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Targeted metabolomics is the quantitative analysis of a large number of small molecule metabolites using internal standards. The samples were analysed using the targeted metabolomics AbsoluteIDQ p180 kit (Biocrates Life Sciences AG, Innsbruck, Austria). All the QCs and calibrators with isotope labelled validated internal standards are supplied in the kit. The analysis uses a combined flow injection analysis (FIA)/MS and LC/MS/MS to quantify absolute concentrations (μM) of 184 metabolites, grouped into metabolite classes of amino acids and biogenic amines by LC/MS and acylcarnitines, glycerophospholipids, sphingolipids and hexose by FIA in positive mode.

The mass spectrometer used in this analysis is a triple quadrupole. It is highly selective (targeted), and therefore allows the instrument to be fine-tuned to specifically look for the metabolites in the kit (Lu et al., 2008a). The first confirmation is the multiple reaction monitoring (MRM). The MRM approach consists of three stages (Roberts et al., 2012). In the first stage of the processing, the ions are protonated to facilitate the identification of metabolites in positive mode. These are often referred to as parent or mother ions. On entering the quadrupoles, the first quadrupole (Q1) measures the parent weight (mass to charge ratio). In the second quadrupole (Q2), molecular /parent ion collides with an inert gas (Argon in this case) and form fragment/daughter ions and the third quadrupole (Q3) identifies the daughter ions and detects subtle differences in the fragments (the daughter ions).

Single quadruple mass spectrometer instruments often measure amino acids and lipids in positive and negative mode because a single quadrupole does not have the MRM (protonation) capability. Measuring the samples using a triple quadrupole does not require the measurement in positive and negative mode because the majority of analytes prefer to be protonated. Measuring in both modes would result in less sensitivity, as the MRM does not double the processing time, instead it allocates half the scan window to the measurement in positive mode and half in negative mode.

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The second confirmation of the accurate detection of the metabolites is the internal standards provided with the kit. The chemically similar, deuterated internal standards have a slightly different mass/charge ratio but have the same retention time window.

The p180 Kit measures free metabolites in plasma, which in general means all non-covalent bound metabolites. Noncovalent bonds are relatively weak chemical bonds that do not involve an intimate sharing of electrons. Macromolecule structures (proteins and lipoproteins) may be stabilised by multiple noncovalent bonds. Lipoprotein phospholipids are measured in this analysis as they are non-covalently bound to structures or compartments present in the sample. During the preparation of the FIA plate, the methanol step precipitates approximately 95% of lipids, which are caught in the filter plate. During the PITC (phospho-isothiocyanate) incubation, all primary amino functional groups are derivatised; all amino acids and biogenic amines (except creatinine). PITC is also known as Edman's Reagent. Only derivatised amino acids and biogenic amines (+ creatinine) are analysed with the p180 Kit. The Q1 mass is the mass of the metabolite + PITC + adduct, e.g. Ala + PITC + H+ _(Biocrates).

Plasma samples (10 µl) were extracted and derivatised (Section 2.3.3.1) and then analysed on a Waters Xevo TQ-S (Triple quadrupole mass spectrometer (MS)). TargetLynx software (Waters) was used to integrate the peaks of the LC/MS analysis. MetIDQ software (Biocrates Life Sciences, Innsbruck, Austria) was used to register the samples before analysis and validate and produce concentrations (μM) of both LC/MS and FIA procedures. The data were then exported to Microsoft Excel for further analysis.

The samples were analysed on four 96-well plates, each plate contained at least one complete time series from one participant from each group with an extra participant from one other group (totalling 4 participants per plate). The samples from all four participants were randomised on the plate to minimise any effect of drift. One plate was measured on the MS each week and all assays were completed within one month.

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2.3.3.1 Sample preparation

All LC/MS Optima grade solvents were manufactured by Fisher Chemicals (Fisher Scientific Loughborough, U.K) unless otherwise specified. The plasma samples were removed from the -80°C freezer and defrosted on ice. The temperature of the centrifuge (5430 R Eppendorf, Stevenage, U.K.) was set to 4°C. The 96-well plate, the standards, the QCs and the calibrators were removed from the -20°C freezer. LC/MS Optima grade water was added to the internal standard (ISTD), QCs and calibration standards immediately before use. The tubes, supplied with the kit were vortexed and then shaken for 15 minutes at 1200 rpm. The QCs and thawed samples were centrifuged at 4°C for 5 minutes at 2750 g. The plate, which consisted of an upper filter plate and a lower collection plate taped together, was prepared horizontally. Firstly 10 µl of internal standard (ISTD) was added to all wells except the blank (well position A1). Then 10 µl zero samples (phosphate buffered saline (PBS)) was added to well positions A2-A4, calibration standards 1- 7 were added to well positions A5- A11, QC1 was added to well position A12, QC1 was added to well position B1, QC2 was added to well position B2 and QC3 was added to well position B3. B4 wells onwards were filled with samples, with additional QC2s in wells C11, E8 and G5. The samples were then dried at room temperature under a stream of nitrogen for 30 minutes.

For derivatisation of the amino acids and biogenic amines a derivatisation pre-mix was prepared (1900 µl ethanol with 1900 µl LC/MS grade water and 1900 µl pyridine, vortexed for 10 seconds) The pre-mix was added to 300 µl of 5% v/v phenylisothiocyanate (PITC) derivatisation solution. The solution was vortexed until it became clear. The PITC solution was used immediately and 50 µl was pipetted into each well. The plate was covered with the plastic lid and incubated for 20 minutes at room temperature. The lid was removed and the plate was dried again under the nitrogen evaporator for 60 minutes. The extraction solvent (300 µl) (Biocrates) was added to each well and the plate was covered with the plastic lid and shaken at 450 rpm for 30 minutes. The plate was centrifuged for 2 minutes at 100 g to transfer the samples from the filter plate to the lower capture plate. 50 µl from each well of the capture plate was transferred to the empty 96-well

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plate with the label “Use for LC”. Then 450 µl of LC/MS Optima grade 40% methanol (MeOH) was added to each well of the LC plate and covered with the silicone mat. This was shaken for 2 minutes at 450 rpm before transferring to the autosampler on the LC/MS. The neat plate, containing the samples for the FIA run, was placed in the fridge at 4°C until the following day. The LC plate was prioritised on the LC/MS because the biogenic amines are unstable after the water has been added.

The following day the FIA running solution was prepared by mixing the Biocrates ‘solvent 1’ ampoule with 290 ml Optima LC/MS grade methanol in a 500 ml Duran and shaken thoroughly. From the original neat plate 20 µl was transferred from each well, to the empty 96-well plate labelled “Use for FIA”. FIA running solvent was added to each well (380 µl), the plate was sealed with a silicone mat then the plate was shaken for 2 minutes at 450 rpm before it was placed in the autosampler on the LC/MS.

A method set up from a previous Biocrates assay containing the retention times of the metabolites, and the sample codes was imported into the LC/MS using TargetLynx. When the LC and FIA runs were complete the data were imported into MetIDQ (Biocrates).

2.3.3.2 Measurement of the samples in MetIDQ and TargetLynx

The acquired data from the LC/MS were loaded into TargetLynx (Waters) for further analysis. Files were saved as a qld. file that could be edited.

2.3.3.3 Standard curve

The kit contained a mixture of chemically homologous internal standards. The 7- point serial dilution (0.1, 0.2, 1, 2, 4, 6 and 8) of calibrators generated standard curves for the LC analysis of amino acids and biogenic amines. An R2 _{value closest}

to 1 was used as a measure of accuracy of each standard curve to which the sample concentration was calculated. Therefore, the first stage of data preparation was to exclude any outliers in the standard curve and maximise the R2 _value.

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2.3.3.4 Integrating peaks

The spectrum data from the LC/MS amino acid and biogenic amines were imported into TargetLynx. The first stage was to modify the settings in the method to automatically capture the metabolite peaks for each plate individually as this can vary slightly from plate to plate. After the method was amended, peaks that were not detected were integrated manually.

2.3.3.5 Validating and exporting the data

The validation process automatically assesses the quality of the calibration standards, QCs and internal standards. Options for export including (i) QC2 correction, which automatically smooths intraplate and interplate variation, (ii) blank corrected, which considers the blanks in the FIA run and (iii) 35 metabolic indicators using the MetIDQ RatioExplorer (predefined ratios and sums of related metabolites associated with a metabolic pathway or a disease state named metabolic indicators). Concentration values (µM) are produced which are exported as an excel file.

2.3.3.6 Quality controls (QCs)

The QCs, comprising of human plasma, were provided with the kit. They contained 3 concentration levels (low, medium and high) of the all metabolites measured in the kit and are used to verify the performance of the assay and the MS. According to the manufacturer’s instructions the medium QC (QC2) was repeated every 20 samples on the assay plate. The QC2 repeats were used to calculate the intraplate and the interplate %CV. Figure 2.8 illustrates how the MetIDQ software normalised the data from more than two plates. A mean of the four QC2s on each plate was calculated. The mean of the QC2s on all plates was calculated. The mean from the plate was divided by the mean of all plates creating a correction factor. Each sample on the plate was then divided by this correction factor for the normalised value used in further analysis.

69 Plate 1 (μM) Plate 2 (μM) Plate 3 (μM) Plate 4 (μM) All plates (μM) QC 1 10 9 11 12 QC 2 11.5 10 12 12 QC 3 12 10 11 12.5 QC 4 9 11 9.5 11.5 mean 10.6 10.0 10.9 12.0 10.9 Sample 495 466

Figure 2.8 Normalisation of samples from multiple plates. Normalisation is carried out using QC2 repeats. Using the formulas A1/B = C1, where A = the plate mean, B = mean of

all plates and C = the correction factor and S1 _normalised = S1/C1, where S = sample and S1

normalised = normalised sample.

2.4 Data preparation and processing