Analysis of formose sugar and formaldehyde by high-performance liquid chromatography

Formose sugar and formaldehyde (HCHO) were analyzed using high-performance liquid chromatography (HPLC) utilizing a CarboPac PA1 column (Dionex) and pulsed amperometric detection. This HPLC system was unsuitable for the analysis of formose sugar and HCHO and thus reducing sugars and unconverted HCHO were determined by endowing them with charges through a derivatization method using 2,4-dinitrophenylhydrazine. The separation and detection of compounds were performed by three Chromolith RP-C18 columns (Merck) and diode array detection, at a wavelength of 360 nm ultraviolet light, respectively. Lower sugars (except HCHO) showed some instabilities when the derivatized samples were kept for the extended periods of time. For C₅ and consecutive higher sugars, a certain derivatization time was necessary. In the present case (formose reaction with partial HCHO conversion), approximately 18 h may be a reasonable compromise for the derivatization reaction. A derivatization agent to compound mole ratio of up to 100:1 was required to complete the derivatization of C₄ and higher sugars. However, the analysis of C₄ and consecutive higher sugars is problematic for example due to overlapping of peaks or branched-chain sugars.     

A novel approach for enantioseparation as applied to (RS)‐etodolac from pharmaceutical formulations: LC MS and density functional theory support for confirmation of diastereomers so separated

In the present studies formation of diastereomers of (RS)‐etodolac was confirmed using LC‐MS when [M + H]⁺ or [M]⁺ were recorded for the diastereomers. The lowest energy optimized structures of two diastereomers were drawn, which confirmed the three‐dimensional geometry of the diastereomers. This supports the optimized analytical separation conditions. In addition, separation of diastereomers was successful using a C₁₈ column and a binary mixture of methanol and triethyl ammonium phosphate buffer of pH 4.5 (80:20, v/v) as mobile phase at a flow rate of 1 mL min^?1 and UV detection at 223 nm. The separation method was validated as per International Conference on Harmonization guidelines. (RS)‐Etodolac was isolated from commercial tablets and purified and characterized to be used as racemic standard. Three pairs of diastereomers were synthesized using enantiomerically pure amines, namely, (R)‐(+)‐α‐methyl benzyl amine, (S)‐(?)‐α,4‐dimethylbenzylamine and (R)‐(?)‐1‐cyclohexylethylamine. Derivatization reactions were carried out under conditions of stirring at room temperature (30 °C for 2 h) as well as under microwave irradiation (MWI), and the two types of diastereomers were compared. Reaction conditions for derivatization were optimized with respect to mole ratio of chiral derivatizing agent and (RS)‐etodolac and MWI time. No racemization was observed throughout the study. Copyright © 2015 John Wiley & Sons, Ltd.     

Quantitative TLC Analysis of Sterol (24β-Ethylcholesta-5,22E,25-triene-3β-ol) in Agnimantha (Clerodendrum phlomidis Linn)

A quantitative method using silica gel 60F₂₅₄ high performance thin layer chromatography plates, automated bandwise sample application, and automated visible mode densitometric method has been developed for the determination of 24β-ethylcholesta-5,22E,25-triene-3β-ol (ECTO) in the aerial part of Clerodendrum phlomidis. ECTO was used as a chemical marker for the standardization of C. phlomidis plant extracts. The separation was performed on silica gel 60F₂₅₄ TLC plates using chloroform-methanol (98.5: 1.5, v/v) as mobile phase. The quantitation of ECTO was carried out using the densitometric reflection/absorption mode at 650 nm after post chromatographic derivatization with anisaldehyde reagent. A precise and accurate quantification can be performed in the linear working concentration range of 150–400 ng band⁻¹ with good correlation (r ² = 0.996). The method was validated for peak purities, precision, robustness, limit of detection (LOD) and quantitation (LOQ), etc. as per ICH guidelines.     

Capillary zone electrophoresis of basic drugs in non-aqueous acetonitrile with buffers based on a conventional pH scale

Summary The pK _a ^* values of 10 nitrogen-containing basic drugs in non-aqueous acetonitrile were determined from the pH^* dependence of their electrophoretic mobilities. The pH^* scale in the organic solvent was established using background electrolytes with known conventional pK _a ^* values, making further calibration with reference pH electrodes unnecessary. In acetonitrile the pK _a ^* values of analytes (or their conjugated cation acids, BH⁺, respectively) were 5.2±8.9 pK units>those in water. The observed change in pK _a ^* values of cationic analytes was, however, much less than the known respective change for neutral acids type HA. From the pK _a ^* values and the actual mobilities, it is possible to predict pH^* conditions to enable separation of analytes, and this was demonstrated for two pairs of common drugs.     

Determination of galacturonic acid content in pectin from fruit juices by liquid chromatographydiode array detection-electrospray ionization tandem mass spectrometry

A reversed-phase high-performance liquid chromatographic (HPLC) method is applied for the determination of galacturonic acid (GA) of pectins in different commercial fruit juices. The separation was carried out on a C₁₈ column using precolumn derivatization with p-aminobenzoic acid (p-ABA) and UV detection at 304 nm. The identification of GA was confirmed by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) in positive ion mode. The concentration of GA in the samples analyzed ranged from 12.9 ± 0.5 to 49.4 ± 0.5 mg_GA L⁻¹. Amongst the samples analyzed, mango juice was found to be richest in GA content, and therefore a good source of pectins. Detection and quantification limits of the described methodology were 1.2 and 3.9 mg L⁻¹, respectively. Quantitative GA recoveries in the beverages had a range between 90 and 98%. The results showed that the HPLC method proposed was precise and suitable for the identification and quantification of GA in commercial fruit juices.     

Determination of Free Fatty Acids in Bryophyte Plants and Soil by HPLC with Fluorescence Detection and Identification by Online MS

A method for the determination of long and short chain free fatty acids (FFAs), using 1-[2-(p-toluenesulfonate)-ethyl]-2-phenylimidazole-[4,5-f]-9,10-phenanthrene (TSPP) as labeling reagent, has been developed. Identification of FFA derivatives was carried out by HPLC-MS with atmospheric pressure chemical ionization (APCI) in positive ion mode. Gradient elution on an Agilent Eclipse XDB-C₈ column gave good separation of the derivatives. Excellent linear responses were observed and good compositional data could be obtained from as little as 200 mg of bryophyte plants and soil samples. Facile TSPP derivatization coupled with HPLC-APCI-MS analysis allowed the development of a highly sensitive method for the quantitative analysis of trace level of FFAs from biological and natural environmental samples.     

Capillary zone electrophoresis with pre-column NDA derivatization and amperometric detection for the analysis of four aliphatic diamines

A method was developed for the analysis of four aliphatic diamines by capillary zone electrophoresis using pre-column derivatization with naphthalene-2,3-dicarboxaldehyde (NDA)/CN⁻ and amperometric detection. The pre-column derivatization reaction conditions including the molar ratio of NDA to amines, the cyanide concentration, the pH value of derivatization buffer, and the reaction time, were investigated. The separation of four derivatives of aliphatic diamines has been optimized by capillary zone electrophoresis (CZE) using end-column amperometric detection with a carbon fiber microelectrode, at a constant potential of 0.7 V versus SCE. The optimum conditions for the separation were 10 mM Tris-H₃PO₄ (pH 4.0) for the running buffer solution, 15 kV for the separation voltage. The detection limits for diaminopropane, putrescine, cadaverine, diaminohexane were 6.7×10⁻⁸, 5.1×10⁻⁸, 1.9×10⁻⁷ and 3.8×10⁻⁷ M, respectively (S/N=3). The proposed method was applied to the determination of aliphatic diamines in a lake water sample by the standard addition method. The recovery of these amines in water was 89.9-107%.     

Analysis of Phytochelatins and Other Thiol-Containing Compounds by RP-HPLC with Monobromobimane Precolumn Derivatization

In this article, a method for quantitative determination of phytochelatins (PC _n being the classic example) and other thiol-containing compounds in mixed standard solution and plant tissues is presented. Thiols were converted to fluorescent derivatives by precolumn derivatization with monobromobimane. The results showed that PC _n and other thiol-containing compounds in standard mixed solutions were rapidly separated within 15 min by using a ACN 0.1% trifluoroacetic acid binary gradient elution. Glutathione was representatively selected to test the precision of this method. The calibration curve was linear in the range of 1.25–160 ng μl⁻¹ (regression coefficient r ²=0.9999). It was confirmed that this method was rapid, simple, highly sensitive, stable, and had the property of simultaneous determination of PC _n and other thiol-containing compounds. This method was applied to determine PC _n and other thiol-containing compounds in a Cd hyperaccumulator Sedum alfredii in response to Cd. It was found that no PC _n was detected in any tissue at any Cd treatment, suggesting that Cd hyperaccumulation and detoxification in this plant is not based on PC synthesis. Translated from Journal of Nanjing University (Natural Science Edition), 2005, 41(3) (in Chinese)     

铂纳米颗粒的可见光化学制备及其对p-硝基苯酚催化还原反应

在可见光照射下,以乙二醇（EG）作为还原剂和稳定剂,在多壁碳纳米管（MWCNTs）上一步合成了铂纳米颗粒,成功制备Pt/MWCNTs复合材料,并通过p-硝基苯酚（p-NP）的催化还原反应研究了Pt/MWCNTs的催化性能。用傅里叶变换红外光谱（FT-IR）、X射线衍射（XRD）、透射电子显微镜（TEM）和X射线光电子能谱（XPS）对所制备材料的形貌和晶体结构进行了表征。实验结果显示,可见光照射促进了EG水溶液中[PtCl₄]^2-前驱体的水解。通过金属界面的电子效应,铂前驱体被还原成了均匀分散的平均直径2.1 nm的超小颗粒Pt（Pt ultra-small particles,Pt UPs）。所制备的Pt/MWCNTs能有效地催化p-NP还原为p-氨基苯酚（p-AP）,表现出较高的催化性能,其表观速率常数为0.25 min^-1。Pt/MWCNTs多次使用后没有显著的活性损失,显示出了良好的稳定性。上述实验结果证明,除了传统的紫外光照射等手段以外,可见光照射也同样是制备铂金属催化剂非常有效的方法。而且,催化剂的形貌控制也完全可以通过简单而非复杂的实验条件加以实现。     

Simultaneous Analysis of Zofenopril and Its Active Metabolite Zofenoprilat in Human Plasma by LC–ESI-MS Using Pre-Column Derivatization with p-Bromophenacyl Bromide

Zofenoprilat is an active metabolite of zofenopril, which is very unstable in plasma because of oxidative degradation of its thiol group. In this method, p-bromophenacyl bromide was used as derivatization reagent, immediately after plasma separation, to react with the free thiol group of zofenoprilat and form the derivative zofenoprilat-p-BPB. After acidification with 50% acetic acid, the derivatized plasma samples were extracted with methyl tert-butyl ether and separated on a C₁₈ column with 40:60 (v/v) 10 mM ammonium acetate buffer solution containing 0.1% formic acid–acetonitrile as mobile phase. Calibration plots were linear over the concentration range 1–500 ng mL⁻¹ for zofenopril and 2–1,800 ng mL⁻¹ for zofenoprilat. The method was successfully used to study the bioavailability of zofenopril calcium capsules relative to that of zofenopril calcium tablets in healthy Chinese volunteers.

     

Determination of Aldoses, Deoxy-aldoses and Uronic Acids Content in a Pectin-Rich Extract by RP-HPLC-FLD after p-AMBA Derivatization

A reversed-phase high-performance liquid chromatographic method with fluorometric detection is proposed for the simultaneous determination of different classes of neutral sugars, such as hexoses (galactose, glucose and mannose), pentoses (arabinose and xylose), deoxy-hexoses (fucose and rhamnose), as well as acidic sugars (galacturonic and glucuronic acids). The separation is carried out on a hydrophilic end capped C₁₈ column following a pre-column derivatization with p-aminobenzoic acid. The fluorometric detection of the derivatives has shown a strong dependency with the mobile phase pH. The performance of the proposed methodology was evaluated and the prerequisites of linearity (r-value > 0.999), precision (intra-day CV < 6 % and inter-day CV < 11 %) and recovery (between 77 ± 7 and 103 ± 3 %) were satisfied. To our knowledge, the obtained values of limit of detection for neutral sugars (within the range 6.1–28 μg L^?1) are the lowest reported using this derivatizing agent. In order to better judge the methodology presented herein, neutral sugars of a pectin-rich orange extract were also analysed by the conventionally used GC-FID (gas-chromatography with flame ionization detector) method of alditol acetate derivatives. A statistical test (paired t test) has proved that no significant differences (α = 0.05) were observed between these two methods.     

Determination of biogenic amines in fermented beverages and vinegars by pre-column derivatization withpara-nitrobenzyloxycarbonyl chloride (PNZ-Cl) and reversed-phase LC

Summary The reagentp-nitrobenzyloxycarbonyl chloride (PNZ-CI) was used for pre-column derivatization of biogenic amines (BAs) at ambient temperature followed by reversed-phase, liquid-chromatographic separation of the derivatives. Optimized derivatization of samples was achieved within 10 min in borate buffer by adding PNZ-Cl in acetonitrile (MeCN). Excess reagent was scavenged by subsequent addition of glycine in water. For LC a Superspher^? RP-18e column and gradient elution using a ternary gradient system containing sodium acetabe buffer (pH 6.1), sodium acetate buffer (pH 4.3) and MeCN, were used. The PNZ-derivatives were quantified by their UV-absorption at 265 nm. Detection limits of BAs were approximately 62–1000 μg L⁻¹ (injected amounts: 53–850 pg) at a signal-to-noise ratio of 3:1. The coefficients of determination were 0.9906–0.9992. Diaminohexane was used as internal standard. Recoveries of BAs ranged from 78–93% depending on the food matrix. This method was applied to the quantitative determination of 2-phenylethylamine, tryptamine, serotonin, putrescine, histamine, cadaverine, tyramine, spermidine, and spermine, in beer, wine, vinegars, and lactic fermented cabbage juice. Parts of the results were presented at the 35th Congress of the “Deutsche Gesellschaft für Ern?hrung”, Kiel, 19^th–20^th March 1998, and the “Regionaltagung der Lebensmittelchemiker”, Giessen, 9^th–10^th March 1998     

Ion chromatography with the indirect ultraviolet detection of alkali metal ions and ammonium using imidazolium ionic liquid as ultraviolet absorption reagent and eluent

Indirect ultraviolet detection was conducted in ultraviolet‐absorption‐agent‐added mobile phase to complete the detection of the absence of ultraviolet absorption functional group in analytes. Compared with precolumn derivatization or postcolumn derivatization, this method can be widely used, has the advantages of simple operation and good linear relationship. Chromatographic separation of Li⁺, Na⁺, K⁺, and NH₄⁺ was performed on a carboxylic acid base cation exchange column using imidazolium ionic liquid/acid/organic solvent as the mobile phase, in which imidazolium ionic liquids acted as ultraviolet absorption reagent and eluting agent. The retention behaviors of four kinds of cations are discussed, and the mechanism of separation and detection are described. The main factors influencing the separation and detection were the background ultraviolet absorption reagent and the concentration of hydrogen ion in the ion chromatography‐indirect ultraviolet detection. The successful separation and detection of Li⁺, Na⁺, K⁺, and NH₄⁺ within 13 min was achieved using the selected chromatographic conditions, and the detection limits (S/N = 3) were 0.02, 0.11, 0.30, and 0.06 mg/L, respectively. A new separation and analysis method of alkali metal ions and ammonium by ion chromatography with indirect ultraviolet detection method was developed, and the application range of ionic liquid was expanded.     

Simultaneous determination of dihydroxybenzene and phenylenediamine positional isomers using capillary zone electrophoresis coupled with amperometric detection

In general capillary zone electrophoresis (CZE) separation models, o‐, m‐, and p‐phenylenediamine isomers can be separated in a weak acidic running buffer for their pK_a values being 4.52, 5.64, 6.04, respectively, while o‐, m‐, and p‐dihydroxybenzene isomers can be separated in a weak basic buffer for their pK_a values being 9.40, 9.40 and 10.04, respectively. So, it is hard to find a suitable running buffer at a fixed pH in normal CZE for simultaneous separation of these two groups of positional isomers. In this paper, a novel method based on alternately running two different pH buffers in CZE coupled with amperometric detection (CZE‐AD) was designed to simultaneously determine these two groups of positional isomers. It is found that when two different pH running buffers were employed alternately under appropriate order and time, the six analytes could be separated perfectly in less than 20 min and the detection limits were as low as 10^–7 mol/L. Furthermore, the effects of working electrode potential, pH and concentration of running buffer, separation voltage and injection time on CZE–AD were investigated. Experimental results demonstrated that the introduced method was practical to simultaneously determine two groups of positional isomers with different pK_a and had some advantages of high sensitivity, good repeatability and small sample requirement.     

Analysis of carbohydrates by HPLC with post-column derivatization

Post-column derivatization of oligosaccharides after HPLC separation with a variety of systems has been achieved either by reaction with thymol in concentrated sulfuric acid or after hydrolysis of saccharides with hydrochloric acid and derivatization of the resulting monosaccharides with p-aminobenzoic acid hydrazide. With both reactions the detection of reducing and non-reducing sugars is possible even at trace levels. The latter reaction, despite the two reaction steps required, is much more convenient and practical, whereas with the first reaction it is also possible to determine alkylpolyglucosides.     

Liquid chromatographic enantioseparation of three beta‐adrenolytics using new derivatizing reagents synthesized from (S)‐ketoprofen and confirmation of configuration of diastereomers

Diastereomers of racemic β‐adrenolytic drugs [namely (RS)‐propranolol, (RS)‐metoprolol and (RS)‐atenolol] were synthesized under microwave irradiation with (S)‐ketoprofen based chiral derivatization reagents (CDRs) newly synthesized for this purpose. (S)‐Ketoprofen was chosen for its high molar absorptivity (ε_o ~ 40,000) and its availability as a pure (S)‐enantiomer. Its ‐COOH group was activated with N‐hydroxysuccinimide and N‐hydroxybenzotriazole; these were easily introduced and also acted as good leaving groups during nucleophilic substitution by the amino group of the racemic β‐adrenolytics. The CDRs were characterized by UV, IR, ¹H‐NMR, HRMS and CHNS. Separation of diastereomers was achieved by RP HPLC and open column chromatography. Absolute configuration of the diastereomers was established with the help of ¹HNMR supported by developing their optimized lowest energy structures using Gaussian 09 Rev. A.02 program and hybrid density functional B3LYP with 6‐31G* basis set (based on density functional theory), and elution order was established. RP HPLC conditions for separation were optimized and the separation method was validated. The limit of detection values were 0.308 and 0.302 ng mL^?1. Copyright © 2016 John Wiley & Sons, Ltd.     

NBD-Cl as a Post-Column Reagent for Primary and Secondary Amines after Separation by Ion-Exchange Chromatography

In this study 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) is proposed as a post-column derivatization (PCD) reagent for the fluorescence detection of aliphatic primary and secondary amines after HPLC separation. Five primary (methylamine, isoamylamine, 2-phenylethylamine, putrescine, and histamine) and one secondary amine (dimethylamine) were separated isocratically on a cation-exchange column using HNO₃ (5 × 10^?3 mol L^?1) as the mobile phase. Post-column derivatization was based on two steps: 1) the derivatization of amines with NBD-Cl in alkaline medium, and 2) the acidification of the resulted mixture in order to minimize the background signal of the reagent and improve dramatically the sensitivity and determination range. The variables of the post-column reaction (concentration of NBD-Cl, buffer concentration and pH, reaction temperature, concentration of HCl, flow rates of the reagents) were thoroughly investigated. Critical chromatographic parameters such as the concentration of HNO₃, the percentage of organic solvent, and the column temperature were also examined to achieve adequate separation. An internal standard of 1,7-diaminoheptane was used. The developed post-column method provides the ability for a fully automated analysis, low detection limits (LODs 20–100 µg L^?1 with S/N = 3), and it requires less sample preparation. The applicability of the proposed analytical scheme was demonstrated by the determination of histamine (HIS) in tuna fish tissues according to US Food and Drug Administration (FDA) guidelines.     

Determination and Validation of an LC Method for Fluvoxamine in Tablets

A new, simple, rapid and specific reversed-phase high-performance liquid chromatography (HPLC) method was developed and validated for the determination of fluvoxamine in pharmaceutical dosage forms. The HPLC separation was achieved on a C₁₈ μ-Bondapack column (250 mm × 4.6 mm) using a mobile phase of acetonitrile–water (80:20, v/v) at a flow rate of 1 mL min⁻¹. Proposed method is based on the derivatization of fluvoxamine with 1,2-naphthoquinone-4-sulphonic acid sodium salt (NQS) in borate buffer of pH 8.5 to yield a orange product. The HPLC method is based on measurement of the derivatized product using UV-visible absorbance detection at 450 nm. The method was validated for specificity, linearity, precision, accuracy, robustness. The degree of linearity of the calibration curves, the percent recoveries of fluvoxamine, the limit of detection and quantification, for the HPLC method were determined. The assay was linear over the concentration range of 45–145 ng mL⁻¹ (r = 0.9999). Limit of detection and quantification for fluvoxamine were 15 and 50 ng mL⁻¹, respectively. The results of the developed procedure (proposed method) for fluvoxamine content in tablets were compared with those by the official method. The method was found to be simple, specific, precise, accurate, reproducible and robust.     

Automated high-performance liquid chromatographic determination of amphetamine in biological fluids using column-switching and on-column derivatization

Summary A rapid and simple liquid-chromatographic method has been developed for on-line quantification of amphetamine in biological fluids. Untreated samples (20 μL) are injected directly into the chromatographic system and purified on a 20 mm×2.1 mm i.d. pre-column packed with 30 μm Hypersil C₁₈ stationary phase. After clean-up the analyte is transferred to the analytical column (125 mm×4 mm i.d., 5 μm LiChrospher 100 RP₁₈) for derivatization and separation using a mixture of acetonitrile and the derivatization reagent (o-phthaldialdehyde andN-acetyl-L-cysteine) as the mobile phase. The experimental conditions for on-line derivatization and resolution of the amphetamine have been optimized, and the results have been compared with those obtained by derivatizing the analyte in pre-column mode. The method described has been applied to the determination of amphetamine in plasma and urine. Good linearity and reproducibility were obtained in the 0.1–10.0 μg mL⁻¹ concentration range, and limits of detection were 25 ng mL⁻¹ and 10 ng mL⁻¹ with UV and fluorescence detection, respectively. The procedure described is very simple and rapid, because no off-line manipulation of the sample is required; the total analysis time is approximately 8 min.     

Separation of Amines as their 6-Methyl-2-phenyl-4-quinolinecarboxylic Acid N-Hydroxysuccinimide Ester Derivatives by High Performance Liquid Chromatography

6-Methyl-2-phenyl-4-quinolinecarboxylic acid N-hydroxysuccinimide ester (MPQC-OSu) has been developed for precolumn derivatization of aliphatic amines in HPLC. The great difference between the fluorescence quantum yield of MPQC-OSu derivative and that of MPQC-OSu hydrolysate, 6-methyl-2-phenyl-4-quinolinecarboxylic acid (MPQC) makes this reagent suitable for amine-labeling, followed by chromatographic separation. In pH8.5 borate buffer, MPQC-OSu reacted with amines at 60°C for 12 min to form highly fluorescent carboxamides and excess reagent hydrolysed to MPQC. The chromatographic behavior of amine derivatives with MPQC-OSu has been investigated using HPLC on C₁₈ and C₈ columns, respectively, with fluorescence detection at 340–402nm. A baseline separation of isopropanolamine, methylamine, ethylamine, n-propylamine, n-butylamine, n-amylamine and n-hexylamine was obtained in 25min using isocratic elution on a C₁₈ column using methanol-water (70:30, v/v) as eluent. Detection limits were in the range 0.13–1 nM.Acknowledgements This research was supported by the National Natural Science Foundation of China, the Foundation of Education Ministry of China and Chengguang Project of Wuhan, China.