EXACT MASS GC/MS ANALYSIS OFAMINE MONOMERS USED IN

COMBINATORIAL LIBRARY PRODUCTION

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Robert L. Johnson1, Robert Wiethe2, William Stuart2, Peter Hancock3 and Anthony Newton3

1GlaxoSmithKline, Computational Analytical and Structural Sciences, RTP, NC, USA.2GlaxoSmithKline, Chemistry and Screening, RTP, NC, USA. 3Waters Corporation, Manchester, UK.

Introduction

Monomers are the low molecular weight buildingblocks used in the construction of combinatoriallibraries. Confirmation of the identity and purity ofthese compounds, prior to using them in synthesis, isimportant in order to ensure that a synthetic route toan identified ‘active’ can be recreated. In addition,the requirement for post-purification of the finalproduct is significantly reduced if not eliminated.1,2,3

In many cases, however, the production ofcombinatorial libraries has sometimes proceeded withlittle regard to the purity of starting material. This hasmore to do with the complexity of analyzing largenumbers of compounds than with a lack of desirefrom the synthetic chemist. While much time has beenspent in developing LC/MS techniques and softwareto analyze the products of a combinatorial synthesis,little has been done to speed the process of analyzing

small molecules not easily seen by LC/MS. GC/MSanalysis in both electron impact (EI) and chemicalionization (CI) modes has long been used for theanalysis of small, volatile compounds.

In this note, monomer confirmation using the GCT™

orthogonal Time-of-Flight (TOF) MS detector combinedwith OpenLynx™ high throughput software is described.The GCT produces exact mass data for elementalcomposition calculation and strucural elucidation.OpenLynx provides a true ‘walk up and use’ interfacewith flexible data browsing and reporting.

Ninety-five (95) primary amines from the MonomersStore facility at GlaxoSmithKline RTP were analyzedby exact mass measurement using the GCT acquiringboth EI and CI mass spectra.

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Methodology

Experiments were performed using a Micromass GCTTM oa-TOF mass spectrometer operated in positive ionelectron impact (EI+) and positive ion chemical ionisation (CI+) modes with a one second acquisition over amass range of 35-500Da. The source temperature was set to 180 °C in EI+ and 100 °C in CI+. Ammoniareagent gas at a pressure of 2 x 10-4 mbar was used for the CI analysis.

GC analyses were performed using a HP6890 gas chromatograph split/splitless injector. A J & W ScientificDB5-MS, 15m x 0.53mm ID column with a 1m x 0.1mm ID fused silica restrictor at the GC transfer line wasused in constant flow mode with 1 mL/min Helium flow. The GC temperature program was 150 °C (2 mins) to250 °C (4 mins) at 50 °C /min. This gave a run time of 8 minutes and injection-to-injection time of 11 minutes.

Results

The EI and CI mass spectra of the 95 amines revealed only eight that did not contain the desired compound (vial74 was broken). The amount of the desired compound present determined from the CI Total Ion Chromatogram(TIC), ranged from less than a few percent to 100%. This calculation was made using OpenLynx, comparing thepeak area of the compound of interest to the total area under the chromatogram. The results shown in Figure 1were extracted from the OpenLynx Browser file shown in Figure 2.

In addition to this purity estimate, the identity of the impurities can also be determined from the data as shown inthe following examples.

Name Well, Expected Formula Target Found Estimated %BUTYLAMINE 1, C4H11N 74.0970 YES 70SEC-BUTYLAMINE 2, C4H11N 74.0970 NO 0ISOBUTYLAMINE 3, C4H11N 74.0970 NO 02-METHOXYETHYLAMINE 4, C3H9NO 76.0762 YES 4CYCLOPENTYLAMINE 5, C5H11N 86.0970 YES 63N-AMYLAMINE 6, C5H13N 88.1126 YES 243-AMINOPENTANE 7, C5H13N 88.1126 YES 112-METHYLBUTYLAMINE 8, C5H13N 88.1126 NO 0ISOAMYLAMINE 9, C5H13N 88.1126 YES 100NEOPENTYLAMINE 10, C5H13N 88.1126 NO 01,2-DIMETHYLPROPYLAMINE 11, C5H13N 88.1126 YES 99N,N-DIMETHYLETHYLENEDIAMINE 12, C4H12N2 89.1079 YES 391,2-DIAMINO-2-METHYLPROPANE 13, C4H12N2 89.1079 YES 193-METHOXYPROPYLAMINE 14, C4H11NO 90.0919 YES 742-AMINO-1-METHOXYPROPANE 15, C4H11NO 90.0919 YES 15FURFURYLAMINE 16, C5H7NO 98.0606 NO 02,2,2-TRIFLUOROETHYLAMINE 17, C2H4F3N 100.0374 NO 0CYCLOHEXYLAMINE 18, C6H13N 100.1126 YES 943-AMINO-1-PROPANOL VINYL ETHER 19, C5H11NO 102.0919 YES 77TETRAHYDROFURFURYLAMINE 20, C5H11NO 102.0919 YES 84HEXYLAMINE 21, C6H15N 102.1283 YES 21,3-DIMETHYLBUTYLAMINE 22, C6H15N 102.1283 YES 692-AMINO-3,3-DIMETHYLBUTANE 23, C6H15N 102.1283 YES 55N1,N1-DIMETHYL-1,2-PROPANEDIAMINE 24, C5H14N2 103.1235 YES 43N,N-DIMETHYL-1,3-PROPANEDIAMINE 25, C5H14N2 103.1235 YES 732-AMINO-1-METHOXYBUTANE 26, C5H13NO 104.1075 YES 563-ETHOXYPROPYLAMINE 27, C5H13NO 104.1075 YES 742-AMINOETHYL ISOPROPYL ETHER 28, C5H13NO 104.1075 YES 90AMINOACETALDEHYDE DIMETHYL ACETAL 29, C4H11NO2 106.0868 YES 703-(METHYLTHIO)PROPYLAMINE 30, C4H11NS 106.0690 NO 02-(ETHYLTHIO)ETHYLAMINE 31, C4H11NS 106.0690 YES 79BENZYLAMINE 32, C7H9N 108.0813 YES 784-(AMINOMETHYL)PYRIDINE 33, C6H8N2 109.0766 YES 633-(AMINOMETHYL)PYRIDINE 34, C6H8N2 109.0766 YES 762-(AMINOMETHYL)PYRIDINE 35, C6H8N2 109.0766 YES 655-METHYL-2-FURANMETHANAMINE 36, C6H9NO 112.0762 YES 45EXO-2-AMINONORBORNANE 37, C7H13N 112.1126 YES 762-THIOPHENEMETHYLAMINE 38, C5H7NS 114.0377 YES 58CYCLOHEPTYLAMINE

Name Well, Expected Formula Target Found Estimated %BUTYLAMINE 1, C4H11N 74.0970 YES 70SEC-BUTYLAMINE 2, C4H11N 74.0970 NO 0ISOBUTYLAMINE 3, C4H11N 74.0970 NO 02-METHOXYETHYLAMINE 4, C3H9NO 76.0762 YES 4CYCLOPENTYLAMINE 5, C5H11N 86.0970 YES 63N-AMYLAMINE 6, C5H13N 88.1126 YES 243-AMINOPENTANE 7, C5H13N 88.1126 YES 112-METHYLBUTYLAMINE 8, C5H13N 88.1126 NO 0ISOAMYLAMINE 9, C5H13N 88.1126 YES 100NEOPENTYLAMINE 10, C5H13N 88.1126 NO 01,2-DIMETHYLPROPYLAMINE 11, C5H13N 88.1126 YES 99N,N-DIMETHYLETHYLENEDIAMINE 12, C4H12N2 89.1079 YES 391,2-DIAMINO-2-METHYLPROPANE 13, C4H12N2 89.1079 YES 193-METHOXYPROPYLAMINE 14, C4H11NO 90.0919 YES 742-AMINO-1-METHOXYPROPANE 15, C4H11NO 90.0919 YES 15FURFURYLAMINE 16, C5H7NO 98.0606 NO 02,2,2-TRIFLUOROETHYLAMINE 17, C2H4F3N 100.0374 NO 0CYCLOHEXYLAMINE 18, C6H13N 100.1126 YES 943-AMINO-1-PROPANOL VINYL ETHER 19, C5H11NO 102.0919 YES 77TETRAHYDROFURFURYLAMINE 20, C5H11NO 102.0919 YES 84HEXYLAMINE 21, C6H15N 102.1283 YES 21,3-DIMETHYLBUTYLAMINE 22, C6H15N 102.1283 YES 692-AMINO-3,3-DIMETHYLBUTANE 23, C6H15N 102.1283 YES 55N1,N1-DIMETHYL-1,2-PROPANEDIAMINE 24, C5H14N2 103.1235 YES 43N,N-DIMETHYL-1,3-PROPANEDIAMINE 25, C5H14N2 103.1235 YES 732-AMINO-1-METHOXYBUTANE 26, C5H13NO 104.1075 YES 563-ETHOXYPROPYLAMINE 27, C5H13NO 104.1075 YES 742-AMINOETHYL ISOPROPYL ETHER 28, C5H13NO 104.1075 YES 90AMINOACETALDEHYDE DIMETHYL ACETAL 29, C4H11NO2 106.0868 YES 703-(METHYLTHIO)PROPYLAMINE 30, C4H11NS 106.0690 NO 02-(ETHYLTHIO)ETHYLAMINE 31, C4H11NS 106.0690 YES 79BENZYLAMINE 32, C7H9N 108.0813 YES 784-(AMINOMETHYL)PYRIDINE 33, C6H8N2 109.0766 YES 633-(AMINOMETHYL)PYRIDINE 34, C6H8N2 109.0766 YES 762-(AMINOMETHYL)PYRIDINE 35, C6H8N2 109.0766 YES 655-METHYL-2-FURANMETHANAMINE 36, C6H9NO 112.0762 YES 45EXO-2-AMINONORBORNANE 37, C7H13N 112.1126 YES 762-THIOPHENEMETHYLAMINE 38, C5H7NS 114.0377 YES 58CYCLOHEPTYLAMINE 39, C7H15N 114.1283 YES 923-METHYLCYCLOHEXYLAMINE 40, C7H15N 114.1283 YES 642-METHYLCYCLOHEXYLAMINE 41, C7H15N 114.1283 YES 814-METHYLCYCLOHEXYLAMINE 42, C7H15N 114.1283 YES 861-(2-AMINOETHYL)PYRROLIDINE 43, C6H14N2 115.1235 YES 563-AMINOHEPTANE 44, C7H17N 116.1439 YES 71N,N-DIETHYLETHYLENEDIAMINE 45, C6H16N2 117.1392 YES 634-DIMETHYLAMINOBUTYLAMINE 46, C6H16N2 117.1392 YES 583-ISOPROPOXYPROPYLAMINE 47, C6H15NO 118.1232 YES 804-METHYLBENZYLAMINE 48, C8H11N 122.0970 YES 100

39, C7H15N 114.1283 YES 923-METHYLCYCLOHEXYLAMINE 40, C7H15N 114.1283 YES 642-METHYLCYCLOHEXYLAMINE 41, C7H15N 114.1283 YES 814-METHYLCYCLOHEXYLAMINE 42, C7H15N 114.1283 YES 861-(2-AMINOETHYL)PYRROLIDINE 43, C6H14N2 115.1235 YES 563-AMINOHEPTANE 44, C7H17N 116.1439 YES 71N,N-DIETHYLETHYLENEDIAMINE 45, C6H16N2 117.1392 YES 634-DIMETHYLAMINOBUTYLAMINE 46, C6H16N2 117.1392 YES 583-ISOPROPOXYPROPYLAMINE 47, C6H15NO 118.1232 YES 804-METHYLBENZYLAMINE 48, C8H11N 122.0970 YES 100

Name Well, Expected Formula Target Found Estimated %3-METHYLBENZYLAMINE 49, C8H11N 122.0970 YES 772-METHYLBENZYLAMINE 50, C8H11N 122.0970 YES 70DL-ALPHA-METHYLBENZYLAMINE 51, C8H11N 122.0970 YES 66(R)-(+)-1-PHENYLETHYLAMINE 52, C8H11N 122.0970 YES 82L-(-)-ALPHA-METHYLBENZYLAMINE 53, C8H11N 122.0970 YES 84PHENETHYLAMINE 54, C8H11N 122.0970 YES 544-(2-AMINOETHYL)PYRIDINE 55, C7H10N2 123.0922 YES 503-(2-AMINOETHYL)PYRIDINE 56, C7H10N2 123.0922 YES 532-(2-AMINOETHYL)PYRIDINE 57, C7H10N2 123.0922 YES 664-FLUOROBENZYLAMINE 58, C7H8FN 126.0719 YES 313-FLUOROBENZYLAMINE 59, C7H8FN 126.0719 YES 272-FLUOROBENZYLAMINE 60, C7H8FN 126.0719 YES 15N-(3-AMINOPROPYL)IMIDAZOLE 61, C6H11N3 126.1031 YES 792-(1-CYCLOHEXENYL)ETHYLAMINE 62, C8H15N 126.1283 YES 77THIOPHENE-2-ETHYLAMINE 63, C6H9NS 128.0534 YES 59CYCLOOCTYLAMINE 64, C8H17N 128.1439 YES 362,3-DIMETHYLCYCLOHEXYLAMINE 65, C8H17N 128.1439 YES 97(R)-(-)-1-CYCLOHEXYLETHYLAMINE 66, C8H17N 128.1439 YES 95(S)-(+)-1-CYCLOHEXYLETHYLAMINE 67, C8H17N 128.1439 YES 952-(2-AMINOETHYL)-1-METHYLPYRROLIDINE 68, C7H16N2 129.1392 YES 772-(AMINOMETHYL)-1-ETHYL-PYRROLIDINE 69, C7H16N2 129.1392 YES 541-PYRROLIDINEPROPANAMINE 70, C7H16N2 129.1392 YES 881-(2-AMINOETHYL)PIPERIDINE 71, C7H16N2 129.1392 YES 622-AMINOOCTANE 72, C8H19N 130.1596 YES 802-AMINO-6-METHYLHEPTANE 73, C8H19N 130.1596 YES 100N-(2-AMINOETHYL)MORPHOLINE 75, C6H14N2O 131.1184 YES 88N,N-DIMETHYLNEOPENTANEDIAMINE 76, C7H18N2 131.1548 YES 1003-DIETHYLAMINOPROPYLAMINE 77, C7H18N2 131.1548 YES 86ETHYL 3-AMINOBUTYRATE 78, C6H13NO2 132.1025 NO 03-BUTOXYPROPYLAMINE 79, C7H17NO 132.1388 YES 744-AMINOBUTYRALDEHYDE DIMETHYL ACETAL 80, C6H15NO2 134.1181 YES 87AMINOACETALDEHYDE DIETHYL ACETAL 81, C6H15NO2 134.1181 YES 741-AMINOINDANE 82, C9H11N 134.0970 YES 672-AMINOINDAN 83, C9H11N 134.0970 YES 932-(P-TOLYL)ETHYLAMINE 84, C9H13N 136.1126 YES 24BETA-METHYLPHENETHYLAMINE 85, C9H13N 136.1126 YES 62ALPHA-ETHYLBENZYLAMINE 86, C9H13N 136.1126 YES 653,4-DIMETHYLBENZYLAMINE 87, C9H13N 136.1126 YES 693-PHENYLPROPYLAMINE

Name Well, Expected Formula Target Found Estimated %3-METHYLBENZYLAMINE 49, C8H11N 122.0970 YES 772-METHYLBENZYLAMINE 50, C8H11N 122.0970 YES 70DL-ALPHA-METHYLBENZYLAMINE 51, C8H11N 122.0970 YES 66(R)-(+)-1-PHENYLETHYLAMINE 52, C8H11N 122.0970 YES 82L-(-)-ALPHA-METHYLBENZYLAMINE 53, C8H11N 122.0970 YES 84PHENETHYLAMINE 54, C8H11N 122.0970 YES 544-(2-AMINOETHYL)PYRIDINE 55, C7H10N2 123.0922 YES 503-(2-AMINOETHYL)PYRIDINE 56, C7H10N2 123.0922 YES 532-(2-AMINOETHYL)PYRIDINE 57, C7H10N2 123.0922 YES 664-FLUOROBENZYLAMINE 58, C7H8FN 126.0719 YES 313-FLUOROBENZYLAMINE 59, C7H8FN 126.0719 YES 272-FLUOROBENZYLAMINE 60, C7H8FN 126.0719 YES 15N-(3-AMINOPROPYL)IMIDAZOLE 61, C6H11N3 126.1031 YES 792-(1-CYCLOHEXENYL)ETHYLAMINE 62, C8H15N 126.1283 YES 77THIOPHENE-2-ETHYLAMINE 63, C6H9NS 128.0534 YES 59CYCLOOCTYLAMINE 64, C8H17N 128.1439 YES 362,3-DIMETHYLCYCLOHEXYLAMINE 65, C8H17N 128.1439 YES 97(R)-(-)-1-CYCLOHEXYLETHYLAMINE 66, C8H17N 128.1439 YES 95(S)-(+)-1-CYCLOHEXYLETHYLAMINE 67, C8H17N 128.1439 YES 952-(2-AMINOETHYL)-1-METHYLPYRROLIDINE 68, C7H16N2 129.1392 YES 772-(AMINOMETHYL)-1-ETHYL-PYRROLIDINE 69, C7H16N2 129.1392 YES 541-PYRROLIDINEPROPANAMINE 70, C7H16N2 129.1392 YES 881-(2-AMINOETHYL)PIPERIDINE 71, C7H16N2 129.1392 YES 622-AMINOOCTANE 72, C8H19N 130.1596 YES 802-AMINO-6-METHYLHEPTANE 73, C8H19N 130.1596 YES 100N-(2-AMINOETHYL)MORPHOLINE 75, C6H14N2O 131.1184 YES 88N,N-DIMETHYLNEOPENTANEDIAMINE 76, C7H18N2 131.1548 YES 1003-DIETHYLAMINOPROPYLAMINE 77, C7H18N2 131.1548 YES 86ETHYL 3-AMINOBUTYRATE 78, C6H13NO2 132.1025 NO 03-BUTOXYPROPYLAMINE 79, C7H17NO 132.1388 YES 744-AMINOBUTYRALDEHYDE DIMETHYL ACETAL 80, C6H15NO2 134.1181 YES 87AMINOACETALDEHYDE DIETHYL ACETAL 81, C6H15NO2 134.1181 YES 741-AMINOINDANE 82, C9H11N 134.0970 YES 672-AMINOINDAN 83, C9H11N 134.0970 YES 932-(P-TOLYL)ETHYLAMINE 84, C9H13N 136.1126 YES 24BETA-METHYLPHENETHYLAMINE 85, C9H13N 136.1126 YES 62ALPHA-ETHYLBENZYLAMINE 86, C9H13N 136.1126 YES 653,4-DIMETHYLBENZYLAMINE 87, C9H13N 136.1126 YES 693-PHENYLPROPYLAMINE 88, C9H13N 136.1126 YES 263-METHOXYBENZYLAMINE 89, C8H11NO 138.0919 YES 694-METHOXYBENZYLAMINE 90, C8H11NO 138.0919 YES 462-METHOXYBENZYLAMINE 91, C8H11NO 138.0919 YES 702-PHENOXYETHYLAMINE 92, C8H11NO 138.0919 YES 184-FLUOROPHENETHYLAMINE 93, C8H10FN 140.0876 YES 963-FLUOROPHENETHYLAMINE 94, C8H10FN 140.0876 YES 582-FLUOROPHENETHYLAMINE 95, C8H10FN 140.0876 YES 434-FLUORO-ALPHA-METHYLBENZYLAMINE 96, C8H10FN 140.0876 YES 31

Figure 1. Results of Monomer Screening

Example 1

Cyclopentylamine, m/z 86.097, is present at 63% (Figure 3).Several impurities are present with a nominal mass of 102, butwith different elemental compositions. Two of these impuritieswere identified as saturated oxidized impurities of amines(Figure 4). Impurities at 104 and 106 were also identified assaturated oxidized impurities (Figure 4).

Figure 2. Example of OpenLynx™ Browser Results

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100

%

0

100

%

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%

0

%

Devel105 TOF MS CI+86.0967.45e4

3.93

Devel105 TOF MS CI+TIC

4.47e53.93

5.254.084.18

4.274.484.87

NH2

Mass Calc. Mass mDa PPM DBE Formula

86.0962 86.0970 -0.8 -9.0 0.5 C5H12N

35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 155 160m/z0

100

%

Devel105 62 (4.032) TOF MS CI+7.85e386.0962

35.0611

79.021556.0493

102.1109

98.060087.0999

104.1072

116.9949116.1427 132.9658119.1541

129.9865 147.9936134.9627 151.9876

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%

0

%

3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50Time

TOF MS CI+86.0967.45e4

3.93

TOF MS CI+TIC

4.47e53.93

5.254.084.18

4.274.484.87

NH2

35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 155 160m/z

Devel105 62 (4.032) TOF MS CI+7.85e386.0962

35.0611

79.021556.0493

102.1109

98.060087.0999

104.1072

116.9949116.1427 132.9658119.1541

129.9865 147.9936134.9627 151.9876

35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 155 160m/z

Devel105 62 (4.032) TOF MS CI+7.85e386.0962

35.0611

79.021556.0493

102.1109

98.060087.0999

104.1072

86.0962

35.0611

79.021556.0493

102.1109

98.060087.0999

104.1072

116.9949116.1427 132.9658119.1541

129.9865 147.9936134.9627 151.9876

Figure 3. Cyclopentylamine

%

Devel105 TOF MS CI+102

1.95e4

0

100 3.90 4.00

4.13

a b

c

0

100

%

0

100

%

0

100

%

0

100

%

Devel105 TOF MS CI+106

2.09e43.97

4.20 4.50

Devel105 TOF MS CI+104

1.64e43.98

3.95

Mass Calc. Mass mDa PPM DBE Formula106.0860 106.0868 -0.8 -7.6 -0.5 C4H12NO2

Mass Calc. Mass mDa PPM DBE Formula104.1074 104.1075 -0.1 -1.3 -0.5 C5H14NO

Mass Calc. Mass mDa PPM DBE Formulaa 102.0917 102.0919 -0.2 -1.9 0.5 C5H12NOb 102.1047 102.1045 0.2 2.3 0.0 C6H14Oc 102.1273 102.1283 -1.0 -9.5 -0.5 C6H16N

Devel105 TOF MS CI+102

1.95e4

0

100 3.90 4.00

4.13

0

100

%

0

100

%

0

100

%

Devel105 TOF MS CI+106

2.09e4

TOF MS CI+106

2.09e43.97

4.20 4.50

TOF MS CI+104

1.64e4

TOF MS CI+104

1.64e4

a

Figure 4. Impurities in Cyclopentylamine

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Example 2

Three isomeric fluorobenzylamines were analyzed invials (4-fluorobenzylamine), (3-fluorobenzylamine), (2-fluorobenzylamine) and showed vastly differentimpurity profiles. The accurate mass data confirmedthat the indicated GC peaks at 4.42 minutes retentiontime (Figure 5) were due to the desired compounds.The amount of desired compound was 31%, 27%,and 15% respectively. A combination of CI and EIspectra and accurate mass data were used todetermine the structure of these impurities. Thecompounds were from three different vendors andcontained one common impurity at approximately 7.5minutes retention time shown in Figure 5. Thiscompound was determined to be an isomer of thedifluorobenzylimine shown in Figure 6. The electronimpact spectrum showed only the molecular ion atm/z 231 and a fragment at m/z 109 due to thefluorobenzyl ion. Since these three compounds arefrom three different vendors, this impurity is probablydue to degradation of the amine.

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100

%

0

100

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0

100

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0

100

%

4.423.67 3.78 7.42

6.376.185.584.90 5.72 5.90 6.726.52

7.55

4.43

4.08

7.50

5.885.50

4.70 5.185.68

6.186.00

6.37 6.756.53 7.306.95

4.42

4.02

7.50

5.53

5.07 5.88 6.18

Mass Calc. Mass mDa PPM DBE Formula126.0717 126.0719 -0.2 -1.6 3.5 C7H9NF

NH2

F

NH2

F

NH2

F

Figure 5. 4-, 3- and 2-Fluorobenzylamine andCommon Impurity

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%

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%

4.42

3.673.78

7.42

6.376.18

5.584.90 5.725.90

6.72

6.52

7.55

Mass Calc. Mass mDa PPM DBE Formula248.0878 248.0887 -0.9 -3.6 8.5 C14H12NOF2

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290m/z0

100

%

265.1155

248.0879

102.1079

98.059635.0604 104.1075119.1548 184.0172132.9665 167.9992

247.0835232.0935233.0980

249.0912

250.0962

266.1189

292.1516267.1219

NH

F F

O

(M+H)+

(M+NH4)+

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%

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%

4.42

3.673.78

7.42

6.376.18

5.584.90 5.725.90

6.72

6.52

7.55

a

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290m/z0

100

%

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290m/z0

100

%

265.1155

248.0879

102.1079

98.059635.0604 104.1075119.1548 184.0172132.9665 167.9992

247.0835232.0935233.0980

249.0912

250.0962

266.1189

292.1516267.1219

NH

F F

O

(M+H)+

(M+NH4)+

Figure 5. 4-, 3- and 2-Fluorobenzylamine andCommon Impurity

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100

%

232.0935

166.102935.0611126.0715

225.1387

233.0975

235.1612

Mass Calc. Mass mDa PPM DBE Formula232.0935 232.0938 -0.3 1.2 8.5 C14H12NF2

N

F

F

Figure 6. Identification of Common Impurity

The major impurity in 4-fluorobenzylaminewas determined to be an amide as shownin Figure 7 (CI spectrum) and Figure 8 (EIspectrum). The two impurities present in 2-fluoro-benzylamine (Figure 9) are notrelated to the desired compound - Figure10 shows the CI spectra and the elementalcomposition results. From the exact massmeasured fragments in the EI spectra, thestructures were determined to be as shownin Figure 11.

F

O+

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260m/z0

100

%

247.0807123.0237

95.0290

75.023069.026256.050241.0381

83.0298 109.0448122.0397

246.0730124.0435

166.9912125.0456

150.0402 170.9627 229.0736

248.0846

249.0895258.1081

F

+F

O+

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260m/z0

100

%

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260m/z0

100

%

247.0807123.0237

95.0290

75.023069.026256.050241.0381

83.0298 109.0448122.0397

246.0730

247.0807123.0237

95.0290

75.023069.026256.050241.0381

83.0298 109.0448122.0397

246.0730124.0435

166.9912125.0456

150.0402 170.9627 229.0736

248.0846

249.0895258.1081

F

+

Figure 8. Major Impurity in 4-Fluorobenzylamine (EI+ spectrum)

3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00 7.25 7.50 7.75Time0

100

%

4.42

4.02

7.50

5.53

5.07 5.88 6.18

NH2

F

3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00 7.25 7.50 7.75Time0

100

%

4.42

4.02

7.50

5.53

5.07 5.88 6.18

Figure 9. Impurities in 2-Fluorobenzylamine

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240m/z0

100

%

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240m/z0

100

%

0

100

%

0

100

%

228.1496

117.1390

116.1434102.091986.096652.088143.0423 56.0482

80.0477 135.0915118.1404

184.0185167.9992147.9941226.1343

229.1537

230.1563242.1606

228.1136

184.0185116.995292.997088.0800

52.0878 80.0512 106.0645123.0905

132.9654 167.9994147.9937

194.1334227.1066

229.1167

230.1203242.1264

Mass Calc. Mass mDa PPM DBE Formula228.1496 228.1501 -0.5 -2.5 7.5 C14H18N3

Mass Calc. Mass mDa PPM DBE Formula228.1136 228.1137 -0.1 -0.4 8.5 C13H14N3O

Figure 10. Impurities in 2-Fluorobenzylamine (CI+ spectra)

Applic

ation

NO

TE

Conclusions

Of the 95 compounds analyzed in this experiment,only eight were found not to contain the desiredcompound. The accuracy of masses measured for thecompounds and impurities presented was calculated.(Figure 12). These masses were measured using asingle lock mass in both EI and ammonia CI. Theexact mass data can also be used to calculateelemental composition, thus enabling the identificationof impurities in the starting material. The use of purerstarting materials can reduce the time necessary forpurification and quantitation of final library members.Time can also be saved if it can be demonstrated thatthe impurities are not likely to lead to impurities in thefinal product. In the case of the benzylamines, forexample, the amine present in all three sampleswould not present a problem if the primary aminewas linked to a solid phase resin. The impuritieswould not react and could be rinsed from the resinafter the coupling reaction had been performed. Thistype of analysis is easily automated using OpenLynx,a sophisticated batch-processing engine allowingchemists easy access to GCT. This streamlines theanalysis of large batches of samples for screeningand automatically verifies that a compound of thedesired formula is present at each well location andthat the purity of each targeted compound exceeds auser-defined threshold.

The combination of GCT and OpenLynx has beenshown to be ideal for the high throughput screeningof monomers for combinatorial library production.

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240m/z0

100

%

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240m/z0

100

%

0

100

%

0

100

%

135.0916

106.064986.0960

58.064942.0340 78.0341

93.0570

99.1045121.0764

136.0952166.9910149.0927

170.9618182.9615

227.1411233.0991

121.0760

93.056978.0338

51.0227 65.0383

79.0411

91.0455

106.0351

94.0639116.9948

135.0554

122.0797 150.0719149.0708 166.9911 182.0835

227.1049225.1235 228.1099 239.0032

N

NH

N

CH3

O

NNH

N

CH3

N

O+

N+

NH

N

CH3

+

N+

Figure 11. Structures of Impurities in 2-Fluorobenzylamine (from EI+ spectra)

Compound Mass CalcMass

mDa PPM DBE Formula

Cyclopentylamine 86.0962 86.0970 -0.8 -9.0 0.5 C5H12NImpurity 1 102.0917 102.0919 -0.2 -1.9 0.5 C5H12NOImpurity 2 102.1047 102.1045 0.2 2.3 0.0 C6H14OImpurity 3 102.1273 102.1283 -1.0 -9.5 -0.5 C6H16NImpurity 4 106.0860 106.0868 -0.8 -7.6 -0.5 C4H12NO2Impurity 5 104.1074 104.1075 -0.1 -1.3 -0.5 C5H14NO

2-,3-and 4-Fluorobenzylamine

126.0717 126.0719 -0.2 -1.6 3.5 C7H9NF

Common Impurity 232.0935 232.0938 -0.3 -1.2 8.5 C14H12NF24- Impurity 248.0878 248.0887 -0.9 -3.6 8.5 C14H12NOF2

2- Impurity 1 228.1496 228.1501 -0.5 -2.5 7.5 C14H18N32- Impurity 2 228.1136 228.1137 -0.1 -0.4 8.5 C13H14N3O

Mass Measurement Accuracy RMS 0.57 4.89

Figure 12. GCT Mass Accuracy

References

1. Van Hijfte, Luc; Marciniak, Gilbert; Froloff, Nicolas. J.Chromatogr., B: Biomed. Sci. Appl. (1999), 725(1), 3-15

2. Volonterio, Alessandro; Bravo, Pierfrancesco; Zanda,Matteo. Tetrahedron Letters (2001), 42, 3141-3144

3. Bookser, Brett C.; Zhu, Shirong. J.Comb. Chem., (2001),3(2), 205-215

Applic

ation

NO

TE

Waters and Micromass are registered trademarks of Waters Corporation.GCT, and OpenLynx are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.©2002 Waters Corporation, October 2002 (AN259 Version 1) 720000531ENFor research use only. Not for use in diagnostic procedures.

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