Ampicillin

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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Ampicillin

C₁₆H₁₉N₃O₄S (anhydrous) 349.41

4-Thia-1-azabicyclo[3.2.0]heptane-2 carboxylic acid, [6-(aminophenylacetyl)amino]-3,3-dimethyl-7-oxo-, [2S-[2α,5α,6β(S*)]]-;

(2S,5R,6R)-6-[(R)-2-Amino-2-phenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid

CAS RN®: 69-53-4; UNII: 7C782967RD.

Trihydrate 403.46 CAS RN®: 7177-48-2; UNII: HXQ6A1N7R6.

1 DEFINITION

Ampicillin is anhydrous or contains three molecules of water of hydration. It contains NLT 900 µg/mg and NMT 1050 µg/mg of ampicillin (C₁₆H₁₉N₃O₄S), calculated on the anhydrous basis.

2 IDENTIFICATION

Spectroscopic Identification Tests 〈197〉, Infrared Spectroscopy: 197K: Except that where the specimen under test is the trihydrate, both it and the USP Ampicillin Trihydrate RS are undried.

3 ASSAY

Procedure

Solution A: 6.54 g/L of monobasic potassium phosphate and 0.34 g/L of dibasic potassium phosphate, adjusted with 1 N sodium hydroxide or 1 N phosphoric acid to a pH of 5.5 before final dilution

Solution B: Acetonitrile and Solution A (2:23)

Solution C: Acetonitrile and Solution A (3:7)

Mobile phase: See Table 1.

Table 1

Solution D: 46.3 g/L of monobasic potassium phosphate and 27.8 g/L of dibasic potassium phosphate, adjusted with 1 N sodium hydroxide or 1 N phosphoric acid to a pH of 6.5 before final dilution

System suitability solution: 0.5 mg/mL of USP Ampicillin RS and 0.1 mg/mL of USP Amoxicillin RS in acetonitrile, water, and Solution D (4:91:5)...

Standard solution: 0.5 mg/mL of USP Ampicillin RS in acetonitrile, water, and Solution D (4:91:5)...

Sample solution: 0.5 mg/mL of Ampicillin in acetonitrile, water, and Solution D (4:91:5)...

Chromatographic system

Mode: LC

Detector: UV 230 nm

Column: 4.6-mm × 15-cm; 5-µm packing L1

Flow rate: 1.5 mL/min

Injection volume: 20 µL

System suitability

Resolution: NLT 10 between ampicillin and amoxicillin

Tailing factor: NMT 1.4 for ampicillin

Relative standard deviation: NMT 2.0%, Standard solution

Analysis

Calculate the quantity, in µg, of ampicillin (C₁₆H₁₉N₃O₄S) in each mg of Ampicillin taken:

Result = (rᵤ/rₛ) × (Cₛ/Cₓ) × P

Where rᵤ = peak response from the Sample solution, rₛ = peak response from the Standard solution,

Cₛ = concentration of USP Ampicillin RS in the Standard solution (mg/mL),

Cₓ = concentration of Ampicillin in the Sample solution (mg/mL),

P = potency of USP Ampicillin RS (µg/mg)

Acceptance criteria: 900–1050 µg/mg on the anhydrous basis

4 IMPURITIES

Organic Impurities, Procedure 1

Organic Impurities, Procedure 1 is recommended when the impurity prole includes ampicillin thiazepine.

Solution A, Solution B, Solution C, Mobile phase, Solution D, System suitability solution, Sample solution, and Chromatographic

system: Prepare as directed in the Assay.

Standard stock solution: Prepare as directed for the Standard solution in the Assay.

Standard solution: 0.005 mg/mL of ampicillin in Solution D and water (1:19) from Standard stock solution. Transfer an aliquot of the Standard

stock solution to a suitable volumetric ask, add Solution D, using about 5% of the nal volume, and dilute with water to volume. Analyze

immediately after preparation.

Sensitivity solution: 0.5 µg/mL of ampicillin in Solution D and water (1:19) from the Standard solution. Transfer an aliquot of the Standard

solution to a suitable volumetric ask, add Solution D, using about 5% of the nal volume, and dilute with water to volume.

System suitability

Samples: Sensitivity solution, System suitability solution, and Standard solution

Suitability requirements

Signal-to-noise ratio: NLT 3, Sensitivity solution

Resolution: NLT 10 between ampicillin and amoxicillin, System suitability solution

Tailing factor: NMT 1.4 for ampicillin, System suitability solution

Relative standard deviation: NMT 10.0%, Standard solution

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of each impurity in the portion of Ampicillin taken:

Result = (r_u /r_s ) × (C_s /C_u ) × P × F × 100

r_u = peak response of each impurity from the Sample solution

r_s = peak response of ampicillin from the Standard solution

C_s = concentration of USP Ampicillin RS in the Standard solution (mg/mL)

C_u = concentration of Ampicillin in the Sample solution (mg/mL)

P = potency of USP Ampicillin RS (µg/mg)

F = conversion factor, 0.001 mg/µg

Acceptance criteria: See Table 2.

a (R)-2-Amino-2-phenylacetic acid.

b (2S,5R,6R)-6-Amino-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0] heptane-2-carboxylic acid.

c (4S)-2-{[(R)-2-Amino-2-phenylacetamido](carboxy)methyl}-5,5-dimethylthiazolidine-4-carboxylic acid.

d(S)-6-[(R)-2-Amino-2-phenylacetamido]-2,2-dimethyl-7-oxo-2,3,4,7-tetrahydro-1,4-thiazepine-3-carboxylic acid.

e (4S)-2-(3,6-Dioxo-5-phenylpiperazin-2-yl)-5,5-dimethylthiazolidine-4-carboxylic acid.

f (4S)-2-{1-[(R)-2-Amino-2-phenylacetamido]-2-[(1R)-2-{carboxy[(4S)-4-carboxy-5,5-dimethylthiazolidin-2-yl]methylamino}-2-oxo-1-phenylethylamino]-2-oxoethyl}-5,5-dimethylthiazolidine-4-carboxylic acid.

g (2S,5R,6R)-6-{(R)-2-[(R)-2-Amino-2-phenylacetamido]-2-phenylacetamido}-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2- carboxylic acid.

h (2S,5R,6R)-6-[(2R)-2-{2-[(R)-2-Amino-2-phenylacetamido]-2-[(4S)-4-carboxy-5,5-dimethylthiazolidin-2-yl]acetamido}-2-phenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid.

i (4S,4'S)-2,2'-{(1R,7R,13R)-1-Amino-14-[(2S,5R,6R)-2-carboxy-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptan-6-ylamino]-2,5,8,11,14-pentaoxo-1,7,13-triphenyl-3,6,9,12-tetraazatetradecane-4,10-diyl}bis(5,5-dimethylthiazolidine-4-carboxylic acid).

Organic Impurities, Procedure 2, Dimethylaniline 〈223〉: Meets the requirements

Organic Impurities, Procedure 2 is recommended when dimethylaniline is used during the production of Ampicillin.

Organic Impurities, Procedure 3

Organic Impurities, Procedure 3 is recommended when the impurity prole includes phenylpyrazinol, pivaloyl phenylglycine, pivaloyl aminopenicillanic acid, diphenyldiketopiperazine, and open ring dimer.

Solution A: 4 g/L of monobasic sodium phosphate dihydrate adjusted with 1 N sodium hydroxide to a pH of 5.0

Solution B: Acetonitrile

Mobile phase: See Table 3.

Diluent: Acetonitrile and Solution A (2:98)

System suitability solution: 1.5 mg/mL of USP Ampicillin System Suitability Mixture RS in Diluent Standard solution: 15 µg/mL of USP Ampicillin RS in Diluent

Sample solution: 1.5 mg/mL of Ampicillin in Diluent. Store the sample in the refrigerator, and discard after 60 min. Chromatographic system

(See Chromatography 〈621〉, System Suitability.) Mode: LC

Detector: UV 220 nm

Column: 4.6-mm × 15-cm; 5-µm packing L7 Column temperature: 40°

Flow rate: 1.5 mL/min Injection volume: 20 µL Autosampler temperature: 4°

System suitability

Samples: System suitability solution and Standard solution Suitability requirements

Resolution: NLT 1.5 between the pivaloyl phenylglycine and diphenyldiketopiperazine peaks, System suitability solution Tailing factor: NMT 2.0, Standard solution

Relative standard deviation: NMT 5.0%, Standard solution Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of each impurity in the portion of Ampicillin taken:

Result = (r_U/r_S) × (C_S/C_U) × P × F × 100

r_U = peak response of each impurity from the Sample solution

r_S = peak response of ampicillin from the Standard solution

C_S = concentration of USP Ampicillin RS in the Standard solution (mg/mL)

C_U= concentration of Ampicillin in the Sample solution (mg/mL)

P = potency of ampicillin in USP Ampicillin RS (µg/mg)

F = conversion factor, 0.001 mg/µg

Acceptance criteria: See Table 4 and Table 5. The limits in Table 5 are to be used only where Ampicillin is intended for use in preparing

veterinary products.

a (R)-2-Amino-2-phenylacetic acid.

b (2S,5R,6R)-6-Amino-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid.

c (4S)-2-{(R)-2-Amino-2-phenylacetamido

methyl}-5,5-dimethylthiazolidine-4-carboxylic acid.

d The system resolves the two isomers of ampicilloic acid. The sum of the two isomers is reported.

e (2S,5R,6R)-6-[(S)-2-Amino-2-phenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid.

f (4S)-2-{[(R)-2-Amino-2-phenylacetamido]methyl}-5,5-dimethylthiazolidine-4-carboxylic acid.

g The system resolves the two isomers of ampilloic acid. The sum of the two isomers is reported.

h (4S)-2-(3,6-Dioxo-5-phenylpiperazin-2-yl)-5,5-dimethylthiazolidine-4-carboxylic acid.

i The system resolves the two isomers of ampicillin rearrangement product. The sum of the two isomers is reported.

j 3-Phenylpyrazin-2-ol.

k (R)-2-Phenyl-2-pivalamidoacetic acid.

l 3,6-Diphenylpiperazine-2,5-dione.

m (4S)-2-{1-[(R)-2-Amino-2-phenylacetamido]-2-[(1R)-2-{carboxy[(4S)-4-carboxy-5,5-dimethylthiazolidin-2-yl]methylamino}-2-oxo-1-phenylethylamino]-2-oxoethyl}-5,5-dimethylthiazolidine-4-carboxylic acid.

n (2S,5R,6R)-6-{(R)-2-[(R)-2-Amino-2-phenylacetamido]-2-phenylacetamido}-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid.

o (2S,5R,6R)-3,3-Dimethyl-7-oxo-6-pivalamido-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid.

p (4S)-2-{1-[(R)-2-amino-2-phenylacetamido]-2-[(1R)-2-{[(4S)-4-carboxy-5,5-dimethylthiazolidin-2-yl]methylamino}-2-oxo-1-phenylethylamino]-2-oxoethyl}-5,5-dimethylthiazolidine-4-carboxylic acid.

q The system may resolve the three isomers of open ring dimer. The sum of the three isomers is reported.

r (2S,5R,6R)-6-[(2R)-2-{2-[(R)-2-Amino-2-phenylacetamido]-2-[(4S)-4-carboxy-5,5-dimethylthiazolidin-2-yl]acetamido}-2-phenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid.

s (R)-2-((2S,5R,6R)-6-((R)-2-Amino-2-phenylacetamido)-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxamido)-2-phenylacetic acid.

t (4S,4'S)-2,2'-{(1R,7R,13R)-1-Amino-14-[(2S,5R,6R)-2-carboxy-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptan-6-ylamino]-2,5,8,11,14-pentaoxo-1,7,13-triphenyl-3,6,9,12-tetraazatetradecane-4,10-diyl}bis(5,5-dimethylthiazolidine-4-carboxylic acid).

Table 5

a (R)-2-Amino-2-phenylacetic acid.

b (2S,5R,6R)-6-Amino-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid.

c (4S)-2-{(R)-2-Amino-2-phenylacetamido

methyl}-3-formyl-5,5-dimethylthiazolidine-4-carboxylic acid.

d (4S)-2-{(R)-2-Amino-2-phenylacetamido

methyl}-5,5-dimethylthiazolidine-4-carboxylic acid.

e The system resolves the two isomers of ampicilloic acid. The sum of the two isomers is reported.

f (2S,5R,6R)-6-[(S)-2-Amino-2-phenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid.

g (4S)-2-{[(R)-2-Amino-2-phenylacetamido]methyl}-5,5-dimethylthiazolidine-4-carboxylic acid.

h The system resolves the two isomers of ampilloic acid. The sum of the two isomers is reported.

i (4S)-2-(3,6-Dioxo-5-phenylpiperazin-2-yl)-5,5-dimethylthiazolidine-4-carboxylic acid.

j The system resolves the two isomers of ampicillin rearrangement product. The sum of the two isomers is reported.

k 3-Phenylpyrazin-2-ol.

l (R)-2-Phenyl-2-pivalamidoacetic acid.

m 3,6-Diphenylpiperazine-2,5-dione.

n (4S)-2-{1-[(R)-2-Amino-2-phenylacetamido]-2-[(1R)-2-{carboxy[(4S)-4-carboxy-5,5-dimethylthiazolidin-2-yl]methylamino}-2-oxo-1-phenylethylamino]-2-oxoethyl}-5,5-dimethylthiazolidine-4-carboxylic acid.

o (2S,5R,6R)-6-{(R)-2-[(R)-2-Amino-2-phenylacetamido]-2-phenylacetamido}-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid.

p (2S,5R,6R)-3,3-Dimethyl-7-oxo-6-pivalamido-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid.

q (4S)-2-{1-[(R)-2-amino-2-phenylacetamido]-2-[(1R)-2-{[(4S)-4-carboxy-5,5-dimethylthiazolidin-2-yl]methylamino}-2-oxo-1-phenylethylamino]-2-oxoethyl}-5,5-dimethylthiazolidine-4-carboxylic acid.

r The system may resolve the three isomers of open ring dimer.

s (2S,5R,6R)-6-[(2R)-2-{2-[(R)-2-Amino-2-phenylacetamido]-2-[(4S)-4-carboxy-5,5-dimethylthiazolidin-2-yl]acetamido}-2-phenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid.

t (R)-2-((2S,5R,6R)-6-((R)-2-Amino-2-phenylacetamido)-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxamido)-2-phenylacetic acid.

u (4S,4'S)-2,2'-{(1R,7R,13R)-1-Amino-14-[(2S,5R,6R)-2-carboxy-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptan-6-ylamino]-2,5,8,11,14-pentaoxo-1,7,13-triphenyl-3,6,9,12-tetraazatetradecane-4,10-diyl}bis(5,5-dimethylthiazolidine-4-carboxylic acid).

Organic Impurities, Procedure 4

Organic Impurities, Procedure 4 is recommended when the impurity prole includes ampilloyl aminopenicillanic acid and penicillanyl

ampicillinamide.

Solution A: 3.4 g/L of dibasic sodium phosphate dodecahydrate and 1.4 g/L of monobasic potassium phosphate adjusted with phosphoric

acid to a pH of 5.5

Solution B: Acetonitrile

Mobile phase: See Table 6

Standard solution: 30 µg/mL of USP Amoxicillin Related Compound A RS, 30 µg/mL of d-phenylglycine, and 25 µg/mL of USP Ampicillin RS in

Solution A

Sample solution: 2.5 mg/mL of Ampicillin in Solution A. Store the Sample solution in the refrigerator, and use within 9 h.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 220 nm

Column: 4.0-mm × 15-cm; 3-µm packing L1

Column temperature: 40°

Flow rate: 1.3 mL/min

Injection volume: 5 µL

Autosampler temperature: 4°

System suitability

Sample: Standard solution

Suitability requirements

Resolution: NLT 1.5 between d-phenylglycine and amoxicillin related compound A

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of each impurity in the portion of Ampicillin taken:

Result = (r_U/r_S) × (C_S/C_U) × P × F × 100

r_U = peak response of each impurity from the Sample solution

r_S = peak response of ampicillin from the Standard solution

C_S= concentration of USP Ampicillin RS in the Standard solution (mg/mL)

C_U= concentration of Ampicillin in the Sample solution (mg/mL)

P = potency of ampicillin in USP Ampicillin RS (µg/mg)

F = conversion factor, 0.001 mg/µg

Acceptance criteria: See Table 7. Disregard any peak with an area less than 0.03 times the area of the ampicillin peak in the System suitability

solution.

a (R)-2-Amino-2-phenylacetic acid.

b (2S,5R,6R)-6-Amino-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid.

c (4S)-2-{(R)-2-Amino-2-phenylacetamido

methyl}-5,5-dimethylthiazolidine-4-carboxylic acid.

d (S)-6-[(R)-2-Amino-2-phenylacetamido]-2,2-dimethyl-7-oxo-2,3,4,7-tetrahydro-1,4-thiazepine-3-carboxylic acid.

e These impurities are listed for information only. They are not to be reported. They are not to be included in total impurities.

f (2S,5R,6R)-6-[(S)-2-Amino-2-phenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid.

g (2S,5R,6R)-6-{2-[(R)-2-Amino-2-phenylacetamido]-2-[(4S)-4-carboxy-5,5-dimethylthiazolidin-2-yl]acetamido}-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid.

h (4S)-2-{[(R)-2-Amino-2-phenylacetamido]methyl}-5,5-dimethylthiazolidine-4-carboxylic acid.

i (4S)-2-(3,6-Dioxo-5-phenylpiperazin-2-yl)-5,5-dimethylthiazolidine-4-carboxylic acid.

j (R)-2-Phenyl-2-pivalamidoacetic acid.

k 3-Phenylpyrazin-2-ol.

l 3,6-Diphenylpiperazine-2,5-dione.

m (2S,5R,6R)-3,3-Dimethyl-7-oxo-6-pivalamido-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid.

n (2S,5R,6R)-6-{(R)-2-[(R)-2-Amino-2-phenylacetamido]-2-phenylacetamido}-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid.

o (2S,5R,6R)-6-[(2R)-2-{2-[(R)-2-Amino-2-phenylacetamido]-2-[(4S)-4-carboxy-5,5-dimethylthiazolidin-2-yl]acetamido}-2-phenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid.

p (2S,5R,6R)-6-{(2S,5R,6R)-6-[(R)-2-Amino-2-phenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxamido}-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid.

q (R)-2-((2S,5R,6R)-6-((R)-2-Amino-2-phenylacetamido)-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxamido)-2-phenylacetic acid.

5 SPECIFIC TESTS

Sterility Tests 〈71〉

Sample solution: Dissolve 6 g in 800 mL of Fluid D containing sucient sterile penicillinase to inactivate the ampicillin, and swirl the vessel

until dissolution is complete before ltering.

Acceptance criteria: Where the label states that Ampicillin is sterile, it meets the requirements when tested as directed for Test for Sterility of

the Product to Be Examined, Membrane Filtration.

Crystallinity 〈695〉: Meets the requirements

pH 〈791〉

Sample solution: 10 mg/mL

Acceptance criteria: 3.5–6.0

Water Determination, Method I 〈921〉: NMT 2.0% where it is labeled as Ampicillin (anhydrous); between 12.0% and 15.0% where it is labeled as

Ampicillin (trihydrate)

Bacterial Endotoxins Test 〈85〉: Where the label states that Ampicillin is sterile or must be subjected to further processing during the

preparation of injectable dosage forms, it contains NMT 0.15 USP Endotoxin Unit/mg of ampicillin.

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in tight containers.

Labeling: Label to indicate whether it is anhydrous or is the trihydrate. Where the quantity of ampicillin is indicated in the labeling of any

preparation containing Ampicillin, this shall be understood to be in terms of anhydrous ampicillin (C H N O S). Where it is intended for use in

preparing injectable dosage forms, the label states that it is the trihydrate and that it is sterile or must be subjected to further processing during

the preparation of injectable dosage forms.

If a test for Organic Impurities other than Procedures 1 and 2 is used, then the labeling states with which Organic Impurities test the article

complies. Where it is intended for use in preparing veterinary products, the label so states.

Change to read:

USP Reference Standards 〈11〉

USP Amoxicillin RS

USP Amoxicillin Related Compound A RS

(2S,5R,6R)-6-Amino-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid.

▲C H N O S 216.26▲ (ERR 1-Apr-2021)

USP Ampicillin RS

USP Ampicillin System Suitability Mixture RS

This is a mixture which contains ampicillin, pivaloyl phenylglycine [(R)-2-phenyl-2-pivalamidoacetic acid; C H NO ; 235.28],

diphenyldiketopiperazine (3,6-diphenylpiperazine-2,5-dione; C H N O ; 266.29), and other related compounds.