Method for determining binding rate of bismuth and plasma protein in bismuth potassium citrate

Abstract

The invention discloses a method for determining the binding rate of bismuth and plasma protein in bismuth potassium citrate, which is characterized by comprising the steps of pretreatment of a plasma sample and determination of the sample. The determination method provided by the invention can solve the stability problem of bismuth potassium citrate in plasma, enables protein binding to be more sufficient, effectively promotes dissociation of bismuth and plasma protein, enables the detected protein binding rate to be closer to a true value, has great significance for guiding clinical medication, is simple and easy to operate, can realize rapid detection, and has good application prospects. And the dependence on instruments is low.

Description

Technical Field

[0001] The invention relates to the field of drug analysis, and in particular to a method for determining the binding rate of bismuth to plasma protein in bismuth potassium citrate. Background Art

[0002] Gastric ulcer is a type of digestive system ulcer disease, which refers to ulcers occurring on the inner wall of the stomach. The gastric mucosa on the inner wall of the stomach has a strong protective effect. Once the gastric mucosa is damaged due to various reasons, ulcers will appear at the damaged site, which is a clinical gastric ulcer. Duodenal ulcer is a local inflammatory lesion caused by excessive secretion of gastric juice or damaged function of the duodenal mucosa, which leads to corrosion of the duodenal mucosa by gastric juice. The occurrence of gastric and duodenal ulcers is caused by the superposition of many risk factors. Among them, Helicobacter pylori is one of the main pathogenic factors. Helicobacter pylori is a very common gastrointestinal pathogen, and its infection rate gradually increases with the improvement of economic level.

[0003] Potassium bismuth citrate is a bismuth-containing complex of indefinite composition, which is a complex salt of basic bismuth citrate. Potassium bismuth citrate is a white powder with a salty taste. It dissolves very slightly in ethanol and is very easily soluble in water, forming a solution after dissolving. Potassium bismuth citrate is taken orally after being dissolved in a certain amount of warm water. After being hydrolyzed by gastric juice, it can form a solid bismuth trioxide colloidal precipitate. The colloidal precipitate forms a diffuse protective layer covering the ulcer surface, isolating the erosion of gastric acid, pepsin and food on the ulcer mucosa. Potassium bismuth citrate can also stimulate the release of endogenous prostaglandins, promote the regeneration of ulcer mucosa and ulcer healing. In the acidic environment of the stomach, a diffuse protective layer is formed to cover the ulcer surface, preventing gastric acid, enzymes and food from invading the ulcer, reducing the activity of pepsin, increasing mucin secretion, and promoting the release of prostaglandins from the mucosa, thereby protecting the gastric mucosa. The structural formula of potassium bismuth citrate is:

[0004]

[0005] As a heavy metal, once excessive absorption by the human body occurs, bismuth will lead to toxic reactions and damage to the kidneys, bones and joints, and the central nervous system. Bismuth-containing preparations such as potassium citrate granules, potassium citrate bismuth capsules, and potassium citrate bismuth tablets dissolve to form free bismuth, which mostly forms a diffuse protective layer covering the ulcer surface in the acidic environment of the stomach to produce pharmacological effects, and a small amount is free in the human digestive system. If the content of free bismuth in the raw materials and preparations is too high, it will inevitably lead to an increase in the total amount of free bismuth entering the human digestive system. From a safety perspective, this is a potential safety hazard.

[0006] Since most drugs exist in two states, protein-bound and free, after being absorbed into the blood through various pathways, bound drugs do not exert pharmacological effects, only free drugs exert pharmacological effects. Therefore, the plasma protein binding rate of drugs has a great influence on the efficacy of the drugs.

[0007] The methods currently used to determine bismuth concentrations include colorimetric analysis, oscillographic polarography, carbon paste electrode oscillographic polarography anodic stripping, and atomic absorption spectrophotometry. Chinese patent CN202010316090.0 discloses a method for detecting bismuth concentration in human serum using inductively coupled plasma mass spectrometry. Its pretreatment process is complicated, and the evaporation and cooling process takes a long time, which is not suitable for the detection requirements of large quantities of biological samples; during the sample pretreatment process, 4mL of concentrated nitric acid was first added, and after heating and evaporation in a 75°C oven, 5mL of 2% nitric acid was added. The nitric acid evaporation process is not only harmful to the human body and the environment, but also long-term injection of high-concentration nitric acid will corrode the injection cone and interceptor cone, reducing the life of the instrument. In addition, during the sample pretreatment process, the subject was a beagle dog, and the amount of serum used was 200μL. The amount of serum used was large and difficult to obtain, which greatly increased the difficulty of detecting large quantities of serum samples.

[0008] Chinese patent CN202210628850.0 also discloses a method for detecting bismuth concentration in human serum by inductively coupled plasma mass spectrometry. Although the sample amount used is small, the analysis time is short, the linear range is wide, and the quantitative limit is low, it uses inductively coupled plasma mass spectrometry, which requires the use of a special mass spectrometer and the conditions of use are limited.

[0009] The above method determines the binding rate of bismuth to plasma protein, which is highly dependent on the instrument, and the operation is complicated, and the accuracy is low. The method currently used to detect the binding rate of plasma protein is to separate plasma protein by ultracentrifugation, detect the free drug concentration in the ultrafiltrate, and then extract the drug from the whole plasma to detect the whole plasma drug concentration. The difference between the two is the protein-bound drug concentration, and the protein binding rate = (whole plasma drug concentration-free drug concentration) / whole plasma drug concentration × 100%. Plasma stability and the degree of dissociation of the bound drug from the protein will become the detection result affecting the protein binding rate. The present invention, starting from the deficiencies in the prior art, provides a bismuth-potassium citrate binding rate of plasma protein, and its detection data is closer to the true value, which is of great significance for guiding clinical medication. Summary of the invention

[0010] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a plasma protein binding rate of bismuth in potassium bismuth citrate, wherein the detection data is closer to the true value, as shown in that the method can accurately determine the plasma protein binding rate of bismuth in potassium bismuth citrate, and is used for clinical pharmacokinetic sample monitoring, which is of great significance for guiding clinical medication.

[0011] The above-mentioned object of the present invention is achieved by the following technical scheme: A method for determining the binding rate of bismuth to plasma protein in potassium bismuth citrate, characterized in that it includes plasma sample pretreatment and sample determination steps,

[0012] 1) Sample pretreatment:

[0013] a. Add an equal volume of polylactic acid-polyethylene glycol copolymer solution to the plasma to be tested;

[0014] b. Incubate the test plasma containing polylactic acid-polyethylene glycol in a water bath at a constant temperature;

[0015] c. Refrigerate the incubated plasma.

[0016] 2) Sample determination:

[0017] The sample was placed in an ultrafiltration centrifuge tube, ultrafiltered, the filtrate was dried with nitrogen, added with acetone: water mobile phase and re-dissolved as the sample to be tested, and detected by HPLC-UV method.

[0018] In the present invention, the addition of polylactic acid-polyethylene glycol copolymer is one of the important technical keys for determining the binding rate of bismuth to plasma protein in bismuth potassium citrate. Plasma stability and the degree of dissociation of the bound drug from the protein will become the test results affecting the protein binding rate. Since bismuth potassium citrate contains a large number of ester groups or lactone groups in its structure, it is easily degraded in plasma, thereby affecting the accuracy of the measurement results. The addition of polyethylene glycol can solve the problem of poor stability of bismuth potassium citrate. As a stabilizer, polyethylene glycol has excellent rheological properties, biocompatibility and biodegradability, which can increase the stability of bismuth potassium citrate and reduce its degradation in plasma.

[0019] The degree of dissociation between the bound drug and the protein will also affect the detection results of the protein binding rate. Chinese patent CN202311645237.0 discloses a method for processing plasma samples when detecting the plasma protein binding rate of voriconazole. This method uses phosphatidic acid to pre-treat the plasma sample before extraction, which effectively promotes the dissociation of voriconazole and plasma proteins. The inventors found that the binding of phosphatidic acid to plasma proteins is not higher than that of potassium bismuth citrate, so potassium bismuth citrate and plasma proteins cannot be dissociated. The inventors were pleasantly surprised to find that the binding of polylactic acid to plasma proteins is much higher than that of potassium bismuth citrate. Therefore, if polylactic acid can be used to pre-treat the plasma sample before extraction, it will promote the dissociation of potassium bismuth citrate and plasma proteins, making the test data closer to reality.

[0020] When detecting the binding rate of bismuth to plasma protein in bismuth potassium citrate, the use of polylactic acid and polyethylene glycol alone will interfere with each other, and the error between the amounts of the two will have a greater impact on the results when the two are taken separately. The inventors found that the use of polylactic acid-polyethylene glycol copolymer can obtain the stabilizer effect of polyethylene glycol and the effect of polylactic acid in promoting dissociation, while avoiding mutual interference between the two, and taking them at one time can avoid the impact caused by the error between the amounts when the two are taken separately.

[0021] In the present invention, the molecular weight of polylactic acid-polyethylene glycol copolymer is one of the important technical keys for determining the binding rate of bismuth to plasma protein in bismuth potassium citrate. When the molecular weight is low, polylactic acid-polyethylene glycol copolymer is liquid, which is inconvenient to use. When the molecular weight is high, the water solubility of polylactic acid-polyethylene glycol copolymer gradually deteriorates, and the dissolution with plasma is reduced, resulting in a large error. Preferably, in the present invention, the molecular weight of polylactic acid-polyethylene glycol copolymer is 2000-3000. More preferably, the molecular weight of polylactic acid-polyethylene glycol copolymer is 2000.

[0022] In the present invention, the concentration of polylactic acid-polyethylene glycol copolymer solution is one of the important technical keys for determining the binding rate of bismuth to plasma protein in potassium bismuth citrate. Based on the environment of plasma, polylactic acid-polyethylene glycol copolymer solution is dissolved with dichloromethane as solvent, which is conducive to the dissociation of potassium bismuth citrate and plasma protein. When the polylactic acid-polyethylene glycol copolymer solution is too high, polyethylene glycol will affect the effect of polylactic acid promoting dissociation, so that the dissociation effect of bismuth and plasma protein in potassium bismuth citrate is poor, and when the polylactic acid-polyethylene glycol copolymer solution is too low, part of potassium bismuth citrate will be degraded by plasma, and at the same time, the effect of polylactic acid promoting dissociation is weakened, affecting the accuracy of the measurement result. Preferably, polylactic acid-polyethylene glycol copolymer solution is to dissolve polylactic acid-polyethylene glycol with dichloromethane, and the concentration is 2.0-2.4mg / ml. More preferably, the polylactic acid-polyethylene glycol copolymer solution is polylactic acid-polyethylene glycol dissolved in dichloromethane, and the concentration is 2.2 mg / ml. The above concentration is the concentration of the polylactic acid-polyethylene glycol copolymer solution, and the polylactic acid-polyethylene glycol copolymer solution at this concentration can promote the dissociation of potassium bismuth citrate from plasma proteins while maintaining the stability of potassium bismuth citrate in plasma, thereby improving the accuracy of detection.

[0023] In the present invention, incubation conditions are one of the keys to the technical effect of determining the binding rate of bismuth to plasma protein in potassium bismuth citrate. The incubation temperature is 36.5°C-37.5°C, which can simulate the human body temperature environment. After incubation for a period of time, the free drug concentrations on both sides of the two compartments are equal, and the bound drug still stays on the protein solution side. The plasma protein binding rate can be calculated by measuring the drug concentrations on both sides. If the incubation time is too long, the liquid can be moved from the buffer side to the protein side under the action of colloid osmotic pressure, thereby diluting the protein concentration and affecting the binding rate. Preferably, in step b, the plasma to be tested of polylactic acid-polyethylene glycol is incubated at a constant temperature in a water bath, and the temperature is 36.5°C-37.5°C, and the incubation time is 30-60min. More preferably, in step b, the temperature is 37.0°C and the incubation time is 40-50min.

[0024] In the present invention, refrigeration conditions are one of the keys to the technical effect of determining the binding rate of bismuth to plasma proteins in potassium bismuth citrate. Refrigeration before testing can place the plasma to be tested in suitable conditions to avoid plasma deterioration and affect the test effect. When the temperature is too low, the plasma coagulates and re-thaws, which will affect the test effect. When the temperature is too high, the plasma deteriorates, which also affects the test effect. If the refrigeration time is too short or too long, the plasma may deteriorate. Only in suitable refrigeration conditions can it be beneficial to detect the binding of bismuth to plasma proteins in potassium bismuth citrate. Preferably, the plasma refrigeration temperature is 0°C-4°C, the refrigeration time is 2-5h, and more preferably, the refrigeration time is 3h.

[0025] In the present invention, test conditions are one of the factors for determining the technical effect of bismuth and plasma protein binding rate in potassium bismuth citrate. Among them, centrifugal conditions are one of the factors, short centrifugal time, insufficient centrifugal force, poor ultrafiltration effect, free drug and bound drug cannot be effectively separated, long centrifugal time, excessive centrifugal force, plasma protein is damaged, affecting the detection effect. Preferably, the ultrafiltration temperature is 15-30 ° C, 6000-10000g centrifugal ultrafiltration 20-40min, more preferably, the ultrafiltration temperature is 25 ° C, 8000g centrifugal ultrafiltration 30min. The type and ratio of the re-dissolving solvent are also one of the factors. When the re-dissolving solvent is acetone: water, the plasma to be tested can be fully dissolved. Preferably, the volume ratio of acetone to water is acetone: water = 1: 2-5, more preferably, acetone: water = 1: 4, under this condition, less solvent can be used to completely re-dissolve the plasma to be tested, and the impact on the test result is small.

[0026] In a preferred embodiment of the present invention, a method for determining the binding rate of bismuth to plasma protein in bismuth potassium citrate is provided, which is characterized by comprising plasma sample pretreatment and sample determination steps.

[0027] 1) Sample pretreatment:

[0028] a. Add an equal volume of polylactic acid-polyethylene glycol copolymer solution to the plasma to be tested;

[0029] b. Incubate the test plasma containing polylactic acid-polyethylene glycol in a water bath at a constant temperature of 37.0°C for 40-50 minutes;

[0030] c. Refrigerate the incubated plasma at 0℃-4℃ for 3h.

[0031] 2) Sample determination:

[0032] The sample was placed in an ultrafiltration centrifuge tube, centrifuged at 8000 g for 30 min at 25°C, and the filtrate was dried with nitrogen and added into a mobile phase of acetone: water = 1:4 to re-dissolve as the sample to be tested, and detected by HPLC-UV method.

[0033] The molecular weight of the polylactic acid-polyethylene glycol copolymer is 2000;

[0034] The polylactic acid-polyethylene glycol copolymer solution is prepared by dissolving polylactic acid-polyethylene glycol in dichloromethane, with a concentration of 2.2 mg / ml.

[0035] The present invention provides a method for determining the binding rate of bismuth to plasma protein in potassium bismuth citrate, which is applicable to potassium bismuth citrate raw materials or preparations thereof, including but not limited to potassium bismuth citrate granules, potassium bismuth citrate capsules, and potassium bismuth citrate tablets.

[0036] The present invention has the following beneficial effects compared with the prior art:

[0037] The invention provides a method for determining the binding rate of bismuth to plasma proteins in bismuth potassium citrate. The method adopts polylactic acid-polyethylene glycol copolymer to pre-treat the plasma sample before extraction. Experiments show that the method can solve the stability problem of bismuth potassium citrate in plasma, make the protein binding more sufficient, and effectively promote the dissociation of bismuth and plasma proteins, so that the detected protein binding rate is closer to the true value, which is of great significance for guiding clinical drug use. At the same time, the method is simple and easy to operate, can realize rapid detection, and has low dependence on instruments. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 HPLC analysis of Example 2 of the present invention

[0039] Figure 2 HPLC analysis of Example 3 of the present invention

[0040] Figure 3 HPLC analysis of Example 4 of the present invention

[0041] Figure 4 HPLC analysis spectrum of comparative example 3 of the present invention

[0042] in, Figure 1 No internal standard was added. Figure 2-Figure 4 Thallium as internal standard DETAILED DESCRIPTION

[0043] The present invention is further described in detail below with reference to examples, but the embodiments of the present invention are not limited thereto.

[0044] Embodiment 1:

[0045] Method for establishing an in vitro plasma protein binding model of potassium citrate bismuth

[0046] Step 1: Prepare bismuth potassium citrate stock solution

[0047] Precisely weigh potassium bismuth citrate, dilute it with acetone: water = 1:4, and accurately prepare a solution containing 500 μg / ml potassium bismuth citrate as a stock solution;

[0048] Step 2:

[0049] In vitro binding

[0050] Take 490 μL of healthy human plasma, add 10 μL of reserve solution, set it on a 37°C constant temperature shaker, and perform in vitro binding at a shaking speed of 50 rpm for 30 minutes to obtain an in vitro binding model of potassium citrate bismuth plasma protein.

[0051] Example 2 Detection of free drug concentration

[0052] Take a portion of the sample that has completed in vitro binding in Example 1, place it in a Centrifree series ultrafiltration centrifuge tube, ultrafilter at a centrifugal force of 8000×g for 30 minutes, take the filtrate and blow dry it with nitrogen, add 500 μL of the mobile phase of acetone: water = 1:4 to re-dissolve it as a sample to be tested, and detect it by HPLC-UV method;

[0053] Chromatographic conditions: high performance liquid chromatography-PDA (Photo-Diode Array, diode array) detector, Diamonsil C18 column (4.6mm×200mm, 5μm, Guangzhou Dima Company) chromatographic column, acetone: water = 1:4 as the mobile phase, 30℃ column temperature, retention time of potassium citrate bismuth and internal standard within 15min, two peak theoretical plate number greater than 5000 were selected as chromatographic conditions.

[0054] Example 3: Detection of drug concentration in whole plasma

[0055] Take a portion of the sample that completed in vitro binding in Example 1, add 500 μL of dichloromethane, place it in a Centrifree series ultrafiltration centrifuge tube, ultrafilter at a centrifugal force of 8000×g for 30 minutes, blow the filtrate dry with nitrogen, add 500 μL of acetone: water = 1:4 mobile phase to dissolve it as a sample to be tested, and detect it by HPLC-UV method, and the chromatographic conditions are the same as above.

[0056] Example 4: Whole plasma drug concentration pre-treated by the method of the present invention

[0057] (1) Precisely prepare a 2.2 mg / ml polylactic acid-polyethylene glycol copolymer solution (molecular weight 2000, purity 99%) with dichloromethane for later use;

[0058] (2) Take a portion of the sample that has completed in vitro binding, add 500 μL of the polylactic acid-polyethylene glycol copolymer solution, mix well, and incubate at a constant temperature of 37.0°C in a water bath for 50 minutes; then refrigerate the incubated plasma at 0-4°C for 3 hours to complete the pretreatment.

[0059] The sample was placed in a Centrifree series ultrafiltration centrifuge tube and ultrafiltered at 8000×g for 30 min at 25°C. The filtrate was dried with nitrogen and 500 μL of a mobile phase of acetone: water = 1:4 was added to reconstitute the sample as the test sample. The HPLC-UV method was used for detection, and the chromatographic conditions were the same as above.

[0060] Example 5: Whole plasma drug concentration pre-treated by the method of the present invention

[0061] (1) Precisely prepare a 2.4 mg / ml polylactic acid-polyethylene glycol copolymer solution (molecular weight 3000, purity 99%) with dichloromethane for later use;

[0062] (2) Take a portion of the sample that has completed in vitro binding, add 500 μL of the polylactic acid-polyethylene glycol copolymer solution, mix well, and incubate at a constant temperature of 37.5°C in a water bath for 60 minutes; then refrigerate the incubated plasma at 0-4°C for 5 hours to complete the pretreatment.

[0063] The sample was placed in a Centrifree series ultrafiltration centrifuge tube and ultrafiltered at 30°C with a centrifugal force of 10,000×g for 40 min. The filtrate was dried with nitrogen and 500 μL of a mobile phase of acetone: water = 1:2 was added to reconstitute the sample as the test sample and detected by HPLC-UV under the same chromatographic conditions as above.

[0064] Example 6: Whole plasma drug concentration pre-treated by the method of the present invention

[0065] (1) Precisely prepare a 2.0 mg / ml polylactic acid-polyethylene glycol copolymer solution (molecular weight 2000, purity 99%) with dichloromethane for later use;

[0066] (2) Take a portion of the sample that has completed in vitro binding, add 500 μL of the polylactic acid-polyethylene glycol copolymer solution, mix well, and incubate at a constant temperature of 36.5°C in a water bath for 30 minutes; then refrigerate the incubated plasma at 0-4°C for 2 hours to complete the pretreatment.

[0067] The sample was placed in a Centrifree series ultrafiltration centrifuge tube and ultrafiltered at 15°C with a centrifugal force of 6000×g for 20 min. The filtrate was dried with nitrogen and 500 μL of a mobile phase of acetone: water = 1:5 was added to reconstitute the sample as the test sample and detected by HPLC-UV under the same chromatographic conditions as above.

[0068] Example 7: Drug concentration in whole plasma pre-treated by the method of the present invention

[0069] (1) Precisely prepare a 2.3 mg / ml polylactic acid-polyethylene glycol copolymer solution (molecular weight 3000, purity 99%) with dichloromethane for later use;

[0070] (2) Take a portion of the sample that has completed in vitro binding, add 500 μL of the polylactic acid-polyethylene glycol copolymer solution, mix well, and incubate at a constant temperature of 37.0°C in a water bath for 55 minutes; then refrigerate the incubated plasma at 0-4°C for 4 hours to complete the pretreatment.

[0071] The sample was placed in a Centrifree series ultrafiltration centrifuge tube and ultrafiltered at 7000×g for 25 min at 20°C. The filtrate was dried with nitrogen and 500 μL of a mobile phase of acetone: water = 1:3 was added to reconstitute the sample as the test sample. The HPLC-UV method was used for detection, and the chromatographic conditions were the same as above.

[0072] Example 8: Whole plasma drug concentration pre-treated by the method of the present invention

[0073] (1) Precisely prepare a 2.1 mg / ml polylactic acid-polyethylene glycol copolymer solution (molecular weight 2000, purity 99%) with dichloromethane for later use;

[0074] (2) Take a portion of the sample that has completed in vitro binding, add 500 μL of the polylactic acid-polyethylene glycol copolymer solution, mix well, and incubate at a constant temperature of 37.0°C in a water bath for 40 minutes; then refrigerate the incubated plasma at 0-4°C for 3 hours to complete the pretreatment.

[0075] The sample was placed in a Centrifree series ultrafiltration centrifuge tube and ultrafiltered at 9000×g for 35 min at 25°C. The filtrate was dried with nitrogen and 500 μL of a mobile phase of acetone: water = 1:4 was added to reconstitute the sample as the test sample. The HPLC-UV method was used for detection, and the chromatographic conditions were the same as above.

[0076] Comparative Example 1: Pre-treated whole plasma drug concentration

[0077] (1) Precisely prepare a 2.5 mg / ml polylactic acid-polyethylene glycol copolymer solution (molecular weight 4000, purity 99%) with dichloromethane for later use;

[0078] (2) Take a portion of the sample that has completed in vitro binding, add 500 μL of the polylactic acid-polyethylene glycol copolymer solution, mix well, and incubate at a constant temperature of 38.0°C in a water bath for 70 minutes; then place the incubated plasma in a refrigerator at 10°C for 6 hours to complete the pretreatment.

[0079] The sample was placed in a Centrifree series ultrafiltration centrifuge tube and ultrafiltered at 35°C with a centrifugal force of 12000×g for 60 min. The filtrate was dried with nitrogen and 500 μL of a mobile phase of acetone: water = 1:1 was added to reconstitute the sample as the test sample and detected by HPLC-UV method. The chromatographic conditions were the same as above.

[0080] Comparative Example 2: Pre-treated whole plasma drug concentration

[0081] (1) Precisely prepare a 1.8 mg / ml polylactic acid-polyethylene glycol copolymer solution (molecular weight 1000, purity 99%) with dichloromethane for later use;

[0082] (2) Take a portion of the sample that has completed in vitro binding, add 500 μL of the polylactic acid-polyethylene glycol copolymer solution, mix well, and incubate at a constant temperature of 36.0°C in a water bath for 20 minutes; then place the incubated plasma in a -5°C refrigerator for 1 hour to complete the pretreatment.

[0083] The sample was placed in a Centrifree series ultrafiltration centrifuge tube and ultrafiltered at 10°C with a centrifugal force of 5000×g for 15 min. The filtrate was dried with nitrogen and 500 μL of a mobile phase of acetone: water = 1:6 was added to reconstitute the sample as the test sample and detected by HPLC-UV under the same chromatographic conditions as above.

[0084] Comparative Example 3: Pre-treated whole plasma drug concentration

[0085] (1) Using a solution of ethyl acetate:n-hexane = 4:1, an extract containing 1.2 mg / ml of phosphatidic acid (also known as lysophosphatidic acid, molecular weight 510, purity 99%) is accurately prepared for use;

[0086] (2) Take a portion of the sample that has completed in vitro binding, add 500 μL of the phosphatidic acid-containing extract, mix well, and incubate at a constant temperature of 37.0°C in a water bath for 50 minutes; then refrigerate the incubated plasma at 0-4°C for 3 hours to complete the pretreatment.

[0087] The sample was placed in a Centrifree series ultrafiltration centrifuge tube and ultrafiltered at 8000×g for 30 min at 25°C. The filtrate was dried with nitrogen and 500 μL of a mobile phase of acetone: water = 1:4 was added to reconstitute the sample as the test sample. The HPLC-UV method was used for detection, and the chromatographic conditions were the same as above.

[0088] Comparative Example 4: Pre-treated whole plasma drug concentration

[0089] (1) Precisely prepare 2.2 mg / ml of polylactic acid solution (molecular weight 1000, purity 99%) with dichloromethane for later use;

[0090] (2) Take a portion of the sample that has completed in vitro binding, add 500 μL of polylactic acid solution, mix well, and incubate at a constant temperature of 37.0°C in a water bath for 50 minutes; then refrigerate the incubated plasma at 0-4°C for 3 hours to complete the pretreatment.

[0091] The sample was placed in a Centrifree series ultrafiltration centrifuge tube and ultrafiltered at 8000×g for 30 min at 25°C. The filtrate was dried with nitrogen and 500 μL of a mobile phase of acetone: water = 1:4 was added to reconstitute the sample as the test sample. The HPLC-UV method was used for detection, and the chromatographic conditions were the same as above.

[0092] Comparative Example 5: Pre-treated whole plasma drug concentration

[0093] (1) Use dichloromethane to accurately prepare 2.2 mg / ml of polyethylene glycol solution (molecular weight 2000, purity 99%) for later use;

[0094] (2) Take a portion of the sample that has completed in vitro binding, add 500 μL of polyethylene glycol solution, mix well, and incubate at a constant temperature of 37.0°C in a water bath for 50 minutes; then refrigerate the incubated plasma at 0-4°C for 3 hours to complete the pretreatment.

[0095] The sample was placed in a Centrifree series ultrafiltration centrifuge tube and ultrafiltered at 8000×g for 30 min at 25°C. The filtrate was dried with nitrogen and 500 μL of a mobile phase of acetone: water = 1:4 was added to reconstitute the sample as the test sample. The HPLC-UV method was used for detection, and the chromatographic conditions were the same as above.

[0096] The test results are shown in Table 1:

[0097] Table 1: Drug concentration in plasma μg / ml

[0098]

[0099]

[0100] According to the formula of protein binding rate = (total plasma pressure drug concentration - free drug concentration) / total plasma drug concentration × 100%, the plasma protein binding after pretreatment and without pretreatment is shown in Table 2:

[0101] Table 2: Plasma protein binding

[0102] sample Plasma protein binding rate (%) Example 3 66.06 Example 4 90.92 Example 5 90.71 Example 6 90.41 Example 7 90.68 Example 8 90.36 Comparative Example 1 77.24 Comparative Example 2 80.42 Comparative Example 3 78.13 Comparative Example 4 83.67 Comparative Example 5 84.05

[0103] As can be seen from Table 2, the binding rate of bismuth to plasma protein in the bismuth potassium citrate measured by Examples 4-8 is higher than 90%, which is consistent with the situation in the U.S. specification that the binding rate of bismuth to plasma protein is greater than 90%. Embodiment 2 is a plasma sample without pre-treatment, and bismuth potassium citrate has unstable and insufficient dissociation in plasma, so the binding rate of bismuth to plasma protein is relatively low. In comparative example 1, the polylactic acid-polyethylene glycol copolymer used has a large molecular weight, poor water solubility, and is reduced in dissolution with plasma. When the polylactic acid-polyethylene glycol copolymer solution is too high, polyethylene glycol can affect the effect of polylactic acid promoting dissociation, so that the dissociation effect of bismuth and plasma protein in bismuth potassium citrate is poor, the incubation time is long, and the refrigeration temperature is high, the centrifugal force is large, and the like factors destroy the environment of some plasma proteins, therefore, the binding rate of bismuth to plasma protein is low, and its true binding situation cannot be reflected. In comparative example 2, the molecular weight of the polylactic acid-polyethylene glycol copolymer used is relatively small, and the error of taking is relatively large. When the polylactic acid-polyethylene glycol copolymer solution is too low, bismuth and plasma protein fail to dissociate well, so that the dissociation effect of bismuth and plasma protein in potassium bismuth citrate is poor, the incubation time is relatively short, and the refrigeration temperature is relatively low, the centrifugal force is insufficient, and the dissociation effect of bismuth and plasma protein is further deteriorated, therefore, the binding rate of bismuth to plasma protein is relatively low, and its true binding situation cannot be reflected either. In comparative examples 3-5, phosphatidic acid, polylactic acid, and polyethylene glycol are used to pre-treat the plasma to be tested, respectively, and the plasma stability and dissociation problems of potassium bismuth citrate cannot be effectively solved, therefore, the plasma protein highly binding rate is relatively low. Examples 4-8 are preferred embodiments of this solution, and various parameters in Example 4, including the molecular weight of the polylactic acid-polyethylene glycol copolymer, the solution concentration, and the incubation, refrigeration, and test conditions, are all the most preferred. Therefore, potassium bismuth citrate has better stability in plasma, and is more thoroughly dissociated from plasma proteins. The detected binding rate is closer to the true value, which is of great significance for guiding clinical medication.

[0104] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for determining the binding rate of bismuth to plasma protein in bismuth potassium citrate, characterized in that: Including plasma sample pretreatment and sample measurement steps, 1) Sample pretreatment: a. Add an equal volume of polylactic acid-polyethylene glycol copolymer solution to the plasma to be tested; b. Incubate the test plasma containing polylactic acid-polyethylene glycol in a water bath at a constant temperature; c. Refrigerate the incubated plasma; 2) Sample determination: The sample was placed in an ultrafiltration centrifuge tube, ultrafiltered, the filtrate was dried with nitrogen, added with acetone: water mobile phase and re-dissolved as the sample to be tested, and detected by HPLC-UV method.

2. The measuring method according to claim 1, characterized in that In the step a, the molecular weight of the polylactic acid-polyethylene glycol copolymer is 2000-3000, preferably 2000.

3. The measuring method according to claim 1, characterized in that In the step a, the polylactic acid-polyethylene glycol copolymer solution is prepared by dissolving polylactic acid-polyethylene glycol in dichloromethane, with a concentration of 2.0-2.4 mg / ml.

4. The measuring method according to claim 3, characterized in that In the step a, the polylactic acid-polyethylene glycol copolymer solution is prepared by dissolving polylactic acid-polyethylene glycol in dichloromethane, with a concentration of 2.2 mg / ml.

5. The measuring method according to claim 1, characterized in that In the step b, the polylactic acid-polyethylene glycol plasma to be tested is incubated in a water bath at a constant temperature of 36.5° C.-37.5° C. for 30-60 minutes.

6. The measuring method according to claim 5, characterized in that In the step b, the temperature is 37.0° C. and the incubation time is 40-50 min.

7. The measuring method according to claim 1, characterized in that In the step c, the plasma is refrigerated at a temperature of 0°C-4°C for 2-5 hours, preferably for 3 hours.

8. The measuring method according to claim 1, characterized in that In the sample determination, the ultrafiltration is performed at 15-30° C., 6000-10000 g centrifugal ultrafiltration for 20-40 min, and acetone: water = 1:2-5.

9. The measuring method according to claim 8, characterized in that In the sample determination, ultrafiltration was performed at 25° C., 8000 g centrifugal ultrafiltration for 30 min, and acetone: water = 1:

4.

10. A method for determining the binding rate of bismuth to plasma protein in bismuth potassium citrate, characterized in that: Including plasma sample pretreatment and sample measurement steps, 1) Sample pretreatment: a. Add an equal volume of polylactic acid-polyethylene glycol copolymer solution to the plasma to be tested; b. Incubate the test plasma containing polylactic acid-polyethylene glycol in a water bath at a constant temperature of 37.0°C for 40-50 minutes; c. Refrigerate the incubated plasma at 0℃-4℃ for 3h; 2) Sample determination: Place the sample in an ultrafiltration centrifuge tube, ultrafilter at 25°C, 8000g for 30 min, blow dry the filtrate with nitrogen, and add acetone: The sample was reconstituted with the mobile phase of water = 1:4 and detected by HPLC-UV method; The molecular weight of the polylactic acid-polyethylene glycol copolymer is 2000; The polylactic acid-polyethylene glycol copolymer solution is prepared by dissolving polylactic acid-polyethylene glycol in dichloromethane, with a concentration of 2.2 mg / ml.

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