Nama NPM

: Gladyola Ayu Mauliarhani : 260110110002

1. Analysis Of Doxepin Meta olites !n "uman #rine Materials and Methods Ultraviolet analyses were perfomed with a Cary Model 11-A recording spectrophotometer. Reagents

Spectroquality n-he ane. !otassium permanganate" saturated aqueous solution. Standards.

Stoc# solution was prepared $y dissolving 1%% mg of do epin hydrochloride per 1%% milliliters in water. &e diluted the stoc# with human urine to provide wor#ing standars. Method 'ive milliliters of urine are pipetted into a (%-ml centrifuge tu$e followed $y 1.) ml of the saturated *Mn+( solution. ,he tu$e is sha#en to mi thoroughly and allowed to stand for ) mm. ,he *Mn+( o idi-es do epin to its corresponding #etone" the." .-dimethyl--y-propylammne side-chain $eing replaced $y /%. 0o epin #etone is e tracted from the o idi-ed urine sample $y sha#ing for 1% mm with 1% ml of n-he ane. ,he tu$es are centrifuged to sharply separate the phases" and the he ane e tract is removed for ultraviolet analysis. 1e ane e tracts are

scanned in the ultraviolet region from 23% to 4%% nm in spectrophotometer cells with a 2%-mm light path. Spectroquality n-he ane is used as the solvent $lan#. A$sor$ance at 25) nm 6trough7 is su$tracted from the a$sor$ance at 233 nm 6pea#7 to o$tain net a$sor$ance.

2. Analysis Of Doxepin Meta olites !n "uman "air $ample ,he samples 6appro imately 2%% rag" 1 cm in length7 were collected $y clipping hair from the same area on the $ac# of the head appro imately 1 cm from the s#in. Samples were placed in plastic $ags until analy-ed. .egative hair samples were washed in deioni-ed water" dried at room temperature" and pulveri-ed. $tandards and %ontrols ,he do epin and desmethyldo epin standard stoc# solutions 61 mglm87 were diluted with methanol to concentrations of 1%" 2" and 1 pg9m8. ,he do epind4 standard stoc# solution 61%% :;g9m87 was diluted with methanol to 1% pg9m8. Si -point standard curves were prepared for do epin and desmethyldo epin $y using )%-mg aliquots of the negative hair spi#ed with methanolic solutions of $oth drugs to achieve the following concentrations of standard hair preparations< %.2)" %.)%" 1.%" ).%" 1%.%" and 2%.% ng9mg. :n addition" two levels of controls were prepared. ,he low controis 62 ng9mg7 were made $y adding )% :;8 of $oth 2-1;g9m8 do epin and desmethyldo epin solutions to )% mg of pulveri-ed negative hair samples. ,he high controls 61) ng9mg7 were prepared $y adding =) :;8 of the l+-pg9m8 solutions of $oth do epin and desmethyldo epin to )% mg of pulveri-ed negative hair.

Analyti%al pro%edure ,he patient>s hair samples were washed in deioni-ed water and allowed to dry at room temperature. ,hey were then pulveri-ed" and )%-mg aliquots were analy-ed in triplicate. ,wenty microliters of the 1%-l;g9m8 solution of the internal standard 6do epind47 was added to the hair samples" standards" and control preparations. ,his was followed $y the addition of %.1M 1Cl 64 m87. ,he test tu$es were capped" vorte mi ed" and incu$ated overnight 61? to 2( h7 at )%@ ,he tu$es were removed from the heating $loc# and allowed to cool down $efore $eing centrifuged for ) rain 6(%% g7. ,he supernatant was transferred to clean tu$es" 1 m8 1.54M acetic acid was added to each test tu$e" and they were vorte mi ed. ,en milliliters of deioni-ed water was then added to each tu$e. ,he 1CA columns were conditioned as follows< methanol 64 m87" deioni-ed water 64 m87" and 1.54M acetic acid 61 m87. ,he samples were added to the columns and slowly drawn through $efore drying 1-2 min. ,he columns were washed with 4 m8 of deioni-ed water 6dried 1-2 rain7" 1 m8 of %.1M 1C: 6dried 12 min7 and 4 m8 of methanol 6dried ) min7. ,he do epin" desmethyldo epin" and do epin-d4 were eluted $y allowing 4 m8 of the methylene chloride9isopropanol9ammonium hydro ide 6=?<2%<2"vh@9v7 mi ture to pass through the columns. All samples were then evaporated to dryness under a stream of air. ,he drug residue was reconstituted in acetonitrile 6(% p87. ,he samples were transferred to autosampler vial inserts" and the vials were crimped. ,he samples were derivati-ed $y adding BS,'A with 1C ,MCS 6(% p87 with a syringe and vorte mi ed. After incu$ating for 1) rain at 3%@ the samples were cooled and analy-ed using DC-MS. &hromato'raphi% method ,he inEector was wor#ing in the splitless mode" and a 1-p8 volume was inEected. ,he inEector temperature was 2=% oC ,heflow rate of the cartier gas

6helium7 was 1.2 m89min. ,he initial DC oven temperature of 14% oC was held for 1 min" then it was increased at the rate of 12 oC9min until it reached 2?% oC.,his temperature was maintained for 4 min. ,he MS ion source temperature was 24%@ and the quadrupole temperature was 1)% oC ,he instrument operated in selected ion monitoring 6S:M7 mode" and the following ions were monitored< for do epin m/z )? and 2=5" for do epin-d4 m/z 31 and 2?2" and for desmethyldo epin m9- 113 and 44=. ,he following ions were used for quantitation< m/z )? for do epin" rn/z 31 for do epin-ds" and rn/z 113 for desmethyldo epin. ,he dwell tirnes were )% ms for m9- )?" 31"2=5" and 2?2 ions and 1%% ms for m9- 113 and 44= ions. (. Analysis Of Doxepin Meta olites !n Other )issue $ample 1uman Mil#

Methods 0iagnosed as having a maEor depressive disorder. ,reatment with o a-epam initiated $y the o$stetrician was unsuccessful and caused some drowsiness in the infant. +n admission to a psychiatric inpatient unit 4% days postpartum she was treated with 1)% mg do epin at night. ,he acute episode $egan to resolve after 2 wee#s" after which she was discharged and then treated as an outpatient for some 3 months. 0o epin and .-desmethyldo epin concentrations in the mil# and plasma were analysed $y high performance liquid chromatography 6h.p.l.c.7. An aliquot of $iological fluid 61 ml7 was placed in a polypropylene tu$e" 2%% ng of amitriptyline was added as internal standard and the sample was al#alinised $y the addition of

%.1 ml 1 M .a+1. ,he sample was e tracted with 1% ml he ane containing 1C isoamyl alcohol $y sha#ing for ) min. After centrifugation" 5 ml of the organic phase was transferred to a clean polypropylene tu$e and the compounds of interest were $ac# e traced $y sha#ing with %.2 ml of %.%) M 1Cl. After a further centrifugation" the organic phase was aspirated and discarded and )% "ul aliquots of the acidic e tract were inEected onto the h.p.l.c. column was used with a mo$ile phase of (%C acetonitrile in an aqueous solution of %.%1C 14!+( and %.%1C .aCl 6flow rate / 2 ml miMf17. !ea#s were detected $y their ultraviolet a$sor$ance at 21% nm. *nown concentrations of do epin and .-desmethyldo epin in $oth mil# and plasma were put through the a$ove procedure and test samples were quantified from a plot of pea# height ratio 6drug9intemal standard7 vs drug concentration. ,he intra-assay coefficient of variation for do epin in plasma and mil# was tested at )% and 2%% "ug 1-1 and ranged from 1.4 to 4.3C 6n / )7F coefficients of variation for .-desmethyldo epin at similar plasma concentrations ranged from %.3 to 4.?C 6n / )7. :nter-assay coefficient of variation" assessed from the slope of the standard curve on different days was 3.2C for do epin and ?.=C for .desmethyldo epin in mil# 6n / (7 and 3.2C for do epin and ?.(C for .- desmethyldo epin in plasma 6n / =7. *. Analysis Of Doxepin Meta olites !n "uman +lood $ample

Materials and methods Chemicals and reagents

All chemicals were analytical grade with purity greater than 5?C and were supplied $y Sigma. 0o epin" sulpiride and S*')2)A standards 61 mg9m8 in ethanol7 were o$tained from :nstitute of 'orensic Science Ministry of !u$lic security !.R.C.

Sample preparation and e traction +ne milliliter of $lood" $ile and one gram of the other samples were $rought

with distillate water to a volume of 4 m8" and then homogeni-ed in a 1% m8 tu$e of centrifuge 64%%%Gg" 1% min7. !rior to vorte for mi ed" internal standard solution 6S*')2)A7was added for final concentration of 4Hg 9m8. ,he solution was $rought to p1 of 1% with the addition of .a+1 2 M. ,o each sample" )ml of ethyl ether was added. After hori-ontal agitation during 1% min and centrifugation at 4%%% rpm for ) min" the organic e tract was transferred into a glass tu$e then evaporated to dryness at (%IC under a gentle stream of air. ,he residues were reconstituted $y )%H8 of ethanol" one micro liters were inEected into the DC9 MS and two micro liters were inEected into the 8C-JS:-MS9MS. :nstrumentation and MS9MS conditions ,he do epin analyses were performed with a ,hermo-'isher gas chromatography9mass spectrometry system< DC90SK. Capillary column 0B)-MS 4% mG %.2) mm G %.2) mm were used for analysis. Carrier was helium at the flow rate of 1 ml9min. :nEector temperature" 2?%IC" inEection mode was split" split ratio" )%<1. ,ransfer line temperature" 2)%IC. :on source temperature" 2)%IC. ,he column temperature program used for do epin was< initial temperature 1%%IC for 1 min" 2%IC9min to 2?%IC" maintained for ( min. ,he instrument was used in full scan J: 6=% eL7 mode" scanning in the range $etween (%M3)% m9-.

,he sulpiride were performed with a &aters ACKU:,N U!8C9,K 0J,JC,+R" ion mode< JS-" capillary voltage 1.=%#v" cone voltage 4?v" e tractor 2v" Rf voltage %.) v" desolvation gas flow< ?%? 891r" cone gas flow< )4 891r" source temp< 12%IC" cone temp< 4)%IC" ion energy< %.)I,he chromatographic separation was achieved on an &aters ACKU:,N U!8C BJ1 C1?" 1.=Hm" 2.1G)%mm column" thermostated at 4%IC. Mo$ile phase A was methanol" B were %.2C ammonium acetate and %.1C formic acid 6v9v7.