Determination of Roxithromycin Residues in Milk by UV-1500 Spectrophotometry - Master's thesis - Dissertation

Spectrophotometric determination of roxithromycin residues in milk. Keywords: spectrophotometry; milk; roxithromycin; analytical instrument; UV-1300. **Objective:** This experiment aims to master the principle and application of UV spectrophotometry for quantifying roxithromycin in milk samples. The method involves colorimetric detection based on a chemical reaction between roxithromycin and p-dimethylaminobenzaldehyde. **Principle:** Roxithromycin, a derivative of erythromycin, can be hydrolyzed in concentrated HCl under glacial acetic acid conditions. When reacted with p-dimethylaminobenzaldehyde, it forms a colored compound that exhibits maximum absorbance at 486 nm. By comparing the absorbance values of the sample with those of standard solutions, the concentration of roxithromycin can be determined. **Instruments and Reagents:** 1. **Instruments:** UV-1300 spectrophotometer, centrifuge, ultrasonic cleaner. 2. **Reagents:** - Glacial acetic acid, concentrated HCl, 95% ethanol, p-dimethylaminobenzaldehyde. - HCl-glacial acetic acid mixture (2:1 by volume). - Color reagent (0.5% p-dimethylaminobenzaldehyde in glacial acetic acid). - Standard solution of roxithromycin (0.8 mg/mL) prepared in 95% ethanol. 3. **Sample:** Fresh milk (2–3 types), 50 mL each. **Procedure:** 1. **Sample Preparation:** Weigh 1.00 g of milk sample into a 100 mL stoppered conical flask. Add 10 mL of 95% ethanol, shake thoroughly, then sonicate for 20 minutes. Centrifuge at 4000 rpm for 10 minutes. Collect the supernatant as the sample extract. 2. **Standard Curve Preparation:** Prepare standard solutions by adding 0, 0.5, 1.0, 1.5, 2.0, and 2.5 mL of roxithromycin standard solution to 50 mL volumetric flasks. Add 20 mL of glacial acetic acid, 5.0 mL of color reagent, and dilute to the mark with HCl-glacial acetic acid mixture. Mix well and allow to stand for 15–35°C for 15 minutes. Measure absorbance at 486 nm and plot a standard curve. 3. **Sample Analysis:** Take 2 mL of the sample extract and follow the same procedure as for the standards. Measure the absorbance at 486 nm and determine the concentration from the standard curve. Perform a reagent blank for accuracy. **Calculation:** The concentration of roxithromycin in the sample is calculated using the formula: x = (c × V) / m Where: - x = content of roxithromycin in the sample (mg/kg) - c = concentration of roxithromycin in the extract (μg/mL) - V = volume of the extract (mL) - m = mass of the sample (g) **Precautions:** Ensure that the volume of the sample extract is adjusted according to the expected concentration of roxithromycin. Avoid contamination during all steps. Maintain consistent temperature and timing during the color development process. Keywords: spectrophotometry; milk; roxithromycin; analytical instrument; UV-1300.

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