The occurrence of antibiotics in seafood has received worldwide interest over Crenolanib mouse the last few years.3, 4, 5 and 6 Analysis of antibiotics such as tetracyclines,7 and 8 sulfonamides,9 and 10 chloramphenicol11 macrolide antibiotics and avermectins12 and quinolones13 and 14 in seafood by using immunoassay, HPLC and LC–MS/MS has been reported for various species from different countries. No method has been reported for analysis of antibiotics in seafood found in India.

So we aimed to determine tetracycline antibiotics (Tetracycline (TC), Oxytetracycline (OTC), Chlortetracycline (CTC) and Doxycycline (DOC)) in prawns obtained from the coastal regions of south India by using LC–MS/MS. Prawns (Penaeus monodon) were collected from Tamil Nadu (Sample-1), Andhra Pradesh (Sample-2), Karnataka (Sample-3) and Kerala (Sample-4). The collected samples, around 500 gm each were stored in the refrigerator at −20 °C. Chromatographic

separation was carried out by using LC–MS/MS (LC-Agilent 1020 series; MS-Applied Biosystem/MDS/Sciex, API-3000; Analyst 1.4.2 software; Electron spray ionization; Chem detector). Separation was carried out by using reverse phase Zorbax Eclipse Plus C18 (5 μ particle size, 4.6 × 100 mm). The mobile phase consists of 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in inhibitors methanol (mobile phase B). Gradient elution technique was used for separation, Regorafenib purchase at a flow rate 400 μl/min, injection volume 20 μl and column temperature 40 °C. Tetracycline antibiotics were monitored by 2 MRM (Multiple reaction monitoring) transitions

(one for conformation and one for quantitation). To optimize the method, tissues of prawns were spiked with all tetracycline antibiotics which were dissolved in 4 ml of methanol and shaken well to make uniform distribution of spiked compounds. Collected samples were cleaned thoroughly, cut in to small pieces and homogenized. Homogenized portion was added with HPLC grade methanol and centrifuged for 15 min at 3000 rpm; supernatant fluid is collected and evaporated to dryness. Dry substance is dissolved in mobile phase (0.1% formic acid in methanol) and filtered through 0.22 μ membrane filter and 20 μl was injected. The proposed oxyclozanide method was validated for selectivity, sensitivity (limits of detection and quantification), accuracy, precision, recovery and robustness according to 2002/657/EC Decision.15 Good reproducibility was achieved by using mobile phase 0.1% formic acid in water (phase A) and 0.1% formic acid in methanol (phase B). The gradient elution results are provided in Table 1. Tetracycline antibiotics were monitored by 2 MRM, the mass(es) precursor ion (m/z) and quantitative ions (m/z): TC: 445.0/410.1 + 445.0/427.0; OTC: 461.1/426.2 + 461.1/442.9; CTC: 479.2/444.0 + 479.1/154.0; DOC: 445.2/428.4 + 445.2/154.0. Quantitation of antibiotics was carried out by external calibration method and the results are given in Table 2.