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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

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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


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Abstract

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

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

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

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

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

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

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

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

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

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

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Keywords:

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


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Introduction

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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consumed through a regular diet.7

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

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

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

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

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



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

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

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

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

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rheumatoid arthritis, hypertriglyceridemia and psoriasis.8, 13

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

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

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

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

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

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

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

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

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

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

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enriching animal food products.

14, 15, 16, 17

A limiting feature of

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

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

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

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

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


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

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also been reported.25, 26

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Materials and Methods

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Chemicals, Reagents and Instrumentation

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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

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

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

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

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

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

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

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

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

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

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software AB Sciex, Concord, ON, Canada).

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Preparation of Calibration Standards. and Quality Control Samples

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

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

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

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

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

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

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

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



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

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

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

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

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

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

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Sample Preparation

141 35, 32

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The sample preparation procedure was based on a previously described method

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

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

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

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

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

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

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

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

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

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

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with some

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Liquid Chromatographic Conditions

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

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

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

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

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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,

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

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

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

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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-

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4.5 70% B.

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MS/MS Conditions

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

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

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

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

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

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15 V (DHA-D5).

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Validation and Verification Procedures

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

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

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

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

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

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

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

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

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

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

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

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

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

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Calibration Curve and Linearity

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

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

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

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

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



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%RE=

(back calculated concentration – nominal concentration) x100 nominal concentration

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Precision and Accuracy and Lower limit of Quantitation

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

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

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

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least 5:1 for LLOQ QC samples.

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Selectivity

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

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

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

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guideline state that any interference should be

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