Retinoids internal standards

Cullum, M. E. and Zile, M. H. (1986) Quantitation of biological retinoid by high-pressure liquid chromatography primary internal standardization using tritiated retinoids. Anal. Biochem. 153, 23-32. 

When measuring the level of nonpolar retinoids in blood, a modified method of Thompson et al, (1971) could be used (see also Varma and Beaton, 1972 Bieri et al., 1979 Ross, 1981). Briefly, the plasma or serum (100-200 xl) is mixed with 1 volume of internal standard solution (e.g., retinyl acetate) in ethanol. To the mixture is added 1-4 volumes of hexane the contents are mixed well and then centrifuged. The hexane (upper phase) is carefully removed and used for chromatography or other analyses. For added protection against oxidation, butylated hydroxytoluene (100 xg/ml) can be added to the hexane. Other solvents used for extraction of retinol and its esters from serum include chlo-roform methanol (see, for example, DeRuyter and De Leenheer, 1978) and hexane methylene chlorideiisopropanol (Besner /., 1980). Recoveries, when reported, range near 100%. 

Internal standards the synthetic retinoids used as internal standards, Ro-13-4306 see Note 2) and Ro-23-5525 can be obtained from Hoffmann-La Roche (Basel, Switzerland or Nutley, NJ), retinyl acetate is obtainable from Sigma... 

In the procedures described above, the hexane extraction of the alkalinized-aqueous phase serves not only to recover the neutral retinoids, but to remove many neutral lipids that could interfere with the RA assay. If retinol and retinyl esters are the only target analytes, a simpler procedure can be used Extract 0 25 mL of human plasma by adding 0.5 mL ethanol, 100 ng of retinyl acetate (as internal standard), and then vortexing twice with 2 mL of hexane each time (6) Evaporate the solvents from the combined hexane extracts to dryness under a gentle stream of nitrogen. 

Alternatively, it is possible to inject the solvent extract directly for HPLC analysis. For good quantitation this method requires an internal standard or very careful measurement of liquid volumes. McClean et al. (69) and Nierenberg and Lester (70) used acetonitrile to denature plasma proteins and then solubilized retinol with ethyl acetate 1-butanol (1 1) for direct injection Siddiqui et al. precipitated proteins from serum by addition of acetonitrile, centrifuged, and then injected the supernatant (71). Retinoids and carotenoids can be efficiently extracted from one volume of serum or plasma with three volumes of 2-propanol dichloromethane (2 1) followed by centrifugation an aliquot can be directly analyzed by HPLC (72,73). The risks of sample loss and degradation during processing can thus be avoided. 

Retinyl acetate is sometimes used as internal standard for retinol analysis it is not ideal because it is an ester, not a free alcohol (and is hydrolyzed by saponification processes), but superior in principle to the use of tocol or other nonretinoid forms for retinoid quantitation. Substituted retinal oximes have been used as internal standards (93). 15-Methylretinol (94) is conceptually an excellent internal standard for retinol analysis, because of its very similar chemical structure and properties. 

Meyer et al. analyzed plasma retinol plus endogenous aW-trans and i-cis retinoic acid isocratically by normal-phase HPLC, using hexane 2-propanol acetic acid as mobile phase (90). Extraction of the retinoic acids required acidification of the sample however, too much acid can result in dehydration of retinol to anhydroretinol, and hydrolysis of endogenous retinoyl P-glucuronide (90). A synthetic retinoid sulfonic acid was used as internal standard. By using absorbance at 350 nm, limits of detection were 1.7 nM (0.5 (ig/L) for retinoic acid isomers, 35 nM (10 Xg/L) for retinol. 13-Demethyl retinoic acid has also been used as internal standard (142). Lanvers et al. used normal-phase HPLC with gradient elution (hexane 2-propanol glacial acetic acid) to analyze 13-c/x retinoic acid, 9-cis retinoic acid, aW-trans retinoic acid, retinol, and the 4-oxo metabolites of the retinoic acid isomers (143). Plasma samples were treated with ethanol to denature proteins, and then were extracted with hexane after addition of saturated ammonium sulfate solution (pH 5). 

Figure 4 Reversed-phase HPLC elution profiles of tocopherols (panel A), retinoids (B), and carotenoids (C) present in human plasma (200 pL). Blood was collected 3 h after an oral dose of retinoic acid. The chromatogram was obtained by use of gradient elution (Table 3). Peak identification 2, 4-oxo-retinoic acid 4, retinoyl P-glucuronide 7, retinoic acid 8, retinol 9, retinyl acetate (internal standard) 15, butylated hydroxy toluene 16, y-tocopherol 17, a-tocopherol 18, free bilirubin 19, lutein 20, zeaxanthin 21, 2, 3 -anhydrolutein 22, P-cryptoxanthin 23, lycopene 24, a-carotene 25, P-carotene. (From Ref. 73.)...

Wyss pioneered use of automated column-switching techniques for analysis of retinoic acid and other polar retinoids (156-160). In general, a plasma sample is diluted with acetonitrile ethanol (95 5) containing an appropriate internal standard the supernatant is passed across a preconcentration column (such as Bonda-pak C18 Corasil, 14 mm X 4.6 mm ID), and the preconcentration column is rinsed with ammonium acetate acetonitrile. The retinoids were eluted from the preconcentration column with a gradient of ammonium acetate acetonitrile acetic acid, and analyzed on a Spherisorb ODS 1 column by gradient elution (159). 

Retinoic acid and other retinoid carboxylic acids, however, can be readily converted to derivatives that are suitable for gas chromatography. Many of these applications have used mass spectrometry for detection (see below). Diazomethane is used, at room temperature, to prepare the methyl esters without apparent isomerization. Pentafluorobenzyl esters of retinoic acid and its analogs have also been prepared for GC-MS (280) or HPLC-MS (281). Deuterated analogs of retinoic acid or other retinoid carboxylic acids have been used as internal standards, with mass spectrometric detection (88,282) (reviewed by Napoli [283] and by De Leenheer and Lambert [89]). The pentafluorobenzyl ester of a synthetic retinoid, Ro 13-7410, was analyzed by column switching the peak of interest from a SE54 colunm was cut to an OV 240 column, with subsequent detection by negative ion chemical ionization mass spectrometry (280). 

The advantages and disadvantages of candidate internal standards for tocopherols have been discussed in III.A.4. In practice, tocol and a-tocopheryl acetate were used in the majority of cases, followed by retinyl acetate in assays including also retinoids and carotenoids. 

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