Determination of intracellular glutathione and glutathione disulfide using high performance liquid chromatography with acidic potassium permanganate chemiluminescence detection

Measurement of glutathione (GSH) and glutathione disulfide (GSSG) is a crucial tool to assess cellular redox state. Herein we report a direct approach to determine intracellular GSH based on a rapid chromatographic separation coupled with acidic potassium permanganate chemiluminescence detection, which was extended to GSSG by incorporating thiol blocking and disulfide bond reduction. Importantly, this simple procedure avoids derivatisation of GSH (thus minimising auto-oxidation) and overcomes problems encountered when deriving the concentration of GSSG from 'total GSH'. The linear range and limit of detection for both analytes were 7.5 × 10(-7) to 1 × 10(-5) M, and 5 × 10(-7) M, respectively. GSH and GSSG were determined in cultured muscle cells treated for 24 h with glucose oxidase (0, 15, 30, 100, 250 and 500 mU mL(-1)), which exposed them to a continuous source of reactive oxygen species (ROS). Both analyte concentrations were greater in myotubes treated with 100 or 250 mU mL(-1) glucose oxidase (compared to untreated controls), but were significantly lower in myotubes treated with 500 mU mL(-1) (p < 0.05), which was rationalised by considering measurements of H(2)O(2) and cell viability. However, the GSH/GSSG ratio in myotubes treated with 100, 250 and 500 mU mL(-1) glucose oxidase exhibited a dose-dependent decrease that reflected the increase in intracellular ROS.     

Screening for antioxidants in complex matrices using high performance liquid chromatography with acidic potassium permanganate chemiluminescence detection

The use of high performance liquid chromatography with acidic potassium permanganate chemiluminescence detection to screen for antioxidants in complex plant-derived samples was evaluated in comparison with two conventional post-column radical scavenging assays (2,2-diphenyl-1-picrylhydrazyl radical (DPPH) and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS(+))). In this approach, acidic potassium permanganate can react with readily oxidisable compounds (potential antioxidants), post-column, to produce chemiluminescence. Using flow injection analysis, experimental parameters that afforded the most suitable permanganate chemiluminescence signal for a range of known antioxidants were studied in a univariate approach. Optimum conditions were found to be: 1×10(-3)M potassium permanganate solution containing 1% (w/v) sodium polyphosphates adjusted to pH 2 with sulphuric acid, delivered at a flow rate of 2.5 mL min(-1) per line. Further investigations showed some differences in detection selectivity between HPLC with the optimised post-column permanganate chemiluminescence detection and DPPH and ABTS(+) assays towards antioxidant standards. However, permanganate chemiluminescence detection was more sensitive. Moreover, screening for antioxidants in green tea, cranberry juice and thyme using potassium permanganate chemiluminescence offers several advantages over the traditional DPPH and ABTS(+) assays, such as faster reagent preparation and superior stability; simpler post-column reaction manifold; and greater compatibility with fast chromatographic separations using monolithic columns.     

Determination of catecholamines by ion chromatography coupled to acidic potassium permanganate chemiluminescence detection

A simple,fast,sensitive,highly selective and eco-friendly analytical method for the determination of catecholamines in human urine by ion chromatography（IC） with chemiluminescence（CL） detection was described in this paper.Using 12 mmol/L H₂SO₄ without any organic additive as eluent,three catecholamines including epinephrine（EP）,norepinephrine（NE） and dopamine（DA） were well separated on a cation-exchange column.The CL detection was based on the reaction of analytes with acidic potassium permanganate in the presence of formaldehyde as an enhancer.The absence of methanol and acetonitrile in eluent made the proposed method more sensitive and eco-friendly.Under the optimal conditions,the linear range of the proposed method was in the range of 0.02-0.5μg/mL.The limit of detection（LOD） was in the range of 0.6 and 5.1μg/L.The relative standard deviations （RSD） for 0.1μg/mL mixed standard solution were in the range of 0.8-1.9%（n = 11）.The method has been applied to the determination of catecholamines in human urine successfully.Excellent spiked recoveries were achieved for catecholamines ranged from 91.2%to 112.7%.     

Determination of Citrus aurantium protoalkaloids using HPLC with acidic potassium permanganate chemiluminescence detection

Acidic potassium permanganate chemiluminescence was explored as a sensitive and selective mode of detection for phenolic phenethylamines (adrenergic amines) in consumer products containing Citrus aurantium extracts. Nine commercially available weight-loss products were analysed using rapid reversed-phase chromatography with a monolithic column (separation time of 4 min). The results were in good agreement with package labelling, with some notable exceptions. The products contained a wide concentration range of synephrine and total adrenergic amines, and the difference in consumer intake was even greater when the manufacturers’ recommended daily consumption was considered. The quantity of the extract, often specified on the packaging as equivalent grams of dry C. aurantium fruit, was a poor indicator of the concentration of the active ingredients. Methionine, a thioether amino acid contained in some weight-loss products, was identified as a potential interferent for this mode of detection.     

Determination of morphine, oripavine and pseudomorphine using capillary electrophoresis with acidic potassium permanganate chemiluminescence detection

A simple and robust capillary electrophoresis chemiluminescence detection system for the determination of morphine, oripavine and pseudomorphine is described, based upon the reaction of these analytes with acidic potassium permanganate in the presence of sodium polyphosphate. The reagent solution was contained in a quartz detection cell which also held both the capillary and the anode. The resultant chemiluminescence was monitored directly using a photomultiplier tube mounted flush against the base of the detection cell. To ensure that no migration of the permanganate anion occurred, the anode was placed at the detector end whilst the electroosmotic flow was reversed by the addition of hexadimethrine bromide (0.001% m/v) to the electrolyte. The three analytes were separated counter to the electroosmotic flow via their interaction with alpha-cyclodextrin. The methodology realised detection limits (3 x S/N) of 2.5 x 10(-7) M for both morphine and oripavine and 5 x 10(-7) M for pseudomorphine. The relative standard deviations of the migration times and the peak heights for the three analytes ranged from 0.6 up to 0.8% and from 1.5 up to 2.1%, respectively.     

Screening of cannabinoids in industrial‐grade hemp using two‐dimensional liquid chromatography coupled with acidic potassium permanganate chemiluminescence detection

Widely known for its recreational use, the cannabis plant also has the potential to act as an antibacterial agent in the medicinal field. The analysis of cannabis plants/products in both pharmacological and forensic studies often requires the separation of compounds of interest and/or accurate identification of the whole cannabinoid profile. In order to provide a complete separation and detection of cannabinoids, a new two‐dimensional liquid chromatography method has been developed using acidic potassium permanganate chemiluminescence detection, which has been shown to be selective for cannabinoids. This was carried out using a Luna 100 Å CN column and a Poroshell 120 EC‐C₁₈ column in the first and second dimensions, respectively. The method has utilized a large amount of the available separation space with a spreading angle of 48.4° and a correlation of 0.66 allowing the determination of more than 120 constituents and mass spectral identification of ten cannabinoids in a single analytical run. The method has the potential to improve research involved in the characterization of sensitive, complex matrices.     

Determination of 1-nitropyrene metabolites by high-performance liquid chromatography with chemiluminescence detection

Three metabolites of 1-nitropyrene, i.e., 3-hydoxy-1-nitropyrene (3-OHNP), 6-hydoxy-1-nitropyrene (6-OHNP) and 8-hydoxy-1-nitropyrene (8-OHNP), were sensitively and selectively detected by HPLC with peroxyoxalate chemiluminescence (CL) detection. In the system, the three OHNPs were reduced to their corresponding amino derivatives through a reduction column packed with Pt/Rh-coated alumina and then concentrated on a concentrator column. By rotating a switching valve, the analytes were eluted into a separator column (ODS), separated and then detected by the bis(2,4,6-trichlorphenyl)oxalate-H(2)O(2) CL system. The detection limits (signal-to-noise ratio=3) were in the range from 5fmol (6-OHNP and 8-OHNP) to 12fmol (3-OHNP) per injection. To demonstrate the proposed method, it was used to determine three compounds in the incubation mixture of 1-nitropyrene and rat S9 mix.     

Post-column detection of benzenediols and 1,2,4-benzenetriol based on acidic potassium permanganate chemiluminescence

Based on the sensitizing effect of formic acid on the chemiluminescence (CL) reaction of polyhydroxylbenzenes with acidified potassium permanganate and the combination technique of high-performance liquid chromatography (HPLC), a sensitive, selective and simple post-column CL detection method for simultaneously determining catechol, resorcinol, hydroquinone and 1,2,4-benzenetriol is described. The optimal conditions for the CL detection and HPLC separation were carried out. The linear ranges were: 6.0 × 10⁻³-1.5 mg/L for hydroquinone, 8.0 × 10⁻³-1.5 mg/L for 1,2,4-benzenetriol, 1.0 × 10⁻²-2.0 mg/L for resorcinol and 1.0 × 10⁻²-2.5 mg/L for catechol, respectively. The detection limits are: 3.2 × 10⁻³ mg/L for hydroquinone, 3.9 × 10⁻³ mg/L for 1,2,4-benzenetriol, 4.7 × 10⁻³ mg/L for resorcinol and 5.2 × 10⁻³ mg/L for catechol, respectively. Combining with solid phase extraction, the proposed method has been successfully applied to the determination of the polyhydroxylbenzenes in river water. The recoveries for three benzentriols were 92.1-95.4% and 82.0% for 1,2,4-benzenetriol, respectively.     

Determination of total iron in fresh waters using flow injection with potassium permanganate chemiluminescence detection

A flow injection method is described for the determination of iron in fresh water based on potassium permanganate chemiluminescence detection via oxidation of formaldehyde in aqueous hydrochloric acid. Total iron concentrations are determined after reducing Fe(III) to Fe(II) using hydroxylamine hydrochloride. The detection limit (three standard deviations of blank) is 1.0 nM, with a sample throughput of 120 h⁻¹. The calibration graph was linear over the range (2–10) × 10⁻⁷ M (r ² = 0.9985) with relative standard deviations (n = 5) in the range 1.0–2.3%. The effect of interfering cations (Ca(II), Mg(II), Zn(II), Ni(II), Co(II), Fe(III), Mn(II), Pb(II), and Cu(II)) and common anions (Cl⁻, SO ₄ ²⁻ , PO ₄ ³⁻ , NO ₃ ⁻ , NO ₂ ⁻ , I⁻, F⁻, and SO ₃ ²⁻ ) was studied at their maximum admissible concentrations in fresh water. The method was applied to fresh-water samples from the Quetta Valley, and the results obtained (0.04 ± 0.001–0.11 ± 0.01 mg/L Fe(II)) were in reasonable agreement with those obtained using the spectrophotometric reference method (0.05 ± 0.01–0.12 ± 0.02 mg/L Fe(II)). The text was submitted by the authors in English.     

Determination of selected neurotransmitter metabolites using monolithic column chromatography coupled with chemiluminescence detection

The oxidation of selected clinically important neurotransmitter metabolites with acidic potassium permanganate in the presence of polyphosphates evokes chemiluminescence of sufficient intensity to enable the sensitive determination of these species. Limits of detection for 5-hydroxyindole-3-acetic acid (5-HIAA), vanilmandelic acid (VMA; α,4-dihydroxy-3-methoxybenzeneacetic acid), 4-hydroxy-3-methoxyphenylglycol (MHPG), homovanillic acid (HVA, 4-hydroxy-3-methoxyphenylacetic acid) and 3,4-dihydroxyphenylacetic acid (DOPAC) were between 5 × 10⁻⁹ and 4 × 10⁻⁸ M, using flow-injection analysis methodology. In addition, we demonstrate the rapid determination of homovanillic acid and 5-hydroxyindole-3-acetic acid in human urine - without the need for extraction procedures - using monolithic column chromatography with chemiluminescence detection.     

Determination of phenolic compounds using high-performance liquid chromatography with Ce-Tween 20 chemiluminescence detection

A novel method for the simultaneous determination of phenolic compounds such as salicylic acid, resorcinol, phloroglucinol, p-hydroxybenzoic acid, 2,4-dihydroxybenzoic acid, and m-nitrophenol by high-performance liquid chromatography (HPLC) coupled with chemiluminescence (CL) detection was developed. The procedure was based on the chemiluminescent enhancement by phenolic compounds of the cerium(IV)-Tween 20 system in a sulfuric acid medium. The separation was carried out with an isocratic elution or with a gradient elution using a mixture of methanol and 1.5% acetic acid. For six phenolic compounds, the detection limits (3σ) were in the range 1.40-5.02 ng/ml and the relative standard deviations (n=11) for the determination of 0.1 μg/ml compounds were in the range 1.9-2.9%. The CL reaction was well compatible with the mobile phase of HPLC, no baseline drift often occurred in HPLC-CL detection was observed with a gradient elution. The method has been successfully applied to the determination of salicylic acid and resorcinol in Dermatitis Clear Tincture and p-hydroxybenzoic acid in apple juices.     

Determination of polyamines by high-performance liquid chromatography with chemiluminescence detection

A method was developed for the determination of polyamines (PA) by high-performance liquid chromatography with chemiluminescence detection. It is based on the unsaturated complex of PA with Cu(II) which had a strong catalytic effect on the luminol-H₂O₂ chemiluminescence reaction. The separation of PA was carried out on a reveres phase C18 column using methanol/water (25/75, v/v) as a mobile phase. The method was applied to the analysis of putrescine and the total amount of spermine and spermidine in apple leaves and strawberry fruit. The results indicated that the method is practical and useful.     

Determination of psilocin and psilocybin using flow injection analysis with acidic potassium permanganate and tris(2,2'-bipyridyl)ruthenium(II) chemiluminescence detection respectively

A simple, rapid and sensitive method for the determination of psilocin and psilocybin is described. This is the first report on the determination of psilocin and psilocybin using flow injection analysis with acidic potassium permanganate and tris(2,2′-bipyridyl)ruthenium(II) chemiluminescence. The limits of detection (signal-to-noise ratio = 3) are 9 × 10⁻¹⁰ M and 3 × 10⁻¹⁰ M for psilocin and psilocybin, respectively.A concise synthetic route for psilocin in three steps from readily available starting materials is also described. The structures were elucidated on the basis of spectroscopic data.     

Quantification of rat brain neurotransmitters and metabolites using liquid chromatography/electrospray tandem mass spectrometry and comparison with liquid chromatography/electrochemical detection

Analytical methodology based on solid-phase extraction, polar reversed-phase liquid chromatography, and electrospray tandem mass spectrometry (LC/MS/MS) with isotope dilution was developed and validated for quantifying the neurotransmitters, dopamine and serotonin, and their major metabolites in brain tissue. Limits of detection (0.1-20 pg/mg tissue) were sufficient for analysis of multiple neurotransmitters in rat brain regions, including parietal cortex, hypothalamus, pituitary, substantia nigra, and striatum. Method performance was compared with contemporaneous measurements using a well-established procedure based on ion-pairing reversed-phase liquid chromatography and amperometric detection. The principal advantages of the LC/MS/MS method include a more robust sample purification procedure, an optimized chromatographic separation, and the qualitative and quantitative assurance that comes from coeluting isotopically labeled internal standards; however, sensitivity did not consistently improve upon that provided by amperometric detection. This methodology may be particularly useful for applications in which simultaneous determinations are required for drugs and their affected neurotransmitters in specific brain regions.     

Determination of flavanones in Citrus juices by means of one- and two-dimensional liquid chromatography

1-D and 2-D comprehensive (LC×LC) liquid chromatography methods have been developed and compared for the separation and quantification of flavanones in various Citrus juices. 1-D analyses were carried out on a superficially porous C18 column, whereas the 2-D LC approach was composed of a polyethylene glycol silica narrow-bore column packed with totally porous particles in the first dimension (D1) and a superficially porous C18 column in the second dimension (D2). Low-selectivity correlations were ensured by the complementary separation mechanisms offered by the D1 and D2 columns. Quantification was carried out both manually and by means of a software capable of detecting and quantifying each peak from the 2-D plot. Limit of detection (LOD) values as low as 0.023 μg/mL were obtained for hesperidin used as reference material for 1-D LC analyses, whereas values as high as 0.432 μg/mL were obtained by comprehensive LC. This discrepancy can be traced back to the minor sensitivity experienced in comprehensive LC due to both sample dilution in D1 and the high flow rates employed in D2. On the other hand, the separation capabilities of the LC×LC approach allowed to reduce the interferences coming from the matrix and to achieve the separation of some critical pairs, e.g. hesperidin/naringin difficult to accomplish in 1-D LC.     

Determination of atmospheric nitrobenzanthrones by high-performance liquid chromatography with chemiluminescence detection.

A method using high-performance liquid chromatography with chemiluminescence detection was developed for analyzing mutagenic nitrobenzanthrone (NBA) isomers in airborne particulates. The method was a modification of our previously described method for analyzing nitropolycyclic aromatic hydrocarbons (NPAHs). The pretreatment and reducing conditions for 1-, 2-, 3- and 10-NBAs were the same as those for NPAHs. In order to separate these NBA isomers, we used a polymeric-type ODS column (Cosmosil 5C-18MS); a mixture of 40% acetonitrile and 60% 10 mM imidazole-HClO4 buffer was employed as the mobile phase at a flow rate of 1 mL/min. The isomers of 1-, 2-, 3- and 10-NBA were determined in chemiluminescence with linear calibration graphs from 0.1 to 4 pmol, from 200 to 4000 pmol, from 1 to 50 pmol and from 10 to 400 pmol, respectively. The detection limits (S/N = 3) of 1-, 2-, 3- and 10-NBA isomers were 0.02 pmol, 35 pmol, 0.3 pmol and 3 pmol, respectively. The method was used to analyze airborne particulates at a heavy traffic site in Kanazawa. 2- and 3-NBAs were detected in the extracts of the particulates, while 1-NBA and 10-NBA were not detected. The atmospheric concentrations of 2- and 3-NBAs were 1.83 pmol/m3 and 24.7 fmol/m3, respectively.     

Determination of polyphenols by high-performance liquid chromatography with inhibited chemiluminescence detection.

A chemiluminescence reaction detector was developed for the detection of polyphenols separated by HPLC based on the inhibition of chemiluminescence from the luminol-potassium hexacyanoferrate(III) reaction by polyphenols. The separation was carried out on a RP-C18 column at 37 degrees C by using stepwise gradient elutions. The detection limits are in the range of 6.8 x 10(-7)-2.0 x 10(-9) g/ml for catechol, protocatechuic acid, chlorogenic acid, rutin, resorcinol, hydroquinone and p-tert.butylpyrocatechol. The method is sensitive, selective, fast and simple. It has been successfully applied to the determination of chlorogenic acid and rutin in real tobacco samples.     

Determination of cobalt at picogram levels by high-performance liquid chromatography with chemiluminescence detection

A simple cation-exchange chromatographic system was used to separte cobalt from other metal ions. The cobalt thus separated was detected by the luminol chemiluminescence reaction using a spectrofluorimeter as the detector. A 3σ detection limit of 0.5 ng l^?1 was achieved, without the need for preconcentration, with a 200-μl sample. The system provided a linear analytical working range from 5 ng l^?1 to 10 μg l^?1. The cobalt content of a rice flour certified reference material was determined using the system and gave good agreement with the certificate value.