QSight™ LC-MS/MS Method for Antibiotic Analysis in Milk

Sulfonamides are typically referred to as “sulfa drug” - derivatives of sulfanilamide (p-amino benzene sulfonamide). They are frequently employed as antibacterial drugs in the veterinary treatment of common bacterial diseases due to their antibacterial properties, low cost and low toxicity.2

The uncontrolled use of veterinary drugs and failure to comply with the withdrawal period can lead to drug residues remaining in animal tissues after treatment. These residues can potentially be passed on to humans via the animal’s milk.3,4

It is common practice to use various antibiotics in dairy cattle for different treatments, but this eventually leads to the accumulation of antibiotic residues in their milk, potentially causing allergic reactions and toxic effects when consumed by humans.

These treatments can also promote the development of resistant strains of bacteria.5,6

Regulatory agencies in the European Union have published a range of official documents with a view of better regulating the control of veterinary drugs in order to protect consumers. Council Directive 96/23/EC7 outlines the control of veterinary residue in food producing animals.

In India, FSSAI (gazette notification dated 20th July 2018) and EIC (RMP for Aquaculture, Egg, Honey, Milk Poultry etc.) mandate the early and frequent analysis of antibiotics. EIC and FSSAI establish maximum residue limits (MRLs) for a range of specific antibiotic residues often found in food matrices from animals.

It is vital that milk is tested for sulfonamide residues in order to maintain consumers’ safety and prevent their exposure to veterinary drugs. This article outlines a rapid, sensitive LC-MS/MS method for the quantitative analysis of sulfonamide antibiotics in milk.

Experimental

Hardware and Software

A PerkinElmer LX-50 UHPLC System was used for chromatographic separation while detection was conducted via a PerkinElmer QSight™ 220 triple quadrupole mass spectrometer, fitted with ESI and APCI ionization sources.

Instrument control, data acquisition and data processing were all performed using the Simplicity 3Q™ software in one window. Table 1 provides LC parameters, including the column and mobile phase gradient program.

Table 1. UHPLC parameters. Source: PerkinElmer Food Safety and Quality

LC Column Univ C18AQ 100 mm X 3 mm X
2.1μm (P/N N9304784)
Mobile Phase A 0.1% Formic acid in water
Mobile Phase B 0.1% Formic acid in Acetonitrile
Mobile Phase Gradient
Sr. No. Time %A %B
1 0.00 98 2
2 1.50 98 2
3 6.00 4 96
4 6.50 4 96
5 7.00 98 2
6 7.50 98 2
Column Oven Temperature 40ºC
Auto sampler Temperature 15ºC
Injection Volume 10 μL
Flow 0.5 mL/Min
Run Time 7.5 minutes
ESI Voltage (+Ve) 5300
Drying Gas 110
Nebulizer Gas 300
Source Temperature 300
HSID Temperature 220

 

Materials

Reagents and Chemicals

All CRM, reagents and chemicals were NIST traceable and LC-MS/MS grade. This study also utilized Type 1 water.

Method

Stock Solution and Calibration Standard Preparation

A stock solution was prepared of 10 mg/L of 10 mL standard mixture of all analytes. This was achieved by appropriate volumes from mother stock to a 10 mL volumetric flask before making this up with an appropriate solvent for each antibiotic analyte.

Standard solutions were all stored at -20 ºC. Calibration standards were prepared by serially diluting working solutions (5, 10, 25, 50, 100 and 200 ng/ml) of the stock solution with water:acetonitrile (80:20, v/v).

Sample Extraction Procedure

  • A total of 2 g ± 0.1 g of sample was weighed into a 50 mL PTFE centrifuge tube.
  • A total of 4 mL of water was added before this was shook in a vortex 30 second and left for 30 minutes.
  • A total of 10 mL of ethyl acetate was added before this was shook in a vortex for 2-3 minutes to ensure proper interaction of solvent and analytes.
  • The sample was centrifuged for 10 minutes at 10000 rpm.
  • A total of 5 mL of supernatant was added to evaporating tube and evaporated up to dryness under a nitrogen evaporator at 40 ºC.
  • The sample was reconstituted with 1 mL of acetonitrile:water (80:20, v/v) and vortexed.
  • The sample was then filtered through 0.2 µm filter paper.

Result and Discussion

The linearity study covers concentration levels of 5 µg/L to 200 µg/L via six calibration points. The method demonstrated excellent linearity with R2 >0.99 for all antibiotics studied in the milk matrix. A limit of quantification (LOQ) of 10 µg/L was achieved for all antibiotics.

Table 1 shows the LC method & MS source parameters, while Table 3 shows the TMRM mode transitions of the studied antibiotics.

Table 2. Matrix effect. Source: PerkinElmer Food Safety and Quality

Analyte Solvent std Area. Matrix-matched Area. % ME
Sulfamethoxypyridine 50655 80960 60
Sulfathiazole 38731.8 43709 13
Sulfadiazin 10873.6 43955.7 304
Sulfapyridine 18719.4 56815 204
Sulfamethizole 76718.8 122715 60
Sulfachloropyridazine 72406.6 111414.3 54
Sulfaisoxazole 94119.2 154994.2 65
Sulfaquinoxazole 30548.2 37611.5 23
Sulfaquinoxaline 29353 37376 27

 

Table 3. MRM Transitions and Retention time of analytes. Source: PerkinElmer Food Safety and Quality

Analyte Precursor Product 1 CE 1 Product 2 CE 2 RT
Sulfamethoxypyridine 281 156 -23 65 -78 3.68
Sulfathiazole 256 156 -20 65 -73 6.07
Sulfadiazin 251 156 -22 108 -34 3.08
Sulfapyridine 250 156 -22 184 -23 3.24
Sulfamethizole 271 156 -19 92 -41 3.50
Sulfachloropyridazine 285 156 -20 108 -38 3.76
Sulfaisoxazole 268 156 -19 113 -20 3.95
Sulfaquinoxazole 301 156 -23 92 -48 4.15
Sulfaquinoxaline 301 156 -23 208 -25 2.15

 

Matrix Effect

Responses of matrix-matched standards (peak area of pre-extraction spike) were compared against corresponding peak areas of standards in solvent. This was done across six replicates.

This process enabled the matrix effect (ME) to be quantified as the average percent suppression or enhancement in the peak area using the area of matrix standard and the area of solvent standard (Table 2). Negative ME values indicate matrix-induced signal suppressions, while positive values indicate signal enhancement.

Recovery Study

The recovery of all compounds was determined by spiking at 10, 25 and 50 µg/kg level in milk sample across six replicates.

Recovery for all analytes was found to be between 80% and 120%, while percentage RSD for all compounds was found to be below 20% - acceptable values that are in line with the regulatory requirements.

Tables 4, 5 and 6 represent the three-level recovery with recovery percentage and RSD. Data was analyzed in six replicates after completing a calculation of the final dilution factor.

Table 4. Recovery at 10 μg/kg spike with 5 times dilution at actual spike. Source: PerkinElmer Food Safety and Quality

Analyte Rec 1 Rec 2 Rec 3 Rec 4 Rec 5 Rec 6 Avg Stdev %RSD % Recovery
Sulfamethoxypyridine 10.73 10.61 10.61 10.00 10.60 10.89 10.57 0.30 2.88 105.72
Sulfathiazole 10.60 9.39 8.98 7.92 6.88 7.73 8.58 1.34 15.58 85.83
Sulfadiazin 9.51 8.84 9.38 10.97 10.10 9.16 9.66 0.76 7.92 96.60
Sulfapyridine 10.05 8.33 9.54 10.55 8.86 9.56 9.48 0.80 8.42 94.81
Sulfamethizole 10.25 9.88 9.95 9.28 9.10 8.94 9.57 0.53 5.52 95.65
Sulfachloropyridazine 10.86 10.27 10.75 10.06 10.48 10.54 10.49 0.30 2.83 104.94
Sulfaisoxazole 10.29 10.39 10.08 9.81 9.81 9.93 10.05 0.25 2.45 100.53
Sulfaquinoxazole 10.42 10.31 10.39 9.27 9.19 9.92 9.92 0.56 5.64 99.16
Sulfaquinoxaline 10.41 10.35 10.71 9.67 9.81 10.63 10.26 0.43 4.19 102.64

 

Table 5. Recovery at 25 μg/kg spike with 5 times dilution at actual spike. Source: PerkinElmer Food Safety and Quality

Analyte Rec 1 Rec 2 Rec 3 Rec 4 Rec 5 Rec 6 Avg Stdev %RSD % Recovery
Sulfamethoxypyridine 26.00 26.84 26.07 19.43 18.88 19.35 22.76 3.89 17.11 91.04
Sulfathiazole 21.19 20.56 23.42 22.02 22.80 21.75 21.96 1.04 4.75 87.83
Sulfadiazin 24.78 24.87 23.56 22.85 21.04 21.84 23.16 1.55 6.69 92.63
Sulfapyridine 25.99 27.17 28.12 22.12 21.31 23.61 24.72 2.79 11.27 98.88
Sulfamethizole 24.29 24.83 24.69 17.75 17.86 17.47 21.15 3.79 17.92 84.60
Sulfachloropyridazine 26.18 26.12 26.49 19.90 19.55 19.31 22.93 3.67 15.99 91.70
Sulfaisoxazole 25.94 26.04 26.06 18.64 18.46 18.93 22.35 4.02 18.00 89.39
Sulfaquinoxazole 26.54 26.41 25.50 18.67 18.18 19.83 22.52 4.03 17.88 90.08
Sulfaquinoxaline 24.80 25.24 25.31 18.85 18.99 17.89 21.85 3.61 16.51 87.38

 

Table 6. Recovery at 50 μg/kg spike with 5 times dilution at actual spike. Source: PerkinElmer Food Safety and Quality

Analyte Rec 1 Rec 2 Rec 3 Rec 4 Rec 5 Rec 6 Avg Stdev %RSD % Recovery
Sulfamethoxypyridine 47.45 48.29 48.24 53.66 54.03 54.35 51.00 3.32 6.50 102.00
Sulfathiazole 55.00 58.66 57.69 46.03 42.92 44.31 50.77 7.12 14.03 101.54
Sulfadiazin 50.20 50.65 49.93 52.42 53.82 53.27 51.71 1.67 3.23 103.43
Sulfapyridine 47.09 53.56 48.14 41.95 47.72 49.41 47.98 3.75 7.82 95.95
Sulfamethizole 48.12 49.35 48.48 56.60 56.17 56.57 52.55 4.29 8.17 105.10
Sulfachloropyridazine 48.23 49.33 47.41 55.44 54.52 54.99 51.65 3.71 7.18 103.31
Sulfaisoxazole 49.05 48.30 47.64 56.79 54.55 55.72 52.01 4.11 7.91 104.01
Sulfaquinoxazole 49.91 47.25 47.99 55.20 54.52 54.91 51.63 3.67 7.10 103.26
Sulfaquinoxaline 48.85 48.02 48.46 54.06 54.54 56.22 51.69 3.64 7.04 103.38

 

Conclusion

The results obtained throughout this study highlight the QSight™ LC-MS/MS method’s capacity for efficient, routine antibiotic analysis in milk. The results demonstrated excellent chromatographic repeatability. Sample analytes were identified and positively confirmed via their qualifier and quantifier ion ratios.

A rapid, reliable UHPLC-MS/MS method was devised to enable the simultaneous estimation of sulfonamides in milk matrix. LOQ for all the analytes was found to be 10 µg/kg, while linearity was found to range between 5 mg/kg and 200 mg/kg with a regression coefficient of > 0.99.

The LOQs attained using this method are confidently below the regulated level, indicating that the PerkinElmer QSight™ 220 LC-MS/MS System is a sensitive and robust platform ideally suited for the analysis of sulfonamides in milk.

References

  1. I A Unsal, M Tasan, T Gokcen and A C Goren. Determination of sulfonamides in milk by ID-LC-MS/ MS, 2018, 12 (1):70-78.
  2. W. Baran, E. Adamek, J. Ziemiańska and A. Sobczak (2011). Effects of the presence of sulfonamides in the environment and their influence on human health, J. Hazard. Mater. 196, 1–15.
  3. W. Zhu, J. Yang, Z. Wang, C. Wang, Y. Liu and L. Zhang (2016). Rapid determination of 88 veterinary drug residues in milk using automated TurborFlow online clean-up mode coupled to liquid chroma- tography-tandem mass spectrometry, Talanta. 148, 401–411.
  4. S. R. Parab and P. N. Amritkar (2012). Development and validation of a procedure for determination of sulfonamide residues in pasteurized milk using modified QuEChERS method and liquid chromatog- raphy/tandem mass spectrometry, J. AOAC Int. 95, 1528–1533.
  5. Wassenaar, T. M. (2005). Use of Antimicrobial Agents in Veterinary Medicine and Implications for Human Health. Critl Rev Microbiol 31, 155 – 169.
  6. Barlow, J. (2011). Antimicrobial resistance and the use of antibiotics in the dairy industry: facing con- sumer perceptions and producer realities. WCDS Advances in Dairy Technology 23, 47 – 58.
  7. European Parliament and of the Council of European Union (1996). Council Directive 96/23/EC of 29th April 1996 on measures to monitor certain substances and residues thereof in live animals and animal products and repealing Directives 85/358/EEC and 86/469/EEC and Decision 89/187/EEC and 91/664/EEC. OJ L125, 10 – 32.

Acknowledgments

Produced from materials originally authored by Narayan Kamble, Ph.D Kailas Gavhane and Umesh Talekar , Ph.D from PerkinElmer (India) Pvt. Ltd.

This information has been sourced, reviewed and adapted from materials provided by PerkinElmer Food Safety and Quality.

For more information on this source, please visit PerkinElmer Food Safety and Quality.

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