Derivatization High-performance Liquid Chromatography Research Articles

Evaluation of selenocysteine contents in Se-enriched yogurt by Utilizing online post column derivative − High-Performance liquid chromatography

This study demonstrated a novel method using online post-column derivatization high-performance liquid chromatography (PCD-HPLC) to evaluate selenocysteine (SeC) in Se-enriched yogurt. The liquid chromatography process utilized a C18 column. The mobile phase A was composed of 4.0 mM borate buffer adjusted to pH 5.0, while mobile phase B was a 50:50 (v/v) mixture of methanol and acetonitrile. The mobile phases were combined at a ratio of 70:30, with a flow rate of 1 mL min−1. SeC was derivatized to bis-Fmoc selenocystine after separation, using a PCD solution (0.03 mM Fmoc, pH 10, 20 mM borate buffer, and 1.0 mL min−1 flow rate) and detected with a photodiode array detector (DAD) for sensitivity enhancement. The method exhibited excellent linearity (0.023 to 0.20 mg L-1, r2 = 0.9996), with a low LOD and LOQ of 0.018 mg L-1 and of 0.040 mg L-1, respectively. Repeatability and reproducibility were satisfactory, with recoveries of 95.50 % ± 1.49 % (the proposed PCD-HPLC) and 94.33 % ± 0.44 % (sample preparation). This method effectively analyzed the conversion of selenite ions (SeO32-) to SeC by lactic acid bacteria in yogurt, highlighting Se-enriched yogurt as a valuable dietary source of SeC. However, the synthesis efficiency of SeC decreased at inorganic Se supplementation levels above 1.0 mg Se L-1, potentially leading to the formation of less toxic elemental Se (Se0). Therefore, the proposed online PCD-HPLC method proves reliable for SeC analysis in Se-enriched yogurt, providing insights into Se speciation and optimizing Se enrichment processes.

Determination of sodium cyclamate in Baijiu by pre-column derivatization-high performance liquid chromatography with fluorometric detection

Sodium cyclamate in Baijiu is a key item in the China National Food Safety Supervision and Inspection Plan. A simple, economical, sensitive, and reliable method is urgently needed for routine analysis and internal quality control. A method based on high performance liquid chromatography with fluorescence detection (HPLC-FLD) was developed for the determination of sodium cyclamate in Baijiu by o-phthalaldehyde derivatization. First, the sodium cyclamate in the sample solution was converted into amino compounds using the desulfurization reaction under acidic conditions. Next, 400 g/L sodium hydroxide solution was added to the sample solution for neutralization. The amino compounds in the sample solution were then derivatized with o-phthalaldehyde to produce indole-substituted derivatives that are capable of producing fluorescence signals. Separation was carried out on a C18 column (250 mm×4.6 mm, 5 μm) in isocratic elution mode using a mobile phase consisting of acetonitrile and phosphate buffer. Finally, the eluate was monitored using a fluorescence detector, and an external standard method was used for quantification. A good linear relationship was obtained in the range of 0.1-2.0 mg/L, with correlation coefficients greater than 0.999. The average recoveries of sodium cyclamate spiked at levels of 0.1-1.0 mg/kg in Baijiu samples ranged from 90.7% to 100.9%, with relative standard deviations (RSDs) of 3.5%-5.6% (n=6). The limits of detection and quantification were 0.03 and 0.10 mg/kg, respectively. Nine Baijiu samples collected from the market were tested, and the results demonstrated that the contents of sodium cyclamate detected by the developed method were consistent with those obtained using the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method described in GB 5009.97-2016 (the third method). The proposed method is economical, sensitive, specific, and accurate; thus, it provides a basic approach for the determination of sodium cyclamate in Baijiu samples and has great potential for routine analysis in foodstuffs.

A simplified small-scale workflow for determination of complete protein-bound amino acids using pre-column derivatization HPLC method

Existing quantification methods of complete protein-bound amino acids profile of a food or feed often involve complicated hydrolysis setup and multiple hydrolysate treatment steps. Herein, we present a simplified workflow requiring only two steps: hydrolysis (in milligram scale) and direct sample dilution (in microliter scale), well adapted for automated pre-column O-phthaldialdehyde (OPA)/ 9-fluorenylmethyl chloroformate (FMOC) derivatized reverse phase high performance liquid chromatography (HPLC) method (the concentration range was established between 0.005 and 0.50 mM). This new workflow significantly reduces the analysis time (by up to 12 h for every 36 samples) while achieving over 90 % recovery for total protein-bound amino acids, including tryptophan, cysteine, and methionine. The easy hydrolysis setup eliminates the need for special hydrolysis tube or explosive reagents and substitutes laborious sample preparation steps like dry-down, reconstitution, neutralization with voluminous diluents and pH adjustment, with straightforward dilution using 0.1 N hydrochloric acid and deionized water. This method accommodates a small sample load, addressing concerns of limited sample availability. Robustness of this established protocol was validated by bovine serum albumin, and further confirmed by several plant proteins and casein standard. Good correlation of our results with an ISO accredited laboratory was verified (r = 0.992).

Tetrodotoxin Derivatization with a Newly Designed Boron Reagent Leads to Conventional Reversed-Phase Liquid Chromatography.

Tetrodotoxin (TTX) is a representative natural toxin causing pufferfish food poisoning, which is especially prominent in East and Southeast Asia, including Japan. TTX has been analyzed through post-column derivatization high-performance liquid chromatography (HPLC), ion-pair LC-MS(/MS), and hydrophilic interaction liquid chromatography (HILIC)-MS(/MS) as alternatives to the mouse bioassay method. However, post-column derivatization requires a system for online derivatization reactions, and with the ion-pair LC-MS approach, it is difficult to remove residual ion-pair reagents remaining in the equipment. Moreover, HILIC-MS provides poor separation compared to reversed-phase (RP) HPLC and requires a long time to reach equilibration. Therefore, we decided to develop a TTX analytical method using pre-column derivatization and RP HPLC for the rapid assessment of outbreak samples, including food remnants. In this study, we focused on the vic-diol moiety of TTX and designed a new derivatization reagent coded as NBD-H-DAB. This NBD-H-DAB was synthesized from 4-hydrazino-7-nitro-2,1,3-benzoxadiazole (NBD-H) and 3-fluoro-2-formylphenylboronic acid (FFPBA) with a simple reaction system and rapidly converted to its boronate form, coded NBD-H-PBA, in an aqueous reaction solution. The NBD-H-PBA demonstrated appropriate hydrophobicity to be retained on the RP analytical column and successfully detected with a UV spectrometer. It was easily reacted with the vic-diol moiety of TTX (C6 and C11) to synthesized a boronic ester. The derivatized TTX could be detected using the RP HPLC-UV, and the limit of detection in the fish flesh samples was 0.06 mg/kg. This novel pre-column derivatization of TTX with NBD-H-PBA proves capable for the analysis of TTX.

Optimization of hydrolysis conditions of alginate based on high performance liquid chromatography

Alginate is the most abundant polysaccharide compound in brown algae, which is widely used in various fields. At present, the determination of the content of alginate is mostly carried out using sulfuric acid and trifluoroacetic acid hydrolysis followed by the determination of the content, but the results are not satisfactory, and there are problems such as low hydrolysis degree and low recovery rate. Therefore, in this study, based on the optimization of high performance liquid chromatographic conditions for pre-column derivatization of 1-phenyl-3-methyl-5-pyrazolone (PMP), the hydrolysis effects of sulfuric acid, trifluoroacetic acid (TFA), oxalic acid, and formic acid were compared and the hydrolysis conditions were optimized. The results showed that formic acid was the best hydrolyzing acid. The optimal hydrolysis conditions were 95 % formic acid at 110 °C for 10 h. The hydrolysis effect was stable, with high recovery and low destruction of monosaccharides, which made it possible to introduce formic acid into the subsequent polysaccharide hydrolysis. The pre-column derivatization high performance liquid chromatography method established in this study was accurate and reliable, and the hydrolysis acid with better effect was screened, which provided a theoretical basis for the subsequent determination of alginate content.

Assay of amino acids in leaves of Eucommia ulmoides under arbor forest mode and leaf-oriented cultivation mode by pre-column derivatization HPLC

This study aims to develop the pre-column derivatization high performance liquid chromatography(HPLC) method for the determination of 16 kinds of amino acids in Eucommia ulmoides leaves, and compare the content of amino acids in the leaves harvested at different time and under leaf-oriented cultivation mode(LCM) and arbor forest mode(AFM). The HPLC conditions are as below: phenyl isothiocyanate(PITC) as pre-column derivatization agent, Agilent ZORBAX C_(18 )column(4.6 mm×250 mm, 5 μm), mobile phase A of acetonitrile-water(80∶20), mobile phase B of 0.1 mol·L~(-1) sodium acetate solution-acetonitrile(94∶6), gradient elution, flow rate of 1.0 mL·min~(-1), injection volume of 5 μL, column temperature of 40 ℃, and detection wavelength of 254 nm. The HPLC profile indicated well separation of 16 kinds of amino acids and the amino acid content in E. ulmoides leaves was up to 16.26%. In addition, the amino acid content in leaves of E. ulmoides under LCM was higher than under AFM. The amino acid content varied with the harvesting time. Through orthogonal partial least squares discriminant analysis, the amino acids of E. ulmoides under LCM and AFM were compared, which can distinguish the leaves under LCM from those under AFM. Principal component analysis was applied to comprehensively score the amino acids of E. ulmoides leaves. The results showed that the score of leaves under LCM was higher than that under AFM. Nutritional evaluation results indicated that the proteins in E. ulmoides leaves belonged to high-quality vegetable proteins. The established method for the determination of amino acid content is reliable. With the amino acid content as index, the leaf quality of E. ulmoides under LCM is better than that under AFM. This study lays a theoretical basis for the promotion of LCM for E. ulmoides and the development of medicinal and edible products from E. ulmoides leaves.

Research on Detection Methods and Applications of Natural Whitening Components in Cosmetics

With the improvement of people's living standards, cosmetics have been widely used. The addition of whitening ingredients in cosmetics is necessary. Natural whitening ingredients have the advantages of small side effects, strong efficacy and high safety, but excessive addition is harmful to the human body. Therefore, the detection of natural whitening ingredients in cosmetics is very important. This paper summarizes the research on natural whitening ingredients detection technology, and compares the advantages and disadvantages of high-performance liquid chromatography, pre-column/ post-column derivatization high performance liquid chromatography, thin layer chromatography and thin layer scanning method. We extract polyphenols from natural plants and thymine from natural animals as ingredients of cosmetics.

Precolumn Derivatization High-Performance Liquid Chromatography for Determination of Perfluorocarboxylic Acids in Catalytic Degradation Solutions.

Perfluoroalkyl carboxylic acids (PFCAs), a series of ubiquitous contaminants in the global environment, attracted much attention due to their potential for high bioaccumulation and toxicity to various organisms. There are a lot of measurement requests in currently increasing degradation studies of PFCAs, which usually rely on expensive liquid chromatography-mass spectrometry (LC-MS). The degradation solutions containing high-concentration PFCAs can easily cause the pipeline pollution of the LC/MS instrument, which is usually used for trace analysis of environmental samples. In this study, a simple and reliable precolumn derivatization LC method coupled with an ultraviolet detector (UV) was developed for the determination of the main PFCAs (C4-9) of environmental concern. These PFCAs in degradation solutions were crosslinked to UV-responsive 3, 4-diphenylamine (DCA) by a carbodiimidization method, followed by a simple solid-phase extraction (SPE) cleanup, and quantitatively measured using a conventional LC-UV instrument. Compared to previously reported precolumn derivatization methods, this new derivatization approach has the advantages such as mild reaction conditions, easy operation, enhanced stability of derivatives, and low cost. The instrumental limits of detection (ILDs) for the targeted PFCAs in organic and aqueous mediums were 0.2–0.5 and 0.6–1.5 mg/L, respectively. The method has been successfully applied to the determination of PFCAs in catalytic degradation solutions and recommended for use in other assays involving relatively high-concentration PFCAs.

Determination of Methyl Methanesulfonate and Ethyl Methylsulfonate in New Drug for the Treatment of Fatty Liver Using Derivatization Followed by High-Performance Liquid Chromatography with Ultraviolet Detection.

A new derivatization high-performance liquid chromatography method with ultraviolet detection was developed and validated for the quantitative analysis of methanesulfonate genotoxic impurities in an innovative drug for the treatment of non-alcoholic fatty liver disease. In this study, sodium dibenzyldithiocarbamate was used as a derivatization reagent for the first time to enhance the sensitivity of the analysis, and NaOH aqueous solution was chosen as a pH regulator to avoid the interference of the drug matrix. Several key experimental parameters of the derivatization reaction were investigated and optimized. In addition, specificity, linearity, precision, stability, and accuracy were validated. The determined results of the samples were consistent with those obtained from the derivatization gas chromatography–mass spectrometry analysis. Thus, the proposed method is a reliable and practical protocol for the determination of trace methanesulfonate genotoxic impurities in drugs containing mesylate groups.

Extraction, preparation, and carboxymethyl of polysaccharide from Lotus root

In this study, we extracted lotus root polysaccharide (LRP) and synthesized carboxymethylated lotus root polysaccharide (CM-LRP) using response surface methodology (RSM). The monosaccharide component of LRP was analyzed by pre-column derivatization high performance liquid chromatography (PCD-HPLC). The polysaccharide was characterized by ultraviolet (UV) spectroscopy scan, Fourier transform infrared spectroscopy (FTIR), circular dichroism spectrometer (CD), and scanning electron microscopy (SEM). It was found that the main monosaccharide component of LRP was glucose, accounting for more than 98% of the total polysaccharide component. RSM analyses revealed that the optimal conditions for CM-LRP synthesis were a reaction duration of 2.22 h, the chloroacetic acid dosage is 2.02 g, and a temperature of 46.87 °C. Under these conditions, the predicted degree of substitution (DS) was determined to be 0.5101. The superoxide anion, ferrous ion and hydroxyl radical antioxidant system were constructed to study the antioxidant activity of LRP and CMLRP. The activity of CMLRP to remove ferrous ions and hydroxyl radicals were significantly stronger than that of LRP, and it also showed a certain concentration-dependent. LRP has a better scavenging ability than CMLRP in scavenging superoxide anion free radicals. Our data revealed that CM-LRP is a promising natural antioxidant with potential value as a food supplement.

Protective Role of a New Polysaccharide Extracted from Lonicera japonica Thunb in Mice with Ulcerative Colitis Induced by Dextran Sulphate Sodium.

Lonicera japonica Thunb is a traditional Chinese herbal medicine for treating intestinal inflammation. The extraction method of Lonicera japonica Thunb polysaccharide (LJP) has been developed previously by our research group. In this study, a Fourier transform infrared spectrometer (FT-IR) was used to perform a qualitative analysis of LJP and a precolumn derivatization high-performance liquid chromatography (HPLC) ((Palo Alto, CA, USA) method was used to explore the monosaccharide composition of LJP. Then, we studied the effect of LJP on the intestinal flora and immune functions of dextran sulfate sodium- (DSS-) induced colitis ulcerative mouse models. The results showed that LJP was consisted of 6 types of monosaccharides and had the characteristic absorption of typical polysaccharides. LJP can increase significantly the weight, organ index, serum cytokines (interleukin, tumor necrosis factor, and interferon-γ), secretory immunoglobulin A (SIgA) concentration, and natural killer (NK) cell and cytotoxic lymphocyte (CTL) activities in DSS-treated mice. The results of intestinal flora showed that a high dose (150 mg kg−1) of LJP had the best effects on improving the intestinal probiotics (Bifidobacterium and Lactobacilli) and antagonizing the pathogenic bacteria (Escherichia coli and Enterococcus). In addition, the measurement results of the spleen lymphocyte apoptosis confirmed from another perspective that LJP had protective effects of immune cells for DSS-treated mice.

An efficient method for determination of components in docosahexaenoic acid-phosphatidylcholine using pre-column derivatization HPLC

The content of docosahexaenoic acid-phosphatidylcholine (DHA-PC) in the phospholipid product is closely related to its nutritional value and health function. An efficient high performance liquid chromatography (HPLC) was firstly proposed and successfully applied to the determination of DHA-PC. DHA-PC was pre-treated by alkaline hydrolysis, acidification and then the obtained components underwent derivatization using α-bromoacetophenone as a derivatization reagent, triethylamine as a catalyst, to make them have ultraviolet (UV) absorption. Major components of DHA-PC could reach baseline separation within a short time (50 min). A gradient elution method using acetonitrile and methanol/water (1:1, volume ratio) at 0.55 mL/min, UV detection at 244 nm and a column temperature of 50 oC was found to optimally separate the components of DHA-PC. The intra-assay precisions (n = 6) and stabilities (n = 6) relative standard deviations (RSDs) for the major components were 2.5%-3.2% and 1.9%-4.0%, respectively. The average recoveries were 97.8%-106%. The results indicated that this method was rapid, accurate and reliable to analyze the content of DHA-PC in the phospholipid product. This method could be used not only to detect the content of DHA-PC in the phospholipid product, but to analyze the content of various fatty acids in mixed PC.

Characterization of guava (Psidium guajava Linn) seed polysaccharides with an immunomodulatory activity

To clarify the property of a novel guava seed polysaccharide (GSPS), GSPS was subjected to purify using Sepharose 6B gel filtration chromatography and further characterize the property of each individual isolated fraction. GSPS further resolved into three purified fractions, guava seed polysaccharide fraction 1 (GSF1), GSF2 and GSF3. Isolated GSF1, GSF2 and GSF3 were respectively subjected to high performance size exclusion chromatography; molecular weights of three polysaccharide fractions were determined. GSPS, GSF1, GSF2 and GSF3 were suggested to be proteopolysaccharides or glycoproteins. GSPS, GSF1, GSF2 and GSF3, particularly GSF3, were found to have a Th2-inclination property and anti-inflammatory potential. Heated GSF3 did not significantly (P > .05) decreased its immunomodulatory activity, suggesting that GSF3 is a proteopolysaccharide. The deproteinated GSF3 markedly lost its immunomodulatory activity, suggesting that both protein and carbohydrate moiety in GSF3 are essential to its immunomodulatory function. Analyses of monosaccharides composition in GSF3 using a pre-column derivatization high performance liquid chromatography exhibited that GSF3 was composed of glucuronic acid (3.28%), galacturonic acid (28.13%), galactose (14.88%), mannose (3.96%), glucose (22.99%), arabinose (7.31%), ribose (1.55%), xylose (14.81%), fucose (1.68%) and rhamnose (1.43%). Overall, we evidence that GSF3 is a low molecular weight proteopolysaccharide with potent anti-inflammatory and immunomodulatory effects.

Development and Validation of a Pre-Column Derivatization HPLC Method for the Assay of Amikacin Sulfate in Pure and Parenteral Dos age Forms

Background: Amikacin sulfate (AMK) belongs to the class of aminoglycoside antibiotics. It is effective against the infections caused by Gram-negative and positive bacteria. AMK lacks a chromophore group in its structure and, therefore, it does not absorb light in the 200-800 nm region which makes it a difficult molecule to analyze by UV detector using high performance liquid chromatography (HPLC). Objective: This study has been carried out to develop and validate a relatively simple, accurate, precise, rapid, economical, and stability-indicating pre-column derivatization HPLC method for the determination of AMK in pure and parenteral dosage forms. Methods: The stock solution of AMK was derivatized prior to its analysis. The mobile phase used for the analysis was acetonitrile and water in the ratio of 50:50 (v/v) at pH 6.0. The method has been validated according to the guideline of International Council for Harmonization (ICH) and different parameters such as linearity, range, accuracy, precision, sensitivity, robustness, solution stability, specificity and system suitability have been studied. AMK was subjected to stress degradation studies including thermolysis, humidity exposure, acid-base hydrolysis, and oxidation in order to determine the specificity of the test method. Results: The retention time of AMK has been found to be 4.7 min. The results indicated that the method is linear in the concentration range of 12.5-125% and possesses high accuracy (99.88±0.42%), precision ((<1.2%) and robustness (<0.5%). The obtained results are compared statistically with a reference method. Conclusion: It was observed that the stress degradation studies do not affect the accuracy of the method. Hence the proposed method can be used for the assay of AMK and its parenteral dosage form.

Determination of immunogenic proteins in biopharmaceuticals by UHPLC\u2013MS amino acid analysis

Nowadays, there is a growing interest in innovative and more efficient therapeutics—biopharmaceuticals, based on peptides or proteins. There are increased demands on quality control of such therapeutics. One of the methods usually used for characterization and quantification of biopharmaceuticals is amino acid analysis. In this work, a modern advanced analytical method based on precolumn derivatization and reversed-phase ultra high-performance liquid chromatography in combination with single quadrupole mass spectrometer was developed for amino acid analysis in different protein samples—model sample of bovine serum albumin, sample of strong immunogenic protein keyhole limpet hemocyanin, and sample of drug etanercept present in commercially available biopharmaceutical Enbrel. The method used isotopically labeled internal standards and was validated according to the International Council for Harmonisation guideline. The developed method was characterized by favorable performance and validation parameters, such as time of analysis (6 min), specificity, linearity (r2 ≥ 0.99), limit of detection (0.009–0.822 µM), limit of quantification (1–2.5 µM), accuracy (recovery in the range 90–102.8%), intra-day (RSD in the range 0.25–11.97%) and inter-day precision (RSD in the range 1.67–11.57%), or stability (RE ≤ 12%). According to these findings, the developed amino acid analysis approach is suitable for routine use in areas of peptide/protein quantification, such as quality control laboratories of biopharmaceutical companies.

Front Cover: A method based on precolumn derivatization and ultra high performance liquid chromatography with high‐resolution mass spectrometry for the simultaneous determination of phthalimide and phthalic acid in tea

Front Cover: A method based on precolumn derivatization and ultra high performance liquid chromatography with high‐resolution mass spectrometry for the simultaneous determination of phthalimide and phthalic acid in tea

Wound Healing Activity of a Skin Substitute from Residues of Culinary-Medicinal Winter Mushroom Flammulina velutipes (Agaricomycetes) Cultivation.

A skin substitute TG05 obtained from residues of the culinary-medicinal mushroom Flammulina velutipes cultivation process was developed in this study for the first time. Pre-column derivatization high-performance liquid chromatography fingerprints analysis revealed that TG05 was composed of water-insoluble fibers containing xylose (57%), glucose (19.5%), and arabinose (16.3%) as major monomers. Porous and opaque structure of TG05 was demonstrated by scanning electron microscopy. Animal experiments conducted on mice and rats indicated that TG05 notably accelerated the wound-healing process. In addition, TG05 induced proliferation and migration of human keratinocytes time- and dose-dependently. Taken together, the skin substitute TG05 with new structure promotes wound healing in vitro and in vivo. This study provided a novel method to produce functional biomaterial from abundant and low-cost agricultural residues generated during edible mushroom cultivation.

Multiple fingerprint profiles and chemometrics analysis of polysaccharides from Sarcandra glabra

Multiple techniques including high performance size-exclusion chromatography (HPSEC), Fourier-transform infrared spectroscopy (FT-IR) and pre-column derivatization high-performance liquid chromatography (PCD-HPLC) were applied to the fingerprint analysis of the polysaccharides from Sarcandra glabra (SGPs) in different regions. Chemometrics was used to evaluate the similarity and differences of SGPs from different regions based on their fingerprints. The results of the present study showed that polysaccharides from 18 batches of Sarcandra glabra had a high degree of similarity based on the HPSEC, PCD-HPLC, and FT-IR fingerprints. The samples from different regions could be classified by clustering analysis based on their nuances. The five monosaccharides (Gal, Rha, Xyl, GlcA, and Glc) and the wavelengths of FT-IR (3371 cm−1 and 1411 cm−1) could be selected as herb markers for the quality control of Sarcandra glabra.

Immunomodulatory activity of a novel polysaccharide from Lonicera japonica in immunosuppressed mice induced by cyclophosphamide.

Lonicera japonica is a typical Chinese herbal medicine. We previously reported a method to isolate polysaccharides from Lonicera japonica (LJP). In this study, we first performed a qualitative analysis of LJP using the Fourier Transform Infrared Spectrometer (FT-IR) and explored the monosaccharide composition of LJP using the pre-column derivatization high performance liquid chromatography (HPLC) method. We then investigated the immunomodulatory function of LJP in cyclophosphamide (CTX)-induced immunosuppressed mouse models. The results showed that LJP had the characteristic absorption of typical polysaccharides consisting of 6 types of monosaccharides. In addition, LJP can increase significantly the organ index, splenic lymphocyte proliferation, macrophage phagocytosis, and natural killer (NK) cell activity in CTX-treated mice. LJP could also restore the levels of serum cytokines interleukin (IL-2), tumor necrosis factor (TNF-α) and Interferon-γ (IFN-γ) in the CTX-treated mice. Finally, the results on measuring the T-lymphocytes subsets of spleen also confirmed LJP-induced immunomodulatory activity in immunosuppressed mice from another perspective. Therefore, LJP could be used as a potential immunomodulatory agent.

High-performance liquid chromatographic determination of 2-aminoethylphosphonic acid and 2-amino-3-phosphonopropionic acid in seawater matrix using precolumn fluorescence derivatization with o-phthalaldehyde-ethanethiol

2-Aminoethylphosphonic acid (2-AEP) and 2-amino-3-phosphonopropionic acid (2-AP3) are two types of abundant and ubiquitous naturally occurring phosphonates used as sources of phosphorus by many prokaryotic lineages. The potential utilization mechanism of 2-AEP and 2-AP3 in eukaryotic phytoplankton is currently under investigation. However, the lack of suitable analytical methods in saline samples are the limitation of such researches. Herein, a high-performance liquid chromatography (HPLC) method for monitoring 2-AEP and 2-AP3 using precolumn fluorescence derivatization with o-phthalaldehyde-ethanethiol (OPA-ET) in seawater matrix was developed. The derivatization procedure and HPLC conditions were carefully examined, which included optimization of the fluorescence excitation and emission wavelengths, the ammonium acetate concentration and pH of the mobile phase, the OPA-ET reagent content and composition and derivatization time. Because increasing salinity was observed to lower the derivatization efficiency, working standards were freshly prepared in artificial seawater with the same salinity as that of the samples for the quantification of 2-AEP and 2-AP3. The developed HPLC method showed a wide linear response with high linearity (R2 > 0.999) and high repeatability at three concentration levels. The relative standard deviation was less than 4.1% for 2-AEP and less than 1.7% for 2-AP3 (n = 7). The limits of detection for 2-AEP and 2-AP3 in artificial seawater matrix were both 12.0 μg/L. The recoveries were 83.0–104% for 2-AEP and 72.6–98.6% for 2-AP3 in different aqueous samples, including algal culture medium prepared with filtered seawater. These results indicated the matrix effect of this method was insignificant.