Quantification of Polymorphic Impurities in APIs Using XRPD: Case Study and 5+ FAQs

To quantify polymorphic impurities using X-ray Powder Diffraction (XRPD), a calibration curve must first be established by analysing physical mixtures containing known proportions of the polymorphic forms. This process involves identifying distinctive diffraction peaks for each polymorph, measuring their peak intensities or integrated areas, and plotting these values against the known concentrations. A linear regression equation of the form y = mx + C is then derived, where y represents the measured intensity or area, x is the impurity concentration, m is the slope, and C is the intercept. This calibration curve is subsequently used to determine the concentration of the polymorphic impurity in unknown samples with high accuracy and reproducibility.

Solid-state characterisation is critical to ensure drug efficacy, safety, and stability of an API. One important aspect of this characterisation is the detection and quantification of polymorphic impurities in Active Pharmaceutical Ingredients (APIs). Polymorphs—different crystalline forms of the same compound—can exhibit significant differences in solubility, bioavailability, and stability. Detecting even minor polymorphic impurities is essential to meet regulatory standards and guarantee consistent therapeutic performance.

In this article, I will discuss a stepwise analytical approach using X-Ray Powder Diffraction (XRPD)—a gold-standard technique for characterising crystalline materials—for the quantitative analysis of polymorphic impurities in APIs.

Why XRPD?

X-Ray Powder Diffraction is a non-destructive, highly specific technique that provides direct information about the crystal structure of materials. Unlike other techniques (like DSC or FTIR), XRPD can distinguish between polymorphs based on their unique diffraction patterns, making it ideal for both qualitative identification and quantitative analysis.

Stepwise Approach for Quantification of Polymorphic Impurities

Step 1: Sample Preparation

Proper sample preparation is critical. The API must be:

• Finely ground to ensure homogeneity
• Free from moisture and contaminants
• Packed in a consistent and reproducible manner

Expert Tip: Poor sample handling can lead to peak broadening, preferred orientation, or inaccurate results.

Step 2: Reference Polymorph Selection

Identify and obtain pure forms of all relevant polymorphs, including:

• The desired polymorph (typically the most stable or bioavailable)
• Any known or suspected impurities

These references will be used to generate calibration standards.

Step 3: XRPD Data Collection

Set the instrument parameters for optimal resolution:

• Use Cu Kα radiation (commonly used source)
• Scan range: typically 5° to 40° 2θ
• Step size: ~0.02° with sufficient counting time

Ensure that the baseline is flat and peaks are sharp for accurate quantification.

Step 4: Peak Identification

Use software or databases (e.g., ICDD PDF) to match peaks and identify:

• Characteristic peaks unique to each polymorph
• Overlapping peaks (if any)

Choose non-overlapping peaks for quantification whenever possible.

Step 5: Calibration Curve Preparation

Prepare physical mixtures of the reference polymorphs in known proportions (e.g., 0%, 1%, 5%, 10%, 25%, 50%). Then:

• Acquire XRPD patterns for each blend
• Measure peak intensities (or area under selected peaks)
• Plot a calibration curve of peak intensity vs. concentration

This allows you to establish a quantitative relationship.

Step 6: Sample Quantification

Measure the sample’s XRPD pattern and compare it against the calibration curve to determine:

• The percentage of polymorphic impurity present

If advanced deconvolution is needed (e.g., for overlapping peaks), use techniques like:

• Rietveld refinement
• Principal Component Analysis (PCA)

Step 7: Method Validation

Validate the method according to ICH guidelines:

• Accuracy (spike recovery)
• Precision (repeatability and intermediate precision)
• Linearity (of calibration curve)
• Limit of Detection (LOD) and Limit of Quantification (LOQ)

This ensures regulatory compliance and reproducibility.

Case Study: Quantitative Analysis of Polymorphic Impurities in Carbamazepine Using XRPD

Objective

To detect and quantify a known polymorphic impurity (Form II) in batches of Carbamazepine API, where Form III is the therapeutically approved and stable form. The study aims to ensure batch consistency and regulatory compliance using X-Ray Powder Diffraction (XRPD).

Background

Carbamazepine, an anticonvulsant and mood-stabilising drug, exists in multiple polymorphic forms (Form I to Form IV). Form III is considered the stable and pharmaceutically acceptable form. However, Form II, a metastable polymorph, can appear during certain crystallisation or milling processes. Even trace amounts of Form II may impact solubility, dissolution rate, and long-term stability.

Materials and Methods

1. Sample and Reference Materials

• API Test Samples: Three batches of Carbamazepine labelled A, B, and C
• Reference Standards: Pure polymorphic forms—Form III (desired) and Form II (impurity)

2. Sample Preparation

• Finely ground powders packed into low-background sample holders
• All samples stored in a desiccator prior to analysis to avoid hydration or phase transformation

3. Instrumentation

• X-Ray Diffractometer: Bruker D8 Advance
• Radiation: Cu Kα (λ = 1.5406 Å)
• Scan range: 5° to 35° 2θ
• Step size: 0.02°, Time per step: 1s

4. Calibration Curve Setup

• Prepared binary physical mixtures of Form II and Form III in the following proportions:
  0%, 1%, 2%, 5%, 10%, 20%, and 50% Form II
• Selected a characteristic peak of Form II at 15.2° 2θ
• Measured peak intensity (height and area) and plotted against % concentration

5. Data Analysis

• Used OriginPro and HighScore Plus for curve fitting
• Linear regression provided the calibration equation
• Applied to batch samples to quantify impurity

Results

Calibration Curve

• R² = 0.998 for intensity vs. concentration of Form II
• LOD: 0.3%
• LOQ: 1.0%

Batch Analysis

Result Summary

• Batch A: Passed—no detectable Form II
• Batch B: Passed—impurity within acceptable limits (< 5%)
• Batch C: Flagged—exceeds internal specification (max 2%)

XRPD successfully detected and quantified polymorphic impurities down to 1% concentration, providing a non-destructive and reproducible method for quality control.

Formula for Quantification of Polymorphic Impurities

How to Obtain m(slope) and I₀​:

You create a calibration curve by preparing mixtures of known proportions of the impurity with the main polymorph (spiked sample with impurity), such as, 0%, 1%, 5%, 10%, etc., then:

• Measure the intensity (area) of the impurity polymorph peak at each concentration
• Plot the curve between concentration/known % impurity (x-axis) and intensity /area (y-axis)
• Calculate the Slope (m) and Intercept (I₀) from the linearity curve using Excel
• Then apply the above formula to your test sample’s peak intensity to calculate the unknown % impurity.

Example:

Suppose Slope (m) is 22.5 and Intercept (I₀) is 5.0, then your calibration curve equation is:

I=22.5×C+5.0 (Equation-1)

Where:

• I=peak intensity/area of the impurity peak in the test sample
• C=concentration of impurity (%) in sample

If your test sample gives I_sample=27.5 then Polymorphic Impurity (C, which is unknown) can easily be calculated from equation 1 in the following way:

27.5=22.5xC+5.0

or C (%)= (27.5-22.5)/5=1%

Advantages

• XRPD is a powerful tool for routine polymorph monitoring in API manufacturing.
• Even low-level polymorphic impurities can be accurately quantified with a proper calibration curve.
• Method validation is essential to ensure accuracy, precision, and regulatory compliance.

Advanced Tools and Techniques

• Rietveld Refinement: For full-pattern fitting and quantification
• Multivariate Analysis (PCA, PLS): To resolve complex or overlapping peaks
• Synchrotron XRPD: For higher resolution in trace-level impurity detection

Conclusion

The quantification of polymorphic impurities in APIs using XRPD is a robust and reliable method when implemented carefully. A stepwise approach—from sample preparation to validation—not only improves accuracy but also ensures regulatory compliance and product consistency.

As pharmaceutical regulations continue to emphasise solid-state control, mastering XRPD-based polymorph quantification becomes a valuable skill for analytical scientists and formulation developers alike.

You may like:

1. Relative Response Factor (RRF) in Pharmaceutical Analysis
2. How To Control Impurities In Pharmaceuticals: Get Mastery In 11 Minutes
3. How To Calculate Potency, Purity and Assay In Pharmaceuticals

FAQs

Further reading:

• Grzesiak, A. L., Lang, M., Kim, K., & Matzger, A. J. (2003).
  Comparison of the four anhydrous polymorphs of carbamazepine and the crystal structure of form I.
  Journal of Pharmaceutical Sciences, 92(11), 2260–2271.
• Florence, A. J., Johnston, A., Fernandes, P., & Shankland, N. (2006).
  Quantitative phase analysis using X-ray powder diffraction.
  Journal of Pharmaceutical and Biomedical Analysis, 41(4), 892–897.