RU2740535C1 - Method of measuring weight fraction of l-carnitine and d-carnitine in fodders, formulated feed, feed additives and medicinal agents for animals by high-performance liquid chromatography with fluorescent detection - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to analytical chemistry, in particular to analysis of feed quality, feedstuffs, fodder additives, medicinal agents. Method of measuring weight ratio of D- and L-carnitine in fodder, formulated feed, feed additives and medicinal agents for animals by chiral high-performance liquid chromatography with fluorescent detection involves preparation of a sample, centrifuging sample extract at 17,608 g for 5 minutes; 30 cm³ of upper layer of extract is transferred into volumetric flask with capacity of 5 cm³, 30 cm³ of carbonate buffer (0.05 mol/dm³) is added, 80 mm³ solution of 9-fluorenylmethoxycarbonyl chloride (FMOC chloride) in concentration of 5 mg/cm³, stirred, flask is held in a water bath at 45 °C for one hour, cooled to room temperature, brought to mark with acetate buffer with molar concentration of 0.05 mol/dm³, in a volumetric flask of 10 cm³ 1 cm³ was transferred resulting solution volume was adjusted to the mark with acetate buffer (0.05 mol/dm³) chromatography under the following conditions: column connected in series: 1) SPHERIOSORB SCX 250 × 4.60 mm, 5 microns; 2) Astec Chirobiotic TAG 250 × 4.60 mm, 5 microns; temperature of columns 25 °C; separation is carried out in mode of gradient elution (phase A - phosphate buffer with triethylamine (6.8 cm³ of triethylamine per 1 dm³ of deionised water, buffer pH is brought with orthophosphoric acid to pH 2.6), phase B - acetonitrile; at ratio of phase A to phase B - 80:20), signal is recorded at excitation light wavelength 260 nm, emission - 310 nm.

EFFECT: technical result is possibility to separate isomers of D- and L-carnitines in samples, ensuring selectivity, linearity, repeatability and reproducibility of results.

1 cl, 3 tbl, 1 dwg

Description

The invention relates to the field of pharmaceutical chemistry, pharmaceutical and food industries, in particular to the analysis of the quality of feed, compound feed, feed additives, veterinary drugs by the method of chiral high-performance liquid chromatography (hereinafter - HPLC).

The proposed method can be used to measure the mass fraction of L-carnitine and D-carnitine (including in the case of joint presence) by HPLC with fluorescence detection and is used to identify products containing D-carnitine and having a toxic effect in feed, mixed feed, feed additives, veterinary medicines. The method is intended for quantitative control of the quality of drugs, feed additives in testing laboratories and in production, as well as in any other areas where HPLC is used.

L-carnitine is known to be a coenzyme involved in the transport of activated long-chain fatty acids into the mitochondria, thereby providing the body with energy. L-carnitine deficiency leads to impaired beta-oxidation of fatty acids, which has a negative effect on the animal body. Several syndromes of secondary carnitine deficiency have been described, which may result from defects in intermediate metabolism and changes mainly associated with mitochondrial oxidative pathways. An acute shortage of L-carnitine is observed with metabolic disorders, resulting in muscle weakness, rapid fatigue, hypotension, nervous exhaustion, and disorders of the cardiovascular system and liver. More than 75% of the daily requirement for L-carnitine must come from food.

The introduction of L-carnitine into the diet of livestock and poultry leads to an increase in the body's endurance, improved heart function, a decrease in body fat, faster recovery due to a general improvement in metabolic processes in cells, improves the physiological and biochemical status and the state of natural resistance, reproductive functions. This allows you to significantly increase the digestibility of feed nutrients, which affects the level and quality of the products obtained.

Carnitine has an optically active center and exists in the form of two spatial isomers - L-carnitine and D-carnitine, having the same chemical composition, but different spatial configuration. Only L-carnitine, which is found in natural sources, has a biologically active effect.

D-carnitine competes with L-carnitine for the same transport systems, disrupts its synthesis in the liver, prevents penetration into the myocardium, muscle tissue, and blocks the transport of L-carnitine in the small intestine and reverse reabsorption in the kidneys. D-carnitine does not affect the intramitochondrial oxidation of long-chain fatty acids and, causing L-carnitine deficiency, can lead to a decrease in the intramitochondrial concentration of fatty acids, as a result of which - to a decrease in energy production [1].

The results of experimental and clinical studies indicate the pronounced toxic effects of D-carnitine on the part of skeletal and cardiac muscles, manifested by myasthenia gravis and arrhythmias.

These symptoms disappear after the administration of L-carnitine. Only L-forms should be used, since synthetically obtained can be either a pure enantiomer or a mixture of L- and D-enantiomers. Based on the data presented in the scientific literature, D- and DL-carnitine cannot be considered as completely safe substances for use in veterinary and medical practice, or as additives to diets [2].

With the advent of effective methods for producing L-carnitine and due to the negative effect of D-carnitine on the human body, the American Food and Drug Administration (FDA) banned the distribution of D-carnitine and DL-carnitine in the USA in 1986 [2, 3, 4 ].

In practice, pharmaceutical compositions containing not L-carnitine, but a racemic mixture of D- and L-carnitine, are still legally used. At the same time, the pharmacopoeias of the European Union and the United States do not allow the content of D-carnitine in medicinal products to exceed 4% [5, 6].

The known methods for the determination of L- and D-carnitine isomers are rather complicated and laborious. They are performed using tandem mass spectrometry (HPLC-MS), which requires the use of expensive mass spectrometry equipment, which is often not available in most laboratories.

As a prototype was chosen a method for the determination of carnitine, which consists in the study of samples using HPLC. Existing in the Russian Federation GOST R 53185-2008, p. 4.4 [7] establishes a method for the quantitative determination of L-carnitine in non-alcoholic and low-alcohol tonic drinks using high-performance liquid chromatography using refractometric detection. This method does not allow the separation of L-carnitine and D-carnitine when they are present together.

Another prototype (BY 17743 C1, 2013.12.30, priority date 12/30/2013 "Method for the determination of N-acetyl-L-carnitine"), in which a sample of the test sample is analyzed by high performance liquid chromatography on a chromatograph equipped with a column with a C18 sorbent and a spectrophotometric detector does not allow the separation of enantiomers due to the weak absorption of the analytes under study in this spectral region.

Thus, it is obvious that the search for a unified method for the detection of carnitine isomers, which will allow the detection of feed additives and drugs for animals containing D-carnitine and having a toxic effect, and create more effective feed additives and drugs with the lowest toxicity, is obvious.

Technical result: The inventive method provides selectivity, linearity, precision (repeatability and reproducibility) and accuracy, which is achieved due to the fact that the sample is processed with 9-fluorenylmethoxycarbolnyl chloride (FMOC chloride) at a concentration of 5 mg / cm ³ . Samples in the form of tablets or granules are ground in a mortar, feed and feed additives are ground in a laboratory mill. Mix the pasty, powdery and liquid samples thoroughly. High-fat samples are defatted. The sample extract is centrifuged at 17608 g for 5 min. In a volumetric flask with a capacity of 5 cm ^3, transfer 30 mm ^{3 of the} upper layer of the extract, add 30 mm ^{3 of} carbonate buffer (0.05 mol / dm ³ ), 80 mm ^{3 of a} solution of 9-fluorenylmethoxycarbolnyl chloride (FMOC chloride) at a concentration of 5 mg / cm ³ ), mix. The flask is kept in a water bath at 45 ° C for one hour, cooled to room temperature, adjusted to the mark with acetate buffer with a molar concentration of 0.05 mol / dm ³ . In a volumetric flask with a capacity of 10 cm ^3, transfer 1 cm ^{3 of the} resulting solution, the volume is brought to the mark with acetate buffer (0.05 mol / dm ³ ). Chromatography conditions:

Series connected columns:

- 1st - SPHERIOSORB SCX 250 × 4.60 mm, 5 microns;

- 2nd - Astec Chirobiotic TAG 250 × 4.60 mm, 5 microns;

Column temperature 25 ° C; the separation is carried out in the gradient elution mode (phase A - phosphate buffer with triethylamine (6.8 cm ^{3 of} triethylamine per 1 dm ^{3 of} deionized water, the pH of the buffer is adjusted with phosphoric acid to pH 2.6), phase B - acetonitrile; with the ratio of phase A to phase B - 80:20). Signal registration is carried out at a wavelength of excitation light 260 nm, emission - 310 nm.

The mass fraction of L-carnitine and D-carnitine C _x in the analyzed sample is calculated by the formula,%:

Where:

C _gr - mass concentration of L-carnitine and D-carnitine in the extract of the analyzed sample, μg / cm ³ ;

m _x is the mass of the analyzed sample, g;

V is the volume to which the sample is diluted, cm ³ ;

10 - coefficient taking into account the dilution of the analyzed sample;

K ₁ - coefficient taking into account the dilution of the analyzed sample if the mass concentrations of analytes in the extract of the analyzed sample are outside the range of the calibration characteristic;

K = 1, when presenting the analysis result in mass fractions of L- and D-carnitine; or

K = 1.23, when presenting the analysis result in mass fractions of L- and D-carnitine hydrochloride;

0.0001 - conversion factor of the final measurement result in percent.

Using the proposed method, the measurement range was determined from 0.1% to 50% in feed, compound feed, feed additives, medicines. The expanded uncertainty of measurement results is in the range from 12 to 35% for L-carnitine and from 10 to 13.5% for D-carnitine.

The claimed method of the invention is based on the separation of the enantiomers of L- and D-carnitines with preliminary derivatization. As a derivatizing agent, 9-fluorenylmethoxycarbolnyl chloride (FMOC chloride) is used, when interacting with which, the enantiomers of L- and D-carnitines form derivatives with greater hydrophobicity and fluorescence, which makes it possible to further analyze them using HPLC with fluorescence detection. In addition, pre-column derivatization can increase the separation specificity. We carried out separation by ion-pair chromatography using a mobile phase with a phosphate buffer with triethylamine (6.8 cm ³ per 1 dm ^{3 of} deionized water. Adjust the pH of the solution with phosphoric acid, pH 2.6), which contributed to an increase in the retention of derivatives of L - and D-carnitine on a column with a sorbent based on silica gel with grafted benzene sulfonic acid, used as a strong cation exchanger and providing good resolution (for example, Waters SPHERIOSORB SCX250 × 4.60 mm with a particle size of 5 μm, or similar). The sequential attachment of a chiral column containing teicoplanin aglycone as a chiral selector (for example, SUPELCO Astec Chirobiotic TAG 250 × 4.60 mm with a particle size of 5 μm, or similar) allowed us to achieve the necessary separation of L- and D-carnitine enantiomers.

Chromatographic conditions: column temperature 25 ° C; the separation is carried out in a gradient elution mode (phase A - phosphate buffer with triethylamine (6.8 cm ³ per 1 dm ^{3 of} deionized water; pH 2.6), phase B - acetonitrile), in accordance with Table 1.

Under these conditions, the release time of analytes is (approximately): L-carnitine - 59.5 minutes; D-carnitine - 61.0 minutes; the flow rate of the eluent is 0.6 cm ³ / min; the volume of the injected sample - 20.0 mm ³ . Signal registration is carried out at a wavelength of exciting light - 260 nm, emission - 310 nm.

Samples in the form of tablets or granules are ground in a mortar, feed and feed additives are ground in a laboratory mill. Mix the pasty, powdery and liquid samples thoroughly. High-fat samples are defatted. In volumetric flasks with a capacity of 100 cm ³ , 50 cm ³ , 25 cm ³ or 5 cm ^3, from 0.5000 to 1,000 g of the sample to be measured was weighed (depending on the expected content of L- and D-carnitine), deionized water was added to the mark, placed for 10 minutes in an ultrasonic bath for extraction in cold mode (at room temperature). The extract was transferred to a 1.5 cm ³ microcentrifuge tube and centrifuged at 17608 g for 5 min. In a volumetric flask with a capacity of 5 cm ^{3 with a} pipette was transferred 30 mm ^{3 of the} upper layer of the extract, 30 mm ^{3 of} carbonate buffer (0.05 mol / dm ³ ), 80 mm ^{3 of a} solution of 9-fluorenylmethoxycarbolnyl chloride (FMOC chloride) at a concentration of 5 mg / cm were added ³ was mixed. The flask was kept in a water bath at 45 ° C for one hour, cooled to room temperature, and made up to the mark with acetate buffer (0.05 mol / dm ³ ). In a volumetric flask with a capacity of 10 cm ³ transferred 1 cm ^{3 of the} resulting solution, the volume was brought up to the mark with acetate buffer (0.05 mol / dm ³ ). The solution was filtered through a 0.45 μm syringe filter into a vial and used for chromatographic measurement. The test is carried out by HPLC with fluorescence detection. Figure 1 shows a chromatogram of a sample containing a mixture of L- and D-carnitine in a ratio of 100: 1 on Waters SPHERIOSORB SCX 250 × 4.60 mm, 5 micron columns and SUPELCO Astec Chirobiotic TAG 250 × 4.60 mm, 5 mcn using a fluorescent detector. Gradient elution mode with the ratio of phosphate buffer to triethylamine, pH 2.6 and acetonitrile (80:20). The flow rate of the eluent is 0.6 cm ³ / min.

Processing of measurement results. The retention times of L-carnitine and D-carnitine obtained from the analysis of calibration chromatograms are used to identify peaks in the chromatogram of the analyzed sample. The areas of the peaks identified by the analytes are determined and, using the calibration characteristics, the mass concentration of these analytes in the extract of the analyzed sample is found.

The mass fraction of L-carnitine and D-carnitine C _x in the analyzed sample is calculated by the formula,%:

Where:

C _gr - mass concentration of L-carnitine and D-carnitine in the extract of the analyzed sample, μg / cm ³ ;

m _x is the mass of the analyzed sample, g;

V is the volume to which the sample is diluted, cm ³ ;

10 - coefficient taking into account the dilution of the analyzed sample;

K ₁ - coefficient taking into account the dilution of the analyzed sample if the mass concentrations of analytes in the extract of the analyzed sample are outside the range of the calibration characteristic;

K = 1, when presenting the analysis result in mass fractions of L- and D-carnitine; or

K = 1.23, when presenting the analysis result in mass fractions of L- and D-carnitine hydrochloride;

0.0001 - conversion factor of the final measurement result in percent.

Calculations are carried out with an accuracy of two decimal places and are expressed as a percentage. The final measurement result is rounded to the first decimal place and expressed as a percentage. Using the proposed method, the measurement range was determined from 0.1% to 50% in feed, compound feed, feed additives, medicines. The expanded uncertainty of measurement results is in the range from 12 to 35% for L-carnitine and from 10 to 13.5% for D-carnitine.

Metrological characteristics. This technique provides measurements of the mass fraction of L- and D-carnitine with an expanded uncertainty of the results of analytical measurements at a coverage factor of k = 2, indicated in Table 2.

принимают среднее арифметическое значение результатов двух параллельных определений, которое вычисляют по формуле:For the result of measuring the mass fraction of L-carnitine and D-carnitine

take the arithmetic mean of the results of two parallel determinations, which is calculated by the formula:

where x ₁ and x ₂ are the values of the mass fraction of L-carnitine and D-carnitine, calculated by the formula (1) for parallel determinations, the discrepancy between which should not exceed the value of the repeatability limit r (%) indicated in Table 3.

If condition (3) is not met, the experiment is repeated, thus obtaining x ₃ and x ₄ .

The results of measurements x ₁ , x ₂ , x ₃ , x ₄ are recognized as acceptable, provided that:

where x _max and x _min are the maximum and minimum values, respectively, from four parallel definitions (%);

CR _0.95 (4) is the value of the critical range for four parallel determinations at a confidence level of P = 0.95 (%), indicated in Table 3.

принимают среднее арифметическое значение результатов четырех параллельных определений, которое вычисляют по формуле:When condition (4) is met, for the final measurement result of the mass fraction of L-carnitine and D-carnitine in the sample

take the arithmetic mean of the results of four parallel determinations, which is calculated by the formula:

принимают значение медианы результатов четырех параллельных определений, которое рассчитывают следующим образом:If condition (4) is not met for the final result of measurements of the mass fraction of L-carnitine and D-carnitine in the sample

take the median value of the results of four parallel determinations, which is calculated as follows:

- order the results of parallel determinations in ascending order of the L-carnitine and D-carnitine mass fraction values:

where x ₍₁₎ , x ₍₂₎ , x ₍₃₎ , x ₍₄₎ are four results of parallel determinations, arranged in ascending order of the mass fraction of L-carnitine and D-carnitine;

- discard the smallest and largest results of single measurements, the two remaining measurement results are averaged:

In addition, the reasons for the appearance of unacceptable results of parallel definitions are determined and eliminated.

The values of the accuracy indicator of the technique are used when:

- registration of measurement results issued by the laboratory;

- assessment of the possibility of using the measurement results when implementing the measurement procedure in the laboratory.

The result of the analysis in the documents providing for its use is presented in the form:

- среднее арифметическое значение результатов n определений, признанных приемлемыми, %;Where

- the arithmetic mean of the results of n definitions recognized as acceptable,%;

U is the value of the relative expanded uncertainty,% (in accordance with Table 2).

Monitoring the stability of measurement results. The stability control of measurement results within the laboratory is carried out in accordance with GOST ISO 5725-6 using Shewhart control charts. The results of the presented metrological evaluation of the method for determining the quantitative content of D- and L-carnitines by HPLC prove the applicability of this method in accordance with the standards set forth in GOST 8.563-2009, GOST R 5725-2002.

The claimed method makes it possible to separate the isomers of D- and L-carnitines in samples of feed, compound feed, feed additives and drugs for veterinary medicine. The range of measurements of the mass fraction for L- and D-carnitine was from 0.1 to 50%. The expanded uncertainty of measurement results is in the range from 12 to 35% for L-carnitine and from 10 to 13.5% for D-carnitine. The claimed method provides selectivity, linearity, precision (repeatability and reproducibility) and accuracy.

The method can be used to ensure the quality and safety of feed, compound feeds, feed additives and medicines for veterinary medicine, as well as in other industries, in addition to the pharmaceutical and food industries, for example, in chemical production for product quality control.

Bibliography

1. Spasov A.A., Iezhitsa I.N. Stereopharmacological features of carnitine. Russian physiological journal. THEM. Sechenov, No. 12, 2005.

2. Preedy V.R. Reviews in Food and Nutrition Toxicity, CRC Press, 2005, v. 3, p. 288.

3. Virmani A, Pinto L, BiniendaZ, Ali S. Food, nutrigenomics, and neurodegeneration-neuroprotection by what you eat! Mol Neurobiol., 2013, v. 48, p: 353-62.

4. FDA Briefing Document Pharmacy Compounding Advisory Committee (PCAC) Meeting, 2016.

5. EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP). Scientific Opinion on the safety and efficacy of L-carnitine as a feed additive for all animal species based on a dossier submitted by EUROPE-ASIA Import Export GmbH. EFSA Journal 2012; 10 (5): 2677.

6. L-Carnitine. Technical Evaluation Report. Compiled by ICF International for the USDA National Organic Program.

7. GOST P 53185-2008, p. 4.4 Non-alcoholic and low-alcohol tonic drinks. Test methods.

Claims (13)

A method for measuring the mass fraction of D- and L-carnitine in feed, mixed feed, feed additives and medicines for animals by the method of chiral high-performance liquid chromatography with fluorescent detection, including sample preparation, centrifugation of the sample extract at 17608 g for 5 min; in a volumetric flask with a capacity of 5 cm ³ transfer 30 mm ^{3 of the} upper layer of the extract, add 30 mm ^{3 of a} carbonate buffer (0.05 mol / dm ³ ), 80 mm ^{3 of a} solution of 9-fluorenylmethoxycarbolnyl chloride (FMOC chloride) at a concentration of 5 mg / cm ³ , stir, the flask is kept in a water bath at 45 ° C for one hour, cooled to room temperature, adjusted to the mark with an acetate buffer with a molar concentration of 0.05 mol / dm ³ , 1 cm ^{3 of the} resulting product is transferred to a 10 cm ³ volumetric flask. solution, the volume is adjusted to the mark with acetate buffer (0.05 mol / dm ³ ) under the following chromatographic conditions: columns connected in series: - 1st - SPHERIOSORB SCX 250 × 4.60 mm, 5 microns; - 2nd - Astec Chirobiotic TAG 250 × 4.60 mm, 5 microns; column temperature 25 ° C; the separation is carried out in the gradient elution mode (phase A - phosphate buffer with triethylamine (6.8 cm ^{3 of} triethylamine per 1 dm ^{3 of} deionized water, the pH of the buffer is adjusted with orthophosphoric acid to pH 2.6), phase B - acetonitrile; at the ratio of phase A to phase B - 80:20), the signal is recorded at an exciting light wavelength of 260 nm, emission - 310 nm, while the mass fraction of L-carnitine and D-carnitine C _x in the analyzed sample is calculated by the formula,%:

Where: C _gr - mass concentration of L-carnitine and D-carnitine in the extract of the analyzed sample, μg / cm ³ ; m _x is the mass of the analyzed sample, g; V is the volume to which the sample is diluted, cm ³ ; 10 - coefficient taking into account the dilution of the analyzed sample; K ₁ - coefficient taking into account the dilution of the analyzed sample if the mass concentrations of analytes in the extract of the analyzed sample are outside the range of the calibration characteristic; K = 1, when presenting the analysis result in mass fractions of L- and D-carnitine; or K = 1.23, when presenting the analysis result in mass fractions of L- and D-carnitine hydrochloride; 0.0001 is the conversion factor of the final measurement result in percent, the expanded uncertainty of the measurement results is in the range from 12 to 35% for L-carnitine and from 10 to 13.5% for D-carnitine.

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