This document describes a study that aimed to develop a method to determine isoflavones in the sea anemone Aiptasia pallida using solid phase extraction (SPE) and gas chromatography-mass spectrometry (GC-MS). SPE was used to isolate analytes from symbiotic and aposymbiotic anemone seawater. GC-MS analysis with different solvents like methanol, hexane, and dichloromethane showed minor peaks in seawater samples but results were inconclusive. Derivatization of samples with BSTFA prior to GC-MS produced distinguishable peaks, but the method needs further optimization to better separate peaks and identify specific isoflavones. More experimentation is also needed to

1. Method Development for the Determination of Isoflavones in Aiptasia
pallida Using SPE and GC-MS
Maura B. Drewry*, Kiersten N. Rule*, Alison M. Roark**, Nicholas J. Kuklinski*
*Department of Chemistry, Furman University, Greenville, SC
**Department of Biology, Furman University, Greenville, SC
Introduction
GC-MS Results Using BSTFA Derivitization
Silyation Derivitization using BSTFA for GC-MS Analysis
The pale sea anemone, Aiptasia pallida, is a powerful model system to study symbiosis due to its ability
to survive both with and without its intercellular algae. This may play a larger role in the anemone's
ability to reproduce both sexually and asexually. Preliminary evidence shows anemones that have had
their algae removed fail to develop gonads and are capable of only reproducing asexually.
Phytoestrogens such as daidzein, genistein, and biochanin A as well as Estradiol have been discovered
to affect the reproduction of other aquatic species. These species might be used by algal symbionts in
sea anemones to produce the observed reproductive changes.
Solid phase extraction (SPE) was used to isolate analytes in symbiotic and aposymbiotic anemone sea
water. GC-MS was used to investigate the species released by intracellular algae and the role algae play
in the anemones' reproduction system. In GC-MS, a sample is injected and then vaporized at high
temperature and vacuum in the GC column. In order for the system to work, a very volatile solvent
must be used because the system separates and records each individual mass spectra as it passes
through the mass filter and is deposited on the ion collector. During this project several volatile
solvents were tested including methanol, hexane, dichloromethane, and methylene chloride. Notably,
BSTFA, a derivitization agent used in precursory experiments to determine isoflavones, was used to
improve detectability of the analytes during GC-MS analysis.
Symbiotic and Aposymbiotic Seawater Sample Preparation
GC-MS Analysis Methods
Conclusions
References
0
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0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10
Abundance
Time
a. Dichloromethane Blank
0
200000
400000
600000
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10
Abundance
Time
b. Symbiotic Seawater in Dichloromethane
0
15000000
30000000
45000000
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10
Abundance
Time
a. BSTFA Blank
0
15000000
30000000
45000000
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10
Abundance
Time
b. Symbiotic Seawater in BSTFA
Daidzein Genistein Estradiol Biochanin A
An Agilent Technologies 5977E series GC/MSD System which includes a ChemStation Data Analysis was used.
The injection volume varied between 4uL and 8uL. Typically the larger sample volume produced more
conclusive and abundant results, but in some cases a smaller quantity was more practical. The method and
split ratio described are below. For each solvent a stock solution of our standards (daidzein, genistein,
estradiol, and biochanin A) was created. These standards were tested before testing the sea water in the
respective solvent. When testing the sea water, a caffeine standard was added to the sample in solvent to
insure that the GC-MS was indeed registering the sample that was injected. In each case, a blank standard
sample was also injected in order to check if the sample in the solvent differed from the solvent alone.
Photographs of a. symbiotic Aiptasia pallida
anemone that contains algae and b.
aposymbiotic anemone that has been bleached
to remove algae. Note the multiple pedal
lacerates (small tissue masses above parent
anemone).
a b
Solid Phase Extraction: SPE is used to separate components of a liquid in order to isolate a desired analyte.
Seawater was first run through a vacuum filtration. An Oaisis HLB 200 mg Extraction Cartridge was then
prepared, loaded with the seawater sample, and cleaned with 10 mL methanol using the SPE apparatus. The
sample was dried using a water bath and nitrogen gas so that it could be dissolved in the desired solvent.
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0 2 4 6 8 10
Temperature
Time (min)
GC-MS Method With Split Ratio of 50:1
0
20000
40000
60000
80000
0 2 4 6 8 10
Abundance
Time
a. Methanol Blank
GC-MS Analysis of Methanol and Hexane as Solvents
Time Temperature
0 40
1 40
2 40
3 40
4 69
5 140
6 190
7 240
8 240
9 240
10 240
0
40000
80000
120000
0 2 4 6 8 10
Abundance
Time
c. Symbiotic Seawater and Caffeine
Standard in Hexane
0
600000
1200000
1800000
0 2 4 6 8 10
Abundance
Time
b. Caffeine Standard in Hexane Blank
a. Example of a 4 uL injecection of methanol
in GC-MS
b. Example of an 8 uL injection of hexane
with a caffeine standard
c. Example of an 8 uL injection of Symbiotic
seawater with the caffeine standard in
hexane with no significant difference to
the corresponding blank
GC-MS Analysis of Dichlormethane as a Solvent
0
70000
140000
210000
0 1 2 3 4 5 6 7 8 9 10
Abundance
Time
c. Aposymbiotic Seawater in Dichloromethane
Silylation reaction using N,N–bis(trimethylsilyl)trifluoroacetamide (BSTFA): TMS =Si(CH3)3, Y = O, S, NH, NAlk, Nar, COO, or R.
BSTFA is extremely volatile and its byproducts are useful when separating early eluting peaks. BSTFA Derivatization products
are also stable and therefore should yield results that are low in detector noise.
a. Example of 8 uL injection of DCM
b. Example of 8 uL injection of Symbiotic seawater in DCM with minor peaks differences
c. Example of 8 uL injection of Aposymbiotic seawater in DCM with minor peak differences
a. Example of 4 uL injection of BSTFA
b. Example of 4 uL injection of Symbiotic Seawater after BSTFA Derivitization with distinguishable peak
differences
There were no significant peaks found when using methanol as a solvent. Genistein and Biochanin A did not
go into solutions with solvents of hexane, dichloromethane, or methylene chloride. When testing seawater
samples, however, DCM as a solvent. In both the DCM and BSTFA samples, small peaks that differed from the
blank sample of the respective solvent. The peaks were non conclusive and these separation will need to be
further optimized.
Further experimentation is needed to find more conclusive GC-MS data. More experimentation is needed to
improve the derivitization method such that the peaks are better separated. Changes may alternatively
involve altering the initial filtration and extraction process, changing the solvent, or changing the method of
solvation. Another area for more experimentation is the GC-MS parameters. Injection volumes of 8uL yielded
more abundant results than injection volumes of 4uL.
1. http://www.sigmaaldrich.com/Graphics/Supelco/objects/4600/4538.pdf
2. http://orgchem.colorado.edu/Spectroscopy/MS/inletsys.html