HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY METHOD FOR DETERMINING ECDYSONE IN HEMOLYMPH OF MACROBRACHIUM NIPPONENSE IN LATE PRE-MOULT STAGE

Abstract

The present invention discloses a high-performance liquid chromatography-tandem mass spectrometry method for determining the ecdysone in the haemolymph of Macrobrachium nipponense in the late pre-moult stage, which belongs to the field of healthy aquaculture. The method for determining the ecdysone in the haemolymph of Macrobrachium nipponense in the late pre-moult stage provided by the present invention includes aliquoting 0.2-0.4 mL of haemolymph from Macrobrachium nipponense in the late pre-moult stage, centrifuging to obtain the supernatant; adding the stabilizer to the supernatant, then adding methanol and tolbutamide, followed by vortex-mixing uniformly, adding acetonitrile, shaking, and centrifuging again to obtain the supernatant, and performing high-performance liquid chromatography-tandem mass spectrometry detection. After validation, the lower limit of quantification (LLOQ) of the determination method provided by the present invention is 0.01 ng / mL. The present invention provides a new method for determining the ecdysone in the haemolymph of Macrobrachium nipponense in the late pre-moult stage.

Description

TECHNICAL FIELD The present invention relates to the field of healthy aquaculture, in particular to a high- performance liquid chromatography-tandem mass spectrometry method for determining the ecdysone in the haemolymph of Macrobrachium nipponense in the late pre-moult stage. BACKGROUND Macrobrachium nipponense, commonly known as Oriental river prawn, river prawn or grass prawn, has tender meat, delicious taste, rich nutrition and high economic value, and is one type of the freshwater economic prawn species with wide farming areas and large outputs in China. Crustaceans such as prawns and crabs undergo periodic moulting during their growth; each moulting leads to increase in the size and weight. Therefore, moulting is crucial for the healthy growth of crustaceans, especially in the aquaculture process, farmers hope that shrimps / prawns and crabs have high moulting synchronization to obtain relatively synchronized growing sizes. Thus, it is more necessary to pay attention to the occurrence time of moulting in cultured crustacean species. Ecdysone is an important hormone triggering moulting in crustaceans. Its active form is 20- hydroxyecdysone (20E). When the concentration of 20E in crustaceans reaches a certain level, moulting occurs. The moult cycle of crustaceans is generally divided into pre-moult stage, ecdysis, post-moult stage and inter-moult stage; the pre-moult stage may be further divided into early pre-moult stage, middle pre-moult stage and late pre-moult stage, among which the late pre-moult stage is a stage closest to the occurrence of ecdysis. Paying attention to the ecdysone level in the late pre-moult stage may not only sensitively indicate the approaching of moulting in aquaculture processes, but also contribute to the research on exploring moulting mechanisms. Therefore, there is an urgent need to establish an efficient, sensitive and accurate method for determining the ecdysone in the haemolymph in the late pre-moult stage of Macrobrachium nipponense. Currently, the disclosed methods for detecting ecdysone include thin-layer scanning method, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), liquid chromatography method, etc., all of which have certain limitations. For example, the detection sensitivity of both the thin-layer scanning method and liquid chromatography method is relatively lower; the radioimmunoassay may not perform accurate qualitative analysis; the ELISA method is more expensive. Zhao Peng et al. (2011) publicly reported a method for analysing ecdysone in haemolymph and muscle of marine crustaceans by high-performance liquid chromatography-triple quadrupole mass spectrometry, in which the haemolymph of Portunus trituberculatus was sampled from living crabs and then immediately extracted with the acetonitrile. It is suitable for crabs which are convenient for live transportation. Han Xiangning et al. (2018) disclosed a method for extracting ecdysone from Euphausia superba and detecting ecdysone by high-performance liquid chromatography. It is suitable for the entire-body extraction of batches of Euphausia superb. The detection sensitivity of this method is low. Compared with crabs, insects or plant samples, Macrobrachium nipponense demands higher working conditions for live transportation. Transporting live Macrobrachium nipponense to quality inspection agencies for haemolymph extraction and ecdysone detection will increase transportation costs and operation difficulties; moreover, if the haemolymph is not detected immediately after extraction, ecdysone is prone to be oxidized, which affects the accuracy of detection results. Therefore, it is necessary to develop a method which is suitable for pretreating and determining the ecdysone in the haemolymph of Macrobrachium nipponense in the late pre-moult stage. SUMMARY The purpose of the present invention is to provide a high-performance liquid chromatography-tandem mass spectrometry method for determining the ecdysone in the haemolymph of Macrobrachium nipponense in the late pre-moult stage, so as to solve the problems existing in the above-mentioned prior art. After validation, the determination method provided by the present invention reaches a lower limit of quantification (LLOQ) of 0.01 ng / mL, and this present invention provides a new method for determining the ecdysone in the haemolymph of Macrobrachium nipponense in the late pre-moult stage. To achieve the purpose above, the present invention provides the following scheme: the present invention provides a high-performance liquid chromatography-tandem mass spectrometry method for determining the ecdysone in the haemolymph of Macrobrachium nipponense in the late pre-moult stage, including the following steps: aliquoting 0.2-0.4 mL of haemolymph from Macrobrachium nipponense in the late pre- moult stage, and performing the first centrifugation to obtain the supernatant; and adding the stabilizer to the supernatant, then adding the methanol and tolbutamide, vortex- mixing uniformly, adding the acetonitrile, shaking, performing centrifugation again to obtain the supernatant, and conducting the determination using high-performance liquid chromatography- tandem mass spectrometry. The conditions of the high-performance liquid chromatography are as follows: the injection volume is 5 uL; the column temperature is 35°C; the mobile phase A is 0.5% acetic acid; the mobile phase B is acetonitrile; the flow rate is 0.3 mL / min, and a gradient elution is performed. The conditions of the mass spectrometry are as follows: electrospray ionization source, positive ion detection, and multiple reaction monitoring mode; the curtain gas is 35.0 kPa; the ion source temperature is 450°C; the collision gas is 9.0 kPa, and the ion source spray voltage is 5500 V. Optionally, the conditions of the first centrifugation are 4°C, 12000 rpm for 15 min. Optionally, the volume ratio of the supernatant, the stabilizer, the methanol, the tolbutamide and the acetonitrile is 20:5:2:2:51. Optionally, the stabilizer is a phenylthiourea solution. Optionally, the phenylthiourea solution is prepared with 80% methanol as the solvent; the concentration of the phenylthiourea solution is 10 mM. Optionally, the conditions of the centrifugation again are 4°C, 13000 rpm for 5 min. Optionally, the conditions of the gradient elution are as follows: 0-0.5 min, 20%B; 05-28 min, 20%-98%B; 2.8-3.8 min, 98%B; 3.8-4.0 min, 98%-20%B; and 4.0-5.0 min, 20%B. Optionally, in the mass spectrometry, the monitoring ion channels for quantitative analysis are as follows: ecdysone, m / z: 481.4>371.1, collision voltage 20 V, and clustering voltage 10 V; tolbutamide is used as the internal standard, m / z: 271.0>91.0, collision voltage is 20 V, and declustering voltage is 10 V; the monitoring ion channel for qualitative analysis is as follows: ecdysone, m / z: 481 .4>445.1, collision voltage is 20 V, and declustering voltage is 10 V. The present invention also provides an application of the above method in determining the ecdysone in the haemolymph of Macrobrachium nipponense in the late pre-moult stage. The present invention discloses the following technical effects. The present invention provides an efficient, sensitive and accurate sample pretreatment and determination method for ecdysone in haemolymph of Macrobrachium nipponense in the late pre-moult stage. The determination method includes the steps of aliquoting 0.2-0.4 mL of haemolymph from Macrobrachium nipponense in the late pre-moult stage, then centrifuging to obtain the supernatant; adding a stabilizer to the supernatant, then adding the methanol and tolbutamide, and vortex-mixing uniformly; adding the acetonitrile, shaking, centrifuging again to obtain the supernatant; finally performing the supernatant for the high-performance liquid chromatography-tandem mass spectrometry detection. After validation, the lower limit of quantification (LLOQ) of the determination method provided by the present invention is 0.01 ng / mL, and the present invention provides a new method for determining the ecdysone in the haemolymph of Macrobrachium nipponense in the late pre-moult stage. BRIEF DESCRIPTION OF THE FIGURES To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the figures needed in the embodiments will be briefly introduced below. Obviously, the figures attached in the following description are only some embodiments of the present invention, and for those of ordinary skill in the art, other figures may be obtained according to these figures without creative work. Fig. 1 is the standard curve in Embodiment 1; Fig. 2 is the detection results of the sample under the internal standard channel in Embodiment 2; Fig. 3 is the detection results of the sample under the qualitative analysis ion channel in Embodiment 2; Fig. 4 is the detection results of the sample under the quantitative analysis ion channel in Embodiment 2. DESCRIPTION OF THE INVENTION The various exemplary embodiments of the present invention will now be described in details. The detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features and embodiments of the present invention. It should be understood that the terms used in the present invention are only for describing specific embodiments and are not used to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or intermediate value within a stated range and any other stated value or intermediate value within the stated range is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail. Without departing from the scope or spirit of the present invention, various modifications and changes may be made to the specific embodiments of the present invention described in this specification, which will be obvious to those skilled in the art. Other embodiments obtained from the description of the present invention will be obvious to the skilled person. The specification and examples of the present invention are only exemplary. Regarding the terms such as "including", "comprising", "having" and "containing" used herein, they are all open-ended terms, meaning including but not limited to. Embodiment 1 1. Solution preparation 1.1 Preparation of stabilizer phenylthiourea solution Adding the stabilizer phenylthiourea to the extracted prawn haemolymph may prevent the oxidation of the analyte. In the present invention, 80% methanol is used as the solvent, and chromatographically pure phenylthiourea (MCE CAS No. 103-85-5) is used as the solute to prepare a 10 mM phenylthiourea solution as the stabilizer for later use. Store it in a refrigerator at 4°C. 1.2 Preparation of standard solution Weigh 5 mg of 20E reference standard with an electronic balance, and dissolve it with the methanol and dilute it to 5 mL to prepare a 1 mg / mL standard stock solution, which is stored in a refrigerator at 4°C for later use. When preparing the working curve, the stock solution is diluted with the methanol into standard solutions at different concentrations, and the internal standard tolbutamide is added to each standard solution to make its contents reach 500 ng / mL. Tolbutamide is selected as the internal standard in this method because the tolbutamide is easy to be protonated, being suitable for positive ion mode with a moderate retention time. 2. Haemolymph preparation and pretreatment 2.1 Haemolymph preparation Referring to the method of Nan Wu et al. (2025), Macrobrachium nipponense in the late pre-moult stage is selected. A 1 mL syringe is used to extract 0.2-0.4 mL of haemolymph from the pericardial cavity of Macrobrachium nipponense; the haemolymph sample is centrifuged at 12000 rpm for 15 min at 4°C quickly, then the supernatant is taken. 2.2 Haemolymph pretreatment (1) 20 uL of the supernatant is quickly put into a sampling tube containing 5 uL of phenylthiourea solution (10 mM), sealed with a parafilm, and stored at -80°C. (2) 2 uL of methanol and 2 uL of tolbutamide (500 ng / mL) are added to each tube of serum sample, vortex-mixing for 30 s; then add 51 uL of acetonitrile, shake the mixture for 5 min, and centrifuge again at 13000 rpm for 5 min (4°C); finally extract the supernatant, and inject it directly into the LC-MS / MS for determination. According to the ratio of the response intensity of the characteristic ion 20E to that of the internal standard tolbutamide, the content of 20E in the haemolymph is calculated using the working curve method with the internal standard addition. 3. LC-MS / MS detection conditions Chromatography-mass spectrometry conditions: CORTECS C18 chromatographic column (100 um><2.1 mm><1.6 um, Waters Corporation, USA) is used; the injection volume is 5 uL and the column temperature is 35°C; the mobile phase A is 0.5% acetic acid; while the mobile phase B is acetonitrile; the flow rate is 0.3 mL / min; the gradient elution conditions are as follows: 0-0.5 min, 20% B; 05-28 min, 20%-98% B; 2.8-3.8 min, 98% B; 3.8-4.0 min, 98%-20% B; and 4.0- 5.0 min, 20% B. Ionization mode: electrospray ionization (ESI) source, positive ion detection, multiple reaction monitoring (MRM) mode; ion source parameters: curtain gas is 35.0 kPa, ion source temperature is 450°C, collision gas is 9.0 kPa; and ion source spray voltage is 5500 V. Monitoring ion channels for quantitative analysis: 20E, m / z: 481.4>371.1, collision voltage is 20 V, and declustering voltage is 10 V. Tolbutamide (internal standard), m / z: 271.0>91.0, collision voltage is 20 V, and declustering voltage is 10 V. Monitoring ion channel for qualitative analysis: 20E, m / z: 481.4>445.1, collision voltage is 20 V, and declustering voltage is 10 V. 4. Establishment of the standard curve (1) Blank matrix: 10% BSA is used as the blank matrix; (2) preparation of the gradient standard solutions: preparing standard solutions containing 2, 5, 10, 25, 50, 100, and 200 ng / mL of 20E respectively (linear range: 2-200 ng / mL); (3) aliquot 20 uL of the blank matrix, add 2 uL of the methanol, 2 uL of the 20E standard solution and 2 pL of the tolbutamide (500 ng / mL), vortex-mixing for 30 s; and then add 51 uL of the acetonitrile, shake for 5 min, and centrifuge the mix at 13000 rpm for 5 min; extract the supernatant, and inject it into the instrument directly. The results are shown in Table 1. The correlation coefficient R2 of the equation of the standard curve equation is 0.9986; and the limit of detection is 0.01 ng / mL. Table 1 Embodiment 2 The method in Embodiment 1 is used to determine the ecdysone content in 12 samples of the haemolymph of Macrobrachium nipponense in the late pre-moult stage from 6 different batches (numbered 1 - 12). The results show that the method provided by the present invention could stably detect the ecdysone content in haemolymph of Macrobrachium nipponense (Table 2, Figs. 2 - 4). Table 2 ___- ___- ___ The above-mentioned embodiments are only for describing the preferred mode of the present invention, and are not intended to limit the scope of the present invention. Under the premise of not departing from the design spirit of the present invention, various modifications and improvements made by those of ordinary skill in the art to the technical solution of the present invention shall fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for high-pressure liquid chromatography-tandem mass spectrometry determines levels of ecdysone in the hemolymph of Macrobrachium nipponense in the late stage for- based on molting, which method comprises the following steps: the collection of 0.2 - 0.4 ml of hemolymph from Macrobrachium nipponense in the late sta- dium prior to molting, and performing the first centrifugation to upper- to obtain standing liquid; adding the stabilizer to the liquid above; subsequently the addition- mix methanol and tolbutamide evenly with a swirl stirrer; the addition of acetonitrile, shake and centrifuge again to the supernatant to acquire; performing the determination using tandem high-pressure liquid chromatography mass spectrometry, where the conditions of high-pressure liquid chromatography are as follows: the injection volume is 5 µl; the column temperature is 35 °C; the mobile phase A is 0.5% acetic acid, while the mobile phase B is acetonitrile; the flow rate is 0.3 ml / min carries and the gradient elution is performed; and the conditions of mass spectrometry are as follows: Electrospray ionization source, positive ion detection and multiple reaction monitoring torsion mode; the curtain gas is 35.0 kPa, the ion source temperature is 450°C; the collision gas is 9.0 kPa; and the ion source spray voltage is 5500 V.

2. The procedure according to claim 1, whereby the conditions of the first centrifugation entail 4°C, 12,000 rpm for 15 minutes.

3. The method according to claim 1, whereby the volume ratio of the above liquid substance, stabilizer, methanol, tolbutamide and acetonitrile amounts to 20:5:2:2:

51.

4. The method according to claim 1, where the stabilizer is a phenylthiourea solution.

5. The method according to claim 4, whereby the phenylthiourea solution is prepared with 80% methanol as solvent and the concentration of the phenylthiourea solution is 10 mM.

6. The procedure according to claim 1, whereby the conditions for centrifugation are again be 4°C, 13000 rpm for 5 minutes.

7. The method according to claim 1, where the conditions of the gradient elution are: 0 - 0.5 min, 20% of B; 0.5 - 2.8 min, 20% - 98% of B; 2.8 - 3.8 min, 98% of B; 3.8 - 4.0 min, 98% - 20% of B; and 4.0 - 5.0 min, 20% of B. method according to conclusion 1, whereby in the mass spectrometry the monitoring ion cation The values ​​for quantitative analysis are: ecdyson, m / z: 481.4 > 371.1, impact stress is 20 and declustering voltage is 10 V; tolbutamide is used as internal standard, m / z: 271.0 > 91.0, collision voltage is 20 V, declustering voltage is 10 V; and the monitoring- ion channel for qualitative analysis is: ecdyson, m / z: 481 4 > 445.1, collision voltage is V and the declustering voltage is 10 V. An application of the method according to any of claims 1 - 8 at the detecting ecdysone in the hemolymph of Macrobrachium nipponense in the late sta- dium preceding molting. Fig. 1

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