Method and kit for detecting ultrahigh-polarity amphoteric compounds on dried blood spots

Abstract

The invention discloses an analysis method and a kit for ultrahigh-polarity amphoteric compounds on dried blood spots, and the analysis method comprises the following steps: S1, taking a whole blood dried blood spot sample, adding an ammonia water solution, and carrying out vortex uniform mixing, with the mass fraction of the ammonia water solution being 0.5-5% and the dosage being 30-100 [mu] L; s2, methanol is added, vortex mixing is conducted again, and high-speed centrifugation is conducted; s3, taking supernate, blow-drying the supernate in nitrogen, and adding water for redissolving; s4, carrying out LC-MS (Liquid Chromatography-Mass Spectrometer) analysis, wherein a chromatographic column is a C18 chromatographic column; the mobile phase A is methanol, and the mobile phase B is an ammonia water solution with the mass fraction of 0.01%-0.1%; and S5, calculating the content of the ultrahigh-polarity amphoteric compound in the sample based on a standard curve method. By adopting the method disclosed by the invention, ultrahigh-polarity amphoteric compounds such as TFV-DP and the like can be rapidly and accurately measured, the extraction efficiency is high, the chromatographic separation effect is good, and meanwhile, the response and the sensitivity of TFV-DP are greatly improved.

Description

Technical Field

[0001] The invention relates to the technical field of chromatographic analysis, in particular to an analysis method for ultra-high polarity amphoteric compounds on dried blood spots. Background Art

[0002] On the chromatographic analysis technology platform, highly polar amphiphilic compounds are considered to be the most difficult class of compounds to analyze on the chromatographic analysis technology platform. The main reasons are as follows: 1) The chromatographic retention behavior is complex. Furthermore, polarity leads to unstable retention time. a. Reverse phase chromatography (RP-HPLC) mainly relies on hydrophobic interactions to separate compounds. The retention time of highly polar compounds on reverse phase chromatographic columns is usually very short, and they may even pass through the chromatographic column directly without being retained. Polar compounds easily form hydrogen bonds or other strong interactions with polar solvents in the mobile phase, thereby reducing the retention time on the chromatographic column and resulting in poor separation. b. The amphiphilic characteristics lead to complex retention behavior. Amphiphilic compounds may be positively charged, negatively charged or neutral under different pH conditions, and their own structures may also be molecular or ionic due to different pH values. The two have different chromatographic retention behaviors. This makes their retention behavior at different pH values ​​changeable and difficult to predict and control. Slight changes in pH may significantly affect the charge state of amphiphilic compounds, thereby affecting their retention time and peak shape on the chromatographic column. 2) Difficulty in selecting mobile phase. a. Highly polar compounds often require the use of highly polar mobile phases for separation, such as aqueous phases containing more than 95% or 100%. Long-term use of these mobile phases may affect the service life and performance of the chromatographic column. Highly polar mobile phases may cause peak broadening, reduced resolution, and difficulty in achieving good separation effects. b. pH control is complex. Since the charge states of amphoteric compounds are different at different pH values, the pH of the mobile phase needs to be precisely controlled to achieve the best separation effect. The selection and optimization of buffer solutions are complex, and the slightest carelessness may lead to separation failure or poor reproducibility. 3) Column selection and chromatographic system compatibility issues make chromatographic column selection difficult. a. Hydrophilic interaction chromatography columns (HILIC) can be used for the separation of highly polar compounds, but they require the use of a high content of organic phase (such as acetonitrile) and an appropriate amount of aqueous phase, as well as a high concentration of salt solution. The HILIC separation mechanism is complex and requires the optimization of multiple parameters, such as aqueous phase content, pH, and buffer concentration, which increases the difficulty of method development. b. Chromatographic system compatibility issues. Traditional reversed-phase columns (such as C18 columns) can meet the chromatographic retention and separation requirements of most compounds. However, their retention capacity for highly polar and amphoteric compounds is poor, which can easily lead to separation failures. The mobile phase additives used in reversed-phase columns (such as C18 columns) are mostly acid systems or low-concentration salt solutions. High-concentration salt solutions usually affect the service life of reversed-phase columns and reduce column efficiency. The operating environment of HILIC columns relies more on high-concentration salt solutions to achieve the retention and separation of polar compounds. In addition, during use, in order to ensure that the high-concentration salt solution is fully infiltrated into the chromatographic column, a long equilibrium time is often required, which greatly limits the operating throughput when testing a large number of samples. In addition, HILIC columns are relatively more susceptible to potential contamination from system pipelines or samples, resulting in performance degradation.Therefore, when switching between two types of chromatographic columns in the same chromatographic system, the maintenance requirements for the overall chromatographic system are higher, and the solvents of the two types of chromatographic columns should be avoided from interfering with each other. Furthermore, although HILIC can solve the retention of some polar compounds, it can only solve a very limited part of the problem. 4) Detection sensitivity and matrix effect. The problem of low detection sensitivity, a. Poor efficiency of electrospray ionization (ESI). Highly polar and amphoteric compounds are more suitable for dissolving in aqueous solvents. The ionization efficiency in electrospray ionization mass spectrometry (ESI-MS) may be low, especially in negative ion mode. The ionization parameters need to be optimized to improve the detection sensitivity. b. Significant matrix effect. The complex matrix of biological samples brings about complex matrix effects. The complex matrix in biological samples (such as proteins and lipids) may have significant matrix effects on the detection of highly polar and amphoteric compounds, interfering with the ionization process and resulting in inaccurate quantification. Efficient sample cleanup methods (such as solid phase extraction, SPE) and matrix-matched internal standards are needed to reduce the impact of matrix effects.

[0003] Therefore, the analysis of highly polar zwitterionic compounds on the chromatographic analysis technology platform has always been the most difficult problem in chromatographic analysis technology, mainly due to its complex chromatographic retention behavior, high polarity mobile phase requirements, complex mobile phase pH control, difficulty in chromatographic column selection, low detection sensitivity, and significant matrix effects. These characteristics make method development and optimization very complicated, requiring precise control and comprehensive optimization of multiple parameters. Furthermore, in many cases, it is necessary to analyze compounds on a special carrier such as dried blood spots (whole blood dried blood spots, not plasma dried blood spots), and it is necessary to establish an analytical method for ultra-high polarity zwitterionic compounds on dried blood spots.

[0004] The present invention selects the most typical compound with the most urgent detection demand, the active metabolite of tenofovir TFV-DP as the representative of the ultra-high polarity amphoteric compound to solve the problem of difficult analysis of this type of compound. The present invention only takes TFV-DP as an example but is not limited to TFV-DP.

[0005] With the development of drugs and technologies, HIV infection has become preventable: Under the current situation of changing HIV infection risks, taking a small amount of tenofovir (TFV) in advance as a pre-exposure prophylaxis (PrEP) strategy can play a role in preventing HIV infection in advance. Taking tenofovir (TFV) in advance can effectively prevent HIV infection. This method is called pre-exposure prophylaxis (PrEP). Pre-exposure prophylaxis (PrEP) is a strategy for preventing HIV infection, which is suitable for people who have not yet been infected with HIV but are in a high-risk environment. PrEP maintains sufficient drug concentrations in the body by taking antiretroviral drugs regularly, thereby preventing viral replication and preventing infection when HIV is likely to be exposed. Tenofovir is a nucleotide reverse transcriptase inhibitor (NRTI) that is metabolized into its active form, tenofovir diphosphate (TFV-DP) after entering the body. The standard PrEP regimen is to take a tablet containing tenofovir (TFV) and emtricitabine (FTC) daily (such as the trade name Truvada). This regimen has been shown to provide up to 99% protection when taken daily. For people who have difficulty taking daily medication, intermittent medication or on-demand medication (such as taking medication before and after high-risk sexual behavior) can also provide effective protection. For example, a regimen of taking it four times a week has been shown to achieve an effective level of protection. PrEP is mainly suitable for high-risk groups. Several studies have shown that PrEP has a significant preventive effect in high-risk groups. Taking tenofovir (TFV) in advance as a pre-exposure prophylaxis (PrEP) strategy significantly reduces the risk of HIV infection by maintaining effective drug concentrations in the body. PrEP provides an important means of protection in high-risk groups and has been proven to be an effective measure to prevent the spread of HIV. Regular medication and testing are important links to ensure the effectiveness of PrEP.

[0006] But the key point is that in this process, it is necessary to evaluate whether the drug exposure is sufficient, which can be evaluated by detecting the drug concentration in the blood after taking the drug, otherwise it will not play a protective role. When taking tenofovir (TFV) in advance as pre-exposure prophylaxis (PrEP), the reasons for testing TFV-DP (tenofovir diphosphate) instead of TFV (tenofovir) are: 1) The detection of active metabolites more accurately reflects the preventive effect. TFV-DP is an active form: TFV is converted into its active form TFV-DP by intracellular enzymes in the body. Only TFV-DP can effectively inhibit HIV reverse transcriptase and prevent viral replication. Therefore, the concentration of TFV-DP directly reflects the preventive effect of the drug in cells. Long half-life: TFV-DP has a longer half-life in cells (about 7 to 14 days). In contrast, TFV has a shorter half-life in plasma (about 17 hours). The long half-life means that the concentration of TFV-DP in the body is more stable, which can more accurately reflect the individual's long-term drug exposure and preventive effect; 2) Evaluation of medication compliance. Stable drug concentration: Since TFV-DP is more stable in cells, the individual's medication compliance can be more reliably assessed by measuring the TFV-DP concentration. Even if an individual takes medication intermittently, the TFV-DP concentration can still provide information on drug exposure over a longer time span. Reduce the impact of intraday changes: The plasma concentration of TFV is greatly affected by the daily medication time and the individual's metabolic rate, and the test results may fluctuate greatly. However, TFV-DP has higher stability, and the test results are more consistent and reliable; 3) Direct assessment of intracellular drug levels. Actual prevention level: The TFV-DP concentration reflects the level of the drug in target cells (such as peripheral blood mononuclear cells, PBMCs), which is directly related to the effect of HIV prevention. Testing TFV-DP can assess whether the drug has reached the effective concentration required to prevent HIV infection. More direct prevention indication: Compared with the TFV concentration in plasma, the TFV-DP concentration in cells is more indicative of the prevention effect because HIV mainly replicates in cells; 4) Research and clinical support. Clinical research results: Multiple studies and clinical trials have confirmed that intracellular TFV-DP concentrations are highly correlated with the preventive effect of PrEP. For example, studies have shown that a TFV-DP concentration of 700 fmol / million PBMCs can provide significant HIV prevention effects. Standardized testing: The detection of TFV-DP concentrations has been standardized in multiple clinical studies, providing a reliable indicator for evaluating the efficacy of PrEP.

[0007] TFV-DP is the most typical ultra-high polarity amphiphilic compound, and its structural formula is as follows:

[0008]

[0009] The Log P of TFV-DP is ACD / LogP:-4.25; ACD / LogD (pH 5.5):-9.93. Extremely low LogP and LogD values ​​are very rare, and most compounds have LogP and LogD values ​​between -2 and 2. Compounds with LogP values ​​of -4.25 or LogD values ​​of -9.93 are very rare, indicating that TFV-DP is also rare among extremely hydrophilic compounds. Indicates that the compound has extremely high hydrophilicity. Under pH 5.5 conditions, TFV-DP is almost completely ionized, showing more extreme hydrophilicity. The extremely low LogP and LogD values ​​of TFV-DP indicate its extremely high hydrophilicity and low lipid solubility, which poses significant challenges in chromatography and mass spectrometry analysis. These characteristics make the retention behavior of TFV-DP in reversed-phase chromatography extremely limited, and special chromatographic columns and optimized detection conditions are required to overcome these difficulties. Such extremely low values ​​are very rare in compounds, further highlighting the complexity of TFV-DP in the analysis process.

[0010] Furthermore, TFV-DP (Tenofovir Diphosphate) is an amphoteric compound, which contains highly polar phosphate groups and alkaline groups in its structure. These chemical groups make it highly hydrophilic and have complex ionization properties. First, there are two phosphate ester groups (-O-PO3H2) on the structure of TFV-DP. The phosphate groups have a strong negative charge, which gives the molecule a high polarity and hydrophilicity. The phosphate groups are easily ionized in water to form a negative charge, which makes TFV-DP highly soluble in aqueous solutions and exhibits ultra-high polarity. In addition, there is an adenine moiety on TFV-DP. Adenine is a base that exists in nucleotides. The adenine group contains an amino group (-NH2) and a nitrogen atom. These groups can accept protons under appropriate pH conditions and exhibit alkalinity. Therefore, TFV exhibits a high degree of pH dependence, and the ionization state of TFV-DP is different under different pH conditions. Under low pH conditions, the phosphate group may be partially protonated, while under high pH conditions, the adenine group may be partially protonated. This pH-dependent ionization property makes TFV-DP exhibit complex behavior in chromatography and mass spectrometry analysis, which is difficult to predict and control. The highly polar and amphoteric properties of TFV-DP make it a significant challenge in chromatography and mass spectrometry analysis. The phosphate group gives it high hydrophilicity, while the adenine group provides basic properties. These structural features lead to complex retention behavior and low ionization efficiency, making the analysis of compounds such as TFV-DP very difficult.

[0011] Furthermore, the content of TFV-DP (Tenofovir Diphosphate) in the body during pre-exposure prophylaxis (PrEP) is very low, and this low content further increases the difficulty of measurement. 1) The problem of medication dosage and absorption. PrEP programs usually use daily or intermittent small doses to reduce side effects and improve compliance. This leads to a low concentration of TFV-DP in the body. TFV is metabolized into TFV-DP in the body and accumulates mainly in cells (such as peripheral blood mononuclear cells, PBMCs). Whole blood or blood cells need to be measured. Not only is the content in cells low, but the matrix of whole blood or blood cells is much more complex than that of plasma. The high polarity of TFV-DP itself will cause a great influence on the matrix, which further increases the problem of matrix effect in whole blood or blood cells; 2) The problem of metabolism and excretion. TFV-DP is an active metabolite formed by TFV in cells after a series of metabolic steps. The metabolic rate and efficiency will affect its accumulation in the body. Summary of the invention

[0012] The purpose of the present invention is to provide an analysis method for ultra-high polarity amphiphilic compounds. By changing the extraction solvent, extraction process and method, chromatographic column specifications, chromatographic separation method, and mobile phase additives, a high-sensitivity, rapid and accurate determination of the ultra-high polarity amphiphilic compound representative TFV-DP on dried blood spots is achieved, and a detection sensitivity of at least 20 fmol / Punch is obtained, and the operation is simple and fast.

[0013] The objective of the present invention is achieved through the following technical solutions:

[0014] The present invention provides an analysis method for ultra-high polarity amphiphilic compounds, comprising the following steps:

[0015] S1. Take a whole blood dried blood spot sample, add ammonia solution, and vortex to mix;

[0016] S2, add methanol, vortex mix again and centrifuge at high speed;

[0017] S3, take the supernatant and blow dry it under nitrogen, then add water to reconstitute;

[0018] S4, performing LC-MS analysis, using a C18 column;

[0019] S5. Calculate the content of ultra-high polarity amphoteric compounds in the sample based on the standard curve method.

[0020] As some specific embodiments of the present invention, in step S1, the whole blood dried blood spot sample is a blood spot with a diameter of 3 mm punched out from the dried blood spot of the standard curve, quality control or sample of each concentration by a puncher.

[0021] As some specific embodiments of the present invention, in step S1, the mass fraction of the ammonia solution is 0.5-10%, and the amount of the ammonia solution is 30-100 μL. Preferably, the mass fraction of the ammonia solution is 1-10%.

[0022] As some specific embodiments of the present invention, in step S1, the vortex mixing time is 2 to 10 minutes.

[0023] As some specific embodiments of the present invention, in step S2, the amount of methanol used is 100-500 μL, and the volume ratio of methanol to ammonia solution is 2:1-5:1.

[0024] As some specific embodiments of the present invention, in step S2, the vortex mixing time is 2 to 10 minutes;

[0025] And / or, the centrifugal force of the high-speed centrifugation is 8000-10000g, and the time is 3-10min.

[0026] As some specific embodiments of the present invention, in step S3, the supernatant is blown dry under nitrogen, and then 50-200 μL of water is added for redissolution.

[0027] As some specific embodiments of the present invention, in step S4, when performing chromatographic analysis in LC-MS analysis, mobile phase A is methanol, and mobile phase B is an ammonia solution with a mass fraction of 0.01%-0.1%.

[0028] Preferably, the mobile phase B is an aqueous ammonia solution with a mass fraction of 0.05%.

[0029] As some specific embodiments of the present invention, in step S4, when performing chromatographic analysis in LC-MS analysis, the elution gradient is:

[0030] Time (min) Flow rate (mL / min) A％ B％ Initial 0.6 98 2 1.50 0.6 98 2 3.00 0.6 2 98 4.00 0.6 2 98 4.01 0.6 98 2 6.00 0.6 98 2 .

[0031] As some specific embodiments of the present invention, in step S4, when performing mass spectrometry analysis in LC-MS analysis, the conditions of mass spectrometry include:

[0032] Ionization mode: ESI+; collision gas CAD: 8; curtain gas pressure CUR: 40; auxiliary gas Gas1: 65; auxiliary gas Gas2: 65; ionization voltage IS: 5500; source temperature: 650.

[0033] As some specific embodiments of the present invention, the ultra-high polarity amphoteric compound includes any one of tenofovir diphosphate, zoledronic acid, adefovir diphosphate, lamivudine triphosphate and tenofovir alafenamide diphosphate.

[0034] In the specific embodiment of the present invention, only TFV-DP is selected to demonstrate the analysis method of ultra-high polarity amphiphilic compounds. The same method can be used for the detection of other similar compounds, including but not limited to zoledronic acid, adefovir diphosphate, lamivudine triphosphate, tenofovir alafenamide diphosphate, etc.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] 1) TFV-DP has extremely high polarity, unstable ionization properties, and poor chemical stability. It has been clearly reported that TFV-DP is unstable, but TFV-DP mainly exists in cells, not in plasma. Therefore, it needs to be measured from dried blood spots (whole blood dried blood spots, not plasma dried blood spots. Whole blood dried blood spots require that TFV-DP can be fully released from blood cells, while plasma dried blood spots do not have this requirement) or blood cells. Although the extraction of whole blood and blood cells is very difficult compared to plasma, direct extraction from whole blood and blood cells may be relatively simple and easy compared to dried blood spots. However, considering that TFV-DP is usually present in the scenario of HIV prevention in healthy people, dried blood spots are used for measurement in most cases. How to efficiently and stably extract TFV-DP from dried blood spots (whole blood dried blood spots, not plasma dried blood spots) is the core problem that must be solved first. The present invention adopts a two-step method to extract TFV-DP on the dried blood spot, and in the first step, an ammonia solution is added, the mass fraction of the ammonia solution is 1-10%, and the amount added is 30-100 μL; in the second step, methanol is added for a two-step extraction, and the ratio of the amount of methanol added to the amount of ammonia added should be 2:1-5:1, and the order of the two-step method cannot be interchanged. The above extraction method can significantly improve the extraction efficiency of TFV-DP on the blood spot, and avoid other complex operations, such as the SPE process, which increases the process loss due to too many operation steps.

[0037] 2) TFV-DP has extremely high polarity. According to the basic theory and knowledge of chromatography, a normal phase column or a reverse phase column should be preferred, the most typical of which is a HILIC column. However, in fact, a HILIC column cannot solve the chromatographic problem of such highly polar and amphoteric compounds. The particularity of such compounds results in a very poor peak shape on HILIC and strong matrix suppression. By selecting a C18 column to separate TFV-DP, a symmetrical chromatographic peak shape was obtained.

[0038] 3) TFV-DP is a metabolite and its content in the body is extremely low. At the same time, its extremely high polarity causes its ionization efficiency to be very poor. Furthermore, the extreme polarity causes TFV-DP to elute simultaneously with a large amount of polar matrix. The matrix effect further reduces the response of TFV-DP. In addition, under normal circumstances, due to the extreme polarity, TFV-DP cannot be retained on the C18 column at all. By adding 0.01%-0.1% ammonia water to the mobile phase, the properties of the system are changed. First, TFV-DP can be well retained on the C18 column, and the response and sensitivity of TFV-DP are greatly improved.

[0039] 4) The problem of high-sensitivity determination of TFV-DP in dried blood spots (i.e., whole blood, or blood cells) was solved by changing the extraction solvent, extraction process and method, chromatographic column specifications, chromatographic separation method, and mobile phase additives, so that people undergoing pre-exposure prophylaxis can determine TFV-DP with high sensitivity and rapidity, and the operation is simple; the determination of TFV-DP obtained a detection sensitivity of at least 20 fmol / Punch, and there is still no public report. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0041] Figure 1 This is a graph showing the peak area results of the extraction of samples of the same concentration by different volumes of ammonia solution in step 1 of Example 1;

[0042] Figure 2 The detection spectra after the samples of the same concentration are extracted by ammonia solutions of different concentrations in step 1 of Example 1, wherein a is the detection spectrum of 0.5% ammonia concentration, b is the detection spectrum of 1% ammonia concentration, c is the detection spectrum of 5% ammonia concentration, and d is the detection spectrum of 10% ammonia concentration;

[0043] Figure 3 This is a graph showing the peak area results of the extraction of samples of the same concentration by ammonia solutions of different concentrations in step 1 of Example 1;

[0044] Figure 4 and Figure 5 The detection spectra of samples with the same concentration using HILIC chromatographic column and C18 chromatographic column respectively in step 4 of Example 1 are verified;

[0045] Figure 6 This is a graph showing the peak area results of the detection using ammonia solutions of different concentrations as the mobile phase B in the fourth step of the verification of Example 1;

[0046] Figure 7 a and b are the detection spectra when the concentration of ammonia solution in mobile phase B is 0.001% and 0.05%, respectively;

[0047] Figure 8 The linear fitting curve diagram obtained from the detection results of the matrix calibration curve in Example 2;

[0048] Fig. 9 This is a typical chromatogram obtained by testing sample No. 1 in Example 2;

[0049] Fig.10 This is the detection spectrum of using formic acid as mobile phase B in step 4 of Example 1. DETAILED DESCRIPTION

[0050] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements may be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0051] The present invention provides an analysis method for ultra-high polarity amphiphilic compounds, and the steps may be as follows:

[0052] Step 1: Use a puncher to punch a 3 mm diameter blood spot from the standard / quality control / sample dried blood spot, add 30-100 μL of 0.5-10% ammonia solution, and vortex for 2-10 minutes to mix;

[0053] Step 2: Add 100-500 μL methanol, vortex again for 2-10 min to mix, and then centrifuge at 8000-10000 g for 3-10 min;

[0054] Step 3: Take the supernatant and blow dry it under nitrogen, then add 50-200 μL of water to reconstitute;

[0055] Step 4: Perform LC-MS analysis;

[0056] The liquid phase conditions are as follows:

[0057] Chromatographic conditions:

[0058] Chromatographic column: C18 column;

[0059] Mobile phase A: methanol;

[0060] Mobile phase B: 0.01%-0.1% ammonia solution;

[0061] Gradient conditions:

[0062] Time (min) Flow rate (mL / min) A％ B％ Initial 0.6 98 2 1.50 0.6 98 2 3.00 0.6 2 98 4.00 0.6 2 98 4.01 0.6 98 2 6.00 0.6 98 2

[0063] The mass spectrometry conditions were as follows:

[0064] Ionization mode: ESI+; collision gas CAD: 8; curtain gas pressure CUR: 40; auxiliary gas Gas1: 65; auxiliary gas Gas2: 65; ionization voltage IS: 5500; source temperature: 650.

[0065] Step 5: Calculate the content of ultra-high polarity amphoteric compounds in the sample based on the standard curve method.

[0066] Example 1

[0067] Part 1: By changing the extraction solvent, extraction process and method, efficient and rapid extraction of TFV-DP, a representative of ultra-high polarity and amphoteric compounds, was achieved, and extremely high extraction efficiency was obtained.

[0068] The first part involves steps 1 to 3 of the present invention, and the detailed description and data are as follows:

[0069] Step 1: Use a puncher to punch out a 3 mm blood spot from the prepared blood spots of each concentration of standard curve / quality control / sample, add 30-100 μL of 0.5-10% ammonia solution, and vortex for 5 minutes;

[0070] Step 2, add 100-500 μL of methanol, the volume ratio of methanol to ammonia solution is 2:1-5:1, vortex for 5 minutes, and centrifuge at high speed for 5 minutes after sufficient vortexing;

[0071] Step 3: Dry the supernatant from step 2 under nitrogen and add 100 μL of water to reconstitute.

[0072] 1) Function: Enhance the extraction efficiency of amphoteric compounds through aqueous ammonia solution.

[0073] 2) Comparison and difference with traditional methods: Traditional blood spot extraction methods mostly use pure water, pure methanol or methanol-water system to soak the blood spots, rather than using alkaline solvents for extraction.

[0074] 3) Value: Enhance the release of amphiphilic compounds from dried blood spots.

[0075] 4) Key technology: The volume of ammonia solution added in step 1 is 30-100 μL, and the mass fraction concentration is 1-10%. For this key technical point, the following two groups of verification are set up:

[0076] A. According to the above steps, take an equal amount of dried blood spot samples, only change the volume of ammonia solution, and keep the other conditions unchanged. Punch six 3mm dried blood spots from the whole blood dried blood spot with a concentration of 1000fmol / punch, place them in 1.5mL centrifuge tubes, add different volumes of ammonia solution, the mass fraction of ammonia solution is 0.5%, and the specific added volume is shown in Table 1. After vortexing for 5 minutes, add 300μL MeOH, vortex for 5 minutes, centrifuge at 10000g for 5 minutes, take all the supernatant nitrogen blown to dryness, and re-dissolve with 100μL water, vortex evenly, and perform LC-MS analysis and detection, using a C18 chromatographic column, methanol as mobile phase A, and 0.05% ammonia solution as mobile phase B. The peak area display results are as follows Figure 1 As shown in Table 1, the results indicate that the volume of aqueous ammonia needs to be between 30 and 100 μL to achieve optimal extraction.

[0077] Table 1 Test results of different ammonia solution volumes

[0078] serial number Volume of ammonia solution / μL Area 1 10 73780 2 20 74060 3 30 302700 4 50 424300 5 100 341600 6 250 119200

[0079] B. According to the above steps, only the concentration (mass fraction) of the ammonia solution was changed, and the other conditions remained unchanged. Five 3mm dry blood spots were punched from the whole blood dry blood spots with a concentration of 1000fmol / punch, placed in 1.5mL centrifuge tubes, and 50μL of ammonia extracts of different concentrations were added respectively. The specific concentrations are shown in Table 2. After vortexing for 5 minutes, 150μLMeOH was added. After vortexing for 5 minutes, centrifuged at 10000g for 5 minutes, 180μL of the supernatant was blown to dryness with nitrogen, and re-dissolved with 100μL of water, vortexed evenly, and LC-MS analysis was performed. A C18 chromatographic column was used, and methanol was used as mobile phase A, and ammonia solution with a mass fraction of 0.05% was used as mobile phase B. The detection spectra when the ammonia concentration was 0.5%, 1%, 5%, and 10% were as follows: Figure 2 As shown in a, b, c, and d, the peak area display results are as follows Figure 3 And as shown in Table 2.

[0080] Table 2 Concentration of ammonia solution in the extract

[0081]

[0082]

[0083] according to Figure 2 , 3 As can be seen from Table 2, compared with the conventional methanol-water system (ammonia concentration of 0%), extracting TFV-DP with an ammonia solution first and then with a methanol solution can greatly improve its extraction efficiency, and the best sensitivity can be achieved when the ammonia solution concentration is between 1-10%.

[0084] Part II: By changing the chromatographic column specifications, chromatographic separation method, and mobile phase additives, we achieved a high-sensitivity, rapid, and accurate determination of TFV-DP, a representative of ultra-high polarity and amphoteric compounds, and obtained a detection sensitivity of at least 20 fmol / Punch.

[0085] The second part relates to step 4 of the invention, and the detailed description and data are as follows:

[0086] Step 4: Inject into a liquid chromatography-mass spectrometer (LC-MS / MS) for analysis. The chromatographic column is a C18 chromatographic column; the mobile phase A is methanol, and the mobile phase B is an ammonia solution with a mass fraction of 0.01%-0.1%.

[0087] 1) Function: Choose C18 chromatographic column instead of normal phase or reversed phase chromatographic column recommended by theory, and use the reversed phase chromatographic system more commonly used in the laboratory to complete the separation of highly polar amphoteric compounds, reduce the cost of daily system replacement and maintenance, and control the final charge properties of amphoteric compounds by adding ammonia water to improve the mass spectrometry signal;

[0088] 2) Comparison and difference with traditional methods: For positively charged compounds, the traditional method usually involves adding high concentrations of formic acid;

[0089] 3) Value: Effectively improve the charging efficiency of amphoteric compounds;

[0090] 4) Key technologies: The chromatographic column is a C18 column, not a conventional HILIC column; the mobile phase additive is ammonia water, and the added concentration is 0.01%-0.1%. The following three groups of verifications are carried out:

[0091] A. The same concentration of standard / quality control whole blood dry blood spots (concentration of 1000fmol / punch) were detected using HILIC column and C18 column respectively, and the remaining steps were carried out as follows: Step 1, punch 2 3mm standard / quality control whole blood dry blood spots of the same concentration, add 100μL of 5% ammonia solution by mass, and vortex for 5min; Step 2, add 300μL methanol, vortex for 5min, and then centrifuge at 10000g for 5min; Step 3, blow the supernatant liquid nitrogen to dryness, and re-dissolve it with 100μL water, and vortex evenly; Step 4, perform LC-MS analysis and detection, the chromatographic columns are C18 column and HILIC column respectively, the mobile phase A is methanol, and the mobile phase B is 0.05% ammonia solution by mass, and the peak area display results of the HILIC column and C18 column detection are shown as follows: Figure 4 and 5 As shown. Figure 4 and Figure 5It can be seen that when TFV-DP (tenofovir diphosphate) is separated and detected, due to its special ultra-high polarity, it shows a very poor peak shape on HILIC and has strong matrix suppression; while using a C18 column to separate TFV-DP, a symmetrical chromatographic peak shape is obtained, solving the chromatographic problem of this highly polar and amphoteric compound.

[0092] B. Select a C18 chromatographic column and use the traditional method (i.e., the mobile phase additive is formic acid) to detect the standard / quality control whole blood dried blood spots of the same concentration. The specific steps are as follows: Step 1. Punch 6 3mm dry blood spots in the standard / quality control whole blood dried blood spots of the same concentration (concentration is 5000fmol / punch), add 100μL of 5% ammonia solution, and vortex for 5 minutes; Step 2. Add 300μL of methanol, vortex for 5 minutes, and then centrifuge at 10000g for 5 minutes; Step 3. Take the supernatant and blow it dry with nitrogen, and dissolve it with 100μL of water, and vortex evenly; Step 4. Perform LC-MS analysis and detection, the chromatographic column is a C18 chromatographic column, the mobile phase A is methanol, and the mobile phase B is formic acid of a universal concentration (0.1%). The results are as follows: Fig.10 As shown, for DFV-DP, no effective chromatographic peaks can be obtained and only the baseline can be collected. Therefore, the traditional formic acid mobile phase is not suitable for the collection of such strong amphoteric compounds.

[0093] C. Only the concentration of ammonia solution in mobile phase B of step 4 liquid chromatography is changed, and the remaining steps are carried out as follows: step 1, punch 6 3mm dry blood spots in the same concentration of standard / quality control whole blood dry blood spots (concentration is 5000fmol / punch), add 100μL of 5% ammonia solution by mass, and vortex for 5min; step 2, add 300μL methanol, vortex for 5min, and then centrifuge at 10000g for 5min; step 3, blow the supernatant liquid nitrogen to dryness, and re-dissolve it with 100μL water, and vortex evenly; step 4, perform LC-MS analysis and detection, the chromatographic column is C18 chromatographic column, mobile phase A is methanol, and mobile phase B is ammonia solution. The peak area display results are shown in Table 3 and Figure 6 As shown, Figure 7 Figures a and b show the detection spectra when the concentration of ammonia in mobile phase B is 0.001% and 0.05%, respectively.

[0094] Table 3 Concentration of ammonia solution in mobile phase B

[0095] serial number Ammonia concentration Area 1 0.0010％ 748400 2 0.0025％ 792300 3 0.0100％ 5096000 4 0.0500％ 7059000 5 0.1000％ 5438000 6 0.5000％ 4820000

[0096] According to Table 3 and Figure 6 , 7 The results show that the concentration of ammonia in mobile phase B needs to be between 0.01% and 0.1% to achieve the best response and sensitivity.

[0097] Example 2

[0098] The preparation methods of standard curves / quality control dried blood spots of each concentration are as follows:

[0099] 1) Accurately weigh tenofovir diphosphate solid standard and prepare standard stock solution with methanol to a concentration of 0.5 mg / mL;

[0100] 2) Accurately pipette the stock solution prepared in step 1) and dilute it to volume with 50% methanol aqueous solution to prepare 200-120000 fmol / punch standard curve / quality control working solution;

[0101] 3) diluting the standard / quality control working solution obtained in step 2) by 10 times with blank whole blood to obtain a matrix standard / quality control solution with a concentration of 20-12000 fmol / punch;

[0102] 4) Accurately pipette 50-70 μL of the matrix standard / quality control solution in step 3) and drop it onto the filter paper. Dry it in a ventilated and dark place for at least 4 hours to prepare a standard / quality control dried blood spot with a concentration of 20-12000 fmol / punch.

[0103] The concentrations of the matrix standard curve / quality control solutions obtained in step 3) are shown in Tables 4 and 5.

[0104] Table 4 Standard curve configuration concentration table

[0105] Track number Standard curve concentration Matrix standard curve 1 20fmol / punch Matrix Standard 2 100fmol / punch Matrix standard curve 3 500fmol / punch Matrix Standard 4 1000fmol / punch Matrix standard curve 5 3000fmol / punch Matrix Standard 6 6000fmol / punch Matrix standard curve 7 12000fmol / punch

[0106] Table 5 Quality control configuration concentration table

[0107] Quality Control Number Quality control concentration Minimum detection limit 20fmol / punch Quality Control 1 350fmol / punch Quality Control 2 700fmol / punch Quality Control 3 9600fmol / punch

[0108] Take dried blood spot samples from 30 subjects and test the above matrix standard curve and quality control products according to the following steps:

[0109] Step 1: Use a puncher to punch out a 3 mm diameter blood spot from each of the prepared concentrations of the standard curve / quality control and the dried blood spot of the subject, add 100 μL of 5% ammonia aqueous solution, and vortex for 5 minutes;

[0110] Step 2, add 500 μL of methanol, the volume ratio of methanol to ammonia aqueous solution is 5:1, vortex for 5 minutes, and centrifuge at a speed of 10000g for 5 minutes after sufficient vortexing;

[0111] Step 3: Dry the supernatant in step 2 under nitrogen and add 100 μL of water to reconstitute;

[0112] Step 4: Inject into liquid chromatography-mass spectrometry (LC-MS / MS) for analysis; the liquid phase conditions are as follows:

[0113] Chromatographic conditions:

[0114] Chromatographic column: C18 column

[0115] Mobile phase A: methanol

[0116] Mobile phase B: 0.1% ammonia aqueous solution

[0117] The elution gradient conditions are shown in Table 6.

[0118] Table 6 Elution gradient conditions

[0119] Time (min) Flow rate (mL / min) A％ B％ Initial 0.6 98 2 1.50 0.6 98 2 3.00 0.6 2 98 4.00 0.6 2 98 4.01 0.6 98 2 6.00 0.6 98 2

[0120] The mass spectrometry conditions were as follows:

[0121] Ionization mode: ESI+

[0122] Collision gas CAD: 8

[0123] Air curtain pressure CUR: 40

[0124] Auxiliary gas Gas1: 65

[0125] Auxiliary gas Gas2: 65

[0126] Ionization voltage IS: 5500

[0127] Source temperature: 650

[0128] The MRM conditions are shown in Table 7.

[0129] Table 7 Mass spectrometry MRM conditions

[0130] Compound Ion mode MRM Cone hole energy (DP) Collision Energy (CE) TFV-DP ESI+ 448.1→175.9 100 30

[0131] Step 5: Perform linear fitting based on the test results of the matrix standard curve. The results are as follows: Figure 8 As shown. It can be seen that the linear relationship is good in the concentration range of 20-12000fmol / punch, and the linear equation is Y=0.0000587500X+0.00160, r=0.99521. The test results of the quality control product are under control, and the accuracy of the test batch is good.

[0132] Step 6: Calculate the content of the ultra-high polarity amphoteric compound tenofovir diphosphate in the subject sample based on the standard curve for accurate quantification. The test results of the 30 samples are shown in Table 8.

[0133] Table 8 Test results of 30 samples

[0134]

[0135]

[0136] Among them, the typical chromatogram of sample number 1 is as follows Fig. 9 As shown. Fig. 9 It can be seen that the chromatographic peak shape of the test results is good, the signal-to-noise ratio S / N>10, and the test results are true and effective. The method of the present invention has the ability to detect real samples, and can see chromatograms with good peak shapes and sensitivity that meet the basic requirements of quantitative detection (signal-to-noise ratio>10) in real samples. And the test results of the samples can be obtained based on the standard curve of the same batch. Judging from the random test results of 30 samples, the minimum concentration in the random samples is 84.94fmol / punch (sample 25), and the sensitivity of the constructed method (20fmol / punch) is sufficient to meet the detection needs of real samples.

[0137] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for analyzing ultra-high polarity amphipathic compounds on dried blood spots, characterized in that: The steps include: S1. Take a whole blood dried blood spot sample, add ammonia solution, and vortex to mix; S2, add methanol, vortex mix again and centrifuge at high speed; S3, take the supernatant and blow dry it under nitrogen, then add water to reconstitute; S4, performing LC-MS analysis, using a C18 column; S5. Calculate the content of ultra-high polarity amphoteric compounds in the sample based on the standard curve method.

2. The analysis method according to claim 1, characterized in that In step S1, the mass fraction of the ammonia solution is 0.5-5%, and the dosage of the ammonia solution is 30-100 μL.

3. The analysis method according to claim 1, characterized in that In step S1, the vortex mixing time is 2 to 10 minutes.

4. The analysis method according to claim 1, characterized in that In step S2, the amount of methanol used is 100-500 μL, and the volume ratio of methanol to ammonia solution is 2:1-5:

1.

5. The analysis method according to claim 1, characterized in that In step S2, the vortex mixing time is 2 to 10 minutes; And / or, the centrifugal force of the high-speed centrifugation is 8000-10000g, and the time is 3-10min.

6. The analysis method according to claim 1, characterized in that In step S4, when the chromatographic analysis in the LC-MS analysis is performed, the mobile phase A is methanol, and the mobile phase B is an ammonia aqueous solution with a mass fraction of 0.01%-0.1%.

7. The analysis method according to claim 1, characterized in that In step S4, when performing chromatographic analysis in LC-MS analysis, the gradient of elution is: 。 8. The analysis method according to claim 1, characterized in that In step S4, when performing mass spectrometry analysis in LC-MS analysis, the mass spectrometry conditions include: Ionization mode: ESI+; Collision gas CAD: 8; Air curtain pressure CUR: 40; Auxiliary gas Gas1:65; Auxiliary gas Gas2:65; Ionization voltage IS: 5500; Source temperature:

650.

9. The analysis method according to claim 1, characterized in that The ultra-high polarity amphoteric compound includes any one of tenofovir diphosphate, zoledronic acid, adefovir diphosphate, lamivudine triphosphate and tenofovir alafenamide diphosphate.

10. A detection kit for ultra-high polarity amphiphilic compounds, characterized in that: The ultra-high polarity amphiphilic compound is detected and analyzed using the detection method as described in any one of claims 1 to 9.

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