541

A methcl is de~criked f~,r the quantitative andysis aE f3-metfraxy~-suipka~kenyl)- etkylene &cat f&HiPG sulpkate) in kumdn urine, based aa selected ioa monitoring gas ckramatagrapky~ spectrametry and using a hpecifrcally deuterifx&akeIled analogue of MHXS sdpfu~te as int=zenref standard. Tke pracedwe involves extraction of tke wine sampIe an hmbedite PL%D-2. foIIawxE by isaktiion of MIiPG sulpkate bp cakzmrr ckramatagrspky cm Sephaden LEE-20. Ck33vqe of the su~ph&~ coajugixte aclcf formntian of the MEiPG fris(tir- fl~omscetate) derivative are czrried at& h a antitep reactiaa, witkarrt recourse ta eaqmic ky3ralysis.

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-es of the compouds of i&E-%I& ZS irrkEnn& Stim [2Qf. MHPG sulphste may be hyclro~ysed by the a&ion of acid [XBJ. but EiIlce free

NHFG is itself mstabie under these conditions ES,?, 91, n;ethod.s for the mea- surement of the s&ph.&e con&&-e zely cn tie “seIective’* z&ion uf sulphstase enzymes: mcorrjug&ed MEPG in urine is determined before and after hydro‘f- ysis and the levels of ZYEE?G sulphate are obbined by difference. Mtfiough emzymic hy&olysis is a powerftt technique for the qm&ative study of co@- gated met&Mites, the use of enzymes for qua&it&&e determinations on a routine basis mffers from a nu.z~ber of disadvantqzs. First, most SrrEphatase prepazatioos cotataLH low, and ofeen variable, unounts .of j3-ghmzrmidase and, if steps are not t&en to selectively inhibit &&mmmidase actitity [Zlft erro- neously high ~&ES fat MHPG sulphate v&l be ohtied. This problem may, however, be overcome if F&@G tiphate is separizted from the corPe5pondiRg glucuronide prior to enzymie hydm~ysis. Chromatugraphic systems bzzsed on thin-layer 1221, pqer ES] and ion-exchange chroxnsstography [2!2] hzve ken described fop the separzxtion of MXFQ and its conjugates, but these have not been adopt& for routine me.

A second diszdvzntage zssociated with the use of e&n& hydrolysis is that hydrolytic zctivi~ may be diminished by the presence in urine samples of en- dogenous inhibitors of enzyme action Es] or by the use of a large excess of the enzyme in the incubation 141. Problems of this n&ure cm be overcome &is- factorily oF_Iy through the use of m appropri&e, srpIpho-conjugated internal standard. Finally, certzin e-me prepmtions hzve &en shown to contain traces of MHPG itself and of other interfering substances [23], thus necessi- tating the use of a “bkmk’” imubation with etzch batch.

In view of -&e ahove disadvmtages, we have i~~~~tigzt& &ernative ap- proaches‘ to the ana.lysis of IWS?G &z.lphate in human mine. In the present com- munication, we report the deveiopment of zm improved stable isotope dilution may for urinary MHFG sulphate, of which a specific&y det&erium-l&e&d mdOgUe has been synthesised for use 2s inte~~nd st.mdar+ Fol.kmiSg exfmc- tion of MEEG and its conjugates from urine, MHPG sulph&.e is isoWed by !iquid-%el chrom~tqraphy zad is converted to a derivative suitabte for GC- MS LX’I~~YS~S by i: aovef procedure whkh does not entail hydrulysis by an aryl mum,_, -Lm srnpna-me.

54x

544

Tke cieuterzhd sulphate IX was stored at 4%” and stock solutions in dist&d water (concentration approx. 250 ~,&ml) were prepared as required. To aliquots of tkese sofutions wzs added 2 fixed amount of miMeHed MHPG sulpkz&e to serve ES intemal standard and tire mixtures were derivatised witi trBuoroacetic ankydride in etkyl acetate. The resrrEting MH??G fzis-TFA derivtt- tives were analysed by seEe&ed ion man&ring GC-MS, wken tke ratios of peak heights in the zecorbings far m/e 3% and 360 were ES& ‘m tzak&te the concentration of [2M,f-MWG salpkate in the origind sfack solutions. ,

TrAenty-four-kour urine sampEes were collected fmm ktltky laboratory per- somxel who were not receiving any medication, and we= stored at -iCF until itndYS&.

hzary MHPG sulpkcrfe. To an aliquot (5 ml) of a 24-k wine coUection in added E2H&SE’G sulphate (approx. 25 ,ug) in distiUed water (IQ0 .r.@. Tke sample is mixed tkorougkly on 8 Vortex mixer and is passfzd tkrougk a cohmn (PO X 2 em) of Amkedite X&D-Z. Tke cotumn‘is washed in turn witk water (LO ml) and kexane (Z.0 ml) and the vzaskings are discarded. MEFG sulphate is tken eMed witk metkharro! (30 ml) acid tke solvent is evaporated under reduced pressure. Tke residue is taken up in 2 ml cklomfonnetkflanol (P:L,- v/v, 0.01 M with respect to sodium cfrforide) and is applied ta a cok~mn (28 x I cm) of Sepkadex LH-28 (5 g), prepaped ir, and efuted witk tie same solvent system. Tke frzction corresponding ta 10-100 ml of effluent is taken to dryness under reduced pressure and is trsnsferred wttk m&kanoE (two B_S-mt potions)- ts a screw-c~ped Rea&vi&@. The salvent is evaporated under 2 stream of nitrogen and the residue is furtker dried for 2 k in 8 vacuum desiccator izi tke presence of pkospkororrs pa&aide.

546

E+ep;lr;ntion of &he internal stand2Ed, [zH&EB?G Sulph2h, W2S Carried out by modification ofa published procedure for the synthesis ofthhecorrespond- ing unlabelled conjugate [26f. Acetovanilfone (I) xw.s convepted to the bromo derhtive III, v&i,& was then heated under refiux v&h potassium acetate in monodeuteroetianot. This treatment gave the aeetoxy ketone IV with sim&a- IECUS incomoration of two atoms of deutetium in the side-chain. Reduetiorr of IV in situ v&h sodium borodeateride, follower2 by &&ne hydrolysis, afforded the ticleuteeratzd g&cot derikxtive V. AceQlation of V urd subsequent removal of the benzyl protecting group yiekkd [*H&WX?G dkcetate (VEf),wvhiehwas converted to the corresponding dph2te coniu&e VIII as described by Mum2 [29].Thiscompoiundwas purified asi~~pofassium salt by chrom2ta- ghaphy on Sephadex II&I-20 [30] ~3~3 finaEEy hydrolysed under alkz&ne con&

tions~0giveIZH3~Gs~phate(fX).

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REFERENCXS