High-performance Liquid Chromatography System Research Articles

Outlining a Multidimensional Approach for the Analysis of Coffee using HPLC

This study investigated approaches for the profiling of coffee using two multidimensional approaches: (1) a multi-detection process and (2) a multi-separation process employing HPLC. The first approach compared multidetection techniques of conventional High Performance Liquid Chromatography (HPLC) hyphenated with a detector (DPPH•, UV-Vis and MS), and multiplexed mode via HPLC with an Active Flow Technology (AFT) column in Parallel Segmented Flow (PSF) format with DPPH• detection, UV-Vis and MS running simultaneously. Multiplexed HPLCPSF enabled the determination of key chemical entities by reducing the data complexity of the sample whilst obtaining a greater degree of molecule-specific information within a fraction of the time it takes using conventional multi-detection processes. DPPH•, UV-Vis and MS (TIC) were multiplexed for the analysis of espresso coffee and decaffeinated espresso coffee. Up to 20 DPPH• peaks were detected for each sample, and with direct retention time peak matching, 70% of DPPH• peaks gave a UV-Vis response for the espresso coffee and 95% for the decaffeinated espresso coffee. The second approach involved the use of a two-dimensional (2D) HPLC system to expand the separation space and separation power for the analysis of coffee, focusing on the resolution and detection of coeluting and overlapping peaks, which was beyond the limits of conventional HPLC in resolving complex samples. The 2DHPLC analysis resulted with the detection of 176 peaks and a closer observation showed the presence of an additional 17 peaks in a cut section where in 1D mode only one peak was observed.

Determination of nucleosides in Cordyceps sinensis and Ganoderma lucidum by high performance liquid chromatography method.

Background:Nucleosides are supportive in the regulation and modulation of various physiological processes in body, they acts as precursors in nucleic acid synthesis, enhance immune response, help in absorption of iron and influence the metabolism of fatty acids. Cordyceps sinensis and Ganoderma lucidum are well-known for its use in traditional medicine of China, Nepal and India. They are rich in nucleosides such as adenine, adenosine, cordycepin, etc. Hence, a simple, economic and accurate high-performance liquid chromatography (HPLC) analytical method was proposed for determination of adenine and adenosine for the quality control of plants.Materials and Methods:Chromatographic experiments were conducted on YL9100 HPLC system (South Korea). Reversed-phase chromatography was performed on a C18 column with methanol and dihydrogen phosphate as the mobile phase in isocratic elution method at a flow rate of 1.0 mL/min. Detection was carried out at 254 nm, which gives a sharp peak of adenine and adenosine at a retention time of 6.53 ± 0.02 min and 12.41 ± 0.02, respectively.Results and Discussion:Linear regression analysis data for the calibration plot showed a good linear relationship between response and concentration in the range of 25–200 µg/mL for adenosine and 100–800 µg/mL for adenine with regression coefficient of 0.999 and 0.996, respectively. The adenine was found 0.16% and 0.71% w/w in G. lucidum and in C. sinensis, respectively, and adenosine was found to be 0.14% w/w in G. lucidum whereas absent in C. sinensis.Conclusion:The developed HPLC method for the quantification of adenosine and adenine can be used for the quality control and standardization of crude drug and for the different herbal formulations, in which adenine and adenosine are present as major constituents. The wide linearity range, sensitivity, accuracy, and simple mobile phase imply the method is suitable for routine quantification of adenosine and adenine with high precision and accuracy.

Chromatographic analysis of wheatgrass extracts

Aim:Wheatgrass (WG) is the shoot of Triticum aestivum Linn. belongs to the family Gramineae, and possess high chlorophyll content and essential vitamins, minerals, vital enzymes, amino acids, dietary fibers etc., It has been shown to possess anti-cancer, anti-ulcer, antioxidant, and anti-arthritic activity due to the presence of biologically active compounds, and minerals. Therefore, in the present study, high-performance thin layer chromatography (HPTLC), and high-performance liquid chromatography (HPLC) methods for qualitative and quantitative analysis have been proposed, which will help in quality evaluation of wheat grass extract.Materials and Methods:Samples for analysis were prepared in methanol and water simply by sonication. These were applied on pre-coated silica plate and chromatograms were developed using toluene: Ethyl acetate: Formic acid. HPLC analysis was done on Waters HPLC system using water, methanol, and acetonitrile as mobile phase. Merck C18 column has been used.Results:HPTLC finger printing of alcoholic extracts of WG was carried out and found 10–11 spots at different wavelengths 254, 366, and 435 nm. HPLC fingerprinting produced 22 peaks at 256 nm. Quantitative HPTLC analysis was done to determine the gallic acid content, and was found to be 0.077% w/w in aqueous extract. By HPLC, the content of gallic acid and rutin was found to be 0.07%, and 0.04% w/w in aqueous extract of WG.Conclusion:The developed HPLC and HPTLC fingerprinting method can be used for the quality control, and standardization of WG and its extracts used as nutritional supplement.

Hemoglobinopathy screening by osmotic fragility test based on flow cytometer or naked eye.

Diagnosis of hemoglobin (Hb) disorders is based mostly on abnormal red blood cell (RBC) indices, elevated levels of HbA2, HbF, or any other Hb on the Variant high performance liquid chromatography (HPLC) system, and confirmation by molecular methods. However, large scale population screening is of prime importance and requires a simple, accurate, and cost effective technique. We have tried to compare the sensitivity of the widely used Naked Eye Single Tube Red Cell Osmotic Fragility Test (NESTROFT) and the osmotic fragility described as % residual RBCs through flow cytometry for population screening. The count of residual red cells was measured sequentially in real-time using flow cytometry. NESTROFT was performed using a 0.36% buffered saline. HbA2 and HbF levels along with other abnormal Hbs were determined on the Variant HPLC System. Molecular studies were done to confirm the diagnosis. The normal group showed a significantly lower percentage of residual RBCs (48.08 ± 11.87) as compared to cases (β thalassemia trait-82.97 ± 12.20, α thalassemia trait-72.58 ± 8.34, and HbS trait-85.00 ± 4.05). The sensitivity and specificity of NESTROFT was high for both β thalassemia traits (98.33 and 96.72%, respectively) and α thalassemia traits (100 and 96.72%, respectively) but very low sensitivity for HbS traits (54.84%). Flow cytometric osmotic fragility was a more sensitive method to discriminate normal from the group of hemoglobinopathy carriers as compared to NESTROFT which missed majority of HbS carriers. However, in view of feasibility and cost effectiveness, NESTROFT could still be used for population screening of thalassemia. © 2014 International Clinical Cytometry Society.

Determination of five antiarrhythmic drugs in human plasma by dispersive liquid–liquid microextraction and high-performance liquid chromatography

A fast and sensitive high-performance liquid chromatography (HPLC) method with ultraviolet (UV) detection was developed and validated for the simultaneous quantitation of five antiarrhythmic drugs (metoprolol, propranolol, carvedilol, diltiazem, and verapamil) in human plasma samples. It involves dispersive liquid–liquid microextraction (DLLME) of the desired drugs from 660µL plasma and separation using isocratic elution with UV detection at 200nm. The complete separation of all analytes was achieved within 7min. Acetonitrile (as disperser solvent) resulting from the protein precipitation procedure was mixed with 100µL dichloromethane (as an extraction solvent) and rapidly injected into 5mL aqueous solution (pH 11.5) containing 1% (w/v), NaCl. After centrifugation, the sedimented phase containing enriched analytes was collected and evaporated to dryness. The residue was re-dissolved in 50µL de-ionized water (acidified to pH 3) and injected into the HPLC system for analysis. Under the optimal conditions, the enrichment factors and extraction recoveries ranged between 4.4–10.8 and 33–82%, respectively. The suggested method was linear (r2 ≥0.997) over a dynamic range of 0.02–0.80µgmL−1 in plasma. The intra- and inter-days relative standard deviation (RSD%) and relative error (RE%) values of the method were below 20%, which shows good precision and accuracy. Finally, this method was applied to the analysis of real plasma samples obtained from the patients treated with these drugs.

A novel analytical pyrolysis device applicable for measurements of less volatile pyrolyzates

A novel analytical pyrolysis device suitable for the measurements of the less volatile pyrolyzates was developed. This device is comprised of a pyrolysis unit based on the vertical microfurnace pyrolyzer and a movable pyrolysis tube made of stainless steel. The upper side of the pyrolysis tube is first set at the heated center of the pyrolysis unit. A polymer sample in a sample cup is introduced into the heated center and pyrolyzed under the flow of nitrogen carrier gas. The pyrolyzates are then transferred to the bottom side of the pyrolysis tube which is cooled down by air blow and the less volatile pyrolyzates are condensed. After that the pyrolysis tube is moved down out of the pyrolysis unit and blocked from the hot zone by moving the three port valve. Finally the trapped pyrolyzates are flushed by an appropriate solvent into a collection vial. The recovered pyrolyzates in the solution can be then subjected to the measurements suitable for less volatile products such as matrix-assisted laser desorption/ionization mass spectrometry. Moreover, this device is also on-line connected to the injection port of a high performance liquid chromatographic system, which is also appropriate for the measurements of oligomeric products. The basic performance of the developed device was tested by using some polymer samples forming less volatile pyrolyzates such as poly(butylene terephthalate) and poly(2,6-dimethyl-1,4-phenylene ether). In addition, the usefulness of this method was demonstrated through the characterization of the crosslinking sequences in a photocured resin combined with the thermally-assisted hydrolysis and methylation reaction.

In-house development of an optimized synthetic module for routine [11C]acetate production.

[11C]Acetate, a radiotracer for PET imaging, is a promising radiopharmaceutical for overcoming the limitation of 2-deoxy-2-[18F]fluoro-D-glucose in a number of cancers. Here, the optimized automatic synthesis of [11C]acetate using an in-house-developed module under different conditions has been reported for routine production. [11C]CO2 was produced in a 16.4 MeV PETtrace cyclotron, and methyl magnesium chloride was used for synthesis. For product purification, ion-exchange solid-phase extraction cartridges were used, connected in series. High-performance liquid chromatography and gas chromatography were used to measure radiochemical and chemical purity. The Limulus amebocyte lysate test and the fluid thioglycollate medium test were performed for quality control of [11C]acetate. The total reaction time of [11C]acetate was within 15 min, and the overall decay-corrected radiochemical yield was 84.33±8.85%. Radiochemical purity was greater than 98% when evaluated on an analytical high-performance liquid chromatography system. No endotoxins or anaerobic bacteria were seen on quality control checks. Optimized production of [11C]acetate was achieved by the in-house module. Radiochemical and biological properties of the [11C]acetate produced were appropriate for clinical PET study.

Binary electroosmotic-pump nanoflow gradient generator for miniaturized high-performance liquid chromatography.

High-performance liquid chromatography (HPLC) plays an important role in biotechnology, and a majority of chromatographic separations use gradient elution. While gradient generators can be built in different formats, binary pumps or quaternary pumps are most frequently used for gradient generator constructions. We have recently developed a high-pressure electroosmotic pump (EOP); the pump can be manufactured at a cost of a few hundred dollars. However, it is challenging to use this pump to deliver a gradient eluent directly. In this study, we first improve the monolith preparation by applying a pressure to the monomer solution during polymerization. We assemble a binary EOP gradient generator and discuss the relationship between the gradient profile and voltage applied to the EOP. We demonstrate the feasibility of the binary EOP gradient generator for generating a smooth and reproducible nanoflow gradient. After integration of the gradient generator into a miniaturized HPLC system, we use the HPLC system for separating peptide mixtures from trypsin-digested proteins. The performance comparison between the above miniaturized HPLC system and an Agilent 1200 HPLC system exhibits comparable efficiencies, resolutions, and peak capacities.

Biosynthesis of lactosylfructoside by an intracellular levansucrase from Bacillus methylotrophicus SK 21.002

Lactosylfructoside is a functional oligosaccharide consisting of d-glucose, d-galactose, and d-fructose. In this work, lactosylfructoside was biosynthesized from sucrose as a fructosyl donor and lactose as an acceptor by an intracellular levansucrase derived from strain Bacillus methylotrophicus (B. methylotrophicus) SK21.002. The trisaccharide was purified from the product using a high performance liquid chromatography (HPLC) system and was confirmed to be lactosylfructoside by nuclear magnetic resonance (NMR) spectroscopy. The biosynthesis conditions (such as pH, temperature, enzyme dosage, substrate concentrations, and the concentration ratio of the two substrates) for lactosylfructoside production were optimized. The optimum conditions for lactosylfructoside preparation were a pH of 6.5, temperature of 37°C, and enzyme dosage of 8U/g substrates. The concentration of substrates (total lactose and sucrose) was 400mg/mL, and the ratio of lactose to sucrose was 1:1. The optimum time for lactosylfructoside production was 20h, the yield of lactosylfructoside under the optimal conditions was 143mg/mL, and the lactosylfructoside conversion efficiency was 36%.

Separation and Characterization of a New Isomeric Impurity in Cefalotin Sodium by HPLC and MEKC

A micellar electrokinetic chromatography (MEKC) method was developed for the determination of impurities in cefalotin sodium. The optimized separation is performed with the background electrolyte composed of 25 mM sodium tetraborate buffer (pH 9.2) containing 0.2 M sodium dodecylsulfate and an applied voltage of 30 kV in a bare fused-silica capillary (50 µm i.d. × 64.5 cm, 56 cm detection length). The method was validated in terms of linearity, accuracy, precision, robustness, limit of detection, and limit of quantification. Analyzing the commercial cefalotin sodium and cefalotin sodium CRS with the novel MEKC method, a new impurity was detected in the range of 0.67–1.48% level along with the potential known impurities, which was co-eluted with the main product in the pharmacopoeia high-performance liquid chromatography systems. The new impurity was confirmed to be 3-positional isomer of cefalotin by synthesis, 1H-NMR spectrometry, and MEKC techniques.

Abstract 4627: Fasting reduces the systemic exposure to irinotecan and its active metabolite SN-38

Abstract Limitations to the use of chemotherapeutic agents are the often severe adverse side-effects which may lead to early discontinuation of treatment. Most anticancer agents have a narrow therapeutic index. In previous work we have shown that 72 hours of fasting prior to treatment with a high dose of irinotecan prevents the occurrence of adverse side effects in C26 colon carcinoma bearing mice, while the antitumor activity is not abrogated. To elucidate the mechanism of fasting induced resistance against adverse side effects, we have examined the pharmacokinetics of irinotecan in 72 hours fasted mice in plasma and liver. Male BALB/c mice were divided into four groups (n=18/group). Two groups were fasted for 72 hours and two groups were fed ad libitum. After the fasting period, mice were fed ad libitum again. One group of ad libitum and fasted animals was treated with 50 mg/kg and the other with 100 mg/kg irinotecan intraperitoneally. Plasma and liver were collected at 1,4,8,12,24 and 48 hours after irinotecan injection. Tissues were homogenized in lithium heparinized plasma and concentrations of irinotecan (CPT-11) and the active metabolite SN-38 were determined using a validated reversed-phase high-performance liquid chromatography (HPLC) system with fluorescence detection. Pharmacokinetic parameters, including peak concentration, area under the plasma concentration-time curve (AUC), clearance (CL), and the half-life of the terminal disposition phase were calculated. In the fasted group, plasma levels of SN-38 in the 50 mg/kg and 100 mg/kg group showed a 57% and 53% reduction, respectively, compared with ad libitum fed controls (AUC0-inf 5,149 vs. 2,242 ng*h/mL and 7,507 vs. 3,562 ng*h/mL). For CPT-11 the effects of fasting were smaller. An increase of 26% in the 50 mg/kg group and a reduction of 15% in the 100 mg/kg group were observed (AUC0-inf 9,715 vs. 12,235 and 37,360 vs. 31,924 ng*h/mL). In the liver, SN-38 levels in the 50 mg/kg and 100 mg/kg group showed a reduction of 51% and 30%, respectively, in the fasted animals (AUC0-inf 85.4 vs. 41.5 µg*h/g and 126 vs. 87.6 µg*h/g), while CPT-11 levels were reduced with 19% in the 50 mg/kg group and 28% in the 100 mg/kg group (AUC0-inf 218 vs. 177 µg*h/g and 712 vs. 512 µg*h/mg). Our data demonstrate that 72 hours of fasting prior to irinotecan administration induces an important change in its metabolism. Plasma and liver levels of the pro-drug CPT-11 were only slightly reduced in fasted animals. Levels of the active metabolite SN-38 were considerably lower in fasted animals compared to ad libitum fed animals. These data suggest that the reduction of side effects by fasting is due to the lower systemic exposure to SN-38, and may have important clinical implications if also found in humans. Citation Format: Sander A. Huisman, P de Bruijn, I.M. Ghobadi Moghaddam-Helmantel, J.N.M. IJzermans, E Wiemer, A.H.J. Mathijssen, R.W.F. de Bruin. Fasting reduces the systemic exposure to irinotecan and its active metabolite SN-38. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4627. doi:10.1158/1538-7445.AM2014-4627

Separation properties of saccharides on a hydrophilic stationary phase having hydration layer formed zwitterionic copolymer

A novel water-holding adsorbent bonded with a zwitterionic polymer, diallylamine–maleic acid copolymer, was developed. With this adsorbent, hydrophilic solutes are partitioned by a hydration layer that forms on the zwitterions, as a main separating force. When the adsorbent was used to separate saccharides by normal-phase partition chromatography, the saccharides eluted in the order, mono-, di- and trisaccharide. The elution profile for mono- and di-saccharides was similar but not identical to that on anion exchange columns. This indicated that the adsorbent exhibited a complex retention behavior by the existence of both anion and cation exchange moieties in the functional polymer. Selecting Na+ as a counter-ion of the maleate moiety enhanced the retention of saccharide. When used in an high performance liquid chromatography (HPLC) system with gradient elution, the adsorbent enabled the simultaneous analysis of mono-, di- and oligosaccharides.

Ultra-hydrophobic ionic liquid 1-hexyl-3-methylimidazolium tris(pentafluoroethyl)trifluorophosphate supported hollow-fiber membrane liquid–liquid–liquid microextraction of chlorophenols

An ultra-hydrophobic ionic liquid, 1-hexyl-3-methylimidazolium tris(pentafluoroethyl)trifluorophosphate ([HMIM][FAP]) was immobilized in the pores of a polypropylene hollow fiber for liquid–liquid–liquid microextraction (HF-LLLME) of chlorophenols (CPs) (4-chloro-3-methylphenol, 2,4-dichlorophenol and 2,4,6-trichlorophenol). The analytes were first extracted from 10ml of water sample into the ionic liquid membrane, and then were extracted back into 5μl of sodium hydroxide aqueous solution in the hollow fiber channel. After extraction, the acceptor solution was directly injected into a high-performance liquid chromatographic system for analysis. Extraction parameters such as extraction time, salt concentration in the sample, the pH of the sample and acceptor phase, and stirring rate during extraction were investigated. The relative standard deviations of the analytes varied from 4 to 6%. Limits of detection of <0.5ng/ml were obtained for the three analytes. The squared regression coefficients relating to the calibration curve were ≥0.9941. The proposed method was applied to the analysis of CPs in canal water.

Validated Stability-Indicating HPLC and UPLC Assay Methods for the Determination of Entacapone in Pharmaceutical Dosage Forms

In the present study, new isocratic stability-indicating reverse phase chromatographic methods were developed using HPLC and UPLC systems for the determination of entacapone (ENT) in its pharmaceutical dosage forms. Ultra performance liquid chromatography (UPLC) using small sub-2 μm particles and high-performance liquid chromatography (HPLC) were compared for separation and determination of the ENT using Acquity HSS C18 (50 × 2.1 mm, 1.8 µm) and Xbridge C18 (150 × 4.6 mm, 5.0 µm) columns by UPLC and HPLC, respectively. When using UPLC, the run time could be reduced five fold and actually the solvent consumption decreased 10 times. The proposed UPLC method would decrease the consumption of organic solvents and reagents. The present methods were validated according to ICH guideline. All analytical procedures including sample preparation, flow rate, run time of the analytical systems were at low levels. Forced degradation studies were also performed for both compounds in bulk drug samples to demonstrate the specificity and stability-indicating comparative advantage of both methods.

Distribution of Tocopherols and Tocotrienols in Guinea Pig Tissues Following Parenteral Lipid Emulsion Infusion.

Tocopherols and tocotrienols possess vitamin E activity and function as the major lipid-soluble antioxidants in the human body. Commercial lipid emulsions are composed of different oils and supply different amounts of vitamin E. The objective of this study was to measure all 8 vitamin E homologs within 4 different commercial lipid emulsions and evaluate their distribution in guinea pig tissues. The distribution of vitamin E homologs within plasma and guinea pig tissues was determined using a high-performance liquid chromatography (HPLC) system. Lipid hydroperoxides in lipid emulsions were determined using a commercial kit (Cayman Chemical Company, Ann Arbor, MI), and malondialdehyde tissue levels were determined using an HPLC system. The lipid emulsions contained variable amounts of tocopherols, which were significantly different between emulsions. Tocotrienols were present at very low concentrations (≤0.3%). We found no correlation between the amount of vitamin E present in the lipid emulsions and lipid peroxidation. Hydroperoxides were the lowest with an olive oil-based emulsion and highest with a fish oil emulsion. The predominant vitamin E homolog in guinea pig tissues was α-tocopherol. No tissues had detectable levels of tocotrienols. Vitamin E levels (primarily α-tocopherol and γ-tocopherol) were highly variable among organ tissues. Plasma levels were a poor reflection of most tissue levels. Vitamin E levels within different lipid emulsions and plasma/tissues are highly variable, and no one tissue or plasma sample serves as a good proxy for levels in other tissues. All study emulsions were well tolerated and did not significantly increase systemic lipid peroxidation.

Separation, Identification, and Characterization of Degradation Products of Erlotinib Hydrochloride Under ICH-Recommended Stress Conditions by LC, LC-MS/TOF

The present research work was carried out to determine the stability of erlotinib hydrochloride (ERLO) drug under different stress conditions recommended by International Conference on Harmonization (ICH) guideline Q1A (R2). The stability of the drug was studied under hydrolytic, oxidative, photolytic, and thermal stress conditions. The drug was found susceptible to degradation under acidic, basic, and photolytic stress conditions, while it was stable under neutral, oxidative, and thermal stress conditions. A total of three degradation products (DPs) were formed. Separation was carried out by using high-performance liquid chromatography system (HPLC). Better separation was achieved on Kromasil®(150 mm × 4.6 mm, 5 µm) C18 column using gradient elution program. A mixture of stressed samples was subjected to LC-MS/TOF studies to obtain mass spectral data. The information obtained from accurate mass study was first utilized to build a complete mass fragmentation pathway of the drug, and later it was used for its degradants. Structures were proposed for each fragment based on the best possible molecular formula. Three novel DPs 6-(2-hydroxyethoxy)-7-(2-methoxyethoxy)quinazolin-4-amine, 6,7-bis(2-methoxyethoxy)quinazolin-4-amine, and N-(3-ethynylphenyl)-6, 7-bis (2-methoxyethoxy)-2-oxy-quinazolin-4-amine were identified.

Fast method for capsaicinoids analysis from Capsicum chinense fruits

Chili peppers are widely utilized in the world as savory food additives due the pungency induced by the capsaicinoids. Also, these compounds have functional properties as antimutagenic, antitumoral, antioxidant and analgesic. These characteristics increase the interest in this compound class, hence the capsaicinoid analysis must be reproducible and accurate. This study aimed to develop and validate a fast, efficient and reproducible method to analyze capsaicinoids in Brazilian Capsicum chinense fruits. The extracts were obtained after an optimization step that indicated the condition 100% of methanol and 10min on ultrasound assisted extraction. The analyses were carried out in an ultra high performance liquid chromatographic system with detection by a photo diode array and mass spectrometer. The analytical method developed permits the separation of 8 capsaicinoids in 4min of time analysis expending only 2mL of solvent as mobile phase. The validation parameters evaluated for the method show the effectiveness and satisfactory performance to answer the analytical needs of this research area, presenting low values to relative standard deviation in repeatability and reproducibility and recoveries ranged from 88 to 112% for capsaicin and 89 to 109% for dihydrocapsaicin. In the extracts from different accessions of C. chinense fruits analyzed, the contents of capsaicin and dihydrocapsaicin were in the range of 156–1442μgg−1 and 26–478μgg−1 of fresh fruit, respectively, showing the large application of this method for quantification of the two major capsaicinoids in fast routine analysis and may be used to determine the concentrations of other minor capsaicinoids once appropriate standards are available.

Determination of iodate by HPLC-UV after on-line electrochemical reduction to iodide.

In this study, a novel on-line pre-column electrochemical instrument (PECI) coupled with high-performance liquid chromatography (HPLC) was developed, and a novel method based on PEC-HPLC-UV for amplifying the ultraviolet (UV) response of iodate (IO₃⁻) was studied. Iodate undergoes reduction in the PECI, and the resulting I(-) was injected to an HPLC system and detected by a UV detector. For IO₃⁻ analysis, conditions that can influence the reduction efficiency, including applied potential, pH value and salt concentration, were investigated in detail. In an appropriate condition, the UV response of iodate after passing through PECI was almost 10 times more than that of the initial form with good precision (relative standard deviation 2.0-4.3%). The detection limit and quantity limit were 9 and 20 ng, respectively. It can be concluded that the proposed method is simple and highly sensitive.

Tandem mass spectrometric characterization of acetylated polyhydroxy hellebosaponins, the principal steroid saponins in Helleborus niger L. roots(#).

Isolation and extensive nuclear magnetic resonance (NMR) analyses revealed polyhydroxy steroid saponins to be characteristic constituents in Helleborus niger L. roots. A comprehensive study including various multi-stage mass spectrometry (MS(n) ) experiments provided first solid chromatographic and mass spectrometric information facilitating future analysis and structural assessment of polyhydroxy saponins by LC/MS(n) techniques without isolation and NMR analyses. The polyhydroxy saponins were analyzed by direct syringe injection or chromatographically separated on a capillary high-performance liquid chromatography (HPLC) system coupled to an electrospray ionization (ESI) source. MS(n) spectra were recorded on an ion trap mass spectrometer including up to four fragmentation stages (LC/ESI-MS/MS). Additionally, high-resolution mass spectra were recorded on an Orbitrap Fourier transform (FT) mass spectrometer equipped with a nanospray-ESI interface. The polyhydroxy hellebosaponins A and D were discovered to be significant constituents from H. niger roots. Extensive study of their MS(n) data revealed that they readily fragmented in the positive ion mode providing diagnostic fragments for elucidation of the steroidal character and number of OH groups. The negative ion mode yielded valuable information on the [M-H](-) ion, number and location of acetyl groups and sugar units. Additionally, fragmentation pathways for positive and negative ion modes were proposed. These results not only extend the knowledge about H. niger saponins, but also provide a facilitated approach to the analysis of polyhydroxy saponins by LC/MS(n) without prior isolation and extensive NMR identification. Additionally, proposed fragmentation pathways for positive and negative ionization modes provide a solid complementary database for further, more detailed MS(n) studies.

Spatial distributions of and diurnal variations in low molecular weight carbonyl compounds in coastal seawater, and the controlling factors

We studied the spatial distributions of and the diurnal variations in four low molecular weight (LMW) carbonyl compounds, formaldehyde, acetaldehyde, propionaldehyde, and glyoxal, in coastal seawater. The samples were taken from the coastal areas of Hiroshima Bay, the Iyo Nada, and the Bungo Channel, western Japan. The formaldehyde, acetaldehyde, and glyoxal concentrations were higher in the northern part of Hiroshima Bay than at offshore sampling points in the Iyo Nada and the Bungo Channel. These three compounds were found at much higher concentrations in the surface water than in deeper water layers in Hiroshima Bay. It is noteworthy that propionaldehyde was not detected in any of the seawater samples, the concentrations present being lower than the detection limit (1nanomole per liter (nM)) of the high performance liquid chromatography (HPLC) system we used. Photochemical and biological experiments were performed in the laboratory to help understand the characteristic distributions and fates of the LMW carbonyl compounds. The primary process controlling their fate in the coastal environment appears to be their biological consumption. The direct photo degradation of propionaldehyde, initiated by ultraviolet (UV) absorption, was observed, although formaldehyde and acetaldehyde were not degraded by UV irradiation. Our results suggest that the degradation of the LMW carbonyl compounds by photochemically formed hydroxyl radicals is relatively insignificant in the study area. Atmospheric deposition is a possible source of soluble carbonyl compounds in coastal surface seawater, but it may not influence the carbonyl concentrations in offshore waters.