Chapter V Development and validation of an LC -MS/MS...
Transcript of Chapter V Development and validation of an LC -MS/MS...
Chapter V
Development and validation of an LC-MS/MS
method for the determination of Lansoprazole
in human plasma
163
5.1. DEVELOPMENT AND VALIDATION OF AN LC-MS/MS METHOD
FOR THE DETERMINATON OF LANSOPRAZOLE IN HUMAN PLASMA
5.1.1. INTRODUCTION
Drug profile:
Lansoprazole, a benzimidazole derivative, is a proton pump inhibitor that acts on the
membrane H+/K+-ATP (adenosine triphosphatase) in gastric parietal cells133
. It is
effective in the treatment of various peptic diseases, including gastric and duodenal
ulcer, reflux esophagitis, Zollinger–Ellison syndrome134
, and other hyperacidic-related
conditions. Lansoprazole is metabolized in the liver and the main metabolites are 5-
hydroxy lansoprazole and lansoprazole sulphone. Formation of the 5-hydroxy
metabolite is mainly by cytochrome P4502C19 (CYP2C19), whereas CYP3A4 is
involved in the formation of the sulphone135, 136
. It is clinically important to measure
CYP2C19 activity using the hydroxylation and sulfoxidation indexes of lansoprazole,
which reflects phenotype, and genotype of CYP2C19137
. Lansoprazole is active
against Helicobacter pylori. The plasma elimination half-life of lansoprazole does not
reflect its duration of suppression of gastric acid secretion. Thus, the plasma
elimination half-life is less than two hours, while the acid inhibitory effect lasts more
than 24 hours.
Fig.1 Chemical structure of Lansoprazole
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Properties:
Chemical name : 2-[[[3-methyl-4-(2,2,2-trifluoro-ethoxy)-2-
pyridinyl]methyl]sulfinyl]-1H- benzimid-azole
Chemical formula : C16H14F3N3O2S
Molecular weight : 369.3610
CAS Registry Number : 103577-45-3
pKa : 8.73
Solubility : Lansoprazole is freely soluble in dimethyl- formamide; soluble in
methanol.
Physical state : White to brownish-white odorless crystalline powder
Therapeutic Category : Anti ulcer agents, Proton-pump inhibitor(PPI)
Trade names : Prevacid,Lanzol; Lanzopral; Lanzor; Lasoprol
Bioavailability : 80% or more
T ½ : 1-1.5 hours
Plasma protein binding : 97%
Metabolism : Hepatic(CYP3A4 & CYP2C19 mediated)
Elimination : Renal and Fecal
Antisecretory activity:
After oral administration, lansoprazole was shown to significantly decrease the
basal acid output and significantly increase the mean gastric pH and percent of time
the gastric pH was >3 and >4. Lansoprazole also significantly reduced meal-
stimulated gastric acid output and secretion volume, as well as pentagastrin-
stimulated acid output. In patients with hypersecretion of acid, lansoprazole
significantly reduced basal and pentagastrin- stimulated gastric acid secretion.
Lansoprazole inhibited the normal increases in secretion volume, acidity and acid
output induced by insulin.
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Drug interactions:
The absorption of certain drugs may be affected by stomach acidity, and, as a
result, lansoprazole and other PPIs that reduce stomach acid also reduce the
absorption and concentration in blood of ketoconazole and increase the absorption
and concentration in blood of digoxin. This may lead to reduced effectiveness of
ketoconazole or increased digoxin toxicity, respectively.
Side effects:
Lansoprazole like other PPIs were well-tolerated. The most common side
effects are diarrhea, nausea, vomiting, constipation, rash and headaches. Dizziness,
nervousness, abnormal heartbeat, muscle pain, weakness, leg cramps and water
retention rarely occur.
High doses and long-term use (1 year or longer) may increase the risk of
osteoporosis-related fractures of the hip, wrist, or spine. Therefore, it is important to
use the lowest doses and shortest duration of treatment necessary for the condition
being treated.
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Past work on the chromatographic method for lansoprazole:
1. Avgerinos Aetal138
., have reported a simple and rapid (extraction less) high-
performance liquid chromatographic method with UV detection at 230 nm for the
determination of lansoprazole in biological fluids and pharmaceutical dosage.
Niflumic acid was added as internal standard. The separation was performed at
ambient temperature on a C18 Spherisorb column with acetonitrile + 0.1 M sodium
acetate (40:60, v/v, pH 7) as mobile phase. The retention time was 5.2 min for
niflumic acid and 6.7 min for lansoprazole. The detection limit was 20 ng/ml using a
100 µl loop. The method was successfully applied to a pharmacokinetic study of
lansoprazole in humans.
2. Isamu Aokietal139
., have reported a simple and sensitive high-performance liquid
chromatographic method with ultraviolet detection for the simultaneous determination
of lansoprazole and its metabolites in human serum and urine. The analytes in serum
or urine were extracted with diethyl ether—dichloromethane (7:3, v/v) followed by
evaporation, dissolution and injection into a reversed-phase column. The recoveries of
authentic analytes added to serum at 0.05–2 μg/ml or to urine at 1–20 μg/ml were
greater than 88%, with the coefficients of variation less than 7.1%. The minimum
determinable concentrations of all analytes were 5 ng/ml in serum and 50 ng/ml in
urine. The method was successfully applied to a pharmacokinetic study of
lansoprazole in human.
3. A. P. Sherjeetal140
., have reported two simple, accurate and precise
spectrophotometric methods for simultaneous determination of lansoprazole and
domperidone in pharmaceutical dosage form. Method A involves formation of Q-
absorbance equation at 256.0 nm (isoabsorptive point) and at 294.2 nm while method
B is two wavelength method where 277.6 nm, 302.1 nm were selected as λ1 and λ2 for
determination of lansoprazole and 231.3 nm, 292.0 nm were selected as λ1 and λ2 for
determination of domperidone. Both the methods were validated statistically and
recovery studies were carried out. The Beer's law limits for each drug individually
and in mixture was within the concentration range of 5-50 μg/ml. Linearity of
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lansoprazole and domperidone were in the range of 24-36 μg/ml and 8-12 μg/ml,
respectively. The proposed methods have been applied successfully to the analysis of
the cited drugs either in pure form or in pharmaceutical formulations with good
accuracy and precision. The method herein described can be employed for quality
control and routine analysis of drugs in pharmaceutical formulations.
4. Julie De Smetet al141
., have reported a hydrophilic interaction LC method with
MS/MS and validated for the simultaneous quantification of omeprazole and
lansoprazole in human plasma. Chromatographic separation was achieved on a Betasil
silica column using a high organic mobile phase (eluent A: ACN/formic acid
997.5:2.5 v/v; eluent B: water/formic acid 997.5:2.5 v/v) and gradient elution. The
mass spectrometer was operated in the Multiple Reaction Monitoring mode. Prior to
chromatography, liquid–liquid extraction with ethyl acetate was used and the organic
layer was diluted with ACN, allowing direct injection on column. The method showed
acceptable linearity, high precision (RSD%<10.5%), accuracy (88.9–109.3%) and
selectivity in the two concentration ranges studied: 1.5–100 and 5–2000ng/mL. The
LOQ was established at 1.5 and 5ng/mL for the two concentration ranges. Lack of
variability in matrix effects was demonstrated and mean extraction recovery for
omeprazole and lansoprazole was determined in the low (56.3–67.7%) and high
(45.3–44.3%) concentration range, respectively. Additionally, plasma samples were
found to be stable after three freeze–thaw cycles and for at least 15h after extraction.
This assay was successfully applied to a pharmacokinetic omeprazole study in
children with cerebral palsy and mental retardation.
5. Takanori Hishinuma et al142
., have reported a rapid, simple and highly sensitive
method for the quantitative determination of lansoprazole and rabeprazole
concentrations in 20 μL of human serum using high-performance liquid
chromatography/tandem mass spectrometry (LC/MS/MS). Analytes, along with an
internal standard (lansoprazole deuterium derivatives), were separated using a mobile
phase of acetonitrile/1 mM ammonium formate (140/60, v/v) on a C18 analytical
column and analyzed in the selected reaction-monitoring (SRM) mode. The lower
limit of quantification was 0.25 ng/mL. A good linear response was observed for each
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analyte (from 0.25 ngto 2.5 μg/mL). This method was useful for therapeutic drug
monitoring and pharmacokinetic studies.
6. PrasannaReddy.Battuet al143
., have reported a simple, selective, accurate high
Performance Liquid Chromatographic (HPLC) method and validated for the analysis
of Lansoprazole. Chromatographic separation achieved isocratically on a C18
column (Inertsil C18, 5 , 150 mm x 4.6 mm) utilizing a mobile phase of
acetonitrile/phosphate buffer (70:30, v/v, pH 7.0) at a flow rate of 0.8 ml/min with
UV detection at 260 nm. The retention time of lansoprazole was 2.53 min. The
method is accurate (99.15-101.85%), precise (intra-day variation 0.13-1.56% and
inter-day variation 0.30-1.60%) and linear within range 0.1-30µg/ml (R2=0.999)
concentration and was successfully used in monitoring left over drug. The detection
limit of lansoprazole at a signal-to-noise ratio of 3 was 1.80ng/ml in human plasma
while quantification limit in human serum was 5.60 ng/ml. The proposed method is
applicable to stability studies and routine analysis of lansoprazole in pharmaceutical
formulations as well as in human plasma samples.
7. Prasanna Reddy.B, et al144
., have reported a simple, sensitive and precise high
performance liquid chromatographic method for the analysis of Pantoprazole sodium
and lansoprazole , validated and used for the determination of compounds in
commercial pharmaceutical products. The compounds were well separated an
isocratically on a C18 column (Inertsil C18, 5µ , 150 mm x 4.6 mm) utilizing a
mobile phase consisting of acetonitrile: phosphate buffer (60:40, v/v, pH 7.0) at a
flow rate of 1.0 ml/min with UV detection at 230 nm. The retention time of
Pantoprazole sodium and lansoprazole was found to be 2.017 min and 2.538. The
procedure was validated for linearity (Correlation coefficient = 0.999). The study
showed that reversed-phase liquid chromatography is sensitive and selective for the
determination of Pantoprazole sodium and lansoprazole using single mobile phase.
8. Bhavna Patel et al145
.have reported a RP-HPLC method for simultaneous
estimation of lansoprazole and domperidone in combined capsule dosage form. The
mobile phase used was a combination of Acetonitrile: Methanol (81:19). The
detection of the combined dosage form was carried out at 280 nm and a flow rate
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employed was 1 ml/min. The retention time for lansoprazole and domperidone was
found to be 2.8 and 1.57 min respectively. Linearity was obtained in the concentration
range of 8-24µg/ml of lansoprazole and 8-24 µg/ml of domperidone with a correlation
coefficient of 0.9977 and 0.9992. Detector consists of photodiode array detector; the
reversed phase column used was RP-C18 (2.27µm size, 250 mm´4.6 mm i.d.)at
ambient temperature. The developed method was validated according to ICH
guidelines and values of accuracy, precision and other statistical analysis were found
to be in good accordance with the prescribed values. Thus the proposed method was
successfully applied for simultaneous determination of domperidone and lansoprazole
in routine analysis.
9. T. K. Ravi et al146
., have reported a simple, fast, precise and accurate high
performance thin layer chromatographic method for the simultaneous estimation of
lansoprazole and domperidone in tablet formulations. This method allows the
determination of 100-500 ng/spot of lansoprazole and 100-500 ng/spot of
domperidone. The mobile phase composition was n-butanol: glacial acetic acid: water
(9.3:0.25:0.5, v/v/v). Densitometric analysis of lansoprazole and domperidone was
carried out in the absorbance mode at 288 nm. The limit of detection for lansoprazole
and domperidone were found to be 10 and 30 ng/spot, respectively. The limit of
quantification for lansoprazole and domperidone were found to be 40 and 65 ng/spot,
respectively. The amounts of drug present in the tablet and recovery studies were also
carried out. The method was validated for precision, accuracy and reproducibility.
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5.1.2. EXPERIMENTAL & ANALYTICAL METHODOLOGY
i) Instrumentation
The author had attempted to develop a liquid chromatographic method for the
determination of Lansoprazole using an isocratic Shimadzu HPLC equipment
comprising of two LC10AT VP pumps, VP CTO-10AS VP column oven, a Hypurity
advance C18 column (4.6 ID X 50 mm, 5µ) and anAPI 4000 (MDS Sciex) mass
detector was used for chromatographic separation. Data acquisition was done by using
Analyst 1.4.2 software. The details of the instruments employed in the study are as
follows.
HPLC System Shimadzu
Mass Spectrometer API 4000, MDS Sciex
Deep Freezer Sanyo (-86°C) VIP Series
Refrigerated centrifuge Heraeusmegafuge
Microbalance Sartorius
Vibramax Heidolph
Vacuum pump Millipore
Refrigerator Samsung
PH meter Orion
Micropipettes, Multipette and Micro tips Brand and Eppendorf
Vortexer Spinix
Poly propylene tubes Torson’s
Water Purification System Elix 10 / Milli-Q gradient
Ultra sonicator Power Sonic510, (Hwashin Technology)
Nitrogen Evaporator ZymarkTurbovap LV station, Caliper
ii) Drug and Internal standard
The reference samples of lansoprazole and pantoprazole were gifted by
Neucon pharma Pvt Ltd., Goa.
iii) Chemicals and solvents
Lansoprazole reference standard
Pantoprazole reference standard
Methanol (HPLC grade)
Milli-Q water
Ammonium acetate (AR grade)
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Sodium hydrogen carbonate (AR grade)
Methyl tert-butyl ether (TMBE) (HPLC grade)
Human plasma
0.45µ Membrane filter
iv) Preparation of solutions
a) Lansoprazole Stock Solution
About 5 mg of Lansoprazole, working standard was weighed accurately, and
transferred to a 5 mL clean glass volumetric flask, dissolved in HPLC grade methanol
and made up the volume with the same to produce a solution of 1 mg/mL of
Lansoprazole.
The stock solutions were diluted to suitable concentrations using diluent for spiking in
to plasma to obtain calibration curve (CC) standards, quality control (QC) samples
and DIQC samples. All other final dilutions (system suitability dilutions, aqueous
mixture, recovery etc.) of Lansoprazole were prepared in mobile phase.
b) Pantoprazole Stock Solution (Internal Standard)
About 10 mg of Pantoprazole Sodium Reference standard was weighed accurately
and transferred to a 10 mL volumetric flask, dissolved in HPLC grade methanol and
made up the volume with the same to produce a solution of 1 mg/mL of Pantoprazole.
The stock solution was diluted to suitable concentration using diluent for internal
standard dilution and mobile phase for system suitability dilution.
c) Biological Matrix
Six K2 EDTA human plasma lots were screened for selectivity test. All six human
plasma lots were found free of any significant interference for Lansoprazole and
internal standard. Selectivity, Matrix Effect and sensitivity tests were performed.
After bulk spiking, aliquots (sample contained 0.5M Sodium Hydrogen Carbonate:
plasma 1:10) of 600 µL for CCs and 600 µL for QCs of spiked plasma samples were
pipetted out in prelabelled polypropylene RIA vials and then all the bulk spiked
samples were transferred to deep freezer maintained at -70 °C ± 10 °C, except twelve
sets of LQC and HQC, which were transferred to deep freezer maintained at -20 °C ±
5 °C for generation of stability data at -20 °C.
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d) Calibration Curve Standards and Quality Control Samples
Calibration curve standards consisting of a set of nine non-zero concentrations
ranging from 10.02 ng/mL to 1999.21ng/mL for Lansoprazole were prepared. Quality
control samples consisted of Lansoprazole concentrations of 10.13 ng/mL (LLOQ
QC), 30.24 ng/mL (LQC), 503.99 ng/mL (MQC1), 1007.98 ng/mL (MQC2) and
1799.97 ng/mL (HQC) were prepared. These samples were stored below -70 °C until
use. Twelve sets of LQC and HQC were transferred to the -20 °C deep freezer to
check stability at -20 °C.
Standard Concentration Lansoprazole
(ng/mL)
Standard I 2 - 3 times of Cmax 1999.21
Standard H 80% of I 1599.37
Standard G 60% of I 1199.53
Standard F 40 % of I 801.28
Standard E 20% of I 400.64
Standard D 10% of I 200.32
Standard C 5% of I 50.08
Standard B 40% of C conc. 20.03
Standard A 50% of B conc. 10.02
LLOQ QC Conc equal to A 10.13
LQC 2.5-3 times of LLOQ 30.24
MQC 1 50% of I 503.99
MQC 2 50% of I 1007.98
HQC 75- 90% of I 1799.97
e) 5mM Ammonium Acetate Buffer
About 385.00 mg of ammonium Acetate was transferred to a 1000 mL reagent bottle
and dissolved in 1000 mL of Milli Q water. The buffer was mixed well and sonicated
in an ultrasonicator for 3 to 5 minutes. The amount weighed depends on total volume
of Milli Q water to be used for preparation of 5mM ammonium Acetate buffer.
173
Buffer was prepared as and when required and used within a period of 4 days of
preparation.
f) Mobile Phase
800 mL of HPLC Methanol was transferred to a 1000 mL reagent bottle and 200 mL
of 5mM Ammonium Acetate Buffer was added it. It was mixed well, sonicated in an
ultrasonic bath for 3 to 5 minutes. The mobile phase was prepared as and when
required and used within a period of 7 days of preparation.
g) Diluent
A mixture of HPLC grade Methanol and Milli Q water was prepared in the volume
ratio of 60:40 (v/v) as diluent. It was then sonicated in an ultrasonicator for 3 to 5
minutes. The diluent was prepared as and when required and used within a period of
7 days of preparation.
h) 0.5M Sodium Hydrogen Carbonate
About 2g of Sodium Hydrogen Carbonate was transferred to a 50 mL reagent bottle
and dissolved in 50 mL of Milli Q water. The buffer was mixed well and sonicated in
an ultrasonicator for 3 to 5 minutes. The amount weighed depends on total volume of
Milli Q water to be used for preparation of 0.5M sodium hydrogen carbonate. Buffer
was prepared as and when required and used within a period of 4 days of preparation.
i) Rinsing Solution
Diluent was used as rinsing solution
j) System Suitability Solution
A mixture of drug and internal standard was prepared for system suitability test. The
concentration of drug corresponds to mean concentration of 250.18ng/mL and that of
internal standard corresponds to working concentration used for calibration range
(251.66 ng/mL). The same solution was injected as an aqueous mixture. Area Ratio
was considered for System Suitability.
174
5.1.3. ANALYTICAL METHOD DEVELOPMENT
Optimization of the chromatographic conditions
For developing the method for the assay of lansoprazole, a systematic study
of the effect of various factors was undertaken by varying one parameter at a time and
keeping all the other conditions constant. The following studies were conducted for
this purpose. A hypurity advance C18column was chosen as the stationary phase for
this study.
The mobile phase and the flow rate
In order to get sharp peaks and base line separation of the components, the
author has carried out a number of experiments by varying the commonly used
solvents, their compositions and flow rate.
To effect ideal separation of the drug under isocratic conditions, mixtures of
commonly used solvents like water, methanol and acetonitrile with or without buffers
in different combinations were tested as mobile phases on a C18 stationary phase. A
binary mixture of methanol and 5 mM Ammonium acetate buffer in a ratio of 80:20
v/v was proved to be the most suitable of all the combinations since the
chromatographic peaks obtained were well defined and resolved and free from tailing.
A mobile phase flow rate of 0.7 mL/min was found to be suitable.
The drug molecule was tuned on the mass spectrometer for the detection of the
parent ion and the daughter ion (precursor ions) by injecting 0.5 μg/mL concentration.
The tuning was carried out in both positive and negative modes of ionization, but
better sensitivity with more reproducibility was found to be observed in the positive
polarity mode. All the optimized potential parameters in the positive polarity mode
have been given in the following table. The mass spectrum of the drug molecule is
given (Fig.2).
175
Fig. 2 Mass Spectra of lansoprazole for parent & product masses
176
RETENTION TIMES:
Lansoprazole : 1.15 ± 0.3 min
Pantoprazole : 1.20 ± 0.3 min
DETECTION:
Source/ Gas Parameters (positive mode): API 4000
Parameter Value
Lansoprazole Pantoprazole
NEB(psi) 6.00 6.00
CUR(psi) 12 10.00 10.00
CAD(psi) 4 7.00 7.00
IS(v) 5500 5500.00 5500.00
TEM (°C) 400 450.00 450.00
DP(v) 18 30.00 30.00
CE (v) 15 17.00 17.00
CXP(v) 6.0 15.00 15.00
FP(v) 6.0 130.00 130.00
EP(v) 10.0 10.00 10.00
Drug name Parent mass (amu) Product mass (amu)
Lansoprazole 370.00 251.90
Pantoprazole 384.20 200.00
177
Retention time of Lansoprazole
A model chromatogram showing the separation of lansoprazole is presented in
Fig. 3 under the above optimized conditions retention times of 1.15 and 1.20 min
were obtained for lansoprazole and pantoprazole respectively.
Data acquisition and processing
All the integrations were performed by Applied Bio systems Analyst® software
version 1.4.2. The slopes, intercepts, and correlation coefficients were determined by
least squares linear regression analysis using the ratios of drug/internal standard peak
areas of Calibration curve standards. All the QC samples were also calculated by
Applied Biosystems Analyst® Software version 1.4.2.
178
Optimized Chromatographic Conditions
Parameter Value
Column Hypurity advance C18
(4.6 X 50 mm, 5µ)
Mobile phase Methanol: 5mM ammonium
acetate buffer (80: 20 v/v)
Buffer 5mM ammonium acetate buffer
Isocratic/gradient mode Isocratic
Flow rate 0.70 mL/min
Run time 2.5 min
Column oven temperature 40 ± 20C
Auto sampler temperature 150C
Volume of injection 20 μL
Rinsing volume 500 μL
Extraction process of plasma samples and their drying
Four hundred micro liters of spiked plasma calibration curve standards and the
quality control samples were transferred to a set of pre-labeled poly propylene tubes
containing each 25 µL of pantoprazole dilution (internal standard; 10.06 μg/mL). The
tubes were added with 5mL of TMBE solution and vortexed for ten seconds. To each
of the tubes 2.5 mL of extraction solvent was added. The tubes were further vortexed
for 20 min at 200 rpm on a vibramax unit and then were centrifuged at 4000 rpm for
10 min in a refrigerated centrifuge at 40C. From the centrifuged tubes approximately
4.0 mL of the supernatant layer was transferred to each of a new set of pre-labeled
poly propylene tubes. The contents of the tubes were evaporated in a stream of
nitrogen at 400C for 10 min and the residues of the dried tubes were reconstituted with
1000μL of the mobile phase. The contents of the tubes were vortexed and transferred
into auto-sampler vials and then analyzed by LC-MS/MS. An aliquot of 10 μL of the
sample was drawn each time from the vials in the auto sampler.
179
REPRESENTATIVE CHROMATOGRAMS
Fig. 3A Representative Chromatogram of an Aqueous Standard and Internal
Standard Mixture
180
Fig.4A Representative Chromatogram of a Blank Plasma Sample
181
Fig. 5A Representative Chromatogram of Blank Plasma with Internal Standard
Sample
182
Fig. 6A Representative Chromatogram of a LLOQ QC Sample
183
Fig.7 A Representative Chromatogram of a HQC Sample
184
Fig. 8A Representative Chromatogram of extracted Blank Plasma Sample
185
5.1.4. ANALYTICAL METHOD VALIDATION
i) Selectivity
The selectivity of the present method was evaluated by checking the blank
EDTA (Ethylene di-amine tetra acetic acid) plasma (without spiking with
Lansoprazole) obtained. Six different lots of blank plasma were screened and all of
them were found to have no significant endogenous interferences at the retention
times of the analyte and the internal standard. The representative chromatogram of the
extracted blank plasma sample was given in Fig.8. The same human EDTA plasma
lots free of interfering substances were used to prepare the calibration curve standards
and the quality control samples for the validation study.
186
ii) S
ensitiv
ity
The lo
west lim
it of reliab
le quan
tification fo
r Lan
soprazo
le was set at th
e concen
tration o
f the L
LO
Q, 1
0.0
2 n
g/m
L. T
he p
recision
and accu
racy fo
r L
anso
prazo
le at th
is concen
tration w
as found to
be
2.4
5%
and 1
02.3
0%
respectiv
ely. T
he resu
lts are with
in th
e
acceptan
ce limits an
d g
iven
in tab
les 1 &
2.
Accep
tan
ce criteria
:
A
ccurac
y m
ust b
e ±20%
of n
om
inal v
alue &
its precisio
n m
ust b
e ≤20%
67%
of L
LO
Q sam
ples in
jected sh
ould
be w
ithin
±20%
of resp
ective n
om
inal v
alue.
Tab
le -1
Con
centra
tion
Resp
on
se Lin
earity
Data
for M
atrix
effect an
d S
ensitiv
ity o
f Lan
sop
razo
le
C
on
centra
tion
(ng/m
L)
Slo
pe
Intercep
t R
R
2
ST
D-A
S
TD
-B
ST
D-C
S
TD
-D
ST
D-E
S
TD
-F
ST
D-G
S
TD
-H
ST
D-I
ME
CC
#
10.0
2
20.0
3
50.0
9
200.3
4
400.6
9
801.3
7
1199.6
6
1599.5
4
1999.4
3
1
9.9
3
20.2
9
50.4
2
201.0
2
411.4
2
846.9
0
1153.0
9
1542.8
4
1952.1
7
0.0
007
0.0
002
0.9
994
0.9
988
% N
om
inal
99.1
0
101.3
0
100.6
6
100.3
4
102.6
8
105.6
8
96.1
2
96.4
6
97.6
4
187
Table 2
Within Batch Precision and Accuracy for Sensitivity of Lansoprazole
S.No
Conc. (ng/mL)
LLOQ
10.02
1 10.47
2 9.97
3 9.92
4 10.50
5 10.38
6 10.26
AVERAGE 10.250
SD 0.2511
%CV 2.45
% NOMINAL 102.30
iii) Matrix effect
Matrix effect for Lansoprazole was evaluated by analyzing all the six batches of
plasma at low (LQC) and high (HQC) concentrations. No significant matrix effect
was observed in all the six batches (batch no.P220410-284, P050510-286, P050510-
287,P050510-288,P050510-289,P050510-290) of plasma for Lansoprazole at low
(LQC) and high (HQC) concentrations. The precision and accuracy for Lansoprazole
at LQC concentration was found to be 1.95% and 107.10% respectively and at HQC
concentration was found to be 0.73% and 98.45% respectively. The results are within
the acceptable limits and given in tables 1 & 3.
Acceptance criteria:
The matrix effect is nullified if the mean % nominal is within 15% & CV%
≤15% at low & high QC.
188
Tab
le-3
Matrix
Effect o
f Lan
sop
razo
le
Pla
sma (B
atch
No.)
ME
QC
#
Con
centra
tion
(ng/m
L)
Mea
n
ME
QC
#
Con
centra
tion
(ng/m
L)
Mea
n
LQ
C
HQ
C
30.2
6
1801.3
2
P220410
-284
1,2
,3
31.1
5
31.7
2
31.8
7
31.5
8
1,2
,3
1752.7
5
1752.8
8
1758.3
2
1754.6
5
P050510
-286
1,2
,3
31.8
7
31.7
6
32.1
1
31.9
1
1,2
,3
1757.0
8
1776.4
9
1766.3
3
1766.6
3
P050510
-287
1,2
,3
32.1
4
32.0
7
32.0
7
32.0
9
1,2
,3
1763.1
6
1776.7
9
1760.8
5
1766.9
3
P050510
-288
1,2
,3
32.9
0
32.8
8
33.2
1
33.0
0
1,2
,3
1768.9
3
1786.6
8
1779.1
1
1778.2
4
P050510
-289
1,2
,3
32.9
2
32.4
1
32.9
0
32.7
4
1,2
,3
1793.1
4
1774.8
6
1788.2
7
1785.4
2
P050510
-290
1,2
,3
33.2
8
32.9
4
33.1
4
33.1
2
1,2
,3
1784.9
4
1795.4
8
1784.5
3
1788.3
2
Mea
n
32.4
08
1773.3
66
S.D
. 0.6
317
12.8
814
C.V
.(%)
1.9
5
0.7
3
% N
om
inal
107.1
0
98.4
5
N
6
6
189
iv
) Lin
earity
T
he lin
earity o
f the m
ethod w
as determ
ined
by a w
eighted
(1/X
2 w
here X
is concen
tration) least sq
uare reg
ression an
alysis o
f the
standard
plo
ts associated
with
the eig
ht p
oin
t standard
curv
e for L
anso
prazo
le. The calib
ration lin
e was lin
ear in th
e range o
f 10.0
2 to
1999.2
1ng/m
L o
f the d
rug as sh
ow
n in
Fig
.7. A
straight-lin
e fit mad
e thro
ugh th
e data p
oin
ts by least sq
uare reg
ression an
alysis sh
ow
ed
a constan
t pro
portio
nality
with
min
imal d
ata scattering. T
he co
rrelation co
efficient (r) w
as greater th
an 0
.99 an
d ran
ged
from
0.9
942 to
0.9
976 fo
r Lan
soprazo
le.
Accep
tan
ce criteria
:
C
oefficien
t of co
rrelation(r
2) should
be ≥
0.9
8
D
eviatio
n o
f LL
OQ
from
nom
inal v
alue can
be ±
20%
D
eviatio
n o
f standard
s oth
er than
LL
OQ
from
no
min
al valu
e can b
e ±15%
N
o tw
o co
ncecu
tive C
Cs m
ust fail to
meet th
e above criteria.
75%
or atleast 6
non zero
CC
s inclu
din
g L
LO
Q &
hig
hest co
ncen
tration m
ust m
eet above criteria.
190
Tab
le 4
Con
centra
tion
-resp
on
se Lin
earity
Data
of L
an
sop
razo
le
N
om
inal C
on
centra
tion
(ng/m
L)
Slo
pe
Intercep
t R
R
2
ST
D-A
S
TD
-B S
TD
-C
ST
D-D
S
TD
-E
ST
D-F
S
TD
-G
ST
D-H
S
TD
-I
CC
#
10.0
2
20.0
3
50.0
8
200.3
2
400.6
4
801.2
8
1199.5
3
1599.3
7
1999.2
1
1
9.6
3
21.3
2
51.9
7
193.6
5
421.3
8
764.9
2
1275.8
0
1585.7
7
1816.3
0
0.0
006
0.0
004
0.9
980
0.9
960
2
9.7
6
20.9
7
50.6
4
195.3
7
427.8
2
771.0
1
1273.0
1
1589.2
9
1813.6
5
0.0
006
0.0
002
0.9
983
0.9
966
3
9.9
2
20.0
0
53.3
6
187.0
4
422.7
7
822.7
8
1232.4
7
1513.3
5
1913.3
0
0.0
006
-0.0
001
0.9
986
0.9
972
4
9.9
1
19.9
7
53.6
2
187.7
2
421.9
4
806.9
7
1232.4
1
1525.7
8
1927.4
8
0.0
006
0.0
001
0.9
988
0.9
976
6
9.7
0
20.3
6
55.3
6
211.5
6
430.7
8
793.4
8
1075.4
8
1507.5
1
1899.1
8
0.0
006
0.0
004
0.9
971
0.9
942
Mea
n
9.7
84
20.5
24
52.9
90
195.0
68
424.9
38
791.8
32
1217.8
34
1544.3
40
1873.9
82
S.D
. 0.1
282
0.6
002
1.7
828
9.9
062
4.1
468
24.2
253
82.3
040
39.9
945
54.7
952
C.V
.%
1.3
1
2.9
2
3.3
6
5.0
8
0.9
8
3.0
6
6.7
6
2.5
9
2.9
2
% N
om
inal
97.6
4
102.4
7
105.8
1
97.3
8
106.0
6
98.8
2
101.5
3
96.5
6
93.7
4
N
5
5
5
5
5
5
5
5
5
191
Fig. 9A Representative Calibration Curve for Regression Analysis
192
v) Precision and Accuracy
The precision of the assay was measured by the percent coefficient of
variation for QC samples of Lansoprazole. The accuracy of the assay was measured
by computing the ratio of the calculated mean values of the QC samples to their
respective nominal values, expressed as percentage nominal.
Within-batch Precision for Lansoprazole (% CV)
Within-batch precision for LLOQ QC ranged from 1.29 to 3.48%
Within-batch precision for LQC, MQC1, MQC2 and HQC ranged from 0.68 to 2.28%
Intra-day Precision for Lansoprazole (% CV)
Intra-day precision for LLOQ QC was 2.50%
Intra-day precision for LQC, MQC1, MQC2 and HQC ranged from 2.01 to 2.46%
Between-batch Precision for Lansoprazole (% CV)
Between-batch precision for LLOQ QC was 2.56%
Between-batch precision for LQC, MQC1, MQC2 and HQC ranged from 3.42 to
4.93%
Within-batch Accuracy for Lansoprazole (% Nominal)
Within-batch accuracy for LLOQ QC ranged from 104.54 to 108.85%
Within-batch accuracy for LQC, MQC1, MQC2 and HQC ranged from 92.91 to
112.01%
Intra-day Accuracy for Lansoprazole (% Nominal)
Intra-day accuracy for LLOQ QC was 107.15%
Intra-day accuracy for LQC, MQC1, MQC2 and HQC ranged from 94.72 to 101.33%
Between-batch Accuracy for Lansoprazole (% Nominal)
Between-batch accuracy for LLOQ QC was 106.89%
Between-batch accuracy for LQC, MQC1, MQC2 and HQC ranged from 96.95 to
103.68%
193
Acceptance criteria:
Precision: Low, medium & high QC concentrations should be within 15% &
20% for LLOQ conc.
Accuracy: Low, medium & high QC concentrations should be within ±15% &
±20% for LLOQ conc of nominal value.
Table 5
Within Batch Precision and Accuracy of Lansoprazole
Concentration (ng/mL)
LLOQ QC LQC MQC1 MQC2 HQC
QC# 10.13 30.24 503.99 1007.98 1799.97
1 10.66 29.98 504.65 990.79 1714.71
2 10.24 30.31 514.35 1005.27 1714.12
3 11.01 30.94 516.54 1025.05 1734.93
4 11.36 30.18 525.21 1029.88 1750.03
5 10.88 31.53 523.57 1042.3 1751.56
6 10.99 31.59 530.62 1044.67 1758.67
Mean 10.857 30.755 519.157 1022.993 1737.337
S.D. 0.3779 0.7016 9.2512 21.1918 19.3665
C.V.% 3.48 2.28 1.78 2.07 1.11
% Nominal 107.17 101.70 103.01 101.49 96.52
N 6 6 6 6 6
7 10.88 30.90 513.35 1006.75 1697.13
8 11.05 30.20 505.36 999.28 1676.98
9 10.74 29.99 509.55 986.33 1672.60
10 10.73 30.14 491.59 977.67 1639.99
11 10.73 29.71 492.32 977.58 1663.46
12 10.98 30.53 501.42 979.77 1684.49
Mean 10.852 30.245 502.265 987.897 1672.442
S.D. 0.1405 0.4182 8.9342 12.3656 19.5401
C.V.% 1.29 1.38 1.78 1.25 1.17
% Nominal 107.12 100.02 99.66 98.01 92.91
N 6 6 6 6 6
13 11.20 30.74 515.76 1012.14 1710.81
14 10.73 30.36 513.42 994.20 1692.78
15 10.99 30.47 513.32 1005.30 1710.39
16 11.04 30.89 522.71 1013.34 1718.93
17 11.38 31.14 520.72 1003.50 1728.20
18 10.82 31.22 523.89 1007.62 1712.95
Mean 11.027 30.803 518.303 1006.017 1712.343
194
S.D. 0.2396 0.3482 4.7248 6.9308 11.6968
C.V.% 2.17 1.13 0.91 0.69 0.68
% Nominal 108.85 101.86 102.84 99.81 95.13
N 6 6 6 6 6
19 10.41 30.36 513.28 1005.02 1677.72
20 10.76 29.55 522.99 994.90 1692.64
21 10.46 30.23 521.42 997.92 1725.45
22 11.03 29.96 522.78 1028.93 1701.96
23 10.47 29.70 509.83 999.74 1694.72
24 10.41 29.86 515.60 987.87 1691.47
Mean 10.590 29.943 517.650 1002.397 1697.327
S.D. 0.2524 0.3088 5.5407 14.1790 15.8737
C.V.% 2.38 1.03 1.07 1.41 0.94
% Nominal 104.54 99.02 102.71 99.45 94.30
N 6 6 6 6 6
31 10.71 32.87 551.90 1068.35 1895.00
32 10.80 33.66 552.54 1076.24 1892.64
33 10.47 34.22 558.44 1076.13 1906.83
34 11.10 33.70 550.32 1080.33 1908.45
35 10.89 34.30 558.44 1093.48 1903.87
36 10.91 34.49 560.51 1083.86 1930.99
Mean 10.813 33.873 555.358 1079.732 1906.297
S.D. 0.2127 0.5941 4.2619 8.4985 13.6725
C.V.% 1.97 1.75 0.77 0.79 0.72
% Nominal 106.75 112.01 110.19 107.12 105.91
N 6 6 6 6 6
195
Table 6
Intra-day Precision and Accuracy of Lansoprazole
Concentration (ng/mL)
LLOQ QC LQC MQC1 MQC2 HQC
QC# 10.13 30.24 503.99 1007.98 1799.97
1 10.66 29.98 504.65 990.79 1714.71
2 10.24 30.31 514.35 1005.27 1714.12
3 11.01 30.94 516.54 1025.05 1734.93
4 11.36 30.18 525.21 1029.88 1750.03
5 10.88 31.53 523.57 1042.3 1751.56
6 10.99 31.59 530.62 1044.67 1758.67
7 10.88 30.90 513.35 1006.75 1697.13
8 11.05 30.20 505.36 999.28 1676.98
9 10.74 29.99 509.55 986.33 1672.60
10 10.73 30.14 491.59 977.67 1639.99
11 10.73 29.71 492.32 977.58 1663.46
12 10.98 30.53 501.42 979.77 1684.49
Mean 10.854 30.500 510.711 1005.445 1704.889
S.D. 0.2718 0.6117 12.3693 24.6896 38.6340
C.V.% 2.50 2.01 2.42 2.46 2.27
% Nominal 107.15 100.86 101.33 99.75 94.72
N 12 12 12 12 12
196
Table 7
Between Batch / Inter-day Precision and Accuracy of Lansoprazole.
Concentration (ng/mL)
LLOQ QC LQC MQC1 MQC2 HQC
QC# 10.13 30.24 503.99 1007.98 1799.97
1 10.66 29.98 504.65 990.79 1714.71
2 10.24 30.31 514.35 1005.27 1714.12
3 11.01 30.94 516.54 1025.05 1734.93
4 11.36 30.18 525.21 1029.88 1750.03
5 10.88 31.53 523.57 1042.3 1751.56
6 10.99 31.59 530.62 1044.67 1758.67
7 10.88 30.90 513.35 1006.75 1697.13
8 11.05 30.20 505.36 999.28 1676.98
9 10.74 29.99 509.55 986.33 1672.60
10 10.73 30.14 491.59 977.67 1639.99
11 10.73 29.71 492.32 977.58 1663.46
12 10.98 30.53 501.42 979.77 1684.49
13 11.20 30.74 515.76 1012.14 1710.81
14 10.73 30.36 513.42 994.20 1692.78
15 10.99 30.47 513.32 1005.30 1710.39
16 11.04 30.89 522.71 1013.34 1718.93
17 11.38 31.14 520.72 1003.50 1728.20
18 10.82 31.22 523.89 1007.62 1712.95
19 10.41 30.36 513.28 1005.02 1677.72
20 10.76 29.55 522.99 994.90 1692.64
21 10.46 30.23 521.42 997.92 1725.45
22 11.03 29.96 522.78 1028.93 1701.96
23 10.47 29.70 509.83 999.74 1694.72
24 10.41 29.86 515.60 987.87 1691.47
31 10.71 32.87 551.90 1068.35 1895.00
32 10.80 33.66 552.54 1076.24 1892.64
33 10.47 34.22 558.44 1076.13 1906.83
34 11.10 33.70 550.32 1080.33 1908.45
35 10.89 34.30 558.44 1093.48 1903.87
36 10.91 34.49 560.51 1083.86 1930.99
Mean 10.828 31.124 522.547 1019.807 1745.149
S.D. 0.2774 1.5083 18.9660 34.8853 86.0509
C.V.% 2.56 4.85 3.63 3.42 4.93
% Nominal 106.89 102.92 103.68 101.17 96.95
N 30 30 30 30 30
197
vi) Stability
a) Room Temperature Stock Solution Stability
Room temperature stock solution stability was carried out at 0 and 7 hours for
Lansoprazole by injecting six replicates of prepared stock dilutions of Lansoprazole
equivalent to the middle concentration (MQC2). Comparison of the mean area
response of Lansoprazole at 7 hours was carried out against the zero hour value, the
stability was found to be 103.20% for Lansoprazole. The stability of Pantoprazole
was carried out at 0 and 7 hours by injecting 6 replicates of prepared stock dilutions of
Pantoprazole equivalent to working concentration. Comparison of the mean area
response of Pantoprazole at 7 hours was carried out against 0 hour value, the stability
was found to be 101.89% for Pantoprazole. The results are within the acceptable
limits and given in tables 8 & 9.
Acceptance criteria:
%Comparison response must be between 90% & 110% and %CV must be ‹10%
Table 8
Room Temperature Stock Solution Stability of Lansoprazole (0 and 7 Hours)
S.No. Area
0 hr 7 hr
1 4748537 4859540
2 4671954 4803355
3 4749115 4879012
4 4693151 4879191
5 4814485 4978978
6 4683376 4867933
Mean 4726769.7 4878001.5
S.D. 54194.27 56943.82
C.V.% 1.15 1.17
% Stability 103.20
198
Table 9
Room Temperature Stock Solution Stability of Pantoprazole (0 and 7 Hours)
S.No. Area
0 hr 7hr
1 5894085 5954159
2 5764199 5855727
3 5824677 5873905
4 5744427 5951531
5 5795723 5913438
6 5823693 5957598
Mean 5807800.7 5917726.3
S.D. 53001.06 44367.69
C.V.% 0.91 0.75
% Stability 101.89
b) Room Temperature Spiking Solution Stability
Room Temperature Spiking Solution Stability was carried out at 0 and 7 hours
for Lansoprazole by injecting six replicates of prepared Spiking Solution of
Lansoprazole equivalent to the middle concentration (MQC2). Comparison of the
mean area response of Lansoprazole at 7 hours was carried out against the zero hour
value of Room temperature Spiking Solution, the stability was found to be 102.45%
for Lansoprazole. The room temperature spiking solution stability of Pantoprazole
was carried out at 0 and 7hours by injecting six replicates of prepared Spiking
dilutions of Pantoprazole. Comparison of the mean area response of Pantoprazole at 7
hours was carried out against 0 hour value of Room Temperature Spiking Solution,
the stability was found to be 100.93% for Pantoprazole. The results are within the
acceptable limits and given in tables 10 & 11.
199
Table 10
Room Temperature Spiking Solution Stability of Lansoprazole (0 and 7 Hours)
S.No. Area
0 hr 7 hr
1 4748537 4818287
2 4671954 4826900
3 4749115 4782957
4 4693151 4902753
5 4814485 4815843
6 4683376 4908636
Mean 4726769.7 4842562.7
S.D. 54194.27 51166.33
C.V.% 1.15 1.06
% Stability 102.45
Table 11
Room Temperature Spiking Solution Stability of Pantoprazole (0 and 7 Hours)
S.No. Area
0 hr 7hr
1 5894085 5943890
2 5764199 5785749
3 5824677 5940271
4 5744427 5793294
5 5795723 5903994
6 5823693 5802200
Mean 5807800.7 5861566.3
S.D. 53001.06 75769.99
C.V.% 0.91 1.29
% Stability 100.93
200
c) Refrigerated Stock Solution Stability (at 2-8 °C)
Refrigerated stock solution stability of Lansoprazole and Pantoprazole was
carried out by injecting six replicates of stock dilutions. The stock solution was found
to be stable for maximum of 6 days. The six days stock Solution stability of
Lansoprazole and Pantoprazole was found to be 99.45% and 99.41% respectively.
Stability standard stock solution and comparison (fresh) standard stock solution of
Lansoprazole were prepared equivalent to the middle concentration (MQC2) level
during method validation. The response of comparison (fresh) sample was corrected
by multiplying with correction factor.
Correction factor was calculated as follows:
Correction factor = Corrected concentration of stability standard stock solution
Corrected concentration of comparison (fresh) standard stock
solution
Note:
Corrected concentration: Concentration corrected using potency, actual amount
weighed and molecular weight of the compound.
The results are within the acceptable limits and given in tables 12 & 13.
Acceptance criteria:
%Comparison response must be between 90% & 110% and %C.V must be ‹10%
201
Table 12
Refrigerated Stock Solution Stability for Lansoprazole 2-8 °C (6 days)
S.No.
Stability standard
stock
Comparison
standard stock Corrected
Response 1000731.0000 1000641.3600
Area Area
1 3954331 4039479 4039117
2 4048669 4058941 4058577
3 3988335 4048605 4048242
4 4091253 4082588 4082222
5 4043378 4068955 4068591
6 4065024 4027806 4027445
Mean 4031831.7 4054395.7 4054032.5
SD 50887.63 19952.40 19950.61
%CV 1.26 0.49 0.49
N 6 6 6
Correcting factor 0.9999
Mean response of
Standard stock 4031831.7
Mean corrected
response 4054032.5
% Response 99.45
Note:
Corrected Response =Comparison Response x Correction Factor
Correction factor =Corrected conc. of stability standard stock solution
Corrected conc. of comparison standard stock
solution
Correction factor:0.9999
% Comparison Response =Mean Response of Stability Standard Stock x 100
Mean Corrected Response of Comparison
Standard Stock
= 4031831.7X100
4054032.5
% Comparison Response= 99.45
202
Table 13
Refrigerated Stock Solution Stability for Pantoprazole 2-8 °C (6 days)
S.No.
Stability standard
Stock
Comparison
standard stock Corrected
Response 940795.9207 944042.2720
Area Area
1 4309906 4232748 4247354
2 4247266 4384337 4399466
3 4377616 4370316 4385396
4 4310405 4367940 4383012
5 4369414 4270238 4284973
6 4313597 4367993 4383065
Mean 4321367.3 4332262.0 4347211.1
SD 47487.50 63968.04 64188.77
%CV 1.10 1.48 1.48
N 6 6 6
Correcting factor 1.0035
Mean response of
Standard stock 4321367.3
Mean corrected
response 4347211.1
% Response 99.41
Note:
Corrected Response =Comparison Response x Correction Factor
Correction factor=Corrected concentration of stability standard stock solution
Corrected concentration of comparison standard stock
solution
Correction factor: 1.0035
% Comparison Response =Mean Response of Stability Standard Stock x 100
Mean Corrected Response of Comparison
Standard Stock
= 4321367.3x 100
4347211.1
% Comparison Response = 99.41
203
d
) Au
to sa
mp
ler Sta
bility
In assessin
g th
e auto
sampler stab
ility, six
sets each o
f LQ
C an
d H
QC
samples w
ere pro
cessed an
d p
laced in
the au
to sam
pler.
These sam
ples w
ere injected
after a perio
d o
f 49 h
ours an
d q
uan
tified ag
ainst fresh
ly sp
iked
calibratio
n cu
rve stan
dard
s of c
oncen
tration
range eq
uiv
alent to
that u
sed fo
r calculatio
n o
f precisio
n an
d accu
racy, refer, T
able 1
4. T
he resu
lts dem
onstrate th
at the p
rocessed
samples w
ere stable fo
r 49 h
ours. T
he p
ercent n
om
inal o
f LQ
C an
d H
QC
at 49 h
ours w
as 91.3
8%
and 9
1.2
1%
respectiv
ely an
d p
recision
of L
QC
and H
QC
at 49 h
ours w
as 2.2
8%
and 0
.74%
respectiv
ely. T
he resu
lts are with
in th
e acceptan
ce limits an
d g
iven
in tab
les 14 &
15.
Accep
tan
ce criteria
:
M
ean %
nom
inal co
nc at each
QC
sample lev
el must b
e betw
een 8
5%
& 1
15%
and th
e precisio
n m
ust b
e ≤15%
of C
.V%
A
tleast 67%
of Q
Cs in
jected at each
level sh
ou
ld b
e with
in ±
15%
of resp
ective n
om
inal v
alue.
Tab
le 14
Fresh
ly S
pik
ed C
on
centra
tion
-respon
se Lin
earity
Data
for S
tab
ilities of L
an
sop
razo
le
C
on
centra
tion
(ng/m
L)
Slo
pe
Intercep
t R
R
2
ST
D-A
S
TD
-B
ST
D-C
S
TD
-D
ST
D-E
S
TD
-F
ST
D-G
S
TD
-H
ST
D-I
0.0
007
0.0
005
0.9
990
0.9
980
FR
ES
H1 C
C
10.0
2
20.0
4
50.1
1
200.4
3
400.8
7
801.7
3
1199.3
0
1599.0
7
1998.8
3
1
10.2
6
18.7
8
52.2
1
189.4
4
420.9
9
800.9
6
1221.4
5
1604.8
6
1958.7
8
% N
om
inal
102.4
0
93.7
1
104.1
9
94.5
2
105.0
2
99.9
0
101.8
5
100.3
6
98.0
0
204
Table 15
Auto sampler Stability of Lansoprazole in Processed
Human Plasma Samples (49 hours)
Concentration (ng/mL)
LQC HQC
QC# 30.24 1799.97
79 26.82 1646.66
80 27.62 1634.14
81 27.66 1621.47
82 28.68 1644.50
83 27.19 1656.23
84 27.83 1647.13
Mean 27.633 1641.688
S.D. 0.6314 12.1610
C.V.(%) 2.28 0.74
% Nominal 91.38 91.21
N 6 6
e) Bench Top Stability
In short-term room temperature stability, six sets each of LQC and HQC, were
determined at 5 hours. The quality control samples were quantified against the
freshly spiked calibration curve standards of concentration range equivalent to that
used for calculation of precision and accuracy, refer, Table 14 Lansoprazole was
found to be stable upto 5 hours as per the acceptance criteria. The percent mean
nominal of LQC and HQC was 90.37% and 89.30% respectively and the precision of
LQC and HQC was 2.86% and 0.43% respectively. The results are within the
acceptance limits and given in tables 14 & 16.
Acceptance criteria:
Mean % nominal conc at each QC sample level must be between 85% &
115% and precision must be ≤15% of C.V%
Atleast 67% of QCs injected at each level should be within ±15% of
respective nominal value.
205
Table 16
Bench Top Stability of Lansoprazole (5 hours)
Concentration (ng/mL)
LQC HQC
QC# 30.24 1799.97
97 28.86 1596.18
98 27.37 1608.61
99 27.12 1611.18
100 26.74 1616.07
101 26.92 1608.63
102 26.95 1603.38
Mean 27.327 1607.342
S.D. 0.7807 6.8478
C.V.(%) 2.86 0.43
% Nominal 90.37 89.30
N 6 6
f) Freeze-thaw Stability
The stability of QC samples was determined after three freeze-thaw cycles.
Six replicates of LQC and HQC were analyzed after three freeze-thaw cycles. The
freeze-thaw quality control samples were quantified against the freshly spiked
calibration curve standards of concentration range equivalent to that used for the
calculation of precision and accuracy, refer, Table 14. The percent nominal for LQC
& HQC was 92.11% and 90.07% respectively and the precision for LQC & HQC was
2.97% and 0.56% respectively for three freeze-thaw cycles. The results are within the
acceptance limits and given in tables 14 & 17.
Acceptance criteria:
Mean % nominal conc at each QC sample level must be between 85%&115%
and the precision must be ≤15% of the C.V%
Atleast 67% of QCs injected at each level should be within ±15% of the
respective nominal value.
206
Table 17
Freeze Thaw Stability (FT– III Cycle) of Lansoprazole
Concentration (ng/mL)
LQC HQC
QC# 30.24 1799.97
61 26.72 1617.76
62 27.69 1614.42
63 27.92 1608.19
64 29.27 1627.98
65 27.95 1631.07
66 27.57 1628.08
Mean 27.853 1621.250
S.D. 0.8261 9.1408
C.V.(%) 2.97 0.56
% Nominal 92.11 90.07
N 6 6
g) Plasma samples Stability at -200C
Plasma samples stability at -200C was determined for 3 days using six sets
each of LQC and HQC. The quality control samples were quantified against the
freshly spiked calibration curve standards of concentration range equivalent to that
used for calculation of precision and accuracy, refer, Table 14. The mean
concentration of stability QCs were compared against the mean of the of the Ist day
QCs when injected for first time after the bulk spiking (first passing P& A Batch).
Lansoprazole was found to be stable up to 3 days at –200C as per the acceptance
criteria. The percent stability was 88.66% for LQC and 92.72% for HQC and the
precision was 1.91% for LQC and 0.64% for HQC. The results are within the
acceptable limits and given in tables 14, 18 & 19.
Acceptance criteria:
Mean % nominal conc at each QC sample level must be between 85% &
115% and the precision must be ≤15% of C.V%
Atleast 67% of QCs injected at each level should be within ±15% of
respective nominal value.
207
Table: 18Short Term Stability at -20 ˚C Lansoprazole Data for Day 0
Concentration (ng/mL)
LQC HQC
QC# 30.24 1799.97
1 29.98 1714.71
2 30.31 1714.12
3 30.94 1734.93
4 30.18 1750.03
5 31.53 1751.56
6 31.59 1758.67
Mean 30.755 1737.337
S.D. 0.7016 19.3665
C.V.(%) 2.28 1.11
% Nominal 101.70 96.52
N 6 6
Table: 19Short Term Stability at -20 ˚C Lansoprazole Data for Day 3
Concentration (ng/mL)
LQC HQC
QC# 30.24 1799.97
109 27.13 1604.27
110 27.76 1602.93
111 26.66 1627.19
112 27.92 1603.59
113 27.40 1620.48
114 26.73 1606.88
Mean 27.267 1610.890
S.D. 0.5221 10.3365
C.V.(%) 1.91 0.64
% Nominal 90.17 89.50
N 6 6
% Stability 88.66 92.72
% Stability =Mean concentration of stability LQC or HQC sample x 100
Mean concentration of LQC or HQC samples of first day
of stability testing
208
h) Wet-Extract Stability
In assessing the Wet extract stability, six sets of LQC and HQC samples were
processed and placed on the bench at room temperature. These samples were injected
after a period of 23 hours from the first set QC’s samples loaded time and quantified
against freshly spiked calibration curve standards of concentration range equivalent to
that used for calculation of precision and accuracy, refer, Table 14. The results
demonstrate that the processed samples were stable for 23 hours. The percent
nominal at 23 hours was 91.28% and 89.49% respectively and precision was 4.89%
and1.60% respectively for LQC & HQC. The results are within the acceptance limits
and given in tables 14 & 20.
Acceptance criteria:
Mean % nominal conc at each QC sample level must be between 85% &
115% and the precision must be ≤15% of C.V%
Atleast 67% of QCs injected at each level should be within ±15% of
respective nominal value.
Table 20
Wet extract Stability of Lansoprazole (23 hours)
Concentration (ng/mL)
LQC HQC
QC# 30.24 1799.97
103 26.49 1594.07
104 27.16 1622.26
105 27.44 1657.75
106 30.27 1595.76
107 26.92 1590.19
108 27.34 1604.24
Mean 27.603 1610.712
S.D. 1.3498 25.7274
C.V.(%) 4.89 1.60
% Nominal 91.28 89.49
N 6 6
209
i) R
einjectio
n S
tab
ility
In accessin
g th
e reinjectio
n stab
ility, six
sets of L
QC
and H
QC
samples w
ere pro
cessed an
d an
alysed
with
calibratio
n cu
rve
standard
. The Q
C sam
ples w
ere retained
in th
e auto
sampler an
d rein
jected after a p
eriod o
f 28 h
ours an
d q
uan
tified ag
ainst th
e initial
calibratio
n cu
rve d
ata, refer, Tab
le 21
. The m
ean co
ncen
tration o
f reinjected
QC
s was co
mpared
again
st the m
ean o
f the Q
Cs w
hen
injected
for first tim
e. The resu
lts dem
onstrate th
at the rein
jected sam
ples w
ere stable fo
r 28 h
ours. T
he p
ercent stab
ility at 2
8 h
ours w
as
99.1
0%
for L
QC
and 1
00.7
1%
for H
QC
and p
recision w
as 0.5
6%
for L
QC
and 1
.45%
for H
QC
for 2
8 h
ours. T
he resu
lts are with
in th
e
acceptab
le limits an
d g
iven
in tab
les 21, 2
2 &
23
.
Accep
tan
ce criteria
:
M
ean %
nom
inal co
nc at each
QC
sample lev
el must b
e betw
een 8
5%
& 1
15%
and th
e precisio
n m
ust b
e ≤15%
of C
.V%
A
tleast 67%
of Q
Cs in
jected at each
level sh
ou
ld b
e with
in ±
15%
of resp
ective n
om
inal v
alue.
Tab
le 21
Con
centra
tion
-resp
on
se Lin
earity
Data
for R
einjectio
n S
tab
ility (2
6 h
ou
rs)
C
on
centra
tion
(ng/m
L)
Slo
pe
Interce
pt
R
R2
S
TD
-A
ST
D-B
S
TD
-C
ST
D-D
S
TD
-E
ST
D-F
S
TD
-G S
TD
-H
ST
D-I
CC
#
10.0
2
20.0
3
50.0
8
200.3
2
400.6
4
801.2
8 1199.5
3 1
599.3
7 1
999.2
1
1
9.6
3
21.3
2
51.9
7
193.6
5
421.3
8
764.9
2 1275.8
0 1
585.7
7 1
816.3
0
0.0
006
0.0
004
0.9
980
0.9
960
%
No
min
al
96.1
1
106.4
4
103.7
7
96.6
7
105.1
8
95.4
6
106.3
6
99.1
5
90.8
5
210
Table 22
Reinjection Stability Data for Lansoprazole 0 Hours
Concentration
LQC HQC
QC# 30.24 1799.97
1 29.98 1714.71
2 30.31 1714.12
3 30.94 1734.93
4 30.18 1750.03
5 31.53 1751.56
6 31.59 1758.67
Mean 30.755 1737.337
S.D. 0.7016 19.3665
C.V.(%) 2.28 1.11
% Nominal 101.70 96.52
N 6 6
Table 23
Reinjection Stability Data for Lansoprazole 28 Hours
Concentration
LQC HQC
QC# 30.24 1799.97
1 30.69 1755.42
2 29.81 1738.28
3 30.53 1750.80
4 30.61 1742.42
5 31.08 1745.79
6 30.15 1765.60
Mean 30.478 1749.718
S.D. 0.4430 9.8533
C.V.(%) 1.45 0.56
% Nominal 100.79 97.21
N 6 6
% Stability 99.10 100.71
Calculations:
% Stability = Mean concentration of QC re-injected over a period of time x 100
Mean concentration of QCs when injected for the 1st time
211
vii) Recovery
For evaluating the recovery six sets each of quality control samples (LQC,
MQC2 and HQC) were processed and injected (extracted samples). Twenty four
blank matrix samples were processed and four sets of six each blank samples were
reconstituted with the aqueous QC dilutions at low, middle and high concentration
without internal standard, which represents 100 % extraction of analyte(s) (non-
extracted samples). Six blanks were reconstituted with the internal standard solution,
which represents 100 % extraction of internal standard. (Non-extracted sample). The
non-extracted samples were injected. The recovery comparison samples of
Lansoprazole were compared against extracted samples of LQC, MQC2 and HQC of
PA-batch-6. The recovery comparison samples of internal standard were compared
against the response of internal standard in MQC2. The mean overall recovery of
Lansoprazole was 56.65% with a precision ranged from 0.29% to 1.31%. The mean
recovery of internal standard was 80.29 % with a precision ranged from 0.95 to
1.11%. The results are within the acceptance limits and given in tables 24 & 25.
Acceptance criteria:
The C.V% of mean analyte & ISTD recoveries must be ≤15% for each QC
conc level.
The difference of % recovery between the lowest % recovery & highest %
recovery should not be more than 25%
212
Tab
le 24
Reco
very
of L
an
sop
razo
le from
Hu
man
Pla
sma
LQ
C R
espon
se M
QC
2 R
espon
se H
QC
Resp
on
se
Extra
cted Q
C
Non
Extra
cted Q
C
Extra
cted Q
C
Non
Extra
cted Q
C
Extra
cted Q
C
Non
extra
cted
QC
RE
C
QC
#
LQ
C
#(7
-12)
LQ
C
#(1
-6)
MQ
C2
#(7
-12)
MQ
C2
#(1
-6)
HQ
C
#(7
-12)
HQ
C
#(1
-6)
31
70388
130807
2258493
4111253
3927758
6486538
32
72107
128935
2256951
4099403
3926009
6495452
33
71497
133794
2270838
4027678
3852481
6430484
34
70520
131147
2253958
4016691
3869665
6420514
35
71616
132333
2253841
3977586
3808401
6410263
36
70845
132887
2254876
4077625
3828246
6334361
Mea
n
71162.2
131650.5
2258159.5
4051706.0
3868760.0
6429602.0
S.D
. 681.6
3
1727.9
0
6471.4
7
52516.3
1
49615.7
8
58491.2
7
C.V
.(%)
0.9
6
1.3
1
0.2
9
1.3
0
1.2
8
0.9
1
N
6
6
6
6
6
6
% R
ecovery
54.0
5
55.7
3
60.1
7
Overa
ll Reco
very
Mea
n
56
.65
S.D
. 3
.16
05
C.V
. (%)
5.5
8
N
3
% D
ifference
6.1
2
213
Table 25
Recovery of Pantoprazole from Human Plasma
viii) Dilution Integrity
Twelve sets of dilution integrity samples were prepared by spiking 1.60 times
highest standard concentration (3180.56ng/mL). Two sets of six each dilution
integrity samples were processed by diluting them twice and four times. These
quality control samples were analyzed along with a processed calibration curve
standards, refer, Table 26. The quality control sample concentrations were calculated
using appropriate dilution factor. Results demonstrate acceptable dilution integrity
for two times and four times .The within batch precision and accuracy, for a dilution
factor of 2 was 0.58% and 100.22%, for a dilution factor of 4 was 0.85% and 96.56%,
respectively. The results are within the acceptance limits and given in tables 26, 27 &
28.
Acceptance criteria:
Within batch precision of QC having same dilution factors must be ≤15%
Within batch accuracy of QC having same dilution factors must be ±15% of
nominal value.
Atleast 67% of QCs injected at each level should be within ±15% of
respective nominal value.
Internal Standard Response
Extracted MQC2 Unextracted QC
31 3270609 4085700
32 3244425 4028524
33 3264712 4046282
34 3227848 3964647
35 3188915 4062562
36 3218675 3994705
Mean 3235864.0 4030403.3
S.D. 30583.13 44600.07
C.V.(%) 0.95 1.11
N 6 6
% Recovery 80.29
214
Tab
le 26
Con
centra
tion
Resp
on
se Lin
earity
Data
for D
ilutio
n In
tegrity
Con
centra
tion
(ng/m
L)
Slo
pe
Intercep
t R
R
2
ST
D-A
ST
D-B
S
TD
-C
ST
D-D
S
TD
-E
ST
D-F
S
TD
-G
ST
D-H
S
TD
-I
CC
#
10.0
2
20.0
3
50.0
8
200.3
2
400.6
4
801.2
8
1199.5
3 1599.3
7 1999.2
1
2
9.7
6
20.9
7
50.6
4
195.3
7
427.8
2
771.0
1
1273.0
1 1589.2
9 1813.6
5
0.0
006
0.0
002
0.9
983
0.9
966
%
No
min
al
97.4
1
104.6
9
101.1
2
97.5
3
106.7
8
96.2
2
106.1
3
99.3
7
90.7
2
215
Table 27
Dilution Integrity Data of Lansoprazole (2 times)
Concentration (ng/mL)
DI QC# 3180.56
1 3154.47
2 3185.62
3 3198.33
4 3209.78
5 3185.50
6 3192.30
Mean 3187.667
S.D 18.6283
C.V % 0.58
%Nominal 100.22
N 6
Table 28
Dilution Integrity Data of Lansoprazole (4 times)
Concentration (ng/mL)
DI QC# 3180.56
1 3025.80
2 3090.09
3 3064.67
4 3079.50
5 3066.48
6 3100.70
Mean 3071.207
S.D 26.1612
C.V % 0.85
%Nominal 96.56
N 6
216
5.1
.5. S
UM
MA
RY
OF
TH
E R
ES
UL
TS
VA
LID
AT
ION
RE
SU
LT
S
An
aly
tePa
ram
eters L
an
sop
razo
le P
an
top
razo
le
Accep
tan
ce C
riteria
%
Nom
inal
Precisio
n
%
No
min
al
Precisio
n
Bio
logical M
atrix
Plasm
a P
lasma
N/A
P
N/A
P
Detectio
n
370.0
0 (p
arent) an
d 2
51.9
0 (P
roduct)
384.2
0 (p
arent)
and
200.0
0 (p
roduct)
N/A
P
N/A
P
Analy
tical Ran
ge
10.0
2ng/m
L –
1999.2
1n
g/m
L
N/A
P
N/A
P
80%
- 120
% (at L
L)
N/A
P
Min
imum
Quan
tifiable
Concen
tration
10.0
2ng/m
L
N/A
P
N/A
P
≤ 2
0%
Matrix
Effect
N/A
P
85%
-
115%
≤ 1
5%
LQ
C
107.1
0%
1.9
5%
HQ
C
98.4
5%
0.7
3%
Coefficien
t of co
rrelation
0.9
942-0
.9976
N/A
P
r2 ≥
0.9
8
Accu
racy an
d P
recision fo
r
Sen
sitivity
102.3
0%
2.4
5%
N
/AP
80%
-
120%
≤
20%
With
in B
atch A
ccurac
y an
d
Precisio
n
104.5
4%
to 1
08.8
5%
LL
OQ
QC
),
92.9
1%
to 1
12.0
1%
.
(L, M
1, M
2, H
).
1.2
9%
to 3
.48
(LL
OQ
QC
),
0.6
8 %
to 2
.28%
.
(L, M
1, M
2,H
).
N/A
P
85%
-
115%
(L, M
1,
M2, H
)
80%
-
120%
(LL
OQ
QC
)
≤15%
%
(L, M
1,
M2,H
)
≤20%
(LL
OQ
QC
)
With
in B
atch A
ccurac
y an
d
Precisio
n fo
r Stab
ilities (Fresh
ly
Spik
ed Q
C’s)
98.5
4%
(LL
OQ
QC
),
89.9
9%
to 1
05.2
8%
.
(L, M
1, M
2, H
).
2.5
1 (L
LO
Q Q
C),
1.2
3%
to 2
.40%
(L,M
1,M
2,
H).
N/A
P
Intra D
ay P
recision
107.1
5%
(LL
OQ
QC
),
94.7
2%
to 1
01.3
3%
.
(L, M
1, M
2, H
).
2.5
0%
(LL
OQ
QC
),
2.0
1%
to 2
.46%
(L,M
1,M
2,
H).
N/A
P
217
VA
LID
AT
ION
RE
SU
LT
S
An
aly
tePa
ram
eters L
an
sop
razo
le P
an
top
razo
le
Accep
tan
ce C
riteria
Betw
een B
atch / In
ter Day
Accu
racy an
d P
recision
106.8
9%
( LL
OQ
QC
),
96.9
5%
to 1
03.6
8%
(. L, M
1,
M2, H
).
2.5
6%
(LL
OQ
QC
),
3.4
2%
to 4
.93%
(L,M
1,M
2,
H).
N/A
P
Room
Tem
peratu
re Sto
ck
Solu
tion S
tability
(0 &
7 h
ours)
103.2
0%
101.8
9%
%
Com
pariso
n
Resp
onse: 9
0%
- 110%
R
efrigerated
Sto
ck S
olu
tion
Stab
ility (7
days)
99.4
5%
99.4
1%
Room
Tem
peratu
re Spik
ing
Solu
tion S
tability
(7 h
ou
rs) 102.4
5%
100.9
3%
%
Com
pariso
n
Resp
onse: 9
0 %
-
110%
Ben
ch-T
op S
tability
(5 h
rs) 89.3
0%
to 9
0.3
7
0.4
3%
to 2
.86%
N
/AP
85%
-
115%
≤
15%
Auto
Sam
pler S
tability
(49 h
ours)
91.2
1%
to 9
1.3
8%
0.7
4%
to 2
.28%
N
/AP
85%
-
115%
≤
15%
Freeze T
haw
Stab
ility (III c
ycle)
90.0
7%
to 9
2.1
1%
0.5
6%
to 2
.97%
N/A
P
85%
-
115%
≤
15%
R
einjectio
n S
tability
(28 h
ours)
99.1
0%
to 1
00.7
1%
0.5
6%
to 1
.45%
Wet ex
tract stability
(23 h
rs) 89.4
9%
to 9
1.2
8%
1.6
0%
to 4
.89%
Plasm
a samples stab
ility at –
96.5
2%
to 1
01.7
0%
1.1
1%
to 2
.28%
.
Reco
very
56.6
5%
0.2
9%
to 1
.31%
80.2
9
%
0.9
5%
-
1.1
1%
. N
/AP
≤
15%
Dilu
tion In
tegrity
Tw
o tim
e
dilu
tion:
With
in B
atch A
ccurac
y an
d
Precisio
n
100.2
2%
, 0.5
8%
N/A
P
85%
-
115%
≤
15%
218
VA
LID
AT
ION
RE
SU
LT
S
An
aly
tePa
ram
eters L
an
sop
razo
le P
an
top
razo
le
Accep
tan
ce C
riteria
Dilu
tion In
tegrity
four tim
e
dilu
tion:
With
in B
atch A
ccurac
y an
d
Precisio
n
96.5
6%
0.8
5%
5.1
.6. C
ON
CL
US
ION
S
Based
on th
e results o
btain
ed in
this stu
dy, it is co
nclu
ded
that th
e presen
t valid
ated m
ethod can
be su
ccessfully
applied
for th
e
estimatio
n o
f Lan
sop
razole in
hum
an p
lasma o
ver th
e con
centratio
n ran
ge o
f 10.0
2 –
1999.2
1ng/m
L. T
he m
ethod fo
r determ
inatio
n o
f
Lan
sop
razole in
hum
an p
lasma u
sing
HP
LC
w
ith m
ass detectio
n m
et th
e acceptan
ce criteria w
ith resp
ect to selectiv
ity, p
recision,
accurac
y, lin
earity, reco
very
and d
ilutio
n in
tegrity
over a th
eoretical co
ncen
tration ran
ge o
f 10.0
2 –
1999.2
1n
g/m
L fo
r Lan
sop
razole.
Stab
ility ev
aluatio
ns
perfo
rmed
in
k
2 ED
TA
hum
an plasm
a, sto
ck so
lutio
ns
and sto
ck dilu
tions
met
the
acceptan
ce criteria,
dem
onstratin
g in
significan
t deg
radatio
n o
f Lan
sop
razole o
ver th
e specified
storag
e duratio
ns an
d co
nditio
ns.