*2.2.2 Processing of samples of dairy products through HPLC*

## a.Chemicals and Reagents:

AFM1 standards (10 μg/l in acetonitrile), Celite and HPLC grade acetonitrile of Sigma–Aldrich, Steinheim, Germany and Immunoaffinity column AflaTm of VICAM, USA were used.

AFM1 standard curve or linearity curve was prepared by diluting the standards with acetonitrile at 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5 μg/ml concentrations and stored in caped vials in refrigerator at – 4 C.

## b.Samples Extraction:

5 g sample of each products and 5 g Celite mixed with 40 ml of dichloromethane in a 50 ml falcon tube. Then centrifuged at 21,000 rpm for 5 mins. After centrifugation the supernatant was separated and evaporated in water bath at 80 C. After evaporation the beaker was shifted in ultrasonic clean-up for 5 min. Then residues in beaker were dissolved in 10 ml mixture of methanol, water and n-hexane with the ratio of 3:5:2. Then 15 ml of this solution mixed by vortex mixture and again centrifuged at 21,000 rpm for 5 mins. After this, aqueous filtrate was passed through immunoaffinity column. The column was washed with 10 ml of water to remove toxins. After this column again washed with 2.5 ml of acetonitrile to get the final extract. This extract then dry under nitrogen steam at 40 C. After evaporation residues were dissolved with 2 ml mobile phase and mix by using vortex mixture. Finally, 20 μ/l sample was injected in HPLC for the analysis.


**Table 2.**

*Therapeutic trials in different groups.*

c.HPLC Conditions:

The HPLC used for the analysis was a Shimadzu LC-10A series (Japan) with the fluorescence detector (HPLC-FLD) having excitation wavelength of 365 nm and emission wavelength of 435 nm.

### **2.3 Therapeutic trial**

Three different toxin binders were used in respective groups A, B, and C each having 10 AFM1 positive animals. The **Table 2** showed types of toxin binder and their dose rates. Each toxin binder was used on the daily basis for 7 days.

### **2.4 Sample collection and processing after therapeutics**

At days 2nd, 3rd, 4th, and 7th 10 ml milk samples from each animal were collected in plain vacutainers and serum was extracted through centrifugation. After that milk samples were checked by using AFM1 rapid test kit.

### **2.5 Statistical analysis**

Collected data will be statistically scrutinized by SPSS 20.0 software and t-test as well as Chi square test was used to analyze the results.
