SRM detection

With the advent of the practical API-based LC-MS interfaces, the high specificity of mass spectral analysis permitted a radical decrease in the amount of analytical time invested (sample preparation, injection, chromatography) prior to final detection (Hsieh et al., 2006 Maurer, 2007). Although SRM detection as the final step in LC-MS analysis can incorporate several stages of specificity (Chapter 3), some form of sample preparation/extraction is still performed to remove unwanted... 

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. 

Figure 10-6 Mass spectrum of BADGE [M + H2Oj at m/z = 358.1 (upper) and its fragment ion m/z = 191 (lower) selected as daughter ion for SRM detection.

Qj = second mass separating quad, SRM detection (a) or product ion scan (b). 

Table 5.2 Selected-reaction monitoring (SRM) transitions nsed for MS-MS detection of the pesticides studied in the systematic investigations on APCI-MS signal response dependence on eluent flow rate. Reprinted from J. Chro-matogr.. A, 937, Asperger, A., Efer, J., Koal, T. and Enge-wald, W., On the signal response of various pesticides in electrospray and atmospheric pressure chemical ionization depending on the flow rate of eluent applied in liquid chromatography-mass spectrometry , 65-72, Copyright (2001), with permission from Elsevier Science...

Another useful standard Is SRM 1647, priority pollutant polynuclear aromatic hydrocarbons (in acetonitrile). It can be used to calibrate liquid chromatographic Instruments (retention times. Instrument response), to determine percent recoveries, and to fortify aqueous samples with known PAH concentrations. Figure 8 Illustrates the HPLC separation and UV detection (fluorescence is also used extensively) for the 16 priority pollutants. 

Figure 8. Reversed-phase HPLC separation of SRM 1647, priority pollutant polynuclear aromatic hydrocarbons (In acetonitrile), using UV detection.

Figure 9. Reversed-phase HPLC analysis of PAH s extracted from SRM 1649, urban dust/organics, with UV detection, not preceded by normal-phase HPLC clean-up, (Reprinted from reference 72.

The second NIST human DNA SRM is a PCR-based DNA Profiling Standard. The PCR was first described by Saiki et al. (1985,1989). Since then it has developed into a highly versatile and widely used detection, identification, manipulation and analysis tool in molecular biology, including DNA profiling. In brief, two short synthetic oligonucleotides, or primers, are used to define an intervening DNA sequence... 

These three NIST SRMs have a number of important quality control applications for forensic DNA profiling, medical diagnostics and mutation detection. The main applications are summarized below ... 

LC-MS-MS was also the method of choice for the analysis of UV filters in solid matrices. Both LC and UPLC have been applied in three out of the four methods available for the determination of UV filters in sludge. Separation was performed on C8 and C18 LC-chromatographic columns (Zorbax, Eclipse, Vydac, and Purosphere) using binary gradient elution of mobile phases consisting of water/ methanol or water/acetonitrile. MS-MS detection was performed in SRM with ESI and atmospheric pressure photoionization (APPI) in both positive and negative modes. For each compound, two characteristics transitions were monitored. In addition to MS and MS-MS, diode array detection (DAD) was occasionally applied to the determination of OT. Spectra were recorded between 240 and 360 nm and discrete channels at 310 nm. 

Fig. 3.38.The IUPAC names of Sudan azo dyes are as follows Sudan 1 = 1— [(2,4-dimethylphenyl)azo]-2-naphtalenol Sudan II = l-(phenylazo)-2-naphtol Sudan III = l-(4-phenylazophenylazo)-2-naphtol Sudan IV = o-tolyazo-o-tolyazo-beta-naphtol and Disperse Orange 13 = 4-[4-(phenylazo)-l-naphtylazo]-phenol. Azo dyes were separated in an ODS column (250 x 2.1 mm i.d. particle size 5 /xm) at 35°C. The isocratic mobile phase consisted of 0.1 per cent formic acid in methanol-0.1 per cent formic acid in water (97 3, v/v). The flow rate was 200 /xl/min. MS conditions were nebulizing and desolvation gas were nitrogen at the flow rates of 50 and 5551/h, respectively electrospray voltage, 3.0 kV cone voltage 25 V source temperature, 110°C desolvation temperature, 110°C. Azo dyes were extracted from the samples by homogenizing 1 g of sample with 10 ml of acetone, then the suspension was centrifuged and an aliquot of 3 ml of supernatant was mixed with 1 ml of deionized water, filtered and used for analysis. LC-ESI-MS/Ms SRM traces of standards and spiked samples are listed in Fig. 3.39. It was found that the detection and quantitation limits depended on both the chemical structure of the dye and the character of the accompanying matrix. LOD and LOQ values in chilli tomato sauce...

Fig. 12.11. The effect of SRM as compared to SIM in the detection of dextrometorphan. Reproduced from Ref. [25] by permission. Elsevier Science, 2001.

FIGURE 5.4 Comparison of shape selectivity and planar recognition test procedures SRM 869a test (solutes PhPh, TEN, BaP eluent acetonitrile/water 85/15 (v/v), flow 2 ml/min, detection UV 254 nm) and Tanaka test (solutes o-TER, TRI eluent methanol/water 80/20 (v/v), flow 1 ml/min, detection UV 254 nm. (Reproduced from Engelhardt, H., et al., Chromatographia, A%, 183, 1998. With permission.)... 

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