CN105300903A - Determining method for total content of phospholipids in hemoglobin oxygen-carrying medicine - Google Patents
Determining method for total content of phospholipids in hemoglobin oxygen-carrying medicine Download PDFInfo
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Abstract
The invention relates to a determining method for the total content of phospholipids in hemoglobin oxygen-carrying medicine, and belongs to the technical field of medicine. The determining method includes the steps that A, determining operation is performed; B, the total content of phospholipids in a sample is calculated. An external standard method is adopted for replacing an existing HPLC method to determine the total content of phospholipids in the hemoglobin oxygen-carrying medicine. Compared with the existing HPLC method, the detection flux is improved, the detection time of the sample is shortened, the detection cost is lowered, the number of operation steps is decreased, and therefore the total content of phospholipids in the hemoglobin oxygen-carrying medicine is easily, rapidly and flexibly determined.
Description
Technical field
The invention belongs to medical art, be specifically related to the assay method of total phospholipids content in a kind of hemoglobin oxygen carrier medicine.
Background technology
Phosphatide (Phospholipid) also claim phospholipid, Phospholipids, refers to the lipid containing phosphoric acid, belong to complex liped.Phosphatide forms biomembranous principal ingredient, is divided into glycerophosphatide and the large class of sphingomyelins two, is made up of respectively glycerine and sphingol.Phosphatide is amphipathic molecule, and one end is the head of hydrophilic nitrogenous or phosphorus, and the other end is the long hydrocarbyl chain of hydrophobic (oleophylic).Due to this reason, phospholipid molecule water-wet side is close to each other, and hydrophobic side is close to each other, and other molecules such as normal and protein, glycolipid, cholesterol form lipid bilayer, i.e. the structure of cell membrane jointly.At present; human red blood cell membrane phospholipid result of study is comparatively ripe at present; wherein content analysis result can list of references (with reference to VirtanenJA; ChengKH, SomerharjuP.Phospholipidcompositionofthemammalianredcell membranecanberationalizedbyasuperlatticemodel.ProcNatlAc adSciUSA.1998Apr28; 95 (9): 4964-9.).Human red blood cell membrane phospholipid, its Main Ingredients and Appearance is mainly: phosphatid ylcholine (Phosphatidylcholine), sphingomyelins (Sphingomyelin), phosphatidyl-ethanolamine (Phosphatidylethanolamine), phosphatidylserine (Phosphatidylserine), these four kinds of phosphatide account for human red blood cell membrane phospholipid 97.3%.
Take oxygen oxygen carrier as novel hemoglobin, it is quantitatively one of quality control key index that people (animal) blood sources hemoglobin takes oxygen medicine total phospholipids.Current research shows that erythrocyte membrane phospholipid has more complicated physiological function; if remaining phosphatide content overproof; its infusion too high levels; likely can cause bio-toxicity when infusion, research shows (with reference to FeolaM, SimoniJ; CanizaroPC; TranR, RaschbaumG, BehalFJ.Toxicityofpolymerizedhemoglobinsolutions.SurgGyn ecolObstet.1988Mar; 166 (3): 211-22.), using four kinds of ox polymeric hemoglobins and rabbit blood plasma as infusion liquid, to lose blood 1/3rd 5 groups of rabbits as change-blood animal pattern.Found that to there is phosphatidyl ethanolamine and POPS group, the infusion liquid of endotoxin group and super large polymerizable molecular amount group, the fatal rate providing 33% is caused to change-blood model, cause Hemodynamics unstable, breathe and kidney metabolism insufficient, provide liver metabolism enzyme level, decrease of platelet, Neuroleptic Leukocytopenia, disseminated intravascular coagulation and activate pathway of complement activation for subsequent use; Complement pathway in activation experiment animal blood is (with reference to FeolaM; SimoniJ; DobkeM, CanizaroPC.Complementactivationandthetoxicityofstroma-fr eehemoglobinsolutionsinprimates.CircShock.1988Aug; 25 (4): 275-90.); Cause vascular to shrink and activate blood coagulation path, thus causing the confusion of hematological system (with reference to FeolaM, SimoniJ, TranR, LoxCD, CanizaroPC.Toxicfactorsintheredbloodcellmembrane.JTrauma .1989Aug; 29 (8): 1065-75.).Therefore, total phospholipids detection method of content in a kind of quick, highly sensitive hemoglobin oxygen carrier medicine must be set up.
The method multipurpose efficient liquid phase chromatography (HPLC) of current bibliographical information measures single content of phospholipid (with reference to Rabinovich-GuilattL by evaporative light-scattering detector (ELSD); DubernetC; GaudinK, etal.Phospholipidhydrolysisinapharmaceuticalemulsionasse ssedbyphysicochemicalparametersandanewanalyticalmethod.E urJPharmBiopharm.2005Sep; 61 (1-2): 69-76.).Its shortcoming to detect the content of hemoglobin oxygen carrier medicine total phospholipids fast, must detect single content of phospholipid respectively by many experiments, finally adds and become total phospholipids content.Complicated and the detection sensitivity of such operation does not reach requirement, must carry out certain enrichment to detection sample.
Summary of the invention
In order to solve the problems referred to above that prior art exists, the invention provides the assay method of total phospholipids content in a kind of hemoglobin oxygen carrier medicine, to replace existing HPLC method, realize simply, fast, total phospholipids content in Measuring hemoglobin class oxygen carrier medicine delicately.
The technical solution adopted in the present invention is:
An assay method for total phospholipids content in hemoglobin oxygen carrier medicine, comprises the following steps:
A. accurate measuring test sample is in right amount in glass test tube, after the organic solvent volatilization completely in test sample, adds the pure perchloric acid of appropriate top grade and carries out clearing up reaction, change the total phospholipids in sample into Phos, wait clearing up completely, obtains digestion solution; Get appropriate described digestion solution and add appropriate purified water and described Phos nitrite ion reacts completely, obtaining reactant liquor, measuring described reactant liquor and the absorbance as blank coordinative solvent by ultraviolet spectrophotometer.
B. the calculating of total phospholipids content in sample: get potassium dihydrogen phosphate titer and be diluted to different concentration as external standard titer, and the absorbance of each external standard titer recorded with ultraviolet spectrophotometer makes external standard typical curve, calculate the content of inorganic phosphorus in reactant liquor described in 1ml with the absorbance of the described reactant liquor liquid recorded in steps A contrast external standard typical curve, calculate the mensuration content of total phospholipids in sample according to following formula I;
Getting appropriate phosphatidyl-ethanolamine titer joins in test sample as internal standard compound, repeats above-mentioned steps A, is obtained the recovery of this measuring method, calculate total phospholipids content in sample according to following formula II by recovery test;
Formula I: in sample total phospholipids mensuration content=1ml reactant liquor in content of inorganic phosphorus × 26.31;
Formula II: the mensuration content/recovery of total phospholipids in total phospholipids content=sample in sample.
By the measurement operation of steps A, by test sample solution through heating and decompose, after making all organophosphoruss in sample change Phos into completely, obtain digestion solution; Get appropriate digestion solution and add appropriate purified water and Phos nitrite ion, Phos to be transformed and Phos nitrite ion have reacted, being that Phos develops the color completely in ultraviolet spectrophotometer, test sample solution and the absorbance as blank coordinative solvent is measured again by ultraviolet spectrophotometer, the wavelength of ultraviolet spectrophotometer is 560-690nm, thus determine total phospholipids in sample be all transformed into Phos after absorbance.The pure perchloric acid of top grade in step B is the commercially available pure perchloric acid of top grade, and its mass concentration is 70% ~ 72%.
By step B, the external standard titer of different concentration is diluted to potassium dihydrogen phosphate titer, external standard typical curve is drawn out by the absorbance of each external standard titer measured, by the absorbance of test sample solution that records in step B and external standard standard curve control, calculate the content of the Phos in step B in 1ml reactant liquor, calculate the mensuration content of total phospholipids in sample according to formula I.
In order to avoid the error that measuring method produces, join using phosphatidyl-ethanolamine titer as internal standard compound in described test sample solution, obtained the recovery of this measuring method by recovery test, and by formula II, finally obtain total phospholipids content in sample.
Said determination method adopts the content of external standard method hemoglobin oxygen carrier medicine total phospholipids, the content of hemoglobin oxygen carrier medicine total phospholipids is obtained by one-time detection, and many experiments must be passed through when avoiding HPLC method to detect the content of hemoglobin oxygen carrier medicine total phospholipids, detect single content of phospholipid respectively, finally add and become total phospholipids content, therefore improve detection flux, shorten the sample detection time, reduce testing cost, there are good economic benefits.
And external standard method is more more accurate than the measurement result of HPLC method, sensitivity is higher, therefore assay method of the present invention can simply, fast, total phospholipids content in Measuring hemoglobin class oxygen carrier medicine delicately.
Further, the wavelength of described ultraviolet spectrophotometer is 560-690nm.
By to potassium dihydrogen phosphate standard solution full wavelength scanner, record Phos at 560-690nm wavelength place by absorption, therefore, the wavelength of ultraviolet spectrophotometer is 560-690nm, so that the absorbance of Phos in assaying reaction liquid.
Further, the wavelength of described ultraviolet ultraviolet spectrophotometer is 634nm.
The maximum absorption band of Phos is positioned at 634nm wavelength place, and therefore, the wavelength of ultraviolet spectrophotometer is preferably 560-690nm.
Further, the volatilization temperature of the organic solution in the described test sample in steps A is 60 ~ 105 DEG C, and carries out in fuming cupboard.
In order to enable the organic solution in test sample volatilize as early as possible, the volatilization temperature of organic solution is 60 ~ 105 DEG C, and carries out in fuming cupboard, and can prevent organic solution volatilization environmental pollution by fuming cupboard, improves the security of test.
Further, clear up reaction described in steps A to carry out under 160 DEG C of oil bath heating conditions.
The temperature clearing up reaction is preferably 160 DEG C, and oil bath can be adopted to heat, and is that invisible spectro reactant liquor reaches 160 DEG C.
Further, in hemoglobin oxygen carrier medicine, the detection sensitivity lower limit of total phospholipids content is 0.4933ppm.
By analyzing, the lowest detection limit of this assay method is 0.4933ppm, therefore has higher sensitivity.
Further, described assay method also comprises the preparation of described potassium dihydrogen phosphate titer in the preparation of described test sample in steps A and described Phos nitrite ion and step B and described phosphatidyl-ethanolamine titer; The preparation of described test sample comprises the preparation of extract, and the preparation of described Phos nitrite ion comprises the preparation of polysorbas20 solution and storage liquid, and the preparation of described storage liquid comprises the preparation of malachite green solution and the preparation of ammonium molybdate solution;
The preparation of described test sample: add extract in the sample to which, carry out centrifugal after vibration mixing at normal temperatures, after centrifugal, remove the transparent liquid level of the visible levels of haemoglobin, adopt siphonage to remove supernatant, obtain lower floor's total phospholipids extract as test sample, 4 DEG C for subsequent use;
The preparation of described extract: mixed with methyl alcohol by chloroform, is mixed with extract;
The preparation of described Phos nitrite ion: by storage liquid and polysorbas20 solution by volume 3:1 mix, be mixed with Phos nitrite ion; Described Phos nitrite ion is now with the current;
The preparation of described polysorbas20 solution: it is appropriate that precision takes polysorbas20, dissolves with ultrapure water, is mixed with the polysorbas20 solution that weight content is 1.5%;
The preparation of described storage liquid: by malachite green solution and ammonium molybdate solution by volume 3:1 filter after mixing, room temperature keeps in Dark Place 2 ~ 3 weeks, obtains storage liquid;
The preparation of described malachite green solution: it is appropriate that precision takes malachite green, dissolves with ultrapure water, is mixed with the malachite green solution that weight content is 0.4%;
The preparation of described ammonium molybdate solution: it is appropriate that precision takes ammonium molybdate, with the dissolving with hydrochloric acid of 5mol/L, is mixed with the ammonium molybdate solution that weight content is 4.2%;
The preparation of described potassium dihydrogen phosphate titer: it is appropriate that precision takes potassium dihydrogen phosphate standard items, dissolves with ultrapure water, is mixed with the potassium dihydrogen phosphate titer of 2 μ g/ml;
The preparation of described phosphatidyl-ethanolamine titer: it is appropriate that precision takes phosphatidyl-ethanolamine standard items, after dissolving, is mixed with the phosphatidyl-ethanolamine titer that concentration is 0.5mg/ml with described extract.
Completed the preparation of required each solution and test sample in mensuration by the preparation process of solution, be convenient to directly take in mensuration process, save experimental period.
Further, in the preparation of described extract, the volume ratio of described sample and described extract is 1:1 ~ 8.
Further, the volume ratio of described sample and described extract is 1:4 ~ 8, and described extract is precooling extract, and precooling temperature is-20 DEG C.
In extract, the volume ratio of chloroform and methyl alcohol is 1:1 ~ 8.When preparing test sample, after centrifugal, for the ease of removing haemoglobin, preferably, the volume ratio of chloroform and methyl alcohol is 1:4 ~ 8, and through cryogenic freezing process, precooling temperature is preferably-20 DEG C.
Further, the described potassium dihydrogen phosphate standard items in steps A are the potassium dihydrogen phosphate standard items through super-dry process.
The easy moisture absorption of potassium dihydrogen phosphate standard items, in order to make the phosphatidyl-ethanolamine titer of preparation accurate, ensure the accurate of measurement result, before the preparation of phosphatidyl-ethanolamine titer, need to carry out drying process to potassium dihydrogen phosphate standard items, to remove unnecessary moisture content, therefore, the accurate of measurement result is conducive to.
Beneficial effect of the present invention is:
In hemoglobin oxygen carrier medicine provided by the present invention, the assay method of total phospholipids content instead of total phospholipids content in existing HPLC method Measuring hemoglobin class oxygen carrier medicine by adopting external standard method, compared with existing HPLC method, which raises detection flux, shorten the sample detection time, reduce testing cost, decrease operation steps, thus achieve simply, fast, total phospholipids content in Measuring hemoglobin class oxygen carrier medicine delicately.
Accompanying drawing explanation
Fig. 1 is the full wavelength scanner figure of potassium dihydrogen phosphate standard solution;
Fig. 2 is the reaction power curve map that potassium dihydrogen phosphate standard solution records in glass cuvette.
Embodiment
Embodiment 1
The preparation of malachite green solution: precision takes malachite green 0.4mg, dissolves with Pall ultrapure water 100ml, is mixed with the malachite green solution that weight content is 0.4%.
The preparation of ammonium molybdate solution: precision takes ammonium molybdate 1.008g, with the dissolving with hydrochloric acid of 5mol/L, is mixed with the ammonium molybdate solution that weight content is 4.2%.
The preparation of polysorbas20 solution: precision takes 0.375g polysorbas20, dissolves with Pall ultrapure water 25ml, is mixed with the polysorbas20 solution that weight content is 1.5%.
The preparation of storage liquid: by described malachite green solution and described ammonium molybdate solution by volume 3:1 filter after mixing, room temperature keeps in Dark Place 2 ~ 3 weeks, obtains storage liquid.
The preparation of Phos nitrite ion: by described storage liquid and described polysorbas20 solution by volume 3:1 mix, be mixed with Phos nitrite ion; Described Phos nitrite ion is now with the current.
The preparation of potassium dihydrogen phosphate titer: precision weighing potassium dihydrogen phosphate 1.000g, in 10ml small beaker is contained, is then placed in baking oven little of constant weight in 110 DEG C of dryings 1.Fast precise weighs dried potassium dihydrogen phosphate 0.5492g, dissolves be settled to 250ml(0.5mg/ml with ultrapure water); Draw 1ml with pipettor again, add 249ml ultrapure water, be finally mixed with the potassium dihydrogen phosphate titer that 250ml concentration is 2 μ g/ml.
The preparation of phosphatidyl-ethanolamine titer: precision takes phosphatidyl-ethanolamine standard items 5.0mg, after dissolving, is mixed with the phosphatidyl-ethanolamine titer that concentration is 0.5mg/ml with extract described in 10ml;
The preparation of extract: by chloroform and methyl alcohol by volume 2:1 mix, be mixed with extract.
The preparation of test sample solution: get 100 μ l samples in EP pipe, will join mixing in EP pipe at the extract of-20 DEG C of precoolings, in EP pipe, the volume ratio of added extract and sample is respectively 8:1.Then, after vortex shakes 30 seconds, EP pipe is at room temperature put into tabletop refrigerated centrifuge centrifugal 5 minutes, and the rotating speed of hydro-extractor is 5000rpm.Solution suction pipette head after centrifugal is removed haemoglobin, and the transparent liquid level of visible levels, then removes supernatant with siphonage, and residue lower floor total phospholipids extract, prepares test sample solution, and save backup at 4 DEG C.
Embodiment 2
Sample and extract volume ratio Selection experiment: under common room temperature, when sample and extract volume ratio 1:4 and 1:8, easily cause the sex change of hemoglobin solutions sample rapid protein, thus may affect extraction rate was acquired.Therefore, the extract prepared is put in advance profound hypothermia refrigerator pre-freeze 30min.
When sample and extract volume ratio are 1:1,1:2,1:4,1:8, volume ratio is in the experiment of 1:1 and 1:2, along with temperature return becomes room temperature, and hemoglobin solutions pinkiness, haemoglobin is not acutely polymerized sex change; And volume ratio is in the experiment of 1:4 and 1:8, along with temperature return becomes room temperature, hemoglobin solutions is kermesinus, haemoglobin polymerization sex change.Carry out centrifugally operated in room temperature, centrifuge speed is 5000rpm, and centrifugation time is 5min.After centrifugal, volume ratio is in the experiment of 1:1 and 1:2, and haemoglobin is centrifugal not easily to be chosen, and tube wall has minimal residue; And volume ratio is in the experiment of 1:4 and 1:8, haemoglobin is because of centrifugal action, and haemoglobin is compressed into round pie, easily chooses, tube wall noresidue, therefore, preferably, the volume ratio of sample and extract is 1:4 ~ 8, and this extract is the extract through precooling, and precooling temperature is preferably-20 DEG C.
Embodiment 3
Potassium dihydrogen phosphate standard solution full wavelength scanner and reaction power curve: in quartz colorimetric utensil, measure 300 μ l potassium dihydrogen phosphate standard solution, then the 100 pure perchloric acid of μ l top grade and Phos nitrite ion 2ml is added, with Ultrospec6300pro ultraviolet-visible pectrophotometer from 200-900nm full wavelength scanner, scanning wavelength is spaced apart 1nm, and sweep velocity is 2649nm/min; From starting to add Phos nitrite ion, do a full wavelength scanner at interval of 5min, writing time is 0min, 5min, 10min, 15min, 20min later.Measurement result as shown in Figure 1, reaction assay wavelength all may be used for measuring at 560-690nm wavelength, and maximum absorption band corresponds to 634nm wavelength, therefore, when using Ultrospec6300pro ultraviolet-visible pectrophotometer to detect the absorbance of test sample solution and potassium dihydrogen phosphate standard solution, optimal wavelength is 634nm.
Carry out reaction power curve model mensuration with Ultrospec6300pro ultraviolet-visible pectrophotometer, sweep spacing is 2min/ time, sweep time length 1 hour.Measurement result as shown in Figure 2, after visible 20 minutes, two curves all tend to be steady, illustrate that Phos and Phos nitrite ion react completely, therefore use the minute of Ultrospec6300pro ultraviolet-visible pectrophotometer to select within 20 minutes, to carry out adding Phos nitrite ion later.
Embodiment 4
The assay method of total phospholipids content in test sample solution: in Example 1, the half volume of each test sample solution of preparation is placed in glass test tube, putting into fuming cupboard at 60 DEG C makes solution organic solvent volatilize completely, then the pure perchloric acid of 1ml top grade is added after taking out test tube cooling, and clear up in oil bath pan in 160 DEG C, make the organophosphorus composition in the total phospholipids in sample change Phos into.Wait clearing up completely, obtain digestion solution.
Add purified water in vitro to 0.3ml, add above-mentioned digestion solution 0.085ml-0.095ml and Phos nitrite ion 2ml again, obtain reactant liquor, after 20 minutes, determined the absorbance (OD sample) of test sample solution by Ultrospec6300pro ultraviolet-visible pectrophotometer.
Get 0.3ml pure water in test tube, and add the pure perchloric acid of 0.1ml top grade and Phos nitrite ion 2ml as blank, determined the absorbance (OD is blank) of test sample solution by Ultrospec6300pro ultraviolet-visible pectrophotometer.
Embodiment 5
The calculating of total phospholipids content in sample
External standard Specification Curve of Increasing: by the potassium dihydrogen phosphate standard solution of 2 μ g/ml of preparation in embodiment 1, proportional diluted becomes 6 parts, respectively gets 300 μ l, and adds the pure perchloric acid of 0.1ml top grade and Phos nitrite ion 2ml.The amount of the Phos in the potassium dihydrogen phosphate standard solution after dilution is respectively 0.6 μ g, 0.3 μ g, 0.15 μ g, 0.075 μ g, 0.0375 μ g, 0.01875 μ g, and measures the absorbance (OD titer) of the potassium dihydrogen phosphate standard solution after every part of dilution accordingly by Ultrospec6300pro ultraviolet-visible pectrophotometer.Absorbance according to the potassium dihydrogen phosphate standard solution after the absorbance of blank and every part of dilution draws external standard typical curve.
External standard typical curve be plotted as existing method, blank according to △ OD=OD titer-OD; △ OD=OD sample-OD is blank, makes scatter diagram, obtains external standard typical curve.Meanwhile, according to equation of linear regression: y=a*ln (x)+b, R2=C, wherein a, b may be different constants according to each test difference, and when C value is greater than 0.980, description standard curve can use.A=1.0215 is recorded in the present embodiment; B=0.0421; R
2=0.9953 > 0.980, and close to 1, therefore, the potassium dihydrogen phosphate titer absorbance in choice experiment concentration range and concentration are good linear relationship, can adopt external standard standard curve control and the mensuration content of total phospholipids in calculation sample.
By the suction pipe degree of test sample solution that records in embodiment 4 and external standard standard curve control, and calculate the content of inorganic phosphorus in 1ml reactant liquor, calculate the mensuration content of total phospholipids in sample according to following formula I; Get appropriate described phosphatidyl-ethanolamine titer to join in described test sample solution as internal standard compound, obtain the recovery of this measuring method, calculate total phospholipids content in sample according to following formula II;
Formula I: in sample total phospholipids mensuration content=1ml reactant liquor in content of inorganic phosphorus × 26.31;
Formula II: the mensuration content/recovery of total phospholipids in total phospholipids content=sample in sample.
Embodiment 6
The recovery and precision: calculating the assay method recovery is 86.86 ± 3.51%(n=3), when the recovery is greater than 80%, illustrate that this assay method is feasible.In addition, calculate total phospholipids in technological process and remove situation in each step.Result is as shown in table 1 below:
Embodiment 7
Detection limit: the estimated value being obtained detection limit by calculating or extrapolation method, can to a series of close to or equal detection limit sample analysis one by one to prove this estimated value, potassium dihydrogen phosphate standard items constant gradient is diluted to respectively 0.02 μ g/ml, 0.04 μ g/ml, 0.06 μ g/ml, 0.08 μ g/ml, 0.10 μ g/ml, repeatedly measure the absorbance of the potassium dihydrogen phosphate standard items of blank absorbance and 5 gradient concentrations, measurement result such as following table 2(" Mean " refers to average, " SD " refers to standard deviation, and " RSD " refers to relative standard deviation) shown in:
Known according to table 2,0.02 μ g/ml concentration is against regulation; 0.04 μ g/ml, 0.06 μ g/ml, 0.08 μ g/ml, 0.10 μ g/ml concentration conform with the regulations.Therefore, learn that the lower limit of the detection limit of external standard typical curve is 0.0625 μ g/ml, in its sample, the lower limit of the inspection limit of total phospholipids content is 0.4933ppm.
The present invention is not limited to above-mentioned preferred forms; anyone can draw other various forms of products under enlightenment of the present invention; no matter but any change is done in its shape or structure; every have identical with the application or akin technical scheme, all drops within protection scope of the present invention.
Claims (10)
1. the assay method of total phospholipids content in hemoglobin oxygen carrier medicine, is characterized in that, comprise the following steps:
A. accurate measuring test sample is in right amount in glass test tube, after the organic solvent volatilization completely in described test sample, add the pure perchloric acid of appropriate top grade and carry out clearing up reaction, change the total phospholipids in described test sample into Phos, wait clearing up completely, obtain digestion solution; Get appropriate described digestion solution and add appropriate purified water and Phos nitrite ion reacts completely, obtaining reactant liquor, measuring described reactant liquor and the absorbance as blank coordinative solvent by ultraviolet spectrophotometer;
B. the calculating of total phospholipids content in sample: get potassium dihydrogen phosphate titer and be diluted to different concentration as external standard titer, and the absorbance of each external standard titer recorded with ultraviolet spectrophotometer makes external standard typical curve, calculate the content of inorganic phosphorus in reactant liquor described in 1ml with the absorbance of the described reactant liquor recorded in steps A contrast external standard typical curve, calculate the mensuration content of total phospholipids in sample according to following formula I;
Getting appropriate phosphatidyl-ethanolamine titer joins in test sample as internal standard compound, repeats above-mentioned steps A, is obtained the recovery of this measuring method, calculate total phospholipids content in sample according to following formula II by recovery test;
Formula I: in sample total phospholipids mensuration content=1ml reactant liquor in content of inorganic phosphorus × 26.31;
Formula II: the mensuration content/recovery of total phospholipids in total phospholipids content=sample in sample.
2. assay method according to claim 1, is characterized in that, the wavelength of described ultraviolet spectrophotometer is 560-690nm.
3. assay method according to claim 1, is characterized in that, the wavelength of described ultraviolet ultraviolet spectrophotometer is 634nm.
4. assay method according to claim 1, is characterized in that, the volatilization temperature of the organic solution in the described test sample in steps A is 60 ~ 105 DEG C, and carries out in fuming cupboard.
5. assay method according to claim 1, is characterized in that, clears up reaction and carry out under 160 DEG C of oil bath heating conditions described in steps A.
6. assay method according to claim 1 and 2, is characterized in that, in hemoglobin oxygen carrier medicine, the detection sensitivity lower limit of total phospholipids content is 0.4933ppm.
7. assay method according to claim 1, is characterized in that, also comprises the preparation of described potassium dihydrogen phosphate titer in the preparation of described test sample in steps A and described Phos nitrite ion and step B and described phosphatidyl-ethanolamine titer; The preparation of described test sample comprises the preparation of extract, and the preparation of described Phos nitrite ion comprises the preparation of polysorbas20 solution and storage liquid, and the preparation of described storage liquid comprises the preparation of malachite green solution and the preparation of ammonium molybdate solution;
The preparation of described test sample: add extract in the sample to which, carry out centrifugal after vibration mixing at normal temperatures, after centrifugal, remove the transparent liquid level of the visible levels of haemoglobin, adopt siphonage to remove supernatant, obtain lower floor's total phospholipids extract as test sample, 4 DEG C for subsequent use;
The preparation of described extract: mixed with methyl alcohol by chloroform, is mixed with extract;
The preparation of described Phos nitrite ion: by storage liquid and polysorbas20 solution by volume 3:1 mix, be mixed with Phos nitrite ion; Described Phos nitrite ion is now with the current;
The preparation of described polysorbas20 solution: it is appropriate that precision takes polysorbas20, dissolves with ultrapure water, is mixed with the polysorbas20 solution that weight content is 1.5%;
The preparation of described storage liquid: by malachite green solution and ammonium molybdate solution by volume 3:1 filter after mixing, room temperature keeps in Dark Place 2 ~ 3 weeks, obtains storage liquid;
The preparation of described malachite green solution: it is appropriate that precision takes malachite green, dissolves with ultrapure water, is mixed with the malachite green solution that weight content is 0.4%;
The preparation of described ammonium molybdate solution: it is appropriate that precision takes ammonium molybdate, with the dissolving with hydrochloric acid of 5mol/L, is mixed with the ammonium molybdate solution that weight content is 4.2%;
The preparation of described potassium dihydrogen phosphate titer: it is appropriate that precision takes potassium dihydrogen phosphate standard items, dissolves with ultrapure water, is mixed with the potassium dihydrogen phosphate titer of 2 μ g/ml;
The preparation of described phosphatidyl-ethanolamine titer: it is appropriate that precision takes phosphatidyl-ethanolamine standard items, after dissolving, is mixed with the phosphatidyl-ethanolamine titer that concentration is 0.5mg/ml with described extract.
8. assay method according to claim 7, is characterized in that, in the preparation of described extract, the volume ratio of described sample and described extract is 1:1 ~ 8.
9. assay method according to claim 8, is characterized in that, the volume ratio of described sample and described extract is 1:4 ~ 8, and described extract is precooling extract, and precooling temperature is-20 DEG C.
10. assay method according to claim 7, is characterized in that, the described potassium dihydrogen phosphate standard items in steps A are the potassium dihydrogen phosphate standard items through super-dry process.
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