Slide 4

Phone Number: 1-816-650-0774

Validated HPLC Methods
To Tweak or Not to Tweak
Slide 5
Phone Number: 1-816-650-0774
Adjustments of operating conditions
to meet system suitability
requirements may be necessary.
USP 23 p1776
001816S1.PPT
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Phone Number: 1-816-650-0774
What is the line between adjusting conditions
and actually modifying an official or regulatory
method?
This is critical to know because modifying a
method requires validation and ruggedness
testing.
001817S1.PPT
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Phone Number: 1-816-650-0774
Lets examine some proposed
guidelines for this area.
System Suitability Tests in
Regulatory Liquid and Gas Chromatographic
Methods:
Adjustments Versus Modifications
William B. Furman, John G. Dorsey, and Lloyd R. Snyder
Pharmaceutical Technology, June 1998 p. 58-64
001818S1.PPT
Slide 8
Phone Number: 1-816-650-0774
HPLC Method Parameters
That Can Be Varied
The pH of the mobile phase: +/- 0.2 pH units
Concentration of the buffer salts: +/- 10%
(buffer pH must remain same +/- 0.2 pH units)
Ratio of the solvents in the mobile phase: +/- 30% relative or
+/- 2% absolute, whichever is larger, but no change can exceed
10% (based on mobile phase component of 50% or less)
Mobile Phase
001819S1.PPT
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Phone Number: 1-816-650-0774
HPLC Method Parameters
That Can Be Varied
Column
Column length: +/- 70% (250 mm columns may be substituted
over the range 75 425 mm)
Column inner diameter: +/- 25% (if method calls for
3.9 mm id, 3.0, 4.0, or 4.6 mm can be substituted)
Particle size: may be reduced up to 50%
(3 or 3.5 m particles can be used instead of 5 m)
Column temperature: +/- 20C
001820S1.PPT
Slide 10
Phone Number: 1-816-650-0774
HPLC Method Parameters
That Can Be Varied
System
Flow Rate: +/- 50%
Injection Volume:
- Increase up to 2x maintain peak shape, resolution,
retention time, etc.
- Decrease as much as will maintain acceptable
precision and sensitivity
001821S1.PPT
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Phone Number: 1-816-650-0774
Lets look at a problem method
and determine if it can be adjusted
or must be modified.
001822S1.PPT
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Phone Number: 1-816-650-0774
USP Diphenhydramine Hydrochloride Method
Mobile Phase: 50% Acetonitrile:
50% Water: 0.5% Triethylamine
Prepare solution and adjust pH to 6.5
with glacial acetic acid
Column: 4.6 x 250 mm, L10 (CN)
Flow Rate: 1mL/min
Detection: UV 254 nm
System Suitability: Benzophenone and
Diphenhydramine Solution
Specifications: Rs > 2.0, Tf < 2.0 for diphenhydramine
001823S1.PPT
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Phone Number: 1-816-650-0774
Method Problems
The pH drifts and retention changes because of unreliable pH adjustment on
mobile phase with organic present.
The traditional CN column shows more changes than the SB-CN column.
October 28
Apparent pH 6.5
Time (min)
0 2 4 6 8 10
D
November 6
Apparent pH 6.2
November 1
Apparent pH 6.2
B
D
B
D
B
Cyano Cyano
Cyano
SB-CN SB-CN SB-CN
D
B
D
B
D
B
Time (min)
0 2 4 6 8 10
Time (min)
0 2 4 6 8 10
001824S1.PPT
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Phone Number: 1-816-650-0774
Method Adjustment
Adjust pH on aqueous component alone. This is done by measuring
the amount of acid it requires to get to the apparent pH, then adding
this amount to the aqueous component (with TEA). This becomes
the new pH of the mobile phase.
This procedure works best if the mobile phase is actually buffered.
001825S1.PPT
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Phone Number: 1-816-650-0774
Recommendations
Use a proper buffer and make the pH adjustment to the aqueous
portion alone, but keep the mobile phase as similar as possible to
maintain expected behavior.
Select a Rapid Resolution L10 column to minimize analysis time and
maintain resolution.
Use an SB-CN (L10) to improve reproducibility.
001826S1.PPT
Slide 16
Phone Number: 1-816-650-0774
Modified Diphenhydramine
Hydrochloride Method Parameters
Column: 4.6 x 75 mm, 3.5 mm, StableBond SB-CN (L10)
1
Mobile Phase: 55% 25 mM ammonium acetate pH 4.5/
0.5% TEA: 45% Acetonitrile
2
Flow Rate: 1mL/min
Detection: UV 265 nm
2
Temperature: RT
System Suitability: Benzophenone and Diphenhydramine Solution
Specifications: Rs > 2.0, Tf < 2.5 for diphenhydramine
1
method adjustment
2
method modification
001827S1.PPT
Slide 17
Phone Number: 1-816-650-0774
Modified Diphenhydramine
Hydrochloride Method Example
Time (min)
0 5 10
Time (min)
0 1 2 3 4 5
Column: StableBond SB-CN Mobile Phase: 55% 25 mM CH
3
COONH
4
/0.5% TEA : 45% ACN Flow Rate: 1.0 mL/min Temperature: RT
Sample: 1. Diphenhydramine 2. Benzophenone Injection Volume: 10 L Detection: UV 265 nm
4.6 x 250 mm, 5 m
R
s
= 7.5
T
f
= 1.5
4.6 x 75 mm, 3.5 m
R
s
= 5.9
T
f
= 1.4
1
2
1
2
001828S1.PPT
Slide 18
Phone Number: 1-816-650-0774
Break Number 1
For Questions and Answers
Press *1 on Your Phone to
Ask a Question
Slide 19
Phone Number: 1-816-650-0774
Method Validation Requirements
Robustness
Linearity
Accuracy
Precision
Limit of Detection
Limit of Quantitation
Specificity/Selectivity
Range
Ruggedness
001829S1.PPT
Slide 20
Phone Number: 1-816-650-0774
USP Data Requirements for
Method Validation
Impurities Product
Parameter Bulk Drug Degradates Performance
Precision Yes Yes Yes
Accuracy Yes Yes Maybe
Limit of Detection No No Maybe
Limit of Quantitation No Yes Maybe
Specificity/Selectivity Yes Yes Maybe
Range Yes Yes Maybe
Linearity Yes Yes Maybe
Ruggedness Yes Yes Yes
001830S1.PPT
Slide 21
Phone Number: 1-816-650-0774
Robustness Testing
1. pH
2. Temperature
3. % Organic
4. Buffer Concentration
5. Column Lot
Vary Key Method Parameters - meet or exceed method
adjustment recommendations
Use System Suitability Mixture - diphenhydramine/benzophenone
and focus on behavior of diphenhydramine
001831S1.PPT
Slide 22
Phone Number: 1-816-650-0774
pH Variation
pH Time R
s
T
f
A 4.0 1.78 6.2 1.7
B 4.5 1.94 5.5 1.7
C 5.0 2.14 4.0 1.8
Column: SB-CN, 4.6 x 75 mm, 3.5 m Mobile Phase: 55% 25 mM CH
3
COONH
4
/0.5% TEA : 45% ACN Flow Rate: 1.0 mL/min
Temperature: RT Sample: 1. Diphenhydramine 0.5 mg/mL 2. Benzophenone .005 mg/mL Injection Volume: 10 mL
Time (min)
0 1 2 3 4 5
1
2
Tested pH at 4.0, 4.5, and 5.0.
Monitor for substantial changes in retention, resolution,
and peak shape
1
2
1
2
A B C
Time (min) Time (min)
0 1 2 3 4 5 0 1 2 3 4 5
001832S1.PPT
Slide 23
Phone Number: 1-816-650-0774
Buffer Concentration
Column: SB-CN, 4.6 x 75 mm, 3.5 m Mobile Phase: 55% CH
3
COONH
4
(pH 4.5)/0.5% TEA : 45% ACN Flow Rate: 1.0 mL/min
Temperature: RT Sample: 1. Diphenhydramine 0.5 mg/mL 2. Benzophenone .005 mg/mL Injection Volume: 10 mL
Time (min)
0 1 2 3 4 5
1
2
Conc. Time T
f
A 20 mM 1.98 1.8
B 25 mM 1.96 1.8
C 30 mM 1.99 1.8
Tested two additional buffer strengths, 20 mM
and 30 mM
Monitor for changes in retention and peak shape
C
B
A
001833S1.PPT
Slide 24
Phone Number: 1-816-650-0774
Temperature
1
2
Column: SB-CN, 4.6 x 75 mm, 3.5 m Mobile Phase: 55% CH
3
COONH
4
(pH 4.5)/0.5% TEA : 45% ACN Flow Rate: 1.0 mL/min
Temperature: see below Sample: 1. Diphenhydramine 0.5 mg/mL 2. Benzophenone .005 mg/mL Injection Volume: 10 L
C T
f
A RT 1.6 1.8
B 30 1.6 1.7
C 35 1.6 1.7
Tested 3 temperatures Room Temperature, 30C
and 35C
Monitor for changes in selectivity () and peak
shape of diphenhydramine
C
B
A
Time (min)
0 1 2 3 4 5
001834S1.PPT
Slide 25
Phone Number: 1-816-650-0774
% Organic
Time (min)
1
2
Column: SB-CN, 4.6 x 75 mm, 3.5 m Mobile Phase: CH
3
COONH
4
(pH 4.5)/0.5% TEA : ACN Flow Rate: 1.0 mL/min
Temperature: RT Sample: 1. Diphenhydramine 0.5 mg/mL 2. Benzophenone .005 mg/mL Injection Volume: 10 mL
% ACN R
t
k R
s
A 40 2.27 2.0 7.5
B 43 2.07 1.8 6.4
C 45 1.96 1.6 5.0
D 50 1.75 1.3 2.8
Expect retention, selectivity and resolution to
change with change in organic.
Determine which mobile phase meets needs
(adequate retention, resolve matrix components)
without wasting time.
D
C
B
A
0 1 2 3 4 5
001835S1.PPT
Slide 26
Phone Number: 1-816-650-0774
Column Lot
Column: SB-CN, 4.6 x 75 mm, 3.5 m Mobile Phase: 55% CH
3
COONH
4
(pH 4.5)/0.5% TEA : 45% ACN Flow Rate: 1.0 mL/min
Temperature: RT Sample: 1. Diphenhydramine 0.5 mg/mL 2. Benzophenone .005 mg/mL Injection Volume: 10 mL
Time (min)
1
2
Compare three current lots of
material for consistency of
retention (k) and selectivity ().
0 1 2 3
Three Lot Summary
Mean SD RSD
k (D) 1.1 0.01 1.0%
k (B) 2.9 0.06 2.1%
2.6 0.05 1.8%
Lot B98001
Lot B97110
Lot B97077
001836S1.PPT
Slide 27
Phone Number: 1-816-650-0774
Break Number 2
For Questions and Answers
Press *1 on Your Phone to
Ask a Question
Slide 28
Phone Number: 1-816-650-0774
Method Validation Requirements
Robustness
Linearity
Accuracy
Precision
Limit of Detection
Limit of Quantitation
Specificity/Selectivity
Range
Ruggedness
001837S1.PPT
Slide 29
Phone Number: 1-816-650-0774
Linearity
How? Regression analysis of test results vs analyte concentration.
For the bulk substance type of samples we must cover a range of
80 - 120% of the expected concentration.
Diphenhydramine Linearity
R
2
= 0.9999
0
200000
400000
600000
800000
1000000
0 50 100 150
Level (%)
A
r
e
a


Area
Predicted
Area
Linear (Area)
001838S1.PPT
Slide 30
Phone Number: 1-816-650-0774
Accuracy
How? Calculate % recovery of known amounts added to samples
above and below expected levels. We tested the ICH* recommended
3 replicates at 3 different levels one above and two below.
Results
Level Accuracy
125% 99.6 +/- 0.2%
75% 100.3 +/- 0.8%
25% 99.2 +/- 0.7%
* ICH - International Conference on Harmonization
001839S1.PPT
Slide 31
Phone Number: 1-816-650-0774
Precision/Repeatability
How? Calculate (relative) standard deviation of a
sufficient number of sample aliquots. This can be from
three levels three repetitions or 6 determinations at 100%.
Results
Level SD RSD
125% 0.16 0.2%
75% 0.76 0.8%
25% 0.67 0.7%
001840S1.PPT
Slide 32
Phone Number: 1-816-650-0774
Limit of Detection*
Column: SB-CN, 4.6 x 75 mm, 3.5 m Mobile Phase: 55% 25 mM CH
3
COONH
4
, pH 4.5 : 45% ACN
Flow Rate: 1.0 mL/min Temperature: RT Sample: 1. Diphenhydramine
Time (min)
How?
Signal-to-noise ratio of 2:1 or 3:1 is
generally accepted. Compare to
blank. We used a S/N of 3:1.
Result 0.2 ppm (2 ng on column)
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
1
*not required for validation of this method
001841S1.PPT
Slide 33
Phone Number: 1-816-650-0774
Limit of Quantitation*
Column: SB-CN, 4.6 x 75 mm, 3.5 m Mobile Phase: 55% 25 mM CH
3
COONH
4
, pH 4.5 : 45% ACN
Flow Rate: 1.0 mL/min Temperature: RT Sample: 1. Diphenhydramine 2. Benzophenone
Time (min)
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
How?
Determine the standard deviation of
blank response x10. Verify accuracy
and precision with samples close to
the calculated limit.
Result 1.2 ppm (12 ng on column)
with precision = 5.9%
1
2
*not required for validation of this method
001842S1.PPT
Slide 34
Phone Number: 1-816-650-0774
Specificity/Selectivity
Column: SB-CN, 4.6 x 75 mm, 3.5 m Mobile Phase: 55% 25 mM CH
3
COONH
4
, pH 4.5 : 45% ACN
Flow Rate: 1.0 mL/min Temperature: RT Sample: 1. Diphenhydramine
How?
Compare test results from
samples with impurities,
degradation products,
excipients etc. with those
without.
Time (min)
0 1 2 3 4 5
1
1
Fresh Tablet Degraded Tablet
Time (min)
0 1 2 3 4 5
001843S1.PPT
Slide 35
Phone Number: 1-816-650-0774
Range
How? Verify acceptable precision, accuracy, and linearity at the ends of the
range and within the range. Our tested range went up to the 175% level.
Therefore we needed to verify linearity, accuracy, and precision at this level, in
addition to those done previously.
Diphenhydramine Linearity
Extended Range
R
2
= 0.9998
0
500000
1000000
1500000
0 100 200
Level (%)
A
r
e
a
Area
Predicted
Area
Linear (Area)
Level Acc. SD RSD
175% 100.6% 0.40 0.4%
001844S1.PPT
Slide 36
Phone Number: 1-816-650-0774
Ruggedness/Reproducibility
How? Multiple chemists in multiple labs run samples. Results should be
reproducible and can be compared to method precision.
Result Samples were run in 3 labs by 3 chemists on 3 different instruments.
Chemist 1 Chemist 2 Chemist 3
Level Accuracy/RSD Accuracy/RSD Accuracy/RSD
125% 99.6 +/- 0.2% 100.2 +/- 0.8% 99.0 +/- 0.8%
75% 100.3 +/- 0.8% 100.5 +/- 0.0% 100.5 +/- 0.3%
125% 99.2 +/- 0.7% 100.6 +/- 0.0% 101.0 +/- 0.7%
Overall 99.7 +/- 0.9% 100.4 +/- 0.4% 100.2 +/- 1.0%
Method 100.0 +/- 0.9%
001845S1.PPT
Slide 37
Phone Number: 1-816-650-0774
Determining System Suitability Specifications
What type of variation do you see normally and
how much leeway do you want?
What makes accurate chromatographic results
possible?
Try to account for column degradation and
insufficiently tested methods.
001846S1.PPT
Slide 38
Phone Number: 1-816-650-0774
System Suitability
Setting System Suitability Specifications:
Tailing Factor < 2.5 (allows for higher sample load)
Resolution > 2.0 (allows for method variation and
column aging)
RSD of replicate injections < 2.0% (checks system
performance)
001847S1.PPT
Slide 39
Phone Number: 1-816-650-0774
Conclusions
New suggested guidelines may make it easier to determine
what is a method adjustment to meet system suitability
requirements.
When needed method modifications exceed adjustments
then method validation is required.
Method validation requires experimentation to verify that a
method will meet analytical needs.
001848S1.PPT
Slide 40
Phone Number: 1-816-650-0774
Acknowledgments
Agilent Technologies
LFAD, Newport Site
John Henderson
Bud Permar
001849S1.PPT
Slide 41
Phone Number: 1-816-650-0774
Wrap-up E-Seminar Questions