CN110018255A - LC-MS detects kit and its application of PKU, CAH and GAL simultaneously - Google Patents
LC-MS detects kit and its application of PKU, CAH and GAL simultaneously Download PDFInfo
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- CN110018255A CN110018255A CN201910327792.6A CN201910327792A CN110018255A CN 110018255 A CN110018255 A CN 110018255A CN 201910327792 A CN201910327792 A CN 201910327792A CN 110018255 A CN110018255 A CN 110018255A
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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
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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
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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Abstract
The present invention relates to kit and its applications that a kind of LC-MS detects PKU, CAH and GAL simultaneously.Kit of the invention includes following reagent: (a) calibration object, and the calibration object contains phenylalanine, 17 α-hydroxyprogesterone and gala saccharide；(b) extracting solution, the extracting solution are at least one of methanol, ethyl alcohol, isopropanol, acetone, acetonitrile；(c) mobile phase, the mobile phase is made of mobile phase A and Mobile phase B, the mobile phase A and Mobile phase B are made of (I) water, (II) methanol and (III) formic acid or acetic acid, alternatively, the mobile phase A and Mobile phase B are made of (I) water, (IV) acetonitrile and (III) formic acid or acetic acid.It is examined quickly using kit of the present invention detection PKU, CAH and GAL, testing result is accurate and repeatability is high.
Description
Technical field
The present invention relates to a kind of kit and its applications, and in particular to a kind of LC-MS detects PKU, CAH and GAL simultaneously
Kit and its application.
Background technique
The impressive progress that disorder in screening is modern age preventive medicine field is carried out to newborn, is improved the health of the people
Important measures.By the examination of newborn infant diseases, early diagnosis can be made when infant does not have clinical disease manifestation, carried out
There is irreversible damage to avoid infant vitals, ensures the normal development of childhood underweight and intelligence in early treatment.
Phenylketonuria is autosomal recessive hereditary diseases, and sick child is the elder generation of phenylalanine hydroxylase (a kind of special liver enzyme)
It lacks or activity reduces, and causes phenylalanine that cannot normally be hydroxylated into tyrosine, into human circulation, thus blood and cerebrospinal fluid
Middle concentration of phenylalanine is consequently increased, and phenylpyruvic acid is discharged from urine.Tyrosinase is also suppressed, and causes tyrosine metabolism
Obstacle, melanin, which is formed, to be reduced, thus the colour of skin is shallowly white, and hair is yellowish, and iris color is light.Since phenylacetic acid is discharged in sweat and urine,
Therefore children have special musty or mouse taste.Due to the accumulation of phenylalanine, phenylpyruvic acid, phenylacetic acid, make cerebral dysgenesis, myelin shape
At being obstructed, ash, white matter denaturation cause baby early stage feeblemindedness to occur.After sick child's phenylalanine increases, serotonin, γ ammonia
Base butyric acid is reduced, and causes one-year-old preceding often generation epileptic attack, and severe one has the clinical manifestation of similar infantile spasms and electroencephalogram to occur
Hyperarrhythmia.Sick child can also have a dyskinesia, cerebral palsy, mostly dynamic, tremble and vomit, uneasiness, eczema etc. show.If early
Phase gives low-phenylalanine diet treatment, and the phenylalanine in infant blood is made to maintain ideal range [60~120 μm of ol/L (1
~2mg/dL), the generation of the diseases such as intelligence serious hindrance and secondary epilepsy can be prevented.Therefore the neonatal screening of PKU is early
Key point phase discovery infant and treated in time.
Phenylketonuria (phenylketonuria, PKU) be due to caused by enzyme defect in phenylalanine metabolic pathway, because
The metabolites such as a large amount of phenylpyruvic acids are discharged in infant urine and gain the name.PKU is one kind more typical in disorder of amino acid catabolism,
Belong to autosomal recessive inheritance.Its disease incidence is different with race, about l/6000~l/25000, and China's disease incidence is about 1/
16500, it show the difference more than the few north in obvious south.
In recent years it finds, by treatment, intellectual development is normal and with women patient PKU that normal male is got married, if pregnant
Period unreasonably keeps on a diet, miscarries, the not congruent incidence of stillbirth, intrauterine growth is very high, even if its baby does not suffer from PKU,
There are feeblemindedness, the complication such as microcephaly, congenital heart disease, this is because the phenylalanine of parent high concentration can pass through more
Placenta makes fetus be damaged.This kind of baby is referred to as maternal instinct PKU infant.For this purpose, the PKU female patient Ying Youji in breeding time
It keeps on a diet with drawing, preferably starts in pregnant the first half until childbirth, allows Blood phenylalanine control in 2~6mg/dL, make
Fetus is from damage.
17 α-hydroxyprogesterone is generated by adrenal cortex and sexual gland, and gestagen active is very low.17 α-hydroxyprogesterone is through 21-hydroxylase
Effect, generates the precursor compound S (CpS) of cortisol.The heredity congenital adrenal hyperplasia patients serum that 21-hydroxylase lacks
In 17 α-hydroxyprogesterone concentration it is significantly raised, 17 α-hydroxyprogesterone ascensional range is smaller when 11- hydroxylase defect.About 6% adult is more
Hair women has different degrees of 21-hydroxylase to lack.The measurement of 17 α-hydroxyprogesterone is also used for the common Cuo of analysis male and female
Sore, male be bald and the sterility of some unknown causes.
Heredity congenital adrenal hyperplasia disease (congenital adrenal hyperplasia, CAH) is also known as adrenal gland
Genital disease or adrenal sex metamorphosis sign.It is deposited mainly due to necessary enzyme in cortex hormone of aadrenaline biosynthetic process
In defect, cause cortin synthesis abnormal.Majority of cases acth secretion manages C-21 cortico-steroid, manages salt hormone deficiency and male swashs
It is plain excessive, therefore different degrees of hypoadrenocorticism clinically occur, it is manlike with girl, and boy's then expression power
Furthermore precocity still has a variety of syndromes such as low blood sodium or hypertension.
In heredity congenital adrenal hyperplasia disease patient, that most commonly seen is 21-hydroxylase missing disease (about 80%) and 11-
Hydroxylase deficiency disease (about 15%), other situations account for 5%.And when 21-hydroxylase missing and 11- hydroxylase deficiency, can all it cause
The concentration of intracorporal 17 α-hydroxyprogesterone increases.Remaining heredity congenital adrenal hyperplasia disease type, such as 11 B-hydroxylase deletion forms
It is all extremely rare with 17 α-hydroxylase deficiency type.So detection 17 α-hydroxyprogesterone is for judging heredity congenital adrenal hyperplasia
Disease has significant meaning.
Galactosemia (galactosemia, GAL) is a kind of autosomal recessive inheritance disease, disease incidence 1/
50000, it is galactokinase in galactose metabolism, one 1 uridine monophosphate acyltransferase of galactolipin and uridine 5'-diphosphate galactolipin
One 4 one isomery enzyme defects can cause galactosemia.Usually said classical galactosemia is that galactosyltransferase lacks
Galactosemia caused by weary, galactosemia has huge harm to newborn, due to enzymes certain during galactose metabolism
The autosomal recessive disease for lacking and causing galactose utilization obstacle endangers seriously newborn, cannot such as obtain morning
Phase diagnosis and treatment will will lead to feeblemindedness, cataract, liver, renal damage etc..In the 1960s, the screening of newborn's galactosemia by
Progressive to enter people's sight and to be put into neonatal screening project, this provides side for the morning discovery of galactosemia, early treatment
It helps, reduces infant mortality rate caused by galactosemia.
Currently, clinically used by phenylketonuria, using fluorescence analysis, fluorescence analysis half-life short can not
To repeat to detect, it is also easy to the polarity by solution, temperature, paramagnet, the pH value of solution, fluorescent quenching, scattering light etc.
The interference of factor easily causes detection false positive results in this way, causes spirit and financial burden to clinician and patient；Mesh
The preceding kit for clinically having not been used in the galactolipin in detection newborn's blood cake；17 α-hydroxyprogesterone is mainly using the time point
Distinguish the competition law detection of immunofluorescence technique, competition law is there are poor specificity, testing result difference when detecting using different kits
Greatly, the exchange being not easy between laboratory, and detection time is long, generally requires 3-4 hours just to go out as a result, flux is low, every time
Each blood cake can only go out once as a result, sample volume is big, and each sample introduction needs 100uL or so, while also increasing the pain of patient
It is bitter.
Summary of the invention
A kind of LC-MS is provided it is an object of the invention to overcome in place of above-mentioned the deficiencies in the prior art while being detected
The kit of PKU, CAH and GAL and its application are not only examined quickly using the kit detection PKU, CAH and GAL, and detected
As a result accurate, testing result repeatability height.
To achieve the above object, the technical scheme adopted by the invention is as follows: a kind of LC-MS detect simultaneously PKU, CAH and
The kit of GAL comprising following reagent:
(a) calibration object, the calibration object contain phenylalanine, 17 α-hydroxyprogesterone and galactolipin；
(b) extracting solution, the extracting solution are at least one of methanol, ethyl alcohol, isopropanol, acetone, acetonitrile；With
(c) mobile phase, the mobile phase are made of mobile phase A and Mobile phase B, and the mobile phase A and Mobile phase B are by (I)
Water, (II) methanol and (III) formic acid or acetic acid form, and in the mobile phase A, the percent by volume of formic acid or acetic acid is 0.01%-
0.05%, the volume ratio of water and methanol is 9:1~19:1；In the Mobile phase B, the percent by volume of formic acid or acetic acid is
The volume ratio of 0.01%-0.05%, water and methanol is 0:100~1:99；Alternatively,
The mobile phase A and Mobile phase B are made of (I) water, (IV) acetonitrile and (III) formic acid or acetic acid, the mobile phase A
In, the percent by volume of formic acid or acetic acid is 0.01%-0.05%, and the volume ratio of water and acetonitrile is 9:1~19:1；The flowing
In phase B, the percent by volume of formic acid or acetic acid is 0.01%-0.05%, and the volume ratio of water and acetonitrile is 0:100~1:99.
Above-mentioned PKU indicates phenylketonuria, and CAH indicates heredity congenital adrenal hyperplasia disease, and GAL indicates galactosemia
Disease.The substance for reflecting phenylketonuria (PKU) test is phenylalanine, and reflection heredity congenital adrenal hyperplasia disease (CAH) is surveyed
The substance of examination is 17 α-hydroxyprogesterone, and the substance of reflection galactosemia (GAL) test is galactolipin.
Mentioned reagent box through the invention can be used LC-MS while detect PKU, CAH and GAL, and testing result is quasi-
Really, easy to operate, examine quickly, detection flux is high, testing result repeatability is high, testing cost is low, and patient's pain can be mitigated.
Detect the preferred embodiment of the kit of PKU, CAH and GAL simultaneously as LC-MS of the present invention, it is described
Kit is for detecting phenylalanine in Filter Paper Dry Blood piece, 17 α-hydroxyprogesterone and galactolipin.Filter Paper Dry Blood piece can have sampling quantity
Less, many advantages, such as mitigating subject's pain, convenient transportation.Kit and its application method of the invention can industrial application in
The screening of clinical newborn's Filter Paper Dry Blood piece.
Detect the preferred embodiment of the kit of PKU, CAH and GAL simultaneously as LC-MS of the present invention, it is described
Calibration object is the human red cell containing phenylalanine, 17 α-hydroxyprogesterone and galactolipin.
Detect the preferred embodiment of the kit of PKU, CAH and GAL simultaneously as LC-MS of the present invention, it is described
Calibration object is 6 sets, the respectively the 1st, the 2nd, the 3rd, the 4th, the 5th and the 6th set of calibration object；1st set of calibration object contains 0.5mg/dL benzene
Alanine and 17 α-hydroxyprogesterone and galactolipin are free of, the 2nd set of calibration object contain 1.5mg/dL phenylalanine, 17 α of 10nmol/L-
Hydroxyprogesterone and 3mg/dL galactolipin, the 3rd set of calibration object contain 3mg/dL phenylalanine, 17 α of 25nmol/L-hydroxyprogesterone and 8mg/
DL galactolipin, the 4th set of calibration object contain 5mg/dL phenylalanine, 17 α of 50nmol/L-hydroxyprogesterone and 15mg/dL galactolipin, and the 5th
Set calibration object contains 10mg/dL phenylalanine, 17 α of 100nmol/L-hydroxyprogesterone and 25mg/dL galactolipin, and the 6th set of calibration object contains
There are 15mg/dL phenylalanine, 17 α of 250nmol/L-hydroxyprogesterone and 50mg/dL galactolipin.
Detect the preferred embodiment of the kit of PKU, CAH and GAL simultaneously as LC-MS of the present invention, it is described
Extracting solution is any one in following (I)~(IV):
(I) mixture of methanol and acetonitrile；
(II) mixture of methanol, acetone and isopropanol；
(III) mixture of methanol, acetonitrile, acetone and isopropanol；
(IV) mixture of methanol, ethyl alcohol, acetonitrile, acetone and isopropanol.
Studies have shown that in said extracted liquid, methanol, ethyl alcohol, acetonitrile, acetone and isopropanol mixture extraction effect most
It is good.
Detect the preferred embodiment of the kit of PKU, CAH and GAL simultaneously as LC-MS of the present invention, it is described
Extracting solution is made of isometric methanol, ethyl alcohol, acetonitrile, acetone and isopropanol.Wherein, it is described in equal volume refer to methanol, ethyl alcohol,
The volume of acetonitrile, acetone and isopropanol is all the same.
Detect the preferred embodiment of the kit of PKU, CAH and GAL simultaneously as LC-MS of the present invention, it is described
Kit further includes quality-control product, and the quality-control product contains phenylalanine, 17 α-hydroxyprogesterone and galactolipin.
Detect the more preferable embodiment of the kit of PKU, CAH and GAL, institute simultaneously as LC-MS of the present invention
Stating quality-control product is the human red cell containing phenylalanine, 17 α-hydroxyprogesterone and galactolipin；The quality-control product is 2 sets, respectively
1st and the 2nd set of quality-control product, the 1st set of quality-control product contain 2.21mg/dL phenylalanine, 17 α of 20nmol/L-hydroxyprogesterone and 4mg/dL
Galactolipin, the 2nd set of quality-control product contain 5.68mg/dL phenylalanine, 17 α of 100nmol/L-hydroxyprogesterone and 20mg/dL galactolipin.
Detect the preferred embodiment of the kit of PKU, CAH and GAL simultaneously as LC-MS of the present invention, it is described
Kit further includes Isotopic Internal Standard product, and the Isotopic Internal Standard product contain the phenylalanine of isotope labelling, 17 α-hydroxyprogesterone
And galactolipin.It is highly preferred that the Isotopic Internal Standard product are the phenylalanine containing isotope labelling, 17 α-hydroxyprogesterone and gala
Sugared freeze-dried powder.
Detect the preferred embodiment of the kit of PKU, CAH and GAL simultaneously as LC-MS of the present invention, it is described
Kit further includes U-shaped 96 hole microwell plate, 96 hole microwell plate of V-type, silicagel pad and kit operational manual.
In addition, the present invention also provides mentioned reagent boxes for detecting the application in PKU, CAH and GAL simultaneously.
Compared with prior art, the invention has the benefit that phenylalanine currently used for clinically neonatal screening
Assay kit mainly uses fluorescence method analytic approach, fluorescence analysis half-life short, and not reproducible detection is highly prone to solution
The interference of the factors such as polarity, temperature, paramagnet, the pH value of solution, fluorescent quenching, scattering light, easily causes inspection in this way
Result false positive is surveyed, causes spirit and financial burden to clinician and patient；When 17 α hydroxyprogesterone assay kits mainly use
Between the competition law of resolved immuno fluorometric method be easy since competition law is there are poor specificity by kit methodology itself, operation
The interference of person and environmental factor causes the testing result interchangeability between laboratory poor, and the operating time is long, generally at least
Testing result could be gone out after needing 240 minutes, give tester and detected person's all bring inconveniences in this way；Newborn half
Lactose kit for screening current state food pharmaceuticals administration general bureau approval listing kit not yet, present invention rate at home
Kit is first developed using LC-MS, mass spectrography combines high separation capacity and mass spectrum part pair of the chromatographic fraction to substance
The ability of the structure of matter and quality analysis has the characteristics that highly selective, high specific, high sensitivity.Mass spectrum can be by phenylpropyl alcohol
Propylhomoserin, 17 α hydroxyprogesterones and galactolipin are applied to the examination of newborn infant diseases simultaneously, specifically, relatively existing immunofluorescence technique, is adopted
It is had the advantage that with kit of the invention with tandem mass spectrometry detection PKU, CAH and GAL
(1) the kit operating time of the present invention is short: the operating time of kit of the present invention is 40min, immunofluorescence technique inspection
The time for surveying PKU is 120min, and the time for detecting CAH is 190min, and the time for detecting GAL is 95min；
(2) kit of the present invention is easy to operate simple: the process for using of kit of the present invention is that punching → extracting solution → mentions
→ transfer → is taken to detect → result；The process of immuno-fluorescence assay PKU is punching → reagent adding → extraction → transfer → incubation
→ reagent adding → detection, the process of immuno-fluorescence assay CAH are that reagent adding → incubation → reagent adding → sample-adding → is incubated for → washes
Plate → reagent adding → detection, the process of immuno-fluorescence assay GAL, which is punching → reagent adding → drying, → with liquid reagent adding → to be added
Reagent → incubation → reagent adding → detection；
(3) present invention detection flux is high: the present invention once tests at least 3 kinds of markers of detection, immunofluorescence technique and examines every time
Survey only detects single marker；
(4) testing result of the present invention is reproducible: RSD≤10% that the present invention detects, and the RSD of immuno-fluorescence assay≤
15%；
(5) testing result accuracy of the present invention is good: detection accuracy of the present invention is high, detects test substance with tandem mass spectrometry
Mass-to-charge ratio, immuno-fluorescence assay detection accuracy is general, and immunofluorescence technique is more by antigen-antibody or fluorescence disturbing factor；
(6) testing cost of the present invention is lower；
(7) present invention detection sampling amount is few: sampling amount of the present invention is 50uL hereinafter, the sampling amount of immunofluorescence technique is
50uL。
To sum up, not only testing result is accurate, easy to operate, flux is high by the present invention, and only need to adopt one every time and bleed, and is not only
It is convenient that operator provides, and reduces pain to special blood sampling person, has a good application prospect.
Detailed description of the invention
Fig. 1 is the calibration object curve graph of present invention detection phenylalanine；
Fig. 2 is the calibration object curve graph of present invention detection 17 α-hydroxyprogesterone；
Fig. 3 is the calibration object curve graph of present invention detection galactolipin；
Fig. 4 is present invention detection phenylalanine, galactolipin and 17 α-hydroxyprogesterone object result figure.
Specific embodiment
Purposes, technical schemes and advantages in order to better illustrate the present invention, below in conjunction with the drawings and specific embodiments pair
The present invention is described further.
In following embodiments, used instrument and reagent are as follows:
(1) instrument: Guangzhou City Fenghua Biological Engineering Co., Ltd FH-6000MD triple quadrupole bar mass spectrometry system, color
Compose column: Phenomenex Kinere 2.6um XB-C18 (50 × 4.6mm) 100A, Guangzhou City Fenghua Biological Engineering Co., Ltd
FWZ-II constant temperature micro oscillator, 3.2mm newborn Filter Paper Dry Blood piece perforating plier or puncher, Jiangsu Sheng Lan instrument manufacturing are limited
Company KH-500DV type numerical control ultrasonic cleaner；
(2) reagent: methanol (HPLC, Fisher), ethyl alcohol (HPLC, Fisher), acetonitrile (HPLC, Fisher), acetone
(HPLC, Fisher), isopropanol (HPLC, Fisher), first (second) acid (LC-MS/MS, Fisher), standard items, Isotopic Internal Standard
Product (U.S. Cambridge Isotope internal standard laboratory)；
(3) consumptive material: 96 holes are U-shaped and V-type plate (NUNC), deionized water water-making machine (Mi Libo), LC-MS test routine
Consumptive material.
Embodiment 1
LC-MS of the present invention detects a kind of embodiment of the kit of PKU, CAH and GAL, the reagent of the present embodiment simultaneously
Box includes following reagent:
(a) calibration object, the calibration object are that the people containing phenylalanine, 17 α-hydroxyprogesterone and gala saccharide is blood red thin
Born of the same parents；The calibration object is 6 sets, the respectively the 1st, the 2nd, the 3rd, the 4th, the 5th and the 6th set of calibration object；1st set of calibration object contains
0.5mg/dL phenylalanine, 17 α of 0nmol/L-hydroxyprogesterone and 0mg/dL galactolipin, the 2nd set of calibration object contain 1.5mg/dL phenylpropyl alcohol
Propylhomoserin, 17 α of 10nmol/L-hydroxyprogesterone and 3mg/dL galactolipin, the 3rd set of calibration object contain 3mg/dL phenylalanine, 25nmol/L
17 α-hydroxyprogesterone and 8mg/dL galactolipin, the 4th set of calibration object contain 5mg/dL phenylalanine, 17 α of 50nmol/L-hydroxyprogesterone and
15mg/dL galactolipin, the 5th set of calibration object contain 10mg/dL phenylalanine, 17 α of 100nmol/L-hydroxyprogesterone and 25mg/dL half
Lactose, the 6th set of calibration object contain 15mg/dL phenylalanine, 17 α of 250nmol/L-hydroxyprogesterone and 50mg/dL galactolipin；
(b) extracting solution, the extracting solution are made of isometric methanol with acetonitrile；
(c) mobile phase, the mobile phase are made of mobile phase A and Mobile phase B, and the mobile phase A and Mobile phase B are by (I)
Water, (II) methanol and (III) formic acid form, and in the mobile phase A, the percent by volume of formic acid is 0.01%, the body of water and methanol
Product is than being 19:1；In the Mobile phase B, the percent by volume of formic acid is 0.01%, and the volume ratio of water and methanol is 1:99；
(d) quality-control product, the quality-control product are the human serum red blood cell containing phenylalanine, 17 α-hydroxyprogesterone and galactolipin；
The quality-control product be 2 sets, respectively the 1st and the 2nd set of quality-control product, the 1st set of quality-control product contain 2.21mg/dL phenylalanine,
17 α of 20nmol/L-hydroxyprogesterone and 4mg/dL galactolipin, the 2nd set of quality-control product contain 5.68mg/dL phenylalanine, 100nmol/L
17 α-hydroxyprogesterone and 20mg/dL galactolipin；
(e) Isotopic Internal Standard product, the Isotopic Internal Standard product are the phenylalanine containing isotope labelling, 17 α-hydroxyprogesterone
With galactolipin freeze-dried powder；
(f) 10 pieces of U-shaped 96 hole microwell plates, the U-shaped 96 hole microwell plate are dummy plate；
(g) 10 pieces of 96 hole microwell plates of V-type, the 96 hole microwell plate of V-type are dummy plate；
(h) silicagel pad；With
(i) 1 part of kit operational manual.
The application method of the kit of the present embodiment is (using tandem mass spectrometry):
(1) dissolution of phenylalanine, 17 α-hydroxyprogesterone, the quasi- product of galactolipin Isotopic Internal Standard: add into Isotopic Internal Standard product
1.0mL extracting solution, shakes up standing about 2 hours；
(2) extract the preparation of working solution: extracting working solution is to go dilution internal standard with extracting solution and obtain, and extracts working solution
Preparation method is as shown in table 1；
(3) calibration object and quality-control product are prepared: collecting the full yin of hepatitis type B virus HBsAg, anti-HCV, anti-TP, anti-HIV
Whole blood takes lower layer's blood cell spare by the isolated upper plasma of centrifugal treating and lower layer's blood cell, by blood cell physiological saline with
Blood cell ratio 1:1 mixing, washing 3 times, by phenylalanine, 17 α-hydroxyprogesterone, galactolipin calibration object and treated human red cell
It is hybridly prepared into and needs concentration, take 50uL to instil in No. 903 Filter Paper Dry Blood on pieces of Whatma, allow its natural diffuseness to dry, vacuum
It is stored in sealed film bag；Quality-control product is prepared similarly；
(4) it punches: calibration object, quality-control product and sample to be tested is punched, be added in clean U-shaped 96 hole microwell plate, and
100 μ L are added in every hole and extract working solution, cover U-shaped 96 hole microwell plate using silicagel pad, the amount of will volatilize is reduced to minimum；
(5) oscillation/extraction: being 30 DEG C (± 5 DEG C) in temperature, vibration frequency is oscillation incubation 30 under conditions of 750~900rpm
Minute, after having vibrated, silicagel pad is thrown off from the microwell plate of U-shaped 96 hole；
(6) shift: (V-type bottom is heat-resisting micro- for the 96 hole microwell plate of V-type of 75 μ L of every hole transfer extraction acquired solution to corresponding position
Orifice plate) in, microwell plate is covered using silicagel pad, the amount of will volatilize is reduced to minimum；
(7) it sample introduction and detects: the 96 hole microwell plate of V-type with silicagel pad is put into autosampler, it is soft to enable application
Part establishes sample list, selects correct internal standard concentration file and acquisition method, starts to detect in application software；Wherein, it examines
The chromatographic condition of survey is as shown in table 2, and the condition of gradient elution is as shown in table 3, and the Mass Spectrometry Conditions of detection are as shown in table 4；
(8) calculating of testing result: using the mark concentration of 6 calibration objects as abscissa (X), with the reality of 6 calibration objects
The ratio for detecting peak area and respective internal standard peak area is ordinate (Y), draws the recurrence side of standard curve and calculated curve
Journey can calculate the concentration value of phenylalanine in Filter Paper Dry Blood piece, 17 α-hydroxyprogesterone, galactolipin；Wherein, phenylalanine is detected
Calibration object curve as shown in Figure 1, detection 17 α-hydroxyprogesterone calibration object curve as shown in Fig. 2, detection galactolipin calibration object
Curve is as shown in figure 3, detection phenylalanine, 17 α-hydroxyprogesterone, galactolipin object result are as shown in Figure 4.
The preparation method of the extraction working solution of table 1
The chromatographic condition that table 2 detects
The condition of 3 gradient elution of table
The Mass Spectrometry Conditions that table 4 detects
Embodiment 2
LC-MS of the present invention detects a kind of embodiment of the kit of PKU, CAH and GAL, the reagent of the present embodiment simultaneously
Only extracting solution and mobile phase are different from kit described in embodiment 1 for box；In the present embodiment, extracting solution is by isometric methanol, third
Ketone and isopropanol composition, mobile phase are to be made of mobile phase A and Mobile phase B, the mobile phase A and Mobile phase B by (I) water,
(II) methanol and (III) acetic acid form, and in the mobile phase A, the percent by volume of acetic acid is 0.05%, the volume of water and methanol
Than for 9:1；In the Mobile phase B, the percent by volume of acetic acid is 0.05%, and the volume ratio of water and methanol is 0:100.
The application method of the kit of the present embodiment is the same as embodiment 1.
Embodiment 3
LC-MS of the present invention detects a kind of embodiment of the kit of PKU, CAH and GAL, the reagent of the present embodiment simultaneously
Only extracting solution and mobile phase are different from kit described in embodiment 1 for box；In the present embodiment, extracting solution is by isometric methanol, second
Nitrile, acetone and isopropanol composition, mobile phase are to be made of mobile phase A and Mobile phase B, and the mobile phase A and Mobile phase B are by (I)
Water, (II) methanol and (III) formic acid form, and in the mobile phase A, the percent by volume of formic acid is 0.03%, the body of water and methanol
Product is than being 14:1；In the Mobile phase B, the percent by volume of formic acid is 0.03%, and the volume ratio of water and methanol is 0.5:99.5.
The application method of the kit of the present embodiment is the same as embodiment 1.
Embodiment 4
LC-MS of the present invention detects a kind of embodiment of the kit of PKU, CAH and GAL, the reagent of the present embodiment simultaneously
Only extracting solution and mobile phase are different from kit described in embodiment 1 for box；In the present embodiment, extracting solution is by isometric methanol, second
Alcohol, acetonitrile, acetone and isopropanol composition, mobile phase are to be made of mobile phase A and Mobile phase B, the mobile phase A and Mobile phase B
It is made of (I) water, (II) acetonitrile and (III) formic acid, in the mobile phase A, the percent by volume of formic acid is 0.01%, water and second
The volume ratio of nitrile is 19:1；In the Mobile phase B, the percent by volume of formic acid is 0.01%, and the volume ratio of water and acetonitrile is 1:
99。
The application method of the kit of the present embodiment is the same as embodiment 1.
Embodiment 5
LC-MS of the present invention detects a kind of embodiment of the kit of PKU, CAH and GAL, the reagent of the present embodiment simultaneously
Only extracting solution and mobile phase are different from kit described in embodiment 1 for box；In the present embodiment, extracting solution is by isometric methanol, third
Ketone and isopropanol composition, mobile phase are to be made of mobile phase A and Mobile phase B, the mobile phase A and Mobile phase B by (I) water,
(II) acetonitrile and (III) acetic acid form, and in the mobile phase A, the percent by volume of acetic acid is 0.05%, the volume of water and acetonitrile
Than for 9:1；In the Mobile phase B, the percent by volume of acetic acid is 0.05%, and the volume ratio of water and acetonitrile is 0:100.
The application method of the kit of the present embodiment is the same as embodiment 1.
Embodiment 6
LC-MS of the present invention detects a kind of embodiment of the kit of PKU, CAH and GAL, the reagent of the present embodiment simultaneously
Only extracting solution and mobile phase are different from kit described in embodiment 1 for box；In the present embodiment, extracting solution is by isometric methanol, second
Nitrile, acetone and isopropanol composition, mobile phase are to be made of mobile phase A and Mobile phase B, and the mobile phase A and Mobile phase B are by (I)
Water, (II) acetonitrile and (III) formic acid form, and in the mobile phase A, the percent by volume of formic acid is 0.03%, the body of water and acetonitrile
Product is than being 14:1；In the Mobile phase B, the percent by volume of formic acid is 0.03%, and the volume ratio of water and acetonitrile is 0.5:99.5.
The application method of the kit of the present embodiment is the same as embodiment 1.
In embodiment 2~6, the calibration object curve of phenylalanine is detected as shown in Figure 1, detecting the calibration object of 17 α-hydroxyprogesterone
Curve is as shown in Fig. 2, the calibration object curve of detection galactolipin is as shown in Figure 3.
Effect example 1
This effect example has been investigated in kit of the invention, influence of the extracting solution to testing result.This effect example is not using
Same extracting solution (kit other components are with embodiment 1), according to the application method of kit described in embodiment 1, in sample
Phenylalanine, 17 α-hydroxyprogesterone, galactolipin is by being added acetonitrile, acetone, ethyl alcohol, isopropanol, methanol, isometric methanol and second
Mixture of nitriles, isometric methanol, acetone and isopropanol mixture, isometric methanol, acetonitrile, acetone and isopropanol mixture, etc.
Volumes methanol, ethyl alcohol, acetonitrile, the acetone extracting solution different with isopropanol mixture are detected, testing result such as table 5,6,7
It is shown.
Table 5: two kinds of testing result comparisons of phenylalanine
Table 6:17 α-two kinds of hydroxyprogesterone testing result comparison
Table 7: two kinds of testing result comparisons of galactolipin
5,6,7 experimental data of table shows extracting solution ingredient with methanol+ethyl alcohol+acetonitrile+acetone+isopropanol mixing extraction efficiency
Preferably.
Effect example 2
This effect example compares the method for the present invention and immuno-fluorescence assay PKU, CAH and GAL using standard samples recovery
Accuracy and repeatability.Wherein, specific step is as follows for standard samples recovery: taking sample to be tested, every part of sample is divided into 8 parts, 2 parts of use
It is detected in PKU, CAH and GAL detection kit (Liquid Chromatography-Tandem Mass Spectrometry of the present invention), standard items work is not added in a copy of it
For control, in addition the standard items of known concentration are added in portion；Other 6 parts of difference phenylalanine assay kit (fluorescence analysis
Method), 17 α of newborn-hydroxyprogesterone assay kit (Timed resolved fluoroimmunoassay), the total galactose measurment reagent of newborn
Box (fluorescence analysis) detection is equally that standard items are not added as control in a copy of it, and in addition the mark of known concentration is added in portion
Quasi- product compare the testing result that standard items are added and standard items are not added, and the concentration since standard items are added is known, Ke Yizhun
Really calculate recovery of standard addition.
Testing result is as shown in table 8.
Table 8 rate of recovery, two kinds of Comparative results
By table 8 as it can be seen that using kit of the present invention, PKU, CAH and GAL relative immunity fluorescence method are detected using LC-MS
It is with higher accuracy and preferably repeated.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention
The limitation of range is protected, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should
Understand, it can be with modification or equivalent replacement of the technical solution of the present invention are made, without departing from the essence of technical solution of the present invention
And range.
Claims (10)
1. the kit that a kind of LC-MS detects PKU, CAH and GAL simultaneously, which is characterized in that including following reagent:
(a) calibration object, the calibration object contain phenylalanine, 17 α-hydroxyprogesterone and galactolipin；
(b) extracting solution, the extracting solution are at least one of methanol, ethyl alcohol, isopropanol, acetone, acetonitrile；With
(c) mobile phase, the mobile phase are made of mobile phase A and Mobile phase B, the mobile phase A and Mobile phase B by (I) water,
(II) methanol and (III) formic acid or acetic acid form, and in the mobile phase A, the percent by volume of formic acid or acetic acid is 0.01%-
0.05%, the volume ratio of water and methanol is 9:1~19:1；In the Mobile phase B, the percent by volume of formic acid or acetic acid is
The volume ratio of 0.01%-0.05%, water and methanol is 0:100~1:99；Alternatively,
The mobile phase A and Mobile phase B are made of (I) water, (IV) acetonitrile and (III) formic acid or acetic acid, in the mobile phase A, first
The percent by volume of acid or acetic acid is 0.01%-0.05%, and the volume ratio of water and acetonitrile is 9:1~19:1；The Mobile phase B
In, the percent by volume of formic acid or acetic acid is 0.01%-0.05%, and the volume ratio of water and acetonitrile is 0:100~1:99.
2. the kit that LC-MS as described in claim 1 detects PKU, CAH and GAL simultaneously, which is characterized in that the examination
Agent box is for detecting phenylalanine in Filter Paper Dry Blood piece, 17 α-hydroxyprogesterone and galactolipin.
3. the kit that LC-MS as described in claim 1 detects PKU, CAH and GAL simultaneously, which is characterized in that the school
Quasi- product are the human red cell containing phenylalanine, 17 α-hydroxyprogesterone and galactolipin.
4. the kit that LC-MS as described in claim 1 detects PKU, CAH and GAL simultaneously, which is characterized in that the school
Quasi- product are 6 sets, the respectively the 1st, the 2nd, the 3rd, the 4th, the 5th and the 6th set of calibration object；1st set of calibration object contains 0.5mg/dL phenylpropyl alcohol
Propylhomoserin and be free of 17 α-hydroxyprogesterone and galactolipin, it is pregnant that the 2nd set of calibration object contains 1.5mg/dL phenylalanine, 10nmol/L17 α-hydroxyl
Ketone and 3mg/dL galactolipin, the 3rd set of calibration object contain 3mg/dL phenylalanine, 25nmol/L17 α-hydroxyprogesterone and 8mg/dL gala
Sugar, the 4th set of calibration object contain 5mg/dL phenylalanine, 50nmol/L17 α-hydroxyprogesterone and 15mg/dL galactolipin, the 5th set of calibration
Product contain 10mg/dL phenylalanine, 100nmol/L17 α-hydroxyprogesterone and 25mg/dL galactolipin, and the 6th set of calibration object contains 15mg/
DL phenylalanine, 250nmol/L17 α-hydroxyprogesterone and 50mg/dL galactolipin.
5. the kit that LC-MS as described in claim 1 detects PKU, CAH and GAL simultaneously, which is characterized in that described to mention
Taking liquid is any one in following (I)~(IV):
(I) mixture of methanol and acetonitrile；
(II) mixture of methanol, acetone and isopropanol；
(III) mixture of methanol, acetonitrile, acetone and isopropanol；
(IV) mixture of methanol, ethyl alcohol, acetonitrile, acetone and isopropanol.
6. the kit that LC-MS as claimed in claim 5 detects PKU, CAH and GAL simultaneously, which is characterized in that described to mention
Liquid is taken to be made of isometric methanol, ethyl alcohol, acetonitrile, acetone and isopropanol.
7. LC-MS as described in any one of claims 1 to 6 detects the kit of PKU, CAH and GAL simultaneously, feature exists
In further including quality-control product, the quality-control product contains phenylalanine, 17 α-hydroxyprogesterone and galactolipin；Preferably, the quality-control product is
Human red cell containing phenylalanine, 17 α-hydroxyprogesterone and galactolipin；The quality-control product is 2 sets, respectively the 1st and the 2nd set
Quality-control product, the 1st set of quality-control product contain 2.21mg/dL phenylalanine, 20nmol/L17 α-hydroxyprogesterone and 4mg/dL galactolipin, and the 2nd
Set quality-control product contains 5.68mg/dL phenylalanine, 100nmol/L17 α-hydroxyprogesterone and 20mg/dL galactolipin.
8. LC-MS as described in any one of claims 1 to 6 detects the kit of PKU, CAH and GAL simultaneously, feature exists
In further including Isotopic Internal Standard product, the Isotopic Internal Standard product contain the phenylalanine of isotope labelling, 17 α-hydroxyprogesterone and half
Lactose.
9. the kit that LC-MS as described in claim 1 detects PKU, CAH and GAL simultaneously, which is characterized in that further include
U-shaped 96 hole microwell plate, 96 hole microwell plate of V-type, silicagel pad and kit operational manual.
10. kit for detecting the application in PKU, CAH and GAL simultaneously as described in any one of claim 1~9.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910327792.6A CN110018255B (en) | 2019-04-23 | 2019-04-23 | Kit for simultaneously detecting PKU, CAH and GAL by liquid chromatography-mass spectrometry and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910327792.6A CN110018255B (en) | 2019-04-23 | 2019-04-23 | Kit for simultaneously detecting PKU, CAH and GAL by liquid chromatography-mass spectrometry and application thereof |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1461347A (en) * | 2000-08-28 | 2003-12-10 | 札幌免疫诊断研究所 | Method and apparatus for examining diseases with inborn errors of metabolism |
| CN104316692A (en) * | 2014-10-24 | 2015-01-28 | 广州市丰华生物工程有限公司 | Newborn total galactose detection kit, as well as application method and preparation method thereof |
| CN106841427A (en) * | 2016-12-30 | 2017-06-13 | 广州市达瑞生物技术股份有限公司 | A kind of tandem mass spectrum kit of detection PKU and CAH is prepared and its applied |
| CN109030671A (en) * | 2018-07-04 | 2018-12-18 | 易达精准（杭州）科技有限公司 | The tandem mass spectrum kit and detection method of PKU, CAH and G-6PD deficiency disease are detected simultaneously |
-
2019
- 2019-04-23 CN CN201910327792.6A patent/CN110018255B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1461347A (en) * | 2000-08-28 | 2003-12-10 | 札幌免疫诊断研究所 | Method and apparatus for examining diseases with inborn errors of metabolism |
| CN104316692A (en) * | 2014-10-24 | 2015-01-28 | 广州市丰华生物工程有限公司 | Newborn total galactose detection kit, as well as application method and preparation method thereof |
| CN106841427A (en) * | 2016-12-30 | 2017-06-13 | 广州市达瑞生物技术股份有限公司 | A kind of tandem mass spectrum kit of detection PKU and CAH is prepared and its applied |
| CN109030671A (en) * | 2018-07-04 | 2018-12-18 | 易达精准（杭州）科技有限公司 | The tandem mass spectrum kit and detection method of PKU, CAH and G-6PD deficiency disease are detected simultaneously |
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| NAM-HEE KIM ET AL.: "Simultaneous diagnostic method for phenylketonuria and galactosemia from dried blood spots using high-performance liquid chromatography-pulsed amperometric detection", 《JOURNAL OF CHROMATOGRAPHY B》 * |
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