Selected reaction monitoring instrumentation

The instrument scan mode called selected reaction monitoring (SRM) is generally used for quantitative applications. SRM is similar to selected ion monitoring (SIM) in single quadrupole MS. The difference is that a product ion from the decomposition reaction in the collision cell is measured instead of a single ion formed in the... 

The major advantage of this instrument is that qualitative and quantitative analysis can be performed in the same LC-MS run. As an example in a data-dependent experiment, the selected reaction monitoring mode can be used as a survey scan and the enhanced product ion mode (EPI) as a dependent scan. The consequence is that for each quantified analyte a confirmatory MS/MS spectmm can be obtained. 

Li, A. C. Alton, D. Bryant, M. S. Shou, W. Z. Simultaneously quantifying parent drugs and screening for metabolites in plasma pharmacokinetic samples using selected reaction monitoring information-dependent acquisition on a QTrap instrument. Rupid Commun Mass Spectrom 2005, 19, 1943-1950. 

The analysis is performed on a Thermo TSQ Quantum AM instrument. The total analysis time is 8 min. The analytes are quantified by selected reaction monitoring using the decays listed in Table 2.2.5 (See Fig. 2.2.9). 

As stated further above, under all circumstances trueness, accuracy and sensitivity of the assay should be demonstrated on a sufficient number of patient samples. In our view, however, the use of an additional, also easily realizable chromatographic dimension (online-SPE-LC-MS/MS) [67, 68] without a doubt represents the analytical state of the art in immunosuppressant TDM. Nowadays tandem MS instruments are used almost exclusively for the detection and quantification of analytes. The detection of analytes is generally performed in the selected reaction monitoring (SRM, synonym MRM) mode. Depending on which instrumentation is used, an analysis can be completed within two to four minutes. 

The HPLC/MS/MS assays of other CYP enzymes are very similar in principle and use the identical instrumentation but employ different internal standards. As a consequence of the high degree of specificity of MS/MS selected reaction monitoring, batteries of CYP assays can be robotically programmed for high throughput with little additional manpower. 

The use of selected reaction monitoring (SRM) methods for quantitative bioanalysis represents increased dimensions of mass spectrometry analysis. A SRM method that features a tandem quadrupole MS/MS instrument for the quantitative analysis of an antipsychotic agent, clozapine, in human plasma was recently described by Dear et al. l Preclinical development studies of clozapine in rats and dogs used HPLC with fluorescence detection (FLD). With this method, a better limit of quantitation (LOQ) of Ing/ml was obtained. As the compound moved into the clinical stages of development, a more sensitive method of... 

For application with triple-quadrapole and especially (J-LIT instruments, a selected-reaction monitoring (SRM) procedure was developed for the sensitive and selective detection of phosphopeptides in proteomes with known amino-acid sequences [26]. A list of SRM transitions of potential phosphopeptides is generated for all expected tryptic peptides in the mixtme with Ser, Thr, or Tyr and for double-and triple-charge ions in the mass range of m/z 400-1600. The number of transitions included is limited by the maximum cycle time of 10 s, which assures that peptides in a 30 s wide peak are at least analysed twice. The procedure was applied to the cell cycle regulatory protein Cyclin B from Schizosaccharomyces pombe. 

When the first edition of this book was pubhshed early 1992, LC-MS conld already be considered an important and mature analytical technique. However, at that time, the great impact on LC-MS that electrospray and atmospheric-pressure chemical ionization (APCl) would have could already be foreseen. Since then, the versatihty and application of LC-MS really exploded. Numerous LC-MS systems have been sold in the past 6 years and have found their way into many different laboratories, although the pharmaceutical applications of LC-MS appear to be most important, at least in terms of instrument sales. LC-MS-MS in selective reaction monitoring mode has now become the method of choice in quantitative bioanalysis. 

Figure 7 Scan modes for a tandem-in-space instrument, the triple quadruple (QqQ). (a) Full scan all source ions are passed through to Q3 while Q1 and q (collision cell) are set to the RF-only mode, (b) Production scan Qi is set to pass a selected ion (precursor ion). This is fragmented in the collision cell and products are analyzed by scanning Q3. (c) Precursor scan Q1 scans all the source ions into the collision cell for collision-induced dissociation (CID). Q3 is set to pass a selected product ion. A signal recorded at Q3 is correlated with the corresponding precursor ion passing through Q-i. (d) Neutral loss scan Q-i is set to scan ions into the collision cell for CID. The Q3 scan is offset by a specified mass, equal to the mass of the neutral, relative to Qi. (e) Selected reaction monitoring (SRM) an ion selected in Q1 is fragmented and a specific fragment is then recorded after selection by Q3. SRM is commonly used in quantitative work to improve assay selectivity and sensitivity.

In both tandem-in-space and tandem-in-time instruments, the most common experiment is for the first analyzer to select specific ions from the total ion beam arriving from the ion source. Next, the selected ions undergo collision-induced dissociation (CID) in a pressurized cell followed by the analysis of the product ions in the second analyzer. In tandem-in-time the same analyzer is used for both scans, but at different times. The resulting product ion spectra (or precursor and neutral loss spectra in other forms of MS/MS analysis) provide vital structural information for the identification of small molecules (such as drug metabolites) as well as complex biomolecules. Selected reaction monitoring (SRM), another mode of MS/MS operation, provides highly specific and sensitive quantification of target analytes. 

The intensity of the current produced by analyte ions is relevant in quantification. Limits of detection are improved when fragmentation is reduced or eliminated and the ion current, attributable to the analyte, is present as a single species. For instance, using Cl often improves both detection and quantification limits when compared to El, although the controlled fragmentation used in selected reaction monitoring can also improve detection limits. Fragmentation as it applies to specific quantification techniques for small molecules is discussed in connection with the quadrupole family of instruments (Sections 3.3.3.1 and 3.3.5). Quantification for biopolymers, particularly proteins, is presented in Section 3.5.1.9. 

Therefore, to determine if oxidized lipids were formed by enzymic processes or by free radical autoxidation, a first step is to visualize the distribution of products. This step requires previous knowledge of the maximum number of oxidized products, their chromatographic behavior and ions associated with mass spectrometric detection of each product. Quantitative analyses almost always require the use of appropriate, pure standards. For samples from more complex sources where the lipids of interest are present at low concentration there may be many interfering ions. In these instances, tandem mass spectrometry can be used to select pairs of precursor ions and product ions formed by collision-induced dissociation in a procedure called selected reaction monitoring (SRM). This type of analysis usually provides a significant improvement in signal to noise so that the product can be accurately quantified. With modem instruments many, up to hundreds, of these transitions can be measured in a single analysis. In conjunction with retention time... 

Sensitivity in mass spectrometry is determined by the signal-to-noise ratio, which, in turn, has components of instrumental and chemical noise, the latter arising from the sample. Instrumental noise is generally a fixed parameter, but chemical noise can often be reduced by the user, not only by more efficient sample cleanup but by the way in which the instrument is operated. One such method is the technique of selected reaction monitoring, implemented on... 

First, if a multiple mass analyzer instrument is used, the pseudomolecular ion can be colHded with a stream of gas to generate collisionally induced dissociation (CID) and the formation of structurally significant fragment ions. MS/MS with selected reaction monitoring in a triple quadrupole is the method of choice for quantitation. Structure determination or confirmation is done by means of any of the available MS/MS fragmentation techniques triple quadrupole, ion trap, quadrupole-TOF, or FT-ICR. 

---