Innovative food companies have shown an increased interest in developing fast analytical techniques to characterize the flavour/aroma of food products that compare well with sensory analysis by human taste panels. Fast sensory-directed techniques can rely on electronic nose (e-nose) or electronic tongue (e-tongue) technology. Both technologies have been developed by research laboratories and commercial companies during the past 10 years, but still have a long way to go before they are applied in an industrial environment. As the human nose is involved in 80–90% of the total flavour perception, digital odour characterization using electronic nose technology can be used for fast classification of food products in good accordance with sensory analysis. 

Conventional techniques for assessing flavour quality, such as sensory analysis and GC–MS, are often too time-consuming and labour-intensive to be used as quality control (QC) methods within the food industry. The ultimate challenge is to develop fast analytical procedures resulting in classifications of food products that correlate well with sensory panel data. Electronic nose technology seemed to be an ideal approach when introduced a few decades ago.

  In the 1990s an 'electronic nose' was defined by Gardner and Bartlett as "an instrument, which comprises an array of electronic chemical sensors with partial specificity and an appropriate pattern recognition system, capable of recognizing simple or complex odours."

These systems generate a sensor array response to a complete volatile pattern, without separating the aroma compounds and use pattern recognition software for data processing. In the same way that humans use the receptors in the nose to smell a mixture of volatiles and the brain to analyse the data, e-nose systems use sensors and a data processor with pattern recognition software for classification of products.

  Currently, two electronic nose technologies exist on the technology market: conventional electronic nose technology based on gas sensors and the more recently developed electronic nose technology based on MS. It is clear that with artificial olfaction systems the operating principle, the number of sensors and their sensitivity and selectivity is very different from the human perception. The indication of such an analytical system as an 'e-nose' system, is perhaps more appropriate when the pattern recognition data interpretation is considered. As is the case with human perception these systems deliver a comparative rather than a qualitative/quantitative composition of the volatile mixture sent to the sensors. 

Gas Sensor-based E-noses Versus MS-based E-noses

Conventional electronic noses are based on gas sensors. In the sensor array of a gas sensor-based electronic nose the volatiles interact physically and/or chemically and a dynamic equilibrium develops by adsorption and desorption at the sensor surface. Different types of gas sensors have been used in literature: metal oxide semiconductors (MOS), metal oxide semiconductor field effect transistors (MOSFET), surface acoustic wave sensors (SAW), quartz microbalance sensors (QMB), conducting organic polymers (CP).

  Usually 6–12 gas sensors from the same type but with different response characteristics are used in a multi-sensor array. 

Although numerous commercial systems have been introduced and were claimed to be able to replace time-intensive sensory panels, in our laboratory we were not really convinced of the value of these techniques for objective characterization of aroma characters in food products. In our opinion the conventional e-nose technology is a 'black box' system and one does not really know what the signals respond to and what the instrument is really measuring. The speed of the systems is attractive, but they would, in most cases, not replace but complement sensory methods and traditional analytical techniques, such as GC–MS profiling.  

Furthermore, the first generation of instruments used static headspace to deliver the volatiles to the sensors. Aroma chemists know that this aroma isolation technique is certainly not sensitive enough to deal with the complexity of aroma compounds in food materials and with the odour potency of specific compounds. Also, the literature claimed that the gas sensor array systems have not generally lived up to expectations. Problems with drift, instability as a result of humidity (water vapour) or carbon dioxide, the need for frequent calibration, sensor poisoning and poor sensor-to-sensor and instrument-to-instrument reproducibility (transportability) are some of the major disadvantages.

More recently, a new generation of e-nose systems based on mass spectrometry was introduced, also referred to as MS-based electronic nose or MS-nose technology and mass fingerprinting or MS-fingerprinting. This e-nose technology was certainly more compatible with the basic knowledge of aroma chemists concerning the complexity and specificity of odour perception and the relationship with food flavour. In our opinion, from a theoretical point of view, the combination of a mass spectrometer and pattern recognition as a fast method to classify food products in relation to their flavour characters should be much more successful compared with the e-nose approach using gas sensor arrays. 

The majority of the e-nose papers in the field of food analysis deal with conventional e-nose technology based on gas sensors. Only a small part of the literature is dedicated to the application of MS-based electronic nose technology.

Configuration of the MS-based Electronic Nose

The MS-nose system was a hyphenated instrument consisting of a sample preparation autosampler (MultiPurposeSampler or MPS-2, Gerstel, Mülheim an der Ruhr, Germany), a 6890/5973 GC–MS system (Agilent Technologies, Palo Alto, California, USA) and a workstation with instrument software (Agilent Technologies) and Pirouette pattern recognition software for data processing (Infometrix, Woodinville, Washington, USA).

Fully automated sample preparation module for fast aroma isolation

The first module of the mass spectrometry-based electronic nose (MS-nose) configuration was a fully automated sample preparation unit for fast isolation of volatiles from a matrix. Although commercially available e-nose systems generally used static headspace (S-HS) as the isolation technique, some e-nose applications can require other isolation techniques for certain crucial aroma compounds because with S-HS the analyte range is often restricted to high-volatile compounds. The autosampler chosen can perform fully automated isolation of volatiles using both static headspace (S-HS) and solid-phase microextraction (SPME). If necessary, the hyphenated configuration can be expanded with a thermal desorption unit, which opens possibilities for other aroma isolation techniques such as direct thermal desorption (DTD), stir-bar sorptive extraction (SBSE), headspace sorptive extraction (HSSE) and dynamic-headspace (D-HS). 

The basis of the MS-nose instrument was a quadrupole mass spectrometer (MS with a classical split/splitless injector). Consequently, this hyphenated configuration can be used for both GC–MS and MS-nose applications, which is especially useful in an R&D environment. 

For using the hyphenated configuration in the MS-nose mode two options were evaluated. Firstly, the GC column chosen (HP-PONA cross-linked methyl silicone; 50 m × 0.2 mm i.d., 0.5 μm film thickness; Agilent Technologies) can be changed by a deactivated fused-silica capillary without any stationary phase (e.g., 1 m × 100 μm i.d.), which can be used as a transfer line from the GC injector to the MS sensing system. Secondly, the GC column can be held continuously at high temperature (e.g., 250 °C) to minimize the restriction of the volatiles, transferring the isolated aroma compounds directly to the mass spectrometer without chromatographic separation. The configuration with the GC column at high temperature had the advantage of high flexibility because with the same configuration both GC–MS mode and MS-nose mode can be applied and it was quite easy to switch from one mode to another. For this reason the latter flexible configuration with the GC column at high temperature was used. 

The third module was a workstation with instrument software for data acquisition and chemometrical software for data analysis. The basic data acquisition software of the GC–MS instrument was extended with specific ChemSensor software. This software allowed the conversion of a total mass spectrum to a so-called 'mass fingerprint'. Pirouette pattern recognition enabled on-line data processing of the generated mass fingerprints. Within the chemometrical software package exploratory algorithms (e.g., hierarchical cluster analysis or HCA, principal component analysis or PCA) were used to explore the MS-nose data, whereas classification algorithms (e.g., soft independent modelling of class analogy or SIMCA, k-nearest neighbours or KNN) were used to classify samples. 

After the fast isolation of the aroma compounds from a food matrix the isolated volatiles were directly introduced into the source of the mass spectrometer without separation. The mass spectrometer generated a total mass spectrum representing the summation of the mass spectra of all isolated volatiles sent to the MS source. Consequently, the total mass spectrum was transferred using software to a mass fingerprint. Mass fingerprints of different products or replicates of the same product were processed on-line by pattern recognition software. The different steps in an MS-nose analysis are summarized in Figure 1.  

One of the most important steps in an MS-nose analysis is the fast isolation of volatiles from the food matrix. Static headspace (S-HS), commonly used in commercial e-nose systems, is often not appropriate for applications in the food field and for objective characterization of flavour characters in food, as might be expected from basic aroma chemistry knowledge. With S-HS the analyte range is restricted to high-volatile compounds. Flavour characterization often requires isolation of crucial less volatile compounds. Therefore, other recently developed solvent-free sorptive techniques, such as solid-phase microextraction (SPME) and stir-bar sorptive extraction (SBSE), can be explored for fast aroma isolation in combination with mass fingerprinting. 

The sensing system of the MS-based electronic nose was a quadrupole mass spectrometer, which was a part of a classical GC–MS instrument as the mass sensor was coupled to a gas chromatograph in the presented hyphenated configuration. As mentioned before, this hyphenated configuration could be used in two operation modes: GC–MS mode and MS-nose mode, which made this instrument very useful for R&D purposes. 

In the MS-nose mode the GC column was continuously held at 250 °C and the isolated volatiles were directly sent to the quadrupole mass spectrometer without prior chromatographic separation. Helium was used as carrier gas (1 mL/min). With the high temperature of the GC column and the flow conditions the transfer of the volatiles to the MS sensing system was achieved in only 5 min. The classical split/splitless injector and the transfer lines were maintained at 250 °C and 280 °C, respectively. Electron impact was used with electron energies of 70 eV. The total ion current was recorded in the mass range from 40–230 amu (scan mode) and the run time was 5.0 min. The injection profile recorded from the mass spectrometer was a 'total ion chromatogram' (TIC) with only one broad peak of 'co-eluting' volatiles (Figure 2). The electron impact mass spectrum of the injection peak corresponded to the total mass spectrum of all isolated volatile compounds. The obtained total mass spectrum resulted from the fragmentation of all aroma compounds that were introduced into the ionization chamber of the mass spectrometer during the run time.  

From the 'total ion chromatogram' (TIC) of a sample a total mass spectrum of all isolated aroma compounds was obtained and converted by the software to a composite mass fingerprint. Figure 3 shows the conversion of a total mass spectrum to a so-called mass fingerprint, which can be visualized in a line plot format with, in the X-axis, the ion masses (

) of the mass fragments of all isolated aroma compounds within the mass range of the sensing system and, in the Y-axis, the corresponding ion abundances of the mass fragments. Hence, it is possible to relate certain 

 values in a mass fingerprint to typical mass fragments of an aroma compound or a class of aroma compounds. This implies that a mass fingerprint contains chemical information about the analysed sample, which is one of the major advantages of MS-nose technology in comparison with conventional electronic nose technology based on gas sensors.  

Chemometrics play an important role in the data processing of MS-nose data. Chemometry is the field of efficiently extracting easily interpretable and meaningful information from complex multivariate (chemical) data sets using tools of statistics and mathematics. The presented MS-nose system used the chemometrical software that applies different algorithms for exploratory data analysis, classification analysis and regression analysis.

The goal of exploratory data analysis is to recognize patterns of association in multivariate data sets and to perform a quality check on the data, exposing possible outliers (unusual samples). The exploratory algorithms are designed to reduce large and complex data sets into a set of best views of the data with minimal loss of information. These views provide insight into the structure and correlations that exist among the samples and variables in a data set. Mostly used in this perspective as unsupervised pattern recognition techniques are principal component analysis (PCA) and hierarchical cluster analysis (HCA). These exploratory algorithms indicate if the data are appropriate for the set-up of classification models. 

If there is sufficient discriminating power in the generated data, a model can be developed to predict a property of interest (e.g., categorical or continuous property). In the case of digital odour characterization the property of interest is generally a discrete category assignment (e.g., variety 1 or variety 2 or variety 3; good or bad). Then classification analysis is the appropriate approach. After method development classification/discrimination of the data, also known as supervised pattern recognition, is performed. Classification analysis allows to build models by analysing training samples belonging to 

 known classes. The obtained models allow assignment of unknown samples to a certain class. Soft independent modelling of class analogy (SIMCA) and k-nearest neighbours (KNN) are classification algorithms mostly used for this purpose. 

All MS-nose data from one analysis run or sequence generated a full data set (.DAT file) which could be visualized as an overlay of mass fingerprints (Figure 3) or as a data table. Exploratory and classification algorithms could be applied on full data sets or on well-defined sub-data sets or exclusion sets. 

 All generated MS-nose data were normalized by averaging all ion abundances to 100.0. Normalization was performed by dividing each data value by a normalization factor. Normalized MS-nose data from an analysis run can be visualized as an overlay of mass fingerprints (Figure 3) or as a data matrix with, in the rows, the samples or replicates and, in the columns, the ion abundances of all mass fragments within the mass range of the sensing system (e.g., 40–230 amu). 

 After normalization of the MS-nose data feature extraction was performed to eliminate data noise. To improve the data quality some ion masses (

= 73, 133, 147 and 177 (column bleeding); 

= 75 and 173 (PDMS fibre); and in some cases 

= 149 (phthalate). Especially column bleeding could lead to the presence of a 'background'. Without elimination of the features attributed to column bleeding (

= 73, 133, 147 and 177), column bleeding would effect pattern recognition. With static headspace (S-HS) no isolation of high-boiling aroma compounds resulted in feature elimination of ion masses with high 

 values (e.g., 141–230 amu) and consequently acceleration of the computational time. 

 Outliers are extreme observations that are situated outside the general pattern formed by other observations. The presence of outliers could seriously affect the performance of a pattern recognition algorithm. Sometimes visualization of a data set in line plots might indicate anomalous values. Also overlaying line plots of all samples in a data set could give a quick indication of samples that might be outliers. Furthermore, score plots (PCA) or class projection plots (SIMCA) could highlight the presence of possible outliers, especially if replicates of the same sample were not all clustered together. For clear identification of outliers the MS-nose data were generally subjected to so-called outlier diagnostics within the PCA or SIMCA algorithms. Depending on the selected number of factors in PCA or SIMCA processing more or less outlier samples could be diagnosed. After the identification of outliers by outlier diagnostics 'out of spec' samples were excluded from the original data set, generating a so-called exclusion set. 

After normalization, feature selection and elimination of outliers the MS-nose data were subjected to exploratory data analysis, using unsupervised pattern recognition techniques (e.g., hierarchical cluster analysis or HCA, principal component analysis or PCA) or classification analysis, using supervised pattern recognition techniques (e.g., soft independent modelling of class analogy or SIMCA, k-nearest neighbours or KNN).

Validation for Flavour Quality Assessment 

The major advantages of MS-based electronic nose technology compared with conventional electronic nose technology based on gas sensors are listed below:

 MS-nose technology is based on the proven technology of quadrupole mass spectrometry, which has been used for decades now and has become one of the most important and universally used detection and identification tools for analytical instrumentation. 

 One of the major advantages is the chemical information that is generated from MS-nose data. Mass fingerprints and their chemometrical data processing can allow correlation with classical GC–MS data. Certain 

values can be related to typical mass fragments of some aroma compounds or classes of aroma compounds. Especially aroma compounds with specific fragmentation patterns and unique mass fragments, which are not common for other aroma constituents, can contribute to a greater extent to discrimination of samples. 

 In contrast to gas sensor systems, which suffer from long-term drift and significant humidity and temperature dependence, MS-based e-noses are more stable and reliable. MS-noses are unaffected by ambient humidity or ambient temperature fluctuations. No sensor poisoning as a result of the presence of alcohols occurs as is the case with some traditional gas sensors. The influence from potential interference compounds may also be reduced or eliminated by excluding the associated 'sensor' responses (

 values) of interfering compounds [e.g., CO

The mass spectrometric sensing system is universal and can be used for all kinds of samples, without any restrictions, because the sensing system responds to all volatiles. Multiple applications can be performed on the same system. Limitation of the applicability of the MS-nose system for aroma characterization of food products is related to the volatile delivery system. Because, depending on the nature of the food matrix, fast aroma isolation can be performed in a more or less reproducible way.  

 The data range is not restricted to the number of sensors in the sensor array, but is dependent on the mass range of the mass spectrometer (e.g., 2–800 amu). Each 

 value is treated as the equivalent of a sensor response from the conventional electronic noses. This means that there are potentially several hundred 'sensors'. 

 The MS sensing system responds linearly over four orders of magnitude. In contrast, gas sensors have small linear ranges, typically no greater than two orders of magnitude. 

Articles

Coupling Matters

Mass spectrometry-based electronic nose technology (MS-nose technology) is a fast hyphenated technique for digital odour characterization of food and beverage products.

Hyphenated Electronic Nose Technique for Aroma Analysis of Foods and Beverages

The high-throughput analysis of pesticides in raw agricultural commodities has been hindered by the slow and laborious sample preparation stages. Using traditional clean-up procedures, as many as 20 steps are required to get the sample in a convenient form for analysis, which typically limits sample throughput to eight samples per day. This study evaluates a new approach using a novel laboratory mill and pulverizer to extract various pesticide residues from different kinds of plant materials and to identify and quantify the active ingredients by liquid chromatography tandem mass spectrometry (LC–MS–MS). It shows that by automating the procedure, the number of clean-up steps can be reduced significantly, which increases sample throughput over traditional approaches.

Cymoxanil (2-cyano-N-[(ethylamino) carbonyl]-2-(methoxyimino) acetamide) is the active ingredient in a number of pesticides manufactured by DuPont, for the control of various fungal diseases in crops and plants. It is mainly used for late blight potatoes, but is also applied to grapes, tomatoes, cucumbers and leafy vegetables. Its protection mechanism is to penetrate the surface to induce a host defence response to stop lesion growth and sporulation. 

The typical pesticide residue set at DuPont contains eight samples and usually takes an eight-hour workday to complete. The goal of this study is to take advantage of recent developments in sample extraction with a new laboratory grinder-mill and improved instrument sensitivity and selectivity using liquid chromatography tandem mass spectrometry (LC–MS–MS) to increase the number of samples analysed per day by a factor of three. The methodology conforms to the US Environmental Protection Agency (EPA) Office of Prevention, Pesticides and Toxic Substances (OPPTS) Residue Chemistry Test Guidelines — OPPTS 860.1340 Residue Analytical Methods.

  These were developed by the EPA as guidance for developing analytical methods to be submitted to the agency for review under federal regulations and dictate that average recoveries must be 70–120% with good reproducibility. 

Sample preparation for the analysis of pesticides and their residues has typically followed DuPont Report No. AMR 3705-95, "Analytical Method for the Determination of Famoxadone and Cymoxanil Residues in Various Matrices".

  In this procedure, ground-up samples are weighed into extraction jars, followed by the addition of water to allow them to rehydrate before extraction. Acetonitrile is added and samples are ground up with a laboratory homogenizer. The plant matrix is then allowed to settle-out and liquid extracts are filtered and collected in mixing cylinders containing sodium chloride. The mixing cylinders are capped, shaken and inverted to aid in the dissolution of sodium chloride and are then allowed to stand while the acetonitrile (upper layer) and water phases separate. 

Traditional Pesticide Analysis Using Liquid Chromatography

For the analysis of cymoxanil, the acetonitrile aliquot is back extracted with hexane, concentrated, diluted with water and passed through a conditioned strong anion exchange solid-phase extraction (SPE) cartridge stacked on top of a conditioned carbon black SPE cartridge. Cymoxanil passes through the strong anion exchange cartridge and is retained on the carbon black cartridge. It is then selectively eluted off the cartridge and the concentrated cymoxanil residues are dissolved in a hexane–ethyl acetate mixture. The resulting solutions are passed through silica SPE cartridges. Cymoxanil is retained, selectively eluted and concentrated into a methanol–water mixture adjusted to pH 3. Samples are filtered and analysed by high performance liquid chromatography (HPLC) with UV detection. 

Sample Preparation for Detection by LC–MS

There are approximately 20 clean-up steps in the AMR 3705-95 procedure, which limits the number of samples to 8–10 per day. A modification to this procedure, laid out in DuPont 13753, "Analytical Method for the Determination of Cymoxanil and its Metabolites in Leafy Vegetables Using LC–MS",

  significantly reduces the number of clean-up steps. In this procedure, samples are extracted using an acetonitrile–water mixture as described in DuPont Report No. AMR 3705 9, Revision 2. For cymoxanil, sodium chloride is added to an aliquot to separate the aqueous phase from the organic phase. The aqueous phase is discarded and the acetonitrile layer containing cymoxanil is passed through a strong anion exchange SPE column. The extract is then further cleaned up using a hexane liquid–liquid extraction followed by an Envi Carb (Supelco, Bellefonte, Pennsylvania, USA) SPE column. Cymoxanil is not retained on either of these columns, so the eluent is passed directly into an LC–MS system for analysis. Based upon the sensitivity and selectivity of LC–MS, the number of sample clean-up steps is reduced to approximately 10, allowing for the analysis of up to 16 samples per day. 

To further reduce the clean-up steps, a new sample preparation technique was evaluated that used a Geno/Grinder Model 2000 laboratory mill (Spex SamplePrep, Metuchen, New Jersey, USA). This is a laboratory mill or grinder specifically designed for vigorous vertical shaking of deep-well plates. It was originally designed to prepare plant tissue for extractions of nucleic acid, protein and other constituents by shaking the tissue, steel balls and a buffering agent together in each well of a titre plate. The application of this tool can be expanded to include micro-organisms when small silica beads are used instead of steel grinding balls. Microbes can also be disrupted in standard 96-well plates as opposed to deep well plates. Sample material that can be prepared includes bacteria, yeasts, molds, seeds, stems, roots, leaves and certain animal tissue. Because the vertical shaking motion of the equipment is so strong, many seeds and other forms of plant tissue can also be pulverized dry with the help of one or two grinding balls per well. 

For the leaves of watery crops such as tomatoes and potatoes, 10 g of sample was extracted twice with 20 mL of a 90:10 mixture of acetonitrile–water solution. To ensure complete extraction of the compounds from the plant material, steel balls were added to the samples to pulverize the plant material. The samples were then extracted by shaking at high speed at approximately 700–1200 cycles/min, depending upon the sample. The sample extract was brought to 50 mL by adding acetonitrile. Approximately 1 mL of the extract was filtered using strong anion exchange and the SPE process described earlier. A 100 µL volume of sample was diluted to 1.0 mL in an HPLC vial with mobile phase and was analysed directly by LC–MS–MS. This approach cuts down the number of clean-up steps to three compared to 20 with the AMR 3705-95 method and 10 with the DuPont 13753 method. 

To evaluate the efficiency of the sample preparation and clean-up stage described previously, cymoxanil and its various metabolites were determined in a number of wet and dry crop and plant materials. All samples generated were analysed using LC–MS–MS. 

This instrument is coupled with a 1100 Series HPLC system (Agilent Technologies, Santa Clara, California, USA) to first separate the biological species of interest, based upon their elution times off a column. The eluent is then introduced into the mass spectrometer for ionization to identify and quantify the species using triple-quadrupole MS. The principle of this technique is based upon confirmation of a particular molecular ion by the generation of its product species using collisionally induced dissociation or fragmentation.

  This technique, commonly known as multiple reaction monitoring (MRM), uses two resolving quadrupole mass filters, separated by another quadrupole, which is slightly pressurized by the introduction of a collision gas such as hydrogen or helium. In this MS–MS mode, the first quadrupole is used in a mass-resolving mode to select the precursor ion. The second quadrupole (or pressurized multipole collision cell) is used to produce fragmentation of the precursor ion. The final quadrupole is used in a mass-resolving mode to provide mass analysis of the resulting fragmented product ions. These species are then compared with reference spectra-data to produce unambiguous identification of the biomolecules of interest. 

 Table 1: Brief summary of the HPLC and LCâMSâMS instrumental conditions used for this study. 

The benefit of the MS–MS design is that it also can be used in MS mode for quantification purposes. In this mode, the first quadrupole is typically used in the rf-only mode, as a wide bandpass filter to transmit molecular ions of a wide mass range. The collision cell is also used in the rf-only mode, but this time no collision gas is flowing, so the cell is just used to transmit ions to the last quadrupole, which is used in the mass resolving mode. This is known as the selective ion monitoring (SIM) mode. The ability to switch between both modes maximizes the amount of data being generated, particularly when small amounts of sample are being analysed. The higher number of product or fragmented ions identified leads to better confirmation of the parent molecules. This is especially important when analysing data from an LC separation. Cymoxanil, for example, which is an aliphatic nitrogen compound with a formula of C

 and molecular weight of 198, is typically eluted from the column after 10–15 min, over a period of 20–30 s. For this reason, it is very important to be able to switch very rapidly between SIM and MRM mode to collect both the molecular and fragmented information for positive and unambiguous identification of these species. After identification is made in the MS–MS mode, quantification of unknown samples can be performed in either the SIM or MRM modes. 

Reversed-phase LC was used to separate cymoxanil and its metabolites from the rest of the extractants. The LC–MS–MS instrument was operated in single-ion monitoring MS negative-ion mode for quantitative analysis. Peak area was used for quantification. For confirmation of the presence of the analyte in unknown samples, the relative intensities of the fragmented ions were measured using the MRM mode. A brief summary of the HPLC and LC–MS–MS conditions are shown in Table 1. 

A 0.01  ppm cymoxanil LOQ fortification sample (following method DuPont 13753) and control (blank) in the SIM mode is shown in Figure 1. It can be seen very clearly that there is a significant peak eluted at about 15 min, which corresponds to the cymoxanil. 

Confirmation of the compound is then made by measuring the intensities of the fragmented product ions in the MS–MS mode, by comparing them to known ratios in a database of pesticides. The mass spectrum (60–300 Da) of cymoxanil in single MS mode is shown in Figure 2, and the resulting calibration curve for 0.2, 0.5, 1.0, 5.0 and 10.0 µg/L in SIM mode (

Table 2: Percentage recovery data and RSD for a 0.05 ppm (LOQ) and 0.50 ppm (10 x LOQ) reference samples run using three different analytical methods. 

Table 2 shows some typical recovery data and RSD for 0.05 ppm and 0.50 ppm reference samples run using three different analytical methods. 

Sample Preparation for Field-incurred Residues

Once the methodology was optimized for the separation and quantification of cymoxanil, the laboratory mill sample preparation and clean-up procedures were used for comparison purposes with traditional sample preparation methods. The basis of this method was then used to investigate an experimental new insecticide (DuP-1) and to identify and quantify its active ingredient and metabolites in various wet and dry plant and crop materials with field-incurred residues. Rather than fortifying directly onto a control sample in the laboratory, the crops were treated in the field with the insecticide at the timing and rate of typical agricultural practices. The crops were harvested at maturity and all detected metabolites have been incorporated into the plant matrix rather than on the surface, as is the case for laboratory fortifications. The results of this experiment will further test the extraction efficiency of the laboratory mill sample preparation procedure, which is shown in the following:  

Extract twice with acetonitrile–water in the grinder for 2 min at 1200 cycles/min.

Dilute 1 mL of extract to 5 mL with water (adjust to pH 2.5).

Filter through strong anion exchange SPE and collect the extract.

Filter through a PTFE membrane into an LC vial.

The total ion chromatogram (TIC) generated by the LC–MS–MS in MRM mode of the new experimental insecticide under investigation and its metabolites (A–G) is shown in Figure 4. Two pairs of metabolites — A and G, as well as D and E, are eluted from the LC column at the same time, indicating that they have similar chemical properties. A benefit of the LC–MS–MS method is that by monitoring the individual transitions of the MRM for each metabolite, the overlapping peaks can be resolved. Metabolite D with a mass of 444 Da can be identified by monitoring the transition 443 (molecular weight  1) to 298 Da and separated from metabolite E, which has a mass of 458 Da and a transition of 457 to 188 Da. Figure 5 shows the resulting MRM chromatograms for a 0.2 ppm standard of the metabolites D and E (top), together with a tomato sample spiked at the LOQ (bottom) and illustrates good resolution of metabolite D from E. 

Table 3 compares the new sample preparation and clean-up method with a traditional homogenizer probe (Tekmar Tissumizer, Cincinnati, Ohio, USA) sample preparation using the DuPont-13753 method described earlier. It shows quantitative data (parts per million in sample) for the experimental new compound and one of its metabolites (D), for various crop samples. This methodology was then used to analyse a variety of wet, dry, oily and acidic crops for the active ingredient together with its metabolites. Table 4 shows spike recovery and RSD data for one of the watery crops (tomato), showing that the DuP-1 insecticide and all its metabolites are within the guidelines stated in EPA OPPTS 860.1340, which states that test data must have average recoveries of 70–120%. Although they are not presented here, the study also looked at other crops including limes (acidic crop), almonds (oily crop) and wheat straw (dry crop) and achieved similar spike recoveries and precision values. 

Table 3: Comparison between the Geno/Grinder and Dupont-13753 sample prep methods for the quantitation of the experimental new compound (DuP-1) and one of its metabolites (D) in a variety of wet and dry crop samples. 

It has been shown that the extraction of pesticide residues using a novel sample preparation procedure and identification and quantification of its active ingredients and metabolites using LC–MS–MS provides an extremely efficient, rugged and high-throughput analytical method. Compared with the traditional sample preparation, clean-up and detection methods, sample throughput was increased by a factor of three, from 8 samples to 24 samples per day. It also demonstrates that the extraction efficiency of the new procedure is valid for field-incurred residues in addition to laboratory-fortified samples. 

Regular Article

A fast clean and simple method to determine three organotin compounds in environmental samples is described

A Novel Sample Preperation Approach to Increase the Throughput of Pesticide Analysis by LC–MS–MS

Dr A.J.P. Martin, Dr Steve Dal Nogare and Prof. A.I.M. Keulemans. You could not have wished for a better group of mentors. In 1957, I performed my first GC injections. I was a graduate student at Purdue University and had just finished a Masters in electrochemistry. I wanted an exciting new topic for my PhD work. I took a summer job at Amoco's Research Labs in Whiting, Indiana, USA. My job was to screen around 80 new liquid phases for selectivity to separate butane-1 and 

-butylene as well as some C18 unsaturated methyl esters. During the summer job, Nobel Laureate A.J.P. Martin came to the labs to install his new gas density balance detector. 

I was assigned as his "gofor" for the day. It was a memorable experience; he really turned me on to GC with his knowledge and enthusiasm and tips on making tea (heat the cup!). The next summer, I worked with Steve Dal Nogare from DuPont in Wilmington, Delaware, USA and was indoctrinated in temperature-programmed GC. Steve was a great mentor and very enthusiastic about GC. Finally, my Fulbright fellowship with Professor A.I.M. Keulemans in 1959, convinced me I would work on GC for as long as I could. So many of the pioneers in GC came to his lab and I was fortunate to work with many of them — and this fired my enthusiasm for GC. 

What do you see as your greatest achievement in the field?

My greatest achievement is collaborating with my students — undergraduates, graduates, post-doctoral fellows — and visiting professors (easily over 200 total). This group of intelligent, humorous, hard-working people made my research and teaching both interesting and challenging — 

! It would be unfair to mention any single individual as there were too many major breakthroughs from a wide range of people. Highlights would definitely include reporting the first capillary GC–MS results in 1961; introducing temperature programmed LC in 1981; using mobile phase modifiers to stabilize retention times on silica gel; developing the first directly coupled LC/GC experiment using two independent computers in 1981; identifying the role of pH in CZE to add to the electrostatic mobility of highly hydrated protons: and also early work on analysing steroids in urine by GC-TOF-MS. The latter study was one of the earliest demonstrations of the use of TOF-MS coupled to GC for determining traces of steroids in urine and this work was honoured with an "outstanding Ph.D. thesis award" from Virginia Tech. 

A research project in 1976, which correlated pKb's of weak aromatic amines with retention time on acidic silica gel, was recognized by NASA as the second most cited scientific publication for that year. The Viking-Mars Landing was Number One! Our work was one of the first publications on the LC analysis of aromatic diamines. Not only did this class of compounds hold high interest for NASA in relation to polymer research, but they also held high biological significance; thus, many researchers in the biomedical and pharmaceutical communities also paid close attention to this work.  

But equally as important as the quality of the scientific publications was the work ethic, the group effort and strong friendships established while at Virginia Tech. Some of the best memories were the long van rides to Pittcon each year. I was always so proud when one of my students gave a great lecture at some conference. I am still in contact with almost all of my former students. 

Did you consider entering academia after your post-doctoral fellowship?

Not really, I was focused on industry. I had spent five summers working in industry at Phillips Petroleum Co., Amoco Central Research, American Cyananid, DuPont and Esso Analytical R&D. I knew industry had better equipment, strong support staff and services, real significant problems and, of course, much better pay. In fact, when I did come to Virginia Tech in 1968, my plan was to stay only a few years then go back into international marketing, hopefully in Europe, in the scientific instrument business. But after three years, I fell in love with teaching students, choosing my own research projects, being able to play tennis almost any time, any day and Blacksburg was a great place to raise a family. 

 Red Lion Inn, Blacksburg VA: McNair and McNair students versus HPLC short course students in a game of volleyball.			

Incidentally, I did actually get a tennis and a scholastic scholarship for the University of Arizona and played there for two years until the number of Physics and Chemistry labs every week left no time for tennis. I still enjoy hitting and try to play two or three times a week.  

You told me that two of your favourite activities: teaching short courses and writing books came from your industrial experience. Why is this?

After one year with Esso R&D in 1961, I returned to Europe to set up operations for F&M Scientific (later H.P., Avondale, Pennsylvania, USA). Even though Europe had introduced and pioneered the art of GC, there were few progressive instrument manufacturers. F+M Scientific and later Aerograph (Varian) realized the importance of TPGC; both became major leaders in the field based on the quality of their instrumentation. I lived in Amsterdam 1962–64 and European customers were begging for seminars, hands-on training courses, booklets; anything to help them learn about GC. Part of my job was then — and still is — telling everyone how great GC really is. My book 

 originated from a lecture at the University of Athens, Greece in 1963. After one lecture, the faculty begged me to come back the next day. After four lectures in four days, they begged me to write up the lectures, which became the first draft of 

. It is written in straight-forward English for Europeans with English as their second language. It was a simple book, easy-to-read and was eventually translated into eight different languages with over 130000 copies sold. 

After joining Varian in 1965, I set up a one-person unit, GC training, which offered "hands-on" GC training, across the US initially, but later in Europe, Canada, Australia and Mexico. This was a huge success. The unit not only showed a small profit each year, but also, as I recall, about 20% of all attendees bought or had already bought a Varian GC. It was a great marketing success. 

Thus logically, I carried the model for short courses and GC books with me to Blacksburg. The short courses helped train my students, generate money in my earlier academic career, recruit new graduate students and several companies left their best chromatographic systems in my labs.  

What do you see as the most exciting development in separation science at the moment/the future?

LC–MS applied to biological compounds. So many people who are not trained as chemists or chromatographers are buying and using LC–MS regularly to study important problems that involve the analysis of complex mixtures of biomolecules. 

Kevin Schug talks to Harold McNair about his illustrious career in chromatography

Icons of Chromatography: Harold McNair

In 1997, in an article that appeared in what then was 

), I departed from my usual technical writing, which up to then had been primarily for peer review. The 

 editor had requested help recognizing the publication's 10th anniversary. Mass spectrometry (MS) and liquid chromatography–mass spectrometry (LC–MS) in particular, given the rapidly evolving commercial landscape, I thought made investigating the phenomenon worthwhile. Little did I realize how much effort a writer must devote to developing and presenting ideas in finished form. I must have forgotten that lesson when, five years later, I agreed to write the "MS — The Practical Art" regularly. 

Nearly 35 bimonthly columns later, I happened across that initial article, "Commercialization of LC–MS 1987–1997: A Successful Decade in Review" [

(11), 728–737 (1997)]. It made me curious about just how many technological advances the field of MS had witnessed during the ensuing decade and what had not changed. 

Of course, the technological basis, or history, remains unchanged for what was then called the "hyphenated" on-line practice of LC–MS. What has changed, though, is this: as an analytical method, LC–MS has become so ubiquitous in industry and research that more practitioners now approach it as a single, integrated analytical process. Based upon the rapid growth of the American Society of Mass Spectrometry (ASMS) indicated by the annual conference attendance in recent years, the cross-section of who constitutes today's practitioners has broadened as well. We have abandoned the sense of technological duality that the hyphenated usage implied. My 1997 article attempted to outline evolution of the mass spectrometer as it was becoming a true mass detector "mixing significant technological events with significant commercialization initiatives over [those] 10 years." It fleetingly acknowledged important events in the development of LC–MS to that point, such as Blakely and Vestal's 1983 publication on thermospray which, though no longer used, was the first LC–MS enabling technology.

John Fenn (along with Koichi Tanaka and Kurt Wüthrich) received the Nobel Prize in Chemistry, in 2002, for his work on the most significant aspect of LC–MS practice — electrospray ionization (ESI) — based upon work published in the early 1980s.

  Among those receiving credit for commercializing the concept were Bruins and Covey who, along with Henion, published commercially promising ESI designs in 1987.

A harbinger of things to come, the first truly hyphenated system appeared in 1982. Hewlett Packard's mass spectrometry detector (MSD) used a gas chromatograph to introduce analytes into the mass spectrometer's ion source. Of course, the gas chromatograph limited the MSD to analysing only compounds of sufficient vapour pressure. However, no such restriction attended coupling a high performance liquid chromatography (HPLC) system to the mass spectrometer. LC–MS had its own problems: vacuum system and flow-rate requirements evolved large, costly, often unreliable instruments. 

In 1993, Waters (Milford, Massachusetts, USA) introduced a benchtop instrument for LC–MS. Designed to operate using particle beam technology, which in the era of ESI exhibited certain performance issues, it nonetheless deserves footnote-level respect for giving us true electron ionization (EI) spectra for an LC separation. That said, the first LC–MS designed for widespread adoption is probably Vestec's thermospray-equipped, modified HP MSD, which the company offered at the 1986 Pittsburgh Conference and Exposition (Pittcon) for the almost magical price of $100000. Thermospray of course faded quickly and so did the dream of vastly capable but inexpensive LC–MS. 

As is so often the case, the optimism of commercial interests proved unwarranted. In 1988, a year after Extrel Corporation had introduced particle beam technology at Pittcon; Hewlett Packard (now Agilent Technologies, Santa Clara, California, USA) introduced a version derived from the original particle beam instrument. The HP product manager claimed the interfacing of LC and MS brought to the LC–MS market the reproducibility of results and ease-of-use characteristic of gas chromatography (GC)–MS. In those days, industry watchers estimated market sales to be $15 to $20 million, with thermospray instruments accounting for 60–80% of the business.

  Always a point of industry contention and one whose public discussion is typically avoided for reasons of competition, Finnigan (now Thermo Fisher Scientific, Waltham, Massachusetts, USA) management succinctly stated at Pittcon 1993 that estimates of market size were almost "guaranteed" to be incorrect and were "probably rather conservative". Indeed, the total market for 1993 was predicted at $640 million. In the years immediately following, it became obvious that both sides of the argument were incorrect.  

The early 1990s fell far short of sales expectations, the consequence of a general economic slowdown. (Remember, these were the days when costly analytical purchases were driven largely by a rapidly building pharmaceutical industry.) A 1996 American Chemical Society report cited 1991 sales exceeding $450 million, but with the caveat that "growth in these areas is difficult to predict." 

Between 1985 and 2004, it was my good fortune to co-organize the LC–MS interest workshop at the annual conference of ASMS (

). During that time, the success of LC–MS transformed ASMS. Once an annual event for thought leaders and innovators in MS research, the conference also came to represent an educational resource for nonspecialists. In 1987, 2200 members attended. By 1997, that number had doubled. In keeping with the growing interest, attendance at the LC–MS interest workshop grew, by some estimates to more than 1200 from a few hundred. 

I began taking note of changes in abstract topics matched to overall attendance during a stint on the programme committee in 2003–2005 (and in the years since — the conference administrator, Brent Watson, kindly obliges with statistics each year). The ASMS conference has grown in the early years following 2000 to nearly 6000 attendees. Once the LC–MS interest wave crested and LC–MS became the ubiquitous tool it is today, the conference organization reflected that interest. In these much larger conferences, LC–MS is no longer the esoteric focus of a special interest group. Indeed, the technology now appears as a topic or reference in virtually every discussion. As for the LC–MS interest group, it has reverted to a more reasonable size, returning to a more utilitarian, narrow charter. 

In the early years, presentations rarely associated LC columns and separations with LC–MS practice. LC served merely as an "inlet" for compounds dissolved in the condensed phase, and it was the mass spectrometer that bore the brunt of separations work. A survey in 1985 noted that of 400 chemists interviewed, less than one third were "comfortable with prospective applications and operation of LC–MS".

  showing comparative analyte ranges for the major LC–MS interfaces appears quite dated today: particle beam and thermospray, two of the four noted along with atmospheric pressure chemical ionization (APCI) and ESI, pretty much vanished years ago. On the other hand, I noted in my article emerging technologies just gaining ground:  

"Significant recent introductions, with much shorter commercialization histories (as of this date), such as the reengineered ion-trap (commercialized initially by Finnigan, now Thermo Fisher Scientific) and time-of-flight (TOF) instruments also are worth a passing note for their potential role in shaping the future direction for LC–MS detection."

It was obvious by the time the article appeared that a few years earlier, "The interface with the greatest potential impact was... previewed as a commercial possibility in 1983,

  when preformed ions in solution were described... The advantage of electrospray, readily embraced by protein chemists, was the ability to charge multiple sites on... relatively large molecular weights... well above the operating range of the quadrupole." 

Looking back over the 16 years since the article was published, it is obvious that quadrupoles still serve as the analytical backbone of LC–MS practice. Evolution of the technology brought ion traps to prominence from their attendant problems of space-charge effects (too many ions in the trap) and quantification issues to much-improved, linear traps coupled with ion cyclotron devices such as the Orbitrap (Thermo Fisher Scientific) or as an adjunct to tandem designs (MDS Analytical Technologies Sciex, Toronto, Ontario, Canada). 

Consider that in recent years, the ASMS conference attracts about 6000 attendees who submit almost 3000 abstracts for talks and posters (low of 2730, in 2009, and recent high of 2907, in 2008). Granted, we must bear in mind the content in recent years has become heavily weighted away from environmental and industrial applications toward life science (searching for individual instances where an abstract contains the term "peptide" for instance returns 1023 hits out of 2730 abstracts), but the consistency in attendance numbers year-to-year can reveal some shifts in technology adoption trends. For instance, "ion trap" and "quadrupole" are too common (500 or more instances for each across numerous applications). Moreover, the ASMS programme committee rules out trade names as part of the acceptance process. Yet some such terms are unavoidable and unique. "Orbitrap", though a trade name, is an example of commercialized novel technology. Since 2006, Orbitrap cites per abstract rose from 39 at its introduction to ASMS to 429 in 2009. Similarly, we can judge unique entities such as SYNAPT (Waters Corporation) and the ability to combine ion mobility as an orthogonal discriminator (in essence multiplying the resolution or peak capacity of a mass spectrometer), which shows a tripling of its unique instances since 2007. 

The ASMS conference is, of course, an MS venue. With that in mind cites for "UPLC" (Waters' ultra high performance LC system) more than tripled, to 142, since 2006, which is impressive because LC is simply an "inlet" device to a mass spectrometer even today (by comparison, "HPLC", long-established as the inlet technology, returns 540 instances). 

From the highest-level view, major corporate acquisitions and mergers seem to have slowed in recent years, and the rapid emergence of revolutionary technologies has morphed, with a few exceptions, into slower, evolutionary change. Agilent Technologies, with quadrupole and TOF technology, has joined the top tier and continues to promote chip-introduction schemes. Waters Corporation has followed suit with the advent of its TRIZAIC UPLC system. Waters' addition of ion mobility to its MS products provides the means to directly control the sample introduced into the mass spectrometer in an orthogonal fashion, layering a variety of ion manipulation techniques with differentiation of molecules by conformational cross-section. Thermo Scientific has steadily improved the speed of acquisition and resolution of its matched devices, the ion trap and the Orbitrap. The company's recently introduced dual-chamber ion-trap system, the LTQ Velos, begins to address some of the impediments inherent in ion-trap technology like space-charge and quantification limitations. 

Applied Biosystems today no longer exists as a corporation, surviving as a brand in the market as part of Life Technologies, a biotechnology manufacturer, after merging with Invitrogen (San Diego, California, USA). The company maintains its joint venture with MDS Analytical Technologies Sciex, which continues to improve the top-end, tandem quadrupole series. 

Based upon the same slanted attendance profile, we can, with some reservation, look at some year-to-year trends: 								

A data base query for ion suppression returns 69 instances representing unique abstracts, which seems typical (76 in 2008; 64 in 2007; and 87 in 2006). A disproportionately large amount of our analytical effort is based upon condensed phase practice, which is amenable to ESI. But ESI is prone to ion suppression and constantly requires better ways of remediation. 

Metabolomics at 139 hits shows a significant increase. In 2008, 80 was an increase from 46 in 2007 (39 in 2006, 54 in 2005). 

Biomarkers remains fairly constant with 355 instances versus 400 in 2008 and 350 in 2007. 

Informatics as a general term returns only 10 hits, while bioinformatics returns 154, down from 239 in 2008.

Making predictions is a tempting but risky exercise when you are committing to print. Yet in the 1997 article, I did venture to state that the "halcyon days of interface development were over," which appears to be true. A recent study report by Strategic Directions International, Inc., a market research firm that tracks instrument business trends indicates that, as of 2008, four manufacturers (Agilent, Applied Biosystems, Thermo Fisher Scientific and Waters Corporation) comprised 70% of the mass spectrometer market, with between 13% and 25% shares each. 

"Despite being well over a $2 billion market, demand for MS is expected to grow significantly, if not stronger than in recent years. This is despite current weakness in major economies the world over, as well as some pull back in the pharmaceutical industry. The fact that MS is such an important tool in high-end basic research in so many leading-edge industries will help to increase demand at a 9% annual rate through 2012. The market will be led for the foreseeable future by the more advanced methods, including Fourier transform (FT)-MS, tandem LC–MS, and quadrupole time-of-flight QTOF LC–MS."

The same report sees "typical" prospects for GC–MS settling for 5% growth, with half the business resulting from replacement sales of an installed base said to be reaching 30000 units. Improvements in GC-TOF and, to a lesser degree, GC–GC tandem account for increased sales. 

Often overlooked, even in these instances, is the central role single quadrupole instruments still play. A relatively large, installed base (7000 is estimated in this report), has Waters and Agilent competing closely, with equal shares dominating 90% of this market segment. 

QTOF instruments are reported to be at 4000 installed, proving, as I predicted in a 2004 column, that complexity coupled with improvements made this hybrid technology accessible for a variety of quantitative and qualitative applications.

Alan Millar (Senior Manager for QTOF Product Development, Waters) recently compiled an historical overview of the efforts of only a single company during the past decade. Eight nominal mass single-quadrupole instruments have been brought to market in the past 16 years, beginning with the Platform in 1993. A total of 11 triple- or tandem-quadrupole instruments appeared between 1989 and today, beginning with the Quattro model. Hybrid-TOF instruments account for eight unique product developments in 13 years, beginning with the original QTOF with 5000 resolution (FWHM) in 1996 to today's recent G2 introduction capable of 50000 resolution. Adding matrix-assisted laser desorption ionization (MALDI)- and TOF-only designs would easily more than double that number in a similar time span. 

Prices continue to be influenced by the cost of vacuum systems and control capabilities. Except for GC–MS, do not expect to see a high-end MS system for less than $100000. A counter-argument can be made that by any standard, single-quadrupole LC–MS instruments, which do sell typically for much less than $100000, offer state-of-the-art quadrupole technology today, lacking only the extensive additional capabilities of the more complex mass spectrometer. Median, or average, prices often appear in descriptions, except in the case of high-volume instruments (single-quadrupole and ion-trap instruments) and even those are often bundled in special arrangements. But without stating detailed specifications, you will find that the prices of higher-performing instruments overlap. 

As we have seen, technology can have a very brief half-life. Some make a great appearance only to fail to thrive after three or five years. Some, however, need enabling technologies to bolster their utility — TOF waited 60 years before fast electronics, software and much improved engineering design made them the practical analytical tool they are today. Software became more capable and hardware clearly improved in recent years, and an improved understanding of fundamentals often allows us to build on such improvements. 

A couple of recent introductions illustrate how sound fundamentals can reappear to our advantage. Both Bruker (Billerica, Massachusetts, USA) and Waters have recently commercialized an atmospheric pressure GC (APGC) adaptation for their LC–ESI-MS systems. The Waters license derives from DuPont and the work of Charles McEwen (currently Houghton professor of Chemistry and Biochemistry, University of the Sciences, Philadelphia, Pennsylvania, USA). McEwen is perhaps better known these days for his work developing yet another classic fundamental, the atmospheric solids analysis probe (ASAP). The ASAP technology allows an operator to simply place a solid or liquid sample on the end of a tube, insert the tube into a stream of heated nitrogen, where the sample is volatilized, and look at the resulting ions from the mixture. Direct links to Horning's work in the 1970s are readily apparent.

Debate over the exact nature and mechanics of ionization might still persist as it has with ESI while we made good use of it. But in the meantime, extremely fast chromatography demanded faster MS acquisition speeds with good fidelity. Improved software allows us to take advantage of our understanding of isotope characterization and our predictive capability. Better engineered, robust MS platforms displaying our improved understanding of gas dynamics yield sources capable of greater diversity. 

For instance, an aspect of the APGC Xevo design (Waters Corporation) puts a "mini-chamber" in place between the ESI inlet in the MS source and the GC transfer line as an integral part of the changeover from LC to GC. The entire procedure takes only 5 min. When the idea of LC and GC in the same instrument was proposed not too many years ago as a viable return-on-investment argument, it failed because substandard GC performance at any cost is unacceptable. Today we see low-femtogram sensitivity in what is ostensibly an LC source design. Add recent developments in atmospheric pressure photoionization (APPI), which show the potential to deliver EI-like spectra, and you realize that the same level of training and the same software and experimental design we've been accustomed to applying for LC–ESI-MS now covers a much more diverse chemical landscape. 

 is principal scientist, MS technology development at Waters Corp. (Milford, Massachusetts, USA); a former adjunct professor and visiting scientist at Roger Williams University (Bristol, Rhode Island, USA); co-founder and current president of the Society for Small Molecule Science (CoSMoS); and a member of 

Direct correspondence about this column to 

, Advanstar House, Park West, Sealand Road, Chester CH1 4RN, UK or e-mail: 

MS - The Practical Art

"In retrospect, we sometimes see the successful return of an earlier idea on the back of great advances in technology."

Commercialization of LC–MS: The Second Decade

Organotin compounds (OTCs) have been extensively used as biocides, pesticides, fungicides, bactericides and catalysts,

  and also as antifouling paints for the protection of ship bulls in polyvinyl chloride (PVC) stabilization.

  High concentrations of OTC can, therefore, accumulate in the environment and, because they are soluble in lipids, OTC may penetrate into tissues and the central nervous system with a potentially high toxicity to organisms and tendency to bioaccumulation.

  They can cause serious problems such as disturbances in the hormonal system and even the occurrence of imposex,

  so are regarded as endocrinal disrupters. Moreover, they can be potentially transmitted to humans by the trophic chain.

  As a result, most countries have banned the use of antifouling paints containing tributyltin (TBT) on small boats, and it was included in the European Union priority pollutant list for fresh waters: RD 995/2000 defines TBT as a quality parameter and establishes its annual medium value as 0.02 µg/L. 

TBT can be degraded in the environment into more polar metabolites, such as dibutyltin (DBT) and monobutyltin (MBT). The observed toxicity effects strongly depend on the compound and the biological species involved.

  The study of their role in ecosystems is important to assess their environmental impact. It is, therfore,  important to develop highly sensitive, robust, accurate and fast analytical methods to determine OTC in environmental samples. 

Most of the analytical methods used in OTC determination combine a separation technique such as gas chromatography (GC)

  with element-selective detection such as atomic absorption spectrometry (AAS),

  inductively coupled plasma mass spectrometry (ICP-MS),

  or pulsed flame-photometric detection (PFPD)

To apply GC techniques to determine OTCs, a derivatization step is useful to form a key step. The three most common derivatization procedures are hydride generation,

  and ethylation with sodium tetraethylborate (STEB).

  STEB methods have been favoured over Grignard alkylation methods because ethylation with STEB can be performed in aqueous media, and it suffers less interference than the hydride generation method. 

Preconcentration techniques play an important role for the extraction and determination of low OTC concentrations in real environmental samples, especially in water samples because of their low solubility. Solid–liquid extraction (SLE) is the most widely used technique for the preconcentration of OTCs.

  However, it is time-consuming (it requires stirring, shaking, sonication and centrifugation) and it involves the use of large volumes of organic solvents, as well as complexing agents.

  Other techniques used include supercritical fluid extraction (SFE),

  and solid-phase microextraction (SPME),

  which have been developed in recent years. SPME is a solvent-free technique with the advantages of simplicity and low cost. In addition, in the headspace mode (HS), the fibre is protected from matrix compounds, the extraction of non-volatile compounds is avoided and the extraction process can be faster.

  Recently, a review about TBT residues determination in marine environment was published.

In this article we propose a methodology that consists of headspace solid-phase microextraction of the OTCs, previously ethylated with STEB, followed by gas chromatography and flame ionization detection (HS-SPME–GC-FID). We have mainly focused on the evaluation of the parameters of derivatization of OTCs and HS-SPME to reduce the analysis time,

  and the costs, as well as improving the method sensitivity with respect to previously published works.

  The usefulness of the method was demonstrated by evaluating OTC pollution in marine water and sediment samples from Gandia's port area (Valencia, Spain); it is an interesting study because this port combines commercial, fishing and sport areas. 

All the reagents were of analytical grade. Monobutyltin (MBT), dibutyltin (DBT), tributyltin (TBT) and sodium tetraethylborate (STEB) were obtained from Aldrich (Stenheim, Germany). Acetonitrile and methanol were of HPLC grade from J.T. Baker (Deventer, Holland). Hydrochloric acid and sodium acetate trihydrate were obtained from Scharlau (Barcelona, Spain). Stock solutions of organotin compounds were prepared in methanol. Working mixture solutions were prepared by dilution with deionized water. All solutions were stored in the dark at 4 °C. 

The STEB solution [2% (m/v)] was prepared in a glovebag under dry nitrogen, in a degasified 0.1 M sodium hydroxide solution.

  STEB solution aliquots were frozen (stable during a month) until the analysis. 

The SPME holder and fibres [100 µm thickness polydimethylsiloxane (PDMS)] were obtained from Supelco (Bellefonte, Pennsylvania, USA). The gas chromatographic system was equipped with flame ionization detector (FID) (Hewlett-Packard 6890 Series, Little Falls, Delaware, USA). The injection mode was splitless and the temperature was set at 250 °C. The column was a 30 m x 0.32 mm i.d. x 0.25 µm film thickness HP-5 (Agilent Techonologies). The oven was programmed from 40 °C (1 min) at 20 °C/min to 220 °C (3 min) and the carrier gas was helium at a constant pressure of 80 kPa. The detector temperature was set at 250 °C. An ultrasonic cleaner bath and a centrifuge were obtained from J.P. Selecta (Barcelona, Spain). 

 Working aqueous standard solutions were prepared by adding the appropriate volume of the aqueous mixture solution (15 µg/L of each analyte) to 19 mL of miliQ-Water in 40 mL amber vials with silicon septa (Supelco). Then, 1 mL of sodium acetate buffer (pH 5; 0.15 M) and 10 µL of STEB [2% (m/v)] solution were added to derivatize the analytes. The standard concentrations obtained were between 0.05–15 µg/L for each OTC. After 5 min stirring at 800 rpm, the fibre was exposed to the headspace for 15 min at 40 °C. Then, the fibre was withdrawn into the needle of the holder and it was placed into the GC injector to perform the derivatives desorption (3 min). 

 To evaluate the pollution by OTCs in Gandia's port area, three marine water samples were collected from different points of the port. They were filtered through 0.45 µm syringe filters (Teknokroma, Barcelona, Spain). After that, the same analytical procedure described above for aqueous standards was applied to the samples. Each sample was analysed in triplicate.  

Analytical procedure for sediment samples

 Working aqueous standard solutions were prepared by adding 300 µL of the methanolic OTC mixture solutions (5–0.5 µg/mL for MBT, 0.5–0.05 µg/mL for DBT and 0.5–0.05 µg/mL for TBT) to 3 mL of an extractant mixture (concentrated hydrochloric acid–acetonitrile, 1:1, v/v). The mixture was sonicated for 15 min. Then, the pH of 2 mL mixture was fixed at 5 by adding the appropriate volume of sodium hydroxide (10 M) and sodium acetate buffer (pH 5, 1.5 M) solutions to 19 mL. The resulting solution was stirred (40 °C for 3 min). After that, 20 µL of STEB [2%, (m/v)] solution was added to volatilize the derivatives for 5 min, and then the fibre was 5–50 µg/L for MBT and between 0.5–5 µg/L for DBT and TBT.  

 Three marine sediment samples (Gandia's port area) were analysed. 0.5 g of sediment was accurately weighed and then 3 mL of the extractant mixture was carefully added. The mixture was sonicated during 15 min and centrifuged at 3200 rpm for 5 min. After that, the same analytical procedure described above for aqueous standards was applied to the samples. Each sample was analysed in triplicate. 

Optimization of the derivatization and the headspace solid-phase microextraction

The HS-SPME was selected as sampling technique because of its suitability in the determination of butyltin compounds derivatizated with STEB. The parameters evaluated were: STEB solution volume, reaction pH, absorption time, headspace volume, procedure temperature and desorption time. All these parameters were tested for an aqueous standard solution of 15 µg/L of each analyte. 

Before SPME, the analytes were derivatized with STEB using volumes between 10–500 µL. Lower STEB volumes were not assayed to ensure a fifty-times excess over the analyte concentration. It was observed that reducing the STEB volume, the derivatives signals increased and the secondary STEB products were removed. As a result, 10 µL was the optimum volume for water samples and 20 µL for sediment samples. The presence of other organometallic compounds in sediment samples would explain the necessity of increasing the STEB volume in this kind of samples. It is important to note that the STEB concentration used in this work was significantly reduced compared with previous works.

Then, the reaction pH was tested between 3 and 9. At pH ≤ 4 OTCs did not react with STEB and at pH ≥ 7 TBT peak slightly increased, but MBT and DBT peaks decreased. Therefore, pH 5 was selected as optimum to perform the simultaneous derivatization of the three analytes. It was fixed by means of a sodium acetate buffer. These results are in accordance to that obtained in the bibliography.

The volatilization of the derivatives required 5 minutes. After that, the fibre was exposed to the headspace and the absorption time was ranged from 5–25 minutes. It was observed that the signals increased until 15 min and no improvement was observed for longer times (see Figure 1). So, 15 minutes was selected as the optimum absorption time. 

Three vials (2, 20 and 40 mL) were used to study the optimum headspace volume (1, 10 and 20 mL, respectively). All the chromatographic peaks increased with the headspace volume: a headspace volume of 20 mL provided peak areas between 10 and 20 times higher than a headspace of 1 mL. Therefore, 20 mL was selected for further work. 							

This article describes a fast, simple and clean procedure to determine three organotin compounds (monobutyltin, dibutyltyin and tributyltin) in environmental samples.

LCGC Europe-11-01-2009

Hyphenated Electronic Nose Technique for Aroma Analysis of Foods and Beverages

A Novel Sample Preperation Approach to Increase the Throughput of Pesticide Analysis by LC–MS–MS

Icons of Chromatography: Harold McNair

Hyphenated Electronic Nose Technique for Aroma Analysis of Foods and Beverages

Commercialization of LC–MS: The Second Decade

Determining Organotin Compounds in Marine Water and Sediment Samples using HS-SPME–GC–FID

Method or System?

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