756
N. A. Manaf et al.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
Testosterone
Epitestosterone
Androsterone Etiocholanolone 5 alpha Adiol
5 beta Adiol
Peak Intensity (10 6
)
Compound
1-Pentanol
Pentane
Hexane
Octane
Tridecane
1-Hexanol
1-Heptanol
1-Octanol
Fig. 3 Selection of different organic solvent on optimising VALLME. Conditions: sample volume,
5 mL; solvent volume, 150 μL, vortex time, 40 s, centrifuge speed and time, 1000 g for 1 min and
no salt addition needed for the extraction
the floating extractant phase restores its initial single-drop shape for the following
instrumental analysis.
The alcohol group solvents were considered because of their good solubility of
hydroxyl steroids (Qiang et al. 2005). Usually in routine analysis, this endogenous
steroid will be extracted using ter-butyl methyl ether (TBME) or diethyl ether but
when small amount of these solvents are used, they form a miscible layer with water
due to their solubility in water. Methanol is a good solvent for steroid but methanol,
ethanol, propanol and butanol are miscible with water. So the longer carbon chain
solvents such as 1-pentanol, 1-hexanol, 1-heptanol and 1-octanol are suggested. It
is clear that 1-pentanol is the best extraction solvent compared to the other solvents
(Fig. 3). So 1-pentanol was chosen for the rest of the studies.
A few other parameters have been optimised and validated as shown in Table 1. The
adopted extraction conditions for 5 mL sample were: extraction solvent, 1-pentanol;
volume of extractant, 150 μL; vortex time, 40 s; centrifuge speed and time, 1000 g
for 1 min and no salt addition needed for the extraction (Table 1). The VALLME
method is rapid (approximately 20 min) as compared to the standard method based
on SPE and LLE (more than 2 h), excluding hydrolysis time.
8 Conclusion
This study present an interesting approach using VALLME as sample preparation
followed by LC-MS/MS analysis for the simultaneous determination of endogenous
steroid profile in urine. The method is rapid, simple and robust and is for the first
N. A. Manaf et al.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
Testosterone
Epitestosterone
Androsterone Etiocholanolone 5 alpha Adiol
5 beta Adiol
Peak Intensity (10 6
)
Compound
1-Pentanol
Pentane
Hexane
Octane
Tridecane
1-Hexanol
1-Heptanol
1-Octanol
Fig. 3 Selection of different organic solvent on optimising VALLME. Conditions: sample volume,
5 mL; solvent volume, 150 μL, vortex time, 40 s, centrifuge speed and time, 1000 g for 1 min and
no salt addition needed for the extraction
the floating extractant phase restores its initial single-drop shape for the following
instrumental analysis.
The alcohol group solvents were considered because of their good solubility of
hydroxyl steroids (Qiang et al. 2005). Usually in routine analysis, this endogenous
steroid will be extracted using ter-butyl methyl ether (TBME) or diethyl ether but
when small amount of these solvents are used, they form a miscible layer with water
due to their solubility in water. Methanol is a good solvent for steroid but methanol,
ethanol, propanol and butanol are miscible with water. So the longer carbon chain
solvents such as 1-pentanol, 1-hexanol, 1-heptanol and 1-octanol are suggested. It
is clear that 1-pentanol is the best extraction solvent compared to the other solvents
(Fig. 3). So 1-pentanol was chosen for the rest of the studies.
A few other parameters have been optimised and validated as shown in Table 1. The
adopted extraction conditions for 5 mL sample were: extraction solvent, 1-pentanol;
volume of extractant, 150 μL; vortex time, 40 s; centrifuge speed and time, 1000 g
for 1 min and no salt addition needed for the extraction (Table 1). The VALLME
method is rapid (approximately 20 min) as compared to the standard method based
on SPE and LLE (more than 2 h), excluding hydrolysis time.
8 Conclusion
This study present an interesting approach using VALLME as sample preparation
followed by LC-MS/MS analysis for the simultaneous determination of endogenous
steroid profile in urine. The method is rapid, simple and robust and is for the first