Silica-titania xerogel for solid phase spectrophotometric determination of salicylate and its derivatives in biological liquids and pharmaceuticals
© Morosanova and Morosanova. 2015
Received: 30 July 2015
Accepted: 15 November 2015
Published: 25 November 2015
Salicylic acid and its derivatives are widely used drugs with potential toxicity. The main areas of salicylate derivatives determination are biological liquids and pharmaceuticals analysis.
Silica-titania xerogel has been used for solid phase spectrophotometric determination of various salicylate derivatives (salicylate, salicylamide, methylsalicylate). The reaction conditions influence on the interaction of salicylate derivatives with silica-titania xerogels has been investigated; the characteristics of titanium(IV)-salicylate derivatives complexes in solid phase have been described. The simple solid phase spectrophotometric procedures are based on the formation of xerogel incorporated titanium(IV) colored complexes with salicylate derivatives. A linear response has been observed in the following concentration ranges 0.1–5, 0.5–10 and 0.05-4.7 mM for salicylate, salicylamide, and methylsalicylate, respectively. The proposed procedures have been applied to the analysis of human urine, synthetic serum, and pharmaceuticals.
The simple solid phase spectrophotometric procedures of salicylate derivatives determination based on the new sensor materials have been proposed for biological liquids and pharmaceuticals analysis.
Salicylic acid and its derivatives (acetylsalicylic acid, salicylamide, methylsalicylate) are widely used as anti-inflammatory, analgesic drugs . Acetylsalicylic acid is the most commonly used salicylate derivative, it is used as analgesic, antipyretic, and also as an antiplatelet drug. Salicylamide is used as an analgesic and antipyretic in several combination products. It is necessary to control their presence in biological fluids due to the potential toxicity. Methylsalicylate is also used as an anti-inflammatory drug, but it is highly toxic if ingested and is only prescribed for external application. The drugs of this group have common pharmacological effect and all of them are toxic in high concentrations. A plasma level higher than 2.2 mM of salicylate is considered to be toxic , and the level higher than 4.3 mM is regarded as lethal . The therapeutic range (0.5–1.5 mM of salicylate in plasma) is very close to the toxicity level.
Monitoring salicylates concentration in biological liquids is important for controlling the dose and frequency of salicylate derivative drug administration as all the salicylate derivatives mostly convert to salicylate in the organism. Accidental overdoses of salicylates are considered to be common in children. Salicylates are one of the toxicants that must be determined in serum and urine of patients of emergency department . The allergenic capacity of salicylates also dictates the necessity of monitoring their levels in biological liquids. Another essential field for salicylate derivatives analysis is the pharmaceuticals quality control.
Various methods have been proposed for the determination of salicylic acid and its derivatives in biological liquids and pharmaceutical preparations: chromatography [4, 5], spectroscopic [6–9], electrochemical [10–15]. Many spectrophotometric and electrochemical methods of salicylate determination are based on its ability of forming complexes with metals: colored complex of salicylate with iron (III) is used in the classical method of salicylate determination (Trinder test) in biological samples ; complexing reaction with transition metal ions is employed in ion-selective electrodes construction [12–14].
The development of the simple methods of salicylate determination is a high-demand task, considering the wide use of salicylate derivatives in medical practice. The search for new sensor materials with an ability to form complexes with salicylates is a highly perspective goal.
Titanium(IV) forms colored complexes in weakly acidic solutions with some aliphatic and aromatic ligands. For example, salicylic acid and 5-chlorosalicylic acid are widely used for spectrophotometric determination of titanium(IV) .
In our previous works the ability of titanium(IV) embedded in silica-titania xerogel matrix to form complexes with ascorbic acid, polyphenols, dopamine, hydrogen peroxide, and fluoride ions was exploited to develop the solid phase spectrophotometric procedures for these substances determination [17–20].
The aim of the present work was to study the complex forming between the silica-titania xerogel and salicylate, salicylamide, or methylsalicylate and to choose the conditions of these salicylate derivatives and also acetylsalicylic acid determination in pharmaceuticals and salicylate determination in biological liquids.
Results and discussions
Interaction of silica-titania xerogel with salicylate derivatives
In the present work the silica-titania xerogel used for complex formation study was prepared using the sol–gel technology. Tetraethoxysilane and titanium(IV) tetraethoxyde were used as precursors. The xerogel with 12.5 % titanium(IV) tetraethoxyde content was chosen considering the micropore distribution analysis . Then the optimal conditions for the complex forming reaction were investigated.
Characteristics of titanium(IV)—salicylate derivatives complexes in solid phase
Salicylate derivative (L)
lgKeq in solid phase
Selected conditions were applied to solid phase spectrophotometric determination of salicylate derivatives.
Analytical characteristics of solid phase spectrophotometric determination of salicylate derivatives (λ 410 nm, time of contact is 15 min)
Analytical range (mM)
Limit of detection (mM)
A = 150·C
A = 86·C
A = 125·C
Salicylate determination in biological liquids
Recovery test of solid phase spectrophotometric determination of salicylate in biological liquids (n = 3, P = 0.95)
0.49 ± 0.10
0.97 ± 0.15
1.42 ± 0.05
2.09 ± 0.04
0.46 ± 0.07
0.70 ± 0.09
1.08 ± 0.07
The analytical range of the developed procedure made it possible to determine various salicylate levels in the serum samples: below the therapeutic dose (<0.5 mM), the therapeutic dose (0.5–1.5 mM), and overdose (2.0 mM).
The analysis of the real sample of human urine proved that the developed procedure allowed the determination of salicylate processed by the organism which is often required in the medical applications.
Solid phase spectrophotometric determination of salicylate derivatives in real samples using the standard addition method (n = 3, P = 0.95)
Found (independent method)
Found (proposed method)
Deep Heat creama
128 mg/g (DC)
123 ± 8 mg/g
240 mg/tablet (DC)
220 ± 10 mg/tablet
Acetylsalicylic acid tablets Tatkhim
500 mg/tablet (DC)
500 ± 20 mg/tablet
Acetylsalicylic acid tablets Medisorb
500 mg/tablet (DC)
510 ± 20 mg/tablet
Synthetic serumc, containing 1 mM salicylate
1.03 ± 0.05 mM (TT)
0.97 ± 0.15 mM
0.42 ± 0.01 mM (TT)
0.46 ± 0.07 mM
Determination of salicylate derivatives in pharmaceuticals
The determination of salicylate derivatives in pharmaceutical samples always comprises the difficulties of various interferences, as many drugs have complex composition and may contain other active substances in comparable quantities.
Determination of salicylate and methylsalicylate in mixed solutions (n = 3, P = 0.95)
Salicylate 2.5 mM
Methylsalicylate 3.75 mM
Salicylate 2.5 ± 0.2
Methylsalicylate 1.9 mM
Salicylate 5.0 mM
Methylsalicylate 1.97 ± 0.5
Recovery test of solid phase spectrophotometric determination of salicylate derivatives (n = 3, P = 0.95)
Deep heat creama
1.01 ± 0.24
1.85 ± 0.27
2.83 ± 0.24
Acetylsalicylic acid tablets Medisorb
0.57 ± 0.06
1.07 ± 0.06
1.68 ± 0.06
Acetylsalicylic acid tablets Tatkhim
0.69 ± 0.09
1.20 ± 0.09
1.79 ± 0.09
0.49 ± 0.09
1.02 ± 0.15
1.58 ± 0.15
The salicylate derivatives concentrations found in the pharmaceuticals have shown good agreement with the content declared by the manufacturer (Table 4). Among other advantages the determination in the presence of other active substances should be noted. The analgesic, anti-inflammatory pharmaceuticals based on salicylate derivatives often contain other active substances, such as paracetamol, caffeine, or menthol, and the absence of their interference broadens the range of possible applications.
The proposed procedures can be characterized as simple and fast and do not require complex labware or special storage conditions, and the analytical range is suitable for pharmaceuticals analysis. The silica-titania xerogel used as the sensor material is highly stable compared to other sensor materials.
The following reagents were purchased from Acros Organics: hydrochloric acid, sodium tetraborate, sodium salicylate, methylsalicylate, salicylamide, sodium hydrocarbonate, citric acid, sodium chloride, aminoacids, titanium(IV) tetraethoxyde, and tetraethyl orthosilicate. All the reagents were of analytical grade, titanium(IV) tetraethoxyde was of technical grade.
Stock solutions of salicylate and salicylamide were prepared with doubly distilled water. Stock solution of methylsalicylate was prepared with ethanol. Only freshly prepared solutions of these substances were used.
Trinder reagent was prepared as in : 4.0 g of iron (III) nitrate nonahydrate and 5.0 g of trichloroacetic acid were dissolved in 100.0 mL of doubly distilled water.
Silica-titania xerogel was obtained by drying in Ethos microwave complex (Milestone, Italy).
Light absorbance of the xerogels water suspensions was measured using KFK-3 spectrophotometer (ZOMZ, Russia) and glass cuvettes (0.1 cm). Cuvette filled with non-colored xerogel water suspension was used as blank.
Light absorbance of the salicylate derivatives solutions was measured using KFK-3 spectrophotometer (ZOMZ, Russia) and quartz cuvettes (1.0 cm).
The pH value was measured using Expert-001 (Econix Expert, Russia) potentiometer with pH-sensitive electrode.
Surface area and porosity BET analysis was carried by using ASAP 2000 (Micromeritics, Norcross, GA, USA).
Synthesis of silica-titania xerogel
Silica–titania xerogel was obtained using earlier developed procedures : 20.0 mL of 0.05 M hydrochloric acid in 50 % ethanol solution was added to 10.0 mL of the precursors mixture (12.5 % titanium(IV) tetraethoxyde, 87.5 % tetraethoxysilane) while stirring. The wet gel was formed in the next 72 h. The wet gels were dried at 800 W microwave irradiation for 10 min to get dry xerogels.
The main characteristics of the xerogel: BET surface area is 540 m2/g, micropore volume is 0.14 cm3/g.
Interaction of silica-titania xerogel with salicylate derivatives at different pH
0.10 g of silica-titania xerogel was added to 5.0 mL of solution, containing 2 mM salicylate, 3 mM salicylamide, or 2.5 mM methylsalicylate. pH of the solution was adjusted adding 0.01–2.0 mL of 0.5 M sulfuric acid, or 1.0 mL of phosphate buffer (pH 4–8), or 1.0 mL of borate buffer (pH 8–12). The obtained mixture was shaken for 15 min. Then the xerogels light absorbance was measured at 410 nm and pH of the solution was measured.
Interaction of xerogel with salicylate derivatives kinetics studies
0.10 g of silica-titania xerogel was added to 5.0 mL of solution, containing 2 mM salicylate (pH 2), 3 mM salicylamide (pH 2), or 2.5 mM methylsalicylate (pH 7.6).The obtained mixture was shaken for 2–20 min. Then the xerogels light absorbance was measured at 410 nm.
Determination of complexes composition and equilibrium constants
0.10 g of silica-titania xerogel was added to 5.0 mL of solution, containing 0.1–25 mM salicylate (pH 2), 0.5–10 mM salicylamide (pH 2), or 0.05–4.7 mM methylsalicylate (pH 7.6). The obtained mixture was shaken for 15 min. Then the xerogels light absorbance was measured at 410 nm. The solution absorbance was measured at 340 nm for salicylate, 320 nm for salicylamide, 315 nm for methylsalicylate. The concentration of unreacted salicylate derivative left in solution was determined using calibration curve at the corresponding wavelength.
0.10 g of silica-titania xerogel was added to 5.0 mL of solution, containing 0.1–5 mM salicylate (pH 2), 0.5–10 mM salicylamide (pH 2), or 0.05–4.7 mM methylsalicylate (pH 7.6). The obtained mixture was shaken for 15 min. Then the xerogels light absorbance was measured at 410 nm. Calibration curves were obtained using the least squares method.
Determination of salicylate derivatives in synthetic serum
Synthetic serum was prepared as in . 1.0 mL of Trinder reagent was added to 5.0 mL of synthetic serum containing 0.1–2 mM salicylate. After 30 min the colored solution absorbance was measured at 620 nm (l 1.0 cm).
0.10 g of silica-titania xerogel and 0.1 mL of 0.5 M sulfuric acid were added to 5.0 mL synthetic serum containing 0.1–2 mM salicylate, the mixture was shaken for 15 min. After that the xerogel light absorbance was measured.
Determination of salicylate derivatives in human urine sample
The research was carried out according to the World Medical Association Declaration of Helsinki, and informed consent was obtained from the subject. The research was also approved by MSU Bioethics Committee [decision N 23-ch(3)]. Author of this work volunteered for the research: the healthy volunteer received a 1000 mg acetylsalicylic acid dose by oral administration. After 1 h the urine sample was collected.
1.0 mL of 0–3.1 mM salicylate was added to 1.0 mL of urine, and then 2.0 mL of Trinder reagent was added. After 30 min the colored solution absorbance was measured at 620 nm (l 1.0 cm).
2.5 mL of 0–1.3 mM salicylate was added to 2.5 mL of urine, and then 0.10 g of silica-titania xerogel and 0.1 mL of 0.5 M sulfuric acid were added. The mixture was shaken for 15 min and the xerogel light absorbance was measured.
Determination of methylsalicylate in pharmaceutical samples
1.50 g of Deep Heat cream was diluted in ~20 mL of ethanol, and then boiled for 5 min. After cooling the solution was filtered to a 25.0 mL volumetric flask. Then the flask was diluted to the mark with ethanol. 0.1 mL of diluted sample was mixed with 3.75 mL of doubly distilled water, 1.0 mL of borate buffer (pH 8.5), 0.15 mL of standard methylsalicylate solution, and 0.10 g of xerogel, and then shaken for 15 min. After that the xerogel light absorbance was measured. Methylsalicylate concentration was determined using standard addition method.
Determination of acetylsalicylic acid in pharmaceutical samples
Tablets, containing acetylsalicylic acid, were ground to powder, and an amount of powder, containing ~0.05 g of acetylsalicylic acid was weighed. 5.0 mL of 2 M NaOH was added to the powder, and then diluted with ~20 mL of doubly distilled water. The solution was heated for 5 min. After the cooling the solution was filtered to a 50.0 mL volumetric flask. Then the flask was diluted to the mark with doubly distilled water. This acetylsalicylic acid hydrolysis procedure completeness was verified by applying it to standard solutions of acetylsalicylic acid. 10.0 mL of hydrolyzed sample was mixed with 20.0 mL of standard salicylate solution, then pH was adjusted to 6.2, and then the solution was transferred to a 50.0 mL volumetric flask, which was diluted to the mark with doubly distilled water. 2.5 mL of diluted sample was mixed with 2.5 mL of doubly distilled water, 0.1 mL of 0.5 M sulfuric acid, and 0.10 g of silica-titania xerogel, and then shaken for 15 min. After that the xerogels light absorbance was measured. Acetylsalicylic acid concentration was determined using standard addition method.
The reliable and simple method of salicylate derivatives determination based on the xerogel incorporated titanium(IV) complexes with salicylate derivatives has been proposed. In comparison with other methods of salicylate derivatives determination the proposed method key characteristics is its simplicity, whereas the analytical ranges are comparable with other methods [5, 6, 10, 11]. The procedures for solid phase spectrophotometric determination of salicylate derivatives in biological liquids and pharmaceuticals have been proposed. These new sensor materials are stable and ready to use and can be successfully applied to biological liquids and pharmaceuticals analysis.
EIM has designed the study. EIM and MAM have written the paper. MAM conducted the experiments. EIM and MAM have conducted the data analysis. All authors read and approved the final manuscript.
Authors would like to thank A.A. Alekseev for the participation in the experiments. The study was funded by Russian Science Foundation (Grant N 14–23–00012).
The authors declare that they have no competing interests.
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