A highly sensitive high-performance liquid chromatographic procedure for the determination of isocyanates in air

Summary A sensitive high-performance liquid chromatographic procedure is described to analyse hexamethylene diisocyanate (HDI), 2,4- and 2,6-toluene diisocyanate (TDI), and 4,4-diphenylmethane diisocyanate (MDI) in air. The isocyanates are trapped on a sorbent coated with 1-(2-methoxyphenyl)piperazine (MPP). The resulting derivatives are separated using a column switching technique using either a diode array UV detector or an electrochemical detector. Working ranges are 1–5000 and 0.05–400 pmol for UV and EC detection, respectively. Virtually no breakthrough occurs if an air volume of up to 1500 l is sampled, and relative detection limits between 0.1 and 1 ng/m³ can be achieved. The procedure can be used to determine HDI and MDI in work place atmospheres and indoor air.Dedicated to Prof Dr. E. Lahmann on the occasion of this 65th birthday     

微波水解衍生高效液相色谱法测定饲料中的氨基酸

建立了一种微波水解衍生高效液相色谱同时测定饲料中17种氨基酸含量的方法。采用2,4-二硝基氯苯作为柱前衍生试剂,利用C_(18)色谱柱分离。二极管阵列检测器进行检测,检测波长为360 nm,并对微波水解时间及温度进行优化。17种氨基酸在2.5~50 mg/L范围内呈良好线性关系,相关系数为0.990 7~0.999 9;相对标准偏差为0.88%~4.2%;加标回收率为90.6%~107.2%;检出限为0.15~2.37 mg/L。研究结果表明,150℃下微波水解16 min的结果与传统加热水解(110℃,24 h)的效果基本相同。该方法分析时间较短,灵敏度较高,可用于饲料中氨基酸含量的检测。     

Liquid chromatography-electrospray ionisation-mass spectrometry based method for the simultaneous determination of algal and cyanobacterial toxins in phytoplankton from marine waters and lakes followed by tentative structural elucidation of microcystins

A liquid chromatography (LC)-based method with mass spectrometric (MS) detection was developed for simultaneous determination of various algal and cyanobacterial toxins extracted from phytoplankton occurring world-wide in marine waters and lakes. The method enables quantification of saxitoxin, anatoxin-A, domoic acid, nodularin, microcystins, okadaic acid and dinophysistoxin-1 with a single chromatographic run. In addition, the applied chromatographic conditions allow isolation and identification of substances suspected to be "new" microcystins (cyclic peptides) by fraction collection, hydrolysis, derivatisation of resulting free amino acids with the modified chiral Marfey's reagent N-alpha-(2,4-dinitro-5-fluorophenyl)-L-valinamide (L-FDVA) and enantioselective analysis of the amino acid derivatives by LC-ESI-MS.     

Direct RP‐HPLC determination of underivatized amino acids with online dual UV absorbance,fluorescence, and multiple electrochemical detection

The combined use of a dual‐UV detector, a fluorimetric one and of a multiple electrochemical (EC) detector equipped with a dual electrode, consisting of a conventional size 3 mm diameter glassy carbon electrode (GCE) and of a pair of 30 μm thick carbon microfibers, is proposed for the determination of 15 amino acids, two dipeptides and creatinine. This online coupling of the above detection modes could partially replace amino acid analysis by derivatization methods, since it solves problems concerning the direct detection of selected underivatized amino acids. Additionally, it was proved that the use of multiple‐detection allows positive peak identification in a single chromatographic run, yields more information for free amino acids and solves in some cases the problem of chromatographic resolution. In order to optimize the detection conditions of the underivatized amino acids and related compounds by different detectors, their detection characteristics were determined by adequate preliminary experiments. The electro‐oxidation characteristics of the underivatized compounds of interest were determined by hydrodynamic voltammetry using a flow cell with a macrodisc GCE and by ex‐situ voltammetry using both a GCE of conventional size and a carbon fiber disk microelectrode. Important practical advantages of microfiber and microdisk electrodes with respect to macroelectrodes were demonstrated.     

Enantiomeric separations of amino acids with inductively coupled plasma carbon emission detection

A chiral crown ether column with a pH 1.9 perchloric acid buffered aqueous mobile phase is used to separate amino acid enantiomers by high performance liquid chromatography. An inductively coupled plasma atomic emission spectrometer coupled to the chromatographic system is used as a detector by monitoring the carbon atomic emission line at 193.09 nm. Seven underivatized amino acids are separated and detected resulting in an average mass detection limit of 5 ng (2.5 ng carbon). The chiral crown ether column resolves compounds with a primary amino group near the chiral center by forming a complex between the crown ether and an ammonium ion moiety from the sample. The -form amino acid always elutes faster than its antipode. The carbon emission detector provides nearly identical sensitivities and similar detection limits for any compounds with comparable mass percents of carbon. Quantification is performed on unknown ratios of amino acids using an internal standard without the need for a calibration curve. Summing the calculated amounts of and amino acid and comparing to the known mixture quantity results in an average error of 1.0% for the seven amino acids separated.     

A comparative study of commercial liquid chromatographic detectors for the analysis of underivatized amino acids

This study compares the main commercial detectors that can detect amino acids in their underivatized form. The detectors tested are: the chemiluminescent nitrogen detector (CLND), the evaporative light scattering detector (ELSD), the nuclear magnetic resonance spectrometer, conductivity detector, refractive index, UV, and electrospray quadrupole mass spectrometry (in simple and tandem MS mode). As ELSD, CLND and MS require a volatile mobile phase, an ion-pair reversed-phase liquid chromatographic system was selected, consisting of an octadecyl column and an aqueous mobile phase containing pentadecafluorooctanoic acid as volatile ion-pairing reagent. Underivatized taurine, hypotaurine, aspartic acid, hydroxyproline, asparagine, serine, glycine, glutamine, cysteine, glutamic acid, threonine and alanine were simultaneously analysed with each detector. In order to test the applicability of these detectors to "real world" samples, the amino acid stoichiometry of the tetrapeptide Gly-Gly-Asp-Ala was determined with each detector after acid hydrolysis. The detectors were compared in terms of linearity, limit of detection, advantages and disadvantages as well as special features (capacity to provide structural information, specificity, quantification with single calibration curve, etc.).     

Indirect conductimetric detection of amino acids after liquid chromatographic separation

A liquid chromatographic method using indirect conductimetric detection is proposed for the determination of low levels of organic compounds, which does not require any special functional characteristics of the analyte. The signal detected is proportional to the molar concentration of the analyte and independent of its nature. The detector response is linearly dependent on analyte concentrations over at least three orders of magnitude. The basis of the detection is to create a conducting background, which will decrease on elution of the organic compounds. The theory of the method is discussed, with special reference to the quantitative displacement of the conducting species of the mobile phase from the column by the analyte on sample injection. The proposed method has been applied to study the chromatographic behaviour of twenty-one amino acids, where a 5 -μm Econosil CN column was used as the stationary phase with a mixture of water-acetonitrile-tetrahydrofuran (70:20:3) containing 1 mM perchloric acid or trichloroacetic acid as the mobile phase. The proposed method allows as little as 10 ng of each amino acid to be determined.     

Selective detection of underivatized 2,4-dichlorophenoxyacetic acid in soil by supercritical fluid chromatography with ion mobility detection

A simplified procedure was developed for the determination of 2,4-dichlorophenoxyacetic acid (2,4-D) in soils. Soil samples were separated by supercritical fluid chromatography after extraction without derivatization and without the use of column chromatography for cleanup. Interferences in the chromatographic separation were eliminated by using a tunably selective ion mobility detector. An atmospheric pressure ion formed by the free acid was selectively monitored so the detector could monitor 2,4-D in the presence of other electron-capturing compounds. For a randomly chosen soil sample, the level of 2,4-D detected was estimated at 500 ppb.     

Screening method for phenoxy acid herbicides in ground water by high-performance liquid chromatography of 9-anthryldiazomethane derivatives and fluorescence detection

A high-performance liquid chromatographic (HPLC) method for phenoxy acid herbicides using precolumn derivatization with 9-anthryldiazomethane (ADAM) is presented. The phenoxy acid herbicides investigated were (2,4-dichlorophenoxy)acetic acid, (4-chloro-2-methylphenoxy)acetic acid, 2-(4-chloro-2-methylphenoxy)propionic acid and (4-chloro-2-methylphenoxy)butyric acid. These herbicides reacted with ADAM under mild conditions and were converted into the corresponding fluorescent derivatives. The ADAM derivatives were separated by reversed-phase HPLC and determined using a fluorescence detector. The detection limits were about 500 pg per injection. For the application of ADAM to the determination of these herbicides in ground waters, the recoveries were more than 93% and the average relative standard deviation was 6.0% at 0.5 microgram/l. The procedure is useful as a screening method for phenoxy acid herbicides in ground water samples.     

Automated coupling of capillary-HPLC to matrix-assisted laser desorption/ionization mass spectrometry for the analysis of small molecules utilizing a reactive matrix

This study describes the application of a novel, reactive matrix for the mass spectral analysis of steroids by capillary-high performance liquid chromatography (capillary-HPLC) coupled to matrix-assisted laser desorption/ionization (MALDI). The mass spectral analysis of steroids was accomplished after fully automated peak deposition of chromatographic peaks onto MALDI targets. The seven corticosteroids used as test compounds were: triamcinolone, prednisone, cortisone, fludrocortisone, dexamethasone, deoxycorticosterone, and budesonide. They were separated using a PepMap C₁₈ (3 m particle size, 100 Å pore width) column at five different concentration levels of 25, 15, 7.5, 2.5 and 1 ng/L, and the peaks were detected at a wavelength of 237 nm. The column effluent was mixed with 2,4-dinitrophenylhydrazine (DNPH) directly following the UV detector. The chromatographic peaks were then deposited onto the MALDI target with a robotic micro-fraction collector triggered by the UV detector signals. A special hydrophobic surface coating allowed the deposition of up to 4 L (up to 90 % of the chromatographic peak volume) onto one sample spot. The compounds were then identified by MALDI mass spectrometry. Depending on the nature of the analyte, radical cations ([M]^+.) and sodium adduct ions ([M+Na]⁺) of the steroids as well as protonated steroid-dinitrophenylhydrazone derivatives ([M_D+H]⁺) were detected in positive ion mode. The detection limits were between 0.5 and 15 ng injected with capillary-HPLC-MALDI-TOF-MS and between 0.3 and 3 ng on target with MALDI-TOF when deposited manually.     

Voltammetric/amperometric detection for liquid chromatography

A voltammetric/amperometric detector based on a dual-electrode electrochemical detector is described for liquid chromatography. The detector combines the advantages of both voltammetric and amperometric detection. A three-dimensional data array of current response as a function of both time (chromatographic domain) and potential (electrochemical domain) is obtained. From the chromatographic point of view, this allows post-experimental choice of the optimal detection potential. Different detection potentials can even be chosen for each chromatographic peak. Having the voltammetric data as well as the chromatographic data provides ready identification of chromatographically unresolved compounds and the ability to resolve such co-eluting compounds voltammetrically. The voltammetric data also provide a second method of peak identification for greater certainty in peak assignments. Voltammetric detection limits of less than 10 pmol of material injected on the column were achieved with this detection method. From the electrochemical perspective, voltammetric/amperometric detection provides a technique for obtaining hydrodynamic voltammograms with small amounts or small volumes of sample. Voltammograms can also be obtained for the individual components of complex mixtures without the need for isolation steps.     

Monitoring inorganic mercury and methylmercury species with liquid chromatography-piezoelectric detection

A piezoelectric detection system coupled with a liquid chromatographic system is developed for the speciation of inorganic mercury(II) and methylmercury. Piezoelectric detection has been demonstrated to be a very sensitive detection system for total mercury determination when a gold-coated piezoelectric quartz crystal was used. The analytical features of this detection unit make it very suitable to be used as a detector coupled with a liquid chromatographic system for monitoring mercury species. After separation by a chromatography column (Spherisorb ODS-2, 5 μm,  mm i.d.), mercury species are liberated and reduced, by using stannous chloride, and are detected as an amalgam on the gold-coated piezoelectric quartz crystal, the sensor subsequently was regenerated by passing a peroxydisulfate solution. This detector exhibits good sensitivity, it allows the determination of mercury at sub-ppb concentration levels (0.30-1.20 μg l⁻¹). The precision, expressed as relative standard deviation, was ±2.7% (n=11) for a 0.5 μg l⁻¹ total mercury concentration. The proposed method is free of interferences and it allowed the determination of inorganic mercury and methylmercury species in natural waters.     

Rapid and sensitive gas chromatographic determination of diacetylmorphine and its metabolite monoacetylmorphine in blood using a nitrogen detector.

A quantitative gas chromatographic method for the determination of plasma concentrations of diacetylmorphine and its metabolite monacetylmorphine using an alkali flane detector (nitrogen detector) is described. Plasma samples (pH 9.0) containing ethylmorphine acetate as internal standard are extracted with benzene. The dried benzene extracts are analysed as their corresponding acetylated derivatives following treatment with trifluoroacetic anhydride-benzent (1:5). The nitrogen detector permits quantitation of narcotic levels down to 100ng/ml with detection as low as 20 ng/ml. The higher sensitivity and selectivity of the nitrogen detector are compared to those obtained in flame ionization detection. Species differences in the rate of conversion of diacetylmorphine to monacetylmorphine in vitro in blood are also presented.     

Interferences of nitrogen dioxide in the determination of aldehydes and ketones by sampling on 2,4-dinitrophenylhydrazine-coated solid sorbent

Summary The influence of nitrogen oxides on the practicability and accuracy of the determination of aldehydes and ketones in air samples using the DNPH-method was examined. Nitrogen dioxide reacts with 2,4-dinitrophenylhydrazine and the reaction products were identified as 2,4-dinitrophenylazide (main product) and 2,4-dinitrochlorobenzene (by-product). They have a similar chromatographic behaviour in high performance liquid chromatography (HPLC) as formaldehyde-2,4-DNP-hydrazone. The chromatographic separation of the reaction products and formaldehyde-2,4-dinitrophenylhydrazone was performed using different gradient systems. Problems which occur in nitrogen dioxide-containing air samples are discussed.     

高效液相色谱法测定盐地碱蓬汁中7种水溶性维生素和18种氨基酸

采用Ｓｈｉｍ－ｐａｃｋＣＬＣ－ＯＤＳＣ_（１８）色谱柱和ＡＡ０３反相键合相柱,对盐地碱蓬（以下称碱蓬）汁中７种水溶性维生素和１８种氨基酸进行了分离测定,结果令人满意。     

Determination of lewisite oxide in soil using solid-phase microextraction followed by gas chromatography with flame photometric or mass spectrometric detection

A rapid, sensitive, and convenient method is described for determining Lewisite oxide in soil. Samples are initially fortified with phenylarsine oxide (surrogate), then both species are extracted using ascorbic acid solutions containing 1,3-propanedithiol (derivatizing reagent). The corresponding filtered supernatant is sampled using a solid-phase microextraction fiber. Collected analytes are thermally desorbed in a heated gas chromatographic inlet, separated using fused-silica capillary columns ("primary" and "confirmatory"), and detected with either a mass spectrometric (selected ion monitoring mode) or flame photometric (sulfur-selective mode) detector. Two independent statistically-unbiased procedures were used to evaluate the detection limit for Lewisite oxide; the values range between 0.1 and 0.5 microg g(-1) soil.     

Determination of Free and Total Underivatized Amino Acids Including L-Canavanine in Bitter Vetch Seeds Using Hydrophilic Interaction Liquid Chromatography

A new hydrophilic interaction liquid chromatography method coupled with diode-array detector was developed for the determination of 17 underivatized amino acids including L-canavanine in bitter vetch [Vicia ervilia (L.) Willd.] seeds. Amino acids were extracted as free as well as total extracts after acid hydrolysis, followed by chromatographic separation on a Zorbax Rx-SIL column with a mobile phase of acetonitrile/potassium phosphate buffer (12.5?mM; pH 3.0) using gradient elution and detection at 190?nm. The method is characterized by a wide linear range (0.01–200?µg/mL, r?>?0.9987), sufficient accuracy (relative error 86.3–109.1%), and suitable precision for the results (relative standard deviation <4.9% in the case of intra-day and <9.8% in the case of inter-day precision). The limits of detection and quantification for free amino acids ranged from 0.01 to 0.24?mg/g and 0.03 to 0.72?mg/g, respectively, whereas the total amino acids ranged from 0.02 to 0.47?mg/g and 0.07 to 1.43?mg/g, respectively. The mean recoveries of free and total amino acids in spiked samples exceeded 70.3% for most amino acids. The mean total content of free and total amino acids in bitter vetch seeds was 1.71 and 14.88?g/100?g seed, whereas the corresponding values for canavanine were 0.07 and 0.19?g/100?g seed, respectively.     

Synthesis of chiral hydrazine reagents and their application for liquid chromatographic separation of carbonyl compounds via diastereomer formation

Five new chiral derivatizing reagents 5-hydrazino-2,4-dinitrophenyl-l-alaninamide (HDNP-l-Ala-NH2), 5-hydrazino-2,4-dinitrophenyl-l-phenylalaninamide (HDNP-l-Phe-NH2), 5-hydrazino-2,4-dinitrophenyl-l-valinamide (HDNP-l-Val-NH2), 5-hydrazino-2,4-dinitrophenyl-l-leucinamide (HDNP-l-Leu-NH2) and 5-hydrazino-2,4-dinitrophenyl-l-phenylglycinamide (HDNP-d-Phg-NH2) were synthesized by straightforward two-step synthesis starting from 1,5-difluoro-2,4-dinitrobenzene. Nucleophilic substitution of one fluorine atom in DFDNB with different amino acid amides yielded Marfey's reagent (5-fluoro-2,4-dinitrophenyl-l-alaninamide) and its structural variants (5-fluoro-2,4-dinitrophenyl-l-phenylalaninamide, 5-fluoro-2,4-dinitrophenyl-l-valinamide, 5-fluoro-2,4-dinitrophenyl-l-leucinamide and 5-fluoro-2,4-dinitrophenyl-d-phenylglycinamide). Chiral hydrazine reagents were prepared by nucleophilic substitution of remaining fluorine atom in Marfey's reagent and its variants with hydrazine under basic conditions. These reagents react quantitatively with chiral carbonyl compounds under mild conditions (30 degrees C, 30 min) to form hydrazone diastereomers. The labeling reaction occurs only in the presence of acid which has a catalytic action and diastereomers have strong absorbance around 348 nm. The separation of diastereomers was tried on a reversed-phase C18 HPLC column using different binary solvent combinations. Excellent separation was achieved in case of cyclic ketones having substitution at alpha-position. Optimization for derivatization yield, limit of detection, limit of quantification, linearity, accuracy and precision was carried out with respect to HDNP-l-Val-NH2. Studies related to effects of structural modification in reagents and analytes on chromatographic behavior of diastereomers were also analyzed.     

An LCEC method for the analysis of synthetic amino acids in fruit juices

Summary A fast and simple HPLC-method for the determination of synthetic amino acids in adulterated orange juice has been developed. The amino acid enantiomers were derivatized with a chiral reagent and the derivatives separated on a 3 m particle size C₁₈ column. An electrochemical detector operating in the oxidative mode was used for detection. The potential at which the derivatives are oxidized was determined by cyclic voltammetry.By using selective (electrochemical) detection it is possible to reduce the sample clean-up to simple centrifugation and filtration steps.     

Sensitive analysis of amino acids in injection liquor by reversed-phase HPLC

A convenient derivatization method of amino acids with l-fluoro-2,4-dimtrobenzene as reaction reagent and a separation system were described. The derivative amino acids were separated on a specific chemically bonded phase column with a simple linear gradient elution consisting of aqueous buffer and methanol. The eluate was detected by common ultraviolet absorption detector at 360 nm. The detection limits of amino acids were as low as 10 picomole. This method has been successfully applied to assay amino acid injection liquor used in hospital. It has good repro-ducibility and precision. The procedures avoid the requirements of particular derivative equipment and analyzer employed in conventional amino acid analysis.