprolide concentration for the mixture containing oleic acid. In fact, these data indicate that the surface concentration of leuprolide is higher on the Al sample. The discrepancy between TOF-SIMS and XPS data is best explained in terms of the analytical zones for each analysis technique. XPS signals were produced from the top 10 nm of the air-exposed and substrate exposed surfaces of the mixtures while the TOF-SIMS mass spectra are produced from the top one to two monolayers of the polymers along cross sections of the samples. Thus, the differences in relative concentration of leuprolide between the XPS and TOF-SIMS data
+
0.45 0.4 0.35 *.
.-cIn.
0.3
C
- 0.25
Sample 01
E 0
;.
-
0.2
0 Sample
Q
AI
al
a 0.15 0.1 0.05 0
110
112
130
136
159
170 249 284 ion mass(m/z)
353
397
466 1210
Figure 6. Graph of the intensities, normalized as a percent of the total ion intensity of the spectrum, of the peptide peaks for samples A1 and 0 1 . The graph illustrates the greater prominence of the peptide peaks in the oleic acid-containing device, sample 0 1 , compared to the spectrum of sample A l . The greater relative abundance of low-mass fragment ions such as those at mlz 110, 112, 130, and 136 compared to higher mass ions such as the molecular ions at mlz 1210 is also illustrated.
obviously relates to the differences in analysis locations and sampling depths from which the respective data were acquired. The most important observation in this research relates to the differences in relative concentration of leuprolide along cross sections of the two polymer blends. Numerous peaks detected in the spectrum of the pure HPC polymer are also observed in the spectra of the polymer mixed with leuprolide. For example, spectra of the leuprolide/HPC mixture contain peaks at m/z 113, 127, 155, 243, 271, 301, and 359 which, although detected on sample 01, are not as prominent as those in the spectra of sample Al. The relative intensities of these low-mass HPC peaks are 5-10 times larger for sample A1 compared to sample 01. These observations strongly suggest that the near-surface chemistry of the two formulations is significantly different. For example, oleic acid could act as a surface coating if it migrated from the bulk polymer onto the surface and this coating could suppress the signals from HPC. Although oleic acid may be concentrated on the surface of sample 01, the surface is not highly enriched in oleic acid since negative ion spectra of the leuprolide + oleic acid samples have an (M H)- ion for oleic acid at m/z 281.3 which constitutes only 0.1%of the total negative ion intensity. The spectra of the leuprolide oleic acid samples also contain 3 x higher intensities for the various PDMS peaks than the other HPC samples, indicating that the 01 samples have been exposed to siloxanes. The ions detected at m/z 221 could contain both siloxane and the a2 fragment ions of leuprolide. A siloxane coating could suppress ionization of the HPC peaks, but this coating should also reduce the yield of leuprolide peaks. However, since the oleic acid samples produce larger relative abundances of peptide peaks, the data suggest that oleic acid has altered the surface chemistry of the solid. This change could simply be an increase in the surface densitiy of H+ from the acid, or it could be a more complex process involving selective migration of the
+
leuprolide to the polymer surface. Acid is commonly added to the liquid matrix in FAB analyses of peptides to enhance the molecular and fragment ion intensities, and hence, it is likely that the oleic acid simply increases the number of protons at the solid surface. In summary, TOF-SIMS spectra of sample 01 have the most intense peptide peaks while spectra of the polymer mixed with only leuprolide produce intense peaks characteristic of HPC. Only a few of the peaks for the yn ion series observed in FAB analysis of leuprolide are detected in TOF-SIMS analysis of leuprolide in HPC, and the relative intensities of protonated molecular ions of leuprolide are considerably lower in the TOF-SIMS spectra of the polymer/peptide mixture than in FAI3 spectra. The most dramatic differences between the FAB data of leuprolide dissolved in glycerol and the TOF-SIMS spectra of leuprolide in the solid matrices are the greater relative intensities of the w3 and w4 ions in the TOF-SIMS. These peaks have intensities comparable to the protonated molecular ion. Side-chain cleavage to form w-type fragment ions was recently observed in collisions of peptide ions with solid surface^,'^ and the mechanisms of bond breakage in surface-induced dissociation are probably very similar to those induced by ion beam sputtering of a solid. Although the primary ion/solid surface impact energy in these TOF-SIMS analyses is much higher than the center of mass collision energies employed in the surface-induced dissociation study, a sputtering ion beam initiates a collision cascade (bond breakage zone) within the solid and the beam energy is rapidly di~sipated.'~ Thus, at distances several atomic diameters from the initial impact point, the average ~~~
_____
(18) Falick, A. M.; Hines, W. M.; Medzihradszky, K. F.; Baldwin, M. A; Gibson, B. W. J. Am. SOC.Mass Spectrom. 1993,4 , 882. (19) Sigmund, P. In Sputterilrg by Particle Bombardment; Behrisch, R., Ed.;
Springer-Verlag: Berlin, 1981; Vol. 1.
Analytical Chemistry, Vol. 67, No. 21, November 1, 1995
3875
Sample A1 : (M + H)+,Convolved
Sample A1 :Total + Ion Image
mI
Sample A1 : C,H,O’ (mlz 59) Ion Image
Sample A l :
Sum d z 136 + 353 + 466 + Ions. Convolved
I
50 m
Sample A1 : mlz 70 Ion Image, Convolved Figure 7. Five positive ion images acquired simultaneously from one region on a cross section of sample At: a total ion image (A, top left); an image (6,middle lett) of a polymer fragment ion, C3H,0+ at d z 5 9 a convolved image (C, bottom left) of immonium-type’8 arginine-derived C4HaNf ions at “ z 7 0 a convolved image (D, top right) of the protonated molecular ions; and a convolved and summed image (E, middle right) of tymsineiJarived immnium ions at ndz 136 and w3 and wn ions at “1353 and 466, respectively. These images clearly reveal the nonunifonity in the leuprolide distribution. which is more concentrated on the polymerlair side ofthe device on the bottom of the images. The dark regions at the top and bottom are gaps between the cross sections and the metal jaws of the cross-sectionalholder. Some of the ion abundance at m‘z 59 is likely due to immonium-relatedions from arginine as are ions observed at d z 100 and t12.‘8
kinetic energy of the recoiling target atoms is sufiiciently low to permit desorption of intact molecular ions or largemass fragment ions. A fraction of these sputtered or desorbed species could be ionized, thus giving rise to the secondary ion signals.20 (20) Wl!iams. P.AppL Surf. Sci. 1982,13,241.
3876 Analytical Chemistw, Vol. 67, No. 21, November 1, 1995
TOF-SlMS Ion Images. Ion images were acquired from cross sections of the two samples containing leuprolide. The cross d o n s were mounted in specially designed metal holders,
and samples were positioned horizontally such that the Teflon side of the devices was at the top of the images. The orientation of these cross sections was confirmed by the localization of F-
:loo
A
Sample 01: (M + H)', Convolved
Sample 01: Total Ion Image
i
Sample 01: mlz 70 Ion Image, Convolved
r16
/I
Sample 01: Sum m/z 136 + 353 + 466, + ions, Convolved
Figure 8. Four positive ion images acquired simultanwusly from one region on a cross section of sample 01: A total ion image (A, top left); a convolved image of immonium-related ions from arginine (6. bottom left); a convolved image (C, top right) of protonated molecular ions; and a convolved imaas ID. bottom riaht) of summed lvrosine-derived immonium ions, w:, ions at d.?353 and wa ions at "r 466 similar to that shown in Figure%. The relative-uniformity of the~distributionof le'uprolide through the cross seclions of sample 0 1 containing oleic acid is clearly illustrated (compare Figure 7E).
and other negative ions produced from the Teflon substrate. Two different regions on sample Al were analyzed in the positive ion mode, and one region was analyzed in the negative ion mode. Three different regions were analyzed on sample 01, and one of these regions was also analyzed in the negative ion mode. The difterent regions on each device produced very similar results. Images of five positive ions produced from sample A1 are illustrated in E i r e 7; all of these images were acquired from a single region on the cross section. Four different ion images fromsample 01are presented in Fgure 8, and these images were also acquired from one region on this sample. Total secondary ion images of the cross sections illustrated in Fires 7A and 8A reveal the topographic features of the surfaces on a microscopicscale. The top regions of the images correspond to the polymer/substrate side, and the bottom regions are the polymedair interface of the samples. The dark regions at the top and bottom are gaps between the cross sections and metal jaws of the cross section holder. Both images exhibit surface roughness approximately 2-4 pm in depth. This roughness is probably the result of the freezefracturing technique used to prepare the cross sections. The images also indicate that the sample thickness is slightly different for the two materials, where sample A1 is -200 pm thick while sample 01 is 120 pm thick.
The relatively uniform intensity distributionsacross these samples indicate that the sample topography does not seriously effect ion emission. In addition, the total ion images suggest uniform composition of the major constituents (C, H, 0)along the top monolayers of the cross sections. The total ion intensily in the image of sample A1 was 2.8 x lo6counts while the total intensity in sample 01 was 3.6 x lo6 counts. Images of the C3HIO+ ions at m/z 59 (Figure 7B) are diagnostic for the hydroxypropyl cellulose polymer wen though a fraction of the ions at this mass could be immonium-type ions produced from arginine residues of the peptide?8 S i c e other immonium-type ions from arginine (at m/z 87. 100, and 112l4 have very low intensities in this spectrum, immoninm ions probably make only a small contribution to the m/z 59 signal. The intensity distributions of CSH70+ are uniform along the surfaces of both samples and its intensity is a factor of -10 higher for sample AI. The relative distribution of leuprolide in the two cross sections is revealed in the convolved ion images shown in Figure 7C-E for sample Al and F i e 8B-D for sample 01. These images have been convolved using a 3 x 3 pixel kernel at unit weighting for each element. Immonium and immonium-type ions fromthe Analylkal Chemistfy, Vol. 67, No. 21, November 1, 1995
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C-terminal proline and arginine residues of sample A1 are illustrated in Figure 7C. This image clearly shows a nonuniform ion intensity distribution with a greater relative concentration of the peptide at the polymer/air interface of the cross section, which corroborates the XPS results (Table 2). Nonuniform distributions of other peptide peaks on the sample A1 cross section are revealed in the protonated molecular ion image in Figure 7D and the summed ion image shown in Figure 7E. This latter image was created by summing the images of the immonium ion for tyrosine at m / z 136 with images of the w3 (mlz 353) and w4 (mlz 466) ions. The intensity distributions for each of these fragments were similar, and hence, summing the signals produces a more intense image, which better illustrates the distribution of the leuprolide. Imaging low-mass fragment ions could play a significant role in TOF-SIMS imaging of large peptides or proteins since these more massive structures often have low molecular ion yields which produce low contrast images. However, lower mass fragment ions of peptides or proteins are normally produced at adequate intensities for ion imaging, and these fragment ions can be used to determine the surface distribution of parent molecules. Although the images clearly reveal the cross section of the sample, we also observe ion signals arising from regions above and below the actual sample. The total ion image of sample A1 in Figure 7E best illustrates this off-sample signal. These ions are most likely produced by inadvertent smearing of the sample surface onto the sides of the cross-sectional holder during sample mounting. The extent of this smearing is more noticeable in the images of sample Al. The uniformity of leuprolide in the device formulated with oleic acid is clearly shown in the convolved images in Figure 8B-D, where the more uniform distribution of the leuprolide molecular and fragment ions is obvious. In particular, the protonated molecular ion image in Figure 8C shows almost uniform intensity
3878 Analytical Chemistry, Vol. 67, No. 27,November 1, 7995
along the cross section as do the sum of tyrosine immonium ions with the w3 and w4 ions as illustrated in Figure 8D. CONCLUSIONS Molecular ion microscopy provides a sensitive method for determining the distribution of organic molecules in organic matrices. The determination of the drug distribution along the polymer device would be extremely difficult without the molecular visualization provided by this microscopic imaging technique. Ion microscopy also dramatically demonstrates that leuprolide dissolved in HPC, as represented in the cross-sectional surface, is clearly nonuniform and concentrates at the polymer/air interface. By contrast, images of leuprolide ions produced from polymer containing oleic acid demonstrate more uniform cross-sectional distribution of the leuprolide. These ion microscopy results are supported by XPS data, and the increased uniformity of leuprolide is definitely related to the presence of oleic acid although the exact nature of the interaction of the acid with the mixture is not known at this time. However, possible explanations for the detection of higher intensity and more uniform leuprolide peaks from the acidtreated polymer are the reduced surface free energy of leuprolide in the acidic solid or an increased degree of solvation of the peptide in the acidic matrix. ACKNOWLEDGMENT The authors thank Dr. Kenneth Matuszak for suppling the FAB mass spectrum of leuprolide. Received for review May 8, 1995. 1995.e
Accepted July 27,
AC950439N ~
Abstract published in Advance ACS Abstracts, September 1, 1995.
