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:

1. Condition OASIS® HLB, 30g/well SPE plate with methanol
   
2. Condition SPE plate with water
   
3. Load sample (50.0 µL sample and 150 µL IS and 100 µL water) to SPE plate
   
4. Wash SPE plate with water
   
5. Elute with methanol
   
6. 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.