Chemical synthesis of nucleic acids can result in a number of by-products, such as shorter length components and protecting groups. Therefore, adequate separation and purification of the target compounds is a crucial step in the production process. This article introduces an analytical method for the separation of oligonucleotides of different chain length by ion‑exchange chromatography (IEC) using a bio-inert ultrahigh-pressure liquid chromatography (UHPLC) system. The effect of changes in mobile phase pH due to CO2 absorption from ambient air during storage was also investigated.

Risa Suzuki received her Ph.D. at the University of Tokyo, Japan, in 2015, focusing on protein engineering and construction of a universal screening system for elucidating enzyme functions involved in drug metabolism. From 2015 to 2017 she worked as a postdoctoral research associate at Nagoya University and Tokyo Institute of Technology, working on a JST funded ALCA project on microbe reaction engineering. In 2017, she was employed as a project assistant professor in the School of Medicine at the University of Tokyo. Her research interests and expertise encompass the developing and manufacturing of biopharmaceuticals and crystal structure analysis of proteins. She now works for Shimadzu Corporation in Kyoto, Japan, as a member of the Healthcare Solution Unit in the Solutions COE.


Chihiro Hosoi graduated with a master’s of engineering from the Tokyo University of Agriculture and Technology, Japan, in 2019, focusing on interaction between nucleic acids and proteins and developing a biosensing system. In 2017, she studied graphene-based biosensors at the University of California, Riverside, USA. She now works for Shimadzu Corporation in Kanagawa, Japan, as a member of the Healthcare Solution Unit in the Solutions COE.

Chemical synthesis of nucleic acids can result in a number of by-products, such as shorter length components and protecting groups. Therefore, adequate separation and purification of the target compounds is a crucial step in the production process. This article introduces an analytical method for the separation of oligonucleotides of different chain length by ion‑exchange chromatography (IEC) using a bio-inert ultrahigh-pressure liquid chromatography (UHPLC) system. The effect of changes in mobile phase pH due to CO

 absorption from ambient air during storage was also investigated.

Antisense oligonucleotides are a variety of nucleic acid drugs, synthetic molecules with a chemical backbone structure composed of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) bases. Therapeutic oligonucleotides have become more prominent over recent years because they can be designed to accurately target a specific disease gene or its encoded messenger RNAs, providing the next generation of therapeutics. While having the targeted specificity of antibody therapeutics, they can also be synthesized chemically like “low-molecular‑weight” drugs, which reduces manufacturing costs compared to the biological process of protein-based biomedicines. Therapeutic oligonucleotides combine some of the best properties of “low‑molecular-weight” therapies and biopharmaceuticals.

Chemical synthesis of nucleic acid results in a number of by-products, such as shorter length components and protecting groups (1). Therefore, adequate separation and purification of the target compound is a crucial step in the production process.

This article presents an analytical method for the separation of oligonucleotides of different chain length by ion-exchange chromatography (IEC), assuming that shorter length components are impurities derived from the synthesis process. To avoid any adsorption of the metal-coordinating compounds to the wetted surface of the instrument, a bio-inert ultrahigh-pressure liquid chromatography (UHPLC) system was used. The effect of mobile phase pH on the separation was also investigated.

The sequence of the oligonucleotide to be analyzed is shown in Table 1. Target oligonucleotides in 20 mer and four sequences that were deleted from n-1 to n-4 on the 3’ terminus of the target were prepared as impurities derived from the synthesis. All of them were unmodified single-stranded DNA and were synthesized by a solid-phase synthesis (high performance liquid chromatography [HPLC] purification). These five sequences were diluted to 5 μmol/L with water, and an oligonucleotide mixture was prepared.

In ion-exchange chromatography, analytes are separated and eluted using a mobile phase of varying salt concentration or pH. In this analysis, an aqueous solution of sodium hydroxide (A) was used in a gradient with a solution of sodium hydroxide containing sodium perchlorate (B), to gradually increase elution strength for the analytes of interest. A detailed description of analytical conditions is listed below.

 System: Nexera XS inert (Shimadzu); column: 100 mm × 4.6 mm, 5-μm Shim-pack Bio IEX Q-NP (Shimadzu); mobile phase A: 10 mmol/L NaOH, mobile phase B: 10 mmol/L NaOH containing 1 mol/L NaClO

; time program: 25%B–32.5%B (0–15 min) " 100%B (15–20 min) " 25%B (20–25 min); flow rate: 0.8 mL/min; oven temperature: 30 °C; injection volume: 4 µL; detection: UV 260 nm.

Using these IEC conditions, separation occurred based on the difference in negative charge of the analytes, which was equivalent to the number of phosphate groups in the oligonucleotide. Consequently, shorter oligonucleotides eluted first. Figure 1 shows a chromatogram of a five-sequence oligonucleotide mixture; all compounds of interest were separated by their length.

Repeatability of the method was determined based on retention time and peak area from six successive analyses of the 16–20 mer oligonucleotide mixture shown in Table 1. With variation of ≤ 0.5% in retention time and peak area for all analytes, good repeatability of the assaywas established. Detailed results can be found in Table 2.

Sample Analysis: Oligonucleotides in the Presence of By-Products: 

A mixture of five oligonucleotides, at 5 μmol/L concentration each, was prepared (four of them were HPLC-purified while one was only desalted) and IEC results were compared to those of the mixture of all HPLC-purified nucleotides (Figure 2). The target oligonucleotides were completely separated from impurities such as free protecting groups and shorter length oligonucleotides.

Effect of Change in Mobile Phase pH on Retention:

 As described above, in the IEC separation of an oligonucleotide mix, the analytes were separated and eluted using a salt concentration gradient. In an aqueous mobile phase (pH ≥ 7), absorption of carbon dioxide from ambient air can change the effective concentration of a basic eluent due to the formation of carbonic acid (2). In IEC, where separation is based on the analytes’ differences in charge, even a slight change in the pH can have a significant impact on retention and hence the analytical results. It is therefore crucial to prevent possible changes in mobile phases pH, to ensure reliable, stable analytical performance.

In this study, results obtained using mobile phase stored in a bottle closed with a standard screw cap were compared to those achieved with a mobile phase from a bottle with a filter cap that suppresses evaporation of the solvent. Analyses were performed using freshly prepared mobile phase and after two days of storage, both using the conditions described earlier. A comparison of chromatograms obtained are shown in Figure 3. Figure 4 depicts the percentage of change in mobile phase pH during the two days of storage under the respective conditions.

As can be seen in Figure 3, when using a standard mobile phase bottle cap 3), the retention time increased by an average of 8% as the pH changed compared to that on the day of preparation of the mobile phase 1); when using a cap with a filter 2), the retention time did not change significantly. The use of a cap with a filter suppressed the change in the pH of the mobile phase, resulting in stable results, even after two days of mobile phase preparation. Also, the change in pH was measurable in the bottles with a standard cap, with a larger impact seen in mobile phase B containing sodium perchlorate, as shown in Figure 4.

A simple gradient method for the analysis of oligonucleotides by ion-exchange chromatography using a bioinert UHPLC system has been presented. Good separation of the oligonucleotides under investigation was achieved, both from one another and from impurities such as protecting groups and shorter length oligonucleotides, which are common by‑products from chemical synthesis. It was shown that even small changes in eluent pH did affect the analytes’ retention time. Therefore, it is important to carefully control the mobile phase pH to obtain reproducible, reliable results.

C. Hosoi and R. Suzuki, Shimadzu Corporation Application News No. 01-00227-EN (Shimadzu Corporation, February 2022).

Ion Chromatography, Third, completely revised and enlarged edition

graduated with a diploma in chemical engineering from the Technical University NTA in Isny, Germany, in 2004 and as a master of science in pharmaceutical analysis from the University of Strathclyde in Glasgow, UK, in 2005. She worked until 2006 as a consultant in HPLC method development and validation in an analytical laboratory of the FAO/IAEA in Vienna, Austria. She gained her doctorate for research in pharmaceutical sciences at the University of Strathclyde in 2010 and was employed as an HPLC specialist in the R&D department at Hichrom Ltd in Reading, UK, from 2009. Since 2013, she has worked as a HPLC product specialist and since 2015 as HPLC Product Manager in the analytical business unit of Shimadzu Europa in Duisburg, Germany

received her Ph.D. at the University of Tokyo, Japan, in 2015, focusing on protein engineering and construction of a universal screening system for elucidating enzyme functions involved in drug metabolism. From 2015 to 2017 she worked as a postdoctoral research associate at Nagoya University and Tokyo Institute of Technology, working on a JST funded ALCA project on microbe reaction engineering. In 2017, she was employed as a project assistant professor in the School of Medicine at the University of Tokyo. Her research interests and expertise encompass the developing and manufacturing of biopharmaceuticals and crystal structure analysis of proteins. She now works for Shimadzu Corporation in Kyoto, Japan, as a member of the Healthcare Solution Unit in the Solutions COE.

graduated with a master’s of engineering from the Tokyo University of Agriculture and Technology, Japan, in 2019, focusing on interaction between nucleic acids and proteins and developing a biosensing system. In 2017, she studied graphene-based biosensors at the University of California, Riverside, USA. She now works for Shimadzu Corporation in Kanagawa, Japan, as a member of the Healthcare Solution Unit in the Solutions COE.

Articles

An analytical method for the separation of oligonucleotides of different chain length by IEC using a bio-inert UHPLC system is presented.

IEC Analysis of Oligonucleotides and Evaluation of the Effect of Changes in Mobile Phase pH on Separation

This month we interview Alexandre Goyon, Senior Scientist in the Small Molecule Pharmaceutical Sciences Organization of Genentech, about his work focused on the online digestion and analysis of RNA molecules using immobilized RNase cartridges attached to a mDLC–MS system, and why the accurate sequencing of sgRNA is important.

This month we interview Alexandre Goyon, Senior Scientist in the
Small Molecule Pharmaceutical Sciences Organization of Genentech, about his work focused on the online digestion and analysis of RNA molecules using immobilized RNase cartridges attached to a multidimensional liquid chromatography–mass spectrometry (mD‑LC–MS) system, and why the accurate sequencing of single guide RNA (sgRNA) is important.

Q. When did you first encounter chromatography and what attracted you to the subject?

My first experience with chromatography was during an internship in the laboratory of Professor Jessop (Kingston, Canada). The Jessop group is specialized in green chemistry. I analyzed samples using gas chromatography (GC) and liquid chromatography (LC) to conclude my experiments, and I knew that analytical chemistry was an area I would like to explore more. After completing my master’s degree, I joined the Nestle Research Center (Lausanne, Switzerland) where I developed LC–tandem mass spectrometry (MS/MS) methods to quantify food contaminants in various matrices.

Q. Can you tell us more about your Ph.D. thesis?

I performed my Ph.D. thesis at the University of Geneva under the supervision of Professor Jean-Luc Veuthey, Dr Davy Guillarme, and Dr Szabolcs Fekete. It was the first Ph.D. thesis in the laboratory dedicated to the analysis of therapeutic proteins. I have developed non-denaturing size-exclusion chromatography (SEC), cation-exchange chromatography (CEC),capillary zone electrophoresis (CZE), and hydrophobic interaction (HIC) methods to characterize monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs). I had unique opportunities to perform internships in pharmaceutical companies—Novartis (Basel, Switzerland) and Genentech (South San Francisco, USA)—where I developed innovative multidimensional (mD)-LC–MS methods to streamline existing analytical workflows and facilitate the characterization of complex drug modalities.

Q. What chromatographic techniques have you worked with?

I have worked with reversed-phase LC, hydrophilic interaction liquid chromatography (HILIC), SEC, CEC, anion exchange chromatography (AEC), HIC, and mixed mode chromatography (MMC) for the analysis of diverse molecules, including small pharmaceutical molecules, therapeutic antibodies, protein-polymer conjugates, oligonucleotides, and nucleic acids.

Q. You were involved in research that fully sequenced CRISPR/Cas9 single guide RNA (sgRNA) using parallel ribonuclease digestions and HILIC−high-resolution MS analysis (1). Why is accurate sequencing of sgRNA important?

With the CRISPR-Cas technique, a single guide RNA (sgRNA) directs the ribonucleoprotein (RNP) complex to a specific target. The CRISPR-Cas technique has attracted significant interest in the field of cell therapy. For example, T cells can be reengineered using this approach. It is crucial to verify the sequence of the sgRNA is correct to limit off-target effects and ensure the safety of the patients. Regulatory agencies expect investigational new drug (IND) applicants to sequence sgRNAs.

Q. What is novel about the method you developed? What were the main challenges from an analytical perspective and how did you overcome them?

A sequencing method should enable the verification of the correct nucleoside order. Ideally, the sequencing method should also confirm the presence and correct position of the chemical modifications, which is not feasible using next generation sequencing (NGS) approaches due to the loss of information during the library preparation.

To my knowledge, this parallel RNase digestion and HILIC–MS/MS approach was the first to confirm the correct order of the nucleosides and the presence of the chemical modifications. Ideally, the sequencing approach should provide 100% sequence coverage. However, the RNase digestion of 100-mer sgRNAs produces non-unique fragments, which reduces the sequence coverage. The challenge was overcome by using multiple RNases in parallel. Our approach required the development of robust i) digestion procedures, ii) chromatographic separation, and iii) MS detection. It was the first time that an RNase digestion mixture was analyzed by HILIC, which provided both truly MS-compatible conditions in the absence of ion-pairing agents and selectivity to discriminate sgRNA sequences. We found that RNase could contaminate HILIC columns and interfere with the analysis of RNase digestion mixtures.

Q. Is HILIC becoming more commonly used in biopharmaceutical research? If so, why, and in what areas?

In my experience, HILIC is becoming more and more utilized and in particular for the analysis of oligonucleotides. Advances in this field are fast, and I am confident future research works will unleash the potential of HILIC for the characterization of new drug modalities. The pharmaceutical landscape has become incredibly diverse, with the development of complex drug modalities beyond the Lipinski’s rule of five. In parallel, the coupling of MS instrument to LC separation has become essential to identify impurities. In many cases, HILIC is an ideal mode, preventing the use of ion-pairing agents needed for the reversed-phase LC analysis of polar molecules.

Q. You also recently published a paper presenting, for the first time, the online digestion and analysis of an RNA molecule using immobilized RNase cartridges attached to a two‑dimensional (2D)-LC–MS system and three-dimensional (3D)-LC–MS system (2). How did this project arise?

I am convinced that the automated digestion and analysis of large oligonucleotides offer unique benefits for their characterization. Bottom-up MS approaches enable i) the sequencing of large oligonucleotides, and ii) the identification/quantification of impurities at single residue level.

Conventional offline, in-solution digestions require a large amount of sample and intensive laboratory work, which may cause errors. Conversely, the automated digestion and analysis of large oligonucleotides via the use of immobilized RNase cartridge and HILIC analysis streamline the process. The advantage of an online procedure was even greater since three RNases were used for the parallel offline, in-solution digestions of sgRNAs.

A 3D-LC–MS approach enabling the online fractionation and bottom-up analysis of fractions was also developed to understand the cause of a peak tailing observed on the ion-pairing (IP) reversed-phase LC profiles of sgRNAs.

Q. What were your main findings and how does your approach benefit the analyst in practice?

The same sequence coverage was found using the online approach in comparison to the offline one. A 10-fold reduction in digestion time and 20-fold reduction in sample amount was achieved using the online 2D-LC–MS approach in comparison to the offline approach. Due to the limited amount of sgRNA available, the characterization of impurity peaks observed at the single nucleotide level could only be performed when using a 3D-LC method, enabling the automated fraction collection and digestion.

In practice, the analyst can save time and focus on other activities, such as data treatment, when using the online approach, since the digestion and analysis are automated.

Q. Is 2D-LC–MS becoming used more commonly for routine analysis in the pharma/biopharma sector? If so, in what application areas? Is 3D-LC likely to be used more widely?

In the field of pharmaceutical small molecules, 2D-LC–MS is used for the characterization of impurity peaks and/or screening of method conditions, such as mobile phases and LC columns. In the field of large molecules, 2D-LC–MS is also used for the desalting of fractions collected from one-dimensional (1D) analysis for the identification of impurities.

In addition, mD-LC–MS systems enable bottom-up MS characterization and fraction collection to be integrated. We have developed several of these methods to characterize fractions collected from reference purity methods—in the past for therapeutic antibodies and now for large oligonucleotides.

Q. Are chromatographers overcautious about implementing multidimensional methods?

Multidimensional methods may often sound complicated and only ever applied to specific projects. As a general rule, the simpler, the better. However, with the increasing complexity of drug modalities and delivery systems, some challenges cannot be solved using 1D-LC methods.

There is indeed an important challenge when going beyond two dimensions, which is mainly due to limited software solutions providing a user-friendly environment. The broader adoption of mD-LC–MS methods will depend on close collaboration between instrument vendors, pharmaceutical companies, and academic laboratories.

Q. What projects are you working on next?

I am hoping to see the implementation of mD-LC–MS methods to help understand manufacturing and purification processes. In particular, an ideal method would isolate pharmaceutical molecules from a biological matrix prior to their comprehensive impurity characterization. Such a streamlined method would provide multiple quality attributes, including titer and purity, and help guide the process.

A. Goyon, B. Scott, K. Kurita, C.M. Crittenden, D. Shaw, A. Lin, P. Yehl, and K. Zhang,

A. Goyon, B. Scott, K. Kurita, C. Maschinot, K. Meyer, P. Yehl, and K. Zhang, 

is Senior Scientist in the Small Molecule Pharmaceutical Sciences Organization of Genentech. He received a Ph.D. degree in pharmaceutical analytical chemistry in 2019 from the University of Geneva, Switzerland. His team supports early- andlate-stage research programs. They develop innovative multidimensional LC–MS methods and complementary approaches to support new drug modalities and drug delivery systems. He has published 36 papers and book chapters, including 23 as first author.

This month we interview Alexandre Goyon, Senior Scientist in the Small Molecule Pharmaceutical Sciences Organization of Genentech, about his work focused on the online digestion and analysis of RNA molecules using immobilized RNase cartridges attached to a mDLC–MS system, and why the accurate sequencing of sgRNA is important.

Rising Stars of Separation Science: Alexandre Goyon

Siloxane contamination can interfere with low-level analysis of target compounds whether they are discrete peaks or manifested as high background bleed. They are one of the most common types of “ghost peaks” in gas chromatography (GC) and can come from a variety of sources such as inlet septa, columns, valves, solvents, and vial cap seals. While both column bleed and other sources of contamination contain siloxanes, the composition is different. Examining the mass spectrum and structure provides clues to the source of the contamination. This article analyzes both septa bleed and column bleed fragments and reviews best practices in reducing siloxane “ghost peaks”, in addition to understanding the differences in composition.

Siloxanes or polysiloxanes are molecules representing [-Si-O-Si-]n, where the Si has two other organic groups attached. For gas chromatography (GC) stationary phases, those side groups could be methyl (“1”‑type column), phenyl (“5”‑type column), combinations such as phenyl/cyano (“624”-type column), or a variety of other functional groups. These long siloxane chains can fold back upon themselves as temperature increases and depolymerize, yielding cyclosiloxanes, predominately trimers (D3), tetramers (D4), and pentamers (D5) (1). It is important to understand these molecules because they can impact a gas chromatogram in several ways.

Oxygen and moisture can greatly accelerate linear siloxane breakdown, which is observed as bleed in GC. Siloxane compounds can originate from carrier gas line. To reduce this risk it is recommended that purification filters are used on carrier gas lines to reduce column bleed, minimize surface activity, and extend column lifetime. This was demonstrated by Linx Waclaski, who performed an experiment attaching different ball valves behind the GC system without purification filters, and set the oven at 35 °C to collect the off‑gassing from the ball valve lubricant. These silicone-based lubricants make opening and closing the valve easier, prevent leaks, and extend valve lifetime. There was a significant amount of contamination observed, which was eliminated by the addition of a triple filter (hydrocarbon, moisture, and oxygen) (2).

Septum bleed from vial caps is less common but can be difficult to isolate since chromatographically it looks identical to inlet septa bleed. Solvents such as methylene chloride can leach siloxanes from septa, especially if the vial has been penetrated more than once. Amanda Rigdon demonstrated this phenomenon by injecting derivatized amphetamines five consecutive times using the same vial and cap septa. In this case, the septum bleed peaks were almost as large as the analyte peaks (3). In another study, repeated use of the same vial cap and vial with a 50-µL insert resulted in endrin and dichlorodiphenyltrichloroethane (DDT) breakdown, in addition to contamination of the inlet (4). Most vial cap septa are lined with a protective layer of polytetrafluorethylene (PTFE) to prevent direct contact with solvent vapour. Without a protective layer, vial cap septa bleed can be detected after 24 h at ambient temperature (3).

The septum on the top of the injection port of the GC system requires care when handling since the septum can adsorb phthalates, finger oils, perfumes, makeup, skin creams, and hand soap residue. GC inlet septum bleed occurs when volatile degradation products enter the column and is seen as a late eluting series of evenly spaced peaks in a programmed GC analysis. During continuous instrument use, change the septum daily to avoid coring and to stop small pieces of septum from entering into the GC liner (5).

Further advice includes the selection of the correct syringe needle to help minimize coring and septa fragments. One popular choice is the tapered cone style 23s–26s needle. Furthermore, understanding the thermal profiles of injection ports for setting inlet temperatures will help to reduce inlet septa bleed. The temperature at the septum is affected by the heating element and the overall inlet design, which varies significantly among manufacturers. For instance, on most GC systems, an inlet set point of 250 °C results in a septa temperature of 100 °C lower. Compare this to some manufacturers of older instruments where the inlet set point is similar to the septa temperature (6,7,8).

If GC column bleed and septa bleed are both made up of siloxanes, what makes them different? At first glance septa bleed has a base peak of 

 73, while column bleed has a base peak of 

 207. But keep in mind both column bleed and septa bleed are made up of cyclosiloxanes. These differences are investigated below.

The first part of this experiment was the extraction and concentration of both septa bleed and column bleed. A 30 m × 0.25 mm, 1.0-µm polydimethylsiloxane stationary phase column (“1-type”) (Restek) was installed into the GC inlet, with the detector end of the column protruding from the GC oven into 1 mL of methylene chloride. By operating the column at its maximum temperature (340 °C), bleed could be captured and concentrated in the methylene chloride, which was placed in a beaker of ice water to minimize evaporative loss. Bleed was collected for 6 h, with methylene chloride being added to the sample as necessary to maintain a volume of approximately 1 mL. Bleed from GC inlet septa was collected by submerging approximately 5 g of septa in 5 mL of methylene chloride for 6 h. Both septum and column bleed were concentrated 10-fold by evaporation at 60 °C. Samples were analyzed individually and combined for a single analysis to evaluate bleed profiles (Figure 1).

The GC column chosen to examine both septa and column was a “1-type” 30 m × 0.25 mm, 0.25-µm (Restek) installed into a GC–MS system (Agilent 7890 GC, 5975 GC–MS) with a split/splitless inlet and a 10:1 split, flow 2 mL/min, and a GC program of 40 °C (hold 1 min) 15 °C per min to 330 °C (hold for 20 min). The MS scan rate was 

 45 to 550 with a 3-min solvent delay. Compounds associated with bleed profiles were identified using a mass spectra database from the National Institute of Standards and Technology (NIST).

Polydimethylsiloxanes (PDMS), the stationary phase in many GC columns, are long‑chain polymers that slowly decompose into short-chain cyclic molecules. Column bleed had two prominent ions, 

 281, which are the base peaks for hexamethylcyclotrisiloxane (D3) and octamethylcyclotetrasiloxane (D4), respectively. Since bleed occurred throughout the chromatogram (Figure 2) and was a mix of these compounds that were not focused, it was seen as background. To confirm the specific compounds in the bleed profile, it was necessary to collect the column bleed and analyze it. Septa bleed concentrated at the head of the column was seen as chromatographic peaks of cyclosiloxanes, but they were in fact much larger molecules. There were over a dozen of these compounds, such as eicosamethylcylcosiloxane (D10), which has a less stable structure and produces a base peak of 

 73 (Figure 2). Figure 3 presents the spectra of D3 and D4 separately and examines the chromatogram bleed spectrum at 19.00 min, confirming that the bleed is a combination of both compounds. While column bleed also contains D5, D6, and D7, the concentrations of these compounds were less compared to D3 and D4. Since the scan rate was limited to 550 amu, compounds with spectral fragments higher than this scan rate were not able to be correctly identified; however, the fragmentation pattern suggested that they were larger cyclosiloxanes. Figure 4 demonstrates how septum-related bleed compounds were still eluting after 15 min at 330 °C. If the column was not baked out at this temperature, the septum compounds would carryover into the next analysis and would be observed as a bump in the baseline or an increase in background.

One overlooked area for septa bleed is the vial cap septa, and, while less common, it is characterized as sharp repetitive peaks even after several blanks have been analyzed (3). With vial inserts, the issues can be greatly exaggerated and can also degrade target compounds; an example of this would be endrin or DDT (4). Coring, whether from the sample vial or the injection port septum, will result in an increased number of siloxanes that will manifest themselves as “ghost peaks” (5). Whether it is valve bleed, septum vial bleed, or GC inlet septum bleed, there will be late eluting compounds, with 

 73 for many of them. Identifying the source of siloxanes makes troubleshooting much easier. While it’s true that both septa bleed and column bleed are the same—that is, they are both made up of cyclosiloxanes—the molecular weights are quite different, resulting in different spectra.

Special thanks to Jaap de Zeeuw and Erica Pack (Restek) for their technical advice and review.

D. Allan, S.C. Radzinski, M.A. Tapsak, and J.J. Liggat, 

, 553–562 (2016) DOI: 10.1007/s12633-014-9247-6

www.restek.com/en/chromablography/chromablography/are-my-shut-off-valves-contaminated

How to Diagnose GC Septum Bleed Contamination Sources: Could it be Your Vial Cap?

www.restek.com/en/technical-literature-library/articles/how-to-diagnose-gc-septum-bleed-contamination-sources-could-it-be-your-vial-cap

Silicone Autosampler Vial Septa Cause Endrin Breakdown and Sample Contamination

www.restek.com/en/chromablography/chromablography/silicone-autosampler-vial-septa-cause-endrin-breakdown-and-sample-contamination

www.restek.com/globalassets/pdfs/literature/gnar2671-unv.pdf

Thermo Trace 1310 Inlet Temperature Profile vs Agilent 7890 for Split/Splitless Injectors 

www.restek.com/en/chromablography/chromablography/thermo-trace-1310-inlet-temperature-profile-vs-agilent-7890-for-splitsplitless-injectors

 It’s a Matter of Degrees, but Do Degrees Really Matter? 

https://www.restek.com/globalassets/pdfs/literature/gnar1937-unv.pdf

 https://pdf2.chromtech.net.au/580134_ADV07-2_22.pdf

has managed a team of chemists in Restek’s innovations laboratory since 2004. Before taking the reins of the laboratory, he spent seven years as an environmental chemist and was critical to the development of Restek’s current line of volatile GC columns. Prior to joining Restek, he operated a variety of gas chromatographic detectors conducting method development and sample analysis. Chris holds a B.S. in environmental science from Saint Michael’s College, USA.

Identifying the source of siloxanes makes troubleshooting that much easier.

Understanding the Origins of Siloxane Ghost Peaks in Gas Chromatography

As chromatographers, our thoughts turn to the use of hydrogen as
an alternative carrier gas, to ensure continuity of supply and reduce
long-term costs.

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.

As chromatographers, our thoughts turn to the use of hydrogen as an alternative carrier gas, to ensure continuity of supply and reduce long-term costs.

Here we go again, on the brink of yet another helium crisis—the fourth since 2010! Reports abound in the industry press, and suppliers are sending warning letters of supply chain issues, prices rising, and restrictions on order volumes. In contrast to the previous crisis in late 2019 and early 2020, the present situation has seen more suppliers limit allocation (sometimes between 45 and 65% of “typical” use volumes) rather than increase prices, although naturally, the prices are creeping up as supply tightens.

I’ve written two blog entries in the recent past (1,2) regarding the practical implementation of hydrogen carrier gas, and while it is a very convenient alternative to generate your own hydrogen carrier gas within the laboratory using only deionized water as a feedstock, there are drawbacks. Besides the initial purchase costs of the generator(s), the explosive nature of hydrogen in air means there may be safety measures that need to be implemented to detect gas leakage. Methods need to be translated in order to match the retention and selectivity characteristics of the original helium-based method. When using mass spectrometric detection, there are minimum flow requirements to ensure pneumatic controllers are able to cope with low back pressure requirements. Source components may need to be changed to ensure vacuum levels within the detector can be maintained, and one needs to be mindful of the possibility of altered spectral ion ratios and possible hydrogenation reactions within the ion source—both of which can lead to altered qualitative and quantitative results.

All of this being said, hydrogen is successfully used as a carrier gas in a large number of laboratories globally, and it is a viable alternative for helium for a huge number of applications. I’m writing this blog not to highlight the process of converting to hydrogen carrier gas, but to shed further light on the current helium supply chain difficulties, and share research that will hopefully allow a balanced view on the medium- to long‑term sustainability of helium as a critical resource for gas chromatography users.

4He formed through underground radioactive 238U decay is typically released into the atmosphere (and subsequently quickly lost into space) through permeable rock; however, where the rock is impermeable, the helium is trapped alongside natural gas deposits. Cryogenic high‑pressure fractional distillation can be used to separate helium and nitrogen from methane and subsequently cool them in a condenser to separate the crude helium (which contains 50–70% helium, 1–3% residual methane, small quantities of hydrogen and neon, and nitrogen). This crude helium is then cooled to -315 °F where the nitrogen and remaining methane condense and are drained off. Air is then added and warmed in the presence of a catalyst, causing hydrogen to react with oxygen from the air to form water vapour, which can be condensed through cooling and drawn off. A pressure swing adsorption (PSA) unit is then employed to separate high-purity helium (99.99%) from any remaining impurities by selective removal using microporous adsorbents (3). Where helium concentrations within the natural gas deposits are low (typically below 0.3% by volume), companies use more advanced, multi‑bed PSA technology to recover the helium in an economically viable manner (4). As the half‑life of 238U is around 4.5 billion years (the earth is estimated to be around 4.7 billion years old), a vanishingly small amount of “new” helium is produced each year and therefore it is afforded “non-renewable source” status.

Helium is used by a variety of industries, including cryogenic cooling of magnetic resonance imaging (MRI) instruments, as a lifting gas for airships and rockets, for welding, in semi-conductor manufacture, and as a leak detection vehicle; scientific and analytical uses for helium account for approximately 20% of the annual helium consumption. The total global consumption of helium (2021) is estimated at approximately 176 million cubic metres, with the global resource estimated at 5.9 billion cubic metres (5), resulting in a total of 335 years of supply at current consumption levels.

So, given that there are plentiful helium reserves, what are the causes of the current supply issues?

Global helium production by region is shown in Figure 1. The U.S. National (sometimes called 

) Helium Program Reserve, currently administrated by the U.S, Bureau of Land Management (BLM), is held at the Cliffside Storage Facility in a natural underground facility located in the Texas panhandle, near the natural gas fields around Amarillo. While some of the helium is refined in the local gas fields, much is piped over many hundreds of miles from other northern United States refining facilities. The Mineral Leasing Act of the 1920s reserved all helium production for US Government use, while the Helium Acts Amendment of 1960 allowed the U.S. Bureau of Mines to arrange for five private plants to recover helium from natural gas, effectively a government purchase of the helium stockpile. By 1995, the National Helium Reserve contained 1 billion cubic metres of gas, but was $1.4 billion dollars in debt and the Helium Privatization Act of 1996 mandated that the helium reserve should be sold off in order to pay down these debts. The hope was that by 2015 the reserve would have been sold off and that private industry would establish new sources of helium to satisfy demand. Critically, the pricing for the auctions of the reserve was set below market value to ensure that the debt was paid off quickly, keeping global pricing relatively low; however, ongoing costs of production and maintenance of the reserve have led to unpredictable price adjustments up to market levels, affecting the helium price globally.

The interest of private industry in helium production did match the 1996 expectations of the US Government, and in 2013 the Helium Stewardship Act was signed into law, allowing sales from the National Helium Reserve to continue to September 2021 (now September 2022), effectively ending federal involvement with the reserve. This act effectively slowed the sale of federal helium and allowed the BLM to auction the gas at higher prices. The bill prevented the federal government from undercutting private producers, thus encouraging more sources of helium production to go online.

The effective end of public sale of helium from the BLM at Cliffside may herald future supply or pricing issues; however, in the shorter term, the facility has other problems. Following a four‑month shutdown in late 2021, the facility has not returned to production due to the discovery of a major leak, leading to a reduction of 14 million cubic metres per annum in the
global supply.

A new production plant in the Amur region of Russia was due to add a further 20 to 30% capacity (up to 70 million cubic metres) into global supply by 2023; however, a fire in October 2021 followed by an explosion and fire in January 2022 has resulted in zero capacity from this plant. There are no proposed dates from the plant coming online, but I have seen estimates of six months upwards. Of course, the supply from the Amur facility is further compounded by Russia’s war with Ukraine, which has led to an embargo of oil and gas product imports.

The Arzew facility in Algeria usually produces helium as a result of the liquefaction of natural gas, which is transported globally by container ship. This compression or liquefaction process makes the production of helium economically viable at this facility because it liquefies at much higher pressures and lower temperatures than natural gas. Again, due to the war with Ukraine, European natural gas supplies are currently highly restricted and most of the natural gas from Arzew is being transported directly by pipeline into Europe via Spain. As transport by pipeline does not require liquefaction, then the extraction of helium is currently not economically viable, leading to a further reduction in global supply. It is estimated that globally, liquified natural gas production is down 30% from January this year, for similar reasons to those above, representing a reduction of 10% in total global supply.

These are just some of the contributing reasons for the current supply chain issues. Others include an embargo of gas from Qatar passing through Saudi Arabia to a large port in the United Arab Emirates (although this situation is apparently now resolved) and various maintenance and unplanned outages from a range of smaller production facilities globally. This “perfect storm” has resulted in the current supply chain issues, any one of which may have caused a “blip” in helium availability, but collectively are presenting a very challenging supply situation. So, what of the short- and mid-term future for helium supply?

Clearly, the BLS facility at Cliffside needs to come back online as soon as possible, and I have seen reports for production starting again before June. However, the situations in the Amur and Azew facilities do not appear to have a short‑term resolution, given the current and likely the ongoing geopolitical situation.

The medium-to-long-term situation does look brighter. One large gas supplier recently announced the opening of the world’s largest helium storage cavern facility, again in Texas, with supply capacity planned to outstrip that of the BLM plant (6). New companies are forming to extract helium as a “pure play” resource, that is, mining and exploration for pure helium extraction rather than as a side product of natural gas refining. Sites are being developed in Saskatchewan, Utah, and Tanzania that could contribute significant capacity into the system from 2024 onwards.

From current research, I believe the short-term supply issues will not resolve quickly, but that mid-to-long term there is enough commercial and political interest in maintaining a secure supply of helium for scientific and medical diagnostic use. As chromatographers we need to keep a keen eye on the helium supply chain and ensure we plan for shortages and price increases over the next two years, whereafter, hopefully, the supply chain issues will ease. All of that being said, our facilities continue to investigate the suitability of alternative carrier gases, including nitrogen, for our range of applications.

Using Hydrogen Carrier Gas with Mass Spectrometric Detection: https://www.chromatographyonline.com/view/lcgc-blog-using-hydrogen-carrier-gas-mass-spectrometric-detection

Is Hydrogen the Only Viable Gas Chromatography Carrier Gas for the Long-Term?: https://www.chromatographyonline.com/view/lcgc-blog-hydrogen-only-viable-gas-chromatography-carrier-gas-long-term-0

https://phys.org/news/2010-08-world-helium-nobel-prize-winner.html

United States Geological Survey, Mineral Commodity Summaries (2019).

https://www.maximizemarketresearch.com/market-report/global-helium-market/26772/

https://www.gasworld.com/air-products-reveals-worlds-largest-helium-storage-cavern/2022545.article

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.

Not Another  Helium Crisis!

Researchers have developed and validated a sensitive analytical method for the determination of 16 polyﬂuorinated alkyl substances (PFAS) in ﬁne airborne particulate matter (PM

) using online solid‑phase extraction (SPE) coupled with liquid chromatography (LC)–negative electrospray ionization high resolution mass spectrometry (−) (ESI-HRMS).

) using online solid‑phase extraction (SPE) coupled with liquid chromatography (LC)–negative electrospray ionization high resolution mass spectrometry (−) (ESI-HRMS) (1).

, as these small particles can penetrate deep into respiratory systems, leading to cardiovascular and respiratory diseases as well as lung cancer. The World Health Organization (WHO) provides guideline values for PM

 mass concentration alone is not indicative of the risks from exposure to fine particles, as the toxicity of those particles is not accounted for. They are chemically complex and the typical composition of ambient PM

 particles contains thousands of individual organic compounds that can be extremely toxic, even when present at low concentrations. PFAS are one such class of contaminant to be concerned about in this regard. They were widely used in a broad range of consumer products and industrial applications for decades, and persist within an environmental context for a long time—even gaining notoriety as “forever chemicals”. Their use has been phased out in many countries, with less toxic alternatives replacing them; however, even these replacements, such as fluorotelomer sulfonates (FTS), have been found to persist in the environment, and there is emerging evidence that their impact on human health is similar to that of banned PFAS.

The analysis of PFAS in environmental samples is challenging because of trace‑level concentrations requiring highly sensitive analytical methods. Currently, only a few publications have reported analytical methods for the identiﬁcation of PFAS in atmospheric samples; however, there are issues surrounding these methods, such as interference from matrix effects. As such, researchers aimed to develop a sensitive online SPE-LC–HRMS technique for the analysis of PFAS in atmospheric aerosol particles that would not only eliminate issues from previous methodologies but also expand the analyte detection range for 4:2 and 8:2 FTS’s, and utilize the developed method to analyze ambient PM2.5 samples collected in urban environments.

Results indicated that the developed method provided limits of detection (LODs) in the range 0.08–0.5 pg/mL of sample extract. This enabled detection of selected PFAS in aerosol particles at low fg/m3 levels, with a high tolerance to the considered PM matrix. When applied to the analysis of PFAS collected at two urban locations in Ireland, the method found several PFAS above the detection limit as well as FTS’s. The detection of PFAS in the environment despite them being phased out from productions in the European Union (EU) two decades ago only highlights their ability to remain in the environment for long periods of time. The detection of FTS’s also raises concerns about their suitability as alternatives to PFAS, with their potential impact on human health and environmental well-being yet to be fully explored.

Researchers have developed and validated a sensitive analytical method for the determination of 16 PFAS in ﬁne airborne particulate matter (PM2.5) using online SPE coupled with LC–ESI-HRMS.

Airbourne PFA and FTS Detection Using Online SPE-LC–HRMS

 is proud to announce that Szabolcs Fekete is the winner of the 2022 HTC Innovation Award. Fekete was honoured at the Hyphenated Techniques and Separations Technology (HTC-17) conference in Ghent, Belgium on Friday 20 May 2022 for his outstanding contributions to the field of separation science.

Symposium Chair of HTC-17, Deirdre Cabooter, said,“Szabolcs Fekete has developed a complete package of novel approaches to push intact protein and protein subunit chromatography to the next level–including the so-called ‘ultra-short column’, ‘multi-isocratic’, ‘negative gradient segment’, and ‘on-column fractioning’ approaches. With these methods, separation scientists can improve selectivity between closely eluting species for more accurate peak integration/identification or routinely separate critical sample components in less than 30 seconds.

“Szabolcs’ research has challenged the status quo of analytical labs in the biopharmaceutical industry, that for a long time have standardized many of their product characterization and quality control techniques on 50 to 150 mm long columns. Szabolcs has challenged conventional thinking by asking fundamental questions, pushing modern column fabrication techniques, and offering new method ideas that have broken the paradigm of these standardized techniques. He has achieved separations of protein species that had long been elusive, and has collected drug product information in 10x less time versus traditional approaches.”

Szabolcs worked in the pharmaceutical industry in analytical R&D for 10 years, and then moved to the University of Geneva in Switzerland, to work in the group of Jean-Luc Veuthey and Davy Guillarme as postdoc and as scientific collaborator for a decade. In April 2021, he joined Waters Corporation and now works as a consulting scientist. He has contributed to 160+ peer-reviewed journal articles and has co-authored 10+ book chapters and edited two handbooks. His main interests include pharmaceuticals, therapeutic proteins and other biomolecules separations, fundamentals of chromatography, column technology, method development, experimental designs, and modelling. He is well known for his various collaborations with both academic and industrial partners.

“It is a huge honour for me to receive the HTC Innovation Award. I feel myself very lucky because I had many possibilities and opportunities to work or collaborate with several outstanding scientists worldwide. I was involved in many industrial and academic collaborations that were very motivating and inspiring. What we have achieved is due to our teamwork. I would like to especially thank Davy Guillarme, Jean-Luc Veuthey, and Matthew Lauber for their continuous support and encouragement,” said Fekete.

The HTC Innovation Award was launched by 

 and the HTC Scientific Committeeto celebrate a separation scientist who has made pioneering contribution to the field of separation sciences by introducing new methodologies, new instrumentation, or new techniques in the field, with a strong focus on applications that benefit society.

 added, “We are very pleased that the HTC Organizing Committee has awarded the prize to Szabolcs, who clearly demonstrates scientific creativity with innovative and practical solutions for biopharmaceutical analysts.”

LCGC is proud to announce that Szabolcs Fekete is the winner of the 2022 HTC Innovation Award. Fekete was honoured at the Hyphenated Techniques and Separations Technology (HTC-17) conference in Ghent, Belgium on Friday 20 May 2022 for his outstanding contributions to the field of separation science.

Szabolcs Fekete Wins 2022 HTC Innovation Award

Agilent Technologies Inc. (Santa Clara, California, USA) has announced that it has joined the National Institute for Innovation in Manufacturing to Advance Biomanufacturing (NIIMBL), demonstrating Agilent’s commitment to supporting biopharmaceutical and biotherapeutics customer challenges through partnership and innovation using tools and workflows.

NIIMBL’s mission is to accelerate biopharmaceutical innovation, support the development of standards that enable more efficient and rapid manufacturing capabilities, and educate and train a world-leading biopharmaceutical manufacturing workforce, which will fundamentally advance U.S. competitiveness in the industry.

“Agilent has long played a key role in providing trusted answers for the analytical needs of the biopharmaceutical industry,” said Agilent CEO Mike McMullen. “The prominence of biopharmaceuticals coupled with the advent of precision cell and gene biotherapeutics has further spurred the need for innovative measurement tools to help deliver life-saving drugs and diagnostics to patients.

“Agilent’s online HPLC sampling portfolio, cellular imaging, and real-time sensor technologies exemplify how the company can help address current and emerging needs. Additionally, Agilent’s NIIMBL membership will allow us to work closely with all continuum partners to help shape the future of innovation and standards used in bioprocess development
and biomanufacturing.”

For more information, please click here: 

Agilent Technologies Inc. has announced that it has joined the NIIMBL, demonstrating Agilent’s commitment to supporting biopharmaceutical and biotherapeutics customer challenges through partnership and innovation using tools and workflows.

Agilent Joins the National Institute for Innovation in Manufacturing to Advance Biomanufacturing

BioMed X (Heidelberg, Germany) has announced the start of its “Ukraine Refugee Funding Programme” that supports doctoral students and masters-level researchers in the life sciences who cannot continue their work at institutions in Ukraine. 

BioMed X (Heidelberg, Germany) has announced the start of its “Ukraine Refugee Funding Programme” that supports doctoral students and masters-level researchers in the life sciences who cannot continue their work at institutions in Ukraine. In collaboration with some of its partners, BioMed X aims to give these scientists the opportunity to continue their work in a research group at the BioMed X Institute in Heidelberg for a period of up to four years. Candidates should have a master’s degree, as well as research experience, in either molecular/cell biology, bio-organic chemistry, pharmacology, or physics. Knowledge of the German language is not required.

Thomas Rückle, Senior VP and Head of Research at the BioMed X Institute, commented: “The situation in Ukraine is a tragedy that requires a joint effort to offer support and help. Our BioMed X Institute is a place where early-career scientists are given the opportunity to develop themselves. Together with some of our partners we launched this funding programme because we sincerely hope to provide a scientific home for some of the researchers who cannot continue their career in Ukraine.”

For further information, please click here: 

BioMed X has announced the start of its “Ukraine Refugee Funding Programme” that supports doctoral students and masters-level researchers in the life sciences who cannot continue their work at institutions in Ukraine. 

BioMed X Launches Ukraine Funding Programme

We continue our “Critical Evaluation” series with a consideration of liquid chromatography (LC) methods that use mass spectrometric detection.

I’ll concentrate on the detector settings that are typically provided in literature or method specifications for the mass spectrometric detector, to discern what we might learn about potential pitfalls before entering the laboratory.

From the outset, I will state that critical evaluation of LC–MS detector settings is perhaps the most difficult of all the extra-laboratory detective skills to master. This is primarily due to the differences between instrument design, the different nomenclature used by the various manufacturers for the variables associated with the source and analyzer components as well as the recommended values and ranges for the voltages, voltage offsets, gas flows, pressures, and temperatures. Perhaps this is why we, as a community of practitioners, tend to assume that many of the variables associated with our mass spectrometry (MS) detectors for liquid chromatography as “lock and leave,” that is, we set or optimize them once and then assume that these are applicable for all other applications. The notable exception here would be the capillary (sprayer) voltage, which tends to be considered as the universal variable for optimization in API interfaces. However, I would argue that the differences between instrument designs and variables is an even bigger reason to be critical when adopting methods from scientific literature or instrument manufacturers application notes, especially when the instrument used in the literature method differs from your own.

It's outside the scope of this short blog to delve into the theory of atmospheric pressure ionization (API) mass spectrometry, but for the interested reader, a short form description of the relevant processes involved in Electrospray Mass Spectrometry can be found here:

https://www.chromatographyonline.com/view/lcgc-blog-underutilized-methods-optimizing-lc-ms-sensitivity

For all instruments, the following processes are critical to ionisation and ion sampling within electrospray LC–MS:

Impart a charge to analyte molecule—typically through pH adjustment of the eluent or via induced charging or charge transfer in the electric filed at the tip of the capillary (sprayer)

Accumulating an excess of positive or negative charge (depending upon detection mode) into a droplet at the tip of the capillary (sprayer)

Increasing the charge (and hence the repulsive forces) at the surface of the sprayed droplet by solvent evaporation in order to induce droplet jet fission and achieve further charge density accumulation within the offspring droplet

Liberating the charged analyte into the gas phase (several mechanisms are proposed)

Preferential sampling of the charged gas-phase analytes into the vacuum region of the mass spectrometer

Within the mass analyzer, further important processes which are common to all mass analyzing devices include:

Declustering or fragmenting the analyte prior to the (first) mass analysing device

Filtering gas-phase ions based on their mass-to-charge ratio

Fragmenting ions from the first mass analyzing device (for MS/MS instruments) by accelerating the ions through a potential difference within a cell containing a collision gas at a given pressure or flow and temperature

The values for the variables given within LC–MS method specifications will all be associated with one or another of the processes highlighted above, and I will attempt to highlight which variable matches which process as we move through the remainder of this blog.

So, to the method for our current study, which is for a multi-laboratory study of Δ9-tetrahydrocannabinol and its metabolites in whole blood extracts (1).

Mobile phase B: 0.1% formic acid in acetonitrile

Gradient (%B): 55% for 3.5 mins, ballistic ramp to 80% B and hold for 2 min

Mode: Positive electrospray ionization, dynamic multiple-reaction monitoring

So, I will start by saying that this is a literature method and a particularly good one in that it contains all of the information necessary to reproduce the method on a similar instrument or to make important decisions when transferring the method to a different manufacturers instrument. I should also add that I’ve not used this method in anger, so make no claims as to the validity or appropriateness of the settings for this particular application.

All of this being said, let’s take a look at the various settings and see what information the method might yield prior to us donning our laboratory PPE and getting hands-on with our instrument.

This is an electrospray API method (we’ll look at other ionization modes in a later blog), and as such we need to ensure that the eluent flow into the MS interface is appropriate and does not interfere with droplet formation processes at the tip of the capillary (sprayer). If the droplets do not have time to form and accumulate charge prior to being dispelled from the tip then the instrument response will be poor or non-existent. In modern instruments, where nebulizing gas is used to help constrain the droplet growth at the capillary tip in order to accumulate charged analyte, “usual” eluent flow rates range from around 0.4 to 1.0 mL/min and anything outside of this range should be carefully considered and manufacturers literature consulted. It may be that nebulizing gas flow rates can be adjusted to help with higher- or lower-eluent flows, but one needs be prepared to make changes as necessary. At 0.6 mL/min. the eluent flow rate in our method seems to be appropriate.

As I’ve mentioned, the nebulizer gas pressure is used to control the flow of gas around the sprayer capillary and helps to ensure proper droplet formation at the capillary tip, which is essential for good sensitivity. The name of this variable and the appropriate setting and range varies between manufacturer and instrument design, and it’s good practice to check instrument manuals to assess if the setting is to the very high or low end of the range in order to assess the validity of the setting. As noted above, it may be necessary to use more extreme values of nebulizer gas flow to cope with very high or very low eluent flow rates. From personal experience, when performing design of experiment (DoE) based source optimization, the nebulizer gas flow rate doesn’t typically feature as a highly significant variable, and therefore this could be seen as a secondary variable when assessing the suitability of a method.

The Capillary Voltage is widely regarded as a (if not “the”) primary variable in affecting the efficiency and sensitivity of the electrospray process. If one considers the API interface as an electrochemical cell that is completed in the gas phase, the voltage applied to this circuit controls several crucial processes, not least of which is the charge separation that occurs at the capillary (sprayer) tip. The processes are highly analyte and eluent dependent and therefore the voltage applied to the capillary is of crucial importance. The effective capillary voltage range for a particular instrument may be wider than one might appreciate and, even if the instrument is producing a signal, it may be far from optimal and may also produce unstable or erratic results. Where possible, it is always recommended that the applied voltage to the API is optimized for each application. From experience, I know that 3500 V seems an appropriate value for the instrument cited in the application, however I would urge readers to be ready to optimize the applied voltage setting with each application and to take the view that this variable is not a one-size-fits-all parameter. In DoE experiments, this variable is very often identified as being significant, but this is not always the case and it isn’t always the most significant variable. One needs to consider the capillary (sprayer) voltage “in the round” with the other interface parameters and one should not attempt to optimize the experiment using this variable alone.

Drying Gas Temperature and Flow (Gas Temperature & Gas Flow in the method conditions above) are used to control the desolvation of the electrospray droplets as they pass through the ion source. The desolvation process is crucial in accumulating surface charge density that leads to droplet jet fission and ultimately to the release of gas-phase analyte ions into the ion source. The droplet desolvation rate is controlled directly by the drying gas flow rate and temperature, as is the position within the inlet where the analyte ions are liberated into the gas phase. Gas-phase ion liberation needs to occur as close to the sampling “sweet spot” as possible within the interface so that the applied voltage and vacuum can direct the charged analyte ions into the sampling orifice of the mass analyzing device. Unsurprisingly then, drying gas temperature and flow are often significant variables when optimizing interface variables in electrospray ionization experiments. Again, one needs to be aware of the allowable range for these variables and assess where in the range the variables in the method under consideration are set. If the instrument used in the literature method is not the same as yours, then an understanding of where the variables are in the allowable range of the instrument used will help to guide the setup of your own instrument.

The instrument used in the literature method uses sheath gas to form a temperature gradient concentrically around the capillary and within the ion source. This is purported to “focus” the sprayed droplets, aid droplet desolvation and focus analyte ions into the “sweet spot” of gas-phase ion sampling within the ion source. In this case, sheath gas temperature and flow are likely to take precedent over the drying gas as primary source optimization parameters. It’s also worth noting that other manufacturers have similar adaptations and improvements to the original electrospray source designs, and each of these needs to be considered when assessing literature methods.

The fragmentor voltage controls the potential difference applied to the first vacuum region post ion source, which is under only partial vacuum due to the sampling orifice being open to atmospheric pressure in the ion source. Higher potential differences are used to accelerate analyte ions through this region in order to further desolvate analyte ions via collisions with the background air molecules and improve the ion filtering and selection process. At higher potential differences, and therefore rates of acceleration, analyte fragmentation can be induced, which can be a positive or a negative depending upon the type of spectral experiment being undertaken. In multiple reaction monitoring (MRM) experiments, specific precursor and product ions are selected by the first and second mass analyzer in order to increase specificity and sensitivity of the analysis. One needs to take care that the fragmentor voltage is set correctly in order to ensure that the precursor ion selected for the first analyzer is either correctly generated or is not destroyed in first vacuum region. When the instrument used in the literature method is similar to yours, provided that the method states the required fragmentor voltages, then instrument setup should be straightforward. However if the instrument is different to yours, then one needs to ensure that the fragmentor (or equivalent) voltages are appropriate to ensure the optimum production (or preservation) of the precursor ion.

The collision cell voltage controls the acceleration of the precursor ion through this cell which is placed between the first and second mass analyzers. Although in the high vacuum region of the instrument, the cell is filled with a collision gas (typically helium or argon) and accelerating ions through the gas will cause them to fragment through collision with the background gas, similar to the processes discussed in the fragmentor region. The higher the gas pressure (density) and the higher the collision cell voltage, the greater the degree of analyte fragmentation. Again, one needs to optimize the collision cell voltage in order to optimize the production of the analyte product ion and hence the sensitivity of the system. The collision cell energy (voltage) is analyte specific and should be optimized for each analyte and application in same way as described for the fragmentor voltage.

Several instruments provide a software driven optimization routine in which the fragmentor and collision energy voltages can be optimized either on an analyte by analyte basis or by taking into account the “universal” response where standards of all analytes are not available. This point is important as, with all of the actions described above, one may need to assess the response of many target analytes, in which case a general optimum response may need to be considered, rather than having the luxury of optimizing one or two target analytes for which pure standards are available for infusion or injection via the LC system.

In the short space allowed here, I’ve tried to highlight some of the thinking processes which one might adopt when considering the use of a method obtained outside our own laboratory or organization. Hopefully, I have managed to highlight that a working knowledge of the instrument and principles of operation is very important in being able to do this effectively. Knowing the parameter ranges for each of the variables highlighted above is crucial in order to evaluate the method and sometimes this also requires us to know something of the instrument on which the original method was developed, to assess if the variable in question is at the higher or lower end of the range. I should also point out that we have only considered the major variables here. There will be many more associated with the tuning of the analyzing devices, for example, that I have not considered because they will be highly instrument specific. What I know to be the empirical truth is that one needs to pay careful attention to the optimization of all of the ion source and mass analyzer parameters mentioned in order to successfully transfer a method into one’s own laboratory. While we are not all skilled in or have access to DoE approaches, simple evaluation of the literature method against the settings used for other methods in our laboratory can help enormously with intercepting problems before entering the laboratory and troubleshooting them if and when they occur!

https://www.oatext.com/multi-laboratory-validation-of-a-9-tetrahydrocannabinol-lc-ms-ms-test-kit-designed-for-quantifying-thc-and-marijuana-metabolites-in-blood.php

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.

We continue our “Critical Evaluation” series with a consideration of liquid chromatography (LC) methods that use mass spectrometric detection. 



The LCGC Blog: Critical Evaluation of Analytical Methods: Liquid Chromatography with Mass Spectrometric Detection

The 2022 Research Awards, the 2022 Research at PUI Award, and the Postdoctoral Career Development Awards promote the research and career development of academic scientists and postdoctoral fellows.

Franklin Leach III of the University of Georgia, Gloria Sheynkman of the University of Virginia, and Thanh D. Do of the University of Tennessee–Knoxville, are the three awardees of the 2022 ASMS Research Awards. They will be recognized at at the ASMS Conference in Minneapolis, Minnesota, on June 7 at 4:45 pm CST.

Franklin Leach III (left) of the University of Georgia, Gloria Sheynkman (center) of the University of Virginia, and Thanh D. Do (right) of the University of Tennessee–Knoxville.

The ASMS Research Awards promote the research of academic scientists within the first four years of joining the tenure track or research faculty of a North American University at the time the award is conferred. The awards are presented annually and are fully supported by Bruker, Thermo Fisher Scientific, and Waters Corporation. Leach III’s award was sponsored by Bruker, Sheynkman’s award was sponsored by Thermo Fisher Scientific, and Do’s award was sponsored by Waters Corporation.

ASMS also recognizes Mac Gilliland of Furman University for being named the recipient of the 2022 Research at Primarily Undergraduate Institution (PUI) Award. Gilliland will be recognized at ASMS on June 7 at 4:45 pm CST.

The Research at PUI award promotes academic research in mass spectrometry by faculty members and their students at primarily undergraduate institutions (PUIs). The award is presented annually and is fully supported by Agilent Technologies.

Finally, ASMS also recognizes Yangjie Li of Stanford University, Xin Ma of the Georgia Institute of Technology, Melissa Pergande of the University of Wisconsin–Madison, Stephanie Rankin-Turner of Johns Hopkins University, and Hua Zhang of the University of Wisconsin–Madison as the recipients of the 2022 Postdoctoral Career Development Awards. Li, Ma, Pergande, Rankin-Turner, and Zhang will be recognized at ASMS on June 8 at 4:45 pm CST.

Yangjie Li (top left) of Stanford University, Xin Ma (top center) of the Georgia Institute of Technology, Melissa Pergande (top right) of the University of Wisconsin–Madison, Stephanie Rankin-Turner (bottom left) of Johns Hopkins University, and Hua Zhang (bottom right) of the University of Wisconsin–Madison.