RU2469304C1 - Ion-selective electrode membrane for determining cephalosporin antibiotics in medicinal and biological media - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: described is composition of an ion-selective electrode membrane for ionometric determination of cephalosporin antibiotics in medicinal and biological media, containing polyvinyl chloride as a matrix, dibutyl phthalate as a plasticiser and an electrode-active compound, wherein the electrode-active compound used is cefuroxime - dimethyl distearylammonium, with the following ratio of components: polyvinyl chloride 24.31-23.87%, dibutyl phthalate 72.94-71.36%, electrode-active compound 2.74-4.5%.

EFFECT: shorter time for determination owing to use of one ion-selective electrode which is sensitive to the whole group of cephalosporin antibiotics, lower detection limit when determining concentration of cephalosporins in biological media, reduced error of determining the result.

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Description

The invention relates to the field of analytical chemistry and can be used in control and analytical, clinical laboratories to determine the concentration of cephalosporin antibiotics (cefazolin, cefotaxime, ceftriaxone, cefuroxime, cefalexin, cefixime, etc.) in the studied liquid media for standardization and quality control of drugs , to determine antibiotics in biosystems (blood serum, oral fluid, urine, etc.) with the aim of studying pharmacodynamics, drug monitoring to regulate the introduction of optimal doses of antibiotics in the treatment of various infectious diseases.

The inventive device can also be used to determine antibiotics in food products, wastewater of pharmaceutical industries.

A pressing public health issue is the quality control of manufactured medicines. The effectiveness of quality control of drugs, as well as their comprehensive study is ensured by the introduction of modern physicochemical methods into the practice of pharmaceutical production, among which potentiometry with ion-selective electrodes occupies an important place.

The objects of this study are cephalosporin antibiotics of various generations used to treat infectious and somatic pathologies - urinary tract infections, upper and lower respiratory tract infections, etc.

To determine cephalosporins: (cefazolin), it was proposed in the literature to use mass spectrometry (plasma and urine), liquid chromatography (biomedia), spectrofluorimetry (cephalexin in blood plasma, urine), diffusion into agar (cefotaxime in serum and near-orange tissues), ion chromatography - ceftriaxone (in the blood and tissues of healthy rats), high performance liquid chromatography (HPLC) (blood serum, urine) [Kulapina EG, Barinova OV, Kulapina OI et al. Modern methods for determining antibiotics in biological and medicinal media. // Antibiotics and chemotherapy, 2009. - V. 54. - No. 9-10. - S.53].

Many of these methods require expensive equipment, reagents, highly qualified operators, which are of a different duration and are not applicable in clinical and biochemical laboratories for express control over the content of antibiotics.

To determine 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 (EAS). The method is characterized by expressivity, selectivity, simplicity and accessibility of equipment.

To determine cephalosporin antibiotics, the most effective class of electrode-active compounds (EAS) are anion exchangers.

It is known to use benzylpenicillin tetradecylammonium compounds (in electrodes for determining penicillin antibiotics), cefazolinetetradecylammonium, cefotaximtetradecylammonium (in electrodes for determining cefazolin and cefotaxime, respectively) as EAS membranes [Kulapina V.G. Rapid determination of β-lactam antibiotics in biological media using potentiometric sensors. // Antibiotics and chemotherapy, 2007. - No. 9-10, S.14-18].

However, the use of different EAS in the membranes of potentiometric sensors is time-consuming: for almost every membrane, it is necessary to synthesize its own electrode-active compound (EAS), obtain a membrane based on it and produce an ion-selective electrode to determine each specific antibiotic. In addition, multiple blood sampling from a vein of practically healthy individuals and patients at certain intervals is very traumatic. Determination of cephalosporin antibiotics in mixed saliva (oral fluid (ORF)) also requires the development of an effective membrane for determining antibiotic concentrations.

The well-known "Membrane composition of the ion-selective electrode for the determination of diphenhydramine" (AS USSR No. 1749813, G01N 27/333), including polyvinyl chloride as a matrix, dibutyl phthalate as a plasticizer and an electrode-active compound. Moreover, in order to ensure direct potentiometric determination of diphenhydramine in aqueous solutions, the diphenhydramine salt with the anion of 12-molybdophosphoric heteropoly acid was used as the electrode-active compound in the following ratio of membrane components, wt.%: Polyvinyl chloride 53.05-51.68; dibutyl phthalate 41.64-40.57; the salt of diphenhydramine with the anion of 12-molybdophosphoric heteroacid the rest.

The composition of the membrane and the arrangement of electrodes for determining one of the antibiotics of the cephalosporin group, namely, cefuroxime in medicinal media, described in the article by the authors, are also known.

The membrane was made using cefuroximetetraoctylammonium as an EAS, 2-nitrophenyl octyl ether as a plasticizer, and 4-tertoctylphenyl as an additive.

The disadvantage of this membrane is the narrow range of membrane applications - for determining only one type of antibiotic of the cephalosporin group (cefuroxime) and only in medicinal media. In addition, a high detection limit of cefuroxime, namely, 2.8 × 10 ^-4 M, is characteristic, which does not allow the use of an electrode to determine the antibiotic in biological media, which contain lower concentrations of the antibiotic.

The objective of the invention is the creation of an ion-selective electrode membrane (ISE) for the rapid determination of cephalosporin antibiotics, for example cefuroxime, cephalexin, ceftriaxone, cefixime in medicinal and biological media (including oral fluid) and the creation of an ion-selective electrode sensitive to the entire group of cephalosporin antibiotics, for their quantitative determination in both medicinal and biological environments (including ZhRP).

The technical result is to reduce the determination time by using one ion-selective electrode that is sensitive to the entire group of cephalosporin antibiotics, as well as by reducing the detection limit when determining the concentration of cephalosporins in biological media, reducing the error in determining the result.

The invention is illustrated by drawings.

Figure 1 shows the design of the liquid contact electrode, where the positions indicated:

1 - down conductor;

2 - selective membrane;

3 - polyvinyl chloride tube;

4 - internal reference electrode;

5 - internal comparison solution: 10 ^-3 M solution of cefuroxime + 10 ^-1 M NaCl.

Figure 2 shows the dependence of the EMF (mV) on the negative logarithm of the concentration of cephalosporins (pC): 1 - cefazolin, 2 - cefuroxime, 3 - ceftriaxone, 4 - cephalexin EAS cefuroxime-dimethyldistearylammonium (Cefur - DMDSA).

Figure 3 - the dependence of the EMF (mV) from the negative logarithm of the concentration of cefixime (pC).

Figure 4 - Dependence of EMF (mV) on the negative logarithm of the concentration of cefuroxime (pC) for mixed saliva.

The problem is solved in that, as part of the membrane of the ion-selective electrode for ionometric determination of cephalosporin antibiotics in medicinal and biological media, including polyvinyl chloride as a matrix, dibutyl phthalate as a plasticizer and an electrode-active compound, according to the proposed solution, cefistroximamide dimethylamide is used as an electrode-active compound the following ratio of the component:

Polyvinyl chloride 24.31-23.87%, Dibutyl phthalate 72.94-71.36%, Electrode active compound 2.74-4.5%.

As a rule, standard and indicator ion-selective electrodes are used to measure EMF. The design of the indicator ISE contains an electrode housing, which is a polyvinyl chloride tube, an internal reference electrode, which is used as a silver wire coated with a layer of silver chloride, an internal reference solution and a membrane.

The inventive membrane is an elastic film containing a powder of polyvinyl chloride, previously dissolved in cyclohexanone, plasticized with dibutyl phthalate, and EAS, namely one of its classes - anion exchanger - ionic associate of cefuroxime-dimethyldistearylammonium. To ensure contact between the membrane and the internal reference electrode, a solution of sodium chloride and any antibiotic of the cephalosporin group are placed inside the housing.

The choice of the optimal composition of the membrane was carried out through experimental research of the properties of its components when measuring the content of cefuroxime.

The implementation of the membrane using a combination of cefuroxime with dimethyldistearylammonium as the electrode-active compound (EAS) ensures the versatility of the electrode, which can be used to determine not just one species, but the entire group of cephalosporin antibiotics. The choice of cefuroxime as an indicator, and not any other cephalosporin antibiotic, is due to the fact that only cefuroxime is administered both intramuscularly and orally, that is, it can be used to determine antibiotics in the oral cavity, avoiding injections and excessive trauma.

The combination of cefuroxime with dimethyldistearylammonium prevents the leaching of cefuroxime and ensures the stability of the whole compound, its low solubility, which is confirmed by experimental studies and a comparison of the properties of anion exchangers with other tetraalkylammonium salts (see Table 1).

Table 1 EAS K _s Linearity, M Detection limit, M Angular coefficient Cefuroxime + dimethyldistearylammonium (3.5 ± 0.4) · 10 ^-8 1 · 10 ^-4 -1 · 10 ^-1 4.8 · 10 ^-5 54-55 Cefuroxime + benzyldimethyltetradecylammonium (4.6 ± 0.4) · 10 ^-5 1 · 10 ^-3 -1 · 10 ^-1 6.910 ^-4 51 Cefuroxime + benzyldimethyldodecylammonium (1.44 ± 0.2) · 10 ^-4 1 · 10 ^-2 -1 · 10 ^-1 6.3 · 10 ^-3 47 Where K _s is the solubility product of anion exchangers.

As a result of experiments and studying the properties of EAS components, it was concluded that when using, for example, benzyl dimethyl teradecylammonium as an EAS, the results are much worse both in terms of solubility product and detection limit.

Examples of membranes with different concentrations of EAS:

EAS c = 2.75%.

2 ml of a solution of Cefur C = 1.5 · 10 ^-2 M and 4 ml of dimethyldystearylammonium (DMDSA) C = 10 ^-2 were placed in a separatory funnel. The mixture was shaken for 2 hours, the chloroform layer was separated from the aqueous phase in a pre-weighed bucks and chloroform was evaporated in a water bath at a temperature of 50-60 ° C in order to avoid decomposition of the electrode-active substance.

Then made a membrane with an electrode-active substance according to the following technology.

0.7966 g or 72.94% of dibutyl phthalate, 2 ml of cyclohexanone were placed in a 10 ml bottle, and 0.03 g or 2.75% of the cephalosporin-dimethyldisteramylammonium compound were added with constant stirring on a magnetic stirrer with slight heating. 0.2655 g or 24.31% polyvinyl chloride. Stirring was continued until the mixture was completely homogenized. The membrane composition was poured into a Petri dish with a diameter of 67 cm and left in the air until complete removal of cyclohexanone (membrane weight 1.0921 g)

Dibutyl phthalate 72.94% Polyvinyl chloride 24.31% EAS 2.75%

EAS c = 3.63%.

4 ml of a 1.5 · 10 ^-2 M solution of cefuroxime and 8 ml of 10 ^-2 M dimethyldystearylammonium (DMDSA) C = 10 ^-2 M were placed in a separatory funnel, the mixture was shaken for two hours, the chloroform layer was separated from the aqueous phase into a pre-weighed bucks, chloroform was evaporated in a water bath at a temperature of 50-60 ° C.

Then made a membrane with an electrode-active compound (EAS) according to the following technology:

0.7966 g or 72.28% of dibutyl phthalate, 2 ml of cyclohexanone were placed in a 10 ml bottle, and 0.04 g or 3.63% of the cephalosporin-dimethyldystearylammonium compound was added with constant stirring on a magnetic stirrer with slight heating. and 0.2655 g or 24.09% polyvinyl chloride. Stirring was continued until the mixture was completely homogenized. The membrane composition was poured into a Petri dish with a diameter of 67 mm and left in air until cyclohexanone was completely removed. (Membrane mass 1.1021 g).

Dibutyl phthalate 72.28% Polyvinyl chloride 24.09% EAS 3.63%.

EAS c = 4.5%:

4 ml of a solution of Cefur C = 1.5 · 10 ^-2 M and 8 ml of DMDSA C = 10 ^-2 M were placed in a separatory funnel, the mixture was shaken for 2 hours, the chloroform layer was separated from the aqueous phase in a pre-weighed bucks and evaporated chloroform in a water bath at a temperature of 50-60 ° C in order to avoid decomposition of the electrode-active substance.

Then made a membrane with an electrode-active substance according to the following technology.

0.7966 g or 71.63% of dibutyl phthalate, 2 ml of cyclohexanone were placed in a 10 ml bottle, and 0.05 g or 4.5% of the cephalosporin-dimethyldystearylammonium compound was added with constant stirring on a magnetic stirrer with slight heating at 50-60 ° C and 0.2655 g or 23.87% polyvinyl chloride. Stirring was continued until the mixture was completely homogenized. The membrane composition was poured into a Petri dish with a diameter of 67 cm and left in the air until cyclohexanone was completely removed (membrane weight 1.1221).

Dibutyl phthalate 71.63% Polyvinyl chloride 23.87% EAS 4,5%

The obtained membrane composition was used for the manufacture of liquid-contact electrodes: disks with a diameter of 7 mm were cut out and glued to a cleaned and polished polyvinyl chloride case with glue containing 0.5 g of polyvinyl chloride, 0.25 g of dibutyl phthalate, and 5 ml of cyclohexanone. After the glue has dried, 1 ml of a standard 1 · 10 ^-1 M solution of sodium chloride and 1 ml of a 1 · 10 ^-3 M solution of the corresponding antibiotic are poured into the tube (volume ratio 1: 1). The electrodes made in this way were conditioned for 1 day in a 1 · 10 ^-3 M solution of the corresponding antibiotic.

An ionometric measurement of the concentration of antibiotics of the cephalosporin group uses an indicator electrode, which is placed in an antibiotic solution, and then calibrates by measuring the electrode potentials (EMF) of an electrochemical circuit composed of an indicator ISE and a standard silver chloride electrode using antibiotic solutions with a known concentration (standard), for example, cefuroxime.

The dependence of the EMF value on the concentration of cephalosporin solutions was measured at a pH of 6-7.

Figure 2 presents a graph of the dependence of the EMF (mV) on the negative logarithm of the concentration of cephalosporins (pC): 1 - cefazolin, 2 - cefuroxime, 3 - ceftriaxone, 4 - cefalexin) EAS cefuroxime-dimethyldystearylammonium (Cefur - DMDSA).

Moreover, anionic functions are performed in the concentration range of cephalosporins 1 * 10 ^-1 -1 * 10 ^-4 (1 * 10 ^-5 ) M with angular coefficients of 59 ± 2 mV / pC for cefazolin, cephalexin, cefuroxime, cefotaxime, cefixime and 29 ± 3 mV / pC for ceftriaxone (disodium salt containing a doubly charged anion). These indicators indicate that the electrode responds to cephalosporin antibiotics in accordance with the Nernst equation underlying the potentiometric method.

The sensitivity (selectivity) of the electrode to cephalosporin antibiotics and inorganic anions that are part of biological media (oral fluid, blood plasma) is illustrated in Table 2, which shows potentiometric selectivity coefficients: the main ion is cefuroxime, the interfering ions are cefazolin (Cef), cephalexin (Cefl), inorganic ions. EAS Cefur - DMDSA.

table 2 Interfering ion To _sat Cl ^- (6.61 ± 0.25) · 10 ^-2 Br ^- (8.92 ± 0.10) 10 ^-2 NSO ₃ (6.31 ± 0.15) 10 ^-2 H ₂ RO ₄ ^- (7.94 ± 0.21) 10 ^-2 NRA ₄ ^2- (6.31 ± 0.20) 10 ^-2 J ^- (8.32 ± 0.15) 10 ^-2 Cef (1.78 ± 0.10) 10 ^-1 Cefl (6.63 ± 0.20) 10 ^-1

The data in Table 2 indicate the possibility of using an electrode based on the selected EAS for determining cephalosporin antibiotics in biological media.

An example of the use of an electrode with the claimed membrane in medicinal media

The most revealing example is the determination of cephalosporin antibiotics by the example of the determination of cefixime in suspensions in order to establish its shelf life.

To determine cefixime, which is used as a suspension in the treatment of children, an electrode with an EAS of cefuroxime + dimethyldystearylammonium was also used.

A portion weighing 2.835 g of dry cefixime was dissolved in a 25 ml volumetric flask (antibiotic concentration 1 · 10 ^-2 M). Solutions of 1 · 10 ^-3 -1 · 10 ^-5 M were prepared from 1 · 10 ^-2 M solution by successive dilution in volumetric flasks with a capacity of 25 ml. The EMF was measured with indicator and silver chloride electrodes.

Figure 3 shows the dependence of the EMF (mV) on the negative logarithm of the concentration of cefixime (pC).

Determination of cefixime in suspension (SUPRAX). To prepare three samples of different concentrations, 1 was taken; 1.5 and 2 ml of suspension and dissolved in 25 ml volumetric flasks. The EMF was measured with the indicator and silver chloride electrodes, and the concentration of cefixime in the suspension was determined by the dependence of the EMF (mV) on the negative logarithm of the concentration of cefixime (pC). The calculation of the content of cefixime in suspension was carried out according to the formula:

m=C·M,

m = C

where C _x is the concentration of cefixime found according to the calibration graph, M;

V _al - aliquot volume, ml;

M is the molar mass of cefixime, 453.5 g / mol;

m is the calculated mass of cefixime, g.

Thus, the use of cefuroxime + dimethyldystearylammonium as an EAS allows one to determine the main substance in SUPRAX medicines used in the form of a suspension, which is especially important because of the limited shelf life of the suspension.

An example of the use of an electrode with the inventive membrane in the oral fluid

The choice of the optimal membrane composition was carried out by means of an experimental study of the properties of its components when measuring the content of cefuroxime in the oral fluid (LRA) of patients with infectious and somatic pathology.

When using electrodes for the determination of cefuroxime in an RRP, the indicator electrode was preconditioned in the RRR of an almost healthy person for 20-30 minutes. To prepare a 10 ^-2 M solution of cefuroxime, a weighed portion of 0.193 g was dissolved in distilled water in a 25 ml volumetric flask. Solutions of 10 ^-3 -10 ^-5 M were prepared by sequential dilution, 0.3 ml of aqueous cefuroxime solutions were taken, and 3 ml were diluted with ZhRP, mixed and the emf was measured with silver and silver chloride electrodes.

A sample of HRP from practically healthy individuals was taken 1-2 hours after eating, and the mouth cavity was rinsed with water before collection. Mixed saliva was centrifuged for 15 minutes at 3500 rpm. To prepare a series of solutions of antibiotics V = 3 ml against the background of high-fatty acid intake with added antibiotics at a concentration of 5 · 10 ^-5 , 1 · 10 ^-4 , 1 · 10 ^-3 M (taking into account dilution), 2.7 ml of the supernatant were taken, transferred to cups for measurement and 0.3 ml of a solution of the corresponding antibiotic with a concentration of 5 · 10 ^-4 , 1 · 10 ^-3 , 1 · 10 ^-2 M (added additives) were added, mixed and EMF measurements were carried out.

Taking into account the dilution, we plotted the dependence of the emf, mV, on the negative logarithm of the concentration of the antibiotic.

The linearity interval of the electrode function was: 10 ^-3 -10 ^-4 M, and the detection limit of cefuroxime 5 · 10 ^-5 M. The obtained calibration characteristics are reproducible and the increase in conditioning time does not affect them.

A decrease in the interval of linearity and the angular coefficient of the electrode function in the ZhRP due to the high ionic strength of the solution and "protein poisoning" of the membrane surface has been established. The dependence of the EMF (mV) on the negative logarithm of the concentration of cefuroxime (pC) for mixed saliva is presented in Figure 4.

Similarly, the oral fluid was prepared for patients with infectious and somatic pathology, and the antibiotic concentration was determined according to the graph of the EMF (mV) versus the negative logarithm of the cefuroxime concentration (pC). The calculation of the content of cefuroxime in the ZhRP was performed according to the following formula:

C = C _xM 1000

where Cx is the concentration of the antibiotic found according to the calibration graph, M;

M is the molar mass of the antibiotic, 453.5 g / mol.

Given the complexity of the analyzed objects and the analyte itself, the reliability of the results obtained when choosing the optimal membrane composition was evaluated as follows. A certain additive of a standard solution of cefuroxime was introduced into a healthy person’s HRP sample and then the sample was carried out through all sample preparation operations. The relative error of determination did not exceed 4-6%.

Final conclusion

The values obtained potentiometric selectivity coefficients enabled to make a prediction about possibility of application developed ion-selective electrodes based on ion associate of cefuroxime Dimethyldistearylammonium for determining cefuroxime, cefazolin, ceftriaxone, cephalexin, cefixime in the formulations and biological fluids at high concentrations of Cl ions, HCO _3, Br, H ₂ HO ₄ , NRA ₄ .

The resulting ISE membrane due to the properties of the selected EAS components studied as a result of the experiments, is characterized by low solubility of the EAS (solubility product is 3.5 + 0.4) 10 ^-8 ). This is of great importance, since the lower the solubility of the EAS, the higher the sensitivity of the electrode.

The resulting membrane provides a wide range of detectable antibiotic contents, namely 10 ^-4 -10 ^{-1 -1} M. Moreover, the high properties of the EAS used in the membrane revealed as a result of the experiments provide a low detection limit of the antibiotic, namely 4.8 × 10 ^-5 M. This allows the use of an electrode for analysis of an antibiotic not only in drugs where the concentration of the antibiotic is high, but also in biological media (including for studying the pharmacokinetics of cephalosporins), where the concentration of the antibiotic is pre slightly low.

As a result, the angular coefficient of electrode functions in cefuroxime solutions is 54-55 mV / pc.

Thus, the obtained ISE membrane expands the functionality of the rapid determination of antibiotics in dosage forms and biological fluids by expanding the spectrum of the studied objects - cephalosporin antibiotics. The resulting membrane provides rapidity, the ability to determine the active concentration of antibiotics in a wide concentration range, and makes it possible to determine any type of antibiotic of the cephalosporin group in patients with infectious and somatic pathology.

Due to the stability of the obtained EAS, a wide range of linearity of functions and a low detection limit of antibiotics are provided.

Claims (1)

The composition of the membrane of the ion-selective electrode for ionometric determination of cephalosporin antibiotics in medicinal and biological media, including polyvinyl chloride as a matrix, dibutyl phthalate as a plasticizer and an electrode-active compound, characterized in that cefuroxime-dimethyldisteramylammonium is used as the electrode-active compound in the following ratio %:
Polyvinyl chloride 24.31-23.87 Dibutyl phthalate 72.94-71.36 Electrode active compound 2.74-4.5

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