Pre-column Derivatization High-performance Liquid Chromatography Research Articles

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.

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.

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.

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.

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.

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.

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.

Determination of Pb2+ in tea by bis(salicylaldehyde)-o-phenylenediamine pre-column derivatization-high performance liquid chromatography

The contents of Pb2+ in tea was determined by the high performance liquid chromatography (HPLC) method using bis(salicylaldehyde)-o-phenylenediamine (SALOPHEN) as derivatization reagent. The detection wavelength of ultraviolet was 226 nm with Hypersil ODS2 C18 column (250 mm×4.6 mm, 5 μ m) as stationary phase, and pH 10.0 methanol-water (80:20, v/v) as mobile phase. The linear range of Pb2+ was 0.1-30 mg/L and the linear correlation coefficient was 0.9988. The limit of detection was 0.01 mg/L with the recoveries of 91.87%-96.96%. The method has high sensitivity and stability with satisfactory results, which can be used for the detection of Pb2+ in tea samples.

Detection of four different amino acid neurotransmitters in cultured rat neurons and the culture medium by precolumn derivatization high-performance liquid chromatography.

We validated and used a high-performance liquid chromatography procedure for the determination of four different amino acid neurotransmitters in cultured rat neurons and used culture medium. Samples were derivatized using 2,4-dinitrofluorobenzene and the amino acids were separated on a C18 column. The method yielded good reproducibility and sensitivity for the quantification of the four free amino acid neurotransmitters, with average recovery factors of 80.25-118.43%, an intraday precision of 0.09-0.17%, and an interday precision of 0.62-0.74%. The assay method can be readily utilized as a precise, sensitive, and highly accurate method for the determination of concentrations of the four amino acid neurotransmitters in cultured rat neurons and used culture medium. Using the described methods, we found that the aspartate, glutamic acid, glycine, and γ-aminobutyric acid concentrations (µmol/g protein) in cultured rat neurons were 25.23±0.81, 35.16±0.32, 77.56±4.51, and 62.87±3.12, respectively, whereas their concentrations (µM) in the used culture medium were 18.18±0.82, 24.27±1.01, 107.18±9.56, and 35.78±2.98, respectively.

Rapid Determination of the Monosaccharide Composition and Contents in Tea Polysaccharides from Yingshuang Green Tea by Pre-Column Derivatization HPLC

A pre-column derivatization high-performance liquid chromatography (HPLC) method was developed and optimized to characterize and quantify the monosaccharides present in tea polysaccharides (TPS) isolated from Yingshuang green tea. TPS sample was hydrolyzed with trifluoroacetic acid, subjected to pre-column derivatization using 1-phenyl-3-methyl-5-pyrazolone (PMP), and separated on an Agilent TC-C18column (4.6 mm × 250 mm, 5 μm) with UV detection at 250 nm. A mixture of ten PMP derivatives of standard monosaccharides (mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, xylose, galactose, arabinose, and fucose) could be baseline separated within 20 min. Moreover, quantitative analysis of the component monosaccharides in Yingshuang green tea TPS was achieved, indicating the TPS consisted of mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, xylose, galactose, and arabinose in the molar contents of 0.72, 0.78, 0.89, 0.13, 0.15, 0.36, 0.39, 0.36, 0.36, and 0.38 μM, respectively. Recovery efficiency for component monosaccharides from TPS ranged from 93.6 to 102.4% with RSD values lower than 2.5%. In conclusion, pre-column derivatization HPLC provides a rapid, reproducible, accurate, and quantitative method for analysis of the monosaccharide composition and contents in TPS, which may help to further explore the relationship between TPS monosaccharides isolated from different tea varieties and their biological activity.

Fingerprinting profile of polysaccharides from Lycium barbarum using multiplex approaches and chemometrics

Techniques including ultraviolet–visible spectra (UV), high performance size-exclusion chromatography (HPSEC), Fourier-transform infrared spectroscopy (FT-IR) and pre-column derivatization high-performance liquid chromatography (PCD-HPLC) were used in the fingerprinting analysis of Lycium barbarum polysaccharides (LBPs) from different locations and varieties. Multiple fingerprinting profiles were used to evaluate the similarity and classification of different LBPs with the help of chemometrics. The results indicated that sixteen batches of LBPs had good consistency, and fingerprinting techniques were simple and robust for quality control of LBPs as well as related products. In addition, fingerprinting techniques combined with chemometrics could also be used to identify different cultivation locations of LBPs samples. Finally, four monosaccharides (galacturonic acid, glucose, galactose and arabinose) and the absorptions of stretching vibration of ester carbonyl groups as well as NH variable angle vibration of CONH could be selected as herbal markers to distinguish different samples.

Identification of dried shark fins by analysis of seven sugars using pre-column derivatization high-performance liquid chromatography and stable carbon isotope ratio analysis

A simple and sensitive method using pre-column high-performance liquid chromatography (HPLC) was developed for the simultaneous detection of seven sugars, which was also applied for the preliminary identification of dried shark fins.

Determination of theanine and γ-aminobutyric acid in tea by high performance liquid chromatography with precolumn derivatization

A method of precolumn derivatization-high performance liquid chromatography (HPLC) for the determination of theanine and gamma-aminobutyric acid (GABA) in tea was established. o-Phthalaldehyde (OPA) and N-acetyl-L-cysteine (NAC) were chosen as the derivatization reagents. The effects of teapolyphenol (Tp), proline (Pro) and Vitamin C (Vc) on derivatization yields were investigated. The results indicated that Vc not only stabilized the stock solution of OPA, but also enhanced the yield of GABA derivative. However, the yield of theanine derivative was less affected. The HPLC separation system was also optimized. The resolution of the derivatives was improved by adjusting the pH value and phosphate-citric buffer concentration of the mobile phase. The limits of detection (LODs) for GABA and theanine were 3.01 x 10(-5) mmol/L and 7.98 x 10(-5) mmol/L, and the limits of quantification (LOQs) were 9.99 x 10(-5) mmol/L and 2.658 x 10(-4) mmol/L, respectively. The linear ranges of GABA and theanine were 0.01 - 0.4 mmol/L with the correlation coefficient of 0.996 and 0.05 - 0.8 mmol/L with the correlation coefficient of 0.995, respectively. The main recoveries for GABA and theanine in green tea, Oolong tea, and black tea, ranged from 99.29% to 119.60% and from 62.88% to 141.06% respectively. The method with simple procedure and efficient separation was proved to be suitable for the determination of GABA and theanine in tea.

Determination of naftopidil enantiomers in rat plasma using chiral solid phases and pre-column derivatization high-performance liquid chromatography

Two bioanalytical HPLC methods (chiral solid phases (CSPs) HPLC and pre-column derivatization HPLC) were developed and validated for the determination of naftopidil enantiomers in rat plasma. Analytes were extracted from biomaterials by liquid–liquid extraction. The pre-column derivatization HPLC method employed (+)-diacetyl-l-tartaric anhydride (DATAN) as the pre-column derivatization reagent, and subsequent separation of diastereomers was conducted on an Agilent Hypersil ODS column with a mixture of methanol–acetonitrile–phosphate buffer (pH 4.1; 20mM) (40:30:30, v/v/v) flowing at 1mL/min as the mobile phase. The CSPs HPLC method utilized a Chiralpak IA column with a mobile phase of methanol–acetonitrile–acetate buffer (pH 5.3; 5mM) (50:25:25, v/v/v) flowing at 0.5mL/min. In both methods, the analytes were monitored using a fluorescence detector with an excitation wavelength of 290nm and an emission wavelength of 340nm. Both methods were consistent (RSD<15% by the derivatization method and<10% by the CSPs method) and linear (r>9950). Compared to the pre-column derivatization method, the CSPs method had lower quantification limits (10.6/9.6ng/mL of (+)-/(−)-naftopidil by derivatization method and 1.1/1.8ng/mL of (+)-/(−)-naftopidil by CSPs method), and was simpler to carry out. The validated CSPs method was successfully applied in a pharmacokinetic study of naftopidil enantiomers in rats, which showed that pharmacokinetic parameters of (+)- and (−)-NAF after intravenous administration of (±)-NAF were similar.