KATHOLIEKE UNIVERSITEIT LEUVEN
GROUP BIOMEDICAL SCIENCES
FACULTY OF PHARMACEUTICAL SCIENCES
Development of International
Pharmacopoeia monographs for selected
protease inhibitors
J.HOOGMARTENS
Yekkala Raja
Laboratory for Pharmaceutical Analysis
OUTLINE
 Introduction
 Aim of the study
 WHO strategy
 Development of monographs
 Results
Indinavir sulfate
Saquinavir (mesilate)
Nelfinavir mesilate
 Conclusions
- LC method improvement
- Selectivity of C18 columns towards
indinavir sulfate components
- Validation
INTRODUCTION
Current status of protease inhibitors (PIs)
 Relatively new class of drugs
 Extensively used in human (chronic) therapy
eg: highly active anti retroviral therapy (HAART)
 Obtained by synthetic procedures
 Impurities are often closely related substances
- difficult to remove
INTRODUCTION
Current status of protease inhibitors (PIs)
 Change of quality
- transfer of production to other countries with
cheaper production capabilities and less stringent GMP
and environment regulations
- use of different synthetic route
 Impurities in pharmaceuticals can have significant
effect on their quality and safety
INTRODUCTION
Current status of protease inhibitors (PIs)
 Very few papers have been published on LC
methods for assay and purity control
 Some monographs are published in official
compendia such as Indian Pharmacopoeia (IP) and
United States Pharmacopeia (USP)
 Most often these monographs prescribe to use
expensive reference standards
 Simple and better analytical methods needed for assay
and purity control in International Pharmacopoeia
OUTLINE
 Introduction
 Aim of the study
 WHO strategy
 Development of monographs
 Results
Indinavir sulfate
Saquinavir (mesilate)
Nelfinavir mesilate
 Conclusions
- LC method improvement
- Selectivity of C18 columns towards
indinavir sulfate components
- Validation
AIM OF THE STUDY
 HIV infected patients have no cure but their quality of
life can be improved with antiretroviral (HAART) therapy
 The World Health Organization (WHO) is interested in
having analytical methods and specifications for the
International Pharmacopoeia (Int. Ph.)
 Monographs are made available worldwide to evaluate
generic drugs
 Analytical methods and specifications provided by the
laboratories collaborating in this project were used as
starting point
AIM OF THE STUDY
To develop complete monographs for selected PIs
CH3
H 3C
CH3
H 3C
O
O

HO
H
H
N
H
NH2
CH3
CH3
NH
H
H OH
N
N
NH
H 3C
S
CH3

HO
O
H
NH
N
OH
H
CH3
CH3
CH3
Saquinavir (mesilate)
H3 C
CH3
CH3
CH3
S
NH
N
O
N
H
H
N
OH
O
Indinavir sulfate
H3C SO3H
N
OH
O
O
NH
H
N
NH H
N

N
H
HO
o
H2SO4
O
S
O
CH3
H3C
H
SO3H
O
N
CH3
H 3C
H
N
H
CH3
H
N
O
H H OH
H
Nelfinavir mesilate
O
H
N
Ritonavir
N
H
O
S
H3C SO
OUTLINE
 Introduction
 Aim of the study
 WHO strategy
 Development of monographs
 Results
Indinavir sulfate
Saquinavir (mesilate)
Nelfinavir mesilate
 Conclusions
- LC method improvement
- Selectivity of C18 columns towards
indinavir sulfate components
- Validation
WHO STRATEGY
Quality control methods at different levels (step-wise strategy)
1. Basic tests (an indication of identity)
- Colour reactions
- Precipitation reactions
 Simple and readily applicable
 Useful when fully equipped laboratory and/or analytical
expertise are not available and when rapid control is
necessary
 Limited role but helps in primary testing and for
detection of counterfeit drugs
WHO STRATEGY
2. Screening tests (confirmation of identity)
- Thin layer chromatography (TLC*)
 These tests help to detect counterfeit drugs and gross
contamination
 However, screening tests can not replace full analysis
* WHO collaborating Center for Chemical Reference
Substances, Centrallaboratoriet, Kungens Kurva, Sweden
WHO STRATEGY
3. Full analysis (identification, purity testing and assay)
Identification tests
- Infrared spectroscopy (IR)
- UV spectroscopy
Purity testing
- Specific optical rotation
- Limit tests for minerals
Heavy metals and Sulfated ash
- Water content / Loss on drying
- Residual solvents (ICH guidelines)
- Related substances
Assay
- Titration
- UV spectroscopy
OUTLINE
 Introduction
 Aim of the study
 WHO strategy
 Development of monographs
 Results
Indinavir sulfate
Saquinavir (mesilate)
Nelfinavir mesilate
 Conclusions
- LC method improvement
- Selectivity of C18 columns towards
indinavir sulfate components
- Validation
DEVELOPMENT OF MONOGRAPHS
 The ICH guidelines for residual solvents are applicable
and monographs do not repeat this
- A method is prescribed where needed
- Limits are reported only when higher than these of ICH
 A recently adopted approach in the Int. Ph. is
implemented to provide alternative tests
- This approach helps many laboratories in developing countries
who do not have access to the more sophisticated techniques
DEVELOPMENT OF MONOGRAPHS
 When more sophisticated techniques like LC are
prescribed for assay, easier to perform techniques like UV
spectroscopic method or potentiometric titrations are also
developed as alternative
 For related substances, special attention was given to
evaluate the existing LC methods
 The best ones were used as a starting point for further
method development
 To avoid use of expensive reference compounds in
routine analysis, the system suitability tests (SSTs) were
developed by degradation of sample
OUTLINE
 Introduction
 Aim of the study
 WHO strategy
 Development of monographs
 Results
Indinavir sulfate
Saquinavir (mesilate)
Nelfinavir mesilate
 Conclusions
- LC method improvement
- Selectivity of C18 columns towards
indinavir sulfate components
- Validation
INDINAVIR SULFATE
1. Cipla
2. Indian Pharmacopoeia and Ranbaxy
3. USP
4. Farmanguinhos-Fiocruz
INDINAVIR SULFATE
1. Cipla
Stationary phase:
C4 (25 cm x 4.6 mm I.D.) 5 µm column,
at room temperature
Kromasil C4
Mobile phase:
Acetonitrile
0.1 M ammonium phosphate buffer pH 4.8
(containing 0.2 g of sod.1 hept. sulfonate)
Detection:
220 nm
Flow rate:
1.0 ml/min
- 35 vol
- 65 vol
INDINAVIR SULFATE
1. Cipla adapted
Stationary phase:
Hypersil C18
Mobile phase: gradient: faster and more sensitive
Acetonitrile
0.1 M ammonium phosphate buffer pH 4.8
(containing 0.2 g of sod.1 hept. sulfonate)
Water
Detection:
220 nm
Flow rate:
1.0 ml/min
A
25
B
70
25
25
50
5
INDINAVIR SULFATE
Cipla
24.26
19.92
20.28
21.29
14.74
15.22
13.33
10.90
11.19
12.17
9.50
7.70
6.73
5.83
2.73
3.07
3.33
3.88
18.83
2.23
5
2.12
Intensity (mV)
15.81
10
0
0
5
10
15
20
25
30
Retention time (min)
Typical chromatogram of indinavir impure sample (1 mg/ml) using
the optimized Cipla method
INDINAVIR SULFATE
2. Indian Pharmacopoeia and Ranbaxy
Stationary phase:
C8 (20 cm x 4.6 mm I.D.) 5 µm column, at 40 °C
Hypersil C8 (25 cm x 4.6 mm I.D.)
Mobile phase:
acetonitrile
- 40 vol
0.05 M sodium citrate buffer pH 5.0 - 60 vol
Detection:
260 nm
Flow rate:
1.0 ml/min
Base line noise due to the high
absorbance of citrate buffer
at lower wavelength
INDINAVIR SULFATE
3. USP
Stationary phase:
C18 (25 cm x 4.6 mm I.D.) 5 µm column, at room temperature
Hypersil BDS C18
C8
Mobile phases:
A)potassium phosphate buffer
0.54 g K2HPO4 + KH2PO4 in 2 L of water
B) acetonitrile
Gradient:
0-40 min: 20% B, 40-45 min: 20% to 70% B
Detection:
220 nm
Flow rate:
1.0 ml/min
many ghost peaks were observed
could not be continued
INDINAVIR SULFATE
4. Farmanguinhos-Fiocruz
Stationary phase:
Hypersil C18 BDS (250 x 4.6 mm I.D.,) 5 µm column,
at room temperature
Mobile phase:
Acetonitrile
Sodium phosphate buffer pH 7.5
(1.0 g/100 ml)
Water
Detection:
220 nm
Flow rate:
1.0 ml/min
A
30
B
70
5
5
65
25
Gradient: faster elution
better sensitivity
INDINAVIR SULFATE
Farmanguinhos-Fiocruz
23.29
23.89
18.26
19.10
20.09
20.42
20.93
15.93
12.01
12.72
13.12
13.75
7.15
5.97
2.21
3.40
2.75
5
1.95
Intensity (mV)
16.67
10
0
0
5
10
15
20
25
30
Retention time (min)
Typical chromatogram of indinavir impure sample (1 mg/ml) using
the optimized Farmanguinhos-Fiocruz method
INDINAVIR SULFATE
The chromatograms shown were obtained under the same integration
and detection conditions
Adapted Cipla method
- detects more impurities
Adapted Farmanguinhos method
- less tailing for principal peak
- less complex mobile phase
Sample solution: 1.0 mg/ml to 2.0 mg/ml

Column temperature: 30 °C, 35 °C, 40 °C and 45 °C
INDINAVIR SULFATE
25
3
20
6
mV
15
2
4
1. Carboxamide (CAR)
2. Unknown 1 (UNK1)
3. Indinavir (IDV)
4. Unknown 2 (UNK2)
5. Unknown 3 (UNK3)
6. Epoxide (EPO)
1
10
5
B
5
A
0
-5
0
5
10
15
20
25
30
35
40
45
Minutes
Typical chromatograms of 2.0 mg/ml (A) IDV commercial sample
and (B) IDV spiked sample solution
INDINAVIR SULFATE
Proposed system suitability test
200
SSTPK
16.843
indinavir
19.360
300
mV
3.5
100
0
0
5
10
15
20
25
Minutes
30
35
40
45
Typical chromatogram of SST solution prepared by heating 2 ml of
2 mg/ml of indinavir sulfate and 2 ml of sulfuric acid (190 g/l) in
boiling water for 10 minutes
OUTLINE
 Introduction
 Aim of the study
 WHO strategy
 Development of monographs
 Results
Indinavir sulfate
Saquinavir (mesilate)
Nelfinavir mesilate
 Conclusions
- LC method improvement
- Selectivity of C18 columns towards
indinavir sulfate components
- Validation
INDINAVIR SULFATE
Selectivity of C18 columns towards indinavir sulfate
 Hypersil BDS C18 (25 cm x 4.6 mm I.D.) 5 µm
 The manufacturer claims:
- both base-deactivated and end-capped
 Separation was examined on a set of 16 columns
- at least either base-deactivated or end-capped
 The columns were chosen based on a column ranking
system developed in our laboratory
http://pharm.kuleuven.be/pharmchem/columnclassification
 The ranking system is based on four chromatrographic
parameters
INDINAVIR SULFATE
The list of C18 columns (25 cm x 4.6 mm I.D.), 5 µm examined and
their characteristics provided by the manufacturers
No
F-value
Name of the column
End
capped
Base
deactivated
PoreSize (Å)
Manufacturer/supplier
1
0.000
Hypersil BDS C18
+
+
130
ThermoQuest
2
0.436
ACE C18
+
+
100
Advanced Chrom. Tech./Achrom
3
0.480
Discovery C18
+
-
180
Supelco
4
0.667
Supelcosil LC-18 DB
-
+
120
Supelco
5
2.135
Nucleosil HD
+
-
100
Macherey-Nagel/Filter Service
6
2.303
Validated C18
+
-
100
Perkin-Elmer
7
2.813
Platinum C18
+
+
100
Alltech
8
3.030
Symmetry
+
-
100
Waters
9
3.940
Purospher
+
-
80
Merck
10
4.698
Kromasil EKA
+
-
100
Akzo Noble/SerCoLab
11
4.888
Purospher Star
+
+
80
Merck
12
5.456
Alltima C18
+
+
120
Alltech
13
7.162
Platinum EPS C18
-
+
100
Alltech
14
9.146
LiChrospher
-
+
100
Merck
15
10.477
Apex Basic
+
+
100
Jones Chromatography/Sopachem
16
26.256
Apex ODS II
+
-
100
Jones Chromatography/Sopachem
INDINAVIR SULFATE
Quality of the separation was evaluated by both SST and CRF
Chromatographic response function (CRF)
g
f
0 < CRF < 1
co-elution of two or more peaks
CRF = 0
complete baseline separation
CRF = 1
f
g
INDINAVIR SULFATE
Quality of the separation was evaluated by both SST and CRF
Name of the column
SST > 3.5
CRF
0.000
Hypersil BDS C18
7.721
1.00
2
0.436
ACE C18
5.135
1.00
3
0.480
Discovery C18
6.532
0.94
4
0.667
Supelcosil LC-18 DB
4.150
0.86
5
2.135
Nucleosil HD
5.086
0.00
6
2.303
Validated C18
5.445
0.58
7
2.813
Platinum C18
1.907
0.00
8
3.030
Symmetry
6.284
0.91
9
3.940
Purospher
6.411
0.82
10
4.698
Kromasil EKA
7.533
1.00
11
4.888
Purospher Star
8.365
0.97
12
5.456
Alltima C18
8.161
0.65
13
7.162
Platinum EPS C18
6.796
0.00
14
9.146
LiChrospher
5.246
0.00
15
10.477
Apex Basic
1.196
0.00
16
26.256
Apex ODS II
4.398
0.23
Column No
F-value
1
F<2
No column has CRF = 0
2<F<6
2 columns have CRF = 0
F>6
3 columns have CRF = 0
INDINAVIR SULFATE
Supelcosil DB; F = 0.667; CRF = 0.86
Hypersil BDS; F = 0.000; CRF = 1.00
25
25
1.
2.
3.
4.
5.
6.
3
20
6
CAR
UNK1
IDV
UNK2
UNK3
EPO
10
6
2
1
5
1.
2.
3.
4.
5.
6.
15
10
4
3
20
mV
mV
15
CAR
UNK1
IDV
UNK2
UNK3
EPO
4
2
5
5
5
1
0
0
-5
0
5
10
15
20
25
Minutes
30
35
40
-5
0
45
10
15
20
25
Minutes
25
35
40
45
25
3
15
10
CAR
UNK1
IDV
UNK2
UNK3
EPO
15
2
6
10
4
1
5
2
5
5
1
0
1. CAR
2. UNK1
3. IDV
4. UNK2
5. UNK3
6. EPO
3
3
20
mV
1.
2.
3.
4.
5.
6.
6
20
-5
0
30
Apex ODS II; F = 26.256; CRF = 0.23
Validated; F = 2.303; CRF = 0.58
mV
5
4
5
0
5
10
15
20
25
Minutes
30
35
40
45
-5
0
5
10
15
20
25
Minutes
30
35
40
45
Chromatograms for purity control obtained on different columns for a
spiked IDV sample
INDINAVIR SULFATE
Relation between end-capping and/or base-deactivation, SST (≥ 3.5)
and sufficient quality of separation (CRF = 0.80)
number of columns
complying with SST (≥ 3.5)
/number of this type of
columns examined
4/6
number of columns with
CRF > 0.80 /number of this
type of columns examined
Endcapped
Basedeactivated
+
+
+
-
7/7
4/7
-
+
3/3
1/3
3/6
 The probability of obtaining a good separation for indinavir sulfate
and its impurities (CRF > 0.80) was higher on the end-capped columns
than on the base-deactivated columns examined
It is observed that SST criteria do not always give the required
information
OUTLINE
 Introduction
 Aim of the study
 WHO strategy
 Development of monographs
 Results
Indinavir sulfate
Saquinavir (mesilate)
Nelfinavir mesilate
 Conclusions
- LC method improvement
- Selectivity of C18 columns towards
indinavir sulfate components
- Validation
INDINAVIR SULFATE
Robustness
 The influence of 4 chromatographic paramaters on the separation
was investigated using Hypersil BDS C18 column
Chromatographic parameter settings applied:
Parameter
Low value (-)
Central value (0)
High value (+)
Acetonitrile (%)
27
30
33
Buffer (%)
4
5
6
pH
7.2
7.5
7.8
Temperature (°C)
37
40
43
 Central composite design was applied using Modde 5.0
statistical graphic software (Umetrics, Umeå, Sweden)
INDINAVIR SULFATE
Regression coefficient plots
RES5
UNK3-EPO
0,00
-0,40
regression coefficient bar
pH*T
Buf*T
Buf*pH
ACN*T
ACN*pH
ACN*Buf
T*T
ACN*ACN
T
pH
Buf
ACN
pH*pH
error bar
-0,80
Buf*Buf
Regressioncoefficients
0,40
Variables and interactions
Individual and interaction parameter effects on the resolution for the
UNK3-EPO
INDINAVIR SULFATE
Response surface plot
UNK3-EPO
RES5
Response surface plot of UNK3-EPO as a function of acetonitrile in
the mobile phase and temperature of the column
INDINAVIR SULFATE
Quantitative aspects
IDV
R2
S y,x
nc
ni
7489 x + 605115
0.985
883108
11
3
0.18 - 500
11077 x + 56073
0.998
98201
7
3
LOD
LOQ (µg/ml)
Concentrations
Regression equation
(µg/ml)
(n = 6 ; % RSD)
range (µg/ml)
y
0.18 - 2500
0.06
0.18 (1.64)
CAR
0.16
0.50 (4.55)
0.50 - 50
16141 x -331
0.999
489
5
3
EPO
0.04
0.12 (2.92)
0.12 - 50
99217 x + 19187
0.999
31454
5
3
R2: coefficient of determination
Sy,x: standard error of estimate
nc: number of experimental concentrations studied
ni: number of injections for each concentration
y: peak area; x: concentration injected (µg/ml)
INDINAVIR SULFATE
Precision data for IDV and some of its impurities
UNK1
UNK2
UNK3
IDV
0.19
0.16
0.05
99.60
Day 1
0.79
1.29
0.91
0.57
Day 2
0.97
1.58
0.84
0.61
Day 3
0.48
2.20
2.92
0.72
% RSD (n = 27)
Days 1-3
1.15
2.28
3.28
0.69
% RSD (n = 9)
Day 4
0.76
0.67
1.85
0.34
% RSD (n = 18)
Days 3-4
0.81
1.59
3.94
0.57
Level (%)
% RSD (n = 9)
INDINAVIR SULFATE
Analysis of commercial samples
Purity control of IDV samples, expressed as IDV (100 % = 2.0 mg/ml)
Sample No
Sum of impurities (%)
Total number of impurities above
disregard limit (0.05 %)
Number of impurities above 0.1 %
Complies?
1
2
3
4
5
0.25
0.26
0.40
0.06
0.05
3
3
3
1
1
0
1
2
0
0
yes
no
no
yes
yes
OUTLINE
 Introduction
 Aim of the study
 WHO strategy
 Development of monographs
 Results
Indinavir sulfate
Saquinavir (mesilate)
Nelfinavir mesilate
 Conclusions
- LC method improvement
- Selectivity of C18 columns towards
indinavir sulfate components
- Validation
SAQUINAVIR (MESILATE)
Proposed LC conditions
50.153
52.007
41.787
39.100
46.520
(C)
50.167
52.020
(B)
41.787
39.107
30.447
2.847
3.123
3.987
19.460
5
36.127
19.940
10.767
2.973
5.020
5.460
mV
10
30.407
2.387
2.850
3.127
3.983
19.460
15
36.107
20
(A)
20.970
0
-5
0
10
20
30
40
Minutes
50
60
70
Typical chromatogram of 0.5 mg/ml of (A) SQV, (B) SQVM sample 1
and (C) SQVM sample 2 using the proposed experimental conditions
SAQUINAVIR (MESILATE)
20
54.953
19.167
2.0
20.280
0
Saquinavir
13.413
11.400
7.187
5
51.073
38.187
2.390
2.867
3.967
4.833
5.360
mV
14
42.180
10
impurity Ro-31-9532
15
-5
0
10
20
30
40
50
60
70
Minutes
A typical chromatogram of 0.5 mg/ml solution of SQVM capsules
(Invirase) using the proposed experimental conditions
SAQUINAVIR (MESILATE)
Proposed system suitability test
RRT 1.8
RRT 1.9
14
40.117
mV
15
10
42.240
RRT 0.45
20
Saquinavir
25
5
2.0
22.630
10.687
0
-5
0
10
20
30
40
50
60
70
Minutes
Typical chromatogram of SST solutions prepared by heating 2 ml
of 0.5 mg/ml of SQV and 5 ml of sulfuric acid (475 g/l) in boiling
water for 30 minutes
SAQUINAVIR (MESILATE)
Hypersil BDS; F = 0.000; CRF = 1.00
ACE; F = 0.436; CRF = 1.00
Apex ODS II; F = 26.256; CRF = 0.00
Chromatograms for purity control obtained on different columns for a
spiked SQV sample
OUTLINE
 Introduction
 Aim of the study
 WHO strategy
 Development of monographs
 Results
Indinavir sulfate
Saquinavir (mesilate)
Nelfinavir mesilate
 Conclusions
- LC method improvement
- Selectivity of C18 columns towards
indinavir sulfate components
- Validation
NELFINAVIR MESILATE
Proposed LC conditions
50
(H
)(G
)(F)
(E)
(D
)(C
)(B
40
mV
30
20
2
1 3 4
2
10
56 7
9 10
8
11
12
13
2
2
4
)
(A)
6
0
0
10
20
30
40
50
60
70
80
90
M in ute s
Typical chromatograms of (A) a 20 µg/ml of reference impurity
mixture and (B to H) 2 mg/ml of NFVM commercial samples using
the proposed experimental conditions
NELFINAVIR MESILATE
50
40
mV
30
(D
)
(C)
20
10
10
2
13
4
0
0
10
5 6 7
20
8
9
30
40
50
Minutes
11 12
60
(B)
13
(A
)
70
80
90
Typical chromatograms of commercial samples using the proposed
method (A to C) NFVM tablets and (D) NFVM oral powder
NELFINAVIR MESILATE
System suitability test
mV
15
10
RRT 1.9
48.537
54.123
15
Nelfinavir
RRT 0.2
20
RRT 1.8
25
4.0
5
6.097
27.320
0
-5
0
10
20
30
40
50
60
70
80
90
Minutes
Typical chromatogram of SST solutions prepared by heating 2 ml
of 2.0 mg/ml solution of NFVM and 5 ml of sulfuric acid (475 g/l)
in boiling water for 30 minutes.
NELFINAVIR MESILATE
(A)
m V
20
10
0
0
1
2
…3 4
…
..
.
10
8 = NFVM
NFVNFV
MNFVM
56
7
20
30
20
mV
10 11
9
40
13
12
50
M in u t e s
Hypersil BDS
F = 0.000; CRF = 0.90
60
70
80
(B)
8 = NFVM
NFVNFV
MNFVM
10
2
0
0
1 3 4 5
…
…
.. 1 0
.
6
9
7
20
30
40
1110
50
M in u t e s
60
Nucleosil HD
F = 2.135; CRF = 1.00
13
12
70
80
90
(C)
20
mV
90
10
2
1
0
0
3 4
5 6
7
10
20
8 = NFVM
NFVNFV
MNFVM
10
30
40
12 ?
9
50
M i n u te s
11
Apex ODS
F = 26.256; CRF = 0.00
13
60
70
80
90
Chromatograms for purity control obtained on different columns for a
spiked NFVM sample
OUTLINE
 Introduction
 Aim of the study
 WHO strategy
 Development of monographs
 Results
Indinavir sulfate
Saquinavir (mesilate)
Nelfinavir mesilate
 Conclusions
- LC method improvement
- Selectivity of C18 columns towards
indinavir sulfate components
- Validation
CONCLUSIONS
Complete monographs of indinavir sulfate, saquinavir (mesilate), and
nelfinavir mesilate for implementation in the Int. Ph. were developed
System suitability tests (SSTs) were developed by degradation of
sample solutions
Separation of each molecule and its impurities was examined on a
number of columns using the column classification system
developed in our laboratory
Separation of each molecule and its impurities was investigated
based on proposed SSTs and CRF
It is observed that SST criteria do not always provide the required
information
CONCLUSIONS
The SST, developed on a single brand of stationary phase, does
not allow to adequately distinguish between suitable columns and
non suitable columns
Resolution control is better by the use of reference
substances/samples, but this is difficult to realize in the Int. Ph.
context
The best separation for the analysis of both indinavir sulfate and
saquinavir (mesilate) was achieved on end-capped columns
The use of end-capped columns can improve the selection of
suitable columns for separation of nelfinavir mesilate
CONCLUSIONS
It is clear that the selection of suitable column is not easy
The procedure in base-deactivation and end-capping varies from
manufacturer to manufacturer and they do not provide much
information how these modifications are done
As a consequence, it is difficult to assess the properties of a
column based on the information provided
The column characterization and classification procedure as
developed by our laboratory, indicated to be a helpful tool for
problems of this kind
CONCLUSIONS
For each of the separations, a robustness study was performed by
means of experimental design
Proposed methods were found to be robust, except for nelfinavir
mesilate (pH of the buffer should be monitored carefully)
Besides methods for the analysis of the active drug substances,
methods for the analysis of saquinavir (mesilate) and nelfinavir
mesilate drug products (dosage forms) were also developed
Proposed methods showed good selectivity, repeatability, linearity
and sensitivity