Criteria for Performing a Scientifically Meaningful Lipemic Plasma Test during
LC-MS/MS Bioanalytical Method Validation (BMV): Which Plasma Type to Choose?
Laurence Mayrand-Provencher, Jean-Nicholas Mess, Milton Furtado, Josée Michon, Annik Bergeron, Isabelle Dumont, Mireille Nohra and Fabio Garofolo*
Algorithme Pharma Inc., Laval (Montreal), QC, Canada
O
N
O
R
P
O
OH
O
O
Fragment
O
N
O
R1
O
R2
P
O
OH
R, R1 and R2 are aliphatic chains.
LPC
O
PC
LIPEMIA INDEX DETERMINATION
• ROCHE Cobas 6000 Analyzer
• Absorbance measurement that provide a
quantitative representation of the level in lipemia
semi-
CV%
Average
CV%
Average
CV%
23
79
40
82
130
61.8
62.3
95.4
59.2
57.6
4.1
4.6
4.2
4.0
4.1
27.8
19.9
25.4
20.2
28.0
1.0
2.3
1.4
2.4
3.4
30.8
8.3
45.8
50.4
52.5
1.00
1.20
1.06
1.18
1.38
22.1
16.3
21.6
29.5
30.0
14
6
20
3
3
Figure 2: Typical Chromatograms Obtained for LPC
T I C o f + T O F P ro d u c t (5 2 0 . 3 ) : E x p 2 , f r o m
S a m p le
41
( S 0 6 ) o f L P I 7 1 3 - 0 2 . w i f f ( D u o S p ra y () )
M a x . 2 1 2 2 .0
cp s.
Figure 3: Typical Chromatograms Obtained for PC
T I C o f + T O F P ro d u c t (7 0 4 . 5 ) : E x p 6 , f r o m
S a m p le 4 1 ( S 0 6 ) o f L P I 7 1 3 - 0 2 . w i f f ( D u o S p ra y () )
M a x . 3 5 9 8 .0 c p s .
1 .6 e 5
3000
2000
The triglycerides concentration was found to be a good
indicator of overall lipemia. It was strongly correlated to the
phospholipids content (r=73%) and lipemia index (r=89%), but
weakly correlated to the cholesterol concentration (r=25%)
(Figure 5). The average total cholesterol concentration was
similar in all cases (Table 2) and there was no strong
correlation with any other variable tested.
1 .2 e 5
1 .0 e 5
8 .0 e 4
6 .0 e 4
4 .0 e 4
2 .0 e 4
0 .0
3 .0
4 .0
5 .0
m e , m in
TimeT i(min)
Figure 4: Data Normalized to Regular Donors (Normal)
6.00
0 .5
Regular Donors (All)
Postprandial Donors
Variables
High-Triglycerides Donors
4.00
1 .5
2 .0
2 .5
3 .0
3 .5
4 .0
3.00
Lipemia index
0.29
0.89
0.64
0.25
0.65
2.00
Cholesterol
0.29
1.00
Triglycerides
0.89
0.25
Phospholipids
0.64
0.65
0.73
0.00
Lipemia Index
Total
Triglycerides Phospholipids
Cholesterol
(mmol/L)
(mmol/L)
4 .5
5 .0
5 .5
e , m in
TimeT i m(min)
Figure 5: Pearson Correlation Coefficient (r)
Regular Donors (Non-Normal)
5.00
1 .0
Phospholipids
2 .0
Triglycerides
1 .0
Synthetic plasma mixes are not sufficiently concentrated in
phospholipids (Table 2) to be spiked in small volumes (1-2%
v/v) in regular plasma, which is desirable to avoid overdiluting
the matrix. Therefore, synthetic plasma mixes are not
recommended for lipemic plasma test in BMV.
1 .4 e 5
1000
0
The post-prandial plasma purchased was collected 90 min
after a high-fat meal. Plasma lots from high-triglycerides
donors were identified visually by the supplier (they were milky
white in all cases).
On average, the high-triglycerides donors lots tested
contained more triglycerides and more phospholipids than
post-prandial plasma lots (Table 2 and Figure 4).
5000
4000
Among the 20 regular lots tested, 14 were in the normal
concentration ranges for triglycerides (<1.70mM), an important
barometer of metabolic health (Table 2). These 14 “normal”
lots were used as the comparison for the phospholipids and
triglycerides relative content evaluation.
Large inter-lots variability are obtained for triglycerides and
phospholipids content of the post-prandial donors and hightriglycerides donors lots (Table 2).
1 .7 e 5
5595
Cholesterol
Fragment
Table 1: Phospholipids Transitions Monitored
Transition Monitored
Phospholipids
(Q1>TOF)
Lysophosphatidylcholine (18:2)
520.3>184.07
Lysophosphatidylcholine (18:1)
522.4>184.07
Lysophosphatidylcholine (18:0)
524.4>184.07
Lysophosphatidylcholine (20:4)
544.3>184.07
Phosphatidylcholine (30:1)
704.5>184.07
Phosphatidylcholine (34:2)
758.6>184.07
Phosphatidylcholine (34:1)
760.6>184.07
Phosphatidylcholine (36:3)
784.6>184.07
Phosphatidylcholine (36:2)
786.6>184.07
Phosphatidylcholine (38:6)
806.6>184.07
Phosphatidylcholine (38:5)
808.6>184.07
Phosphatidylcholine (38:4)
810.6>184.07
Average
Synthetic Plasma
0.67
13.6
3.5
694
1
Lipisol 10%:Normal Plasma 50:50% v/v
0.71
0.5
2.8
12
1
Intralipid 20%:Normal Plasma 0:100% v/v
0.81(1)
5.2
2.8
204
1
Intralipid 20%:Normal Plasma 1:99% v/v
0.87(1)
10.4
2.8
392
1
Intralipid 20%:Normal Plasma 2:98% v/v
1.00(1)
19.3
2.8
762
1
Intralipid 20%:Normal Plasma 4:96% v/v
1.06(1)
25.1
2.8
972
1
Intralipid 20%:Normal Plasma 5:95% v/v
2.39
122.8
2.3
3366
1
Intralipid 20%:Normal Plasma 25:75% v/v
3.86
242.4
3.1
8952
1
Intralipid 20%:Normal Plasma 50:50% v/v
5.16
5.1
18016
1
Intralipid 20%:Normal Plasma 100:0% v/v
485.2(2)
(1) Data obtained from a linear interpolation (3 points, R2 = 0.9985). (2) Data obtained from a linear extrapolation (7 points, R2 = 1.0000).
Detection
• AB SCIEX TripleTOFTM5600
• Electrospray, positive ionization mode
• DP = 150V, CE = 50V, Mass extraction window = 60mDa
• Twelve transitions between monocharged phospholipids
and their 184.07 m/z fragment were monitored (Figure 1
and Table 1).
Data Processing
• Analyst TF 1.5 software was used for data acquisition.
• Peak integration was completed with the Multiquant
software version 2.1.
• Two peaks were obtained for some phospholipids (most
likely isomers; see Figure 2 and Figure 3). All peaks were
integrated and the peak area was summed.
• All injections were performed in duplicate and the
average peak area was calculated.
CV%
Lipemia index
OH
Chromatography
• Agilent Technology Series 1100 pumps and autosampler
• Halo HILIC, 30×2.1mm, 2.7µm
• 6.0 min isocratic elution of 20 mM ammonium acetate
water (pH 6) and ACN
Average
In te n s ity , c p s
Lipids can potentially cause matrix effect and although
using a stable isotope-labelled internal standard can often
compensate for this effect, it is not always the case, which
can lead to erroneous results if left unaccounted for.
Currently, the type of commercially available lipemic
plasma to be used for bioanalytical assay development
remains unclear, as the differences between them are
not well known. The work reported herein describes the
differences between normal plasma, synthetic lipemic
plasma and two types of natural lipemic plasma.
Figure 1: Structures of lysophosphatidylcholines
(LPC) and phosphatidylcholines (PC).
Non-Synthetic Plasma
Regular Donors (Normal)
Regular Donors (Non-Normal)
Regular Donors (All)
Postprandial Donors
High-Triglycerides Donors
Phospholipids
(Normalized)
Triglycerides
(mM)
Total Cholesterol
(mM)
Lipemia Index
n
Plasma Type
In te n s ity , c p s
For several years, only ANVISA has addressed the testing
of lipemic samples (as part of selectivity). Only recently
EMA has included performing matrix effect on lipemic
plasma as part of their guideline (Feb. 2012). ANVISA
included it in their new (2012) BMV guidance (for both
selectivity and matrix effect). FDA has started focusing
their attention on this subject during inspections as well.
Sample Extraction
• 100 µL of sample was extracted with 400 µL of Methanol,
vortexed and centrifuged. 25 µL of supernatant was diluted
with 475 µL of Acetonitrile before injection.
Intensity (cps)
INTRODUCTION
DISCUSSION
Table 2: Compiled Results
Intensity (cps)
Purpose
- To determine the type of commercially available lipemic
plasma to be used for bioanalytical method validation
(BMV)
Method
- Regular plasma, synthetic lipemic plasma and two types
of non-synthetic lipemic plasma were tested
- Samples were extracted by protein precipitation and
analysed by HILIC chromatography on a Q-TOF mass
spectrometer for semi-quantitation of phospholipids
- Lipemia index, triglycerides and total cholesterol
concentration were also determined
Results
- Plasma from high-triglycerides donors best reflects
lipemic incurred samples that may be obtained as part
of clinical trials
RESULTS
METHODS
PHOSPHOLIPIDS CONTENT EVALUATION
Value Normalized to
Regular Donors (Normal)
OVERVIEW
0.73
Weak correlation:
0.0 < |r| < 0.3
Moderate correlation:
0.3 < |r| < 0.7
Strong correlation:
|r| > 0.7
CONCLUSION
Plasma from high-triglycerides donors best reflects lipemic
incurred samples that may be obtained as part of clinical trials.
- Evaluation of lipids in plasma is not done systematically
on all subject enrolled.
- A volunteer can be enrolled as a healthy volunteer and
present above average levels of triglycerides.
High-triglycerides donors plasma samples should be used to
perform a scientifically meaningful lipemic plasma test for BMV.
- Due to the large inter-lot variability in lipid content, only
those which have a sufficient triglycerides
concentration should be used.
* CORRESPONDING AUTHOR