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

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

2 HPLC in Pharmaceutics Σ

3 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.

4 Accounting for Every Peak in the Related Compounds Profile - Even Down to %Area Compound Retention Time () Area % Signal-to- Noise Impurity Impurity Butamben Butamben Impurity 3 Impurity Impurity >LOQ

5 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 Blank mau ????? Sample mau

6 INJECTION OF PURE SOLVENT: Why would there be peaks? Injection System Peak??? Carry over??? Contamination???

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

8 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.

9 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

10 Injection of free amino acids into mobile phase containing: Ac buffer, CuAc,, and Heptsulfonate. Diluent: Water Levin and Grushka Dr. Shulamit Levin 2005, Medtechnica

11 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

12 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

13 Real System Peaks in Multi-component Mobile Phase H 2 O, MeCN, MeOH, THF Chromatogram and Peaks' UV-VIS Spectra Cpd nm nm nm AU Cpd THF Wavelength: 215nm Diluent does not contain all mobile phase components

14 Contaminations Peaks - Not System Peaks! Phosphate Buffer and Acetonitril Do Not Produce Such System Peaks! Mobile phase: ACN : Phosphate Buffer ph 9.5 1:1 Wavelength: 280 nm AU Sample Name: Diluent

15 Use of Diode-Array Detector for Troubleshooting of Contamination Peaks in Multicomponent Mobile Phase Cpd nm Chromatogram and Peaks' UV-VIS Spectra nm nm Aromatic (not in mobile phase) nm nm Negative UV spectrum AU Cpd Wavelength: nm

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

17 Carry Over in the Injector Washed by Blank Injection A1100 VWD AU - A1100 AU 286 nm AU Blank Injection A1100 VWD AU - A1100 AU 286 nm AU Blank Injection 2 Contamination is cleaned

18 Blank Injections Signals are reduced from Injection to Injection indicate carry over in the injector Series of Blank Injections mau

19 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

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

21 Accumulation on Column AU MAIN1 Sample SampleName: SST; Vial: 1; Injection: 1; Name: MAIN1; Area: AU MAIN1 Blank # SampleName: DILUENT; Vial: 2; Injection: 1; Name: MAIN1; Area: 5063 AU Cleaned Not Cleaned MAIN1 Blank # SampleName: DILUENT; Vial: 2; Injection: 2; Name: MAIN1; Area: 4784

22 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 Aromatic group Aromatic group Diluent at 280 nm

23 Peaks of aromatic compounds in non aromatic mobile phase: Cannot be Legitimate System Peaks Chromatogram and UV -VIS Spectra - of a Blank Run nm AU Sample Name: blank ; Wavelength: 214 Mobile phase: Gradient of 0.1% TFA in water with ACN

24 Contamination Peaks - Residues Contamination on-column and/or in the mobile phase Chromatogram and UV-VIS Spectra of Contaminations Peaks nm Aromatic compounds not legitimate components of the mobile phase AU Blank=Diluent at 220 nm Gradient with TFA

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

26 Contaminations Origin Outside the Sample Non-pure solvents Vials leachables Reservoir s leachables

27 Baseline at Gradient Change of Baseline with Time at Various Wavelengths: Indication for non pure solvent 220 nm AU nm 254 nm 280 nm Gradient s Profile % Composition

28 -2.919PG Non-Pure/Contaminated Solvents Used in Gradients Contaminations can also come from the Parafilm used to seal reservoirs!!! BHA BHT Sample TBHQ Blank

29 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 This is the reason why there are Gradient Grade solvents Blank mau Sample mau

30 Peak s s Origin: Contamination accumulated on pump s s in-line filter Pressure Jump Bubble-Detection Unexpected Baseline Perturbation

31 Contaminations Origin Outside the Sample Non-pure solvents Vials leachables Reservoir s leachables

32 Contamination from Vials Surfaces Non-Certified AU AU Change of vial brand In the same experiment! Certified

33 Contamination is developed during the experiment in complex Diluents (Sample s s Solvent) AU Contamination SampleName: Sample; Vial: 1:F,1; Injection: 1; Name: Contamination AU Contamination SampleName: Diluent ; Vial: 1:E,2; Injection: 1; Name: Contamination

34 Contaminations Origin Outside the Sample Non-pure solvents Vials leachables Reservoir or Column s leachables

35 Contamination Peaks in Size Exclusion Chromatography (SEC) Contamination is NOT a monomer! Chromatogram at 210 nm Sample Contamination Monomer AU Blank Higher Molecular Weight

36 Other Reasons for Extraneous Peaks

37 Column s s Packing Collapse All Peaks are split

38 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

39 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

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

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