The pharmaceutical industry is heavily regulated beyond all other industries because many of the materials produced are potentially dangerous and can be severely toxic if not manufactured with proper care. This level of regulation has led to the mandatory examination of all substances from starting materials to finished products. Generally this includes the proof of product stability, drug-release profiles, Active Pharmaceutical Ingredient (API) form, API quantification, and product safety, for example, sterility (for non-biological materials) and the presence of foreign materials including metal impurities.
One of the most common product safety related analytical tests is the quantification of elemental impurities within a pharmaceutical product. This normally includes the toxic metals, often referred to as heavy metals, such as As, Cd, Hg and Pb. Other metals, such as Fe, Cr, Ni and Zn are also referred to as heavy metals and have well documented health risks. Many APIs are synthesized using metal catalysts, which are invariably left bound to the final product.
Therefore it is also common practice to measure residual metal catalysts, such as Ru, Pd and Pt, which are also well classified toxic elements. Throughout the manufacturing processes there are many potential sources of contamination. Therefore, in addition to measuring the starting materials it is essential to measure all finished products and in some cases intermediates, to demonstrate the process-recovery efficiency and compliancy with the various regulations.
Using energy dispersion X-ray fluorescence (EDXRF) it is possible to measure all (Na – U) elemental concentrations without heating or destroying the sample. Also, unlike time-consuming acid digestions, samples can be measured as loose powders or pressed into pellets and ready for measurement within minutes. XRF can measure larger sample volumes resulting in a better characterization of final products, which are often complex composites numerous excipients and APIs. Furthermore, the XRF technique provides high accuracy and precision with excellent detection limits (0.1 – 1 mg/kg).
Preparation of standards
A common excipient material (pharmaceutical grade wood-cellulose) was chosen as a base material. In-house standards were prepared using ultra- pure commercially available organo- metallic standards representing 6 heavy metals. The elements were chosen to represent a significant proportion of the periodic table ranging from light to heavy transition metals (Cr to Pt). Standard chemical techniques were employed to dope the cellulose and produce a set of standards. The standard concentrations were confirmed by ICP-MS.
All materials, standards and samples, were analyzed as loose powders (approximately 2500 mg) in sample cups. This simplified process reduces sampling and sample preparation errors often compounded when using small masses and dissolution procedures commonly employed by other techniques.
Calibration curves were set up using different conditions with a total measurement time of 30 minutes (real time). The excitation parameters used to cover the whole range of elements from
Ti to Pb are shown in Table 1. If the analysis of few elements is required, some condition sets may be removed, thereby reducing the measurement time.
Table 1: Measurement parameters used in this study
The Epsilon 5 software includes a very powerful deconvolution algorithm, which analyzes the sample spectrum and determines the net analyte peak intensity. The accuracy with which this is carried out is essential to trace element analysis. A spectrum of a standard material containing Ru (21.1 μg/g) and Pd (20.6 μg/g) is shown in Figure 1. This exemplifies the low background and excellent peak fitting (deconvolution) capabilities of the Epsilon 5.
Figure 1: Spectrum of a standard containing 21.1 μg/g Ru and 20.6 μg/g Pd
The method produced highly linear calibration curves; see Table 2 and Figures 2 and 3. Net elemental intensities were ratioed to the intensity of the Compton scatter peak to correct for sample matrix and mass or thickness variations (Table 2).
Calibration graphs (Figures 2 and 3 ) demonstrate the capability of Epsilon 5 in determining heavy metal contaminants from various sources, such as environmental (As), stainless- steel reaction vessels (Cr) and catalyst residuals for the manufacturing of APIs (Ru and Pd).
Figure 2. As Calibration. Figure 3. Pd calibration
Table 2: Calibration coefficients (measured using 300 second condition times)
The calibration root mean square (RMS) values presented in Table 2 demonstrate a high degree of accuracy for the method. The RMS value is a measure of the difference between the calculated concentration and the chemical concentration and is therefore a measure of the accuracy of the method
The precision of the Epsilon 5 for this application was demonstrated by analyzing a single standard against the calibration curve 10 times consecutively, Table 3. The statistical evaluation of the repeated measurements demonstrates excellent precision (standard deviation) for all elements. The average calculated concentrations were very close to the reported ICP-MS values, demonstrating the excellent agreement between the two techniques.
Table 3: Replicate analyses of standard #3
The lower limits of detection (LLDs) are calculated from three times the standard deviation of twenty repeated measurements of a blank sample. An advantage of this method is the capability to achieve lower detection limits by simply extending the measurement time. This is shown in Table 4.
Table 4: Lower limits of detection
Epsilon 5 is a unique instrument and is capable of non-destructively determining metal concentrations in pharmaceutical materials, with minimal sample preparation. This is possible because of its ability to generate high-energy polarized
X-rays without producing heat that would normally damage these sample types. The applied method demonstrates the efficiency of Epsilon 5 to analyze a range of pharmaceutical matrices from excipients, APIs, intermediates and finished products. Furthermore, Epsilon 5 demonstrates a high degree of accuracy, precision and robustness necessary for validation and routine measurement of pharmaceuticals. The calculated detection limits (LLD) and analysis accuracy (RMS) clearly demonstrate that Epsilon 5 can reliably reasure most elements of concern well below 1 µg/g (ppm). Thus Epsilon 5 can be used to meet the requirements of any international pharmacopeia and related regulatory bodies.
Epsilon 5 is fully capable of measuring other elements that are known to be of interest to biologists, food scientists, pharmacologists and health regulators such as Na, Mg, Fe and Co (essentially any element from Na to U). Also, the stability of Epsilon 5 is such that individual calibrations can be used for months, meaning that time-consuming re- standardizations are unnecessary and users can benefit from highly consistent data over the long term.