Susan R. Lane
and James T. Stewart School of Pharmacy University of Georgia Athens, 30602
Titrimetric Determination of Sodium Salicylate in Blood A student laboratory experiment
Knowledge of scientific literature reveals the increasing prominence of the application of analytical techniques to the determination of drugs in biological fluids. Yet the number of experiments designed for students interested in drug analysis is small. Assay methods for drugs in hiological systems usually have not been written for the undergradlate teaching- laboratory. Most lahoratory methods available are found in t o x i c o l o ~manuals or handbooks (I, 2). Therefore, a lahoratory experiment was designed which would demonstrate the analysis of a drug in one type of biological fluid, namely hlood, in a simple and straightforward manner. The exercise and a discussion of its application in an undergraduate analysis course are the subjects of this paper. Diphasic titrimetry is a useful technique for the analysis of drugs that give water insoluble products that ohscure the visual endpoint. In the technique, an aqueous solution of a drug is titrated with standardized acid or base titrant in a separatory funnel containing an immiscible organic solvent. As the titration proceeds, the waterinsoluble product liberated passes into the organic solvent, thus allowing for a more precise observation of the equivalence or endpoint. Sodium salicylate was chosen for analysis since it represents a widely used medicinal agent and is also important as one of the primary products of aspirin metabolism. It yields, upon direct titration with standardized acid solution, an insoluble precipitate that obscures the visual endpoint. However, by utilizing a diphasic titration in which diethyl ether is the immiscible brganic solvent, the titration proceeds in a satisfactory manner. This method has been adopted for the analysis of sodium salicylate in blood. Procedure
Prior to their laboratory session, the students were assigned relevant texts (3, 41 and an adequate time for students' questions was arranged. In the laboratory, the instructor demonstrated the technique of diphasie titrimetry. A 500-mg sample of pure powder (sodium salicylate) was dissolved in 50 ml of water and transferred to a 125ml seoaratorv funnel. Twenty-five milliliters of diethyl ether and 5 drops of brbmaphenol blueindicator were added. b he two-phase solution was titrated with N / 2 hydrochloric acid until a green color was obtained in the aqueous layer. The separatory funnel was stoppered and shaken at intervals during the titration. The aqueous layer was then transferred to a clean separatary funnel and the stem rinsed with 2 ml of water. The remaining ether layer was washed with two 5-ml portions of water. The stem and ether washings were then added to the original water solution. To this aqueous layer was added, with shaking, 20 ml of fresh ether. The titration was continued, shaking well after each addition, until a green color was obtained again in the water layer. A blank determination was also prepared. The percent recovery of sodium salicylate was calculated. Each student was then asked to accurately weigh two quantities (250 mg) of sodium salicylate. One portion was dissolved in 10 ml of freshly collected whale blood1 and the other in 10 ml of serum obtained by centrifugation of the blood. These were placed in 125-ml separatory funnels and assayed by the titrimetric method described above, along with a blank determination. The percent recovery of sodium salicylate in whole hlood versus serum was calculated. 588
/ Journal 01 Chemical Education
Table 1. Analysis of Sodium Salicylate in Serum
Sodium salicylate Sodium salicylate added (me) found (me)
152.6 179.3 199.1 221.1 255.5
160 .O 176.0 192 .O 220.0 248.0 averaze recoverv
% Recoverv
104.80 98.15 96.50 99.50 97.06 99.20 =t3.33
Table 2. Analysis of Sodium Salicylate in Whole Blood
Sodium salicyla$e Sodium salicylate added (mg) found, mg
average recoverv
% Recovery
86.40 + 2.90
Finally, the student supplied to the instructor another sample of serum, to which was added a quantity of sodium salicylate to serve as an "unknown." These solutions were analyzed and the calculated results obtained were placed in a laboratory report which was graded by the instructor according to preset guidelines. Results and Discussion
Thirty students working in groups of six performed the exercise. Each group was led by a graduate student assistant who, in essence, was the lahoratory instructor for his group. Typical results obtained by the students in the analysis of samples of sodium salicylate in whole hlood and serum are given in Tables 1and 2. An evaluation of the data collected by the students shows that the experiment provides a simple approach to demonstrating the analysis of a drug in hlood. The entire exercise is performed with equipment which is readily available and inexpensive. The simplicity of sample preparation permits the students to focus on the analysis, and the fact that the procedure was performed in blood received a positive response from the students. The two-step extraction process is necessary to achieve quantitative results for sodium salicylate. If only a one-step extraction is used, extraction efficiency is on the order of 90-95%. During lecture the laboratory results are discussed within the context of the capabilities, limitations, and applications of diphasic titrations as applied to drug analysis in biological fluids. The concentration of sodium salicylate used in this experiment is an approximate hundred-fold excess over that which would he present a t the maximum therapeutic level in human serum. The experiment is designed to acquaint the student with drug analysis in biological fluid, and diphasic titrimetry was chosen as an analytical tool to help achieve the goal. In actual practice, salicylate 'Bovine hlood supplied by the College of,Veterinary Medicine was used. Human blood can also be used if available.
levels are determined by either ultraviolet or visible spectrophotometry (5, 61. In summary, the exercise introduces the student to the of drug analysis in whole blood and serum without burdening him with excessive theoretical and technical considerations. Literature Cited 111 Sunshine, I. (Editor). "Manual of Analytical Torieoloey," Tho Chemical Rubber
CO., c ~ ~ ~ ~ ~ s ~ 1971. d,ohio, 121 Sunshine. I. IEditurl. "Handboak of Analytical Toxicology." The c h m i e a i ~ u b h e r Co.. Cleveland. Ohio. 1969. I31 C o m m . K. A . "A Textbook of Phannaceuticsl Analysis." John Wilsy & Sons. h e . , New Yak, 1967, pp3-17. I41 ~ ~ k ~A. t H. t,, stenlake, J. B.. -practical ~ h ~ h ~ ~~2ndi ~ Ed.. Part I. The Athlone Press, London. 1968. p 114. 15) Williarns.L.. Lion. R., and^& B.. J ~ a bc.l i n hied.. s.156119~3). (61 T7ind.r. P.. B i o c h m J , 57.301 11954).
Volume51. Numberg, September 1974
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