Tylosin and tilmicosin (T&T) residues in livestock products have received extensive attention from consumers. Time-resolved fluorescence immunochromatographic assay (TRFICA), as a fast, efficient and sensitive immunoassay method, has played an increasingly important role in the food safety field. Therefore, herein a quantitative and visual TRFICA was established for simultaneously detecting T&T in milk in a group-screening manner. Under the optimal conditions, the standard curve range of developed TRFICA based on the T&T was 1.87~7.47 ng/mL, and the half-maximal inhibition concentrations (IC50) were 4.06 ng/mL and 3.74 ng/mL, respectively. The limits of detection (LOD) of the TRFICA method were from 1.72 ng/mL to 1.39 ng/mL, and the visual cut-off values were 31.25 ng/mL and 62.50 ng/mL for T&T in milk, respectively. Moreover, the stability experiments showed that the strips could be stored at 4 °C for more than 6 months, the total detection time was less than 13 min, and the cross-reactivities (CRs) with related compounds were less than 0.1%, which concluded that the developed TRFICA method could be used in real milk sample detection.

INTRODUCTIONAs a typical water-soluble vitamin, vitamin B2 (riboflavin) is an essential micronutrient for human beings. It plays an important role in the human body [1][2][3][4] . Riboflavin deficiency may lead to skin and mucosal disorders, angular cheilitis, anemia or glossitis and seborrheic dermatitis 5 . Therefore, a simple, sensitive and accurate determination of riboflavin is very important. Different methods have been used for the determination of riboflavin, such as high performance liquid chromatography (HPLC)-photodiode array detection 6 , HPLC-fluorescence spectroscopy 7-9 , HPLC-matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (HPLC-MALDI-TOF) 10 and capillary electrophoresis (CE) laser induced fluorescence (LIF) spectroscopy 11,12 . To our best of knowledge, no methods to date have been published for the determination of riboflavin in food by HPLC with in-column optical fiber laser-induced fluorescence detection technology. In this work, the HPLC separation of vitamin B2 in foods and the ICOF-LIF detection system are studied and optimized. This method is simple, high sensitivity and good precision. EXPERIMENTALTangerine and mushroom were purchased from local markets (Chengdu, China). HPLC-grade methanol and VB2