Mass spectrometry (MS) is an analytical technique that provides valuable compound identity information, particularly for substances present at low concentrations such as extractables and leachables (E&Ls) that are often difficult to detect using other methods. In fact, detection of extremely low levels of E&Ls in large-volume parenteral formulations is testing the limits of conventional analytical techniques, and as a result there is growing interest in MS for this application, according to Patrick Bennett, marketing director for pharmaceuticals and biopharmaceuticals at Thermo Fisher Scientific. Advances in technology are not only improving the sensitivity and accuracy of MS analyses, they are also helping to reduce the cost of MS instruments and, therefore, are driving greater use of MS for E&L analysis
Orthogonal methods including MS are essential
To effectively screen for E&Ls, it is essential to use a number of orthogonal analytical methods, or those that will complement each other with respect to the detection of different compound classes, explains Carsten Worsøe, a principal scientist with Novo Nordisk. Such analytical methods include high-performance liquid chromatography-ultraviolet spectroscopy (HPLC–UV), liquid chromatography-mass spectrometry (LC–MS), gas chromatography-mass spectroscopy (GC–MS), gas chromatography-headspace-mass spectroscopy (GC–HS–MS), inductively coupled plasma-atomic emission spectroscopy (ICP–AES), ICP–MS, and ion chromatography (IC). MS is often coupled with chromatographic techniques for the identification and accurate mass detection of organic E&Ls. More recently, gas chromatography time-of-flight (TOF) mass spectrometry and liquid chromatography-nuclear magnetic resonance spectroscopy and liquid chromatography-solid-phase extraction nuclear magnetic resonance spectroscopy have been shown to be valuable techniques for identifying unknown organic E&Ls, says Worsøe.
Many of these latest analytical techniques used for E&Ls determinations are improvements on current technologies; more modern equipment and electronics offer greater sensitivity, while advances in computer technology and computer software capabilities enable the analysis of much more complex data, observes Daniel L. Norwood, a distinguished research fellow with Boehringer Ingelheim Pharmaceuticals. For Dennis Jenke, a distinguished scientist in the Technology Resources Division of Baxter Healthcare Corporation, the coupling of high-efficiency chromatographic techniques with information-rich detectors is proving very useful for E&L analysis. In particular, he notes that the coupling of ultra high-performance liquid chromatography (UHPLC) with high-resolution MS detectors capable of providing accurate mass information enables the determination of the empirical formulas for otherwise unidentified E&Ls is a noteworthy recent advance.
Benefits of MS for E&L analysis
There are multiple benefits of using MS for E&L analysis, according to Wayland Rushing, a senior scientific advisor at ABC Laboratories. Firstly, E&Ls cover a wide range of chemical compounds, some of which have very limited UV absorbance characteristics. Their analysis using traditional HPLC–UV us, therefore, challenging while MS detection occurs independent of chromophoric properties. Secondly, with MS, the information necessary for determining the structure of unknown compounds can be gathered. Third, the detection limits are significantly lower with MS than the levels that can be achieved using gas chromatography-flame ionization detector (GC–FID) or HPLC–UV techniques.
The use of reliable/accurate exact mass instruments has also reduced the time it takes to analyze and identify E&L compounds. “In the past, it would take significant time, because the unknown compounds first had to be isolated before they could be characterized, which was very time-intensive. With the sensitivity and accuracy of the instruments available today, this time can usually be significantly reduced because structure elucidation is now possible without the need to perform laborious isolation work for the individual unknowns,” Rushing explains.
Choice of MS instruments for data generation
Given the wide range of compounds in E&L samples, the increasing choice in MS instruments that can be used in combination with various chromatography/separation systems has increased the applicability of MS in E&L analysis. All of the instruments essentially gather the same basic information mass-spectral data on the compound. It is the type, sensitivity, and accuracy of the data that vary across the different instrument types, Rushing points out.
Exact mass instruments are used for the determination of the chemical formula of E&Ls while fragmentation instruments are used for the determination of functional groups and the structure of analytes. TOF and quadrupole TOF (QTOF) provide exact mass and formula information, with QTOF having high accuracy and sensitivity. Ion trap, on the other hand, provides fragmentation data for the determination of functional groups and chemical structure. Single quad systems provide basic mass information. Triple quads (MS/MS) also provide fragmentation data and are frequently used for quantitation and compound identification; Orbitrap MS systems, meanwhile, offer this functionality at high-resolution accurate mass (HRAM) levels. Matrix-assisted laser-desorption ionization (MALDI)–TOF is useful for large organic molecules and matrices where sample preperation is problematic. “Of all of these instruments, the Orbitrap could be considered the instrument of choice for most E&L work,” Rushing says
It should also be noted that the same type of instruments from different suppliers will provide slightly different results. This fact should not be surprising, according to Norwood, given that the ion sources are based on proprietary technology and as a result compounds may ionize differently even though the process is nominally the same. Furthermore, some instruments are much more robust than others, and are thus more suited to initial sample analyses. “For example, we will screen samples on a more rugged instrument, such as a triple quadrupole, and only use the fourier transform mass spectrometry (FTMS) ion cyclotron resonance instrument with an ion trap for pure samples that require extremely high sensitivity. Injection of samples with large quantities of contaminants is problematic on the FTMS system, and cleaning results in significant downtime for the instrument. TOF MS also tends to be used with UHPLC because it enables the rapid scans required for this very fast chromatographic technique,” Norwood comments.
As a result, most laboratories that do trace analysis work have several different types of mass spectrometers and several of the same types but from different manufacturers to ensure they possess the greatest possible analytical capabilities.
Advances in technology
The application of MS in E&L analysis isn’t new, and in fact has has been used since the early 1980s. Several advances have, however, led to the much wider use of the technology today, according to Norwood. He points to the coupling of liquid-chromatography methods with MS in the early 1990s as a crucial development. “The detection of extractables, and particularly leachables, is a trace analysis problem involving the identification of compounds that exist at trace levels in very complex matrices. The ability to interface MS with chromatography methods has, therefore, been a significant improvement,” he says. New ionization techniques, such as electrospray and atmospheric pressure chemical ionization, have also made a difference. In addition to being easier to interface with HPLC systems, they are easier to use, more rugged, and have much lower noise, and therefore greater sensitivity than older ionization processes, Norwood observes. He also notes that FTMS is perhaps the most significant advance in MS techniques for E&L analysis. “FTMS provides sensitive and accurate mass measurements of compounds present at very low levels as they elute from an HPLC system.”
Cyclotrons, ion traps, and triple quadrupole instruments produce data that used to be very difficult to obtain. However, it is not only advances in ion sources and ionization methods that have enabled the types of E&L analyses commonly seen today. “Without the advances in computer and software technology that have taken place in the last few decades, we would not be able to process the data generated by these mass spec instruments,” Norwood emphasizes. “Computers and software really should be considered analytical tools in their own right,” he adds.
One of the major limitations of MS is the need for skilled, experienced chemists to interpret the data generated by advanced mass spectrometers. “To make sense of the data in the context of the samples being analyzed, a person with training and expertise is still needed. Artificial intelligence systems, library matching, and other techniques are still not able to deal with the potentially very complicated sets of unknowns that can be generated as the result of the interactions of active pharmaceutical ingredients, excipients, and packaging materials,” observes Rushing.
Jenke also cautions that the data obtained from accurate MS analyses must be considered in context. “Accurate mass is not always able to give a single definitive empirical formula. Oftentimes several viable candidates are identified and it is not possible, based on the accurate mass data alone, to establish which candidate formula is the correct one. There can be literally hundreds of compounds with a certain empirical formula but only a few may be toxic. It is necessary to know the structure in order to reach the ultimate end game for E&L, which is safety assessment. Accurate mass can therefore, help shore up a tentative identification and may provide insight into a compound’s identity, but it is only one piece of several pieces of necessary information for elucidating and confirming an identification,” said Jenke.
Wishes for the future
Although there are mass spec instruments that can interface with UHPLC systems, Norwood would like to see improvements in this area. As with any analytical technique, he would also like to see instruments with greater sensitivity. The ultimate mass spectrometer, though, would have separation and mass detection capabilities without the need for chromatography.