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INTERNATIONAL JOURNAL OF RESEARCH ARTICLE PHARMACEUTICAL INNOVATIONS ISSN 2249-1031
48 | P a g e Volume 3, Issue 1, Jan. ₋ Feb. 2013 http://www.ijpi.org
Identification and Reduction of Matrix Effects Caused By
Polyethylene Glycol 400 in Bioanalysis Using Liquid
Chromatography/Tandem Mass Spectrometry
*Vijaya Bhaskar V.
Department of pharmacy, Jagadishprasad Jhabermal Tibrewala University, Jhunjhunu
Abstract
Ion suppression effect of dosing vehicle excipient polyethyleneglycol 400 on the accuracy of
liquid chromatography/tandem mass spectrometry (LC-MS/MS) measurements was studied.
Ion supression cause significant errors in accuracy of the measured concentrations of test
compounds, thereby invalidating the assessment of pharmacokinetic results. Using
polyethyleneglycol 400 as a probe compound, the concentration-time profile of the excipient
in plasma from rats dosed both orally and intravenously was determined. A total of nine
oligomers were identified for PEG 400. The most abundant ions corresponding to PEG 400
oligomers at m/z 327, 371, 432, 476, 520, 564, 608, 652 and 696 with daughter ion at m/z 89
were selected for multiple reaction monitoring (MRM) in electrospray mode of ionisation.
Analyte peak area of the oligomers was summed up to calculate the plasma concentrations of
total PEG 400. Plasma concentrations of PEG 400 ranging from 3-10 mg/mL in the initial
sampling points caused 2-10 fold ion supression on most of the analytes studied. This can
result in false rejection of compounds in a drug discovery screen. Several sample perparation
methods, enhanced chromatographic selectivity, and alternative ionization methods were
investigated as means to avoid or minimize ion suppression effects. The elimination of ion
suppression effects was achieved by Liquid Liquid Extraction (LLE) with hexane as sample
preparation method. The mechanism of ion supression caused by polyethylene glycol 400 in
relation to both liquid and gas phase reactions was discussed.
Key Words: PEG 400, LC-MS/MS, MRM, LLE, Matrix effect
1 INTRODUCTION
High throughput pharmacokinetic
screening plays an important role in
pharmaceutical research to rapidly identify
pharmacokinetic profiles of potent and
selective compounds [1-3]
. Liquid
chromatography/tandem mass
spectrometry (LC-MS/MS) with either
electro spray ionisation (ESI) or
atmospheric pressure chemical ionisation
(APCI) provides a sensitive and selective
detection method for the quantitation of
drug candidates in biological matrices [4]
.
Although LC-MS/MS is extremely
sensitive and robust in terms of
performance there is potential for ion
suppression which leads to incorrect data
*Corresponding Author
Vijaya Bhaskar V.
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interpretation. ESI is more prone to ion
suppression effects than APCI [5, 6]
. Ion
suppression could originate from
endogenous compounds such as
phospholipids [7]
, metabolites, co
administered drugs, internal standards [8]
,
dosing vehicles [9-11]
, mobile phase
additives [12]
and plastic tubes [13]
. In
preclinical pharmacokinetic studies,
formulation excipients typically are used at
high concentrations to facilitate the
dissolution of test compounds in the
formulation solution. Polyethylene glycol
400, a polymeric formulation excipient can
cause significant signal suppression for
certain analytes when minimal sample
cleanup is used. The presence of higher
concentration of formulation excipient in
early time point samples after intravenous
or oral administration can cause significant
ion suppression on the analytes [9, 10, 14]
.
This effect is more pronounced with the
use of ultrafast gradients that causes
coelution of many analytes. Ion
suppression effects are complicated to
handle in a drug discovery environment
where hundreds of molecules with
differing physicochemical properties
(logD7.4, logP, pKa) are handled. These
molecules may be differrentially ion
suppressed depending on their elution on a
typical liquid chromatography (LC)
gradient, compared with ion suppressing
agent and thier ability to compete with
charge from the suppressing agent. The
U.S food and drug administration (FDA)
guidance for industry on bioanalytical
method validation insists upon the
assessment of matrix effects during
method validation for quantitative
bioanalytical LC-MS/MS methods [15]
.
Several approaches investigated so far to
minimize the ion suppression effects by
polyethyleneglycol 400 were LC gradient
manipulation [10, 14]
, alternative column
choice [9]
, different sample preparation
strategies [9, 10, 14]
, sample dilution [16]
and
even the development of novel formulation
agent [17]
. While these strategies are
helpful to solve the ion suppression issues
on few analytes, a unique solution wasn„t
found for wide range of new chemical
entities studied in drug discovery. In this
paper, identification of ion suppression due
to polyethyleneglycol 400 and effective
removal of ion suppression are discussed.
The mechanism of ion suppression has
been proposed and discussed by several
research groups, but is not fully
understood. Various mechanisms by which
formulation excipients cause ion
suppression are: charge competition,
change in droplet surface tension,
preferential ion evaporation, gas phase
deprotonation and coprecipitation [18- 20]
.
The mechanism for polyethyleneglycol
400 related signal interference has been
proposed.
2 EXPERIMENTAL SECTION
2.1 Materials
Reference compounds (atenolol, caffeine,
metaprolol, propranolol, telmisartan,
ketoconazole, diltiazem, ranitidine and
warfarin) were procured from Sigma
Aldrich Co. (St. Louis, MO, USA).
Polyethyleneglycol 400, dimethyl
sulfoxide (DMSO), monobasic sodium
phosphate, dibasic sodium phosphate and
ammonium acetate were procured from
sigma Aldrich Co. (St. Louis, MO, USA).
Acetonitrile, water and methanol (HPLC
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grade) were obtained from Merck
specialities pvt ltd (Mumbai, India).
Formic acid (90% purified) was procured
from Merck specialities pvt ltd (Mumbai,
India). Sprague dawley rats were procured
from Bioneeds ltd (Bangalore, India).
Blood collection vacutainers (Lithium
Heparin as anticoagulant) were sourced
from BD (Franklin lakes, USA).
2.2 Physical parameters (log D at pH
7.40)
The log of octanol-buffer partition
coefficients (log D) was estimated for
reference compounds using saturated
octanol and phosphate buffer (pH 7.40). 15
µL of 10mM stock of reference compound
was spiked in 500 µL of saturated octanol
and vortex mixed at 1200 rpm for 10 min.
Equivalent volume of saturated phosphate
buffer (pH 7.40) was added and vortex
mixed at 1200 rpm for 1 hour. The sample
plate was centrifuged at 4000 rpm for 30
min to ensure complete separation of both
phases. Samples were analyzed on HPLC
(Shimadzu, Japan) at a detection
wavelength of 254 nm. Mobile phases
consisted of 0.1% formic acid in water and
100 % acetonitrile. A C18 (Waters
XBridge, 50 x 4.6 mm, 3.5 µm) column
with a 5 min LC generic gradient (Table 1)
was used. Log D values of reference
compounds were presented in Table 2.
2.3 Plasma Concentrations of PEG 400
2.3.1 Preparation of calibration
standards and quality control
samples
Master stock solution of telmisartan
(internal standard) was prepared in DMSO.
Working standard solutions of
polyethylene glycol 400 were prepared by
serial diluting master stock
(polyethyleneglycol 400 provided by
supplier with density of 1.126 g/mL was
used as master stock) with Acetonitrile:
DMSO: water (2: 2: 1). Working standard
solutions were prepared at 25 fold higher
concentration than plasma calibration
standards and quality control samples. A
total of nine calibration standards and three
quality control samples were prepared.
Plasma calibration standards (1.01, 2.03,
10.14, 50.68, 202.71, 506.76, 810.82,
912.06, 1013.40 µg/mL) and quality
control samples (3.89, 486.43, 810.72
µg/mL) of polyethylene glycol 400 were
prepared by spiking 2 µL of the working
standard solutions into 48 µL of blank rat
plasma. Working stock solution of
telmisartan (100 ng/mL) was prepared by
diluting an aliquot of master stock solution
in acetonitrile. Master and working stock
solutions were stored at 4oc when not in
use.
2.3.2 Animal Dosing
Polyethylene glycol 400 was administered
intravenously (lateral tail vein) and orally
(oral gavage needle) to fasted male
sprague dawley rats at a dose of 3.38 g/kg.
Dose volume administered was 5 mL/kg.
The composition of dosing vehicle used
for the study was DMSO/polyethylene
glycol 400/water (5: 60: 35, % v/v) [21,
22]. Serial blood samples were collected
into vacutainers containing lithium heparin
(anticoagulant) at 0.08, 0.25, 0.50, 1, 2, 4,
8 and 24 h post dose [23]
after intravenous
administration and 0.25, 0.50, 1, 2, 4, 8
and 24 h post dose [23]
after oral
administration. Plasma was separated after
centrifugation and stored at -80oC until
analysis.
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2.3.3 Sample preparation
A 50 µL aliquot of plasma (blank control
plasma, plasma samples from rats dosed
with polyethylene glycol 400, blank
plasma spiked with calibration standards
and QC samples) was pipetted in to a 96
well polypropylene plate and extracted
with 200 µL of acetonitrile containing
internal standard. Samples were vortex
mixed for 10 min at 1200 rpm and
centrifuged at 4000 rpm for 10 min at 4oC.
50 µL of supernatant was pipette
transferred in to a fresh analysis plate and
diluted with 450 µL of methanol: water
(1:1). 10 µL aliquots were injected for LC-
MS/MS analysis.
2.3.4 LC-MS/MS analysis
All mass spectrometric estimations were
performed on a sciex 3200 QTrap triple
quadrupole instrument with turboionspray
(Electrospray Ionization) (AB Sciex,
Toronto, Canada) using C18 column
(Waters XBridge 50 x 4.6 mm, 3.5 µm).
The HPLC system consisted two of
LC20AD UFLC pumps and a SIL HTC
auto sampler (Shimadzu, Kyoto, Japan).
The mobile phase consisted of 0.1%
formic acid in water as aqueous
component and 100% methanol as organic
modifier. A generic gradient LC method
(Table 3) with a short run time of 3.5 min
was used for the analysis of PEG 400 in
plasma samples. The column and auto
sampler were maintained at 40oC and 4
oC
respectively. The turboionspray source
was operated with typical settings as
follows: ionization mode, positive; curtain
gas, 20 psi; nebulizer gas (GS1), 50 psi;
heater gas (GS2), 50 psi; ion spray voltage,
5500V; temperature, 550oC. The mass
spectrometer was set up to perform in
MS/MS mode and to run in MRM mode.
The molecular ions of PEG 400 and
telmisartan were formed using the
declustering potentials of 40V. In MRM
mode the most abundant and informative
molecular ions were selected at m/z 327.3
(Oligomer 1), 371.3 (Oligomer 2), 432.3
(Oligomer 3), 476.3 (Oligomer 4), 520.3
(Oligomer 5), 564.3 (Oligomer 6), 608.3
(Oligomer 7), 652.3 (Oligomer 8), 696.3
(Oligomer 9) and fragmented to identical
daughter ion m/z 89.2 at collision energy
of 30, 32, 35, 38, 40, 42, 45, 48, 50 v
respectively and with medium CAD gas
setting. Molecular ion (m/z, 515.30) of
telmisartan was fragmented to m/z, 276.10
at collision energy of 65 v with medium
CAD gas setting. Peak areas for all
components were automatically integrated
using Analyst software version 1.5.
2.4 Preparation of plasma samples -
PEG 400 investigations
2.4.1 Preparation of master and
working stock solutions
Master stock solutions of atenolol,
caffeine, metaprolol, telmisartan,
propranolol, diltiazem, ketoconazole,
ranitidine and warfarin (1 mg/ml) were
prepared in DMSO. Working standard
solutions of polyethylene glycol 400 were
prepared by serial dilution from master
stock (polyethylene glycol 400 provided
by supplier with density of 1.126 g/mL
was used as master stock) at 25 times
higher concentration than plasma
concentrations in acetonitrile: water:
DMSO (2: 2: 1). A total of twelve working
concentrations of polyethylene glycol 400
were prepared. Plasma concentrations
(0.05, 0.125, 0.25, 0.50, 1.00, 1.25, 2.50,
5.00, 7.50, 10.00, 12.50, 15.00 mg/mL) of
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polyethylene glycol 400 were prepared by
spiking 2 µL of working concentrations in
48 µL of blank rat plasma. Pooled working
stock solution of reference compounds at
1000 ng/mL concentrations was prepared
in acetonitrile: water (1: 1). Master stock
and working stock solutions were stored at
4oC when not in use.
2.4.2 Sample Preparation
2.4.2.1 Protein Precipitation (PPT)
A 50 µL aliquot of plasma (blank plasma,
plasma samples spiked with polyethylene
glycol 400) was pipette transferred in to a
96 well polypropylene plate and extracted
with 200 µL of acetonitrile. Samples were
vortex mixed for 10 min at 1200 rpm and
centrifuged at 4000 rpm for 10 min at 4oC.
150 µL of supernatant was pipette
transferred to a fresh analysis plate and
diluted with 150 µL of pooled working
stock solution of reference compounds. 10
µL were injected for LC-MS/MS analysis.
2.4.2.2 Liquid Liquid Extraction (LLE)
A 50 µL aliquot of plasma (blank plasma,
plasma samples spiked with polyethylene
glycol 400) was pipette transferred in to a
96 well polypropylene plate and extracted
with 1000 µL of ethyl acetate, tert-butyl-
methyl ether (TBME) and hexane
individually. Samples were vortex mixed
for 10 min at 1000 rpm and centrifuged at
4000 rpm for 10 min at 4oC. 800 µL of
supernatant was pipette transferred to a
fresh evaporation plate and evaporated to
dryness under nitrogen at 40oC for 10 min.
After evaporation, samples were
reconstituted with 300 µL of pooled
working stock solution of reference
compounds and 10 µL were injected for
LC-MS/MS analysis.
2.4.3 LC-MS/MS analysis
All mass spectrometric estimations were
performed on a sciex 3200 QTrap triple
quadrupole instrument with turboionspray
(AB Sciex, Toronto, Canada). The HPLC
system consisted two of LC20AD UFLC
pumps and a SIL HTC auto sampler
(Shimadzu, Kyoto, Japan). The stationary
phase was XBridge C18 with 3.5 µm
particle diameter (Waters, Ireland). The
column dimensions were 50 x 4.6 mm.
The mobile phase flow rate was 1.0
mL/min with a split ratio of 1:1 to the
ionization source. The mobile phase
consisted of the following combinations of
aqueous and organic modifiers: 1) 0.1%
formic acid in water, 100% acetonitrile
(FA-ACN) 2) 0.1% formic acid in water,
100% methanol (FA-MEOH) 3) 10mM
ammonium acetate in water, 100%
acetonitrile (AMM. ACET.-ACN) 4)
10mM ammonium acetate in water, 100%
methanol (AMM. ACET.-MEOH). A
generic gradient LC method (Table 3) with
a short run time of 3.5 min was used for
the quantification of reference compounds
and PEG 400. The column and
autosampler were maintained at 40oC and
4oC respectively. The turboionspray source
was operated with typical settings as
follows: ionization mode, positive; curtain
gas, 15 psi; nebulizer gas (GS1), 50 psi;
heater gas (GS2), 50 psi (ESI); ionspray
voltage (IS), 5500V (ESI); nebulizer
current (NC), 5A (APCI); temperature,
550oC. Multiple reactions monitoring
(MRM) mode was employed for the
quantification of reference compounds and
PEG 400. List of MRM used for
quantification was presented in Table 4.
Peak areas for all components were
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automatically integrated using Analyst
software version 1.5.
3 RESULTS AND DISCUSSION
3.1 Plasma concentrations of PEG 400
Plasma samples from both intravenous and
oral routes were analysed with the
developed bioanalytical method using LC-
MS/MS. For calculating the plasma
concentrations of PEG 400 as a whole the
analyte peak areas of each oligomer was
summed up and calibration curve was
built. No interference at the retention times
of PEG 400 (2.05 min) (Figure 1a) was
observed in any of the lots screened as
shown in representative chromatogram of
the extracted blank plasma sample,
confirming the selectivity of the present
method. Representative chromatogram of
PEG 400 at LLOQ was shown in Figure
1b. Representative chromatograms of PEG
400 from intravenous (2.00 hr), oral (2.00
hr) study samples were shown in Figure 1c
and Figure 1d respectively. PEG 400
plasma concentrations following
intravenous administration were high (3-10
mg/mL) in the initial sampling points. The
oral bioavailability of PEG 400 was
measured as 33.84 % with a rapid terminal
half life of 2 hr, which was consistent with
published results [24]. Tmax after oral
administration was 2.00 hr. Mean plasma
concentrations of PEG 400 after
intravenous and oral administration were
shown in Table 5 and Table 6 respectively.
Although, the bioavailability of
polyethylene glycol 400 oligomers will
decrease upon increase in mass of
oligomers, nevertheless the plasma
concentrations observed in oral route of
administration will not be more than
plasma concentrations observed in
intravenous route of administration, when
similar dose was administered. Our
objective in this study is to measure the
maximum physiological concentrations of
excipient in plasma after administration to
rats.
3.2 Preparation of plasma samples -
polyethylene glycol 400
investigations
Samples extracted by protein precipitation
were analyzed with different mobile phase
conditions in ESI mode to check if the
elution pattern of the excipient behaves
differently to that of reference compounds.
As it is well known that APCI was less
prone to matrix effects compared to ESI,
protein precipitated samples were also
analyzed in this mode. Samples extracted
by LLE were analyzed in ESI mode.
Analytical conditions used for analysis of
LLE samples in ESI mode and protein
precipitated samples in APCI mode were
similar to the conditions used for the
analysis of PEG 400 in plasma samples.
Solid phase extraction wasn‟t tried as
alternate extraction technique as this work
was done to provide a unique solution for
nullifying the ion suppression effects
caused by polyethyleneglycol 400 in the
bioanalysis of new chemical entities. For
developing a SPE method, molecular,
physico chemical properties of NCEs
should be known and a lot of time should
be invested on method development which
practically is impossible in drug discovery
where throughput drives the fate of
project.
Peak area of reference compound at each
concentration of polyethyleneglycol 400
spiked in to plasma was compared against
negative control samples to calculate %
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ion suppression. A total suppression of
±15% from the control response was
considered as acceptable according to US
FDA validation guidelines [15]. A detailed
discussion on the results obtained with
different system conditions for each
reference compounds was given below.
3.2.1 Propranolol
At an excipient concentration of 15
mg/mL, propranolol had >75 % ion
suppression using ethyl acetate as
extraction solvent (Figure 2). Sample
preparation with hexane, TBME and
ionization by APCI mode of ionization for
protein precipitation samples proved to be
the best methodologies for the analysis of
propranolol (Figure 2). Ion suppression at
different concentrations of
polyethyleneglycol 400 under different
analytical conditions was shown in Figure
3a.
3.2.2 Caffeine
At an excipient concentration of 15 mg/mL
, caffeine had >80 % ion suppression
using a) APCI mode of ionization b)
different mobile phase combinations c)
ethyl acetate extraction (Figure 2).
Suppression effects weren‟t observed
when samples were extracted using n-
hexane (Figure 2). Ion suppression at
different concentrations of
polyethyleneglycol 400 under different
analytical conditions was shown in Figure
3b.
3.2.3 Ketoconazole
At an excipient concentration of 15
mg/mL, ketoconazole had >80 % ion
suppression using ethyl acetate as
extraction solvent (Figure 2). Sample
preparation with hexane, TBME and
ionization by APCI mode of ionization for
protein precipitation samples proved to be
the best methodologies for the analysis of
ketoconazole (Figure 2). Suppression
effects weren‟t observed when mobile
phase combination of 10mM ammonium
acetate and acetonitrile was used (Figure
2). Ion suppression at different
concentrations of polyethyleneglycol 400
under different analytical conditions was
shown in Figure 3c.
3.2.4 Diltiazem
At an excipient concentration of 15
mg/mL, diltiazem had >65 % of ion
suppression using ethyl acetate as
extraction solvent (Figure 2). Sample
preparation by extraction with hexane,
TBME and ionization by APCI mode of
ionization for protein precipitation samples
proved to be the best methodologies for
the analysis of diltiazem (Figure 2).
Suppression effects weren‟t observed with
mobile phase combinations of a) 10mM
ammonium acetate and acetonitrile b)
10mM ammonium acetate and methanol
(Figure 2). Ion suppression at different
concentrations of polyethyleneglycol 400
under different analytical conditions was
shown in Figure 3d.
3.2.5 Ranitidine
At an excipient concentration of 15
mg/mL, ranitidine had >35% ion
suppression using a) APCI mode of
ionization b) different mobile phase
combinations c) ethyl acetate extraction
(Figure 2). Suppression effects weren‟t
observed when samples were extracted
using n-hexane, TBME (Figure 2). Ion
suppression at different concentrations of
polyethyleneglycol 400 under different
analytical conditions was shown in Figure
4a.
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3.2.6 Atenolol
At an excipient concentration of 15
mg/mL, atenolol had >90 % ion
suppression using mobile phase
combination of 10mM ammonium acetate
and acetonitrile (Figure 2). Sample
preparation by extraction with hexane,
TBME and ionization by APCI mode of
ionization for protein precipitation samples
proved to be the best methodologies for
the analysis of atenolol (Figure 2). Ion
suppression at different concentrations of
polyethyleneglycol 400 under different
analytical conditions was shown in Figure
4b.
3.2.7 Telmisartan
At an excipient concentration of 15
mg/mL, telmisartan had >50 % ion
suppression using mobile phase
combination of formic acid and
acetonitrile (Figure 2). Sample preparation
by extraction with hexane, TBME and
ionization by APCI mode of ionization for
protein precipitation samples proved to be
the best methodologies for the analysis of
telmisartan (Figure 2). Suppression effects
weren‟t observed using mobile phase
combination of 10mM ammonium acetate
and acetonitrile (Figure 2). Ion suppression
at different concentrations of
polyethyleneglycol 400 under different
analytical conditions was shown in Figure
4c.
3.2.8 Metaprolol
At an excipient concentration of 15
mg/mL, metaprolol had >70 % ion
suppression using a) ethyl acetate
extraction b) different mobile phase
combinations (Figure 2). Sample
preparation by extraction with hexane and
ionization by APCI mode of ionization for
protein precipitation samples proved to be
the best methodologies for the analysis of
metaprolol (Figure 2). Ion suppression at
different concentrations of
polyethyleneglycol 400 under different
analytical conditions was shown in Figure
4d.
3.2.9 Warfarin
At an excipient concentration of 15
mg/mL, warfarin had >45 % ion
suppression using a) ethyl acetate
extraction b) mobile phase combination of
10mM ammonium acetate and methanol
(Figure 2). Sample preparation by
extraction with hexane, TBME and
ionization by APCI mode of ionization for
protein precipitation samples proved to be
the best methodologies for the analysis of
warfarin (Figure 2). Ion suppression at
different concentrations of
polyethyleneglycol 400 under different
analytical conditions was shown in Figure
5a.
Using different mobile phase conditions
helped to nullify the ion suppression on
ketoconazole, diltiazem and telmisartan in
ESI mode of analysis. No ion suppression
effects were observed for propranolol,
ketoconazole, diltiazem, ranitidine,
atenolol, telmisartan and warfarin when
samples were extracted using TBME.
However, Ion suppression was observed
for metaprolol and caffeine. LLE with
ethyl acetate was the poor extraction
method with severe ion suppression effects
on all the reference compounds tested.
Sample analysis by APCI mode of
ionization brought down the ion
suppression effects caused by
polyethyleneglycol 400 on propranolol,
ketoconazole, diltiazem, atenolol,
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metaprolol and warfarin. However,
ranitidine and caffeine still had ion
suppression in APCI mode of ionization.
This shows that polyethyleneglycol 400
cause ion suppression both in liquid and
gas phases. ESI is prone to matrix effects
caused by excipients in liquid and gas
phase whereas APCI is resistant to liquid
phase suppression effects.
We proposed various mechanisms by
which polyethyleneglycol 400 cause ion
suppression effects on different analytes:
1. Increase in surface tension and
viscosity of the droplets due to
high concentrations of excipients
leading to insufficient evaporation
(ESI)
2. Charge competition between
analyte and ion suppressing agent
leading to overall reduced
ionization of analyte (ESI/APCI)
3. Co precipitation with non volatile
components (ESI/APCI)
4. Gas phase reactions causing
charge transfer between analytes
and ion suppressing agent
(ESI/APCI)
The only extraction technique that
provided unique solution for all the
reference compounds tested was liquid
liquid extraction with hexane. Monitoring
the excipient levels with the developed
MRM method also helped to take a
decision on the best extraction technique.
Significant levels of polyethyleneglycol
400 were detected with sample preparation
by protein precipitation, LLE with ethyl
acetate and TBME. Polyethyleneglycol
400 levels weren‟t detected when
extraction with hexane was used as sample
preparation method (Figure 5b). The
reason behind lack of ion suppression
effects in the samples extracted with
hexane was due to complete insolubility of
excipient in hexane.
4 CONCLUSION
A MRM based method was developed for
the quantification of polyethylene glycol
400 concentration levels in rat plasma
samples. Based on the physiological
concentration levels of excipient, various
approaches such as a) different mobile
phase conditions b) different extraction
techniques c) different ionization
conditions were tested for finding best
technique that nullifies ion suppression
effects. The approaches for reducing the
ion suppression effects in LC-MS/MS are
largely analyte dependent. However,
sample preparation with hexane as
extraction solvent totally nullified the ion
suppression effects caused by polyethylene
glycol 400. Mechanism of ion suppression
caused by polyethylene glycol 400 was
proposed as a) charge competition b)
increase in surface tension/viscosity of
droplets c) co-precipitation d) gas phase
reactions.
5 REFERENCES
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2. Olah TV, McLoughlin DA, Gilbert
JD. The simultaneous
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candidates by liquid
chromatography/atmospheric
pressure chemical ionization mass
spectrometry as an invivo drug
screening procedure. Rapid
Commun. Mass Spectrom. 1997;
11(1): 17-23.
3. Watt AP, Morrison D, Locker KL,
Evans DC. Higher throughput
bioanalysis by automation of a
protein precipitation assay using a
96 well format with detection by
LC-MS/MS. Anal. Chem.2000; 72:
979-984.
4. Covey TR, Lee ED, Henion JD.
High speed liquid
chromatography/tandem mass
spectrometry for the determination
of drugs in biological samples.
Anal. Chem. 1986; 58: 2453-2460.
5. Pommier F, Frigola R. Quantitative
determination of rivastigmine and
its major metabolite in human
plasma by liquid chromatography
with atmospheric pressure
chemical ionization tandem mass
spectrometry. J. Chromatogr. B
2003; 784: 301-313.
6. Dams R, Huestis MA, Lambert
WE, Murphy CM. Matrix effect in
bioanalysis of illicit drugs with LC-
MS/MS: influence of ionization
type, sample preparation, and
biofluid. J. Am. Soc. Mass
Spectrom. 2003; 14: 1290-1294.
7. Little JL, Wempe MF, Buchanan
CM. Liquid chromatography- mass
spectrometry/mass spectrometry
method development for drug
metabolism studies: examining
lipid matrix ionization effects in
plasma. J. Chromatogr. B 2006;
833: 219-230.
8. Sojo LE, Lum G, Chee P. Internal
standard signal suppression by
coeluting analyte in isotope
dilution LC-ESI-MS. Analyst
2003; 128: 51-55.
9. Tong XS et al. Effect of signal
interference from dosing excipients
on pharmacokinetic screening of
drug candidates by liquid
chromatography/mass
spectrometry. Anal. Chem. 2002;
74: 6305-6313.
10. Shou WZ, Naidong W. Post
column infusion study of the
“dosing vehicle effect” in the liquid
chromatography/tandem mass
spectrometric analysis of discovery
pharmacokinetic samples. Rapid
Commun. Mass Spectrom. 2003;
17: 589-597.
11. Schuhmacher J, Zimmer D, Tesche
F, Pickard V. Matrix effects during
analysis of plasma samples by
electrospray and atmospheric
pressure chemical ionization mass
spectrometry: practical approaches
to their elimination. Rapid
Commun. Mass Spectrom. 2003;
17: 1950-1957.
12. Mallet CR, Lu Z, Mazzeo JR. A
study of ion suppression effects in
electrospray ionization from
mobile phase additives and solid
phase extracts. Rapid Commun.
Mass Spectrom. (2004) 18: 49-58.
13. Mei H, Hsieh Y, Nardo C, Xu X,
Wang S et al. Investigation of
matrix effects in high performance
liquid chromatography/tandem
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mass spectrometric assays:
application to drug discovery.
Rapid Commun. Mass Spectrom.
2003; 17: 97-103.
14. Weaver R, Riley R. Identification
and reduction of ion suppression
effects on pharmacokinetic
parameters by polyethyleneglycol
400. Rapid Comm. Mass Spectrom.
2006; 20: 2559-2564.
15. Guidance for Industry:
Bioanalytical Methods Validation.
US Department of Health and
Human Services, Center for Drug
Evaluation and Research, and
Center for Veterinary Medicine,
May 2001. Available at
http://www.fda.gov/cder/guidance/i
ndex.html.
16. Larger P, Breda M, Fraier D,
Hughes H, James C. Ion
suppression effects in liquid
chromatography tandem mass
spectrometry due to a formulation
agent, a case study in drug
discovery bioanalysis. J. Pharm.
Biomed. Anal. 2005; 39: 206-216.
17. Temesi D, Law B, Howe N.
Synthesis and evaluation of
PEG414, a novel formulation agent
that avoids analytical problems
associated with polydispersive
vehicles such as PEG400. J.
Pharmaceutical Sciences 2003; 92
(12): 2512-2518.
18. Chambers E, Wagrowski DM, Lu
Z, Mazzeo JR. Systematic and
comprehensive strategy for
reducing matrix effects in
LC/MS/MS analyses. J.
Chromatogr. B. Analyt. Technol.
Biomed Life Sci. 2007; 852: 22-34.
19. King R, Bonfiglio R, Fernandez
MC, Miller SC, Olah T.
Mechanistic investigation of
ionization suppression in
electrospray ionization. J. Am. Soc.
Mass Spectrom. 2000; 11: 942-
950.
20. Bonfiglio R, King RC, Olah TV,
Merkle K. The effects of sample
preparation methods on the
variability of the electrospray
ionization response for model drug
compounds. Rapid Commun. Mass
Spectrom. 1999; 13: 1175-1185.
21. Neeravannan S. Preclinical
formulations for drug discovery
toxicology: physicochemical
challenges. 2006; 2(5): 715-731.
22. Sheftel VO. Indirect food additives
and polymers: Migration and
Toxicology. 2000; 1114-1116.
23. Kwon Y. Pharmacokinetic study
design and data interpretation.
Kluwer Academic Publishers, New
York 2002, pp 21-46.
24. He YL, Murby G, Warhurst L,
Gifford D, Walker J, et al. Species
differences in size discrimination in
the paracellular pathway reflected
by oral bioavailability of
poly(ethylene glycol) and D-
peptides J. Pharm. Sci. 1998; 87:
626-633.
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Table 1: LC Generic gradient for measurement of lipophilicity of reference compounds
Time (min) Flow Rate
(mL/min)
%A
(Aqueous Modifier)
%B
(Organic Modifier)
0.01 1.00 95 5
2.00 1.00 35 65
3.00 1.00 5 95
4.00 1.00 5 95
4.01 1.00 95 5
5.00 1.00 95 5
Table 2: Calculated log D values of reference compounds
Compound Name Log D Value
Propranolol 1.27
Caffeine -0.09
Ketoconazole 3.74
Diltiazem 2.05
Ranitidine -0.12
Atenolol -1.29
Telmisartan 1.90
Metaprolol -0.30
Warfarin 0.80
Table 3: Generic gradient method used for the analysis of reference compounds and PEG 400
Time (min) Flow Rate
(mL/min)
%A (Aqueous
Modifier)
%B (Organic
Modifier)
0.01 1.00 95 5
1.50 1.00 5 95
2.50 1.00 5 95
2.60 1.00 95 5
3.50 1.00 95 5
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Table 4: List of MRM used for quantifying the reference compounds and PEG 400
Compound
Name
Q1 Mass
(Da)
Q3 Mass
(Da)
Dwell Time
(m sec)
Declustering
Potential (v)
Collision
energy (v)
Propranolol 260.10 116.20 100 40 25
Caffeine 195.10 137.90 100 45 25
Ketoconazole 531.10 82.10 100 80 80
Diltiazem 415.10 178.10 100 40 32
Ranitidine 315.10 176.10 100 25 22
Atenolol 267.10 145.10 100 40 32
Telmisartan 515.30 276.10 100 65 64
Metaprolol 268.10 116.00 100 50 25
Warfarin 309.20 163.00 100 50 21
PEG 400 520.40 89.20 100 40 29
Table 5: Plasma concentration levels of PEG 400 after intravenous administration at
3.38 g/kg dose
Time (hr)
Concentration (µg/mL)
%CV Rat-1 Rat-2 Rat-3 Mean STDEV
0.08 7866.52 9757.32 7459.52 8361.12 1226.15 15
0.25 4894.70 5400.41 4686.05 4993.72 367.33 7
0.50 3183.35 4141.70 3322.76 3549.27 517.77 15
1.00 1598.45 1600.54 1722.19 1640.39 70.85 4
2.00 799.27 1125.81 810.20 911.76 185.45 20
4.00 420.76 762.33 324.21 502.43 230.20 46
8.00 314.99 265.75 259.02 279.92 30.56 11
24.00 1.54 2.92 4.71 3.06 1.59 52
Table 6: Plasma concentration levels of PEG 400 after oral administration at 3.38 g/kg dose
Time (hr)
Concentration (µg/mL)
%CV Rat-1 Rat-2 Rat-3 Mean STDEV
0.25 249.08 246.85 250.12 248.68 1.67 1
0.50 367.34 261.51 435.77 354.87 87.80 25
1.00 615.31 345.77 589.90 516.99 148.83 29
2.00 804.31 469.78 707.60 660.56 172.15 26
4.00 210.13 465.85 322.94 332.97 128.15 38
8.00 131.07 177.94 117.47 142.16 31.72 22
24.00 BLQ BLQ BLQ NC NC NC
NC - Not Calculated BLQ - Below Limit of Quantitation
INTERNATIONAL JOURNAL OF RESEARCH ARTICLE PHARMACEUTICAL INNOVATIONS ISSN 2249-1031
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Sample Name: "PEG400-IV-2.00HR-2" Sample ID: "" File: "049.wiff"Peak Name: "PEG400-1" Mass(es): "327.3/89.2 Da,371.3/89.2 Da,432.4/89.2 Da,476.4/89.2 Da,520.4/89.2 Da"Comment: "" Annotation: ""
Sample Index: 1
Sample Type: Unknown
Concentration: N/A
Calculated Conc: 0.00 ng/mL
Acq. Date: 12/26/2012
Acq. Time: 1:57:22 PM
Modified: No
Proc. Algorithm: Analyst Classic
Bunching Factor: 1
Noise Threshold: 10.00 cps
Area Threshold: 100.00 cps
,Num. Smooths: 10
Sep. Width: 0.20
Sep. Height: 1.00
Exp. Peak Ratio: 5.00
Exp. Adj. Ratio: 4.00
Exp. Val. Ratio: 3.00
RT Window: 30.0 sec
Expected RT: 1.96 min
Use Relative RT: No
Int. Type: Base To Base
Retention Time: 1.94 min
Area: 782848 counts
Height: 5.48e+004 cps
Start Time: 1.58 min
End Time: 2.38 min
0.5 1.0 1.5 2.0 2.5 3.0Time, min
0.0
2000.0
4000.0
6000.0
8000.0
1.0e4
1.2e4
1.4e4
1.6e4
1.8e4
2.0e4
2.2e4
2.4e4
2.6e4
2.8e4
3.0e4
3.2e4
3.4e4
3.6e4
3.8e4
4.0e4
4.2e4
4.4e4
4.6e4
4.8e4
5.0e4
5.2e4
5.4e4
Inten
sity, cp
s
1.94
Acq. File:
PEG400-CE
ADJUSTED-POS
MRM.dam,..
Sample Name: PEG400-STD-1
Sample Number: Sample 4 of 11
Acq. File:
PEG400-CE
ADJUSTED-POS
MRM.dam,..
Sample Name: PEG400-STD-1
Sample Number: Sample 4 of 11
Acq. File:
PEG400-CE
ADJUSTED-POS
MRM.dam,..
Sample Name: PEG400-STD-1
Sample Number: Sample 4 of 11
Acq. File:
PEG400-CE
ADJUSTED-POS
MRM.dam,..
Sample Name: PEG400-STD-1
Sample Number: Sample 4 of 11
Acq. File:
PEG400-CE
ADJUSTED-POS
MRM.dam,..
Sample Name: PEG400-STD-1
Sample Number: Sample 4 of 11
Acq. File:
PEG400-CE
ADJUSTED-POS
MRM.dam,..
Sample Name: PEG400-STD-1
Sample Number: Sample 4 of 11
Acq. File:
PEG400-CE
ADJUSTED-POS
MRM.dam,..
Sample Name: PEG400-STD-1
Sample Number: Sample 4 of 11
Acq. File:
PEG400-CE
ADJUSTED-POS
MRM.dam,..
Sample Name: PEG400-STD-1
Sample Number: Sample 4 of 11
Acq. File:
PEG400-CE
ADJUSTED-POS
MRM.dam,..
Sample Name: PEG400-STD-1
Sample Number: Sample 4 of 11
Acq. File:
PEG400-CE
ADJUSTED-POS
MRM.dam,..
Sample Name: PEG400-STD-1
Sample Number: Sample 4 of 11
Acq. File:
PEG400-CE
ADJUSTED-POS
MRM.dam,..
Sample Name: PEG400-STD-1
Sample Number: Sample 4 of 11
Sample Name: "PEG400-PO-2.00HR-1" Sample ID: "" File: "064.wiff"Peak Name: "PEG400-1" Mass(es): "327.3/89.2 Da,371.3/89.2 Da,432.4/89.2 Da,476.4/89.2 Da,520.4/89.2 Da"Comment: "" Annotation: ""
Sample Index: 1
Sample Type: Unknown
Concentration: N/A
Calculated Conc: 0.00 ng/mL
Acq. Date: 12/26/2012
Acq. Time: 3:03:10 PM
Modified: No
Proc. Algorithm: Analyst Classic
Bunching Factor: 1
Noise Threshold: 10.00 cps
Area Threshold: 100.00 cps
,Num. Smooths: 10
Sep. Width: 0.20
Sep. Height: 1.00
Exp. Peak Ratio: 5.00
Exp. Adj. Ratio: 4.00
Exp. Val. Ratio: 3.00
RT Window: 30.0 sec
Expected RT: 1.96 min
Use Relative RT: No
Int. Type: Base To Base
Retention Time: 1.93 min
Area: 1379842 counts
Height: 9.74e+004 cps
Start Time: 1.58 min
End Time: 2.38 min
0.5 1.0 1.5 2.0 2.5 3.0Time, min
0.0
2000.0
4000.0
6000.0
8000.0
1.0e4
1.2e4
1.4e4
1.6e4
1.8e4
2.0e4
2.2e4
2.4e4
2.6e4
2.8e4
3.0e4
3.2e4
3.4e4
3.6e4
3.8e4
4.0e4
4.2e4
4.4e4
4.6e4
4.8e4
5.0e4
5.2e4
5.4e4
5.6e4
5.8e4
6.0e4
6.2e4
6.4e4
6.6e4
6.8e4
7.0e4
7.2e4
7.4e4
7.6e4
7.8e4
8.0e4
8.2e4
8.4e4
8.6e4
8.8e4
9.0e4
9.2e4
9.4e4
9.6e4
Intensi
ty, cps
1.93
Acq. File:
PEG400-CE
ADJUSTED-POS
MRM.dam,..
Sample Name: PEG400-STD-1
Sample Number: Sample 4 of 11
Acq. File:
PEG400-CE
ADJUSTED-POS
MRM.dam,..
Sample Name: PEG400-STD-1
Sample Number: Sample 4 of 11
Acq. File:
PEG400-CE
ADJUSTED-POS
MRM.dam,..
Sample Name: PEG400-STD-1
Sample Number: Sample 4 of 11
Acq. File:
PEG400-CE
ADJUSTED-POS
MRM.dam,..
Sample Name: PEG400-STD-1
Sample Number: Sample 4 of 11
Acq. File:
PEG400-CE
ADJUSTED-POS
MRM.dam,..
Sample Name: PEG400-STD-1
Sample Number: Sample 4 of 11
Acq. File:
PEG400-CE
ADJUSTED-POS
MRM.dam,..
Sample Name: PEG400-STD-1
Sample Number: Sample 4 of 11
Acq. File:
PEG400-CE
ADJUSTED-POS
MRM.dam,..
Sample Name: PEG400-STD-1
Sample Number: Sample 4 of 11
Acq. File:
PEG400-CE
ADJUSTED-POS
MRM.dam,..
Sample Name: PEG400-STD-1
Sample Number: Sample 4 of 11
Acq. File:
PEG400-CE
ADJUSTED-POS
MRM.dam,..
Sample Name: PEG400-STD-1
Sample Number: Sample 4 of 11
Acq. File:
PEG400-CE
ADJUSTED-POS
MRM.dam,..
Sample Name: PEG400-STD-1
Sample Number: Sample 4 of 11
Acq. File:
PEG400-CE
ADJUSTED-POS
MRM.dam,..
Sample Name: PEG400-STD-1
Sample Number: Sample 4 of 11
a) b)
c) d)
Figure 1: MRM LC-MS/MS chromatograms of a) PEG 400 in rat blank plasma b) rat
plasma sample spiked with 1.01 µg/mL of PEG 400 (LLOQ) c) plasma sample
obtained 2.00 hr after intravenous administration of PEG 400 to SD rats d) plasma
sample obtained 2.00 hr after oral administration of PEG 400 to SD rats
Sample Name: "BLK" Sample ID: "" File: "002.wiff"Peak Name: "PEG400" Mass(es): "520.4/89.2 Da"Comment: "" Annotation: ""
Sample Index: 1
Sample Type: Unknown
Concentration: N/A
Calculated Conc: No Intercept
Acq. Date: 10/7/2012
Acq. Time: 5:10:34 PM
Modified: Yes
1.0 2.0 3.0Time, min
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
105
110
115
120
Intensi
ty, cps
3.06
2.13
2.23
2.78
2.38
3.26
3.32
Sample Name: "PEG400-STD-1/2" Sample ID: "" File: "005.wiff"Peak Name: "PEG400-1" Mass(es): "327.3/89.2 Da,371.3/89.2 Da,432.4/89.2 Da,476.4/89.2 Da,520.4/89.2 Da"Comment: "" Annotation: ""
Sample Index: 1
Sample Type: Unknown
Concentration: N/A
Calculated Conc: 0.00 ng/mL
Acq. Date: 12/26/2012
Acq. Time: 10:45:34 AM
Modified: No
Proc. Algorithm: Analyst Classic
Bunching Factor: 1
Noise Threshold: 10.00 cps
Area Threshold: 100.00 cps
,Num. Smooths: 10
Sep. Width: 0.20
Sep. Height: 1.00
Exp. Peak Ratio: 5.00
Exp. Adj. Ratio: 4.00
Exp. Val. Ratio: 3.00
RT Window: 30.0 sec
Expected RT: 1.96 min
Use Relative RT: No
Int. Type: Base To Base
Retention Time: 1.96 min
Area: 37422 counts
Height: 2.63e+003 cps
Start Time: 1.64 min
End Time: 2.23 min
0.5 1.0 1.5 2.0 2.5 3.0Time, min
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
2000
2100
2200
2300
2400
2500
2600
2700
2800
2900
Intensi
ty, cps
1.96
2.79
Acq. File:
PEG400-CE
ADJUSTED-POS
MRM.dam,..
Sample Name: PEG400-STD-1
Sample Number: Sample 4 of 11
Acq. File:
PEG400-CE
ADJUSTED-POS
MRM.dam,..
Sample Name: PEG400-STD-1
Sample Number: Sample 4 of 11
Acq. File:
PEG400-CE
ADJUSTED-POS
MRM.dam,..
Sample Name: PEG400-STD-1
Sample Number: Sample 4 of 11
Acq. File:
PEG400-CE
ADJUSTED-POS
MRM.dam,..
Sample Name: PEG400-STD-1
Sample Number: Sample 4 of 11
Acq. File:
PEG400-CE
ADJUSTED-POS
MRM.dam,..
Sample Name: PEG400-STD-1
Sample Number: Sample 4 of 11
Acq. File:
PEG400-CE
ADJUSTED-POS
MRM.dam,..
Sample Name: PEG400-STD-1
Sample Number: Sample 4 of 11
Acq. File:
PEG400-CE
ADJUSTED-POS
MRM.dam,..
Sample Name: PEG400-STD-1
Sample Number: Sample 4 of 11
Acq. File:
PEG400-CE
ADJUSTED-POS
MRM.dam,..
Sample Name: PEG400-STD-1
Sample Number: Sample 4 of 11
Acq. File:
PEG400-CE
ADJUSTED-POS
MRM.dam,..
Sample Name: PEG400-STD-1
Sample Number: Sample 4 of 11
Acq. File:
PEG400-CE
ADJUSTED-POS
MRM.dam,..
Sample Name: PEG400-STD-1
Sample Number: Sample 4 of 11
Acq. File:
PEG400-CE
ADJUSTED-POS
MRM.dam,..
Sample Name: PEG400-STD-1
Sample Number: Sample 4 of 11
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Figure 2: Percent of control response for propranolol, Caffeine, Ketoconazole, Diltiazem,
Ranitidine, Atenolol, Telmisartan, Metaprolol and Warfarin at 15 mg/mL
concentration of PEG 400 under different analytical conditions
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
100.00
% C
on
tro
l re
spo
nse
APCI
FA-ACN
FA-MEOH
AMM ACET-ACN
AMM ACET-MEOH
LLE-EA
LLE-HEX
LLE-TBME
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a) b)
c) d)
Figure 3: Ion suppression effect at different concentrations of PEG 400 on a)
Propranolol b) Caffeine c) Ketoconazole d) Diltiazem
0.00
20.00
40.00
60.00
80.00
100.00
120.00
0 10 20
% C
on
tro
l re
spo
nse
PEG 400 Concentration (mg/mL)
APCI
FA-ACN
FA-MEOH
AMM ACET-ACN
AMM ACET-MEOH
LLE-EA
LLE-HEX
LLE-TBME
0.00
20.00
40.00
60.00
80.00
100.00
120.00
0 10 20
% C
on
tro
l re
spo
nse
PEG 400 Concentration (mg/mL)
APCI
FA-ACN
FA-MEOH
AMM. ACET.-ACN
AMM. ACET.-MEOHLLE-EA
LLE-HEXANE
LLE-TBME
0.00
20.00
40.00
60.00
80.00
100.00
120.00
0 10 20
% C
on
tro
l re
spo
nse
PEG 400 Concentration (mg/mL)
APCI
FA-ACN
FA-MEOH
AMM. ACET.-ACNAMM. ACET.-MEOHLLE-EA
LLE-HEXANE
LLE-TBME
0.00
20.00
40.00
60.00
80.00
100.00
120.00
0 10 20
% C
on
tro
l re
spo
nse
PEG 400 Concentration (mg/mL)
APCI
FA-ACN
FA-MEOH
AMM. ACET.-ACNAMM. ACET.-MEOHLLE-EA
LLE-HEXANE
LLE-TBME
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a) b)
c) d)
Figure 4: Ion suppression effect at different concentrations of PEG 400 on a)
Ranitidine b) Atenolol c) Telmisartan d) Metaprolol
0.00
20.00
40.00
60.00
80.00
100.00
120.00
0 10 20
% C
on
tro
l re
spo
nse
PEG 400 Concentration (mg/mL)
APCI
FA-ACN
FA-MEOH
AMM. ACET.-ACN
AMM. ACET.-MEOH
LLE-EA
LLE-HEXANE
LLE-TBME
0.00
20.00
40.00
60.00
80.00
100.00
120.00
0 10 20
% C
on
tro
l re
spo
nse
PEG 400 Concentration (mg/mL)
APCI
FA-ACN
FA-MEOH
AMM.ACET.-ACN
AMM. ACET.-MEOHLLE-EA
LLE-HEXANE
LLE-TBME
0.00
20.00
40.00
60.00
80.00
100.00
120.00
0 5 10 15 20
% C
on
tro
l re
spo
nse
PEG 400 Concentration (mg/mL)
APCI
FA-ACN
FA-MEOH
AMM. ACET.-ACNAMM. ACET.-MEOHLLE-EA
LLE-HEXANE
LLE-TBME0.00
20.00
40.00
60.00
80.00
100.00
120.00
0 10 20
% C
on
tro
l re
spo
nse
PEG 400 Concentration (mg/mL)
APCI
FA-ACN
FA-MEOH
AMM. ACET.-ACNAMM. ACET.-MEOHLLE-EA
LLE-HEXANE
LLE-TBME
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a) b)
Figure 5: a) Ion suppression effect at different concentrations of PEG 400 on warfarin b)
Peak area counts of PEG 400 quantified under different analytical conditions
0.00
20.00
40.00
60.00
80.00
100.00
120.00
0 10 20
% C
on
tro
l Re
spo
nse
PEG 400 Concentration (mg/mL)
APCI
FA-ACN
FA-MEOH
AMM. ACET.-ACNAMM. ACET.-MEOHLLE-EA
LLE-HEXANE
LLE-TBME
0
5000000
10000000
15000000
20000000
25000000
30000000
35000000
40000000
0 10 20
Pea
k A
rea
Co
un
ts (c
ps)
Concentration (mg/mL)
APCI
FA-ACN
FA-MEOH
AMM. ACET-ACNAMM. ACET.-MEOHLLE-EA
LLE-HEXANE
LLE-TBME