High Performance Liquid Chromatography-hydride Generation-atomic Fluorescence Spectrometry Research Articles

Trimethylselenonium ion determination in human urine by high-performance liquid chromatography-hydride generation-atomic fluorescence spectrometry optimization of the hydride generation step.

This work describes the intricacies of the determination of the trimethylselenonium ion (TMSe) in human urine via high-performance liquid chromatography-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS). By definition, this technique requires that the separated TMSe can be online converted into a volatile compound. Literature data for the determination of TMSe via the hydride generation technique are contradictory; i.e., some authors claim that direct formation of volatile compounds is possible under reduction with NaBH4, whereas others reported that a digestion step is mandatory prior to conversion. We studied and optimized the conditions for online conversion by varying the mobile phase composition (pyridine, phosphate, and acetate), testing different reaction coils, and optimizing the hydride generation conditions, although technically no hydride (H2Se) is formed but a dimethylselenide (DMSe). The optimized conditions were used for the analysis of 64 urine samples of 16 (unexposed) volunteers and the determination of low amounts of TMSe (LOD = 0.2ngmL-1). Total (specific gravity-corrected) selenium concentrations in the urine samples ranged from 7.9 ± 0.7 to 29.7 ± 5.0ngmL-1 for individual volunteers. Four volunteers were characterized as TMSe producers (hINMT genotype GA) and 12 were non-producers (hINMT genotype GG). Urine of TMSe producers contained 2.5 ± 1.7ngmL-1 of TMSe, compared to 0.2 ± 0.2ngmL-1 for non-producers.

Arsenic-Related Health Risk Assessment of Realgar-Containing NiuHuangJieDu Tablets in Healthy Volunteers Po Administration

Realgar, an arsenic-containing traditional Chinese medicine of As2S2, has significant therapeutic effects for hundreds of years. NiuHuangJieDu tablets (NHJDT) is one of the most commonly prescribed realgar-containing preparations for the treatment of sore throat, swelling, and aching of gums. However, realgar-containing TCMs raise great safety concerns due to the adverse effects reported by arsenic poisoning. In this study, the arsenic-related health risk assessment of NHJDT was conducted in healthy volunteers after single and multiple doses oral administration. Blood, plasma, and urine samples were collected after dosing at predetermined time points or periods. Simple, rapid, and sensitive methods were established for the quantification of total arsenic and arsenic speciation in biological samples. The total arsenic and arsenic speciation were determined by hydride generation-atomic fluorescence spectrometry (HG-AFS) and high-performance liquid chromatography–hydride generation–atomic fluorescence spectrometry (HPLC-HG-AFS), respectively. No significant fluctuation of total arsenic was observed in human blood, and no traces of arsenic speciation were found in human plasma. Dimethylarsenic acid was detected as the predominated arsenic species in human urine after dosing. Therapeutic dose administration of NHJDT was relatively safe in single dose for the limited blood arsenic exposure, but long-term medication may still pose health risks due to the accumulation of arsenics in blood and its extremely slow excretion rate. Therefore, arsenic exposure should be carefully monitored during realgar-containing TCM medication, especially for long-term regimen. The results obtained in this study will provide scientific references for the clinical application of realgar and its-containing TCMs.

Pharmacokinetic Characteristics, Tissue Bioaccumulation and Toxicity Profiles of Oral Arsenic Trioxide in Rats: Implications for the Treatment and Risk Assessment of Acute Promyelocytic Leukemia.

Oral arsenic trioxide (ATO) has demonstrated a favorable clinical efficiency in the treatment of acute promyelocytic leukemia (APL). However, the pharmacokinetic characteristics, tissue bioaccumulation, and toxicity profiles of arsenic metabolites in vivo following oral administration of ATO have not yet been characterized. The present study uses high performance liquid chromatography-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS) to assess the pharmacokinetics of arsenic metabolites in rat plasma after oral and intravenous administration of 1 mg kg−1 ATO. In addition, the bioaccumulation of arsenic metabolites in blood and selected tissues were evaluated after 28 days oral administration of ATO in rats at a dose of 0, 2, 8, and 20 mg kg−1 d−1. The HPLC-HG-AFS analysis was complemented by a biochemical, hematological, and histopathological evaluation conducted upon completion of ATO treatment. Pharmacokinetic results showed that arsenite (AsIII) reached a maximum plasma concentration rapidly after initial dosing, and the absolute bioavailability of AsIII was 81.03%. Toxicological results showed that the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and white blood cells (WBC) in the 20 mg kg−1 d−1 ATO group were significantly increased compared to the control group (p < 0.05). The distribution trend of total arsenic in the rat was as follows: whole blood > kidney > liver > heart. Dimethylated arsenic (DMA) was the predominant bioaccumulative metabolite in the whole blood, liver, and heart, while monomethylated arsenic (MMA) was the predominant one in the kidney. Collectively, these results revealed that oral ATO was rapidly absorbed, well-tolerated, and showed organ-specific and dose-specific bioaccumulation of arsenic metabolites. The present study provides preliminary evidence for clinical applications and the long-term safety evaluation of oral ATO in the treatment of APL.

Characteristics of arsenic species in cerebrospinal fluid (CSF) of acute promyelocytic leukaemia (APL) patients treated with arsenic trioxide plus mannitol.

Arsenic speciation in cerebrospinal fluid (CSF) is critical for treatment/prevention of central nervous system (CNS) relapse in acute promyelocytic leukaemia (APL) patients treated with arsenic trioxide (ATO). Previous study showed low total arsenic level in CSF of APL patients. Mannitol infusion was applied to improve blood-brain barrier (BBB) permeability for arsenic. Arsenite (AsIII ), monomethylarsonic acid (MMAV ), dimethylarsinic acid (DMAV ), and arsenate (AsV ) in CSF and plasma were analysed by high performance liquid chromatography-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS). The profile and concentration of arsenic species in CSF from APL patients administered ATO alone and in combination with mannitol were compared. The overall distribution trend of arsenic species in CSF was AsIII , DMAV > MMAV > AsV . Arsenicals accumulated in CSF with administration frequency. The permeability of BBB for AsIII was higher than that for MMAV and DMAV . Arsenic concentration in CSF was much lower than that in plasma. There were significantly higher arsenic species concentrations in CSF of APL patients treated with mannitol than that without mannitol. Mannitol infusion significantly increased AsIII penetration into CSF, which was beneficial to optimize efficacy in APL patients with CNS relapse.

Polymorphisms in arsenic (+ 3 oxidation state) methyltransferase (AS3MT) predict the occurrence of hyperleukocytosis and arsenic metabolism in APL patients treated with As2O3.

Polymorphisms in arsenic (+ 3 oxidation state) methyltransferase (AS3MT) have been shown to be related to interindividual variations in arsenic metabolism and to influence adverse health effects in acute promyelocytic leukemia (APL) patients treated with arsenic trioxide (As2O3). The occurrence of hyperleukocytosis with As2O3 treatment seriously affects the early survival rate of APL patients, but no definite explanation for such a complication has been clearly established. To clarify the causes of this situation, AS3MTpolymorphisms 14215 (rs3740390), 14458 (rs11191439), 27215 (rs11191446), and 35991 (rs10748835) and profiles of plasma arsenic metabolites were evaluated in a group of 54 newly diagnosed APL patients treated with single-agent As2O3. High-performance liquid chromatography-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS) was used to determine the concentrations of plasma arsenic metabolites. Plasma arsenic methylation metabolism capacity was evaluated by the percentage of inorganic arsenic (iAs), monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), primary methylation index (PMI, MMA/iAs), and secondary methylation index (SMI, DMA/MMA). The results showed that APL patients who developed hyperleukocytosis had ahigher plasmaiAs%, but alower MMA% and PMI than those who did not develop hyperleukocytosis during As2O3 treatment. In addition, patients with the AS3MT 14215 (rs3740390) CC genotype had significantly higher plasma iAs% and incidence of hyperleukocytosis, but lower PMI than patients with the CT + TT genotype. Conversely, we did not observe statistically significant associations between the occurrence of hyperleukocytosis and AS3MT 14458 (rs11191439), 27215 (rs11191446), and 35991 (rs10748835) polymorphisms in our study subjects. These results indicated that AS3MT 14215 (rs3740390) might be used as an indicator for predicting the occurrence of hyperleukocytosis in APL patients treated with As2O3.

Application of enzymatic probe sonication for selenium speciation in animal feeds

Enzymatic probe sonication (EPS) was investigated as a novel and alternative technology for the extraction of five Se species SeCys2, MeSeCys, Se (IV), SeMet and Se (VI) from animal feeds. The critical parameters of EPS such as enzyme types, ultrasonic power, sample/enzyme ratio, extraction time and temperature were carefully optimized. Under current conditions, one sample can be extracted in 60s, in contrast to several hours required by the conventional enzymatic methods Moreover, the extraction is performed in pure water, no further temperature control or pH adjustment is needed, and therefore the risk of species interconvertion is drastically reduced or inhibited. The Se species were separated and quantitatively determined by high-performance liquid chromatography-hydride generation atomic fluorescence spectrometry (HPLC-HG-AFS), which allows the separation of organic and inorganic Se species in a single chromatographic run. The newly developed method was successfully applied to the determination of Se species in 18 feed samples collected from markets and local farms. Concerning the feed samples studied, the results of this work suggest that stricter measures should be taken to control the Se-enriched feed supplements in terms of Se species.

HPLC-HG-AFS determination of arsenic species in acute promyelocytic leukemia (APL) plasma and blood cells

Arsenic trioxide (ATO) has been successfully used in the treatment of acute promyelocytic leukemia (APL). To clarify the arsenic species in APL patients, high performance liquid chromatography-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS) and HG-AFS methods were developed and validated to quantify the plasma concentrations of inorganic arsenic (As(III) and As(V)) and methylated metabolites (MMA and DMA), and the total amounts of arsenic in blood cells and plasma. Blood cells and plasma were digested with mixtures of HNO3H2O2 and analyzed by HG-AFS. For arsenic speciation, plasma samples were prepared with perchloric acid to precipitate protein. The supernatant was separated on an anion-exchange column within 6min with isocratic elution using 13mM CH3COONa, 3mM NaH2PO4, 4mM KNO3 and 0.2mM EDTA-2Na. The methods provided linearity range of 0.2–20ng/mL for total arsenic and 2.0–50ng/mL for four arsenic species. The developed methods for total arsenic and arsenic species determination were precise and accurate. The spiked recoveries ranged from 81.2%-108.6% and the coefficients of variation for intra- and inter-batch precision were less than 9.3% and 12.5%, respectively. The developed methods were applied successfully for the assay of total arsenic and arsenic species in 5 APL patients. The HPLC-HG-AFS may be a good alternative for arsenic species determination in APL patients with its simplicity and low-cost in comparison with HPLC-ICP-MS.

Pharmacokinetic properties of arsenic species after oral administration of Sargassum pallidum extract in rats using an HPLC-HG-AFS method

Sargassum pallidum is one of the Traditional Chinese Medicine widely used for phlegm elimination and detumescence. Arsenic is present in high concentration in seaweed belonging to the genus Sargassum. Therefore, the consumption of S. pallidum is a route of exposure to arsenic. Since the toxicity of arsenic is highly dependent on its chemical speciation, the determination of total arsenic is not adequate to assess the risks. Here, a high performance liquid chromatography-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS) was developed for determination of the common arsenic species including arsenite [As(III)], dimethylarsinate (DMA), methylarsonate (MMA) and arsenate [As(V)] simultaneously. This method was applied to study the pharmacokinetic profile of these arsenic species in rats after oral administration of S. pallidum extract at different doses. The described assay was validated for limit of quantification, linearity, intra-day and inter-day precisions, accuracy, extraction recovery and stability according to the FDA validation guidelines. As(III) or MMA was not detected in any samples collected at all time points using the present HPLC-HG-AFS method. As(V) and DMA in the S. pallidum could be readily absorbed and eliminated in rats. A trend of dose-dependence was shown for DMA and As(V) in the drug concentration–time profiles. This study would be helpful for the apprehension of the action mechanism and clinical application of medicinal seaweeds.

Adsorption and photocatalytic decomposition of roxarsone by TiO2 and its mechanism

Roxarsone (3-nitro-4-hydroxyphenylarsonic acid) has been widely used as organic arsenic additive in animal industry. In this study, the adsorption of roxarsone on TiO₂ under dark conditions, the photocatalytic decomposition of roxarsone under UV/TiO₂, and the possible photocatalytic pathway were investigated. At the initial concentration of 5-35 mg/L, the adsorption of roxarsone fitted well with the pseudo-second-order kinetics. The isotherms analysis showed that the Langmuir model was better than the Freundlich and Dubinin–Radushkevich models for describing the adsorption process. After 7 h of photocatalytic decomposition, a complete disappearance of roxarsone was achieved. The pH value has a significant effect on both adsorption and photocatalytic decomposition of roxarsone. The results of high-performance liquid chromatography-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS) and gas chromatography-mass spectrometry (GC/MS) analyses proved the cleavage of the As-C bond during the photocatalytic decomposition process by TiO2 and the intermediates of the decomposition. Based on the results, a possible photocatalytic decomposition pathway was proposed.

Liquid chromatography–hydride generation–atomic fluorescence spectrometry determination of arsenic species in dog plasma and its application to a pharmacokinetic study after oral administration of Realgar and Niu Huang Jie Du Pian

A high performance liquid chromatography-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS) method was developed for the simultaneous determination of four arsenic species (As(III), dimethylarsinic acid (DMA), monomethylarsonic acid (MMA) and arsenate As(V)) in dog plasma. Good separation of the four arsenic species was achieved within 15min on an anion-exchange column with isocratic elution using 15mmol/L KH(2)PO(4) (pH 5.9) as eluent at a flow rate of 1.0mL/min. The assay was linear over the range of 1.25-200, 1.56-200, 1.34-172, and 2.50-200ng/mL with the detection limits of 0.80, 1.00, 0.86 and 2.00ng/mL for As(III), DMA, MMA and As(V), respectively. The method was validated for selectivity, precision, accuracy and recovery and then applied to a comparative pharmacokinetic study of the arsenic species in beagle dogs after a single oral administration of Realgar (24.32mg/kg, equivalent to 11.31mgAs/kg) alone or Niu Huang Jie Du Pian (a patent traditional Chinese medicine (TCM), 380mg/kg, equivalent to 28.45mgAs/kg), respectively. DMA was found to be the predominant species in the dog plasma after dosing, with As(V) appeared as the quickly eliminating one. No traces of MMA and As(III) were detected at any sampling time points. The main pharmacokinetic parameters found for DMA p.o. administration of Realgar and Niu Huang Jie Du Pian were as follows: C(max) (14.7±4.2) and (57.0±32.0)ng/mL, T(max) (2.4±0.5) and (2.5±0.5)h, AUC(0-36) (151.1±12.9) and (635.9±418.2)ngh/mL, AUC(0-∞) (206.0±44.5) and (687.2±425.1)ngh/mL, t(1/2) (16.2±7.9) and (9.4±2.2)h, respectively. The influence of compounding in Niu Huang Jie Du Pian on the pharmacokinetics of arsenics was shown with increased transformation of DMA and its faster elimination rate.

Health implications of the distribution of arsenic species in airborne particulate matter

Airborne particle samples were taken between 2001 and 2008 at an urban site (Huelva) in southwestern Spain. Arsenic was found in the samples due to the presence of a near-by copper smelter, sometimes at concentrations above the target value of 6ngm−3 proposed by EU regulations (annual means from 4.6 to 10.4ngAsm−3 in PM10, and 3.0 to 9.1ngAsm−3 in PM2.5). The results obtained by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) showed that arsenic accumulates preferentially (ca. 70–80%) in the particles with smaller diameter (PM2.5 versus PM10), representing a threat to human health due to the higher capacity of the finer particles to enter the organism through the respiratory system. Moreover, the toxicity of the inorganic arsenic species depends also on the oxidation state, As(III) being more toxic that As(V). The speciation analysis performed with High Performance Liquid Chromatography-Hydride Generation- Atomic Fluorescence Spectrometry (HPLC-HG-AFS) with samples collected between 2006 and 2008, showed that As(V) represented the main arsenic species, but As(III) was also found at significant concentration, representing a 5–10% of the total arsenic content. The results also indicate that the more toxic As(III) tends to concentrate preferentially in the finer fraction PM2.5 in comparison with As(V), thus representing an added health risk for the local population.

Detection of inorganic arsenic in seaweed food using HPLC-HG-AFS

The inorganic arsenic(iAs) in seaweed food is an important safety index in seafood quality detection. High performance liquid chromatography-hydride generation-atomic fluorescence spectrometry(HPLC-HG-AFS) was used to determine the iAs in seaweed food. The concentration of extraction reagents,the extraction time and instrument conditions were optimized. A new method to determine the content of inorganic arsenic in seaweed food using HPLC-HG-AFS was obtained:firstly the seaweeds were extracted with 1.2 mol/L HCl at 70 ℃ for 1 h ,then the extracts were oxidized with hydrogen peroxide,and finally were analyzed by HPLC-HG-AFS under the condition of 15 mmol/L(NH4) 2HPO4 flow phase(pH=6.0). The average recovery rates of 0.10 mg/kg and 1.00 mg/kg AsⅢ in samples were all above 92% and the relative standard deviations were all below 4% with a high accuracy. The present study proved that HPLC-HG-AFS was a reliable method to determine the iAs in seaweed food and provide enough evidences for the establishment of the standard for iAs in seaweed food.

Determination of Arsenic in the Rainbow Trout Muscle and Rice Samples

The content of arsenic was determined in the rainbow trout muscle (&lt;I&gt;Oncorhynchus mykiss) &lt;/I&gt; and in rice samples. The method of atomic absorption spectrometry with hydride generation (HG-AAS) in graphite furnace was used for total arsenic determination. Arsenic speciation was done using the high performance liquid chromatography-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS). The rainbow trout muscle was extracted by ultra pure deionised water; trifluoroacetic acid was used for the extraction of rice samples. Total arsenic values ranged from 60.90 &amp;mu;g/kg to 310.00 &amp;mu;g/kg in rice samples and from 0.72 mg/kg to 2.23 mg/kg in rainbow trout muscle. An inorganic trivalent arsenic As(III) was determined as the main species in rice and non-toxic arsenobetaine in the samples of rainbow trout.

Evaluation of the extraction methods for arsenic speciation in rice straw, Oryza sativa L., and analysis by HPLC-HG-AFS

The most important and difficult step during arsenic speciation analysis is to extract the various arsenic species in the original forms from the samples. In this work, a study has been carried out to compare the different extraction methods by using different extraction solvents including water, methanol, water–methanol, water–ethanol and water–acetonitrile. Different techniques (shaking, ultrasonic, Soxhlet and microwave-assisted extraction) were applied during the procedure. A new, nontoxic and effective extraction method for arsenic speciation in the terrestrial plant material, rice straw, with water–ethanol mixture as extraction solvent using microwave-assisted extraction was found to provide good extraction efficiency without internal species conversion. The extraction of arsenic from the rice straw and subsequent determination of individual arsenic species by high performance liquid chromatography-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS) is described. By using the proposed extraction method, the total arsenic extraction efficiency was 90.3%. The arsenic species in rice straw were analyzed and four normal chemical forms, AsIII, AsV, MMAV and DMAV, in straw shoots were found, but no MMAV was found in the straw roots.

Glutathione-dependent reduction of arsenate in human erythrocytes--a process independent of purine nucleoside phosphorylase.

Reduction of arsenate (AsV) to the more toxic arsenite (AsIII) is toxicologically important, yet its mechanism is unknown. To clarify this, AsV reduction was investigated in human red blood cells (RBC), as they possess a simple metabolism. RBC were incubated with AsV in gluconate buffer, and the formed AsIII was quantified by high performance liquid chromatography-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS). The observations are compatible with the following conclusions. (1) Human RBC reduce AsV intracellularly, because 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, inhibitor of the chloride-bicarbonate exchanger, which also mediates phosphate and AsV uptake), as well as chloride and phosphate, countered AsIII formation. (2) Purine nucleoside phosphorylase (PNP), whose AsV reductase activity has been directly demonstrated, cannot be a physiologically relevant AsV reductase, because its inhibitor (BCX-1777) failed to decrease the basal erythrocytic AsV reduction, although it prevented the increase in AsIII formation caused by artificial activation of PNP with inosine and dithiothreitol. (3) The basal (PNP-independent) AsV reduction requires glutathione (GSH), because the GSH depletor diethylmaleate strongly diminished AsIII formation. (4) The erythrocytic AsV reduction apparently depends on NAD(P) supply, because oxidants of NAD(P)H (i.e., pyruvate, ferricyanide, methylene blue, nitrite, tert-butylhydroperoxide, dehydroascorbate, 4-dimethylaminophenol) enhanced AsIII formation from AsV. The oxidant-stimulated AsV reduction is PNP-independent, because BCX-1777 failed to affect it, but is GSH-dependent, because diethylmaleate impaired it. (5) Pyruvate-induced glucose depletion, which causes NAD enrichment in the erythrocytes at the expense of NADH, enhanced AsV reduction. This suggests that the erythrocytic AsV reduction requires both NAD supply and operation of the lower part of the glycolytic pathway starting from glyceraldehyde-3-phosphate dehydrogenase (GAPDH) that, unlike the upper part, remains fed with substrates originating from the degradation of 2,3-bisphosphoglycerate in RBC depleted of glucose by pyruvate. (6) Fluoride, which arrests glycolysis at enolase and thus prevents NAD formation, inhibited AsV reduction in glucose-sufficient RBC, but increased it in glucose-deficient (NAD-enriched) cells, suggesting that the section of glycolysis coupled to AsV reduction lies between GAPDH and enolase. In conclusion, besides the artificial PNP-dependent AsV reduction, human RBC contain a PNP-independent AsV-reducing mechanism. This appears to require the supply of GSH, NAD, and substrate to one or more of the glycolytic enzymes localized between GAPDH and enolase.