CN115047130A - Analysis method for detecting methyltestosterone and metabolite thereof in animal muscle tissue - Google Patents
Analysis method for detecting methyltestosterone and metabolite thereof in animal muscle tissue Download PDFInfo
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- CN115047130A CN115047130A CN202210718195.8A CN202210718195A CN115047130A CN 115047130 A CN115047130 A CN 115047130A CN 202210718195 A CN202210718195 A CN 202210718195A CN 115047130 A CN115047130 A CN 115047130A
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
Abstract
The invention discloses an analysis method for detecting methyltestosterone and metabolites thereof in animal muscle tissues, which comprises sample extraction, sample purification and sample analysis, wherein a sample with complete pretreatment is analyzed by GC-MS; the analysis method of the invention has good linear relation in the range of 50 ng/mL-500 ng/mL, S/N is 10 as the limit of quantification, and the limits of quantification of methyltestosterone and M2 are both 0.05 mg/kg; taking S/N-3 as a detection limit, the detection limit of methyltestosterone is 0.02mg/kg, and the detection limit of M2 is 0.015 mg/kg; the recovery rate range of the methyltestosterone is between 97.7 and 112.4 percent; the recovery rate range of M2 is between 96.6% and 116.6%, and the results show that the analysis method has high sensitivity and good linear relation, the recovery rate and the precision can meet the requirements of actual detection, and the qualitative and quantitative results are accurate.
Description
Technical Field
The invention relates to a method for detecting and analyzing methyl testosterone and a metabolite thereof, in particular to an analysis method for detecting methyl testosterone and a metabolite thereof in animal muscle tissues.
Background
Methyl testosterone (17 beta-hydroxy-17 alpha-methyl-4-en-3-one, MT) is a synthetic male steroid hormone, can increase muscle protein synthesis, and has growth promoting effect related to the increase of Growth Hormone (GH) metabolism. Methyltestosterone can be effective in a very short time and has a significant effect on the increase in the growth rate of animals. However, in recent years, reports indicate that methyl testosterone and its metabolites remained in foods of animal origin are harmful to the environment and public food safety.
Meanwhile, the metabolic products of the methyl testosterone in vertebrate bodies are found in 7 types, and the results of the pharmacokinetic experiments of the methyl testosterone and the metabolic products thereof on tilapia mossambica show that the main metabolic product of the methyl testosterone is M2(17 alpha-methyl-5 beta-androstan-3 alpha, 17 beta-diol), and the modeling analysis is carried out by using 3p97 software, and the conclusion is that: on the one hand the metabolic pathway of M2 is similar to that of methyltestosterone and on the other hand the half-life of absorption (t) of the metabolite M2 (t) 1/2a ) And elimination half-life (t) 1/2b ) 4.164 hours and 362.740 hours, respectively, indicating that the rate of absorption distribution of M2 in vivo is fast, but the rate of elimination is very slow; peak concentration of M2 at the same level (c) max ) And elimination half-life (t) 1/2b ) 5604.883ng/mL and 362.740 hours respectively, the peak concentration and elimination half-life of the methyltestosterone are 1.032 mug/mL and 18.401 hours respectively, and compared with the methyltestosterone, M2 is higher in content in vivo and longer in retention time. The prior art only detects the methyltestosterone, but does not detect the M2, so the problems of less residual quantity of the methyltestosterone after in vivo metabolism and inaccurate detection exist.
Disclosure of Invention
The purpose of the invention is as follows: the invention provides an analysis method for detecting methyltestosterone and metabolites thereof in animal muscle tissues by double confirmation by taking MT and M2 as high-feature risk substances aiming at the problem that major metabolites of MT have longer metabolic time in the existing national standard or row standard detection method.
The technical scheme is as follows: the invention relates to an analysis method for detecting methyltestosterone and metabolite 17 alpha-methyl-5 beta-androstane-3 alpha, 17 beta-diol (M2) thereof in animal muscle tissues, which comprises the following steps:
(1) sample pretreatment: taking an animal muscle sample, removing water, lipid and protein, and extracting a component to be detected to obtain a sample extracting solution;
(2) sample purification: taking a sample of the extract, and performing solid phase extraction on the sample by using a C18 solid phase extraction column and NH 2 Eluting the solid phase extraction column, removing impurities and preparing a methanol extracting solution of the sample;
(3) and (3) sample analysis: analyzing and detecting the methanol extract obtained in the step (2) by using gas chromatography-tandem quadrupole mass spectrometry, wherein S/N is 10 as a quantitative limit, and the quantitative limits of methyltestosterone and M2 are both 0.05 mg/kg; the detection limit of methyl testosterone is 0.02mg/kg, and the detection limit of M2 is 0.015mg/kg, with S/N being 3.
Preferably, the step (1) is specifically to take an animal muscle sample, add an internal standard deuterated methyl testosterone (D-MT), remove moisture by diatomite, remove lipid impurities by n-hexane, remove protein by beta-glucoside sulfatase serving as a hydrolase, and extract a component to be detected by acetonitrile serving as an extraction solvent.
Preferably, the internal standard deuterated methyl testosterone is a methanol solution of deuterated methyl testosterone.
Preferably, the step (2) is specifically that a sample of the extract is put into a C18 solid phase extraction column, and water, an acetone-water solution and n-hexane are adopted for leaching in sequence; NH connected in series 2 Solid phase extraction column, adopting acetonitrile to elute; removing the solvent by rotary evaporation, and adding methanol to prepare a methanol extracting solution of the sample.
Preferably, the step (3) is specifically carried out by using an Shimadzu SH-Rxi-5MS gas chromatographic column (30m multiplied by 0.25mm multiplied by 0.25 mu m), the sample injection amount is 0.5-2 mu L, the sample injection is not divided, the sample injection inlet temperature is 200-320 ℃, the carrier gas is helium, the column flow is 0.7-1.5 mL/min, the mass spectrum quadrupole rod temperature is 250-350 ℃, and the EI source temperature is 200-280 ℃.
Furthermore, the preferred injection port temperature is 280 ℃, the carrier gas is helium, the column flow is 1mL/min, the mass spectrum quadrupole rod temperature is 280 ℃, and the EI source temperature is 230 ℃.
Preferably, the temperature raising procedure in the step (3) is to keep the temperature at 100-200 ℃ for 1-5 min, raise the temperature at 5-25 ℃/min to 250-280 ℃, raise the temperature at 5-15 ℃/min to 280-330 ℃ and keep the temperature for 1-10 min.
Further, the temperature rising program is preferably to keep the temperature at 170 ℃ for 2min, rise the temperature at 15 ℃/min to 250 ℃, rise the temperature at 5 ℃/min to 280 ℃ and keep the temperature for 2 min.
Preferably, SIM mode is selected, with M/z 305 (quantitation ion), 245, 202 for deuterated methyl testosterone D-MT, M/z 302 (quantitation ion), 124, 229 for methyl testosterone, M/z 230 (quantitation ion), 215, 135 for metabolite M2 as internal standard.
Sample pretreatment: the components in the meat matrix are complex and contain a large amount of water, protein and lipid substances, and if the components are not completely removed, the components are easy to pollute a chromatographic column and an ion source, and the accuracy of an experimental result is reduced. Therefore, the pretreatment operation of the sample is developed for removing various impurities in the sample and extracting all the substances to be detected, diatomite is used for absorbing water in muscle tissues through full grinding, lipid substances generally comprise simple fat free fatty acid, ester formed by free fatty acid and alcohol, and composite fat, and the lipid substances are fatty acid or fatty acid composition, so that the lipid substances in the sample have a long single-chain structure and weak polarity, and the low-polarity n-hexane which is also in the single-chain structure is used for dissolving the lipid substances in the n-hexane layer and removing the lipid substances. M2 is ultimately metabolized, primarily by the formation of a conjugate with glucuronic acid, and thus must be hydrolyzed by the addition of β -glucosinolate sulfatase; the free methyl testosterone after hydrolysis and M2 have high solubility in acetonitrile, and acetonitrile can be selected as an extracting agent. On the other hand, acetonitrile can cause protein denaturation by destroying hydrogen bonds between hydrophilic amino acid residue side chains, because the acetonitrile is provided with a hydrogen bond acceptor or a donor, the acetonitrile can form hydrogen bonds with the hydrogen bond acceptor and the donor on the hydrophilic amino acid side chains, further the hydrogen bonds between amino acids are influenced, the three-dimensional structure is destroyed, hydrophobic groups in the protein are exposed, precipitates are formed, and the precipitates are further removed by centrifugation.
Sample purification: although the sample extraction operation can remove most of the water, protein and lipid substances in the sample, the analysis requirements of GC-MS cannot be met, so that a solid phase extraction column is required to further remove impurities in the extraction liquid. Methyl testosterone, deuterated methyl testosterone and M2 have a steroid mother ring with a four-membered ring and are less polar. According to the separation principle of the reversed-phase chromatographic column, the component to be detected is easy to retain in the C18 solid-phase extraction column, and impurities with polarity stronger than that of the component to be detected are removed in the leaching process. NH (NH) 2 The solid phase extraction column combines polar groups in polar components in the extracting solution through hydrogen bonds, while the steroid compounds with weak polarity are generally difficult to combineAnd (5) reserving. Therefore, the strong polar impurities in the extracting solution can be further remained in the column and removed by using the amino column, so that the component to be detected flows out and is collected.
And (3) sample analysis: the present invention uses Gas chromatography-mass spectrometry (GC-MS) which has a large chromatographic resolution and in which small-molecule compounds eluted by a solvent front in a reversed-phase LC-MS are well retained.
Has the advantages that: compared with the prior art, the invention has the following advantages:
(1) experiments prove that M2 has longer metabolism time and retention time in vivo, MT and M2 are detected simultaneously, so that whether carnivorous animals use methyltestosterone can be more accurately analyzed and judged, M2 is used as another high-characteristic component for monitoring the use of methyltestosterone, and an analysis method for simultaneously monitoring the methyltestosterone and the metabolite M2 in animal-derived food has important significance and application prospect; the invention can simultaneously detect the methyltestosterone and the metabolite 17 alpha-methyl-5 beta-androstane-3 alpha, 17 beta-diol thereof, does not need to perform derivatization on two target substances, and has simple and rapid operation;
(2) the analysis method of the invention has good linear relation in the range of 50 ng/mL-500 ng/mL, S/N is 10 as the limit of quantification, and the limits of quantification of methyltestosterone and M2 are both 0.05 mg/kg; taking S/N-3 as a detection limit, the detection limit of methyltestosterone is 0.02mg/kg, and the detection limit of M2 is 0.015 mg/kg; the recovery rate range of the methyltestosterone is between 97.7 and 112.4 percent; the recovery rate range of M2 is between 96.6% and 116.6%, and the results show that the method has high sensitivity and good linear relation, the recovery rate and the precision can meet the requirements of actual detection, and the qualitative and quantitative results are accurate.
Drawings
FIG. 1 is an analytical mass spectrum of a sample according to the invention, M2: t R 11.11min, deuterated methyl testosterone t R 12.63min, methyl testosterone t R ＝12.66min；
FIG. 2 is a graph showing the standard working curve of methyltestosterone according to the present invention and the standard working curve of M2.
Detailed Description
The invention is further described below with reference to the accompanying drawings and specific embodiments.
Example 1
Investigation of solid phase extraction recovery:
preparing a mixed standard solution with the concentration of deuterated methyl testosterone being 50ng/mL, the concentration of methyl testosterone being 50ng/mL and the concentration of M2 being 50ng/mL as a standard solution, and carrying out GC-MS analysis; a10 mL centrifuge tube was charged with 3mL acetonitrile and 3mL acetonitrile-saturated n-hexane solution, centrifuged at 10000r/min at 4 ℃ for 5min, the lower layer solution was transferred to a 15mL screw cap polypropylene centrifuge tube using a 1mL syringe, and 10. mu.g/mL methyltestosterone solution, 10. mu.g/mL deuterated methyltestosterone solution, and 5. mu.L each of 10. mu.g/mL M2 solution were added to the centrifuge tube and mixed to prepare a sample solution. The sample solution was transferred to an activated C18 solid phase extraction column with a flow rate controlled to not exceed 3 mL/min. Leaching with 5mL of water, 5mL of acetone-water solution (1:4, v/v) and 5mL of n-hexane in sequence, draining the small column after the leaching solution completely passes through the small column, and treating the activated NH 2 The solid phase extraction column is connected below the C18 solid phase extraction column in series, 6mL acetonitrile is used for elution, and the flow rate is controlled to be not more than 3 mL/min. The eluate was collected in a 50mL eggplant-shaped flask, evaporated under reduced pressure with a rotary evaporator, and the solvent was removed. To the eggplant-shaped bottle from which the solvent was removed, 1mL of methanol was added, the residue was dissolved, and a small amount of anhydrous sodium sulfate was added, vortexed for 30s, and extracted with sonication for 1 min. Finally, the methanol solution was aspirated using a 1mL syringe, passed through a 0.22 μm organic microfiltration membrane, and awaited GC-MS analysis. The recovery rate of the solid phase extraction column is 99.4 percent, and the recovery rate is good.
Example 2
Linearity and range:
mixed standard solutions with the concentrations of deuterated methyl testosterone being 50ng/mL, methyl testosterone being 50, 100, 200, 400 and 500ng/mL respectively and M2 being 50, 100, 200, 400 and 500ng/mL respectively are prepared for GC-MS analysis. Drawing a standard working curve of the methyltestosterone by taking the ratio of the area of the methyltestosterone peak to the area of the deuterated methyltestosterone peak as a ordinate and the concentration of the methyltestosterone peak as an abscissa; and drawing a M2 standard working curve by taking the ratio of the M2 peak area to the deuterated methyl testosterone peak area as the ordinate and the M2 concentration as the abscissa. Through GC-MS analysis, M2 was detected first, the retention time was 11.11min, and the retention times of deuterated methyl testosterone and methyl testosterone were 12.63min and 12.66min, respectively. The concentration of the substance to be detected is used as an abscissa, and the ratio of the peak area of the substance to be detected to the peak area of the internal standard is used as an ordinate for plotting, and the linear relation between methyltestosterone and M2 is found to be good in a concentration range of 50ng/mL to 500ng/mL (as shown in the following figure), wherein the standard curve equation of methyltestosterone is that y is 0.0208x +0.2096, R is 0.9995, the standard curve equation of M2 is that y is 0.03x +0.6857, and R is 0.9997.
Example 3
And (3) standard recovery rate and precision:
preparing a mixed standard solution with the concentration of deuterated methyl testosterone being 50ng/mL, the concentration of methyl testosterone being 50ng/mL and the concentration of M2 being 50ng/mL as a standard solution, and carrying out GC-MS analysis under the following mass spectrum conditions: an Shimadzu SH-Rxi-5MS gas chromatographic column (30m multiplied by 0.25mm multiplied by 0.25 mu m) is used, the sample injection amount is 1 mu L, split-flow sample injection is not carried out, the sample injection port temperature is 280 ℃, the carrier gas is helium, the column flow is 1mL/min, the temperature raising program is 170 ℃, the retention time is 2min, the temperature is increased to 250 ℃ at 15 ℃/min, the temperature is increased to 280 ℃ at 5 ℃/min, the retention time is 2min, the mass spectrum temperature is 280 ℃, the EI source temperature is 230 ℃, and the SIM mode is selected. The internal standard D-MT has an M/z of 305 (quantification ion), 245 and 202, methyltestosterone has an M/z of 302 (quantification ion), 124 and 229, and metabolite M2 has an M/z of 230 (quantification ion), 215 and 135.
Approximately 1.00g of raw fish flesh was weighed, and 5. mu.L each of a 10. mu.g/mL methyl testosterone solution, a 10. mu.g/mL deuterated methyl testosterone solution, and a 10. mu.g/mL M2 solution was added to the raw fish flesh. After standing for 15min, approximately 1.0g of diatomaceous earth was added and ground thoroughly. After grinding, the samples were transferred to 10mL centrifuge tubes, 5mL acetonitrile and 60. mu.L beta-glucosinolsulfatase were added, vortexed and mixed for 30s, and incubated overnight at 37 ℃ with shaking. After incubation, the cells were removed, allowed to stand at room temperature, centrifuged at 4000 rpm for 5min at 4 ℃ and the supernatant collected in a clean 50mL eggplant-shaped flask. Adding 3mL acetonitrile into the centrifugation residue, mixing uniformly by vortex for 30s, performing ultrasonic extraction at room temperature for 5min, centrifuging at 4 ℃ and 10000r/min for 5min, collecting supernatant, adding 2mL acetonitrile into the centrifugation residue, repeating the above operation to collect supernatant, mixing the 3 times of extracting solutions, performing reduced pressure evaporation by using a rotary evaporator, and removing the solvent. Adding 1mL of acetonitrile into an eggplant-shaped bottle, dissolving residues, then adding 1mL of acetonitrile saturated n-hexane solution, carrying out vortex mixing for 30s, transferring the liquid in the bottle into a 10mL centrifuge tube, centrifuging for 5min at 4 ℃ at 10000r/min, transferring the lower layer solution into a 15mL spiral cover polypropylene centrifuge tube by using a 1mL syringe, adding 10mL of water, mixing uniformly, and purifying.
Transferring the sample extract to activated C18 solid phase extraction column, and controlling flow rate not to exceed 3 mL/min. And (3) leaching by using 5mL of water, 5mL of acetone-water solution and 5mL of n-hexane in sequence, draining the small column after the leaching solution completely passes through the small column, connecting the activated NH2 solid-phase extraction column below the C18 solid-phase extraction column in series, eluting by using 6mL of acetonitrile, and controlling the flow rate to be not more than 3 mL/min. The eluate was collected in a 50mL eggplant-shaped flask, evaporated under reduced pressure with a rotary evaporator, and the solvent was removed. To the eggplant-shaped bottle from which the solvent was removed, 1mL of methanol was added, the residue was dissolved, and a small amount of anhydrous sodium sulfate was added, vortexed for 30s, and extracted with sonication for 1 min. Finally, the methanol solution was aspirated through a 0.22 μm organic microfiltration membrane using a 1mL syringe.
The purified methanol extract was used for GC-MS analysis, with the following mass spectrometry conditions: an Shimadzu SH-Rxi-5MS gas chromatographic column (30m multiplied by 0.25mm multiplied by 0.25 mu m) is used, the sample injection amount is 1 mu L, split-flow sample injection is not carried out, the sample injection port temperature is 280 ℃, the carrier gas is helium, the column flow is 1mL/min, the temperature raising program is 170 ℃, the retention time is 2min, the temperature is increased to 250 ℃ at 15 ℃/min, the temperature is increased to 280 ℃ at 5 ℃/min, the retention time is 2min, the mass spectrum temperature is 280 ℃, the EI source temperature is 230 ℃, and the SIM mode is selected. The internal standard D-MT has an M/z of 305 (quantification ion), 245 and 202, methyltestosterone has an M/z of 302 (quantification ion), 124 and 229, and metabolite M2 has an M/z of 230 (quantification ion), 215 and 135. The recovery rates were calculated from the peak areas, respectively. The recovery rate of the methyl testosterone of 6 standard samples prepared in parallel is between 97.7% and 112.4%, and the RSD is 5.2%; the recovery of M2 ranged between 96.6% and 116.6%, with RSD 6.8%. The results show that the recovery and precision of methyltestosterone and M2 are good at a spiking level of 0.05 mg/kg.
Example 4
Detection limit and quantification limit:
taking 10 times of signal-to-noise ratio (S/N) as a quantitative limit, and obtaining that the quantitative limits of the methyltestosterone and the M2 are both 0.05mg/kg through experiments; the detection limit of methyl testosterone is 0.02mg/kg and that of M2 is 0.015mg/kg by taking 3 times of S/N as the detection limit.
Claims (7)
1. An assay for the detection of methyl testosterone and its metabolites in animal muscle tissue wherein said metabolites of methyl testosterone are 17 α -methyl-5 β -androstane-3 α,17 β -diol, said assay comprising the steps of:
(1) sample pretreatment: taking an animal muscle sample, removing water, lipid and protein, and extracting a component to be detected to obtain a sample extracting solution;
(2) sample purification: taking a sample of the extract, and performing solid phase extraction on the sample by using a C18 solid phase extraction column and NH 2 Eluting the solid phase extraction column, removing impurities and preparing a methanol extracting solution of the sample;
(3) and (3) sample analysis: analyzing and detecting the methanol extract obtained in the step (2) by using gas chromatography-tandem quadrupole mass spectrometry, wherein S/N is 10 as a quantitative limit, and the quantitative limits of methyltestosterone and M2 are both 0.05 mg/kg; the detection limit of methyl testosterone is 0.02mg/kg, and the detection limit of M2 is 0.015mg/kg, with S/N being 3.
2. The analysis method as claimed in claim 1, wherein the step (1) is specifically that an animal muscle sample is taken, an internal standard deuterated methyl testosterone is added, water is removed by diatomite, lipid impurities are removed by n-hexane, protein is removed by beta-glucoside sulfatase as hydrolase, and acetonitrile is used as an extraction solvent for extracting the component to be detected.
3. The assay of claim 2 wherein the internal standard deuterated methyl testosterone is a methanol solution of deuterated methyl testosterone.
4. The method of claim 1, wherein step (2) comprises taking the sample of the extract solution to a C18 solid phase extractionTaking a column, and leaching the column by adopting water, an acetone-water solution and normal hexane in sequence; NH connected in series 2 Solid phase extraction column, adopting acetonitrile to elute; removing the solvent by rotary evaporation, and adding methanol to prepare a methanol extracting solution of the sample.
5. The analytical method as defined in claim 1, wherein the step (3) is performed by using Shimadzu SH-Rxi-5MS gas chromatography column, the sample injection amount is 0.5-2 μ L, no split injection is performed, the injection inlet temperature is 200-320 ℃, the carrier gas is helium, the column flow rate is 0.7-1.5 mL/min, the mass spectrum quadrupole temperature is 250-350 ℃, and the EI source temperature is 200-280 ℃.
6. The analytical method according to claim 5, wherein the temperature raising procedure is performed by holding at 100 to 200 ℃ for 1 to 5min, raising the temperature at 5 to 25 ℃/min to 250 to 280 ℃, raising the temperature at 5 to 15 ℃/min to 280 to 330 ℃, and holding for 1 to 10 min.
7. The assay of claim 5 wherein the SIM model is selected such that the m/z of deuterated methyl testosterone is 305, 245, 202, the m/z of methyl testosterone is 302, 124, 229, 17 α -methyl-5 β -androstane-3 α,17 β -diol is 230, 215, 135.
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