Determination of (+)‐, (−)‐, and total gossypol in cottonseed by high‐performance liquid chromatography

Abstract

Abstract Gossypol, a pigment in cottonseed, is a polyphenolic, binaphthyl dialdehyde. Due to steric hindrance between the functional groups of the molecule at the bond connecting the two naphthyl rings, gossypol exists as (+)‐ and (−)‐isomers. Gossypol is physiologically active with the (−)‐isomer appearing to be more active and causing temporary infertility in males. It is thus important to know the amounts of isomers in livestock feeds. A quantitative high‐performance liquid chromatography (HPLC) procedure was developed for the separation of (+)‐ and (−)‐gossypol contained in cottonseed. This method involves derivatization of gossypol with ( R )‐(−)‐2‐amino‐1‐propanol followed by HPLC separation employing either a Phenomenex Prodigy (5 µ, ODS‐3, 100 × 3.2 mm) or a MetaChem Inertsil (5 µ, ODS‐3, 100 × 3.0 mm) reversed‐phase column eluted with 80% acetonitrile and 20% 10 mM KH 2 PO 4 adjusted to pH 3.0 with H 3 PO 4 at 1.0 mL/min. The (+)‐ and (−)‐gossypol‐2‐ amino‐1‐propanol complexes eluted at roughly 1.4 and 2.6 min, respectively. It was found that gossypol from Upland ( Gossypium hirsutum ) seed was rich in the (+)‐enantiomer, with the (+)‐ and (−)‐enantiomers in a ratio of about 65:35, respectively, while gossypol from the seed of a Pima ( G. barbadense ) cultivar (S‐6) was slightly richer in the (−)‐enantiomer (46.8:53.2).