Raw Material Specification & Method of Analysis (MOA) / Standard Testing Procedure (STP) for Amoxicillin Trihydrate USP and per USP Volume No. PF 41(4).
RM Specification of Amoxicillin Trihydrate USP
| Sr. No. | Test Parameter | Specification |
| 01 | Description | White, practically odorless, crystalline powder. |
| 02 | Solubility | Slightly soluble in water and in methanol; insoluble in benzene, in carbon tetrachloride, and in chloroform. |
| 03 | Identification
A) By IR B) By HPLC |
The infrared absorption spectrum of the sample is concordant with the spectrum obtained with Amoxicillin Trihydrate or with the reference spectrum of Amoxicillin Trihydrate.
The retention time of the major peak of the sample solution corresponds to that of the Standard solution, as obtained in the Assay. |
| 04 | Organic Impurities | By HPLC. |
| Amoxicillin related compound Ia
(D-hydroxyphenylglycine) |
Not more than 1.0% | |
| Amoxicillin related compound Db,c
(Amoxicillin open ring)
|
Not more than 1.0% (RRT : 0.53)
Not more than 1.0% (RRT : 0.68) |
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| Amoxicillin related compound Ad
(6-aminopenicillanic acid) |
Not more than 0.5% | |
| Amoxicillin related compound Gg
(D- hydroxyphenylglycyl-amoxicillin) |
Not more than 1.0% | |
| Amoxicillin related compound Eh
(amoxicillin penilloic derivative) |
Not more than 1.0% | |
| Amoxicillin related compound Mi (N-(penicillan-6-yl) open ring amoxicillinamide) | Not more than 1.0% | |
| Amoxicillin related compound Cl (amoxicillin rearrangement product) | Not more than 1.0% | |
| Amoxicillin related compound Eh,i (amoxicillin penilloic derivative) | Not more than 1.0% | |
| Amoxicillin related compound Jm (amoxicillin open ring dimer) | Not more than 1.0% | |
| Amoxicillin related compound Ln (N-(penicillan-6-yl) amoxicillinamide) | Not more than 1.0% | |
| Any unspecified individual impurity | Not more than 1.0% | |
| Total Impurities | Not more than 5.0% | |
| 05 | Crystallinity | Particle shows birefringence and extinction position when microscope stage revolved. |
| 06 | Dimethylaniline | Not more than 0.002% (20 ppm). |
| 07 | pH | Between 3.5 and 6.0 |
| 08 | Water Determination | Between 11.5 and 14.5% |
| 09 | Assay (By HPLC) | Not less than 900 µg & not more than 1050 µg/mg of Amoxicillin (C16H19N3O5S), on anhydrous basis. |
Download the Word copy of Specification – Amoxycilline Trihydrate USP
MOA/STP of Amoxicillin Trihydrate USP
1.0 Description
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- Carry out the test on sample powder as per procedure. For detailed Description Test Procedure – Refer GTP – Description / Appearance Test.
2.0 Solubility of Amoxicillin Trihydrate
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- Slightly soluble in water and in methanol; insoluble in benzene, in carbon tetrachloride, and in chloroform.
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- Carry out the test as per procedure of solubility check.
Solubility Table
| Descriptive Term | Parts of Solvent Required for 1 Part of Solute |
| Very soluble | Less than 1 |
| Freely soluble | From 1 to 10 |
| Soluble | From 10 to 30 |
| Sparingly soluble | From 30 to 100 |
| Slightly soluble | From 100 to 1,000 |
| Very slightly soluble | From 1,000 to 10,000 |
| Practically insoluble, or Insoluble | Greater than or equal to 10,000 |
3.0 Identification
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- A. By IR
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- The infrared absorption spectrum of the sample is concordant with the spectrum obtained with Amoxicillin Trihydrate CRS or with the reference spectrum of Amoxicillin Trihydrate. Carry out the test as per procedure.
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- B. By HPLC
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- The retention time of the major peak of the sample solution corresponds to that of the Standard solution, as obtained in the Assay.
4.0 Organic Impurities of Amoxicillin Trihydrate
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Procedure :
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- Solution A : 2.72 g/L of Monobasic potassium phosphate. Adjust with 1 N Potassium hydroxide or 20% Phosphoric acid to a pH of 5.0 ± 0.1.
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- Solution B : Methanol
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- Mobile Phase : See the gradient table below.
| Time (min) | Solution A (%) | Solution B (%) |
| 0 | 97 | 3 |
| 10 | 97 | 3 |
| 22 | 75 | 25 |
| 26 | 97 | 3 |
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- Standard Solution : 12.5 µg/ml of USP Amoxicillin RS in Solution A.
(Dilution: 12.5 mg Amoxicillin WS/RS→ 100 ml→ take 5 ml →50 ml)
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- System Suitability Solution : 12.5 µg/ml each of USP Amoxicillin Related compound A RS and USP Amoxicillin Related compound D RS in Solution A.
(Dilution: about 2.5 mg each of USP Amox. Rel. com. A RS, and USP Amox. Rel. com.D RS → 20 ml
→ take 5 ml →50 ml)
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- Sample Solution : 1.25 mg/ml of Amoxicillin in Solution A. (Note:- Store this solution at 4° and used within 4 h.)
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Chromatographic System :
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- Mode : LC,
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- Detector : UV 210 nm,
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- Coloumn : 4.6 mm × 10 cm; 5 µm packing L1
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- Column temperature : 40°
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- Flow rate : 1.5 ml / min,
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- Injection size : 10 µl
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- Auto sampler temperature : 4°
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- System Suitability :
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- Samples : Standard solution and System suitability solution
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- Suitability Requirements : (Note:- Identify peaks by the relative retention times of Impurity Table – 1).
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- Resolution : NLT 1.5 between Amoxicillin Related compound A and the second peak for Amoxicillin Related compound D, System suitability solution.
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- Relative Standard Deviation : NMT 10%, Standard solution.
Calculate the percentage of each impurity in the portion of Amoxicillin taken :
Result = (rU/rS) × (CS/CU ) × F × 100
Where
rU = peak area response of each impurity from the Sample solution
rS = peak area response of Amoxicillin from the Standard solution
CS = concentration of USP Amoxicillin RS in the Standard solution (μg/ml)
CU = nominal concentration of Amoxicillin in the Sample solution (mg/ml)
F = unit conversion factor (0.001 mg/μg)
(Note:- The reporting limit is 0.03% of the Amoxicillin peak from the Standard solution).
Individual Impurities : See Impurity Table – 1
Total Impurities : NMT 5.0
Impurity Table 1
| Name | Relative Retention Time | Acceptance Criteria NMT (%) |
| Amoxicillin related compound Ia (D-hydroxyphenylglycine) | 0.32 | 1.0 |
| Amoxicillin related compound Db,c (Amoxicillin open ring) | 0.53 | 1.0 |
| 0.68 | 1.0 | |
| Amoxicillin related compound Ad (6-aminopenicillanic acid) | 0.78 | 0.5 |
| Amoxicillin related compound Be,f (L-amoxicillin) | 0.87 | – |
| Amoxicillin | 1.0 | – |
| Amoxicillin related compound Gg (D- hydroxyphenylglycylamoxicillin) | 2.9 | 1.0 |
| Amoxicillin related compound Ehi (amoxicillin penilloic derivative) | 4.5 | 1.0 |
| Amoxicillin related compound Ml (N-(penicillan-6-yl) open ring amoxicillinamide) | 6.0 | 1.0 |
| Amoxicillin related compound Fe,k (phenylpyrazinediol) | 6.3 | – |
| Amoxicillin related compound Cl (amoxicillin rearrangement product) | 6.4 | 1.0 |
| Amoxicillin related compound Eh,i (amoxicillin penilloic derivative) | 6.7 | 1.0 |
| Amoxicillin related compound Jm (amoxicillin open ring dimer) | 8.8 | 1.0 |
| Amoxicillin related compound Ln (N-(penicillan-6-yl) amoxicillinamide) | 9.0 | 1.0 |
| Any unspecified individual impurity | – | 1.0 |
5.0 Crystallinity
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- Carry out the test as per USP general chapter <696>.
6.0 Dimethylaniline
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- Carry out the test by method A or B .
7.0 pH
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- Carry out the test as per procedure. Refer detailed procedure for pH value determination.
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- Sample Solution : 2 mg/ml
8.0 Water Determination
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- Determined on 0.1 g.
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- Carry out the test method I as per procedure. Refer the GTP – Water Content Determination
9.0 Assay of Amoxicillin Trihydrate USP
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Procedure : By HPLC;
- Diluent : 6.8 g/L of monobasic potassium phosphate in water. Adjust with a 45% (w/w) solution of Potassium hydroxide to a pH of 5.0 ± 0.1.
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- Mobile Phase : Acetonitrile and Diluent (1 : 24)
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- Standard Solution : 1.2 mg/ml of USP Amoxicillin RS in Diluent. (Note:– Use this solution with 6 h.)
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- Sample Solution : 1.2 mg/ml of Amoxicillin in Diluent. (Note:– Use this solution with 6 h.)
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- Chromatographic System :
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- Mode : LC,
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- Detector : UV 230 nm,
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- Column : 4 mm × 25 cm; packing L1
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- Flow rate : 1.5 ml / min,
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- Injection size : 10 µl
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- System Suitability :
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- Samples : Standard solution
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- Suitability Requirements :
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- Tailing Factor : NMT 2.5
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- Relative Standard Deviation : NMT 2.0%,
Calculate the quantity, in µg/mg, of C16H19N3O5S in the portion of Amoxicillin taken :
Result = (rU/rS) × (CS/CU ) × P
rU = peak response from the Sample solution
rS = peak area response from the Standard solution
CS = concentration of USP Amoxicillin RS in the Standard solution (μg/ml)
CU = concentration Sample solution (mg/ml)
P = Potency of Amoxicillin in USP amoxicillin RS (µg/mg)
Additional In-house Test:
Water Activity (Free Water Content)
Equipment : Novasina Lab Swift-aw
Safety Precautions : Use Mask, Goggles. Lab Swift-aw system must not be used in hazardous zones or similar areas. Operate the system below 25˚C.
Procedure : Switch on the instrument by pushing the right function key. The system carries out a self-test (“STST”). A start display (self-test) appears shortly and the software version is displayed on the upper display line.
After the self-test the sensor is warming up (“WARMUP”). Has the warm up finished the instruments is ready for the first aw-measurements of a sample.
Note:- The warm-up time mainly depends on the present humidity and the ambient temperature. Generally this time is 2 to 3 minutes.
During the warm-up stage the measurement can be already started. But there will be no output of measurement data until the warm-up is finished.
Fill the sample cup with the material to be measured. The sample cup must be completely clean and dry (rub dry with tissue paper if necessary). Ideally, always use a fresh cup and dispose of it after use.
Fill the sample cup to approximately 2/3 with the product to be measured. Do not overfill the sample cups. While placing the sample in the measuring chamber, open the cover and place the sample dish carefully inside. Be aware about powder samples that the product will not contact the measuring head or measuring cell.
The analysis function starts after closing the measurement chamber. The flashing display shows ‘’ ANALIZING’’.
As long as it is flashing alternately (Analizing /…..˚C) the aw-value and the temperature are not stable. The result can be read at earliest when the ‘’stable’’ value is displayed.
Download the Word copy of Method of Analysis – Amoxicillin Trihydrate USP