B:
Determine the peptide fragments, using the following peptide mapping procedure.
Sulfate buffer—
Mix equal volumes of 2.0 M ammonium sulfate and 0.5 M sulfuric acid, and filter.
Enzyme solution—
Prepare a solution of Staphylococcus aureus V-8 protease in water having an activity of 500 units per mL.
HEPES buffer—
Dissolve 2.38 g of HEPES (N-2-hydroxyethylpiperazine-N¢-2-ethanesulfonic acid) in about 90 mL of water in a 100-mL volumetric flask. Adjust with 5 M sodium hydroxide to a pH of 7.5, dilute with water to volume, and mix.
Solution A—
Prepare a filtered and degassed mixture of 100 mL of acetonitrile, 700 mL of water, and 200 mL of Sulfate buffer.
Solution B—
Prepare a filtered and degassed mixture of 400 mL of acetonitrile, 400 mL of water, and 200 mL of Sulfate buffer.
Mobile phase—
Use variable mixtures of
Solution A and
Solution B as directed for
Chromatographic system. Make adjustments, if necessary (see
System Suitability under
Chromatography 
621
).
Standard digest solution—
Dissolve about 6 mg of USP Human Insulin RS in 3 mL of 0.01 N hydrochloric acid, and transfer 500 µL of the resulting solution to a clean vial. Add 2.0 mL of
HEPES buffer and 400 µL of
Enzyme solution, and incubate at 25
for 6 hours. Quench the digestion by adding 2.9 mL of
Sulfate buffer.
Test digest solution—
To 1 mg of Insulin Human add 500 µL of 0.01 N hydrochloric acid, and mix to dissolve. Proceed as directed for Standard digest solution, beginning with “Add 2.0 mL of HEPES buffer”.
Chromatographic system (see Chromatography 621)—
A liquid chromatograph is equipped with a 214-nm detector and a 4.6-mm × 10-cm column that contains packing L1. The flow rate is about 1 mL per minute. The column temperature is maintained at 40
. The chromatograph is programmed as follows.
Time
(minutes) |
Solution A
% |
Solution B
% |
Elution |
| 0 |
90 |
10 |
equilibration |
| 0–60 |
90®30 |
10®70 |
linear gradient |
| 60–65 |
30®0 |
70®100 |
linear gradient |
| 65–70 |
0 |
100 |
isocratic |
| 70–71 |
0®90 |
100®10 |
linear gradient |
| 71–86 |
90 |
10 |
re-equilibration |
Chromatograph the
Standard digest solution, and record the peak responses as directed for
Procedure: the chromatogram of the
Standard digest solution corresponds to that of the standard chromatogram provided with
USP Insulin Human RS. For the chromatogram of the
Standard digest the tailing factor is not greater than 1.5; and the resolution,
R, is not less than 3.4 for digest fragments II and III.
[N
OTE* —Fragment I elutes at the same time in insulin derived from pork and Insulin Human; Fragment II elutes at the same time in all insulins; and Fragment III elutes at the same time in insulin derived from beef and pork.
]
Procedure—
Using the gradient program, run a blank. Inject equal volumes of the Standard digest solution and the Test digest solution into the chromatograph, and record the chromatograms. The chromatographic profile of the Test digest solution corresponds to that of the Standard digest solution.
;)
