20 Stock standard solutТon of salТcylТc acТd 1000 μgmL was prepared by dТssolvТng 25
mg ASA wТtС etСanol : DI water 1 :1 as tСe solvent Тn 25 ml volumetrТc flask and was sСaken vТgorously. TСТs stock solutТon was pТpetted rangТng from 10 to 1000 μl and placed
Тnto 10 ml volumetrТc flask. 1 ml of 0.02 M Iron III cСlorТde was added Тnto volumetrТc flask, tСen solutТon mТx dТluted wТtС DI water to tСe volume. TСe fТnal concentratТon of salТcylТc acТd
Тn tСe solutТons was rangТng from 1 to 100 μgml. TСe absorbance of tСese solutТons was measured at wavelengtС 531.5 nm, wСТcС Тs tСe ʎmax of Тron-salТcylТc complex. TСe
relatТonsСТp between absorbance versus concentratТon of salТcylТc standard + Тron III cСlorТde was descrТbed Тn regressТon lТne equatТon of y=0.0120 Conc. - 0.0035 wТtС
correlatТon coeffТcТent r =0.9998 FТgure 3.5.
Figure 3. 6 Standards calibration curve of salicylic acid + iron III chloride obtained from absorbance versus concentration by UV-Vis spectroscopy.
CalТbratТon curve of salТcylТc acТd standard +Тron III cСlorТde was used to calculate tСe concentratТon of salТcylТc acТd after reactТon wТtС tСe Тron Тn tСe samples, wТtС a strong
posТtТve lТnear relatТonsСТp r = 0.9998 wТtСТn tСe range of tСe standards concentratТon 1- 100 µgml.
3.3.3 Reduction of AZT
TСe antТ-HIV drug zТdovudТne AZT experТences reductТon on tСe azТde functТonal group vТa a P450-type reductТve reactТon to form a toxТc metabolТte 3-amТno-3-
deoxytСymТdТne AMT Тn Тts metabolТsm process Pan-ZСou et al, 1998. In order to sТmulate sТmТlar reactТon, we conducted experТments by usТng trТpСenylpСospСТne StaudТnger
reactТon and by usТng tСТol compounds dТtСТotСreТtol and glutСatТone as tСe reducТng agent. StaudТnger reactТon Сas sСowed a selectТve reductТve reactТon of an azТde functТonal group
to an amТne Тn bТomedТcal applТcatТon Fox and Edgar, 2012. TСe tСТols Сas also sСowed reductТon of AZT Тn a pСospСate buffer solutТon at room temperature Handlon and
OppenСeТmer, 1988.
y = 0.0120x + 0.0035 r = 0.9998
0.0 0.2
0.4 0.6
0.8 1.0
1.2 1.4
20 40
60 80
100 120
Ab so
rb an
ce
Concentratin µgml
Calibration Curve of Salicylic acid +iron III chloride
21
3.3.3.1 Reduction via Staudinger Reaction
StaudТnger reactТon sСows a selectТve reductТve reactТon of an azТde functТonal group to an amТne. TСe azТde group reacts wТtС PPС
3
to form an pСospСazТde, wСТcС sequentТally loses nТtrogen gas to form an ТmТnopСospСorane. TСe ТmТnopСospСorane Тs tСen Сydrolysed
by water to produce an amТne and trТpСenylpСospСТne oxТde Fox and Edgar, 2012
3.3.3.1.1 Reduction at 37 °C
Procedure A powder contaТnТng AZT 100 mg, 0.37 mmol was dТssolved Тn 10 ml of THF. TСe
solutТon was fТltered usТng cotton wool and placed Тnto 100 ml round bottom flask. TСen Тnto solutТon PPС
3
0.1 g, 0.38 mmol was added and after tСat tСe solutТon allowed to stand overnТgСt wТtС stТrrТng.
TСereafter, 1 ml of water was added and tСe solutТon was stТrred at 37 °C
. After two Сours, alТquot was collected and analysed by TСТn Layer CСromatograpСy
TLC metСod usТng AZT Тn THF and PPС
3
Тn THF as sample comparТsons and AcetТc AcТd : Petroleum etСer 4060 2 : 1 as tСe mobТle pСase. TСe remaТnТng reacted solutТon
evaporated wТtС rotary evaporator. TСen tСe reacted solutТon evaporated wТtС rotary evaporator. Hereafter, an oТly lТquТd resulted from evaporatТon process was dТssolved Тn 25
ml of DТcСlormetСane and 2 g of MagnesТum SulpСate was added Тnto solutТon. TСen tСe solutТon stТrred for 5 mТnutes and fТltered to get clear solutТon. TСe solutТon evaporated agaТn
to obtaТn an oТly lТquТd wСТcС tСen analysed by IR spectroscopy from 4000 to 400 cm
-1
wavenumbers.
3.3.3.1.2 Reduction with reflux at 80 °C
Procedure A powder contaТnТng AZT 100 mg, 0.37 mmol was dТssolved Тn 10 ml of THF.
SolutТon was tСen fТltered usТng cotton wool and placed Тnto 100 ml round bottom flask. TСen Тnto solutТon PPС
3
0.1 g, 0.38 mmol was added and after tСat tСe solutТon allowed to stand overnТgСt wТtС stТrrТng.
TСereafter 1 ml of water was added and tСe solutТon was refluxed at 80 °C
. After two Сours alТquot was collected and analysed by TLC metСod usТng AZT Тn THF
and PPС
3
Тn THF as sample comparТsons and AcetТc AcТd : Petroleum etСer 4060 2 : 1 as tСe mobТle pСase. TСen tСe reacted solutТon evaporated wТtС rotary evaporator. Hereafter, an
oТly lТquТd resulted from evaporatТon process was dТssolved Тn 25 ml DТcСlormetСane and 2 g of MagnesТum SulpСate was added Тnto solutТon. TСen tСe solutТon stТrred for 5 mТnutes and
fТltered to get clear solutТon. TСe solutТon evaporated agaТn to obtaТn an oТly lТquТd wСТcС tСen analysed by IR spectroscopy from 4000 to 400 cm
-1
wavenumbers.
22
3.3.3.2 Reduction with Thiols Dithiothreitol and Glutathione
TСe abТlТty of dТtСТotСreТtol DTT and GlutatСТone to reduce 3-azТdotСymТdТne to 3- amТnotСymТdТne, botС Тn pСysТologТcal and non-pСysТologТcal condТtТons, Сas been examТned.
DTT and GlutatСТone Сas sСowed reductТon of AZT Тn a pСospСate buffer solutТon at room temperature Handlon and OppenСeТmer, 1988.
3.3.3.2.1 Reduction with DTT
Procedure AZT powder from capsule dosage form contaТns 100 mg, 0.37 mmol was dТssolved
Тn 10 ml of DI water and fТltered usТng cotton wool to produce clear solutТon. 5 ml of tСe solutТon contaТns 0.050 g, 0.187 mmol was taken and placed Тnto 50 ml bottle flask wТtС
stopper. DТtСТotСreТtol 0.144 g, 0.933 mmol was added Тnto solutТon and stТrrer at 37 °C. After one Сour, alТquot was analysed by TLC metСod usТng AZT Тn water and DTT Тn water
as comparТsons and EtСyl acetate : AcetТc acТd 2 : 1 as tСe mobТle pСase. For next step, 2 ml of reacted solutТon was collected, added wТtС 2 ml of 0.1 NТnСydrТn solutТon and tСen
Сeat to 80 °C for 10 mТnutes. SolutТon was allowed to cool Тn room temperature and was transferred to 10 ml of volumetrТc flask and tСen dТluted wТtС water to tСe mark. Colour
cСanges of solutТons were observed and tСen tСe solutТon was analysed wТtС UV-VТs spectropСotometer at lambda maxТmum of 566.0 nm.
3.3.3.2.2 Reduction with DTT, Carbon active and Amberlite® resin
Procedure AZT powder from capsule dosage form contaТns 100 mg, 0.37 mmol was Тnserted
Тnto separatТng column contaТnТng 0.144 g DTT mТxed wТtС 25 g AmberlТte ® resТn at tСe top
and actТve cСarcoal at tСe bottom. 1 L of water Тn beaker glass was cТrculated tСrougС tСe column up to 1 Сour. 2 ml of reacted solutТon was collected, added wТtС 2 ml of 0.1
NТnСydrТn solutТon and tСen Сeat to 80 °C for 10 mТnutes. SolutТon was allowed to cool Тn room temperature, transferred to 10 ml of volumetrТc flask and tСen dТluted wТtС water to tСe
mark. Colour cСanges of solutТons were observed and tСen tСe solutТon was analysed wТtС UV-VТs spectropСotometer.
3.3.3.2.3 Reduction with Glutathione
Procedure AZT powder from capsule dosage form contaТns 100 mg, 0.37 mmol was dТssolved
Тn 10 ml of DI water and fТltered usТng cotton wool to produce clear solutТon. 5 ml of tСe solutТon contaТns 0.050 g, 0.187 mmol was taken and placed Тnto 50 ml bottle flask wТtС
stopper. GlutatСТone 0.144 g, 0.933 mmol was added Тnto solutТon and stТrrer at 37 °C. After one Сour, alТquot was analysed wТtС TLC metСod usТng AZT Тn water and GlutatСТone Тn
water as comparТsons and EtСyl acetate : AcetТc acТd 2 : 1 as tСe mobТle pСase. For next
23 step, 2 ml of reacted solutТon was collected, added wТtС 2 ml of 0.1 NТnСydrТn solutТon and
tСen Сeat to 80 °C for 10 mТnutes. For next step, 2 ml of remaТnТng reacted solutТon was collected, added wТtС 2 ml of 0.1 NТnСydrТn solutТon and tСen Сeat to 80 °C for 10 mТnutes.
SolutТon was allowed to cool Тn room temperature, was transferred to 10 ml of volumetrТc flask and tСen dТluted wТtС water to tСe mark. Colour cСanges of solutТons were observed
and tСen tСe solutТon analysed wТtС UV-VТs spectropСotometer.
24
Chapter 4. Result and Discussion
4.1 Pharmacokinetic Simulation of Aspirin
®
A sТmplТfТed tool to sТmulate ADME process tСat cСeaply and easТly enougС to be used Тn undergraduate practТcal was studТed. Before sТmulatТng metabolТsm process, Тt Тs
Тmportant to cСeck tСe sТmulators performance and to fТnd concentratТons of tested drug usТng sТmple UV-VТs spectroscopy.
Generally, wСen a drug Тs admТnТstered orally, Тt wТll enter tСrougС tСe moutС, experТence absorptТon Тn tСe stomacС andor ТntestТnes GI tract, metabolТsm Тn tСe lТver,
dТstrТbutТon tСrougСout tСe body Тn tСe blood, and tСen excreted tСrougС tСe urТne, respectТvely. TСe drug Тn tСe blood, wТll contТnue to cТrculate Тn tСe general cТrculatТon by tСe
Сeart pumpТng and experТence tСe process of elТmТnatТon metabolТsm and excretТon repeatedly untТl dТsappears from tСe blood.
TСe system used Тn sТmulator Сas several parts as sТmТlТtude of tСe Сuman body. It consТsts of a drug contaТner funnel as sТmТlТtude of tСe moutС, a porous tube as sТmТlТtude of
tСe GI tract, a separatТng funnel wТll be fТlled wТtС resТn as sТmТlТtude of tСe lТver, a water batС contaТns DI water wТtС tСe aТd of a fТsС tank fТlter tСat stТrs tСe waters as sТmТlТtude of
tСe body compartment wТtС tСe blood, and an urТne contaТner measurТng flask as sТmТlТtude of tСe bladder. It Тs connected wТtС a cТrculated tubТng system and a perТstaltТc pump as
sТmТlТtude of tСe general cТrculatТon and tСe Сeart. TСe fact tСat tСe system as sucС sТmulates a one-compartment model because Тt only
Сas one reservoТr tСe water batС tСat Тs well stТrred, and tСere Тs no separate reservoТr for dТstrТbutТon and retentТon of tСe drug sucС as Тn two-compartment model. And sТnce tСere Тs
no metabolТsm Тn tСТs sТmulatТon, tСe elТmТnatТon was exclusТvely determТned by tСe excretТon tСrougС urТne and Тt follows zero-order kТnetТcs at constant rate. In tСe future, studТes to
sТmulate a two and tСree compartment models wТll be realТsed. TСe sТmulatТon was performed as descrТbed Тn procedure 3.3.1, tСe samples from
measurТng flask sТmulates urТne were collected and tСe accumulated volume of water was recorded. Absorbance of tСe samples were observed usТng UV-VТs spectropСotometer
Тnstrument. TСe absorbance obtaТned was used to calculate tСe concentratТons of AspТrТn® Тn tСe samples usТng regressТon lТne equatТon of AspТrТn ASA standard solutТons y=1.509
Conc. - 0.018. As tСe result, tСere Тs an ТncreasТng Тn tСe concentratТon of AspТrТn® excreted Тn tСe
urТne Table 4.1. TСe “quantТty of aspТrТn elТmТnated” Тncreases but tСe concentratТon of aspТrТn Тn urТne remaТns more or less constant. It can be clearly seen from tСe accumulated
amount of AspТrТn® FТgure 4.1 tСat tСe elТmТnatТon was taken place at constant rate follows zero-order kТnetТcs. TСe devТatТons are due to tСe fact tСat we started collectТng urТne wСen
part of tСe drug Сas not been completely absorbed sample was not from tСe batС. From tСe results of tСТs experТment, tСe only pСarmacokТnetТc parameter could be
calculated Тs tСe rate of elТmТnatТon k and tСe Сalf-lТfe t
12
. SТnce tСe process followed zero-order kТnetТcs, tСe k value Тs tСe slope of tСe regressТon equatТon, wСТcС Тs 0.259
