HPLC法测定黄连中的小檗碱-上禾生物

1)试剂

a. 乙腈（分析纯）  b. 重蒸水  c. 磷酸（分析纯）  d.十二烷基磺酸钠（SDS）

2)仪器

高效液相色谱仪（Shimadzu）      分析天平（1/10000）   

    超声波清洗仪                    0.45 mm 微孔过滤器        

    50 ml 容量瓶                    带塞小玻璃瓶

3)色谱条件

色谱柱   Hypersil ODS C₁₈ 5 mm  4.6´200mm

波长     282nm

流动相   A:水（含0.02%mol/L磷酸、0.2%十二烷基磺酸钠）  B: 乙腈

A:B=52:48(等梯度)

流速     0.8ml/min

进样量   10 mm

4)样品制备

（1）待测样品  精确称取30mg提取物于50ml容量瓶中，用0.1mol/L磷酸超声溶解，冷却定容，0.45 mm微孔滤膜过滤为供试液。

（2）标样制备  精密称取标样用0.1mol/L磷酸溶解，使其浓度为0.15mg/ml，过0.45mm滤膜，置冰箱贮存。

5)计算

根据各峰峰面积利用外标法计算各种成份的含量。

盐酸小檗碱生产企业-上禾生物

（27） Determination  of  Berberine  by  HPLC

1) Reagents

a. Acetronitrile (HPLC)    b. Nanopure water     c.SDS    d.Phosphate acid(A.R)    

2) Apparatus

HPLC (Shimadzu)            Analytical balance (1/10000)         Ultrasonic cleaner

0.45μm PTFE filter           50ml volumetric flask               Screw cap vials

3) HPLC condition

Column:  Hypersil ODS C₁₈ 5μm , 4.6×200mm

Detector:  UV 282nm

Mobile Phase: A=water ( including 0.02ml/L phosphate acid and 0.2% SDS)  

B= Acetronitrile

A/B=52/48  

Flow Rate: 0.8ml/min

Injection: 10μl

4) Procedure

Samples Preparation

  Accurately weigh 30mg extraction into 50ml volumetric flask. Add 0.1%mol/L Phosphate acid to volume and sonicate to mix, filter through a 0.45μm PTFE filter into a screw cap vial and proceed with the analysis.

Standard preparation /calibration

Prepare a standard solution (approximately 0.6mg/ml in 0.1%mol/L Phosphate acid) using a commercial reference standard of berberine，Sonicate for about 10 minutes and transfer to screw-cap vials and can be stored in the refrigeration.

5) Calculation

Determine the peak areas of the berberine on the sample chromatogram and compare to the corresponding peaks in the chromatogram for the reference standard solution. Calculate the weight percent.