ChromTalks: The Benefits of Hindsight in Chromatography 

Molar Mass Determination of Collagen Peptides

New HPLC, MS, and CDS Products Introduced in 2020–2021

New Gas Chromatography Products for 2020–2021

Combining Dynamic Headspace Sampling, Capillary Gas Chromatography and Time‐of‐Flight Mass Spectrometry

High Analytical Performance for Untargeted Aroma Profiling 

An Inside Look at Single Immunoaffinity Cleanup and Isotope Dilution LC–MS/MS

Analysis of 12 Regulated Mycotoxins in 8 Food Matrices

Tony Taylor is Group Technical Director of Crawford Scientific Group and CHROMacademy. His background is in pharmaceutical R&D and polymer chemistry, but he has spent the past 20 years in training and consulting, working with Crawford Scientific Group clients to ensure they attain the very best analytical science possible. He has trained and consulted with thousands of analytical chemists globally and is passionate about professional development in separation science, developing CHROMacademy as a means to provide high-quality online education to analytical chemists. His current research interests include HPLC column selectivity codification, advanced automated sample preparation, and LCâMS and GCâMS for materials characterization, especially in the field of extractables and leachables analysis.

Most readers will be familiar with the major gas chromatography (GC) variables, setting them into our data acquisition software, and reading them from our SOPs or laboratory work instruction methods on a regular basis. Generally speaking, those variables are not in scope here, other than to consider the lesser-understood effects of some of them.

Instead, I’d like to concentrate on variables that can really impact our chromatography, but may be on hidden, supplementary, or advanced pages of our software, or may appear on the main software acquisitions menus, but are poorly understood or rarely altered. These variables are often not specifically referenced in laboratory methods documents or, if they do appear, are poorly understood. While some of these variables appear to be esoteric, I’ve seen all of these either ruin otherwise perfectly good chromatography or confound users during method troubleshooting.

Autosampler syringe plunger speed and needle depth


Most autosamplers will give the option to change the speed of the plunger within the syringe barrel, both during sample aspiration and injection into the GC. When dealing with viscous samples, the plunger speed is usually set to a slower speed to avoid cavitation of the sample within the needle, and consequently, variable sample amounts being aspirated. The syringe needle depth may also need to be adjusted when dealing with very low sample amounts, vials with different shapes, or multiphasic samples, for example, where the top layer of a biphasic solvent mixture needs to be sampled. The speed of sample ejection into the inlet may also need to be altered with narrow bore inlet liners or some modes of cool on column injection.


Certainly not the most exciting of topics within the GC arena, but wash solvents are highly important, nonetheless. Wash solvents should be chosen on their ability to solubilize the analytes of interest and not just the sample matrix. This fact is often overlooked when choosing appropriate solvents. The solvents should be highly miscible with the GC injection solvent and, where possible, two wash solvent bottles should be used: one “dirtier” and one “cleaner” bottle. The trick is to wash the syringe prior to sample aspiration in the cleaner solvent and then post-injection, wash first in the dirtier solvent followed by a rinse in the cleaner solvent. Where three wash solvent positions are available, then the third solvent should be reserved for the pre-sample aspiration wash only. It’s also a good idea to leave wash-solvent bottles uncapped because any sample residue from the outside of the syringe needle can potentially contaminate the septum or needle guide from the bottle tops, and be resolubilized into the next sample, causing carryover. Always try to aspirate the wash solvent and discard to waste three times in each operation to ensure that the syringe needle does not penetrate any liquid in the waste bottle—meaning, empty the wate bottle regularly! Having a table of conditions within the method that stipulates the wash regime is a great way to highlight the importance of this variable.

Paying attention to the wash-solvent regime will help to improve your quantitative reproducibility by cutting down on carryover from injection to injection.


Again, not the most inspiring of topics, however, one that can be very important in determining peak shape, especially in splitless injection. I realize that most written methods will cite the injection solvent to be used, and this parameter may be reliant upon sample extraction or manipulation operations from a previous step in the analytical workflow. I cite the importance of the injection solvent here because less experienced GC users may not be aware of just how important the selection of injection solvent is. Injection solvent expansion volume plays an important role in carryover with both split and spitless injection. If the injected liquid volume results in a gas volume that exceeds the liner volume under the operating conditions employed, this will potentially contaminate the inlet and associated tubing, which may result in carryover into subsequent injections. Interested readers can look up the phenomenon of “backflash” for more information. In splitless injection mode, the solvent plays a major role in focusing analytes at the head of the analytical column, and so peak shape in the splitless mode, especially for less volatile analytes, will rely on the compatibility of the injection solvent polarity with that of the GC column stationary-phase material. Incompatibility will lead to shouldering and splitting analytes peaks.


The septum purge gas flows directly beneath the under face of the inlet septum and serves to carry any outgassing products and contaminants away from the septum material. It also has a role in controlling the degree of mass discrimination (relative discrimination between high- and low-boiling analytes), depending upon the configuration of the liner. While most modern GC systems will control the septum purge flow, it is encouraging to know what this setting is, and to manually check the flow prior to analysis. It wouldn’t be the first time that I’ve seen an electronic flow (or pressure) controller to be reading the incorrect value, and this problem can be very difficult to troubleshoot unless one knows to look for it! Stating the required value (typically just a few mL per minute) improves awareness for less experienced staff.


This the time at which the split vent is opened after sample injection in splitless injection mode. I only sporadically see the time associated with this variable written into analytical methods, which is surprising because it can critically affect baseline quality, peak shape of the solvent, and early-eluting analyte peaks as well as the quantitative reproducibility of early-eluting analytes. Ideally, the splitless time is empirically optimized such that the vast majority of all analytes have entered the column, and only the lingering excess sample solvent is ejected by opening the split valve. The optimization involves measuring the peak height (or area) of early eluting analytes while lengthening the splitless time. Too short a splitless time and the analyte peaks will continue to grow as the splitless time is lengthened, the peak area will eventually reach a constant value, and the ideal splitless time (typically 0.25 to 1.25 min post injection) is that which is associated with the second constant peak height or area measurement. Whenever specifying a splitless time, it is necessary to also specify the split flow rate or split ratio. A high split ratio is preferred to ensure that the excess sample solvent vapors are quickly ejected from the inlet to avoid an oversize and tailing solvent peak.


In split and splitless injection it is sometimes advisable to apply a higher inlet pressure during the sample introduction phase to reduce backflash (inlet contamination due to overspilled sample vapor) or to increase injection volume for increased sensitivity. The inlet pressures and flows, as well as the time for the increased pressure phase are very important in determining the quantitative accuracy of the method, and these parameters are typically empirically optimized, based on recommended manufacturers settings. The phasing of the pressure reduction with any splitless time is also crucial. It is good practice to lay out all of these variables in a table within the method and to optimize them carefully to avoid poor analyte peak shapes and very poor quantitative reproducibility, especially for early eluting analytes.


This is the time between the GC oven setpoint being reached and detected by the oven thermocouple and the instrument registering a “ready” state to begin the next injection in the sequence. This seemingly innocuous parameter is often ignored but can influence the selectivity and resolution of early-eluting compounds, and I’ve also known it to affect to the quantitative reproducibility. Essentially, just because the air within the GC oven has reached the setpoint temperature to begin the next analysis, the same is not necessarily true for the carrier gas flowing through the column, due to the thermal lag created by the capillary column walls and stationary phase. This is especially true for larger bore (internal diameter) columns and those with thicker stationary-phase films. If the carrier flowing through the column has not reached thermal equilibrium with the air within the oven, the temperature gradient effectively begins at the wrong temperature. This can affect critical processes governing analyte focusing and separation, resulting in irreproducibility. Always pay attention to this parameter and extend the equilibration time, if necessary, to correct any issues with peak shape (typically peak broadening of later eluting analytes) or retention time and resolution of earlier eluting analytes.

Initial oven temperature (splitless injection)


While all methods will cite the initial oven temperature within the temperature program, I wonder if we take the time to properly consider the importance of this parameter in splitless injection methods? To overcome gross peak broadening caused as the analyte slowly enters the column from the splitless inlet (analyte transfer typically occurs at the carrier gas flow rate), the analytes must be focused at the head of the GC column. This is usually achieved by condensation of higher boiling analytes and solvent trapping of the lower boiling analytes. Both processes are directly affected by the initial column temperature, and the general recommendation is that the initial oven temperature is 10–20 oC below the boiling point of the sample injection solvent to avoid peak broadening and shouldering. The time for which the initial oven temperature is held can also be crucial in determining peak shape in splitless injection and is typically empirically optimized. Do all your splitless methods follow the rules? Note that the column Equilibration Time (above) can also play a role in determining the peak shape in splitless injection and should also be empirically optimized.

Initial oven temperature and hold time (split injection)


Contrary to splitless injection, the mechanisms in split injection rely on faster analyte transfer from the inlet to the GC column and rapid onset of analyte migration through the capillary column. Any processes that retard the analyte progress risk the deposition and subsequent broadening of the analyte in the stationary phase film. Therefore, low initial oven temperatures and extended hold times at the initial temperature can affect both the peak shape and selectivity in split injection. These variables are sometimes highly influential of the quality of the separation and sometimes much less so, however, due consideration should be afforded during method development and troubleshooting.

GC column dimensions, carrier gas linear velocity, and head pressure


It’s good practice to cite not only the carrier gas type and flow rate but also the inlet pressure and resulting linear velocity of the carrier in your written methods. If the column dimensions or carrier gas type are accidently incorrectly entered into the instrument acquisition method, then the resulting required carrier head pressure and linear velocity of the carrier will be incorrect, and retention times will vary from those of the reference chromatogram or previous data. Knowing and, critically, checking the instrument head pressure and carrier gas linear velocity can be an excellent troubleshooting tool. The correct linear velocity and head pressure values can be read from the instrument prior to being written into the acquisition method during method development or validation.

Constant carrier gas pressure versus constant flow


Most modern GC instruments will offer the ability to maintain constant carrier gas flow by automatically increasing the head pressure applied during the temperature program. This avoids long elution times and broad peaks in the latter part of the chromatogram. One should ensure that the correct carrier mode is set in the instrument method, otherwise large discrepancies in retention time and peak shape will occur between the analysis obtained and reference chromatograms within the method, or previous chromatograms obtained under the correct carrier gas flow mode.

Variable FID makeup (constant column + makeup flow)


Many modern GCs will offer the ability to apply an increasing amount of Flame Ionization Detector (FID) makeup gas to preserve good peak shape when constant pressure carrier gas flow mode is chosen. The increasing makeup flow ensures the column effluent gas is efficiently transferred into the detector and avoids peak broadening. Again, one needs to ensure that the correct “mode” for the makeup flow is selected in the instrument acquisition method.

MS transfer line/source/analyzer temperatures


I think it would be fair to say that some of the most overlooked variables in GC are the MS detector temperatures, especially when it comes to verification of the temperatures between the written method and the instrument acquisition set points. Perhaps this is because these are often considered “lock and leave” variables. Once a reasonable set of parameters are obtained, they are left without optimization between one method and the next. Perhaps the factory settings or manufacturer-recommended temperatures are never changed. However, there are many circumstances in which these temperatures can have a large effect on the qualitative and quantitative performance of the detector, including the analysis of either very low or very high boiling compounds, when changing EI source conditions, ionization mode, or when switching between carrier gases. Again, it is very important that the temperatures cited in the written method are verified in the instrument acquisition method.


The thermal conductivity detector (TCD) is highly sensitive to gas type and flow rate through the reference and column effluent channels. Typically, the reference gas flow rate is optimized as a ratio to the carrier gas plus the makeup gas flow rate. Your manufacturer should provide information on the optimum ratio based on carrier gas flow rates. This ensures that the optimum detector response is obtained when the reference and column effluent flows are compared via the resistivity of the heated filament within the detector. If the reference gas type is not the same as the makeup and carrier gas, then meaningless measurements will be made. The reference and makeup flows and gas types should be specifically cited within the written method.


Simply put, the sensitivity of the ECD detector is directly affected by the makeup or anode purge flow rate and gas type. When the makeup gas type is different from the carrier, then typically the makeup gas flow should be at least three times that of the carrier flow. Lower makeup gas flows will increase the sensitivity of the detector and should be adjusted according to manufacturers guidelines, with higher makeup gas flows required for very sharp peaks to avoid peak broadening. The makeup gas type and flow rates should be clearly specified within the written method.

NPD air and makeup flow rates and bead type/voltage


Modern Nitrogen Phosphorous detectors use two different bead types, the so-called ceramic and Blos beads. Each of these heating bead types require different applied voltages and makeup gas flow rates for optimum sensitivity and performance. The air flow into the detector is typically much lower than that used for FID detectors to prevent the formation of a flame withing the detector. Any written methods should contain information on the bead type to be used, the required bead voltage, and the detector gas types and flow rates.

Who knew there was so much to properly specifying, understanding, and troubleshooting gas chromatography methods? I’ve cited a mix of variables, some of which are typically written into GC specification methods and a lot of which are typically omitted. In my experience, we need to specify as many variables as possible to assist with both data acquisition and instrument and method troubleshooting. An awareness of the existence and effect of each variable is critical to method development, optimization, and troubleshooting.

Tony Taylor is the Chief Scientific Officer of Arch Sciences Group and the Technical Director of CHROMacademy. His background is in pharmaceutical R&D and polymer chemistry, but he has spent the past 20 years in training and consulting, working with Crawford Scientific Group clients to ensure they attain the very best analytical science possible. He has trained and consulted with thousands of analytical chemists globally and is passionate about professional development in separation science, developing CHROMacademy as a means to provide high-quality online education to analytical chemists. His current research interests include HPLC column selectivity codification, advanced automated sample preparation, and LC–MS and GC–MS for materials characterization, especially in the field of extractables and leachables analysis.

Articles

I’d like to concentrate on variables that can really impact our chromatography, but may be on hidden, supplementary, or advanced pages of our software, or may appear on the main software acquisitions menus, but are poorly understood or rarely altered. These variables are often not specifically referenced in laboratory methods documents or, if they do appear, are poorly understood. 

The LCGC Blog: Killer Gas Chromatography Variables—and Other Insidious Ways to Destroy your Chromatography!

Kevin A. Schug is a Full Professor and Shimadzu Distinguished Professor of Analytical Chemistry in the Department of Chemistry & Biochemistry at The University of Texas (UT) at Arlington. He joined the faculty at UT Arlington in 2005 after completing a Ph.D. in Chemistry at Virginia Tech under the direction of Prof. Harold M. McNair and a post-doctoral fellowship at the University of Vienna under Prof. Wolfgang Lindner. Research in the Schug group spans fundamental and applied areas of separation science and mass spectrometry. Schug was named the LCGC Emerging Leader in Chromatography in 2009, and most recently has been named the 2012 American Chemical Society Division of Analytical Chemistry Young Investigator in Separation Science awardee.

This blog is a collaboration between LCGC and the American Chemical Society Analytical Division Subdivision on Chromatography and Separations Chemistry.

The past year has been a remarkable ride for the oil and gas industry, and for the entire energy sector in general. Extreme volatility in commodity pricing, an oil contango, and an apparent paradigm shift away from fossil fuel-powered vehicles—thanks to Tesla—has all been “par for the course” in the past 12 months. Fortunately, the petroleum industry appears to be making significant strides to improve the environmental stewardship and operational efficiency of energy production by implementing new technologies and capitalizing on previously wasted resources like produced water and flare gas. Nonetheless, many questions still remain about the management of aging or even abandoned infrastructure because abandoned production wells provide a potential for undocumented contamination events. More specifically, how can we monitor the environmental impacts of these neglected sites, and will these impacted sites grow in magnitude over time? To answer these questions, we must first understand what is required to perform such analyses.

Having contributed to numerous high-profile litigations involving alleged oil and gas-related contamination, we never cease to be amazed at how intricate and idiosyncratic these cases can be. A thorough account of historical events and the chronology of nearby anthropogenic activities is certainly helpful, but this information does not tell us about the extent of the problem from an environmental or ecological perspective; and it generally doesn’t provide enough resolution to successful identify the point source. For this latter function, we turn to analytical chemistry to help guide us towards a successful outcome, a process known as 

In many cases, a relatively straightforward analysis of the environment can provide sufficient insight into the extent of environmental damage from a specific source (1). In the case of alleged groundwater contamination, using ion chromatography and inductively coupled plasma–mass spectrometry (ICP-MS), we can characterize various brine elements (chloride and bromide) and pertinent metal ions species, respectively, to better under the extent of fluid migration from flowback and produced water. Concurrently, gas and liquid chromatography–mass spectrometry (GC–MS and LC–MS) can be utilized to characterize the presence of volatile and semi-volatile organic compounds, which collectively can be representative of chemical additives used in the energy production process (that is, hydraulic fracturing) and be a reflection of other natural gas constituents ( C1-C5 hydrocarbon gases) being present. In the event that natural gas is present, as may be evidenced by a strong sulfur-bearing aroma or water effervescence, the characterization of methane, ethane, and propane, and their respective carbon isotopes, can provide insight into the origin of the rogue gas and help differentiate between naturally occurring biogenic (microbial) gas and thermogenic gas that may have made its way into the water via anthropogenic activities.

This is where things get very interesting. In numerous cases, we have seen that the isotopic signature of the rogue natural gas in a water source is a match with the equivalent signature collected from a nearby production well. Case closed, right? Not exactly. In some instances, there may also be a naturally occurring source of shallow gas that is not economically viable to extract, and that has a comparable isotopic signature to the two other thermogenic gases. In situations like these, higher-resolution analytical tools are required to parse out the differences. For example, the relatively recent emergence of noble gas and noble gas isotopic analyses has allowed investigators the opportunity to better perform point source attribution, and simultaneously characterize the migration pathway from the contamination source to the contamination site (2,3). Because noble gases are chemically inert and are not subject to oxidation and microbial degradation, they are ideal tracer elements that can be used to better understand subsurface fluid and gas migration. Using the aforementioned case with three matching methane isotope signatures, the characterization of noble gas analytes allowed us to differentiate water wells that had been inadvertently drilled directly into the intermediate gas layer from those that has been impacted by the activities of a nearby production well (4).

Another tool that is adding a lot of value to environmental investigations, particularly groundwater and surface water analyses, is matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). This particular tool has made a tremendous impact in the clinical setting as a way to screen through a large library of bacterial species that can have deleterious effects to susceptible or immune-compromised patients. MALDI-TOF-MS is now making a big splash in the environmental realm with the ability to screen samples for the presence of thousands of different microbes (5). Our research across Texas using MALDI-TOF-MS has demonstrated the value of comprehensive microbial analyses as a way to characterize bacterial ecology, which can be representative of the current contamination state (6,7). And although the aforementioned noble gas analyses are arguably the most powerful weapon in the environmental arsenal, MALDI-TOF-MS microbial analysis is much more rapid (<48 h) and can detect various indicator species that can suggest the presence or absence of atypical environmental conditions. For example, the detection of extremophilic or halophilic species that are not native to typical, healthy groundwater conditions can be indicative of a much larger problem that may warrant further investigation.

Unconventional oil and gas extraction is a multi-faceted landscape. From assessment of air, soil, and water quality to oilfield wastewater treatment and re-use, there are many places where both routine and advanced chemical analysis can provide insight. Even as some favor the rapid transition to renewable energy resources, the extraction and use of fossil fuels will continue as a pertinent piece of the energy supply puzzle for the foreseeable future. We will continue to be faced with situations where environmental forensics and point source attribution are needed to understand the cause of various contamination events. Even if fossil fuel extraction declines significantly over the next 20 years, we will still be left with the need to ensure abandoned well sites are properly closed and pose no threat to the environment. We have the technology and the know-how, but of course, there are still real limits in the research that can be performed with limited to no investment in such studies from traditional funding agencies.

I.C. Santos, Z.L. Hildenbrand, K.A.Schug, 

T.H. Darrah, A. Vengosh, R.B.Jackson, N.R. Warner, and R.J. Poreda, Proceedings of the National Academy of Sciences, 

C.J.Whyte, A. Vengosh, N.R. Warner, R.B. Jackson, K. Muehlenbachs, F.W. Schwartz, andT.H. Darrah, 

Z.L. Hildenbrand, D.D Carlton Jr., A.P. Wicker, S. Habib, P. Stigler-Granados, K.A. Schug, 

I.C. Santos, Z.L. Hildenbrand, K.A. Schug, 

M.S. Martin, I.C. Santos, D.D. Carlton Jr., P. Stigler-Granados, Z.L. Hildenbrand, K.A. Schug, 

I.C. Santos, M.S. Martin, M.L. Reyes, D.D. Carlton Jr., P. Stigler-Granados, M.A. Valerio, K.W. Whitworth, Z.L. Hildenbrand, K.A. Schug, 

Zacariah L. Hildenbrand is a partner of Medusa Analytical. He sits on the scientific advisory board of the Collaborative Laboratories for Environmental Analysis and Remediation (CLEAR), is a director of the Curtis Mathes Corporation (OTC:TLED) and is a research professor at the University of Texas at El Paso. Hildenbrand’s research has produced more than 60 peer-reviewed scientific journal articles and textbook chapters. He is regarded as an expert in point source attribution and has participated in some of the highest profile oil and gas contamination cases across the United States. Hildenbrand has also provided consultation for several private-sector clients on various water-treatment and hydrocarbon-capturing technologies.

Kevin A. Schug is a Full Professor and Shimadzu Distinguished Professor of Analytical Chemistry in the Department of Chemistry & Biochemistry at The University of Texas (UT) at Arlington. He joined the faculty at UT Arlington in 2005 after completing a Ph.D. in Chemistry at Virginia Tech under the direction of Prof. Harold M. McNair and a post-doctoral fellowship at the University of Vienna under Prof. Wolfgang Lindner. Research in the Schug group spans fundamental and applied areas of separation science and mass spectrometry. Schug was named the 

Emerging Leader in Chromatography in 2009 and the 2012 American Chemical Society Division of Analytical Chemistry Young Investigator in Separation Science. He is a fellow of both the U.T. Arlington and U.T. System-Wide Academies of Distinguished Teachers.

 and the American Chemical Society Analytical Division Subdivision on Chromatography and Separations Chemistry (ACS AD SCSC). The goals of the subdivision include

promoting chromatography and separations chemistry

organizing and sponsoring symposia on topics of interest to separations chemists

developing activities to promote the growth of separations science

increasing the professional status and the contacts between separations scientists.

For more information about the subdivision, or to get involved, please visit 

https://acsanalytical.org/subdivisions/separations/

The petroleum industry appears to be making significant strides to improve the environmental stewardship and operational efficiency of energy production by implementing new technologies and capitalizing on previously wasted resources like produced water and flare gas. Nonetheless, many questions still remain about the management of aging or even abandoned infrastructure because abandoned production wells provide a potential for undocumented contamination events.

The LCGC Blog: The Analytical Arsenal for Point Source Attribution

Examination of extractables and leachables (E&L) is of great importance to patient protection and regulatory requirements. Accurate and rapid identification of unknown compounds in E&L is often difficult due to the wide range of additives, oligomers, and degradation products from the plastic material. Oligomers often exist at high percentages in extracted samples, making them a critical aspect of data analysis. Furthermore, oligomers can exhibit either linear or cyclic forms with a variable ratio of composing monomers, making it difficult to identify oligomers with a general formula. Toward this end, we propose a screening method for categorizing polymeric oligomers, utilizing their common fragment structure ions. First, a total ion chromatogram (TIC) of samples in both MS and elevated energy MS (MS

) mode are acquired. Next, oligomers’ characteristic fragment ions with known mass ion values, are extracted to give extracted ion chromatograms (EICs). These oligomer peaks are quickly categorized and identified without library matching. Known polyethylene terephthalate (PET) fragments including [OCC

 were utilized. Out of a total of nineteen extractable peaks, thirteen PET oligomers were identified with this oligomer screening approach. This new approach greatly optimizes the accurate identification of categorized oligomers and enables a more precise E&L safety assessment.

The implementation of single-use technology (SUT) has been a growing trend across the field of pharmaceutical production, due to cost effectiveness and operating efficiency advantages over traditional stainless-steel systems. At the same time, extractables and leachables (E&L) from plastic single-use components pose risks of leaching into solutions, potentially compromising process conditions and safety for the patient. Bis(2,4-di-tert-butylphenyl) phosphate (bDtBPP), a common antioxidant additive in plastic bioreactors, showed toxicity to cell culture growth. Levels as low as 1 mg/L of bDtBPP in solution led to over 90% cell growth reduction (1). E&L are getting more attention in the biopharmaceutical industry, to the point where general requirements for E&L safety assessments have been mandated by regulatory agencies. USP (2) and BioPhorum Operations Group (BPOG) (3) have published standardized extractables protocols and industry guidelines on E&L testing and safety assessments. With increased concern and regulation, E&L testing systems and safety assessments are being developed by pharmaceutical industry members and CRO companies (4,5). As raw materials and manufacturing processes of single-use components and systems change, it is critical to evaluate the safety profile and potential impacts of new E&L compounds.

As a supplier and integrator of SUT, Pall Corporation takes on increased responsibilities to ensure delivery of both high quality and safe materials to end-users and patients. We established a standardized E&L testing protocol and risk assessment strategy (6–8) based on USP and BPOG guidelines. This protocol covers the extract sample preparation, extraction condition, data recording, and results reporting. A full analytical chemistry testing procedure has been created to analyze organic volatiles and non-volatiles, as well as trace elements. This procedure includes ultraviolet-visible spectrophotometry (UV-vis), Fourier transform infrared spectroscopy (FT- IR), gas chromatography‒mass spectrometry (GC–MS), liquid chromatography‒mass spectrometry (LC–MS), and inductively coupled plasma‒mass spectrometry (ICP-MS).

An array of polymer resins is used for SUT including polyethylene (PE), polypropylene (PP), polycarbonate (PC), polyethersulfone (PES), silicones, and fluoropolymers. Typical E&L are from plastic additives, rinsing solvents, colorants, lubricants, and other compounds related to the materials of construction in single-use components. With such a large pool of E&L compounds, an extensive internal library is needed for analytical screening. As single-use components and systems contain plastics, sterilization procedures, such as heat and gamma irradiation, can cause degradation to these materials. These processes produce oligomers, such as nylon 6,6 oligomers and polyethylene terephthalate oligomers (Figure 1). The degraded compounds may end up in the process solution and pose risks to drug potency and safety.

Figure 1: Examples of oligomers/degradants from plastic resins: (a) polyethylene terephthalate oligomers; (b) nylon 6,6 oligomers.

After gamma irradiation or heat sterilization of the single-use components, these degraded oligomers bear a variety of functional groups. In some cases, there are no comprehensive E&L libraries nor literature resources for these oligomeric structures. Additionally, an interference is seen between the oligomers and other plastic additives in chromatography. All these factors make compound identification and structure elucidation of the oligomers challenging. With the structures of certain E&L compounds remaining uncertain, suppliers and drug manufacturers expend more resources on advanced analytical data, E&L safety assessments, and toxicological studies with patient safety and process efficacy at the forefront. Here, we propose an oligomer screening approach to group and identify these oligomeric compounds based on type of polymer and fragment ions. Unequivocal identification of specific oligomers against the library or by structure elucidation is conducted in the subsequent step. This new approach greatly optimized the accurate identification of categorized oligomers. It increases confident in identification to facilitate toxicological and safety risk assessment of oligomeric extractables. A screening of PET oligomers in a filter extracted sample is used as a case study to demonstrate this approach.

A 50% ethanol solution was selected as extraction solvent for a nylon filter with polyester drainage and support layer. It is an aggressive extraction solvent, where high level of oligomeric species has been found in the extracted samples. The majority of these oligomers come from the filter membranes or the support and drainage layer materials, as they constitute the largest surface area in contact with the extraction solvent. The extraction system is set up with temperature control and proper thermal isolation (Figure 2). Both the pump and tubing are made from inert materials to avoid introducing contamination. The extraction solvent was recirculated through the filter and the system. A separate extraction was concurrently performed without filters to generate a negative control. The extract samples are then injected to Waters Xevo G2S QTOF MS (UHPLC) for further analysis. Chromatograms with MS and MSE information under low and high collision energy (CE) are acquired. The high CE chromatogram with MSE provides us fragmentation details of the analytes.

Figure 2: Pall extraction set-up (PFA= perfluoro-alkoxy alkane); figure from reference (6).

Polyethylene terephthalate (PET) is one of the most common thermoplastic polymer resins of the polyester (PE) family (9), composed of repeating unit C

 (192.0423 Da). As we investigated the mass spectrometry profile of the 50% ethanol extract sample, thirteen structures in a series of PET oligomers have been proposed based on accurate mass of parent ions and fragment ions summarized in Table I with literature backup (10). The mass error between the measured value and calculated value was within 2 mDa. PET cyclic trimer [TG]

 was found at 6.80 min with parent ions of [M+H]

 under low CE mode. Fragment ions of [TG]

 were found with high intensity in the high CE mode (Figure 3). PET cyclic dimer with one ethylene glycol unit inserted into the PET cyclic dimer ([TG]

 adducts under low CE mode, with identical C

 fragments in high CE mode. These three fragments were also seen in several other peaks under high CE mode. A 2 μg/mL PET cyclic trimer [TG]

 authentic standard was spiked and injected. The presence of C

 fragments have further confirmed them as characteristic fragments of PET oligomers.

-G in 50% ethanol extract sample under high collision energy (CE) mode. The x-axis label is observed mass (m/z) and Y-axis is intensity (counts).

Knowing these characteristic fragments, we can rapidly screen out PET oligomers in extract samples of filters comprising its polymer resin. An example is shown in Figure 4. Extracted ion chromatograms (EICs) were obtained by extracting C

 characteristic fragment ions (±0.5 Da range) in the chromatogram, respectively. Comparing the TIC with the EICs, these peaks between 4.8 and 6.6 min in TIC (in green) were shown to have intensive C

 fragment ion peaks in EIC under high CE mode. This comparison demonstrates that they produce characteristic fragments of PET oligomers and bear the oligomeric functional groups in structure. These compounds were then identified to be 

. Further library matching of parent ions and structure elucidation was conducted in a subsequent step to unequivocally identify each PET-related oligomer. Additionally, this oligomer screening approach is not limited to PET oligomers; it also applies to siloxane and nylon oligomers (11).

Figure 4: Chromatograms of an extract sample of filter comprising PET resin: (a) total ion chromatogram under low collision energy (CE); (b) extracted ion chromatogram (EIC) of 149.0231 

A stream map of this oligomer screening approach has been proposed in Figure 5. First, a TIC of samples in both MS and MSE mode are acquired. Second, oligomers’ characteristic fragment ions are proposed from polymer structure or looked up in literature. With known mass ion values of the characteristic fragment ions, EICs can be obtained. The peaks in EIC may suggest the presence of oligomeric functional groups in structure. Further identification of the compounds can be conducted with the help of library search and structure elucidation. If proposed library matches yield incomplete or inconsistent results in the subsequent step, these oligomer peaks can be quickly categorized and identified based on family of polymers, even though it is not an unequivocally ID of each PET oligomer. This new approach significantly decreases processing time on oligomer screening, and optimizes the accurate identification of categorized oligomers. It yields thorough screening and accurate identification of E&L compounds, and then provides meaningful E&L risk assessments. Furthermore, a good E&L risk assessment gives SUS suppliers and their customers confidence in their product portfolio.

Figure 5: A stream map of oligomer screening approach.

Chemical safety risk assessment is the process by which the potential adverse patient safety impact of E&L is determined and quantified. A patient’s total daily intake (TDI) level is determined by considering E&L exposure from the clinical use of the drug product. Based on the leachable’s toxicity, a permitted daily exposure (PDE) is established. If the TDI is less than the PDE, the leachable is deemed to be safe. Equations 1 and 2 above show examples of a safety assessment on PET related oligomer with a total of 4 μg/ mL in the extract sample from an extractables study. The sample was extracted from one filter at 30 °C for 24 h, which brackets end-users process conditions for manufacturing of a drug product intended for 10 mL/day administration. TDI was calculated considering the design of the extraction study, dilution volume in the manufacturing process, and daily dosage (2). Toxicologists determine the PDE for human exposure based on available toxicological information, body weight, and applicable modifying factors (12). In this example, the PDE of a PET oligomer is 2000 μg/day, which is significantly higher than the estimated TDI 16 μg/day. The margin of safety (PDE/TDI) is greater than 1 for a PET-related oligomer identification, yielding a negligible adverse impact. If this compound were an unknown, it would have to be identified as higher than acceptable safety concern thresholds (for example, 1.5 μg/day).

With this oligomer screening approach, 13 PET oligomers and 3 nylon oligomers were identified successfully by extracting common fragments. These oligomer peaks are quickly screened and categorized before library matching. This new approach greatly optimized the accurate identification of categorized oligomers. It enables a more precise and meaningful E&L safety assessment. Our future work is to expand this oligomer screening approach to a wider range of polymeric materials and build a comprehensive E&L library of oligomers from SUT.

The authors would like to thank Gilbert Tumambac for helpful technical reviews of the manuscript. The authors are also grateful to Wenan Lu, Emily Volk, Kuang-Wei Yang and Jin Ren for their contributions in the early stages of this project. The authors would like to give a final thanks to Rebecca Kale for facilitating the revision process.

(1) K. Fujimori, M. Hammond, J. Liu, H. Lee, M. Ronk, H, Nunn, and Y. Samuel, 

United States Pharmacopeia General Chapter <USP 665> Plastic Components and Systems Used to Manufacture Pharmaceutical Drug Products and Biopharmaceutical Drug Substances and Products

BioPhorum Best Practices Guide for Extractables Testing of Polymeric Single-use Components Used in Biopharmaceutical Manufacturing

(5) I. Paul; S. Dorey, M. Barbaroux, B. Lagrange et al., 

(6) G. Tumambac, B. Song, C. Xing, G. Li, W. Lu, L. Liu, C. Shih, and J. Hathcock, 

(7) G, Tumambac, B. Song, X. Li, G. Li, C. Shih, and J. Hathcock, 

(8) W. Ding, G. Madsen, E. Mahajan, S. O’Connor, and K. Wong, 

Polymer Science: A Comprehensive Reference

 (Else- vier, Waltham, Massachusetts, 2012), pp. 311‒328.

(11) T. Abe, L. Ackerman, M. Mutsuga, K. Sato, and T. Begley, 

 are with Pall Corporation in Westborough, Massachusetts. Direct correspondence to 

Analysis of extractables and leachables (E&L) from plastic packaging is of great importance for pharmaceutical product safety. Accurate and rapid identification of unknown compounds in E&L is often complex and challenging. To address this challenge, we demonstrate a quick method for oligomer determination using LC–QTOF-MS.

A Quick Approach to Screen Oligomers from Extractables Studies Using Liquid Chromatography–Quadrupole Time-of-Flight Mass Spectrometry (LC–QTOF-MS)

Photoionization has long been used to probe molecular structure on a fundamental level, but it has also been used in many mass spectrometry applications. This overview describes the mechanisms of and technical requirements for molecular photoionization based on single photon, multiphoton, and ultrashort pulse strategies under analyte conditions ranging from vacuum to atmosphere. One application of photoionization is the use of laser-based single photon ionization to enhance the signal while minimizing differential detection in analyses that probe solid samples by laser desorption or ablation. This laser desorption postionization method offers the possibility of foregoing matrix application to the sample while achieving higher lateral resolution. Results are presented for femtosecond (fs) laser desorption postionization mass spectrometry (LDPI-MS) using 7.9 eV single photon ionization for the detection of drug compounds that have previously been found to have low ionization efficiencies by secondary ion mass spectrometry. Geological applications of LDPI-MS are also discussed. Finally, a few commercial implementations are mentioned.

Short pulse lasers with a wide range of wavelengths have been popular in mass spectrometry for desorption/ablation of solid samples. Supplementing these methods with laser postionization increases ionization yields, improves lateral resolution in mass spectrometry imaging, and generally expands analytical opportunities.

Popular Methods in Mass Spectrometry (MS) Imaging

A major goal in mass spectrometry (MS) imaging is analyzing sub-micrometer features and integrated depth profiling, but these are limited by the trade-off in established methods between chemical information and lateral resolution (1). The most widely used MS imaging technique is matrix assisted laser desorption–ionization (MALDI) (2), which ionizes samples by mixing them with an organic matrix, then irradiating the resultant film with nanosecond (ns) laser pulses. Typical lateral resolution in MALDI is ~20 μm, with no capacity for in situ depth profiling, although a new implementation of atmospheric pressure MALDI has improved the resolution to a few micrometers (3). Although less popular than MALDI, secondary ion mass spectrometry (SIMS) was the first MS imaging method to demonstrate molecular imaging and permit depth profiling (4). Pulsed cluster ion beam sources have pushed the lateral resolution in SIMS of molecular species to slightly below one micrometer. Nano-SIMS uses atomic ion probes that enable much higher lateral resolution of ~50 nm, but is limited to the detection of only atomic ions, CN-, and other small fragment ions that nevertheless have been used for sophisticated molecular analyses (5). Desorption electrospray ionization (DESI) has been limited to ~10 μm lateral resolution without any capability for direct depth profiling (6). MALDI, SIMS, and DESI have all been shown to be quite powerful for MS imaging, but they also suffer from various shortcomings that can sometimes be addressed by the addition of laser postionization (4,7–9).

Volatilizing Analytes by Laser Desorption or Ablation

The molecular analysis of tissue, biofilms, and synthetic organic films can be achieved by laser desorption or ablation coupled with laser postionization to facilitate MS imaging with enhanced detection of neutrals (8,9). We begin by considering the initial step, where the term laser desorption (LD) generally refers to a lower energy emission of analyte into the gas phase whereas the term laser ablation (LA) refers to an explosive ejection. It should be noted that LD and LA are often used interchangeably by the MS community (for example, LD and LA are used interchangeably in MALDI, which proceeds by a more explosive mechanism).

The mechanism of analyte volatilization in LD, LA, MALDI, and laser desorption ionization (LDI) vary with wavelength, fluence, and pulse length, along with the optical properties of the sample (8,9). Pulse lengths can vary from ~10 ns to less than 100 fs; all can be used for ablation or desorption with varying results. Mechanisms depend in part on the optical properties of the target: ultraviolet nanosecond–laser desorption (UV ns-LD) of metal targets often proceeds by laser-induced thermal desorption whereas semiconductor targets can additionally undergo desorption induced by electronic transitions. MALDI most commonly employs UV ns-LD, but proceeds by a more complex desorption–ionization mechanism.

A more universal ablation occurs when utilizing ultrashort, <100 fs laser pulses that can ablate any material regardless of its optical properties while imparting little to no thermal damage to the underlying sample (8,10). These unique characteristics of femtosecond laser ablation (fs-LA) have led to several popular applications. Sub-100 fs lasers are used to perform fs-LA on biological cells and tissues in laser surgery with minimal damage to the adjacent tissue (10). Inductively coupled plasma–mass spectrometry (ICP-MS) also sometimes employs fs-LA to volatilize geological and biological samples for elemental analysis (11). Finally, fs-LA has been coupled to gas chromatography–mass spectrometry (GC–MS) for analyzing petroleum-bearing fluid inclusions (12). These prior applications of fs-LA have inspired its use in sampling for MS imaging, as described below.

Ion Formation and Introduction to Postionization

We have so far ignored the process of ion formation in LD, LA, MALDI, and LDI. Laser desorption, ablation, and ionization are inextricably linked in MALDI and LDI. MALDI and SIMS ion yields are typically <10-3 (4,8,9), leaving behind considerable sample undetected and limiting lateral resolution. MALDI also requires extensive and often complex sample preparation, including matrix application, that often varies with the sample type. Ion formation in other ns-LDI-MS strategies varies widely with the sample type and preparation (8). MS imaging with fs-LDI has also been demonstrated for lipids from pancreatic tissue (13), but limited molecular ion formation makes fs-LDI-MS most promising for elemental analysis (8,14).

LDPI-MS, also known by the terms two laser MS, two-step laser MS, or laser ablation laser ionization MS (7,8,15), is an MS imaging technique that uses one laser pulse to desorb or ablate molecules from a solid sample held under vacuum and a second, temporally delayed laser pulse to photoionize the resultant plume of gaseous species, as shown in Figure 1. Non-optical methods of postionization, such as electrospray, chemical, and plasma-based ionization, have also been coupled with LD and LA to enhance ion yields beyond what is possible with MALDI or ns-LDI (16).

Figure 1: Schematic representation of laser desorption postionization mass spectrometry (LDPI-MS). The purple arrow follows the path of a pulsed laser beam that desorbs a solid sample to volatile neutrals that are postionized by a pulsed laser, as represented by the green arrow.

Laser Postionization via Single Photon Ionization

A common optical postionization strategy has been single photon ionization (SPI), which can occur when the energy of an incident photon exceeds the ionization energy of a gas phase molecule (8). Most organic compounds have ionization energies below ~10 eV and will undergo SPI by irradiation with vacuum ultraviolet (VUV) light of a similar or higher photon energy. One trend within organic homologous compounds is that ionization energies decrease as molecular weight increases (15). SPI has a simple mechanism and relatively low limits of detection that can reach down to the mid-low parts-per-billion range (15).

It is instructive to compare SPI with electron impact (EI), as shown in Figure 2 as coupled to gas chromatography and for SPI, laser sampling. Both methods initially generate radical cations, M

, which have long been considered to be less stable than the protonated molecules, MH

, typically generated by electrospray and chemical ionization. For decades, EI has been used for identifying molecular species by comparison of the mass spectral fragmentation patterns of separated compounds to those of known compounds in EI-MS libraries (17). However, SPI has higher ionization efficiency than EI as well as lower fragmentation when performed with photons no more than a few eV above the ionization energy (15). This may partially explain why there have been few reports of postionization of molecular species by electron impact of molecules volatilized by LD or LA.

Figure 2: This schematic shows formation of a radical cation precursor via (a) electron impact (EI) and (b) single photon ionization (SPI). The diagram represents either gas chromatography (GC) or in the case of SPI, laser desorption, to volatize neutrals for ionization. Figure used with permission from reference (7).

A recent study showed that three drug compounds that could not be easily detected by SIMS were observed using LDPI-MS, where ablation was performed with ~75 fs, 800 nm laser pulses, and SPI with ~7 ns, 7.9 eV laser pulses (see below) (18). The LDPI mass spectrum of imipramine displays higher intact precursor ion M

 compared with its corresponding EI mass spectrum in Figure 3. Furthermore, although the most intense peaks from EI-MS were replicated by LDPI-MS, the latter showed overall less fragmentation. The similarity between LDPI-MS and EI-MS was observed with the two other compounds successfully examined in this study (18). SPI-MS often displays major fragments as similar to those seen in EI-MS, but almost always displays the M

 that can be missing from EI-MS of labile compounds and additionally leads to a lower overall extent of fragmentation (19). This similarity implies that it may be possible to use EI-MS libraries to reliably interpret LDPI-MS performed with SPI.

Figure 3: Femtosecond-LDPI mass spectra of imipramine compared with EI mass spectra. Figure used with permission from reference (18).

The use of SPI for postionization in LDPI-MS is limited by the performance of sources of pulsed VUV radiation. The most convenient laboratory VUV source for LDPI-MS is the 157 nm fluorine excimer laser. Fluorine lasers are reasonably priced and reliable. Their intensities are in the range of ~1 mJ/pulse, allowing high efficiency detection via saturation of SPI. Furthermore, their pulse repetition rate is ~500 Hz, high enough for rapid imaging of relatively large sample areas. However, the fluorine laser photon energy is only 7.9 eV, generally limiting SPI to molecules such as polyaromatic hydrocarbons, tertiary amines, some fused ring drug compounds (15), and certain clustered ion species (20).

SPI of the wider array of organic compounds with higher ionization energies is possible with a VUV source that displays a correspondingly higher photon energy. Third harmonic generation of the 355 nm output from a standard ns pulsed Nd:YAG laser produces 118 nm wavelength (10.5 eV photon energy) laser pulses that serve as the more universal source for SPI alluded to above (15). However, this strategy produces <1 μJ/ pulse that cannot saturate SPI and leads to low overall ionization efficiency. The low repetition rates of the high power Nd:YAG lasers needed for VUV generation also limit imaging speed. Finally, the low intensity VUV beam is difficult to manipulate for inexperienced personnel and requires considerable engineered improvements before it can be readily used for general analytical applications.

Overall, expanding the use of SPI in LDPI-MS requires a more robust laboratory source of pulsed VUV radiation with ~10 eV photon energy, ~1000 pulse rate, and at least 100 μJ/pulse. High harmonic generation from ultrashort pulse lasers might one day fulfill these requirements if considerably higher intensity VUV pulses can be generated (21).

Postionization Using Multiphoton to Strong-Field Ionization

An alternative postionization to SPI is to instead utilize multiphoton strategies. SPI yields are linearly dependent on the number of photons, but resonance-enhanced multiphoton ionization (REMPI) becomes possible with exponentially higher laser intensities, in the range of 10

 (9,22). Furthermore, the excitation outlet pathway in REMPI proceeds through at least one intermediate excited state.

Figure 4a depicts the simplest form of REMPI: Two photon resonant ionization where one photon excites to an intermediate state via a strong optical absorption (which can be observed in UV-vis absorption spectroscopy), then a second photon excites out of the intermediate state to form a radical cation. An example of a resonant two photon ionization would be using 266 nm, ns laser pulses to excite a π-π* transition in a polyaromatic hydrocarbon like anthracene, then excite out of the π* state to form an ion. The two photons collectively impart 9.3 eV of energy to the polyaromatic hydrocarbon, well in excess of its ionization energy. The specific molecular electronic structure determines the types of REMPI pathways possible, the requisite laser wavelengths, and the extent of dissociation of the precursor.

Figure 4: Schematic of the two multiphoton ionization mechanisms: (a) resonance-enhanced multiphoton ionization (REMPI), (b) nonresonant multiphoton ionization (NRMPI) and (c) strong-field ionization, which can occur via ion tunneling.

REMPI is traditionally performed with ns-laser pulses, but transitioning to shorter laser pulses can reduce fragmentation that often occurs through other pathways out of the intermediate states (9,22). Femtosecond-laser MS is performed with pulses shorter than a picosecond that lead to less dissociation in REMPI and can open up new ionization pathways less dependent on specific molecular structure (23). Femtosecond laser MS can also proceed by non-resonant multiphoton ionization (NRMPI) when yet higher laser intensities ~10

 are used and has the added advantage that it does not require the laser wavelength to be resonant with an excited state (Figure 4b) (9).

The higher laser intensities that allow NRMPI, when accessed via sub-100 fs IR laser pulses, can also lead to strong-field ionization (9,24). Strong-field ionization can occur by tunneling through the barrier to ionization via high intensity electric field-induced distortion of molecular electronic states induced by ultrashort laser pulses (9). Pulse duration, laser intensity, wavelength, and molecular electronic structure control how a specific molecule will undergo strong-field ionization. FIGURE 5 shows how structural isomers can be quantified in mixtures using a dual pulse fs IR laser technique in which a pump fs laser pulse ionizes the mixture and creates a radical cation in a ground state, then a time-delayed probe laser pulse (from the same laser) induces dissociation (24). The nitrotoluene ion signal varies with pump-probe delay time for its structural isomers 

-nitrotoluene (ONT, MNT, and PNT, respectively). Preliminary results found that the relative concentration of each isomer in binary and ternary mixtures could be determined to better than 10% accuracy without any preliminary use of chromatographic separation. Such a strategy would be very powerful when applied to LDPI-MS imaging, which like most MS imaging methods lacks a chromatographic front end. Femtosecond-laser MS also potentially allows coupling with sophisticated computational modeling of the ionization event that might find predictive utility in analysis (24).

Figure 5: Time dependent pump-probe experiments used for quantification of structural isomers in a mixture. Nitrotoluene ion signal varies with pump-probe delay time for its structural isomers 

-nitrotoluene (ONT, MNT, and PNT, respectively).

Nanosecond-LDPI-MS has found applications in analysis of biofilms, tissue samples, organic electronics, and geological samples (7,8,15,16). For example, LDPI-MS was able quantify antibiotics in biofilms and differentiate biofilms of different strains of 

A version of LDPI-MS is under consideration for incorporation into space flight instruments being developed for analysis of icy moons, comets, and carbonaceous asteroids (25). The molecular analyzer for complex refractory organic-rich surfaces (MACROS) is an instrument package utilizing ns-pulse lasers that has been developed at the NASA Goddard Space Flight Center. One of MACROS’ four basic operation modes for in situ sample analysis uses ns pulsed 266 nm IR laser for desorption and a ns pulsed laser for postionization via REMPI. L2MS is considered a “detailed” mode in MACROS because it does not require sample preparation, has a lower limit of detection, and is highly sensitive to detection of aromatic organic molecules of great interest to the mission.

The application of LDPI-MS using fs-laser ablation is at an earlier stage (8), but has already been shown to allow imaging of an organic electronic compound at a lateral resolution of 2 μm by taking advantage of the nonlinearity of fs-LA with laser intensity (8). Alternatively, the application of experimentally robust fs laser postionization to LDPI-MS will allow faster imaging and analysis of a wide range of compounds. Femtosecond laser MS has been coupled with gas chromatography for the ultrasensitive analysis of explosives (23,24) and pesticides (26), indicating the promise of its use in postionization for LDPI-MS.

Postionization via Photoionization at Elevated Pressures

We have focused on LDPI-MS performed with the sample, desorption, ablation, and postionization all occurring under high vacuum. However, there are several advantages to performing the analogous analysis at atmospheric pressure (8). The sample does not dehydrate and does not need to undergo the slow evacuation process required in traditional mass spectrometers. The plume of species desorbed or ablated from a sample immediately collides with air molecules, which enhances dissociation of a larger species, slowing down molecular velocities and possibly leading to oxidation (this final event also possibly being a disadvantage). Atmospheric pressure photoionization is most commonly initiated with SPI by VUV radiation and subsequently leads to chemical–ionization-like events that can result in the formation of protonated molecules instead of radical cations (27). Such protonation processes show great potential for expanding the breadth of LDPI-MS applications and may have driven the application of non-laser based postionization (16). Finally, most commercial mass spectrometers currently have atmospheric pressure interfaces, so coupling a LDPI front end to them avails one of their exquisite performance in terms of mass resolution and accuracy as well as tandem capabilities.

Utilizing any analytical method relies on its commercial availability, but such options for LDPI-MS remain limited as of this writing. MALDI-2 is a method that uses a ns-pulse UV postionization laser to induce secondary ionization in the gas phase of plumes ablated by MALDI (28). Postionization in MALDI-2 occurs at intermediate pressures within an ion mobility device, leading to the declustering of higher mass species formed by MALDI. The pressure-dependent mechanism of MALDI-2 is probably more akin to that of atmospheric pressure photoionization than direct laser photoionization of a molecule isolated in vacuum. Perhaps foremost amongst the impressive advantages of UV laser postionization in MALDI-2 is that it enhances signal intensities of membrane lipids and other species detected in a tissue sample by up to two orders of magnitude over those observed by traditional MALDI. This higher signal also allows increased lateral resolution for MS imaging and Bruker is now selling an instrument with a MALDI-2 option.

Another two-laser commercial instrument currently in development by Exum Instruments is laser ablation laser ionization time-of-flight mass spectrometry (LALI-TOF-MS) (29). LALI-TOF-MS utilizes a 213 or 266 nm ns-pulsed laser to ablate into a pressure gradient and a second 266 nm, ns-pulsed laser for multiphoton ionization. The LALI-TOF-MS instrument is initially being developed for elemental analysis but will permit identification of a range of molecular species that can be expanded with the modification of the laser postionization strategy.

The development of LDPI-MS and related methods will continue to be driven by the rapid improvement and lowered costs of sub-ns-pulsed lasers, but requires coupling to higher performance mass analyzers to increase user acceptance. Readers interested in a more rigorous discus- sion of laser postionization are referred to a recent book on the fundamentals and the entire range of applications of photo-ionization in mass spectrometry (9).

Luke Hanley and Teodora Zagorac are supported by grant CHE-1904145 from the U.S. National Science Foundation (LH ORCID: 0000-0001-7856-1869. TZ ORCID: 0000-0001-7467-8736).

(1) R. Trouillon, M.K. Passarelli, J. Wang, M.E. Kurczy, and A.G. Ewing, 

(2) A. Palmer, D. Trede, and T. Alexandrov, 

(3) S. Heiles, M. Kompauer, M.A. Müller, and B. Spengler, 

(4) I.S. Gilmore, S. Heiles, and C.L. Pieterse, 

(5) J. Nuñez, R. Renslow, J.B. Cliff III, and C.R. Anderton, 

(6) R. Yin, J. Kyle, K. Burnum-Johnson, K.J. Bloodsworth, L. Sussel, C. Ansong, and J. Laskin, 

(8) L. Hanley, R. Wickramasinghe, and Y.P. Yung, 

Photoionization and Photo-Induced Processes in Mass Spectrometry: Fundamentals and Applications

(13) A.V. Walker, L.D. Gelb, G.E. Barry, P. Subanajouy, A. Poudel, M. Hara, I.V. Veryovkin, G.I. Bell, and L. Hanley, 

(14) A. Cedeño López, V. Grimaudo, A. Riedo, M. Tulej, R. Wiesendanger, R. Lukmanov, P. Moreno-Garcia, E. Lortscher, P. Wurz, and P. Broekmann, 

, in press (2020) https://doi.org/10.1002/mas.21649

(18) C.L. Pieterse, I. Rungger, I.S. Gilmore, R.C. Wickramasinghe, and L. Hanley, 

(19) G. Isaacman, K.R. Wilson, A.W.H. Chan, D.R. Worton, J.R. Kimmel, T. Nah, T. Hohaus, M. Gonin, J.H. Kroll, D.R. Worsnop, and A.H. Goldstein, 

(20) M.T. Blaze, L.K. Takahashi, J. Zhou, M. Ahmed, G.L. Gasper, F.D. Pleticha, and L. Hanley, 

(21) T. Popmintchev, M.-C. Chen, D. Popmintchev, P. Arpin, S. Brown, S. Ališauskas, G. Andriukaitis, T. Balciunas, O.D. Mucke, A. Pugzlys, A. Baltuska, B. Shim, S.E. Schrauth, A. Gaeta, C. Hernandez-Garcia, L. Plaja, A. Becker, A. Jaron-Becker, M.M. Murnane, and H.C. Kapteyn, 

(25) S.A. Getty, J. Elsila, M. Balvin, W.B. Brinck- erhoff, T. Cornish, X. Li, J. Ferrance, A. Grubisic, and A. Southard, 

Molecular Analyzer for Complex Refractory Organic-Rich Surfaces (MACROS)

. in 2017 IEEE Aerospace Conference. 2017. https://doi.org/10.1109/ AERO.2017.7943706

(26) X. Yang, T. Imasaka, and T. Imasaka,

(27) T.J. Kauppila, J.A. Syage, and T. Benter, 

(28) J. Soltwisch, H. Kettling, S. Vens-Cappell, M. Wiegelmann, J. Müthing, and K. Dreisewerd, 

 are with the Department of Chemistry at University of Illinois at Chicago, in Chicago, Illinois. Direct correspondence to: 

Supplementing short pulse lasers with laser postionization increases ionization yields for desorption and ablation of solid samples in mass spectrometry. Here, we give an overview of the mechanisms and technical requirements for molecular photoionization in femtosecond (fs) laser desorption postionization mass spectrometry (LDPI-MS).

Laser Desorption Postionization Mass Spectrometry

Ion mobility separations are based on fundamental principles that differ from liquid chromatography (LC) in that several parameters of ionized molecules—size, charge, shape, and structure—come into play simultaneously. As a result, analytes that have the same molecular mass can be separated by their shape, charge and collision cross section (CCS). A new development in ion mobility separation, high-resolution ion mobility (HRIM), overcomes the problem of ion loss in other ion mobility separation techniques. HRIM also provides excellent structural resolution and high reproducibility. The technique is particularly well suited to challenging applications in the pharmaceutical and clinical fields, such as glycosylation monitoring of biological drugs and vitamin D analysis.

Truly disruptive advances catapult onto the scientific stage when a convergence between an emerging need and innovation occurs, thus thrusting a new technology into the limelight. High-resolution ion mobility (HRIM) based on structures for lossless ion manipulation (SLIM), originally invented in the laboratory of Dr. Richard D. Smith at Pacific Northwest National Laboratory (PNNL), provides the unprecedented capability to separate and identify molecular structures that are practically indistinguishable using traditional methods such as liquid chromatography (LC). Some of these inherently difficult to detect analyte classes include peptides, proteins, lipids and glycans. In fact, HRIM’s separation capability is powerful enough to separate and detect isobaric molecules; one particularly relevant example of this is glycan analysis of the novel coronavirus spike protein (1).

Classical analytical separation techniques, like LC, are often too slow, too experimentally challenging, or not powerful enough to resolve and structurally characterize molecules to meet the current demands for fast or high throughput commercial applications. HRIM offers an alternative that should be evaluated where appropriate to help meet the demand for fast, efficient analyses, including biologic therapeutic development and biomarker analysis.

Ion mobility separations are based on fundamental principles that differ from liquid chromatography, in that several parameters of ionized molecules—size, charge, shape and structure—come into play simultaneously. As a result, analytes that have the same molecular mass can be separated by their shape, charge and collision cross section (CCS).

Ion mobility separation has been available for decades, but there remains concern about ion loss leading to a reduction in sensitivity, the mass range per analysis is limited, and the length of the separation path is not sufficient enough to achieve high-resolution separation. The distinguishing feature of HRIM compared with other ion mobility separation techniques is that separations are achieved essentially losslessly on very long pathlengths implemented with serpentine electrode patterns on conventional printed circuit board (PCB) technology. Digitizing separations, so to speak, on PCBs with nearly limitless pathlengths addresses the ion loss and resolution limitations of incumbent technology. With HRIM technology, ion manipulation and separation are achieved by applying electric fields to electrodes on two parallel PCBs to create an ion conduct through which ions travel without striking physical surfaces and thereby avoiding neutralization and loss that are typical to other ion mobility technologies.

The serpentine separation path design packs a single pass 40-foot ion path (in the first commercial product) into a device about the size of a laptop, addressing the resolution limitations inherent to shorter pathlength ion mobility instruments, whether multi-pass cyclic or linear, and ultimately achieves performance that enables characterizations that were previously impossible. The long pathlengths achieved with HRIM, and the foundational separation mechanism of ion mobility technology, provide additional structural information not otherwise gleaned from other separation techniques.

The Differentiators Between High-Resolution Ion Mobility and Liquid Chromatography Mechanism of Separation

HRIM separates ions based on the difference in collision cross-section, size, charge density and overall shape. The sample of interest is dispersed in the gas phase using conventional electrospray ionization. As the ions enter the separation chamber, voltages applied to electrodes on the PCBs provide the lossless separation conduit through which the ions traverse along the separation pathlength. Separation is achieved because smaller CCS molecules travel faster than the larger CCS molecules and hit the detector first. Because CCS is an inherent physical property, highly reproducible results are achieved as long as pressure and temperature are controlled within the system. With separation occurring in the gas phase compared to the liquid phase with LC, separations and data collection are achieved in milliseconds not minutes or hours. Thus, dozens of samples could be processed in the time required for a single LC run.

Analyte Agnostic: No Column Changes for Greater Instrument Uptimes

“Those LC guys need to understand a bit of magic,” is the way this author has heard it phrased. LC often necessitates matching column size, length, and packing material to specific separations and their intended purposes. In many cases, columns are dedicated to particular types of analytes. For example, one set of protein samples will be run on a different column than a set of small molecule samples. Similarly, an operator might use different column materials (positively or negatively charged, polar or non-polar) to separate different types of molecules. LC very often relies on adjusting the solvent (liquid) system to the task at hand, which may require complicated calculations, trial runs, algorithms for step or gradient changes, and so on. In reality, scientists are running an entirely different set of experiments, each time, for each analyte type, complete with lengthy experimentation to validate the method development.

Scientists who work with LC are often extremely experienced operators, sometimes like a magician in their ability to utilize workarounds and nuances to overcome stumbling blocks and achieve successful results.

In contrast, HRIM allows scientists to resolve multiple classes of analytes without component changes, as there are no hardware changes to make. Less experience is required to run experiments, and often, with less than a week’s worth of training, the experiments can be up and running. In a laboratory setting where different sample types (such as glycans, peptides, proteins, small molecules) are the mainstay, the same HRIM instrument can be used across the board. Running samples of varying analytes back-to-back is practical and simple, significantly increasing laboratory productivity.

With HRIM, reproducibility and achieving consistent data collection depends less on the experience of the operator. HRIM achieves separation of ionized molecules based on the physical parameters of the analytes themselves.

Since HRIM does not make use of specific columns or solvent systems, and separations are analyte agnostic, there are fewer variables to control, and the process for running HRIM requires much less user input. HRIM technology lends itself to more reproducible results because the separation mechanism is an inherent physical property with fewer variables than LC based methods. Enabling more predictable and more reproducible results across different laboratories, LC’s reproducibility is highly dependent on the preparation of buffers, solutions, and samples, method development, solvent amounts, column, flow rate, and sample size can each affect the reproducibility of the results. In fact, data reproducibility has been the most consistent comment received from HRIM’s beta users.

To put the comparison simply, the biggest difference between HRIM and LC is the level of resolution that is achievable in a short amount of time. In situations where multiple molecules have similar elution times on the chromatogram, LC separation can take more than an hour to distinguish them effectively, whereas HRIM routinely achieves the most challenging separations in two to five minutes. HRIM eliminates the common tradeoff between speed for resolution or vice versa, resolution for speed. With HRIM you can have both speed and resolution without sacrifice.

HRIM outperforms in areas that have been notoriously challenging: specifically, lipid and glycan analysis. In fact, study of these biomolecular classes has suffered from the difficulty to closely follow their biosynthesis, structural distribution, and metabolism, largely due to isomeric natures and vast structural diversity (2–4).

HRIM has come to the forefront to meet these needs. Biosimilar development provides one example of the importance of glycan analysis, where in order to achieve regulatory approval, the biosimilar glycan profile must match the originator (5). The more detailed the glycan profiles for the purpose of comparison, the higher likelihood of approval.

Collectively, scientists using HRIM can achieve unprecedented glycan separation, including distinct elucidation of isomers and other molecules with highly similar composition. Advances in glycan characterization are more important now, more than ever, given the need to understand glycosylation of human and animal viruses such as influenza, HIV, and coronaviruses. HRIM is currently being used to detail the glycosylation microheterogeneity in the spike glycoprotein that decorates the surface of the SARS-CoV-2 (Covid- 19) viral capsid. It is expected that this information will aid understanding of the heterogeneity in glycosylation on this surface protein, help explain how the virus binds to its target, and aid the development of an effective treatment. This particular application of HRIM makes clear its enormous potential, especially in conditions where full resolution and fast analysis are necessary.

Figure 1: HRIM-MS produces higher quality data more quickly, resulting in savings for development costs and improvement of safety and efficacy for potentially faster regulatory approvals. Shown above, a 15-min LC–MS generated chromatogram of IgG control with N-glycan identifications noted, and the extracted ion mobility drift plots (or mobiligrams) from the 2-min HRIM–MS method demonstrating resolution of glycoforms not identified using the LC–MS method.

Method validation is the process of proving that an analytical method is acceptable for use. Verifying that an analysis procedure will accurately and consistently deliver a reliable measurement of an active ingredient in a compounded preparation requires that the method be specific, accurate, and precise over a usable range. Furthermore, it must have an acceptable limit of detection and quantitation, and be robust enough for the demands of the experimental conditions.

Software platforms can assist with method development for LC; however, trial runs and adjustments to a myriad of variables can consume hours, if not days. In the end, the method may not be valid for all sets of conditions, or all the desired analytes.

Using ion mobility, analytes that have the same molecular mass can be separated by their shape, centers of mass, and collision cross section, but challenges such as ion loss can still occur. A new development in ion mobility separation, high-resolution ion mobility (HRIM), addresses such problems, and is particularly well suited to challenging applications, such as glycosylation monitoring of biological drugs and vitamin D analysis.