Isotope-dilution mass spectrometry analytical procedures

Some abuse drugs have been extracted from urine by SFE [viz. cocaine and its metabolites (134) and amphetamine and methamphetamine (135). In the first instance, the levels measured using SFE showed analyte recovery better than 70% for cocaine, better than 40% for benzoylecgonine, and better than 85% for ecgonine methyl ester from whole blood and urine. The limits of detection and quantitation were 1 and 10 ng, respectively, based on a 200-pL sample. Regarding amphetamine (AP) and methamphetamine (MA), an in situ SFE and chemical derivatization procedure followed by GC-isotope dilution mass spectrometry in urine was described. The mean recoveries achieved were 95% (RSD = 3.8%) for AP and 89% (RSD = 4%) for MA. The calibration graphs were linear within 100-500,000 ng/mL, varying the limits of detection and quantitation from 19 to 50 and from 21 to 100 ng/mL, respectively. 

The book offers the reader in its first part a general and as detailed as necessary introduction into the basic principles and methods, starting with sampling, sample storage and sample treatment. These steps are of utmost importance for each analytical procedure. This is followed by the description of the potential of a number of modern trace analytical methods, i.e. atomic absorption and emission spectrometry, voltammetry, neutron activation and isotope dilution mass spectrometry. The latter method is an important reference method within a general concept for quality control and the generation of reference materials which are an absolute must in this context. 

A widely accepted quantification procedure for accurate elemental analysis and isotope ratio measurements is isotope dilution mass spectrometry. In this procedure the sample is spiked with a known amount of a less-abundant enriched isotope of the same element, and the ratio of the unknown number of analyte atoms to the known number of the spike atoms is measured using mass spectrometry. 

Some analytes are difficult to standardize owing to the complexity of developing a primary standard, or analyte inhomogeneity, an International Standard is used to provide comparability. Such standards are based on the principle of transferability. A definitive method, e.g., isotope-dilution mass spectrometry (MS), is used to obtain the best possible estimate of the accurate concentration of the analyte. This value would be transferred to a reference method, which is usually a very carefully documented analytical procedure of known and impeccable performance, e.g., Abel-Kendal assay for cholesterol, and standards are compared in the procedure before being used in the filed. 

Surrogate Internal Standard (SIS), often just referred to as Internal Standard (IS) A compound of known chemical purity that is added, in a known amount, to the sample for analysis before any extraction and work-up procedures used to monitor and correct for analyte losses and variations in instrumental sensitivity with mass spec-Iromelric detection isotope-labeled versions of the analyte (isotopologs) are the best (this procedure is referred to as isotope dilution mass spectrometry, IDMS) but isomers or analogs can be used, see Table 2.1. 

It seems to us that the only method available for the assessment of accuracy is by comparison to a definitive method, which is generally accepted to be isotope dilution mass spectrometry (74). Such methods are not widely available for vitamin D metabolites but will be discussed later on in this chapter. The use of such methodology is also subject to criticism in that the definitive GC-MS procedure may itself be flawed. It may be necessary in stage 3 to demonstrate the identity of the analyte, which can be done by a number of physicochemical means (such as UV and/or infrared spectra, nuclear magnetic resonance [NMR], mass spectrometry, etc.). These techniques have been reviewed (7) and will be dealt with later on in this chapter only insofar as they pertain to the solution of this problem in the field of vitamin D. There are a number of published reviews of vitamin D methodology which deal with both the early methodology and more recent developments (6,7,75-82). 

Mass spectrometry is increasingly used in vitamin Be analysis. Hachey et al. (155) described analysis of Be vitamers in biological samples by isotope dilution mass spectrometry. Deuterated forms of the different Be vitamers were added in the early stages of the sample preparation procedure these deuterated vitamers were used as internal standards to compensate for analytical losses during the isolation and derivatization steps. The Bg vitamers in the homogenized tissue sample were separated by cation exchange HPLC as described previously (76). Acetylation was chosen as derivatization procedure for GLC. However, prior to... 

Of the various analytical techniques, methods using mass spectrometry with isotope dilution play a special role. Initially they were used for determination of inorganic analytes, but later, in the last decades of twentieth century, they were also used for the analysis of organic species. These methods require the use of isotope-labeled compounds, but lead to very good precision and accuracy, as required in definitive procedures [14]. 

Chemical separations may be specific for the analyte of interest (see Chapter 3), such as liquid or gas chromatography, or scavenging (such as by precipitation) to remove the major interfering substances. Addition of carrier, as practiced in radioanalytical chemistry to assist in purifying radionuclides, usually is not appropriate for mass spectrometric analysis. Such addition undermines the isotopic ratio measurements that are often at the heart of this procedure, and also overloads the system for ion generation and peak resolution (but carrier addition is used for accelerator mass spectrometry). Addition of tracers, known as isotope dilution, is often employed for yield determination (see Section 17.2.9). Interferences are distinctly different in radiometric and MS analyses of radionuclides, and may be the deciding factor in selecting one method versus the other. 

Analytical procedures have been developed utilising chemical ionisation mass spectrometry in conjunction with stable isotope labelled amines to obtain quantitative information on the metabolism of (/ -), (.S-), and (R.S-) methyldopa (alpha-methyldopa) and the regional concentrations of dopamine, a-methyl-dopamine, noradrenaline (norepinephrine) and a-methyinoradrenaline in rat brain. C-labelled (5)-a-methyldopa, as well as deuterated amines, were employed. Isotope dilution techniques provided good quantitative data [164]. 

Inductively coupled plasma—mass spectrometry (referred to in this book as ICP-MS), a technique for the elemental chemical characterization of virtually any material, evolved during the late 1990s into a mature analytical procedure. This technique has a variety of characteristics that make it uniquely suited for the solution of chemical analysis problems in many applications. These characteristics include the abihty to precisely identify and measure (quantitate) all elements in the periodic table including the often difficult to analyze refractory elements. In addition to this wide scope of elemental analysis, the technique has the inherent capabihty to perform these determinations in a multielement analysis mode, efficiendy providing comprehensive elemental compositional characterization. The technique also has the powerful ability to measure individual isotopes of the analyte elements, providing a capability that has many useful apphcations ranging from isotope dilution quantitation to stable isotope tracer studies. 

Unlike other isotope dilution analytical methods (i.e., thermal ionization mass spectrometry), separation and isolation of the analyte species are not required, greatly simplifying the determination procedure. 

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