CN112098559B

CN112098559B - Method for detecting content of citrinin

Info

Publication number: CN112098559B
Application number: CN202010999108.1A
Authority: CN
Inventors: 倪莉; 周康熙; 肖炎钦; 吕旭聪
Original assignee: Fuzhou University
Current assignee: Fuzhou University
Priority date: 2020-09-22
Filing date: 2020-09-22
Publication date: 2021-12-28
Anticipated expiration: 2040-09-22
Also published as: CN112098559A

Abstract

The invention discloses a method for measuring citrinin, which adopts an external standard method to measure the content of citrinin and comprises the following steps: (1) preparing a citrinin standard solution with a certain concentration gradient, and drawing a standard curve of concentration-peak area by using liquid chromatography; (2) the sample solution was passed through a 0.22 μm filter and then measured by liquid chromatography, and the concentration was calculated from the standard curve in (1). The citrinin measuring method is suitable for detecting citrinin in monascus fermentation liquor, monascus extracting solution, wheat starter yellow wine or monascus yellow wine, the lowest detection limit and the quantification limit are respectively 3ng/mL and 10ng/mL, the linearity is good within the range of 10-1000 ng/mL, and the interference of other fluorescent substances generated by monascus can be overcome.

Description

Method for detecting content of citrinin

Technical Field

The invention belongs to the technical field of citrinin determination, and particularly relates to a method for detecting the content of citrinin.

Background

The red yeast is obtained by inoculating and fermenting monascus, and can be used as a leaven for brewing red yeast yellow wine. Monascus is a kind of small filamentous saprophytic fungi, and primary metabolites (such as amylase, protease and the like) and secondary metabolites (such as monascus pigment, lovastatin and the like) of monascus are widely applied in the field of food and medicine, but citrinin with nephrotoxicity can be metabolized in the growth process, so that the detection and control of the content of citrinin are particularly important. The China light industry Standard QB/T2847-2007 limits the citrinin content of functional red yeast rice (powder) to 50 mug/kg (50 ppb), the Japan content to 100ppb, and the U.S. food and drug administration FDA content to 20 ppb. The liquid sample with the quantitative limit of the abandoned old national standard (GB/T5009.222-2008 determination of citrinin in red yeast products) is 50ng/mL, the solid sample is 1000 mug/kg, the accuracy is not good enough, and only the sample with the citrinin exceeding the standard can be quantitatively analyzed; the new national standard (GB 5009.222-2016 food citrinin determination) method, namely the method for detecting the monascus citrinin product, is high in sensitivity (detection limit is 25 mug/kg), but an immunoaffinity column with high price is required, and the HPLC method can cause the peak tailing of a standard product and is not beneficial to the citrinin detection of the monascus product. Aiming at the defects of the HPLC detection method of citrinin in the new and old national standards, the invention provides an effective and accurate determination method.

Disclosure of Invention

The invention aims to provide a method for detecting the content of citrinin aiming at the defects of the prior art.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for measuring citrinin content comprises the following steps: the content of citrinin is determined by an external standard method. Preparing a citrinin standard solution with a certain concentration gradient, and drawing a standard curve of concentration-peak area by using liquid chromatography; the sample solution was passed through a 0.22 μm filter and measured by liquid chromatography, and the concentration was calculated from the standard curve. The sample solution is one of monascus fermentation liquor, monascus extracting solution, wheat starter yellow wine or monascus yellow wine.

(1) The preparation method of the citrinin standard solution comprises the following steps: accurately weighing 1mg of citrinin in an electronic analytical balance, adding 10mL of 75% ethanol for full dissolution to prepare 100 mu g/mL of citrinin stock solution, subpackaging into 10 tubes, taking one tube, diluting the tube with 75% ethanol to obtain 1000ng/mL, 500ng/mL, 250ng/mL, 100ng/mL, 50ng/mL and 25ng/mL of citrinin standard solution, and freezing and storing the rest stock solution at-80 ℃. The citrinin standard solution is prepared for use, and the citrinin stock solution can be preserved for 1 year.

(2) The detection conditions of the liquid chromatogram are as follows: a chromatographic column: agilent Zorbax SB-C18 (4.6X 250mm, 5 μ), excitation wavelength 331nm, emission wavelength 500nm, sample size 20 μ L, flow rate 1.0mL/min, detection time 15min, mobile phase A is 100% methanol, liquid B is an aqueous solution containing 0.02% (v/v) triethylamine, 0.32% (v/v) tetrahydrofuran and 0.1% (v/v) phosphoric acid, liquid C is acetonitrile containing 0.1% (v/v) formic acid, and the elution gradient of the mobile phase is shown in Table 1.

TABLE 1 elution gradient conditions for the mobile phase of the liquid phase

The invention has the beneficial effects that:

the method can separate the fluorescent substances metabolized by the citrinin and the monascus, is suitable for detecting the citrinin in monascus fermentation liquor, red yeast rice and yellow wine products, improves the detection sensitivity compared with the old national standard of the citrinin detection in 2008, and has the advantages of earlier peak emergence time, more complete peak shape, no tailing, accurate and feasible method and high reliability compared with the new national standard HPLC method (the method for detecting the monascus products in the first method) for the citrinin detection in 2016.

Drawings

FIG. 1 is a liquid chromatogram of a 500ng/mL citrinin standard in the present invention.

FIG. 2 is a liquid chromatogram of a standard 500ng/mL citrinin product in the national standard of 2008.

FIG. 3 is a liquid chromatogram of a standard 500ng/mL citrinin product in the national Standard of 2016.

FIG. 4 is a liquid chromatogram of a 25ng/mL citrinin standard in the national Standard of 2016.

FIG. 5 is a standard curve of citrinin in accordance with the present invention.

FIG. 6 is a liquid chromatogram of citrinin in the fermentation broth of Monascus purpureus went.

FIG. 7 is a liquid chromatogram of citrinin in the ethanol extract of red yeast rice.

FIG. 8 is the citrinin liquid chromatogram of the red rice yellow wine of the present invention.

FIG. 9 is a liquid chromatogram of citrinin in wheat koji yellow wine.

FIG. 10 is a liquid chromatogram of citrinin from the fermentation broth of Monascus purpureus went under the condition of aQ-C18 column.

Detailed Description

The present invention will be further described with reference to the following examples, but the present invention is not limited to these examples.

Example 1

The invention relates to a method for measuring citrinin content, which specifically comprises the following steps:

(1) preparation of citrinin standard product: accurately weighing 1mg of citrinin in an electronic analytical balance, adding 10mL of 75% (v/v) ethanol for full dissolution to prepare 100 mu g/mL of citrinin stock solution, subpackaging into 10 tubes, taking one tube, diluting the tube with 75% (v/v) ethanol to obtain 1000ng/mL, 500ng/mL, 250ng/mL, 100ng/mL, 50ng/mL and 25ng/mL of citrinin standard solution, and freezing and storing the rest stock solution at-80 ℃. The citrinin standard solution is prepared for use, and the citrinin stock solution can be preserved for 1 year.

(2) Pretreatment of a sample: grinding and crushing solid samples such as red yeast rice and the like, accurately weighing a certain mass of solid powder, adding 75% (v/v) ethanol, extracting at 60 ℃ for 2h, centrifuging at 4500r/min for 15min, and filtering supernate with a 0.22 mu m filter membrane to be detected; and (3) directly centrifuging liquid samples such as monascus fermentation liquor, yellow wine and the like at 4500r/min for 15min, and filtering supernate with a 0.22-micron filter membrane to be detected.

(3) The detection conditions of the liquid chromatogram are as follows: a chromatographic column: agilent Zorbax SB-C18 (4.6X 250mm, 5 mu), excitation wavelength 331nm, emission wavelength 500nm, sample size 20 mu L, flow rate 1.0mL/min, detection time 15min, mobile phase A is 100% methanol, liquid B is an aqueous solution containing 0.02% (v/v) triethylamine, 0.32% (v/v) tetrahydrofuran and 0.1% (v/v) phosphoric acid, liquid C is acetonitrile containing 0.1% (v/v) formic acid, and the elution conditions of the mobile phase are shown in Table 1.

The HPLC method used in this example for the old national standard of 08 years is as follows: liquid chromatography column Luna C18 (2) (4.6 × 150mm, 5 μ), column temperature 28 ℃, excitation wavelength 331nm, emission wavelength 500nm, sample size 20 μ L, flow rate 1.0mL/min, mobile phase a liquid 100% acetonitrile, B liquid pH =2.5 phosphoric acid water, a: b = 35: 65.

the 16-year new national standard HPLC method used in this example is as follows: a liquid chromatography column, Luna C18 (2) (4.6X 150mm, 5 mu), at 30 ℃ of column temperature, excitation wavelength of 350nm, emission wavelength of 500nm, sample size of 20 mu L, flow rate of 0.7mL/min, mobile phase A of 100% acetonitrile, liquid B of 0.1% (v/v) phosphoric acid, mobile phase and gradient elution conditions are shown in Table 2.

HPLC mobile phase gradient elution condition for detecting citrinin in red yeast rice and products thereof in new national standard of table 22016 edition

According to the method, the retention time of a 500ng/mL high-concentration citrinin standard product is advanced to 4.3min (figure 1), the whole peak time is 2.25min, while the retention time of the 500ng/mL citrinin standard product is 10.56min and the whole peak time is seriously trailing by adopting a 08-year old national standard HPLC method (figure 2); in the HPLC method of red yeast rice and products thereof in the new national standard of 16 years, the retention time of 500ng/mL citrinin standard is 9.63min, the whole peak-out time is relatively tail (figure 3), and the base line under the gradient elution condition is not flat (figure 4) for the low concentration citrinin of 25 ng/mL. The citrinin HPLC determination method is superior to the current two national standard methods.

Under the method of the invention, a standard curve of citrinin concentration and a standard substance is drawn, wherein y =60.357x-225.75, R²=0.9999, the linear range is good (fig. 5). The detection limit is 3 times of the baseline signal-to-noise ratio, the detection limit of the method is 3ng/mL, the quantification limit is 10ng/mL when the baseline signal-to-noise ratio is 10 times. The method can realize the measurement of monascus fermentation liquor (figure 6), monascus ethanol extract (figure 7), monascus yellow wine (figure 8) and wheat starter yellow wine (figure 9), wherein the content of citrinin in the four samples is respectively as follows: 2059.44ng/mL, 108.77ng/mL, 180.10ng/mL and 180.97ng/mL, is not interfered by other fluorescent substances in a sample in the determination process, has the detection result deviation of not more than 10 percent compared with the new and old national standard method, and is within the acceptable range defined by the national standard. Herein Monascus hairThe fermentation liquid is a sample obtained by inoculating the monascus purpureus CZ into a fermentation medium for fermentation for 7d, and the formula of the fermentation medium is as follows: 22.33g/L of monosodium glutamate, 9.72g/L of ammonium sulfate, 30.00g/L of long-shaped rice flour, 12.50 g/L of glucose, 9.00 g/L of monopotassium phosphate, 0.21 g/L of manganese sulfate monohydrate and 2.1 g/L of magnesium sulfate heptahydrate; the red yeast is from Fujiangningde, the red yeast yellow wine is from Fujiangningde, and the wheat starter yellow wine is from Zhejiang Shaoxing.

In addition, it is also mentioned in the present example that although the chromatographic columns are all C18 columns, the detection effect may be affected by different brands or fillers, for example, Agilent Zorbax SB-C18 (4.6X 250mm, 5 μ) is replaced by Thermo syncrons aQ C18 (4.6X 250mm, 5 μ), the separation effect on citrinin in the fermentation liquid of Monascus purpureus is poor, and other fluorescent substances interfere (FIG. 10).

Claims

1. A method for measuring citrinin content is characterized by comprising the following steps: the content of the citrinin is determined by adopting an external standard method, and the method comprises the following steps: (1) preparing a citrinin standard solution with a certain concentration gradient, and drawing a standard curve of concentration-peak area by using liquid chromatography; (2) measuring the sample solution by liquid chromatography after passing through a 0.22-micron filter membrane, and calculating the concentration according to the standard curve in the step (1);

the conditions of the liquid chromatography in the steps (1) and (2) are as follows: a chromatographic column: 4.6X 250mm, 5 μ Agilent Zorbax SB-C18, excitation wavelength 331nm, emission wavelength 500nm, sample size 20 μ L, flow rate 1.0mL/min, detection time 15min, mobile phase: phase A is 100% methanol, phase B is an aqueous solution containing 0.02% volume fraction of triethylamine, 0.32% volume fraction of tetrahydrofuran and 0.1% volume fraction of phosphoric acid, phase C is acetonitrile containing 0.1% volume fraction of formic acid, and the elution gradient of the mobile phase is as follows: 0-7 min, 30% of B +70% of C; increasing the liquid A from 0% to 30% and reducing the liquid B from 30% to 0% in 7-10 min, and keeping the liquid C at 70%; reducing the liquid A from 30% to 0%, increasing the liquid B from 0% to 30% and keeping the liquid C at 70% for 10-12 min; 12-15 min, 30% of B +70% of C;

the preparation method of the citrinin standard solution comprises the following steps: accurately weighing 1mg of citrinin in an electronic analytical balance, adding 10mL of 75% volume fraction ethanol for full dissolution, preparing 100 microgram/mL citrinin stock solution, subpackaging into 10 tubes, taking one tube, diluting the tube with 75% volume fraction ethanol into 1000ng/mL, 500ng/mL, 250ng/mL, 100ng/mL, 50ng/mL and 25ng/mL citrinin standard solution respectively, freezing the rest stock solution at-80 ℃, using the citrinin standard solution as it is, and preserving the citrinin stock solution for 1 year;

the sample solution is one of monascus fermentation liquor, monascus extracting solution, wheat starter yellow wine or monascus yellow wine.