On-line separation and identification of inorganic and organic arsenic species in ethanolic kelp and bladderwrack extracts through liquid chromatography/particle beam-electron ionization mass spectrometry (LC/PB-EIMS)

Abstract

Ion-pair, reversed-phase high-performance liquid chromatography (IP-RP-HPLC) and ion exchange chromatography (IEC), coupled to particle beam-electron ionization mass spectrometry (PB-EIMS), are employed for the separation and identification of inorganic and organic arsenic compounds (As (III) chloride, arsenobetaine (AB), dimethylarsinic acid (DMA) and an arsenosugar (oxo-arsenosugar-glycerol, As 328) in commercial ethanolic kelp and bladderwrack extracts. The reversed-phase separation was performed on a C₁₈ column using an isocratic mobile phase composition of water:methanol (96:4) containing 0.1% of trifluoroacetic acid (TFA) as an ion-pairing agent. The flow rate was optimized at 0.7 mL min⁻¹ and the separation accomplished in less than 8 minutes. Baseline resolution was achieved in an elution window of about 4 minutes. The ion-exchange separation was performed on an anion-exchange column using a gradient mobile phase composition of 0.5 mM nitric acid containing 2% methanol and 50 mM nitric acid. The separation was accomplished in less than 8 minutes at a flow rate of 0.9 mL min⁻¹. The eluted species were detected and identified using a PB-EIMS system providing species-specific information. The influence of various instrument parameters was evaluated to achieve optimal mass spectrometric response for the test compounds. The absolute detection limits of arsenic species with HPLC/PB-EIMS were 0.03, 0.05, 0.008 ng and 0.005 (5 µL injection) in the SIM mode and 0.1, 0.14, 0.04 and 0.01 ng in the TIC mode for As (III), DMA, AB and As 328 respectively. These investigations revealed that the majority (90–95%) of the arsenic present in the ethanolic extract samples is in the form of inorganic arsenic, with minor amounts of As (5–10% of the total As) present in the form of DMA. There were no detectable levels of AB and arsenosugars. The total arsenic content in the ethanolic extracts was verified by ICP-OES and validated by analyzing NIST SRM 3241 Ephedra sinica Stapf Native Extract and SRM 3243 Ephedra-Containing Solid Oral Dosage Form. These investigations indicate that PB-EIMS is a viable approach for comprehensive speciation of various botanical extracts.