阅读说明：本技术 人血浆中缬沙坦和沙库巴曲的浓度的测定方法及其用途 (Method for determining concentration of valsartan and sabotarol in human plasma and application thereof ) 是由 余珊 曹媛媛 古珑 房成伟 于 2021-01-22 设计创作，主要内容包括：本发明涉及人血浆中缬沙坦和沙库巴曲的浓度的测定方法及其用途。提供一种人血浆中缬沙坦和沙库巴曲的浓度的测定方法,所述方法包括：血浆样品的配制工序,和高效液相色谱串联质谱分析的工序,其中所述高效液相色谱串联质谱分析采用体积比为90/10的含0.1%甲酸的水-含10%甲醇的乙腈的混合液作为流动相,采用粒径为3.5μm的色谱柱进行梯度洗脱。(The invention relates to a method for determining the concentration of valsartan and sabotarol in human plasma and application thereof. There is provided a method of determining the concentration of valsartan and sabotarol in human plasma, the method comprising: the method comprises a plasma sample preparation process and a high performance liquid chromatography-tandem mass spectrometry analysis process, wherein the high performance liquid chromatography-tandem mass spectrometry analysis adopts 90/10 mixed liquid of water containing 0.1% formic acid and acetonitrile containing 10% methanol as a mobile phase, and adopts a chromatographic column with the particle size of 3.5 mu m for gradient elution.)

1. A method for determining the concentration of valsartan and sabotarol in human plasma, the method comprising:

a procedure for preparing a plasma sample, and

a working procedure of high performance liquid chromatography tandem mass spectrometry,

wherein the HPLC-MS analysis adopts 90/10 volume ratio water containing 0.1% formic acid-acetonitrile containing 10% methanol mixed solution as mobile phase, and adopts chromatographic column with particle size of 3.5 μm for gradient elution.

2. The method of claim 1, wherein in the hplc tandem mass spectrometry the internal standard used is a deuterated derivative of the analyte.

3. The method of claim 2, wherein the internal standard is valsartan-d₃And Shakuba Qu-d₄。

4. The method according to claim 1 or 2, wherein the process of preparing the plasma sample comprises a process of pre-treating the plasma sample.

5. The method of claim 4, wherein the pretreatment process is performed by precipitating proteins in the plasma sample using acetonitrile.

6. The method according to claim 4, wherein the plasma sample preparation process further comprises a dilution process after the pretreatment process.

7. The method according to claim 6, wherein the dilution process is performed by dilution with purified water containing 30% acetonitrile.

8. Use of the method according to any one of claims 1 to 7 in the pharmacokinetic study of valsartan and sabotarol.

Technical Field

The invention relates to a method for detecting the concentration of a medicament in human plasma. Specifically, the invention relates to a method for determining the concentration of valsartan and salvastrexone in human plasma by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).

Background

Angiotensin ii (ang ii) is a polypeptide substance produced by the hydrolysis of angiotensin I by angiotensin converting enzyme. Angiotensin receptors are present on the vascular smooth muscle, adrenal cortico zonal cells, and cells of several parts of the brain, heart and kidney organs of the human body. The angiotensin II is combined with an angiotensin receptor to cause corresponding physiological effects, including that firstly, the arteriole and the vein of the whole body are contracted, the blood pressure is increased, and the quantity of the blood returning to the heart is increased; increasing the release amount of the sympathetic vasoconstrictor fiber transmitter; ③ the sympathetic vasoconstriction center is tensed; stimulating adrenal gland to synthesize and release aldosterone; inducing or enhancing craving and resulting in drinking behavior.

Angiotensin II receptor antagonists (also known as Angiotensin receptor blockers, ARBs) are now widely used in the treatment of clinical hypertension and other cardiorenal diseases such as diabetic nephropathy and congestive heart failure. The pharmacological action of the 'sartan' medicine, namely angiotensin II receptor Antagonist (ARB) antihypertensive medicine is mainly through the interaction with angiotensin type 1 receptor (AT)₁Receptor) to antagonize the hypotensive and target organ damaging effects of Ang II, but antagonize AT₁The receptor varies in strength, selective action, and chemical activity (parent or metabolite thereof). At present, the variety of losartan, valsartan, eprosartan, irbesartan, candesartan and the like which are clinically applied are various, wherein losartan and valsartan are the most widely applied in China.

Valsartan (Valsartan) is a non-peptide, orally active angiotensin II receptor antagonist against AT₁The height of the receptor isSelectively, competitively antagonize without any agonism, and also inhibit AT₁Receptor mediated release of aldosterone by adrenal cells, but no inhibition of potassium release, also suggests that valsartan is AT₁Selective action of the receptor. AT the molecular level, valsartan confers AT₁Receptor blockade, elevated angiotensin II plasma levels, stimulation of unblocked AT₂Receptor for Simultaneous antagonism of AT₁The receptor acts, thereby achieving the effect of expanding blood vessels and lowering blood pressure. In vivo experiments of various types of hypertension animal models also show that valsartan has good blood pressure reducing effect, has no obvious influence on the cardiac contraction function and the heart rate, and does not produce the blood pressure reducing effect on animals with normal blood pressure. Good tolerance, low toxic and side effects and AT₁The highly selective action of the receptor makes valsartan a promising antihypertensive drug.

The valsartan sodium of Sacubitril contains an enkephalinase inhibitor, Sacubitril (sacubitril) and an angiotensin receptor antagonist, valsartan. Valsartan sodium inhibits enkephalinase (neutral endopeptidase; NEP) by LBQ657 (active metabolite of prodrug Sacubitril), and blocks AT of angiotensin II by valsartan₁A receptor. By increasing the levels of peptides (e.g. natriuretic peptides) degraded by enkephalinase LBQ657, while inhibiting angiotensin II action by valsartan, valsartan sodium may produce cardiovascular and renal effects in heart failure patients. Valsartan can selectively block AT₁The receptor inhibits the action of angiotensin II and also inhibits the release of angiotensin II-dependent aldosterone.

Generally, the detection of drug concentrations in blood or other biological samples is the basis for pharmacokinetic, pharmacodynamic studies and clinical treatments. The method for detecting the concentration of a drug in plasma should have high sensitivity, be able to obtain results quickly, and be able to be performed using a small amount of sample. However, since various components including phospholipids, proteins, etc. exist in blood, detection is easily affected by various factors such as matrix effect, etc. and various physicochemical properties of the drug, how to reduce the matrix effect and residue to the maximum extent and obtain the accurate concentration of the drug in blood as much as possible is a subject to be studied intensively in the art.

The high performance liquid chromatography is a common method for detecting the blood concentration at present, and has wide application range. However, the use of chromatographic columns for separation takes a long time and has low sensitivity, and is not suitable for high-throughput analysis of biological samples.

The Ultra Performance Liquid Chromatography-mass spectrometry (UPLC-MS/MS) improves the analysis flux and saves the analysis time of samples and solvents. However, the UPLC-MS/MS has a special requirement for mobile phase, chromatographic column, sample pretreatment, mass spectrum interface, and data acquisition system, i.e., a large number of tests are required for various conditions including sample pretreatment, internal standard, mobile phase, chromatographic column selection, mass spectrum condition setting, etc., so as to realize rapid detection and analysis of UPLC-MS/MS, and the process time is long and complicated.

For example, CN110031568A (patent document 1) provides a method for measuring the concentrations of sabotara, desethylsabotara and valsartan in human plasma by ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). In order to reduce the toxicity to the chromatographic column, the concentrations of three drugs (metabolites) of Sacubitril, desethylSacubitril and valsartan in plasma are simultaneously detected by a series of operations of carrying out sample pretreatment by methanol water, adopting a deuterated derivative of an analyte as an internal standard, adjusting a mobile phase to be 0.1% formic acid water/acetonitrile and the like.

High Performance Liquid Chromatography tandem mass spectrometry (HPLC-MS/MS) has less requirements on a mobile phase, a chromatographic column, sample pretreatment, a mass spectrum interface and a data acquisition system than UPLC-MS/MS, and analysis flux, sample and solvent analysis time are completely in a reasonable requirement range, so that the experimental method has good reproducibility, and has less residue than UPLC-MS/MS when various experimental conditions (such as the mobile phase and complex solution) are fixed. Mass spectrometry detection is different from chromatography detection, and additional attention is also required for various aspects such as mobile phase composition, flow velocity and the like, so that the HPLC-MS/MS method with good separation condition, small residue, high sensitivity and good tolerance can be obtained by specifically and deeply researching experimental conditions such as mobile phase selection, setting of mass spectrometry parameters and the like.

The existing method for detecting the concentrations of valsartan and sabotarol in rat plasma is not suitable for large-batch sample detection of clinical pharmacokinetic research.

Therefore, there is an urgent need in the art for a rapid, sensitive method that can be used to detect the concentration of valsartan and sabotarol in human plasma.

Disclosure of Invention

Problems to be solved by the invention

In view of the above-mentioned major drawbacks of the background art, it is an object of the present invention to provide a method for simply, efficiently and sensitively detecting the concentration of valsartan and sabotarol in plasma.

Bioequivalence studies aimed at obtaining more reliable clinical specimen concentrations for pharmacokinetic studies, and therefore require the establishment of reasonable linear ranges, leaving bioanalytical assays that meet the acceptance criteria (less than the 20% lower limit of quantitation), minimizing sample re-dilution and chromatographic contamination of the sample.

As a result of intensive studies by the present inventors, a rapid and sensitive method for detecting the concentrations of valsartan and sabotara in human plasma by high performance liquid chromatography tandem mass spectrometry has been established. The method of the present invention has advantages including, for example, simple and rapid sample processing, high sensitivity, small residue, etc. In view of the difference of the simultaneous administration and metabolism of the valsartan and the sabotarol, the method realizes the simultaneous detection of the concentrations of the valsartan and the sabotarol, and has important significance.

Means for solving the problems

The invention provides a method for determining the concentration of valsartan and sabotarol in human plasma, which comprises the following steps: the method comprises a plasma sample preparation process and a high performance liquid chromatography-tandem mass spectrometry analysis process, wherein the high performance liquid chromatography-tandem mass spectrometry analysis adopts 90/10 mixed liquid of water containing 0.1% formic acid and acetonitrile containing 10% methanol as a mobile phase, and adopts a chromatographic column with the particle size of 3.5 mu m for gradient elution.

According to the method for determining the concentration of valsartan and sabotarol in human plasma, the internal standard is a deuterated derivative of an analyte in the high performance liquid chromatography tandem mass spectrometry.

According to the method for determining the concentrations of valsartan and sabotarol in human plasma, the internal standard is valsartan-d₃And Shakuba Qu-d₄。

According to the method for determining the concentrations of valsartan and sabotarol in human plasma, the preparation process of the plasma sample comprises a pretreatment process of the plasma sample.

According to the method for measuring the concentrations of valsartan and sabotarol in human plasma of the present invention, the pretreatment process is performed by precipitating the protein in the plasma sample using acetonitrile.

According to the method for measuring the concentrations of valsartan and sabotarol in human plasma, the preparation process of the plasma sample further comprises a dilution process after the pretreatment process.

According to the method for measuring the concentration of valsartan and sabotarol in human plasma of the present invention, the dilution process is performed by dilution with purified water containing 30% acetonitrile.

The invention also provides the use of the method for determining the concentration of valsartan and sabotarol in human plasma as described above in the pharmacokinetic studies of valsartan and sabotarol.

ADVANTAGEOUS EFFECTS OF INVENTION

When the method adopts the mixed solution of water containing formic acid and acetonitrile containing 10 percent of methanol as the mobile phase for gradient elution analysis, the substance to be detected can be quickly and fully separated, good peak shape is obtained, the analysis time is short, the baseline noise is low, and the residue is reduced to a certain extent.

In the invention, the specific chromatographic column is adopted to fully separate the substances to be detected, obtain good peak shape and have short analysis time, and the residue is greatly reduced.

In the invention, valsartan-d is adopted₃And Shakuba Qu-d₄Can provide a detection method with good linear relation and meeting the requirements of precision, matrix effect, extraction recovery rate and stability.

According to the invention, when the protein precipitation method is adopted to pretreat the sample, a surprisingly high recovery rate is obtained, so that a complicated and time-consuming liquid-liquid extraction process can be avoided, high sensitivity can be obtained, a matrix effect can be avoided, and further, the retesting caused by internal standard abnormality in the analysis of a large number of biological samples can be avoided. Further, the present inventors have found that adding purified water containing 30% acetonitrile to dilute after protein precipitation, preferably at a solvent ratio close to that of the mobile phase, can obtain a better peak shape and reduce the residue, and in particular can obtain an improved sensitivity.

The method has good reproducibility, and effectively reduces the occurrence of residual effect. The method is suitable for the bioequivalence research of the 100mg Sacubitril valsartan sodium tablet, effectively avoids the dilution of excessive samples, reduces the workload of sample analysis and does not influence the PK parameters of the samples.

The specificity, matrix effect, extraction recovery rate and stability of the method are verified, and the method is successfully applied to the detection of clinical pharmacokinetic samples.

Drawings

FIGS. 1A-1D show secondary mass spectra. FIG. 1A: a second order mass spectrum of valsartan; FIG. 1B: ion scanning of the Shakuba koji; FIG. 1C: valsartan-d₃A sub-ion scan; FIG. 1D: shakuba Qu-d₄A scan of the daughter ion.

Figures 2A-2F show characteristic chromatograms of blank plasma, blank plasma with internal standard, and low lower limit of quantitation for 4 target compounds. FIG. 2A: the blank matrix map of valsartan has an upper graph corresponding to valsartan and a lower graph corresponding to valsartan-d₃(ii) a FIG. 2B: the upper graph corresponds to Sacubitril and the lower graph corresponds to Sacubitril-d₄(ii) a FIG. 2C: typical chromatogram of blank plasma with added internal standard of valsartan, the upper graph corresponding to valsartan and the lower graph corresponding to valsartan-d₃(ii) a FIG. 2D: typical chromatogram of blank plasma with added internal standard of Sacubitril, with the upper panel corresponding to Sacubitril and the lower panel corresponding to Sacubitril-d₄(ii) a FIG. 2E: the upper graph corresponds to valsartan, and the lower graph corresponds to valsartanIn response to valsartan-d₃(ii) a FIG. 2F: the lowest quantitative limit map of Sacubitril corresponds to Sacubitril at the upper part and Sacubitril-d at the lower part₄。

Fig. 3 is a valsartan standard curve showing an embodiment of the invention.

Fig. 4 is a standard curve of shakubatu showing an embodiment of the present invention.

Detailed Description

In some embodiments, the present invention provides methods for measuring the concentration of valsartan and sabotara in human plasma by high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS), wherein the methods are performed using a mixture of water containing formic acid-acetonitrile containing 10% methanol as the mobile phase.

The inventors have found that although a better response is obtained using methanol as the mobile phase, the baseline noise is also correspondingly higher and the signal of the target compound is higher with more severe residue. When the mixed solution of water containing formic acid and acetonitrile containing 10% of methanol is used as a mobile phase for analysis, the substance to be detected can be quickly and fully separated by elution, a good peak shape is obtained, the baseline noise is low, and the residue is weakened.

In some embodiments, the mobile phase employed in the process of the present invention may have a volume ratio (v/v) of water containing formic acid to acetonitrile containing 10% methanol of 90/10 to 10/90.

In some embodiments, the mobile phase employed in the process of the present invention may be a mixture of water containing 0.1% formic acid (hereinafter referred to simply as 0.1% formic acid water) -acetonitrile containing 10% methanol. In some embodiments, the mobile phase is carried out using the volume ratios described above.

In some embodiments, the methods of the invention may be performed using gradient elution or isocratic elution. In some specific embodiments, the methods of the invention are performed by gradient elution. In some embodiments, the process of the invention is carried out with a flow rate of the mobile phase of 0.3 to 0.8 mL/min. In some preferred embodiments, the mobile phase in the process of the invention is preferably carried out with a flow rate of 0.50 mL/min. In some specific embodiments, the methods of the invention may be performed using the following gradient elution: a: 0.1% formic acid water; b: acetonitrile with 10% methanol, elution gradient: 0-0.3 min: 60% of B; 2.5 min: 85% of B; 2.6-4.0 min: 100% B, 4.1-6.0 min: 60% B, and the elution flow rate was set to 0.5 mL/min.

To improve chromatographic separation selectivity, in some embodiments, mobile phase modifiers or the like are added to the mobile phase to further adjust the polarity of the mobile phase. It has been observed that elution with the mobile phase allows sufficient separation of the substance to be detected, obtaining a good peak shape and a fast analysis time.

In some embodiments, in hplc tandem mass spectrometry of the methods of the invention, it was found that a column with a particle size of 3.5 μm can maximize residue reduction while accounting for chromatographic peak shape. In chromatographic analysis, the selection of a chromatographic column is very important, and the chromatographic column is required to have high column efficiency, good selectivity, high analysis speed and the like. When the inner diameter of the column is within the above range, good separation and peak patterns of valsartan and sabotara can be obtained, and the elution time is appropriate. If the amount is less than the above inner diameter, the retention of the objective compound is poor, the symmetry of the peak shape is poor, and the residue is severe. If it is higher than the above inner diameter, the retention time is reduced and the column efficiency is also reduced. As an example of the column, Agilent ZORBAX SB-Aq (100X 3.0 mm, 3.5 μm) (product name, manufactured by Agilent technologies, Ltd.) can be exemplified.

In some embodiments, the hplc tandem mass spectrometry of the invention employs a deuterated derivative of the analyte as an internal standard. When the internal standard method is adopted, the selection of the internal standard substance is very important work. The ideal internal standard should be added to the sample in an accurate, known amount, and have substantially the same or as consistent physicochemical properties (e.g., chemical structure, polarity, volatility, and solubility in solvents, etc.), chromatographic behavior, and response characteristics as the sample being analyzed; under chromatographic conditions, the internal standard must be sufficiently separated from the components of the sample. In some embodiments, deuterated derivatives of analytes, preferably valsartan-d, are employed₃And Shakuba Qu-d₄Can provide good linear relationGood detection method, and high precision, matrix effect, extraction recovery rate and stability.

In some embodiments, the methods of the invention employ a protein precipitation method for pretreatment. The inventor has found that when the sample is pretreated by liquid-liquid extraction, the operation is complicated and time-consuming, the extraction recovery rate is low, the detection sensitivity is low, and the requirement of detecting the concentrations of valsartan and shakubaqu in human plasma by a large sample in clinical pharmacokinetics cannot be met. The research results of the invention show that the protein precipitation method can be adopted for sample pretreatment under the conditions of the invention, and surprisingly high recovery rate is obtained, so that the complicated and time-consuming liquid-liquid extraction process can be avoided. However, the inventors found that when the plasma sample is directly injected after being precipitated by methanol protein and further diluted by 50% methanol, the residual peak area of the blank sample after the peak of the standard curve is high, and the chromatographic peak has a tail. In the present invention, the protein precipitation treatment is preferably performed by acetonitrile.

In some embodiments, the methods of the invention enable improved recovery. In some embodiments, the methods of the invention achieve recoveries of up to 80% or more. In some embodiments, the methods of the invention achieve recoveries of up to 85% or more. In some embodiments, the methods of the invention achieve recoveries of up to 90% or more. In some embodiments, the methods of the invention achieve recoveries of up to 95% or more. In some embodiments, the present invention enables surprising extraction recoveries of up to 99%. In some embodiments, the method of the invention can achieve extraction rates as high as 99%, and can achieve high sensitivity and avoid matrix effects.

In some embodiments, the human plasma sample to be tested is diluted with purified water containing 30% acetonitrile after pretreatment by protein precipitation. In some embodiments, the human plasma sample to be tested is diluted with purified water containing 30% acetonitrile to a solvent ratio close to that of the mobile phase after being pretreated by a protein precipitation method such as acetonitrile protein precipitation. In some embodiments, dilution is performed by purified water containing 30% acetonitrile to a solvent ratio selected from the group consisting of: water acetonitrile (80/20, v/v) -water acetonitrile (20/80, v/v), preferably water acetonitrile (60/40, v/v) -water acetonitrile (40/60, v/v), more preferably water acetonitrile (47/53, v/v). The inventors have found that the addition of purified water containing 30% acetonitrile after precipitation of the protein for dilution, preferably at a solvent ratio close to that of the mobile phase, allows a better peak shape to be obtained and reduces the residue, and in particular allows an improved sensitivity to be obtained. As can be seen from the examples section which follows, the method of the present invention has a sensitivity of greater than 7.5ng/mL for detection of valsartan in plasma and greater than 5ng/mL for sabotarol.

In some specific embodiments, the pretreatment of a human plasma sample to be detected adopts acetonitrile protein precipitation, and after the human plasma sample is further diluted by purified water containing 30% acetonitrile, the human plasma sample is separated by an Agilent ZORBAX SB-Aq (100 multiplied by 3.0 mm, 3.5 mu m) chromatographic column by a mixed solution of water containing formic acid and acetonitrile containing 10% methanol.

In order to more clearly explain the present invention, the following description will explain embodiments of the present invention in more detail by way of specific examples. It is to be understood, however, that the following specific examples are illustrative of the invention and are not to be construed as limiting the invention in any way, wherein the materials, reagents, equipment and operating conditions are representative and not limiting. From reading the following description, those skilled in the art can make changes and modifications to the present invention without departing from the scope of the invention defined by the appended claims, and such changes and modifications are also within the scope of the invention as claimed.

The inventor has established a method for measuring the concentrations of valsartan and sabotara for high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS), and has investigated methodology according to the verified guiding principle of the quantitative analysis method of biological samples in the Chinese pharmacopoeia 2015.

1. Materials and methods

1.1 reagents and drugs

The valsartan and the Sacubitril standard are provided by Nanjing Youkee biotechnology limited and internally mark valsartan-d₃And Shakuba Qu-d₄Assay of drugs purchased from ChinaThe research institute. High performance liquid chromatography pure grade methanol, isopropanol and acetonitrile were purchased from Merck, high performance liquid chromatography pure grade formic acid was purchased from Sigma, and the experimental water was ultrapure water produced by ELGA Classic UV pure water system.

1.2. Laboratory apparatus and analysis conditions

Liquid chromatograph: high performance liquid chromatography by Shimadzu corporation;

a chromatographic column: ZORBAXSB-Aq (100X 3.0 mm, 3.5 μm) of Agilent;

liquid phase conditions:

mobile phase: 0.1% formic acid water: acetonitrile (90/10, v/v) with 10% methanol;

flow rate: 0.50 mL/min;

sample introduction amount: 5 muL;

column temperature: 40 ℃;

sample injector internal temperature: 5 ℃ is adopted.

Mass spectrum conditions:

analytical detection was carried out by Sciex API 4000 triple quadrupole mass spectrometry. Electrospray ionization (ESI), positive ion mode, multiple reaction monitoring mode (MRM), spray voltage 5500V;

ion source temperature: 600 ℃, gas curtain gas: 30psi, atomizing gas: 40psi, assisting atomising gas: 65 psi.

Valsartan, Sacubitril, internal standard valsartan-d₃And Shakuba Qu-d₄The detected ion pairs are respectively:

m/z 436.4 → 235.4, DP, EP, CXP and CE 60V, 10V, 12V, 28V, respectively;

m/z 412.3 → 309.1, DP, EP, CXP and CE are 80V, 10V, 12V, 26V, respectively;

m/z 439.4 → 235.4, DP, EP, CXP and CE 60V, 10V, 12V, 28V, respectively;

m/z 416.3 → 309.1, DP, EP, CXP and CE 52V, 10V, 12V, 26V respectively.

Valsartan, Sacubitril, internal standard valsartan-d₃And Shakuba Qu-d₄The secondary mass spectra of (A) are shown in FIGS. 1A-1D.

1.3. Preparation of stock solution and working solution

Precisely weighingPutting 2mg of valsartan and Shakubaqu standard substance into a glass bottle with a proper volume, adding a proper amount of methanol/water (4/1, volume ratio) mixed solution, dissolving and uniformly mixing to prepare a stock solution with the concentration of 1 mg/mL. Accurately weighing about 1mg of valsartan-d₃And Shakuba Qu-d₄Putting the standard substance into a glass bottle with a proper volume, adding a proper amount of methanol/water (4/1, volume ratio) mixed solution, dissolving and uniformly mixing the mixed solution to prepare a stock solution with the concentration of 1 mg/mL.

The internal standard working solution is diluted by 50 percent methanol water, and the concentration of the internal standard working solution is 800/200ng/mL of valsartan-d₃/Shakuba Qu-d₄And (4) internal standard working solution.

The valsartan and the Sacubitril stock solution are further diluted by 50% methanol according to a certain proportion to prepare standard curve working solutions with the concentrations of the valsartan and the Sacubitril of 150/100, 300/200, 750/500, 3000/2000, 15000/10000, 30000/20000, 120000/80000 and 150000/10000ng/mL and quality control working solutions of 114000/76000, 12000/8000, 450/300 and 150/100 ng/mL.

The stock solution was stored at-20 ℃ and the working solution at-20 ℃.

1.4. Preparation of plasma samples

Adding 20 mu L of diluent 2 (methanol/water, 50/50, volume ratio) into a 1.5mL EP tube to dilute the obtained working solution and 380 mu L of blank K₂Obtaining a plasma sample from EDTA plasma, adding 50 muL of the plasma sample and 50 muL of internal standard working solution into a 96 pore plate, adding 500 muL of acetonitrile, carrying out vortex mixing for 15min, 6000g and 5 ℃ centrifugation for 10min, taking 100 muL of supernatant into a new 96 pore plate, adding 200 muL of 30% acetonitrile for dilution, carrying out vortex centrifugation for 5min at 15min, 6000g and 5 ℃, and taking 5 muL for sample injection detection.

Methodology validation

The detection method is verified according to the 'biological sample quantitative analysis method verification guiding principle' in 'Chinese pharmacopoeia' in 2015, and the verification contents comprise specificity, precision, sensitivity, a standard curve, extraction recovery rate, matrix effect and stability.

(1) Specificity of

Respectively taking 50 muL of blank plasma of 6 individuals, adding 50 muL of 50% methanol, then operating under the item of '1.4 plasma sample treatment', and carrying out sample injection detection to obtain blank plasma maps (fig. 2A and 2B).

Respectively taking 50 muL of 6 individual blank plasma, and carrying out sample injection detection according to the operation under the item of '1.4 plasma sample treatment' to obtain blank plasma maps only added with internal standards (fig. 2C and 2D).

Respectively taking 380 muL of 6 individual blank plasma, adding 20 muL 150/100ng/ml of standard curve working solution, then adding 50 muL of internal standard working solution, and then performing sample injection detection to obtain a map of a low quantitative lower limit according to the operation under the item of '1.4 plasma sample treatment' (fig. 2E and 2F).

As can be seen from FIGS. 2A to 2F, the retention time of valsartan is 1.736min, the peak shape is symmetrical, the signal to noise ratio of the chromatographic peak of the lower limit of quantitation (7.5ng/mL) is greater than 5, the interference of the isotope internal standard (concentration 800ng/mL) of valsartan on the object to be detected after pretreatment and dilution is less than 20% of the lower limit of quantitation, and as can be seen from FIG. 2A, a blank K₂In the chromatogram of EDTA plasma, valsartan and valsartan-d₃No interference peak influencing the valsartan quantification is found at the retention time of the chromatographic peak, so the valsartan and the valsartan-d in the method of the invention₃The selectivity and the specificity are good; the retention time of the Sacubitril is 1.839min, the peak shapes are symmetrical, the signal-to-noise ratio of a chromatographic peak at the lower limit of quantification (5ng/mL) is more than 5, the interference of an isotope internal standard (the concentration is 200ng/mL) of the Sacubitril on a substance to be detected after pretreatment and dilution is less than 20% of the lower limit of quantification, and as can be seen from a graph 2B, a blank K₂In the chromatogram of EDTA plasma, Sacubitril and Sacubitril-d₄No interference peak influencing the quantitative determination of the Shakubaqu is observed at the retention time of the chromatographic peak. Shakubaqu and Shakubaqu-d in the method of the invention₄The selectivity and the specificity of the kit are good, and the kit meets various requirements of quantitative detection of biological analysis.

(2) Precision and accuracy

380 μ L of blank plasma was added with working solution of corresponding concentration of 20 μ L to prepare 1 each of 7.5/5ng/mL (valsartan/Sacubitril, LLOQ, hereinafter also applicable), 22.5/15ng/mL (valsartan/Sacubitril, LQC, hereinafter also applicable), 600/400ng/mL (valsartan/Sacubitril, MQC, hereinafter also applicable), 5700/3800ng/mL (valsartan/Sacubitril, HQC, hereinafter also applicable) plasma samples, each of which was added to 6 wells of a 96-well plate as a sample of precision and accuracy, followed by treatment under the term "1.4 plasma sample treatment". And (3) measuring the samples of the batches on different days, substituting the ratio of the area of the valsartan/sabotara peak to the area of the internal standard peak into the standard curve on the day to obtain the measured concentration, and calculating the precision and accuracy of the samples in the batches and between the batches. The accuracy deviation and the precision are respectively expressed by RE% and RSD%, the standard deviation is expressed by SD, the accuracy deviation and the precision absolute value of the low-concentration quality control sample (LQC), the medium-concentration quality control sample (MQC) and the high-concentration quality control sample (HQC) are less than 15%, and the accuracy deviation and the precision absolute value of the quantitative lower limit quality control sample (LLOQ) are less than 20%. The results are shown in tables 1A and 1B.

Table 1A valsartan batch-to-batch precision and accuracy.

Table 1B intra-and inter-batch precision and accuracy of shakubama bats.

Compared with the prepared concentration, the precision and the accuracy of the method are obtained by adopting a one-factor variance analysis method, and the table 1A shows that the intra-batch RE of the valsartan is less than or equal to 3.6 percent, the intra-batch RSD is less than or equal to 8 percent, the inter-batch RE is less than or equal to 2.6 percent and the inter-batch RSD is less than or equal to 6 percent; table 1B shows that the intra-batch RE of the Sacubitril is less than or equal to 4.5%, the intra-batch RSD is less than or equal to 11.2%, the inter-batch RE is less than or equal to 5.3%, and the inter-batch RSD is less than or equal to 6.5%, all the results meet pharmacopoeia acceptance standards.

(3) Standard curve

380 muL of blank plasma is added with standard curve working solution with corresponding concentration of 20 muL to prepare simulated plasma samples with concentration of valsartan and sabotaxus in the plasma of 7.5/5, 15/10, 37.5/25, 150/100, 750/500, 1500/1000, 6000/4000 and 7500/5000ng/mL respectively, and then the item of 'treatment of 1.4 plasma samples' is followedAnd (5) performing sample injection detection. Verifying three batches, taking the ratio (X) of the concentration of valsartan/sabotazole in plasma to the concentration of an internal standard as an abscissa and the peak area ratio f of the valsartan/sabotazole to the internal standard as an ordinate, and weighting (W is 1/X)²) And performing regression operation by using a least square method, wherein the obtained linear regression equation is the standard curve. The valsartan standard curve is shown in figure 3 and the sabotarg standard curve is shown in figure 4.

As can be seen from FIGS. 3 and 4, in the method of the present invention, valsartan is within the range of 7.50 to 7500ng/mL of plasma, and sabotara is in good linear relationship within the range of 5.00 to 5000ng/mL of plasma.

(4) Extraction recovery and matrix effects

Preparation of control (SET1) sample: adding 380 muL of diluent 2 (methanol/water, 1/1, volume ratio) into a 1.5mL centrifuge tube, adding 20 muL of quality control mixed working solution (low-concentration quality control working solution/medium-concentration quality control working solution/high-concentration quality control working solution) to obtain sample solutions with the concentrations of valsartan and shakubata being 22.5/15ng/mL, 600/400ng/mL and 5700/3800ng/mL respectively, adding 50 muL of sample solution and 50 muL of internal standard solution into a 96-well plate, precisely adding 500 muL of 30% acetonitrile aqueous solution, carrying out vortex mixing, carrying out centrifugation at 6000g for 10min, sucking 100 muL of supernatant and adding into a new 96-well plate, adding 200 muL of 30% acetonitrile water for dilution, mixing uniformly by vortex, centrifuging for 5min at 6000g and 5 ℃, and taking 5 muL for HPLC-MS/MS analysis. Preparing 3 samples per concentration, recording chromatogram, recording the peak area (A) of valsartan, calculating the average value As and the peak area (B) of Sacubitril, calculating the average value Bs, and recording the internal standard valsartan-d of each group₃The peak area (C) is obtained, and the average value (Cs) of the peak area (C) and the peak area (C) is obtained₄The peak area (D) was averaged to obtain the average value (Ds).

Preparation of control (SET2) sample: accurately adding 50 mu L of 6 different individual blank plasmas into a 1.5mL Ep tube, adding 500 mu L of acetonitrile, carrying out vortex oscillation for 15min, centrifuging for 10min at 6000g, sucking 100 mu L of supernatant, adding the supernatant into a new 96-well plate, adding the supernatant in the preparation of a 100 mu L control group (SET1) sample, adding 100 mu L of 30% acetonitrile water for dilution, carrying out vortex mixing, centrifuging for 5min at 6000g and 5 ℃, and taking 5 mu L for HPLC-MS/MS analysis. Recording the chromatogramRecording the peak area Am of valsartan at each concentration, and calculating the average value Am ', the peak area Bm of Sacubitril and the average value Bm'; recording internal standard valsartan-d in all concentrations₃The peak area Cm is calculated, the average value Cm' is calculated, and the Shakuba koji-d is calculated₄The peak area Dm is the average Dm'.

Preparation of experimental group (SET3) samples: according to a determination method of a 'plasma sample standard curve', 6 parts of low, medium and high plasma samples with the plasma concentrations of valsartan and sabotara respectively being 22.5/15ng/mL, 600/400ng/mL and 5700/3800ng/mL are prepared, HPLC-MS/MS analysis is carried out according to the operation under the item of 'treatment of 1.4 plasma samples', and chromatograms are recorded. Recording the peak area Ai of valsartan and the peak area Bi of sabotarol of each concentration sample, and recording all internal standards of valsartan-d₃Peak area Ci, Shakubatu-d₄The peak area Di is obtained by respectively substituting Ai, Bi, Ci, Di and As, Bs, Cs and Ds into the following formula to obtain valsartan and sabotara, and internally marking valsartan-d₃And Shakuba Qu-d₄Absolute extraction recovery (AR%):

valsartan: AR% ═ Ai/As × 100%;

shakuba koji: AR% ═ Bi/Bs × 100%;

internal standard valsartan-d₃：AR%＝Ci/Cs×100%；

Shakuba Qu-d₄：AR%＝Di/Ds×100%；

Substituting Ai, Bi, Ci, Di, Am ', Bm', Cm 'and Dm' into the following formula to obtain valsartan, Sacubitril and internal standard valsartan-d₃And Shakuba Qu-d₄Relative extraction recovery (RR%):

valsartan: RR% ═ Ai/Am' x 100%;

shakuba koji: RR% ═ Bi/Bm' x 100%;

internal standard valsartan-d₃：RR%＝Ci/Cm′×100%；

Internal standard Shakuba Qu-d₄：RR%＝Di/Dm′×100%。

Substituting Am, Bm, Cm, Dm, As, Bs, Cs and Ds into the following formula to obtain valsartan and sabotara, and internally labeling valsartan-d₃And Shakuba Qu-d₄Of (2) a substrateFactor (MF%):

valsartan: MF% ═ Am/As × 100%;

shakuba koji: MF% ═ Bm/Bs × 100%;

internal standard valsartan-d₃：MF%＝Cm/Cs×100%；

Internal standard Shakuba Qu-d₄：MF%＝Dm/Ds×100%。

The ratio of the matrix factors of the valsartan and the sabotarol to be detected to the matrix factor of the internal standard is internal standard normalized matrix factor (MF%), and the internal standard normalized matrix factor CV of 6 individuals is less than 15%.

The results are shown in tables 2A, 2B, 2C, 2D, 2E, 2F, 2G, and 2H.

Table 2A valsartan extraction recovery.

Table 2B sakuba extraction recovery.

TABLE 2C Valsartan-d₃And (5) extracting and recovering rate.

TABLE 2D Shakubaqu-D₄And (5) extracting and recovering rate.

Table 2E valsartan matrix effect.

Table 2F shakubatu matrix effect.

TABLE 2G Valsartan-d₃Matrix effect.

TABLE 2H Shakubaqu-d₄Matrix effect.

The results in table 2A show that after the plasma sample of valsartan is extracted by methanol, the recovery rate of the high-concentration quality control sample (HQC) is 92.2%, the recovery rate of the medium-concentration quality control sample (MQC) is 91.5%, the recovery rate of the low-concentration quality control sample (LQC) is 89.2%, the average recovery rate of the low, medium and high concentrations is 91.0%, the relative variation coefficient is 1.7%, and the isotope internal standard valsartan-d is₃The recovery rate of the high-concentration quality control sample (HQC) was 100.9%, the recovery rate of the medium-concentration quality control sample (MQC) was 99.1%, the recovery rate of the low-concentration quality control sample (LQC) was 98.8%, the average recovery rates of the low, medium and high concentrations were 99.6%, the relative variation coefficient was 1.1%, and isotopic Neigosaur batu-d was obtained after methanol extraction of the plasma sample of Sacabaqu₄The recovery rate of the valsartan/sabotara is 100.1 percent, the recovery rate of the valsartan/sabotara in the method is high, the variation coefficient of the recovery rate of low, medium and high concentrations is small, and the requirements of pharmacopoeia are met.

Tables 2E-2H show that the matrix factors of valsartan/sabotarol are all between 0.85 and 1.15, which indicates that the analysis method of the invention is slightly influenced by individual matrix difference and has high method reliability.

(5) Stability survey

Stability studies include: the plasma sample is placed for 17h at room temperature, the stability of repeated freeze thawing (freezing at 70 ℃ and thawing at room temperature) is obtained for 6 times, and the sample injector is placed for 72h after treatment. Preparing 6 low-concentration and high-concentration quality control samples respectively, placing the samples under corresponding environmental conditions, processing the samples, performing sample injection detection, quantifying the samples according to a freshly prepared standard curve and the quality control samples, and calculating the deviation between the measured concentration and a theoretical value. The results are shown in tables 3A-3F.

Table 3A: short term stability of valsartan biological samples.

The test conditions are as follows: short term stability of 17 hours at room temperature.

Table 3B: short term stability of the biological sample of Sacubitril.

The test conditions are as follows: short term stability of 17 hours at room temperature.

Table 3C: and (3) freeze-thaw stability of the valsartan.

The test conditions are as follows: frozen in a super-low temperature freezing refrigerator (-70 ℃) for at least 24 hours for the first time, and then thawed at room temperature for at least 30 minutes for at least 12 hours.

Table 3D: and (5) freeze-thaw stability of the Shakuba koji.

The test conditions are as follows: frozen in a super-low temperature freezing refrigerator (-70 ℃) for at least 24 hours for the first time, and then thawed at room temperature for at least 30 minutes for at least 12 hours.

Table 3E: stability of the sample after valsartan treatment.

The test conditions are as follows: stability after 72 hours at 5 ℃.

Table 3F: stability of the sample after treatment with Shakuba koji.

The test conditions are as follows: stability after 72 hours at 5 ℃.

The analysis method provided by the invention verifies that the valsartan/sabotara plasma sample is stable after being placed for 17h at room temperature, 6 times of freeze-thaw stability (-70 ℃ freezing/room temperature thawing), and 72h after treatment.

The verification of the methodology is that the method of the invention conforms to the rules of pharmacopoeia and is suitable for high-throughput analysis of biological samples.

Example (b): human body bioequivalence research of Sacubitril valsartan sodium tablets

Taking Chinese healthy volunteers as test subjects, evaluating the relative bioavailability of Sacaba valsartan calcium sodium tablets (100 mg: 1 tablets, tested preparations) produced by a certain fasting oral company and Sacaba valsartan sodium tablets produced by Nowa company under the trade name of Noxintuo (100 mg: 1 tablets, reference preparations), and evaluating whether the tested preparations and the reference preparations have or possibly have bioequivalence. The study was a single-center, randomized, open, two-dose, single-dose, three-cycle partially-repeated crossover, fasting, postprandial bioequivalence experiment in healthy subjects.

Designing a blood sampling point: venous blood was collected at 20 time points of 0 hour (within 60 minutes before administration) and 0.167(10min), 0.333(20min), 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 12, 24, 36, 48, 72 hours after administration, and approximately 4mL of each blood draw was placed in a K chamber₂All centrifuged plasma samples were split into two in EDTA anticoagulant vacuum blood collection tubes and stored in an ultra-low temperature freezer (freezer temperature range-80 ± 10 ℃) for pharmacokinetic analysis.

HPLC-MS/MS analysis was performed according to the blood collection time point and the administration dose of the bioequivalence test, under the item of "treatment of 1.4 plasma samples".

In this example, a mass spectrometer (AB sciex 4000) was used to optimize and fix a mobile phase gradient of 0.1% formic acid water using a specific chromatographic column (Agilent ZORBAX SB-Aq, 100 x 3.0 mm, 3.5 μm): a specific detection range (the linear range of valsartan is 7.5-7500ng/mL, and the linear range of sabotara is 5-5000ng/mL) is designed at a flow rate of 0.5mL/min for a mixed solution (90/10, v/v) of acetonitrile containing 10% methanol, a plurality of independent sabotara sodium tablets for human body bioequivalence research are efficiently carried out, detection and analysis of ten thousand clinical samples are realized, and the clinical sample reanalysis (ISR) is 100% passed.

Reference example

In the same manner as in example 8 and example 1 described in patent document 1(CN110031568A), the human bioequivalence of the sodium sarkubara valsartan tablet was measured and used as a reference example of the present invention.

The measurement conditions in patent document 1 are shown below, and the entire contents thereof are incorporated herein by reference.

Liquid phase conditions: mobile phase: acetonitrile-0.1% aqueous formic acid (v/v,60:40), flow rate: 0.4 mL/min.

Mass spectrum conditions: a TRIPLE quadrupole tandem mass spectrometer model AB, TRIPLE QUAD 5500, was used (equipped with an electrospray ionization ESI source, Analyst 1.6.3 software).

Plasma sample pretreatment: methanol is adopted for pretreatment.

An advantage of embodiments of the present invention is that the target compound can be better separated at a flow rate of 0.5mL/min, and a good peak shape and reasonable analysis time are obtained. In the detection of a large number of clinical samples, the interference of unknown metabolites in the clinical samples is effectively avoided through the reasonable chromatographic gradient program of the method, and the accuracy and the authenticity of the concentration monitoring of the clinical samples are ensured. The method for determining the concentrations of valsartan and sabotarol by HPLC-MS/MS has the advantages of simple and quick sample treatment and suitability for large-flux clinical sample detection. Compared with a reference example, the method adopts a more common mass spectrum model (AB sciex 4000) and a conventional type chromatographic column (Agilent ZORBAX SB-Aq, 100 x 3.0 mm and 3.5 mu m), namely, the method realizes the human body bioequivalence research of the Sacuba varix sodium tablets by using a conventional low-end mass spectrometer, and is more practical. In addition, the pretreatment protein precipitator is optimized, acetonitrile (high removal efficiency of acetonitrile phospholipid) is used for replacing methanol in a reference example, so that the absolute matrix factor of valsartan/sabotara is (0.97-1.07), and the absolute matrix factor in the reference example is (0.788-1.23), and therefore the method has higher specificity and selectivity and is less influenced by individual difference.

The detection range of the valsartan/Sacubitril designed by the method effectively avoids dilution of a large number of samples, ensures that the concentration of the elimination phase sample can still be detected, and effectively monitors the accuracy of the clinical sample because the concentration of the quality control sample designed by the method covers the concentration distribution of the clinical sample.

The present invention is not intended to be limited in scope by the specifically disclosed embodiments, which are provided to illustrate aspects of the present invention. Various modifications to the methods described will become apparent from the description and teachings of the invention. Such variations may be practiced without departing from the true scope and spirit of the invention, and are intended to fall within the scope of the invention.