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Factor (A): Digestion with acids
D1: Wet digestion with diacid (HNO3:HCLO4)
D2: Diacid digestion after ashing at 550 0C
D3: Wet digestion with HNO3 plus H2O2
Factor (B): Quantification with instruments
M1: ICP-OES
M2: AAS
CRD and Descriptive statistics
REPLICATION
OBSERVATION TO BE RECORDED
1. Concentration of microelements (mg/kg):
Fe, Mn, Zn, Cu, Ni in accordance with treatment combinations
2. Standardization of methods
(a) Specificity
(b) Linearity
(c) Accuracy (% recovery)
(d) LOD and LOQ
(e) Precision (% RSD)
3. Economic analysis
(a) Cost of analysis
(b) Time of analysis
Prior to analyze the honey samples, the analytical methods will be standardized (ICH, 2007) by determining linearity, LOD and LOQ, accuracy and precision.
After standardization, the honey samples will be subjected to different digestion and instrument techniques and most efficient methodology in terms of easiness, accuracy, cost and time will be adopted for real sample analysis.
Following points will be considered for performing the present experiment
- Honey will be collected from different sources viz., Apis mellifera, A. cerana and Tetragonula spp. (Stingless bee)
- Recovery study will be carried out at level X, 5X and 10X of LOQ
- Percent RSD of recovery at different fortification levels will be used to assess precision
- Specificity will be determined with spiked samples/ matrix blanks
- To determine the Limit of Detection (LOD) and Limit of Quantitation (LOQ), calibration curve approach will be used and calculating them based on the standard deviation of the response (σ) and the slope of the calibration curve (S) using the formulas: LOD = 3.3σ/S and LOQ = 10σ/S
Linearity will be assessed by preparing a series of calibration standards with known analyte concentrations, analyzing them, and plotting the results against the expected concentrations. Then, statistical analysis, such as linear regression will be performed to assess the relationship and determine if the method exhibits a linear response within the tested range.
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