Table 2: Sample preparation, instrumental analysis, validation parameters and application of sent standard mixture for laboratory coded 2
COMPOUND
CP
SAMPLE PREP
Sample preparation
(sample pre-treatment: pH adjustment, filter
pore size, material and trade name,
extraction method and conditions, additional
clean-up, derivatisation, internal standard
…)
extraction: June 10, 2013
1º) Samples A, B, C, D thawed (1h; 40°C)
and 10 min sonicated (sonication bath)
2º) Samples divided into two aliquots
(2x300ml); isotopically labelled internal
standards (surrogate standard, CP-4D)
added into one of the aliquots
3º) Filtration of samples through glass
microfibres filter (100% borosilicate glass;
1.2 µm; 47mm)
4º) Solid phase extraction SPE of the
samples; Strata-X (Phenomenex,
200mg/6ml); cartridges conditioned with
methanol (5ml) and then with acetate buffer,
5 mM, pH = 4 (5ml), no backpressure
applied during conditioning; sample
application (300 ml, flow rate 1-2 ml/min);
column dried for 40 min (air passing with
backpressure); extract eluted by 5 ml of
methanol/acetonitrile (1:1 v/v)
5º) Solvent evaporated under gentle stream
of nitrogen until the last drop
6º) Extracts dissolved in 1ml of acetate
INTRUM ANALYSIS
Instrumental analysis
(operational parameters: separation
and detection condition,
…)
analyses: 06/17/2013
- Analysis by LC-MS/MS
- Equipment: Agilent system 1200
coupled with a 6410 Triple-Quad
MS (Agilent Technologies Inc.,
USA), with an ESI operating in a
positive ion mode.
- LC separation of 10 μl of the
sample on a reversed-phase
column Zorbax SB-C18 column
(2.1 × 30 mm, 3.5 μm; Agilent
Technologies Inc., Wilmington,
DE, USA) maintained at 30°C.
Acetate buffer 5 mM, pH = 4 (A)
and acetonitrile (B) were used as
mobile phase (flow rate 0.3 mL
min-1). LC linear gradient: 0-6
min, 10%-25% B; 6-7 min, 25%80% B; 7-10 min, 80%B; 10-15
min, 10% B; retention time of CP
(CP-4D) 6.2 ± 0.1 min;
calibration curve ranged from
0.36 to 36 ng/ml of CP.
- Internal standard (CP-4D in
acetate buffer) was added by
automated injection programme at
HPLC (100 pg/column)
- MS/MS conditions: positive ESI;
VALIDATION PARAM
Validation parameters
(sensitivity, accuracy, recovery,
reproducibility, repeatability,
…)
- Quantification were
performed using an
external calibration
procedure of CP
considering internal
standard (CP-4D)
- Calibration curves prepared
in acetate buffer (5mM, pH
4)
- Linearity: determination
coefficient r2 = 0.9992
- Sensitivity: method limit of
quantification (s/n 10) = 0.8
ng/L in surface water and
in wastewater.
- Accuracy: relative recovery
(calculated with respect to
the IS CP-4D, at 100 ng/L
spiking level, in surface
and wastewater, n=4) = 87120%.
- Repeatability: relative
standard deviation (RSD, in
surface and wastewater,
spiking level 100 ng/L,
n=4) = 11%.
Application of sent mixture
a. Standard mixture was used
for calibration
b. Standard mixture was used
for check-up
c.Standard mixture was not
used
Standard mixture was used only
for comparison purposes_
STD MIX LAB2 0,51mg/ml
with CP monohydrate was
diluted with acetate buffer and
analysed (determined
concentration of CP
monohydrate was 0.425
mg/ml).
buffer (5 mM, pH = 4) using sonication in
bath (3 min); residual particles observed in
sample D removed by filtration (0.45 µm,
nylon)
7º) Extracts frozen at -18°C prior analyses
and then analyzed as described
8º) For the samples where CP was out of the
range of the calibration curve were further
diluted in the acetate buffer and re-analyzed
MRM mode with collision energy
22 eV; the capillary and cone
voltage 2000 V and 130 V,
respectively. The m/z transitions
261.1>140.0 for CP and
265.1>144.0 for CP-4D