CPSA Digest 2003

Day 3

WeOB2

Matrix Effect in LC-MS/MS Analysis

Background
When matrix components are not efficiently removed from a sample they are contained in the isolated supernatant or filtrate. These endogenous materials may adversely affect detection. In MS/MS detection systems, matrix contaminants have been shown to reduce the efficiency of the ionization process using atmospheric pressure ionization (API) techniques. A loss in response is observed and this phenomenon is referred to as ionization suppression or ³matrix effect². This effect can lead to decreased reproducibility and accuracy for an assay, failure to reach the desired limit of quantitation (LOQ), and curve splitting (especially when analogue internal standards are used) due to accumulation of matrix on the analytical column which can affect the internal standard (IS) and drug differently. The matrix effect is always present and its reduction is one of the most challenging aspects of the method development process.

The FDA Guidance from May 2001 requires that the matrix effect be thoroughly evaluated during the method development and validation process. Specifically, the guidance requires the analyst to:

• Quantitatively measure matrix effect in assay validation.
• Evaluate blank matrix effect from minimum of six lots of blank matrices.
• Ensure lower limit of quantitation (LLOQ) precision and accuracy in minimum of six lots of blank matrices.

Sources of Matrix Effect

• Endogenous matrix components (proteins, salts, etc.).
• Other potential interference substances (metabolites, decomposition products, concomitant medication, exogenous xenobiotics).
• Matrix from the formulation (e.g., surfactants).

Type I and Type II Matrix Effects

• Type I Matrix Effect: Suppression or enhancement of the analyte signal by the matrix
  • Suppression Effect (in most cases)
    • Results from competitive ionization in source area (universal suppression).
    • Mass specific suppression caused by isobaric components which produce different product ions from the analyte.
  • Enhancement Effect
    • Formation of adduct(s) with analyte that make the analyte more volatile or easier to ionize.
• Type II Matrix Effect: Interference peak from matrix in the retention region of the analyte
  • Interference peaks
    • Arise from endogenous substances.
    • Isobaric components can generate same product ion(s) as the analyte.

How to Measure the Matrix Effect

• Unextracted vs. Neat
  • Analysts use a post-extraction spiked matrix blank and compare the results with an analytical standard in neat solution to determine the influence of the matrix on the analysis. A matrix blank is a representative biological sample that is free of the target analytes. A spiked matrix blank is a control sample that has been fortified with the target analytes at a defined, relevant level.
  • Matrix Effect = (Response of post-extracted spike)/(Response of unextracted sample).
  • The absence of a matrix effect is indicated by a ratio of 1.0. Suppression of ionization results in reduced analyte response (ratio < 1.0). Total matrix suppression yields a value of zero. Signal enhancement may also result (ratio > 1.0).
• Post-Column Infusion
  • Post column infusion of analyte (neat solution) yields a steady signal followed by an injection of an extracted blank matrix through the column. A comparison is made between the signal from infusion and the signal at the retention time of the analyte after injection of extracted blank matrix.

Remedies for Matrix Effects

• HPLC Conditions
  • Early Eluents (matrix from same injection)
    • Phenomena: Signal is steadily low or variable peak area did not proportionally increase with the increase of injection volume.
    • Remedy: Better separation of analyte from matrix (slower gradient, different mobile phase, longer retention time, different column).
  • Late Eluents (matrix carried over from previous injections)
    • Phenomena: Signal decreases after first few injections of extracted sample and increases after inject a few neat solutions or by flushing column.
    • Remedy: Increase column flushing time, use higher organic flush, higher flow rate, different column or different mobile phase.
  • Interference Peaks
    • Remedy: Obtain better separation on the HPLC column (slower gradient, longer retention time, different column, different mobile phase).
  • Mass Spectrometry Conditions
    • Interference Peak
      • Increase mass spec resolution (narrow the molecules reaching Quadrupole 1).
      • Use different mass transitions (different daughter ion)
    • Mass Specific Suppression (rare, but possible)
      • Use different mass transition (use in source fragmentation and MRM transition).
    • APCI vs. ESI
      • APCI tends to demonstrate less matrix effect.
  • Extraction Scheme to Obtain Cleaner Samples
    • Liquid-Liquid Extraction (LLE)
      • Use a selective extraction solvent.
      • Add buffer (pH adjustment depends on analyte pKa).
    • Solid Phase Extraction (SPE)
      • Use a selective sorbent chemistry and reduced sorbent mass.
      • Modify the loading/washing/eluting conditions
    • Protein Precipitation Extraction (PPT)
      • Modify the reagent composition and volume.
    • Combination(s) of the Above
      • e.g., use PPT followed by SPE
  • A More Appropriate Internal Standard (IS) - Cannot directly change matrix effect, but can cancel out most matrix effects or solve curve splitting issue caused by matrix effect.
    • Use an isotope labeled IS, if available.
    • Use a structurally related analogue.

  Matrix Effect Tests in Method Validation

  • Quantify Matrix Effect (Unextracted Vs. Neat)
    • Unextracted: analyte and IS spiked into extracted blank matrix.
    • Neat: Analyte solution without any matrix.
  • Blank Specificity
    • Check the Type II matrix effect (use minimum of six different lots of matrices).
    • Criteria: Interference peak area <= 20% of the LLOQ standard peak area and <5% of internal standard peak area.
  • Spike-in Specificity
    • Check the Type I matrix effect (LLOQ standard spiked in minimum of six different lots of matrices).
    • Criteria: Accuracy within 100±20% of the nominal concentration.
    • Precision <= 20%.

  
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