CN113109491A - Universal method for detecting toxic drugs from biological samples - Google Patents
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Abstract
The invention relates to a general method for detecting toxic medicine from a biological sample, which comprises the following steps of sample pretreatment: adding a blood sample to be detected into a centrifugal tube; adding SKF 525A; adding acetonitrile, oscillating and mixing uniformly; adding ethyl acetate, oscillating, mixing uniformly, and performing liquid-liquid separation; taking the supernatant to another test tube, and volatilizing at normal temperature; adding methanol into the residue for redissolving, oscillating and uniformly mixing, and then carrying out solid-liquid separation; taking the separated supernatant for detection. The pretreatment step of the invention is used for treating the blood sample to be detected, so that the acidic, neutral and alkaline toxic (drug) can be detected and extracted simultaneously, the detection for multiple times by adopting different technical specifications is not needed, the required sample amount is less, the efficiency is improved, and the manpower and material resources are saved.
Description
Technical Field
The invention relates to the technical field of toxic drug analysis, in particular to a universal method for detecting a toxic drug from a biological sample.
Background
Various drugs, such as pesticides, rodenticides, herbicides and other chemicals have great harm to human bodies, so that poisoning accidents such as poison throwing, suicide and mistaking food caused by the drugs happen. In such cases, due to intentional concealment or after-concealment of the suspect, it is often difficult for the police or the toxicant analyst to clearly identify the direction in advance, and therefore, the screening detection method is mainly used in cases of this kind.
In the detection of toxic drugs and drug cases, toxicant analysis workers are often faced with extremely complex in vivo biological detection materials, and the biological detection materials contain a large amount of endogenous substances such as proteins, metabolites and the like, which can interfere the detection of the toxic drugs and damage instruments. Therefore, the biological samples need to be pretreated before the instrumental analysis. The quality of the sample pretreatment not only directly affects the final analysis result, but also affects the service life of the analysis instrument. The pretreatment of the sample is a very key and important link in-vivo toxic drug analysis, and a toxic analysis worker can select different pretreatment modes according to the physicochemical properties (mainly acidity, neutrality and alkalinity) of a target compound to be detected, so that the effects of separating, extracting, purifying and enriching the target compound to be detected are achieved, and the subsequent instrument analysis is facilitated.
The ministry of justice appraisal and management bureau of the ministry of justice of the people's republic of China issued multiple technical specifications in 4 months 2010, 9 months 2016 and 11 months 2018 respectively to solve the problem of extracting and detecting acidic, neutral and alkaline toxic drugs from biological test materials, and each standard name and specific operation steps are as follows.
Technical specification 1 measurement liquid chromatography-tandem mass spectrometry (SF/Z JD 0107009-2010) for aconitine, mesaconine and hypaconitine in biological detection materials solves the problems of extraction and detection of alkaline toxic alkaloid aconitine in biological detection materials, and comprises the following specific steps: 6.1.1 taking 0.5mL of blood or urine, placing in a 10mL centrifuge tube with a plug, adding 1.0mL borax buffer solution, extracting with diethyl ether 3mL, vortex mixing, centrifuging, transferring the organic layer to another centrifuge tube, volatilizing in a water bath at 60 ℃, dissolving the residue with 200 μ L acetonitrile, mobile phase buffer solution (70:30), and analyzing by LC-MS/MS. 6.1.2 tissue was minced or homogenized, 0.5g was weighed into a 10mL centrifuge tube with a stopper, and after soaking in 1.0mL boric acid buffer for half an hour, extraction was performed with 3mL diethyl ether, as described below under 6.1.1.
Technical specification 2 determination of barbiturates in biological samples liquid chromatography-tandem mass spectrometry SF/Z JD0107008-2010 solves the problems of extraction and detection of barbiturates in biological samples, and comprises the following specific steps: 6.1.1 direct extraction of blood or urine 1mL, 1. mu.g of internal standard acetylsalicylic acid was added, placed in a 10mL centrifuge tube, 2 drops of 0.1mol/L HCl were added, 3.5mL of diethyl ether was added, vortex mixing, centrifugation was carried out to separate layers, the diethyl ether layer was transferred to another centrifuge tube, water bath at about 60 ℃ was evaporated, 100. mu.L of acetonitrile was added, the residue was dissolved in mobile phase buffer (70:30), and 5. mu.L of acetonitrile was taken and charged to LC-MS/MS. 6.1.2 extraction of gastric content or tissue 1g of gastric content or minced tissue (or homogenate) was weighed and added with 1. mu.g of internal standard acetylsalicylic acid, following the same procedure as in 6.1.1.
The technical specification 3 detection liquid chromatography tandem mass spectrometry for 238 kinds of toxic (medicine) substances in blood and urine SF/Z JD0107005-2016 solves the extraction and detection problems of neutral and alkaline toxic substances and medicines in biological detection materials, and comprises the following specific steps: taking 1mL of blood or urine, adding 10 mu L of diazepam-d 5 and SKF525A internal standard solution (1 mu g/mL), adding 2mL of boric acid buffer solution with pH9.2, extracting with 3.5mL of diethyl ether, performing mixed rotation, and centrifuging. The supernatant was evaporated in a water bath at 60 ℃ and 200mL of mobile phase was added to the residue for reconstitution, and 10. mu.L was taken for LC-MS/MS analysis.
Technical specification 4 liquid chromatography-tandem mass spectrometry detection method of 13 anticoagulant raticides such as bromadiolone in blood SF/Z JD0107018-2018 solves the extraction and detection problems of weak acid rodenticides in biological detection materials, and comprises the following specific steps: taking 1mL of blood, adding 3mL of ethyl acetate, carrying out vortex mixing, centrifuging at 2500r/min for 3min, transferring the supernatant into another centrifuge tube, adding 3mL of ethyl acetate, repeatedly extracting once, combining organic phases, drying under 60 ℃ water bath air flow, and redissolving the residue by 100 mu L of methanol for instrument analysis.
Technical specification 5 liquid chromatography-tandem mass spectrometry detection method of gelsemide, gelsemine and gelsemine in biological detection material SF/Z JD0107021-2018 solves the extraction and detection problem of alkaline gelsemin toxic alkaloids in biological detection material, and comprises the following specific steps: 6.1.1.1.1 transferring blood or urine sample, adding 50 μ L of 1% NaOH solution, mixing by vortex for 1min, adding 3mL of ethyl acetate, extracting by vortex for 2min, and centrifuging at 3000r/min for 3 min. The supernatant was dried under 55 ℃ water bath air flow, and the residue was redissolved with 100. mu.L of the mixed solution and mixed well for instrument analysis. 6.1.1.1.2 tissue sample is prepared by weighing 0.5g of minced tissue, adding 800 μ L of 1% NaOH solution, mixing by vortex for 1min, adding 3mL of ethyl acetate, extracting by vortex for 3min, and centrifuging at 3000r/min for 3 min. The supernatant was dried under 55 ℃ water bath air flow, the residue was redissolved with 200. mu.L of a mixed solution [ methanol: [20mmol/L ammonium acetate solution (containing 0.1% formic acid and 5% acetonitrile) (volume ratio 7:3) ], vortexed uniformly and transferred to a 1.5mL centrifuge tube, placed in a refrigerator at-20 ℃ for 30min, centrifuged at 13000r/min for 2min, and the supernatant was taken for instrumental analysis.
It can be seen from the above technical specifications that different pretreatment modes need to be adopted according to the difference of the properties (mainly, acidity and alkalinity) of different objects to be detected, and when detecting various substances in the case that the detection indexes cannot be clearly determined in advance, pretreatment needs to be performed under various conditions by referring to a plurality of technical specifications, so that huge waste exists in time, labor and material resources. Meanwhile, the detection material for toxicant analysis is usually disposable and can not be collected repeatedly, and in judicial identification activities, a certain amount of detection material is required to be reserved for rechecking and rechecking, so that the situation of repeated sampling exists when detection is carried out by referring to a plurality of technical specifications, and the situation that the sample cannot be reserved for rechecking and rechecking (especially blood and urine samples) can be caused due to the relatively large sampling volumes (0.5mL or 1mL of blood and urine and 0.5g or 1g of tissue) in the technical specifications, so that certain influence is brought to the subsequent progress of a case.
Disclosure of Invention
The invention aims to overcome the defect that multiple detections need to be carried out according to multiple technical specifications when multiple substances are detected in the prior art, and provides a method for detecting a toxic medicament from a biological sample, which can realize extraction and detection only once.
In order to achieve the above object, the embodiments of the present invention provide the following technical solutions:
a general method for detecting a toxic drug from a biological sample, comprising a pre-treatment step of the sample, the pre-treatment step comprising the following processes in sequence:
sample adding: adding a biological sample to be tested (blood or urine or tissue homogenate) into a test tube;
internal standard: adding SKF 525A;
and (3) precipitation: adding acetonitrile, and uniformly mixing by oscillation;
and (3) extraction: adding ethyl acetate, oscillating, mixing uniformly, and performing liquid-liquid separation;
enrichment: taking the separated supernatant into another test tube, and volatilizing at normal temperature;
redissolving: adding the complex solution into the residue for redissolving, oscillating and uniformly mixing, and then carrying out solid-liquid separation;
collecting: taking the supernatant after redissolution and solid-liquid separation for detection.
In a further optimized scheme, in the sample adding step, the amount of the added blood or urine sample is 0.1-0.5ml, or the tissue homogenate is 0.1-0.5 g.
In a further optimized scheme, in the extraction step, the amount of the added ethyl acetate is 2-4 times of the amount of the biological sample.
In a further preferred embodiment, the method further comprises, after the pre-processing step, a detection step, where the detection step uses two mobile phases, each of which is: mobile phase A: 5mmol/L ammonium acetate + 0.1% formic acid; mobile phase B: and (3) acetonitrile.
In a further preferred embodiment, the detection step uses a gradient elution mode.
Compared with the prior art, the invention has the beneficial effects that:
(1) the pretreatment conditions adopted by the invention can simultaneously extract aconitine, barbiturates, anticoagulant raticide, gelsemin and various compounds such as neutral and alkaline poison medicines in the technical specifications SF/Z JD0107005-2016 of the detection liquid chromatogram tandem mass spectrometry method for 238 kinds of poison (medicines) in blood and urine in multiple technical specifications, so that the pretreatment process is simpler and more efficient, and a large amount of time, manpower and material resources are saved. Meanwhile, due to the uniform operation, the condition of adding wrong reagents can not occur by referring to different technical specifications.
(2) The sample size used by the method is only 0.1-0.5mL (or 0.1-0.5 g), and is saved by about 1-10 times compared with the sample size used in each technical specification (0.5mL and 1mL, 0.5g and 1.0 g); meanwhile, along with the reduction of the sample amount, various solvents used in subsequent pretreatment are saved by about 1-10 times, the pretreatment process is more environment-friendly, and the samples for rechecking and rechecking can be better retained in the judicial identification process. When a plurality of technical specifications are used for detection (for example, when one detection material needs to simultaneously detect barbiturates, benzodiazepines, aconitines, gelsemins and anticoagulant rodenticides), the use amount of the detection material and related reagents is saved more obviously.
(3) The mobile phase A used in the invention is 5mmol/L ammonium acetate containing 0.1% formic acid, has lower concentration of ammonium acetate than that in the mobile phase A used in the technical specification (20mmol/L ammonium acetate containing 0.1% formic acid or 20mmol/L ammonium acetate solution (containing 0.1% formic acid and 5% acetonitrile) or 10mmol/L ammonium acetate solution (containing 5% methanol)), does not contain organic solvent, and is more economical and environment-friendly. The mobile phase B used in the present invention is acetonitrile, and the mobile phase B in the specification is methanol or acetonitrile. Because the mobile phase is unified, the detection is more convenient, and the condition of using the mobile phase in a mixed way can be avoided.
(4) The invention adds 4 novel insecticides of acetamiprid, imidacloprid, pyridaben and chlorpyrifos which are widely used at present, two novel poisons of LSD and carbazone, two hypnotic sedatives of nitrazepam and chlordiazepoxide and the extraction and detection of isoniazid antituberculosis drugs, thereby enlarging the detection range.
(5) The detection limits for barbital, phenobarbital, amobarbital, secobarbital, morphine, monoacetylmorphine, methadone, codeine, pethidine, amphetamine, methamphetamine, A, ephedrine, ***e, benzoylekanine, methcathinone, diazepam, nitrazepam, clonazepam, flunitrazepam, lorazepam, midazolam, estazolam, alprazolam, triazolam, clozapine, amitriptyline, atropine, dimethoate, omethoate, acephate, parathion, malathion, meturon, aconitine, gelseminal, leptine, gelsemin, hispidem, flocoum, crimson, rodenticide, clomazone, warfarin and the like detection indices in the present invention are all lower than the detection limits in the prior art specifications. The detection limit refers to the lowest concentration of the indexes which can be detected by the method, because the method of the invention improves the recovery rate of partial indexes and has lower matrix effect, and simultaneously, because a gradient elution procedure is adopted to separate various substances as much as possible, the ion inhibition effect can be reduced in the ionization process to ensure that the ionization efficiency is higher, so the lower detection limit can be realized.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
Figure 1 is a graph of the external standard-correction profile of morphine in experimental example 1.
Figure 2 is a graph of the external standard-correction profile of morphine in experimental example 2.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. The components of embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations. Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without making any creative effort, shall fall within the protection scope of the present invention.
The embodiment of the invention provides a method for detecting a toxic medicament from a biological sample, which comprises a sample pretreatment step and a sample detection step, wherein the detection steps are different aiming at different targets, but the sample pretreatment steps are the same.
Specifically, the pretreatment steps of the sample are as follows:
sample adding: adding 0.1-0.5ml of a blood or urine sample to be detected (if the sample is a tissue, homogenizing the sample, and then adding 0.1-0.5 g of tissue homogenate) into a 15ml centrifuge tube;
internal standard: adding 0.01mL SKF525A (Prodilene hydrochloride) with the concentration of 1 mug/mL;
diluting: adding ultrapure water (water with resistivity of 18M omega cm (25 ℃) in an amount which is 1-2 times the volume of the blood sample (for example, the blood sample is 0.3ml, and the ultrapure water is 0.6ml), and uniformly mixing by oscillation (the urine or tissue homogenate is not required to be diluted by the ultrapure water);
and (3) precipitation: adding acetonitrile with the volume 2-3 times of that of the sample, and oscillating and uniformly mixing;
and (3) extraction: adding ethyl acetate with the volume 2-4 times of that of the sample, oscillating, uniformly mixing, and centrifuging for a certain time, such as 5 minutes;
enrichment: separating supernatant into another centrifuge tube, volatilizing at normal temperature to volatilize solvent to achieve the purpose of concentration;
redissolving: adding 20% methanol with the same volume as the sample into the residue for redissolving, shaking and mixing uniformly, and centrifuging at high speed (such as the rotating speed of more than 10000 rpm) for 5 minutes; here, in addition to methanol, the reconstitution can also be carried out using acetonitrile and a mobile phase as the reconstitution liquid.
Collecting: taking the supernatant after redissolution to another centrifuge tube, and centrifuging at high speed for 5 minutes again;
and (3) detection: the second supernatant (i.e., the supernatant after the second centrifugation) was taken ready for LC-MS/MS detection.
In the pretreatment step, the extraction solvent is volatilized to achieve the purpose of concentrating the sample, and the ethyl acetate entering the equipment can be removed, so that the phenomenon that the emulsification phenomenon occurs in the chromatographic column and the efficiency of the chromatographic column is influenced is prevented. After concentration, the solution is redissolved by 20% methanol, and the redissolved solution contains a plurality of solid impurities which can not be dissolved and enter (the impurities can also damage equipment), so the impurities are removed by a centrifugal mode. A small amount of solid impurities may remain in the supernatant after centrifugation, so that the solid impurities can be removed by centrifugation again, the solid impurities can be effectively removed after twice centrifugation, and the supernatant is taken again for analysis, so that the damage of the impurities to an instrument can be reduced, and the accuracy of analysis can be improved.
In the pretreatment step of the sample, after the blood sample and the internal standard are added, ultrapure water is added to dilute the blood sample, so that large protein sediments can be prevented from embedding a target object during subsequent protein precipitation, the emulsification phenomenon in the liquid-liquid extraction process can be reduced, and the subsequent compound extraction can be facilitated. The acetonitrile added in the precipitation process can precipitate protein, further reduce the emulsification phenomenon in the liquid-liquid extraction process, and also can extract the target object from blood, so that the aim of carrying out the same pretreatment on the blood sample by detecting different target objects can be fulfilled without regulating acid or alkali of the blood sample. And finally, transferring the target object from the mixed liquid of the acetonitrile and the biological sample to an organic phase by using ethyl acetate in a liquid-liquid extraction mode, so that the subsequent concentration and enrichment are facilitated.
In the pretreatment step, the ultrapure water is added for the purpose of diluting the blood sample to facilitate the extraction of the target substance, but the dilution may be performed without adding ultrapure water. In addition, liquid-liquid separation is carried out in a centrifugal mode in the extraction step, and other modes such as standing can achieve the same effect. The solid-liquid separation is also performed by a centrifugal method in the steps of redissolution, collection, etc. to separate solid impurities, but of course, other methods such as filtration by a filtration membrane may be used. In addition, the supernatant obtained by two times of centrifugation is extracted in the collection step, and the purpose of performing two times of centrifugation is to more reliably ensure that impurities are removed more thoroughly so as to avoid the influence of the impurities on the extraction of the target substance, but it is easy to understand that only one centrifugation treatment is performed, or more centrifugation treatments are also allowed. The following experimental examples are described and compared in terms of the amount and concentration of each substance, and if there is no specific description, there is no specific requirement, for example, there is no specific requirement for the concentration and amount of SKF525A used in the internal standard process, and the influence on the detection result is small.
Compared with a plurality of technical specifications in the prior art, the pretreatment step is an optimized pretreatment method, when the toxic drugs in the biological sample are detected, the pretreatment step is adopted to treat the biological sample, so that the acidic, neutral, alkaline and amphoteric toxic drugs can be detected simultaneously, namely, the biological sample can be detected after being treated by the pretreatment step aiming at the toxic drugs with different pH values, and the extraction and detection for a plurality of times are not needed, so that the detection efficiency can be greatly improved, the investment of manpower and material resources is reduced, the waste of the sample and various substances needed by the detection is avoided, and the guarantee is provided for reserving the sample for rechecking and rechecking. In addition, the optimized pretreatment method enables the recovery rate of most substances in the biological sample to be improved or the matrix effect to be reduced, so that the dosage of the blood or urine sample only needs 0.1-0.5ml, and the tissue sample only needs 0.1-0.5 g to complete the detection, compared with the prior art which needs 0.5ml and 1ml or 0.5g and 1g, the dosage of the sample is reduced, and the pretreatment process of the method is more environment-friendly.
The method also has more environmental protection performance, and is embodied in the following three aspects: 1. the sample used in the method is 0.1-0.5ml (or 0.1 g-0.5 g), and the standard uses 0.5ml and 1ml or 0.5g and 1g, so the method uses less organic solvent, and is more environment-friendly; 2. when some samples need to detect multiple types of indexes, multiple specifications are used for extraction, and then more samples are extracted and detected under extraction conditions of different specifications to meet detection requirements (for example, benzodiazepines and barbiturates are detected simultaneously, 1 sample is taken to be subjected to alkali adjustment and then extracted to complete detection of the benzodiazepines, and another sample is taken to be subjected to acid adjustment and then extracted to complete detection of the barbiturates). 3. The method unifies the used mobile phase, changes the 20mmol/L ammonium acetate solution containing 0.1% formic acid or 20mmol/L ammonium acetate solution (containing 0.1% formic acid and 5% acetonitrile) or 10mmol/L ammonium acetate solution (containing 5% methanol) from the specification into unified 5mmol/L ammonium acetate solution containing 0.1% formic acid, uses lower amount of ammonium acetate, and does not mix with organic solvent (such as 5% acetonitrile or 5% methanol), thereby being more environment-friendly.
The following will describe the results of the same pretreatment as described above for different substances in a biological sample, by referring to several test examples.
The detection device comprises: LC-MS/MS
Liquid chromatography conditions:
gradient elution procedure:
ion source conditions: configuring ESI sources
Target ion pair parameters:
except for newly added detection indexes such as acetamiprid, imidacloprid, pyridaben, chlorpyrifos, LSD, carbazone, nitrazepam, chlordiazepoxide and isoniazid, the other detection indexes are the same as the ion pair information of each detection index in the standard 'liquid chromatogram-tandem mass spectrometry for measuring aconitine, new aconitine and hypaconitine' SF/Z JD0107009-2010 ', the' liquid chromatogram-tandem mass spectrometry for measuring barbiturates in biological detection materials 'SF/Z JD 0107008-2010', the 'liquid chromatogram-tandem mass spectrometry for detecting 238 kinds of poisons (drugs) in blood and urine' SF/Z JD0107005-2016 ', the liquid chromatogram-tandem mass spectrometry detection method for 13 kinds of anticoagulant raticides such as bromadiolone in blood' SF/Z JD0107018-2018 'and the' liquid chromatogram-tandem mass spectrometry detection method for gelsemine, gelsemine and gelsemine 'SF/Z JD 0107021-2018'.
The newly added detection index ion pair parameters are as follows:
note: is a quantitative ion pair.
Experimental example 1
(1) Blood samples to be tested for morphine (amphoteric substance) were taken in two aliquots, each 0.3mL, and the two blood samples were pre-treated according to sample treatment method 1 in the following table, and the supernatants were extracted and tested under the above test conditions.
(2) Simultaneously preparing 0.3ml of blank blood sample (namely the blood sample without any toxic medicine), 7 blank blood adding samples with the morphine concentrations of 1ng/ml, 5ng/ml, 10ng/ml, 20ng/ml, 30ng/ml, 50ng/ml and 100ng/ml in blood and quality control samples with the morphine concentration of 10ng/ml in blood, wherein each sample is 0.3ml, pretreating and detecting the blank blood sample, 7 blank blood adding samples, 1 quality control sample and two blood samples to be detected according to the same method, and establishing an external standard-correction curve of the mixed standard substance. The calibration curve (partial least squares) is obtained by linear regression of the quantitative ion peak area of each index with different concentrations to the corresponding concentration, and the external standard-calibration curve is shown in fig. 1.
(3) Morphine component is detected from the blood sample to be detected, and the content is 35.63 ng/ml.
The step (1) is to directly detect the sample, and the step (2) is to control the negative and positive quality of the sample while detecting the sample, so as to ensure that the whole detection process is not influenced by external pollution, matrix effect and the like. The calibration curve was established in order to determine whether a sample contained morphine and to calculate the morphine content. If only step (1) is carried out, only one chromatographic peak with certain response intensity appears in each of two channels of morphine, but the chromatographic peaks in the two channels are generated by morphine and are also generated by other metabolites in the blood sample, and the chromatographic peaks are unknown, so that the interference generated by other metabolites in the blood is eliminated by using blank blood in step (2), and the retention time and abundance ratio of morphine in blank addition are used for determining whether the two peaks appearing in the sample to be tested are generated by morphine, so as to confirm that the blood sample to be tested contains morphine. And meanwhile, calculating the concentration of the morphine in the sample to be detected according to different responses (namely peak areas) generated by the concentrations in the correction curve. The quality control sample in the step (2) is used for quality control to ensure that the concentration calculated by the external standard-correction curve is accurate, namely the concentration deviation of the quality control sample calculated by the external standard-correction curve is within +/-20% of 10ng/ml (namely 8 ng/ml-12 ng/ml), and the standard curve is indicated to be used for the quantitative analysis.
Experimental example 2
(1) Blood samples to be tested for morphine (amphoteric substance) were taken in two aliquots, each 0.3mL, and the two blood samples were pre-treated according to sample treatment method 2 in the following table, and the supernatants were extracted and tested under the above test conditions.
(2) Simultaneously preparing 0.3ml of blank blood sample, 7 blank blood adding samples with the morphine concentrations of 1ng/ml, 5ng/ml, 10ng/ml, 20ng/ml, 30ng/ml, 50ng/ml and 100ng/m in blood and one quality control sample with the morphine concentration of 10ng/ml in blood, wherein each 0.3ml of blank blood sample, 7 blank blood adding samples, 1 quality control sample and two blood samples to be detected are pretreated and detected according to the same method, and an external standard-correction curve of the mixed standard substance is established. The calibration curve (partial least squares) is obtained by linear regression of the quantitative ion peak area of each index with different concentrations to the corresponding concentration, and the external standard-calibration curve is shown in fig. 2.
(3) Morphine component is detected from the blood sample to be detected, and the content is 36.41 ng/ml.
As can be seen from Experimental examples 1 and 2, different doses of ethyl acetate have little influence on the detection result, and the dosage of ethyl acetate can be 2-4 times of the dosage of the blood sample.
Experimental example 3
(1) Taking two blood samples of which the concentration is 0.1mL and of which the concentration needs to be detected, preprocessing the two blood samples by referring to a sample processing method 3 in the following table, extracting supernatant and detecting according to the detection conditions.
(2) Simultaneously preparing 0.1ml of blank blood sample, 10ng/ml, 50ng/ml, 100ng/ml, 200ng/ml, 300ng/ml, 500ng/ml and 1000ng/ml of object to be detected, mixing the blank blood adding sample with one part of quality control sample with the concentration of organophosphorus in the blood being 300ng/ml, and pretreating and detecting the blank blood sample, 7 parts of blank adding sample, 1 part of quality control sample and two parts of blood sample to be detected according to the same method, and establishing an external standard-correction curve of the mixed standard substance.
(3) The dichlorvos component is detected from the blood sample to be detected, and the content is 936.0 ng/ml.
Experimental example 4
(1) Taking two blood samples of which the concentration is 0.5mL for detecting organophosphorus insecticides (neutral substances), pretreating the two blood samples by referring to a sample treatment method 4 in the following table, extracting supernatant, and detecting according to the detection conditions.
(2) Simultaneously preparing 0.5ml of blank blood sample, 10ng/ml, 50ng/ml, 100ng/ml, 200ng/ml, 300ng/ml, 500ng/ml and 1000ng/ml of object to be detected, mixing the blank blood adding sample with one part of quality control sample with the concentration of organophosphorus in the blood being 300ng/ml, and pretreating and detecting the blank blood sample, 7 parts of blank adding sample, 1 part of quality control sample and two parts of blood sample to be detected according to the same method, and establishing an external standard-correction curve of the mixed standard substance.
(3) The dichlorvos component is detected from the blood sample to be detected, and the content is 940.7 ng/ml.
The blood sample consumption is too much, which causes waste of the blood sample, and the detection result is possibly not accurate enough when the blood sample consumption is too little, the blood sample consumption can be within the range of 0.1-0.5ml after the analysis and test of the extracting solution processed by the pretreatment step, the detection of the concentration of the toxic medicament is not greatly influenced, and the blood sample consumption is most preferably 0.3ml, which can give consideration to both the accuracy and the sample saving.
Experimental example 5
(1) Taking two blood samples of which the concentration is 0.2mL each for detecting novel drugs (alkaline substances), pretreating the two blood samples according to a sample treatment method 5 in the following table, extracting supernatant, and detecting according to the detection conditions.
(2) Simultaneously preparing 0.2ml of blank blood sample, 1ng/ml of specimen to be detected, 5ng/ml of specimen to be detected, 10ng/ml of specimen to be detected, 20ng/ml of specimen to be detected, 30ng/ml of specimen to be detected, 50ng/ml of specimen to be detected and 100ng/ml of specimen to be detected, preprocessing and detecting the blank blood sample, 7 blank samples of specimen to be detected, 1 mass of quality control sample and two specimens of specimen to be detected according to the same method, and establishing an external standard-calibration curve of the mixed standard substance.
(3) LSD (lysergic acid diethylamine) component with the content of 48.71ng/ml is detected from the blood sample to be detected.
Experimental example 6
(1) Taking two blood samples of which the concentration is 0.2mL each for detecting novel drugs (alkaline substances), pretreating the two blood samples according to a sample treatment method 6 in the following table, extracting supernatant, and detecting according to the detection conditions.
(2) Simultaneously preparing 0.2ml of blank blood sample, 1ng/ml of specimen to be detected, 5ng/ml of specimen to be detected, 10ng/ml of specimen to be detected, 20ng/ml of specimen to be detected, 30ng/ml of specimen to be detected, 50ng/ml of specimen to be detected and 100ng/ml of specimen to be detected, preprocessing and detecting the blank blood sample, 7 blank samples of specimen to be detected, 1 mass of quality control sample and two specimens of specimen to be detected according to the same method, and establishing an external standard-calibration curve of the mixed standard substance.
(3) LSD (lysergic acid diethylamine) component was detected from the blood sample to be tested, and the content was 46.55 ng/ml.
Experimental examples 5 and 6 show that for the detection of the same substance, when the using amount of ultrapure water can be 1-2 times of that of a blood sample, the samples are diluted by using ultrapure water with different doses, so that the detection result is not greatly influenced.
Experimental example 7
(1) Taking two blood samples of which the concentration is 0.3mL for detection of the nicotine pesticide (alkaline substance), pretreating the two blood samples according to a sample treatment method 7 in the following table, extracting supernatant, and detecting according to the detection conditions.
(2) Simultaneously preparing 0.3ml of blank blood sample, 1ng/ml of specimen to be detected, 5ng/ml, 10ng/ml, 20ng/ml, 30ng/ml, 50ng/ml and 100ng/ml of specimen mixed blank blood adding sample and 0.3ml of quality control sample with the novel drug concentration of 5ng/ml in blood respectively, pretreating and detecting the blank blood sample, 7 blank adding samples, 1 quality control sample and the two specimen blood samples according to the same method, and establishing an external standard-correction curve of the mixed standard substance.
(3) The acetamiprid component is detected from the blood sample to be detected, and the content is 3.69 ng/ml.
Experimental example 8
(1) Taking two blood samples of which the concentration is 0.3mL for detecting the nicotine pesticide (alkaline substance), pretreating the two blood samples by referring to a sample treatment method 8 in the following table, extracting supernatant, and detecting according to the detection conditions.
(2) Simultaneously preparing 0.3ml of blank blood sample, 1ng/ml of specimen to be detected, 5ng/ml, 10ng/ml, 20ng/ml, 30ng/ml, 50ng/ml and 100ng/ml of specimen mixed blank blood adding sample and 0.3ml of quality control sample with the novel drug concentration of 5ng/ml in blood respectively, pretreating and detecting the blank blood sample, 7 blank adding samples, 1 quality control sample and the two specimen blood samples according to the same method, and establishing an external standard-correction curve of the mixed standard substance.
(3) The acetamiprid component is detected from the blood sample to be detected, and the content is 3.52 ng/ml.
Experimental examples 7 and 8 show that for the detection of the same substance, when the amount of acetonitrile is 2-3 times of the amount of a sample, the detection result is not greatly influenced by using acetonitrile precipitated proteins with different doses.
Experimental example 9
(1) Taking two blood samples of which each volume is 0.4mL and which need to be detected, preprocessing the two blood samples according to a sample processing method 9 in the following table, extracting supernatant and detecting according to the detection conditions.
(2) Simultaneously preparing 0.4ml of a blank blood sample, 1ng/ml of a substance to be detected, 5ng/ml, 10ng/ml, 20ng/ml, 30ng/ml, 50ng/ml and 100ng/m of the substance to be detected, mixing the blank blood adding sample and the benzodiazepine quality control sample with the concentration of 50ng/ml in the blood, preprocessing and detecting the blank blood sample, 7 blank adding samples, 1 quality control sample and the two blood samples to be detected according to the same method, and establishing an external standard-correction curve of the mixed standard substance.
(3) The eszolam component is detected from the blood sample to be detected, and the content is 90.05 ng/ml.
Experimental example 10
(1) Two blood samples to be tested for barbiturates (acidic substances) were taken, each 0.4mL, and the two blood samples were pretreated according to the sample treatment method 9, and then the supernatant was extracted and tested under the above test conditions.
(2) Simultaneously preparing 0.4ml of a blank blood sample, 10ng/ml, 50ng/ml, 100ng/ml, 200ng/ml, 300ng/ml, 500ng/ml and 1000ng/ml of a sample to be detected, mixing the blank blood additive sample with the barbiturate concentration in the blood of 500ng/ml, and respectively preparing 0.4ml of each sample, pretreating and detecting the blank blood sample, 7 blank additive samples, 1 quality control sample and two samples to be detected according to the same method, and establishing an external standard-correction curve of the mixed standard substance.
(3) The phenobarbital component is detected from the blood sample to be detected, and the content is 336.4 ng/ml.
In each of the above experimental examples, the step of blank blood sample was adopted to eliminate the external interference, and the external standard-calibration curve method was used to determine the concentration of the target substance. For the determination of the concentration of the target, there are also internal standard-calibration curve methods, external standard-single point calibration methods and internal standard-single point calibration methods, which are also suitable for the present invention. By improving the pretreatment steps, the invention can detect the toxic drugs with various pH (acid-base properties) by adopting the same pretreatment method, namely, the multiple detection is not required to be carried out by adopting multiple technical specifications, thereby greatly improving the detection efficiency and reducing the dosage of samples. In addition, the pretreatment method of the invention also reduces the sample dosage required by single detection.
Experimental examples 9 and 10 show that the method can extract and detect substances with different acid-base properties.
It should be noted that, in this embodiment, only the blood sample is taken as an example, and the extraction and detection of the contained toxic drug from the blood sample are described. It is to be understood and clearly determined that the method of the present embodiment is also applicable to the detection and extraction of toxic drugs in urine and tissues. That is, the sample may be blood, urine, tissue, or the like.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (7)
1. A general method for detecting toxic drugs from a biological sample is characterized by comprising the steps of preprocessing the sample, wherein the preprocessing steps sequentially comprise the following processes:
sample adding: adding a biological sample to be tested (blood or urine or tissue homogenate) into a test tube;
internal standard: adding SKF 525A;
and (3) precipitation: adding acetonitrile, and uniformly mixing by oscillation;
and (3) extraction: adding ethyl acetate, oscillating, mixing uniformly, and performing liquid-liquid separation;
enrichment: taking the separated supernatant into another test tube, and volatilizing at normal temperature;
redissolving: adding the complex solution into the residue for redissolving, oscillating and uniformly mixing, and then carrying out solid-liquid separation;
collecting: taking the supernatant after redissolution and solid-liquid separation for detection.
2. The method of claim 1, wherein in the sample application step, the amount of the blood or urine sample applied is 0.1 to 0.5ml, and the amount of the tissue homogenate is 0.1 to 0.5 g.
3. The method of claim 1, further comprising, prior to the precipitating step: ultrapure water was added and mixed by shaking, wherein the urine and tissue homogenates did not need to be diluted in this step.
4. The method according to claim 1, wherein in the sample application step, the amount of acetonitrile added is 2 to 3 times of the amount of the biological sample.
5. The method according to claim 1, wherein the amount of ethyl acetate added in the extraction process is 2-4 times of the amount of the biological sample.
6. The method according to claim 1, further comprising, after the pre-processing step, a detection step in which two mobile phases are used, respectively: mobile phase A: 5mmol/L ammonium acetate + 0.1% formic acid; mobile phase B: and (3) acetonitrile.
7. The method of claim 6, wherein the detecting step comprises gradient elution.
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