Effects of functional groups of acrylic acid derivatives as derivatization reagents for thiol compounds on molecular ion responses in electrospray ionization-mass spectrometry

JOURNAL OF MASS SPECTROMETRYJ. Mass Spectrom. 33, 1199È1208 (1998)

E†ects of Functional Groups of Acrylic AcidDerivatives as Derivatization Reagents for ThiolCompounds on Molecular Ion Responses inElectrospray Ionization-Mass Spectrometry

Kenji Matsuura* and Hideo TakashinaDevelopmental Research Division, Santen Pharmaceutical Co. Ltd, 9È19, Shimoshinjo 3-chome, Higashiyodogawa-ku, Osaka5338651, Japan

In order to explore the sensitivity enhancement of derivatization reagents for thiol compounds with respect to liquidchromatography-mass spectrometry (LC-MS) analysis, the electrospray ionization (ESI)-MS of tiopronin (TP)derivatives (TP-MA, TP-IBA, TP-AMD, TP-DMAE, TP-TFE, TP-AA and TP-AMDSA; Fig. 1) obtained fromthe Michael addition reaction using seven structurally diverse acrylic acid analogues was investigated. This studyfocused on the molecular ion species of TP derivatives and those response characteristics observed in LC-MS usingfour mobile phases that contained commonly used modiÐers (triÑuoroacetic acid, acetic acid, ammonium acetateand ammonium hydroxide). It was found that TP-DMAE gave an intense [M + H ]‘ ion in all the mobile phasesin the positive ion mode and its response was the highest among all of the TP derivatives owing to the high protonaffinity of a dimethylamino group. The other TP derivatives gave di†erent molecular ion species and lowerresponses based on the mobile phase used. The formation of a dominant [M Ô H ]— ion of TP-AMDSA wasobserved in all the mobile phases owing to the high acidity of a sulfonic acid group in the negative ion mode.TP-TFE also gave strong ion responses because it contains a high electronegative group in the negative ion mode.The ion intensities of most of the TP derivatives could be enhanced by the addition of acetic acid and ammoniumhydroxide to the mobile phase and suppressed by the addition of triÑuoroacetic acid and ammonium acetate in bothionization modes. These di†erences based on the mobile phase modiÐers used were strongly related to the ESIspray current on the ES capillary. 1998 John Wiley & Sons, Ltd.(

KEYWORDS: electrospray ; liquid chromatography-mass spectrometry ; thiol ; acrylic acid derivatives ; derivatization

INTRODUCTION

Thiol-containing compounds are labile due to the highreactivity of the thiol group and are easily transformedinto mixed disulÐdes.1 Therefore, in biological samplesit is important to stabilize thiol groups by immediatelyderivatizing them with appropriate reagents. Previousstudies demonstrated that acrylate derivatives, such asmethyl acrylate and isobutyl acrylate, are suitable sta-bilizers for thiol groups and show high reactivity forthiol groups. In addition resulting reaction compoundsare sufficiently volatile in gas chromatographicanalysis.2,3 The application of acrylate derivatives tothe stabilization of thiol-containing compounds has alsobeen described. Recently, a liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS)assay of thiol compounds using methyl acrylate for the

* Correspondence to K. Matsuura, Developmental Research Divi-sion, Santen Pharmaceutical Co. Ltd, 9È19, Shimoshinjo 3-chome,Higashiyodogawa-ku, Osaka 5338651, Japan.

stabilization was reported,4,5 but it may not be theoptimum reagent for thiol group stabilization inLC-MS analysis. Although methyl acrylate o†ers thevolatility, it does not contribute to enhance the sensi-tivity of MS detection. The desirable features of a deri-vatization reagent are rapid reactivity for thiol groupstogether with enhancement of the sensitivity of MSdetection.

ESI-MS is the most versatile soft ionization techniqueand allows direct interfacing with HPLC.6,7 The useful-ness of ESI in the analysis of biological samples hasbeen demonstrated for a wide range of molecules,ranging from proteins8,9 to small polar compounds.10Recently drug metabolism studies of pharmaceuticalchemicals have been performed on ESI-MS.11h13 TheESI process is strongly dependent on the solution chem-istry, the electrophoretic migration and the surfacebehavior of analyte ions.14h17 The formation of particu-lar ion species also depends on the ion affinity and themolecular structure of an analyte and LC-MS condi-tions used. The polar functional groups incorporatedinto molecules are particularly relevant to the responseof the molecular ion species produced. Moreover, the

CCC 1076È5174/98/121199È10 $17.50 Received 5th March 1998( 1998 John Wiley & Sons, Ltd. Accepted 20th August 1998

1200 K. MATSUURA AND H. TAKASHINA

composition of the mobile phase a†ects spray per-formance and ion production at the ESI interface, andthus has a major impact on the ionization process.18h20

We report here our Ðndings in an investigation of theionization response of seven tiopronin derivatives pre-pared by the Michael addition reaction of tioproninwith acrylic acid derivatives which have structurallydiverse functional groups. The study focuses on theresponse characteristics of molecular ion species in bothpositive and negative ion modes in ESI-MS with respectto the e†ect of mobile phase modiÐers such as variousacids, base and salt which change the pH of the mobilephase.

EXPERIMENTAL

Materials

Tiopronin (TP) is a product of Santen Pharmaceutical(Osaka, Japan). Methyl acrylate (MA), isobutyl acrylate(IBA), triÑuoroethyl acrylate (TFE), 2-(NN-dimethyl-amino)ethyl acrylate (DMAE), acrylamide (AMD),acrylic acid (AA) and 2-acrylamino-2-methyl-propanesulfonic acid (AMDSA) were purchased fromTokyo Chemical Industry (Tokyo, Japan). Methanol(HPLC grade), acetonitrile, acetic acid, triÑuoroaceticacid (TFA) and ammonium acetate were from WakoPure Chemical Industries Ltd. (Osaka, Japan). A 25%ammonia solution was purchased from Nacalai TesqueINC (Kyoto, Japan). Water was puriÐed using a Milli-Qsystem (Millipore, Bedford, MA, USA).

Preparation of TP-derivatives

The reaction product of TP with DMAE was preparedusing the following procedure : a 32 mg (0.2 mmol) ofTP were dissolved in 1 ml of acetonitrile and 0.2 ml ofwater. A 30 ll aliquot of triethylamine was added to thesolution and then 180 ll (2 mmol) of DMAE was addeddropwise with stirring. The reaction product wasallowed to stand for one hour with stirring. The reac-tion mixture was further puriÐed using silica gel columnchromatography and the desired derivative wasobtained. The other derivatives were prepared in thesame manner.

The methyl esters of TP-acrylate derivatives were pre-pared by the addition of a diazomethane solution to amethanol solution of TP acrylate derivatives (1 lmol).The reaction mixture was allowed to stand for 5 minand was then dried under a stream of nitrogen.

Liquid chromatograph-mass spectrometry

ESI-MS was performed on a Finnigan TSQ-7000 triple-stage quadrupole mass spectrometer (San Jose, CA,

USA). A Hewlett-Packard 1050 HPLC system (PaloAlto, CA, USA) was used for solvent delivery. The ESIspray voltage was set to 4.5 kV. Nitrogen was used as asheath gas with a pressure setting of 60 psi and an aux-iliary gas was not used. The heated capillary tem-perature was set to 240 ¡C.

For HPLC conditions, a gradient elution wasemployed using a combination of (B1) methanol :50 mM acetic acid (85 : 15) and (B2) methanol : 50 mMacetic acid (15 : 85). Initially, 100% of the mobile phase(B1) was delivered and then the proportion of the mobilephase (B2) was increased up to 100% in 5 min, and main-tained there until 15 min. The HPLC column used wasDevelosil ODS-HG5 (5 lm, 150] 2.0 mm I.D.,Nomura Chemical Co., Ltd., Aichi, Japan). The columntemperature was set to 40 ¡C and the Ñow-rate was0.2 ml/min.

Flow injection analysis

A volume of 2 ll of TP derivative (1 nmol) in methanolwas injected via a Rheodyne Model 7125 injector valveÐtted with a 20-ll loop. The Ñow rate was set at 0.2 ml/min. The following four mobile phases were used : (A)methanol : 10 mM TFA (1 : 1, pH 2.0), (B) methanol :50 mM acetic acid (1 : 1, pH 3.6), (C) methanol : 50 mMammonium acetate (1 : 1, pH 7.6), (D) methanol : 10 mMammonia solution (1 : 1, pH 10.8).

Q1MS spectra were acquired over a m/z range of100È600 with a scan speed of 1 s in the positive andnegative ion modes. The MS data were averaged fromtwo repeat injections. The peak area of the protonatedmolecule in the positive ion mode was calculated fromthe mass trace of the protonated molecule ([M ] H]`)ion. The peak area of the molecular ion species was cal-culated by displaying the reconstructed mass tracespeciÐed by all of the molecular ion species (e.g.[M] H]`, [M] Na]` etc.) observed[M] NH4]`,in the spectrum. The peak areas from mass traces of thedeprotonated molecule and the molecular ion species inthe negative ion mode were acquired in the samemanner.

Reactivity of acrylic acid derivatives with tiopronin inaqueous solution

A 50 ll of TP solution (12 lmol/ml) was added to0.1 ml of 50 mM TrisÈHCl bu†er (pH 9.1), and then50 ll of acrylic acid derivative solution (0.6 mmol/ml ;50 eq) were immediately added to the solution andmixed by vortex. After 10 min, a volume of 1 ll of thereaction mixture was injected into the LC-MS. Noanalysis column was applied and the mobile phase (B)was used. The protonated molecules were monitored forTP-MA, TP-IBA, TP-DMAE and TP-TFE and thedeprotonated molecules were for TP-AMD, TP-AA andTP-AMDSA. The reactivity was calculated by compar-ing the peak area of the TP-derivative observed withthat of the standard compound.

( 1998 John Wiley & Sons, Ltd. J. Mass Spectrom. 33, 1199È1208 (1998)

ESIMS OF ACRYLIC ACID DERIVATIVES 1201

RESULTS AND DISCUSSION

The addition of modiÐers to the mobile phase serves toimprove the LC separation. ModiÐers such as aceticacid, triÑuoroacetic acid, ammonium acetate andammonium hydroxide are commonly used in LC-MSanalysis. These modiÐers signiÐcantly a†ect the forma-tion of molecular ion species and the signal responsesobserved. Therefore, the signal responses of the molecu-lar ion species of seven TP derivatives (Michael addi-tion reaction products of TP with seven acrylic acidderivatives, Fig. 1) were investigated in four commonlyused mobile phases with di†erent solution pHs.

Signal responses of TP derivatives in positive ion mode

The peak areas obtained from the mass trace of theprotonated ions ([M ] H]`) and the reconstructedmass trace as the sum of the intensities of the molecularion species for seven TP derivatives in the positive ionmode are summarized in Fig. 2(I) and (II). The responseof the [M] H]` ion of TP-DMAE was signiÐcantlyhigher than that of the other derivatives in all of themobile phases tested. That of TP-IBA was the nexthighest followed by TP-TFE and TP-MA. On the otherhand, [M] H]` ion responses of TP-AMDSA andTP-AA were much lower than TP-MA. The responsesof the molecular ion species of TP derivatives wereobserved in the same order found in the [M ] H]` ionresponses [Fig. 2(II)]. In the ESI mass spectra ofTP-DMAE in the four mobile phases [Fig. 3(A)È(D)],the dominant [M ] H]` ion and the much less domi-nant sodium adduct ion ([M ] Na]`) were observed in

Figure 1. Structures of tiopronin and tiopronin derivativesobtained from reactions with acrylic acid analogues.

each mass spectrum. Compounds which have a func-tional group with high proton affinity, especially higherthan the proton affinity of ammonia, produced thedominant [M] H]` ion in the positive ion mode inESI-MS.18,19,21,22 Because TP-DMAE has a tertiaryamino group with high proton affinity in the mol-ecule, an intensive [M] H]` signal was formed in allof the mobile phases. A weak sodium adduct ion but noammonium adduct ion was detected owing to its highproton affinity. Weak [M ] Na]` ion of TP-DMAE isproduced in mobile phases (A) and (B). Compoundsthat have basic functional groups due to amide nitro-gens are often seen as sodium ion adducts.22 Although

Figure 2. Effect of the mobile phase composition on the ion response of tiopronin derivatives in the positive ion mode in ESI-MS: (I) peakarea from mass traces of ÍM½HË½ ions ; (II) peak area from mass traces as sum of signal intensities of molecular ion species. Mobile phases :(A) methanol : 10 mM TFA (1 : 1) (pH 2.0) ; (B) methanol : 50 mM acetic acid (1 : 1) (pH 3.6) ; (C) methanol : 50 mM ammonium acetate(1 : 1) (pH 7.6) ; (D) methanol : 10 mM ammonium hydroxide (1 : 1) (pH 10.8).



sodium has not been deliberately added to the mobilephase, it is generally present in pure solvents such aswater and methanol and results in the formation of[M] Na]` ions.22 For TP-IBA the [M] H]` ionwas observed as a base peak and the [M ] Na]` ionwas observed when mobile phases (A) and (B) contain-ing TFA and acetic acid were used [Fig. 4(A) and (B)].On the other hand, the ammonium adduct ion ([M

was dominant instead of [M] H]` ion in] NH4]`)mobile phases (C) and (D) containing ammoniumacetate and ammonium hydroxide [Fig. 4(C) and (D)].It was found that the response of the molecular ionspecies varied depending on the modiÐer added to themobile phase for all TP derivatives except TP-DMAE.The mass spectra of pharmaceutical chemicals whoseactive thiol groups were derivatized with methyl acry-late and isobutyl acrylate indicated similar responses forthe molecular ion species.4

The ESI process involves two steps ; the formation ofhighly charged droplets and the production of a gasphase ion.15,23,28 The ions observed in the ESI sourcedepend on the compounds ionized in the solvent beingsprayed, and hence the of compounds and the pHpKaof the solvent used strongly a†ects the ion responses.Moreover, the ion signal detected by MS may not resultfrom the ions at the droplet surface alone. The gasphase ions could be modiÐed in the ion sampling

regions of the mass spectrometer by an ion-moleculereaction.16 Therefore ion competition in the dropletsurface and gas phase ion-molecule reactions wouldcontribute to signal intensities. Some observations sug-gested that the ion produced from the electrosprayedcharged droplet did not reÑect the equilibrium concen-trations of ions in solution.29,30 For instance, the inten-sities of the [M] H]` ion of amino acids observedover the pH range 3È11 varied by factors of ca. 3, never-theless the calculated variation of equilibrium concen-tration in the bulk solution was expanded to severalorders. The formation of intense [M ] H]` ionthroughout the wide pH range was interpreted asfollows : solvated ions (e.g. in theH3O`, CH3OH2`)acetic acid solution and ion in the ammoniumNH4`hydroxide solution were produced in the evaporationprocess of solvent molecules from the highly chargeddroplet and then the subsequent proton transfer reac-tion to the sample molecule occurred and formed[M] H]` ion. Provided that this interpretation couldbe expanded to small organic compounds, the protonaffinity of sample molecule to be ionized would be themost important factor to predict the production of thedominant [M ] H]` ion. As described above, theproton affinity of TP-DMAE is higher than that ofammonia. Accordingly, the dominant ion formed is the[M] H]` ion in all of the mobile phases. DMAE has a

Figure 3. Positive ion ESI spectra of TP-DMAE in the four mobile phase systems; (A) methanol : 10 mM TFA (1 : 1, pH 2.0), (B) methanol :50 mM acetic acid (1 : 1, pH 3.6), (C) methanol : 50 mM ammonium acetate (1 : 1, pH 7.6), (D) methanol : 10 mM ammonium hydroxide(1.1, pH 10.8).



Figure 4. Positive ion ESI mass spectra of TP-IBA in the four mobile phase systems (see Fig. 3).

tertiary amino group whose value is estimated topKabe 10È11. Therefore, the calculated equilibrium concen-trations of TP-DMAE in the case of mobile phases (B,pH 3.5) and (D, pH 10.8) are di†erent, but for observedpeak areas from the [M] H]` ion in mobile phase (B)are a factor of 1.5 higher than those in mobile phase(D). This result supported the earlier reports that theESI mass spectra did not reÑect the equilibrium concen-tration of ions in solution. The suppression of the for-mation of protonated molecules for TP-IBA observed inmobile phases (C) and (D) containing ammonium ions[Fig. 4(C) and (D)] is due to the lower proton affinityof TP derivatives except TP-DMAE than thatof ammonia, nevertheless the dissociation of

adduct ion into neutral ammonia and the[M] NH4]`formation of [M ] H]` ion were thermochemicallyfavored.29 A possible explanation for the di†erent ionresponses of the TP derivatives other than TP-DMAEis that the proton affinity of the other TP derivativesmay be higher than that of water, methanol, acetic acidand TFA and lower than that of ammonia. The protonaffinity of most organic compounds containing oxygenor nitrogen atoms is generally higher than that ofwater.24 Hence, the proton transfer reaction to samplemolecules does not completely proceed and results inthe formation of the more intensive ion[M] NH4]`instead of the [M ] H]` ion.

Comparing the responses of the molecular ion speciesof TP derivatives in the four mobile phases, mobile

phase (B) signiÐcantly enhanced the total signal inten-sities for the vast majority of TP derivatives tested. Onthe other hand, mobile phase (A) strongly suppressedthe ion signals. These observations are comparable tothose in earlier reports.19,20,29 Di†erences in the ionresponses were also found between the two mobilephases containing ammonium ions (mobile phases (C)and (D)). The mass spectral proÐles of the ion species[Fig. 3(C) and (D) and Fig. 4(C) and (D)] were essen-tially the same, but the ion intensities observed formobile phase (D) were almost twice as high as those formobile phase (C). Concerning the concentration ofmodiÐers used, a signiÐcant decrease in analyte ionintensity occurs when ammonium acetate is present atconcentrations in the 10~2È10~1 M ranges.16 The ESIspray current from the ES capillary indicates the rate atwhich excess electrolyte ions leave the capillary.17 Themeasured spray current is composed of the real spraycurrent, due to the electrochemical oxidation or the oxi-dation of hydroxide ion at needle, and the currentÑowing through the mobile phase back into thegrounded parts of ESI Ñange.31 The current leakingback into the Ñange is dependent on the conductivity ofan electrolytic nature and most of the high spraycurrent observed consisted of the current leakingthrough the mobile phase. Because the observed currentis proportional to the total droplet charge, the extensivespray current indicates that the excess electrolyte ionsare present on the charged droplet surface and may



Figure 5. Effect of the mobile phase composition on ion response of tiopronin derivatives in the negative ion mode in ESI-MS: (I) peakarea from mass traces of ÍMÉHËÉ ions ; (II) peak area from mass traces as sum of signal intensities of molecular ion species.

a†ect ion competition on the droplet surface controllingthe ionization process. Under the present experimentalconditions, the ESI current is \1.2 lA in mobile phases(B) and (D), but it is about 15 lA in mobile phases (A)

and (C). Mobile phases which resulted in a high ESIspray current tended to suppress the overall signalintensities. This phenomenon may be caused since theelectrolyte ions in the mobile phase compete with the

Figure 6. Negative ion ESI spectra of TP-TFE in the four mobile phase systems (see Fig. 3).



Figure 7. Effect of the mobile phase composition on the ion response of tiopronin derivatives (methyl ester) in the negative ion mode inESI-MS: (I) peak area from mass traces of ÍMÉHËÉ ions ; (II) peak area from mass traces as sum of signal intensities of molecular ionspecies.

sample molecule to be ionized in the conversion processto gas-phase ions. The suppression e†ect of the highconcentration of electrolyte ions in the mobile phasecan be explained by the equations for a two-electrolytesystem.31,32 It is likely that the ESI spray current wouldbe useful for the determination of the concentration ofmodiÐers to be added and using a mobile phase withextensive high spray current should be avoided.

Signal responses of TP derivatives in negative ion mode

The peak areas obtained from the mass trace of the de-protonated molecule ([M[ H]~) and the reconstructedmass trace as the sum of the intensities of the molecularion species for TP derivatives in the negative ion modeare plotted in Fig. 5(I) and (II). The responses of the[M[ H]~ ion and the molecular ion species ofTP-TFE and TP-IBA were much higher than those ofthe other derivatives in all of the mobile phases. Theresponses for TP-MA were the next highest. Theresponses for TP-AMDSA were particularly strong inmobile phase (B). However, signiÐcant di†erences in theion responses based on the functional groups of theacrylic acid derivatives observed in the positive ionmode were not found in the negative ion mode. Com-pounds containing acidic groups (e.g. carboxylic acidand sulfonic acid) are e†ectively ionized in the negativeion mode.25,26 Because TP employed as a test com-pound has a carboxylic acid group in the molecule, allof the TP derivatives have an acidic group, which mayexplain why large di†erences in signal intensities amongthe TP derivatives could not be found. In the ESI massspectra of TP-TFE [Fig. 6(A)È(D)], only the adduct ion[M] TFA[ H]~ and no [M[ H]~ ion were foundin mobile phase (A), but the [M[ H]~ ion was domi-

nant in mobile phases (B), (C) and (D). Spectral di†er-ences based on the mobile phases used were also foundin the case of TP-IBA. As mentioned in positive ionmode, the absolute intensities of [M ] H]` ions varyslightly with pH and do not depend on the equilibriumconcentrations of ions in the bulk solution. Vice versa,the production of [M [ H]~ ion from the electro-sprayed droplets does not reÑect the equilibrium con-centrations. The proposed mechanism is interpreted asthe major ions produced in the charged droplet,hydroxide ion in basic solution or AcO~ ion in aceticacid solution, take part in the deprotonation reaction ofthe sample molecule.29,30 Moreover, the dissociation of[M] AcO]~ adduct ion to form [M [ H]~ ion isthermochemically favored.29 Considering the ionresponse characteristics of the [M[ H]~ ion of TPderivatives, the formation of intense [M [ H]~ ion inmobile phases (B), (C) and (D) is reasonable in view ofthe mechanism. The majority of ion intensities of[M[ H]~ ion in mobile phases (B) and (D) werealmost the same [Fig. 5(I)] and did not reÑect the con-centration of ions in the bulk solution equilibria despiteTP derivatives having a of 3.6 which is due topKathe carboxylic acid group. The observation of[M] TFA[ H]~ adduct ion in mobile phase (A) canbe explained by the equilibrium concentration based onthe value of the analyte and the pH of the mobilepKaphase, provided that the deprotonation from samplemolecule do not function in the TFA solution or theextensive lower pH solution and the ion production iscontrolled by the ions at the droplet surface. In mobilephase (A) (pH 2.0), the dissociation of TP-TFE was sup-pressed because the pH was lower than its hencepKa ,TP-TFE was emitted as the [M] TFA[ H]~ adduction and observed in the spectrum. On the other hand,the [M[ H]~ ion of TP-AMDSA was observed in allof the four mobile phases owing to the low value ofpKa



Figure 8. Negative ion ESI spectra of methyl esters of TP-AMDSA (A) and TP-TFE (B) in mobile phase B; methanol : 50 mM acetic acid(1 : 1) (pH 3.6).

TP-AMDSA based on the sulfonic acid group. Compar-ing the ion responses of the TP derivatives in the mobilephases tested, a similar tendency was observed in thepositive ion mode. Namely the ionization of analyteswas suppressed in mobile phases (A) and (C), andenhanced in mobile phases (B) and (D). The ESI spraycurrent observed in each mobile phase in the negativeion mode was almost the same as that in the positiveion mode. Increasing the pH of the mobile phase by theaddition of ammonia hydroxide was more e†ective forthe enhancement of signal intensities in the negative ionmode. This observation is consistent with those ofearlier reports.19,20

Signal responses of methyl esters of TP derivatives innegative ion mode

The TP derivatives possess a carboxylic acid group,which a†ect the evaluation of ion response character-istics based on the functional group of acrylic acidderivatives in the negative ion mode. To evaluate theactual di†erences in ion responses, TP derivatives wereconverted into their corresponding methyl esters andthe ion responses were determined under the same

Table 1. Reactivity of acrylic acid deriv-atives with tiopronin in 50 mMTris–HCl (pH 9.1) in 10 min

Acrylic acid derivatives Reactivity (mean ÀS.D.)a (%)

MA 95.9 À3.8

IBA 95.6 À1.6

DMAE 95.9 À3.5

TFE 97.8 À3.1

AMD 94.5 À8.4

AMDSA 17.8 À0.3

AA 5.7 À0.5

a n ¼4.

mobile phase conditions. As TP-AA gives the same pro-ducts as TP-MA, the preparation of the methyl ester ofTP-AA was not carried out.

The responses of the [M [ H]~ ion and the sum ofthe intensities of all molecular ion species observed arepresented in Fig. 7(I) and (II). A strong [M [ H]~ ionresponse for TP-AMDSA due to the presence of a sul-fonic acid group [Fig. 8(A)] and a weak [M[ H]~ ionresponse for TP-AMD were observed, but no molecularion response was observed for the other TP derivatives.On the other hand, the dominant adduct ion,[M] AcO]~, of TP-TFE [Fig. 8(B)] was observed inmobile phases (B) and (C), and its responses was muchstronger than that of TP-AMD and the other deriv-atives. Applying high voltages to the ESI capillary oftenleads to a corona discharge before a sufficient numberof charged droplets are produced in the negative ionmode.26 Electrons generated by the corona are readilycaptured by electronegative species such as oxygen toform the superoxide anion. This anion is the majorreactant negative ion, has a low electron affinity andwill transfer its charge to compounds with higher elec-tron affinities.24 TP-TFE contains a high electronega-tive group in the molecule, and hence it may contributeto the intense ionization of TP-TFE in the negative ionmode.

Reactivity of acrylic acid derivatives with tiopronin inaqueous solution

As shown in Table 1, TP quantitatively reacted within10 min with acrylic acid derivatives, except AMDSAand AA which have an acidic functional group in themolecule. The reactivity with AMDSA and AA did notincrease when the reaction mixtures were left up to30 min and were \25% for AMDSA and 7% for AA.Changes in the pH on addition of the acrylic acid deriv-atives to the reaction mixture were not observed. There-fore, the low reactivity of AMDSA and AA did notresult from decreasing of the solution pH. The derivat-ization conditions to improve the reactivity of the deriv-atives are under investigation.



Figure 9. Mass chromatograms of protonated molecule for standard solutions of TP derivatives (200 ng of each compound) on reversedphase LC condition.

Mass Chromatograms obtained from the trace of theprotonated molecule for standard solutions of TP deriv-atives (200 ng of each compound) are shown in Fig. 9.All TP derivatives were retained and separated onreversed-phase column in the mobile phase system (amixture of methanol and 50 m acetic acid solution).

CONCLUSION

The response characteristics of the molecular ion speciesof TP derivatives with structurally diverse functionalgroups were investigated. It is obvious that the ionresponse depends strongly on the functional group ofacrylic acid derivatives and the functional group whichexhibits the intensive responses di†ers in accordancewith the polarity of the detection mode. The selection ofan acrylic acid derivative to be used for the derivatiza-tion of thiol groups should be carefully carried outaccording to the functional characteristics of test com-pounds analyzed, and the best selection of the reagentallows the sensitive LC-ESI-MS analysis. Most acrylic

acid derivatives immediately react with TP in aqueoussolution (Table 1) and the resulting TP derivatives areseparable under reversed phase LC conditions (Fig. 9).The quantitative reactivity of acrylic acid derivativeswith a thiol compound in the biological material hadbeen demonstrated.2,3 Therefore, the several acrylic acidderivatives investigated here could be the valuable deri-vatization reagents in LC-ESI-MS analysis. Furtherpractical applications of the quantitative analysis forthiol compounds using acrylic acid derivativesdescribed in the present paper will be reported in a sub-sequent publication.27

ModiÐers added to the mobile phases function notonly to adjust the pH of the mobile phase and to be thecounter ion for the separation of analytes on LC butalso to be the gas-phase reactant ion, and lead to theformation of di†erent molecular ion species. The selec-tion and the concentration of the modiÐers used signiÐ-cantly a†ect the sensitivity of detection of MS.

Acknowledgement

The authors thank Dr K.-I. Harada of the Meijo University forreviewing the manuscript prior to publication.



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Effects of functional groups of acrylic acid derivatives as derivatization reagents for thiol compounds on molecular ion responses in electrospray ionization-mass spectrometry