CN102221588A - Method for quickly determining trace albumin in urine by adopting high performance liquid chromatography - Google Patents
Method for quickly determining trace albumin in urine by adopting high performance liquid chromatography Download PDFInfo
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Abstract
The invention provides a method for quickly determining trace albumin in urine by adopting high performance liquid chromatography (HPLC). The method comprises the following detailed operation conditions: a. adopting a molecular sieve chromatographic column (Agilent Zorbax GF-250250mm*4.6mm i.d, 4 microns); b. preparing a flow phase by mixing 0.1% aqueous formic acid with acetonitrile by the ratio of 17:3 (volume ratio); c. flowing speed: 1ml/min; column temperature: 30 DEG C; and d. detection wavelength: 205nm. The method can be operated simply, does not require special reagents, and has exact results, high sensitivity and wide linear scope. Compared with other HPLC methods, the method has the outstanding advantages that the separation is quick and the detection time is shortened greatly.
Description
Technical field
Introduction of the present invention be the method for measuring microalbumin in the urine, what be specifically related to is with the microalbumin in the high performance liquid chromatography fast measuring urine.
Background technology
Exist albumin in normal person's urine, but content is very low, is generally less than the 30mg/24h urine.When kidney generation reversibility was damaged, the albumin in the urine can increase, and generally in the 30-300mg/24h urine, be called microalbuminuria this moment, and its mensuration is significant to the early stage injury of kidney of human body.Initial urine albumin detection by quantitative depends on radiommunoassay, and this method need utilize radioelement as tracer agent, influences the healthy of proofer, and serious environment pollution.A large amount of subsequently immunological methods are adopted by clinical labororatory, this method is highly sensitive, precision is good, but can only detect the immuno-chemical reaction albumin, when this is interpreted as albumin through nearly bent renal tubule, because the effect of renal tubule lysosomal enzyme, albuminous a part of epitope are modified or conformational change taken place, albumin antibody can not react with it and cause underestimating its content.High performance liquid chromatography with microalbumin in the molecular sieve chromatography post analysis urine, it is a kind of analytical technology of separating and measuring that integrates, have good sensitivity and accuracy, immuno-chemical reaction albumin and the non-reacted albumin of immunochemistry in the urine can be detected, microalbumin in the more urine can be detected than immunological method.The used moving phase of having reported at present of high performance liquid chromatography mainly contains the gradient elution system and the sodium sulphate-phosphate buffer of NaCl-phosphate buffered solution, trifluoroacetic acid-acetonitrile solution composition, and these moving phases preparations are loaded down with trivial details relatively time-consuming.Albuminous appearance time is all longer, and is the longest at 14.1min
[1], be 13.1min secondly
[2], the shortest 9.5min that also wants
[3]And this law adopts aqueous formic acid-acetonitrile as moving phase, and preparation simply saves time, and albumin just goes out the peak about 1.7min, shortened the detection time of sample greatly, has realized fast measuring.
List of references:
[1] Li Meiai, Wan Genping, Luo Mingyong waits the .HPLC method to measure the urine non-reacted albumin of immunochemistry and diagnoses meaning in children's diabetic nephropathy in early days. Chinese Journal of Pathophysiology, 2009,25 (8): 1625-1627.
[2] Chen Xiaofei, Yang Jianrong, Chen Liling waits the .HPLC method to measure the foundation and the Preliminary Clinical of urinary albumin method. Chinese laboratory medicine magazine, 2007,30 (1): 37-40.
[3]Osicka?TM，Comper?WD.Characterization?of?Immunochemically?NonreactiveUrinary?Albumin.Clin?Chem.2004，50(12)：2286-2291.
Summary of the invention
The method that the purpose of this invention is to provide microalbumin content in a kind of high performance liquid chromatography express-analysis urine.Use the molecular sieve chromatography post, the moving phase that aqueous formic acid-acetonitrile is formed, ultraviolet wavelength 205nm detects, and reaches the purpose that the assay method result is accurate, highly sensitive and shorten detection time.
For achieving the above object, adopt following technical scheme:
A, usefulness molecular sieve chromatography post Agilent Zorbax GF-250 250mm * 4.6mm i.d, 4 μ m;
The composition of b, moving phase: 0.1% aqueous formic acid and acetonitrile are in the preparation of 17: 3 ratios (volume ratio), and the percent concentration of described 0.1% aqueous formic acid is a percent by volume;
C, flow velocity: 1ml/min, column temperature: 30 ℃, sample size: 20 μ l;
D, detection wavelength: 205nm;
The processing procedure of e, urine sample is: the centrifugal 10min of urine sample 3000g/min, get supernatant then in glass sample introduction bottle, the phosphate buffer that adds the 0.2mol/L pH7.0 of 2 times of volumes again, whirlpool mixing 30s moves to sample introduction 20 μ l in the sample bottle through the filtration of needle-based filter.
The processing of f, albumin standards: earlier albumin standards is diluted to series concentration with ultrapure water, again it is done volume ratio dilution in 1: 2 with the 0.2mol/L phosphate buffer of pH7.0 respectively during sample introduction.Concrete dilution process is: 1 part of titer getting series concentration adds 2 parts of the 0.2mol/L phosphate buffers of pH7.0 again in glass sample introduction bottle, and whirlpool mixing 30s filters through the needle-based filter and to move to sample introduction 20 μ l in the sample bottle.
The processing of g, blank sample: get the 0.2mol/L phosphate buffer of 1 part of ultrapure water and 2 parts of pH7.0, whirlpool mixing 30s moves to sample introduction 20 μ l in the sample bottle through the filtration of needle-based filter.
Relatively waiting of h, production standard curve, precision evaluation, recovery test, detection lower bound, the range of linearity and method.
Compared with prior art, the advantage of this method has:
1, this law albumin appearance time about about 1.7min, has shortened the detection time of sample greatly greatly, has realized fast measuring.
2, this law is simple to operate, detects with common ultraviolet wavelength, does not need special reagent, and the moving phase preparation is simple.
3, through repetition test, find out the top condition that a cover is measured microalbumin in the urine, comprise that phosphate buffer is used for the dilution of titer and urine sample, the selection of chromatographic column and moving phase, setting of flow velocity, column temperature, sample size, detection wavelength or the like.
4, linear test and detection lower bound result show that the range of linearity is wide, and albumin standards concentration is in the 2000mg/L scope, and the linear relationship of concentration and peak area is (R well
2=0.9969); Highly sensitive, the detection lower bound of urine sample is 2mg/L.
5, this law is measured the variation within batch coefficient of microalbumin in the urine sample and interassay coefficient of variation all less than 5%; The recovery is 102.3%, 98.1%, 97.2%.
6, this law and immune scattering turbidimetry are carried out paired sample t check, and difference has statistical significance (P＜0.05), can think two kinds of methods to albuminous measurement result difference in the urine, and the high effective liquid chromatography for measuring result is apparently higher than immune scattering turbidimetry.High performance liquid chromatography can detect more albumin, and is higher than the susceptibility of immune scattering turbidimetry.
Description of drawings
Fig. 1 is the blank sample chromatogram;
Fig. 2 is the albumin standards chromatogram;
Fig. 3 is the urine sample chromatogram;
Fig. 4 is a canonical plotting.
Embodiment
Following embodiment is in order to describe the present invention in detail, and can not influence its claimed scope.
Key instrument equipment and chromatographic condition:
Agilent Technologies 1200 series high performance liquid chromatographs, G1367C automatic sampler, G1316B column oven, the online degasser of G1379B, G1312B double base pump, G1314A UV-detector, Agilent Chemstation analysis software.Immunity scattering turbidimetry instrument is the IMMAGE of U.S. BECKMAN-COULTER company 800.
Chromatographic column: Agilent Zorbax GF-250250mm * 4.6mm i.d, 4 μ m; Moving phase: the aqueous formic acid of percent by volume 0.1% and acetonitrile are in the preparation of 17: 3 ratios (volume ratio); Flow velocity: 1ml/min; Column temperature: 30 ℃; Sample size: 20 μ l; Detect wavelength: 205nm.
Main agents:
The albumin standard items are available from Randox company;
Formic acid, analyze pure, available from Tianjin section close europeanized reagent development centre;
Acetonitrile, chromatographically pure is available from CNW Technologies GmbH;
The reagent and the standard items of immunity rate scattering turbidimetry are Beckman Coulter company microalbumin kit.
One, the preparation of the foundation of chromatographic condition and titer
(1), the selection of high-efficient liquid phase chromatogram condition
1, moving phase is selected
Moving phase at first adopts constant current and gradient elution system to measure with ammonium formate and the methyl alcohol of 10mmol/L to albumin standards, then use 20mmol/L ammonium formate and methyl alcohol as moving phase, adopt gradient elution system, use 20mmol/L ammonium formate and acetonitrile then, adopt constant current and gradient elution system, all can not separate the albumin peak well, determine that at last 0.1% aqueous formic acid and acetonitrile can obtain desirable blank solution chromatogram in 17: 3 ratio (volume ratio) preparation moving phase, titer chromatogram and sample chromatogram, albumin standards concentration becomes good linear relationship with peak area.
2, detect the selection of wavelength
The protein peptide bond energy absorbs near the spectrum the 215nm strongly, respectively with 254nm, 220nm, 215nm, 210nm, 205nm, 200nm, 195nm as detecting wavelength, albumin standards and 1 part of urine sample to 20mg/L, 200mg/L, 2000mg/L variable concentrations are measured, albumin standards is all bigger at 205nm wavelength place's peak height and peak area as a result, urine sample also has chromatographic peak more clearly at the 205nm place, so select 205nm as detecting wavelength.
3, flow velocity is to the influence of separating effect
Adopt 0.5ml/min, 1ml/min, 1.5ml/min, the flow rate of mobile phase that 2.0ml/min is different is tested.Experiment shows that when 0.5ml/min, wash-out is insufficient, and at 1.5ml/min and 2.0ml/min, peak area response reduces, and the back pressure of chromatographic system increases.And when 1ml/min, the complete wash-out in albumin peak, peak area are of moderate size, so adopt 1ml/min as flow velocity.
(2), the preparation of titer
Getting albumin standard items freeze dried powder adds ultrapure water 5ml by operational manual and is made into the albumin standards that concentration is 42.5mg/L to dissolving fully.
Embodiment 2
One, the detection of sample
1, the detection of blank sample
Get the 0.2mol/L phosphate buffer of 1 part of ultrapure water and 2 parts of pH7.0, whirlpool mixing 30s moves to sample introduction 20 μ l in the sample bottle through the filtration of needle-based filter.High-efficient liquid phase chromatogram condition: that chromatographic column is selected for use is Agilent Zorbax GF-250250mm * 4.6mm i.d, 4 μ m chromatographic columns; Moving phase: the aqueous formic acid of percent by volume 0.1% and acetonitrile are in the preparation of 17: 3 ratios (volume ratio); Flow velocity: 1ml/min; Column temperature: 30 ℃; Sample size: 20 μ l; Detect wavelength: 205nm.
Fig. 1 is a sample introduction albumin blank sample chromatogram.
2, the detection of titer
Earlier albumin standards is diluted to variable concentrations with ultrapure water, again it is done dilution in 1: 2 with the 0.2mol/L phosphate buffer of pH7.0 respectively during sample introduction, whirlpool mixing 30s moves to sample introduction 20 μ l in the sample bottle through the filtration of needle-based filter.High-efficient liquid phase chromatogram condition: that chromatographic column is selected for use is Agilent Zorbax GF-250250mm * 4.6mm i.d, 4 μ m chromatographic columns; Moving phase: the aqueous formic acid of percent by volume 0.1% and acetonitrile are in the preparation of 17: 3 ratios (volume ratio); Flow velocity: 1ml/min; Column temperature: 30 ℃; Sample size: 20 μ l; Detect wavelength: 205nm; Appearance time: 1.72min.Fig. 2 is a sample introduction 1000mg/L albumin standards chromatogram.
3, the detection of urine sample
With 2ml freshly voided urine sample balance to 37 ℃, the centrifugal 10min of 3000g/min gets supernatant 200 μ l in glass sample introduction bottle then, adds the phosphate buffer 400 μ l of 0.2mol/L pH7.0 again, whirlpool mixing 30s moves to sample introduction 20 μ l in the sample bottle through the filtration of needle-based filter.High-efficient liquid phase chromatogram condition: that chromatographic column is selected for use is Agilent ZorbaxGF-250250mm * 4.6mm i.d, 4 μ m chromatographic columns; Moving phase: the aqueous formic acid of percent by volume 0.1% and acetonitrile are in the preparation of 17: 3 ratios (volume ratio); Flow velocity: 1ml/min; Column temperature: 30 ℃; Sample size: 20 μ l; Detect wavelength: 205nm; Appearance time: 1.72min.Fig. 3 is a certain urine sample chromatogram of sample introduction.
4, quantitative test
Carry out quantitative test with the external standard method peak area.Promptly under identical chromatographic conditions, measure albumin standard items and urine sample, the chromatographic peak area of albuminous chromatographic peak area in the urine sample and albumin standard items is compared try to achieve albuminous content in the urine sample.Be formulated as:
Annotate: albumin peak area in the Au=urine
As=albumin standards peak area
Cs=albumin standards concentration
5, the making of typical curve
Prepare albumin standards 2mg/L, 10mg/L, 50mg/L, 200mg/L, 500mg/L, 1000mg/L, the 2000mg/L of a series of concentration.Each concentration is made parallel 4-5 pipe, and getting its mean value respectively is horizontal ordinate with the albumin concentration, and peak area is the ordinate mapping, and getting equation is Y=1.1872X+21.286, coefficient R
2=0.9984.Fig. 4 is the albumin canonical plotting.
6, the mensuration of the range of linearity
Preparation series concentration albumin standards 0mg/L, 2mg/L, 2.5mg/L, 5mg/L, 10mg/L, 20mg/L, 25mg/L, 50mg/L, 100mg/L, 200mg/L, 250mg/L, 500mg/L, 1000mg/L and 2000mg/L, each sample feeding 3 times, getting its mean value respectively is horizontal ordinate with the albumin concentration, peak area is the ordinate mapping, getting equation of linear regression is: Y=1.1927X+18.977, coefficient R
2=0.9969, show that in the 2000mg/L scope, the urinary albumin measurement result is extraordinary linear relationship.
7. detect the mensuration of lower bound
Get the blank sample replication 20 times, get 20 peak absorption values, calculate its mean value and add 3 times of standard deviations, obtaining detecting lower bound is 2mg/L.
8, the evaluation of precision
Select the sample of basic, normal, high (20mg/L, 200mg/L, 2000mg/L) three kinds of concentration to distinguish replicate determination 20 times, try to achieve x, s, obtaining crowd interior CV is 3.99%, 3.60%, 4.74%.Same sample with these three kinds of concentration is respectively surveyed in every morning, afternoon 1 time, does altogether 20 times 10 days, try to achieve criticize between CV be respectively 4.01%, 3.55%, 4.65%.The sample of three kinds of concentration, in its batch and betweenrun precision all less than 5%, as shown in table 1.
Batch interior and betweenrun precision of three kinds of concentration samples of table 1
9, recovery test
Getting the normal person urinates compound sample urine sample 3.6ml and is equally divided into four groups, the 1st group adds the 0.1ml ultrapure water as basic sample, 2nd, 3,4 groups add the albumin standards 0.1ml that concentration is 100mg/L, 500mg/L and 1500mg/L respectively, its mean value calculation recovery is got in every group of concentration replicate determination three times.The results are shown in Table 2.
Table 2 determination of recovery rates test findings
10, methodology comparison test
Fresh urine sample 56 examples of outpatient service of picked at random Xiangya Hospital, Central-South China Univ. and inpatient, measure with high performance liquid chromatography and immune scattering turbidimetry respectively, the measurement result of two kinds of methods is carried out paired sample t check, difference has statistical significance (P＜0.05), can think that two kinds of methods are inconsistent to albuminous measurement result in the urine, the high effective liquid chromatography for measuring result is apparently higher than immune scattering turbidimetry.High performance liquid chromatography can detect more albumin, and is higher than the susceptibility of immune scattering turbidimetry.
Claims (3)
1. the method with microalbumin in the high effective liquid chromatography for measuring urine is characterized in that, the actual conditions of described method operation is as follows:
A, usefulness molecular sieve chromatography post Agilent Zorbax GF-250250mm * 4.6mm i.d, 4 μ m;
B, moving phase: prepare by 17: 3 volume ratios according to 0.1% aqueous formic acid and acetonitrile, the percent concentration of described 0.1% aqueous formic acid is a percent by volume;
C, flow velocity: 1ml/min, column temperature: 30 ℃;
D, detection wavelength: 205nm.
2. a kind of method according to claim 1 with microalbumin in the high effective liquid chromatography for measuring urine, it is characterized in that, the pre-treatment process of urine sample is: the centrifugal 10min of urine sample 3000g/min, get supernatant then in glass sample introduction bottle, the phosphate buffer that adds the 0.2mol/LpH7.0 of 2 times of volumes again, whirlpool mixing 30s moves to sample introduction in the sample bottle through the filtration of needle-based filter.
3. a kind of method with microalbumin in the high effective liquid chromatography for measuring urine according to claim 1 is characterized in that, uses the 0.2mol/L phosphate buffer dilution of pH7.0 before mensuration again with the series standard liquid after the ultrapure water dilution.
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Citations (4)
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|---|---|---|---|---|
| JPS6383100A (en) * | 1986-09-26 | 1988-04-13 | Green Cross Corp:The | Production of albumin in human urine |
| US5246835A (en) * | 1991-05-24 | 1993-09-21 | Wakamoto Pharmaceutical Co., Ltd. | Method of diagnosing renal diseases |
| CN1520462A (en) * | 2001-06-28 | 2004-08-11 | Ī��ʲ��ѧ | Method for kidney disease detection by protein profiling |
| CN1902495A (en) * | 2003-12-30 | 2007-01-24 | 英特尔公司 | Methods for using Raman spectroscopy to obtain a protein profile of a biological sample |
-
2011
- 2011-03-25 CN CN2011100729018A patent/CN102221588A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6383100A (en) * | 1986-09-26 | 1988-04-13 | Green Cross Corp:The | Production of albumin in human urine |
| US5246835A (en) * | 1991-05-24 | 1993-09-21 | Wakamoto Pharmaceutical Co., Ltd. | Method of diagnosing renal diseases |
| CN1520462A (en) * | 2001-06-28 | 2004-08-11 | Ī��ʲ��ѧ | Method for kidney disease detection by protein profiling |
| CN1902495A (en) * | 2003-12-30 | 2007-01-24 | 英特尔公司 | Methods for using Raman spectroscopy to obtain a protein profile of a biological sample |
Non-Patent Citations (6)
| Title |
|---|
| BABIC T ET AL,: "Application of Liquid Chromatography-Mass Spectrometry Technology for Early Detection of Microalbuminuria in Patients with Kidney Disease", 《CLINICAL CHEMISTRY》 * |
| STEVEN P ET AL,: "The analysis and characterization of immunounreactive urinary Albumin in healthy volunteers", 《CLINICAL BIOCHEMISTRY》 * |
| SVIRIDOV D ET AL,: "Coelution of Other Proteins with Albumin during Size-Exclusion HPLC: Implications for Analysis of Urinary Albumin", 《CLINICAL CHEMISTRY》 * |
| 吴学飞 等,: "尿微量白蛋白的测定及其临床意义", 《临床和实验医学杂志》 * |
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