Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: This study focused on the development of an analytical method whereby creatinine, creatine and caffeine could be determined quantitatively. Urine is the preferred body fluid for the analysis of metabolites that the body excretes after administration of medicinal and illicit drugs. The detection of these metabolites depends on the volume of water the patient has drunk or, in criminal cases, the amount of water the suspect may deliberately add to their urine to dilute it. Creatinine, whose concentration in urine has been found to correlate with muscle mass, is chosen as an endogenous control substance against which the metabolite concentration is compared. While high performance liquid chromatography with ultraviolet detection (HPLC–UV) is commonly selected for the analysis, the quality of chromatography is affected by the fact that creatinine, being highly polar, is not retained in the reversed-phase columns. Furthermore, urine contains many polar substances that elute with the solvent front along with creatinine, thereby grossly affecting HPLC measurements. Hydrophilic interaction chromatography (HILIC) is a good alternative, although these methods generally require extensive sample preparation. Direct infusion electrospray ionization mass spectrometry (DI–ESI–MS) is ideally suited to highly polar compounds and was selected for this work. Pneumatically assisted ESI is preferred above the standard ionization method of atmospheric pressure chemical ionization (APCI) since pneumatically assisted ESI disperses the solution into ion-containing aerosol droplets which do not promote online conversion of creatinine to creatine. The objective of this study was to develop a simple and sensitive DI–ESI–MS method for the determination of various compounds in urine with creatinine as analytical reference compound and internal standard (IS). The analytical method development includes addition of 1-methyl-3-phenylpropylamine as a primary IS to standard solutions as well as to urine samples, followed by direct infusion of the sample into a mass spectrometer to determine the absolute concentrations of creatinine, creatine and caffeine. After appropriate instrument conditions were established, linear graphs of analyte-IS signal intensity ratios were obtained. The ratio of the concentration of the analyte (drug or metabolite) to that of creatinine (as IS) may be used to determine analyte concentration in artificial samples and/or urine. This method is not affected by change in fluid volume or adulteration of urine samples because the analyte-to-creatinine ratio remains unchanged. As part of this study, the developed DI–ESI–MS method was compared with an LC–UV–MS method developed for this purpose. / AFRIKAANSE OPSOMMING: Hierdie studie fokus op die ontwikkeling van ‘n analitiese metode waardeur kreatinien, kreatien en kaffeïen kwantitatief bepaal kan word. Uriene is die voorkeur liggaamsvloeistof vir die analise van metaboliete wat deur die liggaam, na administrasie van mediese en onwettige middels, uitgeskei word. Die deteksie van hierdie metaboliete hang van die volume water af wat die pasiënt gedrink het, of in strafbare gevalle, die hoeveelheid water wat verdagtes met opset by hul uriene gevoeg het ten einde dit te verdun. Daar is bevind dat die konsentrasie van kreatinien in uriene met spiermassa korreleer, derhalwe is kreatinien as ‘n interne kontrolemiddel gekies waarmee die metaboliet-konsentrasie vergelyk kan word. Hoë-druk vloeistofchromatografie met ultravioletdeteksie (HPLC–UV) word algemeen vir die analise van kreatinien ingespan, maar die gehalte van die chromatografie word deur die hoogs polêre aard van kreatinien beïnvloed en het swak retensie in omgekeerde-fasekolomme tot gevolg. Bowendien, uriene bevat groot hoeveelhede polêre middels wat saam met kreatinien in die oplosmiddelfront elueer en sodoende HPLC-bepalings uitermatig beïnvloed. Hidrofiliese interaksiechromatografie (HILIC) is ‘n goeie alternatief, ofskoon omvangryke monster-voorbereidings algemeen vereis word. Direkte inspuitelektrosproei-ionisasiemassaspektrometrie (DI–ESI–MS) is ideaal geskik vir hoogs polêre stowwe en is vir hierdie studie gekies. Pneumatiese hulp-ESI word bo die standaard ionisasie-metode van lugdruk chemiese ionisasie (APCI) verkies weens pneumatiese hulp-ESI se vermoë om die oplosmiddel in aërosoldruppels wat ione bevat, te versprei – sonder die aanlynomskakeling van kreatinien na kreatien. Die doel van hierdie studie was om ‘n eenvoudige en sensitiewe DI–ESI–MS-metode te ontwikkel wat verskeie stowwe in uriene kan bepaal deur kreatinien as analitiese verwysingsmiddel en interne standaard (IS) vir die opstelling van ‘n IS-kalibrasiekurwe te gebruik. Die analitiese metode-ontwikkeling sluit die gebruik van 1-metiel-3-fenielpropielamien as primêre IS in. Die IS word tot standaard oplossings en urienemonsters gevoeg, gevolg deur direkte inspuiting van die monster in ‘n massaspektrometer om die absolute konsentrasies van kreatinien, kreatien en kaffeïen te bepaal. Lineêre kurwes van die seinintensiteitsverhouding van analiet tot IS is verkry na gepaste instrumentkondisies vasgestel is. Die verhouding van konsentrasie van die analiet (middel of metaboliet) tot dié van kreatinien (as IS) mag gebruik word om die analietkonsentrasie in die standaard oplossings en/of urienemonster te bepaal. Die metode word nie deur veranderinge in die vloeistofvolume of verwatering van urienemonsters beïnvloed nie, weens die analiet-tot-kreatinienverhouding wat onveranderd bly. ‘n LC–UV–MS-metode is voorts ontwikkel om die ontwikkelde DI–ESI–MS-metode se data te vergelyk.

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: See item for full text / AFRIKAANSE OPSOMMING: Sien item vir volteks

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: This study describes: · Development of analytical tools to investigate the light struck flavour (LSF) in beer by Gas chromatography mass spectrometry (GCMS) and by liquid chromatography mass spectrometry/mass spectrometry (LCMS/MS). Development of a high performance liquid chromatography (HPLC) method to analyse carbohydrates in beer. · The efficiency a pilot scale ultraviolet (UV-C) system at 254 nm to inactivate spoilage microorganisms spiked in commercial beer. Bacteria test were Lactobacillus brevis, Acetobacter pasteurianus and Saccharomyces cerevisiae · A pilot scale UV treatment of commercial and non-commercial lager beers at UV dosage of 1000 J/L. Following the UV treatment, the correlation between chemical analyses and sensory tests conducted by consumers’ tasters were investigated. · A pilot scale UV treatment of non-commercial beer brewed with reduced hops iso-α-acids (tetrahydro-iso-α-acids) at UV dosage of 1000 J/L. Sensory changes and chemical properties were investigated. · The development and optimisation of an UV light emitting diodes (UV-LED) bench scale apparatus. Chemical and microbiological tests were conducted to investigate the effect of UV-LEDs on beer at 250 nm and 275 nm wavelengths. / AFRIKAANSE OPSOMMING: Hierdie studie beskryf: · Die ontwikkeling van analitiese toerusting om die invloed van lig op die smaakontwikkeling in bier te bestudeer m.b.v gaschromatografie massa spektrometrie (GCMS) en vloeistofchromatografie massa spektrometrie/massa spektrometrie, asook die ontwikkeling van ‘n hoë druk vloeistofchromatografiese metode vir die analise van koolhidrate in bier. · Die doeltreffendheid van ‘n toetsskaal ultraviolet (UV-C) sisteem om die nadelige mikroorganismes waarmee die bier geïnnokuleer was, by 254 nm te inaktiveer.. Toetse is uitgevoer met die volgende bakterieë, Lactobacillus brevis, Acetobacter pasteuriants en Saccharomyces cerevisiae. · ‘n Toetsskaal UV behandeling van kommersiële en nie-kommersiële lager biere by ‘n UV dosering van 1000 J/L. Na UV behandeling is die verwantskap tussen chemiese analises en ‘n reeks sensoriese toetse deur vebruikers proeërs ondersoek.. · ‘n Toetsskaal UV behandeling van ‘n nie-kommersiële bier gebrou met verlaagde hops-iso-α-sure (tetrahidro-iso-α -sure) by UV dosering van 1000 J/L. Sensoriese veranderinge asook chemiese eienskappe is ondersoek. · Die ontwikkeling en optimalisering van ‘n UV-lig emissie diodes bankskaal apparaat. Chemiese en mikrobiologiese toetse is uitgevoer om die effek van UV lig op bier by 250 nm en 275 nm te ondersoek.

Lipids are important for the structural and physiological functions of neuronal cell membranes. Alterations in their lipid composition may result in membrane dysfunction and subsequent neuronal deficits that characterise various disorders. This study focused on profiling lipids of aged and LPS-treated rat brain and liver tissue with a view to explore the effect of atorvastatin in neuroinflammation, and examining lipid changes in different areas of rat brain of the Flinders Sensitive Line (FSL) rats, a genetic model of depression. Lipids and other analytes extracted from tissue samples were analysed with proton nuclear magnetic resonance spectroscopy (1H-NMR), gas chromatography (GC) and liquid chromatography-tandem mass spectroscopy (LC/ESI-MS/MS). Changes in the lipid profiles suggested that brain and liver responded differently to ageing and LPS-induced neuroinflammation. In the aged animals, n-3 PUFA were reduced in the brain but were increased in the liver. However, following treatment with LPS, these effects were not observed. Nevertheless, in both models, brain concentration of monounsaturated fatty acids was increased while the liver was able to maintain its monounsaturated fatty acid concentration. Atorvastatin reversed the reduction in n-3 PUFA in the aged brain without reducing brain and liver concentration of cholesterol. These findings further highlight alterations in lipid metabolism in agerelated neuroinflammation and show that the anti-inflammatory actions of atorvastatin may include a modulation of fatty acid metabolism. When studying the FSL model, there were differences in the lipid profile of different brain areas of FSL rats compared to Sprague-Dawley controls. In all brain areas, arachidonic acid was increased in the FSL rats. Docosahexaenoic acid and ether lipids were reduced, while cholesterol and sphingolipids were increased in the hypothalamus of the FSL rats. Furthermore, total diacylglycerophospholipids were reduced in the prefrontal cortex and hypothalamus of the FSL rats. These results show differences in the lipid metabolism of the FSL rat brain and may be suggestive of changes occurring in the brain tissue in depression.

published_or_final_version / abstract / Chemistry / Doctoral / Doctor of Philosophy

<p>This thesis covers sampling and analytical procedures for isocyanates (R-NCO) and amines (R-NH₂), two kinds of chemicals frequently used in association with the polymeric material polyurethane (PUR). Exposure to isocyanates may result in respiratory disorders and dermal sensitisation, and they are one of the main causes of occupational asthma. Several of the aromatic diamines associated with PUR production are classified as suspected carcinogens. Hence, the presence of these chemicals in different exposure situations must be monitored. </p><p>In the context of determining isocyanates in air, the methodologies included derivatisation with the reagent di-<i>n</i>-butylamine (DBA) upon collection and subsequent determination using liquid chromatography (LC) and mass spectrometric detection (MS). A user-friendly solvent-free sampler for collection of airborne isocyanates was developed as an alternative to a more cumbersome impinger-filter sampling technique. The combination of the DBA reagent together with MS detection techniques revealed several new exposure situations for isocyanates, such as isocyanic acid during thermal degradation of PUR and urea-based resins. Further, a method for characterising isocyanates in technical products used in the production of PUR was developed. This enabled determination of isocyanates in air for which pure analytical standards are missing. Tandem MS (MS/MS) determination of isocyanates in air below 10^-6 of the threshold limit values was achieved.</p><p>As for the determination of amines, the analytical methods included derivatisation into pentafluoropropionic amide or ethyl carbamate ester derivatives and subsequent MS analysis. Several amines in biological fluids, as markers of exposure for either the amines themselves or the corresponding isocyanates, were determined by LC-MS/MS at amol level. In aqueous extraction solutions of flexible PUR foam products, toluene diamine and related compounds were found. </p><p>In conclusion, this thesis demonstrates the usefulness of well characterised analytical procedures and techniques for determination of hazardous compounds. Without reliable and robust methodologies there is a risk that exposure levels will be underestimated or, even worse, that relevant compounds will be completely missed.</p>

The Supercritical Fluid Extraction (SFE) technique was used to fractionate Suwannee River reference fulvic acid (FA). The fractions were characterized by gas chromatography (GC) and reversed-phase high performance liquid chromatography (RP-HPLC). A SFE fractionation method was developed using stepwise gradient of supercritical CO₂ and methanol. Three FA fractions were separated. The average mass recovery was 102% with the coefficient of variation of 2.8%. The fractionation dynamics and the difference in the ratios of UV absorption to fluorescence emission indicate the real fractionation of the FA. The HPLC chromatographic peak patterns and the spectra of the corresponding peaks were almost indistinguishable. The overall results of this research support the argument that FA exhibits polymer-like molecular structure.

The spread of tetracyclines through agricultural systems is causing the present bacteria to develop antibiotic resistance. The spread of this bacteria, as well as the tetracycline antibiotics in the environment is dangerous because these antibiotics pose health hazards for humans. The overuse of antibiotics, which are added to livestock feed, results in the antibiotics being released into the environment via animal feces. In this research, we have attempted to design an analytical method to isolate antibiotics from agricultural wastes with subsequent detection using liquid chromatography and mass spectrometry (LC-MS). The antibiotics investigated in this study were tetracycline, chlortetracycline, and oxytetracycline. The analytical procedure involves mixing the agricultural samples with an organic solvent, such as methanol, which solubilizes these antibiotics. Next, samples are centrifuged to remove solid particulates. A polymeric weak cation cartridge was used to concentrate and separate the antibiotics from the unwanted organic chemical compounds found in the samples. The antibiotics were released with methanol with small amounts of acid and then detected and quantified using LC-MS and high performance liquid chromatography with a photodiode array detector (HPLC-PDA).

Lignin, one of main components in the woody cell walls, is a complex heterogeneous biopolymer, which provides structural support and transportation of water in plants. It is highly recalcitrant to degradation (both chemically and environmentally) and protects cellulose from being degraded/hydrolyzed. Due to the structural complexity of native lignin, complete characterization and elucidation of lignin’s structure remains very challenging. The overarching goal of this work is to develop mass spectrometry based analytical methods to contribute to a better understanding of lignin structures. This dissertation will focus on the development and application of High-Resolution Accurate-Mass (HRAM) Mass Spectrometry (MS) as main analytical technique for studying lignin model compounds, including understanding the ionization behavior, studying corresponding fragmentation patterns and extracting structural information for structural elucidation eventually. Analytical methods were also developed to study the post-pretreatment products of the synthetic trimeric model compound using High-Performance Liquid Chromatography (HPLC) coupled with High-Resolution Accurate Mass (HRAM) Mass Spectrometry (MS). The first project of this dissertation focuses on mass spectral the characterization of lignin models from the in vitro oxidative coupling reactions. Three specific trimeric compounds were isolated and their ionization behaviors were investigated using HRAMMS via electrospray ionization (ESI). The reaction parameters of the in vitro oxidative coupling reaction were critical in alternating the linkage profiles of resulting dehydrogenation polymers (DHPs). Reaction parameters were tuned to obtain desired DHP linkages profile. Upon the isolation of three different trimeric compounds, a systematic comparison of ionization efficiency of three trimeric compounds was carried out using ESI-HRAM-MS under different ionization conditions. The second project was aimed to design a synthetic route for a lignin model compound that will be a good representation for native lignin during the pretreatment process. The model compound of interest has not been obtained previously through chemical synthesis. Due to the reactivity of cinnamyl alcohol, which contains the unsaturated side chain, this new synthesis strategy was developed based on the known aldol-type reaction route. A versatile synthesis procedure for preparation of β-O-4 oligomeric compounds was designed and implemented to include the most important functional groups (phenolic alcohol, aryl glycerol β-aryl ether bond and unsaturated side chain) in the resulting model compound. This new synthesis route also allowed incorporation of different monolignols. In the third project, Fenton chemistry was applied to a synthetic lignin model compound. Due to the non-specificity in the post pretreatment product profile, nontargeted analytical strategy was developed and applied to study the post-pretreatment products of the model compound using HPLC-HRMS. The results from this dissertation showed a significant difference in ionization behavior between three structurally different model compounds and indicated that primary structures of lignin compounds can largely affect corresponding electrospray ionization properties as well as fragmentation pattern. The work in this dissertation provides analytical techniques for non-targeted analysis of complex lignin samples and an insightful understanding of Fenton’s reaction pretreatment upon lignin model compound.

<p>Dosada&scaron;nji objavljeni literaturni podaci ukazuju da aflatoksini, usled njihovog dokazanog toksičnog, mutagenog, teratogenog i kancerogenog efekta na organizam ljudi i životinja, predstavljaju najpoznatiju i najrizičniju grupu mikotoksina. Nasuprot navedenom, dostupna naučna i stručna literatura iz Republike Srbije ukazuje na nedostatak sistematski sprovedenih monitoringa učestalosti pojave aflatoksina. Kao sirovine za ispitivanje pojave aflatoksina, u ovoj doktorskoj disertaciji, odabrani su kukuruz i mleko. Kukuruz je odabran jer predstavlja jednu od najzastupljenijih žitarica u Srbiji. Navedena činjenica dodatno povećava potrebu za istraživanjem, jer pojava aflatoksina u velikoj meri utiče i na kvalitet kukuruza, kao i smanjenje prinosa i ekonomskog profita zemlje. Cilj da se ispita mleko proistekao je iz činjenice da ono usled velike frekventnosti u ishrani, predstavlja namirnicu sa najvećim potencijalnim rizikom za unos aflatoksina u organizam ljudi.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Analiza pojave aflatoksina u kukuruzu obuhvatila je 1000 uzoraka, sakupljenih tokom perioda od četiri godine (2009-2012.). Dobijeni rezultati ukazali su da aflatoksini nisu detektovani u uzorcima kukuruza iz 2009., 2010. i 2011. godine. Međutim, od 700 analiziranih uzoraka kukuruza iz 2012., čak 553 (79%) bilo je kontaminirano aflatoksinima. Značajne razlike u pojavi aflatoksina u kukuruzu, iz različitih proizvodnih godina, mogu se objasniti izuzetno toplim i suvim vremenskim uslovima zabeleženim u 2012. godini. Su&scaron;ni vremenski uslovi bili su povoljni za razvoj plesni i sintezu aflatoksina u kukuruzu, a takođe su u velikoj meri doprineli i visokom nivou pojave aflatoksina M1 (AFM1) u mleku. Analiza 200 uzoraka različitih kategorija kravljeg mleka pokazala je da je AFM1 sa koncentracijama od 0,01 do 1,20 ug/kg detektovan u 198 (98,7%) uzoraka. Na osnovu određenih koncentracija AFM1 u uzorcima mleka, prikupljenih informacija o prosečnom unosu mleka, telesnoj težini različitih starosnih kategorija i polova 1500 ispitanika, procenjen je rizik od unosa AFM1. Dobijeni rezultati ukazali su da su sve ispitane starosne kategorije, a posebno deca, izložena visokom riziku od unosa AFM1 u organizam preko kontaminiranog mleka.<br />Usled svega navedenog, proizilazi potreba za kontinuiranim praćenjem pojave aflatoksina u kukuruzu i AFM1 u mleku, sa ciljem za&scaron;tite stanovni&scaron;tva od rizika povezanih sa njihovim dokazanim toksičnim i kancerogenim efektima. Tokom poslednjih godina, do&scaron;lo je do povećane tražnje za razvojem osetljive, tačne, jednostavne i brze metode, čijom primenom će se obezbediti pouzdano određivanje različitih aflatoksina pri niskim koncentracijama. Usled toga, ELISA metode, kao i metode tečne hromatografije sa različitim detektorima (HPLC-FLD, HPLC-UV light-FLD, LC-MS/MS) su razvijene, optimizovane i validovane. Validacija je izvr&scaron;ena prema smernicama odgovarajućih evropskih Regulativa, analizom sertifikovanih referentnih materijala i spajkovanih uzoraka. Dobijeni parametri validacije potvrdili su da se sve primenjene metode sa visokom pouzdano&scaron;ću mogu koristiti za određivanje aflatoksina u kukuruzu, odnosno AFM1 u mleku. Validovane metode su zatim primenjene za analizu aflatoksina i AFM1 u prirodno kontaminiranim uzorcima kukuruza i mleka. Analizom prirodno kontaminiranih uzoraka kukuruza uočeno je da određena koncentracija aflatoksina u velikoj meri zavisi od neravnomerne raspodele aflatoksina unutar čvrstih uzoraka. Pored toga, analiza uzoraka kukuruza iz skladi&scaron;ta, takođe je ukazala na neravnomernu raspodelu ukupnih plesni, vrste Aspergillus flavus, kao i aflatoksina. S druge strane, dobijeni koeficijenti korelacije (&gt; 0,9) između primenjenih metoda za određivanje AFM1 u mleku, ukazali su da među dobijenim rezultatima postoji snažna korelacija, koja može poticati i od relativno homogenije raspodele AFM1 unutar tečnih uzoraka.</p> / <p>Recent studies have demonstrated that аflatoxins (AFs) represent the best known and most intensively researched mycotoxins in the world, with proven toxic, mutagenic, teratogenic and carcinogenic effects on human and animal health. However, up to the author&#39;s knowledge, there are no investigations concerning the occurrence of AFs in food or feed from Republic of Serbia. For that purpose, maize and cow&acute;s milk samples were chosen as matrixes for investigation. Maize is selected because it represents one of the major crops grown in Serbia and presence of AFs leads to reduction of maize quality as well as significant economical losses. On the other hand, milk has the greatest demonstrated potential for AFs introducing into the human diet since it represents one of the main foodstuffs in human nutrition.<br />An investigation regarding occurrence of AFs in maize included 1000 samples collected during four years period (2009-2012). Obtained results indicate that AFs were not detected in maize samples collected during 2009-2011 period. However, among 700 analyzed maize samples from 2012 even 553 (79%) samples contained AFs. Significant differences in occurrence of AFs in maize could be contributed to extremely hot and dry weather conditions noted during maize growing season 2012. Described weather conditions were favorable for mould growth and AFs synthesis in maize and also could be the possible reason for high contamination frequency of aflatoxin M1 (AFM1) in milk. Analysis of 200 cow&acute;s milk samples showed that AFM1 were detected in 198 (98.7%) samples in concentrations ranged from 0.01 to 1.20 &mu;g/kg. On the basis of the obtained AFM1 concentrations in cow&rsquo;s milk samples, collected information about average milk intake and mean body weight (bw) for different age&rsquo;s categories, mean ingestion of AFM1 in ng/kg per bw per day were estimated. Obtained results showed that all age&rsquo;s categories, especially children, are exposed with high risk related to presence of AFM1 in milk.<br />Based on everything stated above, presence of AFs in maize and AFM1 in milk should be continuously controlled in order to protect the population from risks associated with its proven toxicity and carcinogenicity. During recent years, there has been an increase in demand for development of sensitive, accurate, simple and fast method which is reliable to detect AFs and AFM1 at low concentrations. For that purpose, enzyme linked immunosorbent asssay (ELISA), high performance liquid chromatography with fluorescence detector (HPLC-FLD; HPLC-UV light-FLD) and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) were optimized and validated. The method validation was performed according European Union Regulative using certificated reference material as well as spiked blank maize and milk samples. The obtained validation parameters indicate that all applied methods are suitable for determination of AFs in maize as well as AFM1 in milk samples. Afterwards, validated methods were applied for analysis of natural contaminated maize and milk samples. Analysis of natural contaminated maize samples indicate that determined AFs concentration mostly depends of uneven distribution of AFs in solid samples. Furthermore, analysis of maize samples from different warehouses indicates an uneven distribution of moulds, Aspergillus flavus and AFs. On the other hand, obtained correlation coefficients (&gt;0.9) indicate that strong correlation exists between applied methods for AFM1 determination. Relatively homogeneous distribution of AFM1 in liquid samples could be the possible reason for obtained strong correlation between applied methods.</p>