Application of Atmospheric Pressure Ionization Liquid

Chapter 25

Application of Atmospheric Pressure Ionization Liquid Chromatography—Tandem Mass Spectrometry for the Analysis of Flavor Precursors

Downloaded by UNIV OF IOWA on September 4, 2016 | http://pubs.acs.org Publication Date: August 13, 1996 | doi: 10.1021/bk-1996-0637.ch025

Markus Herderich, RenéRoscher, and Peter Schreier Lehrstuhl für Lebensmittelchemie der Universität Würzburg, Am Hubland, 97074 Würzburg, Germany The development of techniques utilizing atmospheric pressure ionization, namely atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI), has pioneered the coupling of liquid chromatography (HPLC) with mass spectrometry (MS) in recent years. Both ESI and APCI generate ions from polar and labile biomaterials with remarkable ease and efficiency. Particularly the use of liquid chromatography together with tandem mass spectrometry (MS/MS) opens further dimensions in the field of bioorganic analysis. Thus, HPLC-MS/MS provides the tools to elicit structure and variety of flavor precursor compounds directly in complex matrices. In order to develop efficient and straightforward strategies for the analysis of flavor precursor systems this chapter outlines the potential and limitations of those hyphenated analytical techniques. Finally, current applications demonstrate the successful analysis of glycoconjugates by atmospheric pressure ionization HPLC-MS/MS. In the past the efficient on-line characterization of flavor progenitors by mass spectrometry has been limited by the polar and labile character of these compounds. Combining HPLC with mass spectrometry could have been the method of choice for the analysis of glycoconjugates. But in order to transform molecules from solution to ions in the gas-phase one had to deal with three major incompatibilities (1): (i) the apparent problem of maintaining the high vacuum of the mass spectrometer after coupling with HPLC systems delivering flow rates up to 1 ml/min; (ii) the "soft" ionization of non-volatile and/or thermally labile compounds in order to obtain molecular weight information and (iii) the efficient transfer of the analytes as ions into the mass analyzer required for the analysis of trace compounds. For achieving the above mentioned goals researchers have developed a variety of methods like direct liquid introduction, thermospray ionization and the particle 0097-6156/96/0637-0261$15.00/0 © 1996 American Chemical Society

Takeoka et al.; Biotechnology for Improved Foods and Flavors ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

262

BIOTECHNOLOGY FOR IMPROVED FOODS AND FLAVORS

beam interface. It was only recently that the rediscovery of techniques utilizing ionization at atmospheric pressure, namely atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI), has directed the application of HPLC-MS to an ever-growing role in analytical chemistry.


Electrospray ionization (ESI) Based on earlier experiments of Dole and co-workers (2) and the work of Iribarne and Thompson (3, 4), Fenn and colleagues developed the technology of electrospray ionization in the mid-eighties (5,tf,7). In an electrospray interface the column effluent is nebulized into the atmospheric pressure region as a result of a strong electric field resulting from the potential difference between the narrow-bore spray capillary and a counter electrode. The field at the capillary tip charges the surface of the emerging liquid mainly by electrophoretic processes and not by electron transfer (8). As a result a Taylor cone is formed by the interaction of surface tension and coulombic forces from which a fine spray of charged droplets disperses. The droplets evaporate neutral solvent molecules until their surface charge density reaches the Rayleigh limit. Then the electrostatic forces overpower the surface tension resulting in a "Coulomb explosion" that produces an array of charged microdroplets which also evaporate until they "explode" themselves. As the last solvent molecules evaporate the charge would be retained by the analyte molecule to produce a free ion (7). Whether this "charged residue" mechanism actually applies is still under debate. An alternative explanation for the formation of single ions represents the "ion evaporation model proposed by Iribarne and Thompson which describes the direct emission of desolvated ionsfrommicrodroplets (4). While pure electrospray nebulization is only capable of flow rates up to 20 pl/min, the development of pneumatically assisted electrospray is compatible with flow rates exceeding 200 pl/min (9). In order to successfully ionize a compound by the electrospray process the analyte should dissociate in solution to form solvated ions prior to nebulization. Ion formation from species that are not ions themselves always requires the presence of a polar atom or functional group to which solute cations or anions can be attached by ion-dipole forces. Biopolymers carrying multiple functional groups will form multiple charged ions, an observation that has had an outstanding impact on the analysis of peptides, proteins and oligonucleotides (7). 11

Atmospheric pressure chemical ionization (APCI) In an APCI interface the column effluent enters a heated nebulizer where the pneu matically assisted desolvation process is almost completed. While still in the spray chamber, ionization of analytes is initiated by corona discharge. The ionization mechanisms in APCI are almost identical to those in conventional medium pressure chemical ionization (7). Positive ion formation can be achieved by proton transfer, adduct formation or charge exchange reactions, while in the negative mode ions are formed due to proton abstraction, anion attachment and electron capture reactions. The APCI interface is compatible with flow rates exceeding 1 ml/min and will


25. HERDERICH ET AL.

Flavor Precursor Analysis Using HPLC-MS/MS

263

generate molecular ions beside some thermal degradation products. Thus, APCI possesses a wider range of application for structure elucidation of smaller molecules, while ESI is the method of choice for the analysis of biopolymers, in particular, if the amount of sample is limited (Table I).

Table I. Comparison of APCI and ESI APCI

ESI

no restriction, „active" ionization

chargeable functional groups and preionization in solvent required

> 1000u

> 100000 u

side reactions

low to moderate thermal stress, avoid low pH of solvent

neglible after optimization of ionization conditions

HPLC solvent:

aqueous solvents

aqueous solvents as well as polar organic solvents

max. flow rate:

1 ml/min (4 mm columns)

0.2 ml/min (2 mm columns)


analyte: upper MW limit:

Application of tandem mass spectrometry for the analysis offlavorprecursors Both ESI and APCI generate molecular ions from polar and labile biomaterials with remarkable ease and efficiency. But the amount of structural information that can be deduced from ESI spectra, in particular, is rather limited. Thus, only the use of tandem mass spectrometry (MS/MS) together with liquid chromatography opens further dimensions in the field of bio-organic analysis (Fig. 1). Beside retention data and UV spectra one can obtain both molecular mass and substructure specific information of any analyte out of a complex matrix. Precursor and neutral loss scanning can be used to identify molecular masses of compounds with specific substructures. Subsequent structure elucidation usually involves generation of product ion spectra which reveals thefragmentationpatterns of the various molecular ions. Selective reaction monitoring by tandem mass spectrometry is the equivalent to single ion monitoring in conventional GC-MS and LC-MS analysis for the detection of known analytes in complex matrices. A compound is only positively identified by SRM, if it exhibits the correct molecular mass together with the characteristic fragment ion at a specific retention time. Thus, the two-dimensional spectral filter in SRM experiments combines highest structural selectivity with sensitivity of detection for the analysis of minor constituents and allows drastically simplified sample preparation procedures. Current examples of the successful application of HPLC-APCI/ESI-MS/MS during our studies on the generation of flavor compounds include the analysis of


264


scan mode

symbolism

product ion scan #^^?*

identification of compounds

precursor ion scan { ^ ^ 9 ==


mfbnnation

screening for substructures

neutral loss scan • ^i

screening for substructures

selected reaction % —* # monitoring srm

combining highest selectivity and sensitivity of detection

Figure 1. MS/MS experiments.

m/z 442: [perac.-M + NH ] 4

r

1:40

100l

111111111111111111111 5:00 6:40 8:20 10:00 time (min)

l I I I I M I I I I I I I M i l l I I I l l | l l l l l M i l l II I I

3:20

II l l | I

11:40

rn/z257: [M + H]

r

E+03 4.027

50

ioo m/z274: N HJ j 174: [M + NH 1

. E+03 1.768

,

4

50 0

100

JL RIC: 5 pg phenyl-p-D-glc

1UV 254 nm:

r

E+05 5.903

50

:50

1:"40

2:So3:'204:'lo"

time (min) Figure 2. Influence of peracetylation on ionization response. Sample: free and peracetylated phenyl P-D-glucopyranoside; ESI positive; HPLC: RP-18; 200 pl/min MeOH-H 0-5 mM NH Ac. 2

4


25.

HERDERICH ET AL.

Flavor Precursor Analysis Using HPLC-MS/MS

terpene phosphates in green tea leaves (70) as well as studies on the formation of fatty acid hydroperoxides and their related secondary metabolites catalyzed by lipoxygenase and allene oxide synthase (77). As to glycoconjugates, structure elucidation of O-glycosides (72), N-glycosides and esterified glycoconjugates (75,14) has been performed.


Analysis of glycosylated flavor precursors by APCI/ESI-MS/MS The application of both ionization techniques together with tandem mass spectrometry yields complementary information that allows localization and structure elucidation of glycosylated flavor precursors. Fig. 2 outlines the influence of introducing functional groups on the ionization response during the analysis of glycoconjugates. While free phenyl P-D-glucopyranoside is not ionized by ESI at all, its tetraacetate exclusively forms the ammonium adduct as molecular ion. For the analysis of acetylated glycoconjugates low detection limits in the sub-pmol range were established, thus demonstrating the excellent sensitivity of ESI. In contrast P-D-glucopyranosyl-anthranilate isolatedfromthe tropical pinuela fruit is instantaneously ionized by ESI without derivatization. Collision induced dissociation of the protonated molecular ion m/z 300 produces a product ion spectrum which is dominated by ions like m/z 138 characterizing the nitrogen containing aglycone (Fig. 3). Thus, a screening method yielding molecular mass information of glycoconjugates based on anthranilic acid can be developed by the subsequent analysis of the corresponding precursor ions. Acetylation of P-D-glucopyranosyl-anthranilate did not improve the ionization response and the corresponding product ion spectrum is less informative as most ions are formed by the consecutive loss of acetic acid (-60 u) and ketene (-42 u), respectively. Neutral loss of the aglycone results in an ion m/z 331 typical for peracetylated hexose moieties. Thefragmention m/z 162 appears to be solely derived from the acetylated aglycone (Fig. 3). In order to gain a better insight in the structure of more complex glycans, permethylation combined with ESI-MS/MS of adduct ions obtained by addition of metal ions to the solvent appears to be favorable (75). In the course of our studies on glycoconjugates derived from alcohols we observed that uponfragmentationinduced by low energy collision activation the substructure of interest (i.e. the aglycone) did not carry the charge. In that case the neutral loss scan can be used to screen for flavor progenitors derivedfromcompounds like phenylethanol and geraniol. Furthermore, underivatized glycoconjugates like the hexose derivative in Fig. 3 are characterized by the neutral loss of the carbohydrate moiety, thus opening a way for the specific detection of glycosides based on the sugar attached to the aglycone. Profiling flavor progenitors Substructure profiling by APCI/ESI-MS/MS provides structure specific information useful for analyzing the heterogeneity of glycoconjugated flavor precursors and allows selective localization of hitherto unknown compounds. For example, in


265

266


S

:

product ions of m/z 300 [M+Hf; R=H


i38.i

[aglycone + H]

+

„ B+04 7.48

282.5 2^3.9

162.0 ^8J).l

227.2

26.3.6

26d

117

lid

m/z product ions of m/z 510 [M+H] ; R=CH CO +

3

E+04 2.87

RO OR OR NHR

i|.9

27U?

2J0

3J0

1

m/z

389.9

W

OR

450.1

6

Application of Atmospheric Pressure Ionization Liquid

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