RU2851164C1 - Method for determining tetracycline using polymethacrylate matrix - Google Patents

Abstract

FIELD: analytical chemistry.

SUBSTANCE: invention relates to methods for the quantitative and test determination of tetracycline and can be used for its determination in various types of water, for quality control of honey for tetracycline content, and for analysis of human and animal urine. The method for determining tetracycline using a polymethacrylate matrix involves the interaction of the analysed solution with a polymethacrylate membrane in a tetraborate buffer medium, measurement of the analytical signal, and determination of the tetracycline content. The polymethacrylate matrix is used simultaneously as a solid-phase extractant of tetracycline for its concentration from solutions with a pH of 7.5-9.5 in the concentration range of 0.0025-2.0 mg/l and as a medium for forming an analytical signal, whereby the tetracycline content is determined by its own yellow-green fluorescence directly in the solid phase of the polymethacrylate matrix at a radiation wavelength of 520 ± 5 nm, the contact time of the analysed solution with the polymethacrylate membrane is 30-60 minutes at a temperature of 20-25°.

EFFECT: ability to determine tetracycline in various objects, in a wide range of detectable concentrations.

1 cl, 2 dwg, 1 tbl

Description

The invention relates to the field of analytical chemistry, namely to methods for the quantitative and test determination of tetracycline, and can be used for its determination in various types of water, in monitoring the quality of honey for tetracycline content, as well as for the analysis of urine in humans and animals.

A known method for determining tetracycline involves treating the analyzed sample with an ammonia buffer solution (pH 10.0-10.5) followed by photometric measurement of the colored complex (Author's Certificate SU 1059491, G01N21/78, published 07.12.83). The proposed method is simple to implement, but has a significant drawback - low sensitivity (detection limit 3.6 mg/L), which limits its use for analyzing trace amounts of tetracycline.

A sensitive method for determining tetracycline in milk and dairy products is known (RU Patent 2566422, G01N30/00, published October 27, 2015). The method is based on preliminary sorption concentration of tetracycline with a natural zeolite, subsequent desorption of the analyte from the sorbent surface, and its determination by high-performance liquid chromatography (HPLC) with ultraviolet detection at a wavelength of 350 nm. The proposed method exhibits high sensitivity due to the use of sorption preconcentration on a natural zeolite and subsequent analysis by HPLC with UV detection, as well as selectivity, since HPLC allows for the effective separation and identification of tetracycline even in complex matrices (milk, dairy products).

The disadvantages of the proposed method for tetracycline determination include labor intensity, due to the fact that the process includes several stages (sorption, desorption, chromatographic analysis), which increases the analysis time; possible loss of analyte during the desorption process (part of the tetracycline may remain on the sorbent, which reduces accuracy); the need for complex and expensive equipment (HPLC with a UV detector is not available in all laboratories).

A fluorescence method for determining tetracycline is known (Li-Ming Shen, Ming-Li Chen, Xu-Wei Chen. A novel flow-through fluorescence optosensor for the sensitive determination of tetracycline // Talanta. 2011. Vol. 85. Pp. 1285-1290), which is based on the effect resulting from the sorption of a fluorescent tetracycline derivative encapsulated in cetyltrimethylammonium bromide (CTAB) micelles on the surface of Sephadex G-50 microgranules. The analysis procedure includes a tetracycline hydrolysis step, for which the analyzed sample is heated in a boiling water bath in an alkaline medium (0.05 M NaOH) with CTAB for 40 min to obtain a fluorescent derivative. The hydrolyzed solution is then passed through a flow cell filled with Sephadex G-50 microbeads at a flow rate of 5 μL/s, or 20 mg of microbeads are mixed with 3 ml of the hydrolyzed solution and incubated at room temperature for 40 minutes. After removing the liquid phase, the microspheres are transferred to a quartz cuvette. The fluorescence signal of the tetracycline derivative adsorbed on the carrier surface is then recorded at excitation and emission wavelengths of 340 and 410 nm, respectively. The analytical signal is determined by the fluorescence intensity, which is proportional to the tetracycline concentration. The range of detectable concentrations was 3-500 μg/L with a detection limit of 1.0 μg/L. The method was tested on pharmaceuticals and surface water samples.

The proposed method for tetracycline determination provides high sensitivity, but is time-consuming and labor-intensive due to the need for multi-step sample preparation of the test solution. The sorbent also requires preparation for analysis by soaking it in deionized water to allow it to swell. Furthermore, this method for tetracycline determination requires a specialized flow-through system (FIA) and a fluorimeter with a flow cell. Manual sorbent preparation (swelling and column packing) reduces reproducibility.

Classical analytical methods (HPLC, fluorimetry, capillary electrophoresis) require expensive equipment, complex sample preparation, and highly qualified personnel, limiting their use in routine research. In contrast, solid-phase-based sorption-colorimetric methods combine ease of implementation, low cost, and the ability to determine analytes without the use of complex instrumentation. This makes them promising for the development of modern test systems. In situations where mass analysis is required (for example, environmental monitoring or food safety control), such methods are the optimal solution.

A method for express sorption-colorimetric determination of tetracycline in urine is known (RU Patent 2350950, G01N33/15, published on March 27, 2009), which is promising for the analysis of biological environments. The method is based on the sorption of tetracycline on silica gel, followed by the formation of a colored coordination complex upon interaction with iron(III) chloride and potassium hexacyanoferrate(II) (K ₄ [Fe(CN) ₆ ]). The color of the sorbent (from pale blue to green) is assessed visually or using a color test scale. The range of detectable tetracycline concentrations in urine is 0.25-10 μg/cm³. The proposed method does not require expensive equipment, replacing them with standard laboratory procedures (centrifugation, visual assessment).

The disadvantages of the proposed method include the multi-stage nature and complexity of the tetracycline determination procedure, which requires repeated centrifugation and washing of the precipitates with acetic acid. Another significant drawback is the subjectivity of the visual assessment. Furthermore, the total analysis time is over 1 hour, making this method unsuitable for rapid analysis.

A known method for determining tetracyclines in water and milk by the sensitized fluorescence of europium on its hydroxide using digital colorimetry (V. G. Amelin et al. Using a smartphone to determine tetracyclines in water and milk by the sensitized solid-phase fluorescence of europium on its hydroxide // Journal of Analytical Chemistry. 2021. Vol. 76. No. 10. pp. 952-958), which is promising for rapid analysis and the closest analogue to the claimed invention. The method is based on the formation of fluorescent complexes of tetracyclines with europium(III) ions in an alkaline medium (with the addition of sodium tetraborate); upon the formation of a europium hydroxide precipitate, its fluorescent properties do not change. The sediment is concentrated by centrifugation at the bottom of the test tube, and the pink fluorescence of the solid phase, excited by UV light (365 nm), is recorded using a smartphone as a color recording device. The analytical signal is calculated based on the change in the color parameters (RGB) of the sediment relative to the blank sample. The range of detectable tetracycline concentrations is 0.005-1 μg/mL (two linear regions: 0.005-0.1 and 0.08-1 μg/mL) with detection limits of 0.001 and 0.005 μg/mL, respectively. The method is applicable to the analysis of natural and wastewater, as well as milk.

The proposed method is simple and accessible due to the use of a smartphone instead of expensive equipment. High sensitivity for tetracycline determination is achieved through the fluorescence of europium complexes on its hydroxide. However, the use of europium(III) increases the cost of analysis due to the high price of rare earth-based reagents, especially for large-scale testing, making the proposed method less accessible to routine laboratories. Furthermore, when performing this tetracycline determination method, it is necessary to ensure that the precipitate after centrifugation is dense and uniform at the bottom of the tube; otherwise, inhomogeneity can lead to signal variations, and light scattering by particles can increase noise.

A good alternative to the above-described method is the direct fluorescence of tetracyclines (without the use of europium salts), which will simplify and reduce the cost of the method, since tetracyclines are able to fluoresce under UV radiation without additional reagents.

The objective of the present invention is to develop a simple, economical and accessible method for determining tetracycline in various objects, in a wide range of detectable contents (the use of a polymethacrylate matrix makes it possible to expand the range of detectable contents of tetracycline, to reduce the lower and increase the upper limits of detectable contents by 2 times).

The solution to the specified problem is achieved in that in the method for determining tetracycline using a polymethacrylate matrix, including the interaction of the analyzed solution with a polymethacrylate membrane in a tetraborate buffer medium, measurement of the analytical signal and determination of the tetracycline content, what is new is that the polymethacrylate matrix is used simultaneously as a solid-phase extractant of tetracycline for its concentration from solutions with a pH of 7.5-9.5 in the concentration range of 0.0025-2.0 mg/l and a medium for forming an analytical signal, while the tetracycline content is determined by its own yellow-green fluorescence directly in the solid phase of the polymethacrylate matrix at an emission length of 520 ± 5 nm, the contact time of the analyzed solution with the polymethacrylate membrane is 30-60 minutes at a temperature of 20 - 25°C.

The essence of the claimed method is as follows: when the test solution interacts with a polymethacrylate matrix, a change in the intensity of the intrinsic yellow-green fluorescence of tetracycline in the polymethacrylate matrix is observed upon irradiation of the matrix with ultraviolet light. The fluorescence intensity is proportional to the tetracycline concentration in the test solution. This effect is observed during interaction of the polymer membrane with the analyte solution at pH 7.5-9.5.

Fig. 1 shows the fluorescence spectra of tetracycline in a polymethacrylate matrix after contact of the matrix with a tetracycline solution, which demonstrate an increase in the fluorescence intensity of tetracycline in the polymethacrylate matrix depending on its concentration in the analyzed solution.

A polymethacrylate matrix was added to the test solution containing tetracycline, thoroughly mixed for 30-60 minutes, removed and irradiated with UV light.

Using a smartphone, we photographed the plates and measured the colorimetric characteristics of yellow-green fluorescence. The analytical signal was then correlated with the tetracycline content in the analyzed solution and evaluated. The calculated ΔE (color difference) value in the RGB system was used as the analytical signal.

Examples of the implementation of the claimed invention are presented below.

Example 1. Measurement of the colorimetric characteristics of yellow-green fluorescence of tetracycline in a polymethacrylate matrix and determination of its content using a calibration graph.

A polymethacrylate matrix plate was placed in 4 ml of the test solution containing tetracycline (0.1-80.0)⋅10 ^-4 mg with a pH of 7.5-9.5 and stirred for 30-60 min at a temperature of 20-25°C, then removed and irradiated with UV light (365 nm). Using a smartphone, a photo of the plate was taken and the colorimetric characteristics of yellow-green fluorescence (520 ± 5 nm) were measured, followed by establishing the dependence of the analytical signal on the tetracycline content in the test solution and its evaluation. ΔE (color difference) in the RGB system was used as the analytical signal, which was calculated using the formula: ΔE = ((RR ₀ ) ² +(GG ₀ ) ² +(BB ₀ ) ² ) ^1/2 ,

where R, G, B, R ₀ , G ₀ , B ₀ are the intensity values of red, green, blue colors of the analyzed and blank samples, respectively.

The tetracycline content was determined using a calibration curve constructed under similar conditions. The calibration curve has two linear sections in the ranges (0.25-10.00)⋅10^-2mg/l with the calibration equation: ΔE = 1150·c (r = 0.995) and (0.25-2.00) mg/l with the calibration equation: ΔE = 74·c + 158 (r = 0.994), where c is the tetracycline concentration, mg/l. The detection limits calculated using the 3s-criterion were 0.001 and 0.210 mg/l, respectively.

Fig. 2 shows photographs of polymethacrylate matrices after contact with tetracycline solutions of different concentrations: a - without irradiation; b - with irradiation with UV light (365 nm).

Example 2. Determination of tetracycline content in different types of water.

When determining tetracycline in bottled and river drinking water, no preliminary sample preparation was required. To analyze samples for tetracycline content, an aliquot of 0.5–3.6 ml of the analyzed samples was placed in a 5 ml test tube, 0.4 ml of tetraborate buffer was added, the sample was diluted to a volume of 4 ^cm3 with distilled water, and a PMM plate was added. The tube contents were stirred for 60 min at a temperature of 20–25°C, then removed and irradiated with UV light (365 nm). The procedure was then as described in Example 1. The results of tetracycline determination in water samples are presented in Table 1.

Example 3. Determination of tetracycline content in human urine samples.

When analyzing human urine for tetracycline content, sample preparation was performed in advance to remove proteins from the urine. For this purpose, 10 ml of urine and 0.1 ml of a 20% trichloroacetic acid solution were added to a 15 ml plastic graduated test tube, mixed on a rotary shaker for 5 min, and then centrifuged for 5 min at 4000 rpm. The centrifugate was transferred to another test tube. An aliquot of 0.5 - 3.6 ml of the centrifugate was transferred to a 5 ml test tube, 0.4 ml of tetraborate buffer was added, the mixture was diluted to a volume of 4 cm ³ with distilled water, and a PMM plate was added. The contents of the test tube were stirred for 30 min at a temperature of 20 - 25 °C, then removed and irradiated with UV light (365 nm). The procedure was then followed as described in Example 1. The results of tetracycline determination in urine samples are presented in Table 1.

Example 4. Determination of tetracycline content in a honey sample.

When analyzing a honey sample for tetracycline content, it was pre-prepared. For this, 20 g of sample was placed in a 50 ml plastic graduated test tube, and distilled water was added to bring the total volume to 45 ml. The sample was heated in a water bath for 10 min at 50°C and then centrifuged for 10 min at 4000 rpm. The sample was then filtered, a 3.6 ml aliquot of the filtrate was transferred to a 5 ml test tube, 0.4 ml of tetraborate buffer was added, and a PMM plate was inserted. The contents of the test tube were stirred for 60 min at 20-25°C, then removed and irradiated with UV light (365 nm). The procedure was then as described in Example 1. The results of tetracycline determination in urine samples are presented in Table 1.

Table 1 - Results of determination of tetracycline in real objects.

Object Entered, mg/l Found, mg/L S _r , % σ, % Natural drinking water "Juno" 0 < PO - - 0.050 0.053 ± 0.007 6 106 0.100 0.095 ± 0.012 8 95 River water 0 < PO - - 0.050 0.046 ± 0.006 5 92 0.10 0.12 ± 0.05 16 116 Human urine 0 < PO - - 15 16 ± 6 15 109 35 34 ± 9 10 97 Mixed-herb honey 0 < PO - - 0.10 0.11 ± 0.03 10 106

The results obtained, presented in Table 1, for all analyzed samples, indicate the correctness and repeatability of the proposed method for determining tetracycline.

The advantage of the claimed invention over the prior art is its wide range of detectable concentrations (the use of a polymethacrylate matrix allows for an expanded range of detectable tetracycline concentrations, lowering the lower limit of detection and increasing the upper limit by a factor of two), and the ease of tetracycline determination. A significant advantage is the use of a polymethacrylate matrix, which allows for the combined extraction of tetracycline and its concentration in the matrix phase, enabling quantitative determination of the antibiotic directly in the solid phase by its intrinsic fluorescence without the use of expensive auxiliary reagents.

Claims (1)

A method for determining tetracycline using a polymethacrylate matrix, which includes the interaction of the test solution with a polymethacrylate membrane in a tetraborate buffer medium, measuring the analytical signal and determining the tetracycline content, characterized in that the polymethacrylate matrix is used simultaneously as a solid-phase extractant for tetracycline for its concentration from solutions with a pH of 7.5–9.5 in a concentration range of 0.0025–2.0 mg/l and a medium for forming an analytical signal, wherein the tetracycline content is determined by its own yellow-green fluorescence directly in the solid phase of the polymethacrylate matrix at an emission wavelength of 520 ± 5 nm, the contact time of the test solution with the polymethacrylate membrane is 30–60 minutes at a temperature of 20–25 °C.