High-Performance Liquid Chromatography (HPLC) in Pediatrics
High-Performance Liquid Chromatography in Pediatrics
Key Points
- Gold standard for many biochemical analyses
- High sensitivity and specificity
- Requires minimal sample volume
- Essential for newborn screening programs
- Valuable in therapeutic drug monitoring
Primary Uses
- Hemoglobinopathy screening
- Metabolic disorder diagnosis
- Drug level monitoring
- Vitamin analysis
- Amino acid quantification
Advantages in Pediatrics
- Small sample requirements (ideal for neonates)
- Rapid turnaround time
- Multiple analyte detection
- High accuracy and precision
Basic Principles of HPLC
Components
- Mobile Phase:
- Liquid solvent mixture
- Carries sample through system
- Composition affects separation
- Stationary Phase:
- Column packing material
- Different types for various applications
- Determines separation mechanism
- Detection Systems:
- UV-visible spectrophotometry
- Fluorescence detection
- Mass spectrometry coupling
- Electrochemical detection
Types of HPLC
- Reverse Phase:
- Most common in clinical applications
- Best for polar compounds
- Normal Phase:
- Used for lipophilic compounds
- Less common in clinical settings
- Ion Exchange:
- For charged molecules
- Used in amino acid analysis
Clinical Applications
Newborn Screening
- Amino Acid Disorders:
- Phenylketonuria
- Maple syrup urine disease
- Homocystinuria
- Hemoglobinopathies:
- Sickle cell disease
- Thalassemias
- Variant hemoglobins
Therapeutic Monitoring
- Anticonvulsants:
- Phenytoin
- Carbamazepine
- Valproic acid
- Antibiotics:
- Aminoglycosides
- Vancomycin
Hemoglobin Analysis by HPLC
Clinical Indications
- Newborn screening
- Anemia evaluation
- Family studies
- Pre-conception screening
Normal Values
- Newborn:
- HbF: 65-95%
- HbA: 20-40%
- HbA2: <1%
- 6 months:
- HbF: <8%
- HbA: >90%
- HbA2: 2-3.5%
Pathological Patterns
- Sickle Cell Disease:
- HbS: >40%
- Absence of HbA
- Beta Thalassemia:
- Elevated HbA2 (>3.5%)
- Variable HbF
Metabolic Disorders
Amino Acid Analysis
- Sample Requirements:
- Plasma: 0.5-1.0 mL
- Urine: 24-hour collection
- CSF: 0.5-1.0 mL
- Common Disorders:
- PKU: Elevated phenylalanine
- MSUD: Branched-chain amino acids
- Tyrosinemia: Elevated tyrosine
Organic Acid Analysis
- Indications:
- Metabolic acidosis
- Hyperammonemia
- Unexplained encephalopathy
- Key Disorders:
- Methylmalonic acidemia
- Propionic acidemia
- Isovaleric acidemia
Therapeutic Drug Monitoring
Anticonvulsant Monitoring
- Phenytoin:
- Therapeutic range: 10-20 mg/L
- Sampling: Trough level
- Carbamazepine:
- Therapeutic range: 4-12 mg/L
- Monitor active metabolite
- Valproic Acid:
- Therapeutic range: 50-100 mg/L
- Protein binding affected
Antibiotic Monitoring
- Aminoglycosides:
- Peak and trough levels
- Narrow therapeutic window
- Vancomycin:
- Trough: 10-20 mg/L
- Timing critical
Result Interpretation
Pre-analytical Factors
- Sample Collection:
- Timing
- Storage conditions
- Transport temperature
- Patient Factors:
- Age
- Feeding status
- Recent transfusions
Quality Control
- Internal Standards
- Calibration Curves
- Reference Materials
- Method Validation
Common Pitfalls
- Sample Degradation
- Matrix Effects
- Interfering Substances
- Timing of Collection
