An Improved High-Throughput Lipid Extraction Method for the Analysis of Human Brain Lipids

We have developed a protocol suitable for high-throughput lipidomic analysis of human brain samples. The traditional Folch extraction (using chloroform and glass–glass homogenization) was compared to a high-throughput method combining methyl-tert-butyl ether (MTBE) extraction with mechanical homogenization utilizing ceramic beads. This high-throughput method significantly reduced sample handling time and increased efficiency compared to glass–glass homogenizing. Furthermore, replacing chloroform with MTBE is safer (less carcinogenic/toxic), with lipids dissolving in the upper phase, allowing for easier pipetting and the potential for automation (i.e., robotics). Both methods were applied to the analysis of human occipital cortex. Lipid species (including ceramides, sphingomyelins, choline glycerophospholipids, ethanolamine glycerophospholipids and phosphatidylserines) were analyzed via electrospray ionization mass spectrometry and sterol species were analyzed using gas chromatography mass spectrometry. No differences in lipid species composition were evident when the lipid extraction protocols were compared, indicating that MTBE extraction with mechanical bead homogenization provides an improved method for the lipidomic profiling of human brain tissue.     

固相微萃取技术及其应用

固相微萃取（solid phase microextraction,SPME)技术是近年来发展起来的一种样品分析预处理的新方法，与传统的溶剂萃取技术相比，该技术具有快速，高效 ，简便，园需溶剂，易于自动化操作等优点，目前已经被应用于环境分析和药品检测等许多领域，本文简介了固相微萃取技术的原理以及该技术在生产，生活领域上的应用。     

Plasma Lipid Profiling Contributes to Untangle the Complexity of Moyamoya Arteriopathy

Moyamoya arteriopathy (MA) is a rare cerebrovascular disorder characterized by ischemic/hemorrhagic strokes. The pathophysiology is unknown. A deregulation of vasculogenic/angiogenic/inflammatory pathways has been hypothesized as a possible pathophysiological mechanism. Since lipids are implicated in modulating neo-vascularization/angiogenesis and inflammation, their deregulation is potentially involved in MA. Our aim is to evaluate angiogenic/vasculogenic/inflammatory proteins and lipid profile in plasma of MA patients and control subjects (healthy donors HD or subjects with atherosclerotic cerebrovascular disease ACVD). Angiogenic and inflammatory protein levels were measured by ELISA and a complete lipidomic analysis was performed on plasma by mass spectrometry. ELISA showed a significant decrease for MMP-9 released in plasma of MA. The untargeted lipidomic analysis showed a cumulative depletion of lipid asset in plasma of MA as compared to HD. Specifically, a decrease in membrane complex glycosphingolipids peripherally circulating in MA plasma with respect to HD was observed, likely suggestive of cerebral cellular recruitment. The quantitative targeted approach demonstrated an increase in free sphingoid bases, likely associated with a deregulated angiogenesis. Our findings indicate that lipid signature could play a central role in MA and that a detailed biomarker profile may contribute to untangle the complex, and still obscure, pathogenesis of MA.     

The Lipid Asset Is Unbalanced in Peripheral Nerve Sheath Tumors

Peripheral nerve sheath tumors (PNSTs) include schwannomas, neurofibromas (NFs), and plexiform neurofibromas (PNFs), among others. While they are benign tumors, according to their biological behavior, some have the potential for malignant degeneration, mainly PNFs. The specific factors contributing to the more aggressive behavior of some PNSTs compared to others are not precisely known. Considering that lipid homeostasis plays a crucial role in fibrotic/inflammatory processes and in several cancers, we hypothesized that the lipid asset was also unbalanced in this group of nerve tumors. Through untargeted lipidomics, NFs presented a significant increase in ceramide, phosphatidylcholine, and Vitamin A ester. PNFs displayed a marked decrease in 34 out of 50 lipid class analyzed. An increased level of ether- and oxidized-triacylglycerols was observed; phosphatidylcholines were reduced. After sphingolipidomic analysis, we observed six sphingolipid classes. Ceramide and dihydroceramides were statistically increased in NFs. All the glycosylated species appeared reduced in NFs, but increased in PNFs. Our findings suggested that different subtypes of PNSTs presented a specific modulation in the lipidic profile. The untargeted and targeted lipidomic approaches, which were not applied until now, contribute to better clarifying bioactive lipid roles in PNS natural history to highlight disease molecular features and pathogenesis.     

固相微萃取技术及其在环境分析中的应用

固相微萃取技术是一种新型的样品前处理技术,它将取样、萃取、浓缩等过程合并在一个操作步骤中完成,不需要有机萃取剂,是一种简便快捷的样品前处理技术。文章在系统地介绍SPME的发展、原理、装置、影响因素的基础上,阐述了近年来固相微萃取技术在环境分析中的应用,并预测了今后的发展趋势。     

Dynamic Assessment of Functional Lipidomic Analysis in Human Urine

The development of enabling mass spectrometry platforms for the quantification of diverse lipid species in human urine is of paramount importance for understanding metabolic homeostasis in normal and pathophysiological conditions. Urine represents a non‐invasive biofluid that can capture distinct differences in an individual's physiological status. However, currently there is a lack of quantitative workflows to engage in high throughput lipidomic analysis. This study describes the development of a MS/MS^ALL shotgun lipidomic workflow and a micro liquid chromatography–high resolution tandem mass spectrometry (LC–MS/MS) workflow for urine structural and mediator lipid analysis, respectively. This workflow was deployed to understand biofluid sample handling and collection, extraction efficiency, and natural human variation over time. Utilization of 0.5 mL of urine for structural lipidomic analysis resulted in reproducible quantification of more than 600 lipid molecular species from over 20 lipid classes. Analysis of 1 mL of urine routinely quantified in excess of 55 mediator lipid metabolites comprised of octadecanoids, eicosanoids, and docosanoids generated by lipoxygenase, cyclooxygenase, and cytochrome P450 activities. In summary, the high‐throughput functional lipidomics workflow described in this study demonstrates an impressive robustness and reproducibility that can be utilized for population health and precision medicine applications.     

Lipidomic Analysis: From Archaea to Mammals

Lipids are among the most important organic compounds found in all living cells, from primitive archaebacteria to flowering plants or mammalian cells. They form part of cell walls and constitute cell storage material. Their biosynthesis and metabolism play key roles in faraway topics such as biofuel production (third‐generation biofuels produced by microorganisms, e.g. algae) and human diseases such as adrenoleukodystrophy, Zellweger syndrome, or Refsum disease. Current lipidomic analysis requires fast and accurate processing of samples and especially their characterization. Because the number of possible lipids and, more specifically, molecular species of lipids is of the order of hundreds to thousands, it is necessary to process huge amounts of data in a short time. There are two basic approaches to lipidomic analysis: shotgun and liquid chromatography–mass spectometry. Both methods have their pros and cons. This review deals with lipidomics not according to the type of ionization or the lipid classes analyzed but according to the types of samples (organisms) under study. Thus, it is divided into lipidomic analysis of archaebacteria, bacteria, yeast, fungi, algae, plants, and animals.     

Comparison of two methods for extraction of volatiles from marine PL emulsions

The dynamic headspace (DHS) thermal desorption principle using Tenax GR tube, as well as the solid phase micro‐extraction (SPME) tool with carboxen/polydimethylsiloxane 50/30 µm CAR/PDMS SPME fiber, both coupled to GC/MS were implemented for the isolation and identification of both lipid and Strecker derived volatiles in marine phospholipids (PL) emulsions. Comparison of volatile extraction efficiency was made between the methods. For marine PL emulsions with a highly complex composition of volatiles headspace, a fiber saturation problem was encountered when using CAR/PDMS‐SPME for volatiles analysis. However, the CAR/PDMS‐SPME technique was efficient for lipid oxidation analysis in emulsions of less complex headspace. The SPME method extracted volatiles of lower molecular weights more efficient than the DHS method. On the other hand, DHS Tenax GR appeared to be more efficient in extracting volatiles of higher molecular weights and it provided a broader volatile spectrum for marine PL emulsion than the CAR/PDMS‐SPME method.     

The Metabolic Impact of Two Different Parenteral Nutrition Lipid Emulsions in Children after Hematopoietic Stem Cell Transplantation: A Lipidomics Investigation

Hematopoietic stem cell transplantation (HSCT) involves the infusion of either bone marrow or blood cells preceded by toxic chemotherapy. However, there is little knowledge about the clinical benefits of parenteral nutrition (PN) in patients receiving high-dose chemotherapy during HSCT. We investigated the lipidomic profile of plasma and the targeted fatty acid profiles of plasma and erythrocytes in children after HSCT using PN with either a fish oil-based lipid emulsion or a classic soybean oil emulsion. An untargeted liquid chromatography high-resolution mass spectrometry platform connected with a novel in silico annotation algorithm was utilized to determine the most relevant chemical subclasses affected. In addition, we explored the interrelation between the lipidomics profile in plasma, the targeted fatty acid profile in plasma and erythrocytes, several biomarkers of inflammation, and antioxidant defense using an innovative data integration analysis based on Latent Components. We observed that the fish oil-based lipid emulsion had an impact in several lipid subclasses, mainly glycerophosphocholines (PC), glycerophosphoserines (PS), glycerophosphoethanolamines (PE), oxidized PE (O-PE), 1-alkyl,2-acyl PS, lysophosphatidylethanolamines (LPE), oxidized PS (O-PS) and dicarboxylic acids. In contrast, the classic soybean oil emulsion did not. Several connections across the different blocks of data were found and aid in interpreting the impact of the lipid emulsions on metabolic health.     

Lipidomic profiling and discovery of lipid biomarkers in snow alga Chlamydomonas nivalis under salt stress

The snow alga Chlamydomonas nivalis is a typical microalgal species that can adapt and resist to natural habitats in the polar region and similar extreme environments. In response to various stress conditions, the change of lipid profile in cell membrane systems is known to play a vital role in cell survival and reproduction. In the present work, to elucidate the involvement of intracellular lipids in the molecular mechanism of cell response and adaptation to salt stress, C. nivalis was cultured and treated with different sodium chloride (NaCl) concentrations in different time courses. The ultra performance liquid chromatography/quadrupole‐TOF‐MS (UPLC/Q‐TOF‐MS)‐based approach was developed for lipidomic profiling followed by multivariate statistical analysis including partial least squares discriminant analysis and orthogonal projection on latent structure discriminant analysis for data classification and potential biomarkers selection. Seven types and 35 kinds of polar lipid molecules were selected and identified as biomarkers, including monogalactosyldiacylglycerol, digalactosyldiacylglycerol, l, 2‐diacylglyceryl‐3‐O‐4′‐(N,N,N‐trimethyl)‐homoserine, phosphatidylglycerol, sulfoquinovosyl‐diacylglycerol, phosphatidylethanolamine, and phosphatidylinositiol. Their functions in relation to the regulation of cell membrane stability, signal transduction and photosynthesis efficiency under salt stress are also discussed in this paper. This is the first study using UPLC/Q‐TOF‐MS‐based lipidomic profiling with multivariate statistical analysis for lipid biomarkers discovery from microalgae in response to stress conditions.     

Metabolic Profiles of Brain Metastases

Metastasis to the brain is a feared complication of systemic cancer, associated with significant morbidity and poor prognosis. A better understanding of the tumor metabolism might help us meet the challenges in controlling brain metastases. The study aims to characterize the metabolic profile of brain metastases of different origin using high resolution magic angle spinning (HR-MAS) magnetic resonance spectroscopy (MRS) to correlate the metabolic profiles to clinical and pathological information. Biopsy samples of human brain metastases (n = 49) were investigated. A significant correlation between lipid signals and necrosis in brain metastases was observed (p < 0.01), irrespective of their primary origin. The principal component analysis (PCA) showed that brain metastases from malignant melanomas cluster together, while lung carcinomas were metabolically heterogeneous and overlap with other subtypes. Metastatic melanomas have higher amounts of glycerophosphocholine than other brain metastases. A significant correlation between microscopically visible lipid droplets estimated by Nile Red staining and MR visible lipid signals was observed in metastatic lung carcinomas (p = 0.01), indicating that the proton MR visible lipid signals arise from cytoplasmic lipid droplets. MRS-based metabolomic profiling is a useful tool for exploring the metabolic profiles of metastatic brain tumors.     

Characterization of Lipids in Saliva,Tears and Minor Salivary Glands of Sjögren’s Syndrome Patients Using an HPLC/MS-Based Approach

The diagnostic work-up of primary Sjögren’s syndrome (pSS) includes quantifying saliva and tear production, evaluation of autoantibodies in serum and histopathological analysis of minor salivary glands. Thus, the potential for further utilizing these fluids and tissues in the quest to find better diagnostic and therapeutic tools should be fully explored. Ten samples of saliva and tears from female patients diagnosed with pSS and ten samples of saliva and tears from healthy females were included for lipidomic analysis of tears and whole saliva using high-performance liquid chromatography coupled to time-of-flight mass spectrometry. In addition, lipidomic analysis was performed on minor salivary gland biopsies from three pSS and three non-SS females. We found significant differences in the lipidomic profiles of saliva and tears in pSS patients compared to healthy controls. Moreover, there were differences in individual lipid species in stimulated saliva that were comparable to those of glandular biopsies, representing an intriguing avenue for further research. We believe a comprehensive elucidation of the changes in lipid composition in saliva, tears and minor salivary glands in pSS patients may be the key to detecting pSS-related dry mouth and dry eyes at an early stage. The identified differences may illuminate the path towards future innovative diagnostic methodologies and treatment modalities for alleviating pSS-related sicca symptoms.     

Tumor Resection Induces Alterations on Serum Phospholipidome of Liver Cancer Patients

Hepatocellular carcinoma and cholangiocarcinoma are the most common primary malignant liver tumors. Since the liver plays a key role in lipid metabolism, the study of serum phospholipid (PL) profiles may provide a better understanding of alterations in hepatic lipid metabolism. In this study, we used a high-resolution HILIC-LC–MS lipidomic approach to establish the serum phospholipidome profile of patients with liver cancer before (T0) and after tumor resection (T1) and a control group (CT) of healthy individuals. After the analysis of PL profiles, we observed that the phospholipidome of patients with liver cancer was significantly modified after the tumor resection procedure. We observed an upregulation of some phosphatidylcholine (PtdCho) species, namely, PtdCho(36:6), PtdCho(42:6), PtdCho(38:5), PtdCho(36:5), PtdCho(38:6) and choline plasmalogens (PlsCho), and/or 1-O-alkyl-2-acyl-glycerophosphocholine (PakCho) in patients with liver cancer at T0 compared to the CT group, and a downregulation after tumor resection (T1) when compared to T0. These results show that LC–MS can detect different serum PL profiles in patients with liver cancer, before and after tumor resection, by defining a specific PL fingerprint that was used to determine the effect of tumor and tumor resection on lipid metabolism.     

Ultra high performance liquid chromatography–mass spectrometric analysis of oxidized free fatty acids and acylglycerols

A fast ultra high performance liquid chromatography (UHPLC)–electrospray ionization (ESI)–mass spectrometric (MS) method was developed for simultaneous analysis of free fatty acids (FFAs), monoacylglycerols (MAG), diacylglycerols (DAG), triacylglycerols (TAG), and their oxidized equivalents. Effect of elevated column temperature was studied in order to optimize the chromatography of closely eluting peaks and to reduce high back pressure formed in UHPLC. The elevated temperature enabled high flow rate, better mass transfer, and therefore more narrow peaks and better separation of the analytes. The new method was applied to the analysis of total lipid extracts of lipolysis samples prepared by an artificial digestion model in order to investigate oxidized lipids and changes in their profiles in the chyme. Over 150 compounds were identified from the extracts. The UHPLC–ESI–MS method was proved to be fast, highly selective, and sensitive. Compared to a previously used high performance LC–ESI–MS method, the new UHPLC–ESI–MS method was over five times faster and consumed one tenth of the solvents while producing comparable quantitative results. Practical applications: Edible oils and fats contain mainly TAGs, the lipolysis of which produces FFAs and MAGs with minor DAG components. These compounds are susceptible to oxidation in the stomach, and therefore the analysis of the oxidation products is important. Fast determination of FFAs and acylglycerols is also important in quality control of biodiesel. Our new method enables accurate and sensitive determination of different molecular species present in digested and processed samples with minimal sample preparation requirements. In this respect, the new method is applicable to large scale and fast screening of biological samples for lipidomic and metabolomic studies.     

Comprehensive and quantitative profiling of lipid species in human milk,cow milk and a phospholipid‐enriched milk formula by GC and MS/MSALL

Here we present a workflow for in‐depth analysis of milk lipids that combines gas chromatography (GC) for fatty acid (FA) profiling and a shotgun lipidomics routine termed MS/MS^ALL for structural characterization of molecular lipid species. To evaluate the performance of the workflow we performed a comparative lipid analysis of human milk, cow milk, and Lacprodan^® PL‐20, a phospholipid‐enriched milk protein concentrate for infant formula. The GC analysis showed that human milk and Lacprodan have a similar FA profile with higher levels of unsaturated FAs as compared to cow milk. In‐depth lipidomic analysis by MS/MS^ALL revealed that each type of milk sample comprised distinct composition of molecular lipid species. Lipid class composition showed that the human and cow milk contain a higher proportion of triacylglycerols (TAGs) as compared to Lacprodan. Notably, the MS/MS^ALL analysis demonstrated that the similar FA profile of human milk and Lacprodan determined by GC analysis is attributed to the composition of individual TAG species in human milk and glycerophospholipid species in Lacprodan. Moreover, the analysis of TAG molecules in Lacprodan and cow milk showed a high proportion of short‐chain FAs that could not be monitored by GC analysis. The results presented here show that complementary GC and MS/MS^ALL analysis is a powerful approach for characterization of molecular lipid species in milk and milk products. Practical applications : Milk lipid analysis is routinely performed using gas chromatography. This method reports the total fatty acid composition of all milk lipids, but provides no structural or quantitative information about individual lipid molecules in milk or milk products. Here we present a workflow that integrates gas chromatography for fatty acid profiling and a shotgun lipidomics routine termed MS/MS^ALL for structural analysis and quantification of molecular lipid species. We demonstrate the efficacy of this complementary workflow by a comparative analysis of molecular lipid species in human milk, cow milk, and a milk‐based supplement used for infant formula. A workflow for milk lipid analysis based on combined gas chromatography and high‐resolution shotgun lipidomics. In‐depth structural characterization and quantification of molecular lipid species in milk and milk products.     

A Distinct Cytokine Profile and Stromal Vascular Fraction Metabolic Status without Significant Changes in the Lipid Composition Characterizes Lipedema

Lipedema is an adipose tissue disorder characterized by the disproportionate increase of subcutaneous fat tissue in the lower and/or upper extremities. The underlying pathomechanism remains unclear and no molecular biomarkers to distinguish the disease exist, leading to a large number of undiagnosed and misdiagnosed patients. To unravel the distinct molecular characteristic of lipedema we performed lipidomic analysis of the adipose tissue and serum of lipedema versus anatomically- and body mass index (BMI)-matched control patients. Both tissue groups showed no significant changes regarding lipid composition. As hyperplastic adipose tissue represents low-grade inflammation, the potential systemic effects on circulating cytokines were evaluated in lipedema and control patients using the Multiplex immunoassay system. Interestingly, increased systemic levels of interleukin 11 (p = 0.03), interleukin 28A (p = 0.04) and interleukin 29 (p = 0.04) were observed. As cytokines can influence metabolic activity, the metabolic phenotype of the stromal vascular fraction was examined, revealing significantly increased mitochondrial respiration in lipedema. In conclusion, despite sharing a comparable lipid profile with healthy adipose tissue, lipedema is characterized by a distinct systemic cytokine profile and metabolic activity of the stromal vascular fraction.     

The plasma phospholipidome of Tursiops truncatus: From physiological insight to the design of prospective tools for managed cetacean monitorization

Plasma biochemical analysis remains one of the established ways of monitoring captive marine mammal health. More recently, complementary plasma lipidomic analysis has proven to be a valid tool in disease diagnosis and prevention, with the potential to validate and complement common biochemical analysis, providing a more integrative approach. In this study, we thoroughly characterized the plasma polar lipid content of Tursiops truncatus, the most common cetacean species held under human care. Our results showed that phosphatidylcholine, lysophosphatidylcholine, and sphingomyelins (CerPCho) are the most represented phospholipid classes in T. truncatus plasma. Palmitic, oleic, and stearic acids are the major fatty acid (FA) present esterified to the plasma polar lipids of this species, although some n-3 species are also remarkably present, namely eicosapentaenoic and docosahexaenoic acids. The polar lipidome identified by HILIC LC–MS allowed identifying 304 different lipid species. These species belong to the phosphatidylcholine (103 lipid species), lysophosphatidylcholine (35), phosphatidylethanolamine (71), lysophosphatidylethanolamine (20), phosphatidylglycerol (13), lysophosphatidylglycerol (5), phosphatidylinositol (15), lysophosphatidylinositol (3), phosphatidylserine (6) lysophosphatidylserine (1), and sphimgomyelin (32) classes. This was the first time that the dolphin plasma phospholipid profile was characterized, providing a knowledge that will be important to further understand lipid metabolism and physiological regulation in small cetaceans. Furthermore, this study proved the practicability of the use of plasma lipid profiling for health assessment in marine mammals under human care.     

Lipidome of Atherosclerotic Plaques from Hypercholesterolemic Rabbits

The cellular, macromolecular and neutral lipid composition of the atherosclerotic plaque has been extensively characterized. However, a comprehensive lipidomic analysis of the major lipid classes within atherosclerotic lesions has not been reported. The objective of this study was to produce a detailed framework of the lipids that comprise the atherosclerotic lesion of a widely used pre-clinical model of plaque progression. Male New Zealand White rabbits were administered regular chow supplemented with 0.5% cholesterol (HC) for 12 weeks to induce hypercholesterolemia and atherosclerosis. Our lipidomic analyses of plaques isolated from rabbits fed the HC diet, using ultra-performance liquid chromatography (UPLC) and high-resolution mass spectrometry, detected most of the major lipid classes including: Cholesteryl esters, triacylglycerols, phosphatidylcholines, sphingomyelins, diacylglycerols, fatty acids, phosphatidylserines, lysophosphatidylcholines, ceramides, phosphatidylglycerols, phosphatidylinositols and phosphatidylethanolamines. Given that cholesteryl esters, triacylglycerols and phosphatidylcholines comprise greater than 75% of total plasma lipids, we directed particular attention towards the qualitative and quantitative assessment of the fatty acid composition of these lipids. We additionally found that sphingomyelins were relatively abundant lipid class within lesions, and compared the abundance of sphingomyelins to their precursor phosphatidylcholines. The studies presented here are the first approach to a comprehensive characterization of the atherosclerotic plaque lipidome.     

Lipidomic Profiling of Chylomicron Triacylglycerols in Response to High Fat Meals

Using lipidomic methodologies the impact that meal lipid composition and metabolic syndrome (MetS) exerts on the postprandial chylomicron triacylglycerol (TAG) response was examined. Males (9 control; 11 MetS) participated in a randomised crossover trial ingesting two high fat breakfast meals composed of either dairy-based foods or vegetable oil-based foods. The postprandial lipidomic molecular composition of the TAG in the chylomicron-rich (CM) fraction was analysed with tandem mass spectrometry coupled with liquid chromatography to profile CM TAG species and targeted TAG regioisomers. Postprandial CM TAG concentrations were significantly lower after the dairy-based foods compared with the vegetable oil-based foods for both control and MetS subjects. The CM TAG response to the ingested meals involved both significant and differential depletion of TAG species containing shorter- and medium-chain fatty acids (FA) and enrichment of TAG molecular species containing C16 and C18 saturated, monounsaturated and diunsaturated FA. Furthermore, there were significant changes in the TAG species between the food TAG and CM TAG and between the 3- and 5-h postprandial samples for the CM TAG regioisomers. Unexpectedly, the postprandial CM TAG concentration and CM TAG lipidomic responses did not differ between the control and MetS subjects. Lipidomic analysing of CM TAG molecular species revealed dynamic changes in the molecular species of CM TAG during the postprandial phase suggesting either preferential CM TAG species formation and/or clearance.     

A new analytical method to monitor lipid peroxidation during bleaching

Whereas solid phase microextraction (SPME) combined with gas chromatography is a wide‐spread technique in certain fields of food analysis this technique is quite new for the analysis of vegetable oils. The method is sensitive enough to follow changes in the oxidative state of vegetable oils by measuring the amount of volatile materials produced during storage and the refining process. In the present study degummed rapeseed oil was bleached using different activated bleaching earths applied in four dosages. Their effect on lipid degradation was determined both by traditional methods (e.g. UV absorbance, p‐anisidine value) and by the SPME‐HS method. Although the p‐anisidine value (p‐AV) gives only the concentration of β‐unsaturated aldehydes it correlates well to the amount of total volatile substances as determined by SPME at the headspace of the sample. The extracted volatile materials were separated and identified by gas chromatography combined with mass spectrometry. SPME gives more information about the stage of oxidation and the applied bleaching earth by quantifying the volatile compounds. Additionally SPME does not require any toxic reagent such as p‐methoxy aniline which is used to determine the p‐AV. Although bleaching is very important it was disregarded in recent years. Therefore one of the aims of the present study is to draw back more attention towards bleaching.