Sipunculus nudus is a species of unsegmented marine worms, also known as peanut worms. The body of the adult worm is around 15 centimetres (5.9 in) in length but can reach up to 25 cm (9.8 in) in some cases.
The worm is commonly found on subtidal zones of sandy shores to seabeds 900 metres (3,000 ft) in depth in temperate or tropical waters. The worm hides in sand burrows which it makes by itself during the day and may extend its tentacles out of the burrow to feed at night. Its diet consists of plant or animal tissue fragments and any surrounding sand it may ingest with it.
Recent research indicates that it is a complex of similar species around the world rather than one species, with at least "five distinct lineages identified by phylogenetic analyses".
The species is collected and sold as a model organism for various fields of science, as fish bait, or for human consumption.
In particular, S. nudus is collected, cleaned of its innards, and eaten as a delicacy in some areas such as Vietnam where it is sometimes used to cook pho stock. S. nudus is also considered nourishment food for the royal family. The Vietnamese gather S. nudus in islands in Quang Ngai. It is also sold and exported as a dried seafood product.
The worms are also consumed in the southern Chinese provinces of Guangdong, Hainan, Guangxi, and Fujian. The worms are local delicacies in Beihai, Guangxi, where Běihǎi shāchóng (北海沙虫, lit. "Beihai sandworm") is cooked by various methods. In Xiamen, Fujian, the species is called tǔsǔn (土笋, lit. "earth bamboo shoot") and served as a gelatin(t土笋凍,s土笋冻,tǔsǔndòng) in local restaurants.
Sipunculus nudus, a Sipunculid species, known as sandworm or haichangzi, has a practically global distribution, except for polar waters. They are unsegmented wormlike animals, comprising two sections namely the trunk (main body) and an introvert (extending and contracting neck-like “feeler”) . Called “marine Cordyceps sinensis” by local residents, it has crisp, tender, fresh and sweet taste, can nourish internal organs and clear the internal heat , and is ranked as a valuable seafood and senior supplement [3,4]. However, since the 1980s, S. nudus has suffered from predatory and unhindered exploration stimulated by the market price, combined with more and more serious pollution of oceans which have led to a sharp decrease in the availability of the wild resource and therefore, more and more attention has been paid to artificial cultivation of the worms . As the technology has developed, the amount of S. nudus has increased rapidly and how to utilize the resource reasonably has become a major study topic.
In the last decades, S. nudus extract was reported to be rich in a variety of nutritional and functional components consisting of free amino acids, fatty acids, polysaccharides, mineral elements and so on. [6,7,8,9]. As we all know, the free amino acids associated with many functional foods such as Ziziphus jujube, royal jelly and Calculus bovis have received considerable attention [10,11,12]. In recent years, nucleosides and nucleobases have also been proven as important nutritional and functional foodstuffs related to multiple properties such as modulation of the immune response, metabolism, angiocarpy and nervous system as well as antimicrobial and antiviral effects . However, there has been no report about the nucleosides and nucleobases in S. nudus so far. Therefore, in order to compile comprehensive information about the nutritional and functional components in S. nudus, we performed a preliminary experiment to detect the nucleosides and nucleobases in S. nudus and found such constituents were abundant in S. nudus water extract.
In the past years, many researches have been carried out on free amino acids, nucleosides and nucleobases as quality control markers of several functional foods such as Geosaurus, brown seaweeds, royal jelly, Ganoderma lucidum and so on [7,14,15,16,17]. However, there are no definite quality control markers for S. nudus, although the free amino acids of S. nudus have been detected with low sensitivity by visible spectrophotometry after derivatization and complex pretreatment procedures . There have no reports about the contents and proportion of free amino acids, nucleosides and nucleobases in S. nudus till now, making it very necessary to develop a fast, convenient and efficient method to precisely measure the amount of these nutritional constituents in S. nudus extract, which will be beneficial for expanding its potential value as well as quality control.
In the traditional way, when S. nudus is consumed in dishes , the internal parts including the intestine and coelomic fluid are usually removed, and then it is cooked alone or with other food materials. The useful constituents in S. nudus may be broken down because of high temperature suing during the processing. Therefore, it is necessary to determine the content variation of free amino acids, nucleosides and nucleobases in different parts of S. nudus with different methods of sample preparation for the sake of best developing S. nudus as a functional seafood.
Ultra-high performance liquid chromatography (UPLC), coupled with mass spectrometry (MS) detection is an important analytical method that has been developed in recent years [19,20,21]. Due to its efficient separation, high selectivity and high sensitivity, it has been widely used for the quantification and qualitative analysis of active components in biological fluids, medicinal materials and so on [22,23,24,25,26].
In our previous research, the amino acids, nucleosides and nucleobases in another seafood were analyzed by using hydrophilic interaction ultra-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry . Since the method is simple and accurate, in this study, we also used such a method for simultaneous identification and quantification of 25 free amino acids and 16 nucleosides and nucleobases in different parts of S. nudus collected from four habitats with different preparation methods. Then, the data were further handled by a PCA scatter plot to compare the content variation of the samples. The determination of these important components in S. nudus could be vital to quality control as well as tapping its full nutritional and functional value.
2. Results and Discussion
2.1. Sample Preparation Optimization
To identify as many target components as possible in different parts of S. nudus, the extraction solvent (water, aqueous methanol of different concentrations), solvent volume (10, 20, 30, 40, 50, and 60 mL), extraction temperature (20, 40, 60, 80, 100 °C), extraction method (refluxing and ultrasonication) and extraction time (10, 20, 30, 40, 50 and 60 min) conditions of the samples from different parts (1.0 g, SE, SI, SC) were optimized. All of parameters were investigated by a univariate method using peak area as a measurement. It was found that the best extraction conditions were ultrasonication at 40 °C for 60 min with 40 mL water as solvent. However, to evaluate the free amino acids, nucleosides and nucleobases of S. nudus extracted in the traditional way, the extraction method of refluxing for 60 min was applied to imitate water decoction.
2.2. UPLC-TQ-MS/MS Conditions Optimization
In preliminary tests two columns, an Acquity BEH C18 (100 mm × 2.1 mm, 1.7 μm) and an Acquity BEH Amide (100 mm × 2.1 mm, 1.7 μm), were compared to obtain chromatograms with better resolution of adjacent peaks, improved peak shape and shortest peak appearance time. On account of the fact that free amino acids, nucleosides and nucleobases are hydrophilic components with high polarity, the results showed that the latter one had a stronger retention ability as well as better resolution under the same mobile phase and other instrument condition circumstances.
As for the mobile phase, as a general rule acetonitrile is known as a polar aprotic solvent with better elution ability, separation selectivity and peak shape compared to methanol, and has been proven to be suitable organic solvent for hydrophilic interaction liquid chromatography with short analysis times. Therefore, a high concentration of acetonitrile was used as organic phase and the concentration was decreased in a gradient. The ammonium acetate and ammonium formate dissolved in acetonitrile are highly volatile, and they can improve the separation of amino acids, nucleosides and nucleobases in the UPLC analysis process . Different mobile phases including independent solutions with different concentration and mixed solutions with different concentration of components were compared. The results showed that a mixed solution containing ammonium formate and ammonium acetate as mobile phase salt additives could increase the sensitivity and improve the peak shapes for these components. The retention times and peak shapes of the compounds were influenced by the different concentrations of ammonium formate and ammonium acetate, consequently, 5 mmol/L ammonium formate and ammonium acetate in the organic phase and 1 mmol/L ammonium formate and ammonium acetate in the aqueous phase produced the best shaped peaks in the shortest time. Meanwhile, formic acid is also used to inhibit solute ionization to improve the peak shape so different concentrations of formic acid were added and compared. Eventually, it was determined that the mobile phase should be composed of A (5 mmol/L ammonium formate, 5 mmol/L ammonium acetate and 0.2% formic acid in aqueous solution) and B (1 mmol/L ammonium formate, 1 mmol/L ammonium acetate and 0.2% formic acid in acetonitrile) with gradient elution. As regard to flow rate and column temperature, the ranges were both optimized, and the results show that the best mobile phase flow rate was 0.4 mL/min and the column temperature was maintained at 35 °C. Analytical chromatograms of the mixed standards and Sample F1 are presented in Figure 1.
For the best MS/MS condition of each analyte, all of the compounds were examined separately in direct infusion mode by a full-scan MS method in both positive and negative ionization modes. The results show that both higher sensitivity and clearer mass spectra were obtained in the positive ion mode compared to the negative ion mode. The free amino acids, nucleosides and nucleobases could combine with H+ to give [M + H]+ quasi-molecular ions in ESI+ mode. MRM mode was applied in the experiment, and the influence of nucleosides could be minimized because the peak could appear only when the parent and daughter ions were both detected by choosing the appropriate parent and daughter ions. To obtain the best ion pairs, at least two precursor/product ion pairs were chosen for each analyte for quantitative research and the most sensitive and specific ion pairs were selected for the MRM determination. For the nucleosides, [M + H]+ were selected as parent ions and [M + H − deoxyribose]+ and [M + H − ribose]+ was selected as daughter ions . As for nucleobases, we have tried to split these compounds before, however, the abundance of product ions for these compounds is too low to be detected by MRM. Consequently, we chose [M + H]+ as both parent and daughter ions for these compounds. For most α-amino acids, [M + H − HCOOH]+ were rearranged to [R − CH = NH2]+ as daughter ions. For γ-aminobutyric acid, [M + H − NH3]+ was rearranged to [R − CH − COOH]+ as daughter ion.
For glutamine and asparagine, [M + H − HCOOH − NH3]+ were selected as daughter ions. For alkaline amino acids such as arginine, lysine, citrulline and so on, their daughter ions could be affected by the presence of the amino groups of every amino acid (Table 1). Then we optimized cone voltage and collision energy by the function of Intellistart software in the Waters XevoTM TQ MS system.
2.3. Method Validation
The established chromatographic method was validated by determining the linearity, LOD, LOQ, intra- and inter-day precisions, repeatability, stability, and recovery. The correlation coefficient values (r2) showed all calibration curves exhibited good linear regressions (r2 > 0.9919) within the determination range of the 41 investigated compounds. The LODs (S/N = 3) and LOQs (S/N = 10) of the 41 compounds ranged from 0.003–0.229 μg/mL and 0.008–0.763 μg/mL, respectively. The intraday precisions were investigated by determining analytes in six replicates at known concentration during a single day while the interday precisions were determined during three successive day. And the RSDs serve as the measure of precisions. The results showed that RSD values for the intraday precisions were <3.72%, and for the interday precisions were <3.42%. The repeatability was evaluated by analyzing six samples processed by the same methods, and the RSDs of the repeatability were <4.48%. The storage stability of the sample was measured by analyzing the same sample at 0, 2, 4, 8, 12, 24 h within one day, and the RSDs of the storage stability were <4.92%. The recovery was performed by adding known amount of individual standards into an accurately weighed sample, and the mixture was processed and analyzed by the same methods of the samples. The recoveries were in the range of 94.03% and 106.33% for the 41 compounds and the RSDs were <3.76%. Besides, no significant matrix effects were noted in relatively complex functional food matrices within 24 h. All of above indicated that the established method was accurate enough for the determination of the 41 amino acids, nucleosides and nucleobases in S. nudus (Table 2). We have compared the determination results with and without internal standards in our preliminary experiments. The results showed that the optimized conditions of sample preparation, chromatogram and mass spectrum were stable and the contents of the components tested in the samples were quite identical, besides there was little difference between the errors of the methodology both with and without internal standards, so consequently, we decided not to use internal standards by reference to relevant methods in the field [29,30].
2.4. Sample Analysis
The method was applied to analyze 25 free amino acids and 16 nucleosides and nucleobases in different parts of S. nudus