REVIEW
A review of analytical methods for the determination of 5-fluorouracil in biological matrices
Massimo Breda &Simona Barattè
Received:25May 2009/Revid:24February 2010/Accepted:3March 2010/Published online:11April 2010#Springer-Verlag 2010
Abstract 5-Fluorouracil (5-FU)is a cytostatic agent that has been widely ud in the treatment of various solid tumours for more than 20years,and is still considered to be among the most active antineoplastic agents in advanced colorectal cancer and malignancies of the head and neck.A large number of non-chromatographic and chromatographic methods for the quantitation of 5-FU,related prodrugs and their metabolites in biological matrices have been developed in the last 30years to support preclinical and clinical studies.However,5-FU monitoring has not been widely ud,at least not in the USA,and certainly not outside the clinical rearch tting,given the abnce of simple,fast and inexpensive testing methods for 5-FU monitoring.Recent developments with testing bad on liquid chromatography –tandem mass spectrometry and a nanoparticle antibody-bad immunoassay may facilitate routine monitoring of 5-FU in daily clinical practice.In this review the advantages and disadvan
tages of the bioanalytical methods developed and ud for 5-FU,its metabolites and related prodrugs are discusd.Keywords 5-Fluorouracil .Analytical methods .Biological matrices
Introduction
5-Fluorouracil (5-FU;Fig.1)is an analogue of uracil which differs from uracil by virtue of a fluorine atom in place of the hydrogen at the carbon-5position of the pyrimidine ring.For
the past 20years 5-FU has been uful as a cytostatic agent in the treatment of solid tumours,including breast cancer,gastrointestinal adenocarcinoma and squamous cell carcinomas of the head and neck [1–4].Recently 5-FU in association with irinotecan and radiotherapy has been ud for the treatment of rectal cancer [5].Metabolism
Toxicity remains an important parameter to be considered when 5-FU is administered,as 5-FU often caus vere gastrointestinal toxicity and myelosuppression [6].For this reason toxicity has been investigated in association not only with the pharmacokinetics of the parent compound but also with the drug ’s metabolism.The mechanism of action of 5-FU is bad on anabolic conversion into cytotoxic nucleotides,using veral pyrimidine metabolic pathway enzymes.As illustrated in Fig.2,the nucleotides can be formed by conversion and quential conversion of 5-FU to 5-fluorou
ridine 5′-monophosphate and by quential conver-sion of 5-FU to 5-fluoro-2′-deoxyuridine 5′-monophosphate (5-FdUMP)[7,8].The antitumour activity results from inhibition of thymidylate syntha,an enzyme required for de novo pyrimidine synthesis,by 5-FdUMP,as well as from incorporation of 5-FU active metabolites into RNA and DNA.Only a small part of the 5-FU do is activated via the routes,as in humans 80–90%of the administered do is degraded mainly in the liver to 5,6-dihydro-5-fluorouracil (5-FUH 2)by dihydropyrimidine dehydrogena [9],the rate-limiting step in 5-FU catabolism.5-FUH 2is further cleaved by dihydropyrimidina to form α-fluoro-β-ureidopropionic acid.α-Fluoro-β-ureidopropionic acid appears as a transient intracellular catabolite and is then converted to α-fluoro-β-alanine (FBAL),carbon dioxide and ammonia by β-ureidopropionate.In addition to the relea of small amounts
M.Breda (*):S.Barattè
l.,Viale Pasteur 10,20014Nerviano,Italy
e-mail:massimo.breda@accelera
Anal Bioanal Chem (2010)397:1191–1201DOI 10.1007/s00216-010-3633-8
of free fluoride anion from FBAL,further metabolites of FBAL have been found,including N -carboxy-α-fluoro-β-alanine,three conjugates of FBAL with bile acids and two metabolites of FBAL produced by transamination (2-fluoro-3-hydroxypropanoic acid and fluoroacetate)(Fig.3).
Many authors have propod,although with controversial results,asssing the toxicity by the expression of the main catabolic enzyme,dihydropyrimidine dehydrogena [10,11],or by evaluating the plasma levels of the metabolites 5-fluorouridine (5-FUrd),5-fluoro-2′-deoxyuridine (5-FdUrd)and 5-FUH 2.
To overcome the toxicity,many derivatives and related compounds have been synthesized,such as tegafur (Fig.1),which acts as a prodrug of 5-FU with a broad spectrum of antitumour activity [12]and comparatively reduced myelo-depression [13].
Chemical properties,sample handling and storage stability 5-FU (molecular weight 130.08)is a white to nearly white crystalline powder with a solubility that decreas with decreasing polarity of the solvent.In aqueous solution,5-FU exists as veral ionic species with p K a1=8and p K a2=13.Solutions of 5-FU are stable if the pH is below 9.For instance,5-FU is stable in 1N HCl at 100°C [14]but in alkaline solution it is degraded to urea,fluoride and an
N H NH O O
F
N NH O O
F
O 5-FU
Tegafur Fig.1Chemical structure of 5-fluorouracil (5-FU )and tegafur
Fig.2Pathways for the anabolic metabolism of 5-FU.PRPP phosphoribosylpyrophosphate
1192
M.Breda,S.Barattè
aldehyde derivative[15].5-FU solution should be stored in the abnce of light since UV irradiation results in changes in the UV spectrum[16,17].5-FU shows significant absorption on glass from methanol solution,although there is no indication that this also occurs with aqueous solutions[18]. However,as a precaution,it is recommended that only plastic or silanized glassware be ud in analytical work.
5-FU was found to be stable in aqueous stock solutions for6h at room temperature,for22months at-80°C[19] and for3months when stored at4°C[20]or5°C[21]. Aqueous stock solutions of5-FU,5-FUH2,5-FUrd and5-FdUrd were stable for at least5–6months if they were refrigerated in the dark[22].Tegafur was found to be stable in methanol stock solution for3months at5°C[21].5-FU has been shown to be unstable in whole blood,primarily due to dihydropyrimidine dehydrogena activity,and parating cells from plasma is critical for obtaining accurate results[23].At room temperature there was a steady loss of94%of5-FU over24h in whole blood.5-FU was found to be mo
re stabler in whole blood and plasma samples kept on ice;over24h of storage in the conditions, the loss was only30and10%,respectively[23].Recently, Beumer et al.[24]reported that blood should be placed on ice immediately and the plasma parated from the blood cells within1h after collection from patients.Care should be taken to ensure that the plasma is free of blood cells and that there is no haemolysis.Contamination of the plasma with blood cells can cau degradation of5-FU at room temperature.Degradation is minimized when the plasma is stored at4°C or below.If the plasma is free of blood cells or initially frozen,samples can be stable at room temperature for veral days without degradation of5-FU[24].Stability in human plasma has been demonstrated for up to6h at20and 4°C for5-FU and its metabolites5-FUrd and FdUrd[20]. The majority(90%)of the compound was still prent in rat rum and different kinds of rat tissues spiked with5-FU after48h at room temperature,and the test compound was stable for up to2–3weeks at4°C[25].In contrast,the
H
CH F C O
Fig.3Pathways for the
catabolic metabolism of5-FU
A review of analytical methods for the determination of5-fluorouracil in biological matrices1193
concentrations of 5-FUrd and 5-FdUrd decread slowly after 1h at 20°C.In human plasma,5-FU was found to be stable for at least five freeze –thaw cycles [26].At least three freeze –thaw cycles can be performed without a loss of more than 10%of 5-FUrd and 5-FdUrd [20],whereas 5-FUH 2was found to be stable after two freeze –thaw cycles [22,26].If reanalysis of plasma samples is required,only samples that have been thawed once should be ud.The obrved freeze –thaw instability for 5-FUH 2also has implications for the conditions during transport,and for this reason it is important to transport plasma samples on dry ice to prevent thawing.Degradation was obrved with the metabolites 5-FUH 2,5-FUrd and 5-FdUrd when human plasma samples were stored at room temperature and in particular at 37°C [22];however,the storage time was not reported for tho conditions.Owing to 5-FUH 2instability at room temperature,as shown in a previous paper [27],caution should be taken during the storage of final extract samples in the autosampler [26],so cooling of the instrument compartment is recom-mended.5-FU was found to be stable in human plas
ma for up to 12months [28]and in human urine for up to 3months [21]when stored at -80°C.5-FU was stable in human plasma for 3–4weeks when stored at -28°C [22]and for up to 61days when stored at -20°C [29].When 5-FU,5-FUrd and 5-FdUrd in human plasma samples were stored at -30°C,the stability was demonstrated for up to 2months [20].In rat rum and tissues,stability of the analytes for up to 3months at -20°C was found [25].A summary of some stability data for 5-FU is given in Table 1.
Recently,Alsarra et al.[30]reported that,owing to the photolability of 5-FU,special caution should be taken to protect samples from photooxidation.For this reason all manipulations of the samples should be performed in a dark room.Wrapping the samples in aluminium foil was found to improve the stability significantly.
Several techniques have been developed in the last 25years for the quantitation of 5-FU,related prodrugs and
their metabolites in biological matrices.In this review the advantages and disadvantages of non-chromatographic and chromatographic methods for the determination of 5-FU and related compounds in biological matrices are discusd.
Non-chromatographic assay procedures
The earlier methods for 5-FU assay in biological material were microbiological,using Enterococcus faecalis [31,32]or Escherichia coli [33].A spectrophotometric method has already been described [34],bad on the formation of an ion pair complex of the monoion with alkylammonium ion extraction into an organic solvent,the addition of perchlorate and re-extraction of liberated 5-FU into the aqueous pha (range 0–100µg/mL plasma).A method has been described for the analysis of 5-FU in saliva using surface-enhanced Raman spectroscopy [35].A personalized chemotherapy management immunoassay for the determination of 5-FU in human plasma has recently been developed [24].Novel,highly lective monoclonal antibodies for 5-FU were developed and then covalently attached to 200-nm nanoparticles,which rve as a label in a homogeneous competitive immunoassay format.The specificity of the antibodies for 5-FU was confirmed when it was shown that cross-reactivity was less than 1%for 5-FUH 2,one of the main catabolites for 5-FU,0.05%for capecitabine and 0.23%for tegafur.For structurally related compounds the cross-reactivity in the assay for the compounds was generally below 0.1%,with the exception of uracil,thymine,and eniluracil,which resulted in respons of 9.9,2.0and 0.6%,respectively.Coadministered drugs and unrelated medications tested had a cross-reactivity of 1%in the assay.Only theophylline showed a cr
oss-reactivity of 1.5%.This immunoassay requires only a small amount of plasma (less than 10μL);takes only about 11min to perform and,when an Olympus AU400ImmunoAnalyzer is ud,up to 400patient samples can be analyd per hour.The lower
Storage temperature
Storage time References Aqueous stock solution Room temperature 6h
[19]Aqueous stock solution –80°C 22months [19]Aqueous stock solution +4/+5°C
3months [20,21]Aqueous stock solution Refrigerated in the dark 5–6months
[22]Human plasma –80°C
5freeze/thaw cycles [26]Human plasma Room temperature 6h [20]Human plasma +4°C 6h
[20]Human plasma –20°C 2months [29]Human plasma –28°C 3–4weeks [22]Human plasma –30°C 2months [20]Human plasma –80°C 12months [28]Human urine –80°C
3months
[21]
Table 1Stability of 5-fluorouracil (5-FU)in solutions and in human biological samples
1194M.Breda,S.Barattè
limit of quantitation(LLOQ)was determined to be86ng/mL. This was the concentration where the coefficient of variation was not greater than10%for samples to which known amounts of5-FU had been added.Beumer et al.[36] measured5-FU drug levels in patients with colorectal cancer and head and neck cancer who were being treated with5-FU-bad regimens and found that the immunoassay results were consistent with tho generated by other validated methods, including high-performance liquid chromatography(HPLC) and HPLC–tandem mass spectrometry(MS/MS).The avail-ability of this new immunoassay method allows important advantages to be obtained in terms of speed,small samples size,minimal sample handling and rapid and automated u of instrumentation.
Chromatographic methods
In this ction various chromatographic methods for the analysis of5-FU and its metabolites in biolog
ical matrices are described,including methods using HPLC,gas chromatogra-phy(GC),GC–mass spectrometry(MS)and HPLC-MS. Table2shows a comparison of more recent HPLC-UV methods.
Sample preparation
5-FU and its metabolites have a high aqueous solubility that makes them difficult to extract into organic solvents.In the literature,liquid–liquid extraction(LLE)has been described, using different types of solvents,such as2-propanol/diethyl ether[37–39],1-propanol/diethyl ether[37,38,40,41],2-propanol/ethyl acetate[42,43],ethyl acetate[19,21,26,28, 29,44],ethyl acetate/2-propanol/dichloromethane[45]ethyl acetate/methanol[46,47]or petroleum/propanol[48].Since the p K a of the acidic compound5-FU is approximately8,an extraction pH of approximately6has to be ud to ensure that it would exist in the unionized form[49].Plasma acidification has already been applied in veral methods [40,42].Escoriaza et al.[49]reported the acidification by adding5%orthophosphoric acid and considered this step very important,taking into account that whereas the plasma obtained from healthy subjects has a mean value of6.5–7, plasma from cancer patients often has pH values higher than 8.Maring et al.[26]reported that the pH of the aqueous layer can vary freely between2and8without affecting the extraction efficiency;therefore,pH stabilization with an extraction buffer was considered unnecessary.Moreover, the extraction recovery depends on
veral factors,such as the type of solvent and the ratio of the volumes of the aqueous and organic solvents.Gamelin et al.[50]tested different solutions and volumes:chloroform,16%2-propanol in diethyl ether,16%2-propanol in ethyl acetate,20%diethyl ether in ethyl acetate.Sixteen percent2-propanol in ethyl acetate gave the best extraction percentage.Chloroform and propanol/diethyl ether gave poor recoveries,20and40%, respectively.The minimum volume of solvent needed to achieve a5-FU recovery of more than70%starting from 0.5mL of plasma was1.2mL[50].Over1.4mL,the recovery gain was negligible.V ortex-mixing was also an esntial step in the extraction[50];however,over2min of vortex-mixing the recovery gain was insignificant[49].A summary of some LLE procedures and the percentage recovery obtained for5-FU are reported in Table3.
To increa the recovery and reduce the detectable amounts of interfering substances,different strategies were adopted.A quential cationic and anionic procedure[51]or an anion-exchange column before the extraction procedure[52]was ud.Different authors have reported a double or back extraction[14,38,40,43,53]or a clean-up procedure by Sep-Pak after the extraction[37].
LLE,using0.03mL of sodium acetate buffer(1M,pH 5.3),0.3mL of a1.4M solution of anhydrous sodium sulphate and5.4mL of a mixture containing1-propanol and diethyl ether(16:84,v/v),was ud to extract5-FU from0.6mL of human plasma,red blood cells or whole blood[40]. The u of a smaller vol
ume of both the organic solvent for extraction and the phosphate buffer(pH11)for back extraction as well as a change in the sodium acetate buffer from pH4.8to5.3improved the nsitivity(10ng/mL)in comparison with that for the original method reported by Christophidis et al.[38].
Other papers described protein precipitation using tri-chloroacetic acid[54,55],ethanol[41],perchloric acid[25] or acetonitrile[56–58]to provide faster sample pretreatment. As reported by Torano et al.[55],trichloroacetic acid resulted in superior recoveries as compared with deproteini-zation methods with methanol or acetonitrile and using salts such as zinc sulphate and aluminium sulphate.To improve the recovery and to obtain cleaner chromatograms,a combination of protein precipitation and LLE was applied. The procedures ud acetonitrile[26],ammonium sulphate [10,22,59,60]or sodium sulphate[40]as the precipitating agent before LLE of5-FU(Table3)and its metabolites in human plasma.As already reported[50],the protein precipitation with ammonium sulphate did not affect the recovery in plasma samples,and the quality of the chromato-grams was improved.The protein precipitation usually performed with trifluoroacetic acid or trichloroacetic acid lowered the recovery becau5-FU coprecipitated with the chemicals.Ice-cold ethanol has no effect on the recovery. Casale et al.[22]found using protein precipitation coupled with LLE that the recovery was acceptable(about60%)for5-FU,5-FdUrd and5-FUH2,but poor(about30%)for5-FUrd. The volume of ex
tractant and vortex-mixing were possibly important in the extraction becau5-FU and its metabolites
A review of analytical methods for the determination of5-fluorouracil in biological matrices1195
T a b l e 2C o m p a r i s o n o f s o m e h i g h -p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y (H P L C )–U V m e t h o d s f o r t h e d e t e r m i n a t i o n o f 5-F U ,i t s m e t a b o l i t e s a n d t e g a f u r i n p l a s m a s a m p l e s
A n a l y t e C o l u m n M o b i l e p h a s e
l (n m )S a m p l e p r e p a r a t i o n T y p e
V o l u m e o f p l a s m a (m L )L L O Q (n g /m L )R u n t i m e (m i n )R e f e r e n c e
5-F U A t l a n t i s c 18
1.5m M K 3P O 4p H 5.8/C H 3O H (99:1,v /v )266
P P +L L E A c e t o n i t r i l e +E t A c 0.24024[26]
5-F U G e n e s i s c 18
C H 3O H /H 2O (10:90,v /v )p H 3.2H C l O 4
梦见很多狗260
L L E E t A c 0.53012.5[30]
5-F U L i c h r o s p h e r 100R P -18
K H 2P O 4p H 3/C H 3O H g r a d i e n t
266
L L E E t A c /2-p r o p a n o l :/C H 2C l 20.512.528
[45]
T e g a f u r 5005-F U S p h e r i r o r b O D S 2
10m M a c e t i c a c i d /C H 3O H (90:10,v /v )
260
P P +L L E (N H 4)2S O 4+E t A c /2-p r o p a n o l 85:15(v /v )0.5425
[59]
负利率
T e g a f u r
4805-F U μB o n d a p a c k c 1850m M K H 2P O 4
260
L L E +S P E E t A c +S A X c o l u m n s 0.53
11
[44]
人多嘴杂5-F U S p h e r i r o r b O D S 1
1.5m M K 3P O 4/C H 3C N /H 3
P O 4p H 5
210P P +L L E (N H 4)2S O 4+2-p r o p a n o l /d i e t h y l e t h e r (80:20,v /v )
0.550023
[22]
5-F U H 2
1,000
5-F U r d
3,000
5-F d U r d
3,000
5-F U S y m m e t r y C 8
20m M K H 2P O 4p H 7w i t h 10%K O H 266
P P 10%T C A
0.5
50
12买卖二手
[55]
5-F U I n e r t i s i l O D S 3
H 2O p H 2w i t h H C l O 4
266
L L E E t A c
0.1
26
20
[28]
5-F U L i c h r o s p h e r 100R P -18
50m M K H 2P O 4p H 3
266A c i d i f i e d p l a s m a w i t h 5%H 3P O 4+L L E 2-P r o p a n o l /d i e t h y l e t h e r (16:84,v /v )
0.5
2
15
[49]
5-F U μB o n d a p a c k c 181%C H 3C N i n 20m M a c e t i c a c i d 275
L L E
E t A c
1
25
33
[78]美女美女我爱你
U r a c i l
5-F U O D S H y p e r s i l
C H 3O H /H 2O /a c e t i c a c i d (3:96.95:0.05,v /v /v )
254
P P
10%T C A
0.1
10015
[25]
5-F U r d
100
5-F d U r d
100
5-F U S p h e r i r o r b O D S 210m M K H 2P O 4p H 3
260
P P +L L E (N H 4)2S O 4+E t A c /2-p r o p a n o l (85:15,v /v )0.5
12.5
17
[50]
5-F U K r o m a s i l c 18C H 3O H /H 2O (3:97)
268S P E C h e m E l u t C 18
0.52512[20]
5-F U r d
25
5-F d U r d
25
5-F U O D S H y p e r s i l 50m M K H 2P O 4p H 3
270
L L E 2-P r o p a n o l /d i e t h y l e t h e r (16:84,v /v )0.6
25
12
[40]
5-F U S p h e r i r o r b O D S 11.5m M K 3P O 4p H 8
somogyi现象268
P P +L L E
50%T C A +E t A c
1
2610
[76]
5-F U H 2
26
5-F U
Y M C O D S -A Q c 1810m M K 3P O 4p H 5.5/C H 3O H g r a d i e n t 266
P P +L L E
N a 2S O 4+E t A c
1
25
20
[29]
5-F U
A m i n e x H P X -87H 0.005M s u l f u r i c a c i d
265
女生适合的专业P P
10%T C A
0.15
25
25
[54]
5F U H a m i l t o n P R P -15m M t e t r a b u t y l a m m o n i u m h y d r o x i d e (p H 11.1)/C H 3C N (84:16,v /v )
254P P
I c e -c o l d a c e t o n i t r i l e
0.2
10018
[72]
5-F d U r d
313
250
普通话考试成绩T e g a f u r
254
250
L L O Q l o w e r l i m i t o f q u a n t i t a t i o n ,5-F U H 25,6-d i h y d r o -5-f l u o r o u r a c i l ,5-F U r d 5-f l u o r o u r i d i n e ,5-F d U r d 5-f l u o r o -2′-d e o x y u r i d i n e ,P P p r o t e i n p r e c i p i t a t i o n ,S P E s o l i d -p h a s e e x t r a c t i o n ,L L E l i q u i d –l i q u i d e x t r a c t i o n ,T C A t
r i c h l o r o a c e t i c a c i d ,E t A c E t h y l A c e t a t e
1196
M.Breda,S.Barattè
