Determination of Erythromycin, Spiramycin, Tilmicosin and Tylosin in Animal Feedingstuffs by Liquid Chromatography-Tandem Mass Spectrometry

Abstract

Detection of macrolides residues in animal feedingstuffs to ensure food safety is very important issue. For this purpose, a liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination of residues of 4 macrolides (erythromycin, spiramycin, tilmicosin and tylosin) in animal feedingstuffs. Roxithromycin (ROX), a macrolide not used in veterinary medicine, was used as internal standard. The mass spectral acquisition was done in the positive-ion mode applying multiple reaction monitoring with the following ions (mass-to-charge ratio, m/z): m/z 837.4→679.2; m/z 734.4→576.2; m/z 422.4→174.1; m/z 869.1→696.1 and m/z 916.4→772.4 for roxithromycin (ROX), erythromycin (ERY), spiramycin (SPI), tilmicosin (TIL), and tylosin (TYL), respectively. Good repeatibility, reproducibility and recovery values was obtained. Average recoveries ranged from 98.9% (TIL) to 101% (ERY) and an overall mean of 99.9%. Method limits of detection (LODs) of ERY, SPI, TIL and TYL 5.1, 6.6, 6.7 and 7.5 µg kg-1 were achieved respectively. Method limits of quantification (LOQs) were 8.5, 11.1, 11.2 and 12.4 µg kg-1 for the same drugs, respectively. Satisfactory decision limits (CCα) and detection capabilities (CCβ) were also attained. The method is simple, rapid, sensitive and suitable for the simultaneous determination of macrolide antibiotics in animal feedingstuffs. Keywords: Macrolides, Feedingstuffs, Validation, LC-MS/MS Hayvan Yemlerinde Eritromisin, Spiramisin, Tilmikosin ve Tilosinin LC-MS/MS İle Tespiti ÖzetMakrolid grubu antibiyotiklerin hayvan yemlerinde tespit edilmesi gıda güvenliğinin sağlanması açısından çok önemli bir konudur. Bu amaçla, hayvan yemlerinde kullanılan 4 adet makrolid (eritromisin, spiramisin, tilmikosin ve tilosin)'in tespiti için sıvı kromatografi-tandem kütle spektrometrisi (LC-MS/MS) metodu valide edilerek kullanılmıştır. Roksitromisin (ROX), veteriner hekimlik alanında kullanılmayan bir makroliddir ve bu yüzden çalışmada iç standart olarak kullanılmıştır. Kütle spektrası uygulaması pozitif-iyon modunda ve çoklu reaksiyon izleme modunda gerçekleştirilerek, (kütle/şarj oranı, m/z) roksitromisin (ROX) için m/z 837.4→679.2; eritromisin (ERY) için m/z 734.4→576.2; spiramisin (SPI) için m/z 422.4→174.1; tilmikosin (TIL) için m/z 869.1→696.1 ve tilosin (TYL) için m/z 916.4→772.4 taranmıştır. İyi bir tekrarlanabilirlik, üretilebilirlik ve geri kazanım değerleri elde edildi. Hesaplanan ortalama geri kazanım değerleri %98.9 (TIL)'den %101'e (ERY) kadar değişmekle beraber, toplam ortalama %99.9 olarak gerçekleşti. Metodun tespit limitleri (LOD) ERY, SPI, TIL ve TYL için sırasıyla, 5.1, 6.6, 6.7 and 7.5 µg kg-1 olarak hesaplandı. Metodun sayı verilebilir limitleri aynı sırayla 8.5, 11.1, 11.2 and 12.4 µg kg-1 olarak gerçekleşti. Validasyon sonucunda belirtilen bu dört makrolid için memnun edici karar limitleri (CCα) ve karar verebilme kapasitelerine (CCβ) de ulaşılabildi. Validasyon sonuçlarına bakıldığında basit, hızlı, duyarlı ve uygun olan bu metot yukarıda belirtilen dört...

Challenges for the determination of spiramycin in aqueous matrices using LC-MS/MS: evidence for the solvent intrusion on the molecule integrity

“…In most of the previous studies conducted for the determination of spiramycin residue in milk samples, the pure standard solutions of spiramycin were prepared in methanol or water, 15,16,[18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35] and in a few studies, the standard solutions were prepared in acetonitrile. [36][37][38] The results of our study revealed that there is a possibility for the intrusion of protic solvents into the spiramycin molecules during the preparation of standard solutions such as stock solutions and working standard solutions or even during the preparation of the sample.…”

Design of Novel Haptens and Development of Monoclonal Antibody-Based Immunoassays for the Simultaneous Detection of Tylosin and Tilmicosin in Milk and Water Samples

“…Thus, our first attempt was to use the reported Shiff base hapten strategy, but this failed to obtain an antibody against tylosin and tilmicosin because of the poor and unstable immune response. We speculated Currently, methods for simultaneous detection of tylosin and tilmicosin mainly include high-performance liquid chromatography (HPLC), high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS), and other large-scale chromatographic methods [18][19][20]. These large-scale equipment methods are highly sensitive and accurate, but are expensive, time-consuming, have low sample throughput, and are inconvenient for detection in the field.…”