| Abstract | Ciljevi istraživanja: Ispitati kratkotrajnu stabilnost etanola u poslijesmrtnim uzorcima krvi, pohranjenim na -20 0 C, korištenjem GC/FID metode u vremenskom razdoblju od 6 mjeseci, usporediti dobivene rezultate u pohranjenim poslijesmrtnim uzorcima krvi sa i bez dodanog antikoagulansa te usporediti dobivene rezultate i procijeniti stabilnost poslijesmrtnih uzoraka krvi pohranjenih u spremnicima različitih volumena. Ustroj istraživanja: eksperimentalna studija Mjesto istraživanja: Kemijsko-toksikološki laboratorij Kliničkog odjela za sudsku medicinu Zavoda za patologiju, sudsku medicinu i citologiju, Kliničkog bolničkog centra u Splitu. Materijali i metode: Za vrijeme obdukcije izuzeti su poslijesmrtni uzorci krvi, negativni na prisustvo etanola. U dio ispitivanih uzoraka za analizu dodan je 2% antikoagulans- NaF (V 2 ), dok je drugi dio uzoraka (V 1 ) uzorka pohranjena i analiziran bez dodatka antikoagulansa.Tako podijeljeni uzorci alikvotirani su u spremnike manjeg (A1 i BA1) i većeg volumena (A2 i BA2), te pohranjeni na -20 0 C do analize. U uzorke pohranjene u spremnike manjeg volumena (1 mL) dodane su različite vrijednosti etanola: 0.5, 1, 1.5 i 2.5 g/kg, dok je u uzorke pohranjene u spremnike većeg volumena, ispunjene do vrha (15 mL), dodan etanol masene koncentracije 1 g/kg. Nakon analize standardnih otopina etanola i izrade umjerne krivulje, pripremljeni su uzorci za kvantitativno određivanje masene koncentracije alkohola dodatkom 1 mL tercijarnog butanola, internog standarda, i 300 µL uzorka poslijesmrtne krvi u staklenu bočicu s magnetiziranim čepom. Stabilnost alkohola navedenih uzoraka krvi ispitivana je ponovljenom analizom istih uzoraka nakon pohrane tijekom šest mjeseci (30 tjedana), koristeći se GC/FID tehnikom. Ukupno je provedeno devet analiza, 0.dan, 1., 2, 4., 7., 11., 15., 21 i 30. tjedan. Rezultati: GC/FID analizom uspješno je određena kratkotrajna stabilnost etanola poslijesmrtnih uzoraka pohranjenih u spremnicima većeg volumena (15 mL) u kojima je uočena veća stabilnost i sljedivost, u odnosu na poslijesmrtne uzorke pohranjene u spremnicima manjeg volumena (1 mL). Utvrđeno je da ne postoji razlika u kratkotrajnoj stabilnosti etanola tijekom svih 6 mjeseci pohrane uzoraka poslijesmrtne krvi u koje je dodan antikoagulans (2% NaF), u odnosu na one bez dodanog antikoagulansa, neovisno o volumenu spremnika uzoraka. Sudeći prema našim rezultatima dobivenim iz uzoraka pohranjenih u manjim spremnicima, u kojima je načelno koncentracija etanola pokazala određenu nestabilnost, ipak su uzorci iz spremnika s dodanim antikoagulansom dali nešto bolje rezultate. Kod uzoraka pohranjenih u spremnicima većeg volumena, potvrđena je kratkotrajna stabilnost etanola, te značajniji utjecaj dodanog antikoagulansa na dobivene rezultate nije uočen. Zaključak: Kvantitativnom analizom bioloških uzoraka poslijesmrtne krvi, koristeći GC/FID tehniku, uspješno je određena kratkotrajna stabilnost etanola. Uzorci pohranjeni u spremnicima većeg volumena (15 mL) dali su bolju sljedivost rezultata te se pokazali stabilnijima u odnosu na one pohranjene u spremnicima manjeg volumena (1 mL). Utjecaj antikoagulansa potvrđen je analizom uzoraka pohranjenih u spremnicima manjeg volumena, dok kod uzoraka pohranjenih u spremnicima većeg volumena nije uočen. Kako bi se izbjegao utjecaj ventilacije na stabilnost uzoraka te smanjio utjecaj postotka zraka (engl. chamber air, CA% ), preporučeno je koristiti do vrha ispunjene epruvete većeg volumena (15 mL), napravljene od stakla ili plastike. |
| Abstract (english) | Objectives: Investigate the short-term stability of ethanol in post-mortem blood samples stored at -20 ° C using a GC / FID method over a 6- month period, to compare the results obtained with stored post-mortem blood samples with and without added anticoagulants and to compare the results obtained and evaluate the stability of post-mortem blood samples stored in containers of different volumes. Design: Experimental study Settings: Laboratory of toxicology, Department of pathology, medicine and cytology, University Hospital of Split Materials and methods: Post-mortem blood samples were excluded during autopsy, negative to the presence of ethanol. 2% anticoagulant NaF was added to the one part of the test sample (V2), while the other part of the sample (V1) was stored and analyzed without the addition of anticoagulants. Divided samples were aliquoted into a smaller (A1 and BA1) and larger volumes ( A2 and BA2), and stored at -20 ° C until next analysis. In to the samples stored in smaller volumes (1 mL), different ethanol values were added: 0.5, 1, 1.5 and 2.5 g/kg, while to the samples stored in larger volumes, which were filled up to the top (15 mL), ethanol mass concentration of 1 g/kg was added. After analyzing standard ethanol solutions and making calibration curves, samples were prepared for quantitative determination of the mass concentration of alcohol by addition of internal standard- 1 mL tertiary butanol and 300 μL of post-mortem blood sample in to a glass container with a magnetized cap. The stability of the alcohol in the blood samples was tested by repeated analysis of the same samples after storage for six months (30 weeks), using GC/FID technique. In total, nine analyzes were conducted: day 0, week 1, 2, 4, 7, 11, 15, 21 and 30. Results: GC/FID analysis was successfully used to determine the short-term stability of the ethanol in the post-mortem samples that were stored in larger volumes (15 mL), in which greater stability and traceability was observed, compared to a post-mortem samples stored in smaller volumes (1 mL). It was found that there was no difference in the short-term stability of ethanol during all 6 months of storage of post-mortem blood samples in which the anticoagulant (2% NaF) was added, compared to those without added anticoagulants, desregardnig of the sample tank volume. Judging by our results obtained from samples stored in smaller containers, in which the ethanol concentration has generally shown a certain instability, however, the samples from the containers with the added anticoagulant gave some better results. The short-term stability of ethanol was verified in the samples stored in the larger volumes, and the significant influence of the added anticoagulant on the obtained results was not observed. Conclusion: Short-term stability of ethanol was successfully determined by quantitative analysis of biological samples of post-mortem blood, using GC / FID technique. Samples stored in larger volumes (15 mL) gave better traceability of the results and proved to be more stable compared to those stored in smaller volumes (1 mL). The effect of anticoagulant was confirmed by the analysis of samples stored in smaller volumes, while it was not detected in samples stored in larger volumes. In order to avoid the effect of ventilation on the stability of the samples and to reduce the effect of the air intake ("chamber air", CA%), it is recommended to use containers (filled up to the top) with larger volumes (15 mL), made of glass or plastic. |
