Application of Mass Frontier Fragment Ion Search Function (FISh) in the Identification of Strychnine Metabolites

Zhou Zhejiang

Thermo Fisher Scientific (China) Co., Ltd.

Keywords: fragment ion retrieval, FISh, metabolite, LTQ Orbitrap

1. Introduction

Strychnos chinensis is a dry mature seed of Strychnos nux-Vomical L., whose main active ingredient is alkaloid, which has the effects of passing through meridians, reducing swelling and pain, and relieving pain. It is a commonly used drug in traditional Chinese medicine. The main active ingredient, strychnine, also known as saponin, is a central nervous system stimulant. It can selectively excite the spinal cord and enhance the tension of skeletal muscle. It is clinically used for hemiplegia, sputum and injection of streptomycin. Skeletal muscle relaxation, amblyopia, etc. and barbiturate poisoning. However, such ingredients are highly toxic and must be strictly controlled as a pharmaceutical. In addition, the addition of strychnine feed bait can be used to eliminate rodent pests such as rats. However, the residues of these animals are transmitted through the food chain, affecting the entire ecosystem. Therefore, it is necessary to establish a rapid, sensitive and accurate method to detect such drugs, and provide reliable analytical tools and data for clinical drugs, environmental protection, and judicial departments.

In this paper, high performance liquid chromatography-ion trap electrostatic field orbitrap high-resolution mass spectrometry was used to comprehensively analyze strychnine and its metabolites, and the target compound was subjected to MSn multi-stage fragmentation by linear ion trap technique. Orbitrap electrostatic field orbital technology acquires high resolution mass spectral information for both primary and multistage mass spectrometry. Mass Frontier 7.0 structural analysis software has a rich database of cracking mechanisms for accurate fragmentation. Its unique Fragment Ion Search (FISh) function combines the common drug and metabolite shared fragments and metabolic pathways. Comprehensive information on metabolites can be detected.

2. Experimental conditions

2.1 liquid chromatography conditions:

Instrument: Accela HPLC system liquid chromatograph

Column: Thermo Scientific Hypersil Gold C18 (100 × 2.1 mm, 1.9 μm) mobile phase: A is aqueous phase: 0.05% formic acid 2 mM aqueous solution of ammonium formate, B is organic phase: 0.05% formic acid 2 mM ammonium formate acetonitrile solution

Gradient conditions:

Flow rate 0.2 mL/min, injection volume: 5 μL, column temperature 30 °C

2.2 Mass spectrometry conditions

Instrument: Thermo LTQ Orbitrap XL

Mass spectrometry ion source parameters: ESI Spray voltage: +4.0 KV; Sheath Gas Pressure: 35 arb; Aux Gas Pressure: 8 arb; Capillary Temp: 275 ° C; Scan mode: Full MS with data-dependent MSn; CID (collision energy 35%) , activation time 30 ms); Scan range: m/z 200~700.

2.3 sample preparation

The male rats were orally administered at a dose of 5 mg/kg. After 6 hours, the urine samples of the rats were taken and purified by SPE solid phase extraction. After appropriate dilution, the samples were analyzed.

3. Results

3.1 representative component search

The representative components in the mass spectrometry data were searched using Mass Frontier's Joint Components Detection algorithm. Based on the statistical analysis of the maximum values ​​of all ion profile maps, by extracting the peak abundance profiles of each mass spectrum, a “pure” mass spectrum tree of the strychnine parent drug and other possible metabolites can be created and the corresponding peak shape is generated (Fig. 1 ).

Figure 1. Joint Components Detection algorithm to find representative components

Analysis of the structure of 3.2 sinning fragments

After obtaining the Shih Ning “pure” mass spectrometer tree, the Mass Frontier Fragmentation Library can be used to quickly locate the strychnization fragment structure, which covers almost all published literature, so it is quickly resolved. At the same time as the fragmentation, a complete fragmentation path can be obtained, and the literature referenced can be conveniently called to ensure the accuracy of fragmentation analysis (Fig. 2).

Figure 2. The Fragmentation Library is attributed to the strychnine fragment and the cleavage pathway.

The complete cracking mechanism of 3.3

The linear ion trap of LTQ Orbitrap XL has higher ion capacity than traditional three-dimensional ion trap, so it has excellent multi-stage mass spectrometry capability, can efficiently capture precursor ions for fragmentation, and obtain highly sensitive fragment ion information. In the process of collecting the precursor ions of the sinensis, the four-stage effective mass spectrometry information (MS4) was obtained. Through multi-stage mass spectrometry analysis, the complete cleavage mechanism of strychnine was summarized, which provided valuable information for the retrieval of unknown metabolites (Fig. 3).

Figure 3. Strychnine MS4 ion tree and its complete cleavage mechanism

3.4 fragment ion retrieval (Fragment Ion Search, FISh) workflow

The fragment ion retrieval function utilizes the structural correlation between the parent drug and the metabolite, and uses the characteristic mass spectrometry fragment of the parent drug to combine the metabolic pathway to complete the retrieval of the unknown metabolite. Using this function, after completing the mass spectrometry cleavage law of the prasenin parent drug, the characteristic fragments were introduced into the FISh module, and the possible metabolic pathways were selected, and the representative components were searched to retrieve the comprehensive information of the unknown metabolites. (Figure 4).

Figure 4. Fragment ion retrieval of the unknown metabolite workflow

3.5 fragment ion retrieval (Fragment Ion Search, FISh) results

Obtain accurate and comprehensive unknown metabolite information through fragment ion retrieval, including unknown metabolite chromatographic information, primary multi-stage mass spectrometry data, unknown metabolite metabolic pathways, etc., while FISh interpretation function can also pass the parent drug and metabolite multi-stage mass spectrometry. The alignment between the information further speculates on the metabolic site (Fig. 5).

Figure 5. Unknown metabolite search and identification results of strychnine

Taking the unknown metabolite of RT 11.5 min as an example, the unknown metabolite was obtained as a hydroxylated metabolite by FISh calculation, and the characteristic fragments such as 264.1, 280.0, 323.1, 334.2, 238.0 m/z were interpreted by FISh, and it was inferred. The metabolite is 16-Hydroxystrychnine (Figure 6).

Figure 6. Strychnine metabolite 16-Hydroxystrychnine

Using the same treatment method, a total of 26 metabolites of strychnine were detected, including 22 metabolites in one phase and 4 metabolites in the second phase, as shown in Table 1.

Table 1. Identification results of strychnine metabolites

4. Summary:

4.1 The Thermo LTQ Orbitrap XL mass spectrometer combines a linear ion trap with high-resolution electrostatic field orbital Orbitrap detection technology to deliver superior performance and versatility. The higher ion capacity of the linear ion trap can efficiently capture the precursor ions for multi-stage fragmentation and obtain high-quality multi-stage mass spectrometry. In this experiment, the complete cracking mechanism of strychnine is analyzed by linear ion trap stepwise cracking. Orbitrap technology has the ability of high resolution and high precision (HR/AM). In this experiment, the quality deviation of the mineralization product of Shishi Ning is <3 ppm, which can effectively eliminate the interference and obtain the accurate elemental composition of the metabolite.

4.2 Mass Frontier is Thermo's professional structure analysis software. With its rich Fragmentation Library, it can quickly guess and assign the multi-level fragment structure of Strychn, which can be obtained while quickly parsing the fragments. Complete fragmentation route, and easy to call to view the literature cited, to ensure the accuracy of fragmentation analysis. There is a structural correlation between the parent drug and the metabolite. The mass spectrometry may generate a common fragment after fragmentation. The fragment ion search (FISh) function combines this correlation with the possible metabolic pathways organically, from the complex matrix sample mass spectrometry signal. The accurate screening of unknown metabolite information, through the comparison of the parent drug and metabolite multi-stage mass spectrometry information, FISh interpretation function can further speculate the metabolic site.