Preparation of Nitrogen-Containing Derivatives for Mass Spectrometry of Fatty Acids

Transcription

1 Preprtion of derivtives for mss spectrometry of ftty cids FATTY ACIDS AD MASS SPECTRMETRY Preprtion of itrogen-contining Derivtives for Mss Spectrometry of Ftty Acids The methodology, including prcticl detils, is given here for preprtion of the nitrogencontining derivtives of ftty cids, i.e. 3-pyridylcrbinol ('picolinyl') esters, 4,4-dimethyloxzoline (DMX) derivtives nd pyrrolidides, which re most useful for mss spectrometric nlysis. Preprtion of methyl ester derivtives is described on seprte webpge. ther useful derivtiztion techniques, e.g. hydrogention, deutertion, etc., re described in the section of these pges deling with Mss spectrometry of methyl esters further derivtiztion. I must lso recommend my book [1]. 1. Hydrolysis For some derivtives, it cn be better to prepre the free cids first by hydrolysis of the lipids with dilute queous ethnolic lkli s described here [1]. Method: A solution of 0.1M potssium hydroxide in 90% queous ethnol is prepred immeditely before use by dding 1 volume of stndrd 1M queous potssium hydroxide to 9 volumes of ethnol. To the lipid smple (up to 6 mg) in stoppered centrifuge tube is dded 0.1M potssium hydroxide in 90% queous ethnol (0.25 ml per mg smple). Leve t 50 C in wter bth or heting block for 3 hours. Remove from wter bth nd dd 2M hydrochloric cid (0.05 ml per mg smple) using Psteur pipette. Add isohexne (3 ml), diethyl ether (3 ml) nd distilled wter (2 ml). Shke thoroughly nd, if necessry (if there re not two distinct lyers nd/or n emulsion is formed), centrifuge for 5 min (bout 400 rpm). Put upper orgnic lyer through short (3 cm) column of nhydrous sodium sulfte (prepred in Psteur pipette, plugged with smll piece of cotton wool, nd pre-wshed with 3 ml isohexne-diethyl ether (1:1, by vol.) prior to use) to dry. Collect in test tube. Repet extrction of lower queous lyer with isohexne-diethyl ether (3 ml, 1:1, by vol.) nd put the orgnic lyer through the sodium sulfte column. Wsh smple through the column into the tube with more isohexne-diethyl ether (3 ml). Tke smple to dryness in gentle strem of nitrogen on heting block t 30 C Pyridylcrbinol ('picolinyl') Esters 3-Pyridylcrbinol esters, commonly but incorrectly known s picolinyl esters, re most esily prepred in good yield from the RCH + SCl 2 RCCl free ftty cids prepred s bove following CH 2 H CH 2 CR ctivtion by conversion to the cid + RCCl chlorides. Rection with thionyl chloride is rpid nd efficient [2], but the milder method of rection with oxlyl chloride overnight is lso suitble [3]. Dry solvents nd fresh regents re required becuse the rections re sensitive to moisture. W.W. Christie lipidlibrry.ocs.org 1

2 Preprtion of derivtives for mss spectrometry of ftty cids Method: The rections re crried out in 0.3 ml screw cp vils. The cid (20 µg) is heted with thionyl chloride (20 µl) for 10 min. t 100ºC. Excess regent is evported in strem of nitrogen, nd solution of 20% 3-(hydroxy-methyl)pyridine in cetonitrile (10 µl) is dded; the mixture is heted for 1 min. t 100ºC. An liquot cn be injected directly onto the GC column. A mild quntittive method developed for derivtizing sensitive polyunsturted ftty cids contining epoxyl groups involving n imidzolide intermedite is useful lterntive [4]. It is simple nd rpid, involving brief rections with first crbonyldiimidzole then 3- (hydroxymethyl)pyridine nd ctlyst. RCH + C R C + H C 2 crbonyldiimidzole CH 2 H CH 2 CR 3-pyridylcrbinol ester + H The rection, s detiled below, is moisture sensitive nd ll regents should be protected from moisture (i.e. by storing in the presence of desiccnt). Fresh crbonyldiimidzole solution should be prepred ech time set of smples is derivtized. Method: The derivtizing regent is prepred by dissolving 3-(hydroxymethyl)pyridine (900 µl) in dichloromethne (9 ml) nd triethylmine (9 ml). It cn be used for up to one month if kept in seled tube (with PTFE-lined screw top) with little dded nhydrous sodium sulfte s drying gent in refrigertor. The free ftty cid smple (up to 1 mg), in centrifuge tube, is dissolved in dichloromethne (100 µl)nd solution of 1,1'- crbonyldiimidzole (100 µl, 10 mg/ml in dichloromethne, freshly prepred) is dded. Leve 1 min. (not longer) t room temperture before proceeding to the next step. Add the 3-pyridylcrbinol regent (200 µl). Agitte nd leve for 10 min. t 37 C. At the end of this time dd cetic cid (25 µl). Tke to dryness on heting block t 30 C with gentle strem of nitrogen. Add isohexne (5 ml) nd distilled wter (2 ml). Shke thoroughly, then put upper isohexne lyer through short (4 cm) column of nhydrous sodium sulfte in Psteur pipette (with smll cotton wool plug nd pre-wshed with isohexne) to dry. Re-extrct the queous lyer with fresh isohexne (2 ml), nd pss through the column lso. If necessry, little crystlline potssium chloride cn be dded to brek emulsions. Finlly, wsh through column with isohexne (1 ml) nd evporte the combined isohexne eluents. Dissolve smple in n pproprite mount of isohexne for GC nd GC-MS nlyses. It is lso possible to prepre 3-pyridylcrbinol esters directly from intct lipids or methyl esters by trnsesterifiction with 3-(hydroxymethyl)pyridine with potssium tert-butoxide in tetrhydrofurn s ctlyst [5]. This rection my indeed be useful for the preprtion of vriety of different ester derivtives by substituting the pproprite lcohol. The regent must be very dry, otherwise n irreversible hydrolysis rection cn occur. It is best to mke fresh solution of potssium tertbutoxide in dry tetrhydrofurn ech time it is required. W.W. Christie lipidlibrry.ocs.org 2

3 Preprtion of derivtives for mss spectrometry of ftty cids CH 2 H _ C(CH RCX + 3 ) 3 where X = CH 3, glycerol, etc CH 2 CR Method: A solution of potssium tert-butoxide in dry tetrhydrofurn (0.1 ml, 1.0 M) is dded to 3-(hydroxymethyl)pyridine (0.2 ml). After mixing, the lipid smple (up to 10 mg) in dry dichloromethne (1 ml) is dded, nd the solution is held t 40 C for 30 min in closed vil. After cooling to room temperture, wter (2 ml) nd hexne (4 ml) re dded, nd the orgnic phse is collected, dried over nhydrous sodium sulfte, nd evported. The smple is dissolved in isohexne for GC-MS nlysis. 3-Pyridylcrbinol esters cn be converted bck to methyl esters by conventionl cidic or bsic trnsesterifiction procedures. 3. Dimethyloxzoline (DMX) Derivtives DMX derivtives re prepred in simple one-pot rection by rection of lipids with 2-mino-2- methyl-1-propnol in nitrogen tmosphere t 180 C ( minimum of 2 hours for free cids, or up to 18 hours for methyl esters nd intct lipids [6]. H 2 H + HCR H H CR - H 2 + H 2 R Method: To the lipid smple (up to 2 mg) in test tube is dded 2-mino-2-methyl-1-propnol (0.25 g). The vessel is flushed with nitrogen, stoppered, nd plced in heting block, t 190ºC overnight. Flush the tube with nitrogen when the tube is up to temperture to help to eliminte moisture nd minimize utoxidtion. A little glycerol dded to the well of the heting block ensures good contct nd tht the correct temperture is mintined. ext dy, remove the test tube from the heting block, llow to cool to room temperture nd wsh off ny glycerol on the externl surfce with wter. Add diethyl ether-isohexne (1:1, v/v; 5 ml) to the tube, wshing down the internl surfce, followed by wter (5 ml). Shke thoroughly nd llow lyers to settle. If necessry, ddition of little sodium or potssium chloride will usully brek up ny interfcil lyers. Trnsfer the orgnic lyer with psteur pipette to fresh test-tube, nd re-extrct the queous lyer with fresh solvent (2 ml). Add distilled wter (3 ml) to the combined solvent lyers, shke nd llow to settle. Trnsfer the solvent lyer to fresh tube with psteur pipette nd dd nhydrous sodium sulfte (bout 1 ml). Leve for one hour with occsionl vigorous shking, then pss the solvent lyer through short (3 cm) column of nhydrous sodium sulfte (prepred in Psteur pipette, plugged with smll piece of cotton wool, nd pre-wshed with c. 3 ml isohexne prior to use) to dry, into fresh test tube. Wsh smple from the sodium sulfte tube through column with isohexne (2 ml). Tke smple to dryness in gentle strem of nitrogen on heting block t 30ºC. Dissolve the smple in n pproprite mount of isohexne for GC nd GC-MS nlyses. Adding few crystls of nhydrous sodium sulfte stbilizes the derivtive for storge. W.W. Christie lipidlibrry.ocs.org 3

4 Preprtion of derivtives for mss spectrometry of ftty cids Unfortuntely, we hve occsionlly observed incomplete rection nd ppernce of the uncyclized intermedite, which elutes lter from GC columns (nd gives mss spectrum lmost identicl to tht of the required derivtive). It is importnt to keep the product nd rection mixtures dry, otherwise trces of moisture cn cuse ring opening, even on storge in inert solvents t low tempertures [1]. For the sme reson, it is not possible to purify DMX derivtives by dsorption chromtogrphy. The prolonged high temperture required for the preprtion of DMX derivtives gives cuse for concern, nd there must be some risk to polyunsturted ftty cids or ny other compound with lbile functionl group. For exmple, trns-3-hexdecenoic cid, common in plnt photosynthetic tissue, ws found to hve isomerized lrgely to cis-2-hexdecenoic cid during the rection [7], nd crepenynic cid ws found to undergo cycliztion rection [8]. We hve lso observed tht linoleic cid cn be prtilly isomerized to conjugted isomers ( CLA ) if the rection is crried out crelessly. An lterntive two-step rection hs been described tht my be sfer in some circumstnces [8]. It involves prepring the simple mide vi the cid chloride, followed by cycliztion with trifluorocetic nhydride. As with 3-pyridylcrbinol esters, I prefer to prepre DMX derivtives vi the free cids rther thn from intct lipids, but this is not lwys prcticl or convenient. RCH + ClC.CCl RCCl CH 3 CH 3 CCH 2 H H 2 trifluorocetic nhydride CH 3 CH 3 CCH 2 H R HCR Method: The ftty cid must first be converted to the cid chloride s in the methods for preprtion of 3-pyridylcrbinol esters vi the cid chloride. This is used immeditely for the preprtion of the required derivtives. A solution of 2-mino-2-methylpropnol in dichloromethne (10 mg/ml) is prepred nd stored over nhydrous sodium sulfte. It is stble in the refrigertor for bout 1 month. This solution (0.5 ml) is dded to the freshly prepred cid chloride, cooled in n ice bth, nd the mixture is then left to wrm up to room temperture for n hour. The solvent is removed in strem of nitrogen, nd trifluorocetic nhydride (0.5 ml) is dded. After 1 hour on heting block t 50 C, or 3 hours t room temperture, the regent is removed in strem of nitrogen. Isohexne (5 ml) is dded followed by wter (2 ml), nd the product is obtined s in the previous rection, tking cre to dry the product thoroughly. A further two-step procedure tht is suitble for the preprtion of both DMX nd other simple oxzoline derivtives hs been described, nd the GC-MS properties of the ltter pper excellent [9]. However, few published spectr re vilble. f the mny lterntive nitrogen-contining derivtives for mss spectrometry tht hve been described in the literture, the ltter pper to me to be the only ones tht might be worth pursuing. Similrly, new mild procedure for prepring DMX derivtives nd pyrrolidides from free cids using Deoxo-Fluor TM regent to ctivte the ftty cids, which should lso be pplicble to pyridylcrbinol esters, hs been described [10,11]. We hve yet to try it out so would be interested in lerning of the experience of others. DMX derivtives cn be converted bck to methyl esters by methyltion procedures used for sphingolipids, e.g. by rection with methnol contining hydrochloric cid [1]. W.W. Christie lipidlibrry.ocs.org 4

5 Preprtion of derivtives for mss spectrometry of ftty cids 4. Pyrrolidides Pyrrolidides re prepred simply by recting methyl esters of ftty cids with pyrrolidine in the presence of cetic cid [12]. RC CH 3 + H RC + CH 3 H Method: The ftty cid methyl ester (up to10 mg) is dissolved in freshly distilled pyrrolidine (1 ml), cetic cid (0.1 ml) is dded, nd the mixture is heted t 100 C for 1 hour. Excess pyrrolidine is evported in strem of nitrogen t 50 C, nd then the residue is tken up in hexne-diethyl ether (1:1, v/v; 8 ml) nd is wshed three times with wter (4 ml portions). After drying over nhydrous sodium sulfte, the required product is obtined on evportion of the solvent. The rection tkes plce under bsic conditions nd is reltively mild so my be sfer thn for DMX derivtives with ftty cids contining lbile functionl groups. Alterntive procedures re lso vilble to prepre pyrrolidides directly from tricylglycerols nd presumbly other glycerolipids [13] or from free ftty cids [10,14,15], the ltter in mild one-pot rection. References 1. Christie, W.W. nd Hn, X. Lipid Anlysis - Isoltion, Seprtion, Identifiction nd Lipidomic Anlysis (4 th edition), 446 pges (ily Press, Bridgwter, U.K.) (2010). 2. Hrvey, D.J. Picolinyl esters s derivtives for the structurl determintion of long chin brnched nd unsturted ftty cids. Biomed. Mss Spectrom., 9, (1982). 3. Mttson, F.H. nd Volpenhein, R.A. Synthesis nd properties of glycerides. J. Lipid Res., 3, (1962). 4. Blzy, M. nd ies, A.S. Chrcteriztion of epoxides of polyunsturted ftty cids by mss spectrometry vi 3-pyridinylmethyl esters. Biomed. Environ. Mss Spectrom., 18, (1989). 5. Destillts, F. nd Angers, P. ne-step methodology for the synthesis of FA picolinyl esters from intct lipids. J. Am. il Chem. Soc., 79, (2002). 6. Fy, L. nd Richli, U. Loction of double bonds in polyunsturted ftty cids by gs chromtogrphymss spectrometry fter 4,4-dimethyloxzoline derivtiztion. J. Chromtogr. A, 541, (1991). 7. Lmberto, M. nd Ackmn, R.G. Positionl isomeriztion of trns-3-hexdecenoic cid employing 2- mino-2-methylpropnol s derivtizing gent for double bond loction by GC/MS. Anl. Biochem., 230, (1995). 8. Christie, W.W. Mss spectrometry of ftty cids with methylene-interrupted ene-yne systems. Chem. Phys. Lipids, 94, (1998). 9. Kuklev, D.V. nd Smith, W.L. A procedure for prepring oxzolines of highly unsturted ftty cids to determine double bond positions by mss spectrometry. J. Lipid Res., 44, (2003). 10. Kngni, C.. nd Kelley, D.E. ne pot direct synthesis of mides or oxzolines from crboxylic cids using Deoxo-Fluor regent. Tetrhedron Letts., 46, (2005). 11. Kngni, C.., Kelley, D.E. nd Evns, R.W. Synthesis nd mss spectrometry of benzoxzoline, dimethyloxzoline nd 4-phenyloxzoline derivtives of polyunsturted ftty cids. Rpid Commun. Mss Spectrom., 21, (2007). 12. Andersson, B.A. nd Holmn, R.T. Pyrrolidides for mss spectrometric determintion of the position of double bonds in monounsturted ftty cids. Lipids, 9, (1974). W.W. Christie lipidlibrry.ocs.org 5

6 Preprtion of derivtives for mss spectrometry of ftty cids 13. Vetter, W. nd Wlther, W. Pyrrolidides s derivtives for the determintion of the ftty cids of tricylglycerols by gs-chromtogrphy. J. Chromtogr. A, 686, (1994). 14. Vetter, W. nd Wlther, W. Preprtion of pyrrolidides from ftty cids vi trimethylsilyl esters for GC- MS nlysis. J. Chromtogr. A, 513, (1990). 15. Kngni, C.., Kelley, D.E. nd DeLny, J.P. ew method for GC/FID nd GC-C-IRMS nlysis of plsm free ftty cid concentrtion nd isotopic enrichment. J. Chromtogr. B, 873, (2008). Willim W. Christie Jmes Hutton Institute (nd Mylnefield Lipid Anlysis), Invergowrie, Dundee (DD2 5DA), Scotlnd Lst updted: Februry 20 th, 2014 W.W. Christie lipidlibrry.ocs.org 6