タイトル imtakt hplc seminar lc-ms applications. prodline
TRANSCRIPT
IMTAKT HPLC IMTAKT HPLC SEMINARSEMINAR
LC-MS ApplicationsLC-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
0.0 2.5 5.0 7.5 10.0 12.5 min
0
25000
50000
75000
100000
125000
150000
175000
200000
225000
250000
275000
300000
325000
350000
584.20582.20
0.0 2.5 5.0 7.5 10.0 12.5 min
0
25000
50000
75000
100000
125000
150000
175000
200000
225000
250000
275000
300000
325000
350000
584.20582.20
Streptomycin ( Shimadzu LCMS-2020 )Mass chromatograms
Streptomycin20 ng
Streptomycin20 ng
Dihydrostreptomycin20 ng
Dihydrostreptomycin20 ng
Imtakt Scherzo SM-C18 (2.0 mmI.D. x 150 mmL., 3 µm)20 mmol/L ammonium acetate/20 mmol/L acetic acid, gradient
ODS (2.0 mmI.D. x 150 mmL., 3 µm)5 mmol/L heptafluorobutyric acid/acetonitrile = 85/15
0.0 2.5 5.0 7.5 10.0 12.5 15.0 min
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
2.50
2.75
3.00
3.25
3.50
3.75
4.00
4.25
4.50
4.75(x10,000,000)
1:615.30(+)1:TIC(+)
Neomycin
Neomycin (Shimadzu LCMS-8030)
Q3 scanSample: 1 mg/mL
LCColumn: Imtakt Scherzo SM-C18 (2.0 mmI.D. x 150 mmL., 3 µm)Mobile phase A: 20 mmol/L ammonium acetate - waterMobile phase B: 20 mmol/L acetic acid - waterGradient program: 10%B(0 min)-90%B(14.0 min)-10%B(14.01-24 min)Flow rate: 0.2 mL/minInjection volume: 2 µLColumn temperature: 40 ºC
MSMS : SHIMADZU LCMS-8030Nebulizing gas flow: 1.5 L/minDrying gas flow: 10 L/minDL temperature: 250 ºCBH temperature: 400 ºCIonization: ESI(+)
Mass chromatograms
250 500 m/z0. 0
1. 0
2. 0
3. 0
4. 0
I nten. (x100,000)
161.1
293.0203.1
277.0 615.1455.2125.2
200 300 400 500 600 700 m/z0. 0
1. 0
2. 0
3. 0
4. 0
5. 0
6. 0
7. 0
8. 0I nten. (x1,000,000)
308.2 615.3
[M+H]+[M+2H]2+
Q3 Scan
Product ion scanPrecursor ion: m/z 615.3
HO
O
ONH2
OH2N
OH
OH
O CH2NH2
H2N
O
H2N
OHOH
OH
CH2OH
OCH2NH2
Mass spectra
455
161
293
Neomycin (Shimadzu LCMS-8030)
LCMS-テトラサイクリン
0.0
5.0
(x100,000)
0.0
1.0
2.0
(x100,000)
0.5
1.0
(x10,000,000)
0.0
1.0
2.0
3.0(x100,000)
2.5 5.0 7.5 10.0 min0.0
2.5
(x100,000)
Sample: 1 µg/mL
LCColumn: Imtakt Scherzo SM-C18 (2.0 mmI.D. x 150 mmL., 3 µm)Mobile phase A: 5mmol/L ammonium formate - waterMobile phase B: 200mmol/L ammonium formate + 0.1% formic acid - acetonitrileGradient program: 0%B(0 min)-100%B(10.0 min)-0%B(10.01-15 min)Flow rate: 0.2 mL/minInjection volume: 2 µLColumn temperature: 40 ºC
MSMS: SHIMADZU LCMS-8030Nebulizing gas flow: 1.5 L/minDrying gas flow: 10 L/minDL temperature: 250 ºCBH temperature: 400 ºCIonization: ESI(+, -)
5:Dalapon140.90>97.10(-)
4:Fosetyl109.00>81.05(-)
3:Acephate184.00>143.00(+)
2:Diquat dibromide184.05>158.05(+)
1:Glufosinate182.00>136.00(+)
AgliChem (shimadzu LCMS-8030)
MRM Chromatograms
LCMS-Wvit
W. Vitamin (Shimadzu LCMS-8030)
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min-100000
0
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
1200000
1300000
1400000
1500000
1600000
1700000
1800000
1900000
2000000
2100000
2200000
2300000
2400000
8:245.10>227.10 7:377.20>242.80 6:678.55>146.80 5:220.10>90.00 4:123.10>80.0
3:124.20>80.0 2:170.10>152.20
1:265.10>122.10
9:440.20>295.10
1: Thiamin
2: Pyridoxin
3: Nicotinic acid
4: Nicotinamide
5: Pantothenic acid
6: Cyanocobalamin
7: Riboflavin
8: Biotin
9: Folic acid
Sample: 10 µg/mL
LCColumn: Imtakt Scherzo SM-C18 (2.0 mmI.D. x 150 mmL., 3 µm)Mobile phase A: 5mmol/L ammonium formate + 0.1% formic acid - waterMobile phase B: acetonitrileGradient program: 0%B(0 min)-50%B(10.0 min)-0%B(10.01-20 min)Flow rate: 0.2 mL/minInjection volume: 1 µLColumn temperature: 40 ºC
MSMS : SHIMADZU LCMS-8030Nebulizing gas flow: 1.5 L/minDrying gas flow: 10 L/minDL temperature: 250 ºCBH temperature: 400 ºCIonization: ESI(+)
MRM Chromatograms
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
2: Pyridoxin 3: Nicotinic acid 4: Nicotinamide 5: Pantothenic acid
6: Cyanocobalamin 7: Riboflavin 9: Folic acid
1: Thiamin
8: Biotin
0 250 ”Z“x0.00
0.25
0.50
0.75
1.00
–ÊÏ(x1,000,000)
0 250 ”Z“x0.0
2.5
5.0
7.5
–ÊÏ(x100,000)
0 500 ”Z“x0.0
0.5
1.0
1.5
–ÊÏ(x100,000)
0 500 ”Z“x0.0
2.5
5.0
7.5
–ÊÏ(x100,000)
0 50 ”Z“x0.0
1.0
2.0
–ÊÏ(x10,000)
0 5000 ”Z“x0.0
1.0
2.0
3.0
4.0
–ÊÏ(x100,000)
0 500 ”Z“x0.0
0.5
1.0
1.5
2.0
–ÊÏ(x10,000)
0 2500 ”Z“x0.0
1.0
2.0
–ÊÏ(x1,000,000)
0 2500 ”Z“x0.0
0.5
1.0
1.5
–ÊÏ(x100,000)
Calibration curves
W. Vitamin (Shimadzu LCMS-8030)
7.50 7.75 8.00 8.25 8.50 8.75 min
250
500
750
1000
1250
1500
9:442.20>295.10(+)
7.00 7.25 7.50 7.75 8.00 8.25 min
0
500
1000
1500
2000
2500
3000
3500
8:245.10>227.10(+)
6.5 7.0 7.5 8.0 min
0
2500
5000
7500
10000
12500
15000
17500
20000
22500
250007:377.20>242.80(+)
6.0 6.5 7.0 7.5 min
0
25
50
75
100
125 6:678.55>146.80(+)
5.0 5.5 6.0 6.5 min
0
10000
20000
30000
40000
50000
60000
70000
80000
5:220.10>90.00(+)
4.00 4.25 4.50 4.75 5.00 5.25 5.50 min
0
50000
100000
150000
200000
250000
300000
350000
400000
450000 4:123.10>80.00(+)
3.50 3.75 4.00 4.25 4.50 4.75 min100
125
150
175
200
225
3:124.00>80.00(+)
2.50 2.75 3.00 3.25 3.50 3.75 min
0
25000
50000
75000
100000
125000
150000
175000
200000
225000
250000 2:170.10>152.00(+)
1.50 1.75 2.00 2.25 2.50 2.75 min
0
25000
50000
75000
100000
125000
150000
1750001:265.10>122.10(+)
測定値 表記値Thiamin 15.6mg 5.25mg
Pyridoxin 11mg 10mg
Nicotinic acid N.D.
Nicotinamide 43.4mg 50mg
Panthothenic acid 20.6mg 10mg
Cyanocobalamin 23.3µg 25µg
Riboflavin 6.9mg 6mg
Biotin 35.6µg 60µg
Folic acid 218.4µg 400µg
2: Pyridoxin 3: Nicotinic acid
4: Nicotinamide 5: Pantothenic acid 6: Cyanocobalamin
7: Riboflavin 9: Folic acid
1: Thiamin
8: Biotin
10 tablets ↓Add 10 mL diluting Solution*1 and mix for 30 secondsthen 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 acid=94/5/1*2:2 times for mix & heat
Sample Preparation MRM Chromatograms
W. Vitamin (Shimadzu LCMS-8030)
LCMS-WvitUSDA
LCMS-ViDOH
LCMS-PhosLipid
H2Blocker (Shimadzu LCMS-2020)
0.0 1.0 2.0 3.0 4.0 5.0 6.0 min
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
2.50
2.75
3.00
3.25
3.50
3.75
4.00
4.25
4.50
(x1,000,000)
338.00(+)(2.00)315.00(+)253.00(+)TIC(+)
200 300 400 500 m/z0.0
0.5
1.0
Inten. (x1,000,000)
253
275 505527 561
200 300 400 500 m/z0.0
0.5
1.0
1.5
2.0
Inten. (x1,000,000)
315
200 300 400 500 m/z0.0
2.5
5.0
Inten. (x100,000)
338
360259189 455
Cimetidine
Ranitidine
Famotidine
Cimetidine
Ranitidine
Famotidine
Imtakt Scherzo SM-C18, (2.0 mmI.D. x 150 mmL., 3 µm)40 mmol/L ammonium acetate/acetonitrile = 80/200.3 mL/min, 40 ºC100 µg/mL, 1 µL
Mass chromatograms Mass spectra
NHN
H3C CH2SCH2CH2NHC
NCN
NHCH3
O(H3C)2NH2C CH2SCH2CH2NHC
CHNO2
NHCH3
S
N CH2SCH2CH2CNC
H2N
H2N
NSO2NH2
NH2
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