European Journal of Scientific Research - EuroJournals

A Comparative Analysis of Gibberellic Acid Content with Respect to Tuber
Induction in Potato Plants Grown Under Differential Photoperiod and Temperature 410
GC-MS Analysis
GC-MS was completed with a Hewlett-Packard 5890 gas chromatograph interfaced with a Hewlett-
Packard 5970 mass selective detector (Hewlett-Packard, Wilmington, DE, USA) working in the
electron-impact ionization mode at 70 eV and ionization current of 600 µA. Samples were injected
split-less into a methylsilicone fused-silica capillary column (25 m x 0.25 mm i.d.; 0.25 µm
methylsilicone film thickness; Quadrex Corporation, New Haven, CT). The carrier gas was helium
with a flow rate of 2.5 ml/min and the mass spectrometer was operated in the “scan” mode. The
injection port and detector temperatures were 260ºC and 300ºC, respectively. The column temperature
was programmed from 120ºC to 220ºC at 15ºC min -1 , then at a rate of 6ºC min -1 to 270ºC with
isothermal hold at this temperature for 4 minutes. The retention times of GA3 and 7-hydroxycoumarin-
4-acetic acid were 8 and 6 minutes respectively. Peak identification was based upon mass spectra
comparison between endogenous and standard compounds.
Calibration curves
The instrument calibration standard was first prepared by dissolving 100 mg of GA3 in 1.0 ml CH2Cl2.
By serial dilution, 1.0-ml stock solutions were then prepared that contained 1/2, 1/4, 1/8, 1/16, 1/32,
and 1/64 of this concentration. To each of these solutions was added 300 µl of CH2Cl2 containing
500.0 ng of 7-hydroxycoumarin-4-acetic acid as internal standard solution. The standard solutions
contained concentrations of GA3 that bracketed the estimated concentrations of GA3 in the potato
tissue extract after an initial analysis of the potato extracts. Each solution was prepared in triplicate (3
per day) and over 3 different days to determine both intra-assay and inter-assay precision of retention
time and peak area of GA3 relative to internal standard. For each solution analyzed, three replicate
injections into the gas chromatograph were made. The solutions were stored in a refrigerator at 4ºC
when not in use. The mean peak area of the analyte divided by that of internal standard was plotted
versus amount of GA3 divided by internal standard amount. The correlation coefficients for these three
standard curves were 0.9997, 0.9997 and 0.9995. Data collected for each amount for all three curves
were averaged and re-plotted to yield a composite standard curve. This curve used to calculate the GA3
content in the tissue extracts was expressed by the following linear equation:
Y = 12556 X + 91.8; R 2 = 0.9996
Where y is the integration unit (peak area) and X is the GA3 amount in nanograms.
The intra-day repeatability and inter-day reproducibility of retention times and peak areas
showed mean coefficients of variation of less than 3.0% for all samples.
Quantification of gibberellic acid in plant tissue samples
Potato tissue samples were subjected to the extraction procedure as described above. The method
proposed was then used to determine endogenous GA3 in potato tissue samples. The peak identified as
GA3 from authentic standard solutions was observed in extracts of leaves and roots from solanum
tuberosum. Estimations were based on the average of three independent analyses of three replicate
samples.
Quantitative analysis (including recovery) of GA3 in the extraction procedure was obtained by
three ways: (1) a homogenized potato tissue sample spiked before the extraction procedure with a
known amount of internal standard (500.0 ng) and synthetic GA3 (500.0 ng); (2) the extract of a second
sample from the same tissue spiked after the extraction procedure, and before the GC-MS analysis,
with the same amount of the internal standard and synthetic GA3; (3) the extract of a third sample from
the same tissue plus the same amount of internal standard without added GA3. Quantitative
determinations performed in these three extracts gave recoveries for the extraction procedures as well
as the amount in the original sample of potato tissue.