RU2318216C2 - Method for detection of water-soluble vitamins - Google Patents

Abstract

FIELD: analytical chemistry, in particular HPLC method for detection of water soluble vitamins in feeds, polyvitamin blends, body fluids (blood serum) and other biological samples.

SUBSTANCE: claimed method includes extraction of tested vitamin from sample of analyzed product, filtration, extract centrifugation and chromatography by HPLC method by using of precolumn, degasser, and thermostat for column, wherein as eluent mixture of methanol and acetate buffer solution with predetermined pH value is used. Methanol/acetate buffer ratio is selected on the base of column type, and wave length is 275 nm for group B vitamins, and 295 nm for vitamin C.

EFFECT: method of increased concentration range and improved accuracy.

8 cl, 1 tbl, 12 dwg, 5 ex

Description

The invention relates to the field of analytical chemistry and can be used in medical, veterinary and other biological studies in the chromatographic determination of water-soluble vitamins in feeds and multivitamin mixtures (blends) for animals, as well as in foods, vitamin-containing drugs, biological fluids (blood serum) and other biological objects.

Known from patent RU No. 2082170 (publ. 1997.06.20), a method for determining vitamin B ₅ in biological material, comprising isolating vitamin B ₅ from the test material by acid hydrolysis at 121 ° C, filtering the hydrolyzate, chromatography on a sorbent, followed by spectrophotometry, and hydrolysis spend 12 N. H ₂ SO ₄ for 30-60 minutes, the hydrolyzate is deproteinized, Silabsorb SPH C 18 is used as the sorbent, and the elution of vitamin B _{5 is} carried out with a mixture of acetic acid: 25% aqueous solution of trimethylamine: water in a volume ratio of 3: 2: 95 The measurements are carried out using a UV detector at a wavelength of 260 nm. The sample volume for analysis is 6 μl, the flow rate of the eluent to the column is 100 μl / min. The time spent on analysis, including hydrolysis, is 1.5 hours. The disadvantage of this method is the limited range of analyzed objects (materials) and the vitamins defined in them.

Known from RF patent No. 2148883 (publ. 1999.08.20) a method for determining the composition of a dosage form containing vitamins, in which the separation is carried out by high performance liquid chromatography (HPLC), and the chromatography is carried out on a Lichrosorb RP-18 in a gradient mode with using methanol in phosphate buffer at pH 7 in the following proportions: 3% methanol -1000 μl, of which 200 μl go to the regeneration of the column, then 10, 30 and 40% of 400 μl. All water-soluble vitamins are determined at 260 nm, only calcium pantothenate (vitamin B ₃ ) at 210 nm. The sequence of output of the peaks of vitamins from the column is: for ascorbic acid (vitamin C) = 1.33 min, for calcium pantothenate (vitamin B ₃ ) = 2.17 min, pyridoxine hydrochloride (vitamin B ₆ = 7.67 min, nicotinamide (vitamin PP) = 10.78 min, thiamine hydrochloride (vitamin B ₁ ) = 17.67 min, riboflavin (vitamin B ₂ ) = 19.33 min.

The disadvantage is a narrow range of detectable concentrations and the use of a phosphate buffer solution in the analysis, which leads to an increase in the viscosity of the mobile phase. In addition, the phosphate buffer solution is an excellent medium for the rapid multiplication of microorganisms, the formation of their colonies, which negatively affects the work of the analytical column and significantly reduces its service life.

The closest in technical essence is known from GOST R 50929 - 96 ("Premixes. Methods for the determination of B vitamins", UDC 636.085.3: 006.354, Gosstandart of Russia, IPK Standards Publishing House, Moscow, 1996) a method for determining group vitamins by HPLC In premixes, according to which vitamins B ₁ , B ₂ and B ₅ are extracted from a sample with a hydrochloric acid solution, followed by centrifugation for 3 minutes at 5000-6000 rpm and their determination on a liquid chromatograph with a spectrophotometric detector. For chromatography, a column with a height of 150 mm and a diameter of 4 mm is used, filled with sorbent Silabsorb S-18 or Separon S-18. The composition of the eluent for B ₁ , B ₂ : 120 cm ³ acetonitrile, 880 cm ³ distilled water, 2.5 cm ³ triethylamine, 0.5 g sodium octyl sulfonate, phosphoric acid to pH 7.6. The elution rate for B ₁ , B ₂ 1.2 ml / min, and the working wavelength of 254 nm.

The composition of the eluent for B ₅ : acetic acid, distilled water and triethylamine in a volume ratio of 3: 95: 2. The elution rate for B _{5 is} 80-100 μl / min, and the working wavelength is 260 nm.

Vitamin determination range:

For B ₁ 50-500 mg / kg

For B ₂ 100-2000 mg / kg

For B ₅ 200-4000 mg / kg

These methods provide for the determination of a narrow circle of water-soluble vitamins in a small range of their content.

In addition, a drawback of the existing GOST is that different methods of sample preparation for chromatographic determination and various chromatographic conditions are used to determine various water-soluble vitamins.

The basis of the invention is the task of developing a method for effectively determining the content of a complex of water-soluble vitamins by a chromatographic method that provides high sensitivity, accuracy, degree of separation and selectivity of determination while reducing the duration of the analysis and selecting universal conditions for its implementation. The objective of the invention is also to increase the number of determined vitamins in the sample and the expansion of the group of analyzed objects.

The problem is solved in that in the method for determining water-soluble vitamins, in which the determined vitamin is extracted from a sample of the analyzed product, the extract is filtered and centrifuged, and the sample is chromatographed using high-performance liquid chromatography with column separation of the selected type and spectrophotometry at the selected working wavelength, new is that they use the ion-pair reversed-phase method of high-performance liquid chromatography in chromatography A pre-column, a degasser, and a column thermostat are used in the process, a ground analyzed product is used as a sample, a mixture of methanol with an acetate buffer solution having a hydrogen pH selected from a predetermined range is used as an eluent, the ratio of methanol and acetate the buffer solution is selected depending on the type of column, while the selected working wavelength for B vitamins is 275 nm, and for vitamin C 295 nm.

It is advisable that the composition of the acetate buffer solution included deionized water, acetic acid, triethylamine and sodium octyl sulfonate, preferably in a percentage ratio, respectively 98.13: 1.00-1.15: 0.13: 0.64-1

Preferably, the pH of the acetate buffer is from the range of 2.8-3.

It is advisable to grind the product to be analyzed and select fractions with a size of not more than 0.2 mm for weighing.

Preferably, the extraction is carried out by heating to a temperature of 70-80 ° C, followed by cooling on a shaking device.

It is desirable to carry out centrifugation for 4-5 minutes at 7000-8000 rpm, and it is possible to carry out a single wash by centrifugation.

It is advisable to carry out the filtration using a membrane filter with a pore diameter of 0.45 μm.

It is preferable to use a column PE activity reduced column C-8, length 83 mm, diameter 4.6 mm, filled with octodecyl silane with a grain size of 3 μm, and the volume ratio of methanol and buffer solution is 15:85, respectively, and the elution rate is 1.5 ml / min

It is advisable to use a Pecosphere 3CR C8 PE column, 83 mm long, 4.6 mm in diameter, filled with a monofunctional base deactivated 3 μm 80 Å as a column; the volume ratio of methanol and buffer solution is 16:84, respectively, and the elution rate is 1.5 ml / min with an increase after 8 minutes to 2 ml / min.

It is possible to choose products from the range containing premixes, compound feeds, multivitamin mixtures, vitamin-containing medicinal preparations, biological materials, and food products as the analyzed products.

It is advisable to reduce the temperature of the thermostat at approximately 35 ° C to reduce the viscosity of the mobile phase and accelerate sorption processes on the analytical column.

It is advisable to use a Scavenger C18 as a precolumn, 33 mm long, 4.6 mm in diameter and 10 microns in grain size.

The following examples illustrate the implementation of the present invention.

Example 1

For the preparation of standard solutions of vitamins, the following vitamins are used in the analysis:

Thiamine hydrochloride (Vitamin B ₁ ), Sigma-Aldrich CAS.No. 67-03-8;

Riboflavin (Vitamin 82), Sigma-Aldrich CAS. No. 83-88-5;

Nicotinic acid or nicotinamide (vitamin B ₅ ), Sigma-Aldrich CAS. No. 59-67-6.98-92-0;

Pyridoxine hydrochloride (Vitamin B ₆ ), Sigma-Aldrich CAS. No. 58-56-0;

Folic Acid (Vitamin B ₉ ), Sigma-Aldrich CAS. No. 75708-92-8.

Cyanocobalamin (Vitamin B ₁₂ ), Sigma-Aldrich CAS. No. 68-19-9.

Ascorbic acid (Vitamin C), Sigma-Aldrich CAS. No. 50-81-7.

To prepare standard solutions of vitamins in the analysis of compound feeds and premixes, weighed portions of vitamins (B ₁ - 0.1 g, B ₂ - 0.25 g, B ₅ - 1 g, B ₆ - 0.2 g) with an accuracy of 0.0005 g in a 100 ml volumetric flask, and then add deionized water with a specific resistance of 18 MOhm / cm ³ and after dissolution, bring the same water to the mark. Each solution is prepared in a separate volumetric flask (respectively, solutions No. 1, 2, 3,4). A working standard solution of vitamins is prepared from the resulting solutions. A 1 ml aliquot was taken from each flask and transferred to a separate 100 ml flask and adjusted to the mark with deionized water. 1 ml of the resulting solution contains 0.01 mg of vitamin B ₁ , 0.025 mg of vitamin B ₂ , 0.1 mg of vitamin B ₅ and 0.02 mg of vitamin B ₆ (solution No. 5).

Vitamin B ₉ is prepared in a separate flask. Take a sample of 0.05 g with an accuracy of 0.0005 g, transfer to a 100 ml volumetric flask and add a 0.1 M solution of sodium bicarbonate in water, after dissolution, bring the same solution to the mark (solution No. 6). From the resulting solution, an aliquot of 1 ml is transferred into a 100 ml volumetric flask and adjusted to the mark with sodium bicarbonate solution. 1 ml of the obtained working standard solution contains 0.005 mg / ml of folic acid (solution No. 7).

For the preparation of standard solutions of vitamins in the analysis of multivitamin mixtures (blends), an aliquot of 5 ml is taken from the basic solution No. 1, 8 ml from the solution No. 2, 5 ml and No. 5 5 ml. Transfer to a 100 ml volumetric flask and make up to the mark with deionized water. 1 ml of the obtained working standard solution contains 0.05 mg of vitamin B ₁ , 0.2 mg of vitamin B ₂ , 0.5 mg of vitamin B ₅ and 0.1 mg of vitamin B ₆ (solution No. 8).

To prepare a working solution of folic acid from solution No. 6, an aliquot of 4 ml was transferred to a 100 ml volumetric flask and adjusted to the mark with a 0.1 M sodium hydrogen carbonate solution in water (solution No. 9).

Basic solutions (No. 1, 2, 3, 4 and 6) are stored in the refrigerator for no more than 2 weeks. Working standard solutions (No. 5,7,8 and 9) are prepared on the day of analysis.

The amount of sample taken for analysis and the final dilution factor depend on the theoretical content of vitamins and on the nature of the sample. Therefore, a sample of the sample is selected based on the formulation of its preparation, since the content of vitamins in compound feeds, premixes and vitamin concentrates is in a very wide concentration range.

The exact mass is calculated so that the concentration of vitamins in the final extract is as close as possible to the value of their concentrations in standard solutions. The approximate weight of the sample for feed and vegetable premix is 1 g, for multivitamin blends - 0.1 g.

The analyzed product is ground in a Pulverisette 14 rotary mill to a dusty state and the sample is weighed with an error of not more than 0.0005 g. For weighing, take the fraction passed through a 0.2 mm sieve.

The extraction of vitamins B ₁ B ₂ In ₅ B ₆ are 15 ml of 0.01 mol / DM ³ HCl.

Extraction of vitamin B _{9 is} 15 ml with a 0.1 M solution of sodium bicarbonate in water.

Extraction is carried out in a 100 ml conical flask, heating it to 70-80 ° С and subsequent cooling for 15 minutes on a mixing device model 6410 M "Ekros" (it is possible to use a magnetic stirrer).

Centrifugation is carried out on an OPN-8 centrifuge apparatus for 4-5 minutes at 7000-8000 rpm, after which the centrifuge is poured into a 25 ml volumetric flask. Washing by centrifugation is carried out 1 time with 10 ml of extractant. The combined centrifuge is adjusted to the mark with deionized water and filtered to remove any mechanical impurities.

When performing a qualitative analysis of the product, it is possible to carry out the extraction of vitamin B ₉ under conditions of extraction of vitamins B ₁ B ₂ B ₅ B ₆ and their joint centrifugation.

Chromatographic analysis is carried out by ion-pair reversed-phase high-performance liquid chromatography under the following conditions.

A mobile phase acetate buffer solution for elution is prepared on the basis of deionized water with a specific resistance of 18 MΩ / cm ³ obtained on a Simplisity (Millipore) apparatus for producing deionized water. A 0.432 g sample of sodium octyl sulfonate was added to a 1000 ml volumetric flask, 950 ml of deionized water was added, stirred, 10 ml of acetic acid and 1.32 ml of triethylamine were added, and then the pH of the solution was measured. If the pH value is outside the range of 2.8-3.0, then the adjustment is carried out with acetic acid and deionized water is added to V = 1000 ml. The resulting solution (solution No. 10) is filtered using a Supelco filter apparatus through a membrane filter with a pore diameter of 0.45 μm.

Series 200 liquid chromatograph (Perkin Elmer) with a photometric detector (wavelength range from 190 to 700 nm, wavelength accuracy ± 1 nm, relative standard deviation of the output signal - not more than 1.5%) and an autosampler (accuracy not worse than 0.5 % Standard deviation by peak area). A chromatography column (PE reduced activity C-8, length 83 mm, diameter 4.6 mm), filled with octodecyl silane with a grain size of 3 μm, is placed in a thermostat.

To protect the analytical column from particles contained in samples of complex multicomponent composition, a Scavenger C18 pre-column is used, 33 mm long, 4.6 mm in diameter, 10 μm granulation

The mobile phase (eluent) is methanol grade HPLC grade (for HPLC) with a solution of No. 10 in a volume ratio of 15:85.

To ensure the stability of the zero line of the detector and extend the life of the analytical column, a high degree of degassing of the eluent with a Series 200 vacuum degasser (Perkin Elmer) is carried out.

Before chromatography, several volumes of the mobile phase are passed through the column to establish equilibrium in the chromatographic column (stable baseline). To saturate the column, several measurements of the standard working solution of vitamins are performed until reproducible peak areas and retention times of the analyzed vitamins are obtained. Then chromatographic solution of the analyzed product under the same conditions, while:

operating wavelength at a UV detector of 275 nm for B ₁ V ₂ V ₅ B ₆ B ₉ ;

elution rate - 1.5 ml / min;

column thermostat temperature - 35 ° С;

the volume of injected extract is 10 μl.

The chromatographic determination time is 10 minutes.

The chromatogram of the premix extract is presented in figure 1.

Chromatograms of the premix P-5-1-2 (for birds) are presented in figure 2 and figure 3.

It is possible to prepare standard solutions and samples using distilled water, but for greater affinity of the injected sample and eluent, it is advisable to use deionized water for all solutions.

In order to optimize the analysis conditions, special studies were carried out on the influence of the temperature of the extraction, which amounted to (70-80 ° C); extraction time, which amounted to 10-20 minutes; centrifugation time, which amounted to 4-5 minutes; temperature control, which amounted to 34-36 ° C, as well as the number of washes of the residue after centrifugation, centrifugation speed, pH of the euent, the percentage of its components, the amount of ion-pair reagent, affecting the completeness of extraction.

Percentage of components:

deionized water: acetic acid: triethylamine: sodium octyl sulfonate is 98.13: 1.00-1.15: 0.13: 0.64-1, respectively.

It is sufficient to rinse the residue after centrifugation once.

When processing the results, the construction of a calibration graph for the working standard solutions was carried out using the TotalChrom Workstation software, and the verification was performed by the addition method.

For chromatographic measurements, the method of comparing peak areas for the analyzed sample and the standard solution was used. The content of water-soluble vitamins X _i , mg / g, calculated by the formula:

C _S - the concentration of vitamin in the working standard solution, mg / ml,

V _P - volume of premix extract, ml,

S _x , S _S - peak areas of vitamins in the chromatogram of the sample and standard, mAU · c,

m is the mass of the sample, g

The arithmetic mean of the results of two parallel determinations (X ₁ and X ₂ ) is taken as the result of measuring the vitamin content in the sample.

Example 2

The sample preparation conditions corresponded to the sample preparation conditions described in Example 1.

For chromatography, a Pecosphere 3CR C8 PE column was used, 83 mm long, 4.6 mm in diameter, monofunctional base deactivated 3 μm 80 Å;

The volume of injected extract is 10 μl;

Acetate Buffer Solution:

0.432 g of sodium octyl sulfonate,

1000 ml of deionized water,

10 ml of acetic acid

1.33 ml of triethylamine,

The pH of the solution is 3.0.

Mobile phase (eluent): methanol with acetate buffer in a volume ratio of 16:84.

The operating wavelength at the UV detector is 275 nm for B ₁ B ₂ B ₅ B ₆ B ₉ .

The elution rate is 1.5 ml / min; after 8 minutes, a jump to 2 ml / min;

Column thermostat temperature 35 ° С;

The chromatogram exit time was 10 min.

The chromatogram of the premix extract is presented in figure 4

Example 3

Under the conditions of sample preparation (extraction with hydrochloric acid), chromatography and signal detection, similar to example 1 (or 2), the content of vitamin B ₁₂ in the test sample was analyzed. The working wavelength at the UV detector is 275 nm.

The chromatogram of the vitamin mixture "Sodrovit Blend Z" is presented in figure 5.

Example 4

Under chromatographic separation conditions similar to Example 1, a qualitative analysis of vitamin C content in dosage forms, biological samples, and food products was performed at a working wavelength of 295 nm.

Figure 6 presents the chromatogram of ascorutin.

Figure 7 presents the chromatogram of a tablet of ascorbic acid with glucose.

On Fig presents a chromatogram of filtered serum of a pig.

Figure 9 presents the chromatogram of baby food "Apple-peach puree."

Figure 10 presents the chromatogram of fruit juice J7 orang.

The results obtained are comparable with the relevant data indicated in the quality certificates or annotations.

Example 5

Under chromatographic separation conditions similar to Example 1, the vitamin preparation Decamevit was analyzed. A chromatogram for B vitamins (working wavelength 275 nm) is shown in FIG. 11. A chromatogram for vitamin C (working wavelength 295 nm) is shown in FIG. 12.

The chromatograms presented in the examples illustrate the complete separation of the components, as regards the analyzed samples of medicinal vitamin-containing preparations, and sufficient, as regards the samples of food products and complex biological protein samples.

The range of defined vitamins according to the proposed method is presented in the table.

B ₁ (3.0-100000) mg / kg In ₂ (1.5-30000) mg / kg At ₅ (1.5-200000) mg / kg B ₆ (1.25-100000) mg / kg At ₉ (1.5-20000) mg / kg B ₁₂ (2.0-10000) mg / kg C (1.5-200000) mg / kg

The proposed method compared to the prototype allows you to get more information about the composition of the complex mixture of interest, expand the range of the contents of the determined vitamins, eliminates the need for analysis of vitamins by various methods, which requires additional time, labor and material costs.

The selection of universal conditions allows for chromatographic separation in a narrow time frame and reduces the duration of the whole analysis.

The proposed method has high sensitivity, selectivity of determination, accuracy and reproducibility, allows to achieve a high degree of extraction, given the complexity of the composition of the analyzed objects.

Using the method allows to increase the service life of analytical columns, thereby reducing the cost of depreciation of expensive chromatographic equipment.

Claims (8)

1. The method of determining water-soluble vitamins, which carry out the extraction of the determined vitamin from a sample of the analyzed product, filtering and centrifuging the extract, as well as chromatography of the sample by high performance liquid chromatography with separation on the column of the selected type and spectrophotometry at the selected working wavelength, characterized in that at chromatography uses a pre-column, a degasser and a column thermostat; a crushed analyzed product is used as a hinge t, to determine various water-soluble vitamins, a mixture of methanol with an acetate buffer solution having a pH of pH selected from a predetermined range is used as the eluent, the ratio of methanol to acetate buffer solution being selected depending on the type of column, while the selected working wavelength for vitamins group B is 275 nm, and for vitamin C 295 nm. 2. The method according to claim 1, characterized in that the composition of the acetate buffer solution includes deionized water, sodium octyl sulfonate, acetic acid and triethylamine. 3. The method according to claim 2, characterized in that the percentage of deionized water, acetic acid, triethylamine and sodium octyl sulfonate is 98.13: 1.00-1.15: 0.13: 0.64-1, respectively. 4. The method according to claim 1, characterized in that the pH of the buffer solution is selected from the range of 2.8-3. 5. The method according to claim 1, characterized in that the crushed analyzed product with a fraction size of not more than 0.2 mm is used as a sample. 6. The method according to claim 1, characterized in that the filtration is carried out using a membrane filter with a pore diameter of 0.45 μm. 7. The method according to claim 1, characterized in that the centrifugation is carried out for 4-5 minutes at 7000-8000 rpm 8. The method according to claim 1, characterized in that the washing by centrifugation is carried out once.

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