CN103278584B - Detection kit based on tandem mass spectrum non-derivatization method - Google Patents
Detection kit based on tandem mass spectrum non-derivatization method Download PDFInfo
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Abstract
The invention relates to the technical field of chemical analysis and detection, and in particular relates to a detection kit based on a tandem mass spectrum non-derivatization method. The detection kit comprises an isotope labeled amino acid standard substance mixture, an isotope labeled acyl carnitine standard substance mixture, an extract liquor and a flow phase. The detection kit based on the tandem mass spectrum non-derivatization method is characterized in that the extract liquor consists of methyl alcohol, water and saturated monocarboxylic acid, wherein the volume ratio of the methyl alcohol to the water to the saturated monocarboxylic acid is 1000: (1-50): (0.01-2). The detection kit based on the tandem mass spectrum non-derivatization method provided by the invention is high in flexibility and accuracy rate, is simple to operate, and has important significance for improving the diagnosis level of metabolic diseases, lowering the burden of society and families and improving the quality of population.
Description
Technical field
The present invention relates to chemical analysis detection technique field, be specifically related to a kind of tandem mass spectrum non-derivative method detection kit.
Background technology
In blood, several amino acids and fatty acyl carnitine are the mark products of human amino acid, organic acid and fatty acid metabolism; the change of aminogram and fatty acyl carnitine spectrum can occur in the blood of metabolic disease, and liquid phase tandem mass spectrum coupling technique (LC-MS/MS) has been applied to the diagnosis of the examination of neonate's Inherited Metabolic Disorders and clinical metabolic disease.
Once second order ms detection is carried out after tandem mass spectrum (MS/MS) i.e. two mass spectrometer series connection, utilize the tandem mass spectrum technology of hypersensitivity, high specific, high selectivity and quick test, tens kinds of Methanogenesis can be carried out to 1 sample in 2 ~ 3min, by the analysis to these products, examination and diagnosis can be carried out to about 40 kinds inherited metabolic diseases (comprising disorder of amino acid metabolism, metabolism of organic acids disorder and fatty acid metabolism disorder disease).
Because amino acid classes in blood is a lot, there is acidity, chemical property that neutrality is different with alkalescence.The coupling of liquid phase tandem mass spectrum detects several amino acids simultaneously and the many need of multiple fatty acyl carnitine carry out derivatization pre-treatment.Derivatization complex operation, takes time and effort, and has pollution to environment, and derivative product can not direct represented amino acid itself, and same problem also occurs in the detection of carnitine and multiple fatty acyl carnitine.
Summary of the invention
Technical matters to be solved by this invention is to provide a kind of tandem mass spectrum non-derivative method detection kit, to solve the defect in existing LC-MS/MS detection.
For the above-mentioned technical matters solved; the invention discloses a kind of tandem mass spectrum non-derivative method detection kit; it comprises isotope labeling amino acid standard items potpourri, isotope labeling fatty acyl carnitine standard items potpourri, extract, mobile phase; it is characterized in that described extract is made up of methyl alcohol, water, saturated monocarboxylic acid, methyl alcohol: water: saturated monocarboxylic acid volume ratio is 1000: 1 ~ 50: 0.01 ~ 2.
In certain embodiments, described saturated monocarboxylic acid is formic acid, acetic acid, propionic acid or butyric acid, is wherein preferably formic acid.
In certain embodiments, described mobile phase is 80% methyl alcohol, 75% ~ 80% acetonitrile or the methyl alcohol containing 1% formic acid, wherein be preferably methyl alcohol, water, saturated monocarboxylic acid mixed liquor, wherein methyl alcohol: water: saturated monocarboxylic acid volume ratio is 1000: 1 ~ 50: 0.01 ~ 2.
In certain embodiments, described tandem mass spectrum non-derivative method detection kit also comprises 96 hole check-out consoles, viscosity extraction plate pad pasting and check-out console aluminium foil at the bottom of the heat-resisting 96 hole extraction plate in the U-shaped end, V-type.
In certain embodiments, described tandem mass spectrum non-derivative method detection kit also comprises amino acid and fatty acyl carnitine neonatal screening quality-control product.
On the other hand, the invention also discloses the using method of described tandem mass spectrum non-derivative method detection kit, comprise the following steps:
1. mark liquid preparation in
A. use the extract of 1.0ml to add in amino acid in target freeze-dried powder bottle, cover tightly bottle cap, concussion, makes powder wherein dissolve completely, dissolves at need, can put in 37 DEG C of baking boxs after 3 minutes, continues concussion, is dissolved to thoroughly.Preferably configuration in 1 day before use.
B. use the extract of 1.0ml to add in fatty acyl carnitine in target freeze-dried powder bottle, cover tightly bottle cap, concussion, makes powder wherein dissolve completely.
C. in, mark liquid can+2 ~+8 DEG C of Refrigerator stores 30 days.
2. working fluid preparation
A. interior mark liquid is put room temperature 20 minutes
B. with every hole 100 μ l extract consumption, calculate and measured sample and the extract total amount needed for charge the same day.
C. add in extract, amino acid to mark in liquid and fatty acyl carnitine with volume ratio 108:1:1 and mark liquid.Such as survey 100 samples, join 11ml containing interior target extraction working fluid.Get 10.8ml extract, add mark liquid in the amino acid interior mark liquid of 0.1ml and the fatty acyl carnitine of 0.1ml respectively, be namely made into working fluid.
D. working fluid is stablized for 24 hours.
3. sample
Quality Control blood sheet, sample blood sheet are sorted at 96 orifice plates.By 3.2mm card punch order, blood sheet is squeezed in the heat-resisting 96 hole extraction plate in the U-shaped end.
4. specimen extraction
100 μ l are added in the reacting hole of 96 orifice plates containing interior target extraction working fluid respectively, extraction sample.With viscosity pad pasting sealing orifice plate.45 DEG C hatch in oscillator, 700rpm shakes 45 minutes.
5. machine testing on sample
Throw off diaphragm seal.Shift out 80 μ l supernatants with the every hole of pipettor, order inserts clean 96 orifice plates.With foil sealing 96 orifice plate.Put into mass spectrum automatic sampler pallet, for Mass Spectrometer Method, sample introduction 20ul, sample introduction speed: 0 ~ 0.2min are 0.12ml/min, 0.2 ~ 1.2min be 0.015ml/min, 1.2 ~ 1.5min is 0.5ml/min.Every this 1.7min of increment.Adopt tandem mass spectrometer electrospray positive ion mode.Ion source temperature 120 DEG C, dissociation temperature 350 DEG C, spray voltage 3.5KV, nitrogen 650l/h, taper hole gas 50l/h.Multiple-reaction monitoring (MRM) pattern collects data.Each ion pair 0.05ms is cycle detection ion pair successively, the detection signal of every part of sample collection 0.3-1.3min time period, and superposed signal intensity is for quantitative.Sample can preserve about 12 hours for Mass Spectrometer Method.
On the other hand, the invention also discloses described tandem mass spectrum non-derivative method detection kit in the purposes detecting amino acid and fatty acyl carnitine in sample.
Described sample is body fluid, and described body fluid is one of in blood, blood plasma, urine or saliva.
Tandem mass spectrum non-derivative method of the present invention detection kit sensitivity and accuracy rate high, simple to operate, to improving the diagnostic level of metabolic disease, improving infant metabolic disease screening efficiency, reducing burden on society, improve the health of the people significant.
Accompanying drawing explanation
Fig. 1 .13 seed amino acid and isotopic standard ion current stacking diagram thereof.
Fig. 2 .13 seed amino acid and isotopic standard total ion current figure thereof.
Fig. 3. carnitine and short chain and medium chain acyl carnitine and standard total ion current figure thereof.
Fig. 4. the total ion current figure of long acyl carnitine and standard items thereof.
Fig. 5 .13 seed amino acid and isotopic standard multiple-reaction monitoring mass spectrogram thereof.
Fig. 6. carnitine and in, short chain acyl carnitine and isotopic standard multiple-reaction monitoring mass spectrogram thereof.
Fig. 7. long acyl carnitine and isotopic standard multiple-reaction monitoring mass spectrogram thereof.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.These embodiments are only not used in for illustration of the present invention and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, the usually conveniently conditioned disjunction condition of advising according to manufacturer.Unless otherwise defined, all specialties used in literary composition and scientific words and one skilled in the art the meaning be familiar with identical.In addition, any method similar or impartial to described content and material all can be applicable in the inventive method.The use that better implementation method described in literary composition and material only present a demonstration.
Instrument, material and reagent
Tandem mass spectrometer (Waters Quatto micro API, Manchester, UK), efficient liquid phase (Waters1525U, Milfort, USA) auxiliary sample introduction, automatic sample handling system (Waters2777Samplemanager, USA), hatch oscillator (PerkinElmer, TriNEST, Wallac OY, TURKU, Finland).
U.S. Cambridge Isotope Laboratories neonatal screening amino acid isotopic standard suit A; Carnitine and multiple fatty acyl carnitine isotopic standard suit B (Andover, MA, USA); U.S. CDC tandem mass spectrum detects several amino acids and fatty acyl carnitine neonatal screening quality-control product; The pure level methyl alcohol of liquid chromatography mass spectrometric is Shanghai ANPEL Scientific Instruments Corporation CNW; The pure level water of liquid chromatography mass spectrometric is PROLABO (Leuven.EC) product; Formic acid 98-100% is MERCK product (Damstadt, Germany).Pidolidone, L-Aspartic acid, glycocoll, L-arginine, L-PROLINE, L-Orn, METHIONINE, L-Leu, ALANINE, Valine, L-Phe, TYR is Chemical Reagent Co., Ltd., Sinopharm Group's product (chromatography assay is qualified); Cit is SIGMA-ALDRICH product.S & S903 filter paper (Schleicher & Schuell BioScience GmbH, New Hampshire, USA).Transparent 96 orifice plates of Nunc polystyrene PS and Nunc acrylic acid 96 orifice plate, polyester sealant pad pasting aluminium foil Nunc(Thermoscientific).
The using method of kit, comprises the following steps:
1. mark liquid preparation in
A. use the extract of 1.0ml to add in amino acid in target freeze-dried powder bottle, cover tightly bottle cap, concussion, makes powder wherein dissolve completely, dissolves at need, can put in 37 DEG C of baking boxs after 3 minutes, continues concussion, is dissolved to thoroughly.Preferably configuration in 1 day before use.
B. use the extract of 1.0ml to add in fatty acyl carnitine in target freeze-dried powder bottle, cover tightly bottle cap, concussion, makes powder wherein dissolve completely.
C. in, mark liquid can+2 ~+8 DEG C of Refrigerator stores 30 days.
2. working fluid preparation
A. interior mark liquid is put room temperature 20 minutes
B. with every hole 100 μ l extract consumption, calculate and measured sample and the extract total amount needed for charge the same day.
C. add in extract, amino acid to mark in liquid and fatty acyl carnitine with volume ratio 108:1:1 and mark liquid.Such as survey 100 samples, join 11ml containing interior target extraction working fluid.Get 10.8ml extract, add mark liquid in the amino acid interior mark liquid of 0.1ml and the fatty acyl carnitine of 0.1ml respectively, be namely made into working fluid.
D. working fluid is stablized for 24 hours.
3. sample
Quality Control blood sheet, sample blood sheet are sorted at 96 orifice plates.By 3.2mm card punch order, blood sheet is squeezed in the heat-resisting 96 hole extraction plate in the U-shaped end.
4. specimen extraction
100 μ l are added in the reacting hole of 96 orifice plates containing interior target extraction working fluid respectively, extraction sample.With viscosity pad pasting sealing orifice plate.45 DEG C hatch in oscillator, 700rpm shakes 45 minutes.
5. machine testing on sample
Throw off diaphragm seal.Shift out 80 μ l supernatants with the every hole of pipettor, order inserts clean 96 orifice plates.With foil sealing 96 orifice plate.Put into mass spectrum automatic sampler pallet, for Mass Spectrometer Method, sample introduction 20ul, sample introduction speed: 0 ~ 0.2min are 0.12ml/min, 0.2 ~ 1.2min be 0.015ml/min, 1.2 ~ 1.5min is 0.5ml/min.Every this 1.7min of increment.Adopt tandem mass spectrometer electrospray positive ion mode.Ion source temperature 120 DEG C, dissociation temperature 350 DEG C, spray voltage 3.5KV, nitrogen 650l/h, taper hole gas 50l/h.Multiple-reaction monitoring (MRM) pattern collects data.Each ion pair 0.05ms is cycle detection ion pair successively, the detection signal of every part of sample collection 0.3-1.3min time period, and superposed signal intensity is for quantitative.Sample can preserve about 12 hours for Mass Spectrometer Method.
Tandem mass spectrum detects the multiple-reaction monitoring condition of several amino acids and fatty acyl carnitine in table 1
Table 1
Table 1(continues)
The determination of embodiment 1, extract formula
Get same Quality Control blood sheet, adopt the extract of formula described in table 2 respectively, detect the content of amino acid, carnitine and fatty acyl carnitine according to above-mentioned tandem mass spectrum non-derivative method, mobile phase is methyl alcohol: water: formic acid volume ratio is 1000: 10: 0.5.
Table 2(volume ratio)
| Extract | Methyl alcohol | Water | Formic acid |
| Formula 1 | 1000 | 5 | 2 |
| Formula 2 | 1000 | 50 | 1 |
| Formula 3 | 1000 | 1 | 0.5 |
| Formula 4 | 1000 | 10 | 0.01 |
| Formula 5 | 1000 | 100 | - |
| Formula 6 | 1000 | 100 | 5 |
Result: extract is methyl alcohol: water: formic acid volume ratio is 1000: 1 ~ 50: 0.01 ~ 2 can obtain desirable effect (table 3,4); haemolysis, albumen are separated out suitably (Fig. 8); the recovery and accuracy rate can reach U.S. CDC Quality Control requirement; exceed this formula range as filled a prescription 5,6; haemolysis kicks the beam or overweight, causes partial amino-acid or the fatty acyl carnitine recovery reduce or raise.
Table 3
| ALA | ARG | CIT | GLY | LEU | MET | |
| CDC Quality Control gathers 95% fiducial limit | 604-1078 | 213-369 | 727-960 | 283-536 | ||
| Formula 1 | 1120.74 | 139.26 | 266.93 | 500.68 | 969.49 | 361.43 |
| Formula 2 | 917.16 | 113.81 | 222.83 | 505.49 | 777.45 | 290.8 |
| Formula 3 | 715.4 | 170.73 | 240.77 | 472.27 | 618.26 | 233.83 |
| Formula 4 | 972.19 | 102.11 | 214.74 | 433.16 | 759.2 | 302.74 |
| Formula 5 | 539.52 | 128.54 | 193.62 | 406.94 | 494.75 | 184.91 |
| Formula 6 | 4208.21 | 2692.53 | 1933.21 | 2934.84 | 6622.05 | 1913.09 |
| ORN | PHE | PRO | TYR | VAL | ||
| CDC Quality Control gathers 95% fiducial limit | 390-648 | 400-734 | 682-932 | |||
| Formula 1 | 276.41 | 756.07 | 230.65 | 823.72 | 1229.41 | |
| Formula 2 | 240.84 | 508.18 | 174.97 | 562.81 | 771.81 | |
| Formula 3 | 181.22 | 333.04 | 195.23 | 503.34 | 810.51 | |
| Formula 4 | 211.05 | 489.34 | 200.19 | 504.65 | 942.75 | |
| Formula 5 | 153.33 | 257.4 | 140.85 | 417.31 | 605.45 | |
| Formula 6 | 2141.83 | 2931.64 | 1023.03 | 5240.11 | 8181.45 |
Table 4
| C0 | C2 | C3 | C4 | C3DC_C4OH | |
| CDC Quality Control gathers 95% fiducial limit | 39.2-57.8 | 27.3-39.6 | 9.6-17.1 | 3.1-4.7 | 4.4-7.4 |
| Formula 1 | 62.07 | 40.13 | 12.44 | 4.35 | 6 |
| Formula 2 | 45.66 | 30.46 | 10.22 | 3.21 | 5.41 |
| Formula 3 | 50.93 | 36.46 | 9.59 | 3.53 | 2.87 |
| Formula 4 | 37.31 | 24.27 | 8.18 | 2.72 | 2.8 |
| Formula 5 | 45.26 | 32.98 | 9.11 | 3.06 | 2.96 |
| Formula 6 | 27.51 | 21.02 | 5.25 | 2.08 | 1.85 |
| C5 | C6 | C8 | C10 | C5DC_C6OH | |
| CDC Quality Control gathers 95% fiducial limit | 1.7-3.1 | 1.5-2.4 | 1.8-2.9 | 1.8-3.0 | 1.2-2.0 |
| Formula 1 | 2.85 | 2.37 | 2.9 | 2.7 | 1.99 |
| Formula 2 | 2.24 | 1.94 | 2.22 | 2.06 | 1.79 |
| Formula 3 | 2.69 | 2.18 | 2.39 | 2.04 | 2.1 |
| Formula 4 | 1.79 | 1.59 | 1.77 | 1.59 | 1.31 |
| Formula 5 | 2.52 | 1.86 | 2.19 | 1.69 | 2.32 |
| Formula 6 | 1.81 | 1.14 | 1.52 | 1.1 | 1.61 |
| C12 | C14 | C14:1 | C16 | C16-OH | |
| CDC Quality Control gathers 95% fiducial limit | 1.4-2.5 | 2.0-3.6 | 7.1-11.5 | 0.6-1.9 |
| Formula 1 | 2.05 | 2.57 | 0.02 | 9.49 | 1.6 |
| Formula 2 | 2.02 | 2.34 | 0.04 | 9.19 | 0.55 |
| Formula 3 | 1.55 | 1.98 | 0.02 | 7.57 | 1.18 |
| Formula 4 | 1.8 | 2.05 | 0.02 | 8.32 | 0.51 |
| Formula 5 | 1.35 | 1.83 | 0.24 | 6.1 | 1.67 |
| Formula 6 | 2.05 | 2.57 | 0.02 | 9.49 | 1.6 |
| C16:1 | C18 | C18:1 | |||
| CDC Quality Control gathers 95% fiducial limit | 3.2-5.7 | ||||
| Formula 1 | 0.05 | 5.79 | 1.17 | ||
| Formula 2 | 0.04 | 4.28 | 0.87 | ||
| Formula 3 | 0.03 | 5.5 | 0.69 | ||
| Formula 4 | 0.03 | 3.29 | 0.66 | ||
| Formula 5 | 0.02 | 4.89 | 0.73 | ||
| Formula 6 | 0.52 | 4.69 | 1.9 |
The determination of embodiment 2, mobile phase formula
Get same Quality Control blood sheet, detect amino acid content according to above-mentioned tandem mass spectrum non-derivative method, except mobile phase difference, adopt the mobile phase of formula described in table 5 respectively, extract is methyl alcohol: water: formic acid volume ratio is 1000: 10: 1.Meanwhile, PerkinElmer(NeoBase is adopted) company's non-derivative method kit detects same Quality Control blood sheet, obtains 95% fiducial limit (PE kit 95% fiducial limit) of each object composition
Table 5(volume ratio)
Result: the ionization of the scale effect determinand of acid and water in mobile phase.Mobile phase is methyl alcohol: water: formic acid (1000:1 ~ 50:0.01 ~ 2) can obtain desirable Detection results; the recovery relative equilibrium of each seed amino acid and fatty acyl carnitine can reach U.S. CDC Quality Control requirement; go beyond the scope; signal intensity step-down; the recovery of partial amino-acid and fatty acyl carnitine can lower or raise, and accuracy declines (table 6).
Table 6
Embodiment 3, a kind of tandem mass spectrum non-derivative method detection kit
3.1 1 kinds of tandem mass spectrum non-derivative method detection kit comprise:
1 bottle, isotope labeling amino acid reference substance potpourri;
1 bottle, isotope labeling acetylcarnitine reference substance potpourri;
Extract (methyl alcohol: water: formic acid volume ratio is 1000: 1: 0.1) 1 bottle (150ml);
Mobile phase (methyl alcohol: water: formic acid volume ratio is 1000: 1: 0.1) 1 bottle (400ml);
The heat-resisting 96 hole extraction plate 10 pieces in the U-shaped end;
96 hole check-out console 10 pieces at the bottom of V-type;
Viscosity extraction plate pad pasting 20;
20, check-out console aluminium foil.
3.2 1 kinds of tandem mass spectrum non-derivative method detection kit comprise:
1 bottle, isotope labeling amino acid reference substance potpourri;
1 bottle, isotope labeling acetylcarnitine reference substance potpourri;
Extract (methyl alcohol: water: formic acid volume ratio is 1000: 50: 2) 1 bottle (150ml);
Mobile phase (80% acetonitrile) 1 bottle (400ml);
The heat-resisting 96 hole extraction plate 10 pieces in the U-shaped end;
96 hole check-out console 10 pieces at the bottom of V-type;
Viscosity extraction plate pad pasting 20;
20, check-out console aluminium foil;
Amino acid neonatal screening Quality Control blood sheet 10;
Fatty acyl carnitine neonatal screening quality-control product 10.
3.3 1 kinds of tandem mass spectrum non-derivative method detection kit comprise:
1 bottle, isotope labeling amino acid reference substance potpourri;
1 bottle, isotope labeling acetylcarnitine reference substance potpourri;
Extract (methyl alcohol: water: formic acid volume ratio is 1000: 10: 1) 1 bottle (150ml);
Mobile phase (80% acetonitrile) 1 bottle (400ml);
The heat-resisting 96 hole extraction plate 10 pieces in the U-shaped end;
96 hole check-out console 10 pieces at the bottom of V-type;
Viscosity extraction plate pad pasting 20;
20, check-out console aluminium foil.
3.4 1 kinds of tandem mass spectrum non-derivative method detection kit comprise:
1 bottle, isotope labeling amino acid reference substance potpourri;
1 bottle, isotope labeling acetylcarnitine reference substance potpourri;
Extract (methyl alcohol: water: acetic acid volume ratio is 1000: 1: 0.01) 1 bottle (150ml);
Mobile phase (methyl alcohol: water: formic acid volume ratio is 1000: 10: 2) 1 bottle (400ml);
The heat-resisting 96 hole extraction plate 10 pieces in the U-shaped end;
96 hole check-out console 10 pieces at the bottom of V-type;
Viscosity extraction plate pad pasting 20;
20, check-out console aluminium foil.
Embodiment 4, kit evaluation experimental
4.1 linear evaluation experiments
By detecting the blood sheet made of blood that adds the various determinand of gradient concentration, to carry out to kit prepared by embodiment 3 evaluations linear, and discovery linear concentration scope contains clinical reference value, comprises the base concentration of blood and the intercepting value of medical diagnosis on disease.Owing to making the limitation of concentration gradient sample, the range of linearity is not ultimate value, just will contain blood preparation basic value and cutting in span of various disease is verified.
The each seed amino acid of table 7 and fatty acyl carnitine linear quantitation range
4.2 method susceptibility
Diluted by sample proportion, the minimum measured value of each index is much smaller than basic value in blood.(table 7)
4.3 repeatability and degree of accuracy
By reclaiming the detection of sample to U.S. CDC Quality Control sample and partial amino-acid self-control, repeatability and accuracy reach the requirement that examination detects.See the following form 8,9
Table 8 blood basis, low concentration, middle concentration and high-purity amino acid criticize interior error
| Sample | ALA | CIT | LEU | MET | PHE | TYR | VAL |
| Base concentration average | 427.90 | 33.71 | 232.69 | 23.63 | 77.01 | 56.30 | 156.91 |
| Error in batch | 0.12 | 0.15 | 0.06 | 0.06 | 0.03 | 0.05 | 0.21 |
| Low concentration average | 620.89 | 88.64 | 439.82 | 104.39 | 207.06 | 253.19 | 328.27 |
| Error in batch | 0.13 | 0.08 | 0.08 | 0.10 | 0.08 | 0.10 | 0.14 |
| Middle concentration average | 734.10 | 189.30 | 684.77 | 235.49 | 393.83 | 413.34 | 540.39 |
| Error in batch | 0.10 | 0.06 | 0.04 | 0.03 | 0.02 | 0.01 | 0.09 |
| High concentration average | 912.51 | 318.79 | 995.23 | 437.83 | 580.80 | 570.84 | 830.71 |
| Error in batch | 0.07 | 0.04 | 0.03 | 0.03 | 0.02 | 0.02 | 0.07 |
| Sample | ARG | ORN | GLY | PRO | ASP | GLU | |
| Base concentration average | 7.34 | 217.84 | 647 | 203 | 365 | 445 | |
| Error in batch | 0.08 | 0.16 | 0.12 | 0.10 | 0.11 | 0.08 | |
| Low concentration average | 24.27 | 274.71 | 1012 | 773 | 880 | 987 | |
| Error in batch | 0.07 | 0.17 | 0.04 | 0.08 | 0.06 | 0.07 | |
| Middle concentration average | 53.04 | 331.28 | 1476 | 1351 | 1290 | 1516 | |
| Error in batch | 0.03 | 0.14 | 0.04 | 0.08 | 0.07 | 0.05 | |
| High concentration average | 165.75 | 392.83 | 1842 | 1900 | 1710 | 2009 | |
| Error in batch | 0.02 | 0.09 | 0.07 | 0.08 | 0.07 | 0.05 |
Table 9 blood basis, low concentration, middle concentration and high concentration fatty acyl carnitine criticize interior error
Error, the recovery between 4.4 batches
Detection U.S. CDC Internal Quality Control product and partial amino-acid self-control recovery sample are criticized an error, the recovery and are met situation with CDC and see the following form 10,11
The table 10 several amino acids recovery and batch between error
The Quality Control of table 11 many kinds of fatty acyl carnitine U.S. CDCs meets situation and the recovery
Embodiment 5 examination and clinical samples checking
The standard items of kit can not comprise whole test item; some disease can cause unsaturated lipid fatty acyl carnitine (as pole long chain acyl Co A Dehydrogenase Deficiency; C14:1 increases), hydroxyl fatty acyl group carnitine increase (as three functional protein defects; C14OH; C16OH; C16:1OH, C18OH, C18:1OH increase).By examination and clinical verification, the method for this kit can detect that these have standard items Quality Control and the Indexes Abnormality without standard items Quality Control completely.The partial results of disease indicators exception sees attached list 12.
Table 12
| SAMPLE | ALA | ARG | ARG/ORN | CIT | GLY | LEU | VAL |
| MUSD | 146.51 | 16.36 | 0.22 | 15.03 | 331.32 | 2771.3 | 530.62 |
| HPA | 484.63 | 48.13 | 0.12 | 40.18 | 705.67 | 266.7 | 284.29 |
| PKU | 122.32 | 14.31 | 0.43 | 18.84 | 279.14 | 129.04 | 159.62 |
| CIT | 226.88 | 33.05 | 0.42 | 107.48 | 284.72 | 132.01 | 143.46 |
| 3MCC,BKT,HMG | 159.81 | 12.7 | 0.26 | 12.71 | 196.92 | 111.69 | 104.28 |
| SCAD | 141.75 | 37.78 | 0.83 | 34.75 | 166.44 | 149.25 | 191.68 |
| GA2 | 247.3 | 11.98 | 0.1 | 17.98 | 377.77 | 111.8 | 75.59 |
| GA1 | 232.21 | 17.29 | 0.26 | 22.97 | 260.63 | 157.77 | 227.99 |
| IVA | 231.87 | 53.39 | 0.84 | 25.18 | 401.08 | 144.39 | 111.86 |
| MCAD | 282.57 | 5.75 | 0.03 | 27.75 | 358.31 | 249.66 | 377.63 |
| VLCAD | 209.14 | 34.66 | 0.37 | 18.77 | 406.99 | 107.06 | 110.89 |
| TFP,LCHAD | 642.55 | 8.68 | 0.09 | 17.53 | 589.72 | 208.13 | 138.52 |
| SAMPLE | MET | ASP | ORN | PHE | PRO | Phe/Tyr | TYR |
| MUSD | 10.84 | 169 | 75.6 | 31.23 | 99.15 | 0.52 | 44.63 |
| HPA | 34.51 | 256 | 323.18 | 504.79 | 364.98 | 5.28 | 96.54 |
| PKU | 15.49 | 321 | 33.78 | 1278.54 | 75.68 | 42.55 | 25.12 |
| CIT | 6.5 | 197 | 76.9 | 26.95 | 271.62 | 0.41 | 59.05 |
| 3MCC,BKT,HMG | 4.19 | 154 | 47.82 | 31.71 | 200.23 | 0.99 | 28.74 |
| SCAD | 8.07 | 380 | 44.98 | 37.91 | 277.66 | 0.66 | 51.37 |
| GA2 | 8.81 | 318 | 119.19 | 21.14 | 338.01 | 0.43 | 44.42 |
| GA1 | 9.69 | 280 | 65.94 | 54.53 | 208.39 | 0.45 | 108.59 |
| IVA | 10.88 | 228 | 62.53 | 40.05 | 208.29 | 0.62 | 58.03 |
| MCAD | 25.59 | 454 | 176.65 | 84.91 | 618.46 | 0.72 | 105.65 |
| VLCAD | 16.24 | 329 | 92.34 | 44.84 | 359.05 | 0.47 | 86.11 |
| TFP,LCHAD | 17.28 | 323 | 91.57 | 53.57 | 576.52 | 0.62 | 77.51 |
| SAMPLE | GLU | C0 | C2 | C3 | C3/C2 | C3DC_C4OH | C4 |
| MUSD | 326 | 23 | 10.12 | 0.53 | 0.05 | 0.09 | 0.11 |
| HPA | 456 | 46.32 | 17.49 | 1.76 | 0.1 | 0.31 | 0.31 |
| PKU | 236 | 15.41 | 27.07 | 0.88 | 0.03 | 0.32 | 0.27 |
| CIT | 288 | 64.29 | 1.31 | 0.16 | 0.12 | 0.06 | 0.08 |
| 3MCC,BKT,HMG | 138 | 36.87 | 0.76 | 0.2 | 0.26 | 0.07 | 0.18 |
| SCAD | 187 | 44.29 | 2.35 | 0.66 | 0.28 | 0.08 | 1.64 |
| GA2 | 437 | 70.26 | 4.24 | 0.33 | 0.08 | 0.09 | 1.1 |
| GA1 | 312 | 95.65 | 6.62 | 1.66 | 0.25 | 0.12 | 0.42 |
| IVA | 214 | 60.02 | 6.56 | 0.95 | 0.15 | 0.21 | 0.23 |
| MCAD | 505 | 26.17 | 2.34 | 0.51 | 0.22 | 0.12 | 0.07 |
| VLCAD | 590 | 19.97 | 8.56 | 0.66 | 0.08 | 0.33 | 0.12 |
| TFP,LCHAD | 680 | 21.44 | 10.9 | 0.84 | 0.08 | 0.29 | 0.16 |
| SAMPLE | C4DC_C5OH | C5 | C5:1 | C5DC_C6OH | C5OH/C8 | C6 | C6DC |
| MUSD | 0.2 | 0.05 | 0.01 | 0.13 | 3.18 | 0.03 | 0.11 |
| HPA | 0.2 | 0.28 | 0.02 | 0.16 | 1.44 | 0.09 | 0.13 |
| PKU | 0.35 | 0.12 | 0.03 | 0.06 | 3.37 | 0.16 | 0.23 |
| CIT | 0.13 | 0.14 | 0.01 | 0.17 | 1.73 | 0.03 | 0.18 |
| 3MCC,BKT,HMG | 32.99 | 0.17 | 0.03 | 0.2 | 340.28 | 0.03 | 0.12 |
| SCAD | 0.2 | 0.2 | 0.01 | 0.17 | 1.63 | 0.04 | 0.13 |
| GA2 | 0.21 | 0.32 | 0.02 | 0.19 | 0.14 | 0.53 | 0.27 |
| GA1 | 0.38 | 0.17 | 0.01 | 3.11 | 4.02 | 0.05 | 0.18 |
| IVA | 0.57 | 2.12 | 0.01 | 0.14 | 3.79 | 0.06 | 0.11 |
| MCAD | 0.21 | 0.18 | 0.02 | 0.21 | 0.07 | 0.21 | 0.22 |
| VLCAD | 0.16 | 0.08 | 0.01 | 0.15 | 1.05 | 0.05 | 0.13 |
| TFP,LCHAD | 0.19 | 0.2 | 0.02 | 0.14 | 1.13 | 0.05 | 0.12 |
| SAMPLE | C8 | C8:1 | C10 | C10:1 | C10:2 | C12 | C12:1 |
| MUSD | 0.03 | 0.14 | 0.05 | 0.08 | 0.03 | 0.05 | 0.04 |
| HPA | 0.11 | 0.24 | 0.1 | 0.08 | 0.02 | 0.07 | 0.04 |
| PKU | 0.09 | 0.12 | 0.13 | 0.09 | 0.05 | 0.1 | 0.12 |
| CIT | 0.05 | 0.1 | 0.03 | 0.03 | 0.01 | 0.04 | 0.02 |
| 3MCC,BKT,HMG | 0.07 | 0.09 | 0.05 | 0.05 | 0.01 | 0.04 | 0.02 |
| SCAD | 0.08 | 0.08 | 0.11 | 0.03 | 0.02 | 0.04 | 0.03 |
| GA2 | 0.97 | 0.4 | 0.44 | 0.31 | 0.04 | 0.2 | 0.09 |
| GA1 | 0.06 | 0.14 | 0.04 | 0.04 | 0.02 | 0.04 | 0.03 |
| IVA | 0.1 | 0.07 | 0.09 | 0.06 | 0.02 | 0.05 | 0.03 |
| MCAD | 2.07 | 0.16 | 0.16 | 0.23 | 0.02 | 0.02 | 0.02 |
| VLCAD | 0.11 | 0.04 | 0.1 | 0.02 | 0.01 | 0.19 | 0.17 |
| TFP,LCHAD | 0.11 | 0.06 | 0.24 | 0.13 | 0.03 | 0.66 | 0.29 |
| SAMPLE | C14 | C14:1 | C14:1/C16 | C14:2 | C16 | C16-OH | C16-OH/C16 |
| MUSD | 0.07 | 0.05 | 0.04 | 0.02 | 1.1 | 0.01 | 0.01 |
| HPA | 0.11 | 0.05 | 0.06 | 0.02 | 0.86 | 0.01 | 0.01 |
| PKU | 0.05 | 0.15 | 0.22 | 0.06 | 0.66 | 0.01 | 0.01 |
| CIT | 0.11 | 0.03 | 0.01 | 0.01 | 0.93 | 0.02 | 0.02 |
| 3MCC,BKT,HMG | 0.06 | 0.04 | 0.02 | 0.02 | 0.65 | 0.01 | 0.02 |
| SCAD | 0.11 | 0.04 | 0.01 | 0.01 | 1.41 | 0.02 | 0.01 |
| GA2 | 0.19 | 0.1 | 0.06 | 0.05 | 0.5 | 0.01 | 0.02 |
| GA1 | 0.11 | 0.04 | 0.02 | 0.02 | 0.65 | 0.02 | 0.03 |
| IVA | 0.18 | 0.06 | 0.01 | 0.03 | 1.7 | 0.02 | 0.01 |
| MCAD | 0.11 | 0.03 | 0.02 | 0.01 | 0.42 | 0.01 | 0.03 |
| VLCAD | 0.35 | 0.31 | 0.03 | 0.02 | 3.13 | 0.05 | 0.02 |
| TFP,LCHAD | 1.55 | 1.09 | 0.08 | 0.16 | 4.37 | 2.26 | 0.47 |
| SAMPLE | C16:1 | C16:1OH | C18 | C18-OH | C18:1 | C18:1-OH | C18:2 |
| MUSD | 0.06 | 0.03 | 0.4 | 0.01 | 0.7 | 0.02 | 0.18 |
| HPA | 0.04 | 0.02 | 0.23 | 0.01 | 0.72 | 0.01 | 0.23 |
| PKU | 0.07 | 0.03 | 0.52 | 0 | 1.01 | 0.01 | 0.26 |
| CIT | 0.03 | 0.03 | 0.23 | 0.01 | 0.69 | 0.02 | 0.18 |
| 3MCC,BKT,HMG | 0.03 | 0.04 | 0.51 | 0.01 | 0.79 | 0.02 | 0.21 |
| SCAD | 0.08 | 0.11 | 1.35 | 0.02 | 1.62 | 0.04 | 0.29 |
| GA2 | 0.05 | 0.02 | 0.32 | 0.01 | 0.57 | 0.01 | 0.22 |
| GA1 | 0.07 | 0.03 | 0.43 | 0.01 | 2.5 | 0.05 | 0.65 |
| IVA | 0.06 | 0.12 | 1.66 | 0.01 | 1.96 | 0.05 | 0.44 |
| MCAD | 0.03 | 0.05 | 0.27 | 0.01 | 0.4 | 0.02 | 0.09 |
| VLCAD | 0.32 | 0.06 | 0.67 | 0.03 | 1.3 | 0.04 | 0.07 |
| TFP,LCHAD | 1.15 | 0.61 | 1.42 | 0.67 | 2.64 | 1.46 | 0.9 |
Claims (10)
1. a tandem mass spectrum non-derivative method detection kit; it comprises isotope labeling amino acid standard items potpourri, isotope labeling fatty acyl carnitine standard items potpourri, extract, mobile phase; it is characterized in that described extract is made up of methyl alcohol, water, saturated monocarboxylic acid, methyl alcohol: water: saturated monocarboxylic acid volume ratio is 1000: 1 ~ 50: 0.01 ~ 2.
2. tandem mass spectrum non-derivative method detection kit as claimed in claim 1, is characterized in that described saturated monocarboxylic acid is one of in formic acid, acetic acid, propionic acid or butyric acid or their combination.
3. tandem mass spectrum non-derivative method detection kit as claimed in claim 1, is characterized in that described saturated monocarboxylic acid is formic acid.
4. tandem mass spectrum non-derivative method detection kit as claimed in claim 1, it is characterized in that described mobile phase is methyl alcohol, water, saturated monocarboxylic acid mixed liquor, wherein methyl alcohol: water: saturated monocarboxylic acid volume ratio is 1000: 1 ~ 50: 0.01 ~ 2.
5. tandem mass spectrum non-derivative method detection kit as claimed in claim 4, is characterized in that described saturated monocarboxylic acid is formic acid.
6. tandem mass spectrum non-derivative method detection kit as claimed in claim 1, is characterized in that described tandem mass spectrum non-derivative method detection kit also comprises 96 hole check-out consoles, viscosity extraction plate pad pasting and check-out console aluminium foil at the bottom of the heat-resisting 96 hole extraction plate in the U-shaped end, V-type.
7. tandem mass spectrum non-derivative method detection kit as claimed in claim 1, is characterized in that described tandem mass spectrum non-derivative method detection kit also comprises amino acid and fatty acyl carnitine neonatal screening quality-control product.
8. tandem mass spectrum non-derivative method detection kit described in claim 1 is detecting the purposes of amino acid and fatty acyl carnitine in sample.
9. purposes as claimed in claim 8, is characterized in that described sample is body fluid, and described body fluid is one of in blood, blood plasma, urine or saliva.
10. the using method of tandem mass spectrum non-derivative method detection kit described in claim 1, comprises the following steps:
A) mark liquid preparation in: use the extract of 1.0ml to add in amino acid in target freeze-dried powder bottle, cover tightly bottle cap, concussion, makes powder wherein dissolve completely; Use the extract of 1.0ml to add in fatty acyl carnitine in target freeze-dried powder bottle, cover tightly bottle cap, concussion, makes powder wherein dissolve completely;
B) working fluid preparation: interior mark liquid is placed in room temperature 20 minutes; With every hole 100 μ l extract consumption, calculate the extract total amount measured the same day needed for sample and Quality Control; Add in extract, amino acid to mark in liquid and fatty acyl carnitine with volume ratio 108:1:1 and mark liquid, be namely made into working fluid;
C) sample: Quality Control blood sheet, sample blood sheet are sorted at 96 orifice plates; By 3.2mm card punch order, blood sheet is squeezed in the heat-resisting 96 hole extraction plate in the U-shaped end;
D) specimen extraction: 100 μ l are added in the reacting hole of 96 orifice plates containing interior target working fluid respectively, extraction sample; With viscosity pad pasting sealing orifice plate, 45 DEG C hatch in oscillator, 700rpm shakes 45 minutes;
E) machine testing on sample: throw off diaphragm seal, 80 μ l supernatants are shifted out with the every hole of pipettor, order inserts clean 96 orifice plates, with foil sealing 96 orifice plate, puts into mass spectrum automatic sampler pallet, for Mass Spectrometer Method, sample introduction 20ul, sample introduction speed: 0 ~ 0.2min are 0.12ml/min, 0.2 ~ 1.2min is 0.015ml/min, 1.2 ~ 1.5min is 0.5ml/min, every this 1.7min of increment; Adopt tandem mass spectrometer electrospray positive ion mode, ion source temperature 120 DEG C, dissociation temperature 350 DEG C, spray voltage 3.5KV, nitrogen 650l/h, taper hole gas 50l/h; Multiple-reaction monitoring pattern collects data, and each ion pair 0.05ms is cycle detection ion pair successively, the detection signal of every part of sample collection 0.3-1.3min time period, and superposed signal intensity is for quantitative.
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| US20170115269A1 (en) * | 2014-03-31 | 2017-04-27 | Sekisui Medical Co., Ltd. | Acylcarnitine composition |
| CN104237442B (en) * | 2014-10-11 | 2015-10-07 | 杨绪庆 | The method of kit and application and joint-detection several amino acids and carnitine |
| CN107621500A (en) * | 2016-07-14 | 2018-01-23 | 上海可力梅塔生物医药科技有限公司 | Amino acid and carnitine tandem mass spectrum derivatization detection method |
| CN113049692A (en) * | 2016-08-31 | 2021-06-29 | 武汉生物技术研究院 | Quantitative detection method for free amino acid in clinical sample |
| CN108152380A (en) * | 2016-12-05 | 2018-06-12 | 山东国际生物科技园发展有限公司 | Tandem mass spectrum non-derivative fado kind amino acid and carnitine detection kit |
| CN110187012A (en) * | 2018-02-23 | 2019-08-30 | 上海可力梅塔生物医药科技有限公司 | A kind of non-derivative method detection kit |
| CN108676843A (en) * | 2018-05-21 | 2018-10-19 | 易达精准（杭州）科技有限公司 | The tandem mass spectrum non-derivative detection kit of glucose-6-phosphate dehydrogenase (G6PD) and its application |
| CN109324134B (en) * | 2018-11-12 | 2022-07-15 | 上海市儿童医院 | Application of C26: 0-acylcarnitine as brain X-ALD disease screening marker |
| CN109540622A (en) * | 2018-12-06 | 2019-03-29 | 扬州诺明哲天医学检验实验室有限公司 | A variety of amino acid and carnitine releasing agent detection pre-treating method and mass spectrometric analysis method |
| CN110133124A (en) * | 2019-04-29 | 2019-08-16 | 天津中医药大学 | The content assaying method of 18 kinds of amino acid in SHUXUETONG ZHUSHEYE |
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