Journal of Analytical Toxicology, Vol. 14, July/August 1990

Note

A Rapid and Simple Color Test for Detection of Salicylate in Whole Hemolyzed Blood W. M i c h a e l

Asselin and Jeffrey

D. Caughlin

Royal Canadian Mounted Police, Forensic Laboratory, 5201 Heather Street, Vancouver, British Columbia, Canada VSZ 3L7

Materials

I Abstract I Acetylsallcylic acid (ASA) Is still one of the most commonly used therapeutic agents. Salicylic acid, the major metabolite of ASA, can be detected easily In urine using simple chemical spot teats such as ferric chloride or Trlnder'a reagent. In forensic cases, urine Is often not available and the rapid detection of sallcylate in whole hemolyzed blood can be difficult. This report describes the rapid and simple detection of aallcylate using ferric chloride and a methanollc extract of whole blood. The color test is rapid and can detect aalicylate at mid-therapeutic concentrations of 5 mg/dL. As little as 300/~L of whole blood is required and no equipment is needed. The color teat can also be used with serum or plasma.

Introduction Acetylsalicylic acid (ASA) is one of the most commonly used therapeutic drugs. ASA has been responsible for numerous salicylate poisonings, particularly in children. The major metabolite of ASA is salicylic acid. The need for rapid detection of salicylate in suspected poisoning cases is obvious. In forensic cases, the need to detect and confirm therapeutic concentrations of salicylate is also important. The presence of therapeutic concentrations of salicylate in a murder victim may or may not corroborate the accused person's testimony. The presence of therapeutic concentrations of salicylate may also be of interest in sexual assault and impaired driving cases. Combination drug preparations containing ASA and CNS depressants such as butalbital or codeine are common in these types of cases. Salicylate is easily detected in-urine using classical color tests such as ferric chloride or Trinder's reagent. Cases in which urine is not available, such as impaired driving and sexual assault cases, can pose a problem. This report describes a modification of the classical ferric chloride test for use with whole hemolyzed blood, serum, or plasma. Our laboratory routinely analyzes a methanolic extract of whole hemolyzed blood using 14 separate EMIT urine and serum assays (1) when urine is not available. Currently, there is no available EMIT assay for the detection of salicylate. We were able to use this same methanolic extract of whole blood to rapidly test for the presence of saticylate.

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Methanol and ferric chloride were reagent grade. Ferric chloride was prepared as a 0.50/0 by weight solution in distilled water. Salicylic acid was obtained from the Department of Health and Welfare, Ottawa and was prepared as a l - m g / m L stock solution in distilled water. The blood, which had been frozen, was bovine blood containing 1% sodium fluoride and 0.25% potassium oxalate.

Procedure

For the EMIT analysis using 14 separate assays and for the test for salicylate, 1.5 mL of whole blood was added to a 13-mL plastic centrifuge tube. Three mL of methanol was added to the blood. The mixture was vortexed vigorously for 3 rain and then centrifuged at 3000 rpm for 5 rain at - 2 0 ~ The clear supernatant (approximately 1.5-2.0 mL) was filtered through a 0.45-/~m disposable-filter assembly (Gelman Acrodisc, Product No. 4184) using a 3-cm 3 disposable syringe (Becton Dickinson). As much sample as possible was removed from the filter assembly by purging with air. This supernatant was then analyzed directly by EMIT (l). The remainder of this supernatant was used for the salicylate test. The sample volume of the methanolic extract required for the salicylate test is 200/~L. If only the salicylate test was to be performed, the above procedure would be carried out with 300/~L of whole blood and 600/~L of methanol. In either case, 200/~L of methanolic extract was transferred by Pasteur pipette to a small test tube (Kimble, borosilicate culture tube 6 x 50 ram). To this was added 50/~L of 0.50/o by weight ferric chloride in water. The ferric chloride fell to the bottom of the tube. As it did, the ferric chloride mixed with the methanolic extract and a diffuse interface formed between the two solutions. A negative control sample prepared from bovine blood was analyzed at the same time. A positive control sample of bovine blood spiked with salicylate to a final concentration of l0 mg/dL was analyzed similarly. The negative control and the case samples were compared against a whitecolored background. The presence of a violet tint at the interface indicated the probable presence of salicylate.

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Journal of AnalyticalToxicology, Vol. 14, July/August 1990

Results and D i s c u s s i o n Ferric chloride has been used as a classic direct test of urine for the presence of phenolic compounds such as salicylate (2). Typically, one or two drops of an aqueous 5% by weight solution of ferric chloride in water is added to approximately 1 mL of urine. A dark violet color indicates the probable presence of salicylate, the major metabolite of ASA. Our initial approach was to use this procedure with methanolic extracts of whole blood. The 5% solution of ferric chloride resulted in a strong yellow color for both negative controls and spiked standards between 0 and 25 mg/dL salicylate. The background yellow color made it very difficult to distinguish the violet color produced by salicylate. Attempts were made to separate the violetcolored complex from the yellow of the ferric chloride. We tried using a water-immiscible organic solvent to separate the two colors. Ferric chloride is readily soluble in ether and ether is virtually insoluble in water. However, ether was very soluble when mixed with the methanolic extract from blood, which contains 66% methanol by volume. Another attempt to minimize the yellow background color was to add the ferric chloride to a 60% by volume solution of glycerine in water. Results were encouraging, but the intensity of the violet color produced varied depending on how much the glycerine and sample layers were mixed and was not easily reproducible. Optimal results were achieved by using a more dilute solution of ferric chloride in water containing 0.5% ferric chloride by weight. Using this solution, the intensity of the yellow background color was greatly reduced. This allowed the violet tint produced from a 10 mg/dL standard of salicylatr in whole blood to be easily observable when compared to a negative control. The minimum detectable concentration of salicylate in whole blood was determined to be 5 mg/dL. This corresponds to a mid-therapeutic concentration (3). The test is semiquantitative in that the intensity of the violet color increases with an increase in salicylate concentration. The other classical color test for the detection of salicylate in urine is Trinder's reagent. This reagent can also be used with plasma and can be used quantitatively (4). Our attempts to use Trinder's reagent failed. The color produced by Trinder's reagent was much less intense than that produced by ferric chloride. As such, using the procedure as described, Trinder's reagent was simply not as sensitive as ferric chloride. Currently, our laboratory is screening blood for salicylate using the above method whenever urine is not available. When urine is available, it is tested directly with ferric chloride. In cases where the urine ferric chloride test has gone positive, the proposed method is used with whole blood from the same case. Quite often, the blood tests negative for salicylate despite a positive urine test. This situation simply reflects the latter stages of metabolism and excretion of salicylate. Normally, following a positive urine test for salicylate, the salicylate in blood would be quantified. If it turned out that no salicylate was detected in

blood during the quantification, the analyst would have wasted both time and sample. The ability to rapidly test blood for salicylate to therapeutic concentrations can clearly be beneficial. Blood samples that test positive for salicylate by the proposed method are quantified and confirmed. Our laboratory routinely uses either an HPLC acid/neutral drug screen modified from that of Chart and Chan (5) or a differential UV method (6).

Summary

Advantages of the proposed method include the following: (1) It allows the rapid and simple screening of whole blood for salicylate when urine is not available; (2) when urine is available, the blood can also be screened for salicylate and if salicylatc is not detected in blood, no time or sample need be wasted attempting to quantify the salicylate in blood; (3) only 300 ~L of whole blood is required to detect a mid-therapeutic salicylate concentration (5 mg/dL); (4) sample preparation is easy to perform and no instrumentation is required; and (5) the methanolic extract from whole blood used for this test can also be analyzed directly by at least 14 separate EMIT d.a.u, and serum assays.

9A c k n o w l e d g m e n t s The authors wish to thank their colleagues in the Toxicology Section for their assistance and support. Sincere thanks are extended to Mrs. D. Asselin for her word processing skills.

References 1. W.M. Asselin, J.M. Leslie, and B. McKinley. Direct detection of drugs of abuse in whole hemolyzed blood using the EMIT d.a.u, urine assays. J. Anal. Toxlcol. 12:207-15 (1988). 2. In Clarke's Isolation and Identification of Drugs, 2nd ed., A.C. Moffat, Ed., The Pharmaceutical Press, London, England, 1986, p. 133. 3, In Disposition of Toxic Drugs and Chemicals in Man, 2nd ed., R.C. Baselt, Ed., Biomedical Publications, Davis, California, 1982, pp. 15-19. 4. R Trinder. Rapid determination of salicylate in biological fluids. Biochem. J. 57:301-303 (1954). 5. E.M. Chan and S.C. Chart. Screening for acidic and neutral drugs by high performance liquid chromatography in postmortem blood. J. Anal Toxicol. 6:173-76 (1984). 6, In Methodology for Analytical Toxicology, t. Sunshine, Ed., C.R.C. Press, Cleveland, Ohio, 1975, pp. 343-46. Manuscript received November 27, 1989.

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A rapid and simple color test for detection of salicylate in whole hemolyzed blood.

Acetylsalicylic acid (ASA) is still one of the most commonly used therapeutic agents. Salicylic acid, the major metabolite of ASA, can be detected eas...
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