European Journal of Scientific Research - EuroJournals

411 Ahmed Malkawi
The results obtained for all samples had a deviation below 3% suggesting a high precision in
the quantification. The percentage mean recovery from extracts spiked with authentic GA3 was 94.0 ±
2.0 % determined in 6 runs. All measured amounts were corrected for the % recovery.
Method validation
7-Hydroxycoumarin-4-acetic acid, which was not detected in potato extract, was selected as internal
standard for endogenous GA3 because it contains structural features that are similar to those present in
GA3 and thus its extraction behavior resembles that of GA3. Consequently, the recovery of this
compound is indicative of the recovery of GA3 during the analytical procedure.
The composite calibration graph was used to determine GA3 in synthetic samples of known
concentrations to further determine the accuracy and precision of the method. On the other hand, to
assess the effect of the sample matrix on accuracy and precision, known amounts of GA3 spiked in
potato tissues were separately determined. Results are shown in Table 1. Each result was obtained from
3 independent determinations (triplicate samples per day X 3 injections per sample).
Table 1: Precision (as RSD %) and accuracy (as relative Error %) of the proposed method for GA3 analysis
Matrix Spiked amount (ng) Determined amount(ng) RSD (%) Error (%)
Solvent 100.0 98.0 3.0 -2.0
Solvent 150.0 147.6 1.3 -1.6
Solvent 200.0 202.8 2.9 1.4
Spiked tissue 100.0 98.7 3.2 -1.3
Spiked tissue 150.0 146.6 1.5 -2.3
Spiked tissue 200.0 203.6 3.1 1.8
Mean of 3 independent analyses in triplicate over 3 days (one replicate X 9 injections each day)
RSD % = relative standard deviation of the mean
The precision expressed as the relative standard deviation (RSD %) and the accuracy (% error)
of the method were less than 3% for all samples. Such relatively low deviations and errors indicate a
high precision and accuracy in the quantification method.
The determined amount of GA3 in potato tissues was directly obtained from the measurement
of its peak area and the calibration equation. The amount of GA3 spiked in the tissue extract just before
GC-MS analysis was obtained from the total determined amounts of GA3 after spiking minus the
determined original amount of GA3 in the tissue. Peak purity by GC-MS showed peak homogeneity
thereby excluding the possibility of the presence of interfering components and demonstrating the
specificity of the method.
Results and Discussion
I. Levels of gibberellic acid in potato tissues
In this study we measured the levels of GA3 in potato (Solanum tuberosum cultivar “Russet Burbank”)
plants grown differentially under tuber inducing and non-inducing conditions.
We specifically chose to analyze GA3 for two reasons. First, it is the most widely known GA
with inhibitory effect on tuberization established by experimentation with exogenous application to
plant systems. Secondly, it is reported that GA3 promotes root growth in plants (Tanimoto 2005) and at
the same time gibberellins are thought to be responsible for stolon growth in potato plants (Smith and
Rappaport, 1969; Kumar and Wareing, 1972). Consequently, a screening of such hormone for its
endogenous levels in the whole plant system within a time course experiment may be advisable.
The effect of induction on endogenous levels of GA3 as influenced by photoperiod and
temperature, induction duration, and tissue location (aerial and underground) can be inferred from the
data listed in Table 2. Our data support the hypothesis that day length and temperature control the