UPLC-MS/MS method for determination of retinol and α-tocopherol in serum using a simple sample pretreatment and UniSpray as ionization technique to reduce matrix effects.

Background Our goal was to develop a simple, rapid and precise ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the determination of retinol and α-tocopherol in serum. Currently published LC-MS/MS methods either require complex extraction procedures (liquid-liquid or solid-phase) or do not meet desirable specifications for imprecision in serum (coefficient of variation [CV] <6.8% and 6.9%, respectively). Methods Sample preparation consisted of a simple protein precipitation with ethanol and acetonitrile. Stable isotope-labeled internal standards (IS) and a homemade calibration curve were used for quantification. The analysis was performed using an Acquity I-class Xevo TQ XS LC-MS/MS. Chromatographic runtime was 6.0 min using a reversed phase gradient elution. UniSpray (US) as an ionization technique was compared to electrospray ionization (ESI). Analytical validation included matrix effect, recovery and trueness compared to National Institute of Standards and Technology (NIST) standards and United Kingdom National External Quality Assessment Service (UK NEQAS) samples. Results Intra- and inter-run CVs were <4.9% for retinol and <1.7% for α-tocopherol, both complying with desirable specifications for imprecision. Bias compared to NIST standards was <3.1% for both compounds. The method was linear over the entire tested range. The lower limit of quantification (LLOQ) with US was lower than with ESI for both retinol (0.022 vs. 0.043 mg/L) and α-tocopherol (0.22 vs. 0.87 mg/L). Matrix effects were not significant (<15%) for retinol. However, for α-tocopherol matrix effects of on average 54.0% were noted using ESI, but not with US. Conclusions We developed a fast, precise and accurate UPLC-MS/MS method for the determination of retinol and α-tocopherol in human serum using a single-step sample pretreatment. Ionization using US eliminated the matrix effects for α-tocopherol.

Simple GC-FID method development and validation for determination of alpha-tocopherol (vitamin E) in human plasma.

This paper describes the development and validation of a novel GC-FID method for the determination of alpha-tocopherol concentration in human plasma which does not requires derivatization. The standard solutions and the plasma working solutions were prepared in absolute ethanol. To determine the concentration of alpha-tocopherol in human plasma, an aliquot of the plasma sample was deproteinized with ethanol. alpha-tocopherol was extracted with a mixture of hexane and dichloromethane (9:1). GC separation was performed using a HP-5 capillary column. Nitrogen was used as carrier gas at a flow-rate of 2 ml min(-1). Calibration curves were linear over the concentration range 1-30 microg ml(-1) (for standard solutions and solutions without endogenous alpha-tocopherol in plasma) and 5-34 microg ml(-1) (for solutions with endogenous alpha-tocopherol in plasma). Absolute recovery, precision, sensitivity and accuracy assays were carried out. The analytical recovery of alpha-tocopherol from plasma averaged 97.44%. The limit of quantification (LOQ) and the limit of detection (LOD) of method for standard samples were 0.35 microg.ml(-1) and 0.30 microg.ml(-1), respectively. Within-day and between-day precision, expressed as the relative standard deviation (RSD) were less than 4%, and accuracy (relative error) was better than 8%. This novel method, developed and validated in our laboratory, could be successfully applied to the in-vivo determination of alpha-tocopherol. The endogenous alpha-tocopherol amounts in blood of twelve healthy volunteers with no vitamin drug usage were measured with this method.

Improvement of content uniformity of d-alpha-tocopheryl acetate as an oily drug in granules by emulsification.

To elucidate the effects of an oily drug emulsification on its content uniformity in granules obtained by wet granulation with a high-shear mixer, d-alpha-tocopheryl acetate (VE) was emulsified with hydroxypropylcellulose (HPC-L) solution (mean diameter of the VE droplets was 1.3 microm). When VE was added to the mixing powder as the emulsion, nuclei rich in VE were not formed and then the content of VE was fairly uniform throughout the granules even at 2 min granulation. We found that the oily drug poor content uniformity could be improved significantly by adding an emulsified drug to the powder in granulation process.

[Tocopherol Acetate Reference Standard (Control 021) of National Institute of Health Sciences].

The raw material of tocopherol acetate was examined for the preparation of the "Tocopherol Acetate Reference Standard (Control 021)", The analytical data obtained were: UV spectrum, lambda max of 278.5 and 284.8 nm and specific absorbance in ethanol at 284 nm = 42.9; IR spectrum, same as that of the Tocopherol Acetate Reference Standard (Control 001); thin-layer chromatography, no impurities were detected until 50 micrograms; high-performance liquid chromatography, total amount of impurities estimated to be less than 0.6%. Based on the above results, the raw material was authorized as the Japanese Pharmacopoeia Tocopherol Acetate Reference Standard (Control 021) of the National Institute of Health Sciences.

[Tocopherol Acetate Reference Standard (Control 001) of National Institute of Health Sciences].

The raw material of tocopherol acetate was examined for the preparation of the "Tocopherol Acetate Reference Standard (Control 001)". Analytical data obtained were: IR spectrum, same as that of the Tocopherol Acetate Reference Standard (Control 974); specific absorbance, E/cm% (284 nm) = 43.7; thin-layer chromatography, no impurities were detected until 50 micrograms of the loaded raw material; high-performance liquid chromatography (HPLC), total amount of impurities estimated to be less than 0.6%; assay by HPLC, 101.7%. Based on the above results, the raw material was authorized as the Japanese Pharmacopoeia Reference Standard (Control 001).