High Performance Liquid Chromatographic Technique for the Simultaneous Determination of Lactone and Hydroxy Acid Forms of Camptothecin and SN-38 in Tissue Culture Media and Cancer cells

Gary Boyd, John F. Smyth, Duncan I. Jodrell, Jeffrey Cummings

Research output: Contribution to journalArticle

Abstract

Analysis of camptothecins in biologic media is hampered by chemical hydrolysis of the parent lactone (form I) to an inactive hydroxy acid (form II). A solid-phase extraction (SPE) method utilizing C2-bonded silica particles (100 mg, 1 ml) is presented for simultaneous determination of forms I and II of camptothecin (CPT) and SN-38 (active metabolite of clinically used CPT-11) in culture media and cell lysates. A new HPLC separation is described that efficiently resolves all four compounds employing gradient elution with 10 mM ammonium acetate, increasing methanol (20–80% over 15 min), and a 15-cm by 3-mm Symmetry Shield (RP8) column. Components were detected by fluorescence at an excitation wavelength of 380 nm and emission wavelength of 423 nm. Lactones were shown to be unstable at alkaline pH and hydroxy acids unstable at alkaline pH while the following conditions preserved the chemical equilibrium in specimens: samples kept on ice, final pH of eluates 7.4, autosampler temperature 4°C, and analysis cycle <4 h. Quantitative recovery of lactones was achieved from RPMI culture medium over a wide concentration range (93.5–111.6% for 1–400 ng/ml) although greater variability was noted with the hydroxy acids (59.6–110.3%, 1–400 ng/ml). Limit of quantitation (precision and accuracy <20%) was 0.2 ng/ml for CPT lactone, 0.5 ng/ml for SN-38 lactone, and 2 ng/ml for the two hydroxy acids. The method was applied to quantitate the accumulation of SN-38 and CPT (form I and II) in HT29 and HCT116 human colon cancer cells.
Original languageEnglish
Pages (from-to)15-24
JournalAnalytical Biochemistry
Volume297
Issue number1
DOIs
Publication statusPublished - Oct 2001

Fingerprint

irinotecan
Camptothecin
Tissue culture
Hydroxy Acids
Lactones
Culture Media
Cells
Liquids
Neoplasms
Wavelength
Solid Phase Extraction
Ice
Metabolites
Silicon Dioxide
Colonic Neoplasms
Methanol
Hydrolysis
Fluorescence
High Pressure Liquid Chromatography
Recovery

Keywords

  • camptothecin
  • SN-38
  • HPLC
  • solid-phase extraction
  • colon cancer cells

Cite this

@article{40a489b1be5c4b18b5c97d7020d6d1b7,
title = "High Performance Liquid Chromatographic Technique for the Simultaneous Determination of Lactone and Hydroxy Acid Forms of Camptothecin and SN-38 in Tissue Culture Media and Cancer cells",
abstract = "Analysis of camptothecins in biologic media is hampered by chemical hydrolysis of the parent lactone (form I) to an inactive hydroxy acid (form II). A solid-phase extraction (SPE) method utilizing C2-bonded silica particles (100 mg, 1 ml) is presented for simultaneous determination of forms I and II of camptothecin (CPT) and SN-38 (active metabolite of clinically used CPT-11) in culture media and cell lysates. A new HPLC separation is described that efficiently resolves all four compounds employing gradient elution with 10 mM ammonium acetate, increasing methanol (20–80{\%} over 15 min), and a 15-cm by 3-mm Symmetry Shield (RP8) column. Components were detected by fluorescence at an excitation wavelength of 380 nm and emission wavelength of 423 nm. Lactones were shown to be unstable at alkaline pH and hydroxy acids unstable at alkaline pH while the following conditions preserved the chemical equilibrium in specimens: samples kept on ice, final pH of eluates 7.4, autosampler temperature 4°C, and analysis cycle <4 h. Quantitative recovery of lactones was achieved from RPMI culture medium over a wide concentration range (93.5–111.6{\%} for 1–400 ng/ml) although greater variability was noted with the hydroxy acids (59.6–110.3{\%}, 1–400 ng/ml). Limit of quantitation (precision and accuracy <20{\%}) was 0.2 ng/ml for CPT lactone, 0.5 ng/ml for SN-38 lactone, and 2 ng/ml for the two hydroxy acids. The method was applied to quantitate the accumulation of SN-38 and CPT (form I and II) in HT29 and HCT116 human colon cancer cells.",
keywords = "camptothecin, SN-38, HPLC, solid-phase extraction, colon cancer cells",
author = "Gary Boyd and Smyth, {John F.} and Jodrell, {Duncan I.} and Jeffrey Cummings",
year = "2001",
month = "10",
doi = "10.1006/abio.2001.5317",
language = "English",
volume = "297",
pages = "15--24",
journal = "Analytical Biochemistry",
issn = "0003-2697",
publisher = "Elsevier B.V.",
number = "1",

}

High Performance Liquid Chromatographic Technique for the Simultaneous Determination of Lactone and Hydroxy Acid Forms of Camptothecin and SN-38 in Tissue Culture Media and Cancer cells. / Boyd, Gary; Smyth, John F. ; Jodrell, Duncan I. ; Cummings, Jeffrey.

In: Analytical Biochemistry, Vol. 297, No. 1, 10.2001, p. 15-24.

Research output: Contribution to journalArticle

TY - JOUR

T1 - High Performance Liquid Chromatographic Technique for the Simultaneous Determination of Lactone and Hydroxy Acid Forms of Camptothecin and SN-38 in Tissue Culture Media and Cancer cells

AU - Boyd, Gary

AU - Smyth, John F.

AU - Jodrell, Duncan I.

AU - Cummings, Jeffrey

PY - 2001/10

Y1 - 2001/10

N2 - Analysis of camptothecins in biologic media is hampered by chemical hydrolysis of the parent lactone (form I) to an inactive hydroxy acid (form II). A solid-phase extraction (SPE) method utilizing C2-bonded silica particles (100 mg, 1 ml) is presented for simultaneous determination of forms I and II of camptothecin (CPT) and SN-38 (active metabolite of clinically used CPT-11) in culture media and cell lysates. A new HPLC separation is described that efficiently resolves all four compounds employing gradient elution with 10 mM ammonium acetate, increasing methanol (20–80% over 15 min), and a 15-cm by 3-mm Symmetry Shield (RP8) column. Components were detected by fluorescence at an excitation wavelength of 380 nm and emission wavelength of 423 nm. Lactones were shown to be unstable at alkaline pH and hydroxy acids unstable at alkaline pH while the following conditions preserved the chemical equilibrium in specimens: samples kept on ice, final pH of eluates 7.4, autosampler temperature 4°C, and analysis cycle <4 h. Quantitative recovery of lactones was achieved from RPMI culture medium over a wide concentration range (93.5–111.6% for 1–400 ng/ml) although greater variability was noted with the hydroxy acids (59.6–110.3%, 1–400 ng/ml). Limit of quantitation (precision and accuracy <20%) was 0.2 ng/ml for CPT lactone, 0.5 ng/ml for SN-38 lactone, and 2 ng/ml for the two hydroxy acids. The method was applied to quantitate the accumulation of SN-38 and CPT (form I and II) in HT29 and HCT116 human colon cancer cells.

AB - Analysis of camptothecins in biologic media is hampered by chemical hydrolysis of the parent lactone (form I) to an inactive hydroxy acid (form II). A solid-phase extraction (SPE) method utilizing C2-bonded silica particles (100 mg, 1 ml) is presented for simultaneous determination of forms I and II of camptothecin (CPT) and SN-38 (active metabolite of clinically used CPT-11) in culture media and cell lysates. A new HPLC separation is described that efficiently resolves all four compounds employing gradient elution with 10 mM ammonium acetate, increasing methanol (20–80% over 15 min), and a 15-cm by 3-mm Symmetry Shield (RP8) column. Components were detected by fluorescence at an excitation wavelength of 380 nm and emission wavelength of 423 nm. Lactones were shown to be unstable at alkaline pH and hydroxy acids unstable at alkaline pH while the following conditions preserved the chemical equilibrium in specimens: samples kept on ice, final pH of eluates 7.4, autosampler temperature 4°C, and analysis cycle <4 h. Quantitative recovery of lactones was achieved from RPMI culture medium over a wide concentration range (93.5–111.6% for 1–400 ng/ml) although greater variability was noted with the hydroxy acids (59.6–110.3%, 1–400 ng/ml). Limit of quantitation (precision and accuracy <20%) was 0.2 ng/ml for CPT lactone, 0.5 ng/ml for SN-38 lactone, and 2 ng/ml for the two hydroxy acids. The method was applied to quantitate the accumulation of SN-38 and CPT (form I and II) in HT29 and HCT116 human colon cancer cells.

KW - camptothecin

KW - SN-38

KW - HPLC

KW - solid-phase extraction

KW - colon cancer cells

U2 - 10.1006/abio.2001.5317

DO - 10.1006/abio.2001.5317

M3 - Article

VL - 297

SP - 15

EP - 24

JO - Analytical Biochemistry

JF - Analytical Biochemistry

SN - 0003-2697

IS - 1

ER -