15
A FAIMS scan of 2,4,6-TMA and N,N-DMT at DF 230 Td (Figure 2 insert) shows that it is possible to select each PGI at a static CF while filtering out interference from the other. The appropriate FAIMS conditions can be combined with thermal desorption for the rapid detection of the PGIs – demonstrated here in a surrogate API (starch). The thermal desorption peak for the FAIMS-selected PGI can be integrated and quantified (Figure 2). Selective detection of N,N-DMT and 2,4,6-TMA provides limits of quantification at 0.13 and 0.19 ppm (w/w) respectively, approximately an order of magnitude lower than the required 1.5 ppm, the threshold of toxicological concern assuming a 1 g per day dose (European Medicines Agency). Precision was evaluated at 1 ppm, giving RSDs of 7.5 and 8.4% for N,N-DMT and 2,4,6-TMA. The requirements for screening PGIs are easily met by combining FAIMS with TD-MS with significantly less sample preparation and analysis times.
The complexity of biological matrices makes the detection of compounds at low levels challenging even for UHPLC separation. Ibuprofen 1-β-o-acyl glucuronide (IAG), a metabolite of ibuprofen, was spiked into a pooled urine sample (healthy male and female urine) and filtered before injection onto a C-18 extended Zorbax column (4.6 mm x 50 mm, 1.8 µm). The UHPLC-MS analysis of IAG in urine results in chemical interference from the urine matrix co-eluting with the IAG peak in the selected ion chromatogram (m/z 381), making peak integration challenging and increasing the limit of quantification (Figure 4a). The introduction of more sample preparation steps to remove the matrix interference, changing the chromatography to increase the IAG elution time or using a narrower mass window could resolve IAG from the chemical interference. However, the former two options are often undesirable because they increase analysis time, and the latter option failed to remove chemical interference without also reducing the IAG response. The application of static FAIMS separation upon peak elution reduces the interference observed in the selected ion chromatogram (Figure 4b) without prolonging the analysis time.
Figure 2. Thermal desorption profile for FAIMS-selected N,N-DMT and (insert) FAIMS spectra for 2,4,6-TMA and N,N-DMT at DF 230 Td. (Reprinted with permission from reference [4]. Copyright 2013, Royal Society of Chemistry).
Reducing Sample Complexity and Chemical Noise
Polyethylene glycol is a component of many pharmaceutical formulations, added to act as solubilizers, dispersants and lubricants, a polyethylene glycol (PEG) matrix gives a complex mass spectrum, masking analytes of interest (Figure 3a). A pharmaceutical intermediate, 2-hydroxy-(4-octyloxy) benzophenone (HOBP) is close enough in mass (17.7 ppm mass difference) to a PEG400 (n=7) peak, which cannot be resolved by a TOF mass analyser (requiring a resolving power of ~130K). A FAIMS scan of HOBP and PEG (n=7) at DF of 48 kV/cm shows 2 peaks for the selected ion response m/z 327.2 (Figure 3.b insert); HOBP is sufficiently separated from the PEG matrix to improve the mass accuracy for HOBP from 11.9 ppm for the overlapping HOBP/PEG peak to 3.3 ppm for the FAIMS-selected HOPB peak (Figure 2b). The removal of the PEG matrix also gives a simpler mass spectrum of HOBP [2].
Figure 4. Selected ion chromatograms (m/z 381 ±0.02) for IAG (highlighted) spiked into urine (0.55 µg/ml) analysed by (a) UHPLC-MS (FAIMS off); (b) UHPLC-FAIMS-MS (FAIMS on) with selective transmission of IAG (DF 260 Td, CF 2.2 Td); and (b insert) CF spectrum of IAG (m/z 381, solid line) and ibuprofen (m/z 205, dashed line). (Reprinted with permission from reference [3]. Copyright 2013, Elsevier).
The quantitative performance observed for UHPLC-MS and UHPLC-FAIMS-MS is compared in Table 1. Despite a loss in signal, the limit of quantification for FAIMS-selected IAG is reduced 2-fold, increasing the linear dynamic range to 3 orders of magnitude; intra-day precision is also improved. The addition of a FAIMS separation was found to increase the selectivity and sensitivity of a UHPLC-MS system by significantly reducing chemical noise from the urine matrix.
Table 1. Quantitative UHPLC-MS and UHPLC-FAIMS-MS performance for IAG. (Reprinted with permission from reference [3]. Copyright 2013, Elsevier).
LC-MS
LOQ (µg/ml) LDR (µg/ml) R2
Intra-day (% RSD)
LC-FAIMS-MS 0.018
0.018-11 0.9991 5.0
0.010
0.010-11 0.9987 2.7
Combining FAIMS with In-source CID
FAIMS-selected in-source collision-induced dissociation-mass spectrometry (FISCID- MS) is a process of fragmenting FAIMS-selected ions via in-source CID followed by the mass analysis of the fragments. In-source CID is not a commonly used method for product ion analysis because the absence of precursor ion selection results in complex product ion spectra making interpretation difficult. Product ion analysis is therefore usually performed on instruments with precursor ion mass-selection capabilities (e.g. a triple quadrupole or quadrupole-TOF). However, the addition of a FAIMS device to a single mass analyser has the added benefit of increasing selectivity and making product ion analysis possible by combining FAIMS precursor ion selection and in-source CID.
Figure 3. Mass spectra of a mixture of HOBP and PEG 400 (1:20 molar ratio): (a) without FAIMS separation; (b) FAIMS-selected HOBP ion (CV = 0.6-0.7V); and (b insert) CV spectrum (m/z 327.2). (Reprinted (adapted) with permission from reference [2]. Copyright 2012, American Chemical Society).
The FAIMS-selected transmission of HOBP from a PEG matrix was presented earlier (Figure 3). Increasing the voltage of a skimmer (fragmentor) lens causes fragmentation of FAIMS-selected HOBP to give easily-identifiable, characteristic fragments which would have otherwise been hidden by interfering fragments from multiple precursors (Figure 5). FISCID-MS was also used to determine fragments of IAG, where the relative intensity of characteristic fragments increased 2-fold (data not shown). FAIMS removes multiple precursors to simplify fragment spectra and aid structural identification of the FAIMS-selected analyte.
WWW.LABMATE-ONLINE.COM
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72 |
Page 73 |
Page 74 |
Page 75 |
Page 76 |
Page 77 |
Page 78 |
Page 79 |
Page 80 |
Page 81 |
Page 82 |
Page 83 |
Page 84 |
Page 85 |
Page 86 |
Page 87 |
Page 88 |
Page 89 |
Page 90 |
Page 91 |
Page 92 |
Page 93 |
Page 94 |
Page 95 |
Page 96
