Technical Notes Anal. Chem. 1994, 66, 3505-351 1
Determination and Characterization of Unreacted Hydroxyl Groups in a Cross-Linked Poly(ortho ester) John D. Stong' and Leonore C. Witchey-Lakshmanan Merck Research Laboratories, West Point, Pennsylvania 19486
A method is presented for the identification and quantitative determination of unreacted hydroxyl groups in a cross-linked poly(ortho ester) derived from the diketene acetal 3,9-bis(ethylidene-2,4,8,1O-tetraoxaspiro[5.5]undecane),(A)-1,2,6hexanetriol, 1,641exanedio1, and tetraethylene glycol. This method involves exhaustive derivatizationof unreactedhydroxyl groups in the intact polymer in advance of mild hydrolysis followed by gradient HPLC analysis. Each of the derivatized polymer components was synthesized and characterized, and evidence is presented demonstrating that exhaustive derivatization has occurred. Bioerodible implants based on polymeric ortho esters have shown great promise and, indeed, utility in sustained drug delivery. Controlled release of an incorporated drug is most desirably achieved through surface erosion of the polymer, which results in a steady zero-order release of The advantages of such a delivery device have prompted considerable work in recent years, especially with regard to the synthesis of new polymers and the design of the delivery device and its behavior in vivo and in vitro.' Both linear and cross-linked poly(ortho esters) (POE) derived from 3,9-bis(ethylidene-2,4,8,l0-tetraoxaspiro[ 5.51undecane) (DETOSU) and di- and trifunctional alcohols have been described for use as bioerodible matrices for the delivery of bioactive compounds. The difunctional alcohols serve as monomers and chain ends, while the trifunctional alcohols can also serve as branch points. Variation of polymer structure (with associated variations in physical and chemical properties) can be achieved by varying the types of alcohols and their stoichiometry.6 The alcohols used to prepare the poly(ortho ester) that is thesubject ofthis workare 1,6-hexanediol(HD), tetraethylene glycol (TEG), and (*)-1,2,6-hexanetriol (HT). (Henceforth it will be understood that the hexanetriol used in this work is (1) Heller, J.; Fritzinger, B. K.; Ng, S. Y.;Penhale, D. W. H. J. Controlled Release 1985, I , 233. (2) Shih, C.; Higuchi, T.; Himmelstein, K. J. Biomaterials 1984, 5, 237. (3) Sparer, R. V.; Shih, C.; Ringeisen, C. D.; Himelstein, K. J. J. Controlled Release 1984, I , 23. (4) Heller, J.; Ng, S. Y.; Penhale, D. W.; Fritzinger, B. K.;Sanders, L. M.; Burns, R. A.; Gaynon, M. G.; Bhosale, S . S . J. Controlled Release 1981, 6,217. ( 5 ) Heller, J.; Penhale, D. W. H.; Helwing, R. F. J. Polym. Sci., Polym. Lett. Ed. 1980, 18, 619. ( 6 ) Heller, J.; Penhale, B. K.; Fritzinger, B. K.; Rose, J. E.; Helwing, R. F. Contracept. Deliu. Syst. 1983, 4, 43.
0003-2700/94/0366-3505$04.50/0 63 1994 American Chemical Society
a racemic mixture, and the (k)designation will be dropped.) Presented in Figure 1 is a schematic representation of the reactions between the alcoholsand DETOSU and the structure of a hypothetical POE segment, together with a representation of the present analytical method for the determination and identification of the unreacted hydroxyl groups in this hypothetical segment. We would like to obtain as much information as possible concerning the chemical microstructure of these poly(ortho esters). This will provide more detailed correlations of such factors as reaction conditions, stoichiometry, drug loading, physical properties, and in vivo performance with chemical structure and polymerization kinetics. Presented in this paper is an analytical procedure for the determination of the quantity and identity of unreacted hydroxyl groups in this class of poly(ortho esters). The method presented here is based on a method for the determination of cross-linking developed by Shih et al.' These methods afford a measurement of the degree of cross-linking by reporting on the number of moles per sample of trifunctional monomer incorporated as a branch point. The present method also reports on the number and identity of chain ends and the relative reactivities of the individual hydroxyl groups. A brief mathematical formalism is presented together with some calculated properties which we define in the Appendix of this paper.
EXPERIMENTAL SECTION Materials. 1,6-Hexanediol,1,2,6-hexanetriol,tetraethylene glycol, and dibutyltin diacetate were all from Aldrich and were distilled under reduced pressure. They were stored in vacuo over PzOS. Triethylamine (Aldrich) was distilled and stored over P2O5. Phenyl isocyanate (Aldrich) was distilled and stored over CaC12. DETOSU was obtained from Merck and Co. and was stored in sealed 1-L bottles at -15 OC. 1,4Diazabicyclo[2.2.2]octane(DABCO) was from Aldrich and was used as received. All other reagents and solvents were used as received. Apparatus. The analytical HPLC equipment consisted of a Hewlett-Packard 1090M solvent delivery system equipped with a Hewlett-Packard 1040A diode array detector, a Shimadzu SIL-6A autoinjector, and a Rainin Model I11 (7) Shih, C.; Waldron, N.; Traugott, C. Q.J. Appl. Polym. Sci. 1993,49, 2221.
Analytical Chemlstty, Vol. 66,No. 20, October 15, 1994 3505
a
HCIO,/H20
I I
HCIOJHZO
HT-2
t
""XI; t
CH3CHzCOOH
HT-2,6
t
underivatizedalcohols
OH
Figure 1. Schematic representation of the reaction of DETOSU with 1,6hexanedioi (HD), tetraethylene glycol (TEG), and 1,2,&hexanetriol (HT) and the structure of a hypothetical poly(ortho ester) fragment. Schematic representationof the derivatization scheme of thls fragment is also shown.
column oven. Absorption spectra were measured with a Hewlett-Packard HP8450 diode array spectrophotometer. A preparative HPLC system controlled by a DEC MINC-11 computer was constructed and used for the isolation of several compounds. Synthesesand Isolation of QualitativeStandards. The basic principle of the derivatization is the reaction of the isocyanate with hydroxyl groups remaining unreacted after the polymerization of the poly(ortho ester). The polymer is then hydrolyzed, and the carbamate derivatives are analyzed via HPLC. In order to identify and quantitate these derivatives, qualitative standards were prepared by reacting the monomers with the phenyl isocyanate. The synthetic procedures used to make the qualitative standards were intended to be the most direct and obvious methods for the production of amounts sufficient for structure determination and identification of the derivatized POE hydrolysate. No attempt was made to maximize yields of these standards, which were very small (probably less than 3506
AnalyticalChemistry, Vol. 66, No. 20, October 15, 1994
1%) for 1,2,6-hexanetriol (HT) mono- and disubstituted derivatives and 1,6-hexanediol (HD) and tetraethylene glycol (TEG) monosubstituted derivatives to about 75-90% for the completely substituted derivatives of HT, HD, and TEG. For simplicity, the following acronyms will be used to indicate the various derivatives: I-(N-phenylcarbamoy1)hexane-2,6-diol (HT- 1); 2-(N-phenylcarbamoyl)hexane- 1,6diol (HT-2); 6-(N-phenylcarbamoyl)hexane-1,2-diol (HT-6); 1,2-bis(N-phenylcarbamoyl)hexan-6-01(HT- 1,2); 2,6-bis(Nphenylcarbamoy1)hexan-1-01 (HT-2,6); 1,6-bis(N-phenylcarbamoyl) hexan-2-01 (HT- 1,6); 1-(N-phenylcarbamoyl) hexan6-01 (HD- 1); 1-(N-phenylcarbamoy1)tetraethylene glycol (TEG-I); 1,2,6-tris(N-phenylcarbamoyl)hexane(HT-1,2,6); 1,6-bis(N-phenylcarbamoyl)hexane (HD-1,6); 1,8-bis(Nphenylcarbamoy1)tetraethylene glycol (TEG- 1$). The monosubstituted N-phenylcarbamates of 1,2,6-hexanetriol, 1,6-hexanediol, and tetraethylene glycol are most conveniently prepared by carefully titrating an excess of the alcoholwith small amounts of phenyl isocyanateusing DABCO
as catalyst. The various derivatives are subsequently examined by HPLC analysis of an aliquot of the reaction mixture. At low concentrations of phenyl isocyanate, a single peak (for HD and TEG) or a group of three early-eluting peaks (for HT) is seen. The total area of these peaks increases with increasing phenyl isocyanate concentration. As the phenyl isocyanate concentration is increased even further, the areas of the early-eluting HPLC peaks begin to decrease, with a concomitant increase in the areas of late-eluting peaks. These peaks are the disubstituted HD and TEG derivatives or the di- and trisubstituted HT derivatives. It is most convenient to isolate the HT, HD, and TEG monosubstituted derivatives by preparative HPLC. The HT1,6 derivativecan be synthesized and isolated directly by taking advantage of the fact that secondary alcohols react much more slowly with phenyl isocyanate than primary alcohols, in the absence of a catalyst. The inclusion of a catalyst such as DABCO and an excess of phenyl isocyanate, however, afford an almost quantitative yield of the disubstituted derivatives of HD and TEG and of the trisubstituted derivative of HT. The 1,2- and 2,6-disubstituted derivatives of H T are more difficult to prepare. It is necessary to block the C6 or C1 OH group, respectively, prior to reaction with phenyl isocyanate. As would be expected, HT will react with other nucleophiles (including benzoyl and silyl chlorides) exactly as it does with phenyl isocyanate. Thus, addition of a small amount of tertbutyldimethylsilyl chloride to a solution of HT will yield predominantly the monosilyl ethers, which can then be reacted further at the remaining hydroxyl groups with phenyl isocyanate and then hydrolyzed under mild conditions. The hydrolysis removes the silyl ethers, resulting in a mixture of HT-1,2, HT-1,6, and HT-2,6. These are then purified by preparative HPLC. Proton NMR and mass spectral data were entirely consistent with the assigned structures of all the compounds. Preparation of Standards and Determination of Accuracy and Linearity. The N-phenylcarbamate derivatives of methanol (ME-l), 1-hexanol (HX-l), 2-propanol (PR-2), and isobutyl alcohol (BU-i) were prepared by well-known methods8 Each was recrystallized from hexane. HT-1, HT-6, HT-1,6, TEG-1,8, HD-1,6, and HT-1,2,6 were prepared as described above and were recrystallized from acetonitrile. These compounds were dried for 4 days in vacuo at 40 "C. Accurately prepared solutions of PR-2, HX-1, HT-1, HT-6, HT-1,6, TEG-1,8, HD-1,6, and HT-1,2,6 were used to determine the molar absorptivity per N-phenylcarbamate group. Concentrations of the standards (ME-1 and BU-i) were then determined spectrophotometrically using the absorptivity determined as described above, t = (1.676 0.033) X lo4 M-' cm-l. The linearity of instrument response was checked over a large range of concentrations that spanned the concentration range of N-phenylcarbamatederivatives found in the samples. Linearity of representative compounds included ME- 1 (0.033.4 pmol/mL), HT-1 (0.01-1.0 pmol/mL), HT-1,6 (0.0060.6 pmol/mL), and HT-1,2,6 (0.005-0.45 pmol/mL). Plots of HPLC peak area vs concentration were linear, with r2 1 0.999.
*
(8) Shriner, R. L.; Fuson, R. C.; Curtin, D. Y . The Systemuric Identification of Organic Compounds; John Wiley and Sons: New York, 1964, p 246.
Accuracy of the method was estimated by chromatographically determining the concentrations of known solutions of HT-1, HT-6, HT-1,6, TEG-1,8, HD-1,6, and HT-1,2,6 relative to the external standard (ME-l), using the conditions of the present method. Preparation of Poly(ortho ester) Pellets. Poly(ortho ester) pellets measuring 0.5 in. diameter X 1.O in. were prepared in a glovebox under nitrogen. Into a jacketed beaker maintained at 40 OC was added 7.73 g (39.8 mmol) of tetraethyleneglycol, 2.80 g (20.9 mmol) of 1,2,6-hexanetriol, and 0.05 mL (0.36 mmol) of triethylamine, each from a separate tared syringe. In addition, 4.69 g (39.7 mmol) of 1,6-hexanediol was added as a solid. The mixture was stirred at 40 "C until all the H D dissolved. The temperature was then lowered to 25 OC and 23.03 g (108.5 mmol) of DETOSU was added rapidly with stirring from a tared 60-mL syringe. The mixture became cloudy upon addition of DETOSU and was stirred at 25 OC until it cleared (about 15 min). The clear viscous solution was then transferred to a Teflon mold containing 12 equally spaced holes (0.5 in. diameter X 1.O in.) in two rows of 6. The bottom of the mold was a sheet of Teflon 0.25 in. thick and was held in place and sealed by silicone caulk. After filling, the top (also 0.25-in. Teflon sheet) was placed over the mold and secured with equally spaced (3 in. on center) C-clamps. The assembled mold was removed from the glovebox and placed in an oven at 65 OC for curing. Cure time was 72 h. After being cooled to room temperature, the mold was opened and the pellets were removed. The individual pellets were placed in 25-mL vials and stored in a vacuum desiccator over PzOS. Preparation of Samples. The poly(ortho ester) pellets were cut into slices of varying thickness with a fine-tooth saw and a small aluminum miter box, and the "sawdust" produced by this procedure was also collected. The thickness of each slice was measured with calipers, weighed, and placed into 25-mL vials equipped with Teflon/silicone rubber seals. Analytical Procedure. Five milliliters of dry dioxane containing 0.2 g (0.57 mmol) of dibutyltin diacetate/mL was added to each polymer sample, and 0.125 mL (1.14 mmol) of phenyl isocyanate was added. The vials were sealed and the derivatization reaction was allowed to proceed for various times at room temperature. The reaction was quenched by theadditionof0.25mL(l.59mmol) of 1-octanolatthedesired times. This reaction was allowed to proceed overnight in an attempt to eliminate completely any traces of phenyl isocyanate occluded within the polymer matrix. After the quench, the samples were hydrolyzed by addition of 0.15 mL of 3% perchloric acid, and were then placed on a rotary shaker (New Brunswick Scientific) at 250 rpm. Complete dissolution of the polymer occurred within 2-4 h. After the samples were completely hydrolyzed, 0.50 mL of the internal standard solution (isobutyl N-phenylcarbamate, 54.07 pmol/mL) was added and the samples were carefully transferred to 50-mL volumetric flasks and diluted to volume with acetonitrile. The internal standard is used to identify the locations of the analyte peaks by comparing retention times relative to that of the internal standard. HPLC analyses were performed using a Zorbax Rx C8,4.6 mm X 250 mm column at 50 OC and a double gradient, beginning with 0% acetonitrile/2.5% methano1/97.5% water, going to 20% acetonitrile/O% methanol/ Analytical Chemistry, Vol. 66, No. 20, October 15, 1994
3507
~~
Table 1. Equlvalent Akorptlvltles for Several KPhenylcarbamates
compd
1VelN (M-I cm-l/N)a
compd
HT- 1 HT-6 HT- 1,6 TEG- 1,8
1.721 1.708 1.690 1.640
HD-1,6 HT-1,2,6 PR-2 HX- 1
a
1Ve/N (M-I cm-I/N)' 1.639 1.662 1.645 1.699
N, number of N-phenylcarbamates groups per molecule.
20% water in 25 min, and then to 100% acetonitrile in 50 min. Flow rate was 1.OO mL/min, and the reequilibration time was 14 min. Detection was at 233 and 269 nm. The external standard was run as a bracketed standard after every third sample. The content of the ith POE derivative in micromoles per gram is given by
where Aiis thechromatographic peakarea of the ith derivative, CESis the concentration (pmol/mL) of the external standard solution, ks' is the volume (mL) of the sample, AESis the chromatographic peak area of the external standard, ni is the ratio of the number of N-phenylcarbamate groups on the ith derivative to the number of N-phenylcarbamate groups on the external standard, and w is the weight (g) of the polymer sample.
RESULTS Listed in Table 1 are the equivalent absorptivities (molar absorptivity per N-phenylcarbamate group) measured for several N-phenylcarbamates. The averagevalue obtained was (1.676 f 0.033) X lo4 M-' cm-'/N, rsd = 1.97%. The 75-min double gradient produced resolution of all the derivatized POE components and the internal standard. Chromatograms of a mixture of the pure derivatives and the hydrolysate of a derivatized sample are shown in Figure 2. Identification of the derivatives in the POE sample is facilitated by comparing relative retention times of the analytes in a reference solution with those of the sample by use of the internal standard retention time as reference. Moreover, it was found that the relative retention times werevery consistent from run to run and from column to column. The relative retention times for all of the POE-phenyl isocyanate derivatives are provided in Table 2. Also presented in Table 2 is a comparison of the apparent recoveries obtained for solutions of HT- 1, HT-6, HT-1,6, TEG-1,8, HD-1,6, and HT-1,2,6 using the external standard (methyl N-phenylcarbamate). The mean recovery for these compounds is 101.4 f 4.4%. It is assumed that this is representative for all of the components in the sample. The results from the analysis of the POE pellet slices are tabulated in Table 3a for the monosubstituted derivatives and in Table 3b for the di- and trisubstituted derivatives. The average values for the reaction times and each corresponding content of each derivative are listed and compared with the component content for the "sawdust" measured at the same 3508
AnalyticalChemistt-y, Vol. 66,No. 20, October 15, 1994
300,
2001
Flgure 2. HPL chromatograms of a mixture of pure phenyl Isocyanate derivatives(bottom)and of derivatizedPOE hydrolysate(top). IS,Internal standard. Note that in this derlvatized POE sample, as in most, HT-1,0 and HD-1,6 are not present in detectable amounts. Table 2. Relatlve Retention nmes for the POE-Phenyl Isocyanate Derivatives, wlth Internal Standard t,,(Bu-I) = 1.000, and Recoveries of some POE-Phenyl Isocyanate Derlvatlves
compd
mean re1 retention time (min)
std dev (min)
% rsd
rec (%)
HT-2 HT- 1 HT-6 TEG-1 HD- 1 HT-1,2 HT-2,6 HT- 1,6 TEG- 1,8 HD- 1,6 HT-1,2,6
0.662 0.675 0.704 0.718 0.860 0.953 0.968 0.975 0.978 1.117 1.148
0.005 0.005 0.004 0.002 0.002 0.003 0.002 0.001 0.002 0.002 0.003
0.735 0.721 0.544 0.307 0.249 0.339 0.197 0.131 0.164 0.203 0.235
nda 99.6 95.8 nd nd nd nd 103.8 99.7 100.8 108.7
(I
nd, not determined.
time. These data are summarized in Figure 3a, in which the content of the monosubstituted components is presented as a function of time, and in Figure 3b, in which this relationship is presented for the di- and trisubstituted derivatives. For clarity, only the most abundant derivative content from the sawdust is shown in this figure.
DISCUSSION The chemistry of this method is based upon the well-known reaction of phenyl isocyanate with alcohols to form a N-phenylcarbamate (phenylurethane):
y o
t?iCOOR
0
+R-OH
-0
These derivatives are hydrolytically stable and can therefore
Table 3. Results from Derlvatlzatlon Analysls of Poly(ortho ester) Pellets
(a) Monosubstituted Derivatives derivative content (pmollg) thickness (mm) group 1 mean (n = 6) std dev group 1 sda group 2 mean (n = 4) std dev group 2 sd' group 3 mean (n = 3) std dev group 3 sda group 4 mean (n = 4) std dev group 4 sd'
a
HT-2
HT- 1
HT-6
TEG- 1
HD-1
2.92 0.59 0.00
2.91 0.21 2.83
165 44 297
6.45 1.79 10.7
0.605 0.138 0.950
17.8 7.21 37.5
5.58 1.27 9.63
3.55 0.29 0.00
16.33 0.27 16.23
302 9 298
12.6 2.0 12.1
1.ooo 0.126 0.908
31.6 4.19 35.7
10.5 1.6 9.60
3.87 0.23 0.00
45.08 0.12 45.17
299 4 297
12.2 0.3 11.6
0.908 0.030 0.879
30.0 0.8 39.5
9.93 0.34 10.3
3.58 0.08 0.00
144.51 0.32 144.78
317 20 293
11.1 1.5 10.7
0.986 0.046 1.036
32.1 2.9 37.4
10.1 1.1 10.4
thickness (mm) group 1 mean (n = 6) std dev group 1 sd' group 2 mean (n = 4) std dev group 2 sda group 3 mean (n = 3) std dev group 3 sd' group 4 mean (n = 4) std dev group 4 sda
reaction time (h)
(b) Disubstituted and Trisubstituted Derivatives derivative content (Hmol/a) reaction time (h) HT-1,2 HT-2,6 HT-1,6 TEG-1,8
HD-1,6
HT-l,
