5-Fluorouracil (5-FU) is a low-molecular-weight anticancer drug in clinical use for several solid tumors in humans. Currently,
the most widely used methodology for 5-FU quantitation is liquid chromatography–tandem mass spectrometry (LC–MS-MS) with either
liquid–liquid extraction (LLE), protein precipitation, or a combination of both as sample cleanup procedures. Here, we report
a new solid-phase extraction (SPE) method for 5-FU analysis by LC–MS-MS. This process is the first report utilizing strong
anion-exchange SPE cartridges to separate 5-FU from the matrix.
Figure 1: 5-FU deprotonation.
The cytostatic chemotherapeutic agent 5-fluorouracil (5-FU) has been widely used for 50 years for the treatment of several
solid tumors in humans including colorectal, breast, gastrointestinal, and head and neck cancers (1–6). It is still considered
to be one of the most active anticancer drugs for advanced colorectal cancer (7,8) and is a component of FOLFIRINOX, a drug
combination currently used for pancreatic cancer treatment (9). A large number of nonchromatographic and chromatographic methods
for the quantitation of 5-FU have been reported to support preclinical and clinical studies (10). Cell culture–based assays
for 5-FU were first developed in the 1960s (11,12) and were replaced by gas chromatography (GC) in the 1970s (13). Several
years later, high performance liquid chromatography (HPLC) (14,15) and gas chromatography–mass spectrometry (GC–MS) methods
(16–18) were developed and used in more extensive clinical studies. More recently, liquid chromatography–tandem mass spectrometry
(LC–MS-MS) methods (19–24) have been developed. These newer methods are highly specific, sensitive, and rapid and are expected
to replace HPLC–UV, HPLC–fluorescence, and GC–MS for 5-FU analysis. Most of the chromatographic methods developed use either
liquid–liquid extraction (LLE), protein precipitation, or a combination of both as sample clean-up procedures (10,19,21,22),
with only a few reported uses of solid-phase extraction (SPE) for sample preparation (15,25). Our goal was to develop a more
sensitive method to extract 5-FU from plasma matrix and to quantify the amount of 5-FU in novel, nano-sized therapeutic vehicles
that will be used in preclinical animal studies. To measure the smaller amounts of 5-FU in these vehicles, a more sensitive
technique for measuring 5-FU was needed. In this study, we report a new SPE sample cleanup procedure that takes advantage
of the anionic state of 5-FU under basic conditions, followed by LC–MS-MS analysis using a hydrophilic interaction chromatography
(HILIC) column. The resulting method (with lower limit of quantitation [LLOQ] of 0.1 ng/mL) is 10-fold more sensitive than
the most sensitive method reported previously (with LLOQ of 1 ng/mL) (24).
Figure 2: 5-FUH2 deprotonation.
The finalized experimental conditions for the SPE procedure and LC–MS-MS analysis are described below.
Figure 3: Chromatograms of 5-FUH2 before and after SPE. 5-FUH2 was not retained on the SPE cartridge — no peak was detected
in the SPE eluent.
Samples were extracted by adding 600 µL of 20% ammonium hydroxide aqueous solution to 200 µL of mouse plasma containing 10
µL of internal standard 5-chlorouracil (5-CU) at a concentration of 100.0 ng/mL. Samples were added to Oasis Max cartridges
(3 cc, 60 mg, Waters Corporation) after preconditioning the cartridges with 2 × 2 mL of methanol and 2 mL of water. Samples
were drawn through under vacuum and then washed with 3 mL of 5% ammonium hydroxide aqueous solution and 3 mL of methanol.
The samples were eluted with 1 mL of 1% formic acid in 60:40 methanol–water, evaporated on a Speed Vac Concentrator (Savant
SPD121P-115, Thermo Electron Corporation), and reconstituted in 100 µL of 15% ammonium hydroxide in acetonitrile. Then 10
µL of the sample solution was injected into the LC–MS-MS system. Calibration standards for 5-FU were prepared by spiking 5-FU
into 200 µL of blank mouse plasma at the concentrations of 0.1, 0.5, 1.0, 5.0, 10.0, 30.0, and 50.0 ng/mL.
Figure 4: Optimizing the percentage of ammonium hydroxide in plasma samples containing 10.0 ng/mL 5-FU and 50.0 ng/mL 5-CU
before SPE. Error bars represent standard deviations of two replicates.
The LC–MS-MS system consisted of an Agilent 1100 system coupled to a tandem quadrupole 4000 Qtrap system (AB Sciex). Chromatographic
separations were performed on a Phenomenex Luna HILIC column (3 µm, 150 mm × 2.00 mm, 200 Å), which is a silica-based particle
with an ethylene cross-linked diol phase. The column temperature was held at 40 °C. Mobile-phase A was 100 mM ammonium formate
in water, and mobile-phase B was acetonitrile. The gradient was as follows: 90% B for 2.0 min, 90–50% B at 4.0 min, 50% B
until 6.0 min, 50–90% B at 6.1 min, and 90% B until 11 min. The MS system was operated in negative electrospray ionization
mode with an ion spray voltage of 4500 V. Nitrogen was used as collision gas. The optimized source conditions were as follows
(arbitrary units if not specified): Gas 1, 70; Gas 2, 60; CUR, 40; CAD, 10; and TEM, 500. The optimized compound conditions
were as follows: DP, 60; EP, 10; CXP, 5; dwell time, 100 ms for all analytes; and CE, 28 V for 5-FU and 31 V for 5-CU. The
MRM transitions were m/z 129.0→42.0 for 5-FU and m/z 145.2→42.0 for 5-CU. Data were processed with Analyst software version 1.5.1 (AB Sciex).