Development of a Near-Infrared Spectroscopic Method for the Determination of Water in Prozac ®

Neil Blakiston, (a) Dr. R. J. Singer, (b)

(a)
Quality Control Laboratory, Eli Lilly and Co. Ltd., Basingstoke (b)
Kingston University - London

Abstract: Comparison of Equipment: Results/Conclusions:

Objective: To conduct a feasibility study; investigating the use of Near-Infrared (NIR) The Orion Turbo2 TM Karl Fischer and BrukerTM MPA FT-NIR are depicted below; Forty one results, taken as the average of the Karl Fischer replicate analysis, were entered for the
Spectroscopy as a replacement analytical technique for Karl Fischer (KF) volumetric analysis, for respective NIR spectra.
the determination of water in pharmaceutical product. Multivariate analysis and chemometric
tools will be used to produce a NIR calibration model using the KF data as the reference method. R1 – Linear Calibration
A simple plot of predicted value versus true for the
Method: Portions of Prozac® powder blend were dried at 105۫˚C for set periods before being determined peak areas (R 2 = 94.87) gave a good
sealed in NIR tubes and analysed on a BrukerTM MPA FT-NIR, using Opus Quant software. The visual correlation.
samples were then titrated in replicate by the KF reference method using an Orion Turbo2TM
Titrator. The average values obtained for the 41 samples were assigned respectively to the NIR An absolute difference of 0.08% H 2O was determined
sample spectra to produce a calibration model. Three calibration models were compared; Linear between five replicates by KF and five readings by
Regression (R1), Vector Normalized + Principle Component Analysis (R2) and a Quant Disadvantages of KF Analysis: The following list highlights some of the NIR, with the latter having the smaller standard
optimised solution (R3). disadvantages of the method and examples of recent test failures that have led to deviation.
lengthy investigation.
Results: The results were determined using each data point to cross validate the model to
• Can not be automated. • Involves toxic reagents.
produce predicted values. The NIR prediction was statistically compared to the ‘True’ reference
R2 – Vector Normalization +
value.
• Requires ventilated area. • Sample exposed during transfer. Principle Component Analysis
A good visual correlation with
• Calibration models had R 2 values of 94.79, 96.31 and 99.82 respectively.
linear performance >R1
• Predicted values had average residuals of 0.000, -0.022 and 0.018 %H 2O respectively. • Weekly maintenance is required. • Destructive test.
• Predicted values had a maximum variance of 0.933, 1.237 and 0.909 %H 2O respectively. R2 = 96.31 Calibration
• Maximum variance on KF replicates was 0.884 %H 2O. • Lengthy daily calibration required. • In-process control samples required. R2 = 94.42 Cross Validation

Conclusions: The NIR method gave predicted values comparable to the reference method with • Replicate readings required. • Solvents can expire or run out.
regards to precision and accuracy based on the 41 data points. This would improve with
additional calibration samples. Using statistical analysis model R1 proved to be slightly more R3 – Fully Optimised
• Sample may not disintegrate. • Control/Acceptance checks may fail. A good visual correlation with
reliable.
Introduction/Background: • Balance may give false reading. • Balance weight transfer fails.
linear performance >R2
Water can have an adverse effect on the performance of a pharmaceutical product. This
R2 = 99.82 Calibration
includes physical changes; tablet friability, capsule integrity and dissolution profile, or
• Solvents need to be in stock. • Not versatile. R2 = 95.74 Cross Validation
chemical changes such as catalysis of degradation products. Stringent regulatory limits are
applied to many drug products to ensure the Quality, Safety, Integrity, Strength and
Performance of the product during shelf life. • Wrong reagents can be used. • End-point incorrectly determined.
Statistical Analysis: The F-test compared each model to the reference method; R1and R2
To comply with product registration, both in-process and final product monitoring is required. • Control samples not conducted. • Air can enter the system. demonstrated no significant difference from the KF method. The t-test proved that the mean from
A common method for the determination of water content is the Karl Fischer titration method. the reference method and model R1 were from the same population. ANOVA for all four data sets
Developed in 1935 from earlier work by R. W. Bunsen 1, Karl Fischer established a direct demonstrates that they are all from the same population. Model R1 demonstrated a residual
relationship between water content and the consumption of iodine (2:1) in the presence of frequency population nearest to the true value for 16 replicates.
sulphur dioxide and a base. Further development by Smith et. al 2 and Scholz 3 refined the
Advantages of NIR Analysis: This technique has several advantages over the KF
reaction parameters and agreed a 1:1 consumption ratio; Conclusion: Three models demonstrated that the water content of Prozac ® powder blend could be
method.
determined by Near-Infrared Reflectance Spectroscopy. Model R1 (Linear Regression) performed
• Fully automated and rapid testing. • Uses sealed samples.
CH3OH + SO2 + RN → [RNH]SO3CH3 : H2O + I2 + [RNH]SO3CH3 + 2RN → [RNH]SO4CH3 + better statistically to the reference method than models using a multivariate approach, even though
2[RNH]I models R2 and R3 demonstrated a better linear calibration.
• No reagents, therefore no waste. • No sample preparation.
The end-point is determined when the first stable appearance of free iodine is detected across Summary: The adoption of NIR spectroscopy by the pharmaceutical industry for routine analytical
two platinum electrodes (biamperometric). • Can analyse through packaging. • Non-destructive test. testing can deliver great benefits; It is a rapid, versatile, non destructive technique, requiring minimal
training and offers an extremely low risk safety profile. Karl Fischer analysis involves a number of
NIR spectroscopy offers a proven alternative for the determination of water and has been used complicated transfer steps and other routines, which are highly prone to human error.
• Automated calibration. • Fully Versatile.
successfully in cereals and forage samples 4. NIR analysis has only recently been accepted in the
more regulated pharmaceutical industry and guidance on the validation requirements are still
vague.

The method relies on spectral differences between samples due to the concentration of
Analysis time can be reduced significantly using NIR spectroscopy. During this References:
project 12 hours of KF analysis were reduced to 20 minutes by NIR. (1) Bunsen, R., Annalen, 1835, 86, 265.
components or their physical properties. The anharmonic oscillator model predicts departures
(2) Smith , D. M, Bryant, W. M. D., J. Amer. Chem. Soc., (1935), 57, 841.
from Hooke’s Law as the fundamental frequencies, which typically occur in the Mid-Infrared
(3) Scholz, E., “KF Reagents with Pyridine”, Fresenius Z., Anal. Chem., (1980), 303, 203-207.
0.65

region. These small variations give rise to combination NIR Spectra: The two spectra show the full
0.60

(4) Windham, W. R., Barton, F. E., “Moisture in Forage NIR-Reflectance Spectroscopy”, J. Assoc.
and overtone bands which appear at imprecise multiples of the
0.55

window of analysis and an expanded view of the primary Off. Anal. Chem., 74(2), 1991, 325-331.
Absorbance Units
0.50

fundamentals. These are 10 – 100 times weaker than the water band (5450 – 4900 cm -1)
0.45

(5) Burns, D. A., Cuirczak, E. W., “Handbook of NIR Analysis”, Marcel Dekker, New York, 2001.
fundamental transitions and appear as weak broad bands in
0.40
1 .0
0 .9
0 .8
0 .5 0 .6 0 .7
so
Ab nc
rb a n its
e U
0 .4
0 .3
0 .2

1 2
0 0 1 0 0 1 0 0 0 0
9 8 0 7 0 0 0 0
6 0 0
5 4 0
a ve n
W u b
mer cm -1
0.35

the NIR region. Due to the nature of these bands they can be
0.30

difficult to interpret, therefore multivariate manipulation 5600 5500 5400 5300 5200 5100 5000 4900

techniques are employed. Modern NIR equipment can

W avenumber cm-1

Models R1/R2 analysed the primary water band using a

Acknowledgements: The members of Eli Lilly Quality Control Laboratory whose
C:\OP US
C:\OP US 5 .5\W
US 5
5 .5\W O
O RK
O RK \NB
RK \NB Train ing
\NB Train
Train ing \P
\P ozaac
\P roz
roz
roz c .0
ac Dr
2blied
2bl end
end.0_8
_1P.0
_2
_3
_4
_5
_6
_7 oza cP oz P
.0 acroz
P rozDried
Integ
ac 2b
ac 2brati
2b lenng
len d_S_10
d_ 1Int
2
3
4
5
6
7
8 pheegrare tin
(S
Ingpher
In teS
te grph
gr e er
ating
atingMeacS S(Sroph
ph
phsa ere
mp(S
ere
ere le)
Mac
(S ro sam
ph ere
ph ere
ere Maple
Ma cros
cros ) am
am ple ple )))
ple 25 /08
20
20
20 /07 /20
/08 /20 07
07
07
C:\OP 5 .5\W Train ing ac 2bl end _9
_1 .0
0.0
1.0
2.0 P roz
Pro ac
za c2 len
ble d_
nd 9
_11
_12 In te gr
Inte ating
grat ingS ph
S ere
ph ere(S ph
(Sgeph Ma
ere cros
Mac am
ros am ple /08 /20
C:\OP
C:\OP
C:\OP
US
US 5
C:\OP US
US 5
.5\W
5 .5\W
.5\W
O
.5\W O
O
RK
O RK
RK
\NB
\NB Train
RK \NB
\NB Train
ing
Train ing
ing
\P
ing \P
\P
roz
\P roz
roz
ac
roz ac
ac
2bl
ac 2bl
ble
end
ble end
nd_
_1
nd_ _Drie
1.1
2.0
2.1
3.0
3.1
4.0
4.1
5.0
3.0
4.0
5.0
6.0
_UnDr
1.0 d.ied
0 .0P
P
Pro
P roza
roza
roza za
Pcb
cb
cb
c2
roz
Plen
len
len ble
ac2
ro d_
zac
d_
d_
nd
bl
1 2b
2
3
4
_13
_14
_15
_16
endlen_Dri Inted
d_Int
Un
Int egr
egr
egr
Inte
Dried
ati ng
ati
ati ng
nggratSing
Integ
S
S p her
p
p her
her S eph
rati
Ine
e (Sere
ng
teg
(S
(S ratin
ph
ph
ph
(S
S ph
er
er
er e
e
ph
ereSMa
Ma
Maere
p(S
he
cro
cro
cro
Mac
pre
he
samreros
(S
sam
sam
M
ph
plere
pl
pl
am
acr
e)
e)
e) Ma))cro
ple
osa mp sam
le ) pl e) 20
21
21
14
10
14
10
/08
10 /08
/08
/20
/08 /20
/20
07
/20 07
07
07
C:\OP
C:\OP
C:\OP US
US
US 5
5
5 .5\W
.5\W
.5\W O
O
O RK
RK
RK \NB
\NB
\NB Train
Train
Train ing
ing
ing \P
\P
\P roz
roz
roz ac
ac
ac ble
ble
ble nd_
nd_
nd_ 5.1
6.0
6.1
7.0
7.1
8.0
8.1
8.2
8.3 P
P roza
roza cb
cb len
len d_
d_ 5
6
7
8 Int
Int egr
egr ati
ati ng
ng S
S p
p her
her e
e (S
(S ph
ph er
er e
e Ma
Ma cro
cro sam
sam pl
pl e)
e) 14
10
10
14
22 /08
/08
/08 /20
/20
/20 07
07
07
C:\OP
C:\OP US
US 5
5 .5\W
.5\W O
O RK
RK \NB
\NB Train
Train ing
ing \P
\P roz
roz ac
ac ble
ble nd_
nd_ 8.4
8.5
8.6
8.7
8.8
8.9
8.1 0 P
P roza
roza
Proza cb
cb
roz cb len
len
ac len
ble d_d_
d_ 8
8
nd 9
_8 2 Int
Int egr
egr
Inegr
teg ati
ati
ra ng
ng S
S
tiating
ngS Sp
p her
her
phe e
e (S
(S
re(S ph
ph
( Sp er
er e
e Ma
Ma cro
cro sam
sam pl
pl e)
e) 22 /08 /20 07
C:\OP
C:\OP US
US 5
5 .5\W
.5\W O
O RK
RK \NB
\NB Train
Train ing
ing \P
\P roz
roz ac
ac ble
ble nd_
nd_ 9.0
9.1
10
11
12 .0
.1
.0
.1 PP
P roz
roz ac
ac ble
ble nd
nd _1
_1 0
1 Int In
In teati
te gr ng
gr ating p Sher
S phe
ph ere
ere (S he
ph
(S er
phe
ph re
ere
ereMaMMaac rosa
cro
Ma sam
cros
cros mp
pl
am
am le)
e)
ple )))
ple 22
10
14
10
14 /08
/08
/08 /20
/20
/20 07
07
07

perform various chemometric calculations 5 to clean the

C:\OP
C:\OP US
US 5
5 .5\W
.5\W O
O RK
RK \NB
\NB Train
Train ing
ing \P
\P roz
roz ac
ac ble
ble nd_
nd_ 13
14
15
16 .0
.1
.0
.1 P
P
1.0
1.1roz
roz ac
ac ble
blePndnd _1
_1
ro zac
zac 3
4
5
6 bl en d_AIn
en d_A In te
0te gr
51gr ating
ating
452 _1 S S ph
ph ere
ere (S
(S
Inte ph
ph
gratere
ere Ma
ingMa cros
cros
S ph
ph am
eream (S ple
Sple
phe ) re
re M
M acac rosarosa mp
mp le
le ))) 10
14
10
14
22
23 /08
/08 /20
/20 07
07
C:\OP US 5 .5\W O RK \NB Train ing \P roz ac ble nd_ A 051 45 2_
2_ 2.8
2.0
2.1
2.2
2.3 P ro zac bl en d_A 0 51 452 _2 Inte grat ing S ph ere /08 /20 07
C:\OP
C:\OP
C:\OP
C:\OP
US
US
US
US
5
5
5
5
.5\W
.5\W
.5\W
.5\W
O
O
O
O
RK
RK
RK
RK
\NB
\NB
\NB
\NB
Train
Train
Train
Train
ing
ing
ing
ing
\P
\P
\P
\P
roz
roz
roz
roz
ac
ac
ac
ac
ble
ble
ble
ble
nd_
nd_
nd_
nd_
A
A
A
A
051
051
051
051
347
45
45
45
45
57
2_
2_
2_
5_
2.4
2.5
2.6
2.7
2.9
2.1
2.1
1.0
1.1
1.2 0
1
2
3
4
5
P
P
P
ro
ro
P
P
ro zac
roza
roza
zac
bl
bl en
cben
cb
bl d_A
le d_A
le nd_
nd_
0
0
A
A
3
51
51
05
05
47
452
452
145
145
575
_2
_2
2_
2_
_1 2
2
Inte
Inte grat
grat
In tegra
In
Inte tegra
grat
ing
ing
tin g
tin
ing gSS
S ph
Sph
S
ere
ere
p here
p here
ere ((( S S
S phe
phe
phe
(Sphe
(S ph ere
ph
re
re
ere
M
M Ma
Ma
ac
ac rosa
rosa
cros
cros
mp
mp
am
am
le
le
pl
pl e)
e)) 22
22
22
23
23
22
/08
/08
/08
/08
/20
/20
/20
/20
07
07
07
07
C:\OP
C:\OP
C:\OP
C:\OP
US
US
US 5
5
5 .5\W
.5\W
.5\W O
O
O RK
RK
RK \NB
\NB
\NB Train
Train
Train ing
ing
ing \P
\P
\P roz
roz
roz ac
ac
ac ble
ble
ble nd_
nd_
nd_ A
A
A 347
347
347 57
57
57 5_
5_
5_ 1.3
1.4
1.5
1.6
1.7
1.8
1.9
1.1
11 0mi
0 n.4 n.5 P
nute
n.0
n.1 P ro
ro
s.0
P zac
zac
roza bl
bl en
cben d_A
d_A
lePnd_
roz 3
3
A
acb47
47
34 575
575
len
757 _1
_1
d_
5_ A575
1347_1 5720Inte
Inte
5_1In grat
grat
tegra
10 ing
miing
tin gS
nute SS ph
ph ere
ere
s p here ((( S
S(Sphe
phe
Inph
teg
re
re
re
ere
M
M
M
ratiMa
ac
ac
ac
ng
rosa
rosa
rosa
cros
S
mp
mp
mp
p heam
le
le
le
re pl
(Se))))ac
p here M acro 22
22
22 /08
/08
sa/08
/08 /20
/20
mp/20
/20 07
07
le) 07
07
C:\OP US
US 5
5 .5\W
.5\W O
O RK
RK \NB
\NB Train
Train ing
ing \P
\P roz
roz ac
ac ble
ble nd_
nd_ AA 347
347 57
57 5_
5_ 12
12 0mi n.2
n.3 PPro za cbl en d_ A3 47 min Inte grat ing S ph ere ((ph
S phe re M ros am ple ) 23

semi-baseline dropped vertical and multivariate analysis

C:\OP
C:\OP US
US 5
5 .5\W
.5\W O RK
RK \NB
\NB Train
Train ing
ing \P roz
roz ac
ac ble nd_ A 347 57 5_ 13
2.0
20
50
70
90 0mi
min
0mi
min n.6
.0
.1
nute
.0
.1
utes P.6
s.0
.0ro zac
P Pro
roz
roz Pza
ac
bl en
acP
ro cbl
ble
d_A
roz
ble
za en
nd
acb
3 47
nd
cbl d_
_A
_A
endA3
len
57534_A
34 47
75
d_
_2A
75 3575
347_1
75_
75_
475 5720
12
20
50
75 mi
Inte
5_1
mi
_70
_90 min
nm
n 30
gratmiing
inu sSInte
Int
nute
teInt egra
ph
s ere
egra grat
ting
ting
In (Sing
Sphe
In
S
tegph
ph S
ratph
teg ere
re
ereere
rati
M
ing (S
ng
ac
(S
Sp S
S phe
ere
rosa
ph p re
Male
hemp
ere
he re re
Ma
(S pMcros
(S
cros
he )ac ros
p re Mam
am
here
ream pleple
)) ) 23
M rosa
ple
ac acro
rosa 23
22 /08
sa/08
23
22
mp /08
mp
/08 /20
/20 07
le) 07
le)/20
/20 07
07
C:\OP US 5 .5\W O RK \NB Train ing \P roz ac ble nd_ A 347 57 5_ 90 min utes .1
.2
.3
.5 P ro za cbl end _A 3 475 75 _90 m inu te s In teg rat ing Sp he re (S p he M ac 22
mp le)/20
C:\OP
C:\OP
C:\OP US
US
US 5
5 .5\W O
5 .5\W
.5\W O RK
O RK
RK \NB
\NB
\NB Train
Train
Train ing
ing
ing
\P
\P
\P roz
roz
roz ac
ac
ac
ble
ble
ble
_1.
nd_
nd_
nd_
0 un
A 347
dri
Dry_
Dry_
O ed1
ed2
rig 57
1.0
1.1
.0
2.0
drie
ina
2.1
P
5_
d.0
l.0
roza
Undri
c_ 1 PPed
P rrP
ro
P _A
oza
oza
roz
ro .0
zazac
cbl
cbl
cbl
acb end
en
end
bl d_dr
leend
In tegr _Dry
nd_u ngied
_Dry
ati _O _1
S2
nd
_2
rig1
rie
pinhe Int
dalre (SIntIn
phteg
egra
egra
Iner rati
Integ ting
teg ngcr
e ting
ratin
Marati_Und
Sng
SSpph
gosam
ph prie
She
ere
Serere
pher
he d_A
(S
p(S(S ph
p(S
reeph
le) her
(Sere
ere
ph eere
Mac
Ma
p here
Mac Macro
ros
Ma
ros sa
cro
croam
ammpple
sam
sa
plele)
p)) le)
mpl e) 10
14
15
15
14
10
20 /08
/08
/08 /20
/20 07
07
07

spectra and extract the required chemical information. respectively . Model R3 examined the entire spectra technical input and support made this study possible. Dr R. J. Singer and Dr S. Barton of Kingston
from 7500 to 5400cm -1. University for their academic support. My partner Jane, and my children Hermione and Hannah for
Vibrational energy levels for a diatomic their patience during all of my studies.
molecule C:\O P
C:\O
C:\O
C:\O
C:\O
C:\O
C:\O
C:\O
C:\O
C:\O
C:\O
C:\O
C:\O
C:\O
C:\O
C:\O
C:\O
P US
C:\O P
P US
P
P
P
P
P
P
P
P
P
P
P
P
P
P
US
US
US
US
US
US
US
US
US
US
US
US
US
US
5. 5\W
US 5.
5.
5.
5.
5.
5.
5.
5.
5.
5.
5.
5.
5.
5.
5\W O
5. 5\W
US 5.
5. 5\W
5\W
5\W
5\W
5\W
5\W
5\W
5\W
5\W
5\W
5\W
5\W
5\W
5\W
O RK
5\W O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
RK \NB
O RK
RK \NB
RK
RK
RK
RK
RK
RK
RK
RK
RK
RK
RK
RK
RK
RK
\NB
\NB
\NB
\NB
\NB
\NB
\NB
\NB
\NB
\NB
\NB
\NB
\NB
\NB
Tra ini
\NB Tra
Tra
Tra
Tra
Tra
Tra
Tra
Tra
Tra
Tra
Tra
Tra
Tra
Tra
ini ng
ini ng
Tra ini
\NB Tra
Tra ini
ini
ini
ini
ini
ini
ini
ini
ini
ini
ini
ini
ini
ini
ng
ng
ng
ng
ng
ng
ng
ng
ng
ng
ng
ng
ng
ng
\Pooza
ng \Pr
\Pr
ng \Pr
\Pr
\Pr
\Pr
\Pr
\Pr
\Pr
\Pr
\Pr
\Pr
\Pr
\Pr
\Pr
\Pr
za c.0
oza
\Pr oza
\Pr oza
oza
oza
oza
oza
oza
oza
oza
oza
oza
oza
oza
oza
cc2b
Drile
c2b
oza c2b
oza c2ben
c2b
cbl
cbl
cbl
cbl
cbl
cbl
cbl
cbl
cbl
cbl
cbl
cbl
cbl
en
en
en
en
en
en
en
en
en
ed.0
le
le
le
en
en
en
nd
nd
d_
d_
d_
d_
d_
d_
d_
d_
d_
d_
d_
d_
d_
P o za cP oz P
nd _9.0
_1.0
_2.0
_3.0
_4.0
_5.0
_6.0
nd _8.0
le nd _7.0
_10 .0
_11
_12
_13
_14
_15
_16
_Drie
8.2
8.3
8.4
8.5
8.6
8.7
8.8
8.9
8.10
9.0
9.1
10.0
10.1
11.0
11.1
12.0
12.1
13.0
13.1
14.0
14.1
15.0
15.1
16.0
16.1
A0
A0
.0d.0
_UnDri
1.0
1.1
2.0
2.1
3.0
3.1
4.0
4.1
5.0
5.1
6.0
6.1
7.0
7.1
8.0
8.1
51 45
51
ed. P
P
P
P
P
2_1 P
45 2_2
45 2_2
2_2
acroz
P
P
0 ro
P
P
P
ro
ro
ro
ro
ro
P
ro
P
P
P
.0
.1
.2
.3
.4
Dri
roz
Proz
roP za
za
za
za
za
za
za
roz
za
roz
roz
roz
.1roz
Inac
roPr
ro
tegr
ac
ac
za
cb
cb
cb
cb
cb
cb
cb
ac
ac
ac
ed2blatin
2bl
2bl
zaoza
c2b
c2b
Plen
len
roz
len
len
len
len
len
ac len
cb
ac ble
ble
bleP
endg_9
end
end
len
c2b
len
d_1
d_2
d_3
d_4
d_5
d_6
d_7
d_8
d_8
ble d_9
ble d_8
nd_ 12
nd_
nd_
nd_
Pnd_
8
SInte
_1
_2
_3
_4
_5
_6
_7
_8
d_1
acle
d_1
2bl
10
11
13
14
15
16
roz ac
roz ac
ac
p hergra
0_Drie
1
2
3
4
5
ndend
6

bl end
bl
e ting
_Un
Inte
Inte
Inte
Inte
Inte
Inte
Inte
end _A
end _A
(S p her
Inte
Inte
Inte
d gra
gra
gra
gra
InInte
Inte teg
gra
Inte
Inte
_AInte
S
Dried
gra
gra
gra
ph
grat
grat
grat
Inte
Inte
e ere
ting
ting
ting
ting
ting
ting
ting
ratin
ting
grat
grat
grat
grat
0 514
0 514
514 52
Ma cro
ing
ing
ing
grati
grati
InS
gS
ing
ing
ing
52ing
S
S
S
S
S
(Sph
S
S
S
ng
Steg
ng
ph
ph
ph
ph
ph
ph
ph
Sph
_1 S
_2
S
S
S
ph
ph
ph
S
sa
rati
S
ere
ere
ere
ere
ere
ere
ere
p ere
he
ph re
ph
ph
ph
ere
mp(S
ere
ere
ere
phe
Intphe
ng
egra
(S
(S
(S
(S
ere
ere
ere
ereIn
(S
(S
re
re
S
(S ph
(S
(S
(S
ph
ph
ph
ph
ph
ph
(Sph
(S
(S
(S
In(S
le)
Mac

teg
teg
ph ere
ph
ph
(S
pere
(S
tinhe
gphe
ere
ere
ere
ere
ere
ere
p ere
he
ph re
ph
ph
ph
ros am
ere
ere
phe
re
Mp(S
S
M
M
M
M
M
M
M
ere
ere
ere
ere
rati
rati
Mac
Mac
Mac
re
re
ac
ac
ac
ac
ac
ac
ac
Ma
ac
ngMac
ng
Mac
Mac
Mac
M
ple
pros
here Mhere
ros
ros
ros
ros
ros
ros
S phe
S
ac
) am
ros
ros
ros
ac
am
am
am
croros
ros
am ple
am
rosa
(Srosa
am
am
am
am
Ma
ph
sa mp
am
ros
ros
ros
phe
phe
ple
ple
ple
ple
ple
ple
ple
ple
am
am
am
ream
re
ple )))
ple
cro
le)
ple
ple
ple
(Sple
(S
mp sa
))mpMa
ere
)))
))) ))
le) mpl
le)

)) re
p he
p he
he
crose)

re MM acro
M
am ple )

acro s
acro s amp
s amp le)
amp le)
le)
25/ 08/2
20/
20/
20/
21/
10/
14/
10/
14/
10/
14/
10/
14/
22/
22/
22/
10/
10/
14/
10/
14/
10/
14/
10/
14/
22/
23/
22/
07/2 00
08/2
21/ 08/2
08/2
08/2
08/2
08/2
08/2
08/2
08/2
08/2
08/2
08/2
08/2
08/2
08/2
08/2
00 7
00 7
08/2 00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
C:\O
C:\O P
P US
US 5.
5. 5\W
5\W O
O RK
RK \NB
\NB Tra
Tra ini
ini ng
ng \Pr
\Pr oza
oza cbl
cbl en
en d_
d_ A0
A0 51
51 45 2_2 .5
.6
.7
.8
.9
.10
.11 P
P roz
roz
Pro ac
za bl
bl
cb end _A
len d_A
d_A 0
0 514
051 52
52
45 _2
_2
2_2 In
In teg
teg
Inte rati
rati
gra ng
ng
ting S
S phe
Sphe re
re
ph ere
ere (S
(S(S p
p he
ph re
re
ere M acro
Macro ac ro
ro s amp
sam le)
ple 22/
22/ 08/2 00 7
C:\O
C:\O
C:\O P
P US
US 5.
5. 5\W
5\W O
O RK
RK \NB
\NB Tra
Tra ini
ini ng
ng \Pr
\Pr oza
oza cbl
cbl en
en d_
d_ A0
A3 51
47 45
57 2_2
5_1 .12
.13
.14
.15
.0
.1
.2
.3
.4
.5 Pro
roz za
Ps.0 ac cb len
bl end
end _A 051
3cbl
475en45
75d_A 2_2
_1 3 47 575In Inte
In_1teg gra
rati ting
ng SS ph (S(S
re (S p ph
he ere MM
reatin ac sam
s her
amp ple
le) )) 23/
23/
22/ 08/2
08/2
08/2 00
00
00 7
7
7
C:\O P
C:\O
C:\O P US
P
P US 5.
US
US 5. 5\W
5.
5. 5\W O
5\W
5\W O RK
O
O RK \NB
RK
RK \NB Tra
\NB
\NB Tra ini
Tra
Tra ini ng
ini
ini ng \Pr
ng
ng \Pr oza
\Pr
\Pr oza cbl
oza
oza cbl en
cbl
cbl en d_
en
en d_ A3
d_
d_ A3 47
A3
A3 47 57
47
47 57 5_1
57
57 5_1 .7
5_1
5_1
.6
.8 min
.9
.10
10
20
20 min
min uteP
.0
.1
.2
.3
.4
in.0ute
.5
.6
roz
Pro ac
za bl
cb
Proz len
roend
zac _A
P rd_A
oza
ble 3 475
347
nd _A75
573d_A_1
5_1
475 75_ 12 0m teg
Inte 10rati
ingraminng
ting S
ute Sphe
Integ re
s ph ere
rati (S
ng p
In he
ph
tegrre
S ere
phe MM acro
reSgac Sro
(S ps
pp amp
sam
he ree le)
e ple
M(Sacr
ph
) ere
acr osa Ma
mp le )) 22/
cro 22/
sam
23/
23/
08/2
pl e)00
08/2
08/2 00 7
00 7
7
C:\O
C:\O P
P US
US 5.
5. 5\W
5\W O
O RK
RK \NB
\NB Tra
Tra ini
ini ng
ng \Pr
\Pr oza
oza cbl
cbl en
en d_
d_ A3
A3 47
47 57
57 5_1
5_2
5_5
5_7
5_9 2m
30
.0
0m
0m in.1
min
in.0
in.1
inu tes Ps.0
.1
.2 PP
.0roz ac
P ro
bl
roz Pzac
acb
Pro
ror_A
acb ble
len3nd
oza
len
zac d_
cbl
475
d_
ble _A
Aen
A 34
75
34
nd 3 _A475
757
_234
75734375_
5_1
47
5_2
5_5 12
2mIn
75 575
0m
75_ 0m
integ
_1
in
70
90
in
30
mirati
minngute
nute Inte
sShe
Inte Integ
grare
phe
sgra rati
ting
ting
In
ng
(Steg S
In
S S
ph
p ph
he
tegrphe
ere M((re
reatin
ere
rati ng acro
S
S
(S
gpheSp
phe
phe she
re
her
amp
re
re
re
M
M
(Sac
M
ac
le) ros
p(Shere
osa
amp
ph ere
ros
here
mp
MaMa
amp le
cro
le
le ))cro
sa14/23/
22/
sam
23/
22/
08/2
08/2 00
pl e)00
08/2
mpl08/2
e) 00
00 7
7
7
C:\O P US 5. 5\W O RK \NB Tra ini ng \Pr oza cbl en d_ A3 47 57 5_9 0m driinurotes .3
.5
.6 P zac ble nd _A 75 75_ 90 mi nute 08/2 7
C:\O
C:\O
C:\O P
P
P US
US
US 5.
5.
5. 5\W
5\W
5\W O
O
O RK
RK
RK \NB
\NB
\NB Tra
Tra
Tra ini
ini
ini ng
ng
ng \Pr
\Pr
\Pr oza
oza
oza cbl
cbl
cbl
c_1 en
en
en d_
d_
.0d_ A3
drie
Dr
Dr
Ori
und 47
d1
y_1
y_2P57
d2
gin
rie.0
al
.1
ro5_Un
.0.0
d.0za c_1 P
PPed_
ro
ro
P
P A .0
zac
zac
rroz
zac
oza ble
blegra
acb
ble
Inte
cblnd
nd_
le
nd nd
end P
_Dry
dri
_Dry
_O
tin
_u gro
ed
ndSzac
_1 1
2
rigi
_2 ble
l nd
naere
ried
ph _A
Integ
In
(SInteg
ph 34
teg 75
rati
ratin
Inrati
Int
ere tegr
egra 75_
ng
ng
Ma g SUnd
S
atin
tinS
croggphe
phe Spried
psam
She re
re
preher
here _A
pl(((Sesph
Sp
Sp
e) (S(S ere
hephreere
ph
re MMa
ere
M In
ac
acteg
cro
Ma
Ma ros
ros rati
sam
amp
cro
amp
cros
ng
plle
lee)
sam
am
S
ple
phe
))ple))
re (S p Ma cro sa 22/
mpl
23/
10/
15/
14/
10/
20/
e)
08/2
08/2 00
00
00 7
7
7