WO2024036702A1 - 一种检测盐酸氨酮戊酸己酯的方法 - Google Patents

一种检测盐酸氨酮戊酸己酯的方法 Download PDF

Info

Publication number
WO2024036702A1
WO2024036702A1 PCT/CN2022/120786 CN2022120786W WO2024036702A1 WO 2024036702 A1 WO2024036702 A1 WO 2024036702A1 CN 2022120786 W CN2022120786 W CN 2022120786W WO 2024036702 A1 WO2024036702 A1 WO 2024036702A1
Authority
WO
WIPO (PCT)
Prior art keywords
mobile phase
hexyl
detecting
solution
hydrochloride
Prior art date
Application number
PCT/CN2022/120786
Other languages
English (en)
French (fr)
Inventor
龙超
王璐瑶
胡荣军
Original Assignee
广东精观生物医药科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 广东精观生物医药科技有限公司 filed Critical 广东精观生物医药科技有限公司
Publication of WO2024036702A1 publication Critical patent/WO2024036702A1/zh

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/50—Conditioning of the sorbent material or stationary liquid
    • G01N30/52—Physical parameters
    • G01N30/54—Temperature
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/64—Electrical detectors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86

Definitions

  • the present application relates to the field of chemical drug analysis methods, and in particular to a method for detecting hexyl aminolevulinate hydrochloride.
  • Aminolevulinic acid hexyl ester hydrochloride as the ester derivative hydrochloride of 5-aminolevulinic acid, is highly consistent with 5-aminolevulinic acid in its related application fields. It serves as a photosensitizer in photodynamic therapy. It has good applications in the diagnosis and treatment of related tumors, and aminolevulinic acid hexyl ester hydrochloride has the advantages of more stable properties, stronger lipophilicity, and higher bioavailability than 5-aminolevulinic acid. These advantages are bound to promote Aminolevulinic acid hexyl ester hydrochloride has been vigorously developed and applied in related application fields.
  • the instrument and chromatographic column will be greatly damaged, which will virtually increase the related detection costs; hexyl aminolevulinate hydrochloride does not have a fluorescent group, and fluorescence detection requires chemical derivatization to form a compound with a fluorescent group for detection, and The derivatization reaction takes a long time, the reaction temperature is relatively high, and the operation is complicated. Among them, the fluorescence intensity of acetylacetone derivatization will decrease with time.
  • the fluorescence detection method is time-consuming and laborious, has high requirements for derivatization reagents, and is difficult to control the stability.
  • the purpose of this application is to provide a method for detecting hexyl aminolevulinate hydrochloride to solve the problems of insufficient accuracy, complicated operation and high detection cost of existing methods for detecting hexyl aminolevulinate hydrochloride.
  • this application provides a method for detecting hexyl aminolevulinate hydrochloride, which uses high-performance liquid chromatography connected to an electrospray detector to detect hexyl aminolevulinate hydrochloride.
  • the method includes:
  • the detection conditions include: chromatographic column temperature 25°C; injection volume 5 ⁇ L; flow rate 1mL/min; trifluoroacetic acid aqueous solution as Mobile phase A, use acetonitrile as mobile phase B; perform gradient elution.
  • the chromatographic column is Waters XSelect HSS T3, 3.5 ⁇ m, 150mm ⁇ 4.6mm.
  • the chromatographic column is Agilent Poroshell 120 EC-C18, 2.7 ⁇ m, 75mm ⁇ 4.6mm.
  • the chromatographic column is Poroshell 120 Bonus-RP, 2.7 ⁇ m, 75mm ⁇ 4.6mm.
  • the chromatographic column is ACE Excel 3 C18-AR, 3.0 ⁇ m, 50mm ⁇ 2.1mm.
  • the conditions for gradient elution are as follows: within 0 to 3 minutes, the volume ratio of mobile phase A to mobile phase B is 95:5; within 3 to 15 minutes, the volume ratio of mobile phase A to mobile phase B is: 95:5 changes to 50:50; within 15-20min, the volume ratio of mobile phase A and mobile phase B changes from 50:50 to 5:95; within 20-30min, the volume ratio of mobile phase A and mobile phase B remains 5:95; within 30-30.1min, the volume ratio of mobile phase A and mobile phase B gradually changes from 5:95 to 95:5; within 30.1-40min, the volume ratio of mobile phase A and mobile phase B remains at 95:5 .
  • the mobile phase A is an aqueous trifluoroacetic acid solution with a volume ratio of 0.15%.
  • the sampling frequency of the electrospray detector is 2 Hz
  • the peak filtering time parameter is 3.6 seconds
  • the atomization temperature is 55°C.
  • the method for detecting aminolevulinic acid hexyl ester in this application uses high-performance liquid chromatography connected to an electrospray detector to detect aminolevulinic acid hexyl ester, solving the existing problem of detecting aminolevulinic acid hexyl ester.
  • the method of hexyl acid ester has problems such as insufficient accuracy, complicated operation and high detection cost.
  • Figure 1 is a spectrum of the blank solvent of this application.
  • Figure 2 is the positioning map of hexyl aminolevulinate hydrochloride of the present application.
  • Figure 3 is a spectrum of the resolution solution of hexyl aminolevulinate hydrochloride of the present application.
  • Figure 4 is an enlarged overlay view of the blank solution of the present application, the detection limit of aminolevulinic acid hexyl ester hydrochloride, and the quantitative limit of aminolevulinic acid hexyl ester hydrochloride.
  • Figure 5 is a chromatographic column screening overlay diagram of the application's method for related substances of hexyl aminolevulinate hydrochloride.
  • Example 1 This application provides a method for detecting hexyl aminolevulinate hydrochloride using high-performance liquid chromatography connected to an electrospray detector.
  • the selected instruments and equipment include:
  • the chromatographic column is Waters XSelect HSS T3, 3.5 ⁇ m, 150mm ⁇ 4.6mm.
  • the selected reagents include: purified water (H2O), HPLC grade; acetonitrile (ACN), HPLC grade; trifluoroacetic acid (TFA), HPLC grade.
  • test sample of hexyl aminolevulinate hydrochloride dilute and dissolve it with an acetonitrile aqueous solution with a volume ratio of 50% to prepare a test solution.
  • the detection conditions include: chromatographic column temperature 25°C; injection volume 5 ⁇ L; flow rate 1mL/min; trifluoroacetic acid aqueous solution as Mobile phase A, preferably, a trifluoroacetic acid aqueous solution with a volume ratio of 0.15% is used as mobile phase A, and acetonitrile is used as mobile phase B; gradient elution is performed.
  • the detection conditions of the electrospray detector are: the sampling frequency is 2 Hz, the peak filtering time parameter is 3.6 seconds, and the atomization temperature is 55°C.
  • the mobile phase preparation method is:
  • the mobile phase is selected based on the chromatographic column and detector. Such mobile phases are recommended for this column and are suitable for use with electrospray detectors.
  • the gradient elution time is 40 minutes, and the gradient elution process is specifically: within 0 to 3 minutes, the volume ratio of mobile phase A to mobile phase B is 95:5; within 3 to 15 minutes, the volume ratio of mobile phase A
  • the volume ratio of mobile phase A and mobile phase B gradually changes from 95:5 to 50:50 at a constant speed; within 15-20 minutes, the volume ratio of mobile phase A and mobile phase B changes from 50:50 to 5:95 at a constant speed; within 20-30 minutes, the flow
  • the volume ratio of phase A and mobile phase B remains at 5:95; within 30-30.1min, the volume ratio of mobile phase A and mobile phase B gradually changes from 5:95 to 95:5; within 30.1-40min, the volume ratio of mobile phase A and mobile phase B
  • the volume ratio of mobile phase B was maintained at 95:5.
  • the high-performance liquid chromatography conditions and the detection conditions of the electrospray detector are all established on the Thermo UltiMate3000 series high-performance liquid chromatograph with electrospray detector.
  • the trapping column can appropriately adjust the time of the gradient elution process and the equilibrium time during the change process according to the actual situation.
  • the chromatographic column selected is Waters XSelect HSS T3, 3.5 ⁇ m, 150 mm ⁇ 4.6 mm.
  • equivalent chromatographic columns can also be selected.
  • the chromatographic column is Agilent Poroshell 120 EC-C18, 2.7 ⁇ m, 75 mm ⁇ 4.6 mm.
  • the column is Poroshell 120 Bonus-RP, 2.7 ⁇ m, 75mm ⁇ 4.6mm.
  • the chromatographic column is ACE Excel 3 C18-AR, 3.0 ⁇ m, 50mm ⁇ 2.1mm.
  • Reference substance Hexyl aminolevulinic acid hydrochloride reference substance (homemade; batch number: P08907-029-P1), impurity A reference substance (aminolevulinic acid hydrochloride reference substance; homemade; batch number: P08907-014-P1), impurity A Reference substance B (ethyl aminolevulinate hydrochloride reference substance; homemade; batch number: P12537-048-P1), impurity C reference substance (heptyl aminolevulinate hydrochloride reference substance; homemade; batch number: P12537-046-P1) , Impurity D reference substance (2.5-pyrazinedipropionic acid reference substance; homemade; batch number: P12537-082-P1), Impurity E reference substance (2,5-pyrazinedipropionic acid dihexyl reference substance; homemade; batch number) :P12537-090-P2).
  • Blank solvent Use a solution that does not contain hexyl aminolevulinate hydrochloride as a blank reagent.
  • the blank solvent is an acetonitrile aqueous solution with a volume ratio of 50%.
  • Injection washing solution acetonitrile-water solution with a volume ratio of 50%.
  • the concentration of hexyl aminolevulinate hydrochloride in the separation solution of aminolevulinate hexyl ester is 2.5 mg/mL
  • the concentration of impurity B is 0.0125 mg/mL
  • the concentration of other known impurities is 0.025 mg/mL.
  • Other known impurities include Impurity A, Impurity C, Impurity E, and Impurity D.
  • the method for preparing impurities is:
  • the preparation of impurity solution A is to prepare the aminolevulinic acid hydrochloride solution.
  • the specific process is to take about 5 mg of aminolevulinic acid hydrochloride reference substance, accurately weigh it, place it in a 10mL volumetric flask, and add an appropriate amount. Dissolve the diluent (ultrasonicate appropriately if necessary), cool to room temperature, add diluent to dilute to volume, shake well, and mark it as impurity solution A.
  • impurity solution B is an aminolevulinic acid ethyl ester solution made from aminolevulinic acid ethyl ester hydrochloride reference substance
  • impurity solution C is aminolevulinic acid heptyl ester hydrochloride made from aminolevulinic acid heptyl ester reference substance.
  • Impurity solution D is a 2.5-pyrazinedipropionic acid solution made with 2.5-pyrazinedipropionic acid reference substance
  • Impurity solution E is 2, made with 2,5-pyrazinedipropionic acid dihexyl reference substance. Dihexyl 5-pyrazine dipropionate solution.
  • the RS solution is to prepare a resolution solution of hexyl aminolevulinate hydrochloride, and the RS solution is detected using high performance liquid chromatography connected to an electrospray detector.
  • RS solution can be stored at -20°C until use, and should be warmed to room temperature before use. If the RS solution cannot meet the system suitability requirements, it needs to be prepared again.
  • the detection conditions for detecting RS solution are the same as those for detecting hexyl aminolevulinate hydrochloride using high-performance liquid chromatography connected to an electrospray detector.
  • the gradient elution process is also the same, both taking 40 minutes.
  • Figure 3 is a chromatogram of the resolution solution of aminolevulinic acid hexyl ester hydrochloride.
  • the test results of the separation solution of aminolevulinic acid hexyl ester hydrochloride are shown in Table 1.
  • ester and impurity A, impurity B, impurity C, impurity D, and impurity E can all be clearly separated, which proves the good detection effect of high performance liquid chromatography conditions and electrospray detector for hexyl aminolevulinate hydrochloride.
  • acetonitrile aqueous solution as the diluent, take 1mL of SPL-1 solution and add diluent to prepare 100mL of solution, and use this solution as SPL-1-1% solution.
  • ASPK peak area of aminolevulinic acid hexyl ester hydrochloride in SPK solution
  • ASTD-1 Content determination of the average peak area of hexyl aminolevulinate hydrochloride in 6 consecutive injections of the reference solvent
  • WSTD-1 Content determination of hexyl aminolevulinate hydrochloride in reference solvent. Weighed sample amount, mg
  • WSPK Weighed sample amount of hexyl aminolevulinate hydrochloride in SPK solution, mg
  • VSTD-1 dilution volume of STD-1, mL
  • VSPK dilution volume of SPK solution, mL
  • the recovery rates of 9 different hexyl aminolevulinate hydrochloride reference solvents range from 96.3% to 104.7%, and the RSD of the 9 accuracy recovery rates is 3.5%, which is less than 5.0%, consistent with Existing acceptance criteria:
  • the recovery rate of the main component aminolevulinic acid hexyl ester hydrochloride should be in the range of 90.0% to 110.0%, and 9 parts of the accuracy recovery rate RSD 5.0%. Therefore, it shows that the accuracy of this method is high.
  • L-STOCK solution take 50.84 mg of hexyl aminolevulinate hydrochloride reference substance and dilute it with diluent to form a solution, named L-STOCK solution
  • L-STOCK solution takes 50.84 mg of hexyl aminolevulinate hydrochloride reference substance and dilute it with diluent to form a solution, named L-STOCK solution
  • L-STOCK solution takes the L-STOCK solution, add diluent to dilute it and formulate it into L-1 solution, and then Dilute L-1 solution with diluent and prepare it into LOQ solution.
  • LOQ solution dilute it with diluent and prepare it into LOD-1 solution and LOD-2 solution.
  • Table 5 The specific preparation method is shown in Table 5.
  • the results of the detection limit experiment are in line with the existing acceptance standards: the S/N of the main component hexyl aminolevulinate hydrochloride in the LOQ solution N should be ⁇ 10, and the RSD of the peak area of the main component hexyl aminolevulinate hydrochloride for 6 consecutive injections should be ⁇ 15%.
  • the passing of the quantitation limit and detection limit experiments proves that the detection of aminolevulinate hexyl ester hydrochloride using high-performance liquid chromatography connected to an electrospray detector can still be effective even when the concentration level of aminolevulinate hexyl ester hydrochloride is very low. .
  • the method of detecting aminolevulinic acid hexyl ester hydrochloride by high-performance liquid chromatography connected with an electrospray detector has the advantages of high sensitivity and low detection limit, which can reach the nanogram (ng) level.
  • This application also uses high performance liquid chromatography connected to an ultraviolet light detector to detect aminolevulinic acid hexyl ester hydrochloride as a comparative experiment of this application.
  • Table 7 Experimental parameter table of high performance liquid chromatography connected to ultraviolet light detector
  • the present application uses high performance liquid chromatography connected to a UV detector to detect aminolevulinate hexyl ester hydrochloride. Even if the concentration of aminolevulinate hexyl ester hydrochloride is at the nanogram level, aminoketone hydrochloride can still be effectively detected. Hexyl valerate, and has a relatively obvious peak area and good resolution. Therefore, the method used in this application to detect aminolevulinic acid hexyl ester hydrochloride through high-performance liquid chromatography connected to an ultraviolet light detector has the characteristics of high sensitivity, good specificity, high accuracy, simple operation, low test cost, and good robustness. , can support large-volume testing work related to hexyl aminolevulinate hydrochloride.
  • this application uses high-performance liquid chromatography connected to an electrospray detector to detect the content and purity of hexyl aminolevulinate hydrochloride, which can effectively solve the accuracy of the existing methods for detecting hexyl aminolevulinate hydrochloride. Insufficiencies, complex operations and high detection costs.

Landscapes

  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

Abstract

本申请提供了一种检测盐酸氨酮戊酸己酯的方法，利用高效液相色谱连接电雾式检测器对盐酸氨酮戊酸己酯进行检测，称取盐酸氨酮戊酸己酯的供试品适量，使用体积比为50％的乙腈水溶液进行稀释溶解制备出供试品溶液；检测条件包括：色谱柱柱温25℃；进样体积5μL；流速1mL/min；以三氟乙酸水溶液作为流动相A，以乙腈作为流动相B；进行梯度洗脱。本申请利用高效液相色谱连接电雾式检测器对盐酸氨酮戊酸己酯的含量和纯度进行检测，能够有效解决现有检测盐酸氨酮戊酸己酯的方法准确度不足、操作复杂以及检测成本较高的问题。

Description

一种检测盐酸氨酮戊酸己酯的方法
本申请要求了申请日为2022年8月17日，申请号为202210984832.6，发明名称为“一种检测盐酸氨酮戊酸己酯的方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本申请涉及化学药物分析方法领域，尤其涉及一种检测盐酸氨酮戊酸己酯的方法。
背景技术
盐酸氨酮戊酸己酯作为5-氨基酮戊酸的酯类衍生物盐酸盐，在其相关应用领域与5-氨基酮戊酸有着高度的一致性，其作为光敏剂在光动力学疗法及相关肿瘤的诊断和治疗都有着良好的应用，而且盐酸氨酮戊酸己酯相对5-氨基酮戊酸有性质更加稳定，亲脂性更强，生物利用度更高等优势，这些优点势必会推动盐酸氨酮戊酸己酯在相关应用领域的大力发展应用。
由于盐酸氨酮戊酸己酯与5-氨基酮戊酸的结构很相似，参考结合5-氨基酮戊酸的一些检测方式对其检测，主要为高效液相带紫外检测器、二极管阵列检测器和荧光检测器的方法或者高效液相串联质谱技术。但由于盐酸氨酮戊酸己酯紫外吸收弱，紫外检测方法有检出限高，无法满足低浓度化合物样品分析检测的缺点；二极管阵列检测需要使用到离子对试剂，其很难冲洗处理，对仪器、色谱柱损伤很大的，无形中增加相关检测成本；盐酸氨酮戊酸己酯不具有荧光基团，采用荧光检测需要进行化学衍生化，形成具有荧光基团的化合物才能进行检测，且衍生化反应时间较长，反应温度比较高，操作比较复杂，其中乙酰丙酮衍生化其荧光强度随时间延长会降低，荧光检测方法耗时费力，对衍生化试剂要求高，稳定性难以控制，方法的应用受到限制；高效液相串联质谱技术，由于相关仪器价格比较贵，利用率相对不高，使用试剂要求高，很多药厂及研究单位没有配备，导致其方法的应用受到限制。
有鉴于此，确有必要提出一种新的检测盐酸氨酮戊酸己酯的方法，以解决上述问题。
发明内容
本申请的目的在于提供一种检测盐酸氨酮戊酸己酯的方法，以解决现有检测盐酸氨酮戊酸己酯的方法准确度不足、操作复杂以及检测成本较高的问题。
为实现上述目的，本申请提供了一种检测盐酸氨酮戊酸己酯的方法，利用高效液相色谱连接电雾式检测器对盐酸氨酮戊酸己酯进行检测，所述方法包括：
分别称取盐酸氨酮戊酸己酯的对照品和供试品适量，使用体积比为50％的乙腈水溶液进行稀释溶解制备出对照品溶液和供试品溶液；
利用高效液相色谱连接电雾式检测器对盐酸氨酮戊酸己酯样品进行检测，检测条件包括：色谱柱柱温25℃；进样体积5μL；流速1mL/min；以三氟乙酸水溶液作为流动相A，以乙腈作为流动相B；进行梯度洗脱。
作为本申请的进一步改进，色谱柱为Waters XSelect HSS T3，3.5μm，150mm×4.6mm。
作为本申请的进一步改进，色谱柱为Agilent Poroshell 120 EC-C18，2.7μm，75mm×4.6mm。
作为本申请的进一步改进，色谱柱为Poroshell 120 Bonus-RP，2.7μm，75mm×4.6mm。
作为本申请的进一步改进，色谱柱为ACE Excel 3 C18-AR，3.0μm，50mm×2.1mm。
作为本申请的进一步改进，梯度洗脱的条件如下：0～3min内，流动相A和流动相B的体积比为95：5；3-15min内，流动相A和流动相B的体积比由95：5变至50：50；15-20min内，流动相A和流动相B的体积比由50：50变至5：95；20-30min内，流动相A和流动相B的体积比保持5：95；30-30.1min内，流动相A和流动相B的体积比由5：95匀速渐变至95：5；30.1-40min内，流动相A和流动相B的体积比保持95：5。
作为本申请的进一步改进，所述流动相A为体积比为0.15％的三氟乙酸水溶液。
作为本申请的进一步改进，所述电雾式检测器的采样频率为2赫兹，峰过滤时间参数为3.6秒，雾化温度为55℃。
本申请的有益效果是：本申请的检测盐酸氨酮戊酸己酯的方法利用高效液相色谱连接电雾式检测器对盐酸氨酮戊酸己酯进行检测，解决现有检测盐酸氨酮戊酸己酯的方法准确度不足、操作复杂以及检测成本较高的问题。
附图说明
下面结合附图，通过对本申请的具体实施方式详细描述，将使本申请的技术方案及其它有益效果显而易见。
图1是本申请的空白溶剂的图谱。
图2是本申请的盐酸氨酮戊酸己酯的定位图谱。
图3是本申请的盐酸氨酮戊酸己酯的分离度溶液的图谱。
图4是本申请的空白溶液、盐酸氨酮戊酸己酯的检测限与盐酸氨酮戊酸己酯的定量限的放大叠加图。
图5是本申请的盐酸氨酮戊酸己酯有关物质方法色谱柱筛选叠加图。
具体实施方式
为了使本申请的目的、技术方案和优点更加清楚，下面结合附图和具体实施例对本申请进行详细描述。
在此，需要说明的是，为了避免因不必要的细节而模糊了本申请，在附图中仅仅示出了与本申请的方案密切相关的结构和/或处理步骤，而省略了与本申请关系不大的其他细节。
另外，还需要说明的是，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者设备所固有的要素。
实施例1：本申请提供了一种利用高效液相色谱连接电雾式检测器对盐酸氨酮戊酸己酯进行检测的方法。
选用的仪器设备包括：
Thermo UltiMate3000系列高效液相色谱仪，带电雾式检测器；十万分之一电子天平或等效仪器；超声仪；纯水仪；A级玻璃器皿；色谱柱。
色谱柱为Waters XSelect HSS T3，3.5μm，150mm×4.6mm。
选用的试剂包括：纯化水(H2O)，HPLC级；乙腈(ACN)，HPLC级；三氟乙酸(TFA)，HPLC级。
分别称取盐酸氨酮戊酸己酯的供试品适量，使用体积比为50％的乙腈水溶液进行稀释溶解制备出供试品溶液。
利用高效液相色谱连接电雾式检测器对盐酸氨酮戊酸己酯样品进行检测，检测条件包括：色谱柱柱温25℃；进样体积5μL；流速1mL/min；以三氟乙酸水溶液作为流动相A，优选地，以体积比为0.15％的三氟乙酸水溶液量作为流动相A，以乙腈作为流动相B；进行梯度洗脱。
电雾式检测器的检测条件为：采样频率为2赫兹，峰过滤时间参数为3.6秒，雾化温度为55℃。
其中，流动相制备的方式为：
取1.5mL TFA和1000mL H2O于适当体积的流动相瓶中，混匀，超声仪超声脱气，放冷至室温，备用，以此作为流动相A。
量取1000mL ACN于适当体积的流动相瓶中，混匀，超声仪超声脱气，放冷至室温，备用，以此作为流动相B。
需要说明的是：流动相是根据色谱柱和检测器来选。该色谱柱推荐这样的流动相，而且这样的流动相也适用于电雾式检测器。
在本实施例中，梯度洗脱的时间为40min，梯度洗脱的过程具体为：0～3min内，流动相A和流动相B的体积比为95：5；3-15min内，流动相A和流动相B的体积比由95：5匀速渐变至50：50；15-20min内，流动相A和流动相B的体积比由50：50匀速渐变至5：95；20-30min内，流动相A和流动相B的体积比保持5：95；30-30.1min内，流动相A和流动相B的体积比由5：95匀速渐变至95：5；30.1-40min内，流动相A和流动相B的体积比保持95：5。
需要注意的是，高效液相色谱条件和电雾式检测器的检测条件均是在带电雾式检测器的Thermo UltiMate3000系列高效液相色谱仪上建立的，如更换不同类型HPLC仪器或加鬼峰捕集柱，可根据实际情况适当调整梯度脱洗过程的时间及变化过程中的平衡时间。
对盐酸氨酮戊酸己酯的检测：
取盐酸氨酮戊酸己酯的供试品溶液，注入高效液相色谱仪中，记录电雾式检测器检测到 的色谱图，用外标法计算供试品溶液中盐酸氨酮戊酸己酯的含量。
需要知道的是，在本实施例中，选用的色谱柱为Waters XSelect HSS T3，3.5μm，150mm×4.6mm。在其他实施例中，也可以选用等效的色谱柱，例如色谱柱为Agilent Poroshell 120 EC-C18，2.7μm，75mm×4.6mm。或，色谱柱为Poroshell 120 Bonus-RP，2.7μm，75mm×4.6mm。或，色谱柱为ACE Excel 3 C18-AR，3.0μm，50mm×2.1mm。
实施例2：方法的验证
1.溶液的制备：
1.1对照品：盐酸氨酮戊酸己酯对照品(自制；批号：P08907-029-P1)、杂质A对照品(盐酸氨酮戊酸对照品；自制；批号：P08907-014-P1)、杂质B对照品(盐酸氨酮戊酸乙酯对照品；自制；批号：P12537-048-P1)、杂质C对照品(盐酸氨酮戊酸庚酯对照品；自制；批号：P12537-046-P1)、杂质D对照品(2.5-吡嗪二丙酸对照品；自制；批号：P12537-082-P1)、杂质E对照品(2,5-吡嗪二丙酸二己酯对照品；自制；批号：P12537-090-P2)。
1.2稀释剂：体积比为50％的乙腈水溶液。
1.3空白溶剂：以不含有盐酸氨酮戊酸己酯的溶液作为空白试剂。在本实施例中，空白溶剂为体积比为50％的乙腈水溶液。
1.4洗针液：体积比为50％的乙腈水溶液。
1.5分离度溶液配制：
盐酸氨酮戊酸己酯分离度溶液中盐酸氨酮戊酸己酯的浓度为2.5mg/mL，杂质B浓度为0.0125mg/mL，其他已知杂质浓度为0.025mg/mL。其他已知杂质包括杂质A、杂质C、杂质E和杂质D。
其中，制备杂质的方法为：
以制备杂质溶液A为例，制备杂质溶液A即为制备盐酸氨酮戊酸溶液，具体流程为取约5mg盐酸氨酮戊酸对照品，精密称定后，置于10mL容量瓶中，加适量稀释剂溶解(必要时可适当超声)，冷却至室温，加稀释剂稀释至刻度，摇匀，标记为杂质溶液A。
同理可制备浓度均为0.5mg/mL的杂质溶液B、杂质溶液C、杂质溶液D、杂质溶液E。其中，杂质溶液B为以盐酸氨酮戊酸乙酯对照品制作的盐酸氨酮戊酸乙酯溶液；杂质溶液C为以盐酸氨酮戊酸庚酯对照品制作的盐酸氨酮戊酸庚酯溶液；杂质溶液D为以2.5-吡嗪二丙酸对照品制作的2.5-吡嗪二丙酸溶液；杂质溶液E为以2,5-吡嗪二丙酸二己酯对照品制作的2,5-吡嗪二丙酸二己酯溶液。
盐酸氨酮戊酸己酯分离度溶液的制备具体为：
取盐酸氨酮戊酸己酯对照品约50mg，精密称定后，置于20mL容量瓶中，加适量稀释剂溶解，向其中加入杂质溶液A、杂质溶液C、杂质溶液E和杂质溶液D各1mL和杂质溶液B 0.5mL，加稀释剂稀释至刻度，摇匀，标记为RS溶液。RS溶液即为制备盐酸氨酮戊酸己酯分离度溶液，对RS溶液利用高效液相色谱连接电雾式检测器进行检测。
RS溶液可存放在-20℃条件下存储一直使用，使用前回温至室温。如果RS溶液无法满足系统适用性要求，需重新配制。
如图1所示，为了排除空白溶剂的干扰，在正式对盐酸氨酮戊酸己酯供试品溶液进行检测之前，本申请制备了体积比为50％的乙腈水溶液，将该溶液作为空白溶剂，对空白溶剂利用高效液相色谱连接电雾式检测器按相同的检测条件进行了检测，得到了如图1的空白溶剂的图谱。图中，横坐标为时间，纵坐标为峰面积，在该图谱中可以看出，空白溶剂中无盐酸氨酮戊酸己酯，更无杂质A、杂质B、杂质C、杂质D、杂质E存在。
请参阅图2所示，使用高效液相色谱连接电雾式检测器对盐酸氨酮戊酸己酯对照品进行检测，得到了盐酸氨酮戊酸己酯的定位图谱，图中横坐标为时间，纵坐标为峰面积。从该图谱中可知，盐酸氨酮戊酸己酯保留时间为16.12min，峰面积为0.9455pA*min，相对峰面积100％，信噪比(S/N)为2941.2，由图2可以看出，本方法测得的盐酸氨酮戊酸己酯的主峰峰形明显，同时主峰出峰时间无显著性干扰。
对RS溶液进行检测的检测条件与利用高效液相色谱连接电雾式检测器对盐酸氨酮戊酸己酯进行检测的检测条件相同，梯度洗脱过程也相同，均用时40min。
请参阅图3所示，为盐酸氨酮戊酸己酯分离度溶液的图谱。其中盐酸氨酮戊酸己酯分离度溶液检测结果见表1。
表1：盐酸氨酮戊酸己酯分离度溶液检测结果表
Figure PCTCN2022120786-appb-000001
由表1和图1可知，盐酸氨酮戊酸己酯的保留时间为15.934min，峰面积为41.911pA*min，相对峰面积90.614％，分离度(EP)为7.3，信噪比(S/N)为51178.2。根据结果可以看出，由该方法检测得到的盐酸氨酮戊酸己酯分离度溶液中，盐酸氨酮戊酸己酯的主峰峰形好，相对峰面积占比大，盐酸氨酮戊酸己酯与杂质A、杂质B、杂质C、杂质D、杂质E都能明显 分离，证明了对采用高效液相色谱条件和电雾式检测器对盐酸氨酮戊酸己酯良好的检测效果。
实施例3：精密度实验
实验过程为：
以乙腈水溶液作为稀释剂，取50.29mg盐酸氨酮戊酸己酯供试品加稀释剂配置20mL溶液，将该溶液作为SPL-1溶液。
以乙腈水溶液作为稀释剂，取1mL SPL-1溶液加稀释剂配置100mL溶液，将该溶液作为SPL-1-1％溶液。
将SPL-1-1％溶液连续进6针，分别命名为SPL-1-1％-01～SPL-1-1％-06，计算连续6针系统精密度溶液色谱图中主成分盐酸氨酮戊酸己酯峰面积和保留时间的标准偏差(RSD)，最后获得的系统精密度结果见表2。
表2：系统精密度结果表
Figure PCTCN2022120786-appb-000002
由表2可以看出，重复进样6次得到的SPL-1-1％的检测结果接近。对保留时间来说，重复进样6针的RSD＝0.00％；对于峰面积来说，重复进样6针的RSD＝0.7％，符合接受标准：在连续6针对照溶液色谱图中，主成分盐酸氨酮戊酸己酯峰面积的RSD应5.0％，保留时间的RSD应1.0％。证明了采用高效液相色谱条件和电雾式检测器对盐酸氨酮戊酸己酯的检测方法的重现性好，方法的精密度良好。
实施例4：系统准确度实验(回收率试验数据)
实验过程为：
取不同质量的盐酸氨酮戊酸己酯对照品加稀释剂稀释后配置成含有不同含量盐酸氨酮戊酸己酯的盐酸氨酮戊酸己酯对照溶剂，分别命名。具体配制见表3。
表3：盐酸氨酮戊酸己酯对照溶剂配置表
Figure PCTCN2022120786-appb-000003
Figure PCTCN2022120786-appb-000004
将9种不同的盐酸氨酮戊酸己酯对照溶剂各进一针，计算9份盐酸氨酮戊酸己酯对照溶剂中主成分回收率(Recovery)的RSD(％)，用于表征含量方法的准确度，并将准确度结果输入表4。
其中按公式计算各盐酸氨酮戊酸己酯对照溶剂的回收率：
Figure PCTCN2022120786-appb-000005
式中：
ASPK：SPK溶液中盐酸氨酮戊酸己酯的峰面积
ASTD-1：含量测定盐酸氨酮戊酸己酯对照溶剂连续6针中盐酸氨酮戊酸己酯平均峰面积
WSTD-1：含量测定盐酸氨酮戊酸己酯对照溶剂中盐酸氨酮戊酸己酯的称样量，mg
WSPK：SPK溶液中盐酸氨酮戊酸己酯的称样量，mg
VSTD-1：STD-1的稀释体积，mL
VSPK：SPK溶液的稀释体积，mL
P：盐酸氨酮戊酸己酯的含量％
表4：准确度实验方法重复性结果表
Figure PCTCN2022120786-appb-000006
Figure PCTCN2022120786-appb-000007
由表4可以看出，9种不同的盐酸氨酮戊酸己酯对照溶剂的回收率在96.3％～104.7％之间，且9份准确度回收率的RSD＝3.5％，小于5.0％，符合现有的接受标准：9份盐酸氨酮戊酸己酯对照溶剂中，主成分盐酸氨酮戊酸己酯回收率Recovery应在90.0％～110.0％的范围内，且9份准确度回收率的RSD 5.0％。因此说明该方法的准确度高。
实施例5：盐酸氨酮戊酸己酯的定量限(LOQ)和检测限(LOD)实验
实验过程为：
取盐酸氨酮戊酸己酯对照品配制线性溶液。
具体为：取50.84mg盐酸氨酮戊酸己酯对照品加稀释剂稀释后配置成溶液，命名为L-STOCK溶液，然后取L-STOCK溶液加稀释剂稀释后配置成L-1溶液，再取L-1溶液加稀释剂稀释后配置成LOQ溶液，最后取不同剂量的LOQ溶液加稀释剂稀释后配置成LOD-1溶液和LOD-2溶液，具体配制方法见表5。
表5：定量限和检测限实验溶液配制表
Figure PCTCN2022120786-appb-000008
将LOQ溶液连续进样6针，计算连续6针LOQ色谱图中主成分盐酸氨酮戊酸己酯的信噪比(S/N)和峰面积的RSD。将LOD-1和LOD-2溶液各进一针，计算色谱图中主成分盐酸氨酮戊酸己酯的S/N，选择S/N≥3的最小浓度溶液作为LOD。最后结果见表6，同时将本申请的空白溶液、盐酸氨酮戊酸己酯的检测限与盐酸氨酮戊酸己酯的定量限的数据制 成如图4的放大叠加图。
表6：定量限和检测限实验结果表
Figure PCTCN2022120786-appb-000009
由表6可知，在0.02％盐酸氨酮戊酸己酯浓度水平的LOD-1溶液中，仍可检测到盐酸氨酮戊酸己酯，此时主成分盐酸氨酮戊酸己酯的S/N为9，定量限实验的结果符合现有接受标准：LOD溶液中主成分盐酸氨酮戊酸己酯的S/N应≥3。LOQ溶液连续进样6针的实验结果中，S/N最小为22，RSD为8％，检测限实验的结果符合现有接受标准：LOQ溶液中主成分盐酸氨酮戊酸己酯的S/N应≥10，且连续6针主成分盐酸氨酮戊酸己酯峰面积的RSD应≤15％。定量限和检测限实验的通过证明了采用高效液相色谱连接电雾式检测器对盐酸氨酮戊酸己酯进行检测即使在盐酸氨酮戊酸己酯浓度水平很低的情况下依旧可以生效。也就是说，高效液相色谱连接电雾式检测器对盐酸氨酮戊酸己酯进行检测的方法的灵敏度高，检测限低的优点，可以达到纳克(ng)级。
实施例6：对比实验
本申请还通过高效液相色谱连接紫外光检测器对盐酸氨酮戊酸己酯进行检测，以此作为本申请的对比实验。
通过高效液相色谱连接紫外光检测器对盐酸氨酮戊酸己酯进行检测的高效液相色谱仪及紫外光检测器的参数如表7所示：
表7：高效液相色谱连接紫外光检测器实验参数表
Figure PCTCN2022120786-appb-000010
Figure PCTCN2022120786-appb-000011
请参阅图5所示，高效液相色谱连接紫外光检测器对盐酸氨酮戊酸己酯进行检测得到的结果绘制成图5。由于盐酸氨酮戊酸己酯和杂质A两个化合物的紫外最大吸收均在200nm左右，当盐酸氨酮戊酸己酯浓度为10mg/ml，杂质A浓度为1mg/ml，其在图谱中显示响应不高，表明两个化合物紫外吸收弱，方法检出限高，无法满足低浓度盐酸氨酮戊酸己酯样品分析检测的需求。
而本申请通过高效液相色谱连接紫外光检测器对盐酸氨酮戊酸己酯进行检测的方法，即使盐酸氨酮戊酸己酯浓度的浓度为纳克级别，仍可有效检测到盐酸氨酮戊酸己酯，并具有相对较明显的峰面积和较好的分离度。因此，本申请通过高效液相色谱连接紫外光检测器对盐酸氨酮戊酸己酯进行检测的方法具有灵敏度高，专属性好，准确度高，操作简单，试验成本低，稳健度好的特性，能支持大批量的盐酸氨酮戊酸己酯相关检测工作。
综上所述，本申请利用高效液相色谱连接电雾式检测器对盐酸氨酮戊酸己酯的含量和纯 度进行检测，能够有效解决现有检测盐酸氨酮戊酸己酯的方法准确度不足、操作复杂以及检测成本较高的问题。
以上实施例仅用以说明本申请的技术方案而非限制，尽管参照较佳实施例对本申请进行了详细说明，本领域的普通技术人员应当理解，可以对本申请的技术方案进行修改或者等同替换，而不脱离本申请技术方案的精神和范围。

Claims (8)

  1. 一种检测盐酸氨酮戊酸己酯的方法，其特征在于，利用高效液相色谱连接电雾式检测器对盐酸氨酮戊酸己酯进行检测，所述方法包括：
    分别称取盐酸氨酮戊酸己酯的对照品和供试品适量，使用体积比为50％的乙腈水溶液进行稀释溶解制备出对照品溶液和供试品溶液；
    利用高效液相色谱连接电雾式检测器对盐酸氨酮戊酸己酯样品进行检测，检测条件包括：色谱柱柱温25℃；进样体积5μL；流速1mL/min；以三氟乙酸水溶液作为流动相A，以乙腈作为流动相B；进行梯度洗脱。
  2. 根据权利要求1所述的检测盐酸氨酮戊酸己酯的方法，其特征在于：色谱柱为Waters XSelect HSS T3，3.5μm，150mm×4.6mm。
  3. 根据权利要求1所述的检测盐酸氨酮戊酸己酯的方法，其特征在于：色谱柱为Agilent Poroshell 120 EC-C18，2.7μm，75mm×4.6mm。
  4. 根据权利要求1所述的检测盐酸氨酮戊酸己酯的方法，其特征在于：色谱柱为Poroshell 120 Bonus-RP，2.7μm，75mm×4.6mm。
  5. 根据权利要求1所述的检测盐酸氨酮戊酸己酯的方法，其特征在于：色谱柱为ACE Excel 3 C18-AR，3.0μm，50mm×2.1mm。
  6. 根据权利要求1所述的检测盐酸氨酮戊酸己酯的方法，其特征在于：梯度洗脱的条件如下：0～3min内，流动相A和流动相B的体积比为95：5；3-15min内，流动相A和流动相B的体积比由95：5变至50：50；15-20min内，流动相A和流动相B的体积比由50：50变至5：95；20-30min内，流动相A和流动相B的体积比保持5：95；30-30.1min内，流动相A和流动相B的体积比由5：95匀速渐变至95：5；30.1-40min内，流动相A和流动相B的体积比保持95：5。
  7. 根据权利要求1所述的检测盐酸氨酮戊酸己酯的方法，其特征在于：所述流动相A为体积比为0.15％的三氟乙酸水溶液。
  8. 根据权利要求1所述的检测盐酸氨酮戊酸己酯的方法，其特征在于：所述电雾式检测器的采样频率为2赫兹，峰过滤时间参数为3.6秒，雾化温度为55℃。
PCT/CN2022/120786 2022-08-17 2022-09-23 一种检测盐酸氨酮戊酸己酯的方法 WO2024036702A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210984832.6 2022-08-17
CN202210984832.6A CN117630189A (zh) 2022-08-17 2022-08-17 一种检测盐酸氨酮戊酸己酯的方法

Publications (1)

Publication Number Publication Date
WO2024036702A1 true WO2024036702A1 (zh) 2024-02-22

Family

ID=89940487

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/120786 WO2024036702A1 (zh) 2022-08-17 2022-09-23 一种检测盐酸氨酮戊酸己酯的方法

Country Status (2)

Country Link
CN (1) CN117630189A (zh)
WO (1) WO2024036702A1 (zh)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108982679A (zh) * 2017-06-05 2018-12-11 上海奥博生物医药技术有限公司 一种白消安中甲磺酸含量的测定方法
CN208443800U (zh) * 2018-07-27 2019-01-29 中国科学院西北高原生物研究所 一种通用型高灵敏电雾式液相色谱仪
CN114518413A (zh) * 2020-11-18 2022-05-20 江苏天士力帝益药业有限公司 一种卡托普利原料药中脯氨酸的含量测定方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108982679A (zh) * 2017-06-05 2018-12-11 上海奥博生物医药技术有限公司 一种白消安中甲磺酸含量的测定方法
CN208443800U (zh) * 2018-07-27 2019-01-29 中国科学院西北高原生物研究所 一种通用型高灵敏电雾式液相色谱仪
CN114518413A (zh) * 2020-11-18 2022-05-20 江苏天士力帝益药业有限公司 一种卡托普利原料药中脯氨酸的含量测定方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MORROW DESMOND I.J., MCCARRON PAUL A., WOOLFSON A. DAVID, DONNELLY RYAN F.: "Practical Considerations in the Pharmaceutical Analysis of Methyl and Hexyl Ester Derivatives of 5-Aminolevulinic Acid", THE OPEN ANALYTICAL CHEMISTRY JOURNAL, vol. 3, no. 1, 3 August 2009 (2009-08-03), pages 6 - 15, XP093140045, ISSN: 1874-0650, DOI: 10.2174/1874065000903010006 *

Also Published As

Publication number Publication date
CN117630189A (zh) 2024-03-01

Similar Documents

Publication Publication Date Title
WO2017152689A1 (zh) 伊马替尼基因杂质的高灵敏度分析方法
WO2021022876A1 (zh) 测定水合氯醛或其制剂中卤代酸含量的方法
US20100320373A1 (en) Mass spectrometric quantitative detection of methyl malonic acid and succinic acid using hilic on a zwitterionic stationary phase
Wen et al. Development and validation of a hydrophilic interaction ultra‐high‐performance liquid chromatography–tandem mass spectrometry method for rapid simultaneous determination of 19 free amino acids in rat plasma and urine
WO2023065521A1 (zh) 一种测定基因毒杂质1,3-二氯-2-丙醇的气相色谱质谱联用方法
CN116773693A (zh) 液相色谱串联质谱法检测血液中11种抗高血压药物及1种代谢产物的方法及应用
WO2024036702A1 (zh) 一种检测盐酸氨酮戊酸己酯的方法
CN112014510A (zh) 一种hplc法测定马来酸桂哌齐特中间体中氯乙酸残留检测方法
CN114280191B (zh) 一种双半胱氨酸及其制剂中有关物质的检测方法
CN110954629A (zh) 一种呋塞米中糠胺含量测定的控制方法
CN114689737B (zh) 一种s-邻氯苯甘氨酸甲酯酒石酸盐有关物质的分析方法
CN114324642B (zh) 一种氢溴酸右美沙芬有关物质的测定方法
CN109425666B (zh) 一种酰氯衍生物的lc-ms分析方法
CN112924601B (zh) 一种高效液相色谱-蒸发光法检测盐酸林可霉素注射液中的林可霉素杂质e的方法
CN112557520B (zh) Tgr-1中tgr-1-对应异构体的检测方法
CN114544785A (zh) 一种氟比洛芬及其杂质的检测方法
CN112697934A (zh) 一种复方氨基酸注射液中焦谷氨酸含量的检测方法
CN115598267B (zh) 一种格列齐特中潜在遗传毒性杂质的分析方法
CN112578044B (zh) 一种高效液相检测Fmoc-AEEA含量的方法
CN114280190B (zh) 一种用于检测双半胱氨酸有关物质的试剂盒
CN112924602B (zh) 一种高效液相色谱-蒸发光法检测盐酸林可霉素注射液中的林可霉素杂质f的方法
CN116124912A (zh) 17-碘-雄甾-5,16-二烯-3β-醇有关物质的检测方法和应用
Silva et al. Sensitive determination of terazosin by x‐ray fluorescence spectrometry based on the formation of ion‐pair associates with zinc thiocyanate
CN107091895B (zh) 采用hplc分离测定利奥西呱原料药中有关物质的方法
Rezaei et al. Synthesis and Purification of Acamprosate Calcium and its Evaluation by RP-HPLC in Pharmaceutical Dosage Forms: Synthesis and Purification of Acamprosate Calcium and its Evaluation by RP-HPLC

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22955477

Country of ref document: EP

Kind code of ref document: A1