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Väitekirja elektrooniline versioon ei sisalda publikatsioone, Käesoleva töö eesmärk oli laiendada teadmisi uudsete eluendilisandite kasutamiseks bioanalüütilises keemias kasutades vedelikkromatograafia-massispektromeetria (LC-MS) analüüsimeetodit. Nende kahe instrumendi: LC ja MS ühendamine laiendab määratavate analüütide hulka, kuigi seab ka piiranguid mobiilfaasi lenduvuse osas. Seejuures mitmed mittelenduvad eluendilisandid, mis aitavad suurendada LC analüüsi efektiivsust ja on küll laialdaselt kasutatud traditsiooniliste UV/Vis detektori puhul, kuid pole kasutatavad MS detektoritele. Uudsete fluoroalkoholide kasutamine eluendilisanditena võimaldab LC-MS analüüsile paljulubavat tulevikku. Fluoroalkoholid kui nõrgad happelised ühendid, millel on ioonpaaride moodustamise võimekus ja mis soodustavad analüütide ioniseerumist MS detekteerimisel, võimaldavad nii keerulistest segudest paremini komponente eraldada kui ka neid paremini detekteetida. Selleks, et uurida mõju ravimite retentsioonile pöördfaas-vedelikkromatograafias, testiti mitmeid paljulubavaid ühendeid aluselise pH juures. Lisandite uurimiseks kasutati erinevaid analüütilisi kolonne - C18, bifenüül ja pentafluorofenüül (PFP) ning uuriti mudel-analüütide ja üldlevinud ravimite ning narkootikumide käitumist. Töö raames leiti, et kasutatud fluoroalkoholid mõjutavad analüüsi vähendades happeliste analüütide retentsiooni ja suurendades aluseliste analüütide retentsiooni. Uudsed eluendilisandid rakendati ka kahes praktilises rakendusnäites. Esiteks kasutati neid uinutite morfiin, klonidiin ja midasolaam (ning nende metaboliitide) ülitundlikuks mõõtmiseks. Teiseks kasutati uudseid lisandeid südame- ja veresoonkonna ravimite milrinooni ja dobutamiini tundlikkuks mõõtmiseks. Neid kahte bioanalüütilist metoodikat kasutati ravimite ja metaboliitide mõõtmiseks laste vereplasmast farmakokineetiliste ja farmakodünaamiliste uuringute tarbeks. Uudsete eluendilisandite mõju analüütide ioniseerumisele võimaldas mõlema metoodikaga saavutada erakordselt madalaid määramispiire, vajades seejuures minimaalset proovikogust, mis oli sobilik just antud patsientide grupile., The aim of this thesis was to expand knowledge about novel eluent additives used in bioanalytical chemistry using liquid chromatography-mass spectrometry (LC-MS) analysis technique. The combination of the two instruments, an LC and MS expands the limits of analytes that can be detected and quantified. However, it also introduced considerable limitations in terms of mobile phase volatility. Thus, many eluent additives which helped to increase the efficiency of LC analysis and were widely used when UV/Vis was a detector did not pass the criteria anymore. However, a promising perspective in LC-MS analysis is offered by novel fluoroalcoholic eluent additives. They are weakly acidic compounds with ion-pairing capabilities and with a positive influence on analyte ionisation in the MS source. To research the influence on retention patterns of pharmaceuticals, several novel eluent additives were tested in the basic mobile phase pH range. Different analytical column stationary phases - C18, biphenyl and pentafluorphenyl (PFP) - were investigated, both with model analytes and a large set of common toxicology screening compounds. It was observed that the use of these fluoroalcohols influenced the analysis by decreasing retention of acidic analytes and increasing retention for analytes with basic properties. Two practical applications employing novel eluent additives were developed and validated for the pharmaceuticals morphine, clonidine, midazolam, and their metabolites, as well as, for milrinone and dobutamine. The two bioanalytical methods were used to obtain data for pharmacokinetic and pharmacodynamic studies in paediatric patients. Both methods reached exceptionally low limits of quantification, with minimal sample amount used due to the positive influence of novel eluent additives on analyte ionisation

Väitekirja elektrooniline versioon ei sisalda publikatsioone, Aminohapped on looduslikus materjalis esinevad ained, mis annavad maitse-, lõhna- ja muud omadused. Aminohappeline koostis viinamarjades või humalas kandub edasi ka nendest toodetavatesse veinidesse ja õlledesse ning nende sisalduse määramine võimaldab kindlaks teha näiteks joogi päritolu või eristada võltsitud jooke originaalidest. Kuna jookides esineb enamasti mitmeid erinevaid aminohappeid, siis kasutatakse nende määramiseks meetodit, kus esmalt eraldatakse kromatograafia abil aminohapped segust (nt veinist), misjärel antakse neile (ionisatsiooniallikas) laeng ning sellest tekkinud laenguga osakeste hulk registreeritakse massidetektoris. Seejärel saabki juba määrata, millised aminohapped ja millisel hulgal proovis esinevad. Aminohapete analüüsi probleemiks on aga see, et need on väga väikesed molekulid ning samuti esineb neid proovides vähe. Selleks, et need oleks paremini massidetektoris detekteeritavad, kasutatakse keemilist reaktsiooni (derivatiseerimist), et tõsta selektiivsust ja alandada määramispiiri. Veelgi väiksemate hulkade määramiseks on vajalik uurida fragmenteeritud aminohapete derivaate, mis tähendab seda, et enne analüüsi peab olema teada, milliseid aminohappeid proovist otsitakse. Muuhulgas on täpse analüüsi läbiviimiseks vaja proovis sisalduvaid aminohappeid kõrvutada puhaste aminohapetega ehk standardainetega, sest see, kui palju tekib erinevate aminohapete korral laetud osakesi ionisatsiooniallikas, võib erineda tuhandeid kordi. Käesoleva doktoritöö peamine eesmärk oli uurida, kas ja kuidas oleks derivatiseeritud aminohapete määramiseks võimalik kasutada nende standardseid signaaliintensiivsusi ionisatsiooniallikas, et eemaldada standardainete kasutamise vajadus. Doktoritöö raames töötati välja meetod, mis võimaldas veinides ja õlledes standardainetevaba aminohapete määramist ka fragmenteeritud aminohapete derivaatide korral, mis võimaldab selektiivsemat ja madalamate määramispiiridega meetodeid. Töö tulemus avab paljusid võimalusi erinevate rakenduste jaoks, mida on võimalik laiendada ka teistele derivatiseerivatele reagentidele ja analüütidele., Amino acids are substances existing in different natural materials determining their taste, smell and other qualities. Composition of amino acids in grapes, hops and malt also transfers into drinks like wines and beers which are produced from them and determination of these substances enables us to certify the origin of drinks or distinguish counterfeited drinks from originals. Since many different amino acids are simultaneously found in drinks, techniques which allow separating them from mixture (chromatography), giving them a charge (in ionization source) and finally detecting the number of charged particles in mass detector are used. Thereafter, one can already evaluate which amino acids and in what concentration exist in sample. The problem of analyzing amino acids is that they are very small molecules and exist in samples in very low concentration. In order to better detect them in mass detector, chemical reaction (derivatization) is used to enhance selectivity and lower detection limit. To lower detectability even more, it is necessary to study fragmented derivatized amino acids, which means that preselection of compounds to be detected from the sample is needed. It is also inevitable to compare amino acids in the samples with pure amino acids (i.e. standard substances) for precise quantitative analysis, since the yield of generation of charged particles of different amino acids in ionization source can vary over thousands of times. The main goal of this doctoral thesis was to study if and how it possible to use standard signal intensities for derivatized amino acids to eliminate the need of using standard substances. During the study, a method was developed, which allowed to measure fragmented derivatized amino acid concentration in beer and wine without standard substances, allowing to achieve lower quantitation limits and higher selectivity. The result of this study opens up many possibilities for different applications, which could be broaden to other derivative reagents and other analytes., https://www.ester.ee/record=b5359898

Väitekirja elektrooniline versioon ei sisalda publikatsioone., Vedelikkromatograafia-massispektromeetria (LC/MS) on laialt kasutatav meetod väga mitmekesiste analüüside teostamiseks nii rutiin- kui teaduslaborites. LC/MS meetodiga määratakse näiteks taimekaitsevahendeid toiduainetes, erinevaid plastiku komponente sellega kokkupuutuvates produktides, narkootikume veres, seedimise käigus organismis tekkivaid ühendeid, rakkudes leiduvaid valke jne, tegemist on ühe oluliseima meetodiga analüüsimaks komponente segudest. Ionisatsiooniallikas on kahe meetodi (vedelikkromatograafia ja massispektromeetria) ühendamiseks oluline liides, mille abil tekitatakse kromatograafist tulevast vedelikust analüüdimolekulide ioonid, mida analüüsitakse massispektromeetriga. Erinevaid ionisatsiooniallikaid on mitmeid ning pidevalt täiendatakse olemasolevaid ja leiutatakse uusi. Seetõttu on vajalik võrrelda erinevaid uusi ionisatsiooniallikaid, et leida millised on spetsiifilise töö jaoks parimad. Antud töö käigus võrreldi mitmeid uusi ionisatsiooniallikaid kommertsiaalselt levinud elektropihustus ionisatsiooni allikaga (ESI) ning näidati ESI eeliseid võrreldes uudsemate allikatega, mis on popullaarseks muutumas, kuid pole läbinud võrdluse kadalippu, et õigustada nende kasutamist. Samuti oli töö eesmärgiks meie töögrupis eelnevalt väljatöötatud uudse ESI allika pihusti (3R) optimeerimine, mis viidi edukalt lõpule. Optimeerimise tulemused näitavad, uudse 3R pihusti eelised seisnevad peamiselt madalamates avastamispiirides ja robustsuses võrreldes kommertsiaalse allikaga. Samuti täideti edukalt uudse pihustiga seotud teine eesmärk: võrreldi kommertsiaalse pihustiga praktilise analüüsi näitel. 3R pihusti eelised võrreldes kommertsiaalse pihustiga seisnesid väiksemate ainekoguse usaldusväärsemas määramises ning paremas korduvuses. Antud töös leitud erinevate ionisatsiooniallikate võrdlusandmed ning uudse ESI pihusti iseloomustamisel leitud eelised lisavad olulist informatsiooni väga mitmekesisele MS ionisatsiooniallikate temaatikale. Teadlased püüdlevad ühest küljest aina universaalsemate allikate poole, kuid samas otsitakse allikaid väga spetsiifiliste analüüside teostamiseks, mis annaksid parimaid tulemusi. Selleks on vajalik aina uuesti võrrelda praeguseid parimaid allikaid uudsete leiutistega, et leida effektiivseim liides LC/MS meetodile., LC/MS method is widely used in routine analysis and research laboratories. LC/MS method is used for example pesticide analysis in food stuffs, plastic components from the products stored in plastics, toxicological studies in blood, components of human cells and the products of the processes happening inside tissues etc. This is a very small part of the wide range of analysis that rely on LC/MS method. There are numerous novel developments aiming to improve the interface between LC and MS. Novel commercial ion sources need to be compared with each other in order to evaluate their advantages. For some novel ion sources the comparison has been done in this study, occasionally with surprising results. Comparison of the conventional ESI source with some of the newer developments in commercial ionisation sources, such as heated electrospray (HESI), multimode (MMI) source and APPI sources demonstrated that the ESI source is on an average the most robust and sensitive method for generating ions in the interface of LC and MS. The novel 3R nebuliser for ESI source recently developed in our group was compared to the conventional ESI source. Analysis of the main nebuliser parameters affecting the ionisation showed that the inner gas capillary pressure and internal diameter as well as the liquid capillary internal diameter had the strongest influence on the results. The importance of the outer gas capillary pressure, responsible of the nebulisation in the conventional ESI nebuliser, proved to be much less important than the gas pressure in the inner gas capillary, showing the advantage of the novel approach to ESI nebuliser. In a practical comparison – neonicotinoids in honey samples–with the native commercial nebuliser the 3R nebuliser proved to be comparable or better. The aims of the thesis were fulfilled successfully, but many more novel ion source developments need to be evaluated and the work on improving the LC/MS method as such is far from done. This study is expected to add valuable information for the improvement of the LC/MS method and proposes a new direction for the improvement of ESI source.

Submitted by Boris Flegr (boris@uerj.br) on 2021-01-06T14:12:29Z No. of bitstreams: 1 Marcio de Miranda Oliveira.pdf: 7989526 bytes, checksum: 7fa5df0ae275c2ad19b73de13cab9234 (MD5) Made available in DSpace on 2021-01-06T14:12:29Z (GMT). No. of bitstreams: 1 Marcio de Miranda Oliveira.pdf: 7989526 bytes, checksum: 7fa5df0ae275c2ad19b73de13cab9234 (MD5) Previous issue date: 2015-04-15 Endocrine disrupters are compounds micropollutants of deleterious effects that cause effects to animals and humans has brought these problems to several decades related to poor training or infertility of various species. The presence of these compounds in the ng L-1 order is already capable of generating negative effects on the endocrine system. This work aimed at determining the presence and amount of some of these compounds as 17α-ethinylestradiol, bisphenol A and estrone among others, in the waters of the river Arroio Fundo, located in Jacarepaguá in Rio de Janeiro that has a bad water quality index. The first stage of this work was the implementation and validation of a method for determination of 17α-ethinylestradiol by liquid chromatography-fluorescence detection which were established limit of detection, quantification and linearity as directed by Inmetro (2010). Besides the evaluation of the samples through the established methodology. In subsequent step were carried out experiments on the determination of endocrine disruptors by tandem quadrupole mass spectrometry detector, were being performed validation procedures and determination of the compounds bisphenol A and estrone, which were not susceptible to matrix effect during ionization, which prevented the determination of other endocrine disrupters. The results indicate that there is no reduction of the presence of detected endocrine disruptors and other organic compounds, also detected but not identified, between the points upstream and downstream of Arroio Fundo treatment plant. However due to the large matrix effect originating from the fact that the sample be extremely complex, could not perform an identification and quantification of all target compounds. Os desreguladores endócrinos são compostos micropoluentes de ação deletéria que causam efeitos aos animais e aos seres humanos. Estes vêm trazendo a várias décadas, problemas relacionados à má formação ou ainda infertilidade de várias espécies. A presença destes compostos na ordem de ng L-1 já é capaz de gerar efeitos nocivos ao sistema endócrino. Este trabalho visou à determinação da presença e quantidade de alguns destes compostos como 17α-etinilestradiol, bisfenol A e estrona entre outros, nas águas do rio Arroio Fundo, localizado em Jacarepaguá no RJ que possui um índice de qualidade de água ruim. A primeira etapa deste trabalho foi a implementação e validação de metodologia para determinação do 17α-etinilestradiol por cromatografia líquida acoplada a detector de fluorescência onde foram estabelecido limite de detecção, de quantificação e linearidade conforme orientação do Inmetro (2010). Além da avaliação das amostras ambientais através da metodologia estabelecida. Na etapa subsequente foram realizados os experimentos relativos à determinação dos desreguladores endócrinos através da espectrometria de massas por detector tipo tandem quadrupolo, onde também foram realizados os procedimentos de validação e determinação dos compostos bisfenol A e estrona que não foram suscetíveis a efeito de matriz durante a ionização, que acabou impedindo a determinação de outros desreguladores endócrinos. Os resultados indicam que não há redução da presença dos desreguladores endócrinos detectados, e de outros compostos orgânicos também detectados, mas não identificados, entre os pontos a montante e a jusante da Unidade de Tratamento do Rio Arroio Fundo. Todavia em função do grande efeito de matriz oriundo do fato da amostra ser de extrema complexidade, não foi possível realizar uma identificação e quantificação de todos os compostos alvos.

[EN] QTL and candidate genes associated to fruit quality traits have been identified in a tomato genetic map derived from Solanum pimpinellifolium L., providing molecular tools for marker-assisted breeding. The study of genetic, physiological, and molecular pathways involved in fruit development and ripening has considered tomato as the model fleshy-fruited species par excellence. Fruit quality traits regarding organoleptic and nutritional properties are major goals for tomato breeding programs since they largely decide the acceptance of tomato in both fresh and processing markets. Here we report the genetic mapping of single-locus and epistatic quantitative trait loci (QTL) associated to the fruit size and content of sugars, acids, vitamins, and carotenoids from the characterization of a RIL population derived from the wild-relative Solanum pimpinellifolium TO-937. A genetic map composed of 353 molecular markers including 13 genes regulating fruit and developmental traits was generated, which spanned 1007 cM with an average distance between markers of 2.8 cM. Genetic analyses indicated that fruit quality traits analyzed in this work exhibited transgressive segregation and that additive and epistatic effects are the major genetic basis of fruit quality traits. Moreover, most mapped QTL showed environment interaction effects. FrW7.1 fruit size QTL co-localized with QTL involved in soluble solid, vitamin C, and glucose contents, dry weight/fresh weight, and most importantly with the Sucrose Phosphate Synthase gene, suggesting that polymorphisms in this gene could influence genetic variation in several fruit quality traits. In addition, 1-deoxy-D-xylulose 5-phosphate synthase and Tocopherol cyclase genes were identified as candidate genes underlying QTL variation in beta-carotene and vitamin C. Together, our results provide useful genetic and molecular information regarding fruit quality and new chances for tomato breeding by implementing marker-assisted selection., Thanks are due to Dr. Fernando Yuste-Lisbona and Dr. Antonio Monforte for critical review of the manuscript. This work was funded by the ESPSOL project from the Fundacion Genoma of the Spanish Ministerio de Ciencia y Tecnologia. We also thank research facilities provided by the Campus de Excelencia Internacional Agroalimentario (CeiA3).

Con la finalidad de controlar la calidad de alimentos infantiles instantáneos se efectuó la validación de la metodología para la determinación de la vitamina A por cromatografía líquida de alto rendimiento (HPLC). Para la precisión del sistema se evaluaron tres parámetros: tiempo de retención, área y altura, hallándose una desviación estándar relativa (RSD) máxima de 1,78%. En la linealidad se obtuvo un coeficiente de correlación r = 0,99 y una precisión con un RSD de 2,70%. La exactitud del método se evaluó en términos de recuperación mediante la adición de vitamina A al alimento obteniéndose un porcentaje de recuperación de 97,98%; y para la sensibilidad del método se obtuvo un límite de detección (LOD) de 0,06 μg/g y un límite de cuantificación (LOQ) de 0,58 μg/g. Con los resultados obtenidos se demuestra que el método en estudio es preciso, exacto, lineal y altamente sensible para ser utilizado en el control de calidad de alimentos infantiles instantáneos para la determinación de vitamina A. Validation of the methodology used to determine Vitamin A through High Performance Liquid Cromatography (HPLC) was performed to assess instant food for children. Three parameters were assessed in order to determine the system´s accuracy: retention time, area and height and a maximum variation (RSD) of 1.78% was found. A correlative coefficient of r = 0.99 was obtained for the lineal parameter, and an accuracy with 2.70% for RSD. The accuracy of the method was evaluated in terms of recovery through the addition of Vitamin A to the food, the recovery percentage obtained was 97.98%, and for the sensitivity a limit of detection (LOD) 0,06 μg/g was obtained, as well as a limit of quantification (LOQ) 0,58%. The findings demonstrate that the method assessed is accurate, exact, lineal and highly sensitive to be used in quality control of instant food for children for the determination of Vitamin A.

Väitekirja elekrooniline versioon ei sisalda publikatsioone., Maatriksefektid tekivad ESI ionisatsiooniallikas analüüdi ionisatsioonil kui analüüdiga samal ajal elueerub vedelikkromatograafist kaasa mõni maatriksi komponent. Enamasti väljendub maatriksefekt analüüdi ionisatsiooniefektiivsuse languses – st kromatogrammil nähtav analüüdi piik proovi korral on väiksem kui sama kontsentratsiooniga analüüdi standardlahuse puhul. Selline fenomen viib alandatud analüüsitulemuseni. Seetõttu on oluline üritada maatriksefekti vähendada või arvesse võtta. Käesolevas töös esitatakse viis lähenemisviisi maatriksefekti vastu võitlemiseks. Kaks neist annavad võimaluse maatriksefekti vähendamiseks – prooviettevalmistuse optimeerimine ning ESI/MS parameetrite optimeerimine. Kaks meetodit võimaldavad maatriksefekti arvesse võtta – maatriksefekti arvestamine analüüsitulemuse määramatuse koosseisus ning korrigeerimine taustaioonide signaalide kaudu. Samuti esitatakse kombinatsiooniline meetod – ekstrapoleeriv lahjendus. Kõik lähenemisviisid on valideeritud ja nende rakendatavus testitud reaalsetel proovidel. Viiest lähenemisviisist kolm on kirjeldatud esmakordselt., The matrix effect occurs in ESI ionization source in LC/ESI/MS analyses. The ionization efficiency of the analyte changes (usually decreases) due to co-eluting matrix compounds. Therefore underestimated analysis results are observed. In order to improve the accuracy of LC/ESI/MS measurements matrix effect should be reduced (preferably eliminated) or accounted for. In this work altogether five approaches are proposed for combating the matrix effect on the example of pesticide analyses in fruits and vegetables. Two methods – improvement of sample preparation and optimization of ESI/MS parameters – are used to reduce matrix effect. Accounting for matrix effect in the uncertainty of the analytical result and correcting of the analysis result via background ion signals are introduced as methods of accounting for matrix effect. As a combinational method extrapolative dilution is introduced. All the approaches are validated and tested on real samples. Three of the five approaches are presented for the first time.

Made available in DSpace on 2014-06-11T19:28:32Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-01-19Bitstream added on 2014-06-13T20:18:20Z : No. of bitstreams: 1 nogueira_cr_me_araiq.pdf: 3939682 bytes, checksum: f8705affaf8fef2708da3356ae2f0d01 (MD5) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) O presente trabalho descreve o isolamento e identificação dos constituintes químicos de Aristolochia melastoma Manso. O extrato acetônico de caules e raízes foi fracionado por cromatografia em coluna de sílica gel, e as frações obtidas foram submetidas a outros fracionamentos cromatográficos (CCDP, Sephadex LH-20, CC de C-18 e CLAE) e/ou lavagens com solventes orgânicos, resultando no isolamento de vinte compostos conhecidos: aristolactama AII, 9,9'-diacetato-secoisolariciresinol dimetil éter, tirosol, ácido p-hidroxibenzóico, ácido vanilínico, ácidos aristolóquicos I e II, aristolocatos de sódio IIIa e IVa, 4,5-dioxodeidroasimilobíneo, aristolocato de sódio I, sitosterol 3-O-β-D-glucopiranosídeo, glicerol, talictosídeo, uridina, talictricosídeo, icarisídeo D2, adenosina, kaempferol 3-O-β-robinobiosídeo e isorhamnetina 3-O-β-robinobiosídeo. A lignana 9,9'-diacetato-secoisolariciresinol dimetil éter e talictricosídeo são relatados pela primeira vez na família Aristolochiaceae. As identificações desses compostos foram baseadas nas análises dos seus dados espectrométricos (RMN 1D e 2D, UV e EM). Além disso, a análise dos dados obtidos a partir de literatura especializada acerca da ocorrência e biogênese de derivados nitrofeniletílicos sugere que a família Aristolochiaceae pode ter evoluído no sentido de formação de compostos nitrados This work describes the isolation and identification of the chemical constituents of A. melastoma Manso. The acetone extract from stems and roots was fractionated by CC over silica gel and the resultant fractions were subjected to other chromatographic methods (PTLC, Sephadex LH-20, CC RP-18, and HPLC) and/or washed with organic solvents to give twenty known compounds: aristolactam AII, secoisolariciresinol dimethyl ether diacetate, tyrosol, p-hydroxybenzoic acid, vanillic acid, sodium aristolochates I, IIIa, and IVa, 4,5-dioxodehydroasimilobine, aristolochic acids I and II, sitosterol-3-O-β-D-glucopyranoside, glycerol, uridine, thalictoside, icariside D2, adenosine, thalictricoside, kaempferol 3-O-β-robinobioside, and isorhamnetin 3-O-β-robinobioside. Among these, secoisolariciresinol dimethyl ether diacetate and thalictricoside have being reported for the first time in the Aristolochiaceae family. The identifications of these compounds were based on analyses of their spectroscopic data (1D- and 2D-NMR, UV, and MS). Furthermore, an analysis of data obtained from specialized literature on the occurrence and biogenesis of nitrophenylethyl derivatives suggests that the Aristolochiaceae family may have evolved towards nitro compounds

El ácido domoico es una neurotoxina, competidora del glutamato, que se halla en los mares y océanos del mundo. Objetivo: Validar la metodología para determinar ácido domoico (biotoxina ASP) por cromatografía líquida de alto rendimiento (HPLC ). Materiales y métodos: Se analizó una muestra de 1 000 g de moluscos bivalvos (conchas de abanico) extraídos de la zona sur del Perú. Para la precisión del sistema se evaluó: tiempo de retención, área y altura; la exactitud del método se estimó en función de recuperación, analizándose el material de referencia (MUS 1B) y mediante la contaminación de moluscos con solución de ácido domoico. La sensibilidad del método se evaluó por la determinación del límite de cuantificación (LOQ). Resultados: En la detección lineal se obtuvo un coeficiente de correlación r=0,99 y la precisión del método tuvo un coeficiente de correlación de 3,61%. Los porcentajes de recuperación fueron 92,52% y 91,10%, respectivamente, obtuviéndose un límite de cuantificación (LOQ) de 1,30 mg/g AD. Conclusión: Nuestra validación demostró que el método en estudio es preciso, exacto, lineal y altamente sensible para ser utilizado en el control de calidad de moluscos bivalvos. Objective: To validate the methology for the analysis of Domoic Acid (ASP biotoxin) by High performance liquid chroma Objective: To validate the methodology to determine Domoic Acid (ASP biotoxin) by High Performance Liquid Chromatography (HPLC). Material and methods: 1000 g of bivalved mollusks extracted from the south of Peru were analized. Three parameters for a precise system were evaluated: retention time, area and height. Accuracy of the method was determined as a function from recovery, analyzing reference material (MUS 1B) and using mollusks samples with known quantities of standard solution of domoic acid added. Sensibility of the method was evaluated by the determination of quantitive limit (LOQ). Results: The correlation coefficient for the linear analysis was 0,99 and the coefficient of variation (RSD) obtained was 3,61% for the precision of the method. Recovery rates 8% REC) were 92,52% and 91,10% and the quantification limit (LOQ) was 1,3 mg/g AD. Conclusions: The results obtained from the method developed in this study indicate that this method is precise, accurate, selective and highly sensitive and sholud be used in quality control of mollusks samples.