CN104614452A - Method for determining L-hydroxyproline through liquid chromatogram-tandem mass spectrum internal standard process - Google Patents
Method for determining L-hydroxyproline through liquid chromatogram-tandem mass spectrum internal standard process Download PDFInfo
- Publication number
- CN104614452A CN104614452A CN201410748715.5A CN201410748715A CN104614452A CN 104614452 A CN104614452 A CN 104614452A CN 201410748715 A CN201410748715 A CN 201410748715A CN 104614452 A CN104614452 A CN 104614452A
- Authority
- CN
- China
- Prior art keywords
- hydroxyproline
- piperdine
- hydroxy
- carboxylic acid
- liquid chromatography
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention relates to a method for determining L-hydroxyproline through a liquid chromatogram-tandem mass spectrum (LC-MS/MS) internal standard process. 4-hydroxypiperidine-2-carboxylic acid is taken as a detection internal standard, and the internal-standard standard curve process is employed for quantification. Compared with the prior art, the method has the quantitative limit of 1 mg/L, the recovery rate of 80.2-116.5%, and the relative standard deviation (RSD) of 4.2-12.6%. According to the method, the whole process for sample pretreatment and determination is corrected through the internal standard, and the method has relatively high measure precision and accuracy.
Description
Technical field
The present invention relates to internal mark method determination field, especially relate to a kind of method of liquid chromatography-tandem mass spectrometry internal mark method determination L-hydroxyproline.
Background technology
" leather milk " is a kind of by adding leather protein hydrolysate thus the milk of raising protein content Testing index.The objectionable impuritiess such as the heavy metal chromium containing severe overweight in leather protein hydrolysate, the healthy even life security of serious harm consumer.Leather protein hydrolysate also claims Hydrolyzed Collagen, and the L-hydroxyproline distinctive amino acid that is Hydrolyzed Collagen, then do not have in lactoprotein (namely the hydrolysate of Hydrolyzed Collagen contains L-hydroxyproline).Therefore, by measuring the L-hydroxyproline in lactoprotein hydrolyzation sample, the amount of Hydrolyzed Collagen or the leather protein hydrolysate mixed can just be determined.
The mensuration of low content L-hydroxyproline, can improve the detection limit of Hydrolyzed Collagen.Internal standard method is one quantitative analysis tech comparatively accurately at liquid chromatography-tandem mass spectrometry.Pre-treatment for lactoprotein sample is comparatively complicated, comprise proteolysis, sample repeatedly to add water to revolve and steam demineralizing acid etc., at this moment in using, mark is more suitable, can calibrate and eliminate the impact because sample pre-treatments condition and testing conditions fluctuation produce, obtain higher measuring accuracy and accuracy.
Liquid chromatography-tandem mass spectrometry internal standard method is interior mark with L-thiamine, and (Xia Jingen Chen Bo Yao is honest and poor for the L-hydroxyproline in detection protolysate, HPLC-MS measures the hydroxyproline in collagen, chromatogram, 2008,26 (5): 595 ~ 598; Zhao Shanzhen, Deng Xiaojun, Peng Tao etc., hydrophilic interaction chromatography-quadrupole rod linear ion trap mass spectrometry of connecting measures L-hydroxyproline in protein food and remains, analytical chemistry, 2011,39 (9): 1373 ~ 1379).There is amido link (as follows) in the structure of L-thiamine, the peptide bond forming albumen is also amido link in essence, and when being hydrolyzed for a long time in acid, the peptide bond of theanine amido link and albumen is hydrolyzed all simultaneously.Therefore, acid hydrolysis uses theanine to be interior mark and 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 former two only has an amino, and be all the imine structure on ring, without acid amides, 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 larger, and effect is better.
4-hydroxy-piperdine-2-carboxylic acid is not primary amino acid, in the structural unit (amino acid) of lactoprotein and Hydrolyzed Collagen, there is not 4-hydroxy-piperdine-2-carboxylic acid, i.e. the hydrolysate of lactoprotein and Hydrolyzed Collagen, there is no 4-hydroxy-piperdine-2-carboxylic acid.In structure, there is not the key of the instability such as amido link in 4-hydroxy-piperdine-2-carboxylic acid, even if when being hydrolyzed for a long time in acid, is not easy degraded yet.Therefore, mark as in the detection of acid hydrolysis, 4-hydroxy-piperdine-2-carboxylic acid is better than L-thiamine.
Summary of the invention
Object of the present invention is exactly provide the method for liquid chromatography-tandem mass spectrometry (LC-MS/MS) the internal mark method determination L-hydroxyproline of a kind of higher measuring accuracy and accuracy to overcome defect that above-mentioned prior art exists.
Object 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, is characterized in that, the method for detecting interior mark, adopts Internal standard curve method quantitative with 4-hydroxy-piperdine-2-carboxylic acid.Concrete employing following steps:
1) interior target preparation: the 4-hydroxy-piperdine-2-carboxylic acid selecting purity >=99.0%, is placed in volumetric flask, adds acetonitrile and water 1: 1 ~ 20 prepares the solvent obtained by volume, and dissolve, constant volume, shakes up, and 4-hydroxy-piperdine-2-carboxylic/solvent is 1g/L;
2) preparation of standard specimen: the L-hydroxyproline selecting purity >=99.0%, is placed in volumetric flask, adds acetonitrile and water 1: 1 ~ 20 prepares the solvent obtained by volume, and dissolve, constant volume, shakes up, and L-hydroxyproline/solvent is 1g/L;
3) hydrolysis standard specimen prepares: 4-hydroxy-piperdine-2-carboxylic acid and the L-hydroxyproline of selecting purity >=99.0%, be placed in volumetric flask respectively, add 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 be 50mg/L;
4) preparation of samples: sample protein to be measured is placed in tube sealing, add 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, sealing, hydrolysis;
5) sample pre-treatments: step 4) sample temperature be 90 ~ 120 DEG C hydrolysis 4 ~ 24 hours, hydrolyzation sample vacuum distillation removing hydrochloric acid (to pH=5 ~ 7), be placed in volumetric flask, add acetonitrile and water prepares the solvent that obtain by volume at 1: 1, constant volume, shake 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 in above-mentioned working curve mensuration, calculation sample albumen.
Step 6) specifically adopt following steps:
Utilize step 1) and step 2) interior mark and standard specimen prepare 7 samples, in sample, 4-hydroxy-piperdine-2-carboxylic/solvent is 100mg/L, L-hydroxyproline/solvent is 1 ~ 200mg/L; With the area of liquid chromatography tandom mass spectrometry determination L-hydroxyproline and 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), with the area ratio of dependent variable L-hydroxyproline and 4-hydroxy-piperdine-2-carboxylic acid) data, calculate 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 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, determined the area ratio of L-hydroxyproline and 4-hydroxy-piperdine-2-carboxylic acid by retention time, calculate L-hydroxyproline content by working curve.
Liquid phase chromatogram condition is as follows: chromatographic column be Venusil Hilic post (2.1 × 150mm, 5 μm,
), pretreatment column be Venusil Hilic Venusil Hilic 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 following flow process:
| 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 scan ion is 132.1/86.2, and collision energy is 15eV; Second pair of scan ion is 146.1/127.2, and collision energy is 18eV; Scan pattern is single ion detection scanning (SRM).
TSQ Quantum Access high performance liquid chromatography-series connection quadrupole rod GC-MS that liquid chromatography tandom mass spectrometry determination adopts power & light company of the U.S. to make, is furnished with electrospray ionization source and atmosphere pressure chemical ion source.
Described sample protein is lactoprotein.
Described sample protein comprises milk, goat milk, camel milk, people's milk, soymilk or coconut milk.
Described sample protein is milk powder, condensed milk, Yoghourt, cream, cheese, dairy beverage, albumen powder, bean 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 lower, the more important thing is in acid hydrolysis stable, to the overall process of sample pre-treatments and mensuration, by Internal standard correction methods, there are higher measuring accuracy and accuracy, to the L-hydroxyproline in different substrates protolysate, quantitative limit reaches 1mg/L, the recovery 80.2 ~ 116.5%, and relative error (RSD) is 4.2 ~ 12.6%.
Embodiment
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), 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, be 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: only containing interior mark 4-hydroxy-piperdine-2-carboxylic acid, and not containing 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, sealing, hydrolysis;
5) sample pre-treatments: step 4) sample temperature be 110 DEG C hydrolysis 12 hours, hydrolyzation sample vacuum distillation removing hydrochloric acid (to pH=5 ~ 7) is to neutral, be placed in volumetric flask, add acetonitrile: water (volume ratio) solution, dissolve, constant volume, shakes up, being made into 20.0mL, is 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) use the area of liquid chromatography tandom mass spectrometry determination L-hydroxyproline and 4-hydroxy-piperdine-2-carboxylic acid, working curve is that (y is L-hydroxyproline and the area ratio ratio of 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 TSQ Quantum Access 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 scan ion is 132.1/86.2, and collision energy is 15eV; The second pair of scan ion is 104.3/58.5 collision energy is 12eV; Scan pattern is single ion detection scanning (SRM).
7-4) chromatographic condition: chromatographic column be Venusil Hilic post (2.1 × 150mm, 5 μm,
), pre-column be Venusil Hilic Venusil Hilic 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
as table 1shown in; Flow velocity is 300 μ L/min; Column temperature is 30 DEG C; Sample size is 10 μ L.
8) area ratio using liquid chromatography-tandem mass spectrometry internal standard method to detect sample 1, L-hydroxyproline and 4-hydroxy-piperdine-2-carboxylic acid is 0.9811, and the amount calculating L-hydroxyproline is thus 0.4805mg, the recovery 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, being made into 10mL, is sample 2 to be measured; All the other steps are identical with embodiment 1.
The area ratio of the L-hydroxyproline in working sample 2 and 4-hydroxy-piperdine-2-carboxylic acid is 0.8647, and the amount calculating L-hydroxyproline is thus 0.1438mg, the recovery 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, being made into 5.0mL, is sample 3; All the other steps are identical with embodiment 1.
The area ratio of the L-hydroxyproline in working sample 3 and 4-hydroxy-piperdine-2-carboxylic acid is 0.8422, and the amount calculating L-hydroxyproline is thus 0.8234mg, the recovery 82.3%.
Embodiment 4
Measure the L-hydroxyproline in lactoprotein-10% Hydrolyzed Collagen hydrolysate
Similar embodiment 1, wherein step 4) in " adding 10.0mL hydrolysis standard specimen 1 ", change into " add 10.0mL hydrolysis standard specimen 2, add 5.0mg Hydrolyzed Collagen ", by step 5) hydrolysis, after demineralizing acid, being made into 20.0mL, is sample 4; All the other steps are identical with embodiment 1.
The area ratio of the L-hydroxyproline in working sample 4 and 4-hydroxy-piperdine-2-carboxylic acid is 0.9246, and the amount calculating L-hydroxyproline is thus 0.4525mg.L-hydroxyproline content in Hydrolyzed Collagen is 10.0%, and Hydrolyzed Collagen amount is 4.52mg, the recovery 90.4%.
Embodiment 5
Measure the L-hydroxyproline in lactoprotein-2% Hydrolyzed Collagen hydrolysate
Similar embodiment 4, step 4) in " add 5.0mg Hydrolyzed Collagen, add 10.0mL hydrolysis standard specimen 2 " change into " add 1.0mg Hydrolyzed Collagen, add 2.0mL hydrolysis standard specimen 2 "; By step 5) hydrolysis, after demineralizing acid, be made into 5.0mL testing sample 5; All the other are identical with embodiment 4.
The area ratio of the L-hydroxyproline in working sample 5 and 4-hydroxy-piperdine-2-carboxylic acid is 0.8194, and the amount calculating hydroxyproline is thus 0.0801mg.L-hydroxyproline content in Hydrolyzed Collagen is 10.0%, and Hydrolyzed Collagen amount is 0.801mg, the recovery 80.1%.
Embodiment 6
Matrix (lactoprotein) the mark-on hydrolysis recovery
Similar embodiment 1, wherein step 4) in " 50mg albumen adds 10.0mL hydrolysis standard specimen 1 ", change into " 50mg albumen is respectively and adds 0.10mg, 0.25mg, 0.5mgL-hydroxyproline ", after hydrolysis, measure the content of L-hydroxyproline, respectively repeat 6 experiments, result as
following table instituteshow, the recovery of visible L-hydroxyproline is higher.Therefore, mark in detecting with 4-hydroxy-piperdine-2-carboxylic acid, can obtain higher accuracy in detection.
Lactoprotein adds L-hydroxyproline results of hydrolysis
Embodiment 7
Other matrix mark-on hydrolysis recovery
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 goat milk, mass ratio), (protein content is 17.5% to 0.286g milk powder, mass ratio), (protein content is 50.0% to 100.0mg albumen powder, mass ratio), add 0.10mg, 0.167mg, 0.5mgL-hydroxyproline respectively.After hydrolysis, vacuum distillation demineralizing acid is to pH value of solution=5, and PEP solid phase extraction column (200mg, 6mL) crossed by sample, crosses 0.22 μm of hydrophilic PTFE filter membrane, measures L-hydroxyproline, the experimental result that the repetition of each group is 6 times as
following table instituteshow, the recovery of visible L-hydroxyproline is higher.Therefore, mark in detecting with 4-hydroxy-piperdine-2-carboxylic acid, to the detection of different matrix, also can obtain higher accuracy in detection.
Matrix adds L-hydroxyproline results of hydrolysis
Embodiment 8
Hydroxyproline detects
Similar embodiment 1, does not have albumen and hydrolysis, measures Hydroxyproline concentration in preparation sample.Step: accurately take L-hydroxyproline, with acetonitrile: water=1: 1 (volume ratio) prepares L-hydroxyproline 6 samples, and concentration is 1 ~ 150mg/L, measures L-hydroxyproline with Liquid Chromatography-Tandem Mass Spectrometry, the result that each sample repeats 3 tests as
following table instituteshow, the L-hydroxyproline recovery is higher.Visible with mark in the detection of 4-hydroxy-piperdine-2-carboxylic acid, measure L-hydroxyproline, also can 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, is characterized in that, the method for detecting interior mark, adopts Internal standard curve method quantitative with 4-hydroxy-piperdine-2-carboxylic acid.Concrete employing following steps:
1) interior target preparation: the 4-hydroxy-piperdine-2-carboxylic acid selecting purity >=99.0%, is placed in volumetric flask, adds acetonitrile and water prepares the solvent that obtain by volume at 1: 1, and dissolve, constant volume, shakes up, and 4-hydroxy-piperdine-2-carboxylic/solvent is 1g/L;
2) preparation of standard specimen: the L-hydroxyproline selecting purity >=99.0%, is placed in volumetric flask, adds acetonitrile and water prepares the solvent that obtain by volume at 1: 1, and dissolve, constant volume, shakes up, and L-hydroxyproline/solvent is 1g/L;
3) hydrolysis standard specimen prepares: 4-hydroxy-piperdine-2-carboxylic acid and the L-hydroxyproline of selecting purity >=99.0%, be placed in volumetric flask respectively, add 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 be 50mg/L;
4) preparation of samples: sample protein to be measured is placed in tube sealing, 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, sealing, hydrolysis;
5) sample pre-treatments: step 4) sample temperature be 90 DEG C hydrolysis 24 hours, hydrolyzation sample vacuum distillation removing hydrochloric acid, to pH=5, be placed in volumetric flask, add acetonitrile and water prepares the solvent that obtain by volume at 1: 1, 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 utilize the L-hydroxyproline content in above-mentioned working curve mensuration, calculation sample albumen, specifically adopt following steps:
Utilize step 1) and step 2) interior mark and standard specimen prepare 7 samples, in sample, 4-hydroxy-piperdine-2-carboxylic/solvent is 100mg/L, L-hydroxyproline/solvent is 1mg/L; With the area of liquid chromatography tandom mass spectrometry determination L-hydroxyproline and 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), with the area ratio of dependent variable L-hydroxyproline and 4-hydroxy-piperdine-2-carboxylic acid) data, calculate 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 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, determined the area ratio of L-hydroxyproline and 4-hydroxy-piperdine-2-carboxylic acid by retention time, calculate L-hydroxyproline content by working curve.
Liquid phase chromatogram condition is as follows: chromatographic column be Venusil Hilic post (2.1 × 150mm, 5 μm,
), pretreatment column be Venusil Hilic Venusil Hilic 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 following flow process:
| 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 scan ion is 132.1/86.2, and collision energy is 15eV; Second pair of scan ion is 146.1/127.2, and collision energy is 18eV; Scan pattern is single ion detection scanning (SRM).
TSQ Quantum Access high performance liquid chromatography-series connection quadrupole rod GC-MS that liquid chromatography tandom mass spectrometry determination adopts power & light company of the U.S. to make, is furnished with electrospray ionization source and atmosphere pressure chemical ion source.
To test sample protein be lactoprotein, such as milk, goat milk, camel milk, people's milk, soymilk or coconut milk, in addition, can also be milk powder, condensed milk, Yoghourt, cream, cheese, dairy beverage, albumen powder, bean 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, is characterized in that, the method for detecting interior mark, adopts Internal standard curve method quantitative with 4-hydroxy-piperdine-2-carboxylic acid.Concrete employing following steps:
1) interior target preparation: the 4-hydroxy-piperdine-2-carboxylic acid selecting purity >=99.0%, is placed in volumetric flask, adds acetonitrile and water prepares the solvent that obtain by volume at 1: 20, and dissolve, constant volume, shakes up, and 4-hydroxy-piperdine-2-carboxylic/solvent is 1g/L;
2) preparation of standard specimen: the L-hydroxyproline selecting purity >=99.0%, is placed in volumetric flask, adds acetonitrile and water prepares the solvent that obtain by volume at 1: 20, and dissolve, constant volume, shakes up, and L-hydroxyproline/solvent is 1g/L;
3) hydrolysis standard specimen prepares: 4-hydroxy-piperdine-2-carboxylic acid and the L-hydroxyproline of selecting purity >=99.0%, be placed in volumetric flask respectively, add 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 be 50mg/L;
4) preparation of samples: sample protein to be measured is placed in tube sealing, 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, sealing, hydrolysis;
5) sample pre-treatments: step 4) sample temperature be 120 DEG C hydrolysis 4 hours, hydrolyzation sample vacuum distillation removing hydrochloric acid, to pH=7, be placed in volumetric flask, add acetonitrile and water prepares the solvent that obtain by volume at 1: 1, 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 utilize the L-hydroxyproline content in above-mentioned working curve mensuration, calculation sample albumen, specifically adopt following steps:
Utilize step 1) and step 2) interior mark and standard specimen prepare 7 samples, in sample, 4-hydroxy-piperdine-2-carboxylic/solvent is 100mg/L, L-hydroxyproline/solvent is 200mg/L; With the area of liquid chromatography tandom mass spectrometry determination L-hydroxyproline and 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), with the area ratio of dependent variable L-hydroxyproline and 4-hydroxy-piperdine-2-carboxylic acid) data, calculate 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 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, determined the area ratio of L-hydroxyproline and 4-hydroxy-piperdine-2-carboxylic acid by retention time, calculate L-hydroxyproline content by working curve.
Liquid phase chromatogram condition is as follows: chromatographic column be Venusil Hilic post (2.1 × 150mm, 5 μm,
), pretreatment column be Venusil Hilic Venusil Hilic 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 following flow process:
| 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 scan ion is 132.1/86.2, and collision energy is 15eV; Second pair of scan ion is 146.1/127.2, and collision energy is 18eV; Scan pattern is single ion detection scanning (SRM).
TSQ Quantum Access high performance liquid chromatography-series connection quadrupole rod GC-MS that liquid chromatography tandom mass spectrometry determination adopts power & light company of the U.S. to make, is furnished with electrospray ionization source and atmosphere pressure chemical ion source.
To test sample protein be lactoprotein, such as milk, goat milk, camel milk, people's milk, soymilk or coconut milk, in addition, can also be milk powder, condensed milk, Yoghourt, cream, cheese, dairy beverage, albumen powder, bean powder, toffee, milk chocolate or milk chocolate.
Claims (8)
1. a method for liquid chromatography-tandem mass spectrometry (LC-MS/MS) internal mark method determination L-hydroxyproline, is characterized in that, the method for detecting interior mark, adopts Internal standard curve method quantitative with 4-hydroxy-piperdine-2-carboxylic acid.
2. the method for a kind of liquid chromatography-tandem mass spectrometry internal mark method determination L-hydroxyproline according to claim 1, it is characterized in that, the method specifically adopts following steps:
1) interior target preparation: the 4-hydroxy-piperdine-2-carboxylic acid selecting purity >=99.0%, is placed in volumetric flask, adds acetonitrile and water 1: 1 ~ 20 prepares the solvent obtained by volume, and dissolve, constant volume, shakes up, and 4-hydroxy-piperdine-2-carboxylic/solvent is 1g/L;
2) preparation of standard specimen: the L-hydroxyproline selecting purity >=99.0%, is placed in volumetric flask, adds acetonitrile and water 1: 1 ~ 20 prepares the solvent obtained by volume, and dissolve, constant volume, shakes up, and L-hydroxyproline/solvent is 1g/L;
3) hydrolysis standard specimen prepares: 4-hydroxy-piperdine-2-carboxylic acid and the L-hydroxyproline of selecting purity >=99.0%, be placed in volumetric flask respectively, add 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 be 50mg/L;
4) preparation of samples: sample protein to be measured is placed in tube sealing, add 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, sealing, hydrolysis;
5) sample pre-treatments: step 4) sample temperature be 90 ~ 120 DEG C hydrolysis 4 ~ 24 hours, hydrolyzation sample vacuum distillation removing hydrochloric acid, pH=5 ~ 7, be placed in volumetric flask, add acetonitrile and water prepares the solvent that obtain by volume at 1: 1, 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 L-hydroxyproline content in above-mentioned working curve mensuration, calculation sample albumen.
3. the method for a kind of liquid chromatography-tandem mass spectrometry internal mark method determination L-hydroxyproline according to claim 2, is characterized in that, step 6) specifically adopt following steps:
Utilize step 1) and step 2) interior mark and standard specimen prepare 7 samples, in sample, 4-hydroxy-piperdine-2-carboxylic/solvent is 100mg/L, L-hydroxyproline/solvent is 1 ~ 200mg/L; With the area of liquid chromatography tandom mass spectrometry determination L-hydroxyproline and 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), with the area ratio of dependent variable L-hydroxyproline and 4-hydroxy-piperdine-2-carboxylic acid) data, calculate 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 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, determined the area ratio of L-hydroxyproline and 4-hydroxy-piperdine-2-carboxylic acid by retention time, calculate L-hydroxyproline content by working curve.
4. the method for a kind of liquid chromatography-tandem mass spectrometry internal mark method determination L-hydroxyproline according to claim 3,
Liquid phase chromatogram condition is as follows: chromatographic column be Venusil Hilic post (2.1 × 150mm, 5 μm,
), pretreatment column be Venusil Hilic Venusil Hilic 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;
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 scan ion is 132.1/86.2, and collision energy is 15eV; Second pair of scan ion is 146.1/127.2, and collision energy is 18eV; Scan pattern is single ion detection scanning (SRM).
5. the method for a kind of liquid chromatography-tandem mass spectrometry internal mark method determination L-hydroxyproline according to claim 4, TSQ Quantum Access high performance liquid chromatography-series connection quadrupole rod GC-MS that liquid chromatography tandom mass spectrometry determination adopts power & light company of the U.S. to make, is furnished with electrospray ionization source and atmosphere pressure chemical ion source.
6. the method for a kind of liquid chromatography-tandem mass spectrometry internal mark method determination L-hydroxyproline according to claim 1 and 2, described sample protein is lactoprotein.
7. the method for a kind of liquid chromatography-tandem mass spectrometry internal mark method determination L-hydroxyproline according to claim 1 and 2, described sample protein comprises milk, goat milk, camel milk, people's milk, soymilk or coconut milk.
8. the method for a kind of liquid chromatography-tandem mass spectrometry internal mark method determination L-hydroxyproline according to claim 1 and 2, described sample protein is milk powder, condensed milk, Yoghourt, cream, cheese, dairy beverage, albumen powder, bean powder, toffee, milk chocolate or milk chocolate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410748715.5A CN104614452B (en) | 2014-12-09 | 2014-12-09 | A kind of method of liquid chromatography-tandem mass spectrometry internal mark method determination L-hydroxyproline |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410748715.5A CN104614452B (en) | 2014-12-09 | 2014-12-09 | A kind of method of liquid chromatography-tandem mass spectrometry internal mark method determination L-hydroxyproline |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104614452A true CN104614452A (en) | 2015-05-13 |
| CN104614452B CN104614452B (en) | 2016-06-29 |
Family
ID=53148994
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410748715.5A Active CN104614452B (en) | 2014-12-09 | 2014-12-09 | A kind of method of liquid chromatography-tandem mass spectrometry internal mark method determination L-hydroxyproline |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104614452B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105929044A (en) * | 2016-04-14 | 2016-09-07 | 宁波出入境检验检疫局检验检疫技术中心 | Method for rapidly detecting hydroxy proline content of milk and dairy products |
| CN106248775A (en) * | 2016-08-31 | 2016-12-21 | 广西壮族自治区地质矿产测试研究中心 | Quantitative detection method for reducing analysis errors of ICP-MS and ICP-AES whole process |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07313179A (en) * | 1993-09-07 | 1995-12-05 | Kyowa Hakko Kogyo Co Ltd | Production of trans-4-hydroxy-l-proline |
| CN102406749A (en) * | 2010-11-22 | 2012-04-11 | 九芝堂股份有限公司 | HPLC (high-performance liquid chromatography) detection method capable of simultaneously detecting contents of six amino acids in donkey-hide glue blood-supplementing preparation |
| CN102590420A (en) * | 2012-02-15 | 2012-07-18 | 上海德诺产品检测有限公司 | Method for detecting content of L-hydroxyproline in dairy product |
-
2014
- 2014-12-09 CN CN201410748715.5A patent/CN104614452B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07313179A (en) * | 1993-09-07 | 1995-12-05 | Kyowa Hakko Kogyo Co Ltd | Production of trans-4-hydroxy-l-proline |
| CN102406749A (en) * | 2010-11-22 | 2012-04-11 | 九芝堂股份有限公司 | HPLC (high-performance liquid chromatography) detection method capable of simultaneously detecting contents of six amino acids in donkey-hide glue blood-supplementing preparation |
| CN102590420A (en) * | 2012-02-15 | 2012-07-18 | 上海德诺产品检测有限公司 | Method for detecting content of L-hydroxyproline in dairy product |
Non-Patent Citations (5)
| Title |
|---|
| A. CONVENTZ 等: "Simultaneous determination of 3-nitrotyrosine, tyrosine, hydroxyproline and proline in exhaled breath condensate by hydrophilic interaction liquid chromatography/electrospray ionization tandem mass spectrometry", 《JOURNAL OF CHROMATOGRAPHY B》, vol. 860, no. 1, 1 December 2007 (2007-12-01), pages 78 - 85, XP022364475, DOI: doi:10.1016/j.jchromb.2007.10.031 * |
| YASUHIKO KUNII 等: "4-Hydroxypipecolic Acid and Pipecolic Acid in Acacia Species:Their Determination by High-Performance Liquid Chromatography,Its Application to Leguminous Plants, and Configuration of 4-Hydroxypipecolic Acid", 《J. AGRIC. FOOD CHEM》, vol. 44, no. 2, 19 February 1996 (1996-02-19), pages 483 - 487 * |
| 刘思洁 等: "乳与乳制品中L-羟脯氨酸检测技术的研究进展", 《食品安全质量检测学报》, vol. 5, no. 9, 30 September 2014 (2014-09-30), pages 2629 - 2633 * |
| 孙艳亭 等: "L-羟脯氨酸寡肽混合物的高效液相色谱分离与质谱分析", 《色谱》, vol. 25, no. 4, 31 July 2007 (2007-07-31), pages 524 - 527 * |
| 赵善贞 等: "亲水性相互作用色谱-四级杆串联线性离子阱质谱测定蛋白食品中L-羟脯氨酸残留", 《分析化学》, vol. 39, no. 9, 30 September 2011 (2011-09-30), pages 1373 - 1379 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105929044A (en) * | 2016-04-14 | 2016-09-07 | 宁波出入境检验检疫局检验检疫技术中心 | Method for rapidly detecting hydroxy proline content of milk and dairy products |
| CN105929044B (en) * | 2016-04-14 | 2018-08-17 | 宁波出入境检验检疫局检验检疫技术中心 | A kind of method of hydroxyproline content in quick detection milk and milk products |
| CN106248775A (en) * | 2016-08-31 | 2016-12-21 | 广西壮族自治区地质矿产测试研究中心 | Quantitative detection method for reducing analysis errors of ICP-MS and ICP-AES whole process |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104614452B (en) | 2016-06-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US9970942B2 (en) | System and method for the detection of allergens | |
| CN104614479B (en) | A kind of detection method of food vitamins | |
| CN106749402B (en) | A kind of stable isotope labeling chiral phosphorus reagent and the preparation method and application thereof | |
| CN104614466B (en) | The assay method of antiseptic in tobacco juice for electronic smoke | |
| Palagama et al. | Improved solid-phase extraction protocol and sensitive quantification of six microcystins in water using an HPLC-orbitrap mass spectrometry system | |
| CN102507833B (en) | Method for determining vanillin, ethyl vanillin and coumarin in milk by solid phase extraction-liquid chromatogram-electrospray tandem mass spectrometry | |
| CN108956840A (en) | A kind of method that ultra performance liquid chromatography-tandem mass spectrum detects 5 kinds of sweeteners in electronic cigarette liquid | |
| CN104965040A (en) | Detection method of free and combined carboxy methyl lysine in milk and dairy products | |
| CN104614452A (en) | Method for determining L-hydroxyproline through liquid chromatogram-tandem mass spectrum internal standard process | |
| CN106645477B (en) | A kind of remaining method of detection florfenicol amine and application | |
| CN103743844A (en) | Determination method of alcohol content in methanol | |
| CN103175931A (en) | Method for determining harmful aromatic amine by liquid chromatogram-tandem mass spectrometry | |
| CN105866315A (en) | Measuring method for amino acid in electronic cigarette smoke liquid | |
| CN102778527B (en) | A kind of measure the method for diacetyl content in Chinese liquor | |
| CN106053695A (en) | Method for detecting content of ethyl carbamate in soybean-flavor Baijiu | |
| CN109975435A (en) | The measuring method of isopropyl mesylate content in a kind of safinamide | |
| CN104215721A (en) | Application of phthalic acid in amino acid analysis | |
| CN104155378A (en) | Method for rapid analysis of amino acid mixture | |
| CN106248847A (en) | A kind of method of seven kinds of organic acid contents in wine brewing material of mensuration simultaneously | |
| CN109425666B (en) | LC-MS analysis method of acyl chloride derivative | |
| US10436794B1 (en) | Protocol for preconcentration and quantification of microcystins using LC-MS | |
| CN111024872B (en) | Method for rapidly detecting 3-methylthio propanol in sesame-flavor liquor | |
| CN109254086B (en) | HPLC detection method for sodium dodecyl sulfate in cefaclor dry suspension sample | |
| US20200025730A1 (en) | Methods for determination of polar pesticides by chromatography | |
| Sun et al. | Highly sensitive and specific detection of histamine via the formation of a self-assembled magic number cluster with thymine by mass spectrometry |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CP01 | Change in the name or title of a patent holder | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 200062 Shanghai city Putuo District Yunling Road No. 345 Patentee after: Shanghai Chemical Research Institute Co., Ltd. Address before: 200062 Shanghai city Putuo District Yunling Road No. 345 Patentee before: Shanghai Research Institute of Chemical Industry |