tandem mass spectrometry method for quantitative estimation of polyethylene glycol 400 and its applications

Journal of Chromatography B, 926 (2013) 68–76

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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb

Liquid chromatography/tandem mass spectrometry method for quantitative estimation of polyethylene glycol 400 and its applications V. Vijaya Bhaskar a,∗ , Anil Middha b , Sudhir Tiwari a , Savithiri Shivakumar a a b

DMPK Laboratory (Biology Division), GVK BIO, Nacharam, Hyderabad, Andhra Pradesh 500076, India Department of pharmacy, Jagadishprasad Jhabermal Tibrewala University, Vidyanagari, Jhunjhunu, Rajasthan 333001, India

a r t i c l e

i n f o

Article history: Received 20 November 2012 Accepted 21 February 2013 Available online 4 March 2013 Keywords: PEG 400 Oligomers LC–MS/MS NCE Method validation Bioavailability

a b s t r a c t A rapid sensitive and selective MRM based method for the determination of polyethylene glycol 400 (PEG 400) in rat plasma was developed using liquid chromatography/tandem mass spectrometry (LC–MS/MS). PEG 400 and telmisartan (Internal standard) were extracted from rat plasma with acetonitrile and analysed on C18 column (Waters Xbridge, 50 × 4.6 mm, 3.5 ␮m) with the mobile phase (A – 0.1% formic acid in water; B – methanol). A generic gradient method with a short run time of 3.5 min was developed for the analysis of PEG 400. 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. The standard curve was linear (0.9954) over the concentration range of 1.01–1013.40 ␮g/mL. The lower limit of quantitation for PEG 400 was 1.01 ␮g/mL using 50 ␮L plasma. The coefficient of variation and relative error for inter and intraassay at three QC levels were 2.31–13.34 and −7.99 to 0.37, respectively. The method was validated for various parameters such as extraction recovery, matrix effect, autosampler stability, benchtop stability, freeze thaw stability, long term stability and was proved to be consistent across three QC levels with overall %CV less than 15. The developed method was successfully applied to the absolute bioavailability study of PEG 400 in male Sprague Dawley rats. Plasma concentrations of PEG 400 was measured after administration through oral and intravenous routes in male Sprague Dawley rats at a dose of 3.38 g/kg. Pharmacokinetic (PK) parameters were characterised by performing the analysis using Phoenix Winnonlin software (v 6.3). PEG 400 has good oral bioavailability with mean absolute bioavailability of 47.23%. Plasma concentration profile/PK parameters of PEG 400 was established in both intravenous and oral routes, which helps to qualify the analytical batch of NCEs having spiky plasma concentration profiles/erratic results. Purity of the PEG 400 oligomers was estimated using ELSD detection. Differences in pharmacokinetics of oligomers was studied. It was found that with increase in molecular weight of the oligomer, a decrease in absolute bioavailability was observed. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved.

1. Introduction In early stages of drug discovery, rat is the most commonly studied animal in pharmacokineitc and drug metabolism and disposition studies as it is relatively inexpensive and can be easily acquired and handled [1]. In a typical pharmacokientic (PK) study new chemical entities (NCEs) are administered to rats via intravenous and oral routes. Serial blood samples are collected and assayed by liquid chromatography/tandem mass spectrometry (LC–MS/MS) and final pharmacokinetic parameters are calculated.

∗ Corresponding author. Tel.: +91 8143853440. E-mail addresses: [email protected], [email protected] (V. Vijaya Bhaskar).

Some NCEs had spiky plasma concentration profiles and various reasons for such profiles could be due to enterohepatic circulation, or discrepancies in sample collection/sample processing. Spiky profiles in elimination phase will lead to inaccurate quantification of PK parameters. Extensive studies needed to be carried to characterise enterohepatic circulation behavior of test compound. Drugs that undergo enterohepatic cycling to a significant extent include colchicine, phenytoin, leflunomide and tetracycline antibiotics [2]. As formulation vehicles has fixed plasma concentration profiles irrespective of NCEs dosed, monitoring the plasma concentration levels of vehicle along with NCE will help to take a decision on the spiky plasma concentration profiles of NCE. A thoroughly developed and validated bioanalytical method is required to fix the plasma concentration profile and understand the pharmacokinetic disposition of formulation vehicle studied. Integrity of results from

1570-0232/$ – see front matter. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2013.02.021

V. Vijaya Bhaskar et al. / J. Chromatogr. B 926 (2013) 68–76

pharmacokinetic studies can be cross verified if formulation vehicles that had fixed plasma concentration profile/PK parameters is monitored along with the test compound studied. In drug discovery the dose to be administered is prepared as a solution in a suitable vehicle consisting of water and formulation vehicles such as ethanol, dimethylsulfoxide (DMSO), polyethylene glycol (PEG), propylene glycol (PG), Tweens, hydroxypropyl ␤ cyclodextrin (HP␤CD) or their combinations [3]. PEGs are widely used in a variety of pharmaceutical formulations, including parenteral, topical, ophthalmic, oral, and rectal preparations [4]. In particular Polyethylene glycol 400 is widely used for solubility and safety reasons. There exist different analytical methods for the quantitative measurement of polyethylene glycol 400 (PEG 400) in the urine using high performance liquid chromatography (HPLC) [5–13]. But these methods lack detection sensitivity with lower limit of quantitation (LLOQ) values ranging from 50 ␮g/mL to 4 mg/mL. Colorimetric methods [14–16] were reported for the analysis of polyethylene glycols in biological matrices but also suffer from lack of sensitivity with an LLOQ value of 500 ␮g/mL. LC–MS [17] and GC–MS [18] 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 present 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.

69

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 ␮g/mL) and quality control samples (3.89, 486.43, 810.72 ␮g/mL) of PEG 400 was prepared by spiking 2 ␮L of the working standard solutions into 48 ␮L of blank rat plasma. Concentrations for plasma calibration standards and quality control samples for oligomers were derived from PEG 400 concentrations based on the purity of each oligomer. 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 4 ◦ C in polypropylene bottles in the dark when not in use. 2.4. Sample preparation A 50 ␮L 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 in to a 96-well polypropylene plate and extracted with 200 ␮L of acetonitrile containing internal standard. Samples were vortex mixed for 10 min at 1200 rpm and centrifuged at 4000 rpm for 10 min at 4 ◦ C. 50 ␮L of supernatant was pipette transferred in to a fresh analysis plate and diluted with 450 ␮L of methanol: water (1:1) and 5 ␮L aliquots were injected for LC–MS/MS analysis. 2.5. LC–MS/MS analysis

2. Experimental 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 obtained from Merck Specialities Pvt Ltd (Mumbai, India). Formic acid (90% purified) was procured from Merck Specialities Pvt Ltd. 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. 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 ␮m particle diameter (Waters, Ireland). The column dimensions were 250 × 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 40 ◦ C and 4 ◦ C, respectively. The ELSD was operated with typical settings as follows: evaporation temperature, 75 ◦ C; nebuliser temperature, 80 ◦ C; gas, 1.65 SLM.

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 ␮m particle diameter (Waters, Ireland). The column dimensions were 50 × 4.6 mm. The mobile phase flow rate was 1.0 mL/min with a split ratio of 1:1 to the ionisation 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 40 ◦ C and 4 ◦ C, respectively. The turboionspray source was operated with typical settings as follows: ionisation mode, positive; curtain gas, 20 psi; nebuliser gas (GS1), 50 psi; heater gas (GS2), 50 psi; ionspray voltage, 5500 V; temperature, 550 ◦ C. 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 40 V. 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.

2.3. Preparation of calibration standards and quality control samples

2.6. Method validation

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

Method validation was performed for total PEG 400, 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,

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Fig. 1. (a) Chromatogram representing the nine oligomers observed in PEG 400 under reverse phase conditions, (b) parent ion (full scan) scan of PEG 400, and (c) product ion scan of telmisartan (internal standard).

810.82, 912.06, 1013.40 ␮g/mL), were analysed on three different days to complete the method validation. In each batch, QC samples at 3.89, 486.43 and 810.72 ␮g/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 analysing blank plasma samples obtained from

V. Vijaya Bhaskar et al. / J. Chromatogr. B 926 (2013) 68–76 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 3600 Calculated Conc: N/A Acq. Date: 12/26/2012 Acq. Time: 10:36:52 AM 3500

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: ""

(b) 2.53

Modified: Yes 3400 Proc. Algorithm: Analyst Classic Bunching Factor: 1 3300 Noise Threshold: 10.00 cps Area Threshold: 100.00 cps 3200 ,Num. Smooths: 10 Sep. Width: 0.20 3100 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 3000 Exp. Val. Ratio: 3.00 RT Window: 30.0 sec 2900 Expected RT: 2.52 min Use Relative RT: No 2800 Int. Type: Base To Base 2700 Retention Time: 2.53 min Area: 38129 counts 2600 Height: 3.65e+003 cps Start Time: 2.26 min End Time: 2.83 min 2500 2400

1900

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2.79

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200

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100 0

1.96

1 Sample Index: 2900 Sample Type: Unknow n Concentration: N/ A ng/m L Calculated Conc: 0.00 2800 Acq. Date: 12/26/ 201 2 Acq. Time: 10:45: 34 A M 2700 Modified: No Proc. Algorithm: Analyst Classi c Bunching Factor: 1 2600 cp s Noise Threshold: 10.00 100.00 cp s Area Thre shold: ,Num. Smooths: 10 2500 Sep. Width: 0.2 0 Sep. Height: 1.0 0 2400 5.0 0 Exp. Peak Ratio: Exp. Adj. Ratio: 4.0 0 3.0 0 Exp. Val. Ratio: 2300 RT Window: 30.0 se c Expected RT: 1.96 mi n Use Relative RT: No 2200 Int. Type: Base To Bas e Retention Time: 1.96 mi n 2100 count s Area: 37422 Height: 2.63e+0 03 cp s 2000 mi n Start Time: 1.64 2.23 mi n End Time:

0.5

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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: "" 1 Sample Index: 2.76 Sample Type: Unknow n 2.66 Concentration: 1.00 ng/m L 2.5 Calculated Conc: N/ A Acq. Date: 10/7/2 012 2.4 3.09 Acq. Time: 5:10:34 PM 2.4 Modified: Yes 2.3 2.3 2.2 2.1 2.1 2.1 2.0 2.0 1.9 2.26 2.49 1.9 1.8 1.8 1.7 1.7 1.6 1.6 1.5 1.5 1.4 1.4 1.3 1.3 3.15 1.2 0.17 0.92 1.28 1.87 1.2 1.1 1.1 1.0 1.0 0.9 0.9 0.8 0.8 0.7 0.7 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0.0 0.5 1.0 1.5 2.0 2.5 3.0

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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: Unknow n Concentr ation: N/ A Calculat ed Conc: No Inte rcep t 120 Acq. Dat e: 10/7/20 12 Acq. Tim e: 5:10:34 PM 115 Modified: Ye s

3.0

3.06

110 105 100 95 90 85 80 75 70

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71

65 60

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55 50 45 40 35 2.23

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2.78 20

2.38 3.26

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3.32

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Fig. 2. MRM LC–MS/MS chromatograms of (a) telmisartan at 100 ng/mL in rat plasma, (b) rat plasma sample spiked with 1.01 ␮g/mL of PEG 400 (LLOQ), (c) telmisartan in rat blank plasma, and (d) PEG 400 in rat blank plasma.

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Table 1 Calculated concentrations of PEG 400 in calibration standards prepared in rat plasma (n = 3). Concentration (␮g/mL)

Statistical parameters

Actual conc.

Mean

SD

% CV

Relative error (%)

% Accuracy

1.04 1.90 10.32 55.94 212.52 549.07 769.59 832.98 932.96

0.052 0.225 0.531 3.517 14.257 20.307 20.501 32.557 33.633

5.00 11.86 5.15 6.29 6.71 3.70 2.66 3.91 3.60

2.97 −6.40 1.78 10.37 4.84 8.35 −5.08 −8.67 −7.94

102.97 93.60 101.78 110.37 104.84 108.35 94.92 91.33 92.06

1.01 2.03 10.14 50.68 202.71 506.76 810.82 912.06 1013.40

Calculated conc. Set-1

Set-2

Set-3

0.98 2.16 10.07 54.47 219.84 549.41 747.77 804.46 941.05

1.07 1.76 10.93 53.39 221.63 528.6 772.55 826.02 961.81

1.07 1.78 9.96 59.95 196.09 569.21 788.45 868.45 896.02

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 benchtop 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 −80 ◦ C for 60 days before analysis.

administration and 0.25, 0.50, 1, 2, 4, 8 and 24 h post dose [21] after oral administration. At each time point 200 ␮L of blood was collected in to vacutainers. Blood samples were collected using retro orbital puncture method. Plasma was isolated by centrifugation at 10,000 rpm for 10 min and stored frozen at −80 ◦ C until assay. 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%.

2.7. Application

PEG 400 oligomers have very weak ultraviolet (UV) absorbance and need to be separated 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 derivatization and is compatible with gradient elution chromatography. Longer gradient program with maximum % organic ramped to 50% had achieved good separation between oligomers (Fig. 1a). Ramping the organic

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, v/v/v) [19,20], 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 [21] after intravenous

3. Results and discussion 3.1. Purity estimation of PEG 400 oligomers

Table 2 Precision and accuracy of PEG 400 in quality control samples. Type

Statistical parameter

Concentration (␮g/mL) LQC (3.89)

MQC (486.43)

HQC (810.72)

Intra day-set-1 (N = 6)

Mean SD % CV % Accuracy Relative error (%)

3.80 0.30 7.96 97.60 −2.40

447.58 19.04 4.25 92.01 −7.99

742.89 40.04 5.39 91.63 −8.37

Intra day-set-2 (N = 6)

Mean SD % CV % Accuracy Relative error (%)

3.65 0.49 13.34 93.70 −6.30

488.24 18.37 3.76 100.37 0.37

813.21 40.83 5.02 100.31 0.31

Intra day-set-3 (N = 6)

Mean SD % CV % Accuracy Relative error (%)

3.65 0.34 9.38 93.92 −6.08

465.33 19.26 4.14 95.66 −4.34

735.71 30.91 4.20 90.75 −9.25

Inter day (N = 18)

Mean SD % CV % Accuracy Relative error (%)

3.70 0.09 2.31 95.07 −4.93

467.05 20.39 4.36 96.02 −3.98

763.94 42.82 5.61 94.23 −5.77

V. Vijaya Bhaskar et al. / J. Chromatogr. B 926 (2013) 68–76

(a)

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: "" 1 Sample Inde x: Unknown Sample Type: N/A Concentrati on: 0.00 ng/mL Calculated Conc: Acq. Date: 1 2/26/20 12 Acq. Time: 1:57:22 PM Modified: No Proc. Algor ithm: Analyst Classic Bunching Fa ctor: 1 Noise Thres hold: 10.00 cps 100.00 cps Area Thresh old: ,Num. Smoot hs: 10 Sep. Width: 0.20 1.00 Sep. Height: Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 3.00 Exp. Val. Ratio: 30.0 sec RT Window: Expected RT: 1.96 min Use Relative RT: No

Sample Name: "P EG400-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: "" 1 Sa mpl e In dex : Un know n Sa mpl e Type: N/A Co nce ntra tio n: 0.0 0 ng /mL 9.6e4 Ca lcu late d C onc: 12 /26/ 201 2 Ac q. Date : 9.4e4 Ac q. Time : 3: 03:1 0 P M 9.2e4 No Mo dif ied: 9.0e4 Pr oc. Alg ori thm: An alys t C lass ic Bu nch ing Fac tor: 1 8.8e4 No ise Thr esh old: 10.0 0 cps 8.6e4 10 0.00 cps Ar ea Thre sho ld: ,N um. Smo oth s: 10 8.4e4 Se p. Widt h: 0.2 0 1.0 0 Se p. Heig ht: 8.2e4 5.0 0 Ex p. Peak Ra tio: 8.0e4 4.0 0 Ex p. Adj. Ra tio: Ex p. Val. Ra tio: 3.0 0 7.8e4 30.0 sec RT Wi ndow : 1.9 6 min Ex pec ted RT: 7.6e4 No Us e Relat ive RT: 7.4e4 In t. Type : Ba se T o B ase 7.2e4 Re ten tion Ti me: 1.9 3 min Ar ea: 13 798 42 co unts 7.0e4 9.7 4e+0 04 cps He igh t: 6.8e4 St art Tim e: 1.5 8 min En d T ime: 2.3 8 min 6.6e4

1.94 5.4e4 5.2e4 5.0e4 4.8e4 4.6e4 4.4e4 4.2e4

Base To Base Int. Type: Retention Time: 1.94 min Area: 782 848 counts 5. 48e+004 cps Height: Start Time: 1.58 min End Time: 2.38 min

(b)

73

4.0e4 3.8e4

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Fig. 3. MRM LC–MS/MS chromatograms of (a) plasma sample obtained 2.00 h after intravenous administration of PEG 400 to SD rats and (b) plasma sample obtained 2.00 h after oral administration of PEG 400 to SD rats.

phase to higher percentages (55–95%) did not achieve good separation between oligomers 6, 7, 8 and 9. Purity of each oligomer was estimated by area normalisation 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. 3.2. LC–MS/MS analysis The electrospray ionisation 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, 696.40 (Fig. 1b) under positive ionisation conditions. The lower masses at m/z 89.10, 133.20, 177.20, 221.10 correspond to in-source fragmentation of different oligomers and these masses did not 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, 696.40) molecular ions except at m/z 327.30, 371.30. A total of 9 abundant and informative oligomers were identified. The fragment ion at m/z 89.20 (two ethylene oxide units) [22] 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 1.01–1013.40 ␮g/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 ionisation of

telmisartan produced abundant protonated molecules ([MH]+ ) at 515.20 amu and generated an intense fragment at 276.10 amu (Fig. 1c). LC–MS/MS methods operated with the C18 column and a 3.5 min generic gradient LC 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 was not achieved as the response got saturated at higher calibration standards. 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. Interference was not 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 ␮L) is sufficient to obtain an LLOQ of 1 ␮g/mL. Even though the calibration range of 1 ␮g/mL to 1000 ␮g/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

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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 ␮L). Representative chromatogram of Telmisartan at 100 ng/mL spiked concentration was shown in Fig. 2a. Representative chromatogram of PEG 400 (chromatogram representing sum of peaks of all oligomers) at LLOQ was shown in Fig. 2b. No interference at the retention times of telmisartan (2.45 min) (Fig. 2c) and PEG 400 (2.05 min) (Fig. 2d) 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 ␮g/mL for PEG 400 using 50 ␮L of rat plasma. The signal-to-noise ratio for PEG 400 is about 400 at 1.01 ␮g/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.

Table 3 Summary of validation parameters for PEG 400 in rat plasma. Validation parameter

Statistical parameter

Result

Extraction recovery

Mean SD % CV

103.82 3.07 2.96

Matrix factor (matrix effect)

Mean SD % CV

0.97 0.06 6.14

Autosampler stability

Mean SD % CV

102.88 0.35 0.34

Benchtop stability

Mean SD % CV

99.57 6.84 6.86

Freeze thaw stability

Mean SD % CV

104.92 2.85 2.71

Long term stability

Mean SD % CV

102.70 2.08 2.03

3.3. Method validation This method was validated to meet the acceptance criteria of industrial guidance for the bioanalytical method validation [23]. Calibration curves were obtained over the concentration range of 1.01–1013.40 ␮g/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 1). The low CV value for the slope indicated the repeatability of the method (Table 1). Table 2 shows a summary of intra and inter-day precision and accuracy data for QC samples containing

PEG 400. Both intra-and inter-assay CV values ranged from 2.31 to 13.34% at three QC levels. The intra and inter-assay RE values for PEG 400 were −7.99 to 0.37% at three QC levels. These results indicate that the present method has an acceptable accuracy and precision. As shown in Table 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 3) at three QC levels shows that the developed method is totally free of matrix effects for the analysis of PEG 400.

Table 4 Pharmacokinetic parameters of PEG 400 after intravenous administration of PEG 400 at 3.38 g/kg dose in male Sprague Dawley rats. Subject

Kel (h−1 )

T1/2 (h)

RAT-1 RAT-2 RAT-3 Mean SD CV%

0.19 0.24 0.17 0.20 0.04 19.42

3.60 2.86 4.18 3.55 0.66 18.65

C0 (␮g/mL) 7481.25 10,010.89 11,325.88 9606.01 1954.03 20.34

AUClast (h ␮g/mL)

AUCINF obs (h ␮g/mL)

AUC %Extrap obs (%)

Vz obs (L/kg)

Cl obs (mL/min/kg)

MRTlast (h)

7104.53 7995.44 6818.89 7306.29 613.68 8.40

7142.62 8017.00 6858.29 7339.31 603.88 8.23

0.53 0.27 0.57 0.46 0.17 36.13

2.46 1.74 2.97 2.39 0.62 25.90

7.89 7.03 8.21 7.71 0.61 7.95

1.94 1.91 1.94 1.93 0.02 0.95

Table 5 Pharmacokinetic parameters of PEG 400 after oral administration of PEG 400 at 3.38 g/kg dose in male Sprague Dawley rats. Subject

Kel (h−1 )

T1/2 (h)

Tmax (h)

Cmax (␮g/mL)

AUClast (h ␮g/mL)

AUCINF obs (h ␮g/mL)

AUC %Extrap obs (%)

Vz F obs (L/kg)

Cl F obs (mL/min/kg)

MRTlast (h)

F (%)

RAT-1 RAT-2 RAT-3 Mean SD CV%

0.60 0.41 0.21 0.41 0.20 48.04

1.16 1.67 3.35 2.06 1.15 55.64

2.00 2.00 2.00 2.00 0.00 0.00

1118.51 632.78 1057.66 936.32 264.63 28.26

3750.84 2922.42 3521.10 3398.12 427.68 12.59

3803.27 3064.40 3531.80 3466.49 373.74 10.78

1.38 4.63 0.30 2.10 2.25 107.12

1.49 2.66 4.63 2.93 1.59 54.25

14.81 18.38 15.95 16.38 1.82 11.14

2.34 3.10 2.60 2.68 0.39 14.40

51.82 41.75 48.12 47.23 5.09 10.78

Table 6 Mean pharmacokinetic parameters of PEG 400 oligomers after intravenous administration of PEG 400 at 3.38 g/kg in male Sprague Dawley rats. Oligomer #

Kel (h−1 )

T1/2 (h)

C0 (␮g/mL)

AUClast (h ␮g/mL)

AUCINF obs (h ␮g/mL)

AUC %Extrap obs (%)

Vz obs (L/kg)

Cl obs (mL/min/kg)

MRTlast (h)

1 2 3 4 5 6 7 8 9

0.23 0.24 0.21 0.20 0.19 0.17 0.15 0.16 0.18

3.11 3.01 3.42 3.66 3.78 4.04 4.54 4.79 4.27

839.14 1551.30 2040.20 1947.94 1368.95 817.94 383.23 175.05 92.12

909.49 1456.20 1692.11 1471.34 948.81 530.28 230.60 97.72 52.10

913.52 1461.64 1699.17 1477.94 953.32 533.01 232.03 98.44 52.47

0.45 0.38 0.42 0.45 0.48 0.52 0.63 0.75 0.73

1.85 1.79 2.30 2.53 2.73 2.91 3.48 3.80 3.04

6.81 6.84 7.69 7.93 8.25 8.25 8.79 8.99 8.22

2.16 2.10 2.01 1.98 1.85 1.78 1.68 1.50 1.53

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Table 7 Mean pharmacokinetic parameters of PEG 400 oligomers after oral administration of PEG 400 at 3.38 g/kg in male Sprague Dawley rats. Oligomer no.

Kel (h−1 )

T1/2 (h)

Tmax (h)

Cmax (␮g/mL)

AUClast (h ␮g/mL)

AUCINF obs (h ␮g/mL)

AUC %Extrap obs (%)

Vz F obs (L/kg)

Cl F obs (mL/min/kg)

MRTlast (h)

F (%)

1 2 3 4 5 6 7 8 9

0.25 0.25 0.40 0.39 0.38 0.37 0.36 0.36 0.34

2.85 2.79 2.07 2.11 2.13 2.24 2.31 2.29 2.32

1.67 2.00 2.00 2.00 2.67 2.67 2.67 2.67 2.33

188.29 275.69 236.99 168.48 82.33 42.39 13.03 3.90 1.65

581.45 886.25 770.45 556.98 276.20 147.50 47.48 14.77 6.85

582.41 887.83 788.92 571.58 283.41 152.15 49.00 15.21 7.10

0.17 0.18 2.45 2.66 2.72 3.46 3.46 3.40 3.55

2.65 2.71 3.01 3.83 5.23 5.64 8.42 11.71 12.11

10.73 11.27 16.59 20.57 28.03 29.20 42.20 59.82 64.69

2.78 3.07 2.84 2.92 3.03 3.21 3.31 3.42 3.63

63.76 60.74 46.43 38.67 29.73 28.55 21.12 15.45 13.53

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 4 ◦ C for 24 h (autosampler stability) prior to injection, with <0.34% (Table 3) difference from theoretical concentration. Spiked QC samples were stable when stored at room temperature for 8 h (benchtop stability) prior to injection, with <6.86% (Table 3) difference from theoretical concentration. Spiked QC samples were stable for three freeze thaw cycles (freeze thaw stability) with <2.71% (Table 3) difference from theoretical concentration. Long term stability at −80 ◦ C was proved for a period of 60 days with <2.03% (Table 3) difference from theoretical concentration. 3.4. Application study 3.4.1. PEG 400 This 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 Fig. 3a and b, respectively. The Intravenous and oral concentration/time profiles of PEG 400 is represented in Fig. 4a and b, 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 vehicles concentrations from PK study samples acts as quality control check starting from dose preparation to bioanalysis. Intravenous and oral pharmacokinetic parameters of PEG 400 were listed in Tables 4 and 5, respectively. The mean 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 [24]. Mean Tmax and Cmax after oral administration of PEG 400 to Sprague Dawley rats was 2.00 h and 936.32 ␮g/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. 3.4.2. PEG 400 oligomers Mean intravenous and oral pharmacokinetic parameters of PEG 400 oligomers were presented in Tables 6 and 7, respectively. It was found that upon increase in molecular weight of oligomers, there was decrease in absolute bioavailability [24]. This could be attributed to decrease in permeability with increase in 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.

Fig. 4. Mean concentration time profile of PEG400 after (a) intravenous administration at 3.38 g/kg dose to SD rats and (b) oral administration at 3.38 g/kg dose to SD rats.

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

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