A high performance liquid chromatography (HPLC) method for the detection of immunoglobulin G (IgG) in milk powder was established by studying the effects of immunoaffinity column performance, temperature of the sample solution during immunoaffinity column cleanup, and the type of eluent on the detection of the IgG content in milk powder. Samples were extracted with phosphate buffer solution, and the extract was purified by immunoaffinity column chromatography. The eluate was separated on a C₄ reversed-phase column (250 mm × 4.6 mm, 3.5 μm), detected using HPLC-ultraviolet detector, and quantified by an external standard method. The results showed that the method was applicable to milk powder with different IgG contents. The standard curve exhibited good linearity in the concentration range of 10-500 μg/mL. The detection limit of the method was 80 mg/kg, and the quantification limit was 300 mg/kg. The recoveries and precision expressed as relative standard deviations (RSDs) at three spiked levels (300, 600 and 3000 mg/kg) were both aligned with the requirements of the Chinese national food safety standard GB 5009.295-2023. This method is simple, efficient and accurate, providing precise technical support for the evaluation of the nutritional value of new functional dairy products and product claims.

A reversed-phase ultra-high performance liquid chromatography method was established for the determination of 1-deoxynojirimycin (1-DNJ) in milk powder. The sample was extracted ultrasonically using a mixed solution of hydrochloric acid (0.01 mol/L) and methanol (1:1, V/V). After purification via low-temperature centrifugation, the extract was derivatized with 9-fluorenylmethoxycarbonyl chloride under alkaline conditions. Glycine was used to neutralize the derivatives, which was terminated by adding a 1% (V/V) acetic acid solution. The analyte was separated on a reversed-phase C₁₈ column and detected using a diode array detector at a wavelength of 266 nm. Qualitative analysis was performed based on the retention time, and quantitative analysis was conducted using the external standard method. The results showed that the calibration curve exhibited a good linear relationship in the concentration range of 1-50 μg/mL. The limit of quantification was 18.0 mg/kg, and the limit of detection was 6.0 mg/kg. The recoveries of 1-DNJ from spiked samples at concentration levels of 18.0-270.0 mg/kg were 105.4%-106.6%, 94.1%-97.4%, 96.7%-102.7%, and 100.5%-104.5% with relative standard deviations of 1.18%, 1.76%, 2.08%, and 1.57%, respectively. This method is characterized by high sensitivity, accuracy and repeatability, and is suitable for the determination of the content of 1-DNJ in milk powder.

An ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method with solid phase extraction (SPE) was established for the detection of ethephon residue in milk. In the sample pretreatment stage, formic acid solution was selected as the extraction solvent, and the extract was purified on an amino SPE column. The target compound was separated on a Waters Anionic Polar Pesticide column (150 mm × 2.1 mm, 5 μm) using gradient elution with a mobile phase consisting of 0.1% formic acid containing 50 mmol/L ammonium formate and acetonitrile. The detection was performed using electrospray ionization in the negative ion mode with multiple reaction monitoring, and the matrix-matched external standard method was used for quantification. The calibration curve for ethephon showed an excellent linearity (R² > 0.99) in the concentration range of 2 to 100 ng/mL, and the limit of detection and limit of quantification of this method were 0.5 and 1.0 μg/kg, respectively. At spiked concentration levels of 1.0, 2.0 and 10.0 μg/kg, the recoveries were between 92.50% and 113.76%, and the precision, expressed as relative standard deviation, ranged from 3.04% to 4.60%. In conclusion, the UPLC-MS/MS method has the advantages of high accuracy, specificity, and repeatability, and is very suitable for the determination of ethphon in milk.