The influence of storage condition on nitrite, nitrate and vitamin C levels in vegetables

Materials

Chemicals used were analysis grade products from Merck KGaA (Germany): N-(1-naphthyl) ethylenediamine dihydrochloride (NED), sodium nitrite, sulfanilic acid, glacial acetic acid, hydrochloric acid, antipyrine, ferrous sulfate, zinc powder, sodium nitrite, ascorbic acid, metaphosphoric acid, and 2.6-dichlorophenolindophenol.

Samples

The vegetables analyzed in this study were sweet mustard (Brassica rapa chinensis), bokchoy (Brassica rapa L.), spinach (Amaranthus tricolor L), and lettuce (Lactuca sativa L). These vegetables were obtained from a local market in Medan, Indonesia. Samples were stored for 0, 24, until 48 hours at room temperature (±25°C) and in a refrigerator (±5°C).

Determination of absorbance curve of nitrite standard solution

In total, 4 ml of standard solution of nitrite (C=10.0 μg/ml) was transferred into 50 ml volumetric flask, added 2.5 ml sulfanilic acid solution and shaken. After 5 min, 2.5 ml NED reagent was added and made to volume with distilled water and homogenized (C=0.8 μg/ml). Absorbance was measured at wave length of 400–800 nm. Then, absorbance and wave length was plotted to construct absorbance curve. Wave length of maximum absorbance was determined from the absorbance curve¹⁰.

Absorbance stability of derivatized nitrite to determine working time

In total, 4 ml of standard solution of nitrite (C=10.0 μg/ml) was transferred into volumetric flask of 50 ml, to which 2.5 ml of sulfanilic acid and stirred. After 5 min, 2.5 ml NED reagent was added and distilled water was added to make 50 ml. Absorbance was measured at wave-length of maximum absorbance obtained from absorbance curve (540 nm), and stability of absorbance was determined by observing absorbance at every minute for 1 hr. The absorbance was found to be relatively stable within 6 min in 7–12 min¹⁰.

Determination of calibration curve

Standard solution of nitrite (C=10.0 μg/ml) of different volume (0.5, 1, 2, 3, 4 dan 5 ml) were transferred into separated volumetric flasks of 25 ml, then 2.5 ml sulfanilic acid reagent added and stirred to homogenize. After 5 min, 2.5 ml NED reagent was added, then distilled water was added to make volume of 25 ml and homogenized. The series of concentration of prepared solutions were of 0.1 μg/ml, 0.2 μg/ml, 0.4 μg/ml, 0.8 μg/ml, 1.0 μg/ml. Absorbance of each solution was measured at wave-length of 540 nm within 7 min. Calibration curve was made by plotting absorbance versus concentration of each solution. From the graph obtained, then linearity of regression equation and correlation coefficient were calculated (Y=aX+b)¹⁰.

Identification of nitrite and nitrate in vegetables

About 10 g ground sample, using blender, was transferred into a glass beaker. Distilled water was added to about 150 ml, heated in a waterbath (80°C) and shaken for 5 minute then cooled and filtered. The supernatant was transferred into a test tube, then 2.5 ml sulfanilic acid reagent was added and stirred. After 5 minutes, 2.5 ml reagent NED was added. Nitrite was identified using sulfanilic acid and NED solution, and the appearance of a violet color indicated the presence of nitrite. Nitrate was identified by adding several drops ferrous sulfate solution and then slowly adding a few drops of concentrated sulfuric acid. The formation of chocolate ring indicates the presence of nitrate¹⁰.

Quantification of nitrite and nitrate in vegetables

Nitrite. Determination of nitrite was carried out with procedure previously described¹⁰. Around ten (10) gram grounded sample transferred into 250 ml beaker glass to which hot distilled water (± 80ºC) was added about 150 ml. This mixture was homogenized by stirring and heated on waterbath for 15 minute while stirring. Allowed to cool and then transferred quantitatively into 250 ml volumetric flask, distilled water added to volume, then filtered. Ten (10 ml) of filtrate transferred into a volumetric flask of 50 ml, then 2.5 ml sulfanilic acid reagent was added and stirred. After 5 minutes, 2.5 ml reagent NED was added, then distilled water added to make 50 ml, and then homogenized. Absorbance was measured at wavelength of 540 nm after period of 7 to 12 minutes time.

Nitrate. In total, 10 g grounded sample transferred into 250 ml beaker glass to which hot distilled water (± 80ºC) was added to about 150 ml. This mixture was homogenized by stirring and heated on waterbath for 15 minute while stirring. Allowed to cool and then transferred quantitatively into 250 ml volumetric flask, distilled water added to volume, then filtered. 10 ml of filtrate transferred into a volumetric flask of 50 ml, then 0.1 g Zn powder and 1 ml HCl 1 N added and allowed to stand for 10 minutes to reduce nitrate to nitrite, then 2.5 ml sulfanilic acid reagent was added and stirred. After 5 minutes, 2.5 ml reagent NED was added, then distilled water added to make 50 ml, and then homogenized. Absorbance was measured at wavelength of 540 nm after period of 7 to 12 minutes time.

Nitrite concentration from reduction of nitrate into nitrite was calculated:

Nitrite concentration from nitrate reduction = Total nitrite content - Initial nitrite concentration in samples

Nitrate content was calculated:

Nitrite concentration from nitrate reduction = Concentration of nitrate × $\frac{MoleculeWeigtnitrate}{MoleculeWeigtnitrite}$

Identification of vitamin C in vegetables

10 gram grounded sample using blender. About 0.5 ml of sample solution in a test tube was neutralized to a pH of 6–8 with NH₄OH 1 N, three drops of 3% FeCl₃ was added – a purple color indicates the presence of vitamin C.

Analysis of vitamin C in vegetables

10 g grounded sample using blender was transferred into 100 ml volumetric flask, then acetic metaphosphoric acid 3% added to make 100 ml, then homogenized and filtered. Two (2) ml of filtrate was transferred into an erlenmeyer, and then added 5 ml of acetic metaphosphoric acid, then titrated with 2.6-dichlorophenol indophenol solution 0.025% until pink steadily¹¹. The levels of vitamin C was calculated.

Vitamin C (mg/g) = $\frac{(Vt-Vb)\times Equivalence\times VL}{Vp\times Bs}$

Vb = The volume of blank (ml); VL = The volume of volumetric flask (100 ml); Vp = The volume of pipetted sample solution(ml); Bs = Sample weight (g)

Nitrite and nitrate

It is found that samples contain nitrite indicated by the appearance of violet color to prove that all samples contained nitrite. The reaction with antipyrine in dilute hydrochloric resulted in the formation of green color to prove the present of nitrite. Nitrate in samples was identified using ferrous sulfate and concentrated sulfuric acid produced brown ring¹⁰.

Vitamin C

Identification using FeCl₃ 3% reagent generated violet color to prove that all samples contained vitamin C¹¹.

The wave-length of maximum absorption of nitrite derivative

The absorbance curve of the nitrite derivative solution (10 μg/ml) is presented in Figure 1. From Figure 1 it is shown that the maximum absorption was at 540 nm, which is similar to the value previously reported^2,7, which was used to determine the analysis of nitrite and nitrate in samples.

Figure 1. Absorbance curve of nitrite derivative.

Working time for measurement

Working time for nitrite and nitrate analysis was determined to know the period of time within which the absorbance of solution still remains stable. Absorbance of nitrite derivative with Griess reagent presented in Figure 2. Figure 2 shows that absorbance was stable within minute 7 to minute 12 then used in the analysis procedure^2,7.

Figure 2. Absorbance of nitrite derivative with time.

Calibration curve of nitrite derivative

Calibration curve made by plotting absorbance versus concentration of each solution, then linearity of regression equation was determined. The calibration curve presented in Figure 3. Regression equation obtained is Y= 0.58064X + 0.0015 with coefficient correlation (r) of 0.99977(where r > 0.999). Figure 3 shows that the correlation coefficient was high (r=0.999) indicated linearity between concentration and absorbance^2,7.

Figure 3. Calibration curve of nitrite derivative.

Influence of storage condition

The levels of nitrite, nitrate, and vitamin C during storage at ±25°C and ±5°C can be seen in Table 1 and Table 2.

Nitrite and nitrate levels in fresh samples were 15.22 and 22.46 mg/kg (sweet mustard), 12.57 and 6.55 mg/kg (bokchoy), 20.26 and 90.60 mg/kg (spinach), and 18.77 and 32.68 mg/kg (lettuce), respectively. Vitamin C levels in fresh samples were 101.15 mg/100g (sweet mustard), 92.17 mg/100g (bokchoy), 88.95 mg/100g (spinach), and 40.03 mg/100g (lettuce).

From Table 1 can be seen that the levels of nitrite and nitrate also increased with storage time. After storage for 48 hours at ±25°C, nitrite and nitrate levels increased 44.97% and 53.19% (sweet mustard), 46.18% and 62.59% (bokchoy), 43.86% and 16.48% (spinach), and 41.05% and 47.09% (lettuce), respectively. While, vitamin C decreased 67.57% (sweet mustard), 24.68% (bokchoy), 81.25% (spinach), and 79.74% (lettuce).

Table 2 shows that storage at ±5°C, nitrite and nitrate levels increased 27.54% and 35.08% (sweet mustard), 13.75% and 43.51% (bokcoy), 19.59% and 10.60% (spinach), 19.85% and 25.16% (lettuce), respectively. Vitamin C levels decreased 30.88% (sweet mustard), 6.05% (bokcoy), 60.92% (spinach), 74.94% (lettuce), respectively.