CN102213703A - Method for measuring contents of tyrosine, tryptophan and 5-hydroxytryptamine in blood serum by high-efficiency liquid phase chromatography-fluorescence method - Google Patents
Method for measuring contents of tyrosine, tryptophan and 5-hydroxytryptamine in blood serum by high-efficiency liquid phase chromatography-fluorescence method Download PDFInfo
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Abstract
The invention provides a method for measuring the contents of tyrosine, tryptophan and 5-hydroxytryptamine in blood serum by adopting a high-efficiency liquid phase chromatography-fluorescence method. The method is accurate and rapid, and comprises the following steps of: removing proteins from blood serum with perchloric acid with the concentration of 5 percent and detecting by adopting a high-efficiency liquid phase chromatography technology, wherein a chromatography condition is that: a chromatography column is an Atlantis C18 column of the specification of 4.6 millimeters*150 millimeters ID; mixing 0.1 mol/L of KH2PO4 with methanol in the volume ratio 85:15 at the flow rate 1.0 ml/min to obtain a mixed solution serving as a moving phase, wherein the excitation wavelength and the emitting wavelength of a fluorescence detector are 228 nanometers and 306 nanometers respectively within 0-4minutes, 278 nanometers and 338 nanometers within 4-7.5 minutes and 285 nanometers and 353 nanometers after 7.5 minutes; and adjusting the wavelengths within a specific period of time to perform separate detection on the tyrosine, the 5-hydroxytryptamine and the tryptophan.
Description
Technical field:
The invention belongs to the analytical chemistry field, relate to tyrosine in a kind of quantitative measurement serum, tryptophane and serotonin Study on content method, particularly a kind of correlation technique of using high performance liquid chromatography-fluorescence method to measure tyrosine in the serum, tryptophane and serotonin content simultaneously.
Background technology:
Tyrosine (tyrosine, Tyr) belong to the nonessential amino acid of human body, by phenylalanine (phenylalanine, Phe) through phenylalanine hydroxylase (phenylalanine hydroxylase, PAH) sloughing hydroxyl is transformed, be the precursor substance of synthetic catecholamines neurotransmitter-dopamine, adrenaline and norepinephrine, important relationship arranged with neuronic transmission.L-tryptophane (L-tryptophan, L-Trp) be one of essential amino acid in the human body, synthetic and the metabolism adjusting of participant's body protein, be serotonin (5-hydroxytryptamine, precursor substance 5-HT), and 5-HT is an important neurotransmitter in the human body, may be the Neurobiology basis of central nervous system pathological change.Studies show that Tyr, Trp and 5-HT three seed amino acids all play an important role in the human nerve psychiatric system.
At present, report is measured Tyr both at home and abroad, the method of Trp and 5-HT has a lot, wherein, report and maximum measure wherein a kind of or two kinds for utilization HPLC method and measure, but do not see and adopt HPLC-FLD to measure Tyr in the serum simultaneously, the report of Trp and 5-HT content, the present invention utilizes Tyr, Trp and 5-HT have the characteristic of natural fluorescence, a kind of serum T yr that measures is simultaneously at home and abroad disclosed first, the HPLC-FLD of Trp and 5-HT content, get the supernatant direct injected behind the serum specimen deproteinized, Tyr, Trp is with after 5-HT separates by chromatographic column, by wavelength Conversion,, detect with separately the suitableeest fluorescence excitation and emission wavelength in certain period of time.
Summary of the invention:
The objective of the invention is to disclose a kind of HPLC-FLD method that can measure Tyr in the serum, Trp and 5-HT content simultaneously, can satisfy medical clinical position and scientific research and measure for the quick, accurate of Tyr, Trp and 5-HT.
A kind of high performance liquid chromatography-fluorescence method is measured the method for tyrosine in the serum, tryptophane and serotonin content simultaneously: with Atlantis C18 chromatographic column is stationary phase, and described chromatographic column specification is 4.6mm * 150mm i.d., 5 μ m; With 0.1mol/LKH
2PO
4: the methyl alcohol mixed solution is a moving phase, described 0.1mol/L KH
2PO
4With the volume ratio of methyl alcohol be 85: 15; Moving phase pH value is 4.3; Flow velocity is 1.0ml/min; Testing sample after chromatographic column wash-out, separation, is analyzed, measured with fluorescence detector; Fluorescence detector excitation wavelength and emission wavelength 0～4min are respectively 228nm and 306nm, and 4～7.5min is 278nm and 338nm, are 285nm and 353nm behind the 7.5min.
Described fluorescence detector is the multi-wavelength fluorescence detector, has hyperchannel, the characteristics of wavelengthtunable.
Before described moving phase is used, with the filtering with microporous membrane of 0.22 μ M, ultrasonic degas 20min.
The standard operation liquid making method of tyrosine, tryptophane and serotonin is as follows: accurately take by weighing tyrosine, tryptophane and serotonin, with perchloric acid solution dissolving, the mixing of 2.5%v/v; Be mixed with the standard operation liquid of 55 μ mol/L, 49 μ mol/L, 1.176 μ mol/L respectively.
The processing of test serum sample: venous blood 2ml coagulates in the pipe in short on an empty stomach, and the normal fast centrifuging serum of normal temperature is got serum in the eppendorf pipe, add equivalent 5% perchloric acid solution, fully room temperature is placed 10min behind the mixing, and the centrifugal 10min of 10000r/min gets supernatant 20 μ L sample introduction analyses.
The present invention utilizes Tyr, three kinds of ammonia amino acid of Trp and 5-HT all have the characteristic of natural fluorescence, and groping through a large amount of experiment conditions, set up and a kind ofly can measure serum T yr simultaneously, the HPLC-FLD method of Trp and 5-HT content, this method is simple, fast, behind the serum specimen deproteinized, get the supernatant direct injected, Tyr, Trp is with after 5-HT separates by chromatographic column, adopt wavelength Conversion,, detect with the suitableeest exciting separately with emission wavelength in certain period of time, 10min can finish detection, the present invention obviously is better than disclosed technology, and is easy, fast, highly sensitive, need not post before, post-column derivation; Therefore be a technology that is worth very much widespread use, popularization.
Description of drawings:
Fig. 1 is the chromatogram of hybrid standard product Tyr, Trp and 5-HT;
Fig. 2 is the chromatogram of normal adult blood serum sample Tyr, Trp and 5-HT.
Embodiment
Be intended to further specify the present invention below in conjunction with embodiment and embodiment, and unrestricted the present invention.
The process of setting up of the inventive method:
1 instrument and reagent:
Instrument: high performance liquid chromatograph comprises Waters 510 chromatogram pumps and 2475 fluorescence detectors, Milli-Q water purifior, Rheodyne 7725 manual injectors (being with 20 μ L injection annulus), 0.22 μ m filter membrane and nutsch filter thereof.Reagent: L-Tyr, L-Trp, 5-HT, L-Phe, kynurenin, kynurenic acid, 0.1mol/L KH
2PO
4And methyl alcohol (chromatographically pure).
The selection of 2Tyr, Trp and 5-HT assay condition
2.1 detect the selection of wavelength:
The present invention finds, by to Tyr, 5-HT and Trp excitation light wave spectral limit (200nm～450nm) compose (250nm～500nm) scan with the emission light wave, obtain separately the suitableeest excitation wavelength and the suitableeest wavelength of transmitted light, be respectively 228nm and 306nm, 278nm and 338nm, 285nm and 353nm.
2.2 the selection of organic phase methanol content in the moving phase:
The present invention finds, change methyl alcohol percent by volume (5%, 10%, 15%, 20%) in the moving phase, analyze as sample with the hybrid standard working fluid, chromatogram shows, along with the increase of methanol content, the corresponding retention time of Tyr, 5-HT and Trp all shortens.And when the methyl alcohol volume fraction is 5%, analysis time is longer, and when the methyl alcohol volume fraction is 20%, separating effect is not good, in the methyl alcohol volume fraction is that 15% o'clock degree of separation is better, the three can separate in 10min preferably, and degree of separation is good, contains the moving phase that volume fraction is 15% methyl alcohol so adopt.
2.3 the selection of moving phase pH value
The present invention finds, adds glacial acetic acid and regulates PH, transfers to 4.0,5.0 and 6.0 respectively and analyzes, and finds under the different PH conditions that the retention time of Tyr, Trp and 5-HT and peak area obviously do not change, so directly select 0.1mol/LKH
2PO
4: methyl alcohol (85: 15, V: V; PH=4.3) solution is as moving phase.
2.4 the selection of flow rate of mobile phase
The present invention finds, by changing flow velocity 0.6,0.8,0.9,1.0,1.2, the 1.4ml/min of moving phase.The retention time of Tyr, Trp and 5-HT obviously shortens, but long-pending also corresponding the reducing in sharp side simultaneously, and bigger flow velocity also needs the pump pressure that provides bigger simultaneously, makes pillar bear bigger pressure, considers that to sum up selecting flow velocity is the 1.0ml/min optimum.
2.5 the selection of protein precipitant
The present invention finds: select for use 5% perchloric acid, the trichloroacetic acid solution (TCA) of 0.6mol/L, the sulfosalicylic acid solution (SSA) of 0.4mol/L to remove serum proteins as protein precipitant respectively, serum is measured.Found that serum with the SSA deproteinized has Interference Peaks and has a strong impact on the mensuration of Tyr, Trp and 5-HT, and the serum effect of perchloric acid and trichloroacetic acid deproteinized is fine, this experimental selection is done precipitation agent with perchloric acid.
3.Tyr, the qualitative and quantitative analysis of Trp and 5-HT
Tyr, Trp and 5-HT adopt peak retention relative method and method of superposition to carry out qualitative analysis, promptly compare hybrid standard product and blood serum sample chromatographic peak and relative retention time under the same conditions.Experimental result shows, adds in blood serum sample before and after the standard items, and the peak position that goes out of three kinds of Tyr, Trp and 5-HT is consistent.Carry out quantitative test with the external standard method peak area, be formulated as:
Serum T yr, Trp or 5-HT
Au is Tyr, Trp or a 5-HT peak area in the serum;
As is Tyr, Trp or a 5-HT peak area in the hybrid standard liquid;
Cs is Tyr, Trp or a 5-HT concentration in the hybrid standard liquid;
4. evaluation of methodology
4.1 the experiment of precision
Imprecision in a few days: get pooled serum, METHOD FOR CONTINUOUS DETERMINATION is 20 times in the same sample one day; In the daytime imprecision: same sample was surveyed once in one day, and METHOD FOR CONTINUOUS DETERMINATION 20 days is calculated in a few days, imprecision in the daytime.See Table 1
The imprecision in a few days and in the daytime of table 1Tyr, Trp and 5-HT
4.2 recovery experiment
Pooled serum is divided into 4 groups, first group adds the 0.1ml ultrapure water as basic sample, other three groups Tyr, 5-HT and the Trp titer 0.1ml that add variable concentrations (high, medium and low) respectively, mixing, every group of every kind of concentration is all measured three times, gets three recovery and calculates average recovery rate.The average recovery rate of Tyr, Trp and 5-HT is respectively 100.56% (97.0%～104.3%), 97.0% (93.3%～103.5%) and 99.3% (96.3%～103.1%).
4.3 interference test:
The material that has fluorescent characteristic in the serum such as Phe, Kyna, Kyn is diluted to the titer of a series of concentration respectively with 2.5% perchloric acid, sample introduction analysis under this chromatographic condition, Phe, Kyna, Kyn is all undetected, shows that mentioned component is noiseless to measuring Tyr, Trp and 5-HT.
4.4 typical curve:
Measure Tyr, Trp and the 5-HT titer of variable concentrations respectively, each sample feeding three times is got its mean value and is carried out regretional analysis with least square method, and long-pending Y is an ordinate with the sharp side, and concentration X is a horizontal ordinate, and regression equation sees Table 2
The regression equation of table 2Tyr, Trp and 5-HT
4.5 sensitivity:
Being blank with moving phase, is 3 to obtain lowest detectable limit by signal to noise ratio (S/N ratio), and Tyr, Trp and 5-HT lowest detectable limit are respectively 0.014 μ mol/L, 0.0049 μ mol/L and 0.024 μ mol/L.
Embodiment 1
Accurately take by weighing Tyr10.0mg, Trp10.0mg, 5-HT10.0mg, with the dissolving of the perchloric acid solution of 2.5% (v/v) and be diluted to 10ml, be mixed with the standard reserving solution of 5500 μ mol/L, 4900 μ mol/L and 117.6 μ mol/L respectively, be placed in after packing in-20 ℃ of refrigerators standby.Be diluted to the hybrid standard working fluid that contains Tyr (55 μ mol/L), Trp (24.5 μ mol/L) and 5-HT (1.176 μ mol/L) with preceding perchloric acid with 2.5%, stand-by.
(mornings 8～9h), 2ml coagulated in the pipe in short to get research object empty stomach venous blood, the normal fast centrifuging serum of normal temperature, get an amount of serum in the eppendorf pipe, add equivalent 5% perchloric acid solution, fully room temperature is placed 10min behind the mixing, the centrifugal 10min of 10000r/min gets supernatant 20 μ L sample introduction analyses.
The principle of work and the course of work
Draw 20 μ L measured matters with manual injector and enter in the moving phase, measured matter is along with moving phase, 0.1mol/LKH
2PO
4Flow into chromatographic column together with methyl alcohol, Atlantis C18 chromatographic column is opened Tyr, Trp, 5-HT branch, is that they arrive fluorescence detector identification successively, detect.This method sample disposal is simple, can finish detection at sample introduction 10min.
Claims (5)
1. a high performance liquid chromatography-fluorescence method is measured the method for tyrosine in the serum, tryptophane and serotonin content simultaneously, it is characterized in that, and be stationary phase with Atlantis C18 chromatographic column, described chromatographic column specification is 4.6mm * 150mm i.d., 5 μ m; With 0.1mol/L KH
2PO
4: the methyl alcohol mixed solution is a moving phase, described 0.1mol/L KH
2PO
4With the volume ratio of methyl alcohol be 85: 15; Moving phase pH value is 4.3; Flow velocity is 1.0ml/min; Testing sample after chromatographic column wash-out, separation, is analyzed, measured with fluorescence detector; Fluorescence detector excitation wavelength and emission wavelength 0～4min are respectively 228nm and 306nm, and 4～7.5min is 278nm and 338nm, are 285nm and 353nm behind the 7.5min.
2. method according to claim 1 is characterized in that: described fluorescence detector is the multi-wavelength fluorescence detector.
3. method according to claim 1 is characterized in that: before described moving phase is used, and with the filtering with microporous membrane of 0.22 μ M, ultrasonic degas 20min.
4. method according to claim 1 is characterized in that: the standard operation liquid making method of tyrosine, tryptophane and serotonin is as follows: accurately take by weighing tyrosine, tryptophane and serotonin, with perchloric acid solution dissolving, the mixing of 2.5%v/v; Be mixed with the standard operation liquid of 55 μ mol/L, 49 μ mol/L, 1.176 μ mol/L respectively.
5. method according to claim 1, it is characterized in that: the processing of test serum sample: venous blood 2ml coagulates in the pipe in short on an empty stomach, the normal fast centrifuging serum of normal temperature, get serum in the eppendorf pipe, add equivalent 5% perchloric acid solution, fully room temperature is placed 10min behind the mixing, and the centrifugal 10min of 10000r/min gets supernatant 20 μ L sample introduction analyses.
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Cited By (6)
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| CN102323305A (en) * | 2011-05-18 | 2012-01-18 | 北京兴有丰科科技发展有限公司 | Simultaneous determination of L-tyrosine and L-tryptophan in compound amino acid by using chemical oscillation reaction |
| CN102662013A (en) * | 2012-05-18 | 2012-09-12 | 上海市徐汇区中心医院 | Quantitative detection method of sarcosine in urine sample |
| CN102879370A (en) * | 2012-09-27 | 2013-01-16 | 江南大学 | Fluorescence method for determining Sulfamethoxazole and Danofloxacin in milk simultaneously |
| CN104678028A (en) * | 2015-02-12 | 2015-06-03 | 中国人民解放军军事医学科学院毒物药物研究所 | Pretreatment and detection method for biogen amine neurotransmitter and detection kit |
| CN106053676A (en) * | 2016-08-03 | 2016-10-26 | 四川威尔检测技术股份有限公司 | High performance liquid chromatography detection method for tryptophan in fodder |
| CN112362637A (en) * | 2020-12-07 | 2021-02-12 | 福建师范大学 | Method for detecting 5-hydroxytryptamine in serum based on surface enhanced Raman technology |
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| CN101750459A (en) * | 2010-01-19 | 2010-06-23 | 中南大学湘雅二医院 | Method for simultaneously determining tryptophan and tyrosine in blood serum by high performance liquid chromatography and fluorescence method |
| CN101762662A (en) * | 2010-01-21 | 2010-06-30 | 中南大学湘雅二医院 | Method for measuring tryptophan and 5-hydroxytryptamine simultaneously by high-efficiency liquid chromatography-fluorescence method |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102323305A (en) * | 2011-05-18 | 2012-01-18 | 北京兴有丰科科技发展有限公司 | Simultaneous determination of L-tyrosine and L-tryptophan in compound amino acid by using chemical oscillation reaction |
| CN102662013A (en) * | 2012-05-18 | 2012-09-12 | 上海市徐汇区中心医院 | Quantitative detection method of sarcosine in urine sample |
| CN102879370A (en) * | 2012-09-27 | 2013-01-16 | 江南大学 | Fluorescence method for determining Sulfamethoxazole and Danofloxacin in milk simultaneously |
| CN102879370B (en) * | 2012-09-27 | 2015-08-05 | 江南大学 | The fluorescent method of sulfamethoxazole and Danofloxacin in a kind of Simultaneously test milk |
| CN104678028A (en) * | 2015-02-12 | 2015-06-03 | 中国人民解放军军事医学科学院毒物药物研究所 | Pretreatment and detection method for biogen amine neurotransmitter and detection kit |
| CN106053676A (en) * | 2016-08-03 | 2016-10-26 | 四川威尔检测技术股份有限公司 | High performance liquid chromatography detection method for tryptophan in fodder |
| CN112362637A (en) * | 2020-12-07 | 2021-02-12 | 福建师范大学 | Method for detecting 5-hydroxytryptamine in serum based on surface enhanced Raman technology |
| CN112362637B (en) * | 2020-12-07 | 2023-05-23 | 福建师范大学 | Method for detecting 5-hydroxytryptamine in serum based on surface-enhanced Raman technology |
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Application publication date: 20111012 |