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Memo N K31 165049 to:. Dr. W.U.Sardner Froms D.H. ~ord and R.C.Hockett R. A. Re: Site Visit with Drs. D./Petersen and J./Thompson, University of Colorado at Boulder, Col, May 21, 1980. Titleis: "Influence of Genotype, Sex. and Chronic Smoking on Nicotine bietabolism in Mlce." SUMMRfy ~ This program has proceeded in a closely defined fashion to develop techniques fJmeasuring nicotine and its metabolites in blood, liver and brain. The analyttical nEethodolgy appears to be a lmost complete, though Dr. Thompson is= continuing to refine procedures. (It would appear to be the essential nature of this individual to be continuously attempting to improve procedures by either variation in technique or introduction of newly developed equipment). HPLC and GCiM techniques appear to be essentially in an online state forA dectecting nicotine, and,have made cpcnsiderable progress with detection of cotinine. Have started preliminary work on cohditioning mice in the Walton Horizontal Smoke apparatus. (This turns out to beinot as easy as suggested in available literature and notes). Have also set up an animal room system reversing the dark/light phase to to permit them to utilize animals readily in the normal animal wake period. Have ~ k "4 C-o ~we n1 recently changes over i'rom, separationswith glass capillary tab*ag to a fused silica coluan, which has greatly improved sharpness of separation. Plans for second year of study, once all 4"sh technical agpects are resolved will be to initiate the experiments on effects of chronic vs. acute nicotine treatment as compared with smoke exposure on nicotine metabolism in various strains of mouse. De-14, 1s _Progress since our visit Nov. 1979 up to Mgy, 1980 ; Isolation of nicotine spiked into tissue homogenates...effective range of detection; 5 to 50png.igm of tissue. Object has been to maximize nicotine recovery and minimize simultaneous extraction of other subFtancds which mg= might compete with nicotine during separation. Problems encountered: High degree of water solubility of nicotine, stability of nicotine as well as its ubiquitous distribution in surrounding environment (from smokers) + need to develop

2 4- H KIi 1 65C50 ipproprinte internal standards tb use with nicotine separation by GC%;+6. Exttaction of nicotine from tissue homogenates spiked with nicotine: ---------------- - S./,:~,~s Have utilized toluene and ethyl acetae(+ 6 otbers which were notso effective). Figure shoass.mount of nicotine left in tissue following each of 3 extractions. Ethy~ acetate was best solvent used. O /o /~ kev--~ -1 ~ /0 Irl =KIMC r ~~ eThyl .gcef4f< o' ~ h 7. a OY tr2AC?tu. /LO Several substances tested for extraction with nicotine as internal standard references. N-methyPanabasine, 3 N-N-Diethylamino methyl pyridine and Deuterated nicotine or nornicotine all worked N-methyl Anabasine ~---- `~ i well. 3-N-N-Diethylamino methyl pyridine 7 `~~CNs~N3 ~•~ 2- N `` \r~~-Cl" ] j Deuterated nicotine GC recovery of nicotine using a dual standaid technique with quinoline and N-methyl anabasine led to a 669 recovery using glass capillary columes. Then a•btemsted utilized a 0V-101 fused silica capillary column (Drawn quartz) and inprcived efficiency 3056 and attained a much sharper peak with a width of about 2 seconds. The narrowness of this peak required obtaining a new chromato~^+r~ integrator (arranged for by Dr. Erwin). They now feel they are at the peak of the at a level of 100po. art in GC work in identifying r.icotine from tissue homogenates

h KO1 1 E5051 3 Procedure for extracting nicotine from tissue :,iver (brain)---Weight, homogenize 4 in pure H20 (1 ml!gm), Add internal standard extraction with ethyl acetate ~- Aqueous phase ~J JSCA2~ Ip Aqueous phase Combine aqueous phases Saturate with (NI14)CO Extrac~ with toluene & centri#uge Aqueous phase I Reveat extrcction with toluene I -V-Aqueous phase Discard Repeat acetate extraction Discard ethyl acetate fraction -_--I Toluene phase y Concentrate under N at 40°C to 100u1 and place on GC or GClNS column for analysis. Will lose about 10% if evaporated beyond 10eu1; 605, if evaporated to dryness. Z Ethyla acetate phase Back extract with 2m1 0.21M acetate pH 4.8.) Ce.,lc.~'w~t IF Ethyl ac at te phase

4 0'oservatioris on the tecniques developed so far: H11,01165052 1. Nitrogen phosphase detector used with Us= GC /N15 separation of nicotine from liver spiked with nic~ptiee, cotinine and methy3pnabasine provides a clear nicotine peak. 2. Some nicotine shows up in *e IiPLC separation even when there is no nicotine spiked in tissue sample (From environment..emokers in building). This appears to represent beiwes~s=~rawi~~=e#== about 5ng of nicotine and thus represents a base level contamination f or all assays of nicotine in tissue. 3. HA Have developed a standard curve for nicotine at levels of from 25 to 1000ng on GC/M5 as well as with GC using the Bused silica capillary column . The two sepaaations have slightly diffe rent sip slopes. 4. GC1?S separation twice as fast as with GC. Extraction of tissue now major time consuming factor. 5. Accuracy: Rerovery of nucotine on GC or GC/M now close to 100% at nicotine levels spiked in tissue homogenatesof 50, 100, 250 and 500 ng. Recovery of nicotine from brain and liver following I.?. injection (ih v'.vo) Using C3'i adult mice, weight 30.5 + 1.2 gm. i`e } & a: Tissue Conc. `_ nicotine(ng) l;e per gm tissue ~--~ . vd Sc (" )+ to t t V w ws~ Time after injection (r~ ~ w) GljIlq4/:- Curves noted to be slower in younger mic:

5 N{4®1165053 Have just recentiyr started attempting to extract continine #n same t as nicotine, rather than having Ito do them seperately. P1ani1 in future studies to determine NIC ratios. Already have some data obtained using the glass cajillary coldmas with 2 and 8ng of contfnine spiked!gm of tissue. Have utilized the ~ McKennis tecnique to synthesize their own cotinine. Have also tried separat~e nicotine from cotinine on the fused silica column with excellent results, using a deuterated cotinine as internal standardp on GC h^14. With a validated . separation ipt a contine dose range €e of frim 0.3 to 13 hig. P9P9tS=p*Rp6 i Future Plans: 1. Finish methodology a. complete development of methods for isolation and quantification of cotinine b. look for other nicotine metabolites (could use a supply of nornicotine as well as the oxoamide of nicotine). c. perfect the HPLC technique for nornicotine al-i 11 J -N-oxide. 2. Experianntal phase a. obtain kinetic data bn nicotine metabolism in C3'.i, C57, HS and Fl mice, male vs. female. b. Compare 11, acute vs. chronic nicotine treatment with results obtained from smoke exposed animals c. Study nicotine metabolites in perfusion fluid and brain from the isolated perfused mouse brain system of Dr. Erwin. 3. ;4anuscript preparation a. Expect to be writitig up analytical procedures by early summer. Dr. Ho wi= will present this information at the Anatytical Chemistry meeting this summer in Denver. Evaluation: Highly organized, well planned program with technical procedural problems almost all solved in relation to etudging determing levels of nicotine and cotinine in tissues and blood. . Should provide an excel':.ent survey in of rates of nicotine metabolism (at least to contine) in vivo and in vitroI IPMof Erwin). nn..n...n..~. s4 .. . .. ..~'-.... ..........a .. .. S..r~i• e•t..a.. .hn o.i..nb•..n i~.w 4 ..