Pretreatment method for detecting pyrrole alkaloid protein adduct and kit

Abstract

The invention provides a pretreatment method for detecting a pyrrole alkaloid protein adduct and a kit, and the pretreatment method is characterized in that 7, 9-di-EtO-DHP-d3 is used as an internal standard to carry out pretreatment on the pyrrole alkaloid protein adduct. According to the present invention, the 2, 4, 6, 7, 9-di-EtO-DHP-d3 can not be damaged during the preparation process of the sample solution, and has characteristics similar to the characteristics of the object to be detected, such that the error of the whole sample pretreatment process and the detection process can be effectively corrected; the pretreatment method provided by the invention has the advantages of good precision and high accuracy, and is especially suitable for detection of low-concentration pyrrole alkaloid protein adducts.

Description

[0001] This application is a divisional application of the patent application with the application number 202311614055.7 (the filing date of the original application is November 29, 2023, and the invention title is a method for detecting pyrrolizidine alkaloid protein adducts and a kit). Technical Field

[0002] The present invention belongs to the technical field of medical detection, and specifically relates to a pretreatment method for detecting pyrrolizidine alkaloid protein adducts and a kit. Background Art

[0003] Pyrrolizidine alkaloid (PA) is a class of alkaloid components widely distributed in nature and has extremely strong hepatotoxicity. Chinese herbal medicines, as traditional drug resources, have a history of use and cultural inheritance of thousands of years and have been widely used. Some plants containing PA, such as Gynura Root, Senecio scandens, Crotalaria pallida, and Heliotropium europaeum, may cause severe hepatic sinusoidal obstruction syndrome (HSOS) after ingestion. Therefore, in 2017, the "Expert Consensus on the Diagnosis and Treatment of Pyrrolizidine Alkaloid-Related Hepatic Sinusoidal Obstruction Syndrome" was promulgated. The consensus and guidelines recommend that HSOS is mainly PA-HSOS, and the most common cause of PA-HSOS is the ingestion of Gynura Root; the evidence level is A, and the recommendation strength is level 1. In the diagnosis of HSOS, currently, HSOS is mainly diagnosed through biochemical indicators such as plasma bilirubin and imaging methods such as ultrasound, CT, and MRI, lacking a method for directly detecting HSOS markers.

[0004] After the human body takes Chinese herbal medicines containing PA, PA undergoes a series of biotransformations by bioenzymes in the body and finally generates pyrrolizidine alkaloid-protein adducts and pyrrolizidine alkaloid-DNA adducts. Directly detecting these markers not only provides the most direct evidence for the etiology of HSOS, but also provides a data basis for the future research on the in vivo pathological mechanism of PA-HSOS. There have been many reports on the detection of pyrrolizidine alkaloid-protein adducts. However, the reported quantitative detection methods do not use internal standards, such as Ma J, Zhang W, He Y, et al. Clinical application of pyrrole–hemoglobin adducts as a biomarker of pyrrolizidine alkaloid exposure in humans[J]. Archives of Toxicology, 2020, 95(3): 1-7 (Existing Method 1); or, although an internal standard is used, it is added only before LC-MS detection, such as Xia Q, Zhao Y, Lin G, et al. Pyrrolizidine Alkaloid-Protein Adducts-Potential Non-Invasive Biomarkers of Pyrrolizidine Alkaloid-Induced Liver Toxicity and Exposure[J]. Chemical Research in Toxicology, 2016: 1282-1292 (Existing Method 2). During the sample preparation process for detecting pyrrolizidine alkaloid-protein adducts, the pyrrole structure part in the pyrrolizidine alkaloid-protein adducts needs to be released through a chemical reaction first, and then subsequent LC-MS detection and analysis are carried out. The sample solution preparation process of this detection process is complex, and the chemical reaction process will have a great impact on the quantitative results. Neither not adding an internal standard nor adding an internal standard only before LC-MS detection can effectively correct the sample preparation error, resulting in poor repeatability and accuracy of quantitative detection. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a pretreatment method for detecting pyrrolizidine alkaloid-protein adducts and a kit. The pretreatment method provided by the present invention has the advantages of high precision and high accuracy. The internal standard will not be damaged during the sample treatment process and can effectively correct the entire sample pretreatment process.

[0006] To achieve the purpose of this invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a pretreatment method for detecting pyrrolizidine alkaloid protein adducts, and the pretreatment method includes the following steps:

[0008] Mix the sample to be tested with a protein precipitant, then centrifuge to remove the first supernatant to obtain a residue, add the internal standard 7,9-di-EtO-DHP-d3, then mix with a releasing agent, centrifuge, take the second supernatant, mix the second supernatant with a derivatizing agent, and then centrifuge to take the supernatant as the sample to be tested;

[0009] The releasing agent is an ethanol solution containing silver nitrate, the derivatizing agent is an ethanol solution containing DABA, and the sample to be tested is a blood sample or a blood cell sample;

[0010] The structure of the internal standard 7,9-di-EtO-DHP-d3 is as follows:

[0011]

[0012] Preferably, the mixing with the releasing agent is carried out under shaking, and the shaking time is 10 - 30 min.

[0013] Preferably, the mixing of the second supernatant with the derivatizing agent is carried out under shaking, the shaking time is 20 - 30 min, and the shaking is carried out at a temperature of 55 - 65 °C.

[0014] Preferably, the protein precipitant is acetonitrile.

[0015] Preferably, the volume ratio of the sample to be tested to acetonitrile and the ethanol solution containing silver nitrate is 1:(2 - 4):(1 - 3).

[0016] Preferably, the mass fraction of silver nitrate in the ethanol solution containing silver nitrate is 0.8% - 1.2%.

[0017] Preferably, the internal standard 7,9-di-EtO-DHP-d3 is added in the form of a solution containing the internal standard 7,9-di-EtO-DHP-d3, and the concentration of 7,9-di-EtO-DHP-d3 in the solution containing the internal standard 7,9-di-EtO-DHP-d3 is 5 ng / mL to 50 ng / mL.

[0018] Preferably, the volume ratio of the solution containing the internal standard 7,9-di-EtO-DHP-d3 to the volume of the blood sample to be tested is (1 - 5):10.

[0019] Preferably, the volume ratio of the derivatizing agent to the second supernatant is (1 - 2):3.

[0020] Preferably, the concentration of DABA in the ethanol solution containing DABA is 15 mg / mL - 25 mg / mL.

[0021] Preferably, the ethanol solution containing DABA further contains perchloric acid with a mass fraction of 0.8% - 1.5%.

[0022] Preferably, the pretreatment method further includes the preparation of a blank sample solution: replacing the test sample with a blank sample and performing the steps of the pretreatment method as described above; the blank sample is a plasma protein solution of a healthy human or animal.

[0023] Preferably, the pretreatment method further includes the preparation of a calibrator solution: replacing the test sample with a calibrator and performing the steps of the pretreatment method as described above; the calibrator is 7,9-di-EtO-DHP or a solution containing 7,9-di-EtO-DHP.

[0024] In a second aspect, the present invention provides a kit for detecting pyrrolizidine alkaloid protein adducts, and the reagents in the kit include: an internal standard, a releasing agent, a derivatizing agent, and a protein precipitant.

[0025] The internal standard is 7,9-di-EtO-DHP-d3 or a solution containing 7,9-di-EtO-DHP-d3.

[0026] The releasing agent is an ethanol solution containing silver nitrate.

[0027] The derivatizing agent is an ethanol solution of DABA and perchloric acid.

[0028] Preferably, the reagents in the kit further include a calibrator, the calibrator is a solution containing 7,9-di-EtO-DHP, and the concentration of 7,9-di-EtO-DHP in the solution containing 7,9-di-EtO-DHP is in the range of 0.2 nM - 1000 nM.

[0029] Preferably, the concentration of 7,9-di-EtO-DHP-d3 in the solution containing 7,9-di-EtO-DHP-d3 is 5 ng / mL - 50 ng / mL.

[0030] Preferably, the mass fraction of silver nitrate in the ethanol solution containing silver nitrate is 0.8% - 1.2%.

[0031] Preferably, the mass fraction of perchloric acid in the derivatizing agent is 0.8% - 1.5%, and the concentration of DABA is 15 mg / mL - 25 mg / mL.

[0032] Preferably, the protein precipitant is acetonitrile.

[0033] Preferably, the method for using the kit includes the following steps: using the reagents in the kit and performing the treatment according to the pretreatment method as described above.

[0034] In a third aspect, the present invention provides a method for detecting a pyrrolizidine alkaloid protein adduct, and the detection method comprises the following steps:

[0035] Step 1: Preparation of a sample solution: Mix the sample to be tested with a protein precipitant, then centrifuge to remove the first supernatant to obtain a residue, add the internal standard 7,9-di-EtO-DHP-d3, then mix with a releasing agent, centrifuge, and take the second supernatant as the test solution;

[0036] Step 2: LC-MS detection: Take the test solution obtained in Step 1 for LC-MS detection;

[0037] Step 3: Data processing: Calculate the concentration or content of the pyrrolizidine alkaloid protein adduct or the pyrrolizidine alkaloid in the pyrrolizidine alkaloid protein adduct in the blood sample to be tested according to the detection result in Step 2 in combination with the internal standard;

[0038] Wherein, the releasing agent is used to release the pyrrole structural part in the pyrrolizidine alkaloid protein adduct;

[0039] The structure of the internal standard 7,9-di-EtO-DHP-d3 is shown as follows:

[0040] In the formula, D is deuterium hydrogen.

[0041] In some embodiments, the releasing agent is an ethanol solution containing silver nitrate. Preferably, the mixing with the releasing agent in Step 1 is carried out under shaking, and the shaking time is 10 - 30 min, which can be 10 min, 11 min, 15 min, 20 min, 25 min or 30 min, etc.

[0042] In some embodiments, after taking the second supernatant in Step 1, it further includes: mixing the second supernatant with a derivatizing agent, then centrifuging and taking the supernatant as the test solution; preferably, the derivatizing agent is an ethanol solution containing DABA; preferably, the mixing of the second supernatant with the derivatizing agent is carried out under shaking, and the shaking time is 20 - 30 min, which can be 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min or 30 min, etc., and the shaking is carried out at a temperature of 55 - 65 °C, which can be 55 °C, 56 °C, 57 °C, 58 °C, 59 °C, 60 °C, 61 °C, 62 °C, 63 °C, 64 °C or 65 °C, etc.

[0043] In some embodiments, the sample to be tested is a blood sample or a blood cell sample.

[0044] In some embodiments, the protein precipitant is any one or a combination of at least two of methanol, ethanol, acetonitrile, acetone, and isopropanol; preferably, the protein precipitant is acetonitrile.

[0045] In some embodiments, the volume ratio of the blood sample to be tested, acetonitrile, and the ethanol solution containing silver nitrate is 1:(2 - 4):(1 - 3), which can be 1:2:1, 1:2.5:1.5, 1:3:2, 1:3.5:2.5, or 1:4:3, etc.

[0046] Preferably, the mass fraction of silver nitrate in the ethanol solution containing silver nitrate is 0.8% - 1.2%, which can be 0.8%, 0.9%, 1%, 1.1%, or 1.2%, etc.

[0047] Preferably, the internal standard 7,9 - di - EtO - DHP - d3 is added in the form of a solution containing the internal standard 7,9 - di - EtO - DHP - d3. The concentration of 7,9 - di - EtO - DHP - d3 in the solution containing the internal standard 7,9 - di - EtO - DHP - d3 is 5 ng / mL to 50 ng / mL. The volume ratio of the solution containing the internal standard 7,9 - di - EtO - DHP - d3 to the volume of the blood sample to be tested is (1 - 5):10, which can be 1:10, 2:10, 1.5:10, 5:10, etc.

[0048] In some embodiments, the volume ratio of the derivatizing agent in step 1 to the second supernatant is (1 - 2):3, which can be 1:3, 1.2:3, 1.4:3, 1.6:3, 1.8:3, or 2:3, etc. The concentration of DABA in the ethanol solution containing DABA is 15 mg / mL - 25 mg / mL, which can be 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, or 25 mg / mL, etc.

[0049] Preferably, the ethanol solution containing DABA further contains perchloric acid with a mass fraction of 0.8% - 1.5%, which can be perchloric acid with concentrations such as 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, or 1.5%, etc.

[0050] Preferably, the method further includes: preparing a blank sample solution: replacing the sample to be tested with a blank sample and performing the treatment process of step 1; the blank sample is a plasma protein solution of a healthy human or animal.

[0051] Preferably, the method further includes: preparation of a calibrator solution: using the calibrator instead of the sample to be tested and performing the treatment process of step 1; the calibrator is 7,9-di-EtO-DHP (7,9-di-C 2 D 5 O-DHP) or a solution containing 7,9-di-EtO-DHP.

[0052] In some embodiments, the conditions for the LC-MS detection in step 2 include: the injection volume is 1-10 μL, which can be 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL or 10 μL, etc. The column temperature is 45-55 °C, which can be 45 °C, 46 °C, 47 °C, 48 °C, 49 °C, 50 °C, 51 °C, 52 °C, 53 °C, 54 °C or 55 °C, etc. The flow rate is 0.55-0.65 mL / min, such as 0.55 mL / min, 0.56 mL / min, 0.57 mL / min, 0.58 mL / min, 0.59 mL / min, 0.6 mL / min, 0.61 mL / min, 0.62 mL / min, 0.63 mL / min, 0.64 mL / min or 0.65 mL / min, etc.

[0053] Preferably, the conditions for the LC-MS detection further include: the chromatographic column is a Supelco Discovery HS C18 4.6×75 mm, 3 μm or a chromatographic column of the same type.

[0054] Preferably, the conditions for the LC-MS detection further include: the mobile phase consists of mobile phase A and mobile phase B, mobile phase A is a 0.1% formic acid aqueous solution, and mobile phase B is a 0.1% formic acid acetonitrile solution.

[0055] Preferably, the conditions for the LC-MS detection further include: gradient elution is adopted, and the gradient elution process includes: from 0 to 0.3 min, the volume fraction of mobile phase A is 55-65%, and the volume fraction of mobile phase B is 35-45%; from 0.3 to 2 min, the volume fraction of mobile phase A uniformly changes from 55-65% to 0-5%, and the volume fraction of mobile phase B uniformly changes from 35-65% to 95-100%; from 2 to 2.5 min, the volume fraction of mobile phase A is 0-5%, and the volume fraction of mobile phase B is 95-100%; from 2.5 to 2.51 min, the volume fraction of mobile phase A uniformly changes from 0-5% to 55-65%, and the volume fraction of mobile phase B uniformly changes from 95-100% to 35-45%; from 2.51 to 3.5 min, the volume fraction of mobile phase A is 55-65%, and the volume fraction of mobile phase B is 35-45%.

[0056] Preferably, the conditions for LC-MS detection further include: adopting the MRM mode and setting the quantitative ion pairs 344.2-299.2 and 341.2-296.2.

[0057] The above numerical ranges are not limited to the specific examples listed, and other unlisted numerical values within the corresponding numerical ranges are equally applicable.

[0058] In a fourth aspect, the present invention provides a kit for detecting pyrrolizidine alkaloid protein adducts. The kit includes the internal standard 7,9-di-EtO-DHP-d3 and one or more other reagents used in the above detection method. The reagents in the kit can be directly used to detect pyrrolizidine alkaloid protein adducts according to the above detection method, which can shorten the detection time. Combined with the above detection method, the detection of pyrrolizidine alkaloid protein adducts in blood samples can be completed within 1 h, so it can be applied to the detection of a large number of clinical samples.

[0059] Compared with the prior art, the present invention has the following beneficial effects:

[0060] The present invention provides a method for detecting pyrrolizidine alkaloid protein adducts. The selected specific internal standard will not be destroyed during the sample treatment process and has similar characteristics to the analyte, which can effectively correct the entire sample pretreatment process and the subsequent detection process, and thus can improve the precision and accuracy of the detection method. The detection method provided by the present invention can accurately quantify and detect pyrrolizidine alkaloid protein adducts at low concentrations, especially suitable for the situation where the content of pyrrolizidine alkaloid protein adducts in blood samples or blood cell samples is low.

[0061] Compared with the prior art, the present invention has the following beneficial effects:

[0062] The present invention provides a pretreatment method for detecting pyrrolizidine alkaloid protein adducts and a method for detecting pyrrolizidine alkaloid protein adducts. The selected specific internal standard will not be destroyed during the sample treatment process and has similar characteristics to the analyte, which can effectively correct the entire sample pretreatment process and the subsequent detection process, and thus can improve the precision and accuracy of the detection method. The detection method provided by the present invention can accurately quantify and detect pyrrolizidine alkaloid protein adducts at low concentrations, especially suitable for the situation where the content of pyrrolizidine alkaloid protein adducts in blood samples or blood cell samples is low. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 MRM chromatogram of 7,9-di-C2D5O-DHP derivative (ion pair 351.2-301.2);

[0064] Figure 2MRM chromatogram of 7,9-di-EtO-DHP-d3 derivative (ion pair 344.2 - 299.2);

[0065] Figure 3 MRM chromatogram of 7,9-di-EtO-DHP derivative in the test result of plasma sample processed by the method of Example 3 of the present invention using 7,9-di-C2D5O-DHP instead of 7,9-di-EtO-DHP-d3 (ion pair 341.2 - 296.2);

[0066] Figure 4 MRM chromatogram of 7,9-di-C2D5O-DHP derivative in the test result of plasma sample processed by the method of Example 3 of the present invention using 7,9-di-C2D5O-DHP instead of 7,9-di-EtO-DHP-d3 (351.2 - 301.2);

[0067] Figure 5 MRM chromatogram of 7,9-di-EtO-DHP derivative in the test result of plasma sample by the method of Example 3 (ion pair 341.2 - 296.2);

[0068] Figure 6 MRM chromatogram of 7,9-di-EtO-DHP-d3 derivative in the test result of plasma sample by the method of Example 3 (ion pair 344.2 - 299.2). Detailed implementation manners

[0069] The technical solution of the present invention will be further described below through specific implementation manners. Those skilled in the art should understand that the embodiments are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0070] The present invention provides a kit for pyrrolizidine alkaloid protein adducts. The kit includes: an internal standard, a releasing agent. The internal standard is 7,9-di-EtO-DHP-d3 or a solution containing 7,9-di-EtO-DHP-d3. The releasing agent is used to release the pyrrole structural part in the pyrrolizidine alkaloid protein adduct.

[0071] In some embodiments, the releasing agent includes an ethanol solution of silver nitrate. The releasing agent reacts with the pyrrolizidine alkaloid protein adduct to release the pyrrole structural part and combine with the ethoxy group provided by the alcohol.

[0072] In some embodiments, other releasing agents can also be used, such as BF 3 ·(C 2 H 5 ) 2O ethanol solution (see Zhang F, Wang CH, Xiong AZ, Wang W, Yang L, Branford-White CJ, Wang ZT, Bligh SW. Quantitative analysis of total retronecine esters-type pyrrolizidine alkaloids in plant by high performance liquid chromatography. Anal Chim Acta. 2007 Dec 12; 605(1): 94-101. doi: 10.1016 / j.aca.2007.10.021.).

[0073] According to the existing method, such as the existing method 2 in the background art, the PA-protein adduct is detected, and 7,9-di-C 2 D 5 O-DHP is used as the internal standard, and its structure is as follows:

[0074]

[0075] 7,9-di-C 2 D 5 The deuterated hydrogen of 7,9-di-C

[0076] O-DHP is on the ethoxy group. If it is added before the reaction of the releasing agent with the pyrrolizidine alkaloid protein adduct, the deuterated ethoxy group on this internal standard will be replaced by the ethoxy group provided by ethanol in the releasing agent, resulting in the loss of deuterium hydrogen. Therefore, this internal standard is unstable and cannot be added before the release reaction, so the sample preparation error cannot be well corrected.

[0077] 7,9-di-EtO-DHP-d3 used in the present invention can stably exist during the release reaction process and subsequent preparation process, can well correct the sample preparation error, and improve the accuracy and repeatability of the detection results.

[0077] In some embodiments, the solution containing the internal standard is a 7,9-di-EtO-DHP-d3 ethanol solution. Using an ethanol solution makes the solvent consistent with the releasing agent, and 7,9-di-EtO-DHP-d3 also has good solubility in ethanol.

[0078] Preferably, the concentration of 7,9-di-EtO-DHP-d3 in the 7,9-di-EtO-DHP-d3 ethanol solution is 5 ng / ml to 50 ng / mL. This concentration is suitable for the treatment of about 100 μL of plasma samples, and the internal standard concentration can also be adjusted according to actual needs.

[0079] In some embodiments, the mass fraction of silver nitrate in the releasing agent is 0.8% - 2%.

[0080] In some embodiments, the releasing agent may include trifluoroacetic acid, and the concentration of the trifluoroacetic acid is 1% - 5%. Trifluoroacetic acid is beneficial to the reaction of the pyrrole alkaloid protein conjugate of silver nitrate, but it will also inhibit the mass spectrometry response subsequently. Its addition does not significantly improve the method, and it can also be chosen not to be used.

[0081] In some embodiments, the kit further includes a derivatizing agent. The derivatizing agent is used to perform a derivatization reaction with the above-mentioned released pyrrole structural moiety to improve the detection response value.

[0082] In some embodiments, the derivatizing agent includes perchloric acid and DABA (p-diaminobenzaldehyde).

[0083] In some embodiments, the mass fraction of perchloric acid in the derivatizing agent is 0.8% - 1.5%.

[0084] In some embodiments, the concentration of DABA is 15 - 25 mg / mL.

[0085] Preferably, the derivatizing agent consists of 0.8% - 1.5% perchloric acid, 15 - 25 mg / mL DABA, and ethanol.

[0086] In some embodiments, the kit further includes a protein precipitant. Preferably, the protein precipitant is one or more of methanol, ethanol, acetonitrile, isopropanol, and acetone. More preferably, the protein precipitant is acetonitrile, and acetonitrile is used as the protein precipitant. Replacing the conventional solvents such as acetone with acetonitrile can lower the test threshold, and at the same time, the peak area of the target peak detected is the largest, with higher detection sensitivity.

[0087] In some embodiments, the kit further includes a calibrator, and the calibrator includes a reference substance or a solution containing one or more concentrations of the reference substance, with the concentration in the range of 0.2 nM - 1000 nM, and the reference substance is 7,9-di-EtO-DHP.

[0088] In some embodiments, the kit further includes a matrix, and the matrix is human or animal plasma protein. This matrix is used to prepare blank samples and can also be used to prepare calibrator solutions. Preferably, the animal plasma protein is bovine serum albumin (BSA).

[0089] In some embodiments, the kit further includes a mobile phase or reagents for preparing the mobile phase, such as formic acid, acetonitrile, formic acid aqueous solution, and formic acid acetonitrile solution.

[0090] It should be noted that the concentrations or dosages of silver nitrate, perchloric acid, DABA, etc. in this application are obtained through a large number of experiments and optimizations, and are applicable to the detection of pyrrolizidine alkaloid protein adducts in trace blood samples or blood cell samples. The same as 202211512693.3, it will not be elaborated in this application.

[0091] The present invention also provides a method for detecting pyrrolizidine alkaloid protein adducts. This method uses the above-mentioned kit to detect pyrrolizidine alkaloid protein adducts in blood, and specifically includes the following steps:

[0092] Step 1 Preparation of sample solution: Mix the sample to be tested with a protein precipitant, then centrifuge to remove the first supernatant to obtain a residue. Add the internal standard 7,9-di-EtO-DHP-d3 or a solution containing the internal standard 7,9-di-EtO-DHP-d3, then mix with a releasing agent, centrifuge, and take the second supernatant;

[0093] Step 2 LC-MS detection: Take the sample solution obtained in Step 1 for LC-MS detection.

[0094] Step 3 Data processing: Calculate the concentration or content of pyrrolizidine alkaloid protein adducts or the pyrrolizidine alkaloids bound thereto in the blood sample to be tested according to the detection results in Step 2 in combination with the internal standard.

[0095] Among them, the releasing agent is used to release the pyrrole structural part in the pyrrolizidine alkaloid protein adduct.

[0096] In some embodiments, the concentration of 7,9-di-EtO-DHP is calculated through the peak area of 7,9-di-EtO-DHP, the peak area of 7,9-di-EtO-DHP-d3, and the concentration in the sample solution, and then the content of pyrrolizidine alkaloid protein adducts in the blood sample to be tested is calculated, that is, the content of pyrrolizidine alkaloid protein adducts is calculated by the internal standard method.

[0097] In some embodiments, the method further includes: preparing a calibration solution, detecting it with LC-MS, constructing a standard curve, and calculating the content of pyrrolizidine alkaloid protein adducts in the blood sample to be tested through the standard curve. Among them, the calibration solution contains the internal standards 7,9-di-EtO-DHP and 7,9-di-EtO-DHP-d3. Calculate the concentration and peak area ratio of 7,9-di-EtO-DH and 7,9-di-EtO-DHP-d3 in the calibration solution respectively, and construct a standard curve through the ratio of concentrations and the ratio of peak areas. Calculate the concentration of 7,9-di-EtO-DHP in the sample solution through the standard curve, and then calculate the content of pyrrolizidine alkaloid protein adducts in the blood sample to be tested.

[0098] In some embodiments, a derivatizing agent such as DABA can be used to perform a derivatization reaction with 7,9-di-EtO-DHP in the sample and the internal standard 7,9-di-EtO-DHP-d3, and then LC-MS detection is carried out. The ionization effect of the obtained derivative is high, significantly improving the sensitivity of the method, and it can be used for the detection of samples of pyrrolidine alkaloid protein adducts at lower concentrations.

[0099] The technical solutions and technical effects of the present invention are illustrated by specific examples below. Unless otherwise specified, the materials or equipment used in the examples can be obtained through commercial channels, and the technologies not involved in the examples can be realized through the prior art.

[0100] Preparation Example 1

[0101] This preparation example exemplarily provides a preparation method of the internal standard 7,9-di-EtO-DHP-d3, and the specific steps are as follows:

[0102]

[0103] Compound 1, silver carbonate and 1,4-dioxane are mixed and heated to 80 °C, and then 2-propyn-1-ol is slowly added. Stir at 80 °C for 12 h under a nitrogen atmosphere to obtain compound 2 (yellow solid).

[0104]

[0105] Compound 2 is dissolved in dichloromethane (DCM), pyridinium chlorochromate (PCC) is added, and stirred at 20 °C for 12 h to obtain compound 3;

[0106]

[0107] Sodium chlorate and potassium dihydrogen phosphate are dissolved in water, and then mixed with a mixed solution of compound 3 in isopentene and tert-butanol (the volume ratio of isopentene to tert-butanol is 1:1). Stir at 20 °C until the reaction is complete, and separate the aqueous phase and the organic phase. The aqueous phase is acidified with hydrochloric acid and extracted with ethyl acetate. The ethyl acetate layer is combined with the above organic phase, dried and concentrated with a desiccant, and the residue is purified by MPLC (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 0 / 1) to obtain compound 4 (white solid).

[0108]

[0109] At 0 °C, potassium carbonate and methyl iodide (MeI) were added to a N,N-dimethylformamide (DMF) solution of compound 4, and then the mixture was stirred at 20 °C under a nitrogen atmosphere until the reaction was complete. Ice water was added, and DMF was removed by concentration. Then, extraction was carried out with ethyl acetate. The ethyl acetate layer was dried with a desiccant, concentrated, and purified by MPLC (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 0 / 1)

i.e., medium pressure liquid chromatography using SiO 2 packing material, and gradient elution was carried out using petroleum ether and ethyl acetate in a volume ratio of 1 / 0 to 0 / 1

[0110]

[0111] At 0 °C, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, catalyst) and methyl acrylate were added to an acetonitrile (MeCN) solution of compound 5. Then, the mixture was stirred at 20 °C for 10 min, and then heated to 80 °C and stirred until the reaction was complete. Ice water was added, and MeCN was removed by concentration. Extraction was carried out with ethyl acetate. The ethyl acetate layer was washed with 1N hydrochloric acid solution, dried, filtered under reduced pressure, concentrated, and purified by MPLC (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 0 / 1) to obtain compound 6 (white solid).

[0112]

[0113] At 20 °C, potassium tert-butoxide (t-BuOK) was added to a toluene solution (Tol.) of compound 6, and the mixture was stirred for 10 min. Then, it was heated to 110 °C and stirred for 30 min to obtain compound 7. The characterization data are as follows:

[0114] H NMR DMSO-d 6 400 MHz: δ = ppm 6.94 (d, J = 2.48 Hz, 1H), 6.59 (d, J = 2.6 Hz, 1H),

[0115] 4.57 (s, 2H), 4.10 - 4.00 (m, 1H), 3.74 (s, 3H), 3.59 - 3.50 (m, 3H).

[0116]

[0117] At 20 °C, 2M hydrochloric acid was added to a 1,4-dioxane solution of compound 7, and the mixture was stirred at 80 °C for 1 h. Then, 2M hydrochloric acid was added again, and the mixture was stirred at 80 °C until the reaction was complete to obtain compound 8 (480 mg, 2.68 mmol, 33.45% yield). The characterization data are as follows:

[0118] H NMR DMSO-d6 400 MHz: δ = ppm 7.35 (d, J = 2.48 Hz, 1H), 6.90 (d, J = 2.52 Hz,

[0119] 1H), 4.33 (t, J = 6.0 Hz, 2H), 3.74 (s, 3H), 3.05 (t, J = 6.0 Hz, 2H);

[0120]

[0121] At 0 °C, lithium aluminum deuteride was added to a tetrahydrofuran (THF) solution of compound 8, and the mixture was stirred for 1 h to obtain compound 9;

[0122]

[0123] At 0 °C, sodium hydride (NaH) was added to a mixture of compound 9 in tetrahydrofuran (THF) and iodoethane (EtI), and then the temperature was raised to 20 °C and stirred until the reaction was complete. Ice water was added, THF was removed by concentration, and extraction was carried out with DCM. The DCM layer was taken, dried with a desiccant, and then concentrated. The residue was purified by prep-HPLC (column: Waters Xbridge Prep OBD C 18 150 * 40 mm * 10 μm; mobile phase: water (10 mM NH 4 HCO 3 ) - acetonitrile; gradient elution: 0 - 8.0 min, 25% - 55% B) to obtain the target product 7,9-di-EtO-DHP-d3 (yellow gummy solid, 21.81% yield, 95.62% purity), and the characterization data are as follows:

[0124] 1H NMR DMSO-d 6 400 MHz: δ = ppm 6.52 (d, J = 2.4 Hz, 1H), 6.16 (d, J = 2.24 Hz, 1H),

[0125] 4.08 - 3.99 (m, 1H), 3.84 - 3.72 (m, 1H), 3.52 - 3.42 (m, 4H), 2.72 - 2.57 (m, 1H), 2.45 - 2.36 (m, 1H), 1.15 - 1.06 (m, 6H);

[0126] LCMS (ESI+): m / z 213.31 (M + H) + 。

[0127] Example 1

[0128] This example provides a kit, which consists of an internal standard and a releasing agent.

[0129] The internal standard is a solution containing 7,9-di-EtO-DHP-d3 (internal standard solution).

[0130] The preparation process of the internal standard solution is as follows: Weigh 7,9-di-EtO-DHP-d3, dissolve it with ethanol and dilute it to a 10 ng / mL ethanol solution of 7,9-di-EtO-DHP-d3.

[0131] The releasing agent is a silver nitrate ethanol solution.

[0132] The preparation process of the silver nitrate ethanol solution is: Weigh silver nitrate, dissolve it with ethanol under light-shielded conditions, and prepare a 1% silver nitrate ethanol solution. Store it in the dark.

[0133] This example also provides a method for detecting pyrrole alkaloid protein adducts. The method uses the kit of this example, specifically as follows:

[0134] 1. Sample processing method (pretreatment method)

[0135] Take 100 μL of the sample (plasma sample or blood cell sample to be measured), add 300 μL of acetonitrile, vortex, centrifuge at 500 xg for 5 min, discard the supernatant, and obtain the residue.

[0136] (1) Add 20 μL of the internal standard solution to the residue, add 200 μL of 1% silver nitrate ethanol solution, shake at 800 rpm and 20 °C for 60 min, centrifuge at 14000 rpm for 5 min, take the supernatant to obtain the sample solution, and take the sample solution for LC-MS detection.

[0137] 2. LC-MS / MS conditions

[0138] Chromatographic column: Supelco Discovery HS C18 (4.6×75 mm 3 μm); Column temperature: 40 °C; Injection volume: 10 μL; Flow rate: 0.6 mL / min; Phase A: Aqueous solution of 0.1% formic acid and 2 mM ammonium acetate, Phase B: 0.1% formic acid acetonitrile solution.

[0139] Liquid phase gradient:

[0140] Time (min) Phase A Phase B 0.00 85％ 15％ 0.30 85％ 15％ 2.00 5％ 95％ 3.0 5％ 95％ 3.1 85％ 15％ 3.50 85％ 15％

[0141] Mass spectrometry conditions:

[0142]

[0143]

[0144] MRM parameters:

[0145]

[0146] 3. Calculation method

[0147] Based on the detection results, combined with the internal standard peak area and concentration, calculate the concentration of 7,9-di-EtO-DHP in the sample solution, and further calculate the concentration or content of the pyrrole alkaloid bound to the pyrrole protein adduct in the plasma or blood cell sample to be tested, as follows:

[0148] Cs = Ps / (Ci / Pi)

[0149] In the formula, Cs is the concentration of 7,9-di-EtO-DHP in the sample solution; Ps is the peak area of 7,9-di-EtO-DHP; Ci is the internal standard concentration in the sample solution, which is about 10 ng / mL × 20 μL / (200 μL + 20 μL) = 0.4545 ng / mL (about 2 nM) in this example; Pi is the peak area of 7,9-di-EtO-DHP-d3.

[0150] Cb = (Cs × Vs) / Vb

[0151] In the formula, Cb is the concentration (nM) of the pyrrole protein adduct in the plasma or blood cell sample to be tested; Vs is the volume of the sample solution, which is 220 μL in this example; Vb is the volume of the plasma or blood cell sample to be tested, which is 100 μL in this example.

[0152] Example 2

[0153] This example provides a kit, which consists of an internal standard, a releasing agent, and a derivatizing agent.

[0154] The internal standard is a solution containing 7,9-di-EtO-DHP-d3 (internal standard solution).

[0155] The preparation process of the internal standard solution is as follows: Weigh 7,9-di-EtO-DHP-d3, dissolve it with ethanol and dilute it to a 20 ng / mL ethanol solution of 7,9-di-EtO-DHP-d3.

[0156] The releasing agent is an ethanol solution containing silver nitrate. The preparation process is: Weigh silver nitrate, dissolve it with ethanol under light protection conditions, and prepare a 1% silver nitrate ethanol solution. Store it under light protection.

[0157] The derivatizing agent is an ethanol solution containing DABA. The preparation process is: Weigh p-diaminobenzaldehyde ethanol (DABA), add ethanol and perchloric acid, and dissolve it by ultrasonic treatment to prepare an ethanol solution containing 20 mg / mL DABA and 1% perchloric acid.

[0158] This example also provides a detection method for pyrrole alkaloid protein adducts. The method uses the kit of this example, as follows:

[0159] 1. Sample treatment method (pretreatment method)

[0160] Take 100 μL of the sample (plasma sample to be tested or blood cell sample), add 300 μL of acetonitrile, vortex, centrifuge at 500 xg for 5 min, discard the supernatant, and obtain the residue.

[0161] Add 20 μL of internal standard solution to the residue, add 200 μL of releasing agent, shake at 800 rpm at 20 °C for 60 min, centrifuge at 14000 rpm for 5 min, take the supernatant, add 60 μL of derivatizing agent, shake at 1000 rpm at 60 °C for 20 min, centrifuge at 4000 rpm for 5 min, take the supernatant to obtain the sample solution, and perform LC-MS detection.

[0162] 2. LC-MS / MS conditions

[0163] Chromatographic column: Supelco Discovery HS C18 (4.6×75 mm 3 μm); Column temperature: 50 °C; Injection volume: 5 μL; Flow rate: 0.6 mL / min; Phase A: 0.1% formic acid aqueous solution, Phase B: 0.1% formic acid acetonitrile solution.

[0164] Liquid phase gradient:

[0165] Time (min) Phase A Phase B 0.00 60％ 40％ 0.30 60％ 40％ 2.00 0 100％ 2.50 0 100％ 2.51 60％ 40％ 3.50 60％ 40％

[0166] Mass spectrometry conditions:

[0167]

[0168]

[0169] MRM parameters:

[0170]

[0171] 3. Calculation method

[0172] Calculate the concentration or content of pyrrole protein adduct in the plasma sample or blood cell sample to be tested by the internal standard method in the same way as in Example 1.

[0173] Example 3

[0174] This example provides a kit, which consists of an internal standard, a releasing agent, a derivatizing agent and a calibrator.

[0175] The internal standard is a solution containing 7,9-di-EtO-DHP-d3 (internal standard solution).

[0176] The preparation process of the internal standard solution is as follows: Weigh 7,9-di-EtO-DHP-d3, dissolve it with ethanol and dilute it to a 20 ng / mL 7,9-di-EtO-DHP-d3 ethanol solution.

[0177] The releasing agent contains a silver nitrate ethanol solution. The preparation process is as follows: Weigh silver nitrate, dissolve it with ethanol under light-shielded conditions, and prepare a 1% silver nitrate ethanol solution. Store it in the dark.

[0178] The derivatizing agent is a DABA ethanol solution. The preparation process is as follows: Weigh p-diaminobenzaldehyde ethanol (DABA), add ethanol and perchloric acid, and dissolve it by ultrasonic treatment to prepare a 20 mg / mL DABA and 1% perchloric acid ethanol solution.

[0179] The calibrator is a solution containing 7,9-di-EtO-DHP. The preparation process is as follows: Weigh 7,9-di-EtO-DHP, dissolve it with ethanol to prepare a 1 mM 7,9-di-EtO-DHP stock solution, and sequentially dilute it with ethanol to prepare 5 series of calibrators with 7,9-di-EtO-DHP concentrations in the range of 0.2 nM to 1000 nM.

[0180] This embodiment also provides a method for detecting pyrrole alkaloid protein adducts. The method uses the kit of this embodiment, specifically as follows:

[0181] 1. Sample processing method (pretreatment method)

[0182] Take 100 μL of the sample (the plasma sample or blood cell sample to be detected), add 300 μL of acetonitrile, vortex, centrifuge at 500 x g for 5 min, discard the supernatant, and obtain the residue.

[0183] Add 20 μL of the internal standard solution to the residue, add 200 μL of the releasing agent, shake at 800 rpm and 20 °C for 60 min, centrifuge at 14000 rpm for 5 min, take the supernatant, add 60 μL of the derivatizing agent, shake at 1000 rpm and 60 °C for 20 min, centrifuge at 4000 rpm for 5 min, take the supernatant to obtain the sample solution, and perform LC-MS detection.

[0184] Treat and detect the calibrator in the same way as the sample.

[0185] 2. LC-MS / MS conditions

[0186] Chromatographic column: Supelco Discovery HS C18 (4.6 × 75 mm 3 μm); Column temperature: 50 °C; Injection volume: 5 μL; Flow rate: 0.6 mL / min; Phase A: 0.1% formic acid aqueous solution, Phase B: 0.1% formic acid acetonitrile solution.

[0187] Liquid phase gradient:

[0188] Time (min) Phase A Phase B 0.00 60％ 40％ 0.30 60％ 40％ 2.00 0 100％ 2.50 0 100％ 2.51 60％ 40％ 3.50 60％ 40％

[0189] Mass spectrometry conditions:

[0190]

[0191] MRM parameters:

[0192]

[0193]

[0194] 3. Calculation method

[0195] The ratio of the peak area of ​​the 7,9-di-EtO-DHP derivative of the calibrator at 5 series of concentrations to the peak area of ​​the internal standard 7,9-di-EtO-DHP-d3 derivative was calculated, and a standard curve was constructed based on the ratio and the concentration of 7,9-di-EtO-DHP.

[0196] Calculate the ratio of the peak area of ​​the sample solution 7,9-di-EtO-DHP derivative to the peak area of ​​the internal standard 7,9-di-EtO-DHP-d3 derivative, substitute the ratio into the standard curve to obtain the concentration of 7,9-di-EtO-DHP in the sample solution, and then calculate the concentration or content of the pyrrole alkaloid protein adduct in the plasma or blood cell sample to be tested.

[0197] Methodological investigation

[0198] In order to verify the reliability of the results of the method of the present invention for detecting plasma or blood cell samples, a methodological investigation experiment (including quantitative lower limit, linearity, accuracy, precision, etc.) was designed to verify the reliability of the detection method. The results of the methodological investigation of the method in Example 3 are taken as an example below:

[0199] 1.1 Lower limit of quantification

[0200] Referring to the sample processing method of Example 3, the residue was prepared with healthy human plasma, 100 μL of 0.5 nM 7,9-di-EtO-DHP ethanol solution was added to the residue, the LLOQ concentration sample was prepared, and the test was repeated 10 times within the same analytical batch, and the average value and coefficient of variation (CV) were calculated.

[0201] Acceptance criteria: mean bias ≤ ±15%, CV ≤ 20%.

[0202]

[0203]

[0204] The test results showed that at the lower limit of quantitation (LLOQ, 0.5 nM), the mean deviation of the test results was 6.2%, and the precision CV was 10.6%, meeting the acceptance criteria. The method could achieve accurate detection at a concentration of 0.5 nM.

[0205] 1.2 Linearity

[0206] Referring to the sample processing method in Example 3, residues were prepared from healthy human plasma. 100 μL of 7,9 - di - EtO - DHP ethanol solutions with different concentrations were added to the residues to prepare 6 concentration levels, namely 0.2 nM, 2.5 nM, 4 nM, 8 nM, 40 nM, and 100 nM linear verification samples. They were detected within the same analytical run and repeated for 3 runs. The ratio of the peak area of 7,9 - di - EtO - DHP to the peak area of the internal standard was calculated, and a standard curve was fitted with this ratio and the concentration of 7,9 - di - EtO - DHP.

[0207] Acceptance criteria: Linear correlation coefficient r > 0.99.

[0208] The linear equation of the results was Y = 0.04224X + 0.00197, and the correlation coefficient was 0.99918. The linear correlation coefficient > 0.99 indicated that the test method had a good linear relationship within the concentration range of 0.2 nM to 100 nM.

[0209] 1.3 Precision

[0210] Referring to the sample processing method in Example 3, residues were prepared from healthy human plasma. 7,9 - di - EtO - DHP ethanol solutions at two concentrations were added to the residues and detected within the same analytical run. Each concentration was repeated for 10 samples and analyzed for 3 runs (one analytical run per day).

[0211] Acceptance criteria: Repeatability and between - batch difference CV ≤ 15%.

[0212] 1) Intra - day precision (concentration unit: nM)

[0213]

[0214]

[0215] 2) Inter - day precision

[0216] Group Low concentration High concentration Batch-to-batch CV% 3.8％ 3.3％

[0217] The precision verification results showed that both intra - day and inter - day precision CVs were < 15%, indicating that the precision of the method met the requirements for quantitative detection.

[0218] 1.4 Accuracy

[0219] Referring to the sample processing method of Example 1, residues were prepared using healthy human plasma. Different concentrations of 7,9 - di - EtO - DHP ethanol solutions were added to the residues to prepare 2 spiked samples with low (4 nM) and high (8 nM) concentrations. Three replicates were prepared for each concentration and detected within the same analytical batch. The average value was taken, and the recovery rate was calculated according to the following formula:

[0220] Recovery rate = (Average measured concentration / Theoretical concentration) * 100%.

[0221] Acceptance criteria: The recovery rate is in the range of 85% - 115%.

[0222] Recovery rate experiment results

[0223]

[0224] The results of accuracy verification showed that the recovery rates were all in the range of 85% - 115%, indicating that the method had good accuracy and met the requirements for accurate quantification.

[0225] Method comparison study

[0226] Comparative Example 1: The same as the existing method 1 in the background technology, without using an internal standard, to detect pyrrole protein adducts in plasma or blood cell samples.

[0227] Comparative Example 2: The same as the existing method 2 in the background technology, using 7,9 - di - C 2 D 5 O - DHP as an internal standard to detect pyrrole protein adducts in plasma or blood cell samples.

[0228] To compare the differences between the method of the present invention and those of Comparative Example 1 and Comparative Example 2, we conducted parallel comparative tests on accuracy and precision verification, as follows:

[0229] 1. Accuracy comparative test

[0230] 7,9 - di - EtO - DHP stock solutions with different concentrations were added to the plasma and blood cell samples of 10 healthy people respectively to prepare 10 spiked samples. The methods of Comparative Example 1, Comparative Example 2 and Example 3 of the present invention were used for detection respectively, and the recovery rates of the three methods at different concentration levels were investigated. The recovery rate = (Detected value of spiked sample - Detected value of healthy person sample) / Spike amount × 100%. The results of the recovery rate test are shown in the following table.

[0231] Table 1 Results of the recovery rate comparative test for plasma samples

[0232]

[0233] Table 2 Results of the recovery rate comparative test for blood cell samples

[0234]

[0235]

[0236] As can be seen from the experimental results in the above table, the recovery rate of the method of the present invention is closer to 100%, indicating that the accuracy of the method of the present invention is higher. In particular, at lower concentrations (5 nM and below), the recovery rates of Comparative Example 1 and Comparative Example 2 are significantly smaller, while the recovery rate of the method of the present invention still meets the standard requirements of 85% - 115%. This shows that the method of the present invention still has high accuracy at lower concentrations and is particularly suitable for the detection of low-concentration samples.

[0237] 2. Precision comparison test

[0238] 7,9-di-EtO-DHP stock solution was added to 10 plasma and blood cell samples from healthy individuals respectively to prepare plasma and blood cell samples of 7,9-di-EtO-DHP with a concentration of 0.2 nM (lower limit of quantification). Comparative Example 1, Comparative Example 2 and the method of the present invention were used for detection respectively. Each sample was repeatedly measured 10 times within a single analysis batch, and three analysis batches were continuously detected to calculate the within-batch repeatability and between-batch difference, and to investigate the precision of the three methods at a lower concentration level.

[0239] Table 3 Results of repeatability comparison test for plasma samples

[0240]

[0241] Table 4 Results of repeatability comparison test for blood cell samples

[0242]

[0243]

[0244] Table 5 Results of intermediate precision comparison test for plasma samples

[0245]

[0246] Table 6 Results of intermediate precision comparison test for blood cell samples

[0247]

[0248] As can be seen from the above table results, the precision results of the method of the present invention are better than those of the method of Comparative Example 2 and better than those of the method of Comparative Example 1. In addition, at the spiked concentration of 0.2 nM, the concentration of the pyrrole protein adduct detected by using Comparative Example 2 is significantly larger, and its detection result is inaccurate.

[0249] We know that the early metabolism of pyrrolizidine alkaloid-protein adducts in the human body is very fast. The concentration of pyrrolizidine alkaloid-protein adducts in the early stage is high in the blood and decreases rapidly, while the subsequent metabolism is slow. The concentration of pyrrolizidine alkaloid-protein adducts decreases slowly and can exist in the human body at a low concentration for more than 300 days (Gao H, Ruan JQ, Chen J, Li N, Ke CQ, Ye Y, Lin G, Wang JY. Blood pyrrole-protein adducts as a diagnostic and prognostic index in pyrrolizidine alkaloid-hepatic sinusoidal obstruction syndrome. Drug Des Devel Ther. 2015 Aug 25;9:4861-8. doi:10.2147 / DDDT.S87858.). Clinically, from taking foods or drugs containing PA to causing PA-HSOS and then to hospital treatment, a relatively long time has often passed. The concentration of pyrrolizidine alkaloid-protein adducts in the blood is already relatively low. At this time, using the method of the present invention will obtain more accurate test results, which is beneficial to the judgment of the cause and condition. At the same time, using the method of the present invention, the concentration of pyrrolizidine alkaloid-protein adducts in the blood of PA-HSOS patients can be monitored for a long time, providing an index for the clinical treatment effect. In addition, in addition to less commonly used Chinese herbal medicines, PA also exists in the foods (such as honey) we come into daily contact with. The content of these PAs is very low, and the concentration of pyrrolizidine alkaloid-protein adducts formed in the blood is also very low. However, long-term or excessive consumption of foods containing PA may also damage human health, especially for people with poor liver metabolism function. At this time, the method of the present invention can be used to monitor the concentration of pyrrolizidine alkaloid-protein adducts in the blood to timely detect possible risks.

[0250] Internal standard comparison study

[0251] According to the LC-MS conditions of Example 2, 7,9-di-C in Comparative Example 2 2 D 5 O-DHP and 7,9-di-EtO-DHP-d3 of the present invention were derivatized with a derivatizing agent and then injected into LC-MS for detection. The chromatograms of the two internal standard derivatives were collected, and the test results are as Figure 1 (7,9-di-C 2 D 5 O-DHP derivative, ion pair 351.2-301.2) and Figure 2 (7,9-di-EtO-DHP-d3 derivative, ion pair 344.2-299.2). From Figure 1 and Figure 2It can be seen that both internal standard derivatives have mass spectrometry responses.

[0252] Using 7,9-di-C 2 D 5 O-DHP to replace 7,9-di-EtO-DHP-d3, and the same healthy human plasma sample was detected by the method of Example 3. As Figure 3 and Figure 4 shown, they are the chromatographic peaks of 7,9-di-EtO-DHP derivative (ion pair 341.2 - 296.2) and 7,9-di-C 2 D 5 O-DHP derivative (ion pair 351.2 - 301.2) respectively. The chromatographic peaks of 7,9-di-EtO-DHP derivative and 7,9-di-EtO-DHP-d3 derivative obtained by the detection method of Example 3 are respectively as Figure 5 and Figure 6 shown.

[0253] By comparison Figure 3 - Figure 6 it can be seen that Figure 4 there is no peak of 7,9-di-C 2 D 5 O-DHP derivative, Figure 5 there is almost no peak of 7,9-di-EtO-DHP derivative in Figure 3 while there is a very high peak of 7,9-di-EtO-DHP derivative in Figure 5 and it is significantly higher than the peak of 7,9-di-EtO-DHP derivative in Figure 6 . At the same time, 2 D 5 O-DHP is used as the internal standard, after being processed by the method of Example 3, -OC 2 D 5 will be replaced by -OC 2 H 5 . On the one hand, almost no internal standard peak can be detected, and on the other hand, the internal standard will be converted into the peak of the analyte, resulting in false positives in the detection results. When using 7,9-di-EtO-DHP-d3 of the present invention as the internal standard, there is no phenomenon that 7,9-di-EtO-DHP-d3 is converted into the analyte or is unstable during the sample processing.

[0254] Therefore, the existing 7,9-di-C 2 D 5When O-DHP is used for sample treatment according to the method of the present invention, it is almost completely converted into the analyte 7,9-di-EtO-DHP, and the correction of the pretreatment process cannot be achieved. It can only be added after the pretreatment is completed and before the machine detection.

[0255] In addition to the fact that the above-mentioned 7,9-di-EtO-DHP-d3 will not lose deuterium hydrogen during the treatment with silver nitrate ethanol solution, the 7,9-di-EtO-DHP-d3 derivative has a similar mass spectrometry fragmentation pattern to the analyte. Therefore, it is easy to generate fragment ions with relatively high abundances of 299.2 ([M+H-OC 2 H 5 + ) and 255.2 ([M+H-OC 2 H 5 -NC 2 H 6 + ). Selecting these two fragment ions can obtain a higher mass spectrometry response. At the same time, similar to the mass spectrometry response of the analyte, this can also well correct the instrument error, making the method have higher sensitivity and higher accuracy and repeatability at low concentrations.

[0256] In this way, 7,9-di-EtO-DHP-d3 can be used as an internal standard and can be injected without derivatization according to the method of Example 1, or can be injected with derivatization according to the method of Example 2 or Example 3. In addition, after derivatization and injection, both the analyte and the internal standard are more easily ionized, and higher sensitivity can be obtained, and quantitative detection of pyrrolizidine alkaloid protein adducts (0.2 nM) at extremely low concentrations in samples can be carried out. When using other internal standards, such as the non-isotope internal standard benzodiazepine (see Yang X, Li W, Sun Y, Guo X, Huang W, Peng Y, Zheng J. Comparative Study of Hepatotoxicity of Pyrrolizidine Alkaloids Retrorsine and Monocrotaline. Chem Res Toxicol. 2017 Feb 20; 30(2):532-539. doi:10.1021 / acs.chemrestox.6b00260. Epub 2017 Jan 31.), there are significant differences in the structures between this non-isotope internal standard and the analyte, and it cannot undergo a similar derivatization reaction with DABA. When using 7,9-di-C 2 D 5 O-DHP as the internal standard, -OC 2 D 5 in 7,9-di-C 2 D 5 ​​Prone to loss, such as being substituted by -OC in the silver nitrate ethanol solution during the above-mentioned release agent treatment process 2 H 5 Although 7,9-di-C 2 D 5 O-DHP can undergo a similar derivatization reaction with DABA, but -OC 2 D 5 may also be substituted by -OC in the DABA ethanol solution 2 H 5 In addition, -OC 2 D 5 of the 7,9-di-C 2 D 5 O-DHP derivatization product is also prone to loss during the mass spectrometry fragmentation process ([M+H-OC 2 D 5 ) + ), making it equally difficult to obtain an internal standard quantitative ion peak with a similar mass spectrometry response and may interfere with the peak to be measured.

[0257] In summary, the present invention uses 7,9-di-EtO-DHP-d3 as an internal standard for the detection of pyrrolidine alkaloid protein adducts. 7,9-di-EtO-DHP-d3 has similar structural characteristics to the analyte (7,9-di-EtO-DHP) throughout the sample treatment process, does not interfere with the analyte, and has similar mass spectrometry characteristics to the analyte. Therefore, it can effectively correct the entire sample pretreatment process and subsequent detection process, and can greatly improve the accuracy and repeatability of the overall detection method compared with the existing methods.

[0258] The applicant declares that the present invention uses the above-mentioned embodiments to illustrate the pretreatment method and kit for detecting pyrrolidine alkaloid protein adducts of the present invention, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0259] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept scope of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0260] In addition, it should be noted that, in the above specific embodiments, the various specific technical features described can be combined in any appropriate manner without conflict. To avoid unnecessary repetition, the present invention will not separately describe various possible combination manners.

Claims

1. A pretreatment method for detecting pyrrolizidine alkaloid protein adducts, characterized in that, the pretreatment method comprises the following steps: Mix the sample to be tested with a protein precipitant, then centrifuge to remove the first supernatant to obtain a residue, add the internal standard 7,9-di-EtO-DHP-d3, then mix with a releasing agent, centrifuge, take the second supernatant, mix the second supernatant with a derivatizing agent, and then centrifuge to take the supernatant as the sample to be tested; the releasing agent is an ethanol solution containing silver nitrate, the derivatizing agent is an ethanol solution containing DABA, and the sample to be tested is a blood sample or a blood cell sample; the structure of the internal standard 7,9-di-EtO-DHP-d3 is as follows:

2. The pretreatment method according to claim 1, characterized in that, the mixing with the releasing agent is carried out under shaking, and the shaking time is 10 - 30 min; Preferably, the mixing of the second supernatant with the derivatizing agent is carried out under shaking, the shaking time is 20 - 30 min, and the shaking is carried out at a temperature of 55 - 65 °C.

3. The pretreatment method according to claim 1 or 2, characterized in that, the protein precipitant is acetonitrile; Preferably, the volume ratio of the sample to be tested to acetonitrile and the ethanol solution containing silver nitrate is 1:(2 - 4):(1 - 3).

4. The pretreatment method according to any one of claims 1 - 3, characterized in that, the mass fraction of silver nitrate in the ethanol solution containing silver nitrate is 0.8% - 1.2%.

5. The pretreatment method according to any one of claims 1 - 4, characterized in that, the internal standard 7,9-di-EtO-DHP-d3 is added in the form of a solution containing the internal standard 7,9-di-EtO-DHP-d3, and the concentration of 7,9-di-EtO-DHP-d3 in the solution containing the internal standard 7,9-di-EtO-DHP-d3 is 5 ng / mL - 50 ng / mL; Preferably, the volume ratio of the solution containing the internal standard 7,9-di-EtO-DHP-d3 to the volume of the blood sample to be tested is (1 - 5):

10.

6. The pretreatment method according to any one of claims 1 - 5, characterized in that, the volume ratio of the derivatizing agent to the second supernatant is (1 - 2):3; Preferably, the concentration of DABA in the ethanol solution containing DABA is 15 mg / mL - 25 mg / mL; Preferably, the ethanol solution containing DABA further contains perchloric acid with a mass fraction of 0.8% - 1.5%.

7. The pretreatment method according to any one of claims 1 - 6, characterized in that, the pretreatment method further comprises the preparation of a blank sample solution: replacing the sample to be tested with a blank sample and performing the steps of the pretreatment method according to any one of claims 1 - 6; the blank sample is a plasma protein solution of a healthy human or animal. Preferably, the pretreatment method further includes the preparation of a calibrator solution: using the calibrator instead of the sample to be tested, and performing the steps of the pretreatment method according to any one of claims 1-6; the calibrator is 7,9-di-EtO-DHP or a solution containing 7,9-di-EtO-DHP.

8. A kit for detecting pyrrolizidine alkaloid protein adducts, characterized in that the reagents in the kit include: an internal standard, a releasing agent, a derivatizing agent, and a protein precipitant; the internal standard is 7,9-di-EtO-DHP-d3 or a solution containing 7,9-di-EtO-DHP-d3; the releasing agent is an ethanol solution containing silver nitrate; the derivatizing agent is an ethanol solution of DABA and perchloric acid.

9. The kit according to claim 8, characterized in that the reagents in the kit further include a calibrator, the calibrator is a solution containing 7,9-di-EtO-DHP, and the concentration of 7,9-di-EtO-DHP in the solution containing 7,9-di-EtO-DHP is in the range of 0.2 nM - 1000 nM; Preferably, the concentration of 7,9-di-EtO-DHP-d3 in the solution containing 7,9-di-EtO-DHP-d3 is 5 ng / mL to 50 ng / mL; Preferably, the mass fraction of silver nitrate in the ethanol solution containing silver nitrate is 0.8% - 1.2%; Preferably, the mass fraction of perchloric acid in the derivatizing agent is 0.8% - 1.5%, and the concentration of DABA is 15 mg / mL - 25 mg / mL; Preferably, the protein precipitant is acetonitrile.

10. The kit according to claim 9, characterized in that the usage method of the kit includes the following steps: using the reagents in the kit and performing the treatment according to the pretreatment method according to any one of claims 1-7.

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