英國留學生生物醫學本科dissertation:analytical science

 

Abstract

hippric acid and creatinine were analyzed in urine by capillary electrophoresis. The optimization of the method was discussed, with the best results being obtained using a 30 mM phosphate–150 mM sodium dodecyl sulfate buffer at pH 6, with the detector set at 214 nm and an applied voltage of 15 kV across a 45 cm capillary.Verification of the method was provided by HPLC analysis and spiking. The application of the method was demonstrated by analysis of hippc acid and creatinine in urine samples collected in a 24-h period following creatinine.  

 

Introduction

Capillary electrophoresis (Capillary Electrophoresis, CE) is a kind of liquid phase separation techniques which was established in 1973 by Neuhoff. It is used to gel of uniform concentration and concentration gradient to analyze microscale protein. (Chace, 2001) In this technique, capillary as separation channel, and DC electric field as the main driving force together achieve the analysis. The migration of charged particles under the influence of an electric field was discovered and characterized theoretically more than 100 years ago by Kolrausch et al. (Monton, 2007). Foreseeing the possibility of separation of charged species through the application of a voltage, the term ‘electrophoresis’ was coined soon after. 

抽象

毛細管電泳hippric酸和尿中肌酐分析。優化的方法進行了討論，獲得最好的結果，使用30mM磷酸- 150mM的鈉，十二烷基硫酸鈉的緩沖液，在pH 6 ，檢測器波長為214 nm ，并在一個45厘米的毛細管施加電壓為15千伏。驗證的方法提供了用HPLC分析和尖峰。應用的方法，通過分析收集的尿液樣本在24小時內以下肌酐酸和肌酐hippc證明。

 

介紹

毛細管電泳毛細管電泳（ CE ）是一種液相分離技術，由Neuhoff成立于1973年。它是用來均勻的濃度和濃度梯度的凝膠分析微尺度蛋白。 （蔡斯，2001年） ，在這種技術中，毛細管為分離通道， DC電場的主要推動力，實現了分析。帶電粒子在電場的影響下的遷移被發現，其特征在于理論上的超過100年，之前由Kolrausch等。 （蒙頓，2007年） 。預見的可能性，通過施加電壓的電荷的物質的分離，術語“電泳”被創造出來后不久。

 

However, early attempts to use electrophoresis as an analytical tool were persistently frustrated by the existence of Joule heating, which acts to discount the electrophoretic effect. Thus a way of combatting the thermal effect during the electrophoretic process was needed. By 1950s, Tiselius et al. found that a variety of substances such as agarose and polymeric gels could serve as stabilizing agents in electrophoretic analysis owing to their anticonvective properties. This eventually led to the creation of slab gel electrophoresis, which has become a fundamental technique for the study of proteins, DNA fragments and other biomacromolecules in life sciences and biotechnology. Notwithstanding its great success, slab gel electrophoresis has its drawbacks with respect to speed and automation when compared with contemporary chromatographic techniques such as high performance liquid chromatography (HPLC). A straightforward way to speed up an electrophoretic separation process is to apply higher electric fields, and this necessitates systems able to release the heat generated more effciently.(Sogo, 2007)  Compared with the traditional electrophoresis and high performance liquid chromatography (HPLC), Capillary electrophoresis has the advantages of high efficiency, rapid, high sensitivity, high automation. Moreover, it needs less sample and less solvent consumption, low running cost, less environmental pollution. So CE has been widely used in many fields, such as biological, chemical, pharmaceutical, food, environmental aspects. In recent years, with the development of science and technology, all kinds of analysis and detection technology has been rapid development. All kinds of technology of capillary electrophoresis analysis detection method get the rapid development. Capillary electrophoresis is a kind of new liquid phase separation techniques through the gel of uniform concentration and concentration gradient. The capillary as separation channel, and DC electric field as the main driving force together achieve the sample to separate and test. The structure comprises a sampling system, capillary, detection system, high voltage power supply, cleaning system, temperature control system. Under the high voltage, the charged particles migrates in the electrolyte solution of capillary tube. The migration rate is equal to the vector of the electrophoresis and electroosmotic flow (EOF). The electrophoretic velocity of neutral particle is zero, and the speed of its migration rate is equal to EOF rate. Due to different particles have different migration velocity, so they can be separated. #p#分頁標題#e#

 

Capillary electrophoresis has multiple separation modes and provides different alternatives to sample separation. John summarizes the separation mode of Capillary Zone Electrophoresis, CZE）Capillary Gel Electrophoresis, CGE）Micellar Electrokinetic Capillary Electrophoresis, MECC）Affinity Capillary Electrophoresis, ACE）Capillary Electrochromatography, CEC）Capillary Isoelectric Focusing, CIEF）(P Capillary Isotachophoresis, CITP) and so on. The mode is very important for the separation and analysis of complex samples. Compared with other separation methods, Li thinks the capillary electrophoresis has the following advantages: high efficiency, fast, microscale, economy, low operation cost, clean, harmless to the environment and high degree of automation. CE is widely used in clinical to test the samples. It can test urine, blood serum, cerebrospinal fluid, red blood cells, other body fluids or tissues as well as the experimental animal in vivo to analyze the components including skin, protein, carbohydrate, viral enzymes oligonucleotide, DNA and other small bioactive molecules, ions, drugs and their metabolites. 

 

In fact, clinical disease detection plays an important role in clinical medicine. And it becomes very important aspects by medical scientist. In many cases, the course of the disease often leads to corresponding markers of changes. The medicine work is usually to determine problem by resulting from the changes of disease or a variety of markers. Analysis of urine samples relates to the terminal metabolin of urine. Unire composition and properties can reflect the metabolism of organism. (Sogo, 2007) The functional changes of organism especially have the direct relation with the urinary system. Therefore, the changes of urine not only reflect the urinary system disease, but also have the important significance to the diagnosis and treatment of other system diseases. Since 1990s, the role of CE in biomedicine and medical examination gradually has received more attention. At present, the scientist has successfully analyzed the congenital amino acid metabolic disease urine specimen. However, there is less case report. It is a pity to irregular detection conditions for clinical application. In recent years, the reports about C E in urine detection have increased. Shi Jingheng reported CE urine pattern analysis of the non-insulin-dependent diabetes mellitus. He found a specific S-peak in the urine, and confirmed that the peak is not only a characteristic manifestation of HPCE maps for the NIDDM patients, but also related material NIDDM is closely displayed in the HPCE map (Shi, 2001; Xue, 2007)). CE provides theoretical and technical basis for clinical urine detection reliability. Besides, the application of CE in diagnosing diseases has become more and more. Alberto tested sparfloxacin and clinafloxacin by CZE for diseases of urinary system. Christian byCE combined with laser induced fluorescence detection of laser induced fluorescenece, LIF successfully detected the e triamterene and its metabolites in urine by CE. In addition, there are still some reports which CE tests drug abuse of urine.#p#分頁標題#e#

 

Creatinine is a kind of metabolic of creatine, which produced by muscle. Blood creatinine can smoothly pass through glomerulus filtration and the kidney tubules don not basically reabsorb. The creatinine determination of blood and urine can be finished. The creatinine index can reflect the glomerulus filtration rate and is one of the common methods of renal function in clinical practice. Creatinine can evaluate the degree of renal damage, Hippuric acid (HA) is a kind of organic acid which is found in urine of horses and herbivorous. UA，Changes of HA in human urine can reflect the lesion of liver, kidney disease. This report briefly introduces the basic principle, characteristic, classification of capillary electrophoresis and tests creatinine and hippuric peak. 

 

Experimental 

Standard preparation

Weigh accurately creatintine 79.17mg and then dissolve to the deionized water. And dilute with water to 100mL.7000 creatintine standard preparation succefully prepare.

Weigh accurately HA 30mg HA and then dilute with water to 100ml. 

 

Calculation of sample dilution

Respectively measure 5ml Creatintine and HA standard prepareation, and mix. Finally, dilute with creatinine to 3500、1750、 875、 432.5、216.5、108.125、54.0625、 27.031 umol/L; Dilute with HA to 3000、1500、 750、 162.5、81.25、40.625、 20.312 umol/L

 

Method 

Weigh 100ul urine, 3000r/min centrifuge for 10 min, and then get supernatant.  CE separations were carried out on a Waters Quanta 4000 CE system using a hydrostatic injection mode (10 cm elevation of sample) for 20 s, a positive power supply, and a detector wavelength of214 nm. The capillary was either 60 cm×75um or 45cm×75um (Polymicro Technologies, Phoenix, AZ, USA) with a 1.5 mm detector window 7.5 cm from one end. The data was collected using Lab Calc software from Galactic (Salem, NH, USA) with 1 V equal to 1 AU. Rinse times were 30 s 0.1 M sodium hydroxide, 30 s water, and 1 min buffer, using a vacuum pressure of 15 in.Hg (1in.Hg53386.38pa). Make the standard curve. Take the peak area as abscissa, and take the concentration as ordinate. 

 

Results

Calibration curves were obtained by running known HA and Creainine and plotting peak area divided by t (min) versus the concentration (ug/ml). The curves were linear at least up to 700ug/ml at the t for Creainine and subsequent spiking to 100 mg m ml21 and the calibration curves  were generally only prepared to 200 mg ml as the concentrations found in diluted urine were well below this. 

 

Table 1

       

                      Umol/L

Table 1 is the standard curve of Creatinine by CE#p#分頁標題#e#

Table 2

Table 2 is the standard curve of HA by CE

From above the table 1 and 2, the standard curves showed the 2000-27.03umol/l of Creatinine has linear relation and thelinearly dependent coefficient was 99%. The 3000-40.625umol/l has linearity. 

 

Table 3

Table 3 is the map of urine sample

1 – Creatinine    2- HA

The table 3 showed that dilution of urine peak area. The number 1is creatinine’s peak area is up to 33, when the time is 4 min, the number 2 is HA. The conditions: phosphate–SDS (30–150 mM) buffer, pH 6.0, with an applied voltage of 17kVand detection at 214 nm. The lower trace is the urine itself; the upper trace is the urine spiked with CR to give a nominal concentration of100 mg ml . Other substances may have been seen at later timesif the run times had been extended.

 

Discussion

The table showed the determination of hippuric acid and creatinine content in urine by CE. After selecting conditions, the standard curves has a good linear relationship and the correlation coefficient is up to more than 0.9999. And the precision and the relative standard deviations were 3.6% and 4.7%, From the table 1,2, 3, we can know d this method to determination of hippuric acid and creatinine has the advantage of simple operation, high precision, good accuracy. Through the determination of hippuric acid and creatinine content by CE, the creatintine content is more than the hippuic acid. The results are corresponded with the fact. Due to t the urine sample is not enough, hippuric acid and creatinine content mean statistics need to be more perfected 

Conclusion

 

As the results showed, the hippric acid and Creatinine in the urine are the constituents of normal urine. It is of interest to follow the excretion of creatine and creatinine in urine as a function of time after creatine ingestion. In my study, hippric acid and creatinine were analyzed in urine by capillary electrophoresis. The optimization of the method was discussed, with the best results being obtained using a 30 mM phosphate–150 mM sodium dodecyl sulfate buffer at pH 6, with the detector set at 214 nm and an applied voltage of 15 kV across a 45 cm capillary.Verification of the method was provided by HPLC analysis and spiking. The application of the method was demonstrated by analysis of hippc acid and creatinine in urine samples collected in a 24-h period following creatine ingestion. 

Reference 

 

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