CN103837634A - Method for simultaneously detecting contents of various organic acids in urine of human body - Google Patents
Method for simultaneously detecting contents of various organic acids in urine of human body Download PDFInfo
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Abstract
The invention relates to a method for simultaneously detecting the contents of various organic acids in urine of a human body. The method specifically comprises the steps of (1) collecting a sample; (2) implementing measurement: A, adding the urine into an internal standard solution; B, adding a hydroxylamine hydrochloride solution and a sodium hydroxide solution to perform oximation reaction; C, adding concentrated hydrochloric acid, then adding ethyl acetate and diethyl ether, fully mixing to be uniform, performing centrifugation, absorbing an upper layer organic phase, and drying the organic phase through air by nitrogen; D, adding a derivatization reagent, putting the mixture into a nitrogen blowing instrument base or a temperature box, and performing derivatization, wherein the derivatization reagent comprises BSTFA and TMCS; E, performing row analysis. The method can be used for simultaneously detecting 72 organic acids probably appearing in the urine of the human body, is simple, convenient to operate and high in accuracy and sensitivity.
Description
Technical field
The present invention relates to biochemistry detection technical field, be specifically related to the method for multiple organic acid content in a kind of while human body urine.
Background technology
Detect and understand body metabolism situation by organic acid series, with the waste gas that detects automobile discharge, infer that engine condition is similar principles.Health becomes carbohydrates, amino acid, fat from nutrients can absorbing material and the process of refuse, is referred to as " metabolic pathway ".In each stage of this process, the enzyme, the coenzyme that need the elements such as different amino acid, vitamin, mineral matter to form regulate metabolic rate, add sharing out the work and help one another of digestive system.In metabolic pathway, if certain individual event or several element are scarce or few, biochemical reaction will be interrupted or slow down, and the acid product of intermediary metabolism will be discharged in urine, and biochemist is referred to as " organic acid ", can be detected by functional medicine inspection.Therefore, urinary organic acid detects the ability of demand to vitamin and mineral matter of the balance, health that can understand the situation of the three large Nutrition and Metabolisms such as carbohydrates, amino acid, fat and picked-up and the energy-producing efficiency of cell, nerve conduction material, removing toxic substances, gut flora ecology in addition.
The diagnosis of many chronic disease is very difficult, and especially when it, to involve nonspecific symptom such as tired, thinking chaos, DOMS, digestive discomfort, arthralgia, sleep quality good etc.The medical test of general standard is the state for judging disease, and a complete set of metabolic function analysis is not for diagnosing the illness, but finds out pathogenic reason with helping us.This analyzes and helps us to assess specific metabolic disorder, and then we can, according to the personalized nutritional supplementation plan of result custom-made by size, make the normal function of physical recovery for the link of needs help.The object of this analysis is not limited only to the treatment of symptom, but wishes before disease occurs, just to find out the defect of nutrition or metabolism and to be corrected.
Summary of the invention
The object of this invention is to provide the method for multiple organic acid content in a kind of while human body urine, 72 kinds of organic acids that simultaneously may occur in human body urine by the method, the method is simple, easy to operate, and accuracy and highly sensitive.
The object of the invention is to be achieved through the following technical solutions:
A kind of method of multiple organic acid content in while human body urine, described method is to adopt gas chromatography mass spectrometry technology to detect, the method detecting specifically comprises the following steps: 1. collection of specimens: collect urina sanguinis or the random time section urine (urine that this urine is preservative free, if use not in time this urine, will preserve, preferably under the condition of freezing or refrigeration, be kept at-20 ℃); 2. A. gets 1. described urine of a certain amount of step, and adds the inner mark solution of 0.4mg/mL; B. add 0.5mg/mL oxammonium hydrochloride solution and 10mol/L NaOH (NaOH) solution, the organic acid in urine sample is carried out to oximation reaction, mix and leave standstill 1h with the abundant vortex of vortex instrument; C. add 12mol/L concentrated hydrochloric acid, then after adding ethyl acetate and ether fully to mix, centrifugal 5min, mixes with the abundant vortex of vortex instrument again; Draw upper organic phase and put into clean centrifuge tube; Dry up with 99.999% nitrogen; D. add derivatization reagent, put into nitrogen flushing instrument base or incubator, 65 ℃ of condition temperature, derivatization 30min, described derivatization reagent comprises BSTFA (two trimethyl silicane trifluoroacetamide) and TMCS (trimethyl chlorosilane), the ratio of BSTFA and TMCS is 97～99.5: 0.5～3, (the BSTFA: TMCS=99: 1 that is preferably 99: 1, be Sylon-BFT), compound is derivatized reagent BSTFA: TMCS=99: 1 (Sylon-BFT) is converted into trimethyl silicane (TMS) derivant; E. sample introduction, utilizes gas chromatograph-mass spectrometer analysis.
Further, the urine described in steps A is the urine that contains 0.2mg creatinine.
Further, the addition of the inner mark solution described in steps A is 20 μ L, and this inner mark solution is that internal standard compound is dissolved in deionized water and is prepared; Described internal standard compound is preferably 2-phenylbutyric acid (2-PA).
Further, the addition of the oxammonium hydrochloride solution described in step B is 100 μ L, the aqueous solution that this oxammonium hydrochloride solution is oxammonium hydrochloride.The addition of the NaOH of 10mol/L described in step B (NaOH) solution is 300 μ L, the aqueous solution that this sodium hydroxide solution is NaOH.
Further, the addition of the concentrated hydrochloric acid described in step C is 300 μ L, and the addition of described ethyl acetate and ether is 2mL.
Further, the addition of the derivatization reagent described in step D is 100 μ L.Described BSTFA and the ratio of TMCS are 99: 1 (BSTFA: TMCS=99: 1, be Sylon-BFT).
Further, in step e: the design parameter of gas chromatograph-mass spectrometer used: the long 30m of capillary chromatographic column (HP-5MS),
thickness 0.25 μ m, 70 ℃ of column oven initial temperatures, 3min, is warming up to 260 ℃ with 10 ℃/min, and then is warming up to 280 ℃ with 20 ℃/min, 2min, 250 ℃ of injector temperatures; Helium (99.999%) overall flow rate 23.4mL/min, split ratio 20: 1; 280 ℃ of ion source temperatures, sweep limit 50～550m/z (mass-to-charge ratio).
The invention provides the method for multiple organic acid content in a kind of while human body urine, its beneficial effect mainly having is: the method is to adopt gas chromatography mass spectrometry technology to detect, any one or more in 72 kinds of organic acids that simultaneously may occur in human body urine by the method or whole content, the method is simple, easy to operate, and accuracy and highly sensitive, has important potential applicability in clinical practice.
Accompanying drawing explanation
With reference to the accompanying drawings the present invention is described in further detail below.
Fig. 1 is the chromatogram of 72 kinds of organic acid standard model mixed liquors described in the embodiment of the present invention;
Fig. 2 is contained organic acid chromatogram in the urine of the human body A described in the embodiment of the present invention;
Fig. 3 is contained organic acid chromatogram in the urine of the human body B described in the embodiment of the present invention.
Embodiment
The method of multiple organic acid content in a kind of while human body urine described in the embodiment of the present invention: 1, get the urine that contains 0.2mg creatinine; 2, add interior mark 2-PA (0.4mg/mL) 20 μ L; 3, add 100 μ L oxammonium hydrochlorides (0.5mg/mL) and NaOH (10mol/L), the organic acid in urine sample is carried out to oximation reaction, fully vortex mixes and leaves standstill 1h; 4, add 300 μ L concentrated hydrochloric acids (12mol/L); After adding again 2mL ethyl acetate and 2mL ether fully to mix, 3500rpm (rev/min) centrifugal 5min, vortex; 5, draw upper organic phase and put into clean centrifuge tube; Dry up with 99.999% nitrogen; 6, add 100 μ L derivating agent BSTFA: TMCS=(99: 1), put into nitrogen flushing instrument base or incubator, 65 ℃ of condition temperature, derivatization 30min; 7, sample introduction, analyzes at GC/MS instrument.
When concrete enforcement, the organic acid that the method for the invention can detect comprises 72 kinds of organic acids, every kind of organic acid title and number as shown in the table.
Table 1
| Numbering | Organic acid | Numbering | Organic acid | Numbering | Organic acid |
| 1 | Citramalic acid | 25 | Alpha-hydroxybutyric acid | 49 | |
| 2 | 5-hydroxy-methyl furancarboxylic acid | 26 | Succinic acid | 50 | Glutaric acid |
| 3 | 3-ketoglutaric acid | 27 | Fumaric acid | 51 | Ascorbic acid |
| 4 | Furans-2,5-dicarboxylic acid | 28 | Malic acid | 52 | 3-hydroxy-3-methylglutaric acid |
| 5 | Furans phosphinylidyne glycocoll | 29 | α-ketoglutaric acid | 53 | NAC |
| 6 | Tartrate | 30 | Aconitic acid | 54 | Methyl citric acid |
| 7 | Arabinose | 31 | Citric acid | 55 | Pyroglutamic acid |
| 8 | Carboxyl citric acid | 32 | Homovanillic acid | 56 | Orotic acid |
| 9 | Tricarballylic acid | 33 | Vanillylmandelic acid (VMA) | 57 | 2-hydroxyl |
| 10 | 2-Hydroxyphenyl Acetic Acid | 34 | 5-HIAA | 58 | 2-hydroxyl isovaleric acid |
| 11 | 4-hydroxyl phenylacetic acid | 35 | Quinolinic acid | 59 | 2-oxo isovaleric acid |
| 12 | 4-HBA | 36 | Kynurenic acid | 60 | 3-methyl-2-oxy pentanoic acid |
| 13 | 4-hydroxyl hippuric acid | 37 | Uracil | 61 | 2-hydroxyl isocaproic acid |
| 14 | |
38 | Thymine | 62 | 2-Oxoisocaproic acid |
| 15 | 3-indolyl acetic acid | 39 | 3-hydroxybutyrate | 63 | 2-oxygen-4-(methylthio) butanoic acid |
| 16 | Benzoic acid | 40 | Acetoacetate | 64 | Mandelic acid |
| 17 | 3-(3-hydroxy phenyl)-3-hydracrylic acid | 41 | 4 hydroxybutyric acid | 65 | Phenyllactic acid |
| 18 | 4-cresols | 42 | Ethyl malonic acid | 66 | Phenylpyruvic acid |
| 19 | Dihydroxy benzenes propionic acid | 43 | Dimethyl succinic acid | 67 | Alcapton |
| 20 | Glyceric acid | 44 | Hexane diacid | 68 | 4-hydroxyphenyl lactic acid |
| 21 | Glycollic acid | 45 | Suberic acid | 69 | N-acetyl group aspartic acid |
| 22 | Oxalic acid | 46 | Decanedioic acid | 70 | Malonic acid |
| 23 | Lactic acid | 47 | Methylmalonic acid | 71 | 3-methylglutaric acid |
| 24 | Pyruvic acid | 48 | Pyridoxic acid | 72 | Phosphoric acid |
As an example of specific experiment case example, embodiment is described below, should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not intended to limit the present invention.
Embodiment 1
1. key instrument equipment used and reagent: spectrophotometer, hydro-extractor, Nitrogen evaporator, Agilent gas chromatograph-mass spectrometer Agilent GC/MS--7890A/5975C, 99.999% nitrogen and 99.999% helium; Picric acid (15g/L), NaOH (10mol/L), deionized water, ethyl acetate (HPLC level), ether (HPLC level), oxammonium hydrochloride solution (0.5mg/mL), hydrochloric acid (12mol/L), derivatization reagent BSTFA: TMCS=99: 1 (Sylon-BFT), internal standard compound is 2-phenylbutyric acid (2-PA, 0.4mg/mL), 72 kinds of organic acid reference materials as shown above.
2. the preparation of preparation of samples and related solution:
A. collection of specimens: directly collect 5～10mL urina sanguinis or random time section urine, preservative free, preserves at-20 ℃;
B. UCr is measured: adopt picric acid method to measure;
C. the preparation of standard model mixed liquor;
D. inner mark solution preparation: the 2-phenylbutyric acid of getting 20mg is placed in 5mL volumetric flask, uses deionized water constant volume;
E.72 plant the preparation of organic acid standard model mixed liquor: take 72 kinds of organic acids given in table, be placed in respectively 5mL volumetric flask, use deionized water constant volume, numbering is labeled as respectively 1～72, the each organic acid soln 1.0mL drawing respectively again in No. 1-72 is placed in the brown volumetric flask of 50mL, use deionized water constant volume, be 72 kinds of organic acid standard model mixed liquors.
3. organic acid assay method in urine
A. get urine fresh or freezing preservation, adopt picric acid method to measure the wherein content of UCr, generally guarantee the UCr that contains 0.2mg in urine
B. in urine, add 0.4mg/mL2-phenylbutyric acid 20 μ L;
C. add 100 μ L0.5mg/mL oxammonium hydrochlorides and 300 μ L10mol/L NaOH, the organic acid in urine sample is carried out to oximation reaction, fully vortex mixes and leaves standstill 1h;
D. add 300 μ L 12mol/L concentrated hydrochloric acids; After adding 2mL ethyl acetate and 2mL ether fully to mix, 3500rpm (rev/min) centrifugal 5min, vortex mixes;
E. draw upper organic phase and put into clean centrifuge tube, dry up with 99.999% nitrogen;
F. add 100 μ L derivatization reagent BSTFA: TMCS=(99: 1), put into nitrogen flushing instrument base or incubator, 65 ℃ of condition temperature, carry out the derivatization about 30min;
G. sample introduction GC bottle, utilizes gas chromatograph-mass spectrometer (GCMS) (GC-MS:Gas Chromatograph-Mass Spectrometer-computer) to analyze.
4. gaseous mass spectrum stratographic analysis
Analysis condition: the long 30m of capillary chromatographic column HP-5MS, φ=250 μ m, thickness 0.25 μ m, 70 ℃ of column oven initial temperatures, 3min, is warming up to 260 ℃ with 10 ℃/min, and then is warming up to 280 ℃ with 20 ℃/min, 2min, 250 ℃ of injector temperatures; Helium (99.999%) overall flow rate 23.4ml/min, split ratio 20: 1; 280 ℃ of ion source temperatures, sweep limit 50～550m/z (mass-to-charge ratio).
Fig. 1 is the chromatogram of 72 kinds of organic acid standard model mixed liquors.
Internal standard method take 2-phenylbutyric acid as internal standard compound is analyzed the urina sanguinis liquid of 1 routine human body A and 1 routine human body B, the analysis result of human body A is respectively as shown in Fig. 2 table 2, the analysis result of human body B is respectively as shown in Fig. 3 table 3, hippuric acid 14 in Fig. 2 Fig. 3 is made up of two peaks, be 14-1 and 14-2 composition, and 73 refer to interior mark 2-phenyl in Fig. 2 Fig. 3; Every kind of organic acid and numbering shown in corresponding table 1 in Fig. 1～3.
In table 2,3, < DL represents in surveyed urine containing this organic acid or at it below detectability.
Table 2
Table 3
Embodiment 3 recovery and precision
Prepare 8 duplicate samples urines, measure wherein organic acid content by the method for the invention, institute's measured value is designated as result A; And then in 8 described duplicate samples urines, add 72 kinds of organic acid standard model mixed liquors of 500 μ l/L, then measure organic acid content wherein by the method for the invention, institute's measured value is designated as result B; The ratio of 72 kinds of organic acid standard model mixed liquors that then deduct the value of result A gained with result B and add carrys out calculate recovery rate, and calculate precision, result is as shown in following table (i.e. table 4), and from following table, the recovery is 80.4%～95.7%; The coefficient of variation is 1.1%～4.8%, the mark using 2 times of peak heights of noise as detectability, and the lowest detection that records the method is limited to 0.02～0.09mmol/mol-Cr.Accuracy and the precision of the analytical approach of setting up can meet analysis requirement.RSD (Relative Standard Deviation, relative standard deviation).
Table 4
Embodiment 4: the repeatability of method
Get a certain urine sample, adding concentration is 72 kinds of organic acid standard model mixed liquors of 100 μ g/L, carries out the processing such as derivatization by method of the present invention, and continuous sample introduction measures for 8 times, calculates retention time RSD and area peak RSD value.The results are shown in Table shown in 5, is that the relative standard deviation of 72 kinds of organic acid retention times and peak area detects composition.
Table 5
Embodiment 5: sample stability test
1. the stability test under the placement of sample urine room temperature
Get certain sample urine, respectively at room temperature condition transfer set to 0,2,4,8 and 12h after by the method for the invention, sample urine is carried out again the processing such as derivatization, and organic acid content in difference working sample urine, see the following form shown in (i.e. table 6), last hurdle is the RSD value of measuring numerical value under these five times, and result is known thus: sample is stable in room temperature is placed 12h.
Table 6
2. the stability test under sample urine refrigeration placement
Get certain sample urine, transfer and set to 0, after 2,4,6 and 8 days, by the method for the invention, sample urine carried out again the processing such as derivatization respectively at refrigeration (0～6 ℃) condition, and organic acid content in difference working sample urine, see the following form shown in (i.e. table 7), last hurdle is the RSD value of measuring numerical value under these five times, and result is known thus: sample is placed in 8 days stable in room temperature.
Table 7
| Numbering | Organic acid | Refrigerate 0 | Refrigerate | 2 days | Refrigerate 4 days | Refrigerate 6 days | Refrigerate 8 days | RSD |
| ? | ? | mmol/mol | mmol/mol | mmol/mol | mmol/mol | mmol/mol | ％ |
| 1 | Citramalic acid | 3.27 | 3.27 | 3.38 | 3.47 | 3.56 | 3.74 |
| 2 | 5-hydroxy-methyl furancarboxylic acid | 3.02 | 3.03 | 3.10 | 3.11 | 3.17 | 2.01 |
| 3 | 3-ketoglutaric acid | 0.05 | 0.05 | 0.06 | 0.06 | 0.06 | 3.58 |
| 4 | Furans-2,5-dicarboxylic acid | 3.90 | 3.99 | 4.03 | 4.05 | 4.08 | 1.74 |
| 5 | Furans phosphinylidyne glycocoll | 0.11 | 0.11 | 0.12 | 0.12 | 0.12 | 2.54 |
| 6 | Tartrate | 1.67 | 1.68 | 1.68 | 1.72 | 1.74 | 1.79 |
| 7 | Arabinose | ＜DL | ＜DL | ＜DL | ＜DL | ＜DL | ? |
| 8 | Carboxyl citric acid | 1.11 | 1.14 | 1.17 | 1.17 | 1.20 | 2.95 |
| 9 | Tricarballylic acid | 0.29 | 0.29 | 0.29 | 0.30 | 0.31 | 3.02 |
| 10 | 2-Hydroxyphenyl Acetic Acid | 0.98 | 0.98 | 0.99 | 1.00 | 1.00 | 1.01 |
| 11 | 4-hydroxyl phenylacetic acid | 15.63 | 15.70 | 15.77 | 15.84 | 16.09 | 1.12 |
| 12 | 4-HBA | 1.06 | 1.08 | 1.10 | 1.14 | 1.17 | 4.03 |
| 13 | 4-hydroxyl hippuric acid | 4.43 | 4.46 | 4.49 | 4.50 | 4.53 | 0.86 |
| 14 | Hippuric acid | 158.02 | 161.66 | 165.32 | 170.03 | 173.09 | 3.68 |
| 15 | 3-indolyl acetic acid | ＜DL | ＜DL | ＜DL | ＜DL | ＜DL | ? |
| 16 | Benzoic acid | 0.82 | 0.84 | 0.84 | 0.88 | 0.91 | 4.23 |
| 17 | 3-(3-hydroxy phenyl)-3-hydracrylic acid | 41.04 | 41.08 | 41.15 | 41.26 | 41.28 | 0.26 |
| 18 | 4-cresols | 4.17 | 4.18 | 4.18 | 4.28 | 4.36 | 1.97 |
| 19 | Dihydroxy benzenes propionic acid | 3.63 | 3.72 | 3.79 | 3.87 | 3.91 | 2.99 |
| 20 | Glyceric acid | 0.13 | 0.14 | 0.14 | 0.14 | 0.14 | 3.24 |
| 21 | Glycollic acid | 20.34 | 20.48 | 20.76 | 20.87 | 20.90 | 1.20 |
| 22 | Oxalic acid | 18.25 | 18.36 | 18.42 | 18.51 | 18.55 | 0.65 |
| 23 | Lactic acid | 2.16 | 2.22 | 2.29 | 2.33 | 2.36 | 3.59 |
| 24 | Pyruvic acid | ＜DL | ＜DL | ＜DL | ＜DL | ＜DL | ? |
| 25 | Alpha-hydroxybutyric acid | 2.49 | 2.49 | 2.51 | 2.52 | 2.53 | 0.71 |
| 26 | Succinic acid | 3.99 | 4.05 | 4.07 | 4.16 | 4.25 | 2.48 |
| 27 | Fumaric acid | 0.37 | 0.38 | 0.39 | 0.40 | 0.40 | 3.36 |
| 28 | Malic acid | ＜DL | ＜DL | ＜DL | ＜DL | ＜DL | ? |
| 29 | α-ketoglutaric acid | 2.77 | 2.83 | 2.87 | 2.93 | 2.96 | 2.66 |
| 30 | Aconitic acid | 15.96 | 16.01 | 16.08 | 16.14 | 16.21 | 0.62 |
| 31 | Citric acid | 217.70 | 222.97 | 229.07 | 232.37 | 234.39 | 3.03 |
| 32 | Homovanillic acid | 4.84 | 4.89 | 5.00 | 5.04 | 5.04 | 1.85 |
| 33 | Vanillylmandelic acid (VMA) | 5.20 | 5.26 | 5.34 | 5.37 | 5.42 | 1.65 |
| 34 | 5-HIAA | ＜DL | ＜DL | ＜DL | ＜DL | ＜DL | ? |
| 35 | Quinolinic acid | ＜DL | ＜DL | ＜DL | ＜DL | ＜DL | ? |
| 36 | Kynurenic acid | ＜DL | ＜DL | ＜DL | ＜DL | ＜DL | ? |
| 37 | Uracil | 0.16 | 0.16 | 0.17 | 0.17 | 0.16 | 3.34 |
| 38 | Thymine | ＜DL | ＜DL | ＜DL | ＜DL | ＜DL | ? |
| 39 | 3-hydroxybutyrate | 0.54 | 0.55 | 0.56 | 0.60 | 0.60 | 4.96 |
| 40 | Acetoacetate | 3.47 | 3.47 | 3.48 | 3.58 | 3.68 | 2.63 |
| 41 | 4 hydroxybutyric acid | 0.43 | 0.45 | 0.45 | 0.44 | 0.46 | 2.56 |
| 42 | Ethyl malonic acid | ＜DL | ＜DL | ＜DL | ＜DL | ＜DL | ? |
| 43 | Dimethyl succinic acid | 0.85 | 0.85 | 0.85 | 0.86 | 0.86 | 0.64 |
| 44 | Hexane diacid | 1.90 | 1.92 | 1.93 | 1.93 | 1.95 | 0.94 |
| 45 | Suberic acid | 1.20 | 1.20 | 1.21 | 1.21 | 1.23 | 1.01 |
| 46 | Decanedioic acid | ＜DL | ＜DL | ＜DL | ＜DL | ＜DL | ? |
| 47 | Methylmalonic acid | 1.66 | 1.68 | 1.71 | 1.74 | 1.76 | 2.41 |
| 48 | Pyridoxic acid | 2.09 | 2.11 | 2.14 | 2.15 | 2.17 | 1.50 |
| 49 | Pantothenic acid | 1.47 | 1.47 | 1.48 | 1.49 | 1.52 | 1.40 |
| 50 | Glutaric acid | ＜DL | ＜DL | ＜DL | ＜DL | ＜DL | ? |
| 51 | Ascorbic acid | ＜DL | ＜DL | ＜DL | ＜DL | ＜DL | ? |
| 52 | 3-hydroxy-3-methylglutaric acid | 7.98 | 8.04 | 8.30 | 8.33 | 8.56 | 2.86 |
| 53 | NAC | ＜DL | ＜DL | ＜DL | ＜DL | ＜DL | ? |
| 54 | Methyl citric acid | ＜DL | ＜DL | ＜DL | ＜DL | ＜DL | ? |
| 55 | Pyroglutamic acid | 0.84 | 0.85 | 0.87 | 0.88 | 0.90 | 2.75 |
| 56 | Orotic acid | 0.16 | 0.16 | 0.17 | 0.16 | 0.16 | 2.76 |
| 57 | 2-hydroxyl hippuric acid | ＜DL | ＜DL | ＜DL | ＜DL | ＜DL | ? |
| 58 | 2-hydroxyl isovaleric acid | 0.23 | 0.24 | 0.24 | 0.24 | 0.25 | 2.95 |
| 59 | 2-oxo isovaleric acid | 0.59 | 0.60 | 0.60 | 0.61 | 0.61 | 1.39 |
| 60 | 3-methyl-2-oxy pentanoic acid | 0.24 | 0.25 | 0.25 | 0.25 | 0.25 | 1.80 |
| 61 | 2-hydroxyl isocaproic acid | 0.17 | 0.17 | 0.18 | 0.18 | 0.18 | 3.11 |
| 62 | 2-Oxoisocaproic acid | 0.11 | 0.12 | 0.12 | 0.11 | 0.12 | 4.72 |
| 63 | 2-oxygen-4-(methylthio) butanoic acid | ＜DL | ＜DL | ＜DL | ＜DL | ＜DL | ? |
| 64 | Mandelic acid | ＜DL | ＜DL | ＜DL | ＜DL | ＜DL | ? |
| 65 | Phenyllactic acid | ＜DL | ＜DL | ＜DL | ＜DL | ＜DL | ? |
| 66 | Phenylpyruvic acid | 0.33 | 0.33 | 0.34 | 0.35 | 0.35 | 2.94 |
| 67 | Alcapton | ＜DL | ＜DL | ＜DL | ＜DL | ＜DL | ? |
| 68 | 4-hydroxyphenyl lactic acid | ＜DL | ＜DL | ＜DL | ＜DL | ＜DL | ? |
| 69 | N-acetyl group aspartic acid | 0.71 | 0.71 | 0.72 | 0.72 | 0.72 | 0.76 |
| 70 | Malonic acid | 0.21 | 0.21 | 0.21 | 0.22 | 0.22 | 2.56 |
| 71 | 3-methylglutaric acid | ＜DL | ＜DL | ＜DL | ＜DL | ＜DL | ? |
| 72 | Phosphoric acid | 524.87 | 530.06 | 539.31 | 544.23 | 547.58 | 1.78 |
3. the Stability Determination under the freezing placement of sample urine
Get certain sample urine, transfer and set to 0, after 3,6 and 9 days, by the method for the invention, sample urine carried out again the processing such as derivatization respectively at freezing conditions, and organic acid content in difference working sample urine, see the following form shown in (i.e. table 8), last hurdle is the RSD value of measuring numerical value under these four times, and result is known thus: sample is placed in 9 days stable in room temperature.
Table 8
The present invention is not limited to above-mentioned preferred forms, any modification relevant of the present invention or change that anyone does under enlightenment of the present invention, and every have identical with a application or akin technical scheme, within all dropping on protection scope of the present invention.
Claims (10)
1. a method for multiple organic acid content in while human body urine, is characterized in that: the method for detection specifically comprises the following steps:
A. collect the urine of urina sanguinis or random time section, and add the inner mark solution of 0.4mg/mL;
B. add 0.5mg/mL oxammonium hydrochloride solution and 10mol/L sodium hydroxide solution, the organic acid in urine sample is carried out to oximation reaction, fully vortex mixes and leaves standstill 1h;
C. add 12mol/L concentrated hydrochloric acid, then after adding ethyl acetate and ether fully to mix, centrifugal 5min, mixes; Draw upper organic phase and put into clean centrifuge tube; Dry up with 99.999% nitrogen;
D. add derivatization reagent, put into nitrogen flushing instrument base or incubator, 65 ℃ of condition temperature, derivatization 30min, described derivatization reagent comprises BSTFA and TMCS, the ratio of the two is 97～99.5: 0.5～3;
E. sample introduction, utilizes gas chromatograph-mass spectrometer analysis.
2. the method for multiple organic acid content in while human body urine according to claim 1, is characterized in that: the urine described in steps A is the urine that contains 0.2mg creatinine.
3. the method for multiple organic acid content in while human body urine according to claim 1, is characterized in that: the addition of the inner mark solution described in steps A is 20 μ L, and this inner mark solution is that internal standard compound is dissolved in deionized water and is prepared.
4. the method for multiple organic acid content in while human body urine according to claim 3, is characterized in that: described internal standard compound is 2-phenylbutyric acid.
5. the method for multiple organic acid content in while human body urine according to claim 1, is characterized in that: the addition of the oxammonium hydrochloride solution described in step B is 100 μ L, the aqueous solution that this oxammonium hydrochloride solution is oxammonium hydrochloride.
6. the method for multiple organic acid content in while human body urine according to claim 1, is characterized in that: the addition of the sodium hydroxide solution of 10mol/L described in step B is 300 μ L, the aqueous solution that this sodium hydroxide solution is NaOH.
7. the method for multiple organic acid content in while human body urine according to claim 1, is characterized in that: the addition of the concentrated hydrochloric acid described in step C is 300 μ L, and the addition of described ethyl acetate and ether is 2mL.
8. the method for multiple organic acid content in while human body urine according to claim 1, is characterized in that: the addition of the derivatization reagent described in step D is 100 μ L.
9. the method for multiple organic acid content in while human body urine according to claim 1, is characterized in that: the ratio of the BSTFA described in step D and TMCS is 99: 1.
10. the method for multiple organic acid content in while human body urine according to claim 1, is characterized in that: in step e: the design parameter of gas chromatograph-mass spectrometer used: capillary chromatography column length 30m,
thickness 0.25 μ m, 70 ℃ of column oven initial temperatures, 3min, be warming up to 260 ℃ with 10 ℃/min, and then be warming up to 280 ℃ with 20 ℃/min, and 2min, the helium overall flow rate that injector temperature is 250 ℃ 99.999% is 23.4mL/min, 280 ℃ of 20: 1 ion source temperatures of split ratio, sweep limit 50～550m/z.
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104569222A (en) * | 2015-01-29 | 2015-04-29 | 深圳爱湾医学技术服务有限公司 | Urine treatment method |
| CN104713874A (en) * | 2015-03-05 | 2015-06-17 | 杭州欣叶生物科技有限公司 | Breast cancer urine detection reagent, detection kit, as well as preparation and detection methods thereof |
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| CN107607666A (en) * | 2017-10-12 | 2018-01-19 | 福建省医学科学研究院 | The detection method of organic acid in a kind of biological sample |
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| CN115389684A (en) * | 2022-07-05 | 2022-11-25 | 广西精准医学科技有限公司 | Method for simultaneously detecting oxalic acid, citric acid and cystine in 24h urine |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102621247A (en) * | 2012-04-18 | 2012-08-01 | 王益超 | Method for synchronously analyzing base, nucleotide, organic acid, fatty acid, amino acid and saccharide metabolic product with two-step derivation method |
| CN102621249A (en) * | 2012-04-18 | 2012-08-01 | 王益超 | Method for synchronously analyzing base, nucleotide, organic acid, fatty acid, amino acid and saccharide metabolic product with multi-step derivation method |
-
2014
- 2014-03-21 CN CN201410105965.7A patent/CN103837634A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102621247A (en) * | 2012-04-18 | 2012-08-01 | 王益超 | Method for synchronously analyzing base, nucleotide, organic acid, fatty acid, amino acid and saccharide metabolic product with two-step derivation method |
| CN102621249A (en) * | 2012-04-18 | 2012-08-01 | 王益超 | Method for synchronously analyzing base, nucleotide, organic acid, fatty acid, amino acid and saccharide metabolic product with multi-step derivation method |
Non-Patent Citations (5)
| Title |
|---|
| 孙卫华 等: "气-质联用技术测定尿有机酸方法的建立及在遗传代谢病诊断中的应用", 《中华检验医学杂志》 * |
| 宋金青 等: "气相色谱-质谱联用分析在有机酸尿症筛查与诊断中的应用", 《中国医刊》 * |
| 张晓红 等: "比较两种不同预处理法的尿液有机酸分析在遗传代谢性疾病筛查和诊断", 《广州医学院学报》 * |
| 江敏妍 等: "尿GC－MS分析技术在遗传代谢缺陷病筛查诊断中的局限性", 《广东医学》 * |
| 罗小平 等: "尿滤纸片法气相色谱-质谱分析技术在遗传性代谢病高危筛查诊断中的应用", 《中华儿科杂志》 * |
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