Stability of arsenic peptides in plant extracts

off-line versus on-line parallel elemental and molecular mass spectrometric detection for liquid chromatographic separation

Katharina Bluemlein, Andrea Raab, Jorg Feldmann (Corresponding Author)

Research output: Contribution to journalArticle

68 Citations (Scopus)

Abstract

The instability of metal and metalloid complexes during analytical processes has always been an issue of an uncertainty regarding their speciation in plant extracts. Two different speciation protocols were compared regarding the analysis of arsenic phytochelatin ((AsPC)-P-III) complexes in fresh plant material. As the final step for separation/detection both methods used RP-HPLC simultaneously coupled to ICP-MS and ES-MS. However, one method was the often used off-line approach using two-dimensional separation, i.e. a pre-cleaning step using size-exclusion chromatography with subsequent fraction collection and freeze-drying prior to the analysis using RP-HPLC-ICP-MS and/or ES-MS. This approach revealed that less than 2% of the total arsenic was bound to peptides such as phytochelatins in the root extract of an arsenate exposed Thunbergia alata, whereas the direct on-line method showed that 83% of arsenic was bound to peptides, mainly as (AsPC3)-P-III and (GS) (AsPC2)-P-III. Key analytical factors were identified which destabilise the (AsPCs)-P-III. The low pH of the mobile phase (0.1% formic acid) using RP-HPLC-ICP-MS/ES-MS stabilises the arsenic peptide complexes in the plant extract as well as the free peptide concentration, as shown by the kinetic disintegration study of the model compound As-III(GS)(3) at pH 2.2 and 3.8. But only short half-lives of only a few hours were determined for the arsenic glutathione complex. Although (AsPC3)-P-III showed a ten times higher half-life (23 h) in a plant extract, the precleaning step with subsequent fractionation in a mobile phase of pH 5.6 contributes to the destabilisation of the arsenic peptides in the off-line method. Furthermore, it was found that during a freeze-drying process more than 90% of an (AsPC3)-P-III complex and smaller free peptides such as PC2 and PC3 can be lost. Although the two-dimensional off-line method has been used successfully for other metal complexes, it is concluded here that the fractionation and the subsequent freeze-drying were responsible for the loss of arsenic phytochelatin complexes during the analysis. Hence, the on-line HPLC-ICP-MS/ES-MS is the preferred method for such unstable peptide complexes. Since freeze-drying has been found to be undesirable for sample storage other methods for sample handling needed to be investigated. Hence, the storage of the fresh plant at low temperature was tested. We can report for the first time a storage method which successfully conserves the integrity of the labile arsenic phytochelatin complexes: quantitative recovery of AsIIIPC3 in a formic acid extract of a Thunbergia alata exposed for 24 h to 1 mg As-v L-1 was found when the fresh plant was stored for 21 days at 193 K.

Original languageEnglish
Pages (from-to)357-366
Number of pages10
JournalAnalytical and Bioanalytical Chemistry
Volume393
Issue number1
DOIs
Publication statusPublished - Jan 2009

Keywords

  • Phytochelatins
  • Electrospray mass spectrometry
  • Inductively coupled plasma mass spectrometry
  • Speciation
  • Sample preparation
  • Freeze-drying
  • Hyphenated techniques
  • Chromatography
  • Plants
  • glutathione complex-formation
  • heavy-metal detoxification
  • HPLC-ICP-MS
  • phytochelatin complexes
  • speciation analysis
  • helianthus-annuus
  • tolerance
  • toxicity
  • cadmium
  • binding

Cite this

@article{2bea147ba6484ac18de5de5c8c4d5ac6,
title = "Stability of arsenic peptides in plant extracts: off-line versus on-line parallel elemental and molecular mass spectrometric detection for liquid chromatographic separation",
abstract = "The instability of metal and metalloid complexes during analytical processes has always been an issue of an uncertainty regarding their speciation in plant extracts. Two different speciation protocols were compared regarding the analysis of arsenic phytochelatin ((AsPC)-P-III) complexes in fresh plant material. As the final step for separation/detection both methods used RP-HPLC simultaneously coupled to ICP-MS and ES-MS. However, one method was the often used off-line approach using two-dimensional separation, i.e. a pre-cleaning step using size-exclusion chromatography with subsequent fraction collection and freeze-drying prior to the analysis using RP-HPLC-ICP-MS and/or ES-MS. This approach revealed that less than 2{\%} of the total arsenic was bound to peptides such as phytochelatins in the root extract of an arsenate exposed Thunbergia alata, whereas the direct on-line method showed that 83{\%} of arsenic was bound to peptides, mainly as (AsPC3)-P-III and (GS) (AsPC2)-P-III. Key analytical factors were identified which destabilise the (AsPCs)-P-III. The low pH of the mobile phase (0.1{\%} formic acid) using RP-HPLC-ICP-MS/ES-MS stabilises the arsenic peptide complexes in the plant extract as well as the free peptide concentration, as shown by the kinetic disintegration study of the model compound As-III(GS)(3) at pH 2.2 and 3.8. But only short half-lives of only a few hours were determined for the arsenic glutathione complex. Although (AsPC3)-P-III showed a ten times higher half-life (23 h) in a plant extract, the precleaning step with subsequent fractionation in a mobile phase of pH 5.6 contributes to the destabilisation of the arsenic peptides in the off-line method. Furthermore, it was found that during a freeze-drying process more than 90{\%} of an (AsPC3)-P-III complex and smaller free peptides such as PC2 and PC3 can be lost. Although the two-dimensional off-line method has been used successfully for other metal complexes, it is concluded here that the fractionation and the subsequent freeze-drying were responsible for the loss of arsenic phytochelatin complexes during the analysis. Hence, the on-line HPLC-ICP-MS/ES-MS is the preferred method for such unstable peptide complexes. Since freeze-drying has been found to be undesirable for sample storage other methods for sample handling needed to be investigated. Hence, the storage of the fresh plant at low temperature was tested. We can report for the first time a storage method which successfully conserves the integrity of the labile arsenic phytochelatin complexes: quantitative recovery of AsIIIPC3 in a formic acid extract of a Thunbergia alata exposed for 24 h to 1 mg As-v L-1 was found when the fresh plant was stored for 21 days at 193 K.",
keywords = "Phytochelatins, Electrospray mass spectrometry, Inductively coupled plasma mass spectrometry, Speciation, Sample preparation, Freeze-drying, Hyphenated techniques, Chromatography, Plants, glutathione complex-formation, heavy-metal detoxification, HPLC-ICP-MS, phytochelatin complexes, speciation analysis, helianthus-annuus, tolerance, toxicity, cadmium, binding",
author = "Katharina Bluemlein and Andrea Raab and Jorg Feldmann",
year = "2009",
month = "1",
doi = "10.1007/s00216-008-2395-z",
language = "English",
volume = "393",
pages = "357--366",
journal = "Analytical and Bioanalytical Chemistry",
issn = "1618-2642",
publisher = "Springer Verlag",
number = "1",

}

TY - JOUR

T1 - Stability of arsenic peptides in plant extracts

T2 - off-line versus on-line parallel elemental and molecular mass spectrometric detection for liquid chromatographic separation

AU - Bluemlein, Katharina

AU - Raab, Andrea

AU - Feldmann, Jorg

PY - 2009/1

Y1 - 2009/1

N2 - The instability of metal and metalloid complexes during analytical processes has always been an issue of an uncertainty regarding their speciation in plant extracts. Two different speciation protocols were compared regarding the analysis of arsenic phytochelatin ((AsPC)-P-III) complexes in fresh plant material. As the final step for separation/detection both methods used RP-HPLC simultaneously coupled to ICP-MS and ES-MS. However, one method was the often used off-line approach using two-dimensional separation, i.e. a pre-cleaning step using size-exclusion chromatography with subsequent fraction collection and freeze-drying prior to the analysis using RP-HPLC-ICP-MS and/or ES-MS. This approach revealed that less than 2% of the total arsenic was bound to peptides such as phytochelatins in the root extract of an arsenate exposed Thunbergia alata, whereas the direct on-line method showed that 83% of arsenic was bound to peptides, mainly as (AsPC3)-P-III and (GS) (AsPC2)-P-III. Key analytical factors were identified which destabilise the (AsPCs)-P-III. The low pH of the mobile phase (0.1% formic acid) using RP-HPLC-ICP-MS/ES-MS stabilises the arsenic peptide complexes in the plant extract as well as the free peptide concentration, as shown by the kinetic disintegration study of the model compound As-III(GS)(3) at pH 2.2 and 3.8. But only short half-lives of only a few hours were determined for the arsenic glutathione complex. Although (AsPC3)-P-III showed a ten times higher half-life (23 h) in a plant extract, the precleaning step with subsequent fractionation in a mobile phase of pH 5.6 contributes to the destabilisation of the arsenic peptides in the off-line method. Furthermore, it was found that during a freeze-drying process more than 90% of an (AsPC3)-P-III complex and smaller free peptides such as PC2 and PC3 can be lost. Although the two-dimensional off-line method has been used successfully for other metal complexes, it is concluded here that the fractionation and the subsequent freeze-drying were responsible for the loss of arsenic phytochelatin complexes during the analysis. Hence, the on-line HPLC-ICP-MS/ES-MS is the preferred method for such unstable peptide complexes. Since freeze-drying has been found to be undesirable for sample storage other methods for sample handling needed to be investigated. Hence, the storage of the fresh plant at low temperature was tested. We can report for the first time a storage method which successfully conserves the integrity of the labile arsenic phytochelatin complexes: quantitative recovery of AsIIIPC3 in a formic acid extract of a Thunbergia alata exposed for 24 h to 1 mg As-v L-1 was found when the fresh plant was stored for 21 days at 193 K.

AB - The instability of metal and metalloid complexes during analytical processes has always been an issue of an uncertainty regarding their speciation in plant extracts. Two different speciation protocols were compared regarding the analysis of arsenic phytochelatin ((AsPC)-P-III) complexes in fresh plant material. As the final step for separation/detection both methods used RP-HPLC simultaneously coupled to ICP-MS and ES-MS. However, one method was the often used off-line approach using two-dimensional separation, i.e. a pre-cleaning step using size-exclusion chromatography with subsequent fraction collection and freeze-drying prior to the analysis using RP-HPLC-ICP-MS and/or ES-MS. This approach revealed that less than 2% of the total arsenic was bound to peptides such as phytochelatins in the root extract of an arsenate exposed Thunbergia alata, whereas the direct on-line method showed that 83% of arsenic was bound to peptides, mainly as (AsPC3)-P-III and (GS) (AsPC2)-P-III. Key analytical factors were identified which destabilise the (AsPCs)-P-III. The low pH of the mobile phase (0.1% formic acid) using RP-HPLC-ICP-MS/ES-MS stabilises the arsenic peptide complexes in the plant extract as well as the free peptide concentration, as shown by the kinetic disintegration study of the model compound As-III(GS)(3) at pH 2.2 and 3.8. But only short half-lives of only a few hours were determined for the arsenic glutathione complex. Although (AsPC3)-P-III showed a ten times higher half-life (23 h) in a plant extract, the precleaning step with subsequent fractionation in a mobile phase of pH 5.6 contributes to the destabilisation of the arsenic peptides in the off-line method. Furthermore, it was found that during a freeze-drying process more than 90% of an (AsPC3)-P-III complex and smaller free peptides such as PC2 and PC3 can be lost. Although the two-dimensional off-line method has been used successfully for other metal complexes, it is concluded here that the fractionation and the subsequent freeze-drying were responsible for the loss of arsenic phytochelatin complexes during the analysis. Hence, the on-line HPLC-ICP-MS/ES-MS is the preferred method for such unstable peptide complexes. Since freeze-drying has been found to be undesirable for sample storage other methods for sample handling needed to be investigated. Hence, the storage of the fresh plant at low temperature was tested. We can report for the first time a storage method which successfully conserves the integrity of the labile arsenic phytochelatin complexes: quantitative recovery of AsIIIPC3 in a formic acid extract of a Thunbergia alata exposed for 24 h to 1 mg As-v L-1 was found when the fresh plant was stored for 21 days at 193 K.

KW - Phytochelatins

KW - Electrospray mass spectrometry

KW - Inductively coupled plasma mass spectrometry

KW - Speciation

KW - Sample preparation

KW - Freeze-drying

KW - Hyphenated techniques

KW - Chromatography

KW - Plants

KW - glutathione complex-formation

KW - heavy-metal detoxification

KW - HPLC-ICP-MS

KW - phytochelatin complexes

KW - speciation analysis

KW - helianthus-annuus

KW - tolerance

KW - toxicity

KW - cadmium

KW - binding

U2 - 10.1007/s00216-008-2395-z

DO - 10.1007/s00216-008-2395-z

M3 - Article

VL - 393

SP - 357

EP - 366

JO - Analytical and Bioanalytical Chemistry

JF - Analytical and Bioanalytical Chemistry

SN - 1618-2642

IS - 1

ER -