Quantitative Validation2010 QP PDF

Quantitative work in HPLC

Dr. Shulamit Levin

LAYOUT
Standards Considerations
Integration events: Explained and demonstrated
Running sets of samples
Blanks Carry over and system peaks
System Suitability
Calibration and Quantitation
Parameters effecting quantitation and validation
Requirements from chromatographic data systems

System Suitability
REFERENCE STANDARDS:
Standard Information
Standards in the Industry
Primary Standard
Primary Standard
Materials which are accepted without reference to other standards.
If the materials have undergone complete analytical characterization, their identity
must be proven (elucidation of chemical structure) and their purity must be sufficiently
high and stated (>99.0%).
The characterization of primary standards generally involves the following:
Elucidation of chemical structure by IR, UV, H-NMR, C-NMR, MS, CD etc.
Purity determination by HPLC,TLC,GC,GPC,DSC,residue of ignition, water content etc.
Assay: Titration,DSC,Chromatography.
It is acceptable that the manufacturing process of primary references standards
differs from the final processing of the drug substance.
Pharmacopeial Standards
Commonly used for certain tests and assayed to achieve accuracy and precision of analytical results required in
compendia monographs. Pharmacopeial standards are basically regarded as primary standards.
Working Standard
Working Standards
Materials are designed for daily use in instrumental analysis such
as routine quality control.
They are characterized by comparison with Primary or
Pharmacopoeia standards. Their purity corresponds to a "typical
batch".
Information Supporting The Integrity of The

Reference Standard
Tests to characterize the reference standard are different and more
extensive than those used to control the new drug substance as to
Identity, Strength, Quality, and Purity
Physical description
Physical constants (RI, pK, BP, MP, etc.)
Chemical Attributes (Formula, weight)
Appropriate analytical tests used to ID
Data establishing purity

System Suitability
Integration
Measurement of Peak Area - Integration

Area =
End
Abs x dt
Start
Detector
Signal
Time
Measurement of Area:
Peak Integration
Data Bunching
Peak Start
Peak Apex
Peak End
Peak Detection Integration Events

Peak Apex
Peak Height
Constructed
Baseline
Start of
chromatogram
Peak End
Peak Start
Retention Time
Influence of Poor Peak Shape on Integration

(Peak Area)
Tailing Factor = 1.00

Recovered Peak Areas
99.9 %
99.8 %
99.6 %

97.8 %
95.3 %
92.3 %
Peak Integration: Data Bunching
15 = Minimum Number of points to define a peak
B = (WxS)/15
Bunching
B = BUNCHING FACTOR
W=PEAK WIDTH
S = SAMPLING RATE
Data
points
Peak Integration - Peak Start
Slope 2 =
(B3-B2)/(t3-t2)
Slope 1 =
(B2-B1)/(t2-t1)
B3
Average
Slope
B2
B1
Threshold value
Peak Integration - Peak End
Slope 2 =
(B3-B2)/(t3-t2)
Average
Slope
Slope 1 =
(B2-B1)/(t2-t1)
B3
B2
Threshold value
B1
(Touchdown)
Peak Integration - Peak Apex
Slope 1 =
(B2-B1)/(t2-t1)
Slope 2 =
(B3-B2)/(t3-t2)
Proper Integration Events are Required
10
Set Peak Width

Peak Width = 30
0 .0 5
98.972
98.972
Peak Width = 120

1 0 0 .0 0
0.05
0 .0 4
100.00
0.04
8 0 .0 0
80.00
4 0 .0 0
60.00
0.02
AU
AU
0 .0 1
% Compos ition
6 0 .0 0
0 .0 2
0.01
40.00
% Compos ition
0.03
0 .0 3
0.00
0 .0 0
- 0 .0 1
-0.01
2 0 .0 0
20.00
-0.02
- 0 .0 2
0.00
0 .0 0
-0.03
- 0 .0 3
9 8 .0 0
9 9 .0 0
1 0 0 .0 0
1 0 1 .0 0
98.00
1 0 2 .0 0
99.00
100.00
101.00
102.00
Minutes
Min u te s
Peak Threshold
Peak Threshold=300
Peak Threshold=75
100.00
100.00
0.015
0.015
80.00
80.00
0.010
0.000
40.00
0.005
60.00
AU
% Composition
AU
60.00
0.000
40.00
-0.005
-0.005
20.00
20.00
-0.010
-0.010
-0.015
-0.015
0.00
70.00
71.00
72.00
73.00
0.00
70.00
74.00
71.00
72.00
Minutes
Minutes
11
73.00
74.00
% Composition
0.005
71.153
71.152
0.010
47.893
0 .0 3
45.253
43.741
W hen a riding peak needs to be skimmed from the slop

there are two options, exponential or tangential skim:
0 .0 2
AU
0 .0 1
0 .0 0
- 0 .0 1
- 0 .0 2
4 2 .0 0
4 4 .0 0
4 6 .0 0
4 8 .0 0
5 0 .0 0
M in u t e s
0 .0 5
47.893
1 0 0 .0 0
0 .0 4
8 0 .0 0
45.253
0 .0 2
0 .0 3
6 0 .0 0
0 .0 2
AU
AU
0 .0 1
0 .0 1
4 0 .0 0
0 .0 0
0 .0 0
- 0 .0 1
- 0 .0 1
2 0 .0 0
- 0 .0 2
- 0 .0 2
- 0 .0 3
4 2 .0 0
4 4 .0 0
4 6 .0 0
M in u t e s
4 8 .0 0
0 .0 0
4 2 .0 0
5 0 .0 0
4 4 .0 0
4 6 .0 0
4 8 .0 0
5 0. 0 0
M in u t e s
Force Baseline by Time

& Force Baseline by Peak
A gradient chromatogram, where the baseline is drifting:
1 00.0 0
57.786
0.0 05
52.316
52.316
0 .00 5
57.786
Event start and end times (Force Baseline by Time)

Peak start and end points within this range (Force Baseline by Peak)
80.00
8 0.00
4 0.00
60.00
AU
% Composition
6 0.00
-0 .01 0
-0.0 10
40.00
-0.0 15
-0 .01 5
-0 .02 0
-0 .02 5
50 .00
52.00
54.00
Minu tes
5 6.00
2 0.00
-0.0 20
0 .00
-0.0 25
20.00
0.00
50.00
5 8.0 0
12
52.00
54.00
Minutes
56.00
58.00
% Composition
-0.0 05
-0 .00 5
AU
100.00
0.0 00
0 .00 0
% Composition
47.893
45.253
43.741
0 .0 3
43.741
Tangential Skim
Exponential Skim
Force Drop line

Originally
Force Drop line

0.05
0.04
0.04
0.03
0.02
0.02
AU
AU
0.03
31.163
31.453
31.453
0.05
0.01
0.01
0.00
0.00
-0.01
-0.01
-0.02
-0.02
30.50
31.00
31.50
32.00
32.50
30.50
33.00
31.00
31.50
32.00
32.50
33.00
Minutes
Minutes
Valey to Valey
Original
Valey to Valey
0.18
0.18
0.12
80.00
0.10
0.10
60.00
0.06
% Composition
0.08
62.087
0.08
AU
62.087
% Composition
60.00
0.06
40.00
0.04
66.548
0.04
66.548
40.00
61.419
AU
67.321
80.00
65.016
0.14
67.321
65.016
62.773
0.12
62.773
0.16
0.14
0.02
100.00
100.00
0.16
0.02
20.00
20.00
0.00
0.00
-0.02
-0.02
0.00
0.00
62.00
62.00
64.00
66.00
64.00
66.00
68.00
Minutes
Minutes
13
68.00
Asymmetric factor or Tailing factor

Symmetric
Asymmetric
5 % of Peak Height
Sources of Errors in Integration of

Asymmetric Peaks
Slope 2 =
Slope 2 =
(B3-B2)/(t3-t2)
(B3-B2)/(t3-t2)
Slope 1 =
Slope 1 =
(B2-B1)/(t2-t1)
(B2-B1)/(t2-t1)
B3
Average
Average
B3
Slope
Slope
B2
B2
B1
Threshold value
Threshold value
14
B1
Integration of Small Peaks

PW=60
Area=25660
PW=15
Area=23011
-10%
AUFS = 0.003
Peak width changed
Threshold set at 30
3.80
4.20
Minutes
4.60
3.80
4.20
Minutes
4.60
Thresh.= 20
Area=25660
Thresh.=300
Area=23087
AUFS = 0.003
-10%
Peak width set at 30 sec
Threshold changed
3.80
4.20
Minutes
4.60
3.80
4.20
Minutes
4.60

System Suitability
15
Running Sets of Samples in

Regulated Environment
Basic Sequence in HPLC

1.
2.
3.
4.
5.
6.
3.
2.
4.
5.
1.
16
6.
Login.
Instrument Method.
Sequence/Run Samples.
Process Data.
Review/Preview.
Result/Sign-Off.
FDA Guidance
Table 2. Holistic Validation of Computerized LC Systems
_________________________________________________________________
Initial Calibration--Linearity
Use at least 4 standard solutions.
Concentration range of standards must span anticipated results, plus safety margin.
Run standards daily before starting sample analysis.
Initial Calibration--System Precision
Calculate precision daily from at least 6 replicate injections of a standard solution
before starting sample analysis.
Running Calibration
Run a standard solution at specified time intervals or after a specified number of
sample solutions.
Data Processor (Integrator, Computer(
Are the macros coded correctly? Validated? Documented?

System Documentation
Is System Documentation in Place?

System Suitability
17
Quantitative Set of Samples- Running Blanks

VIAL SAMPLE NAME
1
2
INJ VOL
No of Inj
Function
Method
Run Time Sample

Weight
Blank
20.0
Inject Samples
System
Suitability
20.0
Inject Samples
Std1
20.0
Clear
Calibration
5 Inject Standards
LC Demo Method Set
3
4
Std2
20.0
1 Inject Standards
Dilution
LC Demo Method Set
10.00
1.00000
1.00000
SST Method Set
10.00
1.00000
1.00000
LC Demo Method Set
10.00
1.00000
1.00000
LC Demo Method Set
10.00
1.00000
1.00000
Report
LC Calibration Report
Report
Standard Comparison
Std1
20.0
Clear
Calibration
1 Inject Standards
LC Demo Method Set
10.00
1.00000
1.00000
Unk.1
20.0
Inject Samples
LC Demo Method Set
10.00
1.00000
1.00000
Unk.2
20.0
Inject Samples
LC Demo Method Set
10.00
1.00000
1.00000
Unk.3
20.0
Inject Samples
LC Demo Method Set
10.00
1.00000
1.00000
Unk.4
20.0
Inject Samples
LC Demo Method Set
10.00
1.00000
1.00000
Unk.5
20.0
Inject Samples
LC Demo Method Set
10.00
1.00000
1.00000
Unk.6
20.0
Inject Samples
LC Demo Method Set
10.00
1.00000
1.00000
Std1
20.0
1 Inject Standards
LC Demo Method Set
10.00
1.00000
1.00000
1
2
Clear
Calibration
Calibrate
LC Demo Method Set
LC Demo Method Set

LC Demo Method Set
Sample vs. Blank (Diluent)

Unexpected peaks can appear in blanks and in all following injections
In this case they are excluded from the processing of the sample
50.00
40.00
Blank
mAU
30.00
20.00
? ?
10.00
0.00
50.00
40.00
Sample
mAU
30.00
20.00
10.00
0.00
0.00
10.00
20.00
30.00
Minutes
18
40.00
50.00
INJECTION OF PURE SOLVENT:

Why would there be peaks?
System Peak ???
Injection
Carry over ???

Contamination ???
System Peaks
Legitmate System Peaks
Originate from the Mobile Phase Components Going
Through Re-Equilibration
19
CONDITIONS FOR APPEARANCE OF REAL SYSTEM

PEAKS
Mobile phase is multi-component (n=>2)

Mobile phase contains adsorbable components
Mobile phases components respond to the detector
(high background)
Sample or sample-diluent is different from the
mobile phase, enough to create equilibrium perturbation.
Mechanism of System Peaks Formation

Vacancy
EXAMPLE:
Two additives in the mobile phase
Example: k(1
k(1) = 1
k' =
tR - t 0
k =
t0
Cs
Example: k(2
k(2) = 2
Step 1:
Step 1:
Equilibrium:
Cs=1
Cs=
1; Cm = 1
Equilibrium:
Cs=2
Cs=
2; Cm = 1
Step 2:
Injection of Vacancy:
Cs=1
Cs=
1; Cm=0
Cm=0
Cm
Step 2:
Injection of Vacancy:
Cs=2
Cs=
2; Cm=0
Cm=0
Step 3:
Re
Re--equilibration:
Cs=0
Cs=
0.5; Cm=
Cm=0
0 .5
CHROMATOGRAM
Step 3:
Re
Re--equilibration:
Cs=1
Cs=
1.33
33;; Cm=
Cm=0
0.67
t0
k=1
20
k=2
An Example for Real System Peaks in Gradients with Trifluoroacetic Acid (TFA) in the Mobile
Phase
Peptide Peak
Sample=Peptide
Blank = Mobile phase
Dissolved in Water
TFA
Blank = Mobile phase
Zoomed
ACN
Blank =Water
System peaks appear because diluent does not contain TFA
Contaminations Peaks - Not System Peaks!

Phosphate Buffer and Acetonitril Do Not Produce Such System Peaks!
0.006
0.005
Mobile phase: ACN : Phosphate Buffer pH 9.5 1:1

Wavelength: 280 nm
3.404
0.004
AU
0.003
0.000
6.477
4.274
2.373
2.731
0.001
18.274
0.002
-0.001
Sample Name: Diluent
-0.002
-0.003
2.00
4.00
6.00
8.00
10.00
12.00
14.00
Minutes
21
16.00
18.00
20.00
22.00
24.00
26.00
28.00
30.00
Use of Diode-Array Detector for Troubleshooting of Contamination Peaks in

Multicomponent Mobile Phase
Chromatogram and Peaks' UV-VIS Spectra
Cpd 2 - 2.188
250.00 nm
250.00
2.748
nm
300.00
250.00
2.867
nm
300.00
250.00
3.180
nm
300.00
250.00
4.226
nm
300.00
300.00
Aromatic (not in
mobile phase)
Negative UV
spectrum
Cpd 2 - 2.188
0.000
1.00
1.20
1.40
1.60
1.80
2.00
2.20
2.40
2.60
2.80
3.00
3.20
4.226
0.005
Wavelength: 254.0 nm
2.748
2.867
AU
0.010
3.180
0.015
3.40
3.60
3.80
4.00
4.20
4.40
4.60
Minutes
Suggested FlowFlow-Chart for Troubleshooting

In a Regulated Environment (no change in method!)
Injection
System Peak ???

Carry over ???
Contamination ???
Yes?
No
Mobile phase:
Multicomponent?
Adsorbed components (ion pair)?
Response in detector (background)?
Yes?
Legitimate System Peaks

Dissolve samples in mobile phase
Adjust diluent with mobile phase components
22
4.80
5.00
5.20
5.40
5.60
5.80
6.00
Suggested FlowFlow-Chart for Troubleshooting Contd.

Steps: by ease of use
1. Review and revise diluents composition
2. Replace vials batch and/or brand
3. Replace in-line filters
4. Replace solvents batch and/or brand
5. Try a new/fresh column
6. Wash system, especially injector
Not System Peaks?

Not washed by
blanks?
Make sure to try a fresh blank each test!
Peaks still persist?

Replace injectors surfaces
Peaks still persist?
Try another instrument/Operator/Lab
Many times the extraneous peak

disappears on its own and
remains an unsolved mystery
23

System Suitability
Quality Control Testing System Suitability

VIAL SAMPLE NAME INJ VOL
1
No of Inj
Function
Method
Run Time Sample

Weight
Dilution
Blank
20.0
1 Inject Samples
LC Demo Method Set
10.00
1.00000
1.00000
2 System
Suitability
20.0
1 Inject Samples
SST Method Set
10.00
1.00000
1.00000
LC Demo Method Set

LC Demo Method Set
10.00
1.00000
1.00000
LC Demo Method Set
10.00
1.00000
1.00000
Std1
20.0
Clear
Calibration
5 Inject Standards
Std2
20.0
1 Inject Standards
Report
Report
Standard Comparison
Std1
20.0
Clear
Calibration
1 Inject Standards
LC Demo Method Set
10.00
1.00000
1.00000
Unk.1
20.0
2 Inject Samples
LC Demo Method Set
10.00
1.00000
1.00000
Unk.2
20.0
2 Inject Samples
LC Demo Method Set
10.00
1.00000
1.00000
Unk.3
20.0
2 Inject Samples
LC Demo Method Set
10.00
1.00000
1.00000
Unk.4
20.0
2 Inject Samples
LC Demo Method Set
10.00
1.00000
1.00000
Unk.5
20.0
2 Inject Samples
LC Demo Method Set
10.00
1.00000
1.00000
Unk.6
20.0
2 Inject Samples
LC Demo Method Set
10.00
1.00000
1.00000
Std1
20.0
1 Inject Standards
LC Demo Method Set
10.00
1.00000
1.00000
Clear
Calibration
Calibrate
24
LC Demo Method Set
LC Demo Method Set

LC Demo Method Set
System Suitability
Sample Name:
Sample Type:
Vial:
Injection #:
Injection Volume:
Run Time:
AntiOX 10 2d
Unknown
2:A,2
1
2.00 ul
2.0 Minutes
Acquired By:
Sample Set Name:
Acq. Method Set:
Processing Method:
Channel Name:
Proc. Chnl. Descr.:
Date Acquired:
Date Processed:
19/10/2009 16:15:30 IST

14/12/2009 22:39:44 IST
System
Antiox_sampling_rate rsd 2D
Restech_Std_UPLC_10_2d_01
Antioxidants_UPLC 220nm
PDA Ch1 220nm@4.8nm
PDA Ch1 220nm@4.8nm
0.02
IRGAFOS 168 - 1.299
0.04
IRGANOX 1076 - 0.966
AU
0.06
Peak Results
Name
VIT E - 0.731
BHT - 0.193
0.08
ENICAMIDE - 0.303
0.10
IRGANOX 1010 - 0.552
Auto-Scaled Chromatogram
RT
EP USP
USP USP
Area Height Plate Plate
Alfa
Res Tailing
Count Count
1 BHT
0.193
97392 106748
1028
948
2 ENICAMIDE
0.303
88614
74300
1379
1419
3.84
1.34 3.391
4 IRGANOX1010 0.552
77254
44092
2229
2287
6.37
1.21 2.603
5 VIT E
59739
37644
4872
4754
4.03
1.18 1.441
6 IRGANOX1076 0.966 131707
64671
5171
5098
4.87
1.14 1.402
7 IRGAFOS 168
18010
6640
6561
5.64
1.09 1.407
3 IRGANOX3114 0.459
0.731
1.299
43447
0.00
0.00
0.20
0.40
0.60
0.80
Minutes
1.00
1.20
1.46
1.40
Reported by User: System

Report Method: SingleSST
Report Method ID: 6108
6108
Project Name:
Test_Sampling_Rate
Date Printed:
16/04/2010
20:48:52 Asia/Tel_Aviv
PEAK BROADENING
25
System Suitability
is
Based on the Theory of
Chromatography
The Chromatographic Process

B+A
Mobile phase
Stationary
Phase
Abs
A
Area A = 300,000
Area B = 100,000
B - 94.776
K = C s/C m
AU
0.34
0.32
0.30
0.28
0.26
0.24
0.22
0.20
0.18
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
-0.02
A - 93.448
Chromatogram
Distribution:
90.50 91.00 91.50 92.00 92.50 93.00 93.50 94.00 94.50 95.00 95.50 96.00
Elution through the Column-movie
Minutes
Time
26
Mixed--Mode Retention:
Mixed
Ion exchange Interaction
with Charged Sites
Hydrophobic Interaction with

Bonded Phase
Mobile
Phase pH < 3
Si - OH
O- Si
O- Si
OH
O- Si
O- Si
OH
O- Si
OH
O- Si
O- Si
O- Si
OH
O- Si
O- Si
+
HN (CH 3 )
Base
O- Si
O- Si
OO- Si
O- Si
+
O - (CH ) HN
3 2
O- Si
OO- Si
O- Si
O- Si
OO-- Si
O- Si
Mobile
Phase pH > 3
Si O
Base
Influence of Poor Peak Shape on Integration

(Peak Area)

99.9 %
99.8 %
99.6 %
27

97.8 %
95.3 %
92.3 %
System Suitability
System Suitability
The checking of a system, before or during analysis of unknowns, to insure system
performance.
No sample analysis is acceptable unless the requirements for system suitability have
been met. (USP Chapter 621)
Plate Count, Tailing, Resolution
Determination of reproducibility (%RSD)
For %RSD < 2.0%, Five replicates
For %RSD > 2.0%, Six replicates
System Suitability "Sample"
A mixture of main components and expected by-products utilized to determine system
suitability
Whenever There is a Significant change in Equipment or Reagents, System Suitability
Testing Should be Performed (USP Chapter 621)
k' = Capacity Factor = Measure of Retention

t2
t1
t3
t0
TIME
.5
5
k' t1 = 1-0.5= 1
0.5
k' t1 = t1 - t0
t0
K' t2 = 2-0.5= 3
0.5
k' t3 = 5-0.5= 9
0.5
28
Standard Deviations and Areas Under the

Normal Curve
For any normal curve with mean mu () and

standard deviation sigma ():
68 percent of the observations fall within
1 standard deviation of the mean.
95 percent of observation fall within 2
standard deviations.
99.7 percent of observations fall within 3
standard deviations of the mean.
Measuring Peak Widths
29
Efficiency method Vs. Peak width
Application
BY ONE PEAK
PERFORMANCE CRITERIA
RETENTION FACTOR or CAPACITY RATIO

ASYMMETRY FACTOR
k' =
tR - t 0
k =
t0
Af =
B(10% h)
(10%
h)
TAILING FACTOR
0.020
Area:
USP Resolution:
USP Tailing:
K Prime:
Retention Time:
height
0.015
AU
Cs
Cm
28501
4.48
1.11
4.02
0.74
Tf =
(5% h)
A+B
2A
Width (50%)
0.010
0.005
10%h
A
0.000
Width(base)
0.710
0.720
0.730
0.740
0.750
0.760
0.770
Minutes
30
NUMBER OF THEORETICAL PLATES
N = 16 (
tR
w
PERFORMANCE BY TWO PEAKS

SELECTIVITY FACTOR
k' ( 2 )
k' (1)
tR(1)
tR(2)
EXPERIMENTAL RESOLUTION
Rs =
t R (2)
1/2
-t
(w
R(1
+w
)
2
t0
w1
w2
PERFORMANCE BY TWO PEAKS

EXPERIMENTAL RESOLUTION
Rs =
t R (2)
1/2
(w
-t
1
R(1 )
+w
2)
tR(2)
tR(1)
t0
w1
31
w2
Separation Development Sequence

See interactively
FIRST INJECTION
ADJUST k' (Retention)
ADJUST N (Plates)
ADJUST alpha (Separation)
Higher Components ratio Higher Resolution Required
100:1
10:1
Dolan
32
The importance of efficient chromatography

Higher Sensitivity
Separation between related compounds
Higher peak capacity
Shorter run times
Solvents and cost saving
UPLC
HPLC
SELECTIVITY vs EFFICIENCY
Rs =
t R (2) - t R(1)
Same in both
1/2 (w 1 + w 2) cases
LOW SELECTIVITY (
)
HIGH EFFICIENCY (N)
HIGH SELECTIVITY (
)
LOW EFFICIENCY (N)
33
SELECTIVITY vs EFFICIENCY
Rs =
t R (2) - t R(1)
same in both cases
1/2 (w 1 + w 2)
HIGH SELECTIVITY (
)
LOW EFFICIENCY (N)
LOW SELECTIVITY (
)
HIGH EFFICIENCY (N)
Fast Gradient Application

Columns:
XTerra MS C18 2.1 x 20 mm, 2.5 m;

XTerra MS C18 2.1 x 50 mm, 5 m
Mobile Phase: A = 0.1% TFA in water,
B = 0.08% TFA in MeCN
Gradient: 5 - 95% B in 45 seconds
and 120 seconds
Column Temperature: 60 C
Flow Rate: 1.5 mL/min.
Detector: 254 nm
Injection Volume: 1 L
0.50
0.40
0.30
AU
2.5 m
2.1 x 20 mm
HPLC
0.20
0.10
0.00
0.20
0.80
0.60
AU 0.40
0.40
0.60
Minutes
0.80
1.00
1.20
1.40
1.20
1.40
5 m
2.1 x 50 mm
0.20
0.00
0.70
0.50
1.80
1.60
2.00
Fast Isocratic Application

UPLC
Method:
0.40
83::17 Buffer phosphate pH

83
pH4
4
Flow: 0.4 ml/min

Temp: 30
30C
C
Column Acquity 2.1x100 mm
280 nm: 40 pt/sec
1.548
0.10
1.231
0.20
1.091
1.146
0.30
1.002
AU
0.60
1.7 m
2.1 x 100 mm
UPLC
1.00
Minutes
4 - 1.652
0.90
0.80
0.80
2 - 0.803
0.60
3 - 0.901
0.40
1 - 0.707
0.20
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
Minutes
34
1.80
2.00
Recommendations Parameters:
System Suitability
Capacity factor
k' > 2
Precision/Injection repeatability
RSD </= 1%, n >/= 5
Resolution
Rs >/= 2 (Major peak and
closest eluting)
Tailing factor
T </= 2
Theoretical Plates
In general N > 2000

System Suitability
35
Quality Control Test of Precision and Calibration

VIAL SAMPLE NAME
1
2
INJ VOL
No of Inj
Function
Method
Run Time Sample

Weight
Blank
20.0
Inject Samples
System
Suitability
20.0
Inject Samples
Std1
20.0
Clear
Calibration
5 Inject Standards
LC Demo Method Set
3
4
Std2
20.0
1 Inject Standards
Dilution
LC Demo Method Set
10.00
1.00000
1.00000
SST Method Set
10.00
1.00000
1.00000
LC Demo Method Set
10.00
1.00000
1.00000
LC Demo Method Set
10.00
1.00000
1.00000
Report
Report
Standard Comparison
Std1
20.0
Clear
Calibration
1 Inject Standards
LC Demo Method Set
10.00
1.00000
1.00000
Unk.1
20.0
Inject Samples
LC Demo Method Set
10.00
1.00000
1.00000
Unk.2
20.0
Inject Samples
LC Demo Method Set
10.00
1.00000
1.00000
Unk.3
20.0
Inject Samples
LC Demo Method Set
10.00
1.00000
1.00000
Unk.4
20.0
Inject Samples
LC Demo Method Set
10.00
1.00000
1.00000
Unk.5
20.0
Inject Samples
LC Demo Method Set
10.00
1.00000
1.00000
Unk.6
20.0
Inject Samples
LC Demo Method Set
10.00
1.00000
1.00000
Std1
20.0
1 Inject Standards
LC Demo Method Set
10.00
1.00000
1.00000
1
2
Clear
Calibration
Calibrate
LC Demo Method Set
LC Demo Method Set

LC Demo Method Set
Summary Report
Sample Set: PrednisoneNoRecircNoRepl
General Information
Sample Set Name
Sample Set Method
Sample Set Start Date
Sample Set Finish Date
Sample Set Id
Sample Set Altered
SampleName
PrednisoneNoRecircNoRepl
27/10/1999 12:36:43
27/10/1999 15:37:29
2090
No
Sample Type
Vial
Run Time
(Minutes)
Inj #
Injection
Volume
(ul)
Acquistion
Method Set
Sample
Weight
Dilution
Label
Prednisone_Tab
Unknown
11
4.00
20.00
Prednisone Mth Set
1.00000
1.00000
AU0203
Prednisone_Tab
Unknown
12
4.00
20.00
Prednisone Mth Set
1.00000
1.00000
AU0204
Prednisone_Tab
Unknown
14
4.00
20.00
Prednisone Mth Set
1.00000
1.00000
AU0206
Prednisone_Tab
Unknown
16
4.00
20.00
Prednisone Mth Set
1.00000
1.00000
AU0208
Area
Calibration Plot
1.0x10
5.0x10
0.0
0.000
0.002
0.004
0.006
0.008
0.010
Amount
0.012
0.014
Name: Prednisone; Processing Method: Prednisone Proc Mth; Fit Type: Linear (1st
Order); Cal Curve Id: 2973; A: 0.000000e+000; B: 4.867524e+007; C:
0.000000e+000; D: 0.000000e+000; R^2: 1.000000
36
0.016
0.018
0.020
Assay
Calibration Plot
OR:
Area
6
1.0x10
5
5.0x10
0.0
0.0000.0020.0040.0060.0080.0100.0120.0140.0160.0180.020
Related Compounds
MAIN
37
= W Std/AStd
Working Curve
A plot
of
response)
the
analytical
as a function of
signal
(the
analyte
concentration,
instrument
or
detector
using a series
of standards of known concentration.
The working curves are then used to determine

an unknown
sample or to
calibrate
the concentration
of
the linearity of an analytical
instrument.
Choice of Standardization:
External or Internal
External Standard
Response Std
Amount Std
Amount Unk
Response Unk
Amount Std
Amount Unk =
Response Std
Response
Std
Amount
Std
38
Response Unk
Choice of Standardization
Simple formulations and sample
preparation: external standard
Gas chromatography, bio-studies

or complex medium and complex
sample preparation:
internal standard
Choice of Standardization:
Internal Standard
C
Int. Std.
Amount Std
Amount Istd
Response Std
Response Istd
Amount Unk
Response Unk
Response Istd
Amount Istd
39
Internal Standard
12,000/6000
11,000/5800
100/10
Amt B/10
B
Int.
Std.
Int.
Std.
100
10
11,000
10
12,000
5,800
Unknown
6,000
Standard
(11,000/5,800) x (100/10)
----------------------------------- x 10 = Amt B =
12,000/6000
94.8
Standard Addition
Due to matrix effects the analytical response for an
analyte in a complex sample may not be the same as
for the analyte in a simple standard.
Signal
Conc.
40
Summary Report
General Information
Sample Set Name
Sample Set Method
Sample Set Id
Sample Set Altered
SampleName
27/10/1999 12:36:43
27/10/1999 15:37:29
2090
No
Sample Type
Vial
Run Time
(Minutes)
Inj #
Injection
Volume
(ul)
Acquistion
Method Set
Sample
Weight
Dilution
Label
Prednisone_Tab
Unknown
11
4.00
20.00
Prednisone Mth Set
1.00000
1.00000
AU0203
Prednisone_Tab
Unknown
12
4.00
20.00
Prednisone Mth Set
1.00000
1.00000
AU0204
Prednisone_Tab
Unknown
14
4.00
20.00
Prednisone Mth Set
1.00000
1.00000
AU0206
Prednisone_Tab
Unknown
16
4.00
20.00
Prednisone Mth Set
1.00000
1.00000
AU0208
Area
Calibration Plot
1.0x10
5.0x10
0.0
0.000
0.002
0.004
0.006
0.008
0.010
0.012
0.014
0.016
0.018
0.020
Amount
0.000000e+000; D: 0.000000e+000; R^2: 1.000000
Standard Deviation
A measure of the uncertainty due to random
error in a set of data (also: precision of a set of
measurements).
s
N
x
x
= standard deviation,
= the num ber of data points,
i = each individual m easurem ent,
bar = the mean of all m easurem ents.
The value x i - x bar is called the residual

for each measurement.
41
Quality Control Test of Accuracy

VIAL
1
SAMPLE NAME
INJ VOL
No of Inj
Function
Blank
20.0
Inject Samples
2 System
Suitability
20.0
Inject Samples
Std1
20.0
Clear
Calibration
5 Inject Standards
Std2
20.0
1 Inject Standards
Method
Run Time
LC Demo Method Set
Sample
Weight
1.00000
Dilution
10.00
SST Method Set
10.00
1.00000
1.00000
LC Demo Method Set
10.00
1.00000
1.00000
LC Demo Method Set
10.00
1.00000
1.00000
1.00000
LC Demo Method Set
Report
Report
Standard Comparison
Std1
20.0
Clear
Calibration
1 Inject Standards
LC Demo Method Set
10.00
1.00000
1.00000
Unk.1
20.0
Inject Samples
LC Demo Method Set
10.00
1.00000
1.00000
Unk.2
20.0
Inject Samples
LC Demo Method Set
10.00
1.00000
1.00000
Unk.3
20.0
Inject Samples
LC Demo Method Set
10.00
1.00000
1.00000
Unk.4
20.0
Inject Samples
LC Demo Method Set
10.00
1.00000
1.00000
Unk.5
20.0
Inject Samples
LC Demo Method Set
10.00
1.00000
1.00000
Unk.6
20.0
Inject Samples
LC Demo Method Set
10.00
1.00000
1.00000
Std1
20.0
1 Inject Standards
LC Demo Method Set
10.00
1.00000
1.00000
1
2
Clear
Calibration
Calibrate
LC Demo Method Set
LC Demo Method Set

LC Demo Method Set
Control Std2
Std2 (Accuracy/Recovery Test)
42
Standards_Comparison
Processing Method Id 2636
Standards Summary Table

Name : methyl paraben
1
2
3
4
5
6
Mean
% RSD
SampleName
std-1
std-1
std-1
std-1
std-1
std-1
Label
S1
S1
S1
S1
S1
S1
Name
methyl paraben
methyl paraben
methyl paraben
methyl paraben
methyl paraben
methyl paraben
Vial Inj
RT
2
1 2.775
2
2 2.775
2
3 2.777
2
4 2.775
2
5 2.773
2
6 2.775
Area
7928853
7926182
7973699
7993516
7993709
8004153
Amount Area_Amount
44.429
178461
44.429
178401
44.429
179471
44.429
179917
44.429
179921
44.429
180156
179388
0.43
Control Summary Table

Name : methyl paraben
1
2
Mean
SampleName Label
std-2
S2
std-2
S2
Name
methyl paraben
methyl paraben
Vial Inj
RT
1
1 2.783
1
2 2.779
Area
7858421
7907563
Amount Control Value Control_Check

43.807
44.257
101.03
44.081
44.257
100.40
100.7
Standards Summary Table

Name : propyl paraben
1
2
3
4
5
6
Mean
% RSD
SampleName
std-1
std-1
std-1
std-1
std-1
std-1
Label
S1
S1
S1
S1
S1
S1
Name
propyl paraben
propyl paraben
propyl paraben
propyl paraben
propyl paraben
propyl paraben
Vial Inj
RT
2
1 9.067
2
2 9.068
2
3 9.066
2
4 9.064
2
5 9.053
2
6 9.058
Area
670840
670683
673923
678792
676660
678451
Amount Area_Amount
45.014
14903
45.014
14899
45.014
14971
45.014
15080
45.014
15032
45.014
15072
14993
0.54
Control Summary Table

Name : propyl paraben
1
2
Mean
SampleName Label
std-2
S2
std-2
S2
Name
propyl paraben
propyl paraben
Vial Inj
RT
1
1 9.098
1
2 9.075
Area
670235
673646
Amount Control Value

44.703
45.026
44.931
45.026

Report Method: Standards_Comparison_Multi
Report Method ID: 4920
Project Name:
Control_Check
100.72
100.21
100.5
Pharma_QC_calculations_Trima
Date Printed:
16/04/2010
Determination of a Control Standard:
Test of Accuracy
Response Std
Amount Std
Amount Control
Response Unk
Amount Std
Amount Control =
Response Std
x Response Unk
Amount Control Determined
=0.99-1.01
Accuracy:
Amount Control Prepared
43
Running the
Unknowns
Quality Control - Quantification

VIAL SAMPLE NAME INJ VOL
1
No of Inj
Function
Method
Run Time Sample

Weight
Dilution
Blank
20.0
Inject Samples
LC Demo Method Set
10.00
1.00000
1.00000
2 System
Suitability
20.0
Inject Samples
SST Method Set
10.00
1.00000
1.00000
LC Demo Method Set
10.00
1.00000
1.00000
LC Demo Method Set
10.00
1.00000
1.00000
Std1
20.0
Clear
Calibration
5 Inject Standards
Std2
20.0
1 Inject Standards
LC Demo Method Set
Report
Report
Standard Comparison
Clear
Calibration
LC Demo Method Set
Unk.1
20.0
Inject Samples
LC Demo Method Set
10.00
1.00000
1.00000
Unk.2
20.0
Inject Samples
LC Demo Method Set
10.00
1.00000
1.00000
Unk.3
20.0
Inject Samples
LC Demo Method Set
10.00
1.00000
1.00000
Unk.4
20.0
Inject Samples
LC Demo Method Set
10.00
1.00000
1.00000
Unk.5
20.0
Inject Samples
LC Demo Method Set
10.00
1.00000
1.00000
Unk.6
20.0
Inject Samples
LC Demo Method Set
10.00
1.00000
1.00000
Std1
20.0
1 Inject Standards
LC Demo Method Set
10.00
1.00000
1.00000
Clear
Calibration
Calibrate
44
LC Demo Method Set

LC Demo Method Set
Basic Report
Reported by User:
System
Project Name:
SAM PLE
Sample Name:
Sample Type:
Vial:
Injection #:
Injection Volume:
Run Time:
Sample Set Name:
Untitled
Prednisone_Tab
Unknown
16
1
20.00 ul
4.0 Minutes
Dissolution_Default
IN FO R MATION
Acquired By:
Date Acquired:
Acq. Method Set:
Date Processed:
Processing Method:
Channel Name:
Proc. Chnl. Descr.:
System
27/10/1999 14:38:40
Prednisone Mth Set
27/03/2001 14:17:26
Prednisone Proc Mth
2487Channel 1
Single @ 242 nm
Prednisone - 1.791
0.18
0.16
0.14
AU
0.12
0.10
0.08
0.04
0.02
0.573
0.06
0.00
0.50
1.00
1.50
2.00
Minutes
2.50
3.00
3.50
4.00
Peak Results
Name
1
2
Prednisone
RT
Area
Height
0.573
22575
822
1.791
1188824
186546
Amount
Units
0.024
mg/ml
Calibration Procedures
Calibration
Standards at
the beginning
only
Calibration
Standards at the
beginning and
end
Calibration
Standards
Bracketing
45
Calibration
Standards
Bracketting
Calibration
Standards
Bracketting
Calibration
Standards
Bracketting

System Suitability
Parameters To Monitor - Validation

Precision (Ruggedness)
Accuracy
Limit of detection
Limit of quantitation
Linearity (range)
Selectivity
Robustness
46
Example Method Validation Protocol

Robustness
Linearity
Day 1-3
LOQ
LOD
Parameter 1
Min & Max
Level 1: 60%
Bulk & Formul.
Level 1
Bulk Drug
Six Blanks
Baseline Noise
Parameter 2
Min & Max
Level 2: 80%
Bulk& Formul.
Level 2
Bulk Drug
Parameter 3
Min & Max
Level 3: 100%
Bulk & Formul.
Level 3
Bulk Drug
Parameter 4
Min & Max
Level 4: 120%
Bulk & Formul.
Level 4
Bulk Drug
Level 5: 140%
Bulk & Formul.
Level 5
Bulk Drug
LOQ:
Precision
Precision
Day 1-3
Six Reps., 100%
Bulk Drug
LOQ Level
Bulk Drug

Accuracy
Limit of detection
Linearity (range)
Selectivity
Robustness
47
CHARACTERISTICS OF AN ANALYTICAL METHODS
Precision:
The reproducibility of results. The degree to which an
experimental result varies from one determination to
next.
the
Precision is related to random error and Accuracy is related to systematic

error.
Illustrating the difference between accuracy and precision
Low accuracy, high precision
Low accuracy, low precision
High accuracy, low precision
High accuracy, high precision
Precision: Definition
Precision
The measure of the degree of agreement among test results when the method
is applied repeatedly to multiple samplings of a homogeneous sample
Expressed as %RSD for a statistically significant number of samples
48
Precision: Definition
Precision Should Be Performed at Three Levels
Repeatability
Intermediate Precision
Reproducibility
Precision: Definition/Determination
Repeatability (Generally the criterion of concern in USP analytical procedures)
Same operating conditions, short time interval
Intra-assay precision
Minimum of 9 determinations covering specified range of procedure (3
levels, 3 reps each), or
Minimum of 6 determinations at 100% test conc.
Intermediate Precision (Experimental design recommended)

Within-lab variations (Random events)
Different days, analysts, equipment
Reproducibility
Precision between labs
Collaborative studies
49
Ruggedness: Definition
Ruggedness
Degree of Reproducibility of Test Results Under a Variety of
Conditions
Different Laboratories
Different Analysts
Different Instruments
Different Reagents
Different Days
Etc.
Expressed as %RSD
Calculating Precision
N
X
Mean :
Standard Deviation :
Coefficient
of Variation :
CV% =
N 1
50
(100 )
Example Precision Data

Injection
Response
125955
125651
126052
124855
126102
125899
Average
125752
Standard Deviation
466.93
Relative Standard Deviation
0.37%
Precision Test
0.25
0.20
AU
0.15
0.10
0.05
0.00
1.00
2.00
SampleName: SysSuit_A;
3.00
Vial: 5;
Vial: 5;
Vial: 5;
Vial: 5;
Vial: 5;
Vial: 5;
Injection: 1;
Injection: 2;
Injection: 3;
Injection: 4;
Injection: 5;
Injection: 6;
4.00
5.00
Minutes
Channel: 2487Channel 1;
6.00
7.00
8.00
9.00
Component Summary Table

Name : EtP
1
2
3
4
5
6
Mean
Std. Dev.
% RSD
SampleName Inj
Channel
Name
SysSuit_A
1 2487Channel 1 EtP
SysSuit_A
2 2487Channel 1 EtP
SysSuit_A
3 2487Channel 1 EtP
SysSuit_A
4 2487Channel 1 EtP
SysSuit_A
5 2487Channel 1 EtP
SysSuit_A
6 2487Channel 1 EtP

Report Method: Summary
Report Method ID:5761
5761
RT
3.997
4.002
3.978
3.993
3.979
4.010
Area
1659138
1657741
1659683
1659981
1660539
1657722
1659134
1177
0.1
Height Amount Vial

202001 30.210 5
201723 30.210 5
202307 30.210 5
201476 30.210 5
203480 30.210 5
201885 30.210 5
202145 30.210
710
0.000
0.4
0.0
Project Name: MVM_Defaults

Date Printed:
16/04/2010
51
10.00
Date Acquired: 01/08/2005 11:08:21 EDT; Injection Volume10.00

Documenting Precision With Bar Charts

0.90
0.80
0.70
0.60
0.50
%RSD
0.40
0.30
0.20
0.10
0.00
Area
Height
Retention
Time
Resolution
Precision - Acceptance Criteria
Less than 2% relative standard deviation is often recommended.

Less than 5% RSD can be acceptable for minor components.
Up to 10% RSD may be acceptable near the limit of
quantitation.
52
CHARACTERISTICS OF AN ANALYTICAL METHOD
Accuracy:
The degree to which an experimental result
approaches the true or accepted answer.
Ways to Describe Accuracy:
Error:
An experimental measure of accuracy. The difference between the

result obtained by a method and the true or accepted value.
Absolute Error = (X )
Relative Error (%) = 100(X )/
where: X = The experimental result

= The true result
Two types of error: random or systematic
ERRORS
Random Error: results in a scatter of results centered on the true value
for repeated measurements on a single sample.
(SPREAD)
Systematic Error: results in all measurements exhibiting a definite
difference from the true value (BIAS)
Random Error
Systematic Error
plot of the number of occurrences or population of each

measurement (Gaussian curve)
53
CHARACTERISTICS OF AN ANALYTICAL METHOD

Ways to Describe Precision:
Range:
the high to low values measured in a repeat series of experiments.
Standard Deviation: describes the distribution of the measured results about

the mean or average value.
Absolute Standard Deviation (SD):
n
SD =
(X X )
i
/( n 1)
i =1
Relative Standard Deviation (RSD) or

Coefficient of Variation (CV):
RSD (%) = ( SD / X )100

where: n = total number of measurements
Xi = measurement made for the ith trial
X = mean result for the data sample
Overlaid Chromatograms
Good Precision
0.05
0.04
0.03
AU
Peak1
0.02
Peak2
Peak3
0.01
0.00
0.00
1.00
2.00
3.00
4.00
5.00
6.00
Minutes
SampleName 2690_Eau40_60CH3OH_2mm_D Vial 4 Injection 1
54
7.00
8.00
M e th o d s Tra n s fe r: 3 H P L C S ys te m s
Accuracy
3 S yste m s
1 in U S A
2 in E u ro p e
3 C h e m is ts
D ia l-a -m ix , 6 0 :4 0
M e O H :H 2 O
1 m L /m in
3 0C
0.05 0
0.04 0
Limit of detection
0.03 0
AU
0.02 0
Linearity (range)
0.01 0
Selectivity
0.00 0
1 .0
2.0
3 .0
4.0
5 .0
6.0
7 .0
Robustness
M in u te s
Method TransferTransfer- 3 different HPLC Systems
0.050
0.040
0.030
AU
0.020
0.010
0.000
1.0
2.0
3.0
4.0
Minutes
55
5.0
6.0
7.0
Column BatchBatch-to
to--Batch
Reproducibility
0.02
Imp1
Imp2
AU
Imp3
Batch 108
Injection: 150
Imp4
0.00
Sample: AZT
L of 0.5 mg/mL
solution
10
0.02
Batch 109
Imp1
Imp2
AU
20
Minutes
Column: Symmetry C18, 3.9 mm

x 150 mm
Temperature: 45 C
Imp3
Mobile Phase: 6% MeOH/ 6%
Imp4
0.00
THF/ 88%
10
0.02
20
Minutes
10 mM potassium phosphate
buffer, pH 2.5
Imp1
Batch 112
Flow rate: 1.7 mL/min
Imp3
AU
Imp2
Detector: UV at 268 nm
Imp4
0.00
10
20
Minutes
Factors Affecting Precision

Area and Retention Time
Integration parameters
Injector performance
Pump performance
56
Variable Reported Concentrations

Problems with Peak Response
Linearity Test of Concentrations
- Check Injector (Use Standards)
* Multiple Injections - Same Vial -- Syringe Problem
or If Only 1st Injection Low -- Septa Problem
* Different Vials -- Evaporation -- Degradation
* Injection Volume Test (Weight before and after injection)
- Integration Software
* Electronic Peak Generator
* Poor Peak Shape
RSD < 5-15%

0.010
- Detector
* Cell Problem
* Lamp Failing
AU
0.005
0.000
51.8 52.0 52.2 52.4 52.6 52.8
Minutes
Injector Linearity
Response
2,000
1,500
Different
volumes of
the same
standard
1,000
Methods
transfer
R2 = 0.9999
500
10
20
30
40
Injection Volume L
57
50
Accuracy: Definition
The closeness of test results obtained by the method to the true
value.
Established across the concentrations range
Accuracy: Determination
BioBio-active Substance
Analysis of reference material
Bio
Bio--active Formulation
Analysis of synthetic mixtures spiked with known quantities
of components
Impurities (Quantitation)
Analysis of samples (Bio-active substances/Bio-active
formulation) spiked with known amounts of impurities
If impurities are not available, see specificity
58
Accuracy: Determination (Cont.)
Recommended Data
Minimum of 9 determinations over a minimum of 3
concentration levels covering the specified range (e.g. 3
concentrations/3 replicates each)
Reported as % recovery of known, added amount, or
difference between the mean and true value, with
confidence intervals
Accuracy of Measurement
Correlation Coefficient
(Xi
) (Yi Y )
(X X )2 (Y Y )2
X
(X
i,
Yi )
X i , X from method one
Y i ,Y from method two
Error :
% Relative Error :
E = O - T
%RE =
E
T
59
x 100
O = Observed Value
T = True Value
Example Accuracy Data

Target Concentration
Spiked Concentration
(mg/mL)
Measured
Concentration
(mg/mL) Avg N=3
% Recovery
50%
0.50
0.49
98.0
75%
0.75
0.74
98.7
100%
1.00
1.03
103
125%
1.25
1.24
99.2
150%
1.50
1.48
98.7
Average Recovery:
Standard Deviation:
Relative Standard Deviation:
99.52%
1.99
2.00
Documenting Accuracy
with Control Charts
Custom Report Control Chart for: ACE, Channel: 996
411879.8
401879.8
391879.8
Area
381879.8
371879.8
UCL
361879.8
Target
351879.8
LCL
341879.8
331879.8
321879.8
1
Run Number
60
10
11
12

(See Detection Qualification)
Limit of Detection:
h signal = 2 x h noise
Limit of Quantitation: h signal = 10 x h noise
h signal
Accuracy
h noise
Limit of detection
LOQ - RSD < 5-10%
Linearity (range)
0.010
Selectivity
AU
0.005
Robustness
0.000
51.8 52.0 52.2 52.4 52.6 52.8
Minutes
Detection Limit (DL/LOD): Determination

Visual (Non-Instrumental Methods)
Signal To Noise Ratio (3 or 2:1 Generally Accepted)
Detection limit may be based on the standard deviation of the
response and slope:
DL = (3.3)STD/S
STD = standard deviation of the response
S = slope of the calibration curve
61
Quantitation Limit (QL/LOQ): Definition

Limit of Quantitation (LOQ)
Lowest Concentration of Analyte in a Sample That Can Be
Determined With Acceptable Precision and Accuracy Under
Stated Operational Conditions
Expressed as the Concentration of Analyte
Accuracy
Precision
Quantitation Limit (QL/LOQ):

Determination
Visual (Non-Instrumental Methods)
Signal To Noise Ratio (10:1 is Typical)
Quantitation limit may be based on the standard deviation of
the response and slope:
QL = (10)STD/S
STD = standard deviation of the response
S = slope of the calibration curve
Both DL and QL are validated by analyzing a suitable number
of samples. Method should be documented
62
Example LOQ/LOD Data and Calculations

Level 1 Response
Level 2 Response
Level 3 Response
Level 4 Response
Level 5 Response
2085
4105
6288
8333
10499
2106
4235
6103
8287
10305
1955
4188
6337
8399
10386
2199
4063
6129
8156
10355
2077
4005
6254
8247
10462
2155
4284
6301
8178
10219
Mean
2096
4147
6235
8267
10371
STDev.
83.13
106.9
96.53
92.54
102.57
Average Standard Deviation:

Slope of Calibration Curve:
Limit of Detection
Limit of Quantitation:
96.33
9378
0.034 mg/mL
0.103 mg/mL
Effect of HPLC Columnss Performance in

Method Validation: LOQ
0.0005
A) Brand Y C18
Signal to Noise = 11
System Suitability results of

Tailing and Plate Count would
have predicted the differences
AUFS
5.00
Minutes
10.00
0.0005
B) Brand X C18
Signal to Noise = 6.5
AUFS
5.00
Minutes
10.00
63
Sample: 0.25 g/mL

Tamoxifen
Injection volume and linear
flow velocity compensated
for on both columns
3.5
3
Accuracy
Absorbance (AU)
2.5
2
Limit of detection
1.5
1
0.5
0
0
10
20
30
40
50
60
70
80
% Absorbe r in Elue nt
Linearity (range)
Selectivity
Robustness
Linearity and Range: Definition

Linearity
The Ability of the Method to Elicit Test Results That Are
Directly Proportional to Concentration Within a Given
Range
Expressed as the Variance of the Slope of the Regression
Line
Range
Interval between upper and lower levels of analyte
demonstrated by the method
Precision and Accuracy Expressed in the same units as the
test results
64
Linearity: Determination
Established across the Range of the method
Dilutions
Separate Weighings
Evaluate by Appropriate Statistical Methods (e.g. Regression)
Include Correlation Coefficient, y-Intercept, Slope, Residual
Sum of Squares, Plot Itself
Minimum 5 Concentrations
Determination of Appropriate Range

Minimum Specified Ranges
Assay
80-120%
Impurity Test
From QL to 120% of spec.
Toxic or more potent impurities: commensurate with the
controlled level
Content Uniformity
70-130% of test concentration
Dissolution Testing
+/- 20% over specified range
65
Example linearity (Calibration) Plot
Linearity (Calibration) Plot

Linearity (Calibration) Plot for TMT, Day 2
Response
5,000,000
4,000,000
3,000,000
2,000,000
1,000,000
0
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.10
Amount
R-square = 1 # pts = 5
y = -8.95e+005 + 5.4e+007x
Include correlation coefficient, y-intercept, slope, plot itself

Minimum of five concentrations, +/- 20% over specified range
66
0.11
Example for HPLC Method Precision

Repeatability: N = 6 at 100%
Day
%RSD-Time
HCT
%RSD-Time
TMT
%RSD-Area
HCT
%RSD-Area
TMT
1
2
3
0.00
0.05
0.00
0.00
0.00
0.00
0.05
0.16
0.18
0.06
0.07
0.05
Intermediate Precision:
N = 18 (6 inj./day, 3 days) at 100%
Compound
%RSD-Time
%RSD-Area
HCT
0.21
3.24
TMT
0.56
3.97

Peak Purity Analysis
300.00
250.00
Spectra at apex and

inflection points are
displayed
Spectrum at maximum
impurity is different
200.00
nm
0.40
0.20
Maximum
Impurity
Accuracy
AU
0.00
2.20
2.40
2.60
2.80
Limit of detection
3.00
Minutes
Photodiode Array Technology
Linearity (range)
Spectral Analyses
Library Matching
Compound identification
Coelution detection
Selectivity/Specificity
Peak Purity Analysis

Peak purity/peak homogeneity
Coelution detection
Robustness
67
Specificity: Definition
Specificity (Selectivity)
The ability to measure accurately and specifically the analyte in
the presence of components that may be expected to be present
in the matrix
The degree of interference
Active Ingredients
Excipients
Impurities
Degradation Products
Placebo Ingredients
Specificity: Determination
Qualitative Identification Tests
Demonstrate ability to select between compounds of closely
related structure
Assay
Demonstrate that the results are unaffected by spiked impurities
or excipients
Impurities
Spike the drug product/substance with impurities and
demonstrate appropriate accuracy and precision
68
Method Development Challenge

Excipients
Impurities
Degradants
Composite sample
Courtesy of:
Dr. Rudy Sneyers, J&J Belgium
PDA is used mostly for Development and Validation of

STABILITY INDICATING METHODS
69
What is a Stability Indicating

Method?
A Stability Indicating Method (SIM) is:
A validated method that can accurately and precisely quantitate
the decrease of the API content due to degradation.
The method:
Is specific for the drug substance
Shows a decrease in assay value (correlated to drug substance loss) due to
degradation
Has no interference from excipients, impurities or degradation products
Detects and quantitates impurities and degradation products
Why are Stability Indicating Methods

Needed?
Why are Stability Indicating Methods needed?
Predominantly used to support long-term stability testing
How the quality of the drug substance or product changes over
time in response to environmental factors
Temperature
Humidity
Light
Establishes storage and packaging conditions
70
When to Use
Stability Indicating Methods
When are SIMs needed?
Stability studies
API release
Drug product release
Toxicology dosing solutions
Excipient compatibility and pre-formulation
Packaging studies
Line extensions
When are SIMs not needed?

In process controls
Secondary assay for API
Titration
Inorganics
Sample Generation
Forced Degradation (Stress) Studies
Forced degradation or stress testing is undertaken to demonstrate
specificity when developing SIMs
Generates a sample for method development
Performed prior to implementation of stability studies

Why do an intentional degradation study?
Understand the reactive chemistry of the drug substance

Help anticipate future stability issues of both drug substance and drug product
Provides useful information for formulation and stability
May be required for regulatory submissions
71
Sample Generation
Common conditions used in Forced Degradation Studies
Study
Conditions
Acidic pH
0.1N HCl
Neutral pH
pH 7.0 Phosphate Buffer
Basic pH
0.1N NaOH
Oxidation
02 Atmosphere, or H2O2
Photolysis (UV)
1000 Watt h/M2
Photolysis (Fluorescence)
6x106 lux h
Goal: Degrade API 5-10

10%
%
Stability Indicating
Method Development
Manipulate chromatographic selectivity
Column
Mobile phase composition/type
pH
Temperature
Specificity
PDA or MS
Goal:
Baseline Resolution, No CoCo-elutions
72
More Changes Courtesy of ICH: Specificity
PDA and MS Together
+
PDA and MS together can
provide complimentary useful
information.
73
Diode Array Detector
3 dimentional plot of a chromatographic run

using a diode-array detector
0.24
0.22
0.20
0.18
0.16
0.12
0.10
0.08
0.06
0.04
0.02
0.00
260.00
280.00
300.00
320.00
340.00
Wavelength
360.00
(nm)
380.00
1.15
1.20
1.25
1.30
1.35
1.40
1.45
1.50
1.55
1.60
Minutes
74
1.65
1.70
1.75
1.80
1.85
AU
0.14
Typical LC/MS System Components

ANALYZER
ION DETECTOR
+
+
SOURCE
+
+
+
+
DETECTION OF IONS
+
+
SORTING OF IONS
+
+
SAMPLE DESOLVATION
AND IONIZATION
LC/MS
INTERFACE
HPLC
DATA SYSTEM
MASS SPECTRUM
CHROMATOGRAM
3 dimentional plot of a chromatographic run

using a mass spectrometer detector
1.100e+008
1.000e+008
9.000e+007
8.000e+007
6.000e+007
5.000e+007
4.000e+007
3.000e+007
2.000e+007
1.000e+007
0.000e+000
200.00
400.00
600.00
800.00
1000.00
1.00
1.05
1.10
1.15
1.20
1.25
1.30
1.35
1.40
1.45
1.50
1.55
Minutes
75
1.60
1.65
1.70
1.75
1.80
m/z
Intensity
7.000e+007
Spectrum Index Plot in UV
250.00
nm
300.00
nm
300.00
250.00
350.00
1.19
Peak1
0.25
250.00
350.00
nm
300.00
350.00
1.32
Peak 2
1.19
1.75
Peak 3
1.31
1.74
1.20
1.33
1.76
1.18
1.31
1.74
1.21
1.34
1.77
Peak1
Peak 2
Peak 3
AU
0.20
0.15
0.10
0.05
0.00
1.00
1.10
1.20
1.30
1.40
1.50
Minutes
1.60
1.70
1.80
1.90
2.00
Chromatogram of three sulfa drugs at 270 nm and a plot of UV Spectra collected from each peak's Apex, Inflection and two
offset points, to demonstrate comparison of spectra collected at various points across the peak.
Spectrum Index Plot in Mass Spectrometry

SIR Chromatogram
MS Spectra
1.2x107
265.1
8.0x106
1.0x107
Peak 1
6.0x106
5.0x106
4.0x106
2.0x106
5x107
Intensity
Intensity
7
6x10
279.1
4x107
3x107
Peak 2
2x107
529.2
530.1
279.1
2x107
Peak 2
280.2
281.1
557.2
8x107
8x107
311.1
6x107
4x107
Intensity
Intensity
266.1
267.1
4x107
1x107
Peak 3
1.20
1.30
1.40
1.50
1.60
1.70
1.80
311.1
6x107
4x107
2x107
2x107
Peak 1
0.0
0.0
1.10
265.1
Intensity
Intensity
1.0x107
1.5x107
Peak 3
312.2
313.1
0
200.00
300.00
400.00
500.00
m/z
Minutes
SIR chromatograms of the same sulfa drugs and their corresponding mass spectra, obtained by combining
spectra across the entire peak.
76
600.00
MS Sensitivity And Specificity Example

Impurity study of Lansoprazole : UV detection
N
NH
O
0.85
0.80
Lansoprazole
0.75
0.70
UV @ 254nm
O
F
0.65
0.60
0.55
-0.00125
0.50
-0.00130
UV @ 254nm
254nm
0.45
0.03%
AU
-0.00135
0.40
-0.00140
0.35
S/N = 2
-0.00145
0.30
-0.00150
AU
0.25
0.20
0.15
-0.00155
-0.00160
0.10
-0.00165
0.05
-0.00170
0.00
-0.00175
-0.05
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00
Minutes
-0.00180
4.80
5.00
5.20
5.40
Minutes
5.60
5.80
6.00
MS Sensitivity And Specificity Example

Impurity study of Lansoprazole : MS detection (SIR mode)
MW+1= 298.2
N
NH
O
N
0.85
0.80
0.75
0.70
0.65
Lansoprazole
O
UV @ 254nm
SIR @ 298.2 m/z
F
F
0.60
0.55
2.0x106
0.50
MS: SIR 298.

298.2
1.8x106
AU
0.45
0.40
1.6x106
0.35
1.4x106
S/N = 870
Intensity
0.30
0.25
0.20
1.2x106
1.0x106
0.15
8.0x105
0.10
6.0x105
0.05
4.0x105
0.00
2.0x105
-0.05
0.50
1.00 1.50
2.00 2.50
3.00 3.50
4.00
4.50 5.00 5.50 6.00

Minutes
6.50 7.00
7.50 8.00
8.50 9.00
9.50 10.00
0.0
4.80
77
5.00
5.20
5.40
5.60
Minutes
5.80
6.00
Sample Generation
Common conditions used in Forced Degradation Studies
Study
Conditions
Acidic pH
0.1N HCl
Neutral pH
pH 7.0 Phosphate Buffer
Basic pH
0.1N NaOH
Oxidation
02 Atmosphere, or H2O2
Photolysis (UV)
1000 Watt h/M2
Photolysis (Fluorescence)
6x106 lux h
Goal: Degrade API 5-10

10%
%
Chromatography of Degraded Risperidone

Risperidone:: Oxidation
By 31%
31% Peroxide - Isocratic Separation - pH 3
0.12
0.10
Risperidone
XTerra MS C18 3.5 m

80%A: 20%B
0.06
0.04
0.02
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
12.00
Minutes
0.12
Risperidone
0.10
XTerra RP18 3.5 m
AU
0.08
80%A: 20%B
0.06
0.04
0.02
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
12.00
Minutes
0.12
Risperidone
0.10
Symmetry C18 3.5 m
AU
0.08
Conditions:
Column Dimensions: 4.6 x 100 mm
Isocratic separation
A: pH 3.0; 10mM NH4COOH in H2O
B: Acetonitrile
Wavelength: 280nm
Flow rate: 1.4 mL/min
Injection volume: 10.0 L
Temperature: 30 oC
Risperidone concentration:
0.5 mg/ ml in 1.55% H2O2
Risperidone degraded ~ 15 - 30%
82%A: 18%B
0.06
Instrumentation:
Waters Alliance HT 2790
Waters 996 PDA detector
0.04
0.02
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
12.00
0.12
Risperidone
0.10
0.08
AU
AU
0.08
SymmetryShield RP18 3.5 m

83%A: 17%B
0.06
0.04
0.02
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
Minutes
78
10.00
11.00
12.00
Forced Degradation Results

Mass Spectrometry
UVUV-VIS
Specificity: Determination (Cont.)

Impurities Are Available
Demonstrate that the assay is unaffected by the presence of
spiked materials (impurities and/or excipients).
Impurities Are Not Available
Compare test results to a second well-characterized procedure
For Assay, compare the two results
For Impurity Tests, compare impurity profiles
Peak Purity Test ("diode array, MS")
79
MS Detection in Peak Purity Analyses

Enhance 3D PDA information with mass spec information:
Added confidence in methods development (peak confirmation)

Peak homogeneity (detect co-eluting compounds)
Peak tracking
Enhanced sensitivity
Enhanced selectivity
Non-UV absorbing compounds
Contributes to universal detection concept
K' vs Buffer Concentration

4
Accuracy
5N-2,4,6T
4A-6C-1,3B
K'
HCT
Limit of detection
TMT
0
10
30 50 70 90
20 40 60 80 100
Linearity (range)
mM NaH2PO4 pH 5.5
Selectivity
Robustness
80
Robustness: Definition
Robustness
Measure of The Capacity to Remain Unaffected by Small
(Deliberate) Variations in Method Parameters
Indication of Reliability During Normal Use
Robustness: Determination
Consider during development of method
Shows reliability of method with respect to deliberate changes
If measurements are susceptible to variations in analytical
procedures, these conditions should be controlled and a
precautionary statement included.
Establish System Suitability parameters to ensure the validity of the
method
81
Robustness of the chromatographic

method
Parameters Varied
:
Solvent strength in the mobile phase,
Temperature,
Flow rate,
pH of the mobile phase,
Ionic strength in the mobile phase,
Sample diluent,
Injection volume,
Wavelength of detection.
The parameter measured:

Response (area/amount)
Retention time,
Selectivity and/or resolution.
Non--Robust Method
Non
pH=4.5
pH=4.9
Minor Changes in
pH Effect Rs
pH=4.7
82
Robust Method
AZT: Robustness Testing
6% M ethanol, 6% THF
AU
0.010
Imp. 1
pH 2.3
Imp. 3
0.008
Imp. 2
0.006
0.004
Imp. 4
0.002
0.000
-0.002
0
10
15
20
Imp. 1
AU
0.010
Imp. 3
0.008
pH 2.5
Imp. 2
0.006
0.004
Imp. 4
0.002
0.000
-0.002
0
10
15
20
Time [min]
AU
Im p. 1
0.010
Imp. 3
0.008
pH 2.7
Imp. 2
0.006
0.004
Imp. 4
0.002
0.000
-0.002
0
10
20
15
Time [min]
Method Change Versus

Adjustment Proposals
Aqueous Buffer pH
Analytes without ionizable groups: +/- 1 unit
Analytes with basic or acidic groups and the buffer pH =
pKa +/- 2 units: +/- 0.2 unit
Analytes with basic or acidic groups and the buffer pH <
or > pKa +/- 2 units: +/- 1 unit
Column Dimensions
Length: +/- 70%
Inner Diameter: +/- 25%, maintaining a constant linear
velocity
Flow Rate: up to 50%
Reference standards must be used to show that the
chromatography is improved by pH adjustment.
83

System Suitability
Requirements from Chromatographic Data

System (CDS)
84
Basic Sequence in HPLC

1.
2.
3.
4.
5.
6.
Login.
Instrument Method.
Sequence/Run Samples.
Process Data.
Review/Preview.
Result/Sign-Off.
3.
2.
4.
5.
6.
1.
Create a Sequence or a Sample Set/List

Check: Created vs. Accomplished
85
Create and Use Methods

Method Set
Instrumental parameters
Processing parameters
Reporting
Exporting
Instrumental Parameters Collect

Chromatographic Data
Method Set
Reporting
Exporting
86
Instrument Method : 50B_flow 1
Example: Instrumental Parameters
Stored : 14 /07 /2006 11 :13 :57 EDT

Method Information
Method Comments
Method Modified User
Method Locked
Method Id
Method Version
Method Edit User
Source S/W Info
Empower 2 Software Build 2154 DB ID : 689194452
System
No
3767
4
Pump&Autosampler Instrument Setup

W2690/5
Type
Instrument Status
On
Channel Name
2690/5
Description
Use Channel Monitor
Off
Monitor Parameter
System Pressure
Stroke Volume
50uL (flow rates <= 1.23 mL/min)
Chart Out
%A
Syringe Draw Rate
Normal
Depth Of Needle
0.0
Degas Mode
On
Pump Mode
Isocratic
Flow
1.000
%A
50.0
%B
50.0
%C
0.0
%D
0.0
High Limit
4000.0
Low Limit
0.0
Enable Sample Temp False
Enable Column Temp False
Bubble Detect
True
Pre Column Volume
0.0
Sample Temp Target

Sample Temp Range
Sparge A
Sparge B
Sparge C
Sparge D
Column Temp Target
Column Temp Range
Flow Ramp
Column choice
Column Equil Minutes
Needle Wash
Switch 1
Switch 2
Switch 3
Switch 4
Use Events
Solvent A
Solvent B
Solvent C
Solvent D
-1.0
5.0
0.0
0.0
0.0
0.0
-1.0
5.0
2.00
No Change
0.00
Normal
No Change
No Change
No Change
No Change
False
Detector Information
Channel Name
Description
Use Channels
2487Channel1
On
Wavelength
254
Output Mode
Data Mode
Sampling Rate
Filter Type
Absorbance A(Ch1)
Absorbance A(Ch1)
2
Hamming
Aufs
2.0000
Time Constant
1.0
AU Offset
0.000
Voltage Offset
0
Polarity
+
AutoZero Wavelength True
AutoZero Keypad
True
AutoZeroEvent Input True
Process the Data to

Create Results
87
AutoZero Inject
Chart Mark Enable
Ratio AuMinimum
Minimum Ratio
Maximum Ratio
True
True
0.1000
0.00
2.00
Basic Report
Reported by User:
System
Project Name:
SAM PLE
Sample Name:
Sample Type:
Vial:
Injection #:
Injection Volume:
Run Time:
Sample Set Name:
Untitled
Prednisone_Tab
Unknown
16
1
20.00 ul
4.0 Minutes
Dissolution_Default
IN FO R MATION
Acquired By:
Date Acquired:
Acq. Method Set:
Date Processed:
Processing Method:
Channel Name:
Proc. Chnl. Descr.:
System
27/10/1999 14:38:40
Prednisone Mth Set
27/03/2001 14:17:26
Prednisone Proc Mth
2487Channel 1
Single @ 242 nm
Prednisone - 1.791
0.18
0.16
0.14
AU
0.12
0.10
0.08
0.04
0.02
0.573
0.06
0.00
0.50
1.00
1.50
2.00
Minutes
2.50
3.00
Peak Results
Name
1
2
Prednisone
RT
Area
Height
0.573
22575
822
1.791
1188824
186546
Amount
0.024
Units
mg/ml
Processing Method
Method Set
Reporting
Exporting
88
3.50
4.00
Processing Methods Parameters

Method Set
Reporting
Exporting
Processing Methods Parameters
89
Processing Method
Type : LC
: Parabens Ten
Processing Method
Parameters: Example
Stored : 13 /07 /2006 14 :32 :15 EDT
Method Information
Method Comments
Method Modified User
System
Method Locked
No
Method Id
2193
Method Version
12
Method Edit User
Source S/W Info
Beta Empower Software Build
1154 DB ID: 685141106
Integration Algorithm
ApexTrack
Average By
None
RT Window %
5.00
Update RT
Never
CCalRef1
Include Internal Standard Amounts in Amount Calculation
Yes
Vial/Default Value Type
Amount
System Suitability Information
Void Volume Time
1.000(min)
Calculate Suitability
Yes
Flag Outside
Yes
Calculate Unknowns
Yes
Pharmacopoeia
All
Tangent Percent (USP Plate Count)61
Integration Parameters
Minimum Area
0(V*sec)
Minimum Height 0(V)
Integration Start 2.000(min)
Integration End
(min)
Peak Width
11.42(sec)
Detection Threshold 5.000e+002(V)
Tangent Percent (USP Resolution) 50

Calculate EP s/n
No
Noise Value for s/n
Baseline Noise
% Runtime
5.0
Maximum Noise
Maximum Drift
Liftoff %
Touchdown %
Baseline Start
Baseline End
(min)
(min)
0.000(%)
0.500(%)
Processing Method Group

'Integration Events
' table contains no. data
Component Table
Validation
Retention Time
RT Window
Name Component
ChannelPeak Match
Calculate Suit Results
Flag Outside Limits
Y ValueX Value Fit WeightingInternal Std
RT Reference
Type
Target Peak Label (min
)
(min
)
1 MeP Main Component 25.000
2.385
0.196
Closest Yes
Yes
Area AmountLinear None
2 EtP Low Level Impurity
30.000
3.940
0.352
Closest Yes
Yes
3 PrP
8.080
0.618
Closest Yes
Yes
Report Method
Method Set
Reporting
Exporting
90
Basic Report
Reported by User:
System
Project Name:
SAM PLE
Sample Name:
Sample Type:
Vial:
Injection #:
Injection Volume:
Run Time:
Sample Set Name:
Untitled
Prednisone_Tab
Unknown
16
1
20.00 ul
4.0 Minutes
Dissolution_Default
IN FO R MATION
Acquired By:
Date Acquired:
Acq. Method Set:
Date Processed:
Processing Method:
Channel Name:
Proc. Chnl. Descr.:
System
27/10/1999 14:38:40
Prednisone Mth Set
27/03/2001 14:17:26
Prednisone Proc Mth
2487Channel 1
Single @ 242 nm
Prednisone - 1.791
0.18
0.16
0.14
AU
0.12
0.10
0.08
0.06
0.573
0.04
0.02
0.00
0.50
1.00
1.50
2.00
Minutes
2.50
3.00
3.50
4.00
Peak Results
Name
1
2
Prednisone
RT
Area
Height
0.573
22575
822
1.791
1188824
186546
Amount
0.024
Units
mg/ml
Summary Report
General Information
Sample Set Name
Sample Set Method
Sample Set Id
Sample Set Altered
SampleName
27/10/1999 12:36:43
27/10/1999 15:37:29
2090
No
Sample Type
Vial
Run Time
(Minutes)
Inj #
Injection
Volume
(ul)
Acquistion
Method Set
Sample
Weight
Dilution
Label
Prednisone_Tab
Unknown
11
4.00
20.00
Prednisone Mth Set
1.00000
1.00000
AU0203
Prednisone_Tab
Unknown
12
4.00
20.00
Prednisone Mth Set
1.00000
1.00000
AU0204
Prednisone_Tab
Unknown
14
4.00
20.00
Prednisone Mth Set
1.00000
1.00000
AU0206
Prednisone_Tab
Unknown
16
4.00
20.00
Prednisone Mth Set
1.00000
1.00000
AU0208
Area
Calibration Plot
1.0x10
5.0x10
0.0
0.000
0.002
0.004
0.006
0.008
0.010
Amount
0.012
0.014
0.000000e+000; D: 0.000000e+000; R^2: 1.000000
91
0.016
0.018
0.020
Quantitative Results
SAM PLE
I N F O R MATI O N
Sample Name :
Sample Type :
Vial :
Injection #:
Injection Volume :
Run Time :
K -26525 /1-H 2O 2
Unknown
24
1
10 .00 ul
55 .0 Minutes
Acquired By :
Sample Set Name :
Acq . Method Set :
Processing Method :
Channel Name :
Proc . Chnl . Descr .:
Date Acquired :
Date Processed :
14 /09 /2000 01 :22 :17 IDT

17 /04 /2010 07 :56 :42 IDT
Sofia
PDA Report:
Example
Teva _ Method _ Set

PDA _ PRocessing _ Method
PDA Max Plot 190 .0 - 800 .0
PDA MaxPlot (190 .0 nm to 800 .0
nm )
Spectrum Index Plot

6.737
200.00
Deg1 - 8.611
200.00
nm
250.00
Main - 11.663
200.00
nm
300.00
250.00
193.7
250.00
193.7
6.74
6.69
6.81
nm
300.00
291.0 291.0 291.0
300.00
193.7
8.61
8.54
8.71
11.66
11.56
11.84
288.6 288.6 288.6
291.0 291.0 291.0
Main - 11.663
Deg1 - 8.611
0.50
6.737
AU
1.00
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
12.00
13.00
14.00
Minutes
1
2
3
Name
RT
Deg1
Main
6.737
8.611
11.663
Purity1
Angle
1.854
0.261
0.041
Purity1
Threshold
2.483
0.439
0.240
Purity1
Flag
No
No
No
PDA Result Table

Match1
PDA/FLR Match 1
Spect. Name
Lib. Name
Main 100 %
Training _Nov09
Main 100 %
Training _Nov09
Main 100 %
Training _Nov09
92
Match1
Angle
1.732
4.183
0.024
Match 1
Threshold
2.489
1.142
1.008
PDA/FLR Match 1
Flag
No
Yes
No
15.00
Match Plot
Match Plot
193.7
Deg1
Match #1 Angle 4.183 Main 100%
288.6
Main
Match #1 Angle 0.024 Main 100%
288.6 288.6
291.0
SampleName: K-26525/1-H2O2 Vial: 24 Injection: 1 Date

Acquired: 14/09/2000 01 :22:17 IDT Name: Deg1 Match1 Angle:
4.183 Match1 Threshold : 1.142 Match1 Spect. Name: Main
100%

Acquired: 14/09/2000 01 :22:17 IDT Name: Main Match 1 Angle:
0.024 Match1 Threshold : 1.008 Match1 Spect. Name: Main
100%
80.00
Deg1 - 8.611
Purity
Auto Threshold
40.00
30.00
60.00
40.00
AU
0.02
1.00
Degrees
AU
Purity Plot
Purity
Auto Threshold
Main - 11.663
Purity Plot
0.04
0.50
20.00
10.00
20.00
0.00
0.00
8.40
8.60
8.80
PDA Report:
Example
9.00
0.00
0.00
11.40
Minutes
PA: 0.261 TH: 0.439

Acquired: 14/09/2000 01 :22:17 IDT Purity 1 Angle: 0.261
Purity1 Threshold : 0.439
Purity
Auto Threshold
11.60
11.80
12.00
Minutes
12.20
12.40
12.60
PA: 0.041 TH: 0.240

Acquired: 14/09/2000 01 :22:17 IDT Purity 1 Angle: 0.041
Purity1 Threshold : 0.240
Purity
Auto Threshold
Reports to Connect
Data to Decisions
93
Degrees
193.7
Electronic Signatures
The Manual Process
Chromatographic
Data System (CDS)
Additional
Calculations
Performed
Reports Compiled
LIMS
Final Review
Signatures Applied
94
Reports Reviewed
The Manual Process

Chromatography to Calculations
Empower
CDS
Additional
Calculations
Performed
The Manual Process

Review and Signature
Reports Compiled
Reports Reviewed
Signatures Applied
95
Final Review
Electronic Review and

Signature Process

Signature Process
96

Signature Process
97

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