(M + H)+; UV: lmax = 284 nm; IR: n = 3392 (O-H), 1511 (C = C), Arbutin metabolites in urine: Urine samples were diluted to a de-
1101 (C-OH), 823 cm-1 (doop, = C-H); HPLC purity grade: 100.0%.
fined volume resulting in concentrations of the analyte within
the controlled linearity range. Blank urine was diluted 1:100.
Hydroquinone sulphate (6): This was synthesised by modification The samples were membrane filtrated and chromatographed
of a published method [10]. 1560 mg (16.6 mmol) phenol were using an aqueous solution of 5(50 ng/mL) as external standard.
dissolved in 50 mL of aqueous potassium hydroxide solution Mobile phase A: 1 mL acetic acid 1%(v/v) + acetonitrile [92.5+ 7.5
10% (m/v), cooled (108C) and the air removed by a continuous ni- (v/v)]; mobile phase B: 1 mL acetic acid 1%(v/v) + acetonitrile [75
trogen stream. A solution of 4580 mg (16.9 mmol) potassium + 25(v/v)]:
persulphate in 100 mL water was added slowly (about 0.6 mL/
min). After 4 hours a pH of 8 was adjusted with carbon dioxide.
The solution was extracted twice with 100 mL of diethyl ether.
The aqueous layer was evaporated to dryness under vacuum,
the residue pulverised and extracted twice with 40 mL ethanol
90% (v/v). The filtrated extract was evaporated to dryness, the
oily product dissolved in boiling absolute ethanol and filtered
immediately. The precipitated product was re-crystallised sever-
al times: 272 mg (1.2 mmol; 7.2%) yellow-white crystals (from
ethanol/petrol ether); m.p. 2328C (decomposition); anal. calcd.
Gradient:
time
[min]
mobile phase A
[%(v/v)]
mobile phase B
[%(v/v)]
0 ± 11
11 ± 14
14 ± 17
17 ± 23
23 ± 35
100
0
100 ® 0
0
0 ® 100
100
0 ® 100
100
100 ® 0
100
for C6H5KO5S: C 31.57, H 2.21, S 14.05; found C 31.40, H 2.18, S Flow: 0.705mL/min; injection volume: 10 mL; fluorescence de-
1
13.46; H NMR (D2O): d = 6.89 (d, J = 8.97, 2H, C-3,5 ), 7.19 (d, tection: lexc = 290 nm, lem = 326 nm; retention times (tR) and
J = 8.98, 2H, C-2,6); MS (neg.): m/z = 189 (M±); UV: lmax
=
correction factors (f) due to different sensitivity: 2: tR = 6.2
231, 276 nm; IR: n = 335 3 (O-H), 1604 (C = C), 15 12 (C = C), min, f = 3.414; 6: tR = 8.5min, f = 11.220; 5: tR = 10.5min. Lin-
n
d
as = 1255 (S-O), 1209 (S-O), ns = 1044 (S-O), n = 840 (S-O-C), earity, LOD and LOQ, calculated to DIN 32645, and precision
oop = 817 (= C-H), d = 587 (SO3), 564 cm±1 (SO3); HPLC purity were performed on urine containing the metabolites 2 and 6
grade: 97.8%.
and spiked with 5 (Tables 3 and 4). A systematic error of 4% re-
sults from the shift of the parallel calibration curves of 5 in the
absence and in the presence of urine. The concentrations of 2
Assays
Aqueous bearberry leaf extract: 1 part of powdered bearberry leaf and 6 in urine did not significantly change within 8 days at
(250) and 100 parts of water were boiled for 10 min and filtered. room temperature without light protection. The robustness of
The filtrate was diluted 1:10 (v/v) with water and chromato- the HPLC assays was also investigated with respect to the chro-
graphed using an aqueous solution of 1 (100 mg/ml) as external matographic conditions. The separation capacity was not sensi-
standard. Mobile phase A: methanol + water [2.5+ 97.5(v/v)];
mobile phase B: methanol + water [15+ 85(v/v)]:
tive to small deviations from the provided polarity of the mobile
phase, but proved to be sensitive to a significant decrease of the
concentration of acetic acid.
149
Gradient:
time
[min]
mobile phase A
[%(v/v)]
mobile phase B
[%(v/v)]
Pilot pharmacokinetic studies
Three healthy volunteers (female, aged 41, weight 53 kg, height
168 cm; male, aged 29, weight 80 kg, height 181 cm; male, aged
60, weight 88 kg, height 173 cm) received each orally 150 mg of 1
and negligible amounts of 4 and 5 in about 150 mL aqueous bear-
berry leaf extract at 8:00 a.m., 2:00 p.m., 8:00 p.m. and 12:00
p.m. Alcohol, caffeine-containing beverages and nicotine were
prohibited during the study. No food was allowed 12 hours prior
0 ± 7.5
7.5 ± 15
15 ± 28
28 ± 30
30 ± 40
100 ® 82
82 ® 0
0
0 ® 18
18 ® 100
100
0 ® 100
100
100 ® 0
100
Flow: 1.5mL/min; injection volume: 15 mL; detection wave- to the study. At 9:30 a.m. two rolls, at 12:00 a.m. a spaghetti
length: 284 nm; retention times (tR) and correction factors (f) meal and from 2:00±6:00 p.m. two other rolls were given. Din-
due to different sensitivity: 1: tR = 4.5min; 5: tR = 6.0 min, ner of one's own choice was allowed after 8:00 p.m. Study ended
f = 0.368; 4: tR = 18.5min, f = 1.073. Linearity, limit of detec-
at 8:00 a.m. the next morning.
tion (LOD) and limit of quantification (LOQ) defined according
to DIN 32645were investigated with 10 aqueous solutions for 1 Urine was collected immediately before the beginning of the
and with 12 aqueous solutions for 4 and 5 with the results listed study, at 10:00 a.m., 12:00 a.m., 2:00 p.m., 4:00 p.m., 6:00
in Table 1. The results of the control of precision of the assays of 1, p.m., 8:00 p.m., 12:00 p.m. and at 8:00 a.m. next morning be-
4 and 5 of the aqueous bearberry extracts and of the accuracy fore breakfast.
tested as recoveries in 50% of the extract amount provided in
the testing procedure spiked with different concentrations of
the analytes are summarised in Table 2. When protected from Results and Discussion
light, solutions of 1, 4 and 5 did not significantly change within
3 days. The separation capacity of the HPLC assays was neither The reference substances which were used as external standards
sensitive to changes of the temperature nor to small deviations for the HPLC assays and not commercially available were bio-
of the flow rate but proved to be sensitive to changes of the com- chemically prepared or synthesised by modified methods de-
position of the mobile phase.
scribed in the literature.
Quintus J et al. Urinary Excretion of¼ Planta Med 2005; 71: 147±152