Absorption Spectra
Under experimental conditions, the addition reaction of Cys with AnB in a borax sodium hydroxide buffer medium at pH 9.8 produced a colorless thioether derivative, resulting in discoloration of the solution and a decrease in the absorbance of the system. The absorption spectra are shown in Fig. 1. The absorption peaks of the reaction solution and the blank reagent were observed at 584 nm. Therefore, 584 nm was chosen as the detection wavelength for Method A.
Similarly, Cys could also undergo an addition reaction with AFu in a borax sodium hydroxide buffer medium at pH 9.7 to produce a colorless thioether derivative, resulting in discoloration of the solution and a decrease in the absorbance. The absorption spectra are shown in Fig. 2. The absorption peaks of the reaction solution and the blank reagent were observed at 540 nm. Therefore, 540 nm was chosen as the detection wavelength for Method B.
Effect of pH
The influence of the pH of the borax-sodium hydroxide buffer solution in the range of pH 9.3 ~ 10.1 on the absorbance of the AnB and AFU systems was investigated and the results are given in Fig. 3 and Fig. 4. When its pH was 9.8, the AnB system had maximal absorbance, the best scope of the buffer solution dose was in the range of 1.75 ~ 2.25 mL (Fig. 5). When its pH was 9.7, the AFu system had maximal absorbance, the best scope of the buffer solution dose was 2.00 mL (Fig. 6). Therefore, 2.00 mL of pH 9.8 or pH 9.7 borax sodium hydroxide buffer solution was chosen for Method A and Method B, respectively, for subsequent studies.
Effect of dye concentration
In Method A, to identify the optimum concentration of AnB, different volumes of AnB at a concentration of 5×10− 4 moL/L were combined with Cys at a concentration of 2.00 mg/L under optimum conditions and the absorbance was assayed at 584 nm. The concentration of acidic magenta in Method B was identified following the same procedure and the absorbance was assayed at 540 nm. The outcomes are presented in Fig. 7 and Fig. 8. Maximum and steady absorbance was acquired over a volume range of 4.00 ~ 5.00 mL for AnB solution or 2.00 ~ 3.00 mL for AFu solution, correspondingly. Hence, the optimum amount of AnB in Method A was 4.5 mL and the optimum amount of AFu in Method B was 2.5 mL.
Effect of Time
Under the optimal conditions mentioned above, the influence of reaction time on absorbance was investigated in the time range of 0 ~ 30 min at room temperature (28 ± 2 ℃). The results indicated that the addition reactions between Cys and dye were completed rapidly at room temperature, with increasing time leading to a gradual decline in absorbance readings. Thus, both experimental procedures were carried out using the ready-to-measure method, which means that the absorbance was measured immediately after the solution was prepared.
Calibration Curves and Detection Limits
Under the above optimal conditions, the calibration curves were built for the AnB system at 584 nm (Fig. 9) and the AFu system at 540 nm (Fig. 10), separately. The linear regression equation was constructed by the method of least squares. The regression equations and associated parameters are given in Table 1.The linear ranges of Cys were 0.20 ~ 2.40 mg/L for method A and 0.50 ~ 6.00 mg/L for method B, respectively, with the correlation coefficients for both methods being greater than 0.9990.
The limit of detection (LOD) was determined using the following formulae: LOD = 3r/b, where r is the standard deviation of the absorbance of the blank reagent for 11 measurements. The results of the calculations were also summarised in Table 1.
Selectivity
The effect of co-occurring substances in the samples on the determination of Cys at 2.00 mg/L was studied under the optimum experimental conditions described above. When the relative error is within ± 5%, the following ingredients (in multiples) do not disturb the determination of Cys in method A or method B: vitamin C, calcium hydrogen phosphate and sodium carboxymethyl starch (500), pre-gelatinised starch, povidone K30, vitamin B6, microcrystalline cellulose, silicon dioxide and magnesium stearate (1000). The results indicate that the proposed methods are sufficiently selective for the determination of Cys in deietary supplements.
Applications
To validate the application of the two proposed methods, the content of Cys in deietary supplements was determined. L-Cystetine capsules were available from a local pharmacy and L-Cysteine tablets were obtained from Now Foods, USA.
The content of one capsule or tablet was ground, then appropriate amount of obtained powder was dissolved in distilled water(equivalent to 10 mg Cys), transferred to a 50 mL volumetric flask, and the volume was adjusted to the mark with distilled water. The solution was filtered through Whatman Grade. 4 filter paper, the appropriate initial filtrate was discarded and the subsequent filtrate was collected. 10 mL of the filtrate was diluted 10 times to obtain a sample solution containing 20.0 mg/L of cysteine. 1.00 mL of the sample solution was pipetted into a 10 mL cuvette and then analysed by spectrophotometric methods A and B according to the determination procedure described previously. To verify the feasibility of the method, copper(II)-neocuproin reagent spectrophotometry (Tütem and Apak 1991) was used as a comparison experiment. The levels of Cys in the samples are shown in Table 2, showing consistency with the results determined by copper(II)-neocuproin reagent spectrophotometry.
Proposal Of The Reaction Mechanism
Jabbari (Jabbari and Shamsipur 1993) established a spectrophotometric method for the determination of SO32− in fruit juices and beverages based on the addition reaction of methyl green with SO32− in alkaline media. AnB, AFu, and methyl green belong to the same group of triphenylmethane dyes, and therefore the mechanism of their addition reactions is similar to that reported by (Jabbari and Shamsipur 1993) (Scheme 1, Scheme 2). The solution discoloration was due to the formation of a colorless thioether derivative from the addition reaction.
