Determination of Glycerol in Wines Using <Superscript>31</Superscript>P-NMR Spectroscopy

³¹P-NMR spectroscopy was employed to detect and quantify glycerol in red wines from various regions of Greece. This novel analytical method was based on the derivatization of the hydroxyl groups of glycerol with 2-chloro-4,4,5,5-tetramethyl dioxaphospholane, and identification of the phosphitylated compound on the basis of ³¹P chemical shifts. Quantification of glycerol in wines was accomplished by integration of appropriate signals in the ³¹P-NMR spectrum and the use of the phosphitylated cyclohexanol as the internal standard. The method was reproducible (CV (%) = 2.35) and accurate (CV (%) = 1.34). Its applicability to glycerol quantification in wines was tested against a weighted amount of a glycerol-model compound by linear regression analysis (R = 0.999; intercept = 0.074 ± 0.078; slope = 0.998 ± 0.003; p = 0.000). Furthermore, the NMR method was compared to the AOAC official method (HPLC) using the Bland and Altman statistical analysis. The distribution of the data points in the bias plot showed that 100% of the measurements of glycerol in 16 wine samples from various regions of Greece were within the limits of agreement of the two methods.     

TLC and <Superscript>31</Superscript>P-NMR Analysis of Low Polarity Phospholipids

High-performance TLC and ³¹P-NMR were assessed as methods of observing the presence of numerous low polarity phospholipids: bis-phosphatidic acid (BPA), semi-lyso bis-phosphatidic acid (SLBPA), N-acyl phosphatidylethanolamine (NAPE), N-(1,1-dimethyl-3-oxo-butyl)-phosphatidylethanolamine (diacetone adduct of PE, DOBPE), N-acetyl PE, phosphatidylmethanol (PM), phosphatidylethanol (PEt), phosphatidyl-n-propanol (PP), phosphatidyl-n-butanol (PB). Both techniques are non-discriminative and do not require the prior isolation of individual lipids. It appears that 2D TLC is superior to ³¹P NMR in the analysis of low polarity phospholipids. All phosphatidylalcohols were well separated by 2D TLC. However, some compounds which can present difficulty in separation by 2D-TLC (e.g., SLBPA and NAPE; or DOBPE and N-acetyl PE) were easily distinguished using ³¹P NMR so the methods are complimentary. A disadvantage of 2D TLC is that Rf values can vary with different brands and batches of TLC plates. The chemical shifts of ³¹P NMR were less variable, and so a library of standards may not be necessary for peak identification. Another advantage of ³¹P NMR is the ease of quantification of phospholipids. The applicability of the methods was tested on natural extracts of fish brain and cabbage stem.     

Rapid Determination of Free Fatty Acid in Extra Virgin Olive Oil by Raman Spectroscopy and Multivariate Analysis

We introduce a visible Raman spectroscopic method for determining the free fatty acid (FFA) content of extra virgin olive oil with the aid of multivariate analysis. Oleic acid was used to increase the FFA content in extra virgin olive oil up to 0.80% in order to extend the calibration span. For calibration purposes, titration was carried out to determine the concentration of FFA for the investigated oil samples. As calibration model for the FFA content (FFA%), a partial least squares (PLS) regression was applied. The accuracy of the Raman calibration model was estimated using the root mean square error (RMSE) of calibration and validation and the correlation coefficient (R ²) between actual and predicted values. The calibration curve of actual FFA% obtained by titration versus predicted values based on Raman spectra was established for different spectral regions. The spectral window (945–1600 cm⁻¹), which includes carotenoid bands, was found to be a useful fingerprint region being statistically significant for the prediction of the FFA%. High R ² and small RMSE values for calibration and validation could be obtained, respectively.     

Multivariate Analysis of <Superscript>1</Superscript>H-NMR Spectra of Genetically Characterized Extra Virgin Olive Oils and Growth Soil Correlations

Mono-varietal extra virgin olive oils were micro-extracted from drupes that were selectively collected from 28 trees distributed in five different Southern Italian Apulian areas. Nuclear Magnetic Resonance (NMR) profiles of these oil samples were correlated to the genetic (young green material) and soil (samples collected within the foliage projection) data of the tree of origin. Genetic analysis, performed on the samples using SSRs (Simple Sequence Repeats) by 9 microsatellite loci, confirmed the specific cultivar assignment (among Cima di Mola, Coratina, Ogliarola, and Oliva Rossa cultivars). Chemometric methods applied to ¹H-NMR spectroscopic data were used for cultivar and geographical origin discrimination of the studied extra virgin olive oils. Linear Discriminant Analysis (LDA) afforded a high reliability degree for discriminating cultivars (almost 90% of prediction ability), and a good assigning ability for the geographical origin (Ogliarola and Coratina samples used as subsets). Soil analyses were performed for each tree. Regression analysis was applied to soil composition in order to correlate available nutrients and total metals with the content of fatty acids and minor components present in monovarietal extra virgin olive oils. In the case of oleic and linoleic fatty acids, and for some terpenes, B, Cr, Mn, Zn were found to give significant correlations. Zn and Mn were the most significant trace elements for all the correlations found (p < 0.01). The results obtained (genetic, spectroscopic and soil analyses) are discussed as a multidisciplinary approach for setting up a strategy for a cultivar and/or geographic origin certification committed database construction.     

Detection of Soft‐Deodorized Olive Oil and Refined Vegetable Oils in Virgin Olive Oil Using Near Infrared Spectroscopy and Traditional Analytical Parameters

The European Parliament identifies virgin olive oil (VOO) as one of the foods which are often subject to fraudulent activities. Possibilities of adulteration are the application of illegal soft deodorization of extra virgin olive oil (EVOO) or the commercialization of blends of EVOO with soft‐deodorized EVOO or refined vegetable oils. Despite the search for possibilities to prove the illegal soft deodorization of EVOO or the addition of cheaper vegetable oils to EVOO, suitable methods are still missing. Therefore, the aim of the study is to develop a new analytical and statistical approach addressing detection of mild deodorization or addition of refined foreign oils. For this purpose, VOOs are treated in lab‐scale for 1 h up to 28 days at different temperatures (20, 50, 60, 80,100, 110, and 170 °C) in order to simulate and study the effect of heat treatment on known analytical parameters by near infrared spectroscopy (NIR). A logit regression model enabling the calculation of the probability for a heat treatment is developed. This new methodology allows detecting both soft deodorized olive oils and blends of EVOO with cheaper full refined vegetable oils. Adding only 10% of full refined oil could be detected in extra VOO. Practical Applications: NIR methods combined with chemometrics have become one of the most attractive analytical tools to control quality of food. It is a simple, precise, and rapid method. All relevant analytical parameters of oxidative and thermal fat degradation can be determined in a single run and be used to detect adulterated virgin olive oils (VOOs). The use of a simple equation developed from the logistic regression using peroxide value, K‐values, p‐anisidine value, pyropheophytine, 1,2‐diacylglycerols, total polar compounds and monomeric oxidized triacylglycerols, and other well‐known parameters allows to detect mild deodorized olive oils or also blends of VOO with soft‐deodorized ones or the addition of low amounts of foreign vegetable oils. This technique has potential to be used as a screening method for the detection of adulterated olive oils using both the traditional laboratory methods and the corresponding NIR‐methods.     

Rapid Identification and Relative Quantification of the Phospholipid Composition in Commercial Lecithins by <Superscript>31</Superscript>P NMR

³¹P NMR analysis of samples prepared in a sodium cholate detergent system was used as a method for the identification and quantification of enzymatic hydrolysis products of lecithin. To precisely characterize all of the hydrolysis products from commercial lecithin, a series of enzymatic reactions of each phospholipid with phospholipase PLA1 were conducted and monitored by ³¹P NMR at different times. Twenty-six phosphorus-containing hydrolysis products from six classes of phospholipids (PC, PI, PS, PE, PG, PA) were found and determined by ³¹P NMR measurement. The impact of pH on the chemical shift values for these hydrolysis products was observed and reported. To the best of our knowledge, this is the first report of ³¹P-NMR chemical shift values for entire lyso-phospholipids hydrolyzed from 6 classes of phospholipids. Rapid and routine analysis of phospholipid composition in commercial lecithins by ³¹P NMR was achieved without the need of phospholipid standards.     

Novel,Rapid Identification,and Quantification of Adulterants in Extra Virgin Olive Oil Using Near‐Infrared Spectroscopy and Chemometrics

A new, rapid Fourier transform near infrared (FT‐NIR) spectroscopic procedure is described to screen for the authenticity of extra virgin olive oils (EVOO) and to determine the kind and amount of an adulterant in EVOO. To screen EVOO, a partial least squares (PLS1) calibration model was developed to estimate a newly created FT‐NIR index based mainly on the relative intensities of two unique carbonyl overtone absorptions in the FT‐NIR spectra of EVOO and other mixtures attributed to volatile (5280 cm^?1) and non‐volatile (5180 cm^?1) components. Spectra were also used to predict the fatty acid (FA) composition of EVOO or samples spiked with an adulterant using previously developed PLS1 calibration models. Some adulterated mixtures could be identified provided the FA profile was sufficiently different from those of EVOO. To identify the type and determine the quantity of an adulterant, gravimetric mixtures were prepared by spiking EVOO with different concentrations of each adulterant. Based on FT‐NIR spectra, four PLS1 calibration models were developed for four specific groups of adulterants, each with a characteristic FA composition. Using these different PLS1 calibration models for prediction, plots of predicted vs. gravimetric concentrations of an adulterant in EVOO yielded linear regression functions with four unique sets of slopes, one for each group of adulterants. Four corresponding slope rules were defined that allowed for the determination of the nature and concentration of an adulterant in EVOO products by applying these four calibration models. The standard addition technique was used for confirmation.     

Handheld Near‐Infrared Spectroscopy for Distinction of Extra Virgin Olive Oil from Other Olive Oil Grades Substantiated by Compositional Data

Miniaturization of analytical technology has paved the way for in‐situ screening of foods. In the current study, the spectral features of olive oils are examined by handheld near‐infrared spectroscopy to explore the technology's capabilities to distinguish extra virgin olive oil (EVOO) from lower grade oils. Eighty EVOO, forty refined olive oil (ROO), and ten pomace olive oil (POO) samples are analysed for their spectral and compositional features. The latter included analysis of the fatty acids (FAs), the chlorophylls and carotenoids, chromatic coordinates and moisture contents. The 1350–1570 nm wavelength range appeared most suitable for distinction of the oils. One‐class classification models with three different classifiers are subsequently estimated using this range, and their quantitative performance is assessed from probabilistic data. Soft independent modeling of class analogies models appears to predict the identity of the oils with a high success rate. Compared to the other oils, POO comprises a significantly higher and lower proportion of polyunsaturated and monounsaturated FAs, respectively. Higher contents of chlorophylls, carotenoids, and moisture are noted for EVOO. The relevant spectral information for distinction of the oils correlates strongly with the degree of unsaturation of the oils as well as their levels of chlorophylls, carotenoids, and moisture. Practical Applications: The findings of this study demonstrate that the handheld NIRS technique is promising for future rapid screening of olive oil grades. The statistical methods used and the robust validation procedure will help potential users to select the optimal strategy for multivariate data analysis. In addition, the exploration of correlations with compositional characteristics provides insight into the handheld NIRS working mechanism in regard to EVOO authentication.     

Quantification of soybean oil ethanolysis with <Superscript>1</Superscript>H NMR

In this study, proton NMR spectroscopy (200 MHz) was used for quantifying the content of ethyl esters in known mixtures of soybean oil and ethyl soyate (biodiesel). For this purpose, the peak areas of ester ethoxy and glycerol methylenic peaks in the region of 4.05–4.40 ppm were measured and a calibration plot of the respective peak areas vs. the known composition of the oil/ethyl ester mixtures was used. The transesterification values determined in this way were compared with viscosity and total glycerol determinations and a good correlation was obtained. Therefore, for routine analysis, the conversion (in %) of oil to ethyl esters was determined. The methodology presented in this work proved to be quicker and simpler than others reported in the literature, such as GC and/or HPLC.     

Chemical Nature and Diversity of Phosphorus in New Zealand Pasture Soils Using <Superscript>31</Superscript>P Nuclear Magnetic Resonance Spectroscopy and Sequential Fractionation

Information on the phosphorus (P) forms of pasture soils is central to agricultural and environmental sustainability. We used a combination of ³¹P nuclear magnetic resonance (NMR) spectroscopy of NaOH–EDTA extracts and sequential fractionation to investigate P forms, with an emphasis on organic P in relation to environmental and biophysical variables, in 24 diverse pasture soils taken from around New Zealand (organic C 19–102 g kg⁻¹, total P 116–2746 mg kg⁻¹, pH 5.2–7.0). Soils were separated by cluster analysis of soil physicochemical properties and sequentially extracted P pools into those either derived from volcanic-ash materials or not. This separation was also evident for organic P species in NaOH–EDTA extracts, which removed about 75% of total soil organic P. The major organic P compound class was monoesters (24–60% of extracted P), made up of 14 to 91% myo-inositol hexakisphosphate. The next largest organic P class was diesters (0–4% of P extracted), which were enriched in volcanic-ash soils (monoester to diester ratio = 14) compared to non volcanic-ash soils (ratio = 30). Correlation analysis indicated that mean annual temperature had a significant negative and positive effect on monoester and diester concentrations, respectively. This was attributed to better physical protection of monoesters (especially inositol phosphates) from mineralization, and increased diesters from microbial activity and biological turnover. The anomalous behaviour of volcanic-ash soils was attributed to slow mineralization and the sampling of soils at different times of year without full knowledge of the large and highly dynamic microbial biomass P pool which is predominantly diesters.     

Field-assisted diffusion of K<Superscript>+</Superscript>, Rb<Superscript>+</Superscript>, Cs<Superscript>+</Superscript>, Ag<Superscript>+</Superscript>, and Tl<Superscript>+</Superscript> ions in sodium silicate glass

The composition of the diffusion zone formed during the interaction between 20Na₂O · 80SiO₂ glass and molten silver, rubidium, cesium, and thallium nitrates with and without imposition of a constant electric field was determined using X-ray microanalysis. The interdiffusion coefficients and values of electrical mobility were calculated, and the parameters of temperature dependence were determined. The electrical mobility was almost independent of the size and chemical nature of a cation and was determined by the mobility of the cation included into the initial glass.     

Application of FTIR Spectroscopy for the Determination of Virgin Coconut Oil in Binary Mixtures with Olive Oil and Palm Oil

Rapid Fourier transform infrared (FTIR) spectroscopy combined with attenuated total reflectance (ATR) was applied for quantitative analysis of virgin coconut oil (VCO) in binary mixtures with olive oil (OO) and palm oil (PO). The spectral bands correlated with VCO, OO, PO; blends of VCO and OO; VCO and PO were scanned, interpreted, and identified. Two multivariate calibration methods, partial least square (PLS) and principal component regression (PCR), were used to construct the calibration models that correlate between actual and FTIR-predicted values of VCO contents in the mixtures at the FTIR spectral frequencies of 1,120–1,105 and 965–960 cm⁻¹. The calibration models obtained were cross validated using the “leave one out” method. PLS at these frequencies showed the best calibration model, in terms of the highest coefficient of determination (R ²) and the lowest of root mean standard error of calibration (RMSEC) with R ² = 0.9992 and RMSEC = 0.756, respectively, for VCO in mixture with OO. Meanwhile, the R ² and RMSEC values obtained for VCO in mixture with PO were 0.9996 and 0.494, respectively. In general, FTIR spectroscopy serves as a suitable technique for determination of VCO in mixture with the other oils.     

The Use of FTIR Spectroscopy and Chemometrics for Rapid Authentication of Extra Virgin Olive Oil

The authenticity of high value edible fats and oils including extra virgin olive oil (EVOO) is an emerging issue, currently. The potential employment of Fourier transform infrared (FTIR) spectroscopy in combination with chemometrics of multivariate calibration and discriminant analysis has been exploited for rapid authentication of EVOO from canola oil (Ca‐O). The optimization of two calibration models of partial least square (PLS) and principle component regression was performed in order to quantify the level of Ca‐O in EVOO. The chemometrics of discriminant analysis (DA) was used for making the classification between pure EVOO and EVOO adulterated with Ca‐O. The individual oils and their blends were scanned on good contact with ZnSe crystals in horizontal attenuated total reflectance, as a sampling technique. The wavenumbers of 3,028–2,985 and 1,200–987 cm^?1 were used for quantification and classification of EVOO adulterated with Ca‐O. The results showed that PLS with normal FTIR spectra was well suited for quantitative analysis of Ca‐O with a value of the coefficient of determination (R²) > 0.99. The error, expressed as root mean square error of calibration obtained was relatively low, i.e. 0.108 % (v/v). DA can make the classification between pure EVOO and that adulterated with Ca‐O with one misclassified reported.     

Structure of Ca–Sr–Ba Sodium–Borosilicate Glasses according to <Superscript>11</Superscript>B and <Superscript>29</Superscript>Si NMR Spectroscopy

The structure of borosilicate glasses is studied with ¹¹B and ²⁹Si NMR spectroscopy in order to investigate the influence of replacement of Na₂O by oxides of alkali earth metals on their local structure. The quantitative data are analyzed with respect to their correspondence to the criterion of the average charge of the structural unit. The reasons for the deviation between the experimental results and this criterion are considered. It is shown that, in the case of glass devoid of borate structural units with nonbridging oxygen atoms, the corrected contents of these units are consistent with the predictions of thermodynamic modeling of the structure and properties of oxide glasses.     

Investigation of Heat‐Induced Degradation of Virgin Olive Oil Using Front Face Fluorescence Spectroscopy and Chemometric Analysis

A simple setup using a 365‐nm light‐emitting diode coupled to a USB spectrometer through an optical fiber, in a front‐face fluorescence configuration, was used to investigate the heat‐induced deterioration of virgin olive oil at different heating temperatures and times. The samples were heated for 30, 60, 120 and 180 min for every temperature setting of 140, 160 and 180 °C, respectively. Two important results are reported in this article. First, a neo‐formed compound around 665 nm due to the degradation of chlorophyll was observed. This new peak was attributed to pyropheophytins. The second result showed an important rise of the peak around 489 nm, which corresponded to the oxidation products. The correlation obtained between the peroxide value and the 489 nm peak using principal component analysis revealed the mechanism of the oxidation process. It further showed that the peak around 489 nm is a direct consequence of the degradation of hydroperoxide.     

<Superscript>31</Superscript>P NMR quantification and monophasic solvent purification of human and bovine lens phospholipids

Most lipid extraction procedures [Folch, J., Lees, M., and Sloane-Stanley, G.H., (1957) A Simple Method for the Isolation and Purification of Total Lipids from Animal Tissues, J. Biol. Chem. 226, 497–509; Bligh, E.G., and Dyer, W.J. (1959) A Rapid Method of Total Lipid Extraction and Purification, Can. J. Biochem. Physiol. 37, 911–917] employ biphasic solvent mixtures designed to dissolve the lipids in an organic phase and remove impurities in an aqueous phase. However, when applying these protocols to biological matrices such as that of the ocular lens, the formation of an emulsion layer between the organic and aqueous phases causes poor reproducibility in extraction yields and gives only a small amount of the lipid-containing chloroform phase. In this study, we quantified phospholipids at each step of the Folch et al. extraction protocol and compared the yield of human and bovine lens phospholipids obtained by the Folch-based approach and a novel monophasic methanol extraction method designed to circumvent the problems associated with biphasic extraction protocols. A monophasic methanol extraction coupled with ³¹P NMR spectroscopy was found to be the simplest, quickest, and most effective method for quantifying the phospholipid content of the lens.     

Characterisation of Endogenous Peptides Present in Virgin Olive Oil

The low molecular weight peptide composition of virgin olive oil (VOO) is mostly unknown. We aimed to investigate the composition of the endogenous peptides present in VOO, the protein sources from which those peptides originate and their biological activities. A water-soluble extract containing peptides was obtained from VOO. The peptides were separated by size-exclusion using fast protein liquid chromatography, and the low molecular weight fraction (1600–700 kDa) was analysed by nanoscale liquid chromatography Orbitrap coupled with tandem mass spectrometry and de novo sequencing. Nineteen new peptides were identified by Peaks database algorithm, using the available Olea europaea (cv. Farga) genome database. Eight new peptides were also identified by Peaks de novo sequencing. The protein sources of the peptides detected in the database by Peaks DB were identified by BLAST-P search. Seed storage proteins were among the most frequent sources of VOO peptides. BIOPEP software was used to predict the biological activities of peptides and to simulate (in silico) the proteolytic activity of digestive enzymes on the detected peptide sequences. A selection of synthetic peptides was obtained for investigation of their bioactivities. Peptides VCGEAFGKA, NALLCSNS, CPANGFY, CCYSVY and DCHYFL possessed strong ACE-inhibitory and antioxidant activities in vitro. Antioxidant peptides could play a role in VOO quality.     

Visible up-conversion luminescence of CaWO<Subscript>4</Subscript>: Er<Superscript>3+</Superscript>,Yb<Superscript>3+</Superscript> and emission enhancement by tri-doping of Li<Superscript>+</Superscript> ions

Er³⁺,Yb³⁺ co-doped CaWO₄ polycrystalline powders were prepared by a solid-state reaction and their up-conversion (UC) luminescence properties were investigated in detail. Under 980 nm laser excitation, CaWO₄: Er³⁺,Yb³⁺ powder exhibited green UC emission peaks at 530 and 550 nm, which were due to the transitions of Er³⁺ (²H_11/2)→Er³⁺ (⁴I_15/2) and Er³⁺ (⁴S_3/2)→Er³⁺ (⁴I_15/2), respectively. Effects of Li+ tri-doping into CaWO₄: Er³⁺,Yb³⁺ were investigated. The introduction of Li⁺ ions reduced the optimum calcinations temperature about 100 °C by a liquid-phase sintering process and the UC emission intensity was remarkably enhanced by Li⁺ ions, which could be attributed to the lowering of the symmetry of the crystal field around Er³⁺ ions.     

<Superscript>13</Superscript>C-NMR Regioisomeric Analysis of EPA and DHA in Fish Oil Derived Triacylglycerol Concentrates

The regio-isomeric distribution of the omega-3 polyunsaturated fatty acids (PUFA) cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) in the triacylglycerols (TAG) of anchovy/sardine fish oil was determined by ¹³C nuclear magnetic resonance (NMR) analysis under quantitative conditions. From the measurements of sn-1,3 and sn-2 carbonyl peak areas it was established that EPA was mainly located in the sn-1,3 positions, whereas DHA primarily occupied the sn-2 position. Reconstituted TAG prepared by Candida antarctica lipase-B (CALB) glycerolysis of the ethyl ester (EE) or the free fatty acid (FFA) forms of anchovy/sardine fish oil, displayed a different pattern: EPA was equally distributed, while DHA was preferentially attached to the sn-1,3 positions. TAG concentrates of varying EPA and DHA molar fractions were prepared by CALB-catalyzed glycerolysis of the corresponding EE and FFA. ¹³C-NMR analysis of the purified products revealed a lack of CALB regioselectivity for EPA and a slight sn-1,3 regioselectivity for DHA. Since this pattern was observed in all cases of this study, it was concluded that the lipase regioselectivity during TAG synthesis is independent of both the acyl donor type (carboxylic acid or ester) and the fatty acid content of the oil substrate.     

<Superscript>1</Superscript>H and <Superscript>13</Superscript>C NMR characterization and stereochemical assignments of bile acids in aqueous media

The unconjugated bile acids cholic acid, deoxycholic acid, and chenodeoxycholic acid; their glycine and taurine conjugated glycocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, and taurochenodeoxycholic acid; and a taurine conjugated ursodeoxycholic acid, tauroursodeoxycholic acid, were characterized through ¹H and ¹³C NMR in aqueous media under the physiological pH region (7.4±0.1). Assignments of ¹H and ¹³C signals of all the bile acids were made using a combination of several one- and two-dimensional, homonuclear (¹H−¹H) and heteronuclear (¹H−¹³C) correlations as well as spectral editing NMR methods. Stereochemical assignment of the five-membered ring of the bile acids is reported here for the first time. The complete characterization of various bile acids in aqueous media presented here may have implications in the study of the pathophysiology of biliary diseases through human biliary fluids using NMR spectroscopy.