STABILITY OF COCAINE IN BLOOD AND IN OTHER TISSUES

STABILITY OF COCAINE IN BLOOD AND IN OTHER

TISSUES*

Marianna KISZKA, Grzegorz BUSZEWICZ, Roman M¥DRO

Chair and Department of Forensic Medicine, Medical Academy, Lublin

ABSTRACT: The stability of cocaine (C) in blood samples and in homogenised sam-ples of liver, kidney and brain was tested. The influence of time (up to 90 days) andtemperature (+25°C, +4°C, –20°C) were evaluated, and, in the case of blood, the in-fluence of acetic acid and sodium fluoride as well. For isolation of C and benzoylec-gonine the solid phase extraction (SPE) was performed. Quantitative analysis wascarried out with the use of the HPLC method. It was shown that the stability of C in-creased with lowering the temperature of storage of the biological material. Freezingblood and other tissues down to –20°C made C stable up to 90 days. In blood samplesstored at +4°C a similar stability of C was reached by addition of acetic acid(to pH = 5) and NaF.

KEY WORDS: Cocaine; Benzoylecgonine; Stability; Autopsy material; Blood; Liver;Kidney; Brain.

Z Zagadnieñ Nauk S¹dowych, z. XLV, 2001, 16-35Received 28 August 2000; accepted 11 October 2000

INTRODUCTION

Cocaine (C) undergoes many biochemical processes in an organism (hy-drolysis, N-demethylation, N-oxidation, hydroxylation, alkylation, dehydro-benzoilation, esterification and other reactions) which lead to the creation ofmany derivative products [18, 19, 20]. The first step of transformation of C isthe hydrolysis of one or two ester groups to pharmacologically inactive me-tabolites: benzoylecgonine (BE), methylesterecgonine (EME), and further toecgonine (E).

* This article is based upon a presentation given at the 11th Meeting of the Polish Society ofForensic Medicine and Criminology, £ódŸ, 1998. The first author, Dr Marianna Kiszka,received the Memorial Award of Professors Jan Markiewicz and Tadeusz Borkowski of theInstitute of Forensic Research, Cracow. The first part of this work (”Stability of cocaine inphosphate buffer and in urine”) was published in Problems of Forensic Sciences 2000,vol. XLIV, pp. 7–23.

BE is formed on the path of hydrolysis of the methyl ester bond, especiallyif pH > 7 [5, 9, 23, 32]. However, it is generally accepted that enzymes alsotake part in the process [2, 9, 21, 26]. The hydrolysis of the other ester bondin the C molecule, leading to the creation of EME, is, however, catalysed bythe liver and plasma esterases [9, 13, 26, 32].

These processes do not only take place in living organisms. They also goon after death until the exhaustion of buffer systems capacity. Moreover, theaction of bacterial enzymatic systems occurs very early after death [25].Also, the great influence of pH on the stability of C should not be overlooked[10] in the interpretation of results of the study of tissue materials. Althoughinitially after death a gradual acidifying of tissues is observed (with themaximum appearing about 24–48 hours after death), in the following phase(of proteolytic autolysis) pH eventually rises above 7 [25].

Knowledge of the dynamics of the decomposition of C in enzymatically ac-tive biological material is therefore of significant importance for toxicologi-cal expert appraisements, both at the stage of securing and storage of sam-ples and at the stage of interpretation of the obtained analytical results.

Stability of cocaine in blood ... 17

Fig. 1. A diagram of preparation and storage of blood samples.

MATERIALS AND METHODS

The material studied was blood samples collected from 5 corpses andfragments of the liver, kidney and brain secured during the autopsy of3 corpses.

The method of preparation of blood samples is shown in part B of Fig-ure 1. Each blood sample was divided into four portions. NaF in the quantityof 10 µg/ml was added to the first portion, CH3COOH was added to the sec-ond portion until pH ≈ 5 was obtained, both compounds were added to thethird, and none of the preservatives was added to the fourth. Then each por-tion (of each blood sample) was divided into two parts, and 5 µg/ml of C wasadded to the first, whereas no C was added to the second1.

18 M. Kiszka, G. Buszewicz, R. M¹dro

Fig. 2. A diagram of preparation and storage of liver, kidney and brain samples.

1 As a background control sample.

The internal organs (3 livers, 3 kidneys and 3 brain hemispheres) werefrozen at –20°C directly after collecting. Then they were partially thawed,cut into small fragments, treated with liquid nitrogen2 and powdered bygrinding in a porcelain mortar. The powdered tissue was divided into twoparts: to the first, C was added to obtain a concentration of 5 µg/g andthe other was left without addition of C3.

Material prepared in this manner (both the blood and the organs) wasplaced into vials that were stored at three different temperatures (+25°C,+4°C, –20°C) and was analysed (in order to determine the concentrations ofC and BE) immediately after the preparation of samples and after 1, 7, 30, 60and 90 days (in accordance with the scheme shown in parts C and D of Fig-ures 1 and 2).

Xenobiotics were isolated with the method of the solid phase extraction(SPE), using Bond Elut Certify 300 mg/3 ml columns made by Varian [1, 6].

Quantitative analysis was carried out by means of the HPLC method(with a liquid chromatograph manufactured by Gilson; Hypersil ODS 250 ×4.0 mm, 5 µm column; the mobile phase: 0.025 M phosphate buffer with addi-tion of 0.5% triethylamine at pH = 3 – acetonitrile 80–20 in a two pump sys-tem; the flow rate of the eluent was 1 ml/min; the volume of the injected sam-ple was 10 µl; detection at a wavelength of 233 nm). Concentrations of C andBE were determined with the method of the internal standard (lignocaine)using Gilson 715 HPLC software4.

Extracts from blood were characterised by a sufficient level of purity,whereas those from other tissues were significantly contaminated, yet theinfluence of ‘biological background’ was not observed at the C peak in any ofthe biological material. However, due to the interference of endogenic com-pounds with the peak of BE, visible in the first part of the HPLC chroma-togram, BE determination was not carried out in the case of extracts fromother (non-blood) tissues.


2 In this way deactivation of enzymes (which occurs in the case of the use of the bladehomogenisor) and a dilution of the biological material was avoided.

3 See footnote no 1.4 The analytical procedure for cocaine and benzoylecgonine determination was presented in

detail in the paper: Kiszka M., M¹dro R., Buszewicz G., “Analytical problems connected withcocaine determination in tissues”, at the 11th Meeting of the Polish Society of ForensicMedicine and Criminology, £ódŸ, 2–5 September 1998.

RESULTS AND DISCUSSION

The concentrations of C and BE determined in 5 different samples ofblood after 1, 7, 30, 60 and 90 days of storage at the following temperatures:+25°C, +4°C and –20°C with and without addition of preservatives (NaF,CH3COOH and NaF + CH3COOH) were used for calculating mean values,and, further, for calculating the decrease in C and the increase in BE5. Theobtained results are graphically presented in Figure 3. Data concerning themean decrease in concentration of C, which was observed in the homo-genates of three different livers (and also kidneys and brains) stored withoutpreservative at temperatures of +25, +4 and –20°C for 1, 7, 30, 60 and90 days are graphically presented in Figure 4. The summary data concern-ing mean losses in the examined biological material as a function of temper-ature and time of storing (and in the case of blood the influence of preserva-tives as well) are listed in Table I.

TABLE I. MEAN COCAINE LOSS (S) IN TISSUES (EXPRESSED IN %).

Temperature 25°C 4°C

Days 1 7 30 60 90 1 7 30 60 90

Blood

– 70 97 100 100 100 21 65 85 96 97

NaF 9 56 91 100 100 2 3 11 42 49

pH 5 5 55 87 98 100 1 4 14 39 51

NaF + pH 5 2 2 4 14 40 1 0 3 6 5

Brain 10 23 55 95 0 0 13 19 26 39

Liver 22 69 78 90 28 28 48 62 59 68

Kidney 34 66 82 100 23 23 33 47 66 70

Stable (S < 15%) Moderately stable (S < 30%) Unstable (S > 30%)

One can observe in Figure 3 that in the samples of blood frozen withoutaddition of preservatives and stored for 90 days, a little decrease in the levelof C down to 77–90% of the starting concentration occurred, whereas in theother blood samples, stored at –20°C, cocaine showed stability during theentire time of the experiment.

At higher temperatures in the non-preserved blood samples a very fastdecrease in concentration of C was observed. At +4°C the loss of C after 1 dayoscillated in the range of 6–37%, after 7 days in the range 28–90%, and at+25°C after only 1 day it reached about 56–84%. Thus the largest value ofthe mean decrease of C at room temperature occurred after 24 hours, and at


5 Expressed in [%] of the starting C concentration.

+4°C after 1 week. Because at the same time no BE was detected in blood orit appeared only at very low concentrations, it should be assumed that themain product of the decomposition of C in non-preserved blood was probablyEME, i.e. the metabolite that was not analysed in this experiment.

Investigations of other authors [5, 7, 16, 22, 27] also indicate that a de-crease in temperature slows down the decomposition of C in blood, but doesnot protect against losses during long storage. Significant differences in thestability of C were present among results of examinations presented in somepublications, which were probably caused by the use of different concentra-tions of C, examinations of blood taken from live persons or corpses and alsoby the individual properties of the studied blood samples. This has been fullyconfirmed in our experiment since we observed large differences betweenthe velocity of the decomposition of C in autopsy blood taken from differentcorpses that could have resulted from differences of post-mortem changes ofpH in blood as well as post-mortem enzymatic activity.

The value of pH of blood after death can decrease to 5.5–6 or even lowervalues [23, 29], so one should expect that in the experiment the chemical de-composition of C to BE would have been inhibited due to the post-mortem de-crease of pH in blood. And, indeed, in some blood samples without preserva-tives, BE appeared only in insignificant concentrations in comparison to thelosses of C. That would support the opinion that the main product of the de-composition of C in blood after death is methyloecgonine, which was not ana-lysed in this experiment [14, 15, 23]. However, the results mentioned aboveare inconsistent with those obtained by Liu et al. [22], who showed in exami-nations of stability of C in blood (10 µg/ml) stored at +16°C that a decrease inC takes place and, simultaneously, an increase in concentration of BE whichis proportional to the decomposition of C – the sum of the concentrations ofboth compounds remaining constant and being equal to the initial concen-tration of C. It seems that this type of decomposition is possible only in caseswhere C does not decompose to EME. This type of decomposition was ob-served only in blood samples with addition of NaF, which is an inhibitor ofpseudocholine esterase responsible for the conversion of C to EME. Thisopinion is confirmed in the works of Isenschmid et al. [14, 15, 16], who con-sider EME to be the main product of the decomposition of C in autopsy blood.

According to Isenschmid et al. [16], the velocity of the enzymatic hydroly-sis of C does not depend on its concentration in blood in the range0.25–1.0 µg/ml, although they do not exclude such an influence at higherconcentrations. Saady et al. [30] quote the opinion of Shuster that the veloc-ity of conversion of C to EME by plasma esterases is strongly connected tothe concentration of the substrate. Thus, in the case of larger concentrationsof C (after exceeding the efficiency of the enzymatic systems), hydrolysis of C


to BE can also take place, which should explain the occurrence of smallquantities of BE in the samples of blood without preservatives.

From a comparison of the velocity of the decomposition of C in blood withNaF and in blood without preservatives (Figure 3), one can observe a signifi-cant influence of sodium fluoride on the stability of C. The addition of NaF toblood stabilised C for 24 hours at +25°C, and for 1 month, if the sample wascooled down to +4°C. In these conditions (temperature +4°C), the maximumloss of C after 7 days was 9% and after 30 days – 23%. However, McCurdy etal. [27] established that under the same conditions in blood samples, C wasstable only for a few days. Nevertheless, observations resulting from theirexperiment concerning the favourable influence of NaF were consistent withresults contained in other publications [5, 7, 11, 16]. The decomposition of Cwhich took place in spite of the addition of NaF can be explained e.g. by theincomplete blocking of enzyme by fluoride [3] or the temporary character ofthis inactivation [16]. The inhibiting influence (on enzymatic activity) ofphosphoro-organic compounds is more effective than that of NaF, but the ad-dition of these compounds is not advantageous because of their interferencewith the analysed xenobiotics [4, 16].

The decrease in the concentration of C in the blood samples of pH = 5 (Fig-ure 3) was close to that found for samples with addition of NaF. In blood pre-served with acetic acid the occurrence of BE was not observed, whereas inblood treated with fluoride BE concentration increased as the concentrationof C decreased. Following Anderson et al. [29] it should be accepted that inblood after death (even in an acidic environment) C is metabolised to EMEby pseudocholine esterase which is still active. So, just acidifying blood sam-ples, especially if left at room temperature, did not sufficiently stabilise theC contained in them.

A significant increase in the durability of C in the acidified blood samples(in comparison to the samples left without any preservation) seems to con-firm the suggestion by Isenschmid et al. [16] of the influence of pH on theprocess of enzymatic decomposition. The stabilising influence of acidity ofblood (pH = 5.2) was mentioned by Moriya and Hashimoto [29], who ob-served no changes in the concentration of C over 24 hours in experimentallydecomposed homogenates of human blood stored at +25°C and +37°C.

Figure 3 shows unequivocally that cocaine in blood was best stabilised bysimultaneous preservation with sodium fluoride and acidifying to pH = 5.After the addition of NaF and acidifying of blood, cocaine was fairly stablefor 60 days, and a decrease in temperature to +4°C offered good protectionagainst decomposition for 90 days. Moreover, in the set of blood samples pre-pared in such a manner (collected from different corpses) the smallest differ-ences in the velocity of decomposition of C were revealed. However, in theother sets (acidified either with addition of fluoride, or containing no preser-


vatives) individual differences were significant. A similar increase in thestability of C was obtained by Isenschmid et al. [16] and Baselt et al. [4], whoalso preserved blood samples by NaF and acetic acid or oxalic acid.

In the tissues stored at +4°C and +25°C (Figure 4 and Table I), the stabil-ity depended on the passage of time and the internal organ from which thesample was taken. Within the same organ some quite large individual differ-ences occurred, which were especially pronounced in the case of livers andkidneys. These differences could indicate the participation of enzymatic pro-cesses in the post-mortem degradation of C in internal organs and could re-sult from different, individually variable, activity of enzymes in particulartissues. In some samples of liver and kidney at a temperature of +25°C theloss of C after 1 day was significant (even 35–43%), although some samplesshowed high stability. On average, after 7 days about 70% of C underwentdegradation. A somewhat slower velocity of decomposition was observed ata temperature of +4°C: after 1 day about 1/4 of the initial concentration of Cwas decomposed, after 7 days the mean loss of C in the liver and in the kid-ney was 48% and 33%, respectively, and during the first month of storage62% and 47%. In the frozen samples of tissues: liver, kidney and brain (Fig-ure 4) C revealed a high stability (similar to that in blood) for practically thewhole time of the experiment. Only in some cases was a small decrease in theconcentration of C observed, but only after 90 days, and the maximum wasin the range of ten to twenty percent, independent of the kind of tissue.

In the performed experiment (Figure 4) the decomposition of C was a lit-tle slower than that noted by Price, who observed decomposition of morethan 90% of C [17] in the liver of a person who died from cocaine poisoning,after two-month storage at +4°C.

A comparison of data on the velocity of the decomposition of C in blood(Figure 3) and in the liver (Figure 4), i.e. in tissues with high activity of en-zymes, leads to the conclusion that at the same temperature the degradationof C in the liver proceeded slower. This can be explained by the higher de-crease in the activity of enzymes in the liver. It is possible, as was suggestedin relation to blood [16], that the differences are connected to the influence ofpH on the activity of enzymes. Liver tissue contains more compounds, whichcan cause acidity, than blood.

The most stable biological environment was the brain. Even at a temper-ature of +25°C the decomposition of C in the brain after 1 day was insignifi-cant (about 10%), after 7 days was moderate (about 1/4 of C), and after1 month in the tissue there were still, on average, about 45% of xenobioticspresent. A decrease in temperature down to +4°C ensured the stability of Cfor many days, since after 30 days the decrease in its concentration was onaverage about 19% and after 60–90 days about 26–39%.



Fig. 3. Stability of cocaine (C) in blood (expressed in [%] as a mean value of 5 bloodsamples) in relation to temperature, storage periods (days) and preservatives: NaFand CH3COOH; BE – benzoylecgonine.

During analysis of autopsy material, Hernandez et al. [12] observedmuch faster decomposition of C in brain samples stored at temperatures of–80°C and +4°C: the difference between the determined concentrations wasabout 48% after a month and about 90% after 2 months. The authors alsoshowed that even after 144 days of storage of samples at +4°C, C was detect-able in the brain.

The results of our work are therefore closer to those in the study carriedout by Spiehler and Reed [31], who did not reveal any significant changes in


Fig. 4. Stability of cocaine in tissues in relation to temperature and storage periods.

C after one and three months in brain samples stored at a temperature of+8°C to +10°C and at –16°C.

In comparison with blood and other tissues the brain is a very good mate-rial for examination, not only because of the high stability of C in vitro ob-served in this work, but also because, as in the cases of poisoning with co-caine [8, 28], the concentrations of this xenobiotic in the brain were rela-tively high (on average 32.9 µg/g) and 4–8 times higher than its concentra-tion in blood. This aspect of the usefulness of brain for toxicological examina-tions, when death from cocaine overdosing is suspected, was confirmed bySpiehler and Reed [31], according to whom the mean ratio of the concentra-tion of C in the brain to that in blood was 9.60 on average, whereas it wasonly 0.36 in the case of BE. The absence of significant concentrations of BEin the brain confirms the high stability of C in this material in vivo as well[8, 28, 31]. This is also supported by the fact that the mean ratio of the con-centrations C/BE in the brain was 14.70 whereas in blood and in the liver0.64 and 0.50 [31] respectively. A comparison of the concentrations of C inthe brain and in the liver reveals greater concentrations in the brain tissue.

Moriya and Hashimoto [29] did not observe a decrease in C during thefirst day at temperatures of 20–25°C and 37°C in the decomposed homo-genates of liver, brain, muscles or blood – so their results differed from thosepresented within the current work. However, the homogenates used byMoriya and Hashimoto were significantly acidified (down to pH = 4.2–5.2).They therefore came to the conclusion that in tissues other than blood thedecomposition of C can be disregarded, since the pH of samples taken afterdeath decreases quickly to a value of less than 7. The post-mortem decompo-sition of C in the liver and in the brain is most probably mainly the result ofthe activity of enzymes, which was confirmed by the fact that in the liver(containing much more esterases) the metabolism of C is faster than in thebrain. The decreased activity of the enzymes in the decomposing tissues in-hibits the breakdown of C to EME – so the progressive processes of decompo-sition (changes of pH and the decrease of the enzymatic activity) can explainthe clearly visible (Figure 4) slower degradation of C in tissues after the30 day period of the experiment.

To summarise, one can state after Manhoff et al. [24] that relatively ad-vantageous conditions for the stability of C arise in corpses, i.e. a decrease intemperature, the acidity of the environment and also a decrease in the effi-ciency of enzymatic processes. However, as was shown in the performedstudy, the conditions do not stabilise the concentration of C in the post-mor-

tem material to a degree that would be sufficient from the point of view of fo-rensic toxicology.


CONCLUSIONS

1. The cold storage of blood samples at a temperature of –20°C, or the ad-dition of 1% NaF plus acidifying down to pH = 5 plus storage at +4°C,ensure the stability of C in blood for 90 days.

2. The freezing of tissue samples at –20°C ensure the stability of C for90 days.

3. The brain is the best material for post-mortem diagnosis of cocaine poi-soning.

4. The quick decrease of C in non-preserved autopsy blood points to thenecessity of determining its metabolites, and simultaneous analysis ofother tissues.

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TRWA£OŒÆ KOKAINY WE KRWI I INNYCH TKANKACH*

Marianna KISZKA, Grzegorz BUSZEWICZ, Roman M¥DRO

WSTÊP

Kokaina (C) w organizmie podlega wielu przemianom biochemicznym (hydroli-zie, N-demetylacji, N-utlenianiu, hydroksylacji, alkilacji, dehydrobenzoilacji, estry-fikacji i innym procesom), które prowadz¹ do powstania wielu produktów pochod-nych [18, 19, 20]. Pierwszym etapem przemiany C jest hydroliza jednej lub obu grupestrowych do nieaktywnych farmakologicznie metabolitów: benzoiloekgoniny (BE)i estru metylowego ekgoniny (EME), a nastêpnie ekgoniny (E).

BE powstaje na drodze chemicznej hydrolizy metylowego wi¹zania estrowego,zw³aszcza przy pH > 7 [5, 9, 23, 32]. Przyjmuje siê jednak, ¿e w procesie tym bior¹udzia³ tak¿e enzymy [2, 9, 21, 26]. Hydroliza drugiego po³¹czenia estrowego w struk-turze cz¹steczki C z wytwarzaniem EME jest natomiast katalizowana przez esterazyw¹troby i osocza [9, 13, 26, 32].

Procesy te maj¹ miejsce nie tylko w ¿ywym organizmie. Do momentu wyczerpa-nia pojemnoœci systemów buforuj¹cych zachodz¹ tak¿e po œmierci. Ponadto w ma-teriale poœmiertnym doœæ wczeœnie wystêpuje dzia³anie bakteryjnych uk³adów en-zymatycznych [25]. W interpretacji wyników badañ materia³u tkankowego nie mo¿-na równie¿ pomin¹æ du¿ego wp³ywu pH na stabilnoœæ C [10]. Po zgonie obserwuje siêwprawdzie pocz¹tkowo stopniowe zakwaszanie tkanek, którego maksimum przy-pada w czasie ok. 24–48 godzin od œmierci, ale w kolejnej fazie (autolizy proteolitycz-nej) dochodzi ostatecznie do wzrostu pH powy¿ej 7 [25].

Poznanie dynamiki rozk³adu C w aktywnym enzymatycznie materiale biologicz-nym ma wiêc istotne znaczenie dla ekspertyzy toksykologicznej zarówno na etapiezabezpieczania i przechowywania próbek przeznaczonych do analizy, jak i na etapieinterpretacji otrzymanych wyników.

MATERIA£ I METODY

Materia³ do badañ stanowi³y próbki krwi pobrane z 5 zw³ok oraz wycinki w¹tro-by, nerki i mózgu, które zabezpieczono w czasie 3 sekcji zw³ok.

Sposób przygotowywania próbek krwi przedstawia czêœæ B ryciny 1. Ka¿d¹ próbêkrwi dzielono na cztery porcje. Do jednej z nich dodawano NaF w iloœci 10 mg/ml, do

* Niniejszy artyku³ opracowany zosta³ na podstawie referatu pt. „Trwa³oœæ kokainy w materia-le biologicznym”, który uznano za najlepsz¹ pracê przedstawion¹ podczas XI KrajowegoZjazdu Polskiego Towarzystwa Medycyny S¹dowej i Kryminologii w £odzi w 1998 roku, a jejpierwszy autor, dr Marianna Kiszka, otrzyma³a Nagrodê imienia Profesorów InstytutuEkspertyz S¹dowych – Jana Markiewicza i Tadeusza Borkowskiego. Pierwsz¹ czêœæ tegoartyku³u („Trwa³oœæ kokainy w buforze fosforanowym i w moczu”) opublikowano w czaso-piœmie Z Zagadnieñ S¹dowych 2000, z XLIV, s. 7–23.

drugiej CH3COOH do uzyskania pH ≈ 5, do trzeciej obie substancje, a do czwartej niedodawano ¿adnych œrodków konserwuj¹cych. Nastêpnie ka¿d¹ porcjê (ka¿dej z próbkrwi) dzielono na dwie czêœci i do jednej z nich dodano C w iloœci 5 µg/ml, natomiastdrug¹ pozostawiono bez dodatku C1.

Narz¹dy wewnêtrzne (3 w¹troby, 3 nerki i 3 pó³kule mózgowe) zamra¿ano w tem-peraturze –20°C bezpoœrednio po ich pobraniu. Nastêpnie czêœciowo rozmra¿ano,krojono na drobne fragmenty i rozdrabniano do postaci proszku przez ucieraniew porcelanowym moŸdzierzu po zalaniu ciek³ym azotem2. Sproszkowan¹ tkankêdzielono na dwie czêœci. Do jednej z nich dodawano C do uzyskania stê¿enia 5 µg/g,a drug¹ pozostawiono bez dodatku C3.

Tak przygotowany materia³ (zarówno krew, jak i narz¹dy) rozdzielono do probów-ek, które przechowywano w trzech ró¿nych temperaturach (+25°C, +4°C, –20°C)i poddawano analizie (w celu oznaczenia poziomu C i BE) tu¿ po sporz¹dzeniu próbekoraz po 1, 7, 30, 60 i 90 dniach (zgodnie ze schematem przedstawionym w czêœciach Ci D rycin 1 i 2).

Ksenobiotyki izolowano metod¹ ekstrakcji z fazy sta³ej (SPE) na kolumnachBond Elut Certify 300 mg/3 ml firmy Varian [1, 6].

Analizê iloœciow¹ wykonywano metod¹ HPLC (chromatograf cieczowy firmy Gil-son; kolumna Hypersil ODS 250 × 4,0 mm, 5 µm; faza ruchoma: bufor fosforanowy0,025 M z dodatkiem 0,5% trietylaminy o pH 3 – acetonitryl 80–20 w systemie dwóchpomp; przep³yw eluentu 1 ml/min; objêtoœæ wstrzykiwanej próbki – 10 µl; detekcja –233 nm). Stê¿enia C i BE oznaczano metod¹ standardu wewnêtrznego (lidokainy)wed³ug oprogramowania Gilson 715 HPLC System4.

Ekstrakty z krwi charakteryzowa³y siê wystarczaj¹cym stopniem czystoœci.Wyci¹gi z innych tkanek by³y natomiast znacznie zanieczyszczone. W miejscu wy-stêpowania piku C nie obserwowano wprawdzie wp³ywu „t³a biologicznego”, ale in-terferencja endogennych substancji z pikiem BE, widoczna w pocz¹tkowej czêœcichromatogramu HPLC sprawi³a, ¿e w przypadku ekstraktów z tkanek zrezygnowa-no z oznaczania BE.

WYNIKI I DYSKUSJA

Stê¿enia C i BE stwierdzone w 5 ró¿nych próbkach krwi po 1, 7, 30, 60 i 90 dniachich przechowywania w okreœlonej temperaturze (+25, +4, –20°C) bez dodatku œrodk-ów konserwuj¹cych oraz z ich u¿yciem (NaF, CH3COOH i NaF + CH3COOH)pos³u¿y³y do obliczenia wartoœci œrednich, a nastêpnie ubytku C i narastania BE5,


1 Jako kontroln¹ próbkê t³a.2 Unikano w ten sposób dezaktywacji enzymów (jaka ma miejsce w przypadku u¿ycia homoge-

nizatora ostrzowego) oraz rozcieñczenia materia³u biologicznego.3 Zob. przypis nr 1.4 Szczegó³y procedur analitycznych zwi¹zanych z oznaczaniem C i BE przedstawione zosta³y

w trakcie XI Krajowego Zjazdu Polskiego Towarzystwa Medycyny S¹dowej i Kryminologii(£ódŸ, 2–5 wrzeœnia 1998 roku) w referacie: Kiszka M., M¹dro R., Buszewicz G., „Problemyanalityczne zwi¹zane z oznaczaniem kokainy w tkankach”.

5 Wyra¿onego w [%] w stosunku do wyjœciowego stê¿enia C.

czego rezultaty przedstawiono graficznie na rycinie 3. Graficznie przedstawiono rów-nie¿ (na rycinie 4) dane na temat œredniego ubytku stê¿enia C, który obserwowanow homogenatach trzech ró¿nych w¹trób (a tak¿e nerek i mózgu) przechowywanychbez dodatku œrodków konserwuj¹cych w temperaturze +25, +4, –20°C przez 1, 7, 30,60 i 90 dni. Zbiorcze zestawienie œredniej wielkoœci strat wyjœciowego stê¿enia Cw przebadanym materiale biologicznym w zale¿noœci od temperatury i czasu jegoprzechowywania, a w przypadku krwi tak¿e wp³ywu œrodków konserwuj¹cych, za-wiera tabela I.

Z ryciny 3 wynika, ¿e w zamro¿onych próbkach krwi bez dodatku œrodków kon-serwuj¹cych po 90 dniach widoczne by³o niewielkie obni¿enie poziomu C do 77–90%jej pocz¹tkowego stê¿enia, zaœ w pozosta³ych próbkach krwi przechowywanej w tem-peraturze –20°C kokaina wykazywa³a stabilnoœæ przez ca³y czas trwania ekspery-mentu.

W wy¿szej temperaturze we krwi nie konserwowanej obserwowano bardzo szyb-kie zanikanie C. W temperaturze +4°C jej straty po 1 dniu waha³y siê w granicach6–37% i 28–90% po 7 dniach, a w temperaturze +25°C ju¿ po 1 dniu siêga³y a¿56–84%. Najwiêkszy œredni ubytek C w temperaturze pokojowej wystêpowa³ zatemw ci¹gu 24 godzin, a w temperaturze +4°C w ci¹gu 1 tygodnia. Ze wzglêdu na to, ¿ew tym czasie nie stwierdzano we krwi BE lub wystêpowa³a ona w bardzo niskich stê-¿eniach, przyj¹æ nale¿y, ¿e g³ównym produktem rozk³adu C we krwi nie konserwo-wanej by³ prawdopodobnie EME, czyli metabolit, którego nie oznaczano w tym eks-perymencie.

Z badañ innych autorów [5, 7, 16, 22, 27] wynika tak¿e, ¿e obni¿enie temperaturyspowalnia rozk³ad C we krwi, ale nie zabezpiecza przed jej stratami podczas d³u¿sze-go przechowywania. Miêdzy wynikami badañ przedstawionych w niektórych publi-kacjach widoczne by³y istotne ró¿nice stabilnoœci C we krwi, które prawdopodobniewynika³y ze stosowania ró¿nych stê¿eñ C, badania krwi pobranej od osób ¿ywych lubze zw³ok, a tak¿e osobniczych w³aœciwoœci badanych prób krwi. Znajduje to pe³ne po-twierdzenie w przeprowadzonym przez autorów eksperymencie. Obserwowano bo-wiem du¿e ró¿nice miêdzy tempem rozk³adu C we krwi sekcyjnej pobranej z ró¿nychzw³ok, co mog³o wynikaæ z ró¿nic w poœmiertnych zmianach pH krwi i poœmiertnejaktywnoœci enzymatycznej.

Odczyn krwi po zgonie mo¿e obni¿aæ siê do pH = 5,5–6,0 lub nawet ni¿szych war-toœci [23, 29] i w zwi¹zku z tym w eksperymencie nale¿a³o oczekiwaæ, ¿e chemicznyrozk³ad C do BE zostanie zahamowany w wyniku poœmiertnego obni¿ania siê pHkrwi. I rzeczywiœcie, w niektórych próbkach krwi bez dodatku substancji konserwu-j¹cych obserwowano pojawianie siê BE tylko w znikomych stê¿eniach w porównaniuze stratami C. Potwierdza³oby to pogl¹d, ¿e g³ównym produktem rozk³adu C we krwipo zgonie jest metyloekgonina, której nie oznaczano w tym eksperymencie [14, 15,23]. Przytoczone wy¿ej wyniki pozostaj¹ jednak w sprzecznoœci z ustaleniami Liu i in.[22], którzy w badaniach stabilnoœci C we krwi (10 µg/ml) przechowywanej w tempe-raturze +16°C wykazali spadek jej zawartoœci i równoczesny, proporcjonalny dorozk³adu C, wzrost stê¿enia BE, przy czym suma stê¿eñ obu sk³adników by³a sta³a,równa pocz¹tkowej iloœci C. Wydaje siê, ¿e tego rodzaju rozk³ad jest mo¿liwy tylkow przypadku, gdy C nie rozk³ada siê do EME. Taki rozk³ad obserwowano jedyniew próbkach krwi z dodatkiem NaF, który jest inhibitorem pseudocholinesterazy od-powiedzialnej za przemianê C do EME. Pogl¹d ten znajduje potwierdzenie w pracach


Isenschmida i in. [14, 15, 16], którzy za g³ówny produkt rozk³adu C we krwi sekcyjnejuwa¿aj¹ w³aœnie EME.

Wed³ug Isenschmida i in. [16] szybkoœæ enzymatycznej hydrolizy C nie zale¿y odjej stê¿enia we krwi w zakresie 0,25–1,0 µg/ml, chocia¿ nie wykluczaj¹ takiegowp³ywu przy wy¿szych stê¿eniach. Jednak Saady i in. [30] przytaczaj¹ opiniê Shus-tera, ¿e tempo rozk³adu C do EME przez esterazy osocza jest mocno zwi¹zane ze stê-¿eniem substratu. W przypadku wiêkszych stê¿eñ C (po przekroczeniu wydolnoœciuk³adów enzymatycznych) mo¿e zatem tak¿e wystêpowaæ hydroliza C do BE, czymnale¿y t³umaczyæ wystêpowanie niewielkich iloœci BE w próbkach krwi bez dodatkukonserwantów.

Z porównania szybkoœci rozk³adu C we krwi z dodatkiem NaF i krwi bez dodatkusubstancji konserwuj¹cych (rycina 3) wynika wyraŸnie zaznaczony stabilizuj¹cywp³yw fluorku sodu. Dodatek NaF do krwi stabilizowa³ zawart¹ w niej C przez 24 go-dziny w temperaturze +25°C i przez 1 miesi¹c wówczas, gdy próbkê krwi sch³odzonodo +4°C. W tych warunkach (temperatura +4°C) maksymalny ubytek C po 7 dniachwynosi³ 9%, a po 30 dniach – 23%. Tymczasem McCurdy i in. [27] stwierdzili, ¿ew tych samych warunkach w próbkach krwi C by³a stabilna zaledwie przez kilka dni.Wynikaj¹ce z eksperymentu spostrze¿enia dotycz¹ce pozytywnego dzia³ania NaF s¹natomiast zgodne z ustaleniami zawartymi w innych publikacjach [5, 7, 11, 16].Rozk³ad C, który zachodzi³ mimo dodatku NaF, mo¿na wyt³umaczyæ m.in. niepe³nymzablokowaniem enzymu przez fluorek [3] lub przejœciowym charakterem tej inakty-wacji [16]. Hamuj¹ce (aktywnoœæ enzymatyczn¹) dzia³anie zwi¹zków fosforoorga-nicznych jest skuteczniejsze ni¿ NaF, ale dodatek tych substancji nie jest korzystnyze wzglêdu na mo¿liwoœæ ich interferencji z oznaczanymi ksenobiotykami [4, 16].

Obni¿enie stê¿enia C w próbkach krwi o pH = 5 (rycina 3) by³o zbli¿one do wyka-zanego w próbkach z dodatkiem NaF. We krwi konserwowanej kwasem octowym nieobserwowano pojawiania siê BE, której stê¿enie narasta³o w miarê spadku stê¿eniaC we krwi fluorkowanej. Nale¿y zatem przyj¹æ za Andersonem i in. [29], ¿e C we krwipo œmierci (nawet w œrodowisku kwaœnym) jest metabolizowana do EME przez wci¹¿aktywn¹ pseudocholinesterazê. Tak wiêc samo zakwaszenie próbek krwi, zw³aszczapozostawionych w temperaturze pokojowej, nie stabilizowa³o dostatecznie zawartejw nich C.

WyraŸny wzrost trwa³oœci C w zakwaszonych próbkach krwi (w porównaniuz próbkami bez jakiejkolwiek konserwacji) wydaje siê potwierdzaæ sugerowany przezIsenschmida i in. [16] wp³yw pH na enzymatyczny proces rozk³adu. Na stabilizuj¹cywp³yw kwaœnego odczynu krwi (pH = 5,2) zwrócili równie¿ uwagê Moriya i Hashimo-to [29], którzy stwierdzili brak zmian stê¿enia C przez 24 godziny w eksperymental-nie roz³o¿onych homogenatach ludzkiej krwi przechowywanych w temperaturze+25°C i +37°C.

Z ryciny 3 wynika jednoznacznie, ¿e kokainê we krwi najlepiej stabilizowa³a rów-noczesna konserwacja fluorkiem sodu i zmiana odczynu do pH = 5. Po dodaniu NaFi zakwaszeniu krwi w temperaturze +25°C kokaina by³a bowiem doœæ stabilna przez60 dni, a obni¿enie temperatury do +4°C praktycznie zabezpiecza³o j¹ przed rozk³a-dem przez 90 dni. W serii tak zabezpieczonych próbek krwi (z ró¿nych zw³ok) wyka-zano ponadto najmniejsze ró¿nice w szybkoœci rozk³adu C. Natomiast w pozosta³ychseriach (nie zawieraj¹cych konserwantów, tylko zakwaszonych i wy³¹cznie fluorko-wanych) indywidualne ró¿nice by³y znaczne. Podobny wzrost stabilnoœci C uzyskali

Trwa³oœæ kokainy we krwi i innych tkankach 33

Isenschmid i in. [16] oraz Baselt i in. [4], którzy tak¿e konserwowali próbki krwi przyu¿yciu NaF i kwasu octowego lub kwasu szczawiowego.

W tkankach przechowywanych w temperaturach +4°C i +25°C (rycina 4 i ta-bela I) trwa³oœæ C zale¿a³a od up³ywu czasu oraz narz¹du, z którego pobrano ma-teria³. W obrêbie tego samego narz¹du wystêpowa³y doœæ du¿e indywidualne ró¿nice,co by³o widoczne zw³aszcza w przypadku w¹troby i nerki. Ró¿nice te mog³y wskazy-waæ na udzia³ procesów enzymatycznych w poœmiertnej degradacji C w narz¹dachwewnêtrznych i wynikaæ z ró¿nej, zmiennej osobniczo, aktywnoœci enzymów w po-szczególnych tkankach. W niektórych próbkach w¹troby i nerki w temperaturze+25°C ubytek C ju¿ po 1 dniu by³ znacz¹cy (nawet o 35–43%), chocia¿ zdarza³y siêpróbki o du¿ej stabilnoœci, a po 7 dniach degradacji ulega³o œrednio ok. 70% C. Niecowolniejsze tempo rozk³adu obserwowano w temperaturze +4°C, gdy¿ w ci¹gu 1 dobyrozk³ada³a siê w przybli¿eniu 1/4 wyjœciowego stê¿enia C, po 7 dniach œrednie stratyC w w¹trobie i w nerce wynosi³y odpowiednio 48% i 33%, a w ci¹gu pierwszego mie-si¹ca przechowywania próbek 62% i 47%. Natomiast w zamro¿onych próbkach tka-nek: w¹troby, nerki i mózgu (rycina 4) C wykazywa³a wysok¹ stabilnoœæ (podobniejak w przypadku krwi) praktycznie przez ca³y czas doœwiadczenia. Tylko w niektó-rych próbkach obserwowano niewielkie obni¿enie stê¿enia dopiero po 90 dniach,maksymalnie do kilkunastu procent, niezale¿ne od rodzaju tkanki.

W przeprowadzonym doœwiadczeniu (rycina 4) rozk³ad C by³ nieco wolniejszy ni¿wynika³o to z obserwacji Price’a, który w w¹trobie osoby zmar³ej w wyniku zatruciakokain¹ po 2 miesi¹cach jej przechowywania w temperaturze +4°C obserwowa³rozk³ad ponad 90% C [17].

Porównanie danych na temat szybkoœci rozk³adu C we krwi (rycina 3) i w w¹tro-bie (rycina 4), tj. tkankach o du¿ej aktywnoœci enzymów, prowadzi do wniosku, ¿ew tej samej temperaturze degradacja C w w¹trobie przebiega³a wolniej. Mo¿na tot³umaczyæ szybszym spadkiem aktywnoœci enzymów w w¹trobie. Byæ mo¿e, jak to su-gerowano w odniesieniu do krwi [16], ró¿nice te wi¹¿¹ siê z wp³ywem pH na aktyw-noœæ enzymów. Tkanka w¹troby zawiera bowiem wiêcej ni¿ krew substancji, któremog¹ powodowaæ zakwaszenie.

Najbardziej stabilnym œrodowiskiem biologicznym okaza³ siê mózg. Nawetw temperaturze +25°C rozk³ad C w mózgu po 1 dniu by³ bowiem nieznaczny(ok. 10%), po 7 dniach umiarkowany (ok. 1/4 C), a po 1 miesi¹cu w tkance tej pozosta-wa³o nadal œrednio 45% ksenobiotyku. Natomiast obni¿enie temperatury do +4°C za-pewnia³o stabilnoœæ C przez wiele dni, gdy¿ po 30 dniach dochodzi³o do spadku jej stê-¿enia œrednio o 19%, a po 60–90 dniach œrednio o 26–39%.

W trakcie analizy materia³u sekcyjnego Hernandez i in. [12] wykazali o wieleszybszy rozk³ad C w próbkach mózgu przechowywanych w temperaturze –80°Ci +4°C, gdy¿ po miesi¹cu ró¿nica miêdzy wykazanymi w nich stê¿eniami wynosi³aoko³o 48% i ponad 90% po 2 miesi¹cach. Autorzy ci wykazali jednak, ¿e nawet po144 dniach przechowywania próbek w temperaturze +4°C mo¿na by³o jeszcze wykryæC w mózgu. Wyniki przeprowadzonego przez autorów niniejszej pracy eksperymentus¹ zatem bli¿sze badaniom Spiehlera i Reeda [31], którzy nie wykazali znacz¹cychzmian stê¿eñ C po up³ywie jednego oraz trzech miesiêcy w próbkach mózgu prze-chowywanych w temperaturze od +8°C do +10°C i w temperaturze –16°C.

W porównaniu z krwi¹ oraz innymi tkankami mózg jest wiêc bardzo dobrym ma-teria³em badawczym, nie tylko ze wzglêdu na obserwowan¹ w tej pracy du¿¹ stabil-


noœæ C in vitro, ale tak¿e ze wzglêdu na to, ¿e w przypadkach zatruæ kokain¹ [8, 28]stê¿enia tego ksenobiotyku w mózgu by³y stosunkowo wysokie (œrednio 32,9 µg/g)i 4–8-krotnie wy¿sze od stê¿enia C we krwi. Ten aspekt przydatnoœci mózgu do badañtoksykologicznych, gdy podejrzewa siê œmieræ w wyniku przedawkowania kokainy,potwierdzaj¹ Spiehler i Reed [31], wed³ug których stosunek stê¿enia C w mózgu dostê¿enia we krwi wynosi³ œrednio 9,60, a dla BE tylko 0,36. Brak znacz¹cych stê¿eñBE w mózgu potwierdza du¿¹ trwa³oœæ C w tym materiale tak¿e w warunkach in vivo

[8, 28, 31]. Œwiadczy o tym równie¿ œredni stosunek stê¿eñ C/BE, który w mózgu wy-nosi³ 14,70, a we krwi i w w¹trobie odpowiednio 0,64 oraz 0,50 [31]. Porównanie po-ziomów stê¿eñ C w mózgu i w w¹trobie wypada przy tym korzystnie dla tkanki móz-gowej [31].

Moriya i Hashimoto [29] nie stwierdzili zanikania C w ci¹gu 1 doby w temperatu-rze 20–25°C i 37°C w roz³o¿onych homogenatach w¹troby, mózgu, miêœni oraz krwi,czym rezultaty ich badañ ró¿ni¹ siê od przedstawionych w tej pracy. Homogenaty teby³y jednak w znacznym stopniu zakwaszone (do pH = 4,2–5,2), dlatego wspomnianiautorzy doszli do wniosku, ¿e w innych ni¿ krew tkankach, chemiczny rozk³ad Cmo¿e byæ pominiêty, poniewa¿ odczyn próbek pobranych po zgonie obni¿a siê szybkodo wartoœci pH mniejszej ni¿ 7. Poœmiertny rozk³ad C w w¹trobie i mózgu jest praw-dopodobnie g³ównie wynikiem dzia³ania enzymów, co potwierdza fakt, ¿e w w¹trobie(zawieraj¹cej du¿o wiêksz¹ iloœæ esteraz) metabolizm C jest szybszy ni¿ w mózgu. Ob-ni¿ona aktywnoœæ enzymów w gnilnie roz³o¿onych tkankach zwalnia rozk³ad C doEME, a zatem postêpuj¹cymi procesami rozk³adowymi (zmiany pH i spadek aktyw-noœci enzymatycznej) mo¿na wyt³umaczyæ wyraŸnie widoczn¹ (rycina 4) wolniejsz¹degradacjê C w tkankach po 30 dniach trwania eksperymentu.

W podsumowaniu mo¿na powtórzyæ za Manhoffem i in. [24], ¿e w zw³okach wy-stêpuj¹ stosunkowo korzystne warunki dla trwa³oœci C, tj. obni¿enie temperatury,kwaœne œrodowisko, a tak¿e spadek wydolnoœci procesów enzymatycznych. Jednak,jak wykazano w przeprowadzonych badaniach, warunki te nie stabilizuj¹ stê¿eñ Cw materiale poœmiertnym w stopniu zadowalaj¹cym z punktu widzenia toksykologiis¹dowej.

WNIOSKI

1. Zamro¿enie próbek krwi w temperaturze –20°C, a tak¿e dodanie 1% NaFi równoczesne ich zakwaszenie do pH = 5 po³¹czone z przechowywaniemw temperaturze +4°C zapewniaj¹ osi¹gniêcie trwa³oœci C we krwi do 90 dni.

2. Skutecznym sposobem zapewnienia stabilnoœci C w tkankach przez 90 dni jestich zamro¿enie w temperaturze –20°C.

3. Mózg jest najlepszym materia³em do poœmiertnej diagnostyki zatruæ kokain¹.4. Szybkie zanikanie C w nie konserwowanej krwi sekcyjnej wskazuje na ko-

niecznoœæ oznaczania jej metabolitów i równoleg³¹ analizê innych tkanek.

Trwa³oœæ kokainy we krwi i innych tkankach 35

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STABILITY OF COCAINE IN BLOOD AND IN OTHER TISSUES