CN114624376A

CN114624376A - Method for detecting aristolochic acid I-DNA adduct in tissue

Info

Publication number: CN114624376A
Application number: CN202011474001.1A
Authority: CN
Inventors: 任进; 王红阳; 戚新明; 仲云煕; 文文; 陈淑桢; 董亚萍; 邢国振
Original assignee: Third Affiliated Hospital Of Chinese People's Liberation Army Naval Medical University; Shanghai Institute of Materia Medica of CAS
Current assignee: Third Affiliated Hospital Of Chinese People's Liberation Army Naval Medical University; Shanghai Institute of Materia Medica of CAS
Priority date: 2020-12-14
Filing date: 2020-12-14
Publication date: 2022-06-14

Abstract

The invention relates to a method for detecting aristolochic acid I-DNA adduct in tissues. Specifically, the method comprises the following steps: pre-treating, comprising subjecting the tissue sample to tissue digestion in sequence; DNA purification; enzyme incubation and cracking; adding precipitator precipitation protein containing aristolochic acid II internal standard, and taking supernatant as a sample to be detected; performing liquid chromatography, namely performing liquid chromatography separation on the sample to be detected; and mass spectrum, namely, adopting an electrospray ionization source ion source and positive ion multi-reaction monitoring scanning. The detection method has the advantages of strong selectivity, reliability and high detection speed, and can be used for trace detection of aristolochic acid I-DNA adduct.

Description

Method for detecting aristolochic acid I-DNA adduct in tissue

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a liquid chromatography-tandem mass spectrometry method for detecting aristolochic acid I-DNA adduct (dA-ALI) in tissues.

Background

Aristolochic Acid (AA) induces chronic kidney disease and urothelial cancer worldwide. AA mainly induces mutant lesions, and the aristolochic acid-dA adduct generates a unique AT-TA mutation AT adenine, mainly occurs on a non-transcription chain, and has a remarkable peak value AT 5'-CTG-3', while AT present, 24 aristolochiaceae traditional Chinese medicines are still clinically used.

There is currently no reliable dA-ALI bioassay to detect concentrations of dA-ALI in patient tissues to demonstrate that the occurrence and progression of related diseases in patients is associated with dA-ALI exposure.

In view of the above, in order to meet the needs of quantitative detection of dA-ALI concentration in animal and clinical human tissue samples in laboratory, it is necessary to develop a detection method with strong selectivity, reliable method and rapid detection.

Disclosure of Invention

The invention aims to provide a dA-ALI quantitative detection method in a tissue sample, which has strong selectivity, reliable method and rapid detection.

The invention provides a liquid chromatography-tandem mass spectrometry method for detecting aristolochic acid I-DNA adduct (dA-ALI) in a tissue sample, which comprises the following steps:

(i) a pretreatment comprising subjecting the tissue sample to the following steps (i-1) to (i-4) in order:

(i-1) tissue digestion;

(i-2) DNA purification;

(i-3) enzyme incubation lysis; and

(i-4) adding a precipitator containing aristolochic acid II (AAII) internal standard to precipitate protein, and taking supernate as a sample to be detected;

(ii) performing liquid chromatography, namely performing liquid chromatography separation on the sample to be detected, and performing gradient elution by using an ACE C18 chromatographic column (50 multiplied by 2.1mm,5 mu m), wherein a mobile phase A is 0.2% acetic acid aqueous solution, and a mobile phase B is acetonitrile; and

(ii) mass spectrometry, adopting an electrospray ionization source (ESI +) ion source, and positive ion multi-reaction monitoring scanning (MRM); wherein, the response monitoring ion pair of dA-ALI is m/z 543.2 → m/z 427.2 and/or m/z 543.2 → m/z 395.2; internal standard AAII reaction monitors ion pairs of m/z 312.2 → m/z 268.0.

In another preferred embodiment, the dA-ALI response monitoring ion pair is m/z 543.2 → m/z 427.2 and m/z 543.2 → m/z 395.2.

In another preferred embodiment, step (i) has one or more characteristics selected from the group consisting of:

(a) the tissue digestion is enzymatic digestion, preferably, the enzyme is proteinase K;

(b) the DNA purification is to purify the DNA by using Genomic DNA Wash 1 and Genomic DNA Wash 2 and a nucleic acid purification column;

(c) the enzyme incubation cleavage is incubation with DNase I, Nuclease P1, alkaline phosphatase and phosphodiesterase I; and/or

(d) The precipitating agent is selected from the group consisting of: ethanol, methanol, acetonitrile, or a combination thereof, preferably, ethanol;

(e) the precipitant and the sample after enzyme incubation and cracking are mixed in a volume ratio of 1:1-4, preferably 1-1: 2;

(f) in the precipitator containing aristolochic acid II (AAII) internal standard, the concentration of the internal standard is 0.2-5 mug/mL, preferably 0.5-1.00 mug/mL.

In another preferred embodiment, in step (i-4), after the protein is precipitated, the shaking and centrifuging step is further included, preferably, the shaking and centrifuging conditions are that the shaking is performed at 2500rpm for 5min, and the centrifugation is performed at 12000rpm × 5 min.

In another preferred embodiment, in step (ii), the liquid chromatography conditions have one or more characteristics selected from the group consisting of:

(a) the liquid chromatograph is UHPLC;

(b) the sample injector temperature is 4-15 deg.C, preferably 8-10 deg.C;

(c) the column temperature is 30-40 ℃, preferably 35-40 ℃;

(d) the flow rate is 0.2-0.6mL/min, preferably 0.5 mL/min; and/or

(e) The procedure for the gradient elution was:

in another preferred embodiment, in step (iii), the conditions of mass spectrometry have one or more characteristics selected from the group consisting of:

(a) mass spectrometry conditions include DP 55V, CE 65eV when the dA-ALI ion pair is m/z 543.2 → m/z 427.2;

(b) mass spectrometry conditions include DP 55V, CE 65eV when the dA-ALI ion pair is m/z 543.2 → m/z 395.2; and/or

(c) Mass spectrometry conditions for an AAII ion pair of m/z 312.2 → m/z 268.0 include DP 80V, CE 11 eV.

In another preferred embodiment, the conditions of mass spectrometry further comprise one or more conditions selected from the group consisting of: curtain Gas,40psi, colloid Gas,10psi, IonSpray Voltage,5000V, Temperature,550V, Ion Source Gas 1,50psi, Ion Source Gas 2,50psi, Entrance Potential,10V, colloid Cell Exit Potential,15V, or combinations thereof.

In another preferred example, the method further comprises the steps of:

(iv) and (3) calculating by taking the concentration of dA-ALI as a horizontal coordinate and the peak area ratio of dA-ALI and the internal standard AAII as a vertical coordinate, regressing to obtain a corresponding standard curve equation, and calculating the concentration of dA-ALI in the tissue sample.

In another preferred embodiment, the tissue sample is from a mammal, such as a human, rat or mouse.

In another preferred embodiment, the tissue sample is selected from the group consisting of: liver, kidney, or a combination thereof; preferably, the kidney.

In another preferred embodiment, the tissue sample is a tissue slice, preferably, the slice has a thickness of 10-20 μm.

In another preferred embodiment, when the tissue sample is a tissue section, the method further comprises the step of dewaxing the sample before performing step (i-1).

In another preferred embodiment, the amount of DNA in said tissue sample is 1. mu.g or more, preferably 1 to 15. mu.g, more preferably 5 to 10. mu.g.

In another preferred embodiment, the tissue sample is liver, and the mass of the sample (wet weight basis) is 10-30mg, preferably 15-25mg, such as 20mg, such as 15, 16, 17, 18, 19 or 20mg, and the minimum sample size may be 10 mg.

In another preferred embodiment, the tissue sample is kidney, and the mass of the sample (on a wet weight basis) is 5-20mg, preferably 10-15mg, such as 5, 6, 7, 8, 9 or 10mg, and the minimum sample size may be 5 mg.

In another preferred embodiment, the sample to be tested has a sample amount of 10-20 μ L, preferably 15 μ L.

The invention provides a dA-ALI detection kit, which comprises:

(A) a tissue digestion agent comprising: 2 Xdigest Buffer, proteinase K Buffer solution and RNase A solution;

(B) DNA purification reagents, preferably, comprising: genomic lysine Buffer, nucleic acid purification column (e.g., Zymo-Spin)^TMIIC Column), Genomic DNA Wash 1, Genomic DNA Wash 2 and DNA extraction buffer;

(C) enzyme incubation lysis reagent: the method comprises the following steps: DNase I solution, Nuclean P1 solution, alkaline phosphatase solution and phosphodiesterase I solution;

(D) internal standard: aristolochic acid ii (aaii) standard; and

(E) a precipitating agent.

In another preferred embodiment, the kit further comprises:

(F) mobile phase additive: acetic acid.

In another preferred embodiment, the internal standard can be formulated in solution with a precipitating agent, preferably at a concentration of 0.2-5 μ g/mL, more preferably 0.5-1.00 μ g/mL.

In another preferred embodiment, the precipitating agent is selected from the group consisting of: ethanol, methanol, acetonitrile, or a combination thereof, preferably ethanol.

In another preferred embodiment, the kit further comprises one or more of the following:

isopropanol, collection tubes, pasteur pipettes, EP tubes, instructions, or combinations thereof.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 is a flow chart of detection according to one embodiment of the present invention;

FIG. 2 is a chromatogram of a quantitative lower limit sample and a blank sample;

FIG. 3 is a AAI administration protocol for SD rats at different periods;

FIG. 4 is the DNA adduct levels of the liver and kidney of SD rats after AAI administration.

Detailed Description

The present inventors have conducted extensive and intensive studies and, as a result, have provided a method for detecting aristolochic acid I-DNA adducts in tissues through a number of screens and tests. The invention establishes a detection method for accurately quantifying trace dA-ALI in human and animal tissues by using specific reagents and liquid quality detection conditions. The present invention has been completed based on this finding.

Term(s) for

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

The term "room temperature" as used herein means a temperature of 4-40 ℃, preferably 25 ± 5 ℃.

The term "aristolochic acid I-DNA adduct" or "dA-ALI" as used herein refers to an adduct of aristolochic acid I with adenine deoxynucleotide (A).

Detection method

(i-1) tissue digestion;

(i-2) DNA purification;

(i-3) enzyme incubation lysis; and

(ii) mass spectrometry, which adopts an electrospray ionization source (ESI +) ion source and positive ion multi-reaction monitoring scanning (MRM); wherein, the reaction monitoring ion pair of dA-ALI is m/z 543.2 → m/z 427.2 and/or m/z 543.2 → m/z 395.2; internal standard AAII reaction monitors ion pairs of m/z 312.2 → m/z 268.0.

More specifically, the detection method of the present invention may comprise the steps of:

1. pretreatment of

1.1 tissue digestion stage

The tissue samples were incubated with the Digestion digest (DEPC water: 2 Xdigestion Buffer: proteinase K Buffer 4.5: 4.5: 1(v/v/v)) in a water bath (50-55 ℃ C.) for 12-16 h. The digested tissue samples were transferred to a water bath (90-94 ℃) and heated (20-25 min). And immediately adding the RNase A solution after the reaction is finished, uniformly mixing, and standing at room temperature (5-10min) to obtain a digested tissue sample.

1.2DNA purification stage

In the present invention, for the purification of DNA, DNA extraction and purification can be performed by its standard procedures using a DNA extraction kit commonly used in the art, such as an Ezup column animal DNA extraction kit.

More preferably, it is performed by adding Genomic lysine Buffer (3-4 times volume based on the volume of the digested tissue sample) to the digested tissue sample and mixing by vortexing thoroughly; adding isopropanol (1-1.5 vol), mixing, centrifuging (1-2min) in a centrifuge (12000-13000 Xg), transferring supernatant to a small column (Zymo-Spin) with a collection tube^TMIIC Column), centrifuging; changing the collection tube, adding Genomic DNA Wash 1 (3-5 times volume) into the small column with the new collection tube, and centrifuging; absorbing the waste liquid in the collecting pipe, adding Genomic DNA Wash 2 (6-8 times of the volume) into the small column, centrifuging (1-2min) at the rotating speed of 13000-; continuously adding 2-3 times of volume of Genomic DNA Wash 2 into the small column, centrifuging (1-2min) at the rotation speed of 13000-14000g, transferring the small column to a clean EP tube, adding 0.4-0.6 times of volume of DNA extraction buffer into the small column, standing (5-10min), centrifuging (5-6min) at the rotation speed of 16000-17000, and obtaining a purified DNA solution in the EP tube.

1.3 incubation phase

Adding DNase I solution into the purified DNA solution, and then incubating in water bath at 37 ℃ (1.5-2 h); then adding a Nuclean P1 solution to incubate in a water bath at 37 ℃ (3h-3.5 h); finally, alkaline phosphatase solution and phosphodiesterase I solution are respectively added to the DNA sample for incubation in water bath at 37 ℃ (18h-20h), so as to obtain the purified DNA sample after incubation.

1.4 protein precipitation stage

To the incubated purified DNA sample was added a protein precipitant (containing 200ng/mL of internal standard AAII, 1-2 volumes based on the volume of the incubated purified DNA sample) and the protein was precipitated by shaking sufficiently. Centrifuging (12000 rpm. times.5 min), collecting supernatant, (10-15 μ L) and analyzing by LC-MS/MS injection.

2. Liquid chromatography

The liquid chromatograph is Sciex ExionLC^TMAD UHPLC, column ACE C18 column (50X 2.1mm,5 μm). Mobile phase A: 0.2% aqueous acetic acid; mobile phase B: acetonitrile; the flow rate was 0.5 mL/min. The gradient elution procedure is shown in table 1. The column oven was set at 40 deg.C, the autosampler at 8 deg.C, and the sample volume at 15 μ L.

TABLE 1 gradient elution procedure

3. Mass spectrometry

Using Sciex TRIPLE

6500+ data were collected and analyzed in MRM mode and positive ion mode using ESI source. The mass spectrum related parameters were as follows: curtain Gas,40 psi; collision Gas,10 psi; IonSpray Voltage, 5000V; temperature, 550V;

ion Source Gas

1,50 psi;

ion Source Gas

2,50 psi; enterce patent, 10V; mass Spectrometry parameters for collagen Cell extension Potential,15V.dA-ALI and IS are as follows.

TABLE 2

In the tandem mass spectrometry conditions of the invention, for dA-ALI, the reaction monitoring ion pair is m/z 543.2 → m/z 427.2 and/or m/z 543.2 → m/z 395.2. Wherein, m/z 543.2 → m/z 427.2 has the characteristics of strong specificity and high response value, and is suitable for being used as an ion pair for quantification, and on the basis, the detection result of m/z 543.2 → m/z 395.2 is considered, so that the method specificity is increased, and the accuracy is improved, especially under the condition of low sample injection concentration.

Reagent kit

The invention provides a dA-ALI detection kit, which comprises:

(A) a tissue digestion agent comprising: 2 XDigestin Buffer, protease K Buffer solution and RNase A solution;

(C) enzyme incubation lysis reagent: the method comprises the following steps: DNase I solution, Nuclear P1 solution, alkaline phosphatase solution and phosphodiesterase I solution;

(D) internal standard: aristolochic acid ii (aaii) standard; and

(E) a precipitating agent.

In the kit of the present invention, it will be understood by those skilled in the art that, depending on the known properties of each reagent, each reagent may be packaged separately or appropriate reagents may be mixed and then packaged; each reagent may be independently a solid or formulated as a solution.

The main advantages of the present invention include:

1. the invention provides a method for determining the concentration of dA-ALI in human and animal tissues by using AAII as an internal standard and adopting dA-ALI and a specific MRM ion pair of the internal standard, and the methodological verification proves that the method has good reproducibility and high accuracy;

2. according to the invention, after the steps of tissue digestion, DNA purification, enzyme incubation and cracking and protein precipitation are carried out on a sample, the interference of endogenous impurity peaks can be effectively eliminated, so that various examination indexes of methodology are met;

3. the method has strong specificity for detecting human and animal tissue samples (particularly liver and kidney), the detection is completed within 4min by adopting ACE C18 column, and endogenous substances do not interfere with the determination in the separation process under the condition of the invention, so that the high-throughput detection of batch biological samples is met;

4. the method disclosed by the invention has the advantages of good stability, strong selectivity, high sensitivity, rapid detection and small sample dosage, the minimum amount of dA-ALI in human and animal tissue samples can be as low as 1.23pg/mL, the analysis requirements of simple, reliable, high-throughput and condition-controllable clinical tissue samples are met, and the method can be used for evaluating the dA-ALI concentration condition in the tissues of clinical patients.

The invention is further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.

Abbreviations

Materials and reagents

Aristolochic acid I (content: 99.63%), aristolochic acid II (content: 98.8%) were purchased from Nanjing spring and autumn bioengineering Co., Ltd; proteinase K, DNase I (DNase I), Alkaline Phosphatase (AP), nuclease P1(NP1), phosphodiesterase I (PDI), polylysine (P6407-5MG) was purchased from Sigma-Aldrich (Shanghai); ACS reagent grade formic acid (98%), isopropanol, beta-mercaptoethanol, methanol, xylene, 75% ethanol, 95% ethanol, absolute ethanol, glacial acetic acid, hydrochloric acid, deoxyadenosine (dA), deoxyguanosine (dG), deoxycytidine (dC), deoxythymidine (dT), uracil (U) purchased from Shanghai pharmaceutical Limited, China; DEPC water, an Ezup column animal DNA extraction kit, 20 XPBS buffer solution and dimethyl sulfoxide (DMSO) are purchased from Shanghai Biotech limited; methanol and acetonitrile were obtained from merck, germany; PEN film slides were purchased from Leica, germany; the concentrated DAB kit is purchased from China fir Jinqiao limited company in Beijing; PicoGreen was purchased from ThermoFisher Scientific, USA; human clinical samples were from the oriental hepatobiliary surgery hospital of the second university of military medical science. The animal samples were obtained from the institute for drug substance, shanghai, institute of medicine, china, and china.

Example 1

The method flow of the invention can refer to fig. 1:

1. pretreatment of

1.1 tissue digestion stage

2 10 μm slices (total mass of 2 slices is less than 25mg) cut with a paraffin slicer are loaded into a 1.5mL EP tube, centrifuged at 10000 Xg for 30s, 400 μ L of dewaxing agent is added to each sample and vortexed, and the samples are incubated at 55 ℃ for 1 minute for dewaxing. Adding 100 μ L of enzymolysis Digestion solution (DEPC water 45 μ L, 2 Xdigestion Buffer 45 μ L, proteinase K Buffer 10 μ L) into dewaxed tissue, incubating in water bath at 55 deg.C overnight for 16h, transferring digested tissue sample into metal bath, heating at 94 deg.C for 20min, adding prepared RNase A solution immediately once, mixing, and standing at room temperature for 5 min.

1.2DNA purification stage

Adding 350 mu L of Genomic lysine Buffer into the digested tissue sample, fully swirling and mixing uniformly; adding 135 μ L isopropanol, mixing, centrifuging at 12000 Xg for 1min, transferring supernatant to small column with collecting tube (Zymo-Spin)^TMIIC Column), centrifuging at 10000 Xg for 1 min; changing the collecting pipe, adding 400 mu L of Genomic DNA Wash 1 into a small column with a new collecting pipe, and centrifuging at the rotating speed of 10000 Xg for 1 min; sucking the waste liquid in the collecting pipe by using a Pasteur pipette, adding 700 mu L of Genomic DNA Wash 2 into the small column, centrifuging for 1min at the rotating speed of 13000g, and sucking the waste liquid in the collecting pipe by using the Pasteur pipette; 200. mu.L of Genomic DNA Wash 2 was added to the column, centrifuged at 13000g for 1min, transferred to a clean 1.5mL EP tube, 50. mu.L of DNA extraction buffer was added to the column, allowed to stand for 5min, centrifuged at 17000 maximum for 5min, and the purified DNA held in the EP tube was retained.

1.3 incubation phase

mu.L of purified DNA solution was added with 1.5. mu.L of LDNase I solution and incubated for 1.5h in 37 ℃ water bath, then 1. mu.L of Nuclean P1 stock solution was added and incubated for 3h in 37 ℃ water bath, and finally 1.5. mu.L of 30U/mL alkaline phosphatase solution and 8. mu. L0.2U/mL phosphodiesterase I stock solution were added and incubated for 18h in 37 ℃ water bath, respectively.

1.4 protein precipitation stage

60 mu L of ethanol protein precipitant (containing 200ng/mL internal standard, AAII) is added into 60 mu L of the incubated purified DNA sample, the protein is fully precipitated by shaking at 2500rpm for 5min, the mixture is centrifuged at 12000rpm multiplied by 5min, 110 mu L of supernatant is taken, and 15 mu L of LC-MS/MS is subjected to sample injection analysis.

2. Liquid chromatography

The liquid chromatograph is Sciex ExionLC^TMAD UHPLC, column ACE C18 column (50X 2.1mm,5 μm). Mobile phase A: 0.2% aqueous acetic acid; mobile phase B: acetonitrile; the flow rate was 0.5 mL/min. The gradient elution procedure is shown in table 1. The temperature of the column oven was set to 40 ℃, the temperature of the autosampler was set to 8 ℃, and the sample volume was 15 μ L.

TABLE 1 gradient elution procedure

3. Mass spectrometry

Using Sciex TRIPLE

ion Source Gas

1,50 psi;

ion Source Gas

2,50 psi; enterce patent, 10V; mass spectrometry parameters for precision Cell extension Potential,15V. dA-ALI and IS are as follows (where dA-ALI selects two ion pairs).

TABLE 2

4 methodological validation content

4.1 Standard Curve and quality control sample

The linear range of dA-ALI validation was 1.23-30.8 pg/mL, the standard curve contained 8 concentration levels of standard curve samples: 1.23pg/mL (STD1), 10pg/mL (STD2), 20pg/mL (STD3), 50pg/mL (STD4), 75pg/mL (STD5), 100pg/mL (STD6), 150pg/mL (STD7) and 200pg/mL (STD 8).

As shown in FIG. 2, the concentration of the lower limit of quantitation sample (LLOQ) is 1.23pg/mL, and the quality control sample contains 3 concentration levels of standard curve samples: 12.3pg/mL (QC1), 24.6pg/mL (QC2), and 30.8pg/mL (QC 3).

4.2 accuracy and precision

Evaluation of 3 accuracy and precision analysis batch, accuracy and precision analysis batch LLOQ and each concentration level quality control samples were measured in parallel 6 samples. And calculating according to the accompanying standard curve, and solving the precision and accuracy between batches according to the calculation result.

4.3 Selectivity

Respectively inspecting the detection interference of endogenous substances in the blank matrix on the analyte and the internal standard; (2) detecting interference investigation of the internal standard on the analyte; (3) interference of the analyte with the internal standard was investigated.

4.4 recovery

"extracted sample": three concentration level quality control samples of low (QC1), medium (QC2) and high (QC3) were extracted, with 6 replicates for QC samples at each concentration level.

"unextracted sample": the mixed blank matrix was extracted and the same blank sample (DBK) was processed, but the same volume of neat solution containing the analyte and internal standard was added instead of ultrapure water, so that the final concentration of analyte and internal standard in the finally prepared "unextracted sample" was equal to the theoretical concentration of analyte and internal standard after extraction of the "extracted sample", 3 replicates for each concentration level QC.

The ratio of the analyte peak area of the extracted sample to the analyte peak area of the unextracted sample is the analyte extraction recovery rate; the ratio of the internal standard peak area of the extracted sample to the internal standard peak area of the unextracted sample is the internal standard extraction recovery rate.

4.5 matrix Effect

"matrix-containing sample": the blank matrix was extracted and the same blank sample (DBK) was processed, but the same volume of clean solution containing the analyte and internal standard was added instead of ultrapure water, so that the final concentration of analyte and internal standard in the final "matrix-containing sample" was equal to the theoretical concentration of analyte and internal standard after extraction of QC1 or QC 4. Matrix effect 6 different individual blank matrices were examined, each at two low and high concentration levels (QC1 and QC4), respectively, with 3 sample replicates per concentration level.

"matrix-free sample": except that ultrapure water is used for replacing a blank substrate, other pretreatment methods are the same as the treatment of a substrate-containing sample, and 3 repeated samples are prepared at low and high concentrations (QC1 and QC 4).

The ratio of the peak area of the analyte or the internal standard containing the matrix sample to the mean value of the peak area of the analyte or the internal standard containing no matrix sample is the Matrix Factor (MF) of the analyte or the internal standard, and the ratio of the analyte MF to the internal standard MF is the internal standard normalized MF.

4.6 residues

The residue was verified by quantifying the blank sample immediately following the upper limit sample injection.

4.7 sample stability

Low and high two concentration QC samples (QC1 and QC4) were used, 6 replicates each, and analyzed immediately after storage under the conditions evaluated. The stability at room temperature, the stability of a sample injector, the freeze-thaw stability, the freeze-preservation stability and the incubation stability are mainly considered.

5 methodological validation results

5.1 Linearity

For the two ion pairs of dA-ALI, dA-ALI was well linear (weight factor 1/X) in the quantitative range 1.23-30.80pg/mL²)。

TABLE 3

Excess acceptance criteria, no standard curve regression calculations were included.

TABLE 4

Excess accepted standard, no standard curve regression calculation was included.

5.2 accuracy and precision

543.2/427.2 ion pair: the in-batch accuracy is 96.70-109.44%, and the in-batch precision is 6.04-11.72%; the accuracy between batches is 88.37-116.67%, and the precision between batches is 3.01-15.09%.

TABLE 5

543.2/395.2 ion pair: the accuracy in batch is 97.70-104.97%, and the precision in batch is 4.23-18.18%; the accuracy between batches was 101.80-107.43% with an accuracy between batches of 6.47-15.31%.

TABLE 6

5.3 Selectivity

The experimental result shows that the interference of the blank matrix on dA-ALI and IS, and the interference of dA-ALI on IS and the interference of IS on dA-ALI all meet the acceptance standard, and the detection requirement of biological samples IS met.

543.2/427.2：

TABLE 7

543.2/395.2：

TABLE 8

5.4 recovery

For 543.2/427.2 ion pair: the extraction recovery rates of the low, medium and high concentrations meet the acceptance standard, the CV values are respectively 11.15%, 3.66% and 8.15%, and the total recovery rate CV value of the three concentrations is 10.26%; the CV value of the recovery rate of the internal standard extraction was 14.15%.

TABLE 9

For 543.2/395.2 ion pairs: the extraction recovery rates of the aristolochic acid adduct in low, medium and high concentrations meet the acceptance standard, the CV values are respectively 11.15%, 3.66% and 8.15%, and the total recovery rate CV value of the aristolochic acid adduct in three concentrations is 10.26%; the CV value of the recovery rate of the internal standard extraction was 14.15%.

Watch 10

For IS: the average recovery of the low, medium and high concentration levels was 85.40%, 86.13% and 103.29%, respectively, and the total recovery was 91.61%.

5.5 matrix Effect

For 543.2/427.2 ion pair: the normalization factors of the matrix effect internal standard of the lower concentration level and the higher concentration level are respectively 10.20% and 8.66%.

TABLE 11

For 543.2/395.2 ion pairs: the normalization factors of the matrix effect internal standard of the lower concentration level and the higher concentration level are respectively 12.65% and 7.30%.

TABLE 12

5.6 residues

543.2/427.2 and 543.2/395.2 two ion pairs were examined for residues after the highest concentration of aristolochic acid adduct (0.0308ng/mL) was injected. The result shows that the aristolochic acid adduct has no residue and does not influence the detection accuracy of the test sample.

543.2/427.2：

Watch 13

543.2/395.2：

TABLE 14

5.7 stability

Experimental results show that dA-ALI is stable and meets the acceptance standard after test samples are placed for 6 hours at room temperature, placed in a sample injector for 21 hours at 4 ℃, subjected to freeze-thaw cycling for 3 times, placed for 7 days at ultralow temperature for freeze storage and incubated for 22.5 hours at 37 ℃.

Watch 15

TABLE 16

Example 2

6. Qualitative confirmation and quantitative analysis of dA-ALI in human hepatocellular carcinoma samples

FFPE samples of 107 hepatocellular carcinomas were obtained from the oriental hepatobiliary surgery hospital and received ethical approval and patient informed consent. Two 10 μm thick sections of each tissue were taken for DNA isolation, adduct extraction and LC-MS/MS analysis of dA-ALI.

The results are as follows:

TABLE 17

Quantitative analysis of dA-ALI in 107 hepatocellular carcinoma samples

Watch 18

Quantitative analysis of dA-ALI in 107 hepatocellular carcinoma samples

Watch 19

As can be seen from tables 17-19, 8 samples out of 107 showed typical peaks 543.2/427.2 and 543.2/395.2 at 1.21min, and 5 samples out of 8 calculated the concentration of two ion pairs, with the relative abundance between the two ion pairs of only one sample varying by less than 50%.

The method is used for carrying out dA-ALI detection on 107 liver cancer samples. In 23 ion pair 543.2/427.2 positive samples, the calculated concentration range of 4 samples is 0-1 pg/ml, and the calculated concentration of only one sample is more than 5 pg/ml;

of the 10 ion pair 543.2/395.2 positive samples, the calculated concentration of 3 samples was between 0-1 pg/mL, the calculated concentration of 6 samples ranged between 1-5 pg/mL, and the calculated concentration of only one sample was greater than 5pg/mL (same as in 543.2/427.2).

7. dA-ALI assay in rat liver and kidney tissues

7.1 animal grouping and handling

All protocols were approved by the Shanghai institute of drug research animal protection and use Committee. 96 male SD rats were divided into 14 groups of 12 animals in

groups

1,5 and 9, and 6 animals in each of the other groups^[80]. The rationality of the AAI dose is mainly based on the study report on aristolochic acid from the national toxicology program. The specific administration method is shown in figure 3.

Aristolochic Acid I (AAI) preparation frequency and method: the preparation of the stock solution is responsible for the preparation of the stock solution by Shanghai pharmaceutical research institute of Chinese academy of sciences and biological analysis departments. (1) Weighing appropriate amount of NaHCO₃Dissolving in physiological saline with a certain volume, wherein the mass volume ratio is 1%, and uniformly mixing for later use. (2) Weighing appropriate amount of AAI dissolved in a certain volume of 1% NaHCO₃To obtain a stock solution with a final concentration of 5 mg/mL. (3) Before administration, 1% NaHCO was used by the dosing personnel₃The stock solution was diluted to the desired concentration for administration. Formulated once per week. The storage condition of the stock solution is 2-8 ℃. 100mg of 2-acetamidofluorene was weighed into 100mL of corn oil and ground to a uniform suspension. Weighing 1000g of feed in a feeding room, adding 100mL of 2-AAF corn oil suspension in two times, and uniformly stirring for later use. Storing at room temperature. In this study, a dose of 10mg/kg was used as the highest dose in the toxicity test.

In the synergy assay of AAI, multiple doses of 0.1, 1.0 and 10mg/kgAAI were used to evaluate their potential synergy with administration of Diethylnitrosamine (DEN) (fig. 3, # 3-5). As high dose controls for groups 3-5, 2.0, 10 and 20mg/kg AAI (#6-8) were given multiple times to explore potential potency without administration of diethylnitrosamine (FIG. 3, # 6-8). In the experiment for verifying the startability of AAI, AAI (20, 50, 100mg/kg) was administered in a single dose by the gavage method on day 1 to examine its startability in the presence or absence of 2-AAF (FIG. 3, # 11-14). At 3 weeks after initiation, all rats were subjected to a two-thirds liver major resection (removal of the left and middle lobes of the rat liver) to stimulate liver regeneration. After 8 weeks of the experiment, all rats were sacrificed to collect liver and kidney tissues.

7.2 liver and kidney tissues were cut into 2 10 μm slices, and then subjected to LC-MS/MS detection after deparaffinization, and the content of aristolochic acid DNA adduct was measured by the same method as in example 1.

7.3 adduct assay results

The results of the measurements are shown in FIG. 4 and Table 20.

TABLE 20 levels of DNA adducts in rat kidney and liver tissues following AAI administration

Watch 20

As can be seen from FIG. 4 and Table 20, the dA-AL-I content in both the liver and kidney was below the limit of detection for 2-AAF alone; AAI (single)20mg/kg) +2-AAF was not detected in the liver, but could be detected in the kidney. With increasing single dose of AAI (20, 50, 100mg/kg), the amount of dA-AL-I accumulated in the liver and kidney was also significantly increased (A and C in FIG. 4, Table 20).

The number of DNA adducts at the level of dA-AL-I in the kidney was 1.5(20mg/kg), 5.5(50mg/kg) and 13.4(100mg/kg) per 10, respectively⁶A nucleotide adduct; the number of DNA adducts at the level of dA-AL-I in the liver was 0.4(20mg/kg), 0.9(50mg/kg) and 2.0(100mg/kg) per 10, respectively⁶The number of the nucleotide adducts and dA-AL-I measured by the kidney is 6 times of that of the liver, which indicates that the kidney is taken as a sample to be measured, so that the sampling amount can be reduced and/or the detection rate can be improved for the same object.

When AAI doses (2mg/kg, 10mg/kg, 20mg/kg, #6-8) are given multiple times, the kidneyDNA dA-AL-I levels were only 1.5-2 times higher than liver dA-AL-I (FIGS. 4B and D, Table 20); at 1% NaHCO₃Aristolochic acid DNA adducts (dA-AL-I) were not detected in liver and kidney tissues of group, DEN-only group, 2-AAF-only group, and DEN + 2-AAF-only group; under the condition of DEN, dA-AL-I can be detected in liver and kidney tissues after multiple AAI administrations (figure 4B and D, table 20), and the content of dA-AL-I in the liver and kidney tissues is obviously increased along with the increase of the dose of AAI administration, and has obvious dose response relation.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes or modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the appended claims of the present application.

Claims

1. A liquid chromatography-tandem mass spectrometry method for detecting aristolochic acid I-DNA adducts (dA-ALI) in a tissue sample, comprising the steps of:

(i-1) tissue digestion;

(i-2) DNA purification;

(i-3) enzyme incubation lysis; and

(ii) mass spectrometry, which adopts an electrospray ionization source (ESI +) ion source and positive ion multi-reaction monitoring scanning (MRM); wherein, the response monitoring ion pair of dA-ALI is m/z 543.2 → m/z 427.2 and/or m/z 543.2 → m/z 395.2; internal standard AAII reaction monitors ion pairs of m/z 312.2 → m/z 268.0.

2. The method of claim 1, wherein the dA-ALI has a response monitoring ion pair of m/z 543.2 → m/z 427.2 and m/z 543.2 → m/z 395.2.

3. The method of claim 1, wherein step (i) has one or more characteristics selected from the group consisting of:

(e) mixing the precipitant and the sample after enzyme incubation and lysis in a volume ratio of 1:1-4, preferably 1-1: 2; and/or

4. The method of claim 1, wherein in step (ii), the liquid chromatography conditions have one or more characteristics selected from the group consisting of:

(a) the liquid chromatograph is UHPLC;

(b) the injector temperature is 4-15 deg.C, preferably 8-10 deg.C;

(c) the column temperature is 30-40 ℃, preferably 35-40 ℃;

(d) the flow rate is 0.2-0.6mL/min, preferably 0.5 mL/min; and/or

(e) The procedure for the gradient elution was:

5. the method of claim 1, wherein in step (iii), the conditions of mass spectrometry have one or more characteristics selected from the group consisting of:

(c) Mass spectrometry conditions for AAII ion pairs of m/z 312.2 → m/z 268.0 include DP 80V, CE 11 eV.

6. The method of claim 1, wherein the tissue sample is selected from the group consisting of: liver, kidney, or a combination thereof; preferably, the kidney.

7. The method of claim 1, wherein the tissue sample is a tissue section, preferably wherein the section has a thickness of 10-20 μm.

8. A dA-ALI detection kit, comprising:

(C) enzyme incubation lysis reagent: the method comprises the following steps: DNaseI solution, Nuclean P1 solution, alkaline phosphatase solution and phosphodiesterase I solution;

(D) internal standard: aristolochic acid ii (aaii) standard; and

(E) a precipitating agent.

9. The kit of claim 8, wherein the kit further comprises:

(F) mobile phase additive: acetic acid.

10. The kit of claim 8, wherein the precipitating agent is selected from the group consisting of: ethanol, methanol, acetonitrile, or a combination thereof, preferably ethanol.