754
N. A. Manaf et al.
new (or modification of) analytical methodologies through the replacement of toxic
reagents by smaller amounts of safer reagents, preferentially obtained from renewable sources (Gałuszka et al. 2013; Pena-Pereira et al. 2015). Several strategies have
been used in LC–MS/MS, such as the reduction of the internal diameter and particle size (sub-2 μm) of chromatographic columns (to diminish eluent consumption),
and the replacement of conventional mobile phases (consisting of acetonitrile and/or
methanol) by environmental friendly alternatives like water, ethanol, and carbon
dioxide in the particular case of supercritical fluid chromatography (Shaaban and
Gorecki 2015; Shaaban 2016).
The use of smaller internal diameter and particle size (sub-2 μm) of chromatographic columns has contributed to it economic and environmental advantages
(Joseph et al. 2014). As the column diameter decreases, so does the volume of
mobile phase, which can lead to a considerable decrease in operational costs, especially when expensive mobile phase components are used such ultrapure modifiers
or chiral additives. Likewise, reduced volumes of mobile phase obviously also are
less of an environmental burden. As an example, a conventional 4.6-mm column
typically operates at a flow-rate of 1 mL/min and will require about 1.5 L of solvent
a day, a large percentage of which is an organic modifier (Joseph et al. 2014). A
micro-LC system refers to LC using columns whose inner diameter is ≤0.5 mm,
commonly 0.3 mm or using a 75 μm column only requires 25 mL of solvent per day.
Depending on the percentage and type of modifier, this can represent a large cost
saving on a yearly basis.
In this study, chromatographic analysis was performed with Thermo Scientific
TSQ Quantum Access MAX equipped with Accela autosampler and Accela 1250
Liquid-chromatography pump and Xcalibur software (version 2.1) for data processing and analysis. Acetonitrile and formic acid (0.1%) (Mobile phase A) and water and
formic acid (0.1%) (Mobile phase B) was used as the mobile phase in the experiment.
The analytes (10 μL) were separated using Hypersil Gold column (50 × 2.1 mm,
1.9 μm) from Thermo Fisher Scientific with gradient programs and column flow rate
was set at 200 μl min
−1 . No derivatising agent was used in LC-MS/MS analysis. The
50 mm column resulted in total running time for LC-MS/MS at 10 min including
equilibrium time, which is optimal for steroid profile analysis.
7 VALLME as a Sample Preparation Technique
Extraction was performed as shown in Fig. 2 (i) Urine sample (5 mL) which was
added with endogenous steroid and deuterated internal standards was placed in a
round bottom glass tube (10 mL). (ii) A 0.5 mL of E. coli in phosphate buffer
(pH 6.4) was added into each tube and mixed. (iii) The mixture was then placed
in the ultrasonic bath for 30 min at 50 °C for hydrolysis and then allowed to cool
down to room temperature. (iv) Each extracting solvent (150 μL) (1-hexanol, 1heptanol, 1-pentanol, 1-octanol, pentane, hexane, octane and tridecane) was added to
the mixture and vortexed mixed at high speed. (v) The mixture was next centrifuged.
N. A. Manaf et al.
new (or modification of) analytical methodologies through the replacement of toxic
reagents by smaller amounts of safer reagents, preferentially obtained from renewable sources (Gałuszka et al. 2013; Pena-Pereira et al. 2015). Several strategies have
been used in LC–MS/MS, such as the reduction of the internal diameter and particle size (sub-2 μm) of chromatographic columns (to diminish eluent consumption),
and the replacement of conventional mobile phases (consisting of acetonitrile and/or
methanol) by environmental friendly alternatives like water, ethanol, and carbon
dioxide in the particular case of supercritical fluid chromatography (Shaaban and
Gorecki 2015; Shaaban 2016).
The use of smaller internal diameter and particle size (sub-2 μm) of chromatographic columns has contributed to it economic and environmental advantages
(Joseph et al. 2014). As the column diameter decreases, so does the volume of
mobile phase, which can lead to a considerable decrease in operational costs, especially when expensive mobile phase components are used such ultrapure modifiers
or chiral additives. Likewise, reduced volumes of mobile phase obviously also are
less of an environmental burden. As an example, a conventional 4.6-mm column
typically operates at a flow-rate of 1 mL/min and will require about 1.5 L of solvent
a day, a large percentage of which is an organic modifier (Joseph et al. 2014). A
micro-LC system refers to LC using columns whose inner diameter is ≤0.5 mm,
commonly 0.3 mm or using a 75 μm column only requires 25 mL of solvent per day.
Depending on the percentage and type of modifier, this can represent a large cost
saving on a yearly basis.
In this study, chromatographic analysis was performed with Thermo Scientific
TSQ Quantum Access MAX equipped with Accela autosampler and Accela 1250
Liquid-chromatography pump and Xcalibur software (version 2.1) for data processing and analysis. Acetonitrile and formic acid (0.1%) (Mobile phase A) and water and
formic acid (0.1%) (Mobile phase B) was used as the mobile phase in the experiment.
The analytes (10 μL) were separated using Hypersil Gold column (50 × 2.1 mm,
1.9 μm) from Thermo Fisher Scientific with gradient programs and column flow rate
was set at 200 μl min
−1 . No derivatising agent was used in LC-MS/MS analysis. The
50 mm column resulted in total running time for LC-MS/MS at 10 min including
equilibrium time, which is optimal for steroid profile analysis.
7 VALLME as a Sample Preparation Technique
Extraction was performed as shown in Fig. 2 (i) Urine sample (5 mL) which was
added with endogenous steroid and deuterated internal standards was placed in a
round bottom glass tube (10 mL). (ii) A 0.5 mL of E. coli in phosphate buffer
(pH 6.4) was added into each tube and mixed. (iii) The mixture was then placed
in the ultrasonic bath for 30 min at 50 °C for hydrolysis and then allowed to cool
down to room temperature. (iv) Each extracting solvent (150 μL) (1-hexanol, 1heptanol, 1-pentanol, 1-octanol, pentane, hexane, octane and tridecane) was added to
the mixture and vortexed mixed at high speed. (v) The mixture was next centrifuged.