Importance of Performing Incurred Sample Stability (ISS) for having a Rugged
and Accurate Omega-3 Bioanalytical Method
Catherine Dicaire, Mathieu Lahaie, Jean-Nicholas Mess, Milton Furtado, Fabio Garofolo and Christopher Perkin*
Algorithme Pharma Inc., Laval (Montreal), QC, Canada
OVERVIEW
• Purpose
– To investigate the short-term stability of free EPA in
incurred samples.
– To show the necessity of performing incurred sample
stability (ISS).
– To ensure a rugged and accurate bioanalytical assay.
• Method
– An ISS evaluation was performed and the results
triggered an investigation.
– The variation of endogenous levels of free EPA was
monitored in plasma/blood generated from 12 healthy
volunteers under various conditions.
– The stability of incurred samples was compared to the
stability of quality control samples (QCs).
– A stabilization procedure was developed.
• Results
– The endogenous level of EPA increased in incurred
samples, while being stable in QCs.
– The increase was donor dependent.
– This deconjugation mainly occurred during the
short-term stability at 4ºC.
– The anti-coagulant used had an impact on conversion.
– An acid preservative was needed to stabilize the incurred
samples.
INTRODUCTION
Eicosapentaenoic acid (EPA) and docosahexaenoic acid
(DHA) are essential omega-3 fatty acids (Figure 1). They are
important for healthy cardiovascular, brain and retina functions.
These supplements are offered mainly as ethyl esters (EE),
synthetically produced forms of fats. The recent interest in
having these compounds as prescribed drugs rather than
over-the-counter (OTC) supplements is requiring the need to
develop rugged and accurate quantitative bioanalytical assays.
This case study shows an increase of EPA concentration
over-time during stability testing of incurred samples. This
phenomenon was not observed in QCs under the same
conditions, thus explaining the need of performing ISS.
Specific sample handling requirements were established in
order to preserve sample integrity.
Figure 1: EPA and DHA Structures
METHODS
SAMPLE PREPARATION
• Calibration curves were prepared in proxy matrix and QCs
were prepared both in proxy and human plasma due to the
presence of endogenous levels of EPA and DHA.
CHROMATOGRAPHY
• Reverse-phase (C18) gradient elution using HPLC Agilent
Technologies Series 1100 pumps and autosampler
DETECTION
• Turbo Ion Spray ESI(-) AB SCIEX LC-MS/MS API5000
Analyte
SRM transitions (MRM)
EPA
301.2/257.2
EPA-D5
306.3/262.3
DHA
327.2/283.2
DHA-D5
332.3/288.3
Table 2: Investigation Results for EPA Stability
Sample ID
Freeze-Thaw Cycle:
Cummulative ShortTerm (hours):
RESULTS
The analytical method for EPA and DHA was fully validated
including short-term, freeze-thaw and long-term stabilities of
EPA and DHA in proxy and human plasma matrices (K2EDTA
and K3EDTA) (partial results presented in Table 1).
Table 1: Stabilities of EPA and DHA in Human Plasma
(K2EDTA)
EPA
Stability
Concentration
% Nominal
% Deviation
Concentration
% Nominal
% Deviation
Concentration
% Nominal
% Deviation
Short-Term Stability at a
Temperature of 4ºC
Freeze-Thaw Stability
(Frozen at -20ºC and
Thawed at 4ºC)
Long-Term Stability at a
Temperature of -20ºC
Duration
26.4 Hours
3 Cycles
47 Days
DHA
Low QC
High QC
Low QC
High QC
Comp. Stab. Comp. Stab. Comp. Stab. Comp. Stab.
37.9
33.4
103.2 96.0
-6.9%
37.9
781.8
721
98
86.5
-11.7%
104.7
96.2
-8.1%
Donor 03
Donor 04
Donor 05
Donor 06
Donor 07
111.1 99.1
-10.8%
Donor 10
52.5
688.3 746.3 312.4 303.7 2160.0 2044.8
91.8
99.5
8.4%
Donor 08
Donor 09
103.2 95.1
-7.8%
99.6 98.6
-0.7%
Donor 02
178.3 159.0 2154.0 2029.6
100.2 88.3
-11.9%
52.1
Donor 01
781.8 727.7 125.0 110.3 2026.2 1861.6
100.2 88.2
-11.9%
33.4
The results of the investigation confirmed an increase in the
concentrations for EPA. Furthermore, it showed that the
increase was donor dependent. When three freeze-thaw
cycles were performed on the incurred samples for a
cumulative short-term of ~15 hours, the increase in
concentrations varied from 15 – 95% for different donors. In
addition, the freeze-thaw cycles did not have a significant
impact on the conversion since different numbers of cycles
(zero, one or three) with similar short-terms (approximately
five hours) showed similar results. The major factor
influencing the conversion was the short-term period, as a
significant increase was observed for extended short-term
even
when
the
samples
were
left
at
4ºC
(5 hrs vs. 10 hrs vs. 15 hrs). (Table 2)
100
97.2
-2.8%
109.5 103.1
-5.8%
101.4
96.0
-5.3%
Donor 11
Donor 12
At first, incurred samples reanalysis (ISR) was performed
using two different aliquots for both analyses. This evaluation
showed acceptable results. Subsequently, a second ISR was
evaluated using the same aliquot for both analyses.
Accordingly, this ISR measured both the method robustness
and the stability of the samples since the second aliquot used
had undergone minimally an extra freeze-thaw cycle and an
extra short-term and long-term period; thus turning this ISR
into an ISS. The results of this evaluation showed a positive
trend for EPA, but not for DHA. To further investigate this
phenomenon, the blood/plasma of 12 healthy volunteers was
drawn and the stability of EPA and DHA endogenous levels
were monitored.
QC5
QC7
0
Concentration (ng/mL)
(% deviation vs. 0 F/T-0.5 hrs ST)
0
3
1
2
3
0.5 hrs
5 hrs
5 hrs
5 hrs
10 hrs
15 hrs
29.1
34.0
35.5
35.5
37.7
40.8
16.9%
22.1%
21.9%
29.6%
40.3%
136.9
150.2
148.5
157.0
155.0
4.5%
14.6%
13.4%
19.8%
18.3%
123.1
132.6
131.5
138.5
143.0
7.0%
15.3%
14.3%
20.4%
24.3%
123.1
132.6
131.5
138.5
143.0
7.0%
15.3%
14.3%
20.4%
24.3%
37.2
39.0
39.7
46.0
54.3
33.1%
39.5%
41.9%
64.7%
94.4%
363.9
373.2
353.7
384.0
391.0
6.1%
8.9%
3.2%
12.0%
14.1%
38.6
41.7
42.7
47.2
52.3
11.5%
20.7%
23.5%
36.3%
51.3%
122.0
128.4
129.6
129.9
145.0
6.4%
12.0%
13.0%
13.3%
26.4%
60.9
62.6
65.6
68.3
76.7
7.8%
10.7%
16.0%
20.9%
35.7%
80.1
86.5
86.5
93.4
104.6
15.9%
25.2%
25.2%
35.2%
51.4%
59.2
64.2
66.2
68.1
71.2
6.1%
15.0%
18.7%
22.1%
27.6%
113.5
121.1
125.8
133.4
152.0
10.5%
17.8%
22.5%
29.9%
47.9%
N/AV
N/AV
38.6
40.6
39.7
-5.2%
-0.2%
-2.4%
757.8
743.1
732.9
0.2%
-1.8%
-3.1%
131.0
115.0
115.0
28.0
342.8
34.6
114.7
56.5
69.1
55.8
102.7
40.7
756.4
N/AV
N/AV
Furthermore, the stability of incurred samples was compared
with the stability of QC samples. Table 2 shows that the
endogenous level of EPA increased steadily in incurred
samples whereas it stayed constant in QC samples. It is
suspected that the enzymatic activity of the freshly generated
plasma was greater than that of the commercial plasma used
for QC preparation. This difference could either be attributed
to the age of the commercial plasma or the addition of organic
solvent to the spiked samples.
The ISS evaluation clearly showed the need to stabilize the
incurred samples before analysis. First, blood kinetics were
performed in different anti-coagulants, using freshly drawn
blood, to verify whole blood short-term stability. This evaluation
showed that the samples were stable in blood for at least two
hours, which gave enough time to generate the plasma
(Table 3).
Table 3: Human Whole Blood (NaF/KOx vs.K3EDTA)
Kinetic Stability of EPA at a temperature of 4ºC
NaF/KOx
Plasma kinetics were then performed in different
anti-coagulants in freshly generated plasma in order to verify
the short-term stability of EPA before sample stabilization
(Table 4). The results showed that a stabilization procedure
needs to be applied to the incurred samples within six hours.
Furthermore, the results showed a slight difference in stability
for the different anti-coagulants used. The use of sodium
fluoride/potassium oxalate seemed to slightly inhibit the
conversion, which could be attributed to the NaF lipase and
esterase inhibitor properties.
Table 4: Human Plasma (NaF/KOx vs.K3EDTA)
Kinetic Stability of EPA at a temperature of 4ºC
Kinetic
Times (h)
0
1.5
3
4.5
6
Average
concentration
(ng/mL) (n=3)
38.2833
38.6452
40.0865
41.3518
42.1229
% Deviation
N/AP
0.9%
4.7%
8.0%
10.0%
Table 5: Stabilization Tests Results for EPA in human
plasma
K3EDTA
Average Peak
Average Peak
Kinetic
Area Ratio
% Deviation
Area Ratio
% Deviation
Times (min)
(n=3)
(n=3)
0
0.0657
N/AP
0.0826
N/AP
15
0.0645
-1.7%
0.0799
-3.3%
30
0.0671
2.2%
0.0801
-3.1%
60
0.0670
2.1%
0.0809
-2.2%
90
0.0682
3.8%
0.0834
0.9%
120
0.0670
2.0%
0.0851
3.0%
NaF/KOx
A significant difference was observed between the stability of
EPA in NaF/KOx anti-coagulant when compared to K2EDTA or
K3EDTA, although the use of NaF/KOx did not completely
prevent the conversion. The addition of extra enzyme
inhibitors such as PMFS, Physostigmine, Halt™ or
Complete™ did not further stabilize EPA. The acidification of
the plasma (0.5%) showed a significant improvement for the
residual conversion observed in NaF/KOx.
K3EDTA
Average
concentration
(ng/mL) (n=3)
47.1982
49.2925
50.5509
52.5919
57.1200
% Deviation
N/AP
4.4%
7.1%
11.4%
21.0%
In order to further investigate the incurred sample stability of
EPA in human plasma, different stabilization procedures were
tried, i.e. addition of different enzyme inhibitors and
acidification of plasma from different donors. The results of
these tests are presented in Table 5 (one donor). The other
donors showed similar results.
Donor
03
QC5
(40.9 ng/mL)
QC7
(756.4 ng/mL)
Anticoagulant
Preservative
Storage
temperature
Concentration Concentration
(ng/mL)
(ng/mL)
1 F/T Cycle +
Fresh
~6 hr ST
140.2
194.2
175.9
155.7
180.4
139.1
195.5
163.7
103.0
116.7
104.4
118.7
107.4
119.0
107.5
110.8
104.0
105.2
106.2
111.9
106.8
108.0
N/AP
K2EDTA N/AP
P800
N/AP
K3EDTA
N/AP
N/AP
PMFS
Physostigmine
NaF-KOx 0.05% acid
0.5% acid
Halt
Complete
-20ºC
-80ºC
-80ºC
-20ºC
-80ºC
K3EDTA
N/AP
-80ºC
38.3
K3EDTA
N/AP
-80ºC
730.1
-80ºC
38.5%
25.5%
28.7%
40.3%
17.5%
13.3%
15.3%
15.5%
7.6%
2.2%
8.6%
4.9%
Concentration
(ng/mL)
2 F/T Cycles +
~12 hr ST
212.7
190.6
191.7
208.8
173.1
112.2
121.5
112.1
115.9
107.3
109.4
109.3
38.6
-5.7%
757.8
0.2%
% Dev.
51.7%
36.0%
36.8%
49.9%
24.3%
9.0%
18.0%
8.9%
12.6%
4.2%
6.3%
6.2%
Concentration
(ng/mL)
3 F/T Cycles +
~20 hr ST
233.3
198.0
201.6
222.4
206.1
119.5
118.6
134.4
120.7
108.4
114.9
115.6
40.6
-0.7%
39.7
-2.9%
743.1
-1.8%
732.9
-3.1%
% Dev.
% Dev.
66.4%
41.2%
43.8%
59.6%
48.0%
16.0%
15.2%
30.6%
17.2%
5.3%
11.6%
12.3%
The 0.5% acidification procedure using NaF/KOx as
anti-coagulant was chosen as optimal to prevent EPA
deconjugation in incurred samples. The method was fully
validated.
CONCLUSION
•The stability performed on incurred samples showed an
increase in EPA concentration over-time.
•The phenomenon was not observed in QC samples, probably
because of different enzymatic activity between the fresh and
the commercial plasma.
•These results triggered an investigation which consisted of
monitoring the endogenous levels of free EPA in freshly drawn
blood/plasma.
•The investigation demonstrated that an extended short-term
storage of samples at 4ºC generated a considerable increase
in free EPA.
•The phenomenon was shown to be donor dependent.
•The deconjugation was totally prevented in acidified plasma
with NaF/KOx as an anti-coagulant.
•The evaluation of the ISS along with a thorough investigation
of the observed instability led to a better collection procedure
and a more rugged and accurate method for the quantification
of free EPA and DHA in human plasma.
* CORRESPONDING AUTHOR