Development and Application of an MSALL-based Approach

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Article

Development and Application of an MSALL-based Approach for the Quantitative Analysis of Linear Polyethylene Glycols in Rat Plasma by Liquid Chromatography Triple-Quadrupole/Time-of-Flight Mass Spectrometry Xiaotong Zhou, Xiangjun Meng, Longmei Cheng, Chong Su, Yantong Sun, Lingxia Sun, Zhaohui Tang, John Paul Fawcett, Yan Yang, and Jingkai Gu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04058 • Publication Date (Web): 18 Apr 2017 Downloaded from http://pubs.acs.org on April 25, 2017

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Analytical Chemistry

1

Development and Application of an MSALL-based Approach for the Quantitative

2

Analysis

3

Chromatography Triple-Quadrupole/Time-of-Flight Mass Spectrometry

4

Xiaotong Zhou

5

Lingxia Sun a, Zhaohui Tang c, John Paul Fawcett d, Yan Yang a*, Jingkai Gu a,e*

6

a

School of Life Sciences, Jilin University, Changchun, 130012, PR China.

7

b

School of Pharmaceutical Sciences, Jilin University, Changchun 130012, PR China.

8

c

Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry,

9

Chinese Academy of Sciences, Changchun, 130022, PR China.

of

Linear

Polyethylene

Glycols

in

Rat

Plasma

by

Liquid

a,#

, Xiangjun Meng a,#, Longmei Cheng a, Chong Su a, Yantong Sun b,

10

d

School of Pharmacy, University of Otago, Dunedin, P.O. Box 56, New Zealand.

11

e

Clinical Pharmacology Center, Research Institute of Translational Medicine, The

12

First Hospital of Jilin University, Changchun 130061, PR China.

13

*Corresponding authors. E-mail: [email protected], [email protected]

14

Tel.: +86 431 88782100 (J.Gu), +86 431 85155381 (Y. Yang). Fax: +86 431 85155380

15

#

The authors contributed equally to this work.

16

1

ACS Paragon Plus Environment


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ABSTRACT

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Polyethylene glycols (PEGs) are synthetic polymers composed of repeating

19

ethylene oxide subunits. They display excellent biocompatibility and are widely used

20

as pharmaceutical excipients. To fully understand the biological fate of PEGs requires

21

accurate and sensitive analytical methods for their quantitation. Application of

22

conventional liquid chromatography-tandem mass spectrometry (LC-MS/MS) is

23

difficult because PEGs have polydisperse molecular weights (MWs) and tend to

24

produce multicharged ions in-source resulting in innumerable precursor ions. As a

25

result, multiple reaction monitoring (MRM) fails to scan all ion pairs so that

26

information on the fate of unselected ions is missed. This paper addresses this

27

problem by application of liquid chromatography–triple-quadrupole/time-of-flight

28

mass spectrometry (LC-Q-TOF MS) based on the MSALL technique. This technique

29

performs information-independent acquisition by allowing all PEG precursor ions to

30

enter the collision cell (Q2). In-quadrupole collision-induced dissociation (CID) in Q2

31

then effectively generates several fragments from all PEGs due to the high collision

32

energy (CE). A particular PEG product ion (m/z 133.08592) was found to be common

33

to all linear PEGs and allowed their total quantitation in rat plasma with high

34

sensitivity, excellent linearity and reproducibility. Assay validation showed the

35

method was linear for all linear PEGs over the concentration range 0.05-5.0 µg/mL.

36

The assay was successfully applied to the pharmacokinetic study in rat involving

37

intravenous administration of linear PEG 600, PEG 4000 and PEG 20000. It is

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anticipated the method will have wide ranging applications and stimulate the

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development of assays for other pharmaceutical polymers in the future.

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INTRODUCTION

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PEG is a synthetic water-soluble, biocompatible polymer consisting of linear or

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branched chains of ethylene oxide of various lengths and MW. It is a neutral and

45

non-volatile material which remains stable over a wide range of temperature and pH.

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As FDA approved excipients for human use, linear PEGs have many applications

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depending on their MW.1 PEGs of low MW are usually used as solvents or surfactants

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to adjust the solubility and viscosity of other vehicles.2 PEGs with medium or high

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MW are often applied in new formulations as framework material or solid dispersion

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material to control the rate of drug release. They have also been employed as

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stabilizers, lubricants, adhesives, plasticizers and porogenic agents.3

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Covalent attachment of PEG (known as PEGylation) to an active pharmaceutical

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ingredient (API) can significantly improve the physicochemical properties,

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pharmacological behavior and therapeutic effects of the API. It does this by increasing

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the water-solubility of the drug, extending its circulation half-life and reducing its

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immunogenicity and antigenicity. 1,4-7 It can also enable specific targeting of tissues or

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cells which has proven to be one of the most efficient strategies for enhancing the

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therapeutic potential of APIs.8-11

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Although the efficacy and toxicity of pharmaceutical products employing PEG as

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excipient or conjugate are mainly due to the API, it does not mean that PEGs are

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without biological effects. The available literature suggests that PEGs are mainly

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eliminated by glomerular filtration in the kidney, a process that becomes slower as the

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MW increases.1,12 This has the potential to lead to accumulation in tissues with

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potential adverse effects on account of long term administration of PEG.13,14 A full

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characterization of the fate and pharmacokinetic behavior of PEGs after

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administration by various routes is therefore necessary to interpret their preclinical

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and clinical toxicity.

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In the past, several approaches have been developed for the analysis of PEGs in

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biological matrices. Some of them including nuclear magnetic resonance (NMR)15,

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colorimetry16,17, gel permeation chromatography (GPC)18 and high-performance

71

liquid chromatography (HPLC)19-23 have lower limits of quantitation (LLOQs) > 5

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µg/mL, which are inadequate for pharmacokinetic studies of PEGs. Radiolabeling

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with isotopes like 125I or 14C has been commonly used but the technique is potentially

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harmful to patients and the environment.12,24 PEG 400 could be determined in plasma

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and urine (LLOQ 0.4

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ion-monitoring

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time-consuming sample preparation and derivatization and could only be applied to

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short-chain PEGs. ELISA provides good sensitivity for PEGs in indirect assays of

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proteins or antibodies conjugated to PEG with LOD of 58.6 ng/mL for PEG 2000,

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14.6 ng/mL for PEG 5000 and 3.7 ng/mL for PEG 10000 and PEG 20000. However,

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the conjugation is not always specific enough to avoid interference from endogenous

82

proteins or antibodies.26

mass

µg/mL) by capillary gas

spectrometry

(GC-MS)25

but

chromatography-selected the

method

involved

83

In recent years, the rapidly developing technique of LC-MS/MS has raised

84

bioanalysis to a new level. Its first application to PEGs was to measure PEG 400 in

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urine based on direct injection electrospray ionization (ESI) combined with detection

86

by selected ion monitoring (SIM).27 Unfortunately the technique was of limited

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selectivity and prone to interference from endogenous substances. Subsequently we

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have evaluated the use of conventional LC-MS/MS to analyze PEGs and established

89

that PEGs acquire multiple charges in the ESI source and form countless precursor

90

ions with different values of m/z. Thus, since MRM can scan only a limited number of

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ion transitions, it is impossible to analyze all the constituents of a given PEG using

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conventional LC-MS/MS. Bhaskar et.al. recognized this problem and compromised

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by monitoring the 9 most abundant oligomers and summing the analyte peaks to

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estimate the total amount of PEG 400 in plasma, a technique that resulted in an LLOQ

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of 1.01 µg/mL.28 Whilst this approach may be appropriate for low MW PEGs, it is

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unsuitable for high MW PEGs containing innumerable polymers and multicharged

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ions since MRM can scan only a very small portion of the ions and information on the

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fate of the majority is missed. A technique based on MRM and in-source CID29 has

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achieved the simultaneous analysis of PEG and PEGylated proteins with an LOD of

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0.05 µg/mL. The intensity of ions generated by dissociation of PEGs was dependent

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on the decluster potential (DP) in the ionization source, but the low fragmentation

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efficiency was provided by the DP and sensitivity was only achieved by summing the

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intensity of the 5 most abundant ions. Stable isotope dilution LC-MS is a facile and

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practical approach for the analysis of PEGs (MWs of 400, 1500, 3000 and 4000)30 and

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gave LODs in the range 0.4-12 ng/mL. However, the assay required a large volume of

106

urine (500 µL of human urine), sample preparation was extremely tedious involving

107

solid-phase extraction and further concentration, and calibration curves were not

108

linear.

109

Q-TOF MS utilizes a hybrid system consisting of a unit mass resolution

110

quadrupole (Q1), a Q2 and a TOF mass analyzer with high resolution. It has two basic

111

scan modes, namely product ion scan and TOF-MS. The principle of product ion scan

112

is similar to MRM in LC-MS/MS but TOF-MS is a unique mode of data acquisition

113

where the entire production of ions in Q1 is delivered to Q2 and subjected to

114

fragmentation through in-quadrupole CID. The TOF analyzer then scans all product

115

ions and provides a complete set of information. This acquisition procedure is known

116

as MSALL and is a traditional qualitative approach widely applied in drug metabolism,

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metabonomic and proteomic studies.31-34 Given that all precursor ions are fragmented

118

regardless of their m/z, MSALL is an ideal technique to apply to the quantitative

119

analysis of polymers like PEG.

120

In this study, the MSALL strategy was applied for the first time to the quantitation

121

of PEGs with different MWs in a biological matrix. Linear PEGs (PEG 400, PEG 600,

122

PEG 750, PEG 2000, PEG 4000, PEG 5000, PEG 6000, PEG 10000 and PEG 20000)

123

were chosen to evaluate the feasibility and potential advantages of the MSALL strategy.

124

It was found that in-quadrupole CID generated a series of PEG-specific fragments of

125

which the one with 3 repeating monomers (theoretical m/z 133.08592) was common

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to all PEGs and was chosen as the product ion for quantitation. This effective

127

dissociation capacity of in-quadrupole CID combined with the high resolution,

128

excellent mass accuracy and fast acquisition speed of Q-TOF MS provided good

129

selectivity and sensitivity. In addition, matrix interference was largely overcome and

130

sensitivity was enhanced by extracting the ion chromatograms of chromatographic

131

peaks with an optimum mass extraction window (MEW) of ±5.0 mmu. Finally,

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sample preparation by protein precipitation and chromatography by gradient elution

133

on a large pore column contributed to the development of an assay with superior

134

LODs of 20 ng/mL for PEG 20000 and 3 ng/mL for PEG 600 and PEG 4000,

135

sensitivities that were suitable for pharmacokinetic studies of these PEGs in rat.

136

EXPERIMENTAL SECTION

137

Chemical reagents and materials

138

Linear PEGs (PEG 400, PEG 600, PEG 750, PEG 2000, PEG 4000, PEG 5000,

139

PEG 6000, PEG 10000 and PEG 20000) were provided by Changchun Institute of

140

Applied Chemistry. Simvastatin for use as internal standard (IS), HPLC grade

141

acetonitrile and MALDI matrix 2-[(2E)-3-(4-tert-butylphenyl)-2-methylprop-2-enylidene]

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malononitrile (DCTB) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).

143

Ultra-high purity water was prepared using a Milli-Q System. Methanol, isopropanol,

144

formic acid and tetrahydrofuran (THF) were analytical grade and used without further

145

purification.

146

MALDI-TOF/MS Analysis

147

MALDI-TOF MS was used to analyze the distribution of molecular mass in a

148

medium and high MW PEG. Solutions of MALDI matrix DCTB (10 mg/mL) and

149

PEG (4000 and 20000 Da) (1.0 mg/mL) were prepared in THF and water, respectively.

150

For each measurement, solutions of the PEG (0.5 µL) and DCTB (0.5 µL) were mixed,

151

loaded onto a stainless steel plate and allowed to stand in air until solvent had

152

evaporated. The plate was then inserted into an Autoflex speed TOF/TOF MS (Bruker

153

Daltonics, Germany) operated in the linear and positive ion mode with a 355 nm

154

Nd:YAG laser and an acceleration voltage of 19 kV. Each spectrum was obtained

155

using 500 laser shots and a delayed extraction time of 150 ns. Flex Analysis 3.3080

156

Software (Bruker Daltonics) was used to process the data.

157

Preparation of standard solutions and quality control (QC) samples

158

PEG standard solutions (1.0 mg/mL) were prepared in water. Calibration

159

standards were prepared by diluting standard solutions with blank rat plasma to final

160

concentration of 0.05, 0.1, 0.3, 1.0, 3.0 and 5.0 µg/mL. LLOQ (0.05 µg/mL) and QC

161

samples (0.1, 1.0 and 4.0 µg/mL) were prepared independently in a similar manner.

162

Simvastatin (IS) was dissolved in methanol and diluted with methanol:water (50:50,

163

v/v) to give an 0.1 µg/mL IS working solution.

164

Sample preparation

165

Calibration standards, LLOQ, QC samples and samples for analysis (50 µL) were

166

mixed with IS working solution (20 µL) and cold acetonitrile (150 µL, -20°C), vortex

8


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167

mixed for 30 s and centrifuged at 15000 rpm for 5 min using a HERAEUS PICO17

168

centrifuge (Thermo scientific). 30 µL of the supernatant was injected into the

169

LC-Q-TOF MS for analysis.

170

Chromatographic conditions

171

Chromatography was performed on an Agilent 1100 HPLC system (Agilent

172

Technologies, Palo Alto, CA, USA) equipped with a degasser, binary pump,

173

autosampler, column oven and switching valve. Separation of PEGs with MW

174

400-6000 Da was achieved on an XBridgeTM BEH 300 C18 column (2.1×50 mm I.D.,

175

3.5 µm, 300 Å, Waters) maintained at 40°C; separation of PEG 10000 and PEG 20000

176

was achieved on a Venusil ASB C8 column (2.1×50 mm I.D., 5 µm, 300 Å, Agela

177

Technologies) maintained at 55°C. The mobile phase consisted of 0.1% formic acid in

178

water (solvent A) and 0.1% formic acid in acetonitrile (solvent B) delivered at 0.4

179

mL/min. Gradient elution was dependent on PEG MW: for PEGs with MW 400-750

180

Da 0-1 min 10% B, 1-1.5 min 10-90% B, 1.5-4.5 min 90% B, 4.5-4.6 min 90-10% B;

181

for PEGs with MW 2000-20000 Da 0-2 min 20% B, 2-4 min 20-90% B, 4-5.9 min

182

90% B, 5.9-6 min 90-20% B. Each run was followed by a equilibration. The HPLC

183

effluent was introduced directly into the MS system without splitting the stream.

184

Q-TOF MS conditions

185

Given that PEGs contain readily protonated oxygen atoms, they are suitable for

186

ionization in the positive ion mode. Q-TOF MS analysis was carried out on a

187

Triple-TOF 5600 MS (SCIEX, Concord, Canada) equipped with an ESI source. MS

188

parameters optimized by direct infusion of standard solutions via a syringe pump were

189

as follows: Source temperature 500°C; ion spray voltage 5500 V; nebulizer gas (N2)

190

50 psi; heater gas (N2) 50 psi; curtain gas 15 psi; DP and CE values for simvastatin

191

and PEGs of different MW were shown in Table 1. The mass range scanned for PEG

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192

specific fragments covering one ion for quantitation and two ions for monitoring was

193

m/z 88.0-178.0 in the TOF-MS scan mode. IS was scanned in the product ion mode

194

using m/z 419.2 for parent ion and a range of m/z 198.5-199.5 for product ion. Table 1. DP and CE values of PEGs and simvastatin

195

Analyte

DP (V)

CE (eV)

Simvastatin

100

20

PEG 400-750

80

25

PEG 2000-6000

100

28

PEG 10000, PEG 20000

100

31

196 197

Data processing

198

Data acquisition was controlled by Analyst 1.6.1 software. Chromatographic

199

peaks were processed by extracting the ion chromatogram of the PEG-specific

200

fragment at theoretical m/z 133.08592 Da. The MEW for quantitation was optimized

201

at ± 5.0 mmu (133.081 to 133.091) with Peakview 2.0 software. All peaks of low,

202

medium and high MW PEGs were integrated using specific integration parameters in

203

Multiquant 2.0.2. Linear least-squares regression of calibration curves with 1/x2

204

weighting was used to evaluate linearity.

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Assay validation

206

The mass accuracy of PEG fragments was measured at different concentrations

207

(6 replicates of each concentration) and the results compared with theoretical exact

208

masses. Specificity was assessed by analyzing blank rat plasma obtained from 6 rats.

209

Calibration curves were constructed for each batch and 6 replicates of LLOQ and QC

210

samples were assayed to evaluate intra- and inter-day accuracy (as relative error, RE)

211

and precision (as relative standard deviation, RSD). Dilution integrity was assessed by

212

assay of 6 replicates of 40 µg/mL samples (10 times the highest QC concentration)

213

diluted to 4.0 µg/mL with rat plasma. The LLOQ was assessed by analyzing the 10


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lowest concentration of the calibration standards. Carryover was calculated by

215

analyzing extracted blank rat plasma samples after analysis of a sample at the upper

216

limit of quantitation (ULOQ). Crosstalk interference between analyte and IS was

217

evaluated by analyzing an ULOQ sample followed immediately by blank rat plasma

218

spiked with IS working solution. Recovery was determined by comparing peak areas

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of analytes and IS in 6 replicates of QC samples with those of post-extraction blank

220

plasma spiked at the same concentrations. Matrix effects were assessed by comparing

221

average peak areas of analytes in sets of 6 replicates of post-extraction spiked samples

222

with those in neat solutions at the corresponding concentrations. Stability was

223

evaluated in low and high QC samples after storage at -80°C for 7 days, room

224

temperature for 4 h and after three freeze/thaw cycles. Stability of processed samples

225

was assessed after storage in autosampler vials for 4 h.

226

Pharmacokinetic study

227

Three groups of male Wistar rats (n=6 per group, weight 200±10 g), provided

228

by the Experimental Animal Centre of Jilin University, were administered single

229

intravenous 3.0 mg/kg doses of either linear PEG 600, PEG 4000 or PEG 20000.

230

Blood samples (200 µL) were collected from ophthalmic veins into heparinized tubes

231

before dosing and at 0.083, 0.25, 0.5, 0.75, 1, 2, 3, 4, 6, 8, 10, 12, 24, 36 and 48 h

232

after dosing. Plasma was separated after centrifuging at 15000 g for 5 min, subjected

233

to sample preparation and injected into the LC-Q-TOF MS system.

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RESULTS AND DISCUSSION

235

MS analysis of PEGs

236

Characteristics of mass spectra of PEG: PEG 4000 and PEG 20000 were

237

examined by MALDI-TOF/MS to confirm their MW range and distribution. Each

238

PEG possessed normal distributions of ions with mean MWs consistent with their

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239

nominal average values. Each PEG was seen to contain a mixture of polymers with a

240

broad distribution of MWs.

241

Following infusion into the Q-TOF MS from a syringe pump, PEG standard

242

solutions were full scanned in the TOF-MS mode with CE set at the minimum value

243

and DP adjusted manually. For low MW PEGs, a normal distribution of singly

244

charged PEG precursor ions was observed (Fig. 1A) with [M+H]+, [M+Na]+ and

245

[M+K] + ions all present in good agreement with a previous report.27 For medium and

246

high MW PEGs (Fig. 1B and 1C), irrespective of how DP was adjusted, only ions

247

with m/z in the range 400-1400 were observed and virtually no ions with m/z > 2500.

248

This indicated that the ions carried multiple charges. Because of this fragmentation

249

and formation of multicharged ions in the ionization source, numerous precursor ions

250

from different MW PEGs were present leading to complex mass spectra. As a result, it

251

was impossible to determine all precursor ions by LC-MS/MS using MRM where

252

quantitation involves selecting precursor ions in Q1 and their corresponding product

253

ions in Q3. It is generally accepted that, because the duration of each duty cycle must

254

be controlled within a certain range to ensure a chromatographic peak is defined by

255

enough data points, the number of ion pairs that can be scanned in a single duty cycle

256

is limited. In addition, mass spectra are continuous in the mass range m/z 400-1400

257

(Fig. 1B, 1C) and require extensive time and labour to differentiate PEG precursor

258

ions from those formed from other molecules. As a result, it is simply impractical to

259

scan all PEG ion pairs by MRM.

260

With DP > 130 V, a series of singly charged ions (m/z 89.0613, 133.0854,

261

177.1106, 265.1643, 309.1868) differing in MW by multiples of 44 Da was produced.

262

These were clearly fragments of PEG containing different numbers of oxyethylene

263

subunits formed by successive loss of various numbers of subunits from the ends of

12


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264

long chains. Dissociation of PEG in the ionization source above a certain value of DP

265

has been observed previously.29 However, as shown in Fig. 1B and 1D, increasing DP

266

to 300 V did not increase the intensity of signals from fragments formed in-source

267

compared to their intensity at DP of 200 V but the spectrum was regularly arranged

268

according to the amount of charge. This presumably reflected the fact that the higher

269

DP provided sufficient energy to efficiently disassociate aggregates and distribute

270

charge but not enough to fragment the PEG.35 Therefore, the maximum signal

271

intensities of these fragments were relatively low and most PEG molecules remained

272

intact.

273 274

Fig. 1 Full scan mass spectra of various PEGs (concentration 1.0 µg/mL) obtained using different

275

DPs: (A) PEG 600 using DP of 100 V, [M+H] +, [M+Na] + and [M+K] + coexist in the positive ion

276

mode; (B) PEG 4000 using DP of 200 V; (C) PEG 20000 using DP of 200 V; (D) PEG 4000 using

277

DP of 300 V, ions with multiple charges (2 to 6) are present (charge is marked on the mass 13



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278

spectrum).

279

Q-TOF MS analysis of PEGs with MSALL: MSALL achieves rapid

280

information-independent acquisition using the TOF-MS mode of a Q-TOF MS

281

instrument. Here the entire collection of PEG precursor ions formed in the ionization

282

source was allowed to pass through Q1 and ions were subsequently fragmented by

283

in-quadrupole CID in Q2. The TOF analyzer carried out full scan of these fragments

284

and obtained their overall high-resolution spectrum.

285

The first task in evaluating the power of MSALL was to investigate the

286

characteristics of the product ions. When PEG standard solutions were infused into

287

the Q-TOF MS at a fixed value of DP of 100 V and ramping CE (Fig. 2A and 2B), a

288

series of singly charged small MW fragments was formed, the signal intensities of

289

which were some ten times higher than those of the same fragments generated by

290

in-source CID (Fig. 1B and 1C). This indicated that many of the long chains that

291

remain intact in the ionization source were effectively fragmented in the collision cell

292

due to the fact that the efficiency of in-quadrupole CE was some 100 times larger than

293

that of in-source DP.36 Furthermore, the newly designed linear accelerator delivered

294

the entire collection of ions through Q2 thereby increasing the speed of analysis and

295

eliminating crosstalk.37

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296 297

Fig. 2 Full scan mass spectra of PEGs (concentration 1.0 µg/mL) obtained at a fixed DP and

298

ramping CE: (A) PEG 4000 using DP of 100 V and CE of 28.5 eV; (B) PEG 20000 using DP of

299

100 V and CE of 31 eV.

300 301

These results illustrated that using the MSALL technique allowed information to

302

be collected from all fragments in a single duty cycle lasting only 0.25 s. This unique

303

capability was closely related to the fast scan speed of the TOF analyzer. Thus Q-TOF

304

MS with in-quadrupole CID was eminently suitable for the accurate quantitation of

305

synthetic polymers like PEG. With a scan range of m/z 88.0-178.0, we found that three

306

product ions with theoretical m/z values of 89.05971 (2 monomers), 133.08592 (3

307

monomers) and 177.11214 (4 monomers) accounted for the vast majority of fragments

308

generated by all PEGs. It was decided to use this relatively narrow scan range in order

309

to reduce damage to the detector. 15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

310

The shortcoming of MSALL lies in its low selectivity when analysing biological

311

samples with complex matrices. The m/z values of selected product ions are relatively

312

low making it highly likely that ions with similar m/z will cause high background

313

interference and reduced sensitivity. The key advantage of Q-TOF MS is its high

314

resolution which allows a mass error within 5.0 ppm and accuracy < 0.0001 Da. It

315

also compensates for the low selectivity by helping to differentiate analyte from

316

interfering ions in the matrix.38

317

It must be emphasized that the MEW is a critical parameter in improving

318

sensitivity and selectivity. It is utilized in post-acquisition data processing to extract

319

the chromatogram of the target analyte from the total ion chromatogram and thereby

320

ensure high-resolution MS.39 At its optimum size, the MEW should be wide enough to

321

include all analytes but narrow enough to exclude interference from co-eluted ions

322

with similar m/z. Selection of the width of the MEW is more critical when the

323

concentration of analytes is low and the intensity of interference is relatively

324

high.38,40-42 In most cases, interference can be removed with an MEW of ± 15.0 mmu

325

and a range of ±5.0 to ±10.0 mmu can be applied to all studies to improve

326

selectivity.38,43

327

Using PEG 4000 at its LLOQ as an example, decreasing the MEW from ±0.5 mu

328

to ±5.0 mmu caused the signal-to-noise ratio to increase by a factor of 4.8 (Fig. 3) and

329

most interfering peaks around the retention time to disappear. However, when the

330

MEW was further narrowed to 1 mmu, the intensity decreased significantly. Taking

331

these results into consideration, we finally selected an MEW of ±5.0 mmu to exclude

332

interfering ions but maintain signal intensity at low concentrations.

333

The chromatogram of PEG-specific fragments composed of 2-4 monomers was

334

extracted and the sensitivity of each fragment qualified. The summation of these three

16


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335

fragments did not increase the signal-to-noise ratio because of the correspondingly

336

elevated baseline. Of the three fragments, the one composed of 3 monomers

337

(m/z=133.08592) gave the best linearity and reproducibility and was therefore chosen

338

for quantitation.

339 340

Fig. 3 Effect of MEW width on extracting the chromatogram of the PEG-specific fragment

341

composed of three monomers (m/z=133.08592) from an LLOQ sample of PEG 4000.

342

Chromatography development

343

PEGs were divided into three groups based on their MW; low MW group

344

400-750 Da, medium MW group 2000-6000 Da, and high MW group 10000-20000

345

Da. 17



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346

As expected, the chromatography of these groups of PEGs differed from each

347

other due to their different polarities. In each group, optimum resolution with

348

symmetric peak shapes and high MS response was achieved under the same

349

chromatographic conditions. Both methanol and acetonitrile have been previously

350

used as the organic phase in PEG analysis but, in our hands, methanol produced no

351

chromatographic peaks even using different gradients. However, acetonitrile gave

352

satisfactory peaks and was used in combination with 0.1% formic acid and 0.1%

353

formic acid in water for gradient elution. Separation of PEGs was performed on either

354

a BEH 300 C18 column (2.1×50 mm 300 Å, Waters) or a Venusil ASB C8 column

355

(2.1×50 mm 300 Å, Agela Technologies). Both are suitable for hydrophilic

356

compounds, provide higher pH stability and give better separation of PEGs and good

357

peak shape with minimum carryover.44 Their large pore size (300 Å) helps to avoid

358

blockade of the pores by large molecules thereby improving column efficiency and

359

extending column life.

360

Low MW PEGs: PEGs are a mixture of different chain length polymers. Low

361

MW PEGs have high polarity and molecules of different chain length show different

362

chromatographic retention even when they differ by only a single monomer. When

363

using a shallow gradient each oligomer is well separated and gives an individual

364

chromatographic peak which causes difficulty in determining the total amount of PEG.

365

In order to solve this problem, a steep gradient (10-90% acetonitrile in 0.5 min) was

366

used so that all oligomers eluted simultaneously (Fig. 4A).

367

Medium MW PEGs: In this case, total PEG was determined using a gradient

368

increasing from 20% to 90% acetonitrile in 2 min. The peak width of medium MW

369

PEGs was only 0.5 min under these conditions (Fig. 4B).

370

High MW PEGs: High MW PEGs showed strong retention on a C18 column

18


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371

producing a wide peak with extensive tailing and a higher baseline after elution. In

372

contrast, chromatography on a C8 column produced a narrow symmetrical peak.

373

Columns with different pore sizes (300 Å, 500 Å and 1000 Å) showed similar

374

behavior with only a slight difference in retention time. Column temperature affected

375

the chromatographic behavior; at 40°C the baseline increased over the peak but at

376

55°C this was not a problem (Fig. 4C).

377 378

Fig. 4 Chromatograms of different PEGs at their LLOQ (0.05 µg/mL) using Q-TOF MS with an

379

MEW of ±5.0 mmu; (A) PEG 600, (B) PEG 4000, (C) PEG 20000, (D) the chromatogram of IS at

380

a concentration of 0.1 µg/mL.

381

Sample preparation

382

In investigating sample preparation, liquid-liquid and solid phase extraction were

383

evaluated but recovery was < 30% in both cases. In contrast, simple protein

384

precipitation with cold acetonitrile gave good recovery primarily because PEG is

385

reasonably soluble in acetonitrile. Methanol and isopropanol were also tested but cold

386

acetonitrile gave the highest recovery using only a 3:1 ratio of acetonitrile:sample.

387

Assay validation

388

Assay validation included testing mass accuracy, specificity, linear range, LLOQ,

389

accuracy, precision, dilution effect, recovery, carryover, crosstalk, matrix effects and

390

stability. Mass accuracy of analyte at different concentrations was evaluated using

391

Peakview 2.0. The results showed that the mass of the quantitative ion was in the 19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

392

range 133.0852-133.0860 and the biggest mass shift was -5.41 ppm of the theoretical

393

value. This indicated that the m/z of PEG was stable with only a slight mass shift

394

regardless of concentration. Nevertheless, the mass scale was recalibrated before each

395

analytical run using an external mass reference standard. Specificity was tested by

396

analyzing blank plasma after sample preparation. There was no significant peak at the

397

retention time of the analyte indicating the assay was free of interference. Testing of

398

linearity showed the assay was linear over the range 0.05-5.0 µg/mL (r2 0.995) with

399

LODs of 0.02 µg/mL for PEG 20000 and 3 ng/mL for PEG 600 and PEG 4000, which

400

could be further lowered for other application of PEGs. Intra- and inter-day accuracy

401

and precision were all within accepted limits (±15%) at the three concentrations and at

402

the LLOQ. Assay of samples with high concentration after a 10-fold dilution also

403

gave satisfactory results with calculated concentrations in the range 85-115% of the

404

nominal concentrations. Recoveries (%) of PEGs at low, medium and high

405

concentrations were respectively: PEG 600 102.6±5.3, 99.8±6.5 and 96.7±6.4; PEG

406

4000 106.4±2.8, 91.9±3.2 and 98.3±3.6; PEG 20000 92.0±5.6, 95.3±5.9 and 95.1±2.8.

407

Corresponding matrix effects (% of nominal concentrations) were: 101.4±7.4,

408

100.5±10.4 and 99.0±7.8; 115.0±1.9, 116.7±3.0 and 115.8±3.3; 96.3±3.4, 98.3±3.3

409

and 93.6±4.7. These results indicated that ion suppression and enhancement was not

410

significant for PEG 600 and PEG 20000 and that ion enhancement for PEG 4000 was

411

weak. In terms of carryover, sequential analysis of ULOQ and blank plasma samples

412

showed no obvious peak in the latter. In addition, crosstalk between analyte and IS

413

was shown to be absent. Stability evaluations showed that PEG 600, PEG 4000 and

414

PEG 20000 were stable under all the evaluated conditions. Detailed accuracy,

415

precision, recovery, matrix effect and stability results for PEG 600, PEG 4000 and

416

PEG 20000 were given in “Supporting Information”.

20


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417

Pharmacokinetic study in rat

418

Mean plasma concentration-time curves for the three groups of PEGs were

419

shown in Fig. 5. The concentration of PEG 600 was reduced to 28.9 ng/mL at 10 h

420

post-dose indicating an almost complete elimination. However, the concentration of

421

PEG 20000 remained at 300 ng/mL at 48 h post-dose suggesting a small amount of

422

PEG was retained in the body. This observation that a higher MW PEG has a longer

423

elimination half-life was consistent with a previous report14 and was due to saturation

424

of glomerular filtration in the kidney.

425

In regard to other applications of the method, we have successfully applied it to a

426

pharmacokinetic study of PEGylated doxorubicin after intravenous injection in rat, a

427

study which included simultaneous analysis of the intact PEGylated molecule,

428

released PEG and released drug. Furthermore, we have shown that the fragment with

429

m/z 133.08592 was also produced from branched chain PEGs (4 branched chains of

430

PEG 880, 2000 and 10000 and 8 branched chains of PEG 5000 and 10000) indicating

431

the potential application of our LC-Q-TOF MS method to studies of branched chain

432

PEGs.

433 434

Fig. 5 Mean plasma concentration-time curves for linear PEG 600 (triangle), PEG 4000 (square)

435

and PEG 20000 (circle) after single intravenous injections (3.0 mg/kg) to groups of rats (data are

436

mean ± SD, n=6).

21



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437

CONCLUSION

438

This report described the development, validation and application of a method

439

using Q-TOF MS and the technique of MSALL for the quantitative analysis of linear

440

PEGs in rat plasma. PEGs of various MW were shown to undergo in-quadrupole

441

fragmentation and generate common low MW fragments. The most stable fragment

442

with the highest signal response allowed quantitation of total PEGs using all precursor

443

ions produced in-source instead of using only some of the PEGs ion pairs as in MRM.

444

Because of the advanced high-resolution facility and appropriate selection of the

445

MEW, matrix effects and background interference were negligible. The applicability

446

of the method was demonstrated in a rat pharmacokinetic study involving single

447

intravenous doses of PEGs with different MW. It is anticipated this methodology will

448

find wide application in the study of PEGs as excipients and PEGylated drugs

449

particularly those of medium and high MW which generate too many ions to be

450

determined using conventional LC-MS/MS with MRM.

451

ACKNOWLEDGMENTS

452

This work was supported by the National Natural Science Foundation of China

453

(Grant No. 81473142, 81673502, 81430087 and 81673396), the Science and

454

Technology Major Specialized Projects for ‘significant new drugs creation’ of the

455

12th five-year plan (201ZX09303-015), the National Key Technology R&D Program

456

of the Ministry of Science and Technology (2012BAI30B00), CERS-1-70

457

(CERS-China Equipment and Education Resources System), and the Norman Bethune

458

Program of Jilin University (2015317).

459

REFERENCES

460

(1) Veronese, F. M.; Pasut, G. Drug Discov. Today 2005, 10, 1451-1458.

461

(2) Lu, E. X.; Jiang, Z. Q.; Zhang, Q. Z.; Jiang, X. G. J. Control. Release 2003, 92, 375-382.

462

(3) Owens, T. S.; Dansereau, R. J.; Sakr, A. Int. J. Pharm. 2005, 288, 109-122. 22


Page 22 of 24

Page 23 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


463

(4) Knop, K.; Hoogenboom, R.; Fischer, D.; Schubert, U. S. Angew. Chem. 2010, 49, 6288-6308.

464

(5) Li, W.; Chang, Y.; Zhan, P.; Zhang, N.; Liu, X.; Pannecouque, C.; De Clercq, E. ChemMedChem

465

2010, 5, 1893-1898.

466

(6) Francis, G. E.; Fisher, D.; Delgado, C.; Malik, F.; Gardiner, A.; Neale, D. Int. J. Hematol. 1998, 68,

467

1-18.

468

(7) Zhai, Y.; Zhao, Y.; Lei, J.; Su, Z.; Ma, G. J. Biotechnol. 2009, 142, 259-266.

469

(8) Greco, F.; Vicent, M. J. Front. Biosci. 2008, 13, 2744-2756.

470

(9) Hoffman, A. S.; Stayton, P. S. Prog. Polym. Sci. 2007, 32, 922-932.

471

(10) Skinner, S. A.; Tutton, P. J.; O'Brien, P. E. Cancer Res. 1990, 50, 2411-2417.

472

(11) Jayant, S.; Khandare, J. J.; Wang, Y.; Singh, A. P.; Vorsa, N.; Minko, T. Pharm. Res. 2007, 24,

473

2120-2130.

474

(12) Yamaoka, T.; Tabata, Y.; Ikada, Y. J. Pharm. Sci. 1994, 83, 601-606.

475

(13) Webster, R.; Elliott, V.; Park, B. K.; Walker, D.; Hankin, M.; Taupin, P. In PEGylated protein

476

drugs: Basic science and clinical applications; Springer, 2009, pp 127-146.

477

(14) Longley, C. B.; Zhao, H.; Lozanguiez, Y. L.; Conover, C. D. J. Pharm. Sci. 2013, 102, 2362-2370.

478

(15) Yoncheva, K.; Lizarraga, E.; Irache, J. M. Eur. J. Pharm. Sci. 2005, 24, 411-419.

479

(16) Chung, T. W.; Chung, C. H.; Lue, Y. F. Anal. Biochem. 2000, 285, 264-267.

480

(17) Veronese, F. M.; Schiavon, O.; Pasut, G.; Mendichi, R.; Andersson, L.; Tsirk, A.; Ford, J.; Wu, G.;

481

Kneller, S.; Davies, J.; Duncan, R. Bioconjug. Chem. 2005, 16, 775-784.

482

(18) Loret, S.; Nollevaux, G.; Remacle, R.; Klimek, M.; Barakat, I.; Deloyer, P.; Grandfils, C.;

483

Dandrifosse, G.

484

(19) Delahunty, T.; Hollander, D. Clin. Chem. 1986, 32, 351-353.

485

(20) Young, G. O.; Ruttenberg, D.; Wright, J. P. Clin. Chem. 1990, 36, 1800-1802.

486

(21) Ryan, C. M.; Yarmush, M. L.; Tompkins, R. G. J. Pharm. Sci. 1992, 81, 350-352.

487

(22) Schwertner, H. A.; Patterson, W. R.; Cissik, J. H.; Wilson, K. W. J. Chromatogr. 1992, 578,

488

297-301.

489

(23) Kinahan, I. M.; Smyth, M. R.J. Chromatogr. 1991, 565, 297-307.

490

(24) Longley, C. B.; Zhao, H.; Lozanguiez, Y. L.; Conover, C. D. J. Pharm. Sci. 2013, 102, 2362-2370.

491

(25) Fakt, C.; Ervik, M. J. Chromatogr. B Analyt. Technol. Biomed. Life. Sci. 1997, 700, 93-100.

492

(26) Chuang, K. H.; Tzou, S. C.; Cheng, T. C.; Kao, C. H.; Tseng, W. L.; Shiea, J.; Liao, K. W.; Wang,

J. Chromatogr. B Analyt. Technol. Biomed. Life. Sci. 2004, 805, 195-202.

23



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

493

Y. M.; Chang, Y. C.; Huang, B. J.; Wu, C. J.; Chu, P. Y.; Roffler, S. R.; Cheng, T. L. Anal. Chem. 2010,

494

82, 2355-2362.

495

(27) Ashiru, D. A.; Karu, K.; Zloh, M.; Patel, R.; Basit, A. W. Int. J. Pharm. 2011, 414, 35-41.

496

(28) Vijaya Bhaskar, V.; Middha, A.; Tiwari, S.; Shivakumar, S. J. Chromatogr. B Analyt. Technol.

497

Biomed. Life. Sci. 2013, 926, 68-76.

498

(29) Gong, J.; Gu, X.; Achanzar, W. E.; Chadwick, K. D.; Gan, J.; Brock, B. J.; Kishnani, N. S.;

499

Humphreys, W. G.; Iyer, R. A. Anal. Chem. 2014, 86, 7642-7649.

500

(30) Lichtenegger, M.; Rychlik, M. J. Chromatogr. B Analyt. Technol. Biomed. Life. Sci. 2015, 1001,

501

182-190.

502

(31) Zhao, Y. Y.; Liu, J.; Cheng, X. L.; Bai, X.; Lin, R. C. Clin. Chim. Acta 2012, 413, 642-649.

503

(32) Yin, P.; Zhao, X.; Li, Q.; Wang, J.; Li, J.; Xu, G. J. Proteome Res. 2006, 5, 2135-2143.

504

(33) Peng, X. C.; Gong, F. M.; Chen, Y.; Qiu, M.; Cheng, K.; Tang, J.; Ge, J.; Chen, N.; Zeng, H.; Liu,

505

J. Y. J. Proteomics 2016, 132, 85-92.

506

(34) Kim, J. Y.; Park, J. Y.; Kim, O. Y.; Ham, B. M.; Kim, H. J.; Kwon, D. Y.; Jang, Y.; Lee, J. H. J.

507

Proteome Res. 2010, 9, 4368-4375.

508

(35) Xu, Y. F.; Lu, W.; Rabinowitz, J. D. Anal. Chem. 2015, 87, 2273-2281.

509

(36) Wells, J. M.; McLuckey, S. A. Methods Enzymol. 2005, 402, 148-185.

510

(37) Song, F. J. Agric. Food Chem. 2011, 59, 4361-4364.

511

(38) Zhang, N. R.; Yu, S.; Tiller, P.; Yeh, S.; Mahan, E.; Emary, W. B. Rapid Commun. Mass Spectrom.

512

2009, 23, 1085-1094.

513

(39) Zhang, M.; Sun, J.; Chen, P. Anal. Chem. 2015, 87, 9974-9981.

514

(40) Xia, Y. Q.; Lau, J.; Olah, T.; Jemal, M. Rapid Commun. Mass Spectrom. 2011, 25, 2863-2878.

515

(41) Fung, E. N.; Xia, Y. Q.; Aubry, A. F.; Zeng, J.; Olah, T.; Jemal, M. J. Chromatogr. B Analyt.

516

Technol. Biomed. Life. Sci. 2011, 879, 2919-2927.

517

(42) Henry, H.; Sobhi, H. R.; Scheibner, O.; Bromirski, M.; Nimkar, S. B.; Rochat, B. Rapid Commun.

518

Mass Spectrom. 2012, 26, 499-509.

519

(43) Ruan, Q.; Ji, Q. C.; Arnold, M. E.; Humphreys, W. G.; Zhu, M. Anal. Chem. 2011, 83, 8937-8944.

520

(44) Wei, J.; Shen, A.; Yan, J.; Jin, G.; Yang, B.; Guo, Z.; Zhang, F.; Liang, X. J. Sep. Sci. 2016, 39,

521

1073-1081.

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Development and Application of an MSALL-based Approach

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