CN117990842A - Mass spectrum detection method for multiple drugs in urine through solid phase extraction - Google Patents
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Abstract
The invention discloses a mass spectrum detection method for multiple drugs in solid phase extraction urine, which comprises the following steps: s1, preparing calibration solutions with different concentrations; s2, adding an internal standard stock solution into the calibration solution, then performing solid phase extraction, and performing mass spectrometry analysis to perform linear regression on the added standard concentration by using the chromatographic peak area ratio of the drug analyte and the internal standard deuterated quantitative ion to construct a standard curve; s3, taking urine to be detected, and adding an internal standard stock solution to obtain detected urine; s4, performing solid-phase extraction on the detection urine, and performing mass spectrometry analysis to obtain the chromatographic peak area of the drug analyte quantitative ion and the chromatographic peak area of the internal standard deuterated quantitative ion; s5, comparing the ratio of the chromatographic peak area of the drug analyte quantitative ion to the chromatographic peak area of the internal standard deuterated quantitative ion with a standard curve, and calculating the concentration of various drugs in the urine sample. The invention has the characteristics of rapid screening and high detection accuracy and reliability.
Description
Technical Field
The invention relates to a detection method of drugs, in particular to a mass spectrum detection method of multiple drugs in urine by solid phase extraction.
Background
Drug abuse has been a serious social problem, not only causing irreversible health damage to drug abusers, but also causing a number of serious social problems. In the process of striking drug crimes, not only is perfect legal support needed, but also the requirement for on-site rapid detection of drugs is increasingly increased due to the endless new drugs and various drugs under pseudo-loading, and the detection means of drugs and rapid screening technology of drug absorbing staff are required.
However, screening of drug-taking personnel is more difficult than drug constant detection due to the problems of large detection sample size, multiple unknown drug types, complex biological matrix, strong timeliness and the like. Therefore, the establishment of the on-site rapid identification and quantification technology of trace drugs in biological fluids (urine, blood and saliva) is not only an important technical support in the work of detoxification, but also an important basis for checking drug-taking personnel as a screening result of the drug-taking personnel.
The existing drug detection method mainly adopts methods such as chemical analysis, gas chromatography, high performance liquid chromatography, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry and the like to detect trace drugs in biological fluid.
For example: the application number is CN201110075177.4, the liquid chromatography-tandem mass spectrometry detection method of 33 drugs in urine with publication date 2011.09.28 discloses the following technical scheme: extracting and purifying a sample by adopting a liquid-liquid extraction method, and adding sodium hydroxide solution to realize protein denaturation and pH value adjustment, and extracting by adopting ethyl acetate liquid-liquid extraction to obtain a sample to be detected; measuring a sample to be measured by using a liquid chromatograph-tandem quadrupole mass spectrometer; except barbiturates were analyzed using negative ion patterns, the remaining compounds were analyzed using positive ion patterns; the residual quantity of each drug in the sample is calculated by taking the relative retention time of each drug and the abundance ratio of the monitoring ion pair of each drug as qualitative basis and taking the peak area or peak height of each drug as the basis. The method can analyze 33 drugs at one time, has strong pertinence, simple operation, high detection speed and wide detection range, reduces the test cost and is easy to popularize and apply.
However, the detection method has complex and complicated pretreatment steps of the sample, and the sample needs to be transported to a laboratory for detection, so that the requirement of on-site rapid screening cannot be met; moreover, the ethyl acetate liquid-liquid extraction is adopted, so that the removal effect of impurities in urine is poor, and the impurities in urine can interfere with the detection accuracy and reliability.
Disclosure of Invention
The invention aims to provide a mass spectrum detection method for extracting various drugs in urine through a solid phase. The invention has the characteristics of rapid screening and high detection accuracy and reliability.
The technical scheme of the invention is as follows: a mass spectrum detection method for solid phase extraction of multiple drugs in urine comprises the following steps:
s1, preparing a standard solution:
a. preparing a drug stock solution and an internal standard stock solution respectively by using methanol;
b. respectively adding drug stock solutions with corresponding volumes into blank urine to obtain labeled urine with different labeling concentrations, and taking the labeled urine as a calibration solution;
S2, constructing a standard curve:
Respectively adding internal standard stock solutions with the same concentration into calibration solutions with different standard adding concentrations to obtain standard urine, performing solid-phase extraction on the standard urine, detecting by adopting a mass spectrometry, and performing linear regression on the standard adding concentrations to construct a standard curve according to the ratio of the chromatographic peak area of the quantitative ion of the drug analyte to the chromatographic peak area of the quantitative ion of the internal standard deuteration calculated by the signal intensity of the continuous ten seconds of spectrogram;
S3, sample processing:
taking urine to be detected, and adding an internal standard stock solution to obtain detected urine;
S4, sample extraction detection:
Performing solid-phase extraction on the detection urine, and detecting by adopting a mass spectrometry method to obtain the chromatographic peak area of drug analyte quantitative ions calculated by the signal intensity of a continuous ten-second spectrogram and the chromatographic peak area of corresponding internal standard deuterated quantitative ions;
S5, quantitative analysis of samples:
Comparing the ratio of the chromatographic peak area of the drug analyte quantitative ion to the chromatographic peak area of the internal standard deuterated quantitative ion with a standard curve, and calculating and detecting the concentration of various drugs in urine.
In the mass spectrometry detection method for the multiple drugs in the solid-phase extraction urine, the drug stock solution is a mixed stock solution containing 6-acetylmorphine, methamphetamine, 3, 4-methylenedioxymethamphetamine, ketamine, norketamine and cocaine.
In the mass spectrometry analysis of the step S4, the method for detecting the mass spectrum of the plurality of drugs in the urine by solid phase extraction comprises the steps of determining that the quantitative ion of 6-acetylmorphine is m/z 211, the quantitative ion of methamphetamine is m/z 119,3,4-methylenedioxymethamphetamine is m/z 163, the quantitative ion of ketamine is m/z 220, the quantitative ion of norketamine is m/z 207, and the quantitative ion of cocaine is m/z 182.
In the aforementioned mass spectrometry detection method for solid-phase extraction of various drugs in urine, the solid-phase extraction in steps S2 and S4 uses a pipette gun for solid-phase extraction of the filler.
In the above method for detecting mass spectra of various drugs in solid phase extraction urine, the extraction method of the liquid-transfering gun of the solid phase extraction filler comprises the following steps: extracting methanol to moisten and activate the gun head, then balancing with ultrapure water, immersing the gun head into a urine sample, and repeatedly sucking and discharging for a plurality of times; then flushing with ultrapure water; and repeatedly eluting with methanol with the same volume as the eluent, centrifuging the final eluent, and taking the supernatant for detection.
In the above method for detecting mass spectra of various drugs in solid phase extraction urine, the extraction method of the liquid-transfering gun of the solid phase extraction filler comprises the following steps: extracting methanol by a liquid-transferring gun for solid-phase extraction of filling materials to moisten and activate a gun head, then balancing by ultrapure water, immersing the gun head in a urine sample, and repeatedly sucking and discharging for at least 9 times, wherein the time for sucking/discharging each time is 8-12 s; then flushing with ultrapure water; and (3) repeatedly eluting for 2-5 times by using methanol with the same volume as an eluent, wherein the eluting time is 8-12 s each time, centrifuging the final eluent at a speed of 5-1 w for 20-40 s, and taking supernatant for detection.
In the aforementioned mass spectrometry detection method for solid-phase extraction of various drugs in urine, the mass spectrometry in step S4 employs a miniature mass spectrometer.
In the above method for detecting mass spectrum of various drugs in urine by solid phase extraction, in the mass spectrometry in step S4, the sample injection is performed by paper spray ionization sample injection or nano-electrospray ionization sample injection.
In the mass spectrometry detection method for the multiple drugs in the solid-phase extraction urine, when nano-electrospray ionization sample injection is adopted, the spray voltage of 6-acetylmorphine is 1400V, the spray voltage of methamphetamine is 1400V, the spray voltage of 3, 4-methylenedioxymethamphetamine is 1200V, the spray voltage of ketamine is 1200V, the spray voltage of norketamine is 1400V, and the spray voltage of cocaine is 1400V.
In the mass spectrometry detection method for the multiple drugs in the urine through solid phase extraction, when nano-electrospray ionization sample injection is adopted, the energy of ISO1 of 6-acetylmorphine is 8V, the energy of ISO1 of methamphetamine is 6.5V, the energy of ISO1 of 3, 4-methylenedioxymethamphetamine is 7V, the energy of ISO1 of ketamine is 8V, the energy of ISO1 of norketamine is 10V, and the energy of ISO1 of cocaine is 8V; the energy of ISO2 of 6-acetylmorphine is 4V, the energy of ISO2 of methamphetamine is 2.5V, the energy of ISO2 of 3, 4-methylenedioxymethamphetamine is 3V, the energy of ISO2 of ketamine is 3.5V, the energy of ISO2 of norketamine is 3.5V, the energy of ISO2 of cocaine is 4V; the energy of the CID of 6-acetylmorphine was 2V, the energy of the CID of methamphetamine was 1V, the energy of the CID of 3, 4-methylenedioxymethamphetamine was 3.75V, the energy of the CID of ketamine was 1.75V, the energy of the CID of norketamine was 2.25V, and the energy of the CID of cocaine was 2V.
Compared with the prior art, the invention has the beneficial effects that:
According to the invention, by combining a solid phase extraction technology, a mass spectrometry technology and a nano-electrospray technology, a detection sample is subjected to simple and rapid pretreatment by the solid phase extraction technology, the interference of impurities in urine is removed, and the signal value of a target object is greatly improved; by optimizing the extraction and elution conditions in the solid phase extraction process and detection parameters in mass spectrometry detection, the method is simple and energy-saving in operation, and can detect multiple drugs such as 6-acetylmorphine, methamphetamine, 3, 4-methylenedioxymethamphetamine, ketamine, norketamine, cocaine and the like in urine in 5 minutes at one time, thereby realizing rapid and continuous detection screening of multiple drugs and quantitative concentration detection of drugs, and improving detection efficiency, detection accuracy, reliability and stability.
Proved by verification, the LOD of the method on drugs in urine is more than or equal to 1ng mL -1,LOD≥1ng·mL-1, each drug is in the linear range of 1 to 50ng mL -1, and the linear relationship is good (r 2 is more than or equal to 0.99).
Therefore, compared with the traditional mass spectrometry method, the method has the characteristics of simplicity in operation, high sensitivity and strong reliability, is quicker and more convenient in detection and lower in power consumption, realizes the on-site quick detection of drugs in urine of drug absorbing personnel, and plays an important role in drug crime on-site investigation.
Drawings
FIG. 1 is an MS/MS spectrum of 6 drugs in a urine matrix;
FIG. 2 is a chromatogram of methamphetamine at different concentrations in urine;
FIG. 3a is a graph of sample injection contrast signals of 25ng mL -1 methamphetamine urine samples of comparative example 1 and comparative example 2, without solid phase extraction, in nano electrospray ionization and paper spray ionization modes, respectively;
FIG. 3b is a graph of sample injection contrast signals of the solid phase extraction treated 25ng mL -1 methamphetamine urine samples of example 1 and example 2 in nanoelectrospray ionization and paper spray ionization modes, respectively.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to be limiting.
Example 1:
detecting drugs: 6-acetylmorphine, methamphetamine, 3, 4-methylenedioxymethamphetamine, ketamine, norketamine, cocaine;
Internal standard solution: 6-Acetylmorphine-D3, methamphetamine-D5, 3, 4-methylenedioxymethamphetamine-D5, ketamine-D4, norketamine-D4 and cocaine-D3.
The materials used are as follows: chromatographic grade methanol; high purity water (resistivity 18.2mΩ·cm); the paper spray kit is purchased from a science and technology company and is prepared from chromatographic paper, a capillary tube, a plastic clip and a cover; the nanometer electrospray kit is purchased in the clear spectrum technology and comprises a glass spray needle, an external electrode, an internal electrode and a plastic shell; ai Bende GELoader gun heads; blank urine was provided by 10 adult volunteers, who were well-conditioned, were free of history of drug intake and abnormal laboratory test results, and were mixed and stored in a 4 ℃ freezer.
The instrument used is as follows: the portable miniature mass spectrometer is used as an analysis instrument to realize on-site rapid detection screening, such as Mini pi miniature mass spectrometer. The miniature mass spectrometer weighs about 10Kg, and has the length of 40 times the width of 27 times the height of 18cm and the working power of less than 100W. A discontinuous atmospheric pressure sample inlet (DAPI) is used for ion introduction, a Linear Ion Trap (LIT) is used as a mass analyzer, and a vortex pump and a turbomolecular pump are matched to form a vacuum system. The detection can be performed at an ambient temperature of 10 to 30 ℃ and a humidity of less than 85%. The miniature mass spectrometer can use full scanning of m/z 50 to 1000 in positive and negative ion modes (MS 1、MS2、MS3、MS4 and MS 5), with a resolution of less than 0.5Da. Upon analysis of the sample, ions generated by ambient ionization enter the linear ion trap through DAPI and are trapped in the linear ion trap. Instrument control and data acquisition was performed using PMS ClientPro software (clear spectrum technology).
A mass spectrum detection method for solid phase extraction of multiple drugs in urine comprises the following steps:
s1, preparing a standard solution:
Methanol is used to prepare a plurality of drug stock solutions containing 6-acetylmorphine, methamphetamine, 3, 4-methylenedioxymethamphetamine, ketamine, norketamine and cocaine with concentration of 5 mg.mL -1 and an internal standard stock solution with concentration of 5 mg.mL -1 respectively, and the drug stock solution and the internal standard stock solution are stored in an environment of-20 ℃.
The internal standard stock solution is a mixed internal standard stock solution prepared by internal standard substances corresponding to various drugs in the mixed stock solution of the drugs, namely the mixed internal standard stock solution prepared by 6 internal standard substances corresponding to the 6 drugs.
The corresponding volumes of the multiple drug stock solutions were added to the blank urine, respectively, to obtain 7 labeled urine with labeled concentration levels (concentration 0.25ng·mL-1、0.5ng·mL-1、1ng·mL-1、5ng·mL-1、10ng·mL-1、25ng·mL-1 and 50ng·ml -1, respectively) as calibration solutions.
Urine standards containing 2 ng-mL -1、15ng·mL-1 and 40 ng-mL -1 of multiple drugs were prepared in the same manner as the calibration solution and used as quality control samples for method validation. Calibration solutions may also be employed as quality control samples for method validation.
S2, constructing a standard curve:
and respectively adding internal standard stock solutions with internal standard concentration of 25 ng.mL -1 into standard solutions with different standard concentrations to obtain standard urine with different standard concentrations, performing solid-phase extraction on the standard urine according to the solid-phase extraction method in the step S4, detecting the standard urine with different concentrations containing 6 drug analytes and internal standard deuterated by using mass spectrometry, drawing chromatographic peak areas of the chromatogram according to signal intensities of a spectrogram with continuous ten seconds of drug analyte quantitative ions and drawing chromatographic peak area ratio of the chromatogram according to signal intensities of a spectrogram with continuous ten seconds of internal standard deuterated quantitative ions, and performing linear regression on the standard concentrations to construct a standard curve.
S3, sample processing:
Taking 0.995mL of urine to be detected, adding 5 mu L of internal standard stock solution, and swirling for 2 minutes to obtain detection urine with the internal standard concentration of 25 ng.mL -1;
S4, sample extraction detection:
Performing solid-phase extraction on the detection urine by using a pipette tip of a solid-phase extraction filler, extracting various drug analytes and internal standard deuteration thereof from the urine, and performing sample injection analysis and detection on the extracted samples by adopting a mass spectrometry; drawing a chromatogram with the signal intensity of the obtained spectrogram of each drug analyte quantitative ion for ten seconds continuously, and calculating the peak area of the drug analyte chromatogram as the signal intensity of the drug analyte; and (3) drawing a chromatogram by using the signal intensity of the spectrogram of the internal standard deuterated quantitative ion for ten seconds, and calculating the peak area of the internal standard deuterated chromatogram to be used as the internal standard deuterated signal intensity.
The liquid-transferring gun of the solid-phase extraction filler is a C18 solid-phase extraction column liquid-transferring gun, an HLB solid-phase extraction column liquid-transferring gun or an MCX solid-phase extraction column liquid-transferring gun, the solid-phase extraction is adopted, the impurity removing effect in urine is more obvious, the interference of urine matrixes in the extracted solution is greatly reduced, and the detection performance is improved.
In this embodiment, the solid phase extraction is performed by using a C18 pipette tip, and the solid phase extraction process specifically includes:
The C18 pipette tip was attached to a pipette, and then 50. Mu.L of methanol was extracted to wet and activate the tip, followed by equilibration with 300. Mu.L of ultrapure water. Immersing the gun head into 1mL of urine sample, and repeating sucking and discharging for 9 times, wherein the time for sucking/discharging each time is 10s; flushing the loaded pipette tip with 300 mu L of ultrapure water; repeatedly eluting drugs in urine extracted for 3 times by using methanol with the same volume as eluent, wherein each eluting time is 10s, centrifuging the final eluent by using a palm centrifuge at 8000 revolutions for 30s, and taking supernatant for detection, wherein the total time is 3min. The operation makes the signal of the drug in the subsequent detection process stronger, and can ensure the recovery rate of the target compound.
Wherein, sample introduction is: paper spray ionization sample injection is adopted. Paper spray ionization sampling is less susceptible to ion inhibition by the substrate and can be used for sample detection of complex substrates.
The specific operation is as follows: and (3) sucking 10 mu L of the extracted sample by using a pipette, adding the extracted sample into a sample inlet at the front part of a paper spray kit of a miniature mass spectrometer, waiting for about 2 minutes to dry the sample, and adding 100 mu L of methanol to the rear end of the paper spray kit to perform on-machine detection. Under the action of 4000V high pressure, the sample, which was primarily separated on paper chromatography, was ejected along the capillary and ionized at the capillary tip.
S5, quantitative analysis of samples:
comparing the ratio of the chromatographic peak area of the drug analyte quantitative ion to the chromatographic peak area of the internal standard deuterated quantitative ion with a standard curve, and calculating and detecting the concentration of 6 drugs in urine.
Peak areas of chromatographic bees for drug analyte and deuterated analyte ions were calculated using Microsoft Excel 2019 and analyzed for mean, standard deviation, and significance levels.
In the quantitative analysis process, the intensity calculation is to draw a color spectrogram by taking time as an abscissa and the signal intensity displayed by an instrument as an ordinate, intercepting the response combination time of target ions in ten continuous seconds in an experiment, and calculating the peak area of a chromatographic peak.
For detecting urine with different concentrations, the intensity ratio of chromatographic peak areas of continuous ten-second quantitative ions of drug analytes and internal standard deuteration in the standard urine is used for constructing a standard curve, so that the method is more scientific, and the quantitative detection is more stable and accurate.
Example 2:
Example 2 is substantially identical to example 1 except that:
Sample introduction is as follows: nanometer-electrospray ionization sample injection is adopted. Has the characteristics of high sensitivity and stability, and is more energy-saving. And (3) extracting 25 mu L of the extracted sample by using a 20 mu L pipette matched with a Ai Bende GELoader gun head, adding the extracted sample into a glass tube, and combining the glass tube with a nano-electrospray kit to obtain the detection on-machine. The built-in metal wire in the glass tube spray needle pushes the sample to spray out of the tip of the glass tube and is ionized under the action of spray voltage.
The calibration solution was subjected to mass spectrometry analysis in the manner of example 2 to obtain a MS 2 chart of 6 drugs, as shown in FIG. 1.
In FIG. 1, the concentration of 6-acetylmorphine was 100 ng.mL -1; the concentrations of the methamphetamine and the 3, 4-methylenedioxymethamphetamine are 50 ng.mL -1; the concentration of the norketamine is 10 ng.mL -1; the concentrations of ketamine and cocaine were 10 ng.mL -1.
MS 2 of 6-Acetylmorphine As shown in FIG. 1f, the parent ion m/z 328 of 6-Acetylmorphine is fragmented to form child ions m/z 268, m/z211 and m/z 193. The 6-acetylmorphine parent ion [ M+H ] + loses one acetic acid group to form a child ion M/z 268, further removes the H 3C-CH2＝N-CH3 group to form a child ion M/z211, and further removes one water molecule to form a child ion M/z 193. In addition, during the experiment, an impurity peak m/z 269 was observed, which was very close to the daughter ion m/z 268, which resulted in some interference with the analysis of 6-acetylmorphine, and thus m/z211 was chosen as the quantitative ion during the subsequent detection.
As shown in FIG. 1a, the MS 2 of methamphetamine forms a parent ion at m/z 150 after ionization, and several fragment ion peaks are generated after fragmentation, and responses are clearly observed at m/z 121, m/z119 and m/z 91, and also at m/z 94, m/z 133 and m/z 135. Wherein the fragment ion M/z119 is formed by removing one methylamine from methamphetamine parent ion [ M+H ] +, and the ion M/z 91 is methamphetamine ion generated by beta-C-C cleavage. Other peaks presented on the mass spectrum are suspected impurity peaks, which may result from the formation of fragmented impurities in urine having a mass number of m/z 150.
MS 2 diagram of 3, 4-methylenedioxymethamphetamine as shown in FIG. 1b, ion peaks (m/z 194) disintegrated to produce fragments m/z 163, m/z 135, m/z 133, m/z 120 and m/z161 plasma peaks. The ion M/z 163, M/z 133 and M/z 135 are obtained by fragmentation of 3, 4-methylenedioxymethamphetamine by a fragmentation pathway analysis, wherein M/z 163 is formed by removing one methylamine from the 3, 4-methylenedioxymethamphetamine parent ion [ M+H ] +; m/z 135 is due to beta-C-C cleavage of [ M+H ] +; the formation of m/z 133 can be attributed to the ring opening reaction of the product ion methylenedioxy ring of m/z 163 and loss of aH 2 c=o. The product ions m/z 120 and m/z161 are then presumed to be interfering ions.
As shown in FIG. 1d, the MS 2 of ketamine shows fewer interference peaks, except for the ion peaks m/z 220, m/z 207, and m/z 179 generated by the fragmentation of ketamine parent ion m/z 238, there are only two impurity ion peaks m/z 223 and m/z 182, respectively, and their responses are lower than the target ion. The sub ion M/z 220 is formed by removing one water molecule from ketamine parent ion [ M+H ] +; the sub-ion M/z 207 is formed by removing one methylamine from ketamine parent ion [ M+H ] +, and the sub-ion M/z 179 is generated by further removing one CO group from the sub-ion M/z 207.
As shown in FIG. 1c, MS 2 of norketamine showed a lot of impurity ions, except for the peaks at m/z 207, m/z 179 and m/z 125, which are generated by fragmentation of norketamine parent ion (m/z 224), the responses at m/z195, m/z 192, m/z 165, m/z 156 and m/z 128 may all be interferent signals, but these interferent signals are far from the fragment ion and do not affect the detection of the target compound. The M/z 207 ion is formed by removing one NH 2 group from norketamine parent ion [ M+H ] +, removing one CO group to form a child ion M/z 179, and removing C 4H6 group to form a child ion M/z 125.
The MS 2 plot of cocaine is shown in FIG. 1e, where cocaine is fragmented at m/z 304 to form an ion peak at m/z182, while other peaks presented in the mass spectrum are presumed to be impurity ions, including m/z211, m/z 245 and m/z 286. The daughter ion M/z182 is generated by the loss of one benzoic acid group from the neutrality of the cocaine parent ion [ M+H ] +.
Based on the analysis, the target ions in the 6 drugs after the gun head solid phase extraction treatment can be easily identified in the MS 2 graph, and the interference of the impurity ions on the target ions is small, so that the feasibility of the method for analyzing the 6 drugs in urine is proved.
The fragment ion with the strongest response of each drug is taken as the quantitative ion of the experiment, and the quantitative ions of different drugs are shown in table 1.
The spray voltage is one of important factors influencing the spray performance, and is too low to push a sample to generate spray at the tip of the glass tube spray needle, and too high to influence the stability of the spray, and the possibility of damaging the tip of the glass tube spray needle is also provided. When nanometer-electrospray ionization sample injection is performed, a plurality of sample injection sequences are arranged, different voltages are arranged in each sequence, and the optimal spray voltages of different drugs are shown in table 1.
In mass spectrometry, isolation selective coupling (ISO) energy directly relates to the influence of impurities on ionization of a target compound, the ISO energy is too small to cause that impurity ions cannot be completely separated, and in the subsequent detection process of an MS 2 scanning mode, the target ions (M+H) + and the impurity ions are co-fragmented, so that qualitative and quantitative detection results are interfered; too high ISO energies, some of the target ions will also be separated out, negatively affecting sensitivity, resolution and stability. Wherein the ISO1 energy can isolate impurity ions that affect the protonation of the target ion ([ m+h ] +), thereby greatly reducing the likelihood of fragmentation of the impurity ions. When nanometer-electrospray ionization sampling is performed, a plurality of sampling sequences are arranged, and different energies are arranged in each sequence. The best ISO1 and ISO2 for different drugs are shown in Table 1.
In mass spectrometry, collision Induced Dissociation (CID) is the most critical factor in fragment ion generation and affecting signal intensity during MS 2 scanning. The collision energy is too low, the parent ions cannot be completely fragmented, and the obtained qualitative and quantitative signals of the fragment ions are not strong, so that the identification is not facilitated; fragment ions with too high collision energy are further fragmented, so that the signal intensity of the fragment ions is reduced, and the targeting identification is also not facilitated. The spectrum diagrams with different proportions of parent ions and child ions can be obtained by optimizing and adjusting CID energy, and the spectrum diagrams with proper and stable proportion not only have more advantages when qualitatively screening substances with low concentration, but also can be more accurately quantified. When nanometer-electrospray ionization sample injection is performed, a plurality of sample injection sequences are arranged, different energies are arranged in each sequence, and the optimal CID of different drugs is shown in table 1.
Table 1 mass spectrum detection parameter table for different drugs
For the target detection of trace drugs, isolation selective coupling (ISO) energy is required to isolate other interference ions except for ions with target mass-to-charge ratios, and the qualitative and quantitative detection of trace target compounds is realized by matching with an MS 2 scanning mode.
The concentration is an important factor affecting the area of chromatographic peaks, and the signal intensity of chromatograms of drugs with different concentrations is different, and taking methamphetamine as an example, the chromatograms of calibration solutions with different concentrations are shown in figure 2.
As can be seen from fig. 2, the area of the chromatographic peak increases with increasing concentration of the addition target and has a substantially linear trend. The samples of each concentration were repeated 6 times for analysis (RSD < 15%), and the linear regression of the calculated chromatographic peak area average, the target ion chromatographic peak area to internal standard chromatographic peak ratio and concentration showed that 6 drugs had good linear relationship (R 2 > 0.99) within the respective linear range, and the quantitative performance of the gun head solid phase extraction and nano electrospray-micro mass spectrometer on 6 drugs in urine was good, and specific values are detailed in table 2.
Linear, limit of detection and limit of quantification of 26 drugs
The sensitivity of the method is calculated according to the signal-to-noise ratio of LOD being more than or equal to 3 and the signal-to-noise ratio of LOQ being more than or equal to 10.
As can be seen from Table 2, the detection limits of methamphetamine, 3, 4-methylenedioxymethamphetamine, norketamine, ketamine, cocaine, and 6-acetylmorphine were 1, 0.5, 0.25, 1ng mL -1, and the quantitative Limits (LOQ) were 5, 10, 5, 1, and 10ng mL -1, respectively. The results show that the gun head solid phase extraction and the nano electrospray-micro mass spectrometer have high detection sensitivity on 6 drugs in urine.
Mass control testing:
Before urine sample detection, taking a quality control sample for sample injection, comparing the actual concentration with the detection concentration, ensuring that the comparison deviation is not more than 15%, and if the comparison deviation is more than 15%, checking a detection method, instrument performance, the quality control sample and the like. Recovery rates of gun head solid phase extraction and nano electrospray-micro mass spectrometry were tested with quality control samples with labeling concentrations of 2, 15 and 40 ng-mL -1, each sample was sampled 6 times, and the results are shown in table 3.
Table 36 shows stability and recovery rate of drugs
Therefore, the recovery rate of 6 drugs in the respective quality control analysis is good, the whole is kept between 85% and 105%, the stability is good, and the RSD is less than 10%.
In summary, the method has simple operation, high sensitivity and quantitative capability meeting the requirements, and can realize the on-site rapid detection of 6 drugs in urine.
Comparative example 1:
Comparative example 1 is substantially identical to example 1 except that: the urine sample is not subjected to solid phase extraction.
Comparative example 2:
comparative example 2 is substantially identical to example 2 except that: the urine sample is not subjected to solid phase extraction.
The urine samples of example 1, example 2, comparative example 1 and comparative example 2 were subjected to mass spectrometry in nano electrospray ionization and paper spray ionization modes, respectively, and the signal values of the detected targets were compared. The detection results are shown in FIG. 3, FIG. 3a is a sample injection contrast signal diagram of the urine sample of 25 ng.mL -1 methamphetamine which is not subjected to the solid phase extraction treatment in comparative example 1 and comparative example 2 in the nanometer electrospray ionization and paper spray ionization modes respectively, FIG. 3b is a sample injection contrast signal diagram of the urine sample of 25 ng.mL -1 methamphetamine which is subjected to the solid phase extraction treatment in example 1 and example 2 in the nanometer electrospray ionization and paper spray ionization modes respectively, as can be seen by comparison, after the methamphetamine is subjected to solid phase extraction, the overall signal intensity is increased, the signal intensity of the sample applied by a paper spray ionization mode is increased from 1.2e5 to 3e5, and the signal intensity of the sample applied by a nano-electrospray ionization mode is increased from 2e4 to 6e5. Therefore, after the urine sample is subjected to solid-phase extraction treatment by the gun head, the detection performance of paper spray is improved by about 3 times, and the detection performance of nano-electrospray is improved by about 30 times, so that the highest detection performance can be achieved.
Claims (10)
1. A mass spectrum detection method for solid phase extraction of multiple drugs in urine is characterized in that: the method comprises the following steps:
s1, preparing a standard solution:
a. preparing a drug stock solution and an internal standard stock solution respectively by using methanol;
b. respectively adding drug stock solutions with corresponding volumes into blank urine to obtain labeled urine with different labeling concentrations, and taking the labeled urine as a calibration solution;
S2, constructing a standard curve:
Respectively adding internal standard stock solutions with the same concentration into calibration solutions with different standard adding concentrations to obtain standard urine, performing solid-phase extraction on the standard urine, detecting by adopting a mass spectrometry, and performing linear regression on the standard adding concentrations to construct a standard curve according to the ratio of the chromatographic peak area of the quantitative ion of the drug analyte to the chromatographic peak area of the quantitative ion of the internal standard deuteration calculated by the signal intensity of the continuous ten seconds of spectrogram;
S3, sample processing:
taking urine to be detected, and adding an internal standard stock solution to obtain detected urine;
S4, sample extraction detection:
Performing solid-phase extraction on the detection urine, and detecting by adopting a mass spectrometry method to obtain the chromatographic peak area of drug analyte quantitative ions calculated by the signal intensity of a continuous ten-second spectrogram and the chromatographic peak area of corresponding internal standard deuterated quantitative ions;
S5, quantitative analysis of samples:
Comparing the ratio of the chromatographic peak area of the drug analyte quantitative ion to the chromatographic peak area of the internal standard deuterated quantitative ion with a standard curve, and calculating and detecting the concentration of various drugs in urine.
2. The method for mass spectrometry detection of multiple drugs in solid phase extracted urine according to claim 1, wherein the method comprises the following steps: the drug stock solution is a mixed stock solution containing 6-acetylmorphine, methamphetamine, 3, 4-methylenedioxymethamphetamine, ketamine, norketamine and cocaine.
3. The method for mass spectrometry detection of multiple drugs in solid phase extracted urine according to claim 2, wherein the method comprises the following steps: in the mass spectrometry analysis of the step S4, the quantitative ion of 6-acetylmorphine is m/z 211, the quantitative ion of methamphetamine is m/z 119,3,4-methylenedioxymethamphetamine is m/z 163, the quantitative ion of ketamine is m/z 220, the quantitative ion of norketamine is m/z 207, and the quantitative ion of cocaine is m/z 182.
4. The method for mass spectrometry detection of multiple drugs in solid phase extracted urine according to claim 1, wherein the method comprises the following steps: the solid phase extraction in steps S2 and S4 both use a pipette gun for solid phase extraction of the packing.
5. The method for mass spectrometry detection of multiple drugs in solid phase extracted urine according to claim 4, wherein the method comprises the following steps: the extraction method of the liquid-transfering gun for solid-phase extraction of the filler comprises the following steps: extracting methanol to moisten and activate the gun head, then balancing with ultrapure water, immersing the gun head into a urine sample, and repeatedly sucking and discharging for a plurality of times; then flushing with ultrapure water; and repeatedly eluting with methanol with the same volume as the eluent, centrifuging the final eluent, and taking the supernatant for detection.
6. The method for mass spectrometry detection of multiple drugs in solid phase extracted urine according to claim 5, wherein the method comprises the steps of: the extraction method of the liquid-transfering gun for solid-phase extraction of the filler comprises the following steps: extracting methanol by a liquid-transferring gun for solid-phase extraction of filling materials to moisten and activate a gun head, then balancing by ultrapure water, immersing the gun head in a urine sample, and repeatedly sucking and discharging for at least 9 times, wherein the time for sucking/discharging each time is 8-12 s; then flushing with ultrapure water; and (3) repeatedly eluting for 2-5 times by using methanol with the same volume as an eluent, wherein the eluting time is 8-12 s each time, centrifuging the final eluent at a speed of 5-1 w for 20-40 s, and taking supernatant for detection.
7. The method for mass spectrometry detection of multiple drugs in solid phase extracted urine according to claim 1, wherein the method comprises the following steps: the mass spectrometry in step S4 employs a miniature mass spectrometer.
8. The method for mass spectrometry detection of multiple drugs in solid phase extracted urine according to claim 2, wherein the method comprises the following steps: in the mass spectrometry in step S4, paper spray ionization sampling or nano-electrospray ionization sampling is adopted for sampling.
9. The method for mass spectrometry detection of multiple drugs in solid phase extracted urine according to claim 8, wherein the method comprises the steps of: when nano-electrospray ionization sample injection is adopted, the spray voltage of 6-acetylmorphine is 1400V, the spray voltage of methamphetamine is 1400V, the spray voltage of 3, 4-methylenedioxymethamphetamine is 1200V, the spray voltage of ketamine is 1200V, the spray voltage of norketamine is 1400V, and the spray voltage of cocaine is 1400V.
10. The method for mass spectrometry detection of multiple drugs in solid phase extracted urine according to claim 8, wherein the method comprises the steps of: when nano-electrospray ionization sample injection is adopted, the energy of ISO1 of 6-acetylmorphine is 8V, the energy of ISO1 of methamphetamine is 6.5V, the energy of ISO1 of 3, 4-methylenedioxymethamphetamine is 7V, the energy of ISO1 of ketamine is 8V, the energy of ISO1 of norketamine is 10V, and the energy of ISO1 of cocaine is 8V; the energy of ISO2 of 6-acetylmorphine is 4V, the energy of ISO2 of methamphetamine is 2.5V, the energy of ISO2 of 3, 4-methylenedioxymethamphetamine is 3V, the energy of ISO2 of ketamine is 3.5V, the energy of ISO2 of norketamine is 3.5V, the energy of ISO2 of cocaine is 4V; the energy of the CID of 6-acetylmorphine was 2V, the energy of the CID of methamphetamine was 1V, the energy of the CID of 3, 4-methylenedioxymethamphetamine was 3.75V, the energy of the CID of ketamine was 1.75V, the energy of the CID of norketamine was 2.25V, and the energy of the CID of cocaine was 2V.
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