A rapid extraction and purification method of formaldehyde in livestock and poultry blood products and its detection application

Abstract

The application discloses a kind of quick extraction detection method of formaldehyde in livestock and poultry blood products: (1) the sample of blood product to be measured is extracted with water to obtain extract; (2) after adding extract and iodine solution, sodium hydroxide solution is reacted in A test tube, add phosphoric acid solution, sodium thiosulfate solution, mix well;(3) add ether vortex extraction, transfer ether layer to B test tube that has been added with 0.025 mol / L sodium hydroxide solution in advance, vortex and carry out back extraction, discard upper ether layer, add ether in A test tube and repeat operation, absorb all lower solution in B test tube, obtain test sample, and the test sample is quantitatively detected by high performance liquid chromatography-ultraviolet detection method.The application solves the technical blank of the prior art for detecting the formaldehyde content in livestock and poultry blood products, and has strong applicability to various types of commercially available blood product matrix.The linear range of formaldehyde is 5 μg / mL-500 μg / mL, the detection limit is 2.5 μg / g, the recovery rate is greater than 75%, the standard deviation is less than 10%, which is simple, fast, economical and saves.

Description

TECHNICAL FIELD

[0001] The present application relates to the pretreatment and detection method of formaldehyde in livestock and poultry blood products, in particular to the extraction and purification method of formaldehyde in livestock and poultry blood products and its detection application. BACKGROUND

[0002] Formaldehyde is a highly toxic substance, and the accumulation of formaldehyde in the human body to a certain concentration will directly cause cancer. Our country stipulates that formaldehyde and formaldehyde compounds are prohibited in food. However, in the market, unscrupulous vendors use liquid agents containing formaldehyde to soak dry food raw materials, so that the food is preserved and durable, and the color is bright. The water-soaked food containing formaldehyde detected frequently includes shrimp, sea cucumber, pomfret, octopus, cuttlefish, hairtail, squid, jellyfish, and other seafood, as well as duck feet, beef strips, chicken claws, and other snacks. In addition, formaldehyde also has the effect of whitening, so some low-quality flour, rice flour, bean products, and mushroom products also contain formaldehyde.

[0003] Livestock and poultry blood products such as duck blood are prone to bacterial spoilage due to their rich nutrition, and some unscrupulous vendors use formaldehyde for preservation, preservation, and promotion of protein coagulation, increase of toughness, and improvement of taste in livestock and poultry blood products. At present, the formaldehyde detection standards mainly include SC / T 3025-2006 determination of formaldehyde in aquatic products, GB / T 21126-2007 determination of formaldehyde and sodium hydrosulfite content in wheat flour and rice flour and products, and NY / T 1283-2007 determination of formaldehyde content in shiitake mushrooms. There is no suitable detection standard for formaldehyde in livestock and poultry blood products, so it is particularly important to establish a more specific, accurate, rapid, and simple blood product formaldehyde detection method.

[0004] In actual detection, it is found that due to the inconsistency of the matrix, whether SC / T 3025-2006 or GB / T 21126-2007 method is used, the recovery rate of added formaldehyde is low when detecting formaldehyde in blood products, and the detection value is lower than the added amount. The analysis may be that duck blood and other blood products contain ferric ions, which have oxidizing properties, while formaldehyde has reducing properties. According to the existing standard method, water vapor distillation and 60℃ heating reaction are required in the process of formaldehyde extraction and derivation, which leads to serious loss of formaldehyde in the experiment process, resulting in low recovery rate. Therefore, it is of great significance to establish a rapid screening technology for formaldehyde in livestock and poultry blood products to improve the efficiency of supervision and ensure food safety. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a rapid extraction and purification method of formaldehyde in livestock and poultry blood products with high extraction recovery rate and its detection application. The detection method is simple, rapid, sensitive, and accurate.

[0006] The technical problem is solved by the technical solution adopted by the present application, which is:

[0007] A rapid extraction and purification method of formaldehyde in livestock and poultry blood products, the method comprises the following steps:

[0008] (1) weigh the sample to be tested, extract twice with water, combine the aqueous phase and dilute to volume to obtain the extract;

[0009] (2) accurately transfer a certain amount of extract to A test tube, add 1mol / L iodine solution, add 1mol / L sodium hydroxide solution, mix well, stand for 15-20 minutes, then add 1mol / L phosphoric acid solution, add 1mol / L sodium thiosulfate solution, mix well;

[0010] (3) add ether and vortex extract, centrifuge, transfer the ether layer to a B test tube pre-added with 0.025mol / L sodium hydroxide solution, vortex the B test tube for back extraction, then centrifuge, discard the upper ether layer, add ether to the A test tube and repeat the above extraction and back extraction twice, finally, take all the lower solution in the B test tube, filter membrane, to obtain the test sample.

[0011] In step (1), the dilution volume can be determined according to the mass of the sample to be tested and the possible concentration range, and the dilution volume is preferably 2-5mL / g based on the mass of the sample to be tested.

[0012] When the sample to be tested is 6g, the dilution volume is preferably 15mL.

[0013] In step (1), the volume of water used for extraction is generally 0.9-2mL / g based on the mass of the sample to be tested, and preferably 1mL / g. That is, when the sample to be tested is 6g, 6mL of water is used for extraction twice.

[0014] The volume ratio of the extract, 1mol / L iodine solution, 1mol / L sodium hydroxide solution, 1mol / L phosphoric acid solution and 1mol / L sodium thiosulfate solution in step (2) is 100:1:4:7:1.

[0015] In the present application, 1mol / L iodine solution refers to the iodine atom concentration of 1mol / L, which is the expression of iodine solution concentration known to those skilled in the art.

[0016] Further, preferably, the extract in step (2) is 5mL, and for every 5mL of extract, the amount of 1mol / L iodine solution is 50μL, the amount of 1mol / L sodium hydroxide solution is 200μL, the amount of 1mol / L phosphoric acid is 350μL, and the amount of 1mol / L sodium thiosulfate is 50μL.

[0017] The volume ratio of the extraction liquid to ether is 1:2-3, and the volume ratio of ether to 0.025 mol / L sodium hydroxide solution is 12:1-1.5.

[0018] Preferably, when the extraction liquid in step (2) is 5 mL, the amount of ether in step (3) is 12 mL, and the extraction is performed for a total of 3 times; and the volume of the 0.025 mol / L sodium hydroxide solution is preferably 1 mL.

[0019] The volume of the 0.025 mol / L sodium hydroxide solution is actually the volume of the test sample to be measured, that is, the final constant volume.

[0020] In step (3), the vortex extraction time is generally 5 minutes, and the back extraction time is generally 5 minutes.

[0021] Further, in step (2), the extraction liquid is added with 1 mol / L iodine solution and 1 mol / L sodium hydroxide solution, mixed, and placed for 15-20 minutes of reaction. Generally, the iodine solution is relatively excessive to the formaldehyde in the extraction liquid, and the reaction solution is not completely decolorized after the reaction.

[0022] If the iodine in the reaction solution is completely decolorized, it means that the formaldehyde is relatively excessive, at this time, the extraction liquid in step (1) is preferably diluted by a suitable multiple, and then step (2) is repeated to react with the iodine solution, and the reaction solution is required to be not completely decolorized after 15-20 minutes of reaction.

[0023] In the present application, the livestock and poultry blood products refer to edible blood products prepared by using fresh livestock (including pig, cow, and sheep) blood or fresh poultry (including chicken, duck, and goose) blood as raw materials, with or without the addition of seasonings, including pig blood, cow blood, sheep blood, chicken blood, duck blood, and goose blood.

[0024] The test sample to be measured obtained by extraction and purification can be quantitatively detected by high performance liquid chromatography-ultraviolet detection method.

[0025] Further, the present application also provides a rapid extraction and detection method for formaldehyde in livestock and poultry blood products, which comprises the following steps:

[0026] (1) A blood product sample to be measured is weighed, extracted with water twice, the water phases are combined and constant volume is obtained to obtain an extraction liquid;

[0027] The general constant volume is preferably 2-5 mL / g based on the mass of the blood product sample to be measured.

[0028] (2) A certain amount of the extraction liquid is accurately transferred to a test tube A, 1 mol / L iodine solution is added, 1 mol / L sodium hydroxide solution is added, mixed, placed for 15-20 minutes of reaction, then 1 mol / L phosphoric acid solution is added, 1 mol / L sodium thiosulfate solution is added, and mixed;

[0029] The volume ratio of the extraction solution, 1 mol / L iodine solution, 1 mol / L sodium hydroxide solution, 1 mol / L phosphoric acid solution, and 1 mol / L sodium thiosulfate solution in step (2) is 100:1:4:7:1.

[0030] (3) adding ether, vortex extraction, centrifugation, transferring the ether layer to a B test tube pre-added with 0.025 mol / L sodium hydroxide solution, vortexing the B test tube for back extraction, then centrifugation, discarding the upper ether layer, adding ether to the A test tube and repeating the above extraction and back extraction twice, finally, absorbing all the lower solution in the B test tube, filtering the membrane, and obtaining the test sample;

[0031] In step (3), the volume ratio of the extraction solution and ether is 1:2-3, and the volume ratio of ether and sodium hydroxide solution is 12:1-1.5.

[0032] In step (3), the vortex extraction time is generally 5 minutes, and the back extraction time is generally 5 minutes.

[0033] (4) The test sample is subjected to high performance liquid chromatography-ultraviolet detection, and the concentration of formaldehyde in the test sample is detected and quantified by an external standard method to obtain the formaldehyde content in the blood product sample.

[0034] The liquid chromatography conditions of the high performance liquid chromatography-ultraviolet detection are as follows: a Thermo Hypersil Gold C18 column (4.6 mm x 250 mm, 5.0 um), a column temperature of 40°C, a mobile phase A of methanol, a mobile phase B of water, a mobile phase C of a 1.5 g / L diammonium hydrogen phosphate aqueous solution (1.5 g of diammonium hydrogen phosphate is weighed, dissolved in 900 mL of water, and the pH is adjusted to 2.6 with 1 mol / L phosphoric acid, and then diluted to 1000 mL with water), a flow rate of 0.7 mL / min, and gradient elution.

[0035] The gradient elution program is shown in Table 1 below:

[0036] Table 1 HPLC elution program

[0037] Time / min A / ％ B / ％ C％ 0 2 0 98 10 2 0 98 10.1 10 90 0 18 10 90 0 18.1 2 10 98 22 2 10 98

[0038] Ultraviolet detector: wavelength 214 nm.

[0039] Further, the quantitative method is operated as follows:

[0040] The test sample is subjected to high performance liquid chromatography-ultraviolet detection to obtain the liquid chromatogram of the test sample, the peak area is compared with the standard curve, and the concentration of formaldehyde in the test sample is calculated;

[0041] The standard curve is prepared by the following method: preparing water solution of different concentrations of formaldehyde standard, performing steps (2), (3), then performing high performance liquid chromatography-ultraviolet detection under the same conditions of step (4), taking the formaldehyde concentration as the abscissa and the peak area as the ordinate to prepare the standard curve.

[0042] The formaldehyde content in the blood product sample to be tested is obtained according to the following formula: X = C*V*F / m,

[0043] In the formula: X is the formaldehyde content in the blood product sample to be tested, in μg / g;

[0044] C is the formaldehyde concentration in the test sample to be tested, obtained according to the standard curve, in μg / mL

[0045] V is the constant volume of the test sample to be tested, in mL;

[0046] m is the mass of the blood product sample to be tested, in g.

[0047] F is the dilution factor.

[0048] Further, steps (1)-(3) are preferably performed as follows:

[0049] (1) 6 g of the blood product sample to be tested is weighed, extracted with 6 mL of water twice, the water phases are combined and constant volume is added to 15 mL to obtain the extract;

[0050] (2) 5 mL of the extract is accurately transferred to a test tube A, 50 μL of 1 mol / L iodine solution, 200 μL of 1 mol / L sodium hydroxide solution are added, mixed, and left to react for 15-20 minutes, then 350 μL of 1 mol / L phosphoric acid solution and 50 μL of 1 mol / L sodium thiosulfate solution are added, and mixed;

[0051] (3) 12 mL of ether is added, vortexed for 5 minutes, centrifuged, the ether layer is transferred to a test tube B which has been pre-added with 1.0 mL of 0.025 mol / L sodium hydroxide, vortexed for back extraction, then centrifuged, the upper ether layer is discarded, ether is added to test tube A to repeat the above extraction and back extraction twice, finally the whole lower solution in test tube B is sucked and filtered to obtain the test sample.

[0052] In the method, if the formaldehyde content in the blood product sample to be tested detected in step (4) is 0, it is determined as a negative sample, and if the formaldehyde content in the blood product sample to be tested detected is >0, it is determined as a positive sample. Generally, the interference of possible formic acid background in the sample also needs to be excluded. However, in the blood product of livestock and poultry, under normal circumstances, it should also not contain formic acid, but in order to ensure the detection rigor, the background formic acid can also be excluded, therefore the method can also include step (5) for excluding the background formic acid:

[0053] (5) Accurately pipette 5 mL of the extract, add 350 μL of 1 mol / L phosphoric acid, then extract according to the method of step (3), and then detect the background formic acid content according to the method of step (4), so that the actual formaldehyde content in the sample can be obtained.

[0054] Compared with the prior art, the advantages of the present application are that: there is no suitable national standard and industry standard for the detection of formaldehyde in livestock and poultry blood products at present, and the prior art generally adopts a derivatization liquid chromatography fluorescence detection method. In actual detection, the recovery rate cannot meet the detection requirements, the livestock and poultry blood products contain ferric iron which has oxidizing property, and formaldehyde has reducing property, the current formaldehyde detection adopts methods such as distillation extraction and heating derivation to accelerate the oxidation of formaldehyde, which leads to low experimental recovery rate. In the present application, formaldehyde is extracted with water at room temperature, then formaldehyde is oxidized into sodium formate by excessive iodine, the excessive iodine is reacted with sodium thiosulfate, the acidity is adjusted, sodium formate in the solution is converted into formic acid, formic acid is extracted with diethyl ether, sodium hydroxide solution is added to the diethyl ether, formic acid is dissolved in the sodium hydroxide solution again, sodium formate is obtained in the water phase of the test sample solution, then liquid chromatography-ultraviolet detector is used for detection, in the acidic mobile phase, sodium formate is converted into formic acid again, which can be detected by ultraviolet detection.

[0055] In the present application, the target compound is extracted with diethyl ether under acidic conditions, and then re-extracted into the water layer under alkaline conditions, which not only plays a purifying role, but also increases the concentration multiple, and makes up for the deficiency of the ultraviolet detector. In the experimental process, formaldehyde is oxidized into formic acid, and then formic acid is extracted for detection, which avoids the loss of formaldehyde in the experimental process, and greatly improves the recovery rate.

[0056] The present application solves the technical blank of the detection of formaldehyde content in livestock and poultry blood products in the prior art, and first proposes a method for accurately and quantitatively detecting the formaldehyde content in livestock and poultry blood products. The results show that the present method has strong applicability to various types of commercially available blood product matrices, the linear range of formaldehyde is 5 μg / mL-500 μg / mL, the detection limit is 2.5 μg / g, the recovery rate is greater than 75%, and the standard deviation is less than 10%, which is simple, fast, economical, and can well meet the requirements of large-scale and rapid analysis in the laboratory. BRIEF DESCRIPTION OF DRAWINGS

[0057] Figure 1 HPLC spectrum of formaldehyde after reaction according to the extraction and purification process of Example 1.

[0058] Figure 2 HPLC spectrum of reagent blank after reaction according to the extraction and purification process of Example 1.

[0059] Figure 3HPLC chromatogram of the reaction after the extraction and purification process of Example 1 for the non-spiked duck blood sample.

[0060] Figure 4 HPLC chromatogram of the reaction after the extraction and purification process of Example 1 for the spiked duck blood sample. DETAILED DESCRIPTION

[0061] The technical solutions of the present application are described in further detail below in conjunction with examples, but the scope of protection of the present application is not limited thereto.

[0062] Example 1

[0063] 1. Instruments and reagents

[0064] High performance liquid chromatograph LC-20AT (Shimadzu, Japan) equipped with a UV detector. The chromatographic column was Thermo Hypersil Gold C18 (4.6 mm x 250 mm, 5.0 μm). Milli-Q high-purity water generator (Millipore, USA). Refrigerated centrifuge (SIGMA, Germany). Vortex (Heldolph, Germany). Filter membrane (DIKMA, Nylon 0.45 μm).

[0065] Formaldehyde standard was purchased from Dr. Company, 10 mg / mL standard stock solution.

[0066] 1 mol / L iodine solution: weigh 20 g of potassium iodide, dissolve in 10 mL of water, add 6.35 g of iodine, dissolve and dilute to 50 mL with water;

[0067] 1 mol / L sodium thiosulfate: weigh 13 g of sodium thiosulfate (containing 5 crystal waters), dissolve with water, and dilute to 50 mL;

[0068] 1 mol / L phosphoric acid: add 5.35 mL of phosphoric acid to 50 mL of water, mix well, and dilute to 100 mL;

[0069] 1 mol / L sodium hydroxide: weigh 4.0 g of sodium hydroxide, dissolve with water, cool to room temperature, and dilute to 100 mL; 0.025 mol / L sodium hydroxide: take 1.25 mL of 1 mol / L sodium hydroxide, dilute with water to 50 mL.

[0070] 2. One-step extraction and purification process

[0071] The matrix sample was purchased from a supermarket.

[0072] The steps are as follows:

[0073] (1) Take 6 grams of blood product sample to be tested, vortex with 6 mL of water for 5 minutes, centrifuge at 4000 rpm for 5 minutes, transfer the supernatant to another test tube, add 6 mL of water to the residue and repeat the above extraction operation, combine the water phase and dilute with water to 15 mL;

[0074] (2) Accurately transfer 5 mL of the extract to A test tube, add 50 μL of 1 mol / L iodine solution, add 0.2 mL of 1 mol / L sodium hydroxide solution and mix well, stand for 15 minutes. Add 0.35 mL of 1 mol / L phosphoric acid solution and 50 μL of 1 mol / L sodium thiosulfate solution and mix well;

[0075] (3) Add 12 mL of ether and vortex for 5 minutes, centrifuge for 5 minutes, transfer the ether layer to B test tube pre-added with 1.0 mL of 0.025 mol / L sodium hydroxide solution. Vortex the B test tube for 5 minutes, centrifuge at 4000 rpm for 5 minutes, discard the ether layer. Repeat the above extraction and back extraction twice by adding 12 mL of ether to A test tube, finally filter the lower layer solution in B test tube.

[0076] 3. Liquid chromatography-ultraviolet detection

[0077] Liquid chromatography conditions: Thermo Hypersil Gold C18 column (4.6 mm x 250 mm, 5.0 um), column temperature 40℃, mobile phase A: methanol, mobile phase B: water, mobile phase C: 1.5 g / L of aqueous diammonium hydrogen phosphate (weigh 1.5 g of diammonium hydrogen phosphate, dissolve in 900 mL of water, adjust pH to 2.6 with 1 mol / L phosphoric acid, and dilute with water to 1000 mL), flow rate: 0.7 mL / min, gradient elution, elution program as shown in Table 1 below:

[0078] Table 1 HPLC elution program

[0079]

[0080]

[0081] Ultraviolet detector: wavelength 214 nm

[0082] 4. Qualitative and quantitative analysis

[0083] Prepare a series of formaldehyde standard solutions with water, with concentrations of 0 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 500 μg / mL, respectively, take 1 mL of standard solution, dilute with water to 5 mL, process according to the above steps (2), (3), take the concentration as the abscissa and the peak area as the ordinate to make a standard curve; the chromatogram of the standard substance is shown in Figure 1The retention time of formic acid is 5.2 min.

[0084] The HPLC spectrum of the reagent blank after the extraction and purification process according to steps (2) and (3) is shown in the following figure. Figure 2

[0085] The HPLC spectrum of the sample without standard addition after the extraction and purification process according to steps (1), (2) and (3) is shown in the following figure. Figure 3 Figure 2 Figure 3 No formic acid peak appeared.

[0086] 5. Calculation of the concentration of the sample to be tested:

[0087] The content of the substance to be tested in the sample is obtained according to the following calculation formula: X = C * V * F / m,

[0088] In the formula, X is the content of formaldehyde in the blood product sample to be tested, in μg / g;

[0089] C is the formaldehyde concentration in the test sample, which is calculated according to the standard curve, in μg / mL

[0090] V is the constant volume of the test sample, in mL;

[0091] m is the mass of the blood product sample to be tested, in g.

[0092] F is the dilution factor. The dilution factor refers to the dilution factor of the formaldehyde mass in the test sample relative to the blood product sample to be tested. In the present application, it is the constant volume of step (1) divided by the volume of the extraction solution of step (2).

[0093] In the present embodiment, F = 3.

[0094] The experimental results are analyzed as follows:

[0095] 1. Selection of the amount of iodine and sodium thiosulfate

[0096] In the present application, the reaction formula of step (2) is shown in the following formula (1) and (2). First, formaldehyde is oxidized by excess iodine, and the unreacted iodine is reduced by excess sodium thiosulfate.

[0097] CHO + I2 + 3NaOH = HCOONa + 2NaI + 2H2O (1)

[0098] I2 + 2Na2S2O3 = 2NaI + Na2S4O6 (2)

[0099] In reaction formula (1), for 5 mL of extraction solution, it corresponds to 2 g of sample to be tested. Generally, the formaldehyde content in 2 g of sample does not exceed 0.5 mg, and a sufficiently large limit value of formaldehyde concentration is set in the experiment.​​​

[0100] 0.5mg formaldehyde is about 0.0165mmol, 0.033ml of 1mol / L iodine is needed, and 0.0495ml of 1mol / L sodium hydroxide is needed. Considering the need for excess iodine solution to ensure complete reaction of 0.5mg formaldehyde, 0.05ml of 1mol / L iodine is selected for the experiment, and the basic condition is conducive to the reaction. 1mol / L sodium hydroxide is selected as 0.2ml.

[0101] 0.05ml of 1mol / L iodine in reaction formula (2) needs to consume 0.05ml of 1mol / L sodium thiosulfate, so 0.05ml of 1mol / L sodium thiosulfate is selected for the experiment. The acidic condition is conducive to the reaction, and the addition of 1mol / L phosphoric acid in an amount of 0.35ml not only meets the need for neutralizing excess sodium hydroxide, but also ensures the acidic condition, which is conducive to equation (2)

[0102] The reaction proceeds, and the requirement for ether extraction in the next step can also be met.

[0103] 2. Changes in target formic acid in water layer after extraction by ether under different acid and base concentrations.

[0104] 5mL of 0.1mg / mL formic acid solution contains 0, 25, 50, 100, 200, 300, 500μL of 1mol / L phosphoric acid, respectively, and 0.025mol / L and 0.05mol / L sodium hydroxide solution are used as solvents to prepare 0.1mg / mL formic acid solution, respectively. After extraction with 12ml of ether, the content of formic acid in the water layer was detected, and the proportion of formic acid in the water layer after extraction was 38.8%, 44.2%, 48%, 44.8%, 43.8%, 43.8%, 45.3%, 97.0%, 100%, respectively. It can be seen that under alkaline conditions, ether cannot extract formic acid, and formic acid is completely left in the water layer;

[0105] The extraction ratio of ether to formic acid in acidic environment can reach more than 50%, and the extraction efficiency of ether to formic acid does not change significantly under the conditions of adding 100, 200, 300, 500μL of 1mol / L phosphoric acid. Considering the addition of sodium hydroxide in the oxidation reaction solution, 350μL of 1mol / L phosphoric acid is added during the extraction process, and the extraction efficiency of ether can reach 55%. Three times of extraction theoretically can reach 90.9%, and the back extraction uses 0.025mol / L sodium hydroxide, and the back extraction rate reaches 100%.

[0106] 3. Effect of ether to sample ratio on extraction efficiency during extraction process. 5mL of 0.1mg / mL formic acid solution is added with 350μL

[0107] 1 mol / L phosphoric acid, adding different proportions of volume of diethyl ether extraction, measured the content of formic acid in water layer, so as to calculate the extraction rate of formic acid with different amount of diethyl ether, the results are shown in table 2.

[0108] Table 2 the extraction rate of formic acid with different amount of diethyl ether

[0109]

[0110] The above experimental results show that the extraction rate increases not obviously with the increase of the amount of diethyl ether, considering the amount of reagent and the extraction rate, the amount of diethyl ether in the extraction process is selected as twice the volume of the sample, the volume of the sample is 5 mL, and the volume after oxidation reaction is about 6 mL, so the amount of diethyl ether is selected as 12 mL, and the extraction is carried out for 3 times.

[0111] 4、In the back extraction process, 0.1 mg of formic acid is added to different volumes of diethyl ether, 1 mL of 0.025 mol / L sodium hydroxide is used for back extraction, the content of formic acid extracted in the water layer is detected, so as to calculate the extraction rate of formic acid, the results are shown in table 3.

[0112] Table 3 comparison of the extraction rate of formic acid extracted from different volumes of diethyl ether

[0113] Ethyl ether dosage 3 mL 5 mL 7 mL 10 mL 12 mL Extraction rate 95.2％ 96.8％ 99.5％ 102％ 99.5％

[0114] The above data show that the back extraction efficiency of formic acid extracted from 3-12 mL of diethyl ether with 1 mL of 0.025 mol / L sodium hydroxide is not much different, and can meet the experimental requirements, therefore, 1 mL of 0.025 mol / L sodium hydroxide is selected for back extraction of formic acid from 12 mL of diethyl ether in the experiment, and the back extraction rate can reach 99.5%, at the same time, it also plays a role in concentrating the sample to a larger volume, which ensures a lower detection limit.

[0115] 5、Optimization of liquid phase conditions

[0116] Diammonium hydrogen phosphate buffer salt is used as the mobile phase, 1.5 ml of phosphoric acid is added to each liter of the mobile phase, so that the pH value is 2.6, which not only meets the requirements of the pH value used range of the chromatographic column, but also makes the response values of the formic acid standard substance with water and 0.025 mol / L sodium hydroxide basically consistent. In the experiment, the chromatographic behaviors of formic acid when methanol-buffer and acetonitrile-buffer systems are used as the mobile phase are compared, the results show that the peak shape is good when methanol-buffer is used as the mobile phase, and the separation degree of the solvent peak and the target peak is good, therefore, alcohol-buffer is used as the mobile phase in the experiment. In addition, two different types of chromatographic columns are compared: Dikma Plus C18 chromatographic column and Hypersil Gold C18 chromatographic column, it is found that the Hypersil Gold C18 chromatographic column is better in the separation degree. Therefore, the Hypersil Gold C18 chromatographic column is used in the present application.

[0117] 6. Analysis of the recovery of the spiked sample

[0118] Formaldehyde standard was added to the common three kinds of livestock and poultry blood product matrix, and liquid chromatography-ultraviolet detector was used for detection, retention time was used for qualitative analysis, external standard method was used for quantitative analysis, linear range was 5-500 μg / mL, linear equation was Y=6.41e 4 *X-3.36e 3 , correlation coefficient r 2 was 0.997, limit of quantification was 5 μg / g, recovery range of the spiked sample was 75%-86%, standard deviation was within 10%, and the results are shown in Table 4. If a positive sample is detected, theoretically, the detection of formic acid needs to be excluded. After extraction in step (1), 5 mL of the extracted solution can be accurately taken, 350 μL of 1 mol / L phosphoric acid is added, step (3) is operated, and then HPLC analysis is performed to determine whether there is interference of formic acid. If formic acid is detected, the actual content of formaldehyde in the sample can be calculated by subtracting the amount of formaldehyde measured by the method from the amount of formaldehyde.

[0119] However, the present applicant has detected 6 kinds of livestock and poultry blood product samples, and no formic acid background has been detected. Therefore, in the case that the sample basically does not contain formic acid background, the exclusion step of the formic acid background is not necessary.

[0120] The HPLC spectrum of the spiked duck blood sample after the extraction and purification process of steps (1), (2) and (3) is shown in Figure 4 .

[0121] The results show that the present method has strong applicability to various livestock and poultry blood products, and can achieve the purpose of rapid extraction and purification of formaldehyde in livestock and poultry blood products.

[0122] Table 4 Experimental data of the spiked concentration of formaldehyde in duck blood, pig blood and goose blood products and the recovery rate thereof

[0123]

[0124] Formaldehyde was added to duck blood, and the amount of addition was 10 μg / g. The standard method of SC / T 3025-2006 and the standard method of GB / T21126-2007 were used for detection, and the recovery rate of the spiked sample is shown in Table 5.

[0125] Table 5 Comparison of the recovery rate of the spiked formaldehyde in duck blood detected by different methods

[0126] Addition level 10 μg / g Recovery of this method 78.2％ Recovery of SC / T3025-2006 0％ Recovery of GB / T21126-2007 10.6％

[0127] As can be seen from Table 5, the existing standard method of the prior art is not suitable for the detection of formaldehyde in livestock and poultry blood products, and the recovery rate is too low, which is easy to detect false negative results.

[0128] At present, the common detection method of formaldehyde in food adopts derivative method, and the fluorescence detection has high sensitivity, but it is not applicable to the detection of special sample of blood products, and formaldehyde titration method is only applicable to high concentration sample, and the detection limit cannot meet the requirement of residual content. In the paper, the principle of formaldehyde titration method is used, formaldehyde is oxidized into formic acid by using the reducing property of formaldehyde, and is extracted by using ether, and the detection in livestock and poultry blood products is realized by using sodium hydroxide back extraction, the detection limit of the method can meet the detection requirement of 10 μg / g detection requirement specified in GB / T 21126-2007 method. The recovery rate of the experiment is improved, and the accuracy of the detection result is ensured.

[0129] The above description is not a limitation of the present application, and the present application is not limited to the above examples. Changes, modifications, additions or replacements made by ordinary skilled persons in the technical field within the essential scope of the present application shall also belong to the protection scope of the present application.

Claims

1. A rapid extraction and detection method for formaldehyde in livestock and poultry blood products, characterized in that... The method comprises the following steps: (1) weigh the blood product sample to be tested, extract twice with water, combine the aqueous phase and dilute to volume, and obtain an extract; the volume to be diluted is 2-5 mL / g based on the mass of the blood product sample to be tested; (2) accurately take a certain amount of the extract into a test tube A, add 1 mol / L iodine solution, add 1 mol / L sodium hydroxide solution, mix well, stand for 15-20 minutes of reaction, then add 1 mol / L phosphoric acid solution, add 1 mol / L sodium thiosulfate solution, and mix well; the volume ratio of the extract, 1 mol / L iodine solution, 1 mol / L sodium hydroxide solution, 1 mol / L phosphoric acid solution and 1 mol / L sodium thiosulfate solution is 100:1:4:7:1; (3) add ether and vortex extract, centrifuge, transfer the ether layer to a test tube B pre-added with 0.025 mol / L sodium hydroxide solution, vortex the test tube B for back extraction, then centrifuge, discard the upper ether layer, add ether to the test tube A and repeat the above extraction and back extraction twice, finally take all the lower layer solution in the test tube B, filter through a membrane, and obtain the test sample; the volume ratio of the extract and ether is 1:2-3, and the volume ratio of ether and 0.025 mol / L sodium hydroxide solution is 12:1-1.5; (4) perform high performance liquid chromatography-ultraviolet detection on the test sample, quantitatively determine the concentration of formaldehyde in the test sample by an external standard method, and convert the formaldehyde content in the blood product sample to be tested; The liquid phase conditions of the high performance liquid chromatography-ultraviolet detection are as follows: a Thermo Hypersil Gold C18 column, a column temperature of 40°C, a mobile phase A of methanol, a mobile phase B of water, a mobile phase C of a 1.5 g / L diammonium hydrogen phosphate aqueous solution with a pH of 2.6, a flow rate of 0.7 mL / min, and gradient elution; the gradient elution program is shown in the following table: HPLC elution program The wavelength of the ultraviolet detector is 214 nm.

2. The method of claim 1, wherein The steps (1)-(3) are operated as follows: (1) weigh 6 g of the blood product sample to be tested, extract twice with water, combine the aqueous phase and dilute to volume to 15 mL, and obtain an extract; (2) accurately take 5 mL of the extract into a test tube A, add 1 mol / L iodine solution 50 μL, add 1 mol / L sodium hydroxide solution 200 μL, mix well, stand for 15-20 minutes of reaction, then add 1 mol / L phosphoric acid solution 350 μL, and add 1 mol / L sodium thiosulfate solution 50 μL, and mix well; (3) add 12 mL of ether and vortex extract for 5 minutes, centrifuge, transfer the ether layer to a test tube B pre-added with 1.0 mL of 0.025 mol / L sodium hydroxide, vortex the test tube B for back extraction, then centrifuge, discard the upper ether layer, add ether to the test tube A and repeat the above extraction and back extraction twice, finally take all the lower layer solution in the test tube B, filter through a membrane, and obtain the test sample.

3. The method of claim 1, wherein The quantitative method is operated as follows: Perform high performance liquid chromatography-ultraviolet detection on the test sample, obtain the liquid chromatogram of the test sample, compare the peak area with a standard curve, and calculate the formaldehyde concentration of the test sample; The standard curve is prepared by the following method: preparing water solutions of different concentrations of formaldehyde standard, performing steps (2) and (3), and then performing high performance liquid chromatography-ultraviolet detection under the same conditions as step (4), taking the formaldehyde concentration as the horizontal coordinate and the peak area as the vertical coordinate to obtain the standard curve.

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