CN111157649B

CN111157649B - UPLC-MS/MS detection method for adding multiple medicines in scutellaria detoxification powder

Info

Publication number: CN111157649B
Application number: CN202010009928.1A
Authority: CN
Inventors: 魏秀丽; 张传津; 刘霄飞; 杨志昆; 徐恩民; 杨林; 张志民
Original assignee: Shandong Veterinary Drug Quality Examination Institute (shandong Animal Product Quality Testing Center)
Current assignee: Shandong Veterinary Drug Quality Examination Institute (shandong Animal Product Quality Testing Center)
Priority date: 2020-01-06
Filing date: 2020-01-06
Publication date: 2021-07-20
Anticipated expiration: 2040-01-06
Also published as: CN111157649A

Abstract

The invention relates to a UPLC-MS/MS detection method for adding various medicines into scutellaria detoxification powder, belonging to the technical field of detection methods for adding medicines into traditional Chinese medicines. The conditions of the ultra-high liquid phase chromatography are as follows: AB 4000Q TRAP liquid, electrospray ion source, positive ion scan, waters BEH C18 column (2.1mm 50mm 1.8um), flow rate of 0.3ml/min, sample size of 5 ul. Mobile phase: a is acetonitrile (0.1% formic acid); b, water (0.1% formic acid); the invention takes the fragments of the parent ions and the specific daughter ions as the identity characteristics of different compounds, has uniqueness and specificity, has sensitivity of one hundredth to one thousandth of that of liquid chromatography, and has low detection limit.

Description

UPLC-MS/MS detection method for adding multiple medicines in scutellaria detoxification powder

Technical Field

The invention relates to a UPLC-MS/MS detection method for adding various medicines into scutellaria detoxification powder, belonging to the technical field of detection methods for adding medicines into traditional Chinese medicines.

Background

The scutellaria detoxification powder is a common traditional Chinese medicine powder for veterinarians, has the effects of clearing heat and detoxifying, and relieving diarrhea with astringents, and is often used for clinical treatment of piglet diarrhea, colibacillosis and the like.

The scutellaria detoxification powder is approved as three new veterinary drugs from the 1295 bulletin of the 2009 department of agriculture, and can be declared by all veterinary drug enterprises due to the fact that the monitoring and protection period is over. The ingredients of the prescription are 500 g of scutellaria baicalensis, 400 g of humifuse euphorbia herb, 220 g of glossy privet fruit, 400 g of copperleaf herb, 400 g of gerbera pilosa, 100 g of figwort root, 200 g of garden burnet root and 200 g of cherokee rose fruit.

Sulfamethazine, sulfamethoxazole, sulfamonomethoxine, pefloxacin, amantadine, enrofloxacin, sarafloxacin, ciprofloxacin and danofloxacin mesylate are chemical pharmacy, have antibacterial or antiviral effects, can enhance the treatment effect of the scutellaria detoxification powder to a certain extent, and can be illegally added.

Because the detection method of the scutellaria detoxification powder is thin-layer identification and liquid-phase determination of the content of baicalin, the existence of illegal additives is difficult to detect due to the complex components of the traditional Chinese medicine, and the traditional Chinese medicine is often used as a traditional Chinese veterinary medicine for illegal addition of medicines and enhancing the treatment effect by illegal veterinary medicine enterprises.

Disclosure of Invention

Aiming at the problems, the invention provides a UPLC-MS/MS detection method for adding various medicines into the scutellaria detoxification powder. The invention breaks through the defects of microscopic identification and thin-layer identification, adopts ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), can qualitatively and quantitatively detect nine illegally added drugs, namely sulfamethazine, sulfamethoxazole, sulfamonomethoxine, pefloxacin, amantadine, enrofloxacin, sarafloxacin, ciprofloxacin and danofloxacin mesylate by optimizing parent ions and daughter ions and tuning mass spectrum parameters. The technical scheme of the invention is as follows:

a UPLC-MS/MS detection method for adding various medicines into scutellaria detoxification powder specifically comprises the following steps:

(1) taking 100mg of negative scutellaria baicalensis detoxification powder, adding 10mL of mixed solution of acetonitrile and water (0.2% formic acid in volume percentage) into a 15mL centrifuge tube, wherein the volume ratio of the acetonitrile to the water (0.2% formic acid) is 8:2, carrying out ultrasonic treatment for half an hour, centrifuging at 8000rpm/min for 5 minutes, taking supernatant, filtering through a 0.22um organic filter membrane, taking 0.2mL of filtrate, adding the initial mobile phase to 10mL, taking 0.1mL of filtrate, adding the initial mobile phase to 10mL of filtrate, and obtaining a substrate matching blank solution; treating the positive sample by the same method, and testing on a machine;

(2) respectively taking nine medicaments of sulfamethazine, sulfamethoxazole, sulfamonomethoxine, pefloxacin, amantadine, enrofloxacin, sarafloxacin, ciprofloxacin and danofloxacin mesylate as reference substances, respectively taking 10mg of each of the nine medicaments, respectively preparing stock solutions of 0.1mg/mL by using acetonitrile, diluting the stock solutions by using matrix blank solutions in a multiple ratio to prepare 50ng/mL, 20ng/mL, 10ng/mL, 5ng/mL and 2ng/mL, and performing on-machine test;

(3) adding 9 drugs of sulfamethazine, sulfamethoxazole, sulfamonomethoxine, pefloxacin, amantadine, enrofloxacin, sarafloxacin, ciprofloxacin and danofloxacin mesylate into 5g of the scutellaria detoxification powder according to the concentration of 0.2 percent, 0.5 percent and 1 percent (mass percentage), and fully and uniformly mixing; the sample treatment process is the same as that of a negative sample;

(4) the conditions of the ultra-high liquid chromatography are as follows: AB 4000Q TRAP liquid, waters BEH C18 column (2.1mm 50mm 1.8um), flow rate 0.3ml/min, sample volume 5 ul. Mobile phase: a is acetonitrile (0.1% formic acid); b, water (0.1% formic acid); chromatographic elution conditions are shown in table 1:

TABLE 1

Tandem mass spectrometry conditions: using an electrospray ion source, positive ion scan mode, ion spray voltage (IS) 5500V; ion source temperature: 550 ℃; air curtain gas (CUR) of 20L/min; atomizing gas (GS 1): 55L/min; assist gas (GS 2): 55L/min.

The parent ion, the daughter ion and the mass spectrum parameters of various medicaments of sulfamethazine, sulfamethoxazole, sulfamonomethoxine, pefloxacin, amantadine, enrofloxacin, sarafloxacin, ciprofloxacin and danofloxacin mesylate are shown in Table 2:

TABLE 2

(5) And (4) measuring.

The detection limit in the detection method of the invention is as follows: the signal-to-noise ratio is more than or equal to 3 and is 0.2 percent, and the quantitative limit is as follows: the signal-to-noise ratio is more than or equal to 10 and is 0.5 percent.

Compared with the prior art, the invention has the following advantages:

in the prior art, a liquid chromatography method is simply adopted, different compounds have different chromatographic conditions, different mobile phases and different maximum absorption wavelengths, different mobile phases need to be prepared by different types of medicines, and different chromatographic conditions are set, so that the method is very complicated. The invention adopts an ultra-high performance liquid chromatography-tandem mass spectrometry mode, takes the parent ions and the specific daughter ion fragments as the identity characteristics of different compounds, has uniqueness and specificity, has sensitivity of one hundredth to one thousandth of the liquid chromatography, and has low detection limit.

Drawings

FIG. 1 is a quantitative ion pair mass spectrum of sulfadimidine;

FIG. 2 is a qualitative ion pair mass spectrum of sulfadimidine;

FIG. 3 is a sulfamethoxazole quantitative ion pair mass spectrum;

FIG. 4 is a sulfamethoxazole qualitative ion pair mass spectrum;

FIG. 5 is a quantitative ion pair mass spectrum of sulfamonomethoxine;

FIG. 6 is a qualitative ion pair mass spectrum of sulfamonomethoxine;

FIG. 7 is a pefloxacin quantitative ion pair mass spectrum;

FIG. 8 is a qualitative ion pair mass spectrum of pefloxacin;

FIG. 9 is an amantadine quantitative ion pair mass spectrum;

FIG. 10 is a qualitative ion pair mass spectrum of amantadine;

FIG. 11 is an enrofloxacin quantitative ion pair mass spectrum;

FIG. 12 is a qualitative ion pair mass spectrum of enrofloxacin;

FIG. 13 is a quantitative ion pair mass spectrum of sarafloxacin;

FIG. 14 is a qualitative ion pair mass spectrum of sarafloxacin;

FIG. 15 is a quantitative ion pair mass spectrum of ciprofloxacin;

FIG. 16 is a qualitative ion pair mass spectrum of ciprofloxacin;

FIG. 17 is a quantitative ion pair mass spectrum of danofloxacin mesylate;

FIG. 18 is a qualitative ion mass spectrum of danofloxacin mesylate.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. The examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

Example (c): a UPLC-MS/MS detection method for adding various medicines into scutellaria detoxification powder specifically comprises the following steps:

(1) taking 100mg of negative scutellaria baicalensis detoxification powder, adding 10mL of mixed solution of acetonitrile and water (0.2% formic acid) into a 15mL centrifuge tube, wherein the volume ratio of the acetonitrile to the water (0.2% formic acid) is 8:2, carrying out ultrasonic treatment for half an hour, centrifuging at 8000rpm/min for 5 minutes, taking supernate, filtering through a 0.22um organic filter membrane, taking 0.2mL of the supernate out of the filtrate, adding the initial mobile phase to 10mL, taking 0.1mL of the supernate out, adding the initial mobile phase to 10mL, and obtaining a matrix matching blank solution; treating the positive sample by the same method, and testing on a machine;

(4) the conditions of the ultra-high liquid chromatography are as follows: AB 4000Q TRAP liquid, waters BEH C18 column (2.1mm 50mm 1.8um), flow rate 0.3ml/min, sample volume 5 ul. Mobile phase: a is acetonitrile (0.1% formic acid); b, water (0.1% formic acid); chromatographic elution conditions are shown in table 3:

TABLE 3

Time (Min)	A acetonitrile (0.1% formic acid)%	B water (0.1% formic acid)%
			0.1	5	95
1	5	95
			4	60	40
5	95	5
			6	95	5
6.2	5	95
			7	5	95

Tandem mass spectrometry conditions were: using an electrospray ion source, positive ion scan mode, ion spray voltage (IS) 5500V; ion source temperature: 550 ℃; air curtain gas (CUR) of 20L/min; atomizing gas (GS 1): 55L/min; assist gas (GS 2): 55L/min.

The parent ions, the daughter ions and the mass spectrum parameters of the drugs sulfamethazine, sulfamethoxazole, sulfamonomethoxine, pefloxacin, amantadine, enrofloxacin, sarafloxacin, ciprofloxacin and danofloxacin mesylate are shown in Table 4:

TABLE 4

(5) And (4) measuring.

Detection limit: the signal-to-noise ratio is more than or equal to 3 and is 0.2 percent, and the quantitative limit is as follows: the signal-to-noise ratio is more than or equal to 10 and is 0.5 percent.

The peak areas, retention times and linear relations of the nine drugs sulfamethazine, sulfamethoxazole, sulfamonomethoxine, pefloxacin, amantadine, enrofloxacin, sarafloxacin, ciprofloxacin and danofloxacin mesylate are shown in Table 5

TABLE 5 ion pairs Peak area, Retention time, Linear equation Table

Name of Compound	Retention time	Peak area	Linear equation of equations
				Sulfamethazine	2.72	180000；43200	y＝15000+4880(r＝0.934)
Sulfamethoxazole	3.57	44500；18300	y＝4150x+805(r＝0.986)
				Sulfamonomethoxine	3.24	14900；13800	y＝977x+1100(r＝0.957)
Pefloxacin	2.73	20900；14400	y＝1260x+346(r＝0.968)
				Amantadine	2.57	193000；19200	y＝13800x+22000(r＝0.981)
Sarafloxacin hydrate	3.32	61800；43300	y＝4220x+3170(r＝0.959)
				Danofloxacin mesylate	2.93	51900；6990	y＝3810x+935(r＝0.9462)
Enrofloxacin	3.03	105000；44400	y＝7440x+7070(r＝0.963)
				Ciprofloxacin	2.76	41100；23000	y＝2890x+2350(r＝0.962)

And (3) detection results:

the quantitative ion pair mass spectrum and the qualitative ion pair mass spectrum of the nine medicines of sulfamethazine, sulfamethoxazole, sulfamonomethoxine, pefloxacin, amantadine, enrofloxacin, sarafloxacin, ciprofloxacin and danofloxacin mesylate are shown in figures 1-18.

The recovery rate of the three additive concentrations is 95-101%, the parallelism is good, the reproducibility is good, and the variation coefficient among batches is less than 10%. The recovery rates of the nine drugs sulfamethazine, sulfamethoxazole, sulfamonomethoxine, pefloxacin, amantadine, enrofloxacin, sarafloxacin, ciprofloxacin, danofloxacin mesylate are shown in table 6:

TABLE 6 recovery Rate conditions

Claims

1. The UPLC-MS/MS detection method for adding various medicines into the scutellaria detoxification powder is characterized by comprising the following specific steps:

(1) taking 100mg of negative scutellaria baicalensis detoxification powder, adding a mixed solution of 10mL of acetonitrile and water containing 0.2% formic acid into a 15mL centrifuge tube, wherein the volume ratio of the acetonitrile to the water containing 0.2% formic acid is 8:2, carrying out ultrasonic treatment for half an hour, centrifuging at 8000rpm/min for 5 minutes, taking the supernatant, filtering through a 0.22-micron organic filter membrane, taking 0.2mL of the supernatant from the filtrate, adding the initial mobile phase to 10mL, taking 0.1mL of the supernatant again, adding the initial mobile phase to 10mL to obtain a substrate matching blank solution; treating the positive sample by the same method, and testing on a machine;

(2) respectively taking nine medicaments of sulfamethazine, sulfamethoxazole, sulfamonomethoxine, pefloxacin, amantadine, enrofloxacin, sarafloxacin, ciprofloxacin and danofloxacin mesylate as reference substances, respectively taking 10mg of each of the nine medicaments, respectively preparing stock solutions of 0.1mg/mL by using acetonitrile, diluting by using a matrix blank solution in a multiple ratio, preparing into 50ng/mL, 20ng/mL, 10ng/mL, 5ng/mL and 2ng/mL, and performing on-machine test;

(3) adding 9 drugs of sulfamethazine, sulfamethoxazole, sulfamonomethoxine, pefloxacin, amantadine, enrofloxacin, sarafloxacin, ciprofloxacin and danofloxacin mesylate into 5g of the scutellaria detoxification powder according to the mass percentages of 0.2%, 0.5% and 1%, and fully and uniformly mixing; the sample treatment process is the same as that of a negative sample;

(4) the conditions of the ultra-high liquid chromatography are as follows: adopting AB 4000Q TRAP liquid, waters BEH C18 chromatographic column, 2.1mm 50mm 1.8 μm, flow rate 0.3ml/min, sample amount 5 μm; mobile phase: a is acetonitrile containing 0.1 percent of formic acid; b, water containing 0.1 percent of formic acid; the chromatographic elution conditions were as follows:

keeping phase A at 5% and phase B at 95% for 0.1-1 min;

1-4min, increasing phase A from 5% to 60%, and decreasing phase B from 95% to 40%;

4-5min, increasing phase A from 60% to 95%, and decreasing phase B from 40% to 5%;

5-6min, keeping 95% of phase A and 5% of phase B;

6-6.2min, reducing the A phase from 95% to 5%, and increasing the B phase from 5% to 95%;

6.2-7min, keeping phase A at 5% and phase B at 95%;

tandem mass spectrometry conditions were: using an electrospray ion source, positive ion scan mode, ion spray voltage (IS) 5500V; ion source temperature: 550 ℃; air curtain gas (CUR) of 20L/min; atomizing gas (GS 1): 55L/min; assist gas (GS 2): 55L/min;

the parent ions and the daughter ions of the drugs sulfamethazine, sulfamethoxazole, sulfamonomethoxine, pefloxacin, amantadine, enrofloxacin, sarafloxacin, ciprofloxacin and danofloxacin mesylate and the mass spectrum parameters are as follows:

sulfadimidine, wherein when the parent ion is 279.1 and the daughter ion is 186, the DP is 83 and the collision energy is 25; when the parent ion is 279.1 and the daughter ion is 156, the DP is 83 and the collision energy is 26;

sulfamethoxazole, the parent ion is 254.1, when the daughter ion is 156, the DP is 97, and the collision energy is 22; the parent ion was 254.1, the daughter ion was 108, the DP was 97, and the collision energy was 32;

sulfamonomethoxine, wherein the parent ion is 281.1, the DP is 104 when the daughter ion is 156.1, and the collision energy is 26; 281.1 for the parent ion and 126.2 for the daughter ion, DP was 104 and collision energy was 29;

pefloxacin, with a parent ion of 334.1 and a daughter ion of 316.1, DP of 113, collision energy of 28; when the parent ion is 334.1 and the daughter ion is 290.2, the DP is 113 and the collision energy is 26;

amantadine, parent ion 152.1, daughter ion 135.2, DP 103, collision energy 18; when the parent ion is 152.1 and the daughter ion is 107.2, the DP is 103 and the collision energy is 37;

when enrofloxacin has 359.9 as parent ion and 316.2 as child ion, DP is 80 and collision energy is 27; the parent ion is 359.9, when the daughter ion is 245.2, the DP is 80, and the collision energy is 37;

sarafloxacin, wherein the parent ion is 386.1, the child ion is 342.2, the DP is 87, and the collision energy is 26; 386.1 for parent ion, 299.1 for daughter ion, 87 for DP and 38 for collision energy;

ciprofloxacin, when the parent ion is 332.3 and the child ion is 288.3, the DP is 80 and the collision energy is 26; the parent ion is 332.3, the child ion is 245.1, the DP is 80, and the collision energy is 35;

danofloxacin mesylate, wherein when the parent ion is 358.2 and the daughter ion is 340.1, the DP is 80 and the collision energy is 22; the parent ion is 358.2, when the daughter ion is 255, the DP is 80, and the collision energy is 46;

(5) and (3) determination:

the detection limit in the detection method is as follows: the signal-to-noise ratio is more than or equal to 3 and is 0.2 percent, and the quantitative limit is as follows: the signal-to-noise ratio is more than or equal to 10 and is 0.5 percent.

2. The use of the method of claim 1 for detecting the content of sulfamethazine, sulfamethoxazole, sulfamonomethoxine, pefloxacin, amantadine, enrofloxacin, sarafloxacin, ciprofloxacin, danofloxacin mesylate in a Chinese medicinal composition or a Chinese medicinal preparation.