A method for rapidly detecting sufentanil content in human saliva

Abstract

The present invention relates to the field of drug detection, and discloses a method for rapidly detecting the content of sufentanil in human saliva, comprising the following steps: (1) selection of qualitative ions and quantitative ions; (2) construction of a standard curve; (3) pretreatment of a saliva sample: taking a saliva sample, adding an internal standard, adding an alkali solution to adjust the pH, adding anhydrous magnesium sulfate and ethyl acetate, centrifuging, and adding methanol; (4) pulsed direct current electrospray ion source mass spectrometry to determine the content of sufentanil in the sample solution to be tested. Compared with the prior art, the present invention develops a simple pretreatment method for mass spectrometry detection in saliva samples, which can effectively eliminate the interference of impurities in saliva, increase detection sensitivity and stability, and is suitable for rapid detection of samples; a complete method for detecting the content of sufentanil in saliva samples by pulsed direct current electrospray ion source mass spectrometry is developed, which has a fast analysis speed and is easy to operate.

Description

Technical Field

[0001] The present invention relates to the field of drug detection, and in particular to a pulsed direct current electrospray ion source for rapidly detecting the content of sufentanil in human saliva. Background Art

[0002] Sufentanil (N-(4-methoxymethyl-1-[2-(2-thiophene)ethyl]-4-piperidinyl)-N-phenylpropionamide) is a synthetic opioid receptor agonist. Its structure is an N-4 substituted derivative with a polar group introduced at the 4-position of fentanyl. It is widely used in clinical anesthesia and analgesia. Sufentanil has a strong analgesic effect, which is 5 to 10 times stronger than fentanyl and 1000 times stronger than morphine. Abuse of fentanyl drugs can cause serious harm to health, and its adverse reactions include dizziness, nausea, respiratory depression, coma, and even death. On the other hand, due to its lipophilicity, it is easy to enter the central nervous system and activate the opioid receptors in the human body, and it has a cheerful and comfortable stimulating effect, which can cause analgesia and pleasure, and is very addictive, thus causing major social and medical problems. Therefore, in order to reduce the morbidity and crime rate associated with fentanyl drugs, rapid and accurate measurement of fentanyl drug concentration is of great significance to public safety and clinical practice.

[0003] At present, the main methods for determining sufentanil in saliva are gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. The judicial administration industry standard of the People's Republic of China uses a method of enrichment and concentration after liquid-liquid extraction to treat saliva, and then uses a liquid chromatography-tandem mass spectrometer for detection. The literature reports the use of ultra-high performance liquid chromatography-tandem mass spectrometry to analyze fentanyl drugs in saliva. For example, Chinese patent publication number CN112782305A discloses a sensitive and rapid method for analyzing sufentanil concentration in plasma suitable for pharmacokinetic research, using: 1. pretreatment; 2. chromatographic separation; 3. mass spectrometry detection method to detect sufentanil in plasma. This type of analysis method often requires the use of complex sample pretreatment methods such as solid phase extraction, enrichment and concentration, and chromatographic separation also takes a long time, which cannot meet the needs of on-site, fast, accurate, and high-throughput analysis of illegal drugs. In addition, the instruments and equipment used are relatively expensive, which is not conducive to the popularization and promotion of the method. Summary of the invention

[0004] The present invention aims to overcome the problems of complicated pre-treatment, long analysis time and difficulty in popularization and promotion of existing detection technologies, and provides a method for rapidly detecting the content of sufentanil in human saliva, which has the advantages of fast analysis speed, simple operation, real-time in-situ, low cost, high sensitivity and good specificity, etc. It can achieve long-term, stable and high-intensity ionization of trace samples (picoliter level) and meet the analysis requirements of small batch samples.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A method for rapidly detecting the content of sufentanil in human saliva comprises the following steps:

[0007] (1) Selection of qualitative and quantitative ions;

[0008] (2) Construction of standard curve;

[0009] (3) Pretreatment of saliva samples: (A) Take a saliva sample, add an internal standard to prepare a positive saliva, and add an alkali solution to adjust the pH; (B) Add anhydrous magnesium sulfate, vortex, add ethyl acetate, centrifuge, and extract the supernatant; (C) Add 0.03-0.08 μL / μL of methanol to the supernatant to obtain a sample solution to be tested;

[0010] (4) Determination of the sufentanil content in the sample solution by pulsed direct current electrospray ionization source mass spectrometry.

[0011] Pulsed DC electrospray ion source mass spectrometry is a new type of mass spectrometry analysis technology that can quickly perform mass spectrometry analysis on-site, in situ and with only a small amount of pretreatment. Pulsed DC electrospray ion source is a portable ion source dedicated to rapid screening and drug analysis of drug users and dealers. Pulsed DC electrospray technology can complete the sampling, injection, ionization and mass spectrum of biological samples within thirty seconds, and analyze the chemical composition and structure of the sample from the mass spectrum of the obtained product. This method has the advantages of fast analysis speed, simple operation, real-time in situ, low cost, high sensitivity and good specificity. The ion source is designed with a disposable sampling kit, which is easy to operate and eliminates cross infection. It can achieve long-term, stable, high-intensity ionization of trace samples (picoliter level) and meet the analysis requirements of small batch samples.

[0012] For mass spectrometry detection technology, for different samples and detection substances, appropriate pre-treatment steps must be performed on the sample to be tested to ensure that the subsequent mass spectrometry detection is carried out smoothly and obtain ideal results. At the same time, different mass spectrometry detection technologies also have certain differences in the pre-treatment requirements of samples. For the pulsed DC electrospray ion source mass spectrometry technology adopted by the present invention, for saliva samples, the substances in its environment are relatively complex, and the main organic components are proteins, which may interfere with the detection of substances. Therefore, it is necessary to take suitable pre-treatment before detection. After a large number of experiments, the inventor found that by adjusting the pH to ensure the stability of the sample and the internal standard, by adding anhydrous magnesium sulfate inorganic salts, the protein in the sample is precipitated to reduce interference, and by adding methanol to reduce the sample polarity, it is conducive to the sample in the subsequent process Full ionization, improve the detection accuracy. The pre-treatment method has clear steps and simple operation, and can make the saliva sample obtain an ideal detection effect in the pulsed DC electrospray ion source mass spectrometry.

[0013] Preferably, in step (A), the alkali solution is 8-12% sodium hydroxide solution, and the pH range is 11-13.

[0014] Preferably, in step (A), the concentration of the internal standard in the positive saliva is 80-120 ng / mL.

[0015] Preferably, in step (B), the amount of anhydrous magnesium sulfate used is 0.4-0.6 mg / μL supernatant; the amount of ethyl acetate used is 0.2-0.4 μL / μL supernatant.

[0016] Preferably, in step (B), the vortex time is 40-80 s, the centrifugation time is 2-4 min, and the centrifugal speed is 12000-15000 r / min.

[0017] Preferably, in step (C), the amount of methanol added is 0.04-0.06 μL / μL supernatant.

[0018] Preferably, the conditions for the pulsed DC electrospray ion source mass spectrometry detection are: scanning mode: positive ion selection scanning mode; electrospray voltage: 3-5KV; transmission capillary temperature: 250-300°C; transmission capillary voltage: 30-40V; ion extraction lens voltage: 100-120V; collision gas: high-purity helium; collision energy is 20-40eV.

[0019] Preferably, the conditions for the pulsed DC electrospray ion source mass spectrometry detection are: scanning mode: positive ion selection scanning mode; electrospray voltage: 4KV; transmission capillary temperature: 275°C; transmission capillary voltage: 35V; ion extraction lens voltage: 110V; collision gas: high-purity helium; and collision energy of 30eV.

[0020] Preferably, the qualifier ion is the qualifier ion of sufentanil, and the quantifier ion is the quantifier ion of sufentanil and the quantifier ion of D5-fentanyl.

[0021] Preferably, the qualifier ion of sufentanil is m / z 355.30, the quantifier ion of sufentanil is m / z 238.20, and the quantifier ion of D5-fentanyl is m / z 188.10.

[0022] Therefore, the present invention has the following beneficial effects: (1) a simple pretreatment method is developed for mass spectrometry detection in saliva samples, which can effectively eliminate the interference of impurities in saliva, increase the detection sensitivity and stability, and is suitable for rapid detection of samples; (2) a complete method for detecting the content of sufentanil in saliva samples by pulsed direct current electrospray ion source mass spectrometry is developed, which has fast analysis speed and simple operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the secondary mass spectrum of sufentanil standard (50 ng / mL).

[0024] Figure 2 It is the selected ion chromatogram of sufentanil standard (50 ng / mL).

[0025] Figure 3 It is the secondary mass spectrum of D5-fentanyl standard (50 ng / mL).

[0026] Figure 4 It is the selected ion chromatogram of D5-fentanyl standard (50 ng / mL).

[0027] Figure 5 This is the secondary mass spectrum of positive saliva.

[0028] Figure 6 It is the selected ion chromatogram of positive saliva.

[0029] Figure 7 This is the low concentration working curve of sufentanil.

[0030] Figure 8 This is the high concentration working curve of sufentanil. DETAILED DESCRIPTION

[0031] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments.

[0032] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the embodiments, unless otherwise specified, are all conventional methods in the art.

[0033] Overall embodiment

[0034] A method for rapidly detecting the content of sufentanil in human saliva comprises the following steps:

[0035] (1) Selection of qualitative ions and quantitative ions: A micro-volume of 30-70 ng / mL sufentanil standard solution was drawn into the sample by a nanoliter electrospray nozzle, and the precursor ions and fragment ions of the sufentanil standard were obtained by online detection by a mass spectrometer, and the quantitative ions and qualitative ions were selected from the fragment ions. D5-fentanyl was used as an internal standard, and a micro-volume of 30-70 ng / mL D5-fentanyl standard solution was drawn into the sample by a nanoliter electrospray nozzle, and the precursor ions and fragment ions of the D5-fentanyl standard were obtained by online detection by a mass spectrometer, and the quantitative ions were selected from the fragment ions;

[0036] (2) Construction of standard curve: Several groups of standard working solutions containing analytes sufentanil and D5-fentanyl at different concentrations were detected by mass spectrometry. The mass spectrometry was a pulsed direct current electrospray ion source mass spectrometry in a positive ion selective scanning mode to obtain the precursor ions and fragment ions of the samples. The precursor ions and fragment ions were compared with the standard mass spectra for qualitative analysis. Then, the average value of the peak intensity ratio of the analyte and the internal standard ion was used as the Y-axis, and the concentration of the analyte in saliva was used as the X-axis. The standard curve was constructed by averaging multiple groups of experimental data.

[0037] (3) Pretreatment of saliva samples: (A) Take a saliva sample, add an internal standard to a positive saliva with a concentration of 80-120 ng / mL, add 8-12% sodium hydroxide solution to adjust the pH to 11-13; (B) Add 0.4-0.6 mg / μL of anhydrous magnesium sulfate to the supernatant, vortex for 40-80 s, add 0.2-0.4 μL / μL of ethyl acetate to the supernatant, centrifuge for 2-4 min, and extract the supernatant at a centrifugal speed of 12000-15000 r / min; (C) Add 0.03-0.08 μL / μL of methanol to the supernatant to obtain a sample solution to be tested;

[0038] (4) Determination of the sufentanil content in the sample solution by pulsed direct current electrospray ion source mass spectrometry. The conditions for pulsed direct current electrospray ion source mass spectrometry detection are: scanning mode: positive ion selection scanning mode; electrospray voltage: 3-5KV; transmission capillary temperature: 250-300°C; transmission capillary voltage: 30-40V; ion extraction lens voltage: 100-120V; collision gas: high-purity helium; collision energy is 20-40eV. Substitute the peak intensity ratio of the quantitative ion of the analyte and the internal standard into the obtained standard curve to calculate the sufentanil content in saliva.

[0039] Example 1

[0040] Step 1: Preparation of standard solution

[0041] The standard solutions of sufentanil and D5-fentanyl were serially diluted with methanol containing 0.1% formic acid, wherein the sufentanil standard solution was diluted to 5 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, and 5 μg / mL, and the D5-fentanyl standard solution was diluted to 100 ng / mL. The chemical structure of sufentanil is shown below:

[0042]

[0043] Step 2: Negative saliva treatment and working solution preparation

[0044] Prepare eight centrifuge tubes of the same specifications, draw 400 μL of human negative saliva into each centrifuge tube, and then add 8 μL5ng / mL, 4 μL50ng / mL, 8 μL50ng / mL, 8 μL100ng / mL, 4 μL500ng / mL, 8 μL500ng / mL, respectively. ng / mL, 4μL5μg / mL, 8μL5μg / mL sufentanil standard solutions were placed in different centrifuge tubes, in which 8μL100ng / mL D5-fentanyl standard solution was added to each centrifuge tube, followed by 200μL ethyl acetate, and finally vortexed for 60 seconds, centrifuged at 13500r / min for 3 minutes, and the upper layer was taken to obtain eight different concentrations of sufentanil standard working solutions containing internal standards (0.1ng / mL, 0.5ng / mL, 1ng / mL, 2ng / mL, 5ng / mL, 10ng / mL, 50ng / mL, 100ng / mL) for constructing the working curve, and the concentration of the internal standard solution remained unchanged (2ng / mL). Before analysis, all spiked sample solutions were freshly prepared.

[0045] Step 3: Selection of qualitative and quantitative ions for sufentanil

[0046] A trace volume of 50 ng / mL sufentanil standard solution was drawn with a nanoliter electrospray nozzle. During operation, a metal electrode was inserted into the nanoliter electrospray nozzle. The electrode and the solution were not in direct contact, and a distance of about 5 mm was maintained. The nozzle was aligned with the mass spectrometer cone. After the electrode was energized, the nozzle generated pulsed electrospray, and the substances in the solution were ionized. The results were obtained by online detection with a mass spectrometer. Figure 1 The secondary mass spectrum of sufentanil shown in FIG. 1 is a fragment ion with the highest signal-to-noise ratio, m / z 238.20, as the quantitative ion and m / z 355.30 as the qualitative ion. The structure is shown in FIG. Figure 1 shown. Figure 2 Selected ion chromatogram of sufentanil standard.

[0047] Step 4: Selection of quantitative ions for D5-fentanyl

[0048] A trace volume of 50 ng / mL D5-fentanyl standard solution was drawn with a nanoliter electrospray nozzle. During operation, a metal electrode was inserted into the nanoliter electrospray nozzle. The electrode and the solution were not in direct contact, and a distance of about 5 mm was maintained. The nozzle was aligned with the mass spectrometer cone. After the electrode was energized, the nozzle generated pulsed electrospray, and the substances in the solution were ionized. The results were obtained by online detection with a mass spectrometer. Figure 3 The secondary mass spectrum of D5-fentanyl shown in FIG. 1 is a fragment ion with the highest signal-to-noise ratio, m / z 188.10, as the quantitative ion according to the mass spectrometry detection results. The structure is shown in FIG. Figure 3 shown. Figure 4 This is the selected ion chromatogram of D5-fentanyl.

[0049] Step 5: Methodological Investigation

[0050] (1) Limit of detection and limit of quantification

[0051] The limit of detection (LOD) and limit of quantification (LOQ) were determined by comparing the signal-to-noise ratios of the sufentanil qualifier and quantifier ions m / z 238.20 and m / z 355.30 determined in step 3 in different concentrations of working solution with those in blank matrix. Blank matrix was a blank sample without any standard added but with the same pretreatment method. LOD and LOQ were defined as the concentration of the analyte at which the signal-to-noise ratio reached 3 and 10, respectively.

[0052] (2) Preparation of working curve

[0053] The average value of the response ratio of the quantification ion of the analyte and the internal standard was taken as the Y-axis, and the concentration of the analyte in saliva (0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL) was taken as the X-axis. Each curve contained at least 5 calibration points. By averaging the data of three sets of parallel experiments, working curves were constructed at 0.1-5 ng / mL and 2-100 ng / mL, respectively. The results are shown in Figure 7 , Figure 8 As shown. Finally, the linear regression equation is obtained through linear regression.

[0054] (3) Accuracy and precision

[0055] Take low, medium and high concentration working solutions (0.1ng / mL, 2ng / mL, 100ng / mL)

[0056] Intra-day precision: Three experiments were conducted in the morning, afternoon and evening. Five parallel experiments were performed on each concentration of working solution. One injection was made for each concentration. Accuracy was defined as the ratio of the measured concentration (Cm) to the actual concentration of the analyte in the sample (Ca), calculated as follows: (Cm / Ca) × 100%. Precision was expressed as relative standard deviation (RSD). The precision of the three concentrations, high, medium and low, was calculated respectively.

[0057] Inter-day precision: The degree of agreement between the results of repeated measurements of the same analyte within six days.

[0058] The mass spectrometry conditions are as follows: scanning mode: positive ion selection scanning mode; electrospray voltage: 4KV; transmission capillary temperature: 275°C; transmission capillary voltage: 35V; ion extraction lens voltage: 110V; collision gas: high-purity helium; and collision energy of 30eV.

[0059] The linearity, detection limit and quantification limit of the method were investigated, and the results are shown in Table 1. As can be seen from Table 1, sufentanil has good linearity in the range of 0.1-100 ng / mL, and R 2 (Correlation coefficient) is satisfactory.

[0060] Table 1 Linearity, detection limit and quantification limit of sufentanil

[0061]

[0062] The reproducibility and precision of the method were investigated, and the results are shown in Table 2. As can be seen from Table 2, the reproducibility and precision of sufentanil were good, and the RSDs of the three concentrations of low, medium and high were all within 10%.

[0063] Table 2 Reproducibility and precision of sufentanil

[0064]

[0065] The spiked recovery rate of this method was investigated, and the results are shown in Table 3. As can be seen from Table 3, the spiked recovery rates of sufentanil are between 91.22% and 107.75%.

[0066] Addition concentration (ng / mL) Average measured concentration (ng / mL) Average spike recovery% 0.1 0.10163003 101.63％ Day 1 2 1.92336283 96.17％ 100 100.979818 100.98％ 0.1 0.10541307 105.41％ Day 2 2 2.07040418 103.52％ 100 107.754696 107.75％ 0.1 0.09715466 97.15％ Day 3 2 2.09330565 104.67％ 100 102.203024 102.20％ 0.1 0.09849964 98.50％ Day 4 2 1.99322121 99.66％ 100 100.641587 100.64％ 0.1 0.10352368 103.52％ Day 5 2 2.14843015 107.42％ 100 98.0737968 98.07％ 0.1 0.10333338 103.33％ Day 6 2 1.92084412 96.04％ 100 91.2243296 91.22％

[0067] Combined with the results in Tables 1, 2 and 3, it is believed that this method has good accuracy and can be used to quickly detect the content of sufentanil in human saliva.

[0068] Step 6: Detection and analysis of sufentanil in positive saliva samples

[0069] The positive saliva samples were pre-processed as follows:

[0070] (A) 600 μL of saliva sample was drawn, and internal standard was added to prepare positive saliva. Sodium hydroxide solution was added to adjust the pH to 12;

[0071] (B) Add excess anhydrous magnesium sulfate, vortex for 1 min, add 200 μL of ethyl acetate, centrifuge at 13500 r / min for 3 min, and extract the supernatant;

[0072] (C) Add 30 μL of methanol to the supernatant to obtain the sample solution to be tested.

[0073] The sample solution to be tested is sucked and injected with a nanoliter electrospray head. The injection steps are the same as step 3. The pulsed DC electrospray ion source mass spectrometer is used for rapid detection to obtain the following Figure 5 The secondary mass spectra of the positive saliva shown and Figure 6 The selected ion chromatogram of the positive saliva is shown; the qualitative analysis is compared with the mass spectrum of the negative saliva and the qualitative ion pairs of the standard, and the internal standard method is used for quantitative analysis. The results are shown in Table 4.

[0074] sample Negative samples Positive samples Concentration (ng / mL) Not detected 8.68

[0075] Table 4 shows that the detection method provided by the present invention can detect the content of sufentanil in saliva samples.

[0076] Example 2

[0077] The difference between this embodiment and embodiment 1 is that the mass spectrometry conditions are: electrospray voltage: 3KV; transmission capillary temperature: 250°C; transmission capillary voltage: 40V; ion extraction lens voltage: 100V; collision gas: high-purity helium; and collision energy of 40eV.

[0078] Example 3

[0079] The difference between this embodiment and embodiment 1 is that the mass spectrometry conditions are: electrospray voltage: 5KV; transmission capillary temperature: 300°C; transmission capillary voltage: 30V; ion extraction lens voltage: 120V; collision gas: high-purity helium; and collision energy of 20eV.

[0080] Comparative Example 1 (pH not adjusted in pretreatment)

[0081] The difference between this comparative example and Example 1 is that the pre-treatment method in step 6 is:

[0082] (A) 600 μL of saliva sample was drawn and internal standard was added to prepare positive saliva; (B) excess anhydrous magnesium sulfate was added, vortexed for 1 min, 200 μL of ethyl acetate was added, centrifuged at 13500 r / min for 3 min, and the supernatant was extracted; (C) 30 μL of methanol was added to the supernatant to obtain the sample solution to be tested.

[0083] Comparative Example 2 (no anhydrous magnesium sulfate added in pretreatment)

[0084] The difference between this comparative example and Example 1 is that the pre-treatment method in step 6 is:

[0085] (A) Take 600 μL of saliva sample, add internal standard to make positive saliva, add sodium hydroxide solution to adjust the pH to 12; (B) Add 200 μL of ethyl acetate, centrifuge at 13500 r / min for 3 min, and extract the supernatant; (C) Add 30 μL of methanol to the supernatant to obtain the sample solution to be tested.

[0086] Comparative Example 3 (pretreatment without methanol)

[0087] The difference between this comparative example and Example 1 is that the pre-treatment method in step 6 is:

[0088] (A) 600 μL of saliva sample was drawn, and an internal standard was added to prepare positive saliva, and sodium hydroxide solution was added to adjust the pH to 12; (B) an excess of anhydrous magnesium sulfate was added, vortexed for 1 min, 200 μL of ethyl acetate was added, and centrifuged at 13500 r / min for 3 min, and the supernatant was extracted to obtain the sample solution to be tested.

[0089] Comparative Example 4 (adding a small amount of methanol in pretreatment)

[0090] The difference between this comparative example and Example 1 is that the pre-treatment method in step 6 is:

[0091] (A) Take 600 μL of saliva sample, add internal standard to make positive saliva, add sodium hydroxide solution to adjust the pH to 12; (B) Add excess anhydrous magnesium sulfate, vortex for 1 min, add 200 μL of ethyl acetate, centrifuge at 13500 r / min for 3 min, and extract the supernatant; (C) Add 5 μL of methanol to the supernatant to obtain the sample solution to be tested.

[0092] Comparative Example 5 (adding a large amount of methanol in pretreatment)

[0093] The difference between this comparative example and Example 1 is that the pre-treatment method in step 6 is:

[0094] (A) Take 600 μL of saliva sample, add internal standard to make positive saliva, add sodium hydroxide solution to adjust the pH to 12; (B) Add excess anhydrous magnesium sulfate, vortex for 1 min, add 200 μL of ethyl acetate, centrifuge at 13500 r / min for 3 min, and extract the supernatant; (C) Add 100 μL of methanol to the supernatant to obtain the sample solution to be tested.

[0095] A number of saliva with a sufentanil added concentration of 10 ng / mL was taken, and the sufentanil concentration was determined under the mass spectrometry parameter conditions of Examples 1-3 and Comparative Examples 1-3, respectively. Five parallel samples were made for each group of experiments, and the final results were averaged to compare the response value and signal-to-noise ratio of the qualifier ion m / z 238.20 in 10 ng / mL saliva under different mass spectrometry parameter conditions.

[0096] Group background m / z 238.20 response Signal-to-Noise Ratio Example 1 64.1 24571 393.92 Example 2 266 20475 329.18 Example 3 65.3 4707 72.08 Comparative Example 1 66.5 23700 356.39 Comparative Example 2 67.4 23800 353.12 Comparative Example 3 66.5 21100 317.29 Comparative Example 4 63.9 20098 314.52 Comparative Example 5 67.1 17100 267.61

[0097] For mass spectrometry detection technology, for different samples and test substances, appropriate pre-treatment steps must be performed on the sample to be tested to ensure that the subsequent mass spectrometry detection is carried out smoothly and obtain the desired results. Example 1 avoids a very complicated sample pre-treatment process by optimizing mass spectrometry parameters and appropriate simple pre-treatment steps. The pulsed DC electrospray ion source mass spectrometry can quickly and highly sensitively detect the sufentanil content in human saliva, and the detection method has good precision and accuracy. Comparative Example 1 does not adjust pH during the pre-treatment process, which reduces the sample stability and causes a decrease in the signal-to-noise ratio during the subsequent detection process. Comparative Example 2 does not precipitate protein during the pre-treatment process, so protein is easy to interfere with the detection of sufentanil in the subsequent mass spectrometry detection process, so that the result has a certain error. Comparative Example 3 does not add methanol during the pre-treatment process, and the sufentanil detection signal-to-noise ratio and response value of Comparative Example 3 are much lower than those of Example 1. The reason may be that methanol can reduce the polarity of the saliva sample, making the saliva sample more easily ionized during the detection process, improving the detection accuracy and accuracy. In Comparative Example 4, only a small amount of methanol was added during the pretreatment process, and the detection response value of sufentanil was significantly lower than that of Example 1, which also illustrates the importance of adding methanol for detection accuracy. In Comparative Example 5, a large amount of methanol was added during the pretreatment process, and the addition of excessive methanol reduced the signal-to-noise ratio, which was also not conducive to the accurate detection of the content of sufentanil.

[0098] In the parameter setting of pulsed DC electrospray ion source mass spectrometry, there are multiple groups of adjustable parameters that cooperate with each other, and there are large differences in the parameter setting for different sample types and test substances. And there is a large influence between each group of adjustable parameters in the mass spectrum, so it is necessary to develop specific detection methods for different samples and test substances. Example 1 avoids a very complicated sample pretreatment process by optimizing mass spectrometry parameters and appropriate simple pretreatment steps. The sufentanil content in human saliva can be detected quickly and with high sensitivity using pulsed DC electrospray ion source mass spectrometry, and the detection method has good precision and accuracy. Example 2 and Example 3 have made minor adjustments to the mass spectrometry parameters, and the response value and signal-to-noise ratio of sufentanil detection are lower than Example 1, which proves that the reasonable setting of mass spectrometry parameters has an important influence on the detection of sufentanil. The detection parameters of Example 1 are the results obtained by the inventor after a lot of debugging.

[0099] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for rapidly detecting the content of sufentanil in human saliva, characterized in that: The following steps are involved: (1) Selection of qualitative and quantitative ions; (2) Construction of standard curve; (3) Pretreatment of saliva samples: (A) Take a saliva sample, add internal standard D5-fentanyl to prepare positive saliva, and add alkaline solution to adjust the pH range to 11-13; (B) Add anhydrous magnesium sulfate, vortex, add ethyl acetate, centrifuge, and extract the supernatant; (C) Add 0.03-0.08 μL / μL of methanol to the supernatant to obtain a sample solution to be tested; (4) The sufentanil content in the sample solution was detected by pulsed direct current electrospray ion source mass spectrometry, the scanning mode was positive ion selection scanning mode; the electrospray voltage was 3-5KV; the transmission capillary temperature was 250-300°C; the transmission capillary voltage was 30-40V; and the ion extraction lens voltage was 100-120V; The collision gas is high-purity helium; the collision energy is 20-40eV.

2. A method for rapidly detecting the content of sufentanil in human saliva according to claim 1, characterized in that: In step (A), the alkali solution is a sodium hydroxide solution with a mass fraction of 8-12%.

3. The method for rapidly detecting the content of sufentanil in human saliva according to claim 1, characterized in that: In step (A), the concentration of the internal standard in the positive saliva is 80-120 ng / mL.

4. The method for rapidly detecting the content of sufentanil in human saliva according to claim 1, characterized in that: In step (B), the amount of anhydrous magnesium sulfate used is 0.4-0.6 mg / μL supernatant; the amount of ethyl acetate used is 0.2-0.4 μL / μL supernatant.

5. The method for rapidly detecting the content of sufentanil in human saliva according to claim 1, characterized in that: In step (B), the vortex time is 40-80s, the centrifugation time is 2-4min, and the centrifugal speed is 12000-15000r / min.

6. The method for rapidly detecting the content of sufentanil in human saliva according to claim 1, characterized in that: In step (C), the amount of methanol added is 0.04-0.06 μL / μL supernatant.

7. The method for rapidly detecting the content of sufentanil in human saliva according to claim 1, characterized in that: The conditions for the pulsed direct current electrospray ion source mass spectrometry detection are: scanning mode: positive ion selection scanning mode; electrospray voltage: 4KV; transmission capillary temperature: 275°C; transmission capillary voltage: 35V; ion extraction lens voltage: 110V; collision gas: high-purity helium; and collision energy of 30eV.

8. The method for rapidly detecting the content of sufentanil in human saliva according to claim 1, characterized in that: The qualifier ion is the qualifier ion of sufentanil, and the quantifier ion is the quantifier ion of sufentanil and the quantifier ion of D5-fentanyl.

9. A method for rapidly detecting the content of sufentanil in human saliva according to claim 8, characterized in that: The qualifier ion of sufentanil is m / z 355.30, the quantifier ion of sufentanil is m / z 238.20, and the quantifier ion of D5-fentanyl is m / z 188.10.

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