Retinoic acid quantification

Quantification of Endogenous Retinoic Acid in Human Plasma by Liquid Chromatography/Mass Spectrometry... 

Retinoic acid, an endogenous retinoid, is a potent inducer of cellular differentiation. Because cancer is fundamentally a loss of cellular differentiation, circulating levels of retinoic acid could play an important role in chemoprevention. However, physiological concentrations are typically below the limits of HPLC detection. Sensitive techniques, such as negative chemical ionization (NCI) GC/MS have been employed for quantification, but cause isomerization and also fail to resolve the cis and trans isomers of retinoic acid. Normal phase HPLC can resolve the cis and trans isomers of retinoic acid without isomerization, and mobile phase volatility makes it readily compatible with the mass spectrometer. Based on these considerations, a method combining microbore normal phase HPLC separation with NCI-MS detection was developed to quantify endogenous 13-cis and all-trans retinoic acid in human plasma. The limit of detection was 0.5 ng/ml, injecting only 8 pg of retinoic acid onto the column. The concentration of 13-cis retinoic acid in normal, fasted, human plasma (n=13) was 1.6 +/- 0.40 ng/ml. 

Most assays for the quantification of endogenous levels of 13-cis and all trans retinoic acid utilize GC/MS. This technique is highly sensitive, but GC isomer resolution is an inherent problem (22). Therefore, unequivocal quantification of cis and trans retinoic acid levels is impossible. The use of HPLC can eliminate isomerization, but lacks sensitivity. Therefore, HPLC in combination with MS should provide a highly sensitive method of quantification without isomerization. In this report, we describe the use of microbore normal phase HPLC/NCI-MS to quantify endogenous levels of 13-cis and all trans retinoic acid in human plasma. 

This observation most likely leads to a slight underestimation of endogenous all trans retinoic acid in plasma, when using the 13-cis retinoic acid internal standard for quantification. 

Several investigators have developed assays for the quantification of all trans retinoic acid in human blood, plasma or serum. Nelson et al. (35) developed a colorimetric assay, but could not detect retinoic acid under physiological conditions. DeRuyter et al. (36) found 1 to 3 ng/ml of all trans retinoic acid in fasted human serum using normal phase HPLC. Chiang (37) validated an assay for all trans retinoic acid in plasma, but could not detect any under normal physiological conditions using 10 ml of plasma. 

Napoli et al. (23) developed a sensitive assay based on negative chemical ionization mass spectrometry to quantify retinoic acid in human plasma. Endogenous levels of all trans retinoic acid in plasma were 4.9 ng/ml, using a 0.1 ml sample. The limit of detection was less than 1 ng/ml. Direct quantification of 13-cis retinoic acid was impossible due to the inability of the GC to resolve the isomers. Barua and Olson (33) described a method to quantify all trans retinoic acid in serum using reverse phase HPLC. They detected 1.8 ng/ml of the all trans isomer, using a 2 ml serum sample and a non-acidic extraction procedure. 

We have developed a very sensitive assay which can quantify both 13-cis and all trans retinoic acid in the same plasma sample. Only 1 ml of plasma is necessary for analysis, with a limit of quantification of 0.5 ng/ml. The assay is linear for both cis and trans retinoic acid, and there is virtually no interconversion of the two isomers by assay manipulations. However, the assay does slightly underestimate the amount of all trans retinoic acid present due to the differential recovery of this isomer from plasma as opposed to recovery from PBS. This will be corrected in future work by the addition of a stable isotope labelled all trans retinoic acid internal standard for quantification. 

Napoli, J L (1986) Quantification of physiological levels of retinoic acid. ods Enzymol. 123, 112-124. 

Napoli, J. L., Pramanik, B. C, Williams, J. B, Dawson, M. I., and Hobbs, P D. (1985) Quantification of retinoic acid by gas-liquid chromatography/mass spectrometry total vs. all-tra s-retinoic acid in human plasma. J Lipid. Res. 26, 387-392. 

Miyagi, M. Yokoyama, H. Shiraishi, H. Matsumoto, M. Ishii, H. Simultaneous quantification of retinol, retinal, and retinoic acid isomers by high-performtuice hquid chromatography with a simple gradiation, J.Chromatogr.B, 2001, 757, 365-368. 

Wang X-D, Krinsky NI (1997) Identification and quantification of retinoic acid and other metabolites from P-carotene excentric cleavage in human intestine in vitro and ferret intestine in vivo. Methods Enzymol 282 117-130... 

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