CN105403637A - High-flux mass spectral method for detecting multiple amino acids in human body blood plasma - Google Patents

Abstract

The invention discloses a high-flux mass spectral method for detecting multiple amino acids in human body blood plasma. The method comprises precipitating protein in blood plasma by employing an organic solvent, adding a stable isotope internal standard, performing high-speed centrifugation to extract amino acids contained in the supernatant, and directly injecting a sample to detect the multiple amino acids in the blood plasma. A 96-well plate sample pretreatment process is employed, protein precipitation, internal-standard addition and centrifugation are performed in a 96-well plate, then the 96-well plate is directly connected with an LC-MS automatic sample-injection device, 96 samples can be processed and analyzed at a time, the detection efficiency is substantially improved, also sample loss and personal error caused by repeated transfer of samples are avoided, and labor cost is reduced. At present, detection on amino acids is mainly applied to screening and diagnosis on inherited metabolic diseases, along with more and more attention of people, more and more clinic detection samples are provided. The method is a rapid high-flux detection method for detecting a large amount of clinic samples.

Description

The mass spectrometry method of several amino acids in a kind of high flux human body blood plasma

Technical field

The invention belongs to chemical analysis detection field, can fast high-flux carry out sample pre-treatments, substantially increase detection efficiency, reduce cost of labor, be specifically related to the mass spectrometry method of several amino acids in a kind of high flux human body blood plasma.

Background technology

Inherited Metabolic Disorders refers to that participating in the encoding gene such as certain enzyme, transporter, membrane receptor of internal metabolism undergos mutation, the product dysfunction making it encode and cause one group of not normal disease of metabolism.Many Inherited Metabolic Disorders are relevant with Aminoacidopathy, and usually relevant with several amino acids.Not only sick kind quantity is various, pathogenesis is complicated for amino acid Inherited Metabolic Disorders, and its clinical manifestation has diversity and lacks specificity.Therefore, a kind of method that simultaneously can detect blood several amino acids and mesostate thereof is sought after clinically, to be applied to the Diagnosis and Treat of disease.The liquid phase tandem mass spectrum coupling technique (LC-MS/MS) carried out in recent years, can carry out qualitative and quantitative analysis to tens of kinds of materials contained in a sample simultaneously.Abroad have been reported and adopt LC-MS/MS to detect amino acid concentration in blood plasma, carry out Inherited Metabolic Disorders examination and diagnosis.In recent years, along with people are to the attention of Inherited Metabolic Disorders examination and diagnosis, the clinical sample that amino acid detects gets more and more, and the detection method of fast high-flux is more and more important.

At present, the derivatization method that the sample-pretreating method that in blood plasma, amino acid detects is mainly traditional, sample handling processes more complicated, but not derivatization method because of Pretreated simple and easy to operate, more and more be applied to the various amino acid contents detected in infant blood, and be used for guiding clinical diagnosis and treatment.But along with the increase of sample size, current sample-pretreating method still can not meet the detection demand of clinical a large amount of sample, clinically in the urgent need to the detection method of a kind of high flux, low cost.

Summary of the invention

The invention provides the mass spectrometry method of several amino acids in a kind of high flux human body blood plasma, the method adopts the sample-pretreating method of 96 orifice plates, in 96 orifice plates protein precipitation, add interior mark, centrifugal after get supernatant and be directly connected with the automatic sampling apparatus of LC-MS, can single treatment, analyze 96 samples, substantially increase detection efficiency, and the sample loss avoiding repeatedly transfer sample and cause and personal error, reduce cost of labor.The present invention is that the detection of a large amount of clinical sample provides a kind of quick, high-throughout detection method.

Particularly, the present invention relates to the following:

1. the mass spectrometry method of several amino acids in high flux human body blood plasma, said method comprising the steps of:

1) by human plasma organic solvent as the protein precipitation such as methyl alcohol, acetonitrile, centrifugally obtain containing amino acid whose supernatant to be measured;

2) method of supernatant LC-MS is carried out qualitative and quantitative analysis.

2. several amino acids according to claim 1 is respectively: (abbreviation is respectively: Met, Leu, Asn for methionine, leucine, asparagine, alanine, tyrosine, phenylalanine, threonine, serine, arginine, isoleucine, valine, tryptophane, glutamic acid, glutamine, proline, lysine and glycocoll, Ala, Tyr, Phe, Thr, Ser, Arg, Ile, Val, Trp, Glu, Gln, Pro, Lys, Gly).

3. according to the step 1 in claims 1) described in, it is characterized in that the step of sample pre-treatments is carried out in 96 orifice plates, step is as follows:

1) in 96 orifice plates, add the serum of 20-100 μ L, add amino acid whose internal standard compound to be measured, then add 5 times of organic solvents to 10 times of volumes and comprise methyl alcohol, acetonitrile etc.

2) by step 3) the mixed solution vortex 1-5min that obtains, 4 DEG C of refrigerators leave standstill 10-30min.

3) by step 4) mixed solution that processes is at the centrifugal 5-15min of 3000r/min.

4) the supernatant volley of rifle fire after centrifugal is transferred in the sample introduction plate in 96 holes.

4. according to the step 2 in claim 1), it is characterized in that 96 orifice plate sample introductions directly used by sample, can single treatment 96 samples.

5. according to the step 2 in claim 1), it is characterized in that the mobile phase A of liquid phase is the first aqueous acid of ammonium acetate containing 10mM and 0.5%, Mobile phase B is ammonium acetate, the formic acid of 0.5% and the acetonitrile solution of 10% water containing 10mM;

Accompanying drawing explanation

Fig. 1 is the high-efficient liquid phase chromatogram of 17 seed amino acid standard items in embodiment 1.

Fig. 2 is the high-efficient liquid phase chromatogram of 17 seed amino acids in the sample 1 of embodiment 2.

Embodiment

Embodiment 1

1. plasma sample pre-treatment step:

In 96 orifice plates, each hole adds mark, 50 μ L water and 900 μ L acetonitriles in 50 μ L test plasma sample, 5 μ L respectively.By above-mentioned mixed solution vortex 3min, then leave standstill 30min at 4 DEG C of refrigerators, by the solution centrifugal 10min under 3000r/min after process, with the volley of rifle fire, supernatant is transferred in the sample introduction plate in 96 holes.Wherein, 17 seed amino acids to be measured and interior mark thereof see the following form:

Sequence number	Amino acid	Abbreviation	MW	Isotopic Internal Standard
					1	Methionine	Met	150	D3
2	Leucine	Leu	137	D3
					3	Asparagine	Asn	132	D3
4	Alanine	Ala	90.2	D4
					5	Tyrosine	Tyr	181	13C6
6	Phenylalanine	Phe	165.3	13C6
					7	Threonine	Thr	119	-
8	Serine	Ser	105	D3
					9	Arginine	Arg	174	13C6
10	Isoleucine	Ile	131	13C6
					11	Valine	Val	117	D8
12	Tryptophane	Trp	204	D5
					13	Glutamic acid	Glu	147	D3
14	Glutamine	Gln	146	D5
					15	Proline	Pro	115	-
16	Lysine	Lys	146	13C6
					17	Glycocoll	Gly	75	D5

2. optimize Mass Spectrometry Conditions:

Directly use pin pump sample introduction with the standard items of 17 seed amino acids and the isotope configuration standard solution 1 μ g/mL of correspondence thereof, the parameter optimizing mass spectrum multiple-reaction monitoring is as follows:

3. optimize liquid-phase condition:

Mobile phase comprises A and B, and mobile phase A is the first aqueous acid of ammonium acetate containing 10mM and 0.5%, and Mobile phase B is ammonium acetate, the formic acid of 0.5% and the acetonitrile solution of 10% water containing 10mM.Flow velocity 0.6mL/min, column temperature 35 DEG C, chromatographic column: XbridgeBEHAmide2.1 × 100mm, 2.5 μm of particle diameters.The gradient condition optimized is as follows:

Time (min)	Mobile phase A %	Mobile phase B %
			0	0	100
2	10	90
			3	40	60
6	40	60
			8	0	100
15	0	100

4. standard items and interior target detect

Detect the standard items of 17 seed amino acids and interior mark according to the liquid phase optimized and Mass Spectrometry Conditions, as shown in Figure 1, visible Most amino-acids can be separated result completely.

5. the mensuration of typical curve

According to the liquid phase optimized and Mass Spectrometry Conditions, the mensuration of typical curve is carried out to the standard items of 17 seed amino acids.The concentration preparing 17 amino acidity scale product is as follows: 0.0625 μ g/mL, 0.3125 μ g/mL, 1.5625 μ g/mL, 3.125 μ g/mL, 6.25 μ g/mL, 12.5 μ g/mL, 25 μ g/mL, 50 μ g/mL.

Embodiment 2

The mensuration of actual sample

According to the method for the sample pre-treatments in embodiment 1,96 human normal plasma's samples (hospital general provides by Beijing Aviation) are carried out albumen precipitation and amino acid extraction, amino acid extract is proceeded in 96 hole sample introduction plates, the mass spectrum optimized according to embodiment 1 and liquid-phase condition are analyzed and data processing sample, calculate the concentration of every seed amino acid according to internal standard method, following table lists wherein 5.Fig. 2 is the high-efficient liquid phase chromatogram of 17 seed amino acids in sample 1.

Claims (5)

1. the mass spectrometry method of several amino acids in high flux human body blood plasma, said method comprising the steps of:

1) by human plasma organic solvent as the protein precipitation such as methyl alcohol, acetonitrile, centrifugally obtain containing amino acid whose supernatant to be measured;

2) method of supernatant LC-MS is carried out qualitative and quantitative analysis.

2. 17 seed amino acids according to claim 1 are respectively: (abbreviation is respectively: Met, Leu, Asn for methionine, leucine, asparagine, alanine, tyrosine, phenylalanine, threonine, serine, arginine, isoleucine, valine, tryptophane, glutamic acid, glutamine, proline, lysine and glycocoll, Ala, Tyr, Phe, Thr, Ser, Arg, Ile, Val, Trp, Glu, Gln, Pro, Lys, Gly).

3. according to the step 1 in claims 1) described in, it is characterized in that the step of sample pre-treatments is carried out in 96 orifice plates, step is as follows:

3) in 96 orifice plates, add the serum of 20-100 μ L, add amino acid whose internal standard compound to be measured, then add 5 times of organic solvents to 10 times of volumes and comprise methyl alcohol, acetonitrile etc.

4) by step 3) the mixed solution vortex 1-5min that obtains, 4 DEG C of refrigerators leave standstill 10-30min.

5) by step 4) mixed solution that processes is at the centrifugal 5-15min of 3000r/min.

6) the supernatant volley of rifle fire after centrifugal is transferred in the sample introduction plate in 96 holes.

4. according to the step 2 in claim 1), it is characterized in that 96 orifice plate sample introductions directly used by sample, can single treatment 96 samples.

5. according to the step 2 in claim 1), it is characterized in that the mobile phase A of liquid phase is the first aqueous acid of ammonium acetate containing 10mM and 0.5%, Mobile phase B is ammonium acetate, the formic acid of 0.5% and the acetonitrile solution of 10% water containing 10mM.