CN114295741A

CN114295741A - Method for simultaneously quantifying travoprost and travoprost acid contained in solution sample

Info

Publication number: CN114295741A
Application number: CN202111551205.5A
Authority: CN
Inventors: 刘继东; 杨强; 赵欣; 王小桐; 祖嶶
Original assignee: Shenyang Xingqi Pharmaceutical Co Ltd
Current assignee: Shenyang Xingqi Pharmaceutical Co Ltd
Priority date: 2021-12-17
Filing date: 2021-12-17
Publication date: 2022-04-08

Abstract

The invention discloses a method for simultaneously carrying out quantitative analysis on travoprost and travoprost acid contained in a solution sample, which is characterized in that the quantitative analysis is carried out by adopting a high performance liquid chromatography-secondary mass spectrometry combined technology. The method of the invention has the following advantages: simple operation, sensitivity, rapidness, high specificity, strong specificity, high accuracy, good reproducibility and short analysis time. The method of the invention can obtain a chromatogram with satisfactory sample peak shape and chromatographic retention time, and particularly can realize simultaneous quantitative analysis of travoprost and active metabolite travoprost acid in a biological tissue sample added with an enzyme inhibitor, and can calculate the influence of the enzyme inhibitor on the conversion rate of the travoprost.

Description

Method for simultaneously quantifying travoprost and travoprost acid contained in solution sample

Technical Field

The present invention relates to the field of pharmacokinetic studies. In particular, the present invention relates to a method for quantitatively analyzing a drug in a solution sample, and more particularly, to a method for simultaneously quantitatively analyzing travoprost and travoprost acid contained in a solution sample.

Background

Travoprost is a prostaglandin F2 alpha analog useful for the treatment of open angle, closed angle glaucoma and ocular hypertension. It is a highly selective and high affinity prostaglandin FP receptor full agonist that lowers intraocular pressure by a mechanism that increases aqueous outflow through the uveoscleral pathway. It is an ester precursor drug of travoprost acid (isopropyl travoprost), can be quickly hydrolyzed into free travoprost acid with biological activity by corneal esterase after being absorbed by cornea, and plays a role in reducing intraocular pressure. The structural formulas of travoprost and travoprost acid are shown below:

travoprost

Travoprost acid

During pharmacokinetic studies of drugs, rapid quantitative analysis of the drug contained in a solution sample is often required. In the context of the present invention, the solution samples include, in particular, biological samples from organisms, in addition to solutions in the general sense in the chemical field.

Usually, the collection process of the biological sample itself affects the quantitative determination result of the drug. For example, to study the pharmacokinetics of a drug in animal ocular tissue, it is necessary to harvest the animal ocular tissue. Since the biological barrier of the eyeball is lost immediately after the death of the animal and the enzyme in the eye tissue can still exert the biological activity after the removal of the eyeball, the eyeball is required to be kept in a frozen state during the dissection process and the sample is required to be rapidly measured.

Experiments have shown that adding travoprost to rabbit aqueous humor, plasma and whole blood, respectively, and standing at about 4 ℃ for about 2 hours after vortexing, travoprost degrades by about 30% in aqueous humor, and even up to about 80% in whole blood and plasma, respectively.

The inventors of the present invention have achieved control of the degradation of travoprost during the collection of biological samples and during the processing of biological samples after collection by adding enzyme inhibitors to biological samples comprising travoprost. The enzyme inhibitor may comprise methanesulfonyl fluoride, physostigmine, dichlorvos, paraoxon, or any mixture of the foregoing. The inventor of the present invention also investigated the effect of various factors such as different enzyme inhibitors, enzyme inhibitor concentrations, temperature and pH conditions on the degradation of travoprost in biological samples, i.e. investigated the effect of these factors on the conversion of travoprost to the corresponding acid in biological samples.

In the prior art, there is no report on a method for simultaneously and quantitatively analyzing travoprost and travoprost acid contained in a solution sample, and particularly, there is no report on a biological analysis method for simultaneously and rapidly measuring travoprost and travoprost acid in a sample solution after an enzyme inhibitor is added to a biological sample.

Disclosure of Invention

Technical problem to be solved by the invention

The invention aims to provide a method for simultaneously realizing rapid quantitative analysis of travoprost and travoprost acid contained in a solution sample. In particular, it would be desirable to provide a method for the simultaneous rapid quantitative analysis of travoprost and its active metabolite travoprost acid in a biological sample containing an enzyme inhibitor. The quantitative analysis method enables the study of the influence of various factors such as different enzyme inhibitors, enzyme inhibitor concentrations, temperature and pH conditions on the conversion of travoprost to travoprost acid during and after the collection of biological samples.

Technical scheme for solving technical problem

In order to solve the technical problem, the invention provides the following technical scheme:

scheme 1: a method for simultaneously performing quantitative analysis on travoprost and travoprost acid contained in a solution sample, the method being characterized in that the quantitative analysis is performed by using a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS) technique, preferably a HPLC-MS technique.

Scheme 2: the method according to scheme 1, wherein the solution sample is obtained by a method comprising the steps of:

i. administering travoprost to an animal and extracting animal tissue from the animal after administration;

placing the animal tissue from step i in an aqueous solution containing an enzyme inhibitor and a pH modifier, preferably comprising citric acid, at a temperature of about 0 ℃ to about 4 ℃ and pre-treating the biological sample, centrifuging and taking a supernatant sample, the pH of the supernatant sample being about 4.0 to about 6.0, preferably about 5.0;

adding the ethoxybenzene salicylamide and travoprost acid-D4 to the sample from step ii and mixing well to dissolve,

adding an organic solvent to the sample from step iii in a volume ratio of about 1:2 to about 1:9, preferably about 1:4, and mixing thoroughly, wherein the organic solvent preferably comprises acetonitrile, methanol, ethanol or any mixture thereof, most preferably acetonitrile, and the mixed sample is centrifuged to obtain a supernatant as the solution sample.

Scheme 3: the method according to scheme 1, wherein the solution sample is obtained by a method comprising the steps of:

extracting animal tissue from an animal to which travoprost has not been administered;

subjecting the animal tissue from step i' to a biological sample pretreatment at a temperature of about 0 ℃ to about 4 ℃, centrifuging and taking a supernatant sample, adding an enzyme inhibitor, travoprost acid and a pH adjuster, said sample having a pH of about 4.0 to about 6.0, preferably about 5.0, said pH adjuster preferably comprising citric acid;

adding the ethoxybenzene salicylamide and travoprost acid-D4 to the sample from step ii' and mixing well to dissolve;

adding an organic solvent to the sample from step iii' in a volume ratio of about 1:2 to about 1:9, preferably about 1:4, to the organic solvent, preferably comprising acetonitrile, methanol, ethanol or any mixture thereof, most preferably acetonitrile, and mixing the sample to obtain a supernatant as the solution sample after centrifugation.

Scheme 4. the method according to scheme 3, wherein the concentration of travoprost in the supernatant sample in step ii' is from about 10 to about 1000ng/mL and the concentration of travoprost acid is from about 2 to about 200 ng/mL.

Scheme 5. the method according to any of schemes 2 to 4, wherein the animal tissue comprises aqueous humor, plasma or whole blood.

Scheme 6. the method according to any of schemes 2 to 5, wherein the enzyme inhibitor comprises dichlorvos, paraoxon, phenylmethylxanthyl fluoride, physostigmine and combinations of one or more thereof, with paraoxon being especially preferred.

Scheme 7. the method according to any of schemes 2 to 6, wherein the concentration of the enzyme inhibitor in the supernatant in step ii or ii' is from about 1 to about 20mM, preferably from about 2 to about 10mM, more preferably about 5 mM.

Scheme 8. the method according to any of schemes 2 to 7, wherein in step iii or iii', the concentration of ethoxybensalamine in the mixed sample is preferably from about 50 to about 200ng/mL, preferably about 100ng/mL, and the concentration of travoprost-D4 is from about 10 to about 30ng/mL, preferably about 20 ng/mL.

Scheme 9: the method according to any one of schemes 1 to 8, wherein the chromatographic conditions in the LC-MS/MS technology comprise that a chromatographic column with octadecylsilane chemically bonded silica as a filler is adopted, and a mixed solution of aqueous formic acid solution and acetonitrile formic acid solution is used as a mobile phase for elution, wherein the elution is carried out, wherein

Setting the flow rate of the mobile phase to about 0.2 mL/min to about 0.4 mL/min, preferably about 0.3 mL/min;

the aqueous formic acid solution is from about 0.09 to about 0.11 volume percent, preferably about 0.1 volume percent, aqueous formic acid solution;

the formic acid acetonitrile solution is about 0.09 to about 0.11 volume percent formic acid, preferably about 0.1 volume percent acetonitrile solution; and the elution is preferably performed by gradient elution, and the procedure of the gradient elution is preferably set as follows:

from about 0 to about 0.8 minutes, maintaining a mobile phase consisting of from about 85 to about 95 volume percent, preferably about 90 volume percent, of said aqueous formic acid and from about 5 to about 15 volume percent, preferably about 10 volume percent, of said acetonitrile solution of formic acid;

from about 0.8 to about 1.2 minutes, allowing the mobile phase to change from a uniform velocity consisting of from about 85 to about 95 volume percent, preferably about 90 volume percent, of said aqueous formic acid and from about 5 to about 15 volume percent, preferably about 10 volume percent, of said acetonitrile solution of formic acid to a uniform velocity consisting of from about 5 to about 15 volume percent, preferably about 10 volume percent, of said aqueous formic acid and from about 85 to about 95 volume percent, preferably about 90 volume percent, of said acetonitrile solution of formic acid;

from about 1.2 to about 2.0 minutes, maintaining a mobile phase consisting of from about 5 to about 15 volume percent, preferably about 10 volume percent, of said aqueous formic acid and from about 85 to about 95 volume percent, preferably 90 volume percent, of said acetonitrile solution of formic acid;

from about 2.0 to about 3.5 minutes, maintaining a mobile phase consisting of from about 85 to about 95 volume percent, preferably 90 volume percent, of said aqueous formic acid and from about 5 to about 15 volume percent, preferably about 10 volume percent, of said acetonitrile solution of formic acid.

Scheme 10. the method according to any of schemes 1 to 9, wherein the mass spectrometric conditions in the LC tandem mass spectrometry technique comprise simultaneous detection of positive and negative ions in a multiple reaction monitoring mode using an electrospray ion source, wherein the mass spectrometric conditions comprise setting of the following parameters:

positive ion mode: internal standard: about 4500V to about 5500V, preferably about 5000V; air curtain air: about 30 to about 40psi, preferably about 35 psi; gas 1: about 60 to about 70psi, preferably about 65 psi; gas 2: about 60 to about 70psi, preferably about 65 psi; collision gas: medium;

negative ion mode: internal standard: about-3500V to about-4500V, preferably about-4000V; air curtain air: about 30 to about 40psi, preferably about 35 psi; gas 1: about 60 to about 70psi, preferably about 65 psi; gas 2: about 60 to about 70psi, preferably about 65 psi; collision gas: medium;

ion source temperature: from about 400 to about 500 deg.C, preferably about 450 deg.C.

Technical effects achieved by the invention

The purpose of the invention is achieved through the technical schemes of the invention. In order to study the effect of enzyme inhibitors on the degradation of travoprost during and after the collection of biological samples during their processing, the present invention established a sensitive, rapid and specific (ultra) high performance liquid chromatography-tandem mass spectrometry (HPLC/UPLC-MS/MS) method that allows the simultaneous determination of the concentration of travoprost and its active metabolite travoprost acid in animal tissues, in particular plasma, whole blood, ocular tissues.

As a combined technology of (ultra) high performance liquid chromatography tandem mass spectrometry (HPLC/UPLC-MS/MS), the method of the invention has the following advantages: simple operation, sensitivity, rapidness, high specificity, strong specificity, high accuracy, good reproducibility and short analysis time. The method of the invention can obtain a chromatogram with satisfactory sample peak shape and chromatographic retention time, and particularly can realize simultaneous quantitative analysis of travoprost and travoprost acid which is an active metabolite in a biological tissue sample, and can calculate the conversion rate of the travoprost.

In particular, the process of the invention has the following advantages, among others:

(1) the sampling amount is less: determining that a sample amount can be less than about 0.1mL, or even only about a few μ L for analysis;

(2) the pretreatment is simple and convenient: after a sample is treated by an enzyme inhibitor and optional citric acid, only an organic solvent is needed for precipitating protein, and the HPLC/UPLC-MS/MS sample injection analysis can be directly carried out after vortex centrifugation, so that the method is simple and easy to implement, and is suitable for conventional quantitative detection;

(3) the sensitivity is high, the detection sensitivity of the determination is obviously improved through mass spectrum detection, and the quantitative lower lines of travoprost and travoprost acid are respectively 10ng/mL and 2 ng/mL;

(4) the selectivity is strong, and endogenous substances in a blank biological sample do not interfere with the determination of the drug and the internal standard;

(5) the determination time is short: the entire chromatographic assay process may be no more than about 3.5 minutes;

(6) the linear ranges of travoprost and travoprost acid are respectively 10-1000 ng/mL and 200ng/mL, the linear range span is large, and the dynamic analysis change determination of the drug in vivo can be well met;

(7) the recovery rate is stable, and the precision (relative standard deviation, RSD) in the day and the day is less than 15%; and

(8) the matrix effect is stable, and the matrix effect of the low, medium and high concentrations is between about 80% and about 120%.

Drawings

In order to more clearly illustrate the present invention, the following description and drawings of the present invention will be described and illustrated. It should be apparent that the drawings in the following description illustrate only certain aspects of some exemplary embodiments of the invention, and that other drawings may be derived therefrom by those skilled in the art without the exercise of inventive faculty.

FIG. 1 IS a mass scan of travoprost (A) and ethoxybenzene salicylamine (IS) (B) using mass spectrometry conditions in the methods of the present invention.

FIG. 2 IS a mass spectrometry scan of travoprost acid (A) and travoprost acid D4(IS) (B) using mass spectrometry conditions in the methods of the invention.

FIG. 3 is an ion detection (MRM) chromatogram of a blank biological sample using mass spectrometry conditions in a method of the invention, wherein the upper panel is a positive ion mode ion detection (MRM) chromatogram of the blank biological sample, and the lower panel is a negative ion mode ion detection (MRM) chromatogram of the blank biological sample.

FIG. 4 is an ion detection (MRM) chromatogram of a blank biological sample supplemented with travoprost (10ng/mL) and travoprost acid (2ng/mL) using the mass spectrometry conditions of the method of the invention, wherein the two plots labeled I and II are the negative ion mode ion detection (MRM) chromatograms of travoprost acid and internal standard travoprost acid D4, respectively; and the two graphs labeled IV and V are positive ion mode ion detection (MRM) chromatograms of travoprost and the internal standard ethoxybenzene salicylamine, respectively.

FIG. 5 is an ion detection (MRM) chromatogram of an actual biological sample using mass spectrometry conditions in the method of the present invention, wherein the two figures labeled I and II are negative ion mode ion detection (MRM) chromatograms of travoprost acid and internal standard travoprost acid D4, respectively; and the two graphs labeled IV and V are positive ion mode ion detection (MRM) chromatograms of travoprost and the internal standard ethoxybenzene salicylamine, respectively.

Detailed Description

Hereinafter, the present invention will be described in detail.

The invention mainly relates to a method for simultaneously carrying out quantitative analysis on travoprost and travoprost acid contained in a solution sample, which is characterized in that the quantitative analysis is carried out by adopting a (ultra) high performance liquid chromatography-mass spectrometry combined technology.

In the present invention, as can be understood by a person skilled in the art, the expression "(ultra) high performance liquid chromatography" includes "high performance liquid chromatography" and "ultra high performance liquid chromatography". Indeed, the terms "high performance liquid chromatography" and "ultra high performance liquid chromatography" are used interchangeably herein.

In the method of the present invention, the hplc-tandem mass spectrometry is preferably hplc-tandem secondary mass spectrometry.

In the method of the present invention, the solution sample may be an aqueous solution, a non-aqueous solution, or a solution containing both water and an organic solvent.

In a preferred embodiment of the method of the invention, the solution sample is obtained by a method comprising the following method steps i to iv:

step i: travoprost is administered to an animal and animal tissue is extracted from the animal after administration.

In the above step i, the administration is not particularly limited herein. In the context of the present invention, it may include, but is not limited to, injection administration, oral administration, transdermal administration, mucosal administration, and the like.

Step ii: placing the animal tissue from step i in an aqueous solution containing an enzyme inhibitor and optionally a pH modifier at a temperature of about 0 ℃ to about 4 ℃ and pre-treating the biological sample, centrifuging and taking a supernatant sample.

Step iii: adding an internal standard to the sample from step ii and mixing it well to dissolve.

Step iv: adding an organic solvent to the sample obtained in step iii and mixing it well, and taking the supernatant as the solution sample after centrifugation of the mixed sample.

In another preferred embodiment of the method of the invention, the solution sample is obtained by a method comprising the following method steps i 'to iv':

step i': animal tissue was extracted from animals not administered travoprost.

Step ii': pre-treating the animal tissue from step i' with a biological sample at a temperature of about 0 ℃ to about 4 ℃, centrifuging and collecting the supernatant sample, and adding an enzyme inhibitor, travoprost acid, and optionally a pH adjusting agent.

In step ii' above, the concentration of travoprost and the concentration of travoprost acid in the supernatant sample are preferably within respective linear ranges of measurement. For example, the concentration of travoprost may be from about 10 to about 1000 ng/mL; the concentration of travoprost acid may preferably be from about 2 to about 200 ng/mL.

In the above step ii', travoprost and travoprost acid may be added as a solid in the form of a pure substance or as a liquid in the form of a solution. Preferably, a liquid in the form of a solution, more preferably an aqueous solution, is used.

Step iii': adding an internal standard to the sample from step ii' and mixing it well for dissolution.

Step iv': adding an organic solvent to the sample obtained from step iii' and mixing it thoroughly, the mixed sample taking the supernatant as the solution sample after centrifugation.

In step i or i' above, the animal is preferably a mammal, including but not limited to rabbits, cats, dogs, mice, monkeys, etc., preferably a rabbit, more preferably a new zealand rabbit.

In the above step i or i', the animal tissue is not particularly limited, and may include, but is not limited to, aqueous humor, plasma, whole blood, or the like.

In step ii or ii' above, the pretreatment of the biological sample is a homogenization treatment method well known to those skilled in the art. The biological sample pretreatment process employed in the present invention can be found, for example, in the prior art documents: velagaleti P.R., Buonarti M.H., Challeges and Strategies in Drug resource Measurement (Bioanalysis) of Ocular Tissues [ J ]. Methods in Pharmacology & Toxicology,2014,33-52, the disclosure of which is incorporated herein by reference.

In the above step ii or ii', the enzyme inhibitor is not particularly limited as long as the enzyme inhibitor can inhibit the enzyme in the animal tissue from hydrolyzing travoprost. For example, in a preferred embodiment of the method of the present invention, the enzyme inhibitor comprises dichlorvos, paraoxon, phenylmethylxanthyl fluoride, physostigmine, and combinations of one or more thereof. In a more preferred embodiment, the enzyme inhibitor comprises paraoxon.

In step ii or ii', the enzyme inhibitor may be added in the form of a pure substance or a solution of the enzyme inhibitor. Preferably, a solution of the enzyme inhibitor is used in the form of a solution, more preferably an aqueous solution.

In the above step ii or ii', the concentration of the enzyme inhibitor in the supernatant is not particularly limited as long as the enzyme inhibitor at the concentration can inhibit the enzyme in the animal tissue from hydrolyzing travoprost in the sample. For example, in a preferred embodiment of the invention, the concentration of the enzyme inhibitor in the supernatant is preferably from about 1 to about 20mM, more preferably from about 2 to about 10mM, and most preferably about 5 mM.

In step ii or ii' above, the pH adjusting agent is optionally present. If necessary, the pH adjusting agent adjusts the pH of the sample in which the enzyme inhibitor, travoprost and travoprost acid are dissolved to weak acidity, and for example, the sample may be adjusted to a pH of about 4.0 to about 6.0, preferably about 5.0. Here, the pH adjuster preferably includes citric acid.

In this case, the pH adjustor to be added may be a pH adjustor in a pure solid form, or may be a pH adjustor solution in a solution form. Preferably, a solution of the pH adjusting agent in the form of a solution, preferably an aqueous solution, is used.

In step iii or iii' above, the internal standards are ethoxybenzene salicylamide and travoprost acid-D4. In this case, the concentration of ethoxybenzene salamide in the mixed sample is preferably about 50 to about 200ng/mL, preferably about 100ng/mL, and the concentration of travoprost-D4 is about 10 to about 30ng/mL, preferably about 20 ng/mL.

The internal standard added here may be a pure internal standard or an internal standard solution in the form of a solution. Preferably, an internal standard solution in the form of a solution, preferably a methanol solution or an aqueous solution, is used.

In the above step iv or iv', the organic solvent is not particularly limited. The purpose of the organic solvent added here is to precipitate out the proteins in the sample.

Preferably, the organic solvent may adopt acetonitrile, methanol, ethanol or any mixture thereof, and particularly preferably the organic solvent comprises acetonitrile or is acetonitrile.

In the context of the present invention, the intimately mixed dissolution may be achieved by means commonly used in the art, as will be appreciated by those skilled in the art. This can be achieved, for example, by means such as oscillation, vortexing, and the like (preferably vortexing).

In the method of the present invention, the chromatographic conditions in the liquid chromatography-tandem mass spectrometry technique include using a chromatographic column using octadecylsilane chemically bonded silica as a filler, and eluting with a mixed solution of aqueous formic acid solution and acetonitrile formic acid solution as a mobile phase.

In a preferred embodiment, the flow rate of the mobile phase may be set to about 0.2 mL/min to about 0.4 mL/min, preferably about 0.3mL/min.

In a preferred embodiment, the aqueous formic acid solution is preferably an aqueous solution of from about 0.09 to about 0.11 volume percent, preferably about 0.1 volume percent, of formic acid; the formic acid acetonitrile solution is preferably about 0.09 to about 0.11 volume percent, preferably about 0.1 volume percent, acetonitrile solution of formic acid.

In addition, the conditions for the elution are not particularly limited as long as the conditions can separate travoprost and travoprost acid, impurities, and the like in the solution sample.

In a preferred embodiment, the elution is preferably performed by a gradient elution procedure, wherein the gradient elution procedure is preferably set to:

In the method of the present invention, the mass spectrometry conditions in the liquid chromatography-tandem mass spectrometry technology include simultaneous detection of positive and negative ions in a multiple reaction monitoring mode by using an electrospray ion source.

In a preferred embodiment, the mass spectrometry conditions comprise setting the following parameters:

positive ion mode: internal Standard (IS): about 4500V to about 5500V, preferably about 5000V; air Curtain gas (curtaingas): about 30 to about 40psi, preferably about 35 psi; gas 1(Gas 1): about 60 to about 70psi, preferably about 65 psi; gas 2(Gas 2): about 60 to about 70psi, preferably about 65 psi; collision Gas (Collision Gas): medium;

negative ion mode: internal Standard (IS): about-3500V to about-4500V, preferably about-4000V; air Curtain gas (curtaingas): about 30 to about 40psi, preferably about 35 psi; gas 1(Gas 1): about 60 to about 70psi, preferably about 65 psi; gas 2(Gas 2): about 60 to about 70psi, preferably about 65 psi; collision Gas (Collision Gas): medium;

Detailed Description

The invention and its advantages will be explained in more detail below by means of exemplary embodiments. The description of the exemplary embodiments is merely illustrative and is in no way intended to limit the disclosure, its application, or uses. The present disclosure may be embodied in many different forms and is not limited to the embodiments described herein. These embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art. It should be noted that: unless otherwise indicated, the relative arrangement of parts and steps, the composition of materials, numerical expressions and values, etc., set forth in these embodiments should be construed as merely illustrative, and not a limitation.

Materials and instruments used:

animals: new Zealand rabbits weighing about 1.5 to about 2.5kg, commercially available from Qingdao Kangda Biotech, Inc.;

dichlorvos: purity ≥ 98.0%, commercially available from Sigma, USA;

phenylmethylsulfonyl fluoride: purity 98.5%, commercially available from Sigma company, usa;

paraoxon: the purity is more than or equal to 98.5 percent, and the product is purchased from a Chinese standard product information network;

physostigmine: the purity is more than or equal to 99.0 percent, and the product is purchased from China standard product information network;

citric acid: 99.5% pure, commercially available from lao nan er kang pharmaceutical ltd;

travoprost: 97.0% purity, commercially available from USP corporation, usa;

travoprost acid: purity 98.9%, commercially available from TLC company, canada;

ethoxybenzene salicylamine: purity 99.9%, commercially available from china institute for pharmaceutical and biological products;

travoprost acid-D4: purity 96.0%, commercially available from TLC, canada;

acetonitrile: chromatographically pure, commercially available from Sigma company, usa;

formic acid: chromatographically pure, commercially available from Sigma company, usa;

methanol: chromatographically pure, commercially available from Sigma company, usa;

water: the chromatogram is pure and self-made;

ultra-high performance liquid chromatography system: under the trade name "ACQUITY Ultra Performance LC^TM"commercially available from Waters corporation, USA; equipped with a universal type binary high-pressure pump and an injector;

a secondary mass spectrometer: a triple quadrupole tandem mass spectrometer, commercially available from AB Sciex corporation, usa under the trade designation "Qtarp 5500", equipped with an electrospray ion source (ESI ion source);

a chromatographic column: octadecylsilane chemically bonded silica gel as filler with size of 2.1mm × 50mm, BEH C18 as filler, particle diameter of 1.7 μm, and trade name of "ACQUITY

"commercially available from Waters corporation, USA.

And (3) biological sample treatment:

test samples were prepared in the following general manner. The general method comprises the following steps:

(1) extracting animal tissue (aqueous humor, plasma or whole blood) from a New Zealand rabbit as a biological sample;

(2) placing the biological sample obtained in the step (1) into a sample collection pipe containing enzyme inhibitors with different concentrations and optionally containing citric acid at a set temperature, adding a determined amount of ultrapure water for pretreatment of the biological sample, adding citric acid according to experimental requirements, wherein the citric acid is added so as to enable the pH value of the sample to reach about 5.0, and centrifuging by using a centrifuge to obtain a supernatant sample;

(3) optionally adding travoprost and/or travoprost acid to the sample from step (2) to a set concentration according to experimental requirements, vortexing for about 1 minute and then allowing to stand for about 15 minutes;

(4) adding a mixed internal standard working solution consisting of a methanol solution of ethoxybenzene salicylamine and a methanol solution of kovoprost-D4 to the sample from step (3) such that the concentration of ethoxybenzene salicylamine in the final biological sample is about 100ng/mL and the concentration of kovoprost-D4 is about 20 ng/mL;

(5) adding acetonitrile to the sample from step (4) in a volume ratio of sample to acetonitrile of about 1:4, vortexing for about 1 minute, centrifuging (about 14500rpm) for about 10 minutes, and taking the supernatant, about 7.0. mu.L of this solution was taken as a test sample for the liquid chromatography tandem secondary mass spectrometry technique as described below.

HPLC/UPLC-MS/MS analysis conditions:

chromatographic conditions

A chromatographic column: a column as described in the materials and instruments section above was used.

Column temperature: at 30 ℃.

Mobile phase: a mixture of a 0.1 vol% aqueous solution (A) of formic acid and a 0.1 vol% acetonitrile solution (B) of formic acid,

and (3) an elution mode: gradient elution, the change in flow match ratio over time was set as follows:

0 to 0.8 min, 90% a;

after 0.8 to 1.2 minutes, the 90 percent A is changed to 10 percent A at a constant speed;

1.2 to 2.0 minutes, 10% a;

2.0 to 3.5 min, 90% a.

Flow rate: 0.3mL/min.

Sample introduction amount: 7.0. mu.L.

Second order Mass Spectrometry Condition

Adopting an electrospray ionization source (ESI ionization source) and a multi-reaction monitoring mode (MRM) to simultaneously detect positive ions and negative ions; the ion pairs for quantitative analysis were:

positive ion mode: travoprost, m/z 501.3 → 207.2; ethoxybenzene salicylamide (IS), m/z 258.1 → 121.1;

negative ion mode: travoprost acid, m/z 457.2 → 161.0; travoprost acid D4(IS), m/z 461.2 → 161.0.

The selection of the positive and negative ion modes described above is determined from the mass spectra shown in figures 1 and 2 of the present specification.

The mass spectrometry conditions were set to include the following parameters:

positive ion mode, Internal Standard (IS): 5000V; air Curtain gas (curtaingas): 35 psi; gas 1(Gas 1): 65 psi; gas 2(Gas 2): 65 psi; collision Gas (Collision Gas): medium;

negative ion mode, Internal Standard (IS): -4000V; air Curtain gas (curtaingas): 35 psi; gas 1(Gas 1): 65 psi; gas 2(Gas 2): 65 psi; collision Gas (Collision Gas): medium;

ion source temperature: at 450 ℃.

It should be noted that the chromatographic conditions and the secondary mass spectrometry conditions are typical conditions, and those skilled in the art can adjust the parameters appropriately according to the disclosure of the present invention to obtain the best effect according to different characteristics of the used instrument in practical application.

HPLCUPLC-MS/MS measurement method:

(1) specificity

In the method, the specificity of the object to be measured and the internal standard is evaluated by comparing the lowest concentration point of the standard curve with a blank biological sample and an actual biological sample operated by the same method.

The blank biological samples were as described above in the "biological sample treatment" section, except that new zealand rabbits to which no travoprost was administered were used, and no travoprost and travoprost acid were added in step (3). The actual biological samples were as described in the "biological sample treatment" section above, except that the New Zealand rabbits to which travoprost had been administered were used, and no travoprost and travoprost acid were added in step (3).

Description figures 3, 4 and 5 show blank biological samples, travoprost (10ng/mL) and travoprost acid (2.0ng/mL) added to blank biological samples, ion detection (MRM) chromatograms of actual biological samples, respectively. As can be seen from these figures, endogenous substances in the blank biological sample and the actual biological sample do not interfere with the determination of travoprost, travoprost acid and their mixed internal standards.

(2) Standard curve

A mixed standard curve series solution of travoprost and travoprost acid was prepared from acetonitrile.

Blank biological samples were taken as described above, and mixed standard curve series solutions of travoprost and travoprost acid were added to prepare standard biological samples containing travoprost at concentrations of 10, 20, 40, 100, 250, 500, 1000ng/mL and travoprost acid at concentrations of 2, 4, 8, 20, 50, 100, 200 ng/mL.

Taking 7 mu L of the standard biological sample to carry out UPLC-MS/MS analysis, and carrying out regression analysis by adopting a weighted least square method to obtain a standard curve, wherein the equation of the standard curve is as follows: travoprost, y is 0.000302x-0.000908(r is 0.9945), travoprost acid, y is 0.281x-0.0344(r is 0.9990), y represents the ratio of the peak areas of the test substance and the internal standard, and x represents the concentration of the test substance. The linear range of the quantitative determination of the travoprost in the method is 10-1000 ng/mL, and the lowest limit of quantitation is 10 ng/mL; the linear range of the assay for travoprost acid was 2 to 200ng/mL with a minimum quantitation limit of 2 ng/mL.

(3) Precision and accuracy

Preparing low, medium and high concentrations of travoprost of 20, 250 and 800ng/mL respectively according to the standard biological sample preparation method of the standard curve part; quality Control (QC) standard biological samples containing travoprost acid at low, medium and high concentrations of 4.0, 50 and 160ng/mL respectively, each concentration was analyzed 6 times, the sample was continuously measured for three days, the method precision was evaluated by the obtained day and day RSD (%), the accuracy was evaluated by the ratio of the actually measured value to the theoretical value, and the analysis results are shown in Table 1.

Table 1: accuracy and precision of travoprost and travoprost acid in biological samples

As shown in table 1, the precision of travoprost in day and in the middle of day is 8.6% to 9.5% and 6.3% to 8.1%, respectively, with an accuracy range of-4.9% to 0.7%; the precision of the travoprost acid is respectively 5.4 to 7.9 percent and 1.4 to 7.9 percent in the day and the day, and the accuracy range is-6.2 to-3.3 percent; all reach the standard of < + > -15%, which shows that the method has good precision and accuracy.

(4) Extraction recovery rate

Quality Control (QC) samples were prepared at low, medium and high concentrations of travoprost, 20, 250, 800ng/mL respectively, and at low, medium and high concentrations of travoprost acid, 4.0, 50, 160ng/mL respectively, following the procedure described above for the series of standard solution formulations in the "standard curves" section, 6 samples were analyzed at each concentration and the chromatographic peaks recorded.

The standard biological samples were prepared with the concentrations of travoprost at 20, 250, and 800ng/mL for the low, medium, and high concentrations, and 4.0, 50, and 160ng/mL for the low, medium, and high concentrations of travoprost acid, respectively, by performing 6 sample analyses at each concentration, and recording the chromatographic peaks, according to the standard biological sample preparation method described in the section "standard curve" above.

And dividing the peak area of each concentration standard biological sample by the peak area of the quality control sample to respectively calculate the recovery rate of the medicine. The analysis and measurement results are shown in table 2:

table 2: recovery of travoprost and travoprost acid (n ═ 6)

As shown in table 2, the extraction recoveries of travoprost at the three concentrations were 92.5%, 99.0% and 94.8%, respectively, and the extraction recoveries of travoprost acid at the three concentrations were 94.9%, 108.8% and 99.5%, respectively, indicating that the method of the present invention has good extraction recoveries for different concentrations of travoprost and travoprost acid.

(5) Matrix effect

Quality control samples were prepared with three concentrations of trovoprost, low, medium and high, 20, 250, 800ng/mL respectively, and trovoprost acid, low, medium and high, 4.0, 50, 160ng/mL respectively, by operating under the conditions of the serial standard solution preparation methods in the "standard curve" section above, 6 samples were analyzed for each concentration, and chromatographic peaks were recorded.

The matrix effect was examined as the ratio of the two peak areas. The analysis and measurement results are shown in Table 3.

Table 3 matrix effects of travoprost and travoprost acid (n ═ 6)

As shown in table 3, the matrix effect of travoprost was between 94.7% and 107.0% at the three concentrations; the matrix effect of travoprost acid at the three concentrations ranged from 91.0% to 100.5%, indicating that the results of the method of the invention with respect to matrix effect were satisfactory for different concentrations of travoprost and travoprost acid.

(6) Stability of

Stability samples with a low and high concentration of travoprost of 20 and 800ng/mL, respectively, and a low and high concentration of travoprost acid of 4.0 and 160ng/mL, respectively, were prepared for examination by following the procedure described in the section "Standard Curve" above for the preparation of the series of Standard solutions. The investigation items comprise 6 hours of placement at room temperature, 48 hours of placement after biological sample treatment, three cycles of freeze thawing and long-term freezing stability at-80 ℃, and 3 samples are analyzed at each concentration. The stability test results are shown in table 4.

TABLE 4 stability results for travoprost and travoprost acid

As shown in Table 4, the deviation of the measured values from the theoretical values of travoprost and travoprost acid is less than +/-15%, which indicates that the method has good stability.

Effect of enzyme inhibitor, enzyme inhibitor concentration, pH and temperature on travoprost conversion test samples were prepared as described in the "biological sample treatment" section. Wherein, the new zealand rabbit is not administrated, and the trovoprost acid are added in the step (3). And (3) changing different inhibitors and concentrations thereof, carrying out quantitative analysis on the contents of travoprost and travoprost acid in each biological sample, and calculating the conversion rate of the travoprost.

The results are shown in tables 5 to 7 below, where the control samples were samples prepared without addition of enzyme inhibitors.

Table 5: effect of different enzyme inhibitors on the conversion of travoprost in Rabbit aqueous humor

Table 6: effect of different enzyme inhibitors on the conversion of travoprost in Rabbit plasma

Table 7: effect of different enzyme inhibitors on the conversion of travoprost in Whole Rabbit blood

From the results of tables 5 to 7 above, it can be seen that 1, 2, 5, 10 and 20mM of the enzyme inhibitors dichlorvos, phenylmethylsulfonyl fluoride, paraoxon and physostigmine were able to significantly inhibit the hydrolysis of travoprost in the aqueous humor, plasma and whole blood of new zealand rabbits, compared to the controls. Further, as can be seen from a review of the data in 3 of Table 1, paraoxon generally exhibits better inhibitory effects at all concentrations and in all tissue samples than other enzyme inhibitors, and thus is preferred as an enzyme inhibitor in the present method.

The above process was repeated using dichlorvos and paraoxon as enzyme inhibitors, wherein the process was carried out under the following three conditions, respectively:

1) the temperature in the step (2) is 4 ℃, citric acid is not added to adjust the pH value,

2) the temperature in step (2) is 4 ℃, citric acid is added to adjust the pH to 5.0, and

3) the temperature in step (2) was room temperature, and citric acid was added to adjust the pH to 5.0.

The test results are shown in table 8.

Table 8: effect of pH and temperature on conversion of travoprost in biological samples

As can be seen from the data of Table 8, combining the data in aqueous humor, plasma and whole blood, under the conditions of temperature of 4 ℃ and pH adjustment to 5.0 by addition of citric acid in step (2), paraoxon better inhibited the hydrolysis of travoprost.

In view of the above, the inventors of the present invention have sufficiently demonstrated that the method of the present invention can achieve the object of the present invention, and those skilled in the art can recognize that the method can be fully applied to simultaneous rapid quantitative analysis of travoprost and travoprost acid in an actual biological sample.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein, and any reference signs in the claims are not intended to be construed as limiting the claim concerned.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art. These other embodiments are also covered by the scope of the present invention.

It should be understood that the above-mentioned embodiments are only for explaining the present invention, and the protection scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical scope of the present invention and the equivalent replacement or change of the technical solution and the inventive concept thereof in the technical scope of the present invention.

The use of the word "comprising" or "comprises" and the like in the present invention means that the element preceding the word covers the element listed after the word and does not exclude the possibility of also covering other elements. The term "about" as used herein has the meaning well known to those skilled in the art, and preferably means that the term modifies a value within the range of ± 50%, ± 40%, ± 30%, ± 20%, ± 10%, ± 5% or ± 1% thereof.

All terms (including technical or scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs unless specifically defined otherwise. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

Techniques, methods, and apparatus known to those of ordinary skill in the relevant art may not be discussed in detail but are intended to be part of the specification where appropriate.

The disclosures of the prior art documents cited in the present description are incorporated by reference in their entirety and are therefore part of the present disclosure.

Claims

1. A method for simultaneously performing quantitative analysis on travoprost and travoprost acid contained in a solution sample, the method being characterized in that the quantitative analysis is performed by using a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS) technique, preferably a HPLC-MS technique.

2. The method according to claim 1, wherein the solution sample is obtained by a method comprising the steps of:

adding and thoroughly mixing the ethoxybensalamine and travoprost-D4 to the sample obtained from step ii and dissolving, iv adding and thoroughly mixing an organic solvent to the sample obtained from step iii, wherein the volume ratio of the sample obtained from step iii to the organic solvent is about 1:2 to about 1:9, preferably about 1:4, the organic solvent preferably comprises acetonitrile, methanol, ethanol or any mixture thereof, most preferably acetonitrile, and the supernatant of the mixed sample after centrifugation is taken as the solution sample.

3. The method according to claim 1, wherein the solution sample is obtained by a method comprising the steps of:

subjecting the animal tissue from step i' to a biological sample pretreatment at a temperature of about 0 ℃ to about 4 ℃, centrifuging and collecting the supernatant, and adding an enzyme inhibitor, travoprost acid and a pH adjusting agent, wherein the pH of the sample is about 4.0 to about 6.0, preferably about 5.0, and the pH adjusting agent preferably comprises citric acid;

4. The method according to claim 3, wherein the concentration of travoprost in the supernatant sample in step ii' is from about 10 to about 1000ng/mL and the concentration of travoprost acid is from about 2 to about 200 ng/mL.

5. The method according to any one of claims 2 to 4, wherein the animal tissue comprises aqueous humor, plasma or whole blood.

6. The method according to any one of claims 2 to 4, wherein the enzyme inhibitor comprises dichlorvos, paraoxon, benzylxanthyl fluoride, physostigmine and combinations of one or more thereof, especially preferably paraoxon.

7. The method according to any one of claims 2 to 4, wherein the concentration of the enzyme inhibitor in the supernatant in step ii or ii' is from about 1 to about 20mM, preferably from about 2 to about 10mM, more preferably about 5 mM.

8. The method according to any one of claims 2 to 4, wherein in step iii or iii', the concentration of ethoxybensalamine in the mixed sample is preferably from about 50 to about 200ng/mL, preferably about 100ng/mL, and the concentration of travoprost-D4 is from about 10 to about 30ng/mL, preferably about 20 ng/mL.

9. The method according to any one of claims 1 to 4, wherein the chromatographic conditions in the LC-MS technique comprise elution with a chromatographic column using octadecylsilane chemically bonded silica as a filler and a mixed solution of aqueous formic acid solution and acetonitrile formic acid solution as a mobile phase, wherein the elution is carried out with the mixed solution of aqueous formic acid solution and acetonitrile formic acid solution as a mobile phase, and the elution is carried out with the mixed solution of aqueous formic acid solution and acetonitrile formic acid solution as a mobile phase

the formic acid acetonitrile solution is about 0.09 to about 0.11 volume percent formic acid, preferably about 0.1 volume percent acetonitrile solution; and

the elution is preferably performed by gradient elution, and the procedure of the gradient elution is preferably set as follows:

10. The method according to any one of claims 1 to 4, wherein the mass spectrometric conditions in the LC tandem mass spectrometry technique comprise simultaneous detection of positive and negative ions in a multiple reaction monitoring mode using an electrospray ion source, wherein the mass spectrometric conditions comprise setting the following parameters: