Vitamin is one of five nutrients needed by animals, among which vitamin A is an important part of rhodopsin (photopigment), which has functions of maintaining normal vision, promoting growth and development, maintaining integrity of epithelial structure and enhancing animal immunity. In order to meet needs of animal growth, it is often added to animal feed.

The current effective national standard for the determination of vitamin A in feed is GB/T 17817-2010 "Determination of vitamin A in feed - High performance liquid chromatography", and its pretreatment methods include saponification extraction method and direct extraction method. The saponification extraction method is suitable for the determination of vitamin A in compound feed, concentrated feed, compound premix feed and vitamin premix feed. The direct extraction method is only suitable for the determination of vitamin A in vitamin premix feed with high vitamin A content. At present, most of laboratories in saponification extraction process have built their own water bath reflux devices, which are cumbersome and labor-intensive. This experiment compares self-built water bath reflux device with a set of array water bath reflux device with integrated heating, magnetic stirring, automatic lifting, timing start and stop, and hot and cold water double bath.

In comparison, the test proves that integrated array water bath reflux instrument effectively optimizes pretreatment process of saponification, and achieves the purpose of simplifying the experimental process, reducing manual operation and improving the experimental efficiency.

 1.  Materials and methods

1.1 Instruments

High performance liquid chromatography (Agilent 1100), array water bath refluxer  (AF6DM), array rotary evaporator(minirotar 500s), analytical balance.

1.2 Standards and reagents

Vitamin A acetate standard, content ≥ 99%.

Anhydrous ether, analytically pure, no peroxide after inspection; absolute ethanol, analytically pure; methanol, chromatographic grade; 2,6-di-tert-butyl-p-cresol (BHT), analytically pure; anhydrous sodium sulfate, analytically pure ; nitrogen (purity 99.9%); potassium iodide solution 100g/L; starch indicator 5g/L; sodium thiosulfate solution 50g/L; potassium hydroxide solution 500g/L; L-ascorbic acid solution 5g/L; phenolphthalein indicator 10g/L.

1.3 Standard solution configuration

Vitamin A standard reserve solution: Weigh 34.3mg (accurate to 0.0001g) of standard vitamin A acetate in a saponification bottle, carry out saponification and extraction according to the pretreatment method, concentrate and evaporate ether extract to dryness,dissolve residue with n-hexane, put which into a 100mL brown volumetric flask and dilute to the mark, mix well, and store at 4°C.

Vitamin A standard working solution: Accurately pipette 1.00mL of vitamin A standard stock solution into a 100mL brown volumetric flask, dry with nitrogen, dilute to volume with methanol, and mix well.

1.4 Pre-processing method

Weigh 10g of compound feed into a 250mL round-bottom flask, add 50mL L-ascorbic acid solution to completely disperse and wet the sample, add 10mL potassium hydroxide solution, and mix well. Put it on a boiling water bath to reflux for 30min, shake from time to time to prevent the sample from adhering to the bottle wall, and when the saponification is over, rinse inside with 5mL absolute ethanol and 5mL water from the top of the condenser tube respectively, take out the flask and cool it to 40°C in a cold water bath.

Quantitatively transfer all the saponification solution into a 500mL separatory funnel filled with 100mL anhydrous ether, rinse the round-bottomed flask with 50mL water 3 times and incorporate it into separatory funnel, then cover, deflate, then mix, vigorously shake for 2min, stand for Layered. Transfer aqueous phase to the second separatory funnel, repeat extraction twice with 100 mL and 60 mL of ether, discard aqueous phase, and combine ether phases three times. The ether extract was washed to neutrality with 100 mL each of water and was dehydrated by anhydrous sodium sulfate, transferred to a 250mL brown volumetric flask, dissolved in 100mg BHT, and made up to volume with ether.

Divide an appropriate volume from ether extract to dryness, dissolve the residue in methanol, filter it through a 0.45um membrane, and inject it for testing.

1.5 Instrument Conditions

Chromatographic column: C18 type column;

Mobile phase: methanol + water (95:5); flow rate: 1.0 mL/min;

Column temperature: room temperature; injection volume: 20ul;

Detection wavelength: 326nm.

2. Results and Discussion

2.1 Comparison of pre-saponification treatment devices

Saponification pretreatment is the key pretreatment process for vitamin A testing. Traditionally, a water bath reflux device is built manually, which is cumbersome and labor-intensive. During the pre-saponification process, it is necessary to shake from time to time to prevent the sample from adhering to the bottle wall.

Disassemble the round-bottomed flask, shake it and then install it for water bath reflux. The array water bath recirculation instrument can process 6 samples at the same time. By equipped with intermittent magnetic stirring, the manual oscillation process is eliminated, and the functions of timing start and stop and automatic lifting and lowering can be realized without personnel on duty. The experiment compared the saponification pretreatment process of the traditional artificially built water bath reflux device and the array water bath reflux instrument, and proved that the array water bath reflux instrument can effectively optimize and simplify the experimental process, reduce manual operation, and improve the saponification pretreatment efficiency.

Table 1  Technical Parameters

 2.2 Sample spike recovery experiment 

Weigh 12 samples of 10g blank compound premix feed, add 10mg vitamin A acetate standard, use a self-built water bath reflux device and an array water bath reflux device for pre-saponification treatment, after extraction and concentration, test on the machine, calculate the two. device recovery rate. The recovery rate of the self-built water bath reflux device is between 94.1% and 97.8%.The recovery rate of the array water bath reflux instrument is between 97.3% and 99.8%, and the recovery rate of array water bath refluxer instrument is slightly better than that of self-built water bath refluxer. The test results are shown in Table 2.

Table 2 Recovery rate of different saponification units

 2.3 Method repeatability

The same feed sample was pre-treated by using a self-built water bath refluxer and an array water bath refluxer and the two devices were used for 6 parallel determinations. The relative standard deviation of the self-built water bath refluxer 6 in parallel tests was 3.75%, and that of the array water bath refluxer was 1.74%.The repeatability of  saponification pretreatment with array water bath refluxer is better than that of self-built water bath refluxer. The results are shown in Table 3.

  Table 3 Repeatability of different saponification devices 

3. Conclusion

In the experiment, self-built water bath refluxer and array water bath refluxer were used to determine the vitamin content in feed. From the whole measurement process,  array water bath refluxer integrated saponification pretreatment operation, especially  intermittent stirring function,the automatic lifting function greatly simplifies the experimental process and reduces manual operation. Simultaneously, automatic simultaneous processing of multiple samples is superior to manual construction of a water bath reflux device in accuracy and repeatability of  samples. The array water bath refluxer can be well applied to the determination of vitamin A in feed.