CN113049692A - Quantitative detection method for free amino acid in clinical sample - Google Patents
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Abstract
The invention discloses a quantitative detection method of free amino acid in a clinical sample, belonging to the field of analysis and detection. The method utilizes acidified methanol solution to extract free amino acid in clinical samples, then changes the property of the amino acid through alcohol derived carboxyl, and uses deuterated reagent derived amino acid to replace deuterated amino acid as an internal standard so as to be beneficial to chromatographic separation and mass spectrum quantitative detection. The method is simple to operate, can realize basic separation of amino acid in a short time, greatly reduces interference of a matrix, has low cost and good specificity, and can be used for quantitative detection of free amino acid in clinical samples.
Description
Technical Field
The invention relates to a quantitative detection method of free amino acid in a clinical sample, belonging to the field of analysis and detection.
Background
Amino acid is one of three nutrients of a living body and is also a necessity of life activities, can provide nutrition required for metabolism, ensures the growth, development and reproduction of organisms, and promotes the development of brains and intelligence. Amino acids play a crucial role in energy metabolism, signal pathway conduction, nerve conduction, and lipid transport. Their existence in vivo is classified into two types, one in the blood in a free state and the other in a bound state. In a normal state, the content of amino acids in blood is in an equilibrium state, and when an abnormality occurs in a living body, metabolic disorder of amino acids in blood is caused. Defects in amino acid metabolism resulting from a congenital deficiency in amino acid catabolic enzymes or a defect in amino acid transport are referred to as amino acid catabolic disorders. In recent years, many novel amino acid metabolic diseases are discovered, common congenital amino acid metabolic defect diseases comprise histidine syndrome, tyrosinemia, phenylketonuria and the like, the diseases are autosomal recessive inheritance, and most of the diseases can be preliminarily diagnosed by detecting free amino acids in blood.
Currently, the detection of amino acids can be mainly divided into direct detection and post-derivation detection. The direct detection of the matrix has serious interference and influences the accuracy of the quantification. The derivatization methods can be divided into derivatization of amino groups and derivatization of carboxyl groups: for amino derivatization, the detection sensitivity is high, the specificity is good, but the derivatization reagent is complex and difficult to obtain; for carboxyl derivatization, normal butanol is commonly used, but the existing detection method has no chromatographic column separation during detection, direct sample injection, serious matrix interference and easy instrument pollution, and deuterated amino acid is used as an internal standard, so that the cost is high.
Disclosure of Invention
The invention aims to provide a detection method which has strong specificity, high sensitivity and low cost, can quickly separate and accurately quantify free amino acid in a clinical sample, and aims to overcome the defects in the prior art.
The method comprises the following steps:
extracting free amino acid in a clinical sample, then adopting alcohols, deriving carboxyl through esterification reaction to change the property of the extracted amino acid, adopting corresponding amino acid standard products as internal standards after being derived through deuterated alcohols, and carrying out high performance liquid chromatography separation and mass spectrum quantitative detection.
The method comprises the steps of extracting free amino acid in a clinical sample by using an acidified methanol solution, then adopting n-butyl alcohol, deriving carboxyl through an esterification reaction to change the property of the extracted amino acid, then adopting a corresponding amino acid standard substance as an internal standard after being derived through deuterated n-butyl alcohol, and carrying out ultra-high performance liquid chromatography separation and mass spectrum quantitative detection.
The detection method provided by the invention comprises the following steps:
(1) vortex extracting free amino acid from clinical sample with formic acid/methanol (0.5%, v/v) solution;
(2) drying the extractive solution, adding 37 wt% HCl/n-BuOH (1:4, v/v) solution, deriving at 75 deg.C for more than half an hour, centrifuging, and drying;
(3) adding a redissolution containing a derivatized deuterated internal standard into the blow-dried sample, fixing the volume, separating by high performance liquid chromatography, and measuring free amino acid in a clinical sample by mass spectrometry.
In the step (1), the clinical sample is a blood sample, a urine sample or a tissue sample.
In the step (3), the complex solution is methanol/water (5%, v/v). The deuterated internal standard is deuterated n-butanol-derived amino acid, and the derivation conditions are the same as those of the non-deuterated internal standard in the step (2).
In the step (3), in the case of chromatography, separation was performed by using a C18+ column from Waters. The sample size was 5. mu.L. The mobile phase consisted of aqueous phase A (0.5 vol% formic acid in 20mM ammonium formate in water) and organic phase B (0.5 vol% formic acid in methanol); the flow rate was 0.4 mL/min. The gradient elution procedure was as follows: 0-1min 5 vol% B, 1-3min 5-30 vol% B, 3-4.5min30 vol% B, 4.5-5.5min30-50 vol% B, 5.5-6min 50-80 vol% B, 6-7min 80 vol% B, 7-7.2min80-5 vol% B, 7.2-11min 5 vol% B.
The invention also provides a quantitative detection method of acyl carnitine in a clinical sample, which comprises the following steps: extracting acyl carnitine in a clinical sample by using an acidified methanol solution, then adopting an alcohol reagent, changing the property of the extracted acyl carnitine by deriving carboxyl through an esterification reaction, and then adopting a corresponding acyl carnitine standard substance as an internal standard after being derived through deuterated alcohol for mass spectrum quantitative detection.
The method does not directly provide clinical diagnosis basis, and is only used as a detection method for further diagnosis reference. The invention has the following advantages: (1) the deuterated alcohol derivative amino acid is used as an internal standard instead of the deuterated amino acid, so that the cost is greatly reduced; (2) when the amino acid is derived from the alcohol, the acyl carnitine in a clinical sample can be derived and quantitatively detected; (3) alcohols are used as derivatization reagents, are cheap and easy to obtain, are easy to remove, cannot interfere detection, and can avoid polluting instruments; (4) the introduction of the C18+ chromatographic column of Waters company is combined with a gradient elution program, so that the effective separation of 15 amino acids can be realized in a short time, the detection interference is further reduced, and a detection method which has strong specificity and high sensitivity, can be quickly separated and accurately quantifies is provided for the detection of free amino acids in clinical samples.
Drawings
FIG. 1 is a secondary mass spectrum of several amino acids and acylcarnitines after derivatization.
FIGS. 2-5 are graphs of Multiple Reaction Monitoring (MRM) for detecting free amino acids in blood samples using the present invention. In which the detection is divided into four periods, fig. 2: 0-2min (arginine, glycine, serine, lysine, histidine), fig. 3: 2-3.5min (threonine, proline, alanine), FIG. 4: 3.5-5min (tyrosine, methionine, valine), fig. 5: 5-8min (tryptophan, phenylalanine, isoleucine, leucine).
Detailed Description
The method comprises the steps of extracting free amino acid in a clinical sample by using an acidified methanol solution, then adopting n-butyl alcohol, deriving carboxyl through an esterification reaction to change the property of the extracted amino acid, then adopting a corresponding amino acid standard substance, deriving the amino acid standard substance by using deuterated n-butyl alcohol to serve as an internal standard substance, and carrying out ultra-high performance liquid chromatography separation and mass spectrum quantitative detection.
Clinical samples, including blood, urine, or tissue samples. The sample generally contains arginine, glycine, serine, lysine, histidine, threonine, proline, alanine, tyrosine, methionine, valine, tryptophan, phenylalanine, isoleucine, and leucine. N-butyl alcohol is adopted, carboxyl is derived through esterification reaction to change the properties of the amino acids, then corresponding amino acid standards are purchased, deuterated n-butyl alcohol is used as an internal standard after being derived, and the internal standard is separated through ultra-high performance liquid chromatography and mass spectrum quantitative detection.
Example 1
Sample treatment:
dripping blood extracted from a human body on card paper, and drying at room temperature overnight;
secondly, punching blood spots with the diameter of about 3.0mm by using a puncher, placing the blood spots in a 1.5mL centrifuge tube, and performing vortex extraction on 200 mu L formic acid/methanol (0.5%, v/v) solution for 30 min;
thirdly, drying 4 mu L of the extracting solution obtained in the second step, adding 50 mu L of 37 wt% HCl/n-BuOH (1:4, v/v) solution, performing derivation in water bath at 75 ℃ for 30min, centrifuging and drying;
and fourthly, re-dissolving 200 mu L of methanol/water (5 percent, v/v) (containing 2ng/mL of deuterated internal standard), separating by using a C18+ chromatographic column, and detecting by mass spectrum.
Wherein the deuterated internal standard is: deuterated n-butanol-derived amino acid standards (purchased from Sigma, usa) and deuterated n-butanol-derived acylcarnitine standards (purchased from PerkinElmer, usa). The conditions of derivation were the same as those of the blood samples.
Separation conditions gradient elution procedure was as follows: 0-1min 5 vol% B, 1-3min 5-30 vol% B, 3-4.5min30 vol% B, 4.5-5.5min30-50 vol% B, 5.5-6min 50-80 vol% B, 6-7min 80 vol% B, 7-7.2min80-5 vol% B, 7.2-11min 5 vol% B; the flow rate is 0.4 mL/min; the sample size was 5. mu.L. The equipment used for separation is an ultra-high performance liquid chromatograph of Shimadzu corporation, and the instrument model is Nexera UHPLC LC-30A.
Mass spectrum parameters: the atomizer and auxiliary gas both used nitrogen, the air curtain gas (CUR) was 30psi, the atomizing gas (GS1) and auxiliary gas (GS2) were 50psi and 45psi, respectively, the spray voltage was 5500V, the atomization temperature was 550 ℃, the positive ion scanning, Multiple Reaction Monitoring (MRM) mode. The equipment used for detection was a triple quadrupole mass spectrometer from SCIEX corporation, instrument model TripleQuad 4500.
Table 1 shows the qualitative and quantitative ion pairs and deuterated internal standard parameters for the corresponding Multiple Reaction Monitoring (MRM) mode after derivatization of 15 amino acids and 2 acylcarnitines. Using the method provided by the present invention, matrix effects derived in blood sample extracts and in solution were calculated (shown in Table 2), and relative standard deviations and recoveries of three amino acid spikings at low (6.25 ng/. mu.L), medium (31.25 ng/. mu.L), and high (93.75 ng/. mu.L) concentrations were added to blood samples (shown in Table 3). As can be seen by combining the matrix effect values provided in table 2 with the smooth baseline in the blood sample separation chromatograms of fig. 2-5, the present invention greatly reduces matrix interference. As can be seen from Table 3, the quantitative detection method of the present invention has high accuracy and good repeatability.
TABLE 1 parameters of amino acids and acylcarnitines after derivatization in Multiple Reaction Monitoring (MRM) mode
a: quantitative ion pair
N/A: without separation
TABLE 2 derivatization in blood extracts and matrix effects derivatized in solution
TABLE 3 spiked relative standard deviation and recovery of three concentrations of amino acids in blood samples
Example 2
Sample treatment:
adding 200 mu L of formic acid/methanol (0.5%, v/v) solution into 10 mu L of urine, whirling for 1min, centrifuging, taking supernatant, and drying;
② adding 50 μ L of 37 wt% HCl/n-BuOH (1:4, v/v) solution, deriving for 30min in 75 ℃ water bath, centrifuging and drying;
200 μ L methanol/water (5%, v/v) (containing 2ng/mL deuterated internal standard) was reconstituted, separated on a C18+ column, and detected by mass spectrometry. The separation conditions and mass spectrometry parameters were the same as in example 1.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (9)
1. A method for quantitatively detecting free amino acids in clinical samples is characterized by comprising the following steps:
extracting free amino acid in a clinical sample, then adopting alcohols, deriving carboxyl through esterification reaction to change the property of the extracted amino acid, adopting corresponding amino acid standard products as internal standards after being derived through deuterated alcohols, and carrying out high performance liquid chromatography separation and mass spectrum quantitative detection.
2. The detection method according to claim 1, characterized in that:
extracting free amino acid in a clinical sample by using an acidified methanol solution, deriving carboxyl by using n-butyl alcohol through an esterification reaction to change the property of the extracted amino acid, deriving a corresponding amino acid standard substance by using deuterated n-butyl alcohol to serve as an internal standard, and performing ultra-high performance liquid chromatography separation and mass spectrum quantitative detection.
3. The detection method according to claim 1 or 2, characterized by comprising the steps of:
(1) vortex free amino acids in 0.5% (v/v) formic acid/methanol solution from clinical samples;
(2) drying the extractive solution, adding 37 wt% HCl/n-BuOH (1:4, v/v) solution, deriving at 75 deg.C for more than half an hour, centrifuging, and drying;
(3) adding a redissolution containing a derivatized deuterated internal standard into the blow-dried sample, fixing the volume, separating by high performance liquid chromatography, and measuring free amino acid in a clinical sample by mass spectrometry.
4. The detection method according to claim 3, characterized in that: in the step (1), the clinical sample is a blood sample, a urine sample or a tissue sample.
5. The detection method according to claim 3, characterized in that: in the step (3), the complex solution is 5% (v/v) methanol/water solution.
6. The detection method according to claim 3, characterized in that: in the step (3), in the case of chromatography, the sample was separated by using a C18+ column from Waters, and the amount of the sample was 5. mu.L.
7. The detection method according to claim 3, characterized in that: the mobile phase consists of an aqueous phase A and an organic phase B, wherein A is an aqueous solution containing 0.5 vol% of formic acid and 20mM ammonium formate, and B is a methanol solution containing 0.5 vol% of formic acid; the flow rate was 0.4 mL/min.
8. The detection method according to claim 7, wherein the gradient elution procedure is as follows: 0-1min 5 vol% B, 1-3min 5-30 vol% B, 3-4.5min30 vol% B, 4.5-5.5min30-50 vol% B, 5.5-6min 50-80 vol% B, 6-7min 80 vol% B, 7-7.2min80-5 vol% B, 7.2-11min 5 vol% B.
9. A method for quantitatively detecting acyl carnitine in a clinical sample is characterized by comprising the following steps:
extracting acyl carnitine in a clinical sample by using an acidified methanol solution, then adopting an alcohol reagent, changing the property of the extracted acyl carnitine by deriving carboxyl through an esterification reaction, and then adopting a corresponding acyl carnitine standard substance as an internal standard after being derived through deuterated alcohol for mass spectrum quantitative detection.
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