same intervals as reagent volume. A

same intervals as reagent volume. As shownin Fig. 2D, the net signal
increased with increasing volume of standard/sample up to 100L.
After that, the net signal plateaued. Hence, a standard/sample volume
of 100L was used in the present work.
3.2. Optimization of variables in the determination of creatinine
3.2.1. Influence of sodium picrate concentration
Initially, the volume of sodium picrate solutionwas set at 200L.
A 2% sodium hydroxide concentration was adopted based on a
study in a previouswork [30]. The optimal concentration of sodium
picrate solutionwas studied over the range from0.01 to 0.035Mat a
creatinine concentration of 100mg L−1 (100L). A concentration of
0.03M was chosen as the optimal concentration (Fig. 3A) because
higher concentrations did not improve the sensitivity. Moreover,
sodium picrate solution at concentrations higher than 0.35M led
to precipitation of sodium picrate. Similar results were obtained at
a creatinine concentration of 50mgL−1 (data not shown).
3.2.2. Influence of sodium hydroxide
The influence of sodium hydroxide for the proposed assay was
assessed using the same concentration of creatinine as mentioned
in Section 3.2.1. Apparently, the presence of sodium hydroxide was
essential for the formation of the products. Hence, sodium picrate
solution was treated with several concentration of sodium hydroxide
(final concentration ranged from0.5 to 3.0%). Sodium hydroxide
higher than 2.5% resulted in the precipitation of sodium picrate as
well. Fig. 3B indicates that the absorbance increases with increasing
sodium hydroxide concentration from 0.5 to 2.0%. In this work, 2%
sodium hydroxide was used in order to maximize sensitivity.
3.2.3. Influences of reagent and standard/sample volume
To minimize the consumption of reagent volumes while maintaining
the highest sensitivity (peak height) and precision, these
parameters were optimized. The volumes of reagent solution and
standard/sample were studied systematically. When varying the
volume of solution of interest, another volumewas kept constant at
0.03M sodium picrate solution + 2% sodium hydroxide. The results
are given in Fig. 3C and D. The influences of the picrate and standard/
sample volumes were examined between 50 and 300L at
50L intervals. It can be observed that the maximal response was
obtained at a volume of 200L for reagent volume. This volume
also yielded the best precision (0.13% R.S.D.). For standard/sample
volume, it was found that the absorbance increased up to 150L
and remained almost constant afterwards. A volume of 100Lwas
selected as an optimal standard/sample volume for subsequent
measurements because this volume gave a smooth baseline in SIA
grams and the best precision (2.27% R.S.D.).
3.3. Number of flow reversals
The mixing of inline reagents within the SIA system was important
for the production of an albumin or a creatinine complex.
The effect of the number of flow reversals was investigated. It was
found that absorbance increased slightly when the number of flow
reversals was increased to three rounds. After that, the absorbance
gradually decreased due to dilution. Hence, three rounds of flow
reversal were used in the proposed method (data not shown).
3.4. Analytical performances
Using the optimized parameters listed above, the SIA system
was evaluated for its response to different concentrations of standard
albumin and creatinine solutions. Absorbance peak heights of
albumin and creatinine standards and a typical calibration curve
are displayed in Fig. 4. Under the optimal conditions, the calibration
curve for albumin was linear between 0 and 20mgL−1, with
the following calibration equation: y = 0.0182x, with a correlation
coefficient (R2) of 0.9984. For creatinine measurement, the calibration
was linear up to 100mg L−1 with the calibration equation:
y = 0.0044x, with a correlation coefficient (R2) of 0.9981, where Y
and X represent the SIA signal as peak height, and albumin or creatinine
concentrations in mgL−1, respectively. The detection limits
(S/N = 3,  is the standard deviation of the blank (n = 10)) were