CN102621267B - Method for measuring D-sorbitol in plasma or urine - Google Patents
Method for measuring D-sorbitol in plasma or urine Download PDFInfo
- Publication number
- CN102621267B CN102621267B CN201210104661.XA CN201210104661A CN102621267B CN 102621267 B CN102621267 B CN 102621267B CN 201210104661 A CN201210104661 A CN 201210104661A CN 102621267 B CN102621267 B CN 102621267B
- Authority
- CN
- China
- Prior art keywords
- glucitol
- urine
- concentration
- ion
- blood plasma
- 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.)
- Expired - Fee Related
Links
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a method for measuring D-sorbitol in plasma or urine with the liquid chromatography-mass spectrometry, which comprises the following steps: taking a to-be-tested sample, adding a certain amount of an organic solvent protein precipitation for protein precipitation, separating via a chromatographic column after pretreatment, and detecting with a mass spectrometric detector. The method disclosed by the invention is rapid, accurate, high in sensitivity and convenient in operation, and is suitable for measuring concentration of D-sorbitol in plasma and urine.
Description
Technical field
The present invention is a kind of Pharmaceutical Analysis technology, relates to the analysis determining method of drug disposition, is specifically related to a kind of method of measuring D-glucitol concentration in blood plasma or urine.
Background technology
Liver function volume of blood flow refers to flows through hepatic sinusoid and brings into play the part hepatic blood flow of function, only has at present by the method for clearance rate and measures.Clinically, D-glucitol clearance rate can reflect THBF, and its change can reflect the variation of hepatic blood flow reliably, thus reflection hepatic functional reserve.So D-glucitol hepatic clearance is to select the reliability index of the result for the treatment of of therapeutic modality, evaluation in cirrhosis disease treatment.By measuring the plasma concentration of D-glucitol and global purge rate and the renal clearance that urine concentration can calculate sorbierite, because sorbierite is removed (take liver removing as main) outward without liver, kidney, therefore the clearance rate of sorbierite liver is the poor of global purge rate and renal clearance.
About the assay method of D-glucitol in biological specimen, being mainly enzyme spectrophotometric method at present, there is the defects such as sensitivity is low, quantitatively inaccurate, complex operation in this method.
Summary of the invention
Goal of the invention: the object of the present invention is to provide a kind of method of measuring D-glucitol concentration in blood plasma or urine.
Technical scheme: the object of the invention is to realize by following scheme:
Measure a method for D-glucitol concentration in blood plasma or urine, adopt LC-MS system measurement, comprise the following steps: (1) blood plasma or urine sample pre-service; (2) D-glucitol carries out liquid chromatography separation; (3) mass spectroscopy; (4) calculate.
The method of D-glucitol concentration in said determination blood plasma or urine,
Step (1) blood plasma or urine sample pre-service: draw and gave the dog of D-glucitol or people's blood plasma or urine sample, add quantitative methyl alcohol or acetonitrile protein precipitant to carry out albumen precipitation;
Step (2) D-glucitol carries out liquid chromatography separation: the Lichrospher NH of chromatographic column Wei Han nation in chromatographic condition
2post; Mobile phase: the methanol-water solution that volume ratio is 70: 30; Flow velocity: 0.6-1.0mLmin
-1, split ratio 1: 3; Column temperature: 30-40 ℃; Type of elution is isocratic elution;
Step (3) mass spectroscopy, condition is: ion gun: electro-spray ionization ionization source ESI; Ion detection mode: selectivity ion detection SIM; Ion polarity: negative ion; Capillary voltage: 3.0KV; Ion source temperature: 80-120 ℃; Desolventizing temperature: 340-360 ℃; Desolventizing gas: 380L/h; Taper hole gas: 30L/h; Desolventizing gas and taper hole gas are high pure nitrogen; The ion selector channel of detected object: D-glucitol, [M-H]-, m/z 180.9, and Dwell 0.2, and Cone 30; Interior mark Tinidazole, [M-H]-, m/z 246.0, and Dwell 0.2, and Cone 30;
Step (4) is calculated: adopt internal standard method, with the peak area ratio substitution typical curve equation calculating D-glucitol concentration of D-glucitol and interior mark Tinidazole.
The method of D-glucitol concentration in said determination blood plasma or urine,
Step (2) D-glucitol carries out liquid chromatography separation: the Lichrospher NH of chromatographic column Wei Han nation in chromatographic condition
2post; Mobile phase: the methanol-water solution that volume ratio is 70: 30; Flow velocity: 0.8mLmin
-1, split ratio 1: 3; Column temperature: 35 ℃; Type of elution is isocratic elution;
Step (3) mass spectroscopy, condition is: ion gun: electro-spray ionization ionization source ESI; Ion detection mode: selectivity ion detection SIM; Ion polarity: negative ion; Capillary voltage: 3.0KV; Ion source temperature: 100 ℃; Desolventizing temperature: 350 ℃; Desolventizing gas: 380L/h; Taper hole gas: 30L/h; Desolventizing gas and taper hole gas are high pure nitrogen; The ion selector channel of detected object: D-glucitol, [M-H]-, m/z 180.9, and Dwell 0.2, and Cone 30; Interior mark Tinidazole, [M-H]-, m/z 246.0, and Dwell 0.2, and Cone 30.
The method of D-glucitol concentration in said determination blood plasma or urine, in step (1), plasma sample: get blood plasma 100 μ L, add 900 μ L methanol extractions, vibration 1min, the centrifugal 10min of 12000rpm, get supernatant 50 μ L, adding 50 μ L methyl alcohol, 400 μ L concentration is 2.174 μ gmL
-1tinidazole methyl alcohol inner mark solution, mix; Urine samples: get urine 10 μ L, add methyl alcohol 990 μ L, vibration 1min, the centrifugal 10min of 12000rpm, gets supernatant 50 μ L, and adding 950 μ L concentration is 2.174 μ gmL
-1tinidazole methyl alcohol inner mark solution, mix.
The method of D-glucitol concentration in said determination blood plasma or urine, in step (2), in chromatographic condition, chromatogram column length is 250mm, and internal diameter is 4.6mm, and particle diameter is 5 μ m.
The method of D-glucitol concentration in said determination blood plasma or urine, in step (1) D-glucitol give with blood plasma or urine sample sampling method be: vein persistent instillation 5%D-sorbitol solution 3h, dosage is 150mgkg
-1, in the rear 3h that instils, gather peripheric venous blood 3ml, inject in calparine pipe, prepare blood plasma or collect urine in the rear 3h that instils, metering, standby survey.
The method of D-glucitol concentration in said determination blood plasma or urine, described blood plasma or urine are dog or people's blood plasma or urine.
Beneficial effect:
(1) preprocess method is easy: a step organic solvent precipitation of protein, is applicable to conventional determining.(2) specificity is strong: adopt Lichrospher NH
2chromatographic column is as fixing phase, and the mixed liquor of first alcohol and water is as mobile phase, isocratic elution, and D-glucitol retention time is 4.83min left and right, and interior mark Tinidazole retention time is 4.13min left and right, within 6.5 minutes, completes mensuration, and endogenous material is not disturbed the mensuration of the two.(3) highly sensitive: the minimum 1.25 μ gmL that are quantitatively limited to of blood plasma
-1, the minimum 0.125 μ gmL that is quantitatively limited to of urine
-1.(4) the inventive method is quick, accurate, highly sensitive, easy and simple to handle, for the clinical blood urine determination of drug concentration of sorbierite provides foundation, has the prospect of clinical practice.The blood plasma typical curve range of linearity of this method is 1.25～125 μ gmL
-1, the urine typical curve range of linearity is 0.125～12.5 μ gmL
-1, be in a few days all less than 5% with day to day precision RSD.
Accompanying drawing explanation:
Fig. 1 is the mass spectrogram of dog blank plasma
Fig. 2 is that dog blank plasma adds the mass spectrogram after standard items
Fig. 3 is the mass spectrogram that dog gives plasma sample after D-glucitol
Fig. 4 is the mass spectrogram of methyl alcohol
Fig. 5 is the mass spectrogram that methyl alcohol adds standard items
Fig. 6 is the mass spectrogram that dog gives urine samples after D-glucitol
Note: figure intermediate ion selector channel 1 is D-glucitol, [M-H]-, m/z 180.9, and retention time is 4.83min left and right; Ion selector channel 2 is interior mark Tinidazole, [M-H]-, m/z 246.0, and retention time is 4.13min left and right.
Embodiment
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand, the described particular content of embodiment is only for the present invention is described, and should also can not limit the present invention described in detail in claims.
Embodiment 1: the mensuration of D-glucitol concentration in dog plasma
One, experiment material and instrument
D-glucitol reference substance: be purchased from Nat'l Pharmaceutical & Biological Products Control Institute; Lot number: 100555-200306 Tinidazole reference substance (interior mark): be purchased from Nat'l Pharmaceutical & Biological Products Control Institute; Lot number: 100336-200402 test water: ultrapure water; Methyl alcohol: chromatographically pure (Merck Company).
ZQ2000LC/MS LC-MS instrument (U.S. Waters company, it is Waters 2695 that high efficiency liquid phase instrument is joined by institute); Masslynx 4.0 data handling systems; The miniature vortex mixed instrument of WH-2 (Shanghai Hu Xi analytical instrument factory); AE240 electronic balance (Shanghai Mettler-Toledo, Inc.); Millipore Drict-Q5 water purification machine (French Millipore company); Biofuge PrimoR high-speed refrigerated centrifuge (German Heraeus company).
Two, liquid matter condition
1. liquid phase chromatogram condition
Chromatographic column: the LichrospherNH2 of Chinese nation post (dp 5mm, 250mm ID * 4.6mm); Mobile phase: methanol-water solution (70: 30, v/v); Flow velocity: 0.8mLmin
-1(split ratio 1: 3); Column temperature: 35 ℃.
2. mass spectrum condition
Ion gun: electro-spray ionization ionization source ESI; Ion detection mode: selectivity ion detection SIM; Ion polarity: negative ion; Capillary voltage: 3.0KV; Ion source temperature: 100 ℃; Desolventizing temperature: 350 ℃; Desolventizing gas: 380L/h; Taper hole gas: 30L/h; Desolventizing gas and taper hole gas are high pure nitrogen; The ion selector channel of detected object: D-glucitol, [M-H]-, m/z 180.9, and Dwell 0.2, and Cone 30; Interior mark Tinidazole, [M-H]-, m/z246.0, Dwell 0.2, and Cone 30.
Three, experimentation:
The preparation of 1.D-sorbierite standard solution:
Precision takes D-glucitol 13.96mg, is placed in 10mL volumetric flask, adds methyl alcohol to dissolve and is settled to scale, shakes up, and obtains 1.396mgmL
-1the storing solution of D-glucitol.Precision measures appropriate storing solution and dilutes successively with methyl alcohol, is made into concentration and is respectively 2.5,5,10,25,50,100,250 μ gmL
-1d-glucitol standard solution.
2. the processing of dog plasma sample:
Get 3 dog vein persistent instillation 5%D-sorbitol solution 3h, dosage is 150mgkg
-1.After instiling, 3h gathers peripheric venous blood 3ml, inject standby survey in calparine pipe, prepare blood plasma, get dog plasma 100 μ L, disposal route is: add 900 μ L methanol extractions, vibration 1min, the centrifugal 10min of 12000rpm, get supernatant 50 μ L, add 50 μ L methyl alcohol, 400 μ L Tinidazole methanol solution (interior mark, 2.174 μ gmL
-1), mix, 5 μ L sample introductions (seeing Fig. 3), the ratio f1 of calculating D-glucitol and interior mark peak area, substitution typical curve calculating concentration, then be multiplied by corresponding extension rate (10), obtain the concentration of D-glucitol in blood plasma.If concentration exceeds standard curve range, sample dilutes certain multiple sample introduction again.
3. specificity:
Get the dog blank plasma 100 μ L that do not give D-glucitol, add 900 μ L methanol extractions, vibration 1min, the centrifugal 10min of 12000rpm, gets supernatant standby.Get this supernatant 50 μ L, add methyl alcohol 450 μ L, mix, 5 μ L sample introductions (seeing Fig. 1).Separately get this supernatant 50 μ L, add D-glucitol standard solution 50 μ L, add 400 μ L Tinidazole methanol solution (interior mark, 2.174 μ gmL
-1), mix 5 μ L sample introductions (seeing Fig. 2).
Result demonstration, under the LC-MS condition adopting in this experiment, plasma sample causes interference without assorted peak to detected components, and D-glucitol retention time is in 4.83min left and right, and interior mark Tinidazole retention time is in 4.13min left and right.D-glucitol and interior mark do not interfere with each other, and peak shape is good, and baseline is steady.
4. linear test
Dog plasma typical curve: get that not give the dog blank plasma of D-glucitol appropriate, within 1: 9 by volume, add methanol extraction, vibration 1min, the centrifugal 10min of 12000rpm, gets supernatant standby.Get respectively the D-glucitol standard solution 50 μ L of variable concentrations, add the supernatant 50 μ L after blank plasma is processed, the concentration being made into containing D-glucitol is respectively 1.25,2.5,5,12.5,25,50,125 μ gmL
-1plasma containing drug sample, add 400 μ L Tinidazole methanol solution (interior mark, 2.174 μ gmL
-1), mix 5 μ L sample introductions.Calculate the ratio f1 of D-glucitol and interior mark peak area, with peak area ratio f1, blood concentration C is done to return and calculate, obtain regression equation: f1=0.9646 * C+0.1192 (r=0.9994), D-glucitol plasma concentration is at 1.25～125 μ gmL
-1scope internal linear relation is good.The minimum 1.25 μ gmL that are quantitatively limited to
-1.
5. accuracy and precision
According to the preparation of dog plasma typical curve method, containing D-glucitol concentration, be 2.5,12.5,50 μ gmL
-1basic, normal, high containing D-glucitol plasma sample, by " processing of dog plasma sample " disposal route, process respectively.Do continuously 3 days, every day, each concentration was respectively done 5 duplicate samples, calculate the ratio f of D-glucitol peak area As and interior mark peak area Ai, in the blood plasma typical curve on substitution same day, try to achieve D-glucitol measured concentration, by measured concentration calculate in a few days, day to day precision and relative standard deviation (RSD), measured concentration with add the ratio of concentration to be accuracy.Result demonstration, is in a few days all less than 5% with day to day precision RSD, and accuracy meets the requirements.
6. measurement result
In above-mentioned 3 dog plasmas that gave D-glucitol, D-glucitol content is respectively 70.0,60.2,65.4 μ gmL
-1.Embodiment 2: the mensuration of D-glucitol concentration in dog urine
One, experiment material and instrument
D-glucitol reference substance: be purchased from company of Nat'l Pharmaceutical & Biological Products Control Institute; Lot number: 100555-200306 Tinidazole reference substance (interior mark): be purchased from Nat'l Pharmaceutical & Biological Products Control Institute; Lot number: 100336-200402 test water: ultrapure water; Methyl alcohol: chromatographically pure (Merck Company).
ZQ2000LC/MS LC-MS instrument (U.S. Waters company, it is Waters 2695 that high efficiency liquid phase instrument is joined by institute); Masslynx 4.0 data handling systems; The miniature vortex mixed instrument of WH-2 (Shanghai Hu Xi analytical instrument factory); AE240 electronic balance (Shanghai Mettler-Toledo, Inc.); Millipore Drict-Q5 water purification machine (French Millipore company); Biofuge PrimoR high-speed refrigerated centrifuge (German Heraeus company).
Two, liquid matter condition
1. liquid phase chromatogram condition
Chromatographic column: the LichrospherNH2 of Chinese nation post (dp 5mm, 250mm ID * 4.6mm); Mobile phase: methanol-water solution (70: 30, v/v); Flow velocity: 0.8mLmin
-1(split ratio 1: 3); Column temperature: 35 ℃.
2. mass spectrum condition
Ion gun: electro-spray ionization ionization source ESI; Ion detection mode: selectivity ion detection SIM; Ion polarity: negative ion; Capillary voltage: 3.0KV; Ion source temperature: 100 ℃; Desolventizing temperature: 350 ℃; Desolventizing gas: 380L/h; Taper hole gas: 30L/h; Desolventizing gas and taper hole gas are high pure nitrogen; The ion selector channel of detected object: D-glucitol, [M-H]-, m/z 180.9, and Dwell 0.2, and Cone 30; Interior mark Tinidazole, [M-H]-, m/z246.0, Dwell 0.2, and Cone 30.
Three, experimentation:
The preparation of 1.D-sorbierite standard solution:
Precision takes D-glucitol 13.96mg, is placed in 10mL volumetric flask, adds methyl alcohol to dissolve and is settled to scale, shakes up, and obtains 1.396mgmL
-1the storing solution of D-glucitol.Precision measures appropriate storing solution to dilute successively with methyl alcohol, is made into concentration and is respectively 2.5,5,10,25,50,100,250 μ gmL
-1d-glucitol standard solution.
2. the processing of dog urine samples:
Get 3 dog vein persistent instillation 5%D-sorbitol solution 3h, dosage is 150mgkg
-1.Collect urine in the rear 3h that instils, metering, standby survey, draw the dog urine 10 μ L that gave D-glucitol, disposal route is: add methyl alcohol 990 μ L, vibration 1min, the centrifugal 10min of 12000rpm, get supernatant 50 μ L, add 950 μ L Tinidazole methanol solution (interior mark, 2.174 μ gmL
-1), mix, 5 μ L sample introductions, the ratio f2 of calculating D-glucitol and interior mark peak area, substitution typical curve calculating concentration, then be multiplied by corresponding extension rate (100), obtain the concentration (seeing Fig. 6) of D-glucitol in urine samples.If concentration exceeds standard curve range, sample dilutes certain multiple sample introduction again.
3. linear test:
Dog urine typical curve: get respectively the D-glucitol standard solution 50 μ L of variable concentrations, add 950 μ L Tinidazole methanol solution (interior mark, 2.174 μ gmL
-1), the concentration being made into containing D-glucitol is respectively 0.125,0.25,0.5,1.25,2.5,5,12.5 μ gmL
-1pastille urine sample, mix 5 μ L sample introductions.Calculate the ratio f2 of D-glucitol and interior mark peak area, with peak area ratio f2, drug usine level C is done to return and calculate, obtain regression equation: f2=0.4395 * C-0.07559 (r=0.9992), D-glucitol urine concentration is at 0.125～12.5 μ gmL
-1scope internal linear relation is good.The minimum 0.125 μ gmL that is quantitatively limited to
-1.
4. specificity
Because urine samples has diluted 1000 times, measure, matrix effect can be ignored, so the test of urine samples specificity detects (seeing Fig. 4, Fig. 5) with methyl alcohol as medium.
Result demonstration, under the LC-MS condition adopting in this experiment, urine samples causes interference without assorted peak to detected components, and D-glucitol retention time is in 4.83min left and right, and interior mark Tinidazole retention time is in 4.13min left and right.D-glucitol and interior mark do not interfere with each other, and peak shape is good, and baseline is steady.
5. accuracy and precision
According to the preparation of dog urine typical curve method, containing D-glucitol concentration, be 0.25,1.25,5 μ gmL
-1basic, normal, high containing D-glucitol urine sample, by " processing of dog urine samples " disposal route, process respectively.Do continuously 3 days, every day, each concentration was respectively done 5 duplicate samples, calculate the ratio f of D-glucitol peak area As and interior mark peak area Ai, in the urine typical curve on substitution same day, try to achieve D-glucitol measured concentration, by measured concentration calculate in a few days, day to day precision and relative standard deviation (RSD), measured concentration with add the ratio of concentration to be accuracy.Result demonstration, is in a few days all less than 5% with day to day precision RSD, and accuracy meets the requirements.
6. measurement result
In above-mentioned 3 dog urines that gave D-glucitol, the content of D-glucitol is respectively 3.60,4.98,5.39 μ gmL
-1.
Embodiment 3: the mensuration of D-glucitol concentration in dog plasma
With reference to embodiment 1, get 3 dog vein persistent instillation 5%D-sorbitol solution 3h, dosage is 150mgkg
-1.After instiling, 3h gathers peripheric venous blood 3ml, injects standby survey in calparine pipe, prepares blood plasma, in sample preparation, adopt acetonitrile protein precipitant to carry out albumen precipitation, all the other conditions are identical, measure, and in result dog plasma, the content of D-glucitol is respectively 53.1,88.2,54.4 μ gmL
-1.
Embodiment 4: the mensuration of D-glucitol concentration in dog urine
With reference to embodiment 2, get 3 dog vein persistent instillation 5%D-sorbitol solution 3h, dosage is 150mgkg
-1.Collect urine in the rear 3h that instils, metering, standby survey, adopt acetonitrile protein precipitant to carry out albumen precipitation in sample preparation, and all the other conditions are identical, measure, and in result dog urine, the content of D-glucitol is respectively 3.87,4.96,5.96 μ gmL
-1.
Embodiment 5: the mensuration of D-glucitol concentration in human plasma
With reference to 1,3 people's vein persistent instillation 5%D-sorbitol solution 3h of embodiment, dosage is 150mgkg
-1.After instiling, 3h gathers peripheric venous blood 3ml, injects standby survey in calparine pipe, prepares blood plasma, in sample preparation, adopt Methanol Protein precipitation agent to carry out albumen precipitation, all the other conditions are identical, measure, and in result human plasma, the content of D-glucitol is respectively 74.1,61.5,64.1 μ gmL
-1.
Embodiment 6: the mensuration of D-glucitol concentration in human urine
With reference to 2,3 people's vein persistent instillation 5%D-sorbitol solution 3h of embodiment, dosage is 150mgkg
-1.Collect urine in the rear 3h that instils, metering, standby survey, adopt Methanol Protein precipitation agent to carry out albumen precipitation in sample preparation, and all the other conditions are identical, measure, and in result human urine, the content of D-glucitol is respectively 3.68,4.96,5.59 μ gmL
-1.
Embodiment 7: the mensuration of D-glucitol concentration in human plasma
With reference to 1,3 people's vein persistent instillation 5%D-sorbitol solution 3h of embodiment, dosage is 150mgkg
-1.After instiling, 3h gathers peripheric venous blood 3ml, injects standby survey in calparine pipe, prepares blood plasma, in sample preparation, adopt acetonitrile protein precipitant to carry out albumen precipitation, all the other conditions are identical, measure, and in result human plasma, the content of D-glucitol is respectively 62.7,64.2,55.7 μ gmL
-1.
Embodiment 8: the mensuration of D-glucitol concentration in human urine
With reference to 2,3 people's vein persistent instillation 5%D-sorbitol solution 3h of embodiment, dosage is 150mgkg
-1.Collect urine in the rear 3h that instils, metering, standby survey, adopt acetonitrile protein precipitant to carry out albumen precipitation in sample preparation, and all the other conditions are identical, measure, and in result human urine, the content of D-glucitol is respectively 3.76,4.43,5.63 μ gmL
-1.
Embodiment 9: the mensuration of D-glucitol concentration in dog plasma
With reference to embodiment 1, get 3 dog vein persistent instillation 5%D-sorbitol solution 3h, dosage is 150mgkg
-1.After instiling, 3h gathers peripheric venous blood 3ml, injects standby survey in calparine pipe, prepares blood plasma, adopts acetonitrile protein precipitant to carry out albumen precipitation in sample preparation, and in liquid chromatography separation condition, flow velocity is 0.6mLmin
-1, column temperature is 30 ℃; Mass spectroscopy, condition intermediate ion source temperature is 80 ℃; Desolventizing temperature: 340 ℃; All the other conditions are identical, measure, and in result dog plasma, the content of D-glucitol is respectively 63.8,82.1,59.4 μ gmL
-1.
Embodiment 10: the mensuration of D-glucitol concentration in dog urine
With reference to embodiment 2, get 3 dog vein persistent instillation 5%D-sorbitol solution 3h, dosage is 150mgkg
-1.Collect urine in the rear 3h that instils, metering, standby survey, adopt acetonitrile protein precipitant to carry out albumen precipitation in sample preparation, and in liquid chromatography separation condition, flow velocity is 1.0mLmin
-1, column temperature is 40 ℃; Mass spectroscopy, condition intermediate ion source temperature is 120 ℃; Desolventizing temperature: 360 ℃; All the other conditions are identical, measure, and in result dog urine, the content of D-glucitol is respectively 3.75,4.53,5.62 μ gmL
-1.
Embodiment 11: the mensuration of D-glucitol concentration in human plasma
With reference to 1,3 people's vein persistent instillation 5%D-sorbitol solution 3h of embodiment, dosage is 150mgkg
-1.After instiling, 3h gathers peripheric venous blood 3ml, injects standby survey in calparine pipe, prepares blood plasma, adopts acetonitrile protein precipitant to carry out albumen precipitation in sample preparation, and in liquid chromatography separation condition, flow velocity is 0.6mLmin
-1, column temperature is 30 ℃; Mass spectroscopy, condition intermediate ion source temperature is 80 ℃; Desolventizing temperature: 340 ℃; All the other conditions are identical, measure, and in result human plasma, the content of D-glucitol is respectively 73.8,89.1,69.4 μ gmL
-1.
Embodiment 12: the mensuration of D-glucitol concentration in human urine
With reference to 2,3 people's vein persistent instillation 5%D-sorbitol solution 3h of embodiment, dosage is 150mgkg
-1.Collect urine in the rear 3h that instils, metering, standby survey, adopt acetonitrile protein precipitant to carry out albumen precipitation in sample preparation, and in liquid chromatography separation condition, flow velocity is 1.0mLmin
-1, column temperature is 40 ℃; Mass spectroscopy, condition intermediate ion source temperature is 120 ℃; Desolventizing temperature: 360 ℃; All the other conditions are identical, measure, and in result human urine, the content of D-glucitol is respectively 5.75,4.83,5.92 μ gmL
-1.
Claims (4)
1. measure a method for D-glucitol concentration in blood plasma or urine, it is characterized in that adopting LC-MS system measurement, comprise the following steps: (1) blood plasma or urine sample pre-service; (2) D-glucitol carries out liquid chromatography separation; (3) mass spectroscopy; (4) calculate,
Step (1) blood plasma or urine sample pre-service: draw and gave the dog of D-glucitol or people's blood plasma or urine sample, add quantitative methyl alcohol or acetonitrile protein precipitant to carry out albumen precipitation;
Step (2) D-glucitol carries out liquid chromatography separation: the chromatographic column Wei Han LichrospherNH of nation in chromatographic condition
2post; Mobile phase: the methanol-water solution that volume ratio is 70:30; Flow velocity: 0.6-1.0mLmin
-1, split ratio 1:3; Column temperature: 30-40 ℃; Type of elution is isocratic elution;
Step (3) mass spectroscopy, condition is: ion gun: electro-spray ionization ionization source ESI; Ion detection mode: selectivity ion detection SIM; Ion polarity: negative ion; Capillary voltage: 3.0KV; Ion source temperature: 80-120 ℃; Desolventizing temperature: 340-360 ℃; Desolventizing gas: 380L/h; Taper hole gas: 30L/h; Desolventizing gas and taper hole gas are high pure nitrogen; The ion selector channel of detected object: D-glucitol, [M-H]-, m/z180.9, Dwell0.2, Cone30; Interior mark Tinidazole, [M-H]-, m/z246.0, Dwell0.2, Cone30;
Step (4) is calculated: adopt internal standard method, with the peak area ratio substitution typical curve equation calculating D-glucitol concentration of D-glucitol and interior mark Tinidazole;
In step (1), plasma sample: get blood plasma 100 μ L, add 900 μ L methanol extractions, vibration 1min, the centrifugal 10min of 12000rpm, gets supernatant 50 μ L, and adding 50 μ L methyl alcohol, 400 μ L concentration is 2.174 μ gmL
-1tinidazole methyl alcohol inner mark solution, mix; Urine samples: get urine 10 μ L, add methyl alcohol 990 μ L, vibration 1min, the centrifugal 10min of 12000rpm, gets supernatant 50 μ L, and adding 950 μ L concentration is 2.174 μ gmL
-1tinidazole methyl alcohol inner mark solution, mix.
2. according to the method for measuring D-glucitol concentration in blood plasma or urine in claim 1, it is characterized in that:
Step (2) D-glucitol carries out liquid chromatography separation: the chromatographic column Wei Han LichrospherNH of nation in chromatographic condition
2post; Mobile phase: the methanol-water solution that volume ratio is 70:30; Flow velocity: 0.8mLmin
-1, split ratio 1:3; Column temperature: 35 ℃; Type of elution is isocratic elution;
Step (3) mass spectroscopy, condition is: ion gun: electro-spray ionization ionization source ESI; Ion detection mode: selectivity ion detection SIM; Ion polarity: negative ion; Capillary voltage: 3.0KV; Ion source temperature: 100 ℃; Desolventizing temperature: 350 ℃; Desolventizing gas: 380L/h; Taper hole gas: 30L/h; Desolventizing gas and taper hole gas are high pure nitrogen; The ion selector channel of detected object: D-glucitol, [M-H]-, m/z180.9, Dwell0.2, Cone30; Interior mark Tinidazole, [M-H]-, m/z246.0, Dwell0.2, Cone30.
3. according to the method for measuring D-glucitol concentration in blood plasma or urine in claim 1, it is characterized in that in the middle chromatographic condition of step (2), chromatogram column length is 250mm, internal diameter is 4.6mm, and particle diameter is 5 μ m.
4. according to the method for measuring D-glucitol concentration in blood plasma or urine in claim 1, it is characterized in that described blood plasma or urine are behaved or blood plasma or the urine of dog.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210104661.XA CN102621267B (en) | 2012-04-09 | 2012-04-09 | Method for measuring D-sorbitol in plasma or urine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210104661.XA CN102621267B (en) | 2012-04-09 | 2012-04-09 | Method for measuring D-sorbitol in plasma or urine |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102621267A CN102621267A (en) | 2012-08-01 |
| CN102621267B true CN102621267B (en) | 2014-08-06 |
Family
ID=46561306
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210104661.XA Expired - Fee Related CN102621267B (en) | 2012-04-09 | 2012-04-09 | Method for measuring D-sorbitol in plasma or urine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102621267B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016013115A1 (en) * | 2014-07-25 | 2016-01-28 | 株式会社 リージャー | Analysis method for diluted biological sample component |
| CN109444301A (en) * | 2018-12-18 | 2019-03-08 | 江苏省中医院 | A kind of method of general reed Ka Bili concentration in measurement blood plasma |
| CN112485340A (en) * | 2019-11-27 | 2021-03-12 | 南京品生医学检验实验室有限公司 | Method for detecting 1, 5-sorbitan in plasma by ultra-high performance liquid chromatography tandem mass spectrometry |
-
2012
- 2012-04-09 CN CN201210104661.XA patent/CN102621267B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN102621267A (en) | 2012-08-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107367562B (en) | Analysis and detection method and application of polymyxin B sulfate | |
| Vaidya et al. | LC–MS–MS determination of olmesartan in human plasma | |
| Korecka et al. | Review of the newest HPLC methods with mass spectrometry detection for determination of immunosuppressive drugs in clinical practice | |
| Salama | Simultaneous HPLC–UV analysis of telmisartan and hydrochlorothiazide in human plasma | |
| CN105136957A (en) | Detection method for simultaneously measuring OXC in human plasma and metabolite MHD and MHD-G | |
| CN102175778A (en) | Method for synchronously measuring blood drug concentrations of multiple antidepressants | |
| CN102621267B (en) | Method for measuring D-sorbitol in plasma or urine | |
| CN106248809A (en) | A kind of method concurrently separating the content measuring three kinds of aminoglycoside antibioticss | |
| Koryagina et al. | Chromatography–mass spectrometry determination of alkyl methylphosphonic acids in urine | |
| CN100580447C (en) | Method for determining human plasma antiviral drug concentration | |
| RU2715378C1 (en) | Method for quantitative gas-chromatographic analysis of chloroacetophenone in water using an internal standard | |
| CN105424853A (en) | LC-MS/MS kit for detecting nicotine and its metabolites in saliva | |
| Studzińska et al. | New approach to the determination phosphorothioate oligonucleotides by ultra high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry | |
| CN107957467A (en) | A kind of method of lysophosphatidyl choline in separation determination pharmaceutical preparation | |
| CN105974016A (en) | Method for simultaneously detecting fosaprepitant and aprepitant in plasma | |
| CN103940918A (en) | A method of simultaneously detecting the content of artesunate and the content of dihydroartemisinin in animal blood plasma | |
| CN106153766B (en) | A kind of method of 8- table diosbulbin E Acetate concentrations in measurement blood plasma | |
| CN104849381A (en) | High-performance liquid chromatography-charged aerosol detector law-based method for simultaneously determining seven astragaloside components | |
| CN102565252B (en) | Method for detecting content of homocysteine in blood or urine | |
| CN111579684B (en) | Method for measuring content of total capsaicin in capsule wall material of capsule | |
| CN104101665B (en) | Method for detecting chaetoglobosin concentration in blood plasma | |
| CN100414293C (en) | Method for determining blood drug level of mizoribine | |
| Zheng et al. | Development and validation of an UPLC-MS/MS method for determination of jujuboside B in rat plasma and its application in pharmacokinetic and bioavailability studies | |
| CN107202837A (en) | A kind of analysis method for being used to detect animal muscle veterinary drug residue thing | |
| Zhurkovich et al. | Determination of buprenorphine and naloxone in patient blood plasma using HPLC-MS |
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 | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140806 Termination date: 20170409 |