Isranalytica2006Unexpected Peaks in Chromatograms - Are They Related Compounds, System Peaks or Contaminations? From the Diary of an HPLC Detective
[email protected] SHULAMIT LEVIN, MEDTECHNICA © Dr. Shulamit Levin 2005, Medtechnica Isranalytica2006 HPLC in Pharmaceutics Σ © 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 Isranalytica2006 System Peaks “Legitmate” System Peaks Originate from the Mobile Phase Components Going Through Re-Equilibration © Dr. Shulamit Levin 2005, Medtechnica 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, 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’ =φ 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 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 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 system’s 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 © Dr. Shulamit Levin 2005, Medtechnica 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 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 © 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 system’s 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 Isranalytica2006 Peaks of aromatic compounds in non aromatic mobile phase: Cannot be Legitimate System Peaks Chromatogram and UV -VIS Spectra of a Blank Run 1.066 350.00 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 Isranalytica2006 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 system’s surfaces © Dr. Shulamit Levin 2005, Medtechnica Isranalytica2006 Contaminations Origin – Outside the Sample q Non-pure solvents q Vials’ leachables q Reservoir’s leachables © Dr. Shulamit Levin 2005, Medtechnica Isranalytica2006 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 Gradient’s 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 Isranalytica2006 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 © Isranalytica2006 Peak’s Origin: Contamination accumulated on pump’s in-line filter Pressure Jump Bubble-Detection Unexpected Baseline Perturbation © Dr. Shulamit Levin 2005, Medtechnica Isranalytica2006 Contaminations Origin – Outside the Sample q Non-pure solvents q Vials’ leachables q Reservoir’s leachables © Dr. Shulamit Levin 2005, Medtechnica Isranalytica2006 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 (Sample’s 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 © Dr. Shulamit Levin 2005, Medtechnica Isranalytica2006 Contaminations Origin – Outside the Sample q Non-pure solvents q Vials’ leachables q Reservoir or Column’s leachables © Dr. Shulamit Levin 2005, Medtechnica Isranalytica2006 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 Isranalytica2006 Column’s Packing Collapse All Peaks are split © Dr. Shulamit Levin 2005, Medtechnica Isranalytica2006 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 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 System Peaks? Not washed by blanks? Peaks still persist? Replace injector’s 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