Determination of Denatonium Benzoate (Bitrex) in Denatured Spirit Preparations
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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 elab o rate a chro matographic method for de ter mi nation of denatonium ben zoate (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, dis in - 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 ta tive 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 pos i 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 cul ture 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 u bility in water is lo- Guinness Book of Records as the bitter est com pound wer (4.5). It was syn thesised 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 – lidocaine. 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 benzyl group 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 ad dition of denatonium benzo ate at a con- centra tion of 30–50 ppm with the in tention of reduc ing the fre quency of se ri ous pae di at ric in tox i ca tions with these prod ucts [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 teris - 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 taste, its low tox icity and of stan dard 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 usu ally 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, paints, coat ing 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].