Pre-column Derivatization-High Performance Liquid Chromatography for the Detection of Monensin in Livestock and Poultry Meat

Jiao WANG Xiujuan WANG Lingtong HU Guixia YANG Dandan HU

Abstract ［Objectives］ A method for the detection of monensin in poultry and livestock meat by pre-column derivatization-high performance liquid chromatography was established.

［Methods］The sample was extracted with chloroform, derivatized with trichloroacetic acid and 2, 4-dinitrophenylhydrazine, and centrifuged to obtain a purified solution. A C18 chromatographic column (4.6 mm × 150 mm, 5 μ m) was used for separation with (1.5%) acetic acid water∶methanol (volume ratio)=1∶9 as the mobile phase using a DAD detector for detection, and the external standard method was adopted for peak area quantification.

［Results］ Monensin had good linearity in the concentration range of 5.00-200 mg/L, with the linear correlation coefficient r2>0.999; the detection limit was 5.00 mg/kg; the relative standard deviation was smaller than 10%; and the recoveries of standard addition experiment were in the range of 75%-110%.

［Conclusions］The method has the advantages of simple pretreatment operation, good derivatization effect and fast detection speed, and is suitable for detecting monensin in poultry and livestock meat.

Key words Pre-column derivatization-high performance liquid chromatography; Livestock and poultry meat; Monensin

Received: December 12, 2020  Accepted: February 21, 2021

Jiao WANG (1994-), female, P. R. China, devoted to research about food additives inspection and testing.

*Corresponding author. E-mail: 1971512510@qq.com.

Monensin, also known as "rumensin", can regulate rumen microflora, improve feed conversion rate, and control rumen fermentation, and is thus widely used in production practice［1-3］. However, there are also some problems in the use process, such as mixed use, abuse and other issues. Announcement No.194 of the Ministry of Agriculture and Rural Affairs stated that "90% of the total amount of antibiotics consumed in the world each year are used on food-borne animals, resulting in increasingly prominent problems such as bacterial resistance and drug residues." In this context, the domestic supervision of monensin use also has higher requirements, so more and more complete methods are needed as support. At present, the domestic detection methods for monensin are mostly concentrated on the post-column derivatization method of liquid chromatography［4-5］, which has brought great obstacles to some laboratories that do not have the conditions for post-column derivatization, which in turn adds to the difficulty of food safety supervision. This study was aimed at establishing an efficient and fast pre-column derivatization-high performance liquid chromatography method to improve detection efficiency, and providing strong guarantees for related researchers and food regulators.

Materials and Methods

Instruments and reagents

High performance liquid chromatograph (1260 Infinity II, Agilent, USA); vortex mixer (XW-80A, Shanghai Chitang); centrifuge (3-18KS, Thermo Fisher, Germany); constant temperature water bath (HHS-21-8, Shanghai Boxun); tissue homogenizer (T25, IKA).

Ultrapure water (made in laboratory); methanol (chromatographically pure); trichloroacetic acid (analytically pure); 2,4-dinitrophenylhydrazine (analytically pure); acetic acid (analytically pure); monensin standard product (purity≥98%).

Experimental methods

Preparation of solutions

Monensin standard solution (1 mg/ml): A certain amount of monensin (0.010 g) was accurately weighed into a 10 ml volumetric flask, dissolved with methanol and diluted to constant volume, obtaining the standard solution, which was stored in a refrigerator at 4 ℃ and diluted with methanol to the required concentration before use.

Trichloroacetic acid solution (500 mg/ml): A certain amount of trichloroacetic acid (25 g) was accurately weighed into a 50 ml volumetric flask, dissolved with water and diluted to constant volume.

2,4-Dinitrophenylhydrazine solution (1mg/ml): A certain amount of  2,4-dinitrophenylhydrazine (0.10 g) was accurately weighed into a 100 ml volumetric flask, dissolved in methanol and diluted to constant volume.

Acetic acid aqueous solution (1.5%): A certain amount of acetic acid (15 ml) was pipetted and added with water to 1 L.

Preparation of sample solution

A certain amount of sample (2.5 g) was weighed into a 50 ml centrifuge tube, added with 25 ml of chloroform, and homogenized for 2 min. The treated sample was centrifuged at 5 000 r/min for 5 min. The supernatant was reserved for later use.

Pre-column derivatization［6］

Standard derivatization: At first, 0, 10, 20, 50, 200, 400 μl of the standard solution were pipetted, respectively, into a 10 ml centrifuge tube with a stopper, and then diluted with methanol to 1.0 ml. Then, 400 μl of trichloroacetic acid solution was added into each centrifuge tube, and each solution was vortex-mixed for 30 s, and stood for 10 min. Next, 600 μl of 2,4-dinitrophenylhydrazine solution was added into each centrifuge tube, and each solution was vortex-mixed for 30 s, heated in a constant temperature water bath at 55 ℃ for 30 min, and taken out and cooled to room temperature. After cooling, each solution was filtered through a 0.22 μm filter membrane for later loading into the machine.

Sample derivatization: At first, 1.0 ml of the supernatant was pipetted into a 10 ml centrifuge tube with a stopper, Then, 400 μl of trichloroacetic acid solution was added into each centrifuge tube, and the solution was vortex-mixed for 30 s, and stood for 10 min. Next, 600 μl of 2,4-dinitrophenylhydrazine solution was added into each centrifuge tube, and each solution was vortex-mixed for 30 s, heated in a constant temperature water bath at 55 ℃ for 30 min, and taken out and cooled to room temperature. After cooling, each solution was filtered through a 0.22 μm filter membrane for later loading into the machine.

For blank experiment, the derivatization method was the same as the sample treatment. Meanwhile, a standard addition experiment was carried out. Three parallel samples were weighed, added with 5, 12.5, 25 μl of monensin standard solution (1 mg/ml), and added with the standard to 2, 5, and 10 mg/kg, respectively, added with 25 ml of chloroform, and homogenized for 2 min. The treated sample was centrifuged at 5 000 r/min for 5 min. The obtained solutions were subjected to derivatization together with the sample treatment.

Chromatographic conditions

Chromatographic column: C18 chromatographic column (150 mm×4.6 mm, 5 μ m); detection wavelength: 392 nm; column temperature: 25 ℃; injection volume: 10 μl; mobile phase: 1.5% acetic acid water-methanol (1∶9); flow rate: 1ml/min; isocratic elution with the chromatographic time of 25 min.

Results and Analysis

Standard curve

The standard chromatogram of monensin is shown in Fig. 1. The concentration-peak area standard working curve equation was y=1.69x-1.47, with the linear range of 5.00-200 mg/L, and R2=0.999 8.

Blank experiment

The blank solution had no absorption peak at the peak time of monensin, indicating that the reagents used in the experiment basically did not interfere with the detection results.

Detection limit of the method

The detection limit of the method was calculated by signal-to-noise ratio (S/N). The detection limit was calculated from the corresponding concentration when the signal-to-noise ratio (S/N) was equal to 3. It can be concluded that when the sample weight was 2.5 g and the constant volume was 25 ml, the detection limit of the method was 2 mg/kg.

Method accuracy and precision

Through the standard addition experiment, the average recovery rate of the experiment was 88.3%, with the relative standard deviation RSD (%) of 1.1, so the accuracy and precision of the method were good.

Conclusions

This study showed that the pre-column derivatization-high performance liquid chromatography method for detecting monensin in poultry and livestock meat has the advantages of simple operation, cost saving, easy preparation of the mobile phase, easy operation of chromatographic conditions, good linearity, stable detection results, and good application prospects, and is thus an efficient and fast detection method.

References

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