CN107462647A - Pheochromocytoma diagnostic kit and its application method - Google Patents

Pheochromocytoma diagnostic kit and its application method Download PDF

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Publication number
CN107462647A
CN107462647A CN201710716931.5A CN201710716931A CN107462647A CN 107462647 A CN107462647 A CN 107462647A CN 201710716931 A CN201710716931 A CN 201710716931A CN 107462647 A CN107462647 A CN 107462647A
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pheochromocytoma
methanol
solution
mobile phase
standby
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CN107462647B (en
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丁敏
邹文毕
甄乾娜
张晓清
吕文静
陈霄
韩润川
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International Institute Of In Vitro Diagnostics Chongqing Medical University
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Chongqing Medical University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/74—Optical detectors

Abstract

The invention belongs to detection technique field, and in particular to a kind of pheochromocytoma diagnostic kit and its application method.The technical scheme is that using methoxyepinephrine (Normetanephrine in high performance liquid chromatography detection random urine, NMN), metanephrine (Metanephrine, MN), 3 methyltyramine (Methoxytyramine, 3 MT) and creatinine (Creatinine, Cr), pheochromocytoma is diagnosed using the ratio of first three determinand and creatinine peak area.Kit of the present invention includes following reagent：Sample pretreatment reagent, standard items storing solution, mobile phase.The present invention diagnoses pheochromocytoma by directly using the ratio between peak area of three kinds of determinands and creatinine, is that a kind of reagent is simple, cheap, easy to use, cost-effective pheochromocytoma diagnostic kit.

Description

Pheochromocytoma diagnostic kit and its application method
Technical field
The present invention provides a kind of kit, relates generally to the diagnosis of pheochromocytoma, belongs to detection technique field.
Background technology
Pheochromocytoma is initiated by the endocrine tumors of adrenal medella and the outer stomodaeal nervous system of adrenal gland, with a large amount of Catecholamine secretion is characterized, and typical performance is that " palpitaition, profuse sweating, headache " three is sought peace secondary hypertension etc..The disease is maximum Harm be Complicated with Hypertension crisis, acute left heart failure and cerebral hemorrhage, severe patient's threat to life.Pheochromocytoma and catecholamine Metabolism it is closely related.Catecholamine is a kind of neural class material containing catechol and amido, including norepinephrine (Norepinephrine, NE), adrenaline (Epinephrine, E) and dopamine (Dopamine, DA), in catechol-O- DA, NE and E are metabolized as 3- respectively under the catalytic action of transmethylase (Catechol-O-methyltransferase, COMT) Methyltyramine (Methoxytyramine, 3-MT), methoxyepinephrine (Normetanephrine, NMN) and methoxy Base adrenaline (Metanephrine, MN).Three kinds of determinand structure below figure (R1:NMN,R2:MN,R3:3-MT).
Pheochromocytoma mainly clinically is diagnosed by detecting NMN the and MN levels of sequestered in blood plasma at present, but it is false Positive rate is higher.Research report 3-MT pheochromocytomas outer to marrow and malignant pheochromocytoma are related.Compared with blood plasma, random urine Diagnosis of the middle sequestered NMN and MN to pheochromocytoma has higher specificity, and random urine sample collecting is convenient, noninvasive Wound property, patient compliance are good.Therefore detect the content of NMN, MN, 3-MT in random urine have to diagnosis pheochromocytoma it is important Clinical value.
The content of the invention
It is an object of the invention to provide a kind of pheochromocytoma diagnostic kit and its application method.
In the present invention, the pheochromocytoma diagnostic kit includes following reagent：
(1) sample pretreatment reagent：
1. 10% ammonia water-methanol solution (volume ratio 3:1)；
2. 0.18mol/L potassium hydroxide methanol solutions；
3. 10mmol/L acetic acid-methanol solution (volume ratio 9:1)；
4. 10mmol/L ammonium phosphate solutions；
5. 0.20mol/L acetums.
(2) standard items storing solution：Acetum containing NMN, MN, 3-MT, Cr standard items.
(3) mobile phase：
Mobile phase A：70mmol/L NaH2PO4Solution；
Mobile phase B：Methanol (chromatographically pure).
Wherein, the detection sample is random urine specimen, collection capacity 5mL.Research object is apprised of 5 before sample collection It forbids taking acetylamino phenols and antidepression class medicine, sample collection previous late fasting banana, tea and coffee etc..Collect Sample, handled using solid phase extraction, selected solid-phase extraction column is that SampliQ Silica SCX extract pillar (100mg/mL, Agilent companies of the U.S.).
Wherein, the solid phase extraction method, specifically includes following steps：500 μ L random urines are taken, add 5mL ultra-pure waters, 200 μ L 0.20mol/L acetic acid, mix.With 5mL10% ammonia water-methanol (volume ratio 1:3), 2mL0.18mol/L potassium hydroxide first Alcoholic solution, 2mL ultra-pure waters activated solid extraction pillar, then above-mentioned sample is mixed into loading, coutroi velocity 1-2mL/ successively min.With 4mL 10mmol/L acetic acid-methanol (volume ratio 9:1), 4mL10mmol/L ammonium phosphate, 4mL ultra-pure waters elute small successively Post.2mL ammonia water-methanol solution elutes, and is redissolved after eluent vacuum is spin-dried for 200 μ L 0.20mol/L acetums.
Wherein, the technical scheme is to use NMN, MN, 3-MT and Cr in high performance liquid chromatography detection random urine.Wherein, NMN, MN, 3-MT are detected using fluorescence detector, excitation wavelength：278nm, launch wavelength：320nm, creatinine use ultraviolet detection Device detects, Detection wavelength：235nm.
Wherein described chromatographic column model Shim-Pack VP-ODS (150mm × 4.6mm, 4.6 μm), column temperature are 25 DEG C.
Wherein, the mobile phase A is 70mmol/L NaH2PO4Solution, Mobile phase B is methanol (chromatographically pure), using gradient Elution process, described gradient elution program are shown in Table 1, and flow rate of mobile phase 0.8mL/min, sample size is 50 μ L.In the condition Under, determinand can be realized and efficiently separated.
The eluent gradient elution parameters of table 1
The preparation method of above-mentioned kit, including：
(1) sample pretreatment reagent：
1. prepare 10% ammonia water-methanol solution (volume ratio 3:1)；0.22 μm of organic membrane filter, it is standby to be placed in 4 DEG C of refrigerators.
2. prepare 0.18mol/L potassium hydroxide methanol solutions；0.22 μm of organic membrane filter, it is standby to be placed in 4 DEG C of refrigerators.
3. prepare 10mmol/L acetic acid-methanol solution (volume ratio 9:1)；0.22 μm of organic membrane filter, it is standby to be placed in 4 DEG C of refrigerators With.
4. prepare 10mmol/L ammonium phosphate solutions；0.45 μm of water system membrane filtration, it is standby to be placed in 4 DEG C of refrigerators.
5. prepare 0.20mol/L acetums；0.45 μm of water system membrane filtration, it is standby to be placed in 4 DEG C of refrigerators.
(2) standard items storing solution：Standard items storing solution：The standard items that NMN, MN, 3-MT are prepared with 0.20mol/L acetic acid store up Standby liquid (10g/L), Cr standard reserving solution (50g/L) is placed in -20 DEG C, standby.
(3) mobile phase：
Mobile phase A：Prepare 70mmol/L NaH2PO4Solution；0.45 μm of water system membrane filtration, it is standby to be placed in 4 DEG C of refrigerators.
Mobile phase B：Methanol (chromatographically pure).
Pheochromocytoma diagnostic kit advantage of the present invention is：The inventive method is directly by determinand and creatinine The ratio between peak area, the diagnosis for pheochromocytoma.Kit has higher diagnostic, without adding exogenous internal standard Quantified, and standard curve need not be drawn, enormously simplify detecting step, be that a kind of reagent is simple, cheap, user Just, cost-effective pheochromocytoma diagnostic kit.
Brief description of the drawings
The high-efficient liquid phase chromatogram of the random urine of Patients With Pheochromocytoma described in Fig. 1 embodiments 3；
The high-efficient liquid phase chromatogram of the random urine of healthy control group described in Fig. 2 embodiments 3；
Cr high-efficient liquid phase chromatogram in the random urine of Patients With Pheochromocytoma described in Fig. 3 embodiments 3；
Cr high-efficient liquid phase chromatogram in the random urine of healthy control group described in Fig. 4 embodiments 3；
NMN ROC curve described in Fig. 5 embodiments 5；
MN ROC curve described in Fig. 6 embodiments 5；
3-MT ROC curve described in Fig. 7 embodiments 5.
Embodiment
Embodiments of the invention are only for illustrating to realize technical scheme, and these embodiments are not to the present invention Form and further limit.In following examples, the high performance liquid chromatograph used is the type high performance liquid chromatography of Agilent 1100 Instrument, the high performance liquid chromatograph of other producers and model can also be used to be attained by the purpose of the present invention.
Embodiment 1:The preparation of pheochromocytoma diagnostic kit
First, instrument and equipment
1. assay balance：Sensibility reciprocal is 0.0001g
2. miillpore filter：0.22 μm of aperture
The type high performance liquid chromatographs of 3.Agilent 1100
The ultrapure water filtering devices of 4.Millipore and Millipore vavuum pumps (Millipore companies of the U.S.)
The low temperature desk centrifuges of 5.HIMAC-cf 15
2nd, reagent and solution：Kit components are shown in Table 2
The kit component of table 2
The preparation method of mentioned reagent box, is specifically included：
(1) sample pretreatment reagent：
1. prepare 10% ammoniacal liquor methanol solution (volume ratio 3:1)；0.22 μm of organic membrane filter, it is standby to be placed in 4 DEG C of refrigerators.
2. prepare 0.18mol/L potassium hydroxide methanol solutions；0.22 μm of organic membrane filter, it is standby to be placed in 4 DEG C of refrigerators.
3. prepare 10mmol/L acetic acid-methanol solution (volume ratio 9:1)；0.22 μm of organic membrane filter, it is standby to be placed in 4 DEG C of refrigerators With.
4. prepare 10mmol/L ammonium phosphate solutions；0.45 μm of water system membrane filtration, it is standby to be placed in 4 DEG C of refrigerators.
5. prepare 0.20mol/L acetums；0.45 μm of water system membrane filtration, it is standby to be placed in 4 DEG C of refrigerators.
(2) standard items storing solution：NMN, MN, 3-MT standard reserving solution (10g/L), Cr are prepared with 0.20mol/L acetic acid Standard reserving solution (50g/L), be placed in -20 DEG C it is standby.
(3) mobile phase：
Mobile phase A：Prepare 70mmol/L NaH2PO4Solution；0.45 μm of water system membrane filtration, it is standby to be placed in 4 DEG C of refrigerators.
Mobile phase B：Methanol (chromatographically pure).
Embodiment 2：The use of pheochromocytoma diagnostic kit
1. urine specimen pre-processes
500 μ L random urines are taken, 5mL ultra-pure waters is added, 200 μ L 0.2mol/L acetic acid, mixes.With 5mL10% ammoniacal liquor-first Alcohol (volume ratio 1:3), 2mL0.18mol/L potassium hydroxide methanol solutions, 2mL ultra-pure waters after activated solid extraction pillar, are incited somebody to action successively Above-mentioned sample mixes loading, coutroi velocity 1-2mL/min.With 4mL 10mmol/L acetic acid-methanol (volume ratio 9:1), 4mL10mmol/L ammonium phosphate, 4mL ultra-pure waters elute pillar successively.2mL ammonia water-methanol solution is eluted, and eluent vacuum is spin-dried for Redissolved afterwards with 200 μ L0.20mol/L acetums.
2. liquid phase chromatogram condition
Mobile phase：Mobile phase A is 70mmol/L NaH2PO4Solution, Mobile phase B are methanol (chromatographically pure), are washed using gradient Desorption method, described gradient elution program are shown in Table 3.
The eluent gradient elution parameters of table 3
Flow velocity：0.8mL/min.
Column temperature：25℃.
Fluorescence detector：Excitation wavelength is 278nm, launch wavelength 320nm.
UV-detector：Detection wavelength is 235nm.
Sample size：50μL.
Embodiment 3
The present embodiment is to investigate the precision that kit of the present invention is used to determine NMN, MN, 3-MT in random urine. The investigation of precision is carried out using authentic specimen, that is, chooses pheochromocytoma patient urine and normal human urine, in a few days repeats to survey It is fixed 5 times, METHOD FOR CONTINUOUS DETERMINATION 5 days, the ratio of determinand and creatinine is calculated, precision assessment is carried out, the results are shown in Table 4.The coefficient of variation is equal Equal to or less than 5.6%, show that the present invention has good precision.
The precision of table 4 (n=5)
Embodiment 4
The present embodiment is to investigate kit of the present invention to do for determining the anti-of NMN, MN, 3-MT and Cr in random urine Disturb ability.Ascorbic acid and uric acid are endogenous interfering materials common in urine sample.Interference test is faced according to American National The evaluation of programme that the bed laboratory standard committee formulates is carried out, and measure does not add the random urine testing concentration (X of chaff interferenceC) and Testing concentration (the X added after chaff interferenceT), interference value (XT-XC) in 1.96S i.e. (the 95% confidence level model of the inventive method Enclose) in be without interfering significantly with and (represented with N), if interference value is more than 1.96S, as interferes significantly with and (represented with I).It the results are shown in Table 5, the ascorbic acid and uric acid of two kinds of concentration have preferable antijamming capability to testing concentration without influence, the present invention.
The interference test of table 5
Embodiment 5
The present embodiment is to investigate diagnostic of the kit of the present invention to pheochromocytoma.
Choose Patients With Pheochromocytoma 37, normal person 56, primary hypertension patient 53, non-pheochromocytoma kidney Upper gland occupy-place patient 55.The selection of pheochromocytoma and non-pheochromocytoma Adrenal masses patient are according to pathological examination. The selection of primary hypertension patient need to meet 2 aspects：1) the Secondary cases height caused by Other diseases is excluded through auxiliary examination Blood pressure；2) patient do not occur in subsequent 1 year the positive related result of any pheochromocytoma (iconography, biochemistry detection, Pharmacological evaluation etc.).Research object forbids taking acetylamino phenols medicine and antidepression class medicine for 5 days before sample collection, Sample collection previous late fasting banana, tea and coffee etc..5mL random urines are gathered, -80 DEG C is placed in and saves backup.As a result：Random urine Three kinds of determinands are shown in Table 6 to the diagnostic of pheochromocytoma in liquid, directly with the ratio between peak area of three kinds of determinands and creatinine, Diagnosis is carried out to pheochromocytoma has higher sensitivity and specificity.The invention provides a kind of reagent is simple, price is low Honest and clean, easy to use, the pheochromocytoma diagnostic kit of high performance-price ratio can be as the reliable method of diagnosis pheochromocytoma.
The diagnostic for comparing pheochromocytoma of 6 three kinds of determinands of table and creatinine peak area
Although being above described in detail with a general description of the specific embodiments to the present invention, It can make some modifications or improvements on the basis of the present invention and with used for other purposes.This is apparent to those skilled in the art 's.Therefore, modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to claimed model Enclose.

Claims (8)

  1. A kind of 1. pheochromocytoma diagnostic kit, it is characterised in that：The kit includes following reagent：
    (1) sample pretreatment reagent：
    1. 10% ammonia water-methanol solution (volume ratio 3:1)；
    2. 0.18mol/L potassium hydroxide methanol solutions；
    3. 10mmol/L acetic acid-methanol solution (volume ratio 9:1)；
    4. 10mmol/L ammonium phosphate solutions；
    5. 0.20mol/L acetums.
    (2) standard items storing solution：Contain methoxyepinephrine (Normetanephrine, NMN), methoxyl group adrenal gland Plain (Metanephrine, MN), 3- methyltyramines (Methoxytyramine, 3-MT) and creatinine (Creatinine, Cr) mark The acetum of quasi- product.
    (3) mobile phase：
    Mobile phase A：70mmol/L NaH2PO4Solution；
    Mobile phase B：Methanol (chromatographically pure).
  2. 2. the pheochromocytoma diagnostic kit as described in claim 1, it is characterised in that：The sample detected is random urine sample This, collection of specimens amount is 5mL.5 days taboos take acetylamino phenols medicine and antidepression class medicine before advising detected object collection of specimens, Collection of specimens previous late fasting banana, tea and coffee etc..Sample pretreating method used is solid phase extraction, selected solid phase Extraction column is that SampliQ Silica SCX extract pillar (100mg/mL, Agilent companies of the U.S.).
  3. 3. the solid phase extraction method as described in claim 2, it is characterised in that：500 μ L random urines are taken, add 5mL ultra-pure waters, 200 μ L 0.20mol/L acetic acid, mix.With 5mL10% ammonia water-methanol (volume ratio 1:3), 2mL0.18mol/L potassium hydroxide first Above-mentioned sample after activated solid extraction pillar, is mixed loading, coutroi velocity 1-2mL/min by alcoholic solution, 2mL ultra-pure waters successively. With 4mL 10mmol/L acetic acid-methanol (volume ratio 9:1), 4mL10mmol/L ammonium phosphate, 4mL ultra-pure waters elute pillar successively. 2mL ammonia water-methanol solution elutes, and is redissolved after eluent vacuum is spin-dried for 200 μ L 0.20mol/L acetums.
  4. 4. the pheochromocytoma diagnostic kit as described in claim 1, it is characterised in that：Technical scheme is to use efficient liquid phase NMN, MN, 3-MT and Cr in chromatogram detection random urine.Wherein, NMN, MN, 3-MT are detected using fluorescence detector, excitation wave It is long：278nm, launch wavelength：320nm.Cr is detected using UV-detector, Detection wavelength：235nm.
  5. 5. the high performance liquid chromatography as described in claim 4, it is characterised in that：Chromatogram column type number Shim-Pack VP-ODS (150mm × 4.6mm, 4.6 μm), column temperature are 25 DEG C.
  6. 6. high-efficiency liquid chromatography method for detecting according to claim 4, it is characterised in that：Mobile phase A is 70mmol/ LNaH2PO4Solution, Mobile phase B are methanol (chromatographically pure), and using gradient elution method, described gradient elution program see the table below, Flow rate of mobile phase is 0.8mL/min, and sample size is 50 μ L.Efficiently separating for determinand can be realized under the conditions of this.
  7. 7. the preparation method of the kit as described in claim 1~6 any one, it is characterised in that：
    (1) sample pretreatment reagent：
    1. prepare 10% ammonia water-methanol solution (volume ratio 3:1)；0.22 μm of organic membrane filter, it is standby to be placed in 4 DEG C of refrigerators.
    2. prepare 0.18mol/L potassium hydroxide methanol solutions；0.22 μm of organic membrane filter, it is standby to be placed in 4 DEG C of refrigerators.
    3. prepare 10mmol/L acetic acid-methanol solution (volume ratio 9:1)；0.22 μm of organic membrane filter, it is standby to be placed in 4 DEG C of refrigerators.
    4. prepare 10mmol/L ammonium phosphate solutions；0.45 μm of water system membrane filtration, it is standby to be placed in 4 DEG C of refrigerators.
    5. prepare 0.20mol/L acetums；0.45 μm of water system membrane filtration, it is standby to be placed in 4 DEG C of refrigerators.
    (2) standard items storing solution：NMN, MN, 3-MT standard reserving solution (10g/L), Cr mark are prepared with 0.20mol/L acetic acid Quasi- storing solution (50g/L), be placed in -20 DEG C it is standby.
    (3) mobile phase：
    Mobile phase A：Prepare 70mmol/L NaH2PO4Solution；0.45 μm of water system membrane filtration, it is standby to be placed in 4 DEG C of refrigerators.
    Mobile phase B：Methanol (chromatographically pure).
  8. 8. pheochromocytoma diagnostic kit according to claim 1, it is characterised in that：The present invention directly by determinand with The ratio between peak area of creatinine, the diagnosis for pheochromocytoma.The kit has higher diagnostic, without adding external source Property internal standard is quantified, and need not draw standard curve, be enormously simplify detecting step, is that a kind of reagent is simple, cheap, Pheochromocytoma diagnostic kit easy to use, cost-effective.
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CN114544826A (en) * 2020-11-24 2022-05-27 重庆医科大学 Application of reagent for detecting histidine in blood plasma in preparation of depression detection kit
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CN116908325A (en) * 2023-07-10 2023-10-20 昆明医科大学第一附属医院 Sample pretreatment liquid, method and kit capable of simultaneously detecting 12 catecholamines, metabolites and creatinine in human urine
CN117092263A (en) * 2023-08-15 2023-11-21 合肥歆智医疗器械有限公司 Free catecholamine and metabolite detection kit, determination method and application

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