The Validation and Verification of an LC;MS ... - ACS Publications

(LC) methods with UV detection have also been published for the analysis of fatty acids. 22, 23, 24. 64. Page 4 of 37 ... major pool of accepted and v...
7 downloads 8 Views 665KB Size
Subscriber access provided by READING UNIV

Article

The Validation and Verification of an LC;MS Method for the Determination of Total Docosahexaenoic Acid (DHA) in Pig Serum Gerald Dillon, Geoff Wallace, Alexandros Yiannikouris, and Colm Moran J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04791 • Publication Date (Web): 02 Feb 2018 Downloaded from http://pubs.acs.org on February 2, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 37

Journal of Agricultural and Food Chemistry

The Validation and Verification of an LC;MS Method for the Determination of Total Docosahexaenoic Acid (DHA) in Pig Serum

Gerald Patrick Dillon1*, Geoff Wallace2, Alexandros Yiannikouris3, Colm Anthony Moran4

* Corresponding author Email: [email protected] Tel: +353 1 8252244 Fax: +353 1 8252251

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 2 of 37

3 1

Abstract

2 3

The paper presents the validation and verification of an analytical method for the determination

4

of total DHA in pig serum by LC-ESI-MS/MS. The characteristics studied during the validation

5

included; precision and accuracy, LOQ, selectivity, calibration range & linearity, parallelism and

6

stability. A separate verification study was also performed. The method was linear over the

7

range. Precision and accuracy met acceptance criteria at all levels and the LOQ was determined

8

as 1 µg/mL. Parallelism experiments were conducted to show that there was no bias introduced

9

in using a surrogate matrix to quantify DHA. Recoveries of free DHA were obtained for quality

10

control samples and stability studies were conducted over 24 hours, 7, 31 and 180 days. The

11

results of the verification study were in line with the validation study and in conclusion, the

12

method was deemed fit for purpose for measuring total DHA in pig serum.

13 14 15 16

Keywords:

17

DHA; enrichment; LC;MS; serum; analytical method; validation and verification

ACS Paragon Plus Environment

Page 3 of 37

Journal of Agricultural and Food Chemistry

4 18

Introduction

19 20

Over the past number of decades, there has been a growing awareness and appreciation in

21

scientific and legislative communities, as well as the public consumer at large, as to the

22

importance of long chain polyunsaturated omega-3 fatty acids (LC PUFA n-3) in the human

23

diet.1, 2 This has resulted in a growth of research into the nutritional and health benefits of LC

24

PUFA n-3 and the enrichment of food with LC PUFA n-3. 3, 4, 5

25 26

Long chain fatty acids typically have between 16 and 26 carbon atoms. Poly-unsaturated fatty

27

acids (PUFA) have at least two or more double bonds and are named depending on the number

28

of carbon atoms in the chain, the number of double bonds and the number of atoms from the

29

terminal methyl group. Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) can be

30

synthesized from the precursor α-linolenic acid (ALA).6 LC PUFA n-6 compounds such as

31

arachidonic acid (AA) are derived from linoleic acid (LA). As ALA and LA are not synthesized

32

endogenously in the body, they are considered ‘essential fatty acids’ as they need to be

33

consumed in the diet. However, with regards to DHA and EPA, recent studies have shown that

34

they are not easily converted from their precursors and, it is therefore imperative that they are

35

consumed through a regular diet.7

36 37

The health benefits of LC PUFA n-3 can be considered from alternative perspectives. Firstly, the

38

human brain and central nervous system are known to be major sites of LC PUFA n-3

39

accumulation, particularly of DHA.8 LC PUFA n-3 are known to be involved in brain structure,

40

brain development and cognitive function as well as optimal pre- and post-natal growth.9,10 In

41

addition, reduced brain DHA is associated with aging and the onset of dementia. There is also

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 4 of 37

5 42

evidence that DHA plays a role in mental health, specifically in depression including postnatal

43

depression, bipolar disorder and other behavioral disorders.11, 12 Secondly, LC PUFA n-3 have

44

been linked to reducing the risk of certain diseases such as cardiovascular disease (CVD), cancer

45

and type-2 diabetes and also are linked in their ability to impact inflammatory ailments like

46

rheumatoid arthritis, hypertriglyceridemia and psoriasis.8, 13

47 48

Being mindful of the potential health benefits which can be offered by DHA, the enrichment of

49

meat has become the focus of much scientific research, where alternative feeding strategies with

50

a host of LC PUFA n-3 rich ingredients, has been investigated.

51

enrichment studies is that animal tissues can only be determined for DHA content at the

52

termination of life. Serum, however, can be analysed as a biomarker throughout the course of a

53

study in assessing DHA status and absorption and hence, the supplementation strategy. If end

54

studies can be performed to measure the accumulation of PUFAs in biological tissues, it is also

55

extremely relevant to evaluate the transient absorption, half-life and distribution of DHA in

56

biological fluids. This approach enables to monitor over time, in a less invasive way, the transfer

57

of dietary DHA to the blood stream, to estimate the success of a feeding-based strategy aiming at

58

enriching animal food products.

14, 15, 16, 17

A limiting feature of

59 60

Fatty acids are conventionally quantified by first extracting them from their relevant matrix using

61

the method developed by Folch et. al.18 (1957) and then analyzing them by Gas Chromatography

62

(GC).19 Variations of GC methodology to include ionic liquids have been reported and provide a

63

more rapid analysis with improved separation and resolution.20, 21 Several liquid chromatography

64

(LC) methods with UV detection have also been published for the analysis of fatty acids.22, 23, 24

ACS Paragon Plus Environment

Page 5 of 37

Journal of Agricultural and Food Chemistry

6 65

Alternative LC methodologies with refractive index detection and light-scattering detection have

66

also been reported.25, 26

67 68

The analysis of PUFA is challenging in many instances because of the inherent properties of

69

PUFAs in terms of solubility, instability and isobaric forms. If GC-FID methods constitute the

70

major pool of accepted and validated methods for lipid analysis in biological tissues and foods,

71

the advent of electrospray ionization technologies (ESI) have enabled mass spectrometry to

72

become a technique of choice, especially when sensitivity and selectivity is needed, for

73

biological fluids, cells or microorganisms. 27, 28, 29 LC has also become a tool of choice because it

74

can easily be interfaced with an ESI-MS. The advent of ultra-pressure liquid chromatography

75

(UPLC) has also enabled an increase in separation capability and chromatographic performance.

76

Using the innovations of triple quadrupole systems and the development of different mass

77

analyzers modes, MS systems dramatically increase the sensitivity, selectivity and accuracy of

78

the detection owing to multi-reaction monitoring specifically aiming at analytical targets selected

79

based on their m/z ratio of the parent ions and specific fragments in the context of targeted

80

lipidomics. Finally, limiting the manipulation of the sample and the use of derivatization agents

81

also represents a key advantage in terms of analyte recovery and precision compared to GC

82

approaches. Therefore, by drawing on advances in recent analytical chemistry, the aim of this

83

paper is to describe the validation and verification of an analytical method for the determination

84

of total DHA in pig serum by LC-ESI-MS/MS. 30

85 86

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 6 of 37

7 87

Materials and Methods

88 89

Chemicals, Reagents and Instrumentation

90 91

For the validation study performed at LGC (Cambridgeshire, UK), DHA was purchased from

92

Matreya LLC (Pennsylvania, USA) and docosahexaenoic acid –D5 (DHA-D5) was purchased

93

from Cayman Chemical (Michigan, USA) for use as an internal standard (IS). HPLC grade

94

acetonitrile, HPLC grade hexane, analytical reagent grade (~37%) hydrochloric acid and

95

laboratory reagent grade acetic acid (glacial) were purchased from Fisher Scientific

96

(Loughborough, UK). Ultrapure water was obtained from a Duo Ultrapure unit from TripleRed

97

(Buckinghamshire, UK). Phosphate buffered saline (PBS) tablets (Dulbecco A) were purchased

98

from Oxoid Ltd (Basingstoke, UK). Tween® 80 was purchased from Acros Organics (Geel,

99

Begium). Bovine serum albumin (BSA), heat shock fraction, protease free, fatty acid free,

100

essentially globulin free was purchased from Sigma-Aldrich (Dorset, UK). Control porcine

101

whole blood (Yorkshire strain) containing lithium heparin anticoagulant and control porcine

102

serum (Yorkshire strain) was purchased from B&K Universal Ltd (Hull, UK). All experiments

103

were performed on an Acquity UPLC® system (Waters Corporation, Hertfordshire, UK) coupled

104

to a Sciex API 4000™ mass spectrometer (Sciex, Warrington, UK). Data was acquired and

105

integrated using Analyst® 1.5.2 (Sciex, Warrington, UK) and calculated concentrations were

106

determined using Watson LIMS™ software version 7.2 (Thermo, Loughborough, UK).

107 108

The verification study was performed at Silliker JR Laboratories, (Burnaby, Canada). For these

109

studies, DHA was purchased from Sigma Aldrich (St. Louis, USA) and DHA-D5 was purchased

ACS Paragon Plus Environment

Page 7 of 37

Journal of Agricultural and Food Chemistry

8 110

from Santa Cruz Biotechnology (Dallas, USA). HPLC grade acetonitrile and hexane and

111

analytical reagent grade (~37%) hydrochloric acid and laboratory reagent grade acetic acid

112

(glacial) were purchased from Fisher Scientific (Ontario, Canada). Phosphate buffered saline

113

(PBS) tablets were purchased from Sigma Aldrich (St. Louis, USA). Tween® 80 (polysorbate

114

80) was purchased from Sigma Aldrich (St. Louis, USA). Bovine serum albumin (BSA), heat

115

shock fraction, protease free, fatty acid free was purchased from Sigma Aldrich (St. Louis,

116

USA). Control pig serum was purchased from Life Technologies Inc. (Burlington, Ontario,

117

Canada). All experiments were performed on an Agilent 1100 HPLC system (Agilent

118

Technologies, Mississauga, ON, Canada) coupled to an API 4000 mass spectrometer (AB Sciex,

119

Concord, ON, Canada). Data was acquired and integrated using Analyst® 1.5 (AB Sciex,

120

Concord, ON, Canada) and calculated concentrations were determined using MultiQuant

121

software AB Sciex, Concord, ON, Canada).

122

123

Preparation of Calibration Standards. and Quality Control Samples

124 125

Stock solutions of DHA were prepared in acetonitrile at 10 mg/mL and DHA-D5 was supplied as

126

a 500 µg/mL solution in ethanol. Calibration, quality control (QC) and IS working solutions

127

were prepared by diluting stocks in acetonitrile. All solutions were stored in amber glass vials at

128

-20 °C. 50 mg/mL fatty acid free BSA in PBS containing 0.1% Tween 80 was used as surrogate

129

matrix, based on work by Bowen et al. (2010), 31 in which fatty acid free human serum albumin

130

was used as surrogate matrix. Calibration standards were prepared at 1, 2, 5, 15, 50, 175, 450 and

131

500 µg/mL by adding 5 µL of each calibration solution to 95 µL of surrogate matrix. DHA QC

132

samples were prepared at 1 (LLOQ), ~3.1 (QCL), ~26.4 (QCM) and ~410 (QCH) µg/mL,

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 8 of 37

9 133

depending on the endogenous DHA content of the serum, and were stored at -20 °C. QC LLOQ

134

was prepared by adding 10 µL of spiking solution to 190 µL of surrogate matrix. QCL was

135

prepared by diluting control pig serum with surrogate matrix to give ~ 3.1 µg/mL DHA

136

(typically ~1:2.5, v/v). QCM and QCH were prepared by adding 10 µL of QC solution to 190 µL

137

of control pig serum. The mean endogenous DHA level of the control pig serum was determined

138

by analyzing 12 replicates and was used to calculate QC concentrations.

139

140

Sample Preparation

141 35, 32

142

The sample preparation procedure was based on a previously described method

143

modification. 25 µL of sample was added to a 2 mL, screwcap, polypropylene tube, 20 µL of IS

144

working solution (10 µg/mL) was added and the tubes were vortex mixed. 150 µL of

145

acetonitrile:hydrochloric acid ~37% (80:20, v/v) was added and the tubes were sealed with screw

146

caps containing an EPDM O-ring, to ensure a tight seal. Tubes were vortex mixed briefly and

147

incubated at 90 °C for 3 hours to hydrolyze the samples, releasing free DHA from bound forms

148

such as phospholipids and glycerides. After cooling to room temperature, 200 µL of water was

149

added and free DHA was extracted with 1 mL of hexane. The tubes were rotary mixed and

150

centrifuged before 10 µL of the hexane layer was transferred to a 96 deep well plate containing

151

glass inserts. This was evaporated under nitrogen at 40 °C and reconstituted in 500 µL of

152

acetonitrile:0.1% acetic acid (aq) (70:30 v/v).

153

154

ACS Paragon Plus Environment

with some

Page 9 of 37

Journal of Agricultural and Food Chemistry

10 155

Liquid Chromatographic Conditions

156 157

The UPLC system utilized a 50 mm x 2.1 mm, 1.7 µm BEH C18 column (Waters Corporation,

158

Milford, USA) maintained at 40 °C. The sample tray was maintained at 4 °C. The mobile phase

159

flow rate was 0.6 mL/min and consisted of mobile phase A: 0.1% acetic acid (aq) and mobile

160

phase B: acetonitrile. The gradient profile was as follows: 0.0-1.0 min 72% B, 1.0-1.1 100% B,

161

1.1-1.3 100%B, 1.3-1.4 72% B, 1.4-1.7 72% B. For the verification study, a 50 mm x 2.1 mm,

162

3.6 µm XB-C8 column was used (Phenomenex, Torrance, USA). The sample tray was

163

maintained at 4 °C. The flow rate was 0.4 mL/min and, as with the validation procedure, the

164

mobile phase consisted of A: 0.1% acetic acid (aq) and mobile phase B: acetonitrile. The

165

following gradient profile was used: 0.0-0.8 min 70% B, 0.8-1.1 100% B, 1.1-1.2 100% B, 1.2-

166

4.5 70% B.

167

168

MS/MS Conditions

169 170

An API 4000 mass spectrometer was operated in negative TurboIonSpray mode and used

171

multiple reaction monitoring transitions m/z 327.3  283.0 and m/z 332.4  288.1 for DHA and

172

DHA-D5 respectively. Source conditions were as follows: Temperature 500 °C, Curtain gas 30

173

psi, Collision gas 6, GS1 60 psi, GS2 40 psi, ionspray voltage -4500 V. The remaining

174

conditions were: Declustering potential -85 V; Collision energy -16 eV; CXP -13 V (DHA) and -

175

15 V (DHA-D5).

176

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 10 of 37

11 177

ACS Paragon Plus Environment

Page 11 of 37

Journal of Agricultural and Food Chemistry

12 178

Validation and Verification Procedures

179 180

Method validation was carried out in LGC’s small molecule bioanalysis laboratory and follows

181

an in-house validation SOP based on procedures outlined in the European Medicines Agency

182

guideline on bioanalytical method validation (EMA, 2011)33 and with reference to guidance from

183

the Food and Drug Administration (FDA, 2001)34. The EMA guidelines does not provide criteria

184

for biomarker assays so precision and accuracy criteria were increased from ≤15%, ±15% to

185

≤20%, ±20% for QCL, QCM and QCH levels respectively, due to the increased complexity of

186

endogenous assays. Likewise, stability acceptance was increased from ±15% to ±20%. As

187

calibration standards and QCLLOQ samples were prepared in surrogate matrix the EMA

188

guideline criteria were retained. Solution stability acceptance and the parallelism test (which

189

replaced the conventional matrix effect test) are not described in the guideline. Criteria tested

190

during validation include: precision and accuracy, LLOQ, selectivity, calibration range &

191

linearity, parallelism and stability. For the verification study, the following parameters were

192

examined: calibration range & linearity, precision and accuracy, sensitivity and selectivity.

193

194

Calibration Curve and Linearity

195 196

Calibration curves were constructed by plotting the DHA: IS peak area ratio of the calibration

197

standards against DHA concentration. Linear regression was performed using a 1/x2 weighting

198

and the correlation coefficient (R2), slope and intercept determined. Acceptance criteria for the

199

LLOQ calibration standards were a relative error (%RE) of ±20% with a minimum signal to

200

noise ratio of 5:1. For all other concentrations the acceptance criteria was ±15% RE.

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 12 of 37

13

%RE=

(back calculated concentration – nominal concentration) x100 nominal concentration

201 202

Precision and Accuracy and Lower limit of Quantitation

203 204

Inter- and intra-assay precision (%CV) and accuracy (%RE) were determined by the analysis of

205

LLOQ, QCL, QCM and QCH quality control samples on 3 separate occasions with 6 replicates

206

per level. The acceptance criteria were %RE ±20%, %CV ≤20% and a signal to noise ratio of at

207

least 5:1 for LLOQ QC samples.

208 209

Selectivity

210 211

Selectivity was assessed in pig serum from six individuals. Due to the endogenous nature of

212

DHA, selectivity was only assessed for the internal standard. The peak area of any co-eluting

213

interference was compared to the average IS response from the QCM samples. The EMA

214

guideline state that any interference should be