CN109521125B - Detection method of anti-microbial mildew preventive in bamboo and wood products - Google Patents
Detection method of anti-microbial mildew preventive in bamboo and wood products Download PDFInfo
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Abstract
The invention relates to the technical field of detection, in particular to a detection method of an antibacterial and mildewproof agent in bamboo and wood products. Cutting and shearing a solid sample into particles with the particle size of less than 5mm x 5mm, weighing 4g of crushed sample in a 50mL centrifuge tube, adding 20mL of methanol, screwing down a cover, uniformly mixing by vortex, soaking for 60min, then carrying out ultrasonic extraction for 10min, centrifuging for 5min at 3000r/min, transferring supernatant into a 50mL volumetric flask, repeatedly extracting for 1 time, fixing the volume to the scale, transferring into a 125mL heart-shaped flask, evaporating to be nearly dry by using a rotary evaporator, fixing the volume to 1mL by using methanol, passing through a 0.45 mu L organic filter membrane, and inputting into a high performance liquid chromatograph equipped with a diode array detector for detection. The detection method provided by the invention can be used for quickly and accurately detecting the 15 antibacterial and mildewproof agents in the bamboo and wood products, so that the effective monitoring on the bamboo and wood food related products is enhanced.
Description
Technical Field
The invention belongs to the technical field of detection, and particularly relates to a detection method of an antibacterial and mildewproof agent in bamboo and wood products.
Background
The antibacterial and mildewproof agent is actually a bactericide, and the action mechanism of the antibacterial and mildewproof agent mainly has three aspects: inhibiting protein synthesis to solidify thallus; inhibiting synthesis of ergot crab alcohol, and inactivating enzyme; preventing metabolism function, and inhibiting spore production or spore germination. The application range of the mildew preventive is gradually expanded, the requirements of various countries in the world on environmental safety are gradually strict, and the mildew preventive serving as a good industrial mildew preventive has the following characteristics: high efficiency, broad spectrum, strong antibacterial power and effective to various mildews and putrefaction fungi; low toxicity, safety and reliability to human, livestock and surrounding environment; the stability is good, the applicable p H value and temperature range are wide, and the effective duration is sufficient; the use is convenient and the material performance is not damaged; wide source and low price.
Bacterial mould spores are ubiquitous, the severe environment can still grow tenaciously, and in the backsouth season of the south of China, the temperature and the humidity provide good external environment for bacterial moulds. Food contact materials are more susceptible to bacterial mold contamination and spoilage because of more or less high nutrient content in the food.
The final action product of the anti-bacterial and anti-mildew additive is edible for human, and the safety regulation requirement is strict, so that the variety of the anti-bacterial and anti-mildew agent for food can be approved, and is not many in various countries in the world, for example, only 5 types of U.S. food bactericides are allowed to be used, wherein 4 types of the anti-bacterial and anti-mildew agent are bleaching powder, the other type of the anti-bacterial and anti-mildew agent is hydrogen peroxide, and the preservative is dehydroacetic acid, sorbic acid, benzoic acid vinegar and the; the Japanese approved products include sorbic acid, p-hydroxybenzoic acid vinegar, dehydroacetic acid, hydrogen peroxide and other traditional old products; the feed anti-bacteria mildew preventive is only 83 in Japan, and the mildew preventive varieties in the United states and the like are only chloropropelin, chlorophyllin, and sulfanilamide mosquito line and the like. The antibacterial and antifungal agent is used in various production links, and is added in each process from an initial raw material to a semi-finished product and a final shaping finished product to ensure the stable quality of the final product, such as small daily wooden products, raw wood is treated by the antibacterial agent such as metal salt, phenols and the like, the antibacterial and antifungal agent is added in an adhesive used during processing and molding, the synthetic resin of the raw material of the adhesive is also added, and the coating for surface treatment of the final product is also added, so the antibacterial and antifungal chemical agent is a procedure for maintaining the product quality.
The bamboo and wood antibacterial mildew inhibitor has corresponding detection methods for only thiabendazole, o-phenylphenol, biphenyl, imazalil, pentachlorophenol and the like in the national standard regulation. The effective component of bamboo defence No. II used in practical production uses bactericide MBT as main body, and adds a certain quantity of pesticide SFS, etc. to form compound preparation. In addition, in the field investigation of bamboo and wood food related products, manufacturers can fumigate bamboo with antimildew II, chlorfenapyr and sulfur dioxide, even household workshop-type small manufacturers use unknown antimildew and insect-proof agents, but 3-iodine-2-propylbutyl methyl carbamate, didecyl dimethyl ammonium chloride, 2, 5-dichloro-3-bromophenol, sodium pentachlorophenate, chlorothalonil, carbendazim, imazalil, thiabendazole, tebuconazole, dimethyl fumarate, lindane, thiocyanogen, sodium benzoate, potassium sorbate, 8-hydroxyquinoline copper, natamycin, captan, sulfur dioxide, salicylanilide, o-phenylphenol, thiabendazole, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, butyl p-hydroxybenzoate, formaldehyde, glutaraldehyde, benzyl alcohol, biphenyl and other common antimildew and mildew-proof agents are not provided with relevant standards and corresponding detection methods at present, therefore, the prior bamboo and wood food related products cannot be effectively monitored, so that great potential safety hazards exist.
Disclosure of Invention
In order to solve the problem that the conventional detection means can only detect a plurality of conventional antibacterial preservatives and cannot effectively monitor related bamboo and wood food products, the invention provides a detection method of an antibacterial and mildewproof agent in a bamboo and wood product, which can simultaneously detect a plurality of antibacterial preservatives in the bamboo and wood food related products.
A detection method of an antibacterial and mildewproof agent in bamboo and wood products comprises the following steps:
s1, sample pretreatment: taking a proper amount of solid sample, cutting and shearing the solid sample into particles with the particle size of less than 5mm x 5mm, weighing 4g of crushed sample in a 50mL centrifuge tube, adding 20mL of methanol, screwing down a cover, uniformly mixing by vortex, soaking for 60min, then carrying out ultrasonic extraction for 10min, centrifuging at 3000r/min for 5min, transferring supernatant into a 50mL volumetric flask, repeatedly extracting for 1 time, fixing the volume to the scale, transferring the supernatant into a 125mL heart-shaped flask, evaporating the supernatant to be nearly dry by using a rotary evaporator, fixing the volume to 1mL by using methanol, and passing through a 0.45 mu L organic filter membrane to be detected.
S2, sample detection: inputting the sample extracting solution into a high performance liquid chromatograph equipped with a diode array detector for detection, wherein the detection conditions comprise that a chromatographic column: agilent ZORBAX SB-C18; the mobile phase A is ammonium acetate, the mobile phase B is methanol, and the mobile phase A contains 0.03% of glacial acetic acid; column temperature: 35-45 ℃.
Further, the gradient elution conditions of the mobile phase were:
wherein the molar concentration of the ammonium acetate is 20mmol/L, and the mobile phase A and the mobile phase B are mixed according to volume percentage.
Further, the gradient elution conditions of the mobile phase were:
wherein the molar concentration of the ammonium acetate is 20mmol/L, and the mobile phase A and the mobile phase B are mixed according to volume percentage.
Preferably, the gradient elution conditions of the mobile phase are:
further, the column size was 5 μm, 250 mm. times.4.6 mm.
Further, the column temperature in step S2 was 40 ℃.
Further, the sample amount in step S2 is 20 μ L.
Further, the detection wavelength of the high performance liquid chromatograph is 230nm and/or 254 nm.
Further, the antibacterial preservative comprises any one or more of benzoic acid, sorbic acid, benzyl alcohol, dimethyl fumarate, imazalil, biphenyl, carbendazim, methyl paraben, ethyl paraben, propyl paraben, butyl paraben, thiabendazole, natamycin, o-phenylphenol and pentachlorophenol.
Specifically, when the detection wavelength is 230nm, measuring benzoic acid, sorbic acid, benzyl alcohol, dimethyl fumarate, imazalil and biphenyl; when the detection wavelength is 254nm, the carbendazim, the methyl paraben, the ethyl paraben, the propyl paraben, the butyl paraben, the thiabendazole, the natamycin, the o-phenylphenol and the pentachlorophenol are measured.
Compared with the prior art, the invention has the following beneficial effects:
(1) according to the detection method provided by the invention, the sample is crushed into small particles in the pretreatment stage, and then the antibacterial preservative in the sample is quickly and fully extracted through quantitative methanol soaking for a specific time, so that the recovery rate of the antibacterial preservative is greatly improved compared with the existing extraction mode.
(2) In the detection process of the detection method provided by the invention, when a mixed sample enters the inlet of the chromatographic column along with methanol and ammonium acetate at the starting point of the gradient, strong sodium pentachlorophenate, biphenyl, imazalil and the like can be retained near the inlet of the chromatographic column due to high distribution coefficient (k value) and hardly move because the content of the strong elution solvent methanol in the mobile phase at the starting point is low; after a certain period of time, the methanol is increased to a certain value, the k value of the compound is small enough, the moving speed of the compound in the column is obviously increased, and the moving speed of the compound is increased rapidly along with the increase of the methanol until the compound flows out of the chromatographic column, the average k value of the compound is small although the moving speed is far longer than the retention time of the compound at the same degree, the compound is detected by a detector and recorded into a chromatographic peak, and the chromatographic peak is not widened, so that the sensitivity is at a high level.
In conclusion, the detection method provided by the invention can be used for effectively, quickly and accurately detecting 15 common antibacterial and antifungal agents and pesticide insect-resistant agents in bamboo and wood food related products, such as benzoic acid, sorbic acid, benzyl alcohol, dimethyl fumarate, imazalil, biphenyl, carbendazim, methyl paraben, ethyl paraben, propyl paraben, butyl paraben, thiabendazole, natamycin, o-phenylphenol and pentachlorophenol, and is beneficial to providing an effective monitoring means for bamboo and wood food related products.
Description of the drawings:
the present invention will be described in further detail with reference to the accompanying drawings and examples.
FIG. 1 is a high performance liquid chromatogram of example 1 at a wavelength of 230 nm;
FIG. 2 is a high performance liquid chromatogram of example 1 at a wavelength of 254 nm;
FIG. 3 is a high performance liquid chromatogram of example 2 at a wavelength of 230 nm;
FIG. 4 is a high performance liquid chromatogram of example 2 at a wavelength of 254 nm;
FIG. 5 is a high performance liquid chromatogram of example 3 at a wavelength of 230 nm;
FIG. 6 is a high performance liquid chromatogram of example 3 at a wavelength of 254 nm.
Detailed Description
The invention is further illustrated by the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it is to be understood that various changes or modifications may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present application.
The following details are described with respect to the present invention:
1. reagents and materials
Unless otherwise stated, all reagents were analytically pure, water was tertiary water as specified in GB/T6682;
15 standard products of the antibacterial preservative: benzoic acid, sorbic acid, benzyl alcohol, dimethyl fumarate, imazalil, biphenyl, carbendazim, methyl paraben, ethyl paraben, propyl paraben, butyl paraben, thiabendazole, natamycin, o-phenylphenol and pentachlorophenol, wherein the concentration of the standard is 6.6 mg/L.
Preparation of a standard solution: the above 5 standard samples were prepared from CNW company of Germany by preparing the above 15 substances with methanol into single standard stock solutions with a concentration of 1000 mg/L. Taking 1mL of each of 15 single-standard stock solutions, and mixing uniformly to obtain a standard intermediate solution with the concentration of 66 mg/L.
2. Apparatus and device
High performance liquid chromatograph: equipped with a diode array detector (SHIMADZU corporation, SHIMADZU, japan); analytical balance (weight instruments for sheep, constant-maturity city, ltd): the sensory amounts are 0.0001g and 0.01 g; vortex oscillator (IKA, germany); an ultrasound extractor (Guangzhou Jeep ultrasonic electronics, Inc.); rotary evaporator (tokyo physical & chemical company, japan).
Example 1
Determination of antibacterial preservative in bamboo chopsticks in Jiangmen City of Guangdong province
Taking a proper amount of solid sample, cutting and shearing the solid sample into particles with the particle size of less than 5mm x 5mm, weighing 4.00g (accurate to 0.01g) of crushed sample in a 50mL centrifuge tube, adding 20mL of methanol, screwing down a cover, uniformly mixing by vortex, soaking for 60min, then carrying out ultrasonic extraction for 10min, centrifuging for 5min at 3000r/min, transferring supernatant into a 50mL volumetric flask, repeatedly extracting for 1 time, fixing the volume to scale, transferring to a 125mL heart-shaped flask, evaporating to near dryness by using a rotary evaporator, fixing the volume to 1mL by using methanol, passing through a 0.45 mu L organic filter membrane, entering a high performance liquid chromatograph, and respectively measuring at the wavelength of 230nm and 254 nm.
The detection conditions include chromatographic column: agilent ZORBAX SB-C18; the mobile phase A is ammonium acetate, the mobile phase B is methanol, and the mobile phase A contains 0.03 percent of glacial acetic acid; column temperature: at 40 ℃. The gradient elution conditions for the mobile phase are shown in table 1. Table 1 shows the ratio adjustment of mobile phase a to mobile phase B in each time period in example 1.
The results are shown in FIGS. 1 and 2. Wherein FIG. 1 is a high performance liquid chromatogram at a wavelength of 230nm of the present example, and FIG. 2 is a high performance liquid chromatogram at a wavelength of 254nm of the present example.
Example 2
Determination of antibacterial preservative in bamboo food steamer of Rouding City, Guangdong province
Taking a proper amount of solid sample, cutting and shearing the solid sample into particles with the particle size of less than 5mm x 5mm, weighing 4.00g (accurate to 0.01g) of crushed sample in a 50mL centrifuge tube, adding 20mL of methanol, screwing down a cover, uniformly mixing by vortex, soaking for 60min, then carrying out ultrasonic extraction for 10min, centrifuging for 5min at 3000r/min, transferring supernatant into a 50mL volumetric flask, repeatedly extracting for 1 time, fixing the volume to scale, transferring to a 125mL heart-shaped flask, evaporating to near dryness by using a rotary evaporator, fixing the volume to 1mL by using methanol, passing through a 0.45 mu L organic filter membrane, entering a high performance liquid chromatograph, and respectively measuring at the wavelength of 230nm and 254 nm.
The detection conditions include chromatographic column: agilent ZORBAX SB-C18; the mobile phase A is ammonium acetate, the mobile phase B is methanol, and the mobile phase A contains 0.03 percent of glacial acetic acid; column temperature: at 40 ℃. The gradient elution conditions for the mobile phase are shown in table 2. Table 2 shows the ratio adjustment of mobile phase a to mobile phase B in each time period in example 2.
The results are shown in FIGS. 3 and 4. FIG. 3 is a high performance liquid chromatogram at a wavelength of 230nm of the present example, and FIG. 4 is a high performance liquid chromatogram at a wavelength of 254nm of the present example.
Example 3
Determination of antibacterial preservative in bamboo chopping block of Rouding City, Guangdong province
The detection method of this example is substantially the same as that of example 2, except that the mobile phase has different gradient elution conditions, which are shown in table 3. Table 3 shows the adjustment of the ratio of mobile phase a to mobile phase B at each time point in example 3.
The results are shown in FIGS. 5 and 6. FIG. 5 is a high performance liquid chromatogram of the present example at a wavelength of 230 nm; FIG. 6 is a high performance liquid chromatogram of the present example at a wavelength of 254 nm.
The above description is only a preferred embodiment of the present invention, and not intended to limit the present invention, and the scope of the present invention is defined by the appended claims, and all changes that come within the meaning and range of equivalency of the specification are therefore intended to be embraced therein.
Claims (3)
1. A detection method of an antibacterial and mildewproof agent in bamboo and wood products is characterized by comprising the following steps:
s1, sample pretreatment: taking a proper amount of solid sample, cutting and shearing the solid sample into particles with the particle size of less than 5mm x 5mm, weighing 4g of crushed sample in a 50mL centrifuge tube, adding 20mL of methanol, screwing down a cover, uniformly mixing by vortex, soaking for 60min, then carrying out ultrasonic extraction for 10min, centrifuging at 3000r/min for 5min, transferring supernatant into a 50mL volumetric flask, repeatedly extracting for 1 time, fixing the volume to the scale, transferring the supernatant into a 125mL heart-shaped flask, evaporating the supernatant to be nearly dry by using a rotary evaporator, fixing the volume to 1mL by using methanol, and passing through a 0.45 mu m organic filter membrane to be detected;
s2, sample detection: inputting the sample extracting solution into a high performance liquid chromatograph equipped with a diode array detector for detection, wherein the detection conditions comprise that a chromatographic column: agilent ZORBAX SB-C18; the mobile phase A is ammonium acetate, the mobile phase B is methanol, and the mobile phase A contains 0.03% of glacial acetic acid; column temperature: 40 ℃;
when the detection wavelength of the high performance liquid chromatograph is 230nm, measuring benzoic acid, sorbic acid, benzyl alcohol, dimethyl fumarate, imazalil and biphenyl simultaneously;
when the detection wavelength of the high performance liquid chromatograph is 254nm, simultaneously detecting carbendazim, methyl paraben, ethyl paraben, propyl paraben, butyl paraben, thiabendazole, natamycin, o-phenylphenol and pentachlorophenol;
wherein the gradient elution conditions of the mobile phase are as follows:
wherein the molar concentration of the ammonium acetate is 20mmol/L, and the mobile phase A and the mobile phase B are mixed according to volume percentage.
2. The detection method according to claim 1, wherein the size of the chromatographic column is 5 μm, 250mm x 4.6 mm.
3. The detection method according to claim 1, wherein the sample amount in the step S2 is 20 μ L.
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| CN104198621A (en) * | 2014-09-26 | 2014-12-10 | 云南中烟工业有限责任公司 | Method for simultaneous determination of special migration volume of multiple preservatives in paper and paperboard |
| CN108169371A (en) * | 2017-12-21 | 2018-06-15 | 上海微谱化工技术服务有限公司 | The method for quantitatively determining of fungicide in a kind of coating |
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| CN104198621A (en) * | 2014-09-26 | 2014-12-10 | 云南中烟工业有限责任公司 | Method for simultaneous determination of special migration volume of multiple preservatives in paper and paperboard |
| CN108169371A (en) * | 2017-12-21 | 2018-06-15 | 上海微谱化工技术服务有限公司 | The method for quantitatively determining of fungicide in a kind of coating |
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| Development and Validation of an HPLC/UV/MS Method for Simultaneous Determination of 18 Preservatives in Grapefruit Seed Extract;MARKUS GANZERA等;《J. Agric. Food Chem.》;20060509;第54卷;第3768-3772页 * |
| 高效液相色谱法同时测定果蔬中10种防腐杀菌剂;杨涛等;《理化检验-化学分册》;20101231;第46卷(第10期);第1189-1192页 * |
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