Simultaneous Quantitation of Free Amino Acids, Nucleosides and Nucleobases in Sipunculus nudus by Ultra-High Performance Liquid Chromatography with Triple Quadrupole Mass Spectrometry

To evaluate the nutritional and functional value of Sipunculus nudus, a rapid, simple and sensitive analytical method was developed using ultra-high performance liquid chromatography coupled with a triple quadrupole mass detection in multiple-reaction monitoring mode for the simultaneous quantitative determination of 25 free amino acids and 16 nucleosides and nucleobases in S. nudus within 20 min, which was confirmed to be reproducible and accurate. The limits of detection (LODs) and quantification (LOQs) were between 0.003–0.229 μg/mL and 0.008–0.763 μg/mL for the 41 analytes, respectively. The established method was applied to analyze 19 batches of S. nudus samples from four habitats with two different processing methods. The results showed that S. nudus contained a variety of free amino acids, nucleosides and nucleobases in sufficient quantity and reasonable proportion. They also demonstrated that the contents of these compounds in different parts of S. nudus were significantly discriminating, which were in the order: (highest) coelomic fluid > body wall > intestine (lowest). The method is simple and accurate, and could serve as a technical support for establishing quality control of S. nudus and other functional seafoods. Moreover, the research results also laid foundation for further exploitation and development of S. nudus.


Introduction
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") [1]. Called "marine Cordyceps sinensis" by local residents, it has crisp, tender, fresh and sweet taste, can nourish internal organs and clear the internal heat [2], 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 Molecules 2016, 21, 408; doi:10.3390/molecules21040408 www.mdpi.com/journal/molecules therefore, more and more attention has been paid to artificial cultivation of the worms [5]. 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 [13]. 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 [18].
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 [2], 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 [27]. 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.

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.

UPLC-TQ-MS/MS Conditions Optimization
In preliminary tests two columns, an Acquity BEH C18 (100 mmˆ2.1 mm, 1.7 µm) and an Acquity BEH Amide (100 mmˆ2.1 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 [28]. 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 glutamine and asparagine, [M + H´HCOOH´NH 3 ] + 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. The peaks of guanine (15) and xanthine (16) were overlapped.

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 (r 2 ) showed all calibration curves exhibited good linear regressions (r 2 > 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].

Sample Analysis
The method was applied to analyze 25 free amino acids and 16 nucleosides and nucleobases in different parts of S. nudus collected from four different habitats in China with different sample preparation methods. The results demonstrated that all the samples were rich in these 41 compounds, and the total contents of these investigated compounds varied from 111.70 mg/g to 268.55 mg/g. From the data, it was found that total contents of free amino acids, nucleosides and nucleobases in S. nudus extract processed by both ultrasonication and refluxing were pretty much the same or just a little decreased during by the refluxing procedure, the details of which are shown in Table 3. However, the total contents of three parts in S. nudus were different, and in the order: (highest) coelomic fluid > body wall > intestine (lowest). As for the habitats, the results showed that S. nudus collected from Hong Kong was low in nutritional value compared to the other habitats, while the qualities of the other three habitats are not too different from each other. From the perspective of shape and appearance, it was also proved that the worms inhabiting the waters surrounding Hong Kong are thinner, shorter, have a darker color and contain more silt (Figure 2).
For specific compounds determined in the experiments, remarkable differences were also observed. The contents of free amino acids except GABA were of milligram magnitude, while the contents of nucleosides or nucleobases were mostly of microgram magnitude. The contents of nucleosides or nucleobases were in the following order: nucleobases > deoxynucleosides > ribonucleosides. The concentrations of 2 1 -deoxyguanosine and its corresponding base were greater than any other nucleosides or nucleobases in all the parts.
2 1 -Deoxyguanosine is recognized as an oxidative damage biomarker of DNA [31]. The high contents of 2 1 -deoxyguanosine in the worms may be a result of physical and chemical factors such as foreign chemicals or ionizing radiation besides body metabolism. Additionally, the content of xanthine, reported to be bronchodilator, is of milligram magnitude and second to 2 1 -deoxyguanosine, which is extremely interesting and worthy of further study. As for free amino acids, S. nudus contains all 20 proteinogenic amino acids and five non-protein amino acids. The percentage of eight essential amino acids in the 20 proteinogenic amino acids varied from 23.91% to 33.11%, and the percentage in body wall and intestine was higher than in coelomic fluid. The total contents of five non-protein amino acids in the samples, including GABA, citrulline, hydroxyproline, ornithine and taurine, varied from 7.77 mg/g to 22.90 mg/g, among which the taurine had the highest percentage which varied from 84.60% to 94.60%. Taurine has been reported to improve aerobic endurance, mental performance, concentration and memory [32], and we suggest that it may play an active role in the reported learning and memory enhancing pharmacological action as well as the anti-fatigue and anti-anoxia action of S. nudus [33,34]. Notably, the contents of fresh flavor amino acids including glycine, alanine, glutamate, aspartate and so on were fairly high, especially the contents of glycine which were the highest of all the free amino acids in S. nudus according to the assays, and all of these fresh flavor amino acids above fully explain its reputed good taste (Table 3).  The data was presented as average of three batches of samples. a Analyte number; b Below the limit of quantitation; c Total content of nucleosides and nucleobases; d Total content of amino acids.  (3), body wall, refluxing (4) and intestine, refluxing (5). The color A stands for total contents of amino acids, and the color N stands for total contents of nucleosides and nucleobases.
2′-Deoxyguanosine is recognized as an oxidative damage biomarker of DNA [31]. The high contents of 2′-deoxyguanosine in the worms may be a result of physical and chemical factors such as foreign chemicals or ionizing radiation besides body metabolism. Additionally, the content of xanthine, reported to be bronchodilator, is of milligram magnitude and second to 2′-deoxyguanosine, which is extremely interesting and worthy of further study. As for free amino acids, S. nudus contains all 20 proteinogenic amino acids and five non-protein amino acids. The percentage of eight essential amino acids in the 20 proteinogenic amino acids varied from 23.91% to 33.11%, and the percentage in body wall and intestine was higher than in coelomic fluid. The total contents of five non-protein amino acids in the samples, including GABA, citrulline, hydroxyproline, ornithine and taurine, varied from 7.77 mg/g to 22.90 mg/g, among which the taurine had the highest percentage which varied from 84.60% to 94.60%. Taurine has been reported to improve aerobic endurance, mental performance, concentration and memory [32], and we suggest that it may play an active role in the reported learning and memory enhancing pharmacological action as well as the anti-fatigue and anti-anoxia action of S. nudus [33,34]. Notably, the contents of fresh flavor amino acids including glycine, alanine, glutamate, aspartate and so on were fairly high, especially the contents of glycine which were the highest of all the free amino acids in S. nudus according to the assays, and all of these fresh flavor amino acids above fully explain its reputed good taste (Table 3).
To evaluate the variation of samples, PCA was performed on the basis of the contents of 41 tested compounds from UPLC-TQ-MS profiles. The first three principal components (PC 1, PC 2 and PC 3) with > 83.70% of the whole variance were extracted. Among them, PC 1, PC 2 and PC 3 accounted for 60.63, 17.16 and 5.91% of total variance, respectively. The remaining principal components, which had a minor effect on the model, were discarded. According to their loadings, PC 1 had good correlations with 2′-deoxyinosine, 2′-deoxyguanosine, guanine, xanthine, glycine, isoleucine, threonine, glutamate, proline and histidine, among which the correlation of glycine, threonine and glutamate are over 95%. And PC 2 had good correlation with analytes of 2′-deoxyadenosine-5′-monophosphate and citrulline whereas PC 3 had good correlation with analytes of inosine and taurine (Table 4). In the scatter plot, each sample is represented as a marker. It was noticeable that the samples were clearly clustered into three groups: cluster A (sample H2, F2, G2, S2, H5, F5, G5, S5), cluster B (H1, F1, G1, S1, H4, F4, G4, S4) and cluster C (F3, G3, S3) ( Figure 3). This result indicated there are significant differences in the proportions and quantity of the 41 compounds in different parts in S. nudus while the impact of the extraction procedure method used was not significant.  (2), coelomic fluid, ultrasonication (3), body wall, refluxing (4) and intestine, refluxing (5). The color A stands for total contents of amino acids, and the color N stands for total contents of nucleosides and nucleobases.
To evaluate the variation of samples, PCA was performed on the basis of the contents of 41 tested compounds from UPLC-TQ-MS profiles. The first three principal components (PC 1, PC 2 and PC 3) with > 83.70% of the whole variance were extracted. Among them, PC 1, PC 2 and PC 3 accounted for 60.63, 17.16 and 5.91% of total variance, respectively. The remaining principal components, which had a minor effect on the model, were discarded. According to their loadings, PC 1 had good correlations with 2 1 -deoxyinosine, 2 1 -deoxyguanosine, guanine, xanthine, glycine, isoleucine, threonine, glutamate, proline and histidine, among which the correlation of glycine, threonine and glutamate are over 95%. And PC 2 had good correlation with analytes of 2 1 -deoxyadenosine-5 1 -monophosphate and citrulline whereas PC 3 had good correlation with analytes of inosine and taurine (Table 4). In the scatter plot, each sample is represented as a marker. It was noticeable that the samples were clearly clustered into three groups: cluster A (sample H2, F2, G2, S2, H5, F5, G5, S5), cluster B (H1, F1, G1, S1, H4, F4, G4, S4) and cluster C (F3, G3, S3) ( Figure 3). This result indicated there are significant differences in the proportions and quantity of the 41 compounds in different parts in S. nudus while the impact of the extraction procedure method used was not significant.      The S. nudus samples collected from the different habitats were identified by Prof. Ding Shaoxiong (Xiamen University, Fujian Province, China). After collection, the animals were kept in a −80 °C refrigerator. Voucher specimens were deposited in Nanjing University of Chinese Medicine, China. After defrosting at the temperature of 4 °C and removing the excreta, the S. nudus were dissected into three parts, including body wall (SE), intestine (SI), coelomic fluid (SC) ( Figure 5). The S. nudus samples collected from the different habitats were identified by Prof. Ding Shaoxiong (Xiamen University, Fujian Province, China). After collection, the animals were kept in a´80˝C refrigerator. Voucher specimens were deposited in Nanjing University of Chinese Medicine, China. After defrosting at the temperature of 4˝C and removing the excreta, the S. nudus were dissected into three parts, including body wall (SE), intestine (SI), coelomic fluid (SC) ( Figure 5). All three parts were freeze-dried in a vacuum freeze drier system (Labconco, Kansas City, MO, USA), then weighed and smashed, respectively. One gram of each dry sample was accurately weighed into a 50 mL conical flask, then 40 mL distilled water was added. All of the mixtures were placed into an ultrasonic bath (40 kHz) for 60 min at room temperature. Meanwhile, the same samples were refluxed for 60 min. Water was added to both sets oif samples to compensate for any lost during extraction. After centrifugation (1,3000 r/min) for 10 min, the protein in the supernatants was removed by adding acetonitrile to double the volume and then stored at 4 °C and filtered through 0.22 μm cellulose membrane filters prior to injection.
The mass spectrometry detection was performed by using a Xevo TM Triple Quadrupole MS (Waters Corp.) equipped with an electrospray ionisation (ESI) source operating in positive ionisation mode. The desolvation gas flow rate was set to 1000 L/h at a temperature of 350 °C, the cone gas flow rate was set at 20 L/h and the source temperature was set at 120 °C. The capillary voltage was set to 3000 V, the cone voltage and collision energy were set depending upon the MRM for each compound. Data were collected in MRM mode by screening parent and daughter ions simultaneously ( Table 1). The raw data were acquired and processed with the Waters MassLynx 4.1 software (Waters China, Shanghai, China).

Data Processing
The raw data were processed by the MassLynx 4.1 software, and the identification of free amino acids, nucleosides and nucleobases was carried out by comparing the retention time of target peaks with those of the standards, and the quantification was calculated by linear calibration plots of the peak. The statistical analysis was performed using the SPSS 17.0 software (SPSS, Chicago, IL, USA). All three parts were freeze-dried in a vacuum freeze drier system (Labconco, Kansas City, MO, USA), then weighed and smashed, respectively. One gram of each dry sample was accurately weighed into a 50 mL conical flask, then 40 mL distilled water was added. All of the mixtures were placed into an ultrasonic bath (40 kHz) for 60 min at room temperature. Meanwhile, the same samples were refluxed for 60 min. Water was added to both sets oif samples to compensate for any lost during extraction. After centrifugation (1,3000 r/min) for 10 min, the protein in the supernatants was removed by adding acetonitrile to double the volume and then stored at 4˝C and filtered through 0.22 µm cellulose membrane filters prior to injection.
The mass spectrometry detection was performed by using a Xevo TM Triple Quadrupole MS (Waters Corp.) equipped with an electrospray ionisation (ESI) source operating in positive ionisation mode. The desolvation gas flow rate was set to 1000 L/h at a temperature of 350˝C, the cone gas flow rate was set at 20 L/h and the source temperature was set at 120˝C. The capillary voltage was set to 3000 V, the cone voltage and collision energy were set depending upon the MRM for each compound. Data were collected in MRM mode by screening parent and daughter ions simultaneously ( Table 1). The raw data were acquired and processed with the Waters MassLynx 4.1 software (Waters China, Shanghai, China).

Data Processing
The raw data were processed by the MassLynx 4.1 software, and the identification of free amino acids, nucleosides and nucleobases was carried out by comparing the retention time of target peaks with those of the standards, and the quantification was calculated by linear calibration plots of the peak. The statistical analysis was performed using the SPSS 17.0 software (SPSS, Chicago, IL, USA).

Method Validation
The linearity was verified by plotting the peak areas versus the corresponding concentrations of each analyte. The limit of detection (LOD) and limit of quantification (LOQ) were obtained by diluting the mixed standard working solution to the appropriate concentrations until the S/N for each compound was about 3 and 10, respectively. The intra-and inter-day precisions were examined by analyzing the mixed standard solutions six times in a day and repeatedly in three consecutive days. To confirm the repeatability, six sample solutions from the same sample were prepared and analyzed in parallel. To evaluate the stability of the components, one of the sample solutions was analyzed in various periods (0, 2, 4, 6, 12, and 24 h). Besides, there was also a recovery test was used to evaluate the accuracy of this method. It was performed by adding corresponding marker compounds with high (120%), medium (100%), and low (80%) levels into accurately weighed samples, and then they were processed and analyzed with the same methods above. We prepared two groups of samples which were the standard solution and the matrix matching standard solution adding appropriate amounts of standards to the samples. Then we used the radios (the peak area of matrix matching standards/the peak area of standards) to investigate the matrix effects.

Conclusions
In this study, a simple, rapid and reliable UPLC-TQ-MS/MS method was developed and applied to simultaneously determine 25 free amino acids and 16 nucleosides and nucleobases in S. nudus extracts within 20 min. The method had acceptable intra-and interday precision (RSD < 3.72%, RSD < 3.42%). The LODs and LOQs ranged from 0.003-0.229 µg/mL and 0.008-0.763 µg/mL, respectively. Recoveries were between 94.03% and 106.33% with RSDs in the range of 0.64%-3.76% for all target compounds. Real sample data demonstrate that S. nudus is an excellent source of free amino acids, nucleosides and nucleobases with great nutritional and functional value. The contents of these compounds in different parts were significantly different, and in the order: (highest) coelomic fluid > body wall > intestine (lowest). As the contents of xanthine, 2 1 -deoxyguanosine, taurine and glycine were the highest in each category, all of them could be proposed as markers for quality control of S. nudus. Moreover, the research results also provide a firm basis for further exploitation and development of S. nudus.