ISSN:0003-2654    

DOI:10.1039/AN95277FX009

出版商:RSC    

年代:1952

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN95277BX011

出版商:RSC    

年代:1952

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN95277FP023

出版商:RSC    

年代:1952

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN95277BP031

出版商:RSC    

年代:1952

数据来源: RSC

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MARCH, 1952 Vol. 77, No. 912 THE ANALYST PROCEEDINGS OF THE SOCIETY OF PUBLIC ANALYSTS AND OTHER ANALYTICAL CHEMISTS INFORMAL DINNER A DELEGATION appointed by the Council of the Society, consisting of the President, Dr. J. R. Nicholls, C.B.E., Mr. R. C. Chirnside, Vice-president, and Dr. K. A. Williams, Honorary Secretary, attended the XIIth International Congress of Pure and Applied Chemistry and the 75th Anniversary Meeting of the American Chemical Society held in New York in September, 1951. A report of these meetings was given by the members of the Delegation at an Informal Dinner held at the Park Lane Hotel, Piccadilly, London, W.l, on Tuesday, December 18th, 1951. NEW MEMBERS Sydney Abbey, B.Eng.Chem. (McGill) ; George Albert Barber, B.Sc. (Manc.) ; Jack Harold Defrates; Marcus Alan Ellis, M.P.S., Ph.C. ; John Stanley Lea, B.Sc. (Lond.), A.R.I.C. ; Bernard Milgrom; Brian Walter Mitchell, B.A. (Cantab.), B.Sc. (Lond.), A.R.I.C. ; James Harry Shelton, A.R.I.C. DEATH Archibald Robert Jamieson WE regret to record the death of

ISSN:0003-2654    

DOI:10.1039/AN952770111a

出版商:RSC    

年代:1952

数据来源: RSC

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118 BOND : THE Staphylococcus aureus [Vol. 77 The Staphylococcus aureus Plate Assay of Penicillin : Effect of Sugars on Zone Edges and Accuracy of Results BY C. R. BOND The presence of sugars in penicillin test solutions effects markedly the definition at the edges of the zones of inhibition in the cylinder plate assay method. The incorporation of 0.1 per cent. of sucrose in a typical Staphylococcus aureus nutrient agar medium greatly improves the definition of the zone edges. The presence of sugars in penicillin test solutions increases the zone diameters and may cause fictitiously high results to be recorded. For accurate work the standards must be compensated by incorporating appropriate sugars in similar concentrations to those present in the test solutions. The regression line obtained by plotting the logarithms of the concentrations against the zone diameters in a medium containing 0.1 per cent.of sucrose has a slight positive curvature, approximating to a maximum error of 3 per cent. if the curvature is ignored in calculating results over the range 2 to 8 units per ml. IN assaying penicillin by the plate method it is of the greatest importance that the edges of the zones of inhibition shall be sharply defined so that their diameters can be accurately measured, for very small differences in zone diameters correspond to large differences in potency of the test solution. When Bacillus subtilisl is used as the test organism, the edges of the zones are extremely well defined and eminently suitable for accurate measurement. According to the British Pharmacopoeia and the Food and Drug Administration of the U.S.A.? StaphyZococcus aureus has to be used as the test organism in penicillin assays, although the strain to be used is not stipulated in the British Pharmacopoeia. The most commonly recognised are the Oxford strain NCTC 6571 and the American F.D.A.strain NRRL 313. The former usually gives poorly defined zones and the latter somewhat better zones when used as a surface inoculum. Surface inoculation, however, needs considerable care if accurate results are to be obtained and is uneconomical in time and effort, especially when used on a large scale in industrial laboratories. The strains of S. aurem mainly used are therefore those that best lend themselves to bulk inoculation and at the same time give the required sensi- tivity.Under routine working conditions, however, even the best strains of s. aureus do not give zones of inhibition equal to those obtained with B. subtilis; the most commonly occurring type of zone possesses a halo and some of them resemble Liesegang rings. These phenomena are no more than a nuisance to the analyst, but have led to much interesting work, notably by Pulvertaft, Greening and Haynes3 and more recently by Ingram*; so far, however, no specific effect appears to have been traced to any particular substance in the test solutions or in the nutrient agar. In the course of routine assay work we observed that the edges of the zones of inhibition varied in definition and general appearance with different samples. The possibility that these effects were produced by differences in working conditions such as variation in inoculum cul- ture, degree of refrigeration, rate of warming in an incubator and so on, were eliminated by producing various types of zones on the same plate with penicillin solutions that contained different substances.Further work was therefore undertaken to trace the agents causing variation and to find suitable agents for the production of zones of inhibition with sharply defined edges. It is well known that when certain dissolved substances are present in penicillin test solutions larger zones are obtained than when they are absent; these larger zones give fictitiously high assay results if normal simple solutions of penicillin are used as standards. In practice, the effect of dissolved substances on results has been largely ignored with such products as penicillin lozenges,2 perhaps because it was assumed that the degree of dilution was sufficiently large to reduce errors from this source to negligible amounts. The work described in this paper shows that this is not always so, and that sometimes the assay results can be significantly too high if the effect of sugars is ignored.In the course of work onMarch, 19521 PLATE ASSAY OF PENICILLIN : EFFECT OF SUGARS 119 lozenges it was observed that the test solutions frequently produced zones of inhibition with well defined edges that often consisted of a single ring of increased bacterial growth permitting measurement of the zone diameters to be accurately made.Preliminary investigation of this phenomenon showed that the causal agent was sucrose. Work was carried out to determine more precisely the effects of the commonly used sugars, sucrose, lactose and dextrose, on the size of the zones of inhibition and on the definition of the zone edges. EXPERIMENTAL The cylinder plate assay method was used, The medium was as follows- Agar . . .. .. .. . . . . 2.0 per cent. Lemco . . .. .. .. . . 0-3 ** Marmite (yeast extract) . . .. . . 0.15 99 Sodium chloride . . .. .. . . 0-3 99 Peptone (Oxoid) . . .. .. . . 1.0 9) Tap water to .. . . .. .. 100 9, The pH was adjusted to 7.5 before sterilising. THE EFFECT ox ZONE DIAMETERS AND ZONE EDGES OF SUGARS IN TEST SOLUTIONS- Solutions containing 0.5 unit of benzyl penicillin (sodium salt) per ml and 0.05, 0.1, 0.2 or 0.4 per cent.of sucrose, lactose or dextrose in 0.01 M phosphate buffer were compared with a standard solution (0.5 unit per ml) of peniciHin in 0.01 M phosphate buffer on each of twenty plates. Typical results in terms of mean zone diameters together with observations on the zone edges are shown in Table I. TABLE I MEAN DIAMETERS OF ZONES OF INHIBITION OF SOLUTIONS OF DIFFERENT SUGARS EACH CONTAINING 0.5 UNIT OF PENICILLIN PER MILLILITRE Concentration of sugar, % Szmose- 0.0 0.05 0.10 0.20 0.40 Lactose- 0.0 0.05 0.10 0.20 0.40 Dext yose- 0.0 0.05 0.10 0-20 0.40 Zone diameter * Expt. 1, Expt. 2, mm mm 12.81 13.22 13-08 13.31 13.17 13-75 13.46 14.10 14-27 14.91 13.41 16.62 13.43 15.58 13-45 16-65 13.35 15.80 13-38 15.84 13.50 14.34 13-46 14.69 13.53 14.76 13-98 15-16 14.55 15.56 Mean increase in zone diameter, mm.0.17 0.45 0.77 1-58 - - - 0.04 0.04 0.06 0.10 - 0.16 0.23 0.65 1.14 Appearance of zone edges Standards had haloes with diffuse inner edges. Sucrose solutions gave zones with very strong inner edges, decreasing in intensity with decreasing concentrations of shcrose Standards and lactose solutions occasionally gave zones alike in appearance, but on other occasions well defined zones resulted, especially with higher concentra- tions of lactose The zones of the dextrose solutions had more sharply defined edges than the standards but were less pronounced than those of the sucrose solutions. In some experi- ments three Liesegang rings were observed It can be seen from these figures that a marked increase in zone diameter occurred with sucrose and glucose in solution.The zone diameter increased with increase in concentration of the sugar. It is interesting to note that lactose gave no significant increase in zone diameter at any of the concentrations used. The effect varied according to the nature of the sugar used and also according to its concentration. It was found to be important to make use of efficient refrigeration between the operations of pouring and potting the plates, or ill-defined zone edges mostly resulted; many of these zone edges had a miscel- laneous variety of Liesegang ring effects. Under well controlled conditions it was found that The effect of sucrose was a little greater than that of glucose. The effect of the sugars on the zone edges was remarkable.120 BOND : THE Sta@hylococcus auureus [Vol.77 sucrose generally gave clearly defined circles, often with a well defined edge of heavy bacterial growth. Glucose produced a similar effect, but the circles were usually less well defined than those obtained with sucrose, and occasionally Liesegang rings were observed. Lactose usually had less effect, but sometimes zone edges were sharper and occasionally Liesegang rings were formed. Similar effects were produced with concentrations of penicillin of 1.0, 2.0 or 8.0 units per ml, but with 8.0 units per ml the zone edges as a rule were less well defined. Generally an increase of sucrose or glucose concentration had the effect of reducing the width of the halo. EFFECT OF SUCROSE IN THE NUTRIENT MEDIUM ON THE DEFINITION OF ZONE EDGES- In view of the results obtained with sugars present in the test solutions, experiments were made to find out whether sucrose in the medium produces the same effects on zone edges as when present in the test solutions.Various concentrations of sucrose were added to the medium. Media were prepared containing 0.025, 0.05, 0.1 or 0.2 per cent. of sucrose. Plates were prepared from each of these media in the usual way. The cylinders were filled with standard solutions of penicillin, ranging in potency from 0.25 to 4.0 units per ml, in 0.01 M phosphate buffer and the plates were placed in an incubator overnight at 37” C. The plates containing 0.025 or 0.05 per cent. of sucrose in the medium had zones of inhibition with haloes and indistinct edges; those with media containing 0-1 per cent.of sucrose gave zones with haloes, but the inner edges of the zones possessed a very dark narrow ridge of heavy bacterial growth, which was ideal for measuring zone diameters; those with 0-2 per cent. of sucrose in the medium had zones similar to those given by 0.1 per cent. of sucrose, but the inner dark ridge of growth was slightly blurred, although thicker. When a medium containing no sugars is used, the outer edge of the halo is usually easier to read than the inner edge and is regarded as the zone perimeter, but when sucrose is added to the medium the inner edge becomes the easier one to read and in some instances there is no definable outer edge at all. This has the apparent effect of reducing the zone diameter, but the zone diameters of the corresponding edges are greater when sucrose is incorporated in the medium.Further experiments incorporating glucose or lactose into the medium showed that lactose usually improved the zone edges, but glucose, although it gives considerable improve- ment, was not so efficient as sucrose. EFFECT OF SUGARS IN TEST SOLUTIONS ON ZONE DIAMETER WITH XUTRIENT MEDIA CONTAINING This experiment was made to determine whether or not the increase in zone diameters caused by the presence of sugars in the test solutions, with a normal medium, still occurred when a modified medium containing 0.1 per cent. of sucrose was used. Four solutions were prepared each containing 1.0 unit of penicillin per ml in 0.01 A4 phosphate buffer.One of these solutions also contained 0.2 per cent. of glucose, another 0.2 per cent. of sucrose and a third 0.2 per cent. of lactose; the fourth solution was kept for a blank determination. A fifth solution containing 0.5 unit of penicillin per ml in 0.01 M phosphate buffer was prepared to enable the slope of the regression line to be found. The concentration of sugar (0.2 per cent.) in each solution was chosen because it can occur when testing lozenges, but is unlikely to be exceeded in any other normal assay. The results of seventeen replicate tests show that under these conditions the apparent potency of penicillin is 19 per cent. too high in a solution containing 0.2 per cent. of sucrose, and 10 per cent. too high in a solution containing 0.2 per cent.of glucose. No apparent increase was obtained with 0.2 per cent. of lactose. The effect of the sucrose and glucose on the zone edges was marked, the zones being extremely sharply defined in most tests. In order to determine the effects of greater dilutions of sucrose, a further experiment was made in which sucrose solutions each containing 1-0 unit of penicillin per ml were assayed against the 0.01 M buffer containing 1.0 unit of penicillin per ml. The concentrations of sucrose used were 0.4, 0.2, 0.1, 0.05 and 0.025 per cent. and the six solutions were compared on each of twenty plates. The results are shown in Table 11. By plotting the values of “zone diameter” against “concentration of sucrose” a nearly linear regression line is given. It follows, therefore, that the effects of sucrose cannot be eliminated by dilution and con- sequently accurate results can only be obtained by adequately compensating the standards.0.1 PER CENT. OF SUCROSE-March, 19521 PLATE ASSAY OF PENICILLIN: EFFECT OF SUGARS 121 The effect of sucrose in solutions containing higher concentrations of penicillin, viz., 4.0 and 8.0 units per ml, was also determined and the results, together with those at the 1 unit per ml level, are shown in Fig. 1. In these solutions up to 1 per cent. of sucrose was used; the zones produced were exceedingly well defined at these high concentrations, but the effect of sucrose on apparent potency was still present, although relative to the actual potency of the solution the effect becomes smaller with increase in penicillin potency.TABLE I1 EFFECT OF SUCROSE I N TEST SOLUTIONS CONTAINING 1 UNIT OF PENICILLIN PER MILLILITRE ON ZONE DIAMETER Concentration of sucrose, 0.4 0.2 0.1 0.05 0.025 nil % Zone diameter, mm 17.11, 17-31, 16.53 16.62, 16.53, 15.84 16.30, 16.30, 15.47 16.10, 16.11, 15.34 16.08, 15.92, 15-28 15.95, 15.89, 15-18 Mean zone diameter, mm 16.98 16-33 16.02 15.85 15.76 15.67 Mean apparent potency, units per ml 1-47 1.22 1.12 1-06 1.03 1.00 The effect of sucrose on results obtained with normal and compensated standards in assays of lozenges was verified on a number of samples of lozenges in the following manner. Samples of penicillin lozenges were assayed after dissolving approximately 1.2 g of sample in 0-01 M buffer and diluting to 500 ml. As the lozenges were composed chiefly of sucrose, the resulting concentration of sucrose was about 0.2 per cent.The penicillin standard solutions used for the tests contained (a) 0.01 M buffer and (b) 0.01 M buffer and 0-2 per cent. of sucrose. The results obtained for both conditions are given in Table I11 and show the marked difference between normal and compensated standards. I t is apparent from this and the previous work that, in the assay of these lozenges, the standards must contain the same amount of sucrose as the sample. In view of the difference in effect on zone diameter produced by different sugars, it is necessary to know the composition of the lozenges before accurate compensation can be made. For normal production samples this is already known and no difficulty arises, but for other samples, an analysis of the sugars in the lozenge base must be made before the assay of penicillin is attempted.TABLE I11 ASSAYS OF PENICILLIN LOZENGES WITH AND WITHOUT COMPENSATED STANDARDS Potency, units per lozenge I Sample With normal standards A 600 B 640 C 620 D 600 E 650 F 700 635 (Mean) 7 With compensated standards containing sucrose 500 550 500 490 540 600 530 (Mean) LINEARITY OF DOSE - RESPONSE CURVE FOR MEDIUM CONTAINING 0.1 PER CENT. OF SUCROSE- Before accepting the increased accuracy available from the improved definition of the zone edges arising from the use of a nutrient medium containing 0.1 per cent. of sucrose, it was necessary to check the effect of the modified medium on the linearity of the dose - response curve. For this purpose the mean zone diameters obtained over a range of con- centration of penicillin from 0.5 to 16 units per ml were measured. Six concentrations of penicillin, 16, 8, 4, 2, 1 or 0.5 units per ml, were tested on each of twenty plates on several days.The mean diameters measured on each twenty replicate plates were as follows- Concentration of penicillin, units per ml . . 16 8 4 2 1 0.6; +++U+ Mean zone diameter, mm .. .. . . 24.99 23.18 21.31 19.26 16.96 14.26 Difference between intervals, mm . . .. 1-81 1-87 2.05 2.30 2.70122 BOND : THE Staphylococcus aweus [Vol. 77 The regression line exhibits a considerable degree of curvature, but if a narrow range only is used in the assay the error from this source is small. With standards of 2 and 8 units per ml a working range for the assay of 1.5 to 16 units per ml is possible with a maximum error due to curvature of less than 4 per cent.; if three standards, viz., 2 , 4 and 8 units per ml are taken, and the mean of the three is used in calculating the potency of the unknown, the error due to curvature becomes less than 3 per cent.Practical tests with the second of these ! Fig. 1. Effect of sucrose in solution on apparent potency of penicillin as determined by the S . aureus plate assay. Full lines determined by experiment; broken line indicates extrapolation two procedures, made by repeated assays on a large number of samples, show that the standard error of the method with six plates each carrying three zones of unknown and one of each of the three standards is approximately 4 per cent.When compensated standards are used for tests on solutions containing considerable amounts of sugars (up to 1 per cent.), a much smaller “slope” is obtained over the range 2 to 8 units per ml, so that the method becomes less accurate. It follows, therefore, that solutions should if possible be assayed at such a concentration of penicillin that excessively high concentrations of sugars are avoided. CONCLUSIONS Sugars in penicillin solutions assayed by the cylinder plate method have in some instances the effect of increasing the zone diameters produced by penicillin. This leads to fictitiously high results if ordinary penicillin standards are used. Of the three sugars examined, sucrose produces the largest effect and dextrose a somewhat smaller effect; up to 0.4 per cent.of lactose is almost without effect on the size of the zones. Consequently in the assay of penicillin lozenges or other samples containing sugars the standards used must be compensated by adding the appropriate sugars. The presence of sucrose in assay solutions gives an improved zone edge in an ordinary nutrient agar medium. A similar effect can be obtained by incorporating 0.1 per cent. of sucrose into the nutrient agar. This greatly increases the accuracy with which the zone diameters can be measured and consequently also the accuracy of the method as a whole. The curvature of the regression line for zone diameter on the logarithm of the concentrationMarch, 19521 PLATE ASSAY OF PENICILLIN: EFFECT O F SUGARS 123 in the sucrose medium is sufficiently small over the range 2 to 8 units per ml to permit calcula- tion of results on a theoretically linear relationship without introducing serious error. The maximum error from this source is about 3 per cent. Compensation of the standards for the presence of sugars in lozenges or other samples must be made by adding amounts of the appropriate sugars to make the concentrations the same as in the test solutions. REFERENCES 1. 2. Foster, J. W., and Woodruff, H. B., J . Bact., 1944, 47, 43. “Compilation of regulations for tests and methods of assay and c@fication of antibiotic and antibiotic-containing drugs. Federal Security Agency, Food and Drug Administration, 1951, p. A.34. Pulvertaft, R. J. V., Greening, J. R., and Haynes, J. A., J . Path. Bact., 1947, 59, 293. Ingram, G. I. C., J . Gen. Microbiol., 1951, 5, 22. Vol. I, Tests and methods of assay. 3. 4. IMPERIAL CHEMICAL INDUSTRIES LTD. DYESTUFFS DIVISION TRAFFORD PARK WORKS MANCHESTER , 17 November, 1951

ISSN:0003-2654    

DOI:10.1039/AN9527700118

出版商:RSC    

年代:1952

数据来源: RSC

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March, 19521 PLATE ASSAY OF PENICILLIN: EFFECT O F SUGARS 123 The Determination of p-Nitrophenol and p-Nitrophenyl-0-S-diethyl Thiophosphate in Parathion BY J. C. GAGE A method is described for the analysis of a mixture of parathion, the S-ethyl isomer of parathion and p-nitrophenol. The mixture is reduced, the p-aminophenol and reduced S-ethyl isomer are successively extracted, coupled with o-cresol and estimated colorimetrically as an indophenol dye. The reduced parathion is extracted, diazotised, coupled with N-sulphato- ethyl-m-toluidine and determined colorimetrically. IN a previous communication1 a method was described for the determination of the insecticide 0-$-nitrophenyl-0-0-diethyl thiophosphate, usually known by the names parathion or E605. In that method parathion was reduced to the corresponding amino compound, which was then diazotised and coupled to give an azo dye, the intensity of which was measured in a suitable colorimeter.The method is not specific for parathion; a similar colour is given by many nitro compounds that react in the same manner, some of which occur in the commercial product as impurities or may be derived from parathion by chemical or physical treatment or by metabolic processes. An investigation to discover a method that would be more specific for parathion and would enable the compounds associated with it or derived from it to be separately determined was undertaken in connection with studies of residual parathion on treated crops and with metabolism experiments on laboratory animals. This paper describes a method for determining P-nitrophenol and 0-9-nitrophenyl-0-S-diethyl thiophosphate, herein termed the S-ethyl isomer of parathion.$-Nitrophenol is derived by hydrolysis from parathion slowly in aqueous solution, but more rapidly in the presence of alkali2; 0-@nitro- phenyl-0-S-diethyl thiophosphate is obtained from parathion by isomerisation above 140” C.3 METHOD REAGENTS- Toluene-“Sulphur-free” toluene purified as described below. Hydrochloric acid-Concentrated and 0.5 N solutions. o-Cresol-A 2 per cent. w/v solution in 0.2 A7 sodium hydroxide. Sodiwn acetate-Analytical reagent quality. iso-Amyl alcohol-Analytical reagent quality. Reduction of nitro groztp-Boil “sulphur-free” toluene under reflux for 20 minutes with Cool, filter PROCEDURE- 2.5 per cent.of benzoic acid, 2-5 per cent. of zinc dust and a trace of aniline.124 GAGE : THE DETERMINATION OF P-NITROPHENOL APiD [Vol. 77 and wash well with 0-5 N hydrochloric acid. This procedure is designed to remove impurities that not only produce a high blank value but also inhibit colour development; the toluene retains sufficient benzoic acid to be suitable for the reduction without further addition of acid. Dissolve the sample to be analysed in the prepared toluene. Add about 0.5 g of zinc dust to 20 ml of the toluene solution and boil gently under an air condenser for 20 minutes. Cool and filter through a plug of cotton wool into a separating funnel; wash the residual zinc with a few millilitres of toluene and pass the washings through the filter.Shake the filtrate for 3 minutes with 10 ml of 0.5 N hydrochloric acid and transfer the aqueous layer into a 6 x 1-inch tube; wash the toluene layer with a few millilitres of water and add the washings to the tube. Determination of p-nitrophenoZ-The reduced P-nitrophenol is coupled with o-cresol in presence of alkali to give an indophenol dye, which is determined colorimetrically.4 Add 1 ml of 2 per cent. o-cresol solution and 2 ml of ammonium hydroxide, sp.gr. 0-880, to the acid extract obtained after the reduction procedure. After 10 minutes pour back the whole of the solution into the separating funnel with the toluene used in the reduction; shake for 3 minutes, add 2 ml of 20 per cent. sodium hydroxide solution and finally shake for 1 minute. By this treatment the blue indophenol colour that develops if $-amino- phenol is present remains in the aqueous phase, and other compounds that are to be subsequently determined pass into the toluene layer.Run the aqueous layer into a 25-ml flask, and wash the toluene layer with a few millilitres of water. Make the contents up to volume and, 30 minutes after the addition of sodium hydroxide, read the optical density at 615 mp against a reagent blank. Determine the amount of nitrophenol present by com- paring the reading of the optical density with a calibration curve made from readings with standard solutions of nitrophenol subjected to the same procedure. Determination of S-ethyl isomer-Extract the original toluene layer with 10 ml of 0-5 N hydrochloric acid by shaking for 3 minutes in a separating funnel, run the lower layer into a tube and wash the toluene layer with a few millilitres of water.Add 1 ml of o-cresol solution and 2 ml of 20 per cent. sodium hydroxide solution to the acid solution. If the S-ethyl isomer is present a blue indophenol colour develops owing to hydrolysis of the reduced compound to p-aminophenol by the alkali. The colour reaches its maximum in about 45 minutes. After 1 hour transfer the solution back to the separating funnel containing the toluene, shake for 3 minutes, run the lower layer into a 25-1111 volumetric flask and make up to volume as before. Read the optical density at 615 mp against a reagent blank and determine the amount of S-ethylisomer present by comparing the reading with a calibration curve made for this component.Determination of parathion-Reduced parathion is not hydrolysed by the alkaline con- ditions in the previous stages and is determined by extracting the resulting toluene layer with 0.5 N hydrochloric acid and subjecting the acid layer to the diazotisa.tion and coupling procedure previously described.l Reduced parathion is not completely extracted from toluene by the procedure used in the previous stages and it is necessary to extract several times with dilute acid. Successively extract the toluene layer with 20, 20 and 10 ml of 0.5 N hydro- chloric acid; collect the aqueous layers in a 50-ml volumetric flask and make up to 50 ml. If the total amount of parathion present is known to be greater than 100 pg, add 1 ml of 0.25 per cent. sodium nitrite to 10 ml of this acid solution and, after 15 minutes, 2 ml of 1 per cent.N-sulphato-ethyl-m-toluidine and 4 ml of 30 per cent. w/v sodium acetate solution, After 15 minutes acidify the solution with 0-5 ml of concentrated hydrochloric acid, add 5ml of ethanol and make up to 25ml with distilled water. If the amount of parathion present is less than 100 pg, diazotise and couple the whole of the acid solution with five times the amount of reagents stated above, and after acidification extract the azo dye into analytical reagent isoamyl alcohol, with two successive portions of 10 and 5ml; pour these extracts into a 15-ml volumetric flask and make up with ethanol. With either method read the optical densities of the solutions a t 510 mp against a reagent blank and determine the amount of parathion present by comparing the reading with a calibration curve made for solutions of parathion subjected to the appropriate procedure AMOUNT TAKEN FOR ANALYSIS- For the investigation a Unicam DG spectrophotometer was used with 1-inch cylindrical cells. A suitable concentration range in the acid solution after reduction was found to beMarch, 19521 p-NITROPHENYL-O-S-DIETHYL THIOPHOSPHATE IN PARATHION 125 between 1 and 5 pg per ml for parathion and P-nitrophenol, and between 2 and 10 pg per ml for the S-ethyl isomer.When mixtures are being analysed, it may be that the concentration necessary for an adequate optical density from a minor component will involve an excessive concentration of a major component. It is possible to obtain an approximation to the amount of the major component by diluting its solution after colour development, but a more accurate procedure is to dilute the acid solution at the appropriate stage with 0.5 N hydrochloric acid before colour development and add proportionally larger amounts of reagents.After the completion of the reaction the whole solution is again extracted with toluene if a further stage is required, and the aqueous phase is diluted to the appropriate volume. RESULTS The S-ethyl isomer undergoes a slow hydrolysis in the ammonia solution used during the determination of p-nitrophenol and if much is present a high value for P-nitrophenol will be obtained. The extent of this has been shown to be of the order of 2 per cent. by repeating this stage of the estimation instead of passing on to the determination of S-ethyl isomer.A correction for this has been applied in the results given below. Samples of parathion and S-ethyl isomer have been kindly supplied by Albright and Wilson Ltd.; parathion had been purified by a chromatographic procedure and the S-ethyl isomer synthesised from P-nitrophenyl ethyl chlorophosphate and sodium ethyl mercaptide. The sample of parathion when received was found to contain 0.1 per cent. of p-nitrophenol and 0.4 per cent. of S-ethyl isomer; four months later the p-nitrophenol content had risen to 1-7 per cent. I am indebted to Mr. B. Topley for the method used by Albright and Wilson Ltd. to establish the purity of the sample of S-ethyl isomer. The elementary analyses for phosphorus and sulphur were correct, and the absence of a P = S group was demonstrated by the nitric acid oxidation r n e t h ~ d .~ The theoretical amount of p-nitrophenol could be obtained by alkaline hydrolysis, and when the extent of hydrolysis at a fixed pH value was measured at progressive time intervals, the calculated hydrolysis constant showed no variation. By the procedure described above the sample was found to contain 3.7 per cent. of $-nitro- phenol and 2.5 per cent. of parathion, or of substances with similar properties calculated as parathion. Mixed solutions of these two samples together with P-nitrophenol in toluene were prepared and analysed; Table I shows that the calculated results for three such mixtures agree well with those expected. TABLE I ANALYSES OF MIXTURES OF P-NITROPHENOL, S-ETHYL ISOMER AND PARATHION p-Nitrophenol S-Ethyl isomer Parathion - & & expected, found, expected, found, expected, found, mg per mg Per mi? Per mg Per mg Per mg per 100 ml 100 ml 100 ml 100 ml 100 ml 100 ml 0.86 0.98 1.12 1.08 9.80 9.76 0.207 0-235 1.52 1-37 5.2: 5-08 11.5 11-7 4.2 5.3 50.8 50.1 DISCUSSION The method described in this paper was that used by Diggle and Gage5 in their study of the inhibition in vitro of cholinestera-se by a variety of samples of parathion; inhibition was found to be proportional to the content of S-ethyl isomer in the samples.As the S-phenyl isomer of parathion, paroxon and bis-$-nitrophenyl ethyl thiophosphate behave in a manner similar to parathion in the procedure described above, they will, if present, be reported as parathion.bis-p-Nitrophenyl ethyl thiophosphate is stated to be present in the commercial parathion manufactured in this country, and some experimental work has been directed toward its determination. After reduction, bis-p-nitropher,yl ethyl thiophosphate gives an azo colour, as does parathion in the above procedure; if the acid solution of the reduced compound is made alkaline and heated it still produces an azo colour of about the same intensity, although it can be shown that the alkali treatment splits off P-aminophenol if zinc dust is added to prevent oxidation. I t is probable that the course126 GAGE [Vol. 77 of the reaction is as follows, and that the final ionised thiophosphoric acid derivative is stable to further alkaline hydrolysis- NO, 0 O/ \OC2H, N H 2 0 O/ \OC,H, When reduced parathion solution is heated with alkali, the greater part is hydrolysed to p-aminophenol, but in all samples examined the solution still gives an azo colour equivalent to about 10 per cent.of the parathion. If a solution of reduced S-ethyl isomer is heated with alkali the proportion apparently stable is about 25 per cent., although if the same sample is subjected to the complete analytical procedure described above, only a negligible trace of azo colour can be developed if the acid solution for the determination of parathion is heated with alkali. It seems likely, therefore, that with these two compounds t'he alkali not only attacks the nitrophenyl group but also the ethoxy or the ethylmercaptide group; this reaction would give an anion similar to that suggested for bis-$-nitrophenyl ethyl thiophosphate.When a solution of S-ethyl isomer is submitted to the analytical procedure described above, the optical density reading, if applied to a P-nitrophenol standard curve, is equivalent to about two-thirds of the calculated figure for combined P-nitrophenol. It is probable that a dual hydrolytic attack on the molecule occurs in the presence of alkali in the determination of S-ethyl isomer stage and forms, in part, an alkali-stable component, which does not give an indophenol colour and which, by virtue of the presence of an ionisable hydrogen atom, is not extracted into toluene from alkaline solution. It has been found that if at this stage o-cresol is not added to a sample of reduced S-ethyl isomer, an appreciable azo colour can be developed in the solution after it has been extracted with toluene. It has been shown that this is not due to the presence of a component with an appreciable partition between the two phases, as the intensity of the azo colour is not significantly changed if the volume of the toluene is increased five-fold. As the analytical results with the S-ethyl isomer are repro- ducible, it can be assumed that the ratio of the end-products is not influenced by normal variations in experimental conditions. REFERENCES 1. 2. 3. 4. 5. Gage, J . C., Analyst, 1950, 75, 189. Peck, D. R., Chem. and Ind., 1948, 526. British Objectives Sub-committee Report No. 1808, p. 9. Fancutt, F., and Twiselton, M. S. J., J . SOC. Clzem. Ind., 1943, 62, 205. Diggle, W. M., and Gage, J. C., Biochem. J., 1951, 49, 491. INDUSTRIAL HYGIENE RESEARCH LABORATORY IMPERIAL CHEMICAL INDUSTRIES LIMITED WELWYN, HERTS. First submitted, May, 1951 Amended, Sefxtember, 1951

ISSN:0003-2654    

DOI:10.1039/AN9527700123

出版商:RSC    

年代:1952

数据来源: RSC

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March, 19521 SHAW AND WILKINSON 127 The Colorimetric Estimation of Small Quantities of Prof lavine Hemisulphate BY W. €3. C. SHAW AND G. WILKINSON A method is described for the colorimetric estimation of proflavine hemisulphate (2 : 8-diaminoacridine hemisulphate) by conversion to the quinone-imine form of 2-aminoacridyl diazonium chloride. A proflavine solution is treated with a limited excess of nitrous acid, under controlled conditions of pH and temperature, and the excess of nitrous acid is subse- quently removed by sulphamic acid. Coupling in acid solution with N- ( 1-naphthy1)-ethylenediamine dihydrochloride then gives a stable purple colour. This procedure is an improvement on the use of the weak and un- stable purple colour of the quinone-imine itself, which has been previously proposed for the estimation of 2 : 8-diaminoacridines.The method detailed gives a rectilinear relation of optical densities and concentrations for quantities of proflavine hemisulphate up to 300 pg and it can be applied without modification to euflavine and acriflavine in similar amounts. Details are also given for the application of the method to pharmaceutical preparations. THE development of a purple colour by interaction of nitrous acid and 2 : 8-diaminoacridine (proflavine) was reported by Grandmougin and Smirous,l who attributed this effect to the formation of a quinone-imine- In alkaline solution this coupled with R-salt to give a red dye, whereas excess of nitrous acid was considered to lead at least to partial formation of the bis-diazo compound- SchulteZ has described a colorimetric method for the determination of acriflavine based on the development of a purple colour produced by addition of nitrous acid, but the method has the disadvantage that the colour fades gradually, even in the dark.The object of the work described here was to apply the reaction with nitrous acid to the estimation of small quantities of proflavine hemisul.phate with the elimination, if possible, of the disadvantages of Schulte's method. EXPERIMENTAL FORMATION OF QUINONE-IMINE- A standard solution containing the equivalent of 0.02 per cent. w/v of anhydrous pro- flavine hernisulphate was prepared from a sample of proflavine hemisulphate B.P. Suitable quantities of this solution were treated under different conditions with sodium nitrite solution, and the optical densities of the resultant solutions were measured on a Spekker absorptio- meter with a 1-cm cell and No.3 orange - yellow glass filters. These filters were selected because comparable amounts of both proflavine hemisulphate solution and of the completely decomposed quinone-imine gave readings close to zero, while the purple quinone-imine ex- exhibited appreciable absorption. Efect of concentration of nitrous acid-A series of solutions was prepared in 50-ml graduated flasks. Each solution contained 5 ml of 0-02 per cent. w/v proflavine hemisulphate solution diluted to about 43 ml. Five millilitres of 0.1 N hydrochloric acid were added to each flask to ensure a final (the optimum) acid concentration of 0.01 N and the solutions were cooled to 0" C.Various quantities of 0.1 N or 0.01 N sodium nitrite solution were128 SHAW AND WILKINSON : THE COLORIMETRIC ESTIMATION OF added and the solutions diluted to 50 ml. The values of the optical densities of the resulting solutions, measured at intervals from the time of mixing, are given in Fig. 1 and show the formation and decomposition of the quinone-imine colour. The optimum a.mount of nitrite was found to be contained in 1 ml of 0.01 N sodium nitrite solution. Efect of variation of pH-The development of colour with 0.01 N nitrite solution in the presence of various concentrations of hydrochloric acid was examined in a similar experi- ment. A series of curves like those shown in Fig. 1 led to the conclusion that 0.01 N was the most effective concentration of acid.[Vol. 77 I I 0.30 h -3 020 ,Q 0" 01 U - U 0.10 Y OOh tb io io i o ;o Qo 90 11 Time, minutes Fig. 1. Effect of concentration of nitrous acid Curve A, 0.6 ml of 0.01 N sodium nitrite; curve B, 1.0 ml of 0.01 N sodium nitrite; curve C, 0.5 ml of 0-1 N sodium nitrite; curve D, 1.0 ml of 0.1 N sodium nitrite; curve E, 2.0 ml of 0.1 N sodium nitrite E'ect of temperatztre-Experiments with 1 ml of 0.01 N sodium nitrite solution in the presence of 0.01 N hydrochloric acid at 0" and 10" C showed that development of colour was complete after 40 minutes and that there was no significant effect of temperature within this range. Efect of light-It was found that the purple diazotised solution, prepared. under the opti- mum conditions given above and exposed to direct sunlight or ultra-violet radiation faded rapidly to a fluorescent yellow colour, closely resembling that of unchanged proflavine hemi- sulphat e soh t ion.COUPLING OF QUINONE-IMINE- Since the results of the experiments up to this point showed that the quinone-imine alone did not provide a sufficiently stable colour, attention was given to the possibility of coupling with suitable compounds. To this end preliminary experiments were made with R-salt and with N-sulphato-ethyl-m-toluidine. The former, in sodium carbonate solution, gave a red colour, but it was found difficult to obtain reproducible results. The latter was even less satisfactory, as only weak colours were given in acid, acetate-buffered or alkaline solutions. A satisfactory coupling component was, however, found in N-( 1-naphthy1)-ethylene- diamir~e.~ This necessitated first removing from the diazotised solution the excess of nitrous acid by means of sulphamic acid. The components were then coupled to give a purple dye, the optical density of which was found suitable for the colorimetric estimation of quant- ities of proflavine hemisulphate up to 0.3 mg.In addition the colour from a given amount of proflavine hemisulphate was more intense than that of the corresponding uncoupled quinone-imine.March, 19521 SMALL QUANTITIES OF PROFLAVINE HEMISULPHATE 129 A typical calibration graph for the Spekker photo-electric absorptiometer with the method described below was a straight line passing through the origin ; the reproducibility for a series of test solutions is shown in Table I.TABLE I DETERMINATION OF PROFLAVINE HEMISULPHATE IN AQUEOUS SOLUTION BY THE PROPOSED METHOD Proflavine hemisulphate- Added, mg per 100ml . . 4.60 5-41 4.04 3.12 2.13 1-63 1.27 2.37 Found, mg per 100 ml . . 4.56 5-36 3.96 3.06 2.08 1-60 1-38 2.30 The transmission curves of proflavine hemisulphate, of the quinone-imine f o m of 2- amino-acridyl diazonium chloride derived from the proflavine hemisulphate after diazotisation and of the dye obtained by coupling the quinone-imine form of 2-aminoacridyl diazonium chloride with N-( 1-naphthy1)-ethylenediamine dihydrochloride are shown in Fig. 2. DISCUSSION Temperature, pH, light and concentration of nitrous acid have all been shown to affect both the rate of formation of the quinone-imine from 2 : 8-diaminoacridine and its subsequent stability.To secure maximum stability the excess of nitrous acid and the acidity must each be reduced to the least value consistent with a reasonable rate of reaction. Rise of temperature increases the rate of formation and decreases the stability; light must be excluded. Optimum conditions are therefore somewhat arbitrary and those selected must be rigidly controlled to ensure reproducible results. The observed deterioration in colour with excess of nitrite and low pH may be caused by the partial formation of the yellow acridine-2 :&bis- diazonium chloride, which is known to be formed in sulphuric acid solution on treatment with sodium nitrite.l Although Schulte2 measured the intensity of the colour of the quinone-imine compound itself, it has been found that the inherent instability of the quinone-imine, and particularly its susceptibility to decomposition on exposure to the light required for the measurement of its intensity, make it unsatisfactory for estimation.A stable red colour was obtained on coupling with R-salt (2-naphthol-3 : 6-sodium disulphonate) in sodium carbonate solution. Results by this method, however, were erratic, probably because of the decomposition of the diazonium compound by local excesses of carbonate on first mixing the solutions and before complete coupling had taken place. After removal of the excess of nitrite, the diazonium chloride couples with N-( l-naphthy1)- ethylenediamine dihydrochloride to form a purple dye stable within the pH range 1 to 2.The intensity of the colour produced in this way by a given weight of proflavine hemisulphate is greater than that of the original quinone-imine colour and so gives rise to an over-all increase in sensitivity. The proposed method gives a linear calibration graph within the range 0 to 300 pg of proflavine hemisulphate. METHOD REAGENTS- be stored in a refrigerator and discarded when yellow. be used within 14 days of preparation. hemisulphate (C13H11N3),H,S0,, which must be stored in the dark. N-(l-~aj5hthyE)-ethylenediamine dihydrochloride-A 0.1 per cent. w/v solution, which must Szzlphamic acid (or ammonium su1phamate)-A 0.5 per cent. w/v solution, which must Sodium nitrite-A freshly prepared 0.01 N solution. ProJlavine hemisulphate-A standard solution containing 0-02 per cent.w/v of proflavine PROCEDURE- Place in a 100-ml graduated flask an aqueous solution of the material under test contain- ing up to 300 pg of anhydrous proflavine hemisulphate. Add 5.0 ml of 0.1 N hydrochloric acid and make up with water to 50 ml. Cool the solution to 10" & 2" C, add 1.0 ml of 0.01 N130 SHAW AND WILKINSON THE COLORIMETRIC ESTIMATION OF [Vol. 77 sodium nitrite solution, mix thoroughly and maintain at 10" 2" C for 40 minutes in a water- bath, protecting the solution from light throughout this period. Subsequent operations (until coupling has taken place) must be carried out in subdued light. After 40 minutes add 1.0ml of sulphamic acid solution, mix thoroughly and allow to stand for 1 minute. Add 2.0 ml of N-( 1-naphthy1)-ethylenediamine dihydrochloride solution, mix thoroughly and allow to stand for 5'minutes.Add 5-Om1 of 0.1 N hydrochloric acid Wavelength, my Fig. 2. Transmission curves Curve A, proflavine hemisulphate, 2 mg in 50ml of hydrochloric acid (l.O-cm cell) ; curve B, quinone-imine form of 2-aminoacridyl diazonium chloride derived from the proflavine hemisulphate after diazotisation with 1.0 ml of 0.01 N sodium nitrite (1-0-cm cell) ; curve C, dye obtained by coupling 25 ml of the quinone- imine form of 2-aminoacridyl diazonium chloride with 2 ml of 0.1 per cent. N- (1-naphthy1)-ethylenediamine dihydrochloride, diluted to 50 ml (0.5-cm cell). I 0.01 N and make up to 100 ml with water. Prepare similarly a blank solution without the sample under test.Measure the optical density of the solution in a 1-cm cell by comparison with the blank, using a Spekker photo-electric absorptiometer and Ilford 605 filters, or another suitable combination of absorptiometer and filters. By the same method arid with suitable dilutions of standard proflavine prepare a calibration graph covering the required range and read from the graph the amount of proflavine hemisulphate contained in the test solution. APPLICATIONS ACRI FLAVI NE , E UFLAVI NE- The method is applicable without modification to both these compounds, which give a linear response with quantities of either up to 300 pg. Small deviations in calibration with different batches of both' compounds may be anticipated, owing to the varying proportions of methylated and unmethylated proflavine present.March, 19.521 SMALL QUANTITIES OF PROFLAVINE HEMISULPHATE 131 Under the conditions of assay no reaction appears to occur' between aminacrine and AMINACRINE HYDROCHLORIDE (5-AMINOACRID1NE)- nitrous acid, so that the method is inapplicable.PREPARATIONS CONTAINING PROFLAVINE HEMISULPHATE- pessary, solution and solution tablets. The method can be applied directly to the following B.P.C. 1949 preparations; eye-drops, Typical results on pessaries are shown in Table 11. TABLE I1 ESTIMATION O F PROFLAVINE HEMISULPHATE IN A PESSARY MASS CONTAINING 0.209 PER CENT. W/W OF THE ANHYDROUS FLAVINE PREPARED ACCORDING TO B.P.C. 1949 Weight taken, c g 0.0486 0.1334 0-1259 0.0675 0.1219 Proflavine content P :alculated, found, mg mg 0.1017 0.100 0,2793 0-270 0.2636 0.260 0,1413 0.139 0.255 0.249 Anhydrous proflavine, 0.206 0.202 0.207 0.206 0.204 % w/w Difficulty is sometimes experienced in recovering the last traces of proflavine in the presence of fatty matter and extraction with an immiscible solvent may be necessary.Ethyl- ene dichloride is preferable to other common organic solvents and losses of proflavine can be reduced to a minimum by maintaining the aqueous phase at an acidity of about 0.1 N with hydrochloric acid. Table I11 shows the results obtained, by the method outlined below, for a sample of proflavine hemisulphate cream B.P.C. containing 0.1 16 per cent. w/w of anhydrous proflavine hemisulphate. PROCEDURE- Weigh accurately 0.1 to 0.2g of the sample into a boiling tube provided with a lip.Warm on a water-bath, dissolve in 2 ml of ethylene dichloride, add 5-0 ml of 0.1 N hydro- chloric acid and maintain at a temperature of about 50" C for 5 minutes, with occasional TABLE I11 PROFLAVINE HEMISULPHATE IN PROFLAVINE HEMISULPHATE CREAM B.P.C." Proflavine hemisulphate - g mi3 mg 0.1178 0.1367 0.133 0-1851 0.2 146 0.207 0-1501 0.1741 0-168 0.1 149 0.133 0.131 0-1543 0.1 789 0.174 Weight taken, calculated, found, Anhydrous proflavine hemisulphate, 0.1 13 0.1 12 0.1 12 0.1 14 0.1 13 % w/w * Proflavine hemisulphate cream B.P.C. containing 0.116 per cent. w/w of active agent. shaking. Transfer the contents to a 15-ml graduated centrifuge tube and add more ethylene dichloride if necessary; wash the boiling tube with two successive 2-ml quantities of warm water, and add the washings to the centrifuge tube.Close the tube by means of a well-fitting rubber stopper and shake vigorously for 30 to 60 seconds. Remove the stopper and rinse any adhering solution back into the original boiling tube. Centrifuge for 5 to 10 minutes at 2500r.p.m. or until a clear upper layer is obtained. By means of a Pasteur pipette, transfer the bulk of the aqueous layer to a 100-ml volumetric flask, rinse the pipette with two portions of distilled water and add the washings to the flask. Wash the lower layer remaining in the centrifuge tube with the rinsings from the boiling tube, using a total of 8 ml132 SCANDRETT : A MODIFIED MICRO-DIFFUSION METHOD FOR [Vol. 77 of warm water. Shake, centrifuge and transfer the upper layer to the flask as before, and repeat the washing procedure. Dilute the contents of the flask to 50 ml and complete the determinations by the general method, as described on p. 129. The above procedure is applicable to ointments with a water-miscible base. The mean error, approximately -3 per cent. (see Table 111), is small and the method is regarded as sufficiently accurate for routine estimations. REFERENCES 1. 2. 3. Grandmougin, E., and Smirous, K., Ber., 1913, 46, 3427. Schulte, M. J., Pharm. Weekblad, 1930, 65, 809. Bratton, A. C., and Marshall, E. K., Jun., J . Biol. Chem., 1939, 128, 537. PHARMACEUTICAL RESEARCH AND SERVICE LABORATORIES IMPERIAL CHEMICAL INDUSTRIES LIMITED HEXAGON HOUSE, BLACKLEY, MANCHESTER July, 1951

ISSN:0003-2654    

DOI:10.1039/AN9527700127

出版商:RSC    

年代:1952

数据来源: RSC

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132 SCANDRETT : A MODIFIED MICRO-DIFFUSION METHOD FOR [Vol. 77 A Modified Micro-Diffusion Method for the Determination of Ethyl Alcohol in Blood BY F. J. SCANDRETT A method is described for the micro-determination of ethyl al.coho1 in blood or urine by means of a new micro-diffusion procedure that gives results in 30 minutes over the range of 80 to 300 p g per 0.1 ml of blood or urine with an accuracy of better than -1-3 per cent. BAHNER,~ working in this laboratory, has devised a convenient micro-diff usion apparatus that permits the separate temperature control of the two chambers. The apparatus, illustrated in Fig. 1, can be applied to many analytical methods in which diffusion at a uniform tempera- ture is slow or incomplete. The lower vessel, which contains the sample for determination, has a flat base with a surface area of 2-5 sq.cm and is attached to the chimney part ofthe “mushroom” by a B19 standard joint. The “mushroom” is surrounded by a simple form of condenser that allows water at 50” C to circulate over and under it. The flat base of the “mushroom,” which has a surface of 10.0 sq. cm, contains the dichromate - sulphuric acid mixture. The first use made of this apparatus, apart from that by Bahner who designed it for the determination of acetone and “ketone bodies” in blood, was in the determination of ammonia (Scandrett, unpublished). By the boric - hydrochloric acid procedure, full recov- eries were regularly obtained in 10 minutes with a solution containing 112 pg of ammonia (NH,) per 0.5 ml, which demonstrated the efficiency of the micro-diffusion in this apparatus.EXPERIMENTAL Preliminary observations indicated that, if the apparatus was to be successfully used for the determination of ethyl alcohol in blood, certain limiting factors would have to be considered and, if necessary, modified to meet the requirements of the unit. The concentrations of potassium dichromate and sulphuric acid appeared to be critical, especially that of the sulphuric acid, as incomplete absorption and oxidation resulted if the concentration of sulphuric acid fell below 50 per cent. As the iodine titration depends on the pH of the solution (the optimum value is about pH 1.4), which involves a considerable dilution immediately before titration, and as the capacity of the “mushroom” is limited, only a small quantity, 1.0m1, of original reagent could be used.This quantity was found just to cover the base of the “mushroom,” which from the point of view of absorption is ideal, since the rate of absorption is inversely pro- portional to the volume (Conway2).March, 19521 THE DETERMINATION OF ETHYL ALCOHOL IN BLOOD 133 Preliminary experiments with 1.0 ml of dichromate - sulphuric acid mixture indicated that the absorption and oxidation is complete in 30 minutes, as against 2 hours by the Wid- mark r n e t h ~ d . ~ A 0.005 N solution of sodium thiosulphate is used for alcohol concentrations up to 200 mg per 100 ml; for higher concentrations, i.e., between 200 and 500 mg per 100 ml, 0.01 N sodium thiosulphate is used. : , . . . . . . . . Fig. 1. View and sections of apparatus -4, lower vessel; B, “mushroom” receiver; C, rubber bung; D, condenser jacket From a consideration of the above conditions and a large number of trials it was evident that the quantities and concentrations of reagents used by Widrnark3 were the most suitable, and this was proved by subsequent experiments. METHOD REAGENTS- used for blood containing less than 200 mg of ethyl alcohol per 100 ml.used for blood containing between 200 and 500 mg of ethyl alcohol per 100 ml. Potassium dichromate, 0.02 N , in concentrated sulphuric acid-This concentration is Potassium dichromate, 0.05 N , in concentrated sulphuric acid-This concentration is Potassium iodide-A 5 per cent. solution. Sodium thiosulphate-0.01 N and 0.005 N solutions. Starch-A 1 per cent.solution. All reagents should be of recognised analytical quality. With a Bang burette, which is available in most analytical laboratories, the strengths of the sodium thiosulphate solutions can be reduced. This was not done in the investigation described here.134 SCANDRETT : A MODIFIED MICRO-DIFFUSION METHOD FOR [Vol. 77 PROCEDURE- Transfer 1.0 ml of the dichromate - sulphuric acid mixture to the inverted “mushroom” by means of a 1.0-ml slow-delivery bulb pipette. I t is advisable to blow the liquid out and to touch the surface of the dichromate - sulphuric acid mixture with the tip six times, to hold the “mushroom” against a white background and to watch the introduction of the liquid and the withdrawal of the pipette by looking down the chimney.Preliminary experi- ments with this procedure for delivery of 1.0 ml of dichromate - sulphuric acid mixture showed that the average error of ten such measurements is less than 1 per cent. Using pipettes, place in the bottom vessel 0.2 ml of water and 0.1 ml of blood, the 0.1-ml blood pipette being rinsed out twice and the water and blood thoroughly mixed. Invert the “mushroom” and, with a cork-screw motion, attach the bottom vessel tightly to the chimney, TABLE I RECOVERY OF ETHYL ALCOHOL ADDED TO FRESHLY WITHDRAWN CITRATED BLOOD Additions made as aldehyde-free ethyl alcohol iri distilled water Alcohol per 0.1 ml of blood Standard r \ deviation Added, Found, Mean, of mean 80.0 78.0, 83.0, 80.8, 82.0, 82.0, 84.2, 80.3 - + 2.97 160-0 161.5, 158.0, 161.6, 160.4, 159.2, 162.0 160.4 5 1.56 300.0 300.0, 305.0, 300.0, 300.0, 305.0, 305.0 302.5 & 2.70 Blanks at each level were: 0 pg added, 0 pg found.A P8 Pg 80-8, 76.8, 77.5, 76.3, 77.5, 84-6 TABLE I1 RECOVERY OF ETHYL ALCOHOL ADDED TO FRESHLY VOIDED URINE MADE ALKALINE WITH SODIUM HYDROXIDE Alcohol per 0.1 ml of urine Standard f \ deviation Added, Found, Mean, of mean 300.0 301.7, 299.6, 296.0, 301-7, 304-0, 298.5 & 2.80 Blank: 0 pg added, 0 pg found. A PE: Pg CLQ 298.0, 294.0, 296.0, 296.0, 298.4 taking care to see that the joints are perfectly dry. Suspend the bottom vessel, held in position in this way, in a bath of boiling water almost up to the joint, while the “mushroom,” enclosed by the condenser, has water at 50” C flowing over and under it. The rate of flow through the condenser may conveniently be 1 litre every 10 minutes, but this is not critical.After 30 minutes disconnect the unit, drain the condenser of water and take it off. Disconnect the bottom vessel, invert the “mushroom” and cool it under the tap. Add 25.0 rnl of water to the “mushroom,” again cool (preferably in ice) and add 0-5ml of 5 per cent. potassium iodide before titrating with sodium thiosulphate in a 5-0-ml micro-burette. Experiments were also made to determine the effect of added water on the rate of absorption and oxidation by the dichromate - sulphuric acid mixture. The following figures show that additions of water did not affect the acid mixture, as full recoveries in each experiment were obtained at the end of 30 minutes. To 1-0-ml portions of dichromate - sulphuric acid mixture were added 0.1, 0.2 or 0.3ml of water.The amounts of alcohol found in 0-1-ml portions of blood containing 80.0 pg of ethyl alcohol per 0.1 ml, when acted upon by these solutions, were 7843, 79.3 and 80-0 pg, respectively. It was not possible to use 0.2 ml of blood, as recoveries were always poor, probably because the heat-coagulated protein formed too great a barrier for the free diffusion of the alcohol vapour through the mixture. The “mushroom” easily holds 25.0ml of water and the constricted opening and the chimney are advantageous in iodimetric titrations.March, 19521 THE DETERMINATIOIS OF ETHYL ALCOHOL I N BLOOD 135 RESULTS AND DISCUSSION Accurate results were obtained throughout the range of 80 to 300 pg of alcohol per 0.1 ml of blood and urine, as shown in Tables I and 11, the standard deviation being less than Ifr 3.0 pg per 0.1 ml of blood.The method reduces the time for a single diffusion to 30 minutes, as against 2 hours with the Widmark3 and W i n n i ~ k ~ ~ ~ 9 ~ methods. A large number of determinations can be carried out simultaneously. Further advantages are that, as a result of the large excess of water in the “mushroom” receiver, there is no possibility of losing the liberated iodine, the end-point is not so abrupt and the subsequent titration and shaking are, therefore, easy to control and manipulate. By increasing the temperature gradient over which absorption takes place, it is possible to increase the range over which diffusion techniques can be used, so that the versatility of the apparatus is increased. Experiments made with blood containing 300 mg of alcohol per 100 ml, stored at room temperature for one week, did not show any diminution of alcohol concentration. Normal blood used as a control and kept under the same conditions did not give a “blank.” To Dr. C. P. Stewart and Dr. F. W. R. Bahner, who have given me advice and helpful criticism, I wish to tender my thanks. REFERENCES 1. 2. 3. 4. 5 . 6. Bahner, F. W. R., personal communication. Conway, E. J., “Micro-Diffusion Analysis and Volumetric Error,” Third Edition, Crosby Lock wood & Son, Ltd., London, 1951. Widmark, E. M. P., “Die theoretischen Grundlagen und die praktische Verwendbarkeit der gerichtlichmedizinischen Alkoholbestimmung,” Urban & Schwarzenberg, Berlin-Wien, 1932. Winnick, T., J. Biol. Chem., 1941, 141, 115. - , Ibid., 1942, 142, 451; 461. - , Ind. Eng. Chem., Anal. Ed., 1942, 14, 523. DEPARTMENT OF CLINICAL CHEMISTRY ROYAL INFIRMARY EDINBURGH June, 1961

ISSN:0003-2654    

DOI:10.1039/AN9527700132

出版商:RSC    

年代:1952

数据来源: RSC

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March, 19521 THE DETERMINATIOIS OF ETHYL ALCOHOL IN BLOOD 135 The Isolation, Identification and Determination of Amphetamine in Viscera BY E. RATHENASINKAM* A scheme is described for the isolation, identification and determination of amphetamine. By combining the Stas-Otto process with a steam distilla- tion method a purer isolation product is obtained than formerly. A number of methods of identification are given and the amount of the alkaloid present is determined by precipitating it as amphetamine oxalate. A comparison of the results obtained by determining the amphetamine by this method with those obtained by a volumetric method is shown. METHOD OF ISOLATION AMPHETAMINE can be isolated from viscera either by the Stas-Otto process or by steam- distillation in an alkaline medium.Both amphetamine and its. acetate are volatile, so viscera should be extracted with alcohol acidified with tartaric acid. Steam-distillation of viscera in an alkaline medium is not possible as excessive frothing ensues. The isolation of amphetamine from viscera is best effected by a combination of the two methods. Steam- distillation has the advantage of giving a purer product than that obtained by extraction with immiscible solvents in the Stas-Otto process. Procedzcre-Extract the minced organs thoroughly with alcohol acidified with tartaric acid. Evaporate the alcohol from the combined alcoholic extracts, take up the residue *Temporary address: 28, West Cromwell Road, London, S.W.5.136 RATHENASINKAM THE ISOLATION, IDENTIFICATION AND [Vol. 77 in about 100 ml of water and transfer to a wide-mouthed distillation flask.Add sufficient sodium chloride to saturate the liquid, make alkaline to litmus with sodium hydroxide solution and steam-distil. Distil about 400 ml into a receiver containing 50 ml of 0.5 N hydrochloric acid. Evaporate the distillate to a small bulk and make up to a definite volume. Discard the ether extract. Make alkaline to litmus with sodium hydroxide solution and extract twice with chloroform. Wash the combined chloroform extracts with a little water and shake the chloroform solution with 10 ml of 0.1 N hydrochloric acid. Draw off the acid layer and evaporate to dryness. The tests for amphetamine are made on the residue obtained. Take an aliquot portion and extract once with ether. METHODS OF IDENTIFICATION Amphetamine gives the general reactions for alkaloids with Kraut's, Wagner's and M ayer ' s reagents.COLOUR TESTS- (a) With Marquis's reagent amphetamine gives a brick-red precipitate changing to brown and finally to a dirty olive-green. Adrenaline gives a brown to reddish-brown colour changing to a dirty violet. The violet tinge is first observed around the edge of the drop. Ephedrine gives only a light brown colour. (b) In Mohler's test as modified by Illingl amphetamine gives a purple colour. Ephedrine and adrenaline under the same conditions give a deep yellow and a slight yellow colour respectively. (c) Beyer and Skinner2 devised a colour test to determine amphetamine in urine during their investigation on the detoxication and excretion of amphetamine. On coupling amphet- amine with diazotised p-nitroaniline a red colour is given that can be intensified by extraction with n-butanol.(d) A further test is given by the colour reaction3 of the nitro-compounds formed by the nitration of amphetamine. Procedure-Nitrate the alkaloid as in Mohler's test. Dilute the product with water and extract once with chloroform. Discard the chloroform extract. Make the aqueous layer alkaline to litmus with ammonium hydroxide solution and extract once with chloroform. Evaporate the chloroform extract to dryness. Dissolve the residue in 1 to 2 ml of acetone, transfer to a test tube, add 1 to 2 drops of a 10 per cent. solution of sodium hydroxide and mix well. Amphetamine gives a purple colour changing to violet.The purple colour develops slowly. Ephedrine under the same conditions gives an immediate violet colour changing to magenta. Adrenaline gives no colour. The test will detect 0.1 mg of amphetamine. DERIVATIVES- determined. The benzoyl derivative4 (m.p. 134" to 135" C) may be prepared and its melting-point MICROCHEMICAL TESTS- (a) In the test described by Cannon5 a 1 per cent. solution of amphetamine in dilute sulphuric acid was used; more dilute solutions (0-2 per cent.) may be used. with a slightly modified technique. Procedwe-Evaporate on a microscope slide a drop of a solution of amphetamine hydro- chloride in water. To the residue add half a drop of dilute sulphuric acid and a drop of 5 per cent. platinic chloride solution, mix and set aside for a few minutes.At. first, interlaced needles form around the edge of the drop. The crystals gradually spread out in the shape of a holly leaf. (b) In the test described by Keenad about 1 mg of amphetamine sulphate was used. Smaller amounts of amphetamine can be made to react satisfactorily by reducing to a fraction of a drop the amount of gold reagent used. Procedure-Evaporate a drop of a 0.2 per cent. solution of amphetamine hydrochloride in water on a microscope slide. To the residue, when cold, add a fraction of a drop of 5 per cent. gold chloride solution and stir vigorously with a glass rod till crystals begin to appear. Square yellow crystals of various sizes are formed.March, 19521 DETERMINATION OF AMPHETAMINE I N VISCERA METHODS FOR THE DETERMINATION OF AMPHETAMINE 137 VOLW METRIC DETERMINATION- Ether is used for extracting amphetamine in the B.P.(1948) method for the assay of amphetamine sulphate; Reznek' has shown that instead chloroform can be used to advantage. The chloroform extracts are then shaken with an excess of standard sulphuric acid, the excess of acid being titrated, with methyl red as indicator. 1 ml of 0.02 N acid = 2.70 mg of amphetamine or 3-68 mg of amphetamine sulphate. GRAVIMETRIC DETERMINATION- The determination can be made by benzoylation5Ts or by precipitation as oxalate. On adding a solution of oxalic acid in ether to a solution of 0.75 mg of amphetamine in 100 ml of ether, amphetamine oxalate is precipitated after a few hours; the amphetamine oxalate is almost insoluble in ether. This is the basis of a new gravimetric method for the determination of amphetamine.A number of determinations were made with various amounts of amphetamine and it was found that the weight of the oxalate precipitate was always greater than the theoretical value, assuming that two molecules of amphetamine react with one molecule of oxalic acid. The ratio of the theoretical weight to the weight found was on average 4:4.94. It was thought that the difference between the weight found and the theoretical value was caused by occlusion of oxalic acid by the oxalate precipitate. The oxalate precipitate, after drying and weighing, was dissolved in water; the free acid present was determined by titration with standard sodium hydroxide solution. The weight of free acid, calculated as anhydrous oxalic acid, was always about one-fifth of the weight of the precipitate; the average value obtained was 0-192.From these observations it was inferred that amphetamine is precipitated as the acid oxalate and not as the normal oxalate. Procedwe-To an aliquot portion of the distillate, containing about 50 mg of amphet- amine, add sufficient sodium chloride to saturate the liquid, make alkaline to litmus with sodium hydroxide solution and extract three times with 30-ml portions of ether. Wash the combined ether extracts with 15 ml of a saturated salt solution and filter through a layer of anhydrous sodium sulphate placed over a plug of cotton, into a glass-stoppered conical flask. Extract the wash solution twice with ether and pass the ether through the same filter into the flask.Add 10ml of a saturated solution of oxalic acid in ether, shake occasionally and put the flask and contents aside overnight. Filter the solution rapidly through a sintered-glass crucible with suction, wash the precipitate twice with 25-ml portions of ether, dry at 100" C and weigh the oxalate. Weight of precipitate x 0.6002 = Weight of amphetamine. Weight of precipitate x 0.8180 = Weight of amphetamine sulphate. The ether used to prepare the oxalic acid solution and to wash the precipitate should be dried over anhydrous sodium sulphate. Visceral matter on steam distillation in an alkaline medium gives small quantities of volatile basic substances that would be estimated as amphetamine in the volumetric method described, which is accordingly not applicable to distillates obtained from viscera.Amphet- amine, if present in weighable amounts, can be determined by the benzoylation or oxalate method. COLORIMETRIC METHODS FOR THE DETERMINATION OF TRACES OF AMPHETAMINE- Illing'sl modification of Mohler's test is capable of determining amounts of amphetamine of the order of 1 mg. The diazotisation method of McNally, Bergman and POllig is based on the colour reaction devised by Beyer and Skinner.3 Amphetamine is isolated by steam-distilling a sulphuric acid - tungstic acid filtrate of the minced organs in an alkaline medium. Amphetamine is extracted from the distillate with ether and the ether extract is shaken with three successive portions of 0-5 N hydrochloric acid. The combined acid extracts are evaporated to dryness.The residue is taken up in 1 ml of water and coupled with diazotised 9-nitroaniline. The transmission is measured on a spectrophotometer, at 530 mp, and the amount of amphetamine read from a standard graph. The method is capable of estimating an amount of amphetamine as small as 0.03 mg in 25 g of tissue. The red colour formed is extracted with n-butanol.138 RATHENASINKAM [Vol. 77 RESULTS A stock solution of amphetamine sulphate was prepared to contain 49.3 mg of amphet- Some experimental results are shown in Table I. amine sulphate in 25ml of the solution. TABLE I COMPARISON OF RESULTS BY THE PROPOSED METHOD AND THE VOLUMETRIC METHOD Amphetamine found , calculated as amphetamine sulphate By volumetric By oxalate method, method, mg mg 49.3 - 49-3 -L - 48.5 48.1 48.7 A f \ 48.1 47-0 48.0 47-0 48.6 45.4 45.8 42.5 46.9 43.2 2-3 No ppt.Details of procedure with 25-ml portions of stock solution containing 49.3 mg of amphetamine sulphate in each Extracted with chloroform Extracted with ether and precipitated as oxalate. The oxalate precipitate was dried, weighed and dissolved in water ; the amphetamine was re-extracted with chloroform and deter- mined volumetrically Steam distilled and titrated. Amphetamine was re-extracted with ether from the titrated solution and precipitated as the oxalate 1OOg of liver were extracted with alcohol; the alcohol was evaporated and the residue was taken up in 75 ml of water. Stock solution was added to this and steam distilled. Amphet- amine was re-extracted with ether from the titrated solution and precipitated as oxalate Stock solution was added to 100 g of liver, which was cut into small pieces and extracted twice with alcohol (extraction was not complete). Amphetamine was re-extracted with ether from the titrated solution and precipitated as oxalate Blank on 100 g of liver calculated as amphetamine sulphate 1. 2. 3. 4. 5. 6. 7. 8. 9. REFERENCES Illing, E. T., Analyst, 1940, 65, 3. Beyer, H. K., and Skinner, J. T., J . Pharmacol., 1940, 68, 419. Rathenasinkam, E., Analyst, 1951, 76, 115. British Pharmacopoeia, 1948, p. 60. Cannon, J. H., J . Ass. O f . Agric. Chem., 1941, 24, 803. Kennan, G. L., Ibid., 1942, 25, 830. Reznek, S., Ibid., 1942, 25, 746. Cannon, J. H., Ibid., 1942, 25, 788. McNally, W. D., Bergman, W. L., and Polli, J. F., J . Lab. CZin. Med., 1947, 32, 913; abstracted in Analyst, 1948, 73, 345. GOVERNMENT ANALYST’S LABORATORY COLOMBO, CEYLON August, 1951

ISSN:0003-2654    

DOI:10.1039/AN9527700135

出版商:RSC    

年代:1952

数据来源: RSC