CN110441416B - Method for measuring bitter agents naringin and denatonium benzoate in children finger print inkpad by using ultra-high performance liquid chromatography - Google Patents

Method for measuring bitter agents naringin and denatonium benzoate in children finger print inkpad by using ultra-high performance liquid chromatography Download PDF

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CN110441416B
CN110441416B CN201910680959.7A CN201910680959A CN110441416B CN 110441416 B CN110441416 B CN 110441416B CN 201910680959 A CN201910680959 A CN 201910680959A CN 110441416 B CN110441416 B CN 110441416B
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望秀丽
卫碧文
高欢
倪彬彬
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Shanghai Customs Mechanical And Electrical Products Testing Technology Center
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Abstract

The invention discloses a method for measuring bitter agents naringin and denatonium benzoate in children finger-painting inkpad by using ultra-high performance liquid chromatography, wherein methanol is used for being 0.02mol/L KH2PO4(30: 70, v/v) is used as a solvent, 2 bittering agents in the children fingerprinted inkpad sample are extracted by an ultrasonic extraction mode, and the quantification is carried out by an external standard method and the UPLC analysis is carried out. The linear range of the naringin and the denatonium benzoate is 0.1-5mg/L (R)2Not less than 0.99), detection limit (S/N)>3) 0.75 and 1.15mg/kg, respectively. The standard recovery rate ranges of naringin and denatonium benzoate in the finger-print inkpad for children at three concentration levels are 86.7-108.4%; the relative standard deviation (RSD, n ═ 6) is less than 5.0%.

Description

Method for measuring bitter agents naringin and denatonium benzoate in children finger print inkpad by using ultra-high performance liquid chromatography
Technical Field
The invention relates to a detection method, in particular to a method for measuring bitter agents naringin and denatonium benzoate in children finger-painting inkpad by using ultra-high performance liquid chromatography.
Background
Bittering agents (Aversive agents), also known as Aversive agents, deterrents, are widely used chemicals added to products to impart a bitter taste or odor to prevent people, especially children, from inhaling or ingesting toxic, harmful substances, rodents and birds from damaging facilities. Denatonium benzoate, also known as picrin (Bitrex), whose structure consists of a benzoic acid group and a denatonium group, is currently the most bitter compound known and is typical of bittering agents. The bitter essence has the characteristics of stable chemical property, low price, easy obtainment, easy water solubility and the like, in addition, the Median lethality (LD 50) of the mammal is 841mg/kg of body weight, the food inhibition effect can be achieved only by adding a very small amount (0.00001%), the bitter essence is safe and nontoxic under the common use condition, and the bitter essence is a common bitter taste agent in daily consumer products, for example, the bitter essence is added for preventing children from ingesting toxic but sweet antifreeze. Naringin is a dihydro flavonoid compound separated from grapefruit peel, has bitter taste, is taken as a bittering agent, and has the advantages of edible property, safety, no toxicity, wide source and the like. In order to minimize the risk to children of potentially intentionally or unintentionally putting the finger-paint in the mouth, the world's multiple standards require that a bittering agent be added to the finger-paint in both compounds. European Union Standard EN 71-7:2014+ A2:2018 International Standard ISO 8124-7:2015 is consistent with the requirements of national painting pigment product Standard GB6675.14-2014 on bittering agents. The above criteria give reference additive concentrations as: 1% naringin, 0.0004% denatonium benzoate (4 mg/kg).
At present, the national and international detection standards related to naringin and denatonium benzoate in consumer products include: the ASTM D7304-2014 concrete method is that after a cooling liquid sample is diluted by a potassium dihydrogen phosphate solution (pH 4.6) with the concentration of 0.1mol/L, the cooling liquid sample is directly analyzed by HPLC, and the quantitative limit of the method is 6.6 mg/kg; the specific method of EN 1214:1997 is that the sample to be tested is mixed with ammonium oxalate and dimethyl formamide uniformly, diluted, heated for 10min at 90 ℃, cooled to room temperature, filtered and analyzed by HPLC. There is a small amount of literature on the method of assaying denatonium benzoate at home and abroad, and Henderson et al report a method of analyzing denatonium benzoate in Oregon consumer products by HPLC, Chemosphere,1998,36(1):203 in Analysis of denatonium benzoate by high performance liquid chromatography, which comprises diluting a sample with methanol, centrifuging, filtering, and then measuring by HPLC, with a limit of quantitation of 1.25 mg/L. The pretreatment process of the sample in the standard and literature method for measuring the denatonium benzoate mainly comprises solid-phase extraction separation and purification or HPLC analysis after directly diluting with a mobile phase.
The literature relating to the measuring method of naringin at home and abroad mainly focuses on the traditional Chinese medicine aspect, and the domestic literature reports methods for analyzing the naringin in the compound Chinese angelica capsule, the lung ventilating and regulating granule, the thoroughfare bitter orange and the indigestion removing powder by the high performance liquid chromatography. A method for simultaneously measuring 14bioactive compounds in Shenzhu decoction by HPLC-DAD and HPLC-MS is reported by Weon et al in Simultaneous determination of 14bioactive compounds in Samchulkunbi-measuring using HPLC-DAD and LC-MS, anal. methods,2014,6: 6023. In the literature method for determining naringin, the sample pretreatment process mainly comprises sample crushing, ultrasonic extraction by adopting a methanol solvent and HPLC or/and HPLC-MS analysis.
The children's finger-print inkpad has complex substrate, and needs to add extraction solvent with high dilution factor to extract in order to avoid interference, while the standard recommended addition concentration of denatonium benzoate is 4mg/kg, which is a challenge to the sensitivity of instruments and methods, and the sensitivity of the instruments and the determination method of the denatonium benzoate cannot meet the requirement.
The children's finger print inkpad is one of finger print pigments, and children dip in the pigment on the inkpad with the finger and create, children's consciousness is weak, if children's inkpad product does not add bittering agent or addition volume does not accord with standard requirement, easily causes serious harm through licking children's physical and mental health. The ultra-high performance liquid chromatography has the advantages of high analysis speed, high separation efficiency and the like, and reports on the analysis of bitter agents naringin and denatonium benzoate in the children's finger-painting inkpad by the ultra-high performance liquid chromatography are not available at present.
Disclosure of Invention
In order to solve the problems, the invention provides a method for measuring the bitter agents naringin and denatonium benzoate in the children finger-painting inkpad by using ultra-high performance liquid chromatography.
The technical scheme of the invention is as follows:
a method for measuring bitter agents naringin and denatonium benzoate in children finger-painting inkpad by using ultra-high performance liquid chromatography comprises the following steps:
(1) preparing a test solution: precisely weighing a sample to be detected in a conical flask with a plug, and adding methanol-0.02 mol/L KH2PO4Extracting a solvent, wherein the volume of the extraction solvent is 50 times of the mass of the sample, performing ultrasonic extraction, filtering clear liquid by using a filter membrane to obtain filtrate, and using the filtrate as a test solution for ultra-high performance liquid chromatography analysis;
(2) preparing a standard solution, namely preparing mixed standard solutions of naringin and denatonium benzoate with different concentration gradients;
(3) performing determination by adopting ultra-high performance liquid chromatography, wherein the chromatographic conditions are as follows:
a chromatographic column: c18;
column temperature: 30-40 ℃;
mobile phase: mobile phaseA is acetonitrile; the mobile phase B is 0.02mol/L KH2PO4，pH≥4.3；
Flow rate: 0.2-0.5 mL/min;
(4) quantitative analysis: respectively injecting the standard solution and the test solution with the same volume into the ultra-high performance liquid chromatography, and quantifying by adopting an external standard method.
Preferably, the step (4) further comprises injecting the sample solution into an ultra-high performance liquid chromatography, scanning the ultraviolet absorption spectrum of naringin, benzoic acid and denatonium ions by a diode array detector, selecting different detection wavelengths for qualitative analysis, and quantitatively analyzing after the qualitative analysis.
Preferably, the detection wavelength of naringin is 282nm, the detection wavelength of benzoic acid ion is 230nm, and the detection wavelength of denatonium ion is 210 nm.
Preferably, the mobile phase gradient: 0min, 10% mobile phase a; when the time is 2min, the solution is raised to 30 percent of mobile phase A and kept for 3 min; and 5.01min, reducing to 10% of mobile phase A, and keeping for 2 min.
Preferably, the standard solution prepared in step (2) has a concentration of 0.1mg/L, 0.2mg/L, 0.5mg/L, 1mg/L, 2mg/L and 5mg/L, respectively.
Preferably, the extraction solvent has a methanol content of 30% by volume.
Preferably, the mobile phase has a pH of 4.3.
Preferably, the mobile phase flow rate is 0.3 mL/min.
Preferably, the column temperature of the column is 40 ℃.
Preferably, C18 is used in a 2.1mm X50 mm, 1.7 μm column.
Compared with the prior art, the invention has the following beneficial effects:
the volume of the extraction solvent is 50 times of the mass of the sample for extraction, so that the interference of the matrix is eliminated;
aiming at the problem that denatonium benzoate is a water-soluble compound and is dissociated into benzoic acid ions and denatonium ions in water, and naringin needs to be dissolved in an organic solvent, the extraction efficiency and the elution capacity of a mobile phase are comprehensively considered by optimizing the proportion of methanol in a sample extraction solvent, and meanwhile, the recovery rate of 90 percent of the naringin and the denatonium benzoate is realized;
higher sensitivity is achieved by optimizing mobile phase composition and gradient, mobile phase flow rate and column temperature to meet the separation.
Of course, it is not necessary for any product in which the invention is practiced to achieve all of the above-described advantages at the same time.
Drawings
FIG. 1 is a UV spectrum of naringin, benzoic acid and denatonium for an embodiment of the present invention;
FIG. 2 is a chromatogram of naringin, benzoic acid and denatonium at various mobile phase pH values for an embodiment of the invention;
FIG. 3 is a chromatogram of different mobile phase flow rates versus naringin, benzoic acid and denatonium for an embodiment of the present invention.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. In practice, the invention will be understood to cover all modifications and variations of this invention provided they come within the scope of the appended claims.
Examples
A method for measuring bitter agents naringin and denatonium benzoate in children finger-painting inkpad by using ultra-high performance liquid chromatography comprises the following steps:
(1) preparing a test solution: precisely weighing a sample to be detected in a conical flask with a plug, and adding methanol-0.02 mol/L KH2PO4Extracting a solvent, wherein the volume of the extraction solvent is 50 times of the mass of the sample, performing ultrasonic extraction, filtering clear liquid by using a filter membrane to obtain filtrate, and using the filtrate as a test solution for ultra-high performance liquid chromatography analysis;
(2) preparing a standard solution, namely preparing mixed standard solutions of naringin and denatonium benzoate with different concentration gradients;
(3) performing determination by adopting ultra-high performance liquid chromatography, wherein the chromatographic conditions are as follows:
a chromatographic column: c18;
column temperature: 30-40 ℃;
mobile phase:the mobile phase A is acetonitrile; the mobile phase B is 0.02mol/L KH2PO4，pH≥4.3；
Flow rate: 0.2-0.5 mL/min;
(4) quantitative analysis: respectively injecting the standard solution and the test solution with the same volume into the ultra-high performance liquid chromatography, and quantifying by adopting an external standard method.
And (4) injecting the test solution into an ultra-high performance liquid chromatography, scanning the ultraviolet absorption spectra of naringin, benzoic acid and denatonium ions by a diode array detector, selecting different detection wavelengths for qualitative analysis, and performing quantitative analysis after the qualitative analysis.
The method is adopted to carry out analysis experiments
1 experimental part
1.1 instruments and reagents
Ultra high performance liquid chromatography (Waters corporation); centrifuge (eppendorf, germany); ultrasound machine (Elma, germany).
Methanol (chromatographically pure, chemical reagents of national drug group, ltd); acetonitrile (chromatographically pure, ANPEL); naringin (CAS: 10236-47-2) (purity not less than 97%, m/m, Shanghai echei Seiei chemical industry development Co., Ltd.), denatonium benzoate (CAS: 3734-33-6) (purity not less than 95%, m/m, Germany Dr. Ehrenstorfer Co., Ltd.); KH (Perkin Elmer)2PO4(Millipore); water (Millipore); 0.22 μm organic filtration membrane (Millipore).
1.2 UPLC analysis conditions
Chromatographic conditions are as follows: a chromatographic column: c18(2.1 mm. times.50 mm, 1.7 μm); column temperature: 40 ℃; flow rate: 0.3 mL/min; sample introduction amount: 10 mu L of the solution; the mobile phase A is acetonitrile, and the mobile phase B is 0.02mol/LKH2PO4(pH 4.3): gradient of mobile phase: increasing to 30% mobile phase A at 0min and 10% mobile phase A at 2min, maintaining for 3min, and maintaining to 10% mobile phase A at 5.01min for 2 min.
1.3 sample pretreatment
About 0.2g (to an accuracy of 0.1mg) of sample was weighed into a stoppered conical flask and 10mL of methanol-0.02 mol/L KH was added2PO4(30: 70, v/v) extracting solvent with water solution, shaking for 1min to fully soak the sample, performing ultrasonic extraction for 20min, cooling to room temperature, and standing16000g for 15min, and the supernatant was filtered through a 0.22 μm filter and used for UPLC analysis.
2 results and discussion
2.1 establishment of UPLC conditions
2.1.1 selection of detection wavelength
Under the chromatographic condition, a Diode Array Detector (DAD) scans an ultraviolet absorption spectrogram of naringin, benzoic acid and denatonium ions as shown in figure 1, the maximum absorption wavelength of naringin is 282nm, the maximum absorption wavelength of benzoic acid ions is 230nm, the maximum absorption wavelength of denatonium ions is near 210nm, but more ultraviolet absorption substances are in the vicinity of 210nm and are easy to generate interference, and the sensitivity of other target substances at 282nm is too low, so that different detection wavelengths are selected for qualitative and quantitative determination. The detection wavelength of naringin is 282nm, that of benzoic acid ion is 230nm, and that of denatonium ion is 210 nm. 2.1.2 selection of the Mobile phase
The experiment compares methanol-KH2PO4Aqueous solution, acetonitrile-KH2PO4Influence of elution effects of 3 mobile phases of aqueous solution and acetonitrile-ammonium acetate aqueous solution. For naringin and denatonium ion, the 3 flows have small influence on the retention time of naringin and denatonium ion, and the response intensity is in acetonitrile-KH2PO4Is superior to methanol-KH in aqueous solution mobile phase system2PO4Aqueous solution and acetonitrile-ammonium acetate aqueous solution mobile phase system. methanol-KH is used2PO4The water solution and the acetonitrile-ammonium acetate water solution system are eluted, and no benzoic acid ion chromatographic peak appears, so the acetonitrile-KH is comprehensively considered and selected2PO4The aqueous solution is a mobile phase system.
The pH of the mobile phase has an effect on the chromatographic behavior of naringin, benzoic acid and denatonium ions, so that a better separation is achieved by adjusting the pH of the mobile phase. The mobile phase KH is used according to the applicable pH range (1-12) of a chromatographic column and the pKa value (4.05) of a denatonium benzoate compound combined with the literature2PO4The fixed concentration of naringin and denatonium benzoate was 10mg/L, and gradient elution was performed according to section 1.2, and the pH of the mobile phase was examined to be 3.0,The effects on retention time and peak area of naringin, benzoic acid and denatonium ions at 4.3, 4.6 and 6.0 are shown in fig. 2, where a: pH 3.0, b: pH 4.3, c: pH 4.6, d: pH 6.0. The test result shows that the pH of the mobile phase is 3.0, and no peak is generated in benzoic acid ions; when the pH of the mobile phase is 4.3 or more, a chromatographic peak appears in benzoic acid ions. The cause of the above phenomenon is presumed: the change in chromatographic behavior of benzoic acid correlates with its pKa value. Under strong acid condition, benzoic acid ion is protonated, and no absorption is generated at more than 200 nm; the pKa value of the denatonium benzoate compound is 4.05, the denatonium benzoate compound mainly exists in an ionic state when the pH of a mobile phase is 4.3 or more, the ionic content is increased along with the gradual increase of the pH, the peak area is increased, the polarity is enhanced, the retention capacity of a reversed phase chromatographic column is weakened, and the retention time is reduced. When the mobile phase pH is more than 4.3, the chromatographic peak of naringin and denatonium ion is increased along with the increase of the mobile phase pH, the peak height is reduced, the peak shape is widened, and the symmetry is deteriorated. The pH of the mobile phase was chosen to be 4.3, taking into account the sensitivity, reproducibility and symmetry of the chromatographic peaks, and the denatonium benzoate was qualitatively and quantitatively determined using the denatonium ion.
2.1.3 selection of flow rates of the mobile phase
The influence of the flow rates of the mobile phase on the chromatographic separation efficiency when the flow rates are respectively 0.2mL/min, 0.3mL/min, 0.4mL/min and 0.5mL/min is examined in the experiment, and the result is shown in FIG. 3, wherein a: 0.2mL/min, b: 0.3mL/min, c: 0.4mL/min, d: 0.5 mL/min. When the flow rate of the mobile phase is increased from 0.2mL/min to 0.5mL/min, the analysis time of naringin and denatonium ions is reduced from 6.3min to 3.2min, but the phenomenon of tailing of the chromatographic peak of the denatonium ions is serious at the same time. The flow rate of the mobile phase is selected to be 0.3mL/min by comprehensively considering the analysis efficiency and the symmetry of chromatographic peaks.
2.1.4 selection of column temperature
In this test, under the condition of a flow rate of 0.3mL/min, factors of separation and tolerance of the chromatographic column are comprehensively considered, and column temperatures of 30 ℃, 35 ℃ and 40 ℃ are selected for comparison respectively. The analysis time of naringin and denatonium ion is reduced as the column temperature rises from 30 ℃ to 40 ℃, and the chromatographic peak shape of denatonium is sharp and the symmetry is good at 40 ℃, and the column temperature of the chromatographic column is selected to be 40 ℃ mainly in consideration of the analysis efficiency and the tolerance of the chromatographic column.
2.2 Standard Curve and detection Limit
Mixed standard solutions of naringin and denatonium benzoate were prepared at concentrations of 0.1mg/L, 0.2mg/L, 0.5mg/L, 1mg/L, 2mg/L and 5mg/L, respectively, and were tested under the optimum test conditions described previously. The linear relationship between the peak area (ordinate) and the mass concentration (abscissa) of the obtained compound is shown in table 1.
TABLE 1 Linear equation, Retention time, correlation coefficient and detection limits for naringin and denatonium
Figure BDA0002144762670000081
Y: peak area; x: mass concentration, mg/L; linear range: 0.1-5mg/L.
The linear correlation coefficients are all above 0.99, which shows that the linear relation is good. Each concentration standard sample is injected 7 times, and the relative standard deviation is 2.14-3.32%. The method carries out 6 times of reagent blank tests, and takes the mass concentration when the signal-to-noise ratio is more than 3 as the detection Limit (LOD) of the compound, and the detection limits (S/N >3) of naringin and denatonium benzoate are respectively 0.75 mg/kg and 1.15 mg/kg. The lower limit of quantitation for naringin and denatonium benzoate by the UPLC method was 2.5 and 4mg/kg, respectively.
2.3 extraction solvent
Experiments compare methanol with volume fractions of 10%, 20% and 30% in different proportions-0.02 mol/LKH2PO4Effect of extraction solvent on recovery. Using a blank sample for labeling, the recovery rates of naringin and denatonium benzoate were 25.0% and 101%, respectively, when the methanol content in the extraction solvent was 10%, 68.3% and 99.7%, respectively, when the methanol content in the extraction solvent was 20%, and 93.2% and 101%, respectively, when the methanol content in the extraction solvent was 30%. Along with the increase of the proportion of methanol in the extraction solvent, the extraction efficiency of naringin is increased, the extraction efficiency of denatonium benzoate is kept stable,comprehensively considering extraction efficiency and elution capacity of mobile phase, and selecting methanol-0.02 mol/L KH as extraction solvent2PO4(30: 70, v/v). Because the mobile phase A is acetonitrile and the mobile phase B is 0.02mol/L KH2PO4(pH 4.3): gradient of mobile phase: increasing to 30% mobile phase A at 0min and 10% mobile phase A at 2min, maintaining for 3min, and maintaining to 10% mobile phase A at 5.01min for 2 min. The experiment result shows that the phenomenon of insufficient elution of a mobile phase, more impurity peaks and interference in measurement can be caused when methanol with higher proportion (such as 40 percent and 50 percent) is added into an extraction solvent.
2.4 recovery and precision
3 concentration levels (the concentration after constant volume is 0.5mg/L, 1mg/L and 5mg/L) are respectively added to the blank sample, and the blank sample is parallelly measured for 5 times, wherein the addition concentration corresponds to the average standard recovery rate range of 86.7-108.4%, and the average relative standard deviation (RSD, n is 6) is less than 5.0%, so that the requirement of quantitative analysis can be met.
2.5 actual sample detection
Naringin and denatonium benzoate were measured in 10 samples of finger-print paste obtained by on-line purchase under the optimized chromatographic conditions described above. They were pretreated as described in section 1.3. None of naringin and denatonium benzoate was detected, as determined under optimal chromatographic conditions in section 1.2, and did not meet national mandatory standard GB6675.14-2014 part 14 of toy safety: the technical requirements of finger painting pigments and the requirements of test methods. The reason for the phenomenon is probably that although the standard of home and abroad finger painting pigments forcibly requires the addition of bittering agents to avoid children from mishaps, no corresponding detection method is issued, and relevant management departments and standards committees need to accelerate the departure progress of corresponding standards so that production enterprises can design and produce according to the standard of standards, thereby protecting the legitimate rights and interests of minors in China and protecting the health of children in China.
The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise embodiments disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims (7)

1. A method for measuring bitter agents naringin and denatonium benzoate in children finger-painting inkpad by using ultra-high performance liquid chromatography is characterized by comprising the following steps:
(1) preparing a test solution: precisely weighing a sample to be detected in a conical flask with a plug, and adding methanol-0.02 mol/L KH2PO4Extracting a solvent, wherein the volume of the extraction solvent is 50 times of the mass of the sample, performing ultrasonic extraction, filtering clear liquid by using a filter membrane to obtain filtrate, and using the filtrate as a test solution for ultra-high performance liquid chromatography analysis; wherein, the volume content of methanol in the extraction solvent is 30 percent;
(2) preparing a standard solution, namely preparing mixed standard solutions of naringin and denatonium benzoate with different concentration gradients;
(3) performing determination by adopting ultra-high performance liquid chromatography, wherein the chromatographic conditions are as follows:
a chromatographic column: c18;
column temperature: 30-40 ℃;
mobile phase: the mobile phase A is acetonitrile; the mobile phase B is 0.02mol/L KH2PO4，pH＝4.3；
Flow rate: 0.2-0.5 mL/min;
wherein, the gradient of the mobile phase: 0min, 10% mobile phase a; when the time is 2min, the solution is raised to 30 percent of mobile phase A and kept for 3 min; reducing to 10% mobile phase A after 5.01min, and keeping for 2 min;
(4) quantitative analysis: respectively injecting the standard solution and the test solution with the same volume into the ultra-high performance liquid chromatography, and carrying out quantitative analysis by adopting an external standard method.
2. The method of claim 1, wherein the step (4) further comprises injecting the sample solution into ultra high performance liquid chromatography, scanning ultraviolet absorption spectra of naringin, benzoic acid and denatonium ions by a diode array detector, selecting different detection wavelengths for qualitative analysis, and after the qualitative analysis, performing quantitative analysis.
3. The method of claim 2, wherein the naringin is detected at 282nm, the benzoic acid ion is detected at 230nm, and the denatonium ion is detected at 210 nm.
4. The method of claim 1, wherein the standard solution prepared in step (2) has a concentration of 0.1mg/L, 0.2mg/L, 0.5mg/L, 1mg/L, 2mg/L and 5mg/L, respectively.
5. The method of claim 1, wherein the mobile phase flow rate is 0.3 mL/min.
6. The method of claim 1, wherein the column temperature of the chromatography column is 40 ℃.
7. The method of claim 1, wherein C18 is applied to a 2.1mm x 50mm, 1.7 μm column.
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