Separation of near-infrared fluorescent conjugates of dATP and related compounds by capillary electrophoresis

Jennifer L. Brocky, David S. Hage

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2 Citations (Scopus)

Abstract

Capillary electrophoresis was examined as a means for the separation and quantitation of deoxyadenosine triphosphate (dATP) and other nucleotides that were labeled with the near-infrared fluorescent dye IRD700 or related tags. Under the final optimized conditions the labeled dATP was separated from several possible impurities, including the unconjugated forms of IRD700 and dATP, as well as dADP, dAMP and their corresponding IRD700 conjugates. The assay was performed under two sets of conditions. First, the sample was injected onto a 50 cmx75 μm I.D. fused-silica capillary at 25 kV in the presence of a pH 9.5, 140 mM borate running buffer. The resulting peaks were monitored at both 254 and 680 nm, where the latter wavelength was used to identify any species that contained the IRD700 label. A second injection was then performed under the same conditions but with a fixed concentration of dTTP now being added to the running buffer; this resulted in the formation of a complex between the dTTP and any dATP, dADP or dAMP-containing components, which changed their rates of migration and allowed them to be differentiated from unconjugated IRD700 or dye contaminants. Only 6 nl of a 1:10 diluted sample were required per analysis. The limit of detection at this injection volume was approximately 1.0 μM (or 6.10-15 mol for a 6-nl injection) for each monitored component. The linear range extended up to at least 80 μM. The analysis time was 20 min per injection and the day-to-day precision was ±2-3%. The same method was also found to be useful in examining related conjugates, such as those based on the dye IRD40. Copyright (C) 2000 Elsevier Science B.V.

Original languageEnglish (US)
Pages (from-to)315-321
Number of pages7
JournalJournal of Chromatography B: Biomedical Sciences and Applications
Volume744
Issue number2
DOIs
StatePublished - Jul 21 2000

Fingerprint

Capillary electrophoresis
Infrared radiation
Buffers
Coloring Agents
Impurities
Borates
Fused silica
Fluorescent Dyes
Labels
Assays
Nucleotides
2'-deoxyadenosine
triphosphoric acid
IRD 700
Wavelength

Keywords

  • Deoxyadenosine triphosphate
  • Nucleotides

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

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title = "Separation of near-infrared fluorescent conjugates of dATP and related compounds by capillary electrophoresis",
abstract = "Capillary electrophoresis was examined as a means for the separation and quantitation of deoxyadenosine triphosphate (dATP) and other nucleotides that were labeled with the near-infrared fluorescent dye IRD700 or related tags. Under the final optimized conditions the labeled dATP was separated from several possible impurities, including the unconjugated forms of IRD700 and dATP, as well as dADP, dAMP and their corresponding IRD700 conjugates. The assay was performed under two sets of conditions. First, the sample was injected onto a 50 cmx75 μm I.D. fused-silica capillary at 25 kV in the presence of a pH 9.5, 140 mM borate running buffer. The resulting peaks were monitored at both 254 and 680 nm, where the latter wavelength was used to identify any species that contained the IRD700 label. A second injection was then performed under the same conditions but with a fixed concentration of dTTP now being added to the running buffer; this resulted in the formation of a complex between the dTTP and any dATP, dADP or dAMP-containing components, which changed their rates of migration and allowed them to be differentiated from unconjugated IRD700 or dye contaminants. Only 6 nl of a 1:10 diluted sample were required per analysis. The limit of detection at this injection volume was approximately 1.0 μM (or 6.10-15 mol for a 6-nl injection) for each monitored component. The linear range extended up to at least 80 μM. The analysis time was 20 min per injection and the day-to-day precision was ±2-3{\%}. The same method was also found to be useful in examining related conjugates, such as those based on the dye IRD40. Copyright (C) 2000 Elsevier Science B.V.",
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T1 - Separation of near-infrared fluorescent conjugates of dATP and related compounds by capillary electrophoresis

AU - Brocky, Jennifer L.

AU - Hage, David S.

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N2 - Capillary electrophoresis was examined as a means for the separation and quantitation of deoxyadenosine triphosphate (dATP) and other nucleotides that were labeled with the near-infrared fluorescent dye IRD700 or related tags. Under the final optimized conditions the labeled dATP was separated from several possible impurities, including the unconjugated forms of IRD700 and dATP, as well as dADP, dAMP and their corresponding IRD700 conjugates. The assay was performed under two sets of conditions. First, the sample was injected onto a 50 cmx75 μm I.D. fused-silica capillary at 25 kV in the presence of a pH 9.5, 140 mM borate running buffer. The resulting peaks were monitored at both 254 and 680 nm, where the latter wavelength was used to identify any species that contained the IRD700 label. A second injection was then performed under the same conditions but with a fixed concentration of dTTP now being added to the running buffer; this resulted in the formation of a complex between the dTTP and any dATP, dADP or dAMP-containing components, which changed their rates of migration and allowed them to be differentiated from unconjugated IRD700 or dye contaminants. Only 6 nl of a 1:10 diluted sample were required per analysis. The limit of detection at this injection volume was approximately 1.0 μM (or 6.10-15 mol for a 6-nl injection) for each monitored component. The linear range extended up to at least 80 μM. The analysis time was 20 min per injection and the day-to-day precision was ±2-3%. The same method was also found to be useful in examining related conjugates, such as those based on the dye IRD40. Copyright (C) 2000 Elsevier Science B.V.

AB - Capillary electrophoresis was examined as a means for the separation and quantitation of deoxyadenosine triphosphate (dATP) and other nucleotides that were labeled with the near-infrared fluorescent dye IRD700 or related tags. Under the final optimized conditions the labeled dATP was separated from several possible impurities, including the unconjugated forms of IRD700 and dATP, as well as dADP, dAMP and their corresponding IRD700 conjugates. The assay was performed under two sets of conditions. First, the sample was injected onto a 50 cmx75 μm I.D. fused-silica capillary at 25 kV in the presence of a pH 9.5, 140 mM borate running buffer. The resulting peaks were monitored at both 254 and 680 nm, where the latter wavelength was used to identify any species that contained the IRD700 label. A second injection was then performed under the same conditions but with a fixed concentration of dTTP now being added to the running buffer; this resulted in the formation of a complex between the dTTP and any dATP, dADP or dAMP-containing components, which changed their rates of migration and allowed them to be differentiated from unconjugated IRD700 or dye contaminants. Only 6 nl of a 1:10 diluted sample were required per analysis. The limit of detection at this injection volume was approximately 1.0 μM (or 6.10-15 mol for a 6-nl injection) for each monitored component. The linear range extended up to at least 80 μM. The analysis time was 20 min per injection and the day-to-day precision was ±2-3%. The same method was also found to be useful in examining related conjugates, such as those based on the dye IRD40. Copyright (C) 2000 Elsevier Science B.V.

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