Metabolomics (2012) 8:5063 DOI 10.

1007/s11306-011-0283-6

ORIGINAL ARTICLE

Development of a gas chromatography/mass spectrometry based metabolomics protocol by means of statistical experimental design
Anders P. H. Danielsson Thomas Moritz Hindrik Mulder Peter Spegel

Received: 11 August 2010 / Accepted: 28 January 2011 / Published online: 11 February 2011 Springer Science+Business Media, LLC 2011

Abstract Metabolomics is a growing research eld where new protocols are rapidly developed and new applications discovered. Common applications include biomarker discovery and elucidation of drug metabolism. However, the development of such protocols rarely includes a systematic optimization followed by validation with real samples. Here a GC/MS-based protocol using methoximation followed by silylation with N-tert-butyldimethylsilyl-N-methyltriuoroacetamide (MTBSTFA) for analysis of blood plasma metabolites is thoroughly developed and optimized from derivatization to detection with statistical design of experiments (DOE). Validation was performed with blood plasma samples and proved the methodology to be efcient, rapid and reliable with a total of 51 analyses performed in 24 h, with linear responses, low detection limits and good precision. The obtained chromatograms were much cleaner, due to the absence of glucose overloading, and the data was found to drift less with MTBSTFA derivatisation than with MTBSTFA derivatisation.
Electronic supplementary material The online version of this article (doi:10.1007/s11306-011-0283-6) contains supplementary material, which is available to authorized users.
A. P. H. Danielsson H. Mulder P. Spegel (&) Unit of Molecular Metabolism, CRC 91:12, Entrance 72, UMAS, Lund University Diabetes Centre, Clinical Research Centre, SE-205 02 Malmo, Sweden e-mail: Peter.spegel@med.lu.se A. P. H. Danielsson Analytical Chemistry, Faculty of Engineering LTH, Lund University, Lund, Sweden T. Moritz Umea Plant Science Center, Swedish University of Agricultural Sciences, Umea, Sweden

Keywords Metabolomics Gas chromatography Mass spectrometry MTBSTFA Blood plasma DOE

1 Introduction Metabolomics aims for an unbiased quantication of all metabolites in a biological sample. As the metabolite levels reects both genetic and environmental effects, metabolomics is an exceptionally useful technique for discovery of potential biomarkers for environmental factors, medical treatments, and diseases (Chorell et al. 2009; Fiehn 2002). However, the metabolome comprises a very large and heterogeneous group of metabolites with concentrations differing several orders of magnitude. Thus, great care must be taken in the development of metabolomics methods to attain a wide selectivity, high efciency and high sample throughput while at the same time avoid introducing excessive biases. To date, no single analytical method exists that is capable of simultaneously measuring all members of the metabolome. Several techniques, including nuclear magnetic resonance spectroscopy (NMR) (Zhang et al. 2008), near-infrared spectroscopy (NIR) (Cozzolino et al. 2006), gas chromatography (GC) (Fiehn 2008; Jiye et al. 2005), liquid chromatography (LC) (Zelena et al. 2009), and capillary electrophoresis (CE) (Lapainis et al. 2009), with the latter three being coupled to mass spectrometry (MS), have been applied in metabolomics. Out of these techniques, the combination of a chromatographic separation with mass spectrometric detection offers a somewhat higher sensitivity than the pure spectroscopic techniques, although sample preparation generally becomes more complicated. Among these techniques, GC/MS, applied in the present investigation, offers the highest separation

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efciency and also yields the cleanest chromatograms due to the absence of interfering peaks from peptides, proteins and other large non-volatile substances. Metabolite derivatisation is an important element of GC/ MS-based metabolomics. Great care must be taken in the development of derivatisation conditions, as it will inuence both the limit of detection (LOD), the sensitivity, and the selectivity of the method. The most common derivatization procedure applied to GC/MS-based metabolomics involves a two-step reaction, comprising a methoximation step and a silylation step (Gullberg et al. 2004). Methoximation reduces the number of sugar tautomers, thus reducing the number of peaks generated from a single sugar metabolite and thereby enhances both separation and quantication (Asres and Perreault 1997; Fiehn et al. 2000; Schweer 1982). Additionally, methoximation protects a-ketoacids from decarboxylation (Fiehn et al. 2000; Tam and Normanly 1998). The silylation reagent most commonly applied in metabolomics, N-methyl-N-trimethylsilyltriuoroacetamide (MSTFA), yields volatile trimethylsilyl (TMS) derivatives of a vast number of metabolites including large multifunctional metabolites such as sugars and their derivatives (Begley et al. 2009; Danielsson et al. 2010; Gullberg et al. 2004). Besides MSTFA, N-tert-butyldimethylsilyl-N-methyltriuoroacetamide (MTBSTFA), has also been applied in metabolomics. The large size of this silylation reagent reduces both the yield and the volatility of glucose resulting in a complete elimination of glucose and other large carbohydrates from the analysis. In blood plasma analysis, glucose is found at high concentrations which generally severely overloads the column and complicating detection and quantication of other metabolites eluted in the same retention interval. Furthermore, the tert-butyldimethylsilyl (TBDMS) derivatives have been shown to offer both improved hydrolytic stability and improved thermal stability over the corre sponding TMS derivatives (Rodrguez et al. 2003; Yu et al. 2007) and generate an intense [M-57]? mass fragment (Fiehn et al. 2000) providing additional information for identication. Although MTBSTFA derivatization has several unique features in comparison to MSTFA derivatization in metabolomics analysis, the derivatization and chromatographic parameters for this derivatization reagent have not yet been thoroughly optimized. In contrast, there are several developed metabolomics protocols based on MSTFA (Begley et al. 2009; Danielsson et al. 2010; Gullberg et al. 2004; Jiye et al. 2005). These protocols are aimed at plant cells (Gullberg et al. 2004), adherent cell cultures (Danielsson et al., 2010) and blood plasma (Begley et al. 2009; Jiye et al. 2005). In the present investigation, a metabolomics protocol for blood plasma analysis based on methoximation and

MTBSTFA derivatization followed by GC/MS is developed and optimized using statistical design of experiments (DOE) (Araujo and Brereton 1996a, b, c). DOE is a multivariate statistical optimization tool that enables efcient experimental planning and identication of both linear effects, interaction effects and higher order non-linear effects with a minimum number of experiments. The twostep derivatization method, injection onto the GC, the chromatographic settings and the mass spectrometer settings were optimized aiming at enhancing the limit of detection (LOD), the sample throughput and the peak capacity. Finally, the developed method was applied to a set of blood plasma samples, and the performance and validity of the developed protocol was assessed.

2 Experimental 2.1 Chemicals Methoxyamine hydrochloride, N-tert-butyldimethylsilylN-methyltriuoroacetamide and N-methyl-N-trimethylsilyltriuoroacetamide were from Aldrich (Steinhein, Germany). Pyridine, heptane, methanol, methyl stearate and the alkane standard mixtures (C8C20 and C21C40) were from Fluka (Buchs, Switzerland). The stable isotope-labeled internal standards; 13C315N alanine, 13C4-succinate, 13C6 phenylalanine, 13C3-serine, 2H7-cholesterol, 13C16-palmitate and 13C5-a-ketoisovalerate were from Cambridge Isotope Laboratories, Inc. (Andover, MA). The stable isotopelabeled standards; 13C915N-tyrosine and 13C18-oleate were from Isotec (Sigma-Aldrich, St. Louis, MA). Water was puried using a Purelab Ultra water purication system (Elga, Gothenburg, Sweden). The metabolites used in the study were all purchased from Sigma-Aldrich. 2.1.1 Blood samples The subjects, four males and two females, were all Caucasian and non-obese (body mass index 22.4 2.4 ranging from 18 to 24). They were 33.8 6.9 (ranging from 28 to 46) years of age, non-smokers and all healthy. None were taking any prescribed medication. Blood samples were drawn after an overnight fast and the subjects were all sitting down during the whole procedure. Blood samples were immediately centrifuged at ?4C and plasma was separated. The blood plasma was subsequently pooled and frozen on dry ice. The samples were kept at -80C until analysis. 2.2 Instrumentation GC/MS was performed on an Agilent 6890N gas chromatograph (Agilent, Atlanta, GA) equipped with an

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Agilent 7683B autosampler (Agilent) and coupled to a Leco Pegasus III TOFMS electron impact, time-of-ight mass spectrometer (Leco Corp.). Two columns were used in the study, a 10 m (ID 180 lm, phase thickness 0.18 lm) and a 30 m (ID 250 lm, phase thickness 0.25 lm), both DB5-MS (J&W Scientic, Folsom, CA). The initial method employed a splitless injection with the injector temperature set to 270C with a purge vent time of 60 s. The ow rate through the column was 1 ml/min, with a three-step temperature program starting with an initial isocratic temperature of 70C kept for 2 min, followed by a gradient rate of 30C/min reaching a nal temperature of 320C kept for a duration of 5 min. The ionization voltage was set at 70 eV and the mass spectra were recorded between 50 and 800 m/z, throughout the study. Data were acquired using the Leco ChromaTof software v. 3.31 (Leco Corp.). Retention indexes (RIs) were calculated based on the elution times of a homologous series of n-alkanes (C8-C40). 2.3 Metabolite cocktail 36 metabolite standards were derivatized and analyzed individually to construct a database (NIST MS Search 2.0) containing retention indexes and mass spectra. The metabolites were selected to cover a broad range of functionalities, metabolic pathways, and retention indexes. Next, a polar and a non-polar model mixture were created from the 36 metabolite standards, isotope labeled internal standards and methyl stearate. The combination of both these standard mixtures is herein referred to as the metabolite cocktail (Table 1) and is used throughout the model optimizations conducted in this paper. 2.4 Raw data pre-treatment and experimental designs Peak integration was performed either directly in Leco ChromaTof 3.31 or after export as NetCDF les to MATLAB 7.0 (Mathworks, Natich, MA) using a hierarchical multivariate curve resolution (H-MCR) script (Jonsson et al. 2006). Peak identication was performed with NIST MS Search 2.0 using mass spectra and retention index from several libraries. For the TBDMS derivatives the database constructed here was used and all metabolites were quantied using only their [M-57]? mass fragment (Table 1). For the TMS derivatives, the NIST mass spectra library, a library developed at the Max Planck Institute in Golm (http://csbdb.mpimp-golm.mpg.de/), and libraries developed in house, both at Umea Plant Science Centre (UPSC) and Lund University Diabetes Centre (LUDC) were used. Experimental designs were created in ModdeTM 8.01 (Umetrics, Umea, Sweden). All responses were centered

and scaled to unit variance (UV) and projections to latent structures (PLS) were used to calculate the models. Prior to evaluation, the models were optimized in a ve-step procedure aimed at reaching the highest possible crossvalidated predictive power (Q2Y), which compares the cross-validated residuals to the total residual for the model (Wold 1978). In the rst step, normalization, when applicable, of the responses was performed. In the second step, responses deviating from the normal distribution were transformed, to avoid erroneous inuence on the model. In the third step, responses with a replicate variance larger than half of the total variance of the response were considered irreproducible and were therefore deleted. In the fourth step responses exhibiting considerable lack-of-t, i.e. responses where the model error variance was equal or larger than the replicate variance, were deleted. In the fth and nal step, linear factors, factor interactions and quadratic factors which reduced Q2Y were identied. These factors and interactions that mainly modeled noise were considered insignicant for the model and consequently deleted. The models were evaluated from coefcient and contour plots. Orthogonal projections to latent structures (OPLS) was performed on centered and UV-scaled data in Simca P? 12.0 (Umetrics, Umea, Sweden). 2.5 Derivatization design A D-optimal design with a quadratic model consisting of 8 factors was constructed to explore the MTBSTFA derivatization conditions using the metabolite cocktail as sample (Table 2). Specically, the temperature, duration and solvent composition used for both methoximation and silylation were varied. The aim was to obtain the maximum yield of metabolite derivatives, and minimum number of artifacts in the shortest possible derivatization time. In total 45 runs were performed including three center point runs. The responses were the peak areas for the metabolites in the metabolite cocktail, normalized to the peak area of the underivatizable methyl stearate, and the number of artifact peaks detected by Leco ChromaTof in the chromatograms. The artifact peaks were quantied as the number of non-metabolite peaks between the rst and last eluted metabolite. 2.6 Injection design A three-level full factorial design with a quadratic model was generated to nd the optimum settings for the injector using the metabolite cocktail as sample (Table 2). The design included 2 factors and the responses consisted of the peak areas of the metabolites in the cocktail. The injection volume was left unchanged at 1 ll throughout the study, to

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Development of a Metabolomic Protocol Table 1 Metabolite and isotope labeled standard derivatives investigated in the present study with their [M-57]? fragments and retention indexes (RIs)

53 [M-57]? 174 188 207 207 202 202 261 260 264 246 288 302 302 290 293 289 286 243 257 302 320 390 346 404 336 419 298b 416 406 453 313 325 484 484 484 431 339 440 591 591 585 375 347 275 411 505 443

Analyte Pyruvate MEOX TBDMS 2-ketobutyrate MEOX TBDMS

13 13

Abbreviation PYR 2KBA AKIX1 AKIX2 AKI1 AKI2 LAC ALA ALAX GLY VAL LEU ILE THR2 SUCX SUC PRO FA11 FA12 T4HP2 MET SER AKG THR3 PHE MAL MEST T4HP3 CYS PEP FA16 FA17 GA3P DHAP1 DHAP2 LYS FA18U HIS CIT ISOC 3PGA TRP SERO NAHT FA23 L5HT CHO

RI 1254.7 1305.5 1321.6 1336 1321.6 1336 1493.1 1541.4 1541.5 1562.1 1662.4 1702.1 1735.7 1747.5 1761.1 1761.1 1775.6 1783.8 1884.3 1970.1 1976.2 1998.5 2011.7 2032.4 2103.5 2117.8 2134.5 2196 2217.1 2239.6 2288.3 2389.5 2346.5 2370.2 2390.2 2390.4 2468.6 2609.6 2632.2 2647.2 2647.6 2708.9 2720.5 2775.7 2999.2 3187.6 3493.8

C5-a-ketoisovalerate MEOX TBDMS peak1 C5-a-ketoisovalerate MEOX TBDMS peak2

a-ketoisovalerate MEOX TBDMS peak1 a-ketoisovalerate MEOX TBDMS peak 2 Lactate 2TBDMS Alanine 2TBDMS
13

C3-15N alanine 2TBDMS

Glycine 2TBDMS Valine 2TBDMS Leucine 2TBDMS Isoleucine 2TBDMS Threonine 2TBDMS
13

C4-succinate 2TBDMS Succinate 2TBDMS Proline 2TBDMS Undecanoate TBDMS Dodecanoate TBDMS (Laurate) Trans-4-hydroxyproline 2TBDMS Methionine 2TBDMS Serine 2TBDMS Alpha ketoglutarate MEOX 2TBDMS Threonine 3TBDMS Phenylalanine 2TBDMS Malate 3TBDMS Methyl Stearate
a

Trans-4-hydroxyproline 3TBDMS Cysteine 3TBDMS Phosphoenolpyruvate 3TBDMS Hexadecanoate TBDMS (Palmitate) Heptadecanoate TBDMS (Margarate) Glyceraldehyde 3-phosphate MEOX 3TBDMS Dihydroxyacetone phosphate MEOX 3TBDMS peak1 Dihydroxyacetone phosphate MEOX 3TBDMS peak2 Lysine 3TBDMS (9Z)-Octadec-9-enoate TBDMS (Oleate) Histidine 3TBDMS Citrate 4TBDMS Isocitrate 4TBDMS 3-Phosphoglycerate 4TBDMS Tryptophan 2TBDMS Serotonin 2TBDMS N-Acetyl 5-hydroxytryptamine TBDMS Tricosanoate TBDMS
a b

Underivatizable standard Quantication mass

L-5 Hydroxytryptophan 3TBDMS Cholesterol TBDMS

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54 Table 2 Summary of the models for derivatization and GC/MS optimization Study Methoximation Design and model D-optimal design, quadratic model Factors Amount Acetonitrile Methoximation (MAC)a Methoximation Temperature (MTE) Methoximation Duration (MDU) Silylation Amount Heptane in Silylation (SHP)b Amount Acetonitrile in Silylation (SAC)b Amount MTBSTFA in Silylation (STB)b Silylation Duration (SDU) Silylation Temperature (STE) Injection Three-level full factorial design, quadratic model Injector Temperature (IJT) Injector Purge Vent Time (PVT) Low 0% 20C 1h 0% 0% 25% 0.5 h 20C 200C 5s High 25% 80C 17 h 75% 75% 100% 4h 100C 320C 115 s 115 s 6 min 90C 40C/ min 3 ml/ min

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Responses Peak areas Artifact peaks

Peak areas

Chromatography Central composite face (CCF) design, Injector Purge vent time (PVT) 45 s quadratic model Initial Gradient Temperature 2 min Duration (ITD) Initial Gradient Temperature (ITE) Temperature Gradient Rate (TGR) Volumetric Flow Rate (VFR) 60C 10C/ min 1 ml/ min

Peak capacity for valine (PxVc) Peak capacity for cholesterol (PxCc) Peak capacity for (9Z)-Octadec9-enoate (PxOc) Average peak capacity (PxMc) Total analysis time (Txc) Sample throughput per 24 h (STxc) Asymmetry factor for valine (AxVc) Peak height for cholesterol (HxC)c

Mass spectrometry
a b c

Three-level full factorial design, quadratic model

Data Acquisition rate (ACQ) Ion source temperature (IST)

10 Hz 50 Hz [M-57] ? Peak area 130C/ 250C/ Ratio of low and high m/zmin min fragment

Pyridine is used as the additional solvent in the methoximation step Formulation factor x refers to the column length and has a value of either 10 or 30 m

minimize liner contamination that is expected from excessive injection of non-metabolite matrix from complex biological samples. 2.7 Gas chromatography design A three-level central composite design (CCF) with a quadratic model was calculated for each column. The two models were then merged to facilitate the interpretation. For each column 5 factors and 8 responses were investigated using the metabolite cocktail as sample (Table 2). The aim was to optimize the separation efciency, peak symmetry, detection limits and analysis time. To achieve this, all parameters of the temperature program were

optimized, including the initial temperature and its duration and the temperature gradient rate. The nal temperature and its isocratic duration were maintained at 320C and 5 min, respectively, due to their insignicant inuence on the selected responses. In addition, also the volumetric ow rate was varied to minimize band broadening and hence optimize the separation efciency. The injector purge vent time was included also in this design to allow for the investigation of sample loading effects on the chromatographic performance. To obtain a representative response for the separation efciency, the peak capacity was calculated from the peak width at 10% of the peak height (w0.1) for three analytes spanning a broad retention window, valine 2TBDMS,

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oleate TBDMS and cholesterol TBDMS, representing the early, intermediate and late portion of the chromatogram, respectively, according to Eq. 1. (Pous-Torres et al. 2008) trcho trval Pc 1 1 w0:1 The elution times for cholesterol TBDMS (tr(chol)) and valine 2TBDMS (tr(val)) dened the retention window. Additionally an average peak capacity was calculated. The asymmetry factor (Kirkland, 1977) (As) for valine 2TBDMS was included as a response as this peak was observed to suffer from asymmetric band broadening, whereas this type of peak distortion was weaker or absent for later eluting metabolites. As was determined according to Eq. 2, As B10% A10% 2

where A10% and B10% are the distances at 10% of the peak height, measured from a line perpendicular to the baseline from the peak apex, to the peak front and back respectively. The noise level was not found to be signicantly affected by the parameter settings and therefore the peak height of cholesterol TBDMS was included as a response reecting the method detection limit. Cholesterol TBDMS was chosen as it is the last eluted metabolite derivative in our cocktail and should therefore be most affected by the longitudinal band broadening which is of primary concern in GC separations. 2.8 Mass spectrometer design A three-level full factorial design with a quadratic model was generated to nd the optimal settings of the mass spectrometer using the metabolite cocktail as sample (Table 2). The responses studied were the area of the [M-57]? fragment peak and the ratio between the [M-57]? fragment peak area and a low weight high intensity fragment peak area. Peak areas of the [M-57]? response were normalized using the [M-57]? peak area showing a close to zero reproducibility as it was assumed that for such a peak the instrumental variability considerably overpowered the systematic variation caused by the varied factors. 2.9 Method validation Pooled blood plasma from 6 healthy individuals was diluted to relative concentrations of 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 (v/v, plasma/plasma ? water). A total of 12 measurements from four preparations measured in triplicates were performed at each concentration to assess both instrumental and preparation variability. The total volume of diluted plasma in each extraction tube was 100 ll. Prior

to extraction 3.75 lg of each stable isotope labeled standard were added to each of the 24 tubes. Extraction of the metabolites was performed according to a previously developed protocol (Jiye et al. 2005). To each tube 900 ll methanol/water mixture (8:1 v/v) was added and the samples were rapidly mixed and kept on ice for 10 min. All tubes were then vigorously extracted using a multitube vortexer (VX-2500 Multi Tube Vortexer, VWR, West Chester, PA) operating at 30 Hz for 3 min. Subsequent the extraction tubes were centrifuged for 10 min at 175309g at 4C. 200 ll of the supernatant was then transferred to a GC vial and evaporated to dryness (miVac Duo concentrator; Genevac, Ipswich, UK). Samples were derivatized and analyzed with the optimized protocol developed here. The results were evaluated with regards to linear range, intra-day precision, limit of detection (LOD) and limit of quantication (LOQ). The peak areas were normalized to the peak areas of the corresponding stable isotope labeled standard, if available. If no corresponding stable isotope labeled standard was available, a stable isotope-labeled standard with similar properties was selected (only performed for determination of linear range and precision). From the normalized peak areas, a linear model was created and analyzed in Modde 8.0.1. The models were tested for lack-of-t and also cross-validated to thoroughly determine the linear range. The precision was calculated as the average relative standard deviation (RSD) for 10 sample runs at 2 different concentrations; 0.1 and 0.4 (v/v, plasma/plasma ? water). The LOD and LOQ were estimated as 3 and 10 times, respectively, the standard deviation for the signal-to-noise ratio (S/N) for a 10 times diluted sample of pooled blood plasma. 2.10 Sample drift comparison Unnormalized data from the method validation runs were further analyzed using OPLS to relate the peak area to the sample run order. An OPLS model was accordingly created for blood plasma samples derivatized with MSTFA according to a protocol developed elsewhere (Jiye et al. 2005) aiming at comparing the drift pattern using these two derivatization protocols.

3 Results and discussion 3.1 Derivatization Initially, a PLS model was calculated from the normalized peak areas of all metabolites in the cocktail. In the loading plot the metabolites were found to cluster roughly

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according to their chemical functionality. However, the model predictive power, Q2Y, was poor which probably was due to the heterogeneity of the responses. Guided by the chemical functionality of the metabolites and their positions in the loading plot, groups of metabolites showing similar behavior were constructed and modeled. All models are summarized in Supplementary material S1. From the linear effects of the three studied methoximation step factors, it was obvious that the settings of the methoximation duration and temperature had the largest inuence on the derivatization yield (Supplementary material S1). For most metabolites, the methoximation duration and temperature were negatively correlated to the peak areas. Furthermore, the methoximation temperature was positively correlated to the number of artifact peaks. Generally, addition of acetonitrile to the methoximation solution did not improve the yield. Next, the linear effects in the silylation step were investigated. It was found that the most inuential factors were the silylation duration and temperature. The silylation temperature was positively correlated with 3-phosphoglycerate 4TBDMS and several amine containing analytes, whereas it was negatively correlated with succinate 2TBDMS, containing two carboxylic acid functionalities. The silylation duration was positively correlated with cysteine 4TBDMS, cholesterol TBDMS and serotonin 2TBDMS but was negatively correlated with isocitrate 4TBDMS, lactate 2TBDMS, succinate 2TBDMS and fatty
Fig. 1 Contour plots describing the effect of methoximation temperature and duration on the normalized peak areas (as numerical values in the plots) for (a) 2-ketobutyrate MEOX TBDMS, (b) a-keto isovalerate MEOX TBDMS, (c) phosphoenolpyruvate 3TBDMS, and (d) histidine 3TBDMS. These contour plots clearly illustrate the complexity involved in optimizing a derivatization method for metabolome analyses

acid TBDMS esters. Thus, a low silylation time and temperature was benecial for carboxylic acids, whereas the opposite improves the yield of amines. The composition of the silylation solvent was generally insignicant. Next, the contour plots were evaluated to characterize the effects of signicant factor interactions and quadratic effects. For the modications of the methoximation solvent, the a-keto acids pyruvate MEOX TBDMS, 2-ketobutyrate MEOX TBDMS and a-ketoglutarate MEOX 2TBDMS showed a negative correlation to the interaction between the added amount of acetonitrile and the methoximation temperature. A moderate temperature of 4055C and no acetonitrile was optimal. Interestingly, the amount of acetonitrile in the methoximation solvent also showed a dependence on the silylation temperature for both leucine 2TBDMS and the number of artifact peaks. The yield for leucine 2TBDMS and the number of artifacts increased with a low fraction of acetonitrile and a high silylation temperature. Only trans-4-hydroxyproline 3TBDMS benetted from a high fraction acetonitrile. Although there are a few benets of using more complex reaction mixtures, the absence of strong effects motivates their removal as a much simplied method will be the result. Therefore, the amount of pyridine in the methoximation step and the amount of MTBSTFA in the silylation step was set to 100%. The interaction between the methoximation time and temperature was signicant for numerous metabolites and the contour plots generally showed that the yield was

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increased with settings in the lower range for both factors. However, there were some exceptions. As noted above the a-keto acids are sensitive to the settings of methoximation temperature and only a-ketoisovalerate MEOX TBDMS beneted from a lower setting while the rest in this group had local maxima for the methoximation temperature in the range of 3570C (Fig. 1a, b). Phosphoenolpyruvate 3TBDMS followed the general trend with a substantial decrease in peak area with higher settings of the methoximation time and temperature (Fig. 1c). Even so, the methoximation temperature was set to room temperature, approximately 20C, which was optimal for most metabolites and resulted in only moderate deviations from the optimum yield for some of the a-keto acids. With respect to the methoximation settings, the majority of metabolites beneted from a short duration, whereas two amino acids with amine-containing side chains, histidine 3TBDMS (Fig 1d) and tryptophan 2TBDMS, were found to have maxima between 5 and 10 h. The settings of the methoximation step has previously been shown to affect also metabolites lacking aldehyde or ketone functionality. (Gullberg et al. 2004) Speculatively, this effect may be related to the solubility of the amino acids in the derivatization solvents and reagents. The methoximation duration was set to 4 h, which allowed for a sufcient yield also for histidine 3TBDMS and tryptophan 2TBDMS. Evaluation of the interactions of the silylation temperature with other factors supports the employment of a high
Fig. 2 PLS loading plot from the injection study, showing the inuence of the factors (lled triangle) and factor interactions (lled diamond) on the metabolite derivatives peak areas (lled circle). The left contour plot shows the behavior of the thermally unstable phosphoenolpyruvate 3TBDMS and the right contour plot shows the behavior of the late eluting cholesterol TBDMS. PVT = purge vent time and IJT = injector temperature
0,8

silylation temperature. This factor was therefore set to 100C, which is within the range of previous studies that have reported 60120C (Birkemeyer et al. 2003; Buscher et al. 2009; Ewald et al. 2009; Fiehn et al. 2000). The silylation duration was set to 170 min, which allowed for sufcient yield of amines, carboxylic acids and cholesterol TBDMS. Quantication of metabolites yielding several different derivatives is difcult as the derivatives may have very different properties and the ratio between derivatives may vary with time (Kanani and Klapa 2007). However, of all investigated metabolites, only two generates multiple derivatives in the form of unsilylated and monosilylated amines. The levels of the unsilylated amines were very low and likely not affecting the quantication of these amino acids to any greater extent. For the same reason, these derivatives could not be accurately modeled due to their low signal-to-noise ratios. With MSTFA, monosilylated and disilylated amines are commonly observed. The partial derivatization of threonine and trans-4-hydroxyproline observed in the present investigation is probably caused by steric hindrance from hydroxyl bound TBDMS which decreases the yield of derivatization of the amine group. The present models support this order of derivatization, with carboxylic acids generally reacting fastest, followed by hydroxyls and amines. Thus, in comparison with TMS derivatization, TBDMS derivatization may reduce both the formation of multiple derivatives and reduce the

0,6

0,4

PVT
0,2

wc[2]

-0,0

-0,2

PVT*PVT

-0,4

IJT*PVT
-0,6

-0,8 -0,2 0,0 0,2

IJT
0,4 0,6 0,8 1,0

wc[1]

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desilylation rate, due to its higher resistance to hydrolysis (Rodrguez et al. 2003; Yu et al. 2007). 3.2 Injection A PLS model was calculated using all metabolite peak areas as responses. The model yielded a total explained variation of 95% (R2Y = 0.954) and a cross-validated predictive power of 93% (Q2Y = 0.934) with 0.846 B R2Y B 0.982 and 0.933 B Q2Y B 0.967 for all individual responses (Supplementary material S2). The loading scatter plot for the rst two PLS components (Fig. 2), the contour plots and the coefcient plot for each of the analytes showed that, for all analytes the peak area was positively correlated with the purge vent time. At this point, an injector purge vent time of 115 s was chosen as optimum and possible column overloading effects will be assessed in the optimization of the GC-settings. The effect of increased injector temperature was for most analytes positively correlated to the yield and there was also a clear trend towards an increasingly positive effect of injector temperature with increasing retention time. For the three latest eluting analytes, having low volatility, the injector temperature was in fact the dominating effect. However, for phosphoenolpyruvate 3TBDMS, threonine 2TBDMS and trans-4-hydroxyproline 2TBDMS, an increase in injector temperature decreased the peak areas, suggesting a degradation of these compounds at higher temperatures. This is illustrated in Fig. 2 for phosphoenolpyruvate 3TBDMS in comparison to cholesterol TBDMS. For these amino acids only the partially silylated 2TBDMS derivatives demonstrated this behavior in contrast to their more abundant 3TBDMS derivatives, clearly showing the improved thermal stability of silylated amines over unsilylated amine. The difference in yield in the investigated temperature interval was for some analytes very large. Increasing the injector temperature from 200 to 320C increased the peak area of cholesterol TBDMS and decreased the peak area for phosphoenolpyruvate 3TBDMS with 90 and 80%, respectively. An injector temperature of 270C was found being a good compromise, resulting in an approximately 45% reduction in peak area of these two analytes with the remaining metabolites close to their maximum values. 3.3 Chromatography A PLS model was calculated, displaying a total explained variation of 93% (R2Y = 0.932) and a cross-validated predictive power of 88% (Q2Y = 0.884) with 0.719 B R2Y B 0.989 and 0.564 B Q2Y B 0.979 for all studied responses (Supplementary material S3).

Fig. 3 Contour plots describing the inuence of the injector purge vent time and temperature gradient rate on the average peak capacity for the 30 m column (a) and 10 m column (b). All factors not presented in the plots are assigned center values

It was found that an increase in injector purge vent time greatly reduced the peak capacity for the shorter column, whereas the effect on the longer column was much smaller (Fig. 3). The main factors affecting the peak asymmetry factor on the 10 m column were purge vent time and ow rate, whereas these factors were insignicant on the 30 m column. It was also observed that the range for the peak asymmetry factor (Kirkland 1977) calculated for valine 2TBDMS was much greater on the 10 m column (0.4 B As B 1.4) than on the 30 m column (0.7 B As B 1.2). The peak height of cholesterol TBDMS was increased for both columns primarily by an increase in temperature gradient rate and/or purge vent time. However, improving this response by increasing these parameters would severely reduce the peak capacity and yield asymmetric peaks, especially on the shorter column. Although the 10 m column may generate faster analyses, only a 17% increase in the number of analyses in 24 h was obtained compared to the 30 m column.

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59

Due to a greatly higher average peak capacity, superior peak symmetry and decreased sensitivity to the factor settings only the 30 m column was further optimized. The purge vent time was set to 115 s, to optimize the chromatography for a high sample load. The ow rate and the gradient rate affected both the analysis time and the peak capacity. The effect of the ow rate on the analysis time was moderate whereas its effect on peak capacity was very pronounced. The ow rate was consequently set to the lower setting of 1 ml/min yielding high peak capacity and only slightly slower analysis. The interaction between the temperature gradient rate and the injector purge vent time was signicant for the peak capacity. At higher gradient rates, an increased purge vent time decreases the peak capacity, whereas at a gradient rate lower than 25C/min, this effect is much less pronounced. The gradient rate was consequently set to 25C/min. Increasing the initial temperature duration decreased the peak capacity for the early portion of the chromatogram while it was increased for the late portion of the chromatogram, suggesting an inuence on the sample zone focusing. However, as the average peak capacity was unaffected, the initial isothermal duration was set to the lowest investigated value, 2 min to increase the sample throughput. The initial temperature was negatively correlated with analysis time and the peak capacity for the early portion of
Fig. 4 PLS loading plot of the model for the normalized peak area of the [M-57]? fragments. The plot shows that all responses are positively correlated with the ion source temperature (IST), whereas the three latest eluting metabolites are strongly and negatively correlated with the acquisition rate (ACQ). The contour plots of cholesterol TBDMS and dihydroxyacetone phosphate MEOX 3TBDMS illustrates the different behavior of these two clusters

the chromatogram, but was positively correlated with the peak capacity for the late portion of the chromatogram. At the highest investigated temperature, 2 min could be cut off the analysis time due to decreased retention during the initial isothermal period. The interaction between the initial temperature and the temperature gradient rate was signicant for the analysis time, but the contour plot showed that at the chosen gradient rate of 25C/min, the setting of the initial temperature was unimportant. The interaction between the initial temperature and the purge vent time was signicant for peak asymmetry for valine 2TBDMS, but as the peak asymmetry factor for valine 2TBDMS was acceptable regardless of the settings, this effect was not further considered. The same interaction also reduced the peak capacity for the intermediate and late portion of the chromatogram, whereas it was insignicant for the early part. A low setting of the initial temperature could therefore be motivated. In conclusion, the 30 m capillary column was optimal together with a 115 s purge vent time, a ow rate of 1 ml/min, an initial temperature of 60C, an initial isothermal time of 2 min, and a gradient rate of 25C/min. With these settings, 51 analyses, including an oven equilibration time of 10 min, can be performed in 24 h. In comparison, the maximum number of analyses achievable with the 30 m column is 68. However, with the fastest method the average peak capacity is severely decreased to 119 compared to the proposed method with which a peak capacity of 220 is achievable.

0,8

Late eluting
0,6

0,4

0,2

wc[2]

-0,0

IST IST*Acq

-0,2

-0,4

Early eluting
Acq*Acq Acq
-0,2 -0,1 -0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1,0

-0,6

-0,8

wc[1]

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60 Fig. 5 PLS loading plot of the peak area ratio between low and high m/z fragments. Contour plots of alpha ketoisovalerate MEOX TBDMS (early eluting), dihydroxyacetone phosphate MEOX 3TBDMS (intermediate eluting) and cholesterol TBDMS (late eluting) illustrate the different behavior of the three clusters

A. P. H. Danielsson et al.

3.4 Mass spectrometry The [M-57]? fragment of a-ketoisovalerate MEOX TBDMS peak 1 was used for normalization as it was found to have a very low reproducibility of 0.005, indicating that the random error greatly overpowered the effects of the factors. The model obtained after normalization yielded R2Y = 0.686 and Q2Y = 0.582 with 0.507 B R2Y B 0.930 and 0.400 B Q2Y B 0.904 for all studied responses (Supplementary material S4). It was found that the ion source temperature was positively correlated with the [M-57]? fragment peak areas of all metabolite derivatives (Fig. 4). The acquisition rate had a low inuence on the majority of responses. However, for the late eluting metabolites, this factor became increasingly important with a negative contribution to the responses. This effect is probably related to the increased peak width with increasing elution times. Although the ion source temperature should be kept high according to the above results, it is likely to affect the fragmentation pattern. To study whether this occurs in the present investigation, a ratio of the unnormalized [M-57]? fragment, and a low m/z-fragment of the metabolite derivatives was modeled. A lower value of this response indicates a more excessive fragmentation. The model yielded R2Y = 0.887 and Q2Y = 0.822 with 0.598 B R2Y B 0.999 and 0.401 B Q2Y B 0.999 for all studied

responses (Supplementary material S5). Interestingly, the PLS loading plot shows distinct clustering of early, intermediate and late eluting metabolites (Fig. 5). The acquisition rate affected the ratio for the early and late eluting metabolites, whereas this effect was insignicant for the metabolites with intermediate elution times. The ratio was furthermore, for all metabolites, declining with increasing ion source temperature. In general, protocols for GC/MS-based metabolomics apply an ion source temperature ranging between 200 and 250C and increasing the temperature further might hamper the construction of common mass spectra databases. For these reasons, the ion source temperature was set to 250C. The acquisition rate was set to 20 Hz. 3.5 Method validation To properly assess the linearity criteria a lack-of-t test, which compares the pure error (random error) to the model error, i.e. the error in the linear t (Araujo, 2009), was included in the linear range determination. For a truly linear response, these errors are of the same magnitude. However, if the model error is signicantly larger than the pure error, the model has lack-of-t and the values do not strictly follow a linear function. For some of the chosen metabolites, lack-of-t due to curvature was detected (Table 3). The curvature was detected at the highest

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Development of a Metabolomic Protocol Table 3 Linear range, LOD and precision calculated from a set of blood plasma samples Metabolite Alanine Cholesterol Citric acid Cysteine Glycine Isoleucine Isocitric acid Lactic acid Leucine Lysine Methionine Oleic acid Phenylalanine Serine Threonine Tyrosine Valine
a b c

61

R2 0.990 0.973 0.987 0.949 0.958 0.976 0.950 0.964 0.980 0.958 0.951 0.961 0.995 0.995 0.957 0.989 0.962

Q2 0.989 0.971 0.986 0.943 0.953 0.973 0.946 0.961 0.978 0.953 0.946 0.958 0.995 0.995 0.953 0.988 0.958

Linear rangea 0.10.8 0.11.0 0.10.8 0.10.8 0.10.8 0.10.8 0.11.0 0.11.0 0.10.8 0.10.8 0.10.8 0.10.8 0.11.0 0.11.0 0.10.8 0.11.0 0.10.8

Internal standard
13 2

LOD (lM) 0.11 14.1 NAb NA

b

LOQ (lM) 0.37 47 NAb NA

b

%RSD (0.1)c 0.8 1.0 6.6 27.8 12.2 10.6 16.1 5.2 9.6 7.4 9.2 6.1 1.4 1.2 9.5 2.9 6.3

%RSD (0.4)c 0.4 5.6 4.6 6.5 4.4 2.3 6.4 3.3 2.3 3.1 2.2 2.7 0.5 0.5 2.7 1.4 1.2

C3-15N alanine C4-succinic acid C3-serine C5-a-ketoisovaleric acid C3-15N alanine C4-succinic acid C4-succinic acid C3-15N alanine C9- N-tyrosine
15

H7-cholesterol

13 13 13 13 13 13 13 13 13

NAb NAb NAb NAb NAb NA

b

NAb NAb NAb NAb NAb NA

b

C3-serine 13 C18-oleic acid

13 13 13 13 13

NAb 2.80 0.05 0.05 NAb 0.07 NAb

NAb 9.3 0.17 0.17 NAb 0.23 NAb

C6-phenylalanine C3-serine C3-serine C9- N-tyrosine C3-15N alanine

15

The linear range is expressed as relative concentrations 0.1, 0.4 and 1.0 (v/v, plasma/plasma ? water) No stable isotope labeled internal standard available Precision expressed as relative standard deviation calculated at relative concentrations 0.1 and 0.4 (v/v, plasma/plasma ? water)

concentration and was therefore concluded to be due to sample overloading. For these analytes the linear range was accordingly decreased. All metabolites investigated were found to be linear at a relative plasma concentration B0.8 (v/v, plasma/plasma ? water). Each linear function was also cross-validated to further ensure that the response is truly linear in the determined range. The LOD and LOQ were estimated for the analytes for which a stable isotope-labeled internal standard was available (Table 3). The LOD and LOQ were found to be in the range of 0.05 and 2.8 lM and 0.17 and 9.3 lM, respectively, for all metabolites except for the latest eluting metabolite, cholesterol, having a LOD and LOQ of 14.1 and 47 lM, respectively. The RSD of the peak areas, reecting the precision of the method, was below 5% for most analytes, although some metabolites exhibited comparatively high standard deviations especially at the lowest concentration (Table 3). This is most likely due to that the LOD for these metabolites are approached at high dilutions of the plasma. Cholesterol, on the other hand, being highly abundant in the plasma has a higher precision at the lowest plasma concentration. Speculatively, this distinct behavior of cholesterol results from a slight overloading at higher plasma concentrations. A chromatogram, illustrated both by the TIC and overlaid SIMs of the investigated metabolites, illustrates the high intensity of the [M-57]? fragments that greatly facilitates the identication and quantication of the metabolites (Fig. 6).

3.6 Sample drift comparison Finally, an orthogonal projections to latent structures (OPLS) model was calculated to relate the metabolite peak area to the run order. The model had one predictive and 5 orthogonal components and yielded R2Y = 0.95 and Q2Y = 0.81. The model indicated that mainly metabolites containing carboxylic acid and hydroxyl functionalities were drifting, with a slight decrease with the run order. The drift was overall very small, as indicated by cholesterol TBDMS possessing the strongest decay with run order (Area = -0.002(run order) ? 1.5, R2 = 0.02). Likewise, a model was calculated on replicates of the same set of plasma samples derivatized with MSTFA (Gullberg et al. 2004; Jiye et al. 2005). This model required one predictive and two orthogonal components, yielding R2Y = 0.90 and Q2Y = 0.76. In that model, several metabolite peak areas were found to increase with run order. Among these metabolites, amino acids with disilylated amines were over-represented indicating a progressing silylation. Glycine 3TMS, having the least sterically hindered amine, was found having the strongest positive loading, and a plot of the raw data indicated a pronounced alteration in peak area over time (Area = 0.11(run order) ? 0.1, R2 = 0.57). Thus, it is evident that the application of a bulkier silylation reagent reduces the number of disilylated amines and that this in turn reduces the drift in the metabolomics data.

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Fig. 6 Chromatogram from pooled blood plasma illustrating the high intensity of the [M-57]? fragments from the metabolites. a TIC (b) SIMs from the [M-57]? fragments of the investigated metabolites

4 Conclusion Optimizing a metabolomics protocol is complicated by the heterogeneity and vastly differing concentration ranges of the metabolites. In an effort to cope with this obstacle when optimizing a metabolomics protocol, sets of metabolites were selected that spanned a large portion of the metabolome. Thus, the individual metabolites investigated here should not be considered being single metabolites but rather representatives of whole groups of metabolites having similar properties. This means that the metabolite cocktail, although containing 36 unique metabolites, is a less complex model of a real plasma sample, where several hundred metabolites normally are detected. Moreover, generating the whole set of responses from all of the metabolites would result in very large data sets, which

would complicate both data generation and interpretation. For this reason, parts of the optimization were only performed on a selected set of representative metabolites. The optimization therefore does not deal with specic co-elution problems that commonly are encountered in metabolomics analyses. Nevertheless, responses related to the overall method performance, such as peak capacity, analysis time, efciency, and detection limits, still are valuable measures, which represent the ability of the method to handle real metabolomics samples. With DOE, the inuence of all major parameters in the metabolomics protocol, spanning derivatization and GC/ MS analysis, could be investigated. The results clearly reected the heterogeneity of the metabolome, with several metabolites showing inversely correlating responses. The response surfaces calculated with DOE were extremely benecial in nding the optimal parameters resulting in sufcient recovery of all metabolites. Although a general metabolomics protocol is presented, the models calculated here may also serve as guidelines to aid the selection of parameters for targeted metabolomics, also known as metabolite proling. The proposed protocol for blood plasma analysis, based on MTBSTFA derivatization, also offers a different selectivity to that of the more frequently used protocols based on MSTFA. Thus, it may serve both as a standard operating procedure for some applications and as a complementary approach for other metabolomics protocols based on MSTFA. The optimal protocol found in the present investigation suggested that methoximation should be performed at 20C for 4 h in 100% pyridine. The silylation should be performed at 100C for 170 min with pure MTBSTFA added the vial. Injection of the samples should be performed at 270C, employing an injector purge vent time of 115 s. The chromatography should be performed on a 30 m capillary column with a ow rate of 1 ml/min, an initial temperature of 60C, an initial isothermal time of 2 min, and a gradient rate of 25C/min. The ion source temperature should be 250C, with an acquisition rate of 20 Hz for the mass spectrometer.
Acknowledgments This work was supported by grants from Swedish Research Council (14196-06-3), the Crafoord Foundation, Lars Hierta, Fredrik and Ingrid Thuring, Ake Wiberg, Albert Pahlsson, O.E. and Edla Johansson Foundations, Knut and Alice Wallenberg Foundation, and the Royal Physiographic Society. Support from Inga and John Hain Foundation to PS is acknowledged.

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