Determination of plasma protein binding rate of Shuganning Injection using equilibrium dialysis and UPLC-MS/MS / 中国中药杂志

Traditional Chinese medicine(TCM) compound preparations have complex compositions. As a widely used TCM injection, Shuganning Injection, its in vivo processes are not yet fully understood. Determining the plasma protein binding rate is of great significance for pharmacokinetic and pharmacodynamic studies. In this experiment, the equilibrium dialysis method combined with UPLC-MS/MS technology was used to determine the plasma protein binding rates of 10 components, including p-hydroxyacetophenone, caffeic acid, baicalein, oroxylin A, geniposide, baicalin, cynaroside, oroxylin A-7-O-β-D-glucuronide, scutellarin, and hyperoside, in Shuganning Injection in rat and human plasma to provide a theoretical basis for further elucidating the in vivo processes of Shuganning Injection and guiding clinical medication. The results showed that, except for baicalein and geniposide, the plasma protein binding rates of the other eight components were higher in human plasma than in rat plasma, and there were interspecies differences. In human plasma, except for geniposide, caffeic acid, and baicalin, the plasma protein binding rates of the remaining seven components were above 80%, with baicalein and oroxylin A exceeding 90%. All components exhibit a high level of binding to plasma proteins, with the exception of geniposide.

Simultaneous determination of LY3214996,abemaciclib,and M2 and M20 metabolites in human plasma,cerebrospinal fluid,and brain tumor by LC-MS/MS / 药物分析学报

A sensitive and rapid liquid chromatography tandem mass spectrometry(LC-MS/MS)method was established for the quantification of total and unbound concentrations of LY3214996,an extracellular signal-regulated kinase inhibitor;abemaciclib,a cyclin-dependent kinase 4/6 inhibitor;and abemaciclib active metabolites,M2 and M20,in human plasma,brain tumor,and cerebrospinal fluid samples.The method was validated over a concentration range of 0.2-500 nM within a total run time of 3.8 min using isocratic elution on a Kinetex? Fs column.Detection was performed on a Sciex QTRAP 6500+mass spectrometer employing multiple reaction monitoring mode under positive electrospray ionization.The intra-and inter-batch accuracy as well as the precision of the method for all matrices was within±20％and≤20％at the lower limit of quantification,and within±15％and≤15％for other quality control levels for all analytes.The unbound fractions of drugs and metabolites in spiked and patient samples were determined using an optimized equilibrium dialysis.The validated method was successfully applied in a phase 0/2 clinical trial to assess the central nervous system penetration of LY3214996 and abemaciclib.

Establishment and application of a method for the determination of plasma protein binding rate of imatinib and its metabolite / 中国药房

OBJECTIVE To establish a method for the determination of plasma protein binding rate of imatinib and its metabolite（N-desmethyl imatinib ）and apply it to patients with gastrointestinal stromal tumor （GIST）.METHODS Using imatinib - d8 as the internal standard ，after being deproteinized methanol ，the sample was determined by equilibrium dialysis combined with liquid chromatography -tandem mass spectrometry . The free concentrations of imatinib and its metabolites in plasma of GIST patients were detected by the same method . RESULTS The protein binding rates of imatinib with albumin ，α1-acid glycoprotein and globulin at 120 ng/mL and 4 000 ng/mL were （92.5±1.0）% and（91.7±0.4）%，（56.6±2.0）% and（62.6±2.6）%，（56.3±3.1）% and （68.0±8.6）% ，respectively. The protein binding rates of N-desmethyl imatinib with albumin ，α1-acid glycoprotein and globulin at 60 ng/mL and 2 000 ng/mL were （90.6±3.5）% and（91.3±1.5）%，（54.1±5.1）% and（63.7±1.3）%，（56.2±7.6）% and（67.5±7.3）%，respectively. Compared with the low concentration group of imatinib （120 ng/mL）and its metabolite （60 ng/mL），the plasma protein binding rate of high concentration of imatinib （4 000 ng/mL）and its metabolite （2 000 ng/mL）with α1-acid glycoprotein and globulin was significantly increased （P＜ Δ基金项目 国家自然科学基金资助项目（No.81503160）；江苏省 0.05），but there was no signifi -cant difference with albumin 卫生健康发展研究中心 2021年度开放课题（No.JSHD2021004）；江苏 （P＞0.05）. In blank plasma ，the protein binding rates of imatinib（4 000 ng/mL）at high concentration and its metabolites（2 000 ng/mL）were significantly lower than those of low （120， 60 ng/mL） and medium （750， 375 ng/mL）concentration （P＜0.01）. Average protein binding rates of imatinib and its metabolite in plasma of GIST patients were （99.0±0.3）% and（99.2±0.3）%，respectively；the correlation coefficients between the concentrations of imatinib and its metabolites and the protein binding rates were －0.298 5 and －3.332 3，respectively（all P＜0.05）. CONCLUSIONS The method for determining the plasma protein binding rates of imatinib and its metabolites is successfully established . The plasma protein binding rates of imatinib and its metabolites in patients with GIST are negatively correlated with drug concentration .

Determination of plasma protein binding rates of nine compounds of Inula cappa extraction based on method of equilibrium dialysis / 中国中药杂志

To determine the plasma protein binding rate of the nine compounds in Inula cappa extraction by the method of equilibrium dialysis. The proteins in plasma samples were precipitated by methanol, and the ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) was developed for determination of the concentrations of the nine active compounds, namely chlorogenic acid, scopolin, neochlorogenic acid, cryptochlorogenic acid, 1,3-O-dicaffeoylquinic acid, galuteolin, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid, with the internal standard of puerarin. We found that all components have a good linearity(r≥0.999), and accuracy, precision, extraction recovery and stability conformed to the requirements of determination, without endogenous compounds disturbing within the range of optimum concentration. This suggested that the method was stable and reliable, and could be used for the determination of the plasma protein binding rates of the nine active compounds in rat and human plasma of I. cappa. The plasma protein binding rates of the nine active compounds in rat and human plasma respectively were(41.07±0.046)%-(94.95±0.008)%, and(37.66±0.043)%-(97.46±0.013)%. According to the results, there were differences in the plasma protein binding rates of the nine compounds in I. cappa extraction between rat and human.

Determination of the protein-binding rates of gardenioside, paeoniflorin and naringin in Weiyanling granules in different species of plasma / 国际中医中药杂志

Objective To study the protein binding rates ofgardenioside,paeoniflorin and naringin in Weiyanling granules in bovine serum albumin,rat plasma and human plasma.Methods The equilibrium dialysis method was carried to determine the binding-rates of three components in Weiyanling granules in bovine serum albumin,rat plasma and human plasma.The HPLC method was used to determine the mass concentration of three components in weiyanling granules in inner and outer dialysis and to calculate the plasma protein-binding rates.Results The linear relationship,precision,extraction recovery and stability of gardenoside,paeoniflorin and naringin all conformed to the methodological requirements.Within the low,middle and high concentration (5.084,10.04 and 20.08 g/L) of Weiyanling granules,the protein-binding rates of gardenioside and bovine serum albumin were 23.77％ ± 13.39％,10.75％ ± 1.34％,5.82％ ± 6.53％,respectively;the protein-binding rates of paeoniflorin were 51.81％ ± 6.53％,30.12％ ± 6.61％,21.39％ ± 9.45％,respectively;the protein-binding rates of naringin were 70.21％ ± 3.31％,67.61％ ± 1.31％,56.00％ ± 3.46％,respectively;the protein-binding rates of gardenioside and rat plasma were 71.56％ ± 3.00 ％,43.56％ ± 12.22％,40.63％ ± 4.73％,respectively;the protein-binding rates ofpaeoniflorin and rat plasma were 55.72％ ± 1.60％,47.59％ ± 6.33％,40.07％ ± 0.72％,respectively;the protein-binding rates of naringin and rat plasma were 85.85％ ± 3.53％,85.38％ ± 0.99％,79.99％ ± 2.57％,respectively;the protein-binding rates of gardenioside and human plasma were 13.56％ ± 2.40％,3.02％ ± 2.41％,5.82％ ± 2.08％,respectively;the protein-binding rates of paeoniflorin and human plasma were 9.49％ ± 5.23％,5.01％ ± 1.10％,3.98％ ± 1.54％,respectively;the protein-binding rates of naringin and human plasma were 25.04％ ± 2.61％,26.09％ ± 13.82％,20.20％ ± 2.24％,respectively.Conclusions Within 5.084,10.04 and 20.08 g/L,the protein binding-rate of gardenioside,paeoniflorin and naringin were shown asfollow,rat plasma ＞bovine serum albumin ＞ human plasma.There were significant differences in species.

Determination of Rat Plasma Protein Binding Rate of Mesaconine by UPLC-MS/MS / 中国药学杂志

OBJECTIVE: To establish an analytical method for determination of mesaconine in rat plasma or dialysate, and study the rat plasma protein binding rate of mesaconine. METHODS: The equilibrium dialysis method was applied to determine the in vitro plasma protein binding rate of mesaconine. A UPLC-MS/MS method was established to determine mesaconine in the intra- and extradialysis samples and then the plasma protein binding rate was calculated. RESULTS: The specificity of the established UPLC-MS/MS method was very good. The calibration curve of mesaconine had good linearity over the range of 0.156 3 - 10 μg•mL-1. The intra-day and inter-day precisions and recovery met the requirements of methodology. The plasma protein binding rates of mesaconine were 18.73%, 20.20%, 26.59%, and 23.68% at the concentrations of 0.2, 0.5, 2.0, and 5.0 μg•mL-1, respectively. CONCLUSION: The method is simple, accurate and sensitive, and can meet the requirement for analysis method of biological samples. Mesaconine has a low rat plasma protein binding rate.

Screening of active components of Jitongning Tablet by liposome equilibrium dialysis and LC-MS analysis / 中草药

Objective: To explore the interaction between the effective components of Jitongning Tablet (JT) and liposome biomembrance, in order to predict in vivo absorption of the effective components. Methods: Liposome as the model of biomembrance, active components were screened by liposome equilibrium dialysis and LC-MS; And the effect of experiment conditions on the interaction of the liposome concentration, and the pH value of buffer were also investigated. Results: There were 11 compounds in JT with obvious interaction with liposome, including 4 flavonoids, 2 triterpenoid, 4 coumarins, and 1 phenylpropionic acid. The concentration of liposome and pH value of buffer remarkably affected the interaction between JT and liposome. Conclusion: The established method contributes to predicting the multiple components absorption in body, indicating the potentially active compounds and providing reference for further research of pharmacodynamic substance basis.

Plasma Protein Binding Characteristics of Neopanaxadiol in Rats by UPLC-MS/MS / 中国药学杂志

OBJECTIVE: To develop an UPLC-MS/MS method for the determination of neopanaxadiol(NPD) in rat plasma sample, and to investigate plasma protein binding rate of NPD in rats. METHODS: Under three different concentrations, equilibrium dialysis method was utilized to imitate the binding process between NPD and plasma protein. The concentration of NPD in and out of the dialysis membrane was determined by UPLC-MS/MS and the plasma protein binding rate of NPD were calculated. RESULTS: Excellent linearity was found between 0.05-8 μg·mL -1. Intra- and inter-day precision values (RSD) of QC samples were both below 15% and the extraction recoveries of NPD from biological matrices were better than 79.37%. The plasma protein binding rates of NPD were (86.55±4.50)%, (76.50±2.61)% and (78.25±1.32)% at low, middle and high concentrations, respectively. There was no significant difference among three concentrations. CONCLUSION: These results indicate the high plasma protein binding rates of NPD in plasma in combination mode.

Determination of plasma protein binding rate of five components in Eucommia ulmoides extract / 中国药理学通报

Aim To determine the plasma protein binding rate of five components of Eucommia ulmoides extract. Methods The equilibrium dialysis method was used to study the plasma protein binding rate. The plasma samples were extracted by protein precipitation with methanol. With the use of puerarin as the internal standard, UPLC-MS/MS was carried out to determine the concentration of the five compounds in and out of the dialysis membrane. Results The average plasma protein binding rates of five compounds on the area of the concentration which was determinate were as fol-lows, respectively: geniposidic acid was ( 25. 77 ± 2. 68 )%, protocatechuic acid was ( 57. 54 ± 3. 79)%, chlorogenic acid was (53. 91 ± 3. 00)%, pinoresinol diglucoside was (24. 15 ± 4. 92)%, and pinoresinol singleglucoside was (49. 78 ± 3. 61)%. Conclusions The results show that the binding percentage of geniposidic acid and pinoresinol diglucoside is relatively low, but the binding rate of the others with rat plasma protein is moderate.

Simultaneous determination of human plasma protein binding of bioactive flavonoids in Polygonum orientale by equilibrium dialysis combined with UPLC-MS/MS / 药物分析学报

A simple and selective ultra performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) assay was developed for the determination of the human plasma protein binding of four bioactive flavonoids (such as orientin and vitexin) in Polygonum orientale. Protein precipitation was used for sample preparation. Equilibrium dialysis technique was applied to determine the plasma protein binding under physiological conditions. The separation was achieved through a Waters C18 column with a mobile phase composed of 0.1%formic acid in acetonitrile and 0.1%aqueous formic acid using step gradient elution at a flow rate of 0.35 mL/min. A Waters ACQUITY?TQD system was operated under the multiple reaction monitoring (MRM) mode of positive electrospray ionization. All of the recovery, precision, accuracy and stability of the method met the requirements. Good correlations (r40.99) of the four compounds were found, which suggested that these compounds can be simultaneously determined with acceptable accuracy. Results showed that the plasma protein bindings of the four bioactive flavonoids were in the range of 74-89% over the six concentrations studied. The binding parameters containing protein binding affinity, protein binding dissociation constant, and protein binding site were studied. The maximum ability to bind with protein was also determined in the assay in order to understand the drug-protein binding of each compound better.

Effect of arsenic on paracetamol binding to bovine serum albumin using site specific probes.

Arsenic contamination in groundwater is a global health challenge. A large number of people worldwide are affected by arsenic poisoning. Paracetamol is a widely used analgesic-antipyretic drug. Effect of arsenic on paracetamol binding to protein has been investigated using two site specific probes and equilibrium dialysis method was used for the experiment. In absence of any site specific probes free concentration of paracetamol bound to bovine serum albumin increased from 3.95 ± 1.164% to 25.36 ± 1.164%. In presence of site-I specific probe warfarin sodium the % release of drug was steady at around 14%. But in presence of site-II specific probe an increment of free drug concen-tration was observed from 14.38 ± 1.164% to 54.72 ± 1.552%. Thus it can be assumed that the free concentration of paracetamol was increased to a greater extent in presence of arsenic and probably arsenic bound to site-II of BSA. Thus arsenic may displace paracetamol by binding with high affinity binding site, site-II in the BSA and probably arsenic has little effect to site-I.

Equilibrium dialysis studies on the binding of anionic surfactants with bovine milk casein.

The binding process of amphiphile molecules with proteins depends on the experimental conditions, such as protein concentration and surfactant type. The proteins may have different binding characteristics, such as specific and cooperative binding. Specific binding is the most important pathway observed at low surfactant concentration at levels of micromolar unity, while the cooperative process becomes the most important at higher surfactant concentration, close to the critical aggregation concentration. At the levels at which specific binding occurs there is competitivity, i.e., the presence of an additional molecules can be induced hydrophobically or electrostatically depending on the characteristics of the molecular structure and experimental conditions. For instance, electrostatic binding is pH dependent and this factor is important for ionic surfactants.

Effect of arsenic on paracetamol binding to bovine serum albumin using site specific probes.

Arsenic contamination in groundwater is a global health challenge. A large number of people worldwide are affected by arsenic poisoning. Paracetamol is a widely used analgesic-antipyretic drug. Effect of arsenic on paracetamol binding to protein has been investigated using two site specific probes and equilibrium dialysis method was used for the experiment. In absence of any site specific probes free concentration of paracetamol bound to bovine serum albumin increased from 3.95 ± 1.164% to 25.36 ± 1.164%. In presence of site-I specific probe warfarin sodium the % release of drug was steady at around 14%. But in presence of site-II specific probe an increment of free drug concen-tration was observed from 14.38 ± 1.164% to 54.72 ± 1.552%. Thus it can be assumed that the free concentration of paracetamol was increased to a greater extent in presence of arsenic and probably arsenic bound to site-II of BSA. Thus arsenic may displace paracetamol by binding with high affinity binding site, site-II in the BSA and probably arsenic has little effect to site-I.

Study on the plasma binding rate of brazilein / 中国药学杂志

OBJECTIVE: To establish an in vitro equilibrium dialysis method for studying the plasma binding rate of brazilein. METHODS: Equilibrium dialysis was used to determine the binding rates of brazilein to bovine serum albumin and rat plasma, and the compound concentration was determined by HPLC. All the biological samples were handled with acetic ether-extraction. RESULTS: The bovine serum albumin binding rates of brazilein at low, medium and high concentrations ranged from 38.5% to 50.8%, and the rat plasma binding rate of brazilein ranged from 86.2% to 97.3%. CONCLUSION: Brazilein has high binding capacity to plasma, and the phospholipid components may have major contribution to the binding between brazilein and plasma. Copyright 2012 by the Chinese Pharmaceutical Association.

In-vitro interaction of verapamil hydrochloride with magnesium sulphate (anhydrous) and its influence on protein binding of verapamil hydrochloride.

The interaction between Verapamil Hydrochloride and Magnesium Sulphate (anhydrous) has been studied in an aqueous system at pH 7.4 and 2.4. From spectrophotometric study, it has been found that Verapamil Hydrochloride form 1:1 complex with Magnesium Sulphate (anhydrous). Spectral studies helps to detect the initial complexation between drug and metal. Job’s plot at 7.4 and 2.4 provides same type of information. The Ardon’s spectrophotometric method confirmed the 1:1 complexation and the value of stability constants was calculated using Ardon’s plot. An in vitro study of protein binding of Verapamil Hydrochloride and their 1:1 mixture with Magnesium Sulphate (anhydrous) has been conducted by equilibrium dialysis method at (37 ± 0.5)0C and at pH 7.4. The Scatchard plots were prepared to reveal the number of binding sites and the affinity for protein binding. It has been found that interaction of the drug with Magnesium Sulphate (anhydrous) results into increasing the affinity and increasing the protein binding of Verapamil Hydrochloride.

Assay of in Vitro Plasma Protein Binding Rate of Mangiferin in Rats / 中国药房

OBJECTIVE:To study the assay method of in vitro plasma protein binding rate of mangiferin in rats.METHODS:The plasma samples which were added with internal standard(rutin)were deproteinized with acetonitrile and the supernatant was assayed by HPLC.The equilibrium dialysis was carried out to determine the plasma protein binding rate of mangiferin.RESULTS:The plasma protein binding rates of mangiferin at low,middle and high concentrations were(50.0?1.9)%,(45.2?2.0)% and(45.9?2.1)%,respectively.CONCLUSION:This assay method is sensitive,reproducible and simple,and it meets the requirement of analysis.

Effects of co-administering probenecid orally on pharmacokinetics of cefaclor in rabbits / 中国临床药理学与治疗学

AIM: To investigate the effects and quantitative relations of co-administering probenecid OF different dosages on pharmacokinetics of cefaclor in rabbits and approach the possible mechanisms involved as well. METHODS: Monitor plasma and urine cefaclor concentrations. 24 male rabbits were randomly divided into 4 groups by Cefaclor 50 mg·kg-1,Cefaclor50 mg·kg-1+Probenecid 100 mg·kg-1,Cefaclor 50 mg·kg-1+Probenecid 250 mg·kg-1 and Cefaclor 50 mg·kg-1+Probenecid 625 mg·kg-1.Blood and urine samples were collected according to the regular time schedule after intragastric administration. The concentration of cefaclor in blood and urine were determined by HPLC. Pharmacokinetic parameters were calculated by DAS (Drug and Statistical) software. Measur plasma protein-binding rate of cefaclor. The experimental groups and drug dosage were same as described above. The blood sample was drawn at 1 hour after administration,and the protein-binding rate of cefaclor was determined by equilibrium dialysis. RESULTS: Within the dosages of probenecid ranged from 0-250 mg·kg-1,T1/2ka,Tmax,Cmax and AUC of cefaclor increased in accordance with increasing dosage of co-administering probenecid while CL/F and Vd/F were decreased(P＜0.01); However,when the dosage of co-administering probenecid was 625 mg·kg-1,Cmax of cefaclor strikingly decreased(P＜0.01),while AUC and CL/F maintained at the levels of those with probenecid250 mg·kg-1.In this experiment, urinary excretive peak time of cefaclor in its prototype pos tponed gradually,biological half life prolonged and urinary excretive accumulation percentage decreased obviously(P＜0.01).To the dosages of probenecid ranging from 0-250 mg·kg-1,protein-binding rate of cefaclor decreased notably(P＜0.01)going with increasing dosages of co-administration probenecid; While the dosage of co-administration probenecid reached 625 mg·kg-1,the protein-binding rate of cefaclor corresponded to that of cefaclor 50 mg·kg-1 without probenecid (P＜0.01).CONCLUSION: Co-administering probenecid can strikingly change pharmacokinetics of cefaclor and the influential degree of pharmacokinetics parameters is dependent on dosages of probenecid used in the experiment. Biological half life prolongs and urinary excretive accumulation percentage of cefaclor decreases obviously.