Isranalytica2006
Unexpected Peaks in Chromatograms - Are
They Related Compounds, System Peaks or
Contaminations?
From the Diary of an HPLC Detective
SHULAMIT LEVIN
©Dr. Shulamit Levin 2005
Isranalytica2006
HPLC in Pharmaceutics
Σ
©Dr. Shulamit Levin 2005
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
Isranalytica2006
Accounting for Every Peak in the Related Compounds’
Profile - Even Down to 0.003 %Area
Butamben
Compound
Retention Time
(Minutes)
Area
%
Signal-toNoise
Impurity 1
2.174
0.0054
4.1
Impurity 2
2.231
0.0229
16.4
Butamben
2.263
99.954
64544
Impurity 3
2.504
0.0037
2.6
Impurity 4
2.549
0.0106
5.8
Impurity 5
2.714
0.0034
2.2
>LOQ
©Dr. Shulamit Levin 2005
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
Blank
mAU
30.00
20.00
?
?
? ?
?
10.00
0.00
50.00
40.00
Sample
mAU
30.00
20.00
10.00
0.00
0.00
10.00
20.00
30.00
40.00
50.00
Minutes
©Dr. Shulamit Levin 2005
Isranalytica2006
INJECTION OF PURE SOLVENT:
Why would there be peaks?
“System Peak” ???
Injection
Carry over ???
Contamination ???
©Dr. Shulamit Levin 2005
Isranalytica2006
System Peaks
“Legitmate” System Peaks
Originate from the Mobile Phase Components Going
Through Re-Equilibration
©Dr. Shulamit Levin 2005
Isranalytica2006
CONDITIONS FOR APPEARANCE OF
REAL 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.
©Dr. Shulamit Levin 2005
Isranalytica2006
Mechanism
Mechanismof
ofSystem
SystemPeaks
PeaksFormation
Formation
Vacancy
EXAMPLE:
Two additives in the mobile phase
k' =
tR - t 0
t0
k’ =φ
Cs
Cm
Example: k’(1) = 1
Example: k’(2) = 2
Step 1:
Step 1:
Equilibrium:
Cs=1; Cm = 1
Equilibrium:
Cs=2; Cm = 1
Step 2:
Step 2:
Injection of
Vacancy:
Cs=1; Cm=0
Injection of
Vacancy:
Cs=2; Cm=0
Step 3:
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
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
Blank = Mobile phase
Water
TFA
Blank = Mobile phase
Zoomed
ACN
Blank =Water
System peaks appear because diluent does not contain TFA
Isranalytica2006
Real System Peaks in Multi-component Mobile Phase
H2O, MeCN, MeOH, THF
Chromatogram and Peaks' UV-VIS Spectra
220.00
240.00 260.00 280.00 300.00 320.00 340.00
Cpd 2 - 2.364
0.00
AU
-0.10
-0.20
-0.30
-0.40
-0.50
1.00 1.20 1.40
1.60 1.80 2.00 2.20
2.817
nm
2.981
nm
220.00
240.00 260.00 280.00 300.00 320.00 340.00
2.817
2.981
220.00
Cpd 2 - 2.364
nm
240.00 260.00 280.00 300.00 320.00 340.00
THF
Wavelength: 215nm
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
Minutes
Diluent does not contain all mobile phase components
5.80 6.00
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
AU
0.003
0.000
6.477
4.274
2.373
2.731
0.001
18.274
0.002
-0.001
Sample Name: Diluent
-0.002
-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
Minutes
©Dr. Shulamit Levin 2005
30.00
Isranalytica2006
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
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
300.00
Aromatic (not in
mobile phase)
0.000
4.226
0.005
3.180
AU
0.010
Wavelength: 254.0 nm
2.748
2.867
0.015
Cpd 2 - 2.188
Negative UV
spectrum
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
Minutes
©Dr. Shulamit Levin 2005
Isranalytica2006
Carry Over:
Chemical Residues in the system
q
Residues in the injector
q
Non-specific irreversible adsorption on column
q
Accumulation on system’s surfaces
©Dr. Shulamit Levin 2005
Isranalytica2006
Carry Over in the Injector
Washed by Blank Injection
A1100 VWD AU - A1100 AU 286 nm
0.0004
0.0003
Blank Injection 1
11.334
AU
0.0002
0.0001
0.0000
-0.0001
A1100 VWD AU - A1100 AU 286 nm
0.0004
0.0003
Blank Injection 2
Contamination is cleaned
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
Minutes
©Dr. Shulamit Levin 2005
Isranalytica2006
Blank Injections
Signals are reduced from Injection to Injection indicate carry over in the injector
100.00
Series of Blank Injections
80.00
mAU
60.00
40.00
20.00
0.00
0.00
10.00
20.00
30.00
40.00
Minutes
©Dr. Shulamit Levin 2005
50.00
Isranalytica2006
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
Isranalytica2006
Carry Over:
Residues in the system
q
Residues in the injector
q
Non-specific irreversible adsorption on column
q
Accumulation on system’s surfaces
Isranalytica2006
Accumulation on Column
AU
MAIN1
0.0010
Sample
0.0005
0.0000
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
Minutes
SampleName: SST; Vial: 1; Injection: 1; Name: MAIN1; Area: 833913
0.0005
Blank #1
MAIN1
AU
0.0010
0.0000
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
Minutes
AU
0.0010
Not Cleaned
Cleaned
0.0005
MAIN1
SampleName: DILUENT; Vial: 2; Injection: 1; Name: MAIN1; Area: 5063
Blank #2
0.0000
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
Minutes
SampleName: DILUENT; Vial: 2; Injection: 2; Name: MAIN1; Area: 4784
14.00
15.00
Isranalytica2006
Contamination Peaks
TFA Containing Mobile Phase: TFA, H2O, MeCN - Do Not Contain Aromatic Components!
Chromatogram and UV-VIS Spectra of Contamination Peaks
Aromatic group
Aromatic group
0.000
-0.001
-0.002
41.142
41.142
AU
0.001
8.510
0.002
11.604
11.604
nm
8.510
340.00
320.00
300.00
280.00
260.00
240.00
220.00
200.00
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
Isranalytica2006
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
1.553
2.362
3.695
6.237
6.418
24.320 26.940 27.026
nm
350.00
300.00
24.320
6.237
6.418
3.695
2.362
26.940
27.026
AU
0.20
1.066
1.553
250.00
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
Isranalytica2006
Contamination Peaks - Residues
Contamination on-column and/or in the mobile phase
Chromatogram and UV-VIS Spectra of Contaminations’ Peaks
1.274
1.514
1.731
8.500
11.633
34.345
36.595
37.612
41.018
42.128
340.00
Aromatic compounds – not legitimate components of the mobile phase
320.00
nm
300.00
280.00
260.00
240.00
220.00
0.025
0.005
8.500
0.010
1.514
1.7311.274
AU
0.015
11.633
Blank=Diluent at 220 nm
0.020
41.018
42.128
0.030
36.595
37.612
34.345
200.00
0.000
5.00
10.00
15.00
20.00
25.00
30.00
Minutes
Gradient with TFA
35.00
40.00
45.00
50.00
55.00
Isranalytica2006
Carry Over:
Residues in the system
q
Residues in the injector
q
Non-specific irreversible adsorption on column
q
Accumulation on system’s surfaces
©Dr. Shulamit Levin 2005
Isranalytica2006
Contaminations
Origin – Outside the Sample
q Non-pure solvents
q Vials’ leachables
q Reservoir’s leachables
©Dr. Shulamit Levin 2005
Isranalytica2006
Baseline at Gradient
Change of Baseline with Time at Various Wavelengths:
Indication for non pure solvent
100.00
0.030
220 nm
0.025
0.020
60.00
AU
0.015
Gradient’s Profile
0.010
0.005
40.00
20.00
230 nm
254 nm
0.000
0.00
280 nm
5.00
15.00
25.00
35.00
45.00
55.00
Minutes
©Dr. Shulamit Levin 2005
% Composition
80.00
Isranalytica2006
Non-Pure/Contaminated
Solvents Used in Gradients
2.919
PG
Contaminations can also come from
the Parafilm used to seal reservoirs!!!
10.633
BHT
-
6.896
BHA
4.525
-
Sample
TBHQ
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
16.00
18.00
20.00
22.00
24.00
26.00
Minutes
©Dr. Shulamit Levin 2005
28.00
30.00
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
mAU
30.00
This is the reason why there are
“Gradient Grade” solvents
Blank
20.00
10.00
0.00
50.00
40.00
Sample
mAU
30.00
20.00
10.00
0.00
0.00
10.00
20.00
Minutes
30.00
40.00
50.00
©Dr. Shulamit Levin 2005
Isranalytica2006
Peak’s Origin:
Contamination accumulated on pump’s in-line filter
Pressure Jump
Bubble-Detection
Unexpected
Baseline Perturbation
©Dr. Shulamit Levin 2005
Isranalytica2006
Contaminations
Origin – Outside the Sample
q Non-pure solvents
q Vials’ leachables
q Reservoir’s leachables
©Dr. Shulamit Levin 2005
Isranalytica2006
Contamination from Vials’ Surfaces
0.0012
0.0010
Non-Certified
0.0008
AU
0.0006
0.0004
0.0002
0.0000
-0.0002
1.00
2.00
3.00
4.00
Minutes
5.00
6.00
7.00
8.00
0.0014
0.0012
0.0010
Certified
AU
0.0008
Change of vial brand
In the same experiment!
0.0006
0.0004
0.0002
0.0000
-0.0002
1.00
2.00
3.00
4.00
Minutes
5.00
6.00
7.00
8.00
Isranalytica2006
Contamination is developed during the experiment in
complex Diluents (Sample’
(Sample’s Solvent)
0.020
Contamination
AU
0.015
0.010
0.005
0.000
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
6.50
7.00
Minutes
SampleName: Sample; Vial: 1:F,1; Injection: 1; Name: Contamination
0.020
Contamination
AU
0.015
0.010
0.005
0.000
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
Minutes
SampleName: Diluent ; Vial: 1:E,2; Injection: 1; Name: Contamination
Isranalytica2006
Contaminations
Origin – Outside the Sample
q Non-pure solvents
q Vials’ leachables
q Reservoir or Column’s leachables
Isranalytica2006
Contamination Peaks in Size Exclusion
Chromatography (SEC)
Contamination is NOT a monomer!
Chromatogram at 210 nm
0.030
Monomer
0.025
Sample
0.020
Contamination
AU
0.015
0.010
0.005
0.000
Blank
-0.005
-0.010
0.00
Higher Molecular Weight
5.00
10.00
15.00
20.00
25.00
Minutes
30.00
35.00
40.00
45.00
50.00
Isranalytica2006
Other Reasons
for Extraneous Peaks
Isranalytica2006
Column’s Packing Collapse
All Peaks are split
©Dr. Shulamit Levin 2005
Isranalytica2006
Suggested Flow-Chart for Troubleshooting
In a Regulated Environment (no change in method!)
“System Peak” ???
Injection
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
Isranalytica2006
Suggested Flow-Chart for Troubleshooting Contd.
Steps: by ease of use
1. Review and revise diluent’s 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
Not System Peaks?
Not washed by
blanks?
Make sure to try a fresh blank each test!
Peaks still persist?
Replace injector’s surfaces
Peaks still persist?
Try another instrument/Operator/Lab
©Dr. Shulamit Levin 2005
Isranalytica2006
Many times the extraneous
peak disappears on its own and
remains an unsolved
mystery…
©Dr. Shulamit Levin 2005