CN114778747B

CN114778747B - Method for simultaneously detecting 16 drugs and metabolites thereof in blood by UPLC-MS/MS method

Info

Publication number: CN114778747B
Application number: CN202210716550.8A
Authority: CN
Inventors: 任晓航; 贾永娟; 刘春冉; 赵金宝; 倪君君
Original assignee: Beijing Harmony Health Medical Diagnostics Co ltd
Current assignee: Beijing Harmony Health Medical Diagnostics Co ltd
Priority date: 2022-06-23
Filing date: 2022-06-23
Publication date: 2022-09-02
Anticipated expiration: 2042-06-23
Also published as: CN114778747A

Abstract

The invention relates to a method for simultaneously detecting 16 drugs and metabolites thereof in blood by a UPLC-MS/MS method, wherein the 16 drugs and the metabolites thereof comprise methotrexate, olanzapine, risperidone, 9-hydroxylrisperidone, amisulpride, ziprasidone, perphenazine, sulindazine, sertindole, chlorprothixene, rosuvastatin, theophylline, escitalopram or citalopram, maprotiline, bupropion, hydroxyamphetazone and azithromycin. The method comprises the following steps: determining a standard curve, processing a blood sample to be detected and detecting the sample to be detected by using a UPLC-MS/MS method. The detection method provided by the invention can be used for rapidly and accurately detecting the contents of 16 medicines and metabolites thereof in blood simultaneously. The pretreatment of the sample adopts a protein precipitation method, the used blood volume is less, and the treatment method is simple and easy to implement. The detection method has the advantages of wide linear range, short analysis time, high sensitivity and good reproducibility, and can analyze 10 groups of samples in 1 hour.

Description

Method for simultaneously detecting 16 drugs and metabolites thereof in blood by UPLC-MS/MS method

Technical Field

The invention relates to the technical field of drug detection, in particular to a method for simultaneously detecting 16 drugs and metabolites thereof in blood by using a UPLC-MS/MS method.

Background

Therapeutic Drug Monitoring (TDM) is a pharmaceutical clinical discipline for studying individualized drug treatment mechanisms, techniques, methods and clinical standards and transforming research results into clinical treatments to maximize rational drug administration. Under the guidance of the principle of Pharmacokinetics (PK), the concentration of the drug and the metabolite thereof in the body fluid of a patient after the drug is treated is quantitatively determined by applying a modern analysis technology, the relationship between the drug and the curative effect is analyzed, and the individual drug administration scheme is adjusted, so that the curative effect of the drug is improved, the toxic and side effects are reduced, and the drug treatment cost is maximally saved by reasonable administration. In the prior art, a method for determining the concentration of a drug in a biological sample is required to be used for performing TDM (time division multiplexing) detection, and the current common TDM method mainly comprises a spectral analysis technology, a chromatographic analysis technology, an immunological detection technology and a liquid mass spectrometry technology. However, due to the complexity of the components in biological samples, the use of liquid mass spectrometry is recommended for drug specificity. The LC-MS/MS method has high sensitivity and strong selectivity, and can simultaneously quantify a plurality of medicines. There are many types of TDM drugs, including antibacterial, antiepileptic, antineoplastic, cardiovascular, antiasthmatic, and antipsychotic drugs.

In order to improve the detection efficiency, the LC-MS/MS method is clinically used for carrying out one-method multiple detection on various medicines. However, considering the physical and chemical properties such as polarity, many tests are generally conducted on the same kind of disease-treating drugs such as immunosuppressant or the same kind of drugs such as aminoglycoside.

In practical applications, TDM may be required daily for a wide variety of drugs, and the clinical requirements for the timeliness of the results require time to switch between different methods. The average length of time per sample when monitoring blood drug levels using mass spectrometry is currently reported to be 5-7 minutes, whereas the time to switch between different methods typically takes 30 minutes. On the other hand, when a plurality of methods are needed for detection, the pretreatment and the standard curve preparation are carried out in multiple times of time, and a plurality of instruments are occupied, so that a larger field is needed. Meanwhile, for some patients, different kinds of medicines are taken at the same time, and if different kinds of blood concentration needs to be detected, the detection time is prolonged and more blood is required to be taken by using multiple methods for measurement.

Therefore, there is a need to provide an assay method capable of simultaneously detecting the above-mentioned multiple drugs efficiently and accurately.

Disclosure of Invention

In order to solve the technical problems, the invention provides a method for simultaneously detecting 16 drugs and metabolites thereof in blood by using a UPLC-MS/MS method.

In a first aspect, the present invention provides a method for simultaneously detecting 16 drugs and their metabolites in blood by UPLC-MS/MS method, comprising the steps of:

(1) preparing at least three standard solutions with different concentrations, and respectively establishing the following standard curves of 16 medicaments and metabolites thereof;

wherein said 16 drugs and their metabolites include methotrexate, olanzapine, risperidone, 9-hydroxyrisperidone, amisulpride, ziprasidone, perphenazine, sulindazine, sertindole, chlorprothixene, rosuvastatin, theophylline, escitalopram or citalopram, maprotiline, bupropion, hydroxybupropion, and azithromycin;

(2) pretreating a blood sample to be detected to obtain a sample to be detected;

the pretreatment method comprises the following steps: mixing serum or plasma of a blood sample to be detected, an internal standard working solution and methanol, centrifuging, and mixing a supernatant with ultrapure water to obtain the sample to be detected;

(3) and (3) detecting the sample to be detected by using UPLC-MS/MS, and quantifying 16 drugs and metabolites thereof in the sample to be detected by using the standard curve established in the step (1).

In the invention, 9-hydroxy risperidone and hydroxy bupropion are active metabolites which have the same therapeutic effect and are generated when risperidone and bupropion are metabolized in vivo; meanwhile, escitalopram and citalopram are stereoisomers of each other, and are generally not taken at the same time in clinic, chromatographic separation is not needed, and the detection of citalopram or escitalopram can be carried out according to the advice of doctors. Thus, the detection of both shares the same ion channel.

The pretreatment method provided by the invention does not need the steps of extraction, nitrogen drying and the like, and can prepare the sample to be detected for subsequent detection only by mixing the protein precipitant with the sample to be detected, centrifuging the mixture and mixing the centrifuged mixture with ultrapure water.

In the present invention, the analytical column used in the UPLC-MS/MS detection was SHIMADZU Shim-pack Velock C18.

In the invention, in the UPLC-MS/MS detection, the liquid chromatography conditions comprise:

mobile phase: a is water containing 0.1% formic acid, 1-10 mmol/L ammonium formate or ammonium acetate, B is methanol containing 0.1% formic acid, 1-10 mmol/L ammonium formate or ammonium acetate;

the remaining conditions are shown in Table 1:

TABLE 1

In the UPLC-MS/MS detection, the mass spectrum conditions are shown in Table 2:

TABLE 2

Parameter(s)	Set value	Parameter(s)	Set value
				CUR	10-20 L/min	TEM	350-600℃
CAD	8-9 L/min	GS1	50-80 L/min
				IS	5000-5500 V	GS2	50-80 L/min

Considering that different types of drugs have large differences in polarity, if one-needle multi-detection is adopted, a part of substances to be detected is not retained on a chromatographic column or is not easily eluted, or the phenomenon that the drugs cannot be successfully separated from the drugs and the matrix due to interference occurs, that is, the accurate quantification of the drug concentration cannot be realized due to the interference between chromatographic peaks corresponding to the drugs or between chromatographic peaks and the matrix, is very easy to occur, so that the currently adopted one-needle multi-detection method is generally used for detecting the same type of drugs. The invention selects the specific analytical chromatographic column and is matched with the specific detection conditions, so that the successful detection of 16 medicaments and metabolites thereof can be realized, the mutual interference does not exist, the detection time is short, the analysis time is short, and the accuracy and the precision are higher.

In a preferred embodiment of the present invention, in the pretreatment method, the volume ratio of the serum or plasma to the protein precipitant is 1 (3-10), for example, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, etc., and the volume ratio of the supernatant to the ultrapure water is 1 (0.5-5), for example, 1:1, 1:2, 1:3, 1:4, etc.

As a preferred technical scheme of the invention, the protein precipitator is methanol or acetonitrile.

As a preferable aspect of the present invention, the pretreatment method includes: and centrifuging a blood sample to be detected, performing vortex mixing on serum or plasma, the internal standard working solution and the protein precipitator, centrifuging to obtain a supernatant, and performing vortex mixing on the supernatant and ultrapure water to obtain the supernatant serving as a sample to be detected.

As a specific embodiment of the present invention, the pretreatment method includes:

transferring 10 mu L of internal standard working solution into a 1.5 mL centrifuge tube by using a liquid transfer gun, then adding 100 mu L of serum or plasma, adding 400 mu L of methanol, carrying out vortex oscillation mixing for 5 min at the rotating speed of 2500 r/min, carrying out high-speed centrifugation for 10 min at 14000 r/min, transferring 50 mu L of supernatant into a 1.5 mL plastic centrifuge tube, adding 100 mu L of ultrapure water, carrying out vortex mixing for 1 min at 2500 r/min, and taking 150 mu L of supernatant to obtain a sample to be detected.

As a preferred embodiment of the present invention, the internal standard substances used for the 16 drugs and their metabolites are specifically shown in table 3:

TABLE 3

In the invention, part of the drugs and metabolites thereof can share the internal standard, the use amount of the internal standard can be reduced by sharing the internal standard, the detection cost is reduced, and meanwhile, the detection method can be simplified by reducing the internal standard, and the detection efficiency is improved.

As a preferred embodiment of the present invention, the preparation method of the standard solution comprises: mixing an internal standard working solution with a standard working solution containing 16 drugs and metabolites thereof, and then sequentially mixing the internal standard working solution with methanol and ultrapure water to obtain the standard solution, wherein the standard working solution comprises at least three levels of concentrations;

the standard working solution with each grade of concentration is obtained by diluting a standard substance intermediate solution with a diluent, the intermediate solution is obtained by diluting a standard substance stock solution with the diluent, and the standard substance stock solution is obtained by dissolving the respective standard substances of the 16 medicines and the metabolites thereof with a solvent.

As a specific embodiment of the present invention, the preparation method of the standard solution comprises:

placing 10 mu L of standard working solution, 10 mu L of internal standard working solution, 90 mu L of ultrapure water and 400 mu L of methanol with at least three different concentrations into a 1.5 mL centrifuge tube by using a pipettor, mixing to prepare at least three standard solutions, shaking and uniformly mixing the standard solutions for 1 min at a rotation speed of 2500 r/min, transferring 50 mu L of uniformly mixed liquid to 1.5 mL centrifuge tube, adding 100 mu L of pure water, and performing vortex mixing for 1 min at 2500 r/min to obtain at least three standard solutions with different concentrations.

As a preferred technical solution of the present invention, the preparation method of the internal standard working solution comprises: and (3) diluting and mixing 15 internal standard stock solutions used by 16 medicines and metabolites thereof by using a diluent to obtain the internal standard working solution.

In a preferred embodiment of the present invention, the diluent is an aqueous methanol solution or an aqueous acetonitrile solution, preferably an aqueous methanol solution or an aqueous acetonitrile solution with a concentration of 50-100%, preferably an aqueous methanol solution with a concentration of 50% or an aqueous acetonitrile solution with a concentration of 50%.

The 50-100% methanol aqueous solution or acetonitrile aqueous solution of the present invention means that the volume ratio of methanol to water in the methanol aqueous solution is 1 (0-1), or the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 1 (0-1).

The diluent is specifically selected, on one hand, the diluent has good solubility for 16 drugs and metabolites thereof and 15 internal standards, and on the other hand, the specific diluent provided by the invention is used for preparing the standard intermediate solution, so that the concentration of the standard intermediate solution can be basically kept unchanged after the standard intermediate solution is frozen for a long time or is repeatedly used for many times, the determination of subsequent standard curves is not influenced, and the detection result is not influenced.

In the invention, the interference of the matrix can be reduced to the maximum extent by selecting the specific internal standard and matching with the specific sample pretreatment condition, and the interference of other impurities in the blood sample can be further avoided by combining the chromatographic column, the detection condition and the like selected by the invention, so that when the standard is established, a blank matrix can not be added, the method for preparing the standard solution is simplified, and the detection accuracy can be ensured.

As a preferred technical scheme of the invention, the blood sample to be detected is blood from a serum blood collection tube, an EDTA plasma blood collection tube and a heparin lithium blood collection tube, and the blood sample is centrifuged to obtain a supernatant so as to obtain the plasma sample or the serum sample.

Compared with the prior art, the technical scheme provided by the embodiment of the invention has the following advantages:

(1) the pretreatment method provided by the invention can shorten the sample pretreatment time within 17 minutes, and the standard curve is established without adding blank blood matrix for pretreatment, so that the detection time from blood sampling to detection result output can be greatly shortened;

(2) the detection method provided by the invention uses less plasma or serum, and can meet the detection requirements of 16 drugs and metabolites by only 100 mu L;

(3) the detection method provided by the invention can be used for simultaneously detecting 16 drugs and metabolites thereof with different polarities and different types in blood, the detection time is short, the analysis time of the method is only 5.5 minutes, and 10 groups of samples can be analyzed within 1 hour. Meanwhile, the use of internal standards is reduced through the sharing of part of internal standards, and the detection cost is reduced;

(4) the detection method provided by the invention has wide standard curve range, can completely cover the reference treatment interval concentration and the warning concentration of 16 blood drugs and metabolites thereof, and avoids the problem that the actual clinical samples possibly exceed the detection linear range as much as possible;

(5) the detection method provided by the invention has the advantages of high detection accuracy, high sensitivity and good reproducibility (stability).

Detailed Description

In order that the above objects, features and advantages of the present invention may be more clearly understood, a solution of the present invention will be further described below. It should be noted that the embodiments of the present invention and features of the embodiments may be combined with each other without conflict.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those described herein; it is to be understood that the embodiments described in this specification are only some embodiments of the invention, and not all embodiments.

Example 1

The embodiment provides a preparation method of a standard solution, which comprises the following steps of preparing a standard stock solution, preparing a standard working solution, preparing an internal standard stock solution, preparing an internal standard working solution and preparing the standard solution:

(1) preparation of a Standard working fluid

A. Diluting standard substance stock solutions corresponding to the 16 medicines and metabolites thereof respectively by using a diluent to obtain a standard substance intermediate solution, wherein the concentrations of the standard substance stock solutions and the standard substance intermediate solution are shown in a table 4;

TABLE 4

Name of substance	Stock solution concentration of Standard substance (μ g/mL)	Concentration of intermediate solution of standard (μ g/mL)
			Methotrexate (MTX)	1943	1080
Rosuvastatin	2274	41
			Risperidone	3719	54
9-Hydroxyrisperidone	1800	54
			Olanzapine	4811	65
Perphenazine	1479	7
			Bupropion derivatives	1055	8
Hydroxy bupropion	5003	675
			Chlorprothixene	1237	203
Sertindole	2078	68
			Azithromycin	10000	1350
Escitalopram	2495	81
			Citalopram	1640	81
Maprotiline	5603	97
			Thilidazine	2277	432
Amisulpride	4035	270
			Ziprasidone derivatives	905	216
Theophylline	5220	3375

B. Mixing the intermediate solution of the standard product, and diluting with a diluent to obtain a standard working solution (L7);

C. diluting the standard working solution by using a diluent to obtain at least three standard working solutions with different concentrations;

the diluent used in this example was an aqueous methanol solution of methanol to water (V: V) =1: 1;

this example provides 7 different concentrations of standard working solutions, and the concentration ranges of 16 drugs and their metabolites (including 9-hydroxyrisperidone and hydroxybupropion) in the standard working solutions are shown in table 5:

TABLE 5

Name of substance	Concentration Range (ng/mL)
		Methotrexate (MTX)	320-40000
Rosuvastatin	12-1500
		Risperidone	16-2000
9-Hydroxyrisperidone	16-2000
		Olanzapine	19.2-2400
Perphenazine	4-500
		Bupropion derivatives	10-1250
Hydroxy bupropion	400-50000
		Chlorprothixene	60-7500
Sertindole	20-2500
		Azithromycin	400-50000
Citalopram/escitalopram	24-3000
		Maprotiline	28.8-3600
Thielidazine	128-16000
		Amisulpride	80-10000
Ziprasidone derivatives	64-8000
		Theophylline	4000-500000

(2) Preparation of internal standard working solution

The internal standard substance stock solution is partially a commercial product, and partially purchased corresponding standard substances are diluted by a diluent, and each internal standard substance stock solution is prepared to have the concentration of 100 mug/mL.

The internal standard stock solution is diluted by using a diluent methanol to water (V: V) =1:1 to obtain an internal standard working solution, and the concentration of the prepared internal standard working solution is shown in Table 6.

TABLE 6

Internal standard substance	Concentration (μ g/mL)	Internal standard substance	Concentration (μ g/mL)
				Methotrexate-d 3	5.56	Chlorprothixene-d 6	0.78
Rosuvastatin-d 6	1.11	Sertindole-d 4	0.56
				Risperidone-d 4	0.22	Azithromycin-d 3	5.56
9-Hydroxyrisperidone-d 4	0.22	Citalopram-d 6	0.06
				Olanzapine-d 8	0.22	Maprotiline-d 5	0.33
Perphenazine-d 4	0.11	Theophylline-d 6	42.51
				Bupropion-d 9	0.22	Ziprasidone-d 8	1.11
Hydroxybupropion-d 6	0.56	/	/

(3) Preparation of Standard solutions

Placing 10 mu L of standard working solution, 10 mu L of internal standard working solution, 90 mu L of ultrapure water and 400 mu L of methanol with at least three different concentrations into a 1.5 mL centrifuge tube by using a pipettor, mixing to prepare at least three standard solutions, shaking and uniformly mixing the standard solutions for 1 min at a rotation speed of 2500 r/min, transferring 50 mu L of uniformly mixed liquid to a 1.5 mL plastic centrifuge tube, adding 100 mu L of ultrapure water, and performing vortex mixing for 1 min at 2500 r/min to obtain at least three standard solutions with different concentrations.

Example 2

This example provides for the use of the apparatus AB SCIEX; the model specification is as follows: AB SCIEX Jasper HPLC MS TRIPLE QUAD 4500MD method for sample detection;

the analytical chromatographic column mobile phases used were: phase A: water (0.1% formic acid +5 mmol/L ammonium formate), phase B: methanol (0.1% formic acid +5 mmol/L ammonium formate), analytical column using gradient elution, the chromatographic conditions are given in table 7:

TABLE 7

For mass spectrometry conditions, the parameters are shown in tables 8-9:

TABLE 8

Parameter(s)	Set value	Parameter(s)	Set value
				CUR	20 L/min	TEM	550℃
CAD	9 L/min	GS1	50 L/min
				IS	5000 V	GS2	80 L/min

TABLE 9

The retention time (peak time) of each target in the chromatogram obtained in example 2 is shown in table 10:

watch 10

Name of substance	Retention time/min	Name of substance	Retention time/min
				Methotrexate (MTX)	1.40	Sertindole	3.00
Rosuvastatin	2.73	Azithromycin	1.96
				Risperidone	1.98	Citalopram/escitalopram	2.10
9-Hydroxyrisperidone	1.90	Maprotiline	2.50
				Olanzapine	1.43	Thielidazine	3.10
Perphenazine	3.20	Amisulpride	1.43
				Bupropion derivatives	2.00	Ziprasidone derivatives	2.08
Hydroxy bupropion	1.93	Theophylline	1.39
				Chlorprothixene	2.80

As can be seen from example 2, the chromatographic column and the chromatographic conditions provided by the present invention can retain all analytes in the chromatographic column and can be completely eluted subsequently, and at the same time, all analytes are successfully separated without interference, so that accurate measurement of the analyte concentration can be achieved.

Example 3

The embodiment provides a method for preparing a sample to be detected from blood of a serum blood collection tube, an EDTA blood collection tube and a heparin lithium blood collection tube, which comprises the following steps:

(1) taking at least 2 mL of blood of a blood collection tube of serum to be detected, an EDTA blood collection tube or a lithium heparin blood collection tube, centrifuging at a centrifugation speed of 3500 r/min for 10 min, taking supernatant to obtain serum or plasma, and freezing the serum or plasma at-20 ℃ for storage until the serum or plasma is reserved before analysis;

(2) transferring 10 mu L of internal standard working solution into a 1.5 mL centrifuge tube by using a liquid transfer gun, then adding 100 mu L of serum or plasma, adding 400 mu L of methanol, carrying out vortex oscillation mixing for 5 min at the rotating speed of 2500 r/min, carrying out high-speed centrifugation for 10 min at 14000 r/min, transferring 50 mu L of supernatant into a 1.5 mL plastic centrifuge tube, adding 100 mu L of ultrapure water, carrying out vortex mixing for 1 min at 2500 r/min, and taking 150 mu L of supernatant to obtain a sample to be detected.

Example 4

The embodiment provides a method for detecting the content of 16 drugs and metabolites thereof in a sample to be detected.

(1) Transferring 150 mu L of a sample to be detected, detecting the sample to be detected according to the reference example 2, wherein the sample injection amount is 10 mu L, and obtaining internal standard chromatograms of 16 drugs and metabolites thereof in the sample to be detected;

(2) and (3) bringing the ratio of the chromatographic peak area of the target object in the chromatogram to the peak area of the internal standard object corresponding to the target object into a standard curve equation corresponding to the target object, calculating to obtain the concentration ratio of the concentration of the target object to the concentration of the internal standard object corresponding to the target object, further calculating to obtain the concentration value of the target object in the sample to be detected, and so on, respectively calculating to obtain the concentrations of the 16 drugs and the metabolites thereof.

Comparative example 1

The comparative example provides a method for detecting the content of 16 drugs and metabolites thereof in a sample to be detected.

This comparative example provides an agilent using an instrument; the model specification is as follows: a method for detecting a sample by LC1260-MS 6410;

the mobile phases used were: phase A: water (0.1% formic acid +2 mmol/L ammonium acetate), phase B: methanol (0.1% formic acid +2 mmol/L ammonium acetate), using gradient elution, the chromatographic conditions are given in Table 11:

TABLE 11

For mass spectrometry conditions, the parameters are shown in tables 12-13:

TABLE 12

Parameter(s)	Set value	Parameter(s)	Set value
				Gas Temp	350℃	Gas Flow	8 L/min
Nebulizer	40 psi	Capillary	4000 V

Watch 13

Name of substance	Precursor Ion (Da)	Product Ion (Da)	Fragmentor (V)	CE (V)	Cell Accelerator Voltage(V)
						9-Hydroxyrisperidone	427.1	207	160	30	7
9-Hydroxyrisperidone-IS	431.1	211	160	30	7
						Bupropion derivatives	240.1	183.9	95	9	7
bupropion-IS	249.1	184.9	100	10	7
						Amisulpride	370.1	242	100	20	7
Azithromycin	749.4	591.3	140	30	7
						Azithromycin-IS	752.4	594.3	140	30	7
Theophylline	181.1	124	100	15	7
						theophylline-IS	187.1	127	100	15	7
Perphenazine	404.1	171.1	170	20	5
						perphenazine-IS	408.1	171.1	170	20	5
Lucilian pyridazine	371	126	160	20	7
						Chlorprothixene	316.1	271	150	20	7
chloroprothixene-IS	322.1	271	150	20	7
						Maprotiline	278.2	250.2	100	10	7
maprotiline-IS	283.1	255.2	100	10	7
						Methotrexate (MTX)	455.2	308.2	120	20	7
methotrexate-IS	458.2	311.2	120	20	7
						Hydroxy bupropion	256.1	238.1	100	10	7
Hydroxy bupropion-IS	262.1	244.1	100	10	7
						Olanzapine	313.1	256.2	120	22	2
olanzapine-IS	321.1	261.2	120	22	2
						Ziprasidone derivatives	413.2	194.1	150	30	7
Risperidone	411.1	191.1	160	30	7
						Risperidone-IS	415.1	195.1	160	30	7
Rosuvastatin	482.1	258.1	140	35	3
						rosuvastatin-IS	488.1	264.2	140	35	3
Sertindole	441.5	113.3	165	40	3
						Sertindole-d 4	445.5	117.3	165	40	3
Citalopram/escitalopram Phthaleins pulchella	325.2	109	120	30	7
						citalopram-IS	331.2	262.1	120	20	7

The method provided by the comparative example 1 needs longer analysis time to detect the substance to be detected; the detection method provided by the comparative example 1 has poor sensitivity, and substances with low concentration can be quantitatively detected only by concentrating in the sample pretreatment process, so that the pretreatment time of the sample is prolonged; the precision of part of the detected substances is poor, and the accuracy of all the substances to be detected cannot be ensured during batch sample detection.

And (3) performance testing: evaluation of the method provided by the invention

(1) Accuracy of

Providing a sample to be tested with known contents of 16 drugs and metabolites thereof, detecting according to the detection method provided in example 4, and calculating to obtain the concentrations of the 16 drugs and the metabolites thereof, wherein the results are shown in Table 14:

TABLE 14

Name of substance	Actual content (ng/mL)	Assay content (ng/mL)	Deviation (%)
				Methotrexate (MTX)	400	387.42	-3.2
Rosuvastatin	15	14.77	-1.58
				Risperidone	20	18.60	-7.25
9-Hydroxyrisperidone	20	19.78	-1.12
				Olanzapine	24	24.69	2.83
Perphenazine	5	4.77	-4.64
				Bupropion derivatives	12.5	11.63	-7.18
Hydroxy bupropion	500	504.00	0.80
				Chlorprothixene	75	72.50	-3.39
Sertindole	25	24.40	-2.43
				Azithromycin	500	480.79	-3.92
Escitalopram	30	29.40	-2.02
				Citalopram	30	29.40	-2.02
Maprotiline	36	38.00	5.41
				Thielidazine	160	176.78	9.97
Amisulpride	100	94.51	-5.65
				Ziprasidone derivatives	80	77.5	-3.71
Theophylline	5000	4987.50	-0.25

As can be seen from Table 14, the method provided by the invention can accurately detect the contents of 16 drugs and metabolites thereof in a sample to be detected, the detection deviation is reduced, and the detection method has higher accuracy within the currently allowed detection deviation range.

(2) Detection limit, quantitation limit, and linear range:

the standard working solution prepared according to the method of example 1 was measured from low to high under the measurement conditions provided in example 2, and plotted by the peak area-concentration of quantitative chromatography to obtain a standard curve, and 100. mu.L of the serum/plasma containing 16 drugs and metabolites thereof prepared above was added with 10. mu.L of the internal standard working solution and processed according to example 3, and the detection limit and the quantification limit were determined from low to high under the measurement conditions provided in example 2, and the results are shown in Table 15:

watch 15

As can be seen from table 15, the detection method provided by the present invention has a low detection limit and a low quantitative limit, can detect the concentrations of the drug and the metabolite thereof under the condition of a low content of the drug and the metabolite thereof, and the low quantitative limit indicates that the accurate detection of the concentrations of the drug and the metabolite thereof can be achieved under the condition of a low content of the drug and the metabolite thereof, and the linear fitting effect is excellent.

(3) Recovery and precision

The standard working solution of 16 drugs and metabolites thereof was prepared into high, medium and low 3 concentrations for sample recovery and precision experiments, and the determination was performed according to the method provided in example 2, and the analysis and determination were repeated for 3 batches, with the recovery and precision as follows, respectively, in table 16 below:

TABLE 16

As can be seen from Table 16, the detection method provided by the invention has the advantages of high detection accuracy, high average recovery rate of 85-115%, high precision and relative standard deviation of 0.00-8.33%.

According to the embodiment and the performance test, the detection method provided by the invention has the advantages that the technical indexes such as detection limit, recovery rate and precision meet the requirements, the concentration of 16 drugs and metabolites thereof in blood can be detected, the reproducibility is good, the sample adding recovery rate is high, and the accuracy of the detection result is high.

It is noted that, in this document, relational terms such as "first" and "second," and the like, may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.

The foregoing are merely exemplary embodiments of the present invention, which enable those skilled in the art to understand or practice the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for simultaneously detecting 16 drugs and metabolites thereof in blood by a UPLC-MS/MS method is characterized by comprising the following steps:

the pretreatment method comprises the following steps: taking serum or plasma of a blood sample to be detected, mixing the serum or plasma with an internal standard working solution and a protein precipitator, centrifuging, taking supernate and mixing with ultrapure water to obtain the sample to be detected;

(3) detecting a sample to be detected by using UPLC-MS/MS, and quantifying 16 drugs and metabolites thereof in the sample to be detected by using the standard curve established in the step (1);

in the UPLC-MS/MS detection, the analytical chromatographic column used was SHIMADZU Shim-pack Velox C182.1 × 100 mm 2.7 μm;

in the UPLC-MS/MS detection, the liquid chromatography conditions include:

column temperature: 35-50 ℃;

gradient elution:

0.00 min：A 80%，B 20%；

0.01-0.50 min：A 60%，B 40%；

0.51-2.00 min：A 40%，B 60%；

3.50-4.00 min：A 0%，B 100%；

4.01 min：A 80%，B20%；

analysis time: 5.50 min;

flow rate: 0.2-0.4 mL/min;

in the UPLC-MS/MS assay, mass spectrometry conditions include:

ion source temperature: 350-600 ℃; collision gas: 8-9L/min; drying gas 1: 50-80L/min; and (3) drying gas 2: 50-80L/min; air curtain air: 10-20L/min; high pressure of ion source: 5000-5500V;

in the pretreatment method, the volume ratio of the serum or the blood plasma to the protein precipitator is 1:3-10, and the volume ratio of the supernatant to the ultrapure water is 1: 0.5-5;

the protein precipitant is methanol or acetonitrile;

the internal standard substances used by the 16 medicines and metabolites thereof comprise methotrexate-d 3, rosuvastatin-d 6, risperidone-d 4, 9-hydroxylrisperidone-d 4, olanzapine-d 8, perphenazine-d 4, bupropion-d 9, hydroxybupropion-d 6, chlorprothixene-d 6, sertindole-d 4, azithromycin-d 3, citalopram-d 6, maprotiline-d 5, ziprasidone-d 8 and theophylline-d 6.

2. The method of claim 1, wherein the internal standard of methotrexate is methotrexate-d 3, the internal standard of olanzapine is olanzapine-d 8, the internal standard of risperidone is risperidone-d 4, the internal standard of 9-hydroxyrisperidone is 9-hydroxyrisperidone-d 4, the internal standards of amisulpride and theophylline are theophylline-d 6, the internal standard of ziprasidone is ziprasidone-d 8, the internal standards of perphenazine and lyridazine are perphenazine-d 4, the internal standard of sertindole is sertindole-d 4, the internal standard of chlorprothixene is chlorprothixene-d 6, the internal standard of rosuvastatin is rosuvastatin-d 6, the internal standards of escitalopram or citalopram are citalopram-d 6, the internal standard of maprotiline is maprotiline-d 5, and the internal standard of bupropion is bupropion-d 9, the internal standard of the hydroxy bupropion is hydroxy bupropion-d 6, and the internal standard of the azithromycin is azithromycin-d 3.

3. The method of claim 1, wherein, in the mass spectrometry condition:

the ion pair used for methotrexate is 455.2, 308.2; olanzapine uses an ion pair of 313.1, 256.2; risperidone used an ion pair of 411.1, 191.1; the ion pair used for 9-hydroxy risperidone is 427.1, 207.0; amisulpride uses ion pairs of 370.1, 242.0; ziprasidone uses ion pairs 413.2, 194.1; the ion pair used for perphenazine was 404.1, 171.1; the ion pair used for ulidazine was 371.0, 126.0; the ion pair used for sertindole is 441.5, 113.3; the ion pair used for chlorprothixene is 316.1, 271.0; rosuvastatin uses an ion pair of 482.1, 258.1; theophylline uses an ion pair of 181.1, 124.0; escitalopram or citalopram using an ion pair 325.2, 109.0; maprotiline uses an ion pair of 278.2, 250.2; bupropion uses an ion pair of 240.1, 183.9; the ion pair used for the hydroxyamphetanones is 256.1, 238.1; the ion pair used for azithromycin is 749.4, 591.3;

methotrexate-d 3 used an ion pair 458.2, 311.2; rosuvastatin-d 6 used an ion pair 488.1, 264.2; risperidone-d 4 used an ion pair 415.3, 195.1; 9-Hydroxyrisperidone-d 4 used an ion pair of 431.1, 211.0; olanzapine-d 8 used an ion pair of 321.1, 261.2; perphenazine-d 4 used ion pairs 408.1, 171.1; bupropion-d 9 used an ion pair of 249.1, 184.9; hydroxyamphetazone-d 6 used an ion pair of 262.1, 244.1; chlorprothixene-d 6 used an ion pair of 322.1, 271.0; sertindole-d 4 used an ion pair of 445.5, 117.3; azithromycin-d 3 uses an ion pair of 752.4, 594.3; citalopram-d 6 used an ion pair of 331.2, 262.1; maprotiline-d 5 used an ion pair of 283.1, 255.2; ziprasidone-d 8 uses an ion pair 421.2, 194.1; theophylline-d 6 used an ion pair of 187.1, 127.0.

4. The method of claim 1, wherein the pre-processing method comprises: and centrifuging a blood sample to be detected, performing vortex mixing on serum or plasma, the internal standard working solution and the protein precipitator, centrifuging to obtain a supernatant, and performing vortex mixing on the supernatant and ultrapure water to obtain the supernatant serving as a sample to be detected.

5. The method of claim 1, wherein the standard solution is prepared by a method comprising: mixing an internal standard working solution with a standard working solution containing 16 drugs and metabolites thereof, and then sequentially mixing the internal standard working solution with methanol and ultrapure water to obtain the standard solution, wherein the standard working solution comprises at least three levels of concentrations;

the standard working solution with each level of concentration is obtained by diluting a standard intermediate solution by using a diluent, the standard intermediate solution is obtained by diluting a standard product stock solution by using the diluent, and the standard product stock solution is obtained by dissolving the standard products of the 16 medicines and the metabolites thereof by using a solvent.

6. The method according to claim 5, wherein the diluent is a 50-100% aqueous solution of methanol or acetonitrile.