ducibility. The range of peak heights for five determinations was 118.4 to 121.5 mm. with a relative standard deviation of 1.1%. Similarly, seven determinations of a standard solution of Tinuvin P ranged in peak height from 94.5 to 97.5 mm. with a standard deviation Of 1.2%.
Polymers containing known amounts of the additives indicated an accuracy of approximately the same degree. LITERATURE CITED
J., Shaw, G. C., Solomon, D. H., ANAL. CHEM.
( 1 ) Cook, C. D., Elgood, E. 34, 1177 (1962).
(2) Metcalfe, L. D., Nature 188, 142 (1960)* (3) Neely, W. B., Nott, J., Roberts, C. B., ANAL.CHEM.34, 1423 (1962). (4) Roberts, C. B., Aerograph Research Notes, Summer Issue, 1963. (5) Spell, H. L., Eddy, R. D., ANAL. CHEM.32,1811 (1960). (6) Stafford, Ibid., 34, 794 (1962). RECEIVEDfor review August 23, 1963. Accepted October 14, 1963.
c.,
Determination of Aspirin by Gas Chromatography RAYMOND C. CRIPPEN Atlas Chemical Industries, Inc., Wilmington, Del.
HENRY C. FREIMUlH Chief Toxicologist, State of Maryland, Baltimore, Md.
,A gas chromatographic method was developed for the determination of aspirin (acetylsalicylic acid) in drug preparations, tissues, and in solutions. The method involves extraction of the drug with hot anhydrous methanol from the dry preparations, followed by addition of a small amount of BF,, and refluxing for 10 to 15 minutes to form the methyl esters. An aliquot i s injected into the gas chromatograph equipped with 2 feet of 3070 Carbowax 20M on chromosorb W (8O-to 100mesh) at a column temperature 175' C. and a helium flow rate of 100 ml./ minute. The retention time of the methyl acetylsalicylate peak was 3.5 minutes. Bound as,pirin may be determined by a modified procedure. Salicylic acid methyl ester was separated from the acetyl salicylate methyl ester by use of lower temperature and longer columns. Tissues or solutions may be analyzed for aspirin by similar techniques. Recoveries were 98.7% with a mean error 3f kl.Oy0, and a precision of =t 1.68%.
A
has been known and used for years, the official analytical methods used to assay preparations or detect its presence are laborious and time consuming. They involved separations with solvents and titration with standiwd acids and bases (5, 20, S I ) ; reaction with bromine or iodine (21) and titration of excess; or gravimetric measurcment after extraction and drying ( 5 ) . Analysis of aspirin in mixtures or compounded in effervescent powders or tablets is more involved and time consuming ( 5 ) . No direct official method as such is available. Many spectrophotometric methods have been developed for analyzing aspirin. 'Many invo ve separations plus color development (2?4-Z6). Others use ultraviolet adsorption (26, 26), and LTHOUGH ASPIRIN
one uses fluorescent measurements (27). A rapid gas chromatographic method was required that gave the lowest possible number of extractions or other manipulations (16,28,29). A solution of aspirin in chloroform did not move on a gas chromatograph under any conditions investigated. Degradation to the free hydrocarbon and measurement as an aromatic component (1, 12, I S ) were considered for quantitative analysis, but were not reproducible. Reduction to a phenolic alcohol and analysis as an oxygenated compound (3, 6, 7 , 9, 19) were also considered, but the manipulations were too involved for a good routine method; chlorination, bromination, or iodination were no improvement ( I O , SO). Other investigators converted fatty acids to methyl esters (6, 12) to aid their movement on the gas chromatograph; still others converted barbiturates and acidic drugs to methyl esters (4, 23); or analyzed mono- and di-carboxylic acids in resinous substances as methyl ester (16,17); determined cyclic (14),phthalic (8, sa), benzoic acids (9, SO), and mixtures of ethers of aromatic acids (18) as esters. Nothing in the literature covers analysis of aspirin by the gas chromatographic method (11). [K'ote: Table 1.
since preparation of this paper, Hoff' man and Mitchell published on the subject in J . Pharm. Sciences 52, 305-6, (1963).] Methyl salicylate (oil of wintergreen, b. p. 222'-9' C.) has a low volatility; methyl acetylsalicylate (b. p. 134'-6' C. a t 9 mm.) is a little less volatile and should move almost as readily on the instrument. This was confirmed-the two components did not separate a t 175' C. They did separate a t lower temperatures and on longer columns. EXPERIMENTAL
BORON TRIFLUORIDE METHYLATINGREAGENT. -4 solution of 20y0 boron trifluoride (BF,) in anhydrous methanol was prepared in a well ventilated Reagents and Apparatus.
hood by bubbling the boron trifluoride gas into methanol weighed in a tared Erlenmeyer flask. The solution must be cooled periodically. When the weight increase corresponds to 20YoJ the gas flow is disconnected quickly t o prevent solvent suck-back, contents are cooled to room temperature, poured into a plastic bottle, and sealed with a plastic cap only. THEPODBIELKIAK CHROMACON thermal conductivity detector gas chromatograph used in this investigation
Analysis of Aspirin-Containing Products
Acetylsalicylic Acid Product Labeled Found (USP) Found (GLC) Aspirin Compound A 3 grains/tablet, 3.55, 3.59 gr. 3.60, 3.66 gr. Aspirin Tablet B 5 grains/tablet 5.27, 5.29 gr. 5.25, 5.30 gr. AsDirin Tablet C 10 erains/tablet 10.30, 10.32 gr. 10.35, 10.30 gr.O Tissue D 24.9, 2 4 . 8 m,* 25 hg./100 g. (added) 24.8, 24.85 mg. 24.8, 24.7 mg.* 25 mg./100 ml. (added) 24.5, 24.3 mg. Blood E Solution F 5 srains/tsD. 5.30. 5.35 pr. 5.28. 5.33 er. Powder G 5 &ns/eivelope 5.201 5.25 gr. 5.22; 5.25 gr. 650 mg./g. 650 mg. 651 mg. Synthetic Mix H a This represents the free and combined acetylsalicylic acid. Direct extraction gives the free aspirin; acidification of the residue and re-extraction gives the combined aspirin. * This method does not differentiate between acetylated and nonacetylated salicylic acid. Lowering the temperature on a larger column assists in the differentiation of nonacetylated salicylic acid. VOL. 36, NO. 2, FEBRUARY 1964
e
273
Table 11.
NO. injections, n
Area generated (cm.2),=zi 30.81 30.25 30.14 30.71 30.14 30.58 31.40 30.13 30.13 30.59
1
2 3 4 5
6
7 8 9 10
Zzi = 304.87
Xi2
949.2561 915.0625 908.4196 943.1041 908.4196 935.1364 985.9600 907.8196 907,8196 935.7481 Zzi2 =9296.7402
,296, 740:0--
92,945.7169
10.4908 cm.2-represents f O .5 mg./ml. (5:305) or 1 1 . 6 8 % precision
=
Represents injection of 5 pl. from 650 mg./25 ml. solution in methanol. Table 111.
Accuracy Studies
Recovery differences (R - X , ) +1.68) +0.951
NO.
runs, n 1
2
n Table I
3
a standard solution made the same way as the sample and of approximately identical concentration. Also prepare standard samples of higher and lower concentration and inject 5.0 pl. into the instrument. Plot the peak areas us. concentration (Figure 2).
Precision Studies
(R- X,)= +7.09
(assuming USP method 100%) X , = % Recovery of aspirin (GLC method) Mean error = =kl.O137, = 1007,
Table IV.
Figure 1. Typical gas chromatograms of methyl acetylsalicylate
was equipped with a 2-foot X '/*-inch 0.d. column of copper tubing packed with 3oy0 Carbowax 20M coated on Chromosorb W (80- to 100-mesh). The instrument oven, block, and column were adjusted to 175O-injection port to 275". Helium flow was adjusted t o 100 ml./minute under 25 p.s.i. gauge pressure. HAMILTON 10-g1. SYRINQE. With fixed needle, calibrated in 1-gl. major divisions and 0.2-g1. minor divisions. SOXHLET EXTRACTOR (MODIFIED). Equipped with a 50-ml. ground glass jointed flask modified for minimum holdup by removal of siphon and replacement with a run-off tube. Procedure. Carefully grind u p at least 2 tablets (5-grain size) or dry a weighed amount of tissue or solution at 60" t o 70" C. (with or without a vacuum) and pulverize t h e residue. Transfer the powder or residue t o a n extraction thimble and extract with 25 ml. of hot anhydrous methanol 5 t o 10 minutes in the 50-ml. modified Soxhlet extractor. To the hot methanol solution, add 20 ml. of 2001, boron trifluoride methylating reagent, change the Soxhlet extractor t o a condenser, and reflux another 5 t o 10 minutes. Cool and transfer t o a 50-ml. volumetric flask, rinse the flask with methanol, and make UD t o volume. Mix thoroughly. Using a 10-gl. syringe, inject 5 gl. of methanol solution above into the gas chromatograph. The retention time of the methyl acetylsalicylate is 31/2 minutes (Figure 1). Now inject
Comparisons of Times on Analytical Methods for Aspirin
USP method Approx. time fbi analysis Elapsed, Man hours hours
GLC4 (elapsed), min.
Method (19) AOAC 32.110 (also NFX) aspirin, phenacetin & caffeine (ext'n.) 2 " 1 30 AOAC 32.113 aspirin, phenacetin & caffeine (columns) 3 = 11/2 35 2 " 1 30 AOAC 32.119 aspirin, phenacetin & salol (ext'n.) 1 30 AOAC 32.129 aspirin (iodimetric) 3 " AOAC 32.131-2 amirin (also USP X V I ) (titrimetric) 1 " = /2 30 AOAC 32.133 aspirin & phenobarbital (spectroph.) 1 " /2 30 AOAC 32.139 aspirin & phenolphthalein (ext'n.) 2 " 1 30 AOAC 32.195 (b). effervescent samples. (ext'n.) (No estimate) 30 GLC method can be extended to salicylates without modification. Advantages of GLC Method: Direct extraction into reactdon media, after esterification; direct injection into GLC instrument without further treatment. Disadvantages of Other Methods : Involve extractions, drying, and weighing of residue or titration or spectrophotometric examination. The more manipulations, invite more chances for errors.
RESULTS AND DISCUSSION
Samples of aspirin-containing preparations were secured from manufacturers and the open market. Results are compared with those of conventional USP methods in Table I. I n effervescent preparations, the free aspirin was lorn-er than the total aspirin, since some of i t reacted with the sodium bicarbonate. The bound aspirin was recovered by acidifying the methanolextracted residue above, drying, and reextracting with methanol. The total aspirin was determined by combining this solution with the original extract. Liquid samples were dried at 60" to 70" C. (with or without vacuum) and prepared for analysis by the standard procedure. I n blood samples, recoveries were good, although the USP method gave decreased values from the proposed method. No free salicylic acid was found in any samples except the blood and tissue samples analyzed by the AOAC method (19). The proposed procedure would differentiate acetvlsalicylic from salicylic acids only if the temperature of 100" to 125" C. and a 4- to 6-foot column were used. The precision of the method was =k1.68% (Table 11). The recoverv was 98.7%,' t h e mean error & l . O % (Table 111) ( 2 ) . Other esters from ethyl to n-butyl did not improve separation of salicylic and acetylsalicylic acids. Other isomeric hydroxy-benzoic or acetyl-hydroxybenzoic acids can be analyzed by this method under slightly modified conditions. Much less time was required to determine routinely aspirin in singlecomponent products by the proposed method (Table IV). It was much faster in analyses of effervescent preparat,ions.
1
I
I~
274
ANALYTICAL CHEMISTRY
Figure 2. Calibration curve of acetylsalicylic acid (methyl ester)
ACKNOWLtiDGMENT
The authors thank the Sel-0-Min Corp., Baltimore, Md., for samples, C . F. Smullin and other members of the Chemical Research Department, Atlas Chemical Industries, Inc., Wilmington, Del., for helpful suggestions and manuscript review. LITERATURE CITED
(1) Am. SOC.Testing Mater., ASTM Std. Method D-1717, Philadelphia, Pa., 1958. (2) Ibid., Method E-122-58, E-177-61T and E-178-61T. (3) “Analysis of Solvents by Gas Chromatography,” Carbide and Carbon Chem. Corp., Res Dept., Charleston, W. VR.. 1960. (4) Andeis, M. W.,Mannering, G. J., J. Chromatog. 7, 253-60 (1962). (5) Assoc. Offic. Agr. Chemists, Official Methods, 9th ed., pp. 481-93, 502. Washington, D. C., 1960. (6) Burchfield, H. I>., Storrs, N. E., “Biochemical Applications of Gas Chromatography,” p. 434, Academic Press, New York, 1962. ( 7 ) ,Cadman, J., Johns, T., Ulrich, W. F., Pittsburgh Confercnce on Analytical
Chemistry and Applied Spectroscopy, paper 115, March, 1958. (8) Coates, V. J., ?gemon, I. S., “Gas Chromatography, p. 149, Academic Press, Sew York, 19.58. (9) .Grippen, R. C., Freimuth, H. C., Division of Analytical Chemistry, 137th Meeting ACS, Cleveland, Ohio, March 1960. (10) Crippen, R. C., Freimuth, H. C., Meeting-in-Miniature. Universitv of MarylaYnd, College Paik, Md., May1960. (11) Dal Nogare, S., Juvet, Jr., R. S., ASAL. CHEY.34, 35R-47R (1962). (12) Desty, D. H., “Vapour Phase Chromatography,” pp. 316-28, Academic Press, New York, 1957. (13) Ibid.,. pp. 291-313. (14) Doenng, W. von E., Mole, T., Tetrahedron 10, 68 (1960). (15) Ellenbogen, W. C., Division of Analytical Chemistry, 141st Meeting, ACS, Washington, D. C., March 1962. (16) Esposito, G. G., Coating & Chem. Aberdeen Lab., Prov. Ground, Md. Rept. PB-181310, Feb. 28, 1962. (17) Esposito, G. G., Swann, M. H., ASAL. CHEM.33, 1854-8 (1961). (18) Komers, R., Razant, V., Doklady Akad. NU&. S.S.S.R. 126, (6), 1268-9 (19.59’1 \ - - - - I -
(19) Kratzl, K., Gruber, K., Holzforsch. Holzverwert. 9, 87-90 (1957).
(20) National Formulary, 11th. ed., p. 19,
Amer. Pharm. Assn., Washington, D. C., 1960. (21) Ibid., p. 20. (22) Parker, K. D., Fontan, C. R., Kirk, P. L., A N A L . CHEM. 34, 757-60 (1962). (23) Parker. K. D.. Kirk. P. L.. Ibid.. 33. ‘ 1378-81, [1961).‘ (24) Snell, F. D., Snell, C. T., “Colorimetric Methods of Analysis,” Vol. 11, p. 385, Van Xostrand, New York, 1946. (25) Ibid., Vol. JII, p. 412-13. (26) Ibid., Vol. I11 A, p. 387-9. (27) Ibid., Vol. 111 A, p. 390. (28) State of Maryland, Office of Med. Examiner, Annual Rept. (1960). (29) Stewart, C. P., Stolman, A,, “Toxicology,” Vol. I, p. 100,Academic Press, New York. 1960. (30) Tuey, 6. A. P., Soap, Perfumery Cosmetics 31, 353-61 (1958). (31) U. S. Pharmacopea, XVI ed., pp. 20-21,USP, Washington, D. C., 1960. I
.
RECEIVED for review September 24, 1962. Resubmitted October 25, 1963. Accepted October 25, 1963. Equipment that initiated this work was paid for by a Public Health Services grant to the University of Maryland Medical School. Analytical Division 142nd Meeting, ACS, Atlantic City, N. J., September 1962.
Quantitation of Estrone, Estradiol, and Estriol on Thin Layer Chromatograms by a Photogram metric Procedure GERT
M. JACOBSOHN
Department of Biochemistry, Hahnemann Medical College, Philadelphia 2, Pa. A method is presented for evaluating estrone, estradiol, and estriol individually in mixtures of the three compounds. The estrogens are separated by thin layer chromatography and made visible by spraying with sulfuric acid. When the plates are illuminated with ultraviolet light, the spots fluoresce. Photographs of the emitted light are tmlarged and the dark areas corresponding to spots are evaluated densitometrically. By analogy with characteriiitic curves of film, it is postulated that the logarithm of light intensity emittisd by each spot i s proportional to the amount of substance for a range of concentration determined experimentally for each substance under a ciiven set of conditions. This applies to determinations of the three estrogens in which a straight-line relation:;hip between density and amount is obtained.
T
SEPARATION of steroid estrogens by means of thin layer chromatography is well established (7, 9). Quantitative evaluation of material
HE
usually requires elution of substances from the adsorbing media. The present report describes a procedure for evaluating estrone (3-hydroxy-17-0~0estra-1,3,5-triene), estradiol (3, 17 P-dihydroxyestra-l,3,5-triene), and estriol (3,16~,17p- trihydroxyestra - 1,3,5- triene), directly on a plate without prior elution. A theoretical treatment is outlined which provides validity to the data obtained. The procedure involves separation by thin layer chromatography, development of color by suitable reagent, photographing of fluorescent spots, and reproduction of the images on a sheet of film so t h a t spots appear dark on a clear background. The dark areas are evaluated densitometrically. EXPERIMENTAL
Thin Layer Chromatography. All solvents were t h e highest quality available commercially a n d were distilled before use. Solutions of standard estrone, estradiol and estriol were prepared to contain mixtures of 0.1 t o 4.0 pg. of estrone and estradiol, and u p t o 10.8 pg. of estriol in 10 pl. of methanol. Any one sample
contained equal amounts of estrone and estradiol and either an equal amount or three times as much estriol. Glass plates, eight X eight inches, were coated uniformly with adsorbent silica gel H (E. Merck, Darmstadt) t o a thickness of 0.25 mm. Thirty grams of adsorbent was stirred while adding slowly 70 ml. of distilled water. A batch sufficed for six plates. After coating, the plates were dried in the atmosphere for one hour, cleaned from the underside and along the edges, and heated in an oven at 100’ C. for a minimum of two hours. Plates were used immediately after cooling or kept in the oven a maximum of two days. More extended periods of heating caused the silica layer t o darken. Plates which did not present a uniform appearance were discarded. Distances of 1.5 cm. were marked off on t h e plates and lines engraved with a Speed-0-Print stylus No. 14. Sample,. of 10 p1. were applied at a point in the center of each lane, 2.5 cm. from the lower edge. A micropipet was used and heat was applied from t h e bottom of t h e plates t o facilitate evaporation of methanol and keep the area small. The area of spots at the origin was 7 mm. or less in diameter. A maximum of 12 VOL. 36, NO. 2, FEBRUARY 1964
275
