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.
Table 1
Regression equations of calibration curves and related parameters
System
|
Wavelength (nm)
|
Regression equation
|
Correlation coefficient
|
LOD (mg/L)
|
AnB system
|
584
|
A=0.337C+0.024
|
0.9995
|
0.20~2.40
|
AFU system
|
540
|
A=0.181C+0.013
|
0.9993
|
0.50~6.00
|
Table 2
Determination of Cys in capsule samples
Batch number of Cys capsule sample
|
Labeled
amount
(mg)
|
Method A
|
Method B
|
copper(II)-neocuproin reagent spectrophotometry (Tütem and Apak 1991)
|
Amount
found (mg)
|
Percentage of labeled value (%)
|
RSD
(%)
|
Amount
found (mg)
|
Percentage of labeled value (%)
|
RSD
(%)
|
Amount
found (mg)
|
Percentage of labeled value (%)
|
RSD
(%)
|
20210301
|
72.0
|
71.7
|
99.58
|
2.1
|
71.4
|
99.17
|
2.3
|
71.5
|
99.31
|
2.2
|
20211001
|
72.0
|
71.5
|
99.31
|
1.9
|
71.6
|
99.44
|
2.0
|
71.3
|
99.03
|
2.5
|
Selectivity
By the composition of the Cys capsule samples, substances that co-exist with Cys include calcium hydrogen phosphate, pre-gelatinised starch, povidone K30, sodium carboxymethyl starch, and magnesium stearate. Under the optimal experimental conditions described above, the impact of these co-occurring substances on the determination of Cys at 2.00 mg/L was studied. 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: calcium hydrogen phosphate (500), pre-gelatinised starch (1000), povidone K30 (1000), sodium carboxymethyl starch (500) and magnesium stearate (1000). The results indicate that the proposed methods are sufficiently selective for the determination of Cys in Cys capsule samples.
Applications
To validate the application of the two proposed methods, the content of Cys in Cys capsule samples was determined. Cys capsules were available from a local pharmacy. Ten capsules were selected and their contents were mixed thoroughly. An accurately weighed amount of the contents equivalent to 100 mg of Cys was transferred into a 50 mL beaker and appropriate water was added to dissolve it. The contents of the beaker were transferred to a 100 mL brown volumetric flask followed by the addition of water to the scale. The contents were mixed thoroughly by shaking and then filtered through Whatman Grade. 4 filter paper. First, an appropriate amount of the initial filtrate was discarded, then 1 mL of the renewed filtrate was measured into a 10 mL brown volumetric flask and fixed with water from which the sample solution was obtained at a concentration of 4.00 mg/L of Cys. 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 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.