The chemical diversity of the phylum Porifera have provided marine natural products (MNPs) such as alkaloids, terpenoids and peptides with ranging biological activity. Every two days a novel compound deriving from sponges are discovered. The aim of this report is to explore the metabolic diversity of deep-sea sponges collected from five different geographical locations. The metabolome of the deep-sea sponges was studied with tandem mass spectrometry (UPLC- MS/MS) and screened for peptides and other major compounds. The aim also includes the selection, isolation and characterisation of a compound obtained from the metabolic profiling of the sponges. The metabolic profiles of 25 sponges were screened. The specimens analysed in this report displayed great metabolic diversity and peptides, halogenated compounds as well as presumable alkaloids were found. The spectra of some specimens displayed low sensitivity which required optimisation such as increase of volume injection. Nevertheless, the report offers novel discoveries for sponges that have never previously been examined. The brominated compound derived from the sponge Desmacella annexa was analysed using large-scale extraction and different analytical techniques such as preparative-HPLC, Q-Tof-MS and NMR. However, the isolation of the target compound was unsuccessful due to co-extractant compounds and insufficient amount of the compound to perform further purification.  Nevertheless, the discoveries made from the metabolic profiles of the sponges may be of assistance when selecting potential bioactive compounds for further studies. Furthermore, bioassays may be performed in the future on the extracts obtained from the sponge Desmacella annexa.

Syftet med detta examensarbete är att förkorta 20-timmars frisättningen för en tablettformulering med förlängd frisättning genom att använda maskininlärning genom multivariat prediktiv modellering, helt automatisera frisättningsmetoden och utvärdera antalet tabletter som kan representera en batch vid frisättningsanalys av en specifik provtablett. Prediktionsmodellerna byggdes i SIMCA 14, som är en programvara för multivariat dataanalys. Två PLS-prediktionsmodeller byggdes som använder sig av de första 8 timmarna av frisättningen som X-variabler för att prediktera mängden aktiv farmaceutisk ingrediens som frisatts vid timme 12 och timme 20. Y-variablerna behandlades med logit-transformation i SIMCA 14. Modellerna bedömdes med en oberoende testset med avseende på linjäritet och precision. Beräkningar visar att prediktionerna av 12 och 20 timmars värden är utmärkta. RMSEP för 12-timmars prediktionen är 0,39 och 1,12 för 20-timmars prediktionen. Detta visar att modellerna korrekt kan prediktera timme 12 och timme 20 efter en 8 timmars frisättningsanalys. En automatiserad 8-timmars frisättningsmetod med alla nödvändiga kommandon har byggts för SingBATH-roboten i SenISS-programvaran. Det är möjligt att koppla modellerna till SingBATH-metoden till SIMCA Quick Predict-modulen som finns i SenISS-programvaran. SingBATH-robotarna är placerade i produktionsanläggningarna, vilket innebär att hela analysen kan överföras från laboratoriet för kvalitetskontroll till produktionen. För att utvärdera möjligheten att använda färre än 6 tabletter vid frisättning av provtabletten för en batch, användes ett testset med data från 73 olika batcher. Flera beräkningar har utförts för att undersöka effekten av att minska antalet tabletter från 6 till 3 tabletter per batch. Det visas att risken för felaktig bedömning av batchens frisättning vid timme 1, 4, 8 och 20 med frisättning av 3 tabletter istället för 6 är mycket liten. Dessutom är den tid och resurser som sparas när antalet testade tabletter per batch minskas mycket stora. En utvärdering av prediktionsmodellerna med 6 och 3 tabletter per batch har också utförts. Resultaten visar att skillnaderna i resultat är små. Genom att flytta analyserna till produktionen, med hjälp av prediktionsmodeller för att minska analystiden till 8 timmar istället för 20 timmar och att minska antalet tabletter som representerar en batchs frisättning till 3 tabletter, från 6 tabletter, innebär en stor tidsbesparing för analys av denna provtablett. / The aim of this thesis degree is to shorten the 20-hour dissolution time of an extended release tablet formulation by using machine learning through multivariate predictive modelling, fully automate the dissolution method and assess the number of tablets needed to have correct test result for a batch of the formulation analyzed in this thesis.  The prediction models were built in SIMCA 14, which is a software for multivariate data analysis. Two PLS prediction models were built using the first 8 hours of dissolution as X-variables to predict the amount active pharmaceutical ingredient dissolved at hour 12 and hour 20. The Y variables were treated with logit transformation in SIMCA 14. The models were assessed with an independent test set with respect to linearity and precision. Calculations show that predictions of the 12 and 20 hour values are excellent. RMSEP for the 12-hour prediction is 0,39 whereas it is 1,12 for the 20-hour prediction. This shows that the models can correctly predict hour 12 and hour 20 after an 8 hours dissolution analysis.  An automated 8 hour dissolution method with all the necessary commands has been built for the SingBATH robot in the SenISS software. It is possible to couple the models to the SingBATH method to the SIMCA Quick Predict module available in the SenISS software. The SingBATH robots are placed in the production facilities, which means that the entire analysis can be transferred from the quality control laboratory to the production. To evaluate the possibility of using fewer than 6 tablets when testing the dissolution profile for a batch, a test set with data from 73 different batches was used. Several calculations have been performed to investigate the effect of reducing the number of tablets from 6 to 3 tablets per batch. It is shown that the risk of incorrectly judging the batch dissolution at hour 1, 4, 8 and 20 associated with testing 3 tablets instead of 6 is very small. Moreover, the time and resources saved when  the number of tablets tested per batch are reduced are very large.  An evaluation of the prediction models with 6 and 3 tablets per batch has also been performed. The results show that the differences in results are minor. By moving the analyses to the production, using prediction models to reduce the analysis time to 8 hours instead of 20 hours, and to reduce the number of tablets representing a batch dissolution to 3 tablets, from 6 tablets, means a great time saving for this analysis.

This project focused on the development and validation of an ultra-performance liquid chromatography tandem mass spectrometer (UPLC-MS/MS) method for the determination of Patulin in apple-based products. Patulin is one of the many mycotoxins that are secondary metabolites from about 60 filamentous fungi. The mold often appears as black or blue on fruit, vegetables or crops. To determine the concentration of Patulin in consumer products is important since it may affect consumer health. The symptoms are often flu-like and can lead to kidney-failure and neurotic damage. The Swedish Food Agency is tasked to analyze consumer products to determine if they are safe to ingest. The European Commission has set maximum residue limits for several toxins that can potentially appear in groceries on the market. Using an UPLC-MS/MS allows for the accurate qualification and quantification of Patulin in apple juice and purees. The method was validated by analyzing several lots of apple juices and a proficiency test from Fapas®. The recovery rate ranged between 70.5-103.8% and were accepted because they met the recovery criteria in Regulation (EC) No. 401/2006 for Patulin.

Vitamin C är en populär ingrediens i hudvårdsprodukter, bland annat på grund av dess positiva effekter på kollagen-produktionen i huden och dess antioxidativa egenskaper. Dessvärre är vitamin C en känslig ingrediens som lätt bryts ned av ljus, syre, hög temperatur eller fel pH-värde. I dagsläget finns inga regleringar för kontroll av nedbrytning av aktiva ingredienser i kosmetiska produkter.  I den här studien har en metod utvecklats med omvänd-fas högupplösande vätskekromatografi (RP-HPLC) och detektor med ultraviolett (UV) ljus för att analysera halten C-vitamin i en kosmetisk testprodukt, hädanefter kallad OTP och en modifierad variant av denna, kallad MTP. En stabilitetstudie gjordes vid olika temperaturer och ljusförhållanden på dessa produkter. En jämförelse gjordes även mellan en högre och lägre syretillgång. I metoden som valdes för stabilitetsanalys använde UV-detektor (254 nm). Den kromatografiska kolonnen innehöll C-18-stationärfasoch hade en längd om - 250x4.6 mm, med 5 µm partikelstorlek, från Chrompack. Mobilfasen bestod av 20 mM lösning av ättiksyra i Milli Q-vatten och metanol i förhållandet 95:5 (v/v) och mobilfashastigheten 2 ml/min, vilket gav en retentionstid på ca 2.4 min för vitamin C. I MTP bröts vitamin C ned snabbare än i OTP. Den lägsta nedbrytningshastigheten kunde ses vid den lägsta temperaturen (4℃) och den lägre syretillgången av dem som studerades. Ett linjärt samband kunde ses mellan förvaringstiden och koncentrationen av vitamin C som fanns kvar i produkten vid detta förvaringsförhållande. / Vitamin C is a popular ingredient in skin care products, due to its positive effect on the collagen production and its antioxidative characteristics. Unfortunately, vitamin C is a sensitive ingredient that is easily degraded by light, oxygen, elevated temperatures, or the wrong pH-value. To today’s date, there are no regulations controlling the degradation of active ingredients in cosmetic products.  In this study, a method has been developed for Reversed Phase-Liquid Chromatography (RP-HPLC) and Ultra Violet (UV)-light detector to analyze the vitamin C content in a cosmetic test product, in this study called OTP and a modified variant of the same product, called MTP. A stability study was made at different temperatures and light conditions. A comparison was also made between higher or lower levels of oxygen present. The method chosen was performed at a wavelength of 254 nm. The column used contained a C-18 stationary phase (250x4.6 mm, 5 µm particle size) from Crompack. The mobile phase consisted of 20mM solution of acetic acid in Milli Q water and methanol 95:5 (v/v) and the flow rate was set to 2 ml/min, which gave a retention time of approximately 2.4 min for vitamin C. Vitamin C was degraded faster in MTP than in OTP. The lowest degradation rate was found at the lowest temperature (4℃) and the lower oxygen content among the ones studied. A linear relationship was found between storage time and the vitamin C concentration left in the product at this storage condition.

Demands for plant-based diets sufficient in all required amino acids are increasing due to health and environmental advantages. As legumes offer a complementing source of quality protein to the already heavily consumed cereals, methods for determining amino acid compositions in legumes are needed. The aim of this thesis was to establish a GC-FID method for analyzing amino acids in legumes and to apply the method to analyze six locally grown legumes: Black beans, brown beans, red kidney beans, white beans, yellow and grey peas. White bean flour was used as an inhouse control sample. Legumes were hydrolyzed with acid and alkaline treatments;hydrolysates were purified and derivatized using an amino acid analysis kit (EZ:faast, Phenomenex) and quantified with GC-FID. All legumes were rich in Asp + Asn, Glu + Gln, Lys and Leu while limited in Met, Cys and Trp. Quality control trials showed a generally good precision of the method (between day CVs were ≤ 10% for 12 of 16amino acids ) and good linearity (r2 were 0,99 or above for 16 of 19 calibration curves). Limits of detection and quantification ranged from 2-46 nmol/mL and 7-154nmol/mL, respectively, depending on the amino acid. No accuracy trial was performed. In conclusion, a rapid GC-FID method for amino acid analysis was established and amino acid compositions of locally grown legumes was tentatively determined. However, further quality control trials are required to validate the method. / Efterfrågan av växt-baserade dieter med komplett innehåll av aminosyror ökar på grund av både hälso- och miljömässiga fördelar. Då baljväxter erbjuder en kompletterande källa till protein av hög kvalitet till cerealier, som redan konsumeras i hög grad, krävs metoder för att analysera aminosyra innehåll i baljväxter. Målet med detta arbete var att etablera en GC-FID metod för att analysera aminosyror i baljväxter och att applicera metoden för att analysera aminosyror för att analysera sex lokala baljväxter: Svarta bönor, bruna bönor, röda kidney bönor, vita bönor, gula och gråa ärtor. Mjöl på vita bönor användes som in-house kontroll prov. Baljväxterna hydrolyserades med både syra och bas behandlingar, hydrolysaterna renades och derivatiserades med ett kit för aminosyraanalys (EZ:faast, Phenomenex) och kvantifierades med GC-FID. Alla baljväxter hade höga halter av Asp + Asn, Glu + Gln, Lys och Leu men låga halter av Met, Cys och Trp. Kvalitetskontroller visade på en generellt hög precision (CV mellan dagar ≤ 10% för 12 av 16 aminosyror) och god linjäritet (r2var 0,99 eller högre för 16 av 19 kalibreringskurvor). Detektions- och kvantifierings-gränser varierade mellan 2-46 nmol / mL respektive 7 – 154 nmol / mL beroende på aminosyran. Inga tester gjordes för att bestämma metodens riktighet.Slutsatsen drogs att en snabb GC-FID metod för aminosyra analys har framtagits och indikativa värden av lokala baljväxters aminosyra innehåll fastställts. Däremot krävs flera kvalitetskontroller för att validera metoden.

In this project, the optimization of obtaining adsorption isotherms for the ion pair reagent, tributylamine was tested. The goal was to have a better understanding of the chromatographic process when separating biomolecules, such as oligonucleotides. To do this one ion pair reagent was tested in different buffers with different compositions of acetonitrile. These solutions adsorbed into a C18 column at different temperatures and stripped into fractions of 35 mL. To analyze the results Liquid-liquid extraction was performed on the fractions and the organic phase was then injected into a gas chromatography. The results showed that at a temperature of 24°C the ion pair reagent adsorbed more to the column than at 37°C and 50°C. For the different compositions of acetonitrile buffers which were tested the one that stood out was the 50% acetonitrile buffert. At all temperature it showed to always be able to adsorb more to the column than the other buffers. To calculate the concentrations of the analytes, standard curves for both tributylamine and dibutylamine were made. For dibutylamine, four unknown samples were provided to test out if the methods could be used to determine the concentrations of dibutylamine in the samples. The methods for acquiring adsorption isotherms and analyzing samples with the gas chromatography showed good results and could be used for more studies. However, to validate the results of the 50% acetonitrile buffer, more work in the future is required.

Hydrophobic interaction chromatography (HIC) is a common purification method for biological drug substances. For continuous monitoring the quality of the purification, a column performance test is carried out to test the packing of the resin. The packing integrity is commonly tested between runs by measuring the Hight Equivalent to a Theoretical Plate (HETP) by injecting a small volume of a tracer solution (spike) that gives a change in conductivity. The column performance test provides useful information regarding the packing quality that could be used for troubleshooting before a purification is performed since a well packed gel is essential for an effective purification. During this thesis project a screening of suitable test conditions for a HETP test for a HIC column was performed. The developed HETP test were then investigated if it could be combined with the storage of the resin or if the packing is affected by the test conditions. Factors such as spike volume, flow rate and the concentration of the elute and spike solution, were considered. 0.1 M NaOH elute and 0.15 M NaCl as spike solution gave the best result based on HETP and the asymmetry of the conductivity peak. A test purification with the new HETP test conditions compared with the old method were then carried out to verify that the quality of the purification is not affected. The purification showed promising result and implementation of a more convenient storage solution and HETP test would be possible without changing the product quality.

Immunoassays have become an essential tool in several fields of bioanalytics with tremendous advances in sensitivity and formats seen through the last three decades. Many diseases are today diagnosed solely based on biomarker concentrations evaluated through immunoassays. More biomarkers will be unravelled for diseases that have low serum concentrations once highly sensitive analyzes can be performed routinely, highlighting the importance of improved sensitivity. The aim of this project was to increase the sensitivity of detection on the Gyrolab immunoassay platform. This was done by optimizing the conditions for fluorescence and by reduction of light scattering in the system. Four red-emitting fluorophores were investigated and assays were performed using additives with the purpose to reduce solvent-assisted quenching by water, and to avoid scattering of light in the system’s column. Deuterated solvents and encapsulation strategies were employed to reduce deactivation processes caused by water, and refractive index matching was used to limit the refraction of light. By using heavy water in assay sensitivity was increased by 17-25% for two biomarkers and with the use of different fluorophores showing consistency for the method. With the use of a refractive index matching liquid, it was possible to increase the depth at which the maximum fluorescence intensity occurred three-fold, using confocal microscopy. However, implementing found advantages in assay proved to be difficult due to mediums being hydrophobic and viscous, highlighting the complexity of the microfluidic system.

All-trans-retinol (vitamin A) används ofta som en aktiv ingrediens i kosmetiska produkter på grund av dess kliniskt validerade anti-age-egenskaper. Däremot är användningen av all-trans-retinol i kosmetiska formuleringar begränsad till följd av den kemiska stabiliteten av all-trans-retinol som påverkas av värme, syre, ljus och syror. Både kompositionen av en kosmetisk formulering, samt lagringsförhållanden och förpackningen av produkten, kan påverka nedbrytningen av all-trans-retinol till mindre aktiva former.  I denna studie har stabilitetstestning genomförts under två månader i syfte att utvärdera inverkan som lagringstemperatur (21°C och 5°C) och produktförpackning (en luftfri behållare och en burk) kan ha på nedbrytningen av all-trans-retinol i en experimentell oljegel. All-trans-retinol extraherades med metanol innehållande 0.5 mg/mL butylhydroxytoluen (BHT) och analyserades med reversed-phase HPLC med 1% ammoniumacetat och metanol (12:88, v/v) som mobilfas. I HPLC-kromatogrammen upptäcktes en okänd nedbrytningsprodukt, vilket möjliggjorde stabilitetstestning av all-trans¬-retinol genom att undersöka förändringar i förhållandet mellan nedbrytningsprodukten och all-trans-retinol. Stabilitetstestningen av de experimentella oljegelerna resulterade i mindre än 4% nedbrytningsprodukt i förhållande till mängden all-trans-retinol efter 56 dagars lagring, oberoende av produktförpackning och lagringstemperatur, med den förslagna analysmetoden. Studien visade också att instabiliteten hos extraherad all-trans-retinol har en dominerande inverkan på resultaten som kan leda till en överskattning av nedbrytningsprodukten på grund av temperaturen under beredning och lagring av förberedda prover. Dessutom utvärderades stabiliteten av all-trans-retinol i metanol och metanol innehållande 0,5 mg/ml butylhydroxytoluen (BHT). Upp till 6% nedbrytning av all-trans-retinol i metanol med BHT kan förväntas om beredda proverna lagras i rumstemperatur, medan mindre än 1% nedbrytningsprodukt detekterades i prover som bevaras kylda under analysen. Av denna anledning har det föreslagits att förhållandena vid beredning, hantering och lagring av extraherad all-trans-retinol bör kontrolleras för att förhindra inverkan från omgivningen, såsom temperatur, ljus och syre. / All-trans-retinol, or vitamin A, is frequently used as an active ingredient in cosmetic products because of its clinically validated anti-aging properties. The use of all-trans-retinol in cosmetic formulations is limited by its susceptibility to heat, oxygen, light, and acids. In a cosmetic product, the composition, as well as the storage conditions and the type of packaging, may influence the degradation of all-trans-retinol into less active forms.  In this study, long-term stability testing was conducted with the purpose to evaluate the effect that storage temperature (21°C and 5°C) and product packaging (an airless container and a jar) may have on the degradation of all-trans-retinol in an experimental oil-gel during a 2-months period. All-trans-retinol was extracted with methanol containing 0.5 mg/mL butylhydroxytoluene (BHT) and was analysed with a reversed-phase HPLC system, using 1% ammonium acetate and methanol (12:88) as the solvent system. The HPLC chromatograms revealed an unknown degradation product, allowing stability testing of all-trans-retinol by detecting variations in the ratio between the degradation product and all-trans-retinol. The stability testing of the experimental oil-gels resulted in less than 4% degradation product in relation to all-trans-retinol after 56 days of storage, independent of the product packaging and storage temperature, with the proposed analytical method. It was also shown that the instability of extracted all-trans-retinol will have a predominant impact on the results causing an overestimation of the degradation product because of the temperature during sample preparation and storage. In addition, the stability of all-trans-retinol in methanol and methanol containing 0.5 mg/mL butylhydroxytoluene (BHT) was evaluated. It was concluded that up to 6% degradation of all-trans-retinol in methanol with BHT can be expected if prepared samples are stored in room temperature, while less than 1% degradation product will be present if prepared samples are kept cold during analysis. It has been proposed that the conditions during preparation, handling, and storage of extracted all-trans-retinol should be controlled to prevent interference of external conditions, such as temperature, light, and oxygen.

Kaffe Bueno, ett bioteknologiskt start-up-företag dedikerat till framsteg inom naturliga och gröna produkter, fokuserar på att utnyttja den outnyttjade potentialen hos kaffebiprodukter genom grön kemi och bioteknik. Då endast 1% av kaffet används för bryggning, extraherar företaget lösliga och olösliga produkter genom en uppdelning mellan ett oljeutvinning och ett efterföljande vattenuttag, vilket bidrar till att sänka CO2-utsläppen per värde och en cirkulär ekonomi. Inom hudvård har Kaffe Bueno tidigare utvecklat en BB-kräm med återvunnen kaffesump för pigmentering och hudvårdsfördelar, men stötte på utmaningar när det gäller täckning jämfört med syntetiska BB-krämer, vilket ledde till målet med detta examensarbete. Målet med denna studie är att formulera en helt växtbaserad BB-kräm med optimal täckning, överträffa standarden satt av syntetiska kemiska BB-krämer. Den centrala fråga som styr forskningen är: "Vilka är de optimala formuleringsrstrategierna och växtbaserade ingredienserna för att uppnå högsta täckning i en helt växtbaserad BB-kräm samtidigt som stabilitet säkerställs?". Denna forskning stämmer överens med den globala kosmetikindustrins växande efterfråga av hållbara, växtbaserade formuleringar, som svar på konsumenternas preferenser för miljövänliga produkter. Forskningen inkluderar utveckling av en växtbaserad vit baskräm utan titanoxid eller zinkoxid, och sedan användning av naturliga pigment som KAFFAGE® för att justera färgen, säkerställa hållbarhet, täckning och stabilitet över tid, vid ljusexponering och användning. Sammanfattningsvis formulerades under denna studie framgångsrikt en växtbaserad BB-kräm med täckning jämförbar med marknadsstandarder, undvikande av traditionella järnoxider och med användning av hållbara ingredienser som KAFFAGE-BD®-pigment, även om användning av titandioxid (TiO2) fortfarande krävs. Forskningen betonade vikten av texturoptimering, färgmatchning och sensorisk perception, vilket positionerar BB-krämen som en pålitlig och mångsidig kosmetisk lösning. Framtida forskning bör prioritera undersökning av alternativ till TiO2, såsom mikrokristallin cellulosa, för att förbättra hållbarheten i BB-krämens formulering. / Kaffe Bueno, a bioscience startup company dedicated to advancing natural and green products, focuses on harnessing the untapped potential of coffee by-products through green chemistry and biotechnology. With only 1% of coffee utilized for brewing, the company extracts soluble and insoluble products through a partion between an oil extraction and a subsequent water extraction, contributing to lowering the CO2 emissions per value from coffee consumption and a circular economy. In skincare, Kaffe Bueno previously developed a Beauty Balm cream using ingredients from upcycled spent coffee grounds for pigmentation and skincare benefits, but faced challenges in achieving coverage comparable to synthetic BB creams, prompting the goal of this master thesis. The objective of this study is to formulate an all plant-based BB cream with optimal coverage, surpassing the standard set by synthetic BB creams. The central question guiding the research is: "What are the optimal formulation strategies and plant-based ingredients for achieving the highest coverage in an all plant-based BB cream while ensuring stability?". This research aligns with the global cosmetic industry's growing demand for sustainable, plant-based formulations, responding to consumer preferences for eco-friendly products. The research includes developing a white plant-based base cream without titanium dioxide or zinc oxide, and then utilizing natural pigments like KAFFAGE® to adjust the tint, ensuring sustainability, up-to-standard coverage, stability over time, light exposition and use. Tests conducted to validate the product’s performance and quality include a stability test, a sensory panel evaluation, a sweat test, and other relevant assessments to ensure the cream's effectiveness, user satisfaction, and compliance with industry standards. In conclusion, this study successfully formulated a plant-based BB cream with coverage comparable to market benchmarks, circumventing traditional iron oxides and relying on sustainable ingredients like KAFFAGE-BD®, even though the use of titanium dioxide (TiO2) is still needed. The research emphasized the importance of texture optimization, color matching, and sensory perception, highlighting the essential attributes for BB creams in general and positioning this particular BB cream as a reliable and versatile cosmetic solution. Future research should prioritize exploring alternatives to TiO2, such as microcrystalline cellulose, to enhance sustainability in BB cream formulations.