ANALYSIS OF A SERIES OF CHLOROGENIC ACID ISOMERS USING ION MOBILITY TANDEM

MASS SPECTROMETRY Jamie Willems

Ph.D. Candidate May 13, 2016

FABS Research Day

Manuscript submitted to Analytic Chimica Acta on March 19, 2016

Chlorogenic Acids ■ Chlorogenic acids are natural components of many fruits and

vegetables and are purported to be of importance for human health

■ The ability to readily identify and differentiate chlorogenic acids plays an important role in their analysis

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Can mass spectrometry be used to reliably differentiate between chlorogenic acid

isomers?

Chlorogenic Acids

■ Chlorogenic acids are phenolic compounds defined by the presence of a cinnamic acid linked to a quinic acid

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5-Caffeoylquinic Acid (5-CQA;

Chlorogenic Acid)

4-Caffeoylquinic Acid (4-CQA;

Cryptochlorogenic Acid)

3-Caffeoylquinic Acid (3-CQA;

Neochlorogenic Acid)

OH

O

HOOC

OH

OH

O

OH

OH

O

OH

COOHOH

O

OH

OH

OHO

O

OH

OH

OH

OH

COOHOH

What is Mass Spectrometry? ■ Analytical technique which helps identify and quantify

compounds based upon the mass-to-charge (m/z) ratio of gas phase ions

■ Requires an ion source, mass analyzer and a detector

Image from: http://www.premierbiosoft.com/tech_notes/mass-spectrometry.html

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Materials & Methods

■ Select chlorogenic acid isomers were analyzed for their fragmentation patterns by:

– Triple quadurpole linear ion trap mass spectrometer (ESI-QqQ-LIT; Q-Trap 4000, Applied Biosystems)

– Quadurpole time-of-flight mass spectrometer (ESI-QqTOF; API QSTAR XL, Applied Biosystems)

– Quadurpole time-of-flight mass spectrometer (ESI-QTOF; 6550 iFunnel, Agilent)

■ The fragmentation patterns of the three CQA isomers were determined in both the positive ion and negative ion modes

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Fragmentation Analysis

■ The fragmentation patterns of the three CQA isomers were examined for diagnostic ions which could be used to identify and differentiate these compounds

■ It was found that the MS/MS of the three compounds differed significantly when analyzed in the negative ion mode

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B

A

C 191

191

191

179

179

173

353

353

353

O

O

OH

OH

OH

OH

COOHOH

O

OH

COOHOH

O

OH

OH

OH

OH

O

HOOC

OH

OH

O

OH

OH

191 179 173

5-CQA ✓

4-CQA ✓ ✓ ✓

3-CQA ✓ ✓

Figure 1. Mass spectra of 5-CQA (A), 4-CQA (B) and 3-CQA (C) (obtained by 6550 iFunnel QTOF).

Table 1. Major MS/MS fragments (counts >5.0x104 by 6550 iFunnel QTOF) used for isomer identification.

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Neg

ativ

e Io

n M

ode

Frag

men

tatio

n A

naly

sis

Figure 2. The proposed generalized negative ion mode MS/MS fragmentation pattern and ESI-QqQ-LIT MS/MS spectrum of 4-CQA.

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High-Field Asymmetric Ion Mobility Spectrometry (FAIMS) ■ HPLC alone or in conjunction with MS is the standard way for

analyzing caffeoylquinic acids – However, HPLC-based methods are time consuming

■ FAIMS is a rapidly growing separation technology that has been successfully interfaced with MS for various applications – Therefore, we employed FAIMS-MS/MS in combination with

the isomer specific MS/MS fragmentation pattern for the separation and identification of mono-caffeoylquinic acids

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High-Field Asymmetric Ion Mobility Mass Spectrometry (FAIMS)

■ FAIMS separates ions based on their mobility in a gas under the influence of an electric field -2000 V

+4000 V

0 V

thigh

tlow

Gas flow

Image modified from Dr. Randy Purves Lecture Notes

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FIAMS Results ■ Optimization of FAIMS was carried out to

ensure reproducible separation of these three isomers

■ To demonstrate the use of FAIMS-MS/MS for the separation of mono-caffeoylquinic acids in real samples, commercial apple and pear juices were analyzed employing the optimized conditions

0

200000

400000

600000

800000

1000000

1200000

1400000

0 5 10 15 20 25

Inte

nsity

Compensation Voltage

173

179

191

3-CQA

4-CQA 5-CQA

A

0

50000

100000

150000

200000

250000

300000

350000

400000

450000

0 5 10 15 20 25

Inte

nsity

Compensation voltage

173

179

191

B

020000400006000080000

100000120000140000160000180000200000

0 5 10 15 20 25

Inte

nsity

Compensation voltage

173

179

191

C

Figure 3. CV spectra for the mixed mono-caffeoylquinic acid isomer standards (A), and commercial apple (B) and pear juices (C).

Conclusions

■ The generalized fragmentation pathways of mono-caffoeylquinic acid isomers were determined in the positive ion and negative ion modes

■ The negative ion mode fragmentation patterns could be used to differentiate between these isomers

■ A rapid (<1 min) method for the separation and identification of mono-caffeoylquinic acids in complex juice samples employing FAIMS-MS/MS was developed

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THANK YOU

Posit

ive

Ion

Mod

e Fr

agm

enta

tion

Ana

lysis

Figure 1. The proposed generalized positive ion mode MS/MS fragmentation pattern and ESI-QqQ-LIT MS/MS spectrum of 5-CQA as a representative spectrum.

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