Generic placeholder image

Current Pharmaceutical Analysis

Editor-in-Chief

ISSN (Print): 1573-4129
ISSN (Online): 1875-676X

Research Article

A Validation and Estimation of Total Eicosapentaenoic and Docosahexaenoic acids Using LC-MS/MS with Rapid Hydrolysis Enzymatic Method for Hydrolysis of Omega Lipids in Human Plasma and its Application in the Pharmacokinetic Study

Author(s): Sekarbabu Viswanathan*, Priya Ranjan Prasad Verma and Muniyandithevar Ganesan

Volume 15, Issue 2, 2019

Page: [172 - 193] Pages: 22

DOI: 10.2174/1573412914666180730094803

Price: $65

Abstract

Background: In this study, we have developed a novel, rapid enzymatic hydrolysis method for conversion of omega lipids (omega fatty acid triglycerides, phospholipids, omega conjugates) in to free fatty acids at room temperature using lipase and esterase enzymes.

Objective: To develop simple enzymatic hydrolysis and rapid sample extraction method for quantification of free (un-esterified) and conjugated (esterified) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) to provide the total EPA and DHA lipids present in human plasma. Quantification of total EPA/DHA was performed using liquid chromatography and tandem mass spectrometer instrument.

Methods: The plasma sample is digested with lipase and esterase enzymes and extracted by using combined precipitation and liquid-liquid techniques. The LC-MS/MS method was optimized using EPA-D5 and DHA-D5 as labeled internal standards for EPA/DHA respectively. The analytical method is validated, utilized for simultaneous quantification of total EPA and DHA lipids in plasma collected from healthy human volunteers clinical study.

Results: The reproducibility of the established enzymatic hydrolysis method was demonstrated by incurred sample reanalysis and the results for total EPA and DHA lipid were 93.33% and 96.67% respectively. The pharmacokinetic and statistical analysis was performed using baseline corrected concentration of total EPA and DHA lipids.

Conclusion: The enzymatic hydrolysis method for conversion of omega fatty acid triglycerides, phospholipids, omega conjugates in to free fatty acid was reported first time for the quantitative application. The shorter time for sample workup procedure, simple enzymatic hydrolysis at room temperature and 3 minutes chromatography run time are well suitable for bioavailability/ bioequivalence studies.

Keywords: Eicosapentaenoic acid, Docosahexaenoic acid, Omega-3-fatty acids, LC-MS/MS, EPA, DHA, Lipase & Esterase.

Graphical Abstract
[1]
Wall, R.; Ross, R.P.; Fitzgerald, G.F.; Stanton, C. Fatty acids from fish: the anti-inflammatory potential of long-chain omega-3 fatty acids. Nutr. Rev., 2010, 68(5), 280-289.
[2]
Kapoor, R.; Patil, U.K. MiniReview: Importance and production of omega-3 fatty acids from natural sources. IFRJ, 2011, 18, 493-499.
[3]
Sujatha, R. Health benefits of plant derived α-linolenic acid. Am. J. Clin. Nutr., 2014, 100(1), 443s-448s.
[4]
Elahe, A.; Sahari, M.A. Long-chain polyunsaturated fatty acid sources and evaluation of their nutritional and functional properties. Food Sci. Nutr., 2014, 2(5), 443-463.
[5]
Danielle, S.; Robert, B.; Mousa, S.A. Omega-3 Fatty Acids EPA and DHA: Health benefits throughout life. Am. Soc. Nutr., 2012, 3, 1-7.
[6]
UK MHRA public assessment report for omega-3-acid ethyl esters 1000 mg soft gelatin capsules. UK/H/4840/001/DC;PL00289/1615. http://www.mhra.gov.uk/home/groups/par/documents/websiteresources/con149815.pdf (Accessed on: September 07, 2016).
[7]
Hazra, A.; Tripathi, S.K.; Ghosh, A. Pharmacology and therapeutic potential of the n-3 polyunsaturated fatty acids, eicosapentaenoic acid EPA) and docosahexaenoic acid (DHA) in fish oils. Indian J. Pharmacol., 1999, 31, 247-264.
[8]
Beckermann, B.; Beneke, M.; Seitz, I. Comparative bioavailability of eicosapentaenoic acid and docosahexaenoic acid from triglycerides, free fatty acids and ethyl esters in volunteers. Arzneimittelforschung, 1990, 40, 700-704.
[9]
Ghasemifard, S.; Turchini, G.M.; Sinclair, A.J. Omega-3 long chain fatty acid “bioavailability” A review of evidence and methodological considerations. Prog. Lipid Res., 2014, 56, 92-108.
[10]
Hussey, E.K.; Portelli, S.; Fossler, M.J.; Gao, F.; Harris, W.S.; Blum, R.A.; Lates, C.D.; Gould, E.; Abu-Baker, O.; Johnson, S. Relative bioavailability of an emulsion formulation for omega-3-Acid ethyl esters compared to the commercially available formulation: a randomized, parallel-group, single-dose study followed by repeat dosing in healthy volunteers. Clin. Pharmacol. Drug Dev., 2012, 1, 14-23.
[11]
Australian Public Assessment Report for Omega-3-acid Ethyl Esters 90, proprietary name: Omacor, Abbott products Pty Ltd, published by Therapeutic Goods Administration (TGA). AUSPAR, https://www.tga.gov.au/sites/default/files/auspar-omacor.pdf (Accessed on: October, 2010).
[12]
Mancini Filho, J.; Melo, I.L.P.; Sattler, J.A.G.; Yoshime, L.T. Bioavailability and the bioequivalence of conjugated fatty acids. BEBA, 2017, 1(1), 000102.
[13]
Ikeda, I.; Sasaki, E.; Yasunami, H. Digestion and lymphatic transport of eicosapentaenoic and docosahexaenoic acids given in the form of triacylglycerol, free acid and ethyl ester in rats. BioChimica et Biophysica Acta, 1995, 1259, 297-304.
[14]
Small, D.M. The effects of glyceride structure on absorption and metabolism. Annu. Rev. Nutr., 1991, 11, 413-434.
[15]
El, Boustani. S.; Colette, C.; Monnier, L. Enteral absorption in man of eicosapentaenoic acid in different chemical forms. Lipids, 1987, 22, 711-714.
[16]
Saghir, M.; Werner, J.; Laposata, M. Rapid in vivo hydrolysis of fatty acid ethyl esters, toxic non-oxidative ethanol metabolites. Am. J. Physiol. Gastrointest. Liver Physiol., 1997, 273, 184-190.
[17]
Gloria, F.L.; Lorena, B.; Alfonso, V.C.; Jose, M.G. Release of omega-3 fatty acids by the hydrolysis of fish oil catalyzed by lipases immobilized on hydrophobic supports. J. Am. Oil Chem. Soc., 2011, 88, 1173-1178.
[18]
Wilson Galvao Morais Junior. Gloria, Fernandez-Lorente.; Jose Manuel Guisan.; Eloizio Julio Ribeiro.; Miriam Maria De Resende. Benevides Costa Pessela. Production of omega-3 polyunsaturated fatty acids through hydrolysis of fish oil by Candida rugosa lipase immobilized and stabilized on different supports. Biocatal. Biotransform., 2017, 35, 63-73.
[19]
Suzana, Ferreira-Dias Georgina, Sandoval.; Francisco, Plou.; Francisco, Valero. The potential use of lipases in the production of fatty acid derivatives for the food and nutraceutical industries. EJB, 2013, 16(3)
[20]
David, C. Concentration of omega-3 fatty acids using enzymes. Agro FOOD industry hi-tech,, Novozymes U.K. Ltd. (DC), HP5 2SG, United Kingdom, 2010, Vol. 21. No. 4.
[21]
Kapoor, R.; Patil, U.K. Mini review importance and production of omega-3 fatty acids from natural sources. IFRJ, 2011, 18, 493-499.
[22]
Halldorsson, A.; Kristinsson, B.; Haraldsson, G.G. Lipase selectivity toward fatty acids commonly found in fish oil. Eur. J. Lipid Sci. Technol., 2004, 106(2), 79-87.
[23]
Tim, Nalder.; Trent, D. Ashton.; Fred, Pfeffer.; Susan N, Marshall.; Colin J, Barrow. 4-Hydroxy-N-propyl-1, 8-naphthalimide esters: New fluorescence-based assay for analysing lipase and esterase activity. Biochimie, 2016, 128, 127-132.
[24]
Dołowy, M.; Pyka, A. Chromatographic methods in the separation of long-chain mono-and polyunsaturated fatty acids. J. Chem.,, Hindawi,. 2015, 1-20.
[25]
Loevenhart, A.S. Are the animal enzymes concerned in the hydrolysis of various esters identical? J. Biol. Chem., 2, 427-460.
[26]
Aslan, M.; Ozcan, F.; Aslan, I.; Yucel, G. LC-MS/MS analysis of plasma polyunsaturated fatty acids in type 2 diabetic patients after insulin analog initiation therapy. Lipids Health Dis., 2013, 12, 169.
[27]
Aslan, M.; Celmeli, G.; Ozcan, F.; Kupesiz, A. LC-MS/MS analysis of plasma polyunsaturated fatty acids in patients with homozygous sickle cell disease. Adv. Clin. Exp. Med., 2015, 15(3), 397-403.
[28]
Sajiki, J.; Yonekubo, J. Determination of free polyunsaturated fatty acids and their oxidative metabolites by high-performance liquid chromatography and mass spectrometry. Anal. Chim. Acta, 2002, 465, 417-426.
[29]
Braeckman, R.A.; Stirtan, W.G.; Soni, P.N. Pharmacokinetics of eicosapentaenoic acid in plasma and red blood cells after multiple oral dosing with icosapent ethyl in healthy subjects. Clin. Pharmacol. Drug Dev., 2014, 3, 101-108.
[30]
Yang, W.C.; Adamec, J.; Regnier, F.E. Enhancement of the LC/MS analysis of fatty acids through derivatization and stable isotope coding. Anal. Chem., 2007, 79(14), 5150.
[31]
Zehethofer, N.; Pinto, D.M.; Volmer, D.A. Plasma free fatty acid profiling in a fish oil human intervention study using ultra-performance liquid chromatography/electrospray ionization tandem mass spectrometry. Rapid Commun. Mass Spectrom., 2008, 22(13), 2125-2133.
[32]
Masoodi, M.; Mir, A.A.; Petasis, N.A.; Serhan, C.N.; Nicolaou, A. Simultaneous lipidomic analysis of three families of bioactive lipid mediators leukotrienes, resolvins, protectins and related hydroxy-fatty acids by liquid chromatography/electrospray ionisation tandem mass spectrometry. Rapid Commun. Mass Spectrom., 2008, 22(2), 75-83.
[33]
Chester, L. Bowen.; Jonathan, Kehler.; Christopher A, Evans. Development and validation of a sensitive and selective UHPLC-MS/MS method for simultaneous determination of both free and total eicosapentaeonic acid and docosahexenoic acid in human plasma. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci., 2010, 878, 3125-3133.
[34]
U.S.F.D.A, Guidelines. Draft Guidance on Omega-3-Acid Ethyl Esters, http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm320011.pdf (Accessed on: August 15, 2016).
[35]
Sekarbabu, V.; Verma, P.R.P.; Muniyandithevar, G.; Jeganathan, M. A novel liquid chromatography/tandem mass spectrometry (LC-MS/MS) based bio analytical method for quantification of ethyl esters of Eicosapentaenoic Acid (EPA) and Docosahexaenoic acid (DHA) and its application in pharmacokinetic study. J. Pharm. Biomed. Anal., 2017, 141, 250-261.
[36]
USFDA Guidelines, U.S.F.D.A. Bio analytical Method Validation: 2013 http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm368107.pdf (Accessed on: August 01, 2016).
[37]
Ghosh, P.K.; Saxena, R.K.; Gupta, R.; Yadav, R.P.; Davidson, S. Microbial lipases: Production and applications. Sci. Prog., 1996, 79, 119-157.
[38]
Iwai, W.; Mieko, J. Microbial lipases: Potential biocatalysts for the future industry. Yushi, 1989, 32, 84-91.
[39]
Bjorkling, F.; Godtfredsen, S.E.; Kirk, O. The future impact of industrial lipases. Trends Biotechnol., 1991, 9, 360-363.
[40]
Marvin, M.N.; Arnold, M.S. Evidence for the specificity of esterase and lipase by the use of three chromogenic substrates. J. Biol. Chem., 1949, 181, 343-355.
[41]
John, A.A.; David, A.P.; Frank, G.S. Activity of microbial lipases on natural fats and synthetic triglycerides. J. Lipid Res., 1964, 5(3), 390-394.
[42]
Chaurasiaa, S.P.; Kriti, B.; Aditi, S.; Ajay, K.D. A review on lipase catalysed synthesis of DHA rich glyceride from fish oils. IJRSI; III(IA), 2016, pp. 9-19.
[43]
Derya, K.; Mia, F.; Sandra, G.; Xuebing, X. Upgrading of farmed salmon oil through lipase-catalysed hydrolysis. Open Biotechnol. J., 2010, 4, 47-55.
[44]
Tushar, R.M.; Avinesh, R.B.; Munish, P.; Colin, B.; Nalam Madhusudhana, R. Selective enrichment of omega-3 fatty acids in oils by phospholipase A1. PLoS One, 2016.
[45]
Macrae, A.R.; Hammond, R.C. Present and future applications of lipases. Biotechnol. Genet. Eng. Rev., 1985, 3, 193-219.
[46]
Iwai, M.; Okumura, S.; Tsujisaka, Y. Synthesis of terpene alcohol esters by lipase. Agric. Biol. Chem., 1980, 44, 2731-2732.
[47]
Nishio, T.; Takanashi, K.; Yoshimoto, T.; Kodess, Y.; Satio, Y.; Inada, Y. Biotechnol. Lett., 1987, 9, 187-190.
[48]
Langrand, G.; Randot, N.; Triantaphylides, C. Baratii. J. Biotechnol. Lett., 1990, 12, 581-586.
[49]
Robert, G.J.; Felice, A.D.; Richard, M.C. Determination of lipase specificity. Lipids, 1983, 18(3), 239-252.
[50]
Thierry, R.; Cecilia, H.; Dominique, L. Fatty acid specificity of hormone-sensitive lipase: implication in the selective hydrolysis of triacylglycerols. J. Lipid Res., 2001, 42, 2049-2057.
[51]
Danielle, B.L.; Laira, P.F.; Luciana, F.F.; Gabriela, A.M. Lipase and esterase - to what extent can this classification be applied accurately. Ciência e Tecnologia de Alimentos, 2011, 31(3), 608-613.
[52]
Susan, N.M.; Fred, P.; Colin, J.B. Characterization of lipase fatty acid selectivity using novel omega-3 pNP-acyl esters: Omega-3 selectivity of some common lipases is determined using these pNP esters. J. Funct. Foods, 2014, 6(1), 259-269.
[53]
Lawson, L.D.; Hughes, B.G. Human absorption of fish oil fatty acids as triacylglycerols, free acids, or ethyl esters. Biochem. Biophys. Res. Commun., 1988, 152(1), 328-335.
[54]
Lawson, L.D.; Hughes, B.G. Absorption of eicosapentaenoic acid and docosahexaenoic acid from docosa oil triacylglycerols or fish oil ethylesters co-ingested with a high-fat meal. Biochem. Biophys. Res. Commun., 1988, 156, 960-963.
[55]
Hansen, J.B.; Grimsgaard, S.; Nilsen, H. Effects of highly purified eicosapentaenoic acid and docosahexaenoic acid on fatty acid absorption, incorporation into serum phospholipids and postprandial triglyceridemia. Lipids, 1998, 33, 131-138.
[56]
Valianpour, F.; Selhorst, J.J.; Van, L.L.E.; Van Gennip, A.H.; Wanders, R.J.; Kemp, S. Analysis of very long-chain fatty acids using electrospray ionization mass spectrometry. Mol. Genet. Metab. Rep., 2003, 79(3), 189-196.
[57]
Lagerstedt, S.A.; Hinrichs, D.R.; Batt, S.M.; Magera, M.J.; Rinaldo, P.; McConnell, J.P. Mol. Genet. Metab. Rep., 2001, 73(1), 38-45.
[58]
Galli, C.; Maggi, F.M.; Rise, P.; Sirtori, C.R. Bioequivalence of two omega-3-fatty acid ethyl ester formulations: a case of clinical pharmacology of dietary supplements. Br. J. Clin. Pharmacol., 2012, 74(1), 60-65.
[59]
Michael, H.D.; Judith, J.; Michael, W.R.; Michael, L.K.; Douglas, F. K. A novel omega-3 free fatty acid formulation has dramatically improved bioavailability during a low-fat diet compared with omega-3-acid ethyl esters: The eclipse (Epanova compared to Lovaza in a pharmacokinetic single-dose evaluation) study. J. Clin. Lipidol., 2012, 6, 573-584.

Rights & Permissions Print Cite
© 2024 Bentham Science Publishers | Privacy Policy