Silver-ion High-performance Liquid Chromatography Research Articles

Impact of column temperature on triacylglycerol regioisomers separation in silver ion liquid chromatography using heptane-based mobile phases

This work presents the investigation of the use of heptane as an alternative and less toxic mobile phase to the most used hexane for triacylglycerols (TAGs) analysis in silver ion high-performance liquid chromatography (Ag+-HPLC). The impact of column temperature (in the 5 °C-35 °C range) on the retention and resolution of five pairs of regioisomers relevant for the confectionery industry was investigated using a heptane-based mobile phase modified with acetonitrile (ACN). The retention behaviour was compared for a standard TAG mixture and an interesterified cocoa butter. The temperature effect previously observed with hexane-based mobile phases was confirmed for this new system, and it was also observed that the ACN concentration had an important impact on the strength of the temperature effect, with a higher ACN concentration leading to a lesser impact of temperature on the TAGs’ elution behaviour. In general, the study allowed to conclude on the equivalence of hexane and heptane for TAGs regioisomers separation in Ag+-HPLC, independently of the used temperature or the ACN concentration. In addition, the applicability of heptane-based mobile phases for the separation of TAGs regioisomers was demonstrated on three other confectionary fat samples, namely palm olein, interesterified palm olein, and interesterified shea olein.

Silver Ion High-Performance Liquid Chromatography-Atmospheric Pressure Chemical Ionization Mass Spectrometry: A Tool for Analyzing Cuticular Hydrocarbons.

Aliphatic hydrocarbons (HCs) are usually analyzed by gas chromatography (GC) or matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. However, analyzing long-chain HCs by GC is difficult because of their low volatility and the risk of decomposition at high temperatures. MALDI cannot distinguish between isomeric HCs. An alternative approach based on silver ion high-performance liquid chromatography (Ag-HPLC) is shown here. The separation of HC standards and cuticular HCs was accomplished using two ChromSpher Lipids columns connected in series. A gradient elution of the analytes was optimized using mobile phases prepared from hexane (or isooctane) and acetonitrile, 2-propanol, or toluene. HCs were detected by atmospheric pressure chemical ionization mass spectrometry (APCI-MS). Good separation of the analytes according to the number of double bonds, cis/trans geometry, and position of double bonds was achieved. The retention times increased with the number of double bonds, and trans isomers eluted ahead of cis isomers. The mobile phase significantly affected the mass spectra of HCs. Depending on the mobile phase composition, deprotonated molecules, molecular ions, protonated molecules, and various solvent-related adducts of HCs were observed. The optimized Ag-HPLC/APCI-MS was applied for characterizing cuticular HCs from a flesh fly, Neobellieria bullata, and cockroach, Periplaneta americana. The method made it possible to detect a significantly higher number of HCs than previously reported for GC or MALDI-MS. Unsaturated HCs were frequently detected as isomers differing by double-bond position(s). Minor HCs with trans double bonds were found beside the prevailing cis isomers. Ag-HPLC/APCI-MS has great potential to become a new tool in chemical ecology for studying cuticular HCs.

Separation and quantification of olefins and diolefins in cracked petroleum fractions using silver-ion high performance liquid chromatography

A method using normal phase high performance liquid chromatography for the analysis of olefins and diolefins in cracked petroleum samples is proposed and validated in this work. A silver-impregnated silica column is used to separate olefin and diolefin species, resulting in accurate quantification of both group-types. Separations are completed using an isocratic hexane mobile phase, with no sample clean up or backflushing required. Results from the analysis of 11 model compounds are shown, as well as quantification data for four representative samples; figures of merit are also described.

Characterization of the Chemical Composition of Chinese Moringa oleifera Seed Oil

AbstractThis work focused on physicochemical property assaying, fatty acid composition, triacylglycerol (TAG) profiles, and unsaponifiable matter composition of the Chinese Moringa oleifera seed oil. The results indicated that there was no significant difference in approximate nutritional components between M. oleifera seeds from China and India, while variations in the mineral element contents are significant. Both the Soxhlet extraction method and the aqueous enzymatic extraction method were adopted to extract oil from Chinese M. oleifera seeds. Oil yield obtained using the Soxhlet extraction method was higher than that obtained using the aqueous enzymatic extraction method. While both the iodine value and unsaponifiable matter content of the aqueous enzymatic extracted oil were a little higher than that of the Soxhlet extracted oil. Both oils possess a very low acid value and peroxide value, suggesting their good quality as edible oil. Fatty acid composition results indicated that this oil was especially high in oleic acid. Characterization of the TAG composition was achieved by a two‐dimensional high‐performance liquid chromatography (HPLC) coupling of nonaqueous reverse‐phase and silver ion HPLC with the atmospheric pressure chemical ionization mass spectrometry method. A total of 22 TAG including 16 regioisomers were determined. Composition results of unsaponifiable matters revealed that this oil possesses a number of phytosterols, in which β‐sitosterol and stigmasterol are most predominant.

Characterization and Determination of Interesterification Markers (Triacylglycerol Regioisomers) in Confectionery Oils by Liquid Chromatography-Mass Spectrometry.

Interesterification is an industrial transformation process aiming to change the physico-chemical properties of vegetable oils by redistributing fatty acid position within the original constituent of the triglycerides. In the confectionery industry, controlling formation degree of positional isomers is important in order to obtain fats with the desired properties. Silver ion HPLC (High Performance Liquid Chromatography) is the analytical technique usually adopted to separate triglycerides (TAGs) having different unsaturation degrees. However, separation of TAG positional isomers is a challenge when the number of double bonds is the same and the only difference is in their position within the triglyceride molecule. The TAG positional isomers involved in the present work have a structural specificity that require a separation method tailored to the needs of confectionery industry. The aim of this work was to obtain a chromatographic resolution that might allow reliable qualitative and quantitative evaluation of TAG positional isomers within reasonably rapid retention times and robust in respect of repeatability and reproducibility. The resulting analytical procedure was applied both to confectionery raw materials and final products.

Application of Silver Ion High-Performance Liquid Chromatography for Quantitative Analysis of Selected n-3 and n-6 PUFA in Oil Supplements.

The aim of this study was to develop a simple method for simultaneous determination of selected cis/cis PUFA-LNA (18:2), ALA (18:3), GLA (18:3), EPA (20:5), and DHA (22:6) by silver ion high-performance liquid chromatography coupled to a diode array detector (Ag-HPLC-DAD). The separation was performed on three Luna SCX Silver Loaded columns connected in series maintained at 10 °C with isocratic elution by 1% acetonitrile in n-hexane. The applied chromatographic system allowed a baseline separation of standard mixture of n-3 and n-6 fatty acid methyl esters containing LNA, DHA, and EPA and partial separation of ALA and GLA positional isomers. The method was validated by means of linearity, precision, stability, and recovery. Limits of detection (LOD) for considered PUFA standard solutions ranged from 0.27 to 0.43 mg L(-1). The developed method was used to evaluate of n-3 and n-6 fatty acids contents in plant and fish softgel oil capsules, results were compared with reference GC-FID based method.

Development of an Offline Bidimensional High-Performance Liquid Chromatography Method for Analysis of Stereospecific Triacylglycerols in Cocoa Butter Equivalents

Acyl migration is a serious problem in enzymatic modification of fats and oils, particularly in production of cocoa butter equivalent (CBE) through enzymatic acidolysis reaction, which leads to the formation of non-symmetrical triacylglycerols (TAGs) from symmetrical TAGs. Non-symmetrical TAGs may affect the physical properties of final products and are therefore often undesired. Consequently, an accurate method is needed to determine positional isomer TAGs during the production of CBE. A bidimentional high-performance liquid chromatography (HPLC) method with combination of non-aqueous reversed-phase HPLC and silver ion HPLC joining with an evaporative light scattering detector was successfully developed for the analysis of stereospecific TAGs. The best separation of positional isomer standards was obtained with a heptane/acetone mobile-phase gradient at 25 °C and 1 mL/min. The developed method was then used in multidimensional determination of the TAG positional isomers in fat and oil blends and successfully identified the TAGs and possible isomers in enzymatically acidolyzed CBE.

Analysis of wax esters by silver-ion high-performance liquid chromatography–tandem mass spectrometry

Wax esters (WEs), esters of long-chain fatty acids and long-chain alcohols, were analysed by Ag-HPLC/APCI-MS/MS. Two ChromSpher Lipids columns connected in series (a total length of 50cm) and hexane–2-propanol–acetonitrile mobile phases were used to achieve good separation of the molecular species. The chromatographic behaviour of WEs was studied under optimised conditions: retention increased with the number of double bonds and with the temperature (15–35°C); retention times were affected by the double-bond position, trans isomers eluted earlier than cis isomers, and the WEs were partially separated depending on the aliphatic-chain length. The WEs provided simple APCI spectra with [M+H]+ ions, the MS/MS spectra showed fragments, which allowed their identification. The method was applied for an analysis of the WE mixtures from jojoba oil and human hair and the results were compared with analogous data from an optimised RP-HPLC system.

Oxidative modifications of conjugated and unconjugated linoleic acid during heating

The oxidative stability of conjugated linoleic (CLA) and linoleic (LA) acids in different chemical forms (free acids, methyl esters and homogeneous triacylglycerols) was compared. All model systems were heated at 180°C for different times (15, 30, 45 and 60min). The primary oxidation products were evaluated by spectrophometric analysis, while the volatile compounds were determined by solid phase micro-extraction (SPME), coupled with gas chromatography–mass spectrometry (HRGC–MS). The isomer profile modifications were investigated by silver-ion high performance liquid chromatography (Ag+-HPLC) equipped with an UV detector. Generally, peroxide values decreased during the heating time. Among the volatiles, saturated aldehydes were the most represented compounds. Isomerization of cis,trans and trans,cis CLA to trans,trans isomers was observed mainly for the methyl form of CLA. The three different chemical forms of LA never showed isomerization phenomena.

Predicting conjugated linoleic acid (CLA) composition in Brazilian dairy products by multiple regression analysis based models

The analysis of conjugated linoleic acids (CLA) in dairy products is of concern for human health, but requires a combination of different analytical methods that are laborious and time consuming. The aims of the present study were to develop mathematical models based on data of the CLA and fatty acid composition of Brazilian dairy products and to apply the developed models for the calculation of CLA based on a basic analysis of fatty acid composition. The fatty acid composition of five types of dairy products was determined by gas chromatography (GC); analysis of CLA was achieved by silver-ion high-performance liquid chromatography and GC-mass spectrometry. Stepwise multiple regression analysis was performed to model the composition of CLA. All the six significant models of CLA composition (total CLA; total cis,trans, trans,cis CLA; cis-9,trans-11 CLA; trans-7,cis-9 CLA; trans-11,cis-13 CLA; and total trans,trans CLA) were physiologically consistent, had appropriate goodness of fit, and were fairly robust. According to the results of Pearson correlation analysis between estimates and experimental data (r = 0.78–0.91 and 0.0000 < P < 0.0028), the models could be used to rank dairy products according to the estimates of total CLA, trans-7,cis-9 CLA; trans-11,cis-13 CLA; total cis,trans, trans,cis CLA; and total trans,trans CLA, but not for cis-9,trans-11. The adjusted regression models allowed the estimation of CLA composition in a variety of Brazilian dairy products.

Characterization of the disappearance and formation of biohydrogenation intermediates during incubations of linoleic acid with rumen fluid in vitro1

Dietary unsaturated fatty acids are extensively hydrogenated in the rumen, resulting in the formation of numerous intermediates that may exert physiological effects and alter the fat composition of ruminant-derived foods. A batch culture method was used to characterize the hydrogenation of linoleic acid (LeA) by strained rumen fluid in vitro. Incubations (n=5) were performed in 100-mL flasks maintained at 39°C containing 400mg of grass hay, 50mL of buffered rumen fluid, and incremental amounts of LeA (0, 1.0, 2.5, 5.0, or 10.0mg) for 0, 1.5, 3.0, 4.5, 6.0, and 9.0h. The fatty acid composition of flask contents was determined using complimentary silver-ion thin-layer chromatography, gas chromatography mass-spectrometry, and silver-ion high-performance liquid chromatography. Linoleic acid was extensively (98.1, 97.6, 98.0, and 89.8% for additions of 1.0, 2.5, 5.0, and 10.0mg of LeA, respectively) hydrogenated over time. Complete reduction of LeA to 18:0 was inhibited in direct relation to the amount of added substrate, the extent of which was greatest for the highest amount of LeA addition. Recoveries of 1.0, 2.5, 5.0, and 10.0mg of added LeA as 18:0 averaged 73.6, 65.0, 57.3, and 10.7%, respectively. Incubation of incremental amounts of LeA resulted in a time-dependent accumulation of geometric isomers of 9,11 and 10,12 conjugated linoleic acid, several nonconjugated 18:2 isomers, and a wide range of cis 18:1 and trans 18:1 intermediates. Several unusual intermediates including cis-6,cis-12 18:2; cis-7,cis-12 18:2; and cis-8,cis-12 18:2, were found to accumulate in direct relation to the amount of added LeA, providing the first indications that hydrogenation of LeA by ruminal bacteria may also involve mechanisms other than hydrogen abstraction or isomerization of the cis-12 double bond. Fitting of single-pool, first-order kinetic models to experimental data indicated that the rate of LeA disappearance decreased with increases in substrate availability. Reduction of 18:1 and 18:2 intermediates occurred at much lower rates compared with conjugated linoleic acid and nonconjugated 18:2 isomer formation. In conclusion, the extent of LeA biohydrogenation in vitro was shown to be time- and dose-dependent with evidence that LeA is hydrogenated by ruminal bacteria via several distinct metabolic pathways. The accumulation of several unusual 18:2 isomers indicates that biohydrogenation of LeA also proceeds via mechanisms other than isomerization of the cis-12 double bond.

Short Communication: Rapid separation of cis9, trans11- and trans7,cis9-18:2 (CLA) isomers from ruminant tissue using a 30 m SLB-IL111 ionic column

Turner, T., Rolland, D. C., Aldai, N. and Dugan, M. E. R. 2011. Short Communication: Rapid separation of cis 9, trans 11- and trans 7,c is 9-18:2 (CLA) isomers from ruminant tissue using a 30 m SLB-IL111 ionic column. Can. J. Anim. Sci. 91: 711–713. Rumenic acid (cis9,trans11-18:2) is the main natural isomer of conjugated linoleic acid (CLA). Rumenic acid has many purported health benefits, but effects of most other CLA isomers are unknown. Typically trans7,cis9-18:2 is the second most abundant CLA isomer, but it co-elutes with rumenic acid on conventional polar gas chromatography (GC) columns, requiring complimentary analysis with silver-ion high performance liquid chromatography (Ag+-HPLC). Herein we report a rapid method for analyzing rumenic acid and trans7,cis9-18:2 using a 30 m ionic-liquid GC column. Optimal resolution of the two CLA isomers was at 145°C and analysis of backfat from barley-fed cattle compared well with GC/Ag+-HPLC (y=0.978x – 0.031, r=0.985, P&lt;0.001).

Characterization of fatty acid and triacylglycerol composition in animal fats using silver-ion and non-aqueous reversed-phase high-performance liquid chromatography/mass spectrometry and gas chromatography/flame ionization detection

Fatty acid (FA) and triacylglycerol (TG) composition of natural oils and fats intake in the diet has a strong influence on the human health and chronic diseases. In this work, non-aqueous reversed-phase (NARP) and silver-ion high-performance liquid chromatography with atmospheric pressure chemical ionization mass spectrometry detection and gas chromatography with flame-ionization detection (GC/FID) and mass spectrometry detection are used for the characterization of FA and TG composition in complex samples of animal fats from fallow deer, red deer, sheep, moufflon, wild boar, cock, duck and rabbit. The FA composition of samples is determined based on the GC/FID analysis of FA methyl esters. In total, 81 FAs of different acyl chain length, double bond (DB) number, branched/linear, cis-/ trans- and DB positional isomers are identified. TGs in animal fats contain mainly monounsaturated and saturated FAs. High amounts of branched and trans-FAs are observed in the samples of ruminants. In NARP mode, individual TG species are separated including the separation of trans- and branched TGs. Silver-ion mode provides the separation of TG regioisomers, which enables the determination of their ratios. Great differences in the preference of unsaturated and saturated FAs in the sn-2 position on the glycerol skeleton are observed among individual animal fats. Unsaturated FAs are preferentially occupied in the sn-2 position in all animal samples except for wild boar with the strong preference of saturated FAs in the sn-2 position.

Regioisomeric analysis of triacylglycerols using silver-ion liquid chromatography–atmospheric pressure chemical ionization mass spectrometry: Comparison of five different mass analyzers

Silver-ion high-performance liquid chromatography (HPLC) coupled to atmospheric pressure chemical ionization mass spectrometry (APCI-MS) is used for the regioisomeric analysis of triacylglycerols (TGs). Standard mixtures of TG regioisomers are prepared by the randomization reaction from 8 mono-acid TG standards (tripalmitin, tristearin, triarachidin, triolein, trielaidin, trilinolein, trilinolenin and tri- gamma-linolenin). In total, 32 different regioisomeric doublets and 11 triplets are synthesized, separated by silver-ion HPLC using three serial coupled chromatographic columns giving a total length of 75 cm. The retention of TGs increases strongly with the double bond (DB) number and slightly for regioisomers having more DBs in sn-1/3 positions. DB positional isomers (linolenic vs. γ-linolenic acids) are also separated and their reverse retention order in two different mobile phases is demonstrated. APCI mass spectra of all separated regioisomers are measured on five different mass spectrometers: single quadrupole LC/MSD (Agilent Technologies), triple quadrupole API 3000 (AB SCIEX), ion trap Esquire 3000 (Bruker Daltonics), quadrupole time-of-flight micrOTOF-Q (Bruker Daltonics) and LTQ Orbitrap XL (Thermo Fisher Scientific). The effect of different types of mass analyzer on the ratio of [M+H−R i COOH] + fragment ions in APCI mass spectra is lower compared to the effect of the number of DBs, their position on the acyl chain and the regiospecific distribution of acyl chains on the glycerol skeleton. Presented data on [M+H−R i COOH] + ratios measured on five different mass analyzers can be used for the direct regioisomeric determination in natural and biological samples.

Fatty acid and CLA composition of Brazilian dairy products, and contribution to daily intake of CLA

In the present study, the conjugated linoleic acid (CLA) contents of dairy products most consumed by the population living in the southeast region of Brazil (whole milk, “Prato” cheese and butter) were determined. In addition, the contribution of these dairy foods to the daily intake of CLA isomers was estimated. Fatty acid composition was determined by gas chromatography. Quantitative analysis of CLA isomers was done by silver-ion high-performance liquid chromatography (HPLC) and their identification was achieved by gas chromatography–mass spectrometry (GC–MS) of dimethyloxazoline (DMOX) derivatives. Eleven CLA isomers were identified and the predominant were cis-9, trans-11 and trans-7, cis-9. The total CLA contents in whole milk, “Prato” cheese and butter ranged from 4.0 to 8.4 mg/g fat and were similar to those reported for dairy products from other countries. Whole milk samples had significantly higher contents of total CLA, when compared to “Prato” cheese and butter, and the total CLA contents were significantly different between the commercial brands of each dairy product. The estimated average daily intake of total CLA in southeastern Brazil was 36 mg/day, which was lower than those of European and North American countries, and milk was the main food source of CLA in the diet.

Separation of isomeric octadecenoic fatty acids in partially hydrogenated vegetable oils as p-methoxyphenacyl esters using a single-column silver ion high-performance liquid chromatography (Ag-HPLC)

A protocol for separation of positionally isomeric trans- and cis-octadecenoic fatty acids as their p-methoxyphenacyl esters, using a single column silver ion high-performance liquid chromatography (Ag-HPLC) with UV detection, is described. The procedure involves hydrolysis of oil to free fatty acids, derivatization to p-methoxyphenacyl esters, purification and separation by Ag-HPLC. The procedure is robust and relatively rapid. In total, it takes about 16 h or overnight and a further 5 h to carry out. Its feature and value consist in the application of p-methoxyphenacyl esters in Ag-HPLC, thus ensuring excellent simultaneous separation of both configurational and positional fatty acid isomers on a single column by a single run. Quantification is not affected by the fatty acid structure, and the method is highly sensitive. As fatty acid isomerization is a strong evidence for adulteration of lipid-containing food products with chemically modified oils and fats, the procedure is especially appropriate for detection and authenticity analysis in many areas of food manufacturing and quality control.

2-Propanol in the mobile phase reduces the time of analysis of CLA isomers by silver ion-HPLC

Individual isomers of octadecadienoic acid (C18:2) with conjugated double bonds (conjugated linoleic acids; CLA) exert different biological activities. Their distribution in food and tissues differs. Therefore, the separation of the various positional and geometric isomers is important. The time of analysis using silver ion-high performance liquid chromatography can extend up to 90 min. The aim of this study was to reduce this time. The time of analysis reduced from ca. 90 min onto 45 to 35 min, respectively, by the addition of 0.05% or 0.1% (v/v) 2-propanol to the mobile phase [acetonitrile (0.1%; v/v) and diethyl ether (0.5%; v/v) in n-hexane]. There was no effect on resolution of the 17 individual CLA isomers of the CLA mixture. Regarding the lowest coefficient of variation and an adequate baseline separation the use of 0.05% 2-propanol in the mobile phase is recommended, without any disadvantages and adverse effects on the service life of columns. In conclusion, adding 0.05% or 0.1% 2-propanol to the mobile phase shortens the time of analysis of CLA isomers, saves solvents and reduces costs.

Oxidative Stability of Conjugated Linolenic Acids

Interest in conjugated linolenic acid (CLnA) and conjugated linoleic acid (CLA) as functional lipids is growing. The present study was (i) to study the oxidative stability of individual CLnA isomers and (ii) to compare the oxidative stabilities of CLnA and CLA with their corresponding nonconjugated counterparts, α-linolenic acid (LN) and linoleic acid (LA). The oxidation was carried out in air at 50 °C and monitored by the gas-liquid chromatography (GC) and the oxygen consumption test. First, it was found that CLnA was most unstable followed by CLA, LN, and LA in decreasing order. Second, analyses of silver ion high-performance liquid chromatography (Ag(+)-HPLC) demonstrated that t,t,t-CLnA isomers had greater stability than c,t,t-CLnA and c,t,c-CLnA isomers. Finally, both green tea catechins (GTCs) and butylated hydroxytoluene (BHT) were capable of preventing the CLnA oxidation, with the former being more effective than the latter.

Regioisomeric Characterization of Triacylglycerols Using Silver-Ion HPLC/MS and Randomization Synthesis of Standards

Silver-ion normal-phase high-performance liquid chromatography (HPLC) provides a superior separation selectivity for lipids differing in the number and position of double bonds in fatty acid chains including the resolution of triacylglycerol (TG) regioisomers under optimized conditions. Our silver-ion HPLC method is based on the coupling of three columns in the total length of 75 cm and a new mobile phase gradient consisting of hexane-acetonitrile-2-propanol which provides better resolution and also reproducibility in comparison to previously used mobile phases. In our work, the chemical interesterification (randomization) of single-acid TG standards is used for the generation of regioisomeric series of TGs, because it provides a random distribution of fatty acids in TGs at well-defined concentration ratios. The baseline separation of regioisomeric TG pairs containing up to three double bonds and the partial separation of TG regioisomers with four to seven double bonds are reported for the first time. Our silver-ion high-performance liquid chromatography/mass spectrometry (HPLC/MS) method is applied for the regioisomeric characterization of complex samples of plant oils and animal fat, where the results clearly demonstrate different preference of sn-2 occupation in plants (mainly unsaturated fatty acids) versus animal fat (mainly saturated fatty acids).

Effect of Latitude and Weather Conditions on the Regioisomer Compositions of α- and γ-Linolenoyldilinoleoylglycerol in Currant Seed Oils

The regioisomer compositions of triacylglycerols (TAG) 18:3(n-3)/18:2(n-6)/18:2(n-6) (Ala/L/L) and 18:3(n-6)/18:2(n-6)/18:2(n-6) (Gla/L/L) in seed oils of black and green currant ( Ribes nigrum L.) and red and white currant ( Ribes rubrum L.) varieties were determined by silver-ion high-performance liquid chromatography and mass spectrometry. The aim was to investigate whether latitude and weather conditions affect the regioisomer compositions of Ala/L/L and Gla/L/L and whether the regioisomerism differs between species and varieties. In R. rubrum the proportion of the symmetric regioisomer LAlaL among Ala/L/L was higher (14.1%) than in R. nigrum (12.1%) (p < 0.001). Generally in currants, the proportion of LAlaL was lower (p < 0.001) in northern Finland (12.1%) than in southern Finland (13.5%), where temperature and radiation sums were higher. In R. rubrum varieties grown in the south, the proportion of LGlaL among Gla/L/L was significantly higher in the years 2005 and 2007 (30.7-32.0%) than in 2006 (24.2-25.4%), when temperature and radiation sums were higher and the amount of precipitation was lower.