CN103439442B - Method for determining content of inosine in blood plasma - Google Patents
Method for determining content of inosine in blood plasma Download PDFInfo
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- CN103439442B CN103439442B CN201310365873.8A CN201310365873A CN103439442B CN 103439442 B CN103439442 B CN 103439442B CN 201310365873 A CN201310365873 A CN 201310365873A CN 103439442 B CN103439442 B CN 103439442B
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Abstract
The invention discloses a method for determining the content of inosine in blood plasma. The method comprises the following steps: (a), adding 25 [mu]l of standard inosine pollution in 250 [mu]l of blank plasma of a human body to obtain at least five parts of inosine blood plasma solutions with different concentrations; then respectively adding 0.1 [mu]g/ml of standard lamivudine solution and 250 [mu]l of 4.25 percent phosphoric acid solution and carrying out eddy mixing to obtain a mixed solution; respectively adding the mixed solution in activated HLB (Hydrophile Lipophile Balance) solid phase extraction columns for elution, discarding an eluant, eluting with 1ml of mobile phase solution, collecting the eluant, centrifuging and obtaining a supernate; (b), carrying out LC/MS/MS (Liquid Chromatography/Mass Spectrometry/ Mass Spectrometry) analysis on the prepared supernate prepared from the step (a) under specific mass spectrometry and chromatography conditions; (c), establishing a standard curve and lower limit of quantitation of the day for calculation by using the same method according to the analysis result obtained from the step (b) to obtain the content of the inosine of the human blood plasma to be determined.
Description
Technical field
The present invention relates to medical science detection field, particularly relate to the assay method of Inosine Content in a kind of human plasma.
Background technology
Inosine (Inosine), also referred to as trophicardyl, inosine etc.Inosine is the normal composition of human body, is the precursor of adenine, and directly permeate through cell membranes enters body cell, participates in the synthetic of nucleic acid in vivo metabolism, energetic supersession and protein.
Studies confirm that: inosine can activate pyruvate oxidase system, improve the activity of coacetylase, activation liver function, and make the histocyte under low energy anaerobic condition proceed metabolism, contribute to the recovery of impaired hepatocyte function.And it is synthetic to participate in human energy metabolism and protein.Inosine can improve ATP level and can change various nucleotide into.Can stimulate in body and produce antibody, also can improve the absorption of enteron aisle to iron, activation liver function, accelerates hepatocellular reparation.There is the effect that strengthens leucocyte hyperplasia.
Summary of the invention
Object of the present invention aims to provide a kind of method of setting up human plasma inosine LC/MS/MS bioassay standard curve and lower limit of quantitation.
Another object of the present invention is to provide a kind of method of measuring Inosine Content in human plasma.
Specifically, first aspect present invention has been to provide a kind of method of setting up human plasma inosine LC/MS/MS bioassay standard curve and lower limit of quantitation, and it comprises the steps:
(a) in human body blank plasma 250ul, add inosine standard solution 25ul, be mixed with the inosine plasma solutions of at least five parts of variable concentrations, add respectively 0.1ug/ml Lamivudine titer 25ul and 4.25% phosphoric acid solution 250ul, eddy current mixes again, and obtains at least five parts of mixed solutions; Described mixed solution is added respectively in activated HLB solid-phase extraction column and carries out wash-out, abandon eluent, then use 1ml mobile phase solution 50mM CH
3cOONH
2: CH
3oH=1:1 wash-out, collects eluent, centrifugal, gets supernatant;
(b) supernatant making through step (a) is carried out to LC/MS/MS analysis under the following conditions
Mass spectrum condition:
Mass spectrum condition:
Compound parameter:
Inosine mothers and sons ion pair: 269.1/137.1, time:400msec
Lamivudine mothers and sons ion pair: 230.2/112.2, time:200msec
Duration：4min
Cycles：393
DP＝26V
CE＝15V
CXP＝4V
CEP＝4V
Q1resolution：Unit
Q2resolution：Unit，
Ion gun/gas parameter:
Ion?Source：Turbo?Spray
Curtain?Gas(CUR)：10psig
Collision?Gas(CAD)：Medium
Ion?Spray?Voltage(IS)：5000V
Temperature(TEM)：700℃
Ion?Source?Gas1(Gas1)：60psig
Ion?Source?Gas2(Gas2)：10psig
Interface?Heater(ihe)：ON，
Liquid phase chromatogram condition:
Chromatographic column: Waters XBridge
tMc18, specification: 3.5um, 2.1mm * 100mm, Part No.:186003022, S/N:014530308140 45
Mobile phase: 50mM CH
3cOONH
2: CH
3oH=1:1
Flow velocity: 0.2ml/min
Sample size: 10ul
Sample injection time: 4min;
(c) analysis result based on step (b), sets up typical curve and lower limit of quantitation that human plasma inosine LC/MS/MS measures.
In a preference, in described step (a), also comprise described mixed solution-70 ℃ frozen step.
In another preference, the inosine plasma solutions of described variable concentrations is six parts, and its concentration is respectively 18.58ug/ml, 37.16ug/ml, 74.31ug/ml, 148.6ug/ml, 297.25ug/ml and 594.5ug/ml.
In another preference, the activation step of described activated HLB solid-phase extraction column comprises: use VARIAN
tMvAC ELUT SPS24 solid-phase extraction device, inserts fixed position by HLB solid-phase extraction column, successively uses 1ml methyl alcohol, 1ml water to carry out wash-out activation.
In another preference, described step (c) comprising: the human plasma inosine concentration to be measured (X) of take is horizontal ordinate, the human plasma inosine chromatographic peak area to be measured (As) of take is ordinate with the ratio (Y=As/Ai) of interior mark chromatographic peak area (Ai), carry out linear regression computing, try to achieve typical curve and lower limit of quantitation that human plasma inosine LC/MS/MS measures.
A second aspect of the present invention has been to provide a kind of method of measuring Inosine Content in human plasma, and it comprises the steps:
(a) in human plasma 250ul to be measured, add 0.1ug/ml Lamivudine titer 25ul and 4.25% phosphoric acid solution 250ul, eddy current mixes, and obtains mixed solution; Described mixed solution is added in activated HLB solid-phase extraction column and carries out wash-out, abandon eluent, then use 1ml mobile phase solution 50mM CH
3cOONH
2: CH
3oH=1:1 wash-out, collects eluent, centrifugal, gets supernatant;
(b) supernatant making through step (a) is carried out to LC/MS/MS analysis under the following conditions
Mass spectrum condition:
Mass spectrum condition:
Compound parameter:
Inosine mothers and sons ion pair: 269.1/137.1, time:400msec
Lamivudine mothers and sons ion pair: 230.2/112.2, time:200msec
Duration：4min
Cycles：393
DP＝26V
CE＝15V
CXP＝4V
CEP＝4V
Q1resolution：Unit
Q2resolution：Unit，
Ion gun/gas parameter:
Ion?Source：Turbo?Spray
Curtain?Gas(CUR)：10psig
Collision?Gas(CAD)：Medium
Ion?Spray?Voltage(IS)：5000V
Temperature(TEM)：700℃
Ion?Source?Gas1(Gas1)：60psig
Ion?Source?Gas2(Gas2)：10psig
Interface?Heater(ihe)：ON，
Liquid phase chromatogram condition:
Chromatographic column: Waters XBridge
tMc18, specification: 3.5um, 2.1mm * 100mm, Part No.:186003022, S/N:014530308140 45
Mobile phase: 50mM CH
3cOONH
2: CH
3oH=1:1
Flow velocity: 0.2ml/min
Sample size: 10ul
Sample injection time: 4min;
(c) analysis result based on step (b), by the described method of setting up human plasma inosine LC/MS/MS bioassay standard curve and lower limit of quantitation, set up typical curve and the lower limit of quantitation on the same day and calculate, obtain the Inosine Content in human plasma to be measured.
In a preference, in described step (a), also comprise described mixed solution-70 ℃ frozen step.
In another preference, the activation step of described activated HLB solid-phase extraction column comprises: use VARIAN
tMvAC ELUT SPS24 solid-phase extraction device, inserts fixed position by HLB solid-phase extraction column, successively uses 1ml methyl alcohol, 1ml water to carry out wash-out activation.
The details of various aspects of the present invention will be able to detailed description in chapters and sections subsequently.By below and the description of claim, feature of the present invention, object and advantage will be more obvious.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise all percentage, ratio, ratio or umber by weight.
Unless otherwise defined, the familiar meaning of all specialties of using in literary composition and scientific words and one skilled in the art is identical.In addition, any method similar or impartial to described content and material all can be applicable in the inventive method.The use that better implementation method described in literary composition and material only present a demonstration.
The above-mentioned feature that the present invention mentions, or the feature that embodiment mentions can combination in any.All features that patent specification discloses can with any composition forms use, each feature disclosing in instructions, can anyly provide the alternative characteristics of identical, impartial or similar object to replace.Therefore apart from special instruction, the feature disclosing is only the general example of equalization or similar features.
Embodiment 1: set up typical curve and lower limit of quantitation that human plasma inosine LC/MS/MS measures
One, instrument and reagent
1. instrument:
1) LC-MS instrument: AB Sciex 3200Q TRAP LC/MS/MS SYSTEM (SER.No:AF22091001), AB SCIEX company
2) solid-phase extraction device: VARIAN
tMvAC ELUT SPS24, WATERS company
3) hydro-extractor: BECKMAN Avanti
tM30Centrifuge (SER.No:GEY95G02), U.S. BECKMAN company
4) electronic balance: Mettler-AE240 double-range analytical balance, Mettler company
5) eddy mixer: XW-80, Instrument Factory, Shanghai Medical Science Univ.
2. reagent, reagent and consumptive material:
1) (O type Rh is positive, Shanghai City Blood Center, lot number: 20130100000031825) for blank plasma
2) inosine standard items (Nat'l Pharmaceutical & Biological Products Control Institute, lot number: 140669-201104)
3) Lamivudine (GlaxoSmithKline PLC, lot number: 12040022)
4) methyl alcohol (chromatographically pure, Tedia company, lot number: 1205441)
5) ammoniacal liquor (the actual company limited in Fauna of Kunshan, Jiangsu gold city, lot number: 040608)
6) phosphoric acid (chromatographically pure, Tedia company, lot number: 101026)
7) acetic acid (Zhen Ya chemical plant, Suzhou, lot number: 010517)
8) redistilled water (benevolence Ji hospital-made)
9) chromatographic column (Waters XBridge
tMc18, specification: 3.5um, 2.1 * 100mm), WATERS company
10) HLB solid-phase extraction column: OASIS
tMhLB Cartridge (Part No.WATO94225), WATERS company
Two, the activation of HLB solid-phase extraction column
Use VARIAN
tMvAC ELUT SPS24 solid-phase extraction device, inserts fixed position by HLB solid-phase extraction column, successively uses 1ml methyl alcohol, 1ml water to carry out wash-out activation, standby.
Three, blank plasma is placed in to normal temperature water-bath 5 minutes, taking out respectively 250ul is put in 6 different test tubes, each test tube adds respectively inosine titer 25ul, be made into successively and be equivalent to the sample that inosine plasma concentration is 18.58ug/ml, 37.16ug/ml, 74.31ug/ml, 148.6ug/ml, 297.25ug/ml and 594.5ug/ml, and add 0.1ug/ml Lamivudine titer 25ul, vortex 30 seconds, then add 4.25% phosphoric acid solution 250ul vortex 30 seconds.Before being added respectively, 6 pipe mixed liquors completed in the HLB pillar of activation, wash-out, and eluent is abandoned it, then uses 1ml mobile phase solution (50mM CH
3cOONH
2: CH
3oH=1:1) wash-out, now collects eluent.To collect liquid 20000rpm high speed centrifugation 10 minutes at 4 ℃, get supernatant, sample and get final product.
With following mass spectrum, chromatographic condition, carry out LC/MS/MS analysis
Mass spectrum condition:
Mass spectrum condition:
Compound parameter:
Inosine mothers and sons ion pair: 269.1/137.1, time:400msec
Lamivudine mothers and sons ion pair: 230.2/112.2, time:200msec
Duration：4min
Cycles：393
DP＝26V
CE＝15V
CXP＝4V
CEP＝4V
Q1resolution：Unit
Q2resolution：Unit，
Ion gun/gas parameter:
Ion?Source：Turbo?Spray
Curtain?Gas(CUR)：10psig
Collision?Gas(CAD)：Medium
Ion?Spray?Voltage(IS)：5000V
Temperature(TEM)：700℃
Ion?Source?Gas1(Gas1)：60psig
Ion?Source?Gas2(Gas2)：10psig
Interface?Heater(ihe)：ON，
Liquid phase chromatogram condition:
Chromatographic column: Waters XBridge
tMc18, specification: 3.5um, 2.1mm * 100mm, Part No.:186003022, S/N:014530308140 45
Mobile phase: 50mM CH
3cOONH
2: CH
3oH=1:1
Flow velocity: 0.2ml/min
Sample size: 10ul
Sample injection time: 4min;
Based on LC/MS/MS analysis result, set up the typical curve of inosine, respectively with the concentration (X of determinand inosine, ug/ml) be horizontal ordinate, take the chromatographic peak area (As) of determinand inosine and the ratio (Y=As/Ai) of interior mark Lamivudine chromatographic peak area (Ai) is ordinate, carry out linear regression computing, try to achieve typical curve Y=0.048 0X-0.064 9.Inosine is at 18.58～594.5ug/ml scope internal linear relation good (r=0.9998).The lower limit of quantitation of inosine is 18.58ug/ml.
Embodiment 2: measure the Inosine Content in human plasma
One, instrument and reagent
1. instrument:
1) LC-MS instrument: AB Sciex3200Q TRAP LC/MS/MS SYSTEM (SER.No:AF22091001), AB SCIEX company
2) solid-phase extraction device: VARIAN
tMvAC ELUT SPS24, WATERS company
3) hydro-extractor: BECKMAN Avanti
tM30Centrifuge (SER.No:GEY95G02), U.S. BECKMAN company
4) electronic balance: Mettler-AE240 double-range analytical balance, Mettler company
5) eddy mixer: XW-80, Instrument Factory, Shanghai Medical Science Univ.
2. reagent, reagent and consumptive material:
1) (O type Rh is positive, Shanghai City Blood Center, lot number: 20130100000031825) for blank plasma
2) inosine standard items (Nat'l Pharmaceutical & Biological Products Control Institute, lot number: 140669-201104)
3) Lamivudine (GlaxoSmithKline PLC, lot number: 12040022)
4) methyl alcohol (chromatographically pure, Tedia company, lot number: 1205441)
5) ammoniacal liquor (the actual company limited in Fauna of Kunshan, Jiangsu gold city, lot number: 040608)
6) phosphoric acid (chromatographically pure, Tedia company, lot number: 101026)
7) acetic acid (Zhen Ya chemical plant, Suzhou, lot number: 010517)
8) redistilled water (benevolence Ji hospital-made)
9) chromatographic column (Waters XBridge
tMc18, specification: 3.5um, 2.1 * 100mm), WATERS company
10) HLB solid-phase extraction column: OASIS
tMhLB Cartridge (Part No.WATO94225), WATERS company
Two, collect human plasma to be measured
From person's upper arm vein to be measured, extract new blood 5ml, the test tube that will have new blood inserts in trash ice, treats that whole blood collects completely, and 4 ℃, 20000rpm high speed centrifugation 10 minutes, get supernatant, obtains experimenter's blood plasma.
Three, the pre-service of human plasma to be measured
Get experimenter's blood plasma 250ul in 1.5mlEP pipe, add 0.1ug/ml Lamivudine titer 25ul, vortex 30 seconds, then add rapidly 4.25% phosphoric acid solution 250ul, vortex 30 seconds, obtains pretreatment sample.EP pipe is placed on test tube rack, is put in low temperature refrigerator-70 ℃ frozen.If get blood scene, there is no low temperature refrigerator, the EP pipe that has pretreatment sample can be put on test tube rack, slowly put into Foam Container (liquid level of liquid nitrogen should not surpass the height of the EP pipe) quick-frozen that has suitable capacity liquid nitrogen, deposit at least 2min.The operating process measure that must properly protect, takes care.(in relatively airtight operation room, and be with Frostbite preventing gloves, slowly put into.) after the complete freeze-drying of liquid to be mixed, EP pipe is put in the Foam Container that is full of dry ice, be transported to rapidly in the laboratory that has low temperature refrigerator-70 ℃ frozen, pretreatment sample is put in time of dry ice should not be over 1 day.
The above-mentioned pretreated sample that is stored in low temperature refrigerator is taken out, be placed in water-bath under room temperature and dissolve 10min.Before until completely dissolved mixed liquor being added, completed in the HLB pillar (with embodiment 1) of activation, wash-out, eluent is abandoned it, then uses 1ml mobile phase solution (50mM CH
3cOONH
2: CH
3oH=1:1) wash-out, and collect.To collect liquid 20000rpm high speed centrifugation 10 minutes at 4 ℃, get supernatant, obtain.
Four, detect the Inosine Content in human plasma to be measured
With above-mentioned mass spectrum, chromatographic condition (with embodiment 1), carry out LC/MS/MS analysis, by the method for embodiment 1, set up typical curve and the lower limit of quantitation on the same day and calculate, obtain the Inosine Content in human plasma to be measured.
Embodiment 3: the methodology checking of inosine assay method of the present invention
3.1. specificity
Get respectively each 250ul of blank plasma of 6 parts of different Healthy Peoples, use instead methanol aqueous solution 25ul except not adding interior mark Lamivudine solution 25ul, all the other press the method operation of embodiment 2; Get the alternative blank plasma that adds of certain density inosine titer 25ul, all the other press methods operation of embodiment 2; Get the plasma sample after experimenter's administration, by the method for embodiment 2, process.Result shows, is selecting under condition, and endogenous substance in plasma does not disturb measured object and interior target to measure, and specificity is good.
3.2. preci-sion and accuracy
By the method for embodiment 1, prepare the quality-control sample that plasma concentration is 28.5ug/ml, 57ug/ml, 228ug/ml, each concentration is carried out 6 increment this analysis, and METHOD FOR CONTINUOUS DETERMINATION 3d, according to typical curve on the same day, calculates the concentration that QC sample records.According to QC sample result, calculate the preci-sion and accuracy of this law, result shows, in the daytime, relative standard deviation (RSD) <15% in a few days, accuracy is all in 85%～115% scope.The preci-sion and accuracy that shows plasma sample analysis is good.Refer to table 1.
The precision of table 1. inosine assay method and accuracy (n=6)
3.3. the recovery and matrix effect
By the method for embodiment 1, prepare the quality-control sample that plasma concentration is 28.5ug/ml, 57ug/ml and 228ug/ml, each concentration is carried out 6 increment this analysis, obtains peak area value Anva and the Arop of inosine and interior mark Lamivudine.
In 1.5ml EP pipe, add respectively 250ul blank plasma, add 4.24% phosphoric acid solution 250ul, vortex 30 seconds.6 pipe mixed liquors are added respectively in the HLB pillar (with embodiment 1.) that has completed activation, wash-out, eluent is abandoned it, then uses 1ml mobile phase solution (50mM CH3COONH2:CH3OH=1:1) wash-out, and collects.To collect liquid 20000rpm high speed centrifugation 10 minutes at 4 ℃, get supernatant and be transferred in another clean 1.5ml test tube, precision adds inosine concentration for (28.5ug/ml, 57ug/ml, 228ug/ml) basic, normal, high contrast liquid 25ul and Lamivudine contrast solution (0.1ug/ml) 25ul respectively, each concentration 6 increment this analysis, vortex 30s, get the analysis of 10ul sample introduction, obtain peak area value Bnva and the Brop of inosine and interior mark Lamivudine.
It is accurate respectively that to add concentration be the inosine titer 25ul of 28.5ug/ml, 57ug/ml and 228ug/ml, add pure water 250ul, each concentration is carried out 6 increment this analysis, all the other press the method operation of embodiment 2, and the analysis of 10ul sample introduction obtains peak area value Cnva and the Crop of inosine and interior mark Lamivudine.
Calculate, the extraction recovery of inosine is 99.69 ± 0.71%, and the average extraction recovery of interior mark Lamivudine is 108.73 ± 4.55%.The average matrix effect of inosine is 91.96 ± 3.53%, and the average matrix effect of interior mark Lamivudine is 99.3 ± 1.50%.Extraction recovery and matrix effect are all in specialized range.
3.4. stability
Study on the stability is prepared the inosine plasma sample of basic, normal, high three concentration (28.5ug/ml, 57ug/ml and 228ug/ml) according to preceding method.Plasma sample is pressed after the method operation of embodiment 2, measures immediately, room temperature places 24h the stability that-70 ℃ of freeze thawing are 3 times after respectively inosine plasma sample being processed.Result shows, it is stable that the concentration of inosine all keeps, and do not occur significant change.(record concentration and add the relative standard deviation of concentration to be all less than ± 15%).
3.5. inosine determination of plasma concentration
Inosine concentration in 2 Healthy People 24h blood plasma is measured, by the method for embodiment 2, carried out operational processes, the inosine concentration range that records first place health volunteer is 33.66～66.70ug/ml; The inosine concentration range of second place is 23.22～59.82ug/ml.
Many aspects involved in the present invention have been done as above and have been set forth.Yet, it should be understood that before not departing from spirit of the present invention and put, those skilled in the art can be equal to and change and modify it, and described change and modification fall into the coverage of the application's claims equally.
Claims (8)
1. a method of setting up human plasma inosine LC/MS/MS bioassay standard curve and lower limit of quantitation, is characterized in that, it comprises the steps:
(a) in human body blank plasma 250 μ l, add inosine standard solution 25 μ l, be mixed with the inosine plasma solutions of at least five parts of variable concentrations, add respectively 0.1 μ g/ml Lamivudine titer 25 μ l and 4.25% phosphoric acid solution 250 μ l, eddy current mixes again, and obtains at least five parts of mixed solutions; Described mixed solution is added respectively in activated HLB solid-phase extraction column and carries out wash-out, abandon eluent, then use 1ml mobile phase solution 50mM CH
3cOONH
2: CH
3oH=1:1 wash-out, collects eluent, centrifugal, gets supernatant;
(b) supernatant making through step (a) is carried out to LC/MS/MS analysis under the following conditions
Mass spectrum condition:
Compound parameter:
Inosine mothers and sons ion pair: 269.1/137.1, time:400msec
Lamivudine mothers and sons ion pair: 230.2/112.2, time:200msec
Duration：4min
Cycles：393
DP＝26V
CE＝15V
CXP＝4V
CEP＝4V
Q1resolution：Unit
Q2resolution：Unit，
Ion gun/gas parameter:
Ion?Source：Turbo?Spray
Curtain?Gas(CUR)：10psig
Collision?Gas(CAD)：Medium
Ion?Spray?Voltage(IS)：5000V
Temperature(TEM)：700℃
Ion?Source?Gas1(Gas1)：60psig
Ion?Source?Gas2(Gas2)：10psig
Interface?Heater(ihe)：ON，
Liquid phase chromatogram condition:
Chromatographic column: Waters XBridge
tMc18, specification: 3.5 μ m, 2.1mm * 100mm, Part No.:186003022, S/N:01453030814045
Mobile phase: 50mM CH
3cOONH
2: CH
3oH=1:1
Flow velocity: 0.2ml/min
Sample size: 10 μ l
Sample injection time: 4min;
(c) analysis result based on step (b), sets up typical curve and lower limit of quantitation that human plasma inosine LC/MS/MS measures.
2. the method for claim 1, is characterized in that, also comprises described mixed solution-70 ℃ frozen step in described step (a).
3. method as claimed in claim 1 or 2, it is characterized in that, the inosine plasma solutions of described variable concentrations is six parts, and its concentration is respectively 18.58 μ g/ml, 37.16 μ g/ml, 74.31 μ g/ml, 148.6 μ g/ml, 297.25 μ g/ml and 594.5 μ g/ml.
4. method as claimed in claim 1 or 2, is characterized in that, the activation step of described activated HLB solid-phase extraction column comprises: use VARIAN
tMvAC ELUT SPS24 solid-phase extraction device, inserts fixed position by HLB solid-phase extraction column, successively uses 1ml methyl alcohol, 1ml water to carry out wash-out activation.
5. method as claimed in claim 1 or 2, it is characterized in that, described step (c) comprising: the human plasma inosine concentration to be measured (X) of take is horizontal ordinate, the human plasma inosine chromatographic peak area to be measured (As) of take is ordinate with the ratio (Y=As/Ai) of interior mark chromatographic peak area (Ai), carry out linear regression computing, try to achieve typical curve and lower limit of quantitation that human plasma inosine LC/MS/MS measures.
6. a method of measuring Inosine Content in human plasma, is characterized in that, it comprises the steps:
(a) in human plasma 250 μ l to be measured, add 0.1 μ g/ml Lamivudine titer 25 μ l and 4.25% phosphoric acid solution 250 μ l, eddy current mixes, and obtains mixed solution; Described mixed solution is added in activated HLB solid-phase extraction column and carries out wash-out, abandon eluent, then use 1ml mobile phase solution 50mM CH
3cOONH
2: CH
3oH=1:1 wash-out, collects eluent, centrifugal, gets supernatant;
(b) supernatant making through step (a) is carried out to LC/MS/MS analysis under the following conditions
Mass spectrum condition:
Compound parameter:
Inosine mothers and sons ion pair: 269.1/137.1, time:400msec
Lamivudine mothers and sons ion pair: 230.2/112.2, time:200msec
Duration：4min
Cycles：393
DP＝26V
CE＝15V
CXP＝4V
CEP＝4V
Q1resolution：Unit
Q2resolution：Unit，
Ion gun/gas parameter:
Ion?Source：Turbo?Spray
Curtain?Gas(CUR)：10psig
Collision?Gas(CAD)：Medium
Ion?Spray?Voltage(IS)：5000V
Temperature(TEM)：700℃
Ion?Source?Gas1(Gas1)：60psig
Ion?Source?Gas2(Gas2)：10psig
Interface?Heater(ihe)：ON，
Liquid phase chromatogram condition:
Chromatographic column: Waters XBridge
tMc18, specification: 3.5 μ m, 2.1mm * 100mm, Part No.:186003022, S/N:01453030814045
Mobile phase: 50mM CH
3cOONH
2: CH
3oH=1:1
Flow velocity: 0.2ml/min
Sample size: 10 μ l
Sample injection time: 4min;
(c) analysis result based on step (b), sets up typical curve and the lower limit of quantitation on the same day and calculates by the method for claim 1, obtain the Inosine Content in human plasma to be measured.
7. method as claimed in claim 6, is characterized in that, also comprises described mixed solution-70 ℃ frozen step in described step (a).
8. the method as described in claim 6 or 7, is characterized in that, the activation step of described activated HLB solid-phase extraction column comprises: use VARIAN
tMvAC ELUT SPS24 solid-phase extraction device, inserts fixed position by HLB solid-phase extraction column, successively uses 1ml methyl alcohol, 1ml water to carry out wash-out activation.
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