284

Characterization of an Unknown CompoundUsing 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 wasprovided and analyzed qualitatively and quantitatively.The following steps were taken to provide data:• Accurately determine the mass of compound X and

determine sum formulae proposals without anyknowledge about the compound prior to mass analysis

• Exclude false positive hits by comparing the proposedsum formulae to the theoretical isotope patterns withoutany knowledge about the compound prior to massanalysis

•equivalent value (RDB equivalent value)

•by MS/MS using CID and HCD by gaining structuralinformation

• Carry out a quantitative analysis by means of dilutionseries of compound X.

The study follows an analytical approach in which thecompound was unknown in the beginning; subsequently it

column.

IntroductionAs a drug, the quaternary ammonium salt Tiotropium is used to prevent wheezing, shortness of breath, and

pulmonary disease (COPD, a group of diseases that affectthe lungs and airways)such as chronicbronchitis (swelling of the air passages thatlead to the lungs) andemphysema (damage to air sacs in thelungs). COPD isconsidered to be

killers after heartattack, stroke, cancer,and Alzheimer'sDisease.

Tiotropium belongs to a class of medications calledbronchodilators which works by relaxing and opening theair passages to the lungs to make breathing easier. Forfurther information please see:http://www .nlm.nih.gov/medlineplus/dr uginfo/medmaster/a604018.html

The pre-charged status of this compound makes itsource-, pH-, and ionization yield- independent and istherefore a ideal candidate to study analyzerperformances.

Methods

LC-ESI-MS and MS/MS Liquid chromatography-electrospray ionization (LC-ESI)mass spectrometry and tandem mass spectrometry incombination with high resolving power and accurate mass

Orbitrap ™ system, a hybrid mass spectrometer consistingof a linear ion trap coupled to an Orbitrap mass analyzer,is used as mass spectrometer.

For quantitative analyses a stock solution was wasprepared 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 µm

dilution experiments. A 20 µL full loop injection and thebelow given gradient were applied to quantitativeexperiments.

Time A% B% µl/min

0.00 95.0 5.0 100.0 1.00 95.0 5.0 100.0 8.00 10.0 90.0 100.010.00 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/MS Orbitrap detection

FTMS + p norm res = 15,000. MS/MS @ 392.1 CIDCollision 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 Energy35.0% Q 0.25 Time 30.0 ms IsoW 3.0 u

285

Qualitative ResultsA Surveyor MS Pump HPLC system with Micro AS ™

autosampler was used for the analyses. The previous given

experiments shown. For qualitative results either directinfusion 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 1 shows the reproducibility of mass measurementsover the LC peak. More than 20 mass measurements canbe performed over the LC peak.

Figure 1: Base peak chromatogram of a 1 pg/µL solution.

Sum Formula Proposals by means of Accurate MassMeasurement, RDBE and Isotope Pattern Evaluation

Compound X was measured at RP 100,000 @ m/z 400 inorder to resolve potential sulfur(s). An inset into a fullscan is given in Figure 2. The inset at m/z 394 reveals theexistence 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 ppmtolerance window into account. For elemental constraintssee Table 2. Assuming the ion observed at m/z 392 is anMH + ion, as this is typical for ESI, RDBE values can onlybe 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.5according to Figure 1 (bottom) simulated with resolution135,000 at 392 @ FWHM.

Sum formula proposals with elemental constraintsaccording to Table 2 are simulated with resolution135,000 at 392 @ FWHM. Proposals without S can beexcluded ( 32S and 34S are responsible for splitting thesignals starting with the A+2 ion) right away, as proposalswith a single S can be excluded (see A+3 ion, A+4 ion) bycomparing to the raw data information. The comparisonreveals that the correct sum formula of compound X as anMH + ion is C 19H 22O 4N 1S2.

A search for the structure of compound X and itsname in a database by use of the previously determinedsum formula C 19H 22O 4NS2 was carried out. A searchagainst http://chem.sis.nlm.nih.gov/chemidplus/ revealsTiotropium as the only hit which exists as a bromide saltat molecular weight 472.42 (Figure 4).

This structure is imported into Mass Frontier ™

MS/MS data (CID and HCD) obtained from compoundX. This is shown in Figure 5.

Collision Induced Dissociation (CID)

Figure 5a: MS/MS @ m/z 392 using Collision InducedDissociation (CID) was a fragmentation techniquesdetected in the Orb itrap analyzer. The accuratelydetermined fragments @ m/z 152 and m/z 170,respectively, come up with a single sum formula proposaleach. The labeling of proposed structures was done usingMass Frontier.

Higher Energy Collision- Trap Dissociation (HCD)

Figure 5b: MS/MS @ m/z 392 using Higher EnergyCollision-Trap Dissociation (HCD) as a fragmentationtechniques detected in the Orbitrap analyzer. Theaccurately determined fragments @ m/z 152 and m/z 170,respectively, come up with a single sum formula proposaleach. In the mass range below m/z 150 some morefragments are observed that can be assigned by sumformulae unambiguously. Labeling of proposed structureswas done by Mass Frontier.

Figure 2: A mass spectrum of the region of interest m/z 391.5 - 395.5 withan inset of m/z 394.

Isotop e Min Max RDB equi v. Mass

14N 0 10 0.5 14.00316O 0 15 0.0 15.99512C 0 30 1.0 12.0001H 0 60 -0.5 1.00832S 0 2 0.0 31.972

Table 2: Elements in use.

m/z Theo. Mass Delta (ppm) RDB equi v. Composition

392.09769 392.09761 0.13 10.5 C18H18O9N1

392.09829 -1.62 6.5 C11H18O7N732S1

392.09696 1.79 1.5 C10H22O11N332S1

392.0984 8 -2.0 9 9. 5 C19H22O4N132S2

392.09644 3.10 19.5 C23H14N532S1

392.09895 -3.28 15.5 C19H14O5N5

392.09579 4.76 10.5 C15H18O2N732S2

Table 3: Elemental composition applying the nitrogen rule.

286

Figure 4: Search for the structure of compound X.

Tiotropium is the only hit with sum formulaC19H 22O 4N 1S2of double bonds / unpaired electron pairs given for thissum formula in Table 3, RDB equiv. = 9.5.

Figure 5a: MS/MS @ m/z 392 using Collision Induced Dissociation (CID) as a fragmentation techniques detected in the Orbitrap analyzer.

Figure 5b: MS/MS @ m/z 392 using Higher Energy C-Trap Dissociation (HCD) as a fragmentation techniques detected in the Orbitrap analyzer.

FTMS + p ESI Full ms2 392.10@cid35.00 [105.00-420.00]

150 200 250 300 350 400m/z

C9H14O1N1

C9H16O2N1

152.106513.5 RDBE-3.2 ppm

170.117112.5 RDBE-2.6 ppm

m/z 392.098

NO

OO

OH

S

Sm/z 392.098

i

m/z 152.107

rHR

NO

+

+

+

+

+

OH

NO

O

O

N

O

OHS

SNO

FTMS + p ESI Full ms2 392.10@hcd100.00 [50.00-1000.00]

50 100 150 200 250 300 350 400m/z

50 60 70 80 90 100 110 120 130 140m/z

C9H14O1N1

C9H16O2N1 C19H22O4N1S2

152.106573.5 RDBE-2.7 ppm

134.096044.5 RDBE-2.9 ppm

122.096083.5 RDBE-2.8 ppm

112.075342.5 RDBE-3.1 ppm

94.064803.5 RDBE-3.4 ppm

91.053904.5 ppm-3.5 ppm

392.09802

9.5 RDBE-1.0 ppm

170.117162.5 RDBE-2.3 ppm

NNO

OH

NO

N

C6H8N1

C6H10N1

C8H12N1

C9H12N1

C7H7

+

+

+

+

NHO

+

+

Proposed fragmentation pathwayfor fragment ion m/z 152.

Figure 3: All seven proposals vs. raw data information. Inset into full isotope pattern (left); insets into the A+1, A+2, A+3 and A+4 ion (right).

287

Figure 4: Search for the structure of compound X.

Tiotropium is the only hit with sum formulaC19H 22O 4N 1S2of double bonds / unpaired electron pairs given for thissum formula in Table 3, RDB equiv. = 9.5.

Figure 5a: MS/MS @ m/z 392 using Collision Induced Dissociation (CID) as a fragmentation techniques detected in the Orbitrap analyzer.

Figure 5b: MS/MS @ m/z 392 using Higher Energy C-Trap Dissociation (HCD) as a fragmentation techniques detected in the Orbitrap analyzer.

FTMS + p ESI Full ms2 392.10@cid35.00 [105.00-420.00]

150 200 250 300 350 400m/z

C9H14O1N1

C9H16O2N1

152.106513.5 RDBE-3.2 ppm

170.117112.5 RDBE-2.6 ppm

m/z 392.098

NO

OO

OH

S

Sm/z 392.098

i

m/z 152.107

rHR

NO

+

+

+

+

+

OH

NO

O

O

N

O

OHS

SNO

FTMS + p ESI Full ms2 392.10@hcd100.00 [50.00-1000.00]

50 100 150 200 250 300 350 400m/z

50 60 70 80 90 100 110 120 130 140m/z

C9H14O1N1

C9H16O2N1 C19H22O4N1S2

152.106573.5 RDBE-2.7 ppm

134.096044.5 RDBE-2.9 ppm

122.096083.5 RDBE-2.8 ppm

112.075342.5 RDBE-3.1 ppm

94.064803.5 RDBE-3.4 ppm

91.053904.5 ppm-3.5 ppm

392.09802

9.5 RDBE-1.0 ppm

170.117162.5 RDBE-2.3 ppm

NNO

OH

NO

N

C6H8N1

C6H10N1

C8H12N1

C9H12N1

C7H7

+

+

+

+

NHO

+

+

Proposed fragmentation pathwayfor fragment ion m/z 152.

Figure 3: All seven proposals vs. raw data information. Inset into full isotope pattern (left); insets into the A+1, A+2, A+3 and A+4 ion (right).

288

Quantitative ResultsThe dilution series applying two different targeted MS/MSmethods was carried out. Results of three data sets areshown in Figure 6a for targeted MS/MS and Orbitrapdetection. The calibration graph on the right gives anoverview over all measurement points (10 fg/µL to 10 pg/µL). The inset (in blue) shows the calibration graphbetween 10 fg/µL (200 fg on column) and 100 fg/µL (2 pg on column) in detail. For details on the graph alsosee the table displayed in Figure 6b. Results for targetedMS/MS and ion trap detection is shown in Figure 7.

Figure 6a: Dilution series applying a targeted MS/MS method, results of three data sets. Targeted MS/MS Orbitrap detection, FTMS + p norm res = 15,000/7,500, MS/MS @ 392.1 CID Collision Energy 35.0% Q 0.25Time 30.0 ms IsoW 3.0 u.

Figure 6b: Result table in detail for the three data sets from Figure 6a.

Figure 7: Quantitation results for targeted MS/MS method, results of a single data sets. 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.

Retrievalfrom 3 data

sets

289

Conclusions• An accurate mass measurement of compound X

combined with RDBE evaluation and isotope patternrecognition revealed a sum formula C 19H 22O 4N 1S2 forcompound X.

• An Internet accessible database search using thepreviously determined sum formula revealed Tiotropiumas single hit.

• MS/MS information obtained by Collision InducedDissociation (CID) and Higher Energy C-trapDissociation (HCD) combined with the fragmentation

Tiotropium. CID and HCD fragments are assigned byMass Frontier.

• A dilution series - applying a targeted MS/MS @ m/z 392 method detecting the fragments at m/z 152 andm/z 170, respectively, in the ion trap or the Orbitrapanalyzer - revealed that reliable calibration curves can be acquired down to total column load of 200 fg of Tiotropium. This is equivalent to a 20 µL full loopload of 10 fg/µL solution of Tiotropium.

290

291

292

293