Determination of O6-benzylguanine in human plasma by reversed-phase high-performance liquid chromatography

Tammy L. Stefan, Stephen T. Ingalls, Stanton L. Gerson, James K V Willson, Charles L. Hoppel

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

A high-performance liquid chromatographic assay for O6-benzylguanine utilizing liquid-liquid extraction and reversed-phase chromatography has been developed. Plasma samples were alkalinized, extracted into ethyl acetate, evaporated, and the residues were reconstituted and chromatographed. Separation was accomplished by gradient elution with a mobile phase of methanol, acetonitrile, and phosphate buffer, pH 3.2. Eluted compounds were detected spectrophotometrically at 280 nm. Sample quantitation was obtained from the regression line of six-point standard curves ranging from 25 to 400 ng/ml. O6-Benzylguanine peak heights were compared to peak heights of O6-(p-chlorobenzyl)guanine (internal standard). The average regression coefficient was 0.999 (n = 4). High concentration (305 ng/ml) and low concentration (38 ng/ml) quality control samples were determined with a day-to-day relative standard deviation of 7 and 8%, respectively (n = 18). The within-day relative standard deviations were 2.7 and 3.0% (n = 18) for the high and low concentration quality control specimens, respectively. Sample quantitation was reliable to 25 ng/ml with a signal-to-noise ratio of 8:1. This method was applied to plasma samples obtained from patients in a clinical trial of O6-benzylguanine.

Original languageEnglish (US)
Pages (from-to)331-338
Number of pages8
JournalJournal of Chromatography B: Biomedical Applications
Volume681
Issue number2
DOIs
StatePublished - Jun 7 1996

Fingerprint

Plasma (human)
High performance liquid chromatography
Quality control
Liquids
Plasmas
Guanine
Chromatography
Methanol
Assays
Signal to noise ratio
Buffers
Phosphates
O(6)-benzylguanine

Keywords

  • O-benzylguanine

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

Determination of O6-benzylguanine in human plasma by reversed-phase high-performance liquid chromatography. / Stefan, Tammy L.; Ingalls, Stephen T.; Gerson, Stanton L.; Willson, James K V; Hoppel, Charles L.

In: Journal of Chromatography B: Biomedical Applications, Vol. 681, No. 2, 07.06.1996, p. 331-338.

Research output: Contribution to journalArticle

Stefan, Tammy L. ; Ingalls, Stephen T. ; Gerson, Stanton L. ; Willson, James K V ; Hoppel, Charles L. / Determination of O6-benzylguanine in human plasma by reversed-phase high-performance liquid chromatography. In: Journal of Chromatography B: Biomedical Applications. 1996 ; Vol. 681, No. 2. pp. 331-338.
@article{8f2e1d47b95245a0baeb3858be3001f4,
title = "Determination of O6-benzylguanine in human plasma by reversed-phase high-performance liquid chromatography",
abstract = "A high-performance liquid chromatographic assay for O6-benzylguanine utilizing liquid-liquid extraction and reversed-phase chromatography has been developed. Plasma samples were alkalinized, extracted into ethyl acetate, evaporated, and the residues were reconstituted and chromatographed. Separation was accomplished by gradient elution with a mobile phase of methanol, acetonitrile, and phosphate buffer, pH 3.2. Eluted compounds were detected spectrophotometrically at 280 nm. Sample quantitation was obtained from the regression line of six-point standard curves ranging from 25 to 400 ng/ml. O6-Benzylguanine peak heights were compared to peak heights of O6-(p-chlorobenzyl)guanine (internal standard). The average regression coefficient was 0.999 (n = 4). High concentration (305 ng/ml) and low concentration (38 ng/ml) quality control samples were determined with a day-to-day relative standard deviation of 7 and 8{\%}, respectively (n = 18). The within-day relative standard deviations were 2.7 and 3.0{\%} (n = 18) for the high and low concentration quality control specimens, respectively. Sample quantitation was reliable to 25 ng/ml with a signal-to-noise ratio of 8:1. This method was applied to plasma samples obtained from patients in a clinical trial of O6-benzylguanine.",
keywords = "O-benzylguanine",
author = "Stefan, {Tammy L.} and Ingalls, {Stephen T.} and Gerson, {Stanton L.} and Willson, {James K V} and Hoppel, {Charles L.}",
year = "1996",
month = "6",
day = "7",
doi = "10.1016/0378-4347(95)00537-4",
language = "English (US)",
volume = "681",
pages = "331--338",
journal = "Journal of Chromatography - Biomedical Applications",
issn = "0378-4347",
publisher = "Elsevier BV",
number = "2",

}

TY - JOUR

T1 - Determination of O6-benzylguanine in human plasma by reversed-phase high-performance liquid chromatography

AU - Stefan, Tammy L.

AU - Ingalls, Stephen T.

AU - Gerson, Stanton L.

AU - Willson, James K V

AU - Hoppel, Charles L.

PY - 1996/6/7

Y1 - 1996/6/7

N2 - A high-performance liquid chromatographic assay for O6-benzylguanine utilizing liquid-liquid extraction and reversed-phase chromatography has been developed. Plasma samples were alkalinized, extracted into ethyl acetate, evaporated, and the residues were reconstituted and chromatographed. Separation was accomplished by gradient elution with a mobile phase of methanol, acetonitrile, and phosphate buffer, pH 3.2. Eluted compounds were detected spectrophotometrically at 280 nm. Sample quantitation was obtained from the regression line of six-point standard curves ranging from 25 to 400 ng/ml. O6-Benzylguanine peak heights were compared to peak heights of O6-(p-chlorobenzyl)guanine (internal standard). The average regression coefficient was 0.999 (n = 4). High concentration (305 ng/ml) and low concentration (38 ng/ml) quality control samples were determined with a day-to-day relative standard deviation of 7 and 8%, respectively (n = 18). The within-day relative standard deviations were 2.7 and 3.0% (n = 18) for the high and low concentration quality control specimens, respectively. Sample quantitation was reliable to 25 ng/ml with a signal-to-noise ratio of 8:1. This method was applied to plasma samples obtained from patients in a clinical trial of O6-benzylguanine.

AB - A high-performance liquid chromatographic assay for O6-benzylguanine utilizing liquid-liquid extraction and reversed-phase chromatography has been developed. Plasma samples were alkalinized, extracted into ethyl acetate, evaporated, and the residues were reconstituted and chromatographed. Separation was accomplished by gradient elution with a mobile phase of methanol, acetonitrile, and phosphate buffer, pH 3.2. Eluted compounds were detected spectrophotometrically at 280 nm. Sample quantitation was obtained from the regression line of six-point standard curves ranging from 25 to 400 ng/ml. O6-Benzylguanine peak heights were compared to peak heights of O6-(p-chlorobenzyl)guanine (internal standard). The average regression coefficient was 0.999 (n = 4). High concentration (305 ng/ml) and low concentration (38 ng/ml) quality control samples were determined with a day-to-day relative standard deviation of 7 and 8%, respectively (n = 18). The within-day relative standard deviations were 2.7 and 3.0% (n = 18) for the high and low concentration quality control specimens, respectively. Sample quantitation was reliable to 25 ng/ml with a signal-to-noise ratio of 8:1. This method was applied to plasma samples obtained from patients in a clinical trial of O6-benzylguanine.

KW - O-benzylguanine

UR - http://www.scopus.com/inward/record.url?scp=0029661926&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029661926&partnerID=8YFLogxK

U2 - 10.1016/0378-4347(95)00537-4

DO - 10.1016/0378-4347(95)00537-4

M3 - Article

VL - 681

SP - 331

EP - 338

JO - Journal of Chromatography - Biomedical Applications

JF - Journal of Chromatography - Biomedical Applications

SN - 0378-4347

IS - 2

ER -