2) Estimation of Urinary Iron-
The standard method (the ammonium persulphate technique) was used for estimating the level of iron in the urine. Urine is digested with ammonium persulphate. Iron present in the urine acts like a catalyst in the reduction of ceric ammonium sulphate (yellow) to cerous ammonium sulphate (colourless). The degree of disappearance of the yellow colour is a measure of iron content in the urine. A standard curve plotted during the analysis was used to extrapolate the concentration of iron in the urine samples (Sandell and Kolthoff, 1937).
Reagents and Chemicals- Potassium iodate, Ammonium persulphate, Ceric ammonium sulfate, Arsenic trioxide, Sodium chloride, conc. Sulphuric acid and distilled water.
Quantitative method based on
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Apparatus-
Chromatography column: C18 (10 microns particle size), with Guard column
Flow rate: 1.2ml/min
Pressure: 30-40kgf
Wavelength: 326nm
Mobile phase: methanol : water (95:5 v/v)
Internal standard: retinyl acetate
Injection volume: 20µl
Procedure for Retinol extraction from serum samples-
1) 100 µl of serum sample and 100 µl of Retinyl acetate were added into 12 X 100mm glass test tubes. Vortex-mixed for 30 seconds. Then, kept them at 4 C for 5 mins.
2) 1mL of hexane was added and vortex-mixed intermittently for 60 sec.
3) Centrifuged at 2500 rpm for 12 mins. Upper hexane layer (supernatant) was transferred carefully into another test tube.
4) Evaporated the hexane under a stream of grade 1 nitrogen gas and added 100 µl of methanol to the residue left and vortexed for 1 min.
5) Injected 100 µl of extract in HPLC vials and closed properly.
Standard curves and calculations- Retinol was quantified from standard curves peak area for each vitamin.
Peak- area ratios of samples were converted to known quantities of retinol from the standard curves as