Improving Selectivity in Quantitative Analysis Using MS 3 on a Hybrid Quadrupole-Linear Ion Trap Mass Spectrometer Overview Increased selectivity is achieved in quantitative analysis by using MS 3. The third stage of MS eliminates interferences, resulting in improved detection limits. Introduction The selectivity of MS/MS in bioanalysis has significantly decreased the analysis time. In the majority of cases, single chromatographic peaks are observed when using unit resolution on triple quadrupole systems. The presence of endogenous species in biological matrix extracts may still lead to interferences in some assays, even in MS/MS mode. In order to improve selectivity, several scenarios can be used to eliminate the interference, and each one of them has their own pros and cons, as stated in Table 1. In this work, MS 3 is employed on a hybrid quadrupole-linear ion trap mass spectrometer to improve selectivity in quantitative applications. The concept of using MS 3 for quantitative analysis is to rely on the selectivity of the fragmentation pathway. This has been demonstrated for the quantitation of mixtures by infusion [1]. Here we extend the concept to LC-MS analysis when interferences are present. Materials and Methods Oxycodone, pamaquin and clonazepam were obtained from Sigma (St Louis, MO). Chromatography was performed using a Luna C18 column (2x50mm, 5µm) from Phenomenex (Torrance, CA) with gradient elution. Samples were injected using Perkin Elmer series 200 autosampler and micro pumps. MS analysis was performed on a 4000 QTRAP system (Applied Biosystems MDS Sciex, Concord, ON) using a TurboIonSpray source operated at 450 o C. For MRM (multiple reaction monitoring) acquisition, optimal collision energy (CE) conditions were used for all fragment ions monitored. For MS 3 acquisition, the excitation time was set to 70msec with 50mV excitation energy, and dynamic fill time (DFT) was activated. Eliminate Interference by Advantage Disadvantage Chromatographically separate interference from analyte Easy to do Extends analysis time Table 1. Potential solutions for eliminating interferences. Use alternate fragment ion Use different collision energy Improve specificity on precursor ion selection LC analysis time unaffected LC analysis time unaffected Easily performed by increasing resolution in Q1 Sensitivity affected Sensitivity affected Reduces sensitivity
Selectivity of MS/MS and MS 3 By relying on the fragmentation pathway in MS 3, it was speculated that a higher level of selectivity could be achieved at unit resolution as opposed to increasing the selectivity on the precursor ion with high resolution in MS/MS mode. In order to evaluate this hypothesis, the NIST database of EI spectra was used to assess MS and MS/MS selectivity as a function of resolution. For single MS, the database was searched to provide a listing of all compounds that have a nominal mass of 315. Out of 109,887 compounds listed in the database, a total of 181 compounds had a precursor ion at nominal mass 315. The exact mass was calculated for each of the compounds retrieved from the database. Figure 1 depicts the probability of observing interference for pamaquin (315.2192) as a function of Q1 resolution. In order to assess the selectivity provided by MS/MS, the database was searched for combinations of precursor mass (315) and a fragment ions present at 10% of the base peak. This number was used to identify the probability of having an interference that would significantly contribute to the signal if a common fragment was present. A total of 11 precursor-fragment combinations were determined and the probability of finding compounds with a significant common fragment (>10%) ranged from 0.02 to 0.07 (average of 0.05). Therefore, using a 0.05 probability of interference by MS/MS and combining it with the data shown in Figure 1, the probability of interference in MS/MS mode as a function of resolution on the precursor ion was calculated and graphed (Figure 2). Probability of Interference 1.20 1.00 0.80 0.60 0.40 0.20 0.00 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 FWHH (amu) m/z 315.2192 Figure 1. The probability of an interference in MS mode as a function of peak width. Based on the 181 compounds listed in the NIST database, the probability of having an interference as a function of peak width was calculated for mass 315.2192 (pamaquin). See text for details. This graph indicates that at unit resolution, there is a 100% probability of finding compounds that will interfere with each other in single MS mode, if chromatography is not employed. Figure 1 also shows that high resolution (<0.1 FWHH) is required to be able to reduce the probability of interference at the 10% level, which would be reasonable under traditional LC conditions, but not for faster- LC analysis (<3min). Probability of Interference 1.0000 0.1000 0.0100 0.0010 0.0001 0.0000 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 FWHH (amu) m/z 315.2192 m/z 315.2192 MSMS m/z 315.2192 MS^3 Figure 2. The probability of an interference as a function of peak width in MS, MS/MS and MS 3. The overall probability of having an interference in MS/MS and MS 3 mode as a function of precursor ion peak width for mass 315.2192 was calculated. The dashed-line arrow indicates the required resolution for precursor ion selection in MS/MS mode to get the same selectivity as MS 3 at unit resolution (0.7amu). It is reasonable to assume that the probability of having compounds that will have common precursor ion, 1 st fragment ion and 2 nd fragment ion would be around 0.05 (same as MS/MS) or even lower. This assumption is based on the fact that MS 3 is achieved by adding another stage of fragmentation to MS/MS. When combined with the other probability of interference as a function of resolution, we get an indication of the relative selectivity of MS,
MS/MS and MS 3 as a function of resolution (Figure 2). This figure indicates that each stage of fragmentation improves the selectivity by at least an order of magnitude. At unit resolution, MS 3 has a 0.0025 (i.e. 0.25%) probability of having an interference if no chromatography is performed. This figure also indicates that the same level of selectivity in MS/MS mode can only be achieved if the precursor ion is selected with a resolution of <0.04amu FWHH. When operating a quadrupole at such resolution, significant losses in transmission are observed leading to a significant loss in sensitivity. Sacrificing sensitivity is a luxury that one can rarely afford in bioanalysis. MS 3 can offer the same selectivity with optimum transmission conditions at unit resolution (0.7 FWHH) on the hybrid quadrupole-linear ion trap mass spectrometer. Results, oxycodone and clonazepam are three compounds that were found to have identical nominal precursor ion mass with common fragment ions. The latter two compounds were used as controlled interferences to demonstrate the selectivity of MS 3 for the quantitative analysis of pamaquin. The chemical formula, exact molecular weight of the precursor ion and, fragment ion information for each of these compounds are listed in Table 2. In the case of pamaquin and oxycodone, the difference in precursor ion mass is 80 mmu, thus indicating that a resolution of 0.08 amu FWHH (or lower) is required in Q1 to eliminate this interference strictly with precursor selection. As stated earlier, under such conditions, significant losses in sensitivity are observed with quadrupoles. When MS 3 is performed on the 243 fragment of pamaquin, the major fragments observed are m/z 241, 187 and 174. For clonazepam, the m/z 241 fragment ion was found to originate from the m/z 270 fragment ion. In the case of oxycodone, the m/z 241 ion was found to originate from more than one fragment ion, none of them common to pamaquin or clonazepam. Thus, distinct fragmentation pathways can be used to eliminate interferences. Standards of pamaquin were prepared in solution containing a high concentration oxycodone (10 ng/µl) as the major interference and clonazepam at 500 pg/µl. The standards where analyzed in MRM mode at unit resolution. The detection limit for pamaquin varied with the fragment ion selected, and the extent of interference present. Figure 3 shows the signal obtained for an injection of 10 pg of pamaquin on column for the 3 MRM signals monitored. With the LC conditions used, there is sufficient separation for the 316-243 MRM transition between pamaquin and oxycodone. However, slight variation in mobile phase conditions or column performance degradation could result in the merging of these 2 peaks as a function of time. It is impossible to identify pamaquin at the 10 pg level if the other two transitions were used, due to the extensive tailing produced by the oxycodone interference. For the other two MRM transitions, the amount of pamaquin injected on column had to be increased to 100 pg and 500 pg on column respectively, to be detectable on top of the tailing portion of the oxycodone signal (Figure 4). Using the same sample set, we performed MS 3 of 316>243 and Compound Formula Exact Mass Mass Difference Common Fragments ( mmu) (optimum CE in ev) C19H29N3O 315.2311 0.0 241 (33) 243 (25) 187 (42) Oxycodone C18H21NO4 315.1471-84.0 241 (40) 243 187 (40) Clonazepam C15H10N3O3Cl 315.0411-190.0 241 (47) 187 (60) Table 2. The chemical formula, exact masses and fragment ion information for pamaquin and 2 controlled interferences, oxycodone and clonazepam.
used extracted ion chromatograms (XIC) of m/z 241 or m/z 187 fragment ion to generate calibration curves. The time required to perform the MS 3 is on the order of 160 msec (including isolation, fragmentation and scanning), which is comparable to the dwell time used for MRM analysis. Figure 5 shows the XIC for fragment m/z 241 and m/z 187 when injected in the presence of oxycodone and clonazepam. It is clear from these chromatograms that the interferences are completely eliminated. Using this approach, the detection limit for pamaquin, in the presence of oxycodone (50 ng on column), was 5 pg on column. This represents a 100x and 20x improvement in detection limit when compared to MRM analysis for the corresponding fragments m/z 241 and m/z 187, respectively. The improvement gains are strictly due to the improved selectivity of MS 3 in the presence of interfering signals (Table 3). When MS 3 is compared with MRM in the absence of interference, the MRM approach still offers the best sensitivity achievable on QqLIT systems [2]. better detection limits can be achieved. The linear dynamic range of this approach is typically only over 2 orders of magnitude. References 1. Simultaneous Quantitation (SRM) and Confirmatory Analysis (MS 3 ) in Human Plasma with Chip-based Infusion, Luc Alexis Leuthold, Chantal Grivet, Mark Allen, Mark Baumert and Gérard Hopfgartner, IMSC 2003, Poster. 2. Multiple Techniques for Qualitative and Quantitative Data Acquisition Using a Hybrid Quadrupole-Linear Ion Trap Mass Spectrometer, Tanya Gamble, Tania Sasaki, Elliott Jones and Gary Impey, ASMS 2004 Poster ThPF 107 MRM without Interference MRM with Interference MS3 Mass Range (pg on column) Mass Range (pg on column) Mass Range (pg on column) 316-243 5-1000 316-243 10-1000 na na 316-241 5-1000 316-241 500-1000 316-243-241 5-500 316-187 5-1000 316-187 100-1000 316-243-187 5-500 Table 3. LOQ and dynamic ranges of each approach. The MS 3 option clearly shows the opportunity to quantitate at low levels in the presence of large concentrations of interferences. Summary It was demonstrated that the selectivity of the fragmentation pathway (MS 3 ) can be used to significantly improve detection limits in the presence of interferences. Using MS 3 for quantitation provides a high degree of selectivity that can be applied in a much more generic fashion than increasing resolution for precursor ion selection. Since both stages of fragmentation are occurring at unit resolution, the loss in transmission is minimized and
Oxycodone Oxycodon MRM 316-243 MRM 316-241 MRM 316-18- 187 (masked) (masked) Clonazepa Clonazepa Clonazepam Clonazepam Clonazepam Figure 3. Chromatograms observed for all 3 MRM ions of. 10pg of was injected on column. A total of 50ng of oxycodone and 2.5ng of clonazepam were injected as interferences. Q1 and Q3 were operated at 0.7amu FWHH. See text for discussion. (A) (B) Oxycodone e MRM 316-241 Oxycodone e MRM 316-187 n n Clonazepam m Clonazepam m Figure 4. LOQ for 241 and 187 fragment ions in MRM mode. (A) 500 pg and (B) 100pg of pamaquin was injected on column. In both cases, 50ng and of oxycodone and 2.5ng clonazepam were injected as interferences. Q1 and Q3 were operated at 0.7amu FWHH. See text for discussion. Sample Peak Name: "_241" 2.0pg/uL_MS3 Mass(es): (4000amu/sec)_WITHInterfere "240.5-240.8 amu" nce" Sample ID:.. Comment: "" Annotation: "" Sample Name: " 2.0pg/uL_MS3 (4000amu/sec)_WITHInterference" Sample ID:.. Peak Name: "_187" Mass(es): "186.8-187.5 amu" 4.8e4 (A) 1.4e4 (B) 4.0e4 3.0e4 XIC of 241 from MS 3 316>243 1.0e4 XIC of 187 from MS 3 316>243 2.0e4 6000 1.0e4 2000.0 0.0 0.5 1.0 1.5 2.0 Time, min 0.0 0.5 1.0 1.5 2.0 Time, min Figure 5. XIC from a 10pg injection of pamaquin acquired in MS3 mode XIC of fragment 241 (A) and 187 (B) from the MS3 of 316>243 acquisition. In both cases, 50ng of oxycodone and 2.5ng of clonazepam were injected as interferences. Q1 and Q3 were operated at 0.7amu FWHH. See text for discussion.
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