Determination of Drugs of Abuse in Hair by LC-MS-MS: Application to Suicide Attempts Investigation.

Considering that the use of psychoactive substances (PSs) is a risk factor to either higher intensity or frequency of suicidal behavior, hair analysis was conducted to investigate the most consumed PSs (opiates, amphetamine stimulants, marijuana, cocaine and heroin) in patients who attempted suicide and received urgent care at emergency service. Hair samples were extracted using methanol and sonicated under heating and then analyzed using liquid chromatography-tandem mass spectrometry. During validation, the method complied with international recommended criteria, with limits of detection between 0.0025 and 0.05 ng/mg and linearity between 0.1 and 4 ng/mg for methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), morphine, amphetamine, 6-acetylmorphine, 3,4-methylenedioxyamphetamine (MDA), fenproporex, diethylpropion and codeine; between 0.025 and 1 ng/mg for tetrahydrocannabinol (THC), benzoylecgonine and cocaethylene and between 0.25 and 10 ng/mg for cocaine and mazindol. A total of 109 hair samples were analyzed and segmented in 404 parts. Among all analyzed samples, 30.3% were positive for at least one PS (n = 33), such as cocaine (90.9%), codeine (12.1%), morphine (3.0%), MDMA (3.0%) and THC (3.0%). In segmental analysis of cocaine positive samples (n = 30), 76.7% of the samples indicated recent exposure to cocaine (<1 month). This same behavior was observed when analyzing codeine (n = 4) and morphine (n = 1). THC positive samples indicated exposure dated ∼4 months prior. In conclusion, the method was validated following international recommendations for the 12 most consumed PSs in Brazil, as well as two of the most common found metabolites.

Rapid and direct detection of artificially aged papers employing easy ambient sonic-spray ionization mass spectrometry.

RATIONALE: The effort to make fake documents look real leads to the use of crickets and beverages to produce artificially aged papers, as land titles, based on yellowing caused by the use of these methods. An old practice in Brazil, called "cricketing", has led to the misappropriation of Brazilian land using these documents. We propose a rapid, simple, instantaneous and non-destructive method to identify artificially aged papers by easy ambient sonic-spray ionization mass spectrometry (EASI-MS) analysis. METHODS: Three typical aging procedures were used to obtain artificially aged papers using coffee, cola drink, and crickets, with the papers being analyzed by EASI-MS. Multivariate statistical analyses were performed on the data to find the sample groups and to study the most relevant ions of each ageing procedure. High-resolution MS (HRMS) was used to obtain the exact masses and attribute formulae to relevant ions present in the samples. RESULTS: The combination of EASI-MS and multivariate statistical analyses allowed us to identify the most relevant ions to classify the adulteration of documents and HRMS identified most of these relevant ions. TMS fingerprinting in combination with multivariate analysis also demonstrated that this approach can qualitatively differentiate all the examined paper samples. CONCLUSIONS: We developed a cheap, fast and easy method that can help to elucidate counterfeit documents that have been artificially aged, helping to identify chemical additives and one that can be used in forensic laboratories.

Development and Validation of a Method for Quantification of 28 Psychotropic Drugs in Postmortem Blood Samples by Modified Micro-QuEChERS and LC-MS-MS.

The development of new sample preparation alternatives in analytical toxicology leading to quick, effective, automated and environmentally friendly procedures is growing in importance. One of these alternatives is the QuEChERS, originally developed for the analysis of pesticide residues, producing cleaner extracts than liquid-liquid extraction, and easier separation of aqueous and organic phases. However, there are few published studies on the miniaturization of this technique for forensic toxicology, especially in postmortem analysis. We developed and validated a modified micro-QuEChERS and LC-MS-MS assay to quantify 16 antidepressants, 7 antipsychotics and 3 metabolites and semi-quantify norfluoxetine and norsertraline in postmortem blood. The calibration curve was linear from 1 to 500 ng/mL, achieved an r > 0.99, with all standards quantifying within ±15% of target except ±20% at the limit of quantification of 1 ng/mL for 26 substances. The F test was applied to evaluate if the variance between replicates remained constant for all calibrators. Six weighting factors were analyzed (1/x, 1/x2, 1/x0,5, 1/y, 1/y2 and 1/y0,5), with the weighting factor with the lowest sum of residual regression errors (1/x2) selected. No endogenous or exogenous interferences were observed. Method imprecision and bias were <19.0% and 19.7%, respectively. Advantages of this method include a low sample volume of 100 µL, simple but effective sample preparation and a rapid 8.5-min run time. The validated analytical method was successfully applied to the analysis of 100 authentic postmortem samples.

LC-MS/MS screening and identification of bioactive compounds in leaves, pulp and seed from Eugenia calycina Cambess.

The Eugenia calycina Cambess, also known as pitanga-do-cerrado, is an unexplored Brazilian fruit used by native people for the treatment of diabetes. The aim of this study was to identify the phenolic compounds from the leaves, pulp and seed of Eugenia calycina by using LC-MS-based targeted and untargeted analysis. The LC-MS-based targeted quantitative analysis showed a high phenolic content in all plant parts in which the ellagic acid was the main phenolic compound with values of 8244.53 µg/g dw (leaves), 5054.43 µg/g dw (pulp) and 715.42 µg/g dw (seed). The leaves, pulp and seed showed a high total phenolic content of 20371.96, 7139.70 and 2204.75 µg/g dw, respectively. In addition, the LC-MS-based targeted analysis showed ellagic acid, myricitrin and epicatechin gallate as the main phenolic compounds in the Eugenia calycina. The LC-MS-based untargeted analysis showed the phytochemical profile of the leaves, pulp and seed in which 153 phytochemicals from different chemical classes were annotated, including organic acids, phenolic acids, flavonoids, and other compounds. The Eugenia calycina has high potential as a plant-based food due to its phytochemical profile and high phenolic content.

Triple quadrupole-mass spectrometry protocols for the analysis of NBOMes and NBOHs in blotter papers.

NBOMe and NBOH are new psychoactive substances with potent activity on serotonin 5-HT2a receptors causing serious toxic effects, including serotonin toxidrome and death. The aim of this work was to develop a comprehensive MS/MS protocol, using triple quadrupole mass spectrometers coupled to LC and GC, for rapid screening and quantitation of NBOMes and NBOHs in seized blotter papers. Different scan methods (neutral loss, precursor ion or multiple reaction monitoring) were used to obtain structural information of phenylethylamine class. The developed protocol was validated for qualitative and quantitative analysis, showing a satisfactory limit of detection (1 ng/mL), with excellent selectivity, imprecision (intra and interday imprecision lower than 1.2 % RSD) and accuracy (between -7.1 and +5.6 %, n = 15), as well as bias values. The analysis of real samples shown that NBOH compounds were the most frequently detected, with concentrations ranging from 0.1 to 1,929 µg per blotter sample. Triple quadrupole mass spectrometers can be a useful tool for identification of new psychoactive substances. A comprehensive protocol using both LC-MS/MS and GC-MS/MS, with different scanning modes, have been developed and showed to be useful to screening NBOMe and NBOH in blotter papers.

Amazon climatic factors driving terpene composition of Iryanthera polyneura Ducke in terra-firme forest: A statistical approach.

Iryanthera polyneura (Myristicaceae) is popularly known as cumala-colorada, and can be found in the Amazon rain forest. The present study aimed the evaluation of the chemical composition of the essential oils (EOs) obtained from the leaves of I. polyneura throughout a two-year period in order to correlate chemical findings with seasonality and climatic variation. Leaves from I. polyneura were collected 15 times from three different individuals, identified as 22EO, 80EO and 530EO, between the years of 2009 and 2011. The EOs were obtained and tested by gas-chromatography-mass spectrometry techniques. ANOVA and multivariate analyses were used to assess the relationship between the percentage of terpenes and seasonal/climatic variations. Fifty-nine compounds were detected in the EOs, of which 44 were identified (74.5%). The main components of the EOs were spathulenol (6.42 ± 1.02%), α-cadinol (5.82 ± 0.40%) and τ-muurolol (5.24 ± 0.03%). Higher levels of spathulenol were observed in 22EO during rainy season, while τ-muurolol occurred in high amounts during the dry season, as did α-cadinol in 22EO and 80EO. Correspondence analysis revealed a distinction between two groups of EOs based on seasonality, whereas a canonic correspondence analysis and ANOVA determined how the major compounds were related to both seasonality and climatic factors. Non-metric multidimensional scaling in association with an analysis of similarities showed that the abundance and composition of terpenes in the samples varied moderately among the three Iryanthera individual plants. Present findings have shown that there is variation in the occurrence of the major compounds spathulenol, τ-muurolol and α-cadinol produced by I. polyneura and that the pattern of variation is related to season and climatic changes.

Interference of Seasonal Variation on the Antimicrobial and Cytotoxic Activities of the Essential Oils from the Leaves of Iryanthera polyneura in the Amazon Rain Forest.

The essential oils (EOs) obtained from the leaves of Iryanthera polyneura Ducke trees was chemically Assessed and tested for the ability of inhibiting the growth of Candida albicans, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus mutans and S. sanguinis. The oil was also tested against breast (MCF-7) and prostate (PC-3) cancer cell lines. Minimum bactericidal concentrations (MBCs) and 50 % inhibition concentrations (IC50 ) values were obtained. EOs were active against Gram-positive bacteria. Spathulenol, α-cadinol and τ-muurolol were major components of EOs. The oils showed a higher cytotoxicity against PC-3 than MCF-7 cells, although the oils were active against both cell types. Oils obtained from leaves collected in the dry season were more active against E. faecalis, S. aureus and PC-3, while the oils obtained from leaves collected in the rainy season were more active against S. mutans, S. sanguinis and MCF-7. The antibacterial and cytotoxic activities of the essential oils from the leaves of I. polyneura are related to the seasonal climate variation and are influenced by compounds that are minor components of the oils.

Characterization of the lipid profile from coconut (Cocos nucifera L.) oil of different varieties by electrospray ionization mass spectrometry associated with principal component analysis and independent component analysis.

Coconut oil (CO) from fifteen different varieties of coconuts (Cocos nucifera L.) and one CO processed on an industrial scale were analyzed by electrospray ionization mass spectrometry (ESI-MS) and the data processed using the chemometric tools principal component analysis and independent component analysis. ESI-MS fingerprinting of lipid compounds showed predominance of diacylglycerols and triacylglycerols, as confirmed by high-resolution MS measurements. Chemometric processing of the ESI-MS data differentiated the coconut oil samples, showing that different coconut varieties/cultivars produce oils with distinguishable abundances of lipidic compounds. Thus ESI-MS analysis followed by data treatment using chemometric tools offers a tool able to classify the industrial coconut oils in a fast, simple and effective way, as well as serving as a potential method to identify the coconut varieties by the CO origin, and the occurrence of any adulteration. The procedure may also be applied for quality control of the industrial processes.

A single administration of fish oil inhibits the acute inflammatory response in rats.

OBJECTIVE: To investigate the antiinflammatory effects of a single administration of fish oil (FO) on the acute inflammatory response. METHODS: The paw edema and pleurisy models were used to evaluate the effects of FO dissolved in olive oil (FOP) orally administered in a single dose in rats. Nitric oxide (NO) concentrations in the pleural exudate were performed according to the Griess method and the cytokine concentrations were determined by Luminex bead-based multiplex assay. RESULTS: FOP treatment (30 and 300 mg/kg) significantly reduced paw edema. FOP treatment at 18.75, 37.5, 75.0, 150.0, and 300 mg/kg decreased both the volume of pleural exudate and cellular migration into the pleural cavity and each of these doses presented the same effectiveness. Treatment with FOP (300 mg/kg) reduced NO, TNF-α, IL-1ß, and IL-6 concentrations in the pleural exudate. CONCLUSIONS: The present data provide evidence that FO has inhibitory effects on the acute inflammatory response when administered in a single dose in rats. This effect might be attributable to a direct inhibitory effect of FO on the production or release of inflammatory mediators that are involved in the pathological processes evaluated herein.

EASI-IMS an expedite and secure technique to screen for 25I-NBOH in blotter papers.

The increasing number of new psychoactive substances (NPS) and their quick worldwide spreading, often only slightly modified in the form of new derivatives and analogues, have brought the need for fast, wide-ranging, and unequivocal identification methods in clinical and forensic investigations. Because it usually provides secure results, gas chromatography coupled to mass spectrometry (GC-MS) has been routinely employed as the standard technique for the detection of NPS in blotter papers. For 25I-NBOH (N-(2-hydroxybenzyl)-2-(4-iodo-2,5-dimethoxyphenyl)ethan-1-aminium), however, GC-MS analysis of an blotter paper extract leads to incorrect results. In this work, we investigated whether easy ambient sonic-spray mass spectrometry imaging (EASI-IMS), and ambient ionization MS method can be applied directly to the surface of the sample requiring therefore no extraction or sample preparations, would serve as an efficient, sensitive, and secure alternative for 25I-NBOH screening.

Forensic Application of X-ray Fluorescence Spectroscopy for the Discrimination of Authentic and Counterfeit Revenue Stamps.

Energy-dispersive X-ray fluorescence (ED-XRF) spectroscopy with data treatment via chemometric tools was explored as an analytical protocol to discriminate between authentic and counterfeit revenue stamps. Untreated samples were directly analyzed, and the discrimination was based on the characterization of constituent elements present in the inks and paper. Authentic samples and samples that were suspected of being counterfeit were analyzed at three different areas on their surfaces: the ink-printed area, the non-printed area, and the holographic area. Principal component analysis (PCA) was applied to the data to discriminate between authentic and counterfeit revenue stamps. Major differences in the elemental composition were noted (according to chemometrics and t-test, p < 0.05), and ED-XRF spectroscopy plus PCA protocol is proposed for use by non-specialist operators to screen for counterfeit stamps.

A dopant for improved sensitivity in easy ambient sonic-spray ionization mass spectrometry.

Recently, 3-nitrobenzonitrile (3-NBN) has been used to improve sensitivity of sonic-spray ionization mass spectrometry. Easy ambient sonic-spray ionization (EASI) is one of the simplest, gentlest and most used spray-based desorption/ionization ambient techniques, but limited sensitivity has been commonly taken as its major drawback. Herein we investigate the use of 3-NBN as a dopant in EASI-MS for improved sensitivity. Using a few typical EASI samples as test cases, the presence of 10 ppm (µg ml(-1) ) of 3-NBN in the spray solvent showed two to fourfold gains in EASI-MS sensitivity as measured both by total ion current and S/N ratios, accompanied with significant reductions in chemical noise. Sensitivity for DESI using 3-NBN as a dopant also improved and dopant DESI versus dopant EASI sensitivities were compared. The use of solvent dopants seems therefore to be a promising strategy to improve sensitivity for spray-based ambient MS techniques. Copyright © 2015 John Wiley & Sons, Ltd.

Purple carrot extract protects against cadmium intoxication in multiple organs of rats: Genotoxicity, oxidative stress and tissue morphology analyses.

The aim of this study was to investigate if purple carrot extract is able to protect against the noxious activities induced by cadmium exposure in multiple organs of rats. For this purpose, histopathological analysis, genotoxicity and oxidative status were investigated in this setting. A total of twenty Wistar rats weighing 250g on the average, and 8 weeks age were distributed into four groups (n=5), as follows: Control group (non-treated group, CTRL); Cadmium group (Cd) and Purple carrot extract groups at 400mg/L or 800mg/L. Histopathological analysis revealed that liver from animals treated with purple carrot extract improved tissue degeneration induced by cadmium intoxication. Genetic damage was reduced in blood and hepatocytes as depicted by comet and micronucleus assays in animals treated with purple carrot extract. SOD-CuZn and cytocrome C gene expression increased in groups treated with purple carrot extract. Purple carrot extract also reduced the 8OHdG levels in liver cells when compared to cadmium group. Taken together, our results demonstrate that purple carrot extract is able to protect against cadmium intoxication by means of reducing tissue regeneration, genotoxicity and oxidative stress in multiple organs of Wistar rats.

Subcritical extraction of flaxseed oil with n-propane: Composition and purity.

Flaxseed (Linum usitatissimum L.) oil was obtained via subcritical n-propane fluid extraction (SubFE) under different temperatures and pressures with an average yield of 28% and its composition, purity and oxidative stability were compared to oils obtained via conventional solvent extraction methods (SEMs). When the oxidative stability was measured by differential scanning calorimetry, the oil was found to be up to 5 times more resistant to lipid oxidation as compared to the SEM oils. Direct infusion electrospray ionization mass spectrometry (ESI-MS) analysis showed characteristic and similar TAG profiles for SubFE and SEMs oils but higher purity for the SubFE oil. The flaxseed oil content of ß-tocopherol, campesterol, stigmasterol and sitosterol were quantified via GC-MS. SubFE showed to be a promising alternative to conventional SEM since SubFE provides an oil with higher purity and higher oxidation stability and with comparable levels of biologically active components.

Novel natural food colourant G8000 benefits LDL- and HDL-cholesterol in humans.

The aim of this study was to investigate the phenolic composition of a natural food colourant (G8000™) as well as its effects on plasma markers after 28-day consumption by healthy individuals at a dietary dose (70 g). Parameters of total cholesterol and its fractions, triglycerides and plasma enzymes biomarkers of muscle injury were measured. Major compounds identified in G8000™ by ESI-MS showed the presence of anthocyanins, organic acids, phenolic acids as well as monosaccharides. HDL levels significantly increased from 43 ± 10.2 mg/dL to 95 ± 16.9 mg/dL. LDL levels significantly decreased from 110 ± 40.9 mg/dL to 69 ± 39 mg/dL (p < 0.001). No significant statistical differences (p > 0.05) were observed for total cholesterol, triglycerides and VLDL. After the intake, plasma enzyme CK-MB decreased from 20 ± 12.1 U/L to 10 ± 1.9 U/L while LDH levels increased from 275 ± 124.4 U/L to 317 ± 114.7 U/L (p < 0.005). No significant differences were observed for CK levels. Taken together, dietary intake of natural colourant G8000™ was able to exert beneficial effects on atherosclerosis biomarkers.

Characterization of ANFO explosive by high accuracy ESI(±)-FTMS with forensic identification on real samples by EASI(-)-MS.

Ammonium nitrate fuel oil (ANFO) is an explosive used in many civil applications. In Brazil, ANFO has unfortunately also been used in criminal attacks, mainly in automated teller machine (ATM) explosions. In this paper, we describe a detailed characterization of the ANFO composition and its two main constituents (diesel and a nitrate explosive) using high resolution and accuracy mass spectrometry performed on an FT-ICR-mass spectrometer with electrospray ionization (ESI(±)-FTMS) in both the positive and negative ion modes. Via ESI(-)-MS, an ion marker for ANFO was characterized. Using a direct and simple ambient desorption/ionization technique, i.e., easy ambient sonic-spray ionization mass spectrometry (EASI-MS), in a simpler, lower accuracy but robust single quadrupole mass spectrometer, the ANFO ion marker was directly detected from the surface of banknotes collected from ATM explosion theft.

Composição e estabilidade lipídica da farinha de espinhaço de tilapia / Composition and lipid stability of tilapia fishbone flour

Espinhaços de tilápias (Oreochromis niloticus) são partes do peixe de composição desconhecida. A composição lipídica dos espinhaços não é citada na literatura, bem como a estabilidade da farinha do espinhaço durante o armazenamento. Nesse sentido, realizou se estudo de processamento dos espinhaços envolvendo etapas de cocção, trituração, secagem, peneiramento e armazenamento da farinha. A farinha ficou armazenada sob refrigeração por um período de 90 dias, sendo sua qualidade monitorada por meio da composição em ácidos graxos, índice de acidez e análises microbiológicas. Os resultados da composição centesimal foram de 14,2 por cento (umidade), 40,8 por cento (proteína), 18,3 por cento (resíduo mineral fixo) e 25,3 por cento de lipídios totais. Nos lipídios totais foi identificado um total de 24 ácidos graxos, com predominância dos ácidos graxos (porcentagem média) de 27.4 por cento (ácido palmítico, 16:0), 35,15 por cento (ácido oléico, 18:1n-9) e 11,82 por cento (ácido linoléico, 18:2n-6) e, em menor proporção: 0,88 por cento (ácido alfa-linolênico, 18:3n-3), 0,08 por cento (ácido eicosapentaenóico, 20:5n-3) e 0,59 (ácido docosahexaenóico, 22:6n-3). Durante os 90 dias de armazenamento, foram observadas algumas alterações no índice de acidez e composição de alguns ácidos graxos, no entanto, para 60 dias de armazenamento, não foram observadas alterações na composição de nenhum ácido graxo, do índice de acidez e nas análises microbiológicas.

The composition of the tilapia (Oreochromis niloticus) fishbone is unknown. Lipid composition fishbone is not cited in the literature, and neither is the stability of the flour of the fishbone during storage. We studied the processing of fishbone cooking, grinding, drying, sieving and the storage of the flour. The flour was stored in a refrigerator for a period of 90 days, and its quality was monitored through fatty acid composition, acid index and microbiology control. The results of the proximate composition were of 14.2 percent (moisture), 40.8 percent (protein), 18.3 percent (ash), and 25.3 percent total lipids. In the total lipids identified 24 fatty acids were identified, with predominance of the fatty acids (medium percentage) of 27.4 percent (palmitic acid, 16:0), 35.15 percent (oleic acmid, 18:1n-9) and 11.82 percent (linoleic acid , 18:2n-6) and, in smaller proportion: 0.88 percent (alpha-linolenic acid, 18:3n-3), 0.08 percent (eicosapentaenoic acid, 20:5n-3) and 0.59 (docosahexaenoic acid, 22:6n-3). During the 90 days of storage some alterations were observed in the acid index and composition of some fatty acids, however, for 60 days of storage no alterations were observed in the fatty acids composition, acid index, and microbiology control.

Evaluation of lipid extraction and fatty acid composition of human plasma

AIM: The objective of this study was to compare the efficiency of lipid extraction and to evaluate the fatty-acid composition in total lipids from human plasma, using a new technique and the established Folch, Lees and Stanley (FLS), Bligh and Dyer (BD), Rose-Gottlieb (RG), and Gerber (GM) methods. METHOD: A new technique, the alternative method, to extract total lipids using a microwave was proposed and evaluated. RESULTS: The total lipids extracted from human plasma varied between 0.19 percent and 0.41 percent; the highest total lipid extracted were obtained by the Folch, Lees and Stanley (0.41 percent), alternative method (0.37 percent) and Rose-Gottlieb (0.36 percent) methods. The Gerber method was ineffective to extract total lipids from human plasma. A total of 24 fatty-acid species were quantified by gas chromatography. Of the methods studied, the highest concentrations were found using the Folch, Lees and Stanley method (p < 0.05). CONCLUSION: The alternative method is a fast lipid-extraction technique that can be used for the identification of human plasma fatty acids, but it is not suitable for their measurement.

OBJETIVO: O objetivo desse estudo foi comparar a eficiência da extração lipídica e avaliar a composição em ácidos graxos nos lipídios totais (LT) no plasma humano usando uma nova técnica e os métodos já conhecidos: Folch, Lees e Stanley (FLS), Bligh e Dyer (BD), Rose-Gottlieb (RG) e Gerber (MG). MÉTODO: A nova técnica para extração de lipídios totais utiza o micro-ondas, método Alternativo (MA). RESULTADOS: Os lipídios totais (LT) extraídos do plasma variaram de 0,19 por cento a 0,41 por cento, os maiores teores foram obtidos por FLS (0,41 por cento), MA (0,37 por cento) e RG (0,36 por cento). O MG foi ineficaz na extração dos lipídios totais do plasma. Um total de 24 espécies de ácidos graxos foi quantificado no plasma por cromatografia em fase gasosa. Entre os métodos estudados, as maiores concentrações de ácidos graxos foram obtidas usando-se o método de FLS (p< 0,05). CONCLUSÃO: O método Alternativo foi considerado uma técnica rápida de extração lipídica, a qual poderá ser utilizada somente na identificação de ácidos graxos em plasma humano, mas não adequado para a quantificação dos mesmos.

[Different lipid extraction methods on fatty acids composition in cow milk]. / Avaliação de diferentes métodos de extração lipídica sobre a composição de ácidos graxos poliinsaturados em leite de vaca.

Different lipid extraction methods on fatty acids composition in cow milk. Comparative studies among 4 (four) different methods of total lipids extraction were carried out to evaluate the lipid extraction efficiency and fatty acids contents in cow milk. Total lipids extraction methods were Bligh e Dyer (BD), Folch et al. (FLS), Roese-Gottlieb (RG) and Gerber (GB). There were non-significant (p < 0.05) difference, in total lipids content among the extraction methods. The smallest concentrations of omega-3 (n-3) fatty acids and polyunsaturated fatty acids (PUFA) were observed on method GE, possibly due degradation of PUFA immersed in sulfuric acid used during analysis of total lipids. The highest concentrations of n-3 PUFA were observed by BD method, especially to omega-3/omega-6 ratio and alpha-linolenic acid (LNA, 18:3n-3), eicosapentaenoic (EPA, 20:5n-3) and docosahexaenoic (DHA, 22:6n-3), significant differences were observed among the methods. The results demonstrate that the different extractions influenced decisively on quantitative fatty acids composition and evaluations indicated the methods BD as better and GE as the worst to polyunsaturated fatty acids determination.