Transcript Streptomycin ( Shimadzu LCMS

IMTAKT HPLC SEMINAR
LC-MS Applications
ProdLine
LCMS-メタミドホス
LCMS-ジクロルボス
LCMS-メラミン
Streptomycin
アミノグリコシド系抗生物質
ODS表面での保持が期待できない
吸光団をもたない
アミノ基を有する
→イオンペアクロマトグラフィー/質量分析が利用されてきた
Streptomycin ( Shimadzu LCMS-2020 )
Mass chromatograms
350000
325000
350000
582.20
584.20
325000
Dihydrostreptomycin
20 ng
300000
300000
275000
275000
250000
250000
225000
225000
200000
200000
Streptomycin
20 ng
175000
150000
175000
150000
125000
125000
100000
100000
75000
75000
50000
50000
25000
25000
0
0
0.0
2.5
5.0
7.5
582.20
584.20
10.0
12.5
min
Imtakt Scherzo SM-C18 (2.0 mmI.D. x 150 mmL., 3 µm)
20 mmol/L ammonium acetate
/20 mmol/L acetic acid, gradient
0.0
Dihydrostreptomycin
20 ng
Streptomycin
20 ng
2.5
5.0
7.5
10.0
12.5
ODS (2.0 mmI.D. x 150 mmL., 3 µm)
5 mmol/L heptafluorobutyric acid
/acetonitrile = 85/15
min
Neomycin (Shimadzu LCMS-8030)
Mass chromatograms
(x10,000,000)
4.75 1:TIC(+)
1:615.30(+)
Q3 scan
4.50
Sample: 1 mg/mL
Neomycin
4.25
LC
Column: Imtakt Scherzo SM-C18 (2.0 mmI.D. x 150 mmL., 3 µm)
Mobile phase A: 20 mmol/L ammonium acetate - water
Mobile phase B: 20 mmol/L acetic acid - water
Gradient program: 10%B(0 min)-90%B(14.0 min)-10%B(14.01-24 min)
Flow rate: 0.2 mL/min
Injection volume: 2 µL
Column temperature: 40 ºC
4.00
3.75
3.50
3.25
3.00
2.75
MS
MS : SHIMADZU LCMS-8030
Nebulizing gas flow: 1.5 L/min
Drying gas flow: 10 L/min
DL temperature: 250 ºC
BH temperature: 400 ºC
Ionization: ESI(+)
2.50
2.25
2.00
1.75
1.50
1.25
1.00
0.75
0.50
0.25
0.00
0.0
2.5
5.0
7.5
10.0
12.5
15.0
min
Neomycin (Shimadzu LCMS-8030)
Mass spectra
Inten. (x1,000,000)
8.0
308.2
7.0
615.3
Q3 Scan
[M+H]+
[M+2H]2+
6.0
OH
5.0
H2N
OH
CH2NH 2
O
4.0
NH 2 O
H2N
HO
3.0
2.0
161
O CH OH
2
1.0
0.0
200
300
Inten. (x100,000)
400
500
600
700
O
Product ion scan
Precursor ion: m/z 615.3
161.1
4.0
455
m/z
OH
293
3.0
CH2NH 2
O
H2N
O
OH
OH
2.0
203.1
1.0
125.2
293.0
277.0
455.2
615.1
0.0
250
500
m/z
LCMS-テトラサイクリン
AgliChem (shimadzu LCMS-8030)
1:Glufosinate
182.00>136.00(+)
MRM Chromatograms
Sample: 1 µg/mL
2:Diquat
dibromide
184.05>158.05(+)
3:Acephate
184.00>143.00(+)
4:Fosetyl
109.00>81.05(-)
5:Dalapon
140.90>97.10(-)
LC
Column: Imtakt Scherzo SM-C18 (2.0 mmI.D. x 150 mmL., 3 µm)
Mobile phase A: 5mmol/L ammonium formate - water
Mobile phase B: 200mmol/L ammonium formate + 0.1% formic acid - acetonitrile
Gradient program: 0%B(0 min)-100%B(10.0 min)-0%B(10.01-15 min)
Flow rate: 0.2 mL/min
Injection volume: 2 µL
Column temperature: 40 ºC
MS
MS: SHIMADZU LCMS-8030
Nebulizing gas flow: 1.5 L/min
Drying gas flow: 10 L/min
DL temperature: 250 ºC
BH temperature: 400 ºC
Ionization: ESI(+, -)
LCMS-Wvit
W. Vitamin (Shimadzu LCMS-8030)
MRM Chromatograms
2400000
1: Thiamin
2300000
2200000
2: Pyridoxin
2100000
2000000
1900000
1:265.10>122.10
2:170.10>152.20
3:124.20>80.0
4:123.10>80.0
5:220.10>90.00
6:678.55>146.80
7:377.20>242.80
8:245.10>227.10
9:440.20>295.10
3: Nicotinic acid
1800000
1700000
4: Nicotinamide
1600000
1500000
5: Pantothenic acid
1400000
1300000
1200000
6: Cyanocobalamin
1100000
1000000
900000
7: Riboflavin
800000
700000
8: Biotin
600000
500000
9: Folic acid
400000
300000
200000
100000
0
-100000
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0 min
Sample: 10 µg/mL
LC
Column: Imtakt Scherzo SM-C18 (2.0 mmI.D. x 150 mmL., 3 µm)
Mobile phase A: 5mmol/L ammonium formate + 0.1% formic acid - water
Mobile phase B: acetonitrile
Gradient program: 0%B(0 min)-50%B(10.0 min)-0%B(10.01-20 min)
Flow rate: 0.2 mL/min
Injection volume: 1 µL
Column temperature: 40 ºC
MS
MS : SHIMADZU LCMS-8030
Nebulizing gas flow: 1.5 L/min
Drying gas flow: 10 L/min
DL temperature: 250 ºC
BH temperature: 400 ºC
Ionization: ESI(+)
W. Vitamin (Shimadzu LCMS-8030)
Calibration curves
m/z
range（ ng/mL)
correlation factor (r)
%RSD
Thiamin
266.10>122.10
0.5-500
0.9999
7.8
Pyridoxin
170.10>152.00
1-500
0.9993
7.0
Nicotinic acid
124.00>80.00
5-1,000
0.9993
12.1
Nicotinamide
123.10>80.00
5-1,000
0.9995
9.0
Panthothenic acid
220.10>90.00
5-100
0.9999
9.6
Cyanocobalamin
678.55>146.80
50-10,000
0.9992
6.5
Riboflavin
377.20>242.80
10-1,000
0.9999
12.2
Biotin
245.10>227.10
5-5,000
0.9993
7.7
Folic acid
442.20>295.10
10-5,000
0.9993
9.7
1: Thiamin
2: Pyridoxin
–ʐ
Ï (x100,000)
3: Nicotinic acid
–ʐ
Ï (x1,000,000)
–ʐ
Ï (x100,000)
7.5
5.0
2.0
0.75
1.0
5.0
0.5
2.5
0.50
2.5
0.25
0.0
0
250
”Z“x
0.00
6: Cyanocobalamin
–ʐ
Ï (x100,000)
250
”Z“x
7: Riboflavin
2.0
3.0
1.5
2.0
1.0
1.0
0.5
0.0
0
0.0
0
500
”Z“x
8: Biotin
–ʐ
Ï (x10,000)
4.0
5000
”Z“x
0.0
0.0
1.0
0
500
”Z“x
9: Folic acid
–ʐ
Ï (x1,000,000)
–ʐ
Ï (x100,000)
1.5
2.0
0
5: Pantothenic acid
–ʐ
Ï (x10,000)
7.5
1.5
1.00
4: Nicotinamide
–ʐ
Ï (x100,000)
1.0
1.0
0.5
0
500
”Z“x
0.0
0
2500
”Z“x
0.0
0
2500
”Z“x
0.0
0
50
”Z“x
W. Vitamin (Shimadzu LCMS-8030)
Sample Preparation
MRM Chromatograms
10 tablets
↓
Add 10 mL diluting Solution*1 and mix
for 30 seconds
then heat (65 ºC, 5 min) *2
↓
Mix for 30 seconds,
Centrifuge(12,000 rpm, 5 min)
↓
Centrifuge of supernatant
(3,000 rpm, 10 minutes)
↓
Filtrate (0.45 µm filter) of supernatant
*1:water/acetonitorile/acetic
*2:2
acid=94/5/1
times for mix & heat
1: Thiamin
250000
175000 1:265.10>122.10(+)
Pyridoxin
175000
125000
75000
100000
50000
75000
25000
Panthothenic acid
1.50
1.75
2.00
2.25
2.50
2.75 min
4: Nicotinamide
450000
Cyanocobalamin
Riboflavin
400000
Biotin
Folic acid
250000
50000
200000
40000
150000
30000
100000
20000
50000
10000
60µg
400µg
3.75
4.00
4.25
4.50
4.75 min
6: Cyanocobalamin
6:678.55>146.80(+)
125
100
75
50
25
0
0
5.0
7: Riboflavin
5.5
6.0
6.5
min
6.0
8: Biotin
7:377.20>242.80(+)
8:245.10>227.10(+)
7500
7.5
min
9:442.20>295.10(+)
1250
1000
750
1500
10000
7.0
1500
2000
12500
6.5
9: Folic acid
3500
22500
15000
35.6µg
3.50
60000
300000
2500
218.4µg
3.75 min
70000
350000
3000
6mg
3.50
5:220.10>90.00(+)
17500
6.9mg
3.25
5: Pantothenic acid
20000
25µg
3.00
80000
10mg
23.3µg
2.75
4:123.10>80.00(+)
11mg
10mg
100
2.50
25000
20.6mg
125
0
5.25mg
50mg
150
25000
0
15.6mg
43.4mg
175
50000
表記値
Nicotinamide
200
150000
100000
測定値
N.D.
3:124.00>80.00(+)
225
200000
125000
0
Nicotinic acid
3: Nicotinic acid
2:170.10>152.00(+)
225000
150000
4.00 4.25 4.50 4.75 5.00 5.25 5.50 min
Thiamin
2: Pyridoxin
1000
500
5000
500
2500
250
0
0
6.5
7.0
7.5
8.0
min
7.00
7.25
7.50
7.75
8.00
8.25 min
7.50
7.75
8.00
8.25
8.50
8.75 min
LCMS-WvitUSDA
LCMS-ViDOH
LCMS-PhosLipid
H2Blocker (Shimadzu LCMS-2020)
Mass chromatograms
(x1,000,000)
TIC(+)
4.50 253.00(+)
315.00(+)
4.25 338.00(+)(2.00)
Mass spectra
Inten. (x1,000,000)
Cimetidine
253
1.0
Ranitidine
NCN
H3C
4.00
CH 2SCH 2CH2NHC
0.5
HN
3.75
3.50
Cimetidine
275
0.0
200
3.25
NHCH3
N
505527 561
300
400
500
m/z
3.00
Inten. (x1,000,000)
2.75
Famotidine
Ranitidine
315
2.0
2.50
1.5
2.25
CHNO2
1.0
2.00
(H 3C)2NH2C
O
CH2SCH2CH2NHC
NHCH3
0.5
1.75
0.0
200
1.50
300
400
500
m/z
1.25
Inten. (x100,000)
1.00
H2N
0.50
2.5
0.00
0.0
189
2.0
3.0
4.0
5.0
6.0
min
Imtakt Scherzo SM-C18, (2.0 mmI.D. x 150 mmL., 3 µm)
40 mmol/L ammonium acetate/acetonitrile = 80/20
0.3 mL/min, 40 ºC
100 µg/mL, 1 µL
300
CH2SCH2CH2C
NH2
S
360
259
N
N
H2N
200
1.0
NSO2NH2
C
0.25
0.0
Famotidine
338
5.0
0.75
455
400
500
m/z
LCMS-抗うつ剤
LCMS-モルヒネ
LCMS-マリファナ代謝物
LCMS-Volicitin
LCMS-リン脂質
LCMS-セラミド
LCMS-ホルモン代謝物
LCMS-単糖
LCMS-砂糖
LCMS-配糖体（ソラニン）
LCMS-Metabolomics