Comparison of a new high-resolution monolithic column with core-shell and fully porous columns for the analysis of retinol and α-tocopherol in human serum and breast milk by ultra-high-performance liquid chromatography†

Vitamins A and E are fat-soluble vitamins that play important roles in several physiological processes. Monitoring their concentrations is needed to detect deficiency and guide therapy. In this study, we developed a high-performance liquid chromatography method to measure the major forms of vitamin A (retinol) and vitamin E (α-tocopherol and γ-tocopherol) in human blood plasma. Vitamins A and E were extracted with hexane and separated on a reversed-phase column using methanol as the mobile phase. Retinol was detected by ultraviolet absorption, whereas tocopherols were detected by fluorescence emission. The chromatographic cycle time was 4.0 min per sample. The analytical measurement range was 0.03-5.14, 0.32-36.02, and 0.10-9.99 mg/L for retinol, α-tocopherol, and γ-tocopherol, respectively. Intr-aassay and total coefficient of variation were <6.0% for all compounds. This method was traceable to standard reference materials offered by the National Institute of Standards and Technology. Reference intervals were established using plasma samples collected from 51 healthy adult donors and were found to be 0.30-1.20, 6.0-23.0, and 0.3-3.2 mg/L for retinol, α-tocopherol, and γ-tocopherol, respectively. In conclusion, we developed and validated a fast, simple, and sensitive high-performance liquid chromatography method for measuring the major forms of vitamins A and E in human plasma.

Section: Introductionmentioning

confidence: 99%

Fast, simple, and sensitive high‐performance liquid chromatography method for measuring vitamins A and E in human blood plasma

Yuan

Burgyan

Bunch

et al. 2014

“…methanol) to optimize selectivity . Several methods for the determination of lipovitamins in biological fluids involve chromatographic separation using UV–Vis or fluorescence detection (FLD) . The LODs and LOQs of these methods are usually sufficient for the analysis of carotenoids, retinoids, and tocopherols with appropriate precision and accuracy.…”

Section: Fat‐soluble Vitaminsmentioning

confidence: 99%

“…However, HPLC–FLD methods showed multiple improvements to the sensitivity in comparison with the HPLC–UV methods. For example, LOQ of α‐tocopherol obtained by UV was 0.2 μM , while the value of 21.4 nM was reached by FLD . Measurement of other analytes, such as vitamin K derivatives or vitamin D hydroxy metabolites, is limited because of their low concentrations in the blood.…”

Section: Fat‐soluble Vitaminsmentioning

confidence: 99%

“…For saponification, KOH or isopropanol are used . For further sample purification, LLE is additionally applied using heptane , hexane , ethyl acetate , chloroform , methyl tert ‐butyl ether , or a mixture of hexane/ethanol/acetone/toluene . Deproteinization and LLE resulted in endogenous compounds of the matrix passing into the supernatant, which may affect the separation and determination of the analytes.…”

Section: Fat‐soluble Vitaminsmentioning

confidence: 99%

See 1 more Smart Citation

A review of chromatographic methods for the determination of water‐ and fat‐soluble vitamins in biological fluids

Karaźniewicz−Łada

Główka

2015

Vitamins are an essential element of nutrition and thus contribute to human health. Vitamins catalyze many biochemical reactions and their lack or excess can cause health problems. Therefore, monitoring vitamin concentrations in plasma or other biological fluids may be useful in the diagnosis of various disorders as well as in the treatment process. Several chromatographic methods have been developed for the determination of these compounds in biological samples, including high-performance liquid chromatography with UV and fluorescence detection. Recently, high-performance liquid chromatography with tandem mass spectrometry methods have been widely used for the determination of vitamins in complex matrices because of their high sensitivity and selectivity. This method requires preconditioning of samples for analysis, including protein precipitation and/or various extraction techniques. The choice of method may depend on the desired cost, convenience, turnaround time, specificity, and accuracy of the information to be obtained. This article reviews the recently reported chromatographic methods used for determination of vitamins in biological fluids. Relevant papers published mostly during the last 5 years were identified by an extensive PubMed search using appropriate keywords. Particular attention was given to the preparation steps and extraction techniques. This report may be helpful in the selection of procedures that are appropriate for certain types of biological materials and analytes.

“…Core–shell particle columns can provide a conventional LC system with separation efficiency similar to that of UHPLC systems, but with much lower back pressure . They have been successfully applied to the analysis of biofluids and plant extracts . The only method in which a core–shell particle packed column has been used for macrolide analysis was developed for gradient separation of four macrolides of different chemical structures using UV detection and no real sample application was investigated .…”

Section: Introductionmentioning

confidence: 99%

Combination of a sub‐3 μm superficially porous particle packed column with charged aerosol detection for the simple and sensitive measurement of nine macrolides in human urine

Jia

Song

Jeong

et al. 2014

Due to the lack of chromophores in many macrolides, analytical methods based on mass spectrometry and electrochemical detection coupled to liquid chromatography have been suggested to be suitable for the quantification of macrolides in complex matrices. In this study, a simple and sensitive analytical method was established for the simultaneous measurement of nine macrolides in human urine by combining a sub-3 μm superficially porous particle packed column with charged aerosol detection. After thorough investigation of various sample preparation methods, including two liquid-liquid extraction methods and four solid-phase extraction methods, HLB solid-phase extraction was selected and further optimized. Absolute recovery of the optimized sample preparation method ranged from 99.5-110.2%, indicating its very high extraction/clean-up efficiency. For chromatography, parameters influencing macrolide separation were systematically optimized, and the resulting conditions allowed baseline separation of nine macrolides within 24 min using a very simple mobile phase. The established method was validated for linearity, limit of detection, limit of quantification, absolute recovery, and precision. Based on its limit of detection (0.025-0.100 μg/mL), the method had similar or greater sensitivity than most methods based on electrochemical detection. It was found that the current method was appropriate for application to real human urine samples after drug administration.