CN107290444A - The method for detecting neopterin and biopterin in human urine - Google Patents
The method for detecting neopterin and biopterin in human urine Download PDFInfo
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Abstract
Analyzed the invention discloses a kind of method of neopterin and biopterin in accurate, quick, high flux detection human urine, including by the urine sample after pre-treatment with liquid chromatogram, chromatographic column is reverse bonding silicagel column；Mobile phase is methanol and ammonium acetate buffer, gradient elution；Detected afterwards with fluorescence detector.The inventive method cost is low, and pre-treatment is simple, and batch processing detection can be achieved, and optimization chromatographic process shortens analysis time, greatly improves sample flux, is particularly suitable for use in demand of the clinical and routine testing to chromatographic process.
Description
Technical field
The present invention relates to a kind of detection method, more particularly to a kind of side for detecting neopterin and biopterin in human urine
Method.
Background technology
Neopterin and biopterin are internal GTP (GTP) metabolites, and GTP (GTP) is auxiliary
Important component in enzyme tetrahydrobiopterin (tetrahy drobiopterin, BH4) building-up process, two kinds of pterins are main
By urine drains, professional can effectively distinguish the parting of hyperphenylalaninemia by its content and the ratio of the two.
Therefore, the antidiastole for carrying out early stage BH4 deficiency disease to HPA infants is extremely important.Atypical hyperphenylalaninemia is with passing
System hyperphenylalaninemia has incomplete same therapeutic scheme, and a kind of pre-treatment of suitable clinical medical inspection is set up in exploitation
Method, accurate, quickly, high-throughout chromatographic process is conducive to quickly obtaining accurate data in large quantity, contributes to professional people
Member carries out corresponding assay, significant.
Containing water-soluble highly polar impurity such as a large amount of salt in urine, neopterin and biopterin easily hydrolyze again, preserve
Stability is poor, is not suitable for carrying out great amount of samples detection under the HILIC patterns of high organic Phase Proportion, 25cm is used more conventional method
The enhancing of C18 chromatographic columns retain and separating effect, analysis time is longer, do not meet clinical medical inspection it is quick, it is high-throughout will
Ask.Meanwhile, traditional HILIC patterns, the testing cost of the method such as LC-MS is higher, is unfavorable for promoting the use of.
The content of the invention
It is an object of the invention to provide neopterin and biological butterfly in a kind of accurate, quick, high flux detection human urine
The method of purine.
The technical solution used in the present invention is：
The method of neopterin and biopterin, comprises the following steps in a kind of detection human urine：
1) pre-treatment：Liquid phase detection pre-treatment is carried out to urine；
2) liquid phase is eluted：Urine sample after pre-treatment is analyzed with liquid chromatogram, the analysis condition is：
Stationary phase：Chromatographic column is reverse bonding silicagel column；
Mobile phase：A phases are methanol；B phases be 15~25mmol/L ammonium acetate buffer, and with acetic acid adjust pH 5.5~
7.0；Gradient elution mode；
3) detect：Detected with fluorescence detector.
As the further improvement of the above method, the chromatographic column is Poroshell 120EC-C18；
As the further improvement of the above method, gradient elution mode is：
The column temperature of chromatographic column is 28~32 DEG C；
The flow velocity of mobile phase is 0.3~0.5mL/min.
As the further improvement of the above method, sample size when liquid phase is eluted is 0.5~2 μ L.
As the further improvement of the above method, testing conditions are：A length of 358~the 362nm of excitation light wave, wavelength of transmitted light
For 448~452nm.
As the further improvement of the above method, before pre-treatment step, in addition to pre-treatment step, the pretreatment step
Suddenly it is：5~7M hydrochloric acid is added after urine collecting immediately, freezing is kept in dark place.
As the further improvement of the above method, pre-treatment step comprises the following steps：
1) sample：Place and thaw to room temperature, mix, take sample to add centrifuge tube；
2) aoxidize：Iodine/liquor kalii iodide is added, lucifuge reaction is placed in a centrifuge after concussion；
3) neutralize：Centrifugation, ascorbic acid, sodium hydroxide, are centrifuged after the concussion that is vortexed successively；
4) filter：Supernatant is taken to cross 0.22 μm of aqueous phase filter membrane sample introduction analysis.
As the further improvement of the above method, the concentration of iodine/liquor kalii iodide is 1~5wt%, and addition is 5~15 μ
L/100 μ L samples.
As the further improvement of the above method, the concentration of ascorbic acid is 1~5mg/mL, and addition is 1~5 μ L/100
μ L samples；The concentration of the sodium hydroxide is 5-10mol/L, and addition is 1~5 μ L/100 μ L samples.
It is intermittent that chromatographic column is carried out to rush post when carrying out liquid phase elution as the further improvement of the above method.
As the further improvement of the above method, the temperature of sample injection disc is 2~6 DEG C.
The beneficial effects of the invention are as follows：
The inventive method cost is low, and pre-treatment is simple, and batch processing detection can be achieved, and optimization chromatographic process shortens analysis
Time, sample flux is greatly improved, be particularly suitable for use in demand of the clinical and routine testing to chromatographic process.
Brief description of the drawings
Fig. 1 is the linear regression curves of neopterin；
Fig. 2 is the linear regression curves of biopterin.
Embodiment
The method of neopterin and biopterin, comprises the following steps in a kind of detection human urine：
1) pre-treatment：Liquid phase detection pre-treatment is carried out to urine；
2) liquid phase is eluted：Urine sample after pre-treatment is analyzed with liquid chromatogram, the analysis condition is：
Stationary phase：Chromatographic column is reverse bonding silicagel column；
Mobile phase：A phases are methanol；B phases be 15~25mmol/L ammonium acetate buffer, and with acetic acid adjust pH 5.5~
7.0；Gradient elution mode；
3) detect：Detected with fluorescence detector.
As the further improvement of the above method, the chromatographic column is Poroshell 120EC-C18；
As the further improvement of the above method, gradient elution mode is：
The column temperature of chromatographic column is 28~32 DEG C；
The flow velocity of mobile phase is 0.3~0.5mL/min.
It can be eluted under the conditions of this quickly, high-energy, improve elution effect.
As the further improvement of the above method, sample size when liquid phase is eluted is 0.5~2 μ L.
As the further improvement of the above method, testing conditions are：A length of 358~the 362nm of excitation light wave, wavelength of transmitted light
For 448~452nm.
As the further improvement of the above method, before pre-treatment step, in addition to pre-treatment step, the pretreatment step
Suddenly it is：5~7M hydrochloric acid is added after urine collecting immediately, freezing is kept in dark place.
As the further improvement of the above method, pre-treatment step comprises the following steps：
1) sample：Place and thaw to room temperature, mix, take sample to add centrifuge tube；
2) aoxidize：Iodine/liquor kalii iodide is added, lucifuge reaction is placed in a centrifuge after concussion；
3) neutralize：Centrifugation, ascorbic acid, sodium hydroxide, are centrifuged after the concussion that is vortexed successively；
4) filter：Supernatant is taken to cross 0.22 μm of aqueous phase filter membrane sample introduction analysis.
As the further improvement of the above method, the concentration of iodine/liquor kalii iodide is 1~5wt%, and addition is 5~15 μ
L/100 μ L samples.
As the further improvement of the above method, the concentration of ascorbic acid is 1~5mg/mL, and addition is 1~5 μ L/100
μ L samples；The concentration of the sodium hydroxide is 5-10mol/L, and addition is 1~5 μ L/100 μ L samples.
It is intermittent that chromatographic column is carried out to rush post when carrying out liquid phase elution as the further improvement of the above method.
As the further improvement of the above method, the temperature of sample injection disc is 2~6 DEG C.At this temperature, it is more beneficial for keeping
The stability of sample, while being had no adverse effect substantially to liquid phase elution.
With reference to embodiment, technical scheme is further detailed.
Embodiment 1
1st, the collection of people source urine and Sample storage
Three, freshly voided urine sample is gathered, every sample adds 6M HCl to adjust after pH to 1 or so respectively, and lucifuge is put respectively
Deposit, every 0, each determine once within 1,2,5,8,15,22 days under the conditions of -20 DEG C and -70 DEG C.
2nd, the pre-treatment of urine sample
1) sample：Place and thaw to room temperature, mixing take the μ L of acidified sample 420 to 1.5mL centrifuge tubes；
2) aoxidize：The μ L of 1% iodine/liquor kalii iodide 30 are added, 3min is shaken, lucifuge in 4 DEG C of centrifuges is placed in and reacts
30min；
3) neutralize：Short centrifugation 3 seconds, sequentially adds 5 μ L 2mg/mL ascorbic acid, and 15 μ L 6M sodium hydroxides are vortexed and shaken
3min, 4 DEG C, 12000rpm centrifugations 5min；
4) filter：Supernatant is pipetted using 2.0mL disposable syringes and crosses 0.22 μm of aqueous phase filter membrane sample introduction analysis.
3rd, pattern detection
Liquid-phase condition
Chromatographic column：Poroshell 120EC-C18 (2.1 × 100mm, 2.7 μm)
Mobile phase A=methanol
Mobile phase B=20mmol/L ammonium acetate buffers, acetic acid adjusts pH6.0
Detection wavelength：Ex360nm, Em450nm, peak width 74.07HZ
Column temperature：30℃
Sample size：1μL
Sample injection disc temperature：5℃
Flow velocity：0.4mL/min
Retention time：NP 1.2min, BP 3.3min
Retention time window：5%
Width：0.1
Threshold value：50.
Method validation
Sensitivity for analysis and range of linearity result
Neopterin linear regression curves are as shown in Figure 1；
Biopterin linear regression curves are as shown in Figure 2.
The method degree of accuracy
The rate of recovery of neopterin between 88.1%~114.5%, the rate of recovery of biopterin 93.3%~
107.8%, meet 85%-115% requirement.
Precision
Withinrun precision and betweenrun precision are respectively less than 20%, meet the requirements.
Neopterin Precision Experiment result collects
Biopterin Precision Experiment result collects
Sample shelf stability
- 20 DEG C of storages of lucifuge, neopterin stability data
- 20 DEG C of storages of lucifuge, biopterin stability data
- 70 DEG C of storages of lucifuge, neopterin stability data
- 70 DEG C of storages of lucifuge, biopterin stability data
It was found from the data in table, urine specimen can under the conditions of -20 DEG C of lucifuge stable storage 5 days, -70 DEG C of conditions of lucifuge
Under can stablize storage 15 days.
Claims (10)
1. a kind of method for detecting neopterin and biopterin in human urine, comprises the following steps：
1) pre-treatment：Liquid phase detection pre-treatment is carried out to urine；
2) liquid phase is eluted：Urine sample after pre-treatment is analyzed with liquid chromatogram, the analysis condition is：
Stationary phase：Chromatographic column is reverse bonding silicagel column；
Mobile phase：A phases are methanol；B phases are 15~25mmol/L ammonium acetate buffer, and adjust pH 5.5~7.0 with acetic acid；
Gradient elution mode；
3) detect：Detected with fluorescence detector.
2. according to the method described in claim 1, it is characterised in that：In the liquid phase elution step：
The chromatographic column is the EC-C18 of Poroshell 120；
The gradient elution mode is：
The column temperature of chromatographic column is 28~32 DEG C；
The flow velocity of mobile phase is 0.3~0.5mL/min.
3. method according to claim 1 or 2, it is characterised in that：Sample size when liquid phase is eluted is 0.5~2 μ L.
4. method according to claim 1 or 2, it is characterised in that：Testing conditions are：Excitation light wave a length of 358~
362nm, wavelength of transmitted light is 448~452nm.
5. according to the method described in claim 1, it is characterised in that：Before the pre-treatment step, in addition to pre-treatment step,
The pre-treatment step is：5~7M hydrochloric acid is added after urine collecting immediately, freezing is kept in dark place.
6. method according to claim 1 or 5, it is characterised in that：The pre-treatment step comprises the following steps：
1) sample：Place and thaw to room temperature, mix, take sample to add centrifuge tube；
2) aoxidize：Iodine/liquor kalii iodide is added, lucifuge reaction is placed in a centrifuge after concussion；
3) neutralize：Centrifugation, sequentially adds ascorbic acid, sodium hydroxide, is centrifuged after the concussion that is vortexed；
4) filter：Supernatant is taken to cross 0.22 μm of aqueous phase filter membrane sample introduction analysis.
7. method according to claim 6, it is characterised in that：The concentration of the iodine/liquor kalii iodide is 1~5wt%, plus
Enter amount for 5~15 μ L/100 μ L samples.
8. method according to claim 6, it is characterised in that：The concentration of the ascorbic acid is 1~5mg/mL, addition
For 1~5 μ L/100 μ L samples；The concentration of the sodium hydroxide is 5-10mol/L, and addition is 1~5 μ L/100 μ L samples.
9. according to the method described in claim 1, it is characterised in that：It is intermittent that chromatographic column is carried out when carrying out liquid phase elution
Rush post.
10. according to the method described in claim 1, it is characterised in that：The temperature of sample injection disc is 2~6 DEG C.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109946412A (en) * | 2017-12-21 | 2019-06-28 | 上海产业技术研究院 | A kind of body fluid pterin spectrum detection kit and its use |
| CN111505179A (en) * | 2020-04-07 | 2020-08-07 | 厦门大学 | Method for detecting biopterin in marine water body |
| CN113125611A (en) * | 2021-04-22 | 2021-07-16 | 北京斯利安药业有限公司 | Method for detecting content of impurity 6-formyl pterin folic acid |
| CN114814063A (en) * | 2022-04-08 | 2022-07-29 | 宁波熙宁检测技术有限公司 | Method for detecting tetrahydrobiopterin in human body |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109946412A (en) * | 2017-12-21 | 2019-06-28 | 上海产业技术研究院 | A kind of body fluid pterin spectrum detection kit and its use |
| CN111505179A (en) * | 2020-04-07 | 2020-08-07 | 厦门大学 | Method for detecting biopterin in marine water body |
| CN111505179B (en) * | 2020-04-07 | 2021-07-13 | 厦门大学 | Method for detecting biopterin in marine water body |
| CN113125611A (en) * | 2021-04-22 | 2021-07-16 | 北京斯利安药业有限公司 | Method for detecting content of impurity 6-formyl pterin folic acid |
| CN113125611B (en) * | 2021-04-22 | 2023-04-18 | 北京斯利安药业有限公司 | Method for detecting content of impurity 6-formyl pterin folic acid |
| CN114814063A (en) * | 2022-04-08 | 2022-07-29 | 宁波熙宁检测技术有限公司 | Method for detecting tetrahydrobiopterin in human body |
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