Прасолов Илья Сергеевич Обнаружение стероидов

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Transcript of Прасолов Илья Сергеевич Обнаружение стероидов

02.00.02 –

– 2015
1.1. –– 8
1.2. –– 10
1.3. 15
1.3.1. 18
1.3.2. 22
1.3.3. 26
1.3.3.1. 26
1.3.3.2. 29
1.3.3.3. 30
1.3.3.4. 32
1.3.3.5. 33
1.3.3.6. 35
1.3.3.7. 36
1.4. 42
1.4.1. 44
1.4.2. 44
1.4.4. 45
2. 49
2.1. 49
2.2. 53
2.3. 53
2.4. 54
2.4.2. – 56
2.4.3. 57
2.4.4. 57
2.4.6. 58
2.4.7. – 59
2.4.8. 60
3
2.9.10. 61
2.4.11. 62
3. 65
3.1. – 65
3.2. 68
3.3. 71
3.4. 73
4.2. – 81
4.3. – 85
5. 90
5.1. 90
5.2. 94
5.3. 96
6. 98
6.1. 98
6.2. 1 – 103
6.3. 2 – 111
6.4. 3 – 119
6.5. 4 – 126
6.6. 5 – 5α––3β,17β– 134
6.7. 6 – 1––3β,17β– 141
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δ[‰]
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61 ( 20 40).

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– , , ,
5α– 5β– – – 16–.
1 , [28]
δ(–A) δ(–) δ(–T) δ(–5α) δ(–5β) −0.2 1.1 1.6 1.3 0.4 0.4 0.5 0.9 0.8 0.7 3× 1.1 1.5 2.8 2.4 2.0 . 0.9 2.6 4.4 3.7 2.4
δ(16––A) δ(16––) δ(16––T) δ(16––5α) δ(16––5β) −0.4 1.0 1.4 1.2 0.3 0.7 0.8 1.1 1.0 0.9 3× 2.1 2.5 3.2 2.9 2.6 . 1.7 3.5 4.7 4.0 2.9
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1976 .
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OH

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[32,33].
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[34,35].
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[36,37]. ,
120
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t = 13 ). δ13

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10 [46]
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1 53 180 0.31 –23.6 –22.0 –21.1 2 8 203 0.04 –23.3 –23.3 –21.9 3 89 218 0.43 –21.3 –20.8 –20.4 4 17 219 0.10 –23.4 –20.7 –20.4 5 45 248 0.20 –23.3 –21.9 –21.1 6 139 251 0.60 –26.1 – – 7 43 324 0.15 –23.0 – – 8 155 328 0.61 –22.2 –21.4 –20.2 9 116 405 0.32 –22.3 – –
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.

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“–”
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,
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3α,17β– [48–50].
δ13 = –28.8‰
13 5α–,
5α––3α,17β– [51].
δ13 5α–,

2.9‰. 5 ,
28 78
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,
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, 1997

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δ13, ‰
Dehydroepiandrosterone Tokyo Kasei Kogyo Co., Ltd. –31.4 Dehydroisoandrosterone Sigma Chemical Co. –33.5 Dehydroisoandrosterone Wako Pure Chemical Industries., Ltd. –34.1
New DHEA Leiner Health Products Inc. –30.5 Schiff DHEA Schiff Products –30.6
Dehydroepiandrosterone Acetate
Dehydroisoandrosterone Acetate
Mylis Kanebo Co., Ltd. –27.1
17,
δ13 .
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100 [64]
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(4–––4––3,17–)
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[65,66].

,
[67,68].


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.

2005 [69].
25 150 /,
––.
(30.30 ± 0.16) ‰,
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4 δ13 ,
100 (n=15) [70]
δ13, ‰ , ‰ –23.9 0.15
–22.7 0.10 –23.6 0.35
–24.8 0.49 –21.6 0.44
5α– –23.4 0.48 5β– –22.0 0.43
21 11–
– 11–– ,
38
100 [70].
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,
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() 11–– () ,
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100 ,
30 .
(22.40 ± 0.23 ‰).
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42 , 2.9 ‰.
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(–) [70]
1.3.3.8.
–3,17–) ,
2004 [17]. ( 23) (1,4–
–17β––3–)
17β–() – ,
[71].
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O
40
( 24) 1940

[72–74].
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OH
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,
. in vivo
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[76,77].
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5 , [78].
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5 [78]
δ13, ‰
Vetranal Riedel–de Haen –30.3 Eldorben Squibb –22.7 Ganabol Laboratorios V.M. Ldta –26.3
Boldabolic Asia Pharma –27.1 Boldabol 200 British Dragon Pharmaceutics –27.7

,
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6
,
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42
δ(–) = 4‰ ( 3‰) [78].
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,
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[78]

1 50 50 –18.0 –19.6 –18.1 – –28.7 –29.2 2 5 6 –18.7 –20.0 –18.4 – –27.0 –30.0 3 10 30 –18.4 –20.3 –18.4 –19.5 –30.1 –28.7 4 – 50 –18.7 –23.5 –20.4 –21.5 – –28.6 5 2 12 –20.3 –21.4 –19.9 –21.5 –28.9 –30.8 6 2 12 –19.3 –21.7 –19.9 –21.4 –21.3 –28.7 7 40 17 –22.5 –24.9 –22.5 –24.2 –29.2 –29.1 8 10 30 –19.4 –20.0 –19.3 –19.7 –29.6 –28.9 9 50 120 –20.6 –23.1 –21.2 –24.2 –27.4 –29.1
10 – 25 –19.5 –23.1 –21.7 –21.4 –29.6 –29.4 11 5 6 –22.8 –23.9 –21.9 –22.8 –29.2 –29.3 12 170 170 –21.8 –23.8 –21.9 – –28.1 –28.2
1.4.
––
:
1) ;
2) –
;
, :
(
pH = 6.0 – 7.0 ( ) 1 50–57
pH = 5.5 ( ) 3 55–60
E. Coli
, ,
Helix Pomatia,
.
, , , , .

( 26)

, ,
, [30].
26
5) () –
.
––
.
44

– () [80,81].
(30 )
.
(pH ~ 5) Helix Pomatia.
55 3 ,
(
, 100, 30 ) – ,
. [81]
.
, : = 4 : 1
(2 )
( [81]).
. , (5α–,
5β–, ) ,
(Ac2O) (Py) t oC
12 . ––.
1.4.2.

() [82–84].
/ – . , , ,
(Ac2O, Py, t oC, 12 ),

. , ,
(pH ~ 5.5)
Helix Pomatia.
45
55 3 . ,
,

: = 50 : 50. ,
, ,
17–. ( )
: = 80 : 20. (Ac2O, Py, t oC,
12 ), ––.
1.4.3.

[85–87].
(10 )
.
(pH ~ 7) β– E. Coli.
37 12 [85], 55
3 [86,87].
,
,
( [26]; A, T, 5α– [86,87]) ,
(Ac2O, Py, 70oC, 30 ).
––.
1.4.4.

[88–91].
(10 )
.
(pH ~ 7) β– E. Coli.
60 1 ,

46
,
2 – , – , 16– 5β–
. (Ac2O, Py, 60oC, 1 )

: = 75 : 25 ( ) ACN ( )
. ––.
1.4.5.


[28,92,93].
(10 )
. ,
(pH ~ 7) 5
. β–.
50 1 .
()
.
– .
: 1 – 11β–– 11β––; 2 – ; 3 – 5α– 5β–
; 4 – ; 5 – ; 6 – 16–. ,
(Ac2O, Py, 70oC, 45 ). [28] 2 3
.
––, 1 4 (
).
.

; .
; .
; .
; ; (, 16–, 5α/5β–).
; .
( ); .

; ( ); .
,

,
––.
“ ”


.

30‰ ( ),

. 13/12
,
17 26‰
, 27‰.
48

––
.
,
,

, ,
. ,

, , ,
, 1,4––3,17–, 5α––3β,17β– 1–
–3β,17β– ––.
49

Bond Elut LRC–C18 10 500 (Varian,
) « » (Chromasolv, Merck,
) « » (Biosolve, )
.
(Fisher Scientific, ) – (Acros,
). (...)
(), , – Riedel–de–Haën
(), 4––DL– () – Sigma ().
β–
E. oli (Roche Diagnostics, ).

« » (JT Baker, ) «
» (Biosolve, ).
, , (N––N–
) « »
Macherey–Nagel (). ,
, “”
Sigma (). () (),
– Sigma ().
–– «60»
99.9999% (–), 99.999%
99.995%.
,
,
8–10.
8

,
16– 274

356
52
1
O
O
O
C10H21
2
OH
O
4
O
O
5
OH
OH
H
6
OH
OH
53
(Restek, ),
(Millipore, ).
Agilent 1100 (Agilent Technologies,
) Dionex Ultimate 3000 (Thermo Scientific, ).
, ,
, ,
.
– Agilent 6890N –
5973inert Agilent ALS 7683
(Agilent Technologies, ) Thermo Trace GC –
Thermo DSQ II Thermo TriPlus
AS (Thermo Scientific, ).
–– Thermo Trace GC
Thermo TriPlus AS
Combustion Interface III, – Thermo
Delta V Plus (Thermo Scientific, ).
2.3.
Toledo, .
CP225D (Sartorius, ).

(1–10, 20–200, 100–1000, 500–5000 1000–10000
), Multipette plus
(0.5–2.5 1–5 ) Eppendorf, .
– ()
Glas–Col (Terre Haute, ) Intelli–Mixer RM–
1L (ELMI Ltd., ).
420 (Hettich, ).
–12 (, ).

Binder, .
RapidVap (Labconco Corp., ).

EC2 (VLM, ).
2.4.
2.4.1.
1) . 2
2000 . 200 1 /
1800 . 100 /
;
2) ( ).
2 2000 .
100 1 / 1900 .
50 / ;
3) . 2 5α––3β–
2000 . 600 1 /
1400 . 300 /
;
4) .
2 1000
.
;
55
5) “”. 1
“T”, 18 2 “”
. ;
6) 1 .
1 /,
11,
300 1200 .
(3 ) .
11

5α––3α,17β– 300 5β––3α,17β– 300
150 150
5β––3α,20α– 400 16(5α)––3α– 50
60
7) 2 .
(1 /) (50 )
(150 ), 1600
1200 . (3 ) ;
8) 3 . (1 /)
5α––3α,17β– (640 )
(60 ) 1100 1200 .
(3 ) ;
9) .
1 /
12 280 .
(1 ) .
56
12

5α––3α,17β– 100 5β––3α,17β– 100
100 100 100
16(5α)––3α– 60 5α––3β– 60
10) . 43 Na2HPO4 · 2H2O, 54
KH2PO4 0.8 NaN3, ,
800 pH (~ 6.2–6.4);
11) . 60 K2CO3 KHO3,
, 800
pH (~ 10.0–10.2);

()
( 5 ).
( 4 )
: 10% , –
20% (3 ). ,
. 2, 4 6
.
( 4 ) ,
, 10% 90% , (3
). ,
. 2, 4 6
.
57

() .

Agilent 1100,
.
Waters Sunfire 18 250 × 4.6 , 5.0 Waters
Sunfire 18 20 × 4.6 , 5.0 .
35º.
1.0 /. 192 240 .
«» – “ ”, «» – .
.
10 .
,
.
/.
/, 30/70, 50/50, 60/40 .
2.4.4.
,
– [94]. 50
30 70.

(1:1),
(60, 70 80)
800 .
58
2.4.5.
3 10 5α/5β–
1 / .
1 ,
1 10% “” .
1
2×5 . ,
100 – .
2.4.6.
, ,

.
–– DSQ II.

, – [94].
– (Agilent MSD 5973):
,
,
J&W HP Ultra–1, 17 × 0.20 × 0.11
.
[94].
( ) 1
Hamilton 1:10.
– (Thermo DSQ II):

RTX–35MS, 30 × 0.25 × 0.25
5 (Restek, ) .

(60, 80 120),
59
. ,
: 120º (1 ), 30º/ 260º, 1º/ 280º
(2 ).
– .
1 2
/, 1.7 /.
250º,
1 , , 50 /.
– 300º.
0.4 Hamilton
80 ,
.
, – .
8 .
–– (Thermo Delta V Plus):

, ,
2 , – 2 /,
2.5
.
2.4.7. –
70 , –
230º. 8
22 . 3×105,
– m/z 50–410 ...
5 . – (Thermo DSQ II)

.
940º,
– 600º. :
– 1.2 (
3.5 /), – 0.85 (
1 /), – 0.63 (
, m/z 44 2.0–2.5 ).

(δ13 = – 27.771‰) .

15 . ,

, 7
20 450 ,
– ( ). ,
5
1600 . 5
.
2.4.9. –

, .
120 , – 3 .
m/z – 44, 45 46.

– 10
20 30 .
,
δ13 0.06‰. –
(1–6 )
.
, , ±0.1‰/.
61
2.9.10.
10 , , 1
10 . , 50
2–6 10
, 50 / , 40 .
( 197 )
, .
,
100 (50:50) 80º
2 , , 60
40 , –
( 197 2–4 6, 240 5)
, . ,
, ,
(100 /) ,
1/10 , , 9/10
. –
,

, 50–100 /.
– ––.
1 4 –
10 . 40
780 , 160 40
57º [95].
3 M
(pH 10), 5
10 .
30º
60º 60
40 .
( 197 ) , .
62

80º 2 , ,
60 40 ,
– ( 240 )
, . ,
, ,
(100 /) , 1/10
, , 9/10
. – ––
.
(10–20 )
( – 5 3,
5 2) . ,
, 5

(3 ). 50º 50–100
30 , 1 5
, 5 ,
.
100 β–
1 57 .
1 5 ,
5 , 4 3000 /,
60º. 60
(50 /) 40

.
90 1

± 0.1 .
63
, 90
5
(1 – , 2 – 5α/5β–, 3 – , 4 – , 5 – 16–)
.
3, 4 5 (300 /)
, 1/10 ,
(3):
V = (Vk)/50 (3),
– (/)1, V – ,
(), k – “ ”2,
.
50º 50–100 40 .
(5
) 50 50
, 2 80° .
50º 50–
100 30 . 1 2
60 (50 /)
40 .
4 6
– III,
.
9/10
(100 /)
,
–/––. –
27.
1 ; 2 k = 0.8 , 5α–, 5β–, , , k = 0.6 16–.
64
[β–, 57oC, 1 ]
[pH ~ 9, – Et2O]

[Ac2O, , 80, 2 ]

3.1. –


.

.

, – [94].
1)
10
5 .
,
,
,
, .

13.

5α– 92±5 5β– 91±5
95±4 92±6 94±2 96±5
66
) , 10% –
90% 20% – 80% ( 4 ).
10 .
, ,
,
,
, (
) ,
.

28.

, .
2, 4 6 . 6
.
67
10 .
,
,
. ,

,
,

.
, ,
,
. .

29.

(
) 2, 4 6 . ,
6
, 4
.
3.2.
(
5α– – 5–50 /), (
10000 / ),
.
, ,
.
.

, ,
. ,
(90 ),
( )

. 250 ×
4.6 .
.

,
197 . 197 ,
.
, 90


69
(
50 ,
).
.
, , 70%
, .
/ =
60/40.
14. –
,

( 30).
(
). , ,
,
.

1 , , % , %
0 70 30 20 0 100 30 0 100 35 70 30 40 70 30
70
.
: 1 – , 2 – 5α/5β–, 3 – /, 4 – , 5 – 16–.
– ( )

5α/β–.
,
–.
,
( 14),
240 . 5α/5β–
, 70% ,
( 15).
71

2 , , % , %
0 30 70 33 0 100 38 0 100 43 30 70 48 30 70
16

,
.
16

1, 197
11.60–12.60 5α––3α,17β–
13.20–14.50 5β––3α,17β–
14.80–16.30
16(5α)––3α– 23.40–24.40
1 1, 240 9.50–10.50
2 2, 192
5α––3α,17β–
3.3.
, 5α/5β–,
(2––N,N,N––2––
). [80,81] ,
72
. ,
. ,
(, )
60…