Extraneous Peaks in HPLC

Isranalytica2006Isranalytica2006

© Dr. Shulamit Levin 2005, Medtechnica

Unexpected Peaks in Chromatograms Unexpected Peaks in Chromatograms -- Are Are They Related Compounds, System Peaks or They Related Compounds, System Peaks or

Contaminations? Contaminations?

From the Diary of an HPLC DetectiveFrom the Diary of an HPLC Detective

[email protected]

SHULAMIT LEVIN, MEDTECHNICA



HPLC in HPLC in PharmaceuticsPharmaceutics

Σ



Stability Indicating MethodsStability Indicating Methods

• Accurately quantifies the active pharmaceutical ingredient without interference from:– Impurities– Degradation products– Excipients– Other potential impurities– Other active ingredients

FDA Guidelines:FDA Guidelines:Where an analytical method reveals the presence of impurities in addition to the degradation products (e.g., impurities arising from the synthesis of the drug substance), the origin of these impurities should be discussed.

Extra Sensitive to Extraneous Peaks



2.20.00342.714Impurity 5

5.80.01062.549Impurity 4

2.60.00372.504Impurity 3

6454499.9542.263Butamben

16.40.02292.231Impurity 2

4.10.00542.174Impurity 1

Signal-to-Noise

Area %

Retention Time (Minutes)Compound

Accounting for Every Peak in the Related Compounds’ Profile - Even Down to 0.003 %Area

Butamben

>LOQ



Sample vs. Blank (Diluent)Sample vs. Blank (Diluent)m

AU

0.00

10.00

20.00

30.00

40.00

50.00

mA

U

0.00

10.00

20.00

30.00

40.00

50.00

Minutes

0.00 10.00 20.00 30.00 40.00 50.00

Blank

Sample

Unexpected peaks can appear in blanks and in all following injectionsIn this case they are excluded from the processing of the sample

? ? ? ? ?



INJECTION OF PURE SOLVENT:INJECTION OF PURE SOLVENT:Why would there be peaks?Why would there be peaks?

Injection “System Peak” ???Carry over ???

Contamination ???



System PeaksSystem Peaks“Legitmate” System Peaks

Originate from the Mobile Phase Components Going Through Re-Equilibration



CONDITIONS FOR APPEARANCE OF CONDITIONS FOR APPEARANCE OF REAL SYSTEM PEAKSREAL SYSTEM PEAKS

• Mobile phase is multi-component (n=>2)

• Mobile phase contains adsorbable components

• Mobile phase’s components respond to the detector(high background)

• Sample or sample-diluent is different from the mobile phase, enough to create equilibrium perturbation.



EXAMPLE:Two additives in the mobile phase

CHROMATOGRAM

Mechanism of System Peaks FormationMechanism of System Peaks Formation

Vacancy

Example: kExample: k’’(1) = 1(1) = 1Step 1:Step 1:

Equilibrium:Equilibrium:Cs=1Cs=1; ; CmCm = 1= 1

Step 2:Step 2:Injection of Injection of Vacancy:Vacancy:

Cs=1Cs=1; Cm=0; Cm=0

Step 3:Step 3:ReRe--equilibration:equilibration:Cs=0.5Cs=0.5; ; Cm=0.5Cm=0.5

Example: kExample: k’’(2) = 2(2) = 2Step 1:Step 1:

Equilibrium:Equilibrium:Cs=2Cs=2; ; CmCm = 1= 1

Step 2:Step 2:Injection of Injection of Vacancy:Vacancy:

Cs=2Cs=2; Cm=0; Cm=0

Step 3:Step 3:ReRe--equilibration:equilibration:

Cs=1.33Cs=1.33; ; Cm=0.67Cm=0.67

k' = tR- t 0

t0

k’ = CCssφCm

t0

k’=1 k’=2



Injection of free amino acids into mobile phase containing:Injection of free amino acids into mobile phase containing:Ac buffer, Ac buffer, CuAcCuAc, and , and HeptsulfonateHeptsulfonate. .

Diluent: WaterDiluent: Water

Levin and Grushka



Legitimate System Peaks Result from Mobile phase components Legitimate System Peaks Result from Mobile phase components due to their absence in the injected sampledue to their absence in the injected sample

Injection of Blank = WaterInjection of Blank = Water

Levin and Abu-Lafi

Conclusion: Use mobile phase composition as the diluent



An Example for Real System Peaks in Gradients with An Example for Real System Peaks in Gradients with TrifluoroaceticTrifluoroaceticAcid (TFA) in the Mobile PhaseAcid (TFA) in the Mobile Phase

Blank =Water

Blank = Mobile phase

Blank = Mobile phase

Zoomed

Sample=Peptide Dissolved in

Water

Peptide Peak

TFA

ACN

System peaks appear because diluent does not contain TFASystem peaks appear because diluent does not contain TFA



Chromatogram and Peaks' UV-VIS Spectra

Wavelength: 215nm

nm220.00240.00 260.00 280.00 300.00 320.00 340.00

Cpd 2 - 2.364nm220.00

240.00 260.00 280.00 300.00 320.00 340.00

2.817nm220.00

240.00 260.00 280.00 300.00 320.00 340.00

2.981

AU

-0.50

-0.40

-0.30

-0.20

-0.10

0.00

Minutes

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

Cp

d2

-2.

364

2.81

72.

981

-0.50

-0.40

-0.30

-0.20

-0.10

0.00

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

Real System Peaks in Multi-component Mobile PhaseH2O, MeCN, MeOH, THF

THF

Diluent does not contain all mobile phase componentsDiluent does not contain all mobile phase components



AU

Sample Name: Diluent

2.37

32.

731

3.40

4

4.27

4

6.47

7

18.2

74

-0.003

-0.002

-0.001

0.000

0.001

0.002

0.003

0.004

0.005

0.006

MinutesMinutes

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00

Mobile phase: ACN : Phosphate Buffer pH 9.5 1:1Wavelength: 280 nm

Contaminations Peaks Contaminations Peaks -- NotNot System Peaks!System Peaks!Phosphate Buffer and Acetonitril Do Not Produce Such System Peaks!



Chromatogram and Peaks' UV-VIS Spectra

Wavelength: 254.0 nm

Use of Diode-Array Detector for Troubleshooting of Contamination Peaks in Multicomponent Mobile Phase

nm250.00300.00

Cpd 2 - 2.188nm250.00

300.00

2.748nm250.00

300.00

2.867nm250.00

300.00

3.180nm250.00

300.00

4.226

AU

0.000

0.005

0.010

0.015

Minutes1.00 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

Cp

d2

-2.

188

2.74

8 2.86

7

3.18

0

4.22

6AU

0.000

0.005

0.010

0.015

Minutes1.00 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

Negative UV spectrum

Aromatic (not in mobile phase)



Carry Over:Carry Over:Chemical Residues in the systemChemical Residues in the system

qq Residues in the injectorResidues in the injector

qq NonNon--specific irreversible adsorption on columnspecific irreversible adsorption on column

qq Accumulation on systemAccumulation on system’’s surfacess surfaces



Carry Over in the InjectorCarry Over in the InjectorWashed by Blank InjectionWashed by Blank Injection

A1100 VWD AU - A1100 AU 286 nm

11.3

34

AU

-0.0001

0.0000

0.0001

0.0002

0.0003

0.0004

A1100 VWD AU - A1100 AU 286 nm

AU

-0.0001

0.0000

0.0001

0.0002

0.0003

0.0004

Minutes8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 13.00 13.50 14.00 14.50

Blank Injection 2Contamination is cleaned

Blank Injection 1



Blank InjectionsBlank Injectionsm

AU

0.00

20.00

40.00

60.00

80.00

100.00

Minutes

0.00 10.00 20.00 30.00 40.00 50.00

Series of Blank Injections

Signals are reduced from Injection to Injection indicate carry over in the injector



CarryCarry--Over in the InjectorOver in the InjectorLCLC--MS/MSMS/MS

InjectionInjection--CleanClean--up with up with EDTAEDTA solved the problemsolved the problem

1.1. Peak elutes at RT of main componentPeak elutes at RT of main component2.2. MRMMRM of the main componentof the main component

Blank Sample before wash

Blank Sample after wash



Carry Over:Carry Over:Residues in the systemResidues in the system






Accumulation on ColumnAccumulation on Column

SampleName: SST; Vial: 1; Injection: 1; Name: MAIN1; Area: 833913

AU

0.0000

0.0005

0.0010

Minutes

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

MA

IN1

SampleName: DILUENT; Vial: 2; Injection: 1; Name: MAIN1; Area: 5063

AU

0.0000

0.0005

0.0010

Minutes

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

MA

IN1

SampleName: DILUENT; Vial: 2; Injection: 2; Name: MAIN1; Area: 4784

AU

0.0000

0.0005

0.0010

Minutes

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

MA

IN1

Sample

Blank #1

Blank #2Cleaned Not Cleaned



Chromatogram and UV-VIS Spectra of Contamination Peaks

Contamination PeaksContamination Peaks

nm

200.00220.00240.00260.00280.00300.00320.00340.00

8.510 11.604 41.142A

U

-0.002

-0.001

0.000

0.001

0.002

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00

8.51

0

11.6

04

41.1

42

-0.002

-0.001

0.000

0.001

0.002

Minutes

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00

Diluent at 280 nm

Aromatic group Aromatic group

TFA Containing Mobile Phase: TFA, HTFA Containing Mobile Phase: TFA, H22O, O, MeCNMeCN -- Do Not Contain Aromatic Components!Do Not Contain Aromatic Components!



-Chromatogram and UV -VIS Spectra of a Blank Run

Sample Name: blank ; Wavelength: 214

nm

250.00

300.00

350.00

1.066 1.553 2.362 3.695 6.237 6.418 24.320 26.940 27.0261.

066

1.55

3 2.36

2

3.69

5

6.23

76.

418

24.3

20

26.9

4027

.026

AU

-0.20

0.00

0.20

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00

AU

-0.20

0.00

0.20

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00

Peaks of aromatic compounds in non aromatic mobile phase:Peaks of aromatic compounds in non aromatic mobile phase:Cannot be Legitimate System PeaksCannot be Legitimate System Peaks

Mobile phase: Gradient of 0.1% TFA in water with ACNMobile phase: Gradient of 0.1% TFA in water with ACN



Chromatogram and UV-VIS Spectra of Contaminations’ Peaks

Contamination Peaks Contamination Peaks -- ResiduesResidues

nm

200.00220.00

240.00

260.00280.00300.00320.00340.00

1.274 1.514 1.731 8.500 11.633 34.345 36.595 37.612 41.018 42.128

AU

0.000

0.005

0.010

0.015

0.020

0.025

0.030

Minutes5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00

1.27

41.

514

1.73

1

8.50

0

11.6

33

34.3

45

36.5

9537

.612 41

.018

42.1

28

AU

0.000

0.005

0.010

0.015

0.020

0.025

0.030

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00

Blank=Diluent at 220 nm

Gradient with TFAGradient with TFA

Contamination on-column and/or in the mobile phase

Aromatic compounds – not legitimate components of the mobile phase



Carry Over:Carry Over:Residues in the systemResidues in the system






Contaminations Contaminations Origin Origin –– Outside the SampleOutside the Sample

qq NonNon--pure solventspure solvents

qq VialsVials’’ leachablesleachables

qq ReservoirReservoir’’s s leachablesleachables



AU

% C

om

po

siti

on

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.00

20.00

40.00

60.00

80.00

100.00

Minutes

5.00 15.00 25.00 35.00 45.00 55.00

Gradient’s Profile

220 nm

230 nm

254 nm280 nm

Baseline at GradientBaseline at GradientChange of Baseline with Time at Various Wavelengths: Change of Baseline with Time at Various Wavelengths:

Indication for non pure solventIndication for non pure solvent



NonNon--Pure/Contaminated Pure/Contaminated Solvents Used in GradientsSolvents Used in Gradients

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00

PG

-2.919

TB

HQ

-4.525

BH

A

-6.896 BH

T

- 10.633

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00

PG

-2.919

TB

HQ

-4.525

BH

A

-6.896 BH

T

- 10.633

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00

Blank

Sample

Contaminations can also come from the Parafilm used to seal reservoirs!!!



Sample Sample vsvs BlankBlankm

AU

0.00

10.00

20.00

30.00

40.00

50.00

mA

U

0.00

10.00

20.00

30.00

40.00

50.00

Minutes

0.00 10.00 20.00 30.00 40.00 50.00

Blank

Sample

If Solvents are not entirely pure their impurities are adsorbed on the Stationary Phase at the beginning of the run and then elute from the column as the gradient develops.

This is the reason why there are “Gradient Grade” solvents



PeakPeak’’s Origin: s Origin: Contamination accumulated on pumpContamination accumulated on pump’’s ins in--line filterline filter

Pressure JumpBubble-Detection

Unexpected Baseline Perturbation






qq ReservoirReservoir’’s s leachablesleachables



Contamination from VialsContamination from Vials’’ SurfacesSurfaces

AU

-0.0002

0.0000

0.0002

0.0004

0.0006

0.0008

0.0010

0.0012

0.0014

Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

AU

-0.0002

0.0000

0.0002

0.0004

0.0006

0.0008

0.0010

0.0012

Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

Non-Certified

CertifiedChange of vial brand

In the same experiment!



SampleName: Sample; Vial: 1:F,1; Injection: 1; Name: Contamination

AU

0.000

0.005

0.010

0.015

0.020

Minutes

0.00 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

Con

tam

inat

ion

SampleName: Diluent ; Vial: 1:E,2; Injection: 1; Name: Contamination

AU

0.000

0.005

0.010

0.015

0.020

Minutes

0.00 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

Con

tam

inat

ion

Contamination is developed during the experiment in Contamination is developed during the experiment in complex Diluents complex Diluents (Sample(Sample’’s Solvent)s Solvent)






qq Reservoir or ColumnReservoir or Column’’s s leachablesleachables



Contamination Peaks in Size Exclusion Contamination Peaks in Size Exclusion Chromatography (SEC)Chromatography (SEC)

Chromatogram at 210 nm

AU

-0.010

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

Minutes0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00

Contamination is NOT a monomer!

Blank

MonomerSample

Contamination

Higher Molecular Weight



Other Reasons Other Reasons for Extraneous Peaksfor Extraneous Peaks



ColumnColumn’’s Packing Collapses Packing Collapse

All Peaks are split



Suggested FlowSuggested Flow--Chart for TroubleshootingChart for TroubleshootingIn a Regulated Environment (no change in method!)In a Regulated Environment (no change in method!)

Mobile phase:Multicomponent?

Adsorbed components (ion pair)?Response in detector (background)?

Yes?Yes?

Legitimate System Peaks•Dissolve samples in mobile phase•Adjust diluent with mobile phase components

Injection “System Peak” ???Carry over ???

Contamination ???

Yes?Yes?

NoNo



Suggested FlowSuggested Flow--Chart for Troubleshooting Contd.Chart for Troubleshooting Contd.

Steps: by ease of use1. Review and revise diluent’s composition2. Replace vials’ batch and/or brand3. Replace in-line filters4. Replace solvents batch and/or brand5. Try a new/fresh column6. Wash system, especially injectorMake sure to try a fresh blank each test!

Peaks still persist?Peaks still persist?

Not System Peaks?

Replace injector’s surfaces

Try another instrument/Operator/Lab

Peaks still persist?Peaks still persist?

Not washed by Not washed by blanks?blanks?



Many times the extraneous peak disappears on its own and

remains an unsolved mystery…

Extraneous Peaks in HPLC

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