A method for determining the plasma protein binding rate of bismuth in bismuth potassium citrate

Abstract

The application discloses a method for determining the bismuth plasma protein binding rate of bismuth potassium citrate, and is characterized in that the method comprises plasma sample pretreatment and sample determination. The determination method can solve the stability problem of bismuth potassium citrate in plasma, can make protein binding more sufficient, can effectively promote the dissociation of bismuth and plasma protein, can make the detected protein binding rate closer to the real value, has great significance for guiding clinical medication, is simple and easy to operate, can realize rapid detection, and has low dependence on instruments.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis, specifically to a method for determining the binding rate of bismuth to plasma proteins in potassium bismuth citrate. Background Technology

[0002] Gastric ulcers are a type of ulcer disease of the digestive system, referring to ulcers that occur on the inner wall of the stomach. The gastric mucosa, which forms the inner wall of the stomach, has a strong protective function. Once the gastric mucosa is damaged for various reasons, an ulcer will appear at the site of the damage, which is the clinical gastric ulcer. Duodenal ulcers, on the other hand, are caused by excessive gastric acid secretion or impaired duodenal mucosal function, leading to localized inflammatory damage to the duodenal mucosa caused by the corrosion of the duodenal mucosa by gastric acid. The occurrence of both gastric and duodenal ulcers is due to the combined effect of numerous 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 rising economic levels.

[0003] Bismuth potassium citrate is a bismuth-containing complex of indeterminate composition, a double salt of basic bismuth citrate. It is a white powder with a salty taste, extremely soluble in ethanol, but readily soluble in water, forming a solution. When dissolved in a measured amount of warm water and taken orally, bismuth potassium citrate hydrolyzes in gastric juice to form a strong bismuth trioxide colloidal precipitate. This precipitate forms a diffuse protective layer covering the ulcer surface, isolating it from the erosive effects of gastric acid, pepsin, and food on the ulcer mucosa. Bismuth potassium citrate also stimulates the release of endogenous prostaglandins, promoting ulcer mucosal regeneration and ulcer healing. In the acidic environment of the stomach, it forms a diffuse protective layer covering the ulcer surface, preventing the invasion of gastric acid, enzymes, and food into the ulcer. It can reduce pepsin activity, increase mucin secretion, and promote the release of prostaglandins from the mucosa, thereby protecting the gastric mucosa. The structural formula of bismuth potassium citrate is:

[0004]

[0005] Bismuth, as a heavy metal, can cause toxic reactions if absorbed in excess by the human body, leading to damage to the kidneys, bones, joints, and central nervous system. Bismuth potassium citrate granules, capsules, and tablets, when dissolved, form free bismuth. In the acidic environment of the stomach, most of this forms a diffuse protective layer covering ulcers, producing a pharmacological effect, while a small amount remains free in the digestive system. If the content of free bismuth in the raw materials and formulations is high, the total amount of free bismuth entering the digestive system will inevitably increase, posing a potential safety hazard.

[0006] Since most drugs are absorbed into the bloodstream through various routes, they exist in two states: protein-bound and free. Bound drugs do not exert pharmacological effects, only free drugs do. Therefore, the plasma protein binding rate of drugs has a significant impact on the efficacy of drug treatment.

[0007] Currently, methods for determining bismuth concentration include colorimetric analysis, oscillometric polarography, carbon paste electrode oscillometric polarographic 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 (ICP-MS). However, this method involves a complex pretreatment process and a lengthy evaporation and cooling process, making it unsuitable for detecting large batches of biological samples. In the sample pretreatment, 4 mL of concentrated nitric acid is added first, heated and evaporated to dryness in a 75°C oven, followed by the addition of 5 mL of 2% nitric acid. The nitric acid evaporation process is not only harmful to humans and the environment, but prolonged injection of high-concentration nitric acid can also corrode the injection cone and retrieval cone, reducing the instrument's lifespan. Furthermore, in the sample pretreatment, the subjects are beagle dogs, and the serum volume is 200 μL, a large volume that is difficult to obtain, significantly increasing 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 using inductively coupled plasma mass spectrometry. Although the sample size is small and the analysis time is short, the linear range is wide and the lower limit of quantitation is low, it uses inductively coupled plasma mass spectrometry, which requires a special mass spectrometry instrument and is subject to limitations in terms of usage conditions.

[0009] The methods described above for determining the binding rate of bismuth to plasma proteins are highly instrument-dependent, complex to operate, and have low accuracy. Current methods for detecting plasma protein binding rate utilize ultracentrifugation to separate plasma proteins, measure the free drug concentration in the ultrafiltrate, and then extract the drug from the whole plasma to measure the whole plasma drug concentration. The difference between these two methods represents the protein-bound drug concentration, and the protein binding rate is calculated as (whole plasma drug concentration - free drug concentration) / whole plasma drug concentration × 100%. Plasma stability and the degree of dissociation between the bound drug and protein significantly affect the protein binding rate measurement results. This invention addresses the shortcomings of existing technologies by providing a method for measuring the binding rate of bismuth in potassium citrate to plasma proteins. The measured data more closely approximates the true values ​​and is of great significance for guiding clinical medication use. Summary of the Invention

[0010] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for measuring the binding rate of bismuth in potassium citrate to plasma proteins, with the measured data being closer to the true value. Specifically, this method can accurately determine the binding rate of bismuth in potassium citrate to plasma proteins, which is of great significance for clinical pharmacokinetic sample monitoring and guiding clinical medication.

[0011] The above-mentioned objective of this invention is achieved through the following technical solution: a method for determining the binding rate of bismuth in potassium bismuth citrate to plasma proteins, 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. Then refrigerate the incubated blood plasma.

[0016] 2) Sample determination:

[0017] The sample was placed in an ultrafiltration centrifuge tube, ultrafiltered, and the filtrate was dried by nitrogen. The filtrate was then redissolved in a mobile phase of acetone:water to obtain the sample for testing. The sample was then detected by HPLC-UV method.

[0018] In this invention, the addition of polylactic acid-polyethylene glycol copolymer is one of the key technical aspects for determining the binding rate of bismuth in potassium bismuth citrate to plasma proteins. Plasma stability and the degree of dissociation between the bound drug and protein significantly affect the protein binding rate. Because potassium bismuth citrate contains a large number of ester or lactone groups, it is easily degraded in plasma, thus affecting the accuracy of the measurement results. The addition of polyethylene glycol can solve the problem of poor stability of potassium bismuth citrate. As a stabilizer, polyethylene glycol has excellent rheological properties, biocompatibility, and biodegradability, which can increase the stability of potassium bismuth citrate and reduce its degradation in plasma.

[0019] The degree of dissociation between the drug and protein also affects the detection results of protein binding rate. Chinese patent CN202311645237.0 discloses a plasma sample processing method for detecting the plasma protein binding rate of voriconazole. This method uses phosphatidic acid to pretreat the plasma sample before extraction, effectively promoting the dissociation of voriconazole and plasma proteins. The inventors found that the binding of phosphatidic acid to plasma proteins was not higher than that of bismuth potassium citrate, therefore, it could not cause the dissociation of bismuth potassium citrate and plasma proteins. The inventors were pleasantly surprised to find that polylactic acid (PLA) bound to plasma proteins much higher than that of bismuth potassium citrate. Therefore, if PLA is used to pretreat the plasma sample before extraction, it will promote the dissociation of bismuth potassium citrate and plasma proteins, making the detection data closer to reality.

[0020] When testing the binding rate of bismuth to plasma proteins in bismuth potassium citrate, the use of polylactic acid (PLA) and polyethylene glycol (PEG) alone can cause interference, and errors in the dosage of each can significantly affect the results. The inventors discovered that using a PLA-PEG copolymer can achieve the stabilizing effect of PEG and the dissociation-promoting effect of PLA, while avoiding mutual interference. Furthermore, a single application avoids the effects of dosage errors when using either copolymer alone.

[0021] In this invention, the molecular weight of the polylactic acid-polyethylene glycol copolymer (PLG) is one of the key technical factors in determining the binding rate of bismuth in potassium citrate to plasma proteins. When the molecular weight is low, the PLAG is a liquid, which is inconvenient to handle; when the molecular weight is high, the water solubility of the PLAG gradually decreases, reducing its solubility with plasma and causing larger errors. Preferably, in this invention, the molecular weight of the PLAG is 2000-3000. More preferably, the molecular weight of the PLAG is 2000.

[0022] In this invention, the concentration of the polylactic acid-polyethylene glycol (PLA-PEG) copolymer solution is one of the key technical aspects for determining the binding rate of bismuth in potassium bismuth citrate to plasma proteins. Based on the plasma environment, the PLA-PEG solution is dissolved in dichloromethane, which facilitates the dissociation of potassium bismuth citrate and plasma proteins. When the PLA-PEG solution concentration is too high, polyethylene glycol affects the dissociation-promoting effect of PLA, resulting in poor dissociation of bismuth in potassium bismuth citrate to plasma proteins. When the PLA-PEG solution concentration is too low, some potassium bismuth citrate will be degraded by the plasma, and the dissociation-promoting effect of PLA is weakened, affecting the accuracy of the measurement results. Preferably, the PLA-PEG solution is prepared by dissolving PLA-PEG in dichloromethane at a concentration of 2.0-2.4 mg / ml. More preferably, 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. The concentration described above refers to the concentration of the polylactic acid-polyethylene glycol copolymer solution. At this concentration, the polylactic acid-polyethylene glycol copolymer solution maintains the stability of bismuth potassium citrate in plasma while promoting its dissociation from plasma proteins, thereby improving the accuracy of detection.

[0023] In this invention, the incubation conditions are one of the key factors in determining the binding rate of bismuth in potassium citrate to plasma proteins. An incubation temperature of 36.5℃-37.5℃ simulates the human body temperature environment. After incubation for a period of time, the concentration of free drug on both sides of the two compartments is equal, while the bound drug remains on the protein solution side. The plasma protein binding rate can be calculated by measuring the drug concentration on both sides. Excessive incubation time can cause the liquid to move from the buffer solution side to the protein side under the influence of colloid osmotic pressure, thereby diluting the protein concentration and affecting the binding rate. Preferably, in step b, the polylactic acid-polyethylene glycol plasma to be tested is incubated in a water bath at a constant temperature of 36.5℃-37.5℃ for 30-60 minutes. More preferably, in step b, the temperature is 37.0℃ and the incubation time is 40-50 minutes.

[0024] In this invention, refrigeration conditions are one of the key factors in determining the binding rate of bismuth in potassium bismuth citrate to plasma proteins. Refrigeration before testing places the plasma under suitable conditions, preventing plasma deterioration and affecting test results. If the temperature is too low, the plasma will coagulate, and re-decoction will affect the detection results. If the temperature is too high, the plasma will deteriorate, similarly affecting the detection results. Both excessively short and excessively long refrigeration times can lead to plasma deterioration. Only under suitable refrigeration conditions can the binding of bismuth in potassium bismuth citrate to plasma proteins be detected effectively. Preferably, the plasma refrigeration temperature is 0℃-4℃, and the refrigeration time is 2-5 hours; more preferably, the refrigeration time is 3 hours.

[0025] In this invention, the testing conditions are one of the factors affecting the technical effectiveness of determining the binding rate of bismuth in potassium citrate to plasma proteins. Centrifugation conditions are one such factor. Short centrifugation time and insufficient centrifugal force result in poor ultrafiltration, failing to effectively separate free and bound drugs. Long centrifugation time and excessive centrifugal force damage plasma proteins, affecting the detection results. Preferably, the ultrafiltration temperature is 15-30℃, and the centrifugation is performed at 6000-10000g for 20-40 minutes. More preferably, the ultrafiltration temperature is 25℃, and the centrifugation is performed at 8000g for 30 minutes. The type and ratio of the reconstitution solvent are also factors. When the reconstitution 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, and more preferably, acetone:water = 1:4. Under these conditions, a smaller amount of solvent can be used to completely reconstitute the plasma to be tested, with minimal impact on the detection results.

[0026] In a preferred embodiment of the present invention, a method for determining the binding rate of bismuth to plasma proteins in bismuth potassium citrate is provided, 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℃ for 40-50 min;

[0030] c. Then refrigerate the incubated plasma at 0℃-4℃ for 3 hours.

[0031] 2) Sample determination:

[0032] The sample was placed in an ultrafiltration centrifuge tube and centrifuged at 8000g for 30 min at 25℃. The filtrate was dried under nitrogen and reconstituted with a mobile phase of acetone:water = 1:4. This was used as the test sample, and the sample was detected by HPLC-UV method.

[0033] The polylactic acid-polyethylene glycol copolymer has a molecular weight of 2000;

[0034] The polylactic acid-polyethylene glycol copolymer solution was 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 proteins in bismuth potassium citrate, applicable to bismuth potassium citrate raw materials or their preparations, including but not limited to bismuth potassium citrate granules, bismuth potassium citrate capsules, and bismuth potassium citrate tablets.

[0036] The present invention has the following advantages over the prior art:

[0037] This invention provides a method for determining the binding rate of bismuth potassium citrate to plasma proteins. The method involves pretreating the plasma sample with polylactic acid-polyethylene glycol copolymer before extraction. Experiments have shown that this method can solve the stability problem of bismuth potassium citrate in plasma, allowing for more complete protein binding while effectively promoting the dissociation of bismuth and plasma proteins. This results in a protein binding rate that is closer to the true value, which is of great significance for guiding clinical medication. Furthermore, the method is simple and easy to perform, enabling rapid detection with low dependence on instruments. Attached Figure Description

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

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

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

[0041] Figure 4 This is the HPLC chromatogram of Comparative Example 3 of the present invention.

[0042] in, Figure 1 No internal standard was added. Figures 2-4 Thallium is the internal standard. Detailed Implementation

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

[0044] Example 1:

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

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

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

[0048] Step 2:

[0049] In vitro binding

[0050] Take 490 μL of plasma from healthy individuals, add 10 μL of stock solution, set up a constant temperature shaker at 37℃, and perform in vitro binding at a shaking speed of 50 rpm for 30 min to obtain an in vitro binding model of bismuth potassium citrate plasma protein.

[0051] Example 2: Detection of Free Drug Concentration

[0052] Take one sample from Example 1 that has been successfully bound in vitro, place it in a Centrifree ultrafiltration centrifuge tube, and ultrafilter it for 30 min at a centrifugal force of 8000×g. Take the filtrate, blow it dry with nitrogen, and add 500 μL of mobile phase with acetone:water = 1:4 to reconstitute it as the sample to be tested. Detect it using HPLC-UV method.

[0053] Chromatographic conditions: A high-performance liquid chromatograph with a PDA (Photo-Diode Array) detector and a Diamonsil C18 column (4.6 mm × 200 mm, 5 μm, Guangzhou Dima Co., Ltd.) were selected. The mobile phase was acetone:water = 1:4. The column temperature was set at 30℃. The average retention time of bismuth potassium citrate and internal standard was less than 15 min. The theoretical plate number of the two peaks was greater than 5000. These were the chromatographic conditions.

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

[0055] Take one sample from Example 1 that has been successfully bound in vitro, add 500 μL of dichloromethane, place it in a Centrifree ultrafiltration centrifuge tube, and ultrafilter at 8000 × g for 30 min. Dry the filtrate with nitrogen and add 500 μL of a mobile phase of acetone:water = 1:4 to reconstitute it as the sample to be tested. Detect it using HPLC-UV method under the same chromatographic conditions as above.

[0056] Example 4: Whole plasma drug concentration after pretreatment using the method of the present invention

[0057] (1) Prepare a solution containing polylactic acid-polyethylene glycol copolymer (molecular weight 2000, purity 99%) at a concentration of 2.2 mg / ml using dichloromethane for later use;

[0058] (2) Take one sample that has completed in vitro binding, add 500 μL of polylactic acid-polyethylene glycol copolymer solution, mix well, and incubate at a constant temperature of 37.0℃ for 50 min; then place the incubated plasma in a 0-4℃ refrigerator for 3 h; complete the pretreatment.

[0059] The sample was placed in a Centrifree ultrafiltration centrifuge tube and ultrafiltered at 8000×g for 30 min at 25℃. The filtrate was dried under nitrogen and reconstituted with 500 μL of a mobile phase of acetone:water = 1:4. The sample was then analyzed by HPLC-UV under the same chromatographic conditions as above.

[0060] Example 5: Whole plasma drug concentration after pretreatment using the method of the present invention

[0061] (1) Prepare a solution containing polylactic acid-polyethylene glycol copolymer (molecular weight 3000, purity 99%) at a concentration of 2.4 mg / ml using dichloromethane for later use;

[0062] (2) Take one sample that has completed in vitro binding, add 500 μL of polylactic acid-polyethylene glycol copolymer solution, mix well, and incubate at a constant temperature of 37.5℃ for 60 min; then place the incubated plasma in a 0-4℃ refrigerator for 5 h; complete the pretreatment.

[0063] The sample was placed in a Centrifree ultrafiltration centrifuge tube and ultrafiltered at 30°C with a centrifugal force of 10000×g for 40 min. The filtrate was dried under nitrogen and reconstituted with 500 μL of a mobile phase of acetone:water = 1:2. The sample was then analyzed by HPLC-UV under the same chromatographic conditions as above.

[0064] Example 6: Whole plasma drug concentration after pretreatment using the method of the present invention

[0065] (1) Prepare a solution containing polylactic acid-polyethylene glycol copolymer (molecular weight 2000, purity 99%) at a concentration of 2.0 mg / ml using dichloromethane for later use;

[0066] (2) Take one sample that has completed in vitro binding, add 500 μL of polylactic acid-polyethylene glycol copolymer solution, mix well, and incubate at a constant temperature of 36.5℃ for 30 min; then place the incubated plasma in a 0-4℃ refrigerator for 2 h; complete the pretreatment.

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

[0068] Example 7: Whole plasma drug concentration after pretreatment using the method of the present invention

[0069] (1) Prepare a solution containing polylactic acid-polyethylene glycol copolymer (molecular weight 3000, purity 99%) at a concentration of 2.3 mg / ml using dichloromethane for later use;

[0070] (2) Take one sample that has completed in vitro binding, add 500 μL of polylactic acid-polyethylene glycol copolymer solution, mix well, and incubate at a constant temperature of 37.0℃ for 55 min; then place the incubated plasma in a 0-4℃ refrigerator for 4 h; complete the pretreatment.

[0071] The sample was placed in a Centrifree ultrafiltration centrifuge tube and ultrafiltered at 7000×g for 25 min at 20℃. The filtrate was dried under nitrogen and reconstituted with 500 μL of a mobile phase of acetone:water = 1:3. The sample was then analyzed by HPLC-UV under the same chromatographic conditions as above.

[0072] Example 8: Whole plasma drug concentration after pretreatment using the method of the present invention

[0073] (1) Prepare a solution containing polylactic acid-polyethylene glycol copolymer (molecular weight 2000, purity 99%) at a concentration of 2.1 mg / ml using dichloromethane for later use;

[0074] (2) Take one sample that has completed in vitro binding, add 500 μL of polylactic acid-polyethylene glycol copolymer solution, mix well, and incubate at a constant temperature of 37.0℃ for 40 min; then place the incubated plasma in a 0-4℃ refrigerator for 3 h; complete the pretreatment.

[0075] The sample was placed in a Centrifree ultrafiltration centrifuge tube and ultrafiltered at 25°C with a centrifugal force of 9000×g for 35 min. The filtrate was dried under nitrogen and reconstituted with 500 μL of a mobile phase of acetone:water = 1:4. The sample was then analyzed by HPLC-UV under the same chromatographic conditions as above.

[0076] Comparative Example 1: Drug concentration in pretreated whole plasma

[0077] (1) Prepare a solution containing polylactic acid-polyethylene glycol copolymer (molecular weight 4000, purity 99%) at a concentration of 2.5 mg / ml using dichloromethane for later use;

[0078] (2) Take one sample that has completed in vitro binding, add 500 μL of polylactic acid-polyethylene glycol copolymer solution, mix well, and incubate at a constant temperature of 38.0℃ for 70 min; then place the incubated plasma at 10℃ for 6 h; complete the pretreatment.

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

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

[0081] (1) Prepare a solution containing polylactic acid-polyethylene glycol copolymer (molecular weight 1000, purity 99%) at a concentration of 1.8 mg / ml using dichloromethane for later use;

[0082] (2) Take one sample that has completed in vitro binding, add 500 μL of polylactic acid-polyethylene glycol copolymer solution, mix well, and incubate at a constant temperature of 36.0℃ for 20 min; then place the incubated plasma at -5℃ for 1 h; complete the pretreatment.

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

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

[0085] (1) Prepare an extraction solution containing 1.2 mg / ml of phosphatidic acid (also known as lysophosphatidic acid, molecular weight 510, purity 99%) using an ethyl acetate: n-hexane = 4:1 solution for later use;

[0086] (2) Take one sample that has been bound in vitro, add 500 μL of phosphatidic acid-containing extract, mix well, and incubate at a constant temperature of 37.0℃ for 50 min; then place the incubated plasma in a 0-4℃ refrigerator for 3 h; complete the pretreatment.

[0087] The sample was placed in a Centrifree ultrafiltration centrifuge tube and ultrafiltered at 8000×g for 30 min at 25℃. The filtrate was dried under nitrogen and reconstituted with 500 μL of a mobile phase of acetone:water = 1:4. The sample was then analyzed by HPLC-UV under the same chromatographic conditions as above.

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

[0089] (1) Prepare a solution containing polylactic acid (molecular weight 1000, purity 99%) at a concentration of 2.2 mg / ml using dichloromethane for later use;

[0090] (2) Take one sample that has been bound in vitro, add 500 μL of polylactic acid solution, mix well, and incubate at a constant temperature of 37.0℃ for 50 min; then place the incubated plasma in a 0-4℃ refrigerator for 3 h; complete the pretreatment.

[0091] The sample was placed in a Centrifree ultrafiltration centrifuge tube and ultrafiltered at 8000×g for 30 min at 25℃. The filtrate was dried under nitrogen and reconstituted with 500 μL of a mobile phase of acetone:water = 1:4. The sample was then analyzed by HPLC-UV under the same chromatographic conditions as above.

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

[0093] (1) Prepare a solution containing polyethylene glycol (molecular weight 2000, purity 99%) at a concentration of 2.2 mg / ml using dichloromethane for later use;

[0094] (2) Take one 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℃ for 50 min; then place the incubated plasma in a 0-4℃ refrigerator for 3 h; complete the pretreatment.

[0095] The sample was placed in a Centrifree ultrafiltration centrifuge tube and ultrafiltered at 8000×g for 30 min at 25℃. The filtrate was dried under nitrogen and reconstituted with 500 μL of a mobile phase of acetone:water = 1:4. The sample was then analyzed by HPLC-UV under the same chromatographic conditions as above.

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

[0097] Table 1: Drug Concentration Detection Table in Plasma (μg / ml)

[0098]

[0099]

[0100] Based on the formula (protein binding rate = (total plasma drug concentration - free drug concentration) / total plasma drug concentration × 100%), the protein binding rates of pretreated and untreated plasma are shown in Table 2.

[0101] Table 2: Plasma protein binding rate

[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 shown in Table 2, the binding rates of bismuth potassium citrate to plasma proteins in Examples 4-8 were all above 90%, consistent with the US specification regarding a bismuth binding rate greater than 90%. Example 2 used an untreated plasma sample; bismuth potassium citrate exhibited instability and incomplete dissociation in plasma, resulting in a lower binding rate of bismuth to plasma proteins. In Comparative Example 1, the polylactic acid-polyethylene glycol copolymer used had a large molecular weight and poor water solubility, reducing its solubility in plasma. Furthermore, when the polylactic acid-polyethylene glycol copolymer solution was too hot, polyethylene glycol affected the dissociation-promoting effect of polylactic acid, leading to poor dissociation of bismuth potassium citrate to plasma proteins. The longer incubation time, higher refrigeration temperature, and greater centrifugal force also disrupted the plasma protein environment, resulting in a lower binding rate of bismuth to plasma proteins and failing to reflect the true binding situation. In Comparative Example 2, the polylactic acid-polyethylene glycol copolymer used had a relatively small molecular weight, leading to a large margin of error. Furthermore, when the polylactic acid-polyethylene glycol copolymer solution was too cold, bismuth failed to dissociate well from plasma proteins, resulting in poor dissociation of bismuth from plasma proteins in potassium citrate. Short incubation times, low refrigeration temperatures, and insufficient centrifugation further worsened the dissociation effect, resulting in a low binding rate of bismuth to plasma proteins, which also failed to reflect the true binding situation. In Comparative Examples 3-5, phosphatidic acid, polylactic acid, and polyethylene glycol were used for pretreatment of the plasma samples, respectively. These methods failed to effectively address the plasma stability and dissociation issues of potassium citrate, resulting in low plasma protein binding rates. Examples 4-8 are preferred solutions in this scheme, and the various parameters in Example 4, including the molecular weight of polylactic acid-polyethylene glycol copolymer, solution concentration, and parameters such as incubation, refrigeration, and testing conditions, are all optimal. Therefore, bismuth potassium citrate has better stability in plasma and dissociates more thoroughly from plasma proteins, and the detected binding rate is closer to the true value, which is of great significance for guiding clinical drug use.

[0104] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for determining the binding rate of bismuth to plasma proteins in bismuth potassium citrate, characterized in that, This includes 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. Then refrigerate the incubated blood plasma; 2) Sample determination: The sample was placed in an ultrafiltration centrifuge tube, ultrafiltered, and the filtrate was dried under nitrogen. The filtrate was then redissolved in an acetone:water mobile phase to obtain the final sample for analysis. HPLC-UV method was used for detection. The polylactic acid-polyethylene glycol copolymer has a molecular weight of 2000-3000; 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; In step b, the polylactic acid-polyethylene glycol plasma to be tested is incubated in a water bath at a constant temperature of 36.5℃-37.5℃ for 30-60 minutes. In step c, the plasma is refrigerated at a temperature of 0°C to 4°C for 2 to 5 hours. In the sample determination, ultrafiltration was performed at 15-30℃, centrifuged at 6000-10000g for 20-40 minutes, with an acetone:water ratio of 1:2-5.

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

3. The determination method according to claim 1, characterized in that, In step a, the polylactic acid-polyethylene glycol copolymer solution is polylactic acid-polyethylene glycol dissolved in dichloromethane, with a concentration of 2.2 mg / ml.

4. The determination method according to claim 1, characterized in that, In step b, the temperature is 37.0℃ and the incubation time is 40-50 minutes.

5. The determination method according to claim 1, characterized in that, In step c, the refrigeration time is 3 hours.

6. The determination method according to claim 1, characterized in that, In the sample determination, ultrafiltration was performed at 25°C, centrifuged at 8000g for 30 minutes, and the ratio of acetone to water was 1:

4.

7. A method for determining the binding rate of bismuth to plasma proteins in bismuth potassium citrate, characterized in that, This includes 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℃ for 40-50 min; c. Then refrigerate the incubated plasma at 0℃-4℃ for 3 hours; 2) Sample determination: The sample was placed in an ultrafiltration centrifuge tube and ultrafiltered at 25℃ and 8000g for 30 min. The filtrate was dried by nitrogen and reconstituted with a mobile phase of acetone:water = 1:4 as the sample to be tested. The sample was then detected by HPLC-UV method. The polylactic acid-polyethylene glycol copolymer has a molecular weight of 2000; The polylactic acid-polyethylene glycol copolymer solution was prepared by dissolving polylactic acid-polyethylene glycol in dichloromethane, with a concentration of 2.2 mg / ml.

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