Determination of Methionine Hydroxy Analogue in Feed by Wayeal’s High Performance Liquid Chromatography

Methionine Hydroxy Analogue (MHA) is an organic acid additive widely used in the feed industry as a substitute for methionine. MHA is widely used in the feed of poultry (broilers and layers), swine (piglets and finishing pigs), ruminants (dairy cattle and beef cattle), and aquaculture (fish and shrimp). As a substitute for methionine, it demonstrates significant effects in reducing feed costs, improving the intestinal environment of animals, and enhancing animal immunity.

High performance liquid chromatography coupled with UV detection is well-suited for the analysis of various complex feed samples, owing to its excellent separation efficiency, strong matrix interference resistance, high detection sensitivity, and stable and reliable quantitative results. This experiment was conducted in accordance with the standard "Determination of Methionine Hydroxy Analogue in Feed by High Performance Liquid Chromatography" (GB/T 19371-2025). A Wayeal LC3400 series high performance liquid chromatograph equipped with a UV detector was employed to validate the sample pretreatment procedure and the liquid chromatographic separation and detection system, enabling both qualitative detection and precise quantitative analysis of methionine hydroxy analogue in various types of feed samples.

Keywords: feed; methionine hydroxy analogue; high performance liquid chromatography.

1. Instruments and Reagents

1.1 HPLC Configuration List

Table 1 High Performance Liquid Chromatography System Configuration List

1.2 Reagents and Reference Standards List

Table 2 Reagents and Reference Standards List

1.3 Liquid Chromatography Conditions

Table 3 High Performance Liquid Chromatography Conditions

1.4 Experimental Materials and Auxiliary Equipment

Ultrasonic cleaner;

Vortex mixer;

High-speed centrifuge;

Analytical balance;

Shaker.

2. Experiment Method

2.1 Solution Preparation

2.1.1 Standard Curve Solutions:

Accurately weigh an appropriate amount of Methionine Hydroxy Analogue reference standard and dissolve it in 10% acetonitrile to prepare a series of standard solutions at mass concentrations of 0.5, 1.0, 2.0, 5.0, 10.0, 20.0, 40.0, and 80.0μg/mL. Accurately transfer 5mL of each standard solution into 15mL stoppered test tubes separately. Add 0.1mL of 50% potassium hydroxide solution accurately, and vortex for 15 seconds. Then accurately add 0.2mL of 50% phosphoric acid solution, and vortex for another 15 seconds. Centrifuge at 8000r/min for 5 minutes. Pass the supernatant through a microporous membrane filter before analysis.

2.1.2 Test Sample Solutions:

Weigh 5g of feed sample into a container, add 50mL of 10% acetonitrile solution, and mix for 30s. Place on a shaker and shake for 30 minutes. Centrifuge at 8000r/min for 5 minutes, and retain the supernatant for later use. Accurately transfer 5mL of the above supernatant into a 15mL stoppered test tube. Accurately add 0.1mL of 50% potassium hydroxide solution and vortex for 15s. Then accurately add 0.2mL of 50% phosphoric acid solution and vortex for another 15s. Centrifuge at 8000r/min for 5 minutes. Pass the supernatant through a microporous membrane filter before analysis. Prepare two parallel test sample solutions.

2.1.3 Recovery Test Solutions:

Accurately transfer 2.5mL of the sample supernatant and 2.5mL of each standard series solution into a 15mL stoppered test tube. Accurately add 0.1mL of 50% potassium hydroxide solution and vortex for 15s. Then accurately add 0.2mL of 50% phosphoric acid solution and vortex for another 15s. Centrifuge at 8000r/min for 5 minutes. Pass the supernatant through a microporous membrane filter before analysis.

3. Experiment Result

3.1 Linearity and Range

Table 4 Standard Curve Concentration Table

Fig 1 Standard Curve of Methionine Hydroxy Analogue

Note: Appropriate amounts of the Methionine Hydroxy Analogue reference standard solution were taken and serially diluted to a series of concentrations for the preparation of the standard curve. The linear range was 0.5–80.0 μg/mL, and the correlation coefficient (R) was 0.99983, indicating excellent linearity for the analyte.

3.2 Precision

Fig 2 Overlaid Chromatograms of Precision Test for Reference Standard (20.0μg/mL)

Table 5 Precision Test Data for Reference Standard (20.0μg/mL)

Note: The reference standard of Methionine Hydroxy Analogue (at a concentration of 20.0μg/mL) was continuously injected for 6 replicates. The repeatability (RSD) of retention time was 0.084%, and that of peak area was 0.391%, indicating good instrument precision.

3.3 Recovery

Fig 3 Overlaid Chromatograms of Recovery Test for Methionine Hydroxy Analogue in Feed

Table 6 Recovery Test Data for Methionine Hydroxy Analogue in Feed

Note: The sample was spiked with the reference standard at a concentration of 40.0μg/mL and analyzed following the procedure described above. The recovery obtained was 88.56%, indicating good accuracy of the method.

3.4 Limit of Detection (LOD)

Fig 4 Chromatogram of Methionine Hydroxy Analogue Standard (2.5μg/mL)

Table 7 Test Data for Methionine Hydroxy Analogue Reference Standard (2.5μg/mL)

Note: Based on the test data of the Methionine Hydroxy Analogue reference standard at 2.5μg/mL as shown in the table above, the theoretical limit of detection (LOD) was calculated as 0.033μg/mL at a signal-to-noise ratio of 3 (S/N = 3).

3.5 Test of a Feed Sample

Fig 5 Chromatogram of a Feed Sample

Note: Methionine Hydroxy Analogue was detected in a feed sample at a concentration of 0.402μg/mL.

4. Conclusion

This experiment was conducted in accordance with the standard "Determination of Methionine Hydroxy Analogue in Feed by High Performance Liquid Chromatography" (GB/T 19371-2025), using the Wayeal LC3400 series high performance liquid chromatograph equipped with a UV detector. The experimental results showed that the target peak exhibited good symmetry and high theoretical plate number. Over the concentration range of 0.5–80.0μg/mL, the correlation coefficient (R) was above 0.9998. The RSD values for retention time and peak area of the MHA reference standard (20.0μg/mL) were 0.084% and 0.391%, respectively, indicating excellent instrument precision. The recovery of MHA in feed samples spiked at 40.0μg/mL was 88.56%, demonstrating good method accuracy. The theoretical limit of detection (LOD) for MHA was determined to be 0.033μg/mL. MHA was successfully detected in a feed sample at a concentration of 0.402μg/mL. All the above data meet the requirements of the pharmacopoeia method for instrument performance.