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Clinical Chemistry 54: 1463-1472, 2008. First published July 17, 2008; 10.1373/clinchem.2007.102251
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(Clinical Chemistry. 2008;54:1463-1472.)
© 2008 American Association for Clinical Chemistry, Inc.

Automation and Analytical Techniques

HPLC with UV or Mass Spectrometric Detection for Quantifying Endogenous Uracil and Dihydrouracil in Human Plasma

Ruta Svobaite1,2,1, Isabelle Solassol3,1, Frederic Pinguet3, Liudas Ivanauskas2, Janine Brès1 and Françoise M. M. Bressolle1,3,a

1 Pharmacokinetic Laboratory, Faculty of Pharmacy, University Montpellier I, Montpellier, France; 2 Department of Analytical and Toxicological Chemistry, Kaunas University of Medicine, Kaunas, Lithuania; 3 Oncopharmacology Department, Pharmacy Service, Val d’Aurelle Anticancer Centre, Montpellier, France.

aAddress correspondence to this author at: Laboratoire de Pharmacocinétique Clinique, Faculté de Pharmacie, B.P. 14491, 34093 Montpellier Cedex 5, France. Fax (33) 4 67 54 80 75; e-mail Fbressolle{at}aol.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Background: We developed and compared 2 different methods for quantifying uracil (U) and dihydrouracil (UH2) in BSA and human plasma. Special attention was paid to the selectivity/specificity and the absence of a matrix effect. The UH2/U ratio is intended as a biomarker to identify patients with deficiency in 5-fluorouracil metabolism.

Methods: We quantified U and UH2 with 2 liquid chromatography methods after solid-phase extraction, one with UV detection (LC-UV) and the other with mass spectrometric detection (LC-MS). We selected 2 internal standards to prevent the risk of interferences. Separation was achieved with a Waters Atlantis dC18 column (LC-MS) or a Waters SymmetryShield RP18 column connected with an Atlantis dC18 (LC-UV). Mass spectrometric data were acquired in single-ion monitoring mode.

Results: Assay imprecision in BSA solution was <15% (LC-UV) and <12% (LC-MS); in plasma, assay imprecision was <9.5% and <9.0%, respectively. Recoveries were 88.2%–110% (LC-UV) and 94.8%–107% (LC-MS). Extraction efficiencies were ≥89.0%. In BSA, the lower limits of quantification for U and UH2 were 2.5 µg/L and 6.25 µg/L, respectively, for the LC-UV method and 2.5 µg/L and 3.1 µg/L for LC-MS. The corresponding values in plasma were 11.6 µg/L and 21.5 µg/L, and 4.1 µg/L and 12.1 µg/L.

Conclusions: To estimate endogenous U and UH2 concentrations and their ratio, we recommend the use of a drug-free human plasma pool in which baseline U and UH2 concentrations have previously been measured with the standard-addition method. Our LC-MS method, which has the better test performance and is useful for measuring UH2/U ratios in cancer patients, is preferred when this equipment is available.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
5-Fluorouracil (5-FU)4 is an antineoplasic agent that is widely used alone or in combination chemotherapy regimens for the treatment of advanced gastrointestinal cancer, breast cancer, and several other types of cancer (1). Furthermore, dose-intensification strategies have been justified at least in colorectal cancer and head and neck tumors, because the response to treatment has been correlated with 5-FU exposure (2)(3)(4)(5); however, interpatient variation in 5-FU catabolism has been a critical factor implicated in unexpected toxicities (6)(7). Dihydropyrimidine dehydrogenase (DPD), the rate-controlling enzyme in pyrimidine and fluoropyrimidine catabolism, accounts for 70%–85% of 5-FU clearance and therefore directly influences 5-FU entry into its corresponding anabolic pathways and its cytotoxic effects (8)(9)(10)(11)(12)(13)(14). DPD exhibits genetic polymorphism and shows broad variation among individuals. Partial deficiency and overexpression of the gene encoding DPD have frequently been found in patients, with complete DPD deficiency occurring at a frequency of approximately 3% (12)(13)(14). Thus, DPD polymorphism has been regarded as a potential index for predicting the 5-FU dose for patients with obvious DPD deficiency or overabundance, rather than the patient’s body weight or body surface area (15)(16)(17). A number of procedures exist for evaluating DPD activity, including its assessment in peripheral blood mononuclear cells [which has been correlated with 5-FU plasma systemic clearance (18)], analysis of plasma 5-FU concentrations (6)(9)(10)(11)(19), and genotyping for IVS 14 + 1G->A (DPD*2A), the most commonly observed polymorphism associated with toxicity (20). These measures are time-consuming, however, and not easy to implement on a large scale. Because 5-FU and uracil (U) are metabolized by the same pathway, quantification of the plasma concentrations of U and its dihydrogenated metabolite dihydrouracil (UH2) and the subsequent calculation of UH2/U ratios may be useful for identifying patients with deficient 5-FU catabolism and therefore at risk of toxicity (16)(21)(22)(23)(24)(25).

To date, U and/or UH2 have been measured in biological matrices by gas chromatography–mass spectrometry (21) and by HPLC with UV detection (LC-UV) (26)(27)(28)(29)(30)(31) or mass spectrometry detection (LC-MS) (32)(33)(34). Only 4 of these methods include simultaneous measurement of U and UH2 in plasma (26)(27)(30)(34), but these methods have some limitations. Three of them have used substitution matrices for the validation: 80 g/L BSA (26), 30 g/L BSA (34), or water(27). Substitution of these matrices for human plasma is an issue because of the risk of matrix effects, particularly in LC-MS-MS, and this problem was not carefully evaluated by the investigators in these studies, although according to Jiang et al. (34), the same slopes (calculated from 3 data points) were obtained in plasma and 30 g/L BSA. In the method described by Garg et al. (30), the biological matrix was modified by the use of doubly dialyzed pooled human plasma, whereas Ciccolini et al. (28) simply calculated the U/UH2 ratio from peak area ratios.

LC-MS analysis involves expensive equipment that is not available for therapeutic drug monitoring in all laboratories. Thus, the aim of the present study was to validate 2 different methods, LC-UV and LC-MS, for quantifying U and UH2. To validate the use of a substitution matrix to quantify U and UH2 in human plasma, we fully validated both methods in 80 g/L BSA and in human plasma. We adapted the LC-UV method from that described by Déporte et al. (26) and took care to ascertain the selectivity/specificity of the methods and the absence of matrix effects.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
chemicals and reagents
Uracil, dihydrouracil, internal standards (ISs) 5-chlorouracil (5-CU) and 5-fluorocytosine (5-FC), and BSA were purchased from Sigma–Aldrich. The IS 5-bromocytosine (5-BrC) and ammonium formate were obtained from Acros. The structures of the analytes are shown in Table 1 . Acetonitrile was obtained from Merck, and KH2PO4, orthophosphoric acid, formic acid, acetic acid, and methanol were obtained from Prolabo. All reagents were of HPLC grade or equivalent purity.


Figure 3
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  		Figure 3. Structures of analytes and variations in proportions of solvents A and B used in LC-MS quantification of U and UH2.

Purified water was generated by a Milli-Q reagent water system (Millipore Corporation). Atoll Xtrem Capacity XC 100/1 solid-phase extraction cartridges were purchased from Interchim.

To validate the BSA method, we used different batches. A BSA solution (80 g/L) was prepared daily in purified water. This solution was used to avoid the physiological concentrations of U and UH2 present in human blood. To validate the method in human plasma, we used 6 different batches of pooled drug-free plasma from healthy volunteers (Etablissement Français du Sang). These plasma pools were aliquoted and then frozen at –20 °C. All the human plasma lots contained endogenous U and UH2. Concentrations were measured with a standard-addition method (SAM) that used 7 concentrations and 6–11 replicates per batch.

The same batch was used during the study to prepare calibrators and QC samples. The other batches were used to study the selectivity/specificity of the methods and the matrix effect.

equipment
The LC-UV system included a Shimadzu model LC-10AT solvent-delivery module, a Shimadzu SIL-10AD automatic sample-injection system thermostated at 4 °C, and a Shimadzu model SPD-10AV variable-wavelength UV-visible detector set at 205 nm. Reversed-phase HPLC was performed on a Waters SymmetryShield RP18 column (5 µm, 250 x 4.6 mm) connected with a Waters Atlantis dC18 column (5 µm, 100 x 4.6 mm). The mobile phase was 10 mmol/L potassium phosphate buffer adjusted to pH 3.0 with phosphoric acid and was filtered through a 0.45-µm Magna filter (MSI) before use. Chromatography was performed at 20 °C and a flow rate of 0.6 mL/min. The injection volume was 50 µL.

The LC-MS equipment consisted of a quaternary pumping unit, an autosampler (4 °C) with a 100-µL sample loop, a 1100 quadrupole mass spectrometer with electrospray ionization, and a data-acquisition station to control the LC-MS system and record the output from the detector (all from Agilent Technologies). We optimized the various experimental variables, including the nature of the stationary phase, eluent composition, the nature of the organic modifier, capillary voltage, nebulizer pressure, and sampling cone voltage (data not shown). Analyte separation was performed at room temperature (20 °C) on the Atlantis dC18 column described above. A 12-min mobile-phase gradient was used. The mobile phases consisted of water containing 1 mL/L acetic acid (solvent A) and acetonitrile (solvent B). Table 1Up shows the changes in the proportions of solvents A and B during separation. The mobile phases were deaerated ultrasonically before use and with a stream of helium during use. The injection volume was 5 µL, and the flow rate was 0.6 mL/min. The quadrupole was calibrated in the positive ion mode with a mixture of NaI and CsI (full width at half mass, 0.6–0.7 atomic mass units). Voltages were set at +3.0 kV for the capillary and +0.5 kV for the skimmer lens. The values for the primary spectrometer variables were as follows: source temperature, 100 °C; quadrupole temperature, 99 °C; nebulizing gas (nitrogen), 0.25 MPa; drying gas (nitrogen), 720 L/h. Mass spectra were obtained during all experiments by scanning from m/z 100 to m/z 210. Mass spectrometric data were acquired in single-ion monitoring mode with a single-ion monitoring dwell time of 144 ms. U, UH2, 5-FC, and 5-BrC were characterized from the full-scan spectra by the protonated molecules [M+H]+ at m/z values of 113, 115, 130, and 190, respectively (see Fig. 1 in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol54/issue9 ). The sampling cone voltages were 100, 80, 180, and 140 V for ion masses 113, 115, 130, and 190, respectively.

drug solutions
Individual stock solutions (100 mg/L) of U, UH2, and the ISs were prepared in purified water and stored at –20 °C. These solutions were stable under these storage conditions for at least 1 month (data not shown). Working solutions (100–1000 µg/L) for use in calibration curves and QC samples were prepared daily from the stock solutions by dilution in purified water. Separate stock solutions were used to prepare calibrators and QC samples.

We prepared reference solutions containing U (2.5, 10, and 80 µg/L), UH2 (6.25, 25, and 200 µg/L), and the ISs (30 µg/L) daily in purified water to check the performance of the LC-UV or LC-MS system.

calibration curves and qc samples
The calibrators and QC samples were prepared in drug-free matrices for which the endogenous U and UH2 concentrations had previously been measured by the SAM (plasma matrix) by adding appropriate volumes of working solutions into 1 mL of BSA solution or plasma. For the LC-UV method, the spiked U and UH2 (U/UH2) concentration combinations were 2.5/6.25, 5/12.5, 10/25, 20/50, 40/100, and 80/200 µg/L in 80 g/L BSA and were 0/12.5, 10/25, 20/50, 40/100, 80/200, 120/300, and 160/400 µg/L in plasma. For the LC-MS method, these concentration combinations were 0/3.1, 2.5/6.25, 5/12.5, 10/25, 20/50, 40/100, and 80/200 µg/L in both BSA solution and plasma. QC samples were prepared independently as described above to yield concentrations of 7.5, 30, 60, and 140 µg/L for U and 6.25, 20, 60, 150, and 350 µg/L for UH2. In human plasma, the endogenous U and UH2 concentrations must be added to the supplemented concentrations listed above to obtain the final concentrations.

Calibration curves were generated by plotting the ratio of the peak area of each analyte to that of the IS vs the nominal concentration of each compound. Calibration curves were generated by means of the unweighted least-squares method. The parameters of the linear equations were used (a) to back-calculate concentrations that were statistically evaluated and (b) to obtain concentration values for that day’s QC samples and unknown samples. The lack-of-fit test was used to confirm the linearity of the method.

sample preparation
The method of sample extraction was based on solid-phase extraction with a Vac Elut 20® instrument (Varian). Two ISs were selected to minimize the risk of interferences. We mixed a 0.5-mL aliquot of BSA or plasma sample with 0.5 mL KH2PO4 solution (10 mmol/L, pH 2.0) and then added the IS working solutions (1 mg/L; 30 µL of 5-BrC and 5-CU for UV detection and 10 µL of 5-BrC and 5-FC for mass spectrometric detection). The sample was vortex-mixed for 10 s and centrifuged at 2500g for 5 min at 4 °C. The supernatant was then loaded onto an individual solid-phase extraction column previously conditioned with 1 mL methanol and 1 mL purified water. The loaded sample was washed with 1 mL of 10 mmol/L ammonium formate (pH 5.0). The analytes were eluted with 0.5 mL methanol. The eluate was evaporated to dryness under a nitrogen stream for 20 min at 45 °C. The dried extract was redissolved in 200 µL of mobile phase (LC-UV) or 200 µL of water containing 1 mL/L acetic acid (LC-MS) and transferred to an injection vial.

validation
We validated the methods in compliance with US Food and Drug Administration guidelines (35)(36)(37). To evaluate the performances of the LC-UV and LC-MS methods, we compared spiked and calculated QC concentrations by calculating imprecision values and the mean differences between spiked and calculated concentrations.

specificity/selectivity
We evaluated the specificity/selectivity of the BSA method by analyzing 6 different BSA batches. The retention times of the endogenous compounds in the matrix were compared with those of the compounds of interest.

To ascertain the specificity/selectivity of the plasma method, we analyzed 6 different batches of pooled human plasma without the ISs. The degree of interference was assessed via diode-array and mass spectrometric detection to identify peaks present on the chromatograms and to verify that each observed peak eluted free of any potential interference.

To check for possible interference by drugs commonly used in combination with 5-FU, we analyzed plasma samples from patients who were undergoing such treatment for the following drugs: 5-FU, doxorubicin, cyclophosphamide, L-folinic acid, dexamethasone, granisetron, cis-dichlorodiaminoplatinum, carboplatin. Possible interference by other hydrophilic compounds, such as other pyrimidines, was also checked.

matrix effect
Undetected matrix components coeluting with analytes may adversely affect the reproducibility of analyte ionization in the electrospray source of the mass spectrometer (38). To verify the absence of ion suppression or ion-enhancement effects attributable to the matrix, we performed the following experiments. Six different batches of each of the 2 matrices (80 g/L BSA and human plasma) were extracted by the method described above in duplicate (n = 12 per matrix and per studied concentration). The final dried extracts were reconstituted with 200 µL of acidified water containing U/UH2 concentration combinations equivalent to calibrators of 7.5/20, 20/60, and 60/150 µg/L and the ISs at concentrations of 20 µg/L. At the same time, we prepared reference solutions containing U, UH2, and the ISs at the same nominal concentrations in the acidic medium (for the plasma matrix, the reference solutions take into account the endogenous U and UH2 concentrations). We injected the 36 supernatants and the 3 preparations from the reference solutions onto the analytical column and compared the peak areas for U, UH2, and the ISs for the extracted samples with the means of those produced by the references (each having been injected 5-fold in the LC-MS system). We then calculated the ratios of the mean peak areas in the matrix to those in the references. These ratios must be near 1; an interval of 0.85–1.15 was considered acceptable.

imprecision and recovery
Imprecision and recovery of the method were assessed by analyzing QC samples at the concentrations mentioned above along with a calibration curve. To evaluate within-day imprecision and recovery, we extracted 6 samples of each QC concentration and injected them on the same day. To evaluate between-day imprecision and recovery, we analyzed 1 sample of each QC concentration per day on different days (n = 6–11). Percent recovery was evaluated as: [(mean found concentration)/(nominal concentration)] x 100. Imprecision was expressed as the CV.

extraction recovery and the lower limit of quantification
We measured extraction efficiencies 6 times in BSA and 11 times in plasma at the following U/UH2 concentration combinations: 2.5/6.25, 10/25, and 80/200 µg/L. We evaluated recoveries by comparing peak areas obtained for extracted samples with those obtained by direct injection of purified water solutions containing each of the analytes at the same concentrations (for the plasma matrix, the reference solutions take into account the baseline U and UH2 concentrations). Extraction recoveries were also evaluated for the ISs.

The lower limit of quantification (LLOQ) was defined as the lowest concentration that could be measured on a daily basis within 80%–120% of the target value and with an imprecision of ≤20%. We assessed recovery and imprecision at this concentration by performing replicate analyses of the lowest calibrator and comparing the results with a calibration curve.

uh2/u ratios in cancer patients
We used the LC-MS method we developed to analyze endogenous U and UH2 concentrations in 56 cancer patients. We collected blood into lithium heparin tubes at the same time of day (between 0900 and 1000) to minimize the influence of circadian variation in DPD activity (34) and immediately centrifuged the samples at 2000g and 4 °C for 10 min to isolate plasma. Plasma samples were stored at –20 °C until analysis (i.e., after 2–10 days of storage). U and UH2 concentrations were calculated from the calibration curves produced for the human plasma batch used in the validation after correction for endogenous U and UH2 concentrations.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
The endogenous concentrations of U and UH2 obtained by the SAM for the human plasma batch used to prepare calibrators and QC samples were 1.55 µg/L and 8.99 µg/L, respectively (n = 11; CV, 5.8% and 8.0%).

calibration curves and lloq

lc-uv
In a first step to quantify U and UH2 in human plasma, we tried to use the LC-UV methods described in the literature. Unfortunately, these methods were difficult to reproduce and demonstrated several interferences when applied to plasma samples. The best results were obtained with the method described by Déporte et al. (26), but an endogenous peak was observed at the retention time (9 min) of the IS used by these authors (5-FC), both in BSA and plasma. Thus, we modified this method slightly and used 2 ISs (5-BrC and 5-CU) to prevent potential interferences by endogenous plasma components or coadministered compounds/metabolites. This method gave good results when calibrators in BSA were used, but in plasma the method did not permit reliable quantification at low concentrations of U (3–11.6 µg/L) or UH2 (<21.5 µg/L) (25)(26). Moreover, an interfering peak from the human matrix occurred at the retention time of 5-BrC.

The peak area ratios (i.e., analytes/ISs) varied linearly with concentration over the following ranges (spiked concentrations): 2.5–80 µg/L for U and 6.25–200 µg/L for UH2 in BSA; 10–160 µg/L for U and 12.5–400 µg/L for UH2 in plasma. Mean values for linear regression parameters are presented in Table 2 . CVs and recoveries around the mean back-calculated concentrations were 0.6%–15% and 90%–114%, respectively, in BSA solutions and were 1.9%–12% and 94%–113%, respectively, in plasma (see Table 1 in the online Data Supplement). Typical chromatograms obtained from extracts of drug-free BSA and plasma spiked at the following U/UH2 concentration combinations: 2.5/6.25 µg/L in BSA and 10/25 µg/L in plasma, are shown in Fig. 1 , A and B. The observed retention times (n = 36) were 13.4/11.6 min for the U/UH2 combination, 19.9 min for 5-BrC, and 27.9 min for 5-CU (CVs, 0.28%–1.2%). The LLOQs for the U/UH2 combination were 2.5/6.25 µg/L in BSA and 11.6/21.5 µg/L in plasma (taking into account the endogenous concentrations). At these concentrations, imprecision was <17%, and the deviation from the target was 85%–117%.


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  		Table 2. Mean values of linear regression parameters.1


Figure 1
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  		Figure 1. Typical chromatograms of drug-free BSA solution and human plasma spiked with the following U/UH2 concentration combinations: 2.5/6.25 µg/L (BSA, LC-UV) (A); 10/25 µg/L (human plasma, LC-UV) (B); 2.5/6.25 µg/L (BSA, LC-MS) (C); and 2.5/6.25 µg/L (human plasma, LC-MS) (D).

Peak 1, UH2; peak 2, U; peak 3, 5-BrC; peak 4, 5-CU; peak 5, 5-FC (total ionic current). See instrumentation section for LC-UV and LC-MS conditions. mAu, milliabsorbance unit.

Thus, our LC-UV assay adapted from Déporte et al. (26) was complex and did not allow the quantification of endogenous U and UH2 in plasma at concentrations <11.6 µg/L and <21.5 µg/L, respectively.

lc-ms
The difficulties encountered with the LC-UV method prompted us to develop and validate an LC-MS method in both BSA and plasma. We also used 2 ISs (5-BrC and 5-FC). In the 2 matrices, the calculated peak area ratios and the added concentrations displayed linear relationships over the concentration ranges (spiked concentrations) of 2.5–80 µg/L for U and 3.1–200 µg/L for UH2. The mean values for the linear regression parameters are presented in Table 2Up . The CVs and recoveries around the mean back-calculated concentrations of the U/UH2 combination were 0.9%–15% and 95.5%–107%, respectively, in the BSA solution and 1.0%–9.1% and 92.1%–102% in plasma (see Table 2 in the online Data Supplement). Fig. 1Up , C and D, shows typical chromatograms obtained for extracts of drug-free BSA and plasma spiked at a U/UH2 concentration combination of 2.5/6.5 µg/L. The observed retention times (n = 77) were 4.3/3.8 min for the U/UH2 combination, 7.1 min for 5-BrC, and 3.5 min for 5-FC (CVs, 0.14%–0.70%). LLOQ values for the U/UH2 combination were 2.5/3.1 µg/L in BSA and 4.1/12.1 µg/L in plasma (taking into account endogenous plasma concentrations) (imprecision, <15%; deviation from target, 90%–110%).

An analysis of the influence of the matrix (BSA or plasma) on the linear regression parameters showed statistically significant differences (P values of <0.05 to <0.0001). Thus, BSA should not be used as a matrix in estimating endogenous plasma concentrations of U and UH2.

selectivity/specificity
In BSA, each analyte was well resolved from the endogenous peaks of the matrix (see Fig. 2, A and B, in the online Data Supplement). The human plasma lots tested (n = 6) contained endogenous U and UH2 (see Fig. 2, C and D, in the online Data Supplement). No interfering peaks were found at the retention time of either U or UH2. No interference was found with any of the tested drugs.

matrix effect (lc-ms)
Peak area ratios (i.e., reconstituted extracts/reference solutions) ranged from 0.85–1.11 in BSA and from 0.85–1.07 in plasma. These findings confirmed the absence of matrix effects.

imprecision, recovery, and extraction efficiency
The imprecision of the quantification of endogenous U and UH2 by the SAM was 2%–9%. Imprecision and recovery values for the BSA and plasma methods are presented in Tables 3 and 4 , respectively.


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  		Table 3. Within-day and between-day imprecision and accuracy of the method in 80 g/L BSA.1


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  		Table 4. Between-day imprecision and accuracy of the method in human plasma.1

The mean differences and imprecision values calculated from comparing spiked and calculated QC concentrations indicate that the LC-MS method is more accurate and precise than the LC-UV method and is the preferred method when LC-MS equipment is available (see Table 3 in the online Data Supplement). The LC-UV method showed a small shift in values when 5-BrC was used as the IS.

The mean extraction recoveries of U, UH2, and ISs from BSA and plasma matrices were ≥89% at the concentrations tested (BSA: U, 90%; UH2, 95%; 5-FC, 93%; 5-BrC, 92%; 5-CU, 95%. Plasma: U, 94%; UH2, 97%; 5-FC, 90%; 5-BrC, 98%; 5-CU, 89%. CV range, 1.2%–8.5%).

uh2/u ratios in cancer patients
We used the LC-MS method to measure the UH2/U ratio in 56 cancer patients. As reported by other authors (16)(25)(26), we observed large interpatient variation in this ratio. Of the 56 patients enrolled into the study, 12 patients (21.4%) had UH2/U ratios <2 (2 patients had ratios <0.5). In contrast to the results of previous studies, the ratio seems to exhibit a bimodal distribution (Fig. 2 ) with a mean UH2/U ratio of 8.6, which is similar to ratios previously reported (25)(26). Overly high DPD activity was suspected in 2 patients with the highest UH2/U ratios. A total absence of UH2 was not found in any of these patients, but metabolic deficiency was suspected in 1 patient.


Figure 2
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  		Figure 2. Bimodal distribution of UH2/U ratios in a population of 56 cancer patients.

Thus, the UH2/U ratio may be regarded as a potential diagnostically sensitive biomarker of deficiency in 5-FU metabolism, and measuring this ratio before 5-FU–based therapy may be useful to identify patients at risk of toxicity. Prospective studies are necessary, however, to demonstrate the clinical usefulness of this biomarker.

Only the analytical performance with human plasma is relevant for any clinical application of chromatographic methods for U and UH2. Thus, to estimate endogenous U and UH2 concentrations, we recommend the use of a drug-free plasma pool (for which baseline U and UH2 concentrations have previously been measured with a SAM) to prepare calibration curves and QC samples.


   Acknowledgments
 
Grant/Funding Support: This work was supported by the Ligue Nationale de Lutte contre le Cancer, Montpellier, France.

Financial Disclosures: None declared.

Acknowledgments: The authors thank the Embassy of France in Lithuania for the grant awarded to Ruta Svobaite. The authors express their gratitude to Professor I. Miseviciene (vice-rector of the University of Medicine of Kaunas) for facilitating Ruta Svobaite’s PhD studies in France.


   Footnotes
 
1 Ruta Svobaite and Isabelle Solassol contributed equally to this work.

4 Nonstandard abbreviations: 5-FU, 5-fluorouracil; DPD, dihydropyrimidine dehydrogenase; U, uracil; UH2, dihydrouracil; IS, internal standard; 5-CU, 5-chlorouracil; 5-FC, 5-fluorocytosine; 5-BrC, 5-bromocytosine; LLOQ, lower limit of quantification; SAM, standard-addition method.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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