GBT 18397-2001 Determination of pantothenic acid in mixed feeds by high performance liquid chromatography

GBT 18397-2001 Determination of pantothenic acid in mixed feeds by high performance liquid chromatography

This standard specifies the method for the determination of pantothenic acid content in composite premixed feeds by high performance liquid chromatography.

This standard is applicable to the determination of pantothenic acid in compound premixed feed and vitamin premixed feed. It is also applicable to the determination of pantothenic acid in concentrated feed. The measurement range is 50 mg or more of pantothenic acid per kilogram of sample.

Method principle

The pantothenic acid in the sample was extracted with water, separated by a high performance liquid chromatography C18 column using a phosphate buffer (pH 5.5), and quantitatively determined by an external standard method at 200 nm using a UV detector.

Reagents and materials

Unless otherwise specified, the reagents used in this standard are of analytical grade, and the water is deionized water, which should meet the requirements of GB/T 6682 Class III water use.

1.1 0.1% phosphoric acid solution.

1.2% disodium edetate (EDTA) solution.

1. 3 phosphate buffer: weigh 3.12 g of sodium dihydrogen phosphate dihydrate (NaH2PO4-2H2O) dissolved in 1L of water, adjusted to pH 5.5 with sodium hydroxide solution c (NaOH) = 0.1m ol / L, passed 0.45 The um filter is filtered and degassed for use.

1.4 Pantothenic acid (or calcium pantothenate) standard solution.

1.4.1 Pantothenic acid (or calcium pantothenate) standard stock solution: Accurately weigh 0.02 g of D-pantothenic acid (or D-pantothenate) standard pure product of known purity, put it in 100ml brown volumetric flask, and use 0.1% phosphoric acid solution ( 1.1) Dissolve and dilute to the mark and shake well.

1.4.2 Pantothenic acid (or calcium pantothenate) standard working solution: precision drawing standard stock solution (1.4.1) diluted with water 1:10, the current concentration of the solution is D-pantothenic acid per ml (or D-pantothenic acid) Calcium) 20 ug,

1.5 sodium hydroxide solution: c (NaOH) = 1 mol / L.

1.6 hydrochloric acid solution: c (HaOH) two 1 mol / L.

equipment

pH meter.

Ultrasonic water bath.

Ultrapure water unit (Millipore or full ground glass distiller).

Centrifuge: 3000 r/min,

GE-200 liquid chromatograph: with 8725i injector, UV UV detector.

Sample preparation

Select a representative sample to at least 500g, the quadruple method is reduced to 100g, ground, all passed through a 0.28mm sieve, mixed, packed in a closed container, stored in the dark at low temperature.

Analysis step

2.1 Preparation of test solution

2.1.1 Compound pre-A feed: Weigh 1-2g of sample or 0.25-0.5g of vitamin premixed feed to the nearest 0.0001g and place in a 100mL brown volumetric flask. Add 60 mL of water to wet, shake well, add 10 mL of 1% disodium edetate solution (1.2), mix and place on an ultrasonic water bath for 15 min. Mix well with water until the mark is evenly mixed. Filter or centrifuge. It was filtered through a 0.45 um filter membrane to adjust the concentration of the solution to more than 1 ug/mL for high performance liquid chromatography.

2.1.2 Concentrated feed: Weigh 2-5g of the sample to the nearest 0.0001g and place in a 100m L brown volumetric flask. Add 60m L of water to soak, shake well, add 10mLl% disodium edetate solution, and place on the ultrasonic water bath to extract and extract for 15min. If the extract is turbid, use a few drops of sodium hydroxide solution (1.5) and hydrochloric acid solution (1.6) to adjust the pH to about 4.5, until the solution is clarified (shown on the test paper) to precipitate the protein, and dilute to the mark with water. The following steps are the same as 2.1. .1.

Note: When the pantothenic acid content per kilogram of concentrated feed is less than 50mg, the sample weight can be increased.

2.2 Determination

2.2.1 Chromatographic conditions

Stationary phase: ODS (C18) particle size 5um. Column length 150mm, inner diameter 3.9mm stainless steel column.

Mobile phase: phosphate buffer (1.3),

Flow rate: 1. 0 m L/min

Temperature: room temperature.

Injection volume: 10-20ul

Detector: UV detector (or diode matrix detector PDA) uses a wavelength of 200nm

2.2.2 Quantitative determination

Adjust the operating parameters and sensitivity (AUFS) of the instrument according to the instructions of the high performance liquid chromatograph, and the chromatographic peak separation meets the requirements. Correct the system with two or more corresponding standard working fluids, and inject the corresponding standard working solution of pantothenic acid or calcium pantothenate [1.4.2 (Vst)" and test solution "2.1 (Vi)] to the column to obtain the peak area response value. (Pst, Pi), quantitatively determined by external standard method.

Resulting and writing

3.1 The content of pantothenic acid in the composite premixed feed is calculated according to formula (1): wi = Pi × V × pi × Vsi × f / Pm × m × Vi

Wi-the content of pantothenic acid per kilogram of sample, mg;

m a sample mass g;

The total volume of V-extraction solution, ml;

Pi-pantothenic acid (or calcium pantothenate) standard working solution concentration, ug/mL;

Vsi-pantothenic acid (or calcium pantothenate) standard working fluid injection volume, uL;

Vi-injection volume from the test solution, uL;

The peak area response value corresponding to the standard working solution injection volume Vsi) of Pm;

The peak area response value corresponding to Pi injection volume (Vi) from the test solution;

The f-conversion coefficient, when using pantothenic acid as a standard substance, has a coefficient of 1; when calcium pantothenate is used as a standard substance, it is converted into pantothenic acid with a coefficient of 0.920 (1 mg of pantothenic acid is equivalent to 1.087 mg of calcium pantothenate).

3.2 Parallel measurement results are expressed as arithmetic mean, one bit after the decimal point.

Allowable difference

The relative deviation of the results obtained by parallel analysis of the same sample by the same analyst is shown in Table 1.

Pantothenic acid content per mg of sample. mg relative deviation %

50-2000 ≤Shi 10

> 2000 ≤ 士5.0

There is no international standard for the determination of pantothenic acid in compound premixed feed. This standard is based on the literature reviewed at home and abroad. Refer to the “National Feed Association (NFIA) Analysis Method Summary” method for high performance liquid chromatography. The method was developed for the determination of ascorbic acid, niacin, nicotinamide, calcium pantothenate and panthenol in liquid and solid multivitamin additives.

This standard refers to foreign methods in terms of technical content, and its method principle is the same; the range, sample extraction conditions, sample weight, and HPLC conditions have been improved and clearly defined. The method is simple, fast and accurate.

This standard is proposed and managed by the National Feed Industry Standardization Technical Committee.

This standard was drafted: National Feed Quality Supervision and Inspection Center (Beijing).

The main drafters of this standard: Chen Bifang, Li Lan, Lin Dongxia.