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Click the title above to open the LCGC North America May 2020 issue, Volume 38, Number 5, in an interactive PDF format.

Vol 38 No 5 LCGC North America May 2020 Regular Issue PDF

In reversed-phase liquid chromatography, increasing the column temperature usually results in a decrease in analyte retention. Will this cause problems if a method does not call for control of the column temperature, but the room temperature of the laboratory environment changes?  

	
	In the early days of liquid chromatography (LC), precise control of the column temperature was not a primary consideration in many instrument setups. Unlike gas chromatography (GC), where the column temperature is the single most important experimental variable that dictates retention, in LC the column temperature has a smaller influence on retention compared to the mobile-phase composition (that is, the organic:aqueous ratio for reversed-phase and hydrophilic-interaction chromatography [HILIC]separations, or cation:anion concentration for charge-based separations). As the community’s knowledge about the effects of temperature on LC separations has increased, the need for accurate and precise control of column temperature has increasingly been recognized. For example, we now know much more about the effects of column temperature on the retention of different analyte types (1), the importance of matching the temperature of mobile phase at the column inlet to the temperature of the column itself (2), and the effects of heat flow in columns on apparent column efficiency (3). As a result, users generally pay attention to control of temperature in their LC instruments (and the designs of instrument modules for doing so), and specify a particular temperature or temperature range when developing and validating a method. However, there are still many methods in use that have been developed for use at “room temperature,” where there is no provision for the LC instrument to control the column temperature, and in some cases no precise specification of what is meant by “room temperature” in the method. This leads to the obvious question: With such a method, can one reasonably expect to obtain the same separation in Anchorage, Alaska, as in Mumbai, India? In this installment of “LC Troubleshooting,” I discuss some of the basics of the effect of temperature on retention in LC, and then discuss some experimental data that help us understand when some variation in room temperature might be okay, and when it might seriously affect the performance of a separation.

Basics of the Effect of Column Temperature on Retention in Reversed-Phase LC

	In reversed-phase LC, the influence of temperature on retention is most commonly described using a Van ’t Hoff type of relationship, like that shown in equation 1:

 is a condition-specific parameter related to the entropy of transfer of the analyte from the mobile phase to the stationary phase and the phase ratio (the relationship of stationary phase volume to mobile phase volume), and 

 is related to the enthalpy of transfer of the analyte from the mobile phase to the stationary phase. Figure 1 shows experimental retention data for several neutral small molecules obtained with a C18 column, an acetonitrile:water mobile phase, at different temperatures. The data follow a roughly linear trend, as predicted by equation 1. Recent work by the Weber group has examined a large body of experimental measurements of this type, and concluded that a non-linear version of equation 1 frequently produces a better fit of the data than a linear one (1). Other researchers have argued that the apparent linearity of the data is more of a happy accident than a thermodynamic inevitability (4).

) for several neutral small molecules obtained using a C18 column and mobile phase of 50/50 (v/v) acetonitrile/water: (a) shows toluene, ethylbenzene, and propylbenzene, while (b) shows p-nitrobenzyl chloride and anisole. Dashed lines are linear regression lines. Data are from the PhD dissertation of Yun Mao (6).

	The slope of each line in Figure 1 is related to the enthalpy of transfer of the analyte from the mobile to the stationary phase. For molecules that are chemically similar, the slopes are numerically similar, as shown by the data for toluene, ethylbenzene, and propylbenzene in Figure 1a. In a separation of a mixture of molecules like these, increasing the column temperature will decrease retention, but the relative spacing of the peaks (that is, the selectivity) will not change much, because all of the peaks move in the same direction at about the same rate. However, for molecules with different functional groups, the slopes can be quite different, as shown in Figure 1b for the analytes p-nitrobenzyl chloride and anisole. In a case like this, increasing column temperature will decrease retention for both analytes, but at different rates, such that selectivity and resolution will change. In extreme cases this can even lead to a reversal of elution order (5). Readers interested in exploring these phenomena on their own can do so easily using our web-based simulator that allows the user to change mobile- phase composition and column temperature, and observe the impact on retention and resolution (www.multidlc.org/hplcsim).

	In preparation for writing this installment of “LC Troubleshooting,” I created a microenvironment for one of the LC instruments in my laboratory. I first disconnected the column from the flow path going through the column thermostat compartment we normally use to control the column temperature, such that the mobile phase flow went directly from the autosampler to the column, and then directly on to the detector. Then, I heated or cooled the microenvironment to simulate the impact of changing room temperature on my separation, and measured the ambient air temperature near the column inlet using a small thermocouple. Finally, I recorded the chromatograms for separations of a simple mixture of small molecules (one acid, one base, and two neutrals) at different “room temperatures” after equilibrating the system for about 30 min for each temperature. Three of these chromatograms-recorded with “room temperatures” of 24, 28, and 42 °C-are shown in Figure 2. The vertical dashed line is added as a visual aid, to help quantify the shift in retention that occurs as the temperature is increased. Although there is a shift that is discernible by eye, it is instructive to plot the resulting data as a function of temperature to more precisely understand the effect, particularly for changes in selectivity. Figure 3 shows the effect of temperature on the retention factors of all four probe compounds. In Figure 3a, I have plotted the absolute values of the retention factors, and, in Figure 3b, I have plotted the percent change in retention relative to average retention for each compound over the range of temperatures measured. As expected, the retention of all compounds decreases as temperature increases, and here we have our first important point from this experiment. That changes in “room temperature” can definitely be enough to cause a change in retention on the order of 2% for small molecules, even over a modest range of laboratory room temperatures that might be experienced in the United States. The data point at 42 °C is admittedly a bit extreme, but there are LC laboratories across the globe that do experience this type of extreme condition.

Experimental chromatograms obtained for a test mixture at different room temperatures. The dashed vertical line is added to aid visual comparison of retention times. Conditions: Column, 50 mm x 4.6-mm i.d., Zorbax SB-C18 (5-µm); Flow rate, 1.0 mL/min.; mobile phase, 40:60 (

) acetonitrile:ammonium formate in water (25 mM ammonium/ammonia, 105 mM formic acid:formate, pH 3.2); injection volume, 2 µL.
	Test compounds are 1) acetophenone, 2) nortriptyline, 3) butyrophenone, 4) n-butylbenzoic acid.

	In Figure 3b, we see that the slopes of the trends (% change in 

) are pretty similar, with the exception of nortriptyline, which exhibits a much steeper slope. As discussed earlier, if the retention of all compounds changed at the same rate in response to a change in temperature, this would not be too much of a problem for method implementation, because resolution would not be affected too much. However, it appears from Figure 3b that the rate of retention for nortriptyline is on the order of two-fold higher than that of the other compounds. By plotting the selectivity of the separation against temperature we can obtain a more precise view of these changes in relative retention. Figure 4a shows α values for several pairs of the probe compounds, and Figure 4b shows the percent changes in these values vs. temperature. Here we see that there is no obvious change in the selectivity of the separation toward the neutral compounds (acetophenone and butyrophenone) as the temperature is increased from 24 to 42 °C. Even the selectivity of the separation for butyrophenone and n-butylbenzoic acid does not change, despite the fact that these are chemically quite different molecules. Even though the acid is ionogenic, at pH 3.2 it is mostly protonated and neutral. On the other hand, we see that the selectivity of the separation toward nortriptyline changes substantially over the range of temperature studied here. The alpha values for nortiptyline and butyrophenone (red circles) or butylbenzoic acid (blue circles) change by almost 5%.

(a) Retention factor and (b) percent change in retention factor for the test compounds over the range of room temperatures tested (21–42 °C).

(a) Selectivity and (b) percent change in selectivity for the test compounds over the range of room temperatures tested (21–42 °C).

Implications for Method Development and Implementation

	So far, we have observed that a change in room temperature can have a significant effect on the selectivity of a reversed-phase separation for some, but not all, types of molecules. The practically important question, then, is: Under what circumstances do changes of this kind really matter? The molecules I chose for this study are well separated (Figure 2), and even a 5% change in selectivity is not enough to bring them close enough together that they are no longer separated. Obviously, this will not always be the case. For separations where two neighboring peaks are just barely resolved at one room temperature, changing to a different room temperature could have a big effect on the resolution of that pair. We can quantify this effect using the Purnell equation for resolution, shown in equation 2.

	If we consider a situation with a plate number (

) of 5 for the later eluted of two peaks, and an α of 1.1, the resolution (

) of the two peaks will be about 2.3. We can use equation 2 to calculate the effect of a change in α on the resolution. Figure 5 shows how the resolution decreases as α decreases by up to 5% (that is, from 1.1 down to 1.045). Whereas a resolution of 2.3 corresponds to a nice separation where the valley between the peaks goes to baseline, at a resolution of 1.1 there is significant peak overlap. This overlap can significantly compromise quantitative accuracy of the method, particularly when one of the members of the peak pair is present at a concentration much higher than the other.

 Effect of a decrease in selectivity (α) on the resolution for a pair of closely eluted peaks. Resolution is calculated using the Purnell equation (equation 2) with 

	In this installment of “LC Troubleshooting,” we have examined the impact of a change in room temperature on the performance of reversed-phase LC separations when the column temperature is not controlled, such that the column takes on the temperature of the ambient room air. In most cases, an increase in temperature leads to a decrease in retention for reversed-phase separations. This will not affect resolution of neighboring peaks too much as long as the degree of shift in retention is similar for both peaks. However, this is not always the case, and significant changes in the selectivity of the separation on the order of several percent can occur. These changes in selectivity can in turn lead to a significant decrease (or increase) in the resolution of closely eluting peaks, on the order of 50%! Taken together, this means that:

	Specifying what constitutes “room temperature” during the method development will help prevent problems during method implementation, particularly if the method will be deployed in different laboratory environments where actual room temperatures may be very different.

	During method development, the robustness of the method should be characterized to help understand what the sensitivity of the method performance is to changes in column temperature.

	Controlling the column temperature with some means of thermostatting can help avoid these problems altogether, albeit at the cost of adding an instrument module for this purpose.

	I would like to thank Dr. Ravi Ravichandran for bringing his thought provoking questions about room temperature LC to me, and many subsequent helpful conversations on this topic.

		A.R. Horner, R.E. Wilson, S.R. Groskreutz, B.E. Murray, and S.G. Weber, 

https://doi.org/10.1016/j.chroma.2018.12.055

		J.D. Thompson, J.S. Brown, and P.W. Carr, 

Selectivity Optimization in Liquid Chromatography Using the Thermally Tuned Tandem Column (T3C) Concept

, Ph.D. Dissertation, University of Minnesota, 2001.	 

 is the editor of “LC Troubleshooting.” Stoll is a professor and co-chair of chemistry at Gustavus Adolphus College in St. Peter, Minnesota. His primary research focus is on the development of 2D-LC for both targeted and untargeted analyses. He has authored or coauthored more than 60 peer-reviewed publications and four book chapters in separation science and more than 100 conference presentations. He is also a member of LCGC’s editorial advisory board. Direct correspondence to: 

LC Troubleshooting

There are still many methods in use that have been developed for use at “room temperature.” With such a method, can one reasonably expect to obtain the same separation in Anchorage, Alaska, as in Mumbai, India?

Liquid Chromatography at Room Temperature: When Should the Column Temperature Be Specified in a Method?

This yearly report on new products introduced in the preceding year, since April 2019, covers sample preparation instrumentation, supplies, and accessories.

	
	New sample preparation technologies introduced in the past year were more bountiful than in previous years. Most product introductions were aimed at improved performance of existing technologies, or aimed at specific applications. Sorbents and accessories for solid-phase extraction (SPE) led the way, as did automated, multiplatform integration of sample preparation.

	Our annual review of sample preparation products covers the previous year. In late 2019, the 

 staff submitted a survey to vendors of sample preparation products. Responses to this survey are compiled in this review, as are new product introductions observed during the past 12 months. Additionally, after a keyword search (using the terms “sample preparation equip,” “extraction equipment,” “blend/grind/mix/shake/stir,” “evaporators/evaporation,” “filtration and purification,” and “pipets/pipetters”) of the on-line Pittcon 2020 vendor list, each of these vendors were visited during the conference.

	This review is presented in three sections. First, SPE sorbents and products are discussed. Next, advances in automated, multiplatform systems are presented. Finally, attention is turned to other sample preparation accessories and supporting technologies. To assist the reader with some of the details behind these new products, each section presents a tabular summary of the associated products. In all cases, the new products we uncovered are presented in the annotated table, while the text highlights particularly worthwhile products.

	Mixed-mode SPE has been gaining in popularity in recent years. Phenomenex introduced a new product in that space, under its Strata line, combining reversed-phase sample cleanup and matrix removal. The Strata-X Pro SPE is available in cartridge (1, 3, and 6 mL) and 96-well plate or microelution formats. The company claims time savings of up to 40% via elimination of the conditioning and equilibration steps. Table I summarizes the Strata-X Pro, and other SPE products.

	Orochem introduced two families of SPE products. The Agility Deluxe line of polymeric ion-exchange products is based on divinylbenzene (DVB) with strong- or weak- cation- or anion-exchange capabilities, and is available in cartridge, plate, and microelution formats. The company also introduced a series of SPE disks, featuring its Panthera Deluxe reversed-phase DVB. The thin disks with high sorbent capacity allow fast flow rates and use less elution solvent. The disks are designed for pesticides, herbicides, polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PCAHs), and total petroleum hydrocarbons (TPHs) from aqueous samples.

	Restek introduced several new products in its Resprep SFE line for method-specific performance. The Resprep PLR SPE is available in cartridge and well-plate formats, with proprietary material for sample cleanup. SPE materials feature graphitized carbon for organochlorine pesticide cleanup in soil and water, CarboPrep adsorbent for general utility use, EPH silica for petroleum hydrocarbons, florisil for pesticides, and a multipurpose silica. A novel co-sintered frit design, with the proprietary sorbents immobilized into the rigid, porous structure, allows a stabilized sorbent with high manufacturing control and performance uniformity.

	CDS Analytical acquired the Empore line from 3M during the past year. Styrene-DVB ion-exchange disks and cartridges and 96-well plates with C8, C18, styrene-DVB ion exchange, and a universal resin are available. The key feature of the original disk format is trapping of the sorbent particles within an inert matrix of polytetrafluoroethylene (PTFE) for a denser, more uniform extraction bed.

	Biotage developed the Atlantic ReadyDisk as single-use (disposable) disks with silica or DVB base material and trifunctional C18 or DVB for absorption or sample cleanup of aqueous samples according to the EPA 500, 600, or 8000 series methods.

	Porvair Sciences announced the Microlute PLR as a next generation microplate for phospholipid and protein removal from plasma and serum samples.

	Trapping columns for liquid chromatography and mass spectrometry (LC–MS) analysis were introduced in a variety of dimensions by Optimize Technologies. Opti-Lynx Micro, Opti-Trap, Exp 2 Nano, and Exp 2 Stem traps are placed into LC injector valves for sample concentration or purification. Various sizes accommodate a range of load rates and a number of phases are available.

	With the growing interest in cannabis testing, due to legalization of medicinal marijuana and industrial hemp, Agilent developed cannabis pesticide and mycotoxin testing kits consisting of 6-mL SPE cartridges and associated consumables. The kits includes consumables for processing 400 samples and method guidelines.

	In addition to SPE products, instrumentation to support or perform SPE was also introduced throughout the year. With the acquisition of the Empore SPE products, CDS Analytical introduced a number of related SPE processing equipment. For example, for working with 96-well plates, positive pressure and vacuum manifolds, centrifuges, and liquid handling robots are available. Orochem also introduced a family of positive-pressure processors under the Ezypress name. Sorbent bed drying, as well as faster analyte processing, are the advantages of this processing mode, especially with viscous samples.

	At the end of March 2020, Thermo Scientific-Dionex came out with the AutoTrace 280 per- and polyfluoroalkyl substances (PFAS)SPE instrument. The system is designed to simultaneously process up to six water samples according to EPA method 537.1 in 2 to 3 h, with only about 15 min of manual intervention. Minimization of PTFE-coated components reduces the risk of interferences.

	The Biotage VacMaster Disk is an SPE unit with a single port vacuum manifold to assist extraction of aqueous samples. Multiple units, up to eight, can be combined in parallel. Existing vacuum pumps can be used, and any disk holder or cartridge with luer fittings are allowed. Suggested applications of the VacMaster include EPA methods 625.1, 1664B, and 8270. Biotage also developed the Extrahera GLP software to manage the good laboratory practice (GLP) workflow with audit trail tracking.

	The EZprep123 from Fluid Management Systems processes six samples in parallel in less than 45 min for the isolation of dioxins, polychlorinated biphenyls, and polybrominated diphenyl ethers (PBDEs). This system is a companion to the EZSPE for water and waste water analysis, and the EZPFC for the extraction of polyfluorinated compounds in water and waste water.

	Given the number of steps in a typical sample preparation scheme (1), the role of automated, multiplatform instrumentation is of strong interest to suppliers of chromatography and sample preparation materials, and to the end users working at the laboratory bench. However, defining which sample preparation steps to include, and development of such equipment at an affordable cost, remain significant hurdles in this area. As such instrumentation continues to emerge, issues of reproducibility, labor intensity, and laboratory safety are addressed. The past year saw important new products in this area.

	Last year, Markes International introduced the Centri, a multiplatform sampling and concentration system for gas chromatography. This year, an expansion of the Centri platform was presented. Trap-enabled preconcentration, extraction, and enrichment makes the system attractive to a wider range of laboratories. High capacity sorptive extraction, headspace sampling, thermal desorption, and solid-phase microextraction (SPME), including the SPME Arrow, allow collection and concentration of a broad range volatile and semivolatile organic compounds. On-board diagnostics accommodates system self-checking. Table II presents a compilation of the automated, multiplatform product introductions. Improvements are also noted in the CTC Analytics PAL3 system, particularly in the area of automation. Smart SPME fibers contain a microchip to monitor extraction parameters and usage history. Polydimethylsiloxane (PDMS), acetate, DVB, and carbon fiber coatings are available. The Smart chip is also used in SPME Arrow and liquid handling syringes. The Sirius Automation MicroTasker is a modular platform with a laboratory information management system  interface featuring vial sorting and weighing, liquid handling, capping and uncapping, and barcode reading.

Sample Preparation Accessories and Related Products

	The field of sample preparation is very broad, and, coupled with the fact that there are any number of established and emerging technologies identified as sample preparation, commercial developments and new product introductions are often in seemingly scattered areas. An attempt is made here to unify these varied product offerings.

	Isolation of analytes via dispersion of an SPE-like particle is widely used and enhanced when magnetic particles are used. Phenomenex introduced bioZen streptavidin-coated paramagnetic beads, as depicted in Figure 1. The bead is activated with a biotinylated capture antibody to prepare for immunocapture of monoclonal antibody targets. After analyte isolation, the beads are recovered from the liquid sample by magnetic attraction. Table III provides an overview of the sample preparation accessories and related products.

	While standard centrifuges are available for test tubes and well-plates, MicroSolve Technology developed the Vial Centrifuge for use with 12 x 32 mm glass autosampler vials for applications in metabolomics, LC–MS, and bioanalysis. Heavy-walled glass vials have 1.2 or 1.8 mL capacity, and an air-flow system keeps the sample cool during centrifugation.

	The Gerstel PyroVial was introduced to the European market this past year, and is slated for release in other geographies. The PyroVial performs at up to 800 °C, and can be used as a microscale reaction chamber prior to LC–MS or GC–MS analysis.

	While SPME featured prominently in the automated, multiple-platform instruments previously discussed, stand-alone SPME products were also featured this year. Restek developed a triple-phase coating with DVB, carbon, and PDMS in standard fiber and SPME Arrow formats. A broad selectivity range allows analysis via headspace sampling or direct immersion.

	Agilent also developed a family of SPME fibers to complement its SPME Arrow line. Applications include the determination of a variety of environmental pollutants from water samples using a wide selection of stationary phase coatings.

	Advion worked with Oak Ridge National Laboratory to create the Touch Express Open Port Sampling Interface, which uses a low volume, open port of continuously swept solvent that flows directly into the its Expression compact mass spectrometer. Direct assays, screening, and large molecule determinations are possible.

	QuEChERS, the quick, easy, cheap, rugged, and safe extraction technique originally created for the screening of pesticide residues  in produce, continues to spawn new product introductions for food safety and other applications. Orochem developed a family of QuEChERS products designed for AOAC method 2007.01 products or specialty areas like fruits, pigmented proteins, hemp, and stevia.

	Shimadzu also created a QuEChERS kit designed for microscale analysis of biological or forensic samples. The Micro Volume kits use only 100 mg of extraction salts preloaded into 2.0 mL tubes.

	Supported liquid extraction combines features of liquid–liquid extraction with the surface area advantages of SPE. Agilent has been a major player in this field, and this year introduced Chem Elut S with a synthetic sorbent to replace diatomaceous earth supports. This more-consistent support material is available in 96-well plate, cartridge, and bulk formats. Applications include removal of salts and phospholipids from sample matrices.

	A number of filtration products were introduced in the past year. Agilent produced the Captiva Filter Vials with nylon, PES, PTFE, and RC filters in 0.2 µm or 0.45 µm pore sizes. The product will allow easier and faster filtration of LC or GC samples than using syringes, syringe filters, or standard vials.

	MilliporeSigma created a disposable polypropylene vacuum filtration unit for sample processing.

	Polymer Char’s External Filtration System is an automated device for eliminating carbon black, catalyst residue, and other small particles in polymer samples. The apparatus extends the lifetime of in-line filters and gel permeation columns and reduces noise in light scattering detectors.

	Pall introduced water-wettable PTFE membranes in its Acrodisk syringe filter line designed to protect LC columns from particulate build-up.

	Cornerstone Scientific presented the Antarisz filter media, glass microfiber disks for botanical extracts.

	Ecodyst marketed a family of Ecochyll high-speed solvent evaporators to aid solvent recovery in extraction laboratories. Models range from 5 to 200 L, and are energy efficient. Volatile or gaseous solvents, like butane or carbon dioxide, can be accommodated along with traditional organic solvents.

	We are all familiar with volumetric glassware, but Kimble advanced the state of the art with the first ever heavy-duty, wide-mouth flasks that reduce glass-to-glass contact during dilution workflows. ISO-certified manufacturing and flasks covering the standard range of volumetric flasks are available, with tight tolerances for accuracy and precision.

	Particle size reduction is often needed in the preparation of solid and semi-solid samples to shorten diffusion pathways for extraction, and to increase sample homogeneity. Recent new products in this area include the Pulverisette 14 line from Fritsch. This new sample grinder combines impact and cutting mill processes, with uniform production of particles down to 40 µm. The inclusion of pre-grinding and fine grinding into a single unit allows comminution of soft to medium hard, brittle, fibrous, and temperature-sensitive materials.

	SPEX introduced the Genolyte multipurpose homogenizer to accommodate plant and animal tissues, seeds, soils, minerals, and other samples in 2–12 mL vials. Sample temperatures between 0 and 10 °C are maintained.

	Organomation introduced the Nitro-Gen nitrogen generator. This instrument uses a hollow-fiber membrane to convert compressed air into 95–99% pure nitrogen gas. The generator couples with any of the company’s gas blowdown evaporators.

	No clear trends in new product introductions were readily observed, which is unsurprising, given the diversity of the field. On that basis, one can only expect the trend in coming years to be some combination tool building and problem solving for bioanalysis, food, environmental, and related areas.

“Sample Prep Perspectives” editor Douglas E. Raynie is a Department Head and Associate Professor at South Dakota State University. His research interests include green chemistry, alternative solvents, sample preparation, high-resolution chromatography, and bioprocessing in supercritical fluids. He earned his PhD in 1990 at Brigham Young University under the direction of Milton L. Lee. Raynie is a member of 

’s editorial advisory board. Direct correspondence about this column via e-mail to 

Sample Prep Perspectives

Our annual review of the new sample preparation instrumentation products introduced in the past year.

New Sample Preparation Products and Accessories

“GC Connections” presents the column’s annual review of new developments in the field of gas chromatography seen at Pittcon and other venues in the past 12 months.

	
	I am pleased to present our annual review of new products in gas chromatography (GC), introduced between spring of 2019 and spring of this year. Many of these products were showcased at the 71st meeting of the Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy (Pittcon) at McCormick Place in Chicago, Illinois, from February 29 to March 5, 2020. As has become tradition, the exposition lasted three days. Overall attendance was down significantly, due to coronavirus COVID-19 concerns. The exposition included 542 exhibitors and there were over 9000 attendees. Several international exhibitors canceled due to travel concerns. Four major players in the GC market, Agilent Technologies, Perkin Elmer, Thermo-Fisher Scientific, and Waters, did not exhibit.

	Pittcon 2021 will be held from March 6 to 10, 2021 in New Orleans, Louisiana. I strongly encourage all readers to consider attending a GC-related conference or participate in your local or regional chromatography discussion group in the next year. As you know, 2020 is a rough year for conference organizers and for all of the vendors, contractors, and economies that conferences support. They need your help. Just as importantly, conference and meeting attendance is still the most effective and efficient way to train and educate yourself about GC, and improve your skills and performance.

	Again this year, the exposition included vendor booths displaying new instrumentation and products, along with educational exhibits including Pittcon Park, the Laboratory Gauntlet, a planetarium, demonstration spaces, and a TED-style presentation theater on the exposition floor. Pittcon still has massive excellent content to offer to students and scientists at all levels of expertise and in all roles. In the demonstration spaces, many vendors performed working demonstrations of their products.

	The technical program at Pittcon 2020, which ran over five days from Sunday through Thursday, included a very strong GC presence. Pittcon continues to be a leading showcase for the latest GC and comprehensive two-dimensional gas chromatography (GCxGC) related research. Three major awards were presented to gas chromatographers this year. The Chromatography Forum of the Delaware Valley presented the Dal Nogare Award to Ron Majors, retired from Agilent Technologies and the former “Sample Prep Perspectives” and “Column Watch” editor for 

. Throughout his career, Majors made numerous GC-related contributions. The 

 Lifetime Achievement Award went to Daniel W. Armstrong of the University of Texas at Arlington. Among Armstrong’s many accomplishments is the development of ionic liquid stationary phases for capillary GC. Finally, the American Chemical Society Division of Analytical Chemistry Satinder Ahuja New Investigator Award in Separation Science was presented to. Katelynn Perrault from the University of Honolulu, Hawaii. Perrault is an up-and-coming leader in GCxGC, and a renowned expert in the application of GCxGC in forensic science.

	In addition to the award sessions, there were several symposia, oral sessions, poster sessions, and networking events with significant GC presence, enough to keep attendees very busy on all days. Overall, sessions related to cannabis analysis, biofuels, food and beverages, forensics, and general applications of both GC and GCxGC were offered.

	The information presented in this article is based on vendors’ responses to questionnaires and additional information from press releases, websites, and product literature, not on actual use or experience of the author. Every effort has been made to collect accurate information, but because of the preliminary nature of some of the material, 

 cannot be held responsible for errors or omissions. This column cannot be considered a complete record of all new GC products introduced in the past year, because not all vendors chose to respond to the questionnaire, and not all had a strong presence at Pittcon, nor is all of the submitted information necessarily included here, because of the limited available space and the editors’ judgment as to its suitability. The index in this article provides a listing of the vendors whose new GC products are covered in this review. For a more complete picture of the GC space today, I encourage you to review the two previous years’ reviews by Hinshaw (1,2).

	In new instruments, trends seen over the past few years toward smaller, more automated systems, systems that are more specialized, and spectrometric detectors continue with the product introductions this year. Table I shows a list of new instruments introduced over the past year.

	Capillary columns continue to be updated by most of the column vendors. The three vendors highlighted in Table II provide examples of three important trends in column development: lower prices, specialized columns for specific systems, and new column chemistries geared toward specific problems.

	Developments in new accessories and consumables trend toward simplifying analysis, and include a range of devices from sample vials and sampling devices through data system software. For more information on sample preparation, see the specific sample preparation article by Raynie in this issue of 

(3). As with the GC reviews, it is worthwhile to review the past two years as well for a complete picture of sample preparation products (4,5). Table III provides a listing of accessories and consumables launched over the past year.

	Besides several short courses associated with Pittcon, additional training and educational resources are available for 2020. Opportunities for in-person short courses, seminars, and training are offered by most major instrument vendors and through many conferences and organizations, including the American Chemical Society, Eastern Analytical Symposium (Princeton, New Jersey, November 2020) and local and regional chromatography discussion groups. 

’s on-line training partner, ChromAcademy, has launched a completely revamped on-line platform. A new edition of McNair and Miller’s classic book “Basic Gas Chromatography” with Nicholas Snow added as an author was published this year. New developments in training and education for 2019–2020 are shown in Table IV.

	As GC approaches 70 years since its invention, development and invention are still going strong. As seen here and in 2020, GC-related developments and innovations are ongoing and include all areas: sampling and supplies, new instruments, columns, detectors, data systems, and educational opportunities. I look forward to more innovation and advancements as the decade progresses, and I hope to see many of you at EAS 2020 in my home state of New Jersey, or at Pittcon 2021 in New Orleans.

 is the Founding Endowed Professor in the Department of Chemistry and Biochemistry at Seton Hall University, and an Adjunct Professor of Medical Science. During his 30 years as a chromatographer, he has published more than 70 refereed articles and book chapters and has given more than 200 presentations and short courses. He is interested in the fundamentals and applications of separation science, especially gas chromatography, sampling, and sample preparation for chemical analysis. His research group is very active, with ongoing projects using GC, GC–MS, two-dimensional GC, and extraction methods including headspace, liquid–liquid extraction, and solid-phase microextraction. Direct correspondence to: LCGCedit@mmhgroup.com

GC Connections

Our annual review of new gas chromatography (GC) products introduced in the past 12 months.

New GC Products Released for 2019–2020

The manufacture of pharmaceutical products is classed as critical work, and thus is continuing during the COVID-19 pandemic. With limitations of travel and requirements for social distancing, how should you maintain the calibration and qualification of chromatographs to ensure data integrity of results during this pandemic?

	
	Over the past three months, as coronavirus or COVID-19 has spread from China around the world, many counties have engaged in various forms of lockdown, social distancing, or travel restrictions to prevent the spread of the virus. The focus in the news has naturally been on the number of infections identified, the number of patients in hospitals, and, sadly, on the number of deaths. In the background, the pharmaceutical and medical device industries are still working to produce medicines such as acetaminophen (paracetamol) to alleviate the fever caused by the virus, developing potential vaccines, and conducting research to see if any existing medicines can combat the disease.

	During this pandemic, it can be easy to forget that we are a regulated industry, and, in the laboratory, chromatographs must be qualified, calibrated, and maintained. To ensure that this is the case, inspections are carried out to ensure that laboratories are complying with regulatory requirements. However, the direct impact of the pandemic on regulated laboratories is an important subject that needs to be considered. In some instances, facilities are restricting access to sites to try to minimize potential risk of exposure for critical employees, while, in others, restrictions may prevent service engineers from traveling. Both issues have the potential to affect compliance in a laboratory. In the absence of inspections and service engineers, how will you keep your chromatographs operating reliably and ensuring data integrity?

Why Are Qualification and Calibration Important to Data Integrity?

	A data integrity model has been presented and discussed in an earlier “Data Integrity Focus” article (1), and also in my book, 

	The model consists of four layers. The foundation layers consist of management leadership, quality culture and ethics, procedures, and training. Level 1 requires the right analytical instrument for the job. The importance of Level 1 to data integrity is if you don’t know how an instrument performs, how can you trust the results it generates? Therefore, at Level 1, analytical instruments must be qualified under 

 on Analytical Instrument Qualification (3), and comply with the requirements of 21 

 Chapter 3 clauses 3.34 and 3.35 (5). As the FDA issued 17 (211.63) and 37 (160[b][4]) 483 observations against these two 

 codes in 2019, these areas are on an inspector’s radar (6). We discuss two of these regulations later in this article.

	Let us have a look at what is happening with two regulatory agencies: the Food and Drug Administration (FDA) in the United States, and the Medicines and Healthcare Products Regulatory Agency (MHRA) in the United Kingdom.

	In a press statement issued on March 10th of this year, the FDA announced that they were postponing all foreign inspections due to travel advice from the State Department that prohibits foreign travel by U.S. government employees. In addition, there are actions by non-U.S. governments that require overseas travelers to self-isolate for 14 days on arrival, or prohibit any foreign travelers arriving at all. Therefore, given that the FDA are unable to physically inspect foreign companies, they will gather information under Mutual Recognition Agreements with other regulatory authorities, and revert to physical inspection and possible testing at the U.S. border, as they have done since the start of the outbreak in China when inspections were halted there (7).

	Later, the FDA announced on March 18th that domestic routine surveillance inspections were being postponed for the safety of the inspectors, and also due to industry concerns about visitors. However, for-cause inspections would be risk assessed, and only critical ones would be carried out (8).

	In the United Kingdom, the MHRA posted a statement on the web site on March 20th, stating that they are only conducting essential good practice (GxP) inspections associated with the UK’s response to COVID-19, and all other inspections will be deferred to a later date (9). The same post noted that:

We are expecting organizations to maintain GxP compliance, and will support the industry and NHS to focus on service continuity by using alternative approaches for routine regulatory oversight, such as office-based assessment and the sharing of information within the international regulatory network.

	On March 30th, there was explicit advice posted with specific guidance for 

Still important to maintain compliance with principles of GLP.

	David Churchward expanded the overall approach in an MHRA blog explaining that inspectors were working from home as much as possible. Office-based inspections consisted of a request of documents from a company by e-mail, and, after review, an inspector would follow up with either e-mail questions or a teleconference (11).

	Although you may not see an inspector for a time, which in some circles may be a cause for celebration, there is still the regulatory expectation that you remain in compliance. So that is all well and good.

	However, consider what happens if you have a chromatograph that is due for a preventative maintenance (PM) visit and annual operational qualification (OQ), and the engineer can’t get to you, or your company won’t let them come on-site. Grace periods for instruments typically only last for one to four weeks. Let’s look closer at the details of two regulations to help us.

Regulations for Qualification and Calibration

The calibration of instruments, apparatus, gauges, and recording devices at suitable intervals in accordance with an established written program containing specific directions, schedules, limits for accuracy and precision, and provisions for remedial action in the event accuracy and/or precision limits are not met. 

Instruments, apparatus, gauges, and recording devices not meeting established specifications shall not be used 

This means that the specifications of the chromatograph should be documented in a user requirement specification (URS) or equivalent under 

Repair and maintenance operations should not present any hazard to the quality of the products (5).

	If any repairs or maintenance are made to a chromatograph, then it must be partially or fully requalified to demonstrate fitness for purpose for analysis during the production of investigational medicinal products (IMPs) or pharmaceutical products.

What Happens to Service and Qualification in Lockdown?

	Apocryphally, the way companies perform qualification and maintenance breaks into two groups. Approximately two thirds of companies will use a third party to service, maintain, and qualify their analytical instruments. Larger sites with a lot of instrumentation from a single supplier, or, more likely with a service provider able to service, maintain, and qualify a range of instruments from different suppliers, may have a service engineer stationed permanently on-site. In this case, the engineer is known, and may be treated differently from one who travels to the site. The remainder of companies (approximately one third) use a combination of in-house engineering and metrology groups to service and qualify their instruments, and only use the original supplier if there are major breakdowns (for example, if the instrument cannot be used).

	The problem with the inspectors now comes to service engineers. Are they counted as essential workers when a state or country is in lockdown? Even if they could travel, would an organization allow them on-site, where they could potentially infect staff? Normally, companies may only consider risk to the patient from the impact of errors, mistakes, or problems (which is covered later in this article). In this situation, batches are not released where there is unacceptable risk. However, in this pandemic, failure to supply may represent a greater risk to the patient, than the risks identified if instruments are used with limited compliance. For example, in March 2020, the FDA issued guidance for industry reminding companies of the requirement to notify the FDA of permanent discontinuance or interruption in manufacturing (12). Failure to do so could result in the FDA issuing a noncompliance letter. While failure to supply is a risk to patient, how should firms balance the potential risks of noncompliance? Where does ethics come into this equation?

	Consider what happens if you have a chromatograph that requires a PM followed by an OQ now.

	We must consider a number of risks, and the management of them, in this unusual situation.

	Typically, a laboratory has multiple chromatographs at its disposal. If the PM/OQ dates of the instruments are spread throughout the year, it might be possible to switch from one instrument to another to mitigate the risk associated with the lack of maintenance and qualification. However, some organizations may synchronize their maintenance and qualification visits over a short period of time, to save on an engineer’s travel time and costs. In this situation, all chromatographs can be out of maintenance and qualification at virtually the same time.

	However, if the lockdown continues, it is likely that the dates of maintenance and qualification of all chromatographs will become overdue. Understand that, at the expiry of a PM or OQ due date and any associated grace period, it does not necessarily mean that a qualification pixie waves a magic wand and all chromatographs are no longer calibrated or qualified and they must not be used. However, there is a deviation from the maintenance and qualification schedule that must be documented.

	If chromatographs are dedicated to specific methods, one way to increase flexibility could be to use an instrument for any analysis. If this is the case, a laboratory should check the ranges of qualification performed on the instrument to ensure that they are not exceeded with the new analysis. If this is the case, a laboratory must ensure that the liquid chromatography (LC) system is thoroughly washed and flushed through between different analyses, including autosampler needle washes; otherwise, problems may occur.

	These gaps and the associated risks must be assessed, evaluated, controlled, and, above all, documented. As part of the risk control strategy, overdue maintenance and qualification might be justified and based on data, such as:

		If an LC instrument is not used frequently, it might not require maintenance even if overdue.

		The total number of sequences and injections might increase or decrease the overall risk.

		The type of therapeutic product being analyzed on the instruments in question (and the product’s therapeutic range, range of individual parameters measured, route of administration, age of patients treated, and so on) might also have an impact on the severity of an over- or understrength product being released to the market (if the instrument used to ensure the products’ quality were out of calibration at the time of analysis).

	Trending of test results, as required by EU

 Chapter 6.9 (13), might also support any risk acceptance.

	The risks associated with any action are dependent on the context from which the actions are considered. So, in this instance, the risks to the patient associated with failure to supply pharmaceuticals must be balanced against the risks that result from instruments that cannot be fully maintained or qualified. If the instruments are not in use, and will remain so, the only risks are the possible noncompliance with procedures, such as grace periods associated with maintenance, and qualification and consideration of how to return the instruments to use in a compliant way after the period of disruption. It is probable that, in the post COVID-19 period, the demand for service personnel will spike and will exceed capability. Companies with a service contract will be prioritized over those that request on-demand services. If your laboratory falls into the latter category, be
	prepared for a long wait for service and requalification.

	If a decision is made to continue using instruments outside of maintenance and qualification grace periods, the principal types of risk associated with this action include:

		Breakdown: Instruments not maintained are more likely to break down during use.

		Regulatory: Justification of actions when the next inspection occurs. This can include defending the decision to continue using the instruments, and documenting scientifically that using instruments without maintenance and full qualification were under control and did not impact

		Patient: Potential risk that substandard or over-strength pharmaceutical may be released.

	Any decision to use an instrument with overdue maintenance or qualification must be documented carefully, and in a transparent manner. The worst possible way to document a sensitive decision such as this would be to try to hide it. Historically, companies may have issued a miniature risk assessment or file note that documented sensitive decisions, but hiding where the document is buried with no traceability to it within corporate quality systems. Such action would now be interpreted as running a secondary quality system from a data integrity perspective (14).

What Can Go Wrong with an HPLC Instrument?

	If your laboratory cannot obtain service, and you decide to continue using your chromatographs, you must perform an enhanced risk assessment to support this decision. You need to try to identify problems with a chromatograph before and during any analysis. In 2015, Paul Smith and I published a “Questions of Quality” article in 

 that is the basis of this section about risk assessment of a high performance liquid chromatography (HPLC) instrument (15). The starting point of our discussion was what can go wrong with an operational HPLC system. The risk assessment article was written from an HPLC perspective, but the principles outlined here (such as, for example, considering how an instrument may fail, and taking appropriate actions to minimize the potential risk associated with the failure) apply equally to other instruments. Because failure modes can be different between different analytical technologies, it is likely that risk assessments for each analytical technique may be needed.

	The HPLC risk assessment from 2015 is shown in Figure 1, where a liquid chromatograph consists of four modules: the pump, the injector (autosampler), the column oven, and the detector. The column has been omitted from the figure, as the aim is to look at an LC instrument’s qualification rather than method performance. Underneath each module are listed the main failures that could occur. Note that this list is not exhaustive, and some of the failures could be broken down further. However, to keep the discussion simple, the problem is looked at from a high level. Some failures may not happen if the instrument in your laboratory does not have a particular feature. For example, for an isocratic pump there will not be gradient errors, or with a fixed wavelength detector the wrong wavelength will not be used.

	Having listed the main failures, we now need to consider the circumstances under which these might be detected in an OQ or performance qualification (PQ), as shown in Table I. The PQ is broken down into three areas where a failure could be detected: during a system suitability test (SST), by the instrument itself, and during what is called 

, where parameters can be measured if the SST is designed to include them, and these can be traced back to the instrument’s user specification.

I’m Overdue for a PM and OQ, But No Engineer is Available

	So let’s consider a scenario in which your instrument is overdue for a PM and OQ, and there is no service engineer available for the foreseeable future. Who are you going to call? At this point, the Ghostbusters are probably not a viable option. What are you going to do?

	If no PM occurs then and there, one potential impact is an increased risk of a catastrophic failure of the chromatograph, as shown in Figure 2. This is the simplest situation; the instrument becomes inoperative, and there is no further work possible until it has been repaired and requalified. There is, therefore, no impact if there is no OQ, no PQ, or even if there is a poor SST for the method.

	A more difficult situation arises if there is no PM visit, but the instrument is still operating, because there could be a change in instrument performance over time. Variable performance could result in variable chromatography. Such variability might be detected, but a slow drift in performance that results in method values being outside set points and limits tested during qualification would be much harder to detect, and potentially more serious. As is typical in many laboratories, PM is usually followed by an annual OQ. But in the scenario we are considering, there is no service engineer available, your grace period has expired, probably along with most of the QA department, and no OQ has been performed. This is the situation shown in Figure 3.

	The first issue with no qualification or calibration, per 21 

 211.160[b][4], is there is now an immediate regulatory gap with a likelihood of a citation. In addition, given that there is no confirmation that the system is working as intended, there is a risk of not detecting any instrument performance failure. The next fallback would be if the PQ can demonstrate that the instrument meets its intended use as defined in the laboratory URS.

	As shown in a recent “Focus on Quality” column that I wrote with Paul Smith, where we discussed PQ for infrared instruments (16), and in the third part of a “Questions of Quality” series on 

 for liquid chromatographs (17), PQ is defined by 

PQ is the documented collection of activities necessary to demonstrate that an instrument consistently performs according to the specifications defined by the user, and is appropriate for the intended use.

The PQ verifies the fitness for purpose of the instrument under actual conditions of use. After IQ (instrument qualification) and OQ have been performed, the instrument’s continued suitability for its intended use is demonstrated through continued PQ (3).

	You will see, from the definition above, that the PQ is linked back to the URS. Therefore, in the absence of a PM and a current OQ, demonstrating that your instrument still meets its intended use, as defined in the URS, is now your only way for you to check that a chromatograph is still functioning correctly.

	We will come back to discuss this after finding out what is involved in a PQ. The PQ is one part of the analytical instrument qualification (AIQ) continuum that is shown in Figure 4 to demonstrate that the user specifications are being met throughout the use of an instrument.

		a PQ test plan covering the scope of work to be performed (ideally also risk assessed)

		holistic or method SST with acceptance criteria (3).

	Of the items in the list above, only the SSTs can be used to check that the instrument performs as required by the user specifications.

	Performing a gap analysis between instrument failure modes and potential detectability of the failure (based on the possible impact on SST) should highlight which failure modes will not be detected with current ways of working.

Performance Qualification Comes to the Fore

	In the situation of a missed or delayed OQ due the non-availability of an engineer, the PQ takes on increased significance, as can be seen in Table I and discussed above. Many of the parameters that would be measured directly in the OQ can be indirectly measured in the PQ, as long as the PQ tests applied are related to the laboratory URS, as shown in Figure 4 (17,18). If a laboratory has invested the time and effort in designing appropriate instrument life cycle processes as an integrated AIQ continuum shown in Figure 4, that work will help them remain compliant. For example, if meaningful SST parameters can be linked to the user requirements for the instrument, these companies will be in a stronger position to defend and justify that the potential compliance risks associated with continuing to use instruments outside of grace periods, or even where an OQ is not performed.

	Companies that historically perform what might be considered the bare minimum may find themselves facing interesting times when inspections resume. For example, a company that performs an SST measuring only column plate count will find it difficult to provide scientifically sound evidence to support an impact assessment because they simply don’t have appropriate data to support
	this decision.

	If your approach to SST leans toward the minimum, then, for the period of time the instrument is going to be used outside of grace periods, you should very seriously consider enhancing both chromatography (method) based SST, such as allowed limits on named peaks, and also consider adding non-method-specific point-of-use instrument tests, such as flow rate and temperature measurements.

	As part of an overall approach to PQ, there should be a holistic test that can show the user requirements are still being met. Analytical procedures should routinely be designed to be as robust as possible (19). However, the principle of a good holistic PQ test is to design an analytical procedure that is sensitive to instrument performance (which is the opposite of normal analytical science). This is the approach used for performance verification testing (PVT) of dissolution instruments, which is universally interpreted as a PQ for these instruments.

	Ideally, the procedure must use stable model compounds, with simple, stable, and robust chromatography to minimize analytical variance from the reference materials or use (17). Standard solutions and mobile phases should be prepared gravimetrically, again to reduce variance, so that instrument problems have a greater probability of being detected. The performance of the procedure is therefore dependent on instrument performance, and can then be related to user requirements, as shown in Figure 4 and Table I. These overall holistic standards allow limits to be set for:

Viewpoints

How should you maintain the calibration and qualification of chromatographs during the COVID-19 pandemic? Here, we discuss the details of how to ensure data integrity of results when faced with travel limitations and requirements for social distancing.

COVID-19 and Data Integrity: What Does Lockdown Mean for Chromatograph Qualification and Calibration?

A single laboratory validation study (SLV) was performed using an ultrahigh-pressure liquid chromatography (UHPLC) for the determination of coenzyme Q10 in a variety of formulations (raw materials, soft gel capsules, and in-process blend). The authors first developed an efficient extraction procedure to obtain coenzyme Q10 from finished products, and then used UHPLC to analyze the ingredient. Compared to the traditional high-performance liquid chromatography (HPLC) method, the UHPLC method reduced the total run time from 15 min to less than 2 min. The total amount of solvent usage was also reduced by 90%, which is a big contribution to green chemistry. The established UHPLC method exhibits good linearity, reproducibility, accuracy, recovery, and specificity. The limit of detection (LOD) and limit of quantification (LOQ) were also captured in the study, and have been proven suitable for monitoring the coenzyme Q10 in complex matrices.

	Coenzyme Q10 (Co Q10) is a nutrient that the body produces naturally for cell growth and maintenance. Research have shown that Co Q10 can lower blood pressure slightly (1). Co Q10 is also used to treat heart failure and other heart conditions (2). It acts as an antioxidant that protects cells from the effects of aging, and it is found inside the mitochondria of most cells; it is also believed that Co Q10 improves the tolerability of cancer treatment, and reduces liver toxicity (3). In addition, Co Q10 is used in prevention and treatment of cardiovascular disease, particularly hypertension, hyperlipidemia, coronary artery disease, and heart failure (4). Other research has reported that Co Q10 helps support the production of cellular energy (5–7). In nature, Co Q10 is found in meat, fish, and whole grain. However, the amount of Co Q10 found in these dietary sources is not high enough to significantly increase the Co Q10 levels in the body. Therefore, it is considered beneficial to take Co Q10 as a dietary supplement.

	Co Q10 can exist in three oxidation states: the fully reduced ubiquinol form, the radical semiquinone intermediate form, and the fully oxidized ubiquinone form (8). The structures of each form are shown in Figure 1. Unlike conventional Co Q10, ubiquinol is a very powerful antioxidant by virtue of its two extra electrons. Those electrons are important because they hold the key to neutralizing free radicals (9). Therefore, ubiquinol is a preferred supplement and is used in our products.

	Ultrahigh-pressure liquid chromatography (UHPLC) is a modern technique that has advanced liquid chromatography (10–17). In 2004, separation science was revolutionized with the introduction of UHPLC. Significant advances in instrumentation and column technology were made to achieve dramatic increases in resolution, speed, and sensitivity in liquid chromatography. For the first time, a holistic approach involving simultaneous innovation in practice, technology, and instrument design came together  to meet and overcome challenges in the analytical laboratory.

	Method validation is the process used to confirm that an analytical procedure employed for a specific test is suitable for its intended use. Results from method validation can be used to judge the quality, reliability, and consistency of analytical results. Therefore, it is an integral part of good analytical practice. The U.S. Food and Drug Administration (FDA), the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH), the Environmental Protection Agency (EPA), and other regulatory agencies and industrial committees have clear guidelines on how and when to validate and revalidate a method (18–23).

	In the work presented here, the authors have developed a UHPLC method for quantitatively analyzing Co Q10 raw materials and finished products. This UHPLC method has been validated to prove that it is suitable for its intended use regardless of the matrix. An efficient extraction method was developed to extract Co Q10 from various complex matrices.

 chapter <1225>, Validation of Compendial Procedures. Specific calculations were used for accuracy, precision, linearity, limit of detection (LOD), and limit of quantification (LOQ) through various Excel functions.

	Coenzyme Q10 reference standard (Cat# 1705301) was purchased from USP. The coenzyme Q10 200 mg USP sample (A640, lot# 2253063) and coenzyme Q10 raw material (394403, lot# 1704102-1) were from Pharmavite LLC. Reagent alcohol (HPLC Grade), acetonitrile (HPLC grade), and hexanes (HPLC grade) were purchased from EMD Millipore Corporation.

	A Kinetex C18, 2.1 mm X 50 mm, 2.6-µm column (Cat # 00B-4462-AN) was purchased from Phenomenex, Inc.  The mobile phase is a mixture of 30% acetonitrile and 70% reagent alcohol. The mobile phase flow rate was 0.5 mL/min. Total run time was 2 min.

	All solvents were filtered before use through a 0.45 µm GMF filter from GE Healthcare Whatman.

	A Waters Acquity UHPLC system with a PDA or TUV detector was used. The UHPLC system was operated using Waters Empower 3 software for data acquisition and storage.

	The coenzyme Q10 standard was dissolved in hexane (25% of the flask volume) by shaking with a wrist shaker. The sample was further diluted with reagent alcohol to a final concentration of the standard and sample of 0.04 mg/mL. Sample and standard were then filtered through a 0.45 µm GMF filter before injection into the UHPLC instrument. The sample preparation method for raw materials, in-process blends, and soft gel capsules were the same as for the standard.

	The column efficiency for coenzyme Q10 was >2000 theoretical plates. The USP tailing factor was ≤1.2.

	The following samples were injected: blank, placebo, standard, and sample. Retention time of the Co Q10 was 0.62 min in the chromatogram, which corresponds to those in the chromatogram of the standard solution, and no interfering peaks were present at the retention time of the active peak in the blank and placebo (Figure 2).

	The linear dynamic range established covers a concentration of the standard from 0.02 mg/mL to 0.08 mg/mL, which is 50% to 200% of the sample concentration used in the analyses. The linearity data is presented in Table I and Figure 3. The 

 is not less than 0.9995. The calibration curve was constructed by the plotting peak area of the standard versus concentration (50%, 75%, 100%, 150%, and 200%).

	Detection limit was shown to be sufficiently low, with known concentration above and below the required detection level. The lowest concentration for coenzyme Q10 was 0.0005 mg/mL.  

	The quantitation limit was shown to be sufficiently low, with known concentration above and below the required detection level. The limit was determined by injecting 10 different concentrations of the standard. The lowest concentration by this method was 0.004 mg/mL (Table II, Figure 4), which is within the linearity range of this testing method.  

	Precision was determined by analyzing six separate samples prepared from a homogeneous sample matrix under the prescribed conditions. The results are presented in Table III for raw materials, and Table IV for finished products. The percent  relative standard deviation (% RSD) of six replicate injections for four sets of precision results were from 0.63% to 2.30%, which were well within the acceptance criteria for precision of <3% RSD.

	The accuracy study was performed with nine samples containing three concentration levels (50%, 100%, and 150%). A placebo containing all excipients in the dosage form except for Co Q10 was spiked with freshly prepared concentration levels, according to the sample preparation procedure. The placebo sample was also analyzed to ensure that there was no interference (Table V). The average recovery of Co Q10 from the sample matrix was 99.2%–101.5%, which is within the acceptable range of 90 to 110%.

A fast ultrahigh-pressure liquid chromatography (UHPLC) method, with low solvent consumption, is described for the determination of coenzyme Q10 in a variety of complex formulations. This method exhibits good linearity, reproducibility, accuracy, recovery, and specificity, while resulting in an acceptable limit of detection (LOD) and limit of quantification (LOQ).

LCGC North America-05-01-2020

Vol 38 No 5 LCGC North America May 2020 Regular Issue PDF

Liquid Chromatography at Room Temperature: When Should the Column Temperature Be Specified in a Method?

New Sample Preparation Products and Accessories

New GC Products Released for 2019–2020

COVID-19 and Data Integrity: What Does Lockdown Mean for Chromatograph Qualification and Calibration?

Determination and Validation of a Single Laboratory Method for Coenzyme Q10 (Ubiquinone) by UHPLC