EP2047281A2 - Method for the determination of phospholipids in amniotic fluid samples - Google Patents
Method for the determination of phospholipids in amniotic fluid samplesInfo
- Publication number
- EP2047281A2 EP2047281A2 EP07825973A EP07825973A EP2047281A2 EP 2047281 A2 EP2047281 A2 EP 2047281A2 EP 07825973 A EP07825973 A EP 07825973A EP 07825973 A EP07825973 A EP 07825973A EP 2047281 A2 EP2047281 A2 EP 2047281A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- phospholipids
- amniotic fluid
- samples
- determination
- accordance
- 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.)
- Withdrawn
Links
- 238000000034 method Methods 0.000 title claims abstract description 41
- 150000003904 phospholipids Chemical class 0.000 title claims abstract description 33
- 210000004381 amniotic fluid Anatomy 0.000 title claims abstract description 29
- 210000001006 meconium Anatomy 0.000 claims abstract description 7
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 38
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 36
- 239000002904 solvent Substances 0.000 claims description 14
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 11
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 8
- 238000000605 extraction Methods 0.000 claims description 8
- 238000000926 separation method Methods 0.000 claims description 8
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 claims description 7
- 229910052740 iodine Inorganic materials 0.000 claims description 7
- 239000011630 iodine Substances 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 6
- 229910052757 nitrogen Inorganic materials 0.000 claims description 6
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 claims description 6
- 239000003480 eluent Substances 0.000 claims description 4
- 239000003960 organic solvent Substances 0.000 claims description 4
- 229920006395 saturated elastomer Polymers 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 3
- 210000004027 cell Anatomy 0.000 claims description 2
- 210000003743 erythrocyte Anatomy 0.000 claims description 2
- 239000011261 inert gas Substances 0.000 claims description 2
- 230000003647 oxidation Effects 0.000 claims description 2
- 238000007254 oxidation reaction Methods 0.000 claims description 2
- 239000006228 supernatant Substances 0.000 claims description 2
- 238000013019 agitation Methods 0.000 claims 2
- 238000001035 drying Methods 0.000 claims 2
- 238000010828 elution Methods 0.000 claims 2
- 238000001704 evaporation Methods 0.000 claims 2
- 230000008020 evaporation Effects 0.000 claims 2
- 238000001816 cooling Methods 0.000 claims 1
- 238000012423 maintenance Methods 0.000 claims 1
- 239000002798 polar solvent Substances 0.000 claims 1
- 230000001376 precipitating effect Effects 0.000 claims 1
- 238000012800 visualization Methods 0.000 claims 1
- 210000004369 blood Anatomy 0.000 abstract description 5
- 239000008280 blood Substances 0.000 abstract description 5
- 238000011109 contamination Methods 0.000 abstract description 5
- 238000004809 thin layer chromatography Methods 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 230000008030 elimination Effects 0.000 abstract 1
- 238000003379 elimination reaction Methods 0.000 abstract 1
- ATBOMIWRCZXYSZ-XZBBILGWSA-N [1-[2,3-dihydroxypropoxy(hydroxy)phosphoryl]oxy-3-hexadecanoyloxypropan-2-yl] (9e,12e)-octadeca-9,12-dienoate Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(COP(O)(=O)OCC(O)CO)OC(=O)CCCCCCC\C=C\C\C=C\CCCCC ATBOMIWRCZXYSZ-XZBBILGWSA-N 0.000 description 14
- AWUCVROLDVIAJX-UHFFFAOYSA-N alpha-glycerophosphate Natural products OCC(O)COP(O)(O)=O AWUCVROLDVIAJX-UHFFFAOYSA-N 0.000 description 14
- 238000004458 analytical method Methods 0.000 description 7
- 210000004072 lung Anatomy 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000011156 evaluation Methods 0.000 description 4
- -1 sphingomyelin Chemical compound 0.000 description 4
- 208000032571 Infant acute respiratory distress syndrome Diseases 0.000 description 3
- 206010028974 Neonatal respiratory distress syndrome Diseases 0.000 description 3
- 201000002652 newborn respiratory distress syndrome Diseases 0.000 description 3
- 150000003905 phosphatidylinositols Chemical class 0.000 description 3
- 239000004094 surface-active agent Substances 0.000 description 3
- 208000000203 Hyaline Membrane Disease Diseases 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 239000000356 contaminant Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 206010038687 Respiratory distress Diseases 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 238000002669 amniocentesis Methods 0.000 description 1
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 1
- 229960001231 choline Drugs 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000001934 delay Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000012764 semi-quantitative analysis Methods 0.000 description 1
- 235000011149 sulphuric acid Nutrition 0.000 description 1
- 239000001117 sulphuric acid Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2405/00—Assays, e.g. immunoassays or enzyme assays, involving lipids
- G01N2405/04—Phospholipids, i.e. phosphoglycerides
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/36—Gynecology or obstetrics
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/36—Gynecology or obstetrics
- G01N2800/368—Pregnancy complicated by disease or abnormalities of pregnancy, e.g. preeclampsia, preterm labour
Definitions
- the present invention refers to a method of determination of phospholipids in amniotic fluid samples by high performance thin layer chromatographic techniques (HPTLC). It has application in the pharmaceutical industry (development of kits of diagnosis) in the respiratory deficiency in hyaline membrane disease of newborns. Summary of the Invention
- the aim of the present invention supplies a method for the determination of phospholipids in amniotic fluid samples through the use of techniques of high performance thin layer chromatography (HPTLC, applied to samples of amniotic liquid, allowing the determination of the foetal lung maturity, in this way, presenting as advantage its great easiness of execution and economy).
- HPTLC high performance thin layer chromatography
- Neonatal respiratory distress syndrome by hyaline membrane disease is the main cause of newborn death, the reason why foetal lung maturity is of great importance.
- This syndrome originated for deficient production of surfactant production by the lung in the neonatal period may be evaluated through analysis in amniotic fluid.
- Lipids present in amniotic fluid have been used as indicators of foetal lung maturity, namely the ratio phosphatidylcholine/sphingomyelin ratio (L/S) higher than 2, but the presence of phosphatidylglycerol, however, seems to constitute a more appropriate indicator of lung maturity.
- this method allows eliminate the false results (positive or negative), it uses common reagents and it has the advantage of the result still not be affected by the contamination of the sample with blood or meconium.
- the aim of the present invention supplies a method for the determination of phospholipids in samples of amniotic liquid. For this effect, a sample of the amniotic fluid is collected for analysis.
- the chloroform phase is evaporated to dryness, through the application of an inert gas stream (nitrogen), at room temperature to prevent the oxidation of phospholipids contained in the sample and, after that, is treated with cold acetone (-4 0 C) to precipitate tensioactive phospholipids.
- an inert gas stream nitrogen
- cold acetone 4 0 C
- the plate is developed in an appropriate solvent (e.g.: a mixture of chloroform/methanol/ammonium/water) (2: 1 ; 1 :3).
- an appropriate solvent e.g.: a mixture of chloroform/methanol/ammonium/water (2: 1 ; 1 :3).
- Figure 1 presents an image of a HPTLC plate after being revealed with iodine during
- samples of amniotic fluid obtained by transabdominal amniocentesis are:
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Medicinal Chemistry (AREA)
- Pathology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Endocrinology (AREA)
- Food Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Physics & Mathematics (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention refers to a fast method for the determination of phospholipids in amniotic fluid samples by HPTLC (high performance thin layer chromatography). This in- novative method presents high sensitivity, reproducibility and resolution. It has also the advantage of being of easy laboratorial interpretation, allowing the elimination of false results due to the contamination of the sample with blood or meconium.
Description
Description METHOD FOR THE DETERMINATION OF PHOSPHOLIPIDS
IN AMNIOTIC FLUID SAMPLES
Technical Domain of the Invention
[1] The present invention refers to a method of determination of phospholipids in amniotic fluid samples by high performance thin layer chromatographic techniques (HPTLC). It has application in the pharmaceutical industry (development of kits of diagnosis) in the respiratory deficiency in hyaline membrane disease of newborns. Summary of the Invention
[2] The aim of the present invention supplies a method for the determination of phospholipids in amniotic fluid samples through the use of techniques of high performance thin layer chromatography (HPTLC, applied to samples of amniotic liquid, allowing the determination of the foetal lung maturity, in this way, presenting as advantage its great easiness of execution and economy).
[3] Extraction of phospholipids contained in the sample is performed by share of organic solvent (chloroform, methanol) in adequate proportions to the extraction of ten- sioactive compounds to analyse. Its separation is performed by a mixture of solvent (chloroform, methanol, ammonium and water) with the suitable polarity to a good resolution of the constituents of the sample. Plaque revelation to the interpretation of the results is performed with a universal developer (iodine vapour). This method, whose execution takes about 90 minutes, besides allowing to separate and to identify phospholipids, still has the capacity to eliminate the occurrence of false negatives and false positives. Background of the Invention
[4] Neonatal respiratory distress syndrome by hyaline membrane disease is the main cause of newborn death, the reason why foetal lung maturity is of great importance. This syndrome, originated for deficient production of surfactant production by the lung in the neonatal period may be evaluated through analysis in amniotic fluid. Lipids present in amniotic fluid have been used as indicators of foetal lung maturity, namely the ratio phosphatidylcholine/sphingomyelin ratio (L/S) higher than 2, but the presence of phosphatidylglycerol, however, seems to constitute a more appropriate indicator of lung maturity.
[5] Nowadays, the determination of foetal lung maturity is performed by the identification of p hosphatidylglycerol in amniotic fluid.
[6] The previous methods were based on: a) the evaluation of the surfactant/albumin ratio- described as a fast and reliable
method in diabetics, it has as main inconvenience the necessity of acquisition of specialized equipment (Russell JC: AN1988-015651); b) the evaluation of L/S ratio with detection of phosphatidylglycerol - one is about a difficult technique, taking a long time, that moreover uses reagents not very usual in the analytical laboratory (Rosenthal MA:AN- 1987-207622); c) the evaluation of phosphatidylglycerol (amnioStat-FLM)- a fast technique, reliable in diabetics but with a high incidence of false negative results; d) the evaluation of tensioactivity (stability foam test) - one is about a fast method, but that does not eliminate the interference due to possible contamination of the sample with blood and/or meconium (Clements JA, Platzer AG, Tierney DF, Hobel CJ, Creasy RK, Margolis AJ, et al. Assessment of the risk of respiratory distress by a rapid test for surfactant in amniotic fluid. NEMJ 1972). This method of determination of foetal lung maturity through the identification of phosphatidylglycerol in amniotic fluid is faster and uses reagents of more common use than the technique related in b).
[7] In comparison with the 'amnioStat-FLM' technique, where it is referred an elevated number of false negatives, in the present method this interference is eliminated.
[8] Concerning tensioactivity, determined by the method related in d), this determination does not allow eliminate the interferences due to the contamination with blood or meconium, in contrast of that it happens in the proposed method, where none of these contaminants interferes in the analysis.
[9] In summary, this method allows eliminate the false results (positive or negative), it uses common reagents and it has the advantage of the result still not be affected by the contamination of the sample with blood or meconium.
[10] The technique previously developed in our laboratory takes more time (takes about
120 minutes), needs the acquisition of appropriate material (colunes Extrelut 3), what takes more time and the chromatographic development, although of being performed in the same kind of chromatographic plaque, uses a different mixture of eluents, expressing less sensitivity of the technique (100 times). This method allows only the detection of concentrations of standard around 1 microgram/microlitre, whereas the new one detects concentrations of standard around 0.01 microgram/microlitre. [AEl] The revelation process, aided by sulphuric acid, followed by heating at 125°C, increases in 20 minutes the time of the analysis.
[11] In the figure it can be observed the results obtained for a serie of standards in the cited conditions: sphingomyelin (S); phosphatidy choline (PC); phosphatidylglycerol (PG) analyzed individually or in mixture (x) and still of two samples of amniotic fluid. It is possible to identify the presence of phosphatidylglycerol in the amniotic fluid. Still in this plate in a semiquantitative analysis of the different spots was performed and in the samples where was evident the presence of phosphatidylglycerol, the phos-
phatidycholine/sphingomyelin was higher than 2. General Description of the Invention
[12] The aim of the present invention supplies a method for the determination of phospholipids in samples of amniotic liquid. For this effect, a sample of the amniotic fluid is collected for analysis.
[13] After that, it is proceeded the extraction from phospholipids contained in the sample with resource to the share of solvent organic (chloroform and methanol) in adequate ratios to the extraction of the tensioactives to be analyzed. Its separation is efectuated by mixture of solvent (chloroform, methanol, ammonium and water) with the suitable polarity to a good resolution of the constituents of the sample.
[14] The samples treated to this form are then applied to the high resolution plates of high performed thin layer chromatography (HPTLC), and its revelation performed, in a reversible way, with a universal developer (iodine vapour).
[15] This method, whose execution delays about 90 minutes, besides allowing to separate and to identify phospholipids, has still the capacity to eliminate the occurrence of false negatives and false positives results. Detailed Description of the Invention
[16] The obtained samples of amniotic fluid, by transabdominal amniocentese, are cent- rifugated at low speed (180 g) to eliminate the presence of cells of descamation, erythrocytes and/or meconium, in order to prevent possible contaminations of the sample for the occurrence of false results.
[17] After that, for the purpose of separation of the phospholipids, they are submitted to a process of extraction with solvent organic with different polarities, as for example a mixture of chloroform/methanol (2: 1) adding in a first phase the solvent with the highest polarity, after which the solvent with the lowest polarity is successively added.
[18] The chloroform phase is evaporated to dryness, through the application of an inert gas stream (nitrogen), at room temperature to prevent the oxidation of phospholipids contained in the sample and, after that, is treated with cold acetone (-40C) to precipitate tensioactive phospholipids.
[19] The obtained precipitate, dissolved in the lowest polarity solvent, is then applied on the HPTLC plate of chromatography simultaneously with the standards of phosphatidylcholine, sphingomyelin, phosphatidylglycerol and phosphatidylinositol.
[20] After that the plate is developed in an appropriate solvent (e.g.: a mixture of chloroform/methanol/ammonium/water) (2: 1 ; 1 :3).
[21] The revelation is carried in chamber saturated with iodine vapour, and plate reading performed by comparison with standards: phosphatidylcholine, sphingomyelin, phosphatidylglycerol and phosphatidylinositol.
[22] Face to the methods contained in the state of the technique this invention presents the following advantages:
- to allow the reduction of the analysis time (about 90 minutes);
- to present high precision and reproducibility;
- to eliminate false positives and false negatives, over all due to not interference of meconium or blood (possible contaminants of the sample);
- to be a technique of low cost; therefore only uses common reagents in all laboratory analysis.
Description of the Drawings [23] Figure 1 presents an image of a HPTLC plate after being revealed with iodine during
5 min. S ( sphingomyelin ); PC ( phosphatidylcholine ); PG ( phosphatidylglycerol ); X
(mixture of standards: S+PC+PG); LA (amniotic fluid).
Examples of Application [24] I - In a preferred accomplishment of the present invention, samples of amniotic fluid obtained by transabdominal amniocentesis are:
1. centrifugated ( 14O g during 10 minutes);
2. 1 ml of the supernatant is transferred to a polyethylene centrifuge tube, and 1 ml of methanol added;
3. vortexed during 15 seconds;
4. 1 ml of chloroform is added, vortexed during 15 seconds;
5. centrifugated ( 1000 g during 5 minutes);
6. the inferior phase obtained after centrifugation is transferred to another tube (tube 2);
7. 1 ml of chloroform is added, vortexed during 15 seconds;
8. centrifugated ( 1000 g during 5 minutes);
9. the inferior phase obtained after centrifugation is transferred to another tube;
10. 1 ml of chloroform is added, vortexed during 15 seconds;
11. centrifugated ( 1000 g during 5 minutes);
12. to transfer the inferior phase, obtained after centrifugation to tube 2;
13. to evaporate chloroform phase to dryness in water bath with nitrogen;
14. to cool the tube of operation 13 in ice during 10 minutes;
15. to add XX drops of acetone (cooled about 4°C);
16. keeping at -20° during 20 minutes;
17. to decant and to dry the residue in nitrogen;
18. to dissolve the residue in 1 ml chloroform, apply in the HPTLC plate with a microapplicator at 0.5 cm of edge;
19. apply simultaneously standards of phosphatidylcholine, sphingomyelin, phos-
phatidylglycerol and phosphatidylinositol;
20. develop the plate in horizontal chamber, previously saturated with developing (chloroform/methanol/ammonium/water) (10:5:0.3:0.5) in 6.5 cm distance;
21. evaporate the developing eluent with the aid of a hair drier;
22. reveal the plate in an iodine chamber;
23. interpretation of the results by comparison with the related standards.
Claims
[1] Method for the determination of phospholipids in samples of amniotic liquid characterized for understanding the following steps:
- centrifugation of amniotic fluid sample;
- separation of the phospholipids from the supernatant with resource to sequential extraction with organic solvents, with agitation;
- addition of other solvent organic of lower polarity followed by agitation;
- centrifugation;
- to the inferior phase add the solvent of lowest polarity and agitate;
- 3 repetitions of the previous step;
- drying of the resultant samples of the extraction with the solvent of lowest polarity, by evaporation in nitrogen stream;
- cooling the sample in ice during about 10 minutes;
- addition of cooled acetone to the sample;
- maintenance of the sample at -200C during about 20 minutes;
- to precipitate the sample, and to dry the residue in nitrogen stream;
- to dissolve the residue in the less polar solvent (chloroform);
- to apply the sample in a high performance thin layer chromatographic plate (HPTLC);
- to apply in the plate, of analogous form, the standards phosphatidylcholine, sphingomyelin, phosphatidyglycerol, phosphatidyinositol;
- elution of the plate in appropriate eluent (e.g. chloroform/ methanol/ammonium/water) ;
- to allow the evaporation of the developing solvent;
- revelation in chamber saturated with iodine.
[2] The method for the determination of phospholipids in samples of amniotic fluid, in accordance with claim 1, characterized by the centrifugation of samples of amniotic fluid at low speed (180 g) to eliminate the presence of cells of descamation, erythrocytes and/or meconium.
[3] The method for the determination of phospholipids in samples of amniotic fluid in accordance with claim 1, characterized by the use, in the separation of the phospholipids, of an organic solvent or a mixture of organic solvents.
[4] The method for the determination of phospholipids in samples of amniotic fluid in accordance with the previous claim, is characterized by the use in the separation of the phospholipids a mixture of chloroform/methanol (2:1).
[5] The method for the determination of phospholipids in samples of amniotic fluid in accordance with the previous claim, characterized by the use in the separation of the phospholipids of a mixture of chloroform/methanol (2: 1) adding in a first phase the solvent of higher polarity and after that of lowest polarity.
[6] The method for the determination of phospholipids in samples of amniotic fluid, in accordance with claim 1, characterized by drying the phospholipids with resource to an inert gas stream , such as nitrogen, at ambient temperature in order to prevent the oxidation of the phospholipids.
[7] The method for the determination of phospholipids in samples of amniotic fluid, in accordance with claim 1, characterized for precipitating ten- sioactive phospholipids with resource to addition of cooled acetone at about 4°C.
[8] The method for the determination of phospholipids in samples of amniotic fluid, in accordance with claim 1, characterized by dissolving the obtained precipitated with resource to the solvent addition of lowest polarity, such as chloroform.
[9] The method for the determination of phospholipids in samples of amniotic fluid, in accordance with claim 1, characterized by the application of the standards in HPTLC plate, mentioned in claim 1, and the residue obtained after extraction of the sample in the already described conditions in the previous claims.
[10] The method for the determination of phospholipids in samples of amniotic fluid, in accordance with claim 1, characterized for carrying the elution of the plate in a HPTLC chamber, using an appropriate eluent, as for instance a mixture of chloroform/methanol/ammonium/water (2: 1 ; 1 :3) for the separation of different phospholipids present in amniotic fluid.
[11] The method for the determination of phospholipids in samples of amniotic fluid, in accordance with claim 1, characterized for carrying the revelation in chamber saturated with iodine vapour in order to allow the visualization of phospholipids.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PT10354706A PT103547B (en) | 2006-07-31 | 2006-07-31 | METHOD FOR THE DETERMINATION OF PHOSPHOLIDES IN A SAMPLE OF AMMYTIC LIQUID |
| PCT/IB2007/053011 WO2008015634A2 (en) | 2006-07-31 | 2007-07-31 | Method for the determination of phospholipids in amniotic fluid samples |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| EP2047281A2 true EP2047281A2 (en) | 2009-04-15 |
Family
ID=38997550
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP07825973A Withdrawn EP2047281A2 (en) | 2006-07-31 | 2007-07-31 | Method for the determination of phospholipids in amniotic fluid samples |
Country Status (3)
| Country | Link |
|---|---|
| EP (1) | EP2047281A2 (en) |
| PT (1) | PT103547B (en) |
| WO (1) | WO2008015634A2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101379289B1 (en) * | 2013-08-02 | 2014-03-28 | 라인호(주) | Method and equipment transfering ship movement efficiency is excellent |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0010917A1 (en) * | 1978-10-23 | 1980-05-14 | The University Of Sydney | Method of assessing foetal lung maturity |
| US5443989A (en) * | 1993-10-25 | 1995-08-22 | Beth Israel Hospital Association | Method for assessing fetal lung maturity using amniotic fluid samples |
-
2006
- 2006-07-31 PT PT10354706A patent/PT103547B/en active IP Right Grant
-
2007
- 2007-07-31 WO PCT/IB2007/053011 patent/WO2008015634A2/en not_active Ceased
- 2007-07-31 EP EP07825973A patent/EP2047281A2/en not_active Withdrawn
Non-Patent Citations (1)
| Title |
|---|
| See references of WO2008015634A3 * |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2008015634A2 (en) | 2008-02-07 |
| WO2008015634A3 (en) | 2008-07-03 |
| PT103547A (en) | 2008-01-31 |
| PT103547B (en) | 2011-05-09 |
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