CN113376277B - High-resolution mass spectrometry detection method for fentanyl drugs in health care products - Google Patents
High-resolution mass spectrometry detection method for fentanyl drugs in health care products Download PDFInfo
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Abstract
The invention provides a high-resolution mass spectrometry detection method of fentanyl drugs in a health product, which comprises the following steps: s1, adding an internal standard into a sample to be detected and extracting; enabling the supernatant to pass through an MCX solid-phase extraction column, leaching, eluting, removing the solvent, dissolving the residue, and filtering to obtain a sample solution; s2, diluting the mixed standard working solution and the internal standard working solution to prepare 0.01-10 ng/mL series of standard working solutions; s3, detecting and analyzing the standard working solution and the sample solution; s4, performing linear regression analysis on the peak area ratio of the component to be detected in the standard solution to the internal standard substance and the concentration of the component to be detected; and S5, substituting the peak area ratio of the component to be measured in the sample to the peak area ratio of the internal standard substance into a standard curve linear regression equation, and calculating to obtain the concentration of the component to be measured. The method combines an internal standard method with an MCX cation solid phase extraction column, is beneficial to eliminating the matrix effect influence of the health care product, and realizes accurate quantification by combining a high-resolution mass spectrum with a PRM technology.
Description
Technical Field
The invention relates to the technical field of health product detection, in particular to a high-resolution mass spectrum detection method for fentanyl drugs in health products.
Background
Fentanyl belongs to a large group of opioid compounds. The pharmaceutical composition is approved to be marketed by China as a medicine, and 3 varieties of fentanyl, remifentanil and sufentanil exist, and alfentanil is still in the stage of drug research and development. Internationally, the commonly used dosage forms include injections, transdermal patches, sublingual tablets, nasal sprays and the like. Among them, fentanyl is frequently used in medical clinical practice for narcotic analgesia of advanced cancers. However, fentanyl has the remarkable characteristics of the arabine compound, such as easy addiction, strong dependence and difficult withdrawal, and the use of a large dose can cause coma, respiratory depression and even death. Therefore, the fentanyl substance is an important component in the third generation drug, namely the laboratory drug, after the traditional drug and the synthetic drug, and is listed in the supplementary catalog of non-medicinal narcotic drugs and psychiatric drug control varieties from 5 months and 1 days in 2019, and 25 fentanyl substances are included in the catalog.
At present, a triple quadrupole mass spectrometry detection technology is mostly adopted for detecting fentanyl compounds, samples such as blood, urine, environmental water and the like are mainly detected, and a liquid-liquid extraction mode is mostly adopted for a sample pretreatment method. The components in the health care product are complex, and especially the health care product containing the plant extract can not realize the extraction and purification of the fentanyl drug in the substance to be detected by only liquid-liquid extraction. Therefore, the pretreatment method for samples such as blood, urine, environmental water and the like in the prior art is not suitable for detecting fentanyl drugs in health care products. In addition, the existing triple quadrupole mass spectrometry detection technology is based on the quantitative/qualitative ion pair detection, the mass number is accurate to 1 position behind a decimal point, and certain defects exist in the aspects of false positive and false negative judgment. Therefore, on the premise and background that the cargo inspection batches on the import and export shore are in a continuous growth situation, the research and development of the high performance liquid chromatography-high resolution mass spectrometry screening and confirmation method for 25 fentanyl compounds is of great significance.
Disclosure of Invention
In order to solve the problems, the invention aims to provide a high-resolution mass spectrometry detection method for fentanyl drugs in health care products, which adopts an internal standard method and an MCX cation solid phase extraction column to be combined, is favorable for eliminating the matrix effect influence of the health care products, adopts QE high-resolution mass spectrometry, and realizes accurate quantification by combining a spindle structure with a mass analyzer and a PRM (parallel reaction monitoring) technology.
In order to achieve the above object, the technical scheme of the invention is as follows.
A high-resolution mass spectrometry detection method for fentanyl drugs in health products comprises the following steps:
s1, pretreatment of a sample to be detected
Adding an internal standard into a sample to be detected, and extracting and centrifuging by using acetonitrile aqueous solution; passing the supernatant through an MCX solid phase extraction column, leaching with water and methanol, and removing leacheate; then eluting with ammonia water methanol solution, and collecting the eluent; then removing the solvent in the eluent, dissolving the residue by using an initial mobile phase and filtering to obtain a sample solution;
s2, preparation of standard working solution
Measuring the mixed standard working solution and the internal standard working solution, diluting the mixed standard working solution and the internal standard working solution by using an initial mobile phase to prepare a series of standard working solutions with the mass concentration of 0.01-10 ng/mL, wherein the internal standard substance concentration is 10.0ng/mL;
s3, high performance liquid chromatography-high resolution mass spectrometry detection
Respectively detecting and analyzing the standard working solution of S2 and the sample solution of S1 by using a high performance liquid chromatography-high resolution mass spectrometer to obtain related chromatograms;
s4, drawing a standard curve
Performing quantitative analysis on the standard working solution prepared in the step S2 by adopting an internal standard method, and performing linear regression analysis on the peak area ratio of the component to be detected in the standard solution to the internal standard substance and the concentration of the component to be detected to obtain a standard curve linear regression equation;
s5, calculating the concentration of the component to be measured
And (3) determining the sample solution of S1, determining the peak area ratio of the component to be determined and the internal standard substance in the sample, substituting the peak area ratio into a standard curve linear regression equation, and calculating to obtain the concentration of the component to be determined.
Further, in S1, the internal standard was fentanyl-D5, and the concentration of the internal standard in the sample solution was 10.0ng/mL.
Further, in S1, the volume percentage of the acetonitrile water solution is 75%; the volume percentage of the ammonia water methanol solution is 5 percent.
Further, in S1, the MCX solid phase extraction column was activated with 5mL of methanol and 5mL of water before use.
Further, in S2, the fentanyl drug standard in the mixed standard working solution includes acetylalfafalfentanil hydrochloride, alfentanil hydrochloride, α -methylfentanyl hydrochloride, β -hydroxyfentanyl hydrochloride, β -hydroxy-3-methylfentanyl, (±) -cis-3-methylfentanyl hydrochloride, 3-methylthiofentanyl, p-fluorofentanyl, remifentanil hydrochloride, sufentanil citrate, fentanyl, acetylfentanyl, butyrfentanyl, (±) - β -hydroxythiofentanyl hydrochloride, 4-fluorobutyrfentanyl, isobutyrfentanyl hydrochloride, alfentanil, carfentanil, furfentanyl hydrochloride, acrylfentanyl hydrochloride, valerylfentanyl hydrochloride, 4-fluoroisobutyrfentanyl, N-phenethyl-4-piperidone, 4-anilino-N-phenethylpiperidine, and hydroxylated norfentanyl.
Further, in S3, the conditions of the high performance liquid chromatography are:
the adopted chromatographic column is ThermoAccucorephynyl-Hexyl, the length is 100mm, the inner diameter is 2.1mm, and the particle size of the filler is 2.6 mu m; the column temperature was 45 ℃ and the sample size was 10. Mu.L.
Further, in S3, a gradient elution program is adopted, and a mobile phase A is a mixed solution of formic acid and an ammonium formate solution, wherein the volume percentage of the formic acid is 0.1%, and the concentration of the ammonium formate solution is 2mmol/L; the mobile phase B is a mixed solution of formic acid and a methanol/acetonitrile solution, wherein the volume percentage of the formic acid is 0.1%, and the volume ratio of the methanol/acetonitrile is 50:50;
the gradient elution procedure comprises the following specific steps in percentage by volume: at the time of 0-1.0 min, 5% of mobile phase A and 95% of mobile phase B are adopted, and the flow rate is 0.3mL/min; at the moment of 1.0-10.0 min, 5% of mobile phase A and 95% of mobile phase B are adopted, and the flow rate is 0.3mL/min; at the time of 10.0-11.6 min, 95% of mobile phase A and 5% of mobile phase B are adopted, and the flow rate is 0.3mL/min; at the time of 11.6-15.5 min, 5% of mobile phase A and 95% of mobile phase B are adopted, and the flow rate is 0.3mL/min.
Further, in S3, the high resolution mass spectrometry conditions are:
adopting a heated electrospray positive ion source Q-active; the spraying voltage is 3.5kV, the lens voltage is 55V, the capillary temperature is 350 ℃, and the auxiliary gas temperature is 320 ℃; sheath gas pressure 40arb, auxiliary gas pressure 15arb, primary and secondary scanning resolution 17500, and adopting an ionization mode to simultaneously monitor parallel reactions; AGC1e 5 ，Maximum IT 50ms。
The invention has the beneficial effects that:
1. the method of the invention adopts the combination of an internal standard method and an MCX cation solid phase extraction column, which is beneficial to eliminating the matrix effect influence of the health care product; and QE high-resolution mass spectrometry is adopted, and a mass analyzer of the mass analyzer is mainly characterized by a spindle structure and is combined with a PRM (parallel reaction monitoring) technology to realize accurate quantification. The limit of the invention for the non-liquid sample quantification is 0.2 mug/kg, the liquid sample quantification is 0.05 mug/kg, and the sensitivity is more than 50 times of that of the prior art.
2. The method can realize quantitative and qualitative detection of 25 fentanyl compounds in the health care product by utilizing a Parallel Reaction Monitoring (PRM) technology of high-resolution mass spectrometry, and improve the monitoring, early warning, inspection, identification and hazard evaluation levels of the fentanyl compounds in China.
3. The method aims at the detection of fentanyl drugs in the health care products, the components of the health care products are complex, the active ingredients are mostly plant extracts, the matrix is complex, and the matrix effect influence cannot be eliminated by adopting the external standard method for quantification, so the method adopts the internal standard method for quantification.
4. The structure of the fentanyl compound contains benzene rings and pyridine ring structures, and belongs to alkaline compounds, MCX cation solid phase extraction has obvious advantages for purifying the compounds, the matrix of the invention is a health product, the matrix of the health product is relatively complex, and MCX has advantages for purifying complex matrices, especially matrices containing plant extracts.
Drawings
FIGS. 1-2 are chromatograms of 25 fentanyl compounds and an internal standard in examples of the present invention.
FIGS. 3 to 6 are chromatograms of a liquid sample in the example of the present invention.
FIGS. 7 to 8 are detection limit spiked PRM chromatograms of liquid samples in examples of the present invention.
FIGS. 9 to 12 are chromatograms of solid samples in examples of the present invention.
FIGS. 13 to 14 are detection limit spiked PRM chromatograms of solid samples in examples of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more clearly understood, the present invention is described in further detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The reagents, materials and experimental instruments related to the embodiment of the invention are as follows:
A. reagents and materials
fentanyl-D5 internal standard stock (100. Mu.g/mL, methanol, achemtek, inc.);
health product samples (liquid/solid; all purchased in the market);
methanol, acetonitrile, formic acid, ammonium formate were all chromatographically pure (Merck Corp.); other reagents are analytically pure;
25 fentanyl compound stock solutions (the mass concentration of each compound is 100 mu g/mL, achemtek company);
the remaining reagents and consumables, unless otherwise specified, are commercially available.
B. Laboratory apparatus
Liquid chromatography-high resolution mass spectrometer (Thermo Q-active, siemmer feishel, usa); VORTEX shaker (IKA VORTEX GENIUS3, IKA, germany); high speed refrigerated centrifuge (AllegraTM X-22R centrifuge, BECKMAN, germany); high purity water generators (Milli-Q Advantage-10/Elix, millpore, USA); nitrogen concentrator (TurboVap LV, caliper, usa);
MCX solid phase extraction column (3 mL,60mg, waters corporation, USA); 0.22 μm organic filter (Shimadzu, japan); column ThermoAccucorephynyl-Hexyl (100X 2.1mm,2.6 μm, sammer Feishell, USA).
Example 1
High resolution mass spectrum detection of fentanyl drugs in the health care product, and an internal standard method/MCX solid phase extraction column.
1. Preparation of internal Standard working solution
Internal standard working solution (1.0. Mu.g/mL): measuring 1mL of fentanyl-D5 internal standard stock solution, diluting the internal standard stock solution to a scale with methanol in a 10mL volumetric flask to prepare an internal standard intermediate solution with the concentration of 10.0 mu g/mL, measuring the intermediate solution in the 1.0mL fish 10mL volumetric flask, and shaking up to obtain 1.0 mu g/mL internal standard working solution. Keeping at 0-4 deg.C in dark place, and keeping the shelf life of 6 months.
2. Sample pretreatment
Liquid sample: accurately weighing 5.00g of liquid sample, placing the liquid sample into a 50mL centrifuge tube, adding 40 muL of fentanyl-D5 internal standard working solution (the concentration is 1.0 mug/mL), adding 20mL of acetonitrile aqueous solution (the volume percentage is 75%), carrying out vortex mixing, carrying out shaking extraction for 30min, and centrifuging at 4500r/min for 5min; 5mL of the supernatant was applied to an MCX solid phase extraction column (activated with 5mL of methanol and 5mL of water before use), and the supernatant was applied to the activated MCX solid phase extraction column at a rate of 1.5 mL/min. Leaching with 5mL of water and 5mL of methanol, and discarding the leaching solution; eluting with 10mL of 5% ammonia water methanol solution by volume percentage, and collecting the eluent; blowing nitrogen to near dryness at 40 deg.C, dissolving the residue with 1mL of initial mobile phase (5% mobile phase A +95% mobile phase B), mixing by vortex, filtering with 0.22 μm organic filter membrane to obtain sample solution, and subjecting to liquid chromatography-high resolution mass spectrometry.
Solid sample: 1.00g of solid sample is accurately weighed, treated according to the method of liquid sample and then injected.
3. Preparation of Standard working solutions
Mix standard working solution (1.0. Mu.g/mL): respectively measuring 1mL of 25 fentanyl compound stock solutions, placing the stock solutions in a 10mL volumetric flask, purging with nitrogen in the mixing process, diluting with methanol to scale, preparing a mixed standard intermediate solution with the concentration of 10.0 mu g/mL, measuring 1.0mL of the intermediate solution in the 10mL volumetric flask, and shaking up to obtain a mixed standard working solution (the concentration of each compound is 1.0 mu g/mL). And storing at 0-4 deg.c in dark place for 6 months.
The mixed standard working solution and the internal standard working solution are measured and diluted by an initial mobile phase (5% of mobile phase A +95% of mobile phase B) to prepare a series of standard working solutions with mass concentrations of 0.01, 0.02, 0.05, 0.1, 0.5, 1.0, 2.0, 5.0 and 10ng/mL, wherein the internal standard substance concentrations are all 10.0ng/mL.
4. High performance liquid chromatography conditions
A chromatographic column: thermoAccucorephynyl-Hexyl (100X 2.1mm,2.6 μm, sammer Feishell, USA); the column temperature was 45 ℃ and the sample size was 10. Mu.L. Mobile phase A:0.1% (v/v) formic acid, 2mmol/L ammonium formate solution; b:0.1% formic acid, methanol/acetonitrile (v/v = 50; the gradient elution procedure is shown in table 1.
TABLE 1 gradient elution procedure
| Time/min | Flow rate/mL/min | A/％ | B/％ |
| 0～1.0 | 0.3 | 5 | 95 |
| 1.0～10.0 | 0.3 | 5 | 95 |
| 10.0～11.6 | 0.3 | 95 | 5 |
| 11.6～15.5 | 0.3 | 5 | 95 |
| 15.5～20.0 | 0.3 | 5 | 95 |
5. Conditions of Mass Spectrometry
Ionization mode: heating an electrospray positive ion source, wherein the spraying voltage is 3.5kV, the lens voltage is 55V, the temperature of a capillary tube is 350 ℃, and the temperature of auxiliary gas is 320 ℃; 40arb of sheath gas pressure, 15arb of auxiliary gas pressure, 17500 of primary and secondary scanning resolution, parallel reaction monitoring at the same time by adopting ionization mode, AGC1e 5 Maximum IT 50ms. The mass spectrometry scan parameters are shown in table 2. The retention times for the 25 fentanyl compounds and the internal standard are shown in FIGS. 1-2.
TABLE 2 accurate mass number, retention time and NCE voltage of fentanyl compounds
6. Preparation of standard curve and calculation of concentration of component to be measured
Appropriate amount of mixed standard working solution and internal standard working solution are measured and diluted by initial mobile phase to prepare 0.01, 0.02, 0.05, 0.1, 0.5, 1.0, 2.0, 5.0 and 10ng/mL series standard solution (the concentration of the internal standard substance is 10.0 ng/mL). And (3) drawing a standard curve by taking the ratio of the peak area of the measured component in the standard solution to the peak area of the fentanyl-D5 internal standard substance as a vertical coordinate and the concentration of the standard solution as a horizontal coordinate. Specific results are shown in table 2.
And (3) measuring the prepared sample solution, measuring the peak area ratio of the component to be measured in the sample to the internal standard substance, substituting the peak area ratio into a standard curve linear regression equation, and calculating to obtain the concentration of the component to be measured.
TABLE 2 Standard working curves for 25 fentanyl compounds in the Mixed Standard working solution
As can be seen from the results in Table 2, when the standard curve is used for correction, 25 fentanyl compounds have good linear relation in the range of 0.01-10 ng/mL, and the correlation coefficients are all larger than 0.99.
7. Quantitative limit, detection limit and spiking recovery rate of the method
7.1 quantitation limit, detection limit and spiking recovery of liquid samples
And adding low-concentration 25 fentanyl compounds into the liquid sample, mixing and labeling, repeating the measurement for 5 times, and determining the detection limit, the quantification limit and the labeled recovery rate of the 25 fentanyl compounds in the liquid health care product, wherein the results are shown in figures 3-8 and tables 3-4, wherein figures 3-6 are chromatograms of the liquid sample, and figures 7-8 are the detection limit labeled chromatograms of the liquid sample.
The calculation formula of the standard recovery rate is as follows:
in the formula:
x-residual amount of analyte in a sample in micrograms per kilogram (g/kg);
a-peak area of the substance to be measured in the sample solution;
A iS -peak area of internal standard in sample solution;
A′ iS -peak area of internal standard in standard working solution;
A S -peak area of the substance to be measured in the standard working solution;
C S -the concentration of the substance to be tested in nanograms per milliliter (ng/mL) in the standard working solution;
C′ iS -concentration of internal standard in standard working solution in nanograms per milliliter (ng/mL);
C iS -concentration of internal standard in nanograms per milliliter (ng/mL) in the sample solution;
v is the volume of the sample solution with a constant volume, and the unit is milliliter (mL);
m is the mass of the sample in grams (g).
TABLE 3 detection limits and spiked recoveries of liquid samples
As is clear from the results in Table 3, in the liquid health care product, the detection limits of β -hydroxyfentanyl hydrochloride (No. 4), β -hydroxy-3-methylfentanyl (No. 5), 3-methylthiofentanyl (No. 7), p-fluorofentanyl (No. 8), sufentanil citrate (No. 10), fentanyl (No. 11), (+ -) - β -hydroxythiofentanyl hydrochloride (No. 14), 4-fluorobutyrylfentanyl (No. 15), isobutyrylfentanyl hydrochloride (No. 16), olfentanyl (No. 17), carfentanyl (No. 18), furfentanyl hydrochloride (No. 19), acryloylfentanyl hydrochloride (No. 20), valerylfentanyl hydrochloride (No. 21), 4-fluoroisobutyrylfentanyl (No. 22), 4-anilino-N-phenylethylpiperidine (No. 24) were 0.05. Mu.g/kg; acetylalfafmethylfentanyl hydrochloride (No. 1), alfentanil hydrochloride (No. 2), α -methylfentanyl hydrochloride (No. 3), (±) -cis-3-methylfentanyl hydrochloride (No. 6), acetylfentanyl (No. 12), butyrylfentanyl (No. 13), N-phenethyl-4-piperidone (No. 23), and hydroxylated norfentanyl (No. 25) were found to have detection limits of 0.1 μ g/kg; the detection limit of remifentanil hydrochloride (No. 9) was 0.5. Mu.g/kg.
The blank sample is subjected to 3-level 6-parallel standard recovery rate measurement, the average recovery rate is 70.0-128.0%, and the relative standard deviation is 0.3-3.5%.
TABLE 4 limit of quantitation and recovery from spiking of liquid samples
From the results in Table 4, it is understood that in the liquid health care product, the amounts of- -hydroxyfentanyl hydrochloride (No. 4), - -hydroxy-3-methylfentanyl (No. 5), 3-methylthiofentanyl (No. 7), p-fluorofentanyl (No. 8), fentanyl (No. 11), (+ -.) - - - - - - - - - - - - - - -) hydroxyfentanyl hydrochloride (No. 14), 4-fluorobutyrylfentanyl (No. 15), oxfentanyl (No. 17), carfentanyl (No. 18), furanfentanyl hydrochloride (No. 19), valerylfentanyl hydrochloride (No. 21), 4-fluoroisobutyrylfentanyl (No. 22), 4-anilino-N-phenylethylpiperidine (No. 24) were limited to 0.1. Mu.g/kg, acetylalfentanyl hydrochloride (No. 1), alfentanyl hydrochloride (No. 2), - -methylfentanyl hydrochloride (No. 3), (-) -cis-3-methylfentanyl hydrochloride (No. 6), acetylfentanyl (No. 12), butyrylfentanyl (No. 13), isobutyrylfentanyl (No. 16), N-phenylethyl ketone (No. 23), and N-phenylethyl ketone (No. 25. Mu.g/kg).
The blank sample is subjected to 3-level 6-parallel standard addition recovery rate measurement, the average recovery rate is 70.7-112.6%, and the relative standard deviation is 0.6-4.8%.
7.2 quantitation limit, detection limit and spiking recovery of solid samples
And adding low-concentration 25 fentanyl compounds into the solid sample, mixing and labeling, repeating the measurement for 5 times, and determining the detection limit, the quantification limit and the labeling recovery rate of the 25 fentanyl compounds in the solid health care product, wherein the results are shown in figures 9-14 and tables 5-6, wherein figures 9-12 are chromatograms of the solid sample, and figures 13-14 are the detection limit labeling chromatograms of the solid sample.
TABLE 5 detection limits and recovery of spiked solid samples
As is clear from the results in Table 5, in the solid health products, the detection limits of alfentanil hydrochloride (No. 2), α -methylfentanyl hydrochloride (No. 3), (±) -cis-3-methylfentanyl hydrochloride (No. 6), remifentanil hydrochloride (No. 9), sufentanil citrate (No. 10), fentanyl (No. 11), acetylfentanyl (No. 12), butyrfentanyl (No. 13), 4-fluorobutyrfentanyl (No. 15), isobutyrylfentanyl hydrochloride (No. 16), carfentanil (No. 18), acryloylfentanyl hydrochloride (No. 20), 4-fluoroisobutyrylfentanyl (No. 22), 4-anilino-N-phenylethylpiperidine (No. 24), and hydroxylated norfentanyl (No. 25) were 0.05 μ g/kg; acetylalfafentanil hydrochloride (No. 1), 3-methylthiofentanyl (No. 7), p-fluorofentanyl (No. 8), valerylfentanyl hydrochloride (No. 21), N-phenethyl-4-piperidone (No. 23) had detection limits of 0.1. Mu.g/kg; the detection limits of olfentanil (No. 17) and furfentanil hydrochloride (No. 19) were 0.2. Mu.g/kg, and the detection limits of β -hydroxyfentanil hydrochloride (No. 4), β -hydroxy-3-methylfentanyl (No. 5) and (. + -.) - β -hydroxyfentanyl hydrochloride (No. 14) were 0.5. Mu.g/kg.
The blank sample is subjected to 3-level 6-parallel standard addition recovery rate measurement, the average recovery rate is 70.2-114.0%, and the relative standard deviation is 0.4-4.3%.
TABLE 6 limit of quantitation and recovery normalized for solid samples
As is clear from the results in Table 6, in the solid health products, alfentanil hydrochloride (No. 2), alfentanil hydrochloride (No. 1), α -methylfentanyl hydrochloride (No. 3), remifentanil hydrochloride (No. 9), sufentanil citrate (No. 10), fentanyl (No. 11), acetylfentanyl (No. 12), and butyrylfentanyl (No. 13) were quantitatively limited to 0.10. Mu.g/kg, (. + -.) -cis-3-methylfentanyl hydrochloride (No. 6), 4-fluorobutyrylfentanyl (No. 15), isobutyrylfentanyl hydrochloride (No. 16), carfentanyl (No. 18), acryloylfentanyl hydrochloride (No. 20), 4-fluoroisobutyrylfentanyl (No. 22), 4-anilino-N-phenylethylpiperidine (No. 24), hydroxylated norfentanyl (No. 25) were quantitatively limited to 0.20. Mu.g/kg, 3-methylthiofentanyl (No. 7), p-fluorofentanyl (No. 8), oxofentanyl hydrochloride (No. 17), furylfentanyl hydrochloride (No. 19), and N-pentanone hydrochloride (No. 21. Mu.g-ethyl-20 kg), the detection limits of the beta-hydroxyfentanyl hydrochloride (No. 4), the beta-hydroxy-3-methylfentanyl (No. 5) and the (+ -) -beta-hydroxythiofentanyl hydrochloride (No. 14) were 1.0. Mu.g/kg.
The blank sample is subjected to 3-level 6-parallel standard addition recovery rate measurement, the average recovery rate is 75.0-111.0%, and the relative standard deviation is 0.2-2.2%.
Comparative example 1
A high-resolution mass spectrum detection method for fentanyl drugs in health products, an internal standard method/PRIME HLB solid phase extraction column. This method is substantially the same as that of example 1, except that,
the MCX solid phase extraction column is replaced by a PRIME HLB solid phase extraction column.
When the purification is carried out by adopting a PRIME HLB solid-phase extraction column, the recovery rates of acetylfentanyl, (+/-) -beta-hydroxyfentanyl hydrochloride, carfentanil, valerylfentanyl hydrochloride and hydroxylated norfentanyl are poor, wherein the recovery rates of acetylfentanyl and hydroxylated norfentanyl are lower than 50%, and the recovery rates of (+/-) -beta-hydroxyfentanyl hydrochloride, carfentanil and valerylfentanyl hydrochloride are higher than 150%. Therefore, the PRIME HLB solid-phase extraction column is not suitable for purifying 25 fentanyl compounds.
Comparative example 2
A high-resolution mass spectrum detection method for fentanyl drugs in health products comprises an external standard method/a PRIME HLB solid phase extraction column. This process is substantially the same as that of example 1, except that,
replacing the MCX solid phase extraction column with a PRIME HLB solid phase extraction column, and adopting an external standard method.
Similarly, when the purification is carried out by adopting a PRIME HLB solid-phase extraction column, the recovery rate of the acetylfentanyl, (+/-) -beta-hydroxyfentanyl hydrochloride, the carfentanil, the valerylfentanyl hydrochloride and the hydroxylated norfentanyl is poor, wherein the recovery rate of the acetylfentanyl and the hydroxylated norfentanyl is lower than 50 percent, and the (+/-) -HLB solid-phase extraction column is adopted
The recovery rate of the-beta-hydroxy-thiofentanyl hydrochloride, the carfentanil and the valeryl fentanyl hydrochloride is higher than 150%. Therefore, the PRIME HLB solid-phase extraction column is not suitable for purifying 25 fentanyl compounds. And because the sample matrix is relatively complex, the quantitative workload of each sample matrix adopting a matrix standard curve is relatively large, so an internal standard method is adopted for quantitative analysis in experiments, and the influence brought by the matrix can be completely corrected by the internal standard method.
By the tests of example 1 and comparative example 1, matrix effect examination was performed using an MCX solid phase extraction column and an HLB solid phase extraction column, respectively, thereby demonstrating that MCX is advantageous for the purification of complex matrices, especially matrices containing plant extracts.
By testing the example 1 and the comparative example 2, matrix effect investigation is respectively carried out by adopting an internal standard method and an external standard method, so that matrix effect influence cannot be eliminated by adopting the external standard method, and matrix effect influence can be eliminated by adopting the internal standard method.
The present invention is not limited to the above preferred embodiments, and any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (5)
1. A high-resolution mass spectrometry detection method for fentanyl drugs in health products is characterized by comprising the following steps:
s1, pretreatment of a sample to be tested
Adding an internal standard into a sample to be detected, and extracting and centrifuging by using acetonitrile aqueous solution; passing the supernatant through an MCX solid phase extraction column, leaching with water and methanol, and removing leacheate; then eluting with ammonia water methanol solution and collecting eluent; then removing the solvent in the eluent, dissolving the residue by using an initial mobile phase and filtering to obtain a sample solution;
s2, preparation of standard working solution
Measuring a mixed standard working solution and an internal standard working solution, and diluting the mixed standard working solution and the internal standard working solution by using an initial mobile phase to prepare a series of standard working solutions with the mass concentration of 0.01-10 ng/mL, wherein the internal standard substance concentration is 10.0ng/mL;
s3, high performance liquid chromatography-high resolution mass spectrometry detection
Respectively detecting and analyzing the standard working solution of S2 and the sample solution of S1 by using a high performance liquid chromatography-high resolution mass spectrometer to obtain related chromatograms;
the conditions of the high performance liquid chromatography are as follows:
the adopted chromatographic column is ThermoAccucorephynyl-Hexyl, the length is 100mm, the inner diameter is 2.1mm, and the particle size of the filler is 2.6 mu m;
adopting a gradient elution program, wherein a mobile phase A is a mixed solution of formic acid and an ammonium formate solution, the volume percentage of the formic acid is 0.1%, and the concentration of the ammonium formate solution is 2mmol/L; the mobile phase B is a mixed solution of formic acid and a methanol/acetonitrile solution, wherein the volume percentage of the formic acid is 0.1%, and the volume ratio of methanol/acetonitrile is 50:50;
the gradient elution procedure is specifically as follows, in volume percent: at the moment of 0-1.0 min, 5% of mobile phase A and 95% of mobile phase B are adopted, and the flow rate is 0.3mL/min; at the moment of 1.0-10.0 min, 5% of mobile phase A and 95% of mobile phase B are adopted, and the flow rate is 0.3mL/min; at the time of 10.0-11.6 min, 95% of mobile phase A and 5% of mobile phase B are adopted, and the flow rate is 0.3mL/min; at the moment of 11.6-15.5 min, 5% of mobile phase A and 95% of mobile phase B are adopted, and the flow rate is 0.3mL/min;
s4, drawing a standard curve
Carrying out quantitative analysis on the standard working solution prepared in the step S2 by adopting an internal standard method, and carrying out linear regression analysis on the peak area ratio of the component to be detected to the internal standard substance in the standard solution and the concentration of the component to be detected to obtain a standard curve linear regression equation;
s5, calculating the concentration of the component to be measured
Measuring the sample solution of S1, measuring the peak area ratio of the component to be measured in the sample to the internal standard substance, substituting the peak area ratio into a standard curve linear regression equation, and calculating to obtain the concentration of the component to be measured;
in S2, the fentanyl drug standard in the mixed standard working solution includes acetylalfafalfentanil hydrochloride, alfentanil hydrochloride, α -methylfentanyl hydrochloride, β -hydroxyfentanyl hydrochloride, β -hydroxy-3-methylfentanyl, (±) -cis-3-methylfentanyl hydrochloride, 3-methylthiofentanyl, p-fluorofentanyl, remifentanil hydrochloride, sufentanil citrate, fentanyl, acetylfentanyl, butyrfentanyl, (±) - β -hydroxythiofentanyl hydrochloride, 4-fluorobutyrfentanyl, isobutyrfentanyl hydrochloride, alfentanil, carfentanil, furfentanyl hydrochloride, acrylfentanyl hydrochloride, valerylfentanyl hydrochloride, 4-fluoroisobutyrylfentanyl, N-phenethyl-4-piperidone, 4-anilino-N-phenethylpiperidine, hydroxylated norfentanyl;
in S3, the conditions of the high-resolution mass spectrum are as follows:
adopting a heated electrospray positive ion source Q-active; the spraying voltage is 3.5kV, the lens voltage is 55V, the capillary temperature is 350 ℃, and the auxiliary gas temperature is 320 ℃; sheath gas pressure 40arb, auxiliary gas pressure 15arb, primary and secondary scanning resolution 17500, and adopting an ionization mode to simultaneously monitor parallel reactions; AGC1e 5 ，Maximum IT 50ms。
2. The method for detecting the high-resolution mass spectrum of the fentanyl drugs in the health care products according to claim 1, wherein the internal standard is fentanyl-D5, and the concentration of the internal standard in the sample solution is 10.0ng/mL.
3. The method for detecting the high-resolution mass spectrometry of the fentanyl drugs in the health care products according to claim 1, wherein in the S1, the volume percentage of the acetonitrile water solution is 75%; the volume percentage of the ammonia water methanol solution is 5%.
4. The method for detecting the high-resolution mass spectrum of the fentanyl drugs in the health care products according to claim 1, wherein in the S1, the MCX solid-phase extraction column is activated by 5mL of methanol and 5mL of water before being used.
5. The method for detecting the fentanyl drug in the health care product by the high resolution mass spectrometry as claimed in claim 1, wherein in S3, the conditions of the high performance liquid chromatography are as follows:
the column temperature was 45 ℃ and the amount of sample was 10. Mu.L.
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