Establishment Of A HPLC-MS Method For The Determination Of Anthraquinones And Its Application In The Pharmacokinetics Study On Realgar And Rhei Radix Et Rhizoma Combinations

Objective:Realgar is an arsenic-containing mineral medicine,it is one of the traditional Chinese medicines commonly used in China.Although Realgar is extremely poorly soluble in the gastrointestinal tract and its oral bioavailability is extremely low,incidents of drug-induced arsenic poisoning caused by long-term,excessive,and irregular use of Realgar and Realgar-containing traditional Chinese medicines have been reported.The theory of traditional Chinese medicine combination and detoxification is an important method for the safe and rational application of toxic traditional Chinese medicines.It has been reported that Rhei Radix et Rhizoma can reduce the concentration of soluble arsenic in Realgar,but there is no research on the pharmacokinetics of two medicine.Based on these understandings,this article will explore the mechanism of Realgar-Rhei Radix et Rhizoma combinations from the following aspects:?1?an atomic fluorescence spectrophotometry?AFS?method was used to detect the concentration of soluble arsenic in different proportions of Realgar and Rhei Radix et Rhizoma,then screened the best proportion according to the least soluble arsenic concentration;?2?an AFS was established to investigate the effect on arsenic of Realgar-Rhei Radix et Rhizoma combinations in vivo;?3?a high performance liquid chromatography-mass spectrometry?HPLC-MS?method was established to investigate the effect of Realgar-Rhei Radix et Rhizoma combinations on the fate of anthraquinones in vivo.The study provide an experimental basis and new direction for the mechanism research of the toxicity reducing and efficacy enhancing of Realgar-Rhei Radix et Rhizoma combinations and its clinical applicationMethods:1.An AFS method was established to determine the concentration of soluble arsenic in different proportions of Realgar and Rhei Radix et Rhizoma extract solution extracted by artificial gastric juice.The effects of ultrasonic extraction time and volume of artificial gastric juice on the dissolution of soluble arsenic were also investigated.2.An AFS method was established to determine the concentration of arsenic in blood and applied in the pharmacokinetics study of Realgar and Realgar-Rhei Radix et Rhizoma combinations.3.A HPLC-MS method was established to measure the concentration of anthraquinones in plasma and applied in the pharmacokinetics study of Rhei Radix et Rhizoma and Realgar-Rhei Radix et Rhizoma combinations.The experiment data were expressed as average±standard.Differences were calculated by SPSS19.0 software using One-sample t-test.The significance of differences between two groups was statistically significant.P<0.05 was considered statistically significant.Results:1.When the ultrasonic time was 1.5 h and the volume of artificial gastric juice was 8 mL,Realgar dissolved the most soluble arsenic,so it was the best experimental condition.With the increase of Rhei Radix et Rhizoma proportion,the concentration of soluble arsenic decreased gradually,and when the ratio of Realgar to Rhei Radix et Rhizoma was 1:4,the concentration of soluble arsenic was the lowest.2.The established AFS method can be used for the determination of arsenic in blood,which meet the requirements of biological sample analysis.Both two absorption peaks appeared in the blood concentration-time curves of two groups,and the second peak was lower than the first.Compared with Realgar group,the first C_maxax of arsenic in Realgar-Rhei Radix et Rhizoma group increased significantly?P<0.05?,and the second T_max was significantly shorter?P<0.05?,it indicated that Rhei Radix et Rhizoma accelerated the absorption of arsenic in vivo;the first t_1/2/2 of arsenic in Realgar-Rhei Radix et Rhizoma group is significantly reduced compared with Realgar group?P<0.05?,which indicated Rhei Radix et Rhizoma accelerated the metabolism of arsenic in vivo.3.The established HPLC-MS method for the determination of anthraquinones in plasma was accurate,reliable and simple.The linear ranges of anthraquinones?The linear ranges of aloe emodin,rhein,emodin and chrysophanol were as follows:0.0460-4.60?g/mL?0.610-61.0?g/mL?0.00130-0.130?g/mL and 0.980-98.0?g/mL?were good within the concentration range under investigation?R²>0.99?.The specificity,precision and accuracy,recovery rate,matrix effect and stability of the method were good,which can be used for determining anthraquinones in biological sample analysis.It was suitable for the determination of anthraquinones in pharmacokinetic study.It only detected aloe-emodin,rhein and emodin in both two groups,and they both showed double peaks except aloe-emodin.The AUC_0-t and C_max of rhein were much bigger than aloe-emodin and emodin in both two groups.The AUC_0-t and C_max of the anthraquinones in Realgar-Rhei Radix et Rhizoma group were bigger than Rhei Radix et Rhizoma group?P<0.05?,but the T_max and t_1/2 value was significantly shorter?P<0.05?,which indicated Realgar accelerated the absorption and metabolism of anthraquinones in vivo.Conclusion:1.The established AFS method is simple,fast,sensitive and reproducible,it can be used to determine the concentration of arsenic in medicine and blood.2.The established HPLC-MS method is selective,accurate,sensitive and reproducible,it can be used to determine the concentration of anthraquinones in plasma.3.Rhei Radix et Rhizoma can significantly reduce the dissolution of soluble arsenic in artificial gastric juice and accelerate the absorption and metabolism of arsenic in the blood,therefore it can reduce the toxicity of Realgar.Realgar can increase the concentration of aloe-emodin,rhein and emodin and accelerate the absorption of anthraquinones in vivo.