L-column3 Application Data Sheet for LC
Application No. L3051

Tocopherols
We found that tocopherol, a fat-soluble vitamin, is less polar and less sensitive in ESI. Despite this, in our L3047 application, we
successfully detected tocopherol in FIA-MS by using an ammonia eluent. Additionally, we compared the detection sensitivity oftocopherol in LC-MS/MS analysis with different types of eluents, and also compared ionization using APCI, commonly employed
for low-polarity component mass spectrometry, and ESI.
Key words: Fat-soluble vitamin vitamin E tocopherol LC-MS/MSColumn: USP category: L1[ Analytical conditions ]Column: L-column3 C18 (ODS, 2 μm, 12 nm); 2.1 mm I.D. × 50 mm L.; Cat. No. 813140
Eluent: A: Aqueous eluent; B: 2-Propanol/CH3CN (50/50)
A/B (20/80)
Flow rate: 0.4 mL/min
Temperature: 40 ℃
Detection: ESI-MS/MS (-)
Injection volume : 1 μL
System: LC: Ultimate 3000 Bio RS (Thermo Fisher Scientific K.K.); MS/MS: 3200 QTRAP (SCIEX)
Sample: Tocopherols, Vitamin EFig.1

R1R2m/zα-Tocopherol

CH3CH3429.3/163.0

β-Tocopherol

CH3H415.5/149.1

γ-Tocopherol

HCH3415.5/149.1

σ-Tocopherol

HH

401.5/135.1

Comparison of eluent type and detection sensitivity

Tocopherol, a fat-soluble vitamin, is known to be less polar and less sensitive to mass spectrometry whenusing conventional ESI. However, our study has shown that the addition of ammonia to the eluent cansignificantly improve detection sensitivity. This effect is attributed to the increased pH of the eluent, which
dissociates the phenol groups and enhances ionization of the tocopherol molecule.

L-column3 Application Data Sheet for LC

Application No. L3051

we compared the detection sensitivity of Electrospray Ionization (ESI) and Atmospheric Pressure Chemical Ionization (APCI)
techniques for HPLC analysis. The analysis was carried out in positive mode for APCI, using formic acid as the eluent, and innegative mode for ESI, using ammonia as the eluent.
[ Analytical conditions ]Column: L-column3 C18 (ODS, 2 μm, 12 nm); 2.1 mm I.D. × 50 mm L.; Cat. No. 813140
ESI A: 0.1% NH3 in H2O ; B: 2-Propanol/CH3CN (50/50)
Eluent:APCI A: 0.1% HCOOH in H2O ; B: 2-Propanol/CH3OH (50/50)
A/B (20/80)→(1/99) 0 min→5 min
Flow rate: 0.4 mL/min
Temperature: 40 ℃
Detection: ESI-MS/MS(-) or APCI-MS/MS(+)
Injection volume : 1 μL
System: LC: Ultimate 3000 Bio RS (Thermo Fisher Scientific K.K.); MS/MS: 3200 QTRAP (SCIEX)
Sample: 1 mg/L in CH3CN（each）

140000

ESI

R1

R2

ESI-MS/MS(-)
m/z

APCI-MS/MS(+)
m/z

CH3

CH3429.3/163.0

431.2/165.2

CH3H415.5/149.1

417.2/151.1

HCH3

415.5/149.1

417.2/151.1

HH

401.5/135.1

403.2/129.2

APCI

140000120000120000β, γ100000100000

80000

80000

60000

60000
δ

40000

40000
α20000

20000
000

1

2

34Time(min)

501

2

3

4Time(min)Fig.2 Comparison of detection sensitivity in ESI and APCI

By using an ammonia eluent, we achieved high sensitivity in the analysis of low-polarity tocopherols via ESI. Detection sensitivity
increased with higher concentrations of ammonia in the eluent. Despite the extended use of the ammonia eluent to collect thedata, L-column 3, which is highly durable, was still usable without requiring column replacement.
However, it is important to note that ammonia eluent can be corrosive to glass containers and metal pipes. Its reaction with CO2 in
the air may also result in changes in pH. Therefore, we recommend replacing the ammonia eluent within 1-2 days after use. If thesystem is not utilized for a prolonged period, it is also advisable to replace the ammonia solution with a solvent such as
water/methanol or water/acetonitrile.

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