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

164

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.

165

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.

166

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

167

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

168

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

169

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.

170

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)

171

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.

172

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


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



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.



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.


%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.


%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)


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.


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)


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.


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


206

Table 17

Freeze Thaw Stability (FT– III Cycle) of Lansoprazole


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.



115% and the precision must be ≤15% of C.V%



207

Table: 18Short Term Stability at -20 ˚C Lansoprazole Data for Day 0


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


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.



115% and the precision must be ≤15% of C.V%



Table 20

Wet extract Stability of Lansoprazole (23 hours)


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.


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.


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.



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)


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)


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.

Chapter V Development and validation of an LC -MS/MS...

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Transcript of Chapter V Development and validation of an LC -MS/MS...