CN104807923A - Method for detecting vanillylmandelic acid in human urine - Google Patents

Abstract

The present invention relates to a method for detecting vanillylmandelic acid in human urine. The method comprises: taking human urine, and carrying out acidification corrosion protection with HCl so as to long-timely store at a temperature of -80 DEG C; extracting the urine with ethyl acetate, volatilizing, dissolving with acetic acid, and carrying out other processes so as to obtain a VMA crude extract; and separating the crude extract through HPLC, obtaining the stable and single VMA absorption peak through a UV detector at a wavelength of 280 nm, drawing a VMA standard curve through an external standard method, and finally calculating the VMA content. The method of the present invention has characteristics of low cost and easy operation, and provides a rapid, inexpensive and sensitive method for detecting VMA in human urine.

Description

The method of vanillylmandelic acid (VMA) is detected in a kind of people's urine

Technical field

The present invention relates to medical science, in especially a kind of people's urine, detect the method for vanillylmandelic acid (VMA).

Background technology

Catecholamine is the parahormone secreted by adrenal medella, which includes dopamine, adrenaline and adrenalectomy element.These hormones often because physics or spiritual stress reaction, are released in blood circulation, improve cerebral nerve impulsion and transmit, and release glucose and fatty acid are to provide energy, and bronchiectasis and pupil amplify.Most of catecholamine can be become vanillylmandelic acid (VMA) (Vanilmandelic acid by metabolic degradation, VMA), homovanillic acid (homovanillic acid, HVA), metanephrine (metanephrine) and norepinephrine (normetanephrine), only have a small amount of catecholamine to excrete with urine.

VMA has another name called 3 methoxy 4 hydroxymandelic acid or 4-hydroxy-3-methoxy mandelic acid, is the metabolic product of a kind of catecholamines adrenaline and adrenalectomy element, finally excretes with urine.The content of VMA in urine sample is highly stable, only has content after there is stress reaction just can slightly increase.But neuroblastoma (neuroblastomas), pheochromocytoma (pheochromocytomas) and other neuroendocrine tumor (neuroendocrine tumors) can produce catecholamine in a large number, cause the sharp increase of VMA content.Because a large amount of catecholamine enters blood circulation, make vessel retraction, peripheral resistance increases, and increased heart rate, the hurried rising of paroxysmal appears in blood pressure.The clinical manifestation of these patients is continuation hypertension, palpitaition, n and V, breathe hard, chest constrains, headache, numb, a large amount of perspiration of brothers, eye-blurred etc., the hypertensive crisis such as cerebral hemorrhage or pulmonary edema may be there is in severe patient.Urine VMA too high levels is an important symbol of Secretion of Catecholamine exception, is the first-selected mark of diagnosis of neuroblastoma and pheochromocytoma.

The detection gimmick of current VMA mainly contains colorimetric-hyphenated techniques chromatography, ELISA and HPLC method.

Commercially available VMA detects ELISA kit and generally applies double-antibody sandwich enzyme-labeled immunity assay VMA level.ELISA kit has sensitive, quick feature, but shortcoming to be stability not high, the technology of operating personnel is had higher requirements.And this series products is mainly external import, as Cloud-Clone Corp. (U.S.), Biocompare (U.S.), RDG (U.S.), Eaglebio (U.S.) etc., expensive.Because the core technologies such as antibody are external monopolization, domesticly there is no independent research product, fundamentally cannot reduce reagent cost.

Colorimetric-hyphenated techniques chromatography, the detection kit of current domestic approval is the vanillylmandelic acid (VMA) assay kit (state's food medicine prison No. 2402315th, tool (entering) word 2010) that Spain Biosystems S.A. produces, the method adopts anion exchange resins to be kept on resin by the VMA absorption in urine, other interfering materials then can not be retained and clear, last eluting VMA.The oxidized formation vanilla of VMA is plain in the basic conditions again, carries out quantitative test by ultraviolet spectrometry colourimetry.The method efficiency is high, detect property good, shortcoming be this kit for monopolize product abroad, expensive.

HPLC technology for detection VMA is considered to the most promising VMA detection method.Wherein the research of electrochemical assay is quite ripe, modifies method (Taran), hydrolysis and condensation (Fa μ ler, Lionetto) and capillary electrophoresis technique detection method and also set up one after another after this outer chemical.These method advantages are that highly sensitive, detection speed is fast, but need specific apparatus, and expensive, cost is high, and operating personnel need special training, are difficult to be widely used in clinical.HPLC combined U V detection method rarely has report, and UV detecting device cost is low, all can be equipped with UV detecting device while generally purchasing HPLC.

Summary of the invention

The present invention will solve the shortcoming of above-mentioned prior art, detects the method for vanillylmandelic acid (VMA) in providing a kind of quick, efficient, cheap people to urinate.

The present invention solves the technical scheme that its technical matters adopts: the method detecting vanillylmandelic acid (VMA) in this people's urine, and its step comprises:

1) get human urine HCl acidifying, preserve under-80 DEG C of conditions, urine is extracted with ethyl acetate after melting, volatilizees, and acetic acid dissolves again, can obtain VMA crude extract;

2) above-mentioned crude extract is separated through HPLC, under UV detecting device 280nm wavelength, obtains and stablizes single VMA absorption peak, and draw VMA typical curve by external standard method, finally calculate the content of VMA.

As preferably, wherein step 1) specific as follows:

A) after collecting freshly voided urine, add 5M HCl acidifying, put into-80 DEG C of refrigerators and preserve, until start subsequent experimental;

B) after urine is melted, get 4ml urine and be preinstalled with in the 15ml centrifuge tube of 2g NaCl to one, the mixing 10min that turns upside down forms saturated salt solution;

C) add 4ml ethyl acetate, turn upside down mixing 5min; The centrifugal 5min of 4000g, careful upper organic phase of drawing is in a new 50ml round bottom centrifuge tube;

D) repeat b-c and walk 2 times, obtain upper organic phase 12ml altogether;

E) centrifuge tube metal heater is heated to 45 DEG C, high wind volatilization in vent cabinet, until all ethyl acetate volatilizations completely, obtains yellow or black precipitate;

F) add 400 μ l1% acetums, fully shake, 4 DEG C of dissolvings of spending the night;

G) 15000g centrifugal after, moved on to by supernatant in new 1.5ml centrifuge tube, 4 DEG C of preservations are no more than 1 week, wait for HPLC detect.

As preferably, wherein HPLC condition is:

Pillar: SP-C18 (4.6mm*250mm, 5um);

Mobile phase: A (water: acetic acid=98: 1): B (water: methyl alcohol: acetic acid=68: 30: 2) gradient elution;

Gradient: 0-15min:100%A-20%A

15-20min：20％A

20-21min：20％A-100％A

21-35min：100％A

Flow velocity: 0.8ml/min;

Determined wavelength: UV280nm;

Applied sample amount: 100 μ l.

As preferably, external standard method drafting VMA typical curve step is as follows: take VMA with electronic analytical balance, acetic acid with 1% is configured to the VMA solution of concentration gradient of 1ng/ μ l, 2.5ng/ μ l, 5ng/ μ l, 10ng/ μ l, 20ng/ μ l, 100ng/ μ l, the VMA solution of these concentration gradients successively upper HPLC column is detected, and according to its testing result drawing standard curve.

Inventing useful effect is: cost of the present invention is low, easy and simple to handle, provides the method detecting VMA in a kind of quick, cheap, sensitive people urine.

Accompanying drawing explanation

Fig. 1 is the HPLC peak figure after the VMA loading of 20ng/ μ l;

Fig. 2 is VMA canonical plotting;

Fig. 3 is the VMA detection figure in embodiment 1;

Fig. 4 is the VMA detection figure in embodiment 2;

Embodiment

Below in conjunction with accompanying drawing, the invention will be further described:

With reference to accompanying drawing:

The detection method that this people of the present invention urinates VMA comprises the following steps:

1) preparation of VMA crude extract

A) after collecting freshly voided urine, add 5M HCl acidifying, put into-80 DEG C of refrigerators and preserve, until start subsequent experimental;

B) after urine is melted, get 4ml urine and be preinstalled with in the 15ml centrifuge tube of 2g NaCl to one, the mixing 10min that turns upside down forms saturated salt solution;

C) add 4ml ethyl acetate, turn upside down mixing 5min; The centrifugal 5min of 4000g, careful upper organic phase of drawing is in a new 50ml round bottom centrifuge tube;

D) repeat b-c and walk 2 times, obtain upper organic phase 12ml altogether;

E) centrifuge tube metal heater is heated to 45 DEG C, high wind volatilization in vent cabinet, until all ethyl acetate volatilizations completely, obtains yellow or black precipitate;

F) add 400 μ l1% acetums, fully shake, 4 DEG C of dissolvings of spending the night;

G) 15000g centrifugal after, moved on to by supernatant in new 1.5ml centrifuge tube, 4 DEG C of preservations are no more than 1 week, wait for HPLC detect.

2) above-mentioned crude extract is separated through HPLC, under UV detecting device 280nm wavelength, obtains and stablizes single VMA absorption peak, and draw VMA typical curve by external standard method, finally calculate the content of VMA.

Wherein HPLC condition is:

Pillar: SP-C18 (4.6mm*250mm, 5um);

Mobile phase: A (water: acetic acid=98: 1): B (water: methyl alcohol: acetic acid=68: 30: 2) gradient elution;

Gradient: 0-15min:100%A-20%A

15-20min：20％A

20-21min：20％A-100％A

21-35min：100％A

Flow velocity: 0.8ml/min;

Determined wavelength: UV280nm;

Applied sample amount: 100 μ l.

VMA Specification Curve of Increasing method: take VMA (Sigma with electronic analytical balance, H0131), acetic acid with 1% is configured to the VMA solution of concentration gradient of 1ng/ μ l, 2.5ng/ μ l, 5ng/ μ l, 10ng/ μ l, 20ng/ μ l, 100ng/ μ l, is detected by the VMA solution of these concentration gradients successively upper HPLC column.

HPLC peak figure after the VMA loading of 20ng/ μ l as shown in Figure 1.VMA peak appears at 11.0-11.5min.

The testing result of variable concentrations VMA is as follows:

VMA(μg)	Peak area
		10	8823731
2	1630482
		1	792206
0.5	146109
		0.25	146109
0.1	60764

Drawing standard curve as shown in Figure 2.

The content that can calculate VMA according to peak area is:

VMA content (μ g)=(peak area+132119)/894737

Calculate that the content of VMA in urine is according to the content of VMA in upper sample:

VMA concentration (μ g/ml)=VMA content * 4/4 in people's urine.

The detection case of VMA in people's urine

Embodiment 1:VMA detects figure as shown in Figure 3.

Ret.Time	Conc.	Area	Height
				11.429	1.14448	238426	17914

The content calculated containing VMA in the every 100 μ l of sample on HPLC is 0.414 μ g.

Calculating VMA content in urine is 0.414 μ g/ml.

Embodiment 2:VMA detects figure as shown in Figure 4:

Ret.Time	Conc.	Area	Height
				11.414	2.15421	458154	32338

The content calculated containing VMA in the every 100 μ l of sample on HPLC is 0.659 μ g.

Calculating VMA content in urine is 0.659 μ g/ml.

In addition to the implementation, the present invention can also have other embodiments.All employings are equal to the technical scheme of replacement or equivalent transformation formation, all drop on the protection domain of application claims.

Claims (4)

1. detect a method for vanillylmandelic acid (VMA) in people's urine, its step comprises:

1) get human urine HCl acidifying, preserve under-80 DEG C of conditions, urine is extracted with ethyl acetate after melting, volatilizees, and acetic acid dissolves again, can obtain VMA crude extract;

2) above-mentioned crude extract is separated through HPLC, under UV detecting device 280nm wavelength, obtains and stablizes single VMA absorption peak, and draw VMA typical curve by external standard method, finally calculate the content of VMA.

2. detect the method for vanillylmandelic acid (VMA) in people's urine according to claim 1, it is characterized in that: step 1) specific as follows:

A) after collecting freshly voided urine, add 5M HCl acidifying, put into-80 DEG C of refrigerators and preserve, until start subsequent experimental;

B) after urine is melted, get 4ml urine and be preinstalled with in the 15ml centrifuge tube of 2g NaCl to one, the mixing 10min that turns upside down forms saturated salt solution;

C) add 4ml ethyl acetate, turn upside down mixing 5min; The centrifugal 5min of 4000g, careful upper organic phase of drawing is in a new 50ml round bottom centrifuge tube;

D) repeat b-c and walk 2 times, obtain upper organic phase 12ml altogether;

E) centrifuge tube metal heater is heated to 45 DEG C, high wind volatilization in vent cabinet, until all ethyl acetate volatilizations completely, obtains yellow or black precipitate;

F) add 400 μ l1% acetums, fully shake, 4 DEG C of dissolvings of spending the night;

G) 15000g centrifugal after, moved on to by supernatant in new 1.5ml centrifuge tube, 4 DEG C of preservations are no more than 1 week, wait for HPLC detect.

3. detect the method for vanillylmandelic acid (VMA) in people's urine according to claim 1, it is characterized in that: HPLC condition is:

Pillar: SP-C18 (4.6mm*250mm, 5um);

Mobile phase: A (water: acetic acid=98: 1): B (water: methyl alcohol: acetic acid=68: 30: 2) gradient elution;

Gradient: 0-15min:100%A-20%A

15-20min：20％A

20-21min：20％A-100％A

21-35min：100％A

Flow velocity: 0.8ml/min;

Determined wavelength: UV280nm;

Applied sample amount: 100 μ l.

4. in people's urine according to claim 1, detect the method for vanillylmandelic acid (VMA), it is characterized in that: external standard method drafting VMA typical curve step is as follows: take VMA with electronic analytical balance, acetic acid with 1% is configured to the VMA solution of concentration gradient of 1ng/ μ l, 2.5ng/ μ l, 5ng/ μ l, 10ng/ μ l, 20ng/ μ l, 100ng/ μ l, the VMA solution of these concentration gradients successively upper HPLC column is detected, and according to its testing result drawing standard curve.