RU2422830C1 - Method of quantitative evaluation of blood acetic, propionic, isobutyric, butyric, valeric, isocapronic and capronic acids by gas chromatography analysis - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: invention describes a method of quantitative evaluation of blood acetic, propionic, isobutyric, butyric, valeric, isocapronic and capronic acids by gas chromatography analysis wherein a blood sample is acidified with 1 % sulphuric acid to pH 2-3, evaluated acids are extracted with isobutyl alcohol volume of which is related to the blood sample volume as 1:1. The protein separation is enabled by centrifugation. 2-3 drops of 0.4 % alkali is added, and the extract is evaporated dry, further the solid residue is added consistently with 1 % sulphuric acid and isobutyl alcohol that is followed with gas chromatography separation of the mixed acids in a capillary column with a flame ionisation detector, and the amount of each acid is evaluated by a calibration diagram. ^ EFFECT: higher sensitivity and accuracy of the method of quantitative evaluation of acetic, propionic, isobutyric, butyric, valeric, isocapronic and capronic acids if found in blood together. ^ 5 cl, 1 ex, 4 tbl

Description

The invention relates to medical toxicological studies, in particular to sanitary toxicology, and can be used for the quantitative determination of acetic, propionic, iso-butyric, butyric, valerianic, iso-caproic and caproic acids in the blood for the diagnosis of gastroduodenitis.

A known method for the determination of volatile fatty acids (acetic, propionic, butyric, iso-butyric, iso-valerianic, valerianic, nylon and iso-nylon) and malondialdehyde in biological media on a gas chromatograph with a flame ionization detector with temperature programming from 60 to 220 ° C at a rate of 10 ° C in 1 min. Analysis time 45 min (ed. St. USSR No. 1725095 dated 06/29/89). According to the known method, exudate samples are taken in an amount of 1-2 ml, the sample is placed in a special hermetically sealed bottle filled with oxygen-free gas (a mixture of nitrogen 85%, hydrogen 10% and carbon dioxide 5%). An equilibrium vapor phase analysis is then carried out. For this, crystalline potassium bisulfate is added to the bottle with the test sample, the bottle is closed with a rubber stopper with a fluoroplastic gasket and placed in a special cartridge for sealing. Thermostating is carried out at 90 ° C for 10 minutes. Then, through the metering needle of the pneumatic metering device, the sample is introduced into the column of the chromatograph. Peaks in the chromatograms of the studied exudate samples are identified by comparing the time of their exit with the time of the output of the peaks of the studied acids and malondialdehyde in a similar study of a standard sample.

Also known is a method for the quantitative determination of organic acids and anhydrides when they are together using the gas chromatography method (auth. USSR USSR No. 347656 of 03/11/1969), which consists in the fact that the analyzed sample before chromatography is sequentially treated with an ether solution of diazomethane, and then with a mixture of ethyl alcohol, diethyl sulfate and sulfuric acid when heated. In this case, methyl esters are formed from acids under the action of diazomethane, and ethyl esters are formed from anhydrides under the influence of ethyl alcohol and diethyl sulfate. The resulting mixture was chromatographed.

A known method for the determination of acetic, propionic and butyric acids in saliva for the diagnosis of chronic gastroduodenitis and functional dyspepsia in children (RF patent No. 2270610 from 08/04/2004). According to this method, a saliva sample is prepared by acidifying 1 ml of saliva with one drop of 10% sulfuric acid. Gas chromatographic separation of the analyzed acids is carried out on a glass column 1 m long, 3 mm in diameter, filled with Porapak Q (USA) with phosphoric acid deposited on it, in isothermal mode at a column temperature of 200 ° C, MOX chromatograph - model 3700. Flame ionization detector The carrier gas is helium. As a result of the analysis of saliva in the examined children, the authors found that in healthy children the concentration of acetic acid in the saliva was 0.60 mg / dm ³ , propionic acid 0.296 mg / dm ³ , butyric acid 0.528 mg / dm ³ . In children with functional dyspepsia, the content of acetic acid in saliva was 54.64 mg / dm ³ , propionic acid - 11.11 mg / dm ³ , butyric acid - 1.14 mg / dm ³ . In children with chronic gastroduodenitis, acetic acid in saliva was found at a concentration of 474.3 mg / dm ³ , propionic acid - 49.6 mg / dm ³ , butyric acid - 50.2 mg / dm ³ .

In the abstract for the competition. student degrees of dr. honey. Sciences Bykova I.L. Molecular mechanisms and the pathogenetic role of metabolic disorders of L-carnitine. Novosibirsk, 2006. It is proposed to determine the concentration of short-chain fatty acids in tissues and physiological fluids by gas chromatography after extraction with diethyl ether with n-valerianic acid as an internal standard. The range of detectable concentrations of propionic acid in tissues and blood plasma is 5.03-6.07 mg / dm ³ , of isobutyric acid - 1.41-2.03 mg / dm ³ .

In the source of information: Dawson R., Elliot D., Jones K. Paper and thin-layer chromatography in biochemistry. Separation techniques. Handbook of biochemist. M .: Mir, 1991, p.379 use paper chromatography for the separation of formic, acetic, propionic, butyric, valerianic and caproic acids. Chromatography is carried out in a solvent containing a volatile base, for example MH ₃ , ethylamine. In the case of a mixed solvent, ethanol: 3 M NH ₃ = 95: 6, the retention coefficient is 0.31 for formic acid, 0.33 for acetic acid, 0.44 for propionic acid, 0.54 for butyric acid, 0, 60 for valerianic acid and 0.68 for caproic acid. Acid detection is carried out by pH-indicator methods using a 0.04% solution of bromocresol green in ethyl alcohol or water, or a 0.04% solution of bromocresol purple in a mixture of formalin: ethanol = 1: 5 with pH 5, or 0, 1% aqueous solution of thymol blue. The sensitivity of the determination of 5 μg. The disadvantage of this method is the semi-quantitative determination of acids and the low sensitivity of the determination.

Also known is the method for determining volatile fatty acids (VFA) by gas-liquid chromatography described in the book of authors Mazankova LN, Ilyina NI, Kondrakova OA, Zatevalova A.M. “Assessment of microbiocenosis disorders in acute intestinal infections in children and their correction. Diff The patient. "2004; 2 (9): 11-6. To determine VFA, it is proposed to use a sample infiltrate. Obtaining an infiltrate is carried out according to the following scheme: in a disposable plastic tube, 2-3 grams of sample contents are weighed on an analytical balance accurate to the 3rd sign, 1 ml of 0.02 N hydrochloric acid, 1 ml of distilled water and 1 ml of standard substance are added to the sample . The tube is closed with a ground stopper and the mixture is homogenized by vigorous shaking. If it is necessary to determine the concentration of VFA in the blood, 0.5 ml of 0.01 N perchlorate acid is also added to the mixture. A test tube with a homogeneous mixture is centrifuged for 10 minutes at 6000 rpm. The resulting infiltrate should be transparent, have an acid reaction. Chromatography is carried out at a thermostat temperature of 140 ° C, an evaporator of 240 ° C. A disposable glass capillary is used to take the microquantity of the supernatant of the sample. The capillary is sealed on one side and attached to a special sample injection device, with which the capillary is placed in the evaporator and the chromatogram reading program is launched.

Also known is a method of gas-liquid chromatography for the quantitative determination of acetic, propionic and butyric acids in the blood of children with viral hepatitis (Akayzin E.S., Zolotarev D.V., Akayzina M.A., Balikin V.F. "Short-chain fatty acids in the blood of children with viral hepatitis ", Ivanovo State Medical Academy of the Ministry of Health and Social Development of Russia, Ivanovo, 2005). According to a known method, the studies were carried out on an MOX model 3700 chromatograph with a flame ionization detector. Chromatographic separation of acids was carried out on a glass column 1 m long, 3 mm in diameter, filled with Porapak Q (USA) with phosphoric acid deposited on it. The temperature of the column is 200 ° C, of the evaporator is 250 ° C. The speed of the carrier gas (helium) 25 ml / min. In healthy children, acetic acid in the blood was found in concentrations of 0.040-0.222 mg / dm ³ , propionic acid - 0.0148-0.362, butyric acid - 0.024-0.077 mg / dm ³ . In patients with viral hepatitis, the acid content in the blood increased.

The disadvantage of this method is the low sensitivity of acetic acid in the blood of 0.04 mg / DM ³ , propionic - 0,015 mg / DM ³ , butyric - 0,024 mg / DM ³ . Isobutyric acid interferes with the determination of butyric acid.

The technical result achieved by the invention is to increase the sensitivity and accuracy of the method for determining acetic, propionic, iso-butyric, butyric, valerianic, iso-caproic and caproic acids when they are together in the blood.

The specified technical result is achieved by the proposed method for the quantitative determination of acetic, propionic, iso-butyric, butyric, valerianic, iso-caproic and caproic acids in the blood by gas chromatographic analysis, according to which the blood sample is acidified with a 1% sulfuric acid solution to pH 2-3, the determined acids are extracted with isobutyl alcohol, the volume of which is related to the volume of the blood sample as 1: 1, centrifugation is carried out to separate the proteins, 2-3 drops of a 0.4% alkali solution and the extract is evaporated to dryness, then a 1% solution of sulfuric acid and isobutyl alcohol are successively added to the dry precipitate, and a gas chromatographic separation of the acid mixture is carried out on a capillary column with a flame ionization detector, and the amount of each acid is set according to a calibration graph.

Adding to the dry residue a 1% solution of sulfuric acid is produced in a volume of 1:40, and isobutyl alcohol - 1:20 to the volume of a blood sample, respectively.

The extraction of the determined acids with isobutyl alcohol is carried out for 5-10 minutes.

Centrifugation is carried out at 6,000 rpm for 10 minutes.

Gas chromatographic separation of the acid mixture on a capillary column with a flame ionization detector is carried out using a hardware-software complex based on a Crystal 2000 gas chromatograph with a flame ionization detector on an HP-FFAP capillary column - 50 m * 0.32 mm * 0, 5 µm at temperature conditions: column - from 70 ° С-160 ° С-180 ° С-280 ° С; evaporator - 250 ° C; detector - 280 ° C; carrier gas flow rate 1 (nitrogen) - 40 cm ³ / min; the flow rate of carrier gas 2 (nitrogen) is 14-30 cm ³ / min.

It was found experimentally that the specified technical result is provided precisely by the totality of the proposed features, the implementation of which achieves high sensitivity and accuracy in the determination of these short-chain fatty acids in their joint presence.

In laboratory conditions, a series of experiments was carried out to establish the significance of the characteristics underlying the proposed method.

Example. According to the claimed method, 2 cm ^{3 of} blood was taken, containing acetic, propionic, iso-butyric, butyric, valerianic, iso-caproic and caproic acids, acidified with a 1% sulfuric acid solution to pH 2-3, extracted with 2 cm ^{3 of} isobutyl alcohol within 5-10 minutes To denature the proteins, the extract was centrifuged at 6000 rpm for 10 min, then 2-3 drops (0.1 cm ³ ) of a 0.4% alkali solution (sodium or potassium) were added, evaporated to dryness, the dry residue was acidified 0, 05 cm ^{3 with} 1% sulfuric acid and 0.1 cm ³ of isobutyl alcohol was added. Next, a quantitative determination of acids in the prepared samples was carried out by a gas chromatographic method according to a calibration graph using a hardware-software complex based on a Crystal 2000 gas chromatograph with a flame ionization detector. The completeness of separation of acetic, propionic, iso-butyric, butyric, valerianic, iso-caproic and caproic acids was achieved on an HP-FFAP capillary column - 50 m * 0.32 mm * 0.5 μm at temperature conditions: column - from 70 ° C -160 ° C-180 ° C-280 ° C; evaporator - 250 ° C; detector - 280 ° C; carrier gas flow rate 1 (nitrogen) - 40 cm ³ / min; the flow rate of carrier gas 2 (nitrogen) is 14-30 cm ³ / min.

To build a calibration graph used the following methodology. In a 10 cm ³ volumetric test tube containing iso-butyl alcohol in a volume of 5 cm ³ , 15 mm ³ (μl-microliter) of acetic, 10 mm propionic, iso-butyric and butyric acids and 5 mm ^{3 of} valerian, iso caproic and caproic acids. The content in the initial certified solution is 3120 μg / cm ³ for acetic acid, 1996 μg / cm ^{3 for} propionic acid, 1916 μg / cm ^{3 for} butyric acid, 1918 μg / cm ^{3 for} butyric acid, and 930 μg / for valerianic acid cm ³ , iso-caproic acid - 923 μg / cm ³ , caproic acid - 923 μg / cm ³ . The shelf life of the certified solution was 12 hours.

The calibration characteristic was established by the absolute calibration method. It expresses the dependence of the peak area in the chromatogram (mm ² ) on the mass (μg) and is built on 5 series of working standard solutions. Each series, consisting of 5 standard solutions, is prepared in 10 cm ³ volumetric tubes. For this, acetic acid, propionic acid, iso-butyric acid, butyric acid, iso-caproic acid, caproic acid, valerianic acid are added to each tube in accordance with table 1 and adjusted to 2 cm ³ with iso-butyl alcohol. The obtained extracts - standard solutions are chromatographed on a chromatograph with a flame ionization detector. The development of optimal gas chromatographic parameters for the determination of these acids in the blood was carried out using a hardware-software complex based on a Crystal 2000 gas chromatograph with a flame ionization detector. The completeness of separation of acetic, propionic, iso-butyric, butyric, valerianic, iso-caproic and caproic acids was achieved on an HP-FFAP capillary column - 50 m * 0.32 mm * 0.5 μm at temperature conditions: column - from 70 ° C -160 ° C-180 ° C-280 ° C; evaporator - 250 ° C; detector - 280 ° C; carrier gas flow rate 1 (nitrogen) - 40 cm ³ / min; the flow rate of carrier gas 2 (nitrogen) is 14-30 cm ³ / min.

In the process of research on the choice of extractant (organic solvent) for the extraction of volatile fatty acids from the blood, the following solvents were tested: benzene, ethyl alcohol, diethyl ether, hexane, isobutyl alcohol and inorganic acids: hydrochloric, phosphoric, sulfuric.

The maximum degree of extraction of the studied acids from blood samples was achieved using sulfuric acid, which was used in further studies.

In the course of experimental work on the choice of extractant, a number of organic solvents were used and the dependence of the degree of extraction on the type of inorganic acids and the nature of organic solvents was studied. The results are shown in Table 2.

The data shown in table 2 show that only when using iso-butyl alcohol along with sulfuric acid in the aggregate of all the claimed operations of the proposed method, the maximum extraction (maximum degree of extraction) of these acids from a blood sample is provided under optimal conditions for gas chromatographic analysis.

The proposed method was studied a number of blood samples with different contents of acetic, propionic, iso-butyric, butyric, valerianic, iso-caproic and caproic acids.

As a result of the experiment, it was found that the greatest extraction of the studied acids from the blood occurs during extraction with isobutyl alcohol in an acidic medium (pH 2-3), with the addition of a 0.4% alkali solution (to convert acids to salts), centrifugation, evaporation to dryness, acidification of the evaporated extract with a 1% solution of sulfuric acid, moreover, in a volume ratio to a blood sample as 1:40 and the introduction of isobutyl alcohol in a volume ratio to a blood sample as 1:20. Moreover, a deviation from the indicated volume ratios to a smaller or greater side led to an increase in the determination error.

The measurement methodology using the proposed method provides obtaining measurement results with an error not exceeding the values given in tables 3 and 4. The data shown in tables 3 and 4 show that the sensitivity of determination in the analyzed volume of a blood sample with joint presence was for: acetic acid - 0.003 mcg; propionic acid - 0.002 mcg; iso-butyric acid - 0.001 mcg; butyric acid - 0.001 mcg; valerianic acid - 0.0005 mcg; iso-caproic acid - 0.0005 mcg; caproic acid - 0.0005 mcg.

The determination error for acetic acid was 17.66%; propionic acid 13.93; iso-butyric acid - 19.06%; butyric acid - 11.21%; valerianic acid - 15.64%; iso-caproic acid - 17.56%; caproic acid - 20.76%, even with their joint presence.

The application of the proposed method allows to increase the sensitivity of determination by acetic, propionic acid and butyric acid by 2 times (compared with the known method). It should also be noted that the prior art does not know the method for determining such a circle of acids in the blood with their joint presence.

The proposed method is available in execution and can be recommended for clinical trials in the laboratory.

Table 1 Standard solutions for establishing a calibration graph Mixture number one 2 3 four 5 Acetic acid Volume of the original std. Solution, cm ³ 2 3 5 7 10 The concentration of acetic acid, µg / cm ³ 3.12 4.68 7.8 10.92 15.6 Propionic acid Initial std. Solution, cm ³ 2 3 5 7 10 The concentration of propionic acid, μg / cm ³ 1,996 2.99 4.99 6.99 9.98 Isobutyric acid Volume of the original std. Solution, cm ³ 2 3 5 7 10 The concentration of Isobutyric acid, μg / cm ³ 0.96 2.87 4.79 6.71 9.58 Butyric acid Volume of the original std. solution, cm ³ 2 3 5 7 10 The concentration of Butyric acid, mcg / cm ³ 0.959 2.88 4.79 6.71 9.59 Valerianic acid Volume of the original std. solution, cm ³ 2 3 5 7 10 The concentration of Valerianic acid, μg / cm ³ 0.47 1.39 2,33 3.26 4.65 Iso-caproic acid Initial std. solution, cm ³ 2 3 5 7 10 The concentration of Iso-caproic acid, mcg / cm ³ 0.46 1.39 2,31 3.23 4.62 Caproic acid Initial std. solution, cm ³ 2 3 5 7 10 The concentration of caproic acid, μg / cm ³ 0.46 1.39 2,31 3.23 4.62

table 2 Average completeness of extraction of acetic, iso-butyric, butyric, valerianic, iso-caproic and caproic acids from a blood sample from the nature of organic solvents and the type of inorganic acids Component Organic solvent Hexane Isobutyl alcohol Type of inorganic acid 2% H ₂ SO ₄ 1% H ₂ SO ₄ 0.5% H ₂ SO ₄ 2% H ₂ SO ₄ 1% H ₂ SO ₄ 0.5% H ₂ SO ₄ The degree of extraction,% 1. Acetic acid 25 ± 2,050 25 ± 2.00 20 ± 2,717 59 ± 4,052 60 ± 4,905 55 ± 3,312 2. Propionic acid 39 ± 2,134 35 ± 2,35 30 ± 2.13 50 ± 4.23 65 ± 5.99 45 ± 3,391 3. Isobutyric acid 32 ± 2.25 29 ± 1.95 35 ± 2,99 80 ± 2,756 88 ± 6.82 75 ± 6.73 4. Butyric acid 30 ± 1,299 30 ± 2,052 25 ± 2,120 75 ± 7.42 82 ± 7.5 70 ± 2,251 5. Valerianic acid 35 ± 2.92 33 ± 2.82 39 ± 3.00 82 ± 6.85 91.3 ± 8.13 89 ± 8.1 6. Iso-caproic acid 28 ± 2.12 29 ± 1.95 40 ± 2.95 80 ± 7.21 95.5 ± 8.65 90 ± 8.32 7. Caproic acid 27 ± 1.99 30 ± 2.05 35 ± 2,33 85 ± 7.09 88 ± 7.54 84 ± 7.23

Table 3 The range of measurements, values of indicators of accuracy, repeatability, reproducibility in the implementation of the proposed method The name of the determined component and the measurement range, μg / cm ³ Repeatability index (relative standard deviation of repeatability), σ _r ,%

%Reproducibility ratio (relative standard deviation of reproducibility)

% Accuracy indicator (limits of relative error with probability Р = 0.95), ± δ,% Acetic acid, from 3 to 16 incl. 19.11 4.71 17.66 Propionic acid, from 2 to 10 incl. 4.48 5.99 13.93 Isobutyric acid, from 1 to 10 incl. 6.18 4.46 19.06 Butyric acid, from 1 to 10 incl. 5.61 4.83 11.21 Valerianic acid, from 0.5 to 5.0 incl. 4.47 5.02 15,64 Iso-caproic acid, from 0.5 to 5.0 incl. 3.53 3.93 17.56 Caproic acid, from 0.5 to 5.0 incl. 3.39 4.27 20.76

Table 4 The values of the limits of repeatability and reproducibility in the implementation of the proposed method (with a confidence probability of P = 0.95) The name of the determined component and the measurement range, μg / cm ³ Repeatability limit (relative value of the permissible discrepancy between two results of parallel determinations), r _n ,%

%The limit of internal laboratory reproducibility (the relative value of the permissible discrepancy between the two measurement results obtained in the same laboratory, but under different conditions),

% Acetic acid, 3 to 16 incl. 12.94 1.31 Propionic acid, from 2 to 10 incl. 12.41 1.66 Isobutyric acid, from 1 to 10 incl. from 17.12 1.24 Butyric acid, from 1 to 10 incl. 15,53 1.34 Valerianic acid, from 0.5 to 5.0 incl. 12.39 1.39 Iso-caproic acid, from 0.5 to 5.0 incl. 9.77 1.09 Caproic acid, 0.5 to 5.0 incl. 9.38 1.18

Claims (5)

1. The method of quantitative determination of acetic, propionic, iso-butyric, butyric, valerianic, iso-caproic and caproic acids in the blood by gas chromatographic analysis, characterized in that the blood sample is acidified with a 1% sulfuric acid solution to pH 2-3, carry out extraction of the determined acids with isobutyl alcohol, the volume of which is related to the volume of the blood sample as 1: 1, is carried out by centrifugation to separate the proteins, add 2-3 drops of a 0.4% alkali solution and the extract is evaporated to dryness, then to dry precipitate consequently, a 1% solution of sulfuric acid and isobutyl alcohol are added and gas chromatographic separation of the acid mixture is carried out on a capillary column with a flame ionization detector, and the amount of each acid is set according to a calibration curve. 2. The method according to claim 1, characterized in that the addition to the dry residue of a 1% solution of sulfuric acid is produced in a volume of 1:40, and isobutyl alcohol - 1:20 to the volume of a blood sample, respectively. 3. The method according to claim 1, characterized in that the extraction of the determined acids with isobutyl alcohol is carried out for 5-10 minutes 4. The method according to claim 1, characterized in that the centrifugation is carried out at 6000 rpm for 10 minutes 5. The method according to claim 1, characterized in that the gas chromatographic separation of the acid mixture on a capillary column with a flame ionization detector is carried out using a hardware-software complex based on a Crystal 2000 gas chromatograph with a flame ionization detector on an HP- capillary column FFAP - 50m × 0.32mm × 0.5μm at temperature conditions: column - from 70 ° С-160 ° С-180 ° С-280 ° С; evaporator - 250 ° C; detector - 280 ° C; carrier gas flow rate 1 (nitrogen) - 40 cm ³ / min; the flow rate of carrier gas 2 (nitrogen) is 14-30 cm ³ / min.