Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/50—Conditioning of the sorbent material or stationary liquid
  • G01N30/52—Physical parameters
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
  • G01N30/94—Development
• • 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
  • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography

Definitions

• the chromatogram is preferably developed using a multicomponent solvent mixture containing water, a p 1969 Germany 19 45 471-8 solvent substantially immiscible in water, a solvent miscible with water and the substantially water-immiscible solvent, and UeS- an acid e g chloroform/n.butanol/ethanol/glacia] acetic [51] Int. Cl ..B01d 15/08 acid/water in 3 5/55 10 5/15 volume m; [58] Field of Search ..210/3l, 198
• Catecholamines are understood in clinical chemistry to mean noradrenaline (NA), adrenaline (A), and dopamine.
• the metabolites thereof which are of greatest importance with respect to quantity and, at present, with respect to diagnosis of diseases, are metadrenaline, norrnetadrenaline and the phenolcarboxylic acids 4-hydroxy-3-methoxymandelic acid (HMMS or VMS vanillin mandelic acid), vanillic acid (VS) and homovanillic acid (I-IVS).
• HMMS or VMS vanillin mandelic acid 4-hydroxy-3-methoxymandelic acid
• VS vanillic acid
• I-IVS homovanillic acid
• the excretion of these metabolites is increased in various diseases, for example in case of pheochromocytoma, neuroblastoma and ganglioneuroma.
• HIES 5-hydroxyindoleacetic acid
• HMMS is excreted to an increased extent also during the attack-free interval.
• IIMMS is difficult, because numerous other phenols are also present in the urine, which phenols form colors with the respective reagents, and because the basic ion exchangers do not bind the phenolcarboxylic acids to a particularly great extent in the presence of the uric salts.
• Purely chromatographic methods exhibit the advantage over processes operating with the aid of cation exchangers in that several metabolites can be simultaneously and specifically determined side-by-side.
• several methods using paper and thin-layer chromatography are known for the determination of these metabolites.
• the extraction of the urine is generally followed by the steps of drying, filtering, and concentrating the thus-obtained extract. Thereafter, a separation by paper or thin-layer chromatography is conducted on silica gel or cellulose.
• the detection of the metabolites is effected in all cases in a conventional manner with a color reagent, the color being compared with a standard.
• catecholamine and serotonin metabolites in body fluids are determined by separation by conventional thin-layer chromatography and color development of the chromatogram employing as chromatographic adsorbent cellulose impregnated with polyethylenimine.
• the chromatogram is developed preferably using a solvent mixture described hereinafter.
• the chromatographic adsorbent employed preferably is conventional cellulose impregnated with polyethylenimine for chromatographic purposes (PEI cellulose). It is also possible to impregnate any cellulose of a quality suitable for chromatographic purposes, preferably micro-crystalline cellulose, with polyethylenimine in a conventional manner. See, for example, Biochimica Biophysica Acta, Vol.61, pp.852-854 (1962). This is most simply done using a commercially available polyethylenimine solution which is about 50 percent aqueous. Such a solution is thoroughly mixed with the cellulose, optionally after dilution with deionized water. The thus-obtained suspension can be used as such for coating the thin-layer chromatographic plates in the conventional manner.
• the proportion of polyethylenimine to adsorbent is usually about 0.15 to 10 percent by weight, preferably about 0.5 to 3.5 percent.
• the polyethylenimine-impregnated cellulose should not be a PEI cellulose neutralized with hydrochloric acid, which is the form customarily employed in chromatography. It is essential that a PEI cellulose is employed which does not contain a firmly bound counter-ion. For this reason, the polyethylenimine should be adsorbed by the cellulose in free form, i.e., as the free base, or if it is adsorbed in neutralized form, it should contain only those anions which have a selectivity coefficient lower than the chloride anion, e.g., forrnate, acetate, propionate and butyrate ions. As is known, selectivity coefficient is a measure of the relative affinity of the ions with respect to the ion exchanger.
• the composition of the eluent employed to develop the chromatogram affects the separation achieved on the chromatograms.
• the eluent mixture preferably consists of at least three components.
• the eluent, i.e., mobile phase contains, in addition to at least 5 percent of water, at least one other solvent from each of the following groups:
• C. acids preferably lower alkanecarboxylic acids.
• an acidic multicomponent mixture preferably consisting of at least three components.
• the desired separations can be obtained especially advantageously if eluents are employed containing, in addition to water, at least one solvent which is immiscible or miscible to only an insubstantial extent with water; at least one solvent miscible with both water and with organic solvents; and at least one acid, preferably a lower-alkanoic acid.
• solvents which are immiscible with water, or miscible only to an insubstantial extent are chlorinated hydrocarbons, e.g., chloroform and methylene chloride; aliphatic alcohols containing more than three carbon atoms, e.g., butanols (except tert.butyl alcohol) and amyl alcohols; esters, e.g., esters of acetic acid and other loweraliphatic carboxylic acids, preferably esters of acetic acid, e.g., butyl acetate, and isoamyl acetate; ethers, e.g., diethyl ether; and aromatic hydrocarbons, e.g., benzene, toluene and xylene.
• chlorinated hydrocarbons e.g., chloroform and methylene chloride
• aliphatic alcohols containing more than three carbon atoms e.g., butanols (except tert
• Suitable solvents which are miscible both with water and organic solvents (group B) are lower-aliphatic alcohols, particularly methanol, ethanol, nand iso-propanol; ketones, e.g., dialkyl ketones, especially acetone and methyl ethyl ketone, cycloalkyl ketones, especially cyclohexanone; cyclic ethers, e.g., dioxane and tetrahydrofuran; acetonitrile; pyridine; dimethylformamide; and dimethylsulfoxide.
• lower-aliphatic alcohols particularly methanol, ethanol, nand iso-propanol
• ketones e.g., dialkyl ketones, especially acetone and methyl ethyl ketone, cycloalkyl ketones, especially cyclohexanone
• cyclic ethers e.g., dioxane and tetrahydrofuran
• Preferred acids (group C) are lower-alkanoic acids containing one to four carbon atoms, e.g., formic acid, acetic acid and propionic acid, and substituted carboxylic acids, especially substituted lower-alkanoic acids, e.g., methoxyacetic acid.
• the separations are generally less successful with mineral acids, which are thus not preferred.
• the proportion of the acid in the total mixture normally is between 1 and 40 percent by volume, preferably about 5-20 percent by volume.
• the eluent employed is a mixture of chloroform/n-butanol/ethanol/ glacial acetic acid/water, especially in a volume ratio of about 5/55/10/15/15.
• suitable mixtures are, for example, n-butanol/isopropanol/glacial acetic acid/water, e.g., in a volume ratio of about 60-40/10-30/15/15, e.g., about 60/10/15/15, and about 40/30/15/15; n-butanol/ethyl acetate/glacial acetic acid/water, e.g., in a volume ratio of about 60/10/ 15/ 15; n-butanol/glacial acetic acid/water; e.g., in a volume ratio of about 70/ 15/ 15; and n-butanol/ethanol/glacial acetic acid/water, e.g., in a volume ratio of about 60/5/ /25, respectively.
• the viscosity of the eluent mixture at 20 C. is preferably between 0.2 and 5 centipoises. lts boiling point range is preferably between 30 and 150 C.
• the separation of the components of the body fluid by the eluents is conducted two to four times.
• an alkaline eluent mixture it is important, when the urine is applied without any pretreatment, which is preferred, that the first separation on the PE] cellulose be conducted with an acidic eluent mixture.
• the further treatment of the resulting chromatogram, especially in two-dimensional chromatography, can also be conducted with acidic, neutral or basic eluents.
• acidic, neutral or basic eluents Especially satisfactory separations can be obtained, for example, if the basic eluent mixtures employed are those containing the solvent components mentioned above for the acidic eluents, except the acid component is replaced by a basic component, preferably ammonia.
• the proportion of base in the solvent mixture is normally between 1 and 40 percent by volume, preferably about 5-30 percent by volume.
• the basic eluent mixture in addition to the base, desirably contains about 5 to 20 percent of water; at least one solvent immiscible or miscible to only a slight extent with water (group A); and at least one solvent miscible both with water and with organic solvents (group B).
• group A ethyl acetate/n-butanol/isopropanol/25 percent aqueous ammonium hydroxide, preferably in a volume ratio of about 30/20/25/25.
• one of the solvent groups A or B can be omitted, since, for many problems, a sufficient separation is nevertheless effected.
• Examples for further basic eluent mixtures are the following: Chloroform/nbutanol/ethanol/25 percent aqueous ammonium hydroxide/water, preferably in a volume ratio of about 20/30/30/ 10; ethyl acetate/n-butanol/isoproanol/25 percent ammonium hydroxide, preferably in a volume ratio of about 30/20/25/25;
• n-butanol/acetone/25 percent ammonium hydroxide/water preferably in a volume ratio of 70/ 10/10/ 10; n-butanollmethyl ethyl ketone/25 percent hydroxide/water, preferably in a volume ratio of about 30/40/15/15; isoamyl acetate/isopropanolZS percent ammonium hydroxide, preferably in a volume ratio of 30/45/25; and isopropanol/25 percent ammonium hydroxide, preferably in a volume ratio of /20.
• the production of the thin-layer chromatographic plates to be employed for conducting the process of this invention is effected in a conventional manner. See, e.g., Bobbitt, James M., Thin-Layer Chromatography (1963) Reinhold Publishing Corp., N. Y.
• the adsorbent suspension is applied by means of a conventional spreader to the substrates of glass, synthetic films, metallic foils, or other suitable materials.
• the thickness of the adsorbent layer normally ranges between 50 and 200 u.
• the plates are then dried for several hours at room temperature, or for a shorter period of time at an elevated temperature, e.g., several minutes at 80-l30 C. Since the layer is light-sensitive, it is desirable to store the plates so that they are protected from light.
• the body fluid to be assayed for catecholamine and serotonin metabolites usually is urine. Basically, however, it is also possible to conduct the determinations with other suitable body fluids, e.g., cerebrospinal fluid.
• the novel process is conducted in a conventional manner.
• the urine or other body fluid is applied to the adsorbent layer, preferably without any pretreatment.
• the urine specimen is, in certain cases, diluted, e.g., with deionized water, in order to employ comparable volumes, and to facilitate the mathematical conversion.
• the excretion for a 24 hour period is, in each case, diluted to the nearest full liter, e.g., 1,200 to 1,600 cc of urine would be diluted to 2 liters.
• each urine sample is applied to the adsorbent layer. It is advantageous to conduct an intermediate drying step if more than 2 #1. are applied to one point. It is desirable to apply one standard solution containing known concentrations of the components being assayed for in the specimens alongside each 23 urine specimens.
• one or several are inserted in customary development chambers containing the element mixture. Suitably, the chamber is closed during the developing step by means of a cover plate. Usually, the eluent front reaches the upper rim of the plate in about l-2 hours (height of ascent 8.5 cm., with aplate size of 10 X10 cm. or 20 X10 cm.).
• a second development is then conducted in the same manner after a drying step, optionally using the same or another acidic eluent or a neutral or basic eluent.
• an appropriate color reagent is thereafter sprayed on the developed plate in the usual manner. Normally, these color reagents are employed in concentrations of about 0.1-1 percent, usually in stabilized form.
• diazotized p-nitroanilines are utilized, e.g., pnitrophenyl-diazonium fluoroborate, 4-diazobenzenesulfonic acid, 4diazo-N-monoethyl-orthotoluidine fluoroborate, 4- diazo-N,N-diethylaniline fluoroborate or 4-diazo-N-ethyl-N- (B-hydroxyethyl)-aniline.
• 2,6-dichloroquinone chlorimide has also been employed for this purpose.
• the chromatogram is evaluated in direct illumination and trans-illumination. The height and the color of the spots of the urine chromatograms are compared with those of the standard solutions.
• Vanillic acid, metadrenaline, normetadrenaline and methoxyhydroxymandelic acid exhibit blue colorations and homovanillic acid shows a light-blue coloration when p-nitrophenyl-diazonium fluoroborate is employed as the color reagent.
• hydroxyindoleacetic acid is colored red with this reagent.
• a definite conclusion can then be drawn whether the apparent increased concentration of a metabolite was merely simulated by the presence of another substance with the same R value in the body fluid being examined.
• the two-dimensional chromatography is preferably conducted twice in each dimension.
• the layer is divided into two parts in twodimensional chromatography.
• spots developed from the urine specimens obtained in this fashion can now readily be identified by reference to the two corresponding spots of the standard solutions, i.e., by means of two coordinates. Also, the quantitative assessment of the separated substances can be conducted with great certainty in this manner.
• the present invention it is now possible for the first time to conduct a side-by-side identification and quantitative as sessment of both Catecholamine and serotonin metabolites, without previously having to treat the urine, thereby eliminating a source of error. Because of its simplicity and rapidity of its conductance, the novel process is suitable for series investigations as well as for individual determinations in case of pathological findings. The low detection limits of the process render it useful for a great variety of problems and differential diagnoses.
• the plates are dried in a drying CONDUCT ANCE OF THE PROCESS EXAMPLE 1 Use As A Clinical Test-For The Recognition Of Increased Renal Excretion of Catecholamine and Serotonin Metabolites
• the collected urine of 24 hours of a patient is brought with distilled water to a volume of l, 2, or 3 liters.
• Two pl. of urine per 1 liter of the standardized urine sample is applied in dots to the longitudinal side of a PEI cellulose glass plate of a size of 10 X 20 cm. at a distance of 1.5 cm. from the lower edge. Between the individual applied dots, a spacing of 1.5 cm. is maintained.
• a standard solution see table below for the composition thereof) is applied.
• each urine sample is adjacent a standard solution, and eight urine specimens can be applied to a plate.
• the plate is now developed twice up to the upper edge of the plate with the eluent mixture of chloroform/butanol/ethano l/glacial acetic acid/water (5/55/10/15/15). After the first development, the plate is taken out of the development chamber, is dried in a stream of warm air, and than is placed again into the development chamber.
• the plate is then dried and sprayed with a ODS-molar solution of p-nitrobenzene-diazonium tetratluoroborate in a 0.3- molar aqueous Na cO solution. After a short period of heating, the individual metabolites appear separately as spots of various colors. On the basis of the intensity of the colorations and the size of the spots, the determination is made whether one of the urine specimens contains a metabolite at a higher concentration than the standard.
• the standard is produced by dissolving known amounts of the respective metabolites in standard volume of water.
• concentrations of the metabolites in the standard correspond to the upper normal range, except for metadrenaline and normetadrenaline, which were added in excess amounts to improve recognition.
• the composition of the standards, as well as the ascent levels of the individual standard substances in the eluent employed is set forth in Table l.
• EXAMPLE 3 Two-Dimensional Chromatography
• a X 20 cm. glass plate coated as described above with PEI cellulose is divided by a pencil line into two 10 X 10 cm. halves.
• To the thus-obtained left-hand portion of the plate at least 4 pl. of an untreated urine is applied at a distance of 1.5 cm. from the left-hand and lower edges, respectively.
• On the right-hand half of the plate the application is carried out correspondingly, but in this case 2 pl. of a standard solution (ST-3; Example 2) is also applied at a spacing of mm. from the point of application of the urine sample.
• ST-3 Standard solution
• the plate is developed twice in the usual manner with intermediate drying by ascending chromatography to the upper edge of the plate with the longitudinal side of origin downwardly using as eluent chloroform/butanol/ethanol/ glacial acetic acid/water (5/55/10/15/15).
• the thus-obtained spots are made visible by spraying with a 0.05-molar solution of p-nitrophenyl-diazonium fluoroborate.
• Each spot can be unequivocally characterized in two coordinates by the standard solutions employed in the two-dimensional development procedure.
• EXAMPLE 4 In the manner described in Example 1, I00 24-hour urine specimens were investigated. In a variation from Example 1, the standard solution was replaced by a standard solution containing I-IMMS, HIES, I-IVS, and VS, each in concentrations of 5 mg./l.
• HVS was discovered only in 10 of the 100 urine specimens by the size and intensity of the light-blue spots.
• a substance having a red coloration was found just beneath the I'IVS spots.
• I-IVS 5 mg, respectively, of I-IVS per liter were added to the separate portions of a urine specimen containing this substance. It was found after the development of the chromatogram that the detection of HVS in the process of this invention is not impaired by this substance which assumes a red coloration, because the differences in HVS concentration could be clearly seen.
• a urine sample contained a substance assuming a red color at exactly the development point of l-IVS.
• twodimensional chromatogram of the specimen showed that the specimen did not contain any substantial amounts of HVS.
• the process of this invention did not erroneously indicate a pathological excretion in any of the 100 urine specimens examined, which established that erroneously positive results are not likely. This is even more surprising in view of the fact the urine samples were obtained from patients who were not readied for the test by control of diet or ingestion of drugs. Erroneous negative results are likewise unlikely, due to the high sensitivity of metabolites detection according to the process of this invention.
• EXAMPLE 5 Semiquantitative Assessment of the Renal Excretion of Catecholamine and Serotonin Metabolites Standard solution having the composition and concentration set forth in Example 2 are prepared.
• Example 1 From a urine sample found to have a striking increased content of homovanillic acid, there are applied alternately 2 pl. of urine per one liter of daily excretion and respectively one of the various standard solutions in increasing concentrations, in the manner described in Example 1. The plate is developed and the color reaction carried out as described in Example 1. By reference to the standards of varying concentration, the amounts of the various metabolites in the urine sample can be assessed without difiiculty and with great accuracy.
• the urine specimen was classified between Standard 2 and Standard 3 (light-blue spot), which corresponds to a homovanillic acid content of 25 mg./l. of the unne specimen.
• a process for the determination of catecholamine and serotonin metabolites in body fluids by thin-layer chromatog- 9 raphy which comprises using as the chromatographic adsorbent a cellulose impregnated with polyethylenimine.
• a process according to claim 1 wherein the adsorbent is microcrystalline cellulose impregnated with polyethylenimine.
• eluent solvent is a mixture of chloroform, n-butanol, ethanol, glacial acetic acid and water.
• eluent mixture is a mixture of ethyl acetate, n-butanol, isopropanol and aqueous ammonium hydroxide.

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  • BE BE755803D patent/BE755803A/xx unknown
• 1969
  • 1969-09-09 DE DE1945471A patent/DE1945471C3/de not_active Expired
• 1970
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