Selected Reaction Monitoring Method

To improve the specificity while retaining the high sensitivity, a new method (i.e., selected or multiple reaction monitoring (SRM/MRM, Chapter 2)) is evolved from the SIM technique, which is only focused on one miz of a compound to the SRM that is focused on both a molecular (or precursor) ion and a fragment-ion resultant from the precursor ion. The specific experiment in known as a transition and is usually written as precursor-ion mass fragment-ion mass. The only requirement to perform this technique is that the mass spectrometer employed has to possess the capability to perform MS/MS. 

Unlike the SIM method where analysis is largely dependent on the mIz of the compound, the identity of each individual species can be virtually defined in the SRM experiment by the elution time, the miz of the ion, and the characteristic fragment 

Generally, in LC-MS analysis, a particular pair or pairs of precursor-/product-ion transitions are monitored at a specified elution time. Of course, these transitions at such an elution time should be predetermined utilizing authentic compounds or close analogs. Alternatively, a data-dependent acquisition approach could be set up with a certain type of instruments [60]. In either case, some degree of preknowledge about the individual lipid species present in the samples is required since currently available instruments are still unable to perform an infinite number of transitions at an elusion time due to the limitation of instmmental duty cycle and/or sensitivity. Moreover, similar to the SIM method, the linear dynamic range, limit of detection, and calibration curves of the species of interest should generally be predetermined for quantitative analysis of lipid species. Thus, the constmcted ion peak area of each species can be compared to a standard curve of the species under identical experimental conditions. 

At least one control compound should be included in each sample analyzed. If the sample contains many classes of lipids, one control species for each class should be included in consideration of the differential ionization efiticiencies between classes (see Chapter 15 for further discussion). Each of the reconstituted ion peak areas of the lipid species is then normalized to the control compound of the class prior to comparison with a standard curve for quantification. For instance, Merrill and colleagues have described this approach in detail for the analysis of sphingolipids [41, 61]. 

The LC-ESI-MS/MS approach is commonly used for the analysis of a small number of lipids (e.g., a preseparated lipid class). This is because only a few pairs of ion transitions can be monitored at any elution time due to the limitation of the duty cycle as aforementioned. Moreover, generation of the necessary standard curves for all species in a cellular lipidome is impractical. However, because of the increased specificity and sensitivity of detection, this SRM/MRM method is particularly useful for quantitative analysis of those lipid classes that are present in low or very low abundance in the cellular lipidomes after a few steps of prechromatographic enrichment. One such typical example is the quantitative analysis of fatty acyl amino acids [62]. 

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Figure 5.67 Reconstructed ion chromatograms for Idoxifene and internal standard (ds-Idoxifene using LC-ToF-MS for (a) double-blank human plasma extract, (b) extract of blank human plasma containing internal standard (IS), and (c) control-blank human plasma spiked with Idoxifene at 5 gml , the LOQ of the method. Reprinted from 7. Chromatogr., B, 757, Comparison between liquid chromatography-time-of-flight mass spectrometry and selected-reaction monitoring liquid chromatography-mass spectrometry for quantitative determination of Idoxifene in human plasma , Zhang, H. and Henion, J., 151-159, Copyright (2001), with permission from Elsevier Science.

The standard addition procedure is another method for recognising and overcoming potential matrix effects in quantification. Both alternatives, FIA—MS or FIA—MS—MS, can be performed using this procedure. Despite the increased expenditure because of a multiplication in analyses, the FIA approach combined with standard addition remains the faster technique even with the application of specific analytical MS—MS techniques such as product-, parent- or neutral loss scans applying selected reaction monitoring (SRM). The greatest drawback of this technique is that the compounds to be quantified must... 

A selective, sensitive, and rapid hydrophilic interaction liquid chromatography with electrospray ionization tandem mass spectrometry was developed for the determination of donepezil in human plasma [32], Donepezil was twice extracted from human plasma using methyl-ferf-butyl ether at basic pH. The analytes were separated on an Atlantis HILIC Silica column with the mobile phase of acetonitrile ammonium formate (50 mM, pH 4.0) (85 15, v/v) and detected by tandem mass spectrometry in the selective reaction monitoring mode. The calibration curve was linear (r = 0.9994) over the concentration range of 0.10-50.0 ng/ ml and the lower limit of quantification was 0.1 ng/ml using 200 /d plasma sample. The CV and relative error for intra- and inter-assay at four quality control levels were 2.7% to 10.5% and —10.0% to 0.0%, respectively. There was no matrix effect for donepezil and cisapride. The present method was successfully applied to the pharmacokinetic study of donepezil after oral dose of donepezil hydrochloride (10 mg tablet) to male healthy volunteers. 

A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed [33] and validated for the determination of donepezil in human plasma samples. Diphenhydramine was used as the IS. The collision-induced transition m/z 380 > 91 was used to analyze donepezil in selected reaction monitoring mode. The signal intensity of the m/z 380 —> 91 transition was found to relate linearly with donepezil concentrations in plasma from 0.1 to 20.0 ng/ml. The lower limit of quantification of the LC/MS/MS method was 0.1 ng/ml. The intra- and inter-day precisions were below 10.2% and the accuracy was between 2.3% and +2.8%. The validated LC/MS/MS method was applied to a pharmacokinetic study in which healthy Chinese volunteers each received a single oral dose of 5 mg donepezil hydrochloride. The non-compartmental pharmacokinetic model was used to fit the donepezil plasma concentration-time curve. Maximum plasma concentration was... 

Macek et al. [120] developed a method to quantitate omeprazole in human plasma using liquid chromatography-tandem mass spectrometry. The method is based on the protein precipitation with acetonitrile and a reversed-phase liquid chromatography performed on an octadecylsilica column (55 x 2 mm, 3 /im). The mobile phase consisted of methanol-10 mM ammonium acetate (60 40). Omeprazole and the internal standard, flunitra-zepam, elute at 0.80 0.1 min with a total rim time 1.35 min. Quantification was through positive-ion made and selected reaction monitoring mode at m/z 346.1 —> 197.9 for omeprazole and m/z 314 —> 268 for flunitrazepam, respectively. The lower limit of quantification was 1.2 ng/ml using 0.25 ml of plasma and linearity was observed from 1.2 to 1200 ng/ml. The method was applied to the analysis of samples from a pharmacokinetic study. 

It is important to first note that many classical clinical assays have traditionally measured one metabolite to detect one disease. Consequently, many of the rules of method validation were designed around this premise. MS/MS, as originally designed, detected two classes of compounds, amino acids and acylcamitines, in four to five different MS methods (known as scan modes such as neutral loss, precursor ion, or selected reaction monitoring), for approximately 500 distinct masses, more than 70 known compounds, and 20-30 stable isotope internal standards. How then did one approach such a complicated validation to gain acceptance as a reliable, useful method The answer is quite simple - start simply and compare to what was already established. 

To ensure that appropriate resolution and lack of interference is obtained during method development, it is highly recommended actively monitoring PLs and aware of the retention/elution times and intensity of the PLs relative to the retention/elu-tion times for the analytes of the interest. Table 2 summarizes several representative Selective Reaction Monitoring (SRM) transitions for PLs monitoring purpose during method development. 

At about the same time, our laboratory has reported the development and validation of an LC tandem MS assay for as much as six TKIs simultaneously. The proposed LC-MS/MS method allows the simultaneous determination of clinically relevant ranges of concentrations for the six major TKIs currently in use imatinib, dasatinib, nilotinib, sunitinib, sorafenib, and lapatinib [122], Plasma is purified by acetonitrile protein precipitation followed by reversed-phase chromatographic separation. Analyte quantification is performed by electrospray ionization-triple quadrupole mass spectrometry by selected reaction monitoring (SRM) detection using the positive mode. This was the first broad-range LC-MS/MS assay covering the major currently in-use TKIs. 

No matter which ionization technique or chromatographic method is used, three acquisition modes exist scanning, selected-ion monitoring (SIM) (not to be mistaken with SIMS, which means secondary ion mass spectrometry) and selected-reaction monitoring (SRM). 

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... 

Quantitation of drags and their metabolites in biological matrices currently is one of the most important applications of LC-MS. This can be attributed to the greatly enhanced selectivity and reliability of the analysis, as compared to LC-UV. Selective reaction monitoring (SRM) in tandem mass spectrometry (MS-MS) has become the method-of-choice in quantitative bioanalysis. Ample examples are described in literatnre. Even a larger number of successful examples are hidden in the archives of pharmaceutical companies and contract research organizations. 

Kim et al. [55] developed an LC-MS-MS method, based on positive-ion APCI mode and selected-reaction monitoring (SRM), for the doping analysis of 19-nortestosterone and three of its esters in plasma of race horses. The limits of quantification are 0.16, 0.1, 2.0, and 5.0 ng/ml for 19-nortestosterone, and its phenylpropionate, decanoate, and cyclopentanepropionate ester, respectively, using 2 ml of plasma. The method enabled detection of 19-nortestosterone np to 23 days... 

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. 

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