Erin E. Chambers, Jessalynn Wheaton and Diane M. Diehl,

 Waters Corporation, Milford, Massachusetts, USA. 

Streamlined sample prep, LC and MS method development.

Complete LC and SPE screening protocols provided.

15× sample concentration without evaporation.

Losses due to poor solubility and adsorption make sample concentration without evaporation highly desirable.

Obtaining selectivity for peptide therapeutics in complex biological matrices is crucial in order to minimize matrix effects and reduce variability associated with incurred samples.

Sensitivity by mass spectrometry is often lower for biomolecules than typical small molecules, necessitating sample concentration and use of the highest sensitivity LC and MS methodologies.

An MS system with an adequate mass range is required for detection of higher 

A Peptide Separation Technology (PST) method development kit was was developed and is available for ACQUITY UPLC (part#176001835) or HPLC (part#176001836.) The kit contains a chromatographic column, the Oasis μElution PST method development extraction plate and simple, logical SPE and LC screening protocols. ACQUITY UPLC technology was used to increase sensitivity, resolution and separation speed. Representative LC–MS performance using the UPLC screening protocol from the kit is shown in Figure 1. It should be noted that a critical pair, vasopressin and desmopressin (differing only by an amino group), are well separated using the generic ACQUITY UPLC method.  

Samples were extracted using the Oasis μElution PST method development extraction plate according to the generic screening protocol included in the kit. SPE results are shown in Figure 2. The Xevo TQ MS was chosen for this application because the mass range on both quadrupoles is 

 2048, a key requirement for bioanalysis of a wide range of peptides. Figure 3 demonstrates a representative LLOQ for angiotensin I extracted from human plasma using the generic LC and SPE screening protocols without optimization.  

The Oasis PST μElution method development plate and protocol can greatly simplify extraction method development and rapidly identifies an appropriate SPE sorbent and selective starting protocol for peptide bioanalysis. The combination of ACQUITY UPLC coupled with the mass range, speed and sensitivity of Xevo TQ MS provides the LC–MS performance required for peptide bioanalysis.  

For the complete UPLC application note, visit 

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Simplifying Peptide Bioanalysis

In gas chromatography (GC), the carrier gas serves as the mobile phase and carries (moves) the solutes down the column. The selection and linear velocity (flow rate) of the carrier gas influences efficiency and retention time. A carrier gas must be inert to solutes and stationary phase, must be free of detectable contaminants, must be readily available at a reasonable price, and must have a leak-free and prodigiously precise pressure–flow delivery system for precise qualitative and quantitative data from the gas chromatograph.  

Commonly used and popular carrier gases include helium, hydrogen, and nitrogen, although argon, ammonia, and carbon dioxide also have been used to a minor extent. Also, hydrogen has long been used as a fuel gas for flame ionization detection (FID) as well as other detection methods in GC. In this application, the hydrogen purity with respect to oxygen and water is not particularly critical. However, the hydrocarbon content of this gas must be minimized to keep low background noise. Helium has been the most widely used carrier gas for GC due to its inertness, good purity, excellent performance, and well-established methodologies. In recent years, the demand for helium has outstripped the supply, resulting in limited supplies in certain geographies, increased costs, and uncertain delivery. Normally produced by radioactive elemental decay, it is more expensive to isolate compared to nitrogen or hydrogen. Although it displays the lowest minimum plate height compared with that of helium or hydrogen, nitrogen has a much narrower velocity range and a steeper van Deemter curve, so at higher flow rates, solute efficiency drops off dramatically (Figure 1). With the shortage and expense of helium and the less desirable properties of nitrogen, gas chromatographers have been looking toward hydrogen as a carrier-gas alternative.       

The choice of carrier gas can have an effect on the appearance of the chromatogram and plays an important role in GC optimization. Compared to high performance liquid chromatography (HPLC), in which the mobile phase has profound influence on the chromatography, the carrier gas in GC has a much smaller effect because it rarely interacts chemically with the stationary phase or solutes and serves mainly to volatilize the solutes under the influence of temperature. Two properties of a gas play a role in the chromatographic process: diffusivity and viscosity. The diffusion speed of an analyte in the gas determines the speed of GC. Analytes need to spend time in both the stationary phase and the carrier gas to separate. The diffusivity of hydrogen and helium are roughly the same, but nitrogen has a value that is roughly three to four times lower than helium, which means that the slower diffusion results in longer separation times. If one tries to speed up the flow rate with nitrogen carrier, then the van Deemter curve (Figure 1) shows that the efficiency will fall off dramatically.  

The viscosity of the carrier gas will determine the inlet pressure required for a given gas velocity. High inlet pressure compresses the gas at the column inlet, and as the gas moves through the column, its linear velocity will change. Normally, for capillary columns, one tries to achieve an average linear velocity of around 20–25 cm/s. Hydrogen has about half the viscosity of helium and nitrogen, and at typical column lengths in the range of 15–30 m, it provides the best combination of efficiency and speed over the widest range of linear velocities. Its optimum carrier-gas velocity is around 60 cm/s so, relative to the other popular gases, separations are usually faster. So, if hydrogen has favorable properties and is less expensive, why hasn't it been endorsed universally by gas chromatographers? The answer is "Safety"! 

Safety Aspects of Hydrogen as a Carrier Gas in GC

Almost everybody remembers the vivid picture of the Hindenburg airship exploding above Lakehurst, New Jersey in May 1937. It was filled with hydrogen and even today, what caused the explosion is still debated. So, the fear of hydrogen igniting in an accidental leak of hydrogen in a laboratory environment lingers in the minds of many chemists. In fact, over the many years of the practice of GC in laboratories throughout the world, a relatively low number of documented reports of hydrogen carrier gas causing explosions has been reported (1). Most of the explosions have occurred with older instruments that had high heating power and small heater filaments that turned red hot when heated ballistically. Newer instruments with less heating power and larger heater filaments hardly reach the temperature of hydrogen ignition. Nevertheless, the flammable nature of hydrogen and the high pressure of cylinders (2400 psig) present concerns over cylinder storage and handling in laboratories.  

Hydrogen is potentially explosive in air in concentrations between 4% and 74.2%. It has the highest burning velocity of any gas and a very low ignition energy. Hydrogen that expands rapidly from high pressure can self-ignite. It burns with a nonluminous flame that can be invisible under bright light. 

To be ignited, a mixture of hydrogen and air must be heated to about 630 °C. This value is in contrast to solvent vapors that can ignite at 0.1% concentration and at temperatures as low as 200 °C.  

Let's do a calculation to see how likely it would be for laboratory concentration to build up to an explosive level. Assume that a small laboratory would have the dimensions of 6 m X 4 m X 3 m, which is equivalent to a total volume of 72 m

 . To reach the minimum of 4% by volume or 2.88 m

  (2880 L), for a hydrogen gas leak or flow of 500 mL/min, 5790 min (96 h or 4 days) would be required. This is assuming no ventilation in the laboratory. Most laboratories have a requirement of a minimum acceptable ventilation rate of five air replacements per hour (2). In our example this would amount to 360 m

 /h. It would be very difficult to build up such a concentration in the laboratory as a whole. 

Let's now consider a local concentration buildup. Hydrogen has a diffusion coefficient of 0.61 cm m

 /s in air (about four times greater than the diffusion coefficient of air). Hydrogen has a density of 0.085 kg/m

  at 293 K. Air has a density of 1.202 kg/m

  at 285 K; this density is about 14 times greater than hydrogen. Thus, any hydrogen emitted into the air floats rapidly upward and diffuses quickly — think about a helium-filled balloon. Hydrogen is half as dense as helium, which was one of the reasons it was used in the Hindenburg. 

For small leaks, it would be very difficult to get local buildup of hydrogen concentrations exceeding 4%. Of course, any air movement due to ventilation greatly facilitates this dissipation process by convection.  

Table I: Comparison of properties of hydrogen and natural gas 

It turns out that the natural gas that many use for cooking is far more dangerous than hydrogen. Table I shows some of the data of the two gases. In reality, natural gas has three times the calorific than hydrogen and diffuses three times more slowly in air. It is 10 times as dense and sinks to the floor and collects in "pools" where a spark or static charge could ignite the natural gas. 

However, a third possibility is the leakage of hydrogen into the GC oven due to a faulty connection or breakage of a capillary column. This situation can occur but with modern gas chromatographs with electronic flow-regulated gas controls, a potentially unsafe fault situation that could result in a leakage will shut down the carrier-gas flow automatically. Furthermore, modern GC instruments have spring-loaded, explosion-ready oven doors. Alternative approaches to an oven leakage situation employ a hydrogen sensor fully integrated into the GC oven (SIM, Oberhausen, Germany). The sensor continuously monitors the air inside the oven. If even a small amount of hydrogen gas appears, LEDs will light up. At a threshold value of 10% of the lower explosion limit (LEL), an acoustic signal will be transmitted. If the level goes to 25% of the LEL, an alarm signal will sound and the carrier gas is switched automatically to an inert gas. A similar device is available from Brechbueler (Houston, Texas). Standalone hydrogen sensor systems installable in any gas chromatograph are available on the market. 

When hydrogen is used as a fuel gas for GC–FID, the absolute purity and consistency of purity with respect to oxygen and water is not critical. In the flame ionization detector, oxygen is required for combustion reaction in the detector and water is produced as a by-product. Thus, concentrations of 10 ppm of water and oxygen contaminants are quite acceptable. 

For carrier-gas applications, absolute hydrogen purity and consistency of hydrogen purity is much more critical than fuel-gas applications. Even traces of water and oxygen in the carrier gas will attack GC capillary column stationary phases and degrade them, especially at high temperatures. The result is column bleed that affects the quality of analysis and reduces column life, thereby increasing column costs. The use of ultrahigh-purity (UHP) or research-grade purity hydrogen is recommended. Such gas is generally in the range of 99.9999+ % pure. 

Laboratory hydrogen usually is supplied by one of three means: point-of-use cylinders, multicylinder configurations, and hydrogen generators. A point-of-use cylinder used to supply hydrogen and is located next to the instrument. This approach is used more frequently in smaller laboratories with one or two gas chromatographs. The cylinder must be monitored to ensure that the gas supply does not run out. Alarm systems are available to warn the user that a cylinder is getting low. When changing cylinders, the hydrogen flow must be interrupted. Cylinders must be changed frequently.  

Multiple hydrogen cylinders connected to manifolds and a switchover system are used to supply several gas chromatographs to larger laboratories. Sometimes the supply cylinders are placed outside of the laboratory. The switchover system ensures that a continuous stream of hydrogen gas is available even if one of the cylinders would run out of gas during an actual run or during cylinder changes. 

In both the point-of-use systems and multiple cylinder systems, there is always the possibility of system air contamination occuring during changeout. If air or moisture gets into the column, damage to the stationary phase can occur. With such systems, gas traps are employed frequently. With gas cylinders, there is a great deal of added attention required: inventories must be maintained, rental costs must be accounted for, and cylinder handling is always required. 

Gas generators are becoming an accepted replacement for gas cylinders. A hydrogen generator provides a continuous stream of gas at a flow rate that is required to supply carrier gas to a single GC instrument or multiple GC instruments. The cost of the generator will pay for itself in a relatively short time. Gas generators can supply 99.9999% pure hydrogen sufficient for use as a GC carrier gas or as a fuel gas for the appropriate detector.  

Laboratory hydrogen generators employ one of two technologies: liquid electrolyte systems and solid polymer electrolyte systems. The liquid electrolyte systems generate hydrogen via the electrolytic dissociation of water. For this type of generator, caustic liquid electrolyte must be used. A voltage is applied across a liquid electrolyte, resulting in hydrolysis breaking down the water molecule into hydrogen and oxygen gases that then are separated by a gas permeable membrane. Once separated, the hydrogen gas goes through a specially designed heated palladium membrane that allows only hydrogen to pass through its pores. The end result is the production of ultrapure hydrogen at a steady and sufficient flow rate to use for a carrier gas for GC.  

The solid polymer electrolyte system uses only deionized water as its feedstock. The system employs a proton exchange membrane cell that splits water into oxygen and hydrogen. The hydrogen that is generated is "wet." Thus, downstream purification and removal of water is required. Water is removed in a number of stages ranging from bulk water removal, a drying membrane, to final hydrogen polishing. which removes the last traces of moisture and also any oxygen that might have been present. The hydrogen that is generated is greater than 99.99999% pure with respect to oxygen and 99.9999% pure with respect to water. 

First, in most generators, only a very small volume (50–300 cm

 ) of hydrogen is stored in the generator. The hydrogen is generated only on demand. The oxygen and hydrogen that is generated are kept apart by the cell and water bottle designs. Internally, generators have multiple fail-safe flow and pressure sensors that are located throughout the flow streams to monitor the system for leaks and pressure buildup. Safety alarms linked to sensors for internal leaks, low pressure, over-run, over-pressure, and oxygen vent blockage are present. In addition, the casing of hydrogen generators is designed for explosion control.  

Like any chromatographic technique, GC optimization is achieved by getting the best resolution in the most rapid run time and with the required sensitivity. As is usual, there is always a compromise among these chromatographic parameters. Options for shortening run times include decreasing stationary phase film thickness, changing the temperature program, decreasing column length or column internal diameter, and changing carrier-gas type and linear velocity (head pressure). The choice of carrier gas can influence the separation, as depicted in Figure 2. Here, the same set of polynuclear aromatic hydrocarbons was separated using different carrier gases under optimized conditions. It might have been possible to obtain equivalent resolution with nitrogen but the analysis time would have been greatly increased.  

Using Method Translation software (Agient Technologies, Santa Clara, California), one can calculate equivalent experimental conditions for different column dimensions and carrier gases while keeping the same stationary phase. In the example of Figure 3, spearmint oil was first separated using helium carrier and a 30 m X 0.25 mm, 0.25-µm HP-Innowax capillary column (phase ratio 250) (Agilent Technologies). The separation time was 27.4 min (Figure 3c). By specifying a 20 m X 0.18 mm column (phase ratio 250, which translates to a film thickness of 0.18 µm), the method translation software sets up the appropriate conditions while still using helium as a carrier. Such a change alone can gain some separation time. Now the equivalent conditions for this new column dimension gave a 17.7-min separation time, a savings of 9.7 min over the initial separation (Figure 3b). By merely substituting hydrogen as a carrier and using the recommended conditions calculated by the software, the separation time — with equivalent resolution to the previous separations — turned out to be 10.6 min with a savings of 61% in analysis time over the initial separation (Figure 3a). Table II shows that equivalent or superior resolution was obtained for the critical pairs of analytes in the spearmint oil by switching to a smaller diameter, shorter column and to a hydrogen carrier gas. Again hydrogen proved to be a superior carrier gas and enabled a faster separation.  

We have demonstrated that the use of hydrogen as a carrier gas in GC brings many advantages over other popular carrier gases. When discussing GC–mass spectrometry (MS), however, one must consider an entirely new set of parameters. Most GC–MS separations have relied upon helium and GC–MS performance usually is based upon this carrier gas. In GC–MS, the column outlet is at vacuum rather than ambient pressure, as occurs with conventional detectors. This vacuum reduces the column head pressure required to provide a certain column flow rate (linear velocity). Thus, helium that has a higher viscosity than hydrogen and consequently, a higher head pressure will suppress the expansion of the injection solvent during splitless injection while hydrogen with its lower viscosity will allow a greater and perhaps uncontrolled solvent expansion volume. Peak tailing or broadening can result. Thus, injection with a hydrogen carrier is best performed using a smaller diameter column, a longer column, or some other injection technique such as cold on-column or temperature-programmed injection techniques.  

Other problems can arise when hydrogen is used as a carrier gas in GC–MS. Too high of a flow rate of hydrogen carrier can overload the vacuum system if the pump being used is not capable of removing the excess gas. Another possibility is the occurrence of source and inlet surface chemical reactions with certain chemical compounds. These are sometimes referred to as ion–molecule reactions that can take place in a higher pressure in the ion source because of the decreased pumping speed for hydrogen relative to helium. Of course, an uncontrolled leakage of hydrogen in the MS source can cause combustion. A few words of caution are appropriate. The MS or MS isolation valves can be shut down either deliberately or automatically via an internal or external failure. However, the carrier gas does not shut down and as a result, the carrier gas can accumulate in the mass spectrometer. It is good practice to shut off the hydrogen at its source any time you shut down the GC system or the GC–MS system.  

Hydrogen can act as a scrubber in the flow system because it can displace contaminants that can be adsorbed on roughened or unswept areas when using helium carrier. On the mass spectrometer, this contamination typically looks like hydrocarbon contamination and may take some time to have a completely noise-free system. However, once cleaned, hydrogen seems to keep contaminants from building up on filaments and other areas.  

To demonstrate the usefulness of hydrogen as a carrier gas for GC–MS, a case study of the separation of the U.S. Environmental Protection Agency's 16 priority pollutant polycyclic aromatic hydrocarbons (PAHs) was undertaken (3). The structures of the compounds studied are shown in Figure 4. The chromatogram in Figure 5 was generated for the PAH separation on an HP-5MS capillary column using helium as a carrier gas. The separation of the PAHs was achieved in about 14 min using temperature programming. Figure 6 shows the same PAH separation performed with hydrogen as a carrier gas with a total analysis time of only 10.3 min with comparable results to the helium run. The final conditions for this separation are shown in Table IV. Table III shows the reproducibility of the retention time (

 = 3) and calibration linearity data for these runs. The calibration linearity for two of the PAH compounds is depicted in Figure 7 for different concentrations ranging from 0.02 to 2.0 ppm. In summary, there was a 29% reduction in separation time when hydrogen was used as a carrier rather than helium. The relative standard deviation for retention times was excellent, as was the linearity of the calibration curves.  

Hydrogen can be used as an effective replacement for helium in many GC and GC–MS applications. It has a wide range of applicability, provides good efficiency, and, for the most part, separations are faster than with other popular carrier gases like helium and nitrogen.  

Table III: Retention time precision and calibration linearity for 16 PAHs using helium and hydrogen as carrier gases 

It is much cheaper to purchase than helium and can be generated easily and cheaply by using hydrogen generators with a very high level of purity. Gas filters and scrubbers might be needed to ensure that the hydrogen gas is clean and oxygen–moisture free. Due to its explosive properties, precautions must be observed in its use but this aspect of hydrogen usage might be over-emphasized.  

Column Watch

Hydrogen carrier gas can be an effective replacement for helium in many gas chromatography (GC) and GC–mass spectrometry (MS) applications.

Hydrogen as a Carrier Gas for GC and GC–MS

Technologies for laboratory analysis advance continuously, just as do computer technologies or transportation technologies. Small advances tend to occur fairly often while major new technologies appear less frequently. As new capabilities become available, laboratories must decide whether to acquire them or to defer and continue to use what they already have. Such decisions are reached by considering the roles and requirements of the laboratory, the short and long-term costs of the new technologies, new skills that laboratory workers may have to acquire, and the relative benefits and drawbacks of all of the changes.     

Reasons and justifications for technology upgrades depend upon laboratories' current and future needs. While the benefits of new capabilities are easy to describe, what may not be so evident are the collateral requirements of implementing new technologies in the laboratory. For example, switching to hydrogen carrier gas generation eliminates the costs of carrier-gas cylinders and can yield faster speeds of analysis if hydrogen is not already in use, but the change also invokes some new safety requirements and procedures. This installation of "GC Connections" discusses two related gas chromatography (GC) technologies and their impact on laboratory equipment and procedures. 

Installing carrier- and flame-gas generators is relatively easy, although there are some special considerations for hydrogen. The benefits of zero future gas cylinder costs plus no cylinder transport or demurrage charges yield an attractive return on investment (ROI), especially given the current high cost of carrier-grade helium. Zero-grade air generators are effective as well. The cost of detector-quality cylinder air is much lower than carrier-grade helium, but its "burn rate" is much higher at over 400 mL/min compared to a range of 50–250 mL/min for carrier gas with a split inlet system. Carrier-gas consumption can be reduced by up to 80–90% if a gas-saver pressure-control mode turns off split flow while the inlet is not actively in use. There is no corresponding saver mode for flame ionization detection (FID) air. A flame detector needs to stay lit and stable as long as there are pending analyses. The result is that much more FID air is used in the average laboratory than carrier gas. 

Gas generators have limited flow and pressure ranges that cannot be exceeded. It's a good idea to acquire gas generators that exceed current flow requirements by 25–50% to allow for future expansion. Also, installing gas generators will create a new requirement for regular generator maintenance, although arguably this is less effort than it takes to haul cylinders in and out of the laboratory. 

Generation of hydrogen for carrier and fuel gas invokes some additional concerns. For the majority of GC applications, hydrogen carrier gas can be substituted for helium. The exceptions are for certain fixed-gas separations as well as for some detection methods, such as helium ionization detection (HID) and electron-capture detection (ECD), in which helium actively participates in the detection chemistry. Even in these cases, it is sometimes possible to apply helium as the makeup gas while using hydrogen as carrier gas, which at least will reduce helium consumption. As an alternative, most ECD systems will work with a 5% methane in argon makeup gas mixture, although sensitivities and relative responses will change compared to helium. Mass spectrometry (MS) detectors are generally compatible with hydrogen carrier; some reduced pumping efficiency as well as lower background ionization levels can be expected. Also, some extra attention to proper detector venting is called for when shutting down to avoid hydrogen accumulation inside the detector's vacuum chamber. MS detector manufacturers can provide detailed information about their specific products. For standard GC separations with FID, hydrogen carrier is an attractive choice because the same hydrogen source also can be used for the FID fuel gas. See reference 1 for some additional frequently asked questions about hydrogen carrier gas. 

Switching to generated hydrogen carrier gas is a two-step process. First, if not already using hydrogen, install a tank of high-purity hydrogen, or use the existing FID hydrogen tank if it's pure enough, and validate performance with the new carrier. The column pressure settings will be different. Lower inlet pressures are required for the same average carrier-gas linear velocities, while the optimum velocity for hydrogen is 10–20% higher than for helium. A flame ionization detector requires a constant flow of hydrogen fuel, which means electronic pneumatics will be needed to maintain flow when the column is temperature programmed. Run the carrier pneumatics in constant-flow mode if possible, and establish a constant total FID hydrogen flow. Once the new carrier-gas and detector settings have been validated, then consider switching from cylinders to a gas generator. From a cost point of view, it might be easier to justify a new hydrogen generator if several GC systems can be converted to hydrogen carrier at once.  

Beyond considerations for method parameters, using hydrogen carrier gas will invoke some concerns for the potential burning or explosion hazards. A cylinder of flammable gas represents three distinct hazards. First, the very high pressure in any gas cylinder is a physical endangerment to personnel if not well understood and handled correctly. Second, the cylinder is very heavy and can present a lifting or falling hazard. Third, hydrogen is flammable and becomes explosive when mixed with air at concentrations between the lower and upper explosive limits of 4–74 % by volume. A fully pressurized A-size cylinder at 2600 psig (18 kPa) contains nearly 8 m

  of gas when expanded to room pressure. In a small 20 X 30 X 10 ft (6 X 9 X 3 m) laboratory, the lower explosive limit (LEL) of hydrogen could be reached if the entire contents of a cylinder were released at once. However, this extreme occurrence is very unlikely to take place by accident. Using a hydrogen generator to produce carrier and fuel gas relieves concerns for the release of a tankful of hydrogen — the generators store only a small amount of hydrogen at any time. Small amounts of hydrogen can be released into the laboratory from split vents or during column installation. For peace of mind, it might be a good idea to install a hydrogen sensor near the ceiling of the laboratory. I have one such sensor in my laboratory that gets tested — loudly — once in a while when I change the carrier gas to hydrogen and purge the carrier-gas lines. But I experiment with different carrier gases much more often than would a production laboratory. 

Modern GC systems include some safety features that address hydrogen concerns as well. Any laboratory that is considering hydrogen carrier is strongly urged to use an instrument with an electronic pressure control system that limits the flow of hydrogen carrier gas and detects and shuts off the flow under leakage or out-of-bounds conditions. Today's GC systems feature explosion-safe ovens that, upon the extremely rare occasion of hydrogen accumulation and ignition, will contain the overpressure safely inside the oven. Hydrogen leak detector accessories are available for gas chromatography ovens as well.  

Increase Speed of Analysis with High-Speed Gas Chromatography

Going to faster speeds of analysis with GC doesn't always require a new or upgraded instrument, but not all existing instruments are suitable. It all depends upon how fast a separation is required. A modest increase of two to four times shorter retentions using conventional capillary columns with inner diameters of 200 µm or greater can be achieved quite reasonably on a wide range of conventional laboratory instruments. Very high speeds can achieve separations that previously needed 10 min or longer in less than 1 min, but such a feat requires significant equipment upgrades or complete replacements. Pushing peaks from a column at high speeds places demands upon autosamplers, inlets, detectors, and data-handling systems that existing equipment might not be able to handle adequately. Newer GC systems incorporate high-speed autosampler injection modes, appropriate inlet designs, and fast data acquisition speeds that encompass the requirements for high-speed operation up to a point. Specialized micro GC systems or dedicated rapid column heating accessories are required to go even further. 

Making the decision to go to higher speeds is just the start of what can be an extended method development and validation exercise. A high-speed capable instrument is a platform upon which to deploy a suitable column and method. It might be able to inject and record very narrow peaks while ramping up the column temperature at impressive rates, but without the necessary separation method, it will not deliver the desired results.  

Generally, faster linear velocities and higher temperatures or temperature program rates, as well as reduced column lengths and inner diameters, will achieve a given separation in less time. This is attractive when considering whether to purchase additional GC systems for the laboratory, and when faced with an increasing sample load. Alternatives such as adding a temporary work shift or contracting to an outside laboratory might be good intermediate solutions but in the longer term, increasing the sample throughput capacity of the laboratory is going to be more cost effective. But the route to achieving higher analysis speeds can be difficult to traverse.  

Certainly, hydrogen carrier gas is effective for faster GC separations because it is less viscous than helium and so produces higher linear velocities at the same pressures. The higher "speed limit" for hydrogen becomes important when pushing longer columns with narrower inner diameters to yield faster separations — speeds of analysis are faster as inlet pressures approach their maximum values. Shifting to higher analysis speeds isn't a simple matter of cranking up the inlet pressure, however. A number of other parameters and considerations come into play in the GC fast lane.  

First and foremost, the peaks of interest must be resolved sufficiently under a new set of conditions. If the separation is isothermal, and if there is extra resolution to start with, then achieving higher speeds is relatively simple. Even when increasing pressures to operate well above optimum velocities, in such situations, enough room between the peaks is available to produce adequate resolution at the higher speeds. All of the peaks move in proportion to an increase in velocity; their separations (α-factors) do not change. 

Another approach to high-speed separations involves going to smaller column inner diameters. Reducing the column inner diameter while still operating at close to the optimum velocity will produce more theoretical plates and correspondingly narrower peaks, and it will increase the resolution. Then the chromatographer can choose to crank up the inlet pressure and increase linear velocities away from the optimum, sacrificing the increased resolution of the narrower inner diameter column for more speed. 

Things get more complex with temperature-programmed separations, or when increasing isothermal temperatures. The elution times of all of the peaks decrease while the relative positions of dissimilar peaks shift as the temperature program rate increases, which also will happen when increasing the temperature of an isothermal separation. This behavior stems from the different ways in which solutes interact chemically with the stationary phase as temperatures change. In a temperature-programmed run, modifying the pressure settings, or even changing from constant pressure to constant flow mode, also will change relative peak positions. In this case, solutes experience slightly different temperatures as they move through the column more rapidly, while the oven temperature program profile is unchanged. 

Reducing the column inner diameter is another approach to higher speeds, as already mentioned. However, with temperature programming there exists the possibility that peaks will shift in relation to each other if the column phase ratio (β) is not maintained while reducing the inner diameter. For example, a 530-µm i.d. column with a 1-µm stationary phase film has a phase ratio of  

In a 250-µm i.d. column, with a potential of about a 1.4X higher theoretical plate count, the corresponding available film thickness for the same approximate phase ratio would be 0.5 µm. If the phase ratio is not kept about the same when changing column inner diameter, then dissimilar peaks in a temperature-programmed separation will again experience relative retention shifts. 

This sensitivity of the separation of dissimilar peaks to changing column dimensions, temperatures, and pressure conditions makes it necessary to optimize and validate any significant changes in an effort to achieve higher speeds. Simulation software such as Agilent's Method Translation Software (2) (Agilent Technologies, Santa Clara, California) can significantly reduce the time and number of experiments required to reach a desired combination of speed and resolution from which to start formal method validation. 

Achieving satisfactory results with high-speed GC, then, requires both the right kind of host equipment and a suitably developed and validated separation. Modest speed increases often are possible with existing equipment, but truly high-speed separations will require a commitment to obtaining the equipment and developing the methods. Crossing the bridge from conventional to high speeds can invoke more work than expected, but high-speed separations can be well worth the extra effort when laboratory requirements demand them.  

In both cases presented here — advancing laboratory operations by switching to gas generation or by transitioning to higher-speed analyses — the decision to improve laboratory capabilities will produce a successful outcome only if laboratory personnel fully consider the implications of the changes and perform the necessary predeployment tasks to ensure that the desired goals can be achieved. Changing to hydrogen carrier gas requires careful planning and consideration for both the potential added hazard as well as appropriate GC method changes. Upgrading to high-speed GC is a great idea, but performance requirements can necessitate the purchase of additional or replacement equipment. In any case, upgrading laboratory technologies is neither simple nor easy, but the results can be well worth the effort. 

 "GC Connections" editor John V. Hinshaw is senior Research Scientist at Serveron Corp., Hillsboro, Oregon, and a member of LCGC's editorial advisory board. Direct correspondence about this column to "GC Connections," LCGC, Woodbridge Corporate Plaza, 485 Route 1 South, Building F, First Floor, Iselin, NJ 08830, e-mail 

. For an ongoing discussion of GC issues, visit the Chromatography Forum discussion group at 

(2) Available for download with no cost at 

http://www.chem.agilent.com/cag/servsup/usersoft/files/GCTS.htm

GC Connections

This month in "GC Connections," John Hinshaw discusses issues that can arise when deciding to upgrade or replace gas chromatography technologies.

Upgrading Gas Chromatography

I recently had a question regarding some changes that took place in the 2009 edition of the 

  the adjustments allowed to meet system suitability for liquid chromatography (LC) methods include a change in column internal diameter of ±25%. A change was made in 

  This stated that column internal diameter "can be adjusted provided that the linear velocity is kept constant." The person was not sure how this adjustment of linear velocity should be made and wondered about what effect it would have on column efficiency. 

Much of the band broadening that takes place within an LC column is a result of slow diffusion of the analyte in and out of the pores in the column packing. This means that the mobile phase flow rate can have an affect on the volumetric width of a peak. By volumetric width, I mean the peak width in units of volume (for example, millilitres or microlitres), not time. In the days when 10 μm diameter particles were used as column packing, a noticeable reduction in column efficiency could be observed when the flow rate was increased. 

The smaller the particle diameter, the less the dependence of column efficiency upon flow rate. Although it is easy to demonstrate a loss in efficiency with an increase in flow rate for very well-behaved analytes with 3 and 5 μm diameter particles, a two-fold change in flow rate is rarely noticed with real applications. With sub-3 μm particles, flow rate has little influence on column efficiency, even with well-behaved compounds.  

In this discussion, I have referred to flow rate, but the important variable is not flow rate, but linear velocity. Linear velocity is the speed at which the mobile phase travels through the column, for example, in millimetres per second. For comparison of equivalent conditions between columns of different internal diameters, the linear velocity should be kept constant. To keep linear velocity constant, the flow rate should be adjusted in proportion to the column cross-sectional area, which is directly proportional to the square of the ratio of column diameters. For the current discussion, let's consider two cases. The first is a change from a 4.6 mm i.d. column to a 2.1 mm i.d. column: 

In both cases, we'll consider the approximations as close enough for practical work and certainly easier to remember and use for mental calculations. 

This means that for equivalent linear velocities, a change from a 4.6 mm i.d. column operated at 1 mL/min to a 2.1 mm i.d. column would require a flow rate adjustment of five-fold, or to 1.0/5 = 0.2 mL/min. A change to a 1.0 mm i.d. column would mean a new flow rate of 1.0/20 = 50 μL/min. 

 32 allowed a change in column diameter of ±25%.

  This meant that a 4.6 mm i.d. column could be exchanged for any column in the 3.45–5.75 mm i.d. range. I suspect that this specification was originally written to allow the old Waters μBondapak 300 mm × 3.9 mm column to be used instead of the 4.6 mm i.d. versions. 

 32 also allowed a ±50% change in flow rate. The 3.9 mm i.d. column would require a flow-rate reduction of 30% for constant linear velocity, so the necessary flow-rate change was covered. Note, though, that the flow-rate change was not required by 

, so a significant change in linear velocity was allowed. Today, however, the most popular column internal diameters are 4.6, 2.1 and 1.0 mm, so the ±25% diameter-change allowance eliminated the option of using smaller-internal-diameter columns. It is clear that a change in the 

 32, Supplement 2 at first seems more complicated by introducing the linear velocity,

  but actually, it gives more flexibility and ensures a more equivalent result than the old allowance. Now any column diameter can be used, so the 2.1 and 1.0 mm i.d. columns can be used in addition to the 3.9 mm i.d. ones. In addition, the adjustment in linear velocity means that the columns will be operated under more similar chromatographic conditions than before. By properly adjusting the linear velocity, the column pressure and analyte retention times should be the same when column diameter is changed. 

What wasn't addressed in the original question was why one would want to use a column of different diameter. There are two primary advantages, decreased mobile-phase consumption and a reduction in peak volume. If the flow rate is adjusted for constant linear velocity, as discussed earlier, the retention times should be the same when column diameter is changed, so the sample run time will be unchanged. If the run time is the same and the flow rate is reduced, less mobile phase will be used. This is attractive, especially in these days of high acetonitrile prices. 

The peak volume (peak width in volumetric terms) drops with the reduction of the cross-sectional area of the column, or square of the change in diameter. This translates into proportionally taller peaks, assuming that the same mass of sample can be loaded onto the column, which may or may not be true. Thus, moving from a 4.6 mm i.d. column to a 2.1 mm column should reduce peak width by five-fold and increase peak height by the same amount (assuming the same mass of sample is injected).  

This leads to the second part of the original question, regarding how a change in column diameter affects column efficiency. In theory, there should be no dependency of efficiency upon column diameter, but from a practical standpoint, narrower columns are usually less efficient than larger-diameter ones.  

The primary factor in determining the column efficiency is the number of particle diameters in the length of the column. Under ideal conditions, a well-packed column should have a plate height 

 equivalent to approximately two particle diameters 

. The column efficiency, or column plate number 

 is the column length. For a 5 μm particle (

 = 10 μm = 0.01 mm. Packed in a 150 mm-long column, this would give  

You might be able to get 15000 plates for a 150 mm × 4.6 mm column mounted on an high performance liquid chromatography (HPLC) system optimized for column testing and using a well-behaved test compound such as toluene or methyl benzoate. However, for real samples on a typical laboratory's HPLC system, most of us would feel very good to get 10000 plates for this column. Even if we used toluene or methyl benzoate, we might get only 12000 plates on the system in the laboratory. 

What is the reason for the reduced plate number on a real HPLC system? Part of it has to do with extracolumn peak broadening. Here's how it works. The width of a peak in a chromatogram can be expressed as  

 is the observed peak width at baseline, drawn between tangents to the sides of the peak, 

 is the peak broadening that takes place as the analyte travels through the column and 

 is the peak broadening outside the column, often referred to as extracolumn effects. 

We have all observed peak broadening within the column 

. For example, with an isocratic separation, the early-eluted peaks are narrower than later-eluted ones. This is because the longer the compound stays on the column, the broader it is. Some other factors related to on-column peak broadening include the particle diameter, column temperature, mobile phase viscosity and analyte diffusion coefficient.  

Extracolumn effects include all sources of peak broadening excluding the column itself. These include such things as the injection volume and solvent, the length and diameter of connecting tubing between the autosampler and column and between the column and detector, the detector cell volume and time constant and the data rate of the data system. For the present discussion, we'll consider these as a composite influence. For a conventional LC system, such as the ones most of us use, set up for routine analysis with 150 mm × 4.6 mm i.d. columns packed with 5 μm diameter particles, 

It is an interesting exercise to assess the degradation of the column plate number 

, for columns of different diameter. In Table 1 I have summarized data for a peak with the retention factor, 

 = 1. The assumptions in both tables are a column length 

 = 4.6 mm (or as noted); column void volume of 60% of the column volume; flow rate 

 = 2.0 mL/min for the 4.6 mm column; 5 μm particles for a column plate number, 

 = 15000 in absence of extracolumn effects; and an extracolumn volume of 15 μL. Data are shown for 4.6, 2.1 and 1.0 mm i.d. columns. I have rounded the displayed values for clarity, so if you are trying to repeat my calculations in a spreadsheet, keep the maximum number of significant figures for all calculations.  

Table 1: Extracolumn effects for k = 1			

In the second and third columns of Table 1, you can see the column volume 

 calculated for each column diameter and the flow rate adjustments required to maintain a constant linear velocity. For an early-eluted peak with 

 = 1 and a 4.6 mm i.d. column, there is a very small increase in peak width resulting from the extracolumn volume. However, the 15 μL of extracolumn volume increases the peak width by more than three-fold for the 1.0 mm i.d. column (2.9 versus 10 s). This reduces the plate number by more than an order of magnitude, and because resolution is proportional to the square root of the plate number, resolution drops by 71%! Most of us consider a drop in resolution of 5% or less to be of little consequence, but it is obvious from the data in Table 1 that use of a 2.1 or 1.0 mm i.d. column with an early-eluted peak will not produce satisfactory results with this LC system. 

Table 2 summarizes the impact of 15 μL of extracolumn volume on peaks with larger 

-values. It can be seen that more retained peaks are influenced less by extracolumn volume for all column diameters. This is expected, because peak width within the column 

 increases with retention, so with constant 

 will be less affected. However, for the chosen chromatographic conditions, the 2.1 mm i.d. column is acceptable (

-loss < ≈5%) only for well-retained peaks. The 1.0 mm i.d. column does not produce satisfactory results at any retention time.  

Table 2: Extracolumn effects for k = 2, 5, and 10			

A disclaimer should be included here, as on the advertisements for new cars: your mileage may vary. Tables 1 and 2 were calculated based upon the assumption that the 5 μm particle diameter column generated 

 = 15000. As mentioned earlier, this represents the performance of a well-packed column operating under ideal conditions. It is likely that such a column will generate plate numbers in the 10000 range for real compounds under real analytical conditions. This means that the peaks will be broader, even under the best circumstances, so the impact of extracolumn volume will be less than that shown in the tables. However, the general conclusion is the same: you will have to minimize extracolumn effects if you want to get acceptable results with 2.1 mm i.d. columns on a conventional LC system. Furthermore, it is unlikely that you will get satisfactory performance from 1.0 mm i.d. columns unless you take great care. This will mean limiting injection volume, replacing the detector with one with a smaller-volume flow cell and making other changes to reduce extracolumn effects. 

It is fairly simple to make the necessary changes in flow rate to maintain constant linear velocity when changes in column diameter are made. A reduction in column diameter can be useful to conserve solvent and to improve detection limits. However, a drop in system performance, as measured by resolution, might be observed for smaller-diameter columns unless special care is taken to reduce extracolumn effects. From a troubleshooting standpoint, it is tempting to blame poor system performance on the column itself, when the real culprit may be the extracolumn volume of the LC system. A relative insensitivity to extracolumn effects is one reason why the 150 mm × 4.6 mm column packed with 5 μm diameter particles is likely to remain the workhorse column for routine LC work for many years to come. 

 is vice president of LC Resources, Walnut Creek, California, USA; and a member of the Editorial Advisory Board of 

. Direct correspondence about this column to "LC Troubleshooting", 

, Park West, Sealand Road, Chester CH1 4RN, UK. 

For an ongoing dicussion of LC Troubleshooting with John Dolan and other chromatographers, visit the Chromatography Forum discussion group at 

 32 (Supplement 2), General Chapter 621, 4149 (2009). 

LC Troubleshooting

What can be expected when the column diameter is changed?

Column Diameter, Linear Velocity and Column Efficiency

 Spark Holland bv, Emmen, The Netherlands. 

The method developed runs on a fully automated platform integrating solid-phase extraction (SPE) and LC (Symbiosis Pico) without any manual pretreatment step(s). Automatic pretreatment results in high throughput, low CVs and high precision. Sample preparation has been simplified to just one operator step before the system takes over the automated analysis of the samples.  

A Spark Holland Symbiosis PICO system with a Mistral column oven and an Applied Biosystems/MDS Analytical Technologies API 4000 LC–MS–MS system are used for this application. The API 4000 is equipped with a Heated Nebulizer (APCI) probe. 

 is Spark Holland's unique (u)HPLC solution with integrated on-line SPE-LC–MS automation. The system offers large flexibility in processing different types of samples selecting one of the three fully automated operational modes LC; XLC (extraction liquid chromatography) and mXLC (multidimensional on-line SPE) 

Sample preparation consists of diluting a human sample with a special protein disrupting buffer, followed by mixing. Samples are then placed in the Symbiosis autosampler. 100 μL of sample is injected on a Spark HySphere C8EC-SE sorbent SPE cartridge for on-line sample clean-up and concentration.  

The mobile phase consists of formic acid, water and methanol with separation on a Merck KGaA Chromolith SpeedROD RP-18e (endcapped), 50 × 4.6 mm HPLC column. An example chromatogram of the separation achieved is shown in Figure 1.  

A calibration curve was created by injecting a full set of calibration standards ranging from 5 to 500 ng/mL spiked human plasma. The calibration curve is calculated with a R

  of 0.999 with a 1/X weighting for both compounds.  

Table 1: Accuracy and precision of the QC samples.			

This method was evaluated using spiked human sodium heparin plasma samples. The developed on-line SPE-MS–MS method has an absolute recovery of more then 90%, and calibration curve with a linear range from 5–500 ng/mL (R = 0.999 for both compounds). The developed method has an accuracy ranging from 89–117% and precision of 0.1–7.9 %CV. The estimated quantification limits for both analytes are more than sufficient to allow the analytical method to be used for therapeutic drug monitoring. When injecting 100 μL, containing 50 μL of plasma, a detection limit of 5 ng/mL is achieved. 

2. Applied Biosystem Vitamin D i-method (User Contributed i-method by Spark).

For more information please send an e-mail to: 

P. de Keyserstraat 8, PO box 3887800 AJ Emmen, The Netherlands  

The method developed runs on a fully automated platform integrating solid-phase extraction (SPE) and LC (Symbiosis Pico) without any manual pretreatment step(s). Automatic pretreatment results in high throughput, low CVs and high precision. Sample preparation has been simplified to just one operator step before the system takes over the automated analysis of the samples.

Determination of 25-mono-hydroxy-vitamin D2 and D3 in Human Plasma Using a Symbiosis Pico System: ACN Free On-line SPE-LC–MS–MS

The elucidation of oxidative metabolism reactions is a crucial point in drug development. Generally, in vivo or in vitro experiments with liver microsomes or hepatocytes are performed. A complementary, purely instrumental method is presented in this article. In an electrochemical (EC) flow-through cell, the oxidative metabolism of xenobiotics is simulated. The cell is used in an online set-up with liquid chromatography–mass spectrometry (LC–MS), so that a separation with subsequent detection and identification of the metabolites is possible. The article provides an introduction to this new approach. The instrumental set-up is presented and selected applications of EC-LC–MS in drug development processes are discussed.

The elucidation of the metabolic fate of drug candidates in the human body is one of the major challenges in pharmaceutical research (1). The main route of drug elimination is an enzymatic biotransformation. This is frequently initiated by oxidation reactions ("phase I metabolism"), which are catalyzed by enzymes of the cytochrome P450 superfamily (2). With a strongly increasing number of novel chemical entities (NCEs) in recent years, rapid screening techniques that provide both reliable and easily accessible information about the biotransformation of a drug candidate are required. The earlier these tests are performed, the earlier unsuitable drug candidates can be excluded from the development process, thus leading to significant economic advantages in the early stages of drug discovery. For this reason, various model systems, which mimic metabolic processes in vitro have been developed (3). 

The established methods for in-vitro simulation of the oxidative metabolism are generally based upon cytochrome P450 enzymes originating from the liver, including an isolated perfused animal liver, liver slices, hepatocytes or, most importantly, liver-cell microsomes (4). However, these testing schemes are based upon animal experiments and are tedious, time consuming, and of limited reproducibility.   

Electrochemistry (EC) is one of the classical methods to induce oxidation reactions. It thus seems obvious to employ EC as a simulation technique in drug-metabolism studies. Electrochemical flow-through cells coupled online to analysis techniques, such as mass spectrometry (MS) with or without previous liquid chromatographic separation (EC-LC–MS or EC–MS), have now become the technique of choice for these simulation experiments because they may provide exhaustive information about the nature of the electrochemically generated metabolites. The respective experimental approaches, applications and possible future trends are presented in this article.  

The majority of studies towards the electrochemical simulation of the oxidative metabolism has been performed using commercially available coulometric flow-through cells. Besides that, amperometric thin-layer or wall-jet cells have been implemented in a number of studies (5–7). All three types of electrochemical cells are equipped with a three electrode set-up, consisting of a working-electrode, a reference electrode and a counter electrode. The oxidation potential is set between the counter electrode and the working electrode. Polarization effects on the counter electrode, resulting in unstable working potentials, are compensated by the reference electrode, which is most commonly based upon Pd/H

The cell geometries of the amperometric and coulometric cells are presented in Figure 1. The working electrode of the coulometric flow-through cell in Figure 1a consists of porous glassy carbon. The analytes are passed through the electrode and are oxidized at the large surface area. The main feature of the coulometric flow-through cell is the high conversion rate, which is affected by the flow rate (~10–500 μL/min) to only a minor extent. The cell is almost maintenance free and the cleaning procedure simply consists of flushing with aqueous or organic solvent. This fact might be considered as an advantage, however, adsorption or fouling processes on the electrode surface, detectable by changes in back pressure or in the electrochemical signal, cannot be detected visually or be removed by manual cleaning procedures.   

Amperometric cells such as the thin-layer cell in Figure 1b have a planar working electrode surface where the electrochemical conversion takes place. This surface is less affected by adsorption processes and the electrode surface can be cleaned and polished manually. In comparison to the coulometric flow-through cell, lower flow rates have to be used to achieve high conversion rates in the thin-layer cell (~10 μL/min). Therefore, these cell types can hardly be implemented on the high-pressure side of the high performance liquid chromatography (HPLC) system and up to now, this cell type has been mainly used in off-line batch experiments. This disadvantage can be overcome using an experimental set-up including a switching valve as explained below. As an additional feature, the thin-layer cell offers the implementation of different working electrode materials, which might be useful for the investigation of further applications. For example, working electrodes made out of platinum are expected to exhibit less adsorption than glassy-carbon electrodes and boron-doped diamond (BDD) electrodes are highly resistant towards chemicals and offer an enlarged range of oxidation potentials. 

The electrochemical oxidation has to be optimized carefully for each application, regarding the cell type, the oxidation potential, the buffer system and the flow rate in the electrochemical cell. Nonpolar compounds tend to adsorb on the surface of the porous glassy-carbon electrode of a coulometric flow-through cell. Thus, amperometric cells with exchangeable electrode materials are more suited for these applications. The buffer solution acts on the one hand as electrolyte, on the other hand a defined pH value is of particular importance regarding the pH-dependent Pd/H

 reference electrodes. For most applications the buffer system is adjusted to a physiological pH of 7.4. The flow rate in the electrochemical cell influences the conversion rate. Lowering the flow rate increases the conversion. However, too low flow rates can promote adsorption on the electrode surface.  

The best way to optimize the oxidation potential is to record a "mass voltammogram": The outlet of the EC cell is directly connected to the ionization source of the mass spectrometer. The compound of interest is pumped continuously through the EC cell, and the potential at the working electrode is ramped within the desired range. Mass spectra are then recorded in dependence upon the electrochemical potential. From the resulting three-dimensional plot, information about reasonable oxidation potentials for the EC-LC–MS set-up, as well as first insight into the nature of possible oxidation products, can be obtained. As an example, the mass voltammogram of the antimalarial agent amodiaquine (AQ) is shown in Figure 2. AQ itself has, as a protonated molecule, an 

 of 356 in the positive ion (PI) mode. An additional signal resulting from in-source fragmentation of AQ is detected at 

 = 283. From the mass voltammogram, it is obvious that AQ starts to oxidize at about 200 mV versus Pd/H

, first under formation of dehydrogenated AQ (amodiaquine quinoneimine, AQQI) and then, above 600 mV versus Pd/H

, under N-dealkylation. Thus, acquiring mass voltammograms provides insight into the oxidation behavior of the drug of interest. 

While first achievements in coupling EC to MS were obtained without HPLC separation (8–10), the chromatographic separation has proved to be an essential part of the whole system more recently (11–13). As demonstrated in Figure 2, the mass voltammogram measured by EC-MS provides a fast overview of the generated oxidation products. However, no information about different isomers or the polarity of the oxidation products is obtained. Using an advanced set-up, consisting of EC coupled online to LC and MS, the different regioisomers, formed, for example, upon electrochemical hydroxylation of aromatic rings, can be distinguished by LC separation. Furthermore, the retention time provides valuable information on the polarity of the oxidation products and reference compounds can be used to identify oxidation products by comparing the retention times.  

Two different online EC-LC–MS set-ups have been implemented for the simulation of oxidative drug metabolism. In the classic flow-through system, the EC cell is placed directly in front of the column and the analyte is injected by the autosampler of the system (Figure 3a) (11–14). After the injection, the compound of interest is oxidized in the EC cell and the oxidation mixture is separated on the HPLC column and detected by MS. However, in this case, the EC oxidation takes place under the same conditions as are used as starting parameters for the HPLC separation concerning flow rate, ionic strength, and composition and pH of the mobile phase. This is a drawback in many cases, for example, if the oxidation at physiological pH is desired, but the separation of the metabolites requires acidic conditions. Additionally, an organic solvent content of 50% or more is often needed in coulometric EC cells to prevent adsorption at the electrode surface. This high solvent concentration is in most cases not compatible with the HPLC separation of the polar analytes. Furthermore, as already discussed above, thin-layer cells cannot be implemented into this set-up as a result of the high flow rates at the high-pressure side of this system.  

Therefore, an improved set-up was developed using a switching valve for the injection (1). As can be seen in Figure 3b, the compound of interest is pumped at a constant flow rate through the EC cell using a syringe pump. The effluent from the cell is discharged into an injection loop, which is mounted on a 10-port switching valve. The injection is triggered by switching the valve. The major advantage of this set-up is the decoupling of EC oxidation and HPLC separation conditions, which can be optimized independently concerning flow rate, composition of the mobile phase, and pH.  

Basically, nonpolar, reversed-phase LC columns are well suited to separate the drug and the generated oxidation products. Upon reversed-phase separation, oxidation products such as hydroxylation and N-dealkylation products generally are eluted earlier than the drug itself, whereas dehydrogenation products, especially quinones, show an increased retention. C8 and C18 stationary phases are used in a number of studies, including the electrochemical metabolism simulation of the drugs amodiaquine (13) and toremifene (15) as well as for the separation of the pesticide boscalide (16) from its oxidation products. Regarding the often aromatic structure of the drug molecules, a phenyl phase can provide an enhanced selectivity compared to solely alkyl-based phases. Figure 4 shows the separation of the drug tetrazepam and its electrochemically generated metabolites using a Nucleodur Sphinx RP column with a stationary phase consisting of propylphenyl and C18 (Macherey-Nagel, Düren, Germany) (7). The EC-LC–MS measurement of tetrazepam points out the necessity of a chromatographic online separation. Besides dehydrogenation and N-dealkylation products, six different tetrazepam +O products are generated and separated in the EC-LC–MS system. The +O products correlated to a hydroxylation of tetrazepam at six different positions.  

Aiming for the quantification of oxidative metabolites, the hyphenation of EC, LC, and inductively coupled plasma mass spectrometry (ICP-MS) has been proven to be a valuable tool (17). ICP-MS allows an element-specific quantification of drugs containing heteroatoms such as iodine. Using ICP-MS detection, an isocratic LC separation is required, which was in the case of the drug amiodarone achieved by phase-optimized LC (POPLC, Bischoff Analysentechnik, Leonberg, Germany). POPLC enables the combination of different stationary phases, so that the phase can be individually adjusted toward a separation problem. Another promising separation technique for the EC-LC–MS set-up is hydrophilic interaction chromatography (HILIC), which will be especially suited for the separation of highly polar oxidation products. 

During the last decade, significant work has been performed to evalute to what extent the electrochemical oxidation reactions are consistent with oxidation reactions catalyzed by enzymes of the cytochrome P450 superfamily. For these studies, numerous compounds have been examined using EC and isolated human- or rat-liver enzymes. The electrochemically generated metabolites, as well as metabolites generated upon microsomal incubation, have been analyzed by LC–MS. Further identification by fragmentation experiments has been performed for metabolites of the drugs tetrazepam, paracetamol, and clozapine. In case of metoprolol, synthetic reference materials have been used for identification. The results are summarized in Table I (7,11–13,18–22). Even though the mechanism of the enzymatic oxidation and the electrochemical oxidation is quite different, both methods show a good agreement. Besides epoxidation and alcohol/aldehyde oxidation, the typical phase-I reactions are successfully simulated in the EC cell.  

Table I: Typical phase I reactions and the possibility for their electrochemical simulation 

Quantitative data regarding the conversion yield of the microsomal approach and the electrochemical simulation have been estimated for the drugs metoprolol, lidocaine, mephenytoin, and 

-methylthiopurine by peak areas in the base-peak chromatograms. In the case of amiodarone, the yields of the microsomal and the electrochemical method have been determined quantitatively by EC-LC–ICP-MS. Regarding the comparison of these quantitative data, it is important to note that the conversion rate varies for each compound and cannot be given in general for a phase-I oxidation reaction. Furthermore, the electrochemical conversion depends upon the conditions, for example in terms of the flow rate or the buffer system. The conversion yield of the microsomal approach is strongly connected to the expression of different isoforms of cytochrome P450 in the organism. Thus, only a tendency towards the conversion rate for the electrochemical simulation of typical phase-I reactions can be provided. 

The information presented in Table I —  including the type of phase-I reactions being simulated, the conversion rate and the conditions — should be seen as a first guideline for the electrochemical simulation of the oxidative phase-I metabolism, which is still under development. Further studies that include a larger number of compounds and variations in the experimental conditions have the potential to extend the range of applications.  

While in most cases, metabolism is regarded as a detoxification process, bioactivation to reactive metabolites may sometimes occur as well. These reactive metabolites may cause severe toxic side effects because of covalent protein binding. Therefore, it is very important to have a reliable detection and identification method for reactive metabolites. However, the direct detection of reactive metabolites in biological systems — such as the commonly used microsomes or hepatocytes — is in most cases not possible because protein binding occurs immediately after formation of the reactive metabolites. Therefore, indirect detection approaches are used in which the reactive metabolites are trapped with suitable trapping agents, such as glutathione, and in which the trapped metabolites are identified. EC-LC–MS in contrast offers a good alternative to perform a direct detection of reactive metabolites (13).  

As an example, a closer look into the electrochemical metabolism of the antimalarial agent amodiaquine (AQ), which was already discussed in the previous section, shall be taken. AQ is of particular interest because the drug is known for the formation of reactive metabolites. One reactive metabolite of amodiaquine is amodiaquine quinoneimine (AQQI), which is formed by dehydrogenation under cytochrome P450 catalysis. As can be seen from Figure 2, AQQI is also formed by electrochemical oxidation of AQ. Figure 5 shows an HPLC separation of AQ and its electrochemical metabolites. While in the chromatogram of the AQ standard, only one peak resulting from AQ itself is visible, electrochemical oxidation of AQ at 700 mV results in various peaks. Using the MS information, the molecular weight of the oxidation products could be assigned. AQQI, the respective deethylated AQQI-Et, and the deethylated AQ could be identified. The retention times are in good accordance with the theoretically expected retention times, because all quinoid species, being less polar than the respective hydrogenated compounds, have longer retention times than AQ itself.  

From the chromatogram after electrochemical oxidation, it is clearly visible that the reactive quinoid metabolites can be detected directly in EC-LC–MS experiments and that trapping with glutathione or other trapping agents is not necessary. For comparison, Figure 5 also shows the results of standard metabolism approaches using microsomes. In the microsomal incubation mixture without trapping agent, only deethylated AQ was detectable, but no reactive quinoid species with longer retention time. However, this does not mean that no quinoid species were formed upon microsomal incubation, because the same experiment in presence of glutathione as trapping agent yielded an AQ-glutathione adduct at a retention time of 9 min, which can only be formed after activation of AQ to the respective quinoid species AQQI. AQQI, however, is not directly detectable as a result of immediate binding to proteins in the microsomal incubation mixture. This example shows that direct detection of reactive metabolites in biological samples is often not possible and indirect detection after trapping has to be performed. In contrast, EC-LC–MS offers the possibility for an easy and convenient direct detection of reactive metabolites. Figure 6 summarizes the metabolic oxidation of AQ and shows the comparison between microsomal and electrochemical oxidation.  

Not only for the direct detection of reactive metabolites, but also for the identification of their protein adducts, EC offers interesting perspectives. As discussed in the previous section, reactive metabolites may covalently modify proteins, resulting in toxic side-effects of the drug. The identification of these covalently modified proteins in a complex biological mixture is rather laborious. As described in a recent publication using EC–LC–MS (23), drug–protein adducts can easily be generated, the binding site in the protein can be identified and the tendency of a drug to form covalent protein adducts can be evaluated.  

The lack of reference standards is a drawback in the identification and quantification of drug metabolites. With respect to this issue, EC offers interesting possibilities as well: In a recently published approach, it was shown that EC is well-suited for the synthesis of a drug metabolite, which was used as reference substance in screening processes for doping-control purposes (24).  

The online coupling of EC with LC–MS has evolved as a powerful tool for the elucidation and study of drug metabolism reactions in recent years. Oxidative metabolism reactions are simulated electrochemically and metabolites are separated with HPLC and identified with MS. Although standard in vivo and in vitro approaches, such as the use of liver microsomes, can certainly not be replaced by EC, the purely instrumental technique has found its application in several complementary issues. By the hyphenation of EC and MS, so-called mass voltammograms can be generated. The three-dimensional plots provide a very fast overview of the oxidative labile sites in a drug molecule. In contrast to standard approaches, the identification and direct detection of reactive metabolites is possible using online EC-LC–MS. Furthermore, the covalent modification of proteins by reactive metabolites can easily be studied by EC-LC–MS. Using an EC-LC system, metabolites can be eletrochemically synthesized and be isolated for further use as reference substances or for advanced structure elucidation by NMR. Therefore, the use of EC for drug-metabolism studies is an interesting supplemental instrument for drug metabolism studies. 

Uwe Karst is a full professor at the University of Münster, Germany. His research interests are LC–MS, with particular focus on the simulation of oxidative metabolism reactions based upon the analysis of nonpolar analytes by means of LC–MS and speciation analysis using LC with electrospray- and ICP–MS.

Wiebke Lohmann completed her Ph.D. studies in analytical chemistry in October 2008 in the group of Uwe Karst. Her thesis was entitled "Simulation of the Oxidative Metabolism of Pharmaceuticals Using Online Electrochemistry/Liquid Chromotography/Mass Spectrometry". She is currently with Bruker Daltonik GmbH, Bremen, Germany.

Anne Baumann studied chemistry at the University of Münster from October 2003 to December 2007. She is currently a Ph.D. student in the group of Uwe Karst. The main focus of her studies is the simulation of metabolism reactions by electrochemistry hyphenated to LC separation and MS detection.

Drug Metabolism in Drug Design and Development

, D. Zhang, M. Zhu, and W.G. Humphreys, Eds. (John Wiley & Sons, Hoboken, New Jersey, 2008), pp. 3–12. 

(2) P.D. Josephy, F.P. Guengerich, and J.O. Miners, 

Regular Article

This article provides an introduction to electrochemical liquid chromatography mass spectrometry.

LCGC Asia Pacific-03-01-2010

Simplifying Peptide Bioanalysis

Hydrogen as a Carrier Gas for GC and GC–MS

Upgrading Gas Chromatography

Column Diameter, Linear Velocity and Column Efficiency

Determination of 25-mono-hydroxy-vitamin D2 and D3 in Human Plasma Using a Symbiosis Pico System: ACN Free On-line SPE-LC–MS–MS

Electrochemistry and LC–MS for Metabolite Generation and Identification: Tools, Technologies, and Trends