A method for analyzing the content of quercetin-3-ol in a biological matrix

Abstract

The present application belongs to the field of analysis and detection, and particularly relates to a method for analyzing the content of quercetin-3-ol in a biological matrix. The method comprises: mixing a biological matrix sample with a precipitant for protein precipitation treatment, separating to obtain a supernatant, and taking the supernatant as a test solution; and using liquid chromatography-tandem mass spectrometry to analyze the concentration of quercetin-3-ol in the test solution. Through sample separation pretreatment and optimization of liquid chromatography and mass spectrometry parameters, the lower limit of quantification of the method can reach 20 ng / mL, which can meet the detection requirements of trace quercetin-3-ol in vivo; the standard curve is linear in a wide concentration range, and is suitable for sample analysis at different concentration levels; in mass spectrometry detection, the MRM detection mode is used, and the dual selectivity of parent ions and daughter ions is used to effectively eliminate the interference of complex components in the biological matrix, and the method has high selectivity and accuracy.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection, and in particular relates to an analytical method for the content of quinine-3-ol in biological matrices. Background Technology

[0002] Quinolin-3-ol is a key intermediate metabolite in the kynurenine pathway of tryptophan metabolism in the human body. Recent studies have shown that quinolin-3-ol is not only closely related to physiological and pathological processes of the nervous system (such as the regulation of neural excitability), but its abnormal accumulation is also considered a potential biomarker for various neurological diseases such as Alzheimer's disease, Huntington's disease, and depression. Therefore, establishing a method for accurate, rapid, and sensitive quantitative detection of quinolin-3-ol concentration in vivo is of great value for mechanistic research, clinical diagnosis, and drug development of related diseases.

[0003] Currently, common methods for detecting quinine-3-ol include high-performance liquid chromatography (HPLC) combined with fluorescence or ultraviolet detectors, and gas chromatography-mass spectrometry (GC-MS). However, HPLC has relatively low sensitivity and is easily affected by endogenous substances when analyzing complex biological matrices (such as plasma and tissue homogenates), making it difficult to accurately quantify trace levels of quinine-3-ol. While GC-MS has higher sensitivity, it usually requires derivatization of quinine-3-ol to increase its volatility and thermal stability. This step is cumbersome, time-consuming, and may introduce errors, affecting analytical throughput and reproducibility.

[0004] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) combines the high separation efficiency of liquid chromatography with the high selectivity and sensitivity of tandem mass spectrometry. However, the key to successfully applying LC-MS / MS to the quantitative analysis of quinine-3-ol lies in systematically optimizing the sample pretreatment process, chromatographic separation conditions, and mass spectrometry detection parameters based on the physicochemical properties of this compound. Currently, there is a lack of a fully validated, comprehensive LC-MS / MS method that can effectively overcome biomatrix effects and achieve stable and reliable detection. In particular, improvements are still needed in ionization efficiency, selection of characteristic fragment ions, peak shape, and analysis speed. Summary of the Invention

[0005] To address the aforementioned problems in the existing technology, this invention provides an analytical method for the content of quinine-3-ol in biological matrices, which can meet the quantitative detection requirements of free quinine-3-ol in biological matrices. This method is simple, rapid, and highly operable.

[0006] To achieve the above objectives, the technical solution provided by the present invention is as follows:

[0007] This application provides a method for analyzing the content of quinine-3-ol in a biological matrix, comprising: mixing a biological matrix sample with a precipitant for protein precipitation treatment, then separating to obtain a supernatant, using the supernatant as the test solution; and analyzing the concentration of quinine-3-ol in the test solution using liquid chromatography-tandem mass spectrometry.

[0008] Optionally, the liquid chromatography uses a C18 column and performs gradient elution with an aqueous solution containing formic acid as mobile phase A and an acetonitrile solution containing formic acid as mobile phase B; and / or, the mass spectrometry uses an electrospray ionization source, positive ion mode, and multiple reaction monitoring mode to detect quinine-3-ol.

[0009] Optionally, the precipitant is acetonitrile; and / or, the volume ratio of the biological matrix to the precipitant is 1:9.

[0010] Optionally, the biological matrix includes one or more of plasma, serum, whole blood, excrement, and tissue homogenate.

[0011] Optionally, after protein precipitation, the supernatant is obtained by centrifugation, and then the supernatant is diluted to obtain the test solution.

[0012] Optionally, the C18 column has the following specifications: inner diameter 2.1 mm, length 50 mm, and packing particle size 1.7 μm.

[0013] Optionally, the gradient elution procedure is as follows: 0-0.30 min, the volume percentage of mobile phase A is 95%; 0.30-1.80 min, the proportion of mobile phase A decreases from 95% to 5%; 1.80-2.40 min, the proportion of mobile phase A is maintained at 5%; 2.40-2.41 min, the proportion of mobile phase A is increased to 95%; 2.41-3.00 min, the proportion of mobile phase A is maintained at 95%; and / or, the mobile phase flow rate during the elution process is 0.6 mL / min.

[0014] Optionally, the mass spectrometry detection conditions are as follows: ion source temperature 550 °C; ionization voltage 5500 V; curtain gas pressure 40 psi; collision gas pressure 10 psi; nebulizer gas and auxiliary gas pressures 35 psi and 70 psi, respectively; the multiple reaction monitoring ion pair for quinine-3-ol is: parent ion m / z 128.05, daughter ion m / z 109.97, and collision energy 30 eV.

[0015] Compared with the prior art, the present invention has at least the following beneficial effects:

[0016] This invention utilizes sample separation pretreatment and optimized liquid chromatography and mass spectrometry parameters, achieving a method limit of quantitation of up to 20 ng / mL, which meets the detection requirements for trace quinine-3-ol in vivo. The standard curve exhibits good linearity over a wide concentration range, making it suitable for sample analysis at different concentration levels.

[0017] The mass spectrometry detection method of this invention adopts the MRM detection mode, which utilizes the dual selectivity of the parent ion and the daughter ion to effectively eliminate the interference of complex components in the biological matrix, and has high selectivity and accuracy.

[0018] This invention utilizes a protein precipitation pretreatment step combined with a 3-minute rapid chromatographic gradient, significantly shortening the analysis cycle for a single sample. The sample pretreatment eliminates the need for complex steps such as solid-phase extraction or derivatization. Experimental consumables and instruments are readily available, and the operation is simple and safe. This method is applicable not only to plasma and whole blood but also to the analysis of quinine-3-ol in various biological matrices such as excrement and tissue homogenates, demonstrating its wide range of applications. Attached Figure Description

[0019] Figure 1 This is to ensure the accuracy of the standard curve for quinine-3-ol in the embodiments of the present invention;

[0020] Figure 2 This is a typical chromatogram of a sample with a lower limit of quantitation (20 ng / mL) in an embodiment of the present invention. Detailed Implementation

[0021] The present invention will now be described in further detail with reference to the accompanying drawings:

[0022] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0023] All reagents and materials used in this example can be purchased routinely. The quantitative experiments involved in the examples were all repeated at least three times, and the results were averaged.

[0024] Example

[0025] I. Instruments and Reagents

[0026] Instrument: Liquid chromatography-tandem mass spectrometry system, model AB Sciex 7500 Triple Quad mass spectrometer, equipped with electrospray ionization (ESI) source and ultra-high performance liquid chromatography system.

[0027] Chromatographic column: ACQUITY UPLC® BEH C18 column, 1.7 μm, 2.1 mm × 50 mm (Waters).

[0028] Solution preparation: Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: 0.1% formic acid acetonitrile solution.

[0029] II. Biological Sample Pretreatment

[0030] Take 30 μL of blank human plasma (or other biological matrix) and add 270 μL of acetonitrile mixture (containing 5 ng / mL tolbutamide and the remainder acetonitrile). Vortex for 3 minutes to ensure complete protein precipitation. Then, place the mixture in a centrifuge at 4°C and centrifuge at 4000 r / min for 10 minutes. Take 100 μL of the supernatant and mix it with deionized water at a 1:1 volume ratio to obtain the test solution.

[0031] III. Conditions for Liquid Chromatography Processing

[0032] Column temperature: 40℃;

[0033] Autosampler temperature: 4℃;

[0034] Injection volume: 5 μL;

[0035] Runtime: 5.00 min;

[0036] The gradient elution procedure is shown in Table 1.

[0037] Table 1 Gradient elution degree

[0038]

[0039] IV: Mass Spectrometry Conditions

[0040] The mass spectrometry testing conditions are shown in Table 2.

[0041] Table 2 Mass Spectrometry Testing Conditions

[0042]

[0043] 5. Methodological Validation

[0044] According to the requirements of General Chapter 9012 of the Pharmacopoeia, a series of preparations of quinine-3-ol were prepared using blank biological matrix (plasma) as standard curve samples and quality control samples, and analyzed according to the above method. The peak area of ​​quinine-3-ol was plotted as the ordinate (Y), and the corresponding theoretical concentration as the abscissa (X), and linear regression was performed using weighted least squares method (weighting coefficient 1 / X²). The accuracy results of the standard curve are shown in […]. Figure 1 .

[0045] Quality control samples were prepared using blank biological matrix (plasma) at four concentration levels: lower limit of cathodic control (LLOQ, 20.00 ng / mL), low (LQC, 60.00 ng / mL), medium (MQC, 600.00 ng / mL), and high (HQC, 3000.00 ng / mL). Six replicates were prepared and measured at each concentration level. Standard deviation and precision data were calculated. The results are shown in Table 3.

[0046] Through Table 3 and Figure 1 It can be seen that the results obtained by this method are highly accurate and reproducible, meeting the requirements of quantitative analysis.

[0047] Table 3 Precision and Accuracy Results

[0048]

[0049] Figure 2 The chromatogram of the sample at the lower limit of quantitation (20 ng / mL) is shown. The chromatographic peak shape of quinine-3-ol is good, and no obvious matrix interference peaks were observed at the elution position of the target analyte, demonstrating that the method has good selectivity.

[0050] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for analyzing the content of quinine-3-ol in a biological matrix, characterized in that, include: The biological matrix sample was mixed with a precipitant for protein precipitation, and then the supernatant was obtained. The supernatant was used as the test solution. The concentration of quinine-3-ol in the test solution was analyzed by liquid chromatography-tandem mass spectrometry.

2. The method for analyzing the content of quinine-3-ol in a biological matrix according to claim 1, characterized in that, The liquid chromatography uses a C18 column and performs gradient elution with an aqueous solution containing formic acid as mobile phase A and an acetonitrile solution containing formic acid as mobile phase B; and / or, the mass spectrometry uses an electrospray ionization source, positive ion mode, and multiple reaction monitoring mode to detect quinine-3-ol.

3. The method for analyzing the content of quinine-3-ol in a biological matrix according to claim 1, characterized in that, The precipitant is acetonitrile; and / or, the volume ratio of the biological matrix to the precipitant is 1:

9.

4. The method for analyzing the content of quinine-3-ol in a biological matrix according to claim 1, characterized in that, The biological matrix includes one or more of plasma, serum, whole blood, excrement, and tissue homogenate.

5. The method for analyzing the content of quinine-3-ol in a biological matrix according to claim 1, characterized in that, After protein precipitation, the supernatant was obtained by centrifugation, and then the supernatant was diluted to obtain the test solution.

6. The method for analyzing the content of quinine-3-ol in a biological matrix according to claim 2, characterized in that, The specifications of the C18 chromatographic column are: inner diameter 2.1 mm, length 50 mm, and packing particle size 1.7 μm.

7. The method for analyzing the content of quinine-3-ol in a biological matrix according to claim 2, characterized in that, The gradient elution procedure is as follows: 0-0.30 min, mobile phase A volume percentage is 95%; 0.30-1.80 min, mobile phase A percentage decreases from 95% to 5%; 1.80-2.40 min, mobile phase A percentage is maintained at 5%; 2.40-2.41 min, mobile phase A percentage is increased to 95%; 2.41-3.00 min, mobile phase A percentage is maintained at 95%; and / or, the mobile phase flow rate during elution is 0.6 mL / min.

8. The method for analyzing the content of quinine-3-ol in a biological matrix according to claim 1 or 2, characterized in that, The mass spectrometry detection conditions were as follows: ion source temperature was 550 °C; ionization voltage was 5500 V; curtain gas pressure was 40 psi; collision gas pressure was 10 psi; nebulizer gas and auxiliary gas pressures were 35 psi and 70 psi, respectively; the multiple reaction monitoring ion pair of the quinine-3-ol was: mother ion m / z 128.05, daughter ion m / z 109.97, and collision energy 30 eV.

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