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© by the Institute of Forensic Research

Problems of Foren sic Science s 2005, LXIII, 275–287 ISSN 1230-7483

DETERMINATION OF DENATONIUM BENZOATE (BITREX) IN DENATURED SPIRIT PREPARATIONS

Dariusz ZUBA, Czes³awa ŒWIEGODA, Bogumi³a BYRSKA, Wojciech LECHOWICZ

In sti tute of Fo ren sic Re search, Krakow

Ab stract The aim of the study was to elabo rate a chro matographic method for de ter mi nation of denatonium benzo ate (Bitrex) in spir its in- tended for in dustrial pur poses as well as in products prepared on the basis of dena tured spirit, such as windscreen washers, disin - fec tants, al co holic sol vents, or bar be cue light ing fluid. High-per for mance liq uid chro ma tog ra phy (HPLC) was ap plied to detec tion and quanti tative analy sis of denatonium benzo ate in spirit prod ucts. Sepa ra tion was performed on a Chromolith Perfor - mance RP-18e (100 ´ 4.6 mm) col umn, in gra di ent condi tions, with the use of 0.01% aque ous solu tion of phos phoric acid and acetonitrile as the elu ents. Iden ti fi ca tion of com po nents was car ried out us ing a spec tro pho to met ric de tec tor with a di ode-ar ray matrix (DAD). The presence of denatonium benzo ate in spirits was confirmed by mass spectrom e try (MS). Liq uid sam ples were in tro duced directly into the ion isation cham ber of the spectrom e ter. Both posi tive (ES+) and neg ative (ES–) ion isation mode were applied. The cap il lary volt age was 3.0 kV and the CID zone voltage was 15 V. The worked-out pro cedure of spir its exam i - nation for the denatonium benzo ate content is straightfor ward and fast. It en ables exam i nation of colour less liquids without pre - lim i nary sample prep ara tion, or, pos sibly, just with centrifugation of the sam ple. The de ter mined limit of quantitation was 0.3 mg/l, i.e. ten times lower than the min i mal amount of denatonium benzo ate, which the dena tured spir its should contain ac- cord ing to the decree of the Minis try of Agri culture and Rural De velop m ent (0.3 g/100 l of 100% al cohol). The above method is used in rou tine ex am i na tions per formed at the In sti tute of Fo ren sic Re search. Key words De na tured spirit; Denatonium ben zo ate; Pu ri fi ca tion.

Re ceived 9 De cem ber 2005; ac cepted 30 De cem ber 2005

1. In tro duc tion benzo ate is highly solu ble in metha nol (69.0 g per 100 ml at 20°C), etha nol (33.5), chloro form (33.0) and Denatonium benzo ate (Bitrex) is descri bed in the iso-propanol (10.5), whereas sol ubil ity in water is lo- Guinness Book of Records as the bitter est com pound wer (4.5). It was synthe sised in 1958 during resear ch known to man. Adding as little as thirty parts of de - on lo cal an aes thet ics. Denatonium ben zo ate is closely natonium benzo ate to one million parts of a liquid related to one such sub stance – . Its chem i cal makes it too bit ter to be toler ated by most hum an sub- struc ture dif fers only in the addi tion of a jects. to the amino nitro gen. The chem ical structure of de- The chem i cal name of Bitrex is N-benzyl-2-(2,6- natonium benzo ate is shown in Figure 1. dimethylphenylamino)-N,N-di ethyl-2-oxoethanami- Tox i co log i cal data in di cate its low tox ic ity [10]. The nium benzo ate (CAS Regis try Num ber: 3734-33-6). It de ter mined LD50 af ter intragastric ap pli ca tion to an i - occurs as white odourless granules of neutral pH mals are as fol lows: LD50 rat – 820 mg/kg, LD50 mouse – (6.5–7.5), with melting point 163–170°C. Denatonium 865 mg/kg, LD50 rabbit – 580 mg/kg, LD50 Guinea 276 D. Zuba, C. Œwiegoda, B. Byrska et al.

quired the addi tion of denatonium benzo ate at a con- centra tion of 30–50 ppm with the inten tion of reduc ing the fre quency of se ri ous pae di at ric in tox i ca tions with these products [11]. Since that time many papers showing the preven ta tive action of denatonium benzo - ate have been published [3, 9, 15]. Denatonium ben zo ate is a qua ter nary am mo nium, which makes its analy sis diffi cult. In 1991, a poly(vinyl chloride) matrix mem brane Fig. 1. The chemi cal structure of denatonium benzo ate ion-se lec tive elec trode for the de ter mi na tion of the (Bitrex). denatonium ion based on the denatonium salt of tetra- phenylborate was described. The response charac ter is - pig – 800 mg/kg, LD50 rainbow trout – 1000 mg/kg. tics of the electrode for the denatonium ion and for How ever, one should rem em ber that there are signif i - several other com pounds that were quater nary am mo - cant gaps in knowledge in this field, espe cially relat ing nium, were studied. The results of potentiometric mea- to chronic tox ic ity in hu mans, tera to ge nici ty, and hu - surem ents of denatonium benzo ate content in rapeseed man hy per sen si tiv ity po ten tial. oil in the range of 1–10 ppm agreed to within ±5% of Use of denatonium benzo ate is linked to its bitter - the spiked amounts [10]. ness. It is used to dena ture ethyl alco hol that it is not The most com mon method used in practice for its taxed as an al co holic bev erage. In fact, the com mon de ter mi na tion in con sumer prod ucts is high-per for- name for this chem ical, denatonium, alludes to this ap- mance liq uid chro ma tog ra phy (HPLC) [5, 8]. Anal y sis pli cation. The in ten sity of its , its low tox icity and of standard solu tions showed a linear ity in the range of lack of car ci no ge nic ity make denatonium ben zo ate an 1.25 to 50 ppm (produc ers usually add from 10 to ideal addi tive to prevent people from ingest ing harm - 30 ppm). The eluate from the range corre spond ing to ful liq uids such as de ter gents, dis in fec tants, clean ing the recorded peak is col lected and the pres ence of dena - flu ids and gar den and hor ti cul tural prod ucts. Be cause tonium ben zo ate is ver i fied by mass spec trom e try. of the aver sion it engen ders, denatonium benzo ate is The aim of another study concern ing denatonium increas ingly being used as an aversion addi tive. The benzo ate was to elabo rate a quick method for the isola - addi tion of denatonium is credited with saving chil- tion and enrich m ent of this com pound from low-solid - dren and ani m als, who (which) might other wise drink i fi ca tion flu ids ap plied to cool ing of in ter nal-com - sweet anti freeze or wiper fluid and get eth ylene glycol bustion engines (liquids based on ethyl ene and propyl - or meth a nol poi son ing re spec tively. Other uses in- ene glycol). Solid phase extrac tion (SPE) was used for clude deer repel lant, “nail polish” – to dis courage sam ple prep a ra tion and de ter mi na tion was car ried out nail-biting, , coating of electri cal cables to pre- by means of high per for mance liq uid chro ma tog ra phy vent rodents from eating the insu la tion. Very dilute so- in reverse d phase mode (RP HPLC). Thanks to opti m i- lutions are also used to coat children’ s thumbs to sation of the sam ple prepa ra tion method, high re cover - prevent thumb sucking. It should be noted that ani m als ies (about 90%) as well as reproducibility of quan- are known to have differ ent sensi tiv ity to the effects of titative and qual i ta tive re sults of de ter mined analyte denatonium ben zo ate. Dif fer ences in per cep tion of its con cen tra tion (RSD = ±1%) were obtained [6]. bitter taste have also been observe d in children, adults The cap il lary elec tro pho re sis (CE) tech nique has and elderly persons [14]. The data indi cate that addi - also been ap plied to stud ies. Op ti mised sep a ra tion was tion of denatonium benzo ate would be expected to sig - carried out in 0.025 mol/l Tris-phos phate electro lyte nif i cantly re duce the prob a bil ity of ac ci den tal (pH 7.0) using direct UV detec tion at 214 nm. Cali bra - inges tion [2]. When ingested at 10 ppm by hum an be- tion was linear in the range 2–50 mg/l. The detec- tion ings, denatonium benzo ate has a bitter, unpleas ant limit de term ined for a 10 s electrokinetic (5 kV) injec - taste. It has been proven in studies that the addi tion of tion was 0.45 mg/l (3/N). The method was applied to denatonium benzo ate to liquid dish deter gents reduces the de ter mi na tion of Bitrex in var i ous de na tured al co- the amount of acci den tal poison ings by children [7]. hol for mu la tions and the re sults were com pa ra ble with Ore gon was the first state in the United States where those obtained by the HPLC method [13]. man da tory ad di tion of this de na tur ing agent to au to- The cur rently de vel oped tan dem tech nique, i.e. motive products contain ing at least 10% ethyl ene mass spec tros copy with at mo spheric-pres sure chem i- glycol or at least 4% metha nol was intro duced [8]. cal ion isation (APCI-MS) coupled with direct flow in - In 1995, the Toxic Household Products Statute re- jection, was applied to the de term ina tion of Bitrex in

Problems of Fo ren sic Sci ences 2005, LXIII, 275–287 Determination of denatonium benzoate (Bitrex) in denatured spirit... 277 etha nol. Com pounds were identi fied on the ba sis of A/0 % of phase B, 21 min – 100% of phase A/0 % of their total-ion chromatograms collected in the range of phase B and 5 min stabili sa tion in these condi tions. m/z 50–500. Quanti ta tive analy sis was also perform ed Mea sure ments of denatonium ben zo ate con cen tra tion using selected- ion m/z 325 for Bitrex. Oper at ing con- were carried out at two wavelengths: 205 nm and di tions were op ti mised for soft ion is ation (pos i tive 230 nm. ion-mode) with frag men tation lim ited to that nec es- “Val i da tion Man ager” soft ware cre ated by sary for analyte iden ti fi ca tion. The cal i bra tion curve Merck/Eurolab was ap plied when cal cu lat ing val i da- was recti lin ear in the concen tra tion range 2–30 g/ml, tion pa ram e ters. the mea sure ment pre ci sion (RSD) was 11.5% and de- Mass spec trome try (MS) was used for con fir ma tion tection limit was 0.017 g/ml. The method allowed si- of the presence of denatonium benzo ate. A Quattro Mi- mul ta neous quan ti fi ca tion with high sen si tiv ity. The cro API mass spec trom e ter man u fac tured by Micromass lack of any sam ple pre-treatm ent and the use of flow- was applied in the study. Liquid sam ples were intro - in jec tion anal ysis meant that the pro cedure was more duced directly into the ionis ation cham ber of the spec- straightfor war d and rapid than previ ously reported trom eter with a flow veloc ity of 10 µl/min. Both the meth ods [1]. posi tive (ES+) and nega tive (ES–) ionis ation mode were The aim of the study presented in this paper was to used. The capil lary voltage was 3.0 kV and the CID zone de velop a sim ple pro ce dure of spir its ex am i na tion us- voltage was 15 V. Dry ing gas was deliv ered with a veloc - ing chrom atographic techniques in or der to deter m ine ity of 600 l/h at 300oC. The tem pera ture of the ions’ the denatonium benzo ate content. source was 100oC. Ion mass range m/z = 25–400 was mon i tored.

2. Ma te ri als and meth ods 3. Re sults and dis cus sion When devel op ing the method of denatonium ben- zo ate de ter mi na tion, a stan dard of this sub stance man- Denatonium benzo ate is a de natur ing agent for ufac tured by Sigma-Aldrich was applied. Standard ethyl alco hol, listed in the de cree of the Minis try of solu tions of Bitrex were prepared by weighing an ap- Ag ri cul ture and Ru ral De vel op ment of 11 Au gust 2003 propri ate amount of denatonium benzo ate and dissolv - con cern ing agents per mit ted to de na ture ethyl al co- ing in rec ti fied spirit (Polmos Warsaw). The ac cu racy hol [16]. Its addi tion does not af fect the basic phy - of the concen tra tion in the pre pared solu tion was as- sicochemical prop erties of ethyl alco hol, be cause the sessed in inter-lab o ra tory ex am i na tions. amount of denatonium benzo ate that should be added The worked out method was also applied to the accord ing to the above mentioned decree is small and anal y sis of ev i den tial ma te rial. Sam ples of ethyl al co - amounts to 0.3 g per 100 l of 100% ethyl alco hol. hol seized in 2005 and deliv ered to the Alco hol and Bitrex can also be added in the form of 20% alco holic Drugs Section of the Insti tute of Foren sic Resear ch so lu tion. were used for this purpose. The main group com prised Denatonium ben zo ate is a chem i cal of rela tively al co holic bev erages seized in restau rants and from pri- high molec u lar mass. Analys ing its structure, one may vate per sons. More over, al co hol orig i nat ing from observe that it is a quater nary salt contain ing chro- plants deal ing with its process ing was also studied. mophoric groups. Therefore , the method of choice for In order to deter m ine the content of denatonium de ter mi na tion of denatonium ben zo ate is high-per for- ben zo ate in ex am ined liq uids, high-per for mance liq- mance liq uid chro ma tog ra phy (HPLC). uid chro ma tog ra phy (HPLC) was ap plied. Ex am i na- Pre lim inary studies were per formed with the use of tions were per formed by means of a LaChrom a classi cal LiChroCART (125 ´ 4 mm) chrom ato- (Merck/Hitachi) instru m ent, which was equipped with graphic col umn filled with Lichrospher 60 RP – select a di ode-array detec tor (DAD). Sam ples of colourless B, but signif i cantly better results were ob tained using al co hols were analysed directly, or ana lysed after a Chromolith Per form ance RP-18e (100 ´ 4.6 mm) centrifugation. An aqueous solu tion of 85% phos pho - monolithic colum n. The use of a monolithic colum n ric acid at a con cen tration of 0.1 ml/l (phase A) and al lowed us to ob tain a sig nif i cantly higher sen si tiv ity acetonitrile (phase B) were used as eluents. Chrom ato - and se lec tiv ity of de ter mi na tions and sig nif i cantly in - graphic sepa ra tion was perform ed on a Chromolith fluenced the range of appli ca tions of the worked out Per for mance RP-18e (100 ´ 4.6 mm) monolithic col- method. umn, in standard gradi ent condi tions: 0 min – 0% of The con cen tra tion of denatonium ben zo ate de fined phase A/100% of phase B, 20 min – 100% of phase in the decree of the Minis try of Agri cul ture and Rural

Problems of Fo ren sic Sci ences 2005, LXIII, 275–287 278 D. Zuba, C. Œwiegoda, B. Byrska et al.

De vel op ment is equiv a lent to a con cen tra tion of than one peak origi nat ing from the same substance al - 3 mg/l. Be cause chrom ato graphic peaks of a rela tively lows us to increas e the preci sion of de term ina tions. high in ten sity were ob tained in the pre lim i nary ex am i- Because it was im pos sible to de term ine on the ba - na tion of denatonium ben zo ate stan dard so lu tion at sis of the obtained spec tra which chrom atographic this concen tration, it was decided to max im ally sim - peak corre sponds to the partic u lar form of denatonium plify the pro ce dure of sam ple prep a ra tion for anal y sis. benzo ate pres ent in the solu tion, analy sis of a standard Sam ples of ethyl alco hol were placed directly in mea - solu tion of was car ried out. A peak of re- surem ent vials, or, in the sit ua tion where the liquids tention time 9.05 min and two absorp tion maxi m ums were not com pletely transpar ent, they were centri - (about 205 nm and 230 nm) were obtained on the fuged. A chromatogram of standard solu tion of dena- chromatogram of this com pound. Com pari son of this tonium benzo ate (Bitrex) at a concen tra tion of 3.0 mg/l chromatogram with the denatonium benzo ate chro- is shown in Figure 2. matogram indi cated that the first of the peaks origi - The con di tions used in chro mato graphic sep a ra tion nates from benzo ate ions. Thus, analy sis of dena- are described in the “Mate rials and Meth ods” section. tonium ben zo ate con tent in al co holic bev er ages per - formed on the basis of the first of the peaks was an in- di rect anal y sis by de ter mi na tion of the con tent of ben- zoate ions. The use of indi rect methods is a frequent prac tice in tox i co log i cal lab o ra to ries. For ex am ple, de- term ina tion of alco hol in blood by means of the ADH enzy m atic method can be included among such deter - mina tions. In this method, the concen tra tion of alco hol is propor tional to the reduced form of NADH, which is formed as the result of the reac tion of alco hol with NAD+ in the presence of the en zyme ADH. NADH has a spe cific absorp tion at a wavelength of 340 nm. Because the authors did not possess a standard of any other salt contain ing denatonium ions, it was im possi - ble to deter m ine if the chrom atographic peak of re ten- tion time 10.30 min origi nates from denatonium Fig. 2. Chromatogram of standard solu tion of denatonium cations or from undissociated mole cules of dena- benzo ate (Bitrex) at a con centra tion of 3.0 mg/l. tonium ben zo ate. Nev er the less, one can as sume that the signal strength is propor tional to the amount of Dur ing anal y sis of stan dard so lu tions of denatonium denatonium ions in the solu tion. benzo ate in the mentioned condi tions, two chrom ato- Standard solu tions of denatonium benzo ate in recti - graphic peaks were obtained. They proba bly cor re- fied spirit that did not contain this com pound were pre- spond to two out of three possi ble forms of this pared in or der to make the cali bra tion curve. The com pound oc cur ring in aque ous-al co holic so lu tions, con cen tra tion of Bitrex in par tic u lar so lu tions was: 0.0, that is: undissociated forms of denatonium benzo ate 1.5, 3.0, 6.0, 9.0 and 12.0 mg/l. The accu racy of the val- (DB), benzo ate ion (B–) and denatonium ion (D+). ues for the prepared solu tions was assess ed within the The chrom atographic peak of re tention time 9.05 min frame work of inter-lab o ra tory anal y ses con cern ing al- had two absorp tion maxi m ums, about 205 nm and cohol solu tion dena tured by Bitrex at 3.0 mg/l. The re- 230 nm, whereas the peak of re tention time 10.30 min – sult was consis tent with expec ta tions (2.9 ± 0.3 mg/l). only one maxi m um (about 205 nm). Cali bra tion curves were prepared for both chrom ato- The occur rence of two or more chrom atographic graphic peaks. For the peak of reten tion time 9.05 min, peaks origi nat ing from the same substance is not a fre- analy ses were per formed at wave length 230 nm, quent occur rence, but it is possi ble in the case of sub- whereas for the peak of reten tion time 10.30 min – at stances that un dergo dis so ci a tion. Qua ter nary salts are 205 nm. For both peaks, a linear rela tion ship between included in this group. In accor dance with basic chem - their areas and the con cen tration of Bitrex in the tested i cal laws, the con cen tra tions of dis so ci ated ions and range (1.5–12 mg/l) was obtained. Values of the corre - undissociated forms are strictly corre lated with the ini- lation coef fi cient were in both cases higher than 0.999. tial com pound concen tra tion, and therefore each of the Cali bra tion curves are shown in Figure 3. obtained chrom atographic peaks can be used as the ba- As can be seen from Figure 3, a higher sensi tiv ity, sis for quan ti ta tive anal y sis. The oc cur rence of more which can be deter m ined by the slope of the cali bra -

Problems of Fo ren sic Sci ences 2005, LXIII, 275–287 Determination of denatonium benzoate (Bitrex) in denatured spirit... 279

4.5E+05 tio = 121, which corre sponds to the mass of the benzo-

4.0E+05 ate ion, was detected. Moreover, a second char acter is - y = 32310 x + 152 Denatonium 3.5E+05 Benzoate tic ion at m/z = 89 was formed. Mass spectra of a stan-

3.0E+05 dard solu tion of alco hol dena tured by Bitrex at a con-

a centration of 3 mg/l are shown in Figure 4.

e 2.5E+05 r A

k The worked out proce dure was applied to deter m i- a

e 2.0E+05 P na tion of the denatonium ben zo ate con cen tra tion in 1.5E+05 spirit prepa ra tions. These included both consum er al- 1.0E+05 y = 12850 x + 81 coholic bever ages seized in restau rants and pubs and 5.0E+04 from private persons and indus trial products prepared 0.0E+00 on the basis of dena tured alco hol. 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 Concentration [mg/l] The ob tained re sults were sur pris ing. Con sis tency be tween con cen tra tions de ter mined on the ba sis of the Fig. 3. Cal i bra tion curves for denatonium benzo ate in alco - benzo ate ion and the second peak, whose signal was holic bev er ages. depend ent on the concen tra tion of denatonium ions in solu tion, was achieved for only two of the exam ined tion curve, is obtained for the peak cor respond ing to sam ples of dena tured spirit. In most cases, only one denatonium ions. Next, lim its of deter m ina tion (LOD) peak corre spond ing to benzo ate ions was obtained. and lim its of quantitation (LOQ) were also de term ined Further analy ses were carried out on con sumer alco - on the basis of cali bra tion curves. They were inde - holic bever ages in the pos session of the Alco hol and pendent of the peak used for calcu la tions and were: Drug Section, and in none of them was the pres ence of 0.1 mg/l and 0.3 mg/l, respec tively . Sim ilar values of ei ther ben zo ate ions or the an a lyt i cal sig nal de pend ent LOD and LOQ calcu lated on the basis of peaks, for on the concen tra tion of denatonium ions detected. which dif fer ent sen si tiv i ties were ob tained, might Standard solu tions of Bitrex stored for several weeks seem to be odd. The expla na tion for this phenom enon were also sub jected to anal ysis and a decrease in con- is the higher value of random error (lower preci sion) centra tion was not detected of any form of this com - for measure m ents of a peak of higher sensi tiv ity. The pound. Analy sis by means of mass spectrom etry obtained value of LOQ is ten times lower than the min - confir med these results. For most of the sam ples in im al amount of Bitrex that should be added in order to posi tive ionis ation mode, an ion at m/z = 325, that is de na ture spirit. Therefore, it can be stated that the de - origi nat ing from the denatonium ion, was not detected, veloped method can be used for exam ina tion of such whereas an addi tional mass peak at m/z ratio = 360 ap - liq uids. peared. At the same time, the mass spectra in neg ative The use of two chrom atographic peaks to deter - ionis ation mode were trans formed and addi tional mass mine the concen tra tion of a com pound allows better peaks, whose con fig u ra tion could in di cate the pres- esti m ation (two results are obtained during one analy - ence of chloride deriv a tives in the solu tion, appeared. sis). The er ror of denatonium ben zo ate de ter mi na tion The above results and oper a tional data obtained from in the worked out measure m ent range was below 2%. the police allowed us to accept the hy pothe sis that per- The pres ence of denatonium benzo ate in spirit pre- sons who intro duce products prepared on the ba sis of pa ra tions was confir med by means of mass spectrom e- dena tured spirit onto the ille gal market, rem ove the try. Analy ses were per formed under the condi tions Bitrex present in such liquids. This is accom plished mentioned in the “Mate ri als and Methods” sec tion. most often by addi tion of sodium hypochlorite. The Measure m ents were per formed both in posi tive mass spectra of a standard solu tion of alco hol dena - (ES+) and nega tive (ES–) ionis ation mode. Dur ing tured by Bitrex at a concen tra tion of 3 mg/l, with addi - analy ses of dena tured spirits, a mass ion corre spond - tion of sodium hypochlorite in the amount of 50 µl/ml, ing to the molec u lar mass of the com pound, m/z = 446, are shown in Figure 5. was not detected in any of the ionis ation modes (in the As can be seen from Figure 5, the mass spectra of prelim inary study the mass range m/z = 25–500 was dena tured spirit, both in posi tive and nega tive ionis - applied). In posi tive ionis ation mode, the most char ac- ation mode, undergo big changes after addi tion of so - teris tic mass ion turned out to be an ion at m/z ra- dium hypochlorite. The is sue of the influ ence of so- dium tio = 325, whose mass cor responds to denatonium ion. hypochlorite on the content of denatonium benzo ate is Other mass ions of rela tively high inten sity were ions the subject of another paper by the authors [16]. at m/z = 144 and 135. During analy sis in nega tive ion- Disap pear ance of the peak whose signal depends isation mode, the presence of mass ion at m/z ra- on the concen tra tion of denatonium in a sam ple indi -

Problems of Fo ren sic Sci ences 2005, LXIII, 275–287 280 D. Zuba, C. Œwiegoda, B. Byrska et al.

Scan ES+ 63.68 1.39e6 100

68.66 a %

80.79 90.64 109.83 121.79 170.84 192.64 397.02 154.91 184.85 212.88 238.91 243.37 270.95 300.92 326.18 332.04 360.17 387.08 0 m/z 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400

Scan ES+ 68.64 2.54e6 100 63.64 a'

%

325.02 80.72 90.63 134.76 143.79 326.03 62.21 114.71 212.89 238.79 255.91 128.68 170.83174.72 202.81 246.83 278.13 300.88304.74 347.09 360.19 383.10 397.04 0 m/z 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400

Scan ES- 112.71 1.13e5 100

b % 76.54 88.67

90.81 61.42 180.77 102.50 124.60 170.72 194.59 238.79 329.96 399.44 129.39 248.66 281.08 314.65 320.76 342.88 371.80 378.47 0 m/z 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500

Scan ES- 88.78 120.73 9.44e4 100 112.65

% b'

59.34 76.23 90.36 142.86 167.14 82.40 188.81 233.75 68.17 126.77 196.93209.25 241.81 293.98 309.04 323.24 358.99 109.90 264.71 283.06 368.91 385.86 398.91 0 m/z 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 Fig. 4. The mass spectra of rec ti fied spirit (a – pos i tive ion isation mode, b – neg ative ion isation mode) and a standard solu tion of spirit dena tured by denatonium benzo ate at a concen tra tion of 3 mg/l (a’ – posi tive ion isation mode, b’ – neg ative ion isation mode). cates that these ions have been re moved from the solu - in such a way with the concen tra tion deter m ined on the tion. As a conse quence, one should state that the basis of benzo ate ions content may indi cate that at- con cen tration of Bitrex is below the limit of detec tion tempts to rem ove Bitrex from the solu tion were made. of the method. Calcu la tions per formed on the basis of These results can be con firmed by mass spectrom etry. the peak cor respond ing to the concen tra tion of benzo - 20% techni cal solu tions of Bitrex used to dena ture ate ions would lead to dif fer ent conclu sions, be cause al co hol were also analysed at the Insti tute of Foren sic these ions are still present in the so lu tion. Research. The results were also surpris ing. It turned Thus, if the aim of the analy sis is to deter m ine the out that these solu tions contained signif i cantly smaller concen tra tion of Bitrex in the solu tion using the devel - amounts of this com pound than declared. Bitrex con - oped method, the calcu la tion has to be based on the centra tion deter m ined by means of the HPLC method peak of reten tion time 10.30 min, whose concen tra tion in such liquids amounted to 10–12%. Liquids used for is propor tional to the content of denatonium ions in the dena tur ation are exam ined before their use, but the an- so lu tion. A com par i son of the con cen tra tion ob tained alyt i cal methods applied for this pur pose are highly

Problems of Fo ren sic Sci ences 2005, LXIII, 275–287 Determination of denatonium benzoate (Bitrex) in denatured spirit... 281

Scan ES+ 68.54 5.54e6 100 63.54

% 80.54

82.54 359.85 128.69 61.98 70.04 114.78 138.65 163.94 186.67 244.64 314.59 352.90 90.51 196.66200.62 232.87 256.59 260.72 304.57 322.97 362.41 397.08 0 m/z 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400

Scan ES- 82.44 1.39e5 100

188.68 % 92.55 190.63 94.49 140.66 80.60 142.67 98.62 150.52 59.98 67.48 112.57 154.67169.59 210.61216.84 268.43274.55 306.59 316.41 328.38 391.03 131.76 226.70 248.35 338.03 366.57 384.60 0 m/z 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 Fig. 5. Mass spectra of spirit de natured by denatonium benzo ate with the addi tion of so dium hypochlorite. non-selec tive (measure m ent of density is used most uid de ter gent in ges tion by chil dren, Fun da men tals of often). Thus, it is possi ble that the lower content of Ap plied Tox i col ogy 1982, 2, 44–48. Bitrex of ten detected in the exam ined liquids re sults 3. Carnahan R. M., Kutscher E. C., Obritsch M. D. [et al.], from its lower content in the dena tur ing fluid. Acute eth a nol in tox i ca tion af ter con sump tion of hair - spray, Pharmacotherapy 2005, 25, 1646–1650. 4. Dziennik Ustaw z dnia 11 sierpnia 2003 r., nr 163, poz. 1582. 4. Con clu sions 5. Faulk ner A., DeMontigny P., High-per for mance liq uid chro mato graphic de ter mi na tion of denatonium ben zo ate The worked out HPLC method allows de term ina - in eth a nol with 5% polyvinylpyrrolidone, Jour nal of tion of denatonium benzo ate (Bitrex) concen tra tion by Chro ma tog ra phy A 1995, 715, 189. deter m ina tion of benzo ate ions as well as denatonium 6. Gadza³a-Kopciuch R. M., Buszewska T., Buszewski B., ions or mole cules of denatonium benzo ate. Analy sis of Iso la tion and de ter mi na tion of denatonium ben zo ate in the content of these com pounds allows us to estab lish antifreezing agents by solid phase ex trac tion and HPLC, whether the exam ined liquid contains Bitrex, or Jour nal of Liq uid Chro ma tog ra phy & Re lated Tech nol o - whether the liquid was dena tured by Bitrex and then gies 2000, 23, 3133–3142. 7. Hansen S. R., Janssen C., Beasley V. R., Denatonium ben - this agent was re moved. zo ate as a de ter rent to in ges tion of toxic sub stances: tox ic - Mass spectrom etry can be used for confir mation of ity and ef ficacy, Vet er i nary and Hu man Tox i col ogy 1993, the pres ence of denatonium benzo ate in dena tured 35, 234–236. spirits. It also often al lows us to assess whether the evi - 8. Henderson M. C, Neumann C. M., Buhler D. R., Anal y sis dential liquid contains com pounds formed as the result of denatonium ben zo ate in Or e gon con sumer products by of the reac tion between denatonium benzo ate and an HPLC, Chemosphere 1998, 36, 203–210. oxidis ing agent, e.g. sodium hypochlorite. 9. Jack son M. H., Payne H. A., Bittering agents: their po ten - tial ap pli ca tion in re duc ing in ges tions of en gine cool ants and wind shield wash, Vet er i nary and Hu man Tox i col ogy Ref er ences: 1995, 37, 323–326. 10. Klein-Schwartz W., Denatonium benzo ate: review of ef- 1. Alvarez-Piñeiro M. E., López De Alda-Villaizán M. J., ficacy and safety, Vet er i nary and Hu man Tox i col ogy Paseiro-Losada P. [et al.], Deter mi na tion of the alco hol 1991, 33, 545–547. 11. Mullins M. E., Zane Horowitz B., Was it neces sary to add denaturants denatonium ben zo ate (Bitrex) and di ethyl Bitrex (denatonium ben zo ate) to au to mo tive prod ucts?, phthalate by di rect flow injec tion APCI-MS analy sis, Vet er i nary and Hu man Tox i col ogy 2004, 46, 150–152. Jour nal of High Res o lu tion Chro ma tog ra phy 1997, 20, 12. Nambiar O. G., Gosavi K., Ravindranathan T., Plastic 321–324. mem brane ion-se lec tive elec trode for the de ter mi na tion 2. Berning C. K., Grif fith J. F., Wild J. E., Re search on the effec tive ness of denatonium benzo ate as a deter rent to liq -

Problems of Fo ren sic Sci ences 2005, LXIII, 275–287 282 D. Zuba, C. Œwiegoda, B. Byrska et al.

of denatonium ben zo ate (Bitrex), An a lyst 1991, 116, 1011–1012. 13. Pranaitytë B., Daunoravièius Ž., Padarauskas A., Devel - op ment and val i da tion of a cap il lary elec tro pho re sis method for the de ter mina tion of denatonium benzo ate in denaturated al co hol for mu la tions, Chromatographia 2004, 60, 353–357. 14. Schiffman S. S., Gatlin L. A., Frey A. E. [et al.], Taste per- cep tion of bit ter com pounds in young and el derly per sons: re la tion to lipophilicity of bit ter com pounds, Neuro bio - logy of Ag ing 1994, 15, 743–750. 15. Sibert J. R., Frude N., Bittering agents in the pre ven tion of ac ci den tal poi son ing: chil dren’s re ac tions to denatonium ben zo ate (Bitrex), Ar chives of Emer gency Med i cine 1991, 8, 1–7. 16. Zuba D., Œwiegoda C., Byrska B. [et al.], Assess ment of the efectiveness of denatonium benzo ate (Bitrex) re moval from de na tured spirit prep a ra tions us ing so dium hypochlorite, Prob lems of Fo ren sic Sci ences 2005, 63, 289–299.

Cor re spond ing au thor Dariusz Zuba Instytut Ekspertyz S¹dowych ul. Westerplatte 9 31-033 Kraków e-mail: [email protected]

Problems of Fo ren sic Sci ences 2005, LXIII, 275–287 Problems of Foren sic Science s 2005, LXIII, 275–287

OZNACZANIE BENZOESANU DENATONIUM (BITREXU) W SKA¯ONYCH WYROBACH SPIRYTUSOWYCH

1. Wpro wad zenie spo¿ywaæ œrodki odmra¿aj¹ce o s³odkim smaku czy p³y - ny do spry skiwacz y, co mo¿e doprowad ziæ do zatruc ia Ben zoe san de nat oni um (Bi trex) zo sta³ od not owa ny gli kol em etyl eno wym lub me tan olem. Bi trex za wier aj¹ w Ks iêd ze re kor dów Gu inn essa jako naj bard ziej gorz ka ró wnie¿ œr odki od stras zaj¹ce je len ie, la kiery do pa znokci sub stanc ja zna na cz³owiek owi. Do dat ek 0,000030 cz êœci zn iechê caj¹ce do ich gry zien ia, far by, a tak ¿e po krywy ob jêt oœciowych ben zoe sanu de nat oni um po wod uje, ¿e pow³ok ka bli elektryc znych w celu ich ochrony przed p³yn jest zbyt gorz ki dla wiêkszoœc i osób, ¿eby móg³ byæ gry zon iami. Bar dzo roz cieñczone roz twory s¹ rów nie¿ przez nie spo¿yty. sto sow ane w celu zap obi e¿e nia ssa niu kci uków przez Che miczna na zwa Bi trexu to ben zoe san N-ben zylo- dzie ci. Nale ¿y zazn aczyæ, ¿e zwie rzêta re aguj¹ z ró¿n¹ 2-(2,6-di met ylo fen ylo ami no)-N,N-diety lo-2-okso etan o- czu³oœci¹ na ben zoe san de nat oni um. Za uwa ¿ono ta k¿e am i ny (nu mer re jes tru CAS: 3734-33-6). Wy stê puje on ró¿ni ce w percepc ji jego gorz kiego smaku u dzie ci, do - w po staci bez wonn ych gra nul ek, ma od czyn ob ojêtny ros³ych i osób starszy ch [14]. Dane wskazuj¹, ¿e dodat ek (pH 6,5–7,5), a jego tem peratu ra topnien ia wy nosi 163– ben zoe sanu de nat oni um zda je siê w znacz¹cy sposób 170°C. Ben zoe san de nat oni um jest do brze roz puszc zalny zmniejszaæ prawdopodobieñstwo przy padk owe go jego w metan olu (69,0 g na 100 ml w tem peratu rze 20°C), spo¿yci a [2]. Po przy jêciu przez cz³owieka 10 ppm ben- etanolu (33,5), chlorof orm ie (33,0) i izo-propan olu (10,5), zoe sanu de nat oni um wy stê puje w ustach skraj nie gorz ki, na tomiast jego ro zpu szczalnoœæ w wo dzie jest ni ¿ - nie przyj emny smak. W bada niach udow odni ono, ¿e do- sza (4,5). Zosta ³ zsyntez owany w 1958 roku podcza s ba- dat ek ben zoe sanu de nat oni um do p³ynów do my cia na czyñ dañ nad œro dkami s³u¿¹cymi do znie czul ania miej sco we- wp³ywa na liczbê przypad ko wych za truæ u dzieci [7]. go. Ben zoe san de nat oni um jest zb li¿ ony bu dow¹ che- Oregon by³ pierwszy m sta nem ameryka ñskim, który miczn¹ do jednego z takich œrodków – li dokainy ; ró¿ni wpro wad zi³ koni ecz noœæ do daw ania tego œr odka do sto- siê tylko obecnoœc i¹ grupy benzyl owej przy azoc ie grupy sow any ch w sa moc hoda ch prod ukt ów za wier aj¹cych co amin owej. Che miczna struk tura ben zoe sanu de nat oni um najm niej 10% gli kolu etyl enowego lub co najm niej 4% zo sta³a przed staw iona na ry cin ie 1. metan olu [8]. W 1995 roku rozporz¹dz enie doty cz¹ce tok - Dane tok syk olo gic zne wska zuj¹ na nisk¹ toks ycz- s ycz noœci pr o duktów go spod arst wa do mo w ego wy maga³o noœæ tego zwi¹zku [10]. Wyznac zone wa rtoœci LD50 po do datku ben zoe sanu de nat oni um w st ê ¿e niu 30–50 ppm podaniu do¿o³¹dkowym dawki œmiert elnej dla zwierz¹t w celu zmniej szen ia czêsto œci powa¿ny ch zatruæ dzieci by³y nastêpuj¹ce: u szczura – 820 mg/kg, u myszy – tymi produkt am i [11]. Od tego cza su opubli kowano wiele 865 mg/kg, u króli ka – 580 mg/kg, u œwinki morskie j – prac wy kaz uj¹cych pre wenc yjne dzia ³anie omaw iane go 800 mg/kg, u pstr¹ga têczowego – 1000 mg/kg, u krewe - œrodka [3, 9, 15]. tek – 400 mg/kg. Nal e¿y jed nak pam iêtaæ, ¿e w wie lu Ben zoe san de nat oni um jest amin¹ cz war tor zêdow¹, aspekt ach wie dza nie jest wy starc zaj¹co du¿a, sz czegó l- co powod uje, ¿e jego analiza nie jest prosta . W 1991 roku nie w od nies ieniu do chro niczn ej toks yczno œci, tera to- opis ano meto dê oznac zania jonu de nat oni um w oparc iu gen noœci oraz zja wis ka nad wra ¿liwoœci u lu dzi. o sól czte rof eny lob ora now¹ z wy kor zyst aniem mem bra- Za stos owa nie ben zoe sanu de nat oni um jest zwi¹zane n owej elekt rody jo nos ele ktyw nej z ma tryc¹ poli(wi nyl- z jego gorzkim smakiem. U¿ywa siê go do ska¿ania al - och lorkow¹). Ba dana by³a cha rakt ery sty ka sy gna³u elek - koholu etyl owego, za który nie jest pobier ana akcyz a tak trody dla jonu denat oni um i kilku innych zwi¹zków bê- jak dla na poj ów al koh olo wych. W rz ecz ywi sto œci g³ów- d¹cych amin ami czwa rto rzêdowymi. Wy niki po mia rów ny cz³on nazwy tego zwi¹zku pochod zi od angiel skiego po tenc jome try cznych za wart oœci ben zoe sanu denato nium s³owa „denat ura ted”, czy li ska ¿ony (st¹d te¿ pol ska na z - w oleju rzepak owym w zakres ie 1–10 ppm by³o zgodne wa „de nat urat”). In ten syw noœæ jego sma ku, ma³a tok- w ±5% ze wstr zykniêt¹ iloœ ci¹ tego zwi¹zku [12]. Me- sycznoœæ oraz brak dzia³ania rakotwórc zego powod uj¹, tod¹ sto sow an¹ naj czêœciej w prak tyce do jego ozna - ¿e jest idea lnym do datk iem za pob iegaj¹cym przy padk o - czenia w pro dukt ach kon sumpc yjny ch jest wy soko - wem u przyj mowaniu przez ludzi takich p³ynów, jak de- sprawna chro matogr afia cieczowa (HPLC) [5, 8]. Anal i- terg enty, œr odki de zynf ekuj¹ce, p³yny czyszcz¹ce, œrodki za rozt wor ów wzor cow ych wskaza³a na lini owoœæ w za - wy kor zyst ywane w og ród kach do mow ych czy pro dukty kresie 1,25–50 ppm (produc enci zwy kle dodaj¹ 10– ogrodn icze. Ze wzglêdu na awersjê, jak¹ wywo³uje, ben - 30 ppm). Eluat z zakresu odpow iada j¹cego zarej est ro- zoe san de nat oni um jest co raz cz êœciej w tym celu wyko - wanem u pikowi by³ zbierany i obec noœæ benzoe sanu de - rzystywany. Do dat ek ben zoe sanu de nat oni um jest sto - nat oni um by³a zwe ryf iko wana me tod¹ spek trom etr ii so wany w celu ochrony dzie ci i zwierz¹t, które mog¹ ma sow ej. 284 D. Zuba, C. Œwiegoda, B. Byrska i in.

Ce lem in nej pra cy [6] do tycz¹cej ben zoe sanu dena to- ¿enia w sporz¹dzo nym roztwor ze zo sta³a ocen iona w ra- nium by³o opracowa nie szyb kiej metody izol acji i wzbo ga - mach porównañ miêdzy laboratoryjnych. cenia tego zwi¹zku z ni skoz amarzaj¹cych p³ynów stoso- Oprac owan¹ metodê za stos owa no do ba dan ia ma- wa nych do ch³od zen ia si lni ków (p³ynów sporz¹dzo nych teria ³u do wod owe go. Wy kor zyst ano w tym celu pr óbki na ba zie gli kolu etyl eno wego i pro pyl eno wego). Do al koh olu etyl owe go za bezp iecz one w 2005 roku i prze- przy got owa nia prób ki wy kor zyst ano ek str akcjê do fazy kazane do badañ w Pra cowni Badan ia Al koholu i Narko - sta³ej (SPE), a oznac zenie prowad zono metod¹ wysok o - tyk ów In styt utu Eks pert yz S¹do wych. G³ówn¹ gr upê sp rawnej chro mat ogr afii cie czow ej w odw róco nym uk³a- sta nowi³y na poje al koh olo we za kwes tiono wane w re- dzie faz (RP HPLC). Dziêki opty maliza cji metody przy- stauracj ach lub u osób prywatn ych. Ponadto ba dano al - go tow ania pr óbki uzys kano wy sok ie od zys ki (oko³o kohol pochodz¹ cy z zak³adów zaj muj¹cych siê jego prze- 90%) oraz po wta rzalnoœæ ja koœ ciowych i il oœc iowych twó rst wem. wy nik ów uzys kane go stê¿ enia anal itu (RSD = ±1%). W celu wy znac zenia za wart oœci ben zoe sanu dena - Do badañ wy kor zyst ano ró wni e¿ te chn ikê elekt rofo- tonium w badan ych p³ynach opracowa no metodê wyso - rezy ka pil arnej (CE). Zop tym ali zow any roz dzia³ by³ pro- ko sprawnej chro mat ogr afii cie czow ej (HPLC). Ba dan ia wad zony w 0,025 mol/l elekt roli cie Tris-fos for ano wym prze prow adz ono za po moc¹ urz¹dze nia La Chrom (pH 7,0) z za stos owa niem be zpoœ red niej de tekc ji w za - (Merck/Hi tac hi) wy pos a¿onego w de tekt or sze regu diod kres ie UV przy 214 nm. Ka lib racja by³a liniowa w zakre - (DAD). Pró bki bez barwn ych al koh oli by³y pod daw ane s ie 2–50 mg/l. Gra nica wyk ryw alnoœci wy znac zona dla anal izie bezp oœr ednio b¹dŸ po od wir owa niu. Jako elu- 10 s elektroki net ycz nego (5 kV) nastrz yku wy nosi³a enty za stos owa no wod ny roz twór 85% kwa su fos for owe- 0,45 mg/l (3/N). Metoda zosta ³a zastos owana do ozna - go o stê¿en iu 0,1 ml/l (faza A) oraz acet oni tryl (faza B). czania bi trexu w ró ¿nych sk a¿onych pro dukt ach al koh o- Roz dzia³ chro mat ogr afic zny by³ pro wad zony na ko lumn - lowyc h, a jej wy niki by³y porów nywa lne do uzyskany ch ie mo nol ity cznej Chro mol ith Per form ance RP-18e (100 ´ metod¹ HPLC [13]. 4,6 mm) w stan dard owy ch wa runk ach gra dient owy ch: Obecn ie oprac owa no metodê tan dem ow¹, tj. spe ktr o- 0 min – 0% fazy A/100% fazy B, 20 min – 100% fazy skopiê mas z jo niz acj¹ che miczn¹ pod ciœnieniem atm o - A/0% fazy B, 21 min – 100% fazy A/0 % fazy B i 5 min sf ery cznym sp rzê¿ on¹ z anal iz¹ przep³ywowo-wstrzy - sta bil iza cji w tych wa runk ach. Po miary st ê¿en ia benzo - kow¹ do oznaczani a Bi trexu w etanolu. Zwi¹zki zide nty - esanu de nat oni um by³y wy kon ywa ne przy dw óch d³u go œ- fikowano na podsta wie ich widm masow ych zbieran ych ciach fali: 205 nm i 230 nm. w za kres ie m/z 50–500. Ró wnoc zeœnie prze prow adz ano Przy ob lic zaniu pa ram etrów wa lid acy jnych wyko - anal izê iloœciow¹ przy wybran ym jo nie m/z 325 dla Bi - rzystano pro gram kom put ero wy “Va lid ati on Ma nag er” trexu. Wa runki pra cy zo sta³y zop tym ali zow ane dla miê k - fir my Merck/Eu rol ab. ki ej jo niz acji (w jo niz acji do datn iej) z frag ment acj¹ Do po twierd zenia ob ecnoœci ben zoe sanu de nat oni um pro wad zon¹ je dyn ie w za kres ie ko nieczn ym do ident yfi - wy kor zyst ano me todê spek trom etr ii mas (MS). W bada- kac ji anal itu. Krzy wa ka lib racy jna by³a pro stol ini owa niach za stos owa no spek trom etr ma sowy Qu att ro Micro w za kres ie stê¿eñ 2–30 g/ml, pre cyzja (RSD) wynosi³ a API fir my Microm ass. Próbki p³ynu by³y wprowad zane 11,5%, a granica wykryw alnoœ ci 0,017 g/ml. Metoda za- bezp oœr ednio do ko mory jo niz acy jnej spek trom etru pewn ia³a ró wnoc zesn¹ an alizê ja koœc iow¹ i il oœci ow¹ z szy bkoœci ¹ przep³ywu 10 µl/min. Sto sow ano zar ówno pro wad zon¹ z wy sok¹ czu³oœci¹. Brak ja kieg oko lwiek do datni¹ (ES+), jak i ujemn¹ (ES–) jonizacjê. Napiêcie przy got owa nia prób ki oraz za stos owa nie anal izy prze- ka pil ary wy nosi³o 3,0 kV, na tom iast na piê cie w stre fie p³ywowo-wstrzy kow ej oznac za³y, ¿e pro ced ura by³a CID wy nosi³o 15 V. Gaz osuszaj¹ cy podaw any by³ z szy b - prostsza i szybsza ni¿ opisane poprzedn io [1]. koœci¹ 600 l/h w tem peratu rze 300°C. Tem per atu ra Ÿród³a Ce lem ba dañ przed staw iony ch w ni niejs zej pra cy jon ów wy nosi³a 100°C. Mo nit oro wano za kres mas jo - by³o oprac owa nie pro stej pro ced ury ba dan ia wy rob ów nowych m/z = 25–400. spi ryt uso wych pod k¹tem zaw art oœci ben zoe sanu de nat o - ni um z wy kor zyst aniem tech nik chro mat ogr afic znych. 3. Wyniki i ich om ówienie

2. Ma ter ia³ i me tody Ben zoe san de nat oni um jest œr odk iem sk a¿aj¹cym al- koh ol etyl owy wy mien ionym w roz porz¹dze niu Mi nis tra Przy oprac owa niu me tody oznac zania ben zoe sanu Rol nict wa i Rozwoju Wsi z dnia 11 sierpnia 2003 r. de na t oni um wy kor zyst ano wzo rzec tej sub stanc ji zaku- w spra wie œrod ków do puszc zony ch do sk a¿ania al koh olu piony w firm ie Sigm a Aldr ich. Roztwory wzorcowe Bi - etylowe go [4]. Jego dodat ek nie wp³ywa na podsta wowe trexu sporz¹dza no po przez od wa¿enie od pow iedni ej w³asn oœci fi zyk och emic zne al koh olu etyl owe go, poni e- il oœci ben zoe sanu de nat oni um i ro zci eñc zenie spi ryt usem wa¿ iloœæ ben zoe sanu de nat oni um, jak¹ na le¿y do daæ rek tyf iko wan ym (Pol mos War szawa). Dok³adn oœæ stê - zgod nie z ww. roz porz¹dze niem, jest niewielka i wy nosi

Problems of Fo ren sic Sci ences 2005, LXIII, 275–287 Oznaczanie benzoesanu denatonium (Bitrexu) w ska¿onych... 285

0,3 g na 100 l 100% al koholu etyl owego. Bi trex mo¿e byæ sub stanc ji po zwala na zw iêks zenie pre cyz ji ozn acz eñ ni¿ do dany ró wni e¿ w po staci 20% roz tworu al koh olo wego. w przy padku, gdy dwa lub wiêcej pików zosta nie u¿ ytych Ben zoe san de nat oni um jest zwi¹zkiem che miczn ym do wy kon ania ob lic zeñ. o sto sunk owo du¿ ej ma sie cz¹stecz kow ej. Anal izuj¹c Po nie wa¿ na pod staw ie uzys kany ch widm nie by³o jego stru ktu rê, mo¿na zauw a¿yæ, ¿e jest to sól czwa rto - mo¿l iwe okre œlen ie, który pik chro mat ogr afic zny od po - rzêdowa, po siad aj¹ca gru py chro mof oro we. Dla tego me- w iada po szc zegó lnym for mom ben zoe sanu de nat oni um tod¹ z wyboru zastos owan¹ do oznaczeni a benzoe sanu wy stê puj¹cym w roz twor ze, wy kon ano an ali zê roz tworu de nat oni um by³a wy sok osp rawna chro mat ogr afia cie - wzor cow ego kwa su ben zoe sowe go. Dla tego zwi¹zku czowa (HPLC). uzys kano na chro mat ogr amie pik o cza sie re tenc ji Wst êpnie ba dan ia pro wad zono z za stos owa niem kla- 9,05 min i dwóch maksim ach absorpc ji (oko³o 205 nm syczn ej ko lumny chro mat ogr afic znej Li ChroC ART i 230 nm). Jego porówna nie z chrom atogr am ami benzo - (125 ´ 4 mm) z wype³nieniem Lichros pher 60 RP-selec t esanu denat oni um wskaza³ o, ¿e pierwszy z pików po - B, jed nak znacznie lep sze wy niki osi¹gn iêto przy zasto - chod zi od jo nów ben zoe sano wych. A za tem anal iza za - so waniu ko lumny mo nol ity cznej Chro mol ith Perfor man- war toœ ci ben zoe sanu de nat oni um w na poj ach al koh olo - ce RP-18e (100 ´ 4,6 mm). Zastos owanie kolum ny mo- wych wykon ana na posta wie analizy pierwsze go z pików no lit ycznej po zwoli³o na uzys kanie znacz nie wiêks zej by³a poœredni¹ anal iz¹ po przez ok reœlenie za wartoœci jo - czu³oœci i selek tywnoœci oznac zeñ oraz znacz¹co wp³y- nów ben zoe sano wych. nê³o na za kres za stosowañ oprac owa nej metody. Wy kor zyst ywan ie me tod po œrednich jest cz êst¹ prak - St ê¿e nie ben zoe sanu de nat oni um ok reœ lone w roz- tyk¹ w la bor ato ria ch tok syk olo gic znych. Do ta kich oz- porz¹dze niu Mi nis tra Rol nict wa i Roz woju Wsi odpo - naczeñ za liczyæ nale¿y np. oznac zanie zawar toœci alko - wiada st ê¿eniu 3 mg/l. Po nie wa¿ w trak cie wstê pnych holu we krwi metod¹ en zym atyczn¹ ADH, gdzie stê¿enie badañ roz tworu wzor cow ego ben zoe sanu de nat oni um al koh olu jest pro porc jona lne do po wsta³ej w wy niku jego o ta kim st ê¿eniu uzys kano piki chro mat ogr afic zne o sto- re akc ji z NAD+ (w ob ecn oœci en zymu ADH) zre duk owa - sunk owo du¿ ej int ensy wno œci, po stan owi ono maksy mal- nej for my NADH, któ ra wy kaz uje spe cyf iczn¹ abs orp cjê nie upr oœc iæ pro ced urê przy got owa nia prób ek do anal izy. przy d³ugoœci fali 340 nm. Poniew a¿ nie dys ponowa no Prób ki al koh olu etyl owe go umieszc zano bezpoœr ednio wzor cem in nej soli za wier aj¹cej jony de nat oni um, nie w naczy nkach pom iar owych, a w sytua cji, gdy p³yny nie by³o mo ¿li we ro zst rzy gniêcie, czy pik chro mat ogr afic z- by³y ca³ko wic ie kla rowne, od wir owy wano je. Na ryci- ny o czasie retenc ji 10,30 min pochod zi od ka tionów de- nie 2 przed staw iono chro mat ogr am roz tworu wzorco- nat oni um czy te¿ od nie zdys ocj owan ych cz¹ste czek ben - wego ben zoe sanu de nat oni um (Bi trexu) o st ê¿eniu zo esanu de nat oni um. Nie mniej jed nak mo¿ na przyj¹æ, ¿e 3,0 mg/l. wielkoœæ sy gna³u jest proporc jona lna do iloœci jon ów de- Do roz dzia³u chro mat ogr afic znego za stos owa no wa- nat oni um w roz twor ze. runki podane w roz dziale „Mater ia³ i metody”. Pod czas Do sporz¹dze nia krzy wej ka lib racy jnej przy got owa - anal izy rozt wor ów wzor cow ych ben zoe sanu de nat oni um no roz twory wzor cowe ben zoe sanu de nat oni um w spiry - w wy mien iony ch wa runk ach uzys kano dwa piki chro- tusie rek tyf iko wan ym nie za wier aj¹cym tego zwi¹zku. mat ogr afic zne od pow iadaj¹ce praw dop odo bnie dwóm St ê¿en ie Bitrexu w po szczególnych roz twor ach wy - z trzech mo¿liwych form tego zwi¹zku wystêpuj¹cym nosi³o: 0,0; 1,5; 3,0; 6,0; 9,0 i 12,0 mg/l. Dok³adnoœæ w roz twor ach wod no-al koh olo wych, tj. for mie niezdy- sporz¹dzo nych roz tworów wzor cow ych zo sta³a ocen iona socjowanej ben zoe sanu de nat oni um (BD), jo nowi ben- w ram ach ba dañ miêdzy laboratoryjnych dla roztworu al- zoe sano wemu (B–) oraz jo nowi de nat oni um (D+). Pik koholu st ê¿onego Bitrexem w il oœci 3,0 mg/l i uzyskano chro mat ogr afic zny o cza sie re tenc ji 9,05 min wy kaz ywa³ wynik zgodny z oczekiwa nym (2,9 ± 0,3 mg/l). Krzy we dwa maksim a absorpc ji – oko³o 205 nm i 230 nm, nato - ka lib racy jne sporz¹dzo no dla obu pik ów chromatogra- miast pik o cza sie retenc ji 10,30 min mia³ jedno m aksi - ficznych, przy czym dla piku o cza sie re tenc ji 9,05 min mum (oko³o 205 nm). anal izy prowad zono przy d³ugoœci fali 230 nm, natom iast Wys têp owanie dwó ch lub wiê ksz ej ilo œci pik ów dla piku o cza sie retenc ji 10,30 min przy d³ugoœci fali chro mat ogr afic znych po chodz¹cych od jed nej sub stanc ji 205 nm. Dla obu pików uzyskano li niow¹ zale¿ noœæ ich nie jest zjawis kiem czêstym , ale jest to mo¿liwe w przy- powier zchni od stê¿e nia Bitrexu w badan ym zakres ie padku sub stanc ji uleg aj¹cych dy soc jacji, do kt óry ch za- (1,5–12 mg/l). Wa rtoœ ci wspó³czyn nika ko rel acji by³y lic zyæ mo ¿na sole cz war torz êdowe. Po nie wa¿ zgod nie w obu przy padkach wiêksze ni¿ 0,999. Wykresy kali b - z pod staw owy mi pra wami che miczn ymi stê ¿enie zdy - racy jne przed staw iono na ry cin ie 3. socjowanych jon ów oraz for my nie zdys ocj owan ej s¹ Jak wy nika z wykresu 3, wi êksz¹ czu³oœæ, któr¹ mo¿- œciœle sko rel owa ne z wyj œci owym stê ¿en iem zwi¹zku, na ok reœl iæ po przez na chyl enie krzy wej ka lib racy jnej, ka¿ dy z uzys kany ch pików chro mat ogr afic znych móg³ uzys kuje siê dla piku odpow iada j¹cemu jo nowi denat o - byæ u¿yty do wy konania anal iz iloœciowy ch. Wystêp o - nium. Na stê pnie na pod staw ie wy kresu ka lib racy jnego wanie wiêcej ni¿ jednego piku pochodz¹ cego od jednej wy znac zono ró wnie¿ gra nice wyk ryw alnoœci (LOD)

Problems of Fo ren sic Sci ences 2005, LXIII, 275–287 286 D. Zuba, C. Œwiegoda, B. Byrska i in. i ozn acz alnoœci (LOQ), które – niezal e¿nie od piku u¿y- roz twory wzor cowe Bi trexu prze chow ywa ne przez kil ka tego do obl icz eñ – wy nosi³y odpow iedni o: 0,1 mg/l ty godni i nie stwierdzono spadku stê¿enia ¿adnej z form i 0,3 mg/l. Zbli ¿one warto œci LOD i LOQ ob lic zone tego zwi¹zku. Analiza za po moc¹ spek trometr ii mas po - w oparciu o piki, dla których uzyskano ró¿n¹ czu³oœæ, twierdzi³ a te wyniki . Dla wiêks zoœc i próbek przy jo niz- mog¹ wyda waæ siê za stan awi aj¹ce. Wy jaœn ien iem tego acji dodatn iej nie stwier dzano jonu o m/z = 325, czy li zja wis ka jest wiê ksza war toœæ b³êdu przy padk owe go po chodz¹cego od jonu de nat oni um; po jaw ia³ siê na tom - (mniejsza pre cyzja) przy po miar ach dla piku o wiêksz ej iast dodatk owy pik masowy o stosunku m/z = 360. Jed- czu³oœci. Uzyskana wart oœæ LOQ jest dzie siêc iokrotnie nocz eœnie wid ma ma sowe przy jo niz acji ujemn ej ulega³y mniej sza ni¿ minim alna iloœæ Bi trexu, jak¹ nale¿y do - przekszt a³ceniu i pojaw ia³ y siê w nich dodatk owe piki dawaæ w celu sk a¿enia spi rytusu. Nal e¿y zatem stwier - ma sowe, kt óry ch uk³ady mog³y ws kaz ywaæ na ob ecn oœæ dziæ, ¿e oprac owa na metoda mo¿e byæ sto sow ana do w roz twor ze chlo rowc opo cho dnych. Powy ¿sze wy niki badan ia tego typu p³ynów. oraz uzyskane z poli cji dane operacy jne pozwoli ³y na Za stos owa nie dw óch pi ków chro mat ogr afic znych do przy jêcie hi pot ezy, ¿e osoby wpro wad zaj¹ce na niele - wy znac zenia st ê¿en ia zwi¹zku po zwala na jego lep sze osza - galny rynek wy roby sporz¹dzone na ba zie ska¿one go spi- cowa nie (uzys kuje siê dwa wy niki w trak cie jed nej analizy). ryt usu, usuwaj¹ obecny w nich Bitre x. Odbywa siê to B³¹d wy znac zenia st ê¿en ia ben zoe sanu de nat o ni um w opra - na jcz êœciej po przez do dan ie pod chlor ynu sodu. Wid ma c owa nym za kres ie po miar owym wy nosi³ po ni¿ ej 2%. ma sowe roz tworu wzor cow ego spi ryt usu ska ¿on ego Ob ecn oœæ ben zoe sanu de nat oni um w wy rob ach spi- Bitrexem o stê ¿e niu 3 mg/l, do którego dodano podchlo - ryt uso wych po twierd zano za po moc¹ spek trom etr ii mas. ryn sodu w il oœci 50 ml/ml, przed stawiono na ry cin ie 5. Ba dan ia pro wad zono w wa runk ach ok reœl onych w roz- Jak wy nika z ryciny 5, widm a masowe ska¿o nego spi - dziale „Mater ia³ i metody”. Po miary pro wad zono za - ryt usu zarówno przy jo nizacji dodatn iej, jak i ujemnej, równo przy jo nizacji dodatn iej (ES+), jak i ujemnej uleg aj¹ bar dzo du¿ ym zmia nom po do dan iu pod chlor ynu (ES–). Podcza s analiz ska¿onych spiryt usów przy obu ro- sodu. Za gadnieni u wp³ywu podchlor ynu sodu na zawa r- dzaj ach jo niz acji nie stwier dzano jonu ma sow ego od - toœæ ben zoe sanu de nat oni um po œwi êco na zo sta³a od rêbna pow ia da j¹cego ma sie cz¹stecz kow ej zwi¹zku, czy li pra ca aut orów [16]. m/z = 446 (w badan iac h wstêp nych stosow ano za kres Zanik piku, którego sy gna³ zale¿ y od st ê¿enia dena - mas m/z = 25–500). Przy jo nizacji dodatn iej naj bardzie j tonium w próbce, oznacza, ¿e jony te zosta ³y usuniêt e cha rakt ery sty cznym jo nem ma sow ym okaza³ siê jon z roz tworu. W kon sek wencji nal e¿y stw ier dziæ, ¿e stê - o stosunku m/z = 325, mas¹ odpow iada j¹cy jo nowi dena- ¿en ie sa mego Bi trexu jest po ni¿ej gra nicy wyk ryw al - to nium. In nymi jo nami ma sow ymi o sto sunk owo du¿ ej noœci metody . Wykon anie obli czeñ w oparciu o pik odpo- int ensy wnoœci by³y jony o m/z = 144 i 135. Podcza s ana - wia daj¹cy stê ¿eniu jon ów ben zoe sano wych do prow adzi - liz przy jo nizacji ujemnej stwier dzano obecnoœæ jonu ma- ³oby do odm iennego wniosku, poni ewa¿ jony te by³y sowego o stosunku m/z = 121, co odpow iada masie jonu nadal obecne w roz twor ze. A za tem, je œli celem anal izy ben zoe sano wego. Po nadto po wstawa³ dru gi cha rakt ery- jest wy znac zenie st ê¿en ia Bi trexu w roz twor ze przy za - sty czny jon o m/z = 89. Widm a masowe roz tworu wzor- stos owa niu oprac owa nej me tody, ko nieczne sta je siê wy- cow ego spi ryt usu ska ¿on ego Bi trexem o st ê¿eniu 3 mg/l konywa nie obli czeñ w oparciu o pik o cza sie retenc ji przedsta wiono na rycinie 4. 10,30 min, kt órego st ê¿enie jest proporc jona lne do za - Oprac owa na pro ced ura zo sta³a za stos owa na do wy - war toœci jon ów de nat oni um w roz twor ze. Por ówna nie znac zenia st ê¿en ia ben zoe sanu de nat oni um w wy rob ach uzys kane go w ten spos ób stê¿ enia ze stê¿ eniem wyzna - spi ryt uso wych. By³y to zar ówno kon sumpc yjne na poje czonym w oparciu o zawartoœæ jonów benzoe sano wyc h al koh olo we za kwes tiono wane w re staur acj ach, pu bach mo¿e wykaza æ próby usuniê cia Bi trexu z roztworu. Wy - czy u osób fi zyczn ych, jak te¿ wyrob y prze mys³owe niki te mog¹ byæ potwi erdzane metod¹ spektrom etr ii mas. sporz¹dzo ne na ba zie sk a¿onego spi ryt usu. Ba dan iom w In styt ucie Eks pert yz S¹do wych pod - Otrzym ane wy niki by³y zaskak uj¹ce . Tylko dla dwóch dano równie¿ technicz ne 20% roz twory Bi trexu stosow a - ba dan ych pr óbek sk a¿o nego spi ryt usu uzys kano zgod- ne do ska ¿an ia al koh olu. Wy niki ró wni e¿ by³y za ska- noœæ stê¿ eñ wy znac zony ch na pod staw ie jonu benzo- kuj¹ce. Okaza³o siê, ¿e roz twory te za wiera³y znacz nie esowego i drugiego piku, którego sy gna³ by³ za le¿ny od mniej sze jego iloœci ni¿ de klar owa ne. Wy znac zone st ê- st ê¿enia jo nów de nat oni um w roz twor ze. W wiê kszo œci ¿enie bitre xu metod¹ HPLC w ta kich p³ynach wynosi³ o prz ypad ków uzys kiwa no tyl ko je den pik od pow iadaj¹cy 10–12%. P³yny stosow ane do ska¿an ia s¹ badane przed jo nom ben zoe sano wym. Kon trol nie prze bad ano kon - ich u¿yciem, jed nak me tody anal ity czne sto sow ane sumpc yjne na poje al koh olo we bêd¹ce w po siad aniu Pra- w tym celu s¹ wy soce nie selekty wne (naj czêœciej do ta- cowni Ba dan ia Al koh olu i Na rko tyk ów i w ¿a dnym kich ba dañ wy kor zyst uje siê po miar gê stoœci). Mo ¿liwe z nich nie stwierdzono obecnoœci zarówno jonów ben - jest za tem, ¿e ni ¿sza za wartoœæ Bi trexu stwier dzana zoe sano wych, jak i sy gna³u anal ity cznego zale ¿ne go od czêsto w badan ych p³ynach wynika z mniej szej jego stê¿ enia jonów de nat oni um. Ba dan iu pod dano rów nie¿ zawar toœci w p³ynie ska¿aj¹cym.

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4. Pod sum ownie

Oprac owa na me toda HPLC po zwala na wy znac zenie st ê¿en ia ben zoe sanu de nat oni um (Bi trexu) po przez ozna- c zenie zawar toœci jonów ben zoe sano wych, jak rów nie¿ zaw art oœci jon ów de nat oni um lub cz¹ste czek ben zoe sanu de nat oni um. Analiza zawa rto œci tych sk³adni ków po z- wala us taliæ, czy badany p³yn za wiera Bi trex, czy te¿ p³yn ten by³ ska¿ony Bi trexem, a nastê pnie zosta ³ odka¿ony . Me toda spek trom etr ii mas mo¿e byæ za stos owa na do po twierd zania ob ecnoœci ben zoe sanu de nat oni um w sk a- ¿on ych spi ryt usa ch. Po zwala ona ró wnie¿ cz êsto oc eniæ, czy dowod owy p³yn za wiera zwi¹zki powsta ³e w wyniku re akc ji ben zoe sanu de nat oni um z czyn nik iem utle n ia - j¹cym, np. podchlorynem sodu.

Problems of Fo ren sic Sci ences 2005, LXIII, 275–287