Comparison of Sample Preparation Techniques
for Reduction of Matrix Interference in
Biological Analysis by LC-MS-MS
Charles Mi, Michael Ye, and An Trinh
Supelco, Division of Sigma-Aldrich, Bellefonte, PA 16823 USA
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Abstract
Three different sample preparation techniques are evaluated
in this study for effective removal of phospholipids matrix
interference. Sample preparation techniques include protein
precipitation (PPT), solid phase extraction (SPE) and
HybridSPE technique.
The degree of phospholipids interference varied greatly
between the three sample preparation techniques. HybridSPE
technique efficiently removed phospholipids and protein
resulting in the least matrix interference. SPE removed
minimal phospholipids and protein resulting moderate matrix
interference. Protein precipitation removed only gross of levels
of proteins from biological with no removal of phospholipids
resulting in greatest matrix interference.
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Introduction
Matrix effects in biological samples have been shown to be a
source of variability and inaccuracy in liquid chromatography
mass spectrometry (LC-MS). Co-elution of endogenous
phospholipids with analyte can cause matrix effect ionsuppression or enhancement that dramatically impact
quantitative LC-MS-MS. In this study, three different sample
preparation techniques are evaluated for effective removal of
phospholipids matrix interference. HybridSPE method are
optimized in this study and SPE and PPT methods both are
generic methods without further modification.
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Sample Preparation Methods
HybridSPE Techniques:
Protein Precipitation (PPT):
• Load 100 µL rat or dog plasma
• Add 300 µL acetonitrile with 1% FA
• Vortex 1 min. before vacuum
• Analyze the eluent via LC-MS
• Load 100 µL rat or dog plasma
• Add 300 µL acetonitrile with 1% FA
• Mix for 1 min. and centrifuge at 5K RPM for 3 min.
• Analyze the sup via LC-MS
Generic polymeric SPE (60 mg/3 mL):
• Condition with 1 mL methanol and 1 mL water
• Load 500 µL rat or dog plasma
• Wash with 5% methanol in water
• Elute with 1 mL methanol
• Evaporate and recondition with 2 mL of water/acetonitrile with 1% FA (1:3)
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Experiment 1
Study Phospholipids Breakthrough
LC-MS:
column:
mobile phase A:
mobile phase B:
flow rate:
temp.:
injection:
Agilent 1100/ABI Q-trap 3200, Turbo Ion Spray ESI+
Ascentis® Express C18, 5 cm x 2.1 mm I.D.
65% acetonitrile with 0.1% ammonium formate
35% water with 0.1% ammonium formate
200 µL/min.
30 °C
5 µL dog plasma sample prepared by different sample preparation methods
Mass Parameters:
CUR
IS
TEM
GS1
GS2
ihe
CAD
20
3500
500
40
55
ON
Medium
Q1 Mass (amu)
Q3 Mass (amu)
Dwell(msec)
DP
EP
CEP
CE
CXP
184
184
50
100
10
10
29
4
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XIC of +MRM (2 pairs): 184.0/184.0 amu from Sample 2 (090208003) of 090208infu.wiff (Turbo Spray)
Max. 1700.0 cps.
9.4e4
9.0e4
8.5e4
8.0e4
Phospholipids Breakthrough with different
sample preparation Methods
7.5e4
7.0e4
6.5e4
6.0e4
5.5e4
Intensity, cps
5.0e4
PPT method
4.5e4
4.0e4
3.5e4
3.0e4
2.5e4
SPE method
2.0e4
1.5e4
HybridSPE
method
1.0e4
5000.0
0.660.720.80
0.0
0.5
274
1.0
546
1.5
819
2.0
1091
2.5
1364
3.0
1637
Time, min
3.5
1909
4.0
2182
4.5
2454
5.0
2727
5.5
3000
6.0
3272
Almost 100% phospholipids in dog plasma were removed by
HybridSPE technique, moderate phospholipids were removed by
generic polymeric SPE method, and no phospholipids were removed by
traditional PPT.
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Experiment 2
Study Drug Compounds Recovery
LC-MS:
column:
mobile phase A:
mobile phase B:
flow rate:
temp.:
injection:
Agilent 1100/ABI Q-trap 3200, Turbo Ion Spray ESI+
Discovery® HS F5, 10 cm x 2.1 mm I.D.
acetonitrile with 10 mM ammonium formate
water with 10 mM ammonium formate
200 µL/min.
30 °C
5 µL of the rat plasma samples cleanup with different sample
preparation methods
Step
Total Time (min.)
Flow Rate (µL/min.)
A (%)
B (%)
0
0.0
200
75
25
1
2.0
200
95
5
2
4.5
200
95
5
3
5.0
200
75
25
4
7.0
200
75
25
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Mass Parameters
CUR
IS
TEM
GS1
GS2
ihe
CAD
25
4500
450
35
20
ON
Medium
Q1 Mass (amu)
Q3 Mass (amu)
Dwell (msec)
DP
EP
CEP
CE
CXP
255.2
209.10
150
36
12
18
17
4
260.30
116.10
150
41
9.5
14
25
4
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Structures of Compounds in this Study
Ketoprofen
O
Propranolol
CH3
NH
OH
O
OH
O
+MRM: 255.20/209.10 amu
+MRM: 260.30/116.10 amu
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Recovery of Drug Compounds in Rat Plasma
by different Sample Preparation Methods
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Sample Preparation
Method
Ketoprofen
Propranolol
HybridSPE
82.0%
68.0%
Traditional PPT
58.8%
37.0%
Generic polymeric SPE1
78.4%
42.0%
Generic polymeric SPE2
76.4%
44.4%
Recovery of drug compounds in rat plasma
90%
Ketoprofen
80%
Propranolol
Recovery
70%
60%
50%
40%
30%
20%
10%
0%
HybridSPE
SPE1
SPE2
PPT
Sample cleanup methods
HybridSPE method provided highest recovery for both
compounds and generic polymeric SPE method provided
moderate recovery of both compounds while PPT method
provided least recovery of both compounds.
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Ketoprofen and Propranolol in Rat
Plasma cleanup by HybridSPE
XIC of +MRM (2 pairs): 260.3/116.1 amu from Sample 29 (092007007) of 092007.wiff (Turbo Spray)
Max. 273.3 cps.
Ketoprofen and Propranolol in Rat
Plasma cleanup by PPT
XIC of +MRM (2 pairs): 260.3/116.1 amu from Sample 28 (092007009) of 092007.wiff (Turbo Spray)
Max. 166.7 cps.
433
440
420
420
400
400
380
380
360
Hybrid PPt
360
340
340
PPT
320
320
300
300
280
280
3.24
260
260
240
240
220
220
200
200
180
180
3.28
160
160
3.20
140
3.24
140
3.15
120
120
100
100
80
80
3.17
60
60
3.37
40
40
2.10
20
0
0.5
1.0
1.5
2.0
2.55
2.5
2.73
3.70
3.0
3.5
Time, min
5.45
4.0
4.5
5.0
5.5
20
5.75
0
6.0
6.5
1.51
0.03
0.5
1.0
3.10
1.55
1.982.04
0.95
1.5
2.0
3.31
3.40
2.33
2.5
3.0
4.73
3.62
3.5
Time, min
4.0
4.5
4.80
5.0
6.11
5.5
6.0
6.55
6.5
Recovery of propranolol in rat plasma by PPT method are much lower
than the recovery of propranolol by HybridSPE method because of
biological sample matrix interference in PPT sample.
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Reocvery
Recovery of ketoprofen and propranolol in rat plasma
Ketoprofen
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
Propranolol
SPE1-pre
SPE1-post
SPE2-pre
SPE2-post
Pre-spiked and post-spiked rat plasma
However, lower recovery of propranolol in SPE samples was also found.
Some propranolol were found adsorbing on polymeric SPE stationary
phase. Therefore, stronger organic solvent may require to elute the rest
of propranolol from polymeric SPE phase to improve its recovery.
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Pre-spiked Ketoprofen and
Propranolol in Rat Plasma
cleanup by SPE
XIC of +MRM (2 pairs): 260.3/116.1 amu from Sample 24 (092007015) of 092007.wiff (Turbo Spray)
Post-spiked Ketoprofen
and Propranolol in Rat
Plasma cleanup by SPE
Max. 233.3 cps.
XIC of +MRM (2 pairs): 260.3/116.1 amu from Sample 26 (092007013) of 092007.wiff (Turbo Spray)
580
420
550
400
Max. 300.0 cps.
380
500
360
340
450
HL post
320
3.28
300
400
280
260
350
240
220
300
200
180
250
3.28
160
3.22
200
140
120
150
100
3.23
80
3.38
100
3.16
60
3.20
3.36
50
0
40
1.49
0.39
0.5
1.75 1.97
1.29
0.95
1.0
1.5
2.0
2.51
2.61
2.5
2.75
3.07
3.0
3.45
3.73
3.5
Time, min
4.13
4.0
4.29
4.5
4.84 5.03
5.0
5.5
2.92
20
5.80
6.35
5.17
6.0
6.43
6.5
0.22 0.49
0
0.5
3.45
3.63
1.74
1.0
1.5
2.0
2.5
3.0
3.5
Time, min
3.99 4.19
4.0
4.46
4.5
4.87
5.0
5.19
6.48
5.84
5.5
6.0
6.5
Recovery of pre-spiked propranolol in rat plasma by SPE method
are low and recovery of post-spiked propranolol in rat plasma by
SPE method are higher.
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6.56
Matrix Effect on HybridSPE
1.2
w/o plasma
Recovery
1
w/plasma
0.8
0.6
0.4
0.2
0
Ketoprofen
Propranolol
Drug compounds in different matrix
Two compounds with or without rat plasma were studied by
HybridSPE method. There is no big difference in their recovery by
using this method. That means there is no or less matrix effect on
these compounds by HybridSPE technique, but some propranolol
may adsorb on the HybridSPE under current condition.
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Experiment 3
Optimization of HybridSPE Method
Two different acid modifiers were compared in this study. The
acids tested were formic acid (FA) and acetic acid (HA). The
acid concentration was systematically adjusted and measured
against recovery using a representative acidic compound
(ketoprofen) and basic compound (propanolol) diluted in rat
plasma (100 ng/mL).
Ratio of plasma and crashing solvent volume with two different
acid modifiers were also studied. The ratio was adjusted to 1:2,
1:3 and 1:5, respectively.
HPLC and MS condition are the same as for Experiment 2.
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Ketoprofen in Plasma by HybridSPE 1
120%
Recovery
100%
FA
80%
HA
60%
40%
20%
0%
0.0%
0.1%
0.2%
0.5%
1.0%
1.5%
2.0%
Acid % in Acetonitrile
Acid type and amount had great affect on the recovery of
ketoprofen (acidic compounds). 1% acid modifier are optimized
for giving an acceptable recovery of acidic compounds.
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Propranolol in plasma recovery by HybridSPE 1
FA
HA
100%
Recovery
80%
60%
40%
20%
0%
0.0%
0.1%
0.2%
0.5%
1.0%
1.5%
2.0%
Acid % in Acetonitrile
Acid type and amount had little affect on the recovery of
propanolol (basic compounds).
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Ketoprofen in plasma by HybridSPE 2
Recovery
120%
100%
FA
80%
HA
60%
40%
20%
0%
1:2
1:3
1:5
Crash Ratio (w /1% acid in acetonitrile)
Ratio of plasma and crashing solvent with different acid modifiers
had some affect on the recovery of ketoprofen (acidic compounds).
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Propranolol in plasm a recovery by HybridSPE 2
FA
HA
Recovery
100%
80%
60%
40%
20%
0%
1:2
1:3
1:5
Protein crash ratios (1% acid)
Ratio of plasma and crashing solvent with different acid
modifiers had little affect on the recovery of propranolol
(basic compounds). 1:3 protein crash ratio is optimized
for acidic and basic compounds in plasma samples.
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Calibration Curves of Ketoprofen and Propranolol
in Rat Plasma cleanup by HybridSPE
Propranolol Calibration (R = 0.9991)
54a-zrsi-plasma.rdb (propranolol): "Linear" Regression ("No" weighting): y = 87.6 x + 8.68 (r = 0.9991)
Ketoprofen Calibration (R = 0.9995)
54a-zrsi-plasma.rdb (ketoprofen): "Linear" Regression ("No" weighting): y = 96.6 x + 14.1 (r = 0.9995)
9000
1.00e4
8500
9500.00
9000.00
8000
8500.00
7500
8000.00
7000
7500.00
6500
7000.00
6000
6500.00
5500
6000.00
5000
5500.00
4500
5000.00
4000
4500.00
3500
4000.00
3500.00
3000
3000.00
2500
2500.00
2000
2000.00
1500
1500.00
1000
1000.00
500
500.00
5
10
15
20
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25
30
35
40
45
50
55
60
Concentration, ng/mL
65
70
75
80
85
90
95
100
5
10
15
20
25
30
35
40
45
50
55
60
Concentration, ng/mL
65
70
75
80
85
90
95
100
Conclusions
The degree of phospholipids interference varied greatly
between the three sample preparation techniques. HybridSPE
technology efficiently removed phospholipids and protein
resulting in the least matrix interference. SPE removed
minimal phospholipids and protein resulting moderate matrix
interference. Protein precipitation removed only gross levels of
proteins from biological with no removal of phospholipids
resulting in greatest matrix interference.
1:3 ratio of plasma and acetonitrile with 1% formic acid is the
optimized HybridSPE sample preparation condition.
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