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References

the Volumn 2 of Chinese Pharmacopoeia

Focal point

The principal of dual wavelength assay.

Discussion

1. Give an explanation of the basis of the wavelength selection for the determination of

sulfamethoxazole and trimethoprim, respectively.

2. Why is the more diluted solution used for the assay of trimethoprim than that for

sulfamethoxazole?

teaching device

50ml beaker; 10ml centrifuge tube mortar

electronic balance 100ml volumetric flask 50ml volumetric flask

UV spectrometer 1cm cell

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实验四 血浆中阿司匹林的高效液相色谱法测定

教学目的

1. 了解生物样品测定的过程;

2. 掌握阿司匹林血浆样品测定的方法和步骤。 本章讲授提纲及学时分配

1. 标准溶液―――――――――――――溶液配置(60min) 阿司匹林标准溶液(100?g/ml):

OOHOOCH3

邻甲基苯甲酸(内标)标准溶液(50?g/ml):

2. 血浆样品处理――――――――――――(1hour)

阿司匹林血浆样品处理 取冷冻的血浆样品在冰水浴中解冻,精密吸取0.5ml置1.5ml的离心管中,精密加入邻甲基苯甲酸标准溶液10?l,加0.5mol/L盐酸0.1ml和乙腈0.5ml,涡旋1分钟,4℃放置15分钟后,4℃下12000 rpm离心10分钟,分取上清液0.5ml,加氯化钠0.1g,涡旋5秒,4℃静置10分钟后,12000 rpm离心10分钟,分取上清液作为供试液。

3. 标准曲线――――――――――(1hour)

将阿司匹林与内标邻甲基苯甲酸的峰面积比,对血浆中阿司匹林的浓度进行线性回归,即得。

4. 阿司匹林血药浓度测定回收率和精密度试验――――――――――(1hour) 取低、中、高浓度的供试液和对照液,照“阿司匹林血药浓度测定”项下的条件分别测定,按内标法,依不同浓度水平,分别以峰面积比进行计算,即得阿司匹林血浆样品测定低、中、高浓度的回收率和精密度,应符合规定。

5. 阿司匹林血药浓度测定――――――――――(3hour) 照高效液相色谱法(中国药典2000年版二部附录Ⅴ D)测定。

测定法 取阿司匹林血浆样品供试液20?l,注入液相色谱仪,记录色谱图,按内标法,

OOHCH3 10

以标准曲线进行计算即得。 本课程学科的新进展

HPLC-MS的应用。 教学参考书

Analysis and bioanalysis 本章内容的重点 生物样品的处理方法 本章内容的难点 生物样品的处理过程 本章内容及讲授的改进意见 复习思考题

1、 本法血浆蛋白干扰的消除方法是什么?常用的其它方法有哪些?各有什么特点? 2、 阿司匹林标准溶液在4℃储藏的目的是什么?为什么冰冻的阿司匹林血浆样品在冰水

浴中解冻?

3、 阿司匹林血浆样品处理过程中加0.5mol/L盐酸0.1ml的目的是什么? 4、 实验中添加内标物邻甲基苯甲酸的目的是什么? 回收率试验的目的和意义是什么? 教具及教学设备要求

50ml烧杯,10ml离心管,电子天平,100ml容量瓶,移液器,离心机,水浴锅,高效液相色谱仪

Experiment 4 Determination of aspirin(ASA) in human plasma

by HPLC

I. Purpose

1. To learn about the principles and procedures for the determination of drugs in biological

samples.

2. To experiment on the determination of aspirin in human plasma.

2. Contents and teach time assignment

1. Standard solutions--------------------------------------(60min) Aspirin standard solution(ASA,100μg/ml):

OOHOOCH311

2-methyl benzoic acid (internal standard)(MBA,50μg/ml):

2. Sample preparation ------------------------------------(1hour)

Aspirin plasma sample preparation Thaw the frozen plasma sample in an ice-water bath. Transfer accurately 0.5 ml of it to a 1.5 ml microcentrifuge tube and add accurately 10μl of MBA standard solution. Then add 0.1 ml of 0.5 mol/L hydrochloric acid and 0.5 ml of acetonitrile. Vortex the mixture for 1 minute and keep at 4℃ for 15 minutes. Then centrifuge at 12000rpm for 10 min at 4℃. To 0.5 ml of the supernatant add 0.1g of sodium chloride and vortex for 5s and placed at 4℃for 10min before centrifuge at 12,000rpm for 10 min at 4℃. Use the supernatant for the determination of aspirin.

3. Calibration curve------------------------------------------(1hour)

Establish the calibration curve by plotting the peak-area ratios of ASA to internal standard versus its concentrations.

4. Recovery and precision for ASA determination-----------------------------------(1hour) Then make the determination according to the procedures described under the ‘determination of ASA in human plasma’. The mean recovery and precision of different concentrations, calculated by comparison of the peak-area ratios of ASA to MBA in the test solutions with those of standards at the same concentration, must comply with the requirements.

5. Determination of Aspirin in human plasma---------------------------------------3(hour) Determination Inject 20μl of ASA plasma sample into the column, and record the chromatogram. Calculate the ASA concentration in the plasma with respect to the peak area ratios obtained in the chromatogram by the internal standard method according to the calibration curve.

OOHCH3References

Analysis and bioanalysis

Focal point

The pretreat method of biological specimen.

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