Rose Bengal photosensitized flow injection chemiluminescence method is reported using luminol-Cu(II) for the determination of vitamins A and C in pharmaceutical formulations. The reaction is based on the enhancement effect of analyte in the production of anion radicals of Rose Bengal (RB
Vitamins are organic substances present in several foods in low quantities and are indispensable to organism functions. Their systematic absence in the diet can result in deficient growing and development. Vitamin A is a necessary micronutrient in the diet for vision, growth, reproduction, and maintenance of the immune system. Humans require only minute amount of vitamin A in their diets (400 to 1300
All glassware and bottles were cleaned overnight in 2% v/v nutrient free detergent (Neutracon, Decon Laboratories Ltd., Hove, UK), rinsed with ultrahigh purity (UHP) water (0.0167
Luminol stock solution (0.01 M) was prepared by dissolving 0.178 g of luminol (5-amino-2,3-dihydrophthalazine-1,4-dione) in 25 mL of 0.01 M sodium hydroxide, sonicated for 10 min, made up of 100 mL with UHP water, and stored in a polyethylene volumetric flask at 4°C. The working luminol solution (5 × 10−5 M) was prepared by diluting 1.0 mL of the stock solution to 200 mL with 0.125 mol L−1 sodium hydroxide. This solution was stored for at least 24 h to allow removal of trace metal impurities from the alkaline reagent via adsorption on to the walls of the reagent bottle and used as required.
Copper(II) stock solution (0.01 M) was prepared by dissolving the required amount of copper nitrate in 0.1 M HCl. RB stock solution (1 × 10−3 M) was prepared daily by dissolving the required amount in 100 mL water and stored at room temperature. A mixture solution of RB (1 × 10−4 M) and copper nitrate (5 × 10−4 M) was prepared daily by diluting required volume of these stock solutions with pyrophosphate buffer (pH 10.25, 0.01 M).
For the interference study, stock solutions (500 mg L−1) of
The FI-CL manifold used in this work is shown in Figure
FI-CL manifold for the determination of retinol. R1 = pyrophosphate buffer (0.01 M, pH 10.25), R2 = RB (1 × 10−4 M) containing Cu2+ (5 × 10−4 M), R3 = luminol solution (5 × 10−5 M), CR = chart recorder, HV = high voltage power supply, and PMT = photomultiplier tube detector.
The photoreactor used in the proposed studies consisted of PTFE tubing (2.0 m length × 0.8 mm i.d., Fisher, UK) wound around a thin glass plate with exposed area (4.5 × 5.5 cm) placed on a compact 4 W UV lamp (Model UVGL-25, UVP, Upland, USA). The plate was placed at a distance of 1.0 cm from the lamp which produced ultraviolet radiation of wavelength 254 nm. The lamp, glass plate, and PTFE tubing were covered with aluminum foil. The process of photoreaction was carried out at room temperature.
The commercial pharmaceutical formulations containing vitamin A were analyzed by the proposed method. Initially, these samples were saponified by taking an amount of each sample and dissolved in 3 mL methanol in light protected glass tube covered with aluminum foil and then treated with KOH (3 mL, 60%). The mixture was incubated at 45–50°C for 2 h with intermittent mixing. The incubated sample was then extracted four times with n-hexane (5 mL). The organic layer was recovered and dried under nitrogen stream and the residues were redissolved in methanol. After appropriate dilution with pyrophosphate buffer (pH 10.25, 0.01 M), the samples were injected directly to the proposed FI-CL manifold for the determination of retinol.
To establish the optimum conditions for the determination of retinol, the effects of key chemical and physical parameters were investigated using a univariate approach. These include selection of a catalyst, pyrophosphate buffer pH, reagents concentrations (buffer, RB, copper(II), sodium hydroxide, and luminol), flow rates, sample volume, and photoreactor length that were optimized for maximum CL intensity and the results are summarized in Table
Selection of variables.
Variables | Range studied | Optimum |
---|---|---|
Pyrophosphate buffer pH (0.01 M) | 8.5–11.0 | 10.25 |
Rose Bengal (M) | 1 × 10−5–2.5 × 10−4 | 1 × 10−4 |
Copper(II) (M) | 1 × 10−4–1 × 10−3 | 5 × 10−4 |
Luminol (NaOH 0.12 M) | 1 × 10−6–1 × 10−5 M | 5 × 10−5 M |
Total flow rate (mL min−1) | 2–10 | 6.0 |
Sample volume (μL) | 60–300 | 60 |
Photoreactor coil length (m) | 0.5–3.0 | 2.0 |
It has been reported that RB has the ability to generate oxidizing species via metal ion catalyzed free radical decomposition of hydrogen peroxide [
The efficiency of photoproducts by RB in the presence of analyte under UV light was highly dependent upon the pH of pyrophosphate buffer. The optimum pyrophosphate buffer pH was examined over the range of 8.5–11.0. An increase pyrophosphate buffer pH up to 10.25 resulted in a maximum CL response and further increase in pH gave decrease in CL response. Therefore, pyrophosphate buffer of pH 10.25 was selected and used for subsequent experiments. The pyrophosphate buffer concentration (pH 10.25) was then examined over the range of 0.001–0.05 M. Maximum CL intensity was observed at 0.01 M with reproducible signals and further increase in buffer concentration resulted in noisy CL signals and high background. Therefore, pyrophosphate buffer of pH 10.25 with concentration of 0.01 M was selected and used subsequently.
RB is a well-known and widely studied water-soluble photosensitizing dye of xanthine origin with a strong visible absorption band around 500–550 nm [
It is well-known that the luminol CL reaction requires alkaline medium conditions. In the proposed CL system, various alkaline buffer solutions were examined for luminol, including carbonate, borate, and sodium hydroxide to improve the sensitivity of the system. Sodium hydroxide was found the most suitable medium for present CL system. Therefore, the effect of sodium hydroxide concentration was examined over the range 0.05–0.2 M. An optimum CL intensity was observed at 0.125 M sodium hydroxide, above which a decrease in CL intensity was observed with high background and noisy CL signals. Therefore, sodium hydroxide concentration of 0.125 M was used in all subsequent experiments.
The effect of luminol concentration was then examined over the range
The effect of key physical parameters including flow rates, sample volume, and photoreactor coil length was then investigated (Table
Rose Bengal is a xanthine dye with outstanding photosensitizing ability [
The high efficiency of RB as a photosensitizer is because of the high yield of formation of the lowest triplet excited state of RB (RB*(T)) and its long intrinsic life time. It has been reported that RB can efficiently sensitize the generation of singlet oxygen (O2 (
Retinol (analyte) can go through electrons transfer reactions being n-electron donor as reported [
The method is successfully applied to the determination of vitamins A and C and RB in pharmaceutical formulations and results are given in Table
Analytical performance and analyte determination in pharmaceuticals by the proposed method (
Figures of merit | Vitamin A | Vitamin C | Rose Bengal |
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Calibration equation [ |
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Linear range (μg/mL) | 0.05–15 | 0.01–20 | 0.1–50 |
Limit of detection (μg/mL, |
0.008 | 0.005 | 0.05 |
Sample throughput | 70 | 70 | 30 |
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Pharmaceutical formulations | Proposed method results/ |
Proposed method results/ |
Proposed method results/ |
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Capsules | 210 ± 3/(200) | — | — |
Chewable dragee | 9975 ± 5/(9900) | 97 ± 6/(100) | — |
Eye drops (0.6 mL) | — | — | 1 ± 0.1/(1) |
Injections | 3378 ± 4/(3300) | — | — |
Tablets | — | 509 ± 5 mg/(500) | — |
A simple FI-CL method has been established for the determination of vitamins A and C using RB as a photosensitizer for the first time. The method was successfully applied to the determination of retinol, vitamin C, and RB in pharmaceutical formulations and the results obtained gave good agreement between the results obtained and values labeled. We also proposed the scheme for RB sensitization effect on chemiluminescence.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors are grateful to the Higher Education Commission, Pakistan, for financial assistance in the form of Indigenous Ph.D. Scholarship.