A metal dish with 30-35 g sea sand with a lid were dried at 103 oC for 2 hours in the oven and cooled in a desiccator. 1.5 g of the samples were weighed onto the dishes, and mixed with the sand. The dishes were placed in the oven with the loosened covers at 103±2 oC. They were heated for 16 hours (overnight) until constant weight was achieved. The dishes were covered while still in the oven and transferred to the desiccator to be weighed as soon as they reached room temperature (AOAC, 2005.950.46).
Calculation
(1) 100
: sample g
g/100 loss
Weight
m m
W ma b
(2) 100
: sample g
g/100 matter
Dry W
79 Where
m: Weight of sample (g)
ma: Weight of sample with dish and sand before drying mb: Weight of sample with dish and sand after drying 2.18.2 Determination of total ash
A platinum dish was heated in the furnace and cooled in a desiccator to room temperature. 1.5–2.0 g of freeze dried samples were weighed onto the platinum dish. The dish and the sample were heated for 2 hours at 103 oC (until the water was expelled). To remove the volatile organic matter, the platinum dish was heated slowly over the flame until swelling had stopped. The platinum dish was transferred into the furnace and heated at 550±5 oC for 15 hours (overnight). The platinum dish was removed from the furnace and transferred into a desiccator to cool for 30 min. The weight was taken as soon as the dish reached room temperature (AOAC, 2005.923.03).
Calculation
(1) 100
: sample wet g Ash/100
g
m m A mb a
(2) 100
: matter dry g Ash/100
g
DM A where:
m: Weight of sample (g) ma: Weight of platinum dish (g)
mb: Weight of platinum dish with ash (g) DM: Dry matter g/100 g sample
2.18.3 Determination of Vitamin B1 and B2 by HPLC
Determination of Vitamin B1 and B2 from green leafy vegetables was performed with HPLC after acid hydrolysis and enzymatic preparation. Freeze dried vegetable nightshade powder (1.5 g) was weighed into a 150 ml conical flask followed by addition of 10 ml 1 Mol/l hydrochloric acid (Annex 2A) and 50 ml bidistilled water (Tab.2.4.8). The samples were loaded into a basket; the hydrolysation was done by heating the samples in an autoclave at 120 oC for 30 min. Subsequently, the samples were cooled to approximately 20 oC in a cooling water bath and transferred into a 100 ml volumetric flask and filled with bidistilled water (Tab.2.4.8) up to the mark. The sample extracts were filtered through a prepleated filter paper into a polyethylene container with a screw cap. 5.0
80 ml of the extract were transferred into a 20 ml volumetric flask; 7.5 ml of 0.1 Mol/l hydrochloric acid (Tab.2.4.2) were added to bring the total volume to 12.5 ml. The pH was adjusted to 4.0 with 2.5 Mol/l sodium acetate solution (Tab.2.4.3). 2.0 ml Taka-Diastase-suspension (Tab.2.4.7) were added, followed by incubating the samples in a water bath for 18 hour at 45 °C.
After enzymatic treatment, the flasks were removed from the water bath and samples were cooled to 20 oC. The pH was adjusted to 3.0 using 1 Mol/l hydrochloric acid (Annex 2A). The magnetic stirrer bars were removed and the flasks were filled with bidistilled water (Tab.2.4.8) up to 20 ml mark. The solution was thoroughly mixed and 5.0 ml of this mixture were put into a 5 ml syringe fitted with a 0.2 µm membrane filter (Annex 4). The first drops of the filtrate were allowed to run out; approximately 1.5 ml of the samples were filtered into the HPLC vials. Filtration was done in duplicates and an extra vial was frozen for subsequent HPLC analysis.
Determination of vitamin B1 and B2 was performed with an Agilent 1100 series HPLC system with modification by Bognar (1992) to detect both vitamin B1 and B2 simultaneously as described in annex.3H.1.
Four calibration standard solutions (0.25 µg/ml, 0.5 µg/ml, 1.0 µg/ml and 2.0 µg/ml) were used to cover the expected range of riboflavin (vitamin B2) and thiamine (vitamin B1) contents of the measured sample. The measurements were taken at the beginning and end of the HPLC to establish calibration curves. The initial volume of the sample prepared for measurement was 20 ml.
Therefore, the sample conversion in µg/100 g was calculated using the following formula:
W = β x4 x 100 ml x (100 20) x E W = (β x 2000)
E Where:
W: Content of analyte in the starting sample material (g/ 100g) β: Content of analyte in the sample measured solution (g/ 20 ml) E: Initial weight of the starting sample material (g)
81 2.18.4 Determination of ascorbic acid (vitamin C)
Determination of vitamin C was done according to Bognar & Daood (2000). The method can detect ascorbic acid and dehydroascorbic acid. It works by principle of an in-line oxidation of L-ascorbic acid (AA) to L-dehydroL-ascorbic acid (DHAA) and separation is performed on a short column of activated charcoal as shown in figure 2.14. The derivatization of DHAA is done with 1,2-phenylenediamine solution in heated capillary reactor into fluorescent quinoxaline compounds and are detected fluorometrically. Freeze dried nightshade powder (0.5–2.0 g) was weighed in a 100 ml volumetric flask and 40 ml meta-phosphoric/acetic acid (7.5 %/20 %) solution (Tab.2.7.1) (extraction solvent) were added and filled to the mark with bidistilled water (Tab.2.4.8). A magnetic stir bar was added and the solution was mixed for 5 min at a maximum speed. A portion of the sample extract was filtered through a prepleated filter paper into a polyethylene 50 ml conical flask. (Process can be accelerated by centrifugation before filtration). 2.0 ml of the sample extracts were transferred into a 20 ml volumetric flask, followed by the addition of 2.0 ml acetonitrile (Annex 2A), and was then thoroughly mixed. Approximately 4.0 ml of the mixture was transferred into a 5 ml syringe fitted with a 0.2 µm membrane filter (Annex 4). The first drops of the filtrate were allowed to run out, and then approximately 2 ml of the sample was filtered into the HPLC vials for subsequent analysis.
Chromatographic analysis of vitamin C was performed with Shimadzu HPLC system (Tokyo, Japan) according to the description in annex 3H.2.
The calibration curve based on external standards was prepared using three calibration solutions (20 µg/ml, 40 µg/ml and 60 µg/ml) to analyse the expected range of L-ascorbic acid content of the measured sample. The measurements were taken at the beginning and end of the HPLC to establish the calibration line and to calculate the regression factors.
The concentration of vitamin C in mg/100 g in the nightshade samples was calculated according to the formula below.
10 V m WAA yAA
Where:
WAA L-ascorbic acid content in mg/100 g sample
82 V Total volume of the sample extracts solution in 100 ml
M Weight of sample in gram
yAA Total concentration of L-ascorbic acid in µg/ml in the final sample extract determined with external calibration.
Figure 2.14: HPLC-system scheme for the determination of total vitamin C.
2.18.5 Extraction and determination of tocopherol equivalents (vitamin E)
Vitamin E is found in foods in four main forms, α-, β-, γ and δ-tocopherols. Tocopherols and tocotrienols are collectively known as tocochromanols and these are a naturally occurring group of antioxidants. A direct extraction method as described by Knecht et al. (2015) was used to determine
Eluent
HPLC Pump 1
Autosampler
T-piece
Derivatization reagent
HPLC Pump 2
Detector Integrator Thermostat
Reactor (20 mTefzel capillary) tube)
Oxidation column (Activated charcoal) 20 mm x mm
Separation Column: Grom-Sil-NH2 250 mm x 4 mm
83 tocopherols and tocotrienols equivalents. Freeze dried vegetable nightshade powder (0.1 g) was weight into 50 ml centrifuge tubes (PP, Corning Inc. New York, USA) followed by the addition of 10 ml acetone (containing 0.025 % butylhydroxytoluene (BHT) (Annex 2A), the sample was subjected to ultra-sonication for 2 minutes and further extracted for 30 seconds by using a vortex mixer (Corning Inc.) at maximum speed. The sample was centrifuged at 3600 x g for 2 minutes at 8
oC, the acetone extract was collected in 50 ml volumetric flask. The extraction process was repeated two times without initial ultra-sonication. The extracts were combined together and the volume was adjusted up to 50 ml with acetone (containing 0.025 % BHT). This was followed by transferring a 10 ml aliquot into 12 ml glass vial which was then dried under a stream of nitrogen. The dried extract was re-dissolved in 500 µl mixture containing methanol/acetone/water in the ratio of 54:40:6 (v: v) (Annex 2B) respectively. The solution was filtered through PTFE 0.2 µm membrane filter (Annex 4) into the HPLC vial for analysis. HPLC-FLD analysis of tocopherols was performed according to information in annex 3H.3.
The calibration curve based on external standards was prepared with seven calibration solutions (0.1 µg/ml, 0.5 µg/ml, 2 µg/ml, 5 µg/ml, 10 µg/ml, 20 µg/ml and 40 µg/ml) to cover the expected range of tocopherols of the measured sample. The measurements were taken at the beginning and end of the HPLC to establish the calibration curves.
Calculation of results
The β, γ and δ-tocopherols are calculated as α-tocopherol equivalent as shown in the formula below:
Tocopherol (µg/100mg) = (𝐾 𝑥 50 𝑥 0.5 𝑥 100
W )
Where:
K: Sum total α-Tocopherol equivalent (1x α) + (0.4 x β) + (0.1 x γ) + (0.01 x δ) measured in the extract
W: Weight of the sample (mg)
50: Volume used to dissolve the sample (ml) 0.5: Volume used to measure the sample (ml)
84 2.18.6 Determination of raw protein by Kjeldahl-method
The Kjeldahl method (AOAC, 2005.955.04) was used for the determination of crude protein content. The method involved three stages: digestion, distillation and titration. Digestion process involves the conversion of nitrogen into ammonium sulphate by boiling with concentrated H2SO4. Four grams freeze-dried vegetable powder were weighed into 300 ml Kjeldahl digestion flask followed by addition of 15 ml of 98 % N2-free sulphuric acid (Annex 2A) and 1 Kjeldahl-tablet (Annex 2A). The sample was digested by heating the flask in an inclined position at approximately 400 oC for 30 min under fume hood. It was cooled and diluted with 50 ml bidistilled water (Tab.2.4.8) and distilled in Erlenmeyer flask under the condenser distillation unit by adding 25 ml of 2
% boric acid (Tab.2.10.2) and 4 drops of taschiro indicator (Tab.2.10.5). The digestion flask was connected to the distillation unit; 70 ml of 40 % sodium hydroxide (Tab.2.10.1) were added to set free ammonia. The flask was removed and the tip of the condenser was washed with water. Titration was done with 0.05 Mol/l sulphuric acid (Tab.2.10.4) and the readings were taken when the colour changed from green to violet.
Calculation of total nitrogen
(1) 10
4008 . 1
: sample g
g/100 Nitrogen
m N V
(2) 25 . 6
: sample g
g/100
Protein PN
(3) 100
matter.
dry g g/100
Protein
DM P
Where:
V volume of 0.05 M sulphuric acid for titration in ml m weight of sample in g
DM dry matter content of sample in g/100 g 2.18.7 Determination of soluble sugars by HPLC
The extraction and determination of soluble sugars (fructose, glucose and sucrose) from freeze dried grounded African indigenous vegetables were determined according to the method described by Van Den et al. (1986). Two grams of the freeze-dried powder were weighed into a 50 ml volumetric flask followed by the addition of approximately 40 ml bidistilled water (Tab.2.4.8). The
85 samples were mixed and heated at 60 oC for 30 min in a water bath. They were cooled to about 20
oC; bidistilled water was then added to the mark. The mixture was centrifuged at 3000 x g for 4 min. The supernatant was filtered through prepleated filter paper (Annex 4); about 10 ml of the filtrate were stored at –18 oC until further HPLC analysis. Prior to injection, 3 ml of acetonitrile (Annex 2A) were mixed with 1 ml of the sample solution in a 10 ml volumetric flask and filtered through a 0.2 µm membrane filter (Annex 4) directly into the auto sampler vial. The calibration solutions were prepared prior to injection by mixing 300 µl of each calibration solution with 900 µl acetonitrile into an auto sampler vials without filtration.
HPLC analysis was carried out into Agilent 1100 series HPLC system as described in annex 3H.4.Three calibration standard solutions; (0.25 mg/ml, 0.5 mg/ml and 1.0 mg/ml) were used to cover the expected range of fructose, glucose and sucrose concentrations of the measured sample.
Calculation of sugars (fructose/glucose/sucrose):
Conc ( g
100g) = D x 50 x 100 W x 1000 Where:
D: concentration measured in final sample extract (mg/ml) W: weight of the sample (g)
50: volume used to dissolve the sample (ml)