High speed quantitative UPLC-MS analysis of multiple amines in human plasma and

serum via pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl

carbamate: Application to acetaminophen-induced liver failure

Nicola Gray1, Rabiya Zia1 Adam King1, Vishal C Patel3, Julia Wendon3 Mark JW McPhail3

Muireann Coen1, Robert S Plumb1, Ian D Wilson1*, Jeremy K Nicholson1,2*

Manuscript accepted 19th January 2017

1 Division of Computational and Systems Medicine, Department of Surgery and Cancer,

Imperial College London, Exhibition Road, South Kensington, London SW7 2AZ, UK2MRC-NIHR National Phenome Centre, Division of Computational and Systems Medicine,

Department of Surgery and Cancer, IRDB Building, Imperial College London, Hammersmith

Hospital, London, W12 0NN, United Kingdom3 Institute of Liver Studies & Transplantation, Kings College Hospital, Denmark Hill, London

SE5 9RS, United Kingdom

*Corresponding Authors: j.nicholson@imperial.ac.uk, i.wilson@imperal.ac.uk.

Abstract: A targeted reversed-phase gradient UPLC-MS/MS assay has been developed for

the quantification/monitoring of amino acids and amino-containing compounds in human

plasma and serum using pre-column derivatization with 6-aminoquinolyl-N-

hydroxysuccinimidyl carbamate (AccQTag UltraTM). Derivatization of the target amino-

containing compounds reagent required minimal sample preparation and resulted in analytes

with excellent chromatographic and mass spectrometric properties. The resulting method,

which requires only 10 µl of sample, provides the reproducible and robust separation of 66

analytes in 7.5 minutes, including baseline resolution of isomers such as e.g. leucine and

isoleucine. The assay has been validated for the quantification of 33 amino compounds

(predominantly amino acids) over a concentration range from 2-20 and 800µM. Intra- and

inter-day accuracy of between 0.05-15.6 and 0.78 -13.7 % and precision between 0.91-16.9

% and 2.12-15.9 % were obtained. A further 33 biogenic amines can be monitored in samples

for relative changes in concentration rather than quantification. Application of the assay to

samples derived from healthy controls and patients suffering from acetaminophen (APAP,

paracetamol) induced acute liver failure (ALF) showed significant differences in the amounts

of aromatic and branched chain amino acids between the groups as well as a number of other

analytes, including the novel observation of increased concentrations of sarcosine in ALF

patients. The properties of the developed assay, including short analysis time, make it suitable

for high throughput targeted UPLC-ESI-MS/MS metabonomic analysis in clinical and

epidemiological environments.

INTRODUCTION

Untargeted metabolic phenotyping (metabotyping1,2) as performed in metabonomic/

metabolomic studies offers the possibility of discovering new biomarkers3,4. The use of LC-

MS-based techniques for this purpose is now widespread5-7 with an increasingly role evident

in biomarker discovery in large scale epidemiological and personalized medicine studies

(e.g.8-12). However, as is widely appreciated, untargeted methods generally provide relative

changes (fold changes) for metabolites, rather than absolute concentration data. In addition,

despite the application of e.g., high resolution UHPLC separations and the use of

combinations of separation techniques (particularly reversed-phase and HILIC modes of

chromatography12) for sample analysis the coverage of the metabolome remains far from

comprehensive. A consequence of this partial, and qualitative, coverage is there is a need for

subsequent further validation of the changes detected by untargeted methods. For example, a

recent study of plasma amino acid profiles in kidney transplant patients compared the results

obtained using both an untargeted metabolic profiling method against a specific LC-MS/MS

assay showing that, whilst there was overlap between the data obtained by both methods,

there were also significant differences13. Use of a validated targeted assay, based on

optimized sample preparation and bespoke LC-MS conditions, combined with appropriate

internal standards, enables quantitative data to be obtained for the analytes of interest which

is especially valuable in clinical biomarker discovery. In addition, compounds related to the

target analytes that may not have been detected in the original metabotyping study can also

be determined. Amino acids, and amino-containing compounds, are modulated in many

different conditions including e.g., toxicity, cancer, metabolic diseases etc., are and often

seen to vary in epidemiological or clinical metabolic phenotyping studies. As such, amino

compounds represent an obvious class of compounds for targeted analysis. There are of

course innumerable methods for the analysis of complex mixtures of amino acids and

biogenic amines (dating back to the pioneering work of e.g., Martin14 and Dent15 and their co-

workers using 2D paper chromatography). Currently many quantitative methods for amino

compounds use MS for detection (reviewed in e.g.,16,17) including those based on e.g., GC-

MS18,19, CE-MS/MS20,21 and LC-MS/MS22-44. However, for reversed-phase (RP) LC-based

methods the amphoteric nature of the amino acids often results in poor retention, making

direct analysis impractical for all but a few analytes. Alternative modes of LC that improve

retention allowing direct analysis include separations based on strong cation exchange22,

HILIC23,24 or ion-pairing25-32. However, such approaches are not without disadvantages and, as

a result, many methods rely on forming derivatives of amino compounds that enable RPLC to

be performed. A number of reagents are available for the derivatization of amino acids to

facilitate analysis by LC-MS33-44. One of these, 6-aminoquinolyl-N-hydroxysuccinimidyl

carbamate (AccQTag UltraTM), originally applied to the determination of primary and

secondary amines via fluorescence detection e.g.,45-49, has also been employed for sensitive

and specific LC-MS detection41-44. Here a high-throughput, sensitive and selective UPLC-

ESI-MS/MS assay for the targeted analysis of amino acids and biogenic amines in human

plasma or serum based on the AccQTag UltraTM reagent is described. This method forms one

of a suite of targeted50,51 and untargeted12 methods developed, or under development, to

support metabolic phenotyping and biomarker validation for the UK National Phenome

Centre. The method was applied to samples obtained from healthy controls and patients

suffering from acute liver failure (ALF) resulting from acetaminophen (APAP, paracetamol)

overdose.

EXPERIMENTAL SECTION

Chemicals and Reagents. Analyte standards (listed in Table 1) were from Sigma Aldrich

(Gillingham, UK). Isotopically labelled amino acids for use as internal standards (IS) (see

Table 1) were from Cambridge Isotope Laboratories (MA, USA) or QMX Laboratories

(Essex, UK) (see Table S9 caption for details). Optima grade water was obtained from Fisher

Scientific (Leicester, UK), LC-MS grade solvents and formic acid were from Sigma Aldrich

(Gillingham, UK) and the AccQTag UltraTM reagent from Waters Corporation (Milford, MA,

USA).

Samples. Plasma samples were obtained from 14 patients with acetaminophen-induced

acute liver failure (ALF) (2 male, 12 female; 19 to 56 (mean = 38) years of age) and 40

healthy volunteers (20 male, 20 female; 32-41 (mean = 37) years of age) as controls. A Mann

U Whitney test was applied to the subjects ages to ensure the absence of any confounders (p

= 0.0001). Local national research ethics service (NRES) approval was obtained for this

study and patients, or their nominee, provided written informed consent within 24 hours of

admission to Kings College Hospital. Blood samples were obtained within 24 hours of

admission into BD Vacutainer lithium heparin-containing vacuum tubes (Franklin Lakes,

NJ). Plasma was obtained by centrifugation (12,000g, 4°C, 10 min.) within one hour of

sample collection and was then stored at -80°C.

Analytical Procedure. Preparation of Stock Solutions. Calibration and QC samples

were prepared from a standard mixture of neutral, basic and acidic physiological amino acids

(Sigma Aldrich) with the addition of asparagine and glutamine on each day of the validation.

Duplicate working stock solutions (A and B) were made in 50:50 water/methanol (v/v) at a

concentration of 400 µM for each analyte. Dilutions of stock A were used to prepare

calibration standards and dilutions of stock B for QC samples. For validation 16 compounds

were quantified against stable isotope-labelled internal standards with 17 validated using a

surrogate internal standard (see Table 1).

Calibration, Quality Control (QC) and Stable Isotope Labelled (SIL) IS Solutions.

Calibration standards were prepared by dilution with 50:50 water/methanol (v/v) to give

concentrations of 0, 1, 2, 4, 10, 20, 40, 100, 200 and 400 µM. QC samples were prepared by

dilution with 50:50 water/methanol (v/v) to provide concentrations at the lower limit of

quantification (LLOQ) (1, 3 or 10 µM depending upon the analyte), low-level (3, 10 or

30 µM depending upon the analyte), mid-level QC (30 or 150 µM depending on the analyte),

high-level (300 µM) and upper limit of quantification (400 µM) of each amino acid.

Solutions of each SIL amino acid (1 mg/mL) in Optima grade water were combined to

provide a stock solution at a concentration of 10 µg/mL. A 20 µL aliquot of each calibration

and QC standard were transferred to an Eppendorf tube followed by 5 µL of the IS solution

(IS was not added to the blanks), and 40 µL of cold isopropanol (IPA) containing 1 % formic

acid (v/v). After 20 min at -20 °C samples were centrifuged (13 000 g, 10 min) and 10 µL of

supernatant transferred to a glass HPLC vial (or 96-well plate) for derivatization.

Sample Preparation. Human plasma or serum samples were left to thaw at 4 °C then 10

µL of each sample was transferred to an Eppendorf tube to which 10 µL of Optima grade

water was added then 5 µL of the 10 µg/mL IS mixture. Proteins were then precipitated using

40 µL of cold isopropanol (plus 1 % formic acid (v/v)) with vortex mixing. After 20 min at -

20 °C samples were centrifuged 13 000 g, 10 min) and then 10 µL of the supernatant was

transferred to a glass HPLC vial (or 96-well plate) for derivatization as described below.

Derivatization. For derivatization 1 mL of acetonitrile was added to the AccQTag Ultra TM

reagent powder, vortex mixed and dissolved by heating at 55 °C (no longer than 15 min).

Then, 70 µL of borate buffer (pH 8.6) was added to the samples (with vortex mixing)

followed by 20 µL of AccQTag UltraTM derivatizing reagent solution, with further vortex

mixing, and heating at 55°C (10 min). Samples were then diluted 1:100 with Optima grade

water for analysis. Sample preparation is summarized in Supplementary Table S1

UHPLC-MS/MS Analyses. UHPLC-MS/MS analysis was performed using an Acquity

UPLC binary solvent manager, sampler manager and column manager (Waters, Milford, MA,

USA) interfaced with a Xevo TQ-S tandem quadrupole mass spectrometer (Waters,

Wilmslow, UK). MS/MS Detection was via electrospray ionization (ESI) in positive ion

mode using multiple reaction monitoring (MRM) for the quantification of each compound

(see Table 1) (MS conditions for each analyte were determined via direct infusion of

individual derivatives). Nitrogen was used as the desolvation gas and argon was used as the

collision gas. The following generic source conditions were used: capillary voltage, 1.5 kV;

source offset, 50 V; desolvation temperature, 600 °C; source temperature, 150 °C,

desolvation gas flow, 1000 L/hr; cone gas flow, 150 L/hr; nebulizer gas, 7.0 bar; collision

gas, 0.15 mL/min. Compound specific parameters are detailed in Table 1.

The chromatographic separation used reversed-phase gradient chromatography on a HSS

T3 2.1 x 150 mm, 1.8 µm column (Waters). The mobile phase was composed of 0.1 %

formic acid in water (v/v) (A) and 0.1 % formic acid in acetonitrile (v/v) (B). The column

temperature was maintained at 45 °C and linear gradient elution was performed at 0.6

mL/min starting at 4 % B, held for 0.5 min before increasing to 10 % over 2 minutes, then to

28 % over 2.5 min finally increasing to 95 % for 1 min, before returning to 4 % B (1.3 min)

for re-equilibration. The weak and the strong washes were 95:5 water/acetonitrile (v/v) and

100% isopropanol respectively.

Before analysis, injections (2 µL) of a double blank were performed to ensure system

stability and cleanliness followed by a “system suitability test”, performed by injecting the

low-level QC sample containing all the standards and internal standards. After the injection

of a further double blank and a single blank, analysis was started with injections of the

calibration curve (low concentrations to high) followed by a double blank injection. The QC

standards (at least 6 QC samples, 2 at each level) were interspersed evenly throughout the

study samples as shown in Figure 1. Study samples were randomized prior to sample

preparation to minimize bias due to batch effects. Following analysis of all of the samples in

the batch a second set of calibration samples were injected, again using the sequence of low

to high concentrations.

Figure 1. Sequence of analysis for amino acid quantification of randomized samples bracketed by calibration standards and interspersed with QC injections.

Method Validation. Method validation for the method was based as far as practicable on

the FDA “Guidance for industry” on Bioanalytical methods52.

Intra- and Inter-Assay Precision. To determine assay precision calibration standards

prepared from stock A and six-fold replicates from stock B at the same concentration as six

of the calibration standards were analysed as QC samples, in a single batch using the methods

described. Linearity was assessed using the R2 correlation coefficient determined from

calibration standards was required to be >0.99 over the three days of the validation.

The intra-assay variability of the method was determined using the CV for replicate assays

(n=6) for each of the six selected concentrations on a single occasion. Inter-assay precision

was assessed on three separate days using six QC samples at three concentrations assayed

together with a set of calibration standards and biological samples and determined as the CV

for each set of the QC samples (n=18)52. To be accepted a minimum of 67 % of the QC

standards must have had a deviation of no more than 15% from their nominal concentration,

with at least 50 % of the QC injections at each concentration meeting this criterion.

Specificity. Human plasma and serum from six different subjects were tested to determine

matrix interferences using stable isotope labelled analogues.

Carry over. Carry over was assessed with a double blank run immediately after an ULOQ

calibration standard and was accepted if the response was ≤20% of the average response from

the LLOQ standards. Carryover for the IS was acceptable if the response in the double blank

sample that was ≤5% of the average response from the calibration standards (including the

single blank).

Recovery. In the absence of analyte free matrix, recovery from plasma/serum from six

sources was estimated using stable isotope labelled (SIL) compounds. Recovery was

calculated by comparing the responses for six replicates of extracted samples spiked at 1

µg/mL ( 0.24-0.67 µM depending on the analyte) and at 3 µM, with replicates of extracted

blank matrix to which SIL(s) were added post-extraction, at the same nominal concentrations.

Matrix interferences. Matrix to analyte interferences were assessed by analysing six

double blanks for responses at the retention times of the analytes compared to the mean of the

analyte responses in the LLOQ calibration standards. A minimum of five of the six double

blanks had to be less than 20% of the signal in the LLOQ calibration standard. Matrix effects

on the SIL ISs were assessed using the same approach with the acceptance criteria based on

signals being less than 5% of the average IS response of the standards in the calibration curve

in at least five of the six double blanks. For analyte to analyte interference aliquots of the

same lot of blank matrix were spiked with each analyte, in triplicate, at the same

concentration as the highest concentration standard and analysed to assess their potential

interference at the retention time of the other analytes. Interference was considered to be

present if a response ≥20 % to that of another analyte was detected for the LLOQ standard.

For analyte to IS interference the blank matrix was spiked with analytes at the concentration

of the highest concentration standard and responses at the retention time of the IS were

compared to the average response of the IS of all standards accepted in the calibration curve

(including single blank(s)). Interference was considered to be present if a response ≥5.0 %.

Stability. Stock solution stability of the underivatized analytes at ambient temperature (6

hr) or stored frozen at -20 °C (48 hr) was investigated. The stability of the derivatised

analytes (diluted 1:9 v/v with water) was also assessed by storing the QC samples at ambient

temperature (1 week) and in the autosampler (diluted 1:99 v/v with water) and by reanalysis

of the QC samples maintained in the autosampler at 4 °C (36 hr). For the analyte to be

considered stable the difference had to be within +10% of the original value.

Data Analysis. The raw LC-MS data were processed by the TargetLynx application

package within MassLynx software (Waters Corporation). The raw data was mean smoothed

and peak integration was performed using ApexTrak algorithm. Further statistical analysis

was performed on the resulting calculated concentrations (corrected for the 2 fold dilution of

the samples compared to the standard curve) using Prism, where a Mann U Whitney test was

applied to determine if differences observed in concentrations between healthy volunteers

and patients with ALF were statistically significant.

RESULTS AND DISCUSSION.

Method development. The AccQTag UltraTM reagent reacts with primary and secondary

amines44-49 (illustrated in supplementary Figure S1) giving derivatives with good reversed-

phase chromatographic and mass spectrometric properties. Potential problems associated with

the use of the non-volatile borate buffer combined with MS have previously been discussed,

and volatile alternative buffers evaluated43. However, probably as a result of large dilution

factor employed here, we encountered no buffer-related problems were apparent and no

changes were made to the recommended reaction conditions. Appropriate positive ESI MS

conditions for each of the analytes were obtained via direct infusion of the individual

derivatives, as detailed in Table 1 (columns 3, 4, 6, 7, 8 respectively). The AccQTag UltraTM

reagent gives rise to a common fragment ion at m/z 171, generated by a loss of the

aminoquinoline (AMQ) moiety and, as can be seen from Table 1 (columns 3 and 4), in most

cases, the combination of parent ion and this common fragment was selected for detection.

Whilst monoamines react to form a mono ACQ derivative which ionises in positive ESI to

the [M+AccQ+H]+ ion some polyamines (e.g. cystine, lysine) have multiple sites that can

form derivatives resulting in the addition of more than one ACQ unit. The number of ACQ

units is reflected in the charge state of the analyte, and doubly derivatized compounds display

the most intense response. However, a smaller proportion of the singly derivatized and singly

charged and doubly derivatised and singly charged ions are still detected. For example, for

cystine and lysine the most abundant ions have m/z values of 291 and 244 respectively,

corresponding to the [M+2xAcq+2H]2+, while ions at m/z values of 581 ([M+2xAqc+H]+)

and 411 ([M+1xAcq+H]+) for cystine and 487 ([M+2xAqc+H]+) and 317 ([M+1xAcq+H]+)

for lysine were observed at lower abundances (for mass spectra see supplementary Figure

S2). For quantification the most abundant transitions from each of these precursor ions were

used except for arginine, glycine and proline where a more appropriate alternative transition

was selected due to matrix interferences. A chromatographic method was then developed

providing an analysis time of 7.5 min/sample (a mass chromatogram for a range of amino

compound standards is shown in Figure S3 and a similar mass chromatogram for the

isotopically labelled internal standards is shown in Figure S4). The average peak width

observed was 3 seconds at the base giving a peak capacity of ca.120. The separation was

highly reproducible, with CVs for retention time (Table 1 column 5) for the ULOQ QC

sample of < 0.44 %.

A number of the compounds determined in this assay are isomers/isobars e.g., 1-

methylhistidine and 3-methylhistidine; sarcosine, β-alanine and alanine; γ-amino-n-butyric

acid, β-amino-iso-butyric acid and α-amino-n-butyric acid; isoleucine and leucine however,

all were resolved by the chromatographic method (illustrated in Figure S5).

Table 1: Chromatographic and Mass Spectrometric Characteristics of the Analytes

No. CompoundParent (m/z)

Fragment (m/z) RT (min)

Dwell time (s)

Cone voltage

(V)

Collision energy

(eV)

LLOQ(µM)#

1 Creatinine 227.0 158.9 0.51 0.024 30 6 -2 Creatine 302.1 112.1 0.61 0.024 62 20 -3 Galactosamine 350.1 171.1 0.81 0.024 30 32 -A Histidine-d3 329.1 159.1 1.53 0.011 20 10 n/a4 Histidine 326.1 156.1 1.55 0.011 20 10 65 N-methyl-L -phenylalanine 351.1 207.0 1.56 0.013 30 24 n/q6 Phosphoserine*A 356.1 171.1 1.61 0.013 20 18 n/q7 Methylamine 202.1 171.1 1.63 0.010 30 16 -8 3-Methylhistidine*A 340.1 170.1 1.73 0.009 30 18 209 4-hydroxyproline*C 302.0 171.1 1.75 0.011 10 22 210 1-Methylhistidine*A 340.1 124.2 1.84 0.009 30 28 2011 Asparagine*D 303.1 171.1 1.97 0.009 30 22 212 Carnosine*C 397.1 227.2 1.99 0.009 30 14 2013 Arginine 345.1 70.1 2.09 0.009 30 36 2014 Anserine 411.2 241.1 2.03 0.009 68 18 -B Arginine-13C6 351.2 181.2 2.15 0.009 6 18 n/a15 Glycylglycine 303.1 171.1 2.15 0.009 30 22 -16 Taurine*B 296.1 116.3 2.18 0.009 30 60 n/qC Glutamine-d5 322.1 171.1 2.26 0.009 30 24 n/a17 Glutamine 317.1 171.1 2.26 0.009 30 24 2D Serine-d3 279.1 171.1 2.28 0.009 30 20 n/a18 Serine 276.1 171.1 2.28 0.009 30 20 619 Homoserine*C 290.1 171.1 2.42 0.013 10 18 N/Q20 Ethanolamine*D 232.1 171.1 2.43 0.011 10 20 2 E Glycine-d5 248.1 171.0 2.56 0.011 30 20 n/a21 Glycine 246.1 116.1 2.56 0.011 30 40 2022 Aspartic acid 304.1 171.1 2.65 0.011 30 22 2

F Aspartic acid-d3 307.0 171.1 2.66 0.011 30 20 n/a23 Citrulline*D 346.2 171.1 2.75 0.011 30 26 624 Sarcosine*F 260.1 171.1 2.82 0.011 30 20 2 G Glutamic acid-d3 321.0 171.0 2.93 0.011 30 20 n/a25 Glutamic acid 318.1 171.1 2.94 0.011 30 22 226 β-alanine*G 260.1 171.1 3.04 0.011 30 20 N/Q27 Threonine 290.1 171.1 3.07 0.018 30 20 2 H Threonine-13C4,15N 295.1 171.0 3.13 0.024 30 26 n/a28 Ophthalmic acid 460.3 171.0 3.40 0.011 48 36 - I Alanine-d3 263.1 171.0 3.43 0.011 30 16 n/a29 Alanine 260.1 116.1 3.43 0.011 30 44 230 Allantoin 329.0 171.1 3.43 0.011 30 20 -31 γ -amino-n-butyric acid*J 274.1 171.1 3.50 0.009 10 20 632 Aminoadipic acid*J 332.1 171.1 3.59 0.011 30 18 -33 Hydroxylysine*J 333.2 171.1 3.50 0.009 16 16 2034 3-amino-n-butyric Acid 274.1 171.1 3.67 0.009 10 20 635 Cysteine 292.1 171.1 3.75 0.009 30 22 -36 Glutathione 478.0 171.1 3.76 0.009 30 38 -J Proline-d7 293.0 171.1 3.76 0.009 30 24 n/a37 Proline 286.1 116.1 3.79 0.009 30 50 638 3-aminoisobutyric acid*J 274.1 171.1 3.77 0.009 10 2039 Cystathionine*J 282.2 171.0 3.85 0.009 30 14 240 Ornithine*J 303.1 171.1 3.86 0.009 60 22 2041 Epinephrine 354.1 171.1 3.93 0.009 30 22 -42 β-amino-iso-butyric Acid 274.1 171.1 3.97 0.009 10 20 643 5-Aminovaleric acid 288.1 171.1 3.98 0.009 30 16 -44 Octopamine 324.2 171.1 3.98 0.009 30 24 -45 α-amino-n-butyric acid*K 274.1 171.1 4.07 0.009 10 20 646 Cystine*K 291.1 171.1 4.08 0.009 10 12 n/q K Lysine-d4 246.1 171.1 4.10 0.020 30 14 n/a47 Lysine 244.2 171.1 4.10 0.020 30 12 248 Putrescine 429.3 145.1 4.22 0.009 30 28 -49 4-Aminohippuric acid 365.2 171.0 4.36 0.019 6 36 -50 3-Hydroxytyramine 324.1 171.1 4.41 0.008 30 20 -51 Tyrosine*M 352.1 171.1 4.41 0.017 30 24 252 3-Hydroxykynurenic Acid 395.2 171.0 4.57 0.009 60 28 -L Methionine-13C5,15N 326.2 171.0 4.59 0.017 30 22 n/a53 Methionine 320.1 171.1 4.59 0.017 30 22 2M Valine-d8 296.1 171.1 4.61 0.017 30 20 n/a54 Homocysteine 306.0 171.1 4.62 0.017 30 20 -55 Valine 288.1 171.1 4.64 0.017 30 16 256 Tyramine 308.1 171.1 4.82 0.017 30 24 -57 N-Methyl-L-valine 302.0 171.1 4.84 0.017 54 26 -58 Homocystine 439.1 171.1 4.87 0.009 10 24 -59 3-Hydroxyanthranilic Acid 324.2 171.1 4.88 0.017 52 20 -N Isoleucine-d10 312.1 171.0 5.16 0.024 30 20 n/a60 Isoleucine 302.1 171.1 5.22 0.024 30 20 2 O Leucine-d10 312.1 171.0 5.26 0.024 30 20 n/a61 O-Phosphoethanol – 312.1 116.1 5.26 0.024 8 50 -

amine62 Leucine 302.1 171.1 5.26 0.024 30 20 2 P Phenylalanine-d5 341.1 171.1 5.44 0.024 30 22 n/a63 Phenylalanine 336.1 171.1 5.46 0.024 30 22 264 Tryptophan*P 375.1 171.1 5.51 0.024 30 26 265 Tryptamine 331.1 171.1 5.86 0.069 30 28 -

# - based on a 10µl plasma or serum sampleNormal Font – Validated against stable isotope labelled IS of the same structureNormal Font* – Validated against a stable isotope labelled IS of a different structure (indicated by letter after *) but similar retention timeItalic Font – Not validated but monitoredBold Font – Stable isotope labelled IS

Sample preparation was investigated based on either protein precipitation with

sulphosalicylic acid or various organic solvents (acetonitrile, methanol and isopropanol), with

or without acidification using 1% v/v formic acid. This led to the selection solvent

precipitation with two volumes of isopropanol, containing 1% v/v formic acid, to one of

diluted plasma/serum sample based on better sensitivity and reduced matrix effects compared

to e.g., methanol, sulphosalicylic acid etc., (data not shown).

Method Validation. Following the development of a suitable UHPLC-MS/MS system the

suitability of the method for the analysis of amino compounds was then evaluated for selected

analytes (Table 1). In the absence of true biological blank samples, standard curves and QC

samples were prepared in 50:50 water/methanol (v/v). A range of analytical attributes were

investigated, including linearity, lower and upper limits of quantification (LLOQ, ULOQ),

intra- and inter-day accuracy and precision, specificity, carryover, recovery, matrix and other

interferences and analyte and derivative stability. Standard curves were found to be linear

over the range measured 1-400 µM, with correlation coefficients (R) of 0.993 or better as

determined over the three days of the validation (Supplementary Data Table S2). The

concentration range covered by the standard curve equates, in this instance, to 2-800 µM for

plasma/serum due to the 1:1 dilution of the samples prior to protein precipitation. The

underivatised analytes were stable for 6 hr at ambient temperature or frozen at -20 °C for 48

hr (Supplementary Data Tables S3 and S4 respectively). Similarly, the derivatized analytes

were also stable at ambient temperature and in the autosampler at 4 °C for up to 1 week (1

week stability data are provided in Supplementary Tables S5 and S6 respectively).

Carryover was 11% or less for all of the analytes tested whilst that of the ISs was less than

1%.

The intra- and inter-day performance of the assay was evaluated by the analysis of

samples on three separate occasions on three separate days over the concentration range of 2-

800 µM and the method was found to be reproducible and accurate. For both intra- and inter-

day analysis a LLOQ of 2 µM was obtained for 18 of the amino acids (summarized in Table

1). A further 8 of the target analytes (ethanolamine, histidine, proline, serine, β-amino-iso-

butyric acid, citrulline, α-amino-n-butyric acid and ɣ-amino-n-butyric acid) gave acceptable

results using a LLOQ of 6 µM, whilst a further 7 compounds (arginine, carnosine, glycine, 3-

methylhistidine, 1-methylhistidine, hydroxylysine and ornithine) could be quantified using

LLOQs of 20 µM. The ULOQ for all analytes was 800 µM. A summary of the validated

analytical ranges for these analytes is provided in Supplementary Table S7.

The intra- and inter-day precision of the assay was determined by examining the CVs of

the QC standards for each of the analytes and ranged from 0.91-16.9 % for the intraday

determinations and 2.10-15.9 % for the inter-day comparison. The corresponding figures for

intra- and inter-day accuracy were 0.05-15.6 and 0.78-13.7 % respectively. Mean intra- and

inter-day bias were calculated to be 0.04-16.4 % and 0.04-12.5 % respectively. The mean

inter-day concentration and precision data are provided in Table S8. Specificity, in terms of

matrix and other interferences was assessed for matrix-to-analyte (at the LLOQ), matrix-to-

internal standard, analyte-to-internal standard and internal standard-to-analyte interference.

Both matrix-to-analyte and matrix-to-internal standard were < 20%. In the case of internal

standard-to-analyte (at the LLOQ) the acceptance criterion of < 20% was met in all cases

except for glycine-d5. This IS is effectively glycine-d2 as 3 of the deuterium atoms are

exchangeable with water protons on dissolution and, in this instance, the 2 mass unit

difference between analyte and IS was insufficient to ensure specificity and resulted in

internal standard-to-analyte interference. Replacement of glycine-d5 with the alternative IS

glycine-13C2 15N d2 should eliminate this issue. All of the analytes gave values of < 5% when

analyte-to-IS interference was investigated except for glycine and glutamine.

The stable isotope labelled compounds used as internal standards were used to assess

gross matrix interferences following spiking into six different human plasma and serum

samples. Whilst differences were noted matrix matrix interferences were generally < 20 %.

Analyte recoveries, as determined using stable isotope labelled ISs spiked into plasma and

serum were high and repeatable (74-78 and 93-101% respectively at 3µM) (Supplementary

Table S9) were, as would be expected for a method with minimal sample preparation,

Application to Human Subjects. Following validation, the method was then applied to

the analysis of samples obtained from patients suffering from acute liver failure and normal

healthy controls. ALF is characterised by a rapid decline in liver function following a

catastrophic insult to the liver (e.g., acetaminophen overdose or viral hepatitis). The liver

plays a major role in amino acid metabolism and is central to the regulation of metabolic

pathways53 and in liver disease these pathways are known to be affected by depletion of the

branched chain amino acids – valine, leucine and isoleucine and increased concentrations of

aromatic amino acids including phenylalanine and tyrosine (the Fischer ratio)54. Perturbation

of the urea cycle and a limited capacity for detoxification of ammonia with glutamine are

associated with acute liver failure and in particular with hepatic encephalopathy although the

mechanism and prognostic significance remains unclear. Application of the methodology

described above to plasma samples derived from healthy controls (n=40) and patients with

APAP induced ALF (n=14) revealed changes in 15 amino compounds that were subject to

quantification. Changes were also observed in 6 of the monitored compounds (Zia et al, in

preparation). Selected ion chromatograms for a “typical” control and ALF subject are shown

in Figure 4. As expected, significantly elevated concentrations of phenylalanine and tyrosine

were observed in the ALF group with mean concentrations of phenylalanine in controls of

65µM compared to 245µM in ALF, whilst for tyrosine the concentrations were 69

Figure 4. Scaled SRM chromatogram of an ALF (A) and a control (B) subject respectively.

versus 191µM in controls and patients respectively (Figure 5). In the case of isoleucine

concentrations were reduced from 76µM in control subjects to 56µM in patients whilst valine

concentrations declined from 234µM in controls to 188µM in patients. Changes were also

seen for glutamine and glycine, which were elevated in ALF, with the former rising from

427µM to 1123µM and the latter from 249 µM to 693µM in patients (see Figure 5). Further

results for quantifiable amino compounds are provided in Supplementary Figure S6. Effects

were also noted on sarcosine with this analyte not detected in controls but observable with

concentrations over the range 2.4-7.2µM in ALF patients. Changes were also noted in the

relative amounts of aminoadipic acid, β-alanine, β-amino-iso-butyric acid, cysteine,

cystathionine, homoserine, histidine, hydroxylysine, 3-methylhistidine and ɣ-amino-n-butyric

acid, however, whilst detectable, these compounds were below the limits of quantification

(data not shown). Furthermore, a 6-fold increase was observed for 5-aminovaleric acid in

ALF patients

Figure 5 Amino acid concentrations (Log µM) in control (blue) and ALF (red) subjects, Box plots; bar, median; box, interquartile range; whiskers, upper and lower values. A two tailed Mann Whitney U test was applied to test for statistical significance, p-values for these are denoted by **** p <0.0001; ***<0.001; ** <0.01; * 0.1;

relative to controls (Compound 43 see Table 1), an amino compound which was subject to

monitoring but not quantification in the current method. As well as confirming known

effects of ALF, such as the expected changes in aromatic and branched chain amino acids the

application of this UPLC-MS method has illustrated changes in the amounts of other amino-

containing compounds in response to liver injury, including the previously unreported effects

on plasma sarcosine concentrations.

CONCLUSIONS

The use of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AccQTag UltraTM) for

the targeted analysis of amino-containing compounds provides stable derivatives with good

RPLC and MS properties. These derivatives enable the sensitive, reproducible and specific

analysis of these compounds in human serum and plasma, requiring only 10 µl of sample, and

the short analysis time makes the methodology attractive for high throughput targeted UPLC-

ESI-MS/MS analysis. Whilst confirming previous observations application of the method

also revealed novel increases in sarcosine concentrations in patients with ALF as a result of

APAP overdose. This methodology should be of great benefit in supporting metabolic

phenotyping studies in epidemiological and clinical environments.

ASSOCIATED CONTENT

*S Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at

DOI:

Figure S1, the derivatisation reaction between AccQTag UltraTM and amines; Figure S2,

mass spectra of cystine (a) and lysine (b) derivatives; Figure S3, The UPLC separation of

selected standards; Figure S4, separation of stable isotope labelled internal standards; Figure

S5, resolution of the derivatives of isomeric amino acids and isobaric analytes; Figure S6,

results for healthy controls and patients with APAP-induced ALF; Table S1, sample

treatment and derivatization for calibration standards/QCs, single blanks, plasma/serum and

double blanks samples; Table S2, standard curve linearity data obtained during the three day

validation; Table S3, summary of short term stock solution stability; Table S4, freeze thaw

stability of stock solutions; Table S5, bench top stability post-derivatisation; Table S6,

autosampler stability; Table S7, summary of the valid concentration ranges for 38 analytes

subject to quantification; Table S8, inter-day validation data; Table S9, recovery data for

quantified analytes.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: j.nicholson@imperial.ac.uk. Phone: +4420 7594 3195.

*E-mail: i.wilson@imperal.ac.uk. Phone: +4420 7594 0730.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS

The MRC Integrative Toxicology Training Partnership (ITTP) is gratefully acknowledged for

financial support of a PhD studentship (Rabiya Zia). The clinical samples were obtained as

part of a New Investigator Award from the Intensive Care Foundation. The MRC-NIHR

National Phenome Centre is supported by the UK Medical Research Council [in association

with National Institute of Health Research (England) (Grant MC_PC_12025). The financial

support of Bruker Biospin, Waters Corporation, Metabometrix LTD and Imperial College is

also gratefully acknowledged by the NPC. The views expressed are those of the authors and

not necessarily those of the NHS, or NIHR or the Department of Health.

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Supplementary data for

High speed quantitative UPLC-MS analysis of multiple amines in human plasma and

serum via pre-column derivitazation with 6-aminoquinolyl-N-hydroxysuccinimidyl

carbamate: application to acetaminophen-induced liver failure

Nicola Gray1, Rabiya Zia1 Adam King1, Vishal C Patel3, Julia Wendon3 Mark JW McPhail3 Muireann Coen1,

Robert S Plumb1, Ian D Wilson1*, Jeremy K Nicholson1,2*

1 Division of Computational and Systems Medicine, Department of Surgery and Cancer, Imperial College

London, Exhibition Road, South Kensington, London SW7 2AZ, UK2MRC-NIHR National Phenome Centre, Division of Computational and Systems Medicine, Department of

Surgery and Cancer, IRDB Building, Imperial College London, Hammersmith Hospital, London, W12 0NN,

United Kingdom

3 Institute of Liver Studies & Transplantation, Kings College Hospital, Denmark Hill, London SE5 9RS, United

Kingdom

This supplementary data section includes 6 figures: Figure S1. Shows the chromatographic separation of a

range of the analyte standards monitored by the assay (see Table 1 for peak assignments) at 400 µM (240 fmol

on column), Figure S2, which shows the Chromatographic separation of the stable isototope labelled internal

standards used to monitor the analysis, Figure S3 illustrates the derivatisation reaction between AQC reagent

(6-aminoquinolyl-N-hydroxysuccinimidyl carbamate) and primary or secondary amino acids, Figure S4

Chromatographic resolution of the derivatives of the isomeric amino acids A) 1-methylhistidine (1-MHis) and

3-methylhistidine (3-MHis) (the separation of these derivatised amino acids from histidine (His) is also shown),

B) sarcosine (Sar), β-alanine (β-Ala) and alanine (Ala), C) γ-amino-n-butyric acid (GABA), β-amino-iso-butyric

acid (BAIBA) and α-amino-n-butyric acid (Abu) and D) isoleucine (Ile) and leucine (Leu). Figure S5 shows

mass spectra for cystine (a) and lysine (b) following derivatisation with AccQTag UltraTM; and Figure S6)

provides results for the quantification of further amino compounds in the plasma of normal healthy controls

(blue) and patients with APAP-induced liver failure.

There are also 9 tables showing in Table S1 the sample treatment and derivatization for calibration

standards/QCs, single blanks, plasma/serum and double blanks samples. Tables S2 to S8 provide information

on the analytical figures of merit for the assay. Table S2; Standard curve linearity data obtained for the analytes

over the course of the three day validation; Table S3, Summary of Short Term Stock Stability (6 hr at room

temperature); Table S4, Summary of Autosampler Stability Data 4 °C (1 week); Table S5, Summary of

freeze thaw stability of stock solutions (48 hr at -20 °C); Table S6, Summary of bench top stability post-

derivatisation (1 week); Table S7, Summary of the valid concentration ranges of the 38 amino acids considered

for absolute quantification; Table S8, Inter-Day Mean Concentration Data and Coefficients of variation for the

Quantified Analytes and Table S9, Recovery calculated using stable isotope labelled compounds spiked into

protein precipitated human plasma/serum at 1 µg/mL and 3 µM and compared to spiked human plasma (n=6)

prior to protein precipitation.

Supplementary Data – Figures

Figure S1. Derivatisation reaction between AQC reagent (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate) and primary or secondary amino acids.

Figure S2. Chromatographic separation of a range of the analyte standards monitored by the

assay (see Table 1 for peak assignments) at 400 µM (240 fmol on column) (a similar mass

chromatogram for the stable isotope labelled internal standards is given in Supplementary

data Figure S2).

Figure S3. Chromatographic separation of the stable isotope labelled internal standards used to monitor the assay (see Table 1 for peak assignments).

Figure S4. Chromatographic resolution of the derivatives of the isomeric amino acids A) 1-

methylhistidine (1-MHis) and 3-methylhistidine (3-MHis) (the separation of these derivatised

amino acids from histidine (His) is also shown), B) sarcosine (Sar), β-alanine (β-Ala) and

alanine (Ala), C) γ-amino-n-butyric acid (GABA), β-amino-iso-butyric acid (BAIBA) and α-

amino-n-butyric acid (Abu) and D) isoleucine (Ile) and leucine (Leu).

Figure S5. Mass spectra for cystine (a) and lysine (b) showing the formation of multiple derivatives following reaction with AccQTag UltraTM;

Figure S6. Full range of amino acid changes, Boxplots are displayed in μM. Red boxes indicate plasma concentrations obtained for Healthy Controls and blue boxes for patients with APAP-induced ALF. The Middle line in the box represents the median values, the lower box bound the first quartile and the upper box bound the upper quartile and the whiskers the maximum and minimum values. A two tailed Mann Whitney U test was applied to test for statistical significance p-values for these are denoted by **** <0.0001; ***<0.001; ** <0.01; * 0.1

Supplementary Data – Tables

Table S1. Sample treatment and derivatization for Standards/QCs, single blanks, plasma/serum and double blanks samples*.

*Because of the additional 1:1 dilution of the plasma/serum prior to protein precipitation the concentrations determined in the samples are 2x that of the calibration standard concentrations.

Table S2; Standard curve linearity data obtained for the analytes over the course of the three day validation;

Amino Acid

Linearity (R2)

Amino acid

Linearity (R2)

Day 1 Day 2 Day3 Day 1 Day 2 Day3

4-hydroxyproline 0.997 0.997 0.990 Proline 0.998 0.997 0.997

Alanine 0.992 0.997 0.996 Serine 0.999 0.999 0.992

Arginine 0.993 0.986 0.991 Threonine 0.999 0.999 0.998

Aspartic acid 0.994 0.993 0.996 Tryptophan 0.999 0.994 0.993

Asparagine 0.996 0.997 0.998 Tyrosine 0.999 0.999 0.992

Carnosine 0.993 0.999 0.999 Valine 0.999 0.999 0.998

Cystine 0.998 0.998 0.993β-amino-iso-butyric acid 0.993 0.998 0.997

Ethanolamine 0.997 0.999 0.999 Citrulline 0.995 0.998 0.998

Glutamic acid 0.998 0.998 0.997 Cystathionine 0.995 0.996 0.994

Glutamine 0.996 0.998 0.997 3-methylhistidine 0.985 0.990 0.994

Glycine 0.994 0.998 0.998 1-methylhistidine 0.993 0.998 0.994

Histidine 0.991 0.994 0.994 Hydroxylysine 0.989 0.997 0.996

Isoleucine 0.999 0.999 0.998 Ornithine 0.986 0.991 0.988

Leucine 0.998 0.998 0.997 Aminoadipic acid 0.998 0.996 0.998

Lysine 0.999 0.996 0.998 α-amino-n-butyric acid 0.987 0.996 0.993

Methionine 0.999 0.999 0.997 Sarcosine 0.995 0.998 0.993

Phenylalanine 0.999 0.999 0.998 ɣ-amino-n-butyric acid 0.994 0.994 0.997

Table S3, Summary of Short Term Stock Stability (6 hr at room temperature)

AnalytePercentage Difference (%)

1 µM 3 µM 10 µM 300 µM

1-methylhistidine 10.0 -7.82

3-methylhistidine -8.54 -9.14

4-hydroxyproline -0.18 -6.80

Alanine 6.90 -2.37

α-amino-n-butyric acid -7.12 -6.18

Aminoadipic acid 0.91 -1.67

Arginine -2.66 -3.06

Asparagine 0.00 -0.08

Aspartic acid -10.5 -2.19

β-amino-iso-butyric acid 3.99 -2.78

Carnosine 7.69 -6.63

Citrulline -0.62

Cystathionine 5.30 -3.62

Cystine 8.99 -7.94

Ethanolamine -5.95 -3.58

γ-amino-n-butyric acid 8.73 -1.89

Glutamic acid -0.74 -2.70

Glutamine -10.7 -3.60

Glycine 6.57 -0.25

Histidine -3.88 -9.39

Hydroxylysine 6.50 -4.47

Isoleucine -2.94 -2.98

Leucine -3.21 -2.39

Lysine -0.71 -3.28

Methionine -0.25 -3.90

Ornithine 6.15 -6.80

Phenylalanine -3.70 -2.22

Proline -4.70

Sarcosine -4.33 0.16

Serine -2.84

Threonine -3.16 -2.01

Tryptophan -1.38 -3.42

Tyrosine 1.01 -6.55

Valine -2.11 -4.10

Table S4. Summary of freeze thaw stability of stock solutions (48 hr at -20 °C).

AnalytePercentage Difference (%)

1 µM 3 µM 10 µM 300 µM

1-methylhistidine -15.6 -20.9

3-methylhistidine 19.7 -9.27

4-hydroxyproline -13.2 -9.40

Alanine 17.4 -1.22

α-amino-n-butyric acid 1.03 -9.46

Aminoadipic acid 10.9 -1.28

Arginine -19.09 -34.12

Asparagine 2.99 -3.55

Aspartic acid 8.79 -5.37

β-amino-iso-butyric acid 3.40 0.97

Carnosine 3.19 2.19

Citrulline -1.80 -10.5

Cystathionine 12.7 -2.65

Cystine 0.63 -13.3

Ethanolamine 8.72 -0.44

γ-amino-n-butyric acid 4.38 -0.08

Glutamic acid -6.45 -7.86

Glutamine 6.76 -6.42

Glycine -6.65 -4.73

Histidine 3.59 -8.75

Hydroxylysine 7.23 1.57

Isoleucine 0.79 -4.13

Leucine 2.85 -2.44

Lysine -4.56 -4.65

Methionine -1.08 -7.75

Ornithine -5.07 0.22

Phenylalanine 1.15 3.47 3.50 -8.08

Proline 13.7 4.93

Sarcosine 2.27 -1.18

Serine 9.31 -4.83

Threonine -2.12 -7.54

Tryptophan 0.37 -9.98

Tyrosine -7.82 -15.2

Valine 3.61 -4.04

Table S5. Summary of bench top stability post-derivatisation (1 week at room temperature)

AnalytePercentage Difference (%)

3 µM 10 µM 300 µM

1-methylhistidine -14.5 7.75

3-methylhistidine -7.06 0.37

4-hydroxyproline 2.97 2.45 3.76

Alanine 1.83 -7.82 3.81

α-amino-n-butyric acid -6.79 -11.9 -4.49

Aminoadipic acid -4.86 2.64 4.39

Arginine -59.6 -8.01

Asparagine -6.60 -4.93 1.88

Aspartic acid 5.32 -5.54 4.20

β-amino-iso-butyric acid 2.52 1.14 4.80

Carnosine -7.36 6.85

Citrulline -5.71 1.52

Cystathionine 6.99 2.46 6.40

Cystine 9.00 -5.44 -0.82

Ethanolamine 20.3 6.10 8.92

γ-amino-n-butyric acid 5.36 4.47 6.62

Glutamic acid -8.32 -6.47 1.04

Glutamine -1.26 -3.15 -1.25

Glycine 1.78 2.20

Histidine -2.30 -19.53 6.63

Hydroxylysine 3.77 -0.73 3.30

Isoleucine -0.13 -3.40 1.31

Leucine 0.99 -5.42 0.09

Lysine 0.43 -6.01 1.32

Methionine 1.19 -11.2 -2.06

Ornithine 33.3 -2.93 13.6

Phenylalanine 0.46 -7.15 1.30

Proline 11.0 -3.57 4.16

Sarcosine 18.7 -13.0 -3.71

Serine 9.79 -8.86 0.57

Threonine 1.11 -7.57 1.26

Tryptophan 0.14 0.47 14.1

Tyrosine 12.4 -3.31 6.45

Valine 3.70 -6.06 0.73

Table S6, Summary of Autosampler Stability Data (4 °C, 1 week)

AnalytePercentage Difference (%)

3 µM 10 µM 300 µM

1-methylhistidine -2.79 0.69

3-methylhistidine 6.19 -0.97

4-hydroxyproline 4.38 -4.89 1.62

Alanine 0.00 -8.40 0.38

α-amino-n-butyric acid -6.55 -7.15 0.19

Aminoadipic acid -4.15 1.18 4.57

Arginine -6.99 -8.61

Asparagine 7.11 -7.66 4.14

Aspartic acid 14.3 0.78 3.32

β-amino-iso-butyric acid -10.6 -8.75 -0.83

Carnosine -13.6 2.50

Citrulline -8.36 2.86

Cystathionine -9.29 -18.5 -3.13

Cystine 6.50 -4.53 2.18

Ethanolamine 2.85 -8.82 1.01

γ-amino-n-butyric acid -10.2 -5.69 -1.26

Glutamic acid -1.72 -7.14 4.61

Glutamine -5.86 -8.15 -1.49

Glycine -0.12 3.71

Histidine -3.32 -16.7 -4.03

Hydroxylysine 3.77 13.6 2.30

Isoleucine 2.52 -4.45 0.62

Leucine -1.50 -9.30 -1.45

Lysine 3.65 -8.58 -0.42

Methionine 0.41 -11.4 -2.51

Ornithine 33.3 7.41 -0.13

Phenylalanine 1.37 -5.84 0.95

Proline -12.3 -11.5 0.27

Sarcosine -9.03 -20.4 -12.0

Serine 8.89 -7.74 2.98

Threonine 0.64 -5.35 0.15

Tryptophan -53.5 -59.9 -47.9

Tyrosine -9.55 -23.8 -11.0

Valine 2.43 -5.14 -0.29

Table S7. Summary of the valid concentration ranges of the 38 amino acids considered for absolute quantification.

Amino acidAnalytical valid concentration range (µM)

Valid concentration range in plasma/serum* (µM)

Valid concentration range on column (fmol)

4-hydroxyproline 1-400 2-800 0.6-240Alanine 1-400 2-800 0.6-240Arginine 10-400 20-800 60-240Aspartic acid 1-400 2-800 0.6-240Asparagine 1-400 2-800 0.6-240Carnosine 10-400 20-800 60-240Cystine N/Q N/Q N/QEthanolamine 1-400 2-800 0.6-240Glutamic acid 1-400 2-800 0.6-240Glutamine 1-400 2-800 0.6-240Glycine 10-400 20-800 60-240Histidine 3-400 6-800 1.8-240Isoleucine 1-400 2-800 0.6-240Leucine 1-400 2-800 0.6-240Lysine 1-400 2-800 0.6-240Methionine 1-400 2-800 0.6-240Phenylalanine 1-400 2-800 0.6-240Proline 3-400 6-800 1.8-240Serine 3-400 6-800 1.8-240Threonine 1-400 2-800 0.6-240Tryptophan 1-400 2-800 0.6-240Tyrosine 1-400 2-800 0.6-240Valine 1-400 2-800 0.6-240β-amino-iso-butyric acid 3-400 6-800 1.8-240Citrulline 3-400 6-800 1.8-240Cystathionine 1-400 2-800 0.6-2403-methylhistidine 10-400 20-800 60-2401-methylhistidine 10-400 20-800 60-240Homoserine** N/Q N/Q N/QHydroxylysine 10-400 20-800 60-240Ornithine 10-400 20-800 60-240Aminoadipic acid 1-400 2-800 0.6-240α-amino-n-butyric acid 3-400 6-800 1.8-240Phosphoserine** N/Q N/Q N/QSarcosine 1-400 2-800 0.6-240Taurine** N/Q N/Q N/Qβ-alanine*** N/Q N/Q N/Qγ-amino-n-butyric acid 3-400 6-800 1.8-240

*Concentration range in human plasma/serum is 2x that of the calibration standard concentrations since the biological samples have an additional 1:1 dilution prior to protein precipitation.

Table S8. Inter-Day Mean Concentration Data and Coefficients of Variation for the Quantified Analytes

Amino acid Mean Inter-day Concentrations

(Coefficients of Variation (%))

1 µM 3 µM 10 µM 30 µM 150 µM 300 µM 400 µM

4-hydroxyproline 1.05

(11.9)

3.08

(5.64)

10.4

(5.17)

31.0

(4.75)

156

(4.12)

311

(4.88)

415

(4.21)

Alanine 1.02

(10.6)

3.08

(6.18)

10.3

(4.87)

31.7

(3.70)

157

(3.14)

314

(3.79)

417

(3.25)

Arginine - - 10.5

(9.07)

30.3

(7.26)

153

(6.90)

298

(7.77)

394

(6.92)

Aspartic acid 0.97

(15.9)

3.07

(6.44)

10.3

(2.83)

31.4

(4.42)

155

(3.66)

307

(3.33)

411 (4.19)

Asparagine 0.95

(8.41)

2.90

(5.48)

9.79

(7.03)

30.0

(6.54)

151

(6.67)

300

(5.27)

401

(4.61)

Carnosine - - 10.48

(8.14)

31.0

(5.34)

151

(5.66)

305

(4.88)

394

(4.89)

Cystine 1.07

(12.0)

3.24

(11.9)

11.3

(11.0)

33.2

(7.75)

166

(9.16)

323

(8.63)

434

(5.65)

Ethanolamine 1.03

(5.75)

3.12

(4.26)

10.3

(3.01)

31.2

(5.14)

155

(3.22)

303

(4.95)

404

(3.08)

Glutamic acid 1.00

(7.33)

3.04

(5.24)

10.3

(4.75)

31.9

(3.53)

158

(4.35)

312

(3.71)

416

(2.52)

Glutamine 1.07

(8.93)

2.89

(6.00)

9.66

(6.77)

29.4

(5.35)

147

(5.35)

289

(4.74)

383

(6.07)

Glycine - - 10.6

(8.00)

32.1

(4.51)

155

(3.58)

302

(4.19)

399

(3.59)

Histidine - 3.14

(9.47)

10.4

(4.84)

32.3

(4.82)

156

(5.78)

313

(5.51)

417

(6.16)

Isoleucine 1.03

(4.81)

3.07

(3.27)

10.3

(2.90)

31.6

(3.08)

157

(3.16)

312

(3.58)

419

(2.58)

Leucine 1.00

(6.55)

3.02

(4.55)

10.2

(2.28)

31.4

(3.08)

156

(2.96)

315

(3.45)

426

(3.43)

Lysine 1.06 3.12 10.3 31.9 157 312 416

(7.72) (4.45) (5.12) (3.40) (3.30) (3.35) (2.26)

Methionine 1.06

(4.87)

3.10

(3.95)

10.3

(2.98)

31.7

(3.51)

157

(3.31)

309

(3.60)

413

(2.42)

Phenylalanine 1.05

(5.66)

3.07

(3.98)

10.3

(3.27)

31.6

(3.47)

156

(2.56)

309

(4.00)

410

(2.44)

Proline - 3.00

(9.49)

10.0

(7.84)

31.4

(4.80)

157

(5.32)

314

(3.67)

421

(3.65)

Serine - 3.12

(5.14)

10.2

(4.26)

31.6

(2.81)

158

(3.38)

309

(4.22)

417

(3.31)

Threonine 1.04

(7.13)

3.04

(3.79)

10.3

(2.30)

31.8

(3.14)

156

(2.79)

311

(2.81)

414

(2.02)

Tryptophan 1.12

(6.03)

3.21

(3.54)

10.8

(2.12)

33.2

(3.54)

166

(3.42)

324

(4.45)

434

(3.80)

Tyrosine 1.09

(7.73)

3.17

(6.88)

10.6

(7.35)

31.5

(5.07)

162

(4.37)

321

(5.51)

438

(4.34)

Valine 1.03

(4.49)

3.06

(3.27)

10.3

(3.83)

31.4

(2.73)

157

(3.02)

309

(2.90)

417

(2.59)

β-amino-iso-butyric acid - 3.10

(6.97)

10.3

(3.21)

31.3

(3.51)

153

(5.15)

300

(5.06)

396

(4.36)

Citrulline - 3.12

(6.98)

10.3

(4.59)

31.2

(3.70)

158

(4.59)

312

(3.56)

413

(3.11)

Cystathionine 1.06

(11.3)

3.16

(5.53)

10.6

(5.62)

32.0

(4.41)

160

(4.45)

323

(3.66)

431

(3.27)

3-methylhistidine - - 9.90

(10.1)

29.2

(7.36)

145

(8.50)

288

(9.58)

398

(8.35)

1-methylhistidine - - 10.8

(8.96)

30.6

(6.37)

153

(7.42)

305

(6.45)

413

(5.29)

Hydroxylysine - - 10.2

(11.9)

30.6

(5.34)

154

(5.92)

303

(5.08)

404

(3.55)

Ornithine - - 9.53

(9.94)

29.5

(6.53)

151

(7.98)

304

(9.21)

414

(7.19)

Aminoadipic acid 1.07

(4.89)

3.00

(4.42)

10.2

(4.07)

31.8

(4.15)

156

(6.17)

308

(3.90)

406

(2.98)

α-amino-n-butyric acid - 3.12 10.7 32.0 154 295 393

(8.12) (5.85) (3.80) (4.72) (6.65) (3.47)

Sarcosine 1.09

(6.16)

3.15

(6.19)

10.5

(5.90)

31.4

(3.90)

158

(3.38)

316

(3.93)

426

(4.55)

ɣ-amino-n-butyric acid - 3.05

(6.27)

10.2

(3.54)

31.4

(4.60)

154

(5.11)

303

(5.68)

396

(3.95)

Concentration of high QC (HQC)Concentration of mid QC (MQC)Concentration of low QC (LQC)

Table S9 Recovery calculated using stable isotope labelled compounds spiked into protein precipitated human plasma/serum at 1 µg/mL and 3 µM and compared to spiked human plasma (n=6) prior to protein precipitation.

Amino Acid1 µg/mL 3 µM

Recovery (%)

Plasma/ Serum

CV (%)

Plasma/ Serum

Recovery (%)

Plasma/ Serum

CV (%)

Plasma/ Serum

Alanine-d3 76.0/90.4 11.4/5.63 97.3/94.8 24.6/11.1

Arginine-13C6 75.6/99.0 8.96/14.9 101/90.7 28.3/6.34

Aspartic acid-d3 73.7/90.0 10.1/6.24 93.6/91.2 26.8/11.6

Glutamic acid-d3 75.0/86.1 11.5/6.83 95.9/91.3 29.0/11.4

Glutamine-d5 77.3/89.7 13.1/6.62 96.9/92.0 24.1/12.2

Glycine-d5 77.1/93.3 12.4/7.06 95.4/93.7 22.6/10.0

Histidine-d3 78.5/84.8 15.2/7.75 100/97.2 31.2/14.9

Isoleucine-d10 78.0/90.0 11.3/6.49 97.2/94.4 23.9/10.9

Leucine-d10 78.0/89.9 11.5/6.44 94.9/94.1 23.9/10.2

Lysine-d4 75.3/87.0 12.1/7.99 96.4/94.3 26.5/10.7

Methionine-13C5 15N 77.9/91.2 12.1/5.29 95.2/93.7 22.2/9.83

Phenylalanine-d5 79.6/92.0 11.5/5.99 93.1/93.6 18.7/8.64

Proline-d7 77.6/91.3 11.4/8.68 96.6/94.6 24.6/11.5

Serine-d3 76.3/90.2 11.6/6.86 93.9/94.2 23.5/8.49

Threonine-13C4 15N 76.3/89.3 11.1/6.29 96.5/93.8 26.2/10.3

Valine-d8 76.4/90.0 12.0/7.56 96.8/93.0 24.3/10.6

Isotopically labelled amino acids for use as internal standards (IS) including DL-alanine-d3, DL-aspartic acid-d3, DL-glutamic acid-d3, L-glutamine-d5, L-isoleucine-d10, DL-leucine-d10, DL-phenylalanine-d5, DL-proline-d7, DL-serine-d3, L-threonine-13C4, DL-tyrosine-d7 and DL-valine-d8 were purchased from Cambridge Isotope Laboratories (MA, USA). DL-Histidine-d3, DL-lysine-d4 and DL-methionine-d3 were purchased from QMX Laboratories (Essex, UK)