Coupling Of High-performance Liquid Chromatography Research Articles

A sensitive and specific assay for glutathione with potential application to glutathione disulphide, using high-performance liquid chromatography–tandem mass spectrometry

We have utilised the combination of sensitivity and specificity afforded by coupling high-performance liquid chromatography (HPLC) to a tandem mass spectrometer (MS–MS) to produce an assay which is suitable for assaying glutathione (GSH) concentrations in liver tissue. The sensitivity suggests it may also be suitable for extrahepatic tissues. The method has been validated for GSH using mouse liver samples and also allows the assay of GSSG. The stability of GSH under conditions relevant to the assay has been determined. A 20-μl amount of a diluted methanol extract of tissue is injected with detection limits of 0.2 pmol for GSH and 2 pmol for GSSG. The HPLC uses an Altima C 18 (150×4.6 mm, 5 μm) column at 35°C. Chromatography utilises a linear gradient from 0 to 10% methanol in 0.1% formic acid over 5 min, with a final isocratic stage holding at 10% methanol for 5 min. Total flow rate is 0.8 ml/min. The transition from the M+H ion (308.1 m/ z for GSH, and 613.3 m/ z for GSSG) to the 162.0 m/ z (GSH) and 355.3 m/ z (GSSG) fragments are monitored.

Directly Coupled High-Performance Liquid Chromatography and Nuclear Magnetic Resonance Spectroscopic with Chemometric Studies on Metabolic Variation in Sprague–Dawley Rats

We report here the first combined use of NMR-PR (pattern recognition) analysis and directly coupled HPLC–NMR analysis to identify metabolic subpopulations in normal laboratoryanimals and their discriminating endogenous urinary biomarkers. Urine samplesobtained from control Sprague–Dawley rats ( n = 68) were analyzed using 1H NMR spectroscopy and principal components (PC) analysis to investigate physiological variability. Two distinctsubpopulations of animals were classified based on metabolite excretion profiles. Analysis of the PC loadings established the spectral regions that wereresponsible for classification of the subpopulationsand was used to direct the identification of biomarkers using a directly coupled HPLC–NMR analysis. One population had low urinary hippuratelevels together with an increased concentration of 3-(3-hydroxyphenyl)propionic acid (3-HPPA)and 3-hydroxycinnamic acid (3-HCA). The other subpopulation excreted high levels of hippurate. Thus, we report the bimodal occurrence of hippuricacid and chlorogenic acid metabolites in a geneticallyhomogeneous population of rats maintainedunder identical conditions, which may have significance in relation to the understanding of the consequences of biochemical variation in animalsused for drug toxicity testing.

Determination of the enantiomeric composition of gamma-lactones in edible oils by on-line coupled high performance liquid chromatography and gas chromatography.

A new method is proposed for the determination of the enantiomeric composition of gamma-lactones in different vegetable edible oils (i. e., olive oil, almond oil, hazelnut oil, peanut oil, and walnut oil), and its potential for authenticity control is underlined for a limited number of samples. The method is based on the direct injection (i.e., without requiring a sample pretreatment step) in on-line coupled reversed phase liquid chromatography to gas chromatography (RPLC-GC) using a chiral stationary phase in the GC-step. Different experimental values for both speed of sample introduction into GC and volume of the transferred fraction are considered to improve the recoveries obtained. Relative standard deviations lower than 10% and detection limits ranging from 0.06 to 0.22 mg/L were achieved for the investigated gamma-lactones.

Speciation of arsenic compounds by coupling high-performance liquid chromatography with inductively coupled plasma mass spectrometry

There is considerable evidence that toxicity and physiological behavior of arsenic depends on its chemical forms. Arsenic speciation became therefore the subject of increasing interest in recent years. A sensitive method for the determination of arsenic species has been developed. The proposed procedure involves the use of high-performance liquid chromatography and inductively coupled plasma mass spectrometry (HPLC-ICP-MS). Six arsenic compounds were separated by anion-exchange chromatography with isocratic elution using tartaric acid as mobile phase with an elution order: arsenocholine, arsenobetaine, dimethylarsinic acid, methylarsonic acid, arsenous acid and arsenic acid. The chromatographic parameters affecting the separation of the arsenic species were optimized. Analytical characterization of the method has been realized with standard solutions. The detection limits for six arsenic compounds were from 0.04 to 0.6 μg/L as As element. The repeatability (expressed by R.S.D) was better than 7% for all investigated compounds. The HPLC-ICP-MS system was successfully applied to the determination of arsenic compounds in environmental and biological samples in μg/L level.

Migration of Arsenobetaine from Canned Seafood to Brine

A study was carried out to determine arsenobetaine (AB) in brines from canned seafood products by coupling high-performance liquid chromatography (HPLC), microwave-assisted oxidation (MO), and hydride generation-atomic absorption spectrometry (HG-AAS). Conditions were established for cleanup of the brines prior to HPLC determination of AB. The analytical features of the method for AB determination in brines were as follows: the detection limit was 2.0 ng g-1 As (fresh mass), the mean relative standard deviation 3%, and the mean recovery percentage 99 ± 4%. The proposed procedure was used to analyze AB in brines from canned seafood purchased at local retail outlets. The ranges of total arsenic and AB expressed as As (both in μg g-1 of fresh mass) were as follows: total arsenic, 0.06−3.64; AB, 0.05−3.87. The percentage of total arsenic represented by AB in brines varied from 48 to 110%. The migration of AB from muscle tissue to the brine does not rule out the possible presence in brines of other toxic spe...

Feasibility of Identification and Monitoring of Arsenic Species in Soil and Sediment Samples by Coupled High-performance Liquid Chromatography ? Inductively Coupled Plasma Mass Spectrometry

The determination of four arsenic species (AsIII, dimethylarsinic acid, monomethylarsonic acid and AsV) in soil and sediments following a single microwave extraction procedure was investigated using an on-line system involving high-performance liquid chromatography (HPLC) coupled with inductively coupled plasma mass spectrometry (ICP-MS). Phosphoric acid was used in conjunction with an open focused microwave system to extract arsenic compounds. This system was optimised with respect to acid concentration, microwave power and time in order to obtain the maximum rate of recovery whilst retaining the integrity of arsenic species. Using an anion-exchange column and buffered phosphate solution with methanol added as the mobile phase, good separation and sensitivity were achieved. Under these conditions, recoveries between 60 and 80% of the total As content were obtained, and the detection limits were in the range 1–2 mg kg–1 for all species. The HPLC–ICP-MS system was used for the determination of arsenic species in acid extracts of in-house reference materials (soil and sediment samples from polluted areas). Only arsenate was found in soil but arsenite was the main species found in a polluted river sediment. Three certified reference materials were analysed to determine the concentrations of the four arsenic species, and the sum of the arsenic concentration was compared with the certified total arsenic value for each of the reference materials. This method allows the speciation of arsenic species in soil and sediment samples to be determined at natural levels, and enables their behaviour in the environment to be monitored.

Microwave-induced Plasma Atomic Emission Spectrometer Used as the Detector for On-line Coupled High-performance Liquid Chromatography–Gas Chromatography

Atmospheric helium microwave-induced plasma atomic emission spectrometry (MIP-AES) was used successfully as a mode of detection for on-line coupled LC–GC. A loop-type interface was used for the on-line coupling. An early solvent vapor exit was used between the retention gap and the separation capillary column to accelerate solvent evaporation. The interface between MIP-AES and GC was achieved using a TM 010 cavity operated at atmospheric pressure. On-line coupled LC–GC provided a high efficiency separation for analytes in a complex sample matrix using LC as an on-line clean-up and enrichment approach. MIP-AES offered simultaneous multi-element detection ability which provided valuable information for the correct identification of compounds.

Application of Directly Coupled High-performance Liquid Chromatography–Nuclear Magnetic Resonance–Mass Spectrometry to the Detection and Characterisation of the Metabolites of 2-Bromo-4-trifluoromethylaniline in Rat Urine

Directly coupled HPLC–NMR–MS was used to identify the major urinary metabolite of 2-bromo-4-trifluoromethylaniline in the rat as the sulfate conjugate of 2-bromo-4-trifluoromethyl-6-hydroxyaniline. This metabolite could not have been fully characterised using HPLC–NMR or HPLC–MS alone as the sulfate group is ‘NMR silent’ and MS would not have enabled the position of substitution on the aromatic ring to have been assigned. The need to carefully select HPLC solvents for directly coupled HPLC–NMR–MS so that NMR and MS data can both be obtained is also shown.

Directly Coupled High Performance Liquid Chromatography—Inductively Coupled Plasma-Mass Spectrometry for the Determination of Organometallic Species in Tea

Organometallic complexes in three teas (Kenyan, Indian, and Chinese) have been studied by directly coupled high performance liquid chromatography–inductively coupled plasma-mass spectrometry (HPLC–ICP-MS) and a desolvation interface. The use of a polymer-based reverse phase column (Hamilton PRP-1) reduced interactions of the complexed metals with the stationary phase below those in a silica-based column. The optimal chromatography was found to be isocration elution using 90% dilute (2%) aqueous acetic acid and 10% acetonitrile at 1.0 ml min−1. Organometallic forms of Cd, Cu, Hg, Mn, Mo, Ni, Pb, Sr, and Zn were found primarily associated with the flavanoid components. The organic components were confirmed using UV absorption detection. The organometallic complexes in the black teas (Kenyan and Indian) appear to be larger and less hydrophilic than those from the green tea (Chinese).

Optimization of the extraction and determination of monomethylarsonic and dimethylarsinic acids in seafood products by coupling liquid chromatography with hydride generation atomic absorption spectrometry

A study was carried out to develop and optimize a method for determining monomethylarsonic (MMA) and dimethylarsinic (DMA) acids in seafood products by coupling high-performance liquid chromatography with hydride generation atomic absorption spectrometry. Extraction and instrumental determination of MMA and DMA were established in popular seafood products. The analytical features of the method are as follows: detection limits, which vary in accordance with the dilution employed, were: for MMA, 0.2–2 ng g–1 As (fresh mass) and for DMA, 0.3–4 ng g–1 As (fresh mass). The relative standard deviation was 3% for MMA for a concentration level of 11 ng g–1 As and 2% for DMA (88 ng g–1 As). The recovery for MMA was 103 ± 6% and for DMA 99 ± 5%. The analysis of DORM-1 (Dogfish Muscle, National Research Council of Canada, Certified Reference Material) provided a DMA value of 440 ± 40 ng g–1 As (dry mass), which is very close to that obtained by other workers using high-performance liquid chromatography inductively coupled plasma mass spectrometry.

Gas chromatographic-mass fragmentographic quantitation of 3 alpha- hydroxy-5 alpha-pregnan-20-one (allopregnanolone) and its precursors in blood and brain of adrenalectomized and castrated rats

Coupling high performance liquid chromatography with gas chromatography-mass fragmentography has made it possible to simultaneously measure subpicomolar concentrations of allopregnanolone and its precursors, pregnenolone, progesterone and 5 alpha-dihydroprogesterone (5 alpha-DHP) in various brain areas. Allopregnanolone was measured in the brain of adrenalectomized/castrated (ADX/CX) rats in nanomolar concentrations long after peripheral sources of allopregnanolone were removed. A partial decrease (approximately 30%) in the content of allopregnanolone was found in the brains of ADX/CX rats compared to sham-operated rats. Moreover, the content of allopregnanolone in brains of sham-operated as well as ADX/CX rats was nonuniformly distributed (olfactory bulb > striatum > cortex > hippocampus) and was one to two orders of magnitude higher than in plasma or liver. Infusion of pregnenolone sulfate in ADX/CX rats elicited a fourfold increase in 5 alpha-DHP and progesterone content and a seven- to eightfold increase in the content of allopregnanolone in brain but not in liver or plasma. Furthermore, the content of allopregnanolone in brain increased to the same extent in both sham-operated and ADX/CX rats following pregnenolone sulfate infusion. The 5 alpha-reductase inhibitor, (17 beta)17[[bis(1-methylethyl)amino]carbonyl] androstane-3,5-diene-3-carboxylic acid (SKF 105111), reduced the brain content of allopregnanolone and blocked the increased formation of allopregnanolone in brain following pregnenolone sulfate infusion. The results clearly demonstrate that the synthesis of allopregnanolone from 5 alpha-DHP and progesterone occurs in the brain and that a significant amount of allopregnanolone is synthesized locally in brain from its precursors. These experiments suggest that the brain, like adrenals and gonads, is a steroidogenic organ which produces allopregnanolone as one of its own most important physiologically relevant steroids.

Optimization of the extraction, clean-up and determination of arsenobetaine in manufactured seafood products by coupling liquid chromatography with inductively coupled plasma atomic emission spectrometry

A study was carried out to develop and optimize a method for determining arsenobetaine (AB) in seafood products by coupling high-performance liquid chromatography (HPLC) with inductively coupled plasma atomic emission spectrometry (ICP-AES). Extraction, clean-up and instrumental determination of AB were established in real samples representative of fish, molluscs and crustaceans. The analytical features of the method are: detection limit, 0.51 µg g–1 As dry mass (dm) or 0.13 µg g–1 As fresh mass (fm); relative standard deviation (n= 6), 8% for cockles, 5% for prawns, 4% for sole and 2% for the Canada National Research Council Reference Material, DORM-1 (Dogfish Muscle)(n= 3). The percentage recovery for AB is: 87 ± 6 (cockles), 86 ± 2 (sole), 86 ± 9 (prawns). The analysis of DORM-1 provided an AB value of 16.5 ± 0.9 µg g–1 As dm, agreeing with results obtained by other authors using HPLC–ICP-mass spectrometry.

Evaluation of the direct injection nebulizer in the coupling of high-performance liquid chromatography to inductively coupled plasma mass spectrometry

A direct injection nebulizer (DIN) was designed and constructed. It was used in coupling high-performance liquid chromatography to inductively coupled plasma mass spectrometry for the speciation of chromium. Speciation of chromium using a concentric nebulizer and the DIN was investigated. Using a slight modification in the chromatographic conditions, the speciation of chromium was achieved on a microbore column. The absolute detection limit for both CrIII and CrVI was 3 pg. The DIN was used to run the National Institute of Standards and Technology Standard Reference Material (SRM) 2670 Toxic Metals in Freeze-Dried Urine, Elevated Level.

High-performance liquid chromatography—mass spectrometry (pneumatically assisted electrospray) of hydroxy polycyclic aromatic hydrocarbons

Electrospray (ES) in both positive and negative ionization mode and coupling high-performance liquid chromatography (HPLC) to mass spectrometry (MS) for the characterization of hydroxy polycyclic aromatic hydrocarbons (hydroxy-PAHs) are studied in this work. A series of reference hydroxy-PAHs were separated on a reversed-phase column and their ES spectra were analyzed in both ionization modes. [M + H] + and [M − H] − were obtained for positive and negative ES. Reduction of quinones was also observed in negative ES. Suitable masses for single-ion monitoring were used in HPLC—MS analysis. Flow injection of solute standards in isocratic mode and LC analyses in gradient mode using methanol—formic acid/ammonium formate 10 m M, pH 3 as eluent were performed.

Arsenic speciation by coupling high-performance liquid chromatography with inductively coupled plasma mass spectrometry

An ICP-MS detector in combination with HPLC has been evaluated for the analysis of six arsenic compounds. The influence of the presence of an organic modifier in the mobile phase on arsenic response and the quality parameters of the analysis are discussed. Detection limits for arsenic species under study range from 10 to 30 pg. The determination of arsenic compounds in solutions simulating fish or sediment extracts has been used to demonstrate the applicability of this technique.

Determination of the Enantiomers of a New 1,4-Dihydropyridine Calcium Antagonist in Dog Plasma by Achiral/Chiral Coupled High-Performance Liquid Chromatography with Electrochemical Detection

An achiral/chiral coupled high-performance liquid chromatographic method with electrochemical detection was developed for the determination of the enantiomers of a new dihydropyridine calcium blocker, methyl (±)-4,7-dihydro-3-isobutyl-6-methyl-4-(3-nitrophenyl)- thieno[2,3-b]pyridine-5-carboxylate (1), in dog plasma. The assay showed linearity over the range 1-200 ng/mL for the two enantiomers. The limit of determination was 1 ng/mL for each analyte. The assay precision was <9%, with relative errors of -5.7 to +6.9% at 3 and 30 ng/mL levels of both the enantiomers. The method was applied to a pharmacokinetic study of the enantiomers of 1 in beagle dogs after oral administration of racemic 1.

Size analysis and stability study of lipid vesicles by high-performance gel exclusion chromatography, turbidity, and dynamic light scattering

Vesicles of egg phosphatidylcholine (EPC) and phosphatidic acid (EPA) were prepared by reverse-phase evaporation (REV) followed either by sequential extrusion through polycarbonate membranes with pore diameters of 0.8, 0.4, 0.2, 0.1, and 0.05 μm or by filtration through 0.8-μm cellulosic or 0.22-μm polyvinylidene fluoride (PVF) membranes. The resulting vesicles ranging from 130 to 640 nm in mean diameter (REVs) were characterized by high-performance liquid chromatography (HPLC) using a TSK G6000 PW gel exclusion column. The efficiency of this technique to determine vesicle size parameters was studied by the analysis of the chromatograms in combination with dynamic light scattering (DLS) determination of the mean diameters (MD) of the fractionated vesicles in the region of the elution profile maxima. The HPLC TSK G6000 PW gel exclusion provides a reproducible and fast method of size characterization for lipid vesicles having MD up to 1 μm, the best selectivity being obtained in the 20- to 500-nm MD range. HPLC analysis of REV's demonstrates that: (i) both the average size and polydispersity of the vesicles decrease with decreasing pore size of the membranes, cellulosic or PVF “tortuous” ones being less efficient than “straight bores” polycarbonate ones; (ii) mixed EPC EPA REVs sequentially extruded down through 0.2-μm polycarbonate membranes are highly deformable without rupture of the bilayer; and (iii) the mean size of extruded REV's is stable for at least 1 week. The role of EPA on the size stability of mixed EPC EPA vesicles was studied by coupling HPLC gel exclusion and turbidity analysis of pure EPC and EPC EPA (mole ratio: 91 9 ) sonicated small unilamellar vesicles as a function of time. The apparent size variation of EPC vesicles observed over a week, is mainly due to their aggregation which is significantly reduced by the introduction of a small amount of EPA in the vesicle membrane.

Coupling of HPLC and ICP-AES for speciation

The cross-flow nebulizer and the thermospray vaporizer can both be used as an interface for coupling high performance liquid chromatography with inductively coupled plasma-atomic emission spectrometry. The dispersion and the peak asymmetry are acceptable for both. With the thermospray system the sensitivity is threefold higher and the fraction of methanol in methanol/water mixtures introduced into the plasma can be as high as 75%, compared with 25% for the cross-flow nebulizer. Initial results obtained for the speciation of selenium in (CH 3) 3Se +, SeO 3 2− and SeO 4 2− are promising.

Coupled high-performance liquid chromatography—gas chromatography for the determination of pesticide residues in biological matrices

A fully automated high-performance liquid chromatography—gas chromatography (HPLC-GC) network is described. A ten-port valve set up as a loop type LC—GC interface allowed the transfer of large LC effluent fractions into the gas chromatograph by concurrent solvent evaporation. The system performed highly efficient sample enrichment and clean up by LC and on-line GC separation with sensitive electron-capture detection. The efficiency of the system was demonstrated by application to the trace analysis of N-(3-chloro-2,6-dimethylphenyl)-N-(2-oxotetrahydrofuranyl)-2-methoxyacetamide (CGA 80000) in various crops and soil samples. The residue level determined was 0.02 mg/kg for crop samples and 0.01 mg/kg for soil samples. The relative standard deviations of the calibration graphs were in the range 2–5%; the mean recovery was >85%.

Hydrocarbon group-type analyses by on-line multi-dimensional chromatography : II. Liquid chromatography-gas chromatography

An on-line multi-dimensional chromatographic system, coupling high-performance liquid chromatography (HPLC) and capillary gas chromatography (GC) is described. The system is controlled by a microprocessor and can be automated. The application of this system to hydrocarbon group-type analysis is demonstrated by using HPLC on silica gel to fractionate petroleum samples into saturates, unsaturates, aromatics and polar compound, followed by the characterization of these groups by capillary GC with flame-ionization detection. Analyses of gasoline and diesel fuel are demonstrated.