Routine impurity analysis with UPLC/MS and UPC2/MS

©2014 Waters Corporation 1

Routine Impurity Analysis with UPLC/MS,

UPC2 /MS


Outline

Sources of Impurities – Why is it important to be able to: o Separate o Detect o Identify o Quantify o Isolate impurities

Chemical Materials Workflow

Waters Technologies

Application Examples


Sources of Impurities

Either naturally occurring or purposely, accidentally, inevitably, or incidentally added into the substance – Raw materials – Contamination / Degradation o During production /

synthesis o Packaging / storage / use

The presence of impurities, even in small amounts, could affect the efficacy, performance, quality, lifetime and safety of the final product


Chemical Materials Workflow

IDENTIFICATION – UNKNOWN IMPURITIES Structural Elucidation – Exact mass, elemental composition, fragments, MS/MS confirmation

IDENTIFICATION – KNOWN IMPURITIES Calibration against standards User library/database UV, Mass, RT, Fragment

DETECTION

PDA / ELSD / FL MS / MRM / SIR MSE / MS/MS NMR

SEPERATION

HPLC UPLC UPC2 APGC


Separation Technology Overview

Gas Chromatography

Liquid Chromatography

Convergence Chromatography

Separation achieved by a temperature gradient

•High efficiency [N] • Virtually no limitation on column length

•Limited selectivity [α] • Limited stationary phase options

Separation achieved by a solvent gradient

•High efficiency [N] • Limited to pressure drop across column

•Moderate selectivity [α] • Different modes: reversed-phase, normal-phase, SEC, IEX, affinity, ion pair, HILIC, GPC…etc.

Separation achieved by density/solvent gradient

•High efficiency [N] • Very low viscosity enables longer columns and smaller particles

•High selectivity [α] • Wide variety of stationary phase and mobile phase co-solvent and modifier options

GC

LC

CC

APGC

UPLC

UPC2

HPLC


Ultra Performance Liquid Chromatography is a variant of HPLC using columns with particle size <2 µm (typically, 1.8 µm, which provides significantly better separation than the traditional (5 µm ) columns and enables much faster analysis

Higher efficiencies with a much wider range of linear velocities,

flow rates, and back pressures

More resolution and sensitivity

While increasing throughput (faster run times)

Optional elevated temperatures (column oven)

Highly robust, dependable, and reproducible system

ACQUITY UPLC


Convergence Chromatography (UPC2)

Convergence Chromatography (CC) is a normal phase separation technique – Uses carbon dioxide as the

primary mobile phase – Choice of adding an co-solvent

such as methanol or acetonitrile

The Waters UltraPerformance Convergence Chromatography (UPC2) builds upon the potential of CC while using proven and robust Waters UPLC technology


ACQUITY UPC2 Flow Path and Components

Inject valve

Auxiliary Inject valve

Column Manager

PDA detector

Back Pressure Regulator (Dynamic and Static)

Waste Modifier CO2 Supply CO2

Pump Modifier

Pump

mixer Thermo-electric heat exchanger

Make-up Pump

Mass Spec

Splitter


The Key Benefits of UPC²

Simplify the workflow with UPC2

– Combine multiple techniques (LC & GC into CC) – Access robust normal phase separations – Eliminate solvent exchange steps for organic extracts

Deal with compound Similarity challenges – Chiral Separations (enantiomers & diastereomers) – Positional isomers (differ in location of functional groups)

Deliver Orthogonal separations

– Different relative retention helps ensure full characterization – Check method specificity by comparison to a second

procedure – Reveal “hidden” impurity or degradation peaks – Increase confidence in characterization of complex samples

SIMPLICITY

SIMILARITY

ORTHOGONALITY


What are the benefits of using SFC based separations?

Deliver Orthogonal separations – Different relative retention helps ensure full characterization – Check method specificity by comparison to a second

procedure – Reveal “hidden” impurity or degradation peaks – Increase confidence in characterization of complex samples

Simplify the workflow with UPC2

– Combine multiple techniques (LC & GC into CC) – Access robust normal phase separations – Eliminate solvent exchange steps for organic extracts

Deal with compound Similarity challenges – Chiral Separations (enantiomers & diastereomers) – Positional isomers (differ in location of functional groups)

SIMPLICITY

SIMILARITY

ORTHOGONALITY

Built upon proven UPLC Technology – Quantifiable increase in productivity

Robust, Reliable and Reproducible – Modernization of SFC-based technology,

making this technique a viable analytical separations tool


Detection

Tandem Quadrupoles

Single Quadrupoles

SQD2

XEVO TQD

XEVO TQ-S

XEVO G2-XS QToF

Time of Flight (ToF)

ACQUITY UPC2 System

ACQUITY UPLC System

Mass Detector

QDa

Point Detectors ELS PDA RI* FLR* TUV* UV/VIS # *UPLC / HPLC only #HPLC only

SYNAPT G2-Si

XEVO TQ-S micro

Alliance HPLC


Designed as mass detector integrated with a separations system

A robust mass detector that does not require adjustments and optimization for each sample

Provides orthogonal detection for the range of applications that benefit from the extra information

ACQUITY QDa Detector


Automated Calibration Pre-optimized ES Zero Tuning Disposable Sample Cone 50-1250 Da 10,000 Da/s +/- Switching 4 Orders Dynamic Range

Detector Characteristics

PDA

QDa


Waters Fraction Manager - Analytical


Analytical Scale Fraction Collector System Description:

– Integrates seamlessly with ACQUITY H-Class or Alliance Systems

– Empower Control OR Stand-Alone Control – Flow Rate Range: 0.1 – 2 mL/min

o Optional needle for flow rates up to 5 mL/min – pH Operating Range 2 – 12

Developed on FTN Sample Manager Platform – known for innovative, robust technology.

Fast valve switching and movement between vessels enabling collection of narrow UPLC peaks.

Designed for low carryover and high recovery to accommodate limited sample.

Temperature controlled from 4 to 40OC for thermally labile samples.

Waters Fraction Manager - Analytical


Agrochemicals

Application note: 720004824en

PDA

QDa

http://www.waters.com/webassets/cms/library/docs/720004824en.pdf�


Agrochemicals

The use of agricultural chemicals ensures that there is decreased crop damage resulting in a food supply that is plentiful and of high quality

The detection, characterization and quantitation of the active ingredient/s and all other components in the pesticide formulation is necessary to support product development and product registration

The presence of impurities (>0.1%) could potentially reduce the toxicological properties of the final product, potentially causing health and environmental effects


Agrochemicals

Experimental Description: UPLC/PDA/QDa Achiral separation by UPLC, PDA and QDa mass detection were

used to provide a data profile of the pesticide formulations. The ACQUITY QDa mass detector, in combination with PDA,

allowed for low-level components to be detected with increased confidence in the pesticide formulations.

The components were identified as having similar optical and

structural properties to the active ingredients (AI).

Inert formulation components not seen in the UV were readily detected by the ACQUITY QDa Detector.


Agrochemicals AU

0.00

0.20

0.40

0.60

0.80

1.00

AU

-0.12

-0.10

-0.08

-0.06

-0.04

-0.02

0.00

0.02

0.04

0.06

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00

1 2

4 3

Formulation Sample

Propiconazole Standard

**

UPLC/UV Analysis of a Formulation


Agrochemicals AU

0.00

0.20

0.40

0.60

0.80

1.00

QDa 1: MS Scan MS TIC (1: 100.00-1000.00 Da ES+, Continuum, CV=7): not integrated, source of MS spectra

Inte

nsity

0.0

5.0x10 8

1.0x10 9

1.5x10 9

2.0x10 9

2.5x10 9

3.0x10 9

3.5x10 9

4.0x10 9

Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00

UV at 220 nm

QDa TIC

1

2

3 4

1

2

3 4

(

Minutes6.00 6.20 6.40 6.60

UV

Mass Detector

UPLC/UV and QDa Mass Detection


Agrochemicals

UV Chromatogram

Total Ion Chromatogram

Automated Extracted Ion Chromatograms

Combined UV and MS Spectra

1 2 3 4

1 2 3 4

The Empower 3 Mass Analysis Window


Agrochemicals

UV Chromatogram

Total Ion Chromatogram

Automated Extracted Ion Chromatograms

Combined UV and MS Spectra

1 2 3 4

1 2 3 4

The Empower 3 Mass Analysis Window


Agrochemicals

261.9

261.9

268.0

268.0

nm210.00 220.00 230.00 240.00 250.00 260.00 270.00 280.00 290.00 300.00

342

344

342

344

342

344

342

344

nm260.00 280.00 300.00 320.00 340.00 360.00 380.00 400.00

1

2

3

4

1

2

3

4

UV and Mass Spectra of the Detected Components


Agrochemicals

Tebuconazole

Channel Description: PDA 220.0 nm (210-400)nm

2.17

9

4.73

1

5.24

9 6.48

5

AU

-0.20

-0.15

-0.10

-0.05

0.00

0.05

0.10

0.15

QDa 1: MS Scan MS TIC (1: 100.00-600.00 Da ES+, Continuum, CV=5): not integrated, source of MS spectra

Inte

nsity

0

1x108

2x108

3x108

4x108

5x108

6x108

7x108

Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40 7.60 7.80 8.00 8.20 8.40 8.60 8.80 9.00 9.20 9.40 9.60 9.80 10.00

1

2 34

1

23

4

UV

QDa TIC

*UV

QDa TIC

UPLC/PDA/QDa Analysis of a Formulation with two AI’s


Channel Description: PDA 220.0 nm (210-400)nm

2.17

9

4.73

1

5.24

9 6.48

5

AU

-0.20

-0.15

-0.10

-0.05

0.00

0.05

0.10

0.15

Channel Description: QDa 1: MS Scan MS Calculated: 256+281+308 (1: 100.00-600.00 Da ES+, Continuum, CV=5)

Inte

nsity

0

1x10 8

2x10 8

3x10 8

4x10 8

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00

p ( )

UV at 220 nm

XIC

XIC

2 3

1

4

2 31

4

Agrochemicals

UV

QDa XIC

Extracted Ion Chromatograms


Application example – Agrochemicals

2.179 Peak 1

212.4

269.9

4.731 Peak 2220.3

267.4

5.249 Peak 3220.9

269.3

6.485 Peak 4220.9

268.0

nm210.00 220.00 230.00 240.00 250.00 260.00 270.00 280.00 290.00 300.00

256

281

308

308

m/z220.00 240.00 260.00 280.00 300.00 320.00 340.00

1

2

3

4

1

2

3

4

AI

Unknown A

Unknown B

Tebuconazole, AI

UV and Mass Spectra of the Detected Components



Furans in Transfer Oil

http://www.waters.com/waters/library.htm?cid=511436&lid=134699422�


Transformer oil – Highly-refined mineral oil, stable at high temperatures – Excellent electrical insulating and heat transfer properties – Used in oil-filled transformers, high voltage capacitors,

fluorescent lamp ballasts, high voltage switches, and in circuit breakers

Furans

– Originate from thermal depolymerization of cellulose solid insulation

– Degree of degradation eventually renders transformer ineffective – Periodic analysis for furans can be used to assess the degree of

degradation and does not require taking the unit out of service in order to take a sample

Furans in transformer oil


UPLC H-Class System

Mobile phase A: Water (0.1% formic acid)

Mobile phase B: Acetonitrile (0.1% Formic acid)

Inj. volume: 10 µL

Column: ACQUITY BEH C18, 2.1 x 150 mm, 1.7µm

Column temp: 40 °C

Sample temp: 10 °C

Time (min)

Flow Rate (mL/min) %A %B Curve

Initial 0.450 80 20 - 1.50 0.450 80 20 6 2.00 0.450 60 40 6 2.01 0.450 0 100 6 3.00 0.450 0 100 6 3.01 0.450 80 20 6 4.00 0.450 80 20 6



Furans method – ACQUITY PDA Detector

o λ range: 190 – 350 nm o Resolution: 1.2 nm o Sampling rate: 20 points/s


Furans and additives method – Xevo TQ MS

o Ionisation: APCI+ o Acquisition: MRM o Corona current: 20 µA o Source temp: 150 °C o APCI probe temp: 400 °C o Desolvation gas flow: 1000 L/hr o Cone gas flow: 100 L/hr



Chemical Substance CAS

Number RT

(min) UV absorbance

(nm) 5-hydroxymethyl-2-furaldehyde

5H2F 67-47-0 1.02 284

Furfurylalcohol 2FOL 98-00-0 1.28 216 2-furaldehyde 2FAL 98-01-1 1.45 277 2-furylmethylketone 2ACF 1192-62-7 1.77 274 5-methyl-2-furaldehyde 5M2F 620-02-0 2.13 292



2-furaldehyde (277 nm)

5-methyl-2-furaldehyde (292 nm)

2-furylmethyl-ketone (274 nm)

Furfuryl alcohol (216 nm)

5-hydromethyl-2-furaldehyde (284 nm)

B

A

B

A

B

A

B

A

B

A

MAX Plot

A

UPLC/UV chromatograms



UPLC/UV Empower Results

5-methyl-2-furaldehyde



Compound Replicate injection results

(mg/kg) Average recovery

(%) 1 2

5-hydroxymethyl-2-furaldehyde

Blank ND -- -- 2 mg/kg 1.63 1.64 81.9 10 mg/kg 9.37 9.36 93.6

Furfurylalcohol Blank ND -- --

2 mg/kg 1.89 1.87 93.8 10 mg/kg 9.51 9.38 94.5

2-furaldehyde Blank ND -- --

2 mg/kg 1.90 1.91 95.1 10 mg/kg 9.54 9.55 95.4

2-furylmethylketone Blank ND -- --

2 mg/kg 1.94 1.95 97.3 10 mg/kg 9.59 9.58 95.8

5-methyl-2-furaldehyde Blank ND -- --

2 mg/kg 1.86 1.86 93.2 10 mg/kg 9.96 10.01 99.8

UPLC/UV Recovery Results



Chemical Substance RT

(min)

Cone Voltage

(V)

MRM Transition

Collision energy (eV)

5-hydroxymethyl-2-furaldehyde

5H2F 1.05 20 127.0 > 109.0 15 127.0 > 81.0 20

Furfurylalcohol 2FOL 1.30 25 81.1 > 53.0 15

2-furaldehyde 2FAL 1.46 25 97.1 > 69.0 15 97.1 > 41.1 15

2-furylmethylketone 2ACF 1.79 20 111.1 > 43 20

111.1 > 69.1 15

5-methyl-2-furaldehyde 5M2F 2.15 25 111.1 > 55.0 20 111.1 > 83.0 15

Benzotriazole BTA 1.66 40 120.1 > 65.0 20 120.1 > 92.0 20



Benzotriazole

5-Methyl-2-furaldehyde

2-Furylmethylketone

2-Furaldehyde

Furfuryl alcohol

5-Hydroxymethyl-2-furaldehyde

0

A

A

A

A

A

A

B

B

B

B

B

B

UPLC/MS/MS Chromatogram


Impurity profiling of Liquid Crystal intermediates using UltraPerformance Convergence

Chromatography (UPC2) with PDA detection


http://www.waters.com/waters/library.htm?cid=511436&lid=134745520�


Background - Liquid Crystals

Properties: o Some properties of liquids:

• Flow, pour like liquids and take the shape of containers o Some optical properties of solids:

• Birefringence • Optical activity

o React predictably to an electric current, enabling the control of light passage

Liquid crystal intermediates: o Building blocks compounds used to prepare liquid crystals

• Used in mixtures (10 to 20 singles used in a typical mixture)

Uses: o Many electronic displays use liquid crystals:

• Watches, calculators, notebooks, personal digital assistant (PDA), mobile phones, projectors, desktops monitors / TVs, viewfinders on cameras / camcorders…..


Liquid Crystal Intermediate Compounds

Methods UPC2 conditions Run time: 5.00 min CCM back pressure: 2000 psi Sample temp.: 20 oC Column temp.: 50 oC Flow rate: 2.0 mL/min Column: ACQUITY UPC2 CSH Fluoro-phenyl, 3.0 mm x 100 mm, 1.7 µm Mobile phase A: CO2 Mobile phase B: Methanol (2% Formic Acid + 15 mM ammonium acetate) Injection volume: 1 µL PDA conditions UV system: ACQUITY UPC2 PDA Detector Range : 210 to 450 nm Resolution: 1.2 nm Sampling rate: 20 pts/sec Filter time constant: Slow (0.2 sec)





Chemical Substance CAS

Number

Retention time

(minutes)

UV optimum absorbance

(nm)

4,4′-Azoxyanisole-d14 39750-11-3 0.69 346

4-Butylbenzoic acid 20651-71-2 1.39 235

4-Octylbenzoic acid 3575-31-3 1.62 235

4-Cyanobenzoic acid 3575-31-3 1.75 252

4-Butoxybenzoic acid 1498-96-0 1.90 252

4-(Octyloxy)benzoic acid 2493-84-7 2.09 235



Empower calibration curve



UV Chromatograms and UV Spectra


Liquid Crystal Intermediate Compounds Impurity Profiling


Primary Aromatic Amines

Waters® ACQUITY UPLC H-Class / ACQUITY PDA coupled with SQ Detector 2:

Technology brief: 720004062en Application note: 720004151en



Used to produce many commodities – Pharmaceuticals, explosives, epoxy polymers, rubber, aromatic

polyurethane products and azo dyes Found as impurities in the final product

– Due to incomplete reactions, as by-products, or as degradation products PAAs can be produced as by-products of azo-dyes Azo-dyes

– A diverse and widely used group of organic dyes – Wide range of uses

o Specialty paints, printing inks, varnishes and adhesives – Found in many consumer products

o Textiles, cosmetics, plastics Risks to human health

– Proven / suspected carcinogenic nature, highly toxic


SQ Detector 2: Ionization mode: ESI+ Acquisition mode: SIR ACQUITY PDA Detector: Wavelength range: 190 – 500 nm


PAA number Primary Aromatic Amines (PAAs) CAS

Number m/z Retention

time (minutes)

Cone Voltage

(V) 1 Aniline 62-53-3 94 2.17 40 2 o-Toluidine 95-53-4 109 3.80 40 3 1,3-Phenylenediamine 108-45-2 109 0.62 40 4 1,4-Phenylenediamine 106-50-3 109 0.41 43 5 2,4-Dimethylaniline 95-68-1 122 5.58 43 6 2,6-Dimethylaniline 87-62-7 122 5.33 43 7 2,4-Toluenediamine 95-80-7 123 1.64 40 8 2,6-Toluenediamine 823-40-5 123 0.85 40 9 o-Anisidine 90-04-0 124 3.74 45 10 4-Chloroaniline 106-47-8 128 4.6 40 11 2,4,5-Trimethylaniline 137-17-7 136 7.06 40 12 2-Methoxy-5-methylaniline 120-71-8 138 5.36 40 13 4-Methoxy-m-phenylenediamine 615-05-4 139 1.51 36 14 2-Naphtylamine 91-59-8 144 6.18 40 15 3-Amino-4-methylbenzamide 19406-86-1 151 2.19 35 16 3-Chloro-4-methoxyaniline 5345-54-0 158 4.00 40 17 5-Chloro-2-methoxyaniline 95-03-4 158 6.06 40 18 1,5-Diaminonaphtalene 2243-62-1 159 2.52 40 19 2-Methoxy-4-nitroaniline 97-52-9 169 4.37 30 20 4-Aminobiphenyl 92-67-1 170 7.57 43 21 2-Aminobiphenyl 90-41-5 170 7.71 50 22 Benzidine 92-87-5 185 4.01 43 23 4-Chloro-2,5-dimethoxyaniline 6358-64-1 188 5.79 40 24 4-Aminoazobenzol 60-09-3 198 7.84 30 25 4,4'-Methylenedianiline 101-77-9 199 5.64 43 26 4,4'-Diaminodiphenylether 101-80-4 201 4.36 45 27 3,3'-Dimethylbenzidine 119-93-7 213 6.01 43 28 4,4'-Thioaniline 139-65-1 217 6.29 43 29 o-Aminoazotoluene 97-56-3 226 8.28 43 30 4,4'- Diamino-3,3'-dimethylbiphenylmethane 838-88-0 227 7.39 40 31 3-Amino-p-anisanilide 120-35-4 243 6.06 40 32 o-Dianisidine 119-90-4 245 6.00 45 33 3,3'-Dichlorobenzidine 91-94-1 253 7.76 45 34 4,4'- Diamino-3,3'-dichlorobiphenylmethane 101-14-4 267 7.90 60

SIR m/z, expected retention times, and cone voltages


Primary Aromatic Amines TargetLynx Quantify results browser showing the calibration quantitation results, calibration curve and example SIR chromatogram for Aniline


UPLC H-Class System Mobile phase A

10 mL of 1 M aqueous ammonium acetate solution and 990 mL water

Mobile phase B 10 mL of 1 M aqueous ammonium acetate solution and 990 mL methanol

Column: ACQUITY BEH C18, 1.7 mm, 2.1 x 50 mm

Column temperature: 40 oC.

Primary Aromatic Amines SIR chromatograms for 34 PAAs in a mixed 1 µg/mL calibration standard


Primary Aromatic Amines Ink analysis Neat ink diluted 1:100 with 5%

methanol / 95% water

Ink spiked at various levels with selected PAAs, and analyzed without any further cleanup or concentration steps

The efficient recoveries obtained (ranging between 83 to 108%)

– minimal signal enhancement / suppression was observed

Ink spiked with PAAs recovery data.


Primary Aromatic Amines The advantages of mass spectral detection over PDA (UV) detection

– Improved sensitivity and selectivity – Matrix effects can be greatly reduced by using mass spectral detection

a) UV chromatograms for 2-Aminobiphenyl and 3,3’-Dichlorobenzidine in individual solvent standards

b) In a mixed solvent standard

Example 1 (selectivity) Considering the PAAs,

2-Aminobiphenyl and 3,3’-Dichlorobenzidine Maximum UV absorbance

can be found at 295 and 284 nm respectively (retention times of 7.71 and 7.76 minutes respectively)

The two compounds are not completely resolved.

Which could potential lead to misidentification, poor integration, and false positive results



Mass spectral detection The two compounds are

resolved Improved selectivity

Extracted ion chromatograms for 2-Aminobiphenyl and 3,3’-Dichlorobenzidine in fortified ink (containing 4.6 µg/mL PAAs)


Primary Aromatic Amines Improvement in selectivity

– The abilities to measure analytes of interest accurately and specifically in the presence of a complex matrix

– Considering the PAA 2,4,5-Trimethylaniline

o When spiked in ink cannot be distinguished due to other UV absorbing compounds present

– However, mass detection is sufficiently sensitive and selective to enable confident detection and quantification of 2,4,5-Trimethylaniline in an ink matrix

UV and extracted ion chromatograms for the PAA 2,4,5-Trimethylaniline spiked in ink (4.6 µg/mL), solvent

standard (5.0 µg/mL) and a blank ink matrix

Blank ink matrix UV:286 nm

Spiked ink UV:286 nm

Solvent standard UV:286 nm

Blank ink matrix m/z:136

Spiked ink m/z:136

Solvent standard m/z:136



Improvements in sensitivity – Signal-to-noise (S/N),

(comparing the UV and the mass spectral data)

– The increase in S/N and sensitivity when using mass spectral data

UV and extracted ion chromatograms for 4-4’-Diaminodiphenylether


Conclusion

By utilizing Waters technology –Fast, selective, and sensitive methods can be

developed for the analysis of impurities Offering many business benefits using UPLC and UPC2

o Increase in sample throughput o Reduction in toxic solvent usage

Using mass spectral detection over UV detection provides – Improvement in sensitivity and selectivity –Reduced matrix effects

PDA and mass detection provide complementary information for peak assignment and structural confirmation of impurities


Science

Routine impurity analysis with UPLC/MS and UPC2/MS