A Simple Extraction and HPLC Method for the Analysis of
Residual Caprylic Acid in Human Serum Albumin Product
W3046
Jian Zhang, Dintletse Ramatlapeng, Chris Lively
Biopharmaceutical Services, PPD Inc., Middleton, WI
PURPOSE
Human serum albumin (HSA) has been used as a
therapeutic agent for over 50 years for restoration and
maintenance of circulating blood volume in situations
such as burn management, hemorrhages, and liver or
kidney failure. In the production process of HSA,
caprylic acid is used as a stabilizer of HSA during
pasteurization and is a process-related impurity.
Residual caprylic acid in the final HSA product must be
determined for quality control purposes. Traditionally,
the analysis of caprylic acid is based on gas
chromatography (GC), which involves time consuming
derivatization steps and yields poor repeatability.
Here, we describe a simple sample preparation and
high performance liquid chromatography (HPLC)
method for the rapid analysis of residual caprylic acid
in HSA product.
METHODS
Sample Preparation
HSA lyophilized powder (fatty acid free, ≥99%) was
purchased from Sigma. A 50 mg/mL HSA solution was
prepared in 1x phosphate buffered saline (PBS) and
spiked with caprylic acid. Caprylic acid was extracted
from the HSA samples by adding a two-fold sample
volume of 0.04% trifluoroacetic acid (TFA) in
acetonitrile to the sample. The sample was then
vortexed briefly and centrifuged to remove the
precipitated protein. The supernatant containing
caprylic acid was analyzed by HPLC
Figure 1. Chromatogram of Six Replicate Preparation of Spiked HSA Samples
Chromatographic Conditions
HPLC System:
Waters Alliance 2695
Mobile Phase:
Water/ACN 60:40 with 0.04% TFA
Flow Rate:
1 mL/min
Column:
Phenomenex Jupiter C18 5 µm, 300Å,
4.6×250 mm
Column Temperature:
40°C
Autosampler Temperature:
5 ± 3°C
Injector Volume:
40 µL
Detector Wavelength:
215 nm
Run Time:
25 min
Caprylic Acid
Figure 3. Six Replication Injections at 2 μg/mL
RESULTS
Caprylic Acid
The performance of the method was evaluated. The
method was found to be precise and accurate. At
0.9 mg/mL (0.3 mg/mL in working sample), %RSD of
caprylic acid concentration determined from six
replicate sample preparations was less than 1.0%
(Table 1 and Figure 1).
Table 1. Spike Recovery of Carprylic Acid from HSA Samples
Spiked Sample
Concentration
(mg/mL)
0.3
0.9
1.5
The method also offered great sensitivity. The limit of
quantitation was less than 2 μg/mL. The mean (n=6)
signal to noise ratio from the six replicate injections
2 μg/mL standard was 18.9 and the %RSD (n=6) of
the peak area was 3.9 (Figure 3). The limit of
quantitation could be further improved by increasing
the injection volume.
Preparation
%Recovery
1
2
3
1
2
3
4
5
6
1
2
3
98.4
98.1
97.5
97.6
99.1
97.0
97.5
98.8
98.3
95.5
95.9
96.7
Mean
%RSD
98.0
0.5
98.1
0.8
96.0
0.6
Spike recovery of caprylic acid from the HSA sample at
three levels from 0.3 to 1.5 mg/mL was within 95100% (Table 1).
Linearity of the chromatographic method was
evaluated for the range of 0.1–0.5 mg/mL with
coefficient of determination (R2) greater than 0.999
(Figure 2).
Figure 2. Linear Calibration Curve (0.1–0.5 mg/mL)
CONCLUSIONS
A sample preparation/RP-HPLC method was developed
and evaluated for quantitative analysis of caprylic acid
in HSA product. The procedure is simple, fast and
outperforms the traditional GC method. Similar
methodology may also be adapted for analysis of
other mid-chain fatty acids in biopharmaceuticals and
biological samples.