A Pilot Test Studies On The Extraction Of Cholic Acid From Bovine And Ovine Bile And Establishment Of HPLC-MS/MS Method For Determination Of Cholic Acid

Cholic acid(cholic acid,CA),as the main active component in the bile of animals such as cattle and Ovine,is one of the main components of artificial bezoar with antipyretic,antibacterial,anti-inflammatory and so on.The aim of this study was to establish a pilot test technology route which based on the small experiment of extracting and purifying of CA in bovine and Ovine bile,and to study the HPLC-MS/MS method for the determination of CA.The main research contents and results are as follows:A method for the determination of CA by HPLC-MS/MS was established.HPLC-MS/MS conditions were Poroshell 120 EC-C18(3.0 mm ×100mm,2.7μm)chromatographic column,sample size:I L,column temperature:35 degrees C,flow rate:0.5mL · min-1,mobile phase:0.5%formic acid methanol solution(A)-Watsons water(B)as mobile phase(A:B=90:10),elution method:isocratic elution.Ion source:electrospray ionization ion source(ESI),mass spectrometry:multi reaction monitoring mode(MRM),negative ionization mode(Negtive)detection.Atomizing air pressure:4500 v,auxiliary heating pressure 3500v(-),scanning range(10-1000),etc.The results showed that HPLC-MS/MS with high sensitivity,CA 3 concentration levels,days precision was 1.304%(n=5),daytime precision was 48.45%(n=5),linear range is 1.745～152.50pg·mL-1(r=0.999 5),average recovery rate was 94.00%～97.00%,RSD were less than 6.70%(n=5),detection limit is 5 ng·mL-1 and quantitative limit is 550ng·mL-1.Pilot test technology route of extracting and purifying of CA from bovine and ovine bile was established by ethanol crystallization method:bovine and ovine bile(10L)→remove the protein(75%ethanol,centrifuged 10 min,use supernatant)→decolorization(supernatant by adding 7.30%activated carbon,centrifuged 10 min,filtration,taking liquid)-concentrated recycling ethanol→alkaline hydrolysis(add 10%NaOH,alkaline hydrolysis sheep bile 20 hours and bovine bile for 20 hours,T = 100℃-110℃)～acidification(adding 10%hydrochloric acid,adjust the pH =5,overnight,take precipitation)→CA crude(precipitate washed with distilled water,discard the filtrate was pumped to get CA crude,naturally dry)→CA boutique(crude CA addsappropriate amountethanol dissolved,concentrated to get crystallize①,the residue plus appropriate amountethanol dissolved,concentrated to get crystallize ②,① + ②repeatedcrystallization,dried up to get boutique CA).The results of the pilot test show that the average extraction amount of CA in 10L bovine bile was 162.80±5.51 g,RSD =3.38%(n = 3),purity of CA was 98.61±2.40%and RSD = 2.43%(n=3),average extraction of add the recovery rate was 178.77 ±2.30 g and RSD =1.39%(n = 3),add the recovery rate was 79.80 ±11.40%and RSD = 14.30%(n = 3).The average extraction amount of CA in 10L sheep bile was 171.40±9.10 g,RSD=5.35%(n=3);purity of CA was 95.58 ± 5.51%,RSD=5.39%(n=3),average extraction of add the recovery rate was 185.90±2.20 g,RSD=1.24%(n=3),add the recovery rate was 72.50±11.00%,RSD=15.21%(n=3).Conclusion.In this study,using the pilot test extraction technology route of small test magnification technology has matured.Compared with the experimental results of a small test,pilot test of CA has a large amount of extraction,high purity,low production cost,short production cycle,high recovery rate,etc.,at the same time,experimental technology more maturity,which is more suitable for industrial scale production.The method of TLC and HPLC-MS/MS for the detection of CA is simple,rapid,sensitive,specific and stable.It is suitable for qualitative and quantitative determination of CA in bovine and ovine bile.