1. RAPID COMMUNICATION
Determining fatty acids by desorption/ionization mass
spectrometry using thin-layer chromatography substrates
Mario F. Mirabelli1
& Giuseppe Coviello1,2
& Dietrich A. Volmer1
Received: 6 January 2015 /Revised: 9 March 2015 /Accepted: 10 March 2015
# Springer-Verlag Berlin Heidelberg 2015
Abstract In this study, we demonstrate the application of
ambient mass spectrometry for measuring fatty acids from
various biological sample matrices such as olive oil, fish oil,
salmon, and human serum. Optimum performance was ob-
tained after spotting samples onto thin-layer chromatography
(TLC) plates as sample substrates for a custom-built solvent-
assisted desorption/ionization mass spectrometry (DI-MS) in-
terface. Good to excellent linearities (coefficients of determi-
nation, 0.9856 to 0.9977) and reproducibilities (average 6 %
relative standard deviation (RSD) using syringe deposition)
were obtained after application of an internal standard. Signal
suppression phenomena were minimized by separating the
analytes by TLC to some extent prior to DI-MS, leading to a
fourfold increase of signal-to-noise ratios as compared to sin-
gle spot mixture analysis without TLC separation.
Keywords Ambient ionization . Desorption/ionization mass
spectrometry . Thin-layer chromatography . Fatty acids
Introduction
Mass spectrometry (MS) techniques for determining intact
chemical species from surfaces have developed rapidly in re-
cent years, allowing direct analysis of molecules in situ from
the surface they are bound to or the (biological) matrix they
are embedded in. Among these techniques are matrix-assisted
laser desorption/ionization (MALDI) [1, 2], laser ablation in-
ductively coupled plasma (ICP)-MS [3], secondary ion mass
spectrometry (SIMS) [4], desorption electrospray ionization
(DESI) [5], and direct analysis in real time (DART) [6]. When
performed with suitably focused ionization probes, these tech-
niques can be used for mass spectrometric imaging (MSI) of
the distribution of elements or molecules on biological sur-
faces such as tissue or cells. MALDI-MSI has been the most
rapidly developing technology for MSI, which was first de-
veloped in the mid-1990s by Caprioli and coworkers [1]. The
information gained from MSI experiments can be extremely
useful, e.g., for spatial analysis of peptide/protein biomarkers
in biological tissues, to assess the performance of drugs
in vivo or to use protein or metabolite expression as tissue
classifier from disease versus control tissue comparisons.
More recently, ambient ionization techniques such as
DART [7] and DESI [8, 9] have gained the interest of many
analytical scientists because of their simple operation and
straightforward implementation on existing mass spectrome-
try platforms. The surface of almost any material can be in-
vestigated directly at atmospheric pressure without any major
sample preparation, for example plant leaves and biological
tissues. Even functional materials such as thin-layer chroma-
tography (TLC) plates can be directly interrogated. Previous-
ly, compounds had to be scraped off the TLC plate and ex-
tracted into a suitable solvent prior to analysis by mass spec-
trometry. Direct TLC-DESI or TLC-DART analysis avoids
many disadvantages resulting from manual sample transfer
Published in the topical collection Mass Spectrometry Imaging with guest
editors Andreas Römpp and Uwe Karst.
Electronic supplementary material The online version of this article
(doi:10.1007/s00216-015-8630-5) contains supplementary material,
which is available to authorized users.
* Dietrich A. Volmer
dietrich.volmer@mx.uni-saarland.de
1
Institute of Bioanalytical Chemistry, Saarland University, Campus
B2 2, 66123 Saarbrücken, Germany
2
Ospedale Specializzato in Gastroenterologia, Saverio de Bellis, Via
Turi 27, 70013 Castellana Grotte, Bari, Italy
Anal Bioanal Chem
DOI 10.1007/s00216-015-8630-5

2. procedures, in particular the time-consuming sample prepara-
tion and problems with reproducibility and recovery of com-
pounds from TLC plates.
Hyphenation of TLC-DART-MS was first implemented by
Morlock and coworkers [10]. The same group successfully
applied the technique to acrylamide [11], lubricants [12],
and natural products [13] and also presented a technical solu-
tion for concentrating analytes prior to DART [14]. Other
groups have shown TLC-DART-MS analysis of phytochemi-
cals [15–17], pharmaceutical formulations [18], for forensic
identification [19], drugs [20], and pesticides [21].
TLC-DESI-MS has been previously applied for analysis of
dyes [22–24], peptides and proteins [25], pharmaceutical
drugs [26, 27], lipids [28, 29], and natural products [30–33],
both in one-dimensional and two-dimensional formats. In par-
ticular, lipid classes were spatially separated by two-
dimensional high-performance TLC (HPTLC) experiments
using nano-silica plates [28]; in the study, the second separa-
tion dimension allowed for partial separation of different lipid
classes, namely fatty acids, sulfatides, phosphatidylethanol-
amines, phosphatidylinositols, phosphatidylserines, phospha-
tidylcholines, sphingomyelins, and gangliosides. Despite the
two-dimensional separation, the fatty acids on the TLC plate
partially overlapped with sulfatides; the limited resolution
suggested that single fatty acids were not fully resolved. Po-
rous Teflon has also been employed for DESI analysis of
dispensed sample solutions and has been shown to be a very
suited material for quantitative analysis at trace levels [34].
High-throughput DESI-MS analysis of pharmaceutical drugs
using a 96-sample PTFE array support has been described
[35], with limits of detection (LODs) down to 10 fmol for
propranolol.
Finally, a commercial TLC-MS interface is available that
can elute analytes from TLC plates without the need of a high
voltage [36]; the device has been successfully utilized in a
number of applications, for example, for quantitative analysis
of drugs in dried blood spots [37], angiotensin-converting
enzyme inhibitors [38], and glycosphingolipids [39].
In the present work, we describe a rapid method for analy-
sis of fatty acids from human serum as well as food samples,
suitable for rapid profiling applications based on a custom-
built solvent-assisted desorption/ionization (DI) interface,
which uses a solvent sprayer similar to DESI, but without
applying a high voltage. Compared to other studies in the
literature, which have mainly described lipid classes such as
phospholipids and sphingolipids [28, 40, 41], this study fo-
cused on free fatty acids. The study’s main objective was the
optimization of the solvent-assisted DI-MS technique, includ-
ing a comparison of several support materials (TLC plates,
Teflon, glass, paper, metal) with different physicochemical
properties and solvent systems, to improve ionization efficien-
cy and analyte recovery. We believe that this proof-of-
principle study provided a distinct approach for analysis of
the fatty acid analytes from complex biological samples,
which was simpler to perform than DESI-MS but generated
similar analytical figures of merit. Furthermore, as shown in
the paper, desorption and ionization abilities were strongly
enhanced by using TLC plates as sample substrates. The per-
formance of the technique improved further after separating
the analytes on the TLC materials prior to DI-MS analysis.
Experimental
Chemicals and materials
Palmitic acid (C16:0, purity ≥99 %), stearic acid (C18:0,
≥98.5 %), oleic acid (C18:1, ≥99 %), linoleic acid (C18:2,
≥99 %), linolenic acid (C18:3, purity ≥99 %), lauric acid
(C12:0, ≥98 %), ammonium acetate, aqueous ammonium hy-
droxide (30 %), sodium hydroxide, sodium chloride, and
lipid-free human serum were obtained from Sigma-Aldrich
(Steinheim, Germany). Fatty acid stock solutions were pre-
pared at 4 g/L in methanol, stored at −20 °C and used within
1 week after preparation. Mixtures of all six fatty acids at
levels between 500 mg/L and 1 μg/L were prepared from
these solutions. HPLC-grade methanol, acetonitrile, dichloro-
methane, dimethylformamide, butylated hydroxytoluene
(BHT), chloroform, hexane, diethylether, acetic acid, sulfuric
acid, and hydrochloric acid (37 %) were purchased from
Merck (Darmstadt, Germany). Isolute SI solid-phase extrac-
tion (SPE) columns were obtained from Sigma-Aldrich. Ul-
trapure water was obtained from a Purelab Elga device
(Bucks, UK). Silica gel 60 F254 silanized TLC glass plates
(DC-Fertigplatten Kieselgel 60 F254, 20×20 cm, thickness
0.25 mm) and cyano (CN) HPTLC plates F254 pre-coated
(5×10 cm, thickness 0.20 mm) were obtained from Merck
and C-18 Nano-Sil TLC plates (10×10 cm, thickness
0.20 mm) from Macherey-Nagel (Düren, Germany). Cellu-
lose chromatographic paper (Whatman 3MM CHR 11×
14 cm) was obtained from Sigma-Aldrich. Porous Teflon
was purchased from Polymicro Technologies (Phoenix, AZ,
USA). Microscope glass slides were obtained from Karl Hecht
(Sondheim/Rhön, Germany) and MALDI AnchorChips from
Bruker (Bremen, Germany). Fish oil was recovered from gel
capsules of a commercial nutritional supplement; olive oil and
fresh salmon were obtained from a local supermarket.
Sample preparation of serum and salmon samples
Fresh Scottish salmon was cut and finely ground before ex-
traction. Human serum samples were stored at −20 °C and
thawed out at room temperature prior to extraction. Extrac-
tions were performed according to Moilanen and Nikkari [42]
(i.e., a modified Folch method [43]). Briefly, hydrolysis of the
fatty acids of membrane phospholipids was achieved by
M.F. Mirabelli et al.

3. adding 0.9 mL of a 20 mM H2SO4 aqueous solution contain-
ing 0.1 % of NaCl to 0.2 mL of the sample (vortexing, 1 min).
Subsequently, chloroform (3 mL) and methanol (1.5 mL) were
added and centrifuged (15,000 rpm, 10 min). The organic
subnatant phase was removed with a Pasteur pipette and
passed through a drying column made from a Pasteur pipette
filled with cotton wool and 3 cm of sodium sulfate. The ex-
tract was passed through a 6 mL Isolute SI SPE column (silica
gel, 0.5 g) after conditioning with 3 mL hexane (to remove
impurities), solvent evaporated using N2, and analytes re-
dissolved in 0.2 mL of hexane (containing 0.05 % BHT).
Saponification of fish oil and olive oil samples [44]
To 0.5 mL of the sample solution, 1.5 mL of NaOH (20 %, w/
w) was added. The mixture was heated to 40 °C under reflux
conditions and stirred for 35 min, after which 0.5 g of NaCl
was added. The resulting soap was filtered under vacuum, and
2 mL of aqueous HCl (20 %, w/w) was added. Liquid/liquid
extraction (LLE) using 2 mL hexane was performed twice
from the mixture to recover the free fatty acids (FFAs). The
combined organic phases were dried with anhydrous Na2SO4,
and the solvent evaporated using N2. FFAs were re-dissolved
in 0.5 mL of hexane (+0.05 % BHT).
Sample deposition and TLC analyses
For each substrate, different volumes of sample solutions (1–
4 μL) were deposited using either a micropipette or a micro-
syringe. Other parameters, such as heating the substrate mate-
rials during spotting and multiple spotting applications onto
the same spot, were also evaluated. For TLC plates, both the
elution behavior (to chromatographically resolve the analytes)
and the capability as desorption/ionization (DI) substrate were
investigated. A mixture of hexane/diethylether/acetic acid
78:20:2 (v/v/v) was used as running solvent for silica and
CH3OH/CH3CN 1:2 (v/v) for C-18. For both materials, the
samples were deposited at 1 cm from the bottom of the sup-
port and eluted for a solvent migration distance of 8 cm on
10 cm square TLC supports (obtained by cutting the original
20×20 cm plates). A glass elution chamber (Sigma-Aldrich)
of dimensions 10.8×12.1×8.3 cm was used.
DI from the TLC plates after chromatography was per-
formed by positioning the plate on a custom-built x,y stage
and manual movement of the stage in one (x) direction relative
to the bent DI inlet capillary. The position of the stage was
controlled by two digital distance meters (along the two axes)
with a lateral resolution of 1 μm. Constant speed was main-
tained at ca. 330 μm/s by manually moving the stage using a
precision wheel aided by a digital metronome. The correct
metronome timing was calculated by taking into account the
distance covered by the stage for one turn of the precision
wheel and the corresponding digital distance readout of the
metronome. The position along the vertical axis (z-axis) was
maintained during each analysis.
Mass spectrometry
Desorption/ionization MS measurements were conducted on
a Bruker Esquire HCT+ quadrupole ion trap mass spec-
trometer, equipped with a custom-built DI ion source, sim-
ilar to a DESI source [35], but without application of high
voltage (vide infra) (Electronic Supplementary Material
(ESM) Fig. S1). The external capillary had outer and inner
diameters of 320 and 200 μm, respectively; the internal
capillary was 90 and 20 μm, respectively. Solvent was
delivered using a syringe pump. Vertical and horizontal
positions of the spray were varied and measured during
the optimization procedure and maintained at the optimum
values reported below. Full-scan mass spectra were ac-
quired in negative ion mode with a cycle time of 110 ms.
DI operating parameters were as follows: nitrogen sheath
gas pressure, 5.5 bar; incident angle, 54° to surface; tip-to-
surface distance, 1.5 mm; tip-to-inlet distance, 6 mm; and
scattering angle, 5° to surface. The syringe pump flow was
adjusted between 1 and 10 μL/min (8 μL/min optimum
value). Mixtures of methanol, water, and dichloromethane
were tested at different ratios with and without addition of
ammonium acetate, sodium hydroxide, or ammonium hy-
droxide (0.005–0.5 %). The optimized MS parameters were
as follows: drying temperature, 300 °C; drying gas, 10 L/
min; skimmer, −26 V; capillary exit, −102 V; injection
time, 50 ms for 1 microscan, and ion charge control
(ICC) off. Drying gas was only used for heating the capil-
lary and the ion transfer line outside the mass spectrometer
to avoid contamination and solvent adducts to be trans-
ferred into the mass spectrometer. It was diverted from
the sample zone by using a thin Teflon sheet placed
2 cm from the end of the external capillary (a larger dis-
tance can also be used, but the short distance allowed for
better heating of the ion transfer line). Because of the par-
ticular electrical configuration of the Bruker instrument
(i.e., high voltage at the skimmer, sprayer grounded) and
resulting electrical safety concerns, we did not apply high
voltage during the analysis. The use of high voltage could,
in principle, cause discharges between the extended metal
capillary of the MS and the sample support, which was
made of metal in our setup. Ion formation was therefore
believed to be induced by solvent-assisted desorption/
ionization mechanisms, similar to thermospray (TSP) ioni-
zation [45–48], rather than DESI mechanisms. In TSP,
droplets exhibit statistical excess charge from charged sol-
utes present in the solvent; this effect is usually enhanced
by adding volatile salts such as ammonium acetate to the
solvent to enhance TSP response.
Determining fatty acids by DI-MS using TLC substrates

4. Results and discussion
In this study, a rapid assay for fatty acid profiling was devel-
oped by utilizing solvent-assisted DI-MS of sample solutions
deposited onto the surface of suitable substrate materials in-
cluding TLC materials. To obtain sufficiently high ionization
efficiencies for the formation of deprotonated molecules ([M-
H]−
) of fatty acids under DI conditions (Table 1), careful meth-
od optimization was required, which was the primary subject
of this study.
Suitability of different substrate materials
for solvent-assisted DI-MS
Different surface materials were initially evaluated as DI sub-
strates for fatty acid analysis, including smooth (glass, metal)
and rough (filter paper, porous Teflon, different TLC mate-
rials) surfaces. The obvious advantages of smooth materials
are the ability to easily clean the surface and the possibility for
repeated use. Sample deposition can be problematic with
smooth surfaces, however, as samples spread out when pipet-
ted onto the surfaces, especially from solutions with a high
content of organic solvent. Porous or chromatographic (TLC)
materials allow easier deposition of sample solutions (see be-
low) but require more prolonged cleaning procedures to avoid
carryover effects during repeated use, especially when
complex/dirty sample matrices are spotted.
In our experiments, standard solutions and sample extracts
were spotted onto (a) filter paper, (b) porous Teflon, (c) C-18
TLC, (d) silica TLC, (e) cyano HPTLC, (f) glass, (g) metal
(hydrophobic MALDI AnchorChip), and their response be-
haviors compared. The desorption/ionization efficiencies of
the tested substrates are summarized in Fig. 1(a–g).
As is immediately obvious from the comparison, the filter
paper (Fig. 1(a)), the porous Teflon (Fig. 1(b)), and the hydro-
phobic AnchorChip (Fig. 1(g)) provided very low signal in-
tensities (Fig. 1(g)), making the three materials clearly unsuit-
able for fatty acid profiling. Interestingly, the hydrophobic
AnchorChip surface failed for DI-MS, because the gas and
solvent streams caused severe spreading of the deposited sam-
ple on the surface during desorption/ionization, resulting in
low and irreproducible compound recovery from the surface.
The poor result for Teflon was in contrast to a previous DESI-
MS study, where the material was found to be a well-suited
support, giving very low LODs [34] and limited sample cross
contamination. We rationalize the different results by the dif-
ferent ionization processes of DI versus DESI.
Glass (Fig. 1(f)) initially promised good results by showing
high ion abundances. Unfortunately, ion currents exhibited
highly irregular signal profiles from the heterogeneous com-
pound distribution during sample deposition, making this sur-
face material unsuitable (Fig. 2).
Significantly improved desorption/ionization behavior was
seen from the three investigated TLC materials, C-18, silica,
and CN (Fig. 1(c–e)). In particular, silica (Fig. 1(d)) gave
excellent compound desorption, followed by the C-18 and
CN materials. The porous nature and focusing abilities of
the chromatography materials yielded tight spots after sample
deposition, and signal variation was much improved in com-
parison to smooth surface (Fig. 2), because of the lack of
compound spreading during sample deposition. This also
allowed us to use higher solvent flow rates for the sprayer
during the DI process.
In addition to the normalized signal intensities, we also
compared signal-to-noise ratios for the different surfaces and
analytes; these are summarized in ESM Table S1.
Sample deposition technique
Quickly depositing large sample volumes (to increase the
amount of analyte) by pipetting was not beneficial in our ex-
periments, because sample spreading resulted in large spotted
areas, which were then mostly not sampled by the impacting
DI sprayer beam. In our experimental setup, the effective
width of the impacting DI spray on the surface of the moving
TLC plate was ca. 1 mm, as measured with a ruler from the
Table 1 Investigated
fatty acids along with m/z
values for [M-H]−
ions
Fatty acid m/z
Palmitic acid (C16:0) 255
Stearic acid (C18:0) 283
Oleic acid (C18:1) 281
Linoleic acid (C18:2) 279
α-Linolenic acid (C18:3) 277
Lauric acid (C12:0) 199
Fig. 1 Relative DI-MS intensities for C16:0, C18:1, and C18:3 fatty
acids that were deposited on different surfaces (for each spot, 2.5 μL of
a standard solution at 500 mg/L was used). Samples were linearly
rasterized across the spots with the DI sprayer at 330 μm/min. Surface
materials are as follows: (a) filter paper, (b) porous Teflon, (c) C-18 TLC
plate, (d) silica TLC plate, (e) cyano HPTLC plate, (f) glass, and (g) metal
(AnchorChip MALDI plate). (Signals were normalized across all
conducted experiments, and 100 % relative abundance was assigned to
the highest signal with an absolute signal of 1.05×105
counts.)
M.F. Mirabelli et al.

5. liquid solvent trace visible immediately after spraying. The
width could be reduced further (if improved resolution was
required, e.g., for imaging), if lower solvent flow rates were
implemented for the DI sprayer, at the cost of reduced sensi-
tivity. We chose sample volumes of 1 μL per spot, applied via
syringe liquid deposition, which gave compound distributions
within a spot radius of ca. 2 mm. Multiple spotting on the
same spot area could be used to increase the amount of analyte
per spot further, provided one waited a sufficient amount of
time for the previous deposit to dry. The systematic compari-
son of different sample deposition techniques is summarized
in Fig. 3. The data in the figure clearly illustrate the significant
spreading effects seen for larger sample volumes as well as the
differences of pipette versus syringe liquid application, with
the syringe exhibiting improved reproducibility of the
resulting ion current profiles. Statistical data to support this
assessment are summarized in ESM Table S2 (reproducibil-
ities for six fatty acids were between 11.3 and 16.0 % versus
2.9–9.6 % RSD for pipette and syringe application, respec-
tively). In all subsequent experiments, a slow syringe applica-
tion (within 5 s) of 1-μL volumes was chosen. In addition,
during the deposition, the supports were heated at 50 °C on a
hot plate to facilitate solvent evaporation and to obtain smaller
spot sizes.
To avoid cross contamination of sample spots during sam-
ple deposition and/or analysis, it was important to maintain a
sufficient spot-to-spot distance. For 1 μL of the sample solu-
tion deposited with a syringe (spot size, ca. 2 mm), a center-to-
center distance of 5.5 mm was chosen. No carryover effects
were observed between sample spots and adjacent blank space
using these parameters. This is also evident from the rectan-
gular shaped signal profiles obtained for analysis of consecu-
tive spots (Fig. 3) as well as the smooth and constant level of
the background signal between the analyzed spots. These re-
sults further pronounce the importance of a porous material in
avoiding sample spreading during sample deposition and sol-
vent desorption, in particular when high solvent flow rates are
used. Carryover effects for one or several fatty acids are easily
detected by monitoring the background signal between spots,
with a slow positive drift indicating carryover effects. For our
experimental conditions, no such detrimental carryover effects
were observed, even after long-term continuous operation,
viz. 1 week; after which, the inner glass capillary transfer line
of the mass spectrometer was routinely cleaned.
As seen in Fig. 3 (top), the deposition of 1-μL samples on a
silica TLC material within 5 s gives a very small accumulation
of compounds at the border of the spot circumferences, the so-
called “coffee ring” effect [49]. The use of syringes allowed
for precise control of deposition time; together with the simul-
taneous heating of the TLC plate (50 °C), the coffee ring effect
was almost negligible, resulting in very stable signal profiles.
In contrast, a fast deposition using a pipette gave a pronounced
coffee ring effect (Fig. 3(b), bottom).
One further important parameter that significantly affected
the ionization efficiency of the investigated fatty acid analytes
in DI was the solvent composition of the DI spray. The opti-
mum response behavior in terms of signal intensity was ob-
served for a 95:5 (v/v) mixture of CH3OH and CH2Cl2. Be-
cause of the acidic nature of the analytes, the effect of pH and
salt concentration on ionization efficiency was also investigat-
ed, by adding bases (NH4OH and NaOH) or ammonium ace-
tate at 0.005, 0.05, and 0.5 % (w/v) to the spray solvent or
spotted onto the TLC plate before sample deposition. The
highest [M-H]−
signals for all fatty acids were seen when
bases were added directly onto the TLC plate rather than via
the spray, with NH4OH giving slightly higher signal intensi-
ties than NaOH. The optimum concentration was determined
to be 0.5 %, with signal intensities decreasing again at higher
concentrations. Signal intensities increased by 72, 66, and
34 % when 0.5 % NH4OH, NaOH, and ammonium acetate,
Fig. 2 Comparison of total ion current (TIC) signal fluctuations for
different sample substrates. Samples were spotted on the different
substrates and analyzed at a constant speed of ca. 330 μm/s along the
diameter of the spotted samples. Two microliters of the sample (standard
solution of a mixture of the investigated fatty acids at 50 mg/L) was
spotted multiple times with a syringe along a straight line at 1 mm
distances to obtain an even distribution of the compounds on the
surface. The TLC material was then continuously sampled for 0.3 min
(corresponding to a distance of 6 mm) for each surface. Surface materials
are as follows: (a) filter paper, (b) porous Teflon, (c) C-18 TLC plate, (d)
silica TLC plate, (e) cyano HPTLC plate, (f) glass, and (g) metal
(AnchorChip MALDI plate). (All experiments were individually
normalized, and absolute signals cannot be compared between traces
(a–g), but rather signal fluctuations between traces.)
Determining fatty acids by DI-MS using TLC substrates

6. respectively, were added to the TCL plate in comparison to the
experiments without adding base. Addition of base to the TLC
plate prior to deposition of the samples was believed to en-
hance the generation of preformed ions via deprotonation of
fatty acids after spotting. DI signal intensities were therefore
expected to increase as fatty acids were desorbed as [M-H]−
ions by the spray solvent. This effect might also explain the
negligible differences seen in signal intensities between volt-
age (DESI) and no voltage (DI) analysis in this mode of op-
eration, as preformed ions were likely desorbed with ba-
se present on the plate. If charge is carried and trans-
ferred through the sprayer solvent, DESI is expected to
be more efficient than DI, in particular for those com-
pounds that do not exhibit preformed ions on the sam-
ple spots. DESI does not rely only on statistical charge
imbalances within droplets as required for our DI tech-
nique if ions are not preformed, which is thought to
function similar to TSP [45–48] (see Experimental.
Please note that voltage was not routinely applied in
our setup for electrical safety reasons!). In general, the
implemented DI technique in this study is therefore ex-
pected to give better performance for compounds that
can be made to form ions by lowering or raising pH,
similar to electrospray ionization.
Quantification of fatty acids
The proof-of-concept application of this study was the quan-
titative measurement of fatty acids. To demonstrate the tech-
nical feasibility, we used linoleic acid as internal standard for
calibration. Other fatty acids could be readily used for this
purpose, depending on the application and the fatty acids of
interest. Six concentration levels (0.05, 0.5, 5, 50, 250, and
500 mg/L) were investigated for the determination of the other
five fatty acids, while keeping the concentration of internal
standard at 200 mg/L. For linear regression, regular least
squares with 1/x weighing was used, resulting in good linear-
ity for the five analytes between 0.5 and 500 mg/L with coef-
ficients of determination (R2
) in the range of 0.9856 to 0.9977
(an example calibration curve for lauric acid is shown in ESM
Fig. S2; the internal standard was a necessity, as is evident
from the figure). The reproducibilities of peak areas deter-
mined from the extracted ion profiles of the fatty acids were
generally between 3 and 10 % RSD in our experiments. Ad-
ditional improvements can be expected from using a
precision-motorized x,y stage during analyses; the manual
sample movement was likely contributing significantly to
the observed uncertainty of measurement; the errors from
manual operation were sometimes visible in the
Fig. 3 Extracted ion current
(EIC) traces of oleic acid (m/z
281, 50 mg/L) for ten different
spots of a fortified lipid-free
human serum extract (1 μL serum
extract each), spotted using a
micro-syringe within 5 s (top) and
a pipette (center). The bottom
trace shows TLC-DI-MS-
extracted ion current profiles of
oleic acid (m/z 281.3, 50 mg/L)
after sample application via
different deposition techniques
(sample solvent/methanol): (a)
2.5 μL pipette deposition, (b)
5 μL pipette deposition, (c) 5×
1 μL pipette deposition, (d) 1 μL
syringe deposition within 5 s, and
(e) 1 μL syringe deposition within
20 s
M.F. Mirabelli et al.

7. chromatographic peak shapes, where distortions were seen
(data not shown). LODs at S/N=3 were evaluated by analyz-
ing the samples at decreasing concentration levels and found
to be in the 2–10 μg/L range for the five investigated fatty
acids. This range corresponded to absolute values of 2–10 pg
for 1 μL of sample depositions on the support and was com-
parable or lower than that of the results obtained using con-
ventional DESI-MS [34, 35].
The method was subsequently applied to an analysis of an
olive oil (extra virgin, Coratina olive fruit, Apulia, Italy) ex-
tract and compared to results from gas chromatography-flame
ionization detection (GC-FID) analysis, to assess the accuracy
of our data. The two compared samples were for the same
olive fruits but differed in the manufacturing process: the re-
sults for the GC-FID analyses were obtained for the commer-
cial production batch provided by an external laboratory; the
sample analyzed here was a craft production sample. The re-
sults of this comparison are summarized in Table 2, demon-
strating moderate-to-good agreement between the two
methods (the corresponding GC-FID separation is shown in
ESM Fig. S3; the DI-MS mass spectra of the olive oil sample
are shown in ESM Fig. S4). Some of the deviations were
likely due to the different manufacturing process, but also
the calibration procedure used here (see below). Furthermore,
the strong overestimation of the levels measured by DI-MS
over those by GC-FID for very low abundant species (C14:0,
C16:1, C17:0, and C17:1) may be due to signal interferences
from isobaric noise, as no separation was performed for DI-
MS. The DI-MS method was faster than GC-FID; however,
conventional analysis of fatty acids from the olive oil samples
requires saponification of the crude sample, liquid-liquid ex-
traction, and derivatization of the fatty acids to methyl esters,
followed by GC-FID analysis, usually taking several hours.
Our DI-MS assay still required saponification and liquid-
liquid extraction from the crude sample but was followed
directly by rapid DI-MS, necessitating an overall analysis time
of <2 h. In addition, multiple different samples can be spotted/
eluted onto the same TLC plate, allowing for high-throughput
operation if extraction and purification steps are automated.
Since we only compared absolute ion abundances of dif-
ferent fatty acids for the same sample, we did not use an
internal standard for olive oil in this proof-of-concept study.
An internal standard is vital when analyzing samples at differ-
ent concentration levels, to obtain proper calibration curves,
because signals at higher concentration levels were found to
be non-linear with increasing concentration levels (as visible
from ESM Fig. S2). While an internal standard would com-
pensate for this effect, it would not give an advantage in a
single spot analysis, where relative signal ratios are evaluated,
unless absolute quantification is desired.
Furthermore, we also analyzed fish oil samples and found
good agreement between the data reported by the supplier for
the eicosapentaenoic acid/docosahexaenoic acid (EPA/DHA)
ratio (1.5) and our measured values of 1.41 (extracted fish oil)
and 1.54 (crude fish oil; used as is, without extraction). Fur-
thermore, we also investigated the EPA/DHA ratio in salmon
tissue (see Experimental), which was determined to be 0.82 in
the investigated farmed Scottish salmon sample. This ratio
was well within the range reported for farmed Scottish salmon
products of supermarkets; a recent study by Henriques et al.
showed EPA/DHA ratios from 0.67 to 1.0 for different Scot-
tish salmon products from major UK retailers [50]. The most
abundant saturated fatty acid was palmitic acid (C16:0)
followed by stearic acid (C18:0), which was also seen in lit-
erature values [51].
Hyphenated TLC-DI-MS
As shown above, TLC plates provided excellent substrates for
direct DI analysis. Alternatively, they can be used as addition-
al separation stage followed by DI analysis. Here, silica and
C-18 plates were evaluated for separation of fatty acids, but
only the latter provided sufficient separation efficiency. Crude
fish oil and fish oil extracts, salmon, and human serum were
analyzed using the optimized RP-TLC-DI-MS method
(Fig. 4). Importantly, the main advantage of the additional
separation step is the reduction of potential signal suppression
effects from co-components of the complex sample matrices.
TLC gave only modest resolving power under the conditions
used here but provided sufficient efficiency for near baseline
separation of several important fatty acids. In turn, signal-to-
noise ratios for the analytes increased by a factor of 4.5, 4.4,
and 2.7 for C16:0, C18:1, and C18:3, respectively. Absolute
peak areas did not increase, however, indicating that signal
suppression effects between the analytes were minor during
the DI-MS experiments described in the previous section,
Table 2 Comparison of
fatty acid profiles
obtained by TLC-DI-MS
and GC-FID analysis
(percentages were
normalized to 100 % for
the considered fatty
acids)
Fatty acid DI (%) GC (%)
C14:0 1.4 0.01
C16:1 6.3 0.1
C16:0 18.3 10.3
C17:1 0.9 0.1
C17:0 1.8 0.1
C18:3 0.7 0.6
C18:2 5.9 7.6
C18:1 58.5 77.2
C18:0 4.8 2.6
C20:1 0.4 0.4
C20:0 0.5 0.4
C22:0 0.4 0.1
DI-MS was performed on a craft produc-
tion sample of Coratina olive fruit; GC-
FID results are for a commercially produc-
tion batch of the same olive species
Determining fatty acids by DI-MS using TLC substrates

8. where analytes were co-deposited on the TLC plates without
chromatographic separation. Moreover, S/N ratios for the
analytes increased further, when a matrix was present
(Table 3). For experiments using delipidized human serum
fortified with fatty acids, we obtained significant increases of
S/N ranging from approx. 3-fold (for C18:3) to 19-fold (for
C12:0). In comparison to the spot analysis in the previous
section, slightly changed ratios of fatty acids were obtained
for the same samples. For example, the EPA/DHA ratio in
salmon was determined as 0.94 as compared to 0.82 if a
non-separated spot was analyzed, which could, of course, be
related to minor signal suppression effects.
Considering the small number of fatty acids investigated in
this proof-of-concept study, the limited resolving power of
TLC over HPLC was sufficient to demonstrate the beneficial
effects. The aim here was not baseline separation of all fatty
acids but rather to demonstrate the increase of S/N ratios of
deprotonated molecules of fatty acids after TLC as compared
to the single spot mixture analysis above, where matrix effects
can influence detection and quantification.
Conclusions
In this study, we have investigated the application of ambient
mass spectrometry using a simple solvent-assisted DI-MS in-
terface for measuring fatty acids from various biological and
food sample matrices. Optimum performance was obtained
after spotting samples onto TLC plates as substrates for DI-
MS. The use of optimized spotting time, temperature, and type
of substrate allowed for reduction of the so-called coffee ring
effect and provided more stable and reproducible signal pro-
files. In our experiments, DI-MS generally exhibited good-to-
excellent linearities and reproducibilities for the investigated
fatty acids. The signal-to-noise ratios were further increased
by separating the species to some extent by TLC and
performing DI-MS directly from the developed TLC plates.
Limits of detection for the selected fatty acids were compara-
ble to literature results from DESI-MS. The data were
achieved with a manual sample stage, which added some peak
distortion and imprecision to the measurements. Therefore,
the analytical figures of merit will likely further improve by
using a precision-motorized linear stage for sample move-
ment, which will be implemented in the future along with an
automated liquid dispenser for spotting samples onto the TLC
plates. Equally important will be the future implementation of
appropriate internal standards for more precise and accurate
quantitative results.
Table 3 Increase of signal-to-noise ratios for DI-MS analysis of fatty
acids deposited on a C-18 TLC surface (1 μL deposition of 100 ng/μL
solution of fatty acids in lipid-free human serum), analyzed before and
after thin-layer chromatographic separation. Samples were linearly
rasterized across the spots with the DI sprayer at 330 μm/min
Fatty acid Before TLC (single spot) After TLC
C12:0 76 1362
C16:0 262 2550
C18:0 114 1448
C18:1 245 1311
C18:2 365 1071
C18:3 77 228
Fig. 4 TLC-DI-MS EIC profiles of seven fatty acids from different
sample matrices after TLC separation on a C-18 material: (a) 100 mg/L
spotted lipid-free human serum with standard FAs, (b) crude fish oil (no
sample preparation), (c) fish oil extract, (d) salmon extract, and (e) human
serum extract. Lauric acid (C12:0, m/z 199.2), palmitic acid (C16:0, m/z
255.2), linolenic acid (C18:3, m/z 277.2), linoleic acid (C18:2, m/z 279.2),
oleic acid (C18:1, m/z 281.3), stearic acid (C18:0, m/z 283.3),
eicosapentaenoic acid (C20:5, m/z 301.3), docosahexaenoic acid
(C22:6, m/z 327.3)
M.F. Mirabelli et al.

9. Acknowledgments GC acknowledges a fellowship from Rotary
International (District 2120, Molfetta, Bari, Italy) for his research visit
to Saarbrücken. The authors acknowledge Chemia s.r.l. (Bironto, Bari,
Italy) for the GC analyses. DAVis grateful for general research support by
the Alfried Krupp von Bohlen und Halbach-Stiftung.
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