Workflow for the isolation of natural product secondary metabolites using off-line SPE guided by gradient reverse-phase liquid chromatography (LCSPE-fast)

The importance of natural products (NPs) as molecular libraries providing diverse three-dimensional structural features and serving as scaffolds for drug discovery is well recognized. However, the isolation of both targeted and untargeted secondary metabolites remains a major challenge in NP research, despite significant advances in analytical and biotechnological tools. To address this, we report a rapid and efficient method for the semipreparative isolation of secondary metabolites from NP extracts using offline C₁₈ solid-phase extraction (SPE) cartridges, guided by the retention profile of a reverse-phase liquid chromatography gradient (LCSPE-fast). LCSPE-fast proved to be fast, cost-effective, and yielded multi-milligram amounts of target-metabolite-enriched fractions (∼0.40 g of chlorogenic acid and 0.03–0.103 g of 3,4-Di-O-caffeoylquinic acid) from Uncaria guianensis cultivated both in vitro and in situ.

1 Introduction

It is widely acknowledged that natural products (NPs) research has been a major source of novel bioactive molecules with pharmaceutical applications. Researchers in this field remain committed to the continuous discovery and characterization of such compounds. Despite significant advances in analytical and biotechnological tools, the identification of new molecules continues to pose considerable challenges. Approaches such as metabolomics (Caesar et al., 2021; Salem et al., 2020; Floros et al., 2016) and global natural products (GNPS)-based molecular networking (Huo et al., 2023; Khushi et al., 2023; Quinn et al., 2017) have greatly facilitated the prioritization and dereplication of both known and unknown metabolites. Nevertheless, the isolation and unambiguous structural elucidation of individual molecules remain essential for downstream biological assays and for validating their potential bioactivity. One of the main challenges in NP research is that many compounds occur in trace amounts, requiring large quantities of raw material for isolation (Queiroz et al., 2024).

This raises the question: what is the average number of purification steps needed to obtain a “pure compound”? Pauli et al. (2012) conducted a meta-analysis of 2,000 publications on bioactive NPs from 13 journals published in 1998–1999, 2004–2005, and 2009–2010. They found that in approximately three-quarters of cases (76.3%), NPs were isolated and purified in fewer than three steps, indicating that isolation efforts generally target abundant or easily separable compounds. By contrast, low-abundance metabolites, often requiring six to ten purification steps, are rarely pursued. Similarly, an analysis of 4,859 articles published in the Journal of Natural Products between 1996 and 2009 revealed that 91.5% of the reported NPs were hydrophobic, while only 8.5% were hydrophilic. More recently, 862 articles published between 2017 and 2019 reported that high-performance liquid chromatography (LC) was the final isolation step in most cases, with C₁₈ columns being used in 67% of them (Berlinck et al., 2022).

In recent years, solid-phase extraction (SPE) has been increasingly applied for the purification and extraction of diverse analytes from biological matrices such as urine and blood (Gu et al., 2025). The process involves three fundamental steps: conditioning of the sorbent, sample loading, and elution of the target analytes (Bucar et al., 2013). A key advantage of SPE in biological sample analysis is its high recovery rate (80%–100%) with excellent reproducibility, often surpassing liquid–liquid extraction (LLE) methods (Badawy et al., 2022). In NP research, SPE is commonly used as a cleanup step prior to LC analysis (Beltrame et al., 2006; Ranušová et al., 2021) as well as to concentrate samples after LC separation for structure elucidation by nuclear magnetic resonance (LC-SPE-NMR) (Simpson et al., 2004; Thomasi et al., 2017).

Herein, we present a general protocol for the offline semipreparative isolation of secondary metabolites from NP extracts using a multi-milligram C₁₈ SPE cartridge. The method employs a reversed-phase elution gradient by LC to define the percentage of organic modifier required for fractionation. This protocol has been used for a while in our group, and to demonstrate the applicability of this protocol, we selected a crude extract of Uncaria guianensis (Aubl.) J.F.Gmel. (Rubiaceae) cultivated both in vitro and in situ. Our group has been investigating U. guianensis, commonly known as cat’s claw, an Amazonian endemic species valued for its anti-inflammatory and immunostimulatory properties (Honório et al., 2016). These biological activities are associated, at least in part, with quinic acid derivatives such as chlorogenic acid (1) and 3,4-Di-O-caffeoylquinic acid (2) (Figure 1), which were chosen as the target metabolites for isolation.

Figure 1

Figure 1. LC-UV chromatogram of the matrix under gradient elution, with the percentage of MeOH selected according to the retention times of target metabolites 1 and 2. Fraction I (35% MeOH), fraction II (42% MeOH) and fraction III (59% MeOH) were obtained after offline C18 SPE cartridge fractionation.

2 Methods

2.1 Plant material

The specimen was identified by Piero G. Delprete (Herbier de Guyane, Institut de Recherche pour le Développement, Cayenne, French Guiana), and voucher material was deposited in the Herbarium of the Universidade de Ribeirão Preto (UNAERP) under the accession code HPMU 2993 (CGEN/MMA: process number A8ABAE8). Four distinct extracts were obtained from different plant parts and produced through in situ and in vitro approaches: aerial parts (in situ), stem bark (in situ), aerial parts (in vitro plantlets), and adventitious roots of U. guianensis.

2.2 Chemicals

Chlorogenic acid, 3,4-Di-O-caffeoylquinic acid, naphthaleneacetic acid (NAA), benzylaminopurine (BAP), 2-aminoethyl diphenylborinate (NP), sucrose, Murashige & Skoog (MS) basal medium and Woody Plant (WP) basal medium were purchased from Sigma-Aldrich® (St. Louis, MO, United States). Polyethylene glycol (PEG) was purchased from Êxodo® (PG07857RA). LC grade solvents were supplied by J. T. Baker.

2.3 Establishment of U. guianensis plantlets and adventitious roots

Plantlets were developed by Lopes et al., 2019, as follows. The process of micropropagation involved the use of a WP basal medium (McCown and Lloyd, 1981) that had been supplemented with sucrose (30 g.L⁻¹), benzylaminopurine (1 mg.L⁻¹), naphthaleneacetic acid (0.5 mg.L), and Gellan Gum (2.5 g.L⁻¹). The pH of the medium was then adjusted to 6.0. The explants were maintained in this medium for a period of 3 months (Lopes et al., 2019). U. guianensis root cultures derived from micropropagated plantlets were cultivated in Erlenmeyer flasks (250 mL) containing 100 mL of MS basal medium culture (Murashige and Skoog, 1962), with sucrose at a concentration of 3% (w/v), 1 mg.L⁻¹ of 1-naphthaleneacetic acid (NAA) and the pH adjusted to 6.0. These cultures were subjected to orbital agitation at a rate of 110 rpm and sub-cultivated over a period of 3 weeks. Plantlets and adventitious roots were maintained in the growth room, where the temperature was maintained at 25 °C ± 2 °C, the relative humidity was 55%–60%, and the photoperiod was 16 h of photoperiod with an intensity of 40 μmol m⁻²s⁻¹, provided by 85 W cool-white GE fluorescent lamps.

2.4 Preparation of crude extracts from U. guianensis

Aerial parts (in situ), stem bark (in situ), aerial parts (in vitro plantlets) and adventitious roots of U. guianensis were dried at 45 °C in a circulating air oven for 24 h in order to determine the dry weight. Crude extracts were prepared by macerating the dried powdered plant (1 g) and extracting it using methanol (10 mL) for two separate 24 h periods (two times).

2.5 HPTLC chromatography conditions

All samples, along with chlorogenic acid (1) and 3,4-Di-O-caffeoylquinic acid (2) standards, were applied to a chromatographic plate (MACHEREY-NAGEL 306179) using a Linomat 5 applicator (Camag, 022.7808) within an HPTLC (High Performance Thin Layer Chromatography) system. The mobile phase consisted of ethyl acetate, water, and anhydrous formic acid (10:1:1, v/v). Chromatographic plate visualization was carried out with natural products-polyethylene glycol reagent (NP/PEG).

2.6 LC chromatography conditions

All samples were solubilized in methanol and filtered through Millipore® filters (0.45 µm membrane). Crude extract sample analyses were performed using a Shimadzu LC system, consisting of LC-10AD pumps, DGU-20A3 degasser, SCL-10 system controller, SIL-10AD automatic injector, and an SPD-M10A UV/VIS DAD detector, operated by Class VP software. A Phenomenex Kinetex XB-C₁₈ column (250 mm × 4.6 mm, 5 µm) coupled to the respective pre-column was used. The mobile phase composition included water acidified with 0.1% acetic acid (A) and methanol (B). The separation was performed by gradient elution at a flow rate of 1.0 mL.min^–1 with a ΔB of 10%–100% from 2 to 32 min. To ensure column cleanup, 100% MeOH was maintained for an additional 8 min, then returned to 10% MeOH, and the column was equilibrated for 5 min before the next injection. The injection volume used was 20 μL (8 mg.mL^–1), with detection occurring at 330 nm.

Fractionation of the crude extracts using C₁₈ solid-phase cartridge (SPE)

The methanolic extracts were subjected to concentration and fractionation in SPE cartridges. Adventitious roots, aerial parts (in vitro plantlets), aerial parts (in situ), and stem bark (in situ) of U. guianensis were submitted to fractionation using a Supelclean™ LC-18 SPE tube (Supelco, 10 g/800 mg). C₁₈ SPE cartridges were conditioned in 100% MeOH (40 mL), under gentle vacuum, and column conditioning prepared the sorbent for effective interaction(s) with the compounds of interest. Subsequently, C₁₈ SPE cartridges were conditioned in 100% of deionized H₂O (40 mL), the appropriate solvent used for sample preparation. The solvent percentage of each fraction was calculated based on the retention time of each selected metabolites (1 and 2) and separating the sample into three fractions: fraction I (44% of MeOH; 13.34 min), fraction II (53% of MeOH; 16.27 min), and fraction III (59% of MeOH; 18.05 min). For fraction I and fraction II, 80% of the calculated MeOH percentage was applied. For fraction III, the exact solvent composition determined by LC was used. Subsequently, the sample (800 mg in 10 mL of deionized H₂O) was applied, and the cartridge was washed with 35%, 42%, 59% and 100% MeOH. Additionally, ethyl acetate solvent (40 mL) was used in the final wash cartridge. The crude extract was fractionated in a 10 g C₁₈ SPE cartridge, and the void volume (Vm) of 19.38 mL was calculated according to Equation 1 (Dong, 2019). In each solvent system, a total solvent volume equivalent to twice the Vm (40 mL) was selected, rather than three Vm, as indicated by the manufacturer. The same chromatographic workflow called LCSPE-fast was applied to the remaining crude extracts, including aerial parts from in vitro plantlets, aerial parts (in situ) and stem bark (in situ). After fractionation with the C₁₈ SPE cartridge, all samples were evaporated to dryness, reconstituted in 100% of MeOH, and subjected to LC-UV analysis.

$\text{Vm}=0,65{\mathrm{r}}^2\mathrm{L}(1)$

3 Results and discussion

To develop the semipreparative isolation LCSPE-fast protocol, the crude extract of U. guianensis adventitious roots was first analyzed under linear gradient elution with a gradient slope (Gs) of 3%. The resulting chromatogram is shown in Figure 1. Based on this analysis, the organic modifier percentages required for the elution of the target metabolites 1 and 2 were calculated as 44% and 59%, respectively, with retention times of 13.34 and 18.05 min. These retention times values were confirmed by comparison with authentic standards analyzed under the same chromatographic conditions (Supplementary Figure S1). Additionally, an unknown peak at 16.27 min, corresponding to 53% methanol, was also selected for isolation.

For scaling up to the SPE cartridges, 70%–85% of the calculated methanol percentage were evaluated from which 80% was applied to isolate metabolites 1 and the unknown peaks, corresponding to 35% and 42% methanol, respectively. For metabolites 2, the full calculated percentage (59%) was used as the other fractions had already been isolated. Figure 2 summarizes each step of the generic workflow employed.

Figure 2

Figure 2. LCSPE-fast workflows for isolating natural products targets from a complex matrix using LC analysis in combination with offline C₁₈ SPE cartridge.

Fractions I to III, eluted with 35%, 42%, and 59% methanol respectively, were collected and analyzed by LC-UV. The resulting chromatograms confirmed that metabolites 1 and 2 were isolated as the major components of fractions I and III, respectively (Figure 3). The solvent volume required for washing and elution was directly proportional to the sorbent mass in the SPE tube and was calculated using Equation 1. It is worth noting that twice the Vm (40 mL) was sufficient for the complete elution of each fraction. To evaluate the general applicability of the protocol, a crude extract of U. guianensis aerial parts (in vitro) was analyzed under the same gradient conditions. As expected, metabolites 1 and 2 were successfully isolated from the 35% and 59% methanol fractions, respectively (Figure 3). Importantly, the 100% methanol fraction, corresponding to the cartridge washing step, did not contain either of the target metabolites. It is important to mention that metabolites such as chlorophyll are absent from cell and root cultures, facilitating the isolation of target compounds (Karalija et al., 2025; Ochatt et al., 2022).

Figure 3

Figure 3. LC-UV chromatograms of the crude extracts obtained from of in vitro U. guianensis (A) and the SPE isolated fractions I-III (B) under gradient conditions. Fraction I-III were isolated with 35, 42% and 59% of MeOH respectively. The target metabolites, chlorogenic acid (1) and 3,4-Di-O-caffeoylquinic acid (2) are highlighted in the crude extracts and in their respective enriched fractions.

The same workflow was subsequently applied to in situ samples (aerial parts and stem bark). The chromatographic profiles of these samples showed marked differences from the in vitro extracts (Figure 4). For in situ aerial parts samples, chlorogenic acid (1) was isolated from fractions I and II (35% and 43% methanol respectively), while metabolite 2 was isolated from fraction III. HPTLC analysis further evidenced that chlorogenic acid (1) was the predominant metabolite in the in situ aerial parts extract (Supplementary Figure S2). By contrast, metabolite 2 was absent from the in situ stem bark samples, in which chlorogenic acid (1) was obtained also from fraction I.

Figure 4

Figure 4. LC-UV chromatograms of the crude extracts obtained from of in situ U. guianensis (A) and the SPE isolated fractions I-III (B) under gradient conditions. Fraction I-III were isolated with 35%, 42%, and 59% of MeOH, respectively. The target metabolites, chlorogenic acid (1) and 3,4-Di-O-caffeoylquinic acid (2) are highlighted in the crude extract and in their respective enriched fractions.

The isolated yields of the enriched fractions containing target metabolite 1 ranged from 46% to 53%, whereas the fractions containing metabolite 2 ranged only from 4.4% to 12.5% (Table 1). It is important to say that fraction III isolated from in situ stem bark did not contain metabolite 2 (Figure 4)

Table 1

Table 1. Mass and isolated yields of fractions I-III.

Despite the persistent challenges associated with isolating pure NPs from complex mixtures, the protocol described herein demonstrates that the combination of LC with an exploratory gradient and offline C₁₈ SPE fractionation can accelerate the isolation of secondary metabolites. Given that cartridge selectivity is generally lower than that of an LC column, the described workflow is suitable for chromatographic bands that show adequate selectivity, irrespective of the number of peaks in the chromatogram.Target-enriched fractions can subsequently be subjected to isocratic semipreparative runs to obtain compounds of high purity. The herein reported results highlight the efficiency of the protocol for the isolation of target metabolites across all evaluated extracts. It is important to note that 3,4-Di-O-caffeoylquinic acid (2) is a high-value quinic acid derivative. Finally, the established LCSPE-fast protocol provides a rational and broadly applicable approach for the semipreparative isolation of metabolites from crude NP extracts.

4 Conclusion

The LCSPE-fast protocol described herein proved to be fast, efficient, and broadly applicable, enabling the fractionation of crude extracts and the yield of enriched fractions containing the target metabolites. By combining exploratory LC analysis with offline C₁₈ SPE cartridges, this approach offers an environmentally friendly alternative for NPs research, characterized by reduced solvent consumption, and operational simplicity. Importantly, the method was effective across all four Uncaria guianensis extracts evaluated, both in vitro and in situ, despite differences in chemical complexity. To the best of our knowledge, this is the first report demonstrating the use of LC-guided offline SPE fractionation for the isolation of secondary metabolites. The rational workflow established here provides a valuable tool for NPs research and can be readily extended to diverse complex extracts from plants, microorganisms, and marine organisms, thereby accelerating the isolation and characterization of bioactive compounds.

Data availability statement

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

Author contributions

LP: Formal Analysis, Writing – review and editing, Investigation, Methodology. MB: Investigation, Methodology, Formal Analysis, Writing – review and editing. AP: Writing – review and editing, Conceptualization, Methodology, Formal Analysis, Investigation. QC: Formal Analysis, Writing – review and editing, Methodology, Data curation, Investigation, Conceptualization. AL: Writing – review and editing, Methodology, Investigation, Writing – original draft, Conceptualization, Formal Analysis, Data curation.

Funding

The author(s) declared that financial support was received for this work and/or its publication. This research was funded by the Biotechnology Unit, University of Ribeirão Preto (UNAERP). We are grateful to the CNPq (National Council for Scientific and Technological Development) grant (n. 302464/2022-0) awarded to QBC.

Acknowledgements

We gratefully acknowledge Luciano Rodrigues AP for inspiring the development of the protocol.

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

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The author(s) declared that generative AI was not used in the creation of this manuscript.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fntpr.2025.1726849/full#supplementary-material

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