identification and reduction of matrix effects caused by...

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.



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



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.



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 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



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



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 %



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




3b.

3.2.3 Ketoconazole


mg/mL, ketoconazole had >80 % ion



preparation with hexane, TBME and




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


mg/mL, diltiazem had >65 % of ion



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


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



4a.



3.2.6 Atenolol


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



4b.

3.2.7 Telmisartan


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




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



4c.

3.2.8 Metaprolol


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




metaprolol (Figure 2). Ion suppression at




4d.

3.2.9 Warfarin


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




warfarin (Figure 2). Ion suppression at




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,



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.

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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.



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



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



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,..



Acq. File:

PEG400-CE

ADJUSTED-POS

MRM.dam,..



Acq. File:

PEG400-CE

ADJUSTED-POS

MRM.dam,..



Acq. File:

PEG400-CE

ADJUSTED-POS

MRM.dam,..



Acq. File:

PEG400-CE

ADJUSTED-POS

MRM.dam,..



Acq. File:

PEG400-CE

ADJUSTED-POS

MRM.dam,..



Acq. File:

PEG400-CE

ADJUSTED-POS

MRM.dam,..



Acq. File:

PEG400-CE

ADJUSTED-POS

MRM.dam,..



Acq. File:

PEG400-CE

ADJUSTED-POS

MRM.dam,..



Acq. File:

PEG400-CE

ADJUSTED-POS

MRM.dam,..



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


Concentration: N/A


Acq. Date: 12/26/2012


Modified: No


Bunching Factor: 1



,Num. Smooths: 10

Sep. Width: 0.20

Sep. Height: 1.00




RT Window: 30.0 sec


Use Relative RT: No



Area: 1379842 counts



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,..



Acq. File:

PEG400-CE

ADJUSTED-POS

MRM.dam,..



Acq. File:

PEG400-CE

ADJUSTED-POS

MRM.dam,..



Acq. File:

PEG400-CE

ADJUSTED-POS

MRM.dam,..



Acq. File:

PEG400-CE

ADJUSTED-POS

MRM.dam,..



Acq. File:

PEG400-CE

ADJUSTED-POS

MRM.dam,..



Acq. File:

PEG400-CE

ADJUSTED-POS

MRM.dam,..



Acq. File:

PEG400-CE

ADJUSTED-POS

MRM.dam,..



Acq. File:

PEG400-CE

ADJUSTED-POS

MRM.dam,..



Acq. File:

PEG400-CE

ADJUSTED-POS

MRM.dam,..



Acq. File:

PEG400-CE

ADJUSTED-POS

MRM.dam,..



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


Concentration: N/A

Calculated Conc: No Intercept

Acq. Date: 10/7/2012


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


Concentration: N/A


Acq. Date: 12/26/2012

Acq. Time: 10:45:34 AM

Modified: No


Bunching Factor: 1



,Num. Smooths: 10

Sep. Width: 0.20

Sep. Height: 1.00




RT Window: 30.0 sec


Use Relative RT: No



Area: 37422 counts



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,..



Acq. File:

PEG400-CE

ADJUSTED-POS

MRM.dam,..



Acq. File:

PEG400-CE

ADJUSTED-POS

MRM.dam,..



Acq. File:

PEG400-CE

ADJUSTED-POS

MRM.dam,..



Acq. File:

PEG400-CE

ADJUSTED-POS

MRM.dam,..



Acq. File:

PEG400-CE

ADJUSTED-POS

MRM.dam,..



Acq. File:

PEG400-CE

ADJUSTED-POS

MRM.dam,..



Acq. File:

PEG400-CE

ADJUSTED-POS

MRM.dam,..



Acq. File:

PEG400-CE

ADJUSTED-POS

MRM.dam,..



Acq. File:

PEG400-CE

ADJUSTED-POS

MRM.dam,..



Acq. File:

PEG400-CE

ADJUSTED-POS

MRM.dam,..





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



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


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


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


APCI

FA-ACN

FA-MEOH


LLE-HEXANE

LLE-TBME



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


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


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


APCI

FA-ACN

FA-MEOH


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


APCI

FA-ACN

FA-MEOH


LLE-HEXANE

LLE-TBME



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


APCI

FA-ACN

FA-MEOH


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

identification and reduction of matrix effects caused by...

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