Verification and validation of large numbers of low-abundance peptides or proteins in complex biological samples require sensitive and robust high-throughput quantification. To that end, a liquid chromatography–mass spectrometry (LC–MS) method was developed that uses a capillary-flow-rate ultrahigh-performance LC system coupled to a triple-quadrupole mass spectrometer. The combined use of 300-µm i.d. columns and a conventional electrospray ionization probe allowed the easy use of flow rates between 1 and 10 µL/min and limits of detection at the 10 amol level in complex matrices with great robustness and repeatability. Run times in every case were kept to less than 30 min. 

Advanced genomics and proteomics discovery studies have contributed to an increase in the number of potential protein biomarkers and drug candidates. These potential biomarkers and drug candidates need to be fully characterized and validated before entering the development pipeline or going through clinical testing (1). The verification and validation of large numbers of samples require a robust, high-throughput method for the sensitive quantification of low-abundance peptides and proteins in biological matrices. These increased analytical needs in the proteomics and clinical proteomics fields have contributed to the continuous improvement of a number of analytical techniques. Although techniques such as protein arrays and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) have been very popular, their intrinsic limitations have promoted the use of liquid chromatography (LC) coupled to mass spectrometry (MS). Low-flow-rate LC–MS systems, in particular nano LC–MS systems, are nowadays the norm for the sensitive analysis of peptides in biological fluids. Contrary to other techniques (for example, capillary electrophoresis) that are limited in terms of loadability, low-flow LC–MS systems generally incorporate a trap column that enables the injection of relatively large sample volumes (up to 100 µL) in a short time. Additionally, the constant improvement and development of novel LC columns permit faster and more robust analysis to be performed.  

In this study, the detection limit, analytical precision, dynamic range, and robustness for the targeted quantification of peptides using a capillary-flow LC–MS system were evaluated by spiking known amounts of isotopically labeled yeast peptides into a 500-ng yeast-digest matrix. An additional mixture of bovine serum albumin (BSA) and enolase digests was used to evaluate the throughput of the method for less-complex samples.  

An extract of yeast proteins (National Institute of Standards and Technology [NIST]) was used to make the yeast digest matrix. The yeast protein extract was reduced and then alkylated, followed by tryptic digestion. A mixture containing eight isotopically labeled yeast peptides (Thermo Scientific) was used in this study as well. A dilution series was prepared in which the mixture of isotopically labeled yeast peptides was spiked into 500-ng yeast digest matrix at five levels (0.005, 0.05, 0.5, 5, and 50 fmol/µL). 

A BSA digest and an enolase digest (Waters) were mixed together and diluted in the loading solvent at a concentration of 1 fmol/µL. 

A capillary ultrahigh-pressure liquid chromatography (UHPLC) system (Thermo Scientific Dionex UltiMate 3000 RSLCnano system) was used for peptide separation. The LC system can deliver pressures up to 800 bar and supports tool-free column connection.  

Both the trap column and the separation column were packed with C18 material. The trap column (2 cm × 100 µm) was packed with 5-µm Thermo Scientific Dionex Acclaim PepMap particles and loaded at a flow rate of 20 µL/min using 0.05% trifluoroacetic acid in 98:2 water–acetonitrile as the loading solvent. Sample loading was performed for 6 min when analyzing the dilute sample series and 3 min for the mixture of BSA and enolase. The separation column (300 µm i.d.) was packed with 2-µm Acclaim PepMap RSLC particles and was 15 cm long in most experiments. A shorter column (5 cm long) was used for the analysis of simpler mixtures where chromatographic resolution was not as important. In both cases, the analysis was performed at a flow rate of 4 µL/min. The solvents were 0.1% formic acid in water for A and 0.1% formic acid in 20:80 water–acetonitrile for B. A gradient from 5% B to 55% B in 30 min was used for the separation of the dilute sample series while a shorter gradient, 5% B to 35% B in 10 min, was used for the analysis of the mixture of BSA and enolase digests. In all cases, the amount injected was 2 µL in microliter pick-up mode using 0.05% trifluoroacetic acid in 98:2 water–acetonitrile as transport liquid. 

A Thermo Scientific TSQ Vantage triple-quadrupole mass spectrometer equipped with a conventional electrospray ionization (ESI) source was used for all experiments. A 50-µm i.d. metal needle was installed on a conventional Thermo Scientific HESI-II probe for the ionization.  

A 3400-V spray voltage, six arbitrary units of sheath gas, and a 275 °C capillary temperature were used for the ionization. The selectivity for both Q1 and Q3 were set to 0.7 Da (full-width half-maximum). The collision gas pressure of Q2 was set at 1.2 mTorr of argon. The mass spectrometer was operated in selected reaction monitoring (SRM) mode. 

Thermo Scientific Pinpoint software, version 1.2, was used for both assay development and data analysis. An MS-MS spectral library was developed based on previous discovery experiment data to enable transition selection per target peptide.  

The two most intense fragment ions per peptide target were automatically selected for the SRM assay for quantifying eight isotopically labeled target peptides.  

Three peptides that have unique peptide sequences to present the targeted protein of BSA and another three peptides that have unique peptide sequences to present the targeted protein of enolase were selected as quantitative surrogates of the two targeted proteins based on the MS-MS spectral library. The three most intense fragment ions per peptide target were automatically selected for the SRM assay.  

The analysis of targeting the eight peptide standards spiked in the dilute sample series was carried out in timed-SRM (tSRM) mode with 2-min scheduled time windows. The cycle time used was 1.5 s. The targeted peptides, precursor and product ions, and collision energies used are listed in Table I. 

Table I: SRM settings for the eight isolopically labeled peptides 			

The analysis of the mixture of BSA and enolase digests was carried out using an unscheduled SRM mode. Cycle time was 1 s. The targeted peptides representing BSA and enolase, precursor and product ions, and collision energies used are listed in Table II. 

Table II: SRM settings for the "signature" peptides representing BSA and enolase			

Thermo Scientific Xcalibur software, version 2.1, and Pinpoint software, version 1.2, were used for data processing. 

LC–MS using standard reversed-phase high performance liquid chromatography (HPLC) separation conditions (4.6-mm i.d. column and 1.0 mL/min flow rate or 2.1-mm i.d. column and 0.2 mL/min flow rate) has been one of the major approaches for quantification of small molecular compounds from biological fluid samples. However, LC–MS using a standard flow rate separation does not provide the necessary sensitivity for the analysis of many low-abundance proteins and peptides in complex matrices. The intrinsic sensitivity of the applied analytical method can be greatly enhanced by reducing the column dimensions (2).  

Although nano LC coupled to MS by a nano ESI interface provides the greatest sensitivity, it generally involves more training, may show limited robustness, and requires more user intervention than the conventional interface used in this study. The use of capillary columns (100–500 µm i.d.) and flow rates (1–10 µL/min) provides significant improvements in sensitivity compared to standard-flow LC–MS. Although making connections with silica capillaries can be challenging or introduce detrimental dead volumes in the sample flow path, several new types of connections have been brought to the market for finger-tight UHPLC-compatible connections of columns and capillaries. The use of capillary LC requires finding the optimum compromise between gradient delay, band broadening, and back pressure. Connection capillaries of 

50 µm i.d. are generally used pre- and post-column. Capillary LC–MS also shows robustness and quantitative reproducibility that, generally, can only be achieved at more conventional flow rates (hundreds of microliters per minute). This type of pumping system typically has an extended flow range up to 50 µL/min, offering great robustness and flexibility. The stability of the flow delivery and the repeatability of the gradient formation allows for the use of timed segments in targeted MS experiments. 

One of the additional benefits that capillary LC–MS provides is the capability to use the conventional electron spray source for the ionization. By sharing the same ion source with standard LC–MS, the capillary LC–MS set up is much easier and cost effective than nano LC–MS. 

Optimization of Capillary LC–MS Analytical Conditions  

A sample containing the eight spiked peptides at a concentration of 5 fmol/µL was used to assess the influence of the flow rate on the workflow sensitivity. Flow rates of 1, 4, and 6 µL/min were investigated. The intensities of targeted peptides were comparable at flow rates of 1 and 4 µL/min. However, the signal intensity of most targeted peptides started to decrease at flow rates greater than 4 µL/min, and at those flow rates the auxiliary gas becomes necessary to stabilize the electrospray. Therefore, a flow rate of 4 µL/min was chosen for all subsequent experiments as no significant gain in sensitivity was observed by lowering the flow rate from 4 µL/min to 1 µL/min. 

Sensitivity and Linear Dynamic Range Using Capillary Flow Rates   

The sensitivity and linear dynamic range of the workflow were evaluated by injecting the sample series of isotopically labeled yeast peptides spiked in a yeast digest matrix in order of increasing concentration starting with a blank and ending with the most concentrated standard. The gradient went from 5% to 55% B in 30 min. Each sample was run in triplicate. All eight peptides were clearly detected from 10 amol to 100 fmol on column. Figure 1 shows the extracted chromatograms of the peptide SAAGAFGPELSR* spiked into a 500-ng yeast-digest matrix at different concentration levels. Because the labeled peptides cover a relatively large spectrum of varying physicochemical properties, we believe that similar sensitivity could be achieved for most peptides. Naturally, the matrix plays a very important role that would influence sensitivity. 

Figure 1: Extracted chromatograms of the peptide SAAGAFGPELSR* spiked into a 500-ng yeast-digest matrix at different concentration levels.			

Figure 2 shows the calibration curve of the peptide ELASGLSFPVGFK* acquired from the diluted sample sequence. Over four orders of linear dynamic range were observed from 0.01 fmol to 100 fmol, which should be sufficient for many protein or peptide quantification applications. 

Figure 2: Calibration curve of spiked peptide ELASGLSFPVGFK*. Over four orders of linear dynamic range were observed.			

The analytical precision was evaluated by calculating the coefficients of variation for triplicate runs at each concentration level. The observed analytical precision was excellent and able to provide quantitative results even for the least abundant proteins or peptides. The coefficients of variation were less than 15% for the targeted peptides even at 10 amol concentration levels. Table III shows the summary of the percent coefficient of variation (%CV) for the samples at all five concentrations. 

Table III: Coefficients of variation (triplicate runs) for eight spiked isotopically labeled peptides across different concentration ranges			

Retention Time Repeatability and Integrated Peak Area Precision   

Figure 3: Retention-time repeatability over 70 consecutive LCâMS runs.			

To evaluate the robustness of the capillary LC–MS workflow and test the repeatability of both retention times and integrated peak areas, the sample containing the spiked peptide mixture at a concentration of 5 fmol/µL was run 70 times consecutively using the previously described method. Both retention times and integrated peak areas showed excellent repeatability over the 70 LC–MS runs, compared to what can be achieved using a standard flow rate for separation. The %CV of the retention time for the three early-eluted peptides were 0.7–1.1%. Peptides eluted later in the gradient showed even lower variations, and the percentages of coefficient of variation were between 0.3% and 0.5% (Figure 3). Retention times proved to be even more repeatable when this workflow was applied to the analysis of simpler samples such as a mixture of BSA and enolase digests; retention times varied by no more than a second or two (Figure 4). 

Figure 4: Retention-time repeatability of targeted peptide NYNDVIAPAFVK over three repeat runs using a 10-min gradient on a 5-cm column.			

The integrated peak area for each targeted peptide was reproducible across 70 consecutive runs without signal intensity normalization. The most hydrophilic peptide showed a coefficient of variation of 12.0%, while all other peptides showed coefficient of variation of less than 7% (Figure 5). 

Figure 5: Integrated-peak-area repeatability over 70 consecutive LCâMS runs.			

Increased Throughput Using a 5-cm Column and a Fast Gradient for the Analysis of Samples of Lesser Complexity   

For less-complex samples, throughput could be further increased by using a shorter column and a faster gradient. A mixture of BSA and enolase digests was used to evaluate throughput for simple protein digests. The concentration of both BSA and enolase was 1 fmol/µL. Three peptides from BSA and three peptides from enolase were targeted. The SRM quantitative assay was run in triplicate using a short gradient (5–35% B in 10 min) and a short column (5 cm). The total run time was 20 min including sample loading, peptide separation, column washing, and column equilibration. Figure 6 shows the extracted-ion chromatograms for all six targeted peptides. High LC efficiency was maintained with a fast gradient that yielded very sharp peaks and high sensitivity. The retention time was highly reproducible across three runs (Figure 4). Throughput could be further increased by mounting the capillary LC–MS setup in tandem, thus doubling the measurement time. Turn-around time could be as low as 2 min per run for the analysis of simple tryptic digests while still retaining excellent robustness and repeatability (data not shown). 

Articles

The detection limit, analytical precision, dynamic range, and robustness of a method for the targeted quantification of peptides using a capillary-flow LC–MS system were evaluated by spiking known amounts of isotopically labeled yeast peptides into a 500-ng yeast digest matrix.

Targeted Protein Quantification Using High-Throughput Capillary LC–MS

The Xevo G2-S QTof and Xevo G2-S Tof benchtop time-of-flight mass spectrometers from Waters are designed with the company's StepWave ion optics and off-axis ion source technology. According to the company, the systems are as much as 20× more sensitive than earlier-generation instruments and critical components stay clean longer. 

The Method Toolbox software from Shimadzu is designed to assist with method optimization. According to the company, the software allows chemists to analyze up to 96 methods, 16 mobile phase solvents, and six columns, all in one batch. 

VICI Valco's Cheminert C72 ultrahigh-pressure UHPLC injector is designed with a stator coating that permits pressures up to 15,000 or 20,000 psi. According to the company, the instrument is available in four-, six-, eight-, and 10-port configurations, with a bore diameter of 0.004, 0.006, or 0.010 in. 

The PesticideScreener system from Bruker is designed for multiresidue analysis of pesticides in food and feed. According to the company, the system includes UHPLC and QTOF-MS hardware, applications software, and LC–MS methods. The system also includes a database that includes retention times for the matched UHPLC method; adduct information; isomer information; fragment ion information; isotopic confirmation; and qualifier ions for "one-shot" confirmation in broadband MS-MS mode. 

The Agilent 5977A series GC mass selective detector reportedly has a redesigned inert MS ion source that enhances performance with efficient ion collection. According to the company, the detector uses the company's TwisTorr 304 FS turbomolecular pump, which is optimized for light gas operation. 

Thermo Fisher Scientific's Prelude SPLC sample preparation and liquid chromatography system is designed to work with the company's mass spectrometers, automating sample preparation and increasing throughput. According to the company, the system has two channels of HPLC separation, and it is capable of gradient operation at flow rates of 1–2000 μL/min. 

PerkinElmer's Flexar photodiode-array detector is designed to provide a wide detection range, low baseline noise, wavelength accuracy, and a fast data acquisition rate. According to the company, the detector is suitable for tandem use with other detectors and is compatible with third-party chromatography data systems.  

The Tridion-9 portable GC–toroidal mass spectrometry system from Torion is designed for field GC–MS applications. The system reportedly combines an electronic pressure controlled gas chromatograph with an amplitude scanning toroidal ion-trap mass spectrometer. According to the company, the system can be used with SPME syringes for sample introduction. 

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The quantitative estimation of drugs from biofluids in pharmacokinetic studies requires high-throughput bioanalytical methods. We report a rapid, simple, and efficient method for simultaneous quantitative determination of different pharmaceutical drugs directly from urine samples. Acetaminophen, griseofulvin, ampicillin, and verapamil spiked in human urine were characterized directly by matrix-assisted laser desorption–ionization time-of-flight mass spectrometry (MALDI-TOF-MS) followed by tandem MS to confirm their identities. Furthermore, quantitative profiling of drugs was carried out directly from spiked urine samples using MALDI-TOF-MS without any chromatographic separation. Linearity was observed for the relevant concentration ranges of the metabolites found in urine. The quantitative analysis successfully produced acceptable precision and high accuracy of recoveries of drugs. 

Pharmacokinetics plays a very important role in the drug development process for prioritizing lead compounds. It involves the investigation of absorption, distribution, metabolism and excretion (ADME) of drugs from the body, and helps in screening of drug candidates. After a drug is administered into the blood stream orally or intravenously, it is distributed to the site of action and metabolized or biotransformed. Finally, the drug and its metabolites are excreted from the body through the kidneys in the form of urine. Slow or incomplete excretion can lead to the accumulation of higher concentrations of the drug at the site of action. Quantitative estimation of the excreted drug is important to determine the therapeutic dosage of the drug, its toxicological effect, the rate of excretion, and the amount of drug absorbed by the body. Simultaneous analysis and quantitation of different drugs with diversity in their chemical nature, especially from biological fluids, is indeed a challenging task.  

Mass spectrometry (MS) has established itself as a crucial bioanalytical tool in the pharmaceutical industry and the drug development process (1,2). Versatility coupled with high sensitivity, resolution, and dynamic range make MS a method of choice in pharmacokinetic studies. Liquid chromatography–mass spectrometry (LC–MS)-based methods for quantitative determination have been used routinely in the past decade and are now considered benchmarks for bioanalytical analysis (3–6). However, chromatographic techniques require a lot of attention and can be time consuming during analytical method development. In drug development processes where time is a critical factor, any time-consuming methods are undesirable. Often, there are also large sample sets in the analytical pipeline that demand high-throughput analytical techniques (7).  

Direct mass analysis and quantitation is an efficient alternative that is not yet being used to its optimum potential. This is primarily because of concerns about introducing an impure sample without chromatographic separation. Major time-consuming steps and tedious sample preparation can be avoided by introducing the sample directly into the ionization source of a mass spectrometer (8). However, most of the recently developed direct mass analytical techniques that can bypass the chromatographic step have not yet been fully explored for quantitative analysis (9,10). Among the direct mass analytical methods, matrix-assisted laser desorption–ionization mass spectrometry (MALDI-MS) has several advantages. MALDI-MS systems are robust, require minimal sample preparation, are tolerant to salts and buffers, and offer high-throughput analysis (11–15). Furthermore, the generation of mainly singly charged ions with high resolution provides uncomplicated spectra (16). The interference of matrix ions in small molecule regions of the mass spectra can be offset by choosing the matrices carefully and using MS-MS validation of the precursor ions. MALDI-MS has been demonstrated for both qualitative and quantitative analysis of targeted low-molecular-weight compounds, pharmaceuticals, and other small-molecule metabolites (13,17–24). Particularly, the use of MALDI-MS in imaging of drugs distributed in cells has also gained much interest (25–27). Considering the limited time span for bioanalytical study during drug development processes, MALDI-MS is an alternative for drug screening from biological fluids because of its inherent advantages.  

Urine has been routinely used as a biofluid for analytical studies because of its noninvasive sampling, lower protein content, and larger quantities that can be collected for multiple cycles of analysis. Urine excreted from the human body also contains a higher concentration of endogenous and exogenous metabolites. In this study, we explored direct and quantitative analysis of several drugs spiked in blank urine samples using MALDI-MS. A simple and rapid quantitative workflow is demonstrated for the analysis of drugs from human urine without any further sample preparation, along with their characterization with comprehensive collision-induced fragmentation.  

-(4-hydroxyphenyl)acetamide), griseofulvin ((2S,5'R)-7-chloro-3',4,6-trimethoxy-5'-methylspiro[1-benzofuran-2,4'-cyclohex-2-ene]-1',3-dione), ampicillin ((2S,5R,6R)-6-[[(2R)-2-amino-2-phenylacetyl]amino]-3, 3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid), verapamil (2-(3,4-dimethoxyphenyl)-5-[2-(3,4-dimethoxyphenyl)ethyl-methylamino]-2-propan-2-yl-pentanenitrile), 2,4-diamino-6-(2-fluorophenyl)-1,3,5-triazine, 2,5-dihydroxybenzoic acid, trifluoroacetic acid, methanol, and acetonitrile were purchased from Sigma-Aldrich. Deionized water with a specific resistivity of 18.2 MΩ was obtained using an SG ultrapure water unit. 

A 100 mM stock solution of each drug was prepared in methanol, diluted to the desired concentration, and mixed in equal volume. The final concentration of each drug molecule in the mixture was as follows: 2 mM acetaminophen, 40 µM griseofulvin, 1 mM ampicillin, and 2 µM verapamil. 2,4-Diamino-6-(2-fluorophenyl)-1,3,5-triazine was used as an internal standard for quantitative analysis. A 1000 ppm stock solution of internal standard was prepared in 1:1 (v/v) methanol–water and diluted to 10 ppm. 2,5-Dihydroxybenzoic acid (DHB) was used as the MALDI matrix. A 20-mg/mL DHB solution was prepared in 1:1 (v/v) acetonitrile–0.1% trifluoroacetic acid. Later a mixture of DHB and internal standard was prepared for quantitative MALDI-MS. 

Urine was collected from a healthy volunteer and immediately aliquoted and stored at -20 °C. To prepare urine samples spiked with pharmaceutical drugs, 100 µL of urine was mixed with 100 µL of the drug mixture and centrifuged at 4 °C for 1 h at 13,200 rpm. The supernatant was collected in a separate vial. A series of calibrants was prepared from urine samples by spiking varying concentrations of the drug mixture. 

A Synapt MALDI-TOF-MS system (Waters) was used in the positive ion mode for all of the MS experiments. Before the experiments, the instrument was properly tuned and calibrated for mass accuracies with standards and protocols as prescribed. Detector voltage and quadrupole settings were adjusted to obtain maximum sensitivity. The laser energy (Nd:YAG laser, 355 nm) was optimized to achieve a good signal-to-noise ratio above the threshold energy of the MALDI matrix. 

All of the samples were analyzed on standard 96-well stainless steel MALDI target plates that were precleaned thoroughly. Before spotting, the samples were premixed with MALDI matrix (a mixture of DHB and internal standard). The samples (1 µL per spot) were spotted manually in replicates using a micropipette. The quantitative data were acquired by using an instrument in automatic acquisition mode. During automatic acquisition, the instrumental conditions and acquisition time were kept uniform across the MALDI target plate. For detection and identification of drug molecules, precursor ions of each drug molecule were subjected to collision-induced dissociation (CID) fragmentation. 

The qualitative data analysis was performed using Masslynx 4.1 (Waters) and mMass (open source) software. For quantitative data analysis, an in-house developed software, "MQ," was used to generate calibration curves based on ratios of peak intensities of analyte to internal standard. These calibration curves were used to determine the concentration of drugs in quality control samples and drug-spiked urine samples. Six replicates were used for each calibration point and the average was calculated for generating the calibration curve. A linear regression equation was used in calculating the correlation coefficient (

				), slope, intercept, recoveries, and relative standard deviations (RSD).  

Qualitative Determination of Drug Molecules

In this study, commonly used pharmaceutical drugs were selected from different classes of drugs according to their function, structure, and molecular weight (Figure 1). 

Figure 1: Chemical structures of pharmaceutical drugs spiked in urine and the internal standard used in this work.			

Acetaminophen is a frequently used analgesic that has the lowest molecular weight among the drugs selected for this study. Griseofulvin is a metabolic product of 

 and it has been used as an antifungal agent to treat dermatophytic infections. Ampicillin has been used extensively as an antibiotic in bacterial infections. It is a  -lactam antibiotic and belongs to the penicillin family. Verapamil, a derivative of phenylalkylamine, is an effective antiarrhythmic and vasodilator. It is also used as a calcium channel blocker. The qualitative study of drug molecules was carried out in two stages. First, MALDI-MS of the drug mixture premixed with the DHB matrix was performed (data not shown). All the drug molecules showed mainly protonated adduct of the respective molecular ion [M+H]

  and had good mass accuracy (within 10 ppm). DHB matrix peaks were also present in the spectra, but the drug molecule peaks were clearly resolved. Subsequently, the precursor ions of drugs were allowed to fragment in the collision chamber. The fragmentation patterns matched with those reported in previously published data and, thus, corroborated the identification of each drug molecule (28–31). 

Figure 2: MALDI-MS spectra of pharmaceutical drugs spiked in urine and analyzed directly. 2,5-Dihydroxybenzoic acid (DHB) was used as matrix.			

MALDI-MS was subsequently used directly on urine samples spiked with drugs followed by MS-MS validation as described above for the pure standards. Figure 2 shows the representative MALDI-MS of drugs from spiked urine samples. A zoomed-in portion of the spectra with a protonated and sodiated adduct of drug molecules is also shown in the figure. As expected, a very complex spectrum was obtained from spiked urine with many peaks seen across the entire mass range until 

 600. This complexity can be attributed to the presence of various endogenous metabolites along with several possible adducts and fragments thereof. Accurate masses for protonated and sodiated adducts of all the spiked drugs were detected with good resolution and signal-to-noise ratio. MS-MS from precursor ions reveals the identity of analyte molecules unambiguously and is especially useful for analysis from complex samples such as urine. Table I summarizes the CID fragments of each drug molecule in MALDI MS-MS. The overall identification of drugs spiked in urine was performed reproducibly in a rapid manner. It is noteworthy that all the drugs were simultaneously detected directly from the urine without any chromatographic separation or sample preprocessing. 

Table I: Comprehensive CID fragmentation of spiked drugs in urine. The expected masses of fragments were obtained from published reports (28â31).			

Quantitative Determination of Drug Molecules

The quantitative trends of the drug molecules were studied by constructing calibration curves from both pure standards and subsequently from spiked urine samples. Calibration responses were measured as peak height ratios of the sample normalized against an internal standard. The internal standard compensates for the variation in signal response that arises because of inhomogeneity in sample spotting and fluctuation in instrumental parameters over time. The selection of the internal standard was based on the reproducibility of signals, miscibility, and noninterference with the drug (18,32). Isotopically labeled internal standards have been a preferred choice for users, but have disadvantages because of their cost and nonavailability. Nonlabeled standards provide an alternative to isotopically labeled internal standards. Previously published reports have successfully shown the usage of chemically nonanalogous compounds as internal standards (22,24). A symmetric triazine derivative was used as an internal standard, which is nonendogenous to urine metabolites. Table II represents the quantitative statistics of drug molecules directly quantified from a mixture of drugs. All drug molecules showed good linearity with correlation coefficients (

) above 0.97. The calibration curve was prepared by taking average values of six replicates per dilution and the concentration range was started above the limit of quantitation (LOQ). The detection limit of standard solution for each drug molecule was estimated before quantitative analysis by taking those concentrations that showed a signal-to-noise ratio with peaks above 3. Using these criteria, all the drug molecules were obtained with detection limits at the low picomoles per microliter level.

To verify the method, quality control samples containing drugs in low, mid, and high concentration ranges of calibration curves were analyzed on the same target plate. Further recoveries of drugs from quality control (QC) samples were calculated from the linear equation of calibration curve. All the drug molecules showed reproducible recoveries within 15% RSD in most cases, and extending up to 20% in a few, validating the linear relationship between concentrations of drugs and response from MALDI-MS data. 

Table II: Quantitation data for the mixture of drugs from standards			

The quantitative analysis of drugs was further extended to samples present in urine to demonstrate the utility of the method for biological fluids. The blank urine was fortified with drugs, which simulated the presence of a drug in excreted urine after the administration of a drug in the human body. Acetaminophen, griseofulvin, ampicillin, and verapamil were spiked in urine according to previously published reports (Table III) (33–36). Serial dilutions of the drugs were added to urine samples with no further dilution. The limit of detection (LOD) and LOQ of drug molecules were determined from spiked urine. Acetaminophen and ampicillin were detected at 7.6 and 8.7-ng/µL, whereas the detection limits for griseofulvin and verapamil were found to be 0.35 and 0.036 ng/µL. The LOD and LOQ were higher for the urine spikes when compared with the pure standards. Calibration curves were generated by plotting the concentration of drugs spiked and their ratios of peak height with internal standard. Table IV summarizes concentration ranges, linearity equations, correlation coefficients, and recoveries from spiked urine samples analyzed directly using MALDI-MS with no sample preprocessing. Excellent linearity was observed for each drug molecule with high correlation coefficients and reproducibility fulfilling the Food and Drug Administration's criteria of 20% RSD at LOQ and 15% elsewhere in the calibration range. The recoveries from QC samples were calculated as the mean of 12 samples. The recoveries of 91–112% were within 15% RSD, except for ampicillin, for which one of the QC samples had an RSD of 19%. 

Table III: Concentration range used in quantitation of drugs from urine samples (33â36)			

Once again, it is noteworthy that the entire analysis, starting from sample preparation to data analysis, was performed within a few hours and did not necessitate any chromatographic separation. The analysis time can further be reduced using automated spotting. Additionally, accuracy and reproducibility of data can also be improved with automation.  

Until recently, MALDI was not considered a suitable ionization method for small-molecule analysis (<

 500) because of matrix interference in the low-molecular-weight region. However, several recent publications have demonstrated the utility of MALDI-MS in targeted small molecule analysis (22–24). Newer generations of MALDI-MS systems are equipped with high duty cycle analyzers and advanced electronics, which provide high resolution data and, subsequently, mass accuracies less than 10 ppm. An accurate identification of a molecule can be obtained based on peaks with high mass accuracy and subsequent CID fragmentation. Peak area–based quantitation can be challenging for metabolites and other small molecules from biological fluids in the absence of a chromatographic separation, because the contiguous peaks in the mass spectrum often are poorly resolved. Peak intensity–based quantitation demonstrated using large sample sets has previously shown efficiency comparable to that of peak area–based quantitation (22,24), and can be advantageous. This approach is quite useful for MALDI-MS, in which inconsistent desorption–ionization processes may lead to poor quantitation. 

Table IV: Quantitation data for drugs spiked in urine followed by direct MALDI-MS. Recovery was calculated for 12 replicates of each spiked concentration. 			

The application of MALDI-MS in qualitative and quantitative analysis of pharmaceutical drugs spiked in urine was demonstrated. Drug molecules were characterized by MALDI-MS followed by CID fragmentation. High resolution and mass accuracies within 10 ppm along with MS-MS fragmentation patterns corresponding to the analytes of interest aid in unambiguous detection from urine. Peak height–based quantitation performed using a chemically unrelated internal standard on both pure standards and spiked urine samples showed excellent sensitivity, linearity, accuracy, and reproducibility. The method was validated with quality control checks. The method reported establishes the versatility of MALDI-MS in direct analysis from urine with minimal sample processing and without any chromatographic separation. Tolerance to sample impurities along with the higher throughput obtained using MALDI-MS can increase the efficiency of ADME and similar bioanalytical studies. 

This research was funded by CSIR grants and the Department of Biotechnology (India) young investigator award (RGYI Grant 102/IFD/SAN/PR-2062/2009–2012). V.P. acknowledges funding from DBT Ramalingaswamy fellowship. 

An application of MALDI-MS in qualitative and quantitative analysis of pharmaceutical compounds spiked in urine is demonstrated.

Rapid and Direct Quantitation of Pharmaceutical Drugs from Urine Using MALDI-MS

High-resolution time-of-flight mass spectrometry (TOF-MS) was investigated as an alternative to Fourier transform mass spectrometry (FT-MS) for the direct analysis of heteroatomic species in crude petroleum. The practical implications of this technique's advantages and drawbacks were explored. The favorable resolution profile observed for high-resolution TOF-MS versus FT-MS instruments yields enhanced spectral peak capacity at higher mass-to-charge ratios (

), allowing more extensive characterization of heavy components within crude petroleum. 

To study the individual components of a mixture as complex as crude petroleum, an ultrahigh-resolution technique is required. Comprehensive two dimensional gas chromatography (GC×GC) evolved to enable the separation of what had been called the "unresolved chromatographic mass" ubiquitous to one-dimensional petroleum chromatograms. Direct infusion ultrahigh-resolution mass spectrometry (MS) resolves signals of important petroleum constituents that are not amenable to GC such as asphaltenes and high-molecular-weight heteroatomic species (1,2). 

Characterization of heteroatomic species present in crude petroleum is critical to the environmental and the economic impact of petroleum recovery, refining, and use as well as for ascertaining the nature and history of petroleum deposits. Accordingly, considerable resources have been invested in developing analytical technologies and methods for the characterization of heteroatomic content in crude petroleum. 

 (1,2) refers to comprehensive approaches to such characterization as well as the acquisition of other information.   

The first mass spectral technology to allow sufficiently detailed petroleomic characterization was Fourier transformation ion cyclotron resonance (FT-ICR) MS (1,2). The ultrahigh spectral resolution achieved by FT-ICR enables multiple thousands of individual crude petroleum components to be measured simultaneously in a single direct infusion experiment that typically lasted only a few minutes. The magnitude of information content delivered in a short time made FT-ICR the authoritative, and for a long time, the only technology for rapid comprehensive characterization of heteroatomic species in crude petroleum, because of the requirement for resolution approaching 100,000.  

More recently, alternative technologies to FT-ICR have been introduced. Electrostatic ion-trap Fourier transform MS (Orbitrap, Thermo Fisher Scientific) is one such alternative (3). As another FT-based technology, it shares similar strengths and weaknesses with FT-ICR. High-resolution time-of-flight mass spectrometry (TOF-MS) is a more complementary alternative, with different advantages and drawbacks. 

This article presents the application of high-resolution TOF-MS to petroleomics. The basic theory of each type of ultrahigh-resolution mass spectral platform is explained briefly. Results from high-resolution TOF-MS analysis of various crude petroleum samples are presented with a short discussion of the meaning of these results. 

Conventional expressions of spectral resolution have shifted from definitions based on spectral peak width at 5% or 10% of the apex height, to definitions based on spectral peak width at 50% of the apex height, or full width half maximum (FWHM). This was driven by the evolution of mass spectral technology, and possibly also by instrument vendors' desire to present specifications with more impressive-looking numbers.   

The first high-resolution mass spectrometers, magnetic sector instruments, yielded peak shapes very close to Gaussian. Sector instruments still lead all other high-resolution platforms with respect to Gaussian nature of peak shape. This peak shape fidelity made the 10% valley definition of resolving power meaningful and natural. 

Sector instruments were largely replaced by FT-ICR instruments as the latter achieved resolution that was orders of magnitude superior to that achieved by the former. However, apodization and zero-filling applied when transforming the ICR image current to a mass spectrum can significantly impact peak shape, and much higher Lorentzian character can result. As Lorentzian character increases, peaks will appear narrower on top, and mass resolving power expressed at FWHM will increase. The penalty for this inflation of resolving power at FWHM (

) is peaks that are wider near the base and have diminished resolving power expressed using historical definitions.   

Among newer high-resolution platforms, electrostatic FT instruments are generally subject to the same peak shape considerations as ICR instruments, and data treatment can introduce significant Lorentzian character.  TOF instruments can yield spectral peak shapes somewhere between those generated by sector instruments and FT-based instruments, except that ion physics and instrument tuning generally affect peak shape more than mathematical considerations. 

The mass resolving power required to distinguish any pair of isobars depends on their intensities relative to each other. As intensity becomes increasingly disparate, higher mass resolving power is needed to observe separate peak apexes. Most isobars recorded are of unequal intensity, and the ability to separate a small signal from the foot of a large signal is important to maximize the information content of a mass spectrum. Therefore, peak width near the base may be more relevant than peak width at FWHM. 

The mass resolving power required to distinguish any pair of isobars also depends on their mass-to-charge ratio (

values, higher mass resolving power is required. Therefore, an ideal, hypothetical high-resolution mass spectrometer would exhibit mass resolving power that increased linearly with 

Figure 1: Expected Gaussian spectral peak widths for selected mass spectrometers: (a) absolute units (mDa), (b) relative units (ppm).			

Figure 1 shows expected spectral peak width curves at 10% of peak height as a function of 

 for several high-resolution instruments. The peak width curves for the 7-T FT-ICR system and the standard electrostatic ion-trap FT-MS system are taken directly from descriptions of these instruments published by Makarov and colleagues (4). The 0.76-s transient time, the apodization, and zero-filling are exactly as described in this publication. The peak width curves for the high-resolution TOF-MS instrument are calculated from resolving power curves with asymptotic limits of 

Figure 1 also assumes perfectly Gaussian peak shape for all instruments.   

Figure 1 clearly shows that FT-based instruments yield narrower spectral peaks at low 

, and high-resolution TOF-MS instruments yield superior resolution at higher 

values. Crude petroleum contains important analytes of 

 >1000, such as asphaltenes and high-molecular-weight tetracarboxylic acids (ARN acids). The opposing trends of the curves for FT-based instruments and high-resolution TOF-MS instruments renders these technologies complementary. 

Spectral resolution translates into spectral information content. Spectral information content may be expressed as the total number of chemically relevant signals resolvable over a given range of 

values. This number is related to the spectral peak width and to the range of mass defects available for chemically relevant signals.  

The approximate upper limit of chemically possible mass defect is the Kendrick line, or the line of Kendrick mass defect = 0. This line of mass defect is taken from the Kendrick mass scale (5) which defines the mass of 

 C ≡ 12. Only higher isotopologues of saturated hydrocarbons will have a mass defect greater than the Kendrick line. The lower limit of chemically possible mass defect depends significantly on heteroatomic content. Because the constituents of crude petroleum are composed principally of carbon, the line of conventional mass defect = 0 may be used as a rough lower limit of chemically possible mass defect for petroleum constituents. The distance between the Kendrick line and the carbon line increases as nominal mass increases. 

From the curves in Figure 1, and the range of chemically possible mass defects described above, the total number of resolvable signals may be calculated. Figure 2 compares the total possible information content that could be captured by two instruments for three different 

 ranges. Klitzke and colleagues presented empirical verification of information content captured by two instruments similar to those discussed herein (6). Figure 2 reinforces the complementary capabilities of FT-based and high-resolution TOF-MS systems.   

Crude petroleum samples from various sources were diluted to about 2 mg/mL with a 1:1 mixture of toluene and methanol as described previously (6). From preparation to infusion, care was taken that the samples contacted only clean glass, PTFE, or PEEK surfaces, and finally the stainless steel electrospray emitter. 

The prepared samples were infused at a rate of 10 µL/min to a LECO electrospray ionization (ESI) source that was interfaced with a LECO Citius LC-HRT high-resolution TOF-MS system. The instrument was operated in both negative-ion mode and positive-ion mode. Ion source parameters and optics voltages were optimized to maximize total ion transmission for each sample. For most samples, the high-resolution TOF-MS system was operated with the ultrahigh-resolution flight path (

 ≈ 100,000). To study the effect of flight path on the overall mass spectrum, a single sample was acquired in positive-ion mode using all three available flight paths:  nominal resolution (

In ultrahigh-resolution mode, the high-resolution TOF-MS system passes the ions of interest through the full flight path twice. This limits the first acquired time-of-flight and the last acquired time-of-flight to a 1:2 ratio, which corresponds to a 1:4 ratio of acquired 

. In other words, the acquisition is limited to a range of 

 400–1600, or any other 1:4 ratio. Because the most abundant signals in crude petroleum samples typically lie between 

 600, a single acquisition between these endpoints can capture sufficient information to adequately characterize the sample. If a wider spectral range is required, multiple acquisitions can be stitched together. 

All data were acquired and processed with LECO ChromaTOF-HRT software. For most samples, the data acquisition system distilled the profile transient data to centroided stick data before recording to disk. This normal mode of operation records all chemically relevant information without wasting disk space to record meaningless data points in between true signals. In addition to TOF and intensity information, each centroid datum also includes the measured width of the original profile peak. A centroid including TOF, intensity, and spectral peak width information is referred to as stick data. Because profile spectral peak width information is included, reconstructed profile spectra can be easily generated from stick data. To verify fidelity of the stick data, a single sample was acquired as both stick data and full profile data. 

Figure 3: Flight path comparison: (a) ultrahigh resolution (RFWHM = 100,000), (b) high resolution (RFWHM = 50,000), (c) nominal resolution (RFWHM = 1500).			

After acquisition, LECO ChromaTOF-HRT software was used to internally recalibrate the 

 axis and assign chemical formulas. Negative ion data were recalibrated using abundant highly saturated O

 class signals. Positive ion data were recalibrated using abundant highly saturated N class signals. 

Figure 3 compares the positive-ion ESI spectrum of the same sample acquired using three flight paths. No bias of intensity with respect to 

 is introduced by changing the flight path. Accordingly, all other results presented herein come from data acquired in ultrahigh-resolution mode. 

Figure 4: Resolved C30H33N and C27H37NS: (a) profile data, (b) stick data.			

Isobaric critical pairs arising from the exchange of C

 are among the most important signals to resolve in a crude petroleum spectrum, because accurate assessment of sulfur content by direct infusion ESI-MS depends on the ability to measure these signals independently. Figure 4 shows an isobaric doublet attributed to [C

 = 408.27195), from the same sample acquired in profile spectrum mode and stick spectrum mode. For context, Figure 5 shows a wider segment of the profile spectrum and the stick spectrum surrounding these isobars. The data from this pair of acquisitions confirms fidelity of the stick spectra. The most significant difference between the spectra can be seen at the baseline. The profile spectra contain multitudes of data points recorded across segments of mass defect that are devoid of true chemical signal, while the stick spectra exhibit negligible noise across these segments. Accordingly, all other results presented herein come from stick data. 

Figure 5: Wider segments of the spectra shown in Figure 4: (a): profile and (b): stick spectra.			

Negative ion ESI spectra of crude petroleum samples tend to present more heteroatomic classes than positive-ion ESI spectra, and more challenging isobaric doublets can be present. Figure 6 shows resolved M+1 signals from an abundant N

 class carbazole that overlap monoisotopic signals from two less abundant classes: N

. Signals shown in Figure 6 are listed in Table I. In the lower 

 region, as in Figure 6, these classes are resolved or partially resolved, and can be measured accurately. At higher 

 C isotopes of such carbazoles will completely obscure monoisotopic O

 class abundance could be estimated by accurately de-isotoping the data. 

Table I: Resolved to partially overlapping N1, N2, and O3 class signals			

Successfully de-isotoping spectra would require initial data of high isotopic fidelity. Although FTMS provides unmatched resolving power (>1,000,000), isotope intensity and the ability to de-isotope accuractely is a weakness of these systems compared to high-resolution TOF-MS. Figure 7 plots M+1 isotopic fidelity of the 100 most intense O

 class naphthenic acids in a typical negative-ion ESI spectrum. A couple of apparent outliers are attributed to spurious isobars of unknown origin; further investigation was not pursued. 

Characterization and Comparison of Samples

High-resolution MS allows rapid characterization and comparison of crude oil samples. Three samples from different sources, herein arbitrarily designated A, B, and C, were concomitantly analyzed. Figure 8 shows the summed intensity of selected heteroatom classes in each of these samples. Sample A appears richer in nitrogen than the other samples, and sample C appears enriched in some thiophenated species. The summed intensities also suggest that sample A is more acidic than the other samples. 

Figure 7: Isotopic fidelity of 100 naphthenic acids in a crude petroleum negative ion ESI spectrum obtained by high-resolution TOF-MS.			

Rings plus double bond equivalents (RDBE) plots illustrate useful information regarding the species present in each sample. Figure 9 compares the O

 class RDBE profile for each of the three samples. The increased overall intensity of sample A predicts a higher risk of processing equipment corrosion. All three profiles exhibit similar shoulders above an RDBE count of 5, consistent with the presence of oxidized aromatic species, thereby indicating similar extents of degradation. 

Figure 8: Summed intensities of selected heteroatom classes in each of the three different crude petroleum samples. 			

Figure 10 compares the N class RDBE profile for each of the three samples. The intense series of carbazole benzologues observed in sample A, and present relatively weakly in samples B and C, predict different requirements for hydrodenitrogenation in the crude processing. 

Figure 9: O2 class rings plus double bond equivalents (RDBE) comparison of three samples.			

The application of high-resolution TOF-MS to petroleomics is presented, and the basic theory of each type of ultrahigh-resolution mass spectral platform is briefly explained.

High-Resolution Time-of-Flight Mass Spectrometry for Rapid Petroleum Characterization

Two-dimensional gas chromatography–time-of-flight mass spectrometry (GC×GC–TOF-MS) is a valuable analytical method for use with passive sampling techniques for the detection of pollutants in water. With this method, it is possible to separate components that would be coeluted in one-dimensional GC, resulting in improved confidence in identification of pollutants. The combination of passive sampling and TOF-MS ensures high sensitivity, and the information-rich datasets mean that the method can be applied to the detection of nontarget compounds with little additional effort. 

In the last decade, regulators have faced increasing demands to protect and enhance water quality. In the European Union, these demands have led to the Water Framework Directive (WFD) for surface water, groundwater, and coastal waters, and the Marine Strategy Framework Directive for marine waters (1,2). 

The majority of analytes of concern in water are organic compounds, most of which are sufficiently volatile to be amenable to analysis by gas chromatography–mass spectrometry (GC–MS). Clearly, it is desirable to be able to monitor all such analytes in a single run using one analytical method, but the legislative demand for increased sensitivity and wider analyte scope requires a fresh look at the sampling and analytical methods used.  

Passive sampling exhibits considerable promise in this respect by concentrating pollutants within the sampler over time, and thus indirectly lowering detection limits (3,4). It also allows for the detection of trace (yet toxicologically relevant) contaminants over extended periods of time. Samplers can be deployed over extended periods, typically four weeks in a river system, providing a better estimate of a compound's mass loading on a watercourse and also substantially increasing the likelihood of capturing pollution events. 

Previous work examined the use of two passive samplers — a semipermeable membrane device (SPMD) and a polar organic chemical integrative sampler (POCIS) — for determining the presence of pollutants in groundwater, and found that they provided an improvement on the results obtained with conventional grab-sampling techniques (5). Such samplers are thus especially useful in investigative monitoring work, where there is no guarantee that the compounds giving rise to poor water quality will be on a target list. 

Figure 1: Analysis of river water by GCâMS passively sampled by a semipermeable membrane device (SPMD) deployed for 4 weeks. 			

SPMDs are used to monitor nonpolar organic compounds (defined as those with a log 

 >3) with a maximum cross-sectional diameter of 1 nm and a molecular mass less than 600 Da. Hydrophobic compounds such as polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and organochlorine pesticides in the dissolved state (that is, those that are readily bioavailable) will partition into the SPMD and become concentrated over time.  

To demonstrate the high compound loading and complexity of GC–MS chromatograms resulting from this sampling technique, Figure 1 shows the results obtained from GC–MS analysis of water sampled using an SPMD passive sampler from the River Loughor in South Wales, UK.  

The complexity of such extracts presents a tough analytical challenge for one-dimensional (1D) GC, particularly in the detection of closely eluted compounds and the identification of trace-level nontargets ("unknowns"). Clearly, therefore, reaping the full benefits of passive samplers requires a reassessment of the traditional GC–MS approach. 

In recent years, comprehensive two-dimensional GC (2D GC or GC×GC) has received increased attention for the analysis of complex samples, particularly in the areas of petrochemicals (6) and food analysis (7). The greater separation space available provides considerable advantages over 1D GC, and GC×GC is increasingly finding applications in mainstream analytical situations. We therefore realized that GC×GC could be an ideal approach to the problem of analyzing passively sampled pollutants in watercourses. 

Table I: Compounds identified from a deployed SPMD passive sampler, extracted and spiked with 55 pesticides. Present in the sample but not identified by either GCÃGCâTOF-MS or GCâquadrupole MS were dimethoate (60-51-5), cyanazine (21725-46-2), endosulfan I (959-98-8), endosulfan II (33212-65-9), pentachlorobenzene (608-93-5), and triallate (2303-17-5).			

The choice of GC×GC also naturally influences the mass spectrometer used. Although triple-quadrupole instruments are widely used for applications where sensitivity is paramount, time-of-flight (TOF) instruments are widely regarded as preferable for GC×GC applications. This is largely because of their inherently high scan rates (thousands of spectra accumulated per second), which allow them to handle the very narrow peaks produced by the second GC column. For this application, our desire for an analytical system able to identify targets and nontargets constrained our choice of TOF instrument to one able to produce spectra closely matching those in the National Institute of Standards and Technology (NIST) database. Table I gives an example of the sort of enhancement that can be achieved, by comparing the analysis of a spiked blank sample using GC×GC–TOF-MS with that using GC–quadrupole MS.  

This paper discusses the results of our investigations, and examines the benefits of passive sampling combined with GC×GC–TOF-MS for the identification of pollutants in watercourses. 

				A standard solution (10 ng/µL) was prepared from custom mixes of polychlorinated biphenyls (Restek), PAHs (SPEX CertiPrep), and organochlorine, organophosphate, and triazine pesticides (Restek). (See Table I for the full list of compounds in the mixture.)			

Standard-size SPMDs (91.4 cm × 2.5 cm low-density polyethylene tubing of 70–95 µm wall thickness) containing 1 mL of triolein were spiked with fluorene-

 (all at 10 µg) as performance reference compounds (Environmental Service Technologies). The SPMDs were shipped in gas-tight metal tins flushed with argon and were kept at –18 °C until deployed. 

At the sampling site, the SPMD was placed onto a stainless steel holder and then into a stainless steel protective cage for rapid deployment (to minimize exposure to atmospheric contaminants). A field blank was used at the deployment site to account for contamination during transport to and from study sites and exposure to airborne contaminants during the deployment and retrieval periods. 

After four weeks' exposure in the river, the SPMD was retrieved and sealed in the same tin as used for shipping to the field. Exposure periods across sampling sites were recorded. The frozen SPMDs including field blanks were shipped back to the laboratory and stored frozen until extracted. 

Following removal of surface biofouling using 

-hexane, gentle brushing with a soft toothbrush, and immersion in dilute acid, the surfaces of the SPMD were rinsed with water, acetone, and finally propan-2-ol. It was subsequently dialyzed twice in 

-hexane at 18 °C (for 18 h and 6 h), and the combined dialysates were reduced to about 3 mL. 

The dialysates were subjected to cleanup using size-exclusion chromatography (SEC) using an Agilent 1200 series HPLC system (Agilent Technologies). The SEC column was a 300 mm × 21.2 mm PL Gel column (10 µm particle size, 100 Å pore size), equipped with a 50 mm × 21.2 mm PL Gel guard column (Agilent Technologies). The mobile phase was 1:1 (v/v) 

-hexane–dichloromethane, with a flow rate of 5 mL/min. 

SEC-cleaned extracts were evaporated to 500 µL using a Turbovap (Biotage) under a flow of nitrogen at 40 °C, made up to 1 mL using 

-hexane, and split into two equal portions. The first was transferred to a 2-mL GC autosampler vial, and the second was spiked with 5 µL of the standard solution. 

Extracts were analyzed using a BenchTOF-dx TOF-MS system (ALMSCO International) and a model 7890A GC system (Agilent Technologies) fitted with a ZX1 cryogenic modulator (Zoex Inc.). Oven programming and modulation parameters were adjusted to yield a wide analyte volatility range and high peak capacity. 

The system used a PTV injector, a 1.8-mm i.d. baffled liner, helium carrier gas with a constant flow at 1.5 mL/min. The injection was performed in splitless mode for 2 min (then 100 mL/min purge). The temperature program was as follows: 60 °C for 0.05 min, 300 °C/min to 320 °C (1 min), 720 °C/min to 360 °C (cleaning phase) held to end of run. A 3-mL/min septum purge flow was used. 

The first-dimension column was a 30 m × 0.25 mm, 0.25-µm 

 SGE BPX5 column, and the second-dimension column was a 3 m × 0.1 mm, 0.1-µm 

 SGE BPX50 column. The modulation loop was the same as for the second dimension. The column set used equivalent pneumatic impedance to 60 m × 0.2 mm (calculated from impedance factor look-up charts for first- and second-dimension columns used). 

GCևC oven temperature program and modulation was as follows: main oven: 50 °C (2.0 min), 5 °C/min to 320 °C (8 min); secondary oven: not applicable; hot jet: 150 °C (2.0 min), 5 °C/min to 400 °C (hold time matched to total run time); cold jet: Dewar fill high 60%, Dewar fill low 50%; cold jet flow: 15 L/min; modulation period: 5 s; hot-jet pulse 350 ms; total run time: 64 min. 

The TOF-MS settings were as follows: filament voltage: 1.6 V; ion source: 300 °C; transfer line: 300 °C; mass range: 40–500 amu; data rate: 50 Hz. 

The platform-neutral software package GC Image (Zoex, Inc.) was used to process the native TOF-MS data. 

The results of the GC×GC analysis of the spiked and unspiked SPMD extract are shown in Figure 2. In both cases, notice the large number of components present, and the good separation in both dimensions. This allows the identification of compounds that would have been problematic in 1D GC because of coelution. The extra complexity of the spiked sample is demonstrated by the insets. 

Figure 2: Analysis of (a) spiked and (b) unspiked extract from an SPMD passive sampler, with insets showing the same zoomed-in region (with contrast enhanced). In the spiked sample, representative PCBs, PAHs, and pesticides are indicated by red, yellow, and blue circles respectively.			

PCBs were formerly used in many countries for a variety of purposes in industrial processes and in household products. Although now largely banned for many purposes, their continued use in certain situations and their environmental persistence means that contamination of watercourses may still occur. 

Because the PCBs in the SPMD extract are chemically similar to each other, they cover a well-defined region of the 2D chromatogram. Assignment of chlorination level was readily achievable, and Figure 3 shows an overlay of extracted-ion chromatograms for molecular ions of 22 dichloro- to heptachlorobiphenyls. 

Figure 3: Two-dimensional contour plots of the spiked extract from the SPMD passive sampler, with chlorinated biphenyls indicated by circles colored by chlorination level. The circle highlights what would be a coelution in one-dimensional GC.			

It is clear from Figure 3 that the second dimension has produced a significant improvement in separation. This level of speciation is important because of the need to report individual PCB congeners, not just isomeric groups, to environmental regulators. A case in point is the separation of a trichlorobiphenyl and a tetrachlorobiphenyl, which are coeluted (circled). Automated identification of these compounds with a 1D column setup would probably have required multiple runs under different conditions, so as to separate the compounds rather than having to rely on deconvolution. In addition, quantitation using extracted-ion chromatograms would have been compromised by the fact that these compounds have a number of shared ions in their electron ionization mass spectra. 

Figure 4: Three-dimensional surface plot showing the overlaid extracted-ion chromatograms for the molecular ions of dichloro- to heptachloro-biphenyls (m/z 222, 256, 292, 326, 360 and 394, each Â±1 amu) for the spiked watercourse sample.			

An additional feature worth noting is the peak shape (Figure 4), indicating the quality of the chromatography and the column modulation. 

PAHs result from the incomplete combustion of organic substances, and have long been recognized to be harmful to human health. They are prone to bioaccumulate in fatty tissues, and their prescribed maximum values in drinking water are correspondingly low (8). Drinking water can become contaminated with PAHs from a variety of sources, including leachate from linings in water storage tanks and pipelines. 

Figure 2 shows that 36 PAHs were identified in the spiked SPMD extract. As with PCBs, separation of individual compounds and isomers is highly desirable, so it is reassuring that all the PAHs are well separated. 

One feature of the chromatogram is the wrap-around of five PAHs (at the right-hand side of Figure 2). Such wraparound is not an uncommon feature of GC×GC analyses when detecting analytes with high boiling points that are highly retained on the secondary GC column. Although undesirable, wrap-around usually is not detrimental to the quality of the analysis; in most cases it can be remedied by using a separate oven for the second GC column, with an increasing temperature offset during the course of the analytical run, so that higher-boiling compounds do not wrap around. 

A carefully chosen analytical technique can enhance the inherent advantages of passive sampling of watercourses over more traditional "grab" or "spot" sampling.

Special Issues-03-01-2013

Targeted Protein Quantification Using High-Throughput Capillary LC–MS

Products

Rapid and Direct Quantitation of Pharmaceutical Drugs from Urine Using MALDI-MS

High-Resolution Time-of-Flight Mass Spectrometry for Rapid Petroleum Characterization

GCxGC–TOF-MS Complements Passive Sampling for the Screening of Pollutants in Water

Vol 11 No 1 Current Trends in Mass Spectrometry March 2013 Issue PDF