Anal. Chem. 1987, 59, 2050-2054
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12) Malinowski, Edmund R. J . Chemmetrics 1887, 1 , 33-40. 13) Gemperllne, Paul J.; Boyette, Stacey E.: Tyndall. Kimberly Appl. Spectrosc. 1887, 41, 454-459. 14) Rutan. Sarah C.; Brown, Steven D.. Anal. Chim. Acta 1984, 160, 99-1 19. (15) Rutan, Sarah C.: Brown, Steven D. Anal. Chim. Acta 1985, 167, 39-50. (16) Malinowski. Edmund R.: McCue, M. Anal. Chem. 1977, 49, 284-287. (17) Savitzky, Abraham: Golay, Marcel J. E. Anal. Chem. 1984, 3 6 , 1627-1639. (18) Steinier, Jean; Terrnonla, Yves; Deltour, Jules Anal. Chem. 1972, 4 4 , 1906- 1909. (19) Burns. David H.;Callis, James B.; Christian, Gary D. Anal. Chem. 1988, 58, 2805-2811. (20) Gemperline, Paul J. J. Chem. I n f . Comput. Sci. 1884, 2 4 , 206-212.
(21) Lawton, Willlam H.; Sylvestre, Edward A. Technometrics 1871, 73, 61 - .7-833. . - - -. (22) Vandeglnste, Bernard G. M.; Derks, Wilber?; Kateman, Gerrit Anal. Chim. Acta 1885, 173, 253-264. (23) Borgen, Odd S.; Kowalski, Bruce R. Anal. Chim. Acta 1985, 774, 1-26.
RECEIVED for review December 23,1986. Accepted April 22, 1987. This research was supported by the Grants-In-Aid Program for Faculty of Virginia Commonwealth University, the Virginia Commonwealth University Biomedical GrantIn-Aid Program, and the Jeffress Trust.
Silica-Based Size Exclusion Chromatography To Characterize the Decapeptide Nafarelin in a Controlled-Release Pharmaceutical Formulation Richard A. Kenley,*' Karen J. Hamme, Maryann 0. Lee, and John Tom Syntex Research, 3401 Hillview, Palo Alto, California 94304
This report descrlbes a hlgh-performance llquid chromatography (HPLC) method that simultaneously detemdnes peptide concentration and polymer molecular welght dlstrlbutlon in a controlled-release pharmaceutical forquiatlon. The peptlde Is nafarelin (a decapeptlde analogue of lutelnlrlng hormonereleaslng hormone) and the polymer Is poiy(lact1de-co giycoilde), PLGA, a biodegradable polyester. The method uses a TSK 3000SW column with aqueous acetonttrlle mobile phase to separate the peptlde from Its hydrolytic decomposltion products and from PLGA and to fractionate the PLGA on the basis of molecular weight. Increasing mobile-phase ionic strength and decreasing organic fractlon decrease nafarelln retention, Indicating that the peptlde retains via Coulombic Interactions wlth dissociated sllanols on the packlng. PLGA retention Is essentialty lnvarlanl with mobtie phase h l c strength and organic fractlon, Indicating PLGA retention by slze exclusion.
Modern column packing materials have greatly advanced the chromatographic analysis of peptides, proteins, and synthetic macromolecules. Among the important recent advances in stationary-phase technology are controlled-porosity silicas that have been derivatized with hydrophilic organic polymers. Such materials permit size exclusion chromatography of polymers, peptides, and proteins without excessive solute losses via strong interactions with silanols on the solid support surface. The TSK SW-type columns represent a widely used and well-characterized example of silica-based materials designed primarily for purification and analysis of biopolymers. The literature includes many examples of specific applications that use the TSK SW-type columns (1-8) and several reviews are available as well (9-12). The literature reveals that the TSK SW columns feature a proprietary, hydroxylated polyether bonded phase. UnPresent
address:
Travenol Laboratories, 6301 Lincoln Ave.,
Morton Grove, IL 60053.
derivatized silanols contribute to a net negative charge on the solid support surface a t near-neutral pH. As with other examples of organically modified silicas, the TSK SW-type packing materials contribute to retention on the dual bases of ideal and nonideal behavior. Retention solely on the basis of size exclusion represents the ideal component of separation. Nonideal (sorption) retention mechanisms include Coulombic interactions of ionic solutes with dissociated silanols and hydrophobic interactions of nonpolar solutes with the organic bonded phase. Although the literature dedicates considerable discussion to ion exchange and hydrophobic interactions in TSK SW chromatography, the emphasis clearly has been on selecting mobile phase components that reduce multiple retention mechanisms. Less well represented, by far, are literature examples that deliberately manipulate mobile-phase composition to effect separations on the basis of multiple retention mechanisms. This report describes our efforts to exploit the sorption retention mechanisms intrinsic to the TSK SW columns to simultaneously quantitate drug potency and polymer molecular weight distributions in a controlled-release pharmaceutical formulation. Specifically, nafarelin (see structure) is a decapeptide luI
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0003-2700/87/0359-2050$01.50/0 0 1987 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 17, SEPTEMBER 1, 1987
2051
Table I. Chromatographic Columns and Operating Conditions for PLGA and Nafarelin HPLC Analysis parameter
reversed phase
column type(s) mobile phase injection solvent injection vol, mL flow rate, (mL/min) column temp, "C detection, nm
size exclusion
TSK SW
Vydac 4.6 X 250 mm, 5 wm, 300 k, pore size, C18 23:77 acetonitrile/25 mM KHpP04" 25:75 methanol/20 mM KH2P04
p-Styragel, lo4 & lo3 A, 7.8 X 300 mm 2000 SW and 3000 SW 7.5 X 300 mm
0.050
0.200
1.0
1.0
40
225
THFb THF
ambient 230
8515 acetonitrile/l3 mM NaC10, 8590 acetonitrile/l3 mM NaC104 0.050 1.0
ambient 225
With 25 mM dibutvlamine. DH 3 added. *THF = tetrahydrofuran.
dicated for treating endometriosis and prostatic carcinoma (13). T o achieve drug release over a 30- to 60-day period, the controlled-release formulation incorporates nafarelin into lactic/glycolic acid copolymer (PLGA) microspheres (14). Following intramuscular injection, the PLGA microspheres hydrolyze to low molecular weight, water-soluble fragments. As the polymer matrix dissolves, nafarelin enters systemic circulation. Characterizing the nafarelin/PLGA formulation requires quantitating nafarelin concentrations within the microspheres and also determining PLGA molecular weight distributions. Nafarelin concentration and PLGA molecular weight data are necessary to control the potency and purity of the drug product and also to probe the relationships between polymer composition and nafarelin release profiles. We originally developed (15, 16) two independent methods to quantitate nafarelin concentrations and PLGA molecular weight parameters. Although these methods performed satisfactorily, they required two sample sets, two sample workup procedures, and two independent instruments for complete analysis. Clearly, the development of a single assay procedure to simultaneously determine nafarelin content and PLGA molecular weight distributions would afford significant practical advantages. We anticipated that the chemical and physical properties of nafarelin and of PLGA would form the basis for a single chromatographic separation on the TSK SW-type columns. Nafarelin features both a cationic moiety (the guanidinium at position 9) and three highly hydrophobic amino acids (the tyrosyl, tryptophyl, and 1-naphthylalanine groups). Thus, nafarelin should interact strongly with surface silanols and possibly with the organic bonded phase. PLGA, on the other hand, is a relatively hydrophilic, nonionic polymer that should retain via ideal size exclusion. We find that the TSK SW-type columns do, indeed, permit analysis of nafarelin and PLGA in a single chromatographic run. The following paragraphs detail the analytical procedures and compare the performance of the TSK method with independent analyses obtained by using conventional reversed-phase and size exclusion chromatography.
EXPERIMENTAL SECTION Materials. Nafarelin was prepared by the Syntex Institute of Organic Chemistry according to published (13) procedures. PLGA samples were prepared by Syntex Chemicals, Boulder, CO, according to the method of ref 17. The PLGA samples studied all featured a 55:45 1actide:glycolidemonomer ratio and exhibited molecular weights in the 10000-80000 g/mol range. Nafarelin was incorporated at 0.8% (w/w) into PLGA microspheres by using an aqueous emulsion technique described in ref 14.
Acetonitrile (HPLC grade) and tetrahydrofuran (spectroscopic grade) were supplied by Burdick and Jackson. Sodium perchlorate was analytical reagent grade from Fluka Chemicals. Water was purified before use with a Barnstead Nanopure system.
The TSK SW2000 and SW3000 columns were from Toyo Soda, the lo3 and lo4 A pStyrage1 columns were provided by Waters Associates. Reversed-phase chromatography used a Vydac C18 column from The Separations Group, Inc. Monodisperse polystyrene standards were from Waters Associates. Instrumentation. The following components comprised the HPLC system: Waters Model 710A autosampler, Waters M6000 solvent delivery system, Kratos Model 757 variable-wavelength spectrophotometric detector, Spectra-Physics Model SP4000 electronic integrator. Procedures. Table I summarizes columns, mobile phases, and system operating parameters employed in the reported investigation. The pStyragel method has been previously described (15) and employs the "universal calibration" (18)technique to provide accurately known PLGA molecular weight distributions vs. polystyrene standards. Five PLGA samples with M , values ranging between 10000 and 80000 g/mol were characterized by using pStyragel columns as above, and these were used in turn as molecular weight standards for establishing molecular weight calibration curves for TSK SW columns. Suitable control experiments established that the previously described (16)reversed-phase analytical method satisfies the usual criteria for recovery, response linearity, and specificity for nafarelin in the presence of its hydrolysis products. Sample Preparation. PLGA samples for pStyragel analyses were dissolved at 1mg/mL in tetrahydrofuran (THF) with gentle stirring overnight. For reversed-phase chromatography, 25 mg of nafarelin/PLGA microspheres was dissolved in 10 mL of hot acetonitrile. A 10-mL aliquot of internal standard (hydrocortisone, 0.030 mg/mL) solution was added and the sample diluted to 50 mL with 200 mM aqueous KH2P04. The solution was centrifuged and diluted 1:l with a 25:75 mixture of methanol and aqueous 20 mM KH2P04. For TSK SW column analysis, PLGA microspheres were diluted at 0.50 mg/mL in mobile phase and directly injected. Data Treatment. Nafarelin concentrations were calculated by peak height measurements and PLGA molecular weight distributions by electronic area integration. PLGA number-average (M,) and weight-average (M,) molecular weights and distributions were calculated according to the following: M , = Ai/(Ai/Mi) (1)
M , = (Ai X Mi)/Ai (2) where Ai is the ith peak slice area and M iis the molecular weight corresponding to the retention time for the ith peak slice. The molecular weight distributions were corrected for symmetrical and unsymmetrical band broadening (15, 18). PLGA hydrolysis kinetics were characterized as pseudo-firstorder rate constant ( k p L c A ) values for polyester bond cleavage according to the following: (3) In (x) = -kpLGA t where X is the number of bond cleavages per initial number average molecule, given by (4) M,(t)/M,,, = 0 ) = 1/(1 + X ) Our previous work (15) establishes the validity of eq 3 and 4 for PLGA hydrolysis characterization.
2052
ANALYTICAL CHEMISTRY, VOL. 59, NO. 17, SEPTEMBER 1, 1987
280
loo7 95 -
-
90
-
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Figure 1. Dependence of nafarelin retention volume on mobile phase ionic strength. The TSK 3000SW column and 85% acetonitrile were
used. The mobile-phase aqueous fraction contained various concen-
trations of sodium perchlorate to adjust ionic strength.
15
13
11
Nafarelin Retention Volume (mL)
Nafarelin Retention volume (mL)
Figure 2. Dependence of nafarelin retention volume on mobile phase acetonitrile fraction. The TSK 2000SW column and 250 mM sodium perchlorate in the aqueous fraction were used. The mobile phase acetonitrile fraction was varied between 70 % and 95 % .
Nafarelin hydrolysis was characterized similarly as pseudofirst-order rate constant (kNAF)values calculated according to In [nafarelin(,,/nafarelin(, = 0,3 = -kNAFt (5)
z
Unless otherwise noted, experimental uncertainties are expressed throughout as two-tailed f 95% confidence intervals.
RESULTS AND DISCUSSION Method Development. A preliminary attempt to simultaneously separate PLGA and nafarelin on a TSK 2000SW column held mobile phase initially constant a t 100% acetonitrile and then stepped to 30% acetonitrile in 0.15 mM phosphate buffer. With this solvent profile, PLGA eluted as a broad peak a t approximately 5 to 10 min, and nafarelin eluted at 22 min. Because the total permeation volume of the TSK 2000SW column is approximately 13 mL (9)and because the nafarelin retention volume exceeded the total permeation volume, multiple retention mechanisms were responsible for controlling the chromatographic behavior of nafarelin. T o better understand the factors controlling nafarelin retention and to identify an optimal mobile phase/column combination for PLGA and nafarelin chromatography, we further investigated the effects of mobile-phase composition and column selection on nafarelin and PLGA retention. Figure 1demonstrates the dependence of nafarelin retention time on mobile-phase ionic strength. These runs used the TSK 3000SW column and 85% aqueous acetonitrile with added sodium perchlorate a t the concentrations indicated in Figure 1. From the figure, it is clear that low (
