Selenium form analysis method for selenium-rich pet food

Abstract

The invention relates to a selenium form analysis method of selenium-rich pet food, which comprises the following steps: 1) sample treatment: accurately weighing a sample in a centrifuge tube, adding protease XIV, carrying out ultrasonic extraction, carrying out enzymolysis in a water bath oscillator, carrying out post-treatment after enzymolysis, carrying out HPLC-ICP-MS (High Performance Liquid Chromatography-Inductively Coupled Plasma-Mass Spectrometry) determination, and simultaneously carrying out a reagent blank experiment; and 2) quantitative analysis of the selenium form: substituting the peak area of the retention time obtained by detecting a selenium-rich pet food sample to be detected into the corresponding mixed standard sample standard curve of the selenium form, and analyzing and calculating the content of the selenium form in the selenium-rich pet food sample. The method for analyzing the five selenium forms in the selenium-rich pet food by using the high performance liquid chromatography-inductively coupled plasma mass spectrometer has the advantages of good stability, high precision and accurate and reliable result, and is an effective method for measuring the selenium form content in the selenium-rich feed.

Description

Technical Field

[0001] The invention belongs to the field of detection technology, and particularly relates to a selenium form analysis method for selenium-enriched pet food. Background Art

[0002] In recent years, with the rapid development of the pet economy, more and more attention has been focused on functional pet food. The research and application of trace elements as feed additives is one of the most promising research directions in the field of pet nutrition. Selenium has physiological and biochemical functions for pets, such as promoting growth, anti-oxidation, and improving immunity. It is becoming more and more common to add selenium-rich additives to pet food. However, selenium exists in different forms in nature. The biological effectiveness and toxicity of the element depend not only on the total amount of selenium, but also on its chemical form to a greater extent. The traditional method of evaluating product nutrition only by the total amount of selenium can no longer meet people's growing needs. It is of far-reaching significance to establish a simple, fast and accurate method for determining the form of selenium and understand the specific form of selenium in selenium-rich pet food. In recent years, the research on selenium form detection mainly includes high performance liquid chromatography-atomic fluorescence spectrometry (HPLC-AFS), gas chromatography tandem mass spectrometry (GC-ICP-MS), capillary electrophoresis-inductively coupled plasma mass spectrometry (CE-ICP-MS), and high performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS). Among them, HPLC-AFS is widely used in selenium form analysis due to its simple operation and low detection cost, but the sensitivity of the fluorescence detector is relatively low, and there are certain limitations for the detection of low-content selenium samples. HPLC-ICP-MS has become the preferred method in the field of selenium form analysis because it combines the high efficiency separation characteristics of liquid chromatography for specific compounds and the advantages of inductively coupled plasma mass spectrometry (ICP-MS) in elemental analysis, such as high accuracy, wide linear range, and fast analysis speed. At present, the research on selenium form at home and abroad is mainly concentrated on rice, vegetables, tea and seafood, and there are few reports on the research on selenium form in selenium-enriched pet food. Summary of the invention

[0003] In view of the problems existing in the prior art, the purpose of the present invention is to provide a method for analyzing the selenium form of selenium-enriched pet food, which is specifically achieved through the following technical solutions:

[0004] A method for analyzing selenium forms in selenium-enriched pet food comprises the following steps:

[0005] 1) Sample processing: Accurately weigh the sample into a centrifuge tube, add protease XIV, ultrasonically extract and place in a water bath oscillator for enzymatic hydrolysis, and perform post-processing for HPLC-ICP-MS determination after enzymatic hydrolysis, and perform a reagent blank test at the same time;

[0006] 2) Quantitative analysis of selenium forms: Substitute the peak area of ​​the retention time obtained after testing the selenium-enriched pet food sample into the standard curve of the corresponding selenium form mixed standard sample to analyze and calculate the content of the selenium form in the selenium-enriched pet food sample.

[0007] Furthermore, the sample processing in step 1) includes conventional samples and samples with high oil content. The post-processing steps of the conventional samples are: centrifugation after enzymatic hydrolysis, and filtering the supernatant through a 0.45μm microporous water filter membrane for HPLC-ICP-MS determination; the post-processing steps of the samples with high oil content are: adding n-hexane after enzymatic hydrolysis, shaking, collecting the lower layer liquid and centrifuging, taking the supernatant and filtering it through a 0.45μm microporous water filter membrane for HPLC-ICP-MS determination.

[0008] Furthermore, the centrifugal conditions are: 8000r / min centrifugation for 10min; the liquid ratio of protease XIV to n-hexane is 2mg:1mL, and the oscillation time is 1min.

[0009] Furthermore, there are five forms of selenium, namely: selenocystine, selenocysteine, selenous acid, selenomethionine, and selenic acid.

[0010] Furthermore, in step 1), the mass ratio of the sample to protease XIV is 25:1, the ultrasonic extraction time is 30 min, the enzymatic hydrolysis temperature is 37° C., and the enzymatic hydrolysis time is 6 h.

[0011] Furthermore, the chromatographic conditions of the analytical method are: SHIMSEN Ankylo C18 chromatographic column, 150mm×4.6mm, 5μm; column temperature: 25°C; injection volume: 50μL; 0.2mmol / L tetrabutylammonium bromide + 0.015mol / L ammonium acetate + 5% methanol as the mobile phase, the pH value of the mobile phase is adjusted to 5.7 with 20% methanol, and isocratic elution.

[0012] Furthermore, the mass spectrometry conditions of the analysis method are as follows: RF transmission power 1550W; carrier gas: high-purity argon; collision gas: high-purity helium; plasma gas flow rate 18L / min; auxiliary gas flow rate 1.2L / min; nebulizer flow rate 0.98L / min; collision gas flow rate 3ml / min; sampling cone, interceptor cone: platinum cone; nebulization chamber: concentric glass nebulization chamber; detection isotope: 78 Se.

[0013] The invention uses a high performance liquid chromatography-inductively coupled plasma mass spectrometer to analyze five selenium forms in selenium-enriched pet food. The method has good method stability, high precision, accurate and reliable results, and is an effective method for determining the content of selenium forms in selenium-enriched feed. The invention enables the five selenium forms to be separated within 11 minutes, and has a good linear relationship within the detection range of 5-100 μg / L, with a linear correlation coefficient R2 The detection limit was greater than 0.9995, the detection limit was 4.2-16.6 μg / kg, the recovery rate was 81.5-105.3%, the five selenium forms had good reproducibility, and the relative standard deviation RSD was 0.91-4.52%. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is the linear graph of the selenocystine standard curve;

[0015] Figure 2 is the linear graph of the selenocysteine ​​standard curve;

[0016] Figure 3 This is the linear graph of the selenomethionine standard curve;

[0017] Figure 4 This is the linear graph of the selenious acid standard curve;

[0018] Figure 5 This is the linear graph of the selenic acid standard curve;

[0019] Figure 6 Mass spectrum of detected substances in samples. DETAILED DESCRIPTION

[0020] The present invention is further described below in conjunction with the accompanying drawings in order to better understand the technical solution.

[0021] Main instruments and reagents: (PerkinElmer, USA); Flexar high performance liquid chromatograph (PerkinElmer, USA); ST-16R centrifuge (Thermo Fisher Scientific, USA); DS-8510DTH numerically controlled ultrasonic cleaner (Shanghai Shengxi Ultrasonic Instrument Co., Ltd.); BCE224-1CCN electronic balance (Sartorius Scientific Instrument Co., Ltd.).

[0022] Selenomethionine solution Se-Met (GBW10034, 97.9 μg / g), selenocysteine ​​solution Se-Cys (GBW10087, 93.5 μg / g), methyl-selenocysteine ​​solution Se-Mecys (GBW10088, 96.6 μg / g), selenite solution Se(Ⅳ) (GBW10032, 68.9 μg / g), selenate solution Se(Ⅵ) (GBW10033, 75.1 μg / g) were all purchased from the National Institute of Metrology, China; Protease XIV (from Streptomyces griseus type XIV, ≥

[0023] 3.5units / mg), Shanghai Anpu Company; ammonium acetate (superior grade, Shanghai MacLean Biotechnology Co., Ltd.), methanol (chromatographic grade, Thermo Fisher Scientific Co., Ltd.), tetrabutylammonium bromide (analytical grade, Shanghai MacLean Biotechnology Co., Ltd.), hydrochloric acid (superior grade, Xilong Chemical Co., Ltd.), formic acid (analytical grade, Shanghai MacLean Biotechnology Co., Ltd.), and the experimental water was laboratory ultrapure water.

[0024] Selenium-rich feed is purchased from the local feed market, crushed, sieved and stored in a cool and dry place.

[0025] The specific process is:

[0026] Pretreatment: Accurately weigh 0.5g of sample (accurate to 0.001g) into a centrifuge tube, add 20mg of protease XIV, extract in an ultrasonic cleaner for 30min, and place in a water bath oscillator at 37℃ for 6 hours. After enzymatic hydrolysis, centrifuge at 8000r / min for 10min, filter the supernatant through a 0.45μm microporous water filter membrane, and then perform HPLC-ICP-MS determination. At the same time, perform a reagent blank test.

[0027] 1) Chromatographic conditions: SHIMSEN Ankylo C18 column (150 mm × 4.6 mm, 5 μm), mobile phase ammonium acetate (0.015 mol / L) + tetrabutylammonium bromide (0.2 mmol / L) + methanol (5%), pH adjusted to 5.7 with formic acid (20%). Flow rate 1.0 mL / min, injection volume 50 μL.

[0028] 2) ICP-MS conditions: RF transmission power 1550W; carrier gas: high-purity argon; collision gas: high-purity helium; plasma gas flow rate 18L / min; auxiliary gas flow rate 1.2L / min; nebulizer flow rate 0.98L / min; collision gas flow rate 3ml / min; sampling cone, interceptor cone: platinum cone; nebulizer chamber: concentric glass nebulizer chamber; detection isotope: 78 Se.

[0029] Quantification of 5 selenium species in the sample to be tested:

[0030] (1) Preparation of mixed standard stock solution: Accurately prepare 5 kinds of selenium forms of mixed standard solutions with concentrations of 5, 10, 20, 50, and 100 μg / L, and draw a standard curve based on the chromatographic peak area of ​​each concentration and the corresponding mass concentration. Determine the corresponding concentration of each chromatographic peak when the signal-to-noise ratio S / N = 3 by stepwise dilution method, and determine the detection limit of the 5 forms when the chromatographic injection volume is 50 μL and the mass spectrometer uses the He collision cell mode. The results are shown in Table 1, and the linear graph of the standard curve of the 5 kinds of selenium forms is shown in Table 1. Figure 1-5 shown.

[0031] Table 1

[0032]

[0033]

[0034] As shown in Table 1, the five selenium forms in selenium-enriched pet food were separated within 11 minutes and had a good linear relationship in the detection range of 5-100 μg / L. The correlation coefficient R 2 Greater than 0.999. The detection limit is 4.2-16.6μg / kg, and the recovery rate is 81.5-105.3%.

[0035] (2) Determination of selenium-enriched pet food samples: Take the processed samples and inject them for testing. Substitute the peak areas obtained by the test into the standard curve of the corresponding substances, and analyze and calculate the contents of the five selenium forms. The mass spectrum of the detected samples is shown in Figure 6 The results are shown in Table 2 below.

[0036] Table 2: Content of 5 types of selenium in samples (unit: μg / L)

[0037]

[0038] (Note: ND means not detected)

[0039] It can be seen from Table 2 that selenocystine and selenomethionine were detected in selenium-enriched pet food, while the other three selenium forms were not detected.

Claims

1. A method for analyzing selenium forms in selenium-enriched pet food, characterized in that: The following steps are involved: 1) Sample processing: Accurately weigh the sample into a centrifuge tube, add protease XIV, ultrasonically extract and place in a water bath oscillator for enzymatic hydrolysis, and perform post-processing for HPLC-ICP-MS determination after enzymatic hydrolysis, and perform a reagent blank test at the same time; 2) Quantitative analysis of selenium forms: Substitute the peak area of ​​the retention time obtained after testing the selenium-enriched pet food sample into the standard curve of the corresponding selenium form mixed standard sample to analyze and calculate the content of the selenium form in the selenium-enriched pet food sample.

2. The method for analyzing selenium forms of selenium-enriched pet food according to claim 1, characterized in that: The sample processing in step 1) includes conventional samples and samples with high oil content. The post-processing steps of conventional samples are: centrifugation after enzymatic hydrolysis, and filtering the supernatant through a 0.45 μm microporous water filter membrane for HPLC-ICP-MS determination; the post-processing steps of samples with high oil content are: adding n-hexane after enzymatic hydrolysis, shaking, collecting the lower layer liquid and centrifuging, taking the supernatant and filtering it through a 0.45 μm microporous water filter membrane for HPLC-ICP-MS determination.

3. The method for analyzing selenium forms of selenium-enriched pet food according to claim 2, characterized in that: The centrifugal conditions are: 8000 r / min centrifugation for 10 min; the liquid ratio of protease XIV to n-hexane is 2 mg:1 mL, and the oscillation time is 1 min.

4. The method for analyzing selenium forms of selenium-enriched pet food according to claim 1, characterized in that: There are five forms of selenium: selenocystine, selenocysteine, selenous acid, selenomethionine, and selenic acid.

5. The method for analyzing selenium forms of selenium-enriched pet food according to claim 1, characterized in that: In step 1), the mass ratio of the sample to protease XIV is 25:1, the ultrasonic extraction time is 30 min, the enzymatic hydrolysis temperature is 37° C., and the enzymatic hydrolysis time is 6 h.

6. The method for analyzing selenium forms of selenium-enriched pet food according to claim 1, characterized in that: The chromatographic conditions of this analytical method were: SHIMSEN Ankylo C18 column, 150 mm × 4.6 mm, 5 μm; Column temperature: 25°C; injection volume: 50 μL; 0.2 mmol / L tetrabutylammonium bromide + 0.015 mol / L ammonium acetate + 5% methanol was used as the mobile phase, and the pH value of the mobile phase was adjusted to 5.7 with 20% formic acid, and isocratic elution was performed.

7. The method for analyzing selenium forms of selenium-enriched pet food according to claim 1, characterized in that: The mass spectrometry conditions of the analysis method are as follows: RF emission power 1550W; carrier gas: high-purity argon; collision gas: high-purity helium; plasma gas flow rate 18L / min; auxiliary gas flow rate 1.2L / min; nebulizer flow rate 0.98L / min; collision gas flow rate 3ml / min; sampling cone and skimmer cone: platinum cone; Spray chamber: Concentric glass spray chamber; Detection isotope: 78 Se.

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