Controlling elemental impurities is an important part of pharmaceutical material characterization and quality control. Nickel can be present at trace concentrations in starting materials, intermediates, and active pharmaceutical ingredients, making a reliable analytical method essential. X-ray fluorescence spectroscopy, commonly known as XRF, provides both qualitative and quantitative elemental analysis while requiring only a small quantity of material.
This application note examines the use of the Malvern Panalytical Epsilon 4 benchtop energy-dispersive X-ray fluorescence, or EDXRF, spectrometer for trace nickel analysis. The work focuses on a matrix-matching method developed and optimized by Esteve CDMO. The method is designed to compensate for absorption effects in complex organic pharmaceutical materials while preserving the simple sample preparation associated with XRF.
External calibration standards for organic pharmaceutical compounds can be prepared using cellulose as a solid support. Cellulose is useful because its principal elements, carbon, hydrogen, and oxygen, have relatively low X-ray absorption. However, some pharmaceutical materials contain elements with higher atomic numbers. Chlorine, for example, can absorb more of the fluorescence signal and cause the behavior of the sample to differ from that of a cellulose calibration standard.
If this difference is not corrected, matrix effects can reduce quantitative accuracy. Standard addition is one possible approach, but it requires multiple mixtures of the sample and standard for every batch. This additional preparation reduces the speed and simplicity that make XRF attractive for routine analysis.
The method described in the application note uses known X-ray absorption properties to calculate the total absorption of an organic compound. The matrix of the external calibration standards is then adjusted to produce comparable absorption. In the featured case study, cellulose was combined with sodium chloride to create standards that matched the mass attenuation coefficient of a chlorine-containing starting material.
Matching these absorption properties allows the calibration standards and pharmaceutical sample to respond more consistently during measurement. The approach is intended to improve accuracy and reliability without requiring a standard addition calibration for every analytical batch.
The full document gives practical information for scientists evaluating EDXRF for pharmaceutical elemental impurity analysis, including:
The method uses approximately 200 mg of material in a sample holder and performs measurements in air, without requiring a vacuum pump or helium consumption. Because XRF analysis is non-destructive, samples can also remain available for subsequent analysis by other techniques when required.
The case study is relevant to analytical scientists, pharmaceutical quality-control teams, method-development specialists, and laboratories investigating alternatives or complementary approaches to established elemental analysis techniques. It illustrates how matrix composition can be incorporated into calibration design when a conventional cellulose standard does not adequately represent the X-ray absorption of a pharmaceutical sample.
Register to access the complete application note, including the measurement conditions, mass attenuation coefficient data, calibration results, precision measurements, recovery study, and conclusions from the Epsilon 4 nickel analysis.
Elemental impurities in starting materials, intermediates, and active pharmaceutical ingredients (APIs) can be effectively analyzed using X-ray fluorescence spectroscopy (XRF). This technique can be used for qualitative or quantitative types of analysis. Quantitative analysis typically relies on external calibration with standards prepared using the target analyte and cellulose as a solid support. Cellulose is an excellent choice due to its elemental similarity to organic pharmaceutical compounds (mainly C, H, O, N). These elements have low X-ray absorption, which minimizes matrix effects and allows for accurate analysis in most cases.
This application note shows the capabilities of Epsilon 4, a benchtop EDXRF spectrometer, to analyze nickel (Ni) impurities with a matrix-matching method. This method was developed and optimized by Esteve CDMO, a pharmaceutical company with headquarters in Barcelona (Spain).
Measurements were performed using an Epsilon 4 EDXRF spectrometer, equipped with a 15 W, 3 mA, 50 kV Ag anode X-ray tube, primary filters, high-resolution Silicon Drift Detector, a spinner and a sample changer for automatic batch measurements. Measurements were performed in air atmosphere. Therefore, there is no need for any vacuum pump or helium consumption.
![[AN260909-figure1.png] AN260909-figure1.png](https://dam.malvernpanalytical.com/0a35336b-912f-4d38-a10b-b4c00020c027/AN260909-figure1_Original%20file.png)
In some cases, organic compounds contain elements with higher atomic numbers, such as chlorine (Cl). These elements cause more absorption of the fluorescence signal than C, H, O and N, leading to matrix effects that reduce method accuracy and result in recoveries outside the acceptable range (70%–150%) of a typical ICP analysis.
A common alternative is the standard addition method, which requires preparing at least three mixtures of sample and standard, per batch. However, this approach compromises one of XRF’s key advantages: fast and simple sample preparation, with only ~200 mg in a sample holder.
![[AN260909-figure2.jpg] AN260909-figure2.jpg](https://dam.malvernpanalytical.com/41038b98-dd16-4ba4-8c3c-b4c00020c207/AN260909-figure2_Original%20file.jpg)
Since the X-ray absorption of each element is known, the total absorption of a compound can be calculated. Therefore, the proposal is to (a) calculate the total absorption of the organic compound, and (b) adjust the matrix of the external calibration standards to match that absorption, by mixing cellulose with compounds like NaCl or others. This way, the simplicity and speed of XRF sample preparation is preserved by reducing the number of sample preparations per batch. Also, the accuracy and reliability in complex matrices is improved. And lastly, the robustness of the whole method is enhanced, ensuring compliance with regulatory guidelines.
In this study, the matrix-matching method was used for accurately quantifying Ni in organic compounds. According to ICH Q3D, the limit of Ni in solid material is defined as 20 ppm when considering 10 g daily dose. When testing at 30% of the limit, the internal concentration limit was set at 5 ppm for Ni in organic material.
Ni was analyzed in the starting material (SM) that contains 14.1 wt% Cl as it is present as a hydrochloride salt. To correct for the matrix effect, mass attenuation coefficients (MAC) for Ni were calculated using the NIST database. By mixing SM with, in this example, NaCl, the MAC of the mixture can be increased and matched with the MAC of the SM. A ratio of 4.8:1.0 (Cellulose:NaCl) matches the same MAC as for SM (Table 1).
| Matrix | Ni μ (Kα 7.50 keV), cm2/g |
|---|---|
| Cellulose | 10.5 |
| Starting Material (SM) | 23.8 |
| NaCl | 87.2 |
| Cellulose + NaCl (Ratio 4.8:1.0) | 23.8 |
![[AN260909-equation.png] AN260909-equation.png](https://dam.malvernpanalytical.com/808b1b94-6e2a-48bd-b941-b4c00020b7f6/AN260909-equation_Original%20file.png)
Where: μ is the mass attenuation coefficient of each element i.
w is the weight fraction of each element i.
Ni concentration in both standards and samples was measured using the same measurement condition as described in Table 2. The measurement time was 30 minutes per sample. The sample spinner was always on during the measurements to improve the particle statistics of the powder samples. A region of interest (ROI) in the XRF spectrum was defined (between 20.86 and 21.02 kV) to correct the Ni intensities for matrix effects and sample density.
| Condition | Element | kV | mA | Filter | Medium | Meas. Time (min) |
|---|---|---|---|---|---|---|
| 1 | Ni | 50.0 | 0.3 | Ag | Air | 30 |
This approach uses a calibration curve for Ni in the cellulose–NaCl matrix standards (ratio 4.8:1.0). Five in-house standards were produced to cover a concentration range between 5 and 50 ppm Ni. Standards 1 to 4 were measured three times and the average value per concentration is shown in the calibration graph (see Figure 3). In order to determine relative standard deviation (RSD), standard 5 was measured 6 times, resulting in RSD of only 2.2% at 5 ppm concentration (see Table 3).
![[AN260909-figure3.png] AN260909-figure3.png](https://dam.malvernpanalytical.com/d2cd81bc-6316-4d88-ad1c-b4c00020c107/AN260909-figure3_Original%20file.png)
| Standard | Concentration (ppm) | Replicates | RSD (%) |
|---|---|---|---|
| 1 | 49.7 | 3 | 0.4 |
| 2 | 19.8 | 3 | 1.0 |
| 3 | 16.5 | 3 | 1.5 |
| 4 | 9.9 | 3 | 6.7 |
| 5 | 5.3 | 6 | 2.2 |
In order to verify the method, the starting material (SM) was quantified using this matrix matching method. The nickel concentration in the sample was determined to be 2.8 ppm. This is well below the safety limit of 30% of 20 ppm for Ni in organic solid samples according to ICH Q3D.
To test the robustness and to validate the method, three different spiked samples of SM were created with targeted concentrations of 5, 10 and 50 ppm Ni. Those samples were quantified against the calibration graph in Figure 1. For each spiked starting material, replicates were measured (3 and 6 times) to obtain the RSD of the measurement. The results are shown in Table 4.
| Sample | Spiked concentration (ppm) | Average of measured concentration (ppm) | RSD (%) of measured concentration | Recovery (%) |
|---|---|---|---|---|
| SM 1 | 50.3 | 50.9 | 0.1 | 99 |
| SM 2 | 9.9 | 10.6 | 0.2 | 93 |
| SM 3 | 4.9 | 5.9 | 0.5 | 92 |
This matrix-matching method enables a simple and rapid correction of matrix effects without the need for standard addition calibration. The low amount of material required (~200 mg) is favorable for pharmaceutical industry or other areas with low amount of material available to analyze. Since standard addition calibration typically requires at least three spiked samples per batch, the proposed method reduces routine quality control workload significantly.
The data presented in this application note clearly demonstrates that Malvern Panalytical Epsilon 4 XRF spectrometer can analyze nickel impurities well below the trace-level concentration limits required by ICH Q3D and USP<232>, even at 10 g daily dose.
This matrix-matching method was further applied for the Ni analysis of the same starting material in its liquid form, which corresponds to the free base of the previously mentioned hydrochloride salt, by selecting a solvent with similar absorption properties for standard calibration. Also, Cu was analyzed using a different starting material through standard calibration method but this time using a cellulose–NaI mixture.
The non-destructive nature of the Epsilon 4 analysis makes it possible to measure the samples subsequently by other analytical techniques, if required.
Malvern Panalytical acknowledges the method development and corresponding measurements described in this application note, were carried out by Esteve CDMO, Barcelona (Spain).