CN111122714A - Gas-phase mass spectrum combination method for measuring 2-acetyl-1-pyrroline content of fragrant rice leaves by external standard multi-point method - Google Patents
Gas-phase mass spectrum combination method for measuring 2-acetyl-1-pyrroline content of fragrant rice leaves by external standard multi-point method Download PDFInfo
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Abstract
The invention relates to the field of food detection, and discloses a gas-phase mass spectrometry combined method for determining 2-acetyl-1-pyrroline content of a fragrant rice leaf by an external standard multipoint method, which comprises the following steps of S1: sampling; s2: pre-treating blades and weighing; s3: pre-treatment of solid phase micro-extraction; s4: gas chromatography-mass spectrometry analysis; s5: establishing a 2-acetyl-1-pyrroline external standard multipoint method; s6: establishing a relative recovery factor RRF; s7: and (4) formula calculation. The standard deviation of data repeatability (referring to the same extraction head used) of the obtained sample is less than 1.5%, the standard deviation of reproducibility is less than 8.5%, the detected concentration of the sample is 0.0035 mg/Kg, and the quantitative detection limit (S/N = 10) is 0.001 mg/Kg. The method provided by the invention is simple, convenient, rapid, accurate, low in cost and efficient, and has good application prospects in early quality identification of fragrant rice, genetic research of fragrant genes and early generation screening of breeding.
Description
Technical Field
The invention relates to the field of food detection, in particular to a gas-phase mass spectrometry combined method for determining 2-acetyl-1-pyrroline content of a fragrant rice leaf by an external standard multi-point method.
Background
The fragrant rice is sweet and popular with consumers, and the selling price of high-quality fragrant rice in the market is far higher than that of common rice. There are many varieties of Chinese rice, more than 200 varieties of fragrant volatiles in rice, of which 2-acetyl-1-pyrroline (2AP) is the main fragrance character volatile (Lin et al, 1990; Ahmed et al, 1995; Tanchotikul and sieh, 1991; Maraval et al, 2008; Mathure et al, 2014), in popcorn type.
The rice odor analysis has been carried out at home and abroad for more than 20 years, most of documents in the rice odor collection adopt a synchronous distillation-extraction method and an adsorbent trapping method, and the methods have the defects of complicated operation steps, large consumption of samples and reagents, time and labor waste, more target interferents and the like. In recent years, a manual solid phase microextraction technology is adopted, although the technology of directly extracting a target object from a sample matrix, integrating extraction, separation, concentration and sample injection into a whole is adopted, the technology has no organic reagent consumption, and is simple and convenient to operate, the risk of target object loss exists in the transfer process because the target object is collected and then transferred into a gas phase for sample injection, and the concentration of a sample characteristic object is reduced. For example, in the reports of domestic Xinghua et al (academic report of analysis and testing, 2011, 27, 69-71), manual solid-phase microextraction is carried out and then the microextraction is transferred to a gas-phase sample inlet, so that loss is easy to occur in the transfer process; the amount of the sample is 8 g, the extraction time is 0.2 h, the sampling amount is large, insufficient extraction is easy to cause, and the like. And the quantification of the main flavor features 2AP is mostly in the form of rice flour (Yixing et al, chromatogram, 2010, 28(8):782 + 785; Sunje et al, food safety quality test report, 2016, 7(3):2011 + 2015) and quantitative calculation using single- point 2,4,6 Trimethylpyridine (TMP) as an internal standard (Yubi Yu et al, spectroscopic laboratories, 2000, 17(6):638 + 642; Yixing et al, chromatogram, 2010, 28(8):782 + 785; Xixing et al, analytical test report, 2011, 27, 69-71; Huangjinlin et al, Chinese agriculture report, 2012(6):1054 + 1065; Yang juan et al; North China agriculture report, 2014, 29(3):128 + 135; Li Red et al, North Huanong report, 2014, 29(1, 159; Chilobelia Chihao et al, Huanan university, 201536-48; Sun Muyu et al, the food safety quality detection report 2016, 7(3): 2011-.
The TMP is used as an internal standard, the 2AP content is calculated by calculating the area ratio of the 2AP to the TMP internal standard, although the 2AP target and the TMP internal standard generate peaks at different time, no overlapping exists, and the properties and the molecular weight are similar; however, in the case of solid phase microextraction in a headspace vial of a given volume, the concentration of target 2AP volatilized after heating in this vial is not necessarily a fixed ratio to the concentration of the TMP internal standard.
Disclosure of Invention
In order to solve the technical problems, the invention provides a gas-phase mass spectrometry method for determining the content of 2-acetyl-1-pyrroline (2AP) in the leaves of the fragrant rice by an external standard multi-point method, which adopts automatic solid-phase microextraction pretreatment, adopts a leaf form, and quantitatively calculates the content of the 2-acetyl-1-pyrroline in a sample by using a standard curve of the 2-acetyl-1-pyrroline external standard multi-point method, can identify fragrance and non-fragrance of fragrant rice plants at an early stage, and has good accuracy, high precision (the relative standard deviation of continuous precision of the method is less than 1.5 percent, the relative standard deviation of the middle precision is less than 8.5 percent), and the detected concentration of the sample is as low as 0.0035 mg/Kg.
The specific technical scheme of the invention is as follows: a gas-phase mass spectrum combination method for measuring the 2-acetyl-1-pyrroline content of the fragrant rice leaves by an external standard multipoint method comprises the following steps:
s1: sampling;
s2: pre-treating blades and weighing;
s3: pre-treatment of solid phase micro-extraction;
s4: gas chromatography-mass spectrometry analysis;
s5: the 2-acetyl-1-pyrroline external standard multipoint method is established as follows: taking m/z83 target characteristic ion peak areas of a plurality of standard samples with different concentrations obtained in S4 as vertical coordinates, taking corresponding standard sample amount as horizontal coordinates, drawing a standard curve, and obtaining a correlation coefficient and a linear calculation formula;
s6: establishing a Relative Recovery Factor (RRF): directly injecting sample by using a sample injection needle with the middle-level concentration in the concentration range of the multi-point standard sample in S5 to obtain the ratio of the m/z83 peak area to the m/z83 peak area obtained in the corresponding S5, namely RRF;
s7: the 2-acetyl-1-pyrroline content C in the leaves is calculated according to the formula C (wet basis) = R/M RRF,
wherein R is the mass ng calculated according to the S5 standard curve;
RRF is S6 to obtain a value;
m is sample weight, mg;
wherein the unit of C is mg/Kg, namely ppm, and C represents the content of 2-acetyl-1-pyrroline in the wet base of the leaf.
Because there is uncertainty that the concentration of target 2AP volatilized in the extract flask is not necessarily a fixed ratio to the concentration of the TMP internal standard when TMP is used as the quantitation internal standard, there is a quantitative bias. According to the method, the 2AP is directly used as an external standard curve through the automatic solid-phase micro-extraction conditions of the fragrant rice leaves and the parameters of a gas-mass spectrometer, the content of the fragrant characteristic substance 2AP can be quantitatively analyzed, and the method is good in repeatability, low in detection limit and good in application prospect in screening early plants for improving the rice quality and the like. In addition, the invention also overcomes the problems of complicated steps and easy loss of the prior manual solid-phase microextraction and then transfer sample introduction.
Although the external standard multi-point method belongs to the prior art, the method is specifically applied to the quantitative detection of the 2AP of the fragrant rice leaf, and a series of technical problems still need to be overcome in order to realize better repeatability, low detection concentration limit of a sample and the like, for example:
preferably, in S1, the sampling is to collect the top first leaf at the early stage of heading or to collect the sword-like leaf after heading.
The contents of the-acetyl-1-pyrroline in the leaves of the rice plants at different positions in different periods are different. Without consistent sampling rules, there is no comparability between the varieties, and the source of the data difference is not known to be from leaf position or different time periods or differences between the true varieties. Thus, the leaf picking rule is determined, the data difference can explain the difference between rice varieties, and a reliable analysis means is provided for effectively identifying and screening the high-quality fragrant rice varieties.
Preferably, in S2, specifically: after the leaves are picked, the leaves are cut into pieces and immediately ground into powder by liquid nitrogen, and then 50-100 mg of the powder is directly weighed.
Preferably, in S2, the sample is weighed and loaded into a screw-top empty sample vial and screwed down with a magnetic metal cap containing a PTFE/blue silica gel spacer.
Preferably, the specific method of S5 is:
1) if the content of the 2-acetyl-1-pyrroline in the wet base of the leaf is 0.000-0.015mg/Kg, taking the amount of external standard points of a plurality of concentration gradients in the range of 0.00-1.00 ng and the corresponding m/z83 peak area of the target characteristic ion as a calibration curve;
2) if the content of the 2-acetyl-1-pyrroline in the wet base of the leaf is 0.007-0.150 mg/Kg, taking the external standard point quantity of a plurality of gradients in the range of 0.00-10.00 ng and the corresponding peak area of m/z83 of the target characteristic ion as a calibration curve;
3) if the content of the 2-acetyl-1-pyrroline in the wet base of the blade is 0.050-1.500 mg/Kg, taking the external standard point quantity of a plurality of gradients in the range of 0.00-80.0 ng and the corresponding peak area of target characteristic ion m/z83 as a calibration curve;
the r of the established linear equation is more than or equal to 0.9900.
The external standard concentration is selected to correspond to the content of 2-acetyl-1-pyrroline in the leaves. If the 2-acetyl-1-pyrroline content in the leaves is higher or lower than the concentration in the series of outer punctuation, deviations or even errors in the quantification may occur. Therefore, it is necessary to select an appropriate external standard concentration series for analysis at the time of measurement. On the basis of the determination pre-analysis of a large number of leaves, the invention provides the concentrations of external standard curve points corresponding to the 2-acetyl-1-pyrroline with different concentrations of the leaves, but the quantitative analysis of the 2-acetyl-1-pyrroline in all the leaves adopts the same very wide external standard concentration. If the concentration is low and the detection limit is close, if a series of external standard points with large span from low to high is used, the external standard points are easy to be undetected or even have negative values. Or high concentrations, with low to high span series of points, tend to result in low results and poor reproducibility. The invention uses three sections of concentration ranges, adopts three external standard curves, and basically covers the concentration range in the leaves of rice plants aiming at the leaf samples with different detected concentrations of the 2-acetyl-1-pyrroline, thereby having good accuracy, repeatability and reproducibility.
Preferably, the number of the gradients is set to at least 4.
Preferably, the specific process of S6 is:
1) if the wet basis content of the 2-acetyl-1-pyrroline in the leaf is 0.000-0.015mg/Kg, adopting the concentration of an intermediate standard sample, such as 0.04-0.06 ng/mul, of 10 mul, directly feeding the sample to obtain the ratio of the peak area of the characteristic ion m/z83 of the 2-acetyl-1-pyrroline to the peak area after corresponding solid phase microextraction, namely a relative recovery factor;
2) if the wet basis content of the leaves is 0.007-0.10 mg/Kg, adopting the concentration of a middle-level standard sample, such as the concentration of 0.40-0.60 ng/mul, of 10 mul, directly feeding a sample to obtain the ratio of the peak area of the characteristic ion m/z83 of 2-acetyl-1-pyrroline to the peak area after corresponding solid phase microextraction, namely a relative recovery factor;
3) if the wet basis content of the 2-acetyl-1-pyrroline in the leaves is 0.05-1.50 mg/Kg, the relative recovery factor is obtained by directly feeding samples with the concentration of a middle-level standard sample of 10 mul of 4.0-6.0 ng/mul to obtain the ratio of the m/z83 peak-forming ion area to the peak area after corresponding solid phase microextraction.
Considering the extraction efficiency of solid phase micro-extraction, namely the extraction head can not extract hundreds of the amount of 2-acetyl-1-pyrroline in the leaves, the invention introduces a relative recovery factor. Meanwhile, the solid phase micro-extraction relates to the extraction capability of the surface coating material of the extraction head on a target object; the concentration of the target in the upper space of the headspace bottle when the sample is heated to release the target, the balance capability of the extraction head, and the like all affect the extraction efficiency. Therefore, aiming at the contents of the 2-acetyl-1-pyrroline with different concentrations in the leaves, the invention adopts standard samples with different concentrations to measure relative recovery factors so as to more accurately reflect the extraction efficiency.
Preferably, the method further comprises the step of S8: precision test of the method (same extraction head is adopted).
Preferably, the specific process of S3 is:
the first stage is as follows: preheating at 70-85 deg.C for 3-20 min;
and a second stage: the type of the solid phase micro-extraction head adopts a carbon molecular sieve/divinylbenzene/polydimethylsilane (CAR/DVB/PDMS, 50/30 mu m, 1 cm long extraction head), and the extraction is carried out for 30-50min under the conditions of 70-85 ℃ and the rotation speed of 200-250 rpm/min; the distance between the extraction head and the sample inlet bottle is 18-23 mm;
and a third stage: the extract is directly fed into the sample inlet of the gas chromatograph, the sample introduction time is 1.5 min, and the sample introduction temperature is 250 ℃.
Preferably, S4 is specifically: and (3) analyzing in a gas chromatograph-mass spectrometer, wherein the set parameters of the gas chromatograph-mass spectrometer are as follows:
the mass interface temperature was 240 ℃ and the ion source temperature was 210 ℃. The separating column adopts an Rxi-5Sil capillary column with the specification of 30 m multiplied by 0.25 mm multiplied by 0.25 mu m; the temperature of the gas phase is programmed to 50 ℃ and kept for 1min, then the temperature is raised to 120 ℃ at 5 ℃/min, and then the temperature is raised to 250 ℃ at 20 ℃/min and kept for 4 min; the mass spectrum adopts a full-scanning combined ion monitoring mode, the scanning speed is 9900, the m/z range is 50-300, and the scanning interval is 0.06; ion channels selected for ion monitoring are m/z83, 111 and 68, with an interval of 0.2; quantification is based on peak ion area; the target ion m/z83 of the 2-acetyl-1-pyrroline is taken as a quantitative ion, and 111 and 68 are taken as qualitative ions; the carrier gas was 99.999% helium, with a flow rate of 35.0 cm/sec; electron bombardment ion source, ionization energy is 70 eV; the judgment of the 2-acetyl-1-pyrroline substance needs to be matched with the standard mass spectrogram of the 2-acetyl-1-pyrroline substance through NIST library retrieval.
Compared with the prior art, the invention has the beneficial effects that: the method disclosed by the invention not only adopts automatic solid-phase microextraction pretreatment, but also adopts a leaf form, and quantitatively calculates the 2-acetyl-1-pyrroline content in the sample by using a 2-acetyl-1-pyrroline external standard multipoint standard curve method, so that the fragrant and non-fragrant identification can be carried out on the fragrant rice plant at an early stage, the precision of the method is high (the relative standard deviation of the continuous precision of the method is less than 1.5%, and the relative standard deviation of the intermediate precision is less than 8.5%), the sample detection concentration is as low as 0.0035 mg/Kg, and the quantitative detection limit (S/N = 10) is 0.001 mg/Kg. .
Drawings
FIG. 1 is a total ion flow chromatogram of a scented rice leaf in example 1;
FIG. 2 is a mass spectrum of 2-acetyl-1-pyrroline in example 1;
FIG. 3 is an external standard curve and a calibration equation of 2-acetyl-1-pyrroline in example 1;
FIG. 4 is a total ion flow chromatogram of a scented rice leaf of example 2;
FIG. 5 is a mass spectrum of 2-acetyl-1-pyrroline in example 2;
FIG. 6 is an external standard curve and a calibration equation of 2-acetyl-1-pyrroline in example 2;
FIG. 7 is a total ion flow chromatogram of Oryza sativa leaves in example 3;
FIG. 8 is a mass spectrum of 2-acetyl-1-pyrroline in example 3;
FIG. 9 is an external standard curve and scaling equation for 2-acetyl-1-pyrroline in example 3.
Detailed Description
The present invention will be further described with reference to the following examples.
General examples
A gas-phase mass spectrum combination method for measuring the 2-acetyl-1-pyrroline content of the fragrant rice leaves by an external standard multipoint method comprises the following steps:
s1: sampling: collecting the uppermost first full leaf at the early stage of heading of the rice plant, or collecting the sword leaf after heading.
S2: pretreatment and weighing of leaves: the method comprises the following steps of cutting the leaves after collection, immediately grinding the leaves into powder by using liquid nitrogen, directly weighing 50-100 mg of the powder, weighing the sample, putting the sample into a threaded-opening headspace sample bottle, and screwing down the sample by using a magnetic metal cover containing a PTFE/blue silica gel spacer.
S3: solid phase microextraction pretreatment: the type of the extraction head adopts carbon molecular sieve/divinylbenzene/polydimethylsilane (CAR/DVB/PDMS, 50/30 mu m, 1 cm long extraction head).
The first stage is as follows: preheating at 70-85 deg.C for 3-20 min;
and a second stage: extracting for 30-50min at 70-85 ℃ and at the rotation speed of 200-; the distance between the extraction head and the sample inlet bottle is 18-23 mm;
and a third stage: the extract is directly fed into the sample inlet of the gas chromatograph, the sample introduction time is 1.5 min, and the sample introduction temperature is 250 ℃.
S4: gas chromatography-mass spectrometry analysis: the instrument setup parameters were as follows:
the mass interface temperature was 240 ℃ and the ion source temperature was 210 ℃. The separating column adopts an Rxi-5Sil capillary column with the specification of 30 m multiplied by 0.25 mm multiplied by 0.25 mu m; the temperature of the gas phase is programmed to 50 ℃ and kept for 1min, then the temperature is raised to 120 ℃ at 5 ℃/min, and then the temperature is raised to 250 ℃ at 20 ℃/min and kept for 4 min; the mass spectrum adopts a full-scanning combined ion monitoring mode, the scanning speed is 9900, the m/z range is 50-300, and the scanning interval is 0.06; ion channels selected for ion monitoring are m/z83, 111 and 68, with an interval of 0.2; quantification is based on peak ion area; the target ion m/z83 of the 2-acetyl-1-pyrroline is taken as a quantitative ion, and 111 and 68 are taken as qualitative ions; the carrier gas was 99.999% helium, with a flow rate of 35.0 cm/sec; electron bombardment ion source, ionization energy is 70 eV; the judgment of the 2-acetyl-1-pyrroline substance needs to be matched with the standard mass spectrogram of the 2-acetyl-1-pyrroline substance through NIST library retrieval.
S5: the 2-acetyl-1-pyrroline external standard multipoint method is established as follows: drawing a standard curve by taking the m/z83 target characteristic ion peak area of the 2-acetyl-1-pyrroline with different concentration gradients obtained by S4 as a vertical coordinate and the concentration (ng) of the corresponding standard sample 2-acetyl-1-pyrroline as a horizontal coordinate to obtain a correlation coefficient and a linear calculation formula;
specifically, the method comprises the following steps:
1) if the content of the 2-acetyl-1-pyrroline in the wet basis of the leaf is 0-0.015mg/Kg, adopting the range of 0-1.00 ng, and making a calibration curve by using external standard points of a plurality of gradients and the corresponding m/z83 target characteristic ion peak area of the 2-acetyl-1-pyrroline;
2) if the content of the wet 2-acetyl-1-pyrroline in the leaves is 0.007-0.150 mg/Kg, adopting the range of 0-10.0 ng, and making a calibration curve by using external standard points of a plurality of gradients and the corresponding m/z83 target characteristic ion peak area of the 2-acetyl-1-pyrroline;
3) if the content of the wet 2-acetyl-1-pyrroline in the leaves is 0.05-1.50 mg/Kg, taking the external standard point quantity of a plurality of gradients in the range of 0-80.0 ng and the corresponding m/z83 target characteristic ion peak area as a calibration curve;
the r of the established linear equation is more than or equal to 0.9900. Preferably, the number of the gradients is set to at least 4.
S6: establishing a relative recovery factor: adopting 2-acetyl-1-pyrroline corresponding to S5 to directly sample by a sample injection needle, and obtaining an average value of the ratio of the peak area to the peak area obtained in the corresponding S5;
the method comprises the following specific steps:
1) if the wet basis content of the 2-acetyl-1-pyrroline in the leaf is 0-0.015mg/Kg, adopting the concentration of an intermediate standard sample, such as the concentration of 0.04-0.06 ng/mul is 10 mul, directly sampling to obtain the ratio of the peak area of the characteristic ion m/z83 of the 2-acetyl-1-pyrroline to the peak area after corresponding solid phase microextraction, namely a relative recovery factor;
2) if the wet basis content of the leaves is 0.007-0.10 mg/Kg, adopting the concentration of a middle-level standard sample, such as the concentration of 0.4-0.6 ng/mul, of 10 mul, directly feeding a sample to obtain the ratio of the peak area of the characteristic ion m/z83 of 2-acetyl-1-pyrroline to the peak area after corresponding solid phase microextraction, namely a relative recovery factor;
3) if the wet basis content of the 2-acetyl-1-pyrroline in the leaves is 0.05-1.50 mg/Kg, the concentration of a middle-level standard sample is 10 mul, such as 4-6 ng/mul, and the ratio of the area of the m/z83 peak-forming ions to the area of the peak after corresponding solid phase microextraction is obtained after direct sample injection, namely the relative recovery factor.
S7: the 2-acetyl-1-pyrroline content C in the leaves is calculated according to the formula C = R/M RRF,
wherein R is the mass ng calculated according to the S5 standard curve;
RRF is S6 to obtain a value;
m is sample weight, mg;
wherein the unit of C is mg/Kg, namely ppm, and C represents the content of 2-acetyl-1-pyrroline in the wet base of the leaf.
S8: precision test of the method
Adopting the same extraction head, continuously measuring for 11 times in the same day under the same method condition, and obtaining the relative standard deviation of the continuous precision, which is less than 1.50%; the same method, using three different days, each time 7 times of repetition, 21 total determination of the relative standard deviation of the intermediate precision is less than 8.50%.
Example 1
Step 1: the uppermost full leaf of the rice variety 1 is cut into pieces and then ground into powder by liquid nitrogen.
Step 2: the sample was weighed 100mg into a 20 ml screw-top empty sample vial and screwed down with a magnetic metal cap containing a PTFE/blue silica gel spacer.
Step 3, carrying out an automatic solid phase micro-extraction stage, wherein the extraction is divided into three stages:
the first stage is as follows: preheat at 70 ℃ for 5 min.
And a second stage: the model of the extraction head was carbon molecular sieve/divinylbenzene/polydimethylsilane (CAR/DVB/PDMS; 50/30 μm, 1 cm long) at 70 ℃ and 250 rpm/min. The depth distance of the extraction head into the sampling bottle is 18 mm. This extraction phase lasted 30 min.
And a third stage: the extract is directly fed into the sample inlet of the gas chromatograph, the sample introduction time is 1.5 min, and the sample introduction temperature is 250 ℃. Step four: and (3) analyzing in a gas chromatograph-mass spectrometer, wherein the set parameters of the gas chromatograph-mass spectrometer are as follows:
the gas interface temperature was 240 ℃ and the ion source temperature was 210 ℃. The separation column was an Rxi-5Sil capillary column (30 m.times.0.25 mm.times.0.25 μm). The gas phase temperature programmed is kept at 50 ℃ for 1min, then is raised to 120 ℃ at 5 ℃/min, and is raised to 250 ℃ at 20 ℃/min, and is kept for 4 min. The mass spectrum adopts a full scan (scan) combined with a Selective Ion Monitoring (SIM) mode, the scan speed is 9900, the m/z range is 50-300, and the scan interval is 0.06. Ion monitoring selected ion channels were m/z 68, 83, 111 with a spacing of 0.3. The carrier gas was high helium (99.999%) at a flow rate of 35.0 cm/sec. The ion source was bombarded with Electrons (EI) and the ionization energy was 70 eV.
And 5: calibration curve of multi-point external standard method
Selecting 0 ng/μ l, 1.0 ng/μ l, 2.0 ng/μ l, 4.0 ng/μ l, 6.0 ng/μ l, 8.0 ng/μ l, and 6 concentration gradients of 10 μ l each of 2-acetyl-1-pyrroline into a 20 ml headspace bottle, operating according to steps 3 and 4, and making a standard curve of m/z83 target ion peak area and 2-acetyl-1-pyrroline amount, r =0.9948, y =83058 x +359976, wherein x is the m/z83 target ion peak area of the 2-acetyl-1-pyrroline in ng and y is the m/z83 target ion peak area of the 2-acetyl-1-pyrroline. The 2-acetyl-1-pyrroline external standard curve and the scaling equation are shown in FIG. 3.
Step 6: relative Recovery Factor (RRF) establishment
And (3) sampling by taking a direct sampling port with the concentration of 4 ng/microliter and the concentration of 10 microliter, and repeating for 3 times to obtain an average value of the ratio of the peak area to the peak area obtained in the corresponding step 5, wherein the average value is 1.47.
And 7: calculation of 2-acetyl-1-pyrroline content in leaves
The peak areas 1525226, 1422665 and 1485216 of sample 1 were substituted into the formulas to obtain 14.03ng, 12.79 ng and 13.55 ng, respectively, and further calculated, C (wet basis) = R/M (weighed amount) × RRF. 0.206 mg/Kg, 0.188 mg/Kg, 0.199 mg/Kg, average 0.198 mg/Kg, and standard deviation 0.9% were obtained, respectively. The total ion current chromatogram of the leaves of the oryza sativa is shown in FIG. 1, and the retention time of the 2-acetyl-1-pyrroline peak is 6.564 min. The mass spectrum of the 2-acetyl-1-pyrroline is shown in figure 2, the characteristic ion fragments are m/z 68, 83 and 111, and the mass spectrum is matched with the standard mass spectrum of the 2-acetyl-1-pyrroline substance through NIST library retrieval, thereby proving to be the 2-acetyl-1-pyrroline.
Example 2
Step 1: after heading, the plants of rice variety 2 were harvested from flag leaves and ground with liquid nitrogen.
Step 2: the sample was weighed 100mg into a 20 ml screw-top empty sample vial and screwed down with a magnetic metal cap containing a PTFE/blue silica gel spacer.
Step 3, carrying out an automatic solid phase micro-extraction stage, wherein the extraction is divided into three stages:
the first stage is as follows: preheating is maintained at 80 ℃ for 10 min.
And a second stage: the model of the extraction head adopts the interval steps of shaking for 20S and stopping for 5S at 80 ℃ and the rotating speed of 220 rpm/min by adopting carbon molecular sieve/divinylbenzene/polydimethylsilane (CAR/DVB/PDMS; 50/30μm, length of 1 cm). The depth distance of the extraction head into the sampling bottle is 22 mm. This extraction phase lasted 40 min.
And a third stage: the extract is directly fed into the sample inlet of the gas chromatograph, the sample introduction time is 1.5 min, and the sample introduction temperature is 250 ℃.
Step four: entering a gas chromatograph-mass spectrometer for analysis, wherein the set parameters of the gas chromatograph-mass spectrometer are as follows:
the gas interface temperature was 240 ℃ and the ion source temperature was 210 ℃. The separation column was an Rxi-5Sil capillary column (30 m.times.0.25 mm.times.0.25 μm). The gas phase temperature programmed is kept at 50 ℃ for 1min, then is raised to 120 ℃ at 5 ℃/min, and is raised to 250 ℃ at 20 ℃/min, and is kept for 4 min. The mass spectrum adopts a full scan (scan) combined with a Selective Ion Monitoring (SIM) mode, the scan speed is 9900, the m/z range is 50-300, and the scan interval is 0.06. Ion monitoring selected ion channels were m/z 68, 83, 111 with a spacing of 0.3. The carrier gas was high helium (99.999%) at a flow rate of 35.0 cm/sec. The ion source was bombarded with Electrons (EI) and the ionization energy was 70 eV.
Selecting 0 ng/mu l, 0.1 ng/mu l, 0.20 ng/mu l, 0.40 ng/mu l, 0.60 ng/mu l and 1.0 ng/mu l of 6 concentration gradients of 2-acetyl-1-pyrroline, respectively 10 mu l, into a 20 ml headspace bottle, operating according to steps 3 and 4, and making a standard curve by using m/z83 target ion peak area of the 2-acetyl-1-pyrroline and the standard amount of the 2-acetyl-1-pyrroline, wherein r =0.9948 and y =122454.24 x +22855.9, wherein x is the content ng, and y is the m/z83 target ion peak area of the 2-acetyl-1-pyrroline. The 2-acetyl-1-pyrroline external standard curve and the scaling equation are shown in FIG. 6.
Step 6: relative Recovery Factor (RRF) establishment
And (3) sampling by taking a direct injection port with the concentration of 0.2 ng/microliter and the concentration of 10 microliter, wherein the average value of the ratio of the peak area obtained by repeating for 3 times to the peak area obtained in the corresponding step 5 is 1.39.
And 7: calculation of 2-acetyl-1-pyrroline content of sample
The peak areas of sample 1 were 408379, 392911, 412480, respectively, and the substitution into the formulas gave 3.15 ng,3.02ng, 3.18 ng, respectively, and further calculated, C (wet basis) = R/M (weight amount) × RRF, and gave 0.044mg/Kg, 0.042mg/Kg, 0.044mg/Kg, average 0.043 mg/Kg, standard deviation 0.1%, respectively. The total ion current chromatogram of the leaves of the oryza sativa is shown in FIG. 4, and the retention time of the 2-acetyl-1-pyrroline peak is 6.533 min. The mass spectrum of 2-acetyl-1-pyrroline is shown in figure 5, the characteristic ion fragments are m/z 68, 83 and 111, and the mass spectrum is matched with the standard mass spectrum of the 2-acetyl-1-pyrroline substance through NIST library retrieval, thereby proving to be the 2-acetyl-1-pyrroline.
Example 3
Step 1, taking the tassel leaves of the rice variety 3, and grinding the tassel leaves by using liquid nitrogen.
Step 2: the sample was weighed 100mg into a 20 ml screw-top empty sample vial and screwed down with a magnetic metal cap containing a PTFE/blue silica gel spacer.
Step 3, carrying out an automatic solid phase micro-extraction stage, wherein the extraction is divided into three stages:
the first stage is as follows: the pre-heating is maintained at 80 ℃ for 15 min.
And a second stage: the model of the extraction head was carbon molecular sieve/divinylbenzene/polydimethylsilane (CAR/DVB/PDMS; 50/30 μm, 1 cm long) at 80 ℃ and 250 rpm/min. The depth distance of the extraction head into the sampling bottle is 23 mm. This extraction phase lasted 50 min.
And a third stage: the extract is directly fed into the sample inlet of the gas chromatograph, the sample introduction time is 1.5 min, and the sample introduction temperature is 250 ℃.
Analyzing in a gas chromatograph-mass spectrometer, wherein the set parameters of the gas chromatograph-mass spectrometer are as follows:
the gas interface temperature was 240 ℃ and the ion source temperature was 210 ℃. The separation column was an Rxi-5Sil capillary column (30 m.times.0.25 mm.times.0.25 μm). The gas phase temperature programmed is kept at 50 ℃ for 1min, then is raised to 120 ℃ at 5 ℃/min, and is raised to 250 ℃ at 20 ℃/min, and is kept for 4 min. The mass spectrum adopts a full scan (scan) combined with a Selective Ion Monitoring (SIM) mode, the scan speed is 9900, the m/z range is 50-300, and the scan interval is 0.06. Ion monitoring selected ion channels were m/z 68, 83, 111 with a spacing of 0.3. The carrier gas was high helium (99.999%) at a flow rate of 35.0 cm/sec. The ion source was bombarded with Electrons (EI) and the ionization energy was 70 eV.
Selecting 0 ng/mu l, 0.02 ng/mu l, 0.05 ng/mu l, 0.10 ng/mu l, 0.2 ng/mu l, 0.25 ng/mu l, and 6 concentration gradients of 10 mu l of each 2-acetyl-1-pyrroline into a 20 ml headspace bottle, operating according to steps 3 and 4, and drawing a standard curve by using m/z83 target ion peak area and content, wherein r =0.995, y =77958 x +1712.1, wherein x is the content ng, and y is the 2-acetyl-1-pyrroline m/z83 target ion peak area. FIG. 9 shows the external standard curve and calibration equation of 2-acetyl-1-pyrroline.
Step 6: relative Recovery Factor (RRF) establishment
And (3) sampling with a direct sampling port with the concentration of 0.10 ng/microliter of 10 microliter, wherein the average value of the ratio of the peak area obtained to the peak area obtained in the corresponding step 5 is 2.0.
And 7: calculation of 2-acetyl-1-pyrroline content of sample
The peak areas of sample 1 were 48196, 49291, and 48597, respectively, and the substitution into the formulas gave 0.596ng, 0.610ng, and 0.601 ng, respectively, and further calculated as C (wet basis) = R/M (weight amount) × RRF, which gave 0.0119 mg/Kg, 0.0122mg/Kg, 0.0120 mg/Kg, average 0.012 mg/Kg, and standard deviation 0.01%, respectively. FIG. 7 is the total ion flow chromatogram of the leaves of Oryza sativa, with a peak retention time of 6.581 min for 2-acetyl-1-pyrroline. FIG. 8 is a mass spectrum of 2-acetyl-1-pyrroline, with characteristic ion fragments of m/z 68, 83 and 111, and the mass spectrum matched with a standard mass spectrum of 2-acetyl-1-pyrroline substance by NIST library search, and the 2-acetyl-1-pyrroline is proved to be 2-acetyl-1-pyrroline.
The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, alterations and equivalents of the above embodiments according to the technical spirit of the present invention are still within the protection scope of the technical solution of the present invention.
Claims (7)
1. A gas-phase mass spectrum combination method for measuring the 2-acetyl-1-pyrroline content of the fragrant rice leaves by an external standard multipoint method is characterized by comprising the following steps:
s1: sampling;
s2: pre-treating blades and weighing;
s3: pre-treatment of solid phase micro-extraction;
s4: gas chromatography-mass spectrometry analysis;
s5: the 2-acetyl-1-pyrroline external standard multipoint method is established as follows: taking m/z83 target characteristic ion peak areas of a plurality of standard samples with different concentrations obtained in S4 as vertical coordinates, taking corresponding standard sample amount as horizontal coordinates, drawing a standard curve, and obtaining a correlation coefficient and a linear calculation formula;
s6: establishing a relative recovery factor: directly injecting sample by using a sample injection needle with the middle-level concentration in the concentration range of the multi-point standard sample in S5 to obtain the ratio of the m/z83 peak area to the m/z83 peak area obtained in the corresponding S5, namely establishing a Relative Recovery Factor (RRF);
s7: calculating the content C of the 2-acetyl-1-pyrroline in the wet base of the leaf according to the formula C = R/M RRF,
wherein R is the mass ng calculated according to the S5 standard curve;
RRF is S6 to obtain a value;
m is sample weight, mg;
wherein the unit of C is mg/Kg, namely ppm.
2. The gas mass spectrometry combination method for determining the 2-acetyl-1-pyrroline content of the leaves of the fragrant rice plant by the external standard multipoint method as claimed in claim 1, wherein in S1, the sampling refers to the collection of the uppermost first full leaf at the early stage of heading of the rice plant or the collection of the sword leaf after heading.
3. The gas mass spectrometry combination method for determining the 2-acetyl-1-pyrroline content of the oryza sativa leaves by the external standard multipoint method as claimed in claim 1, wherein the specific method of S5 is as follows:
1) if the content of the 2-acetyl-1-pyrroline in the wet base of the leaf is 0.000-0.015mg/Kg, taking the amount of external standard points of a plurality of concentration gradients in the range of 0.00-1.00 ng and the corresponding m/z83 peak area of the target characteristic ion as a calibration curve;
2) if the content of the 2-acetyl-1-pyrroline in the wet base of the leaf is 0.007-0.150 mg/Kg, taking the external standard point quantity of a plurality of gradients in the range of 0.00-10.00 ng and the corresponding peak area of m/z83 of the target characteristic ion as a calibration curve;
3) if the content of the 2-acetyl-1-pyrroline in the wet base of the blade is 0.050-1.500 mg/Kg, taking the external standard point quantity of a plurality of gradients in the range of 0.00-80.0 ng and the corresponding peak area of target characteristic ion m/z83 as a calibration curve;
the r of the established linear equation is more than or equal to 0.9900.
4. The gas mass spectrometry method for determining the 2-acetyl-1-pyrroline content of the fragrant rice leaf by the external standard multipoint method as claimed in claim 3, wherein the gradient is set to be at least 4.
5. The gas mass spectrometry combination method for determining the 2-acetyl-1-pyrroline content of the oryza sativa leaves by the external standard multipoint method as claimed in claim 4, wherein the specific process of S6 is as follows:
1) if the content of the 2-acetyl-1-pyrroline in the wet base of the leaf is 0.000-0.015mg/Kg, adopting 10 mul of a middle-level standard sample with the concentration of 0.04-0.06 ng/mul, and directly feeding a sample to obtain the ratio of the peak area of the m/z83 characteristic ion of the 2-acetyl-1-pyrroline to the peak area after corresponding solid phase microextraction, namely a relative recovery factor;
2) if the content of the leaf wet base 2-acetyl-1-pyrroline is 0.007-0.150 mg/Kg, adopting 10 mul of a middle-level standard sample with the concentration of 0.4-0.6 ng/mul, directly sampling to obtain the ratio of the peak area of the m/z83 characteristic ion of the 2-acetyl-1-pyrroline to the peak area after corresponding solid phase microextraction, namely a relative recovery factor;
3) if the content of the leaf wet base 2-acetyl-1-pyrroline is 0.050-1.500 mg/Kg, 10 mul of the intermediate level standard sample with the concentration of 4-6 ng/mul is adopted, and the ratio of the m/z83 peak-out ion area to the peak area after corresponding solid phase microextraction is obtained after direct sample injection, namely the relative recovery factor.
6. The gas mass spectrometry combination method for determining the 2-acetyl-1-pyrroline content of the oryza sativa leaves by the external standard multipoint method as claimed in claim 1, wherein the specific process of S3 is as follows:
the first stage is as follows: preheating at 70-85 deg.C for 3-20 min;
and a second stage: extracting for 30-50min by adopting a carbon molecular sieve/divinylbenzene/polydimethylsilane and a 50/30 mu m and 1 cm long solid phase micro-extraction head at the temperature of 70-85 ℃ and the rotation speed of 200-; the distance between the extraction head and the sample inlet bottle is 18-23 mm;
and a third stage: the extract is directly fed into the sample inlet of the gas chromatograph, the sample introduction time is 1.5 min, and the sample introduction temperature is 250 ℃.
7. The gas mass spectrometry combination method for determining the 2-acetyl-1-pyrroline content of the oryza sativa leaves by the external standard multipoint method as claimed in claim 1, wherein S4 specifically comprises: and (3) analyzing in a gas chromatograph-mass spectrometer, wherein the set parameters of the gas chromatograph-mass spectrometer are as follows:
the gas interface temperature is 240 ℃, and the ion source temperature is 210 ℃; the separating column adopts an Rxi-5Sil capillary column with the specification of 30 m multiplied by 0.25 mm multiplied by 0.25 mu m; the temperature of the gas phase is programmed to 50 ℃ and kept for 1min, then the temperature is raised to 120 ℃ at 5 ℃/min, and then the temperature is raised to 250 ℃ at 20 ℃/min and kept for 4 min;
the mass spectrum adopts a full-scanning combined ion monitoring mode, the scanning speed is 9900, the m/z range is 50-300, and the scanning interval is 0.06; ion channels selected for ion monitoring are m/z83, 111 and 68, with an interval of 0.2; quantification is based on peak ion area; the target ion m/z83 of the 2-acetyl-1-pyrroline is taken as a quantitative ion, and 111 and 68 are taken as qualitative ions; the carrier gas was 99.999% helium, with a flow rate of 35.0 cm/sec; electron bombardment ion source, ionization energy is 70 eV; the judgment of the 2-acetyl-1-pyrroline substance needs to be matched with the standard mass spectrogram of the 2-acetyl-1-pyrroline substance through NIST library retrieval.
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