Comparison of two data processing approaches for aroma marker identification in different distilled liquors using comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry dataset.

Whisky, brandy, rum, and Chinese baijiu are popular distilled spirits globally, and the volatile components play an essential role in the aroma of these distilled liquors. Volatile compounds in whisky, brandy, rum, and three main aroma types of Chinese baijiu (strong, light, and sauce) were investigated using comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOFMS). Two significant variable detection approaches, the variable importance in the projection (VIP) and nonparametric test (Mann-Whitney U test), were compared to determine the volatile markers among these samples. It was found that the VIP model was more efficient in screening significant variants than the U test. A total of 117 common markers were selected by both the VIP and U test methods with potential aroma contributions. Esters and acids were the main aroma markers for baijiu, diethyl esters were the main aroma markers for brandy, whereras pyrazines, lactones, and furans were the main aroma markers for whisky. Based on the chosen markers, different unknown distilled liquors were successfully classified in the model validation. This study provided a feasible methodology for spirit sample speculation based on volatile composition obtained by GC×GC-TOFMS.

Can Electronic Nose Replace Human Nose?-An Investigation of E-Nose Sensor Responses to Volatile Compounds in Alcoholic Beverages.

Electronic nose (E-nose) technology is frequently attempted to simulate the human olfactory system to recognize complex odors. Metal oxide semiconductors (MOSs) are E-noses' most popular sensor materials. However, these sensor responses to different scents were poorly understood. This study investigated the characteristic responses of sensors to volatile compounds in a MOS-based E-nose platform, using baijiu as an evaluation system. The results showed that the sensor array had distinctive responses for different volatile compounds, and the response intensities varied depending on the sensors and the volatile compounds. Some sensors had dose-response relationships in a specific concentration range. Among all the volatiles investigated in this study, fatty acid esters had the greatest contribution to the overall sensor response of baijiu. Different aroma types of Chinese baijiu and different brands of strong aroma-type baijiu were successfully classified using the E-nose. This study provided an understanding of detailed MOS sensor response with volatile compounds, which could be further applied to improve the E-nose technology and its practical application in food and beverages.

Multidisciplinary evaluation of plant growth promoting rhizobacteria on soil microbiome and strawberry quality.

The natural soil environment is considered one of the most diverse habitats containing numerous bacteria, fungi, and larger organisms such as nematodes, insects, or rodents. Rhizosphere bacteria play vital roles in plant nutrition and the growth promotion of their host plant. The aim of this study was to evaluate the effects of three plant growth-promoting rhizobacteria (PGPR), Bacillus subtilis, Bacillus amyloliquefaciens, and Pseudomonas monteilii for their potential role as a biofertilizer. The effect of the PGPR was examined at a commercial strawberry farm in Dayton, Oregon. The PGPR were applied to the soil of the strawberry (Fragaria × ananassa cultivar Hood) plants in two different concentrations of PGPR, T1 (0.24% PGPR) and T2 (0.48% PGPR), and C (no PGPR). A total of 450 samples from August 2020 to May 2021 were collected, and microbiome sequencing based on the V4 region of the 16S rRNA gene was conducted. The strawberry quality was measured by sensory evaluation, total acidity (TA), total soluble solids (TSS), color (lightness and chroma), and volatile compounds. Application of the PGPR significantly increased the populations of Bacillus and Pseudomonas and promoted the growth of nitrogen-fixing bacteria. The TSS and color evaluation showed that the PGPR presumptively behaved as a ripening enhancer. The PGPR contributed to the production of fruit-related volatile compounds, while the sensory evaluation did not show significant differences among the three groups. The major finding of this study suggests that the consortium of the three PGPR have a potential role as a biofertilizer by supporting the growth of other microorganisms (nitrogen-fixing bacteria) as part of a synergetic effect and strawberry quality such as sweetness and volatile compounds.

Lipid metabolic characteristics and marker compounds of ripened Pu-erh tea during pile fermentation revealed by LC-MS-based lipidomics.

Ripened Pu-erh tea (RPT) is a unique microbial fermented tea. Herein, we investigated the lipid composition of RPT and its metabolic changes during pile fermentation, by nontargeted lipidomics profiling and quantitative analysis using liquid chromatography-mass spectrometry (LC-MS). A total of 485 individual lipid species covering 26 subclasses were detected, and fatty acid ester of hydroxy fatty acid (FAHFA) was detected in tea for the first time. Among them, 362 species were significantly altered during fermentation. Chlorophylls decomposition, phospholipids degradation (especially phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine), formation of free fatty acid (FFA) (especially FFA18:3, FFA18:2), and formation of FAHFA, were annotated as the key pathways. Particularly, FAHFAs were undetected in raw tea and gradually enriched to 227.0 ± 9.6 nmol/g after fermentation (p < 0.001), which could serve as marker compounds of RPT associated with microbial fermentation. This study will advance understanding the lipid metabolic fate in microbial fermentation and its role in RPT quality. Chemical compounds studied in this article: Linolenic acid (PubChem CID: 5280934); Linoleic acid (PubChem CID: 5280450); Oleic acid (PubChem CID: 445639); PS(22:0/18:2) (PubChem CID: 52925820); PS(20:0/18:3) (PubChem CID: 52925629); Pheophytin a (PubChem CID: 135398712); Pheophorbide a (PubChem CID: 253193).

Isolation, Characterization, and Compositional Analysis of Polysaccharides from Pinot Noir Wines: An Exploratory Study.

It has been reported that polysaccharides in wine can interact with tannins and other wine components and modify the sensory properties of the wine. Unfortunately, the contribution of polysaccharides to wine quality is poorly understood, mainly due to their complicated structure and varied composition. In addition, the composition and molecular structure of polysaccharides in different wines can vary greatly. In this study, the polysaccharides were isolated from pinot noir wine, then separated into high-molecular-weight (PNWP-H) and low-molecular-weight (PNWP-L) fractions using membrane-based ultrafiltration. Each polysaccharide fraction was further studied using size exclusion chromatography, UV-Vis, FT-IR, matrix-assisted laser desorption/ionization-high-resolution mass spectrometry, and gas chromatography-mass spectrometry (GC-MS). The results showed that PNWP-L and PNWP-H had different chemical properties and compositions. The FT-IR analysis showed that PNWPs were acidic polysaccharides with α- and ß-type glycosidic linkages. PNWP-L and PNWP-H had different α- and ß-type glycosidic linkage structures. FT-IR showed stronger antisymmetric and symmetric stretching vibrations of carboxylate anions of uronic acids in PNWP-L, suggesting more uronic acid in PNWP-L. The size exclusion chromatography results showed that over 72% of the PNWP-H fraction had molecular sizes from 25 kDa to 670 kDa. Only a small percentage of smaller molecular polysaccharides was found in the PNWP-H fraction. In comparison, all of the polysaccharides in the PNWP-L fraction were below 25 KDa, with a majority distributed approximately 6 kDa (95.1%). GC-MS sugar composition analysis showed that PNWP-L was mainly composed of galacturonic acid, rhamnose, galactose, and arabinose, while PNWP-H was mainly composed of mannose, arabinose, and galactose. The molecular size distribution and sugar composition analysis suggested that the PNWP-L primarily consisted of rhamnogalacturonans and polysaccharides rich in arabinose and galactose (PRAG). In comparison, PNWP-H were mostly mannoproteins and polysaccharides rich in arabinose and galactose (PRAG). Further research is needed to understand the impacts of these fractions on wine organoleptic properties.

Evaluation of Sulfur-Based Biostimulants for the Germination of Sclerotium cepivorum Sclerotia and Their Interaction with Soil.

White rot is an economically significant disease of Allium crops. The pathogen Sclerotium cepivorum produces long-lived sclerotia that germinate in response to sulfur-containing compounds released from Allium roots. Diallyl disulfide (DADS) was the primary organic sulfur compound detected in the rhizosphere soil of two garlic cultivars, "California Early and Late", growing in greenhouse conditions. DADS, dimethyl trisulfide (DMTS), dimethyl disulfide (DMDS), isopropyl disulfide (IPDS), dipropyl disulfide (DPDS), diethyl disulfide (DEDS), together with garlic oil, garlic juice, garlic powder, raw onion pieces, cabbage pieces, and Chinese cabbage pieces were investigated for their activities toward germinating dormant sclerotia. Results showed that DADS and other volatile sulfur compounds could stimulate sclerotial germination, and a dose-response was observed. In addition, garlic juice, powder, raw onion, and the two cabbages could stimulate sclerotial germination. Furthermore, the laboratory soil incubation experiments demonstrated the strong interaction of organic sulfur compounds with soil.

Blue Light-Mediated, Photocatalyst-Free Decarboxylative Alkylation of Heteroaryl Sulfinimines.

We report a diastereoselective, photocatalyst-free decarboxylative alkylation of (hetero)aryl sulfinimines using redox-active esters under blue light. High yields and diastereoselectivities can be achieved under mild conditions, and we demonstrate its utility as a synthetic method, especially for medicinal chemists.

Composition of Pinot Noir Wine from Grapevine Red Blotch Disease-Infected Vines Managed with Exogenous Abscisic Acid Applications.

Grapevine red blotch disease (GRBD) has negative effects on grape development and impacts berry ripening. Abscisic acid (ABA) is a plant growth regulator involved in the initiation of berry ripening. Exogenous abscisic acid application was compared to an unsprayed control on GRBD-positive Pinot noir vines during two vintages, and the total monomeric anthocyanin, total phenolics, phenolic composition, and volatile profile were measured in wines. In addition, untargeted metabolites were profiled using high-resolution LC-MS/MS. Results showed that the wine composition varied by vintage year and was not consistent with ABA application. Wines from the ABA treatment had a lower total anthocyanin and total phenolic content in one year. The untargeted high-resolution LC-MS/MS analysis showed a higher abundance of phenolic compounds in ABA wines in 2019, but lower in 2018. The wine volatile compounds of ABA treatments varied by vintage. There were higher levels of free ß-damascenone, ß-ionone, nerol, and several fermentation-derived esters, acids, and alcohols in ABA wines, but these were not observed in 2019. Lower 3-isobutyl-2-methoxypyrazine (IBMP) was also observed in wines with ABA treatment in 2019. The results demonstrated that ABA application to the fruit zones did not consistently mitigate the adverse impacts of GRBD on Pinot noir wines.

Insight into aroma dynamic changes during the whole manufacturing process of chestnut-like aroma green tea by combining GC-E-Nose, GC-IMS, and GC × GC-TOFMS.

Processing is the crucial factor for green tea aroma quality. In this study, the aroma dynamic changes throughout the manufacturing process of chestnut-like aroma green tea were investigated with gas chromatography electronic nose (GC-E-Nose), gas chromatography-ion mobility spectrometry (GC-IMS), and comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC × GC-TOFMS). GC-IMS identified 33 volatile compounds while GC × GC-TOFMS identified 211 volatile components. Drying exerted the greatest influence on the volatile components of chestnut-like aroma green tea, and promoted the generation of heterocyclic compounds and sulfur compounds which were commonly generated via the Maillard reaction during the roasting stage. A large number of heterocyclic compounds such as 1-methyl-1H-pyrrole, pyrrole, methylpyrazine, furfural, 2-ethyl-5-methylpyrazine, 1-ethyl-1H-pyrrole-2-carboxaldehyde, and 3-acetylpyrrole were newly formed during the drying process. This study also validated the suitability of GC-E-Nose combined with GC-IMS and GC × GC-TOFMS for tracking the changes in volatile components of green tea throughout the manufacturing process.

Volatile Analysis of Wuliangye Baijiu by LiChrolut EN SPE Fractionation Coupled with Comprehensive GC×GC-TOFMS.

Wuliangye baijiu is one of the most famous Chinese liquors with a protected geographical indication. This study used LiChrolut® EN-based solid-phase extraction (SPE) and fractionation combined with comprehensive two-dimensional chromatography-time-of-flight mass spectrometry (GC×GC-TOFMS) to unveil its volatile composition. The volatiles were isolated with LiChrolut® EN-based SPE and traditional liquid-liquid extraction (LLE). The neutral/basic fractions from LLE and the SPE were fractionated on a LiChrolut® EN SPE column and analyzed by comprehensive GC×GC-TOFMS. Compared with LLE, more esters and alcohols were detected in the SPE-based extraction. The SPE fractionation and GC×GC-TOFMS analysis resulted in the identification of about 500 volatile compounds in more than 3000 peaks of the Wuliangye baijiu. The approach simplifies the complex baijiu composition into functional group-based fractions for reliable identification and analysis. This study provided a confidence volatile identification approach for Chinese baijiu based on the SPE fractionation GC×GC-TOFMS.

In vivo and in vitro aroma release in surimi gel with different cross-linking degrees by proton transfer reaction-mass spectrometry.

Transglutaminase-induced cross-linking has been suggested as a strategy to govern surimi gels' texture. To achieve the aroma regulation of surimi gels by cross-links, surimi gels were treated with microbial transglutaminase to get different cross-linking degrees, and in vivo and in vitro aroma releases were investigated by a proton transfer reaction-mass spectrometry (PTR-MS). Seventeen compounds in surimi gels were detected by PTR-MS. The in vitro release curves of odorants were fitted by a pseudo-first-order kinetics model. As the cross-links increased, most aroma compounds' released concentrations and release rates decreased first, and then increased significantly (P < 0.05) when the cross-linking degree exceed around 35.4%, negatively related to the springiness and the gel strength of surimi gels. However, the in vivo aroma release results showed that the harder surimi gel released fewer aroma compounds. In conclusion, texture affected by cross-links could be a strategy to control the aroma release of surimi gels.

Profiling polar lipids in whey protein phospholipid concentrate by LC-HRMS/MS.

Bioactive polar lipids in the milk fat globular membrane can be recovered and enriched during whey protein processing into a co-product called whey protein phospholipid concentrate (WPPC). A food-grade solvent successfully extracted polar lipids from powdered dairy products, and lipids can be fractionated under temperature-induced crystallization. This study investigates the specific lipid species present in ethanol extracted lipid residues from commercially available WPPC using a UPLC-Q-TOF-MS/MS lipidomics method. In general, sphingomyelins and phosphatidylcholines were retained in the polar lipid enriched fraction. Sphingomyelin was found to be a rich source of long chain fatty acids. Several glycosphingolipids, glucosyl-, galactosyl-, lactosyl-, and galabiosylceramide, were also detected in WPPC; these species were observed to crystallize away from other polar lipids during fractionation. Correlation analysis supported the claim that majority of polar lipids recovered in a total lipid extract using ethanol were retained in a polar lipid enriched residue after fractional crystallization.

Untargeted Headspace-Gas Chromatography-Ion Mobility Spectrometry in Combination with Chemometrics for Detecting the Age of Chinese Liquor (Baijiu).

This paper proposes the combination of headspace-gas chromatography-ion mobility spectrometry (HS-GC-IMS) and chemometrics as a method to detect the age of Chinese liquor (Baijiu). Headspace conditions were optimized through single-factor optimization experiments. The optimal sample preparation involved diluting Baijiu with saturated brine to 15% alcohol by volume. The sample was equilibrated at 70 °C for 30 min, and then analyzed with 200 µL of headspace gas. A total of 39 Baijiu samples from different vintages (1998-2019) were collected directly from pottery jars and analyzed using HS-GC-IMS. Partial least squares regression (PLSR) analysis was used to establish two discriminant models based on the 212 signal peaks and the 93 identified compounds. Although both models were valid, the model based on the 93 identified compounds discriminated the ages of the samples more accurately according to the goodness of fit value (R2) and the root mean square error of prediction (RMSEP), which were 0.9986 and 0.244, respectively. Nineteen compounds with variable importance for prediction (VIP) scores > 1, including 11 esters, 4 alcohols, and 4 aldehydes, played vital roles in the model established by the 93 identified compounds. Overall, we determined that HS-GC-IMS combined with PLSR could serve as a rapid and accurate method for detecting the age of Baijiu.

Tandem Solid-Phase Extraction Columns for Simultaneous Aroma Extraction and Fractionation of Wuliangye and Other Baijiu.

Wuliangye baijiu is one of the most famous baijiu in China, with a rich, harmonic aroma profile highly appreciated by consumers. Thousands of volatiles have been identified for the unique aroma profile. Among them, fatty acid esters have been identified as the main contributors to the aroma profile. In addition, many non-ester minor compounds, many of which are more polar than the esters, have been identified to contribute to the characteristic aroma unique to Wuliangye baijiu. The analysis of these minor compounds has been challenging due to the dominance of esters in the sample. Thus, it is desirable to fractionate the aroma extract into subgroups based on functional group or polarity to simplify the analysis. This study attempts a new approach to achieve simultaneous volatile extraction and fractionation using tandem LiChrolut EN and silica gel solid-phase extraction (SPE) columns. A baijiu sample (10 mL, diluted in 40 mL of water) was first passed through the LiChrolut EN (1.0 g) column. The loaded LiChrolut EN column was then dried with air and coupled with a silica gel (5.0 g) SPE column with anhydrous Na2SO4 (10.0 g) in between. The volatile compounds were eluted from the LiChrolut EN column and simultaneously fractionated on the silica gel column based on polarity. The simultaneous extraction and fractionation technique enabled the fractionations of all fatty acid esters into less polar fractions. Fatty acids, alcohols, pyrazines, furans, phenols, hydroxy esters, and other polar compounds were collected in more polar fractions. This technique was used to study the volatile compounds in Wuliangye, Moutai, and Fengjiu baijiu. In addition to fatty acid esters, many minor polar compounds, including 2,6-dimethylpyrazine, 2-ethyl-6-methylpyrazine, 2-ethyl-3,5-dimethylpyrazine, p-cresol, and 2-acetylpyrrole, were unequivocally identified in the samples. The procedure is fast and straightforward, with low solvent consumption.

Smoked-Derived Volatile Phenol Analysis in Wine by Stir Bar Sorptive Extraction-Gas Chromatography-Mass Spectrometry.

Smoke-derived taint has become a significant concern for the U.S. wine industry, particularly on the west coast, and climate change is anticipated to aggravate it. High volatile phenols such as guaiacol, 4-methylguaiacol, 4-ethylguaiacol, 4-ethylphenol, and o-, p-, m-cresols have been suggested to be related to smoke-exposed grape and wine. This paper describes an analytical approach based on ethylene glycol/polydimethylsiloxane (EG/PDMS)-stir bar sorptive extraction-gas chromatography-mass spectrometry (SBSE-GC-MS) to quantify or estimate the concentrations of some smoke-related volatile phenols in wines. Correlation coefficients with R2 ≥ 0.990 were obtained. This method can quantify most smoked-related volatile phenols down to 0.5 µg/L in wine in selective ion monitoring mode. Recovery for the targeted volatile phenols ranged from 72.2% to 142.4% in the smoke-tainted wine matrix, except for 4-vinylguaiacol. The standard deviations of the volatile phenols were from 0 to 23% in smoke-tainted wine. The approach provides another tool to evaluate wine smoke exposure and potential smoke taint.

Sensory characterization of Baijiu pungency by combined time-intensity (TI) and temporal dominance of sensations (TDS).

Pungency is increasingly being recognized as an important factor of overall sensory quality, palatability, and consumer preference of distilled spirits. The characterization of pungency is necessary to evaluate the potential sensory quality of distilled spirits. In this study, the temporal profiles of pungency of Baijiu with different aging times were evaluated using time-intensity (TI) and temporal dominance of sensations (TDS) methods, considering both pungency intensity and pungency sub-qualities. TI results indicated significant differences in release rate of pungency during Baijiu consumption. Compared to young Baijiu, old Baijiu tend to show higher release rate of pungency, the areas under the curve and duration of pungency were significantly decreased in old Baijiu. The TDS results showed significant differences in the combination of dominant sub-qualities, as well as in the maximum dominance rates and the dominant duration of sub-qualities among Baijiu. The young Baijiu were mainly characterized by the dominant sub-qualities of "burning" and "numbing", whereas for old Baijiu, "burning", "prickle", and "drying" were dominant. The application of TI and TDS provided dynamic and temporal profiles of pungency to fully characterize pungency differences of distilled beverages.

Sensory evaluation of fresh/frozen mackerel products: A review.

Mackerel has received considerable attention in the global food market as one of the most important pelagic commercial fish species. The quality of mackerel is influenced by species, season, fishing area, nutritional status, catching method, handling, and storage. Due to the mackerel's perishability, its quality is mainly measured by sensory procedures. Although considerable effort has been made to explore quick and reliable quality analysis, developing a practical and scientific sensory evaluation of mackerel has been an active ongoing study area to meet the quality evaluation demand of the industry. Different sensory evaluation methods have been used to assess the mackerel fish quality, including Palatability and Spoilage test, Torry scheme, EU scheme, Quality Index Method, Catch damage index and Processed fish damage index, Affective test, Discriminative test, and Descriptive test. Each method has its strength and weakness. Despite mackerel sensory evaluation protocols having undergone partial harmonization, specific sample process needs to be carefully followed to minimize the change during sample preparation. This review summarizes the sensory evaluation methods in mackerel research, the factors affecting sensory evaluation, and then updates the latest advances in mackerel sensory evaluation and offers guidance for presenting its application in the mackerel chain. Also, each technique's advantages and limitations are discussed. In our opinion, the future trends for sensory evaluation of mackerel should be consumer-centric.

Characterization of Aroma-Active Compounds in Northern Highbush Blueberries "Bluecrop" (Vaccinium corymbosum "Bluecrop") and "Elliott" (Vaccinium corymbosum "Elliott") by Gas Chromatography-Olfactometry Dilution Analysis and Odor Activity Value.

Aroma-active compounds in northern highbush blueberries "Bluecrop" (Vaccinium corymbosum "Bluecrop") and "Elliott" (Vaccinium corymbosum "Elliott") were isolated using solvent extraction followed by solvent-assisted flavor evaporation. The aroma-active compounds were compared by aroma extract dilution analysis (AEDA) on a polar and a chiral column and odor activity values (OAVs). The results showed that both cultivars had similar aroma-active compounds, but the flavor dilution (FD) factors and OAV values varied, responsible for the subtle aroma differences. AEDA (FD ≥ 64) suggested that the important aroma-active compounds in both cultivars could be ethyl 2-methylbutanoate, ethyl 3-methylbutanoate, (Z)-3-hexenal, 1-octen-3-one, (Z)-3-hexen-1-ol, methional, linalool, (E,Z)-2,6-nonadienal, 2-methylbutanoic acid, α-terpineol, (E,E)-2,4-nonadienal, ß-damascenone, geraniol, geranyl acetone, o-cresol, eugenol, and vanillin. Different isomers can have different sensory thresholds and attributes. Enantiomer analysis revealed that although S-(+)-linalool was dominant in blueberry, R-(-)-linalool had a higher FD value and OAVs than that of the (S)-isomer. The S-(-)-α-terpineol was slightly elevated than R-(+)-α-terpineol in both cultivars.

Direct ethanolic extraction of polar lipids and fractional crystallization from whey protein phospholipid concentrate.

Polar lipids, including sphingomyelin (SM) and other sphingolipids, have nutritional implications in many important metabolic pathways. Milk and whey are valuable sources of these bioactive lipids, differing in SM content compared with other plant-based lecithins in the food industry. Dairy lipids were extracted from whey protein phospholipid concentrate powder by direct exposure to a food-grade solvent, ethanol. A total of 20 mass equivalents of solvent was required to completely recover all lipid material of the original whey protein phospholipid concentrate sample. Using a minimal apparatus, absolute ethanol, and heating to a boiling point, a final total lipid extract composed of 37.7% phospholipids (PL) by mass was achieved. The method was developed with potential pilot-scale and industry application in mind. Investigations into fractional crystallization showed limited success at purifying PL from triglyceride content (<1% increase in percent PL by mass). However, the relative percentage of SM was increased in the fractionated sample compared with the total lipid extract. Fatty acid analysis of the PL fraction reported a composition of 33.3% monounsaturated fatty acids and 9.8% polyunsaturated fatty acids. Additional work is needed to investigate different conditions for lipid recovery, PL recovery, and PL purification by fractional crystallization in ethanol.

Phospholipid analysis in whey protein products using hydrophilic interaction high-performance liquid chromatography-evaporative light-scattering detection in an industry setting.

The main objective of this work was to develop an analytical method that can be used in a dairy manufacturing facility for the quantitation of phospholipids in dairy products. Total lipids from a dairy matrix were obtained first by Folch extraction. The total lipid extract was then applied to a silica gel-based solid-phase extraction column, and triglycerides and other nonpolar lipids were separated from the phospholipids and sphingolipids. Quantitation was performed by hydrophilic interaction HPLC coupled to evaporative light-scattering detection using a quaternary separation method. The method was validated using a commercial whey protein phospholipid concentrate and was used to analyze phospholipid and sphingolipid composition in buttermilk, whey protein concentrate, whey protein phospholipid concentrate, and several other dairy ingredients. This method was sensitive and reproducible and can be used in the dairy industry as a research tool to develop new value-added dairy phospholipid products, then later as a standard protocol for quality assurance analysis of current and future products.