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Increase Data Quality for Sulfonamide Residue Analysis by HPLC and UHPLC Using Unique Biphenyl Column Selectivity

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 concern, as drugs in this antibiotic class are commonly used in feed additives 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 resolution, we chose a Biphenyl column, part of our USLC family of phases, for this work. Due to the column’s unique selectivity and high retention, 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 positive identifications and easy integrations obtained on the Biphenyl column allow more accurate reporting of sulfonamide residues.

In addition to providing improved separation of target analytes, focusing on stationary phase selectivity when choosing the analytical column allowed us to use simple, MS-friendly mobile phases. This approach provides several advantages for sulfonamide residue analysis. First, the separation can be easily transferred from UV to MS without 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 from matrix interferences. Matrix interferences can play a significant role in MS analyses by lowering 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.

Figure 1: Due to their unique selectivity, Biphenyl columns can provide the retention and separation needed for accurate sulfonamides analysis with simple, MS-friendly mobile phases.

PeakstR (min)
1.Sulfanilamide4.40
2.Sulfadiazine10.18
3.Sulfapyridine10.63
4.Sulfathiazole10.99
5.Sulfamerazine11.72
6.Sulfamethazine12.94
7.Sulfachlorpyridazine16.08
8.Sulfadoxine16.42
9.Sulfisoxazole17.65
10.Sulfadimethoxine19.47
11.Sulfaquinoxaline19.86
Sulfur Antibiotics on Ultra Biphenyl
LC_GN0531
ColumnUltra Biphenyl (cat.# 9109565)
Dimensions:150 mm x 4.6 mm ID
Particle Size:5 µm
Pore Size:100 Å
Temp.:25 °C
Sample
Diluent:0.1% Formic acid in water
Conc.:50 µg/mL
Inj. Vol.:10 µL
Mobile Phase
A:0.1% Formic acid in water
B:0.1% Formic acid in acetonitrile
Time (min)%A%B
0.009010
3.09010
20.06040
21.06040
Flow:1.0 mL/min
DetectorUV/Vis @ 265 nm
InstrumentShimadzu UFLCXR

Figure 2: A phenyl hexyl column, used under identical conditions, does not provide adequate retention or selectivity for sulfonamide residue analysis.

PeakstR (min)
1.Sulfanilamide3.07
2.Sulfadiazine7.15
3.Sulfapyridine7.43
4.Sulfathiazole7.96
5.Sulfamerazine8.54
6.Sulfamethazine8.53
7.Sulfachlorpyridazine13.49
8.Sulfadoxine13.63
9.Sulfisoxazole15.41
10.Sulfadimethoxine17.22
11.Sulfaquinoxaline17.45
Sulfur Antibiotics on Waters XSELECT CSH Phenyl-Hexyl
LC_GN0533
ColumnWaters XSELECT™ CSH Phenyl-Hexyl
Dimensions:150 mm x 4.6 mm ID
Particle Size:5 µm
Temp.:25 °C
Sample
Diluent:0.1% Formic acid in water
Conc.:50 µg/mL
Inj. Vol.:10 µL
Mobile Phase
A:0.1% Formic acid in water
B:0.1% Formic acid in acetonitrile
Time (min)%A%B
0.009010
3.09010
20.06040
21.06040
Flow:1.0 mL/min
DetectorUV/Vis @ 265 nm
InstrumentShimadzu UFLCXR

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. While high efficiency separations can help increase sample throughput, efficiency is volume dependent and resolution can be affected by changes in instrumentation. 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.


Figure 3: Ultra selective analysis of sulfonamides on a unique Biphenyl column can be used in conjunction with UHPLC for higher sample throughput.

PeakstR (min)
1.Sulfanilamide1.55
2.Sulfadiazine3.74
3.Sulfapyridine4.09
4.Sulfathiazole4.24
5.Sulfamerazine4.35
6.Sulfamethazine4.91
7.Sulfachlorpyridazine5.87
8.Sulfadoxine5.99
9.Sulfisoxazole6.37
10.Sulfadimethoxine7.14
11.Sulfaquinoxaline7.40
Sulfur Antibiotics by UHPLC on Pinnacle DB Biphenyl
LC_GN0532
ColumnPinnacle® DB Biphenyl (cat.# 9409212)
Dimensions:100 mm x 2.1 mm ID
Particle Size:1.9 µm
Pore Size:140 Å
Temp.:25 °C
Sample
Diluent:0.1% Formic acid in water
Conc.:50 µg/mL
Inj. Vol.:2 µL
Mobile Phase
A:0.1% Formic acid in water
B:0.1% Formic acid in acetonitrile
Time (min)%A%B
0.00955
86040
Flow:0.4 mL/min
DetectorUV/Vis @ 265 nm
InstrumentShimadzu UFLCXR

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 addition, Biphenyl columns in a UHPLC format allow faster sample throughput, while maintaining good separation of target compounds.

For more about the advantages of USLC Biphenyl columns, visit www.restek.com/uslc