Isranalytica2006

Unexpected Peaks in Chromatograms - Are They Related Compounds, System Peaks or Contaminations? From the Diary of an HPLC Detective
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SHULAMIT LEVIN, MEDTECHNICA

Dr. Shulamit Levin 2005, Medtechnica

Isranalytica2006

HPLC in Pharmaceutics

Dr. Shulamit Levin 2005, Medtechnica

Isranalytica2006

Stability Indicating Methods

Extra Sensitive to Extraneous Peaks
Accurately quantifies the active pharmaceutical ingredient without interference from:
Impurities Degradation products Excipients Other potential impurities Other active ingredients

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.

Dr. Shulamit Levin 2005, Medtechnica

Isranalytica2006

Accounting for Every Peak in the Related Compounds Profile - Even Down to 0.003 %Area
Retention Time (Minutes)

Compound Impurity 1 Impurity 2 Butamben Impurity 3

Area %
0.0054 0.0229 99.954 0.0037 0.0106 0.0034

Signal-toNoise 4.1 16.4 64544 2.6 5.8 2.2

2.174 2.231 2.263 2.504 2.549 2.714

Butamben

Impurity 4 Impurity 5

>LOQ

Dr. Shulamit Levin 2005, Medtechnica

Isranalytica2006

Sample vs. Blank (Diluent)

Unexpected peaks can appear in blanks and in all following injections In this case they are excluded from the processing of the sample
50.00 40.00 30.00

Blank
? ? ? ? ?

mAU

20.00 10.00 0.00

50.00 40.00 30.00

Sample

mAU

20.00 10.00 0.00 0.00 10.00 20.00 30.00 40.00 50.00

Minutes

Dr. Shulamit Levin 2005, Medtechnica

Isranalytica2006

INJECTION OF PURE SOLVENT: Why would there be peaks?

Injection

System Peak ??? Carry over ??? Contamination ???

Dr. Shulamit Levin 2005, Medtechnica

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System Peaks
Legitmate System Peaks Originate from the Mobile Phase Components Going Through Re-Equilibration

Dr. Shulamit Levin 2005, Medtechnica

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CONDITIONS FOR APPEARANCE OF REAL SYSTEM PEAKS

Mobile phase is multi-component (n=>2) Mobile phase contains adsorbable components Mobile phases components respond to the detector (high background) Sample or sample-diluent is different from the mobile phase, enough to create equilibrium perturbation.

Dr. Shulamit Levin 2005, Medtechnica

Isranalytica2006

Mechanism of System Peaks Formation Mechanism of System Peaks Formation

Vacancy EXAMPLE: Two additives in the mobile phase Example: k(1) = 1 Example: k(2) = 2
Step 1:

k' =

tR - t 0 t0 Cs Cm

Step 1:

Equilibrium: Cs=1; Cm = 1
Step 2:

Equilibrium: Cs=2; Cm = 1
Step 2:

k =

Injection of Vacancy: Cs=1; Cm=0

Step 3:

Injection of Vacancy: Cs=2; Cm=0

Step 3:

Re-equilibration: Cs=0.5; Cm=0.5

CHROMATOGRAM

Re-equilibration: Cs=1.33; Cm=0.67

t0
k=1 k=2
Dr. Shulamit Levin 2005, Medtechnica

Isranalytica2006

Injection of free amino acids into mobile phase containing: Ac buffer, CuAc, and Heptsulfonate.

Diluent: Water

Levin and Grushka

Dr. Shulamit Levin 2005, Medtechnica

Isranalytica2006

Legitimate System Peaks Result from Mobile phase components due to their absence in the injected sample
Levin and Abu-Lafi

Injection of Blank = Water

Conclusion: Use mobile phase composition as the diluent

Dr. Shulamit Levin 2005, Medtechnica

Isranalytica2006

An Example for Real System Peaks in Gradients with Trifluoroacetic Acid (TFA) in the Mobile Phase
Peptide Peak

Sample=Peptide Dissolved in Water

Blank = Mobile phase

TFA

Blank = Mobile phase

Zoomed
ACN

Blank =Water

System peaks appear because diluent does not contain TFA

Dr. Shulamit Levin 2005, Medtechnica

Isranalytica2006

Real System Peaks in Multi-component Mobile Phase H2O, MeCN, MeOH, THF
Chromatogram and Peaks' UV-VIS Spectra
Cpd 2 - 2.364 nm
240.00 260.00 280.00 300.00 320.00 340.00 220.00 220.00

2.817 nm
240.00 260.00 280.00 300.00 320.00 340.00

220.00

2.981 nm
240.00 260.00 280.00 300.00 320.00 340.00

Cpd 2 - 2.364

0.00 -0.10

2.817 2.981

THF

AU

-0.20 -0.30 -0.40 -0.50 1.00 1.20 1.40

Wavelength: 215nm
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

1.60 1.80 2.00 2.20

2.40 2.60 2.80 3.00

Minutes

Diluent does not contain all mobile phase components

Dr. Shulamit Levin 2005, Medtechnica

Isranalytica2006

Contaminations Peaks - Not System Peaks!

Phosphate Buffer and Acetonitril Do Not Produce Such System Peaks!
0.006

0.005

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

3.404

0.004

0.003 AU 0.002

2.373 2.731

0.000

-0.001

4.274

6.477

0.001

-0.002

Sample Name: Diluent

-0.003 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

Minutes
Dr. Shulamit Levin 2005, Medtechnica

18.274

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Use of Diode-Array Detector for Troubleshooting of Contamination Peaks in Multicomponent Mobile Phase
Chromatogram and Peaks' UV-VIS Spectra
Cpd 2 - 2.188 250.00 nm
300.00

2.748 250.00 nm
300.00

2.867 250.00 nm
300.00

3.180 250.00 nm
300.00

4.226 250.00 nm
300.00

Aromatic (not in mobile phase)

Negative UV spectrum

Cpd 2 - 2.188

0.015

Wavelength: 254.0 nm
2.748 2.867 3.180 4.226
Minutes

0.010

AU

0.005

0.000

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

Dr. Shulamit Levin 2005, Medtechnica

Isranalytica2006

Carry Over:
Chemical Residues in the system
q q q Residues in the injector Non-specific irreversible adsorption on column Accumulation on systems surfaces

Dr. Shulamit Levin 2005, Medtechnica

Isranalytica2006

Carry Over in the Injector Washed by Blank Injection

A1100 VWD AU - A1100 AU 286 nm 0.0004 0.0003 AU 0.0002 0.0001 0.0000 -0.0001 A1100 VWD AU - A1100 AU 286 nm 0.0004 0.0003 AU 0.0002 0.0001 0.0000 -0.0001 8.00 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

11.334

Blank Injection 1

Blank Injection 2 Contamination is cleaned

Minutes

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Isranalytica2006

Blank Injections
Signals are reduced from Injection to Injection indicate carry over in the injector
100.00

Series of Blank Injections

80.00

60.00 mAU 40.00 20.00 0.00 0.00 10.00 20.00 30.00 40.00 50.00

Minutes

Dr. Shulamit Levin 2005, Medtechnica

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Carry-Over in the Injector

LC-MS/MS
1. 2. Peak elutes at RT of main component MRM of the main component

Blank Sample after wash

Blank Sample before wash

Injection-Clean-up with EDTA solved the problem

Dr. Shulamit Levin 2005, Medtechnica

Isranalytica2006

Carry Over:
Residues in the system
q q q Residues in the injector Non-specific irreversible adsorption on column Accumulation on systems surfaces

Dr. Shulamit Levin 2005, Medtechnica

Isranalytica2006

Accumulation on Column
MAIN1
0.0010 AU 0.0005

Sample
10.00 11.00 12.00 13.00 14.00 15.00

0.0000 1.00 2.00 3.00 4.00 5.00 6.00 7.00 Minutes 8.00 9.00

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

0.0010

AU

0.0005

0.0000 1.00 2.00 3.00 4.00 5.00 6.00 7.00 Minutes 8.00

9.00

MAIN1

Blank #1
10.00 11.00 12.00 13.00 14.00 15.00

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

Not Cleaned
MAIN1
0.0010

AU

0.0005

Cleaned

Blank #2
10.00 11.00 12.00 13.00 14.00 15.00

0.0000 1.00 2.00 3.00 4.00 5.00 6.00 7.00 Minutes 8.00 9.00

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

Dr. Shulamit Levin 2005, Medtechnica

Isranalytica2006

Contamination Peaks
TFA Containing Mobile Phase: TFA, H2O, MeCN - Do Not Contain Aromatic Components!

Chromatogram and UV-VIS Spectra of Contamination Peaks 8.510

340.00 320.00 300.00 280.00 260.00 240.00 220.00 200.00 0.002 0.001
8.510

11.604
Aromatic group

41.142

Aromatic group

nm

11.604

0.000

-0.001 -0.002

Diluent at 280 nm
5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00

Minutes

Dr. Shulamit Levin 2005, Medtechnica

41.142

AU

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Peaks of aromatic compounds in non aromatic mobile phase:

Cannot be Legitimate System Peaks
Chromatogram and UV -VIS Spectra of a Blank Run 1.066
350.00

1.553

2.362

3.695

6.237

6.418

24.320 26.940 27.026

nm

300.00 250.00

6.237 6.418

24.320

2.362

0.20

AU

0.00 -0.20 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

Minutes Sample Name : blank ; Wavelength 214 :

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

Dr. Shulamit Levin 2005, Medtechnica

26.940 27.026

1.066 1.553

3.695

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Contamination Peaks - Residues

Contamination on-column and/or in the mobile phase
Chromatogram and UV-VIS Spectra of Contaminations Peaks
1.274 340.00 320.00 300.00 nm 280.00 260.00 240.00 220.00 200.00
41.018 42.128 34.345 Aromatic compounds not legitimate components of the mobile phase

1.514

1.731

8.500

11.633

34.345

36.595

37.612

41.018

42.128

0.030 0.025 0.020 AU 0.015 0.010 0.005 0.000 5.00 10.00 15.00 20.00 25.00 30.00
1.5141.274 1.731

Blank=Diluent at 220 nm
8.500 11.633

35.00

36.595 37.612

40.00

45.00

50.00

55.00

Minutes

Gradient with TFA

Dr. Shulamit Levin 2005, Medtechnica

Isranalytica2006

Carry Over:
Residues in the system
q q q Residues in the injector Non-specific irreversible adsorption on column Accumulation on systems surfaces

Dr. Shulamit Levin 2005, Medtechnica

Isranalytica2006

Contaminations
Origin Outside the Sample
q Non-pure solvents q Vials leachables q Reservoirs leachables

Dr. Shulamit Levin 2005, Medtechnica

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Baseline at Gradient
Change of Baseline with Time at Various Wavelengths: Indication for non pure solvent
0.030 0.025 0.020 60.00 100.00

220 nm
80.00

AU

0.015 Gradients Profile 0.010 0.005 0.000 40.00

230 nm 254 nm 280 nm

5.00 15.00 25.00 35.00 45.00

20.00

0.00 55.00

Minutes

Dr. Shulamit Levin 2005, Medtechnica

% Composition

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Non-Pure/Contaminated Solvents Used in Gradients

Contaminations can also come from the Parafilm used to seal reservoirs!!!
10.633 BHT 10.633 BHT 6.896 BHA 2.919PG 4.525 4.525 TBHQ TBHQ

Sample

0.00

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 Minutes

30.00

Blank
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 Minutes 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00

Dr. Shulamit Levin 2005, Medtechnica

Isranalytica2006

Sample vs Blank
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.
50.00 40.00 30.00

This is the reason why there are Gradient Grade solvents

Blank

mAU

20.00 10.00 0.00 50.00 40.00 30.00

Sample

mAU

20.00 10.00 0.00 0.00 10.00 20.00 30.00 40.00 50.00 Dr. Shulamit Levin 2005, Medtechnica

Minutes

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Peaks Origin: Contamination accumulated on pumps in-line filter

Pressure Jump Bubble-Detection

Unexpected Baseline Perturbation

Dr. Shulamit Levin 2005, Medtechnica

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Contaminations
Origin Outside the Sample q Non-pure solvents q Vials leachables q Reservoirs leachables

Dr. Shulamit Levin 2005, Medtechnica

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Contamination from Vials Surfaces

0.0012 0.0010 0.0008 AU 0.0006 0.0004 0.0002 0.0000 -0.0002 1.00 2.00 3.00

Non-Certified

Minutes

4.00

5.00

6.00

7.00

8.00

0.0014 0.0012 0.0010 0.0008

Change of vial brand In the same experiment!

AU

Certified

0.0006 0.0004 0.0002 0.0000

-0.0002 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

Minutes

Dr. Shulamit Levin 2005, Medtechnica

Isranalytica2006

Contamination is developed during the experiment in complex Diluents (Samples Solvent) (Sample
0.020 0.015

AU

0.010

0.005

0.000 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 Minutes 4.00 4.50 5.00 5.50 6.00 6.50 7.00

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

0.020

0.015

AU

0.010

0.005

0.000 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 Minutes 4.00 4.50 5.00 5.50 6.00 6.50 7.00

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

Contamination

Contamination

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Isranalytica2006

Contaminations
Origin Outside the Sample q Non-pure solvents q Vials leachables q Reservoir or Columns leachables

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Contamination Peaks in Size Exclusion Chromatography (SEC)

Contamination is NOT a monomer!
Chromatogram at 210 nm
0.030 0.025 0.020 0.015

Monomer

Sample
Contamination

AU

0.010 0.005 0.000 -0.005 -0.010 0.00

Blank
Higher Molecular Weight
5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00

Minutes

Dr. Shulamit Levin 2005, Medtechnica

Isranalytica2006

Other Reasons for Extraneous Peaks

Dr. Shulamit Levin 2005, Medtechnica

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Columns Packing Collapse

All Peaks are split

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Suggested Flow-Chart for Troubleshooting

In a Regulated Environment (no change in method!)
Injection

System Peak ??? Carry over ??? Contamination ???

Yes?

No

Mobile phase:
Multicomponent? Adsorbed components (ion pair)? Response in detector (background)?

Yes?

Legitimate System Peaks

Dissolve samples in mobile phase Adjust diluent with mobile phase components

Dr. Shulamit Levin 2005, Medtechnica

Isranalytica2006

Suggested Flow-Chart for Troubleshooting Contd.

Steps: by ease of use 1. Review and revise diluents composition 2. Replace vials batch and/or brand 3. Replace in-line filters 4. Replace solvents batch and/or brand 5. Try a new/fresh column 6. Wash system, especially injector
Make sure to try a fresh blank each test!

Not System Peaks?

Not washed by blanks?

Peaks still persist? Replace injectors surfaces Peaks still persist?

Try another instrument/Operator/Lab

Dr. Shulamit Levin 2005, Medtechnica

Isranalytica2006

Many times the extraneous peak disappears on its own and remains an unsolved mystery

Dr. Shulamit Levin 2005, Medtechnica