Method for identifying sibutramine compounds and recognizing new derivatives of sibutramine by using UPLC-QTOF-MS / MS technology

Abstract

The present application relates to the method for identifying sibutramine compounds and recognizing new derivatives of sibutramine compounds by UPLC-QTOF-MS / MS technology, which comprises sample preparation, detection analysis and identification, and structure deduction steps.Different from the prior art, the application discloses the classification diagnosis ion of sibutramine compounds, the specific diagnosis ion of monochloro-substituted derivatives is m / z 125.01, the specific diagnosis ion of dichloro-substituted derivatives is m / z 158.97, and the two characteristic ions can be used as the core basis for rapid screening, classification identification and structure analysis of the compounds.

Description

Technical Field

[0001] This invention relates to UPLC-QTOF-MS / MS technology, and particularly to a method for identifying sibutramine-like compounds and recognizing novel derivatives of sibutramine-like compounds. Background Technology

[0002] With the continuous improvement of public health awareness, the demand for weight management is growing rapidly, leading to the rapid expansion of the market for weight-loss foods, traditional Chinese medicines, and health foods. In pursuit of rapid weight loss, some unscrupulous merchants illegally add prohibited chemicals such as sibutramine and its derivatives to their products. These additives lack systematic safety and toxicological evaluation, have no standardized dosage control, and are prone to structural modifications and variants, making their illegal addition highly concealed. Long-term intake can easily cause serious health hazards such as cardiovascular damage and liver and kidney failure, posing a significant threat to public food and drug safety. Therefore, developing efficient, accurate, and comprehensive detection technologies for novel derivatives has become an urgent and practical need for food and drug safety supervision.

[0003] Currently, screening for illegal additives in weight-loss products still mainly relies on HPLC and conventional LC-MS / MS. Among them, high-performance liquid chromatography has problems such as cumbersome pretreatment procedures, significant sample matrix interference, insufficient sensitivity, and limited detection throughput; traditional liquid chromatography-tandem mass spectrometry is mostly targeted detection, highly dependent on standard references and existing databases, with a fixed screening range, making it difficult to identify structurally modified novel derivatives and unknown illegally added ingredients.

[0004] Ultra-high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF-MS / MS) has advantages such as high separation efficiency, excellent sensitivity, and rich secondary mass spectrometry information, making it particularly effective in the qualitative identification of unknown substances in complex matrices. However, existing detection schemes mostly rely on standard library matching and lack targeted data post-processing methods. They also have insufficient ability to identify novel sibutramine derivatives not included in the database, failing to meet the requirements of broad-spectrum, high-throughput routine regulatory screening. Summary of the Invention

[0005] In view of the above problems, this application provides a method for identifying sibutramine compounds and recognizing novel sibutramine derivatives using UPLC-QTOF-MS / MS technology.

[0006] The first aspect of this application provides a method for identifying sibutramine-like compounds based on UPLC-QTOF-MS / MS technology, comprising the following steps:

[0007] S1 Sample preparation: Weigh 1 g of the compound to be tested, add 100 ml of methanol, sonicate and filter to obtain the sample.

[0008] S2 Detection and Analysis: The chemical components in the sample were detected and analyzed by ultra-high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF-MS / MS).

[0009] The chromatographic conditions were as follows: C18 column, binary mobile phase A consisting of 0.1 wt% formic acid aqueous solution and phase B consisting of methanol, flow rate of 0.3 mL / min, column temperature of 30℃, and injection volume of 2 μL.

[0010] Mass spectrometry conditions were as follows: ionization was performed using an electrospray ionization (ESI) source, and IDA detection was performed in positive ion mode; spray gas: 50 psi, auxiliary heating gas: 55 psi, curtain gas: 35 psi, ionization voltage (IS): 5500 V; MS 1 Scan range: m / z 50~1000 Da, MS 1 - Declustering voltage DP: 50V, MS 1 - Collision energy CE: 10V; MS 2 -DP:50V, MS 2 -CE± Collision Energy Diffusion (CES): 40±20V, MS 2 Scan range: m / z 30~1000Da;

[0011] Identification of S3 Sibutramine-like compounds

[0012] The total ion chromatogram of the sample collected in positive ion mode was extracted using analysis software; sibutramine-like compounds were screened using m / z 125.01 or / and m / z 158.97 as screening targets.

[0013] Unlike existing technologies, this application specifies that in UPLC-QTOF-MS / MS technology, the specific diagnostic ions for sibutramine-like compounds are: m / z 125.01 for monochloro-substituted derivatives and m / z 158.97 for dichloro-substituted derivatives. These two characteristic ions can serve as the core basis for rapid screening, classification, identification, and structural analysis of these compounds.

[0014] Furthermore, the elution gradient program of the circulating phase is as follows: 0-3 min, 80% A; 3-8 min, 80% A; 8-15 min, 25% A; 15-23 min, 25% A; 23-27 min, 2% A; 27-28 min, 2% A; 28-32 min, 80% A.

[0015] Furthermore, the C18 chromatographic column has dimensions of 2.1 mm × 100 mm and a diameter of 1.8 μm.

[0016] Furthermore, in step S3, if m / z 125.01 or / and m / z 158.97 are present, it indicates that the compound to be tested contains sibutramine-like compounds.

[0017] The second aspect of this application provides a method for deducing novel derivatives of sibutramine compounds based on UPLC-QTOF-MS / MS technology, comprising the following steps:

[0018] S1 Sample preparation: Weigh 1 g of the compound to be tested, add 100 ml of methanol, sonicate and filter to obtain the sample.

[0019] S2 Detection and Analysis: The chemical components in the sample were detected and analyzed by ultra-high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF-MS / MS).

[0020] The chromatographic conditions were as follows: C18 column, binary mobile phase A consisting of 0.1 wt% formic acid aqueous solution and phase B consisting of methanol, flow rate of 0.3 mL / min, column temperature of 30℃, and injection volume of 2 μL.

[0021] Mass spectrometry conditions were as follows: ionization was performed using an electrospray ionization (ESI) source, and IDA detection was performed in positive ion mode; spray gas: 50 psi, auxiliary heating gas: 55 psi, curtain gas: 35 psi, ionization voltage (IS): 5500 V; MS 1 Scan range: m / z 50~1000 Da, MS 1 - Declustering voltage DP: 50V, MS 1 - Collision energy CE: 10V; MS 2 -DP:50V, MS 2 -CE± Collision Energy Diffusion (CES): 40±20V, MS 2 Scan range: m / z 30~1000Da;

[0022] S3 Targeted Screening and Structural Derivation

[0023] S31 uses analysis software to extract the total ion chromatogram of the sample collected in positive ion mode;

[0024] S32 extracts chromatographic peaks containing fragments of m / z 125.01 or / and m / z 158.97, corresponding to the m / z value and secondary spectrum of the parent ion;

[0025] S33 uses the m / z ratio and distribution pattern of fragments on the secondary spectrum to deduce the specific chemical structure of the parent ion, thus obtaining candidate compounds.

[0026] Furthermore, step S33 includes checking whether the fragments on the secondary spectrum contain low-quality fragments of m / z 46.06, m / z 32.04, m / z 60.08, and m / z 91.05, and deriving the corresponding structures.

[0027] Unlike existing technologies, this application identifies m / z 125.01 and m / z 158.97 as core diagnostic feature fragment ions and low-mass differential fragments. By comparing fragments, novel sibutramine derivatives that are not included in the database or regulatory catalog can be deduced and identified. This method is practical and has the ability to identify unknown variants, filling the gap in non-targeted rapid detection technology for this type of substance.

[0028] Furthermore, in step S33, the structure of the compound is determined by the quality of the parent ion and fragments.

[0029] Furthermore, between steps S32 and S33, there is also step S30, which compares the secondary spectrum and retention time of the parent ion with sibutramine-like compound standards to check whether they are compatible.

[0030] Furthermore, the elution gradient program of the circulating phase is as follows: 0-3 min, 80% A; 3-8 min, 80% A; 8-15 min, 25% A; 15-23 min, 25% A; 23-27 min, 2% A; 27-28 min, 2% A; 28-32 min, 80% A.

[0031] Furthermore, the C18 column has dimensions of 2.1 mm × 100 mm and a diameter of 1.8 μm.

[0032] The above description of the invention is merely an overview of the technical solution of this application. In order to enable those skilled in the art to better understand the technical solution of this application and to implement it based on the textual description, and to make the above-mentioned objectives and other objectives, features and advantages of this application easier to understand, the following description is provided in conjunction with the specific embodiments and accompanying drawings of this application. Attached Figure Description

[0033] The accompanying drawings are only used to illustrate the principles, implementation methods, applications, features, and effects of specific embodiments of this application and other related content, and should not be considered as limitations on this application.

[0034] In the accompanying drawings of the instruction manual:

[0035] Figure 1 This is the second-order mass spectrum of sibutramine.

[0036] Figure 2 This is the secondary mass spectrum of N-monodemethylsibutramine.

[0037] Figure 3 This is the secondary mass spectrum of N,N-bisdemethylsibutramine.

[0038] Figure 4 This is the secondary mass spectrum of homositol.

[0039] Figure 5 This is the secondary mass spectrum of benzylsibutramine.

[0040] Figure 6 This is the secondary mass spectrum of chlorinated sibutramine.

[0041] Figure 7 This is a secondary mass spectrum of the dichloroisoamyl substitute for sibutramine.

[0042] Figure 8 This is a diagram showing the inferred fragmentation pathway of sibutramine via mass spectrometry.

[0043] Figure 9 This is the total ion chromatogram of the sample in positive ion mode.

[0044] Figure 10 The filtered spectra are for feature fragments with m / z 125.01 and m / z 158.97.

[0045] Figure 11 The image shows the secondary mass spectrum of component 1 (retention time 8.535 min).

[0046] Figure 12 The image shows the secondary mass spectrum of component 2 (retention time 8.630 min).

[0047] Figure 13 The image shows the secondary mass spectrum of component 3 (retention time 8.331 min).

[0048] Figure 14 The image shows the secondary mass spectrum of component 4 (retention time 25.477 min).

[0049] Figure 15 The image shows the secondary mass spectrum of component 5 (retention time 8.230 min).

[0050] Figure 16 The image shows the secondary mass spectrum of component 6 (retention time 9.856 min). Detailed Implementation

[0051] To illustrate the possible application scenarios, technical principles, implementable specific solutions, and achievable objectives and effects of this application in detail, the following description, in conjunction with the listed specific embodiments and accompanying drawings, provides a detailed explanation. The embodiments described herein are merely illustrative of the technical solutions of this application and are therefore intended to limit the scope of protection of this application.

[0052] In this document, the term "embodiment" means that a specific feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The term "embodiment" appearing in various places throughout the specification does not necessarily refer to the same embodiment, nor does it specifically limit its independence or connection with other embodiments. In principle, in this application, as long as there are no technical contradictions or conflicts, the technical features mentioned in each embodiment can be combined in any way to form corresponding implementable technical solutions.

[0053] Unless otherwise defined, the technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the use of related terms herein is merely for the purpose of describing particular embodiments and is not intended to limit this application.

[0054] In the description of this application, the term "and / or" is used to describe the logical relationship between objects, indicating that three relationships can exist. For example, A and / or B means: A exists, B exists, and A and B exist simultaneously. Additionally, the character " / " in this document generally indicates that the preceding and following objects have an "or" logical relationship.

[0055] In this application, terms such as “first” and “second” are used only to distinguish one entity or operation from another, and do not necessarily require or imply any actual quantity, hierarchy or order relationship between these entities or operations.

[0056] Unless otherwise specified, the use of terms such as “comprising,” “including,” “having,” or other similar expressions in this application is intended to cover non-exclusive inclusion, which does not exclude the presence of additional elements in a process, method, or product that includes the stated elements, such that a process, method, or product that includes a list of elements may include not only those defined elements but also other elements not expressly listed, or elements inherent to such a process, method, or product.

[0057] In this application, expressions such as "greater than", "less than", and "exceeding" are understood to exclude the stated number; expressions such as "above", "below", and "within" are understood to include the stated number. Furthermore, in the description of the embodiments of this application, "multiple" means two or more (including two), and similar expressions related to "multiple" are also understood in this way, such as "multiple groups" and "multiple times", unless otherwise explicitly specified.

[0058] I. Feature Fragment Acquisition:

[0059] 1. Selection of UPLC-QTOF-MS / MS ionization mode

[0060] 1.1 Molecular structure and physicochemical properties of sibutramine

[0061] Sibutramine hydrochloride, chemically named (±)N-{1-[1-(4-chlorophenyl)cyclobutyl]-3-methylbutyl}-N,N-dimethylamine hydrochloride monohydrate, has a molecule composed of a para-chloro-substituted benzene ring, a rigid cyclobutane core, and a terminal tertiary amine basic side chain. The nitrogen atom of the tertiary amine readily protonates under acidic conditions, forming a stable [M+H] group. + Therefore, this study used 0.1% formic acid water as the mobile phase to improve ionization efficiency and signal stability.

[0062] 1.2 Electrospray positive ion (ESI) + Detection mode selection criteria

[0063] The positive ion mode was chosen in this study for two main reasons: First, sibutramine and its derivatives contain basic tertiary amine groups, which can be efficiently protonated under acidic conditions, resulting in strong ion response and high sensitivity. In contrast, the negative ion mode lacks effective ionization sites and produces extremely weak signals, failing to meet the requirements for trace detection. Second, the protonated parent ion is highly stable and can generate characteristic fragments with strong regularity and good reproducibility under collision-induced dissociation. The structurally modified derivatives still retain the core fragmentation pathway, providing a reliable basis for non-targeted screening and structural deduction.

[0064] 2. Mass spectrometric fragmentation patterns of sibutramine and its derivatives

[0065] 2.1 Materials and Instruments

[0066] 2.1.1 Standards: N,N-bis-demethylsibutramine (batch number: 520001-201902), N-monodemethylsibutramine (batch number: 520002-201902), sibutramine (batch number: 2424033), benzylsibutramine (batch number: 2418629), homosibutramine (batch number: H3001355), chlorosibutramine (batch number: CDDM-C370150), and dichloroisoamyl substitute for sibutramine (batch number: 2580217).

[0067] 2.1.2 The test samples acetonitrile, methanol, and formic acid were all of chromatographic grade, other chemical reagents were of analytical grade, and water was self-made ultrapure water.

[0068] 2.1.3 UPLC-QTOF-MS (AB X500R, USA), electrospray ionization source (ESI), SCIEX OS Version 1.7.0.36606 (AB SCIEX); XS-205 electronic balance (Mettler); ultrasonic cleaner (250 W power, 40 kHz operating frequency); KH20R high-speed refrigerated centrifuge (Hunan Kaida).

[0069] 2.2 Test Methods

[0070] 2.2.1 Ultra-high performance liquid chromatography conditions

[0071] Chromatographic column: Agient Eclipse Plus-C18 (2.1 mm × 100 mm, 1.8 μm); binary mobile phase: phase A was 0.1% formic acid, phase B was methanol. Flow rate: 0.3 mL / min, column temperature: 30 ℃, injection volume: 2 μL. The gradient elution program for the mobile phase is shown in Table 1.

[0072] Table 1. Gradient elution program for mobile phase

[0073]

[0074] 2.2.2 Mass Spectrometry Conditions

[0075] Ionization was performed using an electrospray ionization (ESI) source, and IDA detection was performed in positive ion mode; spray gas: 50 psi, auxiliary heating gas: 55 psi, curtain gas: 35 psi, ionization voltage (IS): 5500 V; MS 1 Scan range: m / z 50~1000 Da, MS 1 - Declustering voltage (DP): 50V, MS 1 - Collision energy (CE): 10V; MS 2 -DP:50V, MS 2 -CE± Collision Energy Dissipation (CES): 40±20V, MS 2 Scan range: m / z 30~1000Da;

[0076] 2.2.3 Preparation of standard solutions

[0077] Accurately weigh approximately 10 mg of each of the seven standards and place them separately in 10 mL volumetric flasks. Sonicate the solutions (250 W, 40 kHz) for 15 min, cool to room temperature, dilute to the mark, and mix well to obtain the standard stock solutions. Accurately measure an appropriate amount of each of the above standard stock solutions and place them in the same volumetric flask. Dilute with methanol to prepare a mixed standard solution of 10–200 µg / mL, and then gradually dilute with the initial mobile phase to a mixed standard solution with a concentration of 500 ng / mL.

[0078] 2.2.4 Test Results:

[0079] Electrospray positive ion (ESI) + In the detection mode, mass spectrometry analysis of seven sibutramine compounds and their derivatives was performed. The results showed that all target compounds specifically generated characteristic fragment ions at m / z 125.01 or m / z 158.97. The secondary mass spectra of each compound are shown below. Figure 1-7 The structural formulas and main ion fragments are shown in Table 2, and the predicted mass spectrometry fragmentation pathways are shown in Table 3. Figure 8 .

[0080] Table 2. Structural formulas and major ionic fragments of sibutramine and its derivatives under electrospray positive ion mode.

[0081]

[0082] The cleavage pathway of sibutramine is as follows: The parent ion first undergoes the cleavage of the dimethylamine group, generating two fragment ions with m / z 235.12 and m / z 46.06. The intermediate (m / z 235.12) can continue to cleave through two pathways: first, by removing isobutylene via a 1,3-hydrogen migration reaction, generating a stable fragment ion with m / z 179.06; second, by directly removing the isopentenene group, forming a four-membered ring positive ion intermediate with m / z 165.05. This four-membered ring structure further undergoes ring-opening cleavage, ultimately generating characteristic fragments with m / z 153.05, 139.03, and m / z 125.01, among which m / z 125.01 exhibits extremely high ionic stability.

[0083] The overall fragmentation mechanisms of monodemethylsibutramine, N,N-bisdemethylsibutramine, and homosibutramine are highly consistent with those of sibutramine, but specific differential fragments exist in the low-mass region. Specifically, N-monodemethylsibutramine can generate a monomethylamine characteristic fragment at m / z 32.04, homosibutramine can generate a characteristic fragment at m / z 60.08, and benzylsibutramine generates a stable benzyl characteristic fragment at m / z 91.05. Specific structures are shown in Table 2.

[0084] By relying on the presence of specific differential fragments in the low-quality region, homologues, demethylated metabolites, and structural modifications can be quickly distinguished from mixtures with similar structures, fundamentally avoiding misjudgments and false positives caused by similar molecular weights.

[0085] pass Figure 1-7 The secondary mass spectra revealed that for structurally modified derivatives with halogenated benzene rings and extended alkyl side chains, the fragmentation patterns further validated the conservation of the parent nucleus fragmentation pathway: the quasi-molecular ion and all characteristic fragment ions of the monochloro-substituted benzene ring derivative increased by 34 Da compared to the overall mass-to-charge ratio of sibutramine; dichloroisopentylsibutramine, as an alkyl-extended product of the chloro derivative, showed an increase of 14 Da in its quasi-molecular ion and characteristic fragment ions compared to the monochloro derivative, with the distribution of other fragment ions completely consistent with the fragmentation pattern. These results indicate that structural modifications to the sibutramine parent nucleus, such as benzene ring substitution, alkyl side chain modification, and demethylation, only alter the molecular weight of the compound and do not disrupt the core nucleus fragmentation mechanism; the neutral loss fragments exhibit high homology. Based on the mass difference and the fragmentation conservation principle, the modification type, substitution quantity, and site can be quickly determined, providing a direct basis for the structural deduction of novel unknown derivatives.

[0086] In summary, this study clarifies the diagnostic ions for sibutramine-like compounds: the specific diagnostic ion for monochloro-substituted derivatives is m / z 125.01, and the specific diagnostic ion for dichloro-substituted derivatives is m / z 158.97. These two characteristic ions can serve as the core basis for rapid screening, classification, identification, and structural analysis of these compounds.

[0087] Example

[0088] In this embodiment, the samples to be tested came from the Quzhou Municipal Institute for Food and Drug Control's random inspection samples, commissioned samples, consultation samples, or samples purchased online, totaling 25 batches of samples.

[0089] Acetonitrile, methanol, and formic acid were all of chromatographic grade, other chemical reagents were of analytical grade, and water was self-made ultrapure water.

[0090] Sample preparation: Weigh 0.1 g of the sample to be tested into a 10 ml volumetric flask, add 10 ml of methanol, shake well, and extract by ultrasonication for 20 min (power 250W, working frequency 40kHz). Filter, take an appropriate amount of the filtrate for instrumental analysis.

[0091] Ultra-high performance liquid chromatography conditions:

[0092] Chromatographic column: Agient Eclipse Plus-C18 (2.1 mm × 100 mm, 1.8 μm); binary mobile phase: Phase A was 0.1% formic acid, and Phase B was methanol. Flow rate: 0.3 mL / min, column temperature: 30 ℃, injection volume: 2 μL. The gradient elution program for the mobile phase is shown in Table 1.

[0093] Table 1. Gradient elution program for mobile phase

[0094]

[0095] Mass spectrometry conditions

[0096] Ionization was performed using an electrospray ionization (ESI) source, and IDA detection was performed in positive ion mode; spray gas: 50 psi, auxiliary heating gas: 55 psi, curtain gas: 35 psi, ionization voltage (IS): 5500 V; MS 1 Scan range: m / z 50~1000 Da, MS 1 - Declustering voltage (DP): 50V, MS 1 - Collision energy (CE): 10V; MS 2 -DP:50V, MS 2 -CE± Collision Energy Dissipation (CES): 40±20V, MS 2 Scan range: m / z 30~1000Da.

[0097] Non-targeted screening was performed on 25 batches of weight-loss samples, and one sample was suspected of containing an unknown sibutramine derivative. The total ion chromatogram of the sample in positive ion mode is shown below. Figure 9 Using the characteristic fragment with dichloro-substituted ion at m / z 158.97 and the characteristic fragment with monochloro-substituted ion at m / z 125.01 as filtration benchmarks, six sibutramine-related structural components were preliminarily identified from the total ion current of the sample. The filtration chromatograms are shown below. Figure 10 Secondary atlases are shown below. Figure 11-16 The details are as follows:

[0098] Component 1 (Retention time 8.535 min): The secondary spectrum and retention time of this compound were compared with those of the standard, confirming its structure as a dichloroisoamyl substitute for sibutramine, as shown in Formula I:

[0099]

[0100] Formula I

[0101] Component 2 (retention time 8.630 min): The exact parent ion m / z 314.14 exhibits a fragmentation pattern highly similar to that of dichloroisoamylsibutramine. The key difference lies in the absence of a fragment ion at m / z 46.06, indicating the absence of a dimethylamino group on its nitrogen atom. Combined with the 32 Da mass difference between the fragment ions, this compound is inferred to be an N-demethylated derivative of dichloroisoamylsibutramine, as shown in Formula II.

[0102] Formula II

[0103] Component 3 (retention time 8.331 min): Precise precursor ion m / z 344.15, characteristic fragment ion m / z 46.04 was detected, confirming retention of the dimethyl substituent on the nitrogen atom. Its precursor ion mass is 16 Da higher than that of dichloroisoamylsibutramine, and an m / z of 326.14 (corresponding to a dehydrated 18 Da fragment) was detected in the fragment. Based on these findings, the compound is determined to be an oxidized derivative of dichloroisoamylsibutramine, as shown in Formula III. Due to limitations in mass spectrometry detection principles, the specific substitution site of the oxygen atom cannot be precisely located at this time.

[0104]

[0105] Formula III

[0106] Component 4 (retention time 25.477 min): The exact parent ion m / z 526.15, and its fragment ion distribution pattern is completely matched with that of dichloroisoamylsibutramine. Based on the mass characteristics of the parent ion, it is inferred that its structure is a dimer formed by the dichloroisoamylsibutramine parent nucleus, as shown in Formula IV.

[0107] Formula IV

[0108] Component 5 (retention time 8.230 min): precise parent ion m / z 294.19, and characteristic fragment ion m / z 46. By comparing the mass differences of m / z 125.01 and 158.97, and m / z 139.03 and 172.99, this compound was identified as a monochlorosubstituted derivative of dichloroisoamylsibutramine, as shown in Formula V.

[0109]

[0110] Formula V

[0111] Component 6 (retention time 9.856 min): Precise parent ion m / z 364.27. Secondary fragment ion comparison analysis confirmed that its core structure has one less chlorine atom than that of dichloroisoamylsibutramine, and an isopentyl group is attached to the nitrogen atom. Combining the above mass spectrometry fragmentation characteristics and structural differences, its potential chemical structure was deduced, as shown in Formula VI.

[0112]

[0113] Style VI

[0114] In summary, relying on the two characteristic fragment ions at m / z 125.01 and m / z 158.97, as well as the characteristic fragments in the low-mass region, it is possible to quickly classify and infer the structure of unknown sibutramine derivatives in the absence of reference standards, providing an efficient and feasible technical approach for non-target screening of such illegal additives.

[0115] In summary, this method overcomes the technical limitations of traditional HPLC, such as low sensitivity and weak resistance to matrix interference, as well as the limitations of conventional LC-MS / MS, such as reliance on reference standards, limited targeted screening capabilities, and difficulty in identifying novel derivatives. It enables rapid identification of unknown structural analogs even without reference standards, effectively reducing the risk of missed detections and false positives. In practical application to 25 batches of weight-loss samples, one batch containing a sibutramine dichloroisoamyl substitute was detected, and five novel sibutramine derivatives not yet in the database or regulatory catalog were preliminarily identified. This verifies the method's practicality and ability to identify unknown variants, filling a gap in non-targeted rapid detection technology for such substances.

[0116] The technical system established in this study is adapted to the high-throughput screening needs of daily food and drug supervision, and can provide reliable technical support for risk prevention and control and law enforcement assessment of illegal additives in weight loss products. At the same time, the mass spectrometry fragmentation pattern analysis and characteristic fragment screening approach adopted can also provide a reference for the rapid identification of other prohibited drug derivatives, and help improve and upgrade the detection technology system for illegal additives in food and drugs.

[0117] It should be noted that although the above embodiments have been described in the text and drawings of this application, this should not limit the scope of patent protection of this application. Any technical solutions that are based on the essential concept of this application and utilize the content described in the text and drawings of this application, resulting in equivalent structural or procedural substitutions or modifications, as well as the direct or indirect application of the technical solutions of the above embodiments to other related technical fields, are all included within the scope of patent protection of this application.

Claims

1. A method for identifying sibutramine-like compounds based on UPLC-QTOF-MS / MS technology, characterized in that, Includes the following steps: S1 Sample preparation: Weigh 1 g of the compound to be tested, add 100 ml of methanol, sonicate and filter to obtain the sample; S2 Detection and Analysis: The chemical components in the sample were detected and analyzed by ultra-high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF-MS / MS). The chromatographic conditions were as follows: C18 column, binary mobile phase A consisting of 0.1 wt% formic acid aqueous solution and phase B consisting of methanol, flow rate of 0.3 mL / min, column temperature of 30℃, and injection volume of 2 μL. Mass spectrometry conditions were as follows: ionization was performed using an electrospray ionization (ESI) source, and IDA detection was performed in positive ion mode; spray gas: 50 psi, auxiliary heating gas: 55 psi, curtain gas: 35 psi, ionization voltage (IS): 5500 V; MS 1 Scan range: m / z 50~1000Da, MS 1 - Declustering voltage DP: 50V, MS 1 - Collision energy CE: 10V; MS 2 -DP:50V, MS 2 -CE± Collision Energy Diffusion (CES): 40±20V, MS 2 Scan range: m / z 30~1000Da; Identification of S3 Sibutramine-like compounds The total ion chromatogram of the sample collected in positive ion mode was extracted using analysis software; sibutramine-like compounds were screened using m / z 125.01 or / and m / z 158.97 as screening targets.

2. The identification method according to claim 1, characterized in that, The elution gradient program for the circulating phase is as follows: 0-3 min, 80% A; 3-8 min, 80% A; 8-15 min, 25% A; 15-23 min, 25% A; 23-27 min, 2% A; 27-28 min, 2% A; 28-32 min, 80% A.

3. The identification method according to claim 1, characterized in that, C18 chromatographic column, with dimensions of 2.1 mm × 100 mm and 1.8 μm.

4. The identification method according to claim 1, characterized in that, If m / z 125.01 or / and m / z 158.97 are present in step S3, it indicates that the compound to be tested contains sibutramine-like compounds.

5. A method for deducing novel derivatives of sibutramine compounds based on UPLC-QTOF-MS / MS technology, characterized in that, Includes the following steps: G1 Sample preparation: Weigh 1 g of the compound to be tested, add 100 ml of methanol, sonicate and filter to obtain the sample. G2 Detection and Analysis: The chemical components in the sample were detected and analyzed by ultra-high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF-MS / MS). The chromatographic conditions were as follows: C18 column, binary mobile phase A consisting of 0.1 wt% formic acid aqueous solution and phase B consisting of methanol, flow rate of 0.3 mL / min, column temperature of 30℃, and injection volume of 2 μL. Mass spectrometry conditions were as follows: ionization was performed using an electrospray ionization (ESI) source, and IDA detection was performed in positive ion mode; spray gas: 50 psi, auxiliary heating gas: 55 psi, curtain gas: 35 psi, ionization voltage (IS): 5500 V; MS 1 Scan range: m / z 50~1000Da, MS 1 - Declustering voltage DP: 50V, MS 1 - Collision energy CE: 10V; MS 2 -DP:50V, MS 2 -CE± Collision Energy Diffusion (CES): 40±20V, MS 2 Scan range: m / z 30~1000Da; G3 Targeted Screening and Structural Derivation Total ion chromatogram of the sample collected in G31 positive ion extraction mode; G32 extracts chromatographic peaks containing fragments of m / z 125.01 or / and m / z 158.97, and the corresponding m / z values ​​and secondary spectra of the parent ions; Based on the m / z and distribution patterns of fragments on the secondary spectrum, G33 deduced the specific chemical structure of the parent ion in reverse, thus obtaining candidate compounds.

6. The derivation method according to claim 5, characterized in that, The G33 step includes checking whether the fragments on the secondary spectrum contain low-quality fragments at m / z 46.06, m / z 32.04, m / z 60.08, and m / z 91.05, and deriving the corresponding structures.

7. The derivation method according to claim 5, characterized in that, In step G33, the structure of the compound is determined by the quality of the parent ion and fragments.

8. The derivation method according to claim 5, characterized in that, Between steps G32 and G33, there is also step G30, which compares the secondary spectrum and retention time of the parent ion with sibutramine-like compound standards to check whether they are consistent.

9. The derivation method according to claim 5, characterized in that, The elution gradient program for the circulating phase is as follows: 0-3 min, 80% A; 3-8 min, 80% A; 8-15 min, 25% A; 15-23 min, 25% A; 23-27 min, 2% A; 27-28 min, 2% A; 28-32 min, 80% A.

10. The derivation method according to claim 5, characterized in that, The C18 chromatographic column has dimensions of 2.1 mm × 100 mm and a diameter of 1.8 μm.

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