Method for detecting impurity I in naproxen

Abstract

The invention discloses a method for detecting an impurity I in naproxen. A naproxen raw material medicine and 1, 2, 4, 5-tetracyanobenzene (TCBB) are fully ground and evenly mixed, then a fluorescence signal is detected under the 365 nm ultraviolet irradiation condition, if green fluorescence is generated, it is indicated that the raw material medicine contains the impurity I, and otherwise, it is indicated that the raw material medicine does not contain the impurity I. The fluorescent probe TCNB and the naproxen impurity I can generate specific intermolecular charge transfer interaction, so that a fluorescent signal is generated, and the visual impurity inspection of the naproxen impurity I is realized. According to the method, the probe and the naproxen impurity I can interact with each other through a grinding method which is simple, convenient and easy to operate, so that bright green fluorescence is generated, 0.1% of limited impurities can still be detected, and meanwhile, the probe cannot interact with naproxen, so that the detection of the impurity I is not interfered. Therefore, the impurity detection method provided by the invention has low detection limit, good specificity and anti-interference capability, can effectively analyze the trace impurity I in naproxen without pretreatment, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a visual rapid detection method for impurity I (6-methoxy-2-acetnaphthalene) in naproxen raw material. Background Technology

[0002] Drug impurity testing is a core component in ensuring drug safety and efficacy. Drug impurities (including residual organic solvents, heavy metals, degradation products, and genotoxic impurities) can not only cause direct health risks such as allergic reactions and organ toxicity, but also indirectly reduce efficacy by altering drug stability, solubility, or metabolic pathways. For example, the global recall of valsartan in 2018 due to nitrosamine carcinogenic impurities highlighted the significant hazards of trace amounts of high-risk impurities. Drug impurity testing has significant and multifaceted importance, spanning the entire lifecycle of drug development, production, distribution, and use. Its core value lies in ensuring the safety, efficacy, and quality controllability of drugs. Due to the significant importance and research value of drug testing, the development of new strategies for drug impurity testing is receiving increasing attention. In recent years, how to construct new strategies for drug impurity testing and expand their practical applications has become a hot topic in drug analysis research.

[0003] Currently, the application of drug impurity testing still faces multiple bottlenecks. Chromatography-based methods account for over 70% of drug impurity detection applications, enabling the separation and detection of multiple impurities in samples. However, they suffer from high equipment investment and costs, require specialized operators, and consume large amounts of solvents. Chromatography-mass spectrometry (LC-MS / GC-MS) offers both high resolution and high sensitivity (detection limits down to ppb levels), but its long development cycle makes it difficult to meet rapid screening needs. Compared to traditional methods, fluorescence spectroscopy shows great potential in impurity control due to its high sensitivity, ease of operation, real-time performance, and dynamic response capabilities. However, its anti-interference ability for complex sample analysis is currently relatively limited.

[0004] Organic eutectics can generate new optical, electrical, and magnetic properties through synergistic interactions between monomers. In particular, intermolecular charge transfer (ICT) leads to a reduction in the band gap of the newly formed recombination state, resulting in a significant change in the optical signal. The significant difference in optical signal between ICT eutectics and monomers makes them a promising candidate for applications in drug analysis, amine detection, and chiral analysis. ICT does not require covalent interactions; it can be achieved solely through appropriate spatial interactions, giving this technique a significant advantage in its ease of operation.

[0005] Naproxen (S-NAP) is a nonsteroidal anti-inflammatory drug with anti-inflammatory, antipyretic, analgesic, and antirheumatic effects. 6-Methoxy-2-acetylnaphthalene (MA) is a key intermediate and degradation product in the chemical synthesis of S-NAP, and is also listed as impurity I in the current Chinese Pharmacopoeia. Currently, the main method for detecting MA in S-NAP is high-performance liquid chromatography (HPLC). However, HPLC is highly instrument-dependent and lacks real-time visualization. In contrast, fluorescence-based impurity detection methods offer advantages such as low cost, simple operation, and visualization. However, ordinary fluorescence analysis methods cannot directly detect MA in S-NAP and also face challenges such as specificity and low sensitivity. Therefore, developing a molecular probe that specifically fluoresces with MA to achieve a convenient and visualized method for detecting MA in S-NAP would have significant methodological and practical application value. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method for detecting naproxen impurity I, especially a visual method for detecting impurities, so as to solve the technical problems of the inability to visualize the detection process of naproxen impurity I, the cumbersome detection process, and the weak specificity.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] One objective of this invention is to provide a method for testing naproxen for impurity I, comprising the following steps: thoroughly mixing naproxen raw material and 1,2,4,5-tetracyanobenzene (TCNB) at a molar ratio of 1-20:1; detecting the fluorescence signal of the complex under 365 nm ultraviolet light; if the detection result shows a green fluorescence signal, it indicates that the naproxen raw material contains impurity I, otherwise it does not; the impurity I is 6-methoxy-2-acetnaphthalene. The amount of naproxen raw material (moles) is calculated based on the molar mass of naproxen, i.e., the influence of impurities is ignored.

[0009] Furthermore, the mixing is achieved through grinding.

[0010] Furthermore, the molar ratio of the naproxen active pharmaceutical ingredient to 1,2,4,5-tetracyanobenzene is 2:1.

[0011] Furthermore, the detection method is fluorescence spectroscopy or visual fluorescence analysis.

[0012] Furthermore, the visual fluorescence analysis involves visually observing the presence or absence of green fluorescence. For example, under 365 nm ultraviolet light, if green fluorescence is visually observed, it indicates that the content of impurity I in naproxen exceeds the limit specified in the pharmacopoeia; if no green fluorescence is observed, it indicates that the content of impurity I in naproxen does not exceed the limit specified in the pharmacopoeia. The co-crystal complex formed by impurity I and 1,2,4,5-tetracyanobenzene in the naproxen sample exhibits a significant red shift in absorption and emission, forming a uniform green fluorescent co-crystal, which facilitates the visual analysis of impurity content in the sample. The visual observation method is also highly sensitive to green fluorescence, capable of detecting even very small sample amounts.

[0013] The fluorescence spectroscopy method described above is a more precise fluorescence analysis method, requiring the use of a fluorescence spectrometer. Specifically, the fluorescence intensity at 500 nm is detected using a fluorescence spectrometer. If the ratio of this fluorescence intensity to the fluorescence intensity of pure naproxen (control) is greater than or equal to 6, it indicates that the content of impurity I in naproxen exceeds the limit specified in the pharmacopoeia. If it is less than 6, it indicates that the content of impurity I in naproxen does not exceed the limit specified in the pharmacopoeia. Specifically, the complex is subjected to a fluorescence spectral scan, with the excitation wavelength set to 365 nm, the scanning range to 400-700 nm, the slit width to 5 nm, and the voltage to 250 V. The obtained spectral data shows that if the ratio of the fluorescence intensity at 500 nm to that of the S-NAP control (pure S-NAP) at 500 nm (I1:I0 ≥ 6), it indicates that the MA content in the sample exceeds 0.1%, exceeding the impurity limit in the pharmacopoeia. If I1:I0 < 6 at 500 nm, it indicates that the MA content in the sample is less than 0.1%, not exceeding the impurity limit in the pharmacopoeia.

[0014] A second objective of this invention is to provide a eutectic composite fluorescent material comprising two components, 1,2,4,5-tetracyanobenzene and 6-methoxy-2-acetnaphthalene, obtained by grinding and mixing; the fluorescent material emits yellow or green fluorescence under 365 nm ultraviolet excitation; it emits yellow fluorescence when the molar ratio of 1,2,4,5-tetracyanobenzene and 6-methoxy-2-acetnaphthalene is 1:1, and green fluorescence when the molar ratio is 2:1.

[0015] A third objective of this invention is to provide the application of the aforementioned fluorescent material in the detection of 6-methoxy-2-acetnaphthalene impurities in naproxen. During the detection process, by adjusting the input ratio of probe 1,2,4,5-tetracyanobenzene, two complexes with impurity I are formed, resulting in yellow and green complexes that exhibit two highly specific characteristic fluorescent signals.

[0016] This invention establishes a fluorescence spectrometry method based on ICT (Intermolecular Charge Transfer) to explore the regulation and influence of intermolecular interactions on fluorescence emission. It achieves an effective and pre-treatment-free method for analyzing trace MA impurities in S-NAP (Synthetic Naproxen Propionate). The detection rate can be as low as the pharmacopoeia-specified limit (0.1%) while exhibiting excellent selectivity and anti-interference capabilities. The fluorescent probe TCNB of this invention undergoes a specific intermolecular charge transfer interaction with naproxen impurity I, thereby generating a fluorescence signal and enabling the detection of naproxen impurity I. In particular, the method of this invention allows the probe to interact with naproxen impurity I through a simple and easy-to-operate grinding method, producing bright green fluorescence. Experimental results show that impurities as low as 0.1% can still be detected. Simultaneously, under this grinding method, the probe cannot interact with naproxen, thus not interfering with the detection of impurity I. Systematic methodological studies have shown that this impurity detection method has a low detection limit, good specificity, and anti-interference capabilities, and can effectively analyze trace impurities I in naproxen without pre-treatment. This invention provides a novel ICT method and technology for drug impurity inspection, revealing the application value of intermolecular charge transfer spectroscopy in the field of drug impurity inspection. In particular, it enables visualized impurity inspection, solving the technical problems of the current inability to visualize naproxen impurity I inspection process, cumbersome detection procedures, and weak specificity, and has broad application prospects. Attached Figure Description

[0017] Figure 1 The fluorescence spectra and quantum yields of the monomers and complexes of this invention are shown in the figures (where a) S-NAP, b) MA, c) TCNB, d) S-NAP / TCNB and MA / TCNB, and the insets are the corresponding fluorescence photographs).

[0018] Figure 2 Other spectra of TCNB, MA, S-NAP, MA / TCNB, and S-NAP / TCNB of the present invention (wherein, a) ultraviolet-visible absorption spectrum, b) Fourier transform infrared spectrum, and c) Raman scattering spectrum).

[0019] Figure 3 The fluorescence spectra and corresponding fluorescence photographs of the complexes obtained by grinding S-NAP-TPP dispersion systems with TCNB at different ratios according to the present invention are shown.

[0020] Figure 4 The molecular packing structure of MA and TCNB when mixed in different proportions according to the present invention (wherein, a) the intermolecular structure of MT2 Stacking, b) Intermolecular forces of MT1 (c) Molecular packing structure of MT2 as observed along the crystallographic a-axis; (d) Molecular packing structure of MT1 as observed along the crystallographic a-axis).

[0021] Figure 5 The fluorescence spectra and ultraviolet images of three complexes of S-NAP, MA, and TCNB in ​​different proportions are shown below (where a) different complexes with the TCNB ratio adjusted while keeping the S-NAP and MA ratio constant, and b) different complexes with different MA contents in S-NAP when TCNB is fixed at 1 / 2 of S-NAP).

[0022] Figure 6 This invention relates to the selectivity and anti-interference capability of the TCNB for MA detection (wherein, a) the molecular structures of R-NAP, MNCA, HN-Me, MNAA, and MNE molecules, b) the selectivity of TCNB for MA and representative molecules, and c) the anti-interference capability of TCNB for detecting MA and representative molecules in coexistence). Detailed Implementation

[0023] The present invention will be described in detail below with reference to embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of application of the present invention. The present invention is not limited to the following embodiments or examples. Any modifications and variations made without departing from the spirit of the present invention should be included within the scope of the present invention. Unless otherwise specified, the experimental materials or reagents used in the following embodiments are commercially available.

[0024] Naproxen (S-Naproxen, S-NAP), 6-methoxy-2-acylnaphthalene (MA), and 1,2,4,5-tetracyanobenzene (TCNB) were all purchased from Aladdin Biochemical Technology Co., Ltd., Shanghai, China. S-NAP underwent further purification by column chromatography and recrystallization to obtain high-purity S-NAP before being used in subsequent experiments.

[0025] Example 1: Photophysical characterization of S-NAP / TCNB and MA / TCNB composites

[0026] First, the interactions between MA and S-NAP and the electron acceptor TCNB were investigated separately through grinding. MA and TCNB were thoroughly ground and mixed at a 1:1 molar ratio to obtain the MA / TCNB complex. The S-NAP / TCNB complex was obtained using the same method. Then, the fluorescence spectra and quantum yields of the S-NAP, MA, and TCNB monomers, as well as the S-NAP / TCNB and MA / TCNB complexes, were measured under 365 nm UV light. The results are as follows: Figure 1As shown, the MA / TCNB complex exhibits yellow fluorescence (542 nm), while the fluorescence emission of S-NAP / TCNB shows no significant change compared to the initial state. Quantum yield data indicate that the quantum yield of MA / TCNB is 8.83%, significantly higher than that of MA (0.50%) and TCNB (2.81%). The strong intermolecular interactions of the ICT cocrystal suppress rotational and vibrational non-relaxation of the ground state, thereby improving the quantum yield of the cocrystal. However, the quantum yield of S-NAP / TCNB is 2.98%, showing little change compared to S-NAP (2.05%) and TCNB (2.81%). Figure 1 UV-Vis absorption spectroscopy was also performed on the three monomers and complexes, and the results are as follows: Figure 2 As shown in Figure a, UV-Vis spectroscopy analysis of the MA / TCNB mixed powder revealed a broad absorption band extending from 330 nm to 600 nm. This absorption band differs from the individual spectra of MA or TCNB, indicating that a charge transfer (CT) complex may have formed between MA and TCNB. In contrast, the absorption spectrum of the S-NAP / TCNB complex showed no significant change. This indicates that no significant CT interaction occurs between S-NAP and TCNB, consistent with the absorption and fluorescence characterization results.

[0027] Example 2: Study on the mechanism of action of S-NAP / TCNB and MA / TCNB complexes

[0028] To understand the interactions and luminescence differences in the eutectic, further spectroscopic and structural characterization of the crystals was performed. First, Fourier transform infrared (FTIR) and Raman spectra of the monomers and eutectic were measured to investigate non-covalent interactions in the S-NAP / TCNB and MA / TCNB complexes. Figure 2 As shown in b, in MA / TCNB, the FTIR band of TCNB shows a slight shift (CH stretching vibration: from 3113 cm⁻¹). -1 Moved to 3111 cm -1 CH stretching vibration: from 3047 cm -1 Moved to 3041 cm -1 C≡N stretching vibration: from 2245 cm -1 Moved to 2243 cm -1 The peak shift of the S-NAP / TCNB complex was less pronounced. The overall red shift of the spectral bands indicates an increase in the electron density of the benzene ring in the TCNB molecule. The significant shift in MA / TCNB suggests that the electron-deficient benzene ring is a key interaction site and is crucial to changes in the eutectic properties. From Raman spectroscopy ( Figure 2 c) From this perspective, the ring bending vibration of TCNB in ​​the MA / TCNB eutectic changes from the initial 723 cm⁻¹. -1Blue shifted to 724 cm -1 The cyano bending vibration starts from an initial 2241 cm. -1 Redshifted to 2238 cm -1 The peak shift of the S-NAP / TCNB complex was less pronounced. Therefore, both infrared and Raman spectral data indicate that there should be no significant interaction between S-NAP and TCNB. Figure 2 b; c).

[0029] Example 3: Analysis of the reasons for the difference in luminescence between S-NAP / TCNB and MA / TCNB

[0030] To investigate the reason why S-NAP / TCNB exhibits different luminescence behavior compared to MA / TCNB, S-NAP was uniformly dispersed in triphenylphosphine (TPP) at different molar ratios. Then, different proportions of the S-NAP-TPP dispersion system were repeatedly ground with TCNB until homogeneous. The fluorescence of the resulting complexes after grinding was then detected. Figure 3 The fluorescence spectra and images shown indicate that when the S-NAP concentration in TPP is sufficiently high, S-NAP molecules tend to bind tightly in a self-aggregated manner, thereby inhibiting ICT interactions with TCNB. However, when the S-NAP concentration in TPP is too low, the fluorescence of the S-NAP / TCNB complex is diluted. Only when the S-NAP concentration in TPP is moderate can it effectively interact with TCNB, thus emitting a distinct yellow fluorescence. Figure 3 This suggests that the strong self-affinity of the electron donor may be a key reason preventing S-NAP from forming an ICT interaction with TCNB.

[0031] Example 4: Study on the interaction differences between MA and TCNB when mixed in different proportions

[0032] Dissolve MA and TCNB in ​​acetonitrile at molar ratios of 1:1 and 1:2 (until just completely dissolved). Figure 4 As shown, it was found that MA and TCNB in ​​acetonitrile solvent can self-assemble into two different colored single crystals in ratios of 1:1 and 1:2. Analysis of the single crystal data of MA / TCNB=1 / 1 (MT1) yellow crystal and MA / TCNB=1 / 2 (MT2) green crystal reveals that MT1... The interplanar spacing of the stacked structures is significantly smaller than that of MT2. The smaller interplanar spacing means that MA and TCNB have stronger intermolecular conjugation when forming the MT1 eutectic. The stronger charge transfer effect will cause MT1 to exhibit a more significant fluorescence red shift, thus emitting a longer fluorescence wavelength than MT2.

[0033] Example 5: Obtaining a unique dual-color fluorescence signal by adjusting the TCNB ratio

[0034] TCNB and S-NAP containing MA were ground in different molar ratios, such as... Figure 5 As shown in Figure a, even in the presence of a large amount of S-NAP, the dichroism of the MA / TCNB complex can still be observed by adjusting the amount of TCNB. This dichroism significantly improves the specificity of the TCNB fluorescent probe in detecting MA using this analytical method.

[0035] Example 6: Exploring the Minimum Limits of TCNB Fluorescent Probe for MA Detection

[0036] As mentioned earlier, the pharmacopoeia classifies MA as an impurity I in the nonsteroidal anti-inflammatory drug S-NAP and requires that the MA content in S-NAP not exceed 0.1%. Therefore, the detection limit of the TCNB fluorescent probe for MA was tested to evaluate the sensitivity of this method (the molar ratio of each complex was fixed at 2:1 for S-NAP:TCNB, and the MA content (mole fraction) in S-NAP was 0%, 0.05%, 0.1%, 0.2%, 0.5%, and 1%). The results are as follows: Figure 5 As shown in b, through intermolecular charge transfer and interaction with TCNB, even MA content as low as 0.1% in S-NAP can still emit green fluorescence, indicating that this method can be used for the impurity analysis of trace MA in S-NAP. Further, the complex was subjected to fluorescence spectroscopy scanning with an excitation wavelength of 365 nm, a scanning range of 400-700 nm, a slit width of 5 nm, and a voltage of 250 V. The resulting fluorescence spectrum showed that the ratio of the fluorescence intensity I1 of the 0.1% complex at 500 nm to the fluorescence intensity I0 of the S-NAP standard (i.e., the 0% complex) at 500 nm, I1:I0, was equal to 6.41 (…). Figure 5 b) Therefore, at 500 nm, when I1:I0≥6, it means that the MA content in the sample exceeds 0.1%, which exceeds the impurity limit in the pharmacopoeia; when I1:I0<6, it means that the MA content in the sample is less than 0.1%, which does not exceed the impurity limit in the pharmacopoeia.

[0037] Example 7: Examination of the selectivity and anti-interference ability of the detection method

[0038] To investigate the selectivity and anti-interference ability of TCNB for the detection of MA, representative coexisting molecules (including R-NAP, 6-methoxy-2-naphthylacetic acid (MNCA), 6-methoxy-2-naphthylacetic acid (MNAA), methyl 6-hydroxy-2-naphthoic acid (HN-Me), and 1-(6-methoxy-2-naphthyl)ethanol (MNE)) were determined, with molecular structures as follows: Figure 6 (as shown in a) Changes in intensity at 540 nm in the fluorescence spectrum after interaction with TCNB. Figure 6As shown in b, the fluorescence intensity only changes significantly when MA is added to TCNB, implying that TCNB can be used for selective sensing of MA (MA or the interfering agent interacts with TCNB at a 1:1 molar ratio). Furthermore, anti-interference experiments show that adding other substances does not interfere with the selective response to MA (MA:TCNB:interfering agent molar ratio of 1:1:1). Figure 6 c), which further confirms that TCNB can be used as an excellent fluorescent probe for detecting MA.

[0039] The conventional techniques and solutions not described in detail in the above embodiments are all well known in the art, and therefore will not be elaborated upon here. The above embodiments and / or experimental examples describe the preferred embodiments of the present invention in detail. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A method for detecting impurity I in naproxen, characterized in that, Includes the following steps: Naproxen active pharmaceutical ingredient and 1,2,4,5-tetracyanobenzene were thoroughly mixed at a molar ratio of 1 to 20:1 to obtain a complex. The complex was subjected to fluorescence signal detection under 365 nm ultraviolet light. If the detection result showed a green fluorescence signal, it indicated that the naproxen active pharmaceutical ingredient contained impurity I, otherwise it did not. Impurity I was 6-methoxy-2-acetylnaphthalene.

2. The method as described in claim 1, characterized in that, The mixing is achieved through grinding.

3. The method as described in claim 2, characterized in that, The molar ratio of naproxen active pharmaceutical ingredient to 1,2,4,5-tetracyanobenzene is 2:

1.

4. The method as described in claim 3, characterized in that, The detection method is fluorescence spectroscopy or visual fluorescence analysis.

5. The method as described in claim 4, characterized in that, The visual fluorescence analysis involves visually observing whether green fluorescence is produced. If green fluorescence is observed, it is determined that the content of impurity I in naproxen exceeds the limit specified in the pharmacopoeia. If no green fluorescence is observed, it is determined that the content of impurity I in naproxen does not exceed the limit specified in the pharmacopoeia.

6. The method as described in claim 4, characterized in that, The fluorescence spectroscopy method involves using a fluorescence spectrometer to detect the fluorescence intensity at 500 nm. If the ratio of the fluorescence intensity to that of the control naproxen is greater than or equal to 6, it is determined that the content of impurity I in naproxen exceeds the limit specified in the pharmacopoeia. If the ratio is less than 6, it is determined that the content of impurity I in naproxen does not exceed the limit specified in the pharmacopoeia.

7. A eutectic composite fluorescent material, characterized in that, It consists of two components, 1,2,4,5-tetracyanobenzene and 6-methoxy-2-acetnaphthalene, which are obtained by grinding and mixing.

8. The fluorescent material as described in claim 7, characterized in that, The fluorescent material emits yellow or green fluorescence under 365 nm ultraviolet excitation.

9. The fluorescent material as described in claim 8, characterized in that, The molar ratio of 1,2,4,5-tetracyanobenzene and 6-methoxy-2-acetylnaphthalene is 1:1 or 2:

1.

10. The application of the fluorescent material as described in any one of claims 7-9 in the detection of 6-methoxy-2-acetnaphthalene impurities in naproxen.

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