ADV06-15_1-412

40 / 413

| 1-800-356-1688 or 1-814-353-1300 | Feedback? E-mail 14
By Rick Lake and Ty Kahler
• Improve reporting accuracy with better selectivity and retention.
• Biphenyl column and MS-friendly mobile phases allow easy transfer between UV and MS detection.
• Maximize sample throughput by combining USLC® selectivity with UHPLC speed.
The analysis of antibiotic residues in food-producing animals is
important worldwide for evaluating food safety and maintaining
compliance with export regulations. Sulfonamides are a specific con-
cern, as drugs in this antibiotic class are commonly used in feed addi-
tives for livestock in order to fight infections and maintain desired
growth levels. The analysis of sulfonamides usually involves a liquid
chromatographic separation and detection by either UV or mass
spectrometry. In both cases, the highly selective separation produced
by a Biphenyl HPLC or UHPLC column can significantly improve data
quality and reporting accuracy.
Increase Accuracy With Ultra Selective
Biphenyl Columns
Since selectivity is the most important factor affecting peak resolu-
tion, we chose a Biphenyl column, part of our USLC® family of phases,
for this work. Due to the column’s unique selectivity and high reten-
tion, we were able to develop a very effective HPLC separation of 11
common sulfonamides with complete resolution (Figure 1). Use of
the Biphenyl column produced much better chromatographic data
compared to results obtained from a phenyl hexyl column used
under identical conditions (Figure 2). The fully resolved sulfonamide
analysis obtained on the Biphenyl column allows for more consistent
and accurate integration.
In addition to providing improved separation of target analytes,
focusing on stationary phase selectivity when choosing the analyti-
cal column allowed us to use simple, MS-friendly mobile phases. This
approach provides several advantages for sulfonamide residue analy-
sis. First, the separation can be easily transferred from UV to MS with-
out further method development. Second, the use of simple mobile
phases saves time and money, since they are quick to prepare and do
not require complex additives.
Higher Retention Reduces Matrix Interferences
in MS Detection
When developing a separation for UV detection, selectivity is critical for
positive analyte identification. If MS detection is used, selectivity may
not be required for analyte identification, but it still may be needed for
adequate sensitivity and separation frommatrix interferences. Matrix
interferences can play a significant role in MS analyses by lowering
method sensitivity through suppressing ionization. Ion suppression in
reversed phase mode often occurs with early eluting compounds, so
it is good practice to retain them to a retention factor (k) of 2. In this
example, we can see that the retention factor of sulfanilamide on the
Biphenyl column is approximately twice as high as it is on the phenyl
hexyl column (Figure 2). As a result, sulfanilamide is more susceptible
to sample matrix interference if a phenyl hexyl column is used. The
increased retention provided by the Biphenyl column, in combination
with the MS-friendly mobile phases used here, ensure good sensitivity
and allow easy method transfer between detectors.
Combining USLC® Selectivity and UHPLC Speed—
The Most Powerful Approach
Selectivity has the greatest influence on resolution, but efficiency is the
best tool for decreasing analysis time. By optimizing column selectivity
first, we can then easily transfer a robust separation to UHPLC for faster
analysis. Figure 3 illustrates the power of combining USLC® selectivity
with UHPLC efficiency. By using a 1.9 µm Biphenyl UHPLC column we are
able to fully separate all 11 sulfonamide peaks in a fast, 8-minute analysis.
Conclusion
Focusing first on selectivity when choosing an analytical column for
sulfonamide residue analysis is an easy way to improve data quality.
The unique selectivity and high retention of Biphenyl columns produce
complete separations and benefit both UV and MS detection. In addi-
tion, Biphenyl columns in a UHPLC format allow faster sample through-
put, while maintaining good separation of target compounds.
Increase Data Quality for Sulfonamide Residue Analysis by HPLC and UHPLC
Using Unique Biphenyl Column Selectivity