Pittcon 2012

Changing experimental conditions to decrease analysis time is commonly investigated in analytical laboratories to improve sample throughput. Column dimensions are among the variables that can be optimized to achieve decreased analysis time. Narrow bore columns offer the advantage of higher efficiency and can therefore be used in shorter column lengths without sacrificing resolution. Using 0.1 mm ID columns has been shown to dramatically decrease analysis time, but also requires careful optimization. Higher carrier gas pressures must be in use for most applications, and column overloading can be experienced without careful control of on column sample size.

To still take advantage of the separation efficiency of narrow bore columns, an intermediate solution employing 0.15 mm ID columns can be used. Columns with slightly larger column inside diameters can be run at slightly lower carrier gas pressure and still yield beneficial reductions in analysis time. Increasing the column inside diameter also improves the sample handling capacity of the column. Thicker stationary phase film thicknesses can be manufactured in wider bore columns, yielding improved capacity while still maintaining column efficiency.

This presentation will examine the theoretical and practical aspects of employing 0.15 mm ID columns for Fast GC analysis. Practical examples will augment the theoretical discussion by illustrating reduced run times, accurate peak detection and measurement, and reasonable lower limits of detection by optimizing column geometry and instrument conditions.

Previous work has suggested that the selection of “orthogonal” columns can be predicted using the values determined from the hydrophobic-subtraction model. This presentation will briefly review the model and calculations while relating them to the phase selectivity in reversed-phase liquid separations. The empirical results of the hydrophobic-subtraction model will be used in the selection of appropriate stationary phases for specific phase-solute interactions, ultimately defining a simplified process for column selection. Additionally, selected terms and the respective contributions to solute retention will be explored for substituted alkyl and non-alkyl phases. This presentation will demonstrate how knowledge of reversed-phase liquid chromatography (RPLC) column phases and phase-solute interactions can aid in choosing the most selective columns for column screening, solute confirmation, and method development.

Persistent organic pollutants (POPs) are a group of chemicals that include halogenated pesticides, brominated diphenyl ethers (BDEs), and polychlorinated biphenyls (PCBs). Due to the lipophilic nature of these components, they accumulate in the fatty tissue of animals and bioaccumulate up the food chain. According to the World Health Organization, human breast milk is an ideal matrix to monitor the levels of POPs in not only the mother and infant, but also as a key indicator of the levels of these chemicals in the local environment. Current methodology for the analysis of halogenated pesticides, PCBs, and BDEs can be expensive, solvent intense, and time consuming. The QuEChERS extraction approach coupled to a silica cartridge SPE cleanup may be an attractive sample preparation alternative for biomonitoring efforts for halogenated POPs in milk. Comprehensive two-dimensional gas chromatography (GCxGC) with an electron capture detector (ECD) may also offer a more cost-effective alternative. Method development was done using whole cow milk and later compared to a NIST Standard Reference Material of Human Breast Milk.

Food pesticide residue monitoring has traditionally been performed using gas chromatography (GC), but there is increasing use of liquid chromatography (LC) with tandem mass spectrometry (MS/MS). Liquid chromatography tandem mass spectrometry (LC-MS/MS) is popular for food pesticide residue monitoring because complete chromatographic resolution of compounds is not essential, as multiple reaction monitoring (MRM) transitions are used for pesticide identification and quantification; however, this technique is susceptible to matrix effects resulting in poor data quality. Despite known problems from sample matrix, one benefit often associated with LC-MS/MS is that no sample cleanup is required as would be for GC methods. A well-advertized sample preparation is dilute-and-shoot, which describes the dilution of sample extracts to the level where matrix effects no longer affect data quality.

In this work, both dilute-and-shoot preparation and Quick – Easy – Cheap – Effective – Rugged – Safe (QuEChERS) methods were used for a variety of food types including high water, high pigment, high fat, citrus, and dry foods with subsequent pesticide determinations by LC-MS/MS. Samples were spiked with carbamate, organophosphorus, aniline, conazole, macrocyclic lactone, phenylurea, benzoylphenylurea, and strobilurin pesticides in the ppb range. Matrix effects were determined by comparing calibration curves of solvent standards with dilute-and-shoot and QuEChERS prepared samples for a subset of pesticides having diverse chemistries. In some cases, dilute-and-shoot methods were successful as the matrix effects could be reduced by various dilution factors. There was more difficulty with the high fat and citrus foods, and QuEChERS was beneficial for sample preparation.

Alumina PLOT columns have a long history of being used for the analysis of volatile hydrocarbons. Due to the highly selective retention mechanisms at work in Alumina PLOT columns, saturated and unsaturated hydrocarbons can be fully resolved in a variety of applications. To enhance the performance of Alumina PLOT columns when performing quantitative work at trace level concentrations, Alumina adsorbents must be deactivated with salts like KCl and Na2SO4.

The practical separation behavior of Alumina PLOT columns is highly dependent on a number of factors.

Alumina PLOT columns are susceptible to water contamination and may need periodic thermal conditioning to restore performance. Repeated cycling to high temperatures can alter the selectivity of the adsorbent material coated in Alumina PLOT columns.

Instrument conditions can also strongly affect the separation characteristics of Alumina PLOT columns. Variations in carrier gas flow rate, initial oven temperatures, initial oven hold times, and oven ramp rates can significantly shift retention times and elution orders for volatile hydrocarbons.

The type of salt used for deactivation and the concentration at which it is applied can also influence the response for active compounds like 1,2-butadiene and chlorofluorocarbons.

This presentation will investigate new Alumina PLOT column adsorbents with respect to thermal stability, inertness, and selective retention. Applications will be shown illustrating their performance in the analysis of chlorofluorocarbons and difficult to analyze reactive hydrocarbons.

The production of tobacco, a high-value crop for the United States, is increased by the use of pesticides that are specifically approved for use on tobacco by the Environmental Protection Agency (EPA). Even after the processing of tobacco, some pesticide residues remain on the product, and under its pesticide registration program, the EPA is charged with assessing risks to smokers from exposure to these residues. Because tobacco is a highly complex matrix, the challenges associated with determining pesticides at trace levels are substantial. This includes pesticide extraction from tobacco, and the instrumental analysis step, which can be complicated by matrix interferences.

We used the Quick – Easy – Cheap – Effective – Rugged – Safe (QuEChERS) sample preparation approach to isolate pesticide residues from tobacco leaves, ranging from small, polar pesticides such as methamidophos to relatively nonvolatile, non-polar, large pesticides like deltamethrin. We also explored two extract cleanup methods—dispersive solid phase extraction (dSPE) and cartridge SPE (cSPE)—monitoring their efficiency at pesticide recovery and matrix reduction. Comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry (GCxGC-TOFMS) and liquid chromatography with tandem mass spectrometry (LC-MS/MS) were compared and contrasted for their ability to determine pesticide residues in the resulting extracts.

Polyaromatic hydrocarbon (PAH) analyses are performed regularly throughout the world for both food safety and environmental testing. Many organizations test for PAH residues in food products, but the analyte lists vary by organization and country. For example, in the United States, the EPA, FDA, AOAC, and NOAA all test for PAHs but do not share the same target compound lists. Internationally, the EU and individual countries will test for different PAH compounds. Chromatographic methods are typically tailored to specific target compounds and are not transferable when target compounds change. A further complication is the need to separate interfering compounds that are not considered target compounds but that do occur in samples, like triphenylene, perylene, and benzo[e]pyrene.

In this work, we developed a chromatographic solution that allows the separation of 29 PAH compounds including interfering compounds. This was accomplished by combining two HPLC columns in series: a PAH-specific phase and an aromatic bond selective phase. Separate column selectivities were tested and then combined based on a predicted ratio of the column lengths that would result in no coelutions. Temperature was also determined to play a critical role in the elution order and resolution of the PAHs tested. Time programmed fluorescence detection was optimized to yield the best sensitivity possible (ppb range) with closely eluting compounds.

Abuse of substances marketed as “research chemicals” often sold for research purposes only or added to consumer products labeled “not for human consumption” has become increasingly popular. Cathinones, including mephedrone, methylone, and MDPV, are one class of compounds that have appeared on the market as part of the “research chemical” movement. These compounds are commonly sold as bath salts; however, drug users often snort or ingest these compounds to induce an amphetamine-like high.

If these substances become scheduled drugs, demand will increase for routine testing for cathinones. Since these compounds are structurally similar to amphetamine and may be taken in lieu of amphetamine, adding them to an existing amphetamine screen can save laboratories time and expense. The objective of this project was to develop a fast, quantitative LC-MS/MS method for the simultaneous determination of mephedrone, methylone, MDPV, amphetamine, methamphetamine, MDA, MDMA, and MDEA in urine.

The LC-MS/MS method employed a 5 µm, 50 mm x 2.1 mm Ultra Biphenyl column. Liquid chromatography utilized a Shimadzu UFLC_XR, and tandem mass spectrometry was achieved on an AB SCIEX API 4000™ system operating in ESI positive mode. Mobile phase A consisted of water with 0.1% formic acid, and mobile phase B contained methanol with 0.1% formic acid. The flow rate used for analysis was 0.6 mL/min. The calibration curve used for analysis ranged from 1 ng/mL to 500 ng/mL.

Linearity (r) for all compounds across the calibration range was 0.9980–0.9995. Signal-to-noise for all compounds at 1 ng/mL ranged from 4:1 to 67:1. Accuracy for QC samples (n=3, 40 ng/mL) was 92%–112%. RSD for QC samples ranged from 4%–7%. RSD for internal standard response across all injections was 4%.

The method presented here allows for fast, routine analysis of mephedrone, methylone, and MDPV simultaneously with amphetamines by LC-MS/MS.

Since synthetic cannabinoids are relatively new, limited research has been performed to determine their urine metabolite profiles. Adding to analysis complexity, many metabolites are mono-hydroxylated isomers of their parent compounds. These metabolites are isobaric compounds that share a similar fragmentation pattern. These isomers are sometimes analyzed as one group, with little to no chromatographic resolution, which makes specific metabolite identification impossible. The objective was to develop an analysis method for the primary metabolites of JWH-018 and JWH-073, and to apply the methods for the analysis of these compounds in authentic samples.

The extraction used for all samples employed a high-load, end-capped 500 mg, 6 mL C18 SPE column. SPE resulted in an overall 2x sample concentration. A Shimadzu UFLC_XR equipped with a 1.9 µm, 50 mm x 2.1 mm Pinnacle DB Biphenyl column was employed for chromatographic separation. The detector was an AB Sciex API 4000 MS/MS operated in ESI positive mode. Three multiple reaction monitoring (MRM) transitions were monitored for each metabolite based on spectra from reference standards. Calibration curves ranged from 1–500 ng/mL for all metabolites, and quantification was accomplished using JWH-018 n-pentanoic acid-d4 as an internal standard.

The n-pentanoic acid metabolite of JWH-018 and the 5-hydroxypentyl metabolite of JWH-018 were identified in authentic urine specimens. In addition to these metabolites, two previously undocumented metabolites were observed at substantial levels in the samples. These metabolites were identified as 4-hydroxypentyl metabolite of JWH-018 and the 3-hydroxybutyl metabolite of JWH-073.

The method presented here resolves the clinically significant metabolites for JWH-018 and JWH-073. By using an SPE method rather than liquid/liquid extraction at high pH, the carboxylic acid metabolites of both JWH-018 and JWH-073 were recovered.

In light hydrocarbon analysis, the separation and detection of traces of polar hydrocarbons like acetylene, propadiene, and methylacetylene is very important. Traditional alumina columns deactivated with KCl or Na2SO4 treatments often produce lower peak responses for these active polar hydrocarbons. In addition, the peak response for polar hydrocarbons is not stable over time, with successive analyses showing even more decreases in peak response. To stabilize the response for polar hydrocarbons, different deactivation techniques have been applied to Alumina PLOT columns, but often at the expense of changing other column performance characteristics, such as sample handling capacity or loadability.

Alumina PLOT columns manufactured with this new deactivation technology are shown in this presentation illustrating improved performance for polar hydrocarbons while maintaining and optimizing sample capacity and resolution. Excellent peak shape and consistent response are obtained for polar hydrocarbons such as propadiene, acetylene, and methylacetylene. A comparison of peak response stability on industry standard columns will be shown as well as the applicability of using this new deactivation technology at higher temperatures for the analysis of higher molecular weight hydrocarbons.

Poor peak shape and decreased peak response can commonly be traced to injection port problems. Improper installation distance, contaminated injection port liners, and non-optimized injection techniques can all be sources of poor analytical results. However, contributions of other instrumental sources to overall chromatographic performance are often overlooked.

This study examines different aspects of flame ionization detector performance. Column installation into the detector can critically affect analytical performance. Improper installation distance and positioning can lead to tailing peaks and diminished response that may be indistinguishable from injection port problems. Detector jet installation and condition can also have similar effects. Data will be shown highlighting both detector problems. Troubleshooting guidelines will be discussed to enable analysts to more effectively troubleshoot and resolve chromatographic problems.

Typical non-arylene cyano (624-type) stationary phases for capillary chromatography provided an effective solution for the separation of many common volatile organic compounds, such as those found in EPA Method 8260. However, along with this unique and versatile polymer came some drawbacks, primarily high bleed, which became particularly problematic for those analyses utilizing ion trap MS detection, where excess bleed significantly impairs detector sensitivity as well as increases detector maintenance frequency.

In this work, we examined the effectiveness in utilizing a 624-type capillary column that incorporates a new highly inert deactivation with an improved stationary phase that incorporates aryl groups into the backbone of the polymer for analyzing common volatile organic contaminants in the environment. This new stationary phase significantly reduces column bleed.

After nearly two years, cleanup efforts continue in the gulf region following the second catastrophic oil platform disaster in the gulf. IXTOC I & Deepwater Horizon both are estimated to have dumped nearly 5 million barrels of oil into the gulf region. A year after the Deepwater Horizon spill, significant amounts of heavy oil and tar balls are washing up on the beaches of the Gulf Coast. Tar near where the IXTOC I oil rig sank can still be found by digging a few centimeters into the ocean floor 31 years later.

In 1990, the Oil Pollution Act (OPA) was promulgated under the direction of the National Oceanic and Atmospheric Administration (NOAA) following the Exxon Valdez oil spill in March of 1989. These regulations require a Natural Resource Damage Assessment (NRDA) following a release of oil into the nation’s waterways. Currently NOAA is conducting an NRDA to determine the impact of the Deepwater Horizon oil spill. There are several NRDA technical working groups (TWGs) assembled to determine: baseline conditions before the oil spill, impacts to plant and animals following the spill, and the current conditions of the marine ecosystem. The trustees are also evaluating impacts from the response to include use of dispersants.

There are a variety of methods for the determination of weathered crude oils. This presentation will discuss the analysis and characterization of different crudes for volatiles, semivolatiles, and biomarkers. Tar balls that have been collected on the beaches of Florida from the time the oil first appeared to the present will be evaluated. Attempts will be made to characterize these samples using a variety of techniques to include: Simulated Distillation chromatography, gas chromatography mass spectrometry, and multidimensional gas chromatography.

A lot of attention has been paid recently to 4-methylimidazole (4MI or 4-MEI), a by-product formed during the production of some caramel colorings. From an analytical chemistry perspective, this compound can be very problematic because it is highly polar and water soluble, thus very difficult to analyze using conventional reversed phase LC. To solve this problem, 4MI has traditionally been analyzed by GC-MS with derivatization or by sophisticated ion-pairing techniques. Both of these options, however, are laborious and often less reproducible, adding unwanted complexity to methods. A simpler aqueous normal phase (ANP) analysis can alleviate method complexity by increasing the retention of highly polar and water-soluble compounds using traditional LC columns. In this presentation, we use our Ultra PFP Propyl column to analyze 4MI with typical LC-MS mobile phases, water and methanol with formic acid, and isocratic conditions in ESI+. We also explore the retention mechanism of the PFP Propyl phase that allows the retention of small polar compounds such as 4MI.