Isotope abundance analysis for improved sample identification with tandem mass spectrometry

Tandem mass spectrometry (MS/MS) is widely used for trace level sample analysis in complex mixtures. However, sample identification in MS/MS is challenging and not as trustworthy as with electron ionization (EI) mass spectral libraries. This paper presents a novel method for the combination of isotope abundance analysis (IAA) and EI‐MS/MS for improved sample identification even at trace level in complex matrices. Accordingly, the first quadrupole is scanned in a narrow range around the molecular ion group of isotopomers such as M⁺, [M+1]⁺ and [M+2]⁺, Q2 serves for collision‐induced dissociation to produce product ions while Q3 transfers the major sample product ions with low resolution, thus encompassing and uniformly transmitting all the product ion isotopomers. IAA can then be used to derive elemental formula information from the cleansed experimental data. IAA‐MS/MS was experimentally tested with perfluorotributylamine and a very good matching factor of 995 (out of 1000) was obtained for IAA on m/z 502, 503 and 504 (fragment ion isotopomers) while Q3 transmitted the m/z 264 product ion with a mass window of 6 m/z units. The IAA‐MS/MS method was further tested with the pesticide diazinon on its molecular ions m/z 304, 305 and 306 while Q3 was locked on its m/z 179 product ion with a mass window of 6 m/z units. Again, very good matching factors were obtained, even for 40 pg diazinon on‐column during its GC/MS analysis (match = 981). IAA‐MS/MS combines the traditional benefits of MS/MS in the removal of matrix interferences with the IAA power of elemental analysis. Copyright © 2009 John Wiley & Sons, Ltd.     

Cluster chemical ionization for improved confidence level in sample identification by gas chromatography/mass spectrometry

Upon the supersonic expansion of helium mixed with vapor from an organic solvent (e.g. methanol), various clusters of the solvent with the sample molecules can be formed. As a result of 70 eV electron ionization of these clusters, cluster chemical ionization (cluster CI) mass spectra are obtained. These spectra are characterized by the combination of EI mass spectra of vibrationally cold molecules in the supersonic molecular beam (cold EI) with CI-like appearance of abundant protonated molecules, together with satellite peaks corresponding to protonated or non-protonated clusters of sample compounds with 1-3 solvent molecules. Like CI, cluster CI preferably occurs for polar compounds with high proton affinity. However, in contrast to conventional CI, for non-polar compounds or those with reduced proton affinity the cluster CI mass spectrum converges to that of cold EI. The appearance of a protonated molecule and its solvent cluster peaks, plus the lack of protonation and cluster satellites for prominent EI fragments, enable the unambiguous identification of the molecular ion. In turn, the insertion of the proper molecular ion into the NIST library search of the cold EI mass spectra eliminates those candidates with incorrect molecular mass and thus significantly increases the confidence level in sample identification. Furthermore, molecular mass identification is of prime importance for the analysis of unknown compounds that are absent in the library. Examples are given with emphasis on the cluster CI analysis of carbamate pesticides, high explosives and unknown samples, to demonstrate the usefulness of Supersonic GC/MS (GC/MS with supersonic molecular beam) in the analysis of these thermally labile compounds. Cluster CI is shown to be a practical ionization method, due to its ease-of-use and fast instrumental conversion between EI and cluster CI, which involves the opening of only one valve located at the make-up gas path. The ease-of-use of cluster CI is analogous to that of liquid CI in ion traps with internal ionization, and is in marked contrast to that of CI with most other standard GC/MS systems that require a change of the ion source.     

Isotope dilution gas chromatography/mass spectrometry for platinum determination in urine

The therapeutic importance of platinum (Pt) compounds, the growing accessibility of gas chromatography/mass spectrometry (GC/MS) systems in clinical laboratories, and the lack of a mass spectrometric method for the determination of Pt in biological samples motivated us to develop an isotope dilution GC/MS assay for Pt. The method is based on the use of lithium bis(trifluoroethyl) dithiocarbamate, Li(FDEDTC), as a chelating agent and enriched ¹⁹²Pt for isotope dilution. Conditions were optimized for the precise and accurate determination of isotope ratios of Pt by using a 10-m DB-l fused silica capillary column and a reverse-geometry double-focusing mass spectrometer with selected ion monitoring. An overall precision of 1% was obtained by combining within-run precision and between-run precision at the 10-ng level. No appreciable memory effect was observed when samples with different isotope ratios were analyzed sequentially. The method was validated by the quantitation of Pt in National Institute of Standards and Technology freeze-dried urine sample SRM 2670. A concentration value of 125 ± 6 /Lg/L (n = 6) was obtained by using four different sets of isotope ratios in the molecular ion and supports the National Institute of Standards and Technology recommended value of 120 ± ? μg/L. Limits-of-quantitation, estimated at 3 μg/L, are made possible by the high sensitivity of the method and the low blank value for Pt.     

Classical electron ionization mass spectra in gas chromatography/mass spectrometry with supersonic molecular beams

A major benefit of gas chromatography/mass spectrometry (GC/MS) with a supersonic molecular beam (SMB) interface and its fly-through ion source is the ability to obtain electron ionization of vibrationally cold molecules (cold EI), which show enhanced molecular ions. However, GC/MS with an SMB also has the flexibility to perform 'classical EI' mode of operation which provides mass spectra to mimic those in commercial 70 eV electron ionization MS libraries. Classical EI in SMB is obtained through simple reduction of the helium make-up gas flow rate, which reduces the SMB cooling efficiency; hence the vibrational temperatures of the molecules are similar to those in traditional EI ion sources. In classical EI-SMB mode, the relative abundance of the molecular ion can be tuned and, as a result, excellent identification probabilities and very good matching factors to the NIST MS library are obtained. Classical EI-SMB with the fly-through dual cage ion source has analyte sensitivity similar to that of the standard EI ion source of a basic GC/MS system. The fly-through EI ion source in combination with the SMB interface can serve for cold EI, classical EI-SMB, and cluster chemical ionization (CCI) modes of operation, all easily exchangeable through a simple and quick change (not involving hardware). Furthermore, the fly-through ion source eliminates sample scattering from the walls of the ion source, and thus it offers full sample inertness, tailing-free operation, and no ion-molecule reaction interferences. It is also robust and enables increased column flow rate capability without affecting the sensitivity.     

Hydrocarbons and fuels analyses with the supersonic gas chromatography mass spectrometry--the novel concept of isomer abundance analysis

Hydrocarbon analysis with standard GC-MS is confronted by the limited range of volatile compounds amenable for analysis and by the similarity of electron ionization mass spectra for many compounds which show weak or no molecular ions for heavy hydrocarbons. The use of GC-MS with supersonic molecular beams (Supersonic GC-MS) significantly extends the range of heavy hydrocarbons that can be analyzed, and provides trustworthy enhanced molecular ion to all hydrocarbons. In addition, unique isomer mass spectral features are obtained in the ionization of vibrationally cold hydrocarbons. The availability of molecular ions for all hydrocarbons results in the ability to obtain unique chromatographic isomer distribution patterns that can serve as a new method for fuel characterization and identification. Examples of the applicability and use of this novel isomer abundance analysis (IAA) method to diesel fuel, kerosene and oil analyses are shown. It is suggested that in similarity to the "three ions method" for identification purposes, three isomer abundance patterns can serve for fuel characterization. The applications of the Supersonic GC-MS for engine motor oil analysis and transformer oil analysis are also demonstrated and discussed, including the capability to achieve fast 1-2s sampling without separation for oil and fuel fingerprinting. The relatively fast analysis of biodiesel is described, demonstrating the provision of molecular ions to heavy triglycerides. Isomer abundance analysis with the Supersonic GC-MS could find broad range of applications including petrochemicals and fuel analysis, arson analysis, environmental oil/fuel spill analysis, fuel adulteration analysis and motor oil analysis.     

Isotope dilution gas chromatography/mass spectrometry method for determination of pyrethroids in apple juice

This paper presents the development of a highly precise and accurate analytical method for the determination of three matrix-bound pyrethroids, namely, cypermethrin, permethrin, and bifenthrin, using an isotope dilution gas chromatography/mass spectrometry technique. Identification of the analytes was confirmed under selective ion monitoring mode by the presence of two dominant ion fragments within specific time windows and matching of relative ion intensities of the ions concerned in samples and calibration standards. Quantitation was based on the measurement of concentration ratios of the natural and isotope analogues in the sample and calibration blends. Intraday and interday repeatabilities of replicate analyses of the pyethroids in an apple juice sample were below 0.5%. The expanded relative uncertainty ranged from 3 to 6%, which was significantly lower than the range obtained using internal or external calibration methods. As a labeled analogue is not available for bifenthrin, bifenthrin was determined using labeled cis-permethrin as the internal standard. The results were counterchecked by a gas chromatography-electron capture detection technique using PCB 209 as the internal standard. The method developed was applied to a recent pilot study organized by CCQM and the results were consistent with those of other participants.     

Supersonic gas chromatography/mass spectrometry

A new gas chromatography/mass spectrometry (GC/MS) system was designed and evaluated which we have named 'Supersonic GC/MS'. It is based on a modification of a commercially available GC/MS system to include a supersonic molecular beam (SMB) MS interface. In this system the standard electron ionization (EI) ion source was replaced with a fly-through EI ion source mounted in the path of the SMB. A hyperthermal surface ionization (HSI) ion source combined with a 90 degrees ion mirror (for the EI-produced ions) was also added, and placed inside the quadrupole mass analyzer in place of its original EI ion source. The 'Supersonic GC/MS' system requires 18 cm added bench space plus the addition of an air-cooled 60 L/s diffusion pump and a 537 L/min rotary pump. The system is user friendly since all the gas flow rates, heated zones, sampling and data analysis are performed the same way as the original system and are computer-controlled via the original software. Similar EI sensitivity was obtained as with the original system for hexachlorobenzene and octafluoronaphthalene, while improved EI detection limits were demonstrated for methyl stearate and eicosane due to the significant enhancement of their molecular ion abundances. A GC/MS detection limit of 500 ag for pyrene was demonstrated using HSI. Good supersonic expansion cooling was achieved with large alkanes, despite the use of a rotary pump at the nozzle chamber instead of a diffusion pump. High temperature GC/MS analysis was demonstrated for large polycyclic aromatic hydrocarbons (PAHs) including ovalene and decacyclene (ten rings). Library searches with EI mass spectra are demonstrated, and it is explained why the enhancement of the molecular ion actually improves the library search in most cases. The analysis of large phthalate esters is also described, and the improvement obtained is shown to originate from their enhanced molecular and high mass fragment ions.     

气相色谱-质谱分析血清葡萄糖的前处理方法研究

用气相色谱-同位素稀释质谱法(GC-ID/MS)测定血清葡萄糖,需要对血清样品进行前处理,为确保测定结果的准确性,比较了乙醇、丙酮、乙腈沉淀及微过滤4种去除蛋白的方法.将用上述4种方法处理的血清样品进行衍生,采用GC-ID/MS测定,得到其精密度、相对回收率和绝对回收率,加以比较.结果表明:4种方法中,乙醇去除血清中蛋白的效果最佳.     

Early gas chromatography/mass spectrometry

Spectroscopy Laboratory, The Dow Chemical Company, Midland, Michigan, USA In December 1955 or thereabouts, the authors coupled a homemade gas chromatograph to a research time-of-flight mass spectrometer constructed by W. C. Wiley, I. H. McLaren, and D. B. Harrington. This unique gas chromatography/mass spectrometry (GC/MS) instrument generated mass spectra at a lo-kHz rate for display on an oscilloscope; eluted gas chromate graphic components, such as methanol, acetone, benzene, toluene, and carbon tetrachloride, could be visually identified immediately from the oscilloscope display. Many years of further research and development in many laboratories worldwide were necessary, however, to make continuous on-line GC/MS the uniquely valuable analytical tool that it is today.     

Liquid chromatography mass spectrometry with supersonic molecular beams

A new approach for liquid chromatography mass spectrometry (LC-MS) is described, based on achieving soft thermal vaporization followed by supersonic expansion and direct sample compound ionization, while in a supersonic molecular beam (SMB). The soft molecular vaporization step utilizes spray formation that is continued by fast thermal vaporization inside a channel supersonic nozzle, followed by ultrafast supercooling in a supersonic expansion. The short time (several microseconds) spent by the vaporized compound in the heated nozzle prior to its expansion cooling may result in incomplete vibrational equilibrium and thus reduced degree of dissociation. In addition, even if vibrational equilibrium at the nozzle temperature is obtained, the sample compounds have significantly reduced time for their dissociation, which is thus further minimized (kinetic consideration). As soon as the molecules expand and form a SMB, they are supercooled and any further dissociation is avoided. While in the SMB, the sample molecules can be ionized either by electron ionization as described in this paper or by hyperthermal surface ionization. The major goal of this method is to obtain high quality library searchable electron ionization mass spectra, for a broad range of thermally labile compounds, with higher sensitivity than that achievable by particle beam LC-MS. The soft thermal vaporization nozzle is described and mass spectral results with corticosterone are demonstrated. The potential advantageous features of this new method are discussed.     

A single glucose derivative suitable for gas chromatography/mass spectrometry and gas chromatography/combustion/isotope ratio mass spectrometry

The incorporation of stable isotopes improves the assessment of glucose metabolism and, with some researchers using two tracers, (2)H-glucose assessed by gas chromatography/mass spectrometry (GC/MS) and (13)C-glucose by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS), a common derivative for both is advantageous. The most commonly used derivatives for GC/MS are inappropriate for GC/C/IRMS as additional functional groups dilute the label. We therefore considered the suitability of six derivatives for both GC/MS and GC/C/IRMS. Glucose alkylboronates were prepared by adding the appropriate alkylboronic acid (butyl- or methylboronic acid) in pyridine to desiccated glucose. The derivatisation was completed by reacting this with either (a) acetic anhydride or trifluoroacetic anhydride (acetate derivatives) or (b) bis(trimethylsilyl)trifluoroacetamide BSTFA (TMS derivatives). All six derivatives were assessed using GC/MS and (13)C GC/C/IRMS.Neither TMS derivative exhibited any signal intensity in the molecular ion, although a M-15 ion showed good agreement between experimental and theoretical data and, whilst still low in intensity, could be suitable for isotope work. Similarly, none of the acetate derivatives showed any intensity at the molecular ion although three key fragmentation series were identified. The most attractive sequence, initiated by the loss of 1,2 cyclic boronate, resulted in the main fragment ion of interest, m/z 240, corresponding to the fluorinated methylboronate derivate. Minimal carbon and hydrogen atoms are added to this derivative making it an excellent choice for stable isotope work, while proving suitable for analysis by both GC/MS and GC/C/IRMS.     

Perdeuterated n-alkanes for improved data processing in thermal desorption gas chromatography/mass spectrometry I. Retention indices for identification

The identification of organic compounds by GC/MS is useful in various areas such as fuel, indoor and outdoor air and flavour and fragrance applications. Multi-compound mixtures often contain isomeric compounds which have similar mass spectra and sometimes cannot be unambiguously identified by library search alone. Retention indices can help with confirmation of identification if they are reproducible. Using perdeuterated n-alkanes as a reference series for calculation of retention indices in GC/MS has a clear benefit because of the distinctive ion trace of m/z 34. Thermal desorption is useful for analysis of volatile organic compounds (VOCs) in air after sampling on appropriate sorbent cartridges. Comparison of indices between three systems, consisting of a thermal desorption unit, a gas chromatograph and a mass spectrometer, showed good agreement for compounds with well-defined peaks, whereas retention times varied.     

Isotope dilution high-resolution gas chromatography/high-resolution mass spectrometry method for analysis of selected acidic herbicides in surface water

In this work, an isotope dilution method for determination of selected acidic herbicides by high-resolution gas chromatography/high-resolution mass spectrometry (HRGC/HRMS) was developed for surface water samples. Average percent recoveries of native analytes were observed to be between 70.8 and 93.5% and average recoveries of labeled quantification standards [(13)C(6)]2,4-D and [(13)C(6)]2,4,5-T were 85.5 and 101%, respectively. Using this method, detection limits of 0.05 ng/L for dicamba, MCPA, MCPP, and triclopyr, and 0.5 ng/L for 2,4-D were routinely achieved. The method was applied to measuring the concentration of these analytes in surface water samples collected from five sampling locations in the Lower Fraser Valley region of British Columbia, Canada. All of the herbicides monitored were detected at varying levels in the surface water samples collected. The highest concentrations detected for each analyte were 345 ng/L for 2,4-D, 317 ng/L for MCPA, 271 ng/L for MCPP, 15.7 ng/L for dicamba, and 2.18 ng/L for triclopyr. Average detection frequencies of the herbicides were 95% for MCPA, 80% for MCPP, 70% for dicamba, 65% for 2,4-D, and 46% for triclopyr. Seasonal variations of herbicide levels are also discussed.     

Integration of gas chromatography mass spectrometry methods for differentiating ricin preparation methods

The investigation of crimes involving chemical or biological agents is infrequent, but presents unique analytical challenges. The protein toxin ricin is encountered more frequently than other agents and is found in the seeds of Ricinus communis, commonly known as the castor plant. Typically, the toxin is extracted from castor seeds utilizing a variety of different recipes that result in varying purity of the toxin. Moreover, these various purification steps can also leave or differentially remove a variety of exogenous and endogenous residual components with the toxin that may indicate the type and number of purification steps involved. We have applied three gas chromatography-mass spectrometry (GC-MS) based analytical methods to measure the variation in seed carbohydrates and castor oil ricinoleic acid, as well as the presence of solvents used for purification. These methods were applied to the same samples prepared using four previously identified toxin preparation methods, starting from four varieties of castor seeds. The individual data sets for seed carbohydrate profiles, ricinoleic acid, or acetone amount each provided information capable of differentiating different types of toxin preparations across seed types. However, the integration of the data sets using multivariate factor analysis provided a clear distinction of all samples based on the preparation method, independent of the seed source. In particular, the abundance of mannose, arabinose, fucose, ricinoleic acid, and acetone were shown to be important differentiating factors. These complementary tools provide a more confident determination of the method of toxin preparation than would be possible using a single analytical method.     

Isotope ratio mass spectrometry coupled to liquid and gas chromatography for wine ethanol characterization

Two new procedures for wine ethanol 13C/12C isotope ratio determination, using high-performance liquid chromatography and gas chromatography isotope ratio mass spectrometry (HPLC/IRMS and GC/IRMS), have been developed to improve isotopic methods dedicated to the study of wine authenticity. Parameters influencing separation of ethanol from wine matrix such as column, temperature, mobile phase, flow rates and injection mode were investigated. Twenty-three wine samples from various origins were analyzed for validation of the procedures. The analytical precision was better than 0.15 per thousand, and no significant isotopic fractionation was observed employing both separative techniques coupled to IRMS. No significant differences and a very strong correlation (r = 0.99) were observed between the 13C/12C ratios obtained by the official method (elemental analyzer/isotope ratio mass spectrometry) and the proposed new methodology. The potential advantages of the developed methods over the traditional one are speed (reducing time required from hours to minutes) and simplicity. In addition, these are the first isotopic methods that allow 13C/12C determination directly from a liquid sample with no previous ethanol isolation, overcoming technical difficulties associated with sample treatment.     

A low thermal mass fast gas chromatograph and its implementation in fast gas chromatography mass spectrometry with supersonic molecular beams

A new type of low thermal mass (LTM) fast gas chromatograph (GC) was designed and operated in combination with gas chromatography mass spectrometry (GC-MS) with supersonic molecular beams (SMB), including GC-MS-MS with SMB, thereby providing a novel combination with unique capabilities. The LTM fast GC is based on a short capillary column inserted inside a stainless steel tube that is resistively heated. It is located and mounted outside the standard GC oven on its available top detector port, while the capillary column is connected as usual to the standard GC injector and supersonic molecular beam interface transfer line. This new type of fast GC-MS with SMB enables less than 1 min full range temperature programming and cooling down analysis cycle time. The operation of the fast GC-MS with SMB was explored and 1 min full analysis cycle time of a mixture of 16 hydrocarbons in the C(10)H(22) up to C(44)H(90) range was achieved. The use of 35 mL/min high column flow rate enabled the elution of C(44)H(90) in less than 45 s while the SMB interface enabled splitless acceptance of this high flow rate and the provision of dominant molecular ions. A novel compound 9-benzylazidanthracene was analyzed for its purity and a synthetic chemistry process was monitored for the optimization of the chemical reaction yield. Biodiesel was analyzed in jet fuel (by both GC-MS and GC-MS-MS) in under 1 min as 5 ppm fatty acid methyl esters. Authentic iprodion and cypermethrin pesticides were analyzed in grapes extract in both full scan mode and fast GC-MS-MS mode in under 1 min cycle time and explosive mixture including TATP, TNT and RDX was analyzed in under 1 min combined with exhibiting dominant molecular ion for TATP. Fast GC-MS with SMB is based on trading GC separation for speed of analysis while enhancing the separation power of the MS via the enhancement of the molecular ion in the electron ionization of cold molecules in the SMB. This paper further discusses several features of fast GC and fast GC-MS and the various trade-offs involved in having powerful and practical fast GC-MS.     

Validation of pentaacetylaldononitrile derivative for dual 2H gas chromatography/mass spectrometry and 13C gas chromatography/combustion/isotope ratio mass spectrometry analysis of glucose

A reference method to accurately define kinetics in response to the ingestion of glucose in terms of total, exogenous and endogenous glucose is to use stable‐isotope‐labelled compounds such as ²H and ¹³C glucose followed by gas chromatography/mass spectrometry (GC/MS) and gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) analysis. The use of the usual pentaacetyl (5Ac) derivative generates difficulties in obtaining accurate and reproducible results due to the two chromatographic peaks for the syn and anti isomers, and to the isotopic effect occurring during acetylation. Therefore, the pentaacetylaldononitrile derivative (Aldo) was validated for both isotopes, and compared with the 5Ac derivative. A correction factor including carbon atom dilution (stoichiometric equation) and the kinetic isotopic effect (KIE) was determined. Analytical validation results for the ²H GC/MS and ¹³C GC/C/IRMS measurements produced acceptable results with both derivatives. When ²H enrichments of plasma samples were ≤1 mol % excess (MPE), the repeatability (RSD_{Aldo Intra assay and Intra day} <0.94%, RSD_{5Ac Intra assay and Intra day} <3.29%), accuracy (Aldo <3.4%, 5Ac <29.0%), and stability of the derivatized samples were significantly better when the Aldo derivatives of the plasma samples were used (p < 0.05). When the glucose kinetics were assessed in nine human subjects, after glucose ingestion, the plasma glucose ²H enrichments were identical with both derivatives, whereas the ¹³C enrichments needed a correction factor to fit together. Due to KIE variation, this correction factor was not constant and had to be calculated for each batch of analyses, to obtain satisfactory results. Mean quantities of exogenous glucose exhibit marked difference (20.9 ± 1.3g (5Ac) vs. 26.7 ± 2.5g (Aldo)) when calculated with stoichiometric correction, but fit perfectly when calculated after application of the correction factor (22.1 ± 1.3g (5Ac) vs. 22.9 ± 1.9g (Aldo)). Finally, the pentaacetylaldononitrile derivative, used here in GC/C/IRMS for the first time, enables measurement of ²H and ¹³C enrichments in plasma glucose with a single sample preparation. Copyright © 2009 John Wiley & Sons, Ltd.