Purification of dinosterol from complex mixtures of sedimentary lipids for hydrogen isotope analysis

We present two new methods for purifying dinosterol (4a,23,24-trimethyl-5a-cholest-22E-en-3b-ol) from sediments for the purpose of hydrogen isotope analysis via gas chromatography–isotope ratio mass spectrometry (GC–IRMS). The first method uses reversed phase-high performance liquid chromatography (RP-HPLC) to purify dinosterol from structurally similar 4a-methyl sterols that co-elute on GC analysis. Dinosterol purified from sedimentary sterol/alcohol fractions using this RP-HPLC method demonstrated an average yield of 80%. A very large isotope effect was observed during RP-HPLC purification, with a 560‰ range in dD value between the first and last 5% of a cholesterol standard, which is four times that during normal phase-HPLC (NP-HPLC) purification. However, we show that dinosterol recombined from 3–4 min of eluent during RP-HPLC purification yields highly reproducible and unbiased isotope values. Due to a larger isotope effect and lower sterol recovery during RP-HPLC, NP-HPLC purification is recommended for samples that do not contain 4a-methyl sterols that co-elute with dinosterol during GC. However, for samples that contain a variety of 4a-methyl sterols, RP-HPLC is more likely to yield baseline resolution of dinosterol. In the second method presented, RP-HPLC purification is preceded by NP-HPLC purification. Using this two step procedure, baseline resolution between dinosterol and all other compounds present was achieved for all samples with an average yield of 60% and, in many cases, dinosterol was purified from all other sedimentary lipids. For samples that contain a variety of 4a-methyl sterols and sitostanol concentration > 2 that of dinosterol, the two step purification method is recommended, as neither NP-HPLC or RP-HPLC alone is likely to yield baseline resolution of dinosterol. 2012 Elsevier Ltd. All rights reserved.