الفهرس 
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Abstract
Nicotine is one of the most widely used botanical insecticide due to it can be extracted easily from natural resources. Also, it can be obtained cheaply from the tobacco industry waste. Significant finding of nicotine residue have recently been detected in mushroom samples.
The concentrations of nicotine in dried mushrooms were in the range 0.22 mg/kg to 5.87 mg/kg. The origin of nicotine in these products has not clarified yet. Potential source could be cross contamination during the drying procedure or harvesting, illegal use as a pesticide or natural occurrence in the mushrooms themselves.
Unfortunately, these residues of nicotine cannot be determined easily in mushroom products due to the interference of high protein content. Therefore, there is a need for the rapid and accurate method of analysis for nicotine residues to detect such compounds in mushroom local markets.
Considering the previous background, the present work aimed to:
1- Optimization of analytical method for determination of nicotine in fresh mushroom using LC-MS/MS.
2- Validation of the analytical method for determination of nicotine in fresh mushroom.
3- Monitoring the nicotine residue in fresh mushroom in Egyptian local markets.
The obtained results could be summarized as follows:
I. Developing and validating a reliable LC-MS/MS MRM method for nicotine analysis:
1) The proper HPLC column for nicotine analysis was C18 column with HPLC method as follows:
A flow rate (200 µl/min), mobile phase composition as follows: (A) 10 mM Ammonium formate buffer (pH 3), (B) Methanol,
Table 10: Gradient elution scheme for nicotine separation from mushroom samples
Time (min) Flow rate Buffer % (A) Methanol % (B)
0 .00 200 100 0
3 200 100 0
3.1 200 99.9 0.1
5 200 40 60
10 200 40 60
10.1 200 100 0
15 200 100 0
2) Developing a reliable LC-MS/MS MRM method for nicotine analysis indicated that the most sensitive transitions of nicotine were selecting the mass of parent ion was 163 dalton, selecting the mass of daughter ions was 132,130,117 and 84 dalton, collision energy (29,21,37,25), declustering potential (66), exit potential (22, 22, 18, 14) and entrance potential (10).
3) Developing of extraction method for nicotine analysis in fresh mushroom resulted in choosing extraction solvent (methanol (5% ammonia) for denaturation of protein and extraction of target compounds based on the resulted accepted recovery and precision of fortified and real samples.
4) The recovery percentages were 85.0 % to 105.0 % with CV was less than 6.0 % for all validation levels 0.05 and 0.1 mg/kg including the LOQ level (0.01 mg/kg).
II. Monitoring nicotine in fresh mushroom collected from local markets.
Analysis of nicotine in fresh collected from local markets in Egypt showed that mushroom samples are free from nicotine.