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TRIS BUFFER INSTABILITY AT ELEVATED TEMPERATURES
1199
Buffer Solutions
ultraviolet detection (HPLC±UV) as described
later. The pH of the buffer solution or the recon-
stituted lyophilized buffer was measured for each
sample to verify that pH did not change during
the studies. Deionized H2O was used to recon-
stitute the lyophilized samples. No signi®cant
change of pH was observed for solution and solid-
state samples.
Tris buffer was used throughout the studies. The
buffer concentration was 0.15 M at pH 8.0. Ionic
strength was maintained constant at 0.5 by
adding appropriate amounts of sodium chloride.
The pH of the buffer solutions was measured with
a pH meter (Orion model 410A) equipped with a
Ross combination electrode.
Sample Analysis
Preparation of Formulations in Solution
and Solid State
Analysis of the Asn-hexapeptide and its deamida-
tion products was performed by an isocratic rever-
sed-phase HPLC (RP-HPLC) method with UV
detection. Details of the method have been repor-
ted previously.2 Brie¯y, the method employs an
Econosphere C18 reversed-phase column (4.6 Â
250 mm, 5-mm particle size) from Alltech Associ-
ates, Inc. (Deer®eld, IL) for separation, and a
Shimadzu HPLC system consisting of an SCL-10
Avp system controller, an SPD-10 Avp UV±vis
detector, an SIL-10ADvp auto injector, and an
LC-10ATvp pump. The mobile phase consists of
8% (v/v) acetonitrile and 0.1% (v/v) tri¯uoroacetic
acid in 0.050 M ammonium acetate at pH 4.6. The
¯ow rate was set at 0.8 mL/min and detection was
at 214 nm.
The analysis of the new peptide degradation
product by liquid chromatography±mass spectro-
metry (LC±MS) was performed by the Mass
Spectroscopy Lab at the University of Kansas.
The Hantzsch reaction was conducted to con®rm
the presence of formaldehyde in solution and in
the solid state.3 An HP 8452 diode array spectro-
photometer was employed to obtain the spectrum
of the solution after the addition of ammonium
acetate, glacial acetic acid, and acetyl acetone
(2,4-pentonedione) to complete the Hantzsch
reaction.
First, a 5% (w/v) solution of dialyzed PVP was pre-
pared in the pH 8.0 buffer solution just men-
tioned. Two different formulations containing 0 or
30% (w/v) glycerol were then prepared in solution;
glycerol was used as a plasticizer to lower the
glass transition temperature (Tg) in solid formu-
lations. The peptide was then added to the solu-
tion at a concentration of ꢁ0.5 mg/mL. After the
addition of the peptide, some of the solution was
placed in a 70ꢀC oven to serve as a solution state
control. The rest of the solution was placed into
different 2-mL glass lyophilization vials with
200 mL of solution in each vial. The samples were
then lyophilized with a VirTis BenchTop lyophi-
lizer (Gardner, NY), using the following lyophili-
zation cycle: (i) freezing at a shelf temperature of
35ꢀC for 2 h; (ii) primary drying at a shelf tem-
perature of 35ꢀC for 10 h; (iii) secondary drying
at shelf temperatures of 15, 5, 5, 15, 20, and
25ꢀC, maintained for 10, 8, 6, 6, 12, and 10 h,
respectively.
Accelerated Stability Studies
Because deamidation in the solid state can be very
slow, particularly in glassy solids, all kinetic
studies were conducted at 70ꢀC to accelerate the
reaction. This temperature was also selected to
correspond with that used in our detailed study of
the role of effective pH in peptide deamidation in
PVP formulations.1 Stability studies were con-
ducted for both solid samples and reconstituted
solutions. In the present studies, solution formu-
lations were placed in closed vials. For solid sam-
ples, open sample vials were placed in a desiccator
containing a saturated solution of NaBr to main-
tain relative humidity (RH) constant at 50%. Beca-
use the peptide is most stable between pH 3 and
5,2 triplicate samples were removed and diluted in
pH 4 buffer solution for analysis after speci®c
time intervals. The samples were then analyzed
by high-performance liquid chromatography with
RESULTS AND DISCUSSION
Formation and Identi®cation of a New
Degradation Product
The typical products observed during deamida-
tion of VYPNGA in neutral solution and in the
solid state are the Asp-hexapeptide, the iso-Asp-
hexapeptide, and the cyclic imide (Scheme 1).
However, in our studies, a new unknown sub-
stance was observed when the model peptide was
heated in Tris buffer, pH 8.0, in the presence of
PVP. The concentration of this new degradant
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 8, AUGUST 2001