RU2582960C1 - Method for fluorimetric determination of flunixin - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: in method realisation into acetate-ammonia buffer solution with pH 7,0-7,8 Twin-80 is added to concentration 1·10^-2 M, terbium Tb³⁺ salt to concentration 1·10^-3 M, medication trioctylphosphinoxide to concentration 1·10^-4 M; solution is irradiated by radiation with wavelength λ_exc=347 nm, and presence of fluorescence at wavelength λ_fl=545 nm is used to conclude about presence of flunixin Fluorescence intensity is measured additionally, with concentration of flunixin in medication being determined by the value of intensity with application of preliminarily obtained calibration graph or by method of standard additive.

EFFECT: simplification of analysis.

3 cl, 1 dwg, 8 tbl, 7 ex

Description

The invention relates to analytical chemistry, specifically to a method for fluorimetric determination of flunixin in drugs in determining the active substance and its presence.

Flunixin (2 - [[2-methyl-3- (trifluoromethyl) phenyl] amino] pyridine-3-carboxylic acid)

refers to non-steroidal, anti-inflammatory, analgesic and antipyretic drugs used in veterinary medicine. Control of its content is carried out by methods based on the principles of chromatography-mass spectrometry involving gas, liquid chromatography [Estelle Dubreil-Chéneau, Yvette Pirotais, Mélaine Bessiral, etc. Development and validation of a confirmatory method for the determination of 12 non steroidal anti-inflammatory drugs in milk using liquid chromatography-tandem mass spectrometry. Journal of Chromatography A, 1218 (2011) 6292-6301; Alessandra Gentili, Fulvia Caretti, Simona Bellante, etc. Development and validation of two multiresidue liquid chromatography tandem mass spectrometry methods based on a versatile extraction procedure for isolating non-steroidal anti-inflammatory drugs from bovine milk and muscle tissue. Anal Bioanal Chem (2012) 404: 1375-1388; Tao Peng, Ai-Ling Zhu, Yue-Ning Zhou etc. Development of a simple method for simultaneous determination of nine subclasses of non-steroidal anti-inflammatory drugs in milk and dairy products by ultra-performance liquid chromatography with tandem mass spectrometry. Journal of Chromatography B, 933 (2013) 15-23; Ngaio Richards, Sarah Hall, Karen Scott, etc. First detection of an NSAID flunixin in sheep's wool using GC-MS. Environmental Pollution 159 (2011) 1446-1450]. Chromatography-mass spectrometry allows the separation of the determined components and their analytical signals, complex in composition of the samples in time, and to obtain mass spectra of each compound in the mixture. The area of the chromatographic peak is proportional to the substance content in the analyzed sample, which allows accurate quantitative analysis of the samples. The advantage of this method lies in expressness and high sensitivity, however, it is not suitable for routine analysis of drugs, since it requires the involvement of expensive equipment and highly qualified personnel.

The closest in technical essence is a method for determining flunixin using differential pulse voltammetry using portable miniature disposable graphite electrodes [V. Meuccia, M. Vannia, M. Sgorbinia, etc. Determination of phenylbutazone and flunixin meglumine in equine plasma by electrochemical-based sensing coupled to selective extraction with molecularly imprinted polymers. Sensors and Actuators B 179 (2013) 226-231]. The method includes preliminary solid-phase extraction of flunixin on a column with subsequent reduction on a graphite electrode and is proposed as an alternative to existing chromatographic methods. Significant disadvantages include the use of disposable electrodes, which increases the cost of analysis, as well as the long determination time. More often in practice, in the analysis of drugs, methods based on the measurement of intrinsic fluorescence are used, however, flunixin does not have fluorescence properties, and this is the limitation of the possibilities of fluorimetry. We first proposed a fluorimetric method for determining flunixin, based on the measurement of the sensitized fluorescence of the terbium complex with flunixin in the presence of trioctylphosphine oxide.

The objective of the invention is the development of a simple fluorimetric express method for determining flunixin using an analytical form that provides the ability to determine flunixin in drugs, eliminating the use of expensive equipment and attracting highly qualified specialists.

The technical result consists in simplifying the method for determining flunixin through the use of a fluorimetric method of analysis, which is possible as a result of the interaction of flunixin with terbium (III) ion and trioctylphosphine oxide (TOPO) in Tween-80 micellar solutions.

The specified technical result is achieved by the fact that according to the claimed method for determining the presence of flunixin in a drug, an ammonia acetate buffer solution with a pH of 7.0-7.8 is added to Tween-80 to a concentration in the final solution of 1 · 10 ^-2 M, terbium salt Tb ³⁺ to a concentration of the final solution of 1 · 10 ^-3 M, the drug trioctylphosphine oxide to a concentration of 1 · 10 ^-4 M in the final solution, irradiate the solution with electromagnetic radiation with a wavelength of λ _exc = 347 nm and judge by the presence of fluorescence at a wavelength of λ _ph = 545 nm the presence of flunic ins. In addition, the fluorescence intensity is measured, and the concentration of flunixin in the drug is determined by the intensity value using a previously obtained calibration graph or by the standard addition method.

The invention is illustrated by the drawing, which shows a calibration graph for determining flunixin, where the abscissa indicates the negative decimal logarithm of the concentration of the drug pC (M), and the ordinate axis represents the decimal logarithm of the intensity of the fluorescence signal lgI.

The use of a fluorimetric method for determining flunixin based on measurements of intrinsic fluorescence is impossible, since the analyte does not have fluorescent properties. However, as a result of its interaction with the terbium (III) salt and trioctylphosphine oxide (TOPO) in the Tween-80 micellar solution, a complex is formed that is characterized by the emission of terbium (λ _exc = 347 nm, λ _fl = 545 nm), which can be used as an analytical signal in determining a non-steroidal anti-inflammatory drug.

The method is implemented as follows.

In a test tube with a buffer solution (pH 7.0 - 7.8), Tween-80 solution is added strictly in order to its concentration in the final solution of 1 · 10 ^-2 M, terbium salt Tb ³⁺ to its concentration in the final solution of 1 · 10 ^-3 M, a drug (or other analyzed solution), trioctylphosphine oxide to its concentration in the final solution of 1 · 10 ^-4 M. The resulting final solution is exposed to electromagnetic radiation with an excitation wavelength λ = 347 nm, and the fluorescence signal intensity is measured at a fluorescence wavelength λ _fl = 545 nm, which depends on the end tration flunixin in solution. Using the proposed method for the presence of fluorescence, it is possible to detect flunexin in the concentration range from 1 · 10 ^-7 to 1 · 10 ^-4 M.

To determine the concentration of flunixin, it is possible to use a calibration graph constructed under similar conditions for standard solutions of flunixin in the coordinates of the logarithm of intensity (logI) - the negative logarithm of the concentration (pC) of flunixin, M. To measure the fluorescence signal, the method of fluorescence allowed in time (delay time is 0 , 3 ms). The intensity I of the fluorescence of the solution is determined, then the logarithm of this value is calculated on the basis of 10 lgI and using the calibration graph, pC and the corresponding concentration of flunixin are found as the antilogarithm of the obtained value (see drawing).

To build a calibration graph, prepare a standard aqueous solution of flunixin concentration of 1 · 10 ^-4 M (Sigma-aldrich, the main substance is not less than 98%), an aqueous solution of Tween-80 (Sigma company, the main substance is not less than 99%) concentration 1 · 10-1 M, a solution in ethyl alcohol of trioctylphosphine oxide (Sigma company, the main substance is not less than 99%) of a concentration of 1 · 10 ^-2 M, an aqueous solution of the salt of terbium (III) chloride, six-water ("AcrosOrganics", 99.9 % of the main substance), concentration 1 · 10 ^-2 M, acetate-ammonia buffer solution (pH 7.0-7.8). In seven to ten tubes (or more to increase accuracy), 1 ml of a buffer solution with a pH of 7.0 - 7.8 is added, then 0.4 ml of 1 · 10 ^-1 M Tween-80, 0.4 ml of a solution of salt of terbium (III) 1 · 10 ^{- 2} M. After this, a standard solution of flunixin is added to each tube so that the final concentrations vary from 1 · 10 ^-7 to 1 · 10 ^-4 M. Then, 0.4 ml of ^10-3 M TOFO is added to each tube and the buffer solution is added to the total volume of 4 mL, was stirred and measured the fluorescence intensity I (λ _exc = 347 nm, λ = 545 nm _fl) mode in time-resolved fluorescence tion (delay time is 0.3 ms). For each solution, the fluorescence intensity is measured and the logarithm of this value is calculated on the basis of 10. The calibration graph (see drawing) is plotted in the coordinates logI - pC, where C is the content of flunixin in a standard solution, M.

It can be seen from the calibration graph that the range of determined concentrations is 1 · 10 ^-7 - 1 · 10 ^-4 M. The detection limit is calculated using the 3δ method (Fundamentals of Analytical Chemistry "in 2 books. Book 1. Methods of chemical analysis: textbook for universities / Yu.A. Zolotov, E.N. Dorokhova et al. Edited by Yu.A. Zolotov. - M .: Higher School. 2004, 494 p.) And is 8 · 10-8 M.

To determine the concentration of flunixin, it is possible to use the method of standard additives (Grishaeva T.I. Methods of luminescent analysis. - St. Petersburg: ANO NPO Professional, 2003, p. 107). The addition of a standard solution of flunixin should be characterized by a fluorescence intensity close in value to the analyzed solution. Methodology: 1.5 ml of the analyzed solution is placed in a volumetric flask with a capacity of 25 ml, add bidistilled water to the mark, mix. 1 ml of buffer solution (pH 7.0-7.8), 0.4 ml of 1 · 10 ^-1 M Tween-80, 0.4 ml of terbium (III) salt solution 1 · 10 ^-2 М and 0.2 - 0.5 ml of diluted “Flunex” solution are added to the test tube , 0.2 - 0.4 ml of a standard solution of flunixin concentration of 1 · 10 ^-4 M, add 0.4 ml of TOFO 10 ^-3 M and a buffer solution to a total volume of 4 ml, mix and measure the fluorescence intensity (delay - 0.3 ms). The concentration of flunixin is determined by the formula:

 Cx = Ix · Ca / Ix + a- Ix, where

Ix - fluorescence of the test solution;

Ix + st - fluorescence of the test solution containing the additive;

Cx - determined concentration, (%);

Ca is the concentration of the additive in the test solution (%).

The results of the determination are presented in table 1. The correctness was controlled by the method of "entered-found."

Table 1
The results of the determination of flunixin in the drug "Flunex" (n = 3, P = 0.95, ttabl = 4.3) The claimed content,% Found,% x ± Δx Sr 8.3 8.45 ± 0.08 0.04

table 2
Correctness control determination of flunixin in the Flunex preparation by the “introduced-found” method (n = 3, P = 0.95, ttable = 4.3) Sample number Introduced
mg / l Found
mg / l Sr texper one 1.48 1.51 ± 0.01 0.01 2.27 2 3.65 3.69 ± 0.10 0.04 1,53 3 2.96 3.17 ± 0.04 0.02 2,32

Sr is the relative standard deviation, Sr = S / χ, where S is the standard deviation equal to S = ∑ (χi-χ) 2 / n-1) 1/2, χi is the unit determination result, χ is the average result, n is number of determinations, p - confidence probability, t _expert - Student's coefficient.

Examples of the method.

A solution of the Flunex drug (NITA-PHARM LLC, Saratov) was used as an analyzed sample.

Example 1. The choice of the optimal metal ion to obtain the maximum analytical signal. Other REE ions can also give a fluorescence signal in the presence of flunixin, TOPO in a Tween-80 micellar solution. In four tubes, 1 ml of buffer solution (pH 7.0 - 7.8) is added sequentially, 0.4 ml of 1 · 10 ^-1 M Tween-80 is added. 0.4 ml of a solution of terbium (III) salt of a concentration of 1 · 10 ^-2 M are added to the first test tube, europium (III) chloride, samarium (III) and gadolinium (III) chloride of the same concentration are added to the others, then 0.4 ml is added to each flunixin, 10 ^-4 M, 0.4 ml of TOFO 10 ^-3 M, add a buffer solution to a total volume of 4 ml, mix and measure the fluorescence intensity (λ _exc = 347 nm, λ _fl = 545 nm) in the mode of time-resolved fluorescence. The measurement results are presented in table 3.

Table 3
The effect of the nature of the REE ion on the intensity of the analytical signal REE Terbium (III) Europium (III) Samaria (III) Gadolinium (III) Fluorescence intensity 250 10 5 6

To obtain the maximum fluorescence signal, we used in further studies of terbium (III).

Example 2. Determining the optimal nature of the surfactant to obtain the maximum analytical signal. The effects of cationic (cetylpyridinium chloride, CPC), anionic (sodium dodecyl sulfate, DDS) and nonionic (Tween-80, Bridge-35, Triton X-100) surfactants on the fluorescence intensity of the terbium (III) complex are considered. Five ml add 1 ml of a buffer solution with pH 7.0-7.8, then add 0.4 ml of 1 · 10 ^-1 M CPC to the first test tube, to the second test tube, to DDS, to the third to Tween-80, to the fourth to Bridge-35, and to the fifth - Triton X-100 and then in all tubes add 0.4 ml of a solution of terbium salt 1 · 10 ^-2 M, 0.4 ml of flunixin 10 ^-4 M, 0.4 ml of TOFO 10 ^-3 M, add a buffer solution to a total volume of 4 ml, mix and measure the fluorescence intensity (λexc = 347 nm, λfl = 545 nm) in the mode of time-resolved fluorescence.

Table 4
The effect of the nature of surfactants on the fluorescence intensity of the terbium (III) complex n / a Central heating DDS Twin 80 Bridge 35 TritonX-100 Fluorescence intensity fifteen one hundred 750 16 fifteen

As can be seen from table 4, of all surfactants, the largest increase in fluorescence intensity is observed in the presence of nonionic Tween-80, which we selected for further studies.

The fluorescence intensity of Tb3 + chelate - flunixin - trioctylphosphine oxide depends on the concentration of Tween-80 in the solution.

Example 3. Determination of the optimal concentration of Tween-80 to obtain the maximum analytical signal. Into four tubes add 1 ml of buffer solution (pH 7.0 - 7.8), 0.4 ml of a solution of terbium salt 1 · 10 ^-2 M, 0.4 ml of flunixin 10 ^-4 M, 0.4 ml of TOPO 10 ^-3 M, and 0.4 ml of concentration solutions are added sequentially 1 · 10 ^-6 M, 1 · 10 ^-4 M, 1 · 10 ^-3 M, 1 · 10 ^-2 M Tween-80, buffer solution to a total volume of 4 ml, mix and measure the intensity of the analytical signal (λ _exc = 347 nm, λ _fl = 545 nm).

Table 5
Determination of the optimal concentration of tween-80 n / a one 2 3 four Tween-80, concentration, M 1 · 10 ^-6 1 · 10 ^-4 1 · 10 ^-3 1 · 10 ^-2 Fluorescence intensity 25 thirty thirty 1000

Due to the fact that a solution of Tween-80 of a higher concentration cannot be prepared due to the limited solubility of the reagent in water, 1 · 10-2 M is the optimal concentration.

Example 4. The choice of the second ligand of the TOPO to obtain the maximum analytical signal. Into four tubes add 1 ml of a buffer solution with a pH of 7.0 - 7.8, add 0.4 ml of 1 · 10 ^-1 M Tween-80, 0.4 ml of a solution of salt of terbium (III) 1 · 10 ^-2 M, 0.4 ml of flunixin 1 · 10 ^-4 M. 0.4 ml of TOFO 10 ^-3 M are added to the first test tube, 0.4 ml of sodium ethylenediaminetetraacetic acid (EDTA) 1 · 10 ^-3 M to the second, 0.4 ml of tenoyl trifluoroacetone (TTA) 1 · 10 ^-4 M, c the fourth - 0.4 ml of 1,10-phenanthroline (Phen) 1 · 10 ^-4 M, add a buffer solution to each tube to a total volume of 4 ml, mix and measure the fluorescence intensity (λ _exc = 347 nm, λ _fl = 545 nm) in time-resolved mode f uorestsentsii. The measurement results are presented in table 6.

Table 6
The choice of the optimal second ligand to obtain the maximum analytical signal Second ligand TOFO EDTA TTA Hair dryer Fluorescence intensity 500 25 10 one hundred

As the second ligand, it is possible to use TOPO or Fen, however, the use of TOPO is optimal for obtaining the maximum value of the analytical signal.

Example 5. Determination of optimal acidity to obtain the maximum analytical signal. In each of the five tubes, 1 ml of buffer solution with pH 5.0, 6.0, 7.0, 8.0, 9.0 is added, and 1 · 10 ^-1 M Tween-80, 0.4 ml of terbium (III) salt solution is added to each 0.4 ml of 1 · 10 ^{- 2} M, 0.4 ml of flunixin 1 · 10 ^-4 M, 0.4 ml of TOFO 10 ^-3 M, add the corresponding buffer solution to a total volume of 4 ml, mix and measure the fluorescence intensity (λ _exc = 347 nm, λ _fl = 545 nm) in time resolved fluorescence mode. The measurement results are presented in table 7.

Table 7
Choosing the optimal acidity to get the maximum analytical signal pH 5.0 6.0 7.0 8.0 9.0 Fluorescence intensity 8 fifty one hundred 95 thirty

It was found that the fluorescence intensity of the terbium (III) - flunixin - TOPO system in the presence of Tween-80 significantly depends on the acidity of the medium and the maximum intensity is observed at pH 7-8.

Example 7. The choice of optimal concentrations of system components. To select the optimal concentration of Tb ³⁺ , we studied the dependence of the fluorescence intensity of a multi-ligand chelate on different contents of Tb ³⁺ ions.

Into six test tubes, 1 ml of buffer solution (pH 7.0 - 7.8), 0.4 ml of 1 · 10 ^-1 M Tween-80 was added, a solution of terbium salt was added to each test tube in the concentration range of 5 · 10 ^-6 - 2 · 10 ^-3 М, and then 0.4 ml of 10 ^-4 M flunixin, 0.4 ml of 1 · 10 ^-3 M TOPO and a buffer solution to a total volume of 4 ml, was mixed and the fluorescence intensity (λ _exc = 347 nm, λ _fl = 545 nm) was measured in the mode allowed fluorescence time. The results are presented in table 8.

Table 8
The choice of the optimal concentration of terbium salt and the second ligand to obtain the maximum analytical signal The fluorescence intensity, M 2 · 10 ^-3 1 · 10 ^-3 5 · 10 ^-4 1 · 10 ^-4 5 · 10 ^-5 1 · 10 ^-5 Terbium 830 850 750 400 200 one hundred TOPO precipitation precipitation one hundred 170 one hundred 70

As can be seen from table 8, to obtain the maximum value of the intensity of the analytical signal, it is necessary to use 1 · 10 ^-3 M salt of terbium (III) and 1 · 10 ^-4 M solution of TOPO.

The proposed method allows you to abandon expensive equipment and attract qualified personnel.

Claims (3)

1. A method for determining the presence of flunixin in a drug, characterized in that Tween-80 is added to an acetate-ammonia buffer solution with a pH of 7.0-7.8 to a concentration in the final solution of 1 · 10 ^-2 M, terbium salt Tb ³⁺ to a concentration of in the final a solution of 1 · 10 ^-3 M, a drug, trioctylphosphine oxide to a concentration in the final solution of 1 · 10 ^-4 M, irradiate the solution with electromagnetic radiation with a wavelength λ _exc = 347 nm and judge by the presence of fluorescence at a wavelength λ _fl = 545 nm the presence of flunixin. 2. The method according to claim 1, characterized in that the fluorescence intensity is measured, the concentration of flunixin in the drug is determined by the intensity value using a previously obtained calibration graph. 3. The method according to claim 1, characterized in that the fluorescence intensity is measured, the concentration of flunixin in the drug is determined by the standard addition method.

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