CN111175390A

CN111175390A - Method for detecting ethephon by using ultra-high performance liquid chromatography-tandem mass spectrometry

Info

Publication number: CN111175390A
Application number: CN201911385951.4A
Authority: CN
Inventors: 刘长生; 肖石基
Original assignee: Jiangsu Bioguide Laboratory Co ltd
Current assignee: Jiangsu Bioguide Laboratory Co ltd
Priority date: 2019-12-29
Filing date: 2019-12-29
Publication date: 2020-05-19

Abstract

The invention relates to a method for detecting ethephon by using ultra-high performance liquid chromatography-tandem mass spectrometry, which comprises the steps of placing a sample to be detected in a volumetric flask, adding formic acid-water (0.2-1): 100 to constant volume to 20-50ml, carrying out vortex mixing for 2-10 min, carrying out ultrasonic extraction for 2-10 min to prepare a mother solution 1, then transferring 200 microliters of the mother solution 1, diluting with formic acid-water (0.2-1): 100 to 20-50ml to prepare a mother solution 2, then transferring 200 microliters of the mother solution 2, diluting with formic acid-water (0.2-1): 100 to 20-50ml, carrying out sample introduction, and detecting ethephon in the sample by using an ultra-high performance liquid chromatography-tandem mass spectrometry. According to the invention, the UPLC-MS/MS is used for simply, quickly and efficiently carrying out qualitative and quantitative detection on ethephon in green agricultural products, agricultural chemicals and food, the detection sensitivity of ethephon can be effectively improved, the matrix interference is effectively avoided, the detection limit of ethephon is reduced, good linearity is shown in the concentration range of 36.8 ng/mL-294.7 ng/mL, and the linear correlation coefficient is 0.9998.

Description

Method for detecting ethephon by using ultra-high performance liquid chromatography-tandem mass spectrometry

Technical Field

The invention relates to a ethephon detection method, in particular to a method for detecting ethephon by using ultra-high performance liquid chromatography-tandem mass spectrometry.

Background

With the development of society and the advancement of science and technology. Human beings pay more and more attention to health, and medicines play a vital role in ensuring human health. Therefore, the pharmaceutical industry has become one of the most important areas of society. How to accurately and rapidly detect potentially low-limiting amounts of hazardous substances in pharmaceutical agents is a challenge facing the pharmaceutical industry today. The application of advanced modern instrumental analysis technology to meet the challenge is of great significance to the promotion of the safety evaluation of the medicine and the improvement of the commercial value of the medicine.

The instrument analysis is taken as a modern detection main means and is widely applied to the detection in the fields of chemistry, medicines, foods, cosmetics, agricultural chemicals and the like. Instrumental analysis enables the determination of the composition, content of the components and chemical structure of a substance by measuring several physicochemical properties, parameters and changes thereof. With the progress of the scientific technology of instrumental analysis, more and more advanced instrumental analysis techniques are widely applied to various detection fields, wherein the mass spectrometry detection technique is one of the techniques. The mass spectrometry detection is to ionize each component in a sample in an ion source to generate charged ions with different mass-to-charge ratios, after the ions are formed under the action of an accelerating electric field, the ions enter a mass analyzer, and after mass spectrometry signals are detected by the mass analyzer, visible spectrograms are formed on computer software through electronic conversion to be analyzed and researched by scientific research technicians. In order to combine the advantages of chromatographic separation, mass spectrometry is generally used in conjunction with chromatography, resulting in a chromatography-mass spectrometry technique. The chromatography-mass spectrometry combined technology has the advantages of both chromatography and mass spectrometry, namely good selectivity, high sensitivity, less test dosage, easy operation, high analysis speed, good reproducibility, small error, accuracy, reliability and the like. According to different chromatographic techniques, the chromatographic-mass spectrometry technology can be divided into LC-MS and GC-MS. The UPLC-MS/MS is one of the LC-MS used in the invention, the method adopts the tandem mass spectrum as a mass spectrum detector, and is formed by connecting three stages of mass spectrums in series, so that the selectivity of target ions can be effectively improved, the noise interference can be reduced, the signal-to-noise ratio can be further improved, and the detection limit can be further reduced, and the UPLC has the advantages of high separation efficiency, high separation speed, less used reagent and the like. In view of the great advantages of the UPLC-MS/MS technology, the technology is widely applied to the fields of medicines, chemistry, food science, cosmetics and the like.

Ethephon is an excellent plant growth regulator. It can promote fruit ripening and plays an important role in the fresh-keeping, storage and transportation process of various fruits. But the concentration requirement is higher in the using process, more use of the pesticide can cause phytotoxicity, and less use of the pesticide cannot achieve the expected effect. Therefore, accurate determination of ethephon concentration is an important prerequisite for its application. In order to ensure the effective application of ethephon in the fresh-keeping and ripening of green agricultural products, an accurate and efficient detection method needs to be established to effectively detect ethephon.

Due to the low boiling point, small molecular weight, weak ultraviolet absorption and more heteroatoms of the ethephon, the ethephon concentration in the sample cannot be effectively detected by common detection methods such as GC-FID, HPLC-UV and GC-MS.

At present, an ultra-high performance liquid chromatography-tandem mass spectrometry combined method which has high detection sensitivity, is simple, rapid and easy to operate is lacked for effectively detecting ethephon.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a method for detecting ethephon by using ultra-high performance liquid chromatography-tandem mass spectrometry, which has the advantages of good selectivity, high detection sensitivity and high speed.

The technical scheme for realizing the purpose of the invention is as follows: a method for detecting ethephon by using ultra-high performance liquid chromatography-tandem mass spectrometry comprises the following steps:

putting a sample to be detected into a volumetric flask, adding formic acid-water (0.2-1), namely 100, to fix the volume to 20-50ml, carrying out vortex mixing for 2-10 min, carrying out ultrasonic extraction for 2-10 min to prepare a mother solution 1, then transferring 200 microliters of the mother solution 1, diluting 20-50ml with formic acid-water (0.2-1), namely 100, to prepare a mother solution 2, then transferring 200 microliters of the mother solution 2, diluting 20-50ml with formic acid-water (0.2-1), namely 100, and carrying out detection on ethephon in the sample by sample injection;

step (2), detection conditions: the ethephon is detected by adopting an ultra performance liquid chromatography-tandem mass spectrometry UPLC-MS/MS as a detection instrument.

According to the technical scheme, in the step (1), 0.02-0.05 g of a sample to be detected is taken, and the accuracy is 0.01 mg.

In the step (2), the data acquisition mode of the ultra-high performance liquid chromatography-tandem mass spectrometer is to select reaction detection (SRM) and select mass-to-charge ratio

And m/z is 143.0 → 107.0, 143.0 → 79.0 as qualitative and quantitative ion pair.

In the step (2), the chromatographic column of the ultra-high performance liquid chromatography-tandem mass spectrometer is a high performance liquid chromatographic column taking C18 bonded silica gel as a stationary phase: the length of the amino column is 50-100 mm, the column diameter is 2.1mm, and the particle size of the filler is 1.7 mu m.

In the technical scheme, in the step (2), the flow rate of the ultra-high performance liquid chromatography-tandem mass spectrometry instrument is 0.1-0.5 ml/min.

In the technical scheme, in the step (2), the column temperature of the ultra-high performance liquid chromatography-tandem mass spectrometry combination instrument is 25-45 ℃.

According to the technical scheme, in the step (2), the sample injection volume of the ultra-high performance liquid chromatography-tandem mass spectrometry instrument is 0.5-5 mu L.

In the step (2), the mass spectrum condition of the ultra-high performance liquid chromatography-tandem mass spectrometer is HESI^-The ion source has the capillary voltage of 2500-3500 v, the capillary temperature of 250-380 ℃, the ion source temperature of 100-300 ℃, the atomizing gas is nitrogen, the sheath gas pressure of 30-100 psi, the auxiliary gas pressure of 0-10 psi, the ion transmission voltage of-10-50 v, the collision gas is argon, the collision gas pressure of 1.0-2.0 mtorr, the collision energy of 5 v-30 v respectively, and the scanning time of 0.1-1.0 second.

After the technical scheme is adopted, the invention has the following positive effects:

the detection method has the advantages of good selectivity, high sensitivity, simplicity, rapidness and the like. The invention can quickly and efficiently carry out limit test on ethephon in bulk drugs, pharmaceutical preparations and other samples by using UPLC-MS/MS; the ethephon detection sensitivity can be effectively improved, matrix interference is effectively avoided, the ethephon detection limit is reduced, and the lowest detection limit is 11.0 ng/mL; the limit of quantitation is 36.8 ng/mL; the linear micro-nano-particles show good linearity in the concentration range of 36.8 ng/mL-294.7 ng/mL, and the linear correlation coefficient is 0.9998.

Drawings

In order that the present disclosure may be more readily and clearly understood, reference is now made to the following detailed description of the present disclosure taken in conjunction with the accompanying drawings, in which

FIG. 1 is a qualitative and quantitative ion chromatogram of a ethephon standard of the present invention;

FIG. 2 is a mass spectrum of a ethephon standard of the present invention;

FIG. 3 is a qualitative and quantitative ion chromatogram of a blank solvent of the present invention;

FIG. 4 is a mass spectrum of a blank solvent of the present invention;

FIG. 5 is a qualitative and quantitative ion chromatogram of a blank sample extract according to the present invention;

FIG. 6 is a mass spectrum of a blank sample extract of the present invention;

FIG. 7 is a qualitative and quantitative ion chromatogram of a sample spiked in accordance with the present invention;

FIG. 8 is a mass spectrum of a sample of the present invention with a label;

FIG. 9 is a qualitative and quantitative ion chromatogram of an 11ng/mL ethephon standard of the present invention;

FIG. 10 is a mass spectrum of 11ng/mL ethephon standard of the present invention;

FIG. 11 is a qualitative and quantitative ion chromatogram of a 36.8ng/mL ethephon standard of the present invention;

FIG. 12 is a mass spectrum of a 36.8ng/mL ethephon standard of the present invention;

FIG. 13 is a qualitative and quantitative ion chromatogram of a 73.7ng/mL ethephon standard of the present invention;

FIG. 14 is a mass spectrum of a 73.7ng/mL ethephon standard of the present invention;

FIG. 15 is a qualitative and quantitative ion chromatogram of a 147.4ng/mL ethephon standard of the present invention;

FIG. 16 is a mass spectrum of 147.7ng/mL ethephon standard of the present invention;

FIG. 17 is a qualitative and quantitative ion chromatogram of 221.0ng/mL ethephon standard of the present invention;

FIG. 18 is a mass spectrum of 221.0ng/mL ethephon standard of the present invention;

FIG. 19 is a qualitative and quantitative ion chromatogram of a 294.7ng/mL ethephon standard of the present invention;

FIG. 20 is a mass spectrum of 294.7ng/mL ethephon standard of the present invention;

FIG. 21 is a standard graph of a linear experiment of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. The components of embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.

(example 1)

The invention provides a method for detecting ethephon by using ultra-high performance liquid chromatography-tandem mass spectrometry, which comprises the following steps:

In the step (1), 0.033g of a sample to be detected is taken, the sample is accurately measured to 0.01mg, the sample is placed in an empty volumetric flask, formic acid-water 0.2:100 is added to the empty volumetric flask to achieve a constant volume of 25ml, vortex mixing is carried out for 10min, ultrasonic extraction is carried out for 5min to prepare

mother liquor

1, 200 microliters of mother liquor 1 is transferred, formic acid-water 0.2:100 is used for diluting the mother liquor to 25ml to prepare

mother liquor

2, 200 microliters of mother liquor 2 is transferred, and formic acid-water (0.2-1): 100 is used for diluting the mother liquor to 20-50 ml.

In the step (2), the data acquisition mode of the ultra performance liquid chromatography-tandem mass spectrometer is to select reaction detection SRM, and the mass-to-charge ratio m/z is 143.0 → 107.0, 143.0 → 79.0 as a qualitative and quantitative ion pair. The chromatographic column of the ultra-high performance liquid chromatography-tandem mass spectrometry instrument is a high performance liquid chromatographic column taking C18 bonded silica gel as a stationary phase: the length of the amino column is 50mm, the diameter of the column is 2.1mm, and the grain diameter of the filler is 1.7 mu m. The flow rate of the high performance liquid chromatography-tandem mass spectrometer is 0.2 ml/min. The column temperature of the ultra performance liquid chromatography-tandem mass spectrometry instrument is 30 ℃. The sample injection volume of the ultra-high performance liquid chromatography-tandem mass spectrometry instrument is 1 mu L. The mass spectrum condition of the ultra-high performance liquid chromatography-tandem mass spectrometer is HESI^-The ion source has the capillary voltage of 2500-3500V, the capillary temperature of 250-380 ℃, the ion source temperature of 100-300 ℃, the atomizing gas of nitrogen, the sheath gas pressure of 40psi, the auxiliary gas pressure of 5psi, the ion transmission voltage of-10 to-50V, the collision gas of argon, the collision gas pressure of 1.0-2.0 mtorr, the collision energy of 5-30V respectively and the scanning time of 0.5 second.

The following test samples were provided by shaoxing east lake ltd.

Blank test and specificity test:

as shown in FIGS. 1-2, about 30mg of ethephon standard was weighed into a 25ml volumetric flask, dissolved with formic acid-water 0.2:100 and brought to volume. After the ethephon standard solution is diluted to 36.8ng/mL by formic acid-water 0.2:100, 1.5mL is taken out to be injected into a sample injection vial for analysis, and the ethephon standard chromatogram map is obtained. As shown in fig. 3 to 4, are chromatograms of formic acid-water 0.2:100 solvent. 1ml of formic acid-water 0.2:100 solvent is sampled for analysis. And the non-interference peak is arranged at the position of the checked target peak.

As shown in fig. 5 to 6, about 30mg of solid ethephon raw material is weighed respectively in a volumetric flask of 25ml, a proper amount of formic acid-water 0.2:100 is added, vortex mixing is carried out for 10min, ultrasonic extraction is carried out for 5min, the volume is determined to scale by formic acid-water 0.2:100 to prepare

mother liquor

1, 200 microliters of mother liquor 1 is transferred, the formic acid-water 0.2:100 is diluted to 25ml to prepare

mother liquor

2, 200 microliters of mother liquor 2 is transferred, and the formic acid-water 0.2:100 is diluted to 25 ml. 2ml of sample was taken for analysis. And the non-interference peak is arranged at the position of the checked target peak.

Comparing the blank solvent chromatogram and the blank sample chromatogram with the ethephon standard chromatogram to see that the formic acid-water blank solvent and the sample blank matrix have no interference peak at the target peak position for 0.5min at a ratio of 0.2: 100; thus, it is believed that the solvent and sample matrix do not interfere with ethephon detection.

Linear experiments:

as shown in fig. 11, fig. 13, fig. 15, fig. 17, fig. 19 and fig. 21, about 30mg of ethephon standard is weighed into a 25ml volumetric flask, dissolved with formic acid-water 0.2:100 and fixed to the scale. The ethephon standard solution is diluted by formic acid-water 0.2:100 to prepare a series of standard solutions, and the samples are injected for analysis. The peak area was plotted against concentration to obtain a linear result. The experimental data obtained are shown in table 1 below, and table 1 is the standard sequence experimental determination data:

TABLE 1

As shown in fig. 21, a standard graph of a linear experiment. The response value of the ethephon instrument shows a good linear relation in 36.8 ng/mL-294.7 ng/mL, and the linear correlation coefficient is as follows: 0.9998, and meets the detection requirement.

The detection limit and the quantification limit are shown in figure 9 and figure 11, and the chromatogram and the mass spectrogram of the ethephon standard sample of 11 ng/mL; the signal-to-noise ratio of the detection signal reaches the detection limit requirement of S/N >3 at 0.5min, and the detection signal is measured by a single-chip microcomputer. Chromatogram and mass spectrum of the ethephon standard sample of 36.8 ng/mL; the signal-to-noise ratio reaches 68 and meets the quantitative limit requirement of S/N >10 at 0.5 min.

Precision:

a ethephon concentration point is selected for 3 times of repeated experiments, and 73.7ng/mL is selected as the ethephon standard solution concentration for precision experiments. The specific data are shown in table 2 below, and table 2 shows the experimental data of ethephon precision:

TABLE 2

As can be seen from the data shown in Table 2, the precision of the experiment can meet the detection requirements.

Standard addition recovery experiment:

in the experiment, 1 standard adding concentration is selected to carry out three times of parallel tests, and the standard adding concentrations are respectively as follows: about 73.7 ng/mL. The experimental data are shown in table 3 below, and table 3 is the ethephon spiking recovery experimental data.

TABLE 3

As shown in Table 3, the results of the ethephon standard recovery test show that the recovery rate is 60-130%, and the test requirements are met.

(example 2)

Example 2 differs from example 1 in that: adding 0.3:100 formic acid-water into the mixture in the step (1), fixing the volume to 25ml, carrying out vortex oscillation for 10min, carrying out ultrasonic extraction for 6min, filtering the mixture through a 0.22 mu m filter membrane, and carrying out sample injection to detect ethephon in a sample; taking 0.025g of a product to be detected, accurately measuring the weight of the product to be detected to 0.01mg, placing the product in an empty volumetric flask, adding formic acid-water 0.3:100 to fix the volume to 20ml, carrying out vortex mixing for 10min, carrying out ultrasonic extraction for 6min to prepare a mother solution 1, then transferring 200 microliters of the mother solution 1, diluting the mother solution to 25ml with formic acid-water 0.3:100 to prepare a mother solution 2, then transferring 200 microliters of the mother solution 2, and diluting to 25ml with formic acid-water 0.3: 100.

(example 3)

Example 3 differs from example 1 in that: adding formic acid-water 0.5:100 to 50ml in the step (1), carrying out vortex oscillation for 10min, carrying out ultrasonic extraction for 8min, filtering through a 0.22 mu m filter membrane, and carrying out sample injection to detect ethephon in the sample; taking 0.065g of a sample to be detected, accurately measuring the sample to 0.01mg, placing the sample in an empty volumetric flask, adding formic acid-water 0.5:100 to fix the volume to 50ml, carrying out vortex mixing for 10min, carrying out ultrasonic extraction for 8min to prepare a mother solution 1, then transferring 200 microliters of the mother solution 1 to prepare a mother solution 2, diluting the mother solution to 25ml by using formic acid-water 0.5:100, then transferring 200 microliters of the mother solution 2, and diluting to 25ml by using formic acid-water 0.5: 100.

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention, and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting ethephon by using ultra-high performance liquid chromatography-tandem mass spectrometry is characterized by comprising the following steps:

2. The method for detecting ethephon by using ultra high performance liquid chromatography-tandem mass spectrometry as claimed in claim 1, wherein: in the step (1), 0.02-0.05 g of a sample to be detected is taken, and the accuracy is 0.01 mg.

3. The method for detecting ethephon by using ultra high performance liquid chromatography-tandem mass spectrometry as claimed in claim 1, wherein: in the step (2), the data acquisition mode of the ultra-high performance liquid chromatography-tandem mass spectrometer is to select a reaction detection SRM and select a mass-to-charge ratio

4. The method for detecting ethephon by using ultra high performance liquid chromatography-tandem mass spectrometry as claimed in claim 1, wherein: in the step (2), the chromatographic column of the ultra-high performance liquid chromatography-tandem mass spectrometer is a high performance liquid chromatographic column taking C18 bonded silica gel as a stationary phase: the length of the amino column is 50-100 mm, the column diameter is 2.1mm, and the particle size of the filler is 1.7 mu m.

5. The method for detecting ethephon by using ultra high performance liquid chromatography-tandem mass spectrometry as claimed in claim 1, wherein: in the step (2), the flow rate of the ultra-high performance liquid chromatography-tandem mass spectrometry instrument is 0.1-0.5 ml/min.

6. The method for detecting ethephon by using ultra high performance liquid chromatography-tandem mass spectrometry as claimed in claim 1, wherein: in the step (2), the column temperature of the ultra-high performance liquid chromatography-tandem mass spectrometry instrument is 25-45 ℃.

7. The method for detecting ethephon by using ultra high performance liquid chromatography-tandem mass spectrometry as claimed in claim 1, wherein: in the step (2), the sample injection volume of the ultra-high performance liquid chromatography-tandem mass spectrometry instrument is 0.5-5 muL.

8. The method for detecting ethephon by using ultra high performance liquid chromatography-tandem mass spectrometry as claimed in claim 1, wherein: in the step (2), the mass spectrum condition of the ultra-high performance liquid chromatography-tandem mass spectrometer is HESI^-The ion source has the capillary voltage of 2500-3500 v, the capillary temperature of 250-380 ℃, the ion source temperature of 100-300 ℃, the atomizing gas is nitrogen, the sheath gas pressure of 30-100 psi, the auxiliary gas pressure of 0-10 psi, the ion transmission voltage of-10-50 v, the collision gas is argon, the collision gas pressure of 1.0-2.0 mtorr, the collision energy of 5 v-30 v respectively, and the scanning time of 0.1-1.0 second.