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PEMAKAIAN

   
RADIOAKTIF  ISOTOP    
SEBAGAI  TRACER  

Rahma5na  B.  Herman  


Isotopes  
Are  chemical  elements  that  have:    
!   the  same  atomic  number:    
 the  sum  of  the  number  of  protons  in  
nucleus  
!   but  different  atomic  masses:    
 the  sum  of  the  number  of  protons  and  
neutrons  in  nucleus  
Isotopes..…  
Certain  isotopes  are:  
!   Unstable    
!   Undergo  spontaneous  disintegra5ons  
(transmuta5ons)  
!   Accompanied  by:    
 -­‐  the  emission  of  par5culate  
 -­‐  some5mes  also  electromagne5c  radia5ons  
!   Are  said  to  be  radioac5ve  and  called  radioisotopes  
or  radionuclides  
!   The  presence  of  radioisotopes    may  readily  be  
detected  by  instruments  sensi5ve  to  their  radia5on  
Isotopes..…
!   Generally  an  organism  cannot  dis5nguish  
between  the  stable  and  radioac5ve  forms  of  the  
same  element  so  that  both  are  metabolized  in  
an  iden5cal  manner  
!   That  is  why  radioisotopes  extremely  useful  and  
may  conveniently  be  employed  as  tracers  
Isotopes..…
The  fate  of  a  given  element  (or  molecule)  in  an  
organism  (or  even  in  individual  cell)  may  be  
studied  by  :  
!   Introducing  radioac5ve  form  of  that  element    
!   Following  the  uptake  and  subsequent  
localiza5on  of  the  radioac5vity  
!   Highly  sensi5ve  detector–  small  enough  
quan55es  of  radioisotopes  to  preclude  
significant  damage  to  cell  cons5tuents  by  
radia5on  
Some  radioisotopes  frequently  used  as  tracers  
Radiation Emitted
Isotopes Half-life Used as tracers of
β partic. γ-rays

1H Yes No 12.3 ys Organic compound


14C Yes No 5570 ys Organic compound
24Na Yes Yes 15 hs Salt metab, exchange across membranes
32P Yes No 14.3 ds Nucleic ac met, phospholipid met, salt met
15S Yes No 87.2 ds Protein metabolism
36Cl Yes No 300000 ys Salt metabolism
42K Yes Yes 12.5 hs Salt metab, exchange across membranes
45Ca Yes No 164 ds Salt metabolism, bone deposition
59Fe Yes Yes 45.1 ds Heme synthesis, hemoglobin synthesis
131I Yes Yes 8.1 ds Protein metabolism
Advantage  of  Radioisotope  Technique  
!   Results  obtained  from  experiments  involving  
the  use  of  radioisotopes  are  quan5ta5ve,  since  
the  amount  of  radioac5vity  present  and  
available  for  detec5on  is  directly  propor5onal  
to  the  radioisotope  content  
!   Numerous  biological  studies  carried  out  
rou5nely  using  radioisotopes  can  only  be  
performed  with  great  difficulty  or  are  virtually  
impossible  without  them  
Advantage  of  Radioisotope  Technique…..  
1.  Determina5on  of  molecular  fluxes  under  
condi5ons  of  zero  net  exchange:  
 -­‐  during  dynamic  equilibrium  →  no  net  transfer  of  
 material  occur  between  cell  and  its  surrounding  
 -­‐  although  a  con5nuous  exchange  between  
 one  region  and  another    
 -­‐  concentra5on  within  a  5ssue  /  cell  fairly  
 constant  as  result  of  balanced  biosynthesis  
 and  degrada5on    
 This  situa5on  cannot  be  easily  detected  /  detected  
by  rou5ne  chemical  analyses  
Advantage  of  Radioisotope  Technique…..  
 …..Determina5on  of  molecular  fluxes  under  
condi5ons  of  zero  net  exchange:  
 Using  isotopes  observa5ons  may  provided  great  
impetus  to  the  development  of  concept  concerning:  
 -­‐    the  con5nuous  metabolic  turnover  in  constant  
 concentra5ons  
 -­‐  ac5ve  transport  of  materials  across  cell  
 membrane  against  concentra5on  gradients  
 -­‐  the  rates  at  which  metabolic  turnover:      
   >    within  5ssue  /  cell    
   >    across  cell  membrane  
Advantage  of  Radioisotope  Technique…..  
2.  Simplifica5on  of  chemical  analyses:  
 For  quan5ta5ve  determina5ons  of:    
 -­‐  distribu5on  of  a  given  element  /  compound  in  
 different  5ssues  of  body  or  in  different  parts  of  
 individual  cell  
 -­‐  fate  of  the  element  /  compound    
 Since  radioisotopes-­‐containing  compounds  are  not  
dis5nguished  from  their  stable  (nonradioac5ve)  by  
an  organism  
Advantage  of  Radioisotope  Technique…..  
…..Simplifica5on  of  chemical  analyses:  
 Two  requirements  must  be  fulfilled:  
 1.  The  administra5on  of  labeled  compound  must  
 quickly  be  followed  by  its  uniform  distribu5on  
 among  stable  equivalents  already  present  in  the  
 system  
 2.    Once  a  uniform  distribu5on  is  a^ained,  the    
   specific  ac5vity  of  the  substance  in  ques5on      
 must  be  determined  in  one  of  the  several        
 components  (i.e.:  5ssues,  cell  frac5ons,  etc)    
   to  be  analyzed    
Advantage  of  Radioisotope  Technique…..  

…..Simplifica5on  of  chemical  analyses:  


 -­‐  Specific  ac5vity  is  defined  here  as  the  quan5ty  
 of  radioac5ve  element  /  compound  per  unit  
 weight  of  total  element  /  compound  
 -­‐  If  does  become  uniformly  distributed  in  
 propor5on  to  the  stable  form  already  present  in  
 system,  then  the  specific  ac5vity  will  be  equal  
 throughout  the  system  
   So,  measurement  may  be  made  simply      
Advantage  of  Radioisotope  Technique…..  

3.  “Isotope  Dilu5on”  Methods:  


 To  determine  the  total  quan5ty  /  volume  of  a  
given  material  in  the  body  /  cell  when  quan5ta5ve  
isola5on  for  analysis  is  not  possible.  
 For  example  to  determine    
 -­‐  total  circula5ng  blood  volume  
 -­‐  erythrocyte  mass  
 -­‐  chloride  space  
 -­‐  exchangeable  sodium,  etc  
Advantage  of  Radioisotope  Technique…..  

…..“Isotope  Dilu5on”  Methods:  


 Such  unknown  quan55es  are  measured  using  
radioisotopes  may  be  exemplified  by  considering  the  
following  generalized  case  
 -­‐  X  is  a  material  occurs  in  a  system  (i.e.  5ssue,  
 cell,  etc)  in  unknown  abundance  
 -­‐  X*  is  labeled  from  this  material  (available  
 commercially  or  can  be  prepared  experimentally  
 having  a  known  specific  ac5vity)  
 -­‐  SA  is  for  Specific  Ac5vity    (X*)  
SA  =  
 -­‐  The  equa5on  is  as  followed:     (X*  +  X)  
Advantage  of  Radioisotope  Technique…..  

…..“Isotope  Dilu5on”  Methods:        


 -­‐    Following  this,  the  specific  ac5vity  of  a    
 small  quan5ty  of  the  material  isolated  from  
 the  system  is  determined  
 -­‐  Since  the  X*  originally  introduced  was  uniformly  
 mixed  with  X  already  present,  its  SA  will  have  
 been  reduced  in  direct  propor5on  to  the  total  
 amount  of  X    present  in  the  system,  with  equa5on  
 as  followed:  
Advantage  of  Radioisotope  Technique…..  
…..“Isotope  Dilu5on”  Methods:  
       
   (SA1)  
   XT    =    X*    -­‐  1  
  (SA2)  
                     
 -­‐    XT      =    the  total  amount  of  X  originally  present  in  the  
     system  
 -­‐    X*    =  the  quan5ty  of  labeled  material  added    
 -­‐    SA1  =  the  original  SA  of  the  material  added  
 -­‐    SA2  =  the  SA  of  the  sample  removed  for  analysis  
Advantage  of  Radioisotope  Technique…..  

4.  Precursor-­‐Product  Rela5onship:  


 -­‐  Radioisotopically  labeled  compounds  may  be  used  
 to  determine  which  of  a  variety  of  alterna5ve  
 metabolic  pathways  is  followed  by  a  compound  by  
 examining  the  radioisotope  content  of  metabolic-­‐
 products  
 -­‐  If  several  pathways  are  open  to  the    precursor,  then  
 the  extent  to  which  each  is  followed  under  a  
 variety  of  experimental    condi5ons  may  be  
 determined  from  the  respec5ve  radioisotope  
 contents  of  the  products  
Advantage  of  Radioisotope  Technique…..  

…..Precursor-­‐Product  Rela5onship:  
 -­‐  The  forma5on  of  an  end  product  ogen    involves    
 a  number  of  intermediate  precursors  and  
 products;  that  is:  

   A    →  →  →  →      B    →  →  →  →      C    →  →  →  →      D    
precursor1                    product  1                        product  2              endproduct  
                                                       precursor  2                  precursor  3  
Advantage  of  Radioisotope  Technique…..  

…..Precursor-­‐Product  Rela5onship:  
 -­‐  By  degrading  the  intermediate  and  final  
 products  of  a  series  of  chemical  reac5ons  
 and  determining  the  distribu5on  of  
 radioisotope  among  the  cons5tuent  atoms    
 -­‐  It  is  ogen  possible  to  determine  the  rate  of  
 each  atom  in  the  pathway  
Proper5es  of  Radioac5ve  Isotopes  
!   Most  radia5ons  emi^ed  by  radioisotopes  are  the  
result  of  changes  in  the  arrangement    of  unstable  
atomic  nuclei  
!   Whether  an  atomic  nucleus  is  stable  depends  in  turn  
upon  numbers  of  neutrons  (N)  and  protons  (Z)  that  it  
contain  
!   Isotopes  with  N  and  Z  numbers  outside  of  the  stable  
region  undergo  spontaneous  changes  in  which  nuclear  
neutrons  and  protons  are  interconverted  
!   These  nuclear  transmuta5ons  are  accompanied  by  the  
emission  of  par5culate  and  electromagne5c  radia5on  
Types  of  Radia5on  Emi^ed  by  Radioisotopes  

!   The  most  common  types  of  nuclear  


radia5ons  are:  
 -­‐  Alpha  par5cles  
 -­‐  Posi5ve  beta  par5cles  
 -­‐  Nega5ve  beta  par5cles  
 -­‐  Gamma  rays  
Types  of  Radia5on  Emi^ed  by  Radioisotopes…..  

!   Alpha  par5cles:  
 -­‐  Consist  of  2  protons  (Z)  and  2  neutrons  (N)  
 -­‐  Iden5cal  to  Helium  nuclei  
 -­‐  Emi^ed  by  radioisotopes  of  high  atomic  
 number  such  as  uranium,  polonium,  thorium,  
 and  radium  
 -­‐    Alpha-­‐emikng  radioisotopes  are  rarely  
 used  as  tracers  in  biological  studies  
Types  of  Radia5on  Emi^ed  by  Radioisotopes…..  

!   Posi5ve  beta  par5cles  (positrons):  


 -­‐  are  emi^ed  from  nuclei  in  which  the  N:Z  ra5o  is  
 below  that  which  is  stable  
 -­‐  Nuclear  transmuta5on  involves  the  conversion  of  a  
 proton  (Z)  into  a  neutron  (N) (Z→N),  positron  and  
 neutrino,  the  la^er  two  being  ejected  from  nucleus  
 -­‐  Positron  possesses  a  unit  posi5ve  charge  and  is  equal  
 in  mass  to  an  electron,  while  neutrino  has  neither  
 mass  nor  charge  
 -­‐  Very  few  positron-­‐emi^ed  radioisotopes  are  
 employed  as  biological  tracers  
Types  of  Radia5on  Emi^ed  by  Radioisotopes…..  

!   Nega5ve  beta  par5cles  (negatrons):  


 -­‐  are  emi^ed  from  nuclei  in  which  the  N:Z  ra5o  is  
 above  that  which  is  stable  
 -­‐  Nuclear  change  involves  the  conversion  of  a  neutron  
 (N)  into  a  proton  (Z)  (N→Z),  with  resul5ng  ejec5on  of  
 a  beta  par5cle  and  neutrino  
 -­‐  Nearly  all  radioisotopes  used  as  tracers  by  biologist  
 emit  nega5ve  beta  par5cles    
Types  of  Radia5on  Emi^ed  by  Radioisotopes…..  

!   Gamma  radia5on:  
 -­‐  is  no  par5culate  
 -­‐    It  is  electromagne5c  

!   Beta  par5cles  and  gamma  rays  emi^ed  by  


radioisotope  ogen  used  as  tracers  
Half-­‐Life  
!   The  number  of  atom  including  radioisotopes    
that  disintegrate  during  a  given  5me  interval  
decreases  logarithmically  with  5me  
!   is  unaffected  by  chemical  and  physical  factors  
that  normally  alter  the  rates  of  chemical  
processes  (i.e.  temperature,  concentra5on,  
pressure,  etc)  
!   Radioac5ve  decay  is  therefore  a  classical  
example  of  first-­‐order  reac5on  
Half-­‐Life….  
!   A  convenient  term  used  to  described  the  rate  of  
decay  of  a  radioisotope  is  the  physical  half-­‐life  
(Tp)  
!   That  is  the  amount  of  5me  required  to  reduce  
the  amount  of  radioac5ve  material  to  one-­‐half  
its  previous  value  
!   Each  radioac5ve  isotope  decays  at  a  
characteris5c  rate  and  therefore  has  a  unique  
half-­‐life  
Half-­‐Life….  
!   When  radioisotopes    are  used  in  in-­‐vivo  
experiments  of  extended  dura5on,  the  turnover  
rate  of  the  element  in  the  body  /  in  the  cell  
must  also  be  considered    
!   For  the  rate  of  decrease  of  radioac5vity  will  be  
a  func5on  of  both  radioac5ve  decay  and  
metabolic  turnover  
!   In  these  instances,  a  more  useful  term  is  the  
effec5ve  half-­‐life  (Te)  
Half-­‐Life….  
!   The  effec5ve  half-­‐life  (Te)  is  defined  as  amount  
of  5me  required  to  reduce  the  radioisotope  
content  of  the  body  /  cell  to  one-­‐half  its  original  
value  by  the  combined  effects  of  decay  and  
turnover  
!   The  biological  half-­‐life  (Tb)  is  defined  as  the  
normal  amount  of  5me  required  for  the  
turnover  of  one-­‐half  of  the  body  content  of  a  
given  element  (radioac5ve  or  nonradioac5ve)  
Half-­‐Life….  
                                                                     
                                                                       Tb  x  Tp  
                               Te  =    
                                                         Tb  +  Tp  
!   Te    =    effec5ve  half-­‐life  
!   Tb    =    biological  half-­‐life  
!   Tp    =  physical  half-­‐life  
Physical,  Biological  and  Effec5ve  Half-­‐life    
of  some  radioisotopes  
Radio Half-Life
Element
isotope Physical Biological Effective
Hydrogen 1H 12.3 ys 19 ds 19 ds
Carbon 14C 5,570 ys 180 ds 180 ds
Phosphorous 32P 14.3 ds 3 ys 14.1 ds
Calcium 45Ca 164 ds 73 ys 16.3 ds
Iron 59Fe 45.1 ds 3.4 ys 27.1 ds
Cobalt 60Co 5.3 ys 8.1 ds 8.0 ds
Iodine 131I 8.1 ds 156 ds 7.7 ds
Radium 236Ra 1,620 ys 104 ds 104 ds
Detec5on  and  Measurement  of  Radia5on  

!   The  selec5on  of  instruments  for  the  detec5on  


and  measurement  of  radioisotopes  is  based  
primarily  on  the  type  and  energy  of  the  emi^ed  
radia5on  
!   The  most  commonly  used  detectors  are:  
 -­‐  Geiger-­‐Muller  counters  
 -­‐  Solid  scin5lla5on  counters  
 -­‐  Liquid  scin5lla5on  counters  
Detec5on  and  Measurement  of  Radia5on…..  

!   Geiger-­‐Muller  (G-­‐M)  counters:  


 -­‐  to  be  employed  primarily  with  isotopes  emikng  
 intermediate  or  high  energy  beta  par5cles    
 -­‐  may  also  be  used  at  low  efficiency  for    the  
 measurement  of  gamma  radia5on  
!   Solid  scin5lla5on  counters:  
 -­‐  to  be  employed  generally  with  gamma  ray  emikng  
 isotopes  

!   Liquid  scin5lla5on  counters:  


 -­‐  to  be  used  with  isotopes  emikng  low  energy  
 beta  par5cles  
Autoradiography  
!   Special  technique  modifica5on  of  radioisotopic  
tracer  
!   Used  in  conjunc5on  with  light  and/or  electron  
microscopy  
!   Special  photographic  film  or  emulsion  →  
autoradiogram:  
 -­‐  number  and  distribu5on  within  cell  /    5ssue  
 -­‐  movement  and/or  localiza5on  of  
 metabolic  intermediates  or  products  
Radioimmuno-­‐Assay  
!   For  hormones  assessment  
!   Principle  of  radioimmuno-­‐assay:  
 An5body  (globulin)  which  is  specific  for  the  
hormone  that  will  be  assessed  must  be  produced  
from  the  animal  in  a  great  amount  (commercially  
available)  
!   The  an5body  then  mixed  with:  
 -­‐  animal  serum  which  contain  hormone  to  be  
 assessed  (h)  
 -­‐  pure  standard  hormone  which  has  been  labeled  
 by  radioisotop  (hsr)  (with  known  amount)  
Radioimmuno-­‐Assay..…  
!   An5body  and  hormone  will  be  bound  (ab-­‐h  &  
ab-­‐hsr)  
!   Hormone  to  be  assessed  and  hormone  labeled  
by  radioisotope  compe55vely  binds  the  
an5body  
!   Concentra5on  of  ab-­‐hsr  then  measured,  soon  
ager  the  binding  has  reached  equilibrium,  using  
radioac5ve-­‐coun5ng  technique  
Radioimmuno-­‐Assay..…  
!   To  make  “assay  highly  quan5ta5ve”,  
radioimuno-­‐assay  must  also  be  applied  for  
“standard”  solu5on  from  pure  un-­‐labeled  
hormone  with  some  levels  of  concentra5on  
!   The  results  then  will  be  arranged  in  a  curve  
which  is  called  Standard  Curve  
 
y   =   0 , 8 2 8 7 x

  3500 R 2   =   0 , 9 9 7 2

Height in Chrom atogram


3000

2500

  2000

1500

1000

500

0
0 500 1000 1500 2000 2500 3000 3500 4000 4500

  Concentration of Norepinephrine (pg/m l)


 
 
 
2500 y   =   0 , 5 3 6 2 x
R 2   =   0 , 9 9 7 4
Height in Chrom atogram

  2000

  1500
 
  1000

500
 
 
 
0
0 500 1000 1500 2000 2500 3000 3500 4000 4500

 
Concentration of Norepinephrine (pg/m l)

 
The standard curve for pure NE (above) and for extracted NE (below)
Guideline  on  Bioanaly-cal  
Method  Valida-on  
 
 (Commi6ee  for  Medicinal  Products  for  
Human  Use  /  CHMP,  2011)  
Method Validation
!   The main objective of method validation is to
demonstrate the reliability of a particular
method for the determination of an analyte
concentration in a specific biological matrix,
such as blood, serum, plasma, urine, or saliva
!   If an anticoagulant is used, validation should be
performed using the same anticoagulant as for
the study samples
!   Generally a full validation should be performed
for each species and matrix concerned
Method Validation…..
!   Main characteristics of bioanalytical method that
are essential to ensure the acceptability of the
performance and the reliability of analytical results
are:
- Selectivity
- Lower limit of quantification (LLOQ)
- the response function and calibration range
(calibration curve performance / standard curve)
- Accuracy
- Precision
- Matrix effects
- Stability of the analytes in biological matrix
- Stability of the analytes and of internal standard (IS) in
the stock and working solutions and in extracts under
the entire period of storage and processing
conditions
Method Validation…..
!   During method validation and analysis of
study sample, a blank biological matrix
will be spiked with the analytes of interest
using solutions of reference standards to
prepare calibration, standards quality
control samples and stability samples
!   In addition, suitable internal standards
(IS) can be added during sample
processing in chromatographic method
Selectivity
!   The analytical method should be able to
differentiate the analytes of interest and internal
standard (IS) from endogenous components in
the matrix or other component in the sample
!   Selectivity should be proved using at least 6
individual sources of the appropriate blank
matrix, which are individually analysed and
evaluated for interference
!   Normally, absence of interfering components is
accepted where the response is < 20% of the
lower limit of quantification for the analyte and
5% for the internal standard (IS)
Lower Limit of Quantification (LLOQ)
!   LLOQ is the lowest concentration in a sample
which can be quantified reliably, with an
acceptable accuracy
!   LLOQ is considered being the lowest calibration
standard
!   The analyte signal of LLOQ sample should be at
least 5 times the signal of blank sample
!   LLOQ should be adapted to expected
concentrations and to the aim of study: for
bioequivalence studies LLOQ should be not
higher than 5% of Cmax
!   LLOQ may be not necessary for exploratory
pharmacokinetic studies
Calibration Curve Performance
!   Before carrying out the validation of the analytical
method, it should be known what concentration range
is expected
!   The range should be covered by calibration curve
range, defined by LLOQ being the lowest calibration
standard and the upper limit of quantification (ULOQ)
being the highest calibration standard
!   A minimum of 6 calibration concentration levels should
be used, in addition to the blank sample (processed
matrix sample without analyte and without IS) and a
zero sample (processed matrix with IS)
!   Each calibration standard can be analysed in replicate
Calibration Curve Performance…..
!   The calibration curve parameters should be
reported (slope and intercept in case of
linear fit)
!   The back calculated concentrations of the
calibration standards should be presented
together with the calculated mean accuracy
values
!   All the available (or acceptable) curves
obtained during validation, with a minimum
of a 3 should be reported
Calibration Curve Performance…..
!   The back calculated concentrations should be
within ±15% of the nominal value, except for
LLOQ for which it should be within ±20%
!   At least 75% of calibration standards, with a
minimum of 6 calibration standard levels, must
fulfill this criterion
!   In case replicates are used, the criteria (within
±15% or ±20% for LLOQ) should also be fulfilled
for at least 50% of calibration standards tested
per concentration level
!   In case a calibration standard does not comply
with these criteria, this calibration standard
sample should be rejected
Standard Curve
!   A standard curve is a type of graph used as a
quantitative research technique.
!   A graphic plot of tracer binding versus the
known concentration of test substances in a set
of standards usually prepared by serial dilution
or incremental addition.
!   Multiple samples with known properties are
measured and graphed
!   So then allows the same properties to be
determined for unknown samples by
interpolation on the graph.
Accuracy
!   Within-run accuracy
Determined by analysing in a single run a minimum of 5
samples per level at a minimum 4 concentration levels
which are covering the calibration range.
The mean concentration should be within 15% of
nominal values, except for LLOQ should be 20% of the
nominal value
!   Between-run accuracy
For the between-run accuracy, LLOQ, low, medium and
high QC samples from at least 3 runs analysed on at
least 2 different days should be evaluated
The mean concentration should be within 15% of the
nominal values for the QC samples, except for LLOQ
which should be within 20% of the nominal value)
Precision
!   Within-run precision
For the validation of the within-run precision, there
should be a minimum of 5 samples per
concentration level at LLOQ, low, medium and high
QC samples in a single run
The within-run CV value should not exceed 15% for
QC samples, 20% for LLOQ
!   Between-run precision
For the validation of the between-in run precision,
LLOQ, low, medium and high samples from at least
3 runs analysed on at least 2 different days should
be evaluated
The between-run CV value should not exceed 15%
for QC samples, 20% for LLOQ
Matrix Effects
!   Matrix effects should be investigated when using mass
spectrometric methods
!   Using at least 6 lots of blank matrix from individual
donors
!   For each analyte and IS, matrix factor should be
calculated for each lot of matrix, by calculating the ratio
of peak area in the presence of matrix (measured by
analysing blank matrix spiked after extraction with
analyte), to the peak area in absence of matrix (pure
solution of analyte)
!   The IS normalized MF should also be calculated by
dividing the MF of analyte by the MF of the IS
!   The CV of IS normalized MF calculated from 6 lots of
matrix should not be greater than 15%
!   This determination should be done at a low and at a
high level of concentration (maximum of 3 times LLOQ
and close to ULOQ)
Stability
!   Evaluation of stability should be carried out to
ensure that every step taken during sample
preparation and sample analysis, as well as the
storage conditions used do not affect the
concentration of analyte
!   The following stability tests should be evaluated:
- stability of the stock solution and working solutions of
the analyte and internal standard (IS)
- freeze and thaw stability of the analyte in the matrix
from freezer storage conditions to room temperature or
sampling processing temperature
- short term stability of the analyte in matrix at room
temperature or sampling processing temperature
- long term stability of analyte in matrix stored in freezer
- on-instrument/ autosampler stability of the processed
sample at injector or autosampler temperature
Percentage  recoveries  of  internal  standard  amitriptyline  

Concentration Pure AT Extracted AT Recovery


of amitriptyline (height in chrom.) (height in chrom.) (%)

31485 28922 91.86


20000pg/ml
25358 20594 81.21
28832 26866 93.18
31115 29189 93.81
27719 22865 82.49
28899 21839 75.57
32143 25846 80.41

Mean ± SD: 85.50 ± 7.31


Percentage  recoveries  of  deriva5zed  NE  
Concentration Pure NE Extracted NE Recovery
of NE (height in chrom.) (height in chrom.) (%) Mean ± SD
103455 89075 86.10
109876 90632 82.49
16000 pg/ml 83387 69863 83.78 84.34±1.56
104638 88943 85.00
  53549 44232 82.60
56791 44345 78.08
  pg/ml
8000 42013
52995
35294
45046
84.01
85.00
82.42±3.06
25711 21698 84.39
28903 24346 84.23
4000 pg/ml 20876 17745 85.00 85.41±1.78
26894 23675 88.83
12894 10521 81.59
13764 11230 81.59
2000 pg/ml 11143 8971 80.51 82.50±2.59
13547 11693 86.31
6690 5429 81.15
6902 5885 85.27
1000 pg/ml 5621 4525 80.50 82.57±2.18
6854 5713 83.35
3112 2471 85.83
3487 2959 84.86
500 pg/ml 2876 2386 82.96 85.43±2.13
3269 2879 88.07
1615 1265 78.33
1775 1473 82.99
250 pg/ml 1313 1089 82.94 81.20±2.23
1656 1334 80.56
Above:  Scanning  chromatogram  of  deriva5zed  extracted  NE  (DNE)  and  extracted      
                         amitriptyline  (AT)      
Below:  Selected  ion  monitoring  (SIM)  chromatogram  of  deriva5zed  extracted  NE  (leg);          
                         extracted  amitriptyline  (right)    
Peak  ra5o  of  deriva5zed  NE  and  amitriptyline  (for  standard  curve)  

Peak ratio
Concentra
tion of NE Mean ± SD
(pg/ml)
1st run 2nd run 3rd run 4th run

8000 1.59 1.64 1.72 1.67 1.65 ± 0.05


4000 0.86 0.90 0.81 0.83 0.85 ± 0.04
2000 0.39 0.40 0.41 0.43 0.41 ± 0.02
1000 0.21 0.22 0.19 0.20 0.21 ± 0.01
500 0.117 0.111 0.107 0.099 0.11 ± 0.008
100 (LLOQ) 0.019 0.024 0.026 0.021 0.023 ± 0.003

LLOQ,  lower  limit  of  quan5ta5on  


 
 
 
  y   =   0 , 0 0 0 2 x
2
1 ,8 R   =   0 , 9 9 9 7
 
1 ,6
  1 ,4

  1 ,2
Peak Ratio

  1

  0 ,8

0 ,6
 
0 ,4
 
0 ,2
  0

  0 1000 2000 3000 4000 5000 6000 7000 8000 9000

C o n c e n tra tio n  o f  N o re p in e p h rin e  ( p g /m l)


 
 
The  standard  curve  based  on  peak  ra5o  of  deriva5zed  NE  and  internal    
standard  amitriptyline.  The  concentra5ons  of  NE  were  from  8000  pg/ml    
to  500  pg/ml  and  100  pg/ml,  the  lower  limit  of  quan5ta5on  (LLOQ).    
Accuracy  and  precision  of  the  assay  for  NE  with  amitriptyline  as  internal  standard  (n=4)  

Intra-assay variation Inter-assay variation


(Intra-day variation) (Inter-day variation)

Conc
of Peak ratio Peak ratio
NE CV
(%) CV
Mean ± SD Mean ± SD
(%)
1st 2nd 3rd 4th 1st 2nd 3rd 4th
run run run run run run run run

8000 1.67 1.62 1.70 1.65 1.66 ± 0.03 1.81 1.67 1.59 1.68 1.72 1.66±0.05 3.01
4000 0.90 0.86 0.82 0.88 0.87 ± 0.03 3.45 0.90 0.82 0.86 0.83 0.85±0.04 4.71
2000 0.41 0.42 0.44 0.40 0.42 ± 0.02 4.76 0.41 0.40 0.43 0.44 0.42±0.02 4.76
1000 0.22 0.20 0.19 0.20 0.20 ± 0.01 5.00 0.22 0.20 0.19 0.21 0.21±0.01 4.76
500 0.121 0.118 0.129 0.124 0.123 ± 0.005 4.07 0.121 0.117 0.119 0.127 0.121±0.004 3.31
100 0.024 0.028 0.026 0.021 0.025 ± 0.003 12.00 0.024 0.026 0.027 0.020 0.024 ± 0.03 12.50
(LLOQ)

CV,  Coefficient  of  varia5on;  LLOQ,  lower  limit  of  quan5ta5on;  Conc,  
concentra5on