Development and Validation of a New High Performance Liquid Chromatography Method for the Determination of Sulfonamides in Milk

Öz

In this study, a rapid, sensitive and simple high performance liquid chromatographic (HPLC) method was developed and validated for the analysis of sulfadiazine (SDZ), sulfamerazine (SMR) and sulfamethoxol (SMX) in milk samples which are in the group of sulfonamides (SAs). Protein precipitation method was used in the extraction of SDZ, SMR and SMX from milk. The precipitant reagent type and its amount were optimized in the protein precipitation method. All validation parameters were studied using milk samples in order to observe the effect of the study matrix effect on the analyses. Validation of the method was carried out with linearity, robustness, accuracy and precision in accordance with the ICH guideline. The limits of detection (LOD) of the method developed for the determination of sulfonamides in milk samples were 0.11, 0.10 and 0.14 µg/mL and the limits of quantification (LOQ) were 0.34, 0.30 and 0.43 µg/mL for SDZ, SMR and SMX, respectively. The average recoveries of SAs in milk are ≥ 93.0 ± 4.4%. The intra-day and inter-day reproducibility of the method in terms of % relative standard deviation are ≤ 6 % and ≤5.4%, respectively. The developed method is accurate and reliable and can be used in routine analysis of SAs in milk samples in food control and toxicological analysis laboratories.

Anahtar Kelimeler

• Sulfonamides
• HPLC
• Method validation

Sütteki Sülfonamidlerin Tayini için Yeni bir Yüksek Performanslı Sıvı Kromatografisi Yönteminin Geliştirilmesi ve Valide Edilmesi

Öz

Bu çalışmada sülfonamidler (SAs) grubunda bulunan sülfadiazin (SDZ), sülfamerazin (SMR) ve sülfometaksazol’ün (SMX) süt numunelerinde analizi için hızlı, hassas ve basit bir yüksek performanslı sıvı kromatografi (HPLC) yöntemi geliştirilmiş ve valide edilmiştir. SDZ, SMR ve SMX’in sütten ekstraksiyonunda protein çöktürme yöntemi kullanılmıştır. Protein çöktürme yönteminde çöktürücü reaktif türü ve miktarı optimize edilmiştir. Çalışma matriks etkisinin analizlere olan etkisinin gözlemlenmesi amacıyla tüm validasyon parametreleri süt numuneleri kullanılarak çalışılmıştır. Yöntemin validasyonunda ICH klavuzu uyarınca doğrusallık, sağlamlık, doğruluk ve kesinlik testleri gerçekleştirilmiştir. Süt numunelerinde sülfonamidlerin tayini için geliştirilen yöntemin gözlenebilme sınırları (LOD) SDZ, SMR ve SMX için sırasıyla 0.11, 0.10 ve 0.14 µg/mLve tayin sınırları (LOQ) 0.34, 0.30 ve 0.43 µg/mL’dir. SAs’lerin sütteki ortalama geri kazanımları ≥ %93.0±4.4’tür. Yöntemin gün içi ve günler arası tekrarlanabilirliğini % bağıl standart sapma cinsinden sırasıyla ≤ %6.0 ve ≤% 5.4’dir. Geliştirilen yöntem doğru ve güvenilir olup gıda kontrol ve toksikolojik analiz laboratuvarlarında süt numunelerinin içeriğindeki SAs'lerin rutin analizi için kullanılabilir.

Anahtar Kelimeler

• Sülfonamidler
• HPLC
• Yöntem validasyonu

Kaynakça

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