Unexpected Peaks in Chromatograms - Are They Related Compounds, System Peaks or Contaminations? From the Diary of an HPLC Detective SHULAMIT LEVIN
HPLC in Pharmaceutics Σ
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.
Accounting for Every Peak in the Related Compounds Profile - Even Down to 0.003 %Area Compound Retention Time () Area % Signal-to- Noise Impurity 1 2.174 0.0054 4.1 Impurity 2 2.231 0.0229 16.4 Butamben 2.263 99.954 64544 Butamben Impurity 3 Impurity 4 2.504 2.549 0.0037 0.0106 2.6 5.8 Impurity 5 2.714 0.0034 2.2 >LOQ
50.00 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 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
INJECTION OF PURE SOLVENT: Why would there be peaks? Injection System Peak??? Carry over??? Contamination???
System Peaks Legitmate System Peaks Originate from the Mobile Phase Components Going Through Re-Equilibration
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.
Vacancy Mechanism of of System Peaks Formation EXAMPLE: Two additives in the mobile phase k' = t R - t 0 t 0 k = C f s Cm Example: k (1) k = 1 Step 1: Equilibrium: Cs=1; Cm = 1 Step 2: Injection of Vacancy: Cs=1; ; Cm=0 Step 3: Re-equilibration: equilibration: Cs=0.5; Cm=0.5 Example: k (2) k = 2 Step 1: Equilibrium: Cs=2; Cm = 1 Step 2: Injection of Vacancy: Cs=2; ; Cm=0 Step 3: Re-equilibration: equilibration: Cs=1.33; Cm=0.67 CHROMATOGRAM t 0 k =1 k =2
Injection of free amino acids into mobile phase containing: Ac buffer, CuAc,, and Heptsulfonate. Diluent: Water Levin and Grushka Dr. Shulamit Levin 2005, Medtechnica
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
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
Real System Peaks in Multi-component Mobile Phase H 2 O, MeCN, MeOH, THF Chromatogram and Peaks' UV-VIS Spectra 220.00 Cpd 2-2.364 nm 220.00 240.00 260.00 280.00 300.00 320.00 340.00 2.817 nm 220.00 240.00 260.00 280.00 300.00 320.00 340.00 2.981 nm 240.00 260.00 280.00 300.00 320.00 340.00 AU 0.00-0.10-0.20-0.30-0.40 Cpd 2-2.364 2.817 2.981 THF Wavelength: 215nm -0.50 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 Diluent does not contain all mobile phase components
Contaminations Peaks - Not System Peaks! Phosphate Buffer and Acetonitril Do Not Produce Such System Peaks! 0.006 0.005 0.004 Mobile phase: ACN : Phosphate Buffer ph 9.5 1:1 Wavelength: 280 nm 0.003 3.404 AU 0.002 0.001 0.000 2.373 2.731 4.274 6.477 18.274-0.001-0.002 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
Use of Diode-Array Detector for Troubleshooting of Contamination Peaks in Multicomponent Mobile Phase Cpd 2-2.188 250.00 nm 300.00 Chromatogram and Peaks' UV-VIS Spectra 250.00 2.748 nm 300.00 250.00 2.867 nm 300.00 Aromatic (not in mobile phase) 250.00 3.180 nm 300.00 250.00 4.226 nm 300.00 Negative UV spectrum AU 0.015 0.010 0.005 0.000 Cpd 2-2.188 2.748 2.867 3.180 Wavelength: 254.0 nm 4.226 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
Carry Over: Chemical Residues in the system Residues in the injector Non-specific irreversible adsorption on column Accumulation on system s s surfaces
Carry Over in the Injector Washed by Blank Injection A1100 VWD AU - A1100 AU 286 nm 0.0004 AU 0.0003 0.0002 0.0001 11.334 Blank Injection 1 0.0000-0.0001 A1100 VWD AU - A1100 AU 286 nm 0.0004 AU 0.0003 0.0002 0.0001 Blank Injection 2 Contamination is cleaned 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
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
Carry-Over in the Injector LC-MS/MS 1. Peak elutes at RT of main component 2. MRM of the main component Blank Sample after wash Blank Sample before wash Injection-Clean Clean-up with EDTA solved the problem
Carry Over: Residues in the system Residues in the injector Non-specific irreversible adsorption on column Accumulation on system s s surfaces
Accumulation on Column AU 0.0010 0.0005 MAIN1 Sample 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 SampleName: SST; Vial: 1; Injection: 1; Name: MAIN1; Area: 833913 0.0010 AU 0.0005 0.0000 MAIN1 Blank #1 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 SampleName: DILUENT; Vial: 2; Injection: 1; Name: MAIN1; Area: 5063 AU 0.0010 0.0005 Cleaned Not Cleaned MAIN1 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 14.00 15.00 SampleName: DILUENT; Vial: 2; Injection: 2; Name: MAIN1; Area: 4784
Contamination Peaks TFA Containing Mobile Phase: TFA, H 2 O, MeCN - Do Not Contain Aromatic Components! Chromatogram and UV-VIS Spectra of Contamination Peaks nm AU 340.00 320.00 300.00 280.00 260.00 240.00 220.00 200.00 0.002 0.001 0.000 8.510 11.604 41.142 Aromatic group Aromatic group 8.510 11.604 41.142-0.001-0.002 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
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 27.026 350.00 nm 300.00 250.00 0.20 1.066 1.553 2.362 3.695 6.418 6.237 24.320 26.940 27.026 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 Sample Name: blank ; Wavelength: 214 Mobile phase: Gradient of 0.1% TFA in water with ACN
Contamination Peaks - Residues Contamination on-column and/or in the mobile phase Chromatogram and UV-VIS Spectra of Contaminations Peaks nm 340.00 320.00 300.00 280.00 260.00 240.00 220.00 200.00 1.274 1.514 1.731 8.500 11.633 34.345 36.595 37.612 41.018 42.128 Aromatic compounds not legitimate components of the mobile phase AU 0.030 0.025 0.020 0.015 0.010 0.005 0.000 1.5141.274 1.731 Blank=Diluent at 220 nm 8.500 11.633 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 34.345 36.595 37.612 41.018 42.128 Gradient with TFA
Carry Over: Residues in the system Residues in the injector Non-specific irreversible adsorption on column Accumulation on system s s surfaces
Contaminations Origin Outside the Sample Non-pure solvents Vials leachables Reservoir s leachables
0.030 0.025 Baseline at Gradient Change of Baseline with Time at Various Wavelengths: Indication for non pure solvent 220 nm 100.00 80.00 AU 0.020 0.015 0.010 0.005 0.000 230 nm 254 nm 280 nm 5.00 15.00 25.00 35.00 45.00 55.00 Gradient s Profile 60.00 40.00 20.00 0.00 % Composition
-2.919PG Non-Pure/Contaminated Solvents Used in Gradients Contaminations can also come from the Parafilm used to seal reservoirs!!! -4.525-6.896 BHA -10.633BHT 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 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 28.00 30.00
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
Peak s s Origin: Contamination accumulated on pump s s in-line filter Pressure Jump Bubble-Detection Unexpected Baseline Perturbation
Contaminations Origin Outside the Sample Non-pure solvents Vials leachables Reservoir s leachables
Contamination from Vials Surfaces 0.0012 0.0010 0.0008 Non-Certified 0.0006 AU 0.0004 0.0002 0.0000-0.0002 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 0.0014 AU 0.0012 0.0010 0.0008 0.0006 Change of vial brand In the same experiment! Certified 0.0004 0.0002 0.0000-0.0002 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00
Contamination is developed during the experiment in complex Diluents (Sample s s Solvent) 0.020 0.015 AU 0.010 0.005 0.000 Contamination 0.020 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 SampleName: Sample; Vial: 1:F,1; Injection: 1; Name: Contamination 0.015 AU 0.010 0.005 0.000 Contamination 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 SampleName: Diluent ; Vial: 1:E,2; Injection: 1; Name: Contamination
Contaminations Origin Outside the Sample Non-pure solvents Vials leachables Reservoir or Column s leachables
Contamination Peaks in Size Exclusion Chromatography (SEC) Contamination is NOT a monomer! 0.030 Chromatogram at 210 nm 0.025 0.020 0.015 Sample Contamination Monomer AU 0.010 0.005 0.000-0.005 Blank Higher Molecular Weight -0.010 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00
Other Reasons for Extraneous Peaks
Column s s Packing Collapse All Peaks are split
Suggested Flow-Chart for Troubleshooting In a Regulated Environment (no change in method!) Injection Carry over??? System Peak??? 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
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 Make sure to try a fresh blank each test! Not washed by blanks? Not System Peaks? Peaks still persist? Replace injector s surfaces Peaks still persist? Try another instrument/operator/lab
Many times the extraneous peak disappears on its own and remains an unsolved mystery