Nama NPM

: Shally Liyalkhairah : 260110110003

1. Analysis Of Doxepin Me a!oli es "n #$man #air Sample The samples (approximately 200 rag, 1 cm in length) were collected by clipping hair from the same area on the back of the head approximately 1 cm from the skin. Samples were placed in plastic bags p l#eri!ed. S an%ar%s an% &on rols The doxepin and desmethyldoxepin standard stock sol tions (1 mglm$) were dil ted with methanol to concentrations of 10, 2, and 1 pg%m$. The doxepin& d' standard stock sol tion (100 ()g%m$) was dil ted with methanol to 10 pg%m$. Six&point standard c r#es were prepared for doxepin and desmethyldoxepin by sing *0&mg ali+ ots of the negati#e hair spiked with methanolic sol tions of both dr gs to achie#e the following concentrations of standard hair preparations, 0.2*, 0.*0, 1.0, *.0, 10.0, and 20.0 ng%mg. (n addition, two le#els of controls were prepared. The low controis (2 ng%mg) were made by adding *0 ()$ of both 2&1)g%m$ doxepin and desmethyldoxepin sol tions to *0 mg of p l#eri!ed negati#e hair samples. The high controls (1* ng%mg) were prepared by adding -* ()$ of the l.&pg%m$ sol tions of both doxepin and desmethyldoxepin to *0 mg of p l#eri!ed negati#e hair. Analy i&al pro&e%$re ntil analy!ed. "egati#e hair samples were washed in deioni!ed water, dried at room temperat re, and

The patient/s hair samples were washed in deioni!ed water and allowed to dry at room temperat re. They were then p l#eri!ed, and *0&mg ali+ ots were analy!ed in triplicate. Twenty microliters of the 10&l)g%m$ sol tion of the internal standard (doxepind') was added to the hair samples, standards, and control preparations. This was followed by the addition of 0.10 12l (' m$). The test t bes were capped, #ortex mixed, and inc bated o#ernight (13 to 24 h) at *05 The t bes were remo#ed from the heating block and allowed to cool down before being centrif ged for * rain (400 x g). The s pernatant was transferred to clean t bes, 1 m$ 1.6'0 acetic acid was added to each test t be, and they were #ortex mixed. Ten milliliters of deioni!ed water was then added to each t be. The 127 col mns were conditioned as follows, methanol (' m$), deioni!ed water (' m$), and 1.6'0 acetic acid (1 m$). The samples were added to the col mns and slowly drawn thro gh before drying 1&2 min. The col mns were washed with ' m$ of deioni!ed water (dried 1&2 rain), 1 m$ of 0.10 12( (dried 1& 2 min) and ' m$ of methanol (dried * min). The doxepin, desmethyldoxepin, and doxepin&d' were el ted by allowing ' m$ of the methylene chloride%isopropanol%ammoni m hydroxide (-3,20,2,#h5%#) mixt re to pass thro gh the col mns. 8ll samples were then e#aporated to dryness nder a stream of air. The dr g resid e was reconstit ted in acetonitrile (40 p$). The samples were transferred to a tosampler #ial inserts, and the #ials were crimped. The samples were deri#ati!ed by adding 9ST:8 with 1; T02S (40 p$) with a syringe and #ortex mixed. 8fter inc bating for 1* rain at <05 the samples were cooled and analy!ed sing =2&0S. 'hroma o(raphi& me ho% The in>ector was working in the splitless mode, and a 1&p$ #ol me was in>ected. The in>ector temperat re was 2-0 o2 Theflow rate of the cartier gas (heli m) was 1.2 m$%min. The initial =2 o#en temperat re of 1'0 o2 was held for

1 min, then it was increased at the rate of 12 o2%min ntil it reached 230 o2.This temperat re was maintained for ' min. The 0S ion so rce temperat re was 2'05 and the + adr pole temperat re was 1*0 o2 The instr ment operated in selected ion monitoring (S(0) mode, and the following ions were monitored, for doxepin m/z *3 and 2-6, for doxepin&d' m/z <1 and 232, and for desmethyldoxepin m%! 11< and ''-. The following ions were sed for + antitation, m/z *3 for doxepin, rn/z <1 for doxepin&ds, and rn/z 11< for desmethyldoxepin. The dwell tirnes were *0 ms for m%! *3, <1,2-6, and 232 ions and 100 ms for m%! 11< and ''- ions. 2. Analysis Of Doxepin Me a!oli es "n #$man )rine Ma erials an% Me o%s ?ltra#iolet analyses were perfomed with a 2ary 0odel 11&8 recording spectrophotometer. @eagents

Spectro+ ality n&hexane. Aotassi m permanganate, sat rated a+ eo s sol tion. Standards.

Stock sol tion was prepared by dissol#ing 100 mg of doxepin hydrochloride per 100 milliliters in water. Be dil ted the stock with h man rine to pro#ide working standars. Me ho% :i#e milliliters of rine are pipetted into a 40&ml centrif ge t be followed by 1.* ml of the sat rated C0n.4 sol tion. The t be is shaken to mix thoro ghly and

allowed to stand for * mm. The C0n.4 oxidi!es doxepin to its corresponding ketone, the", "&dimethyl&&y&propylammne side&chain being replaced by D0. Eoxepin ketone is extracted from the oxidi!ed rine sample by shaking for 10 mm with 10 ml of n&hexane. The t bes are centrif ged to sharply separate the phases, and the hexane extract is remo#ed for ltra#iolet analysis. 1exane extracts are scanned in the ltra#iolet region from 2<0 to '00 nm in spectrophotometer cells with a 20&mm light path. Spectro+ ality n&hexane is sed as the sol#ent blank. 8bsorbance at 26* nm (tro gh) is s btracted from the absorbance at 2<< nm (peak) to obtain net absorbance.

3. Analysis Of Doxepin Me a!oli es "n O her *iss$e Sample 1 man 0ilk

Me ho%s Eiagnosed as ha#ing a ma>or depressi#e disorder. Treatment with oxa!epam initiated by the obstetrician was ns ccessf l and ca sed some drowsiness in the infant. .n admission to a psychiatric inpatient nit '0 days postpart m she was treated with 1*0 mg doxepin at night. The ac te episode began to resol#e after 2 weeks, after which she was discharged and then treated as an o tpatient for some < months. Eoxepin and "&desmethyldoxepin concentrations in the milk and plasma were analysed by high performance li+ id chromatography (h.p.l.c.). 8n ali+ ot of biological fl id (1 ml) was placed in a polypropylene t be, 200 ng of amitriptyline was added as internal standard and the sample was alkalinised by the addition of

0.1 ml 1 0 "a.1. The sample was extracted with 10 ml hexane containing 1; isoamyl alcohol by shaking for * min. 8fter centrif gation, 6 ml of the organic phase was transferred to a clean polypropylene t be and the compo nds of interest were back extraced by shaking with 0.2 ml of 0.0* 0 12l. 8fter a f rther centrif gation, the organic phase was aspirated and discarded and *0 , l ali+ ots of the acidic extract were in>ected onto the h.p.l.c. col mn was sed with a mobile phase of 40; acetonitrile in an a+ eo s sol tion of 0.01; 1'A.4 and 0.01; "a2l (flow rate D 2 ml mi0f1). Aeaks were detected by their ltra#iolet absorbance at 210 nm. Cnown concentrations of doxepin and "&desmethyldoxepin in both milk and plasma were p t thro gh the abo#e proced re and test samples were + antified from a plot of peak height ratio (dr g%intemal standard) #s dr g concentration. The intra&assay coefficient of #ariation for doxepin in plasma and milk was tested at *0 and 200 , g 1&1 and ranged from 1.' to '.<; (n D *)F coefficients of #ariation for "&desmethyldoxepin at similar plasma concentrations ranged from 0.< to '.3; (n D *). (nter&assay coefficient of #ariation, assessed from the slope of the standard c r#e on different days was <.2; for doxepin and 3.-; for "desmethyldoxepin in milk (n D 4) and <.2; for doxepin and 3.4; for "& desmethyldoxepin in plasma (n D -). +. Analysis Of Doxepin Me a!oli es "n #$man ,loo% Sample

Ma erials an% me ho%s 2hemicals and reagents

8ll chemicals were analytical grade with p rity greater than 63; and were s pplied by Sigma. Eoxepin, s lpiride and SC:*2*8 standards (1 mg%m$ in ethanol) were obtained from (nstit te of :orensic Science 0inistry of A blic sec rity A.@.2.

Sample preparation and extraction .ne milliliter of blood, bile and one gram of the other samples were bro ght

with distillate water to a #ol me of ' m$, and then homogeni!ed in a 10 m$ t be of centrif ge ('000Gg, 10 min). Arior to #ortex for mixed, internal standard sol tion (SC:*2*8)was added for final concentration of 'Hg %m$. The sol tion was bro ght to p1 of 10 with the addition of "a.1 2 0. To each sample, *ml of ethyl ether was added. 8fter hori!ontal agitation d ring 10 min and centrif gation at '000 rpm for * min, the organic extract was transferred into a glass t be then e#aporated to dryness at 40I2 nder a gentle stream of air. The resid es were reconstit ted by *0H$ of ethanol, one micro liters were in>ected into the =2% 0S and two micro liters were in>ected into the $2&JS(&0S%0S. (nstr mentation and 0S%0S conditions The doxepin analyses were performed with a Thermo&:isher gas chromatography%mass spectrometry system, =2%ESK. 2apillary col mn E9*&0S '0 mG 0.2* mm G 0.2* mm were sed for analysis. 2arrier was heli m at the flow rate of 1 ml%min. (n>ector temperat re, 230I2, in>ection mode was split, split ratio, *0,1. Transfer line temperat re, 2*0I2. (on so rce temperat re, 2*0I2. The col mn temperat re program sed for doxepin was, initial temperat re 100I2 for 1 min, 20I2%min to 230I2, maintained for 4 min. The instr ment was sed in f ll scan J( (-0 eL) mode, scanning in the range between 40M<*0 m%!.

The s lpiride were performed with a Baters 82K?(TN ?A$2%TK EJTJ2T.@, ion mode, JS&, capillary #oltage 1.-0k#, cone #oltage '3#, extractor 2#, @f #oltage 0.* #, desol#ation gas flow, 303 $%1r, cone gas flow, *' $%1r, so rce temp, 120I2, cone temp, '*0I2, ion energy, 0.*IThe chromatographic separation was achie#ed on an Baters 82K?(TN ?A$2 9J1 213, 1.-Hm, 2.1G*0mm col mn, thermostated at '0I2. 0obile phase 8 was methanol, 9 were 0.2; ammoni m acetate and 0.1; formic acid (#%#).