Performance Liquid Chromatography-hydride Generation-atomic Fluorescence Research Articles

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.

Effect of continuous venovenous haemodialysis on outcome and pharmacokinetics of arsenic species in a patient with acute promyelocytic leukaemia and acute kidney injury.

This study presents outcome and pharmacokinetics of arsenic trioxide (ATO) metabolites in patients on continuous venovenous haemodialysis (CVVHD). Of 3 acute promyelocytic leukaemia patients receiving CVVHD in management of acute kidney injury, only 1 patient was included. The patient presented disseminated intravascular coagulation and acute kidney injury before induction therapy was conducted. CVVHD was performed and ATO was initiated. Species of ATO metabolites in plasma and effluent were analysed using high performance liquid chromatography-hydride generation-atomic fluorescence spectrometry. Plasma concentrations of AsIII , monomethylarsonic acid and dimethylarsinic acid with CVVHD were lower than those without CVVHD. Area under the concentration-time curve from 0 to the last sample with quantifiable concentration of AsIII without CVVHD was significantly higher than that with CVVHD (292.10ngh/mL vs 195.86ngh/mL, P=.037), which were not observed for monomethylarsonic acid and dimethylarsinic acid. Dialysate saturation of arsenic species was remarkable, especially for AsIII . Complete remission was achieved and renal function recovered. In this study, ATO can be used safely and effectively to treat acute promyelocytic leukaemia patients undergoing CVVHD without dose adjustment.

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.

Determination of Inorganic Arsenic Speciation in Municipal Solid Waste Incineration Fly Ash by High Performance Liquid Chromatography-Hydride Generation-Atomic Fluorescence Spectroscopy with Phosphoric Acid as Extracting Agent

A method for the determination of arsenic speciation in municipal solid waste incineration fly ash was developed by high performance liquid chromatography (HPLC) coupled with hydride generation atomic fluorescence spectroscopy (HG-AFS). Before determination, various forms of arsenic were extracted with phosphoric acid through microwave digestion. The effect of phosphoric acid in the extracted solution on the speciation analysis of inorganic arsenic (As(III)/As(V)) was studied in detail. The results showed that when using diammonium phosphate as the mobile phase, the determination of As(V) was adversely affected. The increasing flow rate of mobile phase hardly reduced the adverse influence caused by phosphoric acid. The adverse influence was effectively suppressed when a mixed solution of disodium hydrogen phosphate/potassium dihydrogen phosphate (pH = 6.864) was used as the mobile phase. By using the improved method, As(III) and As(V) could be separated in 5 min, and the detection limits of As(III) and As(V) were 0.063 μg L−1 and 0.110 μg L−1, respectively. The recovery rate of As(III) and As(V) in spiked standard sample was satisfactory (86.9%–102.6%). The determination of arsenic species in fly ashes sampled from four different municipal solid waste incineration plants were successfully accomplished by using the method. The results showed that the arsenic was predominantly in the form of As(V).

Speciation of Arsenic in Rice by High-Performance Liquid Chromatography–Hydride Generation-Atomic Fluorescence Spectrometry with Microwave-Assisted Extraction

Arsenic speciation in paddy rice is of considerable interest due to its impact on the food safety and human health. In this study, a simple methodology was developed to simultaneously extract and analyze As species in rice from China. Arsenic species, including arsenite (As(III)), arsenate (As(V)), dimethylarsinic acid (DMA), and monomethylarsonic acid (MMA), were extracted by methanol-water (50:50, v/v) containing 0.02 mol L−1 nitric acid with a microwave-assisted procedure, and then determined by high performance liquid chromatography–hydride generation-atomic fluorescence spectrometry (HPLC–HG-AFS). The results showed that the method has good efficiency (>90%) for rice, indicating that there were no significant losses or transformations of arsenic during sample treatment and analysis. The limits of quantification (LOQ) of the method were 8.0, 20, 12, and 12 ng g−1 for As(III), As(V), DMA, and MMA, respectively. When this method was applied to the analysis of rice, As(III) had the highest concentration, followed by DMA, As(V), and MMA. The estimated weekly intake of inorganic As from rice by Chinese people accounted for 11.83% of the provisional tolerable weekly intake. The As speciation results in this study suggest that the risk associated with As in rice to human health may be negligible.

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.

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.

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.

Simultaneous speciation analysis of Sb(III), Sb(V) and (CH 3) 3SbCl 2 by high performance liquid chromatography-hydride generation-atomic fluorescence spectrometry detection (HPLC-HG-AFS): Application to antimony speciation in sea water

This paper presents an improvement for the simultaneous separation of Sb(V), Sb(III) and (CH 3) 3SbCl 2 species by high performance liquid chromatography (HPLC) and its detection by hydride generation-atomic fluorescence spectrometry (HG-AFS). The separation was performed on an anion exchange column PRP-X100 using a gradient elution program between EDTA/KHP (potasium hydrogen phtalate) as first mobile phase and phosphate solutions solution as the second one. The chromatographic separation and the HG-AFS parameters were optimized by experimental design. The best results were obtained by using an elution program with 20 mmol l −1 EDTA + 2 mmol l −1 KHP solution at pH 4.5, during 1.15 min, then change to 50 mmol l −1 (NH 4) 2HPO 4 solution at pH 8.3, switching back after 4.0 min to the first mobile phase, until 5 min, with a constant flow rate of 1.5 ml min −1. Retention time of Sb(V), Sb(III) and trimethylantimony species were 1.22, 2.31 and 3.45 min and the detection limits were 0.13; 0.07 and 0.13 μg l −1, respectively. Studies on the stability of this antimony species in sea water samples on the function of the elapsed time of storage in refrigerator at 4 °C was performed employing the optimized method. Results revealed that Sb(III) is easily oxidized within some hours to Sb(V) in sea water stored at 4 °C. However, when the sea water was immediately mixed with EDTA no oxidation of Sb(III) was observed up to 1 week of storage. The proposed methodology was then applied to the antimony speciation in sea water samples.

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.

Speciation of the immediately mobilisable As(III), As(V), MMA and DMA in river sediments by high performance liquid chromatography–hydride generation–atomic fluorescence spectrometry following ultrasonic extraction

In this work, a fast method is developed for the speciation of As(III), As(V), MMA and DMA in the immediately mobilisable fraction of river sediments (i.e. water-soluble and phosphate-exchangeable) by high performance liquid chromatography–hydride generation–atomic fluorescence detection (HPLC–HG–AFD) after extraction using focused ultrasound. The influence of relevant parameters influencing an ion-pairing chromatographic separation following isocratic elution (i.e. amount of MeOH in the mobile phase, ion pair reagent concentration, pH, flow rate) was studied. Focused ultrasound transmitted from an ultrasonic probe provided the same extractable contents as conventional extraction with no changes in the species distribution. The effect of the drying step over extraction of As species was investigated. The following drying procedures were compared: freeze-, oven-, microwave- and air-drying. No influence of the drying operation on the water-extractable fraction was observed. However, freeze- and air-drying yielded significantly higher phosphate-extractable amounts of As(III) and As(V) as compared to oven and microwaves. Detection limits for the As species were in the range 1.3–4.1 ng/g for the water-soluble fraction and 1.6–4.8 ng/g for the phosphate buffer exchangeable fraction. The method was applied to the speciation of immediately mobilisable As(III), As(V), DMA and MMA in 11 sediment samples collected along the beds of the Louro River (southern Galicia, Spain).

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.