Derivatization High-performance Liquid 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.

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.

A sensitive simultaneous detection approach for the determination of 30 atmospheric carbonyls by 2,4-dinitrophenylhydrazine derivatization with HPLC-MS technique and its preliminary application.

Atmospheric carbonyls are important precursors of PM2.5 and ground-level ozone, and some carbonyls are toxic and harmful; thus, it is crucial to obtain accurate information on the ambient levels of carbonyls. However, the detection of carbonyls is difficult due to their relatively higher reactivities and chemical instabilities; therefore, accurate determination of atmospheric carbonyls is important. In this study, an analytical method for atmospheric carbonyls with high concentration or reactivity was developed, the precursor ion of each carbonyl compound was selected, and the declustering potential (DP) and entrance potential (EP) for each precursor ion were optimized. A 2,4-dinitrophenylhydrazine cartridge derivatization-high performance liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry (DNPH-HPLC/APCI-MS) method for the determination of 30 carbonyls was established. The results showed that the linear range of 24 carbonyls was 1.2-600 ng/mL, while other 6 carbonyls was 1.2-300 ng/mL, and the detection limits of 30 carbonyls ranged from 0.092 to 0.947ng/mL (0.005-0.049μg/m3 with an ambient air sampling volume of 96L). The intra-day and inter-day repeatability ranges were 0.55-4.20% and 1.40-12.48%, respectively. A preliminary application of the method was carried out in the urban area of Beijing in spring and summer of 2021, and it was found that the mean total mass concentration of 30 carbonyls was 35.894μg/m3. This study provided additional concentration information for 14 atmospheric carbonyls, including mono-, di-, oxygen-containing and heterocyclic carbonyls, which accounted for 38% and 35% of the total mass concentration and OH radical reactivities of 30 carbonyls, respectively. This is the first investigation of simultaneous quantitative analysis of multiple atmospheric carbonyls based on commercial standard derivatives. The optimized method could provide more comprehensive information for atmospheric carbonyls and further support research concerning the role of chemical reaction processes and health effects than traditional measuring techniques.

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.

Research progress on detection of 2-acetyl-1-pyrroline, the characteristic aroma component of fragrant rice

2-Acetyl-1-pyrroline (2-AP) has been identified as the characteristic aroma component of fragrant rice, and its concentration determines the quality and price of the rice. However, obtaining accurate assay results with modern analytical instruments remains a major challenge. The two reasons for this setback are the ultralow concentration of 2-AP in samples and the serious interferences in its determination. In natural fragrant rice, the concentration of 2-AP is very low, at the μg/kg level. The interferences mainly originate from the sample matrix or due to co-elution during chromatographic separation. In the present paper, various methods for the sample pretreatment and instrumental analysis of 2-AP in rice are reviewed. The sample pretreatment methods include distillation, extraction, and headspace enrichment procedures. Common instrumental analytical methods include gas chromatography (GC) or GC-mass spectrometry (MS), GC-olfactometry, and derivatization-high performance liquid chromatography-MS/MS developed by the researchers recently. The present review will provide a reference for the determination of 2-AP in the food trade, the research on fragrant rice breeding as well as the management of water and fertilizers in agriculture, and the development of stabilized flavor compounds of fragrant rice scent in food processing.

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.

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.

Determination of residual 4-nitrobenzaldehyde in chloramphenicol and its pharmaceutical formulation by HPLC with UV/Vis detection after derivatization with 3-nitrophenylhydrazine

4-Nitrobenzaldehyde is the synthetic raw material and an important photodegradation product of chloramphenicol. With a structural “alert” of human genotoxic potential and reported mutagenicity, this compound should be controlled in drug substances as a potential genotoxic impurity. However, current analysis methods require complex pre-treatment processes and/or lack sufficient specificity and sensitivity. Nitrophenylhydrazine is a common carbonyl derivatization reagent used to determine the residual aromatic aldehydes in drug samples. In the present study, we report an unexpected advantage of 3-nitrophenylhydrazine hydrochloride as a derivatization reagent in the derivatization high-performance liquid chromatography-ultraviolet detection method to determine 4-nitrobenzaldehyde in chloramphenicol samples. Compared with other nitro-substituted phenylhydrazines, 3-nitrophenylhydrazine hydrochloride can minimize drug matrix and derivatization reagent interferences, since the maximum absorption wavelength of its derivative is significantly red-shifted to 397 nm. The derivatization conditions have been optimized in terms of reaction efficiency, including reaction temperature, time, and diluting solvent, through a design of experiments. As a result, after reaction with 500 μg mL−1 of 3-nitrophenylhydrazine hydrochloride in acetonitrile-water (70:30, v/v) at 60 °C for 30 min, the developed HPLC method could be used to determine 4-nitrobenzaldehyde with a limit of detection of 0.009 μg mL−1. The method was then validated and applied for the determination of residual 4-nitrobenzaldehyde in chloramphenicol and its eye-drop samples.

A quantitative analytical method for valienone and its application in the evaluation of valienone production by a breakthrough microbial process

AbstractValienone is a significant natural carbasugar member of the C7-cyclitol family as a valuable precursor for glycosidase inhibitor drugs. It is an intermediate of validamycin A biosynthesis pathway and exhibits minimal accumulation in the fermentation broth of the natural Streptomyces producer. A quantitative analytical method is crucial for the development of a breakthrough microbial process overcoming the consumption of the natural metabolic flux. The present study was designed to develop a pre-column derivatization high-performance liquid chromatography method for quantification of valienone and to help establish a straightforward fermentation process for valienone production by metabolically engineered Streptomyces hygroscopicus 5008. Valienone was derivatized by 2, 4-dinitrophenylhydrazine (DNPH) in 10 mmol·L−1 H3PO4 at 37 °C for 45 min and the derivatives were separated on Eclipse XDB-C18 (5 μm, 4.6 mm × 150 mm) column at 30 °C eluted with 50% acetonitrile for 18 min. The derivatives were detected by diode array detector at 380 nm and the configurations of the derivatives were determined by computational studies. The method was shown to be effective, sensitive, and reliable. Good linearity was found in the range of 5–2 000 μg·mL−1. The intra- and inter-day precisions were 1.1%–2.7% and 1.7%–2.2%, respectively. The absolute recovery of the spiked samples was 97.2%–102.6%. To date, this is the first reversed-phase high-performance liquid chromatography detection method for valienone in microbial culture medium. This method successfully helped evaluate the valienone production capability of the engineered Streptomyces hygroscopicus 5008 and could be promising for C7-cyclitol profiling of different engineered mutants combined with the metabonomics methods.

A simple pre-column derivatization method for the determination of mancozeb technical (fungicide) by reverse phase HPLC-UV

A simple and fast pre-column derivatization high performance liquid chromatographic method for the technical analysis of mancozeb was developed and validated in the present study.