RU2180748C1 - Method for quantitatively determining levomycetin in foods and pharmaceutical preparations - Google Patents

Abstract

FIELD: analytical methods in biochemistry. SUBSTANCE: invention provides method that can be applied in determining antibiotic in biological systems (blood, urine, etc.) when performing pharmacokinetic investigations, in toxicological and technical analyzes of drugs, animal feeds, and also can be a basis to create an antibiotic metabolism diagnostic system. Method consists in that levomycetin is first transferred from a sample into solution, after which one performs acid hydrolysis, precipitates protein from hydrolyzate, and determines levomycetin voltammetrically in protein- free hydrolyzate via registration of cathodic peaks of antibiotic on indicator mercury-film or glass-carbon electrodes in differential voltammogram record mode at potentials -(0.67+/-0.05) V and -(0.60+/-0.03) V respectively relative to saturated silver chloride electrode against 0.1 M C₆H₁₄O₇N₂(pH4,7÷5,1) (pH 4.7-5.1) or 0.1 M (NH₄)₂SO₄ to which HCl is added to give pH 5.1, scan velocity of potential being 10 to 25 mV/s. Concentration of levomycetin is determined from peak height utilizing certified mixture addition approach. EFFECT: increased analytical sensitivity, rapidity, and selectivity. 2 tbl, 4 ex

Description

Левомицетин относится к антибактериальным веществам и обладает высокой клинической эффективностью в лечении ряда тяжелых инфекций. Его применяют в ветеринарии и животноводстве в качестве лечебно-профилактических средств. АБ добавляются, как правило, в корм на уровне 50-200 г на 1 т. Левомицетин способен переходить в мясо, молоко животных, яйца птиц, другие продукты и оказывать токсическое действие на организм человека при концентрации более 25 мкг/см³ [Позняковский В. М. Гигиенические основы питания и экспертизы продовольственных товаров. - Новосибирск: Изд-во Новосиб. ун-та, 1996. - 432 с.], что делает потенциально опасным его бесконтрольное применение.The invention relates to the field of analytical chemistry, in particular to the inversion voltammetric method for determining the antibiotic (AB) of chloramphenicol (chloramphenicol), which is
D (-) - threo-2-dichloroacetylamino-1- (4-nitrophenyl) propan-1,3-diol:

Chloramphenicol belongs to antibacterial substances and has high clinical efficacy in the treatment of a number of serious infections. It is used in veterinary medicine and animal husbandry as therapeutic and prophylactic agents. ABs are usually added to feed at a level of 50-200 g per 1 ton. Levomycetin is able to pass into meat, animal milk, poultry eggs, other products and have a toxic effect on the human body at a concentration of more than 25 μg / cm ³ [Poznyakovsky V M. Hygienic fundamentals of nutrition and examination of food products. - Novosibirsk: Publishing House Novosib. Univ., 1996. - 432 p.], which makes its uncontrolled use potentially dangerous.

 Biomedical requirements and sanitary standards for the quality of food raw materials and food products indicate the inadmissibility of residual amounts of chloramphenicol in food products. The maximum level of AB residues is 0.01 U / g, which corresponds to 0.01 mg / kg or 10.0 μg / kg (maximum permissible concentration, MPC) [Hygienic requirements for the quality and safety of food raw materials and food products - Sanitary rules and SanPiN norms 2.3.2.560-96.]. This, in turn, places high demands on the quality control of food raw materials and the improvement of AB determination methods.

 Microbiological methods have found the widest application both in scientific research and in practical institutions [Methodological guidelines for determining the residual amounts of antibiotics in animal products. Min USSR Health. - Moscow, 1985], allowing to determine the minimum concentration of antibiotics in the test material. They are based on the direct biological effect of AB on sensitive strains of microorganisms and are relatively simple, however, they are characterized by insufficient specificity and reproducibility. The methods using them are extremely long (analysis time is more than 36 hours), time-consuming and often used only for a qualitative determination of AB.

 There are practically no instrumental methods for determining chloramphenicol in foods. The known method [Melamed DB, Brick VK, Lyapkov B. G. The method of analysis of chloramphenicol in food products. A.S. SU 1702302 A1, G 01 N 30/90, A 23 L 3/00] high performance liquid chromatography (HPLC) in an acetonitrile-water-decylamine solvent system. To increase the sensitivity, ultraviolet detection is used at λ = 278 nm. Quantitative determination of chloramphenicol is carried out by comparing the peak areas of chloramphenicol - the standard and chloramphenicol in the sample. Despite the high sensitivity of the HPLC method (10 μg / kg), the duration of the analysis, taking into account the time of sample preparation, as well as the high cost of the instruments, significantly limit its use in food control.

 There are many options for optical methods for determining chloramphenicol in dosage forms [Tablets of chloramphenicol. FS 42-3679-98. - p. 8 .; Chloramphenicol solution. FS 42-3425-97. - p.6 .; Tiraspolskaya S.G., Stepanyuk S.N., Filipyeva K.N. Photometric determination of chloramphenicol in dosage forms. // Pharmacy, 1980.-T.29, 6, p. 48-49].

 According to published data, chloramphenicol absorbs in the ultraviolet region from 205 to 400 nm. The maximum light absorption is at 278-280 nm.

A significant place is occupied by colorimetric methods based on various specific color reactions [Tiraspolskaya SG, Stepanyuk SN, Filipyeva K. N. Photometric determination of chloramphenicol in dosage forms. //Pharmacy. -1980.-t.29, 6, p. 48-49]. The sensitivity of these methods is low and is 0.1 mg / cm ³ or more.

An enzyme-linked immunosorbent assay was developed for chloramphenicol in human serum [Kolosova A. Yu., Samsonova Zh.V., Blintsov AN, Egorov AM Enzyme-linked immunosorbent assay of chloramphenicol in human serum. // Questions of medits. Chemistry. - 1998. - v.44., 2, p.194 - 202], based on measuring the optical density of the product of the enzymatic reaction in an antibiotic solution in the concentration range of 10 ÷ 1000 ng / cm ³ . The work used a large number of inaccessible biochemical reagents. The technique requires a lot of labor.

Much attention is paid in the literature to the development of a polarographic method for the determination of chloramphenicol in pharmaceutical preparations (tablets, capsules, eye drops, injection solutions and technical powders) [Miskidzhyan SP, Kravchenyuk LP Polarography of drugs. - Kiev: Publishing house "Vishcha school", 1976. - 230 p.]. The polarographic depolarization of chloramphenicol is based on the reduction of nitro groups and the dichloroacetyl residue on a mercury dripping electrode (r.c.e.). Chloramphenicol formed two waves, the first of which had a half-wave potential of E _1/2 = -0.35 V and corresponded to the four-electron process of irreversible reduction of the nitro group to hydroxylamine. E _1/2 = -1.10 V. To suppress the maxima, thymol or gelatin was used. The most reproducible results of the quantitative determination of chloramphenicol on the river. c. in a pure preparation obtained using gelatin [Passet B.V., Antipov M.A. Workshop on technical analysis and control of the production of chemical and pharmaceutical preparations and antibiotics. - M.: "Medicine", 1981. - 272 p.] (Prototype). The essence of the method consists in dissolving 0.1 g of the drug in 100 ml of 95% alcohol. 1 cm ^{3 was} taken from the resulting solution, an acetate buffer background solution was added to a volume of 10 cm ³ and 1 drop of a 1% gelatin solution. After transmission of hydrogen, a polarogram was recorded starting at 0.0 V. The half-wave potential was recorded at E _1/2 = -0.34 V. A solution of a standard sample of chloramphenicol was prepared in the same way, and its polarogram was removed under the same conditions. The minimum detectable concentration is 1000 mg / kg. Under these conditions, an express quantitative determination of chloramphenicol at the level of requirements defined by regulatory documents (0.01 mg / kg) is impossible.

 One of the limiting factors for the use of polarography (from a safety point of view) is also the use of large quantities of toxic mercury as electrodes in an electrolytic cell. Therefore, the challenge arises of replacing r.ke. to safer ones. Thus, despite the possibility of quantitative determination of chloramphenicol, none of the above polarographic methods are used in food analysis.

 One of the most promising methods for determining AB, in our opinion, is voltammetric (VA) and, first of all, its highly sensitive variants, such as differential voltammetry (TWA). The advantage of VA due to its high sensitivity is the ability to work with small in mass and volume samples in turbid and colored environments. Sample preparation can be greatly simplified due to the optional additional purification of the analyte before the actual electrochemical analysis. Therefore, simpler methods of preliminary separation of compounds from a complex multicomponent system are often acceptable. Voltammetry methods have also not been previously used for CHA of chloramphenicol in food products. This, apparently, is explained by the lag in the electroanalytic practice of food products and the lack of equipment in the laboratories of this industry.

 The task of the invention is to increase the sensitivity, expressivity and selectivity of the determination of chloramphenicol in foods by differential voltammetry.

The problem is achieved by the fact that chloramphenicol is transferred from the sample to the solution, acid hydrolysis is carried out, and protein is precipitated from the hydrolyzate, followed by voltammetric determination of the antibiotic. New in the method is that they carry out acid hydrolysis of 0.10-0.12 mol / dm ³ Hcl at a temperature of 25-30 ^o C for 10-15 minutes, then carry out the precipitation of water-soluble protein with 2.5-3.0 g sulfate ammonium and 3.0-5.0 cm ³ ethanol, separation of the precipitate by centrifugation for 10-15 minutes at a speed of 6000 rpm, followed by adsorption voltammetric determination of chloramphenicol in a protein-free hydrolyzate by recording the cathode peaks of the antibiotic on a mercury-film indicator (RPE) ) or glassy carbon (SU) electrodes in differential In the voltammogram shooting mode at the corresponding potentials - (0.67 ± 0.05) V and - (0.60 ± 0.03) V with respect to the saturated silver chloride electrode (sat. HSE) on the background 0.1 mol / dm ³ ammonium citrate disubstituted With ₆ H ₁₄ O ₇ N ₂ (pH 4.7-5.1) or 0.1 mol / dm ³ (NH ₄ ) ₂ SO ₄ with the addition of HCl to pH 5.1 at a speed potential sweep of 10-25 mV / s.

The prototype describes the use of acetate buffer solution (pH 4.4) as the background. The determination of chloramphenicol under these conditions at a level of 10 μg / kg is difficult due to poor reproducibility and distortion of the shape of the analytical signal associated with a large residual current, and the registration of an additional peak at a potential of E _p = - (0.48 ± 0.05) V. Supposed in the claimed invention, the background is 0.1 mol / dm ³ C ₆ H ₁₄ O ₇ N ₂ (pH 4.7-5.1) allows you to determine chloramphenicol at a level of 2.8-30.0 μg / kg with good reproducibility. The relative standard deviation for the indicated concentration range varies from 0.20 to 0.08. The absolute novelty is the experimentally selected backgrounds: a mixture of (NH ₄ ) ₂ SO ₄ with Hcl (pH 5.1); 0.1 mol / dm ³ C ₆ H ₁₄ O ₇ N ₂ , the established pHl of the solution is 4.7-5.1, on which the quantitative determination of chloramphenicol depends. Background electrolytes (NH ₄ ) SO ₄ and C ₆ H ₁₄ O ₇ N ₂ are simultaneously good precipitants of protein impurities in foods. For the quantitative determination of chloramphenicol, they were not previously used. Of particular importance in the voltammetric analysis of chloramphenicol is the pH of the medium. The optimal indicator is the experimentally established pH value of 4.7-5.1, which corresponds to the isoelectric state of the protein molecules of dairy and meat products. At a pH value of 4.7-5.1, the solubility of protein impurities is reduced and protein molecules do not move under the influence of an external electric field, which helps to stabilize residual protein impurities and obtain well reproducible recovery peaks of chloramphenicol, especially when using the SU indicator electrode. At a pH of 4.7-5.1, one distinct peak is recorded with a potential of (0.67 ± 0.05) and - (0.60 ± 0.03) V at the RPE and SU electrodes, respectively. At 5.1 <pH <4.7, the proteins of residual impurities are charged and move under the influence of an external electric field. This contributes to their adsorption on the electrodes, which degrades reproducibility, reduces the sensitivity of determination and expressity.

The established pH value of 4.7-5.1 depends on the nature of the background electrolytes and allows you to analyze solutions at the level of 2.8 μg / kg (lower limit of the determined contents, C _n ). The detection limit C _min.p calculated according to the 3σ criterion is 1.5 μg / kg. The dependence of the analytical signal on the concentration of chloramphenicol in such solutions is directly proportional to the range of determined contents from 2.8 to 30.2 μg / kg.

Another distinguishing feature is the established conditions of electrochemical accumulation: E _e = - (0.45-0.47) V. At potentials of -0.45 <E _e <-0.47, the value of the recovery current of organic matter decreased, in addition, at E _e <-0.47 V there was a large residual current associated with the evolution of hydrogen in acidic solutions, and the peak height decreased. Without preliminary electrolysis, it is not possible to register a clear peak of the chloramphenicol at a level of 2.8 μg / kg.

 Important for determining chloramphenicol is the choice of the sweep speed of the polarizing voltage. The optimal speed is 10-25 mV / s. An increase in speed of more than 25 mV / s increases the sensitivity, but the residual current increases and the resolution of the method decreases. Using a speed of more than 25 mV / s in food analysis does not completely remove the analyte from the surface of the electrode. This impairs reproducibility and requires additional cleaning of the electrode surface, especially SU. At a speed of less than 10 mV / s, the cathode current decreases and the detection sensitivity decreases.

The pre-electrolysis time (τ _e ) for the concentration range of chloramphenicol 2.8-30.0 μg / kg is 30-60 s, while the maximum value of the recovery current is reached. At 60c <τ _e <30c, the error of determination increases (relative standard deviation, S _r > 0.17 when determining chloramphenicol at a level of 10 μg / kg).

Another distinguishing feature is the use of RPE or SU electrodes as indicator. The mercury-film film is a mercury film 20-50 μm thick deposited on a silver substrate, the surface area is 15.0 mm ² , the working surface of the glassy carbon electrode is 25-30 mm ² (the prototype used a mercury dripping electrode, r.c. .). A significant advantage of the RPE is the possibility of obtaining narrower and higher peaks, which serve as the analytical characteristics of the analyte, due to a significant increase in the ratio of the active surface to the volume of mercury and a decrease in the release time of the analyte from the thin film (two to three times in comparison with the river e.). The use of the SU electrode is due to the high chemical and electrochemical stability of graphite, a wide range of working potentials in both aqueous and non-aqueous media, as well as the simplicity of mechanical surface renewal. The recovery potential of chloramphenicol is determined by the structure, structure and degree of adsorption on graphite hexagons. For ease of recovery, RPE and SU electrodes can be positioned:
RPE
- (0.67 ± 0.05) V
SU
- (0.60 ± 0.03) V
The structural similarity of the electrode material and the specific adsorption, apparently, favor a more reversible reduction of chloramphenicol on the SU electrode than on the RPE. The lower boundary of the determined contents of chloramphenicol when using RPE and SU electrodes is 2-3 orders of magnitude lower than on the river e. , and is 2.8 and 3.4 μg / kg, respectively. The peaks obtained using such electrodes are characterized by good reproducibility, in addition, the RPE is less toxic than r.ke., and SU is not toxic. Both electrodes are convenient in practical work. To determine the chloramphenicol RPE and SU have not been previously used.

 Using the differential mode of recording voltammograms allows you to capture clear narrow peaks, which increases the resolution of the method and facilitates the automation of the electrode process. To determine chloramphenicol differential mode has not been previously used.

Возможны также конформационные изменения белковых макромолекул на поверхности [Тарасович М.Р., Радюшкина К. А. , Богдановская В.А. Электрохимия порфиринов. - М.: Наука, 1991. - 312 с.]. Поэтому присутствие белков затрудняет электродные процессы, снижает чувствительность, воспроизводимость и точность анализа. Для отделения белка и его распада на более простые составные части использовали кислотный гидролиз 0,10-0,12 моль/дм³ НС1 при температуре 25-30^oС в течение 10-15 минут при перемешивании или встряхивании (при анализе мясных продуктов). (В прототипе гидролиз не применяли). Концентрация кислоты, время гидролиза (10-15 минут) и температура (25-30^oС) подобраны экспериментально. Использование НСl с молярной концентрацией с <0,10 моль/дм³ ухудшает условия гидролиза, а при с > 0,15 моль/дм³ ухудшаются условия электрохимического определения из-за большого значения остаточного тока, связанного с выделением водорода на индикаторных электродах. Оптимальное время гидролиза (τ_г): 10-15 минут. При τ_г>15 мин снижается экспресспость анализа, а при τ_г<10 мин ухудшаются условия проведения гидролиза. Оптимальная температура гидролиза (t_г): 25-30^oС. При t_г< 25^oC усиливаются процессы соосаждения антибиотика с нерастворимыми белками, а при t_г>30^oC снижается устойчивость левомицетина в растворе, что приводит к уменьшению высоты пика и снижению точности определения.In the proposed method, the conditions for the preparation of samples of dairy products, eggs, meat and offal of slaughtered animals and birds are established. The constituent components of soluble proteins of food products are adsorbed on carbon and mercury electrodes and are involved in redox transformations. A layer of protein may form on the surface of the electrode.

Conformational changes of protein macromolecules on the surface are also possible [Tarasovich MR, Radyushkina K. A., Bogdanovskaya V.A. Electrochemistry of porphyrins. - M .: Nauka, 1991. - 312 p.]. Therefore, the presence of proteins complicates the electrode processes, reduces the sensitivity, reproducibility and accuracy of the analysis. To separate the protein and its breakdown into simpler components, acid hydrolysis of 0.10-0.12 mol / dm ³ HCl at a temperature of 25-30 ^o C for 10-15 minutes with stirring or shaking (when analyzing meat products) was used. (In the prototype, hydrolysis was not used). The acid concentration, hydrolysis time (10-15 minutes) and temperature (25-30 ^o C) are selected experimentally. The use of HCl with a molar concentration with <0.10 mol / dm ³ worsens the hydrolysis conditions, and with c> 0.15 mol / dm ^{3 the} conditions for electrochemical determination worsen due to the large value of the residual current associated with the evolution of hydrogen on the indicator electrodes. Optimum hydrolysis time (τ _g ): 10-15 minutes. When τ _r> 15 minutes reduced ExpressPay analysis, while for τ _r <10 min deteriorating conditions of the hydrolysis. The optimum hydrolysis temperature (t _g ): 25-30 ^o C. At t _g <25 ^o C, the antibiotic co-precipitates processes with insoluble proteins, and at t _g > 30 ^o C the stability of chloramphenicol in solution decreases, which leads to a decrease in peak height and decrease the accuracy of determination.

A distinctive feature is the established conditions for the deposition of residual soluble protein from the hydrolyzate (in the prototype, precipitation was not used). In the proposed method, the precipitant selected is the ammonium salt of sulfate (NH ₄ ) ₂ SO ₄ in an amount of 2.5-3.0 g with the addition of 3.0-5.0 cm ³ ethanol. The use of large amounts of salt (more than 3.0 g) and alcohol (more than 5.0 cm ³ ) increases the solubility of protein impurities and worsens the conditions for the actual electrochemical reaction on the indicator electrode. With the addition of salt of less than 2.5 g and a volume of alcohol of less than 3.0 cm ^3, precipitation conditions worsen. The protein precipitate may partially re-dissolve when the sample is standing or after the sample has been diluted with water or a background electrolyte solution. Salt and alcohol are added to the hydrolyzate, the pH of which lies in the range of 4.7-5.1, which corresponds to the isoelectric point of the residual amounts of soluble protein impurities. At these pH values, the solubility of proteins is reduced. At 5.1 <pH <4.7, the solubility of residual protein impurities increases, which affects the conductivity of the electrode process.

 After separating the precipitate by centrifugation for 10-15 minutes at a rotation speed of 6000 rpm, the hydrolyzate was filtered through a paper filter, followed by differential voltammetric determination of chloramphenicol in an aliquot of the hydrolyzate. The centrifugation time and rotation speed are established experimentally. Using a centrifugation time of more than 15 minutes reduces expressivity. Using a rotation speed of more than 6000 rpm leads to a partial loss of the centrifuge, and less than 6000 rpm (and time less than 15 minutes) is not enough to completely separate the precipitate.

 Voltammograms of chloramphenicol are recorded at the maximum potential values of - (0.67 ± 0.05) V and - (0.60 ± 0.03) V, respectively, on the mercury-film and glassy carbon electrodes.

 When determining chloramphenicol in drugs (eye drops, tablets, powders), no additional separation of the accompanying substances was required. After transferring the sample to the solution, differential voltammetric determination of the drug was carried out under the established optimal conditions for the electrode process.

 The method of voltammetry for the determination of chloramphenicol in foods and drugs has not previously been used.

 The mass fraction of the antibiotic in the sample is determined by the method of additives of certified mixtures according to the generally accepted method.

 The established analysis conditions in the proposed method for the first time allowed expressly (in 40-60 minutes) quantitative determination of chloramphenicol in food products (table 1) and drugs (table 2) without preliminary separation of water-soluble vitamins of groups B, PP, ascorbic, folic, nicotinic, lemon , uric acids, urea, cystine, cysteine, anions and heavy metal ions.

 Example 1. Determination of chloramphenicol (chloramphenicol) in milk and dairy products.

25.0 cm ^{2 of} milk (or a dairy product weighed to the nearest 0.01 g) and 25.0 cm ² 0.1 mol / dm ³ HCl are introduced into a conical flask with a capacity of 100.0 cm ³ . The mixture is shaken vigorously for 10 minutes. Then 2.5-3.0 g of (NH ₄ ) ₂ SO ₄ and 3.0-5.0 cm ^{3 of} ethanol are added to the mixture. Shake the contents of the flask vigorously again for 5 minutes. The resulting mixture was transferred to centrifuge tubes and centrifuged for 10 minutes at a speed of 6000 rpm. The centrifugate is filtered through a paper filter into a conical flask with a capacity of 100.0 cm ³ , C ₆ H ₁₄ O ₇ N _{2 is} added to pH 4-5 and stirred until complete dissolution. An aliquot of the filtrate with a volume of 10.0 cm ^{3 is} placed in the electrolyzer and voltammetric measurements are carried out under the conditions: electrolysis potential E _e = -0.45 V, electrolysis time τ _e = 30 s, potential sweep speed w = 10 mV / s. The cathode peak of chloramphenicol is recorded using a mercury-film electrode in the potential range of (0.67 ± 0.05) V or a glassy carbon indicator electrode in the potential range of (0.60 ± 0.03) V with a device sensitivity of 1 • 10 ^-9 - 5 • 10 ^-10 A / mm in the differential mode of shooting voltammograms. The antibiotic content is evaluated by the method of additives of certified mixtures. The analysis time for one sample does not exceed 60 minutes.

 Example 2. Determination of chloramphenicol (chloramphenicol) in meat and offal of slaughtered animals.

5.0-10.0 g of finely ground meat, weighed to the nearest 0.01 g, is added to a conical flask with a capacity of 100.0 cm ³ , 20.0 cm ² 0.1 mol / dm ³ Hcl and 5.0 cm are added ³ ethyl alcohol. The mixture is shaken for 10-15 minutes, transferred to centrifuge tubes and centrifuged for 15 minutes at a speed of 6000 rpm. The centrifugate is filtered through a paper filter into a conical flask with a capacity of 100.0 cm ³ and add 3.0 g (NH ₄ ) ₂ SO ₄ . The contents of the flask are shaken for 10-15 minutes. The resulting mixture was transferred to centrifuge tubes and centrifuged again for 15 minutes at a rotation speed of 6000 rpm. The centrifuge is filtered through a paper filter into a conical flask with a capacity of 50.0 cm ³ . For analysis, take an aliquot of the obtained filtrate with a volume of 1.0-5.0 cm ³ , bring it into a quartz glass with a capacity of 15.0-25.0 cm ³ using a pipette and bring with double-distilled water to a volume of 10.0 cm ³ . Then voltammetric measurements are carried out under the conditions: electrolysis potential E _e = -0.45 V, electrolysis time τ _e = 30 s, potential sweep speed w = 20 mV / s. The cathode peak of chloramphenicol is recorded in the potential range - (0.67 ± 0.05) V or - (0.60 ± 0.03) V, respectively, on a mercury-film or glassy carbon electrode with a sensitivity of 1 • 10 ^-9 - 5 • 10 ^-10 A / mm in differential mode for shooting voltammograms. The antibiotic content is evaluated by the method of additives of certified mixtures. The analysis time for one sample does not exceed 60-80 minutes.

 Example 3. Determination of chloramphenicol (chloramphenicol) in a bird's egg.

In a conical flask with a capacity of 100.0 cm ^3, add 5.0-10.0 g of pre-beaten to a homogeneous egg mass, weighed to the nearest 0.01 g, add 5.0 cm ^{3 of} ethanol and 20.0 cm ³ 0.1 mol / dm ³ Hcl. The mixture is gently mixed by shaking for 10-15 minutes, transferred to centrifuge tubes and centrifuged for 15 minutes at a speed of 6000 rpm. The centrifugate is carefully poured into a flask with a capacity of 50.0 cm ³ , add 3.0 g of (NH ₄ ) ₂ SO _{4 in} small portions until the salt is completely dissolved, until egg jelly is formed. The resulting jelly is shaken vigorously until a homogeneous liquid solution is formed, transferred to centrifuge tubes and centrifuged for 15 minutes at a rotation speed of 6000 rpm. The centrifuge is filtered through a paper filter into a conical flask with a capacity of 50.0 cm ³ . For analysis, take an aliquot of the obtained filtrate samples with a volume of 1.0-5.0 cm ³ , bring into a quartz glass with a capacity of 15.0-25.0 cm ³ using a pipette and bring with double-distilled water to a volume of 10.0 cm ³ . Then voltammetric measurements are carried out under the conditions: electrolysis potential E _e = -0.45 V, electrolysis time τ _e = 30 s, potential sweep speed w = 20 mV / s. The cathode peak of chloramphenicol is recorded in the potential range - (0.67 ± 0.05) V on a mercury-film electrode with a device sensitivity of 1 • 10 ^-9 - 5 • 10 ^-10 A / mm in the differential mode of shooting voltammograms. The antibiotic content is evaluated by the method of additives of certified mixtures. The analysis time for one sample does not exceed 70 minutes.

 Example 4. Determination of chloramphenicol (chloramphenicol) in eye drops and tablets.

A sample of eye drops with a volume of 0.2 cm ^{3 is} introduced into a quartz glass with a capacity of 15.0 ÷ 25.0 cm ³ , diluted with bidistilled water to 10 cm ³ . For analysis, take an aliquot of the resulting solution with a volume of 0.2 cm ³ , add it to another polarographically clean quartz glass with a capacity of 15.0 ÷ 25.0 cm ³ and adjust the background electrolyte solution with 0.1 mol / dm ³ (NH ₄ ) ₂ SO ₄ to 10.0 cm ³ and voltammetric measurements are carried out under conditions: electrolysis potential E _e = -0.45 V, electrolysis time τ _e = 30 s, potential sweep speed w = 25 mV / s. The cathode peak of chloramphenicol is recorded in the potential range - (0.67 ± 0.05) V on the mercury-film electrode with the sensitivity of the device (1-5) • 10 ^-8 A / mm in the differential mode of shooting voltammograms. The antibiotic content is evaluated by the method of additives of certified mixtures. The analysis time for one sample does not exceed 10 minutes.

In the analysis of tablets of chloramphenicol, 5 tablets are ground in a mortar, 0.09 g of sample weighed, weighed to the nearest 0.001 g, taken into a volumetric flask with a capacity of 100.0 cm ³ , the sample is dissolved in 5.0 cm ^{3 of} ethanol and adjusted to double mark with bidistilled water . 0.2 cm ^{3 of the} resulting solution is introduced into a quartz glass with a capacity of 15.0-25.0 cm ³ and bidistilled water up to 6.0 cm ^{3 is} added. For analysis, an aliquot of 0.3 cm ^{3 is} taken, the background electrolyte solution is adjusted to 0.1 mol / dm ³ (NH ₄ ) ₂ SO ₄ to 10.0 cm ^3, and then voltammetric measurements are carried out as described in Example 4 above.

 According to the proposed method, metrological certification of the method of quantitative chemical analysis of samples of food products and drugs for the content of mass concentrations of chloramphenicol (chloramphenicol) by differential voltammetry was carried out.

 Metrological certification was carried out in order to establish the attributed characteristics of the error in the results of the analysis, as well as for the appointment of standards for accuracy control. Metrological studies and certification of this method were carried out by the accredited metrological service of the research laboratory of Tomsk Polytechnic University according to the requirements of GOST R 8.563-96 "Measurement Methods" and MI 2336-95 "Characteristics of the error in the results of quantitative chemical analysis. Algorithms for evaluation."

 The characteristic of the random component of the error (reproducibility index) is estimated from the results of analyzes of various objects. From each object, 14 samples were analyzed in two parallel samples. At the same time, the conditions for analysis (time, instruments, temperature, operators, etc.) were varied to take into account all possible influencing factors. The characteristic of the random component of the error in the range of determined mass concentrations of chloramphenicol 3.0-30.0 μg / kg is in the range from 7 to 14 rel.%.

 Evaluation of the characteristics of the systematic component of the error (correctness indicator) was carried out according to MI 2336-95 using control samples and the method of adding a certain substance to the sample. Samples for control according to the first algorithm were prepared from real working samples of the analyzed objects in which the antibiotic was absent, with the addition of a defined substance. Additives of chloramphenicol in the samples were made from pure reagents obtained according to the pharmacopoeial articles of the Ministry of Health of Russia to the stage of sample preparation.

 The characteristic of the total error was estimated by the values of the characteristics of the random and systematic component.

 Thus, a comparison of the characteristics of CHA of chloramphenicol in food products and pharmaceuticals by the proposed method significantly improved the metrological characteristics of the analysis. Significantly increased expressivity. The analysis of pharmaceuticals was reduced by 3 times, the proposed method allows expressly for 1-1.5 hours to determine chloramphenicol in foods. The conditions used in the prototype do not allow the analysis of food products.

 The proposed method is simple, does not require large labor costs, a large number of reagents and can be applied in any chemical laboratory, especially at the present time, when the production of modern computerized analyzers such as STA and TA has been launched. The proposed method can be used to determine the antibiotic in biosystems (blood, urine, etc.) in pharmacokinetic studies, in the toxicological and technical analysis of drugs, in animal feed, and can also serve as the basis for creating a diagnostic system for antibiotic metabolism.

Claims (1)

The method of quantitative determination of chloramphenicol by differential voltammetry, which consists in the fact that chloramphenicol is transferred from the sample to the solution, acid hydrolysis is carried out and the protein is precipitated from the hydrolyzate, followed by voltammetric determination, characterized in that the voltammetric determination of chloramphenicol is carried out by recording the cathodic peaks of the antibiotic film or glassy carbon electrodes in the differential mode of shooting voltammograms with the corresponding their potentials - (0,67 ± 0,05) and B - (0,60 ± 0,03) in respect to saturated silver chloride electrode on the backgrounds of 0.1 mol / dm ³ Ammonium citrate dibasic (pH = 4,7-5, 1) or 0.1 mol / dm ³ (NH ₄ ) ₂ SO ₄ with the addition of HCl to pH 5.1 at a potential sweep speed of 10-25 mV / s and the concentration of chloramphenicol is determined by peak height by the method of additives of certified mixtures.

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