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

7

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

7

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

8

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)

10

11

• 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

Phenylethylamine Drug Group

Amphetamine

Ephedrine Dimethylamylamine (DMAA)

2-Phenylethylamine

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

Products

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

What is the origin of the DMAA in the supplements?

See: Drug Test. Analysis, 4 (2012) 986-990.

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.

The only CSP that will do the separation!

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

Does DMAA really exist in geranium?

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

2011 2012

FDA orders halt to sale of products

containing DMAA, April 24th, 2012