An Inside Look at the Irish Whiskey Industry

Examining Volatile Aromatic Congeners in Irish Whiskey Using GC–MS and GCO

The Role of LC–MS/MS-based Metabolomics in Disease Biology

A Look at Chemometrics and Machine Learning

Approaches to Accelerate LC Method Development in the Laboratory 

A Look at Targeted Multispecific Antibody Cancer Therapeutics

Overcoming Complex Analytical Challenges

Human Body Odors Tested for Mosquito Attraction in Zambia

Patrick Lavery is an Editor for the MJH Life Sciences brands LCGC and Spectroscopy, and their respective websites, 

. He previously spent nearly a decade as a news anchor, reporter, and producer at New Jersey 101.5 FM.

A study written by Guomin Qi, who is affiliated with the College of Chemistry and the Engineering Technology Research Center on Reagent and Instrument for Rapid Detection of Product Quality and Food Safety in Fujian Province, both at Fuzhou University in Fuzhou, China, presents a novel, three-dimensional covalent organic framework (3D-COF) for the purpose of high-performance capture of a marine toxin, okadaic acid (OA) (1).

Mussels, clams, all the fresh sea | Image Credit: © corradobarattaphotos - stock.adobe.com

, the study reviewed the effectiveness of methods such as high performance liquid chromatography (HPLC) and liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS), in terms of both reproducibility and stability, to analyze OA, which is classified as a polyether and polyketide derivative of a C38 fatty acid (1). (And in fact, LC–MS/MS was ultimately used along with a 3D-COF to analyze the toxins identified in this study.) OA is further defined as a lipophilic toxin possessing distributed hydroxyl, polycyclic ether, and carboxyl groups, and may lead to human food poisoning. Covalent organic frameworks (COFs) are considered chemically and architecturally stable, Qi said, and are gaining a reputation as viable sample pretreatment and separation materials because they also offer efficient adsorption, ordered pore distribution, high and specific surface area and low density.

For the experiment authored by Qi, pore-surface engineering was employed to integrate linear building blocks (4,4′-diamino-3,3′-biphenyldiol [BD(OH)

] and benzidine [BD]) with a 3D structural building block backbone (4,4′,4′',4′''-methane-tetrayltetrabenzaldehyde [TFPM]). The ratio of BD(OH)

 was then adjusted to create functional, multicomponent COFs ([OH]

-BD-TFPM) with multiple-mode hydrophobic and hydrophilic groups, where dispersive solid-phase extraction (dSPE) was optimized at x = 25% (1). Essentially, Qi said, this process created content-tunable hydroxyl groups on the pore walls.

Clams and mussels purchased from local markets were the shellfish analyzed in this study. Because OA content in seafood is typically low, an effective pretreatment method has traditionally been sought for high-performance analysis; in this instance, the obtained shellfish muscles were pureed with methanol (MeOH) added into the centrifuge tube, filtered, dried with nitrogen, then redissolved in water and refrigerated (1).

-BD-TFPM COF, coupled with LC–MS/MS, was judged by Qi to be sensitive in the quantitative detection of OA in the clam and mussel samples, and especially so in comparison to previous detection of OA by solid-phase extraction (SPE) materials without hydrophilic interactions; the limit of detection (LOD) in this experiment was established at 0.005 μg/kg (1). Recoveries of OA ranged from 93.9% to 105.1% for the clam samples and 96.7% to 110.2% for the mussels.

Qi compared the robustness of this novel 3D-COF favorably with a previously reported two-dimensional (2D)-COF, particularly in terms of faster adsorption kinetics, better adsorption capacity, and better values for 

, or molal freezing-point depression constant. With a high-performance target capture the end goal of this study, the author concluded that a three-dimensional covalent organic framework SPE was powerful and easy to deploy.

(1) Qi, G. Efficient Capture and Highly Sensitive Analysis of Okadaic Acid by Three-Dimensional Covalent Organic Frameworks with Hydroxyl Surface Engineering. 

Events

Okadaic acid (OA) is considered a marine toxin with distributed hydroxyl, polycyclic ether, and carboxyl groups, easily accumulated in shellfish and known to cause food poisoning in humans.

3D-COFs Assist Capture of Okadaic Acid Toxins in Shellfish

Detecting bioactive compounds is important when studying herbs. In a recent study published in the 

, researchers from China introduced a novel approach that used high performance liquid chromatography coupled with tandem mass spectrometry (HPLC–MS/MS), using a supramolecular solvent (SUPRAS) for extraction (1).

Centella asiatica or gotu kola | Image Credit: © voranat - stock.adobe.com

), commonly known as Gotu Kola, has long been celebrated for its potential health benefits, attributed to bioactive compounds such as madecassoside (MS), asiaticoside (AS), asiatic acid (AA), and madecassic acid (MA) (1). Traditional extraction methods are often complex and time-consuming and this new method aims to simplify this process and reduce the time needed to extract compounds.

The SUPRAS method was created by using a combination of water, tetrahydrofuran (THF), and n-hexanol (1). SUPRAS is an effective method for extracting compounds from the herb. In this study, 4 mL of SUPRAS was applied to the sample for a brief 5-min period, followed by centrifugation to achieve phase separation (1). Then, the researchers used HPLC–MS/MS to analyze the solution.

The SUPRAS method was successful because of several reasons. First, it was able to draw out the bioactive compounds from the herbal material. It accomplished this by using both the dispersion and hydrogen bond interactions (1). Researchers also conducted a comprehensive investigation into various parameters, including the composition of supramolecular solvents, dosage, and vortex time, all of which were found to influence extraction efficiency (1).

Validation of the SUPRAS-based method was conducted by analyzing spiked samples, yielding recoveries ranging from 91.6% to 99.9%, with relative standard deviations between 1.7% and 7.9% (1). These results show that the method was capable of being efficiency, precise, and sensitive (1). There is also the benefit of the method being ecofriendly. The SUPRAS method does not require nitrogen blowing or any onerous clean-up procedures (1).

As a result, the researchers demonstrated how SUPRAS-based extraction was successful in analyzing real herb samples, further highlighting its potential to revolutionize the field of herbal compound analysis (1). The SUPRAS-based approach, coupled with HPLC–MS/MS, represents a new technique in analytical chemistry, with potential applications that could benefit from using this technique include pharmaceuticals, nutraceuticals, and herbal medicine industries.

(1) Yuan, Y.; Qiao, Y.; Zheng, X.; Yu, X. Dong, Y.; Wang, H.; Sun, L. Simultaneous determination of four active compounds in Centella asiatica by supramolecular solvent-based extraction coupled with high performance liquid chromatography-tandem mass spectrometry. 

In a recent study, researchers from China presented a novel approach that uses a supramolecular solvent (SUPRAS) method for detecting bioactive compounds.

Supramolecular Solvent Method Enhances Herbal Compound Analysis Using HPLC–MS/MS

Scientists from Dalian Maritime University in Dalian, China, are testing a new amphetamine-type stimulant extraction system on wastewater and urine samples (1). The scientists used a combination of dummy molecularly imprinted polymers (DMIPs) and molecularly imprinted polymer mixed matrix membranes (MMMs), and their findings were recently published in the Journal

Palm Oil Mill Effluent (POME) wastewater being discharged | Image Credit: © t4nkyong - sto

Amphetamine-type stimulants (ATSs) are synthesized psychoactive substances such as 

 (AMP), methamphetamine (METH), and 3,4-methylenedioxymethamphetamine (MDMA). While gas or liquid chromatography–mass spectrometry are the techniques mostly used for quantifying ATSs, scientists still face challenges finding test methods that are rapid, efficient, involves minimal sample preparation, and can produce improved purification. Solid phase extraction (SPE) does have advantages over traditional sample preparation methods such as low solvent consumption and cost, and from there, multiple solvents, such as magnetic nanoparticles and polymer monolith, have been used to better separate complex substrates such as sewage and blood.

One SPE solvent that is becoming more popular are molecularly imprinted polymers (MIPs). In the study, highly cross-link polymers were prepared in the presence of template molecules, functional 

, and cross-linker. After the template molecules were removed, imprinting sites matching several of their qualities are formed in the polymer matrix, allowing MIPs to specifically recognize the target. This allows for the dummy molecularly imprinted method using structural analogues as virtual templates to be used for building 

 systems. The scientists also looked to a novel extraction method based on molecularly imprinted polymer mixed matrix membranes (MMMs). MMMs are formed by homogeneous MIPs particles dispersed in the polymer matrix phase, with different qualities such as high porosity, thermal reversibility, and good bonding ability being effective detection factors.

The scientists created their DMIPs-MMMs-based method to test its effectiveness in analyzing complex substrates, as well as various individual qualities. 

 (SYN) molecules were first selected as the dummy template of ATSs. The adsorption capacity and selectivity of DMIPs were also investigated. MMMs were then formed by combining the prepared DMIPs with an agarose polymer matrix. Extraction efficiencies of MMMs for ATSs were evaluated by studying the effects of membrane matrix, pH, elution reagents, shaking time, and dissolved organic content. Finally, the optimized DMIPs-MMMs method coupled with HPLC-MS/MS was validated and implemented to extract and detect five ATSs from wastewater and urine samples.

The polymers were irregularly large with a specific surface area of 330 m

. Adsorption experiments found that the imprinting factors for five ATSs (amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, and 3,4-methylenedioxy-N-ethylamphetamine) were 2.3∼3.7. Furthermore, extraction conditions such as membrane matrix, sample pH, dissolved organic matter content, extraction time, and elution reagent were optimized. This enabled the developed MMMs-HPLC-MS/MS method, leading to exhibit low limits of detection (0.1∼3.0ng 

), satisfactory recoveries (91.7∼100%), and good repeatability (RSD < 7%, 

=3). It was then successfully applied to ATSs analysis in wastewater and urine samples. Recoveries of ATSs in spiked wastewater and urine were 82.0∼98.4% and 82.3∼95.7%, respectively.

Based on the study’s results, the developed MMMs-HPLC-MS/MS method could be developed further and used as a feasible strategy to extract and determine trace ATSs in wastewater and urine samples.

(1) Tan, D.; Liang, Y.; Guo, T.; Wang, Y.; Li, Y.; Sun, X.; Wang, D. Dummy molecularly imprinted polymers-agarose gel mixed matrix membrane for extraction of amphetamine-type stimulants in wastewater and urine. J. Chromatogr. A 

https://doi.org/10.1016/j.chroma.2023.464368

Articles

Scientists from Dalian Maritime University in Dalian, China, are testing a new amphetamine-type stimulant extraction system on wastewater and urine samples.

Amphetamine-Type Stimulant Extraction System Tested on Wastewater and Urine

Whiskey's intricate and diverse flavors are the result of a complex interplay of congeners, a group of chemical compounds that are naturally produced during the fermentation and aging process in whiskey production. Congeners are shaped by the type of cereal used, the mash bill, and the stages of malting, mashing, fermentation, distillation, and cask maturation. These congeners serve as biomarkers for quality and authenticity, and scientists have been using analytical techniques to improve whiskey manufacturing (1).

These compounds contribute to the flavor, aroma, and overall character of the whiskey. Congeners can include a wide range of substances, such as aldehydes, esters, higher alcohols, and phenolic compounds, among others.

However, despite the recent advancements in extraction and analytical techniques, there has been limited research on the extensive profiling of congeners specific to whiskey styles, especially within the rapidly expanding Irish whiskey industry. As technology and chromatographic techniques continue to evolve, there is potential to gain deeper insights into the congeners responsible for distinct flavor profiles and their role in authentication (1). 

Professor Kieran N. Kilcawley of Teagasc Food Research Centre and of University College Cork and his colleagues are using gas chromatographic (GC) techniques to explore how to analyze the quality and taste of Irish whiskeys through congener profiles. Research into these congeners is essential for understanding and managing the evolving industry, especially as it rapidly grows (1). 

 sat down with Prof Kilcawley to learn more about the research he and his team have conducted, the conclusions they’ve drawn from their chromatographic analysis, and the role that chromatography will play going forward in the Irish whiskey industry.

Professor Kieran N. Kilcawley of Teagasc Food Research Centre and of University College Cork | Image Credit: © Kieran Kilcawley.

Can you explain what is a congener and why the congener profile of a whiskey is critically important to its quality in whiskey production?

 is a term used uniquely to describe flavor active compounds in alcoholic beverages. The congener profile of a whiskey is formed from the combination of ingredients, and each part of the process, with fermentation and cask maturation assumed to have the greatest impact on the flavor of the final product. Therefore, a congener profile can potentially be used to differentiate styles of whiskey, brands, and even provide information on cask types. Understanding more about congener formation during the process and their association with sensory quality will provide additional information that could be used to improve quality, or direct flavor. Greater information on congener profiles related to specific types of whiskey can also be used as potential biomarkers for authentication.

What type of congener analysis have you and your team been conducting?

We work on samples diluted to 10% alcohol by volume (ABV), as it helps to standardize different samples for comparative analysis, as the ethanol content of whiskies vary depending upon the type, but also in relation to the strength of new make spirit (post distillation prior to addition the cask), cask strength and in the final bottled product. This is also undertaken to reduce the amount of ethanol introduced into the gas chromatography–mass spectrometry (GC–MS) instrument. The main difficulty with whiskey samples is that they have a very high concentration of ethanol compared to all the congeners. At the moment, we have profiled various new make spirits, single malts, pot still, and grain and blended whiskies. To date, we have optimized and evaluated two volatile extraction methods, headspace solid-phase microextraction (HS-SPME) and HS-SPME Arrow, using both conventional GC–MS and two-dimensional GC time-of-flight MS (GCxGC–TOF-MS).

Can you briefly talk about the four types of Irish whiskey and explain how these whiskey types influenced how you and your team conducted your analysis of the congener profiles?

The four types of Irish whiskey are single malt (made from 100% barley in a pot still), pot still (made from no less than 30% malted barley, 30% green barley and up 5% other cereal sources in a pot still), grain (can be any cereal source but mainly maize, wheat or rye in a column still), and blended whiskey (which consists of mainly grain whiskey with either single malt or pot still added, or both). Each single malt, pot still, and grain whiskey can be cask matured in any type of wooden barrel (mainly oak), but it must be for more than three years. Most bottled whiskies are produced from more than one aged cask (for example, the final product may be an amalgamation of numerous casks, with the choice and ratio dependent upon the skill of the master distiller). A single malt may be a blend of various single malts matured in different casks (virgin oak, ex sherry, port, bourbon casks, to name a few) but from the same distillery. In order for Irish whiskey to be labelled as a spirit drink and of Irish origin production must comply to the Irish Whiskey Technical File (2) and certain legal definitions, under EU Regulations EU2019/787 and 2021/1235 associated with its Geographical Indication Status. The final products are quite complex because of the potential diversity in both the production process and in cask maturation. Grain whiskey tends to have a less complex profile as a column still is used in its production, which can generate higher concentrations of pure ethanol, with less congeners (those less volatile than ethanol). All these factors need to be considered when developing a suitable method to extract and identify volatile congeners. A key issue as previously mentioned is the ratio of ethanol to all the other components, thus the samples need to be diluted and a split injection required to minimize the amount of ethanol introduced to the GC–MS.

a dark and moody atmosphere with a glass of sophisticated whiskey, including ice cubes, placed on a wooden table | Image Credit: © HN Works - stock.adobe.com

In a previous study (3), you and your team investigated how barley variety and its geographical location influence the aromatic profile of whiskey. What were the conclusions that you and your team were able to draw from the analyses conducted?

The study was published in 2021 and was on new make spirit (that is, the spirit post distillation before it goes into the cask). In that study, all other factors other than the cereal (barley) were controlled to the best of our ability. We looked at two different varieties of barley grown at two distinct geographical locations in Ireland, with differing soil types and weather conditions. The location of where the barley was grown had a greater impact on the volatile congeners in the resultant new make spirt than the barley variety. This was a first terroir type study for whiskey and highlighted the potential importance of where the barley was grown. Arguably, such differences in the new make spirit maybe reduced or eliminated by cask maturation, but it does provide food for thought and highlights additional factors in the development of whiskey congeners. Most distilleries utilize barley from a host of farms (geographical locations) and therefore any impact of terroir is lost in such an approach. However, some distilleries are now specifically harnessing this potential diversity in barley and terroir as a unique point of difference.

You and your team used GC–MS and gas chromatography olfactometry (GCO) to determine volatile compounds in whiskey. Why was GC–MS and GCO best suited for the analysis that you were conducting?

GC–MS is ideally suited for the analysis of most whiskey congeners because they are volatile. Even though we can identify more than 100 volatile congeners by HS-SPME GC–MS in a single run, we cannot be sure about their actual impact on sensory perception. We can make estimates based on abundance and referenced odor thresholds, but it is not practical to quantify so many volatiles. Therefore, the best way to identify those most likely to impact sensory perception in our experience is GCO. GCO is a relatively simple way of obtaining information about the aroma activity of individual volatile congeners using the human nose as an additional detector. More information relating to intensity and perception of individual congeners can be achieved using the split valve to dilute the amount of extract going to the column, which has the added advantage of eliminating matrix effects if the sample itself was diluted. GCO is relatively easy to perform, especially if you are regularly using the same personal as trained panelists. Obviously, the main disadvantage of GCO is that odors from the synergistic effects of two or more congeners are not captured. Co-elution issues and distinct aromas without corresponding peaks are typical hurdles, but many can be overcome by modifications in the extraction process or via changes in chromatography.

Your 2021 paper talked about exploring terroir in whiskey, stating that it has been explored in the wine industry, but not for other alcoholic beverages (3). Can you explain what terroir is chemically and how this can impact whiskey quality and production?

 is described as the set of all environmental factors that affect a crop’s phenotype, including unique environmental contexts, farming practices, and the crop’s specific growth habitat. In wine, it represents the quality and the unique sensory characteristics associated with its geographical origin and method of production. Therefore, this context was applied to whiskey in terms of where barley was grown in relation to the production new make spirit. Our study highlighted that the volatile congener profile generated in the new make spirit was impacted by the geographical location of where the barley was grown; that is, the local geographical conditions altered the phenotypic expression, which has also recently been shown in beer production (4). Further work is required to evaluate if these differences influence cask matured whiskey.

What challenges did you and your team have to overcome while conducting whiskey analysis using GC–MS and GCO?

Our main challenges to date relate to the use of GCxGC. GCxGC rapidly generates gigabytes of data and we had to invest in dedicated data storage to support this capability. Data processing is also a significant bottleneck because of the complexity and size of the data sets. Further advances in specific data processing software will help GCxGC to become more mainstream as its benefits our obvious. For example, we have investigated HS-SPME Arrow using conventional GC–MS (mid-polar column) with a single quadrupole MS, and identified around 100 congeners, but have also used HS-SPME Arrow with GCxGC (first dimension non-polar, reverse flow modulation and second dimension mid-polar column) with a TOF-MS instrument and typically see more than 200 congeners in the same sample.

Staying on the topic of challenges, what do you see as the biggest challenges in the Irish whiskey industry, and how do you think chromatographic techniques can meet these challenges?

The number of whiskey distilleries in Ireland has increased significantly in the last decade, with exports having risen from ~€200 million in 2010 to >€1 billion in 2022 (5). This rapid growth has resulted in huge increases in the volume and diversity within each whiskey type, but this increase in popularity has also increased the risk of fraudulent products appearing in the global marketplace. The critical and immediate need to create congener databases to aid in quality control, but also to identify biomarkers for authentication purposes. Chromatography has a significant role for such purposes, but also in tandem with other non-destructive spectroscopic techniques to rapidly identify outliers in production or in the marketplace.

Whiskey distilleries are growing in Ireland as Irish whiskeys grow in popularity. Image Credit: © Peter - stock.adobe.com

What are the next steps in your research?

We are keen to expand the use of GCxGC in whiskey analysis and plan to utilize flow modulation to enhance the least abundant (trace) volatile congeners going into the MS while reducing the volume of the most abundant congeners and ethanol to enhance the number we can positively identify. We would also plan to evaluate high capacity sorptive extraction as a direct immersion technique in whiskey research as we have found it very useful in other products, such as skim milk powder, butter, and beef. This approach is more likely to capture some of the least volatile higher molecular weight congeners because of the direct contact between the sample and the sorbent phase. We are also keen to continue our work using GCO to better identify key volatile congeners associated with the quality of specific types of whiskey. In addition, we are keen to expand our collaboration nationally and internationally with others involved in whiskey or other distilled spirits research.

(1) Kelly, T. J.; O’Connor, C.; Kilcawley, K. N. Sources of Volatile Aromatic Congeners in Whiskey. 

(2) Food Industry Development Division, Department of Agriculture, Food, and the Marine, 

Technical File Setting Out The Specifications With Which Irish Whiskey Must Comply

 [Online]; EU Commission Services, 2019. 

http://www.marketaccess.agriculture.gov.ie/media/marketaccess/content/IrishWhiskeyUisceBeathaEireannachIrishWhisky030519%20220623.pdf

(3) Kyraleou, M.; Herb, D.; O’Reilly, G.; Conway, N.; Bryan, T.; Kilcawley, K. N. The Impact of Terroir on the Flavour of Single Malt Whisk(e)y New Make Spirit. 

(4) Herb, D., Filichkin, T., Fisk, S., Helgerson,L., Hayes, P., Meints, B, Jennings, R., Monsour, R. Tynan, S., Vinkemeier, K., Romogosa, I., Moscou, M., Carey, D., Thiel, R., Cistue, L., Martens, C., Thomas, W. Effects of Barley 9Hordeum Vulgare L.) Variety and Growing Environment on Beer Flavor. 

Journal of the American Society of Brewing Chemists, The Science of Beer 

 Report [Online]; Drinks Ireland Ibec, 2022. 

https://www.ibec.ie/drinksireland/news-insights-and-events/news/2023/08/11/irish-spirits-market-report-2022

Professor Kieran N. Kilcawley of Teagasc Food Research Centre and of University College Cork and his colleagues are using gas chromatographic (GC) techniques to explore how to analyze the quality and taste of Irish whiskeys through congener profiles. 

 offers a review of recent developments in chiral stationary phases (CSPs) for pharmaceutical analysis using high performance liquid chromatography (HPLC) (1). Various CSPs, the research team said, are being developed because chiral pharmaceutical separation and analysis rely on them, and so they must be tailored to fit this purpose.

The researchers, from the Chinese Academy of Sciences in Lanzhou, China and Northeastern University in Shenyang, China, reviewed three types of CSPs. One was saccharides, which include amylose, cellulose, chitosan, and cyclodextrin; another, macrocycles, including macrocyclic glycopeptides, pillararenes, and polyamides; and the third being porous organic materials which include covalent organic frameworks (COFs), metal-organic frameworks (MOFs), and porous organic cages (1).

Chirality in drugs is a challenge when the pharmaceutical industry is perpetually seeking more optically pure medications (1). Chiral medications differ in pharmacodynamics, pharmacokinetics, and toxicity, but these authors say that often only one of the two mirror-image enantiomers has a therapeutic effect; the other may even cause side effects. In general, the researchers said, CSPs used in conjunction with HPLC can separate more than 90% of compounds that would be considered optically active.

The research team looked mainly at direct, rather than indirect, methods of HPLC separation of chiral substances. These included the macrocyclic compounds and porous organic frameworks. The direct method is viewed by the authors to be preferred because of its simplicity and convenience, although while commercial CSPs are widely available, they can be expensive (1). What’s more, even with their advantages these CSPs are considered to have limited separation ability, so more efficient, accurate, and economical choices are always being sought.

One objective the researchers had in publishing this study was to draw attention to the lack of reports, up to now, of real samples in previous studies. They said this reflects the current state of poor selectivity of CSPs, with both high selectivity and column capacity of stationary phases (SPs) in general being important in their estimation (1).

In summarizing the three CSP categories they evaluated, the authors began with saccharide-based CSPs, which they said were the first kind of material to be developed, and as a result there are many chiral columns derivative of saccharides in the commercial market (1). Similar to the saccharide cyclodextrin, macrocycles have a cavity structure and many active and easily modifiable sites on their surface. While porous organic frameworks are gaining traction, comparatively few instances currently exist of the likes of COFs and MOFs being used in actual chiral drug separation applications.

Suggestions for further research from the authors included more investigation into the chiral separation process itself, namely interactions between analytes and CSPs. And because pharmacological molecule structures are so typically complicated, they said, more or better calculations or measurements are needed to produce a thorough evaluation.

(1) Liu, H.; Wu, Z.; Chen, J.; Wang, J.; Qiu, H. Recent Advances in Chiral Liquid Chromatography Stationary Phases for Pharmaceutical Analysis. 

This review of pharmaceutical analysis in the last three years includes three categories of chiral liquid chromatography stationary phases: saccharides, macrocyclic substances, and porous organic materials.

Recent Chiral Stationary Phase Advancements in HPLC Analysis

A team of scientists from Universitat Rovira i Virgili in Tarragona, Spain, is investigating a new approach to monitoring water samples for contaminants of emerging concern, with their work being published in the 

Researcher holds a test tube with water in a hand in blue glove | Image Credit: © IVASHstudio - stock.adobe.com

Contaminants of emerging concern (CECs) in water is one of the most vital issues in environmental studies, partly because they can stem from many compound groups, including pharmaceuticals, flame retardants, hormones, and plastic additives, amongst others. Monitoring contaminants in environmental waters can be done in numerous ways. One approach that has recently grown in popularity is the use of passive sampling. Although most analytical methods for contaminant detection are based on discrete grab sampling, passive sampling combines sampling with sample treatment steps, which can provide time-weighted average (TWA) concentration values. This, along with low cost, ease of deployment, capacity for performing multi-site screening, no requirements for power or supervision, easy maintenance, and a reduction of matrix interferences, among other factors, makes passive sampling a good option for monitoring contaminants in water.

To test this method, the scientists used ceramic passive samplers (CPSs) using mixed-mode strong cation-exchange sorbent (Oasis MCX) as retention phase for the determination of 21 different drugs and their metabolites in river water samples, which were previously determined with liquid chromatography-tandem mass spectrometry. The CPSs were calibrated using 10 samplers in a 4L beaker with bottled water spiked at 20 μg/L with a mixture of analytes, before repeating the process with the same conditions, only with river water instead.

According to the researchers, bottled water was used for calibration since it proved more like the river water than Mili-Q ultrapure water. The CPSs were calibrated for 9 days with bottled water and river water, obtaining, for the 19 stable compounds, sample rates (

) ranging between 0.180 and 1.767 mL/day and diffusion coefficients (

/s. After calibration, CPSs were deployed into the Ebre River, located in Spain. Reproducibility was achieved, and multiple analytes were determined, including, but not limited to, gabapentin at 76 ng/L, caffeine at 203 ng/L and diclofenac amine at 57 ng/L.

The stability and calibration studies, despite using different types of water, disclosed similar results, in addition to proving effective in the experiment. “The passive method presented here is simple and feasible for the monitoring of CECs at trace levels in river water. This study opens the possibility of using other mixed-mode sorbents or other types of sorbents as retaining phase on CPSs or other passive samplers,” the scientists wrote in the study (1).

(1) Clivillé-Cabré, P.; Lacorte, S.; Borrull, F.; Fontanals, N.; Marcé, R. M. Evaluation of ceramic passive samplers using a mixed-mode strong cation-exchange sorbent to monitor polar contaminants in river water. 

https://doi.org/10.1016/j.chroma.2023.464348

A team of scientists from Universitat Rovira i Virgili in Tarragona, Spain, is investigating a new method for testing water samples for contaminants of emerging concern.

Passive Sampling Method for Analyzing Water Contaminants

Caroline Hroncich is the associate editorial director for LCGC and Spectroscopy. She can be reached via email at chroncich@mjhlifesciences.com. 

Turmeric and other spices are displayed at a market. 

Turmeric is a popular spice used throughout Southeast Asia. While traditionally used to flavor dishes like curry, turmeric has also become popular in the pharmaceutical and cosmetics industry. 

To better understand the purity of the compounds in this spice, a group of researchers from the National Institute of Pharmaceutical Education and Research (NIPER) Guwahati used repeated flash chromatography to isolate and purify curcuminoids in lakadong turmeric (1). The study was published in the most recent edition of the 

India is one of the largest exporters of turmeric in the world and there is a large demand for pure versions of curcumin because of its anti-inflammatory, anti-microbial, cardioprotective, and neuroprotective qualities, the researchers wrote. The 

 for curcumin was $58.2 million in 2020 and is expected to jump 16.1% between 2020 and 2028.

“The Coronavirus disease 2019 (COVID-19) pandemic has increased the consumption of turmeric as an immunity booster,” the scientists wrote. “It is foreseen that the demand for curcumin will swiftly reach its pre-COVID level, and the market is expected to see an exponential growth rate throughout the projection period, in line with the growing traction for curcumin as a treatment for viral infections around the world.”

The researchers studied lakadong turmeric, which has a high concentration of curcuminoids. These include curcumin, desmethoxycurcumin, and bisdemethoxycurcumin (1). To characterize the curcuminoids, the research team used ultraviolet-visible (UV-vis) Spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), proton nuclear magnetic resonance (1HNMR), carbon-13 nuclear magnetic resonance (13CNMR), high-resolution mass spectrometry (HRMS), and inductively coupled plasma mass spectrometry (ICP-MS).

Using the hot percolation method for 18 hours using 100% ethanol at 40 °C, the researchers extracted and analyzed the curcuminoids. Curcumin, desmethoxycurcumin, and bisdemethoxycurcumin were all analyzed to be more than 99.5% purity using high performance liquid chromatography-diode array detector (HPLC-DAD), they wrote. HPLC-DAD is an analytical technique commonly used in the food and beverage industries for quality control. Using HPLC-DAD, the sample is first analyzed using a chromatography column and then scanned by a diode-array detector.

“This method is economical, and reliable and can be easily scaled up to the industrial level,” the scientists wrote. “These high pure markers have high demand and huge commercial potential as active pharmaceutical ingredients (APIs) or as reference molecules in various industry segments such as pharmaceutical and agro-based industries.”

Vardhini, N. M.; Punia, J.; Jat, S.; Pawar, S. D.; Devi, N.; Radhakrishnanand, P.; Murty, U. S.; Saini, A.; Sethi, K. K.; Kumar, P. Purification and Characterization of Pure Curcumin, Desmethoxycurcumin, and Bisdemethoxycurcumin from North-East India Lakadong Turmeric (Curcuma Longa). 

, 464358. DOI:10.1016/j.chroma.2023.464358.

A group of researchers from India are using analytical methods to analyze the purity of the spice. 

Studying the Chemical Compounds in Turmeric 

Scientists from the National Institute of Pharmaceutical Education and Research (NIPER) in India used a variety of analytical techniques to characterize the medication erdafitinib. The team published their findings in a recent article in 

Erdafitinib is a type of tyrosine kinase receptor inhibitor used to treat a variety of diseases, most notably urothelial carcinoma. The study aimed to examine erdafitinib and its degradation behaviors when under different stress conditions as per the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) (2). The scientists also wanted to test the compatibility with various excipients under accelerated stability conditions.

The scientists used a Phenomenex Gemini C18 column (250 × 4.6 mm; 5 μm) with 10 mM ammonium acetate (pH 4.50) buffer and methanol as mobile phase in gradient elution mode at a flow rate of 1 mL/min. Under hydrolytic, photolytic, and oxidative stress conditions, erdafitinib was found to be labile, meaning that it can change quickly and spontaneously (3). However, when exposed to accelerated stability conditions, it proved incompatible with polyethylene glycol-4000, polyvinyl pyrrolidone-K30, crospovidone, and carboxymethylcellulose. In total, eight novel degradation products and one interaction product were generated.

Two more methods were then used to analyze erdafitinib: liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (LC–Q-TOF-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy. LC–Q-TOF-MS/MS studies were conducted to propose the structures of degradation and interaction products formed by comparing with the fragmentation pattern of the drug, while the NMR study helped confirm the chemical structures of two degradation products (specifically DP7 and DP8).

From there, the degradation pathway of erdafitinib was laid down, and 

 toxicity and mutagenicity data was created using DEREK and Sarah software. All the while, DP1, DP4, DP5, DP6, DP7, and DP9 showed mutagenicity as an endpoint.

(1) Velip, L.; Dhiman, V.; Kushwah, B. S.; Samanthula, G

 Characterization of Degradation Products and Drug–Excipient Interaction Products of Erdafitinib by LC–Q-TOF-MS/MS and NMR. 

https://doi.org/10.1007/s10337-023-04268-x

https://www.medicalnewstoday.com/articles/labile-hypertension

Scientists from the National Institute of Pharmaceutical Education and Research (NIPER) in India used a variety of techniques to characterize erdafitinib and its various products.

Exploring the Characterization of Erdafitinib

Scientists based out of Zhejiang Academy of Agricultural Sciences in Hangzhou, China, recently created a new QuEChERS method for analyzing pesticides in fruit samples, with their work being published in the 

Pesticides play an important role in protecting foodstuffs, but consuming large amounts can be dangerous. There are many ways to analyze for pesticides, but this study involved the establishment of a new QuEChERS method for the determination of multi-pesticide residues in fruits. While QuEChERS has been widely adopted in this field, current methods involve acetonitrile extraction, separation, and dispersed solid-phase extraction (d-SPE), which can limit preprocessing speeds. This approach involves the use of magnetic nanomaterials, which can be easily separated from extracts under an external magnetic field, enabling quick phase separation without additional waiting time. By improving the sample pre-processing program in this way, the scientists believe this will have great advantages in operation process and reagent dosage, among other factors.

To purify the fruit matrices, the scientists created their own poly-dopamine-modified magnetic nanomaterial (Fe3O4-pDA), which was used as a co-adsorbent with 3-(N, n‑diethyl amino) propyl trimethoxy-silane (PSA) in the developed integrated QuEChERS method to purify the fruit matrix, in addition to absorbing saccharides. This method was used on a variety of fruit samples, with grapes being used as a representative sample. Under optimized conditions, the method showed good linearity for 92.6% of pesticides in the concentration range of 1–150 μg L

 with method limit of quantitative (mLOQs) ranged from 10 to 18 μg kg

. From there, tests were conducted on more types of grape and different types of fruit samples, namely apple, watermelon, pear, winter jujube and peach.

Comparison experiments showed that this method, compared to traditional QuEChERS methods, was more convenient to operate, more efficient, and had low reagent consumption. Furthermore, under real sample analysis, the overall detection rate was 52%, with only 2% of samples were exceeding maximum residue limits.

“All results confirmed that the proposed method could be used for the rapid, simple, low-costing and effective analyses of trace multi-pesticides residue in fruit samples,” the scientists wrote in the study (1).

(1) Qi, P.; Wang, J.; Liu, Z.; Zhao, H.; Wang, Z.; Di, S.; Wang, X. Fabrication of poly-dopamine-modified magnetic nanomaterial and development of integrated QuEChERS method for 122 pesticides residue analysis in fruits.

https://doi.org/10.1016/j.chroma.2023.464336

Scientists based out of Zhejiang Academy of Agricultural Sciences in Hangzhou, China, recently used a new QuEChERS method for analyzing pesticides in fruit samples.

QuEChERS Method Proposed to Detect Pesticides in Fruits

, a team of researchers from the Key Laboratory of Pharmaceutical Quality Control of Hebei Province in the College of Pharmaceutical Sciences at Hebei University in Baoding, China introduced three new peptide chiral stationary phases (CSPs) with promising enantioselective capabilities (1). These phases, designed with a main chain comprised of L-proline (L-Pro), L-phenylalanine (L-Phe), L-valine (L-Val), and L-leucine (L-Leu), offer new opportunities in the field of high performance liquid chromatography (HPLC) for resolving chiral compounds. This development addresses a need in both the pharmaceutical and analytical chemistry industries.

AI Generated. AI Generative. Mono single amino acid molecule graphic design deocaration. Chemistry medicine education vibe. Graphic Art | Image Credit: © AkimD - stock.adobe.com

The researchers crafted these stationary phases by immobilizing L-citrulline, L-lysine, and L-tryptophan on 3-aminopropyltrimethoxysilane-modified silica gel (1). To ensure their quality and reliability, the newly developed phases underwent comprehensive analyses and characterizations, including Fourier transform infrared (FT-IR) spectra, elemental analysis, and thermogravimetric analysis.

One of the primary objectives of this study was to evaluate the enantioselective performance of the three peptide stationary phases. This evaluation involved nine racemic compounds and was conducted under normal-phase HPLC conditions (1). The researchers optimized the enantiomeric separation conditions and achieved what they described as impressive results. For instance, on the CSP-1 column, the resolutions of compounds such as flurbiprofen, naproxen, benzoin, 1,1ʹ-bi-2-naphthol, and ketoprofen were measured at 1.61, 2.0, 0.62, 0.52, and 1.20, respectively. These results demonstrated the potential of these novel stationary phases for separating a wide range of chiral compounds effectively.

Furthermore, the study investigated the reproducibility of the CSP-1 column, a crucial factor in chromatography. The findings indicated that these peptide stationary phases exhibit reproducibility with a relative standard deviation (RSD) of just 0.12% (n=5) (1).

The significance of this research lies in its contribution to the field of chromatography, particularly in the realm of chiral separations. Chiral compounds, which exist as mirror-image isomers, often possess distinct biological activities, making their separation a fundamental requirement in pharmaceutical development and chemical analysis. The introduction of these novel peptide chiral stationary phases represents a promising advancement in this domain.

The development and characterization of the L-Pro-L-Phe-L-Val-L-Leu peptide stationary phases exemplifies continuous efforts to advance analytical techniques and contribute to the broader understanding of complex chemical systems.

(1) Guo, X.; Shang, P.; Wei, B.; et al. Preparation and Enantiomeric Separation of L-Pro-L-Phe-L-Val-L-Leu Peptide Stationary Phases. 

Researchers from Hebei University in China have developed and characterized three novel peptide chiral stationary phases (CSPs) for enantiomeric separation, demonstrating their potential in high performance liquid chromatography (HPLC).

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