daniel w. armstrong department of chemistry & biochemistry
TRANSCRIPT
Something New, Something Old in GC: IL Phases for Consumer Product Analysis &
Cyclodextrin CSPs for Performance Enhancing Drug Analyses
Daniel W. Armstrong
Department of Chemistry & Biochemistry
University of Texas at Arlington
How Will Ionic Liquid Stationary Phases Fit into the
Pantheon of GC Columns?
I) New IL stationary phases will be introduced that are engineered to produce
identical separations to current, often flawed commercial stationary phases. Example: The polar stationary phase TCEP does some unique separations, but it
has an upper temperature of 140oC.
II) New IL stationary phases will be introduced that will have completely unique
selectivities compared to any/all commercial columns.
Example: The trigonal, amide-type of tricationic liquids
III) New IL stationary phases of ultra-high thermal stability are at hand.
Caveat: Some multifunctional Ils have greater thermal stability than the
outer polyimide coating of the fused silica capillaries
IV) IL stationary phases should play a significant role in multidimensional
separations because of their unique group selectivity and their natural or
engineered orthogonality to existing stationary phases.
Prior to Ionic Liquids, the most polar GC stationary phase was:
TCEP = (1,2,3-Tris(2-CyanoEthoxy)Propane
• H2C-O-CH2CH2CN * A Highly Polar, Fluid Stationary Phase
*Oxygen and Moisture Sensitive
* Maximum Operating Temp. = 140oC
H2C-O-CH2CH2CN
HC-O-CH2CH2CN
TCEP Mix on TCEP Column at 110 °C
0 10 20
Time (min)
1
2
3
4 5
6
7
8
1. n-Tridecane
2. Toluene
3. Ethylbenzene
4. p-Xylene
5. Isopropylbenzene (Cumene)
6. 1,2,4-Trimethylbenzene
7. 1,2,4,5-Tetramethylbenzene (Durene)
8. Cyclohexanone
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0
Time (min)
TCEP Mix on SLB-IL100
1.0 2.0 3.0
Time (min)
1 3
4 2
5
6
7 8
110 °C
1
3
4
2
5
6
7 8
80 °C
1. n-Tridecane 2. Ethylbenzene 3. p-Xylene 4. Isopropylbenzene (Cumene) 5. 1,2,4-Trimethylbenzene 6. 1,2,4,5-Tetramethylbenzene
(Durene) 7. Toluene 8. Cyclohexanone
•A 1,9-di(3-vinyl-imidazolium)nonane bis(trifluoromethyl) sulfonyl imidate (SLB-IL100) Ionic liquid phase has a polarity and selectivity similar to TCEP.
•SLB-IL100 has an approximate maximum temperature of at least 230 °C which is a significant improvement over the 140 °C maximum temperature of TCEP
6
0 10 20Time (min)
0 10 20Time (min)
0 10 20Time (min)
0 10 20Time (min)
0 10 20Time (min)
0 10 20Time (min)
6
6
6
6
6
7
7
7
7
7
8
8
8
8
8
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8
6
SLB-IL59
SLB-IL61
SLB-IL76
SLB-IL82
SLB-IL100
SLB-IL111
Rapeseed Oil FAMEs on Ionic Liquid Columns
30 m Columns, 180 ºC Isothermal
6=C18:3 7=C20:0 8=C20:1
Increasing polarity
Relative retention of C18:3 increases with polarity while C20:0
decreases
3 double bonds
0 double bonds
1 double bond
Thanks to Len Sidisky for this & subsequent 3 slides
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Cis/ trans FAMES on SLB-IL60 vs. PEG Type Phase
11.0 12.0 13.0 14.0 15.0Time (min)
trans cis
C18:1n9 cis / trans FAMEs @ 180°C
SLB-IL60
21.0 22.0 23.0 24.0Time (min)
PEG
C18:2n6 cis & trans FAME Isomers- 180°C
10 12 14 16 18 20Time (min)
C18:2n6 tt
C18:2n6 cc
26 28 30 32 34Time (min)
C18:2n6 tt
C18:2n6 cc
10 20
Time (min)
PAHs on SLB-IL59
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1
2 3
4
5 6
7 8
9 10
11 12 13
14
15 16 17
Resolution of all isomer sets
column: SLB-IL59, 20 m x 0.18 mm I.D., 0.14 µm oven: 65 ºC (0.5 min), 25 ºC/min to 300 ºC (20 min) inj. temp.: 265 ºC carrier gas: helium, 50 psi constant pressure detector: MSD, full scan, m/z=50 – 500 MS interface: 300 ºC Injection: 1 µL, splitless (1 min) liner: 4 mm I.D. FocusLiner w/taper sample: PAHs, 10 ug/ml in methylene chloride
1. Naphthalene 2. Acenaphthene 3. Acenaphthylene 4. Fluorene 5. Phenanthrene 6. Anthracene 7. Fluoranthene 8. Pyrene 9. Benzo[a]anthracene 10. Chrysene 11. Benzo[b]fluoranthene 12. Benzo[k]fluoranthene 13. Benzo[j] fluoranthene 14. Benzo[a]pyrene 15. Indeno[1,2,3-cd]pyrene 16. Dibenzo[a,h]anthracene 17. Benzo[g,h,i]perylene
These three cannot be resolved on a nonpolar column
Improved resolution compared to a nonpolar column
Very strong retention for these analytes – a shorter column is used for timely elution of the heavier PAHs.
Ionic Liquid Structures
N
S
S
O
CF3
O
O
O CF3
-2N N N N
++
SLB-IL111
+NN N N
+
2NTF2-
SLB-IL 82
+ +
S
N
S CF3
CF3
OO
OO
-2
PP
NH
C5H10P+
O
N
HN C5H10
P+
OHN
C5H10
P+
O
S
N
S CF3
CF3
OO
OO
-3
(NTF 2-)
SLB-IL 59
SLB-IL 76
Separation and Quantification of Alkyl Amines (Fatty Amines) by GC-MS and High-
Performance Liquid Chromatography (HPLC) with Electrospray Ionization–Mass
Spectrometry (ESI–MS)
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• The molecular structure of fatty amines is characterized by one or more C8 to C22 aliphatic alkyl groups, R, with one or more amine or quaternary ammonium functionalities
• Contains both hydrophilic group (nitrogen group) and hydrophobic group (alkyl group) in the same molecule, and is therefore a class of cationic surfactants.
• Adsorptive properties (particularly on solid surface) of cationic surfactants make it suitable for surface modification and have functions such as:
corrosion inhibitors, fabric softeners, hair rinses and hydrophobation.
Alkyl amines (Fatty amines)
12
Analyte Mass Abbrev. Numbers of
C atom Strucutre
Dodecylamine 185.2 Am1 12
Tetradecylamine 213.3 Am2 14
Pentadecylamine 227.3 Am3 15
Hexadecylamine 241.3 Am4 16
Oleylamine 267.3 Am5 18
Octadecylamine 269.3 Am6 18
Didodecylamine 353.4 Am7 24
Tridodecylamine
521.6 Am8 36
H2N
H2N
H2N
H2N
NH2
NH
H2N
N
13
Am1
Am2
Am3 Am4
Am5
Am6
Am7
Am8
0 10 20 30 Time (min)
0 10 20 30 Time (min)
Chromatographic condition: Column: Agilent Poroshell 120 EC-C18 (4.6*50mm, 2.7micron) Solvent A: H2O containing 50mM ammonium formate Solvent B: ACN containing 30mM formic acid Gradient elution: 0-4 min: 5-50% Solvent B 4-15 min: 50-95% Solvent B 15- 30 min: 95% Solvent B Flow rate: 0.5mL/min (0-20min) 1.5mL/min (20-30min) Sample concentration: 100ug/mL Sample volume: 0.5uL
HPLC-MS separation for eight aliphatic amine standards
Total flow rate: 0.5 mL/min Total flow rate: 1.5 mL/min
14
Fatty amines derivatized with TFA work equally well with GC-MS using
the SLB-IL107 stationary phase
Testing for Performance Enhancing Drugs:
Daniel W. Armstrong Robert A. Welch Professor
Department of Chemistry and Biochemistry University of Texas at Arlington
Performance-enhancing drugs are substances used by people to
improve their performance in the sports in which they engage. The
term may also refer to drugs used by military personnel to enhance
combat performance.
BACKGROUND
Mechanism of Action • DMAA works
similarly to norepinephrine
• Norepinephrine – Increasing heart
rate
– Triggering release of glucose
– Increasing blood flow to skeletal muscle
• That makes DMAA an effective ingredient in pre workout supplements.
Zang, P.; Qing, J.; Lu, Q. A study on the chemical constituents of geranium oil. Guizhou Gongxueyuan Xuebao 1996, 25, 82-85
The BALCO (Bay Area Laboratory Co-operative*) scandal is well known.
Most believe it only involved “designer” anabolic steroids (Barry Bonds, Rodger Clemons & many other Olympic
athletes and football players).
In reality it involved other drugs and substances
*Victor Conte, owner of the San Francisco-based “nutrition” company
Most Recent Background
Anabolic-Androgen Steroids (AAS)
The natural anabolic hormone testosterone, 17β-hydroxy-4-androsten-3-one. The name was derived from testicle and sterol, and the suffix of ketone. Widely used by Russian and Eastern European athletes in the 1950’s and 1960’s.
Structure of the synthetic steroid methandrostenolone (Dianabol). See the 17α-methylation (upper-right corner) which enhances oral bioavailability.
Structure of tetrahydrogestrinone (THG), the “designer” anabolic steroid of the BALCO scandal.
We were asked:
• Can you tell the difference between natural and synthetic DMAA?
• Is the DMAA in supplements natural (from geranium oil or plants) or synthetic?
• Is there any significant DMAA in geranium plants and products?
Structure of DMAA
NH2
(R)
(S)
H2N
(S)
(R)
NH2
(S)
(S)
H2N
(R)
(R)
NH2
If synthetic, it will be racemic and have a distinct diastereomeric ratio characteristic of the synthetic process.
Methods
• GC – Quantification of DMAA contents in
supplements
– Stereoisomeric ratios of DMAA in synthetic standards and supplements
• HPLC – Examine presence of DMAA in geranium oils
– Check stereoisomeric ratios in standards & supplements
Separation of DMAA diastereomers in synthetic standards by GC
DMAA diastereomers
Internal standard
A B
Diastereomeric ratio = Peak area A / Peak area B ≈ 1.32 ± 0.09
Supplements Manufacturer Diastereomer
-ic ratio
% DMAA
Dry weight
DMAA per
serving(mg)
Stated DMAA
per serving
(mg)
Labeling DMAA
as
1
1,3-
DIMETHYLAMYL
AMINE
Primaforce 1.23 3.7±0.4 17±2 20 1,3-
dimethylamylamine
2 Speed V2 diet pills LG Science 1.28 0.20±0.06 1.2±0.4 * geranium oil extract
3 ADRALIN
dietary supplement CTD Labs 1.31 2.1±0.3 34±4 *
1,3-
dimethylpentylamine
4 RIPPED JUICE BETANCOUR
T NUTRITION 1.34 11.2±1.0 80±7 * geranamine
5 OxyELITE Pro USPlabs 1.36 10.2±1.7 31±5 *
1,3-
dimethylpentylamine
hydrochloride
6 Jack3d USPlabs 1.43 2.6±0.5 142±25 * geranium stem
7 FlashOver Omega Sports 1.32 2.9±0.5 285±51 20 1,3-
dimethylamylamine
8 OVERDOSE NRGX LABS 1.27 0.11±0.01 217±26 * geranium stem
9 PWR iSatori, LLC 1.28 0.33±0.09 16±4 * 1,3-
dimethylpentylamine
10 1.M.R BPI 1.31 1.1±0.1 85±9 * 1,3-
dimethylamylamine
11 STIM-FORCE LABRADA
NUTRITION 1.31 0.72±0.04 27±1 *
1,3-
dimethylpentylamine
hydrochloride
12 HEMO RAGE NutreX
research, Inc. 1.35 1.03±0.04 33±1 *
1,3-
dimethylpentylamine
13 HYDROXYSTIM MuscleTech 1.25 1.9±0.2 10±1 177 geranium extract
*The amount of DMAA per serving in the supplement was not stated.
Enantiomeric composition of the DMAA in synthetic standards and the supplements by enantioselective
GC (Chiraldex GTA)
pA
4.6
4.8
5.0
5.2
min145 150 155 160 165 170 175 180
pA
4.6
4.8
5.0
5.2
Enantiomers Enantiomers Synthetic DMAA
DMAA from supplement Primaforce
No. Oil Analyzed Manufacturer Origin Extraction method Part of plant
1 Geranium essential oil
Starwest Botanicals, Inc.
(Rancho Cordova, CA,
USA)
China Steam distillation Leaves and branches
2 Geranium essential oil Earth Solutions
(Atlanta, GA, USA) China Steam distillation Flowers and stems
3 Geranium essential oil Aura Cacia
( Urbana, IA, USA) China Steam distillation
Leaves and
flowering branchlets
4 SOMA Geranium oil
Dreaming Earth
Botanicals (Asheville,
NC, USA)
China Steam distillation Leaves and stems
5 Geranium essential oil
Now Foods
(Bloomingdale, IL,
USA)
Egypt Steam distillation Fresh plant
6 Oshadhi Geranium select
Ayus GmgH (Bühl,
Baden-Württemberg,
Germany)
Egypt Steam distillation Leaves
7 Geranium essential oil Lotus Brands, Inc.
(Twin Lakes, WI, USA) Egypt Steam distillation Not labeled
8 Nature's Alchemy
Geranium essential oil
Lotus Brands, Inc.
(Twin Lakes, WI, USA) Egypt Steam distillation Not labeled
Commercial geranium oil products
DMAA in Geranium Oil by LC-ESI-LIT
• Because of large amounts of non-volatile components in geranium oil samples, HPLC was used for the analysis.
• HPLC-ESI-LIT method was used for underivatized DMAA.
DMAA
HPLC-ESI-LIT results
In the 8 geranium oil samples analyzed, no DMAA could be detected in any of them (LOD is 50 ppb).
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Time (min)
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Time (min)
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
A: Now Foods
geranium oil spiked with
100 ppb DMAA
B: Now Foods
geranium oil without
DMAA spike
DMAA in Geranium Oil by LC-ESI-QQQ
• In order to lower the LOD –Geranium oil samples were derivatized –Analyzed by HPLC-ESI-QQQ
DMAA
Dansyl chloride
A: Nature's Alchemy geranium oil
spiked with 30 ppb DMAA
HPLC-ESI-QQQ results
In the 8 geranium oil samples analyzed, no DMAA could be detected in any of them (LOD is 10 ppb).
Time (min)
2.3 2.5 2.7 2.9 3.1 3.3 3.5 3.7 3.9 4.1
0
100
200
300
400
500
600
700 349.00>171.00(+)
Time (min)
2.3 2.5 2.7 2.9 3.1 3.3 3.5 3.7 3.9 4.1
0
100
200
300
400
500
600
700 349.00>171.00(+)
B: Nature's Alchemy geranium oil
without DMAA spike
Conclusions
• DMAA extracted from supplements have the same diastereomeric ratios as synthetic DMAA standards;
• They are all racemic;
• No DMAA was found in 8 geranium oils at 10 ppb
level.
Pelargonium graveolens
Commercial
Supplements