Dual isotope analysis

A dual isotope labeling technique [85] has been used to measure membrane permeability in plant cells, based on the selective permeabiHty of the membranes of living cells to tritiated water and carbon-14 labeled mannitol. Kieran [29] showed that the results of the dual isotope labeling and Evan s Blue staining methods correlated well as indicators of cell viability however, the latter was preferable in terms of reagent cost and ease of analysis. 

The use of carbon isotopes to study DOC is becoming more prevalent due to technological advances in mass spectrometry. DOC generally occurs in natural waters in low concentrations, typically ranging between 0.5 ppm and 10 ppm carbon (Thurman, 1985 see Chapter 5.10). Thus, several liters to tens of liters of water were once necessary to extract enough DOC for conventional dual gas-inlet isotopic analysis. Today, automated total organic carbon analyzers (TOCs) are commercially available, and have been successfully interfaced with continuous flow isotope ratio mass spectrometers (CF-IRMS) for stable isotopic measurements of samples containing ppb concentrations of DOC (e.g., St-Jean, 2003). 

SP-2401" and 3% SP-2250. ° Detectors used by EPA standards procedures, include photoionization (PID)," electron capture (ECD)," Eourier transform infrared spectrometry (PTIR), " and mass spectrometry detectors (MSD)." ° Method 8061 employs an ECD, so identification of the phthalate esters should be supported by al least one additional qualitative technique. This method also describes the use of an additional column (14% cyanopropyl phenyl polysiloxane) and dual ECD analysis, which fulfills the above mentioned requirement. Among MSDs, most of the procedures employ electron impact (El) ionization, but chemical ionization (CI) ° is also employed. In all MSD methods, except 1625, quantitative analysis is performed using internal standard techniques with a single characteristic m/z- Method 1625 is an isotope dilution procedure. The use of a FTIR detector (method 8410) allows the identification of specific isomers that are not differentiated using GC-MSD. 

Cylinders of the flashed gas were analysed for hydrocarbon gas composition on a Carle gas chromatograph (GC) system equipped with both a thermal conductivity detector (TCD) and a flame ionization detector (FID). An offline preparation system and dual inlet mass spectrometer (MS) were used to analyse the carbon and hydrogen isotopic values of hydrocarbon components. A customized Gow Mac GC was interfaced with a vacuum/combustion system to separate hydrocarbons from other components and combust to CO2 and water that were purified and sealed into Pyrex tubes for isotopic analysis. The CO2 was analysed directly on one of three dual inlet mass spectrometers Finnigan Delta S, Finnigan Delta + XL or VG SIRA II. The water was reacted with zinc turnings and converted to hydrogen gas, which was analysed on either the Delta S or Delta + XL MS. 

Radionuclidic purity is only of concern in the context of dual-isotope labeling, or if crosscontamination from a laboratory mishap is suspected. Radionuclidic purity is best measured by liquid scintillation counting modem LSC instruments have detectors and analysis software designed to discriminate quantitatively between the different isotopes used in the life sciences, except at very low counting levels. Radionuclidic purity is entirely distinct from isotopic purity, or content, of compounds labeled with stable isotopes, such as deuterium or carbon-13. Such information may be very important to the utility of stable-labeled compounds such as internal standards for mass spectrometric quantitation assays. ... 

Fig. 6.12 Sample pretreatment and conversion of organic compounds into gases for isotope ratio mass spectrometry (IRMS). The reductive pyrolysis is normally performed on glassy carbon (for a summary see e.g. [ 1291). In case of coupled HPLC-IRMS for determination wet oxidation is used [ 122f In the routine isotope ratio analysis of water often isotope equilibration with gases are used for 0-analysis CO2 [ 130], for S H-analysis H2 gas in the presence of Pt [131]. Recently an on-line method for S O and in water has been described showing dual-inlet like performance [ 132]. Low-temperature pyrolysis in connection with GC is used for compound fragmentation with the aim of partial isotope pattern analysis [133]. TC-EA = thermo conversion elemental analyser...

The reference principle for on-line methods has to be different as compared to classic dual-inlet isotope ratio analysis, as it is not possible to introduce sample and reference gas in exactly the same maimer into the mass spectrometer. Reference gas pulses have to be set at different points during one mn, but also laboratory standards with knovm 8-values have to be analysed periodically and exactly in the same manner and under the same conditions as the samples under investigation (identical treatment principle ]13]). A general problem of on-line HPLC and GC methods in isotope ratio analysis is the lack of international reference materials suitable to fulfil the above mentioned requirements. This implies the necessity to establish suitable laboratory standards by EA (elemental analysis) or classic measurements. Meanwhile, also efforts in supplying suitable international organic standards have been overtaken by the IAEA (e.g. caffeine, glutamic acid ]176, 177]). This programme (benzoic acid reference materials with different 8 0-values) will also support the on-line EA and GC 8 0-meas-urement by reductive pyrolysis (carbon reduction) methods (standardisation problems are compiled in ]178]). 

Lithium metabolism and transport cannot be studied directly, because the lack of useful radioisotopes has limited the metabolic information available. Lithium has five isotopes, three of which have extremely short half lives (0.8,0.2, 10 s). Lithium occurs naturally as a mixture of the two stable isotopes Li (95.58%) and Li (7.42%), which may be determined using Atomic Absorption Spectroscopy, Nuclear Magnetic Resonance Spectroscopy, or Neutron Activation analysis. Under normal circumstances it is impossible to identify isotopes by using AAS, because the spectral resolution of the spectrometer is inadequate. We have previously reported the use of ISAAS in the determination of lithium pharmacokinetics. Briefly, the shift in the spectrum from Li to Li is 0.015 nm which is identical to the separation of the two lines of the spectrum. Thus, the spectrum of natural lithium is a triplet. By measuring the light absorbed from hollow cathode lamps of each lithium isotope, a series of calibration curves is constructed, and the proportion of each isotope in the sample is determined by solution of the appropriate exponential equation. By using a dual-channel atomic absorption spectrometer, the two isotopes may be determined simultaneously. - ... 

Samples are analyzed by either dual inlet isotope ratio MS, or more commonly nowadays by continuous flow isotope ratio MS. The development of the latter technique has greatly increased the number of palaeodietary studies through the reduced analysis time and the resulting greater number of analyses performed. 

With a dual-inlet system, the samples for analysis are converted into gases offline. The offline sample preparation procedure utilizes a specially designed apparatus involving vacuum lines, compression pumps, concentrators, reaction furnaces, and microdistillation equipment. This technique is time consuming, usually requires larger samples, and contamination and isotopic fractionation can potentially occur at each of the steps [9]. 

While the dual inlet technique is generally the most precise method for stable isotope ratio measurements, the continuous-flow technique offers online sample preparation, smaller sample size requirements, faster and simpler analyses, increased cost-effectiveness, and the possibility of interfacing with other preparation techniques, including elemental analysis, gas chromatography (GC), and liquid chromatography (LC). For these reasons, CF-IRMS is the subject of the majority of research currently being conducted in the field of forensic science [1],... 

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