Food safety is an important issue in the EU and a legal framework covering the entire food chain has been established.

				 The central goal of this framework is to guarantee a high level of protection of human health in relation to food. Historically, the analysis of residues in food products is a relatively young discipline. In the Benelux (Belgium, the Netherlands and Luxembourg), residue analysis started in the late 1960s. In most European countries, research on residues and its application in regulatory control of slaughter animals and crop production started even later.  

A residue may be defined as a trace of a substance (µg/kg, ng/kg) present in a matrix (e.g., meat, urine, etc.). For any type of animal or food matrix, there are two main groups of substances that must be monitored according to council directive 96/23/EC:

 group A and B substances. Group A comprises the prohibited growth-promoting agents (e.g., hormones, beta-agonists) and the prohibited substances for which no maximum residue limits (MRLs) could be established (Table 1). Group B comprises all registered veterinary drugs, having an MRL, and other residues as summarized in Table 1. 

Table 1: Group A and Group B substances.			

Control of group A has higher priority than group B because of public-health concern and, therefore, more stringent criteria are implemented. Technical guidelines and performance criteria (e.g., selectivity, specificity) for residue control are described in Commission Decision 2002/657/EC.

 For banned (A) substances the emphasis is on the identification of the substances in a large number of matrices in a concentration as low as possible (zero tolerance principle). In this case, firstly, qualitative methods, targeting multiple residues, need to be developed and, secondly, quantitative methods. For B substances, methods for the quantitative determination of substances in edible matrices need to be developed. 

The development of analytical procedures for sample preparation, detection and separation for A and B substances has evolved substantially and these trends will be discussed in this article. 

In the early 70s thin layer chromatography (TLC) was the method of choice for the qualitative detection of banned substances (thyreostats and certain anabolics at that time). The reasons for this were its specificity, the simplicity of development in two dimensions and the possibility of reaching low limits of detection for an acceptable budget (often using fluorescence detection). The only alternative with acceptable limits of detection — at that time — was gas chromatography with electron capture detection (GC–ECD). High performance liquid chromatography (HPLC) with ultraviolet (UV) detection was introduced in the mid-70s, but the first instruments were expensive and not robust. Also, UV detection did not match the specificity and limits of detection needed for A substances. Fluorescence detectors were only introduced later. However, for the quantitative determination of B substances, UV detection and post-column derivatization was often used. During the 90s, more and more affordable gas chromatography–mass spectrometry (GC–MS) apparatus appeared on the market and the transition from TLC (and HPLC) to GC–MS methods was ongoing. At the end of the 90s, liquid chromatography–mass spectrometry (LC–MS) and LC–MS

 belonged more and more to the mandatory standard equipment of a residue laboratory (Figure 1). 

Figure 1: Evolution of methods used in residue analysis (adapted from De Brabander et al.6)			

 it may be concluded that LC–triple quadrupole tandem MS (QqQ-MS–MS) is currently the preferred method for residue analysis. The majority of current LC–MS-based hormone and veterinary drug residue analyses relies on the high sensitivity and selectivity of the selected reaction-monitoring (SRM) mode of QqQ-MS–MS. The two-stage mass selection provides the selectivity and sensitivity to enable the detection, identification and quantification of preselected targets at the low µg/kg level in complex biological matrices such as urine, faeces, tissue, feed and hair.

 As the cost-effectiveness of analytical procedures is becoming an important parameter for laboratories involved in residue analysis, automation has been introduced to speed up many analytical procedures. Instrumentation to provide for automation is, however, expensive and a distinct number of samples would be required to compensate for the significant capital expenses.  

An alternative way to improve cost-effectiveness is to maximize the number of analytes that may be determined by a single procedure. The recent introduction of ultra-high performance liquid chromatography (UHPLC) and fast-switching QqQ-MS–MS instruments significantly increased the number of analytes that can be detected in one run. Indeed, modern instruments produce high signal-to-noise (S/N) ratios, even when relying on short SRM dwell times. This permits monitoring of an increasing number of transitions. For a number of growth promoters (anabolic steroids), the SRM approach is depicted in Figure 2.  

Figure 2: UHPLCâQqQ-MSâMS chromatogram obtained from a standard solution of ten anabolic steroids; 0.5 ng injected amount of each analyte. SRM transitions monitored for each component are presented to the right.			

Increasing the number of analytes to be monitored further, requires monitoring at specific retention-time windows, also called "timed SRM" or "scheduled SRM". In each retention-time window, the number of transitions to be acquired can be kept relatively low (less than 10). Using this approach, selective and sensitive analysis of a larger number of compounds (maximum 100–120) may be achieved within one single LC–QqQ-MS method. This approach, however, requires frequent readjustment as a result of small shifts in retention time. Another major inherent limitation of targeted LC–MS–MS approaches is the inability to detect residues such as novel illegally administered anabolic steroids designed to escape from control agencies. 

Therefore, an attractive alternative is the use of full-scan MS approaches [e.g., time-of-flight (TOF), Fourier transform ion cyclotron resonance (FT-ICR) or Fourier transform Orbitrap]. Full-scan MS approaches offer the possibility to simultaneously analyse a virtually unlimited number of compounds. Furthermore, the retrospective "post-targeted" evaluation of old data offers the possibility to detect non

selected analytes (i.e., no analyte-specific transitions have to be defined before injecting the sample). Moreover, their accurate-mass capabilities support the reconstruction of highly selective, accurate-mass chromatograms of target residues in complex matrices. To allow the detection of residues at the low ppb (mg/kg) or ppt (ng/kg) concentration ranges demanded by legislation, very sensitive full-scan analysers are required. 

The medium resolution of TOF systems significantly effects the selectivity and, therefore, the sensitivity gain, compared with unit-resolution scanning MS.

 The utility of high performance TOF mass spectrometric applications has been demonstrated for the multi-compound screening of veterinary drugs in different matrices from animal origin,

 but also for doping agents in human urine

 It should, however, be noticed that accurate-mass determination without proper mass-resolution criteria might lead to false compliant (false negative) results, both in MS screening and MS–MS confirmation. 

A lack in mass-resolving power was demonstrated for the anabolic steroid stanozolol analysed using a LC–QTOF-MS.

 Indeed the currently available TOF instruments have mass accuracies of less than 5 ppm and resolutions of maximally 15000 FWHM (full width at half maximum).

				 This resolution may lead to inaccurate mass measurements in complex matrices because of unresolved background matrix interferences. Co-elution of isobaric compounds may cause significant deviations in exact mass measurements. This mass resolution is of crucial importance to successfully identify residues or contaminants in samples containing high amounts of complex matrix co-extracts.

 In this context, the high resolving power (up to 100000 FWHM) of the Fourier transform Orbitrap mass spectrometric technology provides precise mass accuracy (below 2 ppm), resulting in both high selectivity and sensitivity for complex sample analysis. This is demonstrated in Figure 3 for the anabolic steroid trenbolone. A minimum resolution of 30000 was required to separate the trenbolone ion from the interfering matrix ion; while complete separation could only be obtained at a resolution of 100000.  

Figure 3: Full-scan Orbitrap mass spectra acquired at different resolving powers. Separation of the [M + H]+ ion of trenbolone (m/z  = 271.16940) from the ion of a  matrix compound (m/z  = 271.18811). No separation between analyte ions and matrix  compound ions takes place at a resolving power of 10 000.			

Van der Heeft et al. observed a similar trend for the determination of steroid esters in hair.

				 In their study, the mass-resolving power of 10000 provided by a UHPLC–TOF-MS was too low to resolve analyte ions and co-eluting isobaric compounds from the sample matrix, generating mass measurement results frequently differing more than 5 ppm from the expected values.

In contrast, the use of a high resolving power (60000) in the UHPLC–Orbitrap MS allows the removal of matrix interferences from isobaric matrix constituents and produces less than 3 ppm mass errors for all steroid esters at all concentration levels.

 Using high-resolving power mass spectrometers, such as the Orbitrap MS, may further improve the confidence in screening results obtained by full-scan accurate mass LC–MS. Figure 4 depicts the reconstructed ion chromatograms of a selection of hormones with very narrow mass tolerance window (< 5 ppm) at 100000 resolving power. 

Figure 4: Full-scan Orbitrap reconstructed ion chromatograms of seven selected hormones at resolving power 100 000; 0.5 ng injected amount of each analyte.			

Next to improving the detection capability of the instrumentation, the clean-up of the samples before instrumental analysis has also evolved through time. While in the 70s only solvent extraction [liquid–liquid extraction (LLE)] and homemade columns (e.g., silica gel, Al

O3) were used for clean-up, solid-phase extraction (SPE) in the 1980s, immunoaffinity chromatography (IAC) in the 90s and molecular imprinted polymers (MIPs) at the end of the 90s, took over the job.

Today, more than 80% of all published methods to determine veterinary drug residues apply LLE and/or SPE. Specific combinations of LLE and SPE may be very selective for a certain group of veterinary residues. Selective sample pre-treatment and analysis are, however, very time-consuming and demand a great deal of effort. To maintain sample throughput and cost-effectiveness, it will be necessary in the future to develop generic liquid chromatography–mass spectrometry (LC–MS) screening methods for the simultaneous detection and identification of a wide range of banned and novel hormones, growth promoters, registered veterinary drugs and their metabolites. Due to the broad variability in physicochemical properties of the compounds of interest, the application of a simple clean-up or extraction procedure will be critical to maintain the recovery of the broad range of analytes.  

The emerging trend towards accurate-mass and full-scan alternatives, allowing the reconstruction of highly selective accurate-mass chromatograms of target residues in complex matrices, supports this generic sample preparation approach because increased resolving powers allow the discrimination between analytes and matrix interferences. Kaufman et al. described a procedure for the analysis of 100 veterinary drugs in urine and their sample-preparation existed of diluting the urine prior to UHPLC–TOF-MS.

				 This approach is, however, not applicable when very low levels of residues need to be determined or when semi-solid or solid samples need to be analysed. Mol et al. developed and validated a generic procedure for the simultaneous extraction of 136 pesticides, 36 natural toxins and 86 veterinary drugs in feed and honey.

 The extraction procedure basically involved an extraction using acidified organic solvent. It may be expected that future initiatives in this line and their application in routine monitoring programmes will drastically reduce both effort and time in residue analysis.  

In the multi-residue approach using full-scan MS techniques, the LC part of the system may, however, cause certain limitations. As a result of the large number of different analytes to be separated, run times may become relatively long. This can be overcome by using shorter columns or faster gradients, which on its turn may result in a lower chromatographic resolution. The introduction of pressure-stable 1.7 µm particulate packing materials and novel low-dead-volume, ultra high-pressure (until 10

 kPa) LC equipment (UHPLC) provides strategies to improve resolution while maintaining or even shortening run times. 

The higher resolution provided by UHPLC may compensate for the selectivity of currently available full-scan accurate mass instrumentation, which is still less than that provided by monitoring MS→MS transitions. One possible disadvantage of using UHPLC is, however, the limited number of spectra, which may be acquired over the peak at a high resolving power (100000) required for discrimination between isobaric interferences and ions of interest. UHPLC peak widths are often only a few seconds long and the scan cycle time at high resolving powers counts more than 1 s per scan.

 Therefore, for most applications resolving powers need to be traded-off against sensitivity.  

Future trends in residue analysis will encompass an expansion of the application of UHPLC coupled with accurate mass, high-resolution mass spectrometry for the screening of target residues and for accurate mass confirmation of known and identification of unknown residues and metabolites. However, criteria are absent for these techniques.  

  MS methods may be used only as confirmatory methods after chromatographic separation (off- or on-line). For LC–MS the 2002/657/EC decision states that suitable LC columns should be used. However, there is no appropriate definition of what "suitable" is. Nowadays, UHPLC columns may offer a comparable separation power to that of capillary columns. Therefore, the time has come to consider the mandatory use of these columns for confirmation. Also, the tolerance of the correspondence of the relative retention time of the analyte to that of the calibration solution [now ±2.5% for (classical) HPLC] should be revised based on existing knowledge on the reproducibility of the retention times in UHPLC.  

The 2002/657/EC defines high resolution mass spectrometry (HRMS) as MS at a mass resolution of 10000 according to the 10% valley definition.

  This resolution of 10000 according to the 10% valley rule corresponds to a resolution of 20000 FWHM for modern instruments (e.g. TOF-MS, FT-ICR–MS and FT Orbitrap-MS). The 2002/657/EC has also introduced identification points (IPs) and laid down permitted tolerances for the relative intensities of the detected ions. For the confirmation of Group A and Group B substances a minimum of 4 and 3 IPs are required respectively. The criteria for HRMS are as follows: 2.0 IPs are earned for each precursor ion and 2.5 IPs for each product ion. No criteria for mass accuracy are described. Several studies have, however, demonstrated that false compliant (false negative) results can be obtained when the mass resolving power of the MS is insufficient to separate analyte ions from isobaric co-eluting sample matrix ions.

  Based on these data, Nielen et al. proposed additional LC–MS criteria to be implemented in 2002/657/EC (Table 2).

Table 2: Proposal for additional LCâMS criteria to be implemented in 2002/657/EC3. Adapted from Nielen et al (2007).11			

This proposal acknowledges the performance of state-of-the-art LC–TOF-MS instruments by ascribing 1.5 IPs per ion versus 1.0 in conventional low-resolution LC–MS,

  but does not allow the use of only one parent and product ion, which should remain the exclusive domain of HRMS.

  For the identification of metabolites or unknown compounds a higher resolution is warranted when NMR cannot be obtained. According to Nielen et al.

  the proposed resolution of ≥70000 (FWHM), will assure that reliable elemental compositions of product ions differing in one CO, C2H4 or N2 substructure can be obtained until 

 = 400. In some cases a resolution of 100000 may even be necessary to achieve discrimination of product ions from matrix interferences, in particular when using more generic sample preparation procedures and multi-residue procedures.  

There has been an increased interest in methods that simultaneously analyse various classes of veterinary drugs, hormones etc. In one run, such multi-residue analytical procedures may screen for more than 100 compounds, but full-scan accurate mass instruments (e.g., TOF-MS, FT Orbitrap-MS) are required to do this. LC–QqQ-MS (using selected SRM transitions) is still the most frequently applied technique in residue analysis. But today, in theory, all components can be measured by full-scan MS, not requiring previous acquisition of ion transitions and allowing post-acquisition interpretation of data. In the future, the most important part of the method development will, therefore, shift from detection optimization to the development of generic extraction of the compounds of interest from the matrix. 

This will undoubtedly lead to increased innovations in sample preparation techniques. Finally, the introduction of new MS techniques also has consequences for the EU criteria defined for confirmation of compound identity. Identity confirmation is influenced by both mass accuracy and mass resolution. Therefore, a discussion should be initiated, ultimately leading to appropriate criteria for accurate mass techniques and revision of Commission Decision 2002/657/EC.  

 has a PhD in bioscience engineering from the Ghent University and is currently appointed as doctor-assistant at the Department of Veterinary Public health and Food Safety. The chemical analyses of food and the analysis of residues and contaminants in biological matrices relating to human health are her core research objectives. 

 is a last-year PhD student whose research focuses on the analysis and metabolism of phytosterols to elucidate the occurrence of boldenone and related steroids in cattle. 

 is a PhD student whose research deals with the optimization of new analytical approaches for the discrimination of exogenously administered and endogenously formed thyreostats in livestock. 

 is European product specialist at Thermo Fisher Scientific. 

 is professor at the Ghent University and dean of the Faculty of Veterinary Medicine. He has over 35 years experience in residue analysis in animal and food matrices. 

1. White Paper on Food Safety, Commission of the European Communities, Brussels,  

http://ec.europa.eu/dgs/health_consumer/library/pub/pub06_en.pdf

5. A.A.M. Stolker, T. Zuidema and M.W.F. Nielen, 

Articles

Coupling Matters

The trend in residue analysis has changed from target-oriented procedures towards accurate mass full-scan MS techniques. This article describes these developments and addresses the implications of 2002/657/EC.

UHPLC Coupled with Fourier Transform Orbitrap for Residue Analysis

Penicillin G (PG) is widely used for therapeutic or prophylactic purposes to treat cattle diseases. PG might be applied in the form of procaine salt to achieve longer lasting activity. PG procaine (PGp) is a combination of PG with the local anaesthetic agent, procaine, and is slowly absorbed into the circulation and hydrolysed to PG. It is used where prolonged low concentrations of PG are required. 

The Codex (Codex Alimentarium Commission) has set a maximum residue limit (MRL) for PG (including PGp) in beef (cattle muscle) at 0.05 μg/g to ensure that beef products for human consumption are virtually residue-free of these substances.

 The acceptable (or ideal) method for the routine monitoring of veterinary drugs in foods of animal origin must be simple, quick, economical in terms of time and cost, and cause negligible harm to the environment and analyst. 

Eliminating the use of toxic organic solvents and reagents is an important goal in terms of environmental conservation, human health and the economy. Although high performance liquid chromatography (HPLC) techniques with ultraviolet (UV) detection to simultaneously determine PG and PGp in plasma

 have been reported, there is no method that does not use toxic solvents/reagents. This article describes a rapid and inexpensive technique that does not use hazardous chemicals to monitor PG and PGp residues in beef. 

 Penicillin G potassium (PG) and penicillin G procaine (PGp) standards were purchased from Wako Pure Chem. (Osaka, Japan) and Nippon Zenyaku Kyogyo (Koriyama, Japan), respectively.  Ethanol (purity 99.5%) and distilled water were of HPLC grade (Wako). Stock standard solutions of PG and PGp were prepared by dissolving each of the compounds in water to a concentration of 100 μg/mL. Working mixed standard solutions of these compounds were prepared by diluting the stock solutions with water. A handheld ultrasonic-homogenizer (model HOM-100, 2 mm i.d. probe, Iwaki Glass, Funabashi, Japan), a micro-centrifuge (Biofuge fresco, Kendo Lab. Products, Hanau, Germany), and an Ultrafree-MC/PL (Millipore, Bedford, Massachusetts, USA) as a centrifugal ultra-filtration unit were used in the sample preparation. 

The HPLC system included a model PU-980 pump and DG-980-50 degasser (both from Jasco Corp., Tokyo, Japan), as well as a model SPD-M10A VP photo-diode array (PDA) detector (Shimadzu Scientific Instruments, Kyoto, Japan). The analytical column was an Inertsil WC300C4 (100 × 4.6 mm, 5 μm) column (GL Science, Tokyo, Japan). The isocratic mobile phase was 0.04 mol/L phosphoric acid (pH 7.0)–ethanol (8:2, v/v) and the flow-rate was 1.0 mL/min. PDA detector was scanned from 190–350 nm: detecting at 205 nm for PG and 290 nm for PGp, respectively (a maximum UV absorption for each compound). The column temperature was operated at 40 °C The injection volume was 20 μL. 

 An accurately weighed 0.1 g homogenized beef sample was placed in a micro-centrifuge tube and homogenized with the ultrasonic homogenizer for 30 s with 0.6 mL of ethanol. After being homogenized, the capped tube was centrifuged at 12000 g for 5 min. A 50 μL portion of supernatant liquid was placed into an Ultrafree-MC/PL and centrifuged at 5000 g for 5 min. The ultra-filtrate was injected into an HPLC system. 

 The recoveries of PG and PGp from blank beef samples spiked at 0.05, 0.1 and 0.2 μg/g, respectively, were determined. These fortification levels were prepared by adding 10 μL of three working mixed standard solutions, 10, 20 and 40 μg/mL, respectively, to separate 2 g portions of the samples. Fortified samples were allowed to stand at 4 °C for 24 h after the mixed standard addition and then mixed prior to the test. 

Figure 1 displays typical chromatograms for beef samples obtained under the procedure developed here. This figure demonstrates that the present method can provide quantifications and identifications of PG and PGp. 

Figure 1: Chromatograms obtained from the HPLC system. PDA detector set at (a) 205 nm or (b) 290 nm. Upper profile: a beef (cattle muscle) sample spiked with the target compounds (0.2 Î¼g/g). Lower profile: (a) a blank beef sample. Peaks: PG (Retention time) = 2.4 min; 2 = PGp (17.3 min).An arrow in (a) lower profile indicates the peak of PG.			

Table 1 summarizes the main 'Method Validation' parameters. The author generated the spiked recovery graph as a practical calibration curve plotting peak areas of fortified sample extracts ranging from 0.05–1.0 μg/g versus their concentration. The decision limit (CCα, μg/g) and detection capability (CCβ, μg/g) values calculated according to the EU regulation

 are also described in this table. Additionally, the selectivity and cost/time performances are as follows: the target spectra obtained from the beef sample were practically identical to those of the standards. Because of the high absorbance of PG and PGp and the satisfactorily purification, UV detection is possible at trace levels using PDA. 

It is, therefore, instructive to demonstrate the purification effectiveness of the sample preparation. The present HPLC system made it unnecessary to use MS, which is very expensive and is not available in a lot of labs for routine analysis, particularly in developing countries, to analyse the target compounds. The time and budget required for the analysis of one sample were less than 30 min and roughly €3.9 (¥500 in 16 March 2009), respectively. For the sequential sample preparations, a batch of 12 samples can be analysed in less than 4.5 h. 

 Using the complete method, the author analysed the different samples of marketed beef in Osaka, Japan. No beef samples contained detectable concentrations of PG (< 0.022 μg/g) and PGp (< 0.020 μg/g), respectively. The chromatograms were free from interference. 

The proposed method is a useful tool for routine residue monitoring of PG and PGp in beef which is inexpensive, harmless to the environment, quick and delivers reproducible recoveries. 

 is an associate professor with the Graduate School of Human Life Science, Osaka City University, Osaka 558-8585, Japan. 

http://www.codexalimentarius.net/mrls/vetdrugs/jsp/vetd_q-e.jsp

Official Journal of European Communication

Regular article

A method of sample preparation followed by reversed-phase high performance liquid chromatography (RP HPLC) with diode array (DA) detection.

A Non-hazardous Technique to Determine Penicillin G and Penicillin G Procaine in Beef

21st International Ion Chromatography Symposium

This year CASSS, the International Separation Science Society, will be holding the 

 in the heart of Malahide, Dublin, Ireland from 

International Ion Chromatography Symposium

 (IISC) is a yearly meeting of the leaders in this specialized applied technology. Experts gather to report on advances in ion chromatography and innovative applications, as well as future directions of ion chromatography (IC) and capillary electrophoresis (CE). 

Oral presentations will be arranged in two parallel sessions consisting of plenary sessions and keynote lectures from speakers invited from academia, industry and government bodies. Topics covered will include 

(food and beverages, pharmaceuticals, fine chemicals, and nuclear and power generation);

 Capillary, Miniaturized and Chip-based Systems 

; Advances in Detection; Trace analysis and Preconcentration- based methods, Hyphenated techniques and speciation, New column technologies 

, Forensic analysis; New Column Technologies: Environmental Analysis 

As well as the oral and poster sessions, there will be a series of short courses and workshops delivered by experts to encourage the involvement of scientists new to the field of ion analysis. Topics will include an introduction to modern IC, which will cover the fundamentals of instrumentation and column selection, and also relevant applications that demonstrate the benefits of this technique in practice. Recent advances in this technology will also be discussed, focusing specifically on smaller, faster and smarter separations. 

There will also be a short course on detection techniques, reviewing the basic theory of chromatographic detection systems, covering the principles of conductivity detection in IC in detail, as well as providing an overview of advanced detection techniques: postcolumn reaction (PCR), electrochemical (e.g., integrated and 3D pulsed amperometry), and mass spectrometry (MS). 

Additionally, a short course on statistics for analytical chemists will provide an introduction to statistically sound techniques for calibrating when using IC, and calculating measurement uncertainty, detection limits and quantification limits. Spiking studies and recovery calculations will also be included. 

Valve Switching for Automated Sample Preparation and 2D chromatography, Method Development, including eluent generation, column selection and suppression 

 Sample Preparation and Sample Handling for Trace Analysis.

The symposium will also include a three-day exhibition displaying the latest instrumentation and equipment and there will also be a number of social activities tailored to encourage maximum interaction and discussion amongst delegates. 

The town of Malahide, where the symposium is being hosted this year, is a beautiful, bustling, coastal town within easy reach of the airport and the city centre. The Grand Hotel Malahide, where the event will be taking place, is within walking distance of several of the very best pubs, restaurants, golf courses and tourist attractions in County Dublin. 

The 9th International Conference Vitamins, Nutrition and Diagnostics

Mendel University of Agriculture and Forestry, Brno

Radanal Ltd, Society for Nutrition, Prague and Tomas Bata University in Zlín 

Radanal Ltd Okružní 613, 530 03 Pardubice, Czech Republic

Ina Kaehler, Rennweg 3, 6020 Innsbruck, Austria

3 Melbourne Avenue, Sheffield, UK, S10 2QJ

GlaxoSmithKline Medicines Research Centre, Stevenage, Hertfordshire, UK

Departments

This year CASSS, the International Separation Science Society, will be holding the 21st International Ion Chromatography Symposium in the heart of Malahide, Dublin, Ireland from 21–24 September 2009.

Event News

This is the fourth "LC Troubleshooting" column in a series looking at different aspects of the calibration process for liquid chromatography (LC) methods. We first considered whether or not to force a calibration curve through the origin (

 The next stop was a discussion of some techniques to determine the limits of detection and quantification.

 we saw how %-error plots could help us visualize possible problems with calibration curves. The present discussion focuses on three different calibration models: external standardization, internal standardization and the method of standard additions. Next month, we will look at the technique of curve weighting. 

The use of external standards is the simplest and probably the most common method of calibration for quantitative LC methods. The technique simply compares the detector response between known concentrations of analyte with the response for samples containing unknown concentrations. A calibration curve (also called a standard curve or sometimes a "line") is generated by injecting a series of calibration standards. For well-behaved methods, as demonstrated by validation studies and narrow ranges, for example, ±10% in concentration, a single-point calibration can be used. In this technique, the response (area) for a known concentration of reference standard is calculated (area/concentration) to generate a calibration factor. This value is divided into the area for an unknown concentration and the result is the concentration of the unknown. 

More commonly, calibrators are prepared that cover the expected sample concentration range and the response of these calibration standards is used to generate a calibration curve. This is demonstrated with the data of Table 1. The data of Table 1 simulate the use of a method for which all the calibrators are injected both at the beginning (data set 1) and end (data set 2) of a batch of samples. Calibrators were prepared at 1, 2, 5, 10, 20, 50, 100, 200, 500 and 1000 ng/mL and injected with the batch of samples. The results are combined in Table 1. Using the data system software or spreadsheet software, such as Microsoft Excel, a calibration curve can be plotted, as is shown in Figure 1. Here, concentration (

-intercept, so the curve can be forced through zero (

). (See the discussion of reference 1 for more information on zero-intercept decisions.) This regression equation is then rearranged (

/403.7) to calculate the concentration of unknown samples. For example, a sample that generates a peak of 36827 area counts (last line, Table 1) would have a concentration of (36827/403.7) = 91.2 ng/mL. A common sense double-check of the calculation shows that in Table 1, 36827 area counts would fall between 50 and 100 ng/mL, so the result seems reasonable. 

Figure 1: External standard calibration plot from the data in Table 1.			

External standardization works well when sample preparation steps are simple and the injection volume precision is good. For example, if the sample comprises a pharmaceutical tablet that is weighed, dissolved in an aliquot of injection solvent, filtered and injected with a modern autosampler, external standardization is appropriate. When many sample preparation steps are included in the method or there is a question about autosampler precision, an internal standard can improve the precision and accuracy of a method.  

With internal standardization, a second compound, often related to the analyte but never found in the sample, is added at a known concentration to every sample and calibrator. For example, one might pipette 20 μL of a 10 μg/mL solution of internal standard (IS) into 1 mL aliquots of sample and calibrator at the beginning of the sample preparation process. This would mean that each sample would now contain 200 ng/mL of internal standard. If the proper internal standard is chosen, it should track the sample through the sample preparation process, correcting for sample losses because of incomplete extraction, sample loss, or minor differences in reconstitution volume. It is the ratio of the analyte to internal standard that is the critical measurement in an internally standardized method. 

The calibration curve data are generated by injecting calibration samples of different concentration that all contain the same concentration of internal standard. The ratio of analyte area to internal standard area is calculated and plotted as the 

-value against the concentration of the calibrator. Table 1 shows the internal standard areas and analyte/IS ratios for the same data used for the external standard experiments. The calibration curve is plotted in Figure 2. The 

 (0.00011), so the curve is forced through zero. The same unknown used previously (last line of Table 1) generates an analyte/IS ratio of 0.3645. The regression equation is rearranged to 

/0.004037), which allows calculation of the unknown concentration of (0.363551/0.004037) = 90.1 ng/mL. This is approximately the same value as that achieved by external standardization. 

Figure 2: Internal standard calibration plot from data in Table 1.			

Now that we've see the two most common standardization methods for LC, which one should be used? In most cases, precedent will have been set already with similar methods, so the choice should be clear. In other instances, it might not be obvious which calibration type to choose. The simplest way to make the decision is to check the results empirically. Prepare calibrator samples that contain internal standards, as described previously and analyse them. Process the data using both the external standard and internal standard technique. Next, "back-calculate" the calibration samples against the calibration curves and determine the %-error by which each point deviates from the regression line. 

 Sometimes this is called calculating the residuals. For the data sets discussed previously, the errors for each curve type are shown in columns 7 and 8 of Table 1, with the ratio of the errors in the right-hand column. The results can be compared in several ways. You can compare the results visually at each concentration or the ratio of the errors. Alternatively, you can calculate the average of the absolute values (2.74% for external standard and 1.22% for IS) or the sum of the absolute values (54.8% and 24.5%). In each case, you can see that the internal standard method reduces the error by about two-fold. This supports the use of internal standardization for this method. This suggests that there is some kind of physical sample loss or inconsistent volumetric recovery in the sample preparation process that is compensated by the use of an internal standard. If the results were comparable or the external standard method gave smaller errors than the internal standard method, the simpler external standardization technique should be used. 

When either the external or internal standard calibration method is used, it is usually best to prepare matrix-based standards. This means that the calibration standard is prepared in a solution that represents the sample extract in all ways except the presence of the analyte. For pharmaceutical samples, the blank matrix might be a placebo of the formulation. For bioanalytical samples (for example, drugs in plasma), a drug-free plasma sample might be used. For a pesticide measurement in soil or water, pesticide-free soil or water might be used. By using a matrix-based standard, the likelihood of signal suppression or enhancement by the matrix is reduced. A blank matrix sample is usually run to confirm that there are no interfering peaks present in the matrix. 

Sometimes, however, it is impossible to obtain an analyte-free matrix. For example, in the measurement of an endogenous component of blood, such as insulin, it might be impossible to obtain blood without the analyte. Or for the analysis of a waste stream, it might be impossible to formulate a blank sample — that is, the waste stream containing everything except the analyte. When a blank sample cannot be obtained, the method of standard additions can be used to determine the concentration of the analyte in an unknown sample. 

A series of calibration standards is prepared at several concentrations. The standards are then added to aliquots of the sample. For the example of Table 1, calibrators were prepared and spiked into sample aliquots to result in samples that contain 0, 1, 2, 5 and 10 ng/mL of added calibrator. Next, the samples are analysed and the results plotted, as in Figure 3. Note that the plot has a significant 

-intercept (dashed line in Figure 3); this represents the response for the analyte content of the unspiked sample. To determine this concentration, the regression line is extended to the left until it intercepts with the 

-axis (arrow in Figure 3). The x-intercept represents the negative value of the concentration in the unknown sample. This is calculated by taking the regression equation (

 = 1055/402.4 = –2.6. The negative of this value (2.6 ng/mL) is the concentration of analyte in the unknown sample. 

Figure 3: Standard additions calibration plot from data in Table 1.			

We have considered three types of calibration plots this month. You will most commonly encounter the external or internal standard techniques. The choice of the technique can be based upon similar methods in your laboratory, customary usage in your industry, or by empirical testing. Usually, internal standardization, because of its additional complexity, is reserved for methods that require extensive sample preparation, which can result in physical loss of sample in the process. The method of standard additions is usually reserved for cases in which a blank matrix is not available and, therefore, may be rarely, if ever, encountered in many laboratories. 

The number of calibration standards used in a method will also vary depending upon the application and the custom for the laboratory or industry. If the calibration plot is linear and passes through the origin, a single-point calibration curve comprising one standard concentration can be justified. However, this technique is often reserved for methods that cover a narrow range in concentrations, such as ±10%. More commonly, calibration standards are formulated at several concentrations that span the expected concentration range of the samples to be analysed. As a general rule, it is best to bracket the concentration range with calibration standards; extrapolation of data beyond the calibration range, while sometimes justified, adds the potential for error to the method. 

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

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

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

For an on-going discussion of LC Troubleshooting with John Dolan and other chromatographers, visit the Chromatography Forum discussion group at 

LC Troubleshooting

The best results depend on the correct choice of the calibration model.

Calibration Curves, Part 4: Choosing the Appropriate Model

 Professor Edward Bald, Dr Grażyna Chwatko, Dr Paweł Kubalczyk, Dr Rafalł Głowacki, PhD Student Kamila Borowczyk and six MSc students, Department of Environmental Chemistry, Faculty of Chemistry, University of Łódź, Łódź. 

 The aim of this team is to solve environmental problems — specifically in environmental health — by developing new analytical methods to determine exogenous and endogenous sulphur-containing compounds in biological fluids. 

The main areas of research are new thiol-specific derivatization reagents for biological sample analysis by liquid-phase separation techniques and new analytical methods to determine homocysteine and metabolically-related thiols in biological samples. The team also studies the mechanism of atherogenic action of homocysteine on blood vessels in humans and the homocysteinylation of proteins by homocysteine thiolactone. The role of hydrogen sulphide in mammals, the redox status of thiols in physiological liquids in humans and food, and the application of high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) for monitoring endo- and exogenous thiols in physiological liquids in humans, are also researched, as well as on-capillary pre-concentration in CE and clinical chemical analysis and toxicological analysis. The group is equipped with several fully computerized HPLC and CE instruments connected to ultraviolet/visible (UV/vis), fluorimetric, electrochemical and refractometric detectors, as well as fully automated biological sample preparation systems encompassing dialysis, trace enrichment and solid-phase extraction (SPE).  

The staff of the group teach environmental analytical chemistry, including separation techniques, to undergraduate and graduate students. Six Master's theses are submitted annually from this research group. 

 Professor Boguslaw Buszewski (Head of the Chair), Dr Andrzej Chmarzynski, Dr Renata Gadzala-Kopciuch, Dr Ewa Klodzinska, Dr Przemyslaw Kosobucki, Dr Tomasz Kowalkowski, Dr Magdalena Ligor, Dr Tomasz Ligor, Dr Myroslav Sprynskyy, Dr Sylwia Studzin'ska, Dr Michal Szumski, Dr Monika Michel and 8 PhD students; technical staff — Piotr Przyzycki and administrative staff — Agnieszka Reczuch and Joanna Kowalska, Faculty of Chemistry, Nicolaus Copernicus University and the Centre for Education and Research in Separation Methods and Bioanalytics 

 The activities of the group are focused on theoretical and practical research in the fields of physico-chemical separation, pollution analytics, chemical and biological monitoring, waste utilization, xenobiotics bioaccumulation, metabolomics, speciation, sample preparation and chemometrics. 

Thanks to full-scale research in the field of separation and related proficiencies (chromatography, electromigration, spectroscopy and hyphenated techniques) and analytical techniques, cooperation with scientific centres both within the country and abroad is continuously expanding. It allows a simple way to recognize common programmes on a regional, national or European Union basis (such as academic teachers and students exchanges within the TEMPUS, CEEPUS, SOCRATES-ERASMUS, V and VI Framework Programmes).  

Research is focused not only on pure chemistry issues, but also on environmental, physical and analytical chemistry within the Chair. Therefore, the group is involved in experiments and studies that include biology and microbiology (cell, biological membrane, bacteria), medicine (medical diagnostics, cancerous diseases), pharmacy (metabolomics and drug quality control) and nanotechnology (porous polymers, new materials for separation techniques, micro- and nanocolumns). Lately the group has undertaken research concerning fast diagnostic tests for cancer detection. The involvement in this course allows them to prepare new methods for the realization of interdisciplinary researches. This is the result of material studies and devices that are necessary for collecting samples and coupling them with GC–MS, LC–MS, CZE–MS and hybrid systems. This includes successfully implemented new generations of stationary phases and columns. 

The next important aspect of their scientific activities is miniaturization. It offers methods and devices using miniature structures (chip-based methods) to sample separation, clinical applications and biochemistry. This research is realized by both the coupling of techniques of on-line systems and by procedures and method automations to eliminate errors during standard analysis. 

Biodegradation processes and neutralization of waste is among the other subjects of interest. The main aim is related to the negative influence of heavy metals and hence an understanding of their behaviour in the soil (speciation). 

Professor Boguslaw Buszewski is the chairman of the Separation Sciences Committee of Polish Academy of Sciences, Polish representative of separation science in Europe and has also been elected to the Steering Board of the European Society for Separation Sciences (EuSSS).

 Professor Teresa Kowalska, Dr Mieczysław Sajewicz, PhD students (Monika Gontarska, Dorota Kronenbach, Dorota Staszek and Łukasz Wojtal), and an MSc student (Ms Magda Wróbel), Department of General Chemistry and Chromatography, Institute of Chemistry, University of Silesia, Katowice. 

 The main interests of the research group involve developing non-trivial applications of chromatographic techniques to solve challenging physico-chemical and analytical tasks. These techniques include thin layer chromatography (TLC), gas chromatography (GC), high performance liquid chromatography (HPLC), flash chromatography (FC) and certain hyphenated chromatographic techniques, including GC–MS and LC–MS. 

As a result of the group's interests, spontaneous oscillatory reactions of chiral conversion have been discovered with a fairly wide variety of low-molecular-weight chiral carboxylic acids, many of them of vital importance to humans (for example as profen drugs, amino acids and hydroxy acids). This research has a number of exciting aspects; the organic oscillatory reactions discovered so far (which obey the rules of non-linear dynamics) are much less common than the purely inorganic, or mixed oscillatory reactions. So far, chiral conversion of profen drugs, amino acids and hydroxy acids has only been reported for the compounds dissolved in the biotic media, whereas the group claims to have found this for the abiotic systems as well. They have also managed to show the performance of TLC in physico-chemical research dedicated to chirality and also for tracing the dynamics of the oscillatory chiral conversion. 

Within the group's analytical interests, a wide range of chromatographic techniques are used to analyse plant material. Headspace gas chromatography with a mass spectrometry detector (HS-GC–MS) is used to separate and identify the volatile organic compounds (VOCs), whereas flash chromatography (FC), HPLC-DAD (diode array detection) and liquid chromatography mass spectrometry (LC–MS) are used to assess other constituents of the plant material. In this laboratory, there are two main goals to this kind of research. Firstly, there is a need for a new approach to plant systematics and it appears that chemosystematics can be a good alternative to biological systematics, with advanced chromatographic techniques as its most efficient working tools. Secondly, the kingdom of plants can serve as an ingenious blueprint for drug design, challenging other, less nature-inspired approaches to the same task. 

 Professor Roman Kaliszan, Department of Biopharmaceutics and Pharmacodynamics, Medical University of Gdańsk, Al. Gen. Hallera 107, 80-416, Gdańsk. 

 The team led by Prof. Roman Kaliszan combines chromatography with chemometrics in their research. The first two papers, which are thought of as initiating an original research field within the chemometrics field, namely QSRR (Quantitative Structure-Retention Relationships), appeared at the same time in 1977: one by R. Kaliszan in 

 and second by I. Michotte and D.L. Massart  of Vrije Universiteit Brussel in 

. Soon after that, QSRR became a rapidly developing research topic with numerous important publications from various laboratories in Poland and abroad. 

In original reports by the Gdańsk group, the QSRR were demonstrated to provide information on the chromatographed analytes that could be  useful for medicinal chemistry and molecular pharmacology, along with information on fundamental chemical behaviour and practical ways to optimize the conditions of chromatographic separations. 

There are a number of important implications of the QSRR — derived in the Department of Biopharmaceutics and Pharmacodynamics for separation science — such as improved methods and procedures to determine drug and drug candidate lipophilicity (hydrophobicity) by HPLC; the first HPLC method to assess the hydrophobicity of non-dissociated basic (as well as acidic and neutral) analytes by the application of a high pH-resistant polybutadiene-coated alumina stationary phase; and also the first applications of the so-called immobilized artificial membrane (IAM) stationary phase to get biorelevant measures of lipophilicity.  

An original theory and procedure of the pH-gradient HPLC was developed here, in a series of 10 papers, published mostly in 

. A comprehensive, thermodynamically consistent theory of pH-gradient and combined pH/organic modifier-gradient liquid chromatography has been developed, which allowed a fast and simultaneous determination of analyte p

 and lipophilicity. The procedure is applicable to analyte mixtures and requires only minute amounts of substances. It offers new, unique possibilities of efficient separation of ionizable compounds. It may also be a method of choice for high-throughput pre-selection of drug candidates of expected suitable pharmacokinetics from a multitude of compounds provided by current methods of combinational chemistry and automated synthesis. 

From QSRR there came the application of HPLC stationary phases with immobilized biomacromolecules to model interactions with drugs and using the obtained chromatographic data in correlation studies of the structure of drugs. They also elaborated on a new original analytical method for proteomics consisting of applying QSRR to predict the retention of peptides using three simple structural descriptors. 

 Professor Irena Baranowska, Department of Analytical and General Chemistry, Silesian University of Technology, M. Strzody Str. 7, Gliwice. 

 This analytical department is active across the broad spectrum of analytical chemistry, especially preliminary and applications research in bioanalytical chemistry. Their research concerns developing new extraction and determination methods for endogenic compounds (biogenic amines, methylxantines, aminoacids) as well as egzogenic compounds (drugs of different therapeutical groups and heavy metals) in physiological solutions. 

New high performance liquid chromatography (HPLC) methods for dopamine, serotonine and their metabolites, as well as caffeine, teophiline, teobromine and their metabolites in urine samples have been developed, in addition to HPLC and voltamperometric procedures to determine dozens of different drugs and their metabolites in physiological solutions and pharmaceuticals. These procedures and methods may be applied to monitor drug therapy, especially in cases that involve the simultaneous application of different drugs (polyteraphy). 

The group's interests also focus on the extraction methods of flavonoides, as well as methods to determine them in stimulants and physiological fluids using HPLC. The team also conduct research concerning the extraction and determination of pesticides in trophic chain samples. Special emphasis is placed on extraction procedures: liquid–liquid (microwaves and ultrasound assisted) and solid-phase extraction (columns and speedisks). 

The photo features people who work with chromatography. However, other forms of research are also conducted in the department, including comestible immunoenzymatic methods for atrazine (herbicide) determination, and preliminary research concerning electrochemical reactions of different drugs, disinfectant agents and biogenic amines depending on the type of electrode used. The presence of heavy metals by flame atomic absorption spectrometry (FAAS) and graphite furnace atomic absorption spectroscopy (GFAAS), and voltamperometric methods in environmental, comestible and biological samples are also investigated. 

 Professors: Jacek Namieśnik (head of department), Marek Biziuk, Marian Kamiński, Józef Pacyna, Waldemar Wardencki, Bogdan Zygmunt; associate professors: Piotr Konieczka, Żaneta Polkowska, Lidia Wolska; assistant professors: Agata Kot-Wasik, Andrzej Wasik, Bożena Zabiegała; 29 postgraduate (PhD) students. Department of Analytical Chemistry, Gdansk University, Gdansk. 

 The department of analytical chemistry has a broad programme of research in analytical chemistry, with special emphasis on the application of different chromatographic techniques to investigate the state of the environment and the processes taking place in it.  

Current projects include the development of new methods to determine trace components in samples of complex matrices, the design, construction and testing of the analytical characteristics of prototypical monitoring/measurement devices and an assessment of the environmental fate of xenobiotics and of bioaccumulation processes in tissues and organs of living organisms. In addition the group works on the application of state-of-the-art analytical techniques for the quality control of petroleum, industrial and food products, the development of new techniques for producing materials with special properties and/or chromatographically tested high-purity-grade materials, as well as being involved in the organization of inter-laboratory studies and proficiency testing.  

They also work on the construction and testing of standard gas mixture generators and construction of special modules for coupling to commercial monitoring/measurement instruments such as gas/liquid chromatographs, environmental pollutant monitoring devices designed for isolation and/or pre-concentration of analytes, deoxygenation, desiccation and purification of gas streams and the liberation of analytes from traps (thermal desorbers, cryogenic traps).

In addition to these projects, the staff of the Department of Analytical Chemistry is involved in training courses in the operation of analytical instruments (in-service training for faculty personnel), gas and liquid chromatography (for different levels of experience) and sample preparation for analysis. There is also postgraduate study courses in the analytics of food and environmental pollution and quality control (QC) and quality assurance (QA) of analytical measurements, as well as European projects for Master studies including a European Joint Master in Quality in Analytical Laboratories (emQal, 

), and the Euromaster "Analytical Science at the Interface" (

http://www.pg.gda.pl/chem/pl/images/stories/Wspolpraca_Miedzynarodowa/Teaching_Project2008-20094.pdf

The department received significant EU funds and the Centre of Excellence in Environmental Analytics and Monitoring (CEEAM) was established in 2003 (

) with Professor Jacek Namieśnik as its director and Professor Hartmut Frank as head of its international scientific advisory board. The department was also involved in the organization of the 35th International Symposium on Environmental Analytical Chemistry (

) and the 15th International Conference on Heavy Metals in the Environment (to take place in Gdańsk) (

Professor Jacek Namieśnik is an editorial board member of the following analytical journals: 

Journal of Chromatography A, Critical Reviews in Analytical Chemistry, Acta Chromatographica, Chemical Analysis

Toxicological and Environmental Chemistry

. He is also Chairman of the Analytical Chemistry Committee of the Polish Academy of Sciences (

 Tadeusz Dzido (head of Department), Katarzyna Czapińska, Wojciech Markowski, Beata Polak, Tomasz Tuzimski have been involved in liquid chromatography for the longest duration in the Department of Physical Chemistry, Chair of Chemistry, Pharmaceutical Faculty, Medical University of Lublin, Lublin. Other group members (Paweł Płocharz, Krzysztof Ścibiorski, Piotr Ślazak and Andrzej Torbicz) have been working in the Department for several years. Some of them are preparing their PhD theses. Three PhD students and five undergraduate students have also been performing chromatographic research in the department. 

 Research performed by the group has been developed based on the chromatography school created by Professor Edward Soczewiński (middle of the front row, retired for 10 years) who was the head of the Chair of Chemistry for 34 years (1964–1998). The Department of Physical Chemistry, is part of the Chair of Chemistry. Prof Soczewiński, author of the well-known equation describing retention mechanism in normal-phase systems of liquid chromatography (Snyder–Soczewiński model of adsorption), has became the inspiration for the investigation of liquid chromatography including both normal-phase and reversed-phase systems. These investigations have involved both column and planar chromatography.  

The group's research interest has focused on the retention and separation selectivity changes in relation to properties of components of the chromatographic systems, including the correlation of retention parameters and physicochemical properties of solutes, mobile and stationary phases. Correlation of retention and biological activity of compounds is also of interest.  

The group has been involved in the optimization of separation conditions in liquid chromatography systems (HPLC and TLC/HPTLC, thin layer chromatography/high-performance thin layer chromatography) of various classes of solutes, especially biologically active ones, including amino acid enantiomers, pesticides, nucleosides and phenolic acids, using chemometric methods and computer simulations of the chromatographic process with programmes developed in both the department (Dr Markowski) and commercially available systems, such as DryLab and ChromSword.  

Methodological aspects of the investigations in the range of liquid chromatography are also marked; especially concerning planar chromatography. The group has developed procedures for gradient development (continuous and stepwise), preparative separations, multidimensional development of chromatograms and multiple development. They have been performing investigations dealing with hyphenated techniques comprised of HPLC and TLC/HPTLC systems. 

Various horizontal developing DS chambers have been designed in the department. These chambers have been used worldwide. Various methodical possibilities of planar chromatography processes have been studied for horizontal DS chambers. 

From the experience gained constructing the chambers, a device for pressurized planar electrochromatography (PPEC) has been designed and the investigation of various parameters that influence the separation efficiency of compounds, especially with biological activity, have been performed with this method. 

Doug Kitson looks at some of Poland's leading separation science groups.

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