Determination of Sparfloxacin in Human Urine by Reversed-Phase High Performance Liquid Chromatography With Nitrous Acid and Hydroiodic Pre-Column Derivatization

Sparfloxacin can be oxidized by nitrous acid, then react with hydroiodic acid to form a fluorescent derivative. Based on this, a reversed-phase high performance liquid chromatographic pre-column derivatization new method is described for the determination of sparfloxacin in human urine. The linear range is 0.05 mg/L to 4.0 mg/L, the recoveries are 91.5%similar to 95.7% and the RSD is 1.2%similar to4.2%. The results showed that this method is suitable for the determination of sparfloxacin in human urine.

Determination of 18 Kinds of Amino Acids in Fresh Tea Leaves by HPLC Coupled with Pre-column Derivatization

A rapid and accurate quantitative method of high performance liquid chromatography( HPLC) with fluorescence detector has been developed for the analysis of 18 kinds of amino acids in fresh tea leaves. The samples were minced and mixed,and extracted with ultra pure water at 90℃ for 20 min. The 6-aminoquinolyl N-hydroxy-succinimidyl carbamate( AQC) was used as pre-column derivatization reagent. Gradient HPLC separation was performed on a C_(18) column( Symmetry C_(18),3. 9 mm × 15 cm,4 μm). Good linearity between concentrations and peak areas was achieved in the concentration range of 5. 0-250 μmol/L for 18 kinds of amino acids. The method was validated by the analysis of five replicates. The 18 kinds of amino acid standards were spiked in fresh tea leaf samples and the average recovery rate was 86. 25%-109. 05% with relative standard deviations( n = 5) ranging from 6. 03% to 10. 56%. The limit of detection( LOD) for the analytes was0. 05-1. 27 μmol/L. The method was successfully applied to the analysis of the 18 kinds of amino acids in fresh tea leaves from east Dongting and west Dongting mountains in Suzhou. The results indicate that the method is simple,rapid,precise and reliable.

Pre-column Derivatization-High Performance Liquid Chromatography for the Detection of Monensin in Livestock and Poultry Meat

[Objectives]A method for the detection of monensin in poultry and livestock meat by pre-column derivatization-high performance liquid chromatography was established.[Methods]The sample was extracted with chloroform,derivatized with trichloroacetic acid and 2,4-dinitrophenylhydrazine,and centrifuged to obtain a purified solution.A C18 chromatographic column(4.6 mm×150 mm,5μm)was used for separation with(1.5%)acetic acid water∶methanol(volume ratio)=1∶9 as the mobile phase using a DAD detector for detection,and the external standard method was adopted for peak area quantification.[Results]Monensin had good linearity in the concentration range of 5.00-200 mg/L,with the linear correlation coefficient r 2>0.999;the detection limit was 5.00 mg/kg;the relative standard deviation was smaller than 10%;and the recoveries of standard addition experiment were in the range of 75%-110%.[Conclusions]The method has the advantages of simple pretreatment operation,good derivatization effect and fast detection speed,and is suitable for detecting monensin in poultry and livestock meat.

Development of Simultaneous HPLC-Fluorescence Assay of Phenol and Chlorophenols in Tap Water after Pre-Column Derivatization with 3-Chlorocarbonyl-6,7-dimethoxy-1- methyl-2(1H)-quinoxalinone

Chlorophenols (2-chlorophenol, 4-chlorophenol, 2,4-dichlorophenol, 2,6-dichlorophenol and 2,4, 6-trichlorophenol) may be presented in natural waters or drinking water as a result of disinfection processes involving chlorination, or as contaminants derived from domestic products, industrial operations and agricultural chemicals. A previous HPLC-UV method for determination of phenol and five chlorophenols in tap water using 4-fluoro-7-nitro-2,1,3-benzoxadiaole as a UV labeling reagent shows limited sensitivity. Here, we present an improved HPLC-fluorescence detection method for simultaneous determination of phenol and the above chlorophenols in tap water after pre-column derivatization with 3-chlorocarbonyl-6,7-dimethoxy-1-methyl-2(1H)-quino- xalinone (DMEQ-COCl), using a short, narrow column (50 × 2.1 mm i.d., packed with 5 μm particles of C18 material) to improve the sensitivity. Standard samples containing the compounds are derivatized with DMEQ-COCl in borate buffer (pH 9.0) at room temperature for 3 mins. The response is linear in the concentration range of 0.01 - 0.05 to 0.5 mg/L with r2 values ≥0.9967 for all compounds. The lower limits of detection are 0.001 to 0.008 mg/L, and the coefficients of variation are less than 8.8%. The recovery values from tap water spiked with standard samples are satisfactory. The present method is suitable for examining whether or not tap water samples are contaminated with phenol and chlorophenols in excess of regulatory values.

Simple HPLC-Fluorescence Determination of Raspberry Ketone in Fragrance Mist after Pre-Column Derivatization with 4-Hydrazino-7-nitro-2,1,3-benzoxadiazole

Raspberry ketone {RK, 4-(4-hydroxyphenyl)butan-2-one} is a natural compound contained in raspberry, and is added to cosmetics for skin whitening. It is very important to measure the RK level in cosmetics for quality assessment, since RK structurally resembles 4-(4-hydroxyphenyl)-2-butanol, which causes leukoderma on consumers’ skin. Here, we present a simple HPLC-fluorescence method for determination of RK in a fragrance mist by pre-column derivatization with 4-hydrazino-7-nitro-2,1,3-benzoxadiazole hydrazine (NBD-H), which reacts with the carbonyl group of RK. The NBD-RK derivative was eluted from a reversed-phase ODS column, and detected with excitation at 470 nm and emission at 550 nm. The retention time of NBD-RK derivative obtained by reaction with NBD-H at 80°C for 20 min was 10.3 min. The standard curve was linear in the range of 0.2 to 10 μg/mL, with a correlation coefficient (r2) value of 0.9980. The lower limit of detection was 0.018 μg/mL (absolute amount of 1.8 pmol). The coefficients of variation were less than 8.1%. The content of RK in fragrance mist (1.00 mL) was 1.18 ± 0.07 mg (range: 1.12 to 1.28 mg, n = 5). Recovery tests were satisfactory (83.9% ± 3.9%;range: 79.6 to 88.8%, n = 5).

Enantioresolution of a Series of Chiral Benzyl Alcohols by HPLC on a Dinitrobenzoylphenylglycine Stationary Phase after Achiral Pre-Column Derivatization

High performance liquid chromatography method for the separation of a series of chiral benzyl alcohols on N-(3,5-dinitrobenzoyl)-D-phenylglycine stationary phase (Macherey Nagel, Chiral-2) after pre-column achiral derivatization was developed. Cheap and easy available aromatic acid chlorides were used as derivatization agents. Good to excellent separations of the enantiomers were achieved in all cases in relatively short analytical runs. It was shown that the enantiorecognition depends on the substituents both in the starting alcohol and in the acid chloride. The method presents an efficient alternative to the direct analyses on polysaccharide and cyclodextrine-derived stationary phases.

Simple HPLC–UV Analysis of Phenol and Its Related Compounds in Tap Water after Pre-Column Derivatization with 4-Nitrobenzoyl Chloride

The purpose of this study is to develop an HPLC-UV (280 nm) method for simultaneous determination of phenol, five chlorophenols (2-chlorophenol, 4-chlorophenol, 2,4-dichlorophenol, 2,6-dichlorophenol, and 2,4,6-trichlorophenol), and three phenylphenols (2-phenylphenol, 3-phenylphenol, and 4-phenylphenol) in tap water after pre-column derivatization with 4-nitrobenzoyl chloride. Standard curves were obtained after derivatization with 4-nitrobenzoyl chloride in borate buffer (pH 8.5) at 50°C for 1 min. The nine 4-nitrobenzoyl derivatives were well separated in less than 15 min on a Cholester column. Calibration plots were linear in the range of 0.02 ~ 0.12 to 0.9 mg/L, with r2 values ≥0.9928, for all compounds. The lower limits of detection were 0.006 to 0.05 mg/L. The coefficients of variation were less than 12.0%. The recovery values from tap water spiked with a standard mixture of test compounds were satisfactory. While the levels of phenol, five chlorophenols, and three phenylphenols in tap water were below the lower limit of determination, our method is expected to be useful for monitoring and/or identifying environmental water samples that are contaminated with these compounds, i.e., for assessing compliance with the official guidelines of the World Health Organization.

Contents Variation Analysis ofγ-Amino Butyric Acid in Semen sojae praeparatum Fermentation Using Online Pre-Column Derivatization-HPLC

This paper reported the contents variation analysis ofγ-amino butyric acid(GABA)in Semen sojae praeparatum(SSP)which is a famous traditional Chinese medicine.High performance liquid chromatography(HPLC)was used and GABA was derivatized by online pre-column derivatization with o-phthalaldehyde(OPA).To validate this method,the precision,stability,repeatability and recovery were discussed.In the concentration range from 0.0125 to 0.400 mg/m L,the calibration curve for GABA was linear and the regression equation was obtained with correlation coefficient(R2)of 0.9999.Relatively high levels of GABA exist in SSP and the content changes of GABA at different time points during the fermenting process were detected.At the"yellow cladding"stage,GABA level was very low or even undetectable;the"secondary fermentation"stage witnessed a rapid increase of GABA content to 1.39-5.52 mg/g,which remained stable after 18 days of"secondary fermentation".This study demonstrated that GABA was generated at the"secondary fermentation"stage,revealing the significance and rationality of the"secondary fermentation"stage in the fermenting process of SSP.On the other hand,it suggested the downside of taking soy isoflavones as the only measurement in existing quality assessment and optimization approach for the fermenting process of SSP.

DMMIC derivatization-assisted liquid chromatography-mass spectrometry method for metabolite profiling of the glutathione anabolic pathway in esophageal cancer tissues and cells

In this work,a new pyrylium derivatization-assisted liquid chromatography-mass spectrometry(LC-MS)method was developed for metabolite profiling of the glutathione anabolic pathway(GAP)in cancer tissues and cells.The pyrylium salt of 6,7-dimethoxy-3-methyl isochromenylium tetrafluoroborate(DMMIC)was used to label the amino group of metabolites,and a reductant of dithiothreitol(DTT)was employed to stabilize the thiol group.By combining DMMIC derivatization with LC-MS,it was feasible to quantify the 13 main metabolites on the GAP in complex biological samples,which had good linearity(R^(2)=0.99810.9999),precision(interday precision of 1.6%e19.0%and intraday precision of 1.4%e19.8%)and accuracy(83.4%-115.7%).Moreover,the recovery assessments in tissues(82.5%e107.3%)and in cells(98.1%e118.9%)with GSH-^(13)C2,^(15)N,and Cys-^(15)N demonstrated the reliability of the method in detecting tissues and cells.Following a methodological evaluation,the method was applied successfully to investigate difference in the GAP between the carcinoma and para-carcinoma tissues of esophageal squamous cell carcinoma(ESCC)and the effect of p-hydroxycinnamaldehyde(CMSP)on the GAP in KYSE150 esophageal cancer cells.The results demonstrate that the developed method provides a promising new tool to elucidate the roles of GAP in physiological and pathological processes,which can contribute to research on drugs and diseases.

High Performance Liquid Chromatography for the Determina- tion of Metoclopramide in Pharmaceutical Preparations Using Pre-column Derivatization with Fluorescamine

A simple, rapid and sensitive high performance liquid chromatography （HPLC） method was developed for the determination of metoclopramide in pharmaceutical preparation. The method is based on the derivatization of metoclopramide with fluorescamine. The separation was achieved on a C18 column using methanol-water （70 ： 30, V/V） mobile phase. Fluorescence detector was used at the excitation and emission of 403 and 485 nm, respectively. The method was validated for linearity, limit of detection, limit of quantification, precision, accuracy, recovery, robust- ness and system suitability. The assay was linear over the concentration range of 100-2000 ng/mL. The mean recovery was 100.37%. The proposed method was successfully applied to the assay of metoclopramide in tablet preparation. The preparation was also analyzed with an official method and statistical comparison by t- and F-tests revealed that there was no significant difference between the results of the two methods with respect to mean values and standard deviations at the 95% confidence level.

Polarity-regulated derivatization-assisted LC-MS method for amino-containing metabolites profiling in gastric cancer

Amino-containing compounds,including amino acids,aliphatic amines,aromatic amines,small peptides and catecholamines,are involved in various biological processes and play vital roles in multiple metabolic pathways.Previous studies indicated that some amino-containing metabolites are significant diagnostic and prognostic biomarkers of gastric cancer.However,the discovery of precise biomarkers for the preoperative diagnosis of gastric cancer is still in an urgent need.Herein,we established a polarityregulated derivatization method coupled with liquid chromatography-mass spectrometry(LC-MS)for amino-containing metabolites profiling in the serum samples of patients with gastric cancer and healthy controls,based on our newly designed and synthesized derivatization reagent(S)-3-(1-(diisopropoxyphosphoryl)pyrrolidine-2-carboxamido)-N-hydroxysuccinimidyl ester(3-DP-NHS).Enhanced separation efficiency and detection sensitivity for amino-containing metabolites were achieved after derivatization.This method exhibited good linearity,recovery,intra-and inter-day precision and accuracy.Only 5 mL serum is needed for untargeted analysis,enabling 202 amino-containing metabolites to be detected.Statistical analysis revealed altered amino acid metabolisms in patients with gastric cancer.Furthermore,ultra high performance liquid chromatography coupled with mass spectrometry(UHPLCMS/MS)analysis quantification revealed increased serum levels of tryptamine and decreased concentrations of arginine and tryptophan in patients with gastric cancer.Receiver operating characteristic(ROC)curves indicated that an increased tryptamine/tryptophan ratio could serve as a potential biomarker for gastric cancer diagnosis.This study demostrated the possibility of using serum amino acid biomarkers for gastric cancer diagnosis,providing new avenues for the treatment of gastric cancer.

Speciation of organomercury compounds by capillary electrophoresis with pre-column derivatization and on-line stacking

Simultaneous separation and detection of three organomercury species, namely methylmercury(MeHg),ethylmercury(EtHg), and phenylmercury(PhHg), was performed by using capillary electrophoresis(CE)with UV detection. Pre-column derivatization with thiosalicylic acid and on-line salt-induced stacking significantly improved the detection performance. Buffer pH, ion strength, and additive were optimized for CE separation, concentration of NaCl in sample solution and injection time were optimized for on-line stacking. The limits of detection were 76.9,83.0 and 76.4 μg/L for PhHg, EtHg and MeHg, respectively. The developed method was validated by certified reference material and liquid chromatography-atomic fluorescence spectroscopy, which suggests this method could be useful in the speciation of organomercury compounds in biological samples.

HPLC of Amino Acids and Oligopeptides by Pre-Column Fluorescence Derivatization with 9-Acridine Formyl Chloride

A highly sensitive HPLC method for the detection of amino acids and oligopeptides with 9-acridine formyl chloride by pre-column fluorescence derivatization has been developed. Glycine, glycylglycine, histidine, triglycine and glutathione were separated on a reversed-phase C-18 column with methanol-water-triethylamine eluent, derivatization and chromatographic conditions were optimized. The five derivatives were eluted in 28 min with a good reproducibility. Linear range of the calibration graph was 0.08-260 nmol/ml(-1). The relative standard deviations(n=6) are < 5%. Detection limits (signal-to-noise ratio=3) for the five derivatives are 20-40 fmol.

Liquid Chromatography Detertmination of Amino Acids by Pre-Column Fluorescence Derivatization with Acridone-N-acetyl Chloride

A sensitive liquid chromatographic mcthod for the detection of andno acids with precolumn fluorescence derivatization with acridone-N-acetyl chloride has been developed. Andno acid derivatives were eluted in 4o ndn with good reproducibility. The reIative standard deviations (n=6) at an analytical concentration of 50 pmol are < 4%. The method described is also suitable for the analysis of andno acids in different biological samples.

HPLC Determination of Amino Acids by Pre-Column Fluorescence Derivatization with Carbazole-9-yl-Acetyl Chloride

A sensitive HPLC method for the detection of amino acids with pre-column fluorescence derivatization with carbazole-9-yl-acetyl chloride has been developed. This method, in conjunction with a multi-gradient program, offers baseline resolution of the common CRAC-amino acids from a linear acetonitrile gradient. Separation of amino acid derivatives was obtained on a reversed-phase C-18 column. Derivatization and chromatographic conditions were optimized. Amino acid derivatives were eluted in 40 min with good reproducibility. The relative standard deviations (n=6) at an analytical concentration of 100 pmol are < 4%. The methods described are also suitable for the analysis of amino acids in different biological samples.

Precolumn derivatization LC-MS/MS method for the determination and pharmacokinetic study of glucosamine in human plasma and urine

A selective precolumn derivatization liquid chromatography–tandem mass spectrometric (LC–MS/MS) method for the determination of glucosamine in human plasma and urine has been developed and validated. Glucosamine was derivatized by o-phthalaldehyde/3-mercaptopropionic acid. Chromatographic separation was performed on a Phenomenex ODS column (150 mm 4.6 mm, 5 mm) using linear gradient elution by a mobile phase consisting of methanol (A), and an aqueous solution containing 0.2% ammonium acetate and 0.1% formic acid (B) at a flow rate of 1 mL/min. Tolterodine tartrate was used as the internal standard (IS). With protein precipitation by acetonitrile and then the simple one-step derivatization, a sensitive bio-assay was achieved with the lower limit of quantitation (LLOQ) as low as 12 ng/mL for plasma. The standard addition calibration curves suitable for clinical sample analysis showed good linearity over the range of 0.012–8.27 mg/mL in plasma and 1.80–84.1 mg/mL in urine. The fully validated method has been successfully applied to a pharmacokinetic study of compound glucosamine sulfate dispersible tablets in health Chinese volunteers receiving single oral doses at 500, 1000 and 1500 mg of glucosamine sulfate, as well as multiple oral doses of 500 mg t.i.d. for 7 consecutive days.

Determination of Content of Amino Acid in Abalone by Precolumn Derivatization and its Nutritional Value Evaluation

[Objectives] This study was conducted to optimize the determination conditions of amino acids from abalone. [Methods] The sample was treated by acid hydrolysis method and subjected to 2,4-2 nitro fluorobenzene column derivatization. The amino acid content in abalone was determined by HLPC,and the nutritional value of the amino acids was evaluated with egg protein model put forward by Institute of Nutrition and Food Hygiene,Chinese Academy of Preventive Medicine. [Results] Abalone contains full amino acids. According to the FAO/WHO ideal,it is a high-quality protein source and suitable for supplement of protein source for human body. [Conclusions]The experimental method has simple operation and could achieve a good effect with wide linear range and correlation coefficient over 0. 999 8,and the obtained results are satisfactory.

Ionic Liquid as Useful Media for Dissolution, Derivatization, and Nanomaterial Processing of Chitin

This paper reviews studies on dissolution, derivatization, and nanomaterial processing of chitin using an ionic liquid as useful media. Because chitin is the second most abundant polysaccharide on the earth after cellulose, there is major interest in conversion of native chitin resources into various useful materials after proper dissolution in suitable solvents. For the derivatization and nanomaterial processing of chitin, the author has been focusing on ionic liquids because which have been found to be used as good solvents for cellulose in a past decade. The author found that an ionic liquid, 1-allyl-3-methylimidazolium bromide (AMIMBr), dissolved chitin in concentrations up to 4.8 wt% and mixtures of the higher amounts of chitin with AMIMBr gave ion gels. Acetylation, the simplest derivatization, of chitin using acetic anhydride was achieved in the AMIMBr solvent under mild conditions. Furthermore, the chitin nanofibers were fabricated by regeneration technique from the chitin ion gel with AMIMBr using methanol. Moreover, filtration of the chitin nanofiber dispersion with methanol was carried out to give a chitin nanofiber film. The chitin nanofiber-poly(vinyl alcohol) composite film was also prepared from the ion gel by co-regeneration method.

Isolation, identification, derivatization and phytotoxic activity of secondary metabolites produced by Cladosporium oxysporum DH14, a locust-associated fungus

Three main phytotoxic compounds including lunatoic acid A （1）, 5Z-7-oxozeaenol （2） and zeaenol （3） were isolated from the fermentation broth of Cladosporium oxysporum DH14, a fungus residing in the locust （Oxya chinensis ） gut. Two additional derivative compounds, compound la and lb, were synthesized by methylation and chlorination of compound 1, respectively. The structures of such compounds were identified on the basis of spectroscopic analysis and by comparison of the corresponding data to those previously reported in the literature. Compounds 1-3 exhibited significantly phytotoxic activities against the radicle growth of Amaranthus retroflexus L. with the 50% inhibitory concentrations （ICso values） of 4.51, 4.80 and 8.16 μg· mL^-1, respectively, which is comparable to that positive control 2,4-dichlorophenoxyacetic acid （IC50=1.95 μg· mL^-1）. Furthermore, the compound 1 showed selective phytotoxic activity with the inhibition rate of less than 22% against the crops of Brassica rapa L., Sorghum durra, Brassica campestris L,, Capsicum annucm and Raphanus safivus L. under the concentration of 100 μg· mL^-1 Both derivatives of compound 1 had moderate phytotoxic activity against the radicle growth of A. retroflexus L. The findings of our present study suggest that these compounds provide new promising candidates for the potential management strategies of weeds.

Quantitative‑Profling Method of Serum Steroid Hormones by Hydroxylamine‑Derivatization HPLC-MS

A sensitive and rapid high performance liquid chromatography-mass spectrometry(HPLC-MS)method was developed and validated for simultaneous quantifcation of ten steroid hormones,including estrogens,androgens,progesterones,and corticosteroids four classes of steroids.The following ten steroid hormones were analyzed:progesterone,21-deoxycortisol,estrone,4-androstenedione,testosterone,dihydro-testosterone,androstenone,dehydroepiandrosterone,corticosterone and cortisone.Stable deuterated isotopes were used as internal standards for quantifcation.Sample preparation with and without derivatization were performed after liquid-liquid extraction,and the corresponding results were compared according to sensitivity and selectivity.Hydroxylamine derivatization was found to improve the ionization efciency of the analytes for electrospray ionization MS analysis.The gradient of mobile phase and experimental parameters for HPLC separation were optimized.The lower limits of quantifcation were in the range of 0.05-5 ng mL^(−1) with wide linear range for the ten steroid hormones.The intra-day precision<11.1%and recovery of 84.5-120% with negligible matrix efect were achieved,where within the acceptance limits of the FDA guideline.Total HPLC-MS analysis time was 6 min.This method enables simultaneous quantifcation of steroids in human serum.It will be helpful for the serum steroid profling in order to understand various endocrinology diseases.