Pemakaian Radioisotop Sebagai Tracer

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
…..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
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
…..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 aâined, 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
…..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
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
…..“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)

-‐ 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:

(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
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
…..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
…..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 emiêd 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 Emiêd by Radioisotopes
!   The most common types of nuclear

radia5ons are:
-‐ Alpha par5cles
-‐ Posi5ve beta par5cles
-‐ Nega5ve beta par5cles
-‐ Gamma rays
Types of Radia5on Emiêd by Radioisotopes…..
!   Alpha par5cles:
-‐ Consist of 2 protons (Z) and 2 neutrons (N)
-‐ Iden5cal to Helium nuclei
-‐ Emiêd by radioisotopes of high atomic
number such as uranium, polonium, thorium,
and radium
-‐ Alpha-‐emikng radioisotopes are rarely
used as tracers in biological studies
!   Posi5ve beta par5cles (positrons):

-‐ are emiêd 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 laêr 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-‐emiêd radioisotopes are
employed as biological tracers
!   Nega5ve beta par5cles (negatrons):

-‐ are emiêd 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
!   Gamma radia5on:
-‐ is no par5culate
-‐ It is electromagne5c
!   Beta par5cles and gamma rays emiêd 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 emiêd
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

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PEMAKAIAN

Rahma5na B. Herman

1H Yes No 12.3 ys Organic compound

…..Simpliﬁca5on of chemical analyses:

3. “Isotope Dilu5on” Methods:

…..“Isotope Dilu5on” Methods:

…..“Isotope Dilu5on” Methods:

4. Precursor-­‐Product Rela5onship:

! The most common types of nuclear

! Posi5ve beta par5cles (positrons):

! Nega5ve beta par5cles (negatrons):

! Beta par5cles and gamma rays emi^ed by

! The selec5on of instruments for the detec5on

! Geiger-­‐Muller (G-­‐M) counters:

! Liquid scin5lla5on counters:

Height in Chrom atogram

Concentration of Norepinephrine (pg/m l)

Concentration Pure AT Extracted AT Recovery

31485 28922 91.86

Mean ± SD: 85.50 ± 7.31

8000 1.59 1.64 1.72 1.67 1.65 ± 0.05

LLOQ, lower limit of quan5ta5on

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)

Intra-assay variation Inter-assay variation

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3.  “Isotope Dilu5on” Methods:

4.  Precursor-‐Product Rela5onship:

!   The most common types of nuclear

!   Posi5ve beta par5cles (positrons):

!   Nega5ve beta par5cles (negatrons):

!   Beta par5cles and gamma rays emi^ed by

!   The selec5on of instruments for the detec5on

!   Geiger-‐Muller (G-‐M) counters:

!   Liquid scin5lla5on counters: