Indoleamines, a class of specialized metabolites derived from tryptophan, include melatonin (N-acetyl-5-methoxytryptamine, MLT) as a prominent representative, which was first isolated from the pineal gland [1]. In plants, MLT plays crucial roles in stress protection, growth, development, and reproduction. Beyond its phytophysiological functions, MLT exhibits a broad spectrum of biological activities in organisms, such as circadian rhythm regulation, anti-inflammatory effects, scavenging of reactive oxygen species, antioxidant activity, and endocrine modulation [[2], [3], [4]]. Clinically, it can assist in the treatment of various diseases, including allergic reactions, insomnia, cardiovascular diseases, and metabolic diseases. However, most commercially available MLT supplements are marketed as health products with unstandardized MLT content. Although MLT exhibits low toxicity, prolonged excessive intake may lead to adverse effects such as headaches, drowsiness, and depression [5,6]. Fortunately, numerous studies have identified MLT in natural sources like vegetables, fruits, and medicinal herbs, offering a safer alternative to reduce the drug-induced side effects [6]. Consequently, inexpensive and readily accessible juices could serve as excellent dietary sources of MLT. Therefore, accurate quantification of MLT in such food samples is essential for identifying high-yield sources and developing adjuvant therapeutic strategies. However, direct detection of trace MLT in complex food matrices remains challenging due to the serious matrix interference and its extremely low abundance. Thus, establishing an efficient, sensitive, and selective sample pretreatment method is imperative.

At present, many methods have been developed for the separation, purification, and preconcentration of MLT from complex matrices, including solid-phase extraction (SPE) [7], solid-phase microextraction [8], dispersed SPE [9], liquid-liquid extraction (LLE) [10], and dispersed liquid-liquid microextraction (DLLME) [11]. Among these, SPE, which integrates the principles of liquid-solid extraction and liquid chromatography, has become a widely adopted sample pretreatment technique due to its operational simplicity, cost-effectiveness, and high extraction efficiency [5]. The design and preparation of the adsorbent are critical to SPE performance. To date, various adsorbents have been employed for MLT extraction, such as C8 [7] and C18 cartridges [12], GO@SiO2 [13], MLT-PEMATrp [14], and Fe3O4@SiO2−MIP [15]. However, most conventional adsorbents (particularly commercial C8 and C18 adsorbents) primarily rely on hydrophobic effects mediated by the indoleamine benzene ring, offering limited binding sites and insufficient selectivity. To address these limitations, developing novel adsorbents with enhanced adsorption capacity and selectivity is imperative for the efficient extraction of MLT from complex samples.

Microporous organic networks (MONs), which are typically synthesized via Sonogashira-Hagihara coupling of aromatic alkynes and halides [16], represent an emerging class of porous materials. They are characterized by extended π-conjugated frameworks, high surface areas, tunable porosity, and remarkable chemical/thermal stability. These distinctive properties have enabled their widespread application in separation science [[17], [18], [19]], particularly in SPE of diverse compounds [20]. Initially, pristine MON was primarily employed to extract non-polar to weakly polar analytes, relying on hydrophobic and π-π interactions [21]. Subsequently, to broaden their applicability and enhance selectivity, various functionalization strategies have been developed to incorporate diverse functional moieties into MON frameworks [20]. Given the molecular structure of MLT, which contains aromatic indole ring and amide group, we hypothesize that carboxyl-functionalized MONs with maintained hydrophobicity and π-conjugation would be particularly effective for MLT enrichment. This design enables synergistic combination of multiple interaction mechanisms: (1) π-π stacking with the indole ring, (2) hydrogen bonding through the amide and carboxyl groups, and (3) electrostatic attraction, thereby significantly improving the extraction efficiency for MLT.

Beyond chemical functionalization, morphological engineering of MONs into hollow architectures (H-MONs) has emerged as an effective strategy to enhance their SPE performances. The unique hollow morphology can reduce the mass transfer resistance, shorten the diffusion pathways, and significantly improve the extraction kinetics [22,23]. For examples, H-MON [24], H-MON-coated fibers [25], and polyethyleneimine-modified H-MON [26] have demonstrated exceptional extraction capabilities for aflatoxins, short-chain chlorinated hydrocarbons, and phenolic acids in complex matrices, highlighting the great potential of H-MONs in sample pretreatment. Building on these advances, we hypothesize that carboxyl-functionalized H-MON would synergistically combine the benefits of hollow morphology (enhanced mass transfer) and tailored surface chemistry (improved MLT affinity) for efficient MLT extraction from food samples. However, to the best of our knowledge, such integration has not yet been explored so far.

Herein, we report the fabrication of a novel carboxyl group-enriched H-MON (H-MON-2COOH) for the SPE of MLT and two structurally related indoleamine hormones (indole-3-acetamide, IAA; methyl n-acetyl-l-tryptophanate, l-Trp-OMe) from complex fruit juice samples, followed by high-performance liquid chromatographic (HPLC) determination. The MON-2COOH was in-situ grew on SiO2 microsphere and the internal SiO2 was then etched by NaOH to obtain the final H-MON-2COOH (Fig. 1). The resulting H-MON-2COOH exhibits a large surface area, excellent stability, and abundant π-π, electrostatic, and hydrogen bonding interaction sites for targets. The extraction conditions affecting SPE of MLT, IAA, and l-Trp-OMe were optimized in detail, including adsorbent dosage, elution solvent, elution solvent volume, eluent flow rate, sample flow rate, pH, and ionic strength. The established H-MON-2COOH-SPE-HPLC-UV method was thoroughly validated, and the extraction mechanism between H-MON-2COOH and MLT was elucidated. This study reveals that carboxyl-functionalized H-MONs with tailored interaction sites can serve as highly efficient SPE adsorbents for trace indoleamines in complex food samples, opening new avenues for the application of H-MONs in food safety and environmental pollutants monitoring.