Food and Chemical Toxicology 38 (Suppl.

2) (2000) S31S41

www.elsevier.com/locate/foodchemtox

Sucralose Metabolism and Pharmacokinetics in Man

A. ROBERTS*1, A. G. RENWICK1, J. SIMS2 and D. J. SNODIN2
1

University of Southampton, Bassett Crescent East, Southampton SO9 3TU, UK and 2Tate and Lyle Speciality Sweeteners, Whiteknights, Reading, UK

AbstractThe metabolic and pharmacokinetic prole of sucralose was studied in human volunteers. Following a single oral dose of 14C-sucralose (1 mg/kg, 100 mCi) to eight male subjects, a mean of 14.5% (range 8.9 to 21.8%) of the radioactivity was excreted in urine and 78.3% (range 69.4 to 89.6%) in the faeces, within 5 days. The total recovery of radioactivity averaged 92.8%. Plasma concentrations of radioactivity were maximal at about 2 hours after dosing. The mean residence time (MRT) for sucralose was 18.8 hr, while the eective half-life for the decline of plasma radioactivity was 13 hr. Two volunteers given a higher oral dose (10 mg/kg, 22.7 mCi) excreted a mean of 11.2% (9.6 and 12.7%) of the radioactivity in urine, and 85.5% (84.1 and 86.8%) in faeces over 5 days. The total recovery of radioactivity was 96.7%. The radiolabelled material present in faeces was essentially unchanged sucralose. Sucralose was the principal component in the urine together with two more polar components which accounted for only 2.6% of the administered dose (range 1.5 to 5.1% of dose); both metabolites possessed characteristics of glucuronide conjugates of sucralose. # 2000 Elsevier Science Ltd. All rights reserved Keywords: sucralose; articial sweetener; metabolism; pharmacokinetics; humans. Abbreviations: AUC = area under the plasma concentrationtime curve; AUMC = area under the rst moment of the concentrationtime curve; MRT = mean residence time; TLC = thin-layer chromatography; TMS = trimethylsilyl.

INTRODUCTION

Sucralose (1,6 dichloro-1,6-dideoxy-b-D-fructofuranosyl-4-chloro-4-deoxy-a-D-galactopryranoside) is a novel intense sweetener with a potency about 600 times that of sucrose (Hough, 1989). Sucralose is not hydrolysed in the intestinal lumen, is poorly absorbed by experimental animals and is excreted largely unchanged in the faeces (John et al., 2000; Wood et al., 2000). In addition, studies in animals have shown that sucralose has low toxicity and therefore it has been developed as a non-nutritive sugar substitute. Information on the fate of food additives in both animals and humans is essential for the rational interpretation of toxicity data (COT, 1982; JECFA, 1987). A preliminary unpublished study in three male subjects using 14C-sucralose has demonstrated limited absorption, a peak plasma 14C activity at about 2 hr after the oral dose and the absence of 14CO2 in the expired air. The present investigations were undertaken to quantify the metabolic and pharmacokinetic

proles of sucralose using highly puried radiolabelled 14C-sucralose in a larger group of subjects.
MATERIALS AND METHODS

Materials Uniformly labelled 14C-sucralose [batch no. CFQ 4503, sp. act. 5 mCi/mg, radiochemical purity 99% by thin-layer chromatography (TLC); batch no. CFQ 4643, sp. act. 21 mCi/mg, radiochemical purity 99.6% by HPLC] was obtained from Amersham International plc (UK). Batch no. CFQ 4643 was found to have a reduced radiochemical purity on receipt and was puried by TLC using solvent system B (Table 1), to give a radiolabelled purity of 99.5%. Non-radioactive sucralose (batch no. KL/5/ 31; purity 99.2% by HPLC) was supplied by Tate & Lyle Research & Development (Whiteknights, Reading, UK). Amberlite XAD-2 was obtained from BDH Chemicals Ltd (Poole, UK). Pre-layered silica gel 60-F254 (Kieselgel) TLC plates (0.25 mm thickness) were obtained from Merck (Darmstadt, Germany). Enzyme preparations were obtained from Sigma, Poole (Dorset, UK). All other chemi-

*Present address: Tate & Lyle Speciality Sweeteners, Whiteknights, Reading, UK.

0278-6915/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII S0278-6915(00)00026-0

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A. Roberts et al.

cals and reagents were of analytical grade and were obtained from BDH Chemicals Ltd.

Sample analysis Determination of total radioactivity. Concentrations of radioactivity in plasma and urine were measured by mixing duplicate aliquots (2 ml) of plasma and triplicate aliquots (0.5 to 2 ml) of urine with Beckman Ready Solv scintillation uid. Faecal samples were homogenized in measured volumes of water and accurately weighed triplicate samples of the homogenate (up to 1 g) were mixed with 100 ml sodium hydroxide (40%, w/v). The samples were decolorized by the drop-wise addition of 1 ml hydrogen peroxide (100 vols; 30%), with iso-octanol (100 ml) added to prevent frothing. After decolorization, glacial acetic acid (200 ml) was added followed by 15 ml scintillation uid. Radioactivity was measured by scintillation counting using a Packard Tri-carb liquid scintillation spectrometer (Packard Model 3255), with counting eciency corrected by the channels ratio method. Preparation of urine and faecal samples for thinlayer chromatography (TLC). Aliquots (50 ml) of urine samples and of control urine spiked with 14Csucralose were mixed with Amberlite XAD-2 resin (approx. 5 g) and the slurry was added to a column of XAD-2 resin (25 2.5 cm). The column was washed with distilled water (100 ml) and the radioactivity eluted with methanol (150 ml). Virtually all of the applied radioactivity (>90%) was recovered in the methanol eluate which was evaporated to dryness at 378C using a rotary evaporator. The resulting residue was dissolved in distilled water (12 ml) and analysed by TLC. The XAD-2 eluate of control urine spiked with 14C-sucralose was chromatographed simultaneously with the urine samples. Accurately weighed samples of the faecal homogenate were freeze-dried overnight and resuspended in a known volume of methanol. The suspension was centrifuged and the recovery of 14C in the supernatant determined by liquid scintillation counting. The recovery of radioactivity from faecal homogenates was 105.5% 2 8.1 SD (n = 35). Control faecal homogenates collected from undosed subjects were spiked with 14C-sucralose and extracted simultaneously to provide a reference standard for TLC analysis. TLC. The nature of the radioactivity in urine samples collected at 03, 36 and 612 hr after dosing at 1 mg/kg was examined by TLC of urine applied directly to the plates without prior XAD-2 extraction. The concentration of radioactivity in urine samples collected after 12 hr was insucient for direct analysis and, therefore, the eluates from XAD-2 columns were analysed by TLC. Urine samples obtained after an oral dose of 10 mg/kg of sucralose (study 2) were also concentrated using XAD-2 resin prior to TLC analysis. The nature of the 14C activity in urine, XAD-2 eluate from urine and methanolic extracts of faeces was examined by TLC using one or more of the solvent systems

Clinical protocol Eight healthy male subjects, mean age 39 yr (30 48 yr) and mean weight 79 kg (70.588 kg), took part in the rst study which was conducted at an oral dose of 1 mg/kg. All volunteers gave their written informed consent before taking part in the studies which had been approved by the local Ethics Committee and the Administration of Radioactive Substances Advisory Committee. Prior to study entry, each subject provided a blood sample for routine clinical chemistry screening tests and underwent a medical examination. Exclusion criteria included a history of gastrointestinal, hepatic or renal disorders, drug or alcohol abuse, serious illness or the use of enzyme-inducing drugs within 4 wk prior to study commencement and the regular use of any medicines. In a second study, sucralose was administered at an oral dose of 10 mg/kg to two of the eight subjects, selected on the basis of their higher than average urinary excretion of radioactivity in the rst study. Following an overnight fast, each volunteer was given sucralose orally, dissolved in water (100 ml), at a dose of 1 mg/kg body weight and 100 mCi (batch no. CFQ 4503) in study 1, and 10 mg/kg and 22.7 mCi (batch no. CFQ 4643 after purication) in study 2. The volunteers spent the initial 8 hr of each study under clinical supervision. The subjects remained supine for 4 hr after receiving the dose. A drink was provided 2 hr after dosing and a light lunch after 4 hr. Subjects were allowed to return to their normal food consumption and activity patterns after 8 hr, although strenuous exercise was avoided. In the rst study, blood (10 ml) was collected into heparinized tubes immediately before dosing and at 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 12, 24, 30, 36, 48 and 72 hr after dosing. During the rst 8 hr, samples were obtained from a venous cannula kept patent by heparinized isotonic saline, and subsequent samples were taken by individual venipuncture. Blood was centrifuged and the plasma radioactivity determined. The plasma samples were stored at 208C in a glass tube containing no anticoagulant. For both studies, each subject emptied his bladder immediately prior to dosing and a sample of urine was retained. Complete urine collections were made over the following intervals: 03, 36, 612, 1224, 2436, 3648, 4860, 6072, 7296 and 96120 hr. The volumes were recorded and the concentration of radioactivity determined. The samples were stored at 208C prior to TLC analysis. All faeces were collected up to 120 hr after dosing into polyethylene bags which were labelled with the appropriate volunteer's code number, date and time. The samples were frozen, weighed and stored at 208C before analysis.

Sucralose metabolism and pharmacokinetics in man

S33

shown in Table 1. Radioactivity on the chromatograms was detected and quantied using a Berthold LB284 automatic TLC-Linear Analyzer equipped with a high resolution head. Gas chromatographymass spectrometry analysis of urine samples. An aliquot (100 ml) of the 36 hr urine (containing 4.4% of dosed radioactivity) collected from subject 6 was subjected to XAD-2 resin treatment as described above. The methanol eluate was evaporated to dryness on a rotary evaporator and redissolved in 0.5 ml methanol. The methanol fraction was applied to a number of TLC plates and chromatographed in solvent system B (Table 1). The radioactivity was located on each plate using the Berthold Linear Analyzer and the radioactive bands corresponding to sucralose and its metabolites were scraped separately from the plates. The radioactivity was eluted from the silica with methanol (120 ml). After evaporation to dryness, pyridine (100 ml), hexamethyldisilane (30 ml) and chloro-trimethylsilane (20 ml) were added to the residue and the mixture was incubated at 408C for 1 hr in order to produce the trimethylsilyl (TMS) derivatives. Gas chromatographymass spectrometry was performed with a Pye Unicam 104 gas chromatograph coupled to an MS30 mass spectrometer. The TMS derivatives were chromatographed on a column (7 ft long and 0.25 in. in diameter) packed with 3% OV-1 on Diatomite CQ (100120 mesh). The injector temperature was 3203308C, the column temperature 3108C and the helium carrier gas ow rate was 60 ml/min. Mass spectra were recorded under ion impact conditions, with the source temperature at 2408C and the membrane separator at 2308C. Liquid chromatographythermospray mass spectrometry analysis of urine samples. XAD-2 resinconcentrated extracts of 36-hr urines (1 mg/kg dose, subjects 2 and 8) were dissolved in 500 mM ammonium acetate (2 ml). An aliquot (1 ml) of reconstituted extract was injected onto a Waters 5m Nova-Pak C18 reverse phase column (15 0.46 cm). The column was eluted with 100% 50 mM ammonium acetate, pH 6.5, at a ow rate of 1.5 ml/ min for 5 min. A linear gradient to 30% methanol in 50 mM ammonium acetate, pH 6.5, was then applied over 30 min using two Waters Model 510 pumps and a Model 680 gradient controller. Fractions were collected each minute for 40 min and an aliquot (150 ml) of each was dissolved in Insta-Gel and analysed for 14C content by liquid scintillation

counting. Fractions 20 and 23 (Fig. 1), which contained appreciable proportions of the total radioactivity, were selected for analysis by combined HPLCthermospray mass spectrometry. Using identical HPLC conditions to those described above, 0.3-ml aliquots of these fractions and of the remaining crude original resin extracts were injected separately. The HPLC column was coupled directly to a Finnigan 4500 quadrupole mass spectrometer via a Finnigan thermospray interface. The mass spectrometer was set to acquire either positive or negative ion spectra across a mass range of m/z 170900 every 3 sec. Data were acquired and processed using a Finnigan Incos Data System. Temperatures of the vaporizer tube and the ion source block were 130 and 2008C, respectively. An XAD-2 resin-concentrated extract of 36 hr urine (dose 10 mg/kg, subject 2) was dissolved in acetonitrile0.04 M ammonium acetate (1:1, v/v). An aliquot was injected onto a Waters Carbohydrate Column (15 0.76 cm) which was eluted with 1:1 acetonitrile0.04 M ammonium acetate at a ow of 1.5 ml/min. Fractions were collected every 30 sec for 20 min and analysed for 14C activity. Under these conditions sucralose (fractions 57) was eluted before either of its metabolites which were clearly separated (M2, fractions 1314; M1, fractions 1618). The identity of the peaks was conrmed by TLC analysis on the collected fractions. The metabolites collected from the Novapak C18 column (see above) were puried further by sequential HPLC using (a) a Lichrosorb C2 column (25 cm 0.46 cm i.d.) with 50 mM ammonium acetate (pH 6.5) at a ow of 1 ml/min, and (b) a Micropak NH2 column (30 cm 0.46 cm i.d.) with 50 mM ammonium acetate at a ow of 1.8 ml/min and a pH gradient from pH 3.5 to pH 10.5. Separate HPLCMS runs were performed at each of these stages to analyse the radioactive component in each fraction. In addition, the puried fractions were analysed by HPLCMS using the carbohydrate column described above. Preparative TLC separation of sucralose urinary metabolites and enzyme incubations. Urine samples from the two subjects given 14C-sucralose at 10 mg/ kg were combined (71 ml of subject 1, 912 hr urine; 29 ml of subject 2, 03 hr urine). The combined samples were passed through an XAD-2 column as described previously. The methanol eluate which contained 94% of the applied 14C was evap-

Table 1. Thin-layer chromatography solvent systems and Rf values of sucralose and its metabolites Rf Values Solvent system A B C D Diethyletherethylmethylketonewater (25:25:1, by vol). Ethylacetatemethanolwaterconc. ammonia (60:20:10:2, by vol). Ethylacetatemethanolwater (6:2:1, by vol). Butanolacetic acidwater (3:1:1, by vol). Sucralose 0.28 0.50 0.73 0.61 M1 0.03 0.10 0.37 0.39 M2 0.03 0.20 0.51 0.39

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A. Roberts et al.

Fig. 1. HPLC analysis of the XAD-2 extract of the urine from subject 8. The radiolabelled components were separated on a Nova-Pak C18 column by gradient chromatography as described in the text. The eluate was collected every min and analysed for total 14C-activity.

orated to a low volume (approx. 2 ml). The residue was applied to TLC plates which were developed in solvent system B (Table 1). The positions of the 14 C-bands were located with a Berthold Linear Analyzer and the bands corresponding to metabolites M1, M2 and sucralose, were scraped o separately, suspended in methanol and ltered. A total of 93% of the radioactivity applied to the plates was recovered in these extracts. Analysis by liquid scintillation counting showed that approximately 10% of the radioactivity was accounted for by M1, 13% by M2 and the remainder (73%) by sucralose. The methanol extracts were evaporated to dryness

and redissolved in distilled water (200 ml) and an aliquot (100 ml) mixed with 0.1 M ammonium acetate (pH 5.0; 50 ml) or Tris buer (pH 7.0; 50 ml). Enzyme incubations were carried out at 378C in the presence or absence of b-glucuronidase/sulfatase mixture (5000 and 200 units, respectively; Type H2) or pure b-glucuronidase (Type B-1; 5000 units) at pH 5.0 overnight, or aryl sulfatase (0.5 units; Type VI) at pH 7.0 for 60 hr. Aliquots (5 ml) of the incubation mixtures were analysed by TLC using solvent systems B, C and D (Table 1), and by HPLC using a Waters Carbohydrate Analysis liquid chromatography column with a mobile phase comprising 0.04 M ammonium acetate buer (pH 5.5) in 50% acetonitrile at a ow of 1.5 ml/min. Data analysis. A model comprising rst order absorption into a two-compartment system was tted to the plasma 14C activity-time data using the non-linear, least squares regression program NONLIN. Because of the very low concentrations of 14C present in the plasma at 72 hr after dosing, the data were tted between 0 and 48 hr only. The mean residence time (MRT) was calculated as the area under the rst moment of the concentrationtime curve (AUMC) divided by the area under the plasma concentration-time curve (AUC) (Gibaldi and Perrier, 1982). The AUC and AUMC were calculated using the trapezoid rule with extrapolation to innity. The ``eective half-life'' based on the statistical moment theory was calculated as 0.693 MRT (Gibaldi and Perrier, 1982). It should be noted that all of these analyses were performed on total 14C and that the data, which are expressed
14

Table 2. Excretion of radioactivity in urine and faeces following a single oral dose of Subject Time (hr) Urine 03 36 612 1224 2436 3648 4872 7296 96120 Total 0120 Faeces 024 2448 4872 7296 96120 Total 0120 No. of samples Total excreted in urine and faeces 1 1.48 1.76 2.34 1.07 0.91 0.46 0.49 0.16 0.10 8.77 16.1 63.3 10.2 89.6 3 98.4 2 4.67 5.16 3.68 1.67 0.79 0.50 0.68 0.46 0.32 17.93 0.0 0.0 39.8 41.5 81.3 5 99.2 3 2.36 3.02 2.19 1.81 0.67 0.60 0.59 0.12 0.06 11.42 18.3 34.0 23.5 3.4 0.6 79.8 6 91.2 4 3.99 3.33 1.87 0.98 0.53 0.15 0.17 0.08 (0.04 11.14 79.2 2.0 0.3 0.0 81.5 6 92.6 5 3.47 3.10 2.73 1.53 0.53 0.23 0.13 0.08 0.10 11.97 23.7 49.4 2.1 0.4 0.2 75.8 10 87.8

C-sucralose (1 mg/kg) to man

6 6.99 4.41 2.49 1.76 0.52 0.28 0.23 0.10 0.06 16.84 63.4 12.0 0.6 0.5 76.5 5 93.3

7 4.05 3.94 3.02 1.85 1.18 0.68 0.50 0.16 0.10 15.48 30.1 29.7 5.1 7.0 0.7 72.6 10 88.1

8 8.55 6.40 2.57 2.24 0.67 0.47 0.48 0.21 0.07 21.66 8.2 39.5 13.2 6.8 1.7 69.4 8 91.1

Mean2 SD 4.45 2 2.32 3.89 2 1.44 2.61 2 0.55 1.61 2 0.42 0.73 2 0.23 0.42 2 0.18 0.41 2 0.20 0.17 2 0.13 0.11 2 0.09 14.40 2 4.30 N/A N/A N/A N/A N/A 78.3 2 6.2 N/A 92.7 2 4.2

= no sample; SD = standard deviation; N/A = not applicable. The results are given as the % of the administered dose.

Sucralose metabolism and pharmacokinetics in man

Table 3. Excretion of radioactivity in urine and faeces following a single oral dose of 14 C-sucralose (10 mg/kg) to two volunteers Subject Time (hr) Urine 03 36 69 912 1224 2436 3648 4860 6072 7296 96120 Total 0120 Faeces 024 2448 4872 7296 96120 Total 0120 Total in urine and faeces 2 3.82 3.52 1.91 0.96 0.88 0.58 0.31 0.24 0.20 0.23 0.08 12.73 0.04 36.8 25.0 15.7 6.6 84.1 96.8 6 3.50 2.36 1.68(612 hr) 0.72 0.68 0.26 0.20 0.18 0.04 0.01 9.63 0.4 56.2 30.2 86.8 96.4 Mean % 3.66 2.94 2.28(612 hr) 0.80 0.63 0.29 0.22 0.19 0.14 0.05 11.20 0.22 46.5 27.6 85.5 96.7

S35

= no sample produced. The results are given as % of dose.

as ng equivalents of sucralose, represent both parent compound and metabolites. The urinary 14C data were analysed by the sigma-minus method (Gibaldi and Perrier, 1982).
RESULTS

Excretion of radioactivity in urine and faeces After oral administration of 14C-sucralose (1 and 10 mg/kg) to eight and two human subjects, respectively, radioactivity was excreted mainly in the faeces. At a dose of 1 mg/kg from 69.4 to 89.6% of the dose was eliminated in the faeces over 5 days with a mean recovery of 78.3% (Table 2). A similar faecal recovery (average 85.5%) was found at the higher dose of 10 mg/kg (Table 3). The pattern of faecal elimination following the 1 mg/kg dose showed wide inter-subject dierences since one volTable 4. Plasma pharmacokinetics of
14

unteer excreted 63% of the dose within 24 hr while another did not eliminate any radioactivity in the faeces for 3 days. Excretion of radioactivity in the urine following doses of 1 and 10 mg/kg accounted for averages of 12.6 and 9.7% of the dose after 24 hr, respectively, which increased only slightly to 14.5 and 11.2% after 5 days (Tables 2 and 3). The total 14C recovered in urine over 120 hr varied from 8.9 to 21.8%, for the eight subjects dosed at 1 mg/ kg. The total radioactivity excreted in urine over 120 hr by subjects 2 and 6 after a dose of 10 mg/kg was reduced compared with 1 mg/kg. Overall, the total recovery of radioactivity from urine and faeces over 5 days averaged 92.8% (range 87.7 to 99.4%) and 96.7% at doses of 1 and 10 mg/kg, respectively. Plasma radioactivity The peak plasma concentrations occurred 1.5 to 3 hr after an oral dose of 1 mg/kg. The mean peak
14

C activity after oral administration of Subject

C-sucralose (1 mg/kg) to man

1 Cmax (ng/ml) Tmax (hr) AUC0-oo(ng ml/hr) t1/2 - plasma (hr) t1/2 - urine sigma minus (hr) Mean residence time (hr) Mean eective half-life (MRT.693) 140.7 3.0 1783 23.0 25.0 21.4

2 284.6 1.5 2504 24.8 42.5 15.6

3 169.7 2.0 1755 34.0 17.5 25.9

4 242.3 2.0 1948 20.9 23.1 16.9

5 206.1 3.0 2158 15.8 36.4 12.9

6 339.7 1.5 2408 21.7 25.1 13.2

7 256.1 2.0 2722 23.7 25.5 20.2

8 454.8 2.0 3529 32.7 17.2 24.3

Mean 2SD 261.82 100.4 2.12 0.6 23512 589 24.6 2 6.1 26.5 2 8.8 18.8 2 4.9 13.0

The data have been analysed as equivalents of sucralose based on total 14C in either plasma or urine. The terminal half-lives in plasma were calculated using NON-LIN based on 048 hr data since the concentrations at 72 hr were too low for accurate determination.

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A. Roberts et al.

metabolites to parent compound in urine (see below) did not alter greatly from 12 hr onwards, it is likely that the derived values provide a reasonable approximation to the plasma kinetics of sucralose itself. TLC proles of radioactivity in urine and faecal extracts Chromatographic analysis of radioactivity in urine applied directly to the TLC plates and urine concentrated using XAD-2 resin indicated that unchanged sucralose was the major component. The Rf values of pure 14C-sucralose and the major radioactive urinary component were similar in the three solvent systems used (A, B and C). When given at a dose of 1 mg/kg, sucralose accounted for nearly 90% of urinary radioactivity in 03 hr samples, decreasing to around 70% in 1224 hr urines (Table 5). During the rst 48 hr after dosing, sucralose accounted for between 72.9 and 85.8% of the total urinary radioactivity (Table 6). At the higher dose of 10 mg/kg, sucralose represented 80 90% of urinary radioactivity in samples collected up to 3 hr, and 5060% in samples collected up to 12 hr after dosing (Table 7). The remainder of the radioactivity was associated with more polar material which chromatographed generally as a single peak in solvent A (Fig. 3A) but was resolved into two components in solvent systems B and C (Fig. 3B). The more polar metabolite (closer to the origin) was designated as M1, and the less polar metabolite as M2. Following a dose of 1 mg/kg, the polar metabolites (M1 + M2) accounted for about 11% of urinary radioactivity in 03 hr urines, and increased to a maximum of 27% in later urines (Table 5). Over 48 hr, metabolism accounted for 2.6% (range 1.55.1%) of the dose of sucralose (Tables 5 and 6). At a dose of 10 mg/kg, approximately 20% of urinary radioactivity in 012 hr urines was present as sucralose metabolites, which were equivalent to 1.61.9% of dose (Table 7). TLC analysis of the radioactivity in faecal samples showed that essentially all of the radiolabelled material was present as unchanged sucralose (Fig. 4). A few faecal samples were found to contain small amounts (less than 1% of the dose for all subjects) of radioactive material that was less polar than sucra-

Fig. 2. Plasma concentration-time curve of total 14C activity after a single oral dose (1 ml/kg) of 14C-sucralose. The results are the means (with standard errors indicated by vertical bars) for eight human volunteers.

plasma concentration was 262 ng equivalents per ml (range 141 to 455 ng equivalents/ml) (Table 4). The concentration of radioactivity in plasma declined rapidly to a mean of 36 ng equivalents/ml at 12 hr (range 24 to 48 ng equivalents/ml), but thereafter declined more slowly to a mean of 4.7 ng equivalents/ ml at 72 hr (range 2.9 to 7.5 ng equivalents/ml) (data not shown). The plasma concentrationtime curves for each individual followed a biphasic decline in concentration (Fig. 2) which could be described adequately by a two-compartment open model. Analysis of the AUC showed that the area corresponding to the terminal phase accounted for 31%2 10 (range 9% to 40%) of the total AUC and thus was the minor component. Under such circumstances, the terminal half-life does not provide the best indication of the ``eective'' half-life (Gibaldi and Perrier, 1982). The MRT, which takes into account absorption plus both the rapid and slow phases of elimination, was calculated to be 18.8 hr (Table 4) and the corresponding half-life (0.693 MRT) was 13.0 hr. As the proportion of

Table 5. TLC quantitation* of urinary constituents (dose 1 mg/kg) Urine collection period (hr) 03 36 612 1224 2436 3648 Total % of
14

C dose

Sucralose as % dose 3.92 3.04 1.87 1.15 0.58 0.33 (1.97) (1.02) (0.56) (0.25) (0.19) (0.17)

M1 as % dose 0.29 0.48 0.36 0.24 0.07 0.04 (0.22) (0.41) (0.15) (0.15) (0.03) (0.02)

M2 as % dose 0.18 0.32 0.35 0.21 0.06 0.03 (0.15) (0.16) (0.11) (0.09) (0.03) (0.02)

M1 + M2 as % dose 0.48 0.81 0.71 0.44 0.13 0.07 (0.36) (0.56) (0.21) (0.22) (0.04) (0.04)

4.45 3.89 2.61 1.61 0.73 0.38

(2.32) (1.44) (0.55) (0.42) (0.23) (0.25)

13.65 (4.27)

10.85 (3.17)

1.47 (0.92)

1.14 (0.49)

2.63 (1.33)

* = Chromatographed in solvent system B. The results are the mean (with SD in parentheses) for the volunteers.

Sucralose metabolism and pharmacokinetics in man

Table 6. Chromatographic analysis of the urinary
14

S37

C after oral administration of

14

C-sucralose (1 mg/kg) to man % dose as

Subject 1 2 3 4 5 6 7 8 Mean SD

Time 036 048 0-48 0-48 0-48 0-48 0-48 0-48

% dose 7.56 16.47 10.65 10.85 11.59 16.45 14.72 20.90 13.65 4.27

Sucralose 5.93 13.91 8.28 8.89 9.95 12.00 12.28 15.55 10.85 3.17

M1 0.69 1.07 1.31 1.06 0.90 2.63 1.01 3.17 1.47 0.92

M2 0.90 1.30 0.85 0.71 0.61 1.65 1.25 1.95 1.14 0.49

M1 + M2 1.56 2.38 2.16 1.78 1.51 4.28 2.26 5.12 2.63 1.33

The results are for solvent system B following XAD-2 concentration of the radioactivity. The analyses assume that the radioactivity present in the methanol extracts is representative of the total urinary 14C activity.

lose. This unknown material was not found in all subjects or in all samples from any one subject. Gas chromatographymass spectrometry analysis of urine samples The mass spectrum of the TMS derivative of the major radioactive urinary component was virtually identical to that of authentic sucralose (Fig. 5) conrming that the sweetener was excreted largely unchanged. Mass spectra could not be obtained for the fraction corresponding to M1 or M2. Liquid chromatographythermospray trometry analysis of urine samples mass spec-

Liquid chromatography and positive ion thermospray mass spectrometry of the HPLC fractions with highest radioactivity from the urine extracts of subjects 2 and 8 (1 mg/kg dose ) showed an ion cluster at m/z 414/416/418/420 in the intensity ratio of approximately 27:27:9:1. A similar mass spectrum was observed for the major peak present in a urine extract that had not been subjected to prior HPLC purication. The ion cluster was virtually indistinguishable from that generated when auth-

entic sucralose was subjected to similar analysis and is caused by the formation of the ammonium adduct ion [M + NH4]+ of sucralose. The positive ion thermospray mass spectra for fractions no. 20 (subject 8) and no. 19 (subject 2) were dominated by an intense ion at m/z 177. This ion is commonly seen in the thermospray spectra of thermally labile glucuronide conjugates (Liberato et al., 1983). Negative ion spectra on the same fractions contained a three-chlorine cluster at m/z 571/ 573/575/577. This is the expected molecular ion [MH] for a glucuronide conjugate of sucralose. As only two signicant radioactive components were eluted from the reverse phase column, it was concluded that the more polar metabolite, M1, was not retained under the HPLC conditions used. Use of a Waters Carbohydrate Column eluted with acetonitrile0.04 M ammonium acetate (1:1) enabled the more polar metabolite (M1) to be isolated and puried. However, even following further purication by HPLC, thermospray mass spectrometry was unsuccessful in identifying M1, probably due to decomposition of the metabolite under thermospray conditions.

Table 7. TLC quantitation of urinary constituents (dose 10 mg/kg) Urinary constituent concentration as % of urinary 14C (as % of dose) Urine collection period (hr) Subject 2 03 36 912 Total Subject 6 03 36 612 Total % of
14

C dose

Sucralose 79.3(3.03) 79.8(2.81) 61.4(1.17) 7.01 89.8(3.14) 66.6(1.57) 48.2(0.81) 5.52

M1 8.4(0.32) 10.2(0.36) 13.0(0.25) 0.93 5.3(0.18) 17.5(0.41) 17.8(0.30) 0.89

M2 6.5(0.25) 8.2(0.29) 20.0(0.38) 0.92 4.1(0.14) 11.3(0.27) 18.8(0.32) 0.73

Total M1 + M2, % of urinary 14.9(0.57) 18.3(0.65) 33.0(0.63) 1.85 9.3(0.33) 28.8(0.68) 36.5(0.61) 1.62

14

C (as % of dose)

3.82 3.52 1.91 9.25 3.50 2.36 1.68 7.54

Data were obtained from TLC analysis using solvent system C. Note: Subject 2 produced a very large volume of urine between 6 and 9 hr which prevented TLC analysis even after XAD treatment.

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Fig. 3. TLC radiochromatogram proles of the XAD-2 extract of the 612 hr urine from subject 2. (Trace Asolvent A), (Trace Bsolvent B), (Oorigin), (SFsolvent front), (Ssucralose), (M1 metabolite), (M2metabolite).

Preparative TLC separation of urinary metabolites and enzyme incubations The TLC separation of the sucralose metabolites did not result in a complete purication of M1 and M2, and there was some cross-contamination. Quantitation of the incubation mixtures after TLC in solvent system B is shown in Table 8. Neither M1 nor M2 was aected signicantly by incubation with pH 5.0 or pH 7.0 buers. Metabolite M1 was almost completely hydrolysed by both pure b-glucuronidase and by the sulfatase/b-glucuronidase mixture but not by sulfatase. The radioactivity in the peak corresponding to sucralose was increased by an amount similar to that lost from the M1 peak. Metabolite M2 was unaected by any of the enzyme systems used except the sulfatase/bglucuronidase mixture which hydrolysed approximately 50% of the M2 present. The Rf value of the radiolabelled b-glucuronidase hydrolysis product of M1 was similar to that of sucralose in TLC solvent systems B, C and D

(Table 1). This hydrolysis product of M1 eluted from the HPLC Carbohydrate Analysis column in the same fractions as 14C-sucralose.
DISCUSSION

Oral administration of 14C-sucralose at doses of 1 and 10 mg/kg to normal human volunteers showed that the faeces were the major route of elimination, while the urine accounted for between 8.9 and 21.8% of the radioactive dose. This inter-individual variability in urinary excretion is probably caused by dierences in the extent of absorption since individuals with the highest excretion also had the highest plasma concentrations. The slightly lower urinary excretion of radioactivity in the two subjects dosed at 10 mg/kg compared with their data for 1 mg/kg, suggests the possibility of reduced absorption of sucralose at higher doses. The elimination prole in humans is similar to those found in the rat, dog and mouse; the urinary excretion in the

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Fig. 4. TLC radiochromatogram proles of a methanol extract of faecal sample 4 from subject 2. (Trace Asolvent A), (Trace Bsolvent C), (Oorigin), (SFsolvent front), (Ssucralose).

rat was about one-half of that in humans, while faecal excretion was higher (Sims et al., 2000). Essentially all of the radioactivity in the faeces was present as sucralose. In contrast, the urine contained two minor metabolites, in addition to unchanged sucralose, which was the major component. Although the urinary metabolites represented a considerable proportion of the urinary 14 C (up to 40% in some of the later urine samples), these samples contained only a small percentage of the dose. Therefore, overall, the urinary metabolites accounted for a low percentage (mean of 2.6%) of the dose. Owing to the high purity of 14C-sucralose (>99%) administered, it was possible to assign unequivocally these non-sucralose components to the metabolism of sucralose rather than to the presence of an absorbable impurity in the dose material. The identity of the major urinary 14C component was conrmed as sucralose by both gas chromatographymass spectrometry and liquid chromatographythermospray mass spectrometry. A slightly

more polar substance, metabolite M2, representing approximately 10% of the urinary radioactivity, was identied from its positive and negative ion thermospray mass spectra as a glucuronide conjugate of sucralose. However, this metabolite was resistant to hydrolysis on incubation with bglucuronidases, although some hydrolysis appeared to occur with b-glucuronidase/sulfatase. Chromatographic analysis of the urine of dogs, collected following an oral dose of 14C-sucralose, revealed the presence of a polar metabolite which was identied by gas chromatographymass spectrometry, and by direct probe mass spectrometry, as a glucuronide conjugate of sucralose. The site of conjugation was on the 4-chloro-4-deoxygalactopyranosyl moiety (Wood et al., 2000). This conjugate was also resistant to b-glucuronidase hydrolysis. Co-chromatography of human urine with dog urine showed that the conjugate formed in the dog corresponded to metabolite M2 formed in man, indicating that the same glucuronide is formed in both man and dog (Wood et al., 2000).

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Table 8. Quantitation of incubation mixtures

Metabolite M1 incubation Incubation conditions Before incubation Buer, pH 5.0 Sulfatase/b-glucuronidase b-Glucuronidase Buer, pH 7.0 Sulfatase Metabolite M2 incubation Incubation conditions Before incubation Buer, pH 5.0 Sulfatase/b-glucuronidase b-Glucuronidase Buer, pH 7.0 Sulfatase

M1 86.9 83.8 7.5 7.8 80.9 90.3 M1 28.2 27.7 1.3 3.1 29.1 28.8

M2 4.2 4.4 3.1 3.8 3.6 1.6 M2 71.0 69.2 35.9 68.6 57.1 70.2

Sucralose 0 0 82.3 79.2 0 0 Sucralose 0 0 60.6 21.3 0 0

The data are the % of the total 14C activity present since each of the components was assessed by TLC in solvent system B followed by quantitation using the Berthold Linear Analyzer. Residual radioactivity was located mainly at the origin.

The more polar metabolite (M1) could not be identied by liquid chromatographythermospray mass spectrometry probably due to its lability under thermospray conditions. However, this metabolite was readily hydrolysed on incubation with b-glucuronidase. The hydrolysis product was identied as sucralose by comparison of Rf values in three dierent TLC solvent systems and by HPLC. It is concluded that M1 is also a glucuronide conjugate, most probably a mono conjugate owing to reactivity considerations that are discussed below. The hydroxyl group at position 6 in sucralose is the only primary hydroxyl group in the molecule

and is the most reactive (Hough, 1989). Glucuronides of primary alcohols are more stable to hydrolysis than those of secondary alcohols (Marsh, 1966). The less polar glucuronide conjugate (M2), which is present in dog urine and has been shown to be substituted in the 4-chloro-4-deoxygalacto-pyranosyl moiety (Wood et al., 2000) is resistant to hydrolysis by b-glucuronidase (Wood et al., 2000). Therefore, M2 can be identied tentatively as the 6-glucuronide of sucralose. In contrast, metabolite M1 was hydrolysed by bglucuronidase, and therefore is likely to be substituted at a secondary position such as 2, 3, 3' or 4'.

Fig. 5. The mass spectrum of sucralose (standard) and sucralose isolated from human urine. Pure sucralose and the urine extract (subject 6; 36 hr sample) were silylated and analysed by ion-impact gas chromatographymass spectrometry.

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Fig. 6. Plasma concentrationtime curves for total 14C activity in two subjects given 14C-sucralose. ww Subject 2 who showed a delayed faecal elimination of 14C activity (approx. 80% of the dose between 72 and 120 hr). rr Subject 6 who showed very rapid faecal elimination of 14C activity (approx. 63% of the dose in the rst 24 hr).

gut. Evidence for this is that two subjects (2 and 6) who showed marked dierences in the rate of faecal elimination (Table 2) showed very similar plasma concentrationtime curves (Fig. 6). Thus, the slow phase probably represents some rate-limiting systemic process, such as tissue redistribution. Secondly, it is probable that the metabolism occurs within the body tissues rather than the gut lumen. Evidence for this is that at 1 mg/kg there was a signicant correlation (r = 0.84; P < 0.01) between the total %14C dose excreted in the urine and the percentage metabolism, in dierent individuals. This suggests that metabolism is proportional to absorption and that the percentage of the absorbed dose that is metabolized is relatively constant. The fate of sucralose in humans was similar to that in rats (Sims et al., 2000), dogs (Wood et al., 2000) and mice (John et al., 2000). Although minor quantitative and qualitative dierences exist, these three animal species are good metabolic and pharmacokinetic surrogates for humans. The stability and hydrophilic nature of sucralose are reected in its poor absorption, rapid elimination, limited conjugative metabolism of the fraction absorbed, and lack of bioaccumulative potential.
AcknowledgementsWe are grateful to Professor D.S. Davies and Dr G. Taylor of the Royal Postgraduate Medical School, Hammersmith, London, for the analysis of urine extracts and samples by liquid chromatography and thermospray mass spectrometry.
REFERENCES

The possibility that M1 is not a mono- but a di-glucuronide of sucralose is unlikely since a second glucuronide substitution would most probably occur at the reactive 6-position. Such a substituent would not be susceptible to b-glucuronidase hydrolysis and therefore a disubstitute involving the 6 position would yield the 6-monoglucuronide on hydrolysis. Consequently, M1 is thought to be a monoglucuronide conjugate of sucralose with substitution occurring at one of the four secondary hydroxyl positions. The plasma concentrationtime data indicated that 14C-sucralose is absorbed with peak concentrations at about 2 hr, followed by a rapid decrease until 12 hr, and a slower late phase up to 72 hr. The half-life for the terminal phase was approximately 24 hr, but a minor proportion of the absorbed sucralose was excreted in this terminal phase. In such cases, calculation of a half-life derived from the mean residence time is considered a better approach for pharmacokinetic evaluation and a mean value of 13 hr was calculated for man (Table 4). Thus, negligible accumulation of sucralose would occur during regular intake consistent with the normal pattern of use of an intense sweetener. This agrees with clinical data, where repeated blood samples taken during a 13-week trial found no trend towards increasing plasma concentration, and thus no indication of accumulation (McNeil, 1987). The data provide two additional insights into the handling of sucralose in humans. First, the slow phase found in both plasma and urine is probably not related to slow absorption from the terminal

COT (1982) Guidelines for the Testing of Chemicals for Toxicity. Committee on Toxicity of Chemicals in Food, Consumer Products and the Environment. HMSO, London. Gibaldi M. and Perrier D. (1982) Pharmacokinetics, 2nd edn. Marcel Dekker, New York. Hough L. (1989) Sucrose, sweetness and sucralose. International Sugar Journal 91, 2337. JECFA (1987) Principles for the safety assessment of food additives and contaminants in food. Environmental Health Criteria 70, World Health Organization, Geneva. John B. A., Wood S. G. and Hawkins D. R. (2000) The pharmacokinetics and metabolism of sucralose in the mouse. Food and Chemical Toxicology 38 (Suppl. 2), S107S110. Liberato D. J., Fenselau C. C., Vestal M. L. and Yevgey A. L. (1983) Characterisation of glucuronides with a thermospray liquid chromatography/mass spectrometry interface. Analytical Chemistry 55, 17411744. Marsh C. A. (1966) Chemistry of D-glucuronic acid and its glycosides. In Glucuronic Acid Free and Combined, ed. G. J. Dutton, pp. 3136. Academic Press. McNeil Specialty Products Food Additive Petition 7A3987 (1987) (Sucralose). Sims J., Roberts A. and Renwick A. G. (2000) The metabolic fate of sucralose in rats. Food and Chemical Toxicology 38 (Suppl. 2), S115S121. Wood S. G., John B. A. and Hawkins D. R. (2000) The pharmacokinetics and metabolism of sucralose in the dog. Food and Chemical Toxicology 38 (Suppl. 2), S99 S106.