CN117074569A

CN117074569A - Method for detecting cyclohexyl sulfamate in food by liquid chromatography-mass spectrometry/mass spectrometry

Info

Publication number: CN117074569A
Application number: CN202311078862.1A
Authority: CN
Inventors: 李绍波; 李佳佳; 左鑫; 叶梅; 万渝平; 李君�; 周易; 刘嘉睿; 肖全伟; 吴文林; 黄玉英; 乔秀明; 姚蕾珺; 熊梅瑾; 文星; 吕金珍; 陈菲; 刘鹏; 邓思思; 刘玲
Original assignee: Chengdu Institute Of Food Inspection
Current assignee: Chengdu Institute Of Food Inspection
Priority date: 2023-08-25
Filing date: 2023-08-25
Publication date: 2023-11-17

Abstract

The invention provides a method for detecting cyclohexyl sulfamate in food by liquid chromatography-mass spectrometry/mass spectrometry, which comprises the steps of sample preparation, sample extraction and liquid chromatography-mass spectrometry detection. The detection method provided by the invention has the advantages of comprehensive application range and stable relative ion abundance in qualitative confirmation of liquid chromatography mass spectrometry/mass spectrometry.

Description

Method for detecting cyclohexyl sulfamate in food by liquid chromatography-mass spectrometry/mass spectrometry

Technical Field

The invention relates to a method for detecting cyclohexyl sulfamate in food by liquid chromatography-mass spectrometry/mass spectrometry, belonging to the field of food detection.

Background

Sodium cyclamate, also known as sodium cyclamate, is now widely used in the food processing industry, and excessive consumption presents a potential hazard to the human body.

The problems of the current GB 5009.97-2016 'determination of sodium cyclohexylsulfamate in food safety national standard food' mainly appear in two aspects: 1. process applicability, process operability. 1. The method has the following applicability: the applicable range of the current standard only includes sample matrices of GB 2760 which involve limited values, whereas most sample matrices which do not use cyclohexylsulfamate are not in the standard applicable range. 2. The method comprises the following steps: the ion pair of the liquid chromatography-mass spectrometry/mass spectrometry method adopts ESI-178>80 and ESI+202>122, positive and negative ions are required to be scanned simultaneously, when a white wine sample is measured, if alcohol substances in the white wine are not volatilized, the relative ion abundance of the sample liquid can far exceed the maximum allowable deviation in qualitative confirmation, and the confirmation risk is increased. In addition, the detection mechanisms reflect that when other samples are measured by adopting a positive and negative ion simultaneous scanning mode, the difference of the relative ion abundance of qualitative and quantitative ion pairs of the standard solution and the sample solution is also large.

Disclosure of Invention

The invention provides a method for detecting cyclohexyl sulfamate in food by using liquid chromatography-mass spectrometry/mass spectrometry in order to solve the problems of incomplete coverage of the existing standard application range, unstable relative ion abundance during qualitative confirmation of liquid chromatography-mass spectrometry/mass spectrometry and the like.

The invention provides a method for detecting cyclohexyl sulfamate in food by liquid chromatography-mass spectrometry/mass spectrometry, which comprises the following steps:

a. preparing a sample;

b. sample extraction: the sample is divided into chocolate, cream, cheese, milk powder, seasoned flour products, fermented bean curd, fermented soybean, meat products, canned aquatic products containing higher grease, jelly, candy, rice flour, starch products, solid, semisolid samples and liquid samples, and is extracted;

c. measurement

Wherein, the liquid chromatography detection conditions are as follows:

chromatographic column: a surface porous layer packing C18 column, a chromatographic column of 3.0mm x 100mm,2.7 μm or equivalent performance; flow rate: 0.4mL/min;

column temperature: 45 ℃; sample injection amount: 2. Mu.L; mobile phase: a is an aqueous solution containing 0.1% formic acid, B is a methanol solution containing 0.1% formic acid, and the gradient elution procedure is as follows:

time/min	A/％	B/％
			0.0	95	5
2.0	95	5
			3.5	2	98
6.5	2	98
			7.0	95	5
10.0	95	5

The mass spectrum detection conditions are as follows:

ion source: electrospray ion source (ESI-); scanning mode: scanning negative ions; the detection mode is as follows: multiple reaction monitoring mode (MRM); capillary voltage: -2000V; drying gas temperature: 300 ℃; dry air flow: 5L/min; atomization gas pressure: 241kPa; sheath temperature: 250 ℃; sheath air flow: 11L/min; ion pairs and mass spectral parameters were monitored as follows:

the sample preparation method in the step a is as follows:

liquid sample: directly shaking up, namely shaking up a liquid sample containing carbon dioxide after removing bubbles by ultrasound; heterogeneous liquid and semi-solid samples: taking edible parts, homogenizing by a homogenizer, and uniformly mixing;

chocolate, chocolate product, cheese, butter samples: freezing edible part at-18deg.C, pulverizing with pulverizer, and stirring;

solid-state sample: taking edible part, pulverizing with grinder or pulverizer, and stirring.

The sample extraction method in the step b is as follows:

1) Chocolate, cream, cheese, milk powder, seasoned flour products, fermented bean curd, fermented soybean, meat and meat products, and canned aquatic products containing higher oil and fat

Weighing 2g of sample, accurately adding 200 mu L of cyclohexyl sulfamic acid isotope internal standard intermediate solution into a centrifuge tube, adding 20mL of acetonitrile water solution containing 0.1% formic acid, heating chocolate, cream, cheese and milk powder samples in a water bath at 60+/-2 ℃ for 20min, swirling for 5min, ultrasonically extracting for 30min, cooling to room temperature, adding 10mL of dichloromethane, slowly shaking for several times, centrifuging for 5min at 9000r/min, taking supernatant, passing through an organic phase microporous filter membrane, and performing upper machine measurement;

2) Jelly, candy, rice flour, starch product sample

Weighing 2g of sample, accurately adding 200 mu L of cyclohexyl sulfamic acid isotope internal standard intermediate solution into a centrifuge tube, adding 20mL of acetonitrile aqueous solution containing 0.1% formic acid, uniformly mixing, heating in a water bath at 60+/-2 ℃ for 20min, swirling for 5min, ultrasonically extracting for 10min, adding 10mL of dichloromethane, slowly shaking for several times, centrifuging for 5min at 9000r/min, taking supernatant, passing through an organic phase microporous filter membrane, and measuring by an upper machine;

3) Other solid, semi-solid samples

Weighing 2g of sample in a centrifuge tube, accurately adding 200 mu L of cyclohexyl sulfamic acid isotope internal standard intermediate solution, adding 20mL of acetonitrile aqueous solution containing 0.1% formic acid, carrying out vortex for 5min, carrying out ultrasonic extraction for 30min, carrying out 9000r/min for 5min, and carrying out organic phase microporous filter membrane on the sampling solution for upper machine measurement;

4) Liquid sample

Weighing 2g of sample in a centrifuge tube, accurately adding 200 mu L of cyclohexyl sulfamic acid isotope internal standard intermediate solution, diluting to 20mL scale by using acetonitrile aqueous solution containing 0.1% formic acid, uniformly mixing, swirling for 5min, performing ultrasonic extraction for 10min, centrifuging for 5min at 9000r/min, and passing the sampling solution through an organic phase microporous filter membrane for machine measurement.

The detection method further comprises an analysis result, and the analysis method comprises the following steps:

the cyclohexylsulfamic acid content in the sample was calculated according to formula (1):

wherein:

x-the content of cyclohexylsulfamic acid in milligrams per kilogram (mg/kg) in the sample;

ρ -the concentration of cyclohexylsulfamic acid in nanograms per milliliter (ng/mL) in the sample solution was calculated from a standard curve;

v-the volume of the sample is determined in milliliters (mL);

m-sample mass in grams (g).

The calculation results are expressed as arithmetic mean of two independent measurement results obtained under repetitive conditions, and the results retain 3 significant digits.

The ion pair of the current standard GB 5009.97-2016 adopts ESI-178>80 and ESI+202>122, positive and negative ions are required to be scanned simultaneously, and when a sample matrix exists, the relative ion abundance exceeds the maximum allowable deviation of the method in qualitative confirmation. The quantitative ion pair and the qualitative ion pair of the method are both in negative ion mode, so that the relative ion abundance in qualitative confirmation of the object to be detected can be ensured to be within the allowable deviation. In addition, the method adds isotope internal standard for correction, and the result is more accurate. The detection method provided by the invention has the advantages of comprehensive application range and stable relative ion abundance in qualitative confirmation of liquid chromatography mass spectrometry/mass spectrometry.

Drawings

FIG. 1 Cyclohexylsulfamic acid standard solution (100 ng/mL) and Cyclohexylsulfamic acid-D ₄ Internal standard solution (50 ng/mL) Multiple Reaction Monitoring (MRM) chromatogram

FIG. 2 ion-pair chromatograms 178>80, 178>96

Fig. 3, 178, ion scan of parent ions

FIG. 4 schematic representation of the Maillard rearrangement

FIG. 5 schematic of beta cracking

FIG. 6 34S isotope ion scan of cyclohexylsulfamic acid

FIG. 7 chromatogram of object to be measured of ultrapure water (a) and ammonium acetate solution (b) as aqueous phases

FIG. 8 Cyclohexylsulfamic acid and isotope internal standard Multiple Reaction Monitoring (MRM) chromatogram under optimal conditions

FIG. 9 ion Source Condition diagram

Detailed Description

EXAMPLE 1 method of detecting Cyclohexylsulfamate in food by liquid chromatography-Mass Spectrometry/Mass Spectrometry of the invention

Principle of

After the cyclohexyl sulfamate in the sample is extracted by the extracting solution, the liquid chromatography-mass spectrum/mass spectrometer is used for measuring, the retention time and the relative ion abundance are qualitative, and the isotope dilution internal standard method is used for quantifying.

1 reagents and materials

Unless otherwise indicated, the reagents used in the method were all analytically pure, and the water was primary water as specified in GB/T6682.

1.1 reagents

Acetonitrile (CH) ₃ CN): chromatographic purity; formic acid (HCOOH): chromatographic purity; methanol (CH) ₃ OH): chromatographic purity; dichloromethane (CH) ₂ Cl ₂ )。

1.2 preparation of reagents

Acetonitrile in water containing 0.1% formic acid: 1.0mL of formic acid is sucked, 300mL of acetonitrile is added, the volume is fixed to 1000mL by water, and the mixture is uniformly mixed.

Aqueous solution containing 0.1% formic acid: 1.0mL of formic acid was aspirated, diluted to 1000mL with water, and mixed well.

Methanol solution containing 0.1% formic acid: 1.0mL of formic acid was taken up, diluted to 1000mL with methanol, and mixed well.

1.3 Standard substance

Sodium cyclohexylsulfamate standard (C) ₆ H ₁₂ NSO ₃ Na): purity is more than or equal to 99 percent, or standard substance certificate is granted by national certification.

Sodium cyclohexylsulfamate-D ₄ (C ₆ H ₈ D ₄ NSO ₃ Na) isotopic internal standard: purity is more than or equal to 98 percent, or standard substance certificate is granted by national certification.

1.4 preparation of Standard solution

Standard stock solution of cyclohexylsulfamic acid (5 mg/mL): accurately weighing a proper amount of sodium cyclohexylsulfamate standard substance (accurate to 0.1 mg) in a 100mL volumetric flask, dissolving with water, fixing the volume to a scale, and uniformly mixing (the conversion coefficient of sodium cyclohexylsulfamate to cyclohexylsulfamic acid is 0.8907). The solution was transferred to a brown glass vessel and stored in the dark at 4℃for 12 months.

Cyclohexyl sulfamic acid isotope internal standard stock solution (100 μg/mL): accurately weighing a proper amount of sodium cyclohexylsulfamate-D ₄ (accurate to 0.1 mg) in a 100mL volumetric flask, dissolved in water and scaled to the volume, and mixed well (sodium cyclohexylsulfamate-D ₄ Converted into cyclohexylsulfamic acid-D ₄ Is 0.8928). The solution was transferred to a brown glass vessel and stored in the dark at 4℃for 12 months.

Cyclohexylsulfamic acid standard intermediate (10. Mu.g/mL): accurately sucking 0.20mL of the standard stock solution of the cyclohexyl sulfamic acid into a 100mL volumetric flask, metering the volume to a scale with water, and uniformly mixing. The solution was transferred to a brown glass vessel and stored in the dark at 4℃for 1 month.

Cyclohexylsulfamic acid isotope internal standard intermediate (5. Mu.g/mL): accurately sucking 5.0mL of the cyclohexyl sulfamic acid isotope internal standard stock solution in a 100mL volumetric flask, fixing the volume to the scale with water, and uniformly mixing. The solution was transferred to a brown glass vessel and stored in the dark at 4℃for 1 month.

Standard series working solutions of cyclohexylsulfamic acid: accurately sucking 10 mu L, 20 mu L, 50 mu L, 100 mu L, 200 mu L, 500 mu L and 1000 mu L of the standard intermediate solution of the cyclohexyl sulfamic acid into a 10mL volumetric flask respectively, adding 100 mu L of the standard intermediate solution of the cyclohexyl sulfamic acid isotope respectively, and fixing the volume to the scale by using an acetonitrile aqueous solution containing 0.1% formic acid. The concentrations of the cyclohexyl sulfamic acid and the standard working solution are respectively 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, 200ng/mL, 500ng/mL and 1000ng/mL, and the concentrations of the cyclohexyl sulfamic acid isotope internal standard in the standard working solution are 50ng/mL. It is prepared in the immediate use.

1.5 materials

Centrifuge tube: 50mL centrifuge tube with screw cap scale.

Organic phase microporous filter membrane: 0.22 μm pore size.

2 instrument and apparatus

Liquid chromatography-mass spectrometry/mass spectrometry equipped with Electrospray (ESI) ion source.

Analytical balance: the sensing amount is 1mg and 0.1mg.

Vortex mixer.

High-speed centrifuges.

An ultrasonic generator.

Sample pulverizer.

And a constant temperature water bath device.

3 analysis step

3.1 sample preparation

3.1.1 liquid sample

Directly shaking (shaking after removing bubbles by ultrasonic treatment of the sample containing carbon dioxide).

3.1.2 heterogeneous liquid, semi-solid samples

Taking edible part, homogenizing by a homogenizer, and mixing uniformly.

3.1.3 samples of chocolate, chocolate products, cheese, butter, etc

Taking edible part, freezing at-18deg.C, pulverizing with pulverizer, and stirring.

3.1.4 solid samples

Taking edible part, pulverizing with grinder or pulverizer, and stirring.

3.2 sample extraction

3.2.1 samples of chocolate, cream, cheese, milk powder, seasoned flour, fermented bean curd, fermented soybean, meat and meat products, cans for seafood, and the like containing higher oils

Weighing 2g of sample (accurate to 0.001 g) in a centrifuge tube, accurately adding 200 mu L of cyclohexyl sulfamic acid isotope internal standard intermediate solution, adding 20mL of acetonitrile water solution containing 0.1% formic acid, heating the sample such as chocolate, cream, cheese, milk powder and the like in a water bath at 60+/-2 ℃ for 20min, swirling for 5min, ultrasonically extracting for 30min, then cooling to room temperature, adding 10mL of dichloromethane, slowly shaking for several times, centrifuging for 5min at 9000r/min, taking supernatant, passing through an organic phase microporous filter membrane, and loading for measurement.

3.2.2 samples of jelly, candy, rice flour, starch products, etc

Weighing 2g of a sample (accurate to 0.001 g) in a centrifuge tube, accurately adding 200 mu L of an internal standard intermediate solution of cyclohexyl sulfamic acid isotope, adding 20mL of an acetonitrile aqueous solution containing 0.1% formic acid, uniformly mixing, heating in a water bath at 60+/-2 ℃ for 20min, swirling for 5min, ultrasonically extracting for 10min, adding 10mL of dichloromethane, slowly shaking for several times, centrifuging for 5min at 9000r/min, taking supernatant, passing through an organic phase microporous filter membrane, and performing upper machine measurement.

3.2.3 other solid, semisolid samples

Weighing 2g of a sample (accurate to 0.001 g) in a centrifuge tube, accurately adding 200 mu L of cyclohexyl sulfamic acid isotope internal standard intermediate solution, adding 20mL of acetonitrile aqueous solution containing 0.1% formic acid, carrying out vortex for 5min, carrying out ultrasonic extraction for 30min, carrying out centrifugation for 5min at 9000r/min, and subjecting the sampling solution to an organic phase microporous filter membrane for upper machine measurement.

3.2.4 liquid sample

Weighing 2g of a sample (accurate to 0.001 g) in a centrifuge tube, accurately adding 200 mu L of an internal standard intermediate solution of cyclohexyl sulfamic acid isotope, diluting to 20mL of scale by using an acetonitrile aqueous solution containing 0.1% of formic acid, uniformly mixing, swirling for 5min, ultrasonically extracting for 10min, centrifuging for 5min at 9000r/min, and measuring the sample liquid by using an organic phase microporous filter membrane.

3.3 instrument reference conditions

3.3.1 liquid chromatography reference conditions

a) Chromatographic column: surface porous layer filler C ₁₈ Column, 3.0mm x 100mm,2.7 μm or equivalent performance chromatography column;

b) Mobile phase: a is an aqueous solution containing 0.1% formic acid, B is a methanol solution containing 0.1% formic acid, and the gradient elution procedure is shown in Table 1;

c) Flow rate: 0.4mL/min;

d) Column temperature: 45 ℃;

e) Sample injection amount: 2. Mu.L.

TABLE 1 gradient elution procedure

Time/min	A/％	B/％
			0.0	95	5
2.0	95	5
			3.5	2	98
6.5	2	98
			7.0	95	5
10.0	95	5

3.3.2 Mass Spectrometry reference Condition

a) Ion source: electrospray ion source (ESI-);

b) Scanning mode: scanning negative ions;

c) The detection mode is as follows: multiple reaction monitoring mode (MRM);

d) Capillary voltage: -2000V;

e) Drying gas temperature: 300 ℃;

f) Dry air flow: 5L/min;

g) Atomization gas pressure: 241kPa;

h) Sheath temperature: 250 ℃;

i) Sheath air flow: 11L/min;

j) The ion pairs monitored and the mass spectral parameter references are shown in table 2.

Table 2 monitoring ion pairs and mass spectral parameter references

3.4 qualitative determination

3.4.1 retention time

The retention time of the chromatographic peak of the target compound in the sample is compared with the retention time of the corresponding standard chromatographic peak, and the variation range is within +/-2.5 percent.

3.4.2 quantitative ion, qualitative ion, and relative ion abundance ratio

The presence of cyclohexylsulfamic acid in the sample can be determined by determining that the maximum allowable deviation of the relative abundance ratio (k) of the two daughter ions of cyclohexylsulfamic acid is not more than the range specified in table 3, as compared with the relative abundance ratio of the two daughter ions of cyclohexylsulfamic acid in a concentration equivalent to that of the standard working solution.

TABLE 3 maximum allowable deviation of relative ion abundance in qualitative validation

Relative ion abundance (%)	k>50	50≥k>20	20≥k>10	k≤10
					Maximum allowable deviation (%)	±20	±25	±30	±50

3.5 preparation of standard curve

And (3) injecting a standard series of working solutions into the liquid chromatograph-mass spectrometer, taking the ratio of the concentration of the detected component to the concentration of the internal standard as an abscissa, taking the ratio of the chromatographic peak area of the detected component to the chromatographic peak area of the internal standard as an ordinate, and drawing a standard curve.

The cyclohexylsulfamic acid multi-reaction monitoring (MRM) chromatogram is shown in FIG. 1.

3.6 measurement of sample solution

And (3) carrying out sample injection measurement on the sample solution and the standard curve working solution, obtaining the concentration of the object to be measured in the sample solution according to the standard curve, and calculating the content of the object to be measured in the sample.

3.7 blank test

Except that no sample was added, the measurement was performed according to 19.2.

4 representation of the analysis results

wherein:

v-the volume of the sample is determined in milliliters (mL);

m-sample mass in grams (g).

5 precision:

the absolute difference between the two independent measurements obtained under repetitive conditions must not exceed 15% of the arithmetic mean.

6 others

When the sampling amount was 2g, the detection limit of the method was 0.03mg/kg, and the quantitative limit was 0.10mg/kg.

Example 2 selection and optimization of measurement conditions of the liquid chromatography-Mass Spectrometry/Mass Spectrometry Instrument of the invention

1. Selection of a cyclohexylsulfamic acid ion pair

The existing standard GB 5009.97-2016 liquid chromatography-mass spectrometry/mass spectrometry method adopts 1 negative ion pair and 1 positive ion pair as ion pairs, wherein the negative ion pairs are quantitative ion pairs. In standard tracking evaluation work, a plurality of institutions reflect that the relative abundance of qualitative ions is greatly influenced by a matrix, and the deviation of the relative ion abundance easily exceeds an allowable range, so that misjudgment of a result is caused. The analysis is because the matrix effect of the matrix in the sample solution on the positive ions and the negative ions is different, and if the qualitative ion relative abundance of the standard solution is taken as a reference, the qualitative ion relative abundance of the object to be detected in the sample solution easily exceeds the maximum allowable deviation. Therefore, when establishing liquid chromatography-mass spectrometry/mass spectrometry, the same polarity ion pair is selected as much as possible.

Through data query, in NY/T3473-2019 'determination liquid chromatography-tandem mass spectrometry of neotame, alitame, aspartame, sodium cyclamate, acesulfame and saccharin sodium in feed' and a small number of documents, ESI-178>80 is selected as a quantitative ion, and ESI-178>96 qualitative ion pairs are also selected. No ESI-178>96 qualitative ion pair chromatogram is available in the NY/T3473-2019 standard, and the 178>96 qualitative ion pair response cannot be judged, but the 178>80 and 178>96 ion pair chromatograms can be clearly seen from an Article (Detection of 10sweeteners in various foods byliquid chromatography/tandemmass spectrometry) in the journal of SCI journal of Original technology, see fig. 2. It can be seen from the graph that the response of the 178>96 ion pair is only about one percent of the response of the 178>80 ion pair. By sub-ion scanning, it was found that the parent ion of 178 did have only 80, 96 fragments, and the response of 178>96 ion pairs was only about 0.8 percent of the 178>80 ion pair response, see fig. 3. For analytical reasons, fragments of 178 parent ion generation 96 need to undergo a mahalanobis rearrangement reaction (see fig. 4 for schematic diagrams), which is more difficult, so 178>96 ion pairs respond poorly. Whereas fragments of 178 parent ion generation 80 only need to undergo beta fragmentation (see schematic fig. 5), so 178>80 ion pairs have a high response.

From the mass spectrum principle book, the types of ions in the mass spectrum are divided into 4 types, and isotope ions are one of the 4 mass spectrum ion types and are also important ion pair sources. The cyclohexyl sulfamic acid contains sulfur, and the sulfur mainly contains three stable isotopes of 32S, 33S and 34S in the natural world, wherein the natural abundance is 95.02%, 0.75% and 4.21%, and the abundance of 34S is the highest. Therefore, 34S isotope ions of the cyclohexyl sulfamic acid can be selected as parent ions, 34S fragment ions are child ions, and 180>82 ion pairs are obtained, as shown in fig. 6.

Selection of 2 isotope internal standard

Two common cyclohexylsulfamic acid deuterated isotopes are available on the market: sodium cyclohexylsulfamate-D ₄ And sodium cyclohexylsulfamate-D ₁₁ Comparison of Cyclohexylsulfamic acid-D by experiments with labeled recovery ₄ And cyclohexylsulfamic acid-D ₁₁ Is corrected for the internal standard of (C) cyclohexylsulfamic acid-D ₄ Is very close to the true value. In addition, sodium cyclohexylsulfamate-D ₄ The price of (2) is very low, the price of 10mg standard is between one thousand and two thousand yuan, and the isotope internal standard cost of each single sample is only 0.1-0.2 yuan. Combines various advantages and disadvantages, and finally selects the sodium cyclohexylsulfamate-D ₄ As an isotopic internal standard for the present method.

3 chromatography condition investigation

When methanol and acetonitrile are used as the organic phase of the mobile phase, the chromatographic peak shape and the column effect are ideal, and finally, the methanol is used as the organic phase according to the sensitivity of an object to be detected, the matrix effect and the elution condition of fat-soluble impurities in sample liquid (the methanol can dissolve protein deposited in a chromatographic column). The chromatographic condition investigation was performed by selecting an aqueous ammonium acetate solution, ultrapure water, and 0.1% aqueous formic acid solution as subjects, and when an aqueous ammonium acetate solution+methanol, ultrapure water, and methanol were selected as mobile phases, the chromatographic peak width of the test substance was broad (see fig. 7), and the retention time was liable to fluctuate due to the influence of the sample matrix. When 0.1% formic acid aqueous solution and 0.1% formic acid methanol solution are used as the mobile phase, the chromatographic peak width of the object to be detected is narrow and the peak height is increased. Thus, 0.1% formic acid aqueous solution and 0.1% formic acid methanol solution were finally selected as reference mobile phases.

Because the pretreatment of liquid chromatography-mass spectrometry/mass spectrometry is relatively simple, considering the service life of the chromatographic column, the surface porous layer packing C18 column, 3.0mm multiplied by 100mm and 2.7 μm are finally selected as reference chromatographic columns, and chromatographic reference conditions are finally obtained by optimizing the mobile phase elution procedure: the mobile phase is an aqueous solution (A) containing 0.1% formic acid; methanol solution (B) containing 0.1% formic acid; the flow rate is 0.4mL/min; the column temperature is 45 ℃; the sample injection amount is 1-5 mu L; the elution procedure is shown in Table 4. The most optimal conditions of the cyclohexylsulfamic acid and the isotope internal standard Multiple Reaction Monitoring (MRM) chromatogram are shown in FIG. 8.

Table 4 elution procedure

4 examination of matrix effects

32 typical blank samples are selected, and a sample matrix solution is obtained according to a pretreatment method of liquid chromatography-mass spectrometry/mass spectrometry. Matrix standard solutions with a concentration of 50.0ng/mL were prepared with 32 sample Matrix solutions, respectively, and peak areas were compared with 50.0ng/mL standard solutions, and calculated according to the formula of Matrix Effect (%) =b/a×100% (a: response value of analyte in pure solvent B: response value of analyte of the same content added to sample Matrix), as shown in table 5. The results show that a significant matrix effect still exists for some of the pre-treated sample solutions. Therefore, the isotope internal standard correction method is adopted, so that measurement deviation caused by matrix effect can be corrected, and the accuracy of a measurement result is ensured.

TABLE 5 matrix effect results Table

5 examination of Using Effect of method

The application range of the liquid chromatography-mass spectrometry/mass spectrometry of the method is expanded to a solid or semi-solid sample for the first time. Therefore, the influence condition of instruments and consumables in the using process of the method needs to be examined. By observation, after 2800 sample injections, the chromatographic column pressure was varied within 1500PSI, and the column efficiency was not significantly reduced, and no significant contamination of the ion source (solvent delay setting with the instrument) was observed, see FIG. 9. The method has ideal use effect.

Example 3 methods of detection of the invention laboratory verification and inter-laboratory verification

1 method linear range

Liquid chromatography-mass spectrum/mass spectrum normal range

When the cyclohexyl sulfamic acid is in the range of 10.00-1000 ng/mL, the standard curve linear equation of the standard series working solution is as follows: y=9.437516x+0.78494, r is 0.9999, which is more than 0.999, which indicates that the linear relation of the standard series working fluid is good, and the standard requirement can be satisfied.

2 liquid chromatography-Mass Spectrometry/Mass Spectrometry detection Limit and quantitative Limit

The detection limit of the liquid chromatography-mass spectrometry/mass spectrometry is initially determined to be 0.03mg/kg, and the quantitative limit is initially determined to be 0.10mg/kg. The method comprises the steps of performing a standard adding experiment according to the initially determined detection limit of sodium cyclamate in cheese, butter, citrus, raisin, soy sauce, corn soft sweet, green ball, mi Buding, walnut cake, meat sausage, marinated eggs, sugar, soy sauce, sweet flour paste, infant milk powder, infant complementary food (rice flour), white wine, fruit tea substitute, yogurt, dried bean curd, chocolate, steamed bread, lotus root starch, canned fish, honey, wine, jelly, hawthorn strips, popcorn and blank matrix of melon seeds (simultaneously adding according to the content level of neotame 10mg/kg, aspartame 10mg/kg, saccharin sodium 20mg/kg, acesulfame 50mg/kg, aspartame 50mg/kg, benzoic acid 50mg/kg and sorbic acid 50mg/kg, so as to investigate the specificity of the method), wherein the experiment shows that the quantitative ion pairs and the signal to noise ratios of qualitative ion pairs of the substances to be detected in all the matrixes are more than 3, and the peak positions of the substances to be detected are not interfered; and (3) performing a labeling experiment according to the preliminarily determined quantitative limit in the 32 matrixes, wherein the signal to noise ratio of quantitative ion pairs and qualitative ion pairs of the to-be-detected objects in all matrixes is greater than 10. The detection limit of the liquid chromatography-mass spectrometry/mass spectrometry is finally determined to be 0.03mg/kg, and the quantitative limit is finally determined to be 0.10mg/kg.

3 accuracy and precision of liquid chromatography-mass spectrometry/mass spectrometry

Cheese, butter, citrus, raisin, soy sauce pickled vegetables, pickled algae, fermented soybean, corn soft sweet, green ball, mi Buding, walnut cake, meat sausage, marinated eggs, sugar, soy sauce, sweet flour paste, infant milk powder, infant complementary food (rice flour), white wine, fruit tea, yogurt, dried bean curd, chocolate, steamed bread, lotus root starch, fish can, honey, wine, jelly, hawthorn strips, popcorn and melon seed matrix are selected as the study objects, and 1-time, 2-time and 10-time quantitative limit level marking is carried out, and table 6 is shown. The results show that the reproducibility of liquid chromatography-mass spectrometry/mass spectrometry meets the standard requirements.

TABLE 6 precision table of cyclohexylsulfamate of 32 matrices by liquid chromatography-mass spectrometry/mass spectrometry

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4 inspection of working solution stability of standard curve

The gas chromatography, the standard curve working solution derived by the liquid chromatography and the standard curve working solution of the liquid chromatography-mass spectrometry/mass spectrometry are respectively divided into 4 parts, are put into a sample injection bottle and are placed into a sample injection tray at normal temperature, sample injection is carried out at 0h, 8h, 24h and 48h respectively, the concentration of an object to be detected is counted, and the quantitative repeatability is inspected, and is shown in a table 7. The result shows that in the gas chromatography, 4 measured values of each concentration standard curve working solution meet the quantitative repeatability requirement (RSD%. Ltoreq.3%) of the JJJG 700-2016 gas chromatograph; in the liquid chromatography, 4 measured values of each concentration standard curve working solution meet the quantitative repeatability requirement (RSD%. Ltoreq.3%) of a JJG 705-2014 liquid chromatograph; in the liquid chromatography-mass spectrometry/mass spectrometry, 4 measured values of each concentration standard curve working solution meet the quantitative repeatability requirement (RSD%. Ltoreq.10%) of a JF 1317-2011 liquid chromatography-mass spectrometer. The standard curve working solution has no significant change in 48 hours.

Table 7 liquid chromatography-mass spectrometry/mass spectrometry standard curve working solution stability investigation table

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Claims

1. A method for detecting cyclohexyl sulfamate in food by liquid chromatography-mass spectrometry/mass spectrometry, which is characterized by comprising the following steps: it comprises the following steps:

a. preparing a sample;

c. measurement

Wherein, the liquid chromatography detection conditions are as follows:

time/min A/％ B/％ 0.0 95 5 2.0 95 5 3.5 2 98 6.5 2 98 7.0 95 5 10.0 95 5

The mass spectrum detection conditions are as follows:

2. the method of claim 1, wherein: the sample preparation method in the step a is as follows:

3. The method of claim 1, wherein: the sample extraction method in the step b is as follows:

2) Jelly, candy, rice flour, starch product sample

3) Other solid, semi-solid samples

4) Liquid sample

4. A detection method according to any one of claims 1-3, characterized in that: the analysis method comprises the following steps:

wherein:

v-the volume of the sample is determined in milliliters (mL);

m-sample mass in grams (g).