handbook of seafood quality, safety and health applications (alasalvar/handbook of seafood quality,...

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5 Instrumental analysis of seafood flavour

Hun Kim and Keith R. Cadwallader

5.1 Introduction

The complex flavour of seafood is composed of equally important nonvolatile taste- andaroma-active components. Early investigations on seafood flavour focused mainly on thetaste-active components, which are generally non-volatile and low-molecular-weight extrac-tive components. These may be divided into two broad groups: nitrogenous compoundsincluding amino acids, low-molecular-weight peptides, nucleotides, and organic bases; andnon-nitrogenous compounds including organic acids, sugars, and inorganic constituents suchas mineral salts [1,2]. Study of the taste active constituents has attracted considerable atten-tion and their importance to seafood flavour has been thoroughly reviewed [3–6]. The firststudy of this type was conducted during the early 1900s [7]. Since that time, most investiga-tions in this area have involved the quantitative analysis of extractive components (mainlynucleotides and free amino acids) by wet-chemical and/or liquid chromatographic meth-ods, including ion exchange chromatography and high-performance liquid chromatography[6,8–14].

Volatile (aroma) constituents are key to flavour perception. Without aromas, it is verydifficult to identify the flavour of specific food products including seafood [15]. Seafoodaromas can be formed via several mechanisms, which may be subdivided into four categoriesaccording to a precursor-mechanism relationship:

1) enzyme-mediated conversion of lipids to aromas;2) autoxidative degradation of free fatty acids leading to the formation of volatile carbonyls,

acids, and alcohols;3) enzymatic conversion of sulphur- and nitrogen-containing precursors to volatiles includ-

ing dimethyl sulphide; and4) thermal decomposition of precursors upon processing or cooking [16].

The aroma components may contribute to the development of pleasant (characteristic flavour)or off-flavour characteristics of seafood. The characteristic flavour of seafood has beendescribed as green, melon-like, and iodine-like, while off-flavours include musty, fishy,woody, rancid, and petroleum notes [4,17]. Research on the volatile constituents of seafood

Handbook of Seafood Q uality, Safety and Health Applications

Edited by Cesarettin Alasalvar, Fereidoon Shahidi, Kazuo Miyashita and Udaya Wanasundara

© 2011 Blackwell Publishing Ltd. ISBN: 978-1-405-18070-2


Instrumental analysis of seafood flavour 51

has markedly increased since the introduction of gas chromatography (GC) coupled withGC-mass spectrometry (GC-MS) in the mid-1960s. Aroma is one of the most importantdeterminants of seafood quality and can profoundly affect consumer acceptability [1,17,18].As mentioned above, the flavour of seafood is comprised of both volatile aroma-active andnon-volatile taste-active components. The identification of volatile constituents of seafood,therefore, is a key first step to the full understanding of seafood flavour. This chapter focuseson procedures for isolation and extraction of volatile flavour components and describes recentadvances in analytical methodology for characterization of seafood flavour.

5.2 Isolation of volatile flavour compounds

Analysis of volatile flavour components in food is complicated due to the presence ofextremely low levels of volatile solutes in highly complex nonvolatile matrices. Isolation orsampling of volatiles should be conducted by taking advantage of their volatility or nonpolarnature prior to GC analysis [18]. There are numerous methods for isolation of volatiles froma food matrix. Methods most often employed in the analysis of volatile flavour componentsof seafood are summarized in Table 5.1 and discussed below.

5.2.1 Headspace sampling

Headspace sampling techniques take advantage of the volatility of aroma compounds, andinvolve several categories including static headspace, dynamic headspace (purge-and-trap),solid phase microextraction (SPME), solid phase aroma concentration extraction, in-tubesorptive extraction, and headspace sorptive extraction. In each case, however, the same fun-damental principle is employed; only volatile compounds are collected from the atmosphereadjacent to the sample, leaving the actual sample material behind.

5.2.1.1 Static headspace sampling

Static headspace sampling (SHS) is the simplest among the headspace techniques. In SHS,the sample is placed in an airtight vessel (vial) and the volatile components are allowedto come to equilibrium between the sample matrix and the surrounding headspace. Theequilibrium is affected by the temperature of vessel, sample size, and equilibration time, etc.[19]. Following this, the headspace vapour (0.1–2.0 mL) is injected into a GC using a gastightsyringe or by direct transfer to the injection port using a headspace sampler (sampling loop).SHS is covered in greater depth elsewhere [23]. Advantages of SHS include simple samplepreparation, low risk of artifacts, and elimination of reagent or organic solvent. The techniqueallows for the analysis of highly volatile low molecular weight aroma compounds in seafoods,such as acetaldehyde, methanthiol, trimethylamine, dimethyl sulphide, or 2-methylpropanal[36].

SHS has been used to identify volatile compounds in fish oil [37], salmon [38,39], whiteherring [40], and other fish species [41]. However, SHS is mainly used in the field of qualitycontrol or grade classification of seafood products by the analysis of certain target volatilecompounds, such as trimethylamine.


52 Seafood Quality, Safety and Health ApplicationsTa

ble

5.1

Met

hods

used

for

the

isol

atio

nof

the

vola

tile

flavo

urco

nstit

uent

sof

seaf

ood

prod

ucts

Isola

tion

meth

od

Pri

nci

ple

sof

the

tech

niq

ue

Ad

van

tag

es

Dis

ad

van

tag

es

Hea

dsp

ace

sam

pli

ng

Stat

iche

adsp

ace

sam

plin

g(S

HS)

Vola

tile

anal

ytes

cont

aine

din

the

head

spac

ega

sph

ase

unde

req

uilib

rium

are

sam

pled

bya

gast

ight

syrin

geor

othe

rm

eans

and

tran

sfer

red

toth

eG

Cco

lum

nfo

ran

alys

is[2

0].

Dire

ctan

dno

n-de

stru

ctiv

ean

alys

isof

the

vola

tile

anal

ytes

[21]

.

Easy

elim

inat

ion

ofin

terf

eren

ces

from

the

com

plex

sam

ple

mat

rix.

Abi

lity

toan

alyz

elo

wm

olec

ular

wei

ght

vola

tiles

with

outt

hepr

esen

ceof

aso

lven

tpe

ak[2

2].

Rela

tive

low

cost

per

anal

ysis

,eas

yau

tom

atio

n,an

dsi

mpl

ean

dfa

stis

olat

ion

ofth

evo

latil

es[2

1,22

].

With

draw

ing

and

tran

sfer

ring

only

smal

lpo

rtio

nof

the

head

spac

e(1

–2m

L)re

sults

inpo

orse

nsiti

vity

for

trac

ele

velv

olat

ilean

alyt

es[2

2,23

].

Not

suita

ble

for

isol

atin

gof

vola

tiles

with

high

boili

ngpo

ints

[22]

.

Dyn

amic

head

spac

esa

mpl

ing

(DH

S)C

arrie

rga

sco

ntai

ning

the

vola

tile

anal

ytes

abov

eth

esa

mpl

e(h

eads

pace

)is

cons

tant

lysw

eptt

hrou

gha

trap

,and

the

vola

tiles

are

reta

ined

onth

etr

ap,w

hich

resu

ltsin

the

conc

entr

atio

nof

the

anal

ytes

[22]

.

DH

Sw

asde

velo

ped

toov

erco

me

the

sam

ple

size

limita

tion

impo

sed

bySH

S[2

1].

Incr

ease

dto

talv

olum

eof

head

spac

e(1

00m

L–1

L),w

hich

may

resu

ltin

high

erre

cove

ryof

the

anal

ytes

and

prov

ides

grea

ter

sens

itivi

tyth

anSH

S[2

2].

Requ

ires

mor

eco

mpl

exan

dex

pens

ive

inst

rum

ents

,suc

has

addi

tiona

lthe

rmal

deso

rptio

nan

dcr

yofo

cusi

ngsy

stem

s.

Leng

thy

anal

ysis

time

due

tom

ore

anal

ytic

alst

eps,

incl

udin

gsa

mpl

epu

rgin

g,tr

apdr

ying

,tr

aptr

ansf

er,a

ndth

erm

alde

sorp

tion

oftr

ap[2

2].

Solid

phas

em

icro

extr

actio

n(S

PME)

The

anal

ytes

inth

eva

pour

phas

ear

eab

sorb

ed/a

dsor

bed

bya

smal

lvol

ume

ofan

extr

actin

gph

ase

(�1

�L)

,whi

chco

nsis

tsof

thin

poly

mer

icfil

ms

coat

edon

tofu

sed

silic

afib

res

prot

ecte

din

ane

edle

ofa

syrin

ge-l

ike

devi

ce[2

4].

Rapi

d,si

mpl

ean

dea

syto

auto

mat

efo

rth

eex

trac

tion

ofbo

thpo

lar

and

non-

pola

rvo

latil

es.

Hig

her

sens

itivi

tyto

war

dsvo

latil

eor

gani

cco

mpo

unds

com

pare

dw

ithSH

Sor

DH

S[2

5].

Adv

anta

geof

dire

ctth

erm

alde

sorp

tion

into

the

GC

inje

ctio

npo

rt.

Smal

lvol

ume

ofth

eex

trac

tion

film

allo

ws

for

only

vola

tiles

havi

nghi

ghpa

rtiti

onco

effic

ient

tobe

extr

acte

dw

ithhi

ghef

ficie

ncy.

SPM

Efib

res

are

rela

tivel

yex

pens

ive,

and

the

poly

mer

coat

ing

isfr

agile

and

easi

lybr

oken

[26]

.

Lim

ited

lifet

ime

ofth

efib

res

(up

to10

0an

alys

es).

Sam

ple

carr

yove

ris

som

etim

esdi

fficu

ltto

elim

inat

e[2

6],a

ndso

me

extr

aneo

uspe

aks

are

form

eddu

eto

part

iald

ecom

posi

tion

offib

reco

atin

g[2

7].



Sorp

tive

extr

actio

nSi

mila

rpr

inci

ple

toth

atof

SPM

Eex

cept

usin

ghi

gher

mas

sof

poly

mer

icfil

m(2

5–30

0�

L)[2

8].

Ove

rcom

esth

elim

ited

conc

entr

atio

nca

pabi

lity

ofSP

ME

[28]

.

Hig

her

reco

verie

san

dhi

gher

sam

ple

capa

city

,whi

chle

ads

tolo

wer

dete

ctio

nlim

its(c

apab

ility

ofis

olat

ion

oftr

ace

vola

tiles

)and

bette

rre

peat

abili

tyth

anot

her

head

spac

ete

chni

ques

[29]

.

Lim

ited

num

ber

poly

mer

icex

trac

tion

film

sav

aila

ble,

whi

chre

stric

tsth

em

etho

dto

the

isol

atio

nof

mai

nly

non-

pola

rvo

latil

eco

mpo

unds

(i.e.

only

non-

pola

rco

mpo

unds

are

extr

acte

dw

ithth

eav

aila

ble

PDM

Sco

atin

g)[3

0].

Solv

ent

extr

act

ion

an

dd

isti

lla

tio

nex

tra

ctio

nD

irect

solv

ent

extr

actio

n(D

SE)

The

anal

ytes

are

isol

ated

from

food

mat

rixby

extr

actio

nw

ithor

gani

cso

lven

ttak

ing

adva

ntag

eof

the

diffe

renc

ein

pola

rity.

Sim

ple,

none

edfo

rco

mpl

exeq

uipm

ent

and

larg

ese

lect

ivity

and

flexi

bilit

y[3

1].

Requ

iring

addi

tiona

lcle

an-u

pst

epin

orde

rto

rem

ove

non-

vola

tile

resi

dues

.

Emul

sion

form

atio

n,w

hich

may

lead

tolo

ssof

anal

ytes

,and

requ

ires

com

plic

ated

and

time-

cons

umin

gal

tern

ativ

est

eps

topr

even

tor

min

imiz

e[3

1].

Stea

mdi

still

atio

nex

trac

tion

(SD

E)SD

Eta

kes

adva

ntag

eof

vola

tility

ofth

ean

alyt

esan

dno

n-vo

latil

ityof

othe

rm

ajor

food

cons

titue

nts

[32]

.

Hig

hre

cove

ryof

stea

m-d

istil

labl

evo

latil

es[3

3].

Sim

plic

ityof

oper

atio

n,re

prod

ucib

ility

and

appl

icab

leto

broa

dra

nge

ofsa

mpl

es[3

1].

Poss

ible

deco

mpo

sitio

nof

vola

tiles

orpr

oduc

tion

ofar

tifac

tsdu

eto

pres

ence

ofw

ater

and

high

extr

actio

nte

mpe

ratu

re[3

4].

Poor

reco

very

for

pola

ran

dw

ater

-sol

uble

anal

ytes

[35]

.

Hig

hva

cuum

dist

illat

ion

extr

actio

nTh

epr

essu

reab

ove

the

acqu

eous

sam

ple

mix

ture

tobe

dist

illat

edis

redu

ced

tole

ssth

anits

vapo

urpr

essu

reca

usin

gev

apor

atio

nof

vola

tile

anal

ytes

incl

udin

gso

lven

tor

wat

er.

Hig

hyi

eld

ofvo

latil

esin

clud

ing

pola

rvo

latil

es,a

ndre

cove

ryof

auth

entic

flavo

rex

trac

ts[3

5].

Cha

nce

for

loss

ofth

ehi

ghly

vola

tile

trac

ean

alyt

es.


54 Seafood Quality, Safety and Health Applications

5.2.1.2 Dynamic headspace sampling

Dynamic headspace sampling (DHS) or purge-and-trap analysis involves the constant strip-ping of the volatile analytes in the atmosphere surrounding a sample by use of an inertcarrier gas such as nitrogen. The volatiles contained in the carrier gas are then enriched bytrapping onto adsorbent materials (generally porous polymers) or by cryogenic focusing.This technique greatly improves the efficiency of headspace sampling. In general, the term“purge-and-trap” is used when referring to liquid samples analyzed by bubbling the carriergas through the liquid, while DHS is used when the sample is a solid [22]. Tenax

R©(poly-2,6-

diphenyl-p-phenyl oxide) is the most widely used adsorbent material for DHS. However, theadsorbent material can be chosen according to the specificity of the target volatile analytes.The volatile analytes are desorbed (released) by heating the trap (thermal desorption), andthe released volatiles are sent to the analytical GC column for analysis. DHS has many of thesame advantages as SHS. Furthermore, volatiles isolated by DHS may more closely resemblethe actual aroma composition that is perceived during smelling. A major disadvantage ofDHS is that it is not efficient towards components of low volatility [19]. DHS is one of themost popular isolating techniques for seafood flavour analysis. DHS has been used by severalresearchers for the isolation of volatiles from various kinds of seafood, such as sea bream[42], herring [43], cooked lobster tail meat [44,45], boiled crayfish [46] and its waste [47],emerald shiner [48], and pickled fish [49].

5.2.1.3 Solid phase microextraction

Solid phase microextraction (SPME) is a relatively new technique for the rapid, solventlessextraction of volatile compounds based on their partitioning between the sample or sampleheadspace and a polymer-coated fibre. The fibre is attached to a stainless steel plunger,sheathed by a protective needle, which is essentially a modified syringe to enable thermaldesorption of the analytes into a GC injection port. The selectivity of volatile extractionfrom the headspace depends on the choice of the fibre, and two factors, such as polarity andvolatility and molecular weight of target analytes, need to be considered [30]. SPME is anequilibrium technique and therefore the volatile profile one obtains is strongly dependentupon sample composition, and careful control of all sampling parameters is required [15].Comprehensive reviews of SPME have been published elsewhere [50–52]. Recently, solidphase aroma concentration extraction (SPACETM) was introduced as a modification of SPME,with the aim of increasing the area of the adsorbent so as to improve sensitivity (over 30 timesmore than SPME) [30,53]. SPACETM consists of a stainless steel rod coated with a mixtureof adsorbents, mainly graphite carbon [28]. Use of SPME for the analysis of seafood flavouris limited [54], with most applications related to the monitoring of quality control factorssuch as freshness and spoilage indicators rather than analysis of total volatiles [55–60].

5.2.1.4 Sorptive extraction

Among several sorptive extraction methodologies, in-tube sorptive and headspace sorptiveextractions (stir bar sorptive extraction) has recently been employed in the field of foodanalysis [61,62]. Both extraction techniques were developed to overcome the relativelylimited concentration capability of SPME [28].

In-tube sorptive extraction techniques include solid phase dynamic extraction (SPDE),which is also known as “the magic needle” [63]. SPDE employs a thick film (50 �m) of



polymer, which is coated onto the inside wall of the stainless steel needle of a gastightsyringe, in order to increase sensitivity. The analytes are accumulated in the polymer coatingby pulling in and pushing out a fixed volume of headspace to be sampled, through the gastightsyringe for an appropriate number of times within a fixed time. The trapped analytes are thenthermally desorbed into the GC injector [28].

In headspace sorptive extraction or stir bar sorptive extraction (commercialized by Gerstel(Mulheim an der Ruhr, Germany, under the name Twister), the headspace analytes arestatically accumulated by suspending a polydimethylsiloxane (25–250 �L) coated glassmagnetic stir bar in the vapour phase. After sampling, the stir bar is placed in a glass tubeand transferred to a thermo-desorption system where the analytes are thermally recoveredand analyzed by GC or GC-MS [28]. Although these techniques have not been used for theanalysis of seafood flavour, they have good potential for this application, especially if theanalysis is focused on the identification of trace level highly volatile or semi-volatile lowmolecular weight of components.

5.2.2 Solvent extraction and distillation extractions

5.2.2.1 Direct solvent extraction

One of the simplest and most efficient techniques for aroma isolation is direct solvent extrac-tion (DSE). DSE takes advantage of the difference in polarity between aroma compoundsand food matrix; most volatile aroma compounds are considerably less polar than aqueousfood matrix material. Solvent extraction can be as simple as putting a food sample into avessel such as a separatory funnel, adding a solvent (diethyl ether or methylene chlorideare good general purpose solvents), and shaking. The solvent phase is collected, dried withanhydrous salt (e.g. anhydrous sodium sulphate), and then concentrated (using distillationor nitrogen gas purging) prior to GC analysis. Another approach is to use liquid-liquid con-tinuous extractors when relatively large amounts of aqueous samples are available. In thecase of solvent extracts prepared from seafood, an additional clean-up step is often requiredin order to separate nonvolatile residues (e.g. lipid) from the volatile material. This can beaccomplished by steam distillation, high vacuum distillation, or DHS. Milo and Grosch [64]performed direct solvent extraction followed by high vacuum distillation for the isolation ofvolatiles from boiled trout, salmon, and cod. An alternative approach is to isolate the volatilecomponents from the sample by distillation, followed by solvent extraction of the aqueousdistillate [15,19].

5.2.2.2 Steam distillation extraction

The most common steam distillation method employs simultaneous distillation-solvent ex-traction (SDE), which is often called the Likens-Nickerson method. In SDE, volatiles aresteam-distillated from the sample (an aqueous solution or slurry of a solid material in water)by heating a sample flask, and simultaneously the solvent is distilled from another separateflask by mild heating. Vapours condense together on a cold finger where the extractionprocess occurs between both liquid films on the condenser surface. Water and solvent (con-taining volatiles) are collected and decanted in the separator, and are finally returned to theirrespective flasks [65]. SDE is often operated under reduced pressure in order to minimize theformation of thermally-induced artifacts. The aroma isolate prepared by SDE contains nearlyall the volatiles in a sample, but their proportions may only poorly represent the true volatile



profile of the sample. Despite this, the method is still popular due to its ability to recovervolatiles with medium to high boiling points [15]. SDE (atmospheric or reduced-pressureoperation) has been widely used for seafood flavour analysis [45,47,66–73].

5.2.2.3 High vacuum distillation extraction

High vacuum distillation, which is one of the early classical techniques, has been appliedto isolate low level (ppb to ppt) volatile components of food products containing high fatcontent. Volatile analytes are distilled from a sample for several hours under high vacuum(∼10−5 Torr) and mild heat (�60◦C) conditions, with subsequent condensation of volatiles ina series of cold traps. The volatiles compounds are later recovered from the condensed phaseby solvent extraction. Although losses may also occur during extraction and concentrationof solvent extract, this technique enables the isolation of a broad range of mid- to high-boiling trace-level flavour compounds at sufficient quantities for analysis [30]. High vacuumdistillation has been used for the determination of aroma-active compounds in cooked tailmeat of lobster [44]. Engel et al. [74] developed a new technique called solvent-assistedflavour evaporation (SAFE), which allows for faster and more efficient isolation comparedwith classic high vacuum distillation methods. The future prospects are excellent for thewidespread use of SAFE in seafood flavour analysis.

5.3 Instrumental analysis of volatile flavour compounds

Tandem GC-MS has been the technique of choice for the analysis of volatile food flavour. GCis ideally suited to deal with solutes in the vapour phase, such as volatile flavour components[19]. Mass spectrometry is one of the most powerful techniques for identification of unknowncompounds. Most research conducted on seafood flavour in the last few years has dependedon GC-MS as the main analytical tool. The technique is so standard and routine in flavourstudies of seafood that there is no need to describe it any further here [19].

Standard GC-MS used in flavour analysis is considered as fused silica, capillary column GCwith bonded phase, providing high resolution, combined with fast scanning, high-sensitiveMS operating in the electron impact ionization mode [19]. Despite the pre-eminence ofstandard GC-MS in flavour research, there are other approaches, which can provide valuableadditional and/or complementary information to GC-MS. These approaches are summarizedin Table 5.2 and discussed below.

5.3.1 Gas chromatography

5.3.1.1 Gas chromatography-olfactometry (sensory-directed analytical techniques)

A high resolution GC column coupled with a standard GC detector is capable of separatingand detecting hundreds of volatile compounds in a single run. However, it is likely that manyof these components have little or no impact on the actual aroma of the food. The aroma-activecomponents in the volatile isolate can be determined by combining GC with olfactometry(GCO). In GCO, the analytes are first separated by GC and then delivered to an oflactometer(sniffing port) where they are mixed with humidified air. Human “sniffers” continuouslybreathe (nasally) the air emitted from the olfactometer, and record the perceived odourdescriptions and intensities of the detected odorants. There are several excellent reviews



Tab

le5.2

Inst

rum

enta

lmet

hods

used

for

the

anal

ysis

ofth

evo

latil

efla

vour

cons

titue

nts

ofse

afoo

dpr

oduc

ts

An

aly

tica

lm

eth

od

Pri

nci

ple

sof

the

tech

niq

ue

Ad

van

tag

es

Dis

ad

van

tag

es

Ga

sch

rom

ato

gra

ph

y(G

C)

Gas

chro

mat

ogra

phy-

olfa

ctom

etry

(GC

O)

Hum

anpa

nelis

tsar

eus

edas

GC

dete

ctor

s(u

seof

hum

ansu

bjec

tsfo

rsn

iffing

ofG

Cef

fluen

ts)[

15,7

5].

Iden

tifica

tion

ofpo

tent

odou

r-ac

tive

com

pone

nt.

Infe

asib

ility

ofco

ntin

uous

anal

ysis

due

tool

fact

ory

fatig

ue.

Tim

eco

nsum

ing

due

toth

ehi

ghnu

mbe

rof

assa

ys(d

ilutio

ns)r

equi

red.

Nee

dfo

rw

ell-

trai

ned

anal

ysts

.

Mul

tidim

ensi

onal

gas

chro

mat

ogra

phy

Two

GC

colu

mns

ofdi

ffere

ntse

lect

ivity

are

linke

din

serie

s,an

dth

ean

alyt

esar

etr

ansf

erre

dfr

omth

efir

stco

lum

n(p

reco

lum

n)to

the

seco

ndco

lum

n(a

naly

sis

colu

mn)

toim

prov

eth

ese

para

tion

pow

er[3

0].

Gre

ater

relia

bilit

yin

iden

tifica

tion

ofm

inor

com

poun

dsin

com

plex

sam

ples

due

toin

crea

sed

peak

capa

city

and

sepa

ratio

npo

wer

[76]

.

Mor

eco

mpl

exan

dex

pens

ive

inst

rum

ent

com

pare

dto

conv

entio

nalG

C[7

6].

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orat

em

etho

dsde

velo

pmen

t,re

quiri

ngop

timiz

atio

nof

man

yse

para

tion

para

met

ers

[76,

77].

Ma

sssp

ectr

om

etry

(MS)

Hig

hre

solu

tion

mas

ssp

ectr

omet

ry(H

R-M

S)H

R-M

Sha

ving

aco

mbi

natio

nof

elec

tros

tatic

(vel

ocity

sele

ctor

)and

mag

netic

(mom

entu

mse

lect

or)s

ecto

rsfo

cuse

sio

nsac

cord

ing

tobo

thdi

rect

ion

and

velo

city

whi

ledi

sper

sing

acco

rdin

gto

mas

s-to

-cha

rge

ratio

[78]

.

Cap

able

ofac

cura

tem

ass

mea

sure

men

t,w

hich

allo

ws

for

the

dete

rmin

atio

nof

elem

enta

lco

mpo

sitio

n(m

olec

ular

form

ulas

)an

did

entifi

catio

nof

new

com

poun

d[1

5,78

].

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dfo

rex

pens

ive

inst

rum

ents

and

asp

ecia

listf

orth

eop

erat

ion.

Sele

cted

ion

mon

itorin

g(S

IM)m

ass

spec

trom

etry

Onl

yth

ein

tens

ities

ofse

lect

edio

nsar

em

onito

red

rath

erth

anen

tire

mas

ssp

ectr

um.

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ovem

ento

fsen

sitiv

ityfo

rta

rget

com

poun

ds.

Rete

ntio

ntim

e(R

T)-b

ased

tech

niqu

e,w

hich

can

occa

sion

ally

caus

em

isid

entifi

catio

nof

the

targ

etan

alyt

esdu

eto

chan

geof

the

peak

RTdu

ring

anal

ysis

.

(Con

tinue

d)



Tab

le5.2

(Con

tinue

d)

An

aly

tica

lm

eth

od

Pri

nci

ple

sof

the

tech

niq

ue

Ad

van

tag

es

Dis

ad

van

tag

es

Che

mic

alio

niza

tion

mas

ssp

ectr

omet

ry(C

I-M

S)Th

ean

alyt

esar

eio

nize

dby

ion-

mol

ecul

ere

actio

nsw

ithm

ostly

posi

tive

char

ged

reag

entg

asio

ns[7

9].

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muc

hfr

agm

enta

tion

ofan

alyt

es,w

hich

isus

eful

toco

nfirm

rela

tive

mol

ecul

arw

eigh

t[80

].

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ited

amou

ntof

stru

ctur

alin

form

atio

n–

addi

tiona

lana

lytic

alte

chni

que

orus

ein

com

bina

tion

with

EIis

requ

ired

inor

der

toov

erco

me

this

limita

tion

[81]

.

Neg

ativ

eio

nch

emic

alio

niza

tion

mas

ssp

ectr

omet

ryTh

epr

inci

ple

ofth

iste

chni

que

isve

rysi

mila

rto

that

ofpo

sitiv

eio

nC

I-M

Son

ly,

exce

ptfo

rus

ing

nega

tivel

ych

arge

dre

agen

tgas

ions

(suc

has

OH

− ).

Hig

her

softn

ess

and

sens

itivi

tyth

anpo

sitiv

eio

nC

I-M

Sin

man

yre

spec

ts[2

1].

Lim

ited

amou

ntof

stru

ctur

alin

form

atio

n–

addi

tiona

lana

lytic

alte

chni

que

orus

ein

com

bina

tion

with

EIis

requ

ired

inor

der

toov

erco

me

this

limita

tion

[81]

.

Tim

e-of

-flig

ht(T

OF)

mas

ssp

ectr

omet

ryC

reat

edio

nsfr

omio

nso

urce

are

acce

lera

ted

byan

elec

tric

field

and

allo

wed

todr

iftth

roug

han

eval

uate

dfie

ld-f

ree

regi

on(fl

ight

tube

)whe

reth

eyse

para

ted

into

grou

ps(is

omas

spa

cket

s)ac

cord

ing

toth

eir

mas

s-to

-cha

rge

ratio

,an

dth

efli

ghtt

ime

ofio

nsre

quire

dto

reac

hth

ede

tect

orth

roug

hfli

ghtp

ath

ism

easu

red

and

used

toca

lcul

ated

mas

s[8

2].

Mea

surm

ento

fall

ofth

eio

nsac

ross

the

m/z

rang

esi

mul

tane

ousl

y,w

hich

resu

ltsin

high

sens

itivi

ty,m

ass

reso

lutio

nan

dm

ass

accu

racy

[82,

83].

Blin

ding

effe

cton

mul

tiple

conc

urre

ntev

ents

inca

setw

oor

mor

eio

nsar

rive

atth

ear

ray

dete

ctor

atth

esa

me

inst

ant,

resu

lting

elec

tric

alpu

lse

tobe

reco

rded

asif

only

one

ion

had

arriv

ed–

adju

stm

entr

equi

red

toco

rrec

tthi

sef

fect

[80]

.

Elec

tro

nic

no

se(e

-no

se)

The

e-no

sefu

nctio

nsby

anal

ysis

ofth

ere

spon

ses

ofa

sens

orar

ray

toa

com

plet

ear

oma

mix

ture

,whi

chm

eans

ther

eis

nose

para

tion

ofar

oma

com

pone

nts

[15]

.

Rapi

dan

alys

isof

flavo

urs

with

out

sepa

ratio

nst

ep,w

hich

isat

trac

tive

for

qual

ityco

ntro

lin

the

food

indu

stry

[15,

84].

Con

tinuo

usan

alys

isw

ithno

sens

ory

fatig

uelik

ew

ithhu

man

subj

ects

.

Doe

sno

tpro

vide

any

spec

ific

deta

iled

chem

ical

info

rmat

ion

that

ispo

ssib

lew

ithG

C-M

Sm

etho

ds[1

5,84

].

Resp

onse

ofth

ese

nsor

sto

war

dno

n-ta

rget

vola

tiles

(i.e.

wat

erva

pour

orC

O2)m

ayal

ter

sens

orre

spon

sepa

ttern

s[1

5].

Nee

dfo

rse

vera

lsen

sors

(3–1

5)fo

rth

ean

alys

isan

dde

terio

ratio

nof

the

sens

ors

with

time

[15]

.



dedicated to GCO [75,85]. Some common methods based on GCO include aroma extractdilution analysis (AEDA) [86], Charm [87], and Osme [88]. These methods mainly differ inhow GCO data are recorded and analyzed.

Osme (time-intensity measurement) measures the perceived odour intensity of a compoundin the GC effluent. The subject rates the aroma intensity by using a computerized 16-point scale time-intensity device and indicates the corresponding aroma characteristics.This technique provides an FID-style aromagram called an osmegram [75]. AEDA andCharmAnalysis (dilution techniques) both rely on GCO of a serial dilution series of anaroma extract. In AEDA, each odour-active compound is assigned a flavour dilution (FD)factor, which is based on the highest extract dilution at which the odorant was last detected byGCO. FD factors are proportional to the odour unit values (compound concentration/odour-detection threshold). CharmAnalysis differs from AEDA in that the duration of the perceivedodour is taken into consideration in the calculation of odour unit values. AEDA has beenused to determine potent odorants in hake [89], boiled carp fillet [90], cooked turbot [91],skipjack tuna sauce [92], cooked spiny lobster tail meat [45], and boiled cod [93]. The use ofCharmAnalysis [18] and/or Osme [94] for the evaluation of seafood flavour is limited. Othermiscellaneous GCO techniques have also been used in the study of seafood flavour [40,58].

5.3.1.2 Multidimensional gas chromatography

With samples as complex as those encountered in a typical flavour analysis, even with the besthigh-resolution GC column components sometimes co-elute during GC-MS analysis, pro-ducing mixed mass spectra that are difficult to interpret [19]. Multidimensional GC (MDGC),which utilizes two different GC columns (having different selectivities) in series, termed apre-column and an analytical column, can often overcome this problem [19]. A thoroughdiscussion of MDGC can be found elsewhere [95]. Comprehensive two-dimensional GC,a type of MDGC, was recently developed, which allows greater separation efficiency thantraditional MDGC [30]. MDGC has been used in the identification of specific environmentalpollutant (PCB and dioxin) in seafood (Baltic herring) and seafood products (fish oil) ratherthan in the study of seafood flavour [96,97].

5.3.2 Mass spectrometry

Electron impact mass spectrometry (EI-MS) is the most common mass spectral techniqueused in flavour analysis, but alternative forms of MS may be employed following other massspectral techniques for certain specific problems.

5.3.2.1 High resolution mass spectrometry

Mass spectrometers may be classified as low-resolution (LR) or high-resolution (HR) in-struments. The LR instruments provide mass measurements to the closet whole unit mass.Since many combinations of atoms may give the same unit mass, LR-MS may provide themolecular weight of a compound but does not provide elemental composition. HR instru-ments provide sufficiently accurate mass measurements to permit determination of elementalcomposition [15]. HR-MS has not yet been widely exploited in seafood flavour analysis –currently focused on the analysis of environmental pollutants in seafood [98]. However,with the continuous improvements in the performance of commercial magnetic sector and



time-of-flight mass spectrometers, especially with regard to sensitivity at high resolution,this method will become more readily available in future [19].

5.3.2.2 Selected ion monitoring mass spectrometry

In the selected ion monitoring (SIM) mode, the MS continuously measures only selected ionsrepresentative of a specific compound, or group of compounds at very short time intervalsthroughout a GC run. The technique is extremely useful in enabling a very high sensitivityassay for the known component or types of components in question, but it does not contributeto the identification of unknown compounds, since full spectra are not recorded. For instance,geosmin ((E)-1,10-dimethyl-(E)-9-decalol), which is an environmental-related off-flavour inseafood products (described as earthy-musty) [99], can be detected at trace levels by selectingm/z 97, 112, and 125 with SIM mode [100].

An alternative approach is “mass chromatography”, which is useful for deconvolutingco-eluted GC peaks [101]. The difference is that complete mass spectra have been recordedthroughout the GC-MS run, rather than selected ions as in SIM. The data analysis system canthen be instructed to select appropriate specific ions from the full recorded spectra of the peak,with the objective of artificially resolving and recognizing the two (or more) components ofthe peak [19].

5.3.2.3 Chemical ionization mass spectrometry

In conventional EI-MS, sometimes no molecular ion peak is obtained in the mass spectrum ofa compound. This may be due to the instability of the molecular ion under the excessive energyimparted by electron impact (an energy of 70 eV is usually employed in EI-MS). If sometarget compounds in a sample are susceptible to the EI, a softer ionization technique shouldbe employed. Chemical ionization (CI) is the most common alternative, softer ionizationapproach in GC-MS. In CI-MS, a reagent gas, such as methane, isobutene, or ammonia,is introduced into the mass spectrometer source to be ionized by broadly conventional EI.A range of positive ions, such as C2H5

+ from methane, is produced. Sample moleculesare then ionized by ion-molecule reactions with reagent gas species. The result is that so-called pseudo-molecular ions are produced, such as (M+H)+, by proton transfer. Typicallyenergy of only 5 eV is imparted to sample molecules, so usually very little fragmentation isobserved under these conditions. The value of CI-MS in flavour analysis is to complementand supplement the data provided by EI-MS [19]. CI-MS is commonly used in stable isotopedilution analysis and has been applied to the analysis of important seafood aroma compounds[36,64,102].

5.3.2.4 Negative chemical ionization mass spectrometry

In addition to positive ion CI-MS, it is possible to perform negative ion CI-MS, in whichnegatively charged reagent gas ions, such as OH−, undergo similar ion molecule interactionswith sample molecules, but with the result that negatively charged pseudo-molecular ionsare obtained, such as (M-H)−, which is produced by proton abstraction. In many respects,negative ion CI-MS can be superior to positive ion CI-MS, both in terms of sensitivity anddegree of “softness”. Negative ion CI-MS has not been widely used in flavour analysis exceptin the case of target analysis, such as in stable isotope dilution analysis [103].



5.3.2.5 Time-of-flight mass spectrometry

The time-of-flight (TOF) mass spectrometer uniquely offers the ability to take a large numberof spectra across a GC peak. This is because TOF instruments employ a detector array forfull range mass detection, which means TOF-MS does not scan but rather measures all of theions across the m/z range simultaneously with a much faster spectra generation rate (50–500spectra/sec) than other types of spectrometers, such as quadrupoles (5–10 spectra/sec) or iontraps (10–15 spectra/sec). Therefore, TOF-MS has improved sensitivity and detection limits.The ability to take many spectra per unit time offers another advantage in facilitating thedeconvolution of mixed spectra that is resolving the MS data from one compound from amixture of compounds that co-elute. If it is required to resolve one compound from anotherto obtain a MS identification, TOF-MS with proper software is able to make identificationsand quantification frequently without the need for peak resolution [15,82]. TOF-MS hasbeen widely used in flavour analysis of foods, such as cooked beans [104], grains [105,106],wines [107,108], olive oil [109], roasted beef [110], and Cheddar cheese [111]. In the fieldof seafood, it has been mainly used for quality control (fish authentication by analysis ofbiomarkers) [112].

5.3.3 Electronic nose

The electronic nose (e-nose) offers a third technique (between instrumental and sensoryanalyses) for analyzing food aroma. E-nose is based on a process similar to the humanolfactometry system in that both e-nose and human olfactory systems consist of an array ofreceptors (sensors), yielding a pattern (signal) of response to any given aroma. The brain,in the case of humans, and the computer, in the case of e-nose, make judgments based on apattern recognition process as to the aroma and its quality [15].

In e-nose, the sample is placed into a glass vessel. Transfer of the headspace vapour to thesensor array can be achieved either by diffusion or by pumping the vapour to the sensors. Thesensors are key components of the e-nose system. Currently, there are several types of sensors,including semiconductor gas sensors, surface acoustic wave devices, biosensors/enzymesensors (designed to measure a specific compound), conducting polymer sensors, and massspectrometry-based sensors [113,114]. In the case of an MS-based e-nose, the analyst canprogramme the system to detect some target sensory-relevant volatile components. The datataken from the e-nose is usually statistically analyzed using software in order to interpretthe e-nose pattern towards the target analyte. Other detail reviews can be found elsewhere[113,114]. The e-nose has been widely employed in the quality control field of seafoods,such as detection of spoiling Alaska Pollack [115] and octopus [116].

5.4 Conclusions

There are numerous methods for the isolation and analysis of the volatile flavour componentsof seafoods and seafood products. Among the various isolation techniques, headspace sam-pling methods are relatively simpler and faster than solvent or distillation extraction methods.Headspace methods also have advantages for the isolation of analytes with low molecularweights and high volatilities. However, for the exhaustive isolation of seafood flavour com-pounds of intermediate and low volatilities, the later techniques are a better choice. In regardto the instrumental analysis of seafood flavour, the classic tandem GC-MS based method is



predominant. Indeed, for certain specific problems, alternative approaches may sometimesbe superior. Consequently, with a problem as difficult and complex as studying and ana-lyzing the flavour components of seafood and its products, it is recommended to considerall possible techniques and procedures and choose those that are available and which mightyield constructive information.

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