Recommendations for Oil Extraction and Refining Process to Prevent the Formation of Monochloropropane-diol Esters in Sunflower Oil.

The aim of this study was to identify the chlorine source during sunflower oil production and propose mitigation strategies in order to prevent monochloropropane-diol ester (MCPDE) formation. Whole sunflower seeds, the separated kernel, hulls, and pressed cake were studied to pinpoint the location of chlorine donors originating from the crop. Acid-water-based degumming, bleaching, cooling, and heat treatment were performed to mimic the current refining process practices. Various oil extraction and refining scenarios were tested. MCPDE and total monochloropropane-diol (MCPD) content of the heat-treated samples were determined by liquid chromatography-HRMS and by an AOCS Official method. The results show that the oil produced from crop hulls and the bleaching clay used are the strongest chlorine sources boosting the MCPDE formation. Using a mixture of pressed and solvent extracted cake oil as model, total 3-MCPD decreased by a factor of 2 when applying static cooling in combination with a washed bleaching clay.

Mitigating the formation of monochloropropanediol diesters in vegetable oils by removing their residual sediments.

This study investigates whether the formation of monochloropropane diol fatty acid esters (MCPDE) can be mitigated by removing the residual sediments from vegetable oils. Settling and centrifugation were conducted in crude sunflower and palm oil and the purified oils and their sediment-rich fractions were heated and analyzed for their MCPDE content. Increased MCPDE levels by factors of x2 to x6 were found in the sediment-rich fractions of settled sunflower oils compared to the sediment-free oil. The sediment-containing fraction could be however purified by ultracentrifugation resulting in the mitigation of MCPDE levels by a factor of 10. The effect of residual sediment on the MCPDE formation was also confirmed in the case of palm oil showing x2 to x10 more MCPDE formation in the sediment containing fractions compared to the purified oil. These results confirm that the mechanical removal of the trace sediments from crude vegetable oils results in reduced MCPDE levels.

Removal of organochlorines from vegetable oils and its benefits in preventing formation of monochloropropanediol diesters.

This paper reports the first results on depleting certain organochlorines from vegetable oils without the use of any solvent in order to mitigate monochloropropanediol diesters (MCDPE). The concept is based on separating the organochlorines from the bulk oil by using trapping agents (e.g. monoacylglycerols) that can be easily separated from the oil. The process starts by mixing and homogenizing crude vegetable oils with the trapping agent and subsequently separating the trapping agent from the oil bulk via crystallization. The proof-of-concept of the approach is demonstrated on a spiked sunflower model system, solvent extracted crude sunflower oil, industrially produced crude soybean and corn oils. The depletion of organochlorines in the crude oils and its beneficial effect on the MCPDE content in the heat treated samples is measured by LC-MS. The depletion efficacy of the monitored organochlorines was estimated to be in the 60-95 % range. Both the melting point and polarity of the trapping agents affected the depletion efficacy of the organochlorines. Trapping agents with higher melting point and polarity, such as monostearin were more effective in comparison to high melting point but less polar agents such as palm stearin or agents rich in polar but low melting point monolinolein/monoolein. The effect of organochlorine depletion on the subsequent MPCDE levels in heat treated oil was in the range of 60-90 % reduction depending on the type of the studied oil.

Benefits of ion mobility for analysing monochloropropane-diol esters.

This paper reports the first application and benefits of ion mobility in combination with liquid chromatography and a transportable time-of-flight mass spectrometer to the analysis of monochloropropane-diol esters (MCPDE) in vegetable oils. The additional selectivity obtained with the ion mobility allowed the quantitative analysis of MCPDEs as such in their intact form (direct analysis) without any chemical derivatisation and, furthermore, without any enrichment or purification step. This gain in selectivity manifests primarily in the resolution of interferences originating, for example, from the diacylglycerol components of palm oil. In silico calculations confirm that resolution of such interferences would require mass resolutions higher than 200,000 at m/z 600, e.g., in the case of signals of the 41K isotope of the PO DAG and the signals of the sodiated PO MCPD. While such resolution can be obtained on certain state-of-the-art costly and laboratory-exhaustive research instruments, this study demonstrates that even transportable time-of-flight MS can achieve the required selectivity when combined with ion mobility. Further advantage of the described approach is that the applied sample preparation is only dilution with minimum consumable requirements and can be performed quickly even outside laboratories directly in the field. The described results suggest that the application of ion mobility in addition to LC-MS is likely to push the boundaries of contaminant analysis especially for high-throughput screening investigations.

Vitamin E and vitamin E acetate absorption from self-assembly systems under pancreas insufficiency conditions.

We determined the bioavailability of vitamin E from self-assembly structures in patients with diagnosed chronic pancreas insufficiency. Vitamin E solubilized in dispersed inverted bicontinuous cubic phase and in micellar formulation was delivered directly to the small intestine by tube-feeding. A cross-over study with randomization of 6 subjects and 2 treatments including a combined dose of 18 mg (27 IU) of vitamin E (RRR-[5,7-methyl-((2)H6)]-α-tocopherol) and 27 mg (27 IU) vitamin E acetate (RRR-[5-methyl-(2)H3]-α-tocopheryl acetate) was applied over a time period of 1 h. Plasma samples were collected for 56 h and analyzed by liquid chromatography-mass spectrometry. Appearance of labeled tocopherols originating from the treatment started at 25 h and reached Cmax (0.6-4.6 µM depending on subject) in the 7-9 h window. From the Tmax onwards, both forms of tocopherols diminished slowly to 30-50% of their maxima within 56 h. Strong inter-individual variation was observed in the plasma appearance curves (relative standard deviation varied between 38-45%). No significant discrimination was found between the absorption of free or acetylated forms of deuterated α-tocopherol confirming that application of acetylated α-tocopherol provides the same bioavailability as free α-tocopherol. This observation is valid in both dispersed inverted bicontinuous cubic phase and micellar formulations. Furthermore, since the area-under-the-curve values from cubic phase and from micellar formulations are similar, the cubic phase formulation could represent an alternative delivery system for lipophilic micronutrients in conditions or studies where polysorbate-based micelles cannot be generated.

Enrichment and quantification of monoacylglycerols and free fatty acids by solid phase extraction and liquid chromatography-mass spectrometry.

Quantification of monoacylglycerols (MAG) and free fatty acids (FA) is of interest in biological systems, in food, cosmetic and pharmaceutical products. This manuscript describes and validates a reversed phase liquid chromatography-tandem mass spectrometry based approach for simultaneous quantification of these analytes in fats and oils. Purification and concentration of MAG/FA were performed using cation exchange solid phase extraction, which allowed elimination of the abundant triacylglycerols. Following cleanup and concentration, the analytes were separated and detected with the aid of volatile ammonium-formate buffer. MAG were detected in positive ion mode, while FA were detected in negative ion mode. The method was validated by the method of standard additions and using stable isotope labeled internal standards. The results confirm the feasibility of quantifying these two classes of analytes simultaneously without any chemical derivatization. The obtained main quantitative features include: (1) lower limits of quantification 1-30ppm for MAG analytes, (2) lower limits of quantification 90-300ppm for FA analytes, (3) averaged inter-batch precision 6%, and (4) averaged bias -0.2% for MAG and 0.5% for FA. Various animal fat and vegetable oil samples were characterized for their MAG/FA profile indicating the usefulness of the method to address quality and authenticity of fats and oils.

Identification of cholesteryl ester of ferulic acid in human plasma by mass spectrometry.

Epidemiological data suggests that regular consumption polyphenol rich foods and beverages is associated with a reduced risk of certain pathological conditions. While the in vivo "per se" antioxidant benefit of polyphenols still has not been clearly demonstrated, it has been suggested that phenolic acids can be incorporated into low-density lipoproteins (LDL). In the present study, we hypothesized that esterification of phenolic acids - such as ferulic acid - with lipophilic substances such as cholesterol can occur in vivo. To prove this hypothesis, we have synthesized pure cholesteryl-ferulate standard and used gas- and liquid chromatography coupled with mass spectrometry to confirm the presence of endogenous form in human plasma. The detection and identification of cholesteryl ferulate was based on: (1) matching gas- and liquid chromatographic retention time with the reference standard; (2) accurate mass of the molecular ion; (3) matching electron ionization mass spectrum and (4) matching electrospray product ion spectrum. The identified cholesteryl ferulate demonstrated an in vitro antioxidant capacity in various assays. The present study confirmed that phenolic acid can be found in human plasma as lipophilic conjugates which exert antioxidant capacity. These molecules can potentially be involved in the protection of lipoproteins against oxidative damages.

Mapping the regioisomeric distribution of fatty acids in triacylglycerols by hybrid mass spectrometry.

This study describes the use of hybrid mass spectrometry for the mapping, identification, and semi-quantitation of triacylglycerol regioisomers in fats and oils. The identification was performed based on the accurate mass and fragmentation pattern obtained by data-dependent fragmentation. Quantitation was based on the high-resolution ion chromatograms, and relative proportion of sn-1(3)/sn-2 regioisomers was calculated based on generalized fragmentation models and the relative intensities observed in the product ion spectra. The key performance features of the developed method are inter-batch mass accuracy < 1 ppm (n = 10); lower limit of detection (triggering threshold) 0.1 µg/ml (equivalent to 0.2 weight % in oil); lower limit of quantitation 0.2 µg/ml (equivalent to 0.4 weight % in oil); peak area precision 6.5% at 2 µg/ml concentration and 15% at 0.2 µM concentration; inter-batch precision of fragment intensities < 1% (n = 10) independent of the investigated concentration; and averaged accuracy using the generic calibration 3.8% in the 1-10 µg/ml range and varies between 1-23% depending on analytes. Inter-esterified fat, beef tallow, pork lard, and butter fat samples were used to show how well regioisomeric distribution of palmitic acid can be captured by this method.

Characteristics of polyglycerol ester and its different fractions.

Polyglycerol esters obtained from edible oils are commonly used surfactants in the food industry. Despite their widespread application, the composition and properties of these surfactants are still not well characterized. This study reveals the presence of so far unknown tetra-, penta-antennary constituents in polyglycerol esters, which exhibit very strong sodium affinity. The implications of these new insights on surfactant activity were investigated. Liquid-liquid extraction was used to fractionate a polyglycerol ester ingredient in order to link physicochemical behavior to the polarity of the fractions. The most polar fraction showed faster adsorption kinetics and higher elastic moduli than the full mixture, whereas the least polar fraction showed slower adsorption kinetics and lower elastic moduli as compared to the complex mixture. The addition of Na(+) was shown to accelerate the agglomeration of surfactant self-assemblies in bulk solutions and also to increase the elastic modulus at the air-water interface. These observations suggest that the composition of natural polyglycerol esters is more diverse than so far assumed and the overall behavior of these mixtures is determined not only by the amphyphilic interactions between mono- and penta-antennary forms, but also by the endogenous salt content of the ingredient.

Specific dietary preferences are linked to differing gut microbial metabolic activity in response to dark chocolate intake.

Systems biology approaches are providing novel insights into the role of nutrition for the management of health and disease. In the present study, we investigated if dietary preference for dark chocolate in healthy subjects may lead to different metabolic response to daily chocolate consumption. Using NMR- and MS-based metabolic profiling of blood plasma and urine, we monitored the metabolic response of 10 participants stratified as chocolate desiring and eating regularly dark chocolate (CD) and 10 participants stratified as chocolate indifferent and eating rarely dark chocolate (CI) to a daily consumption of 50 g of dark chocolate as part of a standardized diet over a one week period. We demonstrated that preference for chocolate leads to different metabolic response to chocolate consumption. Daily intake of dark chocolate significantly increased HDL cholesterol by 6% and decreased polyunsaturated acyl ether phospholipids. Dark chocolate intake could also induce an improvement in the metabolism of long chain fatty acid, as noted by a compositional change in plasma fatty acyl carnitines. Moreover, a relationship between regular long-term dietary exposure to a small amount of dark chocolate, gut microbiota, and phenolics was highlighted, providing novel insights into biological processes associated with cocoa bioactives.

Absorption of dimethoxycinnamic acid derivatives in vitro and pharmacokinetic profile in human plasma following coffee consumption.

SCOPE: This study reports the 24 h human plasma pharmacokinetics of 3,4-dimethoxycinnamic acid (dimethoxycinnamic acid) after consumption of coffee, and the membrane transport characteristics of certain dimethoxycinnamic acid derivatives, as present in coffee. METHODS AND RESULTS: Eight healthy human volunteers consumed a low-polyphenol diet for 24 h before drinking 400 mL of commercially available coffee. Plasma samples were collected over 24 h and analyzed by HPLC-MS(2) . Investigation of the mechanism of absorption and metabolism was performed using an intestinal Caco-2 cell model. For the first time, we show that dimethoxycinnamic acid appears in plasma as the free aglycone. The time to reach the C(max) value of approximately 0.5 µM was rapid, T(max) = 30 min, and showed an additional peak at 2-4 h for several subjects. In contrast, smaller amounts of dimethoxy-dihydrocinnamic acid (C(max) ∼ 0.1 µM) peaked between 8 and 12 h after coffee intake. In the cell model, dimethoxycinnamic acid was preferentially transported in the free form by passive diffusion, and a small amount of dimethoxycinnamoylquinic acid hydrolysis was observed. CONCLUSION: These findings show that dimethoxycinnamic acid, previously identified in plasma after coffee consumption, was rapidly absorbed in the free form most likely by passive diffusion in the upper gastrointestinal tract.

Identification of O-methyl-(-)-epicatechin-O-sulphate metabolites by mass-spectrometry after O-methylation with trimethylsilyldiazomethane.

(-)-Epicatechin, an abundant dietary polyphenol found mainly in cocoa and tea, is known to extensively undergo metabolism after ingestion giving rise to a complex series of conjugated metabolites including numerous isomers. In the present study, the combination of fractionation, chemical derivatization and various mass spectrometric approaches is described to determine the exact position of sulphate group in methylated epicatechin metabolites. Four O-methyl-(-)-epicatechin-O-sulphate metabolites isolated from human urine samples were derivatized under mild condition using trimethylsilyldiazomethane (TMSD) in the presence of methanol. The resulting methylated reaction products were then analyzed by high resolution and multistage mass spectrometry for the subsequent identification of the sulphate positional isomers. Results show that O-methylation affects the charge delocalization in negatively charged ions and hereby the fragmentation pattern of the sulphate isomers allowing the identification of diagnostic ions. In addition, this study demonstrates that methoxy derivatives of polyphenol metabolites can be prepared using TMSD. Subsequently, the localization of the sulphate group in the polyphenol metabolites can be achieved by analyzing the methoxy derivatives by multistage mass spectrometry. Using an enzymatic reaction for identification of the O-methyl position, and a chemical O-methylation with TMSD follow by high resolution and multistage tandem MS for the identification of the sulphate group position, we were able to identify the previously unknown O-methyl-(-)-epicatechin-O-sulphate. Accordingly, we identified 3'-O-methyl-(-)-epicatechin-5-O-sulphate and 3'-O-methyl-(-)-epicatechin-7-O-sulphate as the main O-methyl-(-)-epicatechin-sulfates(-)-epicatechin metabolites in humans.

Comparative study of serine-plasmalogens in human retina and optic nerve: identification of atypical species with odd carbon chains.

The objective of this work was to detect and identify phosphatidylserine plasmalogen species in human ocular neurons represented by the retina and the optic nerve. Plasmalogens (vinyl-ether bearing phospholipids) are commonly found in the forms of phosphatidylcholine and phosphatidylethanolamine in numerous mammalian cell types, including the retina. Although their biological functions are unclear, the alteration of cellular plasmalogen content has been associated with several human disorders such as rhizomelic chondrodysplasia punctata Type 2 and primary open-angle glaucoma. By using liquid chromatography coupled to high-resolution and tandem mass spectrometry, we have identified for the first time several species of phosphatidylserine plasmalogens, including atypical forms having moieties with odd numbers of carbons and unsaturation in sn-2 position. Structural elucidation of the potential phosphatidylserine ether linked species was pursued by performing MS(3) experiments, and three fragments are proposed as marker ions to deduce which fatty acid is linked as ether or ester on the glycerol backbone. Interpretation of the fragmentation patterns based on this scheme enabled the assignment of structures to the m/z values, thereby identifying the phosphatidylserine plasmalogens.

Factors impacting the formation of monochloropropanediol (MCPD) fatty acid diesters during palm (Elaeis guineensis) oil production.

Recently, organic and inorganic chlorinated compounds were detected in crude and commercially refined palm oils. Further, the predominant formation mechanism of monochloropropanediol (MCPD) diesters at high temperatures (>170-180°C) was revealed. The present study involved the development and comparison of solutions to mitigate MCPD diester levels in oils from various stages of palm oil production. Partially refined palm oil samples and oil extracted from fresh palm fruits were submitted to bench-top deodorisation experiments. Application of glycerol and ethanol as refining aids during the deodorisation of refined-bleached palm oil proved to be moderately effective; about 25%-35% reduction of MCPD diester levels was achieved. Washing crude palm oil with ethanol-water (1:1) prior to deodorisation was also an effective strategy yielding an ∼30% reduction of MCPD diester contents. Washing palm fruit pulp before oil extraction, however, was most impactful, resulting in a 95% reduction of MCPD diesters when compared to the deodorised control oil. This suggests that intervention upstream in the process chain is most efficient in reducing levels of these contaminants in refined oils. Following the study, a root-cause analysis was performed in order to map the parameters potentially responsible for the occurrence of MCPD diesters in refined palm oil and related fractions.

Formation mechanisms of monochloropropanediol (MCPD) fatty acid diesters in refined palm (Elaeis guineensis) oil and related fractions.

Monochloropropanediol (MCPD) fatty acid esters are process contaminants generated during the deodorisation of edible oils. In particular, MCPD diesters are found in higher abundance in refined palm oil than other edible oils. In the present study, a series of model reactions mimicking palm oil deodorisation has been conducted with pure acylglycerols in the presence or absence of either organic or inorganic chlorine-containing compounds. Results showed that the bulk of MCPD diesters are formed above 200°C through the reaction of organochlorines with triacylglycerols (TAG). Additional experiments confirmed that this reaction can be initiated during palm oil deodorisation by hydrogen chloride (HCl) gas evolved through the thermal degradation of organochlorines present in the oil. Therein, the majority of the ultimately produced MCPD diesters are the result of HCl reacting with TAG, via protonation, followed by the elimination of a fatty acid residue. Two possible MCPD diester formation mechanisms are highlighted, both of which involve acyloxonium ion reactive intermediates. Investigations with pure TAG regio-isomers showed that MCPD ester formation is regioselective and the sn-1(3) position of the glycerol backbone is favoured.

Non-covalent binding of proteins to polyphenols correlates with their amino acid sequence.

The present paper describes the assessment of non-covalent binding (NCB) between milk proteins and polyphenols and its correlation with the physicochemical parameters of proteins. A method based on ultrafiltration and liquid chromatography-tandem mass spectrometry was used to analyse free and non-covalently bound polyphenols (ligands) in mixtures with major milk proteins. Binding strength values of individual polyphenols were normalised to those obtained with quercitrin (quercetin-3-O-rhamnoside), used as a reference compound. NCB data acquired by experiments at pH 6.6 without any preliminary protein denaturation were correlated with the physicochemical parameters of ligands and proteins. Unsupervised multivariate analysis revealed that NCB of proteins clustered according to their family (caseins separated from albumins). Based on this model, a predictive relationship was observed between protein-polyphenol binding strength and primary/secondary structure parameters of the proteins e.g. number of charges, proline residues and extended strand. These results confirm that, under the investigated experimental conditions, the NCB between polyphenols and protein mixtures can be predicted and optimised based on the molecular structures.

Glycidyl esters in refined palm (Elaeis guineensis) oil and related fractions. Part II: practical recommendations for effective mitigation.

In a previous work, it was shown that at high temperatures (up to 280°C) glycidyl esters (GE) are formed from diacylglycerols (DAG) via elimination of free fatty acid (FFA). In the present study, the impact of DAG content and temperature on the formation of GE using a model vacuum system mimicking industrial edible oil deodorization is investigated. These deodorization experiments confirmed that the formation of GE from DAG is extensive at temperatures above 230-240°C, and therefore, this value should be considered as an upper limit for refining operations. Furthermore, experimental data suggest that the formation of GE accelerates in particular when the DAG levels in refined oils exceed 3-4% of total lipids. Analysis of the lipid composition of crude palm oil (CPO) samples allowed the estimation that this critical DAG content corresponds to about 1.9-2.5% of FFA, which is the conventional quality marker of CPO. Moreover, high levels (>100ppm) of GE were also found in palm fatty acid distillate samples, which may indicate that the level of GE in fully refined palm oils also depends on the elimination rate of GE into the fatty acid distillate.

Identification of novel circulating coffee metabolites in human plasma by liquid chromatography-mass spectrometry.

This study reports a liquid chromatography-mass spectrometry method for the detection of polyphenol-derived metabolites in human plasma without enzymatic treatment after coffee consumption. Separation of available standards was achieved by reversed-phase ultra performance liquid chromatography and detection was performed by high resolution mass spectrometry in negative electrospray ionization mode. This analytical method was then applied for the identification and relative quantification of circulating coffee metabolites. A total of 34 coffee metabolites (mainly reduced, sulfated and methylated forms of caffeic acid, coumaric acid, caffeoylquinic acid and caffeoylquinic acid lactone) were identified based on mass accuracy (<4 ppm for most metabolites), specific fragmentation pattern and co-chromatography (when standard available). Among them, 19 circulating coffee metabolites were identified for the first time in human plasma such as feruloylquinic acid lactone, sulfated and glucuronidated forms of feruloylquinic acid lactone and sulfated forms of coumaric acid. Phenolic acid derivatives such as dihydroferulic acid, dihydroferulic acid 4'-O-sulfate, caffeic acid 3'-O-sulfate, dimethoxycinnamic acid, dihydrocaffeic acid and coumaric acid O-sulfate appeared to be the main metabolites circulating in human plasma after coffee consumption. The described method is a sensitive and reliable approach for the identification of coffee metabolites in biological fluids. In future, this analytical method will give more confidence in compound identification to provide a more comprehensive assessment of coffee polyphenol bioavailability studies in humans.

Plasma pharmacokinetics of catechin metabolite 4'-O-Me-EGC in healthy humans.

BACKGROUND: Tea is an infusion of the leaves of the Camellia sinensis plant and is the most widely consumed beverage in the world after water. Green tea contains significant amounts of polyphenol catechins and represents a promising dietary component to maintain health and well-being. Epidemiological studies indicate that polyphenol intake may have potential health benefits, such as, reducing the incidence of coronary heart disease, diabetes and cancer. While bioavailability of green tea bioactives is fairly well understood, some gaps still remain to be filled, especially the identification and quantification of conjugated metabolites in plasma, such as, sulphated, glucuronidated or methylated compounds. AIM OF THE STUDY: In the present study, we aimed to quantify the appearance of green tea catechins in plasma with particular emphasis on their methylated forms. RESULTS: After feeding 400 mL of green tea, 1.25% infusion to 9 healthy subjects, we found significant amounts of EC, EGC and EGCg in plasma as expected. EGC was the most bioavailable catechin, and its methylated form (4'-O-Me-EGC) was also present in quantifiable amounts. Its kinetics followed that of its parent compound. However, the relative amount of the methylated form of EGC was lower than that of the parent compound, an important aspect which, in the literature, has been controversial so far. The quantitative results presented in our study were confirmed by co-chromatography and accurate mass analysis of the respective standards. We show that the relative abundance of 4'-O-Me-EGC is ~40% compared to the parent EGC. CONCLUSION: 4'-O-Me-EGC is an important metabolite derived from catechin metabolism. Its presence in significant amounts should not be overlooked when assessing human bioavailability of green tea.

First identification of dimethoxycinnamic acids in human plasma after coffee intake by liquid chromatography-mass spectrometry.

There is a substantial amount of published literature on the bioavailability of various coffee components including the most abundant metabolites, caffeic and ferulic acids. Surprisingly, to date, the appearance of dimethoxycinnamic acid derivatives in humans has not been reported despite the fact that methylated form of catechol-type polyphenols could help maintain, modify or even improve their biological activities. This study reports an LC-MS method for the detection of dimethoxycinnamic acid in human plasma after treatment with an esterase. Liquid chromatography, including the combination of methanol and acetonitrile as organic eluent, was optimized to resolve all interferences and enable reliable detection and identification of 3,4-dimethoxycinnamic and 3,4-dimethoxy-dihydrocinnamic acids. In addition to the good mass accuracy achieved (better than 5 ppm), tandem mass spectrometric and co-chromatography experiments further confirmed the identity of the compounds. The optimized method was applied to analyze samples obtained immediately, 1 and 10 h after coffee ingestion. The results show that in particular 3,4-dimethoxycinnamic acid appears in high abundance (∼380 nM at 60 min) in plasma upon coffee intake, indicating that it is important to consider these derivatives in future bioavailability and bioefficacy studies.