Imported High-quality L-threonine,Content in Aquatic Animal Feed

The content of L-threonine in aquatic animal feed is usually detected by high-performance liquid chromatography (HPLC). The specific detection steps are as follows:

I. Sample Pretreatment

Weigh an appropriate amount of the aquatic animal feed sample accurately to 0.0001 g. Grind and crush the sample and sieve it to make the sample particles uniform, so as to ensure the accuracy and representativeness of subsequent analysis.

Weigh a certain amount of the crushed sample into a stoppered Erlenmeyer flask, and add an appropriate amount of hydrochloric acid solution to release L-threonine from complexes such as proteins in the feed. Generally, the concentration of hydrochloric acid is 6 mol/L, and the liquid-solid ratio (mL/g) is between 10:1 and 20:1.

Place the Erlenmeyer flask in a constant-temperature shaking water bath pot, and heat and hydrolyze it at a certain temperature (such as 110°C) for a certain period of time (usually 24 hours) to completely hydrolyze the protein into amino acids. During the hydrolysis process, pay attention to maintaining the consistency of the hydrolysis conditions to ensure the stability of the hydrolysis degree.

After the hydrolysis is completed, take out the Erlenmeyer flask and cool it to room temperature. Then transfer the hydrolysate to a volumetric flask, dilute it to the mark with deionized water, and shake it well.

Take an appropriate amount of the above solution, filter it with filter paper, discard the initial filtrate, and collect the subsequent filtrate as the test solution. Centrifugation can also be used. Centrifuge at a speed of 3000 - 5000 r/min for 10 - 15 minutes, and take the supernatant as the test solution.

II. Preparation of Standard Solution

Accurately weigh an appropriate amount of L-threonine standard substance accurately to 0.0001 g. Dissolve it in an appropriate amount of hydrochloric acid solution (such as 0.1 mol/L), then transfer it to a volumetric flask, dilute it to the mark with deionized water, and prepare an L-threonine standard stock solution with a concentration of 1.0 mg/mL.

Pipette appropriate amounts of the standard stock solution respectively, and dilute them with deionized water to a series of standard working solutions with different concentrations, such as 0.05 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.5 mg/mL, 1.0 mg/mL, etc., for drawing the standard curve.

III. Derivatization Treatment

Take appropriate amounts of the test solution and the standard working solution respectively, and place them in stoppered test tubes. Add an appropriate amount of derivatization reagent, such as o-phthalaldehyde (OPA)-β-mercaptoethanol reagent. Generally, the volume ratio of the derivatization reagent to the sample solution is between 1:1 and 1:5.

Mix them quickly and react at room temperature for a certain period of time (usually 1 - 5 minutes) to make L-threonine fully react with the derivatization reagent to generate derivatives with fluorescent properties.

IV. Setting of Chromatographic Conditions

Chromatographic Column: Select a chromatographic column suitable for amino acid analysis, such as a C18 reversed-phase chromatographic column. The column length is generally 150 - 250 mm, the inner diameter is 4.6 - 5.0 mm, and the particle size is 5 - 10 μm.

Mobile Phase: Usually, a methanol-water-acetate buffer solution system is used. For example, mobile phase A is a 0.05 mol/L sodium acetate buffer solution (pH = 7.2), and mobile phase B is methanol. A gradient elution program is adopted. Initially, the proportion of mobile phase A is relatively high (such as 95%), and over time, the proportion of mobile phase B is gradually increased to achieve the effective separation of different amino acids.

Flow Rate: Generally set at 1.0 - 1.5 mL/min.

Column Temperature: Keep the column temperature between 30 - 40°C to ensure the stability and separation effect of the chromatographic column.

Detector: Use a fluorescence detector with an excitation wavelength of 340 - 360 nm and an emission wavelength of 450 - 480 nm to detect the derivatized L-threonine.

V. Sample Determination

After the instrument is stable, pipette appropriate amounts of the standard working solution and the test solution in turn and inject them into the high-performance liquid chromatograph, and record the chromatogram.

Draw a standard curve according to the chromatographic peak areas and concentrations of the standard working solutions. Generally, use the concentration as the abscissa and the peak area as the ordinate, and perform linear regression by the least squares method to obtain the equation of the standard curve.

Calculate the concentration of L-threonine in the test solution according to the chromatographic peak area of the test solution through the equation of the standard curve. Then, calculate the content of L-threonine in the aquatic animal feed, expressed as a mass fraction (%), according to parameters such as the weighed amount of the sample and the dilution factor.

During the entire detection process, pay attention to the calibration and maintenance of the instrument to ensure the accuracy and stability of the instrument. At the same time, perform parallel sample determinations to reduce errors, and conduct blank tests to deduct the background interference.