Anal. Chem. 2003, 75, 2950-2958
Simultaneous Time-of-Flight Secondary Ion MS Quantitative Analysis of Drug Surface Concentration and Polymer Degradation Kinetics in Biodegradable Poly(L-lactic acid) Blends Joo-Woon Lee† and Joseph A. Gardella, Jr.*
Department of Chemistry, University at Buffalo, The State University of New York, Buffalo, New York 14260-3000
This paper reports a new quantitative method of analyzing both the earliest stage of degradation of a polymer and the surface concentration of an additive using time-of-flight secondary ion mass spectrometry (TOF-SIMS). The static SIMS spectra of triphenylamine (Ph3N)/poly(L-lactic acid) (PLLA) (20:80 wt %) blend matrixes hydrolyzed in buffered conditions within a short-term (0 h), respectively. On the other hand, the degradation kinetics13,14,18 in a specific base catalysis system is derived on the basis of base-catalytic hydrolysis mechanism. The rate law in base buffer system can be expressed as eq 3. Since [OH-] at buffered pH 10.0 has not
-d[ester]/dt ) k[ester][OH-]
(3)
been changed during the hydrolysis treatment, eq 3 can be derived to a pseudo-first-order reaction (eq 4), where kpH10 ) k0 +
-d[ester]/dt ) kpH10[ester]
(4)
kH+[H+] + kOH-[OH-] ) k0 + kH+Kw/[OH-] + kOH-[OH-] and Kw ) [H+][OH-] = 10-14. Using the relationship (eq 5) between [ester] and Mn, where F is the density of the polymer, integration
[ester] ) F(DP - 1)/Mn
(5)
can lead to eq 6 as the same as eq 2. The average DP at time t is
ln[(DP - 1)/DP] ) - kpH10t + ln[(DPS - 1)/DPS]
(6) ) (2) (6)
defined by eq 7 as the repeating number of Mn of PLLA
DP ) (Mn - 18)/72
(7)
degradation products, where 18 is the mass of both end groups and 72 is the mass of the PLLA repeat unit. RESULTS AND DISCUSSION Positive TOF-SIMS spectra have been analyzed to provide quantitative data for simultaneous detection of Ph3N concentration from and hydrolytic PLLA degradation at the surface of Ph3N/ PLLA blend matrixes. Figure 1 shows the comparison of low (AD) and high (E and F) mass portions of the spectra obtained from pure PLLA (A and C), pure Ph3N (B), and Ph3N/PLLA (20:80 wt %) blend matrixes (D-F) hydrolyzed in various conditions. Figure 1A shows the characteristic PLLA fragment ion,13,18 [C3H4O]•+ ) 56 m/z, generated from the polymer repeat unit through the ionization process during TOF-SIMS measurement. Figure 1B obtained from pure Ph3N shows the most intense protonated molecular ion peak, [Ph3NH]+, at 246 m/z, and the molecular ion (30) Zhu, K. J.; Hendren, R. W.; Jensen, K.; Pitt, C. G. Macromolecules 1991, 24, 1736-1740.
Figure 1. Comparison of low (top) and high (bottom) mass portions in positive TOF-SIMS spectra: (A) characteristic fragment ion of pure PLLA; (B) molecular ion of pure Ph3N; (C) pure PLLA in m/z ) 241-247 after hydrolysis treatment in pH 10.0 buffer for 24 h; (D) [Ph3NH]+ observed at the surface of untreated Ph3N/PLLA (20:80 wt %) blend matrixes; (E) and (F) Ph3N/PLLA (20:80 wt %) blend matrixes hydrolyzed for 24 h at 37 °C in pH 7.4 and pH 10.0 buffers, respectively. Over the mass range m/z ) 600, the intact oligomeric degradation product ions, [nMmon + H2O + Na]+, are marked with an asterisk (/).
([Ph3N]•+), an odd-electron ion, is observed at 245 m/z. However, the peak from pure PLLA matrixes in Figure 1C is overlapped at 245 m/z with comparable intensity. Due to the contribution of PLLA, the most intense peak intensity observed at 245 m/z of Figure 1D from Ph3N/PLLA (20:80 wt %) blend matrixes is attributed to the two different ion species. Therefore, the peak intensity at 246 m/z from the blend matrixes is taken to be representative of the concentration of Ph3N at the surface. The high-mass portion of the spectra in panels E and F of Figure 1 demonstrates the effect of pH buffer medium on hydrolysis treatment of Ph3N/PLLA (20:80 wt %) blend matrixes for 24 h at pH 7.4 and pH 10.0, respectively. The most intense peaks are labeled with an asterisk (/). The spacing between two consecutive peaks is equal to the mass of the PLLA repeat unit (Mmon ) 72.02 Da). This allows the polymer to be identified. / is assigned to Na+-cationized PLLA hydrolysis product as an intact molecular ion, [nMmon + H2O + Na]+. The source of Na+ is the buffered environmental medium. The distribution of peaks in Figure 1F is extended to higher mass than those observed in Figure 1E. The intensities of intact oligomeric hydrolysis product ion peaks in Figure 1F show a typical distribution curve, while the intensities of fragment ion peaks decrease as m/z increases.
This phenomenon indicates that the generation of hydrolysis products increases in the basic buffered condition and the probability of desorption is greater than that of fragmentation in the high-mass region. Hydrolysis treatments in acidic sodium biphthalate-buffered condition (e.g., pH 4.0 at 25 °C from J. T. Baker Inc.) were also carried out in the same way as under neutral and basic conditions. However, the spectra (not shown) show little change in degradation-generated PLLA oligomeric peak distributions and intensities despite increasing hydrolysis time up to 48 h. This observation is contrary to our previous results,14,15 where we reported the acceleration of degradation rate at the same acidic condition for pure PGA matrixes. Therefore, it can be postulated in the present work that little significant change in TOF-SIMS spectra for Ph3N/ PLLA blend matrixes hydrolyzed in acidic conditions might be due to the retardation effect of Ph3N on hydrolytic degradation of PLLA in sodium biphthalate buffer (NaO2CC6H4CO2H, 0.05 M; buffer capacity, 0.016; ionic strength, 0.05). This is because the amine group with a lone pair electrons in Ph3N would react with the carboxylic acid (or sodium salt) of sodium biphthalate. In future work, the detailed chemistry between Ph3N and sodium biphthalate of the acidic buffer will be investigated by quantifying Analytical Chemistry, Vol. 75, No. 13, July 1, 2003
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Figure 2. Integrated relative intensity [Ph3NH]+/[C3H4O]•+ of a series of Ph3N/PLLA blend matrixes as a function of blend ratio.
the change in TOF-SIMS at a certain hydrolysis time of Ph3N/ PLLA blend matrixes as a function of Ph3N concentration incorporated in the blend matrixes. Otherwise, it would be valuable to blend a neutral additive molecule with PLLA in order to better understand how the nature of an additive would affect the hydrolytic degradation process of PLLA blend matrixes in acidic buffer. For the present study, we decided to focus on the quantitative results from neutral and basic conditions. By considering the information in Figure 1, we propose that the low-mass portion of the TOF-SIMS spectra can be used to determine the profile of Ph3N surface accumulation from the blend matrixes and the high-mass portion for the hydrolytic degradation kinetics of PLLA at the surface of Ph3N/PLLA blend matrixes. This combination of quantitative data for both an additive and a biodegradable polymer can be simultaneously extracted from the blend matrixes. Low-Mass Analysis for Ph3N Accumulation Profile. The integrated relative intensity of the protonated Ph3N, [Ph3NH]+, divided by the integral intensity of the peak at 56 m/z, [C3H4O]•+, gives the ratio for the determination of Ph3N concentration accumulated at the surface of Ph3N/PLLA blend matrixes. This is measured as a function of each blend ratio (wt %) to develop a standard calibration curve in Figure 2, where the error bars reflect the standard deviation (SD) at each blend ratio. Good linearity (R2 ) 0.998 35) is obtained from the 10:90 up to the 40: 60 blend (in wt %). At the increased amount of Ph3N (>40 wt % Ph3N) blended in the matrixes, a linear curve fit cannot be applied. This is likely due to the saturation of the surface with Ph3N. In the present study for the surface concentration of an additive and hydrolytic degradation of a polymer, the 20:80 wt % Ph3N/ PLLA blend matrix is chosen and hydrolyzed at various hydrolysis times at two buffered pHs. The Ph3N accumulation profiles are shown in Figure 3. The data points represent the average of integrated relative intensity ratio from up to 5 times measurements, and the error bars reflect the SD at each hydrolysis time. The rate of increase in relative intensity of [Ph3NH]+/[C3H4O]•+ within 24-h hydrolysis time in basic buffered condition (ApH10 ) -2.7 × 10-4) is ∼9 times faster than that in physiological buffer (ApH7.4 ) -0.3 × 10-4) up to 48 h. Saturation of Ph3N at the surface of blend matrixes occurs after 24 h of basic buffered hydrolysis 2954 Analytical Chemistry, Vol. 75, No. 13, July 1, 2003
Figure 3. Profiles for Ph3N surface accumulation from Ph3N/PLLA (20:80 wt %) blend matrixes hydrolyzed in two pH buffered conditions as a function of hydrolysis treatment time. The curves were fit with an empirical exponential expression, [([Ph3NH]+/[C3H4O]•+)]t0t ) Ae(t-t0)/11.56: ApH7.4 ) -0.3 × 10-4 for pH 7.4 and ApH10 ) -2.7 × 10-4 for pH 10.0.
treatment. Therefore, it can be concluded from the above observation that the accumulation profile from the surface of blend matrixes is pH sensitive. To test the effects of hydrolysis on the pure polymer, the ratio of the relative intensity of the peak at 246 m/z from the pure PLLA to [C3H4O]•+ was calculated for hydrolysis in each pH buffer medium at 24 h. The values were 2.62 × 10-3 for pH 7.4 and 2.59 × 10-3 for pH 10.0. These values indicate that for pure PLLA matrixes the relative intensity of the peak at 246 m/z divided by [C3H4O]•+ is independent of pH and hydrolysis time. The details of the pH sensitivity of the Ph3N surface accumulation will be discussed in comparison with the results from the hydrolytic degradation study of PLLA at the surface of Ph3N/PLLA (20:80 wt %) blend matrixes hydrolyzed in the same conditions. High-Mass Analysis for Hydrolytic PLLA Degradation Kinetics. As illustrated in Figures 1 and 4, the comparison of basic buffer to physiological buffer shows that the relative intensity of hydrolysis-generated oligomeric PLLA degradation products increases and their distribution is extended to higher mass with increasing hydrolysis time. We constructed a system to test whether the effect was due to the presence of Na alone. As shown in Figure 4A obtained at pH 10.0, the TOF-SIMS spectrum from untreated Ph3N/PLLA blend (20:80 wt %) matrixes doped with NaCl shows an exponential decrease in intensities as m/z increases. Very little signal is observed over the range from 600 m/z except a noisy background. This observation is likely due to the strong entanglement of the long-chain PLLA, which mediates against desorption of ions with higher mass above 600 Da without multiple bond breaking events along the backbone. Upon exposure to the hydrolysis treatment solution, however, low-MW PLLA oligomers are gradually generated with increasing hydrolysis time as a result of the hydrolytic degradation. These degradationgenerated oligomers are shorter and less entangled; they readily desorb from the surface as secondary ions in a TOF-SIMS experiment. Therefore, the distribution of peak intensities over 600 m/z reflects the hydrolysis-generated oligomeric PLLA degradation products.13-18 In the course of the hydrolytic degrada-
Figure 4. Comparison of MWD of hydrolytic degradation reaction products adsorbed at the surface of Ph3N/PLLA (20:80 wt %) blend matrixes after different hydrolysis treatment times in pH 10.0 buffer solution at 37 °C: (A) 0, (B) 12, (C) 18, (D) 36, (E) 42, and (F) 48 h.
tion at pH 10.0, meanwhile, a double distribution of the intensities starts to develop from 12 h of hydrolysis treatment but disappears at 48 h of hydrolysis. The second distribution of hydrolytic PLLA degradation products has been postulated as being due to the crystalline phase or lower layers of the matrixes.14,15 As mentioned in the discussion of panels E and F of Figure 1, the most intense peaks labeled with / in the high-mass portion represent Na+-cationized intact PLLA degradation products, [nM+ mon + H2O + Na] . A series of ion peaks in the pattern are observed between two consecutive most intense peaks (Figure 5), and the pattern is consistent in all hydrolysis treatments. Figure 5 shows the expansion data from Figure 1F obtained from the blend matrixes hydrolyzed at pH 10.0 for 24 h. The peak labeled with a cross (+) is assigned to Na+-cationized oligomeric polyglycolate sodium salts, [nMmon + OH + 2Na]+. This indicates that one H+ from a carboxylic acid end group of a hydrolysis product is replaced with one Na+. The other five peaks labeled with A-E are identified through characteristic neutral ion dissociation and fragmentation pathways. This ion formation mechanism is likely occurring during the TOFSIMS desorption/ionization of oligomeric intact PLLA hydrolysis
products with the greater repeat numbers as parent ions.13 A series of each mass formula and representative peak assignment are summarized in Table 1 based on the repeat number of each parent ion. The MW average is extracted from the distribution of the hydrolysis-generated oligomeric PLLA degradation products over 600 m/z, by integrating the intensities of seven ion peaks, /(n), A(n), +(n), B(n), C(n), D(n), and E(n), with the same repeat number (n). The resultant sum is used as the total intensity (Ni, i ) n) for the particular oligomeric PLLA hydrolysis product, /(n): Mi ) nMmon + H2O. By summing all intensities of the ions related to the oligomer,13 we overcome a potential obstacle in the quantification attributable to the change in secondary ion yields as surface chemistry changes; and also this enhances the accuracy of the measurement.13 Therefore, the distribution of the oligomeric PLLA hydrolysis products in TOF-SIMS (Figure 6A) can be converted to the corresponding MWD function (Figure 6B) expressed with two terms (Ni vs Mi) of a statistical averaging MW calculation. Using this MWD function, Mn (number average MW) is plotted as a function of hydrolysis time in two pH buffers, respectively in Figure 7, where the error bars also reflect the SD Analytical Chemistry, Vol. 75, No. 13, July 1, 2003
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Figure 5. Characteristic oligomeric ion peak family observed within two consecutive hydrolytic PLLA degradation products of Ph3N/PLLA (20:80 wt %) blend matrixes hydrolyzed in pH 10.0 buffer for 24 h at 37 °C. Top in m/z ) 890-1070 and bottom in m/z ) 1325-1505, where neutral ion dissociation and fragmentation pathways occurring at the surface during TOF-SIMS experiment are proposed. Table 1. Representative Peak Assignment for PLLA Observed within Consecutive Most Intense Peaks
of Mn. The decrease in Mn of hydrolytic PLLA degradation products generated at the surface of blend matrixes is gradually observed with increasing hydrolysis time. This shows that the hydrolytic surface degradation study of PLLA blend matrixes is 2956
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similar to that reported for relatively higher crystalline PGA.14 The difference between results from PLLA and PGA is that for PGA we detected an initial increase followed by decrease in Mn. This difference can be explained by the preferential degradation of surface-segregated (1) amorphous phase of PGA, (2) low-MW fraction resulted from the melt-casting fabrication of PGA, or (3) both. This behavior was not observed for PLLA, likely because of the lower crystallinity and the spin-casting method used for preparation. Using eqs 2 and 6, the semilog term, ln[(DP - 1)/DP], is presented by plotting the average ( SD for a maximum of five replicate measurements at each hydrolysis time of two different pHs in Figure 8. Good linearity (R2 ) 0.998 62) with an excellent adherence to the linear model for the experimental data is obtained at the physiologically buffered pH 7.4. In pH 10.0 basic buffer conditions, however, good linearity (R2 ) 0.990 86) is only obtained from 12- to 36-h hydrolysis treatment. Each slope obtained from the linear curve fitting represents the rate constant of hydrolytic PLLA degradation at the surface of Ph3N/PLLA (20: 80 wt %) blend matrixes. This linear regression relationship in degradation kinetics is representative of the characteristic induction period of the biodegradable poly(R-hydroxy acid) bulk erosion profile.13,14,18 This is because the diffusion19 of hydrolysis products into the external hydrolysis media after the induction period
Figure 6. (A) Positive TOF-SIMS spectrum of Ph3N/PLLA (20:80 wt %) blend matrixes after hydrolysis treatment in pH 7.4 buffered condition for 48 h at 37 °C and (B) corresponding MWD function of PLLA hydrolysis products.
Figure 7. Change in Mn of hydrolytic PLLA degradation reaction products adsorbed at the surface of Ph3N/PLLA (20:80 wt %) blend matrixes as a function of hydrolysis treatment time in two different pH buffered conditions, respectively.
results in the decrease15 in polymer degradation rate at the surface as well as overall polymer weight loss. As a result, the hydrolytic PLLA degradation rate at pH 10.0 (kpH10 ) -1.70 × 10-4) is 2 times faster than that at pH 7.4 (kpH7.4 ) -8.51 × 10-5) during the induction period of bulk erosion of Ph3N/PLLA (20:80 wt %) blend matrixes. This result is due to the favorable base-catalytic hydrolysis of an ester bond. The change in hydrolytic degradation rate from the 42-h exposure at pH 10.0 may be due to (1) surface saturation of Ph3N in top-layer surface analysis using TOF-SIMS, (2) the interference attributed to the increased surface concentration of Ph3N in the secondary ionization process of oligomeric
Figure 8. Semilog plots of (DP - 1)/DP versus hydrolysis treatment time for hydrolytic PLLA degradation reaction products adsorbed at the surface of Ph3N/PLLA (20:80 wt %) blend matrixes in two different pH buffered conditions, respectively.
PLLA degradation products, or (3) the onset of PLLA weight loss in the course of bulk erosion of Ph3N/PLLA (20:80 wt %) blend matrixes. pH Effect on the Rapid Increase in Ph3N Surface Concentration. Both the Ph3N accumulation profile and hydrolytic PLLA degradation kinetics at the surface are pH-sensitive in the two different pH buffered conditions. To better understand the role of the environmental pH on the surface concentration of Ph3N, the surface accumulation profile of Ph3N has been compared with the hydrolytic PLLA degradation kinetics. The extent of initial Analytical Chemistry, Vol. 75, No. 13, July 1, 2003
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increase in surface concentration of Ph3N (ApH10 ≈ 9ApH7.4) is found to be 4.5 times greater than the corresponding change in the rates of hydrolytic PLLA degradation (kpH10 ≈ 2kpH7.4), indicating that the extent of rapid increase in Ph3N surface concentration is attributed to PLLA degradation kinetics during the induction period but not entirely due to polymer degradation. CONCLUSION TOF-SIMS enables the simultaneous acquisition of data on the degradation kinetics of a polymer and the surface accumulation profile of a water-insoluble additive at the earliest erosion stage of the blend matrixes. For Ph3N/PLLA blend matrixes, the results from TOF-SIMS allows an evaluation of the role of environmental pH effects on the initial rapid increase in surface concentration of Ph3N during the induction period of bulk erosion profile of PLLA blend matrixes. In the course of hydrolytic degradation up to for 36 h in each neutral and basic buffer system, good linearity is obtained in the kinetics study of PLLA degradation at the surface. This is responsible for the characteristic induction period of bulk erosion of the PLLA blend matrixes. The reactivity calculated from the slope at pH 10.0 is ∼2 times faster than that at pH 7.4. On the other hand, the integrated relative intensity of
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[Ph3NH]+/[C3H4O]•+ for the quantitative Ph3N assay at the surface indicates that the rate of Ph3N surface accumulation is much more pH-sensitive than the hydrolytic PLLA degradation, comparing the extent of relative increase in the initial surface concentration of Ph3N with the corresponding increase in hydrolytic PLLA degradation rate constant. With reference to the design of new biodegradable polymeric devices for clinical applications, therefore, TOF-SIMS may have considerable potential in studying and initial screening biodegradability, biocompatibility, protein adsorption, and molecular recognition. ACKNOWLEDGMENT The authors acknowledge support from the National Science Foundation Analytical and Surface Chemistry program, Grant CHE 0079114.
Received for review March 27, 2003. Accepted May 22, 2003. AC034305I
