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1、284 Characterization of an Unknown Compound Using the LTQ Orbitrap Donald Daley, Russell Scammell, Argenta Discovery Limited, 8/9 Spire Green Centre, Flex Meadow, Harlow, Essex, CM19 5TR, UK Objectives unknown compound (compound X), compound X was provided and analyzed qualitatively and quantitative
2、ly. The following steps were taken to provide data: Accurately determine the mass of compound X and determine sum formulae proposals without any knowledge about the compound prior to mass analysis Exclude false positive hits by comparing the proposed sum formulae to the theoretical isotope patterns
3、without any knowledge about the compound prior to mass analysis equivalent value (RDB equivalent value) by MS/MS using CID and HCD by gaining structural information Carry out a quantitative analysis by means of dilution series of compound X. The study follows an analytical approach in which the comp
4、ound was unknown in the beginning; subsequently it column. Introduction As a drug, the quaternary ammonium salt Tiotropium is used to prevent wheezing, shortness of breath, and pulmonary disease (COPD, a group of diseases that affect the lungs and airways) such as chronic bronchitis (swelling of the
5、 air passages that lead to the lungs) and emphysema (damage to air sacs in the lungs). COPD is considered to be killers after heart attack, stroke, cancer, and Alzheimers Disease. Tiotropium belongs to a class of medications called bronchodilators which works by relaxing and opening the air passages
6、 to the lungs to make breathing easier. For further information please see: http:/www .nlm.nih.gov/medlineplus/dr uginfo/ medmaster/a604018.html The pre-charged status of this compound makes it source-, pH-, and ionization yield- independent and is therefore a ideal candidate to study analyzer perfo
7、rmances. Methods LC-ESI-MS and MS/MS Liquid chromatography-electrospray ionization (LC-ESI) mass spectrometry and tandem mass spectrometry in combination with high resolving power and accurate mass Orbitrap system, a hybrid mass spectrometer consisting of a linear ion trap coupled to an Orbitrap mas
8、s analyzer, is used as mass spectrometer. For quantitative analyses a stock solution was was prepared in 95 / 5 water / acetonitrile containing 0.1% formic acid. This solution was used to prepare a dilution Surveyor Hypersil GOLD 2.1 mm column, 50 mm length, 1.9 m dilution experiments. A 20 L full l
9、oop injection and the below given gradient were applied to quantitative experiments. Time A% B% l/min 0.00 95.0 5.0 100.0 1.00 95.0 5.0 100.0 8.00 10.0 90.0 100.0 10.00 10.0 90.0 100.0 10.10 95.0 5.0 100.0 12.00 95.0 5.0 100.0 Table 1: LC gradient. MS Methods for Quantitative Experiments Targeted MS
10、/MS Orbitrap detection FTMS + p norm res = 15,000. MS/MS 392.1 CID Collision Energy 35.0% Q 0.25 Time 30.0 ms IsoW 3.0 u Targeted MS/MS Ion Trap detection ITMS + p norm MS/MS 392.1 CID Collision Energy 35.0% Q 0.25 Time 30.0 ms IsoW 3.0 u 285 Qualitative Results A Surveyor MS Pump HPLC system with M
11、icro AS autosampler was used for the analyses. The previous given experiments shown. For qualitative results either direct infusion or injection via the autosampler was chosen. A typical qualitative experiment performed is illustrated in Figure 1. The inset into the base peak chromatogram in Figure
12、1 shows the reproducibility of mass measurements over the LC peak. More than 20 mass measurements can be performed over the LC peak. Figure 1: Base peak chromatogram of a 1 pg/L solution. Sum Formula Proposals by means of Accurate Mass Measurement, RDBE and Isotope Pattern Evaluation Compound X was
13、measured at RP 100,000 m/z 400 in order to resolve potential sulfur(s). An inset into a full scan is given in Figure 2. The inset at m/z 394 reveals the existence of sulfur (S). The sum formula proposals for m/z 392 is taking C, H, N, O and up to 2 S in a 5 ppm tolerance window into account. For ele
14、mental constraints see Table 2. Assuming the ion observed at m/z 392 is an MH +ion, as this is typical for ESI, RDBE values can only be x.5 for a singly charged ion, as displayed in Table 3. These seven proposals are simulated and compared to the Sum formula proposals with RDBE values x.5 according
15、to Figure 1 (bottom) simulated with resolution 135,000 at 392 FWHM. Sum formula proposals with elemental constraints according to Table 2 are simulated with resolution 135,000 at 392 FWHM. Proposals without S can be excluded ( 32S and 34S are responsible for splitting the signals starting with the A
16、+2 ion) right away, as proposals with a single S can be excluded (see A+3 ion, A+4 ion) by comparing to the raw data information. The comparison reveals that the correct sum formula of compound X as an MH +ion is C 19H22O4N1S2. A search for the structure of compound X and its name in a database by use of the previously determined sum formula C 19H22O4NS2was carried out. A search against http:/chem.sis.nlm.nih.gov/chemidplus/reveals Tiotropium as the o
