Development of a new UFHPLC-MS method for determination of different antibiotics in animal feeds

26 July 2021

AperTO - Archivio Istituzionale Open Access dell'Università di Torino

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Development of a new UFHPLC-MS method for determination of different antibiotics in animal feeds

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Development of a new UFHPLC-MS method for determination of

different antibiotics in animal feeds

Federica Dal Bello*

_{, Valentina Santoro*, Ilenia Siciliano*, Francesco Molinaro*, Claudio Medana* and Claudio Baiocchi*}

*Università degli Studi di Torino, Torino, Italy.

_{Corresponding author: federica.dalbello@unito.it}

References

[1] Jensen BB. 1998. J Anim Feed Sci 7: 45-46.

[2] WHO. 2012. “The evolving threat of antimicrobial resistance: options for action”. [3] Landers TF, Cohen B, Wittum TE, Larson EL. 2012. Public Health Reports 127: 4-22.

Overview

• In Europe, antibiotics could be add into animal feeds exclusively for therapeutic use; • Antibiotics in animal feeds enhance human resistance against bacterial infections;

• A rapid and sensitive UFHPLC-MS/MS method to determine different antibiotics classes into animal feed was developed;

• The developed method was applied to bovine meat samples.

Introduction

Since from the middle of the last Century, antibiotics were added at sub-therapeutic doses in animal feed as animal growth promoters.

The possible action mechanism involves the suppression by antibiotics of huge intestinal bacterial populations, which consume energy during fermentation process. So, this remaining energy could be re-used for animal growth.

As consequence, the meat has less fat proportion, enhanced muscle mass and high protein percentage [1].

However, the antibiotics abuse leads to the development of bacterial resistance. Consequently, many infections result incurable [2, 3].

Therefore, from 2006 antibiotics as growth promoters are banned in Europe [4] and they can only be used as therapeutic drugs when infection occurs. Animals under antibiotic treatment could not be slaughtered before an average of 28 days [5, 6]. If this time is not respected, there is a potential risk of finding antibiotics traces in meat.

A new rapid and sensitive UFHPLS-MS/MS method to detect traces of different classes of antibiotics (sulfonamides, β-lactams, lincosamides, pleuromutilins, macrolides and diaminopyridine) in animal feeds and meats was developed. A validation procedure was carried out with definition of LOD, LOQ, precision and repeatability and reproducibility.

[4] Gazzetta Ufficiale Europea L268/29, Regolamento CE N. 1831/2003.

[5] Gazzetta Ufficiale Italiana N. 78 del 3 aprile 1993, S.O., Decreto Legislativo 3 marzo 1993, N.92.

[6] Gazzetta Ufficiale Italiana N. 113 del 17 maggio 2005, Decreto Legislativo 3 marzo 2005.

Materials and Method

Sample Preparation

 Animal Feeds Solid-Liquid Extraction:

• animal feed samples spiked with different amounts of antibiotics mix solution were extracted under stirring with methanol, filtrated and analyzed.

 Bovine Meat SPE Extraction:

• meat samples spiked with different amounts of antibiotics mix solution were flushed with acetone/dichloromethane /acetic acid 50:50:10;

• SPE column (Waters Oasis HLB 60 mg and Agilent Technologies Bond Elute 500 mg) were used as follow: equilibrate with hexane, then acetone/dichloromethane/acetic acid 50:50:10, load the samples, wash with methanol, elute with methanol/ammonium hydroxide 2:1;

• dry the samples under stream of N₂ and re-dissolve in ammonium acetate 5 mM pH 3/Methanol 8:2.

NEXERA LC-30AD (Shimadzu) Conditions

• Kinetex 2.6 µm C18 100Å 100 × 2.1 mm (Phenomenex); • Eluents: Acetonitrile (A) and Formic Acid 0.05% (B);

• Gradient Condition: from 10% to 50% of solvent A in 25 minutes; • Injection volume: 10 µL; Flow rate: 500 µL/min

QTRAP5500 (Turbo Ion Spray, ABSciex) Settings

• MRM are listed in Table 1

Analyte Structural Formula Q1 (m/z) Q3 (m/z) Time (sec) DP EP CE CXP Amoxicillin 366 349 @ 207 30 100 5 10 @ 15 18 @ 11 Ampicillin 350 106 @ 192 30 170 10 20 @ 21 15 @ 12 Lincomycin 407 359 @ 126 50 210 8 24.7 @ 34 23 @ 17 Sulfadiazine 251 156 @ 92 30 70 4 19.7 @ 33 1.5 @ 1.3 Sulfadimethoxine 311 156 @ 108 30 98 6 27 @ 34.6 11 @ 19 Sulfamerazine 265 156 @ 172 50 60 5.7 21.3 @ 21.4 9 @ 19.8 Sulfamethazine 279 186 @ 124 50 80 5.5 24.1 @ 29.6 12 @ 8.7 Tiamulin 494 192 @ 119 30 220 10 27 @ 57 16 @ 13 Tilmicosin 435.1 522 @ 696.5 50 120 10 24 25 @ 30 Trimethoprim 291.3 230 @ 123 30 16.7 10 35.5 @ 54.7 50 @ 14 Tylosin 916.6 174 @ 772.5 50 60 5 47 @ 39 10 @ 34 NH HO N S NH2 O O O HO C16H20N3O5S 366.112367 H NH N S NH2 O O O HO C16H20N3O4S 350.117453 H N NH O H OH OH HO HO S O C18H35N2O6S 407.221583 H S HN N N O O NH2 C10H11N4O2S 251.060272 H S HN N N O O NH2 O O C12H15N4O4S 311.081401 H S HN N N O O NH2 C11H13N4O2S 265.075922 H S HN N N O O NH2 C12H15N4O2S 279.091572 H O OH O S N O C28H48NO4S 494.330406 H O O OH O N O O HO O O OH OH N O O C46H81N2O13 869.573866 H N N NH2 H2N O O O C14H19N4O3 291.145715 H O O O OH O O O O N OH OH HO O O O HO O O C46H78NO17 916.526975 H

Table 1: Multiple Reactions Monitor for analyzed antibiotics.

Conclusions

• The developed UFHPLC-MS/MS method was fruitfully used to qualitatively and quantitatively determine different classes of antibiotics both in animal feeds and bovine meats;

• Solid-liquid extraction protocol for animal feeds and HLB-SPE protocol for bovine meats give good yields, up to 82% and 38% respectively;

• The chromatographic separations of eleven antibiotics is obtained in only 25 minute long run;

• The sensitivity is very high due to the QTRAP5500 intrinsic performance, but especially due to the optimization of MS parameters, such as source gases and temperature, potentials, collision energies and so on.

• The precision, repeatability and reproducibility of the developed method were evaluated and the results are excellent.

Results and Discussion

Analyte Experimental _{LOQ (µg/Kg)}

Amoxicillin 1070 ±22.1 Ampicillin 59 ± 12.6 Lincomycin 55 ± 13.2 Sulfadiazine 50 ± 16.3 Sulfadimethoxine 52 ± 0.8 Sulfamerazine 54 ± 22.6 Sulfamethazine 51 ± 15.6 Tiamulin 62 ± 10.3 Tilmicosin 64 ± 5.3 Trimethoprim 57 ± 5.8 Tylosin 63 ± 29.1

Table 3: List of experimental

LOQ of analyzed antibiotics in animal feeds matrices.

The chromatographic run gave a good separation between the different analytes, with only one co-elution (ampicillin together with sulfamethazine, Rt= 10.8 min). Figure 1 shows a chromatogram of pure standard in aqueous solution.

Figure 1: Chromatographic separation of pure standard in aqueous solution.

The method was validated in terms of linearity, relative standard deviation of inter and intra day precision with pure standards both in water solution and in animal feed after solid-liquid extraction. Table 2 and Figure 2 illustrate the results in water and in animal feeds respectively.

In animal feeds, the limit of quantitation was evaluated, and the values are reported in table 3. Figure 4 illustrates the yields of solid-liquid extraction of antibiotics.

Moreover, the developed method was tested on muscles meat samples from bovine and the yields of two different SPE (Bond Elute, Agilent Technologies; HLB, Oasis Waters) was evaluated (Figure 4). Analyte R² RSD% inter day RSD% intra day LOD (ng/Kg) LOQ (ng/Kg) Amoxicillin 0.9920 31.6 22.4 100 500 Ampicillin 0.9798 23.1 9.0 21 50 Lincomycin 0.9866 13.2 15.6 26 50 Sulfadiazine 0.9849 14.0 22.2 6 50 Sulfadimethoxine 0.9794 11.2 17.1 17 50 Sulfamerazine 0.9872 9.2 11.9 14 50 Sulfamethazine 0.9895 8.4 15.2 6 50 Tiamulin 0.9751 16.0 22.9 5 50 Tilmicosin 0.9574 26.2 33.0 200 500 Trimethoprim 0.9851 10.2 11.9 9 50 Tylosin 0.9851 23.2 20.1 81 500

Table 2: Linearity (R²), relative standard

deviation (RDS%) of inter and intra day of pure standards in water solution.

Figure 2: Linearity (R²) of pure standards in

animal feeds matrices after solid-liquid

extraction. Sulfadiazine - C6H9NO2S C10H11N2O3 207.07 NH HO NH2 O O C16H17N2O5S 349.08 - NH3 NH HO N S O O O HO C16H20N3O5S 366.11 NH HO N S NH3 O O O HO Amoxicillin H2N SO2+ C6H6NO2S 156.17 - C7H7N3 H2N SO2NH2 N N C11H13N4O2S 265.08 - C6H7N + SO2NH N N C5H6N3O2S 172.02 Sulfamerazine C8H16N 126.22 N NH2 O H OH OH HO HO S O C18H35N2O6S 407.22 - CH4S - C10H19NO6S N H C17H31N2O6 359.44 N NH2 O H OH OH HO HO O Lincomycin C10H12N2O2 192.21 NH NH3 O O C7H8N 106.14 NH2 NH N S NH3 O O O HO C16H20N3O4S 350.12 - C6H8NO2S -C9H12N2O4S Ampicillin H3N N N +SO2NH N N H2N SO2NH2 N N C12H15N4O2S 279.09 C6H8N3O2S 186.03 - C6H7N C6H10N3 124.09 - C6H5NO2S Sulfamethazine C5H7N4 123.07 C13H12NO3 230.08 N O O O N N NH3 HN C14H19N4O3 291.14 N N NH3 H2N O O O - C9H12O3 - CH7N3 Trimethoprim - C6H9N3O2 H2N O+_C 6H6NO 108.11 H2N SO2+ C6H6NO2S 156.17 S H2N N N O O NH2 O O C12H15N4O4S 311.08 - C6H9N3O3S Sulfadimethoxine C46H78NO17 917.12 O H3C HO OCH3 OCH3 O OH3C O CH3 H3C OH CH3 CHO O O HO CH3 N OH H3C CH3 O H O HO CH3 N OH H3C CH3 H O H3C HO OCH3 OCH3 O OH3C O CH3 H3C OH CH3 CHO O O HO CH3 N O O CH3 OH OH H3C H3C CH3 O H - C7H12O3 C39H66NO14 772.45 C8H16NO3 174.11 - C31H50O11 Tylosin H2N SO2NH2 N N H2N SO2 + H2N C10H11N4O2S 251.06 C6H6NO2S 156.17 - C4H5N3 C6H6N 92.05 - C4H5N3O2S O O OH OH2 N O HO O O O O C38H66NO10 696.45 O O OH O N O O HO O O OH OH N O O C8H16NO3 - C46H82N2O13 870.58 2H O HO OH N O O OH2 C30H52NO6 522.34 C16H30NO7 - Tilmicosin O OH O S NH O OH S NH O C8H18NO2S 192.10 C28H48NO4S 494.33 OH S OH C4H7O2S 119.01 C24H41NO2 - C20H30O2 - Tiamulin 0 20 40 60 80 100 120 Yi el ds (%) Figure 3: Percentage yields of solid-liquid extraction in animal feeds samples. Figure 4: Percentage yields of SPE extraction in bovine meats samples. 0 20 40 60 80 100 120 Yi el ds ( % ) HLB SCX 0 20 40 60 80 100 120 Yi el ds ( % ) HLB SCX BOND ELUTE 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 0 50 100 150 200 A re a A n al yt e /A re a In te rn al S ta n d ar d Concentration (ng/mL)

Linearity of antibiotics pure standard in animal feeds after solid-liquid extraction

Solid-liquid extraction