8
76
Vol. 56, No. 6
aldosterone provided approximately 10-fold higher ESI re- EtOH (0.4 ml) and to this solution was added 35% HCl (0.1 ml) and the re-
sulting solution was allowed to stand at room temperature for 30 min. After
addition of EtOH (2 ml), the reaction mixture was evaporated to dryness
under reduced pressure at 50 °C to give 10 (5.1 mg, 89.5%) as semi-solid
sponse (judging from their signal-to-noise ratio) in its posi-
tive-LC-ESI-MS/MS (SRM), compared to that of underiva-
tized molecule (m/z 359→m/z 189, negative mode). It was
suggested from these results that atto mole level of aldos-
terone could be analyzed by this SRM. The feasibility and
relevancy of this method in the reliable diagnosis of primary
aldosteronism are currently being evaluated in our labora-
tory.
with a single HPLC peak (t ꢀ3.9 min).
R
1
11b,18-,18x,21-Diepoxypregn-4-ene-3,20-dione (10): H-NMR d: 1.31
3H, s, 19-Me), 4.00 (1H, d, Jꢀ16.1 Hz, 21R-H), 4.09 (1H, d, Jꢀ16.1 Hz,
(
2
1S-H), 4.70 (1H, d, Jꢀ6.3 Hz, 11a-H), 4.98 (1H, s, 18-H), 5.74 (1H, s, 4-
ꢂ
·
H). HR-MS Calcd for C H O : 342.1833 (M ), Found: 342.1828.
2
1
26
4
Picolinyl Derivatives of Aldosterone Aldosterone (1) (5.5 mg) was dis-
solved tetrahydrofuran (THF, 0.1 ml) and to this solution was added the
reagent mixture (0.3 ml) (2-methyl-6-nitrobenzoic anhydride: MNBAn;
30 mg, picolinic acid: PA; 25 mg, 4-dimethylaminopyridine: DAP 25 mg; tri-
Experimental
Materials and Reagents 11b,21-Dihydroxypregn-4-ene-3,20-dion-18- ethylamine: TEA; 0.03 ml in THF 1 ml) and the resulting mixture was al-
1
9—22)
al (aldosterone, 1) was purchased from Steraloids Inc. (Newport, RI, lowed to stand at room temperature for 30 min as previously reported.
2
U.S.A.). [ H ]EtOH (EtOH-d , 99.5 atom % D) was obtained from ISOTEC The reaction mixture was diluted with 5% NaHCO (2 ml) and the resulting
6
6
3
(
Miamisburg, OH, U.S.A.). Picolinic acid, 2-methyl-6-nitrobenzoic anhy- mixture was transferred onto Bond Elut C18 cartridge (60 mgꢄ2, pre-condi-
dride and 4-dimethylaminopyridine were obtained from Tokyo Chemical In- tioned with MeOH 3 ml and H O 3 ml) by two portions. After washing the
2
dustry Co., Ltd. (Tokyo, Japan). Bond Elut C18 and Oasis HLB cartridges
cartridges subsequently with H O (3 ml), 5% HCl (3 ml), H O (3 ml) and
2 2
were obtained from Varian Inc. (Lake Forest, CA, U.S.A.) and Waters (Mil- MeCN–H O (20 : 80, v/v, 3 ml), the products were eluted with MeCN–H O
2
2
ford, MA, U.S.A.). LC-MS grade MeCN, MeOH, ultra-pure water and (80 : 20, v/v, 3 ml). Evaporation of the eluate gave the picolinyl derivative
CH COOH were purchased from Wako Pure Chemical Industries Ltd. (5.7 mg, 80.3%) as a semi-solid consisted of 6 (t ꢀ3.5 min, ca. 60%) and 7
3
R
(Osaka, Japan), and EtOAc and triethylamine from Nacalai Tesque Inc. (tRꢀ4.3 min, ca. 40%) by HPLC.
(
Kyoto, Japan), respectively.
Apparatus H-NMR spectra were recorded in CDCl solution using a
11b,18-,18x,20x-Diepoxy-20x,21-dihydroxypregn-4-ene-3,20-dione 21-
1
1
Picolinate (6): H-NMR d: 1.30 (3H, s, 19-Me), 4.28 (1H, d, Jꢀ11.2 Hz,
3
JNM-LA400 (400 MHz) or a JNM-LA600 (600 MHz) spectrometers (JEOL,
21R-H), 4.53 (1H, d, Jꢀ11.2 Hz, 21S-H), 4.28 (1H, d, Jꢀ5.6 Hz, 11a-H),
Tokyo, Japan) using tetramethylsilane as an internal standard. Low resolu- 5.41 (1H, s, 18-H), 5.74 (1H, s, 4-H), 7.57 (1H, t, Jꢀ7.3 Hz, 21-picolinyl-5-
tion and high resolution (HR) mass spectra were recorded by a JMS-700 H), 7.93 (1H, t, Jꢀ8.3 Hz, 21-picolinyl-4-H), 8.20 (1H, d, Jꢀ7.8 Hz, 21-
double focusing mass spectrometer (JEOL) with an electron ionization mode picolinyl-3-H), 8.85 (1H, d, Jꢀ4.6 Hz, 21-picolinyl-6-H). HR-MS (as a mix-
ꢂ
·
electron voltage: 70 eV). HPLC was run on a Waters 2695 Separation Mod- ture of 6, 7) Calcd for C H NO : 465.2151 (M ), Found: 465.2145.
27 31 6
(
ule equipped with a Waters 2487 Dual l Absorbance UV detector (Waters).
Alkyl Ether-Picolinyl Derivatives of Aldosterone (8, 9) Ethyl ether
The column was a X-Bridge C18 (150 mmꢄ4.6 mm I.D., 5 mm, Waters) and derivative of aldosterone (4, 5.1 mg) was dissolved in THF (0.1 ml) and to
used at an ambient temperature. The mobile phase consisted of MeCN–
H O–CH COOH (45 : 55 : 0.1, v/v/v) was used at flow rate of 1 ml/min with
this solution was added the reagent mixture (0.3 ml) (MNBAn 30 mg, PA
25 mg, DAP 25 mg, TEA 0.03 ml in THF 1 ml), and the resulting mixture
was allowed to stand at room temperature for 30 min. The same work-up of
2
3
an isocratic elution.
LC-Electrospray Ionization-Tandem Mass Spectrometry (ESI- the reaction mixture as described above afforded 8 (5.3 mg, 81.8%) as semi-
MS/MS) A Finnigan TSQ Quantum triple stage quadrupole mass spec- solid with a single HPLC peak (t ꢀ7.6 min). Similarly, treatment of 5
R
trometer (Thermo Electron, San Jose, CA, U.S.A.) equipped with an ESI-ion
(3.9 mg) with the reagent mixture afforded 9 (4.1 mg, 82.2%) as semi-solid
source, a Surveyor MS pump and autosampler (Thermo Electron) was em- with a single HPLC peak (t ꢀ5.4 min).
R
ployed. The column was a X-Bridge (100 mmꢄ2 mm I.D., 5 mm, Waters)
and used at an ambient temperature. The mobile phase consisting of
MeCN–H O–CH COOH (45 : 55 : 0.1, v/v/v) was used at a flow rate of
Alternatively, the picolinyl derivative 6 (4.3 mg) was dissolved in EtOH
(0.5 ml) and to this solution was added 35% HCl (0.1 ml). The reaction mix-
ture was allowed to stand at room temperature overnight, and then diluted
2
3
0
.2 ml/min. The general ESI-MS conditions were as follows: spray voltage, with H O (2 ml). The same work-up of this solution as described above
2
4500 V; sheath gas, nitrogen, 35 arbitrary unit (gas pressure); auxiliary gas, afforded 8 (4.5 mg, 98.7%) as semi-solid with a single HPLC peak
nitrogen, 15 arbitrary unit (gas pressure); ion transfer capillary temperature, (tRꢀ7.6 min). Treatment of the picolinyl derivative
50 °C; collision gas argon, 1.5 mTorr (gas pressure); ion polarity positive. HCl–MeOH gave 9 (3.6 mg, 99.7%) as semi-solid with a single HPLC peak
In selected reaction monitoring, an optimized collision energy and character- (tRꢀ5.4 min).
6 (3.5 mg) with
3
istic product ion was chosen for the picolinyl derivative of aldosterone from
the break down curves of precursor ion.
Alkyl Ether Derivatives of Aldosterone (4, 5) Aldosterone (1) (5.5 Me), 3.34 (1H, m, –OCH CH ), 3.70 (1H, m, –OCH CH ), 4.56 (1H, d,
11b,18-Epoxy-18x-ethoxy-21-hydroxypregn-4-ene-3,20-dione 21-Picoli-
1
nate (8): H-NMR d: 1.13 (3H, t, Jꢀ7.0 Hz, –OCH CH ), 1.27 (3H, s, 19-
2
3
2
3
2
3
mg) was dissolved in EtOH (0.5 ml) and to this solution was added 35% HCl Jꢀ6.6 Hz, 11a-H), 4.60 (1H, s, 18-H), 5.04 (1H, d, Jꢀ15.0 Hz, 21R-H),
0.1 ml) and the resulting solution was allowed to stand at room temperature 5.10 (1H, d, Jꢀ15.4 Hz, 21S-H), 5.72 (1H, s, 4-H), 7.50 (1H, ddd, Jꢀ7.3,
for 20 min. The reaction mixture was diluted with H O (2 ml), the resulting 4.7, 1.1 Hz, 21-picolinyl-5-H), 7.86 (1H, dt, Jꢀ7.7, 1.8 Hz, 21-picolinyl-4-
(
2
solution was transferred onto Bond Elut C18 cartridge (60 mgꢄ2, pre-condi-
H), 8.16 (1H, td, Jꢀ7.8, 1.1 Hz, 21-picolinyl-3-H), 8.78 (1H, d, Jꢀ4.1 Hz,
ꢂ
·
tioned with MeOH 3 ml and H O 3 ml) by two portions. After washing the 21-picolinyl-6-H). HR-MS Calcd for C H NO : 493.2469 (M ), Found:
2
29 35
6
cartridges subsequently with H O (3 ml), 5% NaHCO (2 ml), H O (3 ml)
493.2460.
11b,18-Epoxy-21-hydroxy-18x-methoxy-pregn-4-ene-3,20-dione 21-Pi-
2
3
2
and MeCN–H O (20 : 80, v/v, 3 ml), the derivative was eluted with MeCN–
2
1
H O (80 : 20, v/v, 3 ml). Evaporation of the eluate gave the ethyl ether deriv- colinate (9): H-NMR d: 1.28 (3H, s, 19-Me), 3.28 (1H, s, –OMe), 4.49 (1H,
2
ative 4 (5.8 mg, 97.8%) as semi-solid with a single HPLC peak (t ꢀ4.0
s, 18-H), 4.56 (1H, d, Jꢀ6.3 Hz, 11a-H), 4.99 (1H, d, Jꢀ15.1 Hz, 21R-H),
5.11 (1H, d, Jꢀ15.4 Hz, 21S-H), 5.73 (1H, s, 4-H), 7.50 (1H, ddd, Jꢀ7.3,
4.6, 1.2 Hz, 21-picolinyl-5-H), 7.86 (1H, dt, Jꢀ7.7, 1.7 Hz, 21-picolinyl-4-
H), 8.17 (1H, td, Jꢀ7.8, 1.0 Hz, 21-picolinyl-3-H), 8.78 (1H, d, Jꢀ4.6 Hz,
R
min). Treatment of 1 (4.2 mg) in MeOH and 35% HCl gave the correspon-
ding methyl ether derivative 5 (4.3 mg, 98.6%) as a semi-solid with a single
HPLC peak (t ꢀ2.9 min).
R
1
ꢂ
·
21-picolinyl-6-H). HR-MS Calcd for C H NO : 479.2308 (M ), Found:
28 33 6
1
1b,18-Epoxy-18x-ethoxy-21-hydroxypregn-4-ene-3,20-dione (4): H-
NMR d: 1.07 (3H, t, Jꢀ6.8 Hz, –OCH CH ), 1.26 (3H, s, 19-Me), 3.26 (1H,
479.2317.
Preparation of Internal Standard (IS) Aldosterone (1) (6.5 mg) was
1R-H), 4.45 (1H, dd, Jꢀ17.6, 4.1 Hz, 21S-H), 4.55 (1H, d, Jꢀ6.6 Hz, 11a- dissolved in EtOH-d6 (0.8 ml) and to this solution was added 35% HCl
H), 4.57 (1H, s, 18-H), 5.72 (1H, s, 4-H). HR-MS Calcd for C H O : (0.2 ml) and the resulting solution was allowed to stand at room temperature
2
3
m, –OCH CH ), 3.67 (1H, m, –OCH CH ), 4.09 (1H, dd, Jꢀ17.8, 3.4 Hz,
2
3
2
3
2
2
3
32
5
ꢂ
·
3
88.2250 (M ), Found: 388.2234.
for 30 min. The reaction mixture was diluted with H O (2 ml), the resulting
2
1
1
1b,18-Epoxy-21-hydroxy-18x-methoxypregn-4-ene-3,20-dione (5): H- solution was transferred onto Bond Elut C18 cartridge (60 mgꢄ2, pre-condi-
NMR d: 1.27 (3H, s, 19-Me), 3.22 (1H, s, –OMe), 4.08 (1H, dd, Jꢀ17.5,
tioned with MeOH 3 ml and H O 3 ml) by two portions. After washing the
2
4
4
.6 Hz, 21R-H), 4.38 (1H, dd, Jꢀ17.6, 4.9 Hz, 21S-H), 4.45 (1H, s, 18-H),
.54 (1H, d, Jꢀ6.3 Hz, 11a-H), 5.72 (1H, s, 4-H). HR-MS Calcd for
cartridges subsequently with H O (3 ml), 5% NaHCO (2 ml), H O (3 ml)
2
3
2
and MeCN–H O (20 : 80, v/v, 3 ml), the derivative was eluted with
2
ꢂ
·
C H O : 374.2093 (M ), Found: 374.2078.
MeCN–H O (80 : 20, v/v, 3 ml). Evaporation of the eluate gave the ethyl
2
2
30
5
2
Anhydro-Derivative of Aldosterone (10) 1 (6 mg) was dissolved in ether derivative (IS, 6.8 mg, 95.6%) as semi-solid with a single HPLC peak