Non-aqueous Reversed-phase High-performance Liquid Research Articles

Development and validation of a non-aqueous reversed-phase high-performance liquid chromatographic method for the determination of four chemical UV filters in suncare formulations

A non-aqueous reversed-phase high performance liquid chromatographic method (RP–HPLC) with UV detection at 313 nm was developed and validated for simultaneous determination of methylene bis-benzotriazolyl tetramethylphenol (Tinosorb M) along with three other chemical UV filters, octocrylene (Eusolex OCR), octyl methoxycinnamate (Eusolex 2292) and octyl salicylate (Eusolex OS) in suncare products. An isocratic elution was performed on a Hypersil BDS RP-C 18 column (250mm×4.6 mm), 5 μm particle size, using a mobile phase consisted of methanol–acetonitrile (90:10, v/v) with a flow-rate of 1.5 ml/min. The determination of the four UV filters was not interfered by the excipients in the products. The method of external standard, as well as the standard addition method was used for the determination. The external standard calibration curves were linear for Eusolex OCR, Eusolex 2292, Eusolex OS, and Tinosorb M in the concentration ranges of 0.5–100 μM, 0.5–100 μM, 0.5–200 μM, and 0.2–100 μM, respectively. Day-to-day relative standard deviation of the determination was within 3%. Limits of detection and quantitation of the above compounds were found equal to 36 and 110 nM, 220 and 660 nM, 170 and 520 nM, 44s and 130 nM, respectively. The recovery of these four chemical UV filters from the spiked samples was 96–103%.

Ultrasensitive assays of trans- and cis-β-carotenes in vegetable oils by high-performance liquid chromatography–thermal lens detection

The fact that β-carotene might be the protective factor against various cancers, suggests the need for a rapid reliable assay for this potential marker. We have proposed the method for selective, precise and simple profiling of carotenoids as well as for simultaneous ultrasensitive assaying of trans-β-carotene (TBC) and cis-β-carotene(s) (CBC) in five vegetable oils. The oil samples diluted 20 or 100 times were directly injected and analysed by means of the isocratic non-aqueous reversed-phase high-performance liquid chromatography (HPLC) combined with ultrasensitive thermal lens detection (TLS). Elution of TBC was followed by CBC; both were identified and determined in olive, safflower, sesame, wheat germ and linseed oil by standard addition method. The presence of lutein/zeaxanthin, some xanthophyll and/or early eluting carotene may be also presumed in the aforementioned oils. The examined oils showed different and characteristic carotenoid/carotene profiles and characteristic TBC-to-CBC ratios. Both analytes were selectively detected in the presence of palmitic, oleic, linoleic, linolenic and stearic acid, β-sitosterol and dl-α-tocopherol. This extends applicability of the method to other vegetable oils as well. Favourable analytical performances (high sensitivity, low limits of detection (LODs) and wide linearity span) enabled the direct analyses of highly diluted oils. This resulted in several major benefits of the proposed method, among which (i) reduced risk of stationary phase deterioration, (ii) avoiding the risk of carotenoids transformations and (iii) substantial labour and time savings. The TBC and CBC in diluted vegetable oils were reliably measured at ultratrace level (1–26 ng ml −1) with the S/ N ratio ranging from 4 to 140 and precisely determined (imprecision 0.4–8.3%). The concentrations of TBC+CBC estimated in the original oils were as follows: 90.5+51.2 ng ml −1 in sesame oil, 146.0+164 ng ml −1in safflower oil, 464.6+206.1 ng ml −1in linseed oil, 453.7+143.3 ng ml −1 in olive oil and 2.31+2.63 μg ml −1 in wheat germ oil. The characteristic and variable portion of TBC within total β-carotene may serve as a reliable indicator of both, quality and authenticity of the vegetable oil. The HPLC–TLS assay proposed may therefore be successfully applied in nutritional, agricultural and epidemiological studies.

Separation and identification of perchlorinated polycyclic aromatic hydrocarbons and fullerenes (C 60, C 70) by coupling high-performance liquid chromatography with ultraviolet absorption spectroscopy and atmospheric pressure chemical ionization mass spectrometry

Simultaneous separation and identification of perchlorinated polycyclic aromatic hydrocarbons (PCPAHs) and fullerenes is of practical interest due to the growth mechanism of fullerenes involved with PCPAHs. Non-aqueous reversed-phase high-performance liquid chromatography (HPLC), with an ODS column and a gradient mobile phase of methanol–ethanol–cyclohexane mixtures, was combined with both rapid-scan ultraviolet spectrometry (UV) and atmospheric pressure chemical ionization mass spectrometry (APCI-MS) for the separation and identification of over 80 PCPAHs as well as fullerenes C 60 and C 70, that were synthesized in the discharge reaction of chloroform. PCPAH retention was found to depend on the number of aromatic rings and the degree of non-planarity of PCPAH structure. Based on the isotopic pattern of molecular ion or/and quasi-molecular ion peaks in corresponding mass spectra, molecular compositions of the PCPAH products were unambiguously determined. The results obtained from the HPLC–UV–MS analysis not only are helpful for the understanding of the fullerenes formation mechanism, but also contribute to the analytical technique capable of separating and identifying the complicated mixture of PCPAHs and fullerenes.

Autoxidation products of normal and genetically modified canola oil varieties determined using liquid chromatography with mass spectrometric detection

Normal, high stearic acid and high lauric acid canola oil varieties were heated in the presence of air to allow autoxidation to occur. After the reaction, the oils were analyzed using a non-aqueous reversed-phase high-performance liquid chromatographic separation followed by detection using atmospheric pressure chemical ionization mass spectrometry. Oxidized products were separated and identified. The major autoxidation products which remained intact were epoxides and hydroperoxides. Two classes of epoxy triacylglycerols (TAGs) were formed. One class with the epoxy group replacing a site of unsaturation and one class adjacent to a site of unsaturation, as was previously reported for model TAGs. Intact oxidation products resulted mostly from oxidation of oleic acid, while oxidation products of linoleic and linolenic acid chains decomposed to yield chain-shortened species. Both neutral and polar chain-shortened products were observed. Polar chain-shortened decomposition products eluted at very short retention times and required a different chromatographic gradient to separate the molecules. This class of molecules was tentatively identified as core aldehydes. The high stearic acid canola oil yielded more intact oxidation products containing stearic acid, as expected. The high lauric acid oil produced intact oxidation products which contained lauric acid.

Prediction of isocratic nonaqueous reversed-phase high-performance liquid chromatography retention parameters and response factors of triacylglycerols detected by an ultraviolet-diode array-evaporative light-scattering on-line system.

Nonaqueous reversed-phase high-performance liquid chromatography of 10 homogeneous triacylglycerol molecular species (TAG), both saturated and unsaturated, is carried out. The eluate from the column is detected by an ultraviolet diode array detector (DAD) on-line with an evaporative light-scattering detector (ELSD). The retention parameters (as selectivities, alpha) for 220 TAGs are determined, and the obtained values are related to the following structural parameters: total carbon number; mono-, di-, and triunsaturated fatty acid residues number/molecule; and monounsaturated fatty acid carbon number. Multiple regression analysis is carried out to obtain a relationship for the prediction of alpha values of any TAG when the same experimental conditions are used. In regard to the quantitative analysis of the separated TAG species, the dependence of response of the two on-line detectors on the aforementioned structural parameters is studied. Three different wavelengths (205, 210, and 215 nm) are considered for TAG detection by DAD; in each case, the obtained multiple regression model shows a good correlation between the dependent variable and predictive values of the TAG species (response factors and considered structural parameters, respectively). The ELSD gives responses exponentially related to injected amounts. Also, in this case, an attempt to relate the response factors of each considered detector to some structural parameters of TAG species is carried out. The results of this study are used to analyze the TAG fraction from an olive oil.

Prediction of relative retention times of triacylglycerols in non-aqueous reversed-phase high-performance liquid chromatography

Accurate and simple methods for prediction of relative retention times of triacylglycerols in non-aqueous reversed-phase HPLC are presented here. The prediction is based on a simple calculation using the experimental relative retention time (min) of the triacylglycerol with the closest corresponding structure and taking into account the contributions (min) of functional groups present in the triacylglycerol for which the standard is not available to predict the relative retention time. The contribution of functional groups on the fatty acyl in the s n-2 position were greater than those at the s n-1(3) position, especially for polar functional groups such as the hydroxy group of ricinoleate. Another method of prediction which is based on the experimental relative retention time of tristearin and disregards the difference of s n-1(3) and s n-2 is also presented here.

Distribution of dietary palm carotenes and their metabolites in the rabbit

The distributions of dietary palm carotenes and their metabolites in rabbit's plasma and organs were determined using non-aqueous reversed-phase high performance liquid chromatography and a photodiode array detector. Rabbits fed with diets enriched with palm carotenes 309 mg/kg diet were found to have the different various carotenes in varying amounts in their plasma and organ tissues, mainly in the liver and adrenal glands. Most of the supplemented α- and β-carotenes were metabolized to retinol and retinyl esters, which were mainly stored in the liver and to a lesser extent, the pancreas. Small amounts of other carotenes such as phytoene, phytofluene and ζ-carotene, were present in the rabbit's plasma and organ tissues. The contents of retinol and retinyl esters in the liver of rabbits fed diets supplemented with palm carotenes were respectively 3 and 30 times higher than that in the rabbit groups fed diets without palm carotenes. The major retinyl esters present in the organ tissues were retinyl palmitate, oleate and linoleate, whereas only retinyl palmitate and stearate were found to be in predominant in the plasma. ϵ-Retinyl esters, which are metabolites from α-carotene, were also found to be present in the liver and pancreas. For rabbits fed with diets enriched with palm carotenes but depleted of palm vitamin E, relatively small amounts of carotenes and retinyl esters were detected (only in the plasma and liver), indicating that vitamin E substantially affects the disposition of dietary carotenes and their metabolites in plasma and tissues of rabbits.

Non-aqueous reversed-phase high-performance liquid chromatography of synthetic triacylglycerols and diacylglycerols

Reversed-phase high-performance liquid chromatography (HPLC) methods were developed for the separation of molecular species of 45 synthetic triacylglycerols and diacylglycerols. These methods used linear gradients of methanol-isopropanol and UV detection at 205 nm as well as radioactive flow detection, which we add for metabolic studies. The elution orders of triacylglycerols and diacylglycerols depend on the polarity of their fatty acid constituents with elution time increasing as polarity decreases. The elution orders of triacylglycerols depending on their fatty acid constituents were as follows: ricinoliec acid<linolenic acid<palmitoleic acid<myristic acid<palmitelaidic acid<linoleic acid<linolelaidic acid<oleic acid<palmitic acid<elaidic acid<petroselinic acid<petroselaidic acid<stearic acid, while the elution orders of diacylglycerols were: palmitic acid<oleic acid<stearic acid. For both classes of glycerides, elution corresponded closely with chain length, degree of unsaturation and presence of polar groups and they were similar to the elution orders of fatty acids on an aqueous C 18 HPLC which we reported recently: Other structural features also affect eltion order, as triacylglycerol containing a cis-fatty acid elutes slightly earlier than its isomer containing a trans-fatty acid. Additionally, higher polarity in the sn-2 position causes earlier elution; Diacylglycerol with a hydroxy group at sn-2 position of the glycerol backbone and triacylglycerol with a polar group on the fatty acid chain at sn-2 position elute slightly earlier than their respective sn-1(3) isomers.

Improved NARP-HPLC method for separating triglycerides of palm olein and its solid fractions obtained at low temperature storage

Non-aqueous reversed-phase high-performance liquid chromatography (NARP-HPLC) with refractive index (RI) detection is described and used for palm olein and its fractions obtained at 12.5 °C for 12–24 h. The calculation formula for fatty acid methyl esters (FAMEs) and carbon number (CN) from the data obtained by NARP-HPLC is described and correction factors for all carbon numbers and fatty acids are tabulated. The results were compared with those obtained from FAMEs analysed on 10% SP 2330 Supelco packed column gas-liquid chromatography (GLC) and CN analysed on 3% OV-1 Supelco packed column GLC. The results were found to agree well for C48, C50, C52, C16:0, C18:0 and C18:1 (correction factor ≈ 1.0); however, a slight variation was observed for components C54, C14:0 and C18:2 (correction factor 1.0 ± 0.37).

Retention behaviour of proteins under conditions of column overload in hydrophobic interaction chromatography

The sample solvent has a significant effect on band profiles in preparative liquid chromatography. This effect has been described by Guiochon and Jandera in non-aqueous reversed-phase high-performance liquid chromatography. Similar behaviour was observed in hydrophobic interaction chromatography of cytochrome c and lysozyme under conditions of column overloading. If the sample solvent contains a lower concentration of salt than the starting mobile phase in gradient hydrophobic interaction chromatography, the protein peak will be broken to two peaks. One peak is longer-retarded, another is shorter-retarded by hydrophobic interaction. The latter changes its retention time with the different injection volumes. This results in a loss in the recovery of protein in preparative chromatography. The possible mechanism of the band-splitting was investigated.

Effect of the sample solvent on band profiles in preparative liquid chromatography using non-aqueous reversed-phase high-performance liquid chromatography

If a solvent with a higher elution strength than the mobile phase is used to dissolve poorly soluble samples in preparative chromatography, significant deformation of the band profiles occurs, especially when the column is overloaded. Eventually, band splitting may take place. This behavior was observed in non-aqueous reversed-phase chromatography of cholesterol and other low-polarity sample compounds dissolved in a non-polar solvent, such as dichloromethane, at concentrations exceeding their solubilities in the mobile phase, a higher polarity solvent such as acetonitrile. In this chromatographic system, the dependence of the band deformation and of its splitting on the volume and concentration of the sample, the composition of the mobile phase and the column temperature were investigated. A model taking into account the dependence of the solubility of the sample components in the sample solvent and in the eluent and of the equilibrium isotherm of these components between the stationary and mobile phases on the composition of these two solvents (eluent and sample solvent) was worked out. The possible formation of supersaturated solutions was also considered in the model. This model permits the computer simulation of the band profiles for a single compound or for mixtures dissolved in a solvent different from the mobile phase. The results of the simulations are in qualitative agreement with the behavior observed experimentally.

Analysis of palm oil carotenoids by HPLC with diode-array detection.

Palm oil carotenoids are analyzed by nonaqueous reversed-phase high-performance liquid chromatography (NARP-HPLC) with UV/vis diode-array detection. Isocratic elution with 60% acetonitrile/35% methanol/5% methylene chloride at 2 mL/min on a 25-cm C18 column results in an analysis time of 30 min. Identification is made through absorption spectra and chromatographic elution behaviors, for example, polyenic pi conjugation, dipole moment of end-groups, and oxygen function on the chromophores. At least 12 carotenoids are identified with alpha- and beta-carotene as the dominant carotenoids (1:2 ratio). Several mono- and di-epoxides of alpha- and beta-isomers and hydrocarbon carotenes are found, including the UV-absorbing phytoene identified by spectral substraction. cis-Isomerization is found and discussed in the light of spectral evidence. The effect of saponification time on the amount of extracted carotenes is investigated. Quantitation results in a combined alpha- and beta-carotene concentration of at least 506 ppm. The detection limit for beta-carotene is 31 ng.

Separation of retinyl esters by non-aqueous reversed-phase high-performance liquid chromatography

Rapid separation and sensitive quantitation of vitamin A esters can be achieved by use of an acetonitrile—dichloromethane (80:20) mobile phase with a 5-μm C 18 column (15 cm x 4.6 mm) and absorbance detection at 325 nm. Either a Waters Resolve or a Rainin Microsorb column was used satisfactorily. Retinyl palmitate is eluted at about 7 min (capacity factor, k′ = 5.5) at a flow-rate of 1.5 ml/min; retinyl palmitate and retinyl oleate, which are usually difficult to separate, are well resolved (resolution 1.2). Sensitivity (at a signal-to-noise ratio of 10:1) is 8 pmol retinyl palmitate (equivalent to 2.5 ng retinol). Quantitation of total retinyl esters is identical to that determined by a gradient high-performance liquid chromatographic technique over the range 30–1000 ng retinyl esters. Retinyl ester peaks in rat liver extracts were identified by their characteristic light absorption spectra, susceptibility to saponification, and by co-chromatography with authentic standards. Nine vitamin A ester peaks were identified and quantitated in rat liver extracts. A 10-μm Whatman Partisil 10 25 ODS-2 column was used with the same mobile phase to obtain partial resolution of retinyl esters (resolution 1.05 between retinyl oleate and retinyl palmitate; k′ = 11.0 for retinyl palmitate) and improved retention for retinol ( k′ = 2.5, compared with k′ = 0.6 for retinol on the 5-μm column).