Determination of impurities and their isolation

Identification and determination of structures of impurities is a fairly common task which we have to deal with. All unknown impurities with higher content than is permitted by applicable legislature (typically 0.1%) are potential complications for customer as they can cause an undesirable interest during the registration process.

Knowledge of the structure of an unknown impurity is then often crucial in distinguishing impurities originating in the manufacturing process from impurities resulting from the subsequent degradation of the final product. The first above mentioned impurities can be controlled, for example, by adjusting the production process. Knowing the structure of an impurity of the degradation origin can, in turn, enable us to prepare its standard on the basis of toxicological data or increase the limit for its content. When determining the structure of a substance in practice, we use mass spectrometry together with either gas chromatography (GC-MS) or liquid chromatography (LC-MS). When determining the structure of volatile substances, we have had excellent results with the GC-MS system together with a single quadrupole, which, when used with EI ionization, provides sufficient information leading to the target structure. During interpretation of spectra, the digital Mass Spectral Library (NIST) can also be used successfully.

Many years of experience have demonstrated that the identification of most non-volatile organic impurities is best achieved with the LC-MS system with ESI or APCI ionization and ion trapping. The LC/MS system LTQ (Thermo-Finnigan), which we use for this purpose, is equipped with a linear ion trap that combines the advantages of a classical ion trap with the capabilities of MSn and quadrupole sensitivity. The advantage is above all the high sensitivity, data collection speed and, thanks to the orthogonal construction of the ion source, also resistance to contamination by, for example, the components of the matrix or partially by non-volatile components in the mobile phase.

If the selected LC/MS alone is not enough, we are prepared to isolate the desired impurities using the preparative liquid chromatography and provide, for example, the measurement of the required NMR spectra, which we are then able to interpret ourselves. In connection to the identification of impurities by, for example, LC/MS, we are able to isolate the given substance in larger amounts at the customer’s request. The degree of success depends on the degree of separation of the given component from the rest of the mixture, but above all on its content in the raw material. If the content is too low, we look for ways to enrich it either by suitable stress conditions or pre-separation.

Isolation can be useful when it is either difficult to directly prepare the substance or other techniques than LC/MS (for example NMR) are necessary for its identification.

For these purposes, we use either classical column chromatography or high performance separation on an automated semi-preparative modular HPLC system Waters. Using these techniques, we are able to isolate, depending on the concentration in the raw mixture and the quality of separation, the individual components in the amount of tens to hundreds of milligrams.

Synthesis and certification of reference materials

Following the previous paragraph relating to the determination of structures of impurities, there is very frequently a need to acquire the given substance as an analytical standard in sufficient quantity so that this impurity can be routinely determined in the product using appropriate methods.

Our advantage is that already during the process of identification of the structure, we often develop procedures that can be used to intentionally produce the given substance by synthesis using allowing suitable reagents to interact with for example the active ingredient, or at least to enrich it. Due to this, we can often acquire the desired substance faster than by conventional synthetic procedures, which we, however, also use in appropriate situations partly in cooperation with external synthetic laboratories.

Additional advantage is the fact that the risk of a mismatch between the originally designed structure of the impurity and its actual structure is our responsibility and this may consequently represent a significant savings of time and money for the customer. We will, of course, design and perform the certification of the given analytical standard including the full characterization of the structure, determination of potency and issuance of the analytical certificate.

Accelerated stability and degradation studies

Part of validations of most HPLC methods for the determination of the content of impurities the so-called degradation study (FDS – Forced Degradation Study). FDS is an instrument for evaluation of analytical methods that aspire for the term “stability-inducing”. The principle of FDS is the exposure of the active ingredient, resp. of the medicinal product (and associated placebo) to a series of physical and chemical stresses that can, in a short-term, generate new unknown impurities that may possibly occur in the final product during storage. The physical and chemical stresses include especially:

• Exposure to heat
• Exposure to light (UV/VIS)
• Acidic, alkaline and oxidizing stresses
• Exposure to elevated temperatures and humidity of the environment

It is beneficial, for chemical stresses in particular, to choose the optimum conditions based on a good knowledge of the chemical behavior of the studied active ingredient. Eventual interactions of the active ingredient with the excipients are studied separately within the so-called compatibility studies. Experience has shown that this type of work is best handled by the R&D sections, which conduct accelerated stability, degradation and compatibility studies as a routine part of the development of new analytical methods, but also as a support of the formulation development of the given medicinal product.

Trace analyses using GC/MS and LC/MS

We have extensive experience in the development of methods for determining trace amounts of monitored impurities. For clients we usually develop methods for the determination of toxic impurities at levels many times lower than required by the relevant regulatory authorities. Although these analytes are often reactive and often in an unfavorable combination with a poorly soluble or reactive matrix, we have always reached the required detection limit.

For these purposes, we most often use LC/MS on systems consisting of HPLC 2690 or UHPLC Acquity (both Waters) LTQ mass spectrometer (Thermo-Finnigan) or GC/MS on a 7890A/5975C system (Agilent Technologies).

On all of these instruments we normally achieve quantification limits in ppm, which is usually quite sufficient for pharmaceutical purposes. If necessary we also use other instrumentation from several LC/MS systems equipped with mass and spectrometers with triple quadrupole or HR-MS system OrbiTrap (Thermo).