The method of quantitative determination of azithromycin dihydrate method stripping voltammetry
The invention relates to the field of analytical chemistry and relates to the quantitative determination of antibiotic groups, macrolide azithromycin dihydrate (azithromycin). The essence of the method: azithromycin is transferred from the sample in the solution and hold voltammetric determination, which shall accumulation of the antibiotic in the mixed solution for 30-60 s at the potential electrolysis (0,10-0,25) In a relatively saturated chloridizing electrode on the backgrounds of 0.2 n solution of sodium hydrogen phosphate or buffer solution of Britton-Robinson, pH 8.0, and 9.0, in the presence of 0.1%aqueous ethanol with subsequent registration of the anodic peaks in differential mode shooting voltamperes at scan rate of the potential of 20-30 mV/C. the Concentration of azithromycin is determined by the peak height in the range of potentials from 0.70 to 0.85 In the method of additives certified mixtures. The method has high sensitivity, the detection limit is equal to 1.86·10-10mol/l 1 PL., 1 Il.
The invention relates to the field of analytical chemistry, in particular for Stripping voltammetric method for the determination of antibiotic (AB) azithromycin dihydrate, 2(O):
Azithromycin dehydrate(azithromycin) belongs to a semi-synthetic antibiotic of the second generation and has a high microbiological and clinical efficacy in the treatment of serious infections of the respiratory tract, skin and soft tissues, some of urogenital infections. Determination of macroscopic quantities of azithromycin is important to assess pharmacological actions and the effectiveness of antibiotic therapy, identification of active substances in medicinal forms, and its metabolites in biological matrices. This in turn places high demands on the quality control of medicines and improving methods for the quantitative determination of macrolide antibiotic. An important problem is the development of new, more sensitive and selective methods for their analysis.
Currently, there are relatively few works on the definition of azithromycin. For the quantitative determination of applied microbiological method [Krichhoff R. M., Laufen H., Schacke G., Kirchhoff G., Gallo E. Determination of azithromycin in gastric biopsy samples./Int.J.Clin Parmacol Ther 1999 Jul; 37(7):361-4], and chromatographic methods of analysis (thin layer chromatography (TLC), Visona 1997 ND 42-1205-97].
The authors note interfering solvents that can overlap the peak of azithromycin and reduce the accuracy of the analysis. To increase the detection sensitivity of the HPLC method used chemical ionization mass spectrometry and electrochemical detection for pharmacokinetic studies of the content of the antibiotic in the blood serum in children [Founda H. G., Schneider, R. P. (HPLC) - atmospheric pressure chemical ionization mass spectrometry: correlation with a standard HPLC - electrochemical method./J. Chromatogr A 1998 Jul 3; 812(1-2):287-93]. Despite the rather high sensitivity options HPLC (from 1.3· 10-7mol/l to 3.2· 10-5mol/l azithromycin), the duration of the analysis with respect to time of sample preparation, purification, extraction, chromatographic separation, and the high cost of the devices significantly limit its use in analytical laboratories for the rapid quantitative determination of the antibiotic. Application of HPLC abroad in the analysis of drugs appears to be associated primarily with the intense development of theoretical foundations of the method, the creation and industrial production required sorbents and equipment.
Electrochemical methods, and, in the first place, such vysokochuvstvitel the various and complex systems: medicinal and pharmaceutical preparations natural objects, food. In the inversion variants VA minimum detectable concentration of the organic substances can be up to 10-10-10-11mol/L. of VA Methods allow for serial analyses using modern computerized voltammetric analyzers with high expressnet, selectivity and, often, without prior sample preparation with simultaneous detection of several substances in one sample, which greatly simplifies the analysis. There is limited information on the application of electrochemical methods, including for voltammetric determination of azithromycin.
The closest is the method of cyclic voltammetry (CVA) used for detection of antibiotic after its preliminary allocation of the sample by HPLC [Shepard R. M., Duthu G. S, Ferraina R. A., Mullins, M. A. High-performance liquid chromatographic assay with electrochemical detection for azithromycin in seum and tissues./J. Chromatogr 1991 Apr 19; 565(1-2), 321-337] (the prototype). Cyclic VA determination of azithromycin was performed after dissolution of the drug in the mobile phase (HPLC conditions), which served as a background: a mixture of 0.02 M ammonium acetate and 0.02 M sodium perchlorate - acetonitrile - methanol in the ratio 22:23:45:10, respectively, is at pH 11 for chloridizing electrode when the rate of change of the potential of 5 mV/sec. The minimum detectable concentration of azithromycin ~3· 10-8mol/l To improve the reproducibility and reducing passivation of the electrode was required cleaning of the electrode 6 M nitric acid and subsequent detector settings.
In these conditions, the determination of azithromycin at level n· 10-10mol/l impossible.
The task of the invention is to increase the sensitivity and expressnet definition of azithromycin method adsorptive Stripping differential voltammetry.
This object is achieved in that the method for quantitative determination of azithromycin dihydrate (azithromycin) includes translation of azithromycin from the sample into the solution and voltammetric determination using indicator glassy carbon electrode, characterized in that use Stripping voltammetry, and the accumulation of azithromycin in the mixed solution is carried out in a period when the potential of 30-60 s at the potential electrolysis (0,10-0,25) In a relatively saturated chloridizing electrode on the backgrounds of 0.2 n solution of sodium hydrogen phosphate or buffer solution of Britton-Robinson, pH 8.0-9.0 in the presence of 0.1%aqueous ethyl alcohol and then registratie and the concentration of azithromycin is determined by the peak height in the range of potentials from 0.70 to 0.85 In the method of additives certified mixtures.
In the proposed method, first established the ability of azithromycin to oxidize at different types of graphite electrodes. As the indicator used SU and graphite (G) electrode impregnated with polyethylene and paraffin in a vacuum (in the prototype used only SU face electrode). The use of such electrodes due to the high chemical and electrochemical stability of graphite, a wide range of working potentials, both in aqueous and nonaqueous media, as well as the simplicity of a manual update of the surface and safety. The ability to oxidation of azithromycin depends on the electrode material and the condition of its surface. The maximum value of the detected current with the use of the G electrode is higher (approximately 10-15%), but due to the large residual current it turned out to be less convenient than SU. To determine azithromycin used “needle” in the form of indicator electrodes, after the preliminary electrochemical modification of their surface. This leads to a decrease of the lower border of the designated contents, to improve the reproducibility of voltammetric measurements and expressnet analysis. “Needle” SU and G electrodes is.
The electrooxidation reaction of azithromycin is electrophilic. The role of the electrophilic reagent with electronic deficit performs the anode and the substrate of the organic molecule, the reactivity of which is determined by the electron density relations of atoms and groups in the molecule and is strongly dependent on pH. Preliminary studies showed that the electrochemical oxidation of azithromycin complicated as adsorption, may, and optional chemical stages and represents a complex diffusion-controlled electrode process involving more than one electron.
In the prototype described using as background a mixture of 0.02 M ammonium acetate and 0.02 M sodium perchlorate - acetonitrile - methanol in the ratio 22:23:45:10, respectively, pH 11. Determination of azithromycin in these conditions is difficult. Molecule of azithromycin has two main radical and azithromycin is very sensitive to changes in pH. With the increase of pH buffer Britton-Robinson from 8.0 to 9.0, the peak potential of the oxidation of azithromycin is shifted to more positive potentials from 0,774 to 0,850 Century. apparently, this is due to the fact that the main radicals molecules macrolide antibiotics, including erythromycin and Asians: Rusich, 1998. - S. 166-197]. Therefore, almost at pH more 9,0 molecule of azithromycin is almost predominantly in the ionized state and oxidized at more positive potential. In addition, when the pH is more 9,0 can register additional peak with EP0.90 In that reduces the resolution of the method. At pH less than 8.0 molecule of azithromycin is in the ionized state and therefore is oxidized at less positive potential. PH 8,0-9,0 are optimal for quantitative chemical determination of azithromycin. In the acidic environment at pH less than 6 oxidation peak is not registered, apparently, is the collapse of the molecules of macrolides [Melent'eva, A. Pharmaceutical chemistry. - M.: Medicine, 1976, 828 S.]. Proposed in the claimed invention backgrounds of 0.2 N. (corresponding to pH 8,6) phosphate sodium (Na2HPO4) or the buffer solution of Britton-Robinson (pH 8.0, and 9.0) in the presence of 0.1% (by volume) ethyl alcohol allows you to define azithromycin at the level of 10-10mol/l with good reproducibility. The relative standard deviation (Sr) does not exceed 0.20 and 0.25 when registration anodic peaks, respectively, at the backgrounds of 0.2 N. Na2HPO4and mortar buffer Brittany concentration 1· 10-6-1· 10-7mol/l (table).
The backgrounds of 0.2 N. Na2HPO4and solutions buffer Britton-Robinson chosen experimentally. An absolute novelty is the experimentally observed range of pH from 8.0 to 9.0 and using alcohol water and organic solvent. The presence of 0.1% by volume of ethyl alcohol contributes to a better removal of the antibiotic from the surface of the electrode and leads to stabilization and reproducible anodic peaks of azithromycin. The application of these in the invention the background electrolyte for the first time possible to reduce the detection limit of azithromycin calculated by 3-sigmatau criterion to 1,84· 10-10mol/l (Cmin,p). The minimum detectable concentration (Cn) 2,2· 10-10mol/l on the background of 0.2 N. Na2HPO4and 1.0· 10-9mol/l on the background solution buffer Britton-Robinson pH 8.0, and 9.0. The minimum detectable concentration of azithromycin on these backgrounds in the prototype ~3· 10-8mol/L. by the Authors (prototype) notes the low reproducibility of the oxidation waves of azithromycin and high passivation of the indicator electrode. The Sr values in the prototype is not specified. In addition, the prototype used a highly toxic mixture and the ohms are installed conditions of the electrochemical accumulation: potential electrolysis Ee=(0,10-0,25)C. Experimental data showed the dependence of the current of the oxidation of azithromycin from Fe(drawing). The magnitude of the anodic current increased by about 2.4 times and reached maximum values in the field potentials (0,10-0,25)Century At Ee=(0,10-0,25) decreased the magnitude of the current of the oxidation of the antibiotic. The use of pre-electrolysis when the value of the potential (0,10-0,25) allows you to register voltamperometry with a pronounced maximum.
This allows to improve the accuracy and selectivity of the method and Express to determine the concentrations of azithromycin in less than 1· 10-8mol/L.
Optimal pre-electrolysis (e) is 30-60 sec. Ateless than 30 with reduced detection sensitivity and increases the error of the determination, and whenemore than 60 with reduced expressnet; the current value reached a maximum value ateequal 30-60 C.
Important for the determination of azithromycin in the dierential VA is the speed of the sweep potential. The optimum speed of 20-30 MB/s speed Increase time resolution of the method. Use a speed of less than 30 mV/s reduces the magnitude of the anode current and lowers the detection sensitivity. Use a speed of 5 mV/s (prototype) does not allow to define azithromycin-level (10-9-10-10) mol/L.
The lower boundary of the designated contents of azithromycin depends on the shooting mode volt-ampere curves. The differentiation allows you to record clear peaks even at very low concentrations (10-9-10-10) mol/l, which increases the detection sensitivity. For the determination of azithromycin has not been applied. In a linear superposition of the potential in the integral mode, the determination of concentrations of azithromycin at level (10-8-1010) mol/l is practically impossible due to the large value of the residual current, which leads to registration fuzzy waves of oxidation, and the minimum detectable concentration is 1· 10-7mol/L. Therefore, for quantitative chemical analysis recommended differential mode shooting voltamperes. In the prototype used a variant of the cyclic voltammetry.
The conducting electrode process for the first time allowed the quantification of azithromycin on encantarora of the antibiotic on the surface SU of the electrode. The proposed voltammetric method has significantly improved metrological characteristics analysis of azithromycin; to increase the detection sensitivity (min,p=1,84· 10-10mol/l, Withn=2,2· 10-10mol/l), which is 2 orders of magnitude lower compared to the prototype. The range of detectable concentrations of azithromycin from 2.2· 10-10mol/l to 1.0· 10-4mol/L.
The measurements were carried out on a computerized voltammetric analyzers HUNDRED, YOU (OOO “ITM”, ,Tomsk), as well as universal polarography type PU.
The definition does not interfere with the substance, the presence of which may be biological objects: water-soluble vitamins of group In (b1In2InwithIn6), PP, ascorbic and uric acid in comparable quantities. The matrix composition of medicinal drugs Sumamed capsules, tablets practically no influence on the current of the oxidation of azithromycin, therefore, did not require pre-selection of antibiotic from the matrix to the actual electrochemical analysis.
Example 1. Determination of azithromycin at level (10-9-10-10) mol/L.
In a quartz glass with a capacity of 20 ml is poured to 9.9 ml of 0.2 N. Na 0,001% within five minutes. Stopping stirring, carry out the electrolysis of a solution under the condition: Ee=0.20 In,C=60 C. turn Off the gas and fixed anode differential voltamperometry at scan rate of the potential of 30 mV/s, since the potential of Ebeg=0,20 Century, the Absence of peaks indicates the purity of the background. Then add a few drops with a volume of 0.01 ml certified mixture of azithromycin (2-4)· 10-7-10-6mol/l, mix a solution of 10 with and conduct electrochemical concentration of sediment at Ee=0.20 In,e=60 S. Shooting volt-ampere curve to start with potential 0,20 Century Peak for a given concentration of a substance recorded in the range of potentials from 0.70 to 0.75 In (us.x.with.E.) when the sensitivity of the device(0,5-1)· 10-9A/mm Time of a single analysis does not exceed 10 minutes.
Example 2. Determination of azithromycin tablets “Sumamed”
In the Cup for voltammetric measurements make 10 ml of 0.2 N. Na2HPO4or 10 ml buffer Britton-Robinson, pH 8.0-9.0 and remove oxygen from the solution by passing nitrogen gas for five minutes. Without stopping the stirring, conduct the electrolysis process is programmu background in the range of potentials from 0.20 to 1.00 (us.x.with.E.). The absence of peaks in voltamperometry in the field potentials 0,70-0,85 shows In the cleanliness of the background electrolyte and polarographic Cup.
In the analysis of tablets containing azithromycin, crushed in a mortar 3-6 tablets without shell. Take a portion of the sample 0,200 g, weighed with accuracy of 0.001 g, introduced into the flask with a capacity of 100 ml, dissolve a portion of the sample in 50 ml of 96% ethanol and brought to the mark with bidistilled water. After 3-5 minutes the solution is filtered through a paper filter. Then 0.01 ml of the resulting filtrate make quartz glass with the background solution. Electronicaly and the analytic signal is carried out in the same conditions. The anodic peak of azithromycin is fixed in the range of potentials from 0.75 to 0.85 In the SU electrode when the instrument sensitivity (1-5)· 10-8A/mm in differential mode shooting voltamperes. The mass concentration of azithromycin in the sample evaluated by the method of additives certified mixtures by measuring the height of the anodic peak. The analysis time per sample, with regard to sample preparation in less than 30 minutes.
Thus, the first set of the ability of quantitative chemical analysis of azithromycin on the peaks of oxidation on his SU “needle” e is lnam oxidation using end SU electrode).
The analysis of the characteristics of quantitative chemical determination of azithromycin in the proposed method shows significant improvements in detection sensitivity (by 2 orders of magnitude compared with the prototype and by more than 4 orders of magnitude compared to HPLC) [Founda H. G., Schneider, R. P. (HPLC) - atmospheric pressure chemical ionization mass spectrometry: correlation with a standard HPLC - electrochemical method./J. Chromatogr A 1998 Jul 3; 812(1-2):287-93]. The limit of detection and lower limit of the designated contents are respectively 1,84· 10-10mol/l and 2.2· 10-10mol/l Significantly decreased the analysis time. The conditions used in the prototype, does not allow you to control waste water, air zone enterprises and chemical laboratories at the level of anagrammatic contents (or less) of azithromycin.
The proposed method is simple, does not require a large amount of reagents and labor, and can be applied in any chemical laboratory with polarograph, especially at present, when adjusted for the production of domestic and foreign electrical equipment with computer control and data processing (analyzers type STA, TA and others) the Proposed method can be used in pharmacokinetic and pharmaceutical research, in toxic chemical and pharmaceutical companies, and to develop techniques for the analysis of azithromycin and related compounds in complex multi-component biological systems (blood, urine, plasma and other) and food.
The method of quantitative determination of azithromycin dihydrate (azithromycin), including the translation of azithromycin from the sample into the solution and voltammetric determination using indicator glassy carbon electrode, characterized in that use Stripping voltammetry, and the accumulation of azithromycin in the mixed solution is carried out in a period of 30-60 s at the potential electrolysis (0,10-0,25) In a relatively saturated chloridizing electrode on the backgrounds of 0.2 n solution of sodium hydrogen phosphate or buffer solution of Britton-Robinson, pH 8.0, and 9.0, in the presence of 0.1%aqueous ethanol with subsequent registration of the anodic peaks in differential mode shooting voltamperes at scan rate of the potential of 20-30 mV/s and the concentration of azithromycin is determined by the peak height in the range of potentials 0,70 - 0,85 In the method of additives certified mixtures.