Biomarkers - HPLC (1)

DE MONTFORT UNIVERSITY LEICESTER

  • Title: Bench to bedside: HPLC analysis

  • Instructor: Dr. Dahlia Salman

  • Contact: Dahlia.Salman@dmu.ac.uk

  • Location: 2.25i Hawthorn Building

Learning Outcomes

  • Topics covered in the lecture:

    • Practice-based challenge for home chemotherapy

    • High-Performance Liquid Chromatography (HPLC)

    • How HPLC works

    • Quantitative analysis

    • Beer-Lambert Law

    • Internal standards

Case Study: Soft Tissue Sarcoma

  • Definition: Sarcomas are rare cancers from connective tissues (muscle, bone, nerves, blood vessels).

  • Statistics:

    • Account for 11% of pediatric cancers.

    • Approximately 3800 new cases diagnosed yearly.

  • Treatment:

    • Home chemotherapy with continuous intravenous infusion of Ifosfamide and Mesna over 14 days.

Home Chemotherapy

  • Overview:

    • Safe, effective, and positively received by patients.

    • Improves quality of life, addresses psychological factors, and reduces hospital wait times.

  • Advantages:

    • Use of ambulatory devices like elastomeric pumps that allow prolonged drug infusions.

    • Patients can return home after connection at hospitals, enjoy comfort with care from family and district nursing.

  • Issues:

    • Existing schedules impact patients’ quality of life and incur extra costs.

Challenge

  • Stability Issues:

    • No stability data for chemotherapy regimen using elastomeric pumps for the entire 14-day period.

    • Patients must return on Day 7 for the next dose, disrupting comfort and increasing costs.

  • Stability Records:

    • Ifosfamide stable for 9 days at 25ºC; loss observed at 37ºC (7% on day 9).

    • Ifosfamide stable for 14 days at 8ºC; >98% initial concentration at 7 days at 37ºC.

Analytical Techniques

  • Need for Study:

    • Assess stability of chemotherapy in clinical practice conditions for 14-day home infusion without hospital return.

  • Questions to Address:

    • What analytical technique to use?

    • Mechanism?

    • Calculating actual concentration from results?

High-Performance Liquid Chromatography (HPLC)

  • Definition: A powerful technique for analyzing various compound classes.

  • Methods:

    • Separation via different methodologies (polarity, size, charge).

  • Components:

    • Columns packed with small silica particles and high pressure.

HPLC Components and Functionality

  • System Components:

    • Pump: Forces mobile phase through the column, enhancing speed and efficiency.

    • Column: Contains minimal dead space for better separation efficiency.

    • Detector: Measures compounds as they elute, detecting signals proportional to substance mass (Beer-Lambert Law).

  • Mobile Phase Handling:

    • Appropriate solvents must be selected and degassed to avoid spurious peaks.

    • Techniques for degassing include sonic baths and bubbling helium.

HPLC Columns and Types

  • Dimensions: Typically 10-30 cm long, 3-10 mm diameter, packed with very fine materials (3-5 μm).

  • Efficiency: Small packing gives high theoretical plates (up to 50,000 per meter).

  • Packing Material: Commonly silica, supportive of various stationary phases (non-polar & reverse-phase).

Detectors in HPLC

  • Common Types:

    • UV-Vis Detectors: Use multiple lamps to generate a spectrum of light.

    • Measure absorbance at specific wavelengths tailored to the sample.

Beer-Lambert Law

  • Equation:

    • Absorbance: A = log10(lo/I)

      • lo = Incident light intensity.

      • I = Transmitted light intensity.

  • Relationship:

    • A = εcℓ

      • A = Absorbance

      • c = Concentration (mol/L)

      • ℓ = Pathlength (cm)

      • ε = Molar extinction coefficient

  • Importance: Linear relationship between absorbance and concentration under specific conditions.

Calibration and Internal Standards

  • Internal Standards: A known substance added to the sample, allowing for accurate measurement and calculation of analyte concentration.

  • Recovering Concentrations:

    • Adjust based on differences between injected and measured amounts to account for losses.

Summary

  • This lecture covered:

    • HPLC technique principles (why, how, what)

    • Common columns and detectors in HPLC analysis

    • Quantitative analysis applications

    • Beer-Lambert Law fundamentals

    • Internal standards utilization in HPLC