Analysis of low molecular weight acids by negative mode matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2007; 21: 3295–3300

) DOI: 10.1002/rcm.3216
Published online in Wiley InterScience (www.interscience.wiley.com
Analysis of low molecular weight acids by negative mode

matrix-assisted laser desorption/ionization time-of-flight

mass spectrometry

Rohit Shroff, Alexander Muck and Ales Svatos*Mass Spectrometry Research Group, Max Planck Institute for Chemical Ecology, Hans-Knoll-Str. 8, 07745 Jena, Germany

Received 20 April 2007; Revised 27 June 2007; Accepted 12 August 2007

*CorrespoGroup, MKnoll-StrE-mail: svContract/School an

Free 9-aminoacridine base is demonstrated to be a suitable matrix for negative mode matrix-assisted

laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) analysis of a wide

range of low molecular weight organic acids including aliphatic (from acetic to palmitic acid),

aromatic acids, phytohormones (e.g. jasmonic and salicylic acids), and amino acids. Low limits of

quantitation in the femtomolar range (jasmonic – 250 fmol; caffeic – 160 fmol and salicylic – 12.5 fmol)

and linear detector response over two concentration orders in the pico- and femtomolar range are

extremely encouraging for the direct study of such acids in complex biological matrices. Copyright#

2007 John Wiley & Sons, Ltd.

Since the 1980s, when ultraviolet matrix-assisted laser

desorption/ionization (UV-MALDI) was first introduced

by Karas et al.1 and Tanaka et al.,2 it has been predominantly

used to analyze large biomolecules. For studying analytes

with m/z <600, the matrix-assisted laser desorption/

ionization time-of-flight mass spectrometry (MALDI-

TOFMS) method is rather limited as most conventional

MALDI matrices produce a large number of interfering ions

in the low-mass region. Hence, for studying low molecular

weight analytes like phytohormones, carboxylic acids, etc.,

high-performance liquid chromatography coupled to elec-

trospray ionization (HPLC/ESI-MS)3 or gas chromatography

coupled to chemical ionization mass spectrometry (GC/

CI-MS)4 is widely used. Both LC/ESI-MS and GC/MS

require considerable pretreatment of samples, for example,

desalting (LC/MS) and derivatization (GC/MS) is typically

carried out before analysis. Not only is such pretreatment

time consuming, but every preparation step leads to loss of

analyte. MALDI, in contrast, is considerably more tolerant to

salts, does not frequently require any chromatographic

purification or derivatization steps,5 and allows a large

number of samples to be analyzed in one run, making it a

high-throughput technique. Numerous reports have advo-

cated using alternative MALDI sample plate material for

small molecule analysis, especially the desorption/ioniz-

ation on silicon (DIOS) sample targets introduced by Siuzdak

and coworkers.6 DIOS targets did not need any matrix for

compound desorption/ionization and have also been used to

analyze fatty acids in the negative mode;7,8 however, the

sensitivity is rather poor (high pmol range) and extensive

ndence to: A. Svatos, Mass Spectrometry Researchax Planck Institute for Chemical Ecology, Hans-

. 8, 07745 Jena, [email protected] sponsor: International Max Planck Researchd the Max Planck Society.

formation of alkali metal ion clusters was observed at low

acid concentrations.

Here, we present the application of negativemodeMALDI-

TOFMS to analyze lowmolecular weight acids5,9–13 including

phytohormones or amino acids at physiological concen-

trations, together with a wide range of other low molecular

weight acids. The acids were chosen such that they exhibited

a wide range of chemistry, including small aliphatic

acids, long-chain aliphatic acids, cyclic acids and aromatic

acids.

EXPERIMENTAL

MaterialsThe 9-aminoacridine (9AA) hydrochloride hydrate salt,

acetic, oxalic, palmitic, succinic, rac-jasmonic, phenyllactic,

DL-tartaric, cyclohexanecarboxylic, 2-(4-chlorophenoxy)-2-

methylpropionic, tiglic, crotonic, salicylic, benzoic, 2-bro-

mobenzoic, 2-iodobenzoic, 2-chlorobenzoic, 3-indoleacetic,

indole-3-carboxylic, caffeic, and ascorbic acids, cysteine, and

3,4-dihydroxyphenylalanine (L-DOPA) and HPLC grade

solvents (methanol, ethanol, chloroform, and acetone) were

purchased from Sigma-Aldrich (St. Louis, MO, USA). Poly-

ethylene glycol (PEG) 600 sulfate was purchased from TCI

Europe (Antwerp, Belgium).

Sample preparation9-Aminoacridine hydrochloride hydrate salt was dissolved

in boiling water followed by alkalinization with sodium

carbonate. The alkalinization leads to the precipitation of the

Copyright # 2007 John Wiley & Sons, Ltd.

3296 R. Shroff, A. Muck and A. Svatos

free base as a yellow solid; the base was extracted from the

reaction mixture with chloroform and the solution was then

dried overmagnesium sulfate. After drying, the solutionwas

evaporated and the obtained solid was recrystallized in

acetone to yield fine yellow-colored crystals of the free base.

Potential Acute Health Effects: Hazardous in cases of skin

contact (irritant, permeator), of eye contact (irritant), of ingestion,

or of inhalation. Thematrix solutionwasmade at 15mg/mL in

HPLC grade methanol. Stock solutions of all the acids were

made at 1mg/mL in appropriate solvents (all acids were

dissolved in methanol with the exception of palmitic acid for

which acetone was used). Sample solutions were mixed 1:1

with the matrix solution and 1mL of the mixture was spotted

on a 96-well stainless steel MALDI target plate (Waters/

Micromass, Manchester, UK). Serial dilutions for caffeic acid,

jasmonic acid, and salicylic acid were done in HPLC grade

methanol.

Mass SpectrometryA MALDI micro MX mass spectrometer (Waters/Micro-

mass, Manchester, UK) fitted with a nitrogen laser (337 nm,

4 ns laser pulse duration, max 290mJ per laser pulse, max.

20Hz repletion rate) was used in reflectron mode and

negative polarity for data acquisition. The instrument

operated with �5 kV set on the sample plate, 12 kV on the

extraction grid, pulse and detector voltages of 2.9 and

2.35 kV, respectively. Laser energy was optimized for

Figure 1. Mass spectrum of the free 9-aminoacridine base in

the negative ion mode. The peaks at m/z 193.0 and 96.0

correspond to the singly [M�H]� and doubly charged

[M�2H]2� ions. The insets show the structure of the [M�H]�

� ion and the isotopic pattern of the molecular peak.


individual acids, typically 120mJ per laser pulse was used.

The extraction delay time was set to 250 ns. MassLynx v4.0

software (Waters) was used for data acquisition and each

spectrum was recorded with 10 laser pulses. Spectra

obtained were smoothened, subtracted, centroided and

lock-mass-corrected (m/z 193.0771) using proprietary soft-

ware. A mixture of six different acids (benzoic,

2-iodobenzoic, 2-bromobenzoic, 2-chlorobenzoic, caffeic,

and palmitic) with PEG 600 sulfate (100 nL/mL in methanol)

in a ratio of 4:1 v/v was used for mass-scale calibration

purposes.

RESULTS AND DISCUSSION

9-Aminoacridine9,11 (9AA) was used as the matrix. The

MALDI-TOF mass spectrum of the free 9AA base clearly

shows only two prominent molecular peaks, one atm/z�193

corresponding to [M�H]�, and the other at m/z �96,

corresponding to the doubly charged species [M�2H]2�

(Fig. 1). Because the free 9AA base was used, no additional

[9AAþCl]� cluster ions detected in the original study9 were

observed (see inset). Apart from the above-mentioned peaks,

the spectrum of 9AA offers a very clean zone in the low

molecular weight range from 100–400Da, making it an ideal

Figure 2. MALDI-TOF mass spectra of phytohormones as

obtained on theMALDI micro MX in the negative ion mode: (a)

rac-jasmonic acid, (b) abscisic acid and (c) 3-indoleacetic

acid. The inset shows the chemical structures of the corre-

sponding [M�H]� ions of the acids. ‘�’ indicates peaks corre-

sponding to the matrix ions.


DOI: 10.1002/rcm

Figure 3. MALDI-TOFMS negative ion spectra of 3,4-di-

hydroxyphenylalanine (a), cysteine (b), and ascorbic acid

(c). For cysteine, two peaks, one at m/z 120.0 corresponding

to the [M�H]� ion and one at m/z 238.9 corresponding to

the [2M�3H]� ion, presumably the cystine anion, could be

observed. The inset shows the chemical structures of the

corresponding [M�H]� acid ions. ‘�’ indicates peaks corre-

sponding to the matrix ions.

Figure 4. MALDI-TOF negative ion mass spectra of benzoic (a),

2-(4-chlorophenoxy)-2-methylpropionic (e), caffeic (f), 3-indoleca

methylpropionic acid also shows fragmentation yielding an ion

The inset shows the chemical structures of the corresponding [M

matrix ions.


Analysis of low molecular weight acids by MALDI-TOFMS 3297

matrix for analyzing low molecular weight acidic com-

pounds which yield intensively deprotonated anions.

PhytohormonesThree phytohormones, namely jasmonic acid (JA), 3-

indoleacetic acid (IAA) and abscisic acid (ABA), were

analyzed. Clear signals for the molecular peaks [M�H]�

were observed for all of them (Fig. 2).

Amino acids and vitamin3,4-Dihydroxyphenylalanine (L-DOPA), cysteine and

ascorbic acid were analyzed using the same instrumental

parameters as used for phytohormones. Molecular peaks

[M�H]� were easily observed for all three (Fig. 3). Cysteine

required a higher laser energy of 140mJ per pulse for

ionization to occur. It also showed the formation of a

prominent ion signal atm/z�239 presumably corresponding

to the [M�H]� ion of cystine (MW 240) formed by oxidation

of the cysteine in solution or during the sample preparation.

Aromatic acids or acidswith aromatic substituentsBenzoic acid, its halogen derivatives (2-chloro-, 2-bromo-

and 2-iodobenzoic acid), salicylic (SA), indole-3-carboxylic,

caffeic, and 2-(4-chlorophenoxy)-2-methylpropionic acid

were analyzed. Deprotonated molecular peaks [M�H]�

2-iodobenzoic (b), 2-bromobenzoic (c), 2-chlorobenzoic (d),

rboxylic (g), and salicylic (h) acids. 2-(4-Chlorophenoxy)-2-

at m/z 127.0 corresponding to the chlorophenoxide anion.

�H]� acid ions. ‘�’ indicateds peaks corresponding to the


DOI: 10.1002/rcm


were clearly observed for all of them (Fig. 4). Although the

same laser energy as used before was needed to analyze the

other acids, caffeic acid required much less laser energy,

84mJ per pulse, for efficient ionization to form the

corresponding anion. Interestingly, 2-iodobenzoic acid

showed fragmentation, and a peak at m/z �127 correspond-

ing to I� was seen. However, the other halo derivatives did

not show a similar loss. Iodobenzoic acid has shown similar

fragmentation on ion trap, triple-quadrupole and q-TOF

instruments.14 That a similar pattern in MALDI analysis

occurs only with an iodo derivative and not with chloro

or bromo derivatives is intriguing. 2-(4-Chlorophenoxy)-2-

methylpropionic acid provided pronounced fragmentation

and a 4-chlorophenoxy anion (m/z 127.0) was observed. An

interesting observation was made with SA: although it

ionized well with the matrix, it did the same without any

matrix, i.e. on laser desorption/ionization (LDI).

Aliphatic and cyclic acidsEight different aliphatic acids were studied using our

method. They included acetic, oxalic, cyclohexane car-

boxylic, tiglic, crotonic, tartaric, palmitic, and succinic acids.

Figure 5. MALDI-TOF negative ion mass spectra of cyclohexane

succinic (f), palmitic (g), and oxalic (h) acids. The inset shows the c

indicates the matrix peaks.


The MALDI-TOF spectra showing the deprotonated mol-

ecular [M�H]� peaks are shown in Fig. 5. Interestingly, very

clear signals for themolecular peaks of very small acids (MW

�100Da) like acetic, oxalic, crotonic and tiglic acids were

observed. The efficient analyses of volatile acids (acetic)

could be attributed to presuable formation of salt with the

9AA free base.

Determination of the limits of quantification(LOQs)The LOQ refers to the lowest concentration at which the ion

intensity signal for the analyte can be confidently differ-

entiated from the background. These were determined for

three acids: JA, SA, and caffeic acid. A signal-to-noise ratio of

5:1 was considered for the LOQ studies. All three acids could

be detected in the femtomolar amount range (JA: 250 fmol,

caffeic acid: 160 fmol, SA: 12.5 fmol; Fig. 6). Standard mass

detector response curves (intensity vs. amount of the

compounds on the target) were plotted and good linearity

was obtained over two orders of magnitude, from the

femtomolar to picomolar range. This result is very encourag-

ing with regard to the ability to directly detect these

carboxylic (a), tiglic (b), crotonic (c), acetic (d), DL-tartaric (e),

hemical structures of the corresponding [M�H]� acid ions. ‘�’


DOI: 10.1002/rcm

Figure 6. TOF detector response curves for increasing concentrations of rac-jasmonic (a), caffeic (b), and salicylic (c) acids.

For JA good linearity could be observed from 250 fmol to 1 nmol (a). For caffeic acid linearity could be observed from 800 fmol to

100 pmol (inset, b). For salicylic acid using laser-desorption/ionization (LDI) in negative ion mode linearity was observed from

12.5 fmol to 5 pmol (inset, c). The TOF-detector ion intensity signals for the lowest LOQ of the three acids are shown for

jasmonic (d), caffeic (e), and salicylic (f) acids, respectively.

Analysis of low molecular weight acids by MALDI-TOFMS 3299

compounds in biological samples. Considering the fact that

most biological extracts would contain these acidswithin this

concentration range,15 the acids in crude extracts could

also be quantified by plotting the signal intensity of new

calibration curves obtained from the crude extracts spiked

with available labeled (2H or 13C) standards.

The mass accuracy for all analyzed acids falls within

8–167 ppm after using external calibration. The observed

high mass accuracy of our measurements represents a

promising starting point for analyzing acids in biological

samples.

Many of the acids discussed above play a vital role in

biological phenomena. For example, phytohormones are

crucial signal molecules for plants and play an essential

role in defensive signalling crosstalk between different

plants,16,17 and also in systemic responses within the same

plant (JA).18 SA also plays a crucial defensive role in

plant-pathogen interactions,19 plant cell death,20 and tripar-

tite interactions between the plant, phytopathogenic micro-

organisms and herbivores.21 Many clinically important

metabolites and pharmaceutical compounds are carboxylic

acids.22 Such acids are also involved in various biosynthetic


pathways and their metabolism products play a significant

role in biological functions. Existing analytical methods for

analyzing such compounds such as LC/MS or GC/MS

require considerable sample pretreatment or derivatization,

processes which could lead to a loss of analytes. Our

MALDI-MS method has proven (manuscript in preparation)

the potential for generating excellent results from low

abundance complex samples over a wide mass range with

negligible sample pretreatment. The prime reason for the

efficiency of MALDI for studying these low molecular

weight analytes in low abundance samples is the selective

nature of MALDI matrices. Depending on the class of

compounds to be studied, a ‘compatible’ MALDI matrix can

be selected. The matrix efficiently co-crystallizes only with

the ‘compatible’ analytes in the complex mixtures. This

enables MALDI-TOFMS to tolerate a high level of salt

impurities and other complexities in biological extracts,

hence obviating the need for sample pretreatment.Moreover,

the ability to analyze samples in a 96–400 spot array format

within a short timespan, a totally automated unmanned

operation, gives the technique unmatched throughput when

compared to LC- or GC-based methods.


DOI: 10.1002/rcm


AcknowledgementsR.S. gratefully acknowledges financial support from the

International Max Planck Research School ‘The Exploration

of Ecological Interactions with Molecular and Chemical

Techniques’. The authors equally acknowledge the financial

support from theMax Planck Society andwould like to thank

Dr. Jan Doubsky (Mass Spectrometry Research Group, Max-

Planck Institute for Chemical Ecology, Jena, Germany) for his

help with the purification of the free base from the matrix

salt, and Emily Wheeler for her editorial help.

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DOI: 10.1002/rcm

Documents

Analysis of low molecular weight acids by negative mode matrix-assisted laser desorption/ionization time-of-flight mass spectrometry