Method for determining crisaborole in plasma and application

Abstract

The invention discloses a method for determining crisaborole in plasma and application. The extracted sample is easy to store and relatively pure, the analysis speed is relatively high, and the method is suitable for large-scale clinical research sample analysis; in addition, the detection sensitivity is relatively high, the specificity is high, the sample pretreatment is relatively simple, the quantitative lower limit of the crisaborole is 0.30 ng / mL, and the determination requirement of the blood concentration is met; the method is good in accuracy, high in precision and good in reproducibility.

Description

TECHNICAL FIELD

[0001] The present application relates to biotechnology, in particular to a method for determining crisaborole in plasma and application. BACKGROUND

[0002] A new target for treating atopic dermatitis is intracellular phosphodiesterase 4 (PDE-4). The activity of PDE-4 in inflammatory cells of atopic dermatitis patients is increased, and the level of cyclic adenosine monophosphate (cAMP) is lower than normal, and PDE4 increases pro-inflammatory cytokines by degrading cAMP. Crisaborole ointment is a small molecule, non-hormonal, anti-inflammatory PDE-4 inhibitor, which indirectly reduces the production of pro-inflammatory mediators by increasing intracellular cAMP levels. Crisaborole has the dual advantages of efficacy and safety, and has significant efficacy, stable long-term use effect, good safety, mild adverse reactions, and is suitable for a wide range of people.

[0003] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) technology combines the high separation performance of liquid chromatography and the high sensitivity and high specificity of tandem mass spectrometry, and has become the most recommended quantitative analysis technology in the field of biological medicine. Compared with traditional drugs, crisaborole is a new type of PDE-4 inhibitor, and the related liquid chromatography-mass spectrometry detection research may be relatively less in quantity.

[0004] The information disclosed in this BACKGROUND section is only for the purpose of increasing the understanding of the background of the application and should not be taken as an acknowledgment or any form of suggestion that this information forms prior art that is publicly known. SUMMARY

[0005] The purpose of the present application is to provide a method for determining crisaborole in plasma and application.

[0006] To achieve the above-mentioned purpose, the embodiments of the present application provide a method for determining crisaborole in plasma, comprising obtaining a standard curve drawn based on the peak area ratio of detection; adding an internal standard solution to the plasma sample for vortex, collecting the supernatant after centrifugation, adding an aqueous solution, vortexing to obtain a pretreated sample to be tested; using liquid chromatography-tandem mass spectrometry detection: separating the sample to be tested by liquid chromatography, and then detecting by mass spectrometry, based on the standard curve and the regression equation, calculating the mass concentration of crisaborole in the sample to be tested.

[0007] In one or more embodiments of the present application, the internal standard solution is crisaborole-d4.

[0008] In one or more embodiments of the application, the concentration of the internal standard solution is 50.00 ng / mL of Crisaborole-d4.

[0009] In one or more embodiments of the application, the solvent of the internal standard solution is acetonitrile.

[0010] In one or more embodiments of the application, the aqueous solution is 0.1% 1 M ammonium acetate in water.

[0011] In one or more embodiments of the application, the conditions of the liquid chromatography are: Chromatographic column: Waters Acquity UPLC ® HSS T3 column, 1.8 pm, 50 x 2.1 mm; Mobile phase: Phase A: 0.1% 1 M ammonium acetate in water, Phase B: 0.1% 1 M ammonium acetate 95% acetonitrile 5% in water.

[0012] In one or more embodiments of the application, the elution conditions of the liquid chromatography are: 0-0.40 min, 35% Phase B, 65% Phase A; 0.40-1.00 min, 35% Phase B→55% Phase B, 65% Phase A→45% Phase A; 1.00-1.50 min, 55% Phase B→95% Phase B, 45% Phase A→5% Phase A; 1.50-1.90 min, 95% Phase B, 5% Phase A; 1.90-1.91 min, 95% Phase B→35% Phase B, 5% Phase A→65% Phase A; 1.91-2.50 min, 35% Phase B, 65% Phase A; Elution time: 2.50 min; Flow rate: 0.500 mL / min; Injection volume: 20.00 pL; Auto-sampler temperature: 6 °C; Column temperature: 45 °C.

[0013] In one or more embodiments of the application, the mass spectrometry conditions of the mass spectrometry detection are: Ion source: Electrospray ion source ESI; Spray voltage: -4500 V; Spray gas Gas1: 55 psi; Auxiliary gas Gas2: 60 psi; Detection mode: Negative ion; Ion source temperature: 450 °C; Collision induced dissociation CAD: 8; Curtain Gas: 40 psi.

[0014] In one or more embodiments of the present application, the method for determining the concentration of crizanolc in plasma as described is applied in the auxiliary detection of atopic dermatitis.

[0015] The object of the present application is achieved by the following technical solutions: In one aspect, the present application provides a method for analyzing the concentration of crizanolc in plasma suitable for pharmacokinetic study, which comprises the following steps: An internal standard (crizanolc-d4) solution (acetonitrile) is added to the plasma sample for vortexing, and after centrifugation, the supernatant is collected, 0.1% 1 M ammonium acetate aqueous solution is added, and vortexing is performed to obtain the pretreated sample to be tested; The sample to be tested is subjected to liquid chromatography separation, and then mass spectrometry detection is performed, a standard curve is drawn based on the peak area ratio, a regression equation is obtained, and finally the concentration of crizanolc in the sample to be tested is calculated.

[0016] In the above method, preferably, the internal standard solution is crizanolc-d4.

[0017] In the above method, preferably, the specific method of pretreatment comprises: 50 μL of the plasma sample is added to a 96-well plate, followed by the addition of 200 μL of the internal standard solution, vortexing, and then centrifugation at 4 °C and 4000 r / min for 10 min to collect the supernatant. 100 μL of the supernatant is taken to a clean 96-well plate, followed by the addition of 50 μL of 0.1% 1 M ammonium acetate aqueous solution, vortexing, and then centrifugation at 4 °C and 4000 r / min for 5 min to obtain the sample to be tested.

[0018] In the above method, preferably, the concentration of the internal standard solution is crizanolc-d4 = 50.00 ng / mL.

[0019] In the above method, preferably, the chromatographic column used for liquid chromatography separation is: Waters Acquity UPLC ® HSS T3 chromatographic column, 1.8 μm, 50×2.1 mm; the mobile phase used is: A phase: 0.1% 1 M ammonium acetate aqueous solution, B phase: 0.1% 1 M ammonium acetate 95% acetonitrile 5% aqueous solution.

[0020] In the above method, preferably, the elution conditions for liquid chromatography separation are: Gradient elution: 0 - 0.40 min, 35% Phase B, 65% Phase A; 0.40 - 1.00 min, 35% Phase B → 55% Phase B, 65% Phase A → 45% Phase A; 1.00 - 1.50 min, 55% Phase B → 95% Phase B, 45% Phase A → 5% Phase A; 1.50 - 1.90 min, 95% Phase B, 5% Phase A; 1.90 - 1.91 min, 95% Phase B → 35% Phase B, 5% Phase A → 65% Phase A; 1.91 - 2.50 min, 35% Phase B, 65% Phase A; Elution time: 2.50 min; Flow rate: 0.500 mL / min; Injection volume: 20.00 µL; Auto-sampler temperature: 6 ℃; Column temperature: 45 ℃.

[0021] In the above method, preferably, the mass spectrometry conditions for mass spectrometry detection are: Ion source: Electrospray Ion Source (ESI); Spray voltage: -4500 V; Spray gas (Gas1): 55 psi; Auxiliary gas (Gas2): 60 psi; Detection mode: Negative ion; Ion source temperature: 450 ℃; Collision-induced dissociation (CAD): 8; Curtain gas: 40 psi.

[0022] In the above method, preferably, the mass spectrometry detection uses a quantitative analysis ion pair, and the quantitative analysis ion pair is: Crisaborole m / z 250.0 → 118.0, collision energy (CE) -40 eV, declustering voltage (DP) -110 V, dwell time 200 ms; Crisaborole-d4 m / z 255.1 → 122.1, collision energy (CE) -40 eV, declustering voltage (DP) -110 V, dwell time 200 ms; In the above method, preferably, the standard curve is specifically prepared as: The regression equation is obtained by taking the theoretical concentration of the sample to be tested as the abscissa and the peak area ratio of the sample to be tested to the internal standard as the ordinate.

[0023] In another aspect, the application also provides the use of the above method in analyzing the concentration of crocetinic acid in a plasma sample.

[0024] The application has the following advantages: (1) The application has the characteristics of simple pretreatment operation, and only one extraction and one dilution are needed for analysis. The sample after extraction is easy to store and relatively pure, and the analysis speed is fast, with an analysis time of only 2.50 min. Therefore, the application is suitable for large-scale clinical research sample analysis.

[0025] (2) The application has high sensitivity, with a lower limit of quantification of crocetinic acid of 0.30 ng / mL. The method has good accuracy, high precision, and good reproducibility, and can more accurately analyze the concentration of the drug.

[0026] In the application, the plasma needs to be acidified to ensure the stability of the sample. Before the implementation of the application, the stability of crocetinic acid under acidic conditions was explored, and citric acid was used as the acidic solution. It was finally found that when the volume ratio of crocetinic acid to citric acid is 100:4, crocetinic acid is most stable. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is a product ion scan mass spectrum of crocetinic acid according to an embodiment of the application; Figure 2 is a product ion scan mass spectrum of crocetinic acid-d4 according to an embodiment of the application; Figure 3 is an MRM chromatogram of crocetinic acid (top) and crocetinic acid-d4 (bottom) in a blank plasma sample according to an embodiment of the application; Figure 4 is an MRM chromatogram of crocetinic acid (top) and crocetinic acid-d4 (bottom) in a lower limit of quantification sample according to an embodiment of the application; Figure 5 is a standard curve graph of crocetinic acid in the linear range of 0.30-90.00 ng / mL according to an embodiment of the application; DETAILED DESCRIPTION

[0028] The following detailed description of some embodiments of the application is provided for the purposes of better understanding the application, but the scope of the protection of the application is not limited by the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the application. The process, conditions, reagents, experimental methods, etc. for implementing the application, except for the following specifically mentioned contents, are the general knowledge and common sense in the art, and the application does not have special limitations.

[0029] Unless otherwise explicitly stated, throughout the specification and claims, the term "comprise" or its variants such as "comprises" or "comprising" will be understood to encompass the stated element or components, but not to exclude other elements or components.

[0030] The purpose of the application is achieved by the following technical solutions: In one aspect, the application provides a method for analyzing the concentration of crizanolc in plasma suitable for pharmacokinetic study, comprising the following steps: An internal standard (crizanolc-d4) solution (acetonitrile) is added to the plasma sample for vortexing, and after centrifugation, the supernatant is collected, 0.1% 1 M ammonium acetate aqueous solution is added, and vortexing is performed to obtain the pretreated sample to be tested; Liquid chromatography-tandem mass spectrometry is used for detection, the sample to be tested is separated by liquid chromatography, and then mass spectrometry is performed, a standard curve is drawn based on the peak area ratio, a regression equation is obtained, and finally the concentration of crizanolc in the sample to be tested is calculated.

[0031] In the above method, preferably, the internal standard solution is crizanolc-d4.

[0032] In the above method, preferably, the specific method of pretreatment comprises: 50 μL of the plasma sample is added to a 96-well plate, followed by the addition of 200 μL of the internal standard solution, vortexing, 4 ℃, 4000 r / min centrifugation for 10 min to collect the supernatant, and then 100 μL of the supernatant is taken to a clean 96-well plate, followed by the addition of 50 μL of 0.1% 1 M ammonium acetate aqueous solution, vortexing, 4 ℃, 4000 r / min centrifugation for 5 min to obtain the sample to be tested.

[0033] In the above method, preferably, the concentration of the internal standard solution is crizanolc-d4 = 50.00 ng / mL.

[0034] In the above method, preferably, the chromatographic column used for liquid chromatography separation is: Waters Acquity UPLC ®HSS T3 column, 1.8 μm, 50×2.1 mm; the mobile phase used was: Phase A: 0.1% 1 M ammonium acetate in water, Phase B: 0.1% 1 M ammonium acetate in 95% acetonitrile 5% water.

[0035] In the above method, preferably, the liquid chromatography separation elution conditions are: Gradient elution: 0-0.40 min, 35% B phase, 65% A phase; 0.40-1.00 min, 35% B phase→55% B phase, 65% A phase→45% A phase; 1.00-1.50 min, 55% B phase→95% B phase, 45% A phase→5% A phase; 1.50-1.90 min, 95% B phase, 5% A phase; 1.90-1.91 min, 95% B phase→35% B phase, 5% A phase→65% A phase; 1.91-2.50 min, 35% B phase, 65% A phase.

[0036] Elution time: 2.50 min; Flow rate: 0.500 mL / min; Injection volume: 20.00 μL; Automatic injector temperature: 6 ℃; Column temperature: 45 ℃.

[0037] In the above method, preferably, the mass spectrometry conditions for mass spectrometry detection are: Ion source: electrospray ion source (ESI); Spray voltage: -4500 V; Spray gas (Gas1): 55 psi; Auxiliary gas (Gas2): 60 psi; Detection mode: negative ion; Ion source temperature: 450 ℃; Collision-induced dissociation (CAD): 8; Curtain gas (Curtain Gas): 40 psi.

[0038] In the above method, preferably, the mass spectrometry detection uses a quantitative analysis ion pair, and the quantitative analysis ion pair is: Crisaborole m / z 250.0 118.0, collision energy (CE) -40 eV, de-clustering voltage (DP) -110 V, dwell time 200 ms; Crisaborole-d4 m / z 255.1 122.1, collision energy (CE) -40 eV, de-clustering voltage (DP) -110 V, dwell time 200 ms; In the above method, preferably, the standard curve is specifically prepared as follows: Taking the theoretical concentration of the sample to be tested as the abscissa and the peak area ratio of the sample to be tested to the internal standard as the ordinate, a linear regression equation is obtained by regression analysis calculation.

[0039] In another aspect, the present application also provides the use of the above method in analyzing the concentration of crisaborole in a plasma sample.

[0040] The present application has the following advantages: (1) The present application has the characteristics of simple pretreatment operation, and only one extraction and one dilution are required for analysis. The sample after extraction is easy to store and relatively pure, and the analysis speed is fast, with an analysis time of only 2.50 min. Therefore, the present application is suitable for large-scale clinical research sample analysis.

[0041] (2) The present application has high sensitivity, with a lower limit of quantification of crisaborole of 0.30 ng / mL. The present application has good accuracy, high precision, and good reproducibility, and can more accurately analyze the concentration of the drug.

[0042] The development of a method for detecting the concentration of a drug in plasma by liquid chromatography-tandem mass spectrometry can generally be divided into three parts, namely, an extraction method (i.e., a pretreatment method), a liquid chromatography method, and a mass spectrometry method. Examples

[0043] I. Pretreatment: In the present application, the plasma dosage is 50 µL, which is suitable for clinical research bioanalysis. The extraction method in the present application uses protein precipitation, which has the advantages of simple operation, short extraction time, and no time-consuming concentration step. In combination with a 96-well plate, it is suitable for high-throughput sample pretreatment in clinical research.

[0044] The specific pretreatment method steps are as follows: 1. Add 50 µL of plasma sample and 200 µL of internal standard solution (crisaborole-d4 = 50.00 ng / mL) to a 96-well plate. 2. Vortex mix and centrifuge for 10 min (4 ℃, 4000 rpm); 3. Take 100 µL of supernatant to a clean 96-well plate. 4. Add 50 µL of 0.1% 1 M ammonium acetate aqueous solution; 5. Vortex mix and centrifuge for 5 min (4 ℃, 4000 rpm).

[0045] 6. The injection volume is 20.00 µL.

[0046] II. Chromatographic Analysis: Chromatography was used to separate the sample into liquid phases using Waters Acquity UPLC. ® HSS T3 column, gradient elution [0-0.40 min, 35% B phase, 65% A phase; 0.40-1.00 min, 35% B phase → 55% B phase, 65% A phase → 45% A phase; 1.00-1.50 min, 55% B phase → 95% B phase, 45% A phase → 5% A phase; 1.50-1.90 min, 95% B phase, 5% A phase; 1.90-1.91 min, 95% B phase → 35% B phase, 5% A phase → 65% A phase; 1.91-2.50 min, 35% B phase, 65% A phase]. Mobile phase A was an aqueous solution containing 0.1% 1 M ammonium acetate, and mobile phase B was an aqueous solution containing 0.1% 1 M ammonium acetate, 95% acetonitrile, and 5% acetonitrile.

[0047] Crizotinol has a small molecular weight and is easily interfered with by other endogenous substances in plasma. The chromatographic separation in this invention uses Waters Acquity UPLC. ® The HSS T3 column provides good retention of both analytes and internal standards, with symmetrical peak shapes. In this mode, the instrument offers high throughput with a run time of only 2.50 minutes, enabling rapid detection and making it suitable for large-scale sample analysis in clinical research.

[0048] III. Mass Spectrometry Analysis: Electrospray ionization was used with negative ion detection. The spray voltage was -4500 V, gas 1 (Gas1) was 55 psi, gas 2 (Gas2) was 60 psi, curtain gas was 40 psi, the ion source temperature was 450 ℃, collision-induced dissociation was 8, and criborone was used for quantitative analysis of ion pairs. m / z 250.0 118.0, Collision Energy (CE) -40 eV, Declustering Voltage (DP) -110 V, Dwell Time 200 ms, Criborone-d4 Quantitative Analysis of Ion Pairs m / z 255.1 122.1, Collision Energy (CE) -40 eV, Declustering Voltage (DP) -110 V, Dwell Time 200 ms.

[0049] Example 1: Abbreviation Explanation:

[0050] 1. Materials 1.1 Instruments Chromatograph: LC-30AD rapid liquid chromatography system, Shimadzu Corporation, Japan.

[0051] Mass spectrometer: Model 5500 triple quadrupole tandem mass spectrometer, equipped with an electrospray ionization source (Turbo Ion Spray), Sciex, Canada.

[0052] Data processing was performed using Analyst (version 1.6.3), Sciex Inc., Canada.

[0053] Centrifuge: 5810R multi-functional benchtop centrifuge, Eppendorf GmbH, Germany.

[0054] Analytical balance: XSR205DU analytical balance, Beijing Sartorius Instruments Co., Ltd.

[0055] 1.2 Reference Standards and Reagents Crizoborone (98.3% purity) and crizoborone-d4 (98.8% purity) were purchased from Shanghai Zhenzhun Biotechnology Co., Ltd. Methanol (HPLC grade) and acetonitrile (HPLC grade) were purchased from Honeywell Pharmaceuticals, USA. Ammonium acetate (AR grade) was purchased from Shanghai Titan Technology Co., Ltd., China. Deionized water (18.2 mΩ, TOC ≤ 50 ppb) was prepared using a Milli-Q ultrapure water system. Dimethyl sulfoxide (HPLC grade) was purchased from Honeywell Pharmaceuticals, USA.

[0056] 2 Methods 2.1 Preparation of solutions and samples Standard series samples: Accurately weigh an appropriate amount of criborone reference standard, dissolve and dilute to volume with dimethyl sulfoxide to prepare a stock solution with a criborone concentration of approximately 2.00 mg / mL. Accurately pipette an appropriate amount of the stock solution and serially dilute with blank human plasma to obtain a standard series of samples with a criborone concentration range of 0.30–90.00 ng / mL.

[0057] Quality control samples: Four concentration levels of criborone were prepared using methods similar to those used for the standard series samples. The low quality control (LQC) concentration was 0.90 ng / mL, the accessorial middle quality control (AMQC) concentration was 5.40 ng / mL, the medium quality control (MQC) concentration was 27.00 ng / mL, and the high quality control (HQC) concentration was 67.50 ng / mL.

[0058] Internal standard solution: Accurately weigh criborone-d4 reference standard, dissolve and dilute to volume with dimethyl sulfoxide to prepare a criborone-d4 internal standard stock solution with a concentration of approximately 1.00 mg / mL. Accurately pipette an appropriate amount of the above internal standard stock solution and dilute with acetonitrile to obtain an internal standard solution with a criborone-d4 concentration of 50.00 ng / mL.

[0059] 2.2 Plasma Sample Processing

[0060] 2.3 Chromatographic and Mass Spectrometry Conditions Chromatographic conditions:

[0061] Mass spectrometry conditions:

[0062] 2.4. Standard Curve and Quality Control Standard curve The linear regression equation (weighting factor W=1 / x) is calculated by using the theoretical concentration of the analyte (standard series samples) as the x-axis and the peak area ratio of the analyte to the internal standard as the y-axis. 2 ).

[0063] Precision and accuracy Each analytical batch consists of 6 samples from 4 concentration control samples. The relative standard deviation (RSD) (CV%) of the QC samples at each concentration level must be less than 15% to be acceptable, and the accuracy must be between 85% and 115% to be acceptable.

[0064] 3 Results and Discussion 3.1 Methods Standard curve The ion scanning mass spectra of criborone products and criborone-d4 products are shown below. Figure 1 and Figure 2As shown; the linear range for determining criborone in plasma samples from clinical studies was 0.30 - 90.00 ng / mL. Figure 5 As shown. The typical linear regression equations for the standard curve of the analyte are as follows: Crizoborone: y = 0.056x + (-0.00349) r 2 =0.9998); Lower limit of quantitation The lower limit of quantification (LOQ) concentration of criborone in the sample was 0.30 ng / mL.

[0065] Quality control samples The precision and accuracy results all met the acceptance criteria, as shown in Table 1.

[0066] Table 1 shows the precision and accuracy of determining criborone in human plasma. Table 1:

[0067]

[0068] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for determining criborone in plasma, comprising: Obtain the standard curve plotted based on the detection peak area ratio; Add internal standard solution to plasma sample and vortex, centrifuge and collect supernatant, then add aqueous solution and vortex mix to obtain pretreated test sample; Detection was performed using liquid chromatography-tandem mass spectrometry: the sample to be tested was separated by liquid chromatography and then detected by mass spectrometry. Based on the standard curve and regression equation, the mass concentration of criborone in the sample to be tested was calculated.

2. The method for determining criborone in plasma as described in claim 1, characterized in that, The internal standard solution was criborone-d4.

3. The method for determining criborone in plasma as described in claim 2, characterized in that, The concentration of the internal standard solution was creborol-d4 = 50.00 ng / mL.

4. The method for determining criborone in plasma as described in claim 1, characterized in that, The solvent for the internal standard solution is acetonitrile.

5. The method for determining criborone in plasma as described in claim 1, characterized in that, The aqueous solution is a wt%: 0.1% 1 M ammonium acetate aqueous solution.

6. The method for determining criborone in plasma as described in claim 1, characterized in that, The conditions for the liquid chromatography are as follows: Column: Waters Acquity UPLC ® HSS T3 column, 1.8 μm, 50 × 2.1 mm; Mobile phase: Phase A: 0.1% 1 M ammonium acetate aqueous solution, Phase B: 0.1% 1 M ammonium acetate 95% acetonitrile 5% aqueous solution.

7. The method for determining criborone in plasma as described in claim 6, characterized in that, The elution conditions for the liquid chromatography are as follows: 0-0.40 min, 35% B phase, 65% A phase; 0.40-1.00 min, 35% B phase → 55% B phase, 65% A phase → 45% A phase; 1.00-1.50 min, 55% B phase → 95% B phase, 45% A phase → 5% A phase; 1.50-1.90 min, 95% B phase, 5% A phase; 1.90-1.91 min, 95% B phase → 35% B phase, 5% A phase → 65% A phase; 1.91-2.50 min, 35% B phase, 65% A phase; Wash-off time: 2.50 min; Flow rate: 0.500 mL / min; Injection volume: 20.00 µL; Autosampler temperature: 6 ℃; Column temperature: 45 ℃.

8. The method for determining criborone in plasma as described in claim 1, characterized in that, The mass spectrometry conditions for the mass spectrometry detection are as follows: Ion source: Electrospray ionization (ESI) source; Injection voltage: -4500 V; Gas spray gas 1: 55 psi; Auxiliary gas Gas2: 60 psi; Detection method: negative ions; Ion source temperature: 450 ℃; Collision-Induced Dissociation (CAD): 8; Curtain Gas: 40 psi.

9. The application of the method for determining criborone in plasma as described in any one of claims 1-8 in the auxiliary detection of atopic dermatitis.

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