Detection of Sulfonamides in Food Grade Honey Using a Triple Quadruple Liquid Chromatography Mass Spectrometer

Sulfonamides are synthetic antimicrobial drugs that are generally used for treating bacterial growth because of their broad and high spectrum activity. Sulfonamides are often utilized during the processing of food grade honey and their residues are often present in honey samples, which can pose significant risks to consumers across the globe. This article describes a case study using the Bruker EVOQ Elite triple quadruple liquid chromatography mass spectrometer (LC-MS/MS), and demonstrates superior sensitivity and selectivity in determining the sulfonamides at the required maximum residue limits (MRLs), and corroborates the technique according to criteria specified by the European Union Commission Decision.

Risks of Sulfonamides

Sulfonamides drugs inhibit bacteria from producing folic acid and thus prevent bacterial growth and multiplication.

Despite the fact that sulfonamides are effective against different bacteria, their use is rather limited because they are known for causing extreme allergic reactions in hypersensitive humans and can expose humans to drug-resistant bacteria.

International Regulation of Sulfonamides

Since sulfonamide exposure poses potential risk to consumers, different regions have set their own maximum residue limits (MRLs), minimum performance levels (MRPL), and specific detection criteria to determine sulfonamides. The European Union has established an MRPL for sulfonamides at a level of 10 µg/kg in honey. It has also set common criteria for the interpretation of test results so as to guarantee the quality and comparability of the analytical results produced for official residue control by approved laboratories.

Detection of Sulfonamides

A number of methods are available to determine and quantify sulfonamides in food grade honey products. However, in case of complex food matrices, many techniques provide poor sensitivity and selectivity and hence cannot be utilized to determine the level of sulfonamides in honey. To this end, a combination of liquid chromatography and mass spectrometry can be used to solve this problem.

Sulfonamides are frequently detected in food grade honey.

Figure 1. Sulfonamides are frequently detected in food grade honey.

A case study shows how the EVOQ Elite LC-MS/MS can be utilized to detect sulfonamide levels to 0.1ppb with constant ion ratio of quantifier to qualifier ions. This method is fast yet simple for the quantitation of sulfonamides in honey, and was found to be repeatable and linear with relative standard deviation (RSD) of <12% and regression (R) of =0.996. The EVOQ provides high specificity and sensitivity and is flexible enough to enable high sample throughput for detection and quantification of sulfonamides.

The high sensitivity of EVOQ reflects several hardware innovations in the ion source such as the Active Exhaust that inhibits the recirculation of nebulized gases, thus reducing the chemical noise. This also translates to less downtime and eliminates the time spent on cleaning and running samples, thus providing high productivity and excellent sensitivity in high throughput laboratories.

Additionally, the EVOQ’s hardware is complemented by Bruker’s PACER software, which provides exception based data review to considerably reduce the rate of errors for quantitative analysis. In this way, PACER improves efficiency by reducing time scans affecting data quality and also speeds up sample-to-report times, which is very important for labs working in routine quantitation.

Quantification of Sulfonamides using EVOQ Mass Spectrometer

The EVOQ Elite triple quadruple liquid chromatography mass spectrometer was utilized to measure nine sulfonamides in honey, as shown in the table below:

Table 1. EVOQ tested for nine commonly used sulfonamides.

Sulfadimethoxin (SMM)
Sulfadiazin (SD)
Sulfadoxin (SDM)
Sulfamethazine (SM2)
Sulfadimidine (SDN)
Sulfamerazine (SM1)
Sulfathiozole (ST)
Sulfamethoxypyridazin (SMP)
Sulfamethizole (SMZ)

For sample preparation, a standard mixed solution of all nine sulfonamides at 1000 ng/mL concentration level was prepared by diluting separate sulfonamide stock solutions using triple distilled water and acetonitrile. Then, calibration solutions were prepared by spiking the proper volume of the mixed standard solution in a blank honey matrix. The instrument calibrations used are as follows:

Chromatography Parameters (Advance UHPLC)
Column Bruker Intensity C18 (50 mm x 2.1 mm x 3µm)
Column temperature Ambient
Flow rate 0.3mL/min
Mobile phase A Water with formic acid
Mobile phase B Acetonitrile with 0.1% formic acid
Injection volume 10µL
Gradient conditions 0.000 min 2% B
1.00 min 2% B
2.50 min 10% B
3.50 min 10% B
4.00 min 20% B
6.00 min 20% B
7.00 min 95% B
8.50 min 95% B
8.60 min 2% B
10.00 min 2% B
Mass Spectrometer Parameters (EVOQ Elite)
HESI +4500V
Probe temperature 450°C
Probe gas 45 units
Nebulizer gas 50 units
Cone gas temperature 350°C
Cone gas 30 units
Active exhaust On
Collision gas Argon
Collision gas pressure 1.5mTorr

Results and Discussion

Chromatograms of all nine sulfonamides analyzed at LOQ concentration of 0.1ppb in honey with peak-to-peak S/N ratio.

Figure 2. Chromatograms of all nine sulfonamides analyzed at LOQ concentration of 0.1ppb in honey with peak-to-peak S/N ratio.

Overlaid chromatograms of all nine sulfonamides at LOQ level and the blank honey matrix.

Figure 3. Overlaid chromatograms of all nine sulfonamides at LOQ level and the blank honey matrix.

Figure 2 illustrates chromatograms of all nine sulfonamides examined at 0.1 ppb in honey with peak-to-peak signal-to-noise (S/N) ratio. It was found that the S/N ration for all nine sulfonamides in honey matrix at 0.1 ppb was much higher than 10. Figure 3 clearly demonstrates that the blank honey matrix does not have any of the targeted sulfonamides.

The case study was performed according to the European Union Decision 2002/657/EC, which specifies a number of parameters to confirm detection of the target compounds.

The technique was repeatable with RSDs below 12%, as shown in table 2. Results achieved for all nine sulfonamides at limit of quantification of 0.1 ng/g were well within the satisfactory range of accuracy. Also, the limit of quantifications of 0.1 ng/g obtained for all nine sulfonamides under study were considerably lower than the MRLs stipulated by different regions regulations.

Table 2. Sulfonamides in honey performance summary.

Name Calibration range (ppb) R2 %RSD for 0.1 ppb 10 repeats/day % RSD for 1 ppb 10 repeats/day %RSD for 10 ppb 10 repeats/day
Sulfadimethoxin (SMM) 0.1-10 0.999 11.53 7.30 8.94
Sulfadiazin (SD) 0.1-10 0.998 6.61 5.04 6.45
Sulfadoxin (SDM) 0.1-10 0.996 6.82 11.43 6.65
Sulfamethazine (SM2) 0.1-10 0.999 9.39 7.90 6.85
Sulfadimidine (SDN) 0.1-10 0.999 3.93 7.18 6.77
Sulfamerazine (SM1) 0.1-10 0.999 5.74 11.74 6.35
Sulfathiozole (ST) 0.1-10 0.999 6.84 6.68 5.62
Sulfamethoxypyridazin (SMP) 0.1-10 0.999 5.74 7.00 4.99
Sulfamethizole (SMZ) 0.1-10 0.999 7.84 6.88 7.81

Conclusion

Bruker’s EVOQ Elite liquid chromatography mass spectrometer delivered excellent repeatability and detection limits for all nine sulfonamides under study in the honey matrix. This combined with the PACER software provided consistent results in the fastest time possible. The use of Advance UHPLC also reduced the total chromatographic run time significantly. Thus, the combination of faster sample-to-report times, enhanced sensitivity and low maintenance makes the EVOQ suitable for processing complex samples in high throughput laboratories.

This information has been sourced, reviewed and adapted from materials provided by Bruker - Chemical And Applied Markets.

For more information on this source, please visit Bruker Daltonics.

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