Analysis of nat ive amino acids by liquid chromat ography / elect rospray ionizat ion mass Spect romet ry : compar at ive st udy bet ween t wo sour ces and int er f aces Marine de Person, Pat rick Chaimbault , Claire Elf akir I nst it ut de Chimie Or ganique et Analyt ique, CNRS FR 2708 UMR 60 05, Univer sit é d ’Or léans BP 6759, 45067 Or léans Cedex 2, Fr ance or if ice and f ocusing r ing Q0 IQ1 ST skimmer sample cur t ain plat e cur t ain gas nebulisat ion gas desolvat at ion gas ions t o mass analyser cone gas nebulisat ion gas desolvat at ion gas cône cleanable baf f le « aper t ur e » ion- t unnel 1 (RF Lens 1) ion- t unnel 2 (RF Lens 2) ion block sample ions capillary t o mass analyser T ype, f unct ion, opt imised values and inf luence on t he [M+H] + ion signal of inst r ument al MS paramet ers 2 Dr oplet evaporat ion +++ Desolvat at ion gas temperature = 350 °C +++ Desolvat at ion gas temperature = 350 °C Pr event ion of spect r omet er cont aminat ion, f inal dr oplet evapor at ion + Cone gas temperature = 120 °C - t emper at ur es Dr oplet evaporat ion + Desolvat at ion gas f low = 500 L/ hr 0 Desolvat at ion gas f low = 8000 mL/ hr Declust er ing, pr event ion of cont aminat ion 0 Cone gas f low = 50 L/ hr +++ Cur t ain gas f low = 6 Spray st abilit y Not measurable Nebulisat ion gas f low + N ebulisat ion gas f low = 10 gas I on t r ansmission 0 RF Lens 2 = 0,5 V - I on t r ansmission 0 Aperture = 0,2 eV - I on t r ansmission ++ RF Lens 1 = 0 V + Ent r ance pot ent ial = 10 V I on f ocalisat ion, ion t r ansmission - ++ Focusing pot ent ial = 200 V Declust er ing, CI D f r agment at ion +++ Cone volt age = 31 V ++ Declust ering pot ent ial = 20 V Gener at ion of ion spr ay cur r ent ++ Capillary voltage = 3 kV ++ Capillary voltage = 5 kV pot ent ials Funct ion I nf luence on t he [M+H]+ ion signal Micr omass Quat t ro Ult ima I nf luence on t he [M+H]+ ion signal PE- Sciex API 300 T ype of paramet er 3 I nf luence of t he mobile phase composit ion on t he response of underivat ized amino acids  I n cur t ain gas int er f ace ( Figure 1- a), a nit rogen cur t ain gas (N 2 ) f lows ar ound t he orif ice in order t o pr event clogging of t he or if ice by non- volat ile mat er ial, t o assist as a count er- cur r ent gas in droplet evapor at ion, and t o declust er t he ion- solvent clust er s by dr oplet gas collisions.  I n Z - Spray int erf ace (Figure 1 - b), ions are or t hogonally ext r act ed f rom t he at mospheric pr essur e ionisat ion (API ) sour ce int o a pumped chamber, and subsequent ly drawn int o t he high- vacuum chamber f or mass analysis. 1- a Curt ain gas int erf ace (PE- Sciex API 300) 1- b Z- spray int erf ace (Micromass Quat t ro Ult ima) All t he inst r ument al MS par amet er s (except ed t emper at ur e) have been opt imised by direct inf usion (at 10 μL/ min) of st andar d solut ions. Under t hese MS condit ions, t he pr ot onat ed molecule [M+H] + was t he most abundant ion f ormed in simple MS f or all t he AAs invest igat ed. As can be seen in t able 2 , t he most inf luent paramet ers are : - cur t ain gas f low and desolvat at ion gas t emper at ur e f or PE- Sciex API 300, - cone volt age and desolvat at ion gas t emperat ure f or Micr omass Q uat t r o Ult ima . W hat ever t he MS inst r ument used, t he opt imal t emper at ur e value (inducing t he most abundant [M+H] + ) is equal to 350 °C f or underivatized AA. The relat ive MS responses recorded f or AAs are dif f erent depending on t he nat ure of AA and t he int er f ace design (see Figures 4 - a and 4b).  PE- Sciex API 300 : t he det ect ion sensit ivit y is bet t er f or AAs cont aining a hydrophobic residue (Phe, I le, Leu, Trp, Val, … ) in compar ison wit h AAs cont aining very polar r esidue, except ed Ar g.  Micromass Quattro Ult ima : t he relat ive MS responses are dif f erent . AAs cont aining hydrophobic r esidue (Phe, T r p, Leu and I le) are st ill goodly det ect ed but t he dif f er ence bet ween hydrophobic and very polar AAs is weaker than API 300. Arg has the best response f act or , due t o it s weak dissociat ion in sour ce and in int erf ace. Nevert heless, some hydrophilic AAs such as Gly (2%), Ala (4%) and Ser (13%) remain badly det ect ed in compar ison wit h ot her AAs. The MS response of valine (46%) and Cys (7%) are weaker than those measured with API 300 (67 and 29%, respectively). The limit s of det ect ion (LODs) obt ained wit h API 300 and Quat t ro Ult ima were est ablished in LC- MS. The chr omat ogr aphic syst em consist s of a Pur ospher RP- 18e (125x2 i.d. ) mm oct adecyl column and an aqueous mobile phase cont aining 7 mM HFBA as ion- pair ing agent . Det ect ion limit s varied f rom 58 μM t o 3. 10 -4 μM depending on t he AA, t he scan mode (LODs (MS) > LODs (MS/ MS) ) and t he mass spect r omet er used (LODs (API 300) > LODs (Quattro Ultima) ). 4 Compar ison of MS r esponses f or pr ot eogenic amino acids 1- W hat ever t he MS inst rument used, t he adj unct ion of or ganic acid at 5 mM (HCOOH, CH 3 COOH, TFA, HFBA, NFPA, TDFHA or PDFOA) in an aqueous mobile phase allows t o increase t he ionisat ion ef f iciency ( see Figures 3- a and 3-b).  PE- Sciex API 300 : The signal of AAs is increased wit h the increase of perf luorinat ed chain. T her ef or e, sur f act ant s wit h t he longest side chain (TDFHA and PDFOA) lead to the highest MS r esponse f or all t he AAs invest igat ed.  Micr omass Q uat t r o Ult ima : sur f act ant s wit h t he shor t est side chain (HFBA and NFPA) proved t o be t he t wo most suit able ion- pair ing agent s f or t he det ect ion of AAs. 2- T he inf luence on AA response of N FPA concent r at ion has been also t est ed f rom 1 t o 100 mM (data not shown) :  PE- Sciex API 300 : concave evolut ion wit h an opt imum obt ained at 10- 20 mM of NFPA.  Micr omass Q uat t r o Ult ima : the mor e t he N FPA concent r at ion is import ant ( 100 mM), the more t he AA signal is high. 3- High per cent ages of or ganic modif ier (ACN) in t he mobile phase wer e more f avourable t han low per cent ages t o a bet t er sensit ivit y (dat a not shown). T he use of ACN st abilises t he ion spray and t heref ore impr oves t he backgr ound noise. T hus, t he mobile phase composit ion r equir ed f or t he LC separ at ion will be det er minant t o choose t he mass spect r omet er t he most appr opr iat e. A power f ul t echnique used f or t he separ at ion and ident if icat ion of underivat ized amino acids (AAs) is ion- pair ing reversed phase liquid chromat ography (I P- RPLC) coupled wit h elect r ospr ay ionisat ion mass spect romet ry (ESI - MS). These syst ems used volat ile perf luorinat ed carboxylic acids as ion- pairing agent s and oct adecyl silica [1] or por ous gr aphit ic carbon [2] as st at ionar y phases. I n order to elut e t he most ret ained AAs, an elut ion gradient based on simult aneously incr easing t he concent r at ion of acet onit r ile (ACN) and decr easing t he concent r at ion of t he ion- pairing agent is generally used. I n t he past f ew year s, inst r ument al development s in t he f ield of ESI - MS have been extremely f ast . Today, t he current ESI t echnology compr ises dif f er ent int erf aces which mainly dif f er in t he geomet r y of t he ion sampling device. I n t his work, we present ed a comparat ive st udy bet ween curt ain gas int erf ace (PE- Sciex API 300) and Z- Spray int erf ace (Micromass Quat t ro Ult ima) f or underivat ized AA analysis, in view t o help scient ist s t o choose among t hem, in r elat ion t o t heir analyt ical requirement s. H2O HCOOH TFA (C2) HFBA (C4) CH3COOH PDFOA (C8) TDFHA (C7) N FPA (C5) 0 50 100 relat ive int ensit y (%) PE- Sciex API 300 3- a relat ive int ensit y (%) H2O HCOOH TFA (C2) HFBA (C4) CH3COOH PDFOA (C8) TDFHA (C7) N FPA (C5) 0 50 100 Micromass Q uat t r o Ult ima 3- b Ser int ensit é relat ive des ions [M+H] + (%) 0 10 20 30 40 50 60 70 80 90 10 0 increasing value of Log D Arg Lys Glu His Gly Gln Cys Pro Ala Thr Val Tyr Met I le Leu Phe Trp PE- Sciex API 300 4- a 0 10 20 30 40 50 60 70 80 90 10 0 int ensit é relat ive des ions [M+H] + (%) increasing value of Log D Arg Lys Glu His Gly Ser Gln Cys Pro Ala Thr Val Tyr Met I le Leu Phe Trp Micromass Q uat t r o Ult ima 4- b [1] K. Petritis, P. Chaimbault, C. Elf akir, M. Dreux, J . Chr omat ogr . A, 896 (2000) 253- 263 [2] P. Chaimbault, K. Petritis, C. Elf akir, M. Dreux, J . Chr omat ogr . A, 870 (2000) 245- 254 based on inf ormat ion f rom t he manuf act ur er based on inf ormat ion f rom t he manuf act ur er View publication stats View publication stats