RU2789107C1 - Ion-selective electrode membrane for the determination of ceftriaxone in biosystems - Google Patents

Abstract

FIELD: instrumentation.

SUBSTANCE: invention relates to electrochemical methods of analysis and can be used in control and analytical laboratories, clinical laboratories to determine the concentration of ceftriaxone (Ceftr), an antibiotic of the cephalosporin series, in biosystems (blood serum, saliva, urine, etc.) in order to regulate the administration of optimal doses of the antibiotic in the treatment of various infectious diseases, as well as in the study of pharmacokinetics. Optimization of the membrane composition of the ion-selective electrode was carried out by changing the mass ratios of the membrane components. Essence: the composition of the membrane of the ion-selective electrode for the determination of Ceftr contains an electrode-active component (EAC), a plasticizer: dioctyl sebacinate (DOS), polyvinyl chloride (PVC), while an ion associate (Ceftr)₂⋅ODA is used as an EAC and composition has the following ratio of components: EAC-0.80, DOS-66.14, PVC-33.06.

EFFECT: reduction of the detection time, reduction of the detection limit of ceftriaxone in the presence of a number of anions and some antibiotics of the cephalosporin series, as well as an increase in the range of detectable concentrations with optimization of metrological characteristics.

1 cl, 9 dwg, 2 tbl

Description

To determine cephalosporins in the literature, it is proposed to use mass spectrometry (plasma, urine), liquid chromatography (biomedia), spectrofluorimetry (cephalexin in blood plasma). For the total determination of antibiotics, a piezoelectric immunosensor is proposed; the amount of polyclonal antibodies to tetracyclines and incubated for 2-3 min until the formation of the immunocomplex, injected into the cell for detection in a phosphate buffer solution with a pH of 7.1-7.2. The change in the frequency of the sensor vibration is recorded as a result of the interaction of unbound antibodies with the tetracycline-protein conjugate on the surface of the sensor electrodes. The analytical signal is inversely proportional to the total concentration of tetracyclines in the analyzed sample, the concentration is determined according to the calibration curve, the regeneration of the receptor coating is carried out by applying a 0.1 M solution of diethylamine hydrochloride to the surface. The invention provides a reduction in the detection limit of tetracyclines and an increase in the sensitivity of the determination with high reliability of the results, a decrease in the analysis time, an expansion of the range of linear determination, and measurements with the possibility of regenerating the sensor within 30 measurement cycles [RF patent No. 2687742 "Method for determining tetracyclines using a piezoelectric sensor" priority from 12/29/2017. Authors: Ermolaeva T.N., Farafonova O.V.].

Currently, methods are used to determine the tetracycline group: biological, microbiological.

The method is characterized by using as a test culture spores of Bacillus stearothermophilus strain KK-VKM B-2130D-obligate thermophilic spore-forming microorganism with an optimal growth temperature of 55-60°C, a facultative anaerobe highly sensitive to all widely used antibiotics, or spores of other thermophilic bacteria. The test material is titrated in enzyme immunoassay plates in a colored nutrient medium with an indicator. The spores of the test culture containing 107 spores/ml are added to the wells. Crops are incubated at 55-60°C. Extraneous microflora does not grow at this temperature. The absence of a change in the color of the medium indicates the presence of an antibiotic in the test material. Extraneous microflora does not grow at this temperature. The absence of a change in the color of the medium indicates the presence of an antibiotic in the test material. [Patent RF 2188421 Levi M.I., Suchkov Yu.G. "Method for the rapid determination of antibiotics by the biological method" priority dated 19.05.2000. Authors: Levi M.I., Suchkov Yu.G.] The method is unreliable, it does not provide a sufficiently high sensitivity of the determination.

Fagbamila I. Antimicrobial screening of commercial eggs and determination of tetracycline residues using two microbiological methods / I. Fagbamila, J. Kabir, P. Abdu, G. Omeiza, P. Ankeli, S. Ngulukun, M. Muhammad, J. Umoh / / International Journal of Poultry Science. - 2010. - V. 9 (10). - P. 959-962; Khismatoullin R. Modification of microbiological detection of tetracycline in honey / R. Khismatoullin, R. Kuzyaev, Ya. Lyapunov, E. Elovikova // APIACTA. - 2003. - V. 38. - P. 246-248; Kirbis A. Microbiological screening method for detection of aminoglycosides, b-lactames, macrolides, tetracyclines and quinolones in meat samples / A. Kirbis // Slov Vet Res. - 2007. - V. 44 (1/2). - P. 8-11], the disadvantage of such methods is low sensitivity (detection limit is 30-40 µg/kg), although these tests are considered fast, they take 3-24 hours to work in an incubator, bacterial strains must be constantly monitored, to make sure they don't become resistant to tetracyclines, the interpretation of test results is highly subjective and can lead to false negatives or positives. High performance liquid chromatography [Ahmadi F. Determination of tetracyclines in meat using two phases freezing extraction method and HPLC-DAD / Ahmadi F., Shahbazi Y., Karami N. // Food Analytical Methods. - 2015. - Volume 8, Issue 7, pp 1883-1891; Moats W.A. Rapid HPLC Determination of Tetracycline Antibiotics in Milk / W.A. Moats, R. Harik-Khan // J. Agric. food chem. - 1995. - V. 43 (4). - P. 931-934; Zhou J. Multiresidue determination of tetracycline antibiotics in propolis by using HPLC-UV detection with ultrasonic-assisted extraction and twostep solid phase extraction / J. Zhou, X. Xue, Yi Li, J. Zhang, F. Chen, L. Wu, L. Chen, J. Zhao // Food Chemistry. - 2009. - V. 115. - Is. 3. - P. 1074-1080; Moats W.A. Determination of tetracycline antibiotics in beef and pork tissues using ion-paired liquid chromatography / W.A. Moats // J. Agric Food Chem. - 2000. - V. 48(6). - P. 2244-2248; Pena A. Determination of tetracycline antibiotic residues in edible swine tissues by liguid chromatography with spectrofluorometric detection and confirmation by mass spectrometry / A. Pena, CM. Lino, R. Alonso, D. Barcelo // J. Agric. food chem. - 2007. - V. 55 (13). - P. 4973-4979; A.R. Shalaby Validation of HPLC method for determination of tetracycline residues in chicken meat and liver / A.R. Shalaby, Nadia A. Salama, S.H. Abou-Raya, Wafaa H. Emam, F.M. Mehaya // Food Chemistry. - V. 124, I. 4, 2011, P. 1660-1666]. Disadvantages: duration and rather complicated procedure of sample preparation using solid-phase extraction and the degree of product recovery of about 80%.

For the determination of cephalosporins: (cefazolin), it is proposed in the literature to use mass spectrometry (plasma and urine), liquid chromatography (biomedia), spectrofluorimetry (cephalexin in blood plasma, urine), agar diffusion (cefotaxime in serum and near-wound tissues), ion chromatography - ceftriaxone (in the blood and tissues of healthy rats), high performance liquid chromatography (HPLC) (blood serum, urine) [Kulapina E.G., Barinova O.V., Kulapina O.I. and other Modern methods for determining antibiotics in biological and medicinal environments. // Antibiotics and chemotherapy, 2009. - V.54. - No. 9-10. - S. 53]. Many of these methods require expensive equipment, reagents, highly skilled operators, differ in duration and are not applicable in clinical and biochemical laboratories for express control of antibiotic levels.

For the determination of antibiotics in pharmaceutical forms and biological fluids, the most promising is the use of potentiometric methods using various sensors, for example, ion-selective electrodes (ISE) with various types of electrode-active compounds (EAC). The method is characterized by rapidity, selectivity, simplicity, efficiency and availability of equipment.

Potentiometric sensors based on ionic associates of cefuroxime with tetraalkylammonium cations have been developed. The sensors provide a wide range of determined antibiotics content 1⋅10 ^-4 (8⋅10 ^-5 ) - 1⋅10 ^-2 M; the detection limit for antibiotics is 10 ^-5 (10 ^-6 ). (O.I. Kulapina, M.S. Mikhailova, E.G. Kulapina Ionometric determination of cefuroxime and cefuroxime axetil in biological and medicinal media // Proceedings of the Saratov University. Ser. Chemistry. Ecology. 2013. Vol. 15. issue 3. P. 40-45 The disadvantage of this membrane is a narrow range of detectable concentrations, a high detection limit of cefuroxime does not allow the use of an electrode for determining the antibiotic in biological media containing lower concentrations of cefuraxime, as well as a narrow range of its application.

There is also a method for determining cefazolin in biological media using ion-selective electrodes (O.I. Kulapina, V.V. Baraguzina, N.V. Skoblikova Determination of cefazolin in biological media using ion-selective electrodes // Chemical Pharmaceutical Journal. 2008. T. 42, No. 8. P. 41-44). The ratio of components in the ion associate, solubility, and thermal stability up to 70 degrees were evaluated. It is shown that the dependence of EMF on the concentration of cefazolin is in the range 1⋅10 ^-1 - 1⋅10 ^-5 mol; the slope of the electrode functions is 56±2 mV/decade. However, the selectivity coefficient for many antibiotics that have the same functional groups as cefazolin has not been determined.

In this paper ["Planning an experiment in optimizing the synthesis of vancomycin-selective "artificial receptors"" Author: E.N. Muzyka // Technical Science Scientific Journal "ScienceRise" No. 3/2 (3) 2014. UDC 66.DOI: 10.15587/2313 -8416.2014.27623] used the chemometric approach "experiment planning", implemented using the MODDE 9.0 application software package, to optimize the parameters of the synthesis of vancomycin-selective nanoparticles ("artificial receptors" based on molecularly imprinted polymers) in an automatic reactor in order to obtain their maximum quantitative exit.

The use of natural receptors in modern sensors, as a rule, provides satisfactory analytical characteristics, but their high cost and instability are a problem. The use of a technology known as molecular imprinting can solve or alleviate the above problems. Molecularly imprinted polymers (MIPs), or molecularly imprinted polymers, can be a low cost alternative to natural receptors. “Artificial receptors” (based on MIPs) are obtained as a result of molecular imprinting - copolymerization of functional and crosslinking monomers in the presence of template molecules or imprint molecules. To date, the most widely used method for the synthesis of MIPs is bulk polymerization.

Disadvantages: The resulting polymers have many limitations, including a high level of non-specific binding and poor site accessibility for template molecules, and hence are not used in commercial assays.

Closest to the proposed technical solution (prototype)

The composition of the membrane and the arrangement of electrodes for the determination of one of the antibiotics of the cephalosporin group, namely, cefuroxime in medicinal media, is described in the authors' article:

J.L.F.C Lima, M.C.B.S.M Montenegro, M.G.F Sales. Cefuroxime selective electrodes for batch and FIA determinations in pharmaceutical preparations. Journal of Pharmaceutical and Biomedical Analysis, V. 18, I. 1-2, 1998, P. 93-103, https://doi.org/l0.1016/S0731-7085(98)00162-9.

Various selective electrodes for the determination of cefuroxime without an internal reference solution containing PVC membranes have been designed and evaluated.

The membrane is made using cefuraxime bis(triphenylphosphoranylidene)ammonium and cefuroximetetraoctylammonium as an EAS, di-(2-ethylhexyl) ester of sebacic acid and 2-nitrophenyloctyl ether as a plasticizer, and 4-tertoctylphenyl as an additive. For these electrodes, the linear limit of the determined concentrations of cefuroxime was 2.8⋅10 ^-4 and 1.3⋅10 ^-4 M, the steepness was 50.4 mV/decade with a service life of about 4-5 months, an acidity of 3.5 pH was created by introducing a phosphate buffer solution. Relative standard deviation <3.0%.

The disadvantage of this membrane is the lack of study of the selectivity of antibiotics of the cephalosporin series, with identical functional-analytical groups as in cefuraxime to the cefuroxime-selective electrode. In addition, a high detection limit of cefuroxime is characteristic, namely 2.8×10 ^-4 and 1.3×10 ^-4 M, which does not allow the electrode to be used for determining the antibiotic in biological media containing lower concentrations of the antibiotic.

The objective of the invention is to create an ion-selective electrode (ISE) membrane for the rapid determination of a cephalosporin antibiotic, in particular ceftriaxone in medicinal and biological media (including blood, urine, oral fluid) and to create a ceftriaxone-selective electrode based on the resulting membrane for determining ceftriaxone, given its use as the main antibiotic in the treatment of covid-19, as well as for the purpose of pharmacokinetic studies.

The technology for manufacturing a ceftriaxone-selective (Ceftr-SE) electrode was carried out as follows: an aqueous solution of ceftriaxone with a concentration of 1⋅10 ^-2 M in a volume of 50 ml was mixed with 100 ml of a chloroform solution of octadecylamine (ODA) of the same concentration. Then the solution was stirred at room temperature for 3-4 hours in a flask with a magnetic stirrer (Fig. 1), after which the chloroform layer was taken and evaporated (in a water bath at a temperature of 50-60°C) until the chloroform completely volatilized until the formation of white crystalline precipitate (Fig. 2). The precipitate was dissolved in ethanol and recrystallized by diluting with distilled water (Fig. 3). The purified precipitate (ionic associate) as an EAS (ionophore) was dissolved in dioctyl sebacenate (di-(2-ethylhexyl) ester of sebacic acid), which acts as a plasticizer, polyvinyl chloride (PVC), tetrahydrofuran was added there, and stirred until complete dissolution. Then, 3.8 ml of the resulting membrane composition was taken, poured into glass rings with a diameter of 22-28 mm, and thin membranes were obtained after complete evaporation of tetrahydrofuran (THF). To avoid rapid evaporation of the latter, the cups were covered with paper and glass on top (Fig. 4). The membranes were glued to the ends of polyvinyl chloride tubes by applying 13% PVC in cyclohexanone (CT) in three layers at 20 min intervals. (Fig. 5). Ceftr-SE was soaked in centimol solutions of ceftriaxone, pre-filled with centimol solutions of mixtures of Ceftr and KCI. The calibration of the developed electrode was carried out by placing the indicator and reference electrodes in pre-prepared Ceftr standard solutions of various concentrations (Fig. 6).

Regardless of the nature of the sensor, the potential arising on it obeys the Nernst equation:

where E ^o is the standard potential of the redox system; R is the universal gas constant equal to 8.312 J/mol×K; T - absolute temperature, K; z _i is the valency of the ion; F - Faraday's constant, equal to 96485 C/mol; ax is the activity of the oxidized and reduced forms of the redox system, respectively. The value 2.3RT/z _i F is called the steepness of the electrode function and is usually denoted as S; a _i \u003d c _i f (c _i - concentration of the ion being determined; f - ion activity coefficient).

Potentiometric studies were carried out using an 8-channel computerized system for collecting potentiometric data based on a PC, Ecotest-120, and a KM-8 switch (NPP ECONIKS, Moscow, Russia); (Fig. 7.) A Wirowka MPW-6 centrifuge was used to separate the protein components from the mixed saliva.

The technical result is a reduction in the time of determination, a decrease in the detection limit of ceftriaxone in the presence of a number of anions and some antibiotics of the cephalosporin series, as well as an increase in the range of determined concentrations with optimization of metrological characteristics.

This technical result is achieved by using the lipophilic octadecylamine cation (ODA) and the lipophilic ceftriaxone anion (Ceftr) as the electrode active compound (EAS). :2; dioctyl sebacate (DOS) served as a plasticizer, solvents: tetrahydrofuran and cyclohexanone (CH), with the following ratio of components in mass. %:

Ceftriaxone (Ceftr) is a third generation cephalosporin antibiotic. Commercial name of the drug "Ceftriaxone", manufacturing companies: 1) "Vertex Export", India; 2) OJSC "Biosintez", Moscow. Release form: powder for solution for injection, 1.0 g in glass vials.

(ODA) (Octadecylamine, analytical grade, TU 6-09-07-230-74, Vekton, St. Petersburg, Russia);

The ion associate was formed according to the scheme:

Octadecylamine was dissolved in chloroform and mixed with an aqueous solution of ceftriaxone with concentrations of 1⋅10 ^-2 M. This solution was transferred into a flask, the mixture was shaken on a magnetic stirrer for 3-4 hours. The resulting chloroform layer was separated from the aqueous phase into a pre-weighed bottle. Chloroform was evaporated in a water bath at a temperature of 50-60°C (to avoid decomposition of the electrode-active component) until a white crystalline precipitate formed. In order to obtain a pure precipitate, recrystallization was carried out by dissolving it in pure ethanol and diluting with distilled water. The purified ion associate was used as EAA to obtain membranes. Further, according to the above, film membranes of a ceftriaxone-selective electrode were obtained using dioctyl sebacate (DOS) as a plasticizer and tetrahydrofuran (THF) as a solvent. Below is the ion exchange equation:

The compositions of the studied membranes are presented in Table 1.

Further studies were carried out with the electrode composition of the membrane masses. %: (ODA) ₂ ⁺ ⋅Ceftr ^2- (0.80), DOS (66.14), PVC (33.06) and slope of the electrode function, as close as possible to the theoretical value for a doubly charged ion of 24.9 mV/dec.

The high lipophilicity of the ionophore of the PVC-plasticized membrane limits its release into solution, which contributes to the completeness of the electrode function, as well as to the long life of the ISE. The lipophilicity value was calculated using the ACD/ChemSketch program, which corresponds to 8.4±0.2 for octadecylamine.

The selectivity of the ceftriaxone-selective electrode to some anions and antibiotics of the cephalosporin series was studied. The selectivity of Ceftr-SE based on the ion associate to the most important inorganic cations: K ⁺ , Na ⁺ , Ca ²⁺ showed that these cations and some antibiotics of the cephalosporin series (cefazolin, cefuroxime, cephalexin) do not interfere with the determination of ceftriaxone, and among the anions, iodides, the other anions had no interfering effect.

The resulting membrane provides a wide range of detectable antibiotic concentrations, namely 1⋅10 ^-1 - 1⋅10 ^-5 M. At the same time, the high properties of the EAS used in the membrane, revealed as a result of the experiments, provide a low detection limit of ceftriaxone, namely 8.3⋅10 ^-6 M. This allows the electrode to be used both for the determination of ceftriaxone in biological fluids, where the concentration of the determined is very low, and for the analysis of drugs based on ceftriaxone, as well as for studying the pharmacokinetics of the antibiotic. As a result, the slope of electrode functions in ceftriaxone solutions is 24.9 mV/dec (Fig. 8.).

The thus obtained ISE membrane expands the functionality of the express determination of ceftriaxone in biological fluids, in dosage forms due to a wide range of determined concentrations and the achievement of a constant potential value within one minute.

The dependence of the membrane potential Ceftr - SE on the acidity of the solution was established (Fig. 9.).

At pH 6-9, the potential Ceftr - SE has a maximum solubility and this interval is taken as the pH of the electrode functioning. The main electrochemical characteristics (slope of the electrode function, linear range, limit of detection and response time) are presented in Table 2.

Claims (1)

The composition of the membrane of an ion-selective electrode for the ionometric determination of ceftriaxone in biological fluids, including an electrode-active compound, characterized in that the ion associate lipophilic octadecylammonium cation and lipophilic anion of ceftriaxone with a ratio of 2:1, (ODA) ₂ ⁺ ⋅Ceftr ^2- and in wt.% (ODA) ₂ ⁺ ⋅Ceftr ^2- (0.80), DOS (66.14), PVC (33.06).

Images