Method Development and Validation of Caffeine and Paraxanthine in Human Plasma
Rachel Sun, Orlando J. Bravo, Brian J. Engel
BASi, 2701 Kent Avenue, West Lafayette, IN 47906
Overview
- Caffeine and paraxanthine quantitation method is validated
- The interference of theobromine and theophylline is evaluated
- It is necessary to separate paraxanthine from theophylline
- The method has been applied to several clinical studies successfully
Introduction
Caffeine and its metabolites are ubiquitous in human biological fluids due to dietary consumption. The metabolites of caffeine are paraxanthine, theobromine and theophylline. The ratio of paraxanthine to caffeine may serve as a surrogate measurement of liver function, and thus, there is much interest in monitoring these two analytes simultaneously. Often times, the interference of theophylline to paraxanthine is overlooked for many LC/MS/MS methods although theophylline and paraxanthine have the same MS/MS transition. Thus, chromatographic separation of theophylline and paraxanthine is necessary for accurate quantitation of the primary metabolite.
Method
SPE extraction procedure:
- Condition OASIS® HLB, 30g/well SPE plate with methanol
- Condition SPE plate with water
- Load sample (50.0 μL sample and 150 μL IS and 100 μL water) to SPE plate
- Wash SPE plate with water
- Elute with methanol
- Blow down to dryness, reconstitute and inject on LC/MS/MS system
HPLC conditions:
Column:
Mobile Phase A:
Mobile Phase B:
Run time:
Retention time: |
SymmetryShield RP18
Formic acid in water
Methanol / water / formic acid mixture
6.5 minutes
4.2 min. ( caffeine) and 2.3 min. (paraxanthine) |
Tandem mass spectrometry:
Mass spectrometer:
Source:
Resolution:
|
API 3000
Turbo Ionspray
Unit/Unit
|
| Ions Monitored: |
Compound |
Precursor ion (Q1 m/z) |
Product ion (Q3 m/z) |
| |
Caffeine
Caffeine-d
Paraxanthine
Paraxanthine-d |
195.2
204.2
181.0
184.0 |
138.0
144.0
124.3
124.3 |
Results
Validation summary
| |
|
Caffeine |
Paraxanthine |
Units |
| Standard calibrator range |
Upper limit |
20,000 |
20,000 |
ng/mL |
| |
Lower limit |
25.0 |
25.0 |
ng/mL |
| Quality control sample range |
High |
15,000 |
15,000 |
ng/mL
|
| |
Middle |
7500 |
7500 |
ng/mL
|
| |
Low |
75.0 |
75.0 |
ng/mL |
| Dilution factor |
|
20 |
20 |
- |
| Freeze/thaw stability |
At -80±10°C |
4 |
4 |
cycles |
| |
At -20±10°C |
4 |
4 |
cycles |
| Short-term matrix stability |
At ambient |
25 |
25 |
hours |
| Long-term matrix stability |
At -20±10°C |
88 |
88 |
days |
| |
At -80±10°C |
175 |
175 |
days |
| Processed sample stability |
At ambient |
141 |
141 |
hours |
| Stock solution stability |
At 2-8°C |
53 |
53 |
days |
| Extraction efficiency |
Analyte |
87.8% |
97.6% |
|
| |
ISTD |
83.6% |
94.8% |
|
| Sample volume |
50 μL |
|
|
|
Standard calibrator sample performance
Caffeine
Paraxanthine
Validation sample performance
Typical calibration curve
Caffeine
Paraxanthine
Influence of metabolites
Caffeine
|
Paraxanthine
|
Typical validation chromatograms
Blank matrix:
|
LLOQ (25.0 ng/mL)
|
Chromatograms for interference assessment
High QC spiked to contain theobromine and theophylline at 3000 ng/mL.
|
Low QC spiked to contain theobromine and theophylline at 3000 ng/mL.
|
Typical incurred sample chromatogram
Caffeine
|
Paraxanthine
|
More than 1000 samples have been assayed using the method. Incurred sample reproducibility of the method was demonstrated in one of studies by reassaying 72 incurred samples. The percentage change of each reassay result relative to the mean of the original and the reassay values was less than ±10.3 and ±11.2 % for caffeine and paraxanthine, respectively.
Discussion
Blank matrix
With the ubiquitous presence of caffeine and its metabolites in human biological fluids as a result of dietary consumption, analyte free plasma should be acquired and screened for possible presence of caffeine and paraxanthine prior to use.
Separation/resolution
The resolution between theophylline and paraxanthine should be at least 1.5 to ensure enough separation to provide accurate quantification of paraxanthine.
Conclusion
This method has been successfully validated and applied to several clinical studies to date. For most incurred samples, a theophylline peak eluting immediately after paraxanthine is clearly observed. This observation confirms the necessity to ensure adequate separation between theophylline and paraxanthine to successfully obtain accurate paraxanthine quantification for LC-MS/MS analysis.
|
