Determination of Inosine, 4-acetamidobenzoic Acid, and N,N-dimethylaminoisopropanol by LC/MS/MS in
Human Plasma: Inosine Stability Issue and Endogenous Level Increase over Time
L. Bouchard, V. Montminy, N. Pelletier, S. Lachance, N. Boudreau, A. Levesque (inVentiv Health Clinical, Québec, Canada)
Introduction
Results
Results
Inosine pranobex is a combination of inosine, 4-acetamidobenzoic acid, and N,N-dimethylaminopropan-2ol used as an antiviral drug. The purpose of this work was to develop a method to analyze these molecules
in human plasma and to evaluate stability in whole blood.
Huge variations of the inosine endogenous level in human plasma in function of the anticoagulant used (10
to >5000 ng/mL) were observed. Human EDTA K2 plasma presented the lowest inosine endogenous level.
The endogenous concentration of Inosine measured in plasma is dependent of the incubation
time of the whole blood prior the plasma separation. The use of acid or Phosphatase inhibitors
failed to stabilize the concentration of Inosine over time.
O
CH 3
N
HN
NH
OH
N
N
HO
O
Table 1. Endogenous Concentration of Inosine in Human Plasma in Function of the Anticoagulant
N
H 3C
O
CH3
CH3
N,N-Dimethylaminopropan-2-ol
HO
OH OH
Inosine
O
(DIP)
4-Acetamidobenzoic Acid
C5H13NO
Mw = 103.16 g/mol
(PAcBA)
C10H12N4O5
C9H9NO3
Mw = 179.17 g/mol
Mw = 268.23 g/mol
1Various
Anticoagulant
Inosine Concentration (ng/mL)1
EDTA K2
Na Citrate
ACD
NaF/K oxalate
10 - 130
300 - 5000
20 - 280
290 - 900
In spiked human whole blood EDTA K2, the rate of degradation of Inosine is really high. Despite
a spiking concentration of 500 ng/mL in fresh whole blood, the resulting plasma showed a very
low concentration of Inosine (5-10 ng/mL) after a short period of time (10 min).
Table 3. Stability of Inosine in Human Whole Blood EDTA K2
donors and collection conditions not controlled.
The use of activated charcoal stripped human plasma as surrogate matrix for the calibration standards was
evaluated. The stability of Inosine in this matrix is pH dependent. The matrix had to be acidified at pH 4 or
less. At pH 6, 85% degradation was observed after 24h at 4°C. Inosine was found stable in 2% BSA in
PBS surrogate matrix without pH adjustment.
Method
Spiking level
(ng/mL)
NONE
NONE
NONE
NONE
NONE
NONE
NONE
500
1Holding
Incubation time
(min)1
30
60
120
30
30
30
30
10
Additive to blood
Variation (%)
NONE
NONE
NONE
1M Citric acid (5% v/v)
1M HCl (6% v/v)
Phosphatase inhibitors cocktail 1 (10% v/v)2
Phosphatase inhibitors cocktail 2 (10% v/v)3
NONE
-6.1
26.5
168.4
127.0
39.0
56.8
80.0
Unstable4
time at 4°C prior plasma separation.
2Aqueous
Analytes are extracted by protein precipitation of the plasma at 4°C using acetonitrile. The diluted extract
is analyzed by LC/MS/MS (ESI+), using XBridge Amide column, to determine simultaneously the
concentration of inosine, 4-acetamidobenzoic acid (PAcBA) and N,N-dimethylaminopropan-2-ol (DIP).
solution of Na3VO4, Na2MoO4, Sodium Tartrate, Imidazole
of Cantharidin, (-)-p-Bromolevamisole oxalate and Calyculin A in DMSO
4Spiked Inosine is degraded at >99%
3Solution
LC-MS/MS Analysis
4-Acetamidobenzoic acid
(PAcBA)
Inosine
Matrix Type
N,N-Dimethylaminopropan-2-ol
(DIP)
Human Plasma (2% BSA in PBS for calibration curve)
Analytical Range
5 - 1000 ng/mL
100 – 10 000 ng/mL
50 – 5000 ng/mL
Internal Standard
Inosine-13C2,15N
PAcBA-d7
DIP-d6
Matrix Volume
50 µL
Extraction
Proteins Precipitation with MeCN (4°C)
Analytical Column
XBridge Amide 50 X 3.0 mm, 3.5µm
Elution Type
Table 2. Stability of Inosine in Unstripped Human Plasma EDTA K2
1 mL/min
Mobile Phase
5.00 min
2.50 min
Additive to plasma
Temperature (°C)1
Variation (%)
NONE
NONE
NONE
NONE
NONE
NONE
500
500
NONE
NONE
0.2 M HCl (20% v/v)
1M HCl (4% v/v)
1M HCl (6% v/v)
100mM Na3VO4 (5% v/v)
NONE
NONE
4
RT
4
4
4
4
4
RT
-6.3
-26.4
32.7
42.8
19.6
8.7
-31.2
-63.4
Sciex API 4000
Source
TurboIonSpray (Positive mode)
269  237
© 2012. inVentiv Health. All rights reserved.
180  94
2.60 min
105  71
1Duration
vary from 18 to 24 hrs
Figure 2. Inosine Endogenous Plasma Concentration in Function of Incubation Time of Whole Blood Prior Plasma Separation
Conclusion
Spiking level (ng/mL)
Water/MeCN (5/95) Ammonium Formate 5 mM
Detector
Ion monitored
The stability of Inosine in unstripped Human Plasma is temperature and pH dependent. The endogenous
concentration of Inosine decrease in Human Plasma EDTA K2, but the rate is slowed down by the
temperature. The acidification of the matrix leads to Inosine formation with time. In Spiked Human Plasma
EDTA K2, the rate of degradation of Inosine is higher than unspiked matrix.
Isocratic
Flow Rate
Retention Time
Figure 1. Stability of Inosine in Activated Charcoal Stripped Human Plasma at Various pH
In vitro formation and degradation of Inosine occurs rapidly in whole blood and plasma and
could lead to artifactual results. Varying the anticoagulant and/or acidifying the whole blood
and/or the plasma didn’t solve the stability issues. Metal chelating agent seems to reduce the
endogenous concentration of Inosine, but doesn’t stabilize the concentration over time.
Investigation is still on-going to understand and to control the enzymatic pathways involved in
Inosine reactivity in biological matrices.