This is the fourth instalment in a series on method development for liquid chromatography (LC), with an emphasis on developing trouble-free methods quickly. We started out by considering some of the goals we might have and some method development strategies.

  Next, we selected starting conditions for reversed-phase separations.

  This was followed by a discussion of how to control retention for good chromatographic performance.

  This month, we'll consider how to pull apart those troublesome peak pairs. 

as a guide for the method development process. Here 

 is the retention factor, α is the separation factor and 

 is the column plate number. We looked at ways to adjust the retention factor  

 are the retention time and column dead time, respectively.) A retention factor of 2 < 

 < 20 is satisfactory in many instances. There is a regular change in retention with solvent strength for each analyte according to: 

 is the (extrapolated) retention at 0% organic (100% water or buffer), %B is the percent organic solvent in the mobile phase and 

 is the slope of the plot. The relationship of equation 3 allows us to predict retention for a given analyte based upon just two experiments at different %B-values, because the plot of log(

) versus %B is linear in most instances. 

Compounds that have very similar structures, such as homologs, we will refer to as "regular" compounds. These have very similar plots of log(

) vs. %B, as seen in Figure 1 for a sample of nine triazine herbicides.

  In such instances, the individual plots tend to fan out, with increasing peak spacing for weaker solvents (lower %B-values). This is what is expected from the fundamental resolution equation (Equation 1) — as 

 is increased. However, relative peak spacing doesn't change, so in terms of selectivity, there is little to be gained from a change in the mobile phase strength. As such, a simple change in the mobile phase strength is of little help in pulling apart two peaks that are difficult to separate when samples are all related closely in structure. 

Fortunately, samples comprising entirely "regular" compounds are much less common than those samples whose components differ in functional group types. Such samples we will refer to as "irregular" samples. An example of the retention behaviour of an "irregular" sample is shown in Figure 2 for a mixture of substituted benzoic acids (nitro, chloro, fluoro and so forth) and substituted anilines.

  It is obvious that, although the general slope of the plots is similar to Figure 1, the peak spacing for individual pairs of compounds changes dramatically with a change in the mobile phase strength. In several instances, peak crossovers (retention reversals) occur. It is such changes that we can take advantage of, so that by adjusting the %B, we can adjust the selectivity of the separation and pull specific peaks apart. We can also quickly find conditions to avoid, where the lines cross in the plots and, thus, peaks overlap completely. 

To quantify selectivity, we use the separation factor α: 

 are the retention factors of the first and second peak of a given peak pair. If we have retention behaviour as in Figure 2, 

-values will not change in parallel, so the α-related term 

 of Equation 1 will change, resulting in a change in resolution.  

  we saw that changing the %B was a great way to change the peak spacing for most samples. Although a change in %B is not the most powerful way to change selectivity, it is very easy, robust and is compatible with UV and mass spectral detectors. Samples that contain analytes with different functional groups, such as those of the irregular sample of Figure 2, will respond well to %B as a tool to change peak spacing. These reasons support our decision to change the %B first in our efforts to finetune the selectivity of a given separation. 

We can use plots, such as Figures 1 and 2, to calculate 

 for each peak and, thus, α for each peak pair at any %B. Because these plots are on a semilog scale, they can be hard to interpret visually. A more useful approach is to take advantage of the relationship of Equation 1. From our log(

) versus %B plots, we can get values for the retention (

) terms of Equation 1 for any %B. A value for resolution 

 or α, and this can be obtained by calculating, measuring, or estimating the column plate number 

). If we chose starting conditions with a 150 mm × 4.6 mm column packed with 5 μm particles, the plate number is approximately 10000,

  which is sufficiently close for resolution estimates using Equation 1. Now, we can plot 

 versus %B, as shown in Figure 3 for a mixture of six nitroaromatic compounds. This is called a resolution map and is available in the popular retention modelling software packages (for example, DryLab from Molnar Institute, Berlin, Germany, ChromSword from Merck, Darmstadt, Germany, and ACD/LC Simulator from Advanced Chemistry Development, Toronto, Canada).  

The resolution map is a plot of the resolution of the least-resolved, or "critical", peak pair at every %B-value. This is illustrated with the simulated chromatograms of Figure 4 for several points on the resolution map of Figure 3. At 40% B [Figure 4(a)], the minimum resolution is near zero and we see that the last two peaks are merged into a single peak. These two peaks pull apart as we move to higher %B-values, as for 50% B in Figure 4(b). The maximum overall resolution is at the apex of the plot at 55% B [Figure 4(c)], where the resolution of peaks 2 and 3 is equal to that of peaks 5 and 6. If we continue to move to higher %B-values, peaks 2 and 3 become the critical peak pair and the overall resolution is reduced [Figure 4(d)]. Note how the retention times (and, thus, 

-values) change for the different conditions of Figure 4. So you can see how this powerful tool can lead you quickly to fine-tuned conditions that give the best overall separation — it can save days of trial-and-error experiments. 

The resolution map of Figure 3 is for a variation in the %B, and requires only two experiments to obtain the input data. Similar maps can be made for most of the other variables of Table 1. Resolution as a function of column temperature requires just two input runs, whereas changing solvent type, ion pairing reagent concentration, or pH requires at least three runs to calibrate the retention model. The use of retention mapping in method development can greatly speed-up the development process. It can help quickly identify the best conditions for the separation as well as danger regions to avoid. 

Once we have adjusted the mobile phase strength so that 1 < 

 < 20, to get the retention times in a region that is likely to give good chromatographic performance, we can move on to the adjustment of peak spacing. Because most samples contain analytes with a variety of functional groups, our samples usually fall into the category of "irregular" samples, as illustrated in Figure 2. When this is the situation, adjusting the mobile phase percent organic can move peaks relative to each other so that we can hopefully find conditions where all the peaks are resolved from each other. The use of Equation 1 allows us to generate resolution maps that will help to identify quickly the conditions for the best separation. Because the resolution map is constructed based upon real experiments, it can provide very accurate predictions of resolution. If the relationships between retention and mobile phase conditions are linear (or log-linear), two experimental runs are required. For more complex relationships, such as retention versus pH, more experimental runs might be required, but the resolution mapping concept works just as well for such variables. Any of the continuous variables of Table 1 (all except column type) are amenable to retention mapping. 

 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", 

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

Readers can also direct questions to the Chromatography Forum at 

4. D.P. Herman, H.A.H. Billiet and L. DeGalan, 

Articles

LC Troubleshooting

The Perfect Method, Part 4: Controlling Peak Spacing

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 Dr Audrius Maruska, Vytautas Magnus University, Kaunas, Lithuania. 

 The development and evaluation strategies of new stationary phases and separation methods, particularly microseparations, has been the main focus of Dr Maruska's research group since 1990. The group gained expertise in synthesis and evaluation of the monolithic adsorbents (continuous beds) for capillary format liquid chromatography (cLC) and electrocromatography (CEC). Normal phase capillary electrochromatography (NPCEC) was introduced in 1995 as a suitable method to separate small organic compounds.  

Different techniques, including molecular imprinting and self-assembly of supramolecular structures, were used to synthesize the continuous beds. 

Capillary format monolithic adsorbents as restricted-access media (cLC RAM) for the direct injection of the biological fluids have been investigated since 2003. Thermoreversible gels for reversed-phase capillary electrochromatography (RPCEC) or chip format electrochromatography were reported. Current developments of monolithic stationary phases for capillary chromatography and electrochromatography include reversed-phase (RP) and normal phase (NP) hydrophilic interaction chromatography (HILIC), chiral chromatography, affinity chromatography (AC), restricted access (RAM) and gradient stationary phases for isocratic elution. 

They began extensive research on phytochemical analysis, chemotyping, epigenetic and molecular investigation of plants using different separation methods in 2002. This project investigates the secondary metabolites of plants, evaluation of their radical scavenging activity, and the development and coupling of different analytical methods for on-line analysis of biologically active analytes. The group has organized more than five summer schools and intensive courses on separation methods and their application. In August 2007 the group organized the 4

  Nordic Separation Science Society (NoSSS) International conference at Vytautas Magnus University, Kaunas. 

Facilities at the institute include supercritical fluid extraction (SFC), solid-phase microextraction (SPM), gas chromatography (GC), high performance liquid chromatography (HPLC) using ultraviolet visible (UV/vis), electrochemical detection (ED), refractometric detection (RD), fluorescence (FD), mass spectrometry (MS), electromigration, capillary electrophoresis (CE) and capillary electrochromatography (CEC), UV/vis spectroscopy and fluorescence spectroscopy (FS), microsynthesis, optical and fluorescence microscopy and inverse size exclusion chromatography (/SEC) porosimetry equipment. 

 Dr Susanne K. Wiedmer, Laboratory of Analytical Chemistry, Department of Chemistry, University of Helsinki, Finland. 

 The focus of the Wiedmer research group is to obtain a deeper understanding of the interactions taking place in biomembranes. Specific attention has been given to lipid–analyte interactions, including analytes ranging from low-molar mass (MM) drugs to proteins with molar masses up to 80 kDa. 

In these studies liposomes composed of commercial, natural or synthetic lipids or lipids extracted from biomatrices have been used. Liposomes are microscopic spherical vesicles that form when phospholipids are hydrated. Typically, several of these vesicles will form within each other in diminishing size, creating a multilamellar structure of concentric phospholipid spheres separated by layers of water. The group decided to use unilamellar vesicles with sizes ranging from 30–120 nm prepared by extrusion or probe-sonication.  

The liposomes are made of phosphatidylcholines combined with other phospholipids, sphingolipids, cholesterol and PEGylated-lipids. The techniques for obtaining information on lipid–analyte interactions and on liposomes have included capillary electromigration techniques, field flow fractionation (FFF), fluorescence spectroscopy (FS) and photon correlation spectroscopy (PCS), monolayer measurements, light scattering, quartz crystal microbalance (QCM) and microscopic techniques.  

Capillaries for electrophoresis have been coated with lipids [i.e., capillary electrochromatography (CEC)] or filled with lipid vesicle dispersions [i.e., electrokinetic capillary chromatography (ECC)]. There has been a lot of research on factors affecting the stability of lipid coatings on fused silica capillaries. Studies on the effect of capillary pretreatment, lipid composition, solvent, pH and coadditives on the ability of lipid vesicles to attach to the fused silica capillary wall have been performed. Applications have included interactions between low molar mass (MM) drugs or proteins and lipid membranes, chiral separations of enantiomers by proteins embedded in lipid membranes and study of sterically stabilized PEGylated vesicles. Recent research has focused on the effect of liposome composition and liposome size on the arrangement of lipids and lipid vesicles on silica surfaces.  

 Dr Chem. Helena Kaszoka, Laboratory of Chromatography, Latvian Institute of Organic Synthesis, Riga, Latvia. 

 The Latvian Institute of Organic Synthesis (LIOS) was founded in 1957 to perform research in organic chemistry, molecular biology and bioorganic chemistry. High-performance liquid chromatography (HPLC) is one of the major analytical techniques for the analysis and separation of the organic substances. The Laboratory of Chromatography (LOC) deals with the development of methods for quality control (QC) of organic substances (candidates for new drugs) synthesized in the Institute. This means that HPLC methods for the analysis of these substances are not available in literature. 

The purpose of this group's research is to investigate the relationships that determine the behaviour of solutes under study in different chromatographic systems and to increase separation selectivity. 

Reversed-phase (RP) liquid chromatography (LC) is the most popular LC mode. However, for some samples separation by RP mode is ineffective. Normal-phase (NP) liquid chromatography can be an alternative approach to optimize HPLC separation. The use of NP separations in combination with RP ones can offer additional possibilities in the QC of organic substances. Unfortunately, adsorption is the dominating retention mechanism under NP mode, therefore a common problem with unmodified silica columns is that polar compounds often show broad, tailing peaks, especially for basic components. 

As well as the well-known HPLC modes a new normal-phase mixed partition-adsorption (MPA) mode was developed and used to create HPLC methods. MPA systems show a good peak symmetry on all silica types. In the MPA mode unmodified silica is used as a stationary phase and a mixture of two or three solvents with a limited mutual solubility (a mobile phase is saturated only partially: 50–70%) is used as a mobile phase. It was established that the stationary liquid phase was generated dynamically in the pores of silica, even in the mobile phases that are only partially saturated with the polar component. The retention process cannot be considered as a simple adsorption but a rather complex mixed partition–adsorption process involving liquid–liquid partition into stationary liquid phase developed from the eluent. Such MPA systems are a good choice not only to improve peak symmetry for polar solutes on silica columns but also to enlarge the number of chromatographic systems in HPLC, especially if a system with a specific selectivity is needed. 

 Prof. Audrius Padarauskas, Department of Analytical and Environmental Chemistry, Vilnius University, Vilnius, Lithuania. 

 The primary focus of the group is research in the field of separation science and covers high-performance liquid chromatography (HPLC), capillary electrophoresis (CE) and gas chromatography (GC). There are two main sections in the group led by Prof. Padarauskas (group head) and Prof. Vida Visckasckaite. 

For the past 10 years, Prof. Padarauskas' main scientific interest has focused on the development of capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) techniques for the analysis of inorganic and low-molecular-weight organic compounds with an emphasis on the development of new strategies for selectivity control, derivatization and preconcentration and element speciation. Main projects include the development and application of dual-opposite injection CE for the simultaneous determination of anions and cations, investigation of in-capillary derivatization using a  zone-passing technique for the speciation studies and application of complex-forming equilibria for HPLC and CE separation of metal species.  

Recent work has focused on the investigation of water-soluble polymers as capillary coatings and pseudostationary phases in capillary electrokinetic separations and the development of headspace water-based liquid-phase microextraction (LPME) for the isolation of volatile analytes from complex matrices and the subsequent CE determination of these species.  

 e's research focuses on the development and application of miniaturized sample preparation techniques, such as solid-phase microextraction (SPME) and LPME coupled with GC. Several alternative SPME fibre coatings were investigated including silicone glue, carbon nanotubes and conventional HPLC sorbents. Special focus is given to the investigation of microextraction with in-fibre/in-drop derivatization of amino and fatty acids. 

 Prof. Mihkel Kaljurand, Institute of Chemistry, Faculty of Science, Tallinn University of Technology, Tallinn, Estonia. 

 Prof. Mihkel Kaljurand's research group consists of seven researchers and ten graduate students. It is mainly supported by Estonian government grants and some EU funds. It is focused on the development of separation protocols, instrumental aspects and applications of capillary electrophoresis (CE).  

The group has developed various non-aqueous buffer systems with ionic liquid additives for the separation of non-soluble compounds using capillary elctrophoresis (CE). The group is also involved in the development and application of novel miniaturized sampling systems for CE, which can be used to monitor the reactions in an electrophoresis capillary in real time. 

Another project involves the analysis of bioactive compounds (polyphenols and vitamins) from biological sources (plant and tissue extracts) to determine their antioxidative activity. Methods to determine the composition of the extracts from wild and domestic berries, tomato skin and eggplant using capillary electrophoresis (CE) with diode array detection have been developed. The team has also optimized the extraction process by using novel environmentally friendly solvents such as CO

. The identification of key polyphenols has led to the development of new selective assays for the determination of the antioxidative power of various extracts and these are compared with the results of commonly used, non-selective assays. 

 Carl-Henrik Brogren, Cellular and Metabolic Research Department of Biomedical Sciences, University of Copenhagen, Denmark. 

 This Danish group focuses on two main areas of biomedical science: diabetes and obesity. This involves immunological and biospecific separations using affinity chromatography (AC) with affinity measurements and also microfluidic cell proteomics. 

), founded by Carl-Henrik Brogen in 1996 owns the intellectual property right (IPR) to a monoclonal autoantibody (IC2) that specifically recognizes the pancreatic beta-cell with applications in, for example, medical imaging with PET and SPECT, stem cell research in type-1 diabetes autoimmunity, and the diagnostic 

 non-invasive quantitative estimation of the residual pancreatic beta-cell mass. These immunotechnological-oriented research activities involve various affinity chromatography (AC) systems to purify, analyse and quantify the diabetic IC2 autoantigen as the precise molecular nature and structure is still unknown, and is the major focus for ongoing research. 

The group is also involved in a European consortium aiming to develop medical imaging using the IC2 autoantibody, and is also collaborating with Orsay Physics in Aix en Provence, the University of Rouen and the Ecole Supérieure de Physique et de Chimie Industrielles in Paris to develop a nanofluidic separation and molecular detection method to study cellular proteins from one single cell. This requires expertise in physics, optics, electronics, biology and biochemistry and an international research collaboration is required. Relatively few research groups worldwide have expertise in all of these areas and a virtual network called 

 has been established to combine expertise.  

The first step in this novel proteomic technology involves nanofluidic isotachophoretic separation of the cellular proteins. Directly after this the sample will finally be analysed by nano-scale single ion mass spectroscopy (Nano-SIMS). The post-column molecular mass analysis by NanoSIMS still requires further development. Currently only the microfluidic level has been reached for the isotachophoretic separation using conductivity, optical digital imaging and laser induced fluorescence (LIF) detection. 

 Dr Jolanta Liesiene, Department of Organic Technology, Kaunas University of Technology, Lithuania. 

 The group focuses on the preparation of stationary phases for the low- and medium-pressure preparative chromatography of proteins. A new cellulose-based, semi-rigid matrix named Granocel with pores accessible to macromolecules with molecular masses up to 2 × 10

This matrix was used for the preparation of various packings, such as adsorbents for hydrophobic interaction chromatography (HIC), ion-exchangers, chelating sorbents for metal affinity chromatography (MAC) and biospecific adsorbents. The group also co-operates with biotechnologists to examine the separation of biological active materials, particularly the human growth hormone, α-interferon, using Granocel. The effect of pore size and ligand density on the chromatographic performance is being investigated. 

The group is currently developing packings for high-pressure liquid chromatography (HPLC) of proteins, by coating macroporous silica with hydrophilic polymers, such as cellulose and its derivatives. Porous silica gels have excellent mechanical properties, however, negatively charged silanol groups cause a strong, even irreversible, nonspecific sorption of proteins. To avoid this disadvantage the silica surface is coated with biocompatible polymers. Coated stationary phases for hydrophobic interaction chromatography (HIC) of the proteins and for lectin affinity chromatography (AC) have also been developed. 

 Prof. Staffan Nilsson (research leader), Christian Nilsson [nanoparticle-based capillary electrochromatography (CEC)], Arthur Varga (non-aqueous capillary electrophoresis (NACE) of ethanol biomarkers), Loy Alkhafadji (airborne chemistry), Seyed Tabaei, Andreas Bengtsson, Sandra Lemos, Department of Pure and Applied Biochemistry, Lund University, Sweden. 

 The research group focuses on miniaturized separation techniques. Novel analytical separation methods are being developed for real biological applications. Capillary electrochromatography (CEC) using nanoparticles as a pseudostationary phase for protein separations is one of the main areas of research. This allows the single-use of the stationary phase to avoid carryover effects and long regeneration procedures. Pseudostationary phase capillary electrochromatography (PSP–CEC) enables use of submicron particles, which is not possible in a practical way in packed columns. 

Nanoparticles possess favourable surface area-to-volume-ratio (SA/V) because of their small size, and highly efficient separations in CEC seem possible. The technique has already shown its potential to separate neutral small molecules, such as dialkyl phthalates, with plate numbers as high as 700000 theoretical plates/m. Small neutral molecules have been analysed, but the current main focus is to perform protein separation, protein–protein or protein–ligand interaction studies at physiological conditions in bare fused-silica capillaries. A parallel project involves the development of "monoclonal" plastic antibodies [i.e., molecularly imprinted polymers (MIPs)]. Airborne chemistry is another main area of interest for the group.  

Nordic Focus

A look at the activities of some of the leading research institutes in the Nordic region.

Norse Legends

Mass spectrometry (MS) is almost a hundred years old, but the coupling of MS with liquid chromatography (LC) is still in its youth and began in the mid-seventies. However, the real advances in this hyphenated technique began in the early eighties with the introduction of fast-atom bombardment (FAB) and thermospray ionization (TS), which led to the development of atmospheric-pressure chemical ionization (APCI). 

The most famous ionization sources — electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) — followed five to seven years later. The next fundamental development for the incredible growth of the LC–MS instrumentation was catalysed by the popularization of "lightweight" mass analysers, such as quadrupoles and ion traps, the revival of time-of-flight (TOF) analysers, and the introduction of the new Orbitrap detector (Thermo Fisher Scientific, San Jose, California, USA). 

These analysers are now available in a wide variety of combinations that allow users to work with tandem MS in a simple, problem-free manner. LC–MS has entered the field of organic analytics in a big way and changed the perspectives and direction of many research fields, including the composition of food and beverages. This article looks at the analysis of wine and the new insights that LC–MS has revealed regarding its composition, health aspects and winemaking practices (Figure 1). 

Even to lay eyes, the different colour nuances of young and aged red wines are clearly visible. It has been known for at least 30 years that this colour change results from the reactions of wine pigments called anthocyanidins with other compounds present in wine.

  There are five anthocyanidins present in grapes as conjugates, mainly glycosides (anthocyanins). Their analysis by LC–MS can be accomplished using APCI, ESI or MALDI ionization, and different tandem MS scanning modes allow their determination in complex mixtures.

  The study of anthocyanic composition can be used to characterize the grape/wine variety and addition of hybrid grape varieties.

  The true value of LC–MS, however, lies in the fact that it allows the identification of pigments formed during wine maturation and resulting from the anthocyanin condensation reactions with pyruvic acid, acetaldehyde,

  to name just a few, and also from anthocyanin polymerization.

  ESI, particularly in negative ionization mode, has become indispensable in these studies.

  The new (non-polymeric) pigments have been named pyranoanthocyanins and are chemically more stable than grape anthocyanins, but their structural diversity is enormous and they continue to form endlessly during wine ageing.

  To further complicate matters, a recent LC–ESI–MS analysis indicated that anthocyanin oligomers are already present in grape skin extract.

The colour change of white wines involves a different mechanism: the browning has been shown to be induced by glycoaldehyde, resulting from malolactic fermentation of wine.

A glass of wine a day keeps the doctor away. Or at least that's what we think should be the new maxim for the grown-up population. A vast body of research in polyphenolic compounds, mainly by the aid of LC–MS methods, has resulted in their great popularity as antioxidants and as protective agents for vascular health.

  Wine is also rich in polyphenolics, mainly flavanol monomers, oligomers (procyanidins) and glycosides.

  Several important oligomers with molecular weights (MWs) above 1000 have been identified in grape seed by MALDI-MS,

  but their presence in wine depends greatly on the winemaking practice (e.g., postfermentative maceration).

  Other important phenolic compounds are also flavanonols and phenolic acids, present in significant amounts in grape stems.

  The great structural diversity of polyphenols found in wine makes LC–MS the most suitable method for their identification and quantification.

  Different ionizations are used: FAB, APCI, ESI, MALDI, often in conjunction with tandem MS. Generally, negative ionization (NI) has proven to be more efficient than positive (PI) for these compounds.

  An example of LC–MS–MS multiple-reaction monitoring chromatogram (ESI-NI) of wine phenolics is shown in Figure 2. 

The total polyphenol content in wines is usually determined using the Folin-Ciocalteu method, and some studies have shown good correlation between this method, LC–MS–MS analyses and the antioxidant activity determined by ABTS.

Another group of health-affecting compounds in wine are stilbenoids or phytoalexins, important for their strong antioxidant, antimicrobial and anticancer properties, which are synthesized by plants in defence against pathogens or stress.

  The most important and commonly found is resveratrol with its glycosides, and LC–MS (PI–APCI) seems also to be the most suitable method for its determination.

  Some other physiologically active stilbenes have also been identified in grapes by LC–ESI–MS: δ-viniferin

  previously thought to occur only in non-edible plants. 

LC–MS has enabled the fate of polyphenols and stilbenoids 

 after their ingestion to be followed. Their absorption in the intestines was studied 

  and their metabolites have been identified.

  It has also been shown that polyphenol metabolites cross the blood–brain barrier and can, therefore, also exhibit their protective effects in the brain.

The truth is that although wine has actually been confirmed to be healthy, we still drink it because of its pleasant taste and aroma (among other reasons). Wine aroma is detected primarily through the sense of smell: thus the main constituents are volatile compounds, (e.g., monoterpenes and aldehydes, which are more readily analysed by GC–MS than by LC–MS).

  However, the majority of terpenes and some other odorous compounds are present in grapes in the form of precursors: glycosides and other conjugates. They are only partially released during fermentation. 

Terpene glycosides remain almost unexplored as LC–MS analytes,

  although they are theoretically amenable to it.

  An example of the PI–ESI–MS spectrum of a compound from grape extract, tentatively identified as a terpene glycoside, is shown in Figure 3. Apart from volatile compounds, the perception of wine in the mouth is important for a harmonic sensation. The taste perception of wine is dominated by phenolic compounds.

  Higher MW tannins interact with salivary proteins and cause astringency, while flavanol monomers cause bitterness.

  Most of these compounds have been determined and characterized by LC–MS.

  Bitterness and astringency have also been ascribed to triterpenes, which are along with some tannins extracted from oakwood used for ageing of some wines. Electrospray in NI mode is an effective type of ionization for these compounds and their determination by LC–MS in beverages.

Carboxylic acids have an important contribution to the overall wine flavour. Although they can be determined by other means, ion exclusion chromatography (IEC) with APCI–MS is one of the more selective and sensitive methods.

Recent research in human receptors has led to the discovery of specific triggering "taste molecules". Generally, they are relatively polar and high-MW and are, therefore, excellent candidates for LC–MS determination. One of them, 

-lactoyl ethanolamine, has been identified in wine using LC–MS–MS.

As with anything we eat or drink, special emphasis should be on wine quality and control of potentially harmful substances. The idea of quality encompasses many things: as well as fine flavour and lack of taints a consumer expects wine to be made of a declared grape variety, although sometimes this may not be the case. Identification of grape variety used for the production of a given wine is extremely difficult and relies mainly on aroma compounds determined with GC–MS.

  The first glimpse at a possible solution was an LC–ESI–MS characterization of resistant, haze-forming proteins in wine, which turned out to be pathogenesis-related (PR) proteins, persisting throughout the complete wine-making process and seemingly characteristic of the grape variety.

  The theory was confirmed also for other grape varieties and using different MS approaches: ESI, nano-ESI, MALDI–TOF.

Quite another problem is the presence of harmful substances in wine, be it naturally-occurring or introduced by humans. The list of possible culprits is getting longer every year. The permanent candidates are various pesticides used in the grape-growing practice. Most of them are suitable for GC–MS analysis, but there are also some polar compounds which are better determined with LC–MS after an appropriate sample preparation procedure: azole fungicides (ESI, MS–MS),

  dicarboximide fungicides (APCI), phenylurea and carbamate herbicides (PI and NI APCI or ESI).

  More unusual is the contamination of wine with "alcohol-based brine" (alcohol or glycol for refrigeration), which can be detected through the additive rhodamine by LC–MS–MS.

The most problematic of naturally-occurring toxins in wine are those produced by various fungi growing on grapes (mycotoxins). Ochratoxin A is probably the most frequently present, and several LC–MS procedures have been proposed for its monitoring.

  Its determination, however, is complicated because ochratoxin slowly degrades under the influence of sunlight into several products, which have been identified by LC–ESI–MS–MS (Figure 4).

  Another, less common mycotoxin alternariol has also been detected in wine using LC–MS–MS with APCI or ESI.

Biogenic amines, which exhibit several physiological functions and are also mutagenic, can be found in wine and have been analysed by LC–TSP–MS.

It is known from historical records that grapevine has been grown and wine produced in the Mediterranean basin already for some 5000 years, yet we do not know much more than that. A recent application of LC–MS–MS for identification of traces of wine markers in ancient Egyptian vessels revealed that they had been used for storage of red wine. A common wine marker is tartaric acid, while syringic acid is the product derived from malvidin, a red wine pigment.

 is an associate professor of analytical chemistry at University of Ljubljana, Faculty of Chemistry and Chemical Technology, her research interests include sample preparation, chromatographic and mass spectrometric methods in the field of natural products research, food and environmental analysis. 

 is a professor of analytical chemistry at University of Ljubljana, Faculty of Chemistry and Chemical Technology, where his research encompasses new analytical approaches in characterisation and stabilization of organic materials, environmental assessment and food analysis. 

 is a researcher at University of Ljubljana, Faculty of Chemistry and Chemical Technology, his main research interests are polymer analysis and development of LC–MS methods for a broad range of samples: wine analysis, determination of pharmaceuticals in wastewater and others. 

 is an associate professor at University of Ljubljana, Biotechnical Faculty, where he researches new approaches in grapevine-growing and influence of various parameters during wine production on its quality.  

6.  N.-E. Es-Safi, V. Cheynier and M. Moutounet, 

8.  Y. Hayasaka, G.A. Baldock and A.P. Pollnitz, 

LC–MS

Liquid chromatography–mass spectrometry (LC–MS) is a popular technique for the analysis of wine. This article gives an overview of wine analysis and new insights this technique has revealed regarding the composition of wine, possible health benefits, customer safety and the understanding of winemaking processes.

In Vino Veritas: LC–MS in Wine Analysis

The Tenth International Symposium on Hyphenated Techniques in Chromatography and Chromatographic Analysers (HTC-10) will be held in Bruges, Belgium, from 30 January to 1 February. The event commemorates 20 years of dissemination of scientific knowledge in the field. 

HTC-10 will again cover all the fundamental aspects, instrumental developments and applications of the various hyphenated chromatographic techniques (e.g., coupling of GC or LC to LC, GC and SFC/SFE; MS, FTIR, AED and other techniques coupled with GC, HPLC, SFC, CZE, CEC and FFF; PTV–GC–MS; on-line air traps-, purge- and trap, extractorsand GPC–GC (or LC); LC to NMR, Raman, FIA–DAD, light scattering; ITP–MS etc.). Emphasis will also be placed on sample introduction, miniaturization, micro-fabricate analytical devices and on the design of hyphenated, on-line and at-line chromatographic analysers. 

ExTech-10 is a symposium series highlighting new extraction technologies for chemical and biochemical analysis in laboratory and on-site settings. The general focus is on solvent free sample preparation including methods for rapid and selective enrichment prior to analysis and micro-sample preparation. Major emphasis will be placed on the fundamental understanding of extraction processes that result in greater selectivity and efficiency in method development, and a rationalization of method selection for specific applications. On-site environmental methods and biomedical applications will be covered. The 2008 edition will focus especially on passive sampling methods, use of molecular imprinted polymers, biologically modified sorbents, novel coatings and sorbent materials. 

Additionally, on Wednesday 30 January, a special one day conference on Public Safety and Security emphasizing the Hyphenated Chromatographic approach will be held. On this day, both the sample preparation problems and the analytical methodologies developed for the continuing public protection will be discussed. This symposium will highlight new sampling, sample-preparation, derivatization, sample-introduction and hyphenated chromatographic techniques for forensic research and for the detection and analysis of chemical and biological warfare agents and their degradation products. 

From Wednesday 30 January until Friday 1 February, there will also be a large integrated exhibition of scientific posters and commercial instrumentation, consumables and services. 

Additional information on the exhibition can be obtained from 

Pittcon 2008 will be held at the Morial Convention Centre in New Orleans from 1–7 March. 

The world's largest conference and exposition for laboratory science will showcase all the latest products and technologies, offer practical technical knowledge and provide a stage for global networking opportunities. 

Contact: Jonathan Slater, Zing Conferences, 

  International Symposium on Microscale Bioseperations and Methods for Systems Biology, organized by the ISAS  —Institute for Analytical Sciences, with 

ISAS — Institute for Analytical Sciences, Bunsen-Kirchhoff-Str. 11, 44139 Dortmund, Germany 

Contact: Messe München GmbH, Messegelände, 81823 München, Germany 

32nd International Symposium on High Performance Liquid Phase Separations and Related Techniques 

Contact: Ms Janet Cunningham, Symposium/Exhibit Manager 

Event News

The Tenth International Symposium on Hyphenated Techniques in Chromatography and Chromatographic Analysers (HTC-10) will be held in Bruges, Belgium, from 30 January to 1 February. The event commemorates 20 years of dissemination of scientific knowledge in the field.

Ex-tech-10 and HTC-10

One recent column examined a structured, methodical approach to characterizing suspected counterfeit active pharmaceutical ingredients (API).

  A logical conclusion this month provided by guest contributors from Cardinal Health (Research Triangle Park, North Carolina, USA) takes the methodology a step further. Once a properly manufactured and tested drug has been packaged, there can be anomalies found on reanalysis after storage. The practitioners in these instances need to employ a variety of tools as well as their experience to come up with a confirmed identity for the anomaly as well as a possible source for its presence. Once again, mass spectrometers play a central role in the effort. Identifying drug-product-related impurities is a challenge that all pharmaceutical companies face. Compared with drug-substance-related impurities, which most commonly originate with the synthetic process or subsequent degradation, drug-product-related impurities can originate from several additional sources. Those sources include excipients and interaction with the packaging or container–closure devices. Although we can identify drug-product-related impurities solely via a sound analytical approach,

  considering and understanding all potential sources of those impurities can facilitate the process and expedite a solution.  

Identifying impurities originating from the drug substance or excipients is not necessarily trivial, but at least those things lend a starting point for the investigation. But identifying impurities originating from the packaging or container–closure devices can sometimes prove more challenging for several reasons:   

The plastic and rubber materials used in the packaging and container–closure devices are often proprietary. 

The additives, potential leachables, are not always known.

The manufacture of plastic and rubber materials does not usually proceed according to current good manufacturing (cGMP) conditions and minor changes in process or technique can affect potential leachables significantly.

Impurities can often appear as drug substance degradants that form on stability, which can lead the investigation astray.

Some leachables can migrate through several layers of seemingly impermeable packaging, which can prove surprising, warranting the consideration of all components of the packaging or container–closure devices as potential sources of leachables. 

Several researchers have published studies on impurities related specifically to the packaging of drug products.

After an initial assessment evaluating both ultraviolet and mass spectral data, if an impurity is categorized as unrelated to the drug substance or excipients, you should evaluate the packaging and container–closure devices. Review the literature as we have indicated in the references, or product information sheets (if available) from the manufacturer of the plastic or rubber materials used in the packaging or container–closure devices. Analysing the components of the packaging or container–closure devices is also a viable option that can narrow the investigation, provide a source with a relatively greater amount of the impurity compared to the drug product, or provide a source not hindered by drug product excipients that can interfere with mass spectrometry (MS) analysis.  

Here, we present several case studies illustrating how we identified leachables in drug-product formulations. 

A typical scenario for these situations occurs during stability testing of a drug product when an unknown peak crosses the 0.1% threshold (or other defined threshold) at which impurities must be identified. 

Figure 1 shows a high performance liquid chromatography (HPLC)-UV chromatogram of an active pharmaceutical ingredient (API) stability assay for an injectable drug. In the chromatogram, an unknown peak is eluted at about 38 min, a peak we had not observed during the drug product analyses performed before the drug was stored in its final packaging (amber glass vial with gray, rubber stopper). In this relatively simple packaging scenario, the likely origin of any impurity that is container closure-related is the elastomeric stopper. But an assay solely by HPLC-UV makes it almost impossible to identify the impurity. 

Even with diode-array spectra of the unknown versus the API (which requires minimal effort other than detector substitution), it is nonetheless difficult to discern the identification (Figure 2). Hence, an ideal scenario for a mass spectrometry solution to the problem.  

The particular method involves no involatile buffers (for example, phosphate buffers) and, thus, requires minimal modifying to convert to an LC–MS method. When analysed using negative-ion atmospheric pressure chemical ionization (APCI), a [M–H]

 339, suggesting a molecular weight of 340 Da. LC–MS–MS analysis showed a primary fragment appearing at 

 163, an impurity that corresponds to a well-known antioxidant 2,2'-methylenebis (4-methyl-6-

This month's instalment of "MS in Practice" provides a slightly different view of how practitioners employ the skills of interpretation that have been the focus in recent columns.

LCGC Europe-12-01-2007

The Perfect Method, Part 4: Controlling Peak Spacing

New Technologies

Norse Legends

In Vino Veritas: LC–MS in Wine Analysis

Ex-tech-10 and HTC-10

Identifying Packaging-Related Drug Product Impurities

A Split Decision