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.

Determination of vitamin A in blood serum based on solid-phase extraction using cetyltrimethyl ammonium bromide-modified attapulgite.

Cetyltrimethyl ammonium bromide-modified attapulgite was prepared and utilized as a novel sorbent in a simple solid-phase extraction method for the determination of vitamin A in blood serum. Several factors affecting extraction efficiency were systematically optimized, including the sampling solvent and its volume, as well as the elution solvent and its volume. Under the optimal solid-phase extraction conditions, the adsorption capacity of vitamin A was as high as 28 mg/g according to the Langmuir isotherm model. Based on the developed solid-phase extraction method, the level of vitamin A in 200 µL blood serum sample could be accurately determined by high-performance liquid chromatography. The recoveries of vitamin A spiked in 10% v/v methanol aqueous solutions were in the range of 86.9-92.8%, with the relative standard deviations not more than 8.1%. The method was applied to the determination of vitamin A in serum samples from 20 pregnant women. Compared with the previously reported solid-phase extraction methods for determination of vitamin A in serum, our developed cetyltrimethyl ammonium bromide-modified attapulgite-based solid-phase extraction method used lower serum volume, omitted extra steps (i.e. evaporation and re-dissolution), and eliminated internal standard. The results were promising for it to be used in routine monitoring during pregnancy.

Extraction of Carotenoids and Fat-Soluble Vitamins from Tetradesmus Obliquus Microalgae: An Optimized Approach by Using Supercritical CO2.

In recent years, great attention has been focused on rapid, selective, and environmentally friendly extraction methods to recover pigments and antioxidants from microalgae. Among these, supercritical fluid extraction (SFE) represents one of the most important alternatives to traditional extraction methods carried out with the use of organic solvents. In this study, the influence of parameters such as pressure, temperature, and the addition of a polar co-solvent in the SFE yields of carotenoids and fat-soluble vitamins from T. obliquus biomass were evaluated. The highest extraction of alpha-tocopherol, gamma-tocopherol, and retinol was achieved at a pressure of 30 MPa and a temperature of 40 °C. It was observed that overall, the extraction yield increased considerably when a preliminary step of sample pre-treatment, based on a matrix solid phase dispersion, was applied using diatomaceous earth as a dispersing agent. The use of ethanol as a co-solvent, under certain conditions of pressure and temperature, resulted in selectively increasing the yields of only some compounds. In particular, a remarkable selectivity was observed if the extraction was carried out in the presence of ethanol at 10 MPa and 40 °C: under these conditions, it was possible to isolate menaquinone-7, a homologous of vitamin K2, which, otherwise, cannot not recovered by using traditional extraction procedures.

Assessment of wild mint from Tunceli as source of bioactive compounds, and its antioxidant Activity.

The types of wild mint (Mentha spicata L.) were sampled from different geographical regions in Tunceli (Turkey) in order to find out their vitamin, mineral, phenolic contents and their antioxidant properties. The total phenol varied from 77.7±0.242 to 52.34±0.351 mg of GAEs/g of dry mint. The highest radical effect of scavenging was observed in Mazgirt parting of the ways 7.5 km with 6.17±0.245 mg/mL. The highest reducing power and metal chelating were observed in the mint from Cicekli parting of the ways 6.5 km Demirkapi. Among the various macronutrients which were estimated in the plant samples, potassium was presented in the highest quantity followed by calcium and phosphate. Although rutin and resveratrol were not determined in any samples, kaempferol and catechin levels were found out in almost all samples. The concentrations of vitamin A ranged between 42,14±5.70 and 13.61±3.00 (mg/kg dry weight). These results show that plants of mint are quite rich in phenolic compounds, and these have been appeared to have antioxidant activity, which agrees with this work, since the extract showed a higher content of phenolic compounds and higher antioxidant activity and mint may be considered as a natural alternative source for food, pharmacology and medicine sectors.

Advancing towards practice: A novel LC-MS/MS method for detecting retinol in dried blood spots.

BACKGROUND: To date, clinical laboratories face challenges in quantifying retinol from DBS samples. Disputes arise throughout the whole detection process, encompassing the storage condition, the release strategy as well as the selection of internal standards. METHODS: We incubated DBS with ascorbic acid solution. Then, retinol-d4 in acetonitrile was introduced to incorporate isotopic internal standard and promote protein precipitation. Afterward, sodium carbonate solution was added to ionize cytochromes (such as bilirubin), which amplified the difference of their hydrophobicity to retinol. Subsequently, cold-induced phase separation could be facilitated to separate retinol from the impurities. In the end, the upper layer was injected for LC-MS/MS analysis. RESULTS: By comparing the detected retinol content in whole blood and DBS samples prepared from the same volume, we confirmed the established pretreatment was capable to extract most of retinol from DBS (recovery >90 %). Thereafter, we verified that within DBS, retinol possessed satisfying stability without antioxidation. Indoor-light exposure and storage duration would not cause obvious degradation (<10 %). Following systematic validation, the established method well met the criteria outlined in the relevant guidelines. After comparing with detected DBS results to the paired plasma samples, 54 out of 60 met the acceptance limit for cross-validation of ±20 %. CONCLUSIONS: We realized precise quantification of retinol from one 3.2 mm DBS disc. By circumventing conventional antioxidation, liquid-liquid/solid-phase extraction and organic solvent evaporation, the pretreatment could be completed within 15 min consuming only minimal amounts of low-toxicity chemicals (ascorbic acid, acetonitrile, and sodium carbonate). We expect this contribution holds the potential to significantly facilitate the evaluation of patients' vitamin A status by using DBS samples in the future.

Determination of vitamin A (retinol) in infant and medical nutritional formulas with AOAC method 992.06 using a modified extraction procedure: single-laboratory validation.

The applicability of AOAC Official Method 992.06, vitamin A (retinol) in milk-based infant formula can be extended to specialty infant formulas, and medical and adult nutritional products with a few minor modifications to the sample preparation procedure. Currently, AOAC Official Method 992.06 is only applicable to milk-based infant formulas containing >500 IU vitamin A per reconstituted quart. When this method is used as written to test specialty infant formulas, vitamin A recoveries are low compared to results generated with alternate validated vitamin A methods. AOAC Method 992.06 vitamin A recoveries can be improved significantly in specialty infant formulas if the amount of potassium hydroxide used during the saponification step is doubled. With this one minor modification to the sample preparation procedure, AOAC Method 992.06 demonstrates acceptable precision and accuracy for the quantitation of vitamin A (retinol) in specialty infant formulas, milk- and soy-based infant formulas, and adult and medical nutritionals. Because increasing the amount of potassium hydroxide can cause emulsions to form, 2-4 mL aliquots of reagent alcohol may need to be added to some samples to separate the organic and aqueous layers during the extraction step. A single-laboratory validation of these modifications was completed. During validation, 15 different product matrixes were analyzed. The intermediate precision averaged 2.70% RSD, and spike recovery data averaged 96.3%.

Carotenoids provide the major antioxidant defence in the globally significant N2-fixing marine cyanobacterium Trichodesmium.

Photosynthetic oxygen-evolving microorganisms contend with continuous self-production of molecular oxygen and reactive oxygen species. The deleterious effects of reactive oxygen species are exacerbated for cyanobacterial nitrogen-fixers (diazotrophs) due to the innate sensitivity of nitrogenase to oxygen. This renders incompatible the processes of oxygen-evolving photosynthesis and N-fixation. We examined total antioxidative potential of various diazotrophic and non-diazotrophic cyanobacteria. We focused on Trichodesmium spp., a bloom-forming marine diazotroph that contributes significantly to global nitrogen fixation. Among the species tested, Trichodesmium possessed the highest antioxidant activity. Moreover, while proteins constituted the dominant antioxidative component of all other cyanobacteria tested, Trichodesmium was unique in that small-molecule natural products provided the majority of antioxidant activity, while proteins constituted only 13% of total antioxidant activity. Bioassay-guided fractionation followed by high-performance liquid chromatography profiling of antioxidant purified fractions identified the highly potent antioxidant all-trans-ß-carotene, and small amounts of 9-cis-ß-carotene and retinyl palmitate. Search of the Trichodesmium genome identified protein sequences homologous to key enzymes in the ß-carotene to retinyl palmitate biosynthetic pathway, including 33-37% identity to lecithin retinol acyltransferase. The present study demonstrates the importance of carotenoids in Trichodesmium's arsenal of defensive compounds against oxidative damage and protection of nitrogenase from oxygen and its radicals.

A study on vitamin D and vitamin A in milk and edible oils available in the United Arab Emirates.

Two high-performance liquid chromatography methods have been validated for the determination of vitamin D and vitamin A in milk and edible oils. The percentage recovery of vitamin D added to milk ranged from 89% to 105%, with the repeatability relative standard deviation ranging from 2.78% to 6.11%. Its recovery in oil samples ranged from 90% to 102%, with the repeatability relative standard deviation ranging from 3.97% to 7.54%. The average recovery of vitamin A added to milk was found to be 98.7%. Analytical data for vitamin D in different brands of milk and milk products in the market samples of the United Arab Emirates indicate that 87% of samples contain vitamin D with 39% of samples within the acceptable range (0.8-1.2 microg/100 ml), where as 31% were found to be under-fortified and 30% were over-fortified. Analytical data for vitamin D in edible oils confirm a large variation. All milk samples analyzed contain less than 55 microg/100 ml vitamin A.

Salivary 1,5-Anhydroglucitol and Vitamin Levels in Relation to Caries Risk in Children.

The objective of this study was to evaluate the association between salivary 1,5-anhydroglucitol (AG), vitamins A (VA), C (VC), and E (VE), and caries risk in children. 100 healthy children aged between 6 and 13 years were divided into two equal groups of caries-free (DMFS/dmfs=0) and caries active (DMFS/dmfs>3). Unstimulated midmorning saliva was collected from all the children and the levels of salivary AG and vitamins A, C, and E were measured. Caries risk assessment was done using American Academy of Pediatric Dentistry Caries Assessment Tool. Analysis of salivary AG and vitamins was performed using a commercially available ELISA kit. Low levels of AG were present in caries active and high caries risk groups compared to caries-free and low/medium caries risk groups. This difference is statistically significant (p < 0.05). A strong negative correlation between AG and caries activity was observed in the caries active group. VA was not related to caries activity, while VC and VE displayed a statistically significant correlation (p < 0.05). Similarly, a strong negative correlation was observed between the levels of AG and high caries risk group. Salivary AG, VC, and VE together are related to caries risk in caries active children. These salivary parameters can act as indicator of caries status in children.

Hepatotoxicity of alcohol-induced polar retinol metabolites involves apoptosis via loss of mitochondrial membrane potential.

Chronic alcohol consumption depletes hepatic vitamin A stores. However, vitamin A supplementation is hepatotoxic, which is further potentiated by concomitant alcohol consumption. It was suggested that polar retinol metabolites generated by alcohol-inducible cytochrome P4502E1 aggravate liver damage. However, experimental evidence supporting this hypothesis is lacking. To elucidate the effects of polar retinol metabolites on cultured HepG2 cells and primary rat hepatocytes, polar retinol metabolites were extracted from liver tissues of rats fed either an alcoholic or isocaloric control Lieber-DeCarli diet. Cell toxicity assays included morphology assessment, trypan blue exclusion test, and LDH/AST leakage. Staining for DAPI and acridine orange, FACS analysis, and Western blot for cleaved caspase-9 and -3 were used to detect apoptosis. Polar retinol metabolites caused marked cytotoxicity in a concentration- and time-dependent manner in both cell types reflected by morphological changes, a dramatic increase in trypan blue positive cells, and LDH/AST leakage. Toxicity was due to apoptosis, as demonstrated by a time-dependent increase of sub-G1 cellular events, a rapid loss of mitochondrial membrane potential, and a time-dependent activation of caspase-9 and -3. No toxicity was found with equivalent doses of the control extract from nonalcoholic rats. We demonstrate that polar retinol metabolites cause marked hepatocyte death through the induction of apoptosis.

Microemulsion electrokinetic chromatography for the separation of retinol, cholecalciferol, delta-tocopherol and alpha-tocopherol.

A microemulsion electrokinetic chromatographic method was used to separate fat-soluble vitamins. The separation of retinol, cholecalciferol, and delta- and alpha-tocopherol was performed using a microemulsion containing 0.75% (v/v) n-heptane, 30 mM bis(2-ethylhexyl)sodium sulfosuccinate (AOT), 5% (v/v) 1-butanol, 15% (v/v) 1-propanol and 15% (v/v) methanol in 20mM boric acid-sodium borate buffer. The effect of the different microemulsion constituents was studied, including the type and concentration of surfactant, buffer, oil and co-surfactants. The presence of methanol in the microemulsion was found to be necessary to achieve the separation of the tocopherols. Detection was carried out at 200, 265 and 325 nm for the tocopherols, cholecalciferol and retinol, respectively. Calibration curves and precision data were obtained for each analyte. Good linear relationships were found between the analytical signal and the analytes concentration in the 25-500 mg L(-1) for retinol and cholecalciferol, and 25-300 mg L(-1) for tocopherols ranges. The precision of the method afforded relative standard deviations in the 4.0-10% range.

Simulation and optimization of a supercritical extraction process for recovering provitamin A.

In this work, a simulation procedure of a supercritical extraction process was developed through the use of the commercial simulator HYSYS (Hyprotech Ltd.), adapting the existing units to the operating conditions typical of the supercritical extraction process. The objective is to recover provitamin A (beta-carotene) from palm oil (esterified) using carbon dioxide/ethanol as the supercritical mixed solvent. This example characterizes the problem for recovering high added value product from natural sources, as the palm oil, which is desired by the market. Owing to the fact that esterified palm oil is a complex mixture, made by several components, in order to characterize this system in the simulator, it was necessary to create hypothetical components using the UNIFAC (universal function-group activity coefficients model) group contribution, because they are not present in a conventional database and, then, their physical properties must be estimated and/or predicted before the simulation. The optimization was carried out in each simulation for each equipment, in terms of operating conditions (temperature and pressure), in order to obtain the maximum recovery of carotenes. According to the results, it was possible to concentrate carotenes through two cycles of supercritical extraction with high yield. Furthermore, ethyl esters (biodiesel) were also obtained, as a byproduct of the proposed process, which can also be used as an alternative fuel, with the important characteristic that it is renewable.

A novel high-throughput method for supported liquid extraction of retinol and alpha-tocopherol from human serum and simultaneous quantitation by liquid chromatography tandem mass spectrometry.

BACKGROUND: Measurement of vitamin A (retinol) and E (alpha-tocopherol) in UK clinical laboratories is currently performed exclusively by high-performance liquid chromatography with ultraviolet detection. We investigated whether retinol and alpha-tocopherol could be measured simultaneously by liquid chromatography tandem mass spectrometry. METHODS: Serum samples (100 µL) were extracted using Isolute + Supported Liquid Extraction plates. Chromatography was performed on a Phenomenex Kinetex Biphenyl 2.6 µm, 50 × 2.1 mm column, and liquid chromatography tandem mass spectrometry on a Waters Acquity TQD. Injection-to-injection time was 4.3 min. The assay was validated according to published guidelines. Patient samples were used to compare liquid chromatography tandem mass spectrometry and high-performance liquid chromatography with ultraviolet detection methods. RESULTS: For retinol and alpha-tocopherol, respectively, the assay was linear up to 6.0 and 80.0 µmol/L, and lower limit of quantification was 0.07 and 0.26 µmol/L. Intra and interassay imprecision were within desirable analytical specifications. Analysis of quality control material aligned to NIST SRM 968e, and relative spiked recovery from human serum, both yielded results within 15% of target values. Method comparison with high-performance liquid chromatography with ultraviolet detection methodology demonstrated a negative bias for retinol and alpha-tocopherol by the liquid chromatography tandem mass spectrometry method. Analysis of United Kingdom National External Quality Assurance Scheme samples yielded mean bias from the target value of +3.0% for retinol and -11.2% for alpha-tocopherol. CONCLUSIONS: We have developed a novel, high-throughput method for extraction of retinol and alpha-tocopherol from human serum followed by simultaneous quantitation by liquid chromatography tandem mass spectrometry. The method offers a rapid, sensitive, specific and cost-effective alternative to high-performance liquid chromatography with ultraviolet detection methodology, and is suitable for routine clinical monitoring of patients predisposed to fat-soluble vitamin malabsorption.

A simultaneous quantitative method for vitamins A, D and E in human serum using liquid chromatography-tandem mass spectrometry.

Non-classical roles of fat-soluble vitamins (FSVs) in many pathologies including cancer have been identified. There is also evidence of hormonal interactions between two of these vitamins, A and D. As a result of this enhanced clinical association with disease, translational clinical research and laboratory requests for FSV measurement has significantly increased. However there are still gaps in the analytical methods available for the measurement of these vitamins. This study aimed to develop a method for simultaneous quantification of 25-hydroxyvitamin-D2 (25-OHD2), 25-hydroxyvitamin-D3 (25-OHD3) and its 3-epimer (epi-25-OHD3), retinol and α-tocopherol in human serum using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The procedure was developed and validated across two LC-MS/MS platforms, using commercial calibrators referenced to certified reference materials, controls, and deuterated internal standards. The samples were prepared by liquid-liquid extraction prior to injection and LC separation (using a Pursuit-PFP column) on two Agilent MS/MS systems (6410 and 6490) in electrospray ionisation positive mode with multiple reaction monitoring. Identification and quantification of 25-OHD3 from its 3-epimer as well as 25-OHD2, retinol and α-tocopherol were achieved. The dynamic ranges were 4-160 nmol/L for 25-OHD2 and epi-25-OHD3, 4-200 nmol/L for 25-OHD3, 0.1-4.0µmol/L for retinol and 4-70µmol/L for α-tocopherol with correlation (r(2)) of 0.997-0.998. Based on participation in an external quality assurance program, the overall performance of the simultaneous methods were: imprecision (CV%) and inaccuracy (average bias) 3.0% and 3.2 nmol/L, respectively, for 25-OHD3; 5.0% and 0.04µmol/L, respectively, for retinol; and 4.7% and 0.2µmol/L, respectively, for α-tocopherol. In summary, two simple LC-MS/MS methods were successfully developed and validated for the simultaneous quantification of the three vitamin D metabolites (25-OHD2, 25-OHD3 and 3-epimer of 25-OHD3), vitamin A (retinol) and vitamin E (α-tocopherol) in serum.

An HPLC-MS/MS method for the separation of α-retinyl esters from retinyl esters.

Enzymatic cleavage of the nonsymmetric provitamin A carotenoid α-carotene results in one molecule of retinal (vitamin A), and one molecule of α-retinal, a biologically inactive analog of true vitamin A. Due to structural similarities, α-retinyl esters and vitamin A esters typically coelute, resulting in the overestimation of vitamin A originating from α-carotene. Herein, we present a set of tools to identify and separate α-retinol products from vitamin A. α-Retinyl palmitate (αRP) standard was synthesized from α-ionone following a Wittig-Horner approach. A high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method employing a C30 column was then developed to separate the species. Authentic standards of retinyl esters and the synthesized α-RP confirmed respective identities, while other α-retinyl esters (i.e. myristate, linoleate, oleate, and stearate) were evidenced by their pseudomolecular ions observed in electrospray ionization (ESI) mode, fragmentation, and elution order. For quantitation, an atmospheric pressure chemical ionization (APCI) source operated in positive ion mode was used, and retinol, the predominant in-source parent ion was selected and fragmented. The application of this method to a chylomicron-rich fraction of human plasma is demonstrated. This method can be used to better determine the quantity of vitamin A derived from foods containing α-carotene.

Identification of 3-dehydroretinol (vitamin A2) in mouse liver.

3-Dehydroretinol (vitamin A2) and its long-chain fatty acyl esters have been isolated from hairless mouse liver by high-performance liquid chromatography (HPLC). In adult animals, these compounds amount to 1-2 micrograms/g liver, corresponding to 1-2% of the retinol (vitamin A1) concentration. Studies on the regulation of 3-dehydroretinol levels in liver showed that the age and vitamin A status of the animal affect the levels, but the relative proportions of retinol and 3-dehydroretinol are constant.

Biosynthesis of retinol in bovine corpus luteum tissue.

The bovine corpus luteum was found to contain retinol, as analyzed by fluorometry and the formation of anhydroretinol. After the incubation of beta-[15,15 foot-3H] carotene with slices of corpus luteum tissue the biosynthesis of radioactive retinol was demonstrated. Alumina column chromatography, thin layer chromatography, and high speed liquid chromatography were used to isolate and identify radioactive vitamin A. Retinol was also found to form a polar complex, from which could be released by acid hydrolysis.

Effect of tetrachlorodibenzo-p-dioxin (TCDD) on the glucuronidation of retinoic acid in the rat.

Administration of a single oral dose (10 micrograms/kg) of tetrachlorodibenzo-p-dioxin (TCDD) caused a 33% decrease in retinyl esters in the livers of male rats, but a 13-fold increase in retinyl esters in the kidney and a 3-fold increase in serum retinol. Liver and kidney microsomal uridine diphosphoglucuronosyltransferase (UDPGT) activity toward all-trans-retinoic acid was increased 3.7- and 2.6-fold, respectively, ten days following exposure to TCDD. Verification of the in vitro formation of [3H]retinoyl beta-glucuronide (RG) was by cochromatography with authenic RG on reversed phase high pressure liquid chromatography (HPLC), identification of retinoic acid as the hydrolysis product after beta-glucuronidase treatment, and the characterization of the all-trans-retinoyl glucuronide by negative fragment mass spectroscopy, fast atom bobardment. We conclude that increased retinoic acid glucuronidation may be a contributing factor to the hepatic depletion of vitamin A and the increased excretion of vitamin A metabolites following TCDD exposure.

Separation and determination of Vitamins E and A in multivitamin syrup using micellar liquid chromatography and simplex optimization.

A rapid method has been described for separation and determination of Vitamins A and E using micellar liquid chromatography (MLC). Influence of temperature of column and addition of organic modifiers on separation efficiency was investigated. A temperature of 30 degrees C and 1-butanol modifier was selected. Optimization of the parameters affecting the separation including percent of organic modifier, pH of the mobile phase, concentration of surfactant, and flow rate of the mobile phase was performed simultaneously using the super-modified simplex (SMS) procedure. Results showed that 11.7% 1-butanol, 76.9 mM sodium dodecyl sulphate (SDS), pH of 6.73 and a flow rate of 1.35 ml min(-1) are the best conditions for separation of these compounds. The analytical parameters including linearity, r>0.9990; limit of detection 1.71 and 4.52 microg ml(-1) for A and E, respectively; precision of the method, R.S.D.<2.85%; and recovery, more than 90%, show that the method is useful for measuring these compounds in pharmaceutical preparations.

Robust measurement of vitamin A status in plasma and blood dried on paper.

Vitamin A deficiency is the leading cause of preventable blindness in children and increases the risk of disease and death from severe infections. In addition, fat soluble vitamin A and associated retinoids directly regulate the expression of genes involved in fatty acid metabolism. Conventional methods for measuring vitamin A involve venipuncture, centrifugation and refrigeration all of which make measuring vitamin A in nutritional surveys expensive. We aimed to develop a simple and robust system for measurement of retinol (biomarker for vitamin A) using dried blood spot (DBS) samples. Low recoveries and inconsistent results reported by others were found to be due to poor extraction efficiency rather than retinol instability. Maintaining acid conditions during extraction resulted in recoveries >95% with <6.5% of coefficient of variation. Using isocratic high performance liquid chromatography, separation was achieved in <3.5 min. Detector response was linear (R(2)=0.9939) within a range of 0.05-2 µg/mL, with a limit of quantification of 0.05 µg/mL. Retinol in DBS was shown to be stable (>95%) at room temperature for up to 10 weeks. DBS values for retinol were highly correlated with venous blood samples from 24 healthy subjects (r=0.9724) and were consistent with results from a commercial laboratory. This simple and reliable method for the determination of vitamin A status should prove particularly valuable for population studies and large clinical trials.