Liquid chromatography-mass spectrometry detection method of penicillium subtomentosum acid

Abstract

The invention provides a liquid chromatography-mass spectrometry detection method of penicillium wool acid, which comprises the following steps: carrying out methylation derivation treatment on a sample containing the penicillium wool acid by adopting trimethylsilanized diazomethane to obtain a derivatized sample, and carrying out liquid chromatography-mass spectrometry detection on the derivatized sample to obtain the penicillium wool acid. And obtaining the content of the penicillic acid in the red yeast rice. According to the method disclosed by the invention, hydroxyl (OH) in penicilliic acid is changed into methoxyl (OCH3), chromatographic separation can be realized between two isomers, the specificity is enhanced, a positive ion mode can be used for detection by mass spectrometry, and the sensitivity is greatly improved.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection, and in particular to a liquid chromatography-mass spectrometry detection method for penicillic acid. Background Art

[0002] Puberulic acid has a seven-membered ring phenolic ketone structure and is a metabolite produced by Penicillium contamination during the fermentation of red yeast rice. It has certain antimalarial activity and certain nephrotoxicity. Only one literature report was found by Masato Iwatsuki (Masato I, Shohei T, Mihoko M, et al. In vitro and in vivo antimalarial activity of puberulic acid and its new analogs, viticolins AC, produced by Penicillium sp. FKI-4410. [J]. The Journal of antibiotics, 2011, 64 (2): 183-8.) who used high performance liquid chromatography with ultraviolet detector to chromatographically separate and purify puberulic acid, prepared a high-purity compound, and then used high-resolution mass spectrometry negative ion mode to determine its molecular weight and used nuclear magnetic resonance spectroscopy to identify its structure.

[0003] There are two structures of puberulic acid reported in the literature, which have the same molecular weight and are isomers of each other, and are mainly produced through enol tautomerism (see Masato I, Shohei T, Mihoko M, et al. In vitro and invivo antimalarial activity of puberulic acid and its new analogs, viticolins A-C, produced by Penicillium sp. FKI-4410. [J]. The Journal of antibiotics, 2011, 64 (2): 183-8. and Halton B, Harvey J. Electrocyclic Ring-Opening Reactions of gem-Dibromocyclopropanes in the Synthesis of Natural Products and Related Compounds [J]. Synlett, 2006, 2006 (13): 1975-2000.).

[0004] Since the safety of red yeast rice has attracted great attention, it is necessary to develop highly sensitive and specific analytical testing technology to detect the content of penicillic acid. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a liquid chromatography-mass spectrometry detection method for penicillic acid, which uses trimethylsilylated diazomethane to methylate penicillic acid and can be detected using a liquid chromatography-mass spectrometry device. The derivatization allows it to be retained by a chromatographic column, thereby separating it from other components in red yeast rice, and then using a positive ion mode for detection in a mass spectrometer detector, further improving the sensitivity.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] The invention provides a liquid chromatography-mass spectrometry (LC-MS) detection method for penicillic acid. The LC-MS detection method comprises: using trimethylsilylated diazomethane to perform methylation derivatization treatment on a sample containing penicillic acid to obtain a derivatized sample, and performing liquid chromatography-mass spectrometry detection on the derivatized sample to obtain the content of penicillic acid in the red yeast rice.

[0008] It is worth noting that when penicillic acid is directly detected by mass spectrometry without derivatization, an alkaline mobile phase such as 0.1% ammonia water is required to ionize and generate mass spectrometry signals. However, when an alkaline mobile phase is used, it cannot be retained on the chromatogram and cannot be effectively separated from other components in the sample. Therefore, liquid chromatography-mass spectrometry cannot be used for high-sensitivity and strong-specific qualitative and quantitative detection. The existing literature reports also fail to distinguish between its two configurational isomers.

[0009] The present invention uses trimethylsilylated diazomethane to perform methylation derivatization treatment on a sample containing penicillic acid, which can not only be detected by liquid chromatography-mass spectrometry equipment, but also can be retained by a chromatographic column through derivatization, thereby being separated from other components in red yeast rice, and then using positive ion mode detection in a mass spectrometer to further improve the sensitivity.

[0010] In the present invention, the detection ion pairs are 255.0→225.0 and 255.0→194.0.

[0011] The equations for the methylation derivatization reaction of the two isomers of penicillic acid in the present invention are as follows:

[0012]

[0013] In the above formula, (1) penicillic acid structure I; (2) penicillic acid structure II; (3) methylated derivative of penicillic acid structure I; (4) methylated derivative of penicillic acid structure II.

[0014] Preferably, the methylation derivatization treatment comprises: mixing a trimethylsilylated diazomethane solution and a sample containing penicillic acid to obtain a mixed sample, and sequentially performing shaking, quenching, drying, redissolving and solid-liquid separation, wherein the liquid phase of the solid-liquid separation is the derivatized sample.

[0015] The present invention requires a quenching step, and those skilled in the art know that the quenching step has the effect of terminating the derivatization reaction.

[0016] Preferably, the solvent in the trimethylsilylated diazomethane solution includes n-hexane.

[0017] Preferably, the concentration of trimethylsilylated diazomethane in the trimethylsilylated diazomethane solution is 1 to 3 mol / L, for example, 1 mol / L, 1.3 mol / L, 1.5 mol / L, 1.7 mol / L, 1.9 mol / L, 2.2 mol / L, 2.4 mol / L, 2.6 mol / L, 2.8 mol / L or 3 mol / L.

[0018] Preferably, the volume ratio of the trimethylsilylated diazomethane solution to the sample containing penicillic acid is 1:(2-7), for example, it can be 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5 or 1:7, etc.

[0019] Preferably, the oscillation speed is 100-500 rpm, for example, it can be 100 rpm, 145 rpm, 189 rpm, 234 rpm, 278 rpm, 323 rpm, 367 rpm, 412 rpm, 456 rpm or 500 rpm.

[0020] Preferably, the oscillation temperature is 20-40°C, for example, it can be 20°C, 23°C, 25°C, 27°C, 29°C, 32°C, 34°C, 36°C, 38°C or 40°C.

[0021] Preferably, the shaking time is 10 to 50 min, for example, it can be 10 min, 15 min, 19 min, 24 min, 28 min, 33 min, 37 min, 42 min, 46 min or 50 min.

[0022] Preferably, the quenching agent used in the quenching comprises acetic acid.

[0023] Preferably, the volume ratio of the quencher to the mixed sample is 1:(4-8), for example, it can be 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5 or 1:8, etc.

[0024] Preferably, the drying comprises drying with nitrogen.

[0025] Preferably, the temperature of the nitrogen drying is 35-50°C, for example, it can be 35°C, 37°C, 39°C, 40°C, 42°C, 44°C, 45°C, 47°C, 49°C or 50°C.

[0026] Preferably, the reconstitution solvent comprises methanol-water solution.

[0027] Preferably, the concentration of methanol in the methanol aqueous solution is 5-15wt%, for example, it can be 5wt%, 7wt%, 8wt%, 9wt%, 10wt%, 11wt%, 12wt%, 13wt%, 14wt% or 15wt%.

[0028] Preferably, the volume of the methanol aqueous solution is 250-700 μL, for example, it can be 250 μL, 300 μL, 350 μL, 400 μL, 450 μL, 500 μL, 550 μL, 600 μL, 650 μL or 700 μL.

[0029] Preferably, the preparation of the sample containing penicillic acid comprises: mixing the sample to be tested with a methanol aqueous solution, and sequentially homogenizing and centrifuging, and the supernatant after centrifugation is the sample containing penicillic acid.

[0030] Preferably, the sample to be tested comprises red yeast rice, preferably red yeast rice powder.

[0031] Preferably, the homogenizing rotation speed is 8000-12000 rpm, for example, it can be 8000 rpm, 8400 rpm, 8800 rpm, 9300 rpm, 9700 rpm, 10000 rpm, 10600 rpm, 11000 rpm, 11500 rpm or 12000 rpm.

[0032] Preferably, the homogenization time is 1 to 4 min, for example, it can be 1 min, 1.4 min, 1.7 min, 2 min, 2.4 min, 2.7 min, 3 min, 3.4 min, 3.7 min or 4 min.

[0033] Preferably, the centrifugal rotation speed is 10000-20000 rpm, for example, it can be 10000 rpm, 11100 rpm, 12200 rpm, 13300 rpm, 14400 rpm, 15500 rpm, 16000 rpm, 17700 rpm, 18800 rpm or 20000 rpm.

[0034] Preferably, the centrifugation time is 5 to 15 min, for example, it can be 5 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min or 15 min.

[0035] Preferably, the centrifugation temperature is 1-10°C, for example, it can be 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C or 10°C.

[0036] Preferably, the chromatographic column temperature of the LC-MS detection is 30-40°C, for example, it can be 30°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C.

[0037] Preferably, the chromatographic column for LC-MS detection comprises a C8 chromatographic column.

[0038] Preferably, the specifications of the chromatographic column for LC-MS detection are a particle size of 2.6 μm, an inner diameter of 2.1 mm, and a length of 100 to 150 mm, for example, 100 mm, 106 mm, 112 mm, 117 mm, 123 mm, 128 mm, 134 mm, 139 mm, 145 mm or 150 mm, etc.

[0039] Preferably, the injection volume of the LC-MS detection is 1 to 10 μL, for example, it can be 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL or 10 μL.

[0040] Preferably, the flow rate of the mobile phase for the LC-MS detection is 0.1-0.5 mL / min, for example, it can be 0.1 mL / min, 0.15 mL / min, 0.19 mL / min, 0.24 mL / min, 0.28 mL / min, 0.33 mL / min, 0.37 mL / min, 0.42 mL / min, 0.46 mL / min or 0.5 mL / min, etc.

[0041] Preferably, the mobile phase for the LC-MS detection comprises a first phase and a second phase, wherein the first phase is an aqueous solution of ammonium formate; and the second phase is an aqueous solution of methanol containing ammonium formate.

[0042] The present invention further preferably adopts the mobile phase of the above composition, wherein ammonium formate has the effect of enhancing the mass spectrometry response signal of penicillic acid, and the methanol aqueous solution can enhance the chromatographic retention compared with the use of methanol alone, and can increase the solubility of penicillic acid in the mobile phase compared with the use of water alone, and the detection result is more sensitive and accurate.

[0043] Preferably, the concentration of ammonium formate in the aqueous ammonium formate solution is 5 to 15 mmol / L, for example, it can be 5 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, 13 mmol / L, 14 mmol / L or 15 mmol / L.

[0044] Preferably, the concentration of ammonium formate in the methanol aqueous solution containing ammonium formate is 5 to 15 mmol / L, for example, it can be 5 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, 13 mmol / L, 14 mmol / L or 15 mmol / L.

[0045] Preferably, the volume ratio of methanol to water in the methanol aqueous solution containing ammonium formate is (0.8-1.2):1, for example, it can be 0.8:1, 0.85:1, 0.89:1, 0.94:1, 0.98:1, 1.03:1, 1.07:1, 1.12:1, 1.16:1 or 1.2:1, etc., preferably 1:1.

[0046] Preferably, the volume proportion of the second phase in the mobile phase of the LC-MS detection is 5-100%, for example, it can be 5%, 16%, 27%, 37%, 48%, 58%, 69%, 79%, 90% or 100%.

[0047] Preferably, the volume proportion of the second phase in the mobile phase of the LC-MS detection includes a first stage, a second stage and a third stage as the detection proceeds, wherein the volume proportion of the second phase in the first stage is 5-15%, the volume proportion of the second phase in the second stage is 90-100%, and the volume proportion of the second phase in the third stage is 5-15%.

[0048] The present invention preferably controls the volume ratio of the first phase and the second phase within the above range, which can be more conducive to the separation of the isomer derivatives of penicillic acid, thereby achieving better detection results during mass spectrometry detection.

[0049] Preferably, the time period of the first stage is 0 to 0.8 min, for example, it may be 0 min, 0.1 min, 0.2 min, 0.3 min, 0.4 min, 0.5 min, 0.6 min, 0.7 min or 0.8 min.

[0050] Preferably, the time period of the second stage is 0.5 to 4.2 min, for example, it may be 0.5 min, 1 min, 1.4 min, 1.8 min, 2.2 min, 2.6 min, 3 min, 3.4 min, 3.8 min or 4.2 min.

[0051] Preferably, the time period of the third stage is 4.1 to 5.5 minutes, for example, it may be 4.1 minutes, 4.3 minutes, 4.5 minutes, 4.6 minutes, 4.8 minutes, 4.9 minutes, 5.1 minutes, 5.2 minutes, 5.4 minutes or 5.5 minutes.

[0052] As a preferred technical solution of the present invention, the LC-MS detection method comprises the following steps:

[0053] Mixing red yeast rice powder and methanol aqueous solution, homogenizing at 8000-12000 rpm for 1-4 min and centrifuging at 10000-20000 rpm for 5-15 min at 1-10° C., the supernatant after centrifugation is the sample containing penicillic acid;

[0054] A trimethylsilylated diazomethane solution with a concentration of 1 to 3 mol / L and a sample containing penicillic acid are mixed in a volume ratio of 1:(2 to 7) to obtain a mixed sample, and the mixed sample is shaken at 20 to 40° C. and 100 to 500 rpm for 10 to 50 min, and then acetic acid and the mixed sample are mixed in a volume ratio of 1:(4 to 8) to quench, and dried by blowing with nitrogen at 35 to 50° C., and the obtained solid phase is redissolved with a methanol aqueous solution with a concentration of 5 to 15 wt% of methanol, and subjected to solid-liquid separation, and the liquid phase of the solid-liquid separation is the derivatized sample;

[0055] Performing liquid chromatography-mass spectrometry detection on the derivatized sample to obtain the content of penicillic acid in the red yeast rice;

[0056] The chromatographic column includes a C8 chromatographic column, the chromatographic column temperature is 30-40°C, the chromatographic column specifications are a particle size of 2.6 μm, an inner diameter of 2.1 mm, a length of 100-150 mm, an injection volume of 1-10 μL, and a flow rate of the mobile phase of 0.1-0.5 mL / min;

[0057] The mobile phase for the LC-MS detection comprises a first phase and a second phase, wherein the first phase is an aqueous solution of ammonium formate with a concentration of 5 to 15 mmol / L; the second phase is an aqueous solution of methanol containing ammonium formate with a concentration of 5 to 15 mmol / L; the volume ratio of methanol to water in the aqueous solution of methanol containing ammonium formate is (0.8 to 1.2):1;

[0058] The volume proportion of the second phase in the mobile phase of the LC-MS detection includes a first stage, a second stage and a third stage as the detection proceeds, wherein the volume proportion of the second phase in the first stage is 5-15%, the volume proportion of the second phase in the second stage is 90-100%, and the volume proportion of the second phase in the third stage is 5-15%; the time period of the first stage is 0-0.8 min; the time period of the second stage is 0.5-4.2 min; and the time period of the third stage is 4.1-5.5 min.

[0059] The present invention has no special limitation on the solid-liquid separation in the above process. Any device and method for solid-liquid separation known to those skilled in the art can be used, and can also be adjusted according to the actual process, such as filtration, centrifugation or sedimentation separation, or a combination of different methods.

[0060] Compared with the prior art, the present invention has at least the following beneficial effects:

[0061] (1) The LC-MS detection method of penicillic acid provided by the present invention can separate two isomers of penicillic acid and detect their respective contents;

[0062] (2) The LC-MS detection method for penicillic acid provided by the present invention uses trimethylsilylated diazomethane to methylate and derivatize the penicillic acid-containing sample, which can be retained by the chromatographic column and ionized to generate mass spectrometry signals, thus realizing the qualitative and quantitative detection of penicillic acid in red yeast rice by LC-MS for the first time;

[0063] (3) The LC-MS detection method for penicillic acid provided by the present invention has high sensitivity, and the detection limit can be as low as 1 μg / kg. The sensitivity is more than 1000 times higher than that of the traditional high performance liquid chromatography ultraviolet detector, and the safety of red yeast rice can be detected. The detection repeatability is excellent. Under the optimal conditions, the standard deviation of 5 repetitions is within 1.6%. In addition, the detection accuracy is high. By detecting samples with known concentrations, the final error is within 2.0%. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 This is the mass spectrum obtained by the LC-MS detection method of penicillic acid in Example 1 of the present invention. DETAILED DESCRIPTION

[0065] For the convenience of understanding the present invention, the present invention lists the following embodiments. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0066] It should be understood that the terms "first", "second", etc. are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly indicate the number of technical features indicated. Thus, a feature defined as "first", "second", etc. may explicitly or implicitly include one or more of the features. In the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0067] Example 1

[0068] This embodiment provides a liquid chromatography-mass spectrometry detection method for penicillic acid, which comprises the following steps:

[0069] (1) mixing 1 g of red yeast rice powder and 100 mL of a methanol aqueous solution with a methanol concentration of 50%, and then homogenizing at 10,000 rpm for 2 min and centrifuging at 15,000 rpm for 10 min at 4° C., and the supernatant after centrifugation is the sample containing penicillic acid;

[0070] (2) a 2 mol / L trimethylsilylated diazomethane solution (solvent: n-hexane) and a sample containing penicillic acid were mixed in a volume ratio of 1:5 to obtain a mixed sample, and the mixture was shaken at 30° C. and 300 rpm for 30 min, and then acetic acid and the mixed sample were mixed in a volume ratio of 1:6 to quench the mixture, and dried with nitrogen at 40° C. The obtained solid phase was redissolved with 500 μL of a 10 wt % methanol aqueous solution, and filtered, and the filtered liquid phase was the derivatized sample;

[0071] (3) performing liquid chromatography-mass spectrometry on the derivatized sample to obtain the content of penicillic acid in the red yeast rice;

[0072] The chromatographic column includes a C8 chromatographic column, the chromatographic column temperature is 35°C, the specifications of the chromatographic column are a particle size of 2.6 μm, an inner diameter of 2.1 mm, a length of 100 mm, an injection volume of 2 μL, and a flow rate of the mobile phase of 0.3 mL / min;

[0073] The mobile phase for the LC-MS detection includes a first phase and a second phase, wherein the first phase is an ammonium formate aqueous solution with an ammonium formate concentration of 10 mmol / L; the second phase is a methanol aqueous solution containing ammonium formate with an ammonium formate concentration of 10 mmol / L; the volume ratio of methanol to water in the methanol aqueous solution containing ammonium formate is 1:1;

[0074] The volume proportion of the second phase in the mobile phase of the LC-MS detection includes a first stage, a second stage and a third stage as the detection proceeds, wherein the volume proportion of the second phase in the first stage is 10%, the volume proportion of the second phase in the second stage is 100%, and the volume proportion of the second phase in the third stage is 10%; the time period of the first stage is 0 to 0.5 min (excluding 0.5 min); the time period of the second stage is 0.5 to 4.0 min; and the time period of the third stage is 4.0 to 5.0 min (excluding 4.0 min).

[0075] The specific gradient elution program is shown in Table 1.

[0076] Table 1

[0077] Time [min] Flow rate [mL / min] Second phase ratio [%] 0.0 0.3 10.0 0.5 0.3 100.0 3.0 0.3 100.0 4.0 0.3 100.0 After 4.0 (excluding 4.0) 0.3 10.0 5.0 0.3 10.0

[0078] In this embodiment, the detection ion pairs are 255.0→225.0 and 255.0→194.0.

[0079] The mass spectrum obtained in this embodiment is as follows Figure 1 As shown, A is the peak diagram without penicillic acid, B is the peak diagram of penicillic acid itself, and C is the peak diagram of the red yeast rice sample containing penicillic acid. Figure 1 It can be seen that, by using the method of this embodiment, the peaks of the two isomeric derivatives of penicillic acid are sharp, well separated, and have good mass spectrometry response.

[0080] Example 2

[0081] This embodiment provides a liquid chromatography-mass spectrometry detection method for penicillic acid, which comprises the following steps:

[0082] (1) mixing 1.2 g of red yeast rice powder and 100 mL of a methanol aqueous solution having a methanol concentration of 40%, and homogenizing at 12000 rpm for 1 min and centrifuging at 10000 rpm for 15 min at 2° C., and the supernatant after centrifugation is the sample containing penicillic acid;

[0083] (2) a 3 mol / L trimethylsilylated diazomethane solution (solvent: n-hexane) and a sample containing penicillic acid were mixed in a volume ratio of 1:2 to obtain a mixed sample, and the mixture was shaken at 40°C and 100 rpm for 50 min, and then acetic acid and the mixed sample were mixed in a volume ratio of 1:8 to quench the mixture, and dried with nitrogen at 35°C. The obtained solid phase was re-dissolved in a methanol aqueous solution with a concentration of 15 wt% by 700 μL of methanol, and filtered. The filtered liquid phase was the derivatized sample;

[0084] (3) performing liquid chromatography-mass spectrometry on the derivatized sample to obtain the content of penicillic acid in the red yeast rice;

[0085] The chromatographic column includes a C8 chromatographic column, the chromatographic column temperature is 40°C, the specifications of the chromatographic column are a particle size of 2.6 μm, an inner diameter of 2.1 mm, a length of 120 mm, an injection volume of 10 μL, and a flow rate of the mobile phase of 0.35 mL / min;

[0086] The mobile phase for the LC-MS detection includes a first phase and a second phase, wherein the first phase is an ammonium formate aqueous solution with an ammonium formate concentration of 15 mmol / L; the second phase is a methanol aqueous solution containing ammonium formate with an ammonium formate concentration of 15 mmol / L; the volume ratio of methanol to water in the methanol aqueous solution containing ammonium formate is 0.8:1;

[0087] The volume proportion of the second phase in the mobile phase of the LC-MS detection includes a first stage, a second stage and a third stage as the detection proceeds, wherein the volume proportion of the second phase in the first stage is 12%, the volume proportion of the second phase in the second stage is 98%, and the volume proportion of the second phase in the third stage is 13%; the time period of the first stage is 0 to 0.8 min (excluding 0.8 min); the time period of the second stage is 0.8 to 4.2 min; and the time period of the third stage is 4.2 to 5.2 min (excluding 4.2 min).

[0088] In this embodiment, the detection ion pairs are 255.0→225.0 and 255.0→194.0.

[0089] Example 3

[0090] This embodiment provides a liquid chromatography-mass spectrometry detection method for penicillic acid, which comprises the following steps:

[0091] (1) mixing 0.8 g of red yeast rice powder and 100 mL of a methanol aqueous solution having a methanol concentration of 60%, and homogenizing at 82,000 rpm for 4 min and centrifuging at 20,000 rpm for 5 min at a temperature of 10° C., and the supernatant after centrifugation is the sample containing penicillic acid;

[0092] (2) a 1 mol / L trimethylsilylated diazomethane solution (solvent: n-hexane) and a sample containing penicillic acid were mixed in a volume ratio of 1:7 to obtain a mixed sample, and the mixture was shaken at 20° C. and 500 rpm for 10 min, and then acetic acid and the mixed sample were mixed in a volume ratio of 1:4 for quenching, and dried with nitrogen at 50° C., and the obtained solid phase was redissolved with 600 μL of a 5 wt % methanol aqueous solution, and filtered, and the filtered liquid phase was the derivatized sample;

[0093] (3) performing liquid chromatography-mass spectrometry on the derivatized sample to obtain the content of penicillic acid in the red yeast rice;

[0094] The chromatographic column includes a C8 chromatographic column, the chromatographic column temperature is 30°C, the specifications of the chromatographic column are a particle size of 2.6 μm, an inner diameter of 2.1 mm, a length of 150 mm, an injection volume of 1 μL, and a flow rate of the mobile phase of 0.1 mL / min;

[0095] The mobile phase for the LC-MS detection includes a first phase and a second phase, wherein the first phase is an ammonium formate aqueous solution with an ammonium formate concentration of 5 mmol / L; the second phase is a methanol aqueous solution containing ammonium formate with an ammonium formate concentration of 5 mmol / L; the volume ratio of methanol to water in the methanol aqueous solution containing ammonium formate is 1.2:1;

[0096] The volume proportion of the second phase in the mobile phase of the LC-MS detection includes a first stage, a second stage and a third stage as the detection proceeds, wherein the volume proportion of the second phase in the first stage is 5%, the volume proportion of the second phase in the second stage is 90%, and the volume proportion of the second phase in the third stage is 5%; the time period of the first stage is 0 to 0.5 min (excluding 0.5 min); the time period of the second stage is 0.5 to 4.5 min; the time period of the third stage is 4.5 to 5.5 min (excluding 4.5 min).

[0097] In this embodiment, the detection ion pairs are 255.0→225.0 and 255.0→194.0.

[0098] Example 4

[0099] This embodiment provides a liquid chromatography-mass spectrometry detection method for penicillic acid. The liquid chromatography-mass spectrometry detection method is the same as that of embodiment 1, except that the second phase in step (3) is an aqueous solution with an ammonium formate concentration of 10 mmol / L, and will not be described in detail here.

[0100] Example 5

[0101] This embodiment provides a liquid chromatography-mass spectrometry detection method for penicillic acid. The liquid chromatography-mass spectrometry detection method is the same as that of embodiment 1, except that the ammonium formate in the first phase and the second phase of step (3) is replaced by ammonium acetate (with the same concentration), and the details are not repeated here.

[0102] Example 6

[0103] This embodiment provides a method for detecting penicillic acid by LC-MS. The method is the same as that in Example 1 except that the volume proportion of the second phase in the mobile phase of the LC-MS detection is always maintained at 10%, and will not be described again.

[0104] Example 7

[0105] This embodiment provides a LC-MS detection method for penicillic acid. The LC-MS detection method is the same as that of Example 1, except that the volume proportion of the second phase in the mobile phase of the LC-MS detection is always maintained at 90%, and will not be repeated here.

[0106] Comparative Example 1

[0107] This comparative example provides a liquid chromatography-mass spectrometry detection method for penicillic acid. The liquid chromatography-mass spectrometry detection method is the same as that of Example 1, except that trimethylsilylated diazomethane is replaced by trimethylchlorosilane in step (2), and will not be described in detail here.

[0108] In this comparative example, trimethylsilylated diazomethane was replaced by trimethylchlorosilane. Compared with Example 1, the derivatization reaction of penicillic acid could not occur, that is, the hydroxyl group in the structural formula could not be derivatized into a methoxy group, which ultimately made it impossible to perform LC-MS detection.

[0109] Comparative Example 2

[0110] This comparative example provides a liquid chromatography-mass spectrometry detection method for penicillic acid. The liquid chromatography-mass spectrometry detection method is the same as that of Example 1, except that trimethylsilylated diazomethane is replaced by chloroform in step (2), and will not be described in detail here.

[0111] In this comparative example, when trimethylsilylated diazomethane was replaced by chloroform, compared with Example 1, the derivatization reaction of penicillic acid could not occur, that is, the hydroxyl group in the structural formula could not be derivatized into a methoxy group, which ultimately made it impossible to perform LC-MS detection.

[0112] Comparative Example 3

[0113] This comparative example provides a liquid chromatography-mass spectrometry detection method for penicillic acid. The liquid chromatography-mass spectrometry detection method is the same as Example 1 except that trimethylsilylated diazomethane is replaced by N,O-bistrimethylsilylacetamide in step (2), and will not be described in detail here.

[0114] In this comparative example, when trimethylsilylated diazomethane is replaced by N,O-bistrimethylsilylacetamide, compared with Example 1, penicillic acid cannot undergo a derivatization reaction, that is, the hydroxyl group in the structural formula cannot be derivatized into a methoxy group, which ultimately makes it impossible to perform LC-MS detection.

[0115] Comparative Example 4

[0116] This comparative example provides a liquid chromatography-mass spectrometry (LC-MS) detection method for penicillic acid. The LC-MS detection method is the same as Example 1 except that a n-hexane solution without trimethylsilylated diazomethane is used in step (2), and will not be described in detail here.

[0117] In this comparative example, no derivatization reaction occurs to penicillic acid, that is, the hydroxyl group in the structural formula is not derivatized and converted into a methoxy group, which ultimately makes it impossible to perform LC-MS detection.

[0118] It can be seen from Example 1 and Comparative Examples 1 to 4 that the selection of the derivatization reagent for the derivatization reaction of penicillic acid in the present invention is very critical. The present invention selects trimethylsilylated diazomethane to perform methylation derivatization treatment on the sample containing penicillic acid, which can derivatize the hydroxyl group into methoxy group. The derivatization reagent itself has no negative impact on the subsequent LC-MS detection, thereby achieving effective detection of penicillic acid.

[0119] Test method: The above examples and comparative examples were tested by liquid chromatography-mass spectrometry.

[0120] Preparation of standard curve: dilute penicillic acid with methanol to 10 pg / mL, 30 pg / mL, 100 pg / mL, 300 pg / mL, 1000 pg / mL, 3000 pg / mL and 10000 pg / mL, take 50 μL of each and add it into a 2 mL EP tube, which is equivalent to the supernatant in step (1) of the above embodiment and comparative example as the penicillic acid-containing sample, and use the subsequent steps (2) to (3) of the embodiment and comparative example to prepare the standard curve.

[0121] The R of the standard curve in the above example 2 As shown in Table 2. Sample A and sample B with known sample concentrations of 15.3 μg / kg and 108 μg / kg were tested 5 times to evaluate the accuracy and repeatability of the test.

[0122] The test results are shown in Table 2.

[0123] Table 2

[0124]

[0125] From Table 2, we can see the following points:

[0126] (1) It can be seen from Examples 1 to 3 that the LC-MS detection method for penicillic acid provided by the present invention can realize the LC-MS detection of penicillic acid in red yeast rice, and the standard deviation of 5 repetitions is within 1.6%, the relative deviation is within 2%, and the detection sensitivity can be as low as 1 μg / kg;

[0127] (2) It can be seen from Example 1 and Example 4 that in Example 4, when methanol is not present, the peaks of the two isomeric derivatives of penicillic acid cannot be eluted from the chromatographic column adsorption, and mass spectrometry signals cannot be generated, making it difficult to achieve liquid-mass detection;

[0128] (3) Combining Example 1 and Example 5, it can be seen that when ammonium formate is replaced by ammonium acetate in Example 5, the mass spectrometry response signals of the peaks of the two penicillic acid isomer derivatives are weakened compared with Example 1, by about 5%, and the relative deviation from the known value is as high as 7.8%, which shows that the present invention has better detection sensitivity by selecting ammonium formate as the solute in the mobile phase;

[0129] (4) It can be seen from Example 1 and Examples 6 to 7 that in Example 1, during the LC-MS detection process, the volume proportion of the second phase includes a first stage, a second stage and a third stage as the detection proceeds, wherein the volume proportion of the second phase in the first stage is 10%, the volume proportion of the second phase in the second stage is 100%, and the volume proportion of the second phase in the third stage is 10%. Compared with Example 6 in which the volume proportion of the second phase in the mobile phase is always maintained at 10% and Example 7 in which the volume proportion of the second phase in the mobile phase is always maintained at 90%, the standard deviation of the five repetitions in Example 1 is within 1.2%, and the relative deviation is within 0.9%, while the standard deviation of the five repetitions in Examples 6 to 7 is as high as 10.2% and 9.9%, respectively, and the relative deviation is as high as 21.6% and 28.1%, respectively. This shows that the present invention can better achieve the separation of the peaks of the two isomer derivatives of penicillic acid by preferably controlling the volume proportions of the second phase and the first phase in the mobile phase of the LC-MS detection within different proportion ranges in different stages, and the detection result is more accurate.

[0130] The present invention illustrates the detailed features of the present invention through the above embodiments, but the present invention is not limited to the above detailed features, that is, it does not mean that the present invention must rely on the above detailed features to be implemented. Those skilled in the art should understand that any improvement to the present invention, equivalent replacement of the technical features selected by the present invention, addition of auxiliary technical features, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A liquid chromatography-mass spectrometry method for detecting penicillic acid, characterized in that: The liquid chromatography-mass spectrometry detection method comprises: using trimethylsilylated diazomethane to perform methylation derivatization treatment on a sample containing penicillic acid to obtain a derivatized sample, and performing liquid chromatography-mass spectrometry detection on the derivatized sample to obtain the content of penicillic acid in the red yeast rice.

2. The LC-MS detection method according to claim 1, characterized in that: The methylation derivatization treatment includes: mixing a trimethylsilylated diazomethane solution and a sample containing penicillic acid to obtain a mixed sample, and then shaking, quenching, drying, re-dissolving and solid-liquid separation in sequence, wherein the liquid phase of the solid-liquid separation is the derivatized sample.

3. The LC-MS detection method according to claim 2, characterized in that: The solvent in the trimethylsilylated diazomethane solution includes n-hexane; and / or, the concentration of trimethylsilylated diazomethane in the trimethylsilylated diazomethane solution is 1 to 3 mol / L; And / or, the volume ratio of the trimethylsilylated diazomethane solution to the sample containing penicillic acid is 1:(2-7).

4. The LC-MS detection method according to claim 2, characterized in that: The rotation speed of the oscillation is 100-500 rpm, and the temperature of the oscillation is 20-40° C.; And / or, the quenching agent used for the quenching includes acetic acid, and the volume ratio of the quenching agent to the mixed sample is 1:(4-8).

5. The LC-MS detection method according to claim 1, characterized in that: The preparation of the sample containing penicillic acid comprises: mixing the sample to be tested with a methanol aqueous solution, and sequentially homogenizing and centrifuging, and the supernatant after centrifugation is the sample containing penicillic acid.

6. The LC-MS detection method according to claim 1, characterized in that: The temperature of the chromatographic column for the LC-MS detection is 30-40°C.

7. The LC-MS detection method according to claim 1, characterized in that: The injection volume of the LC-MS detection is 1 to 10 μL; And / or, the flow rate of the mobile phase for the LC-MS detection is 0.1-0.5 mL / min.

8. The LC-MS detection method according to claim 1, characterized in that: The mobile phase for LC-MS detection includes a first phase and a second phase, wherein the first phase is an aqueous solution of ammonium formate; and the second phase is an aqueous solution of methanol containing ammonium formate.

9. The LC-MS detection method according to claim 8, characterized in that: The concentration of ammonium formate in the ammonium formate aqueous solution is 5 to 15 mmol / L; The methanol aqueous solution containing ammonium formate; The volume ratio of methanol to water in the methanol aqueous solution containing ammonium formate is (0.8-1.2):1, preferably 1:

1.

10. The liquid chromatography-mass spectrometry detection method according to claim 9, characterized in that: The volume proportion of the second phase in the mobile phase of the LC-MS detection is 5 to 100%; The volume proportion of the second phase in the mobile phase of the LC-MS detection includes a first stage, a second stage and a third stage as the detection proceeds, wherein the volume proportion of the second phase in the first stage is 5-15%, the volume proportion of the second phase in the second stage is 90-100%, and the volume proportion of the second phase in the third stage is 5-15%; The time period of the first stage is from 0 to 0.8 minutes; The time period of the second stage is from 0.5 to 4.2 minutes; The time period of the third stage is 4.1 to 5.5 minutes.

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