Simultaneous Determination of Methylxanthines and Cotinine in Human Plasma by Solid-Phase Extraction Followed by LC–MS–MS

May 01, 2014

A multi-residue method has been developed and validated for the analysis of methylxanthines (caffeine and its metabolites) and cotinine in human plasma. The method involves off-line solid-phase extraction (SPE) and analysis by liquid chromatography coupled to tandem mass spectrometry LC–MS–MS with electrospray ionization (ESI) in positive mode (PI). The developed and validated method was fast, selective, and convenient for the simultaneous determination of cotinine, caffeine, and its metabolites in human plasma. The method has been successfully applied to the analysis of 500 samples from pregnant women in a clinical study.

Photo Credit: Getty Images/Joey Boylan
The determination of methylxanthines in human plasma or urine is based on liquid chromatography (LC) coupled to spectrophotometric detection (UV) or mass spectrometry (MS). UV detection provides low selectivity since endogenous compounds present in biological fluids may interfere with the target analytes. Liquid chromatography coupled to single mass spectrometry (LC–MS) (1,2) or tandem mass spectrometry (LC–MS–MS) (3,4) have been recently applied for the analysis of methylxanthines in human biofluids.

LC–MS–MS provides better sensitivity and selectivity compared to LC–MS. Published LC–MS and LC–MS–MS methods are based on direct injection of the sample prior to LC column-switching (2) or centrifugation/filtration followed by dilution of the sample to reduce matrix impact on the chromatographic determination (3,4).

The most frequently used technique for the determination of cotinine in human fluids is gas chromatography coupled to mass spectrometry (GC–MS). Recently, LC–MS and LC–MS–MS have been successfully applied for the analysis of cotinine in biological matrices (5).

The analytical method described in this paper is based on solid-phase extraction (SPE) followed by LC–MS–MS analysis with electrospray ionization (ESI) and allows a simultaneous determination of cotinine and four methylxanthines in human plasma, which is useful to assess human exposure to tobacco smoke and coffee consumption. The validated method was successfully applied to the analysis of plasma from pregnant women in a clinic study.


The samples used for these analyses were taken from 500 pregnant women (recruited in the period 1997 to 2002). An ethylenediaminetetraacetic acid (EDTA) blood sample was taken from the women at their first antenatal visit at their general practitioners. The blood sample was forwarded on to the biological bank for the Danish National Birth Cohort (6). After recruitment at the biological bank, the sample was centrifuged and separated in plasma and buffy coat, which were cooled down with liquid nitrogen and stored at -30 °C. The Scientific Ethic Committee of Central Denmark approved the study (M-20080113).

Chemicals: Caffeine, theobromine, 1,7-dimethylxanthine, theophylline, and cotinine were purchased from Sigma Aldrich as neat compounds or solutions in methanol. Theobromine D6, 1,7-dimethylxanthine D6, and cotinine D3 were from Sigma Aldrich. Caffeine 13C3 and theophylline 13C2 15N2 were from Cambridge Isotope Laboratories. HPLC-grade methanol and acetic acid were from Merck. Pure water used in the LC mobile phase and in extraction was purified with Super Q apparatus (Millipore). Primary stock solutions of the analytes were prepared in methanol or methanol:pure water 50:50 (v/v) for theobromine. The internal standard stock solution was prepared in methanol and further diluted with water to obtain a working solution at a concentration of 100 ng/mL. Calibration standards were prepared by spiking 100 µL rabbit serum (Sigma Aldrich) at the following concentrations: 40 ng/mL, 100 ng/mL, 200 ng/mL, 400 ng/mL, and 800 ng/mL. The calibration standards followed the same extraction procedure as the samples and were extracted every second batch of samples.

Instrumentation: The LC–MS–MS system consisted of an Agilent 1200 series (Agilent Technologies) and a QTrap 5500 triple quadrupole mass spectrometer (AB Sciex) equipped with an electrospray ionization source (ESI). The analytes were separated on a 150 × 2.1 mm, 2.7-µm, Ascentis Express RP Amide column (Supelco). Analyst software 1.5.1 (AB Sciex) was used for system control and data processing.

The composition of the mobile phase was 83% A (1% acetic acid in water) and 17% B (methanol) and the flow rate was 0.2 mL/min run isocratically. The injection volume was 10 µL.

Table 1: MS–MS parameters and LC retention time for the target compounds. The most abundant parent ion is the first listed.
The ESI source was operated in positive mode at a temperature of 600 °C and an ion spray voltage of +4500 V. Nitrogen was used as the collision gas. The analyses were performed with a multiple reaction monitoring (MRM) method that monitored two mass transitions (parent ion and product ion) for each analyte, with the exception of theobromine for which only one transition ion was obtained. The values of the voltages applied to the sampling cone, focusing lenses, collision cell, and quadrupoles were optimized in MRM mode by direct infusion of a solution containing the analytes. The precursors and product ions for each analyte, together with the applied collision energy, are summarized in Table 1. Detection was based on retention time and the most abundant mass transition corresponding to an authentic standard. Confirmation of analyte identity was based on the relative response of the secondary mass transition to the primary mass transition.

Sample Preparation: Plasma samples (100 µL) were spiked with the labelled standards to a final concentration of 100 ng/mL and 1 mL pure water was added to the sample. Extraction was performed with Discovery DSC 18 solid phase columns, 500 mg/3 cc (Supelco) previously conditioned with 2 mL of methanol followed by 2 mL of pure water. The analytes were eluted with 2 mL methanol. The solvent was evaporated to dryness using nitrogen at 25 °C and reconstituted in 200 µL HPLC mobile phase.

Quantification and Quality Control: Quantification of the analytes was done using response factors calculated from a five-point calibration curve consisting of blank samples (rabbit serum) spiked with the analytes in the concentration range 20–800 ng/mL and extracted following the same procedure as for the samples. Peak area ratios of target analytes and the respective internal standards were calculated at each concentration. Blank samples consisting of rabbit serum spiked with the isotope-labelled standards were analyzed with each batch of samples. Random samples were extracted and analyzed in duplicate for each batch of samples.

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