Table IV.
Mixture I
I1 I11
Determination of Propylene Oxide in Aldehyde-Oxide Mixtures with Dodecanethiol
A 18,25 45.90
Component, % Weight B C
IV V VI VI1 30.73 VI11 43.26 A . Isobut raldehyde. B. Formahehyde. C. Acetaldehyde. D. Propylene oxide.
49.14 45.95 21.65 49.74
D 81.75 54.10 50.86 54.05 78.35 50.26 69.27 56.74
Oxide Recovery, Eq. /Gram Error, Known Found 97, 0.01409 0.01417 $0.6 0,00933 0.00940 $0.8 0,00876 0.00901 $2.7 0.00933 0.00965 f3.3 0.01351 0.01461 +8.1 0,00901 0.00906 f0.5 0.01194 0.01228 +2.9 0.00979 0,01067 f8.9
titration of the excess dodecanethiol because of the partial reduction of iodine added. However, the high results in Tables I and IV prove that this reaction does not occur under these test conditions. ACKNOWLEDGMENT
The author gratefully acknowledges the assistance of E. D. Williams and J. H. Gardner of Sational Research Corp, and expresses appreciation for their helpful suggestions. LITERATURE CITED
(1) Beesinn. D. IT.. Tvler. TI7. F.. Kurtz.
During the titration with iodine, a fading end point chn be minimized by using a large escess of alcohol. The addition of 100 ml. of 2-propanol should be more than sufficient to give reproducible titers even if a substantial amount of water is present. A general rule is to use 100 ml. of alcohol for aqueous volumetric samples of 20 ml. or less. For larger samples, add an alcohol volume five times as great as the volume of the sample. This end point difficulty is not encountered with nonaqueous epoxide solutions. Flasks used repeatedly should be scrupulously clean and dry, as slight errors can result if traces of iodine
remain. I n an alkaline medium, iodine is readily converted to the iodate ion. I n turn, this will revert to iodine upon acidification, thus providing high values in the final calculations. Normally, low molecular weight aldehydes can be partially oxidized with a bromine-glacial acetic acid mixture. This results from the relatively great oxidizing power of bromine. One would expect that these same organic materials would be susceptible to attack by iodine, but to a lesser degree because of its weaker oxidation capacity. If such were the case, low epoxide values would be obtained, as a greater volume of the reagent, would be required for the
. 3 ) Dal Nogare; S., Oemler, A. N., ASAL.
CHEW. 24.902 11952). (4) Eastham, A.’XI.,-Latremouille, G. A,, Can. J.Research B28,264 (1950). ( 5 ) Hilton, F., Trans. Inst. Rubber Ind. 17,319 (1941). (6) King, G., S u t u r e 164, 706 (1949). ( 7 ) Rainey, J. L., Denoon, C. E., Jr., Chem. Eng. Sews 31, 4521 (1953). (8) Ross, W. C. J., J . Chem. SOC.1950, 22.57 . (9) Swan, J. D., ANAL. CHEX 26, 878 (19%).
RECEIVEDfor review March 3, 1960. Accepted May 18, 1960. Division of Analytical Chemistry, 138th Meeting, ACS, Xew York, K.Y., September 1960.
Rapid Ultraviolet Spectrophotometric Determination of Salicylate in Blood GEORGE W. STEVENSON Department o f Pharmacology and Toxicology, School o f Medicine, University o f Californiu, 10s Angeles 24, Calif.
b Single and double extraction methods for determination of salicylate in blood are presented. Whole blood or any blood fraction containing 0 to 50 mg. % of salicylate i s extracted with a solution of malonic acid in butyl ether. In the single extraction method, the ultraviolet absorption peak at 307 mp in the butyl ether is used for identification and determination. This i s as rapid as, and much more specific than, the colorimetric procedures. In the double extraction method, the butyl ether extract i s extracted with pH 6.86 buffer and the peak absorbance at 296 rnp in the buffer i s measured.
T
determination described is one of a series of analyses of drugs and tosic substances integrating a fern simple and selective extractions HE SALICYLATE
1522
ANALYTICAL CHEMISTRY
with spectrophotometry. V7hen salicylate is known to be present, colorimetric procedures may be satisfactory, but they are not ideal for unknowns. They are not specific and are not capable of detecting and distinguishing other compounds. Fluorimetric (1-3) and ultraviolet spectrophotometric methods (4, 6) are preferable. Since the colorimetric procedures are usually faster than fluorimetric or ultraviolet methods, the latter may not be used even if equipment is available. It n-as found that organic acids were more efficiently extracted from aqueous phases by ethers than by halogenated hydrocarbons. Ether-aqueous partition coefficients of neutral and basic compounds were much lower than the corresponding halogenated hydrocarbon-aqueous partition coefficients, so that a lower proportion of these substances as extracted into the ethers.
Butyl ether was found to be superior in this laboratory to both chloroform and ethylene dichloride for blood salicylate and barbiturate determinations. The advantages of butyl ether which led to its use in this salicylate procedure are its relatively specific extraction of acids, its formation of an upper layer with blood, its low volatility, and its ability to dissolve acid for blood acidification. Other innovations are measurement of ultraviolet absorption in the solvent extract of blood, and acidification simultaneous with blood extraction by malonic acid dissolved in the butyl ether. This method is, therefore, as rapid as the colorimetric ones. It $vas compared with a double extraction method in which the butyl ether was extracted with p H 6.86 buffer. Since the double extraction method does not give appreciably loner blanks and has
few advantages, the single extraction method is usually the method of choice.
Table I. Ultraviolet Absorption in Butyl Ether (A,,,
APPARATUS AND REAGENTS
Wave Length,
h Beckman DU spectrophotometer
Mlr
was used either with 1-em. quartz cells requiring 1 ml. of solution (available from Arthur H. Thomas Co.) or standard 1-cm., 3-ml. cells. Centrifuge tubes used were 13-ml. glass-stoppered tubes similar to Corning No. 8064 but with rounded bottoms. n-Butyl Ether. R a s h t h e ether (Matheson practical grade) with ferrous sulfate t o remove peroxides. Shake with sodium hydroxide pellets t o remove Iyater. Reflux ether with sodium metal overnight. Distill, discarding t h e initial a n d final portions, and collect t h e ether boiling a t 1401' C . Store in amber bottles in t h e d a r k at 0' C. T h e ultraviolet cutoff should not be higher than 277 mp. Used ether, free of other orgdnic solvent, can be re-used. Wash used ether with distilled water, several portions of sodium hydroxide solution, and finally distilled water. Malonic Acid in Butyl Ether. Dissolve 400 mg. of finely ground malonic acid i n 100 ml. of t h e butyl ether purified a s above. Store in t h e dark at 0' C. until used. Warm t o room temperature before pipetting it. pH 6.86 Phosphate Buffer. Dissolve 3.40 grams of reagent grade potassium dihydrogen phosphate and 3.55 grams of reagent grade anhydrous disodium hydrogen phosphate in sufficient water t o make 1 liter of solution. (This S a t i o n a l Bureau of Standards buffer is available in packet form from Beckman.)
378 340 314 307 300 280
PROCEDURE
Single Extraction Method. Pipet 300 pl. of serum or plasma and 3 ml. of malonic acid in butyl ether reagent into a centrifuge tube. Prepare a blank of 300 pL of distilled water and 3 ml. of malonic acid in butyl ether. Rapidly invert the tubes approximately 200 times. Use 300-p1. portions of whole blood or red blood cells, add the ether so as to minimize mixing with the blood, and shake very vigorously for 1 minute right after addition of the ether, If the red blood cells are partially mixed with the reagent before shaking, part of the salicylic and malonic acid is trapped in a gelatinous hemoglobin mass and failure to acidify a11 the sample and to extract all the salicylic acid results. Centrifuge the tubes. Decant the butyl ether layers into absorption cells and read the absorbances a t 378, 314, 307, 300, and 280 mp cs. the ether blank. Return the ether to the centrifuge tubes if the double extraction method is to be used. If hemoglobin containing samples were used, rinse the cells Kith sodium hydroxide solution and discard the rinse. Calculate salicylate concentration using Equations 1 and 2. Double Extraction Method. Pipet 2 ml. of t h e butyl ether blank and of each of t h e butyl ether extracts (absorbances of these need not have
emax
( l M , 1 cm.)
Gentistic Acid
4160
4660
CALCULATIONS
Mg. % salicylic acid = 44.4 (A307 A280 I C ) (1) where k is 0.005 for serum, 0.016 for plasma, 0.034 for whole blood, and 0.021 for red blood cells.
+
70salicylic acid
=
33.6 (A301 - 0.1%
A3ig)
Mg. 70 salicylic acid = 43.7 (A288 Mg. % salicylic acid
(2)
- Asao)
39.7 Alae
(3) (4)
E uations 1 through 4 are derived as folows:
Mg.
70
salicylic acid = dil* R 1,395 106 R ( 5 ) recov.
loo
I
where 100 = factor converting mg./ml. to mg./100 ml. M.W. = molecular weight - of salicylic acid = 138.1 dil. = dilution factor = 10 recov. = fraction recovered from blood = 0.99
-4307
n nnn
...
RESULTS
Ultraviolet Absorption Properties. T h e relation between absorbance and wave length of compounds involved in t h e determination is indicated in Tables I and 11, where t h e absorbances a t t h e peak equal 1.000. Both butyl ether a n d p H 6.86 buffer solutions were prepared, each Jyith a n absorbance of 1.5 (1-cm. path length) The absorbance of each a t the A,,. solution was measured a t various dilutions down to a n absorbance of 0.1. The emax values listed in Tables I and I1 remained constant with dilution. Acetylsalicylate monoanion does not have a peak in the buffer, but has a shoulder a t approximately 276 mp, c = 650. Blanks. Apparent salicylate levels in salicylate-free samples shown in Table 111 were calculated using each of t h e four equations. Random hospital blood samples collected in B-D tubes and barbiturate-containing postmortem blood samples were used. A serum or plasma sample free of hemoglobin gives a blank of less than 1 mg. per cent using Equation 2 even without the correction term. Butyl ether extract of samples containing red blood cells or hemoglobin contains a porphyrin with peak absorbance a t 378
II.
- 0.195 AX8 -
Ultraviolet Absorption in pH 6.86 Buffer (A,,,
e307
Wave Length, Mfi 378
= 1.000)
Salicylate 0.000
Gentisate 0.004
340
0.013
0.464 1.000
30% 296 "0 260
0 .BOG 1.000
0,605
0,909 0.091
0.265 0,040
320
tmnx
For highest accuracy, the constants in the equations should be checked for a
1160
specific spectrophotometer by preparation of blanks and standards in the appropriate media and the carrying out of the procedure.
Table R (Eq. 2) =
Porphyrin from Acetylealicylic Hemoglobin Acid
0.027
been measured) above into centrifuge tubes each containing 2 ml. of the pH 6.86 buffer. Rapidly invert t h e tubes approximately 100 times. Centrifuge. Transfer aqueous layers into 1-ml, cells by pipet. Pass the pipet into the aqueous layer, expel ether from the tip gently with several portions of air, and wipe the tip of the pipet t o remove traces df ether before emptying it. Measure the absorbances at 302, 296, 290, and 260 mp us. the blank buffer. Calculate salicylate concentration using Equations 3 and 4.
Mg.
= 1.0001
Salicylic Acid 0.002
(lJI, 1 em.)
0.164
3510
0.415
4000
VOL. 32, N O . 11, OCTOBER 1960
0
1523
Table V. Analysis of Serum Samples Salicylic Acid in Mg. Per Cent
Table Ill. Salicylic Acid Blanks in Blood in Mg. Per Cent
B1oo.d Fraction Serum Plasma Whole blood Post-mortem whole blood Red blood cells
Mean i Std. Dev. (No. of Samples) Eq. 1 O.l*O.l(9) 0 . 2 i 0 . 3 (8) 0 . 1 i 0 . 1 (8)
Eq. 2 0.2*0.2(22) 0 . 7 i0 . 4 ( 8 ) 0 . 4 i 0 . 3 (8)
Eq. 3 O.l*OO.l(9) 0 . 2 0 . 3 (8) 0 . 2 i 0 . 2 (8)
0 . 2 i 0 . 2 (12) 0 . 1 i 0 . 1 (8)
0 . 2 i 0 . 4 (12) 0 . 2 f 0 . 1 (8)
0 . 1 f 0 . 2 (12) 0 . 9 i 0.6 (12) 0 . 3 f0 . 2 ( 8 ) , 0 . 2 i0 . 2 (8)
*
Eq. 4 0.2iOo.2(20~ 0 . 5 i 0 . 4 (8) 0 . 4 f 0 . 4 (8)
Single Extraction Eq. 1 4.7 5.7 16.7 .
I
.
6.8 , . .
mp derived from hemoglobin as a result of acidification. Peak absorbances are no higher than 1.5 with whole blood, 1.0 with red blood cells, and much lower with serum or plasma containing hemoglobin. The second term of Equation 2, the product of As78 and the relative absorbance a t 307 mp of the porphyrin (Table I), corrects Equation 2 for porphyrin blank. Corrections can be made at' each wave length to give the porphyrin-free absorption curve. Blank due to the porphyrin is more easily eliminated by using the 307- to 280-mp absorbance difference, since this is zero for the porphyrin and 75% of the -4307 of salicylic acid. Extracts of the various blood fractions have slightly higher absorbances a t 280 than a t 307 mp which are compensated for by the factor k in Equation 1. When the double extraction method is used there is no porphyrin in the buffer, since it is precipitated out a t the ether-buffer interface during the second extraction. Recovery Studies. Results indicated in Table IV were obtained after addition of salicylic acid to salicylate-free serum, plasma, whole blood, and red blood cells. Analysis of Clinical Samples. Results of analysis of blood samples after salicylate ingestion are listed in Table V. Since methods were modified with use, d a t a were not available for calculation of some values. Most of t h e blood samples were from children who had ingested aspirin, some were from adults on therapeutic salicylate, and three were post-mortem samples containing salicylate. DISCUSSION
The single extraction procedure is much more rapid than the other ultraviolet procedures cited. A disadvantage of the method of Ungar et al. (4) is that deproteinized serum is used for the absorbance measurements. Some substances not extractable from the serum interfere. The method of Williams et al. (6),patterned after the Goldbaum barbiturate procedure, is not ideal for strong organic acids, since the 0.45Msodium hydroxide 1524
ANALYTICAL CHEMISTRY
Table IV.
Recovery of Salicylic Acid Added to Blood
Blood Fraction Serum Plasma Whole blood
Red blood cells
Concn., Mg. 70 50 5 44.5 22.5 4.45 44.5 36.4 22.25 4.45 44.5 22.25 4.45
Recovery,
7c
100 98 99 98 98-100 98-99 98 98-100 100-101 99-100 100-102 97-101
extraction of the ethylene dichloride also removes weak acids. When strong acids like salicylic are extracted into solvent from acidified blood, they should be removed before the weak acid fraction or there may be mutual interference. As shown by the values in Tables I11 and V, the double extraction method is not appreciably more specific than the single. This is because the butyl ether does not extract interfering neutral and basic substances. I n some instances blanks were higher with the double extraction method, since ionized forms in the aqueous solution had higher absorbances. Blood barbiturate is extracted into butyl ether, but does not have ultraviolet absorption in butyl ether. The low proportion of barbiturate extracted into the p H 6.86 buffer has little ultraviolet absorption at 260 mp or above a t this pH. Ultraviolet absorption of various substances in solvent extract of blood is rarely utilized despite many advantages. Conventional low-boiling solvents are not feasible for such quantitative procedures. Butyl ether is sufficiently nonvolatile, has sufficient extraction capacity for salicylic acid, and has the advantage over ethyl ether of extracting much less of any interfering substances present. The disadvantage of butyl ether is that solvent of sufficiently low ultraviolet absorption is not commercially available. The separate acidification step used in other blood salicylate procedures is eliminated. Malonic acid in the ether
26.2 30.8 13.6 6.8 23.1 5.7 12.7 17.6" 13.3b 3.2c 21.2 27 . O d 56. 6d 66,Od 9.3" 6 .Ob 2.3c 40.1 13. gC a
Eq. 2 4.9 6.9 18.2 23.2 7.4 26.2 27.0 30.9 13.9 7.0 23,l 4.8 13.1 18.1" 12.7& 3.P 21.3 27, 55,Od 64, 2d 9.6" 5.Sb 2.2c 40.4 14.0"
Double Extraction Eq. 3 4.7 5.1 16.7 22.0
...
... ...
. . ,
... ... ... ...
...
17.8"
...
... 21.3 26. 4d 54. Sd 63. 6d 9.2" 6.3b 2.7c 39.6 14. 2c
Eq. 4 4.9 7.1 19.1 24.0 7.5 26.6 27.2 31.5 14.7 6.7 24.0 4.6 12.7 18.6" , . .
... 21.6 29. Sd 55.0d 63. 4d 9.44 6.3b 2.5c 39.1 11.5c
Plasma.
* Whole blood from which above sample
obtained. Red blood cells from same samole above. Post-mortem whole blood.
a8
passes into the blood when the phases are equilibrated, buffering the blood layer a t about p H 3. This technique was introduced to allow the analysis of whole blood and red blood cells, but it also speeds analysis of serum or plasma. These can be acidified with strong acid before extraction but large masses of hemoglobin are precipitated in whole blood or red blood cell samples so that quantitative extraction is unlikely. Extraction is simultaneous in the new technique with hemoglobin precipitation, and after rapid initial suspension in the solvent, blood remains in fine droplets or in a thin film permitting quantitative extraction. No other procedure appears to be applicable to direct analysis of red blood cells. The procedure was designed for concentrations up to 50 mg. %, since most of the samples submitted to the clinical laboratory are within this range. If many samples are expected to fall between 50 and 100 mg. %, 20 volumes of twofold diluted malonic acid in butyl ether solution should be used for one volume of blood and appropriate changes made in Equations 1 through 4. If the p H 6.86 extraction is to be carried out, a double or other appropriate volume ratio of buffer to butyl ether can be used for dilution of concentrated samples. The greater specificity of these methods than of currently used colorimetric and ultraviolet procedures depends upon more specific extraction of
acids into butyl ether and demonstration Blood samples of Table V were analyzed of the correct ultraviolet absorption for acetylsalicylic acid, which is rapidly curve. When Equations 1 and 2 give hydrolyzed to salicylate in the body, nearly the same values, it is unlikely and these metabolites. Acetylsalicylic that appreciable quantities of interacid and gentisic acid can be deterfering compounds are present, since mined by their absorbances a t 280 these must have the same A ~ o ~ / i j - ~ oand 340 mp, respectively, and salicyluric ratios as salicylic acid. Presence in the acid by a simple partition procedure butyl ether extract of acidified blood of involving measurement of the absorbanother substance with the same abance of acid aqueous washes of the sorbance curve as salicylic acid is posbutyl ether extract, all in the presence of sible but very unlikely. Only salicylsalicylate. These substances were not uric acid, one of a large number of comfound in blood, confirming the results of pounds screened, is extractable and others. The samples were not analyzed has a peak within 1 5 mp that of salicylic for glucuronides because they are not acid. But this metabolite of salicylic extractable into butyl ether. acid has not been demonstrated in Use of a recording spectrophotometer blood. -4 large number of phenols or or readings made a t wave lengths in enols which may be present in the addition to those recommended in the blood are determined as salicylate procedure will show primarily the using colorimetric procedures, but have presence or absence of patterns of ababsorbance peaks much lower than 307 sorption due to other compounds. The finding of such a pattern may make mp. Readings should always be made a t 314 and 300 mp to show the pregence possible the identification of the comor absence of the 307-mp salicylic acid pound, whereas the colorimetric propeak. .ill the samples of Table T' cedures would either indicate salicylate showed peaks a t 307 mp in butyl ether or absence of salicylate. This explains and 296 nip in pH 6.86 buffer. in part the higher blanks obtained with Preliminary work on the determinathe colorimetric procedures. It is antion of salicylate and its metabolites in ticipated that both the single and double urine will be published separately. estraction procedures may be used in
the determination of other ultravioletabsorbing acids. ACKNOWLEDGMENT
The author thanks Frank NcKee, Director of the Clinical Laboratories, UCLA Medical Center, for supplying blood samples; Raymond Abernethy, Toxicologist, Los Angeles County coroner's office, for his kind cooperation; and Ronald Stein for carrying out a literature search and pilot study. LITERATURE CITED
(1) Chirigoe, M.
A,, Cdenfriend, S., J . Lab. Clin. Med. 54! 769 (1959). (2) Saltzman, A,, J . Bzol. Chem. 174, 399 (1948). (3) Truitt, E. B., Jr., Morgan, A. M., Little, J. M., J. Am. Pharm. ASSOC., 815.Ed. 44,142 (1955). (4) Un ar G., Damgaard E., Wong, W. h o c . SOC.Exptl. biol. Med. 80, 45 (1952). (5) Williams, L. A., Linn, R. A., Zak, B., J. Lab. Clan. Med. 53,156 (1959). RECEIVEDfor review January 20, 1960. Accepted July 20, 1960. Investigation supported by research grant B-1106 from the National Institute of Neurological Diseases and Blindness of the National Institutes of Health, U.S. Public Health Service.
If,
The Analysis of Metal Chelates by Gas Chromatography SIR: Gas chromatography has been used successfully for the analysis of metals in the form of acetylacetonato complexes. Our studies have shown that the method is particularly well suited for beryllium determination. Previous applications of gas chromatography t o metal analysis have involved the use of volatile inorganic salts ( I , 3 ) . The larger number of volatile p-diketone chelates and the greater stability of this class of compounds with respect to reaction with the constituents of the atmosphere extend and facilitate the use of gas chroniatography in inorganic metal analysis. The combination of solvent extraction and complex formation should make this method of analysis exceedingly useful. This York was done on a Pye argon chromatograph equipped with an ionization detector. Using conventional columns (4 feet X 1/4 inch of 10% Apiezon L on Celite 545, 80- to 100mesh) , separations were achieved only a t temperatures where thermal deconi-
position of the complexes was noticeable. The use of glass bead columns (2) (glass homogenizing beads supplied by the VirTis Co., Inc.) permitted rapid elution of beryllium, aluminum, and chromium acetylacetonato complexes (boiling points 270°, 314', and 340' C.', respectively) a t temperatures well below those a t which thermal decomposition is significant.
COLUMN TEMP. ("C,) 90 100 120 140
80
160
b Figure 1. Gas chromatographic separation of beryllium and aluminum acetylacetonato complexes in acetylacetone Sample size, 0.34 pl. Concentration of bir(acetylacetonato)beryllium (a), 0.217 gram in 3.00 ml. of acetylacetone Concentration of trir(acetylacetonato)aluminum (Ill),saturated solution, at 25' C. Column, 4 feet X VI inch of 0.5% by weight Apiezon 1 on glass beads, 200 microns in diameter Flow rate of argon, 50 cc. per minute Detector voltage, 1500 v. Sensitivity, X3
0
5
10
TIME
15
20
25
30
(MIN.1
VOL. 32, NO. 1 1, OCTOBER 1960
1525
