analytical technologies and compound identification · 2018-04-06 · compound identification...

Analytical Technologies and Compound Identification Daniel L. Norwood, MSPH, PhDSCĪO Analytical Consulting, LLC.

Extractables/Leachables Characterization (USP<1663>)

Characterization is the discovery, identification, and quantitation of each individual organic and inorganic chemical entity present in an extract above a specified level or threshold. Such thresholds can be based on patient safety considerations, materials considerations, the capabilities of analytical technology, etc.

Scouting is the process of acquiring general chemical information that provides insight into the nature and magnitude of extractables.

Discovery is the process of searching for, and ultimately finding, individual organic and inorganic chemical entities present in an extract.

Identification is the process of assigning a molecular structure to an organic extractable, or assigning constituent elements in the case of an inorganic extractable.

Quantitation is the process of measuring the level, or concentration, of an individual organic or inorganic chemical entity contained in an

Identification – Sub-categories

• Qualitative Analysis – A type of chemical analysis in which the analyte or analytes in a sample are identified (YES/NO result).

• Structural Analysis - A type of chemical analysis in which the molecular structure(s) of an analyte(s) is elucidated.

• Quantitative Analysis - A type of chemical analysis in which the amount of each analyte in a sample is determined (numerical result).

3

Compound Specific Detectors(currently available for Trace Organic Analysis)

• Single-crystal X-ray Spectrometer• FTIR (Fourier Transform Infrared

Spectrophotometer)• NMR (Nuclear Magnetic Resonance

Spectrometer)• Mass Spectrometer

• GC/MS• LC/MS

4

5

Why a mass spectrometry centered approach?

• Sensitivity – mass spectrometers can generate structural information from high pg to low ng of analyte.

• Selectivity – mass spectrometers can distinguish between vast numbers of different analytes.

• Duty Cycle – the duty cycle of a mass spectrometer is amenable to the duty cycles of GC and HPLC.

• Sample Quality – mass spectrometers can accommodate complex mixtures of analytes and (in many cases) relatively “dirty” samples.

Mass Spectrometry Nomenclature

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Nominal MassInteger mass of the most abundant stable isotopes

Theoretical Accurate MassCalculated mass of a particular molecular formula

Measured Accurate MassExperimentally determined mass of a particular molecular formula

Protonated Molecular Ion [M+H]+

An ion formed by addition of a proton (either in solution or in the gas phase) to an intact molecule

Deprotonated Molecular Anion [M-H]-

An ion formed by abstraction of a proton (either in solution or in the gas phase) from an intact molecule

Mass Spectrometry Nomenclature

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MS/MSA technique by which a selected precursor ion is fragmented (usually through collisions with neutral gas molecules) to produce structural information

Resolving Power/ResolutionA measure of the ability of a mass spectrometer to separate ions of two different masses

Ionization Processes

• EI - electron ionization• CI - chemical ionization• FAB - fast atom bombardment• LSIMS - liquid secondary ion• TSP - thermospray• ESI - electrospray• APCI - atmospheric pressure CI• MALDI - matrix assisted laser desorption• LMIS - liquid metal ion source

8

Hyphenated Analytical Techniques

• LC/MS LC/NMR GC/MS GC/FTIR• What distinguishes a “Hyphenated Analytical

Technique” from any other analytical technique? What is the “Line of Demarcation”?• e.g. Why is LC/UV not considered to be a hyphenated

analytical technique?• Norwood’s Definition –

• “A hyphenated analytical technique is one in which the detector has significantly greater scientific impact than the sample introduction system.”

9

“Modern” LC/MS SystemsLTQFT Ultra

“FTMS”

API 5000(Triple Quadrupole)

QTOF(SYNAPT G2)

“A good field to get out of.”Bill Haddon, 1982 ASMS Meeting 10

Mass Spectrometry Data Elements

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a. Mass spectrometric fragmentation behavior/expert mass spectrum interpretationb. Confirmation of molecular weightc. Confirmation of elemental compositiond. Mass spectrum matches automated library or literature spectrume. Mass spectrum and chromatographic retention index match authentic reference compoundf. Supporting spectral information from an orthogonal method (e.g., NMR)

LC/MS Instrument Summary

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Instrument MW In-source Frag.

MS/MS MSn Accurate Mass

Single Quad. * *

Triple Quad. * * *

Ion Trap * * * *

TOF * * *

FTMS * * (*) (*) *

QTOF * * * *

Trap-FTMS * * * * *

Quad-FTMS * * * (*) *

Identification Categories

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• A Tentative identification means that data have been obtained that are consistent with a class of molecule only. This is typically the case when only information such as a or d is available.• A Confident identification means that the tentative identification has been bolstered by additional and sufficient confirmatory information to preclude all but the most closely related structures. This would be the case, for example, ifthe tentative information (a and/or d) were augmented by b, c, or f. The more confirmatory information obtained, the greater the level of confidence.• A Confirmed identification means that the preponderance of evidence confirms that the entity in question can only be the identification that is provided. Although it is possible that a highly confident identification may meet the standard implied by the preponderance of evidence (for example, having a, b, c, e, and f), the only means of providing a confirmed identification is via mass spectral and retention time match with an authentic reference compound(item e).

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 3600

1000

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7000

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9000

m/z-->

Abundance

Scan 5446 (21.468 min): 07110301.D88

304

120184

21673

14956 231167 289105 257135 201 366

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 3600

1000

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m/z-->

Abundance

#180831: Zinc, bis(dimethylcarbamodithioato-S,S')-, (T-4)- (88

304

120 1847344 216

289231152137 257

14

Total Ion Chromatogram of Three Representative Extractables/Leachables

4.005.006.007.008.009.0010.0011.0012.0013.0014.0015.0016.0017.00200000

250000

300000

350000

400000

450000

500000

550000

600000

650000

700000

750000

800000

850000

900000

950000

1000000

Time-->

Abundance

TIC: 071019010.D\data.ms

Tetramethylthiourea

DEHP

Irganox 1076

15

Electron Ionization Mass Spectrum of Tetramethylthiourea (130 pg on-column)

40 50 60 70 80 90100110120130140150160170180190200210220230240

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m/z-->

Abundance

Average of 5.742 to 5.801 min.: 071019010.D\data.ms (-)132

88

72

42116

56 15097 207220106 164 231193176 250141

40 50 60 70 80 90100110120130140150160170180190200210220230240

1000

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9000

m/z-->

Abundance

#21710: Thiourea, tetramethyl- (CAS) $$ N,N,N',N'-Tetramethylthiou 132

4488

74

5635 9965 115

S

N N

16

Electron Ionization Mass Spectrum of Bis-2-ethylhexylphthalate (304 pg on-column)

50 100 150 200 250 300 350 400 450 500 550 600 6500

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m/z-->

Abundance


7041

112279

179 405249 325217 492355 431 530 618460 572 644

50 100 150 200 250 300 350 400 450 500 550 600 6500

2000

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8000

m/z-->

Abundance

#230979: 1,2-Benzenedicarboxylic acid, bis(2-ethylhexyl) ester (CAS 149

57

11383 279209 327179

O

O

OO

17

Electron Ionization Mass Spectrum of Irganox1076 (500 pg on-column)

50 100 150 200 250 300 350 400 450 500 550 600 650 7000

1000

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m/z-->

Abundance


57

219

97

147431

263187 315 403 479345373 604569 687646

50 100 150 200 250 300 350 400 450 500 550 600 650 7000

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m/z-->

Abundance

#263312: IRGANOX 1076 $$ 2,6-Di-tert-butyl-4-[(2-octadecyloxycarb 530

57

219

97 147 278189 474374331 430

OH O

O

18

Irganox 1076 APCI Negative Ion FT-ICR Mass Spectrum(1.0 ng on-column, 2.4 ppm mass error)

SP071016021 #264 RT: 5.07 AV: 1 SB: 24 5.22-5.41 , 4.70-4.89 NL: 5.43E5T: FTMS - p APCI corona Full ms [515.00-545.00]

527.0 527.5 528.0 528.5 529.0 529.5 530.0 530.5 531.0 531.5 532.0 532.5 533.0m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relat

ive Ab

unda

nce

529.46280

530.46649

527.44766531.47007

528.45105532.47341530.15963 530.96725528.21492 529.15334

[M-H]-

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ESI+ TOF Mass Spectrum of TMTMS

20

[M+H]+

[M+Na]+

[2M+Na]+

Expanded ESI+ TOF Mass Spectrum of TMTMS

+TOF MS: 0.220 to 0.293 min from tmms01.wiff Agilent Max. 1.3e6 counts.

230.5 231.0 231.5 232.0 232.5 233.0 233.5 234.0m/z, amu

0.0

1.0e5

2.0e5

3.0e5

4.0e5

5.0e5

6.0e5

7.0e5

8.0e5

9.0e5

1.0e6

1.1e6

1.2e6

1.3e6

Intensi

ty, cou

nts

231.0051

233.0006232.0065231.2400

21

[M+Na]+

Elemental Composition Report for TMTMS from ESI+ TOF-MS

Single Mass Analysis - displaying only valid resultsTolerance = 10.0 PPM / DBE: min = -0.5, max = 20.0

Monoisotopic Mass, Odd and Even Electron Ions43463 formula(e) evaluated with 3 results within limits (all results (up to 1000) for each mass)

Maximum: 2.0 10.0 20.0

Mass Calc. Mass mDa PPM DBE Formula

231.0052 231.0052 0.0 0.2 8.0 12C4 1H2 14N9 23Na 32S 231.0051 0.1 0.6 9.5 12C11 1H7 14N2 32S2 231.0060 -0.8 -3.6 1.5 12C6 1H12 14N2 23Na 32S3

22

MS/MS Spectrum of Tetramethylthiuram Disulfide(ESI+; products of m/z 241)

40 60 80 100 120 140 160 180 200 220 240 260m/z0

100

%

T081903002 7 (0.153) Sm (Mn, 2x0.75); Cm (4:18) Daughters of 241ES+ 3.13e588

120

241

23

[M+H]+

Structure and Fragmentation of Tetramethylthiuram Disulfide in Positive Ion

APCI (MS/MS)

SSN S

S

N

SSN

S

N

S

N S SS

24

N

+H+

m/z 241

m/z 196 m/z 120

m/z 88 m/z 44

++

++

LC/MS Analysis of Cyclic Oligomer Co-polymer (COC) Extract

Irganox 10101208 mw

(C)

1120 mw (B)916 mw

(A)UV =280 nm

Negative Ion APCITotal Ion

Chromatogram

Negative Ion APCI Mass Spectrum of Irganox 1010 in the COC Extract

OHO

O

C

OH

OO

OH O

O

OH

OO

[M-H]-

Negative Ion APCI Mass Spectrum of Irganox 1010 Degradation Product A

[M-H]-

O

OH

O

O

OH

O

OH

O

OH

O

Negative Ion APCI Mass Spectrum of Irganox 1010 Degradation Product B

[M-H]-

O

OH

O

O

OH

O

O

OH

O

O

OH

O

Negative Ion APCI Mass Spectrum of Irganox 1010 Degradation Product C

Positive Ion HESI Mass Spectrum of Irganox 1010 Degradation Product C

SP120712006 #869 RT: 11.54 AV: 1 NL: 4.80E5T: FTMS + c ESI Full ms [150.00-1500.00]

500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300m/z

0

5

10

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20

25

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40

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Rel

ativ

e Ab

unda

nce

1226.8095

934.6414716.2295

1256.8475

835.2474 1116.7376550.6283 776.2329

1275.7973

626.2120 1314.77421046.1067 1175.7719985.4962503.8969

Positive Ion HESI Mass Spectrum of Irganox 1010 Degradation Product C

SP120712006 #869 RT: 11.54 AV: 1 NL: 4.80E5T: FTMS + c ESI Full ms [150.00-1500.00]

1210 1215 1220 1225 1230 1235 1240 1245 1250 1255 1260 1265 1270 1275 1280m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

1226.8095

1231.7653 1247.7386

1267.8343

1254.84221242.79971210.8117 1275.79731221.6213 1264.74651238.52301217.0051

(expanded view of molecular ion region)

[M+NH4]+

[M+Na]+ [M+K]+

Negative Ion HESI Mass Spectrum of Irganox 1010 Degradation Product C

SP120711007 #741 RT: 11.57 AV: 1 NL: 4.39E5T: FTMS - c ESI Full ms [150.00-1500.00]

450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400m/z

0

5

10

15

20

25

30

35

40

45

50

55

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85

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100

Rel

ativ

e A

bund

ance

1321.77

915.60

1207.78

987.63711.35633.37

519.28 1021.66 1353.76829.59 947.59 1270.77579.40 1081.62 1404.76775.62 868.61 1181.16676.69 1136.99497.88

[M-H]-

[M+TFA]-

Summary of Mass Spectrometry Data from Irganox 1010 and Related Structures

[M+H]+ 1Adduct Ions (+) 2[M-H]- 3Adduct Ions (-) 4FormulaIrganox 1010 N/A [M+NH4]+ 1194

[M+Na]+ 1199[M+K]+ 1215[M+ACN+NH4]+ 1235

1175 [M+TFA-H]- 1289 C73H112NO12

(0.741 ppm)

A N/A [M+NH4]+ 934[M+Na]+ 939[M+K]+ 955[M+ACN+NH4]+ 975

915 [M+TFA-H]- 1029[M+HAc-H]- 975

C56H88NO10

(0.284 ppm)

B N/A [M+NH4]+ 1138[M+Na]+ 1143[M+K]+ 1159[M+ACN+NH4]+ 1179

1119 [M+TFA-H]- 1233 C69H104NO12

(0.717 ppm)

C N/A [M+NH4]+ 1226[M+Na]+ 1231[M+K]+ 1247[M+EA+H]+ 1254[M+ACN+NH4]+ 1267

1207 [M+TFA-H]- 1321 C73H112NO14

(1.432 ppm)

1From positive ion ESI spectra (ACN: acetonitrile; EA: ethylamine).2From negative ion ESI and APCI spectra.3From negative ion ESI spectra (TFA: trifluoroacetic acid; HAc: acetic acid).4From [M+NH4]+ in positive ion ESI spectra (ppm deviation of experimental mass from calculated mass at 50,000 resolving power).

SPE LC/NMR (in-line) UV chromatogram of Cyclic Olefin Copolymer Extract

trapping

(2H7)

RT(11

.38)

end tra

pping (2

H7) R

T(11.8

3)trap

ping (2H

8) RT

(12.10

)end

trappin

g (2H8)

RT(1

2.52)

trapping

(2H9)

RT(12

.63)

end tra

pping (2

H9) R

T(13.1

5)trap

ping (2H

10) R

T(13.6

3)

end tra

pping (2

H10) R

T(14.4

7)

2 4 6 8 10 12 14 16 Time [min]

-200

0

200

400

Intens.[mAU]

Irganox 1010

1208 mw (C)

1120 mw (B)

916 mw (A)

Flow-Probe 1H-NMR Spectrum of Irganox 1010(2 collections)

0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.5 ppm

OHO

O

C

OH

OO

OH O

O

OH

OO

Flow-Probe 1H-NMR Spectrum of C(10 collections)

Structure of Irganox 1010 Degradation Product C

OHO

O

C

OH

OO

O O

O

OH

OO

O

OH

m/z 915

High-Field Cryoprobe HMBC Spectrum of C

Concluding Thoughts

• There is no such thing as an unknown.• There is only the limit of our need and

motivation to identify.• Mass spectrometry techniques that were

once considered advanced are now becoming routine.

• Please use the standard identification criteria and categories.

Thank You!

analytical technologies and compound identification · 2018-04-06 · compound identification...

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