Supporting Information
1
Method Validation
The method for the quantitative determination of etilefrine and oxilofrine in equine plasma and
urine were validated. The method was validated without enzyme hydrolysis in plasma whereas
in urine it was validated with enzyme hydrolysis.
1.1
Selectivity
The selectivity is the ability to determine and to differentiate the analyte unequivocally in the
presence of matrix components. The selectivity of the method is established by investigating
six different blank plasma and blank urine samples collected from six different horses for peaks
interfering with the detection of the analytes or the IS by the procedure described previously.
The chromatograms were evaluated for the absence of etilefrine, oxilofrine and phenylephrineD3 (IS).
1.2
Linearity
The linearity was determined through the analysis of series of matrix fortified calibration
standards. The plasma calibration standards were prepared in the range of 0.1 to 20 ng/mL and
the urine calibration standards were prepared in the range of 1 to 200 ng/mL for both etilefrine
and oxilofrine. The linearity was assessed by applying weighted least squares models such as
the inverse of the concentration (1/x) or the inverse of the squared concentration (1/x 2) to
adequately compensate the heteroscedasticity.
1.3
Intra-day and inter-day precision and accuracy
Intra-day precision and accuracy were evaluated by analyzing six replicate matrix spiked
samples at three different concentrations levels for etilefrine and oxilofrine on the same day.
For inter-day precision and accuracy, three batches of six replicates matrix spiked samples at
the same concentrations levels as intra-day were analyzed on three different days. The matrix
spikes were prepared at 0.4, 4 and 16 ng/mL levels for plasma and at 4, 16 and 60 ng/mL levels
for urine.
1.4
Limit of detection (LOD)
The LOD of the method is the lowest concentration of an analyte in a sample that the method
can reliably distinguish from background noise. For LOD signal-to-noise (S/N) equal to or
greater than five is chosen (S/N ≥ 5).
1.5
Matrix effects (ion suppression/enhancement)
Suppression or enhancement of analyte ionisation by co-eluting compounds is a well
characterized phenomenon in HPLC-MS-MS analysis. It depends on the sample matrix, the
sample preparation procedure, chromatographic resolution and condition, mobile phase
additives and ionisation type. The matrix effect was determined by comparing peak areas of
neat standard solutions prepared in methanol with those ones prepared in blank matrix
extracts. For plasma 0.4 ng/mL and 16 ng/mL levels were used and for urine 4 ng/mL and 60
ng/mL levels were used for both etilefrine and oxilofrine.
2
Stability
2.1
Stability of etilefrine and oxilofrine in equine Plasma and Urine
To evaluate the stability of etilefrine and oxilofrine in matrix at different storage conditions,
equine plasma and urine were spiked with etilefrine and oxilofrine in triplicates and stored at
room temperature (~25C), refrigerator (2-8C) and in a freezer (-20C). The plasma spikes
were prepared at 1 and 15 ng/mL levels whereas the urine spikes were prepared at 10 and 80
ng/mL levels.
Stability of etilefrine and oxilofrine in equine plasma and urine under different storage
condition
Storage
condition
Matrix
Analyte
At 25C for
24 hours
Plasma
Etilefrine
Oxilofrine
Urine
Etilefrine
Oxilofrine
Nominal
Concentration
(ng/mL)
1
15
1
15
10
80
10
Calculated
Mean Value
(n=3)
0.97
12.2
0.77
13.3
13.5
69.2
10.2
%
Nominal
97%
81%
77%
88%
135%
87%
102%
At 4C for
72 hours
Plasma
Etilefrine
Oxilofrine
Urine
Etilefrine
Oxilofrine
At -20C for Plasma
7 days
Etilefrine
Oxilofrine
Urine
Etilefrine
Oxilofrine
At -20C for Plasma
30 days
Etilefrine
Oxilofrine
Urine
Etilefrine
Oxilofrine
2.2
80
1
15
1
15
10
80
10
80
1
15
1
15
10
80
10
80
1
15
1
15
10
80
10
80
72.7
1.03
10.7
0.97
12.2
9.5
63.7
11.2
68.9
1.4
12.6
0.95
12.7
11.8
76.8
9.8
76.4
1.1
14.8
1.03
14.8
12.1
86
13.1
100
91%
103%
71%
97%
81%
95%
80%
112%
86%
140%
84%
95%
85%
118%
96%
98%
96%
110%
99%
103%
99%
121%
108%
131%
125%
Stability of etilefrine and oxilofrine standard solutions
Stability of etilefrine and oxilofrine standard solutions at different storage conditions was
determined by keeping 10 ng/mL and 100 ng/mL standard solutions prepared in duplicates at
room temperature (25C), refrigerator (4C) and in freezer (-20C) for 24 hours, 4 days and 7
days respectively.
The working standard solutions of etilefrine and oxilofrine are very stable at all storage
conditions studied.
3
Dilution accuracy and precision
To demonstrate the ability to dilute and analyze samples with etilefrine and oxilofrine
concentrations above the upper limit of quantitation, plasma samples spiked with etilefrine and
oxilofrine (60 ng/mL) in triplicates were diluted ten times with blank plasma (to 6 ng/mL) and
urine samples spiked with etilefrine and oxilofrine (15 µg/mL) in triplicates were diluted
hundred times with blank urine (to 150 ng/mL) prior to sample preparation described in this
paper. The calculated mean concentrations from the analysis were further converted to the
original sample concentrations to compare with nominal concentration (60 ng/mL for plasma
and 15 µg/mL for urine) for dilution accuracy and precision.
Dilution accuracy and precision of etilefrine and oxilofrine spikes in equine plasma (15 µg/mL)
and urine (60 ng/mL)
Original
Concentration
Nominal
Concentration
After Dilution
Calculated
Concentration
Conversion to
Original
Concentration
Plasma-
6 ng/mL
5.75 ng/mL
57.5 ng/mL
Etilefrine
(10 times
5.52 ng/mL
55.2 ng/mL
60 ng/mL
dilution)
5.75 ng/mL
57.5 ng/mL
Plasma-
6 ng/mL
6.00 ng/mL
60.0 ng/mL
Oxilofrine
(10 times
5.74 ng/mL
57.4 ng/mL
60 ng/mL
dilution)
6.10 ng/mL
61.0 ng/mL
Urine-
150 ng/mL
155.60 ng/mL
15.56 µg/mL
Etilefrine
(100 times
151.85 ng/mL
15.19 µg/mL
15 µg/mL
dilution)
146.94 ng/mL
14.69 µg/mL
Urine-
150 ng/mL
157.32 ng/mL
15.73 µg/mL
Oxilofrine
(100 times
155.20 ng/mL
15.52 µg/mL
15 µg/mL
dilution)
143.61 ng/mL
14.36 µg/mL
4
Mean
Concentration
%
Accuracy
Precision
(%RSD)
56.7 ng/mL
95%
2.30%
61.0 ng/mL
102%
3.11%
15.1 µg/mL
101%
2.65%
15.2
101%
4.61%
Product ion mass spectra
The enhanced product ion mass spectra of etilefrine and oxilofrine are shown below. The
source and compound dependent mass spectrometric parameters were optimized for the three
most intense product ions. For etilefrine product ions having m/z 135, 109 and 91 were
optimized whereas for oxilofrine product ions having m/z 149.1, 105.2 and 133.1 were
optimized. These optimized transitions were used to develop the MRM method to detect and
quantify etilefrine and oxilofrine in equine plasma and urine. The product ions arising from the
loss of non-specific fragments such as water and ammonia were avoided in the MRM method.
5
EPI scan mass spectrometric parameters used for the identification of etilefrine
metabolites in equine plasma and urine samples
Metabolite Name
Positive Mode ionization
m/z
Collision
Energy (CE)
(V)
Etilefrine glucuronides
358
Etilefrine sulfates
Tetrahydroisoquinoline
Negative Mode ionization
m/z
Collision
Energy (CE)
(V)
30
Collision
Energy
Spread
(CES) (V)
15
356
-30
Collision
Energy
Spread
(CES) (V)
-15
262
30
15
260
-30
-15
194
30
15
370
30
15
368
-30
-15
Metabolites
Tetrahydroisoquinoline
glucuronide Metabolites
Tetrahydroisoquinoline
274
30
15
182
30
5
sulfate Metabolites
Etilefrine (Parent)
272
-30
-15