An internal standard method for the unattended high-performance liquid chromatographic analysis of ascorbic acid in blood components

Mark A. Kutnink, Wayne C. Hawkes, Ellen E. Schaus, Stanley T. Omaye

Research output: Contribution to journalArticle

153 Citations (Scopus)

Abstract

A paired-ion, reversed-phase, high-performance liquid chromatography procedure using electrochemical detection and internal standard quantitation based on isoascorbic acid (IA) is described for the determination of ascorbic acid (AA) in blood cells and plasma. By correcting for vial-to-vial variations in the AA oxidation rate, use of IA as an internal standard overcomes a major problem associated with AA instability and eliminates the necessity of assaying samples immediately after they are prepared for analysis. The ion-pairing agent, dodecyltriethylammonium phosphate, gives improved AA-IA resolution over agents with shorter carbon chains and also eliminates the interference of an unidentified substance extracted with platelet AA. Five percent metaphosphoric acid extracts of mononuclear leukocytes (MN), polymorphonuclear leukocytes (PMN), platelets, or plasma were mixed with the IA internal standard and diluted with an EDTA-cysteine solution. The samples were placed in a refrigerated autosampler at 4°C prior to chromatography on a 5-μm octadecylsilyl column. AA concentrations (mean ± SD) in platelets, MN, and PMN from six healthy volunteers were 0.25 ± 0.05, 15.2 ± 6.28, and 2.43 ± 1.63 μg/108 cells, respectively; the mean plasma AA concentration was 0.97 ± 0.34 mg/dl. All are in good agreement with published values. Refrigerated sample extracts containing the internal standard can be reassayed up to 3 weeks later with negligible change in calculated AA concentration. Up to 70 samples can be assayed per day with a detection limit (3 × SD) and minimum quantifiable level (less than 5% coefficient of variation) of 0.02 and 0.2 ng/injection, respectively.

Original languageEnglish (US)
Pages (from-to)424-430
Number of pages7
JournalAnalytical Biochemistry
Volume166
Issue number2
DOIs
StatePublished - Nov 1 1987
Externally publishedYes

Fingerprint

High performance liquid chromatography
Ascorbic Acid
Blood
High Pressure Liquid Chromatography
Platelets
Mononuclear Leukocytes
Blood Platelets
Plasmas
Neutrophils
Ions
Chromatography
Edetic Acid
Cysteine
Limit of Detection
Blood Cells
Healthy Volunteers
Carbon
Phosphates
Cells
Oxidation

Keywords

  • ascorbic acid
  • blood plasma
  • HPLC techniques
  • leukocytes
  • platelets
  • vitamins

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

An internal standard method for the unattended high-performance liquid chromatographic analysis of ascorbic acid in blood components. / Kutnink, Mark A.; Hawkes, Wayne C.; Schaus, Ellen E.; Omaye, Stanley T.

In: Analytical Biochemistry, Vol. 166, No. 2, 01.11.1987, p. 424-430.

Research output: Contribution to journalArticle

Kutnink, Mark A. ; Hawkes, Wayne C. ; Schaus, Ellen E. ; Omaye, Stanley T. / An internal standard method for the unattended high-performance liquid chromatographic analysis of ascorbic acid in blood components. In: Analytical Biochemistry. 1987 ; Vol. 166, No. 2. pp. 424-430.
@article{40116d397a3e423d9ba32804db30e0a3,
title = "An internal standard method for the unattended high-performance liquid chromatographic analysis of ascorbic acid in blood components",
abstract = "A paired-ion, reversed-phase, high-performance liquid chromatography procedure using electrochemical detection and internal standard quantitation based on isoascorbic acid (IA) is described for the determination of ascorbic acid (AA) in blood cells and plasma. By correcting for vial-to-vial variations in the AA oxidation rate, use of IA as an internal standard overcomes a major problem associated with AA instability and eliminates the necessity of assaying samples immediately after they are prepared for analysis. The ion-pairing agent, dodecyltriethylammonium phosphate, gives improved AA-IA resolution over agents with shorter carbon chains and also eliminates the interference of an unidentified substance extracted with platelet AA. Five percent metaphosphoric acid extracts of mononuclear leukocytes (MN), polymorphonuclear leukocytes (PMN), platelets, or plasma were mixed with the IA internal standard and diluted with an EDTA-cysteine solution. The samples were placed in a refrigerated autosampler at 4°C prior to chromatography on a 5-μm octadecylsilyl column. AA concentrations (mean ± SD) in platelets, MN, and PMN from six healthy volunteers were 0.25 ± 0.05, 15.2 ± 6.28, and 2.43 ± 1.63 μg/108 cells, respectively; the mean plasma AA concentration was 0.97 ± 0.34 mg/dl. All are in good agreement with published values. Refrigerated sample extracts containing the internal standard can be reassayed up to 3 weeks later with negligible change in calculated AA concentration. Up to 70 samples can be assayed per day with a detection limit (3 × SD) and minimum quantifiable level (less than 5{\%} coefficient of variation) of 0.02 and 0.2 ng/injection, respectively.",
keywords = "ascorbic acid, blood plasma, HPLC techniques, leukocytes, platelets, vitamins",
author = "Kutnink, {Mark A.} and Hawkes, {Wayne C.} and Schaus, {Ellen E.} and Omaye, {Stanley T.}",
year = "1987",
month = "11",
day = "1",
doi = "10.1016/0003-2697(87)90594-X",
language = "English (US)",
volume = "166",
pages = "424--430",
journal = "Analytical Biochemistry",
issn = "0003-2697",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - An internal standard method for the unattended high-performance liquid chromatographic analysis of ascorbic acid in blood components

AU - Kutnink, Mark A.

AU - Hawkes, Wayne C.

AU - Schaus, Ellen E.

AU - Omaye, Stanley T.

PY - 1987/11/1

Y1 - 1987/11/1

N2 - A paired-ion, reversed-phase, high-performance liquid chromatography procedure using electrochemical detection and internal standard quantitation based on isoascorbic acid (IA) is described for the determination of ascorbic acid (AA) in blood cells and plasma. By correcting for vial-to-vial variations in the AA oxidation rate, use of IA as an internal standard overcomes a major problem associated with AA instability and eliminates the necessity of assaying samples immediately after they are prepared for analysis. The ion-pairing agent, dodecyltriethylammonium phosphate, gives improved AA-IA resolution over agents with shorter carbon chains and also eliminates the interference of an unidentified substance extracted with platelet AA. Five percent metaphosphoric acid extracts of mononuclear leukocytes (MN), polymorphonuclear leukocytes (PMN), platelets, or plasma were mixed with the IA internal standard and diluted with an EDTA-cysteine solution. The samples were placed in a refrigerated autosampler at 4°C prior to chromatography on a 5-μm octadecylsilyl column. AA concentrations (mean ± SD) in platelets, MN, and PMN from six healthy volunteers were 0.25 ± 0.05, 15.2 ± 6.28, and 2.43 ± 1.63 μg/108 cells, respectively; the mean plasma AA concentration was 0.97 ± 0.34 mg/dl. All are in good agreement with published values. Refrigerated sample extracts containing the internal standard can be reassayed up to 3 weeks later with negligible change in calculated AA concentration. Up to 70 samples can be assayed per day with a detection limit (3 × SD) and minimum quantifiable level (less than 5% coefficient of variation) of 0.02 and 0.2 ng/injection, respectively.

AB - A paired-ion, reversed-phase, high-performance liquid chromatography procedure using electrochemical detection and internal standard quantitation based on isoascorbic acid (IA) is described for the determination of ascorbic acid (AA) in blood cells and plasma. By correcting for vial-to-vial variations in the AA oxidation rate, use of IA as an internal standard overcomes a major problem associated with AA instability and eliminates the necessity of assaying samples immediately after they are prepared for analysis. The ion-pairing agent, dodecyltriethylammonium phosphate, gives improved AA-IA resolution over agents with shorter carbon chains and also eliminates the interference of an unidentified substance extracted with platelet AA. Five percent metaphosphoric acid extracts of mononuclear leukocytes (MN), polymorphonuclear leukocytes (PMN), platelets, or plasma were mixed with the IA internal standard and diluted with an EDTA-cysteine solution. The samples were placed in a refrigerated autosampler at 4°C prior to chromatography on a 5-μm octadecylsilyl column. AA concentrations (mean ± SD) in platelets, MN, and PMN from six healthy volunteers were 0.25 ± 0.05, 15.2 ± 6.28, and 2.43 ± 1.63 μg/108 cells, respectively; the mean plasma AA concentration was 0.97 ± 0.34 mg/dl. All are in good agreement with published values. Refrigerated sample extracts containing the internal standard can be reassayed up to 3 weeks later with negligible change in calculated AA concentration. Up to 70 samples can be assayed per day with a detection limit (3 × SD) and minimum quantifiable level (less than 5% coefficient of variation) of 0.02 and 0.2 ng/injection, respectively.

KW - ascorbic acid

KW - blood plasma

KW - HPLC techniques

KW - leukocytes

KW - platelets

KW - vitamins

UR - http://www.scopus.com/inward/record.url?scp=0023506154&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0023506154&partnerID=8YFLogxK

U2 - 10.1016/0003-2697(87)90594-X

DO - 10.1016/0003-2697(87)90594-X

M3 - Article

C2 - 3434782

AN - SCOPUS:0023506154

VL - 166

SP - 424

EP - 430

JO - Analytical Biochemistry

JF - Analytical Biochemistry

SN - 0003-2697

IS - 2

ER -