Method for determining concentration of monoravir and / or active metabolite beta-d-N4-hydroxycytidine thereof in human body fluid based on liquid chromatography-mass spectrometry after benzoyl chloride chemical derivatization

Abstract

The invention provides a method for determining the concentration of monoravir and / or an active metabolite beta-d-N4-hydroxycytidine thereof in human body fluid based on liquid chromatography-mass spectrometry after benzoyl chloride chemical derivatization, and belongs to the technical field of medical examination. According to the method, benzoyl chloride serving as a derivatization reagent reacts with Monoravir and / or beta-d-N4-hydroxycytidine in a human body fluid sample to generate a corresponding derivatization product, so that the concentration of Monoravir and / or beta-d-N4-hydroxycytidine is indirectly determined. An isotope substituted derivatization reagent benzoyl chloride-D5 reagent reacts with Monoravir and / or beta-d-N4-hydroxycytidine to generate a corresponding deuterated derivatization product as an internal standard. The method solves the problem of lack of a method for determining the monoravir and / or beta-d-N4-hydroxycytidine, which has high sensitivity, a simple and rapid pretreatment process and a simple and universal mobile phase at the same time, at present.

Description

Technical Field

[0001] The present invention belongs to the field of medical testing technology, and specifically relates to a method for determining the concentration of monolavine and / or its active metabolite β-d-N4-hydroxycytidine in human body fluids based on benzoyl chloride chemical derivatization followed by liquid chromatography-mass spectrometry. Background Art

[0003] After literature search, only 4 papers were found. [1～4] Determination of the concentration of monolavin and / or its active metabolite β-d-N4-hydroxycytidine in human plasma, of which only one article simultaneously determined monolavin and its active metabolite β-d-N4-hydroxycytidine. More importantly, a common disadvantage of most of these methods is that in order to achieve higher sensitivity, a drying and re-dissolution process is used in the pretreatment process, which makes the pretreatment operation complicated and time-consuming. Among them, Gouda AS et al. [1] The sample was concentrated by vacuum evaporation followed by reconstitution, which improved the quantification limit of β-d-N4-hydroxycytidine to 20 ng mL -1 However, in the pharmacokinetic analysis of healthy volunteers 0.25 to 12 hours after administration, 11.4% of patients still had blood drug concentrations below the limit of quantification. Komarov T et al. [2] Although the method directly uses acetonitrile protein precipitation with 2 times the sample volume, the pretreatment is relatively simple, but the proportion of protein precipitant used is too low, which may lead to insufficient plasma protein precipitation and easy blockage of chromatographic columns and instrument pipelines. In addition, the sample recovery rate and matrix effect at different concentrations in the study vary greatly, indicating that the pretreatment method is not appropriate.

[0004] References

[0005] [1]Gouda AS,Marzouk HM,Rezk MR,Salem AM,Morsi MI,Nouman EG,AbdallahYM,Hassan AY and Abdel-Megied AM(2022)A validated LC-MS / MS method fordetermination of antiviral prodrug molnupiravir in human plasma and its application for a pharmacokinetic modeling study in healthy Egyptian volunteers.J Chromatogr B Analyt Technol Biomed Life Sci 1206:123363.

[0006] [2]Komarov,T,Karnakova,P,Archakova,O,Shchelgacheva,D,Bagaeva,N,Popova,M,Karpova,P,Zaslavskaya,K,Bely,P,and Shohin,I(2023).Development andvalidation of a high-performance liquid chromatography with tandem massspectrometry(HPLC-MS / MS)method for quantification of major molnupiravirmetabolite(β-D-N4-hydroxycytidine)in human plasma.Biomedicines,11(9),2356.)

[0007] [3]Amara A,Penchala SD,Else L,Hale C,FitzGerald R,Walker L,Lyons R,Fletcher T and Khoo S(2021)The development and validation of a novel LC-MS / MSmethod for the simultaneous quantification of molnupiravir and its metabolitess-d-N4-hydroxycytidine in human plasma and saliva.J Pharm Biomed Anal 206:114356.

[0008] [4] Parsons TL, Kryszak LA and Marzinke MA (2021) Development and validation of assays for the quantification of beta-DN(4)-hydroxycytidine in human plasma and beta-DN(4)-hydroxycytidine-triphosphate in peripheral bloodmononuclear cell lysates. J Chromatogr B Analyt Technol Biomed Life Sci 1182:122921. Summary of the invention

[0009] The present invention is made to solve the above-mentioned problems, and aims to provide a method for determining the concentration of monolavine and / or its active metabolite β-d-N4-hydroxycytidine in human body fluids based on benzoyl chloride chemical derivatization and liquid chromatography-mass spectrometry.

[0010] The present invention provides a method for determining the concentration of monolavir and / or its active metabolite β-d-N4-hydroxycytidine in human body fluids based on benzoyl chloride chemical derivatization followed by liquid chromatography-mass spectrometry, which has the following characteristics: a derivatization reagent benzoyl chloride is reacted with monolavir and / or β-d-N4-hydroxycytidine in a human body fluid sample to generate corresponding derivatization products, thereby indirectly determining the concentration of monolavir and / or β-d-N4-hydroxycytidine in the body fluid sample, and an isotope-substituted derivatization reagent benzoyl chloride-D5 reagent is reacted with monolavir and / or β-d-N4-hydroxycytidine to generate corresponding deuterated derivatization products as internal standards.

[0011] In the method provided by the present invention for determining the concentration of monolavil and / or its active metabolite β-d-N4-hydroxycytidine in human body fluids based on benzoyl chloride chemical derivatization and liquid chromatography-mass spectrometry, the method may also have the following characteristics: wherein the body fluid in the body fluid sample is any one of saliva, urine, plasma, tissue fluid, lymph fluid, and cerebrospinal fluid.

[0012] In the method provided by the present invention for determining the concentration of monolavin and / or its active metabolite β-d-N4-hydroxycytidine in human body fluids based on benzoyl chloride chemical derivatization and liquid chromatography-mass spectrometry, it can also have the following characteristics: wherein the body fluid sample is a sample prepared by mixing monolavin and / or β-d-N4-hydroxycytidine standards with body fluids, or is fresh body fluid obtained from a human body taking monolavin.

[0013] In the method provided by the present invention for determining the concentration of monolavine and / or its active metabolite β-d-N4-hydroxycytidine in human body fluids by liquid chromatography-mass spectrometry after chemical derivatization with benzoyl chloride, the method may also have such a feature that the method comprises the following steps:

[0014] Step 1, prepare internal standard solution:

[0015] Adding the monolavir stock solution and / or the β-d-N4-hydroxycytidine stock solution to the organic reagent, and adding the benzoyl chloride-D5 reagent, vortexing to mix, reacting at a predetermined temperature for a predetermined time, and further adding the organic reagent to dilute to the final concentration;

[0016] Step 2, pretreatment of body fluid samples and derivatization of analytes in samples:

[0017] The body fluid sample is placed in a centrifuge tube, and an organic reagent containing a predetermined concentration of benzoyl chloride is added to precipitate the protein, thereby chemically derivatizing while extracting the monolavin stock solution and / or β-d-N4-hydroxycytidine, and after centrifugation, the quantitative supernatant is drawn, and an internal standard solution and water are added to obtain a mixed solution to be tested;

[0018] Step 3, sample separation:

[0019] The mixed solution to be tested is separated by liquid chromatography using a phenyl high performance liquid chromatography column, the liquid chromatography system comprises a high pressure metering pump, a temperature-controlled column oven and an automatic sample injector, and acetonitrile and water are used as mobile phases for gradient elution to obtain a sample to be tested;

[0020] Step 4, tandem mass spectrometry detection and concentration calculation:

[0021] A tandem mass spectrometer was used to detect the peak area As of the derivatized product of the monolavin stock solution and / or β-d-N4-hydroxycytidine and the peak area Ai of the internal standard in the sample to be tested. The concentration of the monolavin stock solution and / or β-d-N4-hydroxycytidine was used as the X-axis, and the ratio of the peak area As of the derivatized product to the peak area Ai of the internal standard was used as the Y-axis. Weighted regression analysis was performed, and the regression equation of the standard curve and the concentration of the monolavin stock solution and / or β-d-N4-hydroxycytidine were calculated using content analysis software.

[0022] In the method provided by the present invention for determining the concentration of monolavil and / or its active metabolite β-d-N4-hydroxycytidine in human body fluids by liquid chromatography-mass spectrometry after chemical derivatization with benzoyl chloride, the method may also have the following characteristics: wherein, in step 1, the organic solvent is acetonitrile, the predetermined temperature is room temperature, and the predetermined time is 9-12 minutes.

[0023] In the method provided by the present invention for determining the concentration of monolavil and / or its active metabolite β-d-N4-hydroxycytidine in human body fluids by liquid chromatography-mass spectrometry based on chemical derivatization with benzoyl chloride, the method may also have the following characteristics: wherein, in step 2, the organic reagent is acetonitrile, the predetermined concentration is 1% (V / V), and the centrifugal speed is 13000 rpm.

[0024] In the method provided by the present invention for determining the concentration of monolavil and / or its active metabolite β-d-N4-hydroxycytidine in human body fluids by liquid chromatography-mass spectrometry after chemical derivatization with benzoyl chloride, the method may also have the following characteristics: wherein, in step 2, the volume ratio of the body fluid sample to the organic reagent containing a predetermined concentration of benzoyl chloride is 1:3, and the volume ratio of the supernatant, the internal standard solution, and the water is 20:1:10.

[0025] In the method provided by the present invention for determining the concentration of monolavine and / or its active metabolite β-d-N4-hydroxycytidine in human body fluids by LC-MS based on benzoyl chloride chemical derivatization, the method may also have the following characteristics: wherein, in step 3, the phenyl high performance liquid chromatography column is an Agilent Eclipse Plus Phenyl-Hexyl column (4.6×250 mm, 5 μm),

[0026] The mobile phase was acetonitrile: water, with a gradient of: 0.01-0.5 min, 30% acetonitrile, 0.5-8.0 min, 30-58% acetonitrile, 8.0-8.1 min, 58-90% acetonitrile, 8.1-9.0 min, 90% acetonitrile, 9.0-9.6 min, 90-30% acetonitrile, 9.6-10 min, 30% acetonitrile, flow rate 1 mL min -1 .

[0027] In the method provided by the present invention for determining the concentration of monolavin and / or its active metabolite β-d-N4-hydroxycytidine in human body fluids by liquid chromatography-mass spectrometry after chemical derivatization with benzoyl chloride, the method may also have the following characteristics: wherein, in step 4, the tandem mass spectrometer detector is a universal tandem mass spectrometer detector, the mass spectrometer ionization mode is: electrospray ionization source, the scanning mode is: multiple reaction monitoring (MRM) positive ion mode, and the ion channels are: m / z 434.2→105.2 (monolavir derivatization product), m / z 364.2→105.3 (β-d-N4-hydroxycytidine derivatization product), m / z 439.2→110.1 (internal standard, monolavin deuterated derivatization product) and m / z 369.2→110.0 (internal standard, β-d-N4-hydroxycytidine deuterated derivatization product).

[0028] In the method provided by the present invention for determining the concentration of monolavin and / or its active metabolite β-d-N4-hydroxycytidine in human body fluids by liquid chromatography-mass spectrometry based on benzoyl chloride chemical derivatization, the method may also have the following characteristics: wherein, in step 4, the mass spectrometer setting parameters are: electrospray voltage 5500V, ion source temperature 700°C, curtain gas 25Arb, collision gas Ar gas pressure of Medium, ion source gases 1 and 2 are 65psi and 45psi respectively, the DP voltages of monolavin derivatization product, β-d-N4-hydroxycytidine derivatization product, monolavin deuterated derivatization product and β-d-N4-hydroxycytidine deuterated derivatization product are 36eV, 38eV, 36eV and 27eV respectively, the collision energies are 33eV, 31eV, 46eV and 40eV respectively, and the scanning time is 200ms.

[0029] The technical effects of the present invention are as follows:

[0030] Based on previous research, the present invention adopts a benzoyl derivatization method to improve the detection sensitivity of the analyte, and the present invention innovatively adopts acetonitrile containing benzoyl chloride for protein precipitation pretreatment, so that the extraction and derivatization of the analyte are carried out simultaneously, which is simpler and faster than the pretreatment process reported in the existing literature.

[0031] The present invention solves the problem of the lack of a method for determining monolavir and / or β-d-N4-hydroxycytidine that has high sensitivity, a simple and rapid pretreatment process, and a simple and universal mobile phase. The method can be routinely used on instruments with relatively poor sensitivity, and the mobile phase is simple and universal, and can be used in more clinical laboratories to carry out concentration determination of monolavir and / or β-d-N4-hydroxycytidine in patient body fluid samples, especially plasma samples.

[0032] In addition, the method of the present invention not only has high detection accuracy, but also has good stability of monolavir / or its active metabolite β-d-N4-hydroxycytidine after derivatization. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of the derivatization of Monolavin and β-d-N4-hydroxycytidine benzoyl chloride in an embodiment of the present invention.

[0034] Figure 2 It is the MRM diagram of the analyte and the internal standard in the embodiment of the present invention (A: monolavir derivative; B: β-d-N4-hydroxycytidine derivative; C: monolavir deuterated derivative; D: β-d-N4-hydroxycytidine deuterated derivative).

[0035] Figure 3These are the non-derivatized and derivatized chromatograms of Monolavine and β-d-N4-hydroxycytidine at the quantitative lower limit concentration in the examples of the present invention (A: non-derivatized; B: derivatized).

[0036] Figure 4 It is the chromatogram of blank matrix plasma of healthy volunteers and blank matrix plasma with added standard in the embodiments of the present invention (A: Monolavine derivative; B: β-d-N4-hydroxycytidine derivative; C: internal standard Monolavine deuterated derivative; D: internal standard β-d-N4-hydroxycytidine derivative). DETAILED DESCRIPTION

[0037] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the following examples and accompanying drawings specifically illustrate a method of the present invention for determining the concentration of monolavil and / or its active metabolite β-d-N4-hydroxycytidine in human body fluids based on benzoyl chloride chemical derivatization and liquid chromatography-mass spectrometry.

[0038] Example 1 Method for determining the concentration of monolavine and / or its active metabolite β-d-N4-hydroxycytidine in human body fluids by LC-MS after chemical derivatization with benzoyl chloride

[0039] This embodiment provides a method for determining the concentration of monolavin and / or its active metabolite β-d-N4-hydroxycytidine in human body fluids based on benzoyl chloride chemical derivatization followed by liquid chromatography-mass spectrometry (LC-MS / MS), wherein the derivatization reagent benzoyl chloride reacts with monolavin and / or β-d-N4-hydroxycytidine in human body fluid samples to generate corresponding derivatization products, thereby indirectly determining the concentration of monolavin and / or β-d-N4-hydroxycytidine in body fluid samples, and an isotope-substituted derivatization reagent benzoyl chloride-D5 reagent reacts with monolavin and / or β-d-N4-hydroxycytidine to generate corresponding deuterated derivatization products as internal standards. Specifically, the following steps are included:

[0040] Step 1, prepare internal standard solution :

[0041] Add monolavin stock solution and / or β-d-N4-hydroxycytidine stock solution to acetonitrile, add benzoyl chloride-D5 reagent, vortex mix, react at room temperature for 10 minutes, and then further add acetonitrile to dilute, so that the concentration of monolavin deuterated derivative and β-d-N4-hydroxycytidine deuterated derivative after theoretical reaction is complete is 500 ng mL -1 of internal standard solution.

[0042] Step 2, pretreatment of body fluid samples and derivatization of analytes in samples:

[0043] The body fluid sample is placed in a centrifuge tube, and acetonitrile containing 1% (V / V) benzoyl chloride is added to precipitate the protein, thereby chemically derivatizing while extracting the monolavin stock solution and / or β-d-N4-hydroxycytidine. After centrifugation at 13000 rpm, the quantitative supernatant is aspirated, and the internal standard solution and water are added to obtain a mixed solution to be tested.

[0044] The body fluid in the body fluid sample is any one of saliva, urine, plasma, tissue fluid, lymph fluid, and cerebrospinal fluid, preferably plasma. The body fluid sample is a sample prepared by mixing monolavir and / or β-d-N4-hydroxycytidine standard with body fluid, or is a fresh body fluid obtained from a human body taking monolavir.

[0045] The volume ratio of the body fluid sample to acetonitrile containing 1% (V / V) benzoyl chloride was 1:3, and the volume ratio of the supernatant, the internal standard solution, and water was 20:1:10.

[0046] Step 3, sample separation:

[0047] The mixed solution to be tested obtained in step 2 is separated by liquid chromatography using a phenyl high performance liquid chromatography column. The liquid chromatography system comprises a high pressure metering pump, a temperature-controlled column oven and an automatic sample injector, and acetonitrile and water are used as mobile phases, and gradient elution is performed. The chromatographic conditions are: Agilent Eclipse Plus Phenyl-Hexyl (4.6×250 mm, 5 μm) chromatographic column, a column temperature of 40° C., a gradient elution program of 0.01 to 0.5 min, 30% acetonitrile, 0.5 to 8.0 min, 30 to 58% acetonitrile, 8.0 to 8.1 min, 58 to 90% acetonitrile, 8.1 to 9.0 min, 90% acetonitrile, 9.0 to 9.6 min, 90 to 30% acetonitrile, 9.6 to 10 min, 30% acetonitrile, and a flow rate of 1 mL·min -1 , and finally the sample to be tested is obtained.

[0048] Step 4, tandem mass spectrometry detection and concentration calculation:

[0049] A universal tandem mass spectrometer is used to detect the peak area As of the derivatized product of the monolavir stock solution and / or β-d-N4-hydroxycytidine and the peak area Ai of the internal standard in the test sample obtained in step 3. The concentration of the monolavir stock solution and / or β-d-N4-hydroxycytidine is used as the X-axis, and the ratio of the peak area As of the derivatized product to the peak area Ai of the internal standard is used as the Y-axis. A weighted regression analysis is performed, and the regression equation of the standard curve and the concentration of the monolavir stock solution and / or β-d-N4-hydroxycytidine are calculated using content analysis software.

[0050] Among them, the mass spectrometry ionization mode of tandem mass spectrometry detection is: electrospray ionization source; the scanning mode is: ESI positive ion multiple reaction monitoring (MRM) positive ion mode; the ion channels are: m / z 434.2→105.2 (monolavir derivatization product), m / z364.2→105.3 (β-d-N4-hydroxycytidine derivatization product), m / z 439.2→110.1 (internal standard, monolavin deuterated product) and m / z 369.2→110.0 (internal standard, β-d-N4-hydroxycytidine deuterated product).

[0051] The mass spectrometer setting parameters were: electrospray voltage 5500 V, ion source temperature 700°C, curtain gas 25 Arb, collision gas Ar pressure Medium, ion source gas 1 and 2 65 psi and 45 psi respectively.

[0052] The DP voltages of the monolavin derivatization product, β-d-N4-hydroxycytidine derivatization product, monolavin deuterated product and β-d-N4-hydroxycytidine deuterated product were 36eV, 38eV, 36eV and 27eV, respectively, the collision energies were 33eV, 31eV, 46eV and 40eV, respectively, and the scanning time was 200ms.

[0053] Example 2 The sensitivity of the derivatized method for determining monolavir and β-d-N4-hydroxycytidine is significantly better than that of the non-derivatized method

[0054] Monolavin and β-d-N4-hydroxycytidine plasma samples were determined using LC-MS / MS method based on benzoyl chloride derivatization and non-derivatized LC-MS / MS method, respectively. The lower limits of quantification of the two methods and the peak areas of the analytes at the same quality control concentrations of low, medium and high were compared.

[0055] 1. Determination method:

[0056] 1) LC-MS / MS method based on benzoyl chloride derivatization

[0057] The reaction product of monolavin and β-d-N4-hydroxycytidine with benzoyl chloride was used as the analyte, and the reaction product of monolavin and β-d-N4-hydroxycytidine with benzoyl chloride-D5 was used as the internal standard. The derivatization reaction was as follows: Figure 1 The MRM diagram of mass spectrometry scanning is shown in Figure 2 The specific method is shown in Example 4.

[0058] 2) Non-derivatized LC-MS / MS method

[0059] The instrumentation is the same as the derivatization method. The chromatographic and mass spectrometric conditions, sample pretreatment methods, etc. are as follows:

[0060] Chromatographic conditions: Agilent Zorbax SB-Aq C 18 column (4.6×250mm, 5μm), column temperature 40℃, mobile phase acetonitrile:0.1% formic acid water, gradient elution, 0.01~3.1min, 12% acetonitrile, 3.1~8.0min, 12~60% acetonitrile, 8.0~8.1min, 60~12% acetonitrile, 8.1~10.0min, 12% acetonitrile, flow rate 1mL·min -1 The injection volume was 10 μL and the autosampler temperature was set at 5 °C.

[0061] Mass spectrometry conditions: ESI positive ion multiple reaction monitoring (MRM) mode; electrospray voltage 5500V; ion source temperature 700℃; curtain gas 25Arb; collision gas (Ar) pressure is Medium; ion source gas 1 and 2 are 65psi and 45psi respectively. The ion pairs used for quantification are: m / z 330.5→128.1 (monolavir), m / z 260.2→128.0 (β-d-N4-hydroxycytidine), DP voltages of monolavir and β-d-N4-hydroxycytidine are 39V and 50V respectively; collision energy is 23V and 22V respectively, and scanning time is 200ms.

[0062] Plasma sample pretreatment: Take 100 μL of plasma sample, add 300 μL of acetonitrile, vortex for 1 min to mix, centrifuge for 10 min (13000 rpm), take the supernatant and inject 10 μL.

[0063] 2. Comparison of measurement results

[0064] 1) Comparison of lower limit of quantification

[0065] The two methods were compared to determine the concentrations of monolavin and β-d-N4-hydroxycytidine in plasma samples corresponding to the standard that the chromatographic peak of the analyte reached a signal-to-noise ratio (S / N)>10. Results: The lower limits of quantification of monolavin and β-d-N4-hydroxycytidine by the non-derivatized LC-MS / MS method were approximately 10 ng·mL -1 L and 25ng·mL -1 The lower limits of quantification of monolavine and β-d-N4-hydroxycytidine based on LC-MS / MS method with benzoyl chloride derivatization were approximately 1.0 ng·mL -1 and 2.5 ng·mL -1 Therefore, the derivatization method is more sensitive than the non-derivatization method and can achieve a lower limit of quantification. The S / N and peak area of ​​monolavir and β-d-N4-hydroxycytidine by non-derivatization and derivatization methods are shown in Table 1. The chromatograms of the quantitative lower limits of the two methods are shown in Figure 3 .

[0066] Table 1 Comparison of S / N lower limits of quantification of Monolavine and β-d-N4-hydroxycytidine by non-derivatized and derivatized methods (n=6)

[0067]

[0068] 2) Comparison of peak areas of low, medium and high concentration quality control samples

[0069] The concentrations of monolavine / β-d-N4-hydroxycytidine were 2 / 5, 40 / 100 and 1600 / 4000 ng·mL -1 Plasma samples were measured using the non-derivatized and derivatized methods, respectively, and the peak areas of the analytes at the same sample concentration measured by the two methods were compared.

[0070] Results: Monolavin / β-d-N4-hydroxycytidine concentration was 2 / 5 ng·mL -1 When the derivatization method was used, there were no effective chromatographic peaks for Monolavin and β-d-N4-hydroxycytidine. The concentrations of Monolavin were 40 and 1600 ng·mL, respectively. -1 When the derivatization method was used, the peak area of ​​the analyte was 27.6 and 25.0 times larger than that of the non-derivatized method; the concentrations of β-d-N4-hydroxycytidine were 100 and 4000 ng·mL -1 When the peak area of ​​the derivatized method was 58.1 and 41.0 times larger than that of the non-derivatized method, respectively. At the same concentration, the peak area of ​​the derivatized method was larger and the coefficient of variation was smaller than that of the non-derivatized method. The specific peak area data are shown in Table 2.

[0071] Table 2 Peak areas of non-derivatized and derivatized analytes of Monolavine and β-d-N4-hydroxycytidine (n=6)

[0072]

[0073] Note: N / A: No valid chromatographic peak

[0074] Example 3 The method for determining the derivatization of monolavin and β-d-N4-hydroxycytidine of the present invention has the advantages of low instrument requirements, high sensitivity, simple and rapid pretreatment process, simple and universal mobile phase, and the ability to simultaneously determine the concentrations of monolavin and β-d-N4-hydroxycytidine.

[0075] 1. Comparison of instruments and equipment used in this invention and existing literature

[0076] The present invention uses Sciex API 3200 triple quadrupole mass spectrometer (AB Company, USA) as a detector, which can make the lower limit of quantification of monolavin in plasma samples reach 1 ng·mL -1 The lower limit of quantification of β-d-N4-hydroxycytidine reached 2.5 ng·mL -1.

[0077] This patent further summarizes the mass spectrometer models and quantitative limits used in existing literature, as shown in Table 3. Compared with existing literature, each literature generally uses a mass spectrometer that entered the market later and has higher sensitivity. For example, some studies used AB SciexAPI 4500 and Shimadzu LCMS 8040 mass spectrometers, but the quantitative limit of monoclavir was higher than that of the present invention (2.5 ng·mL -1 ), the lower limit of quantification of β-d-N4-hydroxycytidine is the same as or higher than that of the present invention (2.5 ng·mL -1 , 10ng·mL -1 , 20ng·mL -1 ). Although Parsons TL et al. used AB Sciex API 5000 triple quadrupole mass spectrometer to determine β-d-N4-hydroxycytidine, they achieved a lower limit of quantification (1 ng mL -1 ), but simultaneous determination with monolavir was not achieved.

[0078] By analyzing the data of these existing documents, we concluded that: the present invention adopts the method of benzoyl chloride derivatization, so that even if the equipment that was launched earlier and has relatively low sensitivity is used, monolavir and β-d-N4-hydroxycytidine can still reach a lower limit of quantification, and the accuracy and precision meet the requirements. Therefore, the present invention reduces the requirements for instruments and equipment for the method of determining the blood concentration of monolavir and β-d-N4-hydroxycytidine, so that the method can be applied in more laboratories.

[0079] 2. The drug extraction and derivatization are carried out simultaneously, making the pretreatment simpler and faster

[0080] The present invention uses acetonitrile containing benzoyl chloride derivatization reagent as a protein precipitation reagent, and uses a protein precipitation reagent and plasma 3:1 (V / V) to extract monolavir and β-d-N4-hydroxycytidine to simultaneously derive compounds. The differences from the conventional pre-treatment operations in the existing literature are further compared and summarized, and the specific results are shown in Table 3. Compared with the results reported in the existing literature, the sample pretreatment process adopted in this patent does not require a post-drying redissolution operation, which greatly simplifies the operation steps and shortens the sample pretreatment time. Although Komarov T et al. used acetonitrile and plasma 2:1 (V / V) for protein precipitation, the operation is also relatively simple, but the sample recovery rate and matrix effect at different concentrations in their study vary greatly.

[0081] 3. The chromatographic retention capacity of the analyte after derivatization is improved, and the mobile phase requirements are simple

[0082] The present invention uses benzoyl chloride derivatization to improve the retention capacity of the analyte on the chromatographic column and delay the retention time, so that a chromatographic peak with a good peak shape can be obtained by using a simple mobile phase. Compared with the mobile phase added with acid, base or salt used in the literature, the present invention does not need to prepare the mobile phase.

[0083] Table 3 Comparison of the method for determining the concentration of monolavir and / or β-d-N4-hydroxycytidine in human plasma with the method of this patent

[0084]

[0085]

[0086] Note: MPV: Monolavin; NHC: β-d-N4-hydroxycytidine; / : not determined in the literature

[0087] Example 4 Specific method for simultaneous determination of the concentration of monolavine and its active metabolite β-d-N4-hydroxycytidine in human plasma by LC-MS / MS after chemical derivatization with benzoyl chloride

[0088] This example highlights the good selectivity and high accuracy of this method. The analyte after derivatization has good stability. All methodological validation results meet the requirements and can be used for the simultaneous determination of the concentrations of monoclavir and its active metabolite β-d-N4-hydroxycytidine in human plasma.

[0089] 1. Experimental Materials

[0090] 1) Instrument: API 3200 triple quadrupole mass spectrometer (AB, USA) connected in series with LC-20AD high performance liquid chromatograph (Shimadzu, Japan), equipped with electrospray ionization source (ESI); data acquisition software: Analyst

[0091] 1.7.2, electronic analytical balance (Mettler, Switzerland); 5804R high-speed low-temperature centrifuge (EPPENDORF, Germany), etc.

[0092] 2) Materials and reagents: Monolavil standard, batch number F676864, purity 98% (Shanghai Yifei Biotechnology Co., Ltd.); β-d-N4-hydroxycytidine, batch number F850476, purity 98%, (Shanghai Yifei Biotechnology Co., Ltd.); benzoyl chloride and benzoyl chloride-D5, purity 99%, were purchased from Sigma-Aldrich; acetonitrile (chromatographic grade) was purchased from Sigma-Aldrich; ultrapure water was purchased from Wahaha Group Co., Ltd.; blank plasma was obtained from healthy volunteers.

[0093] 2. Experimental Methods

[0094] 1) Preparation of stock solution

[0095] Accurately weigh an appropriate amount of Monolavin standard into a 5 mL volumetric flask and add pure acetonitrile to a concentration of 200 μg mL -1 Accurately weigh an appropriate amount of β-d-N4-hydroxycytidine standard into a 5 mL volumetric flask and add 80% acetonitrile water to a concentration of 200 μg mL -1 Precisely weigh appropriate amounts of Monolavin and β-d-N4-hydroxycytidine standards into 5 mL volumetric flasks and prepare them with pure acetonitrile and 80% acetonitrile water to a concentration of 200 μg mL -1 Quality control stock solution.

[0096] 2) Preparation of internal standard solution

[0097] 10 μL of Monolavit stock solution, 10 μL of β-d-N4-hydroxycytidine stock solution, 20 μL of benzoyl chloride-D5 and 960 μL of acetonitrile were mixed and allowed to stand at room temperature for 10 min, and then 3 mL of acetonitrile was further added to dilute the mixture, so that the concentration of Monolavit deuterated derivative and β-d-N4-hydroxycytidine deuterated derivative was 500 ng mL after the reaction was theoretically complete. -1 of internal standard solution.

[0098] 3) HPLC-MS / MS conditions

[0099] Agilent Eclipse Plus Phenyl-Hexyl (4.6×250mm, 5μm) chromatographic column, column temperature 40℃, mobile phase acetonitrile: water, gradient elution, 0.01~0.5min, 30% acetonitrile, 0.5~8.0min, 30~58% acetonitrile, 8.0~8.1min, 58~90% acetonitrile, 8.1~9.0min, 90% acetonitrile, 9.0~9.6min, 90~30% acetonitrile, 9.6~10min, 30% acetonitrile, flow rate 1mL·min -1 The injection volume was 10 μL and the autosampler temperature was set at 5 °C.

[0100] ESI positive ion multiple reaction monitoring (MRM) mode; electrospray voltage 5500V; ion source temperature 700℃; curtain gas 25Arb; collision gas (Ar) pressure is Medium; ion source gas 1 and 2 are 65psi and 45psi respectively. The DP voltages of monolavir derivatization product, β-d-N4-hydroxycytidine derivatization product, monolavir deuterated derivatization product (internal standard) and β-d-N4-hydroxycytidine deuterated derivatization product (internal standard) are 36V, 38V, 36V and 27V respectively; the collision energy is 33V, 31V, 46V and 40V respectively, and the scanning time is 200ms. The ion reactions used for quantification were m / z 434.2→105.2 (monolavir derivatization product), m / z 364.2→105.3 (β-d-N4-hydroxycytidine derivatization product), m / z 439.2→110.1 (internal standard, monolavin deuterated product) and m / z 369.2→110.0 (internal standard, β-d-N4-hydroxycytidine deuterated product). MRM mass spectra are shown in Figure 2 .

[0101] 4) Pretreatment and derivatization methods

[0102] Take 100 μL of plasma sample, add 300 μL of acetonitrile solution containing 1% (V / V) benzoyl chloride, vortex for 1 min to mix, centrifuge for 10 min (13000 rpm). Take 200 μL of supernatant, mix with 10 μL internal standard solution and 100 μL water, vortex for 1 min, and inject 10 μL.

[0103] 4. Methodological Validation

[0104] 1) Selectivity

[0105] Blank plasma from 6 different sources (from healthy volunteers) was taken and analyzed after pretreatment; a certain concentration of standard solution was added to the blank plasma to prepare a plasma sample with a quantitative lower limit concentration, which was analyzed after pretreatment. The chromatograms of blank plasma samples and quantitative lower limit concentration samples are shown in Figure 4 , endogenous substances have no interference in the peak positions of the analyte and the internal standard.

[0106] 2) Linear

[0107] Take blank plasma, accurately add the standard curve stock solution of Monolavin and β-d-N4-hydroxycytidine, and prepare Monolavin concentrations of 5000, 2000, 500, 100, 20, 5, and 2.5 ng·mL -1 ; β-d-N4-hydroxycytidine concentrations were 2000, 800, 200, 40, 8, 2, and 1 ng·mL -1A series of standard curves of plasma samples; according to the plasma sample pretreatment operation, the sample was processed according to the "pretreatment conditions" and then analyzed to establish a standard curve. The concentration of the analyte in the serum X is the horizontal axis, and the peak area of ​​the analyte and the internal standard Y is the vertical axis. The weighted least squares method (W = 1 / x 2 ) to perform regression operation and obtain the regression equation, i.e., the standard curve. The typical linear regression equation of Monolavin is y=0.00638x+0.00183, r=0.9990; the typical linear regression equation of β-d-N4-hydroxycytidine is y=0.00625x+-0.00125, r=0.9995.

[0108] 3) Accuracy and precision

[0109] Blank plasma was taken and quality control (QC) solutions of Monolavix and β-d-N4-hydroxycytidine were precisely added to prepare monolavix / β-d-N4-hydroxycytidine solutions with concentrations of 1 / 2.5 (LLOQ), 2 / 5 (QCL), 40 / 100 (QCM) and 1600 / 4000 ng·mL -1 The plasma quantitative lower limit samples and quality control samples (QCH) were analyzed according to the operation under "4) Pretreatment and derivatization methods". Six samples were analyzed at each concentration and three batches were measured continuously for at least two days. The results are shown in Tables 5 and 6.

[0110] Table 5 Intra-batch precision and accuracy of the analytes

[0111]

[0112] Table 6 Inter-batch precision and accuracy of the analytes

[0113]

[0114] 4) Extraction recovery and matrix effect

[0115] The sample was protein precipitated with acetonitrile solution containing benzoyl chloride, and monolavin and β-d-N4-hydroxycytidine were extracted and derivatized at the same time. The extraction recovery and matrix effect were found to meet the requirements. The concentrations of monolavin / β-d-N4-hydroxycytidine were 2 / 5 (QCL), 40 / 100 (QCM) and 1600 / 4000 ng·mL -1(QCH) Plasma quality control samples, according to the operation under "4) Pretreatment and derivatization method", 6 samples were analyzed for each concentration, and the peak area Ax was recorded; blank plasma was taken and precipitated with 3 volumes of acetonitrile protein containing 1% (V / V) benzoyl chloride, and the supernatant was taken, and Monolav and β-d-N4-hydroxycytidine stock solutions were accurately added to prepare blank plasma extraction samples of corresponding concentrations, and the samples were allowed to stand for 10 minutes, and 200 μL was taken and mixed with 10 μL internal standard solution and 100 μL water, and 6 samples were analyzed for each concentration, and the peak area As was recorded, and the extraction recovery rate was calculated with the corresponding peak area ratio Ax / As. The recovery rates of Monolav and β-d-N4-hydroxycytidine at three concentrations were within the range of 100.9-107.4% and 107.4-108.4% respectively (Table 7), indicating that the extraction recovery rate of this method meets the requirements.

[0116] Six blank plasma samples from different sources were taken, and protein precipitation was performed with 3 volumes of acetonitrile containing 1% (V / V) benzoyl chloride, and the supernatant was taken to prepare samples; the quality control solution of the corresponding concentration was prepared using blank solvent, and 6 samples were prepared for each concentration. Internal standard normalized matrix effect factor (%) = ratio of peak area of ​​the sample prepared with matrix supernatant after precipitation to that of the sample prepared with blank solvent / ratio of peak area of ​​internal standard of the sample prepared with matrix supernatant after precipitation to that of the sample prepared with blank solvent × 100%. The results showed that the internal standard matrix effect factors of monolavir and β-d-N4-hydroxycytidine were between 92.7 and 101.9%, and the CVs were all less than 9.0% (Table 7), indicating that this method was not affected by the matrix components in human plasma.

[0117] Table 7 Extraction recovery and matrix effect of analytes in plasma

[0118]

[0119] 5) Dilution reliability

[0120] Prepare plasma samples with a concentration of 2 times the upper limit of quantitation of Monolavin and β-d-N4-hydroxycytidine, dilute them 2.5 times and 5 times with blank plasma, and operate according to the "Pretreatment and Derivatization Conditions". The results show that the concentration of samples higher than the upper limit of quantitation is within ±15% of the actual concentration after dilution.

[0121] 6) Residual Effect

[0122] After the high quality control sample injection was completed, the blank plasma sample was injected and repeated 6 times. No interference was found at the peak time of Monolavin, β-d-N4-hydroxycytidine and internal standard in the blank plasma sample. The blank sample signals were all lower than 20% of the lower limit of quantification and 5% of the internal standard.

[0123] 7) Stability

[0124] Prepare three concentrations of Monolavil plasma samples, and examine the stability at room temperature for 1h, 2h, and 3h, and at 4°C for 2h and 3h. Prepare three concentrations of β-d-N4-hydroxycytidine plasma samples, and examine the stability at room temperature and 4°C for 10h and 24h. Prepare three concentrations of quality control samples, and after operating under the "Pretreatment and Derivatization Conditions", examine the stability of the autosampler (5°C) for 5 days.

[0125] The results showed that the relative deviations of the measured concentrations and the labeled concentrations of Monolavin plasma samples after being placed at room temperature for 1 to 3 hours and at 4°C for 2 to 3 hours were both greater than ±15%; the relative deviations of the measured concentrations and the labeled concentrations of β-d-N4-hydroxycytidine plasma samples after being placed at room temperature and 4°C for 10 hours were within ±15%, and the relative deviations of the measured concentrations and the labeled concentrations after being placed at room temperature and 4°C for 24 hours were both greater than ±15%, and the relative deviations of the measured concentrations and the labeled concentrations of Monolavin and β-d-N4-hydroxycytidine after the plasma samples were pretreated and derivatized and then placed in the sampler for 5 days were within ±15% (Table 8, Table 9). This indicates that Monolavin is unstable in plasma, β-d-N4-hydroxycytidine is stable in plasma for at least 10 hours, and the stability of Monolavin and β-d-N4-hydroxy is improved after plasma pretreatment and derivatization, and they are stable in the autosampler for at least 5 days.

[0126] Table 8 Stability of Monolavin under different conditions

[0127]

[0128] Note: NA. The measured concentration is below the lower limit of quantification

[0129] Table 9 Stability of β-d-N4-hydroxycytidine under different conditions

[0130]

[0131] Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for determining the concentration of monolavine and / or its active metabolite β-d-N4-hydroxycytidine in human body fluids based on benzoyl chloride chemical derivatization and liquid chromatography-mass spectrometry, characterized in that: The derivatization reagent benzoyl chloride is reacted with monolavir and / or β-d-N4-hydroxycytidine in human body fluid samples to generate corresponding derivatization products, thereby indirectly determining the concentration of monolavir and / or β-d-N4-hydroxycytidine in the body fluid samples, and the isotope-substituted derivatization reagent benzoyl chloride-D5 reagent is reacted with monolavir and / or β-d-N4-hydroxycytidine to generate corresponding deuterated derivatization products as internal standards.

2. The method for determining the concentration of monolavine and / or its active metabolite β-d-N4-hydroxycytidine in human body fluids based on benzoyl chloride chemical derivatization and LC-MS / MS according to claim 1, characterized in that: in, The body fluid in the body fluid sample is any one of saliva, urine, plasma, tissue fluid, lymph fluid, and cerebrospinal fluid.

3. The method for determining the concentration of monolavine and / or its active metabolite β-d-N4-hydroxycytidine in human body fluids based on benzoyl chloride chemical derivatization and LC-MS / MS according to claim 1, characterized in that: in, The body fluid sample is a sample prepared by mixing monolavir and / or β-d-N4-hydroxycytidine standard with body fluid, or is fresh body fluid obtained from a human body taking monolavir.

4. The method for determining the concentration of monolavine and / or its active metabolite β-d-N4-hydroxycytidine in human body fluids based on benzoyl chloride chemical derivatization and LC-MS / MS according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: Step 1, prepare internal standard solution: Adding the monolavir stock solution and / or the β-d-N4-hydroxycytidine stock solution to the organic reagent, and adding the benzoyl chloride-D5 reagent, vortexing to mix, reacting at a predetermined temperature for a predetermined time, and further adding the organic reagent to dilute to the final concentration; Step 2, pretreatment of body fluid samples and derivatization of analytes in samples: The body fluid sample is placed in a centrifuge tube, and an organic reagent containing a predetermined concentration of benzoyl chloride is added to precipitate the protein, thereby chemically derivatizing while extracting the monolavin stock solution and / or β-d-N4-hydroxycytidine, and after centrifugation, the quantitative supernatant is drawn, and the internal standard solution and water are added to obtain a mixed solution to be tested; Step 3, sample separation: The mixed solution to be tested is subjected to liquid chromatography separation using a phenyl high performance liquid chromatography column, wherein the liquid chromatography system comprises a high pressure metering pump, a temperature-controlled column oven and an automatic sample injector, and acetonitrile and water are used as mobile phases for gradient elution to obtain a sample to be tested; Step 4, tandem mass spectrometry detection and concentration calculation: A tandem mass spectrometer was used to detect the peak area As of the derivatized product of the monolavin stock solution and / or β-d-N4-hydroxycytidine and the peak area Ai of the internal standard in the sample to be tested. The concentration of the monolavin stock solution and / or β-d-N4-hydroxycytidine was used as the X-axis, and the ratio of the peak area As of the derivatized product to the peak area Ai of the internal standard was used as the Y-axis. A weighted regression analysis was performed, and the regression equation of the standard curve and the concentration of the monolavin stock solution and / or β-d-N4-hydroxycytidine were calculated using content analysis software.

5. The method for determining the concentration of monolavine and / or its active metabolite β-d-N4-hydroxycytidine in human body fluids based on benzoyl chloride chemical derivatization and LC-MS / MS according to claim 4, characterized in that: in, In step 1, the organic solvent is acetonitrile, the predetermined temperature is room temperature, and the predetermined time is 9-12 minutes.

6. The method for determining the concentration of monolavine and / or its active metabolite β-d-N4-hydroxycytidine in human body fluids based on benzoyl chloride chemical derivatization and LC-MS / MS according to claim 4, characterized in that: in, In step 2, the organic reagent is acetonitrile, the predetermined concentration is 1% (V / V), and the centrifugal speed is 13000 rpm.

7. The method for determining the concentration of monolavine and / or its active metabolite β-d-N4-hydroxycytidine in human body fluids based on benzoyl chloride chemical derivatization and LC-MS / MS according to claim 6, characterized in that: in, In step 2, the volume ratio of the body fluid sample to the organic reagent containing the predetermined concentration of benzoyl chloride is 1:

3. The volume ratio of the supernatant, the internal standard solution and water is 20:1:

10.

8. The method for determining the concentration of monolavine and / or its active metabolite β-d-N4-hydroxycytidine in human body fluids based on benzoyl chloride chemical derivatization and LC-MS / MS according to claim 4, characterized in that: in, In step 3, the phenyl HPLC column is an Agilent Eclipse Plus Phenyl-Hexyl column (4.6×250 mm, 5 μm). The mobile phase was acetonitrile: water with a gradient of: 0.01~0.5min, 30% acetonitrile, 0.5~8.0min, 30~58% acetonitrile, 8.0~8.1min, 58~90% acetonitrile, 8.1~9.0min, 90% acetonitrile, 9.0~9.6min, 90~30% acetonitrile, 9.6~10min, 30% acetonitrile, Flow rate 1mL·min -1 .

9. The method for determining the concentration of monolavine and / or its active metabolite β-d-N4-hydroxycytidine in human body fluids based on benzoyl chloride chemical derivatization and LC-MS / MS according to claim 4, characterized in that: in, In step 4, the tandem mass spectrometer detector is a universal tandem mass spectrometer detector, The mass spectrometry ionization mode is: electrospray ionization source, Scanning mode: multiple reaction monitoring (MRM) positive ion mode, The ion channels are: m / z 434.2→105.2 (monolavir derivatization product), m / z 364.2→105.3 (β-d-N4-hydroxycytidine derivatization product), m / z 439.2→110.1 (internal standard, monolavin deuterated product) and m / z 369.2→110.0 (internal standard, β-d-N4-hydroxycytidine deuterated product).

10. The method for determining the concentration of monolavine and / or its active metabolite β-d-N4-hydroxycytidine in human body fluids based on benzoyl chloride chemical derivatization and LC-MS / MS according to claim 9, characterized in that: in, In step 4, the mass spectrometer setting parameters are: electrospray voltage 5500V, ion source temperature 700°C, curtain gas 25Arb, collision gas Ar gas pressure Medium, ion source gas 1 and 2 65psi and 45psi respectively, The DP voltages of the monolavin derivatization product, β-d-N4-hydroxycytidine derivatization product, monolavin deuterated product and β-d-N4-hydroxycytidine deuterated product were 36eV, 38eV, 36eV and 27eV, respectively, the collision energies were 33eV, 31eV, 46eV and 40eV, respectively, and the scanning time was 200ms.

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