Determination of picomole amounts of carbonyls as luminarin hydrazones by high-performance liquid chromatography with fluorescence detection

Summary Fluorogenic reagents (luminarin 3, luminarin 11 and luminarin 12), having a quinolizinocoumarin moiety as fluorophore and a carboxylic acid hydrazide function as reacting group, have been developed. These reagents were found to be highly sensitive fluorescence derivatization reagents for aldehydes and ketones in high-performance liquid chromatography. The reagents readily react with carbonyl compounds in aqueous sulphuric acid solution (0.1 M) at room temperature to produce the corresponding hydrazone derivatives, which can be separated on both reversed or normal-phase column. The structures of the derivatives were studied, together with their properties in reversed and normalphase chromatographic systems. UV absorbance, corrected fluorescence spectral data and quantum yields of luminarin 3, luminarin 11 and luminarin 12 are presented. The detection limits (signal to noise ratio=3) for aldehydes and ketones were in the sub-pmol range. Luminarin 3 was also applied to the determination of hydroxymethylfurfural (HMF) in orange juices and concentrates. The method for HMF involves the solid-liquid extraction of the juice by using a C-18 cartridge prior to derivatization and normal-phase separation of the derivative with fluorimetric detection at 387 nmex., 444 nm em. The calibration curve was linear for amounts of HMF ranging from 0.1 to 10 nmol. Intrarun relative standard deviation was 12.8% for 0.1 nmol and 2.6% for 1 nmol. Recovery studies indicated an average of 98.7±1.9% for juice concentrate and 99.8±3.2% for pasteurized juice.     

Rapid screening of selective serotonin re-uptake inhibitors in urine samples using solid-phase microextraction gas chromatography–mass spectrometry

In this paper a solid-phase microextraction–gas chromatography–mass spectrometry (SPME–GC–MS) method is proposed for a rapid analysis of some frequently prescribed selective serotonin re-uptake inhibitors (SSRI)—venlafaxine, fluvoxamine, mirtazapine, fluoxetine, citalopram, and sertraline—in urine samples. The SPME-based method enables simultaneous determination of the target SSRI after simple in-situ derivatization of some of the target compounds. Calibration curves in water and in urine were validated and statistically compared. This revealed the absence of matrix effect and, in consequence, the possibility of quantifying SSRI in urine samples by external water calibration. Intra-day and inter-day precision was satisfactory for all the target compounds (relative standard deviation, RSD, <14%) and the detection limits achieved were <0.4 ng mL^–1 urine. The time required for the SPME step and for GC analysis (30 min each) enables high throughput. The method was applied to real urine samples from different patients being treated with some of these pharmaceuticals. Some SSRI metabolites were also detected and tentatively identified.     

A study of the chemiluminescence behavior of myoglobin with luminol and its analytical applications

A chemiluminescence signal at 425 nm was observed when ferric state myoglobin was mixed with luminol in alkaline medium. Because the signal was remarkably enhanced in the presence of Fe(CN)₆ ^4–, analytical applications were investigated in a flow-injection system. The increase in chemiluminescence was linearly dependent on myoglobin concentration in the range 0.1 to 100 nmol L^–1, and the limit of detection was 0.04 nmol L^–1 with relative standard deviation 3.2% (3). It was also found that binding of Mb with the ligands CN^–, SCN^–, and F^– significantly inhibited the chemiluminescence reaction. The linear dynamic ranges for the ligands were 1.0–300.0, 0.1–3.0, and 0.5–100.0 nmol L^–1, and the limits of detection (S/N=3) 0.4, 0.04, and 0.2 nmol L^–1, for F^–, CN^–, and SCN^–, respectively. The relative standard deviations were 5.32%, 6.13%, and 3.38% for 0.1 nmol L^–1 CN^–, 0.5 nmol L^–1 SCN^–, and 1.0 nmol L^–1 F^–, respectively. At a flow rate of 2.0 mL min^–1 the assay could be accomplished in 1 min, including sampling and washing. The method has been successfully applied to the determination of myoglobin in human urine and F^– in water samples. A possible mechanism of chemiluminescence production by myoglobin and luminol is presented.     

Determination of N-nitrosamines by high-performance liquid chromatographic separation with voltammetric detection

The highly polar, nonvolatile N-nitrosamines, N-nitrosoproline and N-nitrosodiethanolamine, are determined by high-performance liquid Chromatographic separation with electrochemical detection using d.c. voltammetry, normal pulse voltammetry, and differential pulse voltammetry. The influence of flow rate and pulse time are determined, and detection limits on the order of 10^-7 M are obtained.     

Tributyltin speciation in aquatic matrices by CGC-FPD and CGC-MS confirmation

An analytical procedure for the determination of tributyltin (TBT) in seawater, sediments and biota is described. Extraction of TBT as chloride is achieved by hydrochloric acid treatment followed by a liquid extraction using a modified solvent with a metal coordinating ligand, and a Grignard derivatization (CH₃MgCl). The organotin fraction was isolated from the derivatized extract by column chromatography. The final determination was accomplished by on-column capillary gas chromatography (CGC) coupled to a flame photometric detector (FPD) and mass spectrometry (MS) confirmation. The relative detection limits of the analytical procedure were dependent of the environmental compartment, 0.5 ng 1^–1 (as TBT) for seawater, and 0.1 ng g^–1 and 0.4 ng g^–1 for sediments and biota, respectively. The TBT recovery of fortified samples was in the range of 90% for water and biota, and of 60% in case of sediments. The reproducibility (RSD) of the whole procedure for three independent replicates was around 15%.     

Simultaneous Determination of Human Serum Albumin and Low-Molecular-Weight Thiols after Derivatization with Monobromobimane

Biothiols are extremely powerful antioxidants that protect cells against the effects of oxidative stress. They are also considered relevant disease biomarkers, specifically risk factors for cardiovascular disease. In this paper, a new procedure for the simultaneous determination of human serum albumin and low-molecular-weight thiols in plasma is described. The method is based on the pre-column derivatization of analytes with a thiol-specific fluorescence labeling reagent, monobromobimane, followed by separation and quantification through reversed-phase high-performance liquid chromatography with fluorescence detection (excitation, 378 nm; emission, 492 nm). Prior to the derivatization step, the oxidized thiols are converted to their reduced forms by reductive cleavage with sodium borohydride. Linearity in the detector response for total thiols was observed in the following ranges: 1.76–30.0 mg mL⁻¹ for human serum albumin, 0.29–5.0 nmol mL⁻¹ for α-lipoic acid, 1.16–35 nmol mL⁻¹ for glutathione, 9.83–450.0 nmol mL⁻¹ for cysteine, 0.55–40.0 nmol mL⁻¹ for homocysteine, 0.34–50.0 nmol mL⁻¹ for N-acetyl-L-cysteine, and 1.45–45.0 nmol mL⁻¹ for cysteinylglycine. Recovery values of 85.16–119.48% were recorded for all the analytes. The developed method is sensitive, repeatable, and linear within the expected ranges of total thiols. The devised procedure can be applied to plasma samples to monitor biochemical processes in various pathophysiological states.     

Determination of cannabinoids in hemp food products by use of headspace solid-phase microextraction and gas chromatography–mass spectrometry

A fully automated procedure using alkaline hydrolysis and headspace solid-phase microextraction (HS-SPME), followed by on-fiber derivatization and gas chromatographic–mass spectrometric (GC–MS) detection has been developed for determination of cannabinoids in hemp food samples. After addition of a deuterated internal standard, the sample was hydrolyzed with sodium hydroxide and submitted to direct HS-SPME. After absorption of analytes for on-fiber derivatization, the fiber was placed directly into the headspace of a second vial containing N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA), before GC–MS analysis. Linearity was good for ⁹-tetrahydrocannabinol (THC), cannabidiol, and cannabinol; regression coefficients were greater than 0.99. Depending on the characteristics of the matrix the detection limits obtained ranged between 0.01 and 0.17 mg kg^–1 and the precision between 0.4 and 11.8%. In comparison with conventional liquid–liquid extraction this automated HS-SPME–GC–MS procedure is substantially faster. It is easy to perform, solvent-free, and sample quantities are minimal, yet it maintains the same sensitivity and reproducibility. The applicability was demonstrated by analysis of 30 hemp food samples. Cannabinoids were detected in all of the samples and it was possible to differentiate between drug-type and fiber-type Cannabis sativa L. In comparison with other studies relatively low THC concentrations between 0.01 and 15.53 mg kg^–1 were determined.     

Determination of Ionol by Voltammetry and Coulometric Titration

Procedures were developed for determining ionol by voltammetry and by coulometric titration with electrogenerated chlorine using the amperometric indication of the titration end point. Possible mechanisms of ionol oxidation with electrogenerated chlorine and its electrochemical oxidation at a glassy carbon and a gold electrode were discussed. Procedures were developed for determining ionol in mineral oil in analytical ranges from 1.0 × 10^–4 to 1.0 × 10^–2 M (RSD = 9%) and from 3.0 × 10^–5 to 4.0 × 10^–3 M (RSD = 9%) using a glassy carbon and a gold electrode, respectively. The detection limits for ionol at the glassy carbon and gold electrode were 2.8 × 10^–4 and 1.0 × 10^–5 M, respectively. The detection limit in coulometric titration was 20 g/mL.     

Simultaneous speciation of mercury and butyltin compounds in natural waters and snow by propylation and species-specific isotope dilution mass spectrometry analysis

A robust method has been developed for simultaneous determination of mercury and butyltin compounds in aqueous samples. This method is capable of providing accurate results for analyte concentrations in the picogram per liter to nanogram per liter range. The simultaneous determination of the mercury and tin compounds is achieved by species-specific isotope dilution, derivatization, and gas chromatography–inductively coupled plasma mass spectrometer (GC–ICP–MS). In derivatization by ethylation and propylation, reaction conditions such as pH and the effect of chloride were carefully studied. Ethylation was found to be more sensitive to matrix effects, especially for mercury compounds. Propylation was thus the preferred derivatization method for simultaneous determination of organomercury and organotin compounds in environmental samples. The analytical method is highly accurate and precise, with RSD values of 1 and 3% for analyte concentrations in the picogram per liter to nanogram per liter range. By use of cleaning procedures and SIDMS blank measurements, detection limits in the range 10–60 pg L^–1 were achieved; these are suitable for determination of background levels of these contaminants in environmental samples. This was demonstrated by using the method for analysis of real snow and seawater samples. This work illustrates the great advantage of species-specific isotope dilution for the validation of an analytical speciation method—the possibility of overcoming species transformations and non-quantitative recovery. Analysis time is saved by use of the simultaneous method, because of the use of a single sample-preparation procedure and one analysis.     

Analysis of mercury(II), methylmercury and phenylmercury by RPHPLC with micellized dithizone post-column derivatization photometric detection

Post-column derivatization detection system based on dithizone solubilized in cetyltrimethyl-ammonium hydrogenesulfate micellar media at pH 2.0 was devised and evaluated for selective detection of mercury(II), methylmercury and phenylmercury in reversed-phase HPLC system with photometric detection at 500 nm. This reagent solution is fully compatible with acidic organo-aqueous mobile phases generally used in RPHPLC. With the aid of the detection systematic study of the retention behaviour of three mercury species on octadecylsilica sorbent was carried out. Influence of pH, acetonitrile volume fraction, complex forming additives was investigated in detail. In mobile phase consisting of 5–30% of acetonitrile in water at pH 2.0 and 2·10^–4 mol·I^–1 DCTA linear calibration curves were measured in range 20–1000 ppb with correlation coefficient better than 0.99. Detection limits were 1–5 ng for this three mercury species. Interferences of copper(II) and silver(I) are negligible.     

On-line preconcentration and determination of cobalt by DPTH-gel chelating microcolumn and flow injection inductively coupled plasma atomic emission spectrometry

This paper reports the development of a new methodology for the determination of cobalt in biological samples by using a flow injection system with loaded DPTH-gel as solid phase to preconcentrate analytes. The procedure is based on the on-line preconcentration of cobalt on a microcolumn of 1,5-bis(di-2-pyridyl)methylene thiocarbohydrazide immobilized on silica gel (DPTH-gel). The trapped cobalt is then eluted with 1% tartaric acid and 1% citric acid (7.1 mL) and determined by inductively coupled plasma atomic emission spectrometry (ICP-AES). The analytical figures of merit for the determination of cobalt are as follows: detection limit (3S), 8.5 ng mL^–1; precision (RSD), 5.8% for 100 ng mL^–1 of cobalt; enrichment factor, 13 (using 7.3 mL of sample); sampling frequency, 40 h^–1 using a 60-s preconcentration time. For a 120-s preconcentration time (14.6 mL of sample volume) a detection limit of 5.7 ng mL^–1, an RSD under 5% at 50 ng mL^–1, an enrichment factor of 25, and a sampling frequency of 24 h^–1 were reported. The precision and accuracy of the method were checked by analysis of biological certified reference materials.     

Gas Chromatographic–Mass Spectrometric Method for Quantitation of Trimethylsilyl Derivatives of Nerve Agent Degradation Products in Human Plasma, Using Strong Anion–Exchange Solid-Phase Extraction

For unequivocal proof of the use of nerve agents such as sarin, soman, cyclohexylsarin, VX, and Russian VX, a simple and accurate method, gas chromatography–mass spectrometry (GC–MS) after trimethylsilyl derivatization, was explored for simultaneous determination of the corresponding alkyl methylphosphonic acids (AMPAs) and of methylphosphonic acid (MPA) in human plasma. GC–MS analysis was performed after solid-phase extraction, with a strong anion-exchange cartridge, from plasma samples previously deproteinized with mercuric acetate, and then derivatization with bis(trimethylsilyl)trifluoroacetamide containing 5% trimethylchlorosilane. All five AMPA derivatives and the MPA derivative were separated to baseline within 11 minutes without interference. Linear calibration plots were obtained over concentrations ranging from 50 ng mL⁻¹ to 5 µg mL⁻¹. The relative standard deviation of recoveries ranged from 1.9 to 9.7% and detection limits were 22 ng mL⁻¹ or below.Revised: 3 and 23 May 2005     

Organotin speciation in aquatic matrices by CGC/FPD, ECD and MS, and LC/MS

Summary The sensitivity and reproducibility of CGC coupled to selective detectors for the determination of alkyl and aryltin compounds (R_xSnY_z, where R=C₄H₉ or C₆H₅, and Y=CH₃ or Cl) (x+z=4) have been evaluated. In this regard, single or dual flame FPD configurations, with or without interference filter (600 nm) have been compared. The electron impact (EI) selected ion monitoring detection (SIM) CGC-MS was also considered for confirmation of the FPD assignments as well as for quantitative purposes. Alternatively, the accuracy and sensitivity of the direct analysis of organotin chlorides (Y=Cl) by cold on-column injection CGC-ECD or by thermospray LC-MS were determined. According to this, an analytical protocol based on acid digestion, extraction with organic solvents modified with tropolone, derivatization with CH₃MgCl, cleanup with alumina and CGC-FPD analysis has been successfully applied to the characterization of organotin compounds in seawater, sediments and biota. The relative detection limits of the whole procedure were dependent of the tin species and the environmental compartment considered, ranging from 0.5 to 6.5 ng l^–1 for seawater, in the filterless operation mode, and 0.1–2 ng g^–1 and 0.7–8 ng g^–1 for sediments and biota, respectively, using a 600 nm interference filter. Reproducibility was in the range of 15% RSD. Aryl and hydroxyalkyltin were identified for the first time in the aquatic compartments.     

Analysis of Bulk and Powdered Samples Using a LAMAS-10M Laser Ionization Time-of-Flight Mass Spectrometer

The problem of the modernization of a LAMAS-10M laser desorption/ionization time-of-flight mass spectrometer was considered, and the methodological fundamentals of the quantitative analysis of bulk and powdered samples by laser desorption/ionization time-of-flight mass spectrometry were discussed. Basic modifications of certain spectrometer units were described, and the reproducibility and accuracy that can be attained using these modifications were estimated. The possibility of the quantitative analysis of bulk samples was demonstrated. These studies were performed using bulk and powdered standard reference materials. In determining precious metals in geological samples, the detection limit for powdered samples was (20–50) × 10^–9. The relative standard deviation for the repeatability of the results (RSD) of the bronze standard reference sample bronza 663 was in the limits RSD = 0.06–0.4% for the major component and 0.5–4.5% for the elements present in percent concentrations. It was shown that the majority of analytes can be determined at a semiquantitative level without using standard reference materials. The key problems of laser desorption/ionization time-of-flight mass spectrometry were considered and methods were proposed to solve these problems. Ways to further improvement of the characteristics of the mass spectrometer were demonstrated.     

Determination of trace amounts of hydrogen chloride and chloride by GC or AAS

Summary Two improved analytical methods for the determination of chloride (hydrogen chloride) in the ppb-range are presented. One method combines a derivatization of chloride with 7-oxabicyclo(4.1.0)heptane with gas-chromatographic separation and detection. The other one uses a derivatization with phenylmercury nitrate. Reagent and product are separated by extraction with trichloromethane or benzene and the product is determined by atomic absorption spectrometry. The detection limit for the gas-chromatographic method is 10 ng Cl^–/ml or 0.5 ng Cl^– per sample, for the atomic absorption spectrometric method 0.2 ng Cl^–/ml or 0.3 ng Cl^– per sample.

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Bestimmung von Spuren Chlorwasserstoff und Chlorid mit Hilfe der GC oder AAS

Zusammenfassung Es werden zwei hochempfindliche analytische Bestimmungsmethoden für Chlorid (Chlorwasserstoff) im ppb-Bereich vorgestellt. Bei der einen Methode wird eine Derivatisierung von HCl mit 7-Oxabicyclo(4.1.0)heptan durchgeführt und das Reaktionsprodukt gas-chromatographisch bestimmt. Die andere Methode verwendet eine Derivatisierung von HCl mit Phenylquecksilbernitrat. Das Reaktionsprodukt wird vom Reagens durch Extraktion mit Trichlormethan oder Benzol abgetrennt und anschließend atomabsorptionsspektralphotometrisch bestimmt. Die Nachweisgrenze für das gas-chromatographische Verfahren liegt bei 10 ng Cl^–/ml bzw. 0,5 ng Cl^– pro Probe, für die atomabsorptionsspektralphotometrische Methode bei 0,2 ng Cl^–/ml oder 0,3 ng Cl^– pro Probe.

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Prof. Dr. W. Fresenius zum 70. Geburtstag gewidmet     

Determination of Rare-Earth Elements in Sulfide Minerals by Inductively Coupled Plasma Mass Spectrometry with Ion-Exchange Preconcentration

A procedure was developed for determining ultratrace rare-earth elements in sulfide minerals by inductively coupled plasma mass spectrometry with ion-exchange preconcentration. The concentration factor was 200. The found concentrations of rare-earth elements were 6–30 times lower than those in chondrites. For lanthanum and praseodymium, RSD < 10%; for other rare-earth elements, RSD < 6%. The accuracy of the results was verified by the addition of known amounts of Eu, Tb, Tm, and Lu to a chalcopyrite sample at the stage of decomposition with HCl and HNO₃. The calculated yield of rare-earth elements was 94–96%. The detection limit was from 0.06 ng/g (6 × 10^–9%) for lutetium to 5 ng/g (5 × 10^–7%) for cerium. The procedure was used for the determination of rare-earth elements in chalcopyrites, pyrites, and sphalerites.     

Determination of styrene in olive oil by an automatic purge-and-trap system coupled to gas chromatography-mass spectrometry

Summary A new gas chromatographic method using an automatic purge-and-trap system coupled to a GC with mass selective detection to analyze styrene at the parts-per-trillions (ng kg^–1) level is described. The method shows a good sensitivity and the detection limit is 10 ng kg^–1 with a relative standard deviation (RSD) of 4.7% for 164 ng kg^–1 styrene in olive oil. This analytical method has been successfully applied to the analysis of styrene in extra-virgin olive oil from the European market.     

Quantification of Neurotransmitter Amino Acids in Human Serum by Capillary Electrophoresis with Laser-Induced Fluorescence Detection

Capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection is developed as a simple and sensitive method for the quantification of arginine (Arg), tyrosine (Tyr) and glutamic acid (Glu) in human serum. The separation conditions and the derivatization conditions with fluoresceinisothiocyanate (FITC) were investigated. Regression equations revealed a linear relationship (correlation coefficients: 0.9927–0.9998) between the peak area and concentration of each analyte. For the amino acids detected, 10^–10M detection limits were reached, and the levels of these amino acids in human serums were easily determined with recoveries of 93.5–106.5%.     

Study of the feasibility of focused microwave-assisted soxhlet extraction of N-methylcarbamates from soil

A fast microwave-assisted dansylation procedure has been developed for the derivatization of N-nitrosamines prior to high-performance liquid chromatography determination. N-Nitrosomorpholine, N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosopyrrolidine and N-nitrosopiperidine are first denitrosated by hydrobromic acid-acetic acid to produce secondary amines, which are then quantitatively dansylated in 5 min using radiation power of 378 W and a maximum pressure of 1.4 bar inside the reactor. The reaction mixture is separated on a C18 column with acetonitrile-water (55:45, v/v) as mobile phase with fluorimetric detection at 531 nm (excitation at 339 nm). The detection limits range from 8 to 75 pg for N-nitrosomorpholine and N-nitrosodiethylamine, respectively. The method was applied to study the recoveries of N-nitrosamines in beer and their determination in cigarette smoke.     

HPLC fluorescence determination of nitrites in water using precolumn derivatization with 4-methyl-7-aminocoumarin

Summary A rapid, simple, and sensitive method is described for determination of nitrites in water. Nitrite (NO₂–) ions react with coumarin 120^® (4-methyl-7-aminocoumarin) in sulfuric acid medium to give the corresponding 7-diazo compound. After hydrolysis, this latter yields (95%) the highly fluorescent 4-methyl-7-hydroxycoumarin (4-methylumbelliferone) which is fluorimetrically detected at 380 nm after excitation at 325 nm.In order to avoid interference from both excess coumarin 120^® and the trace amounts of 4-methylumbelliferone which occurs in coumarin 120^® as an impurity, use of HPLC is mandatory; a satisfactory separation is obtained on a cyano stationary phase with apolar hexane-isopropanol (955, v/v) as eluent. Under these conditions, linearity of response is obtained from 1 to 30 g.L^–1 of NO₂–; the limit of detection is 0.5 g.L^–1. The repeatability and reproducibility, expressed as RSD %, are 2.5 and 4.7 % respectively, for n=6 and 5 g.L^–, analytical characteristics which demonstrate the reliability of the proposed method.