Derivatives suitable for GC-MS

Chemistry and Phygcz of Hpid~ 21 (1978) 403-416 © ElsevierjNo~h-Hoil~ad Sciewdfic Publishers Ltd.

DERIVATIVES S(~qTABLE FOR GC-MS CJ.W. BROOKS, C.G. EDMONDS, S J . GASKELL ~:nd A.G. SMITH Department of Chemistry, Uni~er~tyof Glasgow, Glasgow,G.128QQ, UK

The preparat~n of deflvatives is often advantageous o~ n e c e ~ y for GC-M$, for ~ vafie~.yof remDm. In respect of GC, these ~dude the need for confezxMgvolatility or stability; ~he Lm~mve. l~1~mtof chtomatograpkic behaviout or sepazation; information or, functional groups; v~ndpr~v~sion for selective detectiom In MS, derb~tfivesare important for pzodu~r.g characteristic mms sh~ftsa~,,M for directiag particular modes of fragmentation trader electron F~,pact or chemical ~3nisa~on. Reagents used for derivative formation may be seneral or s e ~ : amo~ the latte~ c~a~ reagents that yield cyclic derbatives from bffuncti~-~alsubstrates are of sp¢~,-ialvalue° Typical cxmnples are given of the applications of derivatives in the analysis, by GC-MS, of ~ong-chainfatty acids~pro~agltadir~ mono- and di~tycerides, sphingolipids, and sterols. Recent developme~s in btstrar~,entation and in the use of computer data systems are outlined.

I. (~nerai aspects The v o ~ n o f s~mples in stable form" aprerequisite for a n , J y ~ g them - as distinct from their pyrolysis products - by GC-MS. Initially, samp!ies have to be volltilised into the carrier gasetream at about atmospheric pre~ure: c,nc¢ they r~:ach the m ~ ~ r o m e t e r , there is o:Minarfly little risk o f condensation. Accordingl~ th,: mit:sbility o f ssmple$ot their deKvatives for GC is also the ~ajor criterion for GC-MSo Th4~m a i n reasom for using derivatives are as fonows [ 1,2]

L Co" nye,~ent ofvolatUity Comlmunds that ate practically involatfle beca,~xse of strong intennolecular associatio~ include zwitterionic amino acids~d-hydrogen-bonded polyhydroxy compo ~nds.

K/mpm~mentoJ'stabgity Thermal d ~ o f many compounds resultz from the presence of sensitive or interactive fimcttotufl groups !rod c a n be avoided or mitigated by protractive deriw~tives. A J~aple example is the u ~ o f ~ l i m e t h y l ~ l a t i o n t o preserve allylic ~-~coholgzou~ing~L e . i , : ~ 5terols [3] c r p r o s t a # a d i m [4] (fig. 1),

fir. Improvementof chromatographicpropera~ Two Mlport~nt elementsin the establishment of satisfactory gas chrcmatograpL~c 403

C.3. W. B ~ o k s

404

e t al.,

Derivativessuitablefor G C - - M S

Me~SiO

.

.

I

Me~SiO

~

~ H

"

V

'

~

~

OSiMe~

Ref. 4

'

Ref. 3

'

= ::

....

Fig. 1. Protection of allylic alcohols as W,',~t~.~hy~l~ f o t

.....

'

. . . .

gu c.l~rolnato$zaphy.

behaviour are (a) chemical congruency between g~npl¢ and stationw/phase mffici~nt to avoid extremely high or extremdy low partition coefficients, and(b)the ~ n ¢ ~ of significant adsorption effects. ~ : ! : For ionisable samples, ¢omlition (a)canlargely be met by the m e o f special stationary phases e.g. basic phases for a m i n e s , ~ phases for acid~ The risk o f adsorption of such polar semples is high; however, and ~ is aeeordin#ymore eonve~d. ent to transform these into derivatives (amides, e ~ r s etc,)that are mitablefor analysis on generally.used stationary phases. Similarly, aeylation or 'sflylation' of hydroxy-compounds affords derivatives less liable to adsorption. iv.

Improvemem of septmttiom

.

:

~

The judiciou..!tchoice of derivatives and stationaw pharos offers ~ e sco~ for th,,~ control of chrcmatogmphic'~,retention p~am~t~n. Thm ( a ) ~ prolongation Ofret,~n. tion times thro~;lh formation of ~F~flvatives o f ~ c h h i ~ r molecUlar,weight is often useful in movin~ 'the de,.red l~ak fled by the use ofberlzy[ este~ techmque permi~ s a n ~ y t ~ grc non-ketonic ster~,ids [6] ; (e) ~'~! tared, a classic e~ ample behng the their t'~r~ethylsfll,l (TMS) e~hers through deriva*J~ion wi~h a chUal reagent [9]. (~ ~ i~ ' ,~

• ? I!~ ~ ~ i~ ~/:~/:,~!~!~i:i~'ii,i ~ : /i~i~ '

. :

~ .... : ,

~ ....... ii, ~i~ ~, ~i ,.?~iJ,~i,i~, , ~!/, ~,:~,~ILI%~SI~'~I,I',I, ~ ~ , ~ :,~i!:~~:~=~ ~.. ~i ~.,~!,:~::i,~,~:~,~ ~/~:i~.~%~:~i~:,~

(:

•

CJ.~, B ~ k s et aL, Der~ttae~ suitable for GC--MS

405

v. Funa'~anat group a~a~vsis i n addition to the aspects covered under paragraph (iv), the comparative GC of mmaples in ft~e feral and a f a r various treatments for derivative formation can ,~ve valuable information ca the number and type of reactive groups present.

v£ Pm~aon for s e l e ~ e detection (non-mass spectrometric) The scope of the electron capture detector and of detectors responding selectively to particular elements (H,S, P,Lt, etc.) is greatly inc~ased by the use of derivatives designed reproduce sensitive responses. Reagents labelled with radioisotopes are similarly useful. The following app~ cations of derivatives are particularly concerned with GO--MS. There are two genera/ways i~ which derivatives can be used to al!'fect mass spectrao It,. the first, exemplified in patagraph vii, the ~im is not to alter fragmen. ration pathway~ ~ut merely to introduce mass changes (by isotopic or other mi~or mbstitution) that will aM analysis or interpretation. The second approach (paragraph ,¢iii) seeks to induce a p~rticular mode o f fragmentation either to assist structural elucidation o r t o produce ions of specific kinds (or masses) for analytical purpo:~es.

vK I~duetion o f mass shifts, in maa~'spectra The simplest u~;s of mass shifts are in respect of the molecular ion, ~L,ice the observed chsnps ~,e here leaat susceptible to any changes in fragmentation. Typical @plicaZions would include, the assessment of enolisabIe hydrogen by deuterium exchaage, wlflc~ can be effected before GC or in transitu [10], ~nd of functional groups by u~3ag reagents c f different mass (homoiogues, or preferably isotope-labeUed analogues). Deliberate adjustment of the isotopic composition, e.g. by deuterium or X3C labdik~ [11] so as to produce doublets or other weU-defmed patterns of ions is h e l p S , for example, in pharmacological studies where the metabolites arising from a drag labelled ~ ~ fasMon may be more readily de~ected inasmuch as their mass substitution is to pta~vide a compound for ~e as an int~m~ standard in thequantitative malysis o f its unlabeHed parent. Compounds are maially labelled with three or four deuterium atoms at sites that do not seriously affect fresmentation. The complete distinction based on the mass difference between the two compounds aao thek similar retention times; ~ o w s the use of a large amount of imemM s t e n d ~ ~ a "carrier" intended to obviate the effec~ of adsorption on the low Ie~els Of sample b~t~ analysed [12].

'~i¸¸• ,

,•:

.:

~

~

:•

/•

:

D~rivatives arc f~quontIy effective in ~ i d ~

m o I ~ c ~ ions where th~ pa~ent

406

compounds afford these in low abundance or not at all. ~ p r e s e n t use of TMS etal. ~l corresp~: are also t-butyld [14] ;in the alky than that referred from 5]+ TMS ethers and other derivativesused+inGC are+alsoo f great importance byvirtue of their directive effects on mass spectrometric fragmentations, Oeavase of the carbon-carbon bond in the grouping I

[

-C-C~OR

•

• +

is more facile for ethers (R = CHs, Si(CHs)s, etc.) than foralcohols (R = H)andthe ion/"C = ~)Ris particularly, prominent, for example, inthe mass spectra of ThLSethers of secondary a~3dtertiary alcohols. An application is outlined in section IlL wCleavage " also involved in the highly characteristic ~ n t a t i o n o f TMS ethers of 3.hydroxysteroid 5-enes which yields complementary ions of role 129 and [M-129] + [15-17]. The use of derivatives to induce informative fragmentations is of evengreater importance in the characterisation of more complex molecules such as oligopeptides [ 18] and glycosphingolipids;in the latter case the molecu4ar ~eights.,involved may require the direct introduction of samplesinto ~ e mass spectrometer [19] i though GC-MS has been applied to a cerebroside derivative of tool. wt. 1259 [20]. ix. Use of derivatives in con]unction with chemical ionisaffon The availability of instruments suitable for GC-~MS with either electmnrimpaet or chemical ionisation requires the consideration of additional criteria for the selection of derivatives. While chemical ionisation in some in~anceS affords essentiallyo~ly a protonated or related type of molecular ion, it iscleex that thepromotion+~ir,.fonnarive fragmentation is also possible. IIo Reagents for derivative formation

Three broad classes of reagent can be distinguished in relati~:to the' formation of derivatives. (a) Reagents that have the power to form imiividual .functional groups, yet are capable

CJ, t~ B ~ o k s et al., Der~vat~:ve~su~taMe for GC-MS

407

note between many hydroxylic groups ~ st~ro[ds(of.Chambaz and H o ~ n g [2i} ), o~ can be used under f~re~ condifion~ to. y:k,ld enol ethers even ef the :~trongly ~ d e r e d 11-oxosteroids [22a]. Acylating ~ents c ~ show an equal range of ~eactivity; to citeonly one exmmple, selectiveN.,acylat:ion of amino alcohols is h~portant ~ the G C - M S analysisof cateci~olamineL sphLug~smes, hydroxyantino-acids,etc. (b) Reagents that ~mutlly ~ t on a s~dler range of functional g~oupso O~e example would be diazenaethane, which is 1~rgely selective, in practice, for methylation of carboxylic acids, because its numerous other reactions are generally much dower. ~ e a~ ofketones to fbrm Schiffbases, and of CSz to form isothiocyanates by reaction with pri_mar~ a,~Mres, are in this category. (Ketones can also serve as

rble for loaning cyclic derivatives from compounds possessing bl- o multi=Jimct~nal reactr.~ty,, This kind of process is of gnat ~mportance because its selectiw;ty &~pendsupon the particular relative disposition and stereochemistry of the ~eactive groups in the substrate; moreover, the fore, orion of a ring limits the tar,~e of possible conformations ~nd gives rise to distinctive chemical, chta~mstographic and mass-spectrometric properties. Classical examples are the use of aldehydes and ketones for ~onversion of 1,2
408

CJ. W. Brooks et al., DerivarLves suitable )'or GC--MS

3m 1% 0V-17 270"C 50 ml/mln 19.hydroxycholesterol as diTMS ether

After incubationwith cholesterol oxtdese

3535

3260

rains.

Fig. 2. Gas chromatograms of trimeth~!~ilylethers of 19.hydroxycholest~roland of tlte product of its oxidation with cholesteroloxidase. mildness: thus cholesterol oxida~, which converts many 3~-hydroxysteroid 5;~nes into the 4-en.3.ones [29,30], effects this tranaformatton in 19-hydroxyeholeslt,rol without affecting the 19-hydroxy group, as illustn~t~l in fig.2 which shows the g u chromatography of the TMS ether derivatives. Reconversion of 4~en.3.ones ¢o 3~-hydroxy~teroid 5-enes can be accompli~ed by the el¢gant reduction tochntque of A ~ g e r and Eneroth [22b]. " ~H. Derivatives in lipid armiysis Representative examples of the use of derivatives in lhe analysis o f lipids by

C.L ~. Brooks et gl., Oertvatg~:, sta~¢b~ for GC-.MS

409

GC-MS are given in thi,~tsection. A ~eftd review of GC-MS of polar lipids has been p~olitff~ed [3.].

i. Long-cludn fatty acids Location of obffhu'c bond position in unsaturated ~ids (as methyl esters) is achiev.,~ ed by osmium tetroxide hydroxylation followed by GC-MS of the diol (or polyo~) TMS ethers; informative ions arise, by cle~vag~:at the original olefinic sites w~ithcharge retention on ei~,~r ox~¢gonated group [32]. For monoenoic acids, fragme~ttatio~s adjacent to the original olefinic sites occur fl:~the diol acetonides [33] ~d cyclic boro~at~s [34], h a completely different apl?roaeh, pyrrotidides of fatty acids have been found eff,~clivein p~moting simple cleavages indicative of olefinic bonds and branch points [35 ~. For the clharaeta",risation ofpolyols derived from hydroxylation of polyet~oic acid methyl esters, a comparative study has indica, ed that TM$ ethers are generally more effecth,e than methyl ethers and are superior to acetonide ~qd aLka~eboronate derivatives [36].

ii. Prostaglandins These are usually s~udied as derivatives of the methyl esters. Protection of the 15a-hydroxy-13-ene group by trimethylsilyiation has been mentioned above. Similar derivatisation or cyclic bo ronate formation [37] serves for the 9c~, I h~-diol system, while prostaglandins E an; effectively gtabilis~d as alkyloxime TMS ethers [38]. Prosta..glandins ha~e been isolate6 from serni~at fluid after methyloximatign of the biological sample (dil~ated with buffer) to protect the #-ketol system before extractk~r~ [39]; P r o ~ a n d i n s contai_ning the 15~hydroxy group yield abunda~t fragments at ~M---69]*) t,y loss of the terminal CsHtx (or C:-Mg)group; diffvrent ~Mto bring the mass of such ions to a value c,o~veedent for quantitative dete~.minatiom based on "selec*~ed ion monitoring". Thi:~ me~hod w~s used in the identification of prostaglandirl F2a in h~_~manaorta[ tissue [40] : ~he tratm recordeM fox the methyl e|Ctorcyclohexan~boronate 15-TMS ether is ff~ustrated in fig. 3. Prostaglandins of the Ft, F~ arid F~ series can be distinguished by chemicaJ ionisation GC-MS of boronate derivatives which yield major ions retaia~ng the in,at, carbon ~eletons ~dth their olefmic features [41].

iii. blon~ and digt.Vce,rides It is usual to cleave the phosphate groups from phospholipids by e~:ymatic hyd roly~L~b e f o ~ p r o c ~ e ~ wil~hgavpha~ anaiy~o TMS e~her~,lof isomeric 1,2,-and ~~3~ digly~ddes ale ea~sily~:h'!guAshed by GC-MS, the latter beiag eiuted tater a~vd yie!diag~undant [M~H~C~OR]* ions [42]. M~oglyeendes, gJycery~monoetbe~; arM'tong,chain 1,2~i~ts ca~ ~ be convenieatly exam~ed ~s cyclic acet~ or bo~o n a ~ , ~ weU~as by 'tr~aet~ylsilytatioa. Methaneboronate:,~ a~e of ~pec.~.~ v,~d~e fo ° chemical ionisatioa GC-It~: thus lomonostesdn metheneboronate, which i~ e]uted

410

CJ; W, BrooP~ et aL, Derivatives ~dtable ~ r :GC,-MS

0

= I

461 ~-

(a) Stet~lard

(b)

A

PGF=oe

Aorlal PGF2~ i

i

----

L

(c)

Is

b

Io min

:, " '

1'/, 0 V - 1 7

: :

Fig. 3. Chrom~.togramsrecorded by GC-MS Of pro~aglandin F2a methyl e.~er.9~! l.¢ydohexaneboronate 15-trimethylsilyl ether: (a) mo~dl toting role 461 for the sample isolated ftot total ionisation chromatogram e,oneu~zeat, Q (100-120 mesh) was operated at 270* w gas ehromatograph-ma~ speetz~metet (e1¢........... ,eJ ,~,~,~-~. earlier than the b i s - TMS ether on OV-1 (fig. 4)yields ( w i t ~ ! i ~ b u t ~ ¢ U i r e ~ m gas)

abundant ions at role 283 ( M H +) and /e/.67 ( C 1 ~ s ~ O ) , i~:. Sphingoiipids

,'

.....

::., ~ ,~

::

; : ..............

Sphingosines have generally been studi~d as O, TMS,ethe~ 4 3 ] o r ~' N - ~ e t O-TMS-ethers [44]. Major fragment ions :t~rive from C l - C 2 an d C 2 - C 3 cleavqes and are hadic:~five of the structure t o be weak and in this respect cyclic bor i~.~conjunction with further derivative fi five dat~ for ~phhlgoskne derivativesare G C - M S of TMS ethers or cyclic boroti ....... ,:~ .:,-,.- ............ ~ ~,~.:,, ~,:~,~ e×arained m , 0 ~ as TMS ethers [48] or aCe,~tyld e a v ~ f ~ ) [ 4 9 ] / ; a

CJ. W. N~.x:~ et ~o, DeNw¢f~,es su#abie j'ov GC°.,?vIA'

4 ~t

A

.t

10

mi[n.

Fig. 4, Gas ¢h~matoNam of a mNture of l - m o n o ~ r i n metha~eboronate (A) and I .-mop.ostearin bls-la'Jmethyl~rl ether (B). The 1 m column of 1% OV-I on Gas Chrom Q (100-120 mesh) was ope~a~:edat :210" wi~h a nitrogen flow rate of 50 ml/mim

methylation/reduet~ton technique [19] may be envisaged for GC-I~S of g~yco,~y]ceramides and arainogly eosyleeramides of moderate molecular weight.

Monohydroxylic sterol.% with a fi,'w exceptions, can be ~uaty~d as free compounds by GC-.MS, but th~:~u ~ of deriv~,tives is more satisfactory. TrLmethylsflyt and other:

c H-. SH

OH-OH O. '

i~l~ "

C . ~. &H

t ,

iHCOCH~

8H

'

!

H INHC2~ ~

Ct,~~. H--C.H--CH., ;~ C,.,H,~~-4~H.-~H.,B/ C"H"'I%'x~/;--C ~. H

M* ": m/e 367 (2~)

~ ~-t. the i~tmn ~npactt70 ev)mu~, ~,e~ra.

M~'°: m& 353 !IOC~!

412

CJ. W. Brooks et ~ , Deriva~,res suttable for GC-MS

~SiM%

OSiM%l

I

I

m~289: 'el). 1~'0 291 : ~tq), z,i0 293: '80, tt:O

Fig. 6. Mass spectrometric fragmentation of the side-eiaaL,',of 20,22.dihydroxy-cholestetol tr/strimethylsilyl ether affording ions indicative of the isotopic composition of the 20- and 22, oxygen atoms.

silyl ethers are widely used to promote characteristic nuclear fragmentations (e.g. those of 3- hydroxysteroid 5.enes mentioned above) [50,51], Thevirtual absence of adsorption eftbcts in GC of these derivatives is also import~mt in achieving clear ~patations of closely-similar sterols by high resolution GC combined with MS [52,53]. Further improvement of chromatographic efficiency is deSi~rablein view of the occurrence of "non-classical" side-chains, which are particularly prevalent in sterols of marine orgaJaisms [54]. The promotion of weleavage by TMS ether formation was effectively applied in a study of the incorporation of oxygen atoms in the enzymatic formation of (2OR, 22R)-dihydroxyeholesterol from cholesterol [55]. Incubation w~s effeeted in the presence of 1602 and lh32 and the isolated dihydroxy sterol was converted to its c~s-TMS ether for GC-MS. Prominent ions (fig. 6 ) o f role 289, 291 and 293, restdting from cleavage at C(17)--C(20), were observed, their masses and relative abundance showing that the ~'ide.chain oxy~n atoms derived not from intact oxygen molecu|es but from the atomic pool of oxygen, . . . . . . . . . Sterol methyl ethers, which were amon8 the derivatives earlier used for GC of sterols, yield characteristic mass spectra [56], but methylation procedures have not yet attained the efficiency typical of trimethylsilylation. Sterol glucosides can be analysed by GC-MS of their TMS ethers [57];

IV, Recent developments Attention is drawn to two more corapret!ensive reviews of lipid analysis [58,59] argot,to a chapter on the identification of steroids [ ~ ] . A wealth of practical .~rfformation may be found in the 'Handbook o f Derivatives for Ch~matogxaphy'edit~,~b¥ Bla~ and King [61 ] General methods of ~nalym:of ~amor m~-me ~ e ~ o t s ~ d ~ b e d by I)jer~si's group [62t, and elegant isolaticn been applied inter alia to the analyis of 3.ket0 3 iI

C.g.~t B ~ g

et ale, Deri~,eaives s~tmb~e fee GC-4~S

413

recent papel~ on novel alkyNlyl derivatives [64..-66] ;on cyclic berol~ates of spbhngeo ~ues [67] and ceramides [68] ;enncw pros~agtandm derivatives [69t and on bbutyL dimethylsflyle ~ e ~ in ~ e an~lys/~ of thromboxane B~ [70]. Qumox~dinol de~vatives are of v~ue for CC-NS ~ y ~ s o f ~phatic 2-',~xoadds [71 ].

V, ::in~rume~l eon~h~'at~mi A full : d i s ~ i o n o f tn~rumen~tion for GC-NS is beyond tl~e scope of this review but a few obsereations are made here on recent developments. Najor improvements it, gas chromatographic ~solution are ael~eved by the ~lse of open-tubular capi~iary eohmans (preferably gltm) and these are now routinely used in GC-MS. The ~rnproved facility for the separation of complex mixtures implies also a greater confider~ce in the use of gas chromatographic retention time as an identification parameter. The latter consideration assumes lpm~dcularimportance when GC-NS analyses are performed m the ~leeted ion detection mode; in this instance, characterisation of the sample is based on gas chromatographic behaviour and the presence of' one, or a few, character~ istie ions. The scope of multiple selected ion detection during GC-MS has been increased by improved facilities for rapid switching between ions over a wide mass r~x~ge,with little variation in sensitivity. In this respecL instruments equipped with a quadrupole mass analyser are advantageous, although recent developments in the design of ~aminated magnets have imp~roved the position with regard ~o conventional sector instruments.. l~,creases in instrument semitivity have accompanied other developments. Clearly, however, the sensi~vity of, for example, a selected ion detection assay procedure is dependent on both the chemi¢~ procedures employed and on instrtanental considerations, Thus, for e x ~ p l e , use~o f t-butyldimethylsilylether derivatives may markedly improve semit~ivityof,detection [14,72], in some instances by as much as a factor of tea 173]. Alternative methods of ionc!sation now supplement conventional electron impact. Ch.evaiealionisation (in wNeh sample ioaisation occurs at relatively high pressure by eo~idon with ionised r~etant gas)~is of particular ~portance Lu GC-MS and has found wide appUeation in bc,th qu~ative ~nd quantRative analyses. Chemical ionization speeta'a are 1Nnerallynoteworthy for the abund~ce of ions retaining the intact molecule (¢~, MW), h aprovement,,~ m !~c~um technolo~" requh'ed by high pressure ionisatio~ techniques haw also enabled 4ireet eoupl~g of GLC capillary columns to raass s~:ctramete~; o b v i a t ~ the n~d, ha thc~ ~stances, for molecular enr,cho~,~, devices. A NdNN~ 1¢v¢! of molectd',~ ion e ~ e a t a t i o n ~ also acNeved by the ~ie~d io~ation teehnkiue (:~ which ior,J~ation oceu~ m a stroN dtectric field), Se~sitivity is generally lowe~:, however, flaa,-~:wfih~temative io~jsa~ion methods. The re~a~ed ~ u e of fleldld~orption is of p ~ i c N ~ i m p o r t ~ where s~ples, by virtue of i

414

C]~WoBrooks e~u~, Dcr/~atf~ suitable~ r C~C-MS

polarity or low volatility,are not mnenab][e to:C~-MS. ~ The vastdata output of efficiently-usedC ~ - M S m ~ e n t s ~ encouraged the, development of dedicat~ computer data s y s t ~ CalForation........ ~ d ~ ~,a t i o n of spectra me normally pefforme~ c~currently ~ data ~ ¢ l ~ O n , mbsequent processing may include comparimn~ of sample ~ e c t r a w i t h spectra ~ m a standard library or interpretative procedures chemist (e.g. ref. 74). Of particular is

the performance of GC-MS analyses m the rapid r e p ~ ~ ~ vdth computerised acquisitton of data and sub~uent celculationof piers ( ~ a s s ehromatoadrams') of the variation in abundance o f , , ~ d ions during ~ malysis. ~•

Admow[edgments

Much of the research ~"; the authors based on GC-MS has been aided by the Medical Research Council and Science Research Couned, to whom grateful acknowledgment is made•

References [ 1] C.J.W.Brooks and J,A, Zabkie~cz, in: Hormonesin Blood, Vol. 2., ed. by C.H. Gray, AcademicPress, New York (1967) 5 l [2] J. Drozd, J. Chromatogr. 113 (1975) 303 [3] c.J.W. Brooks, Proceu e|ochem, May (1967) 27 [4] M. Bygdemanand B. Samueluon;~ C ~ . Acta 10 (1964) 566 . . . . [5] H.-P.Klem_n~,U. Htntze and G' Gerck~ J. ~ o g z ! 75(1973) 19 [6] P.G.Devaux. M.G. Homing~ E.C. H o n l ~ , ~ i ; ~ . 4 (197I) [7] T. Lunkkaiwm,WJ.A. VendeaHeuvet,E.O~, H ~ ~ F C , H ~ , B i o ~ ; BiophyL Acta 52 (1961) 599 .... • ~ ~ . R. Vil~z, A¢.~ EndocxinoLSuppl. 109 (1966) 1 [9] E. Gil-Avan~ D. Numk. Adv,C~matogt, I0 (1974) 99 ~ [10| H. Elias, Adv.C~matogr. 7 (1968) 24~: [11]

[12~ [131 [14] [15] [161 [17] [181 [19l [201 [21l [22a] [22b]

CJ. W: Brooks et aL, Der~aeDes suitable for GC-&IS

[231 [24] [25] {261 [271 i281 [:z91 [3o1 [311 [321 [331 [341 [3.51

[361 [371 1381

[391 [401

[411 [421 [43] [441 [451 [461 [471 [48] [49.1 1801 1811 [521 183} 1541 1551 [861 1871 [SSl

[ssl 1601 [61]

CJ.W. Brooks arid2[.~A"~.-'.mn, Chem. C o m m u n (1967) 952 C.I.W. Brooks and L Watson, m: Gas Chromatography ed. by C~LoA. Harbourn~ instituteof Petroleum (t969) 129 I. Mexit%J. Lipid lies. 3 (1962) 126 L, Sickmm~n,B. Spiegelhaider ~nd H. Breuer, Z. A~aL Chem. 261 (1972) 377 E. Men[hi and LK. Norym'~ ki, Biochem. J. 95 (1965) 1 G.M. Anthony and C,LW, ~rooks, Rest, Steroids, 3 (1968) 13] A.G. Smith and CJ.W. Brooks, J. Chtomatogro 101 (1974) 373 A.G. Smith and C.I.W. Brooks, J. Steroid Biochem. 7 (1976) 705 C.V. Viswanathan, J. Chromatogr. 98 (1974) 105 P. CapeUa and C.M. Zorzut, Amd. Chem. 40 (1968) 1458 J.A. M¢Closkeyalq~lMJ. McClelland, J. A.n. Chem. Soc. 87 (1965) 5090 CJ.W. Brooks and I. Mac,lean, J. Chromatogr. Sci. 9 (1971) 18 B.A. Andersson and R.T. Holm~m,Lipids 10 (1975) '116 V. Dommes, F. Wirtz-Peitz and W.-H. Kunau, J, Chromatogr. Sci. 14 (1976) 360 C. Pace-Asciakand L.S. Wolfe, J. ChromatogL 56 (1971) 129 K. Gr6en, Chem. Phys. Lipids 3 (1969) 254 P.L. Taylor and R W. Kelly, Nature London 250 (19'74) 665 A.G. Smith, J.D. Gilbert, W.A. Hatland and CJ.W. Brooks, Biochcm. Soc. Transo 4 (1976) 108 A.G. Smith and CJ.W. Brooks, Biomed. Mass Spectrom. 4 (1977) 258 M.G. Homing, G. Casparrini and E.C. Homing, J. Chr~matogr. Sci. 7 (J 969~ 267 K.~A. Kaflsson, Acta Chem. Seand. 19 (1965) 2425 R.C. Gave, and C.C. Swe~My,J. Am. Oil Chem. See. 42 (1965) 294 SJ. Gaskell, C.G. Edmonds and CJ.W. Brooks, Anal. L~tt. 9 (1976) 325 SJ. Gaskell and CJ.W. Brooks, J. ChromatogL 122 (1976) 415 S J. Gaske/l, C.G. Edmond s and C J .W. Brooks, L Chromatogt. 126 (1976 ) 591 K~ ~Bamuelssonaral B. Samuelsson, Biochem Biophys. Res. Comm. 37 (1969) 15 B.~,. Andexmon, K.-A. Karlsson, I. Pascher, B.E. Samuelsson and G.O. Steen Chemo Pl~y:. Lil4d~ 9 (1972) 89 t" Homing and J.S. Young, Lipids 3 (1968) 391 CJ,W. Brooks, E,~. CJ .W. Brooks, W. Henderson and G. Steel, Bioctfim. Biophys. Acta 296 (1973) 431 D.R. Idler, M.W. Khalfl, J,D. Gilbert and C.t.W. Brooks, Steroids 27 (1976) ~55 e.G. Edmonds, A.G. Smith and CJ.W. Brooks, J. Chromatogx. 133 (1977) 372 Y.M, Sheikh and C. Djexassi, Steroids 26 (1975) 129 S. Burstoin, B.S. Middltditch and M. Gut, Biochem. Biophys. Res. Comm. 61 (1974) 69', D.P, Idler, L.M. $;afe and S.H. Safe, Steroids 16 (1970) 251 Ro~. Laine and A.D. Elbein, Biochemistry I0 (1971) 2547 C.C. SwooMy, in: Fundamentals of Lipid ChomLC~ry, eM. by RA4. Burton and F.C. Guerrl , BbScienoe,Webster Grows, Me., (1976) pp. 119-169 R.A. Klein and P. Kemp, in Methods in Membrane Biology, Vol. 8, ¢d. by E.D. Kern, Plenum Press, N~w York & London, (1977) p. 51 B,P. Lisboa, in: Fundamentals of Lipid ~hemistry od. by R,M. Burton and F.C. Gue~a, Bi-S~l Me., (1976) pp 439-473 K. Blau ~book of Derivatives for Chromatography, Heyden, London,

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S J. Gaskc~land CJ.W. Brooks, J. Chmmatogr., 142 (1977~ 469 J. Rosselt6,C. Sufiol, JAI. Tusen and E.Ge~ui, P,iomed;MusSpe~rom.,4 (1977) 237 A.G. Smith, W.A. Harland, and CJ.W. Brooks, J. Cl~omatogr,, 142 (1977) 533 U. Langenbeck, H.-U. M6hring, B. Hinney and M. Spiteller, Biomed. MttssSpeetxom., 4 (1977) 197 [72] G. Phillipou, Steroids 26 (1975)516 [73] SJ. Gaskell, CJ.W. Brooks and SJ3; Matin, BlOmed. ! [74] K.-S., Kwok, R. Venkatazeghav,tn andF.W. Mc~Laffe~ 19.73) 4185