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

Abstract

A straightforward and common analytical method for α-tocopherol (αT) determination in various biological samples, including plasma, red blood cells (RBC), tissues and cultured cell lines, was developed and validated, using a reverse phase-chromatographic method (RP-HPLC). Even though many chromatographic methods for αT determination have been reported, most of them require readjustment when applied to different types of samples. Thus, an effective and simple method for αT determination in different biological matrices is still necessary, specifically for translational research. This method was applied using a C18 column (250 × 4.6 mm, 5 µm particle size) under isocratic elution with MeOH:ACN:H2 O (90:9:1 v/v/v) at a flow rate of 1 mL/min and detected using photodiode array at 293 nm. Linearity (r >0.9997) was observed for standard calibration with inter- and intraday variation of standard <4%. Lower limits of detection and quantification for αT in this assay were 0.091 and 0.305 µg/mL respectively. Validation proved the method to be selective, linear, accurate and precise. The method was successfully applied in great variety of biological samples, that is, human and mouse plasma, RBCs, murine tissues and human/mouse/rat cultured cell lines. More importantly, a single protocol of extraction and detection can be applied, making this method very convenient for standardization of different types of samples.

The detection of age-, gender-, and region-specific changes in mouse brain tocopherol levels via the application of different validated HPLC methods

“…With regard to rodent studies on tocopherol homeostasis, only limited data are available about the effect of aging and gender on plasma or serum and brain tocopherol levels. Most of the available rodent tocopherol studies did not separate animals to the necessary groups for later statistical comparisons [19][20][21][22][23][24][25][26]. Although Gohil et al [27] determined αT level in several brain regions 4 (cerebral cortex, hippocampus, cerebellum, midbrain and the remaining part of the brainstem) of 5 months old C57Bl/6 female (n = 5) and male (n = 3) mice and found significantly higher αT concentrations in all the five regions in females than in males, moreover significantly lower αT level was observed in the cerebellum than those of other examined brain regions, no information was obtained about the effect of aging.…”

The stability of water‐ and fat‐soluble vitamin in dentifrices according to pH level and storage type

“…In several studies, the vitamins in foods, pharmaceuticals, cosmetics or biological samples were analyzed using various liquid chromatography methods (Li, ; Luhua et al ., ; Lebiedzinska et al ., ; Mattila et al ., ; Santos et al ., ; Viñas et al ., , ; Cimadevilla et al ., ). However, analyses of vitamins in dentifrices have rarely been reported.…”

Simultaneous HPLC–DAD Determination of Retinol and Eight Vitamin E Isomers in Human Serum

“…Since then, the method has been constantly improved, and more VE isomers have been included. Many HPLC methods have been reported for the analysis of VA and VE in human serum [25,28] or plasma [22,23,[27][28][29]. In 1999, Yap et al [27] reported a reversed-phase HPLC (RP-HPLC) method for determination of α, γ, and δ-T3 in human plasma.…”

“…In 2007, Siluk et al [23] described a rapid RP-HPLC method for determination of VA and α, γ, and δ-T in human plasma. In 2014, Cimadevilla et al [29] developed an HPLC-UV method for α-T in biological samples including plasma, red blood cells, tissues, and cultured cell lines. Melchert et al [25] achieved complete normal-phase HPLC (NP-HPLC) separation of the four tocopherols in human serum.…”

“…(HPLC) [22][23][24][25][26][27][28][29][30][31][32]. The detectors most often used are the ultraviolet detector (UVD) [22], fluorescence detector (FLD) [23], electrochemical detector (ECD) [24], and mass spectrometer (MS) [25].…”