XRD Method is a widely used Analytical technique in the Pharmaceutical Industry. The full form of the XRD is “X-Ray diffraction” Spectroscopy.
Introduction of XRD Method Development
X-Ray diffraction (XRD) is a powerful analytical tool used for determining the crystal structure of materials. The development of XRD methods involves several steps that can vary depending on the specific research question being addressed. Here are some general steps to consider when developing an XRD method:
- Sample preparation: The first step in XRD method development is preparing the sample. The sample should be finely ground to ensure that it is homogeneous and free from impurities. If the sample is not already in a powdered form, it may need to be crushed and ground to obtain a fine powder. The sample may also need to be mounted on a suitable substrate for analysis.
- Instrument setup: Once the sample is prepared, the instrument must be set up. This involves selecting the appropriate X-ray wavelength, setting the X-ray tube voltage and current, and adjusting the detector settings. The instrument must be calibrated to ensure that it is operating within its specified range.
- Data collection: Data collection involves exposing the sample to X-rays and recording the resulting diffraction pattern. The XRD instrument rotates the sample, and the detector measures the intensity of the diffracted X-rays at each angle. The resulting data can be visualized as a diffractogram, which shows the intensity of the diffracted X-rays as a function of the diffraction angle.
- Data analysis: The diffractogram obtained from the data collection must be analyzed to determine the crystal structure of the sample. The analysis involves comparing the diffractogram to a database of known diffractograms to identify the crystal structure that best matches the experimental data.
- Refinement: Once the crystal structure has been identified, the method may need to be refined to improve the accuracy of the results. Refinement may involve adjusting the instrument settings or changing the sample preparation method.
However, developing an XRD method requires careful attention to sample preparation, instrument setup, data collection, data analysis, and refinement. The goal is to obtain accurate and reproducible results that can be used to answer specific research questions.
X-Ray Diffraction Method – Preliminary Evaluation
As part of the X-Ray Diffraction Method development, analyze all the pure forms as per the regular identification conditions and confirm the pattern as per the patent literature. In case of published forms (or) confirm the pattern with std. conformational data being generated in-house or naval polymorphs. Else check as per the method mentioned in the patent or also use other techniques like IR, DSC, TGA (for hydrates), etc.
Then note down the non-interfering and high-intensity peak for undesired polymorphs with respect to desired polymorphs.
Non-interfering peaks shall be unique with respect to the polymorphic form under consideration.
Select the required scan range in which a non-interfering peak for the undesired form is present and it shall be visually absent in the required form.
Select the reference peak in the required form, which is closer to the peak of the undesired form, and note the scan range for the same.
Scan the pure forms in the selected range by applying appropriate slow scan conditions to inspect the absence of undesired form and also carry out the scan in the selected range of reference peak of the desired form.
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Determination of LOD and LOQ
The XRD instrument limit of detection (LOD) is to be determined by measuring the instrumental noise level in the selected scan range considered for undesired form. This is one of the critical parameters in XRD method development. Carry out the analysis at least with duplicate preparations and note down the average noise in counts (A)
Carry out the analysis by using duplicate preparations for pure undesired form by applying the same instrumental conditions carried out for noise determination and note down the average intensity (B)
The instrumental LOD is calculated by using the following equation
3 X A
LOD ( % ) = ———— X 100
B
Where, A Instrument LOD, in counts
B Average intensity of pure undesired form, it counts
The instrumental LOQ can be calculated as follows
LOQ (%) = 9 X Instrument Noise or 3 X instrument LOD
Note: LOD: Limit of Detection
LOQ : Limit of Quantification
Visual Assessment to confirm the XRD instrumental LOD
Prepare the spiked preparation of undesired form in the desired form at its LOD level and carry out the analysis by applying the finalized instrumental conditions.
Check the peak intensity in counts and the value shall be approximately equal to
(3 X instrumental Noise)
Reproducibility at the below LOD
Prepare the three spiked samples at below LOD level and scan in the selected range to check the possibility of detection.
Reproducibility at LOD
Prepare the three spiked samples at the LOD level and scan in the selected range and visually observe the presence of the peak due to undesired polymorph. Note down the intensity counts and shall be approximately equal to (3 X instrumental Noise)
Repeatability at LOD
Prepare the spiked sample at the LOD level and scan in the selected range three times. Note down the intensity counts and shall be approximately equal to (3 X instrumental Noise)
Confirmation of Instrumental LOD by Different analyst
Check the reproducibility and repeatability at the LOD level in the selected scan range. Note down the intensity counts and shall be approximately equal to (3 X instrumental Noise)
Determination of Linearity (For quantification method development)
Linearity is one of the critical parameters in XRD method development.
Prepare the spiked samples of undesired polymorphic form in the desired form at and above the LOQ level at different concentrations (Minimum of five different concentrations) and carry out the analysis scan in both the ranges (selected scan range for detection as well as ref peak range for desired form).
Determination of XRD Response Factor (K)
To calculate the K Factor, the linearity is to be established by using sample preparations as described in 2.1 and to calculate the ratio of the area corresponding to the undesired form (S2) versus the area of the desired form (S1). The response factor (K) can be determined as follows.
S2 * W1
Response Factor, K = ————-
S1 * W2
Where S1 = Area of the peak corresponds to the desired form
S2 = Area of the peak corresponds to the undesired form
W1 = Wt. of the desired form taken
W2 = Wt. of the undesired form taken
The K factor determined for all the concentrations shall fall within the same range. The K factor shall fall within the range of + 10% of the value obtained with LOQ preparation.
Quantification of Undesired polymorphic form in the desired form
The quantification of the undesired polymorphic form can be carried out by using the below formula. These is very important and critical parameters in XRD method development.
(S2 / S1)
% of Undesired Form = ————————— X 100
K+ ( S2 / S1)
Where S1 = Area of the peak corresponds to the desired form
S2 = Area of the peak corresponds to the undesired form
K = The response factor
Demonstration of Repeatability
Demonstration of Repeatability is one of the critical parameters in XRD method development.
Carry out the analysis for two different known concentrations six times each. (Take LOQ and any other concentration above the LOQ level, that falls in the linear range).
Acceptance criteria: % RSD shall not be more than 15 for each concentration.
Demonstration of Reproducibility at LOQ level
Prepare LOQ-level spiked preparations six times and carry out the analysis.
Acceptance criteria: % RSD shall not be more than 15
Finalize the method based on the outcome and justification.
Precautions for X-Ray diffraction Method Development (XRD Method)
During the XRD Method development, the below precautions shall be considered.
Ensure the uniformity of all the desired and undesired polymorphic forms and sift the material through 60 mesh prior to analysis, if required.
After preparation of the sample, ensure the uniformity of the surface on the sample holder. The irregular surface may cause the preferred orientation
Ground the material uniformly with the mortar and pestle (if it does not affect the polymorphic form stability).
Use a similar type of sample holder for all sample analyses.
Ensure the intensity of the X-Ray source prior to and after completion of the method development/validation.
XRD Method – Important notes
For detection method development, follow the procedure described in 1.0 to 7.0.
For quantification method development, follow the procedure described in 1.0 to 3.0 and 8.0 to 11.0.
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X-Ray Diffractometry Method Reference
Ron Jenkins, Robert L Snyder – A book of “Introduction to X-Ray diffractometry”.
USP General chapters and assays < 941>.
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