Qualitative detection of furosemide in an urinary matrix using the
ThermoQuest LCQ; an example of an ISO quality norms based
approach for method validation
Laboratório de Análises de
Dopagem e Bioquímica
Susana M.S.S.Simões (), Helder Lopes, Douwe de Boer and Lesseps J.A.L. dos Reys
() Laboratório de Análises de Dopagem e Bioquímica, Instituto Nacional do Desporto, Av. Professor Egas Moniz (Estádio
Universitário) 1600-190 Lisboa.
Introduction
Furosemide is, as a doping agent, the most frequently detected diuretic in sport
drug testing programs. In this study a qualitative confirmation method was
developed to detect furosemide in an urinary matrix based on a LC/MS/MS
procedure using the negative Electrospray Ionization mode. The method was
validated according to the EUROCHEM Guide for method validation, which follows
ISO 17025 quality requirements. Parameters, which were considered to be
essential, were extraction recovery, limit of detection (LOD), limit of identification
(LOI) and selectivity.
The LOD proved to be 4 ng/mL. The limit of identification (LOI) was defined as
the lowest amount of the compound of interest in a sample that fulfils in house
validated identification criteria using equipment specific library search algorithm
routines. These routines were applied by comparing the spectrum of interest with
that of a reference spectrum obtained from a blank sample fortified with an
amount for furosemide and analysed under identical experimental conditions as
the spectrum of interest. The LOI for furosemide was identical to the LOD.
D :\D a ta \22J UL\LD (F ur.6ngm L)
22 -J ul-02 03:45:00 P M
R T: 0.00 - 16.00 S M : 9G
Relative Abundance
Experimental
80
60
40
20
1.27 1.74
0
100
Extraction procedure
NL: 1.55E 5
m /z= 28 4.5 0-2 85.50 F : c E S I F ull m s 2
329.00@ 28.0 0 [
200.00-40 0.00 ] M S
LD (F ur.6 ngm L)
8.18
100
2.61 3.44
3.87
4.86 5.4 1
9.01 9.28
6.56 6 .99
10.82
NL: 6.4 5E4
m /z= 28 6.5 0-2 87.50 F : c E S I F ull m s 2
331.0 0@ 28 .00 [
200.00-40 0.00 ] M S
LD (F ur.6 ngm L)
8.20
80
60
40
5 mL of urine with ISTD (mefruside)
↓
0.1 M phosphate buffer pH 6 to adjust to pH 6-7
↓
Condition a BondElut-LRC Certify SPE column with methanol and 0.1 M
phosphate buffer pH 6
↓
Draw the sample through the column
↓
Wash with 0.1 M phosphate buffer pH6/methanol (4:1; v/v), 1.0 M acetic acid
and hexane
↓
Elution with methylene chloride
↓
Evaporate the organic phase to dryness.
Reconstitute in 100 µL of mobile phase and analyse the sample by
LC/MS/MS
20
1.36 1.68
0
Relative Abundance
0
1
2
3.06
3
4.21
4
5.04
5.87
5
6
7.25 7.7 6
7
8.95 9.86 10.29 10 .76
8
T im e (m in )
9
10
11
12
13
14
100
328.88
50
246.99 261.13
0
44
308.98 328.22
286.94
LD(Fur.6ngm L)#409-422 RT:
8.08-8.32 AV: 7 NL: 5.72E4 F:
- c ESI Full m s 2
[email protected] [
200.00-400.00]
20
248.99
240
286.18
260
16
329.47
330.89
204.72
0
200
220
15
LD(Fur.6ngm L)#409-423 RT:
8.06-8.34 AV: 8 NL: 1.30E5 F:
- c ESI Full m s 2
[email protected] [
200.00-400.00]
284.95
300.84 313.09
280
300
m /z
320
340
360
380
400
Figure 2. Chromatograms and spectra of a sample containing furosemide (6 ng/mL).
Selectivity
Instrumental conditions
Selectivity was determined by checking for interferences caused by known and
unknown compounds that could be present in authentic samples. An interference
was defined as a signal of a diagnostic ion with a signal-to-noise ratio > 3 and a
resolution < 1.0. Resolution (RS) was determined by fortifying the sample, which
was associated with the interference, with a certain amount of furosemide. The
following formula was applied:
Instrument: HPLC TSP P4000 with a LCQ Advantage
Column: Hypersil ODS (125mm × 2.1 mm, 5 µm)
Mobile phase: solvent A: 100% CH3CN; B: 2 mM HCOONH4
Flow rate: 0.3 mL/min
Gradient program:
initial composition
10%A
gradient
10%A for 2 min
75%A in 20 min
75%A for 2 min
Mass spectrometric parameters
ionisation
ESI in negative mode
sheath gas flow rate
60 units
aux gas flow rate
20 units
capillary temperature
300 °C
tune method
specific
acquisition mode
MS/MS
number of segments
2
Furosemide conditions
precursor ion
m/z 329; m/z 331
collision energy
28%
isolation width
1 unit
ISTD conditions
precursor ion
m/z 381
collision energy
35%
isolation width
1 unit
RS = 2 ×
(t r2 − t r1 )
(w b1 − w b2 )
R T : 6 .0 0 - 1 0 .0 2
S M: 9 G
100
NL:
7 .0 0 E 5
t r1
95
90
T IC F : - c E S I
F u ll m s 2
3 2 9 .0 0 @ 2 8 .0 0 [
2 0 0 .0 0 - 4 0 0 .0 0 ]
M S B U -1 1 (fu r)
7 .9 1
85
80
75
70
Relative Abundance
65
t r2
60
55
50
8 .4 2
45
40
35
30
25
Results and Discussion
20
15
w b1
10
5
Extraction recovery
6 .0
The extraction recovery obtained using a special developed BondElut-LRC Certify
Solid Phase Extraction (SPE) method was 93%.
Limit of detection (LOD)
The LOD, defined as the lowest amount in a sample, that can be distinguished
from background noise, was calculated based on a calibration curve in the range
of a pre-estimated value of the LOD and linear regression analysis. The following
formula was applied:
LOD =
3.3 × S y/x
b
Relative areas
7 .2 0
6 .7 6
w b2
8 .9 8
6 .5
7 .0
7 .5
8 .0
T i m e ( m in )
8 .5
9 .6 9
9 .0
9 .5
9 .9 3
1 0 .0
Figure 3. An example of the presence of an interference (tr2) in a furosemide sample (tr1)
No known compounds were found to interfere. In relation to unknown compounds,
some interferences were noted, but these did not lead to false suspected
samples.
Conclusion
The LC/MS/MS combined with a SPE method proved to be a suitable qualitative
confirmation method of the furosemide presence in human urine samples.
References
Residuals
0.25
Eurochem Guide – The fitness for purpose of analytical methods (1998)
0.015
0.2
0.01
0.15
0.005
EAL-P11 – Validation of test methods (1997)
PGER-LADB-015 – Validacao de metodos de ensaio
Caslavska J.; Thormann W.. Rapid analysis of furosemide in human urine by capillary electrophoresis with laser-induced fluorescence and
electrospray ionisation-ion trap mass spectrometric detection. In: J. of Chromatography B., 770 (2002) 207-216
0
0.1
y = 0.0075x + 0.0102
R2 = 0.9703
0.05
-0.005 0
10
Conc. (ng/mL)
20
5
10
15
20
25
30
Sanz-Nebot V.; Toro I.; Berges R.; Ventura R.; Segura J.; Barbosa J.. Determination and characterization of diuretics in human urine by liquid
chromatography coupled to pneumatically assisted electrospray ionization mass spectrometry. In: J. of mass spectrom. 36 (2001) 652-657
-0.01
0
0
6 .5 3
0
30
-0.015
Figure 1. Calibration curve and residuals for the limit of detection (LOD)
Acknowledgements
The LADB acknowledge ThermoUNICAM for the financial support, which
made the presentation of this poster possible.
Laboratório de Análises de Dopagem e Bioquímica - Instituto Nacional do Desporto
Centro de Medicina Desportiva Av. Prof. Egas Moniz (Estádio Universitário), 1600-190 Lisbon - Portugal
Tel: +351 217954000 Fax: +351 217977529 [email protected]