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 language | English (US) |
|---|---|
| Pages (from-to) | 424-430 |
| Number of pages | 7 |
| Journal | Analytical Biochemistry |
| Volume | 166 |
| Issue number | 2 |
| DOIs | |
| State | Published - Nov 1 1987 |
| Externally published | Yes |
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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 journal › Article
}
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 -