Spe c ial Iss ue : Review Arti c le

Received 12 August 2010, Revised 10 September 2010, Accepted 10 September 2010 Published online in Wiley Online Library: 6 November 2010

(wileyonlinelibrary.com) DOI 10.1002/bmc.1548

Derivatization reagent s in liquid c hromatography/ele c tro s pray ionization tandem ma ss s pe c trometry

Tomofumi Santa *
ABSTRACT: Liquid c hromatography/ele c tro s pray ionization tandem ma ss s pe c trometry (LC/ESI- MS/MS) is one of the mo s t prominent analyti c al te c hnique s owing to it s inherent s ele c tivity and s en sitivity. In LC/ESI- MS/MS, c hemi c al derivatization is often u s ed to enhan c e the dete c tion s en sitivity. Derivatization improve s the c hromatographi c s eparation , and enhan c e s the ma ss s pe c trometri c ionization e c ien c y and MS/MS dete c tability. In this review, an overview of the derivatization reagent s whi c h have been applied to LC/ESI- MS/MS is pre s ented , fo c u sing on the appli c ation s to low mole c ular weight c ompound s. Copyright 2010 J ohn Wiley & Son s, Ltd . Key word s: derivatization; chromatography; electrospray; tandem mass spectrometry

Introdu c tion
The development of sensitive and selective determination methods for trace level compounds is essential to elucidate their biological roles and functions in living systems. Recently, liquid chromatography/mass spectrometry (LC/MS) has frequently been utilized for this purpose. In particular, liquid chromatography/tandem mass spectrometry (LC/MS/MS) equipped with electrospray ionization (ESI) ion source is the most prominent method, since it requires lower temperature for ionization compared with other ionization methods, and thus it can be used for thermally unstable compounds. Furthermore, the decrease in the noise level by MS/MS detection improves the signal-to-noise ratios and hence enables sensitive detection of the targeted compounds. However, not all compounds can be favorably analyzed by LC/ESI-MS/MS. For example, the ionization eciencies are often extremely low and such compounds cannot be sensitively detected. An analyte should have the following properties to be sensitively analyzed by LC/ESI-MS/MS. Firstly, it must be in its ionic form in the solution phase or be chargeable through adduct formation in gas-phase reaction (Cech and Enke, 2001). Secondly, it is preferable to have a non-polar region, since hydrophobic compounds can be well separated from salts and interfering compounds possessing suppression eects on ESI (Nordstrom et al., 2004). The non-polar ions prefer the dropletair interface and reside at the droplet surface. Consequently these ions enter the gas phase more readily than those in the droplet interior and show a higher ESI response (Cech et al., 2001; Cech and Enke, 2000, 2001; Zhou and Cook, 2001). Furthermore, the hydrophobic compounds are eluted by the mobile phase with the higher organic solvent content on the reversed-phase column. The higher organic solvent content is suitable for the generation of charged droplets by electrospray (Cech and Enke, 2001) and thus gives the higher ESI response (Zhou and Hamburger, 1995). Generally, the chargeability and hydrophobicity of the analytes are the critical factors for the ESI response (Ehrmann et al., 2008; Henriksen et al., 2005; Okamoto et al., 1995; Nord-

strom et al., 2004). Thirdly, it is desirable that the target analyte fragments eciently upon collision-induced dissociation (CID) and generates an intense product ion for the sensitive MS/MS detection.

* Correspondence to: T. Santa, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail: santa@mol.f.u-tokyo.ac.jp Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan Abbreviation s u s ed : 4-APC, 4-[2-(trimethylammonio)ethoxy] benzenaminium halide; APDS, 3-aminopyridyl-N-hydroxysuccimidyl carbamate; 4-APEBA, 4-{2-[(4-bromophenethyl)dimethylammonio] ethoxy}benzenaminium dibromide; APZ, 4-(4-methyl-1-piperazyl)-3nitrobenzoyl azide; AQC, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate; CID, collision-induced dissociation; DAABD-AE, 4-[2-(N,Ndimethylamino)ethylaminosulfonyl]-7-(2-aminoethylamino)-2,1,3benzoxadiazole; DAABD-MHz, 4-[2-(N,N-dimethylamino)ethylaminosulfonyl ]-7-N-methylhydrazino-2,1,3-benzoxadiazole; DMAP-NCS, p(dimethylamino)phenyl isothiocyanate; DMIS, 1,2-dimethylimidazole-4sulfonyl; DNPH, 2,4-dinitrophenylhydrazine; DMAE, dimethylaminoethyl; Dns-Cl, 5-dimethylamino-1-naphthalenesulfonyl chloride; Dns-Hz, 5dimethylaminonaphthalene-1-sulfonyl hydrazine; ESI, electrospray ionization; FMOC, 9-uorenylmethyl chloroformate; FPMTS, 2-uoro-1methylpyridinium p-toluensulfonate; Gir P, 1-(carboxymethyl)pyridium chloride hydrazide; Gir T, (carboxymethyl)trimethylammonium chloride hydrazide; HMP, 2-hydrazino-1-methyl-pyridine; HP, 2-hydrazinopyridine; HTMOB, 4-hydrazino-N,N,N-trimethyl-4-oxobutanaminium iodide; MDMAES, mono-(dimethylaminoethyl) succinyl; NA, isonicotinoyl azide; NBD-F, 4-uoro-7-nitro-2,1,3-benzoxadiazole; NP-NCS, m-nitrophenyl isothiocyanate; 17-OHP, 17-hydroxyprogesterone; PA, 2-picolylamine; PPZ, 1-(2,4dinitro-5-uorophenyl)-4-methylpiperazine; PBS, 4-(1H-pyrazol-1-yl) benzenesulfonyl; PS, pyridine-3-sulfonyl; PrCl, propyl chloroformate; Py-NCS, 3-pyridyl isothiocyanate; SPTPP, 5-N-succinimidoxy-5-oxopententyl) triphenylphosphonium bromide; SRM, selected reaction monitoring; TFA, triuoroacetic acid; TMAE, trimethylaminoethyl; THAS, 4-(trimethylammonium)anilyl-N-hydroxysuccidimidyl carbamate iodide.

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T. Santa Chemical derivatization of the analyte is often used to enhance the detection sensitivity in ESI-MS. In the past decade, it was reported that the chemical derivatization of the analyte by the chargeable compounds improved ESI-MS responses of the target analytes (Quirke et al., 1994; Van Berkel et al., 1994, 1998, 2000). Since then, several conventional reagents originally utilized in ultraviolet or uorescence detection were borrowed for this purpose. These have summarized in several review papers (Gao et al., 2005; Honda et al., 2007a; Higashi and Shimada 2004; Higashi 2006; Johnson 2005; Santa et al, 2007a; Blair 2010). Most of these reagents are aimed to enhance the ESI response but they are not designed to generate a particular product ion by CID. Therefore, the fragment patterns of the derivatives depended on the structures of the generated derivatives. On the contrary, the reagents developed specically for LC/ESI-MS/MS carried a structure suitable for MS/MS detection. They were eciently fragmented by CID to generate intense and specic product ions. In this review, an overview of the derivatization reagents which have been applied to LC/ESI-MS/MS is presented, focusing on the applications to low molecular weight compounds.
Figure 1 . Derivatization reaction for ketones and aldehydes, and the product ion of the derivative: (a) Dns-Hz; (b) HMP; (c) DAABD-MHz.

Derivatization Reagent s
For Ketone s and Aldehyde s Conventional reagent s. Ketones and aldehydes are neutral functional groups. The ionization eciencies in ESI of these compounds are sometimes low. Therefore a chargeable moiety was often introduced to enhance the ionization eciency. Hydroxylamine reacts with ketones to form the corresponding oximes. Hydroxylamine was applied to the analysis of testosterone (Borrey et al., 2007; Kushnir et al., 2006a), adorenal steroids (Kushnir et al., 2006b), altrenogest (a steroid used for the control of estrus in horses; Lampinen-Salomonsson et al., 2006), and succinylacetone (the hallmark of hepatorenal tyrosinemia; Rashed et al., 2005). A signicant increase of detection sensitivity for altrenogest was observed (Lampinen-Salomonsson et al., 2006). The generated derivatives usually gave several product ions dependant on the analyte structure. DNPH (2,4dinitrophenylhydrazine) was used for the determination of aldehydes and ketones in disinfected water (Zwiener et al., 2002). The derivatives were detected in the negative-ion mode. Common product ions such as m/z 163, m/z 152 and m/z 122, derived from the reagent skeleton, were observed. The transitions of the [M - H]- ions to these product ions were used for SRM (selected reaction monitoring). Girards reagent P [1-(carboxymethyl)pyridium chloride hydrazide; Gir P] and Girards reagent T [(carboxymethyl)trimethylammonium chloride hydrazide; Gir T] are reagents that possess a permanent cationic charge. Gir P was used for the derivatization of 17-hydroxyprogesterone (17-OHP), the marker for congenital adrenal hyperplasia. Fragment ions at m/z 80, 93 and 121 were generated by CID. The transition of m/z 299 ([M]2+) to m/z 80 was used for SRM (Lai et al., 2001). Gir T was used for 5-formyl2-deoxyuridine, a major thymidine lesion generated by reactive oxygen species. The generated derivative gave the product ion at m/z 195, by the loss of a trimethylamino moiety (59 Da; Hong and Wang, 2007). Gir T was also applied to the analysis of succinylacetone in urine and dried bloodspots (Johnson et al., 2007). Usually, the product ion spectra of the derivatives with these reagents were not simple and clear.

Conventional reagent s having a s uitable s tru c ture for MS/MS. Dansyl hydrazine (5-dimethylaminonaphthalene-1sulfonyl hydrazine; Dns-Hz) was used for the analysis of malondialdehyde (Lord et al., 2009) and succinylacetone in dried blood spot specimens (Al-Dirbashi et al., 2006). Dansyl moiety is suitable for MS/MS detection. The generated dansyl hydrazone of succinylacetone selectively gave the product ion at m/z 170 by CID, assigned to the cleavage of dimethyaminonaphtyl moiety originating from the reagent (Fig. 1a). The product ion spectra of the derivatives were rather simple and clear. The transition of m/z 462 ([M + H]+) to m/z 170 was used for SRM (Al-Dirbashi et al., 2006). Cyclohexanedione was used for the analysis of aliphatic aldehydes (C3C10), including 4-hydroxynonenal and nonanal, the peroxidation products of fatty acids. Aldehydes were condensed with two molecules of cyclohexanedione in the presence of ammonia to form the tri-cyclic compounds (Hantzsch reaction). These compounds gave the common product ion at m/z 216 by CID, derived from the tri-cyclic structure. The transitions of [M + H]+ ions of the derivatives to m/z 216 were used for SRM (OBrien-Coker and Mallet, 2001). Similarly, 5,5-dimethyl-1,3cyclohexanedione was used for biogenic aldehydes. The derivatives gave the common product ions at m/z 273 or 274 by CID (Williams et al., 2005). The transitions of [M + H]+ ions of the derivatives to m/z 273 or 274 were used for SRM. Reagent s de signed for LC/ESI- MS/MS. One of the prominent reagents for carbonyl compounds is HMP (2-hydrazino-1-methylpyridine; Higashi et al., 2005a). HMP reacted with carbonyl compounds such as keto-steroids at 60C within 1 h in ethanol containing 0.5% triuoroacetic acid (TFA). The ionization eciencies of HMP derivatives were rather high and the generated derivatives gave a product ion at m/z 108 by CID, derived from 1-methylpyrizinoamino moiety (Fig. 1b). HMP was applied to the analysis of keto-steroids such as testosterone, 5a-dihydrotestosterone in prostate and prostatic tissue (Higashi et al., 2005a, b), testosterone in rat serum and brain (Higashi et al.,

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Derivatization reagents in LC/ESI-MS/MS 2006), testosterone in saliva (Shibayama et al., 2009), dehydroepiandrosterone (Higashi et al., 2007a), 5a-reduced pregnane-type neurosteroids in rat brain and serum (Higashi et al., 2007b), 17ahydroxypregnenolone and 17a-hydroxyprogesterone in dried blood spots from low-birthweight infants (Higashi et al., 2008a), and neuroactive androgen (Higashi et al., 2009). HMP is not eective for increasing the detection responses of di-oxo-steroids, since small molecules with a multi-charge are unstable in the gas phase and provided multiple ions. To overcome these problems, HP (2-hydrazinopyridine) was used for di-oxo-steroids such as androsterone and progesterone. The generated derivatives gave the intense product ions at m/z 322 and 348 for the derivatives of androsterone and progesterone, respectively (Higashi et al., 2007c). Although the structures of these ions could not be identied, it was inferred that they were formed by the loss of one HP moiety together with a part of the A-ring. HP was also applied for the analysis of 17a-hydroxyprogesterone (Shibayama et al., 2008). DAABD-MHz {4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7N-methylhydrazino-2,1,3-benzoxadiazole} reacted with aldehydes at 50C within 10 min in acetonitrile containing 0.5% TFA. The generated derivatives gave a predominant product ion at m/z 151 by CID, derived from the protonated (N,N-dimethylamino)ethylaminosulfonyl moiety of the reagent (Fig. 1c; Santa et al., 2008). 4-[2-(Trimethylammonio) ethoxy]benzenaminium halide (4-APC) reacted with aldehydes and generated amines in the presence of NaBH3CN.The generated derivatives gave the product ions at m/z [M - 59]+ and [M - 87]+, derived from the loss of trimethylammonium moiety and trimethylaminoethyl moiety. 4-APC was used for the analysis of aldehydes in biological mixtures (Eggink et al., 2008). Similarly, 4-{2-[(4-bromophenethyl)dimethylammonio]ethoxy} benzenaminium dibromide (4-APEBA) was reported. It contains a bromophenyl group and the isotopic signature of 79Br and 81Br isotope (100:98) provided conrmation of the presence of bromine in the derivatives (Eggink et al., 2010). HTMOB (4-hydrazino-N,N,N-trimethyl-4-oxobutanaminium iodide) was a modied Girard reagent. The derivatives gave a rather simple product ion spectra, derived from the neutral loss of 59 Da (trimethylamino moiety). HTMOB was applied to the proling of ketones, ketoacids and ketodiacids in the childrens urine samples by ESI-MS/MS without chromatographic separation (Johnson, 2007). For Al c ohols Conventional reagent s. Alcohols and phenols are neutral and rather hydrophilic compounds. Therefore, deivatization is often used to enhance the ionization eciency and to increase the hydrophobicity. Simple esterication of the analytes has sometimes been adopted for this purpose. Propionyl and benzoyl anhydride were used for the derivatization of bases, cytokinins, ribosides and intact nucleotides such as AMP, ADP and AMP (Nordstrom et al., 2004). The ESI response was enhanced by the formation of hydrophobic derivatives. In addition, the retention on a reversed-phase column was greatly increased, and the derivatives were separated without the need for an ion paring reagent, known for its unwanted suppression eects on ionization. Similarly, acetyl chloride was used for the derivatization of cholesterol (Liebisch et al., 2006). Conventional reagent s having a s tru c ture s uitable for MS/MS One of the most widely used derivatization reagents for phenols in LC/ESI-MS/MS is dansyl chloride (5-dimethylamino-1-

Figure 2 . Derivatization reaction for alcohols and the product ion of the derivative: (a) Dns-Cl; (b) picolynic acid; (c) NA; (d) MDMAES imidazole.

naphthalenesulfonyl chloride; Dns-Cl). Dns-Cl has a dimethylamino group as an ionization moiety and a hydrophobic aromatic structure. It reacted with phenols at 60C within several minutes in acetone and sodium bicarbonate buer (pH 10.5; Anari et al., 2002). The generated derivatives are easily separated on the reversed-phase column and sensitively detected by ESIMS. The derivatives also gave an almost single product ion at m/z 171 by CID, which was assigned to protonated dimethylaminonaphtyl moiety (Fig. 2a). SRM using the transition of quasimolecular ion of the derivative to m/z 171 or 170 resulted in a sensitive detection of the derivatives. So far, it was used for determination of steroids having hydroxyl group such as estradiol, ethinyl estradiol, estrone and the related compounds (Anari et al., 2002; Xia et al., 2004; Shou et al., 2004; Zhang et al., 2004; Nelson et al., 2004; Xu et al., 2005; Tai and Welch, 2005; Li et al., 2005a; Lin et al., 2007; Pedreira et al., 2007; Kushnir et al., 2008; Farlow et al., 2009; Petucci et al., 2010; Scarth et al., 2010), propofol (veterinary medicine; Beaudry et al., 2005), 1-hydroxypyrene (biomarker to monitor exposure to polycyclic aromatic hydrocarbons; Li et al., 2005b), icariin (Gong et al., 2007), 4-dimethylaminophenol (Zhuang et al., 2008) and multiple classes of phenols in blood (Chang et al., 2010). Picolynic acid was sometimes used for the derivatization of alcohols. It reacted with 7a-hydroxy-4-cholestene-3-one, a biomarker for bile acid biosynthesis, in 30 min at room temperature in the presence of the condensation reagents in tetrahydrofuran. The derivative gave the product ion at m/z 383 by CID, due to the loss of picolinic acid moiety (Fig. 2b). The transition of m/z 506 ([M + H]+) to m/z 383 was used for SRM (Honda et al., 2007b). Picolinic acid was used for the analysis of the steroids having hydroxyl groups (Honda et al., 2008; 2009a; Yamashita et al., 2007ac; 2008ac, 2009a, b). 2-Fluoro-1-methylpyridinium p-toluensulfonate (FPMTS) reacted with hydroxyl group at room temperature for 1 h in dichloromethaneacetonitrile containing

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T. Santa triethylamine. FPMTS was used for the analysis of testosterone, dihydrotestosterone (OBrien et al., 2009) and propofol (Thieme et al., 2009). Allylic isoprenols were derivatized with 3-nitrophtalic anhydride and analyzed by LC/ESI-MS/MS under the negative ion mode. The derivatization reaction proceeded at 70C for 30 min in pyridine. The generated derivatives gave the product ion at m/z 166 by CID. The transitions of [M - H]- to m/z 166 were monitored (Teshima and Kondo, 2008). Reagent s de signed for LC/ESI- MS/MS. Nishio et al. used 1-(2,4dinitro-5-uorophenyl)-4-methylpiperazine (PPZ) and 4-(4methyl-1-piperazyl)-3-nitrobenzoyl azide (APZ) for derivatization of steroids having a hydroxyl group. The generated derivatives were analyzed after the conversion to the quaternary amino compounds with methyl iodide. The quaternary amino compounds provided an almost single product ion by CID; for example, methylated PPZ-estradiol gave the product ion at m/z 504.3 [M 47]+. Although the product ions could not be identied, they were specic ions of the derivatives with satisfactory intensity. The detection responses in LC/ESI-MS/MS were signicantly increased by the conversion to the quaternary amino compounds (Nishio et al., 2007). However, introducing two permanently charged groups to the small molecules did not improve the detection responses, since the multi-charged small molecules are unstable in the gas phase. Therefore, the reagent having a high-proton anity group such as pyridyl group is favorable for the derivatization of di-hydroxysteroids. Higashi et al. used isonicotinoyl azide (NA) for the derivatization of di-hydroxysteroids such as estradiol and 5a-androstane-3a,17bdiol (Higashi et al., 2007c, d, 2008b). NA reacted with two hydroxyl groups of estradiol at 80C for 30 min in benzene. The generated derivative gave the product ion at m/z 139 by CID, which was assigned to the protonated pyridyl carbamic acid (Fig. 2c). The transition of [M + H]+ ion to m/z 139 was usable for SRM analysis (Higashi et al., 2007d). Xu and Spink compared four derivatization reagents having sulfonyl chloride as a reaction group, Dns-Cl, 1,2-dimethylimidazole-4-sulfonyl (DMIS) chloride, pyridine-3-sulfonyl (PS) chloride and 4-(1H-pyrazol-1yl)benzenesulfonyl (PBS) chloride. The product ion spectra of the dansyl and DMIS derivatives were dominated by ions representing derivatization reagent moieties. In contrast, the product ion spectra of PS derivatives and PBS derivative showed analytespecic fragments. These reagents were used for the analysis of steroidal estrogens (Xu and Spink, 2008). MDMAES [mono(dimethylaminoethyl) succinyl] imidazole was used for cholesterol and dehydrocholesterol. MDMAES imidazole reacted with hydroxyl group at 70C for 10 min in dichloromethane containing 1% triethylamine, and generated MDMAES ester. The ester gave the product ion at m/z 369 by CID, due to the neutral loss of MDMAES moiety (189 Da; Fig. 2d). Cholesterol and dehydrocholesterol in dried spots of plasma were analyzed by ESI-MS/MS without chromatographic separation (Johnson et al., 2001a). For Carboxyli c A c id s Con c entional reagent s. Carboxylic acids are detectable in the negative ESI-MS. However their sensitivity is rather poor because of the high background noise. In addition the mobile phases for the carboxylic acids separation are not always compatible with ESI-MS. Therefore carboxylic acids were usually transformed to the hydrophobic derivatives. Butanolic HCl derivatization of carboxylic acids is sometimes useful in LC/ESI-MS/MS. The generated butyl esters are rather hydrophobic and they often gave the product ion at m/z of ([M + H 56]+), due to the loss of C4H8 (56 Da) by CID. Methylmalonic acid is the marker for a group of metabolic disorders caused by deciency in methylmalonyl-CoA mutase or a defect in vitamin B12 metabolism. Its di-butyl ester gave the product ion at m/z 119, due to the loss of 2 C4H8. The transition of m/z 231 ([M + H]+) to m/z 119 was used for SRM (Kushnir et al., 2001; Magera et al., 2000; Schmedes and Brandslund, 2006). Butanolic HCl derivatization is also applied to the analysis of nitrotyrosine, the tyrosine nitration product (Delatour et al., 2002) and homocarnosine, a brain-specic di-peptide and the marker of heritable defect in GABA pathway (Jansen et al., 2006). One of the successful examples for ESI-MS/MS detection of carboxylic acids with butanolic HCl derivatization is the simultaneous analysis of amino acids and acylcarnitines in dried blood spots for newborn screening (Rashed et al., 1994, 1995, 1997). The generated butyl esters of amino acids were introduced to ESIMS/MS without chromatographic separation. Most a-amino acids butyl esters gave the intense product ions corresponding to the loss of HCOOC4H8 (102 Da) by CID. Therefore, the amino acid prole in biological samples can be obtained by the neutral loss scan of 102 Da. Acylcarnitines are the marker metabolites for inherited disorders related to organic acid and fatty acid metabolism (Millington et al., 1990). Acylcarnitines have a quaternary ammonium group and a carboxylic group in their structure. Their butyl esters were introduced to ESI-MS/MS and gave the common product ion at m/z 85 by CID (Rashed et al., 1994). Therefore, acylcarnitine prole can be obtained by precursor ion scan of m/z 85. These methods are currently widely used for the analysis of amino acids and acylcarnitines in urine, plasma, serum or blood (dried blood spot) (Rashed, 2001; Chace et al., 2003; Chace and Kalas, 2005; Garg and Dasouki, 2006; Roschinger et al., 2003; Carpenter and Wiley, 2002). Conventional reagent s having a s tru c ture s uitable for MS/MS. Several conventional reagents suitable for MS/MS were reported. HP (2-hydrazinopyridine) was used for the analysis of chenodeoxycholic acid and its glycine-conjugate. The generated derivatives gave the product ion at m/z 110 by CID, derived from the protonated 2-hydrazinopyridine moiety (Fig. 3a). The transitions to m/z 110 from the [M + H]+ of the derivatives were used for SRM (Higashi et al., 2010a). Higashi et al. reported the simple and practical method for the analysis of carboxylic acids using HP and 2-picolylamine (PA). Several biological important compounds having carboxylic acids were derivatizaed with these reagents. The generated derivatives of HP gave the strong product ion at m/z 110 and those of PA gave the strong product ion at m/z 109 by CID (Higashi et al., 2010b). 3-Hydroxy-1-methyl-piperidine was used for the derivatization of malonic acid (Honda et al., 2009b). The generated ester gave the product ion at m/z 98, derived from protonated 1-methylpiperidine moiety by CID (Fig. 3b). Similarly, 3-(hydroxymethyl)-pyridine was used for the polar molecules in plant extracts (Kallenbach et al., 2009). 4Dimethylaminobenzylamine was used for the analysis of valproic acid and its metabolite. The generated amide derivative gave the product ion at m/z 120 by CID, derived from dimethylamino phenyl moiety of the reagent. The transition of m/z 277 ([M + H]+) to m/z 120 was used for SRM (Cheng et al., 2007). Reagent s de signed for LC/ESI- MS/MS. DAABD-AE {4-[2-(N,Ndimethylamino)ethylaminosulfonyl]-7-(2-aminoethylamino)2,1,3-benzoxadiazole; Santa et al., 2007b} was synthesized and

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Derivatization reagents in LC/ESI-MS/MS the generated derivatives can be more easily separated from the interfering compounds on the reversed-phase column, and can be sensitively detected in ESI-MS. In addition, the increase in the molecular weight decreases in the background noise from the matrix, since the background is generally lower in the higher mass range. Acetyl anhydride was used for polyaromatic amines such as diaminopyrenes and aminonitropyrenes, human carcinogens (Straube et al., 2005). Acetylation improved peak shapes and reproducibility in LC of diaminopyrene, resulting in high signal-to-noise ratios. Acetyl anhydride was also used for sphingosine-1-phosphate (S1P) and dihydrosphingosine-1phosphate, an endogenous sphingolipid and the analog. The transition of m/z 462 ([M - H]-) to m/z 402 for S1P by CID, due to the neutral loss of acetic acid, was used for SRM (Berdyshev et al., 2005). Similarly, pentauoropropionic acid anhydride were used for diamines, markers for the exposure to isocyanates. The [M H]- ion was selected as precursor ion. The product ions of [M - H - 120]- or [M - H - 240]-, due to the neutral loss of one or two CF3CHF2-group, or m/z 119 corresponding to CF3CF2- ion, were monitored (Marand et al., 2004). Uutela et al. reported the comparison of PrCl (propyl chloroformate) and FMOC (9-uorenylmethyl chloroformate) as the derivatization reagents for amino acids (Uutela et al., 2009). FMOC reacted with the amino group, and the generated derivatives gave the product ion at m/z 179 derived from dissociation of FMOC moiety. The cleavage of the FMOC moiety (loss of 222 Da), producing a protonated amino acids, was commonly observed. PrCl reacted with both amino and carboxylic groups. The MS/MS spectra of PrCl derivatives showed more product ions. The loss of propanol (60 Da) was common for all the PrCl-derivatized amino acids. PrCl was used for the amino acids analysis in rat brain microdialysates. The derivatization of amino acids and dipeptides in cerebrospinal uids with PrCl was also reported (Fonteh et al., 2007). NBD-F (4-uoro-7-nitro-2,1,3-benzoxadiazole) was used for biogenic amines such as tryptamine, histamine, N-methylsalsolinol and agmatine. The characteristic product ions were produced for each derivative by CID. In case of agmatine, the transition of m/z 294 ([M + H]+) to m/z 277 was used for SRM (Song et al., 2004). NBD-F was applied to the determination of D-amino acids. The characteristic precursor to product ion transitions, m/z 297279 (NBD-Asp), m/z 269223 (NBD-Ser) and m/z 311293 (NBD-Glu) were monitored for quantication (Song et al., 2007). Conventional reagent s having a s tru c ture s uitable for MS/MS. Dns-Cl was used for musimol and ibotenic acid, bioactive compounds in mushroom. The generated derivatives gave the product ion at m/z 171, derived from protonated dimethylaminonaphtyl moiety of the reagent. The transitions of (M+) ions to m/z 171 were used for SRM (Tsujikawa et al., 2007). Dns-Cl was also used for the analysis of glutamic acid, the excitatory amino acid, in diusion medium (Timperio et al., 2007), a-uoro-balanine, 5-uorourasil, capecitabine (Licea-Perez et al., 2009), gemcitabine and 2,2-diuoro-2-deoxyuridine in human plasma (Bowen et al., 2009). Ethyl bromoacetate was used for the derivatization of trimethylamine, the marker metabolite of trimethylaminuria (Johnson, 2008). The major product ion at m/z 118, derived from loss of 28 Da (ethylene), was observed. Trietylamine in urine was analyzed by a ow injection ESI-MS/MS. Nitrobenzyl chloroformate was used for 3-amino2(S)hydroxypropylmethylphospheric acid, GABAB receptor agonist. The derivative gave the product ion at m/z 152, assigned to

Figure 3 . Derivatization for carboxylic acids and the product ion of the derivative: (a) HP; (b) 3-hydroxy-1-methyl-piperidine; (c) DAABD-AE; (d) TMAE alcohol.

used for the analysis of very long chain fatty acids, the markers of peroxisomal disorders. The generated amide derivative gave the product ion at m/z 151 by CID, derived from the protonated (N,Ndimethylamino)ethylaminosulfonyl moiety of the reagent (Fig. 3c). The transition of [M + H]+ ions to m/z 151 was used for SRM. Compared with standard gas chromatographymass spectrometric methods routinely used for this purpose, this LC/ESIMS/MS method is simpler, saves 75% of instrument time and requires one-tenth of the biological sample volume (Al-Dirbashi et al., 2008). DAABD-AE was also used for the analysis of the dicarboxylic acids such as glutaric acid and 3-hydroxyglutarate, the marker metabolites for glutaric acidemia type 1 in human urine (Al-Dirbashi et al., 2007). TMAE (trimethylaminoethyl) ester derivatives were used for the analysis of very long chain fatty acids, the diagnostic markers for peroxisomal disorders (Johnson et al., 2003). Fatty acids were treated with oxalyl chloride, dimethylaminoethanol, followed by the methylation with methyl iodide. These derivatives gave the product ion by the loss of 59 Da, derived from (CH3)3N moiety of the derivatization reagent (Fig. 3d), and each fatty acid derivative was detected by SRM. The generated derivatives were suitable for MS/MS detection. However, the three step derivatization reaction was tedious for routine assay. TMAE or DMAE (dimethylaminoethyl) ester derivatization of fatty acids and ESI-MS/MS analysis without chromatographic separation was reported (Johnson, 1999, 2000a, b; Johnson et al., 2001b; Johnson and Trinh, 2003). For Amine s Conventional reagent s. The compounds having amino group are easily protonated under acidic conditions and suitable for ESI-MS. However, the analysis of amines is often troublesome because of their high polarity, basicity and high water solubility. Chemical derivatization makes amines more hydrophobic and

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T. Santa duced characteristic fragment ions at m/z 177, derived from the reagent moiety (Fig. 4c). Amino acids were analyzed with the detection limits of atto-mole level (Shimbo et al., 2009a). APDS (3-aminopyridyl-N-hydroxysuccimidyl carbamate) was also designed for LC/ESI-MS/MS and used for the analysis of more than 100 compounds with amino group in biological uid. The generated derivatives were hydrophobic compared with corresponding THAS derivatives, and thus they were suitable for the separation on the reversed phase column. The transition of all the protonated molecular ions to the common fragment at m/z 121 was monitored (Fig. 4d; Shimbo et al., 2009b, 2010). SPTPP [(5-Nsuccinimidoxy-5-oxopententyl)triphenylphosphonium bromide] was used for the analysis of amines and amino acids. The hydrophobicity of the analytes increased by derivatization and the resolution of the analytes on the reversed-phase column was improved dramatically. The product ions derived from the reagent moiety were observed. The limits of detection of the SPTPP were sub-femtomole levels. SPTPP was used for the analysis of neurotransmitter 4-aminobutanolic acid (GABA) and oxidative stress markers in rat serum (Inagaki et al., 2010). The reagent developed for proteomics, iTRAQTM (Ross et al., 2004), was also applicable for amino acid analysis by LC/ESI-MS/MS. A comparison of iTRAQTM-LC-MS/MS, GC-MS and amino acid analyzer was reported (Kaspar et al., 2009).
Figure 4 . Derivatization for amines and the product ion of the derivative: (a) 4-nitrobenzyl chloroformate; (b) 3-pyridyl isothiocyanate; (c) THAS; (d) APDS.

Con c lu sion
The derivatization reagents in LC/ESI-MS/MS applied to the low molecular weight compounds are reviewed. The derivatization improved the separation eciency, ionization eciency and MS/MS detectability of the analytes. Some conventional regents aimed to enhance the ESI response but they were not designed to generate a particular product ion by CID. The fragment patterns of the derivatives and the eciencies depended on the structures of the analytes. On the contrary, the reagents designed for LC/ESI-MS/MS have a suitable structure for MS/MS detection. They were fragmented easily by CID and eciently generated particular, intense product ions. Ester, aromatic sulfonyl compound, urea or thiourea, hydrazone and alkyl quaternary amine were often easily cleaved by CID and were suitable for MS/MS detection. The prominent derivatization regents for LC/ESIMS/MS are still desired for the sensitive and selective detection of the various kinds of the compounds in the elds of biomedical analysis.

4-nitrobenzyl alcohol anion, which was generated by the cleavage of ester bond of the reagent (Fig. 4a). The transition of m/z 371 ([M - H]-) to m/z 152 was used for SRM (Blum et al., 2000). NIT (naphtylisothiocyanate) was used for the determination of eighteen kinds of primary and secondary amines in air samples. The thiourea moiety of the derivatives was easily cleaved by CID and gave a product ion. The derivatives of the primary amines gave the common base peak at m/z 144 and the fragment ion at m/z 127 by CID, whereas those of secondary amines gave the common base peak at m/z 186 and the fragment ion at m/z 128 by CID. This method provided the structural information of the analytes, and was suited for the analysis of complex environmental samples (Claeson et al., 2004). AQC (6-aminoquinolyl-Nhydroxysuccinimidyl carbamate) is the commonly used uorescence derivatization reagent for amino acids. AQC was used for the derivatization of b-N-methylamino-L-alanine and 2,3-diaminobutylic acid, a potential human neurotoxin and its isomer. The urea structure of the derivatives was cleaved by CID and gave the product ion at m/z 145 and 171 derived from the AQC moiety were observed (Spacil et al., 2010). Urea and thiourea moieties were easily cleaved by CID and suitable structure for MS/MS. Conventional isothiocyanates, 3-pyridyl isothiocyanate (Py-NCS), p-(dimethylamino)phenyl isothiocyanate (DMAP-NCS) and m-nitrophenyl isothiocyanate (NP-NCS) were applied to the derivatization of amines. Their generated derivatives gave the intense product ions at m/z 137, m/z 179 and m/z 137, respectively (Fig. 4b; Santa, 2010). Reagent s de signed for LC/ESI- MS/MS. THAS [4(trimethylammonium)anilyl-N-hydroxysuccidimidyl carbamate iodide] was the reagent designed for LC/ESI-MS/MS. It reacted with amino acids to form urea compounds. The derivatives gave the characteristic cleavage at the urea bond by CID, and pro-

A c k nowledgement s
This work was partially supported by a Grant-in-Aid for Scientic Research from Ministry of Education, Culture, Sports, Science, and Technology of Japan. The author thanks Dr Chang-Kee Lim, MRC Bioanalytical Science Group for his kind suggestions and valuable discussion.

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