Finished products · Tablets (paracetamol 500 mg as the worked example)
Related Substances (Impurities) by HPLC
Separates the drug from its process impurities and degradation products and reports each impurity as a percentage of the drug, using a diluted standard and relative response factors. Paracetamol tablets are used as the example, where the key degradation product is 4-aminophenol.
- HPLC (reversed phase, gradient, UV detection)
- HPLC
- UHPLC
- Analytical balance
- Sonicator
Reference: Typical approach based on pharmacopoeial related-substances tests and ICH Q3B(R2). Specified impurities, limits, RRFs and conditions must come from your approved specification and STP.
PRINCIPLE
Why this method works
A concentrated test solution is chromatographed so that impurities at the 0.05% level still give measurable peaks. Each impurity peak is compared with the peak of a diluted reference standard of the drug (for example at 0.1% of the test concentration). Because an impurity may absorb more or less UV light than the drug, its area is corrected with a relative response factor (RRF). Results are compared with the specification and with the ICH Q3B(R2) reporting, identification and qualification thresholds, which depend on the maximum daily dose.
Instruments used
MATERIALS
Reagents & standards
| Reagent | Grade / specification |
|---|---|
| Paracetamol reference standard | Certified purity, for the diluted standard |
| Impurity reference standards | 4-aminophenol and other specified impurities (e.g. 4-nitrophenol, 4'-chloroacetanilide), for identification, system suitability and RRF determination |
| Methanol | HPLC grade |
| Buffer salts | e.g. potassium dihydrogen phosphate, AR grade; ion-pair reagent only if the method specifies one |
| Orthophosphoric acid / potassium hydroxide | For pH adjustment as per the method |
| Water | HPLC grade |
SAMPLE PREPARATION
Preparing the sample
- 01
Weigh & powder
Weigh 20 tablets, find the average weight and powder them.
- 02
Weigh portion
Weigh powder equivalent to about 100 mg paracetamol.
- 03
Dissolve
Transfer to a 100 mL flask with about 70 mL diluent and sonicate.
- 04
Make up
Cool, make up to volume and mix (about 1.0 mg/mL).
- 05
Filter & inject
Filter through 0.45 µm, discard the first few mL and inject promptly.
Procedure
- 1Weigh 20 tablets, calculate the average tablet weight and powder them finely.
- 2Accurately weigh powder equivalent to about 100 mg of paracetamol into a 100 mL volumetric flask.
- 3Add about 70 mL of diluent and sonicate for about 10–15 minutes with intermittent shaking, keeping the bath cool.
- 4Allow to cool, make up to volume with diluent and mix (test solution, about 1.0 mg/mL paracetamol).
- 5Filter through a 0.45 µm membrane filter that has been shown not to adsorb the impurities; discard the first few mL.
- 6Protect solutions from light and inject within the established solution-stability period; 4-aminophenol can form and oxidise in solution.
Standard preparation
- 1Diluted standard (0.1%): weigh about 25 mg paracetamol RS into a 50 mL flask, dissolve and make up (0.5 mg/mL). Dilute 1.0 mL to 100 mL (5 µg/mL), then 2.0 mL to 10 mL (about 1 µg/mL, i.e. 0.1% of the test concentration).
- 2Sensitivity solution: dilute the diluted standard to the reporting threshold (e.g. 0.05%, about 0.5 µg/mL).
- 3System suitability solution: test solution (or paracetamol RS) spiked with 4-aminophenol and the other specified impurities at about their specification level.
- 4Impurity identification: inject individual impurity standards to confirm retention times and relative retention times (RRT).
Notes for analysts
- The chromatogram is an illustrative training example of a spiked test solution on an expanded scale; the paracetamol peak is truncated. Retention times, RRTs and the RRF value of 1.35 are not real method data — use the values established in your validated method.
- ICH Q3B(R2) thresholds depend on maximum daily dose. Reporting: 0.1% (≤ 1 g/day) or 0.05% (> 1 g/day). Identification: from 1.0% (< 1 mg/day) down to 0.10% (> 2 g/day). Qualification: from 1.0% (< 10 mg/day) down to 0.15% (> 2 g/day). Check the current guideline table for the dose bands and µg/day alternatives.
- ICH Q3B(R2) covers degradation products in the drug product. Process impurities from the API synthesis (such as 4'-chloroacetanilide for paracetamol) are controlled in the API specification under ICH Q3A(R2), but may still be monitored in the product.
- Report results below 1.0% to two decimal places (e.g. 0.06%), rounded by conventional rules.
- Identify peaks by RRT against the paracetamol peak; confirm unknowns above the identification threshold by LC-MS and reference standards.
- Pharmacopoeial methods typically apply a correction factor (1/RRF) when an impurity's response differs markedly from the drug (often when the RRF is outside about 0.8–1.2), or use an external impurity standard; ignoring it biases the result.
- Mutagenic impurities (e.g. nitrosamines) are controlled separately under ICH M7, usually by more sensitive LC-MS or GC-MS methods.
TEST CONDITIONS
Method parameters (typical)
| Column | C18 (or C8 as specified), 250 × 4.6 mm, 5 µm |
|---|---|
| Mobile phase A | Aqueous phosphate buffer (pH and any ion-pair reagent as per the validated method), filtered and degassed |
| Mobile phase B | Methanol |
| Gradient (illustrative) | 0–5 min 10% B; 5–25 min 10 → 50% B; 25–30 min 50% B; 30–31 min back to 10% B; 31–40 min re-equilibrate |
| Flow rate | 1.0 mL/min |
| Detection | UV at about 245 nm (PDA recommended for peak purity) |
| Injection volume | 20 µL |
| Column temperature | 35 °C |
| Run time | About 40 minutes (long enough to elute late impurities) |
| Diluent | Mobile phase A : methanol (e.g. 90 : 10) |
BEFORE YOU REPORT
System suitability & sequence
| Parameter | Limit |
|---|---|
| Resolution (paracetamol vs closest specified impurity) | Typically not less than 2.0, as per method |
| %RSD of paracetamol area, diluted standard (6 injections) | Typically not more than 5.0% |
| Signal-to-noise, sensitivity solution | Not less than 10 |
| Tailing factor (paracetamol, diluted standard) | Not more than 2.0 |
| Blank | No peak interfering with any specified impurity |
Typical sequence
- 1Blank (diluent) × 1
- 2System suitability (spiked) solution × 1
- 3Sensitivity solution × 1
- 4Diluted standard × 6
- 5Placebo × 1 (to identify excipient peaks)
- 6Test solution 1 × 1, test solution 2 × 1
- 7Bracketing diluted standard × 1
CALCULATION
Calculation with a worked example
% impurity = (Ai / As) × (Cs / Ct) × (1 / RRF) × 100
Where
- Ai
- Peak area of the impurity in the test solution
- As
- Mean peak area of paracetamol in the diluted standard
- Cs
- Concentration of paracetamol in the diluted standard (mg/mL, corrected for purity) = WS / 50 × 1/100 × 2/10 × P/100
- Ct
- Nominal concentration of paracetamol in the test solution (mg/mL) = WT × LC / Avg. wt / 100
- RRF
- Relative response factor of the impurity (response per unit concentration of impurity ÷ that of paracetamol); 1.0 for unknown impurities
Given
- Ai (4-aminophenol)
- 14,620
- As
- 15,420
- WS
- 25.10 mg
- P
- 99.8%
- WT
- 121.5 mg
- Avg. wt
- 605.0 mg
- LC
- 500 mg
- RRF (4-aminophenol, illustrative)
- 1.35
- Unknown at RRT 1.6 / RRT 2.3
- Areas 9,280 / 4,650; RRF 1.0
Working
- Cs = 25.10 / 50 × 1/100 × 2/10 × 0.998 = 0.0010020 mg/mL (about 1.002 µg/mL)
- Ct = 121.5 × 500 / 605.0 / 100 = 1.00413 mg/mL
- Cs / Ct = 0.0010020 / 1.00413 = 0.00099787
- 4-aminophenol: Ai / As = 14,620 / 15,420 = 0.94812
- 4-aminophenol: 0.94812 × 0.00099787 × (1 / 1.35) × 100 = 0.0701 → 0.07%
- Unknown RRT 1.6: (9,280 / 15,420) × 0.00099787 × 1 × 100 = 0.60182 × 0.099787 = 0.0601 → 0.06%
- Unknown RRT 2.3: (4,650 / 15,420) × 0.00099787 × 100 = 0.0301 → 0.03% (below the 0.05% reporting threshold, not reported)
- Total reportable impurities = 0.07 + 0.06 = 0.13%
4-aminophenol 0.07%; largest unknown 0.06% (RRT 1.6); total impurities 0.13% — compare each with the specification limits
ACCEPTANCE CRITERIA
What result passes
- 4-aminophenol: not more than the limit in the monograph / your specification (pharmacopoeial limits are commonly of the order of 0.1%; check the current monograph).
- Any unspecified degradation product: typically not more than the ICH Q3B(R2) identification threshold for the maximum daily dose (0.10% for paracetamol, maximum daily dose about 4 g).
- Total impurities: not more than the specification limit.
- Peaks below the reporting threshold (0.05% for a maximum daily dose above 1 g) are disregarded; placebo and blank peaks are excluded.
METHOD VALIDATION
Validation parameters (ICH Q2(R2))
| Parameter | Typical requirement |
|---|---|
| Specificity | Impurities, degradation products (from forced degradation) and placebo peaks resolved from each other and from paracetamol; peak purity by PDA |
| LOQ / LOD | LOQ at or below the reporting threshold (S/N about 10); LOD S/N about 3 |
| Linearity | For each specified impurity and the drug, from LOQ to about 120–150% of the specification limit; r typically ≥ 0.99 |
| Relative response factor | RRF = slope of impurity line ÷ slope of drug line, from linearity data at the same wavelength |
| Accuracy | Recovery of impurities spiked into placebo / sample at LOQ, 100% and about 150% of the limit; typically about 80–120% at low levels |
| Precision | Repeatability and intermediate precision on spiked samples; %RSD limits set by level |
| Robustness and solution stability | Small changes in pH, temperature, flow and gradient; stability of test and standard solutions over the analysis period |
In LimsPulse · Sample Management
Run this method in LimsPulse: specifications and STPs under version control, results calculated and checked against limits, and a signed Certificate of Analysis.