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Clinical Chemistry 32: 2137-2142, 1986;
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Clinical Chemistry, Vol 32, 2137-2142, Copyright © 1986 by American Association for Clinical Chemistry

Simultaneous quantification of erythrocyte zinc protoporphyrin and protoporphyrin IX by liquid chromatography

GG Bailey and LL Needham

A simple, rapid, specific, and sensitive isocratic "high-performance" liquid-chromatographic procedure is described for measuring protoporphyrin (PPIX) and zinc protoporphyrin (ZPP) in erythrocytes. A 30-microL whole-blood sample is treated with a solution of formic acid, deproteinized with acetone, and centrifuged. A 20-microL aliquot of the supernate is injected into a system consisting of a stationary phase of mu-Bondapak C18 and a mobile phase of acetone, methanol, water, and formic acid. ZPP and PPIX are detected fluorometrically (lambda ex = 417 nm, lambda em = 635 nm) within 6 min. The range of linearity extends beyond 10 mg/L for ZPP and 580 micrograms/L for PPIX. The detection limits for ZPP and PPIX are 11.9 micrograms/L (6.93 pg) and 2.55 micrograms/L (1.485 pg), respectively. The precision for ZPP and PPIX determinations averaged 2.86 and 5.59%, respectively, for within- day CVs and 4.98 and 8.14, respectively, for among-day CVs. Analytical recoveries averaged 97.2% for ZPP and 101.5% for PPIX. Interferences in the form of fluorescent quenching of ZPP and PPIX by hemin are avoided by chromatographic separation. We also used this method to determine the purity of commercially prepared ZPP, and compared the results obtained with this method with those from an extraction method.


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R. F. Labbe, H. J. Vreman, and D. K. Stevenson
Zinc Protoporphyrin: A Metabolite with a Mission
Clin. Chem., December 1, 1999; 45(12): 2060 - 2072.
[Abstract] [Full Text] [PDF]


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Copyright © 1986 by the American Association for Clinical Chemistry.