Method for simultaneous quantitative analysis of travoprost and travoprost acid contained in a solution sample

Abstract

The present invention discloses a method for simultaneously quantitatively analyzing travoprost and travoprost acid contained in a solution sample, wherein the method is characterized in that the quantitative analysis is performed using high performance liquid chromatography-mass spectrometry. The method of the present invention has the following advantages: simple operation, sensitivity, rapidity, high specificity, strong specificity, high accuracy, good reproducibility, and short analysis time. The method of the present invention can obtain a chromatogram with satisfactory sample peak shape and chromatographic retention time, which in particular enables simultaneous quantitative analysis of travoprost and its active metabolite travoprost acid in a biological tissue sample to which an enzyme inhibitor is added, and thus the effect of the enzyme inhibitor on the conversion rate of travoprost can be calculated.

Description

Technical Field

[0001] The present invention relates to the field of pharmacokinetic research. Specifically, the present invention relates to a method for quantitatively analyzing a drug in a solution sample, and more specifically, the present invention relates to a method for simultaneously quantitatively analyzing travoprost and travoprost acid contained in a solution sample. Background Art

[0002] Travoprost is a prostaglandin F2α analogue that can be used to treat open-angle and angle-closure glaucoma and ocular hypertension. It is a highly selective and high-affinity prostaglandin FP receptor full agonist that reduces intraocular pressure by increasing the outflow of aqueous humor through the uveoscleral pathway. It is an ester prodrug of travoprost acid (travoprost acid isopropyl ester), which is absorbed through the cornea and can be quickly hydrolyzed by corneal esterase to form biologically active free travoprost acid, which exerts its intraocular pressure-lowering effect. The structural formulas of travoprost and travoprost acid are shown below:

[0003] Travoprost

[0004] Travoprost acid

[0005] In the process of studying the pharmacokinetic of drugs, it is often necessary to conduct rapid quantitative analysis of the drugs contained in the solution sample. In the present invention, the solution sample includes not only the solution in the general sense in the field of chemistry, but also the biological sample obtained from the organism.

[0006] Usually, the process of collecting biological samples itself will affect the quantitative measurement results of drugs. For example, in order to study the pharmacokinetics of drugs in animal eye tissues, animal eye tissues need to be collected. Since the biological barrier of the eyeball disappears immediately after the animal dies, and the enzymes in the eye tissue can still exert biological activity after the eyeball is removed, the eyeball often needs to be kept frozen during the dissection process and the samples need to be quickly measured.

[0007] Experiments have shown that when travoprost is added to rabbit aqueous humor, plasma and whole blood and placed at about 4°C for about 2 hours after vortexing, travoprost is degraded by about 30% in aqueous humor and even up to about 80% in whole blood and plasma.

[0008] The inventors of the present invention have achieved control over the degradation of travoprost during the collection of the biological sample and during the processing of the biological sample after collection by adding an enzyme inhibitor to the biological sample containing travoprost. The enzyme inhibitor may include methylsulfonyl fluoride, physostigmine, dichlorvos, paraoxon or any mixture of the above substances. The inventors of the present invention have also studied the effects of various factors such as different enzyme inhibitors, enzyme inhibitor concentrations, temperature and pH conditions on the degradation of travoprost in the biological sample, that is, the effects of these factors on the conversion of travoprost into the corresponding acid in the biological sample.

[0009] There is no report in the prior art on a method for simultaneously quantitatively analyzing travoprost and travoprost acid contained in a solution sample, especially there is no literature report on a bioanalytical method for simultaneously and rapidly determining travoprost and travoprost acid in a sample solution after adding an enzyme inhibitor to the biological sample. Summary of the invention

[0010] Technical Problems to be Solved by the Invention

[0011] The object of the present invention is to provide a method capable of simultaneously and rapidly quantitatively analyzing travoprost and travoprost acid contained in a solution sample. In particular, a method is provided for simultaneously and rapidly quantitatively analyzing travoprost and its active metabolite travoprost acid in a biological sample containing an enzyme inhibitor. The quantitative analysis method enables the study of the effects of various factors such as different enzyme inhibitors, enzyme inhibitor concentrations, temperature and pH conditions on the conversion of travoprost into travoprost acid during the collection of biological samples and during the processing of the biological samples after collection.

[0012] Technical solutions to technical problems

[0013] In order to solve the technical problem, the present invention provides the following technical solutions:

[0014] 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 using high performance liquid chromatography tandem mass spectrometry, and the high performance liquid chromatography tandem mass spectrometry is preferably high performance liquid chromatography tandem mass spectrometry secondary mass spectrometry.

[0015] Scheme 2: The method according to Scheme 1, wherein the solution sample is obtained by a method comprising the following steps:

[0016] i. administering travoprost to an animal, and extracting animal tissue from the animal after administration;

[0017] ii. placing the animal tissue obtained in step i in an aqueous solution containing an enzyme inhibitor and a pH adjuster at a temperature of about 0°C to about 4°C and performing biological sample pretreatment, taking a supernatant sample after centrifugation, wherein the pH value of the supernatant sample is about 4.0 to about 6.0, preferably about 5.0, and the pH adjuster preferably comprises citric acid;

[0018] iii. Add ethoxyfensalamide and travoprost acid-D4 to the sample obtained in step ii and mix them thoroughly to dissolve,

[0019] iv. adding an organic solvent to the sample obtained in step iii and mixing it thoroughly, wherein the volume ratio of the sample obtained in step iii to the organic solvent is about 1:2 to about 1:9, preferably about 1:4, and the organic solvent preferably comprises acetonitrile, methanol, ethanol or any mixture thereof, most preferably comprises acetonitrile, and after centrifugation of the mixed sample, the supernatant is taken as the solution sample.

[0020] Scheme 3: The method according to Scheme 1, wherein the solution sample is obtained by a method comprising the following steps:

[0021] i'. Extracting animal tissue from animals that have not been given travoprost;

[0022] ii'. Pre-treating the animal tissue obtained in step i' at a temperature of about 0°C to about 4°C, taking a supernatant sample after centrifugation, adding an enzyme inhibitor, travoprost, travoprost acid and a pH adjuster, wherein the pH value of the sample is about 4.0 to about 6.0, preferably about 5.0, and the pH adjuster preferably comprises citric acid;

[0023] iii 'to the sample obtained in step ii 'add ethoxybensalamide and travoprost acid -D4 and mix well to dissolve;

[0024] iv'. Add an organic solvent to the sample obtained in step iii' and mix it thoroughly, wherein the volume ratio of the sample obtained in step iii' to the organic solvent is about 1:2 to about 1:9, preferably about 1:4, and the organic solvent preferably comprises acetonitrile, methanol, ethanol or any mixture thereof, most preferably comprises acetonitrile, and the supernatant of the mixed sample is taken as the solution sample after centrifugation.

[0025] Embodiment 4. The method according to embodiment 3, wherein the concentration of travoprost in the supernatant sample in step ii' is about 10 to about 1000 ng / mL, and the concentration of travoprost acid is about 2 to about 200 ng / mL.

[0026] Embodiment 5. The method according to any one of embodiments 2 to 4, wherein the animal tissue comprises aqueous humor, plasma or whole blood.

[0027] Embodiment 6. The method according to any one of Embodiments 2 to 5, wherein the enzyme inhibitor comprises dichlorvos, paraoxon, phenylmethyl yellow yl fluoride, physostigmine, and a combination of one or more thereof, and particularly preferably comprises paraoxon.

[0028] Scheme 7. The method according to any one of schemes 2 to 6, wherein the concentration of the enzyme inhibitor in the supernatant in step ii or ii' is about 1 to about 20 mM, preferably about 2 to about 10 mM, more preferably about 5 mM.

[0029] Scheme 8. The method according to any one of schemes 2 to 7, wherein in step iii or iii', the concentration of ethoxyfensalamide in the mixed sample is preferably about 50 to about 200 ng / mL, preferably about 100 ng / mL, and the concentration of travoprost acid-D4 is about 10 ng / mL to about 30 ng / mL, preferably about 20 ng / mL.

[0030] Scheme 9: A method according to any one of schemes 1 to 8, wherein the chromatographic conditions in the liquid chromatography tandem mass spectrometry technique include using a chromatographic column with octadecylsilane bonded silica gel as a filler and using a mixture of formic acid aqueous solution-formic acid acetonitrile solution as a mobile phase for elution, wherein

[0031] 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;

[0032] The formic acid aqueous solution is an aqueous solution of about 0.09 to about 0.11 volume % of formic acid, preferably about 0.1 volume %;

[0033] The formic acid acetonitrile solution is about 0.09 to about 0.11 volume % of formic acid, preferably about 0.1 volume % of acetonitrile solution; and the elution is preferably carried out in a gradient elution manner, and the program of the gradient elution is preferably set as follows:

[0034] from about 0 to about 0.8 minutes, maintaining the mobile phase consisting of about 85 to about 95 volume %, preferably about 90 volume % of the formic acid aqueous solution and about 5 to about 15 volume %, preferably about 10 volume % of the formic acid acetonitrile solution;

[0035] for about 0.8 to about 1.2 minutes, uniformly changing the mobile phase from about 85 to about 95 volume %, preferably about 90 volume % of the formic acid aqueous solution and about 5 to about 15 volume %, preferably about 10 volume % of the formic acid acetonitrile solution to about 5 to about 15 volume %, preferably about 10 volume % of the formic acid aqueous solution and about 85 to about 95 volume %, preferably about 90 volume % of the formic acid acetonitrile solution;

[0036] for about 1.2 to about 2.0 minutes, maintaining the mobile phase consisting of about 5 to about 15 volume %, preferably about 10 volume % of the formic acid aqueous solution and about 85 to about 95 volume %, preferably 90 volume % of the formic acid acetonitrile solution;

[0037] for about 2.0 to about 3.5 minutes, maintaining the mobile phase consisting of about 85 to about 95 volume %, preferably 90 volume % of the formic acid aqueous solution and about 5 to about 15 volume %, preferably about 10 volume % of the formic acid acetonitrile solution.

[0038] Scheme 10. The method according to any one of schemes 1 to 9, wherein the mass spectrometry conditions in the liquid chromatography tandem mass spectrometry technique include using an electrospray ion source to perform simultaneous detection of positive and negative ions in a multiple reaction monitoring mode, wherein the mass spectrometry conditions include setting the following parameters:

[0039] Positive ion mode: internal standard: about 4500V to about 5500V, preferably about 5000V; curtain gas: about 30 to about 40psi, preferably about 35psi; gas 1: about 60 to about 70psi, preferably about 65psi; gas 2: about 60 to about 70psi, preferably about 65psi; collision gas: Medium;

[0040] Negative ion mode: internal standard: about -3500 V to about -4500 V, preferably about -4000 V; curtain gas: about 30 to about 40 psi, preferably about 35 psi; gas 1: about 60 to about 70 psi, preferably about 65 psi; gas 2: about 60 to about 70 psi, preferably about 65 psi; collision gas: Medium;

[0041] Ion source temperature: about 400 to about 500°C, preferably about 450°C.

[0042] Technical effects achieved by the present invention

[0043] The purpose of the present invention is achieved through the various technical solutions of the present invention. In order to study the effect of enzyme inhibitors on the degradation of travoprost during the biological sample collection process and the biological sample processing process after collection, the present invention establishes a sensitive, rapid and specific (ultra) high performance liquid chromatography-tandem mass spectrometry (HPLC / UPLC-MS / MS) method, which can simultaneously determine the concentration of travoprost and its active metabolite travoprost acid in animal tissues, especially in plasma, whole blood and eye tissues.

[0044] The method of the present invention is a (ultra) high performance liquid chromatography tandem mass spectrometry (HPLC / UPLC-MS / MS) coupling technology, and has the following advantages: simple operation, sensitivity, rapidity, high specificity, strong specificity, high accuracy, good reproducibility, and short analysis time. The method of the present invention can obtain a chromatogram with satisfactory sample peak shape and chromatographic retention time, which in particular enables simultaneous quantitative analysis of travoprost and its active metabolite travoprost acid in biological tissue samples, and thus enables calculation of the conversion rate of travoprost.

[0045] Specifically, the method of the present invention has the following advantages:

[0046] (1) Small sampling volume: The amount of a sample can be less than about 0.1 mL, or even only a few μL;

[0047] (2) Simple pretreatment: After the sample is treated with enzyme inhibitors and optionally citric acid, only an organic solvent is needed to precipitate the protein, and the HPLC / UPLC-MS / MS injection analysis can be directly performed after vortex centrifugation, which is simple and easy and suitable for conventional quantitative detection;

[0048] (3) High sensitivity: mass spectrometry significantly improves the detection sensitivity of the assay, with the quantitative limits of travoprost and travoprost acid being 10 ng / mL and 2 ng / mL, respectively;

[0049] (4) Strong selectivity: endogenous substances in blank biological samples do not interfere with the determination of drugs and internal standards;

[0050] (5) Short measurement time: The entire chromatographic analysis process can take no more than about 3.5 minutes;

[0051] (6) The linear ranges of travoprost and travoprost acid are 10 to 1000 ng / mL and 200 ng / mL, respectively. The linear range span is large and can well meet the dynamic analysis and change determination of drugs in vivo;

[0052] (7) The recovery rate is stable, and the intra-day and inter-day precision (relative standard deviation, RSD) are less than 15%; and

[0053] (8) The matrix effect is stable, and the matrix effects of low, medium, and high concentrations are all between about 80% and about 120%. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] In order to more clearly illustrate the present invention, the following description and drawings of the present invention will be described and illustrated. Obviously, the drawings in the following description only illustrate certain aspects of some exemplary embodiments of the present invention, and it is possible for a person of ordinary skill in the art to obtain other drawings based on these drawings without creative work.

[0055] Figure 1 The mass spectrometry conditions of the method of the present invention are used to scan the mass spectra of travoprost (A) and ethoprost (IS) (B).

[0056] Figure 2 The mass spectrometry conditions of the method of the present invention are used to scan the mass spectra of travoprost acid (A) and travoprost acid D4 (IS) (B).

[0057] Figure 3 It is an ion detection (MRM) chromatogram of a blank biological sample using the mass spectrometry conditions in the method of the present invention, wherein the upper figure is an ion detection (MRM) chromatogram of a blank biological sample in positive ion mode, and the lower figure is an ion detection (MRM) chromatogram of a blank biological sample in negative ion mode.

[0058] Figure 4 The ion detection (MRM) chromatograms are obtained by adding travoprost (10 ng / mL) and travoprost acid (2 ng / mL) to a blank biological sample using the mass spectrometry conditions in the method of the present invention, wherein the two figures marked as 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 figures marked as IV and V are the positive ion mode ion detection (MRM) chromatograms of travoprost and internal standard ethoprostamide, respectively.

[0059] Figure 5 It is an ion detection (MRM) chromatogram of an actual biological sample using the mass spectrometry conditions in the method of the present invention, wherein the two figures marked as I and II are respectively the negative ion mode ion detection (MRM) chromatograms of travoprost acid and internal standard travoprost acid D4; and the two figures marked as IV and V are respectively the positive ion mode ion detection (MRM) chromatograms of travoprost and internal standard ethoprostamide. DETAILED DESCRIPTION OF THE INVENTION

[0061] Hereinafter, the present invention will be described in detail.

[0062] The present invention mainly relates to a method for simultaneously quantitatively analyzing travoprost and travoprost acid contained in a solution sample, wherein the method is characterized in that the quantitative analysis is performed by (ultra) high performance liquid chromatography coupled to mass spectrometry.

[0063] 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". In fact, the terms "high performance liquid chromatography" and "ultra high performance liquid chromatography" are used interchangeably in the present invention.

[0064] In the method of the present invention, the high performance liquid chromatography tandem mass spectrometry technique is preferably high performance liquid chromatography tandem mass spectrometry (HPLC-MS / MS).

[0065] 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.

[0066] In a preferred embodiment of the method of the present invention, the solution sample is obtained by a method comprising the following method steps i to iv:

[0067] Step i: administering travoprost to an animal, and extracting animal tissue from the animal after the administration.

[0068] In the above step i, the administration is not particularly limited. In the case of the present invention, it may include but is not limited to injection, oral administration, transdermal administration, mucosal administration, etc.

[0069] Step ii: placing the animal tissue obtained in step i in an aqueous solution containing an enzyme inhibitor and an optional pH adjuster at a temperature of about 0°C to about 4°C and performing biological sample pretreatment, and taking a supernatant sample after centrifugation.

[0070] Step iii: Add the internal standard to the sample obtained in step ii and mix it thoroughly to dissolve.

[0071] Step iv: adding an organic solvent to the sample obtained in step iii and mixing them thoroughly, and taking the supernatant of the mixed sample as the solution sample after centrifugation.

[0072] In another preferred embodiment of the method of the present invention, the solution sample is obtained by a method comprising the following method steps i' to iv':

[0073] Step i': extracting animal tissue from animals not administered travoprost.

[0074] Step ii': subjecting the animal tissue obtained in step i' to biological sample pretreatment at a temperature of about 0°C to about 4°C, taking a supernatant sample after centrifugation, and adding enzyme inhibitors, travoprost, travoprost acid and an optional pH adjuster.

[0075] In the above step ii', the concentration of travoprost and the concentration of travoprost acid in the supernatant sample are preferably within their respective measurement linear ranges. For example, the concentration of travoprost may be about 10 to about 1000 ng / mL; the concentration of travoprost acid may preferably be about 2 to about 200 ng / mL.

[0076] In the above step ii', the added travoprost and travoprost acid may be solid in the form of pure substance or liquid in the form of solution, preferably liquid in the form of solution, more preferably liquid in the form of aqueous solution.

[0077] Step iii': Add the internal standard to the sample obtained in step ii' and mix it thoroughly to dissolve.

[0078] Step iv': adding an organic solvent to the sample obtained in step iii' and mixing them thoroughly, and centrifuging the mixed sample to obtain the supernatant as the solution sample.

[0079] In the above step i or i', the animal is preferably a mammal, including but not limited to rabbits, cats, dogs, mice, monkeys, etc., preferably a rabbit, and more preferably a New Zealand rabbit.

[0080] 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 or whole blood.

[0081] In the above step ii or ii', the biological sample pretreatment is a homogenization treatment method known to those skilled in the art. The biological sample pretreatment process used in the present invention can be found in the prior art document: Velagaleti PR, Buonarati MH, Challenges and Strategies in Drug Residue Measurement (Bioanalysis) of Ocular Tissues [J]. Methods in Pharmacology & Toxicology, 2014, 33-52, the contents of which are incorporated herein by reference.

[0082] 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 includes dichlorvos, paraoxon, phenylmethyl yellow yl fluoride, physostigmine and a combination of one or more thereof. In a more preferred embodiment, the enzyme inhibitor includes paraoxon.

[0083] In the above step ii or ii', the added enzyme inhibitor may be an enzyme inhibitor in the form of a pure substance or an enzyme inhibitor solution in the form of a solution. Preferably, an enzyme inhibitor solution in the form of a solution is used, and more preferably an aqueous solution.

[0084] 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 this concentration can inhibit the enzyme in the animal tissue from hydrolyzing travoprost in the sample. For example, in a preferred embodiment of the present invention, the concentration of the enzyme inhibitor in the supernatant is preferably about 1 to about 20 mM, more preferably about 2 to about 10 mM, and most preferably about 5 mM.

[0085] In the above step ii or ii', the pH adjuster is optional. If necessary, the pH adjuster adjusts the pH of the sample dissolved with the enzyme inhibitor, travoprost and travoprost acid to a weak acidity, for example, the sample can be adjusted to a pH of about 4.0 to about 6.0, preferably about 5.0. Here, the pH adjuster preferably comprises citric acid.

[0086] In this case, the added pH adjuster can be a pure solid pH adjuster or a solution of a pH adjuster, preferably a solution of a pH adjuster, preferably an aqueous solution.

[0087] In the above step iii or iii', the internal standards are ethoxyfensalamide and travoprost acid-D4. In this case, the concentration of ethoxyfensalamide in the mixed sample is preferably about 50 to about 200 ng / mL, preferably about 100 ng / mL, and the concentration of travoprost acid-D4 is about 10 ng / mL to about 30 ng / mL, preferably about 20 ng / mL.

[0088] Here, the added internal standard can be an internal standard in the form of a pure substance, or an internal standard solution in the form of a solution. Preferably, an internal standard solution in the form of a solution is used, preferably a methanol solution or an aqueous solution.

[0089] In the above step iv or iv', the organic solvent is not particularly limited. The purpose of adding the organic solvent is to precipitate the protein in the sample.

[0090] Preferably, the organic solvent may be acetonitrile, methanol, ethanol or any mixture thereof, and it is particularly preferred that the organic solvent contains acetonitrile or is acetonitrile.

[0091] In the context of the present invention, as those skilled in the art will appreciate, the sufficient mixing and dissolving can be achieved by means commonly used in the art, such as oscillation, vortexing, etc. (preferably vortexing) to achieve this purpose.

[0092] In the method of the present invention as described above, the chromatographic conditions in the liquid chromatography tandem mass spectrometry technique include using a chromatographic column with octadecylsilane bonded silica gel as a filler and using a mixture of formic acid aqueous solution-formic acid acetonitrile solution as a mobile phase for elution.

[0093] In a preferred embodiment, the flow rate of the mobile phase can be set to about 0.2 mL / min to about 0.4 mL / min, preferably about 0.3 mL / min.

[0094] In a preferred embodiment, the formic acid aqueous solution is preferably an aqueous solution of about 0.09 to about 0.11 volume % of formic acid, preferably about 0.1 volume %; the formic acid acetonitrile solution is preferably an acetonitrile solution of about 0.09 to about 0.11 volume % of formic acid, preferably about 0.1 volume % of formic acid.

[0095] In addition, the elution conditions are not particularly limited as long as the conditions can separate travoprost and travoprost acid from impurities in the solution sample.

[0096] In a preferred embodiment, the elution is preferably carried out in a gradient elution mode, wherein the gradient elution program is preferably set as follows:

[0097] from about 0 to about 0.8 minutes, maintaining the mobile phase consisting of about 85 to about 95 volume %, preferably about 90 volume % of the formic acid aqueous solution and about 5 to about 15 volume %, preferably about 10 volume % of the formic acid acetonitrile solution;

[0098] for about 0.8 to about 1.2 minutes, uniformly changing the mobile phase from about 85 to about 95 volume %, preferably about 90 volume % of the formic acid aqueous solution and about 5 to about 15 volume %, preferably about 10 volume % of the formic acid acetonitrile solution to about 5 to about 15 volume %, preferably about 10 volume % of the formic acid aqueous solution and about 85 to about 95 volume %, preferably about 90 volume % of the formic acid acetonitrile solution;

[0099] for about 1.2 to about 2.0 minutes, maintaining the mobile phase consisting of about 5 to about 15 volume %, preferably about 10 volume % of the formic acid aqueous solution and about 85 to about 95 volume %, preferably 90 volume % of the formic acid acetonitrile solution;

[0100] for about 2.0 to about 3.5 minutes, maintaining the mobile phase consisting of about 85 to about 95 volume %, preferably 90 volume % of the formic acid aqueous solution and about 5 to about 15 volume %, preferably about 10 volume % of the formic acid acetonitrile solution.

[0101] In the method of the present invention as described above, the mass spectrometry conditions in the liquid chromatography tandem mass spectrometry technique include using an electrospray ion source to perform simultaneous detection of positive and negative ions in a multiple reaction monitoring mode.

[0102] In a preferred embodiment, the mass spectrometry conditions include setting the following parameters:

[0103] Positive ion mode: internal standard (IS): about 4500V to about 5500V, preferably about 5000V; Curtain gas: about 30 to about 40psi, preferably about 35psi; Gas 1: about 60 to about 70psi, preferably about 65psi; Gas 2: about 60 to about 70psi, preferably about 65psi; Collision Gas: Medium;

[0104] Negative ion mode: internal standard (IS): about -3500 V to about -4500 V, preferably about -4000 V; Curtain gas: about 30 to about 40 psi, preferably about 35 psi; Gas 1: about 60 to about 70 psi, preferably about 65 psi; Gas 2: about 60 to about 70 psi, preferably about 65 psi; Collision gas: Medium;

[0105] Ion source temperature: about 400 to about 500°C, preferably about 450°C. DETAILED DESCRIPTION

[0106] The present invention and its advantages will be specifically described below by exemplary embodiments. The description of the exemplary embodiments is merely illustrative and is by no means intended to limit the present disclosure and its application or use. The present disclosure can be implemented in many different forms and is not limited to the embodiments described herein. These embodiments are provided to make the present disclosure thorough and complete and to fully express the scope of the present disclosure to those skilled in the art. It should be noted that unless otherwise stated, the relative arrangement of the parts and steps, the composition of the materials, the numerical expressions and the numerical values, etc., described in these embodiments should be interpreted as being merely exemplary and not as limiting.

[0107] Materials and instruments used:

[0108] Animals: New Zealand rabbits, weighing about 1.5 to about 2.5 kg, purchased from Qingdao Kangda Biotechnology Co., Ltd.;

[0109] Dichlorvos: purity ≥98.0%, purchased from Sigma, USA;

[0110] Phenylmethylsulfonyl fluoride: purity 98.5%, purchased from Sigma, USA;

[0111] Paraoxon: purity ≥98.5%, purchased from China Standards Information Network;

[0112] Physostigmine: purity ≥ 99.0%, purchased from China Standard Information Network;

[0113] Citric acid: purity 99.5%, purchased from Hunan Erkang Pharmaceutical Co., Ltd.;

[0114] Travoprost: purity 97.0%, purchased from USP, USA;

[0115] Travoprost acid: purity 98.9%, purchased from TLC, Canada;

[0116] Ethoxyphensalamide: purity 99.9%, purchased from the National Institute for the Control of Pharmaceutical and Biological Products;

[0117] Travoprost acid-D4: purity 96.0%, purchased from TLC, Canada;

[0118] Acetonitrile: chromatographically pure, purchased from Sigma, USA;

[0119] Formic acid: chromatographically pure, purchased from Sigma, USA;

[0120] Methanol: chromatographically pure, purchased from Sigma, USA;

[0121] Water: chromatographically pure, homemade;

[0122] Ultra-high performance liquid chromatography system: Under the trade name "ACQUITY Ultra Performance LC TM "Commercially purchased from Waters Company of the United States; it is equipped with a general-purpose binary high-pressure pump and an injector;

[0123] Secondary mass spectrometer: triple quadrupole tandem mass spectrometer, commercially available from ABSciex, USA, under the trade name “Qtarp 5500”, equipped with an electrospray ion source (ESI ion source);

[0124] Chromatographic column: Octadecylsilane bonded silica gel is used as filler, size is 2.1mm×50mm, filler is BEHC18, filler particle size is 1.7μm, trade name “ACQUITY "Commercially available from Waters Company, USA.

[0125] Biological sample processing:

[0126] The test sample was prepared by the following general method. The general method comprises the following steps:

[0127] (1) Extracting animal tissues (aqueous humor, plasma or whole blood) from New Zealand rabbits as biological samples;

[0128] (2) placing the biological sample obtained in step (1) in a sample collection tube containing enzyme inhibitors at different concentrations and optionally containing citric acid at a set temperature, adding a determined amount of ultrapure water for biological sample pretreatment, wherein citric acid is added according to experimental requirements, and the purpose of adding citric acid is to make the pH value of the sample reach about 5.0, and collecting a supernatant sample by centrifugation;

[0129] (3) optionally adding travoprost and / or travoprost acid to the sample obtained in step (2) to a set concentration according to experimental requirements, vortexing for about 1 minute and then leaving for about 15 minutes;

[0130] (4) adding a mixed internal standard working solution consisting of an ethoxyfensalamide methanol solution and a travoprost acid-D4 methanol solution to the sample obtained in step (3), so that the ethoxyfensalamide concentration in the final biological sample is about 100 ng / mL and the travoprost acid-D4 concentration is about 20 ng / mL;

[0131] (5) Add acetonitrile to the sample obtained in step (4) in a volume ratio of about 1:4, vortex for about 1 minute, centrifuge (about 14500 rpm) for about 10 minutes, and take the supernatant. Take about 7.0 μL of this solution as a detection sample for liquid chromatography-tandem mass spectrometry as described below.

[0132] HPLC / UPLC-MS / MS analysis conditions:

[0133] Chromatographic conditions

[0134] Chromatographic columns: The chromatographic columns described in the “Materials and Apparatus Used” section above were used.

[0135] Column temperature: 30℃.

[0136] Mobile phase: a mixture of a 0.1 vol% formic acid aqueous solution (A) and a 0.1 vol% formic acid acetonitrile solution (B).

[0137] Elution mode: gradient elution, the mobile phase ratio changes with time as follows:

[0138] 0 to 0.8 min, 90% A;

[0139] 0.8 to 1.2 minutes, 90% A changes at a constant speed to 10% A;

[0140] 1.2 to 2.0 minutes, 10% A;

[0141] 2.0 to 3.5 minutes, 90% A.

[0142] Flow rate: 0.3mL / min.

[0143] Injection volume: 7.0 μL.

[0144] Secondary mass spectrometry conditions

[0145] Electrospray ion source (ESI ion source) and multiple reaction monitoring mode (MRM) were used for simultaneous detection of positive and negative ions; the ion pairs for quantitative analysis were:

[0146] Positive ion mode: travoprost, m / z 501.3→207.2; ethoxyfensalamide (IS), m / z 258.1→121.1;

[0147] Negative ion mode: travoprost acid, m / z 457.2→161.0; travoprost acid D4 (IS), m / z 461.2→161.0.

[0148] The selection of the positive and negative ion modes is based on the Figure 1 and 2 The mass spectra shown in .

[0149] The mass spectrometry conditions were set to include the following parameters:

[0150] Positive ion mode, internal standard (IS): 5000V; Curtain gas: 35psi; Gas 1: 65psi; Gas 2: 65psi; Collision Gas: Medium;

[0151] Negative ion mode, internal standard (IS): -4000 V; Curtain gas: 35 psi; Gas 1: 65 psi; Gas 2: 65 psi; Collision gas: Medium;

[0152] Ion source temperature: 450°C.

[0153] It should be noted here that the above-mentioned chromatographic conditions and secondary mass spectrometry conditions are typical conditions. In actual applications, those skilled in the art can refer to the contents disclosed in the present invention to appropriately adjust the parameters according to the different characteristics of the instruments used to obtain the best effect.

[0154] HPLCUPLC-MS / MS measurement method:

[0155] (1) Exclusivity

[0156] In this method, the specificity of the analyte and the internal standard is evaluated by comparing the lowest concentration point of the standard curve with blank biological samples and actual biological samples operated in the same way.

[0157] The blank biological sample is as described in the "Biological Sample Processing" section above, except that New Zealand rabbits that have not been administered with travoprost are used, and travoprost and travoprost acid are not added in step (3). The actual biological sample is as described in the "Biological Sample Processing" section above, except that New Zealand rabbits that have been administered with travoprost are used, and travoprost and travoprost acid are not added in step (3).

[0158] Instructions attached Figure 3 , 4 Figures 5 and 6 show the ion detection (MRM) chromatograms of blank biological samples, blank biological samples added with travoprost (10 ng / mL) and travoprost acid (2.0 ng / mL), and actual biological samples, respectively. It can be seen from these figures that the endogenous substances in the blank biological samples and actual biological samples do not interfere with the determination of travoprost, travoprost acid and their mixed internal standards.

[0159] (2) Standard curve

[0160] A mixed standard curve series of travoprost and travoprost acid solutions were prepared in acetonitrile.

[0161] As described above, a blank biological sample was taken and a mixed standard curve series of solutions of travoprost and travoprost acid were added to prepare standard biological samples containing travoprost concentrations of 10, 20, 40, 100, 250, 500, 1000 ng / mL and travoprost acid concentrations of 2, 4, 8, 20, 50, 100, 200 ng / mL.

[0162] 7 μL of the above standard biological sample was taken for UPLC-MS / MS analysis, and the weighted least squares method was used for regression analysis to obtain the standard curve. The standard curve equation was: travoprost, y=0.000302x-0.000908 (r=0.9945), travoprost acid, y=0.281x-0.0344 (r=0.9990), y represents the ratio of the peak area of ​​the analyte to the internal standard, and x represents the concentration of the drug to be tested. The linear range of the quantitative determination of travoprost in the method of the present invention is 10 to 1000 ng / mL, and the minimum limit of quantification is 10 ng / mL; the linear range of the quantitative determination of travoprost acid is 2 to 200 ng / mL, and the minimum limit of quantification is 2 ng / mL.

[0163] (3) Precision and accuracy

[0164] According to the standard biological sample preparation method in the "Standard Curve" section above, quality control (QC) standard biological samples containing low, medium and high concentrations of travoprost at 20, 250 and 800 ng / mL, respectively; and low, medium and high concentrations of travoprost acid at 4.0, 50 and 160 ng / mL, respectively were prepared. Six sample analyses were performed for each concentration for three consecutive days. The precision of the method was evaluated by the intra-day and inter-day RSDs (%), and the accuracy was evaluated by the ratio of the actual measured value to the theoretical value. The analysis results are shown in Table 1.

[0165] Table 1: Accuracy and precision of travoprost and travoprost acid in biological samples

[0166]

[0167] As shown in Table 1, the intra-day and inter-day precisions of travoprost were 8.6% to 9.5% and 6.3% to 8.1%, respectively, with an accuracy range of -4.9% to 0.7%; the intra-day and inter-day precisions of travoprost acid were 5.4% to 7.9% and 1.4% to 7.9%, respectively, with an accuracy range of -6.2% to -3.3%; all met the standard of <±15%, indicating that this method has good precision and accuracy.

[0168] (4) Extraction recovery rate

[0169] According to the preparation method of the series of standard solutions in the "Standard Curve" section above, prepare quality control (QC) samples with low, medium and high concentrations of travoprost at 20, 250 and 800 ng / mL, and low, medium and high concentrations of travoprost acid at 4.0, 50 and 160 ng / mL, respectively. Analyze 6 samples at each concentration and record the chromatographic peaks.

[0170] According to the standard biological sample preparation method in the "Standard Curve" section above, prepare standard biological samples with low, medium and high concentrations of travoprost at 20, 250 and 800 ng / mL, and low, medium and high concentrations of travoprost acid at 4.0, 50 and 160 ng / mL, respectively. Analyze 6 samples at each concentration and record the chromatographic peaks.

[0171] The recovery rate of the drug was calculated by dividing the peak area of ​​each concentration of the standard biological sample by the peak area of ​​the quality control sample. The analysis and determination results are shown in Table 2:

[0172] Table 2: Extraction recovery of travoprost and travoprost acid (n=6)

[0173]

[0174] 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 travoprost and travoprost acid at different concentrations.

[0175] (5) Matrix effect

[0176] According to the preparation method of the series of standard solutions in the "Standard Curve" section above, prepare quality control samples with low, medium and high concentrations of travoprost at 20, 250 and 800 ng / mL, and low, medium and high concentrations of travoprost acid at 4.0, 50 and 160 ng / mL, respectively. Analyze 6 samples at each concentration and record the chromatographic peaks.

[0177] According to the standard biological sample preparation method in the "Standard Curve" section above, prepare standard biological samples with low, medium and high concentrations of travoprost at 20, 250 and 800 ng / mL, and low, medium and high concentrations of travoprost acid at 4.0, 50 and 160 ng / mL, respectively. Analyze 6 samples at each concentration and record the chromatographic peaks.

[0178] The matrix effect was investigated by the ratio of the two peak areas. The analysis and determination results are shown in Table 3.

[0179] Table 3. Matrix effects of travoprost and travoprost acid (n=6)

[0180]

[0181] As shown in Table 3, the matrix effects of travoprost at the three concentrations are between 94.7% and 107.0%; the matrix effects of travoprost acid at the three concentrations are between 91.0% and 100.5%, indicating that the results of the matrix effects of the method of the present invention for travoprost and travoprost acid at different concentrations meet the requirements.

[0182] (6) Stability

[0183] According to the preparation method of the series of standard solutions in the "Standard Curve" section above, the stability samples of low and high concentrations of travoprost were prepared at 20 and 800 ng / mL, and the stability samples of low and high concentrations of travoprost acid were prepared at 4.0 and 160 ng / mL for investigation. The investigation items included 6 hours at room temperature, 48 hours after biological sample treatment, three cycles of freeze-thaw, and long-term freezing stability at -80°C. Three samples were analyzed for each concentration. The results of the stability test are shown in Table 4.

[0184] Table 4. Stability results of travoprost and travoprost acid

[0185]

[0186] As shown in Table 4, the deviations between the measured values ​​and the theoretical values ​​of travoprost and travoprost acid were all <±15%, indicating that the method had good stability.

[0187] Effects of enzyme inhibitors, enzyme inhibitor concentrations, pH and temperature on travoprost conversion efficiency The test samples were prepared as described in the "Biological Sample Processing" section. Unmedicated New Zealand rabbits were used, and travoprost and travoprost acid were added in step (3). Different inhibitors and their concentrations were changed, and the contents of travoprost and travoprost acid in each biological sample were quantitatively analyzed to calculate the conversion efficiency of travoprost.

[0188] The results are shown in Tables 5 to 7 below, wherein the control samples are samples prepared without adding enzyme inhibitors.

[0189] Table 5: Effects of different enzyme inhibitors on the conversion rate of travoprost in rabbit aqueous humor

[0190]

[0191] Table 6: Effects of different enzyme inhibitors on the conversion rate of travoprost in rabbit plasma

[0192]

[0193] Table 7: Effects of different enzyme inhibitors on the conversion rate of travoprost in rabbit whole blood

[0194]

[0195] From the results of Tables 5 to 7 above, it can be seen that, compared with the control, 1, 2, 5, 10 and 20 mM of the enzyme inhibitors dichlorvos, phenylmethylsulfonyl fluoride, paraoxon and physostigmine can significantly inhibit the hydrolysis of travoprost in the aqueous humor, plasma and whole blood of New Zealand rabbits. Further, it can be seen from the data of 3 in Table 1 that, compared with other enzyme inhibitors, paraoxon generally exhibits better inhibitory effects in all concentrations and all tissue samples, so paraoxon is preferably used as an enzyme inhibitor in the method of the present invention.

[0196] The above process was repeated using dichlorvos and paraoxon as enzyme inhibitors, wherein the method was performed under the following three conditions respectively:

[0197] 1) In step (2), the temperature is 4°C and no citric acid is added to adjust the pH.

[0198] 2) in step (2), the temperature is 4°C, citric acid is added to adjust the pH to 5.0, and

[0199] 3) In step (2), the temperature is room temperature, and citric acid is added to adjust the pH to 5.0.

[0200] The test results are shown in Table 8.

[0201] Table 8: Effect of pH and temperature on the conversion of travoprost in biological samples

[0202]

[0203] It can be seen from the data in Table 8 that, based on the data in aqueous humor, plasma and whole blood, when the temperature was 4°C and citric acid was added to adjust the pH to 5.0 in step (2), paraoxon better inhibited the hydrolysis of travoprost.

[0204] In summary, the inventors of the present invention have fully demonstrated that the method of the present invention can achieve the purpose of the present invention, and those skilled in the art can recognize that the method can be fully applied to the simultaneous rapid quantitative analysis of travoprost and travoprost acid in actual biological samples.

[0205] It is obvious to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the main idea or essential features of the present invention. Therefore, from all points of view, the embodiments should be regarded as exemplary and non-restrictive, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention, and any reference numerals in the claims should not be regarded as limiting the claims involved.

[0206] In addition, it should be understood that although this specification is described according to the implementation modes, not every implementation mode includes only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation modes that can be understood by those skilled in the art. These other implementation modes are also covered within the protection scope of the present invention.

[0207] It should also be understood that the specific embodiments described above are only used to explain the present invention, and the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, can make equivalent replacements or changes based on the technical solutions and inventive concepts of the present invention, which should be covered by the protection scope of the present invention / invention.

[0208] The words "include" or "comprising" and the like used in the present invention mean that the elements before the word include the elements listed after the word, and do not exclude the possibility of also including other elements. The term "about" used in the present invention has a meaning well known to those skilled in the art, and preferably refers to the value modified by the term within the range of ±50%, ±40%, ±30%, ±20%, ±10%, ±5% or ±1%.

[0209] All terms (including technical terms or scientific terms) used in the present disclosure have the same meanings as those understood by ordinary technicians in the field to which the present disclosure belongs, unless otherwise specifically defined. It should also be understood that terms defined in general dictionaries should be understood to have meanings consistent with their meanings in the context of the relevant technology, and should not be interpreted in an idealized or extremely formal sense, unless explicitly defined in this article.

[0210] Technologies, methods, and equipment known to ordinary technicians in the relevant field may not be discussed in detail, but where appropriate, the technologies, methods, and equipment should be considered as part of the specification.

[0211] The disclosed contents of the prior art documents cited in the present specification are incorporated into the present invention by reference in their entirety and are therefore part of the disclosure of the present invention.

Claims

1. A method for simultaneously quantitatively analyzing travoprost and travoprost acid contained in a solution sample, wherein the quantitative analysis is performed by high performance liquid chromatography tandem mass spectrometry. The solution sample is obtained by a method comprising the following steps: i. administering travoprost to an animal, and extracting an animal tissue from the animal after administration; ii. placing the animal tissue obtained in step i in an aqueous solution containing an enzyme inhibitor and a pH adjuster at a temperature of 0°C to 4°C and performing biological sample pretreatment, taking a supernatant sample after centrifugation, wherein the pH value of the supernatant sample is 4.0 to 6.0, the pH adjuster comprises citric acid, and the enzyme inhibitor comprises one or more of dichlorvos, paraoxon, benzyl yellow fluoride, and physostigmine; iii. Adding ethoxyfensalamide and travoprost acid-D4 to the sample obtained in step ii and mixing them thoroughly to dissolve, iv. adding an organic solvent to the sample obtained in step iii and mixing it thoroughly, wherein the volume ratio of the sample obtained in step iii to the organic solvent is 1:2 to 1:9, and the organic solvent comprises acetonitrile, methanol, ethanol or any mixture thereof, and the supernatant of the mixed sample is taken as the solution sample after centrifugation; The chromatographic conditions in the liquid chromatography tandem mass spectrometry technology include using a chromatographic column with octadecylsilane bonded silica gel as a filler, and using a mixture of formic acid aqueous solution-formic acid acetonitrile solution as a mobile phase for elution, wherein the elution adopts a gradient elution mode, wherein the formic acid aqueous solution is an aqueous solution of 0.09 to 0.11 volume % of formic acid; the formic acid acetonitrile solution is an acetonitrile solution of 0.09 to 0.11 volume % of formic acid; The mass spectrometry conditions in the liquid chromatography tandem mass spectrometry technique include using an electrospray ion source to simultaneously detect positive and negative ions in a multiple reaction monitoring mode, and the ion pairs for quantitative analysis are: Positive ion mode: travoprost, m / z 501.3→207.2; ethoxyfensalamide, m / z 258.1→121.1; Negative ion mode: Travoprost acid, m / z 457.2→161.0; Travoprost acid D4, m / z 461.2→161.

0.

2. The method according to claim 1, wherein the pH value of the supernatant sample in step ii is 5.

0.

3. The method according to claim 1, wherein in step iv, the volume ratio of the sample obtained from step iii to the organic solvent is 1:

4.

4. The method of claim 1, wherein the organic solvent in step iv comprises acetonitrile.

5. The method according to any one of claims 1 to 4, wherein the animal tissue comprises aqueous humor, plasma or whole blood.

6. The method of any one of claims 1 to 4, wherein the enzyme inhibitor comprises paraoxon.

7. The method according to any one of claims 1 to 4, wherein the concentration of the enzyme inhibitor in the supernatant in step ii is 1 to 20 mM.

8. The method according to claim 7, wherein the concentration of the enzyme inhibitor in the supernatant in step ii is 2 to 10 mM.

9. The method according to claim 8, wherein the concentration of the enzyme inhibitor in the supernatant in step ii is 5 mM.

10. The method according to any one of claims 1 to 4, wherein in step iii, the concentration of ethoprost in the mixed sample is 50 to 200 ng / mL, and the concentration of travoprost acid-D4 is 10 ng / mL to 30 ng / mL.

11. The method according to claim 10, wherein in step iii, the concentration of ethoxyfensalamide in the mixed sample is 100 ng / mL, and the concentration of travoprost acid-D4 is 20 ng / mL.

12. The method according to any one of claims 1 to 4, wherein the aqueous formic acid solution in the mobile phase in the liquid chromatography tandem mass spectrometry technique is a 0.1 volume % aqueous solution of formic acid; and the formic acid acetonitrile solution is a 0.1 volume % acetonitrile solution of formic acid.

13. The method according to any one of claims 1 to 4, wherein the chromatographic conditions in the liquid chromatography tandem mass spectrometry technique include: The flow rate of the mobile phase was set to 0.2 mL / min to 0.4 mL / min. The gradient elution program was set as: 0 to 0.8 minutes, maintaining the mobile phase consisting of 85 to 95 volume % of the formic acid aqueous solution and 5 to 15 volume % of the formic acid acetonitrile solution; 0.8 to 1.2 minutes, changing the mobile phase from 85 to 95 volume % of the formic acid aqueous solution and 5 to 15 volume % of the formic acid acetonitrile solution to 5 to 15 volume % of the formic acid aqueous solution and 85 to 95 volume % of the formic acid acetonitrile solution at a uniform rate; 1.2 to 2.0 minutes, maintaining the mobile phase consisting of 5 to 15 volume % of the formic acid aqueous solution and 85 to 95 volume % of the formic acid acetonitrile solution; for 2.0 to 3.5 minutes, maintaining the mobile phase consisting of 85 to 95 volume % of the formic acid aqueous solution and 5 to 15 volume % of the formic acid acetonitrile solution.

14. The method according to claim 13, wherein the flow rate of the mobile phase is set to 0.3 mL / min, and the gradient elution program is set to: 0 to 0.8 minutes, maintaining the mobile phase consisting of 90 volume % of the formic acid aqueous solution and 10 volume % of the formic acid acetonitrile solution; 0.8 to 1.2 minutes, changing the mobile phase at a uniform rate from 90% by volume of the formic acid aqueous solution and 10% by volume of the formic acid acetonitrile solution to 10% by volume of the formic acid aqueous solution and 90% by volume of the formic acid acetonitrile solution; 1.2 to 2.0 minutes, maintaining the mobile phase consisting of 10 volume % of the formic acid aqueous solution and 90 volume % of the formic acid acetonitrile solution; for 2.0 to 3.5 minutes, maintaining the mobile phase consisting of 90 volume % of the formic acid aqueous solution and 10 volume % of the formic acid acetonitrile solution.

15. The method according to any one of claims 1 to 4, wherein the mass spectrometry conditions in the liquid chromatography tandem mass spectrometry technique include setting the following parameters: Positive ion mode: Internal standard: 4500 V to 5500 V; Curtain gas: 30 to 40 psi; Gas 1: 60 to 70 psi; Gas 2: 60 to 70 psi; Collision gas: Medium; Negative ion mode: Internal standard: -3500 V to -4500 V; Curtain gas: 30 to 40 psi; Gas 1: 60 to 70 psi; Gas 2: 60 to 70 psi; Collision gas: Medium; Ion source temperature: 400 to 500°C.

16. The method according to claim 15, wherein the mass spectrometry conditions include setting the following parameters: Positive ion mode: internal standard: 5000 V; curtain gas: 35 psi; gas 1: 65 psi; gas 2: 65 psi; Collision gas: Medium; Negative ion mode: internal standard: -4000 V; curtain gas: 35 psi; gas 1: 65 psi; Gas 2: 65 psi; Collision gas: Medium; Ion source temperature: 450°C.

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