RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2010; 24: 643–650

) DOI: 10.1002/rcm.4421

Published online in Wiley InterScience (www.interscience.wiley.com

Comprehensive characterization of marine dissolved

organic matter by Fourier transform ion cyclotron

resonance mass spectrometry with electrospray and

atmospheric pressure photoionization

Juliana D’Andrilli1, Thorsten Dittmar2, Boris P. Koch3, Jeremiah M. Purcell4y,

Alan G. Marshall1,4 and William T. Cooper1*1Department of Chemistry & Biochemistry, Florida State University, Tallahassee, FL 32306-4390, USA2Carl Von Ossietzky University, Oldenburg and Max Planck Research Group Marine Geochemistry, D-26111 Oldenburg, Germany3Alfred Wegener Institute for Polar and Marine Research, D-27570 Bremerhaven, Germany4Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, 1800 East Paul Dirac Drive, Tallahassee, FL 32310-4005, USA

Received 25 August 2009; Revised 10 December 2009; Accepted 11 December 2009

*CorrespoBiochemi4390, USAE-mail: cy PresentTechnoloTX 77210

We compare the ultrahigh resolution 9.4T Fourier transform ion cyclotron resonance (FT-ICR) mass

spectra of marine dissolved organic matter (DOM) isolated from two sites in the Weddell Sea

(Antarctica) obtained by complementary electrospray ionization (ESI) and atmospheric pressure

photoionization (APPI). Ions produced by APPI extend to higher carbon unsaturation than those

producedbyESI, indicatedbyhigherdouble-bondequivalents (ringsplusdoublebonds)minusoxygen

(DBE-O) values, whereas ESI-generated ions are more oxygenated. Moreover, many sulfur-containing

compoundswereefficiently ionizedbyESIbutnotdetectedbyAPPI.Because themassspectraobtained

byESI andAPPI are significantly different, both are necessary to obtain amore complete description of

the molecular composition of marine DOM. Copyright # 2010 John Wiley & Sons, Ltd.

Dissolved organic matter (DOM) accounts for most of the

organic carbon stored in the oceans, and it is roughly equal to

the carbon present in the atmosphere as carbon dioxide

(�750 Pg C).1 Marine DOM is a complex, polydisperse mixture

of organic compounds, and previous high-resolution mass

spectrometry studies have detected molecules with masses

ranging from 300 to 700 Da.2 Marine DOM plays a prominent

role in global carbon cycling1,3 and understanding its

molecular composition is a critical aspect of better describing

many biogeochemical processes in the ocean.1

The predominant source of DOM in the oceans is thought to

be marine primary and bacterial secondary production, with

only minor contributions from land.2,4 Due to the extensive

formation of Antarctic bottom water there, the Weddell Sea

(Antarctica) has been the subject of numerous circulatory and

hydrographic investigations.5–10 This Antarctic bottom water

influences all major oceanic basins on earth.11,12 The Antarctic

Circumpolar Current isolates the Weddell Sea from terrestrial

and aeolian influences, making it an excellent target for studies

of DOM produced from purely marine sources.2,3

Marine DOM is a complex mixture of several thousand

compounds consisting of intact or transformed biomolecules

ndence to: W. T. Cooper, Department of Chemistry &stry, Florida State University, Tallahassee, FL 32306-

.ooper@chem.fsu.eduaddress: Shell Global Solutions (US) Inc., Westhollowgy Center, M-1260, 3333 Highway 6, South Houston,, USA.

released from living and decaying biota.13 The complexity of

DOM from any source, including the ocean, makes

comprehensive molecular characterization problematic, as

the common components of marine biomass (carbohydrates,

amino acids, and lipids) represent less than 10% of deep sea

DOM.14–18 Fourier transform ion cyclotron resonance mass

spectrometry (FT-ICR MS) at high magnetic field (>9.4 Tesla)

is a powerful method for the advanced characterization of

DOM.2,13,19–21 High field strength is the key to the ultrahigh

mass resolving power(m/Dm50% >200 000), and mass

accuracy (error <0.5 ppm) required for complete compo-

sitional representation of complex DOM samples without

prior chromatographic separation.2

Electrospray ionization (ESI) and atmospheric pressure

photoionization (APPI) are the soft ionization techniques

most suitable for FT-ICR MS characterization of DOM.13 ESI

has to date been the ionization method of choice for studies of

both terrestrial and marine DOM.2,20–28 ESI creates positively

or negatively charged ions over a wide mass range (10< m/z

<3000)29 and can be easily coupled with ultrahigh-resolution

mass spectrometry.30 However, ESI generates ions only from

polar molecules, ionization efficiency increases with ion

polarity, and salt adducts from sodium and/or potassium

present in a sample are often produced.31 The impact of such

selective ionization on DOM characterization by ESI-FT-ICR

MS has been discussed in a recent review article.13

Unlike ESI, APPI can ionize non-polar molecules through

electron detachment or charge transfer between a photo-

ionized dopant (e.g., toluene) or other analyte and a DOM

Copyright # 2010 John Wiley & Sons, Ltd.

644 J. D’Andrilli et al.

analyte of interest.13,32 APPI is also capable of ionizing polar

species, is more resistant than ESI to chemical noise from

solvents and salts (particularly beneficial for DOM charac-

terization), and exhibits less ion suppression from matrix

effects.33,34 Generally considered to be a soft ionization

technique, APPI may induce more fragmentation than ESI

due to the heated nebulizer source that usually operates at

�3008C, although fragmentation of DOM by either ionization

mode has not been addressed to any significant extent.35

Dopant-assisted APPI can also produce radical cations that

may participate in further gas-phase reactions. An earlier

review suggested some advantages and disadvantages of

both ionization techniques for comprehensive DOM

analysis.13 Two recent reports compared mass spectra of

terrestrial DOM obtained by ESI and APPI.36,37 Marine DOM,

which comprises by far the largest DOM pool on earth, may

be different from terrestrial DOM at the molecular level, in

particular in functional group distribution and aromaticity.38

Therefore, molecular differences between marine and

terrestrial DOM may lead to different ionization profiles.

The objective of this work is thus to critically evaluate ESI

and APPI for use in a comprehensive approach to ultrahigh-

resolution mass analysis for the characterization of marine

DOM.

EXPERIMENTAL

SamplingWeddell Sea samples were collected during the ANT XXII/2

expedition of the research vessel Polarstern in the western

Weddell Sea (6 November 2004 to 19 January 2005).39 The two

sampleschosenfor thisstudywerea highlysalinebrinesample

formed within ice channels of the sea ice at the surface (Brine)

and a Weddell Deep Water (WDW) sample from 700 m water

depth. Sea ice brine accumulates in small niches or pockets in

the floating sea ice and may be in contact with underlying

water. Organisms thrive in these brines, especially during

warmer seasons and with increased light exposure,

resulting in the production of large amounts of DOM that is

probably much younger than the DOM in adjacent bottom

waters.40 WDW is mainly composed of old water masses from

the North Atlantic and Pacific and it is a precursor of Antarctic

Bottom Water.11,12 In the Weddell Sea, it is carried to the sea

surface and modified by the air/surface interface depending

on the amount of sea ice formation and melting.41–43

DOM extraction and sample preparationDOM was isolated from the salty seawater matrix via a new

solid-phase extraction (SPE) method.44 To date, this

improved SPE method is the most efficient technique for

obtaining salt-free DOM samples from seawater. Prior to

extraction each sample was prefiltered through a sequence of

3.0 and 0.2mm Nuclepore filter cartridges. A volume of 20 L

of Weddell Sea water was then acidified with hydrochloric

acid (p.a.) to pH 2 and pumped through a 5 g SPE cartridge

(PPL, Varian Mega Bond Elut, Varian Inc., Palo Alto, CA,

USA) at a flow rate of <50 mL/min.44 The cartridges were

then rinsed with 100 mL of Milli-Q water (water purified

with a Millipore Milli-Q to a purity greater than 18 MV/cm),

acidified to pH 2 to remove any remaining salts, dried with a

Copyright # 2010 John Wiley & Sons, Ltd.

stream of N2 and eluted with 30 mL of methanol (Merck

Lichrosolv) at a flow rate of <10 mL/min. Eluted DOM

samples were stored in the dark at �188C. For a complete

description of the SPE method, see Dittmar et al.44

Prior to ESI and APPI FT-ICR-MS analysis, the samples

were divided in half and diluted to 50:50 methanol/water

(HPLC grade) by volume. The samples prepared for APPI

were subsequently spiked with additional methanol and

toluene (100mL sample, 2 mL methanol, 200mL toluene).

Each sample was vigorously shaken for 1 min prior to

injection. Blank samples consisted of 100mL of 50:50 metha-

nol/water for ESI, and 100mL of 89.1% methanol, 8.7%

toluene, and 2.2% water for APPI-FT-ICR MS. These blanks

were injected before each sample to prevent sample carry-

over. Each sample was analyzed in duplicate and the mass

spectral data processed independently for each.

FT-ICR MSA custom-built 9.4 T FT-ICR mass spectrometer located at the

National High Magnetic Field Laboratory (NHMFL) in

Tallahassee, Florida, USA45,46 was used for all mass spectral

measurements. The 9.4 T instrument routinely produces

mass accuracy of approximately �0.2 ppm up to m/z �800.22

Multiple (150–200) time domain acquisitions were summed

for each sample, Hanning apodized, and zero-filled once,

before Fourier transform and magnitude calculation.47 The

external ion trap and ion guide octopole frequencies were

maintained at 1.5 MHz for all experiments. Each mass

spectrum was internally calibrated with a series of ions

observed and identified in previous studies.48 The mass

accuracy for these internal standards was calculated as

<1 ppm for all singly charged ions. The mass spectra of these

samples produced ions between m/z 300 and 700 for which

elemental compositions could be assigned. All observed ions

were singly charged, based on a separation of m/z 1.0034

between the 12Cn and 12Cn-113C1 isotopic forms of the same

ion.49,50

Electrospray ionizationNegative ions were produced with a custom-built ESI

source51,52 and a 50mm i.d. fused-silica tube. A syringe

pump delivered the analyte solution at a flow rate of 0.5mL/

min. Electrospray conditions were as follows: needle voltage,

�2.5 kV; tube lens, �340 V; and heated metal capillary

operated at �8 W. These conditions were based on previous

humic acid studies23 and optimized for DOM characteriz-

ation by ESI. We focused on negative ionization because it

produces more ions from marine DOM samples than positive

ionization; this is reasonable because of the high content of

carboxyl functional groups in marine DOM.

Atmospheric pressure photoionizationThe APPI source was manufactured by Thermo Scientific

(Waltham, MA, USA) and operates by pumping the

vaporized analyte gas stream in a direction orthogonal to

both the mass spectrometer inlet and a krypton vacuum UV

lamp.53 The ion source is coupled to the first differentially

pumped stage of the 9.4 T FT-ICR mass spectrometer through

a heated metal capillary (stainless steel, 750mm i.d.).53 The

analyte solution was delivered to the heated nebulizer in the

Rapid Commun. Mass Spectrom. 2010; 24: 643–650

DOI: 10.1002/rcm

Figure 1. FT-ICR mass spectra of Weddell Sea Brine DOM.

Top: APPI; Bottom: ESI. Insets are 0.40 m/z units segments

for nominal m/z 503. Peaks labeled with (þ) appear only by

APPI; those labeled with (�) appear only by ESI. Unlabeled

peaks are seen by both APPI and ESI.

Characterization of marine dissolved organic matter 645

APPI source by a stainless steel syringe (8 mL) and syringe

pump (Harvard Apparatus, Holliston, MA, USA).32 The

APPI conditions to produce negative ions were as follows:

solvent flow rate, 50–100mL/min; nebulizer heater tempera-

ture, 280–3008C; sheath gas (CO2) pressure, 550 kPa; and the

auxiliary gas port was sealed.32

Molecular formula assignmentFormula assignment begins by first analyzing ionm/zvalues as

described by Stenson et al.23 Briefly, the data are sorted first

according to odd/even mass, then by Z� (an independent

parameter used to further sort homologous �CH2 groups

based ondegreeofunsaturation),and finallybyKendrick mass

defect (KMD) to enable unambiguous elemental composition

assignment of neutrals.23,54 NHMFL MS software then

calculated all possible elemental compositions within

�1 ppm in mass. The allowed elements and the range of

atoms of that element were: 12C (0-100), 1H (0-200), 14N (0-3),16O (0-30), 32S (0-2), and 13C (0-1). These constraints are similar

to those established by Stenson et al.23 for chemical formula

determination of Suwannee River fulvic acids. A formula was

assigned to each ion peak with a magnitudeat least 10 times the

baseline root-mean-square (rms) noise. This conservative

threshold ensures a high level of confidence in our formula

assignments. For each negative ion observed, the elemental

composition reported is the corresponding neutral whose

mass is the massof the negative ionplusa proton(1.007276 Da).

Molecular formulae were assigned based on mass

accuracy (< �1 ppm), homologous Kendrick series, and13C isotope peak confirmation, which is especially important

at higher masses because mass resolving power and thus

mass accuracy decrease with increasing m/z. Once formulae

had been assigned, the data were then divided into different

categories for interpretation, including: carbon number,

heteroatom class, double-bond equivalents (DBE; number of

rings plus double bonds to carbon),55 and DBE minus oxygen

(DBE-O). Data sorted by class refers to heteroatom content

(numbers of N and S atoms). We do not include the number

of O atoms in the heteroatom count because O is found in all

DOM formulae. DBE is a measure of unsaturation (e.g., a

fully saturated hydrocarbon has a DBE equal to zero), where

c, h, and n are the number of carbon, hydrogen, and nitrogen

atoms in the chemical formula, CcHhNnOoSs.

DBE ¼ c � h=2 þ n=2 þ 1 (1)

Each additional ring or double bond results in a loss of two

hydrogen atoms.32 Here we have ignored the contribution of

S to unsaturation since the valency of S is not known.35

Previous work23 has identified an abundance of carboxyl

groups in DOM and thus the DBE-O parameter, even after

approximating the O valency and ignoring unsaturation due

to S, is nevertheless a useful measure of the hydrocarbon

unsaturation in DOM molecules.

RESULTS

ESI and APPI FT-ICR-MS of Brine DOMAPPI and ESI 9.4 T FT-ICR mass spectra of the Weddell Sea

Brine DOM extract are shown in Fig. 1. Both spectra exhibit a

roughly Gaussian distribution with m/z values ranging from

Copyright # 2010 John Wiley & Sons, Ltd.

300 to 800 that are characteristic of marine DOM spectra.2,28,56

The prominent peak at m/z 557.261 in the ESI spectrum is

from an impurity. The mass difference between the

measured mass and that calculated from the assigned

elemental composition was typically <0.5 ppm and the

resolving power was >500 000 at m/z 500. A 0.4m/z scale-

expanded segment at nominal m/z 503 in each spectrum is

depicted as an inset. Between m/z 503.000 and 503.400, 20

assignable molecular formulae are observed by ESI but only

15 with APPI (Table 1). The insets in Fig. 1 are annotated to

identify the ions observed in only one of the ionization

modes, referred to here as unique peaks. ESI yielded CHO

compounds ranging from O7 to O13 whereas for APPI the

oxygen content varied from O6 to O12. The major difference

between the two spectra is the six unique ion formulae

observed by ESI, five of which contain one sulfur atom.

Within this m/z 0.4 window at m/z 503, no sulfur-containing

ions were detected by APPI. This trend suggests that the

sulfur compounds in marine DOM exist either as a polar

group, i.e., sulfite or sulfate, or are not aromatic and therefore

are not ionized efficiently by APPI.

Rapid Commun. Mass Spectrom. 2010; 24: 643–650

DOI: 10.1002/rcm

Table 1. Chemical formulae derived from ions observed in

Brine DOM spectrum at m/z 503 (Fig. 1). Ions detected by

APPI are denoted by (þ) and those detected by ESI are

denoted by (�)

Method Formula Method Formula Method Formula

þ/� C24H24O12 þ/� C25H32O9N2 þ C30H48O6

þ/� C23H24O11N2 þ/� C23H36O12 � C23H20O13

þ/� C21H28O14 þ/� C27H36O9 � C20H24O13S1

þ/� C25H28O11 þ/� C24H40O11 � C21H28O12S1

þ/� C24H28O10N2 þ/� C28H40O8 � C22H32O11S1

þ/� C22H32O13 þ/� C25H44O10 � C23H36O10S1

þ/� C26H32O10 þ/� C29H44O7 � C24H40O9S1

646 J. D’Andrilli et al.

To extend these observations from this limited 0.4 m/z

mass range to the entire data set, the data generated by ESI

and APPI were evaluated for chemical formula matches of

neutral molecules over the entire mass range of observed

ions. For the Brine DOM sample, 3395 elemental compo-

sitions were identified in both data sets with molecular

masses differing by less than �0.00153 Da (i.e., common

formulae), whereas 2623 formulae were uniquely observed

by ESI and 2618 by APPI (i.e., unique formulae).

Heteroatom (N and S) class abundances for Brine DOM

based on both ionization techniques are displayed in Fig. 2.

In general, heteroatom class abundances were reproducible

to about 10%. The CHO class is the most abundant for both

modes. APPI more efficiently ionized all chemical classes

than ESI except for compounds containing sulfur. It is

informative that of the molecules observed by only one

technique, the class most frequently detected by APPI (CHO)

is different from that most frequently detected by ESI (S1 –

data not shown). This observation supports our contention

that combined ESI and APPI analyses are necessary to

provide a more complete molecular representation of marine

DOM.

Additional information about the selectivities of ESI and

APPI can be obtained from van Krevelen plots (H/C vs. O/C

atomic ratios for each formula).25 Figure 3(a) includes data

for all ions detected by both ionization modes. Figures 3(b)

and 3(c) are van Krevelen diagrams of the molecular

formulae unique to APPI and ESI, respectively. There is

Figure 2. Histogram of relative abundances of all members

of each of the listed heteroatom classes, observed by APPI or

ESI for Brine DOM. All mass spectral peaks above 1% relative

abundance are included in the calculation. Data are averages

of two independent measurements, and error bars represent

the difference between the two measurements.

Figure 3. van Krevelen diagrams for (a) all formulae

observed in Brine DOM by either ionization mode, (b)

formulae observed only by APPI, and (c) formulae observed

only by ESI.

Copyright # 2010 John Wiley & Sons, Ltd.

considerable ‘overlap’ in the elemental ratios of formulae

observed by the two ionization modes (Fig. 3(a)). However,

the data in the three figures taken together suggest that

negative-ion APPI will ionize those molecules with relatively

lower O/C and H/C ratios that are not ionized by negative-

mode ESI. Conversely, ESI produces ions from molecules

with relatively higher O/C and H/C ratios not observed by

Rapid Commun. Mass Spectrom. 2010; 24: 643–650

DOI: 10.1002/rcm

Figure 4. Numbers of formulae as a function of DBE-O value

for Brine DOM, sorted by ionization mode.

Characterization of marine dissolved organic matter 647

APPI. The highest abundance observed by APPI corresponds

to O/C¼ 0.38 and H/C¼ 1.37, whereas in ESI it is shifted to

O/C¼ 0.51 and H/C¼ 1.45, i.e., ionizable compounds

having more oxygen and more hydrogen than for APPI.

These results are in general qualitative agreement with those

of Hertkorn et al.36 Figures 3(b) and 3(c) further emphasize

that there are significant numbers of ions produced by one

ionization mode but not the other. It should be noted that

these van Krevelen diagrams do not have the resolution to

unequivocally identify every peak observed in the FT MS

spectra. While it may seem that formulae with the same O/C

and H/C ratios are being observed in both modes, the data in

Figs. 3(b) and 3(c) do indeed represent formulae unique to

each.

Once molecular formula information is available, each

homologous series can be sorted by class, carbon number,

oxygen number, DBE, and DBE-O. Table 2 reports the

characteristics of the homologous series observed by each

ionization method in both Brine and WDW DOM. A

traditional Kendrick homologous series is defined as organic

compounds that differ by integer multiples of �CH2. We

identify members of homologous series based on the unique

neutral molecular mass, Z�, DBE, and DBE-O using an

original program developed in the PYTHON programming

language (Python Software Foundation). APPI and ESI for

Brine produced 399 and 472 �CH2 homologous series,

respectively. The data in Table 2 reflect a remarkable

similarity in the broad characteristics of molecules observed

by these two ionization methods.

However, further analysis reveals significant differences.

DBE-O distributions from APPI and ESI data are included in

Fig. 4. For APPI, the DBE-O distribution peaks at 0, whereas

�2 is the maximum for the ESI data. The DBE-O distribution

shift to more positive values for APPI suggests that more

highly unsaturated and possibly less polar compounds are

preferentially ionized by APPI relative to ESI.

Although the abundances of sulfur-containing compounds

in the Brine DOM observed by APPI are quite low (i.e., the S1

series in Fig. 2), enough are detected to further probe the

composition of this class. It should be noted that here we

define the S1 series as all compounds with one sulfur atom,

Table 2. Characteristics of �CH2 homologous series ident-

ified in Brine and WDW DOM by APPI and ESI. Note that S1

denotes a compound that contains one sulfur atom; the

remaining constituents are carbon, hydrogen, and oxygen

VariableAPPIBrine

ESIBrine

APPIWDW

ESIWDW

Number of �CH2

homologous series399 472 386 484

Carbon # range 13–44 13–41 13–48 13–43Oxygen # range 4–20 5–19 4–21 6–21DBE range 0–18 2–18 0–19 1–18DBE-O range �15–7 �11–7 �16–7 �10–6Number ofS1-containingcompounds

11 1224 2 855

S1 seriescarbon # range

16–24 13–34 14, 16 15–37

S1 series DBE range 0–1 0–13 1 2–14

Copyright # 2010 John Wiley & Sons, Ltd.

without regard to the presence of oxygen. This designation is

different from that commonly used to describe the

heteroatom content of crude oils, for which S1 refers to

hydrocarbons with one sulfur and no oxygen.32 The data in

Table 2 indicate that ESI ionizes S1 molecules with more

carbon atoms and higher DBE values (0–13) compared with

APPI. An increase in carbon number with a corresponding

increase in DBE is associated with the addition of aromatic

rings rather than additional alkylation. Lower DBE values

(0–1) for the APPI Brine S1 series suggest the presence of

alkylated compounds, not highly aromatic. We speculate

that these sulfur-containing compounds are part of less polar

species that are uniquely ionized by APPI. No S1 series

molecular formulae were observed by both ESI and APPI.

ESI and APPI FT-ICR-MS of WDW DOMMolecular formula analyses of ESI and APPI FT-ICR-MS data

for WDW DOM were carried out in exactly the same fashion

as for the Brine DOM. The results from the deep sea sample

largely confirm our findings obtained on the sea-ice brine.

For WDW DOM, 3248 molecular formulae were common to

both data sets, corresponding to mass differences less than

�0.00118 Da. Of the 4385 formulae not observed by both

methods, 2647 were unique to ESI and 1738 were unique to

APPI. The numbers of formulae sorted by ionization

technique and heteroatom class are summarized in Fig. 5.

Just as for the Brine DOM, CHO is the most abundant class

for both ESI and APPI. All sulfur-containing compounds

were ionized only by ESI, suggesting that sulfur in WDW

Figure 5. Heteroatom class distribution for WDW DOM

observed by APPI or ESI. All mass spectral peaks above 1%

relative abundance are included in the calculation. Data are

averages of two independent measurements, and error bars

represent the difference between the two measurements.

Rapid Commun. Mass Spectrom. 2010; 24: 643–650

DOI: 10.1002/rcm

648 J. D’Andrilli et al.

DOM also exists as polar species or as a part of a largely polar

molecule.

Figure 6 includes van Krevelen diagrams for all observed

ions (Fig. 6(a)) (i.e., those detected by both ionization modes),

ions observed only by APPI (Fig. 6(b)), and ions observed

only by ESI (Fig. 6(c)). The most common formulae unique to

APPI occur at lower O/C elemental ratios and higher H/C

Figure 6. van Krevelen diagrams for (a) all formulae

observed in WDW DOM by either ionization mode, (b)

formulae observed only by APPI, and (c) formulae observed

only by ESI.

Copyright # 2010 John Wiley & Sons, Ltd.

ratios, as for the Brine DOM. The ESI spectrum of WDW

DOM produced 484 homologous alkylation series, whereas

the APPI spectrum produced 386. The gross molecular

features of these series are summarized in Table 2. Figure 7

shows DBE-O distributions for elemental compositions

assigned by APPI and ESI. The trends in that data are

generally similar to those observed for Brine DOM, although

the ESI data does suggest higher relative abundances of

molecules with more negative DBE-O values in the Brine

DOM the than in the WDW DOM. Based on a comparison of

elemental ratios using the data in Figs. 3(c) and 6(c), this

feature appears to be due to the presence of more molecules

with ‘lipid-like’ or ‘protein-like’’’ character; i.e., higher H/C

and moderate O/C ratios.25 These data are consistent with

Brine DOM being somewhat fresher and less degraded than

the WDW DOM.

DISCUSSION

Ultrahigh mass resolution and mass accuracy 9.4 T FT-ICR

MS is an important tool for the advanced characterization of

marine DOM. Previous studies have compared ESI and APPI

coupled to FT-ICR MS in an effort to identify the

characteristics of each ionization method for the analysis

of terrestrial DOM.36,37 Hockaday et al.37 studied DOM

isolated from a lake by use of C18 SPE, and primarily

compared positive-ion ESI and APPI mass spectra. Fewer

than 1000 common ions were observed by positive-ion ESI

and APPI, far less than the number of common ions that we

observed by negative-ion ESI and APPI. Hertkorn et al.36

used ESI, APPI and atmospheric pressure chemical ioniz-

ation (APCI) to generate ultrahigh-resolution mass spectra of

Suwannee River fulvic acids. In that work, more assignable

formulae were identified by ESI than by APPI or APCI, in

general agreement with our results for marine DOM. These

authors also suggested that APPI tended to ionize smaller

and less polar molecules, but both ionization methods

generated ions with high levels of oxygenation. Our results

indicate that ESI produces ions that in general contain more

oxygen atoms.

The selective ionization of heteroatom DOM species

containing S and N is one very interesting result of this

work. Organic S-containing compounds are efficiently

ionized by ESI but almost unobservable by APPI, while N-

containing compounds are preferentially ionized by APPI. It

is important to note that here we define heteroatom classes

Figure 7. Numbers of formulae as a function of DBE-O value

for WDW DOM, sorted by ionization mode.

Rapid Commun. Mass Spectrom. 2010; 24: 643–650

DOI: 10.1002/rcm

Characterization of marine dissolved organic matter 649

(e.g. S1, N1) without regard to the number of oxygen atoms in

a molecule, since all DOM molecules contain oxygen. This

approach is different than that used by Purcell et al.53 who

were looking at heteroatom distributions in crude oils where

many species are not oxygenated. In that work, S1 refers to

the heteroatom class that contains one sulfur atom and no

oxygen. While Purcell et al. did not see any S1 compounds in

those crude oils by ESI, they did observe some S1O1 species,

results which are consistent with our data.

At this point we can only speculate as to the origins of these

observations, since there has been almost no discussion of the

mechanisms of ionization of DOM heteroatom species by

these techniques. However, we can suggest that, based on the

known ionization mechanisms of the two techniques, organic

sulfur compounds exist as polar molecules, probably

sulfates, and the sulfate groups are attached to aliphatic

and not aromatic core structures. Conversely, a substantial

fraction of organic nitrogen compounds appears to be non-

polar and probably a part of core aromatic structures.53

Taken together, the data in Figs. 3(a) and 6(a), as well as in

Figs. 4 and 7, suggest that the DOM in these samples is more

saturated, less aromatic and more oxygenated than in the

terrestrial DOM studied in previous comparisons of APPI

and ESI.36,37 This is not a surprising result, given the large

amounts of lignin, tannins and other highly chromophoric

molecules typically found in terrestrial DOM. Indeed, the

van Krevelen diagrams observed here are quite similar to

those of the refractory, carboxyl-rich alicyclic compounds

observed by Hertkorn et al. in Pacific Ocean marine DOM.28

Furthermore, we have more confidence in our observations

of a lack of highly unsaturated and aromatic compounds in

these samples because of the additional APPI data that we

collected, since APPI would preferentially ionize these types

of compounds.

CONCLUSIONS

We have demonstrated that neither ionization method, ESI or

APPI, can sufficiently ionize all heteroatom classes present

within marine DOM. Nearly all sulfur species were uniquely

detected by ESI, thus supporting the hypothesis that the

majority of sulfur-containing compounds in Brine and WDW

exist as polar species. Purcell et al.32 reported efficient

ionization of non-polar sulfur species in petroleum by APPI,

and we therefore conclude that the sulfur-containing

compounds present in Brine and WDW DOM must be polar

if less than 1% are detected by APPI. Less polar species were

more efficiently ionized by APPI, as reflected in the higher

H/C and lower O/C elemental ratios of the observed

formulae. In summary, these data emphasize the need to

combine as many ionization techniques as possible for a

more accurate representation of marine DOM.

AcknowledgementsThis work was supported by the US National Oceanic and

Atmospheric Administration through Grant No.

NA05OAR4311162, the NSF Division of Materials Research

through DMR-06-54118, and the State of Florida. We

thank Brad Vanmiddlesworth for his contributions to the

Copyright # 2010 John Wiley & Sons, Ltd.

development of data analysis programs written for both

PYTHON and MATLAB.

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