CN104614452B - A kind of method of liquid chromatography-tandem mass spectrometry internal mark method determination L-hydroxyproline - Google Patents
A kind of method of liquid chromatography-tandem mass spectrometry internal mark method determination L-hydroxyproline Download PDFInfo
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Abstract
The present invention relates to a kind of method of L-hydroxyproline in liquid chromatography-tandem mass spectrometry (LC-MS/MS) internal mark method determination milk, with 4-hydroxy-piperdine-2-carboxylic acid for detecting interior mark, adopt Internal standard curve method quantitative.Prior art is compared, and quantitative limit of the present invention reaches 1mg/L, the response rate 80.2～116.5%, and relative error (RSD) is 4.2～12.6%.This method overall process to sample pre-treatments and mensuration, by Internal standard correction methods, has higher certainty of measurement and accuracy.
Description
Technical field
The present invention relates to internal mark method determination field, a kind of method especially relating to liquid chromatography-tandem mass spectrometry internal mark method determination L-hydroxyproline.
Background technology
" leather milk " is a kind of by adding leather hydrolyzed protein thus improving the milk of protein content Testing index.The harmful substance such as heavy metal chromium containing severe overweight in leather hydrolyzed protein, the healthy even life security of serious harm consumer.Leather hydrolyzed protein is also referred to as hydrolytic collagen, and the L-hydroxyproline distinctive aminoacid that is hydrolytic collagen, lactoprotein does not then have (namely the hydrolyzate of hydrolytic collagen contains L-hydroxyproline).Therefore, by measuring the L-hydroxyproline in lactoprotein hydrolyzation sample, it is possible to determine the hydrolytic collagen mixed or the amount of leather hydrolyzed protein.
The mensuration of low content L-hydroxyproline, it is possible to improve the detection limit of hydrolytic collagen.Internal standard method is a kind of accurately quantitative analysis tech at liquid chromatography-tandem mass spectrometry.Pre-treatment for lactoprotein sample is complex, hydrochloric acid etc. is evaporated off including the rotation that repeatedly adds water of Proteolytic enzyme, sample, at this moment in use, mark is more particularly suitable, it is possible to the impact that calibration and elimination produce due to sample pre-treatments condition and testing conditions fluctuation, it is thus achieved that higher certainty of measurement and accuracy.
Liquid chromatography-tandem mass spectrometry internal standard method, with L-thiamine for interior mark, (Xia Jingen Chen Bo Yao is honest and poor for the L-hydroxyproline in detection protolysate, HPLC-MS measures the hydroxyproline in collagen protein, chromatograph, 2008,26 (5): 595～598；Zhao Shanzhen, Deng Xiaojun, Peng Tao etc., hydrophilic interaction chromatography-quadrupole rod linear ion trap mass spectrometry of series connection measures L-hydroxyproline in protein food and remains, analytical chemistry, and 2011,39 (9): 1373～1379).There is amido link (as follows) in the structure of L-thiamine, the peptide bond constituting albumen is substantially also amido link, and when being hydrolyzed for a long time in acid, the peptide bond of theanine amido link and albumen is all hydrolyzed simultaneously.Therefore, acid hydrolysis uses theanine to be interior mark improper.From structure, 4-hydroxy-piperdine-2-carboxylic acid is hexatomic ring, and L-hydroxyproline is five-membered ring, and L-thiamine is fisher's formula；The only one of which amino of the former two, is all the imine structure on ring, and without amide, and L-thiamine has two amino, all different from L-hydroxyproline.Therefore, in structure, 4-hydroxy-piperdine-2-carboxylic acid and L-hydroxyproline similarity degree are much larger than L-thiamine.As interior mark, similarity degree is more big, and effect is more good.
4-hydroxy-piperdine-2-carboxylic acid is not primary amino acid, in the construction unit (aminoacid) of lactoprotein and hydrolytic collagen, it is absent from 4-hydroxy-piperdine-2-carboxylic acid, the i.e. hydrolysate of lactoprotein and hydrolytic collagen, it does not have 4-hydroxy-piperdine-2-carboxylic acid.In structure, 4-hydroxy-piperdine-2-carboxylic acid is absent from the unstable keys such as amido link, even if be hydrolyzed for a long time in acid, is also not easy degraded.Marking accordingly, as in the detection of acid hydrolysis, 4-hydroxy-piperdine-2-carboxylic acid is better than L-thiamine.
Summary of the invention
The purpose of the present invention is contemplated to overcome the defect that above-mentioned prior art exists and the method providing liquid chromatography-tandem mass spectrometry (LC-MS/MS) the internal mark method determination L-hydroxyproline of a kind of higher certainty of measurement and accuracy.
The purpose of the present invention can be achieved through the following technical solutions:
A kind of method of liquid chromatography-tandem mass spectrometry (LC-MS/MS) internal mark method determination L-hydroxyproline, it is characterised in that the method, with 4-hydroxy-piperdine-2-carboxylic acid for detecting interior mark, adopts Internal standard curve method quantitative.Specifically adopt following steps:
1) interior target preparation: select the 4-hydroxy-piperdine-2-carboxylic acid of purity >=99.0%, be placed in volumetric flask, adds acetonitrile and 1: 1～20 prepares the solvent obtained by volume with water, dissolve, constant volume, shake up, and 4-hydroxy-piperdine-2-carboxylic/solvent is 1g/L；
2) preparation of standard specimen: select the L-hydroxyproline of purity >=99.0%, be placed in volumetric flask, adds acetonitrile and 1: 1～20 prepares the solvent obtained by volume with water, dissolve, constant volume, shake up, and L-hydroxyproline/solvent is 1g/L；
3) hydrolysis standard specimen prepares: selects 4-hydroxy-piperdine-2-carboxylic acid and the L-hydroxyproline of purity >=99.0%, is respectively placed in volumetric flask, adds the hydrochloric acid that concentration is 4～12M, dissolve, constant volume, shakes up, and 4-hydroxy-piperdine-2-carboxylic acid concentration and L-Hydroxyproline concentration are all for 50mg/L；
4) preparation of samples: sample protein to be measured is placed in tube sealing, adding hydrolysis standard specimen and concentration is the hydrochloric acid of 4～12M, the ratio of sample protein and hydrolysis standard specimen, hydrochloric acid is: 20～100mg:1～10mL:10～30mL, and nitrogen is replaced, seal, hydrolysis；
5) sample pre-treatments: step 4) sample temperature be 90～120 DEG C be hydrolyzed 4～24 hours, hydrolyzation sample vacuum distilling removes hydrochloric acid (to pH=5～7), it is placed in volumetric flask, add acetonitrile and prepare the solvent that obtain at 1: 1 by volume with water, constant volume, shaking up, controlling 4-hydroxy-piperdine-2-carboxylic/solvent in solution is 0.1～10mg/L；
6) liquid chromatography-tandem mass spectrometry internal standard method is used, in utilizing, the determination content of mark and standard specimen obtains peak area ratio and the standard working curve of L-hydroxyproline and 4-hydroxy-piperdine-2-carboxylic acid, then utilizes the L-hydroxyproline content that above-mentioned working curve measures, calculates in sample protein.
Step 6) specifically adopt following steps:
Utilize step 1) and step 2) interior mark and standard specimen preparation 7 samples, in sample, 4-hydroxy-piperdine-2-carboxylic/solvent is 100mg/L, L-hydroxyproline/solvent is 1～200mg/L；Area with liquid chromatography tandom mass spectrometry determination L-hydroxyproline Yu 4-hydroxy-piperdine-2-carboxylic acid, with independent variable (mass ratio of L-hydroxyproline and 4-hydroxy-piperdine-2-carboxylic acid or mass concentration ratio), area ratio with dependent variable L-hydroxyproline Yu 4-hydroxy-piperdine-2-carboxylic acid) data, calculating obtains working curve y=10.12x+0.00892 (y is the area ratio of L-hydroxyproline and 4-hydroxy-piperdine-2-carboxylic acid, and x is L-hydroxyproline and the mass ratio of 4-hydroxy-piperdine-2-carboxylic acid or mass concentration ratio)；
Utilize the sample of liquid chromatography tandom mass spectrometry determination pre-treatment, retention time determine L-hydroxyproline and the area ratio of 4-hydroxy-piperdine-2-carboxylic acid, calculated by working curve and obtain L-hydroxyproline content.
Liquid phase chromatogram condition is as follows: chromatographic column be VenusilHilic post (2.1 × 150mm, 5 μm,), pretreatment column be VenusilHilicVenusilHilic post (2.1 × 10mm, 5 μm,)；Mobile phase A is acetonitrile: 0.5mmol/L ammonium acetate=9: 1, pH=6.68；Mobile phase B is acetonitrile: 25mmol/L ammonium acetate=6: 4, pH=6.67；Gradient elution, flow velocity is 300 μ L/min；Column temperature is 30 DEG C；Sample size is 10 μ L；
Gradient elution adopts below scheme:
| Time(min) | A% | B% |
| 0 | 95 | 5 |
| 10 | 75 | 25 2 --> |
| 12 | 75 | 25 |
| 12.5 | 95 | 5 |
| 15.0 | 95 | 5 |
Mass Spectrometry Conditions is as follows: spray voltage is 5000V；Sheath air pressure is 172.4kPa；Assist gas pressure is 34.5kPa；Capillary temperature is 275 DEG C；First pair of scanning ion is 132.1/86.2, and collision energy is 15eV；Second pair of scanning ion is 146.1/127.2, and collision energy is 18eV；Scan pattern is single ion detection scanning (SRM).
Liquid chromatography tandom mass spectrometry determination adopts TSQQuantumAccess high performance liquid chromatography-series connection quadrupole rod GC-MS that power & light company of the U.S. makes, and is furnished with electrospray ionization source and atmosphere pressure chemical ion source.
Described sample protein is lactoprotein.
Described sample protein includes milk, sheep milk, camel milk, people's milk, bean milk or Coconut milk.
Described sample protein is milk powder, condensed milk, Yoghourt, butter, cheese, dairy beverage, egg albumen powder, Semen Glycines powder, toffee, milk chocolate or milk chocolate.
Compared with prior art, the interior mark 4-hydroxy-piperdine-2-carboxylic acid price that the present invention adopts is relatively low, the more important thing is in acid hydrolysis stable, overall process to sample pre-treatments and mensuration, by Internal standard correction methods, has higher certainty of measurement and accuracy, to the L-hydroxyproline in different substrates protolysate, quantitative limit reaches 1mg/L, the response rate 80.2～116.5%, and relative error (RSD) is 4.2～12.6%.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in detail.
Embodiment 1
The testing result of lactoprotein-1.0%L-hydroxyproline hydrolysis
1) mark in: the 4-hydroxy-piperdine-2-carboxylic acid of purity >=99.0%, is dissolved in acetonitrile: water=1: in 1 (volume ratio), is made into 4-hydroxy-piperdine-2-carboxylic acid concentration for mark 100mL in 1g/L；
2) preparation of standard specimen: the L-hydroxyproline of purity >=99.0%, is dissolved in acetonitrile: water=1: in 1 (volume ratio), and being made into L-Hydroxyproline concentration is 1g/L standard specimen 100mL；
3) hydrolysis standard specimen 1 prepares: the 4-hydroxy-piperdine-2-carboxylic acid of purity >=99.0% and L-hydroxyproline, is dissolved in the hydrochloric acid that concentration is 6M, be made into 4-hydroxy-piperdine-2-carboxylic acid concentration and and L-Hydroxyproline concentration be all the hydrolysis standard specimen 100mL of 50mg/L；Hydrolysis standard specimen 2 prepares: a containing the internal standard 4-hydroxy-piperdine-2-carboxylic acid, without L-hydroxyproline, all the other are with standard specimen 1；
4) preparation of sample: 50mg albumen, is placed in tube sealing, adds 10.0mL step 3) described hydrolysis standard specimen 10mL, add the hydrochloric acid that 20mL concentration is 6M, nitrogen is replaced, and seals, hydrolysis；
5) sample pre-treatments: step 4) sample temperature be 110 DEG C be hydrolyzed 12 hours, hydrolyzation sample vacuum distilling removes hydrochloric acid (to pH=5～7) to neutral, it is placed in volumetric flask, add acetonitrile: water (volume ratio) solution, dissolve, constant volume, shake up, it is made into 20.0mL, for sample 1 to be measured；
6) with the L-hydroxyproline in liquid chromatography-tandem mass spectrometry internal mark method determination protolysate, L-hydroxyproline and 4-hydroxy-piperdine-2-carboxylic acid is determined by retention time and mass spectrometric data, by peak area ratio and standard working curve, calculate L-hydroxyproline content；
7) with the working curve of liquid chromatography-tandem mass spectrometry internal mark method determination L-hydroxyproline, undertaken by following steps:
7-1) working curve sample preparation: with above-mentioned 1) and 2) interior mark and standard specimen, prepare 7 samples, in sample, 4-hydroxy-piperdine-2-carboxylic acid concentration is 100mg/L, L-Hydroxyproline concentration is 1～200mg/L；
7-2) with the area of liquid chromatography tandom mass spectrometry determination L-hydroxyproline with 4-hydroxy-piperdine-2-carboxylic acid, working curve is that (y is the area ratio ratio of L-hydroxyproline and 4-hydroxy-piperdine-2-carboxylic acid to y=10.12x+0.00892, x is mass ratio or the mass concentration ratio of L-hydroxyproline and 4-hydroxy-piperdine-2-carboxylic acid), (instrument is TSQQuantumAccess high performance liquid chromatography-series connection quadrupole rod GC-MS in coefficient R=0.9999, power & light company of the U.S., is furnished with electrospray ionization source and atmosphere pressure chemical ion source)
7-3) Mass Spectrometry Conditions is as follows: spray voltage is 5000V；Sheath air pressure is 172.4kPa；Assist gas pressure is 34.5kPa；Capillary temperature is 275 DEG C；First pair of scanning ion is 132.1/86.2, and collision energy is 15eV；Second pair of scanning ion be 104.3/58.5 collision energy is 12eV；Scan pattern is single ion detection scanning (SRM).
7-4) chromatographic condition: chromatographic column be VenusilHilic post (2.1 × 150mm, 5 μm,), pre-column be VenusilHilicVenusilHilic post (2.1 × 10mm, 5 μm,)；Mobile phase A is acetonitrile: 0.5mmol/L ammonium acetate=9: 1, pH=6.68；Mobile phase B is acetonitrile: 25mmol/L ammonium acetate=6: 4, pH=6.67；Gradient elution, actual conditions is as shown in table 1；Flow velocity is 300 μ L/min；Column temperature is 30 DEG C；Sample size is 10 μ L.
8) area ratio with liquid chromatography-tandem mass spectrometry internal standard method detection sample 1, L-hydroxyproline with 4-hydroxy-piperdine-2-carboxylic acid is 0.9811, and thus calculating the amount obtaining L-hydroxyproline is 0.4805mg, the response rate 96.1%.
Embodiment 2
The testing result of lactoprotein-0.34%L-hydroxyproline hydrolysis
Similar embodiment 1, wherein step 4) in hydrolysis standard specimen 1 change 3.40mL into, add the hydrochloric acid that 26mL concentration is 6M；By step 5) hydrolysis, after demineralizing acid, it is made into 10mL, for sample 2 to be measured；All the other steps are identical with embodiment 1.
The area ratio measuring the L-hydroxyproline in sample 2 and 4-hydroxy-piperdine-2-carboxylic acid is 0.8647, and thus calculating the amount obtaining L-hydroxyproline is 0.1438mg, the response rate 84.6%.
Embodiment 3
The testing result of lactoprotein-0.10%L-hydroxyproline hydrolysis
Similar embodiment 1, step 4) in hydrolysis standard specimen 1 change 2.0mL into, add the hydrochloric acid that 28mL concentration is 6M；By step 5) hydrolysis, after demineralizing acid, it is made into 5.0mL, for sample 3；All the other steps are identical with embodiment 1.
The area ratio measuring the L-hydroxyproline in sample 3 and 4-hydroxy-piperdine-2-carboxylic acid is 0.8422, and thus calculating the amount obtaining L-hydroxyproline is 0.8234mg, the response rate 82.3%.
Embodiment 4
Measure the L-hydroxyproline in lactoprotein-10% hydrolytic collagen hydrolyzate
Similar embodiment 1, wherein step 4) in " adding 10.0mL hydrolysis standard specimen 1 ", change " adding 10.0mL hydrolysis standard specimen 2, add 5.0mg hydrolytic collagen " into, by step 5) hydrolysis, after demineralizing acid, be made into 20.0mL, for sample 4；All the other steps are identical with embodiment 1.
The area ratio measuring the L-hydroxyproline in sample 4 and 4-hydroxy-piperdine-2-carboxylic acid is 0.9246, and thus calculating the amount obtaining L-hydroxyproline is 0.4525mg.L-hydroxyproline content in hydrolytic collagen is 10.0%, and hydrolytic collagen amount is 4.52mg, the response rate 90.4%.
Embodiment 5
Measure the L-hydroxyproline in lactoprotein-2% hydrolytic collagen hydrolyzate
Similar embodiment 4, step 4) in " adding 5.0mg hydrolytic collagen, add 10.0mL hydrolysis standard specimen 2 " change " adding 1.0mg hydrolytic collagen, add 2.0mL hydrolysis standard specimen 2 " into；By step 5) hydrolysis, after demineralizing acid, it is made into 5.0mL testing sample 5；All the other are identical with embodiment 4.
The area ratio measuring the L-hydroxyproline in sample 5 and 4-hydroxy-piperdine-2-carboxylic acid is 0.8194, and thus calculating the amount obtaining hydroxyproline is 0.0801mg.L-hydroxyproline content in hydrolytic collagen is 10.0%, and hydrolytic collagen amount is 0.801mg, the response rate 80.1%.
Embodiment 6
Substrate (lactoprotein) the mark-on hydrolysis response rate
Similar embodiment 1, wherein step 4) in " 50mg albumen; add 10.0mL hydrolysis standard specimen 1 ", change " 50mg albumen respectively adds 0.10mg, 0.25mg, 0.5mgL-hydroxyproline " into, after hydrolysis, measure the content of L-hydroxyproline, 6 experiments of each repetition, result is as shown in the table, it is seen that the response rate of L-hydroxyproline is higher.Therefore, with mark in the detection of 4-hydroxy-piperdine-2-carboxylic acid, it is possible to obtain higher accuracy in detection.
Lactoprotein adds L-hydroxyproline results of hydrolysis
Embodiment 7
Other substrate mark-on hydrolysis response rate
Similar embodiment 6, wherein step 4) in " 50.0mg albumen " change 1.67g milk into (protein content be 3.0%, mass ratio), (protein content is 4.0% to 1.25g sheep milk, mass ratio), (protein content is 17.5% to 0.286g milk powder, mass ratio), (protein content is 50.0% to 100.0mg egg albumen powder, mass ratio), add 0.10mg, 0.167mg, 0.5mgL-hydroxyproline respectively.After hydrolysis, vacuum distilling demineralizing acid is to pH value of solution=5, and PEP solid phase extraction column (200mg crossed by sample, 6mL), cross 0.22 μm of hydrophilic PTFE filter membrane, measure L-hydroxyproline, the experimental result that each group repeats 6 times is as shown in the table, it is seen that the response rate of L-hydroxyproline is higher.Therefore, with mark, the detection to different substrate, it is also possible to obtain higher accuracy in detection in the detection of 4-hydroxy-piperdine-2-carboxylic acid.
Substrate adds L-hydroxyproline results of hydrolysis
Embodiment 8
Hydroxyproline detects
Similar embodiment 1, it does not have albumen and hydrolysis, measures Hydroxyproline concentration in preparation sample.Step: accurately weigh L-hydroxyproline, with acetonitrile: water=1: 1 (volume ratio) prepares 6 samples of L-hydroxyproline, concentration is 1～150mg/L, L-hydroxyproline is measured with Liquid Chromatography-Tandem Mass Spectrometry, the result that each sample repeats 3 tests is as shown in the table, and the L-hydroxyproline response rate is higher.Visible with mark in the detection of 4-hydroxy-piperdine-2-carboxylic acid, measure L-hydroxyproline, it is also possible to obtain higher accuracy in detection.
L-hydroxyproline determination result
Embodiment 9
A kind of method of liquid chromatography-tandem mass spectrometry (LC-MS/MS) internal mark method determination L-hydroxyproline, it is characterised in that the method, with 4-hydroxy-piperdine-2-carboxylic acid for detecting interior mark, adopts Internal standard curve method quantitative.Specifically adopt following steps:
1) interior target preparation: select the 4-hydroxy-piperdine-2-carboxylic acid of purity >=99.0%, be placed in volumetric flask, adds acetonitrile and prepares the solvent that obtain at 1: 1 by volume with water, dissolve, constant volume, shake up, and 4-hydroxy-piperdine-2-carboxylic/solvent is 1g/L；
2) preparation of standard specimen: select the L-hydroxyproline of purity >=99.0%, be placed in volumetric flask, adds acetonitrile and prepares the solvent that obtain at 1: 1 by volume with water, dissolve, constant volume, shake up, and L-hydroxyproline/solvent is 1g/L；
3) hydrolysis standard specimen prepares: selects 4-hydroxy-piperdine-2-carboxylic acid and the L-hydroxyproline of purity >=99.0%, is respectively placed in volumetric flask, adds the hydrochloric acid that concentration is 4M, dissolve, constant volume, shakes up, and 4-hydroxy-piperdine-2-carboxylic acid concentration and L-Hydroxyproline concentration are all for 50mg/L；
4) preparation of samples: be placed in tube sealing by sample protein to be measured, adds hydrolysis standard specimen and concentration is the hydrochloric acid of 4M, and the ratio of sample protein and hydrolysis standard specimen, hydrochloric acid is: 20mg: 1mL: 10mL, and nitrogen is replaced, and seals, hydrolysis；
5) sample pre-treatments: step 4) sample temperature be 90 DEG C be hydrolyzed 24 hours, hydrolyzation sample vacuum distilling removes hydrochloric acid, to pH=5, it is placed in volumetric flask, add acetonitrile and prepare the solvent that obtain at 1: 1 by volume with water, constant volume, shakes up, and controlling 4-hydroxy-piperdine-2-carboxylic/solvent in solution is 0.1mg/L；
6) liquid chromatography-tandem mass spectrometry internal standard method is used, in utilizing, the determination content of mark and standard specimen obtains peak area ratio and the standard working curve of L-hydroxyproline and 4-hydroxy-piperdine-2-carboxylic acid, then the L-hydroxyproline content utilizing above-mentioned working curve to measure, calculate in sample protein, specifically adopts following steps:
Utilize step 1) and step 2) interior mark and standard specimen preparation 7 samples, in sample, 4-hydroxy-piperdine-2-carboxylic/solvent is 100mg/L, L-hydroxyproline/solvent is 1mg/L；Area with liquid chromatography tandom mass spectrometry determination L-hydroxyproline Yu 4-hydroxy-piperdine-2-carboxylic acid, with independent variable (mass ratio of L-hydroxyproline and 4-hydroxy-piperdine-2-carboxylic acid or mass concentration ratio), area ratio with dependent variable L-hydroxyproline Yu 4-hydroxy-piperdine-2-carboxylic acid) data, calculating obtains working curve y=10.12x+0.00892 (y is the area ratio of L-hydroxyproline and 4-hydroxy-piperdine-2-carboxylic acid, and x is L-hydroxyproline and the mass ratio of 4-hydroxy-piperdine-2-carboxylic acid or mass concentration ratio)；
Utilize the sample of liquid chromatography tandom mass spectrometry determination pre-treatment, retention time determine L-hydroxyproline and the area ratio of 4-hydroxy-piperdine-2-carboxylic acid, calculated by working curve and obtain L-hydroxyproline content.
Liquid phase chromatogram condition is as follows: chromatographic column be VenusilHilic post (2.1 × 150mm, 5 μm,), pretreatment column be VenusilHilicVenusilHilic post (2.1 × 10mm, 5 μm,)；Mobile phase A is acetonitrile: 0.5mmol/L ammonium acetate=9: 1, pH=6.68；Mobile phase B is acetonitrile: 25mmol/L ammonium acetate=6: 4, pH=6.67；Gradient elution, flow velocity is 300 μ L/min；Column temperature is 30 DEG C；Sample size is 10 μ L；
Gradient elution adopts below scheme:
| Time(min) | A% | B% |
| 0 | 95 | 5 |
| 10 | 75 | 25 |
| 12 | 75 | 25 |
| 12.5 | 95 | 5 |
| 15.0 | 95 | 5 |
Mass Spectrometry Conditions is as follows: spray voltage is 5000V；Sheath air pressure is 172.4kPa；Assist gas pressure is 34.5kPa；Capillary temperature is 275 DEG C；First pair of scanning ion is 132.1/86.2, and collision energy is 15eV；Second pair of scanning ion is 146.1/127.2, and collision energy is 18eV；Scan pattern is single ion detection scanning (SRM).
Liquid chromatography tandom mass spectrometry determination adopts TSQQuantumAccess high performance liquid chromatography-series connection quadrupole rod GC-MS that power & light company of the U.S. makes, and is furnished with electrospray ionization source and atmosphere pressure chemical ion source.
Institute's test sample albumen is lactoprotein, such as milk, sheep milk, camel milk, people's milk, bean milk or Coconut milk, in addition to this it is possible to be milk powder, condensed milk, Yoghourt, butter, cheese, dairy beverage, egg albumen powder, Semen Glycines powder, toffee, milk chocolate or milk chocolate.
Embodiment 10
A kind of method of liquid chromatography-tandem mass spectrometry (LC-MS/MS) internal mark method determination L-hydroxyproline, it is characterised in that the method, with 4-hydroxy-piperdine-2-carboxylic acid for detecting interior mark, adopts Internal standard curve method quantitative.Specifically adopt following steps:
1) interior target preparation: select the 4-hydroxy-piperdine-2-carboxylic acid of purity >=99.0%, be placed in volumetric flask, adds acetonitrile and prepares the solvent that obtain at 1: 20 by volume with water, dissolve, constant volume, shake up, and 4-hydroxy-piperdine-2-carboxylic/solvent is 1g/L；
2) preparation of standard specimen: select the L-hydroxyproline of purity >=99.0%, be placed in volumetric flask, adds acetonitrile and prepares the solvent that obtain at 1: 20 by volume with water, dissolve, constant volume, shake up, and L-hydroxyproline/solvent is 1g/L；
3) hydrolysis standard specimen prepares: selects 4-hydroxy-piperdine-2-carboxylic acid and the L-hydroxyproline of purity >=99.0%, is respectively placed in volumetric flask, adds the hydrochloric acid that concentration is 12M, dissolve, constant volume, shakes up, and 4-hydroxy-piperdine-2-carboxylic acid concentration and L-Hydroxyproline concentration are all for 50mg/L；
4) preparation of samples: be placed in tube sealing by sample protein to be measured, adds hydrolysis standard specimen and concentration is the hydrochloric acid of 12M, and the ratio of sample protein and hydrolysis standard specimen, hydrochloric acid is: 100mg: 10mL: 30mL, and nitrogen is replaced, and seals, hydrolysis；
5) sample pre-treatments: step 4) sample temperature be 120 DEG C be hydrolyzed 4 hours, hydrolyzation sample vacuum distilling removes hydrochloric acid, to pH=7, it is placed in volumetric flask, add acetonitrile and prepare the solvent that obtain at 1: 1 by volume with water, constant volume, shakes up, and controlling 4-hydroxy-piperdine-2-carboxylic/solvent in solution is 10mg/L；
6) liquid chromatography-tandem mass spectrometry internal standard method is used, in utilizing, the determination content of mark and standard specimen obtains peak area ratio and the standard working curve of L-hydroxyproline and 4-hydroxy-piperdine-2-carboxylic acid, then the L-hydroxyproline content utilizing above-mentioned working curve to measure, calculate in sample protein, specifically adopts following steps:
Utilize step 1) and step 2) interior mark and standard specimen preparation 7 samples, in sample, 4-hydroxy-piperdine-2-carboxylic/solvent is 100mg/L, L-hydroxyproline/solvent is 200mg/L；Area with liquid chromatography tandom mass spectrometry determination L-hydroxyproline Yu 4-hydroxy-piperdine-2-carboxylic acid, with independent variable (mass ratio of L-hydroxyproline and 4-hydroxy-piperdine-2-carboxylic acid or mass concentration ratio), area ratio with dependent variable L-hydroxyproline Yu 4-hydroxy-piperdine-2-carboxylic acid) data, calculating obtains working curve y=10.12x+0.00892 (y is the area ratio of L-hydroxyproline and 4-hydroxy-piperdine-2-carboxylic acid, and x is L-hydroxyproline and the mass ratio of 4-hydroxy-piperdine-2-carboxylic acid or mass concentration ratio)；
Utilize the sample of liquid chromatography tandom mass spectrometry determination pre-treatment, retention time determine L-hydroxyproline and the area ratio of 4-hydroxy-piperdine-2-carboxylic acid, calculated by working curve and obtain L-hydroxyproline content.
Liquid phase chromatogram condition is as follows: chromatographic column be VenusilHilic post (2.1 × 150mm, 5 μm,), pretreatment column be VenusilHilicVenusilHilic post (2.1 × 10mm, 5 μm,)；Mobile phase A is acetonitrile: 0.5mmol/L ammonium acetate=9: 1, pH=6.68: Mobile phase B is acetonitrile: 25mmol/L ammonium acetate=6: 4, pH=6.67；Gradient elution, flow velocity is 300 μ L/min；Column temperature is 30 DEG C；Sample size is 10 μ L；
Gradient elution adopts below scheme:
| Time(min) | A% | B% |
| 0 | 95 | 5 |
| 10 | 75 | 25 |
| 12 | 75 | 25 |
| 12.5 | 95 | 5 |
| 15.0 | 95 | 5 |
Mass Spectrometry Conditions is as follows: spray voltage is 5000V；Sheath air pressure is 172.4kPa；Assist gas pressure is 34.5kPa；Capillary temperature is 275 DEG C；First pair of scanning ion is 132.1/86.2, and collision energy is 15eV；Second pair of scanning ion is 146.1/127.2, and collision energy is 18eV；Scan pattern is single ion detection scanning (SRM).
Liquid chromatography tandom mass spectrometry determination adopts TSQQuantumAccess high performance liquid chromatography-series connection quadrupole rod GC-MS that power & light company of the U.S. makes, and is furnished with electrospray ionization source and atmosphere pressure chemical ion source.
Institute's test sample albumen is lactoprotein, such as milk, sheep milk, camel milk, people's milk, bean milk or Coconut milk, in addition to this it is possible to be milk powder, condensed milk, Yoghourt, butter, cheese, dairy beverage, egg albumen powder, Semen Glycines powder, toffee, milk chocolate or milk chocolate.
Claims (5)
1. the method for liquid chromatography-tandem mass spectrometry (LC-MS/MS) internal mark method determination L-hydroxyproline, it is characterised in that the method, with 4-hydroxy-piperdine-2-carboxylic acid for detecting interior mark, adopts Internal standard curve method quantitative, specifically adopts following steps:
1) interior target preparation: select the 4-hydroxy-piperdine-2-carboxylic acid of purity >=99.0%, be placed in volumetric flask, adds acetonitrile and prepares, with water 1:1～20 by volume, the solvent obtained, dissolve, constant volume, shake up, and 4-hydroxy-piperdine-2-carboxylic/solvent is 1g/L；
2) preparation of standard specimen: select the L-hydroxyproline of purity >=99.0%, be placed in volumetric flask, adds acetonitrile and prepares, with water 1:1～20 by volume, the solvent obtained, dissolve, constant volume, shake up, and L-hydroxyproline/solvent is 1g/L；
3) hydrolysis standard specimen prepares: selects 4-hydroxy-piperdine-2-carboxylic acid and the L-hydroxyproline of purity >=99.0%, is respectively placed in volumetric flask, adds the hydrochloric acid that concentration is 4～12M, dissolve, constant volume, shakes up, and 4-hydroxy-piperdine-2-carboxylic acid concentration and L-Hydroxyproline concentration are all for 50mg/L；
4) preparation of samples: sample protein to be measured is placed in tube sealing, adding hydrolysis standard specimen and concentration is the hydrochloric acid of 4～12M, the ratio of sample protein and hydrolysis standard specimen, hydrochloric acid is: 20～100mg: 1～10mL: 10～30mL, and nitrogen is replaced, seal, hydrolysis；
5) sample pre-treatments: step 4) sample temperature be 90～120 DEG C be hydrolyzed 4～24 hours, hydrolyzation sample vacuum distilling removes hydrochloric acid, pH=5～7, it is placed in volumetric flask, add acetonitrile and prepare, with water 1:1 by volume, the solvent obtained, constant volume, shakes up, and controlling 4-hydroxy-piperdine-2-carboxylic/solvent in solution is 0.1～10mg/L；
6) liquid chromatography-tandem mass spectrometry internal standard method is used, in utilizing, the determination content of mark and standard specimen obtains peak area ratio and the standard working curve of L-hydroxyproline and 4-hydroxy-piperdine-2-carboxylic acid, then utilizes above-mentioned working curve to measure, calculates L-hydroxyproline content in sample protein；
Wherein, step 6) specifically adopt following steps:
Utilize step 1) and step 2) interior mark and standard specimen preparation 7 samples, in sample, 4-hydroxy-piperdine-2-carboxylic/solvent is 100mg/L, L-hydroxyproline/solvent is 1～200mg/L；Area with liquid chromatography tandom mass spectrometry determination L-hydroxyproline Yu 4-hydroxy-piperdine-2-carboxylic acid, with the mass ratio of L-hydroxyproline and 4-hydroxy-piperdine-2-carboxylic acid or mass concentration ratio for independent variable, the area ratio of L-hydroxyproline and 4-hydroxy-piperdine-2-carboxylic acid is dependent variable data, calculating obtains working curve y=10.12x+0.00892, wherein y is the area ratio of L-hydroxyproline and 4-hydroxy-piperdine-2-carboxylic acid, and x is mass ratio or the mass concentration ratio of L-hydroxyproline and 4-hydroxy-piperdine-2-carboxylic acid；
Utilize the sample of liquid chromatography tandom mass spectrometry determination pre-treatment, retention time determine L-hydroxyproline and the area ratio of 4-hydroxy-piperdine-2-carboxylic acid, calculated by working curve and obtain L-hydroxyproline content；
Liquid phase chromatogram condition is as follows: chromatographic column is 2.1 × 150mm, 5 μm,VenusilHilic post, pretreatment column is 2.1 × 10mm, 5 μm,VenusilHilicVenusilHilic post；Mobile phase A is acetonitrile: 0.5mmol/L ammonium acetate=9:1, pH=6.68；Mobile phase B is acetonitrile: 25mmol/L ammonium acetate=6:4, pH=6.67；Gradient elution, flow velocity is 300 μ L/min；Column temperature is 30 DEG C；Sample size is 10 μ L；
Mass Spectrometry Conditions is as follows: spray voltage is 5000V；Sheath air pressure is 172.4kPa；Assist gas pressure is 34.5kPa；Capillary temperature is 275 DEG C；First pair of scanning ion is 132.1/86.2, and collision energy is 15eV；Second pair of scanning ion is 146.1/127.2, and collision energy is 18eV；Scan pattern is single ion detection scanning (SRM).
2. the method for a kind of liquid chromatography-tandem mass spectrometry internal mark method determination L-hydroxyproline according to claim 1, liquid chromatography tandom mass spectrometry determination adopts TSQQuantumAccess high performance liquid chromatography-series connection quadrupole rod GC-MS that power & light company of the U.S. makes, and is furnished with electrospray ionization source and atmosphere pressure chemical ion source.
3. the method for a kind of liquid chromatography-tandem mass spectrometry internal mark method determination L-hydroxyproline according to claim 1, described sample protein is lactoprotein.
4. the method for a kind of liquid chromatography-tandem mass spectrometry internal mark method determination L-hydroxyproline according to claim 1, described sample protein includes milk, sheep milk, camel milk, people's milk, bean milk or Coconut milk.
5. the method for a kind of liquid chromatography-tandem mass spectrometry internal mark method determination L-hydroxyproline according to claim 1, described sample protein is milk powder, condensed milk, Yoghourt, butter, cheese, dairy beverage, egg albumen powder, Semen Glycines powder, toffee, milk chocolate or milk chocolate.
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