0002 - Complete Monograph Methods - Esomeprazole - MM

Esomeprazole (USP)
Esomeprazole is the S-enantiomer of omeprazole.
Esomeprazole is a proton pump inhibitor and reduces acid secretion through inhibition of the
H+ / K+ ATPase in gastric parietal cells. By inhibiting the functioning of this transporter, the drug
prevents formation of gastric acid. It is used in the treatment of dyspepsia, peptic ulcer disease,
gastroesophageal reflux disease, and Zollinger-Ellison syndrome.
Common commercial brand names: Nexium, Essocam, Esomezol

Esomeprazole was developed by AstraZeneca.
Sales in 2010 were $4.9 billion globally.
Patent expired in 2014
We have followed the experimental conditions in USP37-NF32 for Esomeprazole magnesium and
Esomeprazole magnesium delayed release capsules monographs.
Identification – FTIR (197K)

Identification – AAS (content of magnesium)
Assay and Related Substances – HPLC and UHPLC (both isocratic and gradient methods)
Karl Fischer – water content
Dissolution
Assay and Related Substances (RS) as well as dissolution testing have been carried out with HPLC
using RP-8 and RP-18 endcapped columns with both particulate and monolithic backbones.
Some of the methods are isocratic and were scaled to UHPLC settings relative to the prescribed
HPLC column. Since the situation with monolithic columns is similar to that with core shell
columns it is possible to make adjustments using the calculation of N and to keep this within
−25% to +50%, relative to the prescribed column (see page 9-14).
We transferred the dissolution testing method for Esomeprazole magnesium delayed release
capsules to a monolithic column. The new method is three times faster, having improved
chromatographic resolution, lower column backpressure, and still meeting all method
performance criteria.
Merck Millipore is a division of Merck KGaA, Darmstadt, Germany

www.merckmillipore.com/perfect-solution
29
Esomeprazole Magnesium (USP)
Definition:
Esomeprazole Magnesium contains NLT 98.0% and NMT 102.0% of C34H36MgN6O6S2, calculated on the
anhydrous basis.
Identification
-A. INFRARED ABSORPTION <197K> FTIR
-B. The sample solution, prepared and tested as directed in the test for Content of Magnesium, exhibits a
significant absorption at 285.2 nm. AAS
Assay: HPLC
-Procedure:
Solution A: Dissolve 0.725 g of monobasic sodium phosphate and 4.472 g of anhydrous dibasic sodium
phosphate in 300 mL of water, and dilute with water to 1000 mL. Dilute 250 mL of this solution with water
to 1000 mL. If necessary, adjust with phosphoric acid to a pH of 7.6.
Solution B: Mix 11 mL of 0.25 M tribasic sodium phosphate with 22 mL of 0.5 M dibasic sodium
phosphate, and dilute with water to 100 mL.
Mobile phase: Acetonitrile and Solution A (7:13)
Standard solution: Transfer 10 mg of USP Omeprazole RS to a 200-mL volumetric flask, and dissolve in
about 10 mL of methanol. Add 10 mL of Solution B, and dilute with water to volume.
[Note—This solution contains 0.05 mg/mL of omeprazole.]
Sample solution: Transfer 10 mg of Esomeprazole Magnesium to a 200-mL volumetric flask, and dissolve
in about 10 mL of methanol. Add 10 mL of Solution B, and dilute with water to volume.
[Note—This solution contains 0.05 mg/mL of esomeprazole magnesium. ]
Chromatographic system: (See Chromatography 621, System Suitability.)
Detector: UV 280 nm
Column: 4.0-mm × 12.5-cm or a 4.6-mm × 15-cm; 5 µm packing L7.
[Note—Alternatively, a 3.9-mm × 15-cm column that contains 4 µm packing L1 may be used.]
Flow rate: 1 mL/min
Injection size: 20 µL
We have used a Purospher® STAR RP-8 endcapped (5 µm) 150x4.6 mm (1.51453.0001) for HPLC analysis

30
System suitability
Sample: Standard solution
Suitability requirements: Column efficiency: NLT 2000 theoretical plates
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of C34H36MgN6O6S2 in the portion of Esomeprazole Magnesium taken:
Result = (rU/rS) × (CS/CU) × [Mr1/(2 ×Mr2)] × 100
rU = peak response from the Sample solution

rS = peak response from the Standard solution
CS = concentration of omeprazole in the Standard solution (mg/mL)
CU = concentration of Esomeprazole Magnesium in the Sample solution (mg/mL)
Mr1 = molecular weight of esomeprazole magnesium, 713.12
Mr2 = molecular weight of omeprazole, 345.42
Acceptance criteria: 98.0%–102.0% on the anhydrous basis
Content of Magnesium AAS

Lanthanum solution: Transfer 58.7 g of lanthanum oxide into a 1000-mL volumetric flask, wet the
substance with some water, and dissolve by cautious addition of 250 mL of hydrochloric acid in 20- to
30-mL portions, cooling between the additions. Add water while stirring, cool to room temperature, and
dilute with water to volume.
[Note—Store the solution in a plastic bottle. ]
Standard stock solution: 1000 µg/mL of magnesium in water, from a commercially prepared atomic
absorption standard solution. [Note—Store the solution in a plastic bottle. ]
Standard solution A: Transfer 10.0 mL of Standard stock solution to a 500-mL volumetric flask, add 50
mL of 1 N hydrochloric acid, and dilute with water to volume. Transfer 20.0 mL of this solution to a 200-
mL volumetric flask, and dilute with water to volume.
[Note—This solution contains 2 µg/mL of magnesium. ]
Standard solution B: Combine 5.0 mL of Standard solution A and 4.0 mL of Lanthanum solution, and
dilute with water to 100.0 mL (0.1 µg/mL).

31
Standard solution C: Combine 10.0 mL of Standard solution A and 4.0 mL of Lanthanum solution, and
Standard solution D: Combine 15.0 mL of Standard solution A and 4.0 mL of Lanthanum solution, and
Standard solution E: Combine 20.0 mL of Standard solution A and 4.0 mL of Lanthanum solution, and
Standard solution F: Combine 25.0 mL of Standard solution A and 4.0 mL of Lanthanum solution, and
dilute with water to 100.0 mL (0.5 µg/mL). [Note—Concentrations of the Standard solutions and the
Sample solution may be modified to fit the linear or working range of the instrument. When using
instruments with a linear calibration graph, the number of Standard solutions can be reduced. ]
Blank solution: Transfer 4.0 mL of Lanthanum solution to a 100-mL volumetric flask, and dilute with
water to volume.
Sample solution: Transfer 250 mg of Esomeprazole Magnesium to a 100-mL volumetric flask, add 20 mL
of 1 N hydrochloric acid, swirl until dissolved, and dilute with water to volume. Allow to stand for 30 min.
Transfer 10.0 mL of this solution to a 200-mL volumetric flask, and dilute with water to volume. Transfer
10.0 mL of the solution to another 100-mL volumetric flask, add 4.0 mL of Lanthanum solution, and
Spectrometric conditions (See Spectrophotometry and Light-Scattering <851>) AAS

Mode: Atomic absorption spectrophotometer
Flame: Air–acetylene
Analytical wavelength: 285.2 nm
Analysis
Samples: Standard solution B, Standard solution C, Standard solution D, Standard solution E, Standard
solution F, Blank solution, and Sample solution . Determine the concentration, Cs, in µg/mL, of
magnesium in the Sample solution using the calibration graph.
Calculate the percentage of magnesium in the portion of Esomeprazole Magnesium taken:
Result = (CS/CU) × (100/(100 F)) × 100
CS = content of magnesium in the Sample solution as calculated above (µg/mL)

CU = concentration of Esomeprazole Magnesium in the Sample solution (µg/mL)
F = content of water in Esomeprazole Magnesium, as determined in Specific Tests, Water Determin. (%)
Acceptance criteria: 3.30%–3.55%, on anhydrous basis

32
IMPURITIES - Organic Impurities - Procedure 1 HPLC

Solution A: 0.725 g of monobasic sodium phosphate and 4.472 g of anhydrous dibasic sodium phosphate
in 300 mL of water, and dilute with water to 1000 mL. Dilute 250 mL of this solution with water to 1000
mL. If necessary, adjust with phosphoric acid to a pH of 7.6.
Mobile phase: Acetonitrile and Solution A (11:29).

[Note—To improve the resolution, the composition may be changed to 1:3, if necessary.]
System suitability solution: 1 mg of USP Omeprazole RS and 1 mg of USP Omeprazole Related

Compound A RS in 25 mL of Mobile phase.
[Note—Omeprazole Related Compound A is omeprazole sulfone.]
Sample solution: 4 mg of Esomeprazole Magnesium in 25 mL of Mobile phase.

[Note—Prepare this solution fresh. ]
Chromatographic system (See Chromatography 621, System Suitability.)

Detector: UV 280 nm
Column: 4.0-mm × 12.5-cm or a 4.6-mm × 15-cm; 5 µm packing L7.
[Note—Alternatively, a 3.9-mm × 15-cm column that contains 4 µm packing L1 may be used.]
Flow rate: 0.8–1 mL/min
System suitability
Sample: System suitability solution
[Note—For relative retention times, see Impurity Table below]
Name RRT Acceptance Criteria - NMT (%)
Omeprazole N-oxide (1) 0.45 0.1

Omeprazole sulfone (2) 0.8 0.2
Omeprazole RS A
Any other individual impurities - 0.1
Omeprazole RS 1.0 -
(1) 4-Methoxy-2-[[(RS)-(5-methoxy-1H-benzimidazol-2-yl)sulfinyl]methyl]-3,5-dimethylpyridine 1-oxide.
(2) 5-Methoxy-2-[[(4-methoxy-3,5-dimethylpyridin-2-yl)methyl]sulfonyl]-1H-benzimidazole

33
Suitability requirements
Resolution: NLT 3 between omeprazole related compound A and omeprazole
Analysis
Sample: Sample solution
Record the chromatogram for at least 4.5 times the retention time of the omeprazole peak, and measure
the peak responses. Identify the impurities based on the retention times shown in Impurity Table 1.
Calculate the percentage of any individual impurity in the portion of Esomeprazole Magnesium taken:
Result = (rU/rT) × 100

rU = peak response for each impurity
rT = sum of all peak responses
Acceptance criteria: Individual impurities: See Impurity Table. Total impurities: NMT 0.5%
Procedure 2: Enantiomeric Purity

- This test could not be performed due to unavailability of a suitable chiral column”
Water Determination Karl Fischer

Method I <921>: 6.0%–8.0%
Color of Solution
Sample solution: 20 mg/mL of Esomeprazole Magnesium in methanol, filtered
Analysis: Determine the absorbance of this solution at 440 nm, in 1-cm cells, using methanol as the
blank.
Acceptance criteria: NMT 0.2
ADDITIONAL REQUIREMENTS
Packaging and Storage: Preserve in tight containers, protected from light. Store at room temperature.
USP Reference Standards 11
USP Esomeprazole Magnesium RS
USP Omeprazole RS
USP Omeprazole Related Compound A RS
Omeprazole sulfone, 5-methoxy-2-[[(4-methoxy-3,5-dimethylpyridin-2-yl)methyl]sulfonyl]-1H-benzimidazole.

34
Recommended Merck Millipore products:
FTIR – Identification (197K)

Potassium bromide for IR spectroscopy Uvasol® (1.04907)
KF – Water Determination (921 –Ia)
CombiTitrant 5 one-component reagent for volumetric KF titration 1 ml = ca. 5 mg H2O apura® 1.88005
CombiSolvent methanol-free for volumetric KF titration with one component reagents apura® 1.88008
AAS - Content of Magnesium

Lanthanum(III) oxide for atomic absorption spectroscopy (1.10982)
Hydrochloric Acid (30% Ultrapur 1.01514)
Water (LiChrosolv® 1.15333 or water from a Milli-Q system)
HPLC Assay and Related Substances (API)

Purospher® STAR RP-8 endcapped (5 µm) 150x4.6 mm (1.51453) for HPLC Assay and RS analysis
Purospher® STAR RP-8 endcapped (2 µm) 100x2.1 mm (1.50629) for RS analysis
Chromolith® HighResolution RP-18 endcapped 100x4.6 mm (1.52022) for RS analysis
Sodium dihydrogen phosphate dihydrate for analysis EMSURE® Reag. Ph Eur 106342
di-Sodium hydrogen phosphate dihydrate for analysis EMSURE® 106580
tri-Sodium phosphate dodecahydrate for analysis EMSURE® ACS,Reag. Ph Eur 106578
ortho-Phosphoric acid 85% for analysis EMSURE® ACS,ISO,Reag. Ph Eur 100573
Acetonitrile (isocratic grade for liquid chromatography LiChrosolv® 1.14291
HPLC Assay and Related Substances (Delayed Release Capsules)

Purospher® STAR RP-18 endcapped (5 µm) 150x4.6 mm (1.51455) for assay and dissolution testing
Chromolith® HighResolution RP-18 endcapped 100x4.6 mm (1.52022.0001)
Purospher® STAR RP-18 endcapped (3 µm) 100x4.6 mm 1.50469.001 for RS analysis
Acetonitrile (isocratic grade for liquid chromatography LiChrosolv® 1.14291 )
Acetonitrile (gradient grade for liquid chromatography) LiChrosolv® Reag. Ph Eur 1.00030
Dissolution Testing
Hydrochloric acid (fuming 37% for analysis EMSURE® ACS,ISO,Reag. Ph Eur 100317 )
Sodium hydroxide solution 50% for analysis EMSURE® 158793
Millex PTFE filter

35
Identification (197K)
A. INFRARED ABSORPTION <197K> FTIR

The reference 197K in a monograph signifies that the substance under examination is mixed intimately with
potassium bromide.
We recommend Potassium bromide for IR spectroscopy Uvasol® (1.04907) to be used.

36
B: Content of Magnesium – Atomic Absorption Spectroscopy (AAS)
Sample Solution: Transfer 250 mg of Esomeprazole Magnesium to a 100-mL volumetric flask, add 20 mL
of 1 N hydrochloric acid, swirl until dissolved, and dilute with water to volume. Allow to stand for 30 min.
Transfer 10.0 mL of this solution to a 200-mL volumetric flask, and dilute with water to volume. Transfer
10.0 mL of the solution to another 100-mL volumetric flask, add 4.0 mL of Lanthanum solution, and
Absorption at 285.2  0.563
Lanthanum solution: Transfer 58.7 g of lanthanum oxide into a 1000-mL volumetric

flask, wet the substance with some water, and dissolve by cautious addition of 250 mL of hydrochloric
acid in 20- to 30-mL portions, cooling between the additions. Add water while stirring, cool to room
temperature, and dilute with water to volume. [NOTE—Store the solution in a plastic bottle. ]
Standard stock solution: 1000 μg/mL of magnesium in water, from a commercially prepared atomic
absorption standard solution. [NOTE—Store the solution in a plastic bottle. ]
Standard solution A: Transfer 10.0 mL of Standard stock solution to a 500-mL volumetric flask, add 50
mL of 1 N hydrochloric acid, and dilute with water to volume. Transfer 20.0 mL of this solution to a 200-
mL volumetric flask, and dilute with water to volume. [NOTE—This solution contains 2 μg/mL of
magnesium. ]
We recommend Lanthanum(III) oxide for atomic absorption spectroscopy (1.10982), hydrochloric acid
Ultrapur (1.01514) and Magnesium ICP standard traceable to SRM from NIST Mg(NO3)2 in HNO3 2-3%
1000 mg/l Mg Certipur® (1.70331) to be used.
Solution A Lanthanum oxide solution Dilution Final standard concentration
Standard Solution B 5.0 ml 4.0 ml 100 ml 0.1 µg / ml
Standard Solution C 10.0 ml 4.0 ml 100 ml 0.2 µg / ml

Standard Solution D 15.0 ml 4.0 ml 100 ml 0.3 µg / ml
Standard Solution E 20.0 ml 4.0 ml 100 ml 0.4 µg / ml
Standard Solution F 25.0 ml 4.0 ml 100 ml 0.5 µg / ml

37
Calibration Curve (AAS):
Concentration (µg/ml) Absorbance

0 0.003
0.1 0.148 Absorbance for sample 0.563
0.2 0.294 Conc. calculated for sample 0.390 µg/ml
0.3 0.437
0.4 0.591
0.5 0.752
0,8
0,7 y = 1,4906x - 0,0018

R² = 0,9995
0,6
0,5
Absorbance
0,4
0,3
0,2
0,1
0
0 0,1 0,2 0,3 0,4 0,5 0,6
Concentration (µg/ml)
Result = (CS / CU) X (100 / (100-F)) X 100 = (0.3896/12.516) X (100/ (100 – 8.121) ) X 100 = 3.39 %
The obtained value is within the acceptance criteria: 3.30%–3.55%, on anhydrous basis

38
Water Determination <USP 921>
Pharmaceutical products are often characterized by complex formulations. Difficulties observed during
Karl Fischer determination are often caused by the limited solubility. In some cases side reactions have to
be considered. In dependence of composition and properties of the formulations, various measures are
necessary for an undisturbed Karl Fischer determination.
In the case of Esomeprazole the water determination can be carried out without problems according
to standard methods.
In pharmaceutical guidelines (USP, Ph Eur, DAB) the Karl Fischer titration is described as common method
for water determination. For some substances special procedures can be found. The determination of
mass loss as method for water determination is not recommended.
Titration one component system
Titrant: apura - CombiTitrant 5 (1.88005)

One component reagent for volumetric Karl Fischer titration, 1 mL = approx. 5 mg water
Solvent: apura – CombiSolvent - methanol-free solvent for volumetric Karl Fischer titration with one
component reagents; 50 ml (1.88008)
Titration parameters
Stirring time: 90 s
Default titration settings, e.g.:
I(pol) = 20 - 50 μA, U(EP) = 100 - 250 mV
Stop criterion: drift < 20 μL/min
Sample size: 0.2 g (we used Esomeprazole Magnesium RS)
Result:
Measured water content in Esomeprazole: 7,63% (USP - requirement: 6-8%)
Procedure
The titration medium is first placed into the titration cell and titrated dry by means of the titrant. Then
the sample is added from a weighing boat (exact sample weight determination by weighing of weighing
boat before and after addition) and the titration is started. For complete dissolution of the sample a
stirring time of 90 seconds is recommended.
Product P/N
CombiTitrant 5 one-component reagent for volumetric KF titration 1 ml = ca. 5 mg H2O apura® 1.88005
CombiSolvent methanol-free solvent for volumetric KF titration with one component reagents apura® 1.88008

39
Esomeprazole Magnesium (USP) – Assay
Purospher STAR® RP-8 endcapped HPLC
Chromatographic Conditions
Column: Purospher® STAR RP-8 endcapped (5 µm) 150x4.6 mm 1.51453.0001
Injection: 20 µL
Detection: UV 280 nm
Cell: 10 µL
Flow Rate: 1.0 mL/min
Dissolve 0.725 g of monobasic sodium phosphate and 4.472 g of anhydrous dibasic sodium
Mobile Phase: phosphate in 300 mL of water, and dilute with water to 1000 mL. Dilute 250 mL of this solution
with water to 1000 mL. If necessary, adjust with phosphoric acid to a pH of 7.6.
Mix acetonitrile and Solution A (7:13 v/v)
Temperature: 25˚C
Diluent: Mix 11 mL of 0.25 M tribasic sodium phosphate with 22 mL of 0.5 M dibasic sodium phosphate, and
dilute with water to 100 mL.
Standard Transfer 10 mg of USP Omeprazole to a 200-mL volumetric flask, and dissolve in about 10 mL
Solution: methanol. Add 10 mL of Solution B, and dilute with water to final volume.
Sample Solution: Transfer 10 mg of Esomeprazole Magnesium to a 200-mL volumetric flask, and dissolve in about 10
mL of methanol. Add 10 mL of Solution B, and dilute with water to final volume.
Pressure Drop: 101 Bar (1464 psi)
500 500
Standard Solution
400 Blank Solution 400
300 300
200 200
100 100
0 0
0 2 4 6 8 10 0 2 4 6 8 10
System Suitability requirement: Column efficiency: NLT 2000 theoretical plates
Chromatographic Data:
Compound Retention Time (min) Plates Tailing Factor
Impurity A
Omeprazole 4.2 8269 1.1

40
Linearity: Concentration (ppm) Area Units
0.1 7273
0.5 33723
1 68763
5 331460
10 677630
25 1694075
40 2734520
50 3388150
60 4068780
75 5082225
STEYX 8320
Slope 67841
LOD 0.4
LOQ 1.2
600
y = 67841x + 8320
500 R² = 0.99999
Response (Area unitsx104)
400
300
200
100
0
0 10 20 30 40 50 60 70 80
Concentration (ppm)

41
Esomeprazole Related Substances (USP)
Injection: 50 µL
Cell: 10 µL
Solution A: Dissolve 0.725 g of monobasic sodium phosphate and 4.472 g of anhydrous dibasic sodium phosphate
in 1000mL water. If necessary, adjust with phosphoric acid to a pH of 7.6.
Mobile Phase : Acetonitrile:Solution A 11:29 (v/v)
Temperature: Ambient
Diluent: Mobile phase
SST solution: Dissolve 1.0mg of Omeprazole standard & related compound A in 25 mL of diluent.
Sample solution: Dissolve 4.0mg of sample in 25 mL of diluent.
500
400
Intensity (mV)
300
200
100
0
0 5 10 15 20
Retention time (mins)
Relative retention time (RRT): 0.8 for and 1.0 for omeprazole related compound A and omeprazole, respectively
Chromatographic Data : SST solution

No. Compound Retention Time (min) Resolution RRT
1 Omeprazole Related compound A 7.46 - 0.85
2 Omeprazole 8.69 4.2 1.00

42
100
80 BLANK
Intensity (mV)
60
40
20
0
0 5 10 15 20
20
15
Intensity (mV)
10
0
0 5 10 15 20

1 Related compound A 7.46 - 0.85
2 Esomeprazole 8.69 4.2 1.00

43
Purospher STAR® RP-8 endcapped UHPLC
Injection: 5 µL
Cell: 2.5 µL
Solution A: Dissolve 0.725 g of monobasic sodium phosphate and 4.472 g of anhydrous dibasic sodium phosphate
in 1000mL water. If necessary, adjust with phosphoric acid to a pH of 7.6.
Mobile Phase : Acetonitrile:Solution A (27:73) (v/v)
SST solution: Dissolve 1.0mg of Omeprazole standard & related compound A in 25 mL of diluent.
180
150
Intensity (mV)
120
90
60
30
0
0 2 4 6 8 10

2 Omeprazole 4.1 4.2 1.00

44
Purospher STAR® RP-8 endcapped UHPLC
40
BLANK
36
Intensity (mV)
32
28
24
20
0 2 4 6 8 10
8
Intensity (mV)
0
0 2 4 6 8 10


45
Chromolith HighResolution® RP-18 endcapped HPLC
Column: Chromolith® HighResolution RP-18 endcapped 100x4.6 mm 1.52022.0001
Injection: 20 µL
Cell: 10 µL
Solution A: Dissolve 0.725 g of monobasic sodium phosphate and 4.472 g of anhydrous dibasic sodium
phosphate in 1000mL water. If necessary, adjust with phosphoric acid to a pH of 7.6.
Mobile Phase : Acetonitrile:Solution A 25:75 (v/v)
SST solution: Dissolve 1.0mg of Omeprazole standard and related compound A in 25 mL of diluent.
300
250
200
Intensity (mV)
150
100
50
0
0 5 10 15 20

46
- Delayed Release Capsules
Definition:
Esomeprazole Magnesium Delayed-Release Capsules contain an amount of Esomeprazole Magnesium
equivalent to NLT 90.0% and NMT 110.0% of the labeled amount of esomeprazole (C34H36MgN6O6S2,).
Identification
A. Enantiomeric purity – not performed as it requires a chiral column (4.0×10 mm; 5 µm packing L41)
Assay: HPLC
-Procedure:
Buffer: Prepare a pH 7.3 phosphate buffer by mixing 10.5 mL of 1.0 M monobasic sodium phosphate
buffer and 60 mL of 0.5 M dibasic sodium phosphate buffer, and diluting with water to 1000 mL.
Diluent: Prepare as directed in Identification test A.
Mobile phase: Mix 350 mL of acetonitrile and 500 mL of the Buffer. Dilute with water to 1000 mL.
Standard solution: Transfer 10 mg of USP Omeprazole RS to a 250-mL volumetric flask, and dissolve in
about 10 mL of alcohol. Add 40 mL of Diluent, and dilute with water to volume. This solution contains
0.04 mg/mL of USP Omeprazole RS.
Sample stock solution: Mix the contents of NLT 20 Capsules. Transfer a portion of the Capsule content,
equivalent to 20 mg of esomeprazole, to a 100-mL volumetric flask, add 60 mL of Diluent, and shake for
20 min to dissolve the pellets. Sonicate for a few minutes, if needed, to completely dissolve. Add 20 mL of
alcohol, and sonicate for a few minutes. Cool, and dilute with Diluent to volume.
Pass a portion of the solution through a filter of 1 µm pore size.
Sample solution: 0.04 mg/mL of esomeprazole from the Sample stock solution in water.
Store this solution protected from light.
Chromatographic system: (See Chromatography 621, System Suitability.)

Detector: UV 302 nm
Column: 4.6-mm × 15-cm; 5 µm packing L1.
Flow rate: 1 mL/min

47
System suitability
Sample: Standard solution
Relative standard deviation: NMT 2.0%
Analysis
Calculate the percentage of the labeled amount of esomeprazole (C17H19N3O3S) in the portion of the
Capsules taken:
Result = (rU/rS) × (CS/CU) × 100

CS = concentration of USP Omeprazole RS in the Standard solution (mg/mL)
CU = nominal concentration of esomeprazole in the Sample solution (mg/mL)
Acceptance criteria: 90.0%–110.0%
Dissolution <711> HPLC

Medium: 0.1 N hydrochloric acid; 300 mL. After 2 h, continue with a pH 6.8 phosphate buffer as follows.
To the vessel, add 700 mL of 0.086 M dibasic sodium phosphate, and adjust with 2 N hydrochloric acid or
2 N sodium hydroxide, if necessary, to a pH of 6.8 ± 0.05.
Apparatus 2: 100 rpm

Time: 30 min in a pH 6.8 phosphate buffer
Standard solution: Prepare a solution containing 2 mg/mL of USP Omeprazole RS in alcohol. Dilute this
solution with pH 6.8 phosphate buffer to obtain a solution containing (L/1000) mg/mL, where L is the
label claim, in mg/Capsule. Immediately add 2.0 mL of 0.25 M sodium hydroxide to 10.0 mL of this
solution, and mix.
[Note—Do not allow the solution to stand before adding the sodium hydroxide solution. ]
Sample solution: After 30 min in pH 6.8 phosphate buffer, pass a portion of the solution under test
through a suitable filter. Transfer 5.0 mL of the filtrate to a suitable glassware containing 1.0 mL of 0.25
M sodium hydroxide. Mix well. Protect from light.

48
Buffer, Mobile phase, System suitability, and Chromatographic system: Proceed as directed in the Assay.
Analysis
Calculate the percentage of esomeprazole (C17H19N3O3S) dissolved:
Result = (rU/rS) × (CS/L) ×V× 100

CS = concentration of the Standard solution (mg/mL)
L = label claim (mg/Capsule)
V = volume of Medium, 1000 mL
Tolerances: NLT 75% (Q) of the labeled amount of esomeprazole (C17H19N3O3S) is dissolved.
IMPURITIES - Organic Impurities HPLC

Buffer: Prepare a pH 7.6 phosphate buffer by mixing 5.2 mL of 1.0 M monobasic sodium phosphate
buffer and 63 mL of 0.5 M dibasic sodium phosphate buffer, and diluting with water to 1000 mL.
Solution A: Mix 100 mL of acetonitrile and 100 mL of the Buffer. Dilute with water to 1000 mL.
Solution B: Mix 800 mL of acetonitrile and 10 mL of the Buffer. Dilute with water to 1000 mL.
Mobile phase: See Table. Time (min) Solution A (%) Solution A (%)
0 100 0
10 80 20
30 0 100
31 100 0
45 100 0
Diluent: Prepare as directed in Identification test A.
System suitability stock solution: 1 mg/mL each of USP Omeprazole RS and USP Omeprazole Related
Compound A RS in methanol
System suitability solution: 1 µg/mL each of USP Omeprazole RS and USP Omeprazole Related
Compound A RS from System suitability stock solution, in a mixture of Diluent and water (1:4)
Sample solution: Transfer a portion of the powdered pellets (about 80–90 mg), from the Capsule content,
to a 200-mL volumetric flask, add 20 mL of methanol, and shake for 30 s. Add 40 mL of Diluent, shake for
30 s by hand, and sonicate for a few minutes. Cool, and dilute with water to volume.
Pass a portion of the solution through a filter of 0.45 µm pore size.
[Note—The solution is stable for 3 h if stored protected from light. ]

49
Chromatographic system (See Chromatography 621, System Suitability.)
Detector: UV 302 nm
Column: 4.6-mm × 10-cm; 3 µm packing L1
Flow rate: 1 mL/min
System suitability
Sample: System suitability solution
[Note—See Table 2 for the relative retention times.]
Resolution: NLT 2.5 between omeprazole related compound A and omeprazole
Analysis
Sample: Sample solution
Calculate the percentage of any individual impurity in the portion of the Capsules taken:
Result = (rU/rT) × 100
rU = peak response for each impurity

rT = sum of all peak responses
Acceptance criteria: See Table.

Name RRT Acceptance criteria, NMT
(%)
Omeprazole sulfonea 0.93 0.5
Omeprazole 1.0 -
Any other individual impurity - 0.2
Total impurities - 2
ADDITIONAL REQUIREMENTS
Packaging and Storage: Preserve in tight containers. Store at room temperature.
USP Reference Standards

USP Omeprazole RS
USP Omeprazole Related Compound A RS = Omeprazole sulfone =
= 5-methoxy-2-[[(4-methoxy-3,5-dimethyl-2-pyridinyl)methyl]sulfonyl]-1H-benzimidazole. (C17H19N3O4S)

50
Esomeprazole Magnesium Capsules (USP)
Purospher STAR® RP-18 endcapped – Related Impurities
Injection: 20 µL
Cell: 10 µL
Buffer: Prepare a pH 7.6 phosphate buffer by mixing 5.2 mL of 1.0 M monobasic sodium
Mobile Phase: phosphate buffer and 63 mL of 0.5 M dibasic sodium phosphate buffer diluting with water to
1000 mL.
Solution A: Mix 100 mL of acetonitrile and 100 mL of the Buffer. Dilute with water to 1000 mL.
Solution B: Mix 800 mL of acetonitrile and 10 mL of the Buffer. Dilute with water to 1000 mL.
Gradient: See table
Temperature: 25˚C
Time (min) Solution A (%) Solution B (%)
0.0 100 0
10 80 20
30 0 100
31 100 0
45 100 0
Diluent: Dissolve 5.24 g of tribasic sodium phosphate dodecahydrate in water.

Add 110 mL of 0.5 M dibasic sodium phosphate solution, and dilute with
Standard Solution: 1 µg/mL each of USP Omeprazole and USP Omeprazole Related Compound A in methanol
Sample Solution: Transfer a portion of the powdered pellets (about 80–90 mg), from the capsule content, to a 200-
mL volumetric flask, add 20 mL of methanol shake for 30 s. Add 40 mL of Diluent, shake for 30 s
by hand, and sonicate for a few minutes. Cool, and dilute with water to volume. Pass a portion of
the solution through a filter of 0.45-μm pore size.
Pressure Drop: 149 Bar to 95 Bar (2160 - 1378 psi)

51
30
15
10 System Suitability Solution (SST)

20
5
14 15 16
10
0
0 5 10 15 20 25 30 35 40 45
30
25
20
15
Blank
10
0
0 5 10 15 20 25 30 35 40 45
Retention Time (minutes)
Compound Retention Time (min) RRT Resolution Tailing Factor
Impurity A 15.0 0.96 - 1.1
Omeprazole 15.6 1.00 3.2 1.2

52
Sample Analysis (delayed release capsules)
30
20
Impurity A
10
0
0 5 10 15 20 25 30 35 40 45
Compound Retention Time (min) RRT Resolution Tailing Factor
Impurity A 15.0 0.96 - 1.1
Omeprazole 15.6 1.0 3.2 1.2

53
Purospher STAR® RP-18 endcapped – Dissolution
Injection: 20 µL
Cell: 10 µL
0.1 N hydrochloric acid; 300 mL. After 2 h, continue with a pH 6.8 phosphate buffer as follows.
To the vessel, add 700 mL of 0.086 M dibasic sodium phosphate, and adjust with 2 N hydrochloric
Medium: acid or 2 N sodium hydroxide if necessary, to a pH of 6.8 ± 0.05.
Apparatus 2: 100 rpm (Time: 30 min in a pH 6.8 phosphate buffer)
Mobile phase: Buffer: Prepare a pH 7.3 phosphate buffer by mixing 10.5 mL of 1.0 M monobasic sodium
phosphate buffer and 60 mL of 0.5 M dibasic sodium phosphate buffer, and diluting with water to
1000 mL. Mix 350 mL of acetonitrile and 500 mL of the Buffer. Dilute with water to 1000 mL.
Temperature: 25˚C
Diluent: Dissolve 5.24 g of tribasic sodium phosphate dodecahydrate in water. Add 110 mL of 0.5 M dibasic
sodium phosphate solution, and dilute with water to 1000 mL.
Standard Solution: Transfer 10 mg of USP Omeprazole RS to a 250-mL volumetric flask, and dissolve in about 10 mL
of alcohol. Add 40 mL of Diluent, and dilute with water to volume.
Sample Solution: After 30 min in pH 6.8 phosphate buffer, pass a portion of the solution under test through a
suitable filter. Transfer 5.0 mL of the filtrate to a suitable glassware containing 1.0 mL of 0.25 M
sodium hydroxide. Mix well. Protect from light.
Peak Purity UV Analysis

mAU mAU
Peak 750
0.75 2000
0.50 500
1000
302
0.25
250
0
0.00 Zero Line
0 200 300 400 500 600 700 nm
4.25 4.50 min

54
mV
150
125
100 Capsule 5
75 Capsule 4
50 Capsule 3
Capsule 2
25
Capsule 1
0
-25
0.0 2.5 5.0 7.5 10.0 12.5 15.0

Sample Standard [Standard solution] Label claim Media volume Dissolution

(area units) (area units) (mg/ml) (mg/capsule) (ml) (%)
318234 91.2
312926 89.7
316158 357635 0.041 40 1000 90.6
313776 89.9
311351 89.2
Average 90.1
Calculate the percentage of esomeprazole dissolved: Result = (rU/rS) × (CS/L) ×V× 100 = 90.1%
Acceptance criteria: NLT 75% of the claimed esomeprazole (C17H19N3O3S) is dissolved.

55
Chromolith® RP-18 endcapped – Related Impurities
Column: Chromolith® HighResolution RP-18 endcapped 100x4.6 mm 1.52022.0001
Injection: 5 µL (linear scaling=13 µL but the efficiency is higher than with particle packed column so we reduced it further)
Cell: 10 µL
Medium: 0.1 N hydrochloric acid; 300 mL. After 2 h, continue with a pH 6.8 phosphate buffer as follows.
To the vessel, add 700 mL of 0.086 M dibasic sodium phosphate, and adjust with 2 N hydrochloric
acid or 2 N sodium hydroxide if necessary, to a pH of 6.8 ± 0.05.
Apparatus 2: 100 rpm (Time: 30 min in a pH 6.8 phosphate buffer)
Mobile phase: Buffer: Prepare a pH 7.3 phosphate buffer by mixing 10.5 mL of 1.0 M monobasic sodium
phosphate buffer and 60 mL of 0.5 M dibasic sodium phosphate buffer, and diluting with water to
1000 mL. Mix 350 mL of acetonitrile and 500 mL of the Buffer. Dilute with water to 1000 mL.
Temperature: 25˚C
Diluent: Dissolve 5.24 g of tribasic sodium phosphate dodecahydrate in water. Add 110 mL of 0.5 M dibasic
sodium phosphate solution, and dilute with water to 1000 mL.
Standard Solution: Transfer 10 mg of USP Omeprazole RS to a 250-mL volumetric flask, and dissolve in about 10 mL
of alcohol. Add 40 mL of Diluent, and dilute with water to volume.
Sample Solution: After 30 min in pH 6.8 phosphate buffer, pass a portion of the solution under test through a
suitable filter. Transfer 5.0 mL of the filtrate to a suitable glassware containing 1.0 mL of 0.25 M
sodium hydroxide. Mix well. Protect from light.
175 Standard Solution

150
Intensity (mV))
125
100
75
50
25
0
0.0 1.0 2.0 3.0 4.0 5.0

56
Chromolith® RP-18 endcapped – Related Impurities
250
Intensity (mV))
200
150 Capsule 5
Capsule 4
100
Capsule 3
50 Capsule 2
Capsule 1
0
0.0 1.0 2.0 3.0 4.0 5.0

Sample Standard [Standard solution] Label claim Media volume Dissolution
(area units) (area units) (mg/ml) (mg/capsule) (ml) (%)
671494 91.6
656845 89.6
665258 751234 0.041 40 1000 90.8
658643 89.9
655000 89.3
Average 90.2
Calculate the percentage of esomeprazole dissolved: Result = (rU/rS) × (CS/L) ×V× 100 = 90.2%
Acceptance criteria: NLT 75% of the claimed esomeprazole (C17H19N3O3S) is dissolved.

57

0002 - Complete Monograph Methods - Esomeprazole - MM

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Esomeprazole (USP)

Esomeprazole is the S-enantiomer of omeprazole.

Common commercial brand names: Nexium, Essocam, Esomezol

Identification – FTIR (197K)

Merck Millipore is a division of Merck KGaA, Darmstadt, Germany

Mobile phase: Acetonitrile and Solution A (7:13)

Chromatographic system: (See Chromatography 621, System Suitability.)

Merck Millipore is a division of Merck KGaA, Darmstadt, Germany

Result = (rU/rS) × (CS/CU) × [Mr1/(2 ×Mr2)] × 100

rU = peak response from the Sample solution

Acceptance criteria: 98.0%–102.0% on the anhydrous basis

Content of Magnesium AAS

Merck Millipore is a division of Merck KGaA, Darmstadt, Germany

Spectrometric conditions (See Spectrophotometry and Light-Scattering <851>) AAS

Calculate the percentage of magnesium in the portion of Esomeprazole Magnesium taken:

Result = (CS/CU) × (100/(100 F)) × 100

CS = content of magnesium in the Sample solution as calculated above (µg/mL)

Acceptance criteria: 3.30%–3.55%, on anhydrous basis

Merck Millipore is a division of Merck KGaA, Darmstadt, Germany

IMPURITIES - Organic Impurities - Procedure 1 HPLC

Mobile phase: Acetonitrile and Solution A (11:29).

System suitability solution: 1 mg of USP Omeprazole RS and 1 mg of USP Omeprazole Related

Sample solution: 4 mg of Esomeprazole Magnesium in 25 mL of Mobile phase.

Chromatographic system (See Chromatography 621, System Suitability.)

Name RRT Acceptance Criteria - NMT (%)

Omeprazole N-oxide (1) 0.45 0.1

Merck Millipore is a division of Merck KGaA, Darmstadt, Germany

Result = (rU/rT) × 100

Procedure 2: Enantiomeric Purity

Water Determination Karl Fischer

Acceptance criteria: NMT 0.2

Merck Millipore is a division of Merck KGaA, Darmstadt, Germany

FTIR – Identification (197K)

AAS - Content of Magnesium

HPLC Assay and Related Substances (API)

HPLC Assay and Related Substances (Delayed Release Capsules)

Merck Millipore is a division of Merck KGaA, Darmstadt, Germany

A. INFRARED ABSORPTION <197K> FTIR

We recommend Potassium bromide for IR spectroscopy Uvasol® (1.04907) to be used.

Merck Millipore is a division of Merck KGaA, Darmstadt, Germany

Lanthanum solution: Transfer 58.7 g of lanthanum oxide into a 1000-mL volumetric

Solution A Lanthanum oxide solution Dilution Final standard concentration

Standard Solution B 5.0 ml 4.0 ml 100 ml 0.1 µg / ml

Standard Solution C 10.0 ml 4.0 ml 100 ml 0.2 µg / ml

Standard Solution E 20.0 ml 4.0 ml 100 ml 0.4 µg / ml

Standard Solution F 25.0 ml 4.0 ml 100 ml 0.5 µg / ml

Merck Millipore is a division of Merck KGaA, Darmstadt, Germany

Concentration (µg/ml) Absorbance

0,7 y = 1,4906x - 0,0018

Merck Millipore is a division of Merck KGaA, Darmstadt, Germany

Titration one component system

Titrant: apura - CombiTitrant 5 (1.88005)

Merck Millipore is a division of Merck KGaA, Darmstadt, Germany

Merck Millipore is a division of Merck KGaA, Darmstadt, Germany

Merck Millipore is a division of Merck KGaA, Darmstadt, Germany

Chromatographic Data : SST solution

Merck Millipore is a division of Merck KGaA, Darmstadt, Germany

Chromatographic Data : SST solution

Merck Millipore is a division of Merck KGaA, Darmstadt, Germany