CN106841452B - A kind of method that HPLC combination UPLC-MS detection PQQ acts on lactic acid - Google Patents
A kind of method that HPLC combination UPLC-MS detection PQQ acts on lactic acid Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
A kind of method that HPLC combination UPLC-MS detection PQQ acts on lactic acid, belongs to bioassay technique field.The present invention is reacted using UPLC technique study PQQ and lactic acid, is optimized separation condition, is reached PQQ with multiple reaction products and separate.And qualitative analysis is carried out to its reaction product using UPLC-MS.The present invention studies the reflex action of PQQ and lactic acid for the first time, and 3 kinds of products, quasi-molecular ion [M+H] are had found in reaction solution+Respectively 403,475 and 547.PQQ occurs esterification with a molecule lactic acid, two molecule lactic acid, three molecule lactic acid respectively and obtains product PQQLa1, PQQLa2 and PQQLa3.Mainly exist by the peak area discovery of differential responses time each product with PQQLa1, the product after esterification enters extracellular fluid through blood-brain barrier, thus reduces the content of extracellular fluid lactic acid.To research PQQ in Mechanism of neuroprotective effect, there is particularly important researching value in terms of disease research especially related with aging.
Description
Technical field
A kind of method that HPLC combination UPLC-MS detection PQQ acts on lactic acid, belongs to bioassay technique field.
Background technique
Energy supply in central nervous system relies primarily on the glucose in peripheral blood.Main ingredient in blood-brain barrier
Astroglia absorbs glucose, generates lactic acid through glycolysis and discharges to extracellular fluid, extracellular fluid reflects nerve cell
Living environment.Metabolite of the lactic acid as glycolysis, has always been considered as having detrimental effect to nerve cell.Therefore, lactic acid exists
Effect in nervous system is paid attention to by scientist always.The raising of acute cerebral trauma patient's Cerebrospinal Fluid Lactate In Patients content has been reported；
Normal brain spinal fluid lactic acid content is very low, and lactic acid content is significantly raised in report discovery glioblastoma；Serum lactic conduct
The biomarker of cerebral injury.The rising that American Academy of Sciences PANS also reports lactic acid concn is related with aging, this may
It is the metabolic alterations that mitochondrial DNA dysfunction causes mouse brain, thus changes the table for the specific gene that control lactic acid is formed
It reaches, this variation results in the increase of brain lactic acid concn.Therefore, detection lactic acid concn to detection central nervous system in aging
Related disease is of great significance, and accelerates intracerebral lactic acid metabolism, reduces the content maintenance lactic acid low concentration of lactic acid to be in treatment
One important means of pivot nervous system disease.
Pyrrolo- quinoline purine quinone (PQQ) is a kind of oxidoreducing enzyme prothetic group, structural formula are as follows:
It can reduce nerve cell death, and the generation of neural factor can be promoted to have nerve to nerve cell in brain
Protective effect.Critical function of the PQQ in terms of nervous system may be special with it chemical structure it is related, there are more carbonyls and more
The functional group of carboxyl can react with various active group.For example it can react with a variety of amino acid thus to ammonia
Base acid neurotransmitter has adjustment effect.And whether there is adjustment effect to the key factor lactic acid related with aging gene, until
The present is without relevant report.
The present invention is reacted using UPLC technique study PQQ and lactic acid, optimizes separation condition, makes PQQ and multiple anti-
Product is answered to reach separation.And qualitative analysis is carried out to its reaction product using UPLC-MS.It is previous that researches show that PQQ structures
In two carbonyls and amino acid participate in condensation reaction and generate disliking azole compounds, research PQQ and lactic acid react for the first time herein
Behavior has found 3 kinds of products, quasi-molecular ion [M+H] in reaction solution+Respectively 403,475 and 547.Pass through product
With a molecule lactic acid, two molecule lactic acid, three molecule lactic acid ester occurs for quasi-molecular ion peak and fragment ion preliminary analysis PQQ respectively
Product PQQLa1, PQQLa2 and PQQLa3 are reacted to obtain in change.By the discovery of the peak area of differential responses time each product mainly with
One molecule esterification products PQQLa1 exists, and the product after esterification enters extracellular fluid through blood-brain barrier, thus reduces cell
The content of external solution lactic acid.To research PQQ in Mechanism of neuroprotective effect, there is pole in terms of disease research especially related with aging
For important researching value.
The measurement of lactic acid content has colorimetric method, EDTA titration, HPLC analytic approach, enzyme process, a GC analytic approach, colorimetric method and
EDTA method is simple, quick, but sensitivity and precision are poor；Enzyme process requires experiment condition harsh and costly；Gas phase color
Lactic acid is easily decomposed in spectrometry, is influenced it and is measured accuracy.Lactic acid content is extremely low in brain tissue, the UV absorption pole of lactic acid
Low, HPLC method reported in the literature mostly uses 210nm to detect greatly, and disturbing factor is more, 5 μM of the minimal detectable concentration of report, for
Brain tissue detection of biological samples sensitivity is too low.The present invention uses fluorescence enhancement reagent N BD-COCl(4- (N- chloroformyl methyl-
N- methylamino) -7- nitro -2,1,3- benzoxadiazole), reacted with lactic acid and generate the biggish fluorescent material of response, determined
PQQ is to D-gal(D- galactolipin) influence of lactic acid content, minimal detectable concentration reach in induced rat aging model biological sample
10nM.Aging model rat plasma, brain tissue and extracellular fluid lactic acid caused by measurement result display PQQ can be reduced because of D-gal
The raising of content is of great significance to the mechanism study of PQQ anti-aging.
Summary of the invention
It is anti-to PQQ the object of the present invention is to provide the method that a kind of HPLC combination UPLC-MS detection PQQ acts on lactic acid
The mechanism study of aging is of great significance.
Technical solution of the present invention: a kind of method that HPLC combination UPLC-MS detection PQQ acts on lactic acid, step are as follows:
(1) UPLC analysis condition
ACQUiTY UPLC@BEH C18 column (2.1 × 100mm, 1.7 μm), mobile phase A: H2O+0.2%TFA,
B: methanol；5/95 B/A elutes 5min, 20 min gradient elutions to 75/25 B/A.Detection wavelength 322nm.
(2) determination of wavelength
A certain amount of PQQ and lactic acid are weighed, is dissolved respectively with 0.1M hydrochloric acid solution, is configured to the PQQ solution and 1mM of 1mM
Lactic acid solution, be that 1 ︰ 5 is mixed by v/v, be protected from light, be incubated for for 24 hours in 37 DEG C of water-baths, ice bath terminates reaction.UV scanning figure is shown
322nm UV absorption is higher；Select 322nm as Detection wavelength.
(3) mobile phase composition is to isolated influence
Add quality percentage respectively in mobile phase are as follows: 0.05%, 0.1%, 0.15%, 0.2%, 0.3 %TFA(trifluoroacetic acid)
Test, when TFA content is 0.2%, mobile phase A: H2O+0.2%TFA, B: methanol；5/95 B/A elutes 5min, 20 min gradients
It is eluted to 75/25 B/A.Each component peak type symmetry is preferable, and PQQ and 3 kinds of products are attained by baseline separation.
(4) qualitative analysis of the UPLC-MS to the reaction product of PQQ and lactic acid
Using ACQUiTY UPLC@BEH C18 column (2.1 × 100mm, 1.7 μm), mass detector SQ
Detector 2 and PDA detector.Optimize mass spectrometry parameters:
Desolvation temperature (Desolvation Temp): 400 DEG C；
Desolventizing gas flow (Desolvation Gas Flow): 600 L/h；
Ion source temperature (source temperature): 110 DEG C；
Taper hole throughput (Cone Gas Flow): 50L/h；
Capillary voltage (capillary voltage): 3000 V；
Orifice potential (cone voltage): 30V.
Mass spectrum uses electron spray positive ion mode (ESI+), and mass scan range is m/z 50~800.
Mobile phase A: H2O+0.2%TFA, B: methanol；5/95 B/A elutes 5min, 20min gradient elution to 75/25 B/A.
10 μ L of sample introduction.In the ESI-MS spectrogram of reaction solution, leading ion is 403,475,547 three peaks m/z.Pass through the standard of product
Molecular ion peak and fragment ion preliminary analysis PQQ occur esterification with a molecule lactic acid respectively and obtain product PQQLa1, and 403, M
+ 1 peak, 330, M-72(lactic acid) peak；Esterification occurs with two molecule lactic acid and obtains product PQQLa2,475, M+1 peaks, 402, M-72
(lactic acid) peak；Esterification occurs with three molecule lactic acid and obtains product PQQLa3,547, M+1 peaks.Mass spectrogram see respectively Fig. 3, Fig. 4,
Fig. 5.PQQLa3 content is lower, and Fig. 5 b is Fig. 5 a partial enlarged view.Its chemical equation is shown in Fig. 6.From the integral face of three kinds of products
Product ratio PQQLa1/PQQLa2/PQQLa3 is 9.6/1.6/1.The integral area of three kinds of products is shown in Fig. 7.
(5) 3 kinds of peak areas (AUS) with the reaction time change curve
PQQ and lactic acid are prepared respectively with molar ratio as 1 ︰, 5 hybrid reaction, is protected from light, is sufficiently stirred, be placed in 37 DEG C of water-baths and incubate
It educates, in 4,8,16,24,32, each time point abstraction reaction liquid of 48h, methanol dilution direct injected carries out UPLC-MS analysis.Acquisition
【M+1】+Respectively 403,475,547 integral area AUS, is plotted against time with AUS, sees Fig. 8.Reaction is substantially at afterwards for 24 hours
Stablize.
(6) PQQ is to D-gal(D- galactolipin) the influence measurement of lactic acid content in induced rat aging model biological sample
La content is extremely low in brain tissue, and the UV absorption of La is extremely low, and HPLC method reported in the literature mostly uses 210nm to examine greatly
It surveys, disturbing factor is more, 5 μM of the minimal detectable concentration of report, too low for brain tissue detection of biological samples sensitivity.We adopt
With fluorescence enhancement reagent N BD-COCl(4- (N- chloroformyl methyl-N- methylamino) -7- nitro -2,1,3- benzoxadiazole) reaction
It generates the biggish fluorescent material of response (derivatization reaction is shown in Fig. 9), to detect the extremely low La of content in biological sample, lowest detection
Concentration reaches 10nM.
Chromatographic condition: 600 high performance liquid chromatograph of U.S. WATERS, 2175 fluorescence detectors, chromatographic column Amethyst
C18-H(4.6 × 250mm, 5 μm), 15% acetonitrile-H of mobile phase2O contains 0.05%TFA, flow velocity 1ml/min, fluorescence detection excitation wave
Long 483nm, launch wavelength 530nm.
Blood plasma or extracellular fluid processing :+180 μ L methanol of+10 μ L deionized water of 10 μ L blood plasma (extracellular fluid), it is sufficiently mixed
Even, 15000rpm/4 DEG C of centrifugation 5min, the freeze-drying of 10 μ L supernatants, dry residue, which is dissolved in 10 μ L deionized waters, directly to be used
In derivatization reaction.
Brain tissue processing: brain tissue, which is weighed, takes 0.1g, the physiological saline of addition 0.9mL pre-cooling, electronic homogenate,
15000rpm/4 DEG C of centrifugation 15min takes+180 μ L methanol of+10 μ L deionized water of 10 μ L supernatant, mixes well, 15000rpm/4
DEG C centrifugation 5min, the freeze-drying of 10 μ L supernatants, dry residue, which is dissolved in 10 μ L deionized waters, is directly used in derivatization reaction.
Derivatization reaction :+5 μ L20mM NBD-COCl of+10 μ L 50mM N-methylmorpholine of 10 μ L sample, 60 DEG C of reactions
15min.Add 150 μ L 2%TFA to terminate reaction, 10 μ L is taken directly to carry out HPLC analysis.
Linear relationship and detection limit measurement: precision weighs lactic acid reference substance, and deionized water is added to be made into 0.1,0.5,1,5,10,
20,30,40,50mmol/L contrast solution, by progress HPLC analysis after the reaction of above-mentioned derivatization reaction method.Gained integral area
Linear regression is carried out to concentration, obtains equation y=1602534x+18831, R2= 0.9999.In 0.1~50mmol/L range
Interior linear good (Figure 15).The assay of lactic acid the results are shown in Table 1 in biological sample.Minimum concentrations be 10nM(S/N >=
3).X is concentration mmol/L, and Y is peak area.
The measurement of repeatability and accuracy: measuring under determining chromatographic condition in interior continuous sample introduction 6 times on the same day, according to
Relative standard deviation obtained by calculated by peak area is about 3.8% (n=6), the relative standard deviation of retention time be about 0.84% (n=
6).
The measurement result of 1 lactic acid concn of table
| Group | Blood plasma (mmol/L) | Extracellular fluid | Brain tissue (mmol/g) |
| Normal group | 1.56±0.85 | 4.82±0.98 | 0.38±0.02 |
| D-gal aging model group | 3.87±0.28* | 7.25±1.32* | 0.66±0.05* |
| D-gal+PQQ administration group | 2.05 ± 0.06 ﹟ | 5.03 ± 1.18 ﹟ | 0.42 ± 0.09 ﹟ |
*P < 0.01 compared with the control group p < 0.01, ﹟ compared with model group
It was found that normally group extracellular fluid lactic acid concn is 3.08 times of blood plasma.D-gal induces Aging Mouse Modle blood plasma, brain group
It knits and is significantly increased with lactic acid content in extracellular fluid, PQQ administration group lactic acid content is remarkably decreased.It may be sent out due to PQQ and lactic acid
Raw esterification generates PQQLa, provides new theoretical foundation for the therapy target of central nervous system disease.
Beneficial effects of the present invention: the present invention is reacted using UPLC technique study PQQ and lactic acid, optimizes lightning strip
Part reaches PQQ with multiple reaction products and separates.And qualitative analysis is carried out to its reaction product using UPLC-MS.The present invention
The reflex action for studying PQQ and lactic acid for the first time, has found 3 kinds of products, quasi-molecular ion [M+H] in reaction solution+Respectively
403,475 and 547.By the quasi-molecular ion peak and fragment ion preliminary analysis PQQ of product respectively with a molecule lactic acid, two
Molecule lactic acid, three molecule lactic acid occur esterification and obtain product PQQLa1, PQQLa2 and PQQLa3.It is each by the differential responses time
Mainly with molecule esterification products PQQLa1 presence, the product after esterification enters through blood-brain barrier for the peak area discovery of product
Extracellular fluid thus reduces the content of extracellular fluid lactic acid.To research PQQ in Mechanism of neuroprotective effect, especially with aging
Related pathogenic mechanism research aspect has particularly important researching value.
Detailed description of the invention
The ultraviolet scanning atlas of Fig. 1 PQQ and lactic acid product.
The chromatogram of Fig. 2 PQQ and lactic acid reaction solution.
Fig. 3 product PQQLa1 mass spectrogram.
Fig. 4 product PQQLa2 mass spectrogram.
Fig. 5 product PQQLa3 mass spectrogram, Fig. 5 b are Fig. 5 a partial enlarged view.
Fig. 6 PQQ and lactic acid chemical equation.
The integral area map of tri- kinds of product PQQLa1/PQQLa2/PQQLa3 of Fig. 7.
The integral area of tri- kinds of product PQQLa1/PQQLa2/PQQLa3 of Fig. 8 with the reaction time variation diagram.
Fig. 9 lactic acid derivatization reaction.
Figure 10 blank derivative reagent HPLC chromatogram.
Figure 11 lactic acid compares the HPLC chromatogram after derivatization reaction.
Figure 12 blood plasma derivatization reaction HPLC chromatogram.
Figure 13 brain tissue derivatization reaction HPLC chromatogram.
Figure 14 extracellular fluid derivatization reaction HPLC chromatogram.
Figure 15 canonical plotting.
Specific embodiment
Embodiment 1
A kind of method that HPLC combination UPLC-MS detection PQQ acts on lactic acid, step are as follows:
(1) UPLC analysis condition:
ACQUiTY UPLC@BEH C18 column (2.1 × 100mm, 1.7 μm), mobile phase A: H2O+0.2%TFA,
B: methanol；5/95 B/A elutes 5min, 20 min gradient elutions to 75/25 B/A；Detection wavelength 322nm；
(2) determination of wavelength
A certain amount of PQQ is weighed, lactic acid is dissolved with 0.1M hydrochloric acid solution respectively, is configured to the PQQ solution and lactic acid of 1mM
Solution is 1 ︰ 5 mixing by v/v, is protected from light, and is incubated for for 24 hours in 37 DEG C of water-baths, ice bath terminates reaction；UV scanning figure shows 322nm purple
Outer absorption is higher；Select 322nm as Detection wavelength；
(3) mobile phase composition is to isolated influence
Add 0.05%, 0.1%, 0.15%, 0.2%, 0.3 %TFA(trifluoroacetic acid respectively in mobile phase) test, work as TFA
When content is 0.2%, mobile phase A: H2O+0.2%TFA, B: methanol；5/95 B/A elutes 5min, 20 min gradient elutions to 75/
25 B/A；Each component peak type symmetry is preferable, and PQQ and 3 kinds of products are attained by baseline separation；
(4) qualitative analysis of the UPLC-MS to the reaction product of PQQ and lactic acid
Using ACQUiTY UPLC@BEH C18 column (2.1 × 100mm, 1.7um), mass detector SQ
Detector 2 and PDA detector optimize mass spectrometry parameters:
Desolvation temperature (Desolvation Temp): 400 DEG C；
Desolventizing gas flow (Desolvation Gas Flow): 600 L/h；
Ion source temperature (source temperature): 110 DEG C；
Taper hole throughput (Cone Gas Flow): 50L/h；
Capillary voltage (capillary voltage): 3000 V；
Orifice potential (cone voltage): 30V；
Mass spectrum uses electron spray positive ion mode (ESI+), and mass scan range is m/z 50~800.
Mobile phase A: H2O+0.2%TFA, B: methanol；5/95 B/A elution 5min, 20min gradient elution to 75/25 B/A,
10 μ L of sample introduction, in the ESI-MS spectrogram of reaction solution, leading ion is 403,475,547 three peaks m/z；Pass through the standard of product
Molecular ion peak and fragment ion preliminary analysis PQQ occur esterification with a molecule lactic acid respectively and obtain product PQQLa1, and 403, M
+ 1 peak；330, M-72(lactic acid) peak；Esterification occurs with two molecule lactic acid and obtains product PQQLa2,475, M+1 peaks, 402, M-72
(lactic acid) peak；Esterification occurs with three molecule lactic acid and obtains product PQQLa3,547, M+1 peaks；Mass spectrogram see respectively Fig. 3, Fig. 4,
Fig. 5.PQQLa3 content is lower, and Fig. 5 b is Fig. 5 a partial enlarged view.Its chemical equation is shown in Fig. 6.From the integral face of three kinds of products
Product ratio PQQLa1/PQQLa2/PQQLa3 is 9.6/1.6/1；The integral area of three kinds of products is shown in Fig. 7.
(5) 3 kinds of peak areas (AUS) with the reaction time change curve
It is 1 ︰, 5 hybrid reaction that PQQ and lactic acid are prepared molar ratio respectively, is protected from light, is sufficiently stirred, is placed in 37 DEG C of water-baths and incubates
It educates, in 4,8,15,24,32, each time point abstraction reaction liquid of 48h, methanol dilution direct injected carries out UPLC-MS analysis；Acquisition
【M+1】+Respectively 403,475,547 integral area AUS, is plotted against time with AUS, sees Fig. 8；Reaction is substantially at afterwards for 24 hours
Stablize；
(6) PQQ is to D-gal(D- galactolipin) the influence measurement of lactic acid content in induced rat aging model biological sample
Using fluorescence enhancement reagent N BD-COCl(4- (N- chloroformyl methyl-N- methylamino) -7- nitro -2,1,3- benzo
Oxadiazoles) the reaction generation biggish fluorescent material of response (derivatization reaction is shown in Fig. 9), it is extremely low to detect content in biological sample
La, minimal detectable concentration reach 10nM.
Chromatographic condition: 600 high performance liquid chromatograph of U.S. WATERS, 2175 fluorescence detectors, chromatographic column Amethyst
C18-H(4.6 × 250mm, 5 μm), 15% acetonitrile-H of mobile phase2O contains 0.05%TFA, flow velocity 1ml/min, fluorescence detection excitation wave
Long 483nm, launch wavelength 530nm.
Blood plasma or extracellular fluid processing :+180 μ L methanol of+10 μ L deionized water of 10 μ L blood plasma (extracellular fluid), it is sufficiently mixed
Even, 15000rpm/4 DEG C of centrifugation 5min, the freeze-drying of 10 μ L supernatants, dry residue, which is dissolved in 10 μ L deionized waters, directly to be used
In derivatization reaction.
Brain tissue processing: brain tissue, which is weighed, takes 0.1g, the physiological saline of addition 0.9mL pre-cooling, electronic homogenate,
15000rpm/4 DEG C of centrifugation 15min takes+180 μ L methanol of+10 μ L deionized water of 10 μ L supernatant, mixes well, 15000rpm/4
DEG C centrifugation 5min, the freeze-drying of 10 μ L supernatants, dry residue, which is dissolved in 10 μ L deionized waters, is directly used in derivatization reaction.
Derivatization reaction :+5 μ L20mM NBD-COCl of+10 μ L 50mM N-methylmorpholine of 10 μ L sample, 60 DEG C of reactions
15min.Add 150 μ L 2%TFA to terminate reaction, 10 μ L is taken directly to carry out HPLC analysis.
Linear relationship and detection limit measurement: precision weighs lactic acid reference substance, and deionized water is added to be made into 0.1,0.5,1,5,10,
20,30,40,50mmol/L contrast solution, by progress HPLC analysis after the reaction of above-mentioned derivatization reaction method.Gained integral area
Linear regression is carried out to concentration, obtains equation y=1602534+ 18831, R2= 0.9999.In 0.1~50mmol/L range
Interior linear good (Figure 15).The assay of lactic acid the results are shown in Table 1 in biological sample.Minimum concentrations be 10nM(S/N >=
3).X is concentration mmol/L, and Y is peak area.
The measurement of repeatability and accuracy: measuring under determining chromatographic condition in interior continuous sample introduction 6 times on the same day, according to
Relative standard deviation obtained by calculated by peak area is about 3.8% (n=6), the relative standard deviation of retention time be about 0.84% (n=
6).
The measurement of lactic acid content in biological sample: each group sample is derivative anti-by above-mentioned reaction condition after handling according to the above method
Should after carry out HPLC analysis, external standard method is calculated the content of lactic acid, the results are shown in Table 1.
The measurement result of 1 lactic acid concn of table
| Group | Blood plasma (mmol/L) | Extracellular fluid | Brain tissue (mmol/g) |
| Normal group | 1.56±0.85 | 4.82±0.98 | 0.38±0.02 |
| D-gal aging model group | 3.87±0.28* | 7.25±1.32* | 0.66±0.05* |
| D-gal+PQQ administration group | 2.05 ± 0.06 ﹟ | 5.03 ± 1.18 ﹟ | 0.42 ± 0.09 ﹟ |
*P < 0.01 compared with the control group p < 0.01, ﹟ compared with model group
It was found that normally group extracellular fluid lactic acid concn is 3.08 times of blood plasma.D-gal induces Aging Mouse Modle blood plasma, brain group
It knits and is significantly increased with lactic acid content in extracellular fluid, PQQ administration group lactic acid content is remarkably decreased.It may be sent out due to PQQ and lactic acid
Raw esterification generates PQQLa, provides new theoretical foundation for the therapy target of central nervous system disease.
Claims (1)
1. a kind of method that HPLC combination UPLC-MS detection PQQ acts on lactic acid, it is characterised in that step are as follows:
(1) UPLC analysis condition
ACQUiTY UPLC@BEH C18 column 2.1 × 100mm, 1.7 μm, mobile phase A: H2O+0.2%TFA, B: methanol；
5/95 B/A elutes 5min, 20 min gradient elutions to 75/25 B/A；Detection wavelength 322nm；
(2) determination of wavelength
A certain amount of PQQ and lactic acid are weighed, is dissolved respectively with 0.1M hydrochloric acid solution, the PQQ solution and 1mM of 1mM are configured to
Lactic acid solution is 1 ︰ 5 mixing by v/v, is protected from light, and is incubated for for 24 hours in 37 DEG C of water-baths, ice bath terminates reaction；UV scanning figure is shown
322nm UV absorption is higher；Select 322nm as Detection wavelength；
(3) mobile phase composition is to isolated influence
Add trifluoroacetic acid TFA mass percent respectively in mobile phase are as follows: 0.05%, 0.1%, 0.15%, 0.2%, 0.3 %TFA
Test, when TFA content is 0.2%, mobile phase A: H2O+0.2%TFA, B: methanol；5/95 B/A elutes 5min, 20 min gradients
It is eluted to 75/25 B/A；Each component peak type symmetry is preferable, and PQQ and 3 kinds of products are attained by baseline separation；
(4) qualitative analysis of the UPLC-MS to the reaction product of PQQ and lactic acid
Using 2.1 × 100mm of ACQUiTY UPLC@BEH C18 column, 1.7 μm, mass detector SQ Detector 2
And PDA detector, optimize mass spectrometry parameters:
Desolvation temperature: 400 DEG C；
Desolventizing gas flow: 600 L/h；
Ion source temperature: 110 DEG C；
Taper hole throughput: 50L/h；
Capillary voltage: 3000 V；
Orifice potential: 30V；
Mass spectrum uses electron spray positive ion mode ESI+, and mass scan range is m/z 50~800；
Mobile phase A: H2O+0.2%TFA, B: methanol；5/95 B/A elutes 5min, 20min gradient elution to 75/25 B/A；Sample introduction
10 μ L, in the ESI-MS spectrogram of reaction solution, leading ion is 403,475,547 three peaks m/z；Pass through the quasi-molecule of product
Quasi-molecular ions and fragment ion analysis PQQ occur esterification with a molecule lactic acid respectively and obtain product PQQLa1,403, M+1 peaks,
330, M-72 lactic acid peaks；Esterification occurs with two molecule lactic acid and obtains product PQQLa2,475, M+1 peaks, 402, M-72 lactic acid
Peak；Esterification occurs with three molecule lactic acid and obtains product PQQLa3,547, M+1 peaks；
The integral area ratio PQQLa1/PQQLa2/PQQLa3 of three kinds of products is 9.6/1.6/1；
(5) 3 kinds of peak areas AUS with the reaction time change curve
PQQ and lactic acid are prepared respectively with molar ratio as 1 ︰, 5 hybrid reaction, is protected from light, is sufficiently stirred, 37 DEG C of water-baths is placed in and is incubated for,
In 4,8,16,24,32, each time point abstraction reaction liquid of 48h, methanol dilution direct injected carries out UPLC-MS analysis；Acquire [M+
1】+Respectively 403,475,547 integral area AUS, is plotted against time with AUS, and reaction is substantially at stabilization afterwards for 24 hours；
(6) influence of the PQQ to lactic acid content in D- galactolipin D-gal induced rat aging model biological sample measures
Using fluorescence enhancement reagent 4- (N- chloroformyl methyl-N- methylamino) -7- nitro -2,1,3- benzoxadiazole NBD-COCl
Reaction generates the biggish fluorescent material of response, and to detect the extremely low La of content in biological sample, minimal detectable concentration reaches 10nM；
Chromatographic condition: 600 high performance liquid chromatograph of U.S. WATERS, 2175 fluorescence detectors, chromatographic column Amethyst C18-H
4.6 × 250mm, 5 μm, 15% acetonitrile-H of mobile phase2O contains 0.05%TFA, flow velocity 1mL/min, fluorescence detection excitation wavelength
483nm, launch wavelength 530nm；
Blood plasma or extracellular fluid processing: 10 μ L blood plasma /+180 μ L methanol of+10 μ L deionized water of extracellular fluid mix well,
15000rpm/4 DEG C of centrifugation 5min, the freeze-drying of 10 μ L supernatants, dried object, which is dissolved in be directly used in 10 μ L deionized waters, to spread out
Raw reaction；
Brain tissue processing: brain tissue, which is weighed, takes 0.1g, the physiological saline of addition 0.9mL pre-cooling, electronic homogenate, and 15000rpm/4 DEG C
It is centrifuged 15min ,+180 μ L methanol of+10 μ L deionized water of 10 μ L supernatant is taken, mixes well, 15000rpm/4 DEG C of centrifugation 5min,
The freeze-drying of 10 μ L supernatants, dried object are dissolved in 10 μ L deionized waters and are directly used in derivatization reaction；
Derivatization reaction :+10 μ L 50mM N-methylmorpholine of 10 μ L sample+5 μ L 20mM NBD-COCl, 60 DEG C of reaction 15min,
Add 150 μ L 2%TFA to terminate reaction, 10 μ L is taken directly to carry out HPLC analysis；
Linear relationship and detection limit measurement: precision weighs lactic acid reference substance, and deionized water is added to be made into 0.1,0.5,1,5,10,20,
30,40,50mmol/L contrast solution, by progress HPLC analysis after the reaction of above-mentioned derivatization reaction method；Gained integral area pair
Concentration carries out linear regression, obtains equation y=1602534x+18831, R2= 0.9999；In 0.1~50mmol/L range
Interior linear good, minimum concentrations are 10nM, and X is concentration mmol/L, and Y is peak area；
The measurement of repeatability and accuracy: in 6 measurements of interior continuous sample introduction on the same day under determining chromatographic condition, according to peak face
It is 3.8%, n=6 that product, which calculates gained relative standard deviation, and the relative standard deviation of retention time is 0.84%, n=6；
The measurement of lactic acid content in biological sample: after each group sample presses above-mentioned reaction condition derivatization reaction after handling according to the above method
Carry out HPLC analysis, the content of lactic acid is calculated in external standard method, as a result are as follows: D-gal induce Aging Mouse Modle blood plasma, brain tissue and
Lactic acid content significantly increases in extracellular fluid, and PQQ administration group lactic acid content is remarkably decreased.
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| CN102323340A (en) * | 2011-06-09 | 2012-01-18 | 江苏省原子医学研究所 | Action of pyrro-quinoline quinone and amino acid neurotransmitters |
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