CN115144484A

CN115144484A - Method for determining representative components and metabolites of oral liquid for removing food retention and relieving cough of children in biological sample

Info

Publication number: CN115144484A
Application number: CN202210304839.9A
Authority: CN
Inventors: 王现珍; 姚景春
Original assignee: Shandong New Time Pharmaceutical Co Ltd
Current assignee: Shandong New Time Pharmaceutical Co Ltd
Priority date: 2021-03-30
Filing date: 2022-03-23
Publication date: 2022-10-04

Abstract

The invention relates to a method for determining representative components of an oral liquid for removing food retention and relieving cough of children and metabolites thereof in a biological sample, belonging to the technical field of medicines. The method adopts ultra-high performance liquid chromatography-electrospray ionization tandem mass spectrometry to detect the representative components and metabolites thereof of the oral liquid for removing food retention and relieving cough of children in a biological sample, wherein a mobile phase in a liquid phase condition consists of 0.1% formic acid-5 mM ammonium acetate water and acetonitrile, and gradient elution is carried out. The UPLC-MS/MS measuring method for measuring the representative components of the pediatric food stagnation removing and cough stopping oral liquid in the biological sample, which is established by the invention, meets the analysis requirements of the biological sample in the aspects of accuracy, precision, specificity, stability, extraction recovery rate, matrix effect and the like of 'guide principles of non-clinical pharmacokinetic research of chemical drugs' issued in the Chinese pharmacopoeia 2015 edition and SFDA 2014.

Description

Method for determining representative components and metabolites of oral liquid for removing food retention and relieving cough of children in biological sample

Technical Field

The invention belongs to the technical field of medicines, and relates to a method for measuring typical components of an oral liquid for removing food retention and relieving cough of children and metabolites thereof in a biological sample.

Background

The oral liquid for eliminating food retention and relieving cough for children is prepared by unique company of Lunan Qianpu pharmacy, and has the functions of clearing heat, dispersing lung qi, eliminating food retention and relieving cough. Can be used for treating infantile cough, exacerbation at night, phlegm and tinnitus, abdominal distention, and halitosis due to food stagnation, phlegm-heat accumulation in lung. The components are as follows: parched fructus crataegi, arecae semen, fructus Aurantii Immaturus, folium Eriobotryae, fructus Trichosanthis, parched Raphani semen, parched semen Lepidii, radix Platycodi, fructus forsythiae, and periostracum Cicadae. In the formula, the hawthorn and the betel nut have the effects of promoting digestion, removing food stagnation, eliminating food retention, and causing stagnation of heat and phlegm without causing dependence and heat generation and phlegm generation without source. Loquat leaves, snakegourd fruit, platycodon grandiflorum and fructus forsythiae are used for clearing lung heat and eliminating phlegm, dispersing lung qi, eliminating phlegm and heat, clearing lung heat and descending lung qi, so that cough is self-stopped. Semen Lepidii, semen Raphani, has the effects of purging the lung-heat and relieving asthma, promoting digestion and resolving food stagnation, lowering qi and resolving phlegm, periostracum Cicadae is used for clearing lung heat, dispersing lung heat, and fructus Aurantii Immaturus is used for promoting qi circulation and eliminating phlegm. The platycodon root can guide all the medicines to directly reach the focus of disease in addition to dispersing lung qi, eliminating phlegm and relieving cough. The medicines are combined to achieve the effects of removing food retention, reducing phlegm, ventilating and smoothing lung and relieving cough.

The quality control component of the oral liquid for removing food retention and relieving cough for children is synephrine which is the main component of immature bitter orange, and the research on the metabolism condition in vivo of the synephrine is less after the oral liquid for removing food retention and relieving cough for children is taken at present, and the oral liquid is particularly in the bodies of young animals and faces the problems of difficult sampling and small sample amount. Therefore, it is urgently needed to develop a biological sample measuring method with sufficient sensitivity and accuracy to discuss the in vivo dynamic process of the oral liquid for removing food retention and relieving cough of children after being taken orally.

In 26.11.2020, IARC published a main evaluation on carcinogenicity of arecoline (arecoline), a main active ingredient of arecoline, in lancet-oncology, and the arecoline was identified as a class 2B carcinogen. In view of the fact that the oral liquid for removing food retention and relieving cough of children contains the betel nut which is a medicinal material, the situation that the main active ingredient of the oral liquid is arecoline which is absorbed into blood needs to be examined. Therefore, a stable, accurate and low-detection-limit liquid quality analysis method is urgently needed to be developed, which can not only examine the blood absorption and bleeding condition of the active ingredient synephrine, but also monitor the content of the toxic ingredient arecoline, and has very important significance for determining the effectiveness and safety of the oral liquid for removing food retention and relieving cough for children, guiding the development of new drugs and guiding the clinical reasonable medication.

Disclosure of Invention

The invention aims to provide a method for detecting typical components and metabolites of an oral liquid for removing food retention and relieving cough of children in a biological sample, and particularly relates to a method for detecting the typical components of the oral liquid for removing food retention and relieving cough of children in the biological sample, namely, detecting the typical components of synephrine, arecoline, II-phase metabolites of the synephrine (glucuronic acid combined synephrine) and I-phase metabolites of the arecoline (arecoline) by adopting an ultra-high performance liquid chromatography-electrospray ionization tandem mass spectrometry method, namely an UPLC-MS/MS method; wherein the II-phase metabolite of synephrine (i.e. glucuronic acid-binding synephrine) is realized by determining total synephrine after the biological sample is subjected to enzymolysis, and other components are realized by directly determining the concentration of free drugs in the biological sample.

Namely, the UPLC-MS/MS method is adopted to detect the concentrations of free synephrine, total synephrine, free arecoline and free arecoline in the biological sample.

The above biological samples according to the present invention include, but are not limited to, blood, urine, bile, organs, feces, and the like.

In the method, after a biological sample is collected, 0.1 percent formic acid normal saline is used as a stabilizer, and the concentration of the free drug is measured by adopting an organic solvent direct precipitation method; the total concentration of the medicine adopts a method of enzymolysis and reprecipitation, and acetonitrile is used as a precipitating solvent in the two methods; the deuterium isotope is used as an internal standard for biological sample determination, namely [13C,2H3] synephrine and D5-arecoline hydrobromide are used as internal standards.

The elution conditions of the liquid phase system in the above-mentioned determination method of the present invention are:

the mobile phase is A phase (0.1% formic acid-5 mM ammonium acetate water) -B phase (acetonitrile);

the gradient elution procedure was:

the chromatographic column is HILIC column (50 mm × 2.1mm,1.9 μm);

flow rate: 0.2mL/min;

column temperature: 30 to 40 ℃;

temperature of the sample pan: 4 to 25 ℃;

sample introduction amount: 1-5 μ L.

The mass spectrum system conditions in the above-mentioned determination method of the present invention are:

an ion source: H-ESI; ejection voltage: 3800-4500V; sheath gas: 30-40 Arb; auxiliary gas: 10 to 20Arb; tail blowing: 0Arb; ion transport capillary temperature: 325 ℃; the gasification temperature: 80-200 ℃.

Through optimization, the ion pair parameters of each analyte in the mass spectrum system of the method are respectively as follows: the m/z parent → daughter ion for synephrine is 168.2 → 150.036, the collision energy is 9.212v, and the RF lens is 45v; [13C,2H3] -synephrine m/z parent ion → daughter ion 172.088 → 154.125, collision energy 9.676v, RF lens 30v; the parent ion → the daughter ion of m/z of arecoline is 156.3 → 44, the collision energy is 15v, the RF lens is 45v; the m/z parent ion → daughter ion of arecaidine is 142.2 → 44, the collision energy is 8.54v, and the RF lens is 51v; the m/z parent ion → daughter ion of deuterated arecoline is 161.2 → 48, the collision energy is 15.36v, and the RF lens is 45v.

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

(1) In the liquid phase condition, the mobile phase is 0.1 percent formic acid-5 mM ammonium acetate water and acetonitrile, gradient elution is carried out, the analysis time of a single sample is only 6.0min, the analysis flux is greatly increased, the analysis speed is improved, the rapid analysis of a large batch of samples is facilitated, and the method can also be used for clinically and rapidly monitoring the blood concentration of a patient.

(2) The chromatographic column adopts an HILIC column, and due to the fact that the polarity of the component to be detected is high, the effect is high when a common C18 column is used, the peak area is unstable, the HILIC column can well retain the substance to be detected, and separation of the substance from the biological matrix is achieved.

(3) The determination method established by the invention meets the analysis requirements of the biological sample in the aspects of accuracy, precision, specificity, stability, extraction recovery rate, matrix effect and the like, which are published in 2015 edition of Chinese pharmacopoeia and technical guidance principle for non-clinical pharmacokinetic research of chemical drugs issued in 2014 of SFDA, and is even more excellent.

Drawings

FIG. 1 is a chromatogram of a blank plasma sample from a rat.

FIG. 2 is a chromatogram of a rat blank plasma dosed sample.

FIG. 3 is a graph of the concentration-time curve of the free drug after oral administration of the oral liquid for removing food retention and relieving cough in children in young rats

FIG. 4 is a graph of the concentration-time curve of the total synephrine drug after the oral administration of the oral liquid for removing food retention and relieving cough for young rats

Note: channel I is a total ion flow diagram, channel II is a arecoline chromatographic peak, channel III is a arecoline chromatographic peak, channel IV is a deuterated arecoline chromatographic peak, channel V is a synephrine chromatographic peak, and channel VI is a [13C,2H3] -synephrine chromatographic peak.

Detailed Description

In order to make the present invention more clear to those skilled in the art, the applicant further describes the method for determining the representative components and metabolites thereof of the Xiao 'er Zhi Ke oral liquid in the biological sample of the present invention, wherein the biological matrix examined by the methodology of the detection method is rat plasma as an example, and the specific example is the pharmacokinetic experiment after the Xiao' er Zhi Ke oral liquid is orally taken by young rats as an example, but those skilled in the art can know that the further description and examples do not limit the scope of the present invention in any way.

EXAMPLE 1 methodological examination of the detection method of the invention

1. Preparation of the solution

(1) Preparation of standard series solution

Preparation of synephrine stock solution: weighing about 20mg of synephrine standard substance, placing the synephrine standard substance into a20 mL volumetric flask, adding methanol for dissolving, and fixing the volume to prepare the synephrine stock solution with the concentration of about 1000 mug/mL.

The formula for calculating the mass of synephrine is as follows: synephrine mass = m × 99.5% (m is the weighed amount of synephrine standard).

Preparing arecoline stock solution: weighing arecoline hydrobromide standard substance about 30mg, placing in a20 mL volumetric flask, adding methanol to dissolve and fix the volume, and preparing the arecoline stock solution with the concentration of about 1000 mug/mL.

The mass calculation formula of the arecoline is as follows: the mass of arecoline = m × 155.19/236.12 × 99.8% (m is the weighed amount of arecoline hydrobromide standard).

Preparing a betelnut hypokaline stock solution: weighing about 20mg of the arecoline standard product, placing the weighed sample product into a20 mL volumetric flask, adding methanol to dissolve the sample product and fixing the volume to prepare the arecoline stock solution with the concentration of about 1000 mug/mL.

Preparing a free drug mixed standard curve working solution: respectively taking the synephrine, arecoline and arecaidine stock solutions, taking 80% acetonitrile water as a dilution solvent, and carrying out gradient dilution to the required concentration, wherein the sequence is as follows: 1200. 600, 300, 120, 60.0, 30.0 and 15.0ng/mL, mixing the diluted standard series solution with gradient in equal proportion, and sequentially mixing the mixed working solution according to the concentration from high to low: 400. 200, 100, 40.0, 20.0, 10.0 and 5.00ng/mL.

Preparing a total synephrine standard curve working solution: taking synephrine stock solution, using 80% acetonitrile water as a dilution solvent, and diluting to seven concentrations from high to low as follows: 16000. 8000, 2000, 500, 100, 40.0, 20.0ng/mL

(2) Preparation of internal standard solution

Preparation of [13C,2H3] synephrine stock solution: taking one (1 mg) of [13C,2H3] synephrine standard substance, adding 1mL of methanol for dissolving, and preparing 1000 mu g/mL of [13C,2H3] synephrine stock solution.

Preparation of [13C,2H3] synephrine working solution: taking synephrine internal standard stock solution, and diluting the synephrine internal standard stock solution into internal standard working solution with the concentration of 2000ng/mL by using 80% acetonitrile water.

Preparing D5-arecoline hydrobromide stock solution: dissolving one D5-arecoline hydrobromide standard substance (5 mg) with methanol, transferring into a 10mL volumetric flask, and metering to obtain a D5-arecoline hydrobromide stock solution of 500 μ g/mL.

Preparing D5-arecoline hydrobromide working solution: taking the arecoline internal standard stock solution, and diluting the arecoline internal standard stock solution into internal standard working solution with the concentration of 2000ng/mL by using 80% acetonitrile water.

[13C,2H3] synephrine as internal standard for free and total synephrine assay, and D5-arecoline hydrobromide as internal standard for arecoline and arecoline assay.

(3) Preparation of standard quality control solution

Preparing synephrine quality control stock solution: weighing synephrine standard substance, placing about 20mg of synephrine standard substance in a20 mL volumetric flask, adding methanol for dissolution and metering volume to prepare the synephrine quality control stock solution with the concentration of about 1000 mug/mL.

Preparing an areca catechine quality control stock solution: weighing arecoline hydrobromide standard substance about 30mg, placing in a20 mL volumetric flask, adding methanol to dissolve and fix the volume, and preparing the arecoline quality control stock solution with the concentration of about 1000 mug/mL.

The mass calculation formula of the arecoline is as follows: the mass of arecoline = m × 155.19/236.12 × 99.8% (m is the weighed amount of the standard arecoline hydrobromide).

Preparing a quality control stock solution of the betelnut: weighing about 20mg of the arecoline standard substance, placing the arecoline standard substance into a20 mL volumetric flask, adding methanol for dissolving, and fixing the volume to prepare the arecoline quality control stock solution with the concentration of about 1000 mug/mL.

Preparing a free drug mixed quality control working solution: respectively taking the synephrine, arecoline and arecaidine stock solutions, using 80% acetonitrile as a dilution solvent, and performing gradient dilution to four quality control concentrations of required high, medium, low and quantitative lower limits, wherein the four quality control concentrations are sequentially as follows: 900. 180, 36.0 and 15.0ng/mL, and then mixing the diluted standard series solution with the gradient in equal proportion, wherein the mixed working solution is prepared from the following components in sequence from high concentration to low concentration: 300. 60.0, 12.0 and 5.00ng/mL.

Preparing a total synephrine quality control working solution: taking synephrine stock solution, using 80% acetonitrile as a dilution solvent, and diluting the synephrine stock solution into four concentrations from high to low: 12000. 800, 50.0 and 20.0ng/mL.

Storing the stock solution and the working solution in a refrigerator at 4 deg.C for use.

2. Confirmation of analytical methods

1. Preparation of Standard plasma sample (drug-containing plasma sample)

When the concentration of the plasma free drug is measured, 45 mu L of diluted rat blank plasma is taken, 5 mu L of standard curve or quality control working solution of the object to be measured is added, and the mixture is uniformly mixed.

When the total drug concentration of the blood plasma is measured, 54 mu L of diluted rat blank blood plasma is taken, 6 mu L of standard curve or quality control working solution of the object to be measured is added, mixed evenly, and 20 mu L of the mixture is taken out for standby.

The blank plasma in the method is blank plasma of normal rats diluted by adding 0.1% formic acid physiological saline 1.

2. Plasma sample pretreatment

When the concentration of the plasma free drug is measured, 50 mu L of plasma sample containing the drug is taken, 5 mu L of internal standard working solution and 145 mu L of acetonitrile are added, vortex is carried out, 9600g is centrifuged for 10min, and supernatant is taken to be the plasma sample to be measured.

When the total drug concentration of the blood plasma is measured, 20 mu L of drug-containing blood plasma sample is taken, 20 mu L of 0.1 percent formic acid physiological saline and 20 mu L of 5000U/mL beta glucuronidase solution are added, and after uniform mixing, incubation is carried out for at least 16h at 37 ℃; adding 5 mu L of internal standard working solution and 195 mu L of acetonitrile precipitation solution, vortexing, centrifuging for 10min at 9600g, and taking supernatant as a plasma sample to be detected.

3. Specificity of the method

Taking the measurement of plasma free drug concentration as an example, the method specifically comprises the following steps:

taking 50 mu L of rat blank plasma, adding 5 mu L of mobile phase and 145 mu L of acetonitrile, vortexing, centrifuging for 10min at 9600g, taking supernatant for analysis, and obtaining a chromatogram map of a blank sample (figure 1);

adding the mixed working solution and the internal standard solution with certain concentration into blank plasma, adding 5 mu L of mobile phase and 145 mu L of acetonitrile, vortexing, centrifuging at 9600g for 10min, taking the supernatant for analysis, and obtaining a corresponding sample adding chromatogram (figure 2).

The results show that endogenous substances in plasma do not interfere with the assay.

4. Standard curve and linear range

Preparing a free drug plasma sample under the item of 'preparation of a standard plasma sample', performing double-sample analysis on each concentration according to the item of 'pretreatment of the plasma sample', and recording a chromatogram.

The results show that the linear relation of synephrine, arecoline and arecaidine is good in the range of 0.500-40.0 ng/mL, and the standard curve of a typical rat plasma sample is as follows: synephrine, Y =0.00112387+, 0.00817865X, r =0.9965; arecoline, Y = 0.00149658+0.0316525X, r =0.9968; arecaidine, Y =0.00144206+ 0.0267798X, r =0.9991, with lower limit of quantitation (LLOQ) of 0.500ng/mL.

Preparing a total drug plasma sample under the item of 'preparation of a standard plasma sample', performing double-sample analysis on each concentration according to the item of 'pretreatment of the plasma sample', and recording a chromatogram.

The result shows that synephrine has good linear relation in the range of 2.00-1600 ng/mL, the standard curve of a typical rat plasma sample is Y = -0.129242+, 0.00354463 x, r = -0.9968, and the lower limit of quantitation (LLOQ) is 2.00ng/mL.

5. Precision and accuracy

A sample of quality control and a sample of lower limit of quantitation (0.500 ng/mL) of three concentrations of low (1.20 ng/mL), medium (6.00 ng/mL) and high (30.0 ng/mL) of free drug were prepared from 45. Mu.L of rat blank plasma according to the method under the section of "preparation of Standard plasma samples". The procedure was as in "plasma sample pretreatment", 6 samples were analyzed for each concentration, and 3 batches were tested in series, following the standard curve.

And calculating the concentration of the QC sample according to the standard curve of the day, and performing variance analysis on the result to obtain the precision RSD and the accuracy RE of the method, wherein the diurnal RSD of the two tested components is less than or equal to 12.2 percent, the diurnal RSD is less than or equal to 8.5 percent, and the RE is between 10.6 and 4.5 percent, so that the related standard requirements are met.

54 μ L of rat blank plasma was taken, and Quality Control (QC) samples and lower limit of quantitation (2.00 ng/mL) samples of total synephrine at three concentrations of low (5.00 ng/mL), medium (80.0 ng/mL) and high (1200 ng/mL) were prepared according to the method under the section "preparation of Standard plasma samples". The operation is carried out under the item of 'pretreatment of plasma samples', 6 samples are analyzed at each concentration, the 3 batches were tested in series, following the standard curve.

And calculating the concentration of the QC sample according to the standard curve of the day, and performing variance analysis on the result to obtain the precision RSD and the accuracy RE of the method, wherein the diurnal RSD of the two measured components is less than or equal to 5.3 percent, the diurnal RSD is less than or equal to 6.2 percent, and the RE is between 3.0 and 4.8 percent, so that the requirements of relevant specifications are met.

6. Matrix effect

The matrix effect was investigated using a blank plasma from 6 rats of different origin, formulated at three levels, low, medium and high.

Sample a: and (3) dissociating the drug, taking 45 mu L of purified water to replace plasma, treating the drug in other steps according to the items of 'preparation of a standard plasma sample' and 'pretreatment of the plasma sample', and recording chromatographic peak areas of the object to be detected and the internal standard.

Total synephrine, 54 μ L of purified water was taken in place of plasma, and the other steps were processed under the terms "preparation of standard plasma sample" and "pretreatment of plasma sample", and the chromatographic peak areas for synephrine and internal standard were recorded.

Sample B: dissociating the drug, adding 145 mu L acetonitrile into 45 mu L blank rat plasma, carrying out vortex precipitation on the protein, adding 5 mu L low, medium and high concentration mixed quality control working solution and 5 mu L internal standard working solution, mixing uniformly, centrifuging at 9600g for 10min, and taking the supernatant for LC-MS/MS analysis. The chromatographic peak areas of the test and internal standards were recorded.

Taking 54 mu L of medicine-containing plasma sample, adding 60 mu L of 0.1% formic acid physiological saline and 60 mu L of 5000U/mL beta glucuronidase solution, after mixing, incubation was carried out at 37 ℃ for at least 16h (overnight). Adding 195 mu L of acetonitrile precipitation solution, vortexing, adding 6 mu L of synephrine quality control working solution with low, medium and high concentrations and 15 mu L of internal standard working solution, uniformly mixing, centrifuging at 9600g for 10min, and taking supernatant for sample injection analysis. The chromatographic peak areas of the test and internal standards were recorded.

And the ratio of the peak area measured by the sample B to the peak area measured by the sample A with the corresponding concentration is the matrix effect of the substance to be measured and the internal standard.

The results show that the matrix effects of the free drug on the low, medium and high QC concentration levels after internal standard normalization are respectively 98.7%, 91.5% and 89.4% of synephrine; arecoline 96.6%, 91.0% and 91.4%; 95.7 percent of arecaidine, 91.0 percent of arecaidine and 90.6 percent of arecaidine. The matrix effect of total synephrine on low, middle and high QC concentration levels normalized by internal standard is 99.7%, 94.5% and 91.1%, respectively. Thus, under the chromatographic and mass spectrometric conditions selected in this experiment, the effects of matrix effects on synephrine, arecoline and arecaidine were negligible.

7. Extraction recovery rate

Sample C: preparing quality control samples with high, medium and low concentration levels of free drugs and total synephrine according to the item of 'preparation of standard plasma samples', processing the plasma samples by adopting a 'plasma sample pretreatment' method, performing LC-MS/MS analysis, and recording peak areas of various substances to be detected and an internal standard chromatogram.

And the ratio of the peak area measured by the sample C to the peak area measured by the sample B with the corresponding concentration is the extraction recovery rate of each substance to be detected.

The results show that the recovery rates of the low, medium and high concentrations are respectively as follows: synephrine, 95.2%,100% and 105%; arecoline, 100%,105% and 101%; 101%,100% and 101% of arecaidine; total synephrine, 103%,100% and 101%.

8. Sample stability

The patent investigates the stability of an untreated plasma sample placed for 4 hours at room temperature, the stability of the plasma sample subjected to 3 freezing-unfreezing cycles, and the stability of the treated plasma sample placed for 24 hours at room temperature. For each stability study, QC samples were prepared at two concentrations, low and high, under the "Standard Curve and Linear Range" conditions, and 3 samples were analyzed at each concentration, and the sample concentrations were measured and the relative error (RE%) calculated as per the "pretreatment of plasma samples". The results show that the treated plasma sample is stable within 24h at room temperature, the untreated plasma sample is stable within 4h at room temperature, and the plasma sample is stable after 3 freeze-thaw cycles.

In conclusion, the method for detecting representative components and metabolites of the Xiaoer Zhike oral liquid in rat plasma by the UPLC-MS/MS method established by the method is accurate and reliable, and meets the investigation and limitation requirements of related methodologies in the aspects of specificity, precision, recovery rate, stability, matrix effect and the like. Is suitable for analyzing and detecting related pharmacokinetic test samples.

Example 2 pharmacokinetic pre-test of pediatric food retention-eliminating and cough-relieving oral liquid in young rat

24 SD rats born for 25 days are divided into 2 groups, and each group is divided into two halves. Before administration, the oral liquid for removing food retention and relieving cough of children is concentrated to be 6 times of the concentration sold on the market, and the oral liquid is filled into the stomach according to the volume of 20 mL/kg. The body weight was weighed on the day of administration, and the dose and the corresponding volume were accurately calculated from the body weight. Blank blood is taken from subclavian veins of rats in each group before administration, 0.2mL of blood is taken after 0.5, 1, 2, 3, 4, 5, 6, 8, 12 and 24 hours after administration, and the blood is added with 0.1% formic acid physiological saline with the same volume and mixed evenly. Two groups of rats were sampled in turn to prevent excessive blood loss from the rats, 12 animals per time point. The blood was centrifuged at 4000g for 10min in 2h, and the supernatant clear plasma was taken and stored rapidly in a refrigerator at-20 ℃ to be assayed.

The measurement of free synephrine, total synephrine, free arecoline and free arecoline in the plasma of the rat is carried out by adopting ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS method). The method specifically comprises the following steps:

1. experimental medicine and instrument

1.1 standards and reagents

Synephrine reference substance (purity 99.5%, china institute for testing food and drug, batch No. 110727-201809)

Arecoline hydrobromide control (purity 99.8%, china institute for testing food and drug, batch number 100752-201502) arecoline control (purity 100%, STANDARDPHARM, batch number 20CDX 0168-SL-01)

[13C,2H3] -synephrine reference (internal standard, purity 98.1% or more, ISOREAG, lot # ZZS 19052404)

Deuterated Bicoline hydrobromide (internal standard, purity 95%, TORONTO. RESEARCH CHEMICALS, batch No. 13-GRS-143-1)

Formic acid: HPLC grade, ROE SCIENTIFIC INC.

Methanol: HPLC grade, merk;

acetonitrile: HPLC grade, merk;

physiological saline: medical grade, sichuan Kelun pharmaceutical Co., ltd;

purified water (Shandong New times pharmaceutical Co., ltd.).

1.2 instruments

Analytical balance: BT125D, sartorius, germany;

a high-speed centrifuge: SIGMA 3K15, sigma;

LC-MS/MS instrument composition:

a Waters Acquity UPLC/Thermo TSQ endira MS tandem triple quadrupole mass spectrometer;

a workstation: xcalibur3.0;

1.3 Experimental animals

SD rats are from Experimental animals technology, inc. of Wei Tongli, beijing.

2. Experimental methods and results

2.1 LC-MS/MS analysis conditions

2.1.1 chromatographic conditions:

a chromatographic column: HILIC column 50mm × 2.1mm,1.9 μm;

fluidity: a:0.1% formic acid-5 mM ammonium acetate; b: acetonitrile;

gradient elution. 0-0.5min 90%, 0.5-2min 90% -50% B, 2.0-4.0min 50% B, 4.0-5.5min50% -90% B, 5.5-6.0 90% B.

Flow rate: 200 mul/min;

column temperature: 35 deg.C

Temperature of sample pan: 8 deg.C

Sample introduction volume: 3 μ l.

2.1.2 Mass Spectrometry conditions: the ionization of each object to be detected adopts an electrospray ion source and a positive ion mode. In SRM scanning mode, the protonated parent ion of each analyte is [ M + H ]] ⁺ . The ion pair and collision reaction conditions used are as follows:

an ion source: H-ESI; ejection voltage: 4100V; sheath gas: 35Arb; auxiliary gas: 10Arb; tail gas blowing: 0Arb; ion transport capillary temperature: 325 ℃; the gasification temperature: at 150 ℃.

2.2 Experimental results:

non-atrioventricular model pharmacokinetic analysis of oral pediatric food retention-elimination cough-relieving oral liquid for young rats: the method for determining representative components of infantile food retention removing and cough relieving oral liquid in biological samples established in the item and the applicationThe lower limit of the quantification of the metabolite determination methodology is 0.500ng/mL of free drug concentration and 2ng/mL of total synephrine concentration, so that the young rat can still detect the blood concentration near the quantification limit after orally taking the oral liquid for removing food retention and relieving cough for 24 hours, and the lowest detection concentration is less than C _max 1/20 of (1).

The blood concentration-time curve of the oral liquid for removing food stagnation and relieving cough for children is shown in figure 3 and figure 4. The drug concentrations of free synephrine, arecoline and arecaidine reach peak within 0.5h, the blood drug concentrations of arecoline and arecaidine at 1h later are about 1-5 ng/mL, and the peak reaches about 3 h for total synephrine. C of free synephrine, arecoline and total synephrine _max Respectively reaches 75.0, 11.8, 12.3 and 1217ng/mL, and the AUC of the system exposure _0-t 168, 34.7, 30.5 and 18445ng/mL × h, respectively. The results show that after the oral liquid for removing food retention and relieving cough of children is taken by young rats, the concentrations of free and total medicaments of the synephrine in blood plasma are far higher than those of the arecoline and the arecoline, namely, the blood exposure of effective components is higher, the blood exposure of toxic components is lower, and the effectiveness and the safety of the oral liquid for removing food retention and relieving cough of children are effectively ensured. Specific data results are shown in table 1.

Table 1: main pharmacokinetic parameters of young rats after oral administration of oral liquid for removing food retention and relieving cough

Claims

1. A method for determining typical components of XIAO ER XIAO JI ZHI KE KOU FU YE and its metabolites in biological samples by ultra performance liquid chromatography-electrospray ionization tandem mass spectrometry is provided.

2. The method of claim 1, wherein the mobile phase in the ultra high performance liquid chromatography in the method is: 0.1% formic acid-5 mM ammonium acetate water, acetonitrile.

3. The method of claim 1, wherein the elution conditions in ultra high performance liquid chromatography in the method are:

4. the method of claim 1, wherein the conditions in electrospray ionization tandem mass spectrometry are:

an ion source: H-ESI; ejection voltage: 3800-4500V; sheath gas: 30 to 40Arb; auxiliary gas: 10 to 20Arb; tail gas blowing: 0Arb; ion transport capillary temperature: 325 ℃; the gasification temperature: 80-200 ℃.

5. The method of any one of claims 1 to 4, wherein the biological sample in the method includes, but is not limited to, blood, urine, bile, organs, stool.

6. The method of any one of claims 1-4, wherein the representative component is synephrine or arecoline.

7. The method of any one of claims 1-4, wherein the metabolite is glucuronic acid-bound synephrine, arecaidine.

8. The method of claim 6, wherein the electrospray ionization tandem mass spectrometry has a mother ion → daughter ion of synephrine m/z of 168.2 → 150.036, a collision energy of 9.212v, and an RF lens of 45v; [13C,2H3] synephrine m/z parent ion → daughter ion 172.088 → 154.125, collision energy 9.676V, RF lens 30V; the parent ion → the daughter ion of m/z of arecoline is 156.3 → 44, the collision energy is 15v, the RF lens is 45v; the m/z parent ion → daughter ion of arecaidine is 142.2 → 44, the collision energy is 8.54v, and the RF lens is 51v; the m/z parent ion → daughter ion of deuterated arecoline is 161.2 → 48, the collision energy is 15.36v, and the RF lens is 45v.

9. The method of claim 2 or 3, wherein the chromatographic column in the ultra high performance liquid chromatography in the method is a HILIC column, the flow rate is 0.2mL/min, the column temperature is 30-40 ℃, and the sample tray temperature is 4-25 ℃.