Sequential Injection Analysis Method for the Determination of Glutathione in Pharmaceuticals

Abstract

A sequential injection analysis method for the determination of glutathione (GSH) in pharmaceuticals has been developed. It is based on the reduction of the Cu(II)-neocuproine complex by GSH and the formation of an orange-yellow colored Cu(I)-neocuproine complex with maximum absorbance at 458 nm. Under optimal conditions the method is characterized by a linear calibration range of 6.0 × 10^-7-8.0 × 10^-5 mol L^-1 (A_max = 3270 C_GSH - 0.0010; R² = 0.9983), limit of detection of 2.0 × 10^-7 mol L^-1, limit of quantification of 6.7 × 10^-7 mol L^-1, repeatability (expressed as relative standard deviation) of 3.8%, and sampling rate of 60 h^-1. The newly developed method has been successfully applied to the determination of GSH in pharmaceutical samples with no statistically significant difference between the results obtained and those produced by the standard Pharmacopoeia method.

Keywords: glutathione; neocuproine; pharmaceuticals; sequential injection analysis.

Figure 1

Schematic diagram of the SIA system: SFP—syringe-free pump (M50), SV—10-port selection valve, HC—holding coil, RC—reaction coil, CS—carrier stream (deionized H₂O), RS—reagent stream, FC—flow cell, W—waste.

Figure 2

Siagram of the aspiration sequence of analyte (GSH) and reagent (RS) solutions. Experimental conditions: c(GSH) = 4 × 10⁻⁵ mol L⁻¹, c(Cu²⁺) = 1.0 × 10⁻⁴ mol L⁻¹, c(Nc) = 2.4 × 10⁻⁴ mol L⁻¹, pH = 3.0, temperature = 25 °C, carrier flow rate = 5000 μL min⁻¹, reagent volume = 200 μL, sample volume = 200 μL, volume of the holding coil = 1000 µL, length of the reaction coil = 65 cm.

Figure 3

Effect of the carrier flow rate on peak absorbance (A_{458 nm}). Experimental conditions: c(GSH) = 4 × 10⁻⁵ mol L⁻¹, c(Cu²⁺) = 1 × 10⁻⁴ mol L⁻¹, c(Nc) = 2.4 × 10⁻⁴ mol L⁻¹, pH = 3.0, temperature = 25 °C, reagent volume = 200 μL, sample volume = 200 μL, volume of the holding coil = 1000 μL, length of the reaction coil = 65 cm. Error bars = ±standard deviation (SD) (n = 3).

Figure 4

Influence of the reagent volume on peak absorbance (A_{458 nm}). Experimental conditions: c(GSH) = 4 × 10⁻⁵ mol L⁻¹, c(Cu²⁺) = 1 × 10⁻⁴ mol L⁻¹, c(Nc) = 2.4 × 10⁻⁴ mol L⁻¹, pH = 3.0, temperature = 25 °C, carrier flow rate = 3000 mL min⁻¹, sample volume = 200 μL, volume of the holding coil = 1000 μL, length of the reaction coil = 65 cm. Error bars = ±SD (n = 3).

Figure 5

Effect of the sample volume on peak absorbance (A_{458 nm}). Experimental conditions: c(GSH) = 4 × 10⁻⁵ mol L⁻¹, c(Cu²⁺) = 1 × 10⁻⁴ mol L⁻¹, c(Nc) = 2.4 × 10⁻⁴ mol L⁻¹, pH = 3.0, temperature = 25 °C, carrier flow rate = 3000 mL min⁻¹, reagent volume = 150 μL, volume of the holding coil = 1000 μL, length of the reaction coil = 65 cm. Error bars = ±SD (n = 3).

Figure 6

Effect of the reaction coil length on peak absorbance (A_{458 nm}). Experimental conditions: c(GSH) = 4 × 10⁻⁵ mol L⁻¹, c(Cu²⁺) = 1 × 10⁻⁴ mol L⁻¹, c(Nc) = 2.4 × 10⁻⁴ mol L⁻¹, pH = 3.0, temperature = 25 °C, carrier flow rate = 3000 mL min⁻¹, reagent volume = 150 μL, sample volume = 200 μL, volume of the holding coil = 500 μL. Error bars = ±SD (n = 3).

Figure 7

Siagrams of the spectrophotometric determination of GSH in the concentration range of 6.0 × 10⁻⁷ mol L⁻¹ to 1.0 × 10⁻⁴ mol L⁻¹. Inset: calibration curve for the SIA determination of GSH in the range 6.0 × 10⁻⁷ mol L⁻¹ to 8.0 × 10⁻⁵ mol L⁻¹, obtained under optimal conditions (Table 2).

Figure 8

Siagrams of the spectrophotometric determination of 4 × 10⁻⁵ mol L⁻¹ GSH (1) and 4 × 10⁻⁵ mol L⁻¹ GSH in the presence of 2 × 10⁻² mol L⁻¹ (500 times molar excess) glucose (2), fructose (3), lactose (4), KNO₃ (5), Na₂SO₄ (6), boric acid (7), or 4 × 10⁻⁴ mol L⁻¹ (10 times molar excess) sodium citrate, obtained under optimal conditions (Table 2).