CN111665303B - Kit for detecting anti-platelet drugs in plasma by ultra-high performance liquid chromatography tandem mass spectrometry technology - Google Patents
Kit for detecting anti-platelet drugs in plasma by ultra-high performance liquid chromatography tandem mass spectrometry technology Download PDFInfo
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Abstract
The invention relates to a kit for detecting an anti-platelet drug in blood plasma by an ultra-high performance liquid chromatography tandem mass spectrometry technology, which has the advantages of simple pretreatment process, low cost, high sensitivity and strong specificity, can be used for separating and detecting the anti-platelet drug within 5 minutes, basically meets the requirements on accuracy and precision, can be used for quantitative analysis of the clinical anti-platelet drug, and provides a reliable detection method for monitoring the therapeutic concentration of the clinical anti-platelet drug.
Description
Technical Field
The invention belongs to the technical field of plasma detection, and particularly relates to a kit for detecting an antiplatelet drug in plasma by an ultra-high performance liquid chromatography tandem mass spectrometry technology, which comprises the following specific drugs: ticagrelor (TIC), desoxyethoxyticagrelor (Deshydroxyethoxy Ticagrelor, DHTIC), aspirin (Acetylsalicylic acid, ASA), salicylic Acid (SA), clopidogrel (CLOP), and Clopidogrel carboxylic acid metabolite (Clopidogrel Carboxylic Acid, CLOPC).
Background
Aspirin is a commonly used non-steroidal drug, and is initially used for relieving fever and pain, and later researches show that small dosage of aspirin can inhibit platelet aggregation, so that the adhesion rate of platelets is reduced, and thus thrombus formation is prevented, 75mg of aspirin is taken as a preventive medicine, is widely used for preventing ischemic cardiovascular diseases, and can be used for myocardial infarction patients when being used at a low dosage. Long-term use is often required as a preventive drug, and long-term use of aspirin has been found to increase bleeding and reduce the risk of neutrophils and the like. Aspirin can be rapidly metabolized to salicylic acid in vivo, resulting in lower aspirin levels in vivo, and too large a difference in the concentrations of the two compounds can also make simultaneous detection of the two compounds difficult. Neither the conventional LC-MS method nor the LC-UV method can detect both substances simultaneously. The LC-MS/MS method allows the simultaneous detection of two compounds in vivo.
Ticagrelor (TIC) is a cyclopentyl triazolopyrimidine drug, is a unique platelet aggregation inhibitor, has a strong inhibition effect on a P2Y12 receptor, can generate reversible inhibition effect on platelet aggregation reaction induced by ADP (adenosine diphosphate), and has certain concentration dependence. Is often used for treating platelet resistance, cholesterol reduction, blood sugar control, blood pressure control and the like. TIC and its major metabolite, deoxyethylticagrelor (DHTIC), all exhibit antiplatelet properties, with 30-40% of the absorbed dose of the major metabolite mostly tolerated well by patients, often reported adverse effects including dizziness, nausea, chest pain, dyspnea and bleeding. The combination with statin may lead to an increased risk of muscle-related adverse events. However, the use of a combination with other drugs may have certain effects resulting in life threatening interactions. Thus, investigation of the concentrations of these compounds is necessary in clinical specimens, and therefore there is a need to develop a sufficiently accurate method for simultaneous determination of TICs, and their metabolites.
Clopidogrel (CLOP) is a thienopyridine derivative, can selectively inhibit the combination of Adenosine Diphosphate (ADP) and platelet receptors and the activation of glycoprotein GPI-Ib/IIIa complex mediated by secondary ADP, can also block the enhancement of platelet activity caused by other peucedanum agonists by releasing ADP, and finally achieves the aim of inhibiting platelet aggregation. Is often used in patients with recent strokes, and in patients with myocardial infarction with definite peripheral arterial disease, and ultimately reduces the occurrence of atherosclerotic events. Clopidogrel is a prodrug, the original drug is not active, the prodrug is mainly metabolized through the liver, carboxylic acid metabolites (CLOPC) of the prodrug are 85% of related compounds in plasma traditional Chinese medicine, the highest concentration time is about 1h after taking the drug, and the concentration difference of clopidogrel and the metabolites of the clopidogrel among different individuals is large, so that accurate drug concentration monitoring is needed.
At present, the comprehensive analysis of domestic and foreign documents and patents only detects one or two compounds at the same time, and no report of detecting the six compounds at the same time exists.
Disclosure of Invention
The invention aims to provide a kit for detecting an antiplatelet drug in blood plasma by using an ultra-high performance liquid chromatography-tandem mass spectrometry technology on the basis of the prior art.
The invention also aims to provide the application of the kit in detecting the anti-platelet drugs in the blood plasma by utilizing the ultra-high performance liquid chromatography tandem mass spectrometry technology.
The technical scheme of the invention is as follows:
a kit for detecting anti-platelet drugs in blood plasma by using ultra-high performance liquid chromatography tandem mass spectrometry technology,
the antiplatelet drugs are respectively: ticagrelor (TIC), deshydroxyethoxy ticagrelor (DHTIC), aspirin (ASA), salicylic Acid (SA), clopidogrel (CLOP), and clopidogrel carboxylic acid metabolite (CLOPC);
the isotope internal standard corresponding to the antiplatelet medicine is respectively: ticagrelor-d 7 (TIC-d 7), dehydroxyethoxy Ticagrelor-d 7 (DHTIC-d 7), aspirin-d 4 (ASA-d 4), salicylic acid-d 4 (SA-d 4) and clopidogrel-d 4 (CLOP-d 4).
The kit comprises the following reagents:
(1) Eluent:
eluent a:0.001% -0.01% formic acid aqueous solution; eluent B: acetonitrile;
(2) Calibrator solution:
preparing a mixed standard solution containing 50000ng/mL of ticagrelor, 50000ng/mL of dehydroxyethoxy ticagrelor, 50000ng/mL of aspirin, 50000ng/mL of salicylic acid, 1000ng/mL of clopidogrel and 1000ng/mL of clopidogrel carboxylic acid metabolite into a calibrator solution with seven different concentration points by using a blank plasma matrix, wherein the seven concentration points of the calibrator solution are as follows:
the concentrations of ticagrelor, dehydroxyethoxy ticagrelor, aspirin and salicylic acid are the same, and the seven concentrations are as follows: 2500ng/mL, 1250ng/mL, 250ng/mL, 125ng/mL, 25ng/mL, 12.5ng/mL, and 5ng/mL;
the clopidogrel and clopidogrel carboxylic acid metabolites have the same concentration, and seven concentrations are as follows: 50ng/mL, 25ng/mL, 5ng/mL, 2.5ng/mL, 0.5ng/mL, 0.25ng/mL, and 0.1ng/mL.
(3) Mixing an internal standard solution:
acetonitrile aqueous solution containing 1500ng/mL ticagrelor-d 7, 1500ng/mL of dehydroxyethoxy ticagrelor-d 7, 2000ng/mL of aspirin-d 4, 2000ng/mL of salicylic acid-d 4, and 20ng/mL of clopidogrel-d 4;
(4) Protein precipitant:
methanol and acetonitrile mixed solution;
(5) Quality control product:
the blank plasma matrix containing antiplatelet medicine is divided into low, medium and high concentrations of QC (L), QC (M) and QC (H), wherein,
QC (L) was diluted 5000-fold with blank plasma matrix for the above mixed standard solution;
QC (M) was diluted 500-fold with blank plasma matrix for the above mixed standard solution;
QC (H) was diluted 50-fold with the above mixed standard solution as blank plasma matrix.
In a preferred embodiment, the eluent A is 0.001% to 0.005% formic acid aqueous solution, preferably 0.004% formic acid aqueous solution.
In one scheme, the volume ratio of the methanol to the acetonitrile in the protein precipitant is 1:1-5; preferably, the volume ratio of methanol to acetonitrile in the protein precipitant is 1:4.
The mixed standard solution mentioned in the invention is prepared according to the following method: 2000. Mu.g/mL of ticagrelor standard mother liquor, 1000. Mu.g/mL of noroxyethoxyticagrelor standard mother liquor, 2000. Mu.g/mL of aspirin standard mother liquor, 5000. Mu.g/mL of salicylic acid standard mother liquor, 100. Mu.g/mL of clopidogrel Lei Biaozhun mother liquor and 100. Mu.g/mL of clopidogrel carboxylic acid metabolite standard mother liquor are formulated into a mixed standard solution containing 50000ng/mL of ticagrelor, 50000ng/mL of noroxyethoxyticagrelor, 50000ng/mL of aspirin, 50000ng/mL of salicylic acid, 1000ng/mL of clopidogrel and 1000ng/mL of clopidogrel carboxylic acid metabolite in an acetonitrile aqueous solution.
The mixed internal standard solution mentioned in the present invention is prepared as follows: the isotope mixed internal standard solution containing 1500ng/mL of ticagrelor-d 7, 1500ng/mL of dehydroxyethoxy ticagrelor-d 7, 2000ng/mL of aspirin-d 4, 2000ng/mL of salicylic acid-d 4 and 20ng/mL of clopidogrel-d 4 is prepared from 150 μg/mL of ticagrelor-d 7 isotope internal standard mother solution, 150 μg/mL of dehydroxyethoxy ticagrelor-d 7 isotope internal standard mother solution, 200 μg/mL of aspirin-d 4 isotope internal standard mother solution, 200 μg/mL of salicylic acid-d 4 isotope internal standard mother solution and 2 μg/mL of clopidogrel-d 4 isotope internal standard mother solution by acetonitrile aqueous solution.
When preparing the mixed standard solution and the mixed internal standard working solution, the acetonitrile aqueous solution adopted is 50% -95% acetonitrile aqueous solution; preferably 70% -90% acetonitrile aqueous solution; more preferably an aqueous 80% acetonitrile solution.
In preparing the mixed standard solution, the blank plasma matrix is blank plasma without anti-platelet drugs.
The concentration of acetonitrile in water referred to herein is generally referred to as the volume concentration.
The plasma referred to in the present invention is human or animal plasma.
In a preferred embodiment, the kit for detecting an antiplatelet agent in plasma by ultra performance liquid chromatography tandem mass spectrometry comprises the following reagents:
(1) Eluent:
eluent a:0.004% formic acid aqueous solution; eluent B: acetonitrile;
(2) Calibrator solution:
preparing a mixed standard solution containing 50000ng/mL of ticagrelor, 50000ng/mL of dehydroxyethoxy ticagrelor, 50000ng/mL of aspirin, 50000ng/mL of salicylic acid, 1000ng/mL of clopidogrel and 1000ng/mL of clopidogrel carboxylic acid metabolite by using 2000 mug/mL of ticagrelor standard mother liquor, 1000 mug/mL of noroxyethoxy ticagrelor standard mother liquor, 2000 mug/mL of aspirin standard mother liquor, 5000 mug/mL of salicylic acid standard mother liquor, 100 mug/mL of clopidogrel Lei Biaozhun product mother liquor and 100 mug/mL of clopidogrel carboxylic acid metabolite standard mother liquor with 80% acetonitrile aqueous solution; preparing the mixed standard solution into seven calibrator solutions with different concentration points by using blank plasma without anti-platelet drugs;
(3) Mixing an internal standard solution:
preparing an isotope mixed internal standard solution containing 1500ng/mL of ticagrelor-d 7, 1500ng/mL of dehydroxyethoxy ticagrelor-d 7, 2000ng/mL of aspirin-d 4, 2000ng/mL of salicylic acid-d 4 and 20ng/mL of clopidogrel-d 4 from 150 μg/mL of a ticagrelor-d 7 isotope internal standard mother solution, 150 μg/mL of a dehydroxyethoxy ticagrelor-d 7 isotope internal standard mother solution, 200 μg/mL of an aspirin-d 4 isotope internal standard mother solution and 2 μg/mL of clopidogrel-d 4 isotope internal standard mother solution by using 80% acetonitrile aqueous solution;
(4) Protein precipitant:
the volume ratio of the methanol to the acetonitrile is 1:4;
(5) Quality control product:
preparing the mixed standard solution into three different concentrations of QC (L), QC (M) and QC (H) by using blank plasma without anti-platelet drugs, wherein the corresponding concentrations of the anti-platelet drug substances in QC (L), QC (M) and QC (H) are shown in a table 1;
TABLE 1 platelet-resistant drug substance control corresponding concentration (Unit: ng/mL)
| Numbering device | Component (A) | QC(L) | QC(M) | QC(H) |
| 1 | TIC | 10 | 100 | 1000 |
| 2 | DHTIC | 10 | 100 | 1000 |
| 3 | ASA | 10 | 100 | 1000 |
| 4 | SA | 10 | 100 | 1000 |
| 5 | CLOP | 0.2 | 2 | 20 |
| 6 | CLOPC | 0.2 | 2 | 20 |
QC (L) contains: 10ng/mL ticagrelor, 10ng/mL of desoxyethoxy ticagrelor, 10ng/mL of aspirin, 10ng/mL of salicylic acid, 0.2ng/mL of clopidogrel, and 0.2ng/mL of clopidogrel carboxylic acid metabolite.
The QC (M) includes: 100ng/mL ticagrelor, 100ng/mL of desoxyethoxy ticagrelor, 100ng/mL of aspirin, 100ng/mL of salicylic acid, 2ng/mL of clopidogrel, and 2ng/mL of clopidogrel carboxylic acid metabolite.
The QC (H) includes: 1000ng/mL ticagrelor, 1000ng/mL of desoxyethoxy ticagrelor, 1000ng/mL of aspirin, 1000ng/mL of salicylic acid, 20ng/mL of clopidogrel, and 20ng/mL of clopidogrel carboxylic acid metabolite.
In a more preferred embodiment, the mixed internal standard solution is prepared as follows:
respectively and accurately transferring a certain volume of the isotope internal standard mother solution of the antiplatelet drug, adding 950 mu L of 80% acetonitrile water solution, and uniformly mixing to obtain 1mL of mixed internal standard solution, wherein the concentration is shown in the table 2 below.
Table 2 preparation of mixed internal standard solutions
| Component (A) | Mother liquor concentration (μg/mL) | Remove volume (μL) | Total volume (mu L) | Mixed internal standard solution concentration (ng/mL) |
| TIC-d7 | 150 | 10 | 1000 | 1500 |
| DHTIC-d7 | 150 | 10 | 1000 | 1500 |
| ASA-d4 | 200 | 10 | 1000 | 2000 |
| SA-d4 | 200 | 10 | 1000 | 2000 |
| CLOP- |
2 | 10 | 1000 | 20 |
In a preferred scheme, the protein precipitant containing the internal standard is prepared by mixing the mixed internal standard solution and the protein precipitant in a volume ratio of 1:99 and is used for ultra-high performance liquid chromatography tandem mass spectrometry detection.
In a more preferred embodiment, the calibrator solution is prepared as follows:
accurately transferring a certain volume of standard mother solution of the antiplatelet drug, adding 870 mu L of 80% acetonitrile water solution, and fully and uniformly mixing to obtain 1mL of mixed standard solution, wherein the concentration is shown in the table 3 below.
TABLE 3 preparation of mixed standard solutions
And (3) preparing standard yeast by adopting a gradient dilution method, taking out the mixed standard solution from a refrigerator at the temperature of minus 20 ℃, swirling for 10 seconds, preparing the highest concentration point of the standard yeast by using the mixed standard solution within 2 minutes, and preserving at the temperature of minus 80 ℃ after preparation. The preparation process is as follows:
adding 10 mu L of mixed standard solution into 190 mu L of blank plasma matrix as a first high-value concentration point (S7); taking a first high-value concentration point (S7), and diluting the first high-value concentration point with an equal volume of blank plasma matrix to obtain a second high-value concentration point (S6); taking a first high-value concentration point (S7), and diluting the first high-value concentration point with 9 times of blank plasma matrix to obtain a third high-value concentration point (S5); taking the second high-value concentration (S6) point, and diluting the second high-value concentration point with 9 times of blank plasma matrix to obtain a fourth high-value concentration point (S4); taking a third high-value concentration point (S5), and diluting the third high-value concentration point with 9 times of blank plasma matrix to obtain a fifth high-value concentration point (S3); taking a fourth high-value concentration point (S4), and diluting the fourth high-value concentration point with 9 times of blank plasma matrix to obtain a sixth high-value concentration point (S2); the fifth high-value concentration point (S3) is diluted by 4 times of blank plasma matrix to obtain a seventh high-value concentration point (S1), and the specific process is shown in the following table 4 (unit: ng/mL):
table 4 standard curve preparation
The application of the kit in detecting the anti-platelet drugs in the blood plasma by utilizing the ultra-high performance liquid chromatography tandem mass spectrometry technology is also within the protection scope of the invention.
The specific detection method comprises the following steps:
a method for detecting anti-platelet drugs in plasma by using ultra-high performance liquid chromatography tandem mass spectrometry technology,
the antiplatelet drugs are respectively: ticagrelor (TIC), deshydroxyethoxy ticagrelor (DHTIC), aspirin (ASA), salicylic Acid (SA), clopidogrel (CLOP), and clopidogrel carboxylic acid metabolite (CLOPC);
the isotope internal standard corresponding to the antiplatelet medicine is respectively: ticagrelor-d 7 (TIC-d 7), noroxyethoxyticagrelor-d 7 (DHTIC-d 7), aspirin-d 4 (ASA-d 4), salicylic acid-d 4 (SA-d 4) and clopidogrel-d 4 (loop-d 4);
the method comprises the steps of detecting the antiplatelet drug in pretreated plasma by adopting an ultra-high performance liquid chromatography tandem mass spectrometry technology, separating an object to be detected from an interference component in a plasma matrix by utilizing ultra-high performance liquid chromatography, and establishing a calibration curve by taking the concentration ratio of a standard substance to an internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis by utilizing a mass spectrometry isotope internal calibration method, wherein the specific chromatographic conditions are as follows:
(1) Ultra-high performance liquid chromatography conditions:
mobile phase a:0.001% -0.01% formic acid aqueous solution; mobile phase B: acetonitrile;
chromatographic column model: waters BEH C18 (2.1X100 mm,1.7 μm);
and (3) performing gradient elution by using the mobile phase A and the mobile phase B as mixed mobile phases, wherein the gradient elution process is as follows: the volume ratio of the mobile phase A to the mobile phase B gradually changes from 95:5 to 40:60 at a constant speed within 0.0-1.0 min; the volume ratio of the mobile phase A to the mobile phase B gradually changes from 40:60 to 2:98 at a constant speed within 1.0-2.0 minutes; within 2.0-3.0 minutes, the volume ratio of mobile phase A to mobile phase B is 2:98; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 2:98 to 95:5 at a constant speed within 3.0-5.0 minutes;
(2) Mass spectrometry conditions:
in an electrospray ionization (ESI) mode, adopting multi-reaction monitoring (MRM) to carry out positive-negative switching scanning; the spray voltage was 3.0kV (ESI+)/2.5 kV (ESI-); the desolvation temperature is 120 ℃; the temperature of the atomizing gas is 500 ℃, the flow rate of the atomizing gas is 800L/h, and the flow rate of the taper hole gas is 150L/h; and simultaneously monitoring each target object and the corresponding isotope internal standard.
In order to improve the chromatographic selectivity, it may be considered to adjust the polarity of the mobile phase. According to the invention, formic acid is added into the mobile phase A, so that ionization efficiency of certain target compounds can be effectively improved, and under the cooperation of other conditions, compared with the detection of the antiplatelet drugs in blood plasma by adopting an LC-MS/MS method in the prior art, the separation and detection of the antiplatelet drugs are completed within 5min, and the pretreatment process is simple, the cost is low, the sensitivity is high, and the specificity is strong. In a preferred embodiment, mobile phase A is 0.001% to 0.005% formic acid in water without affecting the effectiveness of the invention. In a more preferred embodiment, mobile phase a is 0.004% formic acid in water.
In chromatography, the choice of the chromatographic column is important, and the requirements for the chromatographic column are: high column efficiency, good selectivity, high analysis speed, etc. The invention adopts 0.001% -0.01% formic acid aqueous solution and acetonitrile as mobile phases, and the chromatographic column model is as follows: watersBEH C18 (2.1X100 mm,1.7 μm) has high sensitivity, strong specificity, low cost and simple pretreatment process, can complete separation and detection within 5.0min, and meets the requirements of precision and accuracy, and endogenous substances do not interfere with the measurement of samples under the cooperation of other conditions.
The selection of the internal standard is a very important task when using the internal standard method. The ideal internal standard should be capable of being added to the sample in accurate, known amounts and have substantially the same or as consistent as possible physicochemical properties, chromatographic behavior and response characteristics as the sample being analyzed; the internal standard must be sufficiently separated from the components of the sample under chromatographic conditions. According to the invention, ticagrelor-d 7 (TIC-d 7), dehydroxyethoxy ticagrelor-d 7 (DHTIC-d 7), aspirin-d 4 (ASA-d 4), salicylic acid-d 4 (SA-d 4) and clopidogrel-d 4 (CLOP-d 4) are respectively adopted as internal standards, the deuterated internal standard and the object to be detected have the same retention time, chemical property and matrix effect, and the repeatability and accuracy are good when the antiplatelet medicine in blood plasma is measured.
In one embodiment, the flow rate is 0.2 to 0.5mL/min, preferably 0.3mL/min.
Further, the column temperature is 35 to 45 ℃, preferably 40 ℃.
Further, the sample volume is 1 to 5. Mu.L, preferably 1. Mu.L.
In a preferred scheme, when the ultra-high performance liquid chromatography tandem mass spectrometry technology is adopted to detect the antiplatelet drugs in the blood plasma, the specific chromatographic conditions are as follows:
(1) High performance liquid chromatography conditions:
mobile phase a:0.004% formic acid-water solution;
mobile phase B: acetonitrile;
chromatographic column model: waters BEH C18 (2.1X100 mm,1.7 μm);
the gradient elution mode is adopted, and the table 5 is shown; the flow rate is 0.3mL/min, the column temperature is 40 ℃, and the sample injection volume is 1 mu L;
TABLE 5 gradient elution parameters for mobile phases
| Time (min) | Flow rate (mL/min) | ％A | ％B | Curve |
| 0.0 | 0.3 | 95 | 5 | - |
| 1.0 | 0.3 | 40 | 60 | 6 |
| 2.0 | 0.3 | 2 | 98 | 6 |
| 3.0 | 0.3 | 2 | 98 | 6 |
| 5.0 | 0.3 | 95 | 5 | 1 |
(2) Mass spectrometry conditions:
in an electrospray ionization (ESI) mode, adopting multi-reaction monitoring (MRM) to carry out positive-negative switching scanning; the spray voltage was 3.0kV (ESI+)/2.5 kV (ESI-); the desolvation temperature is 120 ℃; the temperature of the atomizing gas is 500 ℃, the flow rate of the atomizing gas is 800L/h, and the flow rate of the taper hole gas is 150L/h; at the same time, antiplatelet drugs and their corresponding isotope internal standards are monitored, and the mass spectrum acquisition parameters of each target object to be detected are shown in Table 6.
Table 6 anti-platelet drug mass spectrometry parameters
| Compounds of formula (I) | Parent ion | Ion | Declustering voltage (V) | Collision voltage (V) | ESI(+/-) |
| CLOPC | 308.0 | 198.0 | 4 | 14 | ESI+ |
| CLOP | 322.1 | 212.0 | 4 | 9 | ESI+ |
| CLOP-d4 | 326.1 | 187.9 | 6 | 20 | ESI+ |
| DHTIC | 479.2 | 126.9 | 12 | 24 | ESI+ |
| DHTIC-d7 | 486.2 | 126.9 | 56 | 56 | ESI+ |
| TIC | 523.2 | 127.0 | 20 | 54 | ESI+ |
| TIC-d7 | 530.2 | 126.9 | 56 | 54 | ESI+ |
| ASA | 136.7 | 93.0 | 34 | 14 | ESI- |
| ASA-d4 | 140.6 | 97.0 | 24 | 14 | ESI- |
| SA | 136.9 | 93.0 | 38 | 14 | ESI- |
| SA-d4 | 141.0 | 97.0 | 44 | 16 | ESI- |
The plasma referred to in the present invention is human or animal plasma.
In one version, the pretreated plasma is prepared as follows: adding a protein precipitant containing an internal standard into the blood plasma, oscillating and centrifuging, and taking a supernatant; wherein the protein precipitant is a mixed solution of methanol and acetonitrile.
Preferably, the volume ratio of methanol to acetonitrile in the protein precipitant is 1:1-5, for example, the volume ratio of methanol to acetonitrile in the protein precipitant is 1:4, without affecting the effect of the present invention.
In a preferred embodiment, the pretreated plasma is prepared as follows: 50 mu L of plasma is taken in a 1.5mL centrifuge tube, 200 mu L of protein precipitant containing an internal standard (the volume ratio of methanol to acetonitrile is 1:1-5) is added into the centrifuge tube, and 60 mu L of supernatant is taken after the centrifuge tube is centrifuged for 4-10 min at the temperature of between 1 and 5 ℃ and 12000-15000 r/min. Wherein the protein precipitant containing the internal standard is prepared by mixing a mixed internal standard solution and the protein precipitant, and the ratio of the mixed internal standard solution to the protein precipitant is 0.1-0.3:19.9-19.7.
In a more preferred embodiment, the pretreated plasma is prepared as follows: taking 50 mu L of plasma in a 1.5mL centrifuge tube, adding 200 mu L of protein precipitant containing an internal standard (the volume ratio of methanol to acetonitrile is 1:4) into the centrifuge tube, and oscillating at a high speed (maximum shaking speed) for 5min; centrifuging at 14000r/min and 4deg.C for 5min; transfer 60. Mu.L of supernatant from EP tube to plastic liner tube and sample in 1. Mu.L. The protein precipitant containing the internal standard is prepared by mixing a mixed internal standard solution and the protein precipitant, wherein the ratio of the mixed internal standard solution to the protein precipitant is 0.2:19.8.
In one embodiment, the internal standard-containing protein precipitant is prepared according to the following method:
preparing a mixed internal standard solution containing 1500ng/mL of ticagrelor-d 7, 1500ng/mL of dehydroxyethoxy ticagrelor-d 7, 2000ng/mL of aspirin-d 4, 2000ng/mL of salicylic acid-d 4 and 20ng/mL of clopidogrel-d 4 from 150 μg/mL of a ticagrelor-d 7 isotope internal standard mother solution, 150 μg/mL of a dehydroxyethoxy ticagrelor-d 7 isotope internal standard mother solution, 200 μg/mL of an aspirin-d 4 isotope internal standard mother solution, 200 μg/mL of salicylic acid-d 4 isotope internal standard mother solution and 2 μg/mL of clopidogrel-d 4 isotope internal standard mother solution by 80% acetonitrile aqueous solution;
200 mu L of the mixed internal standard solution is added into 19.8mL of protein precipitant (the volume ratio of methanol to acetonitrile is 1:4), and the protein precipitant containing the internal standard is obtained.
In one embodiment, the standard solution is prepared as follows:
preparing a mixed standard solution containing 50000ng/mL of ticagrelor, 50000ng/mL of dehydroxyethoxy ticagrelor, 50000ng/mL of aspirin, 50000ng/mL of salicylic acid, 1000ng/mL of clopidogrel and 1000ng/mL of clopidogrel carboxylic acid metabolite by using 2000 mug/mL of ticagrelor standard mother liquor, 1000 mug/mL of noroxyethoxy ticagrelor standard mother liquor, 2000 mug/mL of aspirin standard mother liquor, 5000 mug/mL of salicylic acid standard mother liquor, 100 mug/mL of clopidogrel Lei Biaozhun product mother liquor and 100 mug/mL of clopidogrel carboxylic acid metabolite standard mother liquor with 80% acetonitrile aqueous solution;
preparing the mixed standard solution into a calibrator solution with seven different concentration points by using blank plasma without anti-platelet drugs, wherein the seven concentration points of the calibrator solution are as follows:
the concentrations of ticagrelor, dehydroxyethoxy ticagrelor, aspirin and salicylic acid are the same, and the seven concentrations are as follows: 2500ng/mL, 1250ng/mL, 250ng/mL, 125ng/mL, 25ng/mL, 12.5ng/mL, and 5ng/mL;
the clopidogrel and clopidogrel carboxylic acid metabolites have the same concentration, and seven concentrations are as follows: 50ng/mL, 25ng/mL, 5ng/mL, 2.5ng/mL, 0.5ng/mL, 0.25ng/mL, and 0.1ng/mL;
taking 50 mu L of each concentration point sample into a 1.5mL centrifuge tube, adding 200 mu L of protein precipitant containing an internal standard (the volume ratio of methanol to acetonitrile is 1:4), and oscillating at a high speed (maximum vibration speed) for 5min; centrifuging at 14000r/min and 4deg.C for 5min; transfer 60. Mu.L of supernatant from EP tube to plastic liner tube and sample in 1. Mu.L.
In one scheme, the quality control product is prepared according to the following method: the mixed standard solution is prepared into three different concentrations of QC (L), QC (M) and QC (H) by using blank plasma without anti-platelet drugs:
QC (L) contains: 10ng/mL ticagrelor, 10ng/mL of desoxyethoxy ticagrelor, 10ng/mL of aspirin, 10ng/mL of salicylic acid, 0.2ng/mL of clopidogrel, and 0.2ng/mL of clopidogrel carboxylic acid metabolite.
The QC (M) includes: 100ng/mL ticagrelor, 100ng/mL of desoxyethoxy ticagrelor, 100ng/mL of aspirin, 100ng/mL of salicylic acid, 2ng/mL of clopidogrel, and 2ng/mL of clopidogrel carboxylic acid metabolite.
The QC (H) includes: 1000ng/mL ticagrelor, 1000ng/mL of desoxyethoxy ticagrelor, 1000ng/mL of aspirin, 1000ng/mL of salicylic acid, 20ng/mL of clopidogrel, and 20ng/mL of clopidogrel carboxylic acid metabolite.
By adopting the technical scheme of the invention, the advantages are as follows:
when the kit is used for detecting the antiplatelet drugs in the blood plasma, the pretreatment process is simple, the cost is low, the sensitivity is high, the specificity is strong, the separation and detection of the antiplatelet drugs are completed within 5min, the accuracy and the precision basically meet the requirements, the kit can be used for quantitative analysis of the clinical antiplatelet drugs, and a reliable detection method is provided for monitoring the therapeutic concentration of the clinical antiplatelet drugs.
Drawings
FIG. 1 is an extracted ion flow chromatogram of an antiplatelet drug standard;
FIG. 2 is an extracted ion flow chromatogram of an antiplatelet agent in a plasma sample.
Detailed Description
The invention will be better understood from the following examples. However, it will be readily appreciated by those skilled in the art that the description of the embodiments is provided for illustration only and should not limit the invention as described in detail in the claims.
Example 1:
1. experimental materials and instruments
1. Material
The samples were from plasma samples collected from the 2019 12 month clinic of the south kyo drummer hospital.
(1) Instrument: a Xex TQ-S triple quadrupole mass spectrometer (Waters Corporation); UPLC I-Class ultra-high performance liquid chromatography system (auto sampler, waters Corporation); SCILOGEX D2012 high speed table centrifuge (usa); ultrapure water meter (ELGA LabWater, uk); multitube Vortex mixer (Vortex genie2, usa); an adjustable pipette (Eppendorf 0.5-10. Mu.L, 10-100. Mu.L, 100-1000. Mu.L); glassware, measuring cylinders, etc. .
(2) Reagent consumable: MS grade methanol (Fisher, USA); MS grade acetonitrile (Fisher, USA); HPLC grade acetonitrile (Honeywell, usa); MS grade formic acid (Fisher, USA); HPLC grade methanol (Honeywell, usa); chromatographic column: waters BEH C18 (2.1X100 mm,1.7 μm).
(3) Standard substance: standards and their corresponding internal standards are shown in table 7 below.
(4) Quality control product: the blank plasma matrix containing antiplatelet agent was divided into low, medium and high concentrations of QC (L), QC (M) and QC (H), respectively, as shown in table 1.
Adding films on the upper and lower periphery of the kit, carrying out shock prevention and heat preservation, placing eluent A and eluent B on the upper left, and respectively placing 11 ampoule bottles on the lower left, wherein the ampoule bottles are standard solution, mixed internal standard solution and quality control product respectively; 25mL of protein precipitant was placed on the right.
2. Liquid condition
(1) Chromatographic conditions: mobile phase a:0.004% formic acid-water solution; mobile phase B: acetonitrile. Chromatographic column model: waters BEH C18 (2.1X100 mm,1.7 μm) was eluted by gradient, as detailed in Table 5. The flow rate was 0.3mL/min, the column temperature was 40℃and the sample volume was 1. Mu.L.
(2) Mass spectrometry conditions: in an electrospray ionization detection mode, a mass spectrum scanning mode of multi-reaction monitoring is adopted; the spray voltage was 3.0kV (ESI+) and 2.5kV (ESI-); the desolvation temperature is 120 ℃; the temperature of the atomizing gas is 500 ℃, the flow rate of the atomizing gas is 800L/h, and the flow rate of the taper hole gas is 150L/h; while monitoring each target and the isotope internal standard. The mass spectrum acquisition parameters of each target object to be detected are shown in table 6.
2. Experimental procedure
(1) Preparing a standard substance:
2000. Mu.g/mL of ticagrelor standard stock solution, 1000. Mu.g/mL of noroxyethoxyticagrelor standard stock solution, 2000. Mu.g/mL of aspirin standard stock solution, 5000. Mu.g/mL of salicylic acid standard stock solution, 100. Mu.g/mL of clopidogrel Lei Biaozhun stock solution, and 100. Mu.g/mL of clopidogrel carboxylic acid metabolite standard stock solution were formulated as a mixed standard solution containing 50000ng/mL of ticagrelor, 50000ng/mL of noroxyethoxyticagrelor, 50000ng/mL of aspirin, 50000ng/mL of salicylic acid, 1000ng/mL of clopidogrel, and 1000ng/mL of clopidogrel carboxylic acid metabolite in 80% aqueous acetonitrile (see Table 3 for details).
The above mixed standard solution was prepared as a blank plasma matrix (blank plasma without anti-platelet drug) into calibration solutions of seven different concentration points (see table 4 for details), the concentrations of each of the calibration points are listed in table 4. Seven concentration points of the calibrator solution are:
the concentrations of ticagrelor, dehydroxyethoxy ticagrelor, aspirin and salicylic acid are the same, and the seven concentrations are as follows: 2500ng/mL, 1250ng/mL, 250ng/mL, 125ng/mL, 25ng/mL, 12.5ng/mL, and 5ng/mL;
the clopidogrel and clopidogrel carboxylic acid metabolites have the same concentration, and seven concentrations are as follows: 50ng/mL, 25ng/mL, 5ng/mL, 2.5ng/mL, 0.5ng/mL, 0.25ng/mL, and 0.1ng/mL.
(2) Preparation of protein precipitant containing internal standard
150 μg/mL of ticagrelor-d 7 isotope internal standard mother liquor, 150 μg/mL of dehydroxyethoxy ticagrelor-d 7 isotope internal standard mother liquor, 200 μg/mL of aspirin-d 4 isotope internal standard mother liquor, 200 μg/mL of salicylic acid-d 4 isotope internal standard mother liquor and 2 μg/mL of clopidogrel-d 4 isotope internal standard mother liquor were formulated as a mixed internal standard solution containing 1500ng/mL of ticagrelor-d 7, 1500ng/mL of dehydroxyethoxy ticagrelor-d 7, 2000ng/mL of aspirin-d 4, 2000ng/mL of salicylic acid-d 4 and 20ng/mL of clopidogrel-d 4 in 80% acetonitrile aqueous solution (see Table 2 for details). 200 mu L of the mixed internal standard solution is added into 19.8mL of protein precipitant (the volume ratio of methanol to acetonitrile is 1:4), and the protein precipitant containing the internal standard is obtained.
(3) Preparing a quality control product:
the above mixed standard solution was prepared into three different concentrations of QC (L), QC (M) and QC (H) with blank plasma without antiplatelet drug, and the details are shown in Table 1.
QC (L) contains: 10ng/mL ticagrelor, 10ng/mL of desoxyethoxy ticagrelor, 10ng/mL of aspirin, 10ng/mL of salicylic acid, 0.2ng/mL of clopidogrel, and 0.2ng/mL of clopidogrel carboxylic acid metabolite.
The QC (M) includes: 100ng/mL ticagrelor, 100ng/mL of desoxyethoxy ticagrelor, 100ng/mL of aspirin, 100ng/mL of salicylic acid, 2ng/mL of clopidogrel, and 2ng/mL of clopidogrel carboxylic acid metabolite.
The QC (H) includes: 1000ng/mL ticagrelor, 1000ng/mL of desoxyethoxy ticagrelor, 1000ng/mL of aspirin, 1000ng/mL of salicylic acid, 20ng/mL of clopidogrel, and 20ng/mL of clopidogrel carboxylic acid metabolite.
(4) Sample processing
1) Pretreatment of standard products: taking 50 mu L of each concentration point sample into a 1.5mL centrifuge tube, adding 200 mu L of protein precipitant containing an internal standard (the volume ratio of methanol to acetonitrile is 1:4), and oscillating at a high speed (maximum vibration speed) for 5min; centrifuging at 14000r/min and 4deg.C for 5min; transfer 60. Mu.L of supernatant from EP tube to plastic liner tube and sample in 1. Mu.L.
2) Pretreatment of plasma samples: taking 50 mu L of plasma in a 1.5mL centrifuge tube, adding 200 mu L of protein precipitant containing an internal standard (the volume ratio of methanol to acetonitrile is 1:4) into the centrifuge tube, and oscillating at a high speed (maximum shaking speed) for 5min; centrifuging at 14000r/min and 4deg.C for 5min; transfer 60. Mu.L of supernatant from EP tube to plastic liner tube and sample in 1. Mu.L.
3) Pretreatment of quality control products: respectively taking 50 mu L of quality control product solution QC (L), QC (M) and QC (H) in a 1.5mL centrifuge tube, and then consistent with plasma sample pretreatment, which are not described herein.
The components of the assay kit are shown in Table 8.
Table 8 preparation of the components of the anti-platelet drug analysis kit (100 parts)
Remarks: the protein precipitant containing the internal standard is prepared according to the following method: 200 mu L of the mixed internal standard solution is taken and added into 19.8mL of protein precipitant, and the protein precipitant containing the internal standard is obtained after uniform mixing.
4. Method verification
1. Extracting an ion flow chromatogram: the peak shapes of the standard anti-platelet drug and the plasma sample are symmetrical, and no impurity peak interference exists, which indicates that the anti-platelet drug can be well detected under the condition, and FIG. 1 is an extracted ion flow chromatogram of the standard anti-platelet drug; FIG. 2 is an extracted ion flow chromatogram of an antiplatelet agent in a plasma sample.
2. Calibration curve: and (3) using an isotope internal calibration method, using TargetLynx software to establish a calibration curve by taking the concentration ratio of a standard substance to an internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis, and calculating the concentration of an object to be detected in blood plasma. The linear fitting equation of the antiplatelet drugs in the respective concentration ranges has good linearity, the correlation coefficient is more than 0.99, and the quantitative requirement is met, as shown in Table 9.
TABLE 9 anti-platelet drug Linear regression equation and Linear correlation coefficient
| Sequence number | Compounds of formula (I) | Retention time (min) | Linear range (ng/mL) | Linear equation | Correlation coefficient (r) |
| 1 | CLOPC | 1.71 | 0.1-50 | Y＝0.0889533*X+0.00661122 | 0.999 |
| 2 | CLOP | 3.04 | 0.1-50 | Y＝0.492127*X+0.00365337 | 0.999 |
| 3 | DHTIC | 2.53 | 5-2500 | Y＝0.0.00688601*X+0.00134171 | 0.999 |
| 4 | TIC | 2.55 | 5-2500 | Y＝0.0189636*X+0.0180986 | 0.998 |
| 5 | ASA | 1.88 | 5-2500 | Y＝0.0115011*X-0.0183838 | 0.997 |
| 6 | SA | 2.11 | 5-2500 | Y＝0.0234423*X+0.473475 | 1.000 |
3. Accuracy investigation: and (5) evaluating the accuracy of the method by adopting a standard adding recovery rate test. A mixed blank serum sample is prepared, the standard substances with low, medium and high concentrations are respectively added, the treatment and the measurement are repeated for 5 times by the same steps, the result shows that the labeled recovery rate of the anti-platelet drug is between 87.64 and 112.50 percent, the RSD of 5 repeated tests is between 1.81 and 8.01, and the statistical results are shown in Table 10.
Table 10 results of the anti-platelet drug labeling recovery
4. Precision test: taking a non-interference blank plasma sample, adding anti-platelet drug standard substances with different concentrations to obtain plasma samples with low, medium and high concentrations, repeatedly treating for 6 batches in one day, continuously treating for three days, quantitatively measuring the concentration of the anti-platelet drug by an isotope internal standard method, treating for 3 batches in three days, and calculating the precision between batches to be 2.43-14.32%, wherein the result is shown in Table 11.
TABLE 11 results of batch-to-batch precision test (concentration units: ng/mL)
5. Discussion of the invention
The invention establishes a method for measuring the antiplatelet drugs in human plasma by UPLC-MS/MS. The plasma dosage is small (only 50 mu L) and the pretreatment is simple, and the analysis of various substances by one needle is only 5min, so that the method is simple and quick.
The isotope internal standard method is adopted for quantification, so that not only can the matrix interference be greatly eliminated, but also the result is not influenced by the conditions of pretreatment process, instrument response fluctuation and the like, and the accurate quantification can be achieved. The accuracy of the method is evaluated by a labeling recovery rate test, and the result shows that the labeling recovery rate of the anti-platelet drug is 87.64-112.50%, the RSD of 5 repeated tests is 1.81-8.01, and the accuracy is good.
The reproducibility result of the method shows that the accuracy of the antiplatelet medicine in the batch is 2.50-9.32%, the accuracy of the antiplatelet medicine in the batch is 2.43-14.32%, and the reproducibility of the method is good.
In a word, the method has the advantages of high sensitivity, strong specificity, accuracy and simple pretreatment process, can finish separation and detection of the compound within 5min, has accuracy and precision meeting the requirements, can be used for quantitative analysis of the blood plasma antiplatelet drugs clinically, and provides a reliable detection method for monitoring the concentration of the related drugs.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments may be modified or some technical features may be replaced equivalently; such modifications and substitutions do not depart from the spirit of the invention.
Claims (1)
1. An application of a kit in detecting anti-platelet drugs in plasma by utilizing an ultra-high performance liquid chromatography tandem mass spectrometry technology,
the antiplatelet drugs are respectively: ticagrelor, deshydroxyethoxy ticagrelor, aspirin, salicylic acid, clopidogrel, and clopidogrel carboxylic acid metabolites;
the kit comprises the following reagents:
(1) Eluent:
eluent a:0.004% formic acid aqueous solution; eluent B: acetonitrile;
(2) Calibrator solution:
preparing a mixed standard solution containing 50000ng/mL of ticagrelor, 50000ng/mL of dehydroxyethoxy ticagrelor, 50000ng/mL of aspirin, 50000ng/mL of salicylic acid, 1000ng/mL of clopidogrel and 1000ng/mL of clopidogrel carboxylic acid metabolite by using 2000 μg/mL of ticagrelor standard mother solution, 1000 μg/mL of dehydroxyethoxy ticagrelor standard mother solution, 2000 μg/mL of aspirin standard mother solution, 5000 μg/mL of salicylic acid standard mother solution, 100 μg/mL of clopidogrel Lei Biaozhun product mother solution and 100 μg/mL of clopidogrel carboxylic acid metabolite standard solution with 80% acetonitrile aqueous solution; preparing the mixed standard solution into seven calibrator solutions with different concentration points by using blank plasma without anti-platelet drugs; seven concentration points of the calibrator solution are:
the concentrations of ticagrelor, dehydroxyethoxy ticagrelor, aspirin and salicylic acid are the same, and the seven concentrations are as follows: 2500ng/mL, 1250ng/mL, 250ng/mL, 125ng/mL, 25ng/mL, 12.5ng/mL, and 5ng/mL;
the clopidogrel and clopidogrel carboxylic acid metabolites have the same concentration, and seven concentrations are as follows: 50ng/mL, 25ng/mL, 5ng/mL, 2.5ng/mL, 0.5ng/mL, 0.25ng/mL, and 0.1ng/mL;
(3) Mixing an internal standard solution:
preparing an isotope mixed internal standard solution containing 1500ng/mL of ticagrelor-d 7, 1500ng/mL of dehydroxyethoxy ticagrelor-d 7, 2000ng/mL of aspirin-d 4, 2000ng/mL of salicylic acid-d 4 and 20ng/mL of clopidogrel-d 4 from 150 μg/mL of a ticagrelor-d 7 isotope internal standard mother solution, 150 μg/mL of a dehydroxyethoxy ticagrelor-d 7 isotope internal standard mother solution, 200 μg/mL of an aspirin-d 4 isotope internal standard mother solution and 2 μg/mL of a clopidogrel-d 4 isotope internal standard mother solution with 80% acetonitrile aqueous solution;
(4) Protein precipitant:
the volume ratio of the methanol to the acetonitrile is 1:4;
(5) Quality control product:
preparing the mixed standard solution into three different concentrations of L-QC, M-QC and H-QC by using blank plasma without anti-platelet drugs, wherein,
the L-QC comprises: 10ng/mL ticagrelor, 10ng/mL of desoxyethoxy ticagrelor, 10ng/mL of aspirin, 10ng/mL of salicylic acid, 0.2ng/mL of clopidogrel, and 0.2ng/mL of clopidogrel carboxylic acid metabolite;
the M-QC comprises: 100ng/mL ticagrelor, 100ng/mL of desoxyethoxy ticagrelor, 100ng/mL of aspirin, 100ng/mL of salicylic acid, 2ng/mL of clopidogrel, and 2ng/mL of clopidogrel carboxylic acid metabolite;
the H-QC comprises: 1000ng/mL ticagrelor, 1000ng/mL of desoxyethoxy ticagrelor, 1000ng/mL of aspirin, 1000ng/mL of salicylic acid, 20ng/mL of clopidogrel, and 20ng/mL of clopidogrel carboxylic acid metabolite;
the application comprises the following steps: firstly, separating an object to be detected from an interference component in a plasma matrix by utilizing ultra-high performance liquid chromatography, then, utilizing a mass spectrum isotope internal calibration method, taking the concentration ratio of a standard substance to an internal standard substance as an X axis, taking the peak area ratio of the standard substance to the internal standard substance as a Y axis, establishing a calibration curve, and calculating the content of an antiplatelet drug in plasma, wherein the specific chromatographic conditions are as follows:
(1) Ultra-high performance liquid chromatography conditions:
chromatographic column model: waters BEH C18;
and (3) performing gradient elution by using the mobile phase A and the mobile phase B as mixed mobile phases, wherein the gradient elution process is as follows: the volume ratio of the mobile phase A to the mobile phase B gradually changes from 95:5 to 40:60 at a constant speed within 0.0-1.0 min; the volume ratio of the mobile phase A to the mobile phase B gradually changes from 40:60 to 2:98 at a constant speed within 1.0-2.0 minutes; within 2.0-3.0 minutes, the volume ratio of mobile phase A to mobile phase B is 2:98; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 2:98 to 95:5 at a constant speed within 3.0-5.0 minutes;
(2) Mass spectrometry conditions:
in an electrospray ionization (ESI) mode, adopting multi-reaction monitoring (MRM) to carry out positive-negative switching scanning; the spraying voltage is 3.0kV ESI+/2.5kV ESI-; the desolvation temperature is 120 ℃; the temperature of the atomizing gas is 500 ℃, the flow rate of the atomizing gas is 800L/h, and the flow rate of the taper hole gas is 150L/h; simultaneously monitoring each target and the corresponding isotope internal standard thereof;
the pretreated plasma was prepared as follows: taking 50 mu L of plasma in a 1.5mL centrifuge tube, adding 200 mu L of protein precipitant containing an internal standard into the centrifuge tube, centrifuging for 4-10 min at the temperature of 1-5 ℃ and taking 60 mu L of supernatant;
pretreatment of the calibrator was prepared as follows: taking 50 mu L of each concentration point sample into a 1.5mL centrifuge tube, adding 200 mu L of protein precipitant containing an internal standard into the centrifuge tube, and oscillating for 5min at a high speed; centrifuging at 14000r/min and 4deg.C for 5min; transferring 60 mu L of supernatant in the EP pipe into a plastic lining pipe, and feeding 1 mu L of sample;
the pretreatment of the quality control product is prepared according to the following method: respectively taking 50 mu L of quality control product solution L-QC, 50 mu L of M-QC and 50 mu L of H-QC in a 1.5mL centrifuge tube, adding 200 mu L of protein precipitant containing an internal standard into the centrifuge tubes, and oscillating for 5min at a high speed; centrifuging at 14000r/min and 4deg.C for 5min; transferring 60 mu L of supernatant in the EP pipe into a plastic lining pipe, and feeding 1 mu L of sample;
wherein: the protein precipitant containing the internal standard is prepared by mixing a mixed internal standard solution and the protein precipitant, wherein the volume ratio of the mixed internal standard solution to the protein precipitant is 1:99.
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