阅读说明：本技术 一种大鼠血浆中芦荟苦素的lc-ms/ms测定方法 (LC-MS/MS determination method for aloesin in rat plasma ) 是由 谭银丰 李海龙 李友宾 王雪松 崔雪 于 2020-11-27 设计创作，主要内容包括：本发明提供一种大鼠血浆中芦荟苦素的LC-MS/MS测定方法,包括以下步骤：(1)血浆的样品处理：取含芦荟苦素的待测血浆,加入含内标芦荟新苷D的甲醇溶液涡旋沉淀,离心,取上清液为待检测样品；(2)设定液相色谱条件为：采用流动相A和流动相B进行梯度洗脱,流动相A为含0.1～1.0‰(v/v)甲酸水溶液,流动相B为甲醇；(3)设定质谱条件为：电喷雾离子源,负离子检测,喷雾电压4500V,喷雾温度为550℃,采用多反应离子监测扫描模式,用于芦荟苦素定量分析的离子对为m/z 393.1→272.9,内标化合物芦荟新苷D离子对m/z为555.3→144.9。本发明方法可更加快速、灵敏、准确地检测血浆中芦荟苦素。(The invention provides an LC-MS/MS determination method of aloesin in rat plasma, which comprises the following steps: (1) sample processing of plasma: adding aloesin-containing plasma to be detected into methanol solution containing internal standard aloinoside D, performing vortex precipitation, centrifuging, and collecting supernatant as sample to be detected; (2) setting the conditions of the liquid chromatogram as follows: gradient elution is carried out by adopting a mobile phase A and a mobile phase B, wherein the mobile phase A is an aqueous solution containing 0.1-1.0 thousandth (v/v) of formic acid, and the mobile phase B is methanol; (3) setting mass spectrum conditions as follows: electrospray ion source, negative ion detection, spray voltage 4500V, spray temperature 550 deg.C, and multiple reaction ion monitoring scanning mode, wherein the ion pair for quantitative analysis of aloesin is m/z 393.1 → 272.9, and the internal standard compound aloinoside D ion pair m/z is 555.3 → 144.9. The method can detect aloesin in blood plasma more quickly, sensitively and accurately.)

1. An LC-MS/MS determination method of aloesin in rat plasma is characterized by comprising the following steps:

(1) sample processing of plasma: adding aloesin-containing plasma to be detected into methanol solution containing internal standard aloinoside D, performing vortex precipitation, centrifuging, and collecting supernatant as sample to be detected;

(2) setting the conditions of the liquid chromatogram as follows: gradient elution is carried out by adopting a mobile phase A and a mobile phase B, wherein the mobile phase A is an aqueous solution containing 0.1-1.0 thousandth (v/v) of formic acid, the mobile phase B is methanol, and the gradient elution procedure is as follows:

time/min Mobile phase A/%) Mobile phase B/%) 0～0.50 95 5 0.51～3.00 95～5 5～95 3.01～4.00 10 90 4.01～5.00 95 5

(3) Setting mass spectrum conditions as follows: an electrospray ion source and negative ion detection are carried out, the spraying voltage is 4500V, and the spraying temperature is 550 ℃; the ion pair used for aloesin quantitative analysis was m/z 393.1 → 272.9 and the internal standard compound aloinoside D ion pair m/z was 555.3 → 144.9 using a multiple reaction ion monitoring scan mode.

2. The LC-MS/MS method for determining aloesin in rat plasma according to claim 1, wherein in the step (1), the methanol solution containing the internal standard aloinoside D and the plasma are mixed according to a volume ratio of 2-4: 1, mixing.

3. The LC-MS/MS method for determining aloesin in rat plasma according to claim 1, wherein in the step (1), the concentration of the methanol solution containing the internal standard aloinoside D is 50 ng/mL-100 ng/mL.

4. The LC-MS/MS method for determining aloesin in rat plasma according to claim 1, wherein in the step (1), the rotation speed of the vortex is 2000rpm, and the time is 10 min; the conditions of the centrifugation are as follows: centrifuge at 13000rpm and 4 ℃ for 10 min.

5. The LC-MS/MS measurement method for aloesin in rat plasma according to claim 1, characterized in that in the step (2), a chromatographic column with a filler C18 is adopted, and the chromatographic column is one of a Synergi Hydro-Rp chromatographic column, a Luna Omega C18 chromatographic column and a Kinetex Evo C18 chromatographic column, the specification of the chromatographic column is 2.10mm x 50mm, and the particle size of the filler is 2-5 μm.

6. The LC-MS/MS method for determining aloesin in rat plasma according to claim 1, wherein the chromatographic conditions are as follows: the column temperature is 28-40 ℃, the sample injection amount is 1-10 mu L, and the flow rate is 0.40-0.60 mL/min^-1。

7. The LC-MS/MS method of determining aloesin in rat plasma according to claim 1, wherein the mass spectrometry conditions are as follows: GS1 is 30-100psi, GS2 is 30-100psi, curtain gas is 20-50psi, and collision gas is 1-8 psi.

8. The LC-MS/MS method of determining aloesin in rat plasma according to claim 1, wherein the parameters of the multiple reactive ion monitoring are:

DP/V CE/V CXP/V EP/V aloesin 90 29 10 10 Aloe neoside D 115 41 11 10

。

9. The LC-MS/MS method for determining aloesin in rat plasma according to claim 6, wherein the column temperature is 30 ℃, the sample volume is 5 μ L, and the flow rate is 0.50 mL-min^-1。

10. The LC-MS/MS method of determining aloesin in rat plasma according to claim 7, wherein the mass spectrometry conditions are as follows: GS1 was at 50psi, GS2 was at 50psi, curtain air was at 30psi, and bump air was at 2 psi.

Technical Field

The invention relates to the technical field of aloesin, in particular to an LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry) determination method of aloesin in rat plasma.

Background

Aloesin is a chromone glycoside substance, which is present in many plants of the genus Aloe of the family Liliaceae, and is present in Aloe barbadensis Miller at about 3% o. Aloe is a traditional Chinese medicine with a long history of application, and is used as a medicine after being concentrated and dried by using juice of leaves of the aloe, and has the effects of relaxing bowels with purgative, clearing liver-fire, killing parasites and treating malnutrition. Related studies have shown that aloesin is one of the important active substances of aloe. The aloesin has effects of inhibiting tyrosinase activity, preventing melanin synthesis, and whitening skin. Aloesin can regulate the release of cell inflammatory factor and growth factor, and has antiinflammatory effect; induces the activation of Smad and MAPK signaling proteins and promotes wound healing. Aloesin can also activate WNT signal channel, regulate Nortch signal channel, regulate digestive system function, and improve pathological state of rat with colitis.

Aloe extract containing aloesin can promote adiponectin secretion, regulate glycolipid metabolism of experimental animal, and resist diabetes. Toxicological studies have shown that aloesin has substantially no toxic side effects. However, few studies on the pharmacokinetics of aloesin have been reported, no pharmacokinetic parameters have been provided, and the analysis techniques used have been relatively lagged. The inventor strives to establish a faster and more sensitive method for analyzing aloesin and apply the method to pharmacokinetic research.

Disclosure of Invention

In view of this, the invention provides an LC-MS/MS determination method for aloesin in rat plasma, which can detect aloesin in plasma more rapidly, sensitively and accurately and can be better applied to pharmacokinetic research.

The technical scheme of the invention is realized as follows:

an LC-MS/MS determination method of aloesin in rat plasma comprises the following steps:

(1) sample processing of plasma: adding aloesin-containing plasma to be detected into methanol solution containing internal standard aloinoside D, performing vortex precipitation, centrifuging, and collecting supernatant as sample to be detected;

(2) setting the conditions of the liquid chromatogram as follows: gradient elution is carried out by adopting a mobile phase A and a mobile phase B, wherein the mobile phase A is an aqueous solution containing 0.1-1.0 thousandth (v/v) of formic acid, the mobile phase B is methanol, and the gradient elution procedure is as follows:

time/min	Mobile phase A/%)	Mobile phase B/%)
			0～0.50	95	5
0.51～3.00	95～5	5～95
			3.01～4.00	10	90
4.01～5.00	95	5

(3) Setting mass spectrum conditions as follows: an electrospray ion source and negative ion detection are carried out, the spraying voltage is 4500V, and the spraying temperature is 550 ℃; the ion pair used for aloesin quantitative analysis was m/z 393.1 → 272.9 and the internal standard compound aloinoside D ion pair m/z was 555.3 → 144.9 using a multiple reaction ion monitoring scan mode.

Further, in the step (1), the methanol solution containing the barbaloin D as the internal standard and the plasma are mixed according to a volume ratio of 2-4: 1, mixing.

Further, in the step (1), the concentration of the methanol solution containing the barbados D as the internal standard is 50 ng/mL-100 ng/mL.

Further, in the step (1), the rotation speed of the vortex is 2000rpm, and the time is 10 min; the conditions of the centrifugation are as follows: centrifuge at 13000rpm and 4 ℃ for 10 min.

Further, in the step (2), a chromatographic column with a filler of C18 is adopted, and the chromatographic column is one of a Synergi Hydro-Rp chromatographic column, a Luna Omega C18 chromatographic column and a Kinetex Evo C18 chromatographic column, the specification of the chromatographic column is 2.10mm multiplied by 50mm, and the particle size of the filler is 2-5 mu m.

Further, chromatographic conditions: the column temperature is 28-40 ℃, the sample injection amount is 1-10 mu L, and the flow rate is 0.40-0.60 mL/min^-1。

Further, mass spectrometry conditions: GS1 is 30-100psi, GS2 is 30-100psi, curtain gas is 20-50psi, and collision gas is 1-8 psi.

Further, the parameters of the multiple reactive ion monitoring are as follows:

	DP/V	CE/V	CXP/V	EP/V
					aloesin	90	29	10	10
Aloe neoside D	115	41	11	10

。

Further, the column temperature was 30 ℃, the sample injection amount was 5 μ L, and the flow rate was 0.50mL · min^-1。

Further, mass spectrometry conditions: GS1 was at 50psi, GS2 was at 50psi, curtain air was at 30psi, and bump air was at 2 psi.

Compared with the prior art, the invention has the beneficial effects that: the invention develops an LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry) determination method for aloesin in rat plasma, adopts the combination of liquid chromatography-mass spectrometry/mass spectrometry, sets a specific gradient elution program, combines certain mass spectrometry conditions, can quickly, sensitively and accurately detect aloesin in plasma, and can be better applied to pharmacokinetic research.

Drawings

FIG. 1 shows the chemical structure and MS/MS spectra of aloesin and barbaloin D.

FIG. 2 LC-MS/MS chromatograms of aloesin and the internal standard aloinoside D,

blank plasma samples (without internal standard treatment, A),

Lower limit sample for standard curve quantification (aloesin 1 ng. mL)^-1，B)、

Blank plasma samples (treated with internal standard, C), plasma samples after 8h of oral administration (D), plasma samples after 8h of intravenous administration (E).

Detailed Description

In order to better understand the technical content of the invention, specific examples are provided below to further illustrate the invention.

The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.

The materials, reagents and the like used in the examples of the present invention can be obtained commercially without specific description.

Example 1

1 Instrument, reagent and laboratory animal

1.1 instruments

Nexera XR UHPLC liquid chromatograph (Shimadzu, Japan) connects AB-SCIEX API 4000+ quadrupole mass spectrometer in series through a Turbo V ion source interface, and Analyst software controls data acquisition and processing (AB, USA). Model 5922 refrigerated centrifuge (KUBOTA, japan), XS105DU ten thousandth electronic analytical balance (mettler-toledo, switzerland), LabTower EDI15 ultrapure water integrated machine (us thermoelectric), Vibrax peripheral shaker (germany erica). AMS small animal anesthesia machine (Gianedel technologies, Inc., Beijing, Gene & I).

1.2 reagent and laboratory animal

Aloesin control is provided by Haouri chemical (Shanghai) Inc.; barbaloin d (aloeresin d) reference was provided by medoferd biotechnology limited under lot number: 19072605. the purity of the reference substances is more than or equal to 98 percent. Chromatographic grade methanol is supplied by merck, germany. The ultrapure water was prepared by LabTower EDI15 ultrapure water machine. Isoflurane is supplied by the Beijing Jiandel technologies, Inc.

The weight of a clean SD male rat is 243-300 g (provided by Tianjiu biotechnology limited of Changsha, the qualification number of an experimental animal is No.43006700017619, and the license number is SCXK 2014-; feeding conditions are as follows: the temperature is 22 +/-2 ℃, the humidity is 40-70%, and the food and the water can be freely taken and drunk after 12 hours of light and shade alternation.

2 methods and results

2.1 chromatography-Mass Spectrometry conditions

Using Synergi Hydro-Rp (2.10mm × 50mm,4 μm, phenomenex) chromatographic column with column temperature of 30 deg.C and flow rate of 0.50 mL/min^-1The sample amount is 5 mu L, gradient elution is carried out by adopting a mobile phase A and a mobile phase B, the mobile phase A is an aqueous solution containing 0.1 per mill (v/v) of formic acid, the mobile phase B is methanol, and the gradient elution procedure is as follows:

time/min	Mobile phase A/%)	Mobile phase B/%)
			0～0.50	95	5
0.51～3.00	95～5	5～95
			3.01～4.00	10	90
4.01～5.00	95	5

。

Electrospray ion source (ESI), negative ion detection, spray voltage 4500V, GS 150 psi, GS2 50psi, curtain gas 30psi, spray temperature 550 deg.C, collision gas 2 psi.

Multi-reactive ion monitoring (MRM), ion pair for aloesin quantitation was m/z 393.1 → 272.9(DP: -90, CE: -29, CXP: -10, EP: -10), and the internal standard compound aloinoside D was 555.3 → 144.9(DP: -115, CE: -41, CXP: -11, EP: -10).

The chemical structures and mass spectrograms of aloesin and barbaloin D are shown in figure 1.

2.2 preparation of the solution

Precisely weighing appropriate amount of aloesin reference substance, adding methanol to make into 0.1 mg/mL^-1The stock solution of (1). Taking appropriate amount of barbaloin D reference substance, precisely weighing, adding methanol to make into 50ng/mL^-1An internal standard solution. The prepared solution is stored at 4 ℃ for later use.

2.3 plasma sample treatment

Collecting 50 μ L of plasma, placing in a centrifuge tube, adding 50ng/mL of neobarbaloin D containing internal standard^-1The solution was vortexed at 2000rpm for 10min to fully precipitate the protein, centrifuged at 13000rpm (4 ℃) for 10min, and the supernatant was put into a micro-sample tube, and 5. mu.L of the supernatant was injected for LC-MS/MS analysis.

2.4 methodological considerations

And verifying the effectiveness of the analysis method according to the requirement of the quantitative analysis method of the biological samples in the four parts of China pharmacopoeia of 2020 edition.

2.4.1 specialization examination

Taking blank plasma from different rats, blank plasma with aloesin reference substance, plasma sample collected via orbital vein after intravenous administration for 8h, and plasma sample after oral administration for 8h, processing according to sample treatment method under item "2.3", and analyzing according to sample introduction under item "2.1", and the chromatogram is shown in figure 2. The peak emergence time of aloesin and barbaloin D is 1.85 min and 2.67min respectively.

2.4.2 Linear relationship investigation

Taking blank plasma, adding 0.1 mg/mL^-1The aloesin stock solution is prepared into 600, 400, 200, 40, 10, 2, 1 ng.mL^-1For each of the plasma samples of the standard curves of the series of concentrations, 50. mu.L of each plasma sample was treated by the method under item "2.3", and 5. mu.L of each plasma sample was injected and analyzed by the method under item "2.1". Taking the ratio (Y) of the peak areas of the object to be measured and the internal standard as the ordinate, the concentration (X) of the object to be measured as the abscissa, and the weight of 1/X²Linear regression was performed to obtain a regression equation Y of 0.00617X +0.00113(r of 0.9945). Indicating that the aloesin is 1-600 ng.mL^-1The linear relationship within the range is good.

2.4.3 residual Effect

Taking a standard curve quantitative upper limit sample (600 ng. mL < -1 >), processing, injecting and analyzing, then injecting and analyzing 1 blank sample, and inspecting and analyzing residues of aloesin and internal standard barbaloin D in a blank sample chromatogram. As a result, the peak area of aloesin residue is less than 20% of the peak area of the lower limit sample, the residual peak area of the internal standard aloinoside D is less than 5% of the peak area of the internal standard of the quality control sample, and the residual effect meets the requirement.

2.4.4 accuracy and precision

Taking blank plasma, adding 0.1 mg/mL^-1The aloesin stock solution is prepared into low, medium and high concentrations (3.0, 50.0, 500.0 ng/mL)^-1) The aloesin quality control sample and the quantitative lower limit sample (1.0 ng. mL)^-1) 5 parallel samples are set for each concentration, and after treatment according to the method under the item 2.3, sample injection analysis is carried out according to the method under the item 2.1. And (3) calculating the precision and accuracy within the day, measuring 3 batches of samples within 3d, and calculating the precision within the day.

The result shows that the intra-day precision and the inter-day precision RSD are both less than 15 percent, and the repeatability is good; the accuracy is between 96.96% and 107.28%. Specific results are shown in table 1.

TABLE 1 Aloesin quantitation precision and accuracy results

2.4.5 matrix Effect and extraction recovery test

According to the existing literature method, the matrix effect is evaluated by adopting an addition method after extraction, and the extraction recovery rate is investigated at the same time. Preparing high, medium and low 3 concentrations (500ng mL) by using 3 different solvents respectively^-1、50ng·mL^-1、3ng·mL^-1) 5 replicates per concentration were used for aloesin samples.

The Set 1 series sample solvent is methanol;

the Set2 series sample solvent is blank plasma from different rats, and after 3 times volume of methanol precipitation protein, supernatant fluid obtained by centrifugal separation is obtained;

the Set 3 series sample solvent is blank plasma from different rats.

Taking 50 μ L of the above samples, treating according to the method under item "2.3", and analyzing according to the method under item "2.1", to obtain peak areas ofA₁、A₂、A₃The matrix factor MF ═ A₂/A₁X is 100%; extraction recovery rate RE ═ a₃/A₂×100％。

The ratio of aloesin to internal standard aloinoside D matrix factor is normalized matrix factor. The results are shown in Table 2.

Table 2 matrixing effect and recovery of aloesin in rat plasma (Mean ± SD, n ═ 5)

2.4.6 stability Studies

Blank plasma was used to prepare 3 concentrations (500 ng. mL)^-1、50ng·mL^-1、3ng·mL^-1) The stability of the aloesin quality control samples was examined under the following conditions.

(1) After being processed, the sample is placed in an automatic sample injector for 8 hours and then is subjected to sample injection analysis;

(2) the sample is processed after 3 freeze thawing cycles, and is subjected to sample injection analysis;

(3) standing the sample at room temperature for 4h, and then taking out the sample for sample injection analysis;

(4) and (4) storing the sample at-20 ℃ for 15 days, then processing, and carrying out sample injection analysis.

Results the accuracy of the test of each concentration sample was between 94.66% and 109.60%, and the RSD was less than 15%. And the stability is qualified. The results are shown in Table 3.

TABLE 3 stability of aloesin in plasma samples

Example 2

This example differs from example 1 in that the mobile phase a is a 1.0% o (v/v) aqueous formic acid solution. The detection result shows that the aloesin and the new barbaloin D have good peak shapes and no mutual interference, and endogenous substances in blood plasma have no obvious influence on signals of the aloesin and the new barbaloin D.

Example 3

This example differs from example 1 in that the concentration of the methanolic solution containing the internal standard barbaloin D was 100 ng/mL. The detection result shows that the peak height and the peak area of the barbaloin D are increased by about 1 time, and the barbaloin D can be used for effectively quantifying the aloesin.

Example 4

This example differs from example 1 in that the column temperature was 40 ℃ under chromatographic conditions. The detection result shows that the aloesin and the new barbaloin D have good peak shapes and no mutual interference, and endogenous substances in blood plasma have no obvious influence on signals of the aloesin and the new barbaloin D.

Example 5

This example differs from example 1 in that the sample size was 10. mu.L under the chromatographic conditions. The detection result shows that the aloesin and the new barbaloin D have good peak shapes and no mutual interference, and endogenous substances in blood plasma have no obvious influence on signals of the aloesin and the new barbaloin D.

Example 6

This example differs from example 1 in that the chromatographic conditions were those of a Luna Omega C18 column (2.10 mm. times.50 mm,4 μm, phenomenex). The detection result shows that the aloesin and the new barbaloin D have good peak shapes and no mutual interference, and endogenous substances in blood plasma have no obvious influence on signals of the aloesin and the new barbaloin D.

Example 7

This example differs from example 1 in that the chromatographic conditions were performed on a Kinetex Evo C18 column (2.10 mm. times.50 mm,4 μm, phenomenex). The detection result shows that the aloesin and the new barbaloin D have good peak shapes and no mutual interference, and endogenous substances in blood plasma have no obvious influence on signals of the aloesin and the new barbaloin D.

Example 8

This example differs from example 1 in mass spectral conditions, GS1 at 100psi, GS2 at 100psi, curtain gas at 50psi, and collision gas at 8 psi. The detection result shows that the peak heights of aloesin and barbaloin D are slightly reduced, and the signal to noise ratio (S/N) of the lowest quantitative limit sample is more than or equal to 10.

Comparative example 1

This comparative example differs from example 1 in that in step (2), the gradient elution procedure was:

time/min	Mobile phase A/%)	Mobile phase B/%)
			0～1.0	80	20
1.01～2.50	50	50
			2.51～3.50	20	80
4.01～5.00	90	10

The detection result shows that the retention time of the aloesin is about 1min, and a strong impurity peak interference signal is present near the position of a chromatographic peak, so that the lower limit of the quantification of the aloesin in the plasma does not reach 1ng/mL.

Comparative example 2

This comparative example differs from example 1 in that in step (3), the mass spectrometry conditions were set as follows: an electrospray ion source, detecting negative ions, wherein the spraying voltage is 5000V, and the spraying temperature is 500 ℃; the ion pair used for aloesin quantitative analysis was m/z 393.1 → 272.9 and the internal standard compound aloinoside D ion pair m/z was 555.25 → 511.2 using a multiple reactive ion monitoring scan mode. The results show that the signal intensity of aloesin and neobarbaloin D is 2 orders of magnitude lower than that in the positive ion mode.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.