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

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584.20582.20

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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

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0.25

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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

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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

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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

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500

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1500

9:442.20>295.10(+)

7.00 7.25 7.50 7.75 8.00 8.25 min

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8:245.10>227.10(+)

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250007:377.20>242.80(+)

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25

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75

100

125 6:678.55>146.80(+)

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5:220.10>90.00(+)

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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(+)

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250000 2:170.10>152.00(+)

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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

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2.75

3.00

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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