Non-aqueous Reversed-phase HPLC Research Articles

Non-aqueous reversed phase liquid chromatography with charged aerosol detection for quantitative lipid analysis with improved accuracy

There is a great need for efficient analysis of the composition of vegetable oils and fats, since it affects the physical and technical properties. However, due to the complex nature of these kind of samples, it is often difficult and costly. In the present study, we developed a Non-Aqueous Reversed-Phase HPLC method that can be used to separate and quantify different free fatty acids, fatty acid esters, monoacylglycerides, diacylglycerides and triacylglycerides, including regioisomers such as SOS/SSO and 1,2- and 1,3-diolein. Two 25 cm Nucleodur C18 Isis columns in series, sub-ambient column temperature and a mobile phase gradient composed of acetonitrile, acetic acid, isopropanol and heptane were used for the separation. The lipids were detected and quantified using a charged aerosol detector and it was found that the peak shape highly affected the detector response as well as the response uniformity, even when inverse gradient compensation was employed. Thus, calibration and determination of response factors were necessary for reliable quantification. A correlation between response factors and peak width at half peak height was found and used for quantification of non-calibrated components. A quantification approach was suggested including an appropriate selection of calibrated components, depending on sample composition and the accuracy required. It was shown in a complex oil sample that the reduced calibration approach, using only 6 instead of 33 calibrated components, resulted in virtually the same composition, but yielded a more accurate result compared to using relative area that neglects response factors. The method validation showed good reproducibility and accuracy, making it an excellent tool for extensive analysis of complex lipid mixtures.

Cholesteryl esters of ω-(O-acyl)-hydroxy fatty acids in vernix caseosa

Cholesteryl esters of ω-(O-acyl)-hydroxy FAs (Chl-ωOAHFAs) were identified for the first time in vernix caseosa and characterized using chromatography and MS. Chl-ωOAHFAs were isolated using adsorption chromatography on silica gel and magnesium hydroxide. Their general structure was established using high-resolution and tandem MS of intact lipids, and products of their transesterification and derivatizations. Individual molecular species were characterized using nonaqueous reversed-phase HPLC coupled to atmospheric pressure chemical ionization. The analytes were detected as protonated molecules, and their structures were elucidated in the negative ion mode using controlled thermal decomposition and data-dependent fragmentation. About three hundred molecular species of Chl-ωOAHFAs were identified in this way. The most abundant Chl-ωOAHFAs contained 32:1 ω-hydroxy FA (ω-HFA) and 14:0, 15:0, 16:0, 16:1, and 18:1 FAs. The double bond in the 32:1 ω-HFA was in the n-7 and n-9 positions. Chl-ωOAHFAs are estimated to account for approximately 1-2% of vernix caseosa lipids.

Efficient Separation and Analysis of Triacylglycerols: Quantitation of β-Palmitate (OPO) in Oils and Infant Formulas

A high-efficiency, convenient, and reliable method for the separation of structurally similar triacylglycerols is detailed and applied in the quantitative analysis of 1,3-dioleoyl-2-palmitoylglycerol (OPO) in infant formulas and OPO oils. OPO is an important lipid component in "humanized" infant formula. A fast preparative isolation of an OPO-containing fraction from the crude complex mixture, by nonaqueous reversed phase HPLC, followed by Ag(+)-HPLC with detection at 205 nm allowed fine separation and detection of the desired fraction. OPO was quantitated independently of its regioisomer 1,2-dioleoyl-3-palmitoylglycerol (OOP) and isomers of stearoyl-linoleoyl-palmitoyl glycerol that might be present in infant formulas. For samples with low OPO content, an evaporative light-scattering detector (ELSD) was more preferable than UV detection, with a calculated LOD of 0.1 μg of OPO injected and LOQ of 0.3 μg. The method, which showed high reproducibility (RSD < 5%), was suitable for both high OPO content oils and low OPO products such as unenriched infant formula. A number of possible interference issues were considered and dealt with.

Analysis of 1,2-diol diesters in vernix caseosa by high-performance liquid chromatography – atmospheric pressure chemical ionization mass spectrometry

Fatty acid diesters of long-chain 1,2-diols (1,2-DDE), or type II wax diesters, were analyzed in the vernix caseosa of a newborn girl. 1,2-DDE were isolated from the total lipid extract by the semipreparative TLC using plates coated with silica gel. Chromatographic separation of the 1,2-DDE molecular species was achieved on the non-aqueous reversed-phase HPLC with two Nova-Pak C18 columns connected in series (a total length of 45cm) and using an acetonitrile–ethyl acetate gradient. 1,2-DDE eluted from the column in the order of their equivalent chain number. The analytes were detected as ammonium adducts by an ion-trap mass spectrometer equipped with an atmospheric pressure chemical ionization source. Their structures were elucidated using tandem mass spectrometry with MS, MS2 and MS3 steps in a data-dependent mode. More than two thousand molecular species of 1,2-DDE were identified in 141 chromatographic peaks. The most abundant 1,2-DDE were monounsaturated lipids consisting of a C22 diol and a C18:1 fatty acid together with C16:0, C14:0 or C15:0 fatty acids. The positions of double bonds were characterized by the fragmentation of [M+C3H5N]+ formed in the ion source.

Development and optimization of a method for analyzing biodiesel mixtures with non-aqueous reversed phase liquid chromatography

Biodiesel (a mixture of fatty acid esters) is normally analyzed using gas chromatography/flame ionization detection, as specified by the ASTM D6584 and EN14105 standards. This paper proposes a binary gradient method for analyzing biodiesel mixtures using non-aqueous reverse phase HPLC with a UV detector capable of overcoming the drawbacks of the gas chromatographic technique normally used. The new analytical method was developed by means of a statistical sensitivity analysis applied to the main parameters influencing the recording, using the full factorial design method combined with the Yates algorithm and the steepest ascent optimization procedure. The present study shows the influence of the main biodiesel mixture separation analysis parameters. The resulting tool proved valid for analyzing not only biodiesel but also any traces of unreacted oil.

Quantitation of triacylglycerols from plant oils using charged aerosol detection with gradient compensation

Quantitative analysis of triacylglycerols (TGs) in plant oils and animal fats by normalization of peak areas can lead to erroneous results due to the large response differences with common HPLC detectors between the various TGs. The charged aerosol detector (CAD), that generates an almost universal response for non-volatile compounds, was combined with non-aqueous reversed-phase HPLC (NARP-HPLC) to develop a simple quantitative method, without need for RFs, for the analysis of complex natural TG mixtures from plant oils. Two 25 cm Hypersil ODS columns, connected in series, and a mobile phase gradient composed of acetonitrile, 2-propanol and hexane were used. Mobile phase compensation was applied, by mixing of the column effluent with the inversed gradient delivered by a second HPLC pump, for the suppression of the response dependency of the analytes on the mobile phase composition. Calibration curves of 16 saturated (from C7:0 to C22:0) and 3 unsaturated (C18:1, C18:2, C18:3) single-acid TG standards were measured and their RFs were compared with a previously described method using atmospheric pressure chemical ionization-mass spectrometry (APCI-MS). The variation in response of the most common TGs (containing fatty acid chains from 12 to 19 carbons) could be reduced to less than 5% making the combination of NARP-HPLC with CAD and mobile phase compensation an adequate tool for fast quantitative analysis of TGs in common plant oils.

Quantitation of triacylglycerols in plant oils using HPLC with APCI‐MS, evaporative light‐scattering, and UV detection

The main constituents of plant oils are complex mixtures of TGs differing in acyl chain lengths, number and positions of double bonds, and regioisomerism. A non-aqueous reversed-phase HPLC method with acetonitrile-2-propanol gradient and 30 + 15 cm NovaPak C18 columns makes possible an unambiguous identification of the highest number of TGs ever reported for these oils, based on positive-ion APCI mass spectra. A new approach to TG quantitation is based on the use of response factors with three typical detection techniques for that purpose (APCI-MS, evaporative light-scattering detection, and UV at 205 nm). Response factors of 23 single-acid TGs (saturated TGs from C7 to C22, 7 unsaturated TGs), 4 mixed-acid TGs, diolein and monoolein are calculated from their calibration curves and related to OOO. Due to differences between saturated and unsaturated acyl chains, the use of response factors significantly improves the quantitation of TGs. 133 TGs containing 22 fatty acids with 8-25 carbon atoms and 0-3 double bonds are identified and quantified in 9 plant oils (walnut, hazelnut, cashew nut, almond, poppy seed, yellow melon, mango, fig, date) using HPLC/APCI-MS with a response factor approach. Average parameters and relative fatty acid concentrations are calculated with both HPLC/APCI-MS and GC/ FID.

DEVELOPMENT AND VALIDATION OF HPLC METHOD FOR SIMULTANEOUS DETERMINATION OF FAT-SOLUBLE VITAMINS IN CAPSULES

A very simple nonaqueous reverse phase HPLC was developed for determining retinol acetate, retinol palmitate, cholecalciferol, alpha-tocopherol acetate, alphacalcidol, and phylloquinone in capsules without the need for saponification. A reverse phase (Lichrospher C8, 4.6 mm, id.) column was used with a mobile phase of acetonitrile–methanol (95:5, v/v) and flow rate of 1 mL/min. Sample treatment consisted only of the dilution of the capsule contents with n-hexane and methanol. The range of the assay method has been set at 2.5–150% of finished product label claim. The limit of detection of each vitamin was between 0.259 μg/mL (K1) and 2.379 μg/mL (alphacalcidol). The minimum average amount recovered was 98.1% (D3). The precision (relative standard deviation of label claim among six sample preparation as repeatability was not more than 0.82% (A palmitate) and, as intermediate (N = 24), was not more than 1.76% (E acetate). The Standard Preparation (at −15°C) and Sample Preparation (at 4°C) were stable for up to 48 and 24 h, respectively. This method is suitable for routine quantification in industrial quality assurance laboratories. The principal aspects of method validation are examined and discussed, and the validation studies indicate that the method is specific, accurate, precise, and robust over the concentration range investigated.

Simultaneous determination of retinol acetate, retinol palmitate, cholecalciferol, α-tocopherol acetate and alphacalcidol in capsules by non-aqueous reversed-phase HPLC and column backflushing

A very simple non-aqueous reversed-phase HPLC method has been developed for analysis of retinol acetate, retinol palmitate, cholecalciferol, α-tocopherol acetate and alphacalcidol in capsules without the need for saponification. A reversed-phase (LiChrospher C8, 4.6 mm i.d.) column is used with acetonitrile-methanol, 95∶5 (v/v) as mobile phase at flow rate of 1 mL min−1. Sample treatment consists only in dilution of the capsule contents withn-hexane and methanol. This method is suitable for routine quantification in the industrial quality-assurance laboratory.

Silver-phase high-performance liquid chromatography–electrospray mass spectrometry of triacylglycerols

Silver-phase chromatography hyphenated with on-line electrospray mass spectrometry can be applied to characterize triacylglycerols present in vegetable oils with respect to their degree of unsaturation, the position of the most unsaturated fatty acid and the carbon number (CN). The CN information obtained with silver-phase HPLC–ESP–mass spectrometry is complementary to the unsaturation information obtained by silver-phase HPLC-flame ionization detection. Both information is essential to monitor or study modified vegetable oils on the presence of non-natural triacylglycerols. The quantitative results obtained with the method are in agreement with the results obtained in the silver-phase HPLC-flame ionization detection and with theoretical values calculated from the fatty acid distribution of the oil. Silver-phase HPLC–ESP-mass spectrometry gives direct information on fatty acid position and triacylglycerol CN, for each of the triacylglycerols in the sample. This in contrast with non-aqueous reversed-phase HPLC hyphenated with on-line atmospheric pressure chemical ionization-mass spectrometry, which requires a more extensive data processing. The results obtained with silver-phase HPLC–ESP-mass spectrometry can be presented in a three-dimensional overview (relative amount, CN, fatty acid position) serving as a fingerprint for the 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.

Nonaqueous chromatography of biopolymers by means of their poly(ethylene glycol)‐mediated solubilization in alcohols

AbstractFreeze‐drying of aqueous solutions of biopolymers and excess poly(ethylene glycol)s leads to formation of hydrophobic complex. Complexation with PEG increases solubility of low molecular weight DNA, RNA, and several proteins in neat ethanol and methanol. Solubilized nucleic acids can be fractionated, and proteins separated by nonaqueous reversed phase HPLC. © 1995 John Wiley &amp; Sons, Inc.

Simultaneous analysis of individual carotenoids, retinol, retinyl esters, and tocopherols in serum by isocratic non-aqueous reversed-phase HPLC

A sensitive high-performance reversed-phase liquid chromatographic assay which utilizes isocratic elution and detection by two detectors has been developed to measure simultaneously several carotenoids, retinol, retinyl esters, and α- and γ-tocopherols in serum. Although 100 μl of serum or plasma is used routinely, as little as 10 μl can be analyzed. Typical lower limits of detection (5 : 1 signal/noise ratio) are 0.2 pmol for retinol, 6 pmol for α-tocopherol, 1 pmol for β-carotene, and 2 pmol for retinyl palmitate. Although lutein and zeaxanthin were only partially resolved from each other, all other major serum carotenoids including α-carotene from β-carotene and α-cryptoxanthin from β-cryptoxanthin were completely separated. Retinyl hexanoate and β-apo-8'-carotenyl decanoate are convenient, readily prepared internal standards for this assay. Relative retention indices, based on elution times of alkylphenone standards, proved useful both for characterization of compounds and for comparison between columns.

Effect of processing on β-carotene retention in crude palm oil and its products

Crude palm oil ( Elaeis guineensis) can serve as a promising source of β-carotene in developing countries where vitamin A deficiency is prevalent, apart from fulfilling other functions of an edible oil. Non-aqueous reverse phase HPLC analysis with a UV-Vis detector at 450 nm was carried out to estimate the amount of β-carotene in crude palm oil (CPO) produced in India. Isocratic elution with acetonitrile/methanol/dichloromethane (60%/35%/5%) at 1·5 ml/min on a 25 cm C 18 column eluted β-carotene at 14·5 min. β-Carotene content was estimated to be around 370 ppm, which amounts to about 70% of the total carotenoids (540 ppm—estimated spectrophotometrically at 450 nm E 1 cm 1% = 2500). Sensitivity and accuracy of the method was observed to be high as indicated by the standard graph of β-carotene and recovery of standard added to sample (100%). Effect of different cooking methods like baking, seasoning, deep frying and shallow frying on retention of β-carotene was studied and it was observed that 70–88% of it was retained in the cooked foods. Repeated deep frying, using the oil five times consecutively, resulted in a total loss of β-carotene by the fourth frying stage itself and alteration of its organoleptic, physical and chemical properties. Hence, CPO may be suitable for single frying operations only or for preparations which involve a short heating time and completely take up the oil into the cooked product, e.g. ‘Cake’, ‘Upma’, ‘Kichidi’, ‘Suji halwa’. Probably ‘Suji halwa’ can be selected as an ideal choice for vitamin A supplementation to vulnerable children because it is well accepted, works out to be cheap per RDA serving and it retains 70% of its β-carotene after cooking.

The use of large polycyclic aromatic hydrocarbons to study retention in non-aqueous reversed-phase HPLC

Elution strengths of 11 common HPLC solvents on a polymeric C18 phase were compared using a marker set of polycyclic aromatic hydrocarbons. Naphthalene, pyrene, benzo[ghi]perylene, and three larger naphthologues of 8, 10, and 12 rings (constituting a “naphthalene zigzag” series) were chosen because they span the solvent strength range up to and including the strongest solvents, tetrahydrofuran (THF) and chlorobenzene. Four pairs of similarly shaped isomers were used to probe solvent selectivity. With the exception of THF, HPLC solvent strength correlated with observed red shifts of fluorescence band maxima in each solvent. For THF, the pure solvent and blended mixtures behaved quite differently.

315 The binding of Tamoxifen in cytosol samples from human breast cancer

Crude palm oil (Elaeis guineensis) can serve as a promising source of β-carotene in developing countries where vitamin A deficiency is prevalent, apart from fulfilling other functions of an edible oil. Non-aqueous reverse phase HPLC analysis with a UV-Vis detector at 450 nm was carried out to estimate the amount of β-carotene in crude palm oil (CPO) produced in India. Isocratic elution with acetonitrile/methanol/dichloromethane (60%/35%/5%) at 1·5 ml/min on a 25 cm C18 column eluted β-carotene at 14·5 min. β-Carotene content was estimated to be around 370 ppm, which amounts to about 70% of the total carotenoids (540 ppm—estimated spectrophotometrically at 450 nm E1 cm 1% = 2500). Sensitivity and accuracy of the method was observed to be high as indicated by the standard graph of β-carotene and recovery of standard added to sample (100%).Effect of different cooking methods like baking, seasoning, deep frying and shallow frying on retention of β-carotene was studied and it was observed that 70–88% of it was retained in the cooked foods. Repeated deep frying, using the oil five times consecutively, resulted in a total loss of β-carotene by the fourth frying stage itself and alteration of its organoleptic, physical and chemical properties.Hence, CPO may be suitable for single frying operations only or for preparations which involve a short heating time and completely take up the oil into the cooked product, e.g. ‘Cake’, ‘Upma’, ‘Kichidi’, ‘Suji halwa’. Probably ‘Suji halwa’ can be selected as an ideal choice for vitamin A supplementation to vulnerable children because it is well accepted, works out to be cheap per RDA serving and it retains 70% of its β-carotene after cooking.

316 The influence of obstrogen receptor status on the response of metastatic breast cancer to aminoglutehimide

Crude palm oil (Elaeis guineensis) can serve as a promising source of β-carotene in developing countries where vitamin A deficiency is prevalent, apart from fulfilling other functions of an edible oil. Non-aqueous reverse phase HPLC analysis with a UV-Vis detector at 450 nm was carried out to estimate the amount of β-carotene in crude palm oil (CPO) produced in India. Isocratic elution with acetonitrile/methanol/dichloromethane (60%/35%/5%) at 1·5 ml/min on a 25 cm C18 column eluted β-carotene at 14·5 min. β-Carotene content was estimated to be around 370 ppm, which amounts to about 70% of the total carotenoids (540 ppm—estimated spectrophotometrically at 450 nm E1 cm 1% = 2500). Sensitivity and accuracy of the method was observed to be high as indicated by the standard graph of β-carotene and recovery of standard added to sample (100%).Effect of different cooking methods like baking, seasoning, deep frying and shallow frying on retention of β-carotene was studied and it was observed that 70–88% of it was retained in the cooked foods. Repeated deep frying, using the oil five times consecutively, resulted in a total loss of β-carotene by the fourth frying stage itself and alteration of its organoleptic, physical and chemical properties.Hence, CPO may be suitable for single frying operations only or for preparations which involve a short heating time and completely take up the oil into the cooked product, e.g. ‘Cake’, ‘Upma’, ‘Kichidi’, ‘Suji halwa’. Probably ‘Suji halwa’ can be selected as an ideal choice for vitamin A supplementation to vulnerable children because it is well accepted, works out to be cheap per RDA serving and it retains 70% of its β-carotene after cooking.

314 Criterion of ER positive in patients with benign and malignant breast diseases

Crude palm oil (Elaeis guineensis) can serve as a promising source of β-carotene in developing countries where vitamin A deficiency is prevalent, apart from fulfilling other functions of an edible oil. Non-aqueous reverse phase HPLC analysis with a UV-Vis detector at 450 nm was carried out to estimate the amount of β-carotene in crude palm oil (CPO) produced in India. Isocratic elution with acetonitrile/methanol/dichloromethane (60%/35%/5%) at 1·5 ml/min on a 25 cm C18 column eluted β-carotene at 14·5 min. β-Carotene content was estimated to be around 370 ppm, which amounts to about 70% of the total carotenoids (540 ppm—estimated spectrophotometrically at 450 nm E1 cm 1% = 2500). Sensitivity and accuracy of the method was observed to be high as indicated by the standard graph of β-carotene and recovery of standard added to sample (100%).Effect of different cooking methods like baking, seasoning, deep frying and shallow frying on retention of β-carotene was studied and it was observed that 70–88% of it was retained in the cooked foods. Repeated deep frying, using the oil five times consecutively, resulted in a total loss of β-carotene by the fourth frying stage itself and alteration of its organoleptic, physical and chemical properties.Hence, CPO may be suitable for single frying operations only or for preparations which involve a short heating time and completely take up the oil into the cooked product, e.g. ‘Cake’, ‘Upma’, ‘Kichidi’, ‘Suji halwa’. Probably ‘Suji halwa’ can be selected as an ideal choice for vitamin A supplementation to vulnerable children because it is well accepted, works out to be cheap per RDA serving and it retains 70% of its β-carotene after cooking.