Simplified method for vitamin E determination in rat adipose tissue and mammary glands by high-performance liquid chromatography

Abstract

A method for vitamin E (alpha-tocopherol) measurement in rat adipose tissue and mammary gland has been developed and validated. Tissues were homogenized in ethanol-water (1:1) and extracted with n-hexane. Vitamin K1 was used as internal standard. Separation was performed by HPLC with methanol-water (96.5:3.5) as eluent in a Nucleosil C18 column (15 x 0.46 cm) at 40 degrees C. Detection was by fluorescence with excitation at 295 nm and emission at 350 nm for vitamin E and at 330 and 440 nm for vitamin K1. Standards and tissue extracts were checked for linearity giving correlation coefficients over 0.99 in a range of concentrations from 0.56 to 4.51 nmol/g in adipose tissue and from 2.18 to 17.4 nmol/g in mammary gland tissue. Intra-assay precision (R.S.D.) varied between 3 and 4%, whereas inter-assay precision was between 8 and 9%. Recoveries ranged between 95 +/- 3% and 98 +/- 11% for the two tissues, respectively. Vitamin E was measured in rats that had previously received one oral dose of this vitamin. Whereas vitamin E content in adipose tissue did not differ between late-pregnant and virgin rats, it was significantly higher in mammary gland of pregnant rats, and this difference could be related to the enhanced lipoprotein lipase activity in this group.

Effects of tocopherols and 2,2′-carboxyethyl hydroxychromans on phorbol-ester-stimulated neutrophils

“…The dried samples were resuspended in methanol. Samples were injected into an HPLC apparatus (HitachiMerck) coupled to a fluorescence detector (F1050) set at 292 nm for excitation and 325 nm for emission [22]. Compounds were identified by spiking the samples with known amounts of individual pure tocopherols, which were used also to calibrate the assay.…”

Determination of α‐tocopherol by reversed‐phase HPLC in feed and animal‐derived foods without saponification

“…Normal‐phase HPLC offers the advantage that β‐ and γ‐tocopherol can be separated, which is not possible with RP‐HPLC . However, poor recoveries were found by Rupérez et al . when determining vitamin E in adipose tissue.…”

“…Normal-phase HPLC offers the advantage that -and -tocopherol can be separated, which is not possible with RP-HPLC. 12 However, poor recoveries were found by Rupérez et al 7 when determining vitamin E in adipose tissue. On the other hand, reversed-phase columns need a shorter equilibrium and analysis time and show a higher reproducibility of retention times.…”

“…Redissolving, however, is very difficult, if not impossible, with samples containing high amounts of fat. Therefore, the residue is sometimes redissolved in chloroform–methanol (1:1) . However, when using 965 mL L −1 of methanol as HPLC solvent, the injection of samples containing fat will cause precipitation of this fat in the solvent as it is not soluble in methanol.…”

Development and validation of a single HPLC method for determination of α‐tocopherol in cell culture and in human or mouse biological samples

“…In previous studies (Abahusain et al, 1998;Gimeno et al, 2001;Grebenstein and Frank, 2012;Nirungsan and Thongnopnua, 2006;Zivanovic et al, 2000), reversed-phase HPLC based on C 18 stationary phase has been preferred to achieve αT analysis rather than using normal phase (Nagy et al, 2007;Richelle et al, 2003). Unfortunately, as a general rule, different types of biological matrices require specific methods for analyzing αT (Aust et al, 2001;Melichar et al, 2010;Ruperez et al, 1998), which is inconvenient for sample handling standardization and for universalization of chromatographic detection in routine laboratories not specifically focused on chromatographic methods. Unfortunately, as a general rule, different types of biological matrices require specific methods for analyzing αT (Aust et al, 2001;Melichar et al, 2010;Ruperez et al, 1998), which is inconvenient for sample handling standardization and for universalization of chromatographic detection in routine laboratories not specifically focused on chromatographic methods.…”