**2.1 SA in blood**

Examination of the chromatograms of blank serum or plasma from published methods of SA analysis revealed the presence of an unknown substance with a retention time (Rt) similar to SA [5, 6]. While Ruffin et al. [7] reported SA in the plasma from 17 of 53 subjects at baseline there was no information as to how they confirmed identification of the compound.

We examined samples from drug free volunteers: extracts of acidified serum were analysed by high performance liquid chromatography (HPLC) with electrochemical detection. Chromatographic conditions were altered and the Rts of the unknown compounds compared against authentic SA, 2,3 DHBA and 2,5 DHBA. Serum samples (some spiked with SA) were also incubated with a bacterial salicylate hydroxylase and the substance which had a Rt identical to SA disappeared. Finally the trimethylsilyl (TMS) derivative of the unknown and SA had, using gas chromatography–mass spectrometry (GC–MS), a similar retention time and total ion chromatogram [8].

## **2.2 SA and salicyluric acid (SU) in urine**

Armstrong et al. [9] had detected, by paper chromatography, a compound with characteristics similar to those of SU in the urine of 400 people who had not taken salicylate drugs. That was in an admixture of 49 compounds of predominantly, it was suggested, dietary origin. Von Studnitz and colleagues, who also used a paper system, suggested SA might be one of the phenolic acids in the urine of subjects *on a*

#### **Figure 1.**

*Metabolism of aspirin, salicylic acid and benzoic acid. (1) acetylsalicylic acid (aspirin); (2) salicylic acid; (3) salicyluric acid; (4) 2,5 dihydroxybenzoic acid; (5) 2,3 dihydroxybenzoic acid; (6) 2,3,5 trihydroxybenzoic acid; (7) benzoic acid; and (8) hippuric acid.*

*Salicylic Acid Sans Aspirin in Animals and Man DOI: http://dx.doi.org/10.5772/intechopen.91706*

dihydroxybenzoic acids (DHBAs)—**Figure 1**—required a sensitive HPLC assay with appropriate controls. That work revealed the presence of substances which had identical retention times to SA, 2,3 DHBA and 2,5 DHBA in the serum extracts of subjects not taking aspirin. The exclusion of contamination was followed by studies to determine the authenticity of these substances as SA, 2,3 DHBA and

*Drug Repurposing - Hypothesis, Molecular Aspects and Therapeutic Applications*

Examination of the chromatograms of blank serum or plasma from published methods of SA analysis revealed the presence of an unknown substance with a retention time (Rt) similar to SA [5, 6]. While Ruffin et al. [7] reported SA in the plasma from 17 of 53 subjects at baseline there was no information as to how they

We examined samples from drug free volunteers: extracts of acidified serum were analysed by high performance liquid chromatography (HPLC) with electrochemical detection. Chromatographic conditions were altered and the Rts of the unknown compounds compared against authentic SA, 2,3 DHBA and 2,5 DHBA. Serum samples (some spiked with SA) were also incubated with a bacterial salicylate hydroxylase and the substance which had a Rt identical to SA disappeared. Finally the trimethylsilyl (TMS) derivative of the unknown and SA had, using gas chromatography–mass spectrometry (GC–MS), a similar retention time and total

Armstrong et al. [9] had detected, by paper chromatography, a compound with characteristics similar to those of SU in the urine of 400 people who had not taken salicylate drugs. That was in an admixture of 49 compounds of predominantly, it was suggested, dietary origin. Von Studnitz and colleagues, who also used a paper system, suggested SA might be one of the phenolic acids in the urine of subjects *on a*

*Metabolism of aspirin, salicylic acid and benzoic acid. (1) acetylsalicylic acid (aspirin); (2) salicylic acid; (3) salicyluric acid; (4) 2,5 dihydroxybenzoic acid; (5) 2,3 dihydroxybenzoic acid; (6) 2,3,5 trihydroxybenzoic*

2,5 DHBA.

**2.1 SA in blood**

ion chromatogram [8].

**Figure 1.**

**178**

*acid; (7) benzoic acid; and (8) hippuric acid.*

confirmed identification of the compound.

**2.2 SA and salicyluric acid (SU) in urine**

*diet restricted to glucose and citric acid* [10]. Young reported a compound with thin layer chromatographic properties of SU [11] in a single individual on a synthetic diet, while Finnie and co-workers found a similar compound on their paper system in children not taking salicylate drugs [12]. At the time of all these earlier investigations methods for adequately characterising and quantifying the purported SA and SU were not readily available.

Other work [13] designed to assess the dietary importance of salicylates had examined acid-treated urine using HPLC with fluorescence detection but salicylates other than SA, salicylate precursors and structurally related compounds may have been included in the values reported.

We examined 24 h urine samples from 10 volunteers who had not taken any salicylate drugs during the previous 2 weeks. The acid hydrophobic compounds (=organic acids) were separated using HPLC and quantified electrochemically. The Rts of the extracted substances and those of SA and SU were compared under two sets of chromatographic conditions and found very similar to those of authentic substances. The unknown substances, isolated by HPLC, and treated with acetyl chloride in methanol were compared with the methyl esters of SA and SU using GC–MS. After esterification the unknown compounds had mass spectra and Rts comparable to those of methyl-SA and methyl-SU [14].

These findings in serum and urine led us on to explore, in some detail, the likely dietary sources for the salicylate compounds we found and confirmed. At the time interest was re-awakening in the potential benefits of salicylates commonly found in the diet [13]—as opposed to what were considered traditional plant medicine sources.
