KR101933611B1 - Simultaneous detecting method of disinfectants and antiseptics - Google Patents

Abstract

Provided is a method of simultaneously detecting a disinfectant and a sanitizer. The method includes: a step (1) of preparing a specimen for differentiation by mixing a reference substance, a diluted solution, and a differentiation target substance; a step (2) of obtaining a chemical shift and its peak intensity which are expected to be used as search key data by measuring the specimen′s spectra for differentiation with quantitative nuclear magnetic resonance spectrometry (qNMR); a step (3) of designating a chemical shift (δ), which is not overlapped with search key data for the specimen for differentiation among search key data for a standard disinfectant and sanitizer, as a marker; a step (4) of identifying whether the specimen for differentiation contains a disinfectant and a sanitizer or not by using the number of designated markers; and a step (5) of calculating a quantitative curve from a marker of the standard disinfectant and sanitizer, and quantifying the content of the disinfectant and sanitizer by applying the content to the quantitative curve. The search key data for the standard disinfectant and sanitizer are the chemical shift (δ) and the peak intensity which are obtained by measuring quantitative nuclear magnetic resonance spectrometry (qNMR) spectra of the standard disinfectant and sanitizer.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for simultaneous detection of a disinfectant and a disinfectant,

The present invention relates to a method and apparatus for detecting and disinfecting disinfectants and disinfectants using Quantitative Nuclear Magnetic Resonance Spectroscopy (qNMR), and more particularly, to disinfectants and disinfectants added to fragrances, air fresheners, deodorizers, humidifiers, And chemical shift (?) NMR markers for NMR and antiseptics, thereby confirming the presence or absence of bactericides and disinfectants, and measuring the content of the added components.

The major known disinfectants are polyhexamethylene guanidine (PHMG), didecyldimethylammonium chloride (DDAC) and methylisothiazolonone (MIT). This compound not only exhibits biocidal and anti-biofouling, but also plays an important role in detergents and anti-corrosive paints for containers and surfaces. [J. of Nanosci. & Tech. Studies have shown that PHMG and oligo- [2- (2-ethoxy) ethoxyethyl] guanidine (PGH) Curing, cytotoxicity and aging. In addition, a study of [Inhalation Toxicology 23: 476-485] [Experimental and Toxicologic Pathology 62: 643-651] showed that DDAC is a cytotoxic, proinflammatory and total fibrosing agent that induces pulmonary inflammation in mice. It has also been reported that MIT, used as a preservative and biocide, causes allergic reactions [Contact Dermatitis 67: 334-341].

The disinfectant and disinfectant present in the enclosed space can directly enter the bronchi and the lungs to remove the odor and kill the bacteria with the fragrant air freshener containing the disinfectant, adversely affecting respiratory diseases such as asthma, rhinitis, bronchus or pneumonia [J Korean Medical Science 31: 817-821] [Journal of Toxicologic Pathology 29: 95-102] [Environmental Health and Toxicology 31: e2016014-2016010].

Disinfectants such as PHMG, DDAC, and MIT can be analyzed by other conventional methods. For example, PHMG crystals can be prepared using Nestler reagent, UV-Vis spectrometer [Inter. J. Anal. Chem. 2011: 7] and Matrix Assisted Laser Desorption / Ionization Time-of-Flight Mass Spectroscopy (MALDI-TOF) [Rapid Communications in Mass Spectrometry 29: 213-219] [Materials Science and Engineering: C 29: 1776-1780]. DDAC analysis can be analyzed by ion chromatography and liquid chromatography time-of-flight mass spectrometry (LC-TOF / MS) [Journal of Toxicologic Pathology 29: 95-102]. In addition, DDAC can be detected by functionalized gold nanoparticles in aqueous solution [Cardiovascular Toxicology 13: 148-160].

MIT can be quantified using ion pair high performance liquid chromatography (Ion pair-HPLC) [Fresenius' Zeitschrift fur analytische Chemie 333: 806-809] [Perkin Transactions 1: 2245-2251]. However, analysis of disinfectants based on these analytical methods is difficult, difficult, time-consuming, chemical derivatization and skilled professionals due to their low sensitivity and selectivity.

It is an object of the present invention to provide a method for simultaneously detecting a disinfectant and a disinfectant, and to provide a method of identifying whether a disinfectant and a disinfectant are added or not by using quantitative nuclear magnetic resonance spectroscopy (qNMR) and quantifying the same.

Other objects and advantages of the present invention will become apparent from the following description. Further, objects and advantages of the present invention can be realized by the means or method described in the claims, and the combination thereof.

According to one embodiment of the present invention, there is provided a method of preparing a sample for discrimination by mixing a reference substance, a diluent, and a substance to be distinguished, comprising the steps of: (1) preparing a sample for identification; (2) using quantitative nuclear magnetic resonance spectroscopy (2) obtaining a chemical shift value (δ) and its intensity (peak intensity) to be used as search key data by measuring the spectrum of the sample for discrimination, (3) determining a chemical shift (delta) that does not overlap with the search key data as a marker (3); determining whether the discriminating sample contains a disinfectant and a disinfectant through the determined number of markers (4) and a step of obtaining a quantitative curve from the marker of the standard product sterilizing agent, the marker of the standard product sterilizing agent and the disinfectant, and substituting the quantitative curve into the quantitative curve to quantify the content of the sterilizing agent and the disinfecting agent ), And the data of the standard product disinfectant and disinfectant search key includes a chemical shift (?) Obtained by measuring a quantitative nuclear magnetic resonance spectroscopy (qNMR) spectrum of a standard disinfectant and a disinfectant, and a disinfectant And a method for simultaneous detection of disinfectants.

In this case, the reference material is tetramethylsilane (TMS) containing methyl hydrogen in the molecule, and the chemical shift (delta) and the intensity of the peak of the reference sample And a chemical shift (delta) with respect to tetramethylsilane (TMS) at 0 ppm.

Also, the diluent is at least one of methanol-d ₄ , ethanol-d ₆ , deuterium oxide, acetonitrile-d ₃ and acetone-d ₆ , and the content of the substance to be discriminated is 0.001 to 100% It can be diluted.

Also, when the chemical shift (delta) of tetramethylsilane (TMS) relative to methyl hydrogen is set to 0 ppm, the search key data of the marker is obtained from the spectrum of the search key data of the discriminating sample May be a chemical shift (delta) and peak intensity present between 0.5 and 12.0 ppm.

When the chemical shift (delta) of tetramethylsilane (TMS) relative to methyl hydrogen was set to 0 ppm, the marker had intrinsic peaks of the disinfectant and disinfectant of 0.30 ppm, 1.37 ppm, 1.34 to 1.43 ppm , 1.59 ppm, 3.17 ppm, 6.32 ppm, and 8.48 ppm can be used as a search key.

The present invention uses a quantitative nuclear magnetic resonance spectroscopy (qNMR) to simultaneously detect a fungicide and a disinfectant in a sample such as a perfume target object, an air freshener, a deodorant, a humidifier, a cosmetic or a therapeutic agent, There is an advantage that it can be detected quickly and accurately without a preprocessing process.

FIG. 1 is a flowchart briefly showing a simultaneous detection method of a disinfectant and a disinfectant according to an embodiment of the present invention.
Figure 2 is a schematic representation of a well-resolved ¹ H NMR signal of polyhexamethylene guanidine (PHMG), didecyldimethylammonium chloride (DDAC), and methylisothiazolonone (MIT) to be.
Figure 3 shows the effect of the well-resolved ¹ H NMR signals of polyhexamethylene guanidine (PHMG), didecyldimethylammonium chloride (DDAC), and methylisothiazolonone (MIT) As shown in Fig.
Figure 4 shows the ¹ H NMR spectra of the samples with DDAC in the undetected sample (Sample No. 4, a), the detected samples (Sample No. 8, b) and the standard sample (c) It is data showing that it has been confirmed.
Figure 5 shows the MIT peaks at constant chemical shifts relative to the sample number, showing that the MIT was not detected (sample number 14, a), the detected sample (sample number 13, b) and the MIT reference material marker (c) It is the data that confirms that MIT is contained in sample number 19.

Hereinafter, the present invention will be described in detail. Prior to this, terms or words used in this specification and claims should not be construed in a conventional or dictionary sense, and the inventor should properly define the concept of the term to best describe its invention The present invention should be construed in accordance with the meaning and concept consistent with the technical idea of the present invention. Therefore, the embodiments described in the present specification and the configurations shown in the drawings are only the most preferred embodiments of the present invention and do not represent all the technical ideas of the present invention. Therefore, at the time of the present application, It should be understood that various equivalents and modifications are possible.

FIG. 1 is a flowchart briefly showing a simultaneous detection method of a disinfectant and a disinfectant according to an embodiment of the present invention. Referring to FIG. 1, the simultaneous detection method of the disinfectant and the disinfectant of the present invention comprises the steps of (1) preparing a sample for discrimination by mixing a reference substance, a diluent, and a substance to be discriminated, (2) using quantitative nuclear magnetic resonance spectroscopy (2) obtaining a chemical shift value (δ) and its intensity (peak intensity) to be used as search key data by measuring the spectrum of the sample for discrimination, (3) determining a chemical shift (delta) that does not overlap with the search key data as a marker (3); determining whether the discriminating sample contains a disinfectant and a disinfectant through the determined number of markers (5) of obtaining a quantitative curve from the marker of the standard product sterilizing agent and disinfectant, and substituting the quantitative curve into the quantitative curve to quantify the content of the sterilizing agent and the disinfectant, Standard disinfectants and antiseptics search key data is a standard disinfectant and quantitative nuclear magnetic resonance spectroscopy (qNMR) chemical shift values obtained by measuring the spectrum (chemical shift, δ) and the intensity (peak intensity) of the disinfecting agent.

The analysis using nuclear magnetic resonance spectroscopy (NMR) is based on the phenomenon that occurs when a nucleus in a magnetic field resonates with electromagnetic waves of a specific frequency to determine the type of hydrogen and the number of different types of hydrogen chemically and magnetically in the molecule It is an analytical method that can obtain information. Therefore, the use of a nuclear magnetic resonance spectrometer provides information on the surrounding chemical and magnetic environment in the presence of hydrogen in a sample, and thus may provide sufficient information to identify unknown molecular components and structures .

The reference material is tetramethylsilane (TMS) containing methyl hydrogen in the molecule, and the chemical shift (delta) and peak intensity of the discrimination sample are determined by measuring the tetramethyl And a chemical shift (delta) to silane (Tetramethylsilane, TMS) of 0 ppm.

In addition, the diluent is methanol -d _4, ethanol -d _6, deuterium oxide (Deuterium oxide), acetonitrile and acetone -d -d ₃ is at least one of _6, the differential substance 0.001% to 100% by weight Or may be diluted to preferably 0.001 wt% to 95 wt%, more preferably 0.001 wt% to 80 wt%.

The method may further include the step (5) of quantifying the amount of the disinfectant and the disinfectant following the step (4) if it is determined that the substance to be discriminated includes the disinfectant and the disinfectant in the step (4). In step (5), a quantitative curve is obtained from the marker of the standard product sterilizing agent and the disinfectant, and the content of the sterilizing agent and the disinfecting agent can be quantified by substituting the quantitative curve into the quantitative curve.

The chemical shift value (δ) and the intensity of the chemical shift (δ) to be used as the retrieval key data obtained in the step (2) and the chemical shift value obtained by measuring the quantitative nuclear magnetic resonance spectroscopy (qNMR) spectrum of the disinfectant and disinfectant (chemical shift,?) and its intensity (peak intensity) to determine the chemical shift value of the position where the peaks overlap, and determine the number of the specified markers to be 3 or more, preferably 5 or more, If there are more than 6, it is recognized that the disinfectant and disinfectant are contained. At this time, all of the retrieval of the retrieval key data, the selection of the marker through data comparison, and the determination of whether the disinfectant and the disinfectant are contained can be automatically performed by a computer program.

The external standard method can generally be used to quantitate the analyte, and in this method, a calibration curve of the ¹ H NMR peak intensity of the marker position according to the standard concentration of the unknown sample can be confirmed. The above correlation can be calculated as the content indicating the relationship between the standard peak intensity and the disinfectant concentration of the analyte. A linear regression analysis of the calibration curve is then performed. The content of fungicide and disinfectant in the sample to be detected can be obtained by the following equation.

Equation 1

Unknown concentration (μg / ml) = concentration of standard product (μg / ml) x [(TMS peak area ratio included in the corresponding marker and solvent in the sample) / (TMS peak area ratio included in the standard marker and solvent)] x [ Volume of solvent (ml) / Weight of sample (g)]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to examples. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the embodiments described below. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

In an embodiment of the present invention, D ₂ O and DMSO-d ₆ solvents containing a measurement temperature of 25 ° C and 0.05% TMS, a standard product and a sample with a concentration of 25 ° C were subjected to FT-NMR at 600 MHz (manufactured by Agilent ) Equipment. The chemical shift (δ) of the methyl hydrogen peak of TMS was determined to be 0 ppm at the measurement frequency of 32 times, and the chemical shifts obtained from the standard solution and the sample were obtained.

Further, through the following examples, it is possible to obtain a quantitative graph using a non-overlapping inherent chemical shift value and a strength of a disinfectant and a disinfectant compound obtained by measuring NMR spectra of a reference material search key of a disinfectant and a disinfectant, And the content thereof is determined. In addition, identification of the bactericide and disinfectant can be performed using nuclear magnetic resonance spectroscopy ( ¹ H NMR). The bactericide and disinfectant identified in the present example are polyhexamethylene guanidine (PHMG) , Didecyldimethylammonium chloride (DDAC, the following formula B), and methylisothiazolonone (MIT, the following formula C).

A

Formula B

Formula C

Example  1. Using nuclear magnetic resonance spectroscopy PHMG , DDAC  And MIT

Figure 2 is a schematic representation of a well-resolved ¹ H NMR signal of polyhexamethylene guanidine (PHMG), didecyldimethylammonium chloride (DDAC), and methylisothiazolonone (MIT) , And the relative concentration is determined according to the marker signal displayed on D ₂ O.

Figure 3 also shows the well-resolved ¹ H NMR signals of polyhexamethylene guanidine (PHMG), didecyldimethylammonium chloride (DDAC), and methylisothiazolonone (MIT) As a graph of the correlation and quantitation as represented by chemical shifts, the intensity of protons in these spectra show practically different aspects due to the different chemical structures and environments of the major constituents. However, the peaks assigned to samples containing disinfectants and fungicides have been identified in comparison to the theoretical spectrum predicted by standard disinfectants and MestReNova software.

Figure 2a shows that the H-1 polymorphism at 3.17 ppm, H-2 at 1.59 ppm and H-3 at 1.43-1.34 ppm were well resolved for PHMG and can be used as a search key and marker. And FIG. 2B shows that the ¹ H NMR peak of DDAC is decomposed at 1.37 ppm at H-5 to 10 at 1.30 ppm to H-11 and can be used as a search key and a marker.

In Figure 2c, MIT shows the resonance of H-3 at 6.32 ppm at H-2 and 8.48 ppm and can be used as a search key and marker. The relative concentration of the disinfectant corresponding to the chemical shift of the search key and the marker can serve as an indicator of the disinfectant and bactericide concentration associated with the sample.

Example  2. Using nuclear magnetic resonance spectroscopy PHMG , DDAC  And quantification of MIT

Fig. 3 shows the correlation coefficient (r ² ) used as a search key and a marker, and good linearity of more than 0.9986 was obtained for all markers. The regression equations for PHMG (1.59 ppm), DDAC (1.30 ppm) and MIT (6.32 ppm) were y = 0.0030x + 0.0627, y = 0.0022x + 0.0623 and y = 0.0024x + 0.0042, respectively.

The detection limit (LOD) of PHMG, DDAC and MIT can be determined as 1.73, 1.03 and 1.45 μg / mL, respectively, based on chemical shift as a marker that is not superimposed on the chromatogram.

The relative standard deviation (RSD) of PHMG was 2.44 ~ 13.0% at the concentration of 2 ~ 10 μg / mL. In addition, the relative standard deviation (RSD) of intra-day and inter-day DDAC ranges from 6.15 to 10.95% in the same concentration range and the corresponding value in MIT is 3.55 to 12.89%. Daily and weekly accuracy and accuracy were within acceptance criteria. (ICH topic Q2B 1996, Industry Guidance for Industry 2001). This validation data is summarized in Table 1.

Analyte Chemical shift (隆) Correlation coefficient
(r ² ) (n = 3) Regression equation LOD
(μg / mL) LOQ
(LOD
(μg / mL) Intra-day analysis (n = 3) (LOD
(μg / mL) Inter-day (1 ^st , 3 ^rd , 5 ^th day) analysis (n = 3) (LOD (μg / mL) Recovery (%) Precision (% RSD) Recovery (%) Precision (% RSD) 2 5 10 2 5 10 2 5 10 2 5 10 PHMG H-2
(delta 1.59) 0.9987 y = 0.0030x + 0.0627 1.73 5.72 110.16 ±
10.77 127.29 + - 8.65 99.10 + - 2.41 9.78 6.80 2.44 109.26 ± 14.21 106.09 ± 12.21 106.47 ± 4.89 13.00 11.42 4.59 DDAC H-5 ~ 10
(delta 1.30) 0.9986 y = 0.0022x + 0.0623 1.03 3.40 84.07 ± 9.20 115.07 ± 11.25 90.19 + - 8.78 10.95 10.79 9.74 87.61 ± 0.05 107.04 + - 9.78 97.02 + - 10.37 6.15 8.33 10.68 MIT H-3
(delta 6.32) 0.9997 y = 0.0024x + 0.0042 1.73 5.72 90.73 ± 11.69 105.29 ± 11.47 104.82 ± 3.84 12.89 10.90 3.67 90.73 + - 7.34 105.29 + - 7.41 104.82. + -. 3.41 7.53 7.33 3.55

Figure 4 shows the ¹ H NMR spectra of the samples with DDAC in the undetected sample (Sample No. 4, a), the detected samples (Sample No. 8, b) and the standard sample (c) Show that it has been confirmed. Proton markers appear in certain chemical shifts and can be used for quantification.

Figure 5 shows MIT peaks in constant chemical shifts for sample numbers. It can be confirmed that MIT is contained in the sample No. 19 in which the MIT is not detected (Sample No. 14, a), the detected sample (Sample No. 13, b) and the MIT standard substance marker (c).

Example  3. Available on the market Analysis results for samples

Table 2 shows the disinfectant and disinfectant contents contained in the commercially available fragrance and disinfectant. In the 17 samples of aroma agents, disinfectants, air fresheners, deodorants and humidifier solutions that are flowing in the market, the quantitative curves and chemical markers are prepared on the samples through the tests on PHMG, DDAC and MIT as described above. The test was performed using a graph. The DDAC and MIT contents of the samples were estimated to be 0.28 and 0.32%, respectively, but the corresponding MIT contents were 78.89 and 52.3 ppm.

Sample
No. PHMG
(delta 1.58) DDAC
(delta 1.30) MIT
([delta] 8.47) One N.D. N.D. N.D. 2 N.D. N.D. N.D. 3 N.D. N.D. N.D. 4 N.D. N.D. N.D. 5 N.D. N.D. N.D. 6 N.D. N.D. 78.89 7 N.D. 3208.05 N.D. 8 N.D. 2856.68 N.D. 9 N.D. N.D. N.D. 10 N.D. N.D. N.D. 11 N.D. N.D. N.D. 12 N.D. N.D. N.D. 13 N.D. N.D. 52.3 14 N.D. N.D. N.D. 15 N.D. N.D. N.D. 16 N.D. N.D. N.D. 17 N.D. N.D. N.D.

N.D: Not detected

Unit: μg / ml

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention.

Claims (5)

(1) preparing a sample for discrimination by mixing tetramethylsilane (TMS), a diluent, and a substance to be distinguished;
(2) obtaining a chemical shift value (δ) and a peak intensity to be used as search key data by measuring the spectrum of the discriminating sample using quantitative nuclear magnetic resonance spectroscopy (qNMR);
(3) determining, as a marker, a chemical shift (?) Not overlapping with the retrieval key data of the discriminating sample among the standard product sterilizing agent and disinfectant search key data;
Identifying (4) whether the discriminating sample includes a disinfectant and a disinfectant through the number of the predetermined markers; And
(5) determining a quantitative curve from the marker of the standard product sterilizing agent and the disinfectant, and substituting the quantitative curve into the quantitative curve to quantify the content of the sterilizing agent and the disinfectant,
The above-mentioned reference material disinfectant and disinfectant search key data are a chemical shift (δ) and a peak intensity obtained by measuring a quantitative nuclear magnetic resonance spectroscopy (qNMR) spectrum of a standard disinfectant and a disinfectant,
The standard disinfectant and disinfectant are at least one of polyhexamethylene guanidine (PHMG), didecyldimethylammonium chloride (DDAC), and methylisothiazolonone (MIT)
When the chemical shift (delta) of tetramethylsilane (TMS) relative to methyl hydrogen was set at 0 ppm, the marker for polyhexamethylene guanidine (PHMG) had a specific peak of 1.34 ppm 1.43 ppm, 1.59 ppm, or 3.17 ppm is used as a search key and the marker for didecyldimethylammonium chloride (DDAC) is used as a search key for its inherent peak of 1.30 ppm or 1.37 ppm, and methyl isothiazolone methylisothiazolonone, MIT) is used as a search key for its intrinsic peak of 6.32 ppm or 8.48 ppm. delete The method according to claim 1,
The diluent is methanol -d _4, ethanol -d _6, and deuterium oxide (Deuterium oxide), acetonitrile, acetone -d -d ₃ and _6, at least one, to dilute the substance in the differential 0.001% to 100% by weight Lt; RTI ID = 0.0 > of disinfectant < / RTI > delete delete

Images