CN101750459A - Method for simultaneously determining tryptophan and tyrosine in blood serum by high performance liquid chromatography and fluorescence method - Google Patents

Abstract

The invention relates to a method for simultaneously determining tryptophan and tyrosine in blood serum by high performance liquid chromatography and fluorescence method. The method comprises the following steps: adopting a MegresC18 column (a chromatographic column of 250mm*4.6mm i.d. and 5 mu m); taking 10 percent (v/v) acetonitrile as a chromatographic mobile phase, wherein a flow rate is 1.2 ml/min; detecting wavelengths by using fluorescence, wherein within 0 to 5 minutes, the wavelength of exciting light is 228nm and the wavelength of emitted light is 306nm, and after 5 minutes, the wavelength of the exciting light is 285nm and the wavelength of the emitted light is 353nm, and a sample size is 20 mu l; and determining the tryptophan and the tyrosine in the blood serum at the room temperature. The method of the invention has the advantages of simplicity, quickness, accuracy, high sensitivity, high practicability and the capability of meeting the detection requirements on clinical blood serum samples.

Description

A kind of high performance liquid chromatography-fluorescence method is measured the method for serum tryptophane and tyrosine simultaneously

Technical field

The invention belongs to the analytical chemistry field, relate to the method that a kind of high performance liquid chromatography-fluorescence method is measured serum tryptophane and tyrosine simultaneously.

Background technology

The method of the detection Tyr of report and Trp is more both at home and abroad at present, comprises high performance liquid chromatography (HPLC), high performance capillary electrophoresis (HPCE), mass spectroscopy (MS) etc.HPCE is shorter analysis time, and separating power is stronger, but the too late HPLC of detectability (ultraviolet absorption detector), and repeatability is relatively poor; Mass-spectrometric technique also has complicated operation, the relatively poor weak point of repeatability; Comparatively speaking, HPLC uses more extensive.Tryptophane (Trp) and tyrosine (Tyr) have indole structure, and self can produce fluorescence, so can adopt fluorescence method to detect.The report of Tyr and Trp in the independent respectively detection serum of domestic and international existing employing HPLC-fluorescence method (HPLC-FD) or HPLC-ultraviolet method (HPLC-UV), the report that adopts HPLC-UV to measure Tyr and Trp is simultaneously arranged, the report that adopts Tyr and Trp content in the high effective liquid chromatography for measuring cerebrospinal fluid is also arranged, do not adopt HPLC-FD to measure Tyr in the serum and the report of Trp simultaneously but have as yet at present.Recently, Sanchez-Machado etc. measure Tyr and Trp content in the shrimp waste material simultaneously with HPLC-FD, but the range of linearity is narrow, though can satisfy amino acid whose detection needs in the shrimp waste material, can not satisfy the detection requirement of clinical serum sample.

Summary of the invention

The purpose of this invention is to provide a kind of high performance liquid chromatography-fluorescence method and measure the method for serum tryptophane and tyrosine simultaneously, analysis time of the present invention is short, and reagent consumption is few, and is highly sensitive, and specificity is good, can satisfy the detection requirement of clinical serum sample.

The present invention can be achieved through the following technical solutions.

The detecting instrument that the present invention adopts is: 510 chromatogram pumps, 2475 type fluorescence detectors (Waters company), HS2000 chromatographic data workstation (Hangzhou English spectrum scientific and technological development company limited), Rheodyne 7725 manual injectors (being with 20 μ L injection annulus).0.22 μ m filter membrane and nutsch filter thereof (Millipore company); Milli-Q water purifior (Millipore company); TGLL 218 desk type high speed refrigerated centrifuges (Hunan, Hunan instrument hydro-extractor factory); The ultrasonic instrument that degass of SB2200 type (energy company limited must be believed in Shanghai).

Main agents of the present invention has: Tyr, Trp, serotonin (5-HT), kynurenic acid (Kyna), phenylalanine (Phe), kynurenin (Kyn) and creatinine (Cr) are all available from Sigma company; Acetonitrile, methyl alcohol are chromatographically pure, available from U.S. Tedia company; It is pure that zinc acetate, glacial acetic acid etc. are homemade analysis, and perchloric acid is that homemade top grade is pure.

The testing conditions that the present invention adopts is: chromatographic column wherein: Megres C18 post (250mm * 4.6mm i.d., 5 μ m, Hanbon Sci. ﹠ Tech. Co., Ltd.); Moving phase: 10% (v/v) acetonitrile; Flow velocity: 1.2mL/min; The fluoroscopic examination wavelength: 0-5min excitation wavelength 228nm, wavelength of transmitted light are 306nm; Excitation wavelength 285nm behind the 5min, wavelength of transmitted light are 353nm; Sample size: 20 μ l; Measure under the room temperature.

Detection principle of the present invention: tryptophane and tyrosine can produce natural fluorescence, utilize the character selection tryptophane of fluorescence detector wavelengthtunable and the optimum detection wavelength of butyric acid, tryptophane is separated with tyrosine, two seed amino acids after the chromatographic resolution are through the detection and the processing of fluorescence detector and chromatographic data workstation, make chromatogram, finish amino acid whose content analysis and mensuration according to chromatogram then.

The present invention includes following steps:

A, employing Megres C18 post 250mm * 4.6mm i.d., 5 μ m chromatographic columns;

The composition of b, moving phase: 10% (v/v) acetonitrile;

C, flow velocity are 1.2ml/min, sample size: 20 μ l, measure under the room temperature;

D, utilize fluorescence detector scanning to determine the maximum excitation optical wavelength and the wavelength of transmitted light of tryptophane and tyrosine, and then make that two kinds of materials all obtain detecting under its optimal wavelength.Wavelengthtunable is set at: 0-5min excitation wavelength 228nm, wavelength of transmitted light are 306nm; Excitation wavelength 285nm behind the 5min, wavelength of transmitted light are 353nm;

E, method for building up is carried out evaluation of methodology: the evaluation of the detection of hybrid standard product and blood serum sample, typical curve and detectability, precision, the investigation of the recovery and interference test.

Compared with prior art, the advantage and the effect that have of the present invention is as follows:

1, the present invention has successfully set up the method that a kind of HPLC-FD measures Tyr and Trp in the human serum simultaneously first.Tyr and Trp have natural fluorescence, and the present invention utilizes fluorescence detector to change the advantage of exciting light and wavelength of transmitted light automatically, select the optimal wavelength of Trp and Tyr, make the chromatographic peak peak area maximum of Trp and Tyr, and detection sensitivity increases; With report method comparison, analysis time of the present invention is short, and reagent consumption is few, and is highly sensitive, and specificity is good, can satisfy the detection requirement of clinical serum sample.

2, the principle of the invention simple, easy and simple to handle, quick (9min can finish detection), feasibility height.

3, the present invention is highly sensitive, specificity good, can satisfy serum specimen tyrosine and tryptophane is measured requirement, is suitable for clinical and research application.Evaluation of methodology shows that within the specific limits, the linear relationship of the typical curve of two kinds of materials is (r 〉=0.990) well, and the range of linearity is wide; The detectability of two kinds of materials is low under this method.Tryptophane is that the recovery is 88.8%-97.2%, the tyrosine recovery is 90.5%-108.8%, tryptophane and tyrosine in a few days with day to day precision all less than 5%, phenylalanine (Phe), kynurenin (Kyn), kynurenic acid (Kyna), serotonin (5-HT) and creatinine (Cr) are noiseless to measuring.

Description of drawings

Fig. 1 is the chromatogram of hybrid standard product;

Peak sequence number: Trp-tryptophane, Tyr-tyrosine;

Fig. 2 is the chromatogram of the serum sample of the present invention's mensuration;

Peak sequence number: Trp-tryptophane, Tyr-tyrosine.

Embodiment

Following examples are intended to further specify the present invention, and unrestricted the present invention.

Embodiment 1, the preparation of reagent and the collection and treatment of serum sample

1. the preparation of standard solution

Accurately take by weighing Trp10.0mg, Tyr10.0mg with the dissolving of the perchloric acid solution of 2.5% (v/v) and be diluted to 10.0mL, mixing, make 4900 μ mol/LTrp standard reserving solutions and 5500 μ mol/LTyr standard reserving solutions respectively, be placed in after packing in-30 ℃ of refrigerators standby.Getting 2 kinds of standard reserving solutions before the use respectively adds 2.5% perchloric acid and is made into standard operation liquid (containing Trp 24.5 μ mol/L, Tyr 55 μ mol/L).

2. protein precipitant

Perchloric acid solution with ultrapure water preparation 5% (v/v) is made the serum proteins precipitation agent.

3. the preparation of moving phase

With the acetonitrile solution of ultrapure water preparation 10% (v/v), use 0.22 μ m filtering with microporous membrane again, ultrasonic degas 20min before using.

4. interference test sample solution

Prepare the titer of finite concentration Phe (610 μ mol/L), 5-HT (114 μ mol/L), Kyn (1.96 μ mol/L), Kyna (26.17nmol/L) and Cr (88.14 μ mol/L) respectively with the perchloric acid solution of 2.5% (v/v).

5. the collection and treatment of serum sample

Experimenter's empty stomach venous blood 2mL is placed clean short coagulating in the pipe, 3000g centrifuging serum, get 100 μ l serum in the Eppendorf pipe, add equal-volume 5% (v/v) perchloric acid solution, add a cover back mixing on the vortex vortex mixer, place 10min under the room temperature, behind the centrifugal 10min of 10000g (4 ℃) supernatant is changed in another Enpendorf pipe with the protein in the abundant precipitation serum, centrifugal again 10min under the same terms gets supernatant 20 μ L sample introduction analyses.

Embodiment 2, the optimization of chromatographic condition

1. detect the selection of wavelength

Dilute certain density Tyr and Trp standard solution with moving phase, with the blank zeroing of moving phase, on 2475 type fluorescence detectors, excitation spectrum and emission spectrum scanning by to Tyr and Trp obtain the maximum excitation optical wavelength respectively: λ ex _Tyr=228nm, λ ex _Trp=285nm; Emission maximum optical wavelength: λ em _Tyr=306nm, λ em _Trp=353nm.

In the moving phase organic ratio to the influence of retention time

Prepare the moving phase of different ethane nitrile contents (5%, 8%, 10%, 12%), analyze as sample with titer.Along with the increase of ethane nitrile content, the corresponding shortening of peak retention time of Tyr and Trp, and Tyr and the also increase accordingly of Trp peak area response; When ethane nitrile content was 5%, 8%, the analysis time of single sample was oversize; But when ethane nitrile content was 12%, serum sample Tyr peak merged mutually with the assorted peak of front, influences quantitative accuracy.Take all factors into consideration above factor, this research employing second eyeball content is 10% moving phase.

3. flow velocity is to the influence of separating effect

Select for use flow velocity 0.8,1.0,1.2,1.5mL/min to experimentize respectively.When flow velocity was 1.5mL/min, though the retention time of Tyr and Trp shortens, peak area response reduced accordingly, and the back pressure of chromatographic system strengthens; Flow velocity is 0.8, during 1.0mL/min, analysis time is oversize, chromatographic peak broadens.Reach the highest for post is imitated, Tyr is reached fully with Trp two peaks separate, it is 1.2mL/min that flow velocity is adopted in this research.

Embodiment 3, the detection of standard items and serum sample

1. the chromatographic fractionation figure of standard items and serum sample

Hybrid standard product (containing Trp24.5 μ mol/L, Tyr 55 μ mol/L), pooled serum sample deproteinized supernatant 20 μ L sample introductions are measured.Testing conditions is: chromatographic column wherein: MegresC18 post (250mm * 4.6mmi.d., 5 μ m); Moving phase: 10% (v/v) acetonitrile; Flow velocity: 1.2mL/min; The fluoroscopic examination wavelength: 0-5min excitation wavelength 228nm, wavelength of transmitted light are 306nm; Excitation wavelength 285nm behind the 5min, wavelength of transmitted light are 353nm; Sample size: 20 μ l; Measure under the room temperature.Fig. 1, Fig. 2 are respectively the chromatographic fractionation figures of hybrid standard product and normal human serum sample.Tyr and Trp retention time are respectively about 3.4min, 7.6min, and the peak type is stable, and baseline is steady, the inclusion-free Interference Peaks, and the two good separation can be finished mensuration in the 9min.

2.Tyr, the Trp qualitative and quantitative analysis

Tyr and Trp all adopt peak retention relative method and method of superposition to carry out qualitative analysis, promptly compare hybrid standard liquid and blood serum sample chromatographic peak and relative retention time under the same conditions; In blood serum sample, add an amount of various standard substances respectively, compare the chromatogram that does not add standard items under the identical chromatographic conditions and add standard items.Test findings shows, adds before and after the standard items in blood serum sample, and the peak position that goes out of Tyr and Trp is consistent.Carry out quantitative test with the external standard method peak area.Promptly under identical chromatographic conditions, measure hybrid standard product and blood serum sample, Tyr and Trp chromatographic peak area in the chromatographic peak area of Tyr and Trp in the hybrid standard product and the blood serum sample are compared the content of trying to achieve three kinds of materials in the blood serum sample.Be formulated as:

Annotate: Tyr in the Au=serum (Trp) peak area;

Tyr (Trp) peak area in the As=hybrid standard liquid;

Tyr (Trp) concentration in the Cs=hybrid standard liquid.

3. typical curve and detectability

Get Tyr and Trp standard storage liquid, prepare the standard mixed liquor of a series of concentration with 2.5% perchloric acid solution, each sample sample introduction 3 times is got its mean value and is correlated with and regretional analysis with least square method.The two regretional analysis the results are shown in Table 1.Wherein, Y represents peak area (μ V * s), X represents the sample introduction concentration (μ mol/L) of analyte.Being blank with moving phase, is 3 to obtain detectability by signal to noise ratio (S/N ratio).Peak area of the two and sample introduction concentration linear relationship are good, and the range of linearity is wide, and lowest detectable limit is low, and is highly sensitive.

Regretional analysis result and the lowest detectable limit of table 1Tyr and Trp

4. the investigation of precision

Under the above-mentioned chromatographic condition, get the test that pooled serum carries out measuring in the daytime and in a few days precision, the results are shown in Table 2.In Tyr and Trp day (20 times) and in the daytime the standard deviation (RSD) of (20d) measured value all be lower than 5%, can satisfy conventional serum sample analysis requirement.

Table 2Tyr and Trp in the daytime and the precision of in a few days measuring

5. the investigation of the recovery

Get pooled serum sample 1.6mL and be equally divided into 4 groups, the 1st group adds the 0.1mL ultrapure water as basic sample, the 2nd, 3,4 group of hybrid standard liquid 0.1mL that adds high, medium and low 3 kinds of concentration respectively; According to the method described above sample is handled and assay determination, its mean value calculation recovery is got in every group of replicate determination 3 times.The results are shown in Table 3.

The recovery of table 3Tyr and Trp

6. interference experiment

For whether some materials in the discussion human body disturb the mensuration of Tyr, Trp, get Phe, Kyna, Kyn, 5-HT, Cr interference test sample solution, sample introduction analysis respectively is mixed and made into the analysis of mixing sample sample introduction more earlier.The result shows that above several material is all noiseless to this law.

Table 4 interference test testing result

Annotate :-representative does not detect.

Claims (1)

1. a high performance liquid chromatography-fluorescence method is measured the method for serum tryptophane and tyrosine simultaneously, it is characterized in that, said method comprising the steps of:

A, employing Megres C18 post 250mm * 4.6mm i.d., 5 μ m chromatographic columns;

B, moving phase: 10% (v/v) acetonitrile;

C, flow velocity are 1.2ml/min, sample size: 20 μ l, measure under the room temperature;

D, fluoroscopic examination wavelength set are: 0-5min excitation wavelength 228nm, and wavelength of transmitted light is 306nm, excitation wavelength 285nm behind the 5min, wavelength of transmitted light are 353nm.

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