ment is necessary for the proper use of the technique, because microquantities of ~4-3-kctones not definitely detected with the weak ethanolic reagent may be detected n-ith the more concentrated spray. These techniqucs have been of great value in studies of the microbiological dehydrogenation of A4-3-ketones.
LITERATURE CITED
(1) Bodhszky, A,, Kollonitsch, J., Nature 17.5, 729 (1955). (2) Bush, 1.E., Biochem. J . 50,370 (1951). (3) Bush, I. E., Nuture 166, 445 (1950).
(4) de Courcy, Constance, Bush, I. E., Gray, C. H., Lunnon, J. B., J . Endocra'nol. 9, 401 (1953). (5) Dorfman, Louis, Chem. Rev. 53, 47 . (1953). (6) ,Ercoli, A., de Giuseppe, L., de Ruggieri, P., Farm. sci. e tee. (Puvia) 7, 170 '
(1952'1. ACKNOWLEDGMENT
The authors wish to acknowledge the capable assistance of Barbara Laird in performing paper chromatographic separations, and of Walter H. Muller and Irene Palestro in determining the absorption spectra of the steroids studied.
( 7 j Gray, C. H., Green, hI. A. S., Holness, X. J., Lunnon, J. B., Lancet 269, 1067 (1955). (8) Grav, C. H., Green, hI. A. S., Holness, K. J.,"Lunnon, J. B., J . Endocrinol. 14, 146 (1956). (9),Haines, W. J., Drake, PI;. A., Federatzon Proc. 9, 180 (1950). (10) JaoudB, F. A., Kaulieu, E. E., Jayle, hl. F., Acta Endocrinol. 26, 30 (1957).
(11) Reich, Hans, Nelson, D. H., Zaffaroni, Alejandro, J . Biol. Chem. 187, 411 (1950). (12) Schriefers, Herbert, Korus, Wolf-
gang, Dirscherl, Wilhelm, Acta Endocrinol. 26, 331 (1957). (13) Szent-Gyorgi, A, Science, 126, 751 (1957). (14) Szpilfogel, S. A,, Van Hemert, P. A., De Winter, hl. S., Rec. trav. chim. 75, 1227 (1956). (15) Umberger, E. J., ANAL.CHEM.27, 768 (1965). (16) Vermeulen, A., Acta Endocrinol. 23, 113 (1956). (17) Vermeulen, A,, J . Clin. Endocrinol. and Metabolism 16, 163 (1956). (18) von Arx, E., Seher, R., Hela. Chitn. Acta 39,1664 (1956). (19) Teichselbaum, T. E., bfargraf, H. W.,J . Clin. Endocrinol. and Metabolism 17, 959 (1957). RECEIVEDfor reviem April 19, 1958. Accepted August 21, 1958.
Determination of Fluoride in Blood Serum LEON SINGER and W. D. ARMSTRONG Department of Physiological Chemistry, University o f Minnesota, Minneapolis, Minn. Existing methods for fluoride determination have been modified and scaled downward to permit the accurate determination of fluoride in 5 ml. of serum. The sample is ashed under prescribed conditions with specially prepared magnesium oxide. Use of nitrogen gas to sweep the fluoride from the microstill into the receiver markedly reduces the volume of distillate. Each step in the procedure has been evaluated b y the use of radioactive fluoride. The colorimetric method applied to the distillate is compensated for chloride and is applicable in the range from 0.0 to 3.0 y of fluoride.
I
of the biochemistry and toxicity of fluoride JTould be aided by reliable methods for the determination of fluoride in volumes of blood which can be obtained easily and repeatedly from the human. I n this work interest has centered on the fluoride content of plasma (or serum) rather than on that of whole blood employed previously (14). Concentrations of other electrolytes-e.g., chloride, bicarbonate, metal ions, etc.are usually determined in plasma because of their unequal distribution on a volume basis between plasma and red cells. Experiments (3) with radiofluoride as a tracer shon-ed that fluoride rapidly exchanges between plasma and red cells and has a concentration per unit volume in red cells which is about KVESTIGATIONS
30 to 35% that in plasma. Also plasma is a component of extracellular fluid and is the fluid of physiological interest because it bathes cells and bone mineral and is the fluid from which urine is formed. Finally, through the use of plasma (as serum) the destruction by ashing of a large amount of organic material is avoided. The Willard and Winter method (17) for the separation of fluoride by steam distillation from interfering substances has been improved in important particulars (1, d, 5-7, 11, I d ) . Further refinement is required to make it applicable in the range 0.0 t o 2.0 y of fluoride in a complex biological sample. The distillation blank obtained with the Killard and Winter apparatus including many of its modifications is of marked disadvantage when the amount of fluoride is in the microgram range, particularly because this blank is frequently variable. The large volume of aqueous distillate required for complete recovery of the fluoride with most of the forms of apparatus previously described results in a very lorn concentration of fluoride in the distillate, and when the total amount is 2 y or less, its determination is exceedingly difficult. Evaporation of distillates containing small amounts of fluoride to lorr volumes has, in this laboratory, given unreliable results unless platinum vessels are employed. A method of proved reliability for fixing the fluoride during ashing is needed with biological samples. This
step is critical, because variable losses of fluoride, or contamination, may occur. An important requirement is to use a fixative (magnesium oxide) of lorn fluoride content, so that its fluoride contribution will not be large in proportion to that of the sample. Titrimetric and colorimetric methods for the determination of fluoride in solution after separation from the sample have been described. Some methods lack the required sensitivity or precision and others are too sensitive to interferences. Megregian (9) described a spectrophotometric method, based upon the fading by fluoride of the color of a zirconium-Eriochrome Cyanine R lake, m-hich has a low order of sensitivity to foreign ions and appeared adaptable to the present requirements. Kielsen (10) has also adapted the method to a range of fluoride concentrations lower than that originally employed. COLORIMETRIC ANALYSIS
OF
DISTILLATE
The method of Megregian (9) has been modified as to the use of a combined solution of dye and zirconium (reagent-lake), and the hydrochloric acid content of this solution has been reduced to produce a concentration of 0 . 3 s acid in the final solutions prepared for colorimetric readings, instead of 0 . 7 s as employed in the original procedure. The Eriochrome Cyanine R and zirconyl chloride are used in the same ratio as specified in the original procedure, but their concentrations in VOL. 31, NO. 1, JANUARY 1959
a
105
the final solution are reduced to 25% of those employed by Megregian The enhanced sensitivity to fluoride resulting from these changes of the method outweighs the disadvantage of increased sensitivity to some foreign ions. However, because of the chemical interferences, the glassware must be scrupulously clean. The absorbance of the solutions can be measured with commonly available spectrophotometers. The reaction of fluoride with the lake to produce fading of its color is not affected by ordinary temperature fluctuations, does not require prolonged reaction times, and gives results which conform, within the range of fluoride employed, to Beer's law. Because the slope of the standard line is constant with a single batch of reagents it may be anchored by a single well established point. Apparatus and Reagents. A Universal Spectrophotometer (Coleman Instrument Inc.) or equivalent instrument with a cuvette of 5-em. light p a t h and a volume of about 2.4 ml. is used. All glassware and containers, unless otherwise specified, are of borosilicate glass. REDISTILLED WATER. Laboratory distilled water is deionized by passing it through a column (36 X 3.4 cm.) of filter-ion mixed bed resin (La Motte Chemical Products Co.). The water is then redistilled from a n all-glass still, collected, and stored in polyethylene bottles. This water is used in the preparation of all reagents and sample dilutions. SOLUTIONA, 2.1067 grams of Eriochrome Cyanine R (Geigy Chemical CorD.) dissolved and diluted to 1 liter with water. SOLUTION B, 0.3046 gram of zirconyl ochloride octahvdrate dissolved in 1614 ml. of concentrated hydrochloric acid, diluted to 2 liters with water, and stored in a polyethylene bottle. REAGENT-LAKE SOLUTION,one volume of Solution A added to two volumes of Solution B. This solution is prepared fresh daily. FLUORIDE STASDARDS (stored in polyethylene bottles). Fluoride Stock Solution, 0.2210 gram of dry c . P. grade sodium fluoride dissolved in 1 liter of water (100 Y of fluoride per ml.), Fluoride Substock Solution, 200 ml. of fluoride stock solution diluted t o 1 liter (20 y of fluoride per ml.), Large volumes of solutions containing 0, 1, 2, and 3 y of fluoride per 10 ml. for use as standards are prepared from this solution. PERCHLORIC ACID. One liter of 60% c. P. perchloric acid contained in a roundbottomed boiling flask is heated a t 135 to 140" C. for several hours. The acid is agitated with a stream of nitrogen gas, and redistilled water a t the rate of 10 drops per minute is added beneath the acid surface after the temperature reaches 120" to 125" C. The cooled acid is stored in a glass bottle a t lorn temperature. It gives no fluoride blank when used in the distillation procedure. 106
ANALYTICAL CHEMISTRY
Bubbier -Device
Gas
Inlet
A
Figure 1.
Microstill for fluoride distillation
A. Device to conduct nitrogen gas and water to within 0.5 to 1.0 cm. of bottom of distillation tube of still; connected to buret and to nitrogen supply with gum rubber tubing Condenser core is 8 mm. 0.d. Delivery tube ends in 5 mm. 0.d. and 1 mm. i.d. Capillary tubing ground to cone shape on its end
Cleaning Methods, Distillate collection tubes (Corning Glass Works No. 9820), stirring rods and the distillation tubes (Figure l ) of the still are rinsed in t a p water and the groundjoint surfaces of the latter are swabbed with acetone to remove silicone. These items are immersed in 30% c. P. perchloric acid contained in a large beaker, and the acid is slowly brought to a boil, and allowed to cool. The glassware is finally rinsed 8 t o 10 times with ordinary distilled Ivater followed by three to four rinses with redistilled water. All other glassware, including pipets, is subjected to acidchromate cleaning solution, rinsed with tap and distilled water, and immersed in 3Oy0 nitric acid for 30 minutes. I t is then rinsed with distilled and redistilled water in sequence. All glassware, except pipets, is dried a t 110" and stored in a cabinet to protect it from dust. Polyethylene bottles are washed with 3Oy0 nitric acid prior t o rinses n ith distilled and redistilled m t e r . These intricate cleaning methods are imperative to prevent erratic results. Experimental Details. The distillates, obtained as indicated below, or other solutions for analysis and the standard solutions are contained in tared tubes with 1 ml. of 1.25N sodium hydroxide solution. These solutions are titrated to the colorless end
point of phenolphthalein with 1.25N hydrochloric acid. One milliliter of reagent-lake is added to each solution with a Krogh-Kegs syringe pipet and the contents of the tubes are diluted to contain 20 ml. by weight. A beam balance (sensitivity 0.05 gram) is convenient. The solutions are mixed and allowed to stand 1 hour, and the absorbances a t 568 mfi (Coleman instrument) are measured. The cuvette is rinsed twice with the solution whose absorbance is to be measured. After use the cuvette is rinsed with alcohol t o remove traces of dye and lake. Adjustment of the instrument during a series of readings is maintained by an arbitrary purple glass filter mounted in the cuvette carrier in the position ordinarily occupied by the cuvette containing a reference solution. This glass filter was selected to give a light transmittance of about goyo,equivalent t o that of 3 y of fluoride standard, when the zero fluoride standard reads approximately 55y0 transmittance. The filter was more convenient than a standard reference solution and its use eliminated errors from evaporation or contamination of the reference solution. I n the usual procedure, replicate solutions a t two concentrations of standard fluoride are used t o establish the standard curve. ilfter several days'
use of the same batch of reagents, the a\-erage slope of the st'andard line (absorbance us. fluoride content) is calculated and thereafter r e p l h t e standards of zero fluoride content are used to establish a fixed point on the line. The fluoride content of the solution is then: y
fluoride
=
absorbance zero std. absorbance sample slope (absorbance units/? F)
Effect of Variables. There is no variation in absorbance of t h e solutions within 30 t o 180 minutes after adding t h e reagent-lake. KO interference from up to 20 y of sulfate v-as noted and 25 y of phosphat'e behaved as 1 y of fluoride (Figure 2 ) . Plasma and serum like many other animal tissues contain large amounts of chloride. This ion is collected in the distillate in all methods based upon the Killard and \Tinter principle, except when a large amount of silver sulfate is used in the still to retain chloride ( 8 ) . In tlie small still to be described, the presence of a n insoluble substance during distillation was troublesome. Alt'hough a large amount of chloride as hydrochloric acid is contained in the reagent-lake, and hence in the solutions prepared for colorimetric analysis, the influence of added quantities of sodiuni chloride was investigated. Sixty-five milligrams of sodium chloride, equivalent to the chloride contained in 10 nil. of serum, added to sodium-free solutions produced a n apparent fluoride of 0.15 y. The procedure described above results in all solut'ions having t'he same final sodium and chloride cont'ents and eliminates the necessity for special techniques to retain chloride in the still or t'o remove it from the distillate ( I , 2, 8). This is accomplished by neutralizing in each solution, whether s t a n d u d or unknown, a uniform amount of sodium hydroxide with hydrochloric acid. PREPARATION OF SERUM FOR ANALYSIS BY ASHlNG
Ashing of serum a t high temperatures causes large losses of fluoride (18). Results in this laboratory shon-ed losses of more than 50% of the fluoride of serum when no fiutive was used. Smith and Gardner (13) observed losses as iron fluoride n lien n hole blood was ashed and this n a s another reason for the use of 9eruni in this procedure. Fluoride is coiiiplete1,v removed from solution bj- boiling lrith magnesium oxide (15, 16). This indicated t h a t magnesium oxide, TT hich has been employed for this purpose with tissues ( 4 ) would be a satisfactory fixative in the ashing of serum and also suggested a way to obtain a low-fluoride-containing magnesium oxide.
I I
3
5
10
'5
MICROGRAMS PO:
io
30
9 R SO;
Figure 2. Influence of phosphate and sulfate on colorimetric method for fluoride
Special loiv-fluoride-containing magnesium oxide is prepared as follows. Five hundred grams of magnesium nitrate hexahydrate (anal!, tical grade) are dissolved in 3 liters of redistilled water, boiled for 30 minutes with 20 grams of magnesium oxide (analytical grade), and filtered. The hltrate is diluted to its original volume and the treatment n ith a fresh quantity of magnesium oxide is repeated. The filtrate is divided into several aliquots and evaporated to dryness in beakers. The solid residues are heated over a burner and ground to a coarse particle size in a mortar. The mixture of magnesium oxide and nitrate iq transferred to a platinum dish and heated until no further nitric oxide fuim.9 a l e evolved. The residue is suspendid in water and agitated with a stream of carbon dioxide gas washed through a trap containing thorium nitrate (to remove fluoride ion). After the magnesium oxide is dissoh ed, the solution is boiled, and the precipitated magnesium carbonate is removed by filtration. The solid is decomposed in platinum over a burner to gire a finely powdered magnesium oxide. In subsequent preparations, portions of this magnesium oxide are used to purify the magnesium nitrate as described above. The product contains 1 to 2 p.p.m. of fluoride, v, hich is less than t h a t found in conimercially available preparations of magnesium oxide. Magnesium acetate nould be an ideal fixative for these purposes because it is soluble in n-ater and serum and i t was used in some of tlie analyses to be reported. Honever, the ash of magnesium acetate prepared from niagnesium oxide and acetic acid always contained more fluoride than v a s present in the original oxide. Presumably the extra fluoride was derived from the acetic acid even though vacuum redistilled acid n as used. The retention of fluoride during the ashing of serum mas tested in experiments in which 2.0 y of fluoride labeled Fvith radiofluoride n'ere added to 5 ml. of serum with various amounts of niag-
nesiuiii oxide. The results were: 5 nig., 80% recovery of fluoride-18,15 to 20 mg., 90% recovery of fluoride-18, and 75 mg., 97.17 =k 0.927% recovery of fluoride-18. Transfer of a part of the ash to the still requires that the platinum dish be n-ashed with cold perchloric acid. Radiofluoride 11-as also used to detect possible losses of fluoride a t this step. Radiofluoride nas added to 60% perchloric acid a t room temperature and because the radioactivity of the solution n a s not altered for a t least 90 minutes, it was concluded that these steps in the manipulation of the ashed sample did not result in loss of fluoride. The procedures for ashing serum and transferring the ash to the still are as follows : To 75 mg. of magnesium oxide contained in a platinum dish are added 5 to 10 nil. of serum or heparinized plasma and 5 ml. of redistilled w t e r . The mixture is stirred constantly n i t h a glass rod while it is gently boiled over a low flame until the volume is reduced to about 2 ml. The dish is placed in anoven a t 100' to evaporate to dryness. The dish and contents are placed in a cold muffle furnace and heated a t 150" for 30 minutes, 300" for 15 minutes, and finally a t 500" for 7 5 minutes. The bulk of t h e ash is transferred with a funncl to the distillation tube of the still. This tube is cooled in ice water while the remnants of the ash are washed from the platinum dish into the distillation tube, first n i t h two I-ml. volumes of cold 30y0 perchloric acid, and finally n-ith 2 ml. of 60% cold perchloric acid. MICRODISTILLATION
OF
FLUORIDE
The still is illustrated in Figure 1. The important principle of operation, in addition to its reduced size, is t h e use of a stream of nitrogen gas to sn eep the fluoride from the still to the receiver instead of the large volume of steam, equivalent to 100 to 150 nil. of distillate, in the usual application of the IYillard and Kinter procedures. The much reduced aniourit (15 nil.) of water required, n hich determines t h e volume of the distillate, is added dropmise from a buret during the distillation. Four stills are connected through a surge manifold (a large flask) n i t h one nitrogen cylinder. The joint surfaces of the still are lightly lubricated with silicone grease and are held firmly together with springs. The distillation tube is heated in a glycerol bath over a flame. Observations with a thermocouple immersed in the contents of the distillation tube showed that this had a temperature of 135-8" when the temperature of the bath 17-as 145" to 150". The distillations require 50 to 60 minutes and the technique is as follows : After the apparatus is assembled, a tared receiver containing 1 ml. of 1.25N sodium hydroxide and a drop of phenolphthalein is positioned so that the tip of VOL. 31,
NO.
1 , JANUARY 1959
107
Table I.
Microdistillation of 5 y of Fluoride Labeled with Radioactive Fluorine
Recovery F18, % (Radioactive Analysis) .58.02 =l= 10.100 ~. ~. 93.94& 1.635 97.25 =l= 1.480 9 9 . 3 4 f 2.018
Distillate Collected,
KO.of
Trials 2 2 7
hL1.
2.5 5 .0
10.0
15.0
8
Recovery Fig, yo (Chemical Snalysis) 99.37&’i’13(3)“ 98.33 f 1.71 (4)5
Number of trials.
Table II. Fluoride Content of SerumMagnesium Oxide Ash
Aliquot, wt., Rlg.
Total F,
F, P.P.M. 0.10 0.12 0.15 0.12 0.14 0.10 0.13 0.12 & 0.017 std. dev.
Y
158.1 144.0 168.8 147.0 148.2 156.7 156.5
1.65 1.76 2.51 1.79 2.00
1.54 1.99 Mean
Table 111. Serum or
Plasma, M1. 10
Species Bovine
the condenser delivery tube is beneath the liquid. The rate of nitrogen flow is adjusted to about 2 bubbles per second formed in the liquid in the distillation tube. The preheated glycerol bath is raised to a height which immerses about three fourths of the distillation tube, and the temperature of the bath, as indicated by a thermometer, is raised to 145’ to 150”. When the temperature reaches 140°, water is admitted from the buret at about 5 to 6 drops per minute. Increases in volume of the liquid contents of the distillation tube above 4 ml. by too rapid inputs of Rater should be avoided. Marked elevations of temperatures of the reaction mixture, which
Fluoride in Plasma and Serum
MgO Equiv. as Fixative, Mg. 100
ino
10
Mean Result, P.P.M. 0 26 =k 0.029 0.12 f 0 . 0 2 6
No. of Samples 6 66
Human* 10 10oc 19 Dog 5 5 to 75 46 a Xinneapolis resident, 0.1 p,p,m. F- in drinking water. St. Paul resident, 1.0 p,p.m. F- in drinking water. Acetate used as fixative, otherwise MgO used.
Table IV.
Fluoride in 5 MI. of Bovine Serum
No. of detns. F blank (75 mg.
Serum plus 1.00.Y Fluoride 26
Serum 26
WZO), Y 0.15 Total F found (av.), 0.85 f Y 0.161 Total F expected, y ... Expected F found , , , (av.1, % h’ative F (av.), 0.14 % P.P.M. 0.025
Table V.
Tissue Enamel Dentin 108
0.15 1.93 & 0.109 1.85 104.6% 5.62
0 13 =k 0 030 0 21 f 0 057
are denoted by the evolution of white fumes (as yet unidentified, but which are odorless, not acidic, and pass through the condenser and receiver and appear to exert no deleterious effects on the results when produced for brief periods) are reversed by temporary increases in the rate of introduction of water or nitrogen and by lowering the glycerol bath, As the distillation proceeds, the receiver is lowered so as t o keep only the capillary portion of the delivery tube immersed in the liquid of the receiver. After 14 ml. of mater have been delivered from the buret the receiver is loxered, an additional 1 ml. of distillate is collected as free falling drops, and the tip of the delivery tube is rinsed with a small amount of water. The volume of distillate needed was
Fluoride in Human Enamel and Dentin
N o . of
Sample Wt .,
Samples 13 21
RIg.
Y
CI
17.1-26.8 3.9- 8 . 5
0.84-1.31 1.20-2.64
0.0049 f 0.00034 0.031 zk 0.0015
ANALYTICAL CHEMISTRY
Fluoride Found,
Fluoride Content, IC
determined from the observations given in Table I. Five microgram-samples of fluoride labeled with radiofluoride were subjected to distillation in which the indicated distillate volumes were collected. While i t is clear that a distillate volume of 10 to 15 ml. is adequate for complete evolution and collection of the fluoride, 15 ml. have been employed routinely. One milligram of phosphate strongly labeled with radiophosphorus was subjected to routine distillation. The radiophosphorus content of the distillate indicated the presence of 0.045 y of phosphate (0.0045%). The colorimetric method can tolerate 20- to 40-fold this amount of phosphate without error (Figure 2 ) . RESULTS
Thirty-two trials of distillation and analysis of 2 y of fluoride as aqueous sodium fluoride yielded a mean result of 101.0 % 3.101, recovery. Forty similar trials with either 3 or 4 y of fluoride gave a mean result of 100.8 f 3.7% recovery. These results are also taken to indicate that there is no appreciable distillation blank. Table I1 gives the results of repeated analyses of aliquots of a large preparation of the ash of serum with magnesium oxide. Table I11 summarizes individual analyses of serum or plasma which were carried through the entire procedure. Many were obtained during the developmental work and in trials in which magnesium acetate was used as a fixative. Most of these results were obtained with 10 ml. of serum, but 5 ml. is nom used routinely. Table IV gives results of analyses for fluoride and for recovery of 1 y of added fluoride in n-hich 5 ml. of serum vias used in all cases. Table V gives the results of repeated analyses of unashed human dentin and enamel. The samples were weighed in small improvised glass boats which n-ere dropped directly into the distillation tube. ACKNOWLEDGMENT
The assistance of P. Venkateswarlu and Jeanne Sheck is gratefully acknowledged. LITERATURE CITED
(1) Armstrong, IT. D., IND.ENG.CHEM., . ~ N A L . ED.8, 384 (1936).
(2) ilssoc. Offic. Agr. Chemists, “Official Methods of Bnalysis,” 8th ed., 1955. (3) Carlson, G. H., thesis, University of Minnesota, 1959. (4) Eberz, W.F., Laub, F. C., Lachele, C. E., IND. E m . CHEJI., AXAL.ED. 10, 259 (1938). (5) Estill, W. B., Mosier, L. C., ANAL. CHEM.27, 1669 (1955). (6) Frazier, R. E., Oldfield, H. G., Pub. Health Repts. ( U . S.), 68, 729 (1953). (7) Huckabay, W. B., Welch, E. T., Metler, A. V., ANAL. CHEJI. 19, 154 (1947).
(8) hIcClure, F. J., IXD.ENG. CHESI., ANAL.ED. 11, 171 (1939). (9) Megregian, Stephen, ANAL.CHEM.26, 1161 (1954). (10) Nielsen, H. X., Ibid., 30,1009 (1958). (11) Richter, F., Z. anal. Chem. 124, 19% (1942). (12) Samachson, Joseph, Slovik, Norman, Sobel, -4.E., AXAL.CHEM.29,1888 (1957).
(13) Smith, F. A., Gardner, D. E., G. S. Atomic Energy Commission unclassified document, U. R.46 (1948). (14) Smith, F. A., Gardner, D. E., Hodge, H. C., J . Dental Research 29,596 (1950). (15) Venkateswarlu, P., Karayana Rao, D., h A L . CHE~LI. 26, 766 (1954). (16) Venkateswarlu, p., Karayana Rao, D., I n d i a n J . M e d . Research 42, 135 (1953).
(17) Willard, H. H., Winter, 0. B., IXD. ENG.CHEM.,ANAL.ED. 5 , 7 (1933). (18) Wulle, Hertha, Z . physiol. Chena. Hoppe-Seyler's 260, 169 (1939). RECEIVED for review May 24, 1958. Accepted August 2, 1958. Work supported by the Research and Develo ment Division, Office of the Surgeon &eneral, Department of the Army, Contract KO. D A-49-007-MD-390.
Enzymatic Spec t ro photo metric Determinat io n of Sucrose in U.S.P. Sirups J.
P. COMER
and
H. F. BRICKLEY
Analytical Department, Eli M y and Co., Indianapolis, Ind.
b Dilutions of U.S.P. XV sirups were hydrolyzed in hydrochloric acid solution to yield glucose. The glucose was determined colorimetrically by the specific glucose oxidase-horseradish peroxidase-dianisidine system. The average difference of found and actual concentration from 10 U.S.P. XV sirups was -0.270 and the average coefficient of variation of five replicates for each sirup was +0.9370.
T U.
are no methods, with the exception of specific gravity, in S. Pharmacopeia XV for the assay of sucrose in the various sirups (8). Procedures for sucrose generally require modifications for each sirup. Keston (5) described a method for testing for glucose utilizing the two enzymes, glucose oxidase (6, 4 ) and horseradish peroxidase (7'). The oxidation of glucose by oxygen is catalyzed by glucose oxidase, n i t h the production of gluconic acid and hydrogen peroxide. The reaction of hydrogen peroxide with a n organic substrate to form a colored product occurs in the presence of the horseradish peroxidase. The result of this sequence is a very specific reaction for glucose. This reaction has been used in commercial semiquantitative ( 1 ) and qualitative (3) test papers for glucose. Commercially available reagents have been used for the photometric microdrtermination of blood glucose (6). The method reported here is based on the enzymatic determination of glucose following the acid hydrolysis of sucrose. Other sugars may yield glucose on hydrolysis, so the method is not specific for sucrose, b u t i t is more specific than the usual reducing sugar methods following hydrolysis. A combination of glucose and sucrose may HERE
also be assayed by color formation, before and after hydrolysis.
Table 1.
Absorbance of Glucose Solutions
EXPERIMENTAL
Glucose Found Using 0.2 Xfg./Ml. Std.7 % 100
Reagents. Enzyme. Dissolve approximately 2900 units of glucose oxidase (Takamine Laboratory, Clifton, N.J.), 1815 Pz units of horseradish peroxidase (Worthington Biochemical Corp., Freehold. X.J.), 0.125 gram of dianisidine citrate (prepared by addition of citric acid t o a n ether solution of dianisidine), 0.200 gram of citric acid monohydrate, and 1.20 grams sodium citrate dihydrate in 500 ml. of water, and filter before using. The enzyme reagent may be stored for a week if refrigerated. The gradual loss in potency is corrected by standard assays. Glucose Standard Solution. This contains 0.2 mg. of anhydrous dextrose per ml. Allow this solution to stand a t least 1 hour, so that the equilibrium amounts of a- and @-glucose can be established. Glucose oxidase is specific for 8-D-glucose and insufficient standing time for the standard solutions will give low standard readings and erroneously high sucrose results. Sirup Dilution. Dilute the sirups to contain approximately 4 mg. of glucose per ml. If the actual glucose content of a mixture of glucose and sucrose is t o be determined, the initial concentration of glucose should be about 0.2 mg. per ml. for the assay before hydrolysis.
tubes will a1loFv unequal atmospheric oxygen diffusion.
Hydrolysis. Heat 5 ml. of t h e sirup dilution, 25 ml. of distilled water, a n d 5 ml. of concentrated hydrochloric acid for 30 minutes on a steam b a t h . Cool, adjust t o p H 5.6, and adjust t h e volume t o 100 ml. with water. Enzymatic Color Formation. Test tubes of t h e same dimensions should be used. T h e oxygen content of t h e enzyme reagent will regulate t h e rate of color formation, a n d differing surface areas of t h e enzyme reagent i n t h e
Pipet 1 ml. of water into the reagent blank tube, 1 ml. of standard glucose into three tubes, and 1 ml. of the sirup dilutions into an appropriate number of tubes. If a sirup is highly colored, a sirup blank should be made by adding 1 ml. of hydrochloric acid (1 to 1) to another tube labeled sirup blank. At definite timed intervals, pipet 10 ml. of the enzyme reagent into each tube. After exactly 10 minutes, add 1 ml. of hydrochloric acid (1 t o 1) t o each tube in the proper sequence, except for the ones containing the sirup blanks. To
Actual Mg. Glucose/Ml.
Absorbance-
0.1
0.201 0.302
0.15 0 20
0.25
0.30 0.35 0.40 400 mp Table
II.
100
0.401 100 0.507 101 0.607 101 0 699 99.7 0.799 99 6 1-em. cells us. reagent blank. Recovery Data of GlucoseSucrose Mixtures
Total Glucose Recovered after Actual Glucose, yG Hydrolysis, 7 0 From From Analyst Analyst sucrose glucose A B 100 0 100 100 96 2 20 80 97.8 40 60 100 100 100 60 40 99.4 20 100 96.2 80 0 100 99.3 100 98.6 Av. 99.2 f1.5 Std. dev. =tO.88
VOL. 31, NO. 1, JANUARY 1959
109
