Application of a mixed-gas microwave induced plasma as an on-line element-specific detector in high-performance liquid chromatography

Summary The applicability of a microwave induced plasma (MIP) as an on-line element-specific detector in high-performance liquid chromatography (HPLC) was investigated. A mixed gas oxygen-argon MIP sustained in a modified discharge tube consisting of two concentric quartz tubes, was used as an atomic emission detector for different mercury compounds. After passing the UV-vis detection cell of the HPLC system the eluent is nebulized and reaches the plasma without prior desolvation. The plasma tolerates methanol-water mixtures up to a methanol content of 90%. Detection limits for organically bound Hg are in the nanogram range. The sensitivity depends, however, on molecular structures. The capability of the HPLC-MIP is illustrated by the separation of some mercury species as 2-mercapto-ethanol complexes and by investigations of immobile mercury compounds in highland peat bog soil.

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Anwendung eines mikrowelleninduzierten Mischgas-Plasmas als on-line elementspezifischer Detektor in der HPLC

Zusammenfassung Ein mikrowelleninduziertes Plasma (MIP) wurde on-line als elementspezifischer Detektor für durch HPLC getrennte, organische Hg-Verbindungen eingesetzt. Das als atomspektroskopische Anregungsquelle verwendete Sauerstoff-Argon-Plasma erfordert ein modifiziertes Entladungsgefäß mit getrennter Zuführung der beiden Gase. Die Elutionslösung strömt durch einen UV-vis-Detektor, wird dann pneumatisch zerstäubt und mit dem Argonstrom direkt in das Plasma transportiert, das Methanol-Wasser-Gemische mit Methanol-Gehalten von bis zu 90% verträgt. Die Nachweisgrenzen für organisch gebundenes Quecksilber liegen im Nanogrammbereich, hängen jedoch von der Struktur der jeweiligen Verbindung ab. Mit Hilfe des beschriebenen HPLC-MIP-Systems wurden einige Hg-Spezies als 2-Mercapto-ethanol-Komplexe getrennt und detektiert, sowie Untersuchungen über immobile Hg-Verbindungen in Hochmoortorfböden durchgeführt.

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Dedicated to Prof. Dr. K. Laqua on the occasion of his 65. birthday     

Speciation of selenium compounds with ion-pair reversed-phase liquid chromatography using inductively coupled plasma mass spectrometry as element-specific detection

For selenium speciation analysis, the hyphenation of chromatographic separation with element-specific detection has proved a useful technique. A powerful separation system, which is capable of resolving several biologically and environmentally important selenium compounds in a single column, is greatly needed. However, that has been difficult to achieve. In this paper eight selenium compounds, namely, selenite [Se(IV)], selenate [Se(VI)], selenocystine (SeCys), selenourea (SeUr), selenomethionine (SeMet), selenoethionine (SeEt), selenocystamine (SeCM) and trimethylselenonium ion (TMSe+), were separated by using mixed ion-pair reagents containing 2.5 mM sodium 1-butanesulfonate and 8 mM tetramethylammonium hydroxide as a mobile phase. The separation of these anionic, cationic and neutral organic selenium compounds on a LiChrosorb RP18 reversed-phase column took only 18 min at a flow-rate of 1.0 ml/min with isocratic elution, and baseline separation among the six organic Se compounds was achieved. Inductively coupled plasma mass spectrometry (ICP-MS) was employed as element-specific detection. A comparison of ICP-MS signal intensity obtained with a Barbington-type nebulizer and with an ultrasonic nebulizer (USN) was made. Different signal enhancement factors were observed for the various selenium compounds when a USN was used. The speciation technique was successfully applied to the study on chemical forms of selenium in a selenium nutritional supplement. Selenomethionine was found to be the predominant constituent of selenium in the supplement.     

An improved method for simultaneous analysis of aminothiols in human plasma by high-performance liquid chromatography with fluorescence detection

Altered levels of aminothiols in biological fluids are thought to be an important risk indicator for several diseases, and reliable methods for the accurate determination of aminothiols concentrations in plasma are thus required. In this paper ammonium 5-bromo-7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (SBD-BF) is proposed as a convenient fluorogenic derivatizating reagent for the determination of aminothiols (cysteine, cysteinylglycine, homocysteine and glutathione) by HPLC with fluorescence detection. The reactions of SBD-BF with aminothiols at room temperature are about three-times faster than those of ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (the most frequently employed reagent) at 60 °C. The derivatives of SBD-BF with cysteine, cysteinylglycine, homocysteine and glutathione are easily separated by HPLC and their calibration curves show excellent linearity over the range 0.05–20 μmol/L with excellent r² values for all analytes. SBD-BF reacts with thiols under mild conditions, i.e. at 25 °C over about 30 min, and is proposed as a suitable fluorogenic reagent for thiol derivatization to be introduced in analytical clinical chemistry. The detection limits of Cys, Cys-Gly, Hcy and GSH at a signal-to-noise ratio of 5 were 0.1 μM for Cys, 0.01 μM for Cys-Gly and Hcy, and 0.02 μM for GSH. Furthermore, validation parameters of the proposed method are quite satisfactory. As an application of this method the determination of thiol derivatives in human plasma was carried out on a number of samples.     

A simple determination of mizoribine in human plasma by liquid chromatography with UV detection

A simple liquid chromatography (LC) method was developed for determination of the therapeutic level of mizoribine in human plasma. After precipitation of plasma proteins with 6% perchloric acid, mizoribine was determined by LC with spectophotometric detection. The peak height for mizoribine was linearly related to its concentrations, which ranged from 0.09 to 3.13 microg/mL. Therefore, the limit of quantitation was considered to be 0.09 microg/mL. The accuracy was 104.96-107.37%. The intra- and interday relative standard deviation values were in the range of 1.10-3.25%. The detection limit was 0.025 microg/mL, defined as a signal-to-noise ratio of 3. The plasma concentrations of mizoribine were not related to the dosage. Because mizoribine was mainly excreted in the urine, the plasma concentrations of mizoribine might be affected by a change in renal function. Therefore, the mizoribine concentration in blood should be monitored and the dosage adjusted, depending on the condition of renal function. It was suggested that the present method may be applied well in the therapeutic drug monitoring for mizoribine.     

High-performance liquid chromatography with electrochemical detection of gossypol in human plasma

A sensitive and selective high-performance liquid chromatographic method with electrochemical detection for the determination of gossypol in human plasma is described. Glutathione is used as a protective agent and gossypol dimethyl ether as an internal standard. Acetonitrile-treated protein-free plasma sample is first introduced on to a C18 pre-column for enrichment and clean-up. By using a column-switching technique, gossypol and the internal standard are subjected to further separation on a C8 analytical column, while the major interfering components are eliminated before entering the column. Methanol-0.1 M citrate buffer (pH 3.2) (80:20) is used as the mobile phase. The detector potential on the glassy carbon electrode is maintained at +0.6 V vs. an Ag-AgCl reference electrode. The linearity with human plasma ranged from 5 to 250 ng/ml. The absolute recoveries of gossypol and gossypol dimethyl ether were 91.3 and 97.5%, respectively, with a within-day precision of 2.5% and a day-to-day precision of 3.8%. The limit of detection is 5 ng/ml (signal-to-noise ratio = 3:1). The method is considered to be suitable for the clinical pharmacokinetic studies of gossypol.     

Determination of serotonin in plasma by liquid chromatography with electrochemical detection

The four major hydroperoxides derived from autoxidation or lipoxygenase action on linoleic acid or on methyl linoleate and their corresponding alcohol derivatives are resolved by high-performance liquid chromatography on Zorbax SIL 3 micron particulate columns irrigated with hexane-diethyl ether-acetic acid mixtures. The four major linoleic acid hydroperoxides are interconverted at different rates in benzene or carbon tetrachloride solutions but are stable to storage under nitrogen at -70 degrees C for several months.     

Determination of arsenic compounds in marine mammals with high-performance liquid chromatography and an inductively coupled plasma mass spectrometer as element-specific detector

Total arsenic concentrations and the concentrations of individual arsenic compounds were determined in liver samples of pinnipeds [nine ringed seals (Phoca hispida), one bearded seal (Erginathus barbatus)] and cetaceans [two pilot whales (Globicephalus melas), one beluga whale (Deliphinapterus leucus)]. Total arsenic concentrations ranged from 0.167 to 2.40 mg As kg⁻¹ wet mass. The arsenic compounds extracted from the liver samples with a methanol/water mixture (9:1, v/v) were identified and quantified by anion- and cation-exchange chromatography. An ICP–MS equipped with a hydraulic high-pressure nebulizer served as the arsenic-specific detector. Arsenobetaine (0.052–1.67 mg As kg⁻¹ wet mass) was the predominant arsenic compound in all the liver samples. Arsenocholine was present in all livers (0.005–0.044 mg As kg⁻¹ wet mass). The tetramethylarsonium cation was detected in all pinnipeds ( < 0.009 to 0.043 mg As kg⁻¹) but not in any of the cetaceans. The concentration of dimethylarsinic acid ranged from < 0.001 to 0.109 mg As kg⁻¹ wet mass. Most of the concentrations for methylarsonic acid ( < 0.001 to 0.025 mg As kg⁻¹ wet mass) were below the detection limit. Arsenous acid and arsenic acid concentrations were below the detection limit of the method (0.001 mg As kg⁻¹). An unknown arsenic compound was present in all liver samples at concentrations from 0.002–0.027 mg As kg⁻¹. © 1998 John Wiley & Sons, Ltd.     

Determination of the antidepressant paroxetine and its three main metabolites in human plasma by liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method has been developed for the determination in human plasma of the specific serotonin reuptake inhibitor (SSRI) antidepressant paroxetine and its three main metabolites (M1, M2, M3). Fluorescence detection was used, exciting at λ = 294 nm and monitoring emission at λ = 330 nm for paroxetine (λ_exc = 280 nm, λ_em = 330 nm for M1 and M2; λ_exc = 268 nm, λ_em = 290 nm for M3). Separation was obtained on a reversed-phase C18 column using a mobile phase composed of 66.7% aqueous phosphate at pH 2.5 and 33.3% acetonitrile. Imipramine (λ_exc = 252 nm, λ_em = 390 nm) was used as the internal standard. A careful pre-treatment of plasma samples was developed, using solid-phase extraction with C8 cartridges (50 mg, 1 mL). The calibration curves were linear over a working range of 2.5-100 ng mL⁻¹ for paroxetine and of 5-100 ng mL⁻¹ for all metabolites. The limit of detection (LOD) was 1.2 ng mL⁻¹ for PRX and 2.0 ng mL⁻¹ for the metabolites. The method was applied with success to plasma samples from depressed patients undergoing treatment with paroxetine. Hence, the method seems to be suitable for the therapeutic drug monitoring of paroxetine and its main metabolites in depressed patients’ plasma.     

Microcolumn liquid chromatography with inductively-coupled plasma atomic emission spectronometric detection

Summary An ICP detector is described which is compatible with microcolumn liquid chromatography. The total column effluent is concentrically nebulized and aspirated into a plasma flame. The carbon compounds are selectively detected by monitoring the characteristic emission line at 247.9 nm using water or aqueous solutions as the mobile phase. The detection limit for carbon is 500 ng or less, and the response is linear over at least two decades. The potential of this system is illustrated by a few applications.     

Differential signal detection system for improved analysis of overlapping signals in liquid chromatography

We have developed a differential signal detection system for the analysis of liquid chromatographic signals using two or more detectors and a differential amplifier circuit. The proposed detection system is an improvement on conventional chromatography in which signals are detected by means of a single detector. The differential signal detection system eliminates difficulties of isolating minor components of the signal which are masked by the major component, as well as difficulties of separating two components of the signal having similar retention times.     

Determination of diclofenac in plasma by high-performance liquid chromatography with electrochemical detection

A reversed-phase high-performance liquid chromatographic method with electrochemical detection for the quantitative determination of diclofenac potassium in plasma was developed. Naproxen was used as the internal standard. The drug and internal standard were isolated from plasma by extraction with dichloromethane and 2 M hydrochloric acid. Chromatographic separation was performed on a C18 column with methanol-water (68:32, v/v) adjusted to pH 3.2 with phosphoric acid as mobile phase. The oxidation potential for detection was established by constructing a voltammogram for diclofenac. The quantification limit for diclofenac in plasma was 5 ng mL(-1). Linearity of the method was confirmed in the range 5-2000 ng mL(-1), correlation coefficient 0.9998. Within-day relative standard deviations (RSDs) ranged from 0.66 to 14.00% and between-day RSDs from 0.59 to 15.78%. The method was successfully applied for the determination of pharmacokinetic parameters after ingestion of a 50 mg dose of diclofenac. Studies were performed on 18 healthy volunteers of both sexes.     

High-performance liquid chromatography with electrochemical detection for analysis of gliclazide in plasma

A sensitive HPLC-electrochemical detection method was developed for the analysis of gliclazide (GL) in human plasma. After deproteination of 100 microL of plasma by acetonitrile, evaporation, and reconstitution, GL was separated on a C18 column (150 mm x 4.6 mm) by the mobile phase (70 mM disodium tetraborate, pH 7.5, containing 26.5% of acetonitrile). The regression equations were linear (r> 0.9990) over the range of 50 nM to 4.00 microM. The precision and accuracy of intra- and inter-day analysis were less than 5.3 and 0.93% for relative standard deviation and relative error, respectively. The limit of detection for plasma was 10 nM for GL (S/N = 3, 10 microL injection). This newly developed method was applied for monitoring blood levels with one healthy volunteer dosing with a GL tablet.     

Quantitation of homoharringtonine in plasma by high-performance liquid chromatography with amperometric detection

A high-performance liquid chromatographic procedure was developed for the quantitation of homoharringtonine in plasma. Harringtonine was used as an internal standard, and 1 ml of sample was required. The single-step extraction with dichloromethane resulted in almost 100% recovery for homoharringtonine and harringtonine. Analysis was performed on a reversed-phase CN column with amperometric detection. Chromatography was completed in 12 min. At an oxidation potential of +1.0 V, the detection limit was 1 ng/ml at a signal-to-noise ratio of 2. The mean analytical recovery for homoharringtonine was 99.5%. The within-run precision and between-run precision were both less than 11%. The method is equally applicable for plasma or serum, and it has been demonstrated to be applicable for study of the pharmacokinetics of homoharringtonine in patients suffering from acute non-lymphocytic leukaemia.     

Measurement of plasma vanillylmandelic acid by liquid chromatography with electrochemical detection

A liquid chromatographic method with electrochemical detection is described for measuring plasma 3-methoxy-4-hydroxymandelic acid (VMA). Plasma is deproteinized by gel filtration and VMA is extracted into ethyl acetate, which is evaporated. VMA is oxidized to vanillin, which is purified by toluene extraction and quantified by high-performance liquid chromatography. The recovery of VMA through the entire procedure is 52 +/- 10% (mean +/- S.D., n = 19). The plasma VMA concentration in healthy young volunteers varies between 4.39 and 14.6 ng/ml, a range that is in excellent agreement with data obtained with mass fragmentography.