# The valuable role of high-performance thin-layer chromatography in the lipidomic analysis of Thlaspi arvense, an emerging feedstock for biofuel production

José Manuel Escuín, Ana Claver, María Ángeles Luján, Marion Schilling, Juliette Jouhet, María Savirón, M. Victoria López, Carmen Jarne, Miguel Alfonso, Vicente L Cebolla

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## To cite this version:

José Manuel Escuín, Ana Claver, María Ángeles Luján, Marion Schilling, Juliette Jouhet, et al. The valuable role of high-performance thin-layer chromatography in the lipidomic analysis of Thlaspi arvense, an emerging feedstock for biofuel production. Journal of Planar Chromatography, 2025, 10.1007/s00764-025-00346-y. http://dx.doi.org/10.1007/s00764-025-00346-y

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# The valuable role of high-performance thin-layer chromatography in the lipidomic analysis of *Thlaspi arvense*, an emerging feedstock for biofuel production

José Manuel Escuín<fnref n="1" /> D · Ana Claver<fnref n="2" /> D · María Ángeles Luján<fnref n="2" /> D · Marion Schilling<fnref n="3" /> D · Juliette Jouhet<fnref n="3" /> D · María Savirón<fnref n="4" /> D · M. Victoria López<fnref n="5" /> D · Carmen Jarne<fnref n="6" /> D · Miguel Alfonso<fnref n="2" /> D · Vicente L. Cebolla<fnref n="1" /> D

Received: 3 February 2025 / Accepted: 1 May 2025

© Akadémiai Kiadó Zrt 2025

## Abstract

Two different lipidomic approaches, high-performance thin-layer chromatography coupled to electrospray ionization–mass spectrometry (HPTLC–ESI–MS) and liquid chromatography coupled to electrospray ionization–mass spectrometry (LC–ESI–MS) were used for the quantitative analysis of lipid fractions obtained from different seed maturation stages from pennycress (*Thlaspi arvense*), an emerging feedstock for biofuel production. Pennycress seed oil is characterized by its unusually high content in erucic acid (C22:1), mostly accumulated in the form of triacylglycerols (TAG). Their performances were compared under optimal conditions for each, and the coherence of results obtained from both techniques were evaluated. The acquisition of HPTLC–ESI(+)–MS spectra obtained at the same time of ionization for each of the seed maturation stages in a rapid and simple way, revealed two important types of information: first, the normalized profiles of TAG ion abundance, without any calibration step. These results and the results from LC–MS were in agreement concerning erucic acid incorporation and were statistically correlated. Results from both techniques confirm the incorporation of 22:1 into TAG already at the initial stage of seed maturation (TAG 58:5), as well as the increase of 22:1 and 20:1 as maturation progresses (TAG 60:4 and TAG 62:4, which correspond to 20:1/18:2/22:1 and 22:1/18:2/22:1, respectively). Second, it revealed the *sn*-positional analysis by HPTLC–ESI(+)–MS/MS of several relevant TAG species in pennycress seed maturation. This analysis indicated that linoleic acid was esterified at *sn*-2 in all stages of seed maturation of several quantitatively important TAG subclasses and, therefore, suggesting a high specificity of lysophosphatidic acid acyl transferase (*LPAT*) for linoleic acid even in a high-erucic-containing species such as pennycress. All of these results support the utilization of HPTLC as a rapid and reliable methodology, compatible with LC–MS, for the analysis of complex lipid mixtures such as those found in plant seed oils.

**Keywords** *Thlaspi arvense* · Seed oil · Triacylglycerol (TAG) · Erucic acid · Very-long-chain fatty acid (VLCFA) · High-performance thin-layer chromatography (HPTLC) · Mass spectrometry · MS · Liquid chromatography–mass spectrometry (LC–MS) · Lipidomics

<figure><img src="image_3.png" /></figure>

<footnote>Vicente L. Cebolla
vcebolla@icb.csic.es</footnote>

<footnote n="1">¹ Agencia Estatal Consejo Superior de Investigaciones Científicas (CSIC), Instituto de Carboquímica, 50018 Saragossa, Spain</footnote>

<footnote n="2">² Department of Plant Biology, Agencia Estatal Consejo Superior de Investigaciones Científicas (CSIC), Estación Experimental de Aula Dei (EEAD), Avda. Montañana 1005, 50059 Saragossa, Spain</footnote>

<footnote n="3">³ Laboratoire de Physiologie Cellulaire Végétale, Université Grenoble Alpes, CNRS, INRAE, CEA, 38000 Grenoble, France</footnote>

<footnote n="4">⁴ Facultad de Ciencias, Agencia Estatal Consejo Superior de Investigaciones Científicas (CSIC), Universidad de Zaragoza, CEQMA, C/ Pedro Cerbuna 12, 50009 Saragossa, Spain</footnote>

<footnote n="5">⁵ Department of Soil and Water Conservation, Agencia Estatal Consejo Superior de Investigaciones Científicas (CSIC), Estación Experimental de Aula Dei (EEAD), Avda. Montañana 1005, 50059 Saragossa, Spain</footnote>

<footnote n="6">⁶ Facultad de Veterinaria, Departamento de Química Analítica, Universidad de Zaragoza, C/Miguel Servet 177, 50013 Saragossa, Spain</footnote>
# 1 Introduction

Liquid chromatography coupled to mass spectrometry (LC-MS) is the gold standard for lipidomic analysis [1]. When electrospray ionization (ESI) is used in combination with a triple quadrupole (QqQ) as mass detector, LC-MS provides an enormous quantitative information about lipid classes and subclasses, in spite of some inconvenients such as, among others: the consecutive injection of samples; long chromatographic times associated to reversed-phase separation; need of rigorous calibrations at several levels to correct lipid responses; and no interruptions during chromatographic runs [2].

By contrast, high-performance thin-layer chromatography was directly coupled to MS (HPTLC-MS) some time ago [3]. Despite this, the technique has hardly been used in lipidomics and, in most cases, only for identification purposes [4]. In general, there are no systematic studies of the use of both techniques applied to the same lipidomic problem, neither to check whether similar or different results are obtained. In addition, there are no studies to propose different working methods derived from the different formats of both techniques, which would allow us to obtain complementary information.

Since the HPTLC separation of complex lipid samples is usually performed on silica gel plates, it is carried out on the basis of polar group (lipid class) rather than molecular separations as in reversed-phase LC. This fact makes the most favorable mass detector for connection with HPTLC to be ion-trap (IT). This is because all subclasses belonging to a given lipid class are sent together to the detector and analyzed there, also using ESI. In addition to the planar format of HPTLC and its properties of simplicity, speed, and flexibility, there are other properties that should make it possible to develop methods that provide complementary information to that of LC-MS: the application of several samples and standards on the same plate, the absence of a postchromatographic solvent, the selectivity selection of a target band using the interface, and its extraction to send it to MS using a single solvent.

The question underlying this work is: Can HPTLC-MS be really useful and provide quality complementary information that even enriches lipidomics performed with LC-MS? To answer this question, we used both techniques, under the conditions that we considered most favorable to each one, for the lipidomic analysis of the seeds of a plant of high interest as a biofuel feedstock.

In fact, *Thlaspi arvense*, a winter weed plant known as pennycress, is an emerging raw material for biofuel production. This plant has favorable agronomic properties and can be cultivated in arid and semiarid drylands, and its seeds have a high oil content (29–40 wt% [weight

percentage]). Its oil is not edible and its cultivation strategy (cultivation during the fallow period in the traditional cereal rotation in Mediterranean Europe) does not compete with other crops that are edible, such as corn, soybean, or sunflower [5]. However, the main characteristic of pennycress seed oil that determines its extraordinary properties for biofuel production is its enriched composition in erucic acid (C22:1, monounsaturated, long-chain C22Δ¹³) as well as other very-long-chain fatty acids (VLCFAs), particularly 20:1. Up to 35 wt% of fatty acids in pennycress seed are erucic acid [6]. However, C22:1 is absent or barely present in oils from other phylogenetically related species to pennycress, such as cultivated rapeseed or *Arabidopsis thaliana*, or in other species used for biofuel production such as soybean, sunflower, or *Camelina sativa*.

The aims of our work were (1) to apply LC-ESI-MS (QqQ) and HPTLC-ESI-MS (IT) to the lipidomic analysis of seed lipid fractions from pennycress; (2) to compare their performances; and (3) to evaluate the coherence of results from both techniques and evaluate whether they provide different and/or complementary information for this lipidomic analysis. For this purpose, five development stages of seed maturation: green (G), green-yellow (GY), yellow-green (YG), yellow (Y), and mature (M) were defined. Triacylglycerol (TAG) is the main reservoir of erucic acid in pennycress seeds. Between 80% and 90% of total lipids present in seeds are found in this form; an exhaustive lipidomic study was carried out to analyze the distribution of acyl groups in TAG during these different stages of seed maturation.

# 2 Experiment

## 2.1 Plant materials

Pennycress seeds from the SPRING32 germline were used (Nottingham Arabidopsis Stock Center, NASC, UK). Conditions for germination and growth were reported elsewhere [6]. Seeds from five developmental stages were selected for lipidomic analysis, which correspond to green (G, 12 days after flowering, DAF), green-yellow (GY, 19 DAF), yellow-green (YG, 26 DAF), yellow (Y, 33DAF), and mature (M, 45 DAF), according to a previous work [6]. Seeds corresponding to these different maturation stages were harvested, frozen in liquid nitrogen, and stored at -80 °C. A total of two samples per maturation stage (G, GY, YG, Y, and M) were analyzed.

## 2.2 Standards and chemicals

Notation adopted for the identity of a molecular lipid subclass was either (C:n), where *n* is the total carbon number of the molecule and *y* corresponds to the total number of
double bonds; or (c_a:n_a/c_b:n_b/c_c:n_c) where c_a, c_b, and c_c are the carbon number of each fatty acyl in the correspond- ing TAG subclass (with c_a + c_b + c_c = C); n_a, n_b, n_c corre- spond to the number of double bonds of each fatty acyl (n_a + n_b + n_c = n). Phosphatidylethanolamine (PE, 18:0/18:0, 1069–79-0 Chemical Abstracts Service, CAS) and diacylg- lycerol (DAG, 18:0/22:6, 65,886–80-8 CAS) were used as internal standards in LC–MS and purchased from Avanti Polar Lipids (Alabaster, AL, USA). Triheptadecanoin (C17:0/C17:0/C17:0, ≥ 99%, 2438–40-6 CAS) was used as internal standard for gas chromatography with flame- ionization detection (GC–FID) analysis and purchased from Sigma-Aldrich (Madrid, Spain). The following stand- ards were used in HPTLC analysis: 1-oleoyl-*rac*-glycerol (C18:1, ≥ 99%, 111–03-5 CAS); 1,2-dioleoyl-*rac*-glyc- erol (C18:1/C18:1, ≥ 97%, 2442–61-7 CAS); glyceryl trioleate (C18:1/C18:1/C18:1, > 99%, 22–32-7 CAS); trierucin (C22:1/C22:1/C22:1, > 99%, 2752–99-0 CAS); oleic acid (C18:1), ≥ 99%, 112–80-1 CAS); linoleic acid (C18:2, ≥ 99%, 60–33-3 CAS); erucic acid (C22:1, ≥ 99%, 112–86-7 CAS); methyl oleate (99%, 112–62-9 CAS); cho- lesteryl oleate (≥ 98%, 303–43-5 CAS); and cholesteryl stea- rate (96%, 35,602–69-8 CAS), which were purchased from Sigma-Aldrich (Madrid, Spain).

High-performance liquid chromatography (HPLC)-grade *n*-hexane (C6, 99%), *n*-heptane (C7, 99.9%), *tert*-butyl methyl ether (MTBE, 99.9%), glacial acetic acid (AcH, 99.8%), methanol (MeOH, 99.9%), isopropanol (99.9%), dichloromethane (DCM, 99.9%), chloroform (CHCl3, 99%), and tetrahydrofuran (THF, 99.9%, without stabilizer) were purchased from PanReac (Barcelona, Spain). Ammonium acetate for HPLC was purchased from Merck. LC/MS-grade MeOH was purchased from Fisher Scientific (Loughbor- ough, UK).

## 2.3 Lipid extraction and fatty acid analysis

Total lipids were extracted from pennycress seeds (0.1 g) with CHCl3–MeOH (6 mL, 2:1, V/V) by the “Bligh and Dyer” method [7]. For total fatty acid quantification, a method based on a previous work [8] was performed through direct transmethylation using triheptadecanoin (30–35 mg) as internal standard. Fatty acid methyl esters of total lipids were analyzed by GC–FID, as previously described [6].

## 2.4 LC–ESI–MS

Glycerolipids in pennycress seed extracts were separated by LC in mode HPLC and quantified by MS/MS using multiple reaction monitoring (MRM) on the LIPANG platform using a general method previously described [9].

The lipid extracts corresponding to 25 nmol of total fatty acids were dissolved in 100 µL of CHCl3–MeOH (2:1, V/V) containing 125 pmol of each internal standard: PE 18:0/18:0, DAG 18:0/22:6, and sulfoquinovosyl dia- cylglycerol (SQDG) 16:0/18:0. The latter was obtained as previously described, from spinach thylakoid and subse- quent hydrogenation [10].

The LC separation method was adapted from a previous publication [11], using an Agilent 1260 Infinity II HPLC system (Santa Clara, CA, USA). The volume injected was 20 µL. The separation was performed on a diol column (150 mm length × 3 mm i.d., 5 µm particle size) (Mach- erey–Nagel, Düren, Germany), at 40 °C. The mobile phases were: C6–isopropanol–water–ammonium acetate (1 M), pH 5.3 (625:350:24:1, V/V) (A), and isopropanol– water–ammonium acetate 1 M, pH 5.3 (850:149:1, V/V) (B). The flow rate of the mobile phase was 200 µL/min.

The distinct glycerolipid classes were eluted succes- sively as a function of the polar head group. Internal stand- ards were useful to correct the three different response zones of the chromatogram: DAG 18:0/22:6 was used to calibrate the responses of DAG, digalactosyldiacylglyc- erols (DGDG), monogalactosyldiacylglycerols (MGDG), and TAG; PE 18:0/18:0 to PE response. SQDG 16:0/18:0 was used to calibrate to cardiolipins (DPG), phosphatidic acids (PA), phosphatidylcholines (PC), phosphatidylglyc- erols (PG), phosphatidylinositols (PI), phosphatidylserines (PS), and SQDG responses.

Mass spectrometric analysis was done on a 6470 triple quadrupole (QqQ) mass spectrometer (Agilent) equipped with a Jet stream electrospray ion source under the fol- lowing settings: drying gas heater: 260 °C; drying gas flow: 13 L/min; sheath gas heater: 300 °C; sheath gas flow: 11 L/min; nebulizer pressure: 25 psi; capillary volt- age: ± 5000 V; and nozzle voltage: ± 1000 V. Nitrogen was used as the collision gas. The quadrupoles Q1 and Q3 were operated at widest and unit resolution, respectively.

ESI–MS conditions for glycerolipid classes are shown in Supplementary Table 1S (Supplementary Information). Quantification was done by MRM using 30 ms as dwell time for all classes except CL (50 ms). MRM transitions of TAG were adapted from a previous publication [12], and they are detailed in Supplementary Table 2S.

Mass spectra were processed by MassHunter Worksta- tion software (Agilent) for the identification and quantifi- cation of lipids. In addition to lipid amounts (pmol) being corrected for response differences between internal stand- ards and endogenous lipids, instrument responses were also corrected by comparison with an external standard- quality control (QC) that corresponds to a known lipid extract from an *Arabidopsis* cell culture that was previ- ously qualified and quantified by thin-layer chromatogra- phy (TLC) and GC–FID [9].
## 2.5 HPTLC-ESI-MS

### 2.5.1 HPTLC experiments

HPTLC silica gel 60 plates (20×10 cm) without fluorescence indicators were employed, from Merck (Darmstadt, Germany). Before being used, plates were immersed in THF (5 min), dried at 70° C and in a vacuum (50 mbar) for 15 min, and predeveloped up to 90 mm using C7-MTBE-AcH (70:30:1, V/V).

The application of solutions of standards and samples as 4-mm bands was carried out with the Automatic TLC Sampler system (ATS4) from CAMAG (Muttenz, Switzerland), using the ATS4 filling quality method. Solutions of individual standards (0.33–2 mg mL⁻¹), per standard in DCM-MeOH (1:1, V/V), were applied in triplicate on the same plate (3 µL/band). Each sample (4 µL) was applied in triplicate on three different plates. Samples were dissolved (3–4 mg mL⁻¹) in DCM-MeOH (1:1, V/V). The minimal distance between tracks was 6 mm and distance from the lateral and lower plate edges was 10 mm. One or more tracks were left empty, as blanks for chromatography and MS analysis. The plates were developed in a horizontal developing chamber (20×10 cm, CAMAG) up to 70 mm using C7-MTBE-AcH (70:30:1, V/V). Detection was carried out using a TLC Scanner 3 (CAMAG) at 190 nm in absorption mode. winCATS software (v 1.4.3.6336, CAMAG) was used to control and process data from sample application and densitometry.

### 2.5.2 HPTLC-MS

The TLC-MS Interface 2 (CAMAG) was used for an extraction of each TAG band directly from the plate. Details of elution-based interface description and operation can be found elsewhere [13]. The interface was equipped with an oval, 4×2-mm extraction head that was positioned on the corresponding TAG-band center, whose x, y coordinates were provided by densitometry and winCATS software. Then, MeOH was delivered for band extraction at 0.2 mL/min by using a PU-2080 HPLC pump (Jasco, Tokyo, Japan). The eluate was directed through a 2-mm stainless steel frit to remove silica gel and then sent to a mass spectrometer, an ion trap Amazon Speed Spectrometer (Bruker Daltonics, Bremen, Germany). Electrospray ionization in positive ion mode (ESI(+)-MS) was selected for TAG analysis. Electrospray ionization was conducted with capillary and endplate offset voltages of -4500 and -500 V, 36 psi as the pressure of the nebulizer gas (N₂), 6.0 L/min as the flow rate of the drying gas (N₂), and 120 °C as the drying gas temperature. Spectra were acquired in the *m/z* 70–1500 range at the ultra-scan mode. Bruker Daltonics Trap Control software packages v 8.0 and Data Analysis v 5.2 were used to control the

mass spectrometer and process data. For each TAG peak, several HPTLC-ESI(+)-MS experiments were performed from replicate bands, and confirmation of identity was carried out by MS/MS (see Results section). These experiments were performed from different plates.

## 2.6 Statistical analysis

Data from LC-MS are expressed as averages ± standard deviation (SD), with three replicates in each experimental group. The statistical comparisons among the different developmental stages during the seed maturation of pennycress were made using one-way analysis of variance (ANOVA), and averages were compared using Duncan's multiple range test (*P* < 0.05). When data showed nonnormality, log or reciprocal transformations were made, and ANOVA was conducted with the transformed data.

To evaluate the correlation between the results from LC-MS and HPTLC-MS, Pearson and Spearman correlation coefficients were calculated from R Posit Cloud (RStudio Pro 2023.12.1 Build 402.pro1 version, Posit Software, PBC). Scatter graphs were done using ggplot function. The Shapiro-Wilk test was used to evaluate whether sample data fit to a normal distribution.

# 3 Results

## 3.1 Rapid, normalized profiles of TAG ion abundance by HPTLC-ESI(+)-MS

Densitograms of standards corresponding to general glycerolipid classes are shown in Fig. 1A. Figure 1B shows densitograms corresponding to two maturation stages (G and M) of a pennycress seed extract together with the densitogram of trierucin, a TAG standard. TAG bands have a migration distance (m.d.) near to 52 mm (Fig. 1B). As will be discussed in Subsection 3.2, confirmation of TAG identity was performed by ESI(+)-MS/MS. Densitograms corresponding to the other maturation stages can be found in Supplementary Fig. 1S.

Without the need for a quantitative analysis of the separated bands, it is already clear from the densitograms that TAG is the main peak in pennycress seeds, representing between 88% and 96% of the total peak area (Supplementary Fig. 1S). This percentage increases as a function of maturation, from the different stages from G to M, consistent with our previous observations [6].

After HPTLC separation, the band corresponding to TAG was extracted by the TLC-MS interface and sent to an ion trap MS. We obtained rapid, normalized intensity HPTLC-ESI(+)-MS profiles that provided the relative abundance of TAG ions. This was done by recording the MS spectra at

<figure><img src="image_4.png" /></figure>
**Fig. 1** A HPTLC densitograms of glycerolipid standards at ultraviolet (UV) 190 nm. 1: 1-oleoyl-rac-glycerol (MAG) (m.d.: 11.3 mm); 2: 1,2-dioleoyl-rac-glycerol (DAG) (m.d.: 18.3 mm); 3: linoleic acid (FA) (m.d.: 35.2 mm); 4: oleic acid (FA) (m.d.: 35.9 mm); 5: erucic acid (FA) (m.d.: 39.2 mm); 6: trierucin (m.d.: 52 mm); 7: glyceryl trioleate (TAG) (m.d.: 54.4 mm); 8: methyl oleate (m.d.: 58.8 mm); 9: cholesteryl stearate (m.d.: 71.8 mm); and 10: cholesteryl oleate (m.d.: 71.8 mm). B HPTLC–UV (190 nm) chromatograms corresponding to the maturation stages G (14.96 µg) and M (14.78 µg) of a pennycress seed extract. TAG bands and trierucin standard (3 µg) migration at m.d. = 52 mm

<figure><img src="image_5.png" /></figure>

the same ionization time for all samples at each maturation stage. No ESI intensity correction was needed in this case since the mobile phase used for separation evaporated before detection, and the subsequent automatic extraction of the band to obtain the mass spectrum was performed using the same solvent in all cases.

From HPTLC–ESI(+)–MS spectra two important types of information are obtained which are discussed in Subsections 3.2 and 3.3.

## 3.2 Structural and *sn*-positional analysis of several relevant particular TAG species in pennycress seed maturation by HPTLC–ESI(+)–MS/MS

Ion trap–MS is a technology that allows multistage tandem analysis, which involves the successive isolation and fractionation of product ions. Under our chromatographic conditions, $[M+Na]^{+}$ ions are obtained that can be fragmented to obtain structural information, as demonstrated in previous works [13–15]. Sodium adducts from ion trap are adequate

for structural analysis of TAG, as they produced the most consistent level of positional sensitivity for the fragmentation [16].

The structural identification of TAG molecular subclasses was carried out by obtaining MS/MS spectra directly from the separated TAG band on the chromatographic plate by isolation and fragmentation of selected ions. When using an ion trap, this must be done in a very limited period of time to obtain the corresponding MS/MS spectrum. As several repetitions of a given sample can be applied on the same plate and chromatographic development takes place for all applied sample loads simultaneously, HPTLC is the technique of choice to have the chance to isolate and fragment a given ion, thus leading to a rapid MS/MS recording. In case of failure in the capture, there are other sample loads applied to try again. However, using LC–MS, the sample would have to be reinjected into a new run and eluted for 70 min.

In a first step of our research, six ions from the HPTLC–ESI(+)–MS spectrum were selected (m/z 907.8; 935.8; 959.9; 989.8; 1017.9; and 1046.0) and their ion intensities
(Fig. 2A), recorded at the same ionization time. They corre-
sponded to TAG 54:3; 56:3; 58:5; 60:4; 62:4; and 64:4. Sub-
sequently, each ion was isolated and fragmented to obtain
its corresponding MS/MS spectrum (Fig. 3B). Although the
number of ions to be identified is not limited with this tech-
nique, we chose six structures as a compromise between a
reasonably short analysis time and the selection of signifi-
cant structures that would give us useful information about
each stage of maturation.

From the detailed inspection of the MS/MS spectra of
the TAG classes selected in Fig. 2, we observed that three
of them, TAG 60:4 (m/z 989.8), TAG 62:4 (m/z 1017.9) and
TAG 64:5 (m/z 1046.0), correspond to a unique combination
of FA (a single triad), and the other three, TAG 54:3 (m/z
907.8), TAG 56:3 (m/z 935.8), and TAG 58:5 (m/z 959.9),

correspond to several possible combinations of fatty acids.
This is explained below, as well as what positional informa-
tion we infer from this, and why this information is impor-
tant in the study of the incorporation of erucic acid and other
VLCFA into triglycerides in pennycress seeds.

In natural products, many isobaric TAG species coming
from different combinations of FA may complicate MS/MS
spectra interpretation, making positional analysis impossi-
ble, i.e., attribution to *sn*-1, *sn*-2, and *sn*-3 positions. Fig-
ure 2B and Supplementary Table 3S show the different pos-
sibilities of fatty acyls combinations for TAG 54:3, 56:3,
and 58:5 subclasses.

However, there are occasions where only one combina-
tion of fatty acid composition is present. When a single
triad of fatty acids is found in an MS/MS spectrum of a

<figure><img src="image_6.png" /><figcaption>**Fig. 2** A HPTLC−ESI(+)−MS ion intensity percentages of six selected ions, recorded at the same ionization time versus their *m/z* and their corresponding TAG subclasses related to seed maturation stages (G, GY, YG, Y, and M) indicated in colors. **B** Glycerol struc-</figcaption></figure>

ture (red) and possible combinations of fatty acyls of TAG 54:3 sub-class. In this example, there is no positional information: fatty acyls can be in any position

<figure><img src="image_7.png" /><figcaption>**Fig. 3** Identification of the fatty acyl at *sn*-2 position when a single triad of FA is present in a MS/MS spectrum of a parent ion. **A** TAG molecules lose simultaneous and preferentially fatty acyls at positions *sn*-1 and *sn*-3 (indistinguishable): two ions of similar abundance are lost and, in turn, they are more abundant than the ion corresponding to the loss of the fatty acyl at *sn*-2. **B** Example of identification of linoleic acid (18:2) at *sn*-2 position in TAG 60:4 from a single triad of fatty acids. MS/MS spectrum of 989.9 m/z parent ion: three main ion products corresponding to losses of fatty</figcaption></figure>

acyl substituents as fatty acids: at 679.6 m/z, which corresponds to
[M+Na−R₁,₃COOH]⁺, R₁,₃=C(20:1)FA; at 651.5 m/z, which
corresponds to [M+Na−R₃,₁COOH]⁺, R₃,₁=C(22:1)FA; and at
709.6 m/z, which corresponds to [M+Na−R₂COOH]⁺, R₂=C(18:2)
FA. Product ions at low intensities: 657.6 m/z, which corresponds
to [M+Na−R₁,₃COONa]⁺; 629.6 m/z, which corresponds to
[M+Na−R₃,₁COONa]⁺; and 687.7 m/z, which corresponds to
[M+Na−R₂COONa]⁺
parent ion, the intensities of the resulting product ions reflect the fatty acid distribution in the glycerol backbone. In these cases, we were able to identify the fatty acid in the *sn*-2 position (Fig. 3 and Supplementary Fig. 3S). The reason is as follows: it is known that existing fragmentation methods cannot distinguish between *sn*-1 and *sn*-3, as fatty acids from these positions in TAG have identical fragmentation efficiencies. Therefore, TAG molecules lose simultaneously and preferentially fatty acyls at positions *sn*-1 and *sn*-3, regardless of the energy and instrumentation employed (Fig. 3). This implies the formation of two ions of similar abundance and more abundant than the ion corresponding to the loss of the fatty acid position at *sn*-2 [17].

As previously mentioned, three of the six ions studied showed a single triad of fatty acids in their structure (Fig. 2B): TAG 60:4 (m/z 989.8), TAG 62:4 (m/z 1017.9), and TAG 64:5 (m/z 1046.0). An example of the MS/MS spectrum interpretation of m/z 989.8 parent ion is given as follows (Fig. 3B): it corresponds to the sodium adduct of TAG 60:4 [C₆₃H₁₁₄NaO₆]. We can see two abundant ion products (651.5 and 679.6 m/z) corresponding to losses of fatty acyl substituents as fatty acids that correspond to *sn*-1 and *sn*-3. These correspond to the losses of C22:1 and C20:1, although we cannot distinguish between these positions. And there is another less abundant ion (709.6 m/z) that corresponds to the fatty acyl C18:2 at *sn*-2. In the MS/MS spectrum (Fig. 2B), there are also very-low-intensity ion products coming from the losses of the corresponding fatty acyl substituents as their sodium salts. Results are consistent with a TAG structure of 22:1/18:2/20:1 with linoleic acid at the *sn*-2 position. Likewise, in the case of 709.6 m/z ion, 18:2 was always identified at the *sn*-2 position of TAG in all maturation stages.

The same occurs in the cases of ions at 1017.9 m/z, TAG 62:4 [C₆₅H₁₁₈NaO₆]⁺, and at 1046.0 m/z, TAG 64:5 [C₆₇H₁₂₂NaO₆]⁺. Spectra and their interpretation are shown in Supplementary Fig. 3S. In the MS/MS spectrum of the 1017.9 m/z parent ion, there is only an abundant ion product, but with double intensity, coming from the loss of two C22:1 fatty acyls. Therefore, C22:1 is at *sn*-1 and *sn*-3 positions. This TAG is already present at the youngest state, in all stages, and becoming the most important in GY, YG, Y, and M. As in the previous case, linoleic acid is at the *sn*-2 position in all stages of seed maturation.

In the case of the ion at 1046.0 m/z, erucic acid and C24:1 are at *sn*-1/*sn*-3 and 18:2 at the *sn*-2 position at all stages of maturation. The presence of C24:1 in pennycreeves seeds was already detected by GC-FID and reported by our previous work [6].

The importance of these results concerning the *sn*-2 position of fatty acids for TAG biosynthesis is discussed in Section 4.

### 3.3 Comparison of TAG ion profiles by HPTLC-MS and LC-MS

In the case of pennycress seed extracts obtained from the five described stages of development, a complete lipidomic analysis of all glycerolipid classes and their corresponding subclasses were obtained by LC-ESI-MS/MS (QqQ) and reported elsewhere [18]. Although chromatography takes 70 min, MRM is able to identify and quantify quickly all of the corresponding precursor ions from the fragments of each lipid class. To have a complete set of quantitative data, calibration was performed at several levels to control the variability of the ionization efficiency due to: (i) the polar head group of the lipid; (ii) the dependence of the ESI on the retention time due to the variable composition of the solvent gradient used in the chromatography; and (iii) the intrinsic effect of the instrument. In this regard, a quality control sample (QC, *Arabidopsis* cell culture extract of known composition) was used as an additional external standard, according to a previous publication of Jouhet et al. [9]. Results corresponding to TAG were used in this work as a reference to compare and discuss the performances and potentialities of LC-MS and HPTLC-MS in seed lipidomic analysis.

The profiles of ion abundance (HPTLC-ESI(+)–MS) and LC-MS distribution corresponding to the six selected TAG subclasses are shown in Fig. 4. In a first stage of the research, the corresponding profiles (Fig. 4) were compared only for these selected subclasses since they had been structurally identified by both MRM in the case of LC-MS–QqQ and MS/MS in the case of HPTLC-MS–ion trap. Concordant results were observed with both techniques (Fig. 4). Results from both confirm the incorporation of 22:1 into the TAG already at the initial stage of seed maturation (G), as well as the increase of 22:1 and other VLCFAs species 20:1, 22:1, as maturation increases. Thus, at the early G and GY stages, TAG 58:5 was the major TAG subclass. Other important TAG subclasses at early stages of development were TAG 56:3, TAG 60:4, and TAG 62:4. Upon seed maturation, the distribution of TAG subclasses at the YG and Y stages showed a change with respect to the G and GY initial stages: an increase of TAG 60:4 and TAG 62:4, which correspond to subclasses containing 20:1/18:2/22:1 and 22:1/18:2/22:1, respectively. A high increase in TAG 62:4 can be observed with respect to the other species. Finally, at the mature stage (M), TAG subclasses such as 58:5, 60:4, and 62:4 were the most abundant ones. These results indicate that, upon maturing, there is an increase in TAG species containing VLCFAs such as 22:1 or 20:1. By contrast, the presence of FA24:1 in TAG 64:4 is consistent with the fatty acid composition of total lipids in mature seeds (GC-FID) and previous results [5].

Both LC-MS and HPTLC-MS profiles shown in Fig. 5 provided similar results. Likewise, results from both
<figure><img src="image_8.png" /><figcaption>**Fig. 4** Profiles of A concentration percentage (LC-ESI(+)-MS) and B ion abundance percentage (HPTLC-ESI(+)-MS) corresponding to the six selected TAG subclasses in function of pennycress seed maturation. Seed maturation stages (G, GY, YG, Y, and M) are indicated in colors</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>**Fig. 5** A Statistical correlation between normalized distribution percentage obtained from LC-MS (y axis) and normalized ion abundance percentage obtained from HPTLC-MS (x axis). Points for each seed maturation stage are indicated in the colors red (G), dark green (GY), violet (YG), blue (Y), and green (M). B Regression zone for a 95% confidence interval</figcaption></figure>

techniques not only are in agreement but also are statistically correlated. Figure 5A shows the scatter graph, obtained through the R cloud application Posit Cloud, showing the correlation between results from LC-MS (y axis) and

HPTLC-MS (x axis). Points for each seed maturation stage are represented by different colors. Represented results correspond to 30 values, i.e., the six selected structures per five stages of maturation. Each value is the average of three repetitions. Results have been normalized for both techniques with respect to the 23 most intense ions. Figure 5B shows the regression zones for a 95% confidence interval. Only one point is outside the zone. Spearman and Pearson correlation coefficients were 0.91 and 0.84, respectively. This implies a very high correlation between both sets of results (a very high correlation is considered when coefficients are between 0.70 and 1).

To see if this similarity in results held, we decided to extend the HPTLC-MS profiling to all TAG subclasses that had been determined by LC-MS. Thus, the intensities of the corresponding ions in HPTLC-ESI(+)-MS as sodium ions were compared with the concentrations obtained by MRM for the corresponding species in LC-MS as ammonia ions. Results shown in Fig. 6 show that both profiles are similar for all detected TAG subclasses and lead to similar analytical conclusions concerning TAG analysis.

# 4 Discussion

## 4.1 Validation of capacity of HPTLC-MS for lipidomic analysis

The acquisition of HPTLC-ESI(+)-MS spectra obtained at the same time of ionization for each of the pennycress seed maturation stages enables obtaining, in a quick and simple way, two important types of information: first, positional information, obtained through the MS/MS spectra of selected parent ions, which has enabled the elucidation of the positioning of linoleic acid (C18:2) at *sn*-2 in all stages

<figure><img src="image_10.png" /></figure>
**Fig. 6** A TAG subclasses during pennycress seed maturation by LC-ESI(+)-MS as ammonium ions (percentage of distribution). Seed maturation stages (G, GY, YG, Y, and M) are indicated in colors. Different letters indicate significant differences among seed maturation stage, at *P* < 0.05. Values presented are average of three determinations, and error bars represent standard deviation. B TAG subclasses during pennycress seed maturation by HPTLC-ESI(+)-MS as sodium ions (percentages of ion abundance)

<figure><img src="image_11.png" /></figure>

of seed maturation of several quantitatively important TAG subclasses in pennycress seed.

Second, the abundance HPTLC-MS profile obtained for the ions and the corresponding TAG subclasses provided similar results to those obtained from the LC-MS profile, to the point that the results of both techniques are statistically correlated.

It is not surprising that the results of both techniques were similar since both have used normal phase (diol in LC and silica in HPTLC), and, in both cases, TAGs are separated altogether and sent to the mass detectors (QqQ in LC and IT in HPTLC). The slight differences in the results are probably due to the fact that in LC-MS not all of the possible transitions were measured (Supplementary Table 3S). When using an MRM method, transitions must be previously entered, while HPTLC-MS, under the used conditions, is a nontargeted method. All of this shows that the ionization of TAG from a single chromatographic peak does not entail the ionic suppression of these species during ESI ionization, as it is sometimes incorrectly reasoned.

It should be noted that HPTLC is a simple technique that allows for the application of samples and standards at the same time, provides a separation into lipid classes in a short time, and allows a selected band, in this case TAG, to be easily sent

to the MS through the interface. The species grouped under the TAG class will all have very similar responses; therefore, no prior calibration is needed when interpreting the mass spectra.

The lipidomics addressed here show a remarkable complementarity of both techniques, which can be used depending on the level of detail and/or speed required depending on the analysis and the objective of the research. LC-MS (QqQ) provides a huge amount of information through the exhaustive analysis of glycerolipids classes and TAG subclasses, at the cost of working in gradient during the separation that implies long chromatographic times, complex calibrations, and no interruptions in the experiments.

HPTLC can provide rapid initial information to see whether the results of the lipid fractions of any kind are interesting enough to go further through LC-MS or not. For example, if you have a mutant collection, it would be interesting to have a rapid “screening tool” to focus later on those with more interesting data.

## 4.2 Positional analysis of TAG species through HPTLC-MS/MS

Positional analysis of acyl-groups esterified to *sn*-1, *sn*-2, and *sn*-3 positions in TAG is very useful to analyze the
influence of acyl-CoA pools and their incorporation into
lipid species by the different TAG biosynthetic pathways.
In general, it is assumed that acyl groups at the *sn*-1 and *sn*-3
positions of TAG are more related with the composition of
the acyl-CoA pool, while those at *sn*-2 are directly related
to the acyl preference of the *LPAT* enzyme [19]. In the past,
positional analysis was performed through enzymatic diges-
tions using specific lipases following separation and fatty
acid determinations. The HPTLC–ESI–MS approach used
in this work has demonstrated as a relatively simple and reli-
able technique to perform this analysis. Our data indicated
that in most of the major TAG species, 18:2 was detected at
the *sn*-2 position, indicating that pennycress *LPAT* showed
higher preference for 18:2-CoA substrates rather than for
other CoA moieties such as erucoyl-CoA, which should
be highly abundant in the pennycress seed. Similar results
were obtained in *Crambe abyssinica*, another high-erucic
containing species [20]. In fact, very few exceptions such
as *Limnanthes douglasii* with an *LPAT* enzyme with high
preference for 22:1-CoA at *sn*-2 in TAG have been reported
in literature [21]. The reasons of this specificity are not clear,
although some stringency has been proposed in the enzyme
niche [19]. This behavior contrasts with the relative flex-
ibility of *GPAT* or *DGAT* enzymes that seemed to transfer
efficiently different acyl groups (16:0; 18:1, or even 22:1) all
along the seed maturation process.

**5 Conclusions**

The results presented here validate the ability of HPTLC and its combination with LC-MS to easily, quickly, and reliably perform the analysis of TAG species in complex lipidomic samples, as well as to obtain positional information. This opens the door to the precise identification of the acyl groups at *sn*-2, as shown in this work, and also to go further towards the identification of acyl groups esterified in the rest of the positions of TAG, which might be extremely useful in the identification of the different pathways contributing to TAG biosynthesis in oilseeds.

**Supplementary Information** The online version contains supplementary material available at https://doi.org/10.1007/s00764-025-00346-y.

**Acknowledgements** This work was supported by the Spanish Ministry of Research and Innovation (MICINN) and FEDER (grant no. PID2021-1265630-B100) and Gobierno de Aragón (grant nos. A09-20R and E25-23R for Research Groups in Aragón and grant no. LMP194_21, Research in Strategic Lines). The Lipid analysis in Gre-noble (LIPANG) platform, hosted by the LPCV (UMR 5168 CNRS-CEA-INRAE-UGA), is supported by the Rhône-Alpes Region, the funds FEDER and GRAL, and financed within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003).

**Author contributions** José Manuel Escuín: HPTLC-MS methodology and statistical analysis; Ana Claver: extraction, plant materials, and pennycress cultivation; María Ángeles Luján: extraction, plant materials, and pennycress cultivation; Marion Schilling: LC-MS methodology, data analysis, and statistical analysis; Juliette Jouhet: supervision and review and editing; María Savirón: HPTLC-MS methodology; M. Victoria López: plant materials, pennycress cultivation, statistical analysis, and connection with transcriptomics; Carmen Jarne: HPTLC-MS methodology and data analysis; Miguel Alfonso: pennycress cultivation, conceptualization, supervision, and writing—review and editing; and Vicente L. Cebolla: conceptualization, supervision, writing—review and editing, and original draft.

**Data availability** Data will be available on request.

**Declarations**

**Conflict of interest** The authors declare no conflict of interest. One of the coauthors (V.L.C.) is a member of the editorial board of the journal. Therefore, the submission was handled by a different member of the editorial board, and V.L.C. did not take part in the review process in any capacity.

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