Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The wine industry is an important sector of agriculture and wine analysis forms the basis of
assessing compliance of its commodities with regulatory standards and research in this field.
Liquid chromatography (LC) is extensively used for the determination of a wide range of nonvolatile
wine components, but conventional detectors impose performance limitations on the
technique that prevents its application to sophisticated analytical problems. In particular,
conventional detectors for LC often lack the sensitivity and specificity for the determination of
many wine compounds, especially trace level analytes, and furthermore, do not possess
spectral capabilities for compound identification or structure elucidation. The hyphenation of
mass spectrometry (MS) to LC has led to the introduction of a range of detectors that confers
high levels of sensitivity and selectivity to the technique. In addition, a wide variety of MS
architectures are available that are inherently suited for targeted analysis or structure
elucidation studies.
In this dissertation, the potential benefits of liquid chromatography – tandem quadrupole mass
spectrometry (LC-MS/MS) to solve analytical problems relevant to the wine industry are
explored. LC-MS/MS is a particularly versatile analytical technique because both mass
analysers can be operated in full-spectrum mode or selected-ion monitoring, which, together
with optional fragmentation, gives rise to four modes of operation that may be used for highly
specific and sensitive targeted analysis or spectral investigations. In multiple reaction monitoring (MRM) mode, both analysers are set at single ion frequencies
specific for the compound under investigation and one or more of its product fragments,
respectively. MRM mode is ideally suited for trace level analysis in complex mixtures, even in
cases where the target components are not resolved from interferences. In this study, MRM
detection was used to solve challenges relevant to the wine industry for the selective
quantitation of target analytes that could not be analysed by conventional LC methods. The
application of this approach for the analysis of natamycin, ethyl carbamate (EC) and 3-alkyl-2-
methoxypyrazines (MPs) in wine is demonstrated.
Natamycin is an antimicrobial preservative that is not permitted in wine in the European Union.
A rapid and sensitive method for the determination of natamycin was developed, and has been used since 2009 to regulate this vitally important sector of the South African wine export
industry.
EC is a natural carcinogen that occurs at trace level amounts in alcoholic products. It also has
the potential to accumulate in wines and can occur in very high concentrations in some fruit
brandies. The determination of EC is complicated by its physicochemical properties, and
available analytical methods suffer from drawbacks such as the requirement for elaborate
extraction procedures and high solvent consumption. A novel method for the determination of
EC in wines, fortified wines and spirits is described and it was applied to perform an audit of the
South African industry as well as to investigate factors responsible for its accumulation in
alcoholic beverages. This work forms an integral part of the food safety mandate of the State
and it ensures that export products comply with international norms for trade.
MPs are ultra-trace-level aroma compounds that contribute to the varietal character of
Sauvignon blanc wines. Their analytical determination is challenging due to their low levels of
occurrence. The loading capacity of LC combined with the sensitivity and resolving power of MS
was exploited to analyse concentrated extracts, in order to achieve very low limits of detection.
The performance of the LC-MS/MS method enabled the quantitation of these compounds at
their natural levels of occurrence, including the first quantitation and spectral confirmation of 3-
ethyl-2-methoxypyrazine in wine. Extensive data pertaining to South African Sauvignon blanc
wines are reported and statistical analysis is performed, reporting the correlation of variables
such as vintage and origin as well as wine parameters such as malic acid with wine MPs. Furthermore, the application of LC-MS/MS for structural elucidation and screening of target
classes of analytes was demonstrated for the analysis of red wine anthocyanins. The
anthocyanidin-glycosides are responsible for the colour of red grapes and wine, contribute to
the sensory properties of wine, and are also of interest due to their beneficial biological
properties. Their determination is complicated by their large numbers and structural diversity,
further exacerbated by diverse reactions during wine ageing as well as the lack of reference
standards for most members of this class of compounds. Tandem MS in scan mode was used
for the highly selective detection of glycosylated anthocyanins and derivatives, exploiting the
predictable elimination of the sugar moiety in neutral loss mode. Concurrent survey scan
experiments were used to unambiguously identify neutral loss detected compounds. The
method therefore follows a simplified and structured approach for unambiguous peak identification based on elution order and mass spectral information to impart a high level of
certainty in compound identification.
In summary, the work presented in this dissertation demonstrates that LC-MS/MS is a versatile
and powerful analytical approach for the analysis of diverse compounds of relevance to the wine
industry. The sensitivity and specificity of MRM mode, and the selectivity and spectral
capabilities of neutral loss and survey scan modes of MS/MS detection, is amply demonstrated
by the applications presented in the dissertation. / AFRIKAANSE OPSOMMING: Die wynbedryf is ‘n belangrike komponent van landbou en wyn-analise vorm ‘n integrale deel
van gehalteversekering ten opsigte van toepaslike wetlike standaarde. Wyn-analise is ook
belangrik in navorsing oor die samestelling van wyn. Vloeistofchromatografie word dikwels
aangewend vir die bepaling van ‘n wye verskeidenheid nie-vlugtige wynkomponente, maar
konvensionele detektors plaas beperkinge op die aanwending van die tegniek tot
gesofistikeerde analitiese toepassings. Meer spesifiek, konvensionele detektors vir
vloeistofchromatografie beskik nie oor die sensitiwiteit en selektiwiteit vir die bepaling van baie
wynkomponente nie, veral in die geval van spoorvlakanalise, en beskik boonop ook nie oor
spektrale vermoëns vir identifikasie van komponente en struktuurbepaling nie. Die koppeling
van vloeistofchromatografie met massaspektrometrie het ‘n reeks detektors tot die tegniek
toegevoeg wat hoë vlakke van sensitiwiteit en selektiwiteit bied. Verder bied die verskeidenheid
van massaspektrometrie-konfigurasies ook instrumente wat inherent geskik is vir geteikende
analise of struktuurbepaling, afhangende van die doel van die ondersoek.
In hierdie dissertasie word die voordele ondersoek wat verbonde is aan die aanwending van
vloeistofchromatografie – tandem kwadrupool massaspektrometrie om relevante analitiese
vraagstukke in die wynbedryf op te los. Hiedie tegniek is besonder toepaslik aangesien beide
massa-analiseerders in geselekteerde-ioon modus of in volle skandering gebruik kan word.
Tesame met opsionele fragmentasie, gee hierdie uitleg aanleiding tot vier funksionaliteite wat vir
hoogs sensitiewe geteikende analise of spektrale onledings gebruik kan word. Eerstens word beide massa analiseerders vir enkel-ioon frekwensies opgestel, spesifiek tot die
teikenkomponent en een of meer van sy produkfragmente, wat verkry word deur komponentspesifieke
fragmentasie. Hierdie modus is by uitstek geskik vir spoorvlakontleding van
komplekse monsters, selfs wanneer die teikenkomponente nie chromatografies van die matriks
geskei is nie. In hierdie studie is die tegniek aangewend vir die hoogs sensitiewe bepaling van
spoorvlak komponente wat nie met konvensionele detektors gemeet kon word nie. Die
aanwending van hierdie tegniek word gedemonstreer vir die spoorvlakbepaling van natamycin,
etielkarbamaat en 3-alkiel-2-metoksiepierasiene in wyn.
Natamycin is ‘n antimikrobiese preserveermiddel wat ontoelaatbaar is in wyn in die Europese
Unie. ‘n Vinnige en sensitiewe metode vir die bepaling van natamycin is ontwikkel, en word reeds sedert 2009 aangewend om hierdie uiters belangrike sektor van die Suid-Afrikaanse wyn
uitvoerbedryf te reguleer.
Etielkarbamaat is ‘n karsinogeen wat natuurlik voorkom in spoorhoeveelhede in alkoholiese
produkte. Dit kan ook onder sekere omstandighede akkumuleer in wyn en in hoë konsentrasies
voorkom in vrugtebrandewyne. Die bepaling van etielkarbamaat word bemoeilik deur sy
chemiese eienskappe, en gevolglik word analitiese metodes gekenmerk deur uitgebreide,
arbeidsintensiewe monstervoorbereiding en die gebruik van groot hoeveelhede, meestal giftige,
oplosmiddels. ‘n Nuwe metode vir die bepaling van etielkarbamaat in wyn, gefortifiseerde wyn
en spiritualië word beskryf en word aangewend om die faktore vir vorming daarvan te
ondersoek. Die metode word aangewend om die Suid-Afrikaanse bedryf te ouditeer in terme
van die voedselveiligheid mandaat van die Staat, en om te verseker dat uitvoere voldoen aan
standaarde vir internasionale handel. Metoksiepierasiene is vlugtige, ultraspoorvlak wynaromakomponente wat verantwoordelik is vir
die kenmerkede kultivarkarakter van Sauvignon blanc wyne. Hul analitiese bepaling word
bemoeilik deur hulle lae konsentrasies in wyn. Die ladingskapasiteit van vloeistofchromatografie
tesame met die sensitiwiteit en selektiwiteit van massaspektrometrie was benut om hoogs
gekonsentreerde ekstrakte te ontleed. Baie hoë vlakke van sensitiwiteit word sodoende verkry.
Die verrigting van die metode was voldoende om hierdie komponente teen hulle natuurlike
konsentrasies te kwantifiseer, insluitende die eerste kwantifisering en spektrale bevestiging van
3-etiel-2-metoksiepierasien. Omvattende data van die vlakke van hierdie komponente in Suid-
Afrikaanse Sauvignon blanc wyne word getoon en statistiese ontleding is gedoen om
korrelasies tussen veranderlikes soos oorsprong en oesjaar sowel as basiese wyn
veranderlikes soos byvoorbeeld appelsuur, met metoksiepierasienvlakke te ondersoek.
Verder was die toepassing van vloeistofchromatografie – tandem massaspektrometrie tot
struktuurbepaling en skandering vir groepe van komponente gedemonstreer vir die ontleding
van rooiwyn antosianiene. Die antosianien-glukosiede is verantwoordelik vir die kleur van rooi
druiwe en wyn, dra by tot die sensoriese eienskappe daarvan, en is ook relevant as gevolg van
die voordelige biologiese eienskappe daarvan. Die bepaling van hierdie komponente word
gekompliseer deur hulle groot getalle en strukturele diversiteit, verder bemoeilik deur die wye
verskeidenheid van reaksies wat hulle ondergaan tydens veroudering. Daar is ook ‘n gebrek
aan beskikbaarheid van standaarde vir die meeste van die lede van hierdie klas van komponente. Tandem massaspektrometrie was in skanderingsmodus gebruik vir hoogs
selektiewe deteksie van die antosianien-glukosiede deur die voorspelbare eliminasie van die
suiker komponent in neutrale verliesskandering te benut. Gelyktydige skanderings van die
komponente wat met neutraleverliesskandering waargeneem word, is gebruik vir
ondubbelsinnige komponent identifikasie. Die metode volg daarom ‘n eenvoudige en
gestruktureerde benadering vir piek identifikasie wat gebaseer is op chromatografiese orde,
sowel as massaspektrale inligting, om ‘n hoë vlak van sekerheid aan die identifikasie van
komponente te verleen.
Samevattend, word daar getoon deur die werk wat in hierdie dissertasie uiteengesit is dat
vloeistofchromatografie – tandem massaspektrometrie ‘n veelsydige en kragtige tegniek bied vir
chemiese analise relevant tot die wynbedryf. Die sensitiwiteit, selektiwiteit en spektrale
vermoëns van die tegniek word duidelik deur toepassings in die dissertasie getoon.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/71693 |
| Date | 12 1900 |
| Creators | Alberts, P. |
| Contributors | Stander, M. A., De Villiers, A. J., Stellenbosch University. Faculty of Science. Dept. of Chemistry and Polymer Science. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 202 p. : ill. |
| Rights | Stellenbosch University |
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