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PEG 400-LC-MC/MS Chapter 2

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CHAPTER 2

PEG 400-LC-MS/MS


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Liquid chromatography/tandem mass spectrometry method for the quantitative

estimation of polyethyleneglycol 400 and its applications

Table of Contents

2.1 Introduction ........................................................................................................ 31

2.2 Experimental section ......................................................................................... 31

2.2.1 Materials ...................................................................................................... 31

2.2.2 Purity estimation of PEG 400 oligomers ..................................................... 32

2.2.3 Preparation of calibration standards and quality control samples ............... 32

2.2.4 Sample preparation ...................................................................................... 33

2.2.5 LC-MS/MS Analysis ................................................................................... 33

2.2.6 Method Validation ....................................................................................... 34

2.2.7 Application ................................................................................................... 34

2.3 Results and discussion ....................................................................................... 35

2.3.1 Purity estimation of PEG 400 oligomers ..................................................... 35

2.3.2 LC-MS/MS Analysis ................................................................................... 35

2.3.3 Method Validation ....................................................................................... 39

2.3.4 Application study ......................................................................................... 45

2.3.4.1 PEG 400 ............................................................................................... 45

2.3.4.2 PEG 400 Oligomers ............................................................................. 46

2.4 Conclusion .......................................................................................................... 52

2.5 References ........................................................................................................... 53


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2.1 Introduction

In drug discovery the dose of NCEs to be administered is prepared as a solution in a

suitable vehicle consisting of water and formulation excipients such as ethanol,

dimethyl sulfoxide (DMSO), polyethylene glycol (PEG), propylene glycol (PG),

Tweens, Hydroxypropylβcyclodextrin (HPβCD) or their combinations (Fort FL et al,

1984). PEGs are widely used in a variety of pharmaceutical formulations, including

parenteral, topical, ophthalmic, oral and rectal preparations (Rowe RC et al, 2009). In

particular PEG 400 is widely used for solubility and safety reasons. There exists

different analytical methods for the quantitative measurement of polyethylene glycol

400 in the urine using high performance liquid chromatography (HPLC) (Cheng TL et

al, 2011; Delahunty T et al, 1986; Young GO et al, 1990; Schwertner HA et al, 1992;

Ryan CM et al, 1992; Sims GE et al, 1980; Ingham KC et al, 1978; Guermant C et al,

1995; Kinahan IM et al, 1991). But these methods lack detection sensitivity with

lower limit of quantitation (LLOQ) values ranging from 50 µg/mL to 4 mg/mL.

Colorimetric methods (Chung TW et al, 2000; Nag A et al, 1996; Li S et al, 2003)

were reported for the analysis of polyethylene glycols in biological matrices, which

also suffers from lack of sensitivity with an LLOQ value of 500 µg/mL. LC-MS

(Ashiru DAI et al, 2011) and GC-MS (Fakt C et al, 1997) methods for the analysis of

PEG 400 in urine were reported, but these methods were based on selected ion

monitoring (SIM) than multiple reaction monitoring (MRM). MRM was proved to be

more selective and specific than SIM mode of detection. In the present work an

attempt was made to develop and validate bioanalytical method for the quantitative

estimation of PEG 400 using LC-MS/MS and establish the plasma concentration

profile/PK parameters in male sprague dawley rats. Pharmacokinetic parameters for

PEG 400 as such and differences in pharmacokinetics of its oligomers were

established.

2.2 Experimental section

2.2.1 Materials

PEG 400, DMSO and telmisartan (internal standard) were procured from Sigma-

Aldrich Co. (St. Louis, MO, USA). Acetonitrile, water and methanol (HPLC grade)

were procured from Merck Specialities Pvt Ltd (Mumbai, India). Formic acid (90%


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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).

2.2.2 Purity estimation of PEG 400 oligomers

Purity of PEG 400 oligomers was estimated using 385 ELSD (Agilent, USA). The

HPLC system consisted of Agilent 1200 RRLC (Agilent, USA). The stationary phase

was XBridge C18 with 5 µm particle diameter (Waters, Ireland). The column

dimensions were 250 x 3.0 mm. The mobile phase flow rate was 0.5 mL/min. The

mobile phase consisted of 0.1% formic acid in water as aqueous component and 100%

acetonitrile as organic modifier. A generic gradient LC method (Time (min)/%B =

0.01/2, 23.00/50, 23.01/2, 30.00/2) with a run time of 30 min was developed for the

purity analysis of PEG 400 oligomers. The column and autosampler were maintained

at 40oC and 4oC respectively. The ELSD was operated with typical settings as

follows: evaporation temperature, 75oC; Nebulizer temperature, 80oC; gas, 1.65 SLM.

2.2.3 Preparation of calibration standards and quality control

samples

Master stock solution of telmisartan (1 mg/mL) was prepared in DMSO. Working

standard solutions of PEG 400 were prepared by serial diluting from master stock

(PEG 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 PEG 400 were prepared by spiking 2 µL of the working standard solutions

into 48 µL of blank rat plasma. Concentrations for plasma calibration standards and

quality control samples for oligomers were derived from the purity of each oligomer

in total PEG 400. The working solution for internal standard (100 ng/mL) was

prepared by diluting an aliquot of stock solution with acetonitrile. All PEG 400 and

telmisartan solutions were stored at 40C in polypropylene bottles in the dark, when not

in use.


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2.2.4 Sample preparation

A 50 µL aliquot of plasma (blank control plasma, plasma samples from rats dosed

with PEG 400, blank plasma spiked with calibration standards and QC samples) was

pipette transferred 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 3350 g 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) and 5 µL aliquots were injected for LC-MS/MS analysis.

2.2.5 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 autosampler

(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 0.1% formic acid in water as aqueous

component and 100% methanol as organic modifier. A generic gradient LC method

(Time (min)/%B = 0.01/2, 2.00/98, 2.50/2, 3.50/2) with a short run time of 3.5 min

was developed for the analysis of PEG 400 in plasma samples. 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,

20 psi; nebulizer gas (GS1), 50 psi; heater gas (GS2), 50 psi; ionspray 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


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medium CAD gas setting. Peak areas for all components were automatically

integrated using Analyst software version 1.5.

2.2.6 Method Validation

Method validation was performed for PEG 400 as a whole, instead of each oligomer.

Three precision and accuracy batches, consisting of calibration standards (1.01, 2.03,

10.14, 50.68, 202.71, 506.76, 810.82, 912.06, 1013.40 µg/mL), were analyzed on

three different days to complete the method validation. In each batch, QC samples at

3.89, 486.43 and 810.72 µg/mL were assayed in sets of six replicates to evaluate the

intra and inter-day precision and accuracy. The percentage deviation of the mean from

true values, expressed as relative error (RE), and the coefficient of variation (CV)

serve as the measure of accuracy and precision, respectively. The selectivity was

evaluated by analyzing blank plasma samples obtained from different animals.

Extraction efficiency of PEG 400 was determined by comparing peak areas of analyte

spiked before extraction into the six different lots of plasma with those of the analyte

post spiked into plasma extracts. Matrix effect was evaluated from matrix factor

values. Matrix factor was calculated by dividing mean peak areas of analyte post

spiked in to plasma extracts with those of analyte spiked in to neat solutions at three

QC levels. To assess post-preparative stability, six replicates of QC samples at each of

the low, mid and high concentrations were processed and stored under autosampler

conditions for 24 h before analysis. To assess bench top stability, six replicates of QC

samples at each of the low, mid and high concentrations were kept at room

temperature for 8 h before analysis. Freeze thaw stability was assessed at three QC

levels for three freeze thaw cycles. To assess long term stability, six replicates of QC

samples at each of the low, mid and high concentrations were kept at -80oC for

60 days before analysis.

2.2.7 Application

Individual rats (male Sprague-Dawley) were dosed at 3.38 g/kg intravenously (bolus)

through tail vein and 3.38 g/kg orally through oral gavage needle. Dosing volume

administered was 5 mL/kg. The composition of dosing vehicles used for the study was

DMSO: PEG 400: water (5:60:35) (Neervannan S, 2006; Sheftel VO, 2000) 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 (Kwon Y, 2002) after


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intravenous administration and 0.25, 0.50, 1, 2, 4, 8 and 24 h post dose (Kwon Y,

2002) after oral administration. At each time point 200 µL of blood was collected in

to vacutainers. Blood Samples were collected using retro orbital puncture method.

Plasma was isolated by centrifugation at 14,850 g for 10 min and stored frozen at -

80oC until analysis. Pharmacokinetic parameters such as elimination rate constant

(Kel), half life (T1/2), extrapolated drug concentration (C0), AUC0-last, AUC0-inf,

AUC%Extrapolated, volume of distribution (Vd), clearance (Cl), Tmax, Cmax, MRTlast and

absolute bioavailability were calculated using phoenix winnonlin software (v6.3).

Absolute bioavailability was calculated using AUC0-inf values as AUC%Extrapolated was

less than 20%.

2.3 Results and discussion

2.3.1 Purity estimation of PEG 400 oligomers

PEG 400 oligomers have very weak ultraviolet (UV) absorbance and need to be

seperated by gradient elution chromatography. This precludes their detection by UV

and refractive index (RI). RI and low wavelength UV detection are highly subject to

base line drifts with gradients and have limited solvent selection. Conversely, ELSD

allows direct detection of PEGs without derivatisation and is compatible with gradient

elution chromatography. Longer gradient program with maximum % organic ramped

to 50% had achieved good separation between oligomers (Figure 2.1). Ramping the

organic phase to higher percentages (55-95%) didn’t achieve good separation between

oligomer 6, 7, 8 and 9. Purity of each oligomer was estimated by area normalization

method. % Purity of oligomer 1, 2, 3, 4, 5, 6, 7, 8 and 9 was 11.007, 17.695, 23.061,

20.704, 13.865, 7.769, 3.589, 1.551 and 0.760% respectively.

2.3.2 LC-MS/MS Analysis

The electrospray ionization of PEG 400 produced the abundant molecular ions at m/z,

89.10, 133.20, 177.20, 221.20, 327.30, 371.30, 415.30, 432.30, 459.30, 476.30,

503.40, 520.40, 547.40, 564.40, 591.30, 608.50, 635.30, 652.40, 679.40 and 696.40

(Figure 2.2) under positive ionization conditions. The lower masses at m/z 89.10,

133.20, 177.20, 221.10 correspond to insource fragments of different oligomers and

these masses didn’t generate further distinct fragment ions. The higher masses

generated ammonium adduct (m/z, 432.30, 476.30, 520.40, 564.40, 608.50, 652.40,


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696.40) molecular ions except at m/z 327.30, 371.30. A total of 9 abundant and

informative oligomers were identified.

Figure 2.1: Chromatogram representing the nine oligomers observed in PEG 400

sample injected under reverse phase conditions

The fragment ion at m/z 89.20 (two ethylene oxide units) (James L, 2011) was

produced as the prominent product ion for all the selected PEG 400 oligomers. 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. Calibration range of

1.01 µg/mL to 1013.40 µg/mL represents total PEG 400. In order to characterise the

pharmacokinetic differences for PEG 400 oligomers, plasma concentrations of each

oligomer was measured against calibration curves built based on purity of each

oligomer. The electrospray ionization of telmisartan produced abundant protonated

molecules ([MH]+) at 515.20 amu and generated an intense fragment at 276.10 amu

(Figure 2.3). LC-MS/MS methods operated with the C18 column and a 3.5 min

generic gradient method was developed for the analysis of PEG 400 in plasma. Final

mobile phase composition used for the analysis was 0.1% formic acid in water as

aqueous phase and 100% methanol as organic modifier. But if acetonitrile is used as

organic modifier, linearity wasn’t achieved as the response got saturated at higher

calibration standards.


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When organic phase was ramped from 5% organic to 95% organic in 1.5 min and

maintained at 95% to 2.5 min in gradient method, a false peak generated at the

retention time of PEG 400 with peak area counts greater than LLOQ response.

Investigation over carryover, contamination showed that these were not the reasons

for the false peak area. It was found that higher organic % as isocratic portion

(1.50-2.50 min) of the gradient was the reason behind the false peak appearance. So a

second gradient method was designed in such a way that organic phase was ramped

from 2% to 98% organic in 2.0 min and to 2% organic at 2.50 min.

+Q1: 0.000 min from Sampl... Max. 9.3e6 cps.

100 200 300 400 500 600 700 800m/z, Da

0.0

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481.3 591.4437.3327.3265.3 696.4

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309.3283.3 635.5409.3 585.3 740.5629.3397.3365.3249.2 441.3

Figure 2.2: Parent Ion (Full Scan) scan of PEG 400


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+MS2 (515.30) CE (50): 0.1... Max. 6.1e6 cps.

100 200 300 400 500m/z, Da

0.0

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ps276.1

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193.1275.2

165.1317.2 467.2183.2155.2

Figure 2.3: Product Ion scan of telmisartan (Internal standard)


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Interference wasn’t observed at the retention time of PEG 400 with the modified

gradient conditions. Because of the higher sensitivity of LC-MS/MS method

compared to that of HPLC or colorimetric methods, lesser plasma sample volume

(50 µL) is sufficient to obtain an LLOQ of 1 µg/mL. Even though the calibration

range of 1 µg/mL to 1000 µg/mL was higher for analysis on mass spectrometer,

analysis of plasma samples revealed that the plasma concentrations of PEG 400 was

around 1-10 mg/mL in the initial sampling points from intravenous route. Therefore,

if these study samples have to fit in to the low ng/mL standard curve, very high

dilution (100-1000 fold) is required, which requires more blank plasma for dilution,

which practically is a limitation in drug discovery. So rather than developing a

method with high sensitivity, here efforts were put to decrease the sensitivity of

higher calibration range by diluting the precipitated samples 10 fold after precipitation

and injected less volume of sample (5 µL) and analyzing samples on low sensitive

mass spectrometer (3200 QTrap). Representative chromatogram of telmisartan at

100 ng/mL spiked concentration was shown in Figure 2.4.

Representative chromatogram of PEG 400 (chromatogram representing sum of peaks

of all oligomers) at LLOQ was shown in Figure 2.5. No interference at the retention

times of telmisartan (2.53 min) (Figure 2.6) and PEG 400 (1.96 min) (Figure 2.7) 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. The

LLOQ was set at 1.01 µg/mL for PEG 400 using 50 µL of rat plasma. The signal-to-

noise ratio for PEG 400 is about 400 at 1.01 µg/mL. The retention times of PEG 400

and telmisartan were reproducible throughout the experiment and no column

deterioration was observed after analysis of plasma samples.

2.3.3 Method Validation

The developed method was validated to meet the acceptance criteria of industrial

guidance for the bioanalytical method validation (FDA, 2001). Calibration curves

were obtained over the concentration range of 1.01-1013.40 µg/mL of PEG 400 in

plasma. Linear regression analysis with a weighting of 1/(x*x) gave the optimum

accuracy of the corresponding calculated concentrations at each level (Table 2.1). The

low CV value for the slope indicated the repeatability of the method (Table 2.1).

Table 2.2 shows a summary of intra and inter-day precision and accuracy data for QC


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samples containing PEG 400. Both intra and inter assay CV values ranged from 2.31

to 13.34% at three QC levels.

Sample Name: "BLK+IS" Sample ID: "" File: "003.wiff"Peak Name: "TELMISARTAN(IS)" Mass(es): "515.3/276.1 Da"Comment: "" Annotation: ""

Sample Index: 1 Sample Type: Unknown Concentration: 1.00 ng/mL Calculated Conc: N/A Acq. Date: 12/26/2012 Acq. Time: 10:36:52 AM Modified: Yes Proc. Algorithm: Analyst Classic Bunching Factor: 1 Noise Threshold: 10.00 cpsArea Threshold: 100.00 cps,Num. Smooths: 10 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 secExpected RT: 2.52 minUse Relative RT: No Int. Type: Base To Base Retention Time: 2.53 minArea: 38129 countsHeight: 3.65e+003 cpsStart Time: 2.26 minEnd Time: 2.83 min

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Figure 2.4: MRM LC-MS/MS chromatogram of telmisartan at 100 ng/mL

concentration level in rat plasma


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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 cpsArea 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 secExpected RT: 1.96 minUse Relative RT: No Int. Type: Base To Base Retention Time: 1.96 minArea: 37422 countsHeight: 2.63e+003 cpsStart Time: 1.64 minEnd Time: 2.23 min

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Figure 2.5: MRM LC-MS/MS chromatogram of rat plasma sample spiked with

1.01 µg/mL of PEG 400 (LLOQ)


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Sample Name: "BLK" Sample ID: "" File: "002.wiff"Peak Name: "TELMISARTAN(IS)" Mass(es): "515.3/276.1 Da"Comment: "" Annotation: ""

Sample Index: 1 Sample Type: Unknown Concentration: 1.00 ng/mL Calculated Conc: N/A Acq. Date: 10/7/2012 Acq. Time: 5:10:34 PM Modified: Yes

0.5 1.0 1.5 2.0 2.5 3.0Time, min

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3.151.28 1.870.920.17

Figure 2.6: MRM LC-MS/MS chromatogram of telmisartan in rat blank plasma


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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

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Figure 2.7: MRM LC-MS/MS chromatogram of PEG 400 in rat blank plasma


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Table 2.1: Calculated concentrations and statistical parameters of PEG 400

calibration standards prepared in rat plasma (n=3)

Concentration (µg/mL) Statistical parameters

Actual conc.

Calculated Conc. Mean SD

% CV

Relative Error (%)

% Accuracy Set-1 Set-2 Set-3

1.01 0.98 1.07 1.07 1.04 0.052 5.00 2.97 102.97 2.03 2.16 1.76 1.78 1.90 0.225 11.86 -6.40 93.60

10.14 10.07 10.93 9.96 10.32 0.531 5.15 1.78 101.78

50.68 54.47 53.39 59.95 55.94 3.517 6.29 10.37 110.37 202.71 219.84 221.63 196.09 212.52 14.257 6.71 4.84 104.84

506.76 549.41 528.6 569.21 549.07 20.307 3.70 8.35 108.35 810.82 747.77 772.55 788.45 769.59 20.501 2.66 -5.08 94.92

912.06 804.46 826.02 868.45 832.98 32.557 3.91 -8.67 91.33 1013.40 941.05 961.81 896.02 932.96 33.633 3.60 -7.94 92.06

The intra and inter assay RE values for PEG 400 were -9.25 to 0.37% at three QC

levels. These results indicate that the present method has an acceptable accuracy and

precision. As shown in Table 2.3, the overall extraction efficiency of PEG 400 was

103.82%, which was consistent with a total % CV less than 2.96% at three QC

concentration levels. Mean matrix factor values of 0.97 (Table 2.3) at three QC levels

shows that the developed method is totally free of matrix effects for the analysis of

PEG 400. Acceptable matrix factor range for qualifying the method to be free from

matrix effects is 0.85 -1.15. Protein precipitation has been successfully applied to the

extraction of PEG 400 from rat plasma. Extracted QC samples were stable when

stored at 4oC for 24 h (autosampler stability) prior to injection, with <0.35% (Table

2.3) difference from theoretical concentration. Spiked QC samples were stable when

stored at room temperature for 8 h (bench top stability) prior to injection, with

<6.84% (Table 2.3) difference from theoretical concentration. Spiked QC samples

were stable for three freeze thaw cycles (freeze thaw stability) with <2.85% (Table

2.3) difference from theoretical concentration. Long term stability at -80oC was

proved for a period of 60 days with <2.08% (Table 2.3) difference from theoretical

concentration.


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Table 2.2: Precision and accuracy of PEG 400 in quality control samples

Type Statistical parameter

Concentration (µg/mL)

LQC (3.89)

MQC (486.43)

HQC (810.72)

Intra Day-Set-1 (N=6)

Mean 3.80 447.58 742.89 SD 0.30 19.04 40.04 % CV 7.96 4.25 5.39 % Accuracy 97.60 92.01 91.63 Relative Error (%) -2.40 -7.99 -8.37

Intra Day-Set-2 (N=6)

Mean 3.65 488.24 813.21 SD 0.49 18.37 40.83 % CV 13.34 3.76 5.02 % Accuracy 93.70 100.37 100.31 Relative Error (%) -6.30 0.37 0.31

Inter Day-Set-3 (N=6)


Inter Day (N=18)


2.3.4 Application study

2.3.4.1 PEG 400

The developed method has been successfully applied to the bioanalysis of rat plasma

samples in absolute bioavailability study of PEG 400. Representative chromatograms

of PEG 400 from intravenous (2.00 h), oral (2.00 h) study samples were shown in

Figure 2.8 and Figure 2.9 respectively. The intravenous and oral concentration-time

profiles of PEG 400 is represented in Figure 2.10 and Figure 2.11 respectively. As

PEG 400 had a clear absorption and elimination phase in oral route of administration

and clear elimination phase in intravenous route of administration, monitoring PEG

400 along with NCEs helps to take a decision on the spiky profile of NCEs.

Monitoring formulation excipients concentrations in PK study samples acts as quality

control check starting from formulation preparation to the completion of bioanalysis.


Page 46

Intravenous and oral pharmacokinetic parameters of PEG 400 were listed in Table 2.4

and Table 2.5 respectively. The mean absolute oral bioavailability of PEG 400 was

measured as 47.23% with a rapid terminal half life of 2 h, which was consistent with

published results (He YL et al, 1998). Mean Tmax and Cmax after oral administration

of PEG 400 to sprague dawley rats was 2.00 h and 936.32 µg/mL respectively. Mean

residence time of PEG 400 after intravenous and oral administration of PEG 400 to

sprague dawley rats was 1.93 and 2.68 h respectively.

Table 2.3: Summary of validation parameters for PEG 400 in rat plasma

Validation Parameter Statistical parameter

Result

Extraction Recovery Mean 103.82 SD 3.07 % CV 2.96

Matrix factor (Matrix effect) Mean 0.97 SD 0.06 % CV 6.14

Autosampler stability Mean 102.88 SD 0.35 % CV 0.34

Bench Top stability Mean 99.57 SD 6.84 % CV 6.86

Freeze thaw stability Mean 104.92 SD 2.85 % CV 2.71

Long term stability Mean 102.70 SD 2.08 % CV 2.03

2.3.4.2 PEG 400 Oligomers

Mean intravenous and oral pharmacokinetic parameters of PEG 400 oligomers were

presented in Table 2.6 and Table 2.7 respectively. It was found that upon increase in

molecular weight of oligomers, there was decrease in absolute bioavailability (He YL

et al, 1998). This could be attributed to decrease in permeability with increase in


Page 47

molecular weight of oligomer. All the oligomers studied have clear absorption and

elimination phase in oral route of administration and clear elimination phase in

intravenous route of administration. So for qualifying the analytical batches any of the

nine oligomers can be studied along with NCEs or all the oligomers can be monitored

and summed up for reflecting the total PEG 400.

Table 2.4: Pharmacokinetic parameters of PEG 400 after intravenous

administration of PEG 400 at 3.38 g/kg dose to male SD rats

PK Parameters Rat-1 Rat-2 Rat-3 Mean SD CV%

Kel (1/h) 0.29 0.28 0.23 0.27 0.03 12.8

T1/2 (h) 2.38 2.48 3.05 2.64 0.36 13.7

C0 (µg/mL) 9834 12889 9284 10669 1942 18.2

AUClast (h*µg/mL) 8651 10705 8338 9232 1286 13.9

AUCINF_obs (h*µg/mL) 8657 10716 8359 9244 1283 13.9

AUC_%Extrap_obs (%) 0.06 0.10 0.25 0.14 0.10 73.1

Vz_obs (L/Kg) 1.34 1.13 1.78 1.42 0.33 23.4

Cl_obs (mL/min/kg) 6.51 5.26 6.74 6.17 0.80 12.9

MRTlast (h) 2.94 2.83 2.99 2.92 0.08 2.76

Table 2.5: Pharmacokinetic parameters of PEG 400 after oral administration of

PEG 400 at 3.38 g/kg dose to male SD rats

PK Parameters Rat-1 Rat-2 Rat-3 Mean SD CV%

Kel (1/h) 0.60 0.41 0.21 0.41 0.20 48.0

T1/2 (h) 1.16 1.67 3.35 2.06 1.15 55.6

Tmax (h) 2.00 2.00 2.00 2.00 0.00 0.00

Cmax (µg/mL) 1119 633 1058 936 265 28.3

AUClast (h*µg/mL) 3751 2922 3521 3398 428 12.6

AUCINF_obs (h*µg/mL) 3803 3064 3532 3466 374 10.8

AUC_%Extrap_obs (%) 1.38 4.63 0.30 2.10 2.25 107

Vz_F_obs (L/Kg) 1.49 2.66 4.63 2.93 1.59 54.3

Cl_F_obs (mL/min/kg) 14.8 18.4 16.0 16.4 1.82 11.1

MRTlast (h) 2.34 3.10 2.60 2.68 0.39 14.4

F (%) 51.8 41.8 48.1 47.2 5.09 10.8


Page 48

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 cpsArea 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 secExpected RT: 1.96 minUse Relative RT: No Int. Type: Base To Base Retention Time: 1.94 minArea: 782848 countsHeight: 5.48e+004 cpsStart Time: 1.58 minEnd 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

Inte

nsity

, cps

1.94

Figure 2.8: MRM LC-MS/MS chromatogram of plasma sample obtained 2.00 h

after intravenous administration of PEG 400 to SD rats


Page 49

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 cpsArea 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 secExpected RT: 1.96 minUse Relative RT: No Int. Type: Base To Base Retention Time: 1.94 minArea: 782848 countsHeight: 5.48e+004 cpsStart Time: 1.58 minEnd 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

Inte

nsity

, cps

1.94

Figure 2.9: MRM LC-MS/MS chromatogram of plasma sample obtained 2.00 h

after oral administration of PEG 400 to SD rats


Page 50

Figure 2.10: Mean concentration time profile of PEG 400 after intravenous

administration at 3.38 g/kg dose to SD rats

Figure 2.11: Mean concentration time profile of PEG400 after oral administration at

3.38 g/kg dose to SD rats


Page 51

Table 2.6: Mean pharmacokinetic parameters of PEG 400 oligomers after

intravenous administration of PEG 400 at 3.38 g/kg dose to male SD

rats

PK Parameter Oligomer #

1 2 3 4 5 6 7 8 9

Kel (1/h) 0.23 0.24 0.21 0.20 0.19 0.17 0.15 0.16 0.18

T1/2 (h) 3.11 3.01 3.42 3.66 3.78 4.04 4.54 4.79 4.27

C0 (µg/mL) 839 1551 2040 1948 1369 818 383 175 92

AUClast (h*µg/mL) 909 1456 1692 1471 949 530 231 98 52

AUCINF_obs (h*µg/mL) 914 1462 1699 1478 953 533 232 98 52

AUC_%Extrap_obs (%) 0.45 0.38 0.42 0.45 0.48 0.52 0.63 0.75 0.73

Vz_obs (L/Kg) 1.85 1.79 2.30 2.53 2.73 2.91 3.48 3.80 3.04

Cl_obs (mL/min/kg) 6.81 6.84 7.69 7.93 8.25 8.25 8.79 8.99 8.22

MRTlast (h) 2.16 2.10 2.01 1.98 1.85 1.78 1.68 1.50 1.53

Table 2.7: Mean pharmacokinetic parameters of PEG 400 oligomers after

intravenous administration of PEG 400 at 3.38 g/kg dose to male SD

rats

PK Parameter Oligomer #

1 2 3 4 5 6 7 8 9

Kel (1/h) 0.25 0.25 0.40 0.39 0.38 0.37 0.36 0.36 0.34

T1/2 (h) 2.85 2.79 2.07 2.11 2.13 2.24 2.31 2.29 2.32

Tmax (h) 1.67 2.00 2.00 2.00 2.67 2.67 2.67 2.67 2.33

Cmax (µg/mL) 188 276 237 168 82 42 13 3.90 1.65

AUClast (h*µg/mL) 581 886 770 557 276 148 47 14.8 6.85

AUCINF_obs (h*µg/mL) 582 888 789 572 283 152 49 15.2 7.10

AUC_%Extrap_obs (%) 0.17 0.18 2.45 2.66 2.72 3.46 3.46 3.4 3.55

Vz_F_obs (L/Kg) 2.65 2.71 3.01 3.83 5.23 5.64 8.42 11.7 12.1

Cl_F_obs (mL/min/kg) 10.7 11.3 16.6 20.6 28.0 29.2 42.2 59.8 64.7

MRTlast (h) 2.78 3.07 2.84 2.92 3.03 3.21 3.31 3.42 3.63

F (%) 63.8 60.7 46.4 38.7 29.7 28.6 21.1 15.5 13.5


Page 52

2.4 Conclusion

A rapid, sensitive and reliable LC-MS/MS method for the determination of PEG 400

in rat plasma has been successfully developed and validated using protein

precipitation extraction as sample preparation procedure. This assay method

demonstrated acceptable sensitivity (LLOQ: 1.01 µg/mL), precision, accuracy,

selectivity, recovery and stability. The validated method was successfully applied to

assay rat plasma samples and established the plasma concentration profiles/PK

parameters of PEG 400 after intravenous and oral administration to male sprague

dawley rats. Pharmacokinetic parameters of PEG 400 and difference in

pharmacokinetics of its oligomers were developed and established.


Page 53

2.5 References

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� Cheng T.L., Chuang K.H., Chen B.M., Roffler S.R. (2011), Analytical

measurement of PEGylated molecules, Bioconjugate Chemistry, 1-19

� Chung T.W., Chung C.H., Lue Y.F. (2000), A colorimetric method for

determining distearoylphosphatidylethanolamine polyethylene glycol 2000 in

blood suspension, Anal. Biochem., 285., 264-266.

� Delahunty T., Hollander D. (1986), New liquid chromatographic method for

measuring polyethyleneglycol in urine, Clin. Chem., 32., 351-353.

� Fakt C., Ervik M. (1997), Determination of low levels of poly(ethylene glycol)

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� Fort F.L., Heyman I.A., Kesterson J.W. (1984), Hemolysis study of aqueous

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� Guermant C., Brygier J., Baeyens V.D., Nijs M., Vincentelli J., Paul C., Looze

Y. (1995), Quantitative determination of polyethyleneglycol based on its

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� Guidance for Industry-Bioanalytical Method Validation. (2001), US

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� He Y.L., Murby S., Warhurst G., Gifford L., Walker D., Ayrton J., Eastmond

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� Ingham K.C., Ling R.C. (1978), A quantitative assay for poly(ethylene glycol)

without interference by proteins, Anal. Biochem., 85., 139-145.

� James L. (2011), Identification of surfactants by Liquid Chromatography-

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� Kinahan I.M., Smyth M.R. (1991), High performance liquid chromatographic

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� Kwon Y. (2002), Pharmacokinetic study design and data interpretation,

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� Li S., Yang Z., Sun X., Tan Y., Yagi S., Hoffman R.M. (2003), A

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� Rowe R.C., Sheskey P.J., Quinn M.E. (2009), Polyethylene glycols, The

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� Ryan C.M., Yarmush M.L., Tompkins R.G. (1992), Separation and

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� Schwertner H.A., Patterson W.R., Cissik J.H., Wilson K.W. (1992), New

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� Sheftel V.O. (2000), Indirect food additives and polymers: Migration and

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� Young G.O., Ruttenberg D., Wright J.P. (1990), Measurement of

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