177shodhganga.inflibnet.ac.in/bitstream/10603/11199/10...quantitative estimation of EO in biological...

Chapter 6 Esomeprazole- Introduction

177

6.1 Introduction

Esomeprazole Magnesium trihydrate is chemically described as, bis (5-

methoxy-2-[(S) - [(4-methoxy -3,5- dimethyl -2- pyridnyl) methyl] sulphinyl] - H -

benzimidazole -1-yl) magnesium trihydrate compound. The molecular formula is

(C17H18N3O3S) 2 Mg X 3 H2O which corresponds to a molecular weight of 767.2 and

713.1 on anhydrous basis. Esomeprazole is the S-enantiomer of Omeprazole.

Esomeprazole is a proton pump inhibitor which reduces gastric acid secretion through

inhibition of H+/K

+-ATPase in gastric parietal cells by inhibiting the functioning of

this enzyme, the drug prevents formation of gastric acid. Esomeprazole is used in the

treatment of dyspepsia, peptic ulcer disease (PUD), gastroesophageal reflux disease

(GORD/GERD)and Zollinger-Ellison syndrome. 1

Several techniques such as, Liquid chromatography (LC)2-8,14

Supercritical

fluid chromatography13,16

Capillary electrophorcis15

Preparative chiral

chromatography 21

NMR 18

methods have been reported in the literature for the

quantitative estimation of EO in biological fluids 2-14

and pharmaceutical 15-21

compounds. Moreover, Capillary electrophorcis, Preparative chiral chromatography,

Supercritical fluid chromatography involves a tedious extraction procedure involving

too many steps. Quantification of EO in human plasma by using LC-MS/MS 2-4

and

HPLC 6-8,14

were reported. Authors 2-4 could not achieve better results for

quantification of Esomepraozole in terms of Sensitivity, ruggedness, Extraction,

runtime and recovery.


178

Fig.6.1.Chemical structures of Esomeprazole and Omeprazole-D3

The Aim of present research is to develop and validate the simple, sensitive,

selective, rugged and reproducible analytical method for quantification of

Esomeprazole in Human plasma samples by HPLC-MS. Moreover, the analyte is to

be compare with deuterated internal standard, which is most useful in selectivity and

matrix effect experiments by using high performance liquid chromatography with

mass spectrometry (HPLC-MS).

6.2 Experimental Investigations

6.2.1 Materials and reagents

Esomeprazole, Omeprazole-d3 obtained from Dr.Reddy’s Labs,

Hyderabad,India. Human plasma (K2EDTA), obtained from Navjeevan blood bank,

Hyderabad, Ammonium formate, Ammonia solution, Phosphoric acid, Sodium

carbonate anhydrous, Methanol, Acetonitrile obtained from SD- Fine chemicals,

Mumbai. Ultra pure water obtained from Milli-Q System.

6.2.2 Instrumentation and equipment


179

Refer Chapter-3.2.2

6.2.3 Preparation of reagents and solvents

Table 6.1 Preparation of reagents and solvents

Reagents and solvents preparation

5 mM Ammonium formate,

pH 9

Weigh 0.63 g of ammonium formate and dissolve into 2 L of water. Adjust pH

to 9.0 ± 0.05 with ammonia solution.

15% ACN in 5 mM

ammonium formate, pH 9

Mix 150 mL of acetonitrile with 850 mL of 5 mM ammonium formate, pH 9

30% ACN in 5 mM

ammonium formate, pH 9

Mix 300 mL of acetonitrile with 700 mL of 5 mM ammonium formate, pH 9

0.02 M sodium carbonate, pH

9.8

Weigh 2.1 g sodium carbonate anhydrous and dissolve in 1000 mL of water,

adjust pH to 9.8 ± 0.05 with phosphoric acid.

20% MeOH in 0.02 M

sodium carbonate, pH 9.8

Mix 200 mL of methanol with 800 mL of 0.02 M sodium carbonate, pH 9.8

80% Methanol Mix 800 mL of methanol with 200 mL of water.

Mobile phase

5 mM Ammonium formate, pH 9 : Acetonitrile in the ratio of 70:30 and

Filter through 0.45 m filter

(Autosampler wash)

80% Methanol Mix 800 mL of Methanol with 200 mL of water.

6.2.4 Preparation of stock solution

Table 4.2 Preparation of stock solution

Name of the stock

solutions Concentration Volume (ml) Diluent

Esomeprazole 100.0 g/ml 25 ml Methanol

Omeprazole-D3 100.0 g/ml 25 ml Methanol

6.2.5 Preparation of standards and quality control (QC) Samples

Standard stock solutions of Esomeprazole (100.00µg/mL) and Omeprazole-D3

(100.00µg/mL) were prepared in methanol. The spiking solution for Omeprazole-D3


180

was prepared in 20% methanol from respective standard stock solution. Standard

stock solutions and IS spiking solutions were stored in refrigerator conditions (2-8 °C)

until analysis. Standard stock solutions were added to drug-free human plasma to

obtain Esomeprazole concentration levels of 5.0, 10.0, 50.0, 100.0, 200.0, 400.0,

800.0, 1200.0, 1600.0 and 2000.0 ng/mL for Analytical standards and 5.0, 15.0,

700.0, 1400.0 ng/mL for Quality control standards and stored in a -30°C set point

freezer until analysis . The Aqueous standards were prepared in reconstitution

solution (30% Acetonitrile in 5mM Ammonium formate (pH-9.0)) for validation

excercises until analysis.

6.3 Method Development

HPLC-MS has been used as one of the most powerful analytical tools in

clinical pharmacokinetics for its selectivity, sensitivity and reproducibility. The goal

of this work is to develop and validate a simple, sensitive, rapid method for

quantitative estimation of Esomeprazole from plasma samples.

The MS optimization was performed by direct infusion of both the solutions

namely, Esomeprazole and Omeprazole –D3 into the mass spectrometer. The critical

parameters in the ESI source which includes the needle voltage and temperature.

Other parameters, such as the nebulizer and heater gases, DP, EP, CE, CXP

were optimized to obtain a better spray shape, resulting in better ionization of the

protonated ionic Esomeprazole and Omeprazole –D3.


181

A CAD product ion spectrum of Esomeprazole and Omeprazole –D3 formed

with the fragment ions at m/z 198.0 for Esomeprazole and m/z 197.9 for Omeprazole –

D3 (Fig. 6.2 to 6.5).


182

Figure 6.2 Parent ion mass spectra (Q1) of Esomeprazole


183

Figure 6.3 Product ion mass spectra (Q3) of Esomeprazole


184

Figure 6.4 Parent ion mass spectra (Q1) Omeprazole –D3


185

Figure 6.5 Product ion mass spectra (Q3) of Omeprazole –D3

Chromatographic optimization


186

Initially, we tried with different extraction techniques like, SPE, Precipitation

techniques. Finally, Precipitation was selected as suitable extraction for drug and IS

interms of recovery and reproducibility. Chromatographic conditions especially, the

composition and nature of the mobile phase, different columns were optimized

through several trials to achieve best resolution to increase the signal of

Esomeprazole, and Omeprazole –D3. A good separation and elution were achieved

with 5mM Ammonium formate (pH 9.0) : Acetonitrile (70:30 v/v). at 0.6 mL/min

flow and Xbridge C18, 50x 4.6 mm 5 m was selected as the analytical column at

40°C.

Chromatographic conditions

A good separation and elution were achieved using Xbridge C18, 50x 4.6 mm

5 m was selected as the analytical column. The mobile phase composition was

05mM Ammonium formate (pH 9.0) : Acetonitrile at a flow rate of 0.6 mL/min and

10L injection volume was used. Column temperature was set at 30°C. Omeprazole –

D3 was found to be appropriate internal standard. Retention time of EO and OMD3

were found to be 2.7 ± 0.2 min, with overall runtime of 4 min.

Sample preparation

Precipitation extraction method was used to isolate EO and OMD3 from

human plasma. For this, 50 µL of OMD3 (250.0 ng/mL) and 850 µL of plasma

sample (respective concentration) was added into labeled polypropylene tubes and

vortexed briefly about 5 minutes followed by centrifuge at 14000 rpm for

approximately 2 min at ambient temperature. From this, 100µL of supernatant sample

was transferred into labeled polypropylene tubes containing 400µL of 15%


187

Acetonitrile in 5mM Ammonium format (pH 9.0) and vortexed briefly. Finally, these

samples were transferred into auto sampler vials and injected in to LC-MS/MS.

Calibration curve parameters and regression model

The analytical curves were constructed using values ranging from 5.0 to

2000.0 ng/mL of EO in human plasma. Calibration curves were obtained by weighted

1/Con2 linear regression analysis

y = ax + b

where, x = EO concentration in plasma sample,

y = Area ratio of EO and OMD3.

The ratio of EO peak area to OMD3 peak area was plotted against the ratio of

EO concentration in ng. mL-1

. Calibration curve standard samples and quality control

samples were prepared in replicates (n=6) for analysis. Accuracy and precision for the

back calculated concentrations of the calibration points should be within ≤ 15 and ±

15% of their nominal values. However, for LLOQ, the precision and accuracy should

be within ≤ 20 and ± 20%.

Method Development Conclusion

The developed method is suitable for estimation of Esomeprazole

concentrations in plasma as a single analytical run, in clinical samples from

Pharmacokinetic studies. This was followed by method validation.

6.4 Method Validation

The objective of the work is to validate specific HPLC-MS method for the

determination of Esomeprazole in human plasma for clinical / Pharmacokinetic study.


188

Chromatography

Representative chromatograms of Plasma blank, blank +IS, LOQ, ULOQ,

LLOQC, LQC, MQC, HQC, Calibration curve are shown in Figure 6.6 to 6.14.

Figure 6.6. Chromatogram of Blank Human Plasma Sample


189

Figure 6.7. Chromatogram of Blank + IS Human Plasma Sample


190

Figure 6.8: Chromatogram of LOQ Sample (Esomeprazole & IS)


191

Figure 6.9: Chromatogram of ULOQ Sample (Esomeprazole & IS)


192

Figure 6.10: Chromatogram of LLOQ Sample (Esomeprazole & IS)


193

Figure 6.11: Chromatogram of LQC Sample (Esomeprazole & IS)


194

Figure 6.12: Chromatogram of MQC Sample (Esomeprazole & IS)


195

Figure 6.13: Chromatogram of HQC Sample (Esomeprazole & IS)


196

Figure 6.14 Calibration Curve of Esomeprazole


197

Blank Matrix Screening

During validation, blank plasma samples from 10 different lots were processed

according to the extraction procedure and evaluate the interference at the retention

times of analyte and internal standard. The 6 free interference lots were selected from

the 10 lots. Results are presented in table 6.3.

Table 6.3 Screening of Different batches of blank matrix for interference free

Esomeprazole blank plasma (Human K2EDTA Plasma)

Matrix identification

Blank plasma Area

Analyte

(Esomeprazole)

RT

Internal

standard

RT

PL Blank-(K2EDTA-AP/2231/09/10) 0 0










Blank+IS with PL Blank-(K2EDTA-

AP/2231/09/10) 0 179076

LOQ with PL Blank-(K2EDTA-

AP/2231/09/10) 8766 177291

Blank Matrix Specificity and Limit of Quantification

During specificity run, the LLOQ standard was prepared in one of the

screened blank plasma including the spiking of working range of internal standard.

Blank plasma samples from 10 different lots, 6 LLOQ standards were processed

according to the extraction procedure. The responses for the blank plasma from 10

different lots were compared to the LLOQ standard of the analyte and internal


198

standard. No significant response (≤20% for the analyte response and ≤5% of the

internal standard response) was observed at the retention times of the analyte or the

internal standard in blank plasma as compared to the LLOQ standard. Results are

presented in table 6.4.

The specificity experiment shall be considered for calculation of LOQ

experiment. Results are presented in table 6.5

Table 6.4 Specificity of Different batches of blank matrix

(Human K2EDTA Plasma)

Matrix

Identification

LLOQ

Area

Internal

standard

(IS) area

Interference with

Analyte(% of

LLOQ Response)

Interference

with IS(% of IS

Response)

PL Blank-

(K2EDTA-

AP/2231/09/10)

8776 179076 0 0

PL Blank-

(K2EDTA-

AP/2232/09/10)

8468 177291 0 0

PL Blank-

(K2EDTA-

AP/2233/09/10)

8136 179794 0 0

PL Blank-

(K2EDTA-

AP/2234/09/10)

8969 182977 0 0

PL Blank-

(K2EDTA-

AP/2235/09/10)

8706 181358 0 0

PL Blank-

(K2EDTA-

AP/2236/09/10)

8633 171573 0 0

Acceptance criteria:

1. Analyte response should be ≤ 20% of LOQ Response in at least 75% of the

blank.

2. Internal standard response should be ≤5% of mean internal standard response

in at least 75% of the blank.


199

Table 6.5 Limit of Quantification for analyte (Esomeprazole)

Matrix identification Blank plasma area at

Analyte RT

LLOQ

response

LLOQ S/N

RATIO

PL Blank-(K2EDTA-

AP/2231/09/10)

0 8776 61.1

0 8468 73.9

0 8136 59.1

0 8969 77.9

0 8706 46.6

0 8633 71.0

N 6 6 6

Mean 0 8615 64.9

LLOQ was spiked in PL Blank-(K2EDTA-AP/2231/09/10)


1. Mean S/N ratio of LLOQ should be ≥5.

2. S/N ratio is analyst software generated data.

Intra Batch Accuracy and precision

Intra batch accuracy and precision evaluation were assessed by analyzing 1

calibration curve and 6 replicate each of the LLOQ, LQC, MQC, HQC, from

precision and accuracy batch-1.

The Intra batch percentage of nominal concentrations for Esomeprazole was

ranged between 97.90% and 100.70%.

The Intra batch percentage of coefficient of variation is 1.6% to 3.5% for

Esomeprazole.

Results are presented in table 6.6.


200

Table 6.6 Intra batch (Within-Batch) Accuracy and Precision for determination

Esomeprazole levels in human plasma

Analytical

Run ID

LLOQ

2.00 ng/ml

Low QC

6.00 ng/ml

Mid QC

750.00 ng/ml

High QC

1750.00 ng/ml

Conc.

found

%

nominal

Conc.

found

%

nominal

Conc.

found

%

nominal

Conc.

found

%

nominal

P&A Batch 1

5.09 101.80 15.27 101.80 705.77 100.82 1372.79 98.06 4.89 97.80 14.35 95.67 727.87 103.98 1398.56 99.90 4.67 93.40 14.61 97.40 701.14 100.16 1397.38 99.81 5.12 102.40 14.88 99.20 715.62 102.23 1388.96 99.21 4.84 96.80 14.52 96.80 684.29 97.76 1392.9 99.49 4.91 98.20 14.42 96.13 696.69 99.53 1439 102.79

N 6

6

6

6

Mean 4.92 14.68 705.23 1398.27

SD (±) 0.17 0.34 15.16 22.02

CV (%) 3.5 2.3 2.1 1.6

%Accuracy 98.40 97.90 100.70 99.90


1. % CV ≤ 15 % except LLOQ for which it is ≤ 20%.

2. Mean % Nominal (100±15% and for LLOQ 100±20%).

Inter Batch Accuracy and Precision

Inter batch accuracy and precision evaluation were assessed by analyzing 5

sets of calibration curves for Esomeprazole and 5 sets of QC samples, 6 replicates

each of the LLOQ, LQC, MQC and HQC.

The inter batch percentage of nominal concentrations for Esomeprazole was

ranged between 88.40% and 108.00%.

The Inter batch percentage of coefficient of variation is 2.0% to 3.4% for

Esomeprazole.

Results are presented in table 6.7.


201

Table 6.7 Inter batch (Between-Batch) Accuracy and Precision for determination

Esomeprazole levels in human plasma

Analytical

Run ID

LLOQ

5.00 ng/ml

Low QC

15.00 ng/ml

Mid QC

700.00 ng/ml

High QC

1400.00 ng/ml

Conc.

found

%

nominal

Conc.

found

%

nominal

Conc.

found

%

nominal

Conc.

found

%

nominal

P&A

Batch 1

5.09 101.8 15.27 101.80 705.77 100.82 1372.79 98.06 4.89 97.8 14.35 95.67 727.87 103.98 1398.56 99.90 4.67 93.4 14.61 97.40 701.14 100.16 1397.38 99.81 5.12 102.4 14.88 99.20 715.62 102.23 1388.96 99.21 4.84 96.8 14.52 96.80 684.29 97.76 1392.90 99.49 4.91 98.2 14.42 96.13 696.69 99.53 1439.00 102.79

P&A

Batch 2

4.82 96.4 14.38 95.87 674.70 96.39 1373.90 98.14 4.91 98.2 14.58 97.20 694.25 99.18 1379.23 98.52 4.66 93.2 14.08 93.87 689.04 98.43 1335.50 95.39 5.05 101 14.42 96.13 695.24 99.32 1379.32 98.52 4.81 96.2 14.97 99.80 694.88 99.27 1368.38 97.74 4.58 91.6 14.58 97.20 696.58 99.51 1362.80 97.34

P&A

Batch 3

5.1 102 15.28 101.87 668.67 95.52 1396.85 99.78 5.17 103.4 14.78 98.53 675.01 96.43 1405.52 100.39 5.29 105.8 14.73 98.20 699.92 99.99 1369.00 97.79 5.21 104.2 14.7 98.00 713.97 102.00 1380.14 98.58 5.21 104.2 15.05 100.33 699.63 99.95 1357.19 96.94 5.18 103.6 15.06 100.40 702.16 100.31 1418.48 101.32

P&A

Batch 4

4.87 97.4 14.39 95.93 690.27 98.61 1424.98 101.78 5.12 102.4 14.51 96.73 722.33 103.19 1358.72 97.05 4.95 99 14.87 99.13 686.89 98.13 1390.79 99.34 4.94 98.8 14.64 97.60 697.36 99.62 1339.05 95.65 4.97 99.4 14.95 99.67 705.81 100.83 1425.47 101.82 4.95 99 14.25 95.00 692.79 98.97 1396.61 99.76

P&A

Batch 5

4.8 96 15.16 101.07 688.92 98.42 1311.53 93.68 4.91 98.2 14.53 96.87 701.30 100.19 1380.87 98.63 4.77 95.4 15.15 101.00 692.63 98.95 1370.69 97.91 4.93 98.6 14.61 97.40 677.88 96.84 1337.33 95.52 5.02 100.4 14.83 98.87 680.91 97.27 1333.35 95.24 4.92 98.4 14.45 96.33 687.14 98.16 1328.94 94.92

N 30

30

30

30

Mean 4.96 14.70 695.32 1377.14 SD(±) 0.17 0.31 13.61 30.89

CV (%) 3.53 2.12 1.96 2.24

%Nominal 99.11

98.00

99.33

98.37

Acceptance criteria: Same as Table 6.6


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Calibration Curve

Calibration curves are found to be consistently accurate and precise for

Esomeprazole over 5.00 to 2000.00ng/ml for calibration range. The correlation

coefficient is greater than 0.9992 for Esomeprazole. Back calculations were made

from the calibration curves to determine Esomeprazole concentrations of each

calibration standard.

Results are presented in tables 6.8 & 6.9.

Table 6.8 Summary of calibration curve parameters for Esomeprazole in human

plasma

Analytical Run

ID A B

Coefficient of

regression (r2)

P&A Batch-1 0.009398 0.000156 0.9989

P&A Batch-2 0.009551 0.000445 0.9994

P&A Batch-3 0.009546 -0.00342 0.9996

P&A Batch-4 0.00963 0.000965 0.9994

P&A Batch-5 0.009569 -0.00041 0.9992

N 5 5 5

Mean 0.009539 -0.00045 0.9993

SD (±) 8.55E-05 0.001731 0.0003

CV (%) 0.9 -382.7 0

Regression Footnote(s): Resp. = A * (Conc.2) + B * Conc. + C


1. Coefficient of regression (r) ≥0.9992.


203

Table 6.9 Back-calculated standard concentrations from each calibration curve

for Esomeprazole in human plasma.

Analytical Run

ID

Nominal Concentration (ng/ml)

CS1 CS2 CS3 CS4 CS5

5.00 ng/ml 10.00

ng/ml

50.00

ng/ml

100.00

ng/ml

200.00

ng/ml

P&A Batch-1 4.85 10.61 49.9 101.62 205.23

P&A Batch-2 4.97 10.09 49.79 100.1 205.21

P&A Batch-3 4.96 10.18 48.98 102.64 200.72

P&A Batch-4 4.96 10.15 50.15 100.84 208.85

P&A Batch-5 4.93 10.28 49.06 101.25 203.6

N 5 5 5 5 5

Mean 4.93 10.26 49.58 101.29 204.72

SD(±) 0.05 0.21 0.52 0.94 2.95

CV% 1 2 1 0.9 1.4

%Nominal -1.4 2.6 -0.8 1.3 2.4

Analytical run

ID

Nominal Concentration (ng/ml)

CS6 CS7 CS8 CS9 CS10

400.00

ng/ml

800.00

ng/ml

1200.00

ng/ml

1600.00

ng/ml

2000.00

ng/ml

P&A Batch-1 389.73 769.03 1202.3 1623.92 1955.26

P&A Batch-2 411.99 784.04 1143.1 1632.62 1979.72

P&A Batch-3 407.25 798.46 1166.85 1565.46 2027.98

P&A Batch-4 391.75 805.69 1202.77 1579.34 1925.56

P&A Batch-5 410.61 753.46 1192.86 1622.27 1993.96

N 5 5 5 5 4

Mean 402.27 782.14 1181.58 1604.72 1976.5

SD(±) 10.69 21.32 26 30.17 38.77

CV% 2.7 2.7 2.2 1.9 2

%Nominal 0.6 -2.2 -1.5 0.3 -1.2

Acceptance criteria

1. Mean % Nominal (100±15%) except lowest calibration standard.

2. Mean % Nominal (100±20%) for lowest calibration standard (CS1).

3. %CV ≤ 15% except lowest calibration standard (CS1) for which it is ≤20%.


204

Extraction Recovery

The percentage recovery of Esomeprazole was determined by comparing the

mean peak area of Esomeprazole in extracted LQC, MQC, HQC samples with freshly

prepared unextracted LQC, MQC, HQC samples respectively.

The mean % recovery for LQC, MQC, HQC samples of Esomeprazole were

94.47%, 94.18% and 96.60% respectively.

The mean recovery of Esomeprazole across QC levels is 95.08%.

The mean recovery of % CV recovery of Esomeprazole across QC levels is

2.6%.

For the internal standard, mean peak area of 18 extracted samples was

compared to the mean peak area of 18 unextracted IS solution. The mean %

recovery is 95.14%.

The %CV recovery of IS Omeprazole D3 for extracted is 2.4%.

Results are presented in 6.10.


205

Table 6.10: Recovery of analyte (Esomeprazole) and IS (Omeprazole-D3) from

human plasma

Standard

Extracted peak

response

Unextracted peak

response

Drug IS Drug IS

Low QC: 15.00 ng/ml

25501 179076 26302 179220

24414 177291 25254 182381

24661 179794 26937 186938

25047 182977 26375 188642

25438 181358 26451 182429

24084 171573 26537 190830

N 6 6 6 6

% Recovery 94.47

SD (±) 2.173

%CV 2.3

Medium QC: 700.00 ng/ml

1112101 174321 1232523 186120

1145575 177880 1195811 181873

1188568 177985 1216829 187827

1204093 176759 1224597 184162

1141631 171028 1234595 186985

1130395 168733 1246127 187342

N 6 6 6 6

% Recovery 94.18

SD(±) 2.874

%CV 3.1

High QC: 1400.00 ng/ml

2265712 169962 2394566 180724

2373997 176987 2395043 180631

2286299 174996 2393315 186279

2357736 179007 2328634 179201

2250538 173758 2358431 184804

2301223 169993 2453586 188461

N 6 6 6 6

% Recovery 96.60

SD(±) 2.076

%CV 2.1

Drug IS

Mean recovery of across QC levels 95.08 95.14

Mean SD(±) of across QC levels 2.514 2.276

The Mean % CV across QC levels 2.6 2.4


1. The coefficient of variation for mean recovery across LQC, MQC and HQC

shall not exceed 25%.

2. The coefficient of variation for mean recovery of IS shall not exceed 25%.


206

Matrix Effect

Samples were prepared at MQC level in triplicate in each of 6 different lots of

human plasma. A calibration curve and 6 replicates of MQC samples in triplicate for

each matrix were freshly prepared and analyzed in single run.

Percentage bias was calculated for each matrix.

No significant matrix effect found in different sources of human plasma tested

for Esomeprazole, Omeprazole-D3.

Results are presented in table 6.11 and 6.12.

Table 6.11 Assessment of Matrix Effect on determination of Esomeprazole levels

in human plasma

Identification of

matrix

Drug response

in Matrix at

MQC Level

Drug response in

No Matrix at

MQC Level

Matrix factor

AP/2231/09/10 1347595 1456242 0.9254

AP/2231/09/10 1380594 1429543 0.9658

AP/2231/09/10 1397767 1463781 0.9549

AP/2232/09/10 1356261 1482597 0.9148

AP/2232/09/10 1385483 1460368 0.9487

AP/2232/09/10 1367208 1422412 0.9612

AP/2233/09/10 1401142 1450675 0.9659

AP/2233/09/10 1387498 1448002 0.9582

AP/2233/09/10 1351424 1457196 0.9274

AP/2234/09/10 1338303 1441645 0.9283

AP/2234/09/10 1339778 1445972 0.9266

AP/2234/09/10 1360330 1418551 0.9590

AP/2235/09/10 1360050 1443307 0.9423

AP/2235/09/10 1346578 1422749 0.9465

AP/2235/09/10 1351450 1462241 0.9242

AP/2236/09/10 1337477 1487161 0.8993

AP/2236/09/10 1365308 1430290 0.9546

AP/2236/09/10 1337909 1455455 0.9192

N 18 18 18

Grand Mean 0.9401

SD(±) 0.0198

CV (%) 2.11%


1. Mean % Nominal 100±15% of nominal value.

2. %CV ≤ 15%.


207

Table 6.12 Assessment of Matrix Effect on determination of IS

(Omeprazole-D3) levels in human plasma at MQC Level

Identification of matrix Internal

standard

response in

matrix at MQC

level

Internal

standard

response in No

matrix

at MQC level

Matrix factor

AP/2231/09/10 201300 217937 0.9237

AP/2231/09/10 200554 218751 0.9168

AP/2231/09/10 206367 217341 0.9495

AP/2232/09/10 203925 216381 0.9424

AP/2232/09/10 211043 215253 0.9804

AP/2232/09/10 211153 212487 0.9937

AP/2233/09/10 207369 224770 0.9226

AP/2233/09/10 201777 212185 0.9509

AP/2233/09/10 202666 219435 0.9236

AP/2234/09/10 202206 213333 0.9478

AP/2234/09/10 202654 215738 0.9394

AP/2234/09/10 205356 213593 0.9614

AP/2235/09/10 205554 212808 0.9659

AP/2235/09/10 201833 215619 0.9361

AP/2235/09/10 200607 215443 0.9311

AP/2236/09/10 200420 222069 0.9025

AP/2236/09/10 200274 206301 0.9708

AP/2236/09/10 197286 213281 0.9250

N 18 18 18

Grand Mean 0.9435

SD(±) 0.0240

%CV 2.54%

Acceptance criteria: %CV<25%.

Dilution Integrity


208

Dilution integrity experiment was carried out at six replicate of two times

diluted (1 in 2 dilution) and four times diluted of approx 1.5 × ULOQ (1 in 4 dilution)

samples were prepared and concentrations were calculated including the dilution

factor against the freshly prepared calibration curve.

The % accuracy of Esomeprazole nominal concentrations ranged between

104.33% and 106.56% for 1 in 2 dilutions and 1 in 4 dilutions respectively.

The % CV is 1.28% to 1.44%.

Results are presented in tables 6.13.

Table 6.13 Assessment of Dilution integrity for Esomeprazole

at DQC Conc (ng/ml)

DQC

Dilution factor: ½

Nominal conc: 3000.0 ng/ml

DQC

Dilution factor: ¼

Nominal conc: 3000.0 ng/ml

Conc. Found % Nominal Conc. Found %Nominal

3080 102.67 3260 108.67

3130 104.33 3220 107.33

3200 106.67 3210 107.00

3120 104.00 3200 106.67

3140 104.67 3160 105.33

3110 103.67 3130 104.33

N 6

6

Mean

%Nominal 104.33 106.56

SD (±) 1.33 1.53

CV (%) 1.28 1.44


1. % CV ≤ 15%.

2. Mean % Nominal (100±15%).

Whole Batch Reinjection Reproducibility


209

To evaluate the whole batch reinjection reproducibility experiment, samples of

P & A batch-2 were kept at auto sampler temperature for approx 26 hrs after the

initial analysis and were re-injected again after approx 26 hrs. Concentrations were

calculated to determine precision and accuracy after reinjection.

The Accuracy of Esomeprazole QC samples in reinjection was between

97.00% and 98.10%.

The Precision (%CV) of Esomeprazole QC samples in reinjection was

between 2.33 % and 1.76%.

Esomeprazole was found to be stable at autosampler temperature post

extraction (in reconstitution solution) for approx 26 hrs and reproducible after

reinjection.

Results are presented in tables 6.14.

Table 6.14 Assessment of Whole Batch Reinjection Reproducibility during

estimation of Esomeprazole in human plasma

Analytical

Run ID

Low QC 15.00 ng/ml High QC 1400.00 ng/ml

Comp sample Reinjection

sample Comp sample

Reinjection

sample

14.38 14.3 1373.9 1410

14.58 14.1 1379.23 1380

14.08 14.6 1335.50 1350

14.42 14.6 1379.32 1360

14.97 14.6 1368.38 1390

14.58 15.1 1362.80 1350

N 6 6 6 6

Mean 14.50 14.55 1366.52 1373.33 SD(±) 0.29 0.34 16.49 24.22 %CV 2.02 2.33 1.21 1.76

%NOM 96.68 97.00 97.61 98.10


1. % CV≤ 15% Except LLOQ for which it is ≤ 20%.

2. Mean % Nominal (100±15% and for LLOQ 100±20%).

3. 67% 0f the re-injected QCs at each level shall be within ±20% of their

previous concentration.

Ruggedness-Different Analyst


210

To evaluate ruggedness experiment with different analysts, one P&A batch

(P&A-3) was processed by different analyst. The run consisted of a calibration curve

standards and 6 replicates of each LLOQ, LQC, MQC, HQC samples.

The Accuracy of Esomeprazole QC samples within the range of 97.83% to

103.87%.

The Precision of Esomeprazole QC samples within the range of 1.20% to

3.39%.

These results indicated that the method is rugged and reproducible by different

analyst.

Results are presented in tables 6.15

Table 6.15 Ruggedness of the method for estimation of Esomeprazole Plasma

levels in human plasma with different analyst.

Analytical

Run ID

LLOQ

5.00 ng/ml

Low QC

15.00 ng/ml

Mid QC

700.00 ng/ml

High QC

1400.00 ng/ml

Analyst

ID A

Analyst

ID B

Analyst

ID A

Analyst

ID B

Analyst

ID A

Analyst

ID B

Analyst

ID A

Analyst

ID B

P&A Batch

3

5.09 5.1 15.27 15.28 705.77 668.67 1372.79 1396.85 4.89 5.17 14.35 14.78 727.87 675.01 1398.56 1405.52 4.67 5.29 14.61 14.73 701.14 699.92 1397.38 1369 5.12 5.21 14.88 14.7 715.62 713.97 1388.96 1380.14 4.84 5.21 14.52 15.05 684.29 699.63 1392.9 1357.19 4.91 5.18 14.42 15.06 696.69 702.16 1439 1418.48

N 6 6 6 6 6 6 6 6

Mean 4.92 5.19 14.68 14.93 705.23 693.23 1398.27 1387.86

SD (±) 0.17 0.06 0.34 0.23 15.16 17.50 22.02 23.18

CV (%) 3.39 1.20 2.35 1.55 2.15 2.52 1.57 1.67

%NOM 98.40 103.87 97.83 99.56 100.75 99.03 99.88 99.13



2. Mean % Nominal (100±15% & for LLOQ 100±20%).


211

Ruggedness-Different Column

To evaluate ruggedness experiment with different column, samples of P&A

batch-5 were reinjected on different columns with same and specifications,

Concentrations were calculated to determine precision and accuracy.

The Accuracy of Esomeprazole QC samples within the range of 95.99% to

100.75%.

The Precision of Esomeprazole QC samples within the range of 1.22%

to 3.39%.

These results indicated that the method is rugged and reproducible by different

analyst.

Results are presented in tables 6.16

Table 6.16 Ruggedness of the method for estimation of Esomeprazole Plasma

levels in human plasma with different Analytical column

Analytical

Run ID

LLOQ

5.00 ng/ml

Low QC

15.00 ng/ml

Mid QC

700.00 ng/ml

High QC

1400.00 ng/ml

Column

ID

LC/221

Column

ID

LC/165

Column

ID

LC/221

Column

ID

LC/165

Column

ID

LC/221

Column

ID

LC/165

Column

ID

LC/221

Column

ID

LC/165

P&A

Batch 5

5.09 4.80 15.27 15.16 705.77 688.92 1372.79 1311.53 4.89 4.91 14.35 14.53 727.87 701.30 1398.56 1380.87 4.67 4.77 14.61 15.15 701.14 692.63 1397.38 1370.69 5.12 4.93 14.88 14.61 715.62 677.88 1388.96 1337.33 4.84 5.02 14.52 14.83 684.29 680.91 1392.90 1333.35 4.91 4.92 14.42 14.45 696.69 687.14 1439.00 1328.94

N 6 6 6 6 6 6 6 6

Mean 4.92 4.89 14.68 14.79 705.23 688.13 1398.27 1343.79

SD(±) 0.17 0.09 0.34 0.31 15.16 8.40 22.02 26.50

CV (%) 3.39 1.88 2.35 2.10 2.15 1.22 1.57 1.97

%NOM 98.40 97.80 97.87 98.60 100.75 98.30 99.88 95.99



2. Mean % Nominal (100±15% & for LLOQ 100±20%).


212

Bench Top Stability (at room temp for 24.5 hrs)

Spiked LQC and HQC samples were retrieved from deep freezer and were

kept at room temperature for 24.5 hrs and were processed and analyzed along with

freshly prepared calibration standards, comparison LQC and HQC samples.

Concentrations were calculated to determine mean % change during stability period.

The mean Accuracy for LQC & HQC samples of Esomeprazole from

comparison samples were 95.45% and 96.19% respectively.

The plasma samples of Esomeprazole were found to be stable for

approximately 24.5 hrs min at room temperature.

Results are present in table 6.17.


213

Table 6.17 Assessment of stability of Analyte (Esomeprazole) in Biological

matrix at Room temperature


Comparison

samples

(0.00 hr)

Stability samples

(24.5 hrs)

Comparison

samples

(0.00 hr)

Stability samples

(24.5 hrs)

Conc.

found

%

nominal

Conc.

found

%

nominal

Conc.

found

%

nominal

Conc.

found

%

nominal

14.5 96.67 14.4 96.00 1310 93.57 1350.00 96.43

14 93.33 14.8 98.67 1330 95.00 1350.00 96.43

14.7 98.00 13.6 90.67 1330 95.00 1380.00 98.57

14.3 95.33 14.4 96.00 1350 96.43 1320.00 94.29

14.4 96.00 14 93.33 1440 102.86 1350.00 96.43

14.4 96.00 14.7 98.00 1360 97.14 1330.00 95.00 N

Mean 14.38 14.32 1353.33 1346.67 SD(±) 0.23 0.45 45.90 20.66

CV (%) 1.61 3.14 3.39 1.53 %NOM 95.89 95.45 96.67 96.19


1. % change should be ± 15 or % Ratio (stability/comparison) should be within

85-115 %.

2. %CV ≤ 15%.

3. Mean % Nominal (100±15%).

Freeze and Thaw Stability (after 3rd

cycle at -30°C)

Samples were prepared at LQC and HQC levels, aliquoted and frozen at -

30±5°C six samples from each concentration were subjected to three freeze and thaw

cycles (stability samples). These samples were processed and analyzed along with

freshly prepared calibration standards, LQC and HQC samples (comparison samples).

Concentrations were calculated to determine mean % change after 3 cycles.

The mean Accuracy for LQC & HQC samples of Esomeprazole from



214

The plasma samples of Esomeprazole were found to be stable after 3 cycles at

-30±5°C.

Results are present in table 6.18.

Table 6.18 Assessment of Freeze-Thaw stability of Analyte (Esomeprazole) at

-30±5°C


Comparison

samples

Stability sample

at 4th

cycle

Comparison

samples

Stability sample at

4th

cycle

Conc.

found

%

nominal

Conc.

found

%

nominal

Conc.

found

%

nominal

Conc.

found

%

nominal

14.50 96.67 14.50 96.67 1310.00 93.57 1370.00 97.86 14.00 93.33 14.30 95.33 1330.00 95.00 1330.00 95.00 14.70 98.00 14.60 97.33 1330.00 95.00 1360.00 97.14 14.30 95.33 14.20 94.67 1350.00 96.43 1330.00 95.00 14.40 96.00 14.20 94.67 1440.00 102.86 1380.00 98.57 14.40 96.00 14.30 95.33 1360.00 97.14 1360.00 97.14

N 6

6

6

6

Mean 14.38 14.35 1353.33 1355.00 SD(±) 0.23 0.16 45.90 20.74

CV (%) 1.61 1.15 3.39 1.53 %Accuracy

95.89

95.67

96.67

96.79

Acceptance criteria


85-115 %.

2. %CV ≤ 15%.

3. Mean % Nominal (100±15%)


215

Autosampler stability at 2-8°C in autosampler

LQC and HQC samples were prepared and processed. These processed

samples were analyzed and kept in auto sampler for 50.5 hrs at 2-8°C and analyzed

along with freshly prepared calibration standard samples. Concentrations were

calculated to determine mean % change during stability period.

The mean Accuracy change for LQC & HQC samples of Esomeprazole from


Esomeprazole samples were stable for 50.5 hrs at 2-8°C in autosampler.

Results are present in table 6.19

Table 6.19 Assessment of autosampler stability of Analyte (Esomeprazole)

at 2-8°C

Low QC 1500 ng/ml High QC 1400.00 ng/ml

Comparison

samples (0.0 hr)

Stability samples

(50.5 hr)

Comparison

samples (0.0 hr)

Stability samples

(50.5 hr)

Conc.

found

%

nominal

Conc.

found

%

nominal

Conc.

found

%

nominal

Conc.

found

%

nominal

14.40 96.00 14.50 96.67 1420.00 101.43 1380.00 98.57 14.50 96.67 14.20 94.67 1360.00 97.14 1420.00 101.43 14.90 99.33 14.30 95.33 1390.00 99.29 1350.00 96.43 14.60 97.33 14.30 95.33 1340.00 95.71 1370.00 97.86 15.00 100.00 14.40 96.00 1430.00 102.14 1390.00 99.29 14.30 95.33 14.50 96.67 1400.00 100.00 1360.00 97.14

N 6

6

6

6

Mean 14.62 14.37 1390.00 1378.33

SD(±) 0.28 0.12 34.64 24.83

CV (%) 1.91 0.84 2.49 1.80

%NOM 97.44 95.78 99.29 98.45


1. % change should be ± 15 or % ratio (stability/comparison) should be within

85-115 %.

2. %CV ≤ 15%.

3. Mean % Nominal (100±15%).


216

Longterm stability (at -30°C temp for 55 days)

Spiked LQC and HQC samples were retrieved from deep freezer after 55 days

and were processed and analyzed along with freshly prepared calibration standards,

comparison LQC and HQC samples. Concentrations were calculated to determine

mean % change during stability period.

The mean Accuracy for LQC and HQC samples of Esomeprazole from


The plasma samples of Esomeprazole were found to be stable for

approximately 55 days at -30°C temp.

Results are present in table 6.20

Table 6.20 Assessment of long term plasma stability of analyte (Esomeprazole) at

-30°C.


Comparison

samples (0.0 hr)

Stability samples

(55 days)

Comparison

samples (0.0 hr)

Stability samples

(55 days)

Conc.

found

%

nominal

Conc.

found

%

nominal

Conc.

found

%

nominal

Conc.

found

%

nominal

14.50 96.67 14.50 96.67 1310.00 93.57 1360.00 97.14 14.00 93.33 14.90 99.33 1330.00 95.00 1360.00 97.14 14.70 98.00 14.80 98.67 1330.00 95.00 1360.00 97.14 14.30 95.33 14.90 99.33 1350.00 96.43 1400.00 100.00 14.40 96.00 14.20 94.67 1440.00 102.86 1330.00 95.00 14.40 96.00 14.40 96.00 1360.00 97.14 1380.00 98.57

N 6

6

6

6

Mean 14.38 14.62 1353.33 1365.00 SD(±) 0.23 0.29 45.90 23.45 CV (%) 1.61 2.00 3.39 1.72 %Accuracy 95.89 97.44 96.67 97.50



85-115 %.

2. %CV ≤ 15%.

3. Mean % Nominal (100±15%).


217

Short Term Stock Solution Stability of Esomeprazole and Omeprazole D3 at

Room Temperature

Stock solution stability was determined by comparing the peak areas of freshly

prepared stock solutions (comparison samples) with stability stock solutions. Main

Stock solutions of Esomeprazole and Omeprazole-D3 were freshly prepared and

aliquots of stocks were kept at room temperature for 9.5 hr (stability samples).

Aqueous equivalent highest calibration standard of Esomeprazole and solution of

Omeprazole D3 were prepared from the stability samples and analyzed. Areas of

stability samples and freshly prepared samples were compared to determine mean %

change during stability period.

The % CV for of Esomeprazole stock solution from comparison samples was

1.91% and % Ratio (stability/comparison) was 100.65

The % CV for of Omeprazole D3stock solution from comparison samples was

1.64% and % Ratio (stability/comparison) was 100.03.

The % CV for Omeprazole D3 working solution (Internal standard spiking

solution) from comparison samples was 0.22% and % Ratio (stability/

comparison) was 99.35.

Esomeprazole, Omeprazole D3 stock solutions and Omeprazole D3 spiking

solutions were found to be stable at room temperatu99.35re for 9.5 hr.

Results are present in table 6.21 and 6.22.


218

Table 6.21 Assessment of Short term stock solution stability of Analyte

(Esomeprazole) and Internal standard (Omeprazole D3) at Room temperature

Analyte Internal standard Comparison

Standard stock

solution response (0.0

hr)

Stability stock

solution response

(9.5 hr)

Comparison stock

solution response

(0.0 hr)

Stability

Standard stock

Response

(9.5 hr)

1035489 1058116 1785320 1792220 1056046 1029001 1834274 1824381 1038947 1047772 1784390 1861938 1049788 1049988 1863426 1794643 1037931 1014094 1794723 1796432 1032724 1011166 1857934 1846273

N 6 6 6 6

Mean 1041821 1035023 1820011 1819315

SD (±) 9078.16 19815.24 36436.94 29784.55

CV (%) 0.87 1.91 2.00 1.64

%

Ratio 100.65 100.03


1. % change should be ± 5 %

Table 6.22 Assessment of short term solution stability of internal standard

spiking solution (Omeprazole D3) at room temperature

Comparison solution (Internal

standard Spiking solution)

Response (0.0 hr)

Stability solution (Internal

standard spiking solution)

Response (9.5 hr)

1793076 1803754 1767291 1794376 1785453 1795641 1792342 1797835 1786523 1803426 1796543 1796453

N 6 6

Mean 1786871 1798581

SD (±) 10464.13 4040.90

CV (%) 0.59 0.22

% Ratio 99.35


1. % change should be ± 5%


219

Method validation Conclusion

As all the values obtained were within the Acceptance criteria. The method

stands validated and is suitable for estimation of plasma Esomeprazole concentrations

in a single analytical run. The rugged, efficient Precipitation extraction method

provides exceptional sample clean up and constant recoveries using 850µl of plasma.

The high extraction efficiency, low limit of quantification, and wide linear dynamic

range make this a suitable method for use in clinical samples from Pharmacokinetic

studies following oral administration of Esomeprazole fixed dose (40/40 mg) tablets

in healthy human subjects.

6.5 Applications

The above described analytical method was applied to determine plasma

concentrations of Esomeprazole following oral administration in healthy human

volunteers. These volunteers have informed consent before participation of study and

study protocol was approved by IEC (Institutional Ethics Committee) as per ICMR

(Indian Council of Medical Research) guidelines. Each volunteer was administered 40

mg dose (one 40 mg capsule) in 27 healthy human volunteers by oral administration

with 240 mL of drinking water. The reference product Nexium capsules (Astrazenica)

40 mg, USA and test product Esomeprazole tablet 40 mg (Test capsules) was used.

Blood samples were collected as a pre-dose (0 h) 5 min prior to dosing followed by

further samples at 0.75, 1, 1.333, 1.667, 2, 2.333, 2.667, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9,

10.5 and 12 hours. After dosing 5 mL blood was collected each time in vacutainer

containing K2EDTA. A total of 38 (19 time points from test and reference

respectively) time points were collected by using centrifugation at 3200 g.force, 10°C,


220

10 min and stored below -30 °C until sample analysis. Test and reference was

administered to same human volunteers under fasting conditions separately with a gap

of 7 days washing period as per approved protocol.

The Mean Plasma concentration vs. time curve is shown in Figure 6.15 and

table 6.23.

Figure 6.15. Mean Plasma concentration vs. time curve

Table 6.23: Esomeprazole Concentration (ng/ml) data of the subject samples

obtained from the LC-MS/MS

Time in hours Mean Concentration data

Period 1 Period 2 0 0.00 0.00

0.75 15.30 36.47


221

1 104.43 189.10 1.333 341.90 643.22 1.667 641.36 953.99

2 927.61 1186.98 2.333 1177.04 1422.01 2.667 1174.96 1453.15

3 1258.60 1400.32 3.5 1287.51 1245.08 4 1198.15 1103.51

4.5 1044.29 953.41 5 826.96 759.09 6 573.92 521.12 7 385.60 373.35 8 276.47 266.11 9 192.68 191.17

10.5 116.39 118.76 12 73.38 73.42

6.6 Pharmacokinetic Studies

Pharmacokinetic parameters from the human plasma concentration samples

were calculated by a non compartmental statistics model using WinNon-Lin5.0

software (Pharsight, USA). Blood samples were taken for a period of 3 to 5 times of

the terminal elimination half-life (t1/2) and it was considered as area under the

concentration time curve (AUC) ratio higher than 80% as per FDA guidelines20-24

Plasma Esomeprazole concentration-time profiles were visually inspected Cmax and

Tmax values were determined. The AUC0–t was obtained by trapezoidal method.

AUC0-∞ was calculated up to the last measureable concentration and extrapolations

were obtained using the last measureable concentration and the terminal elimination

rate constant (Ke). The terminal elimination rate constant (Ke), was estimated from the

slope of the terminal exponential phase of the plasma of Esomeprazole concentration–

time curve by means of the linear regression method. The terminal elimination half-

life, t1/2, was then calculated as 0.693/Ke. Regarding AUC0–t, AUC0-∞ and Cmax

bioequivalence was assessed by means of analysis of variance (ANOVA) and


222

calculating the standard 90% confidence intervals (90% CIs) of the ratios

test/reference (logarithmically transformed data). The bioequivalence was considered

when the ratio of averages of log-transformed data was within 80 to 125% for AUC0–t,

AUC0-∞ and Cmax. Pharmacokinetic data is shown in Table 6.24 and Table 6.25.

Table 6.24. Esomeprazole Pharmacokinetic data

Esomeprazole Pharmacokinetic data

Pharmacokinetic

Parameter

Test Reference

Mean±SD Mean±SD

Cmax 1287.506 ± 484 1453.151 ± 514

AUC 0-t 6347.388 ±204 6531.622 ± 188

AUC 0-inf 6612.884 ± 532 6818.823 ± 612

tmax 3.5 2.7

T 1/2 2.2 2.3

Table 6.25. Esomeprazole Pharmacokinetic data

Pharmacokinetic

Parameter

Cmax AUC 0-t AUC 0-inf

Test/Reference 88.60 97.17 96.97

Pharmacokinetic Conclusion

The present study provides firm evidence to support that the in house

Esomeprazole 40 mg was not bioequivalent with Nexium capsule (Astrazenica, USA)

40 mg capsule under fasting conditions.

In vivo data was predicted by using Precipitation Extraction procedure and

concentrations were found through Liquid Chromatography Tandem Mass


223

Spectroscopy detection. The Pharmacokinetic parameters assessed were AUC0-t,

AUC0-, Cmax, Tmax, t1/2. The bioequivalence criteria are based on the 90% confidence

intervals whose acceptance range is in between 80%-125%.

The results obtained for Esomeprazole was within the acceptance range.

Therefore, it can be concluded that the two Esomeprazole formulations (reference and

test) analyzed were Bioequivalent terms of rate and extent of absorption.

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