Supporting Information

Calibration curve-free GC-MS method for quantitation of amino and non-amino organic acids in biological samples

Sergey Tumanova,b, Yuri Zubenkoa#, Vladimir Obolonkinc, David R. Greenwooda,d, Vadim Shmanaib, Silas G. Villas-Bôasa*

a School of Biological Sciences, The University of Auckland, 3A Symonds Street, Auckland 1142, New Zealandb National Academy of Science, Institute of Physical Organic Chemistry, 13 Surganova Street, Minsk 220050, Belarusc Livestock Improvement Corporation Ltd., PB 3016, Hamilton 3240, New Zealandd New Zealand Institute for Plant & Food Research Limited, 120 Mt Albert Rd, Sandringham, Auckland 1025, New Zealand# Currently at Independent Petroleum Laboratory, Port Marsden Highway, Ruakaka 0151, Northland, New Zealand

*Corresponding author: Silas Granato Villas-Bôas, Tel.: +64 9 373 7599, E-mail: s.villas-boas@auckland.ac.nz

Content:

S1. Synthesis of deuterium labelled methyl chloroformate

S2. Detailed calculations for metabolite absolute concentration

Figure S1. The two-step reaction scheme used to synthesize d-MCF

Table S1. Analytical characteristics of metabolites used to perform quantitative method on Agilent GC-MS instrument

Table S2. The list of calculated fatty acid response factors and their drifts (%RSD, n=3) within 6 month time

Table S3. The list of calculated correction factors for phosphoenolpyruvic acid, glutamic acid and glutamine within 6 month timeS-1

Table S4. List of the most common metabolites quantified in biological samples used for method validation

S1. Synthesis of deuterium labelled methyl chloroformate

Due to the high toxicity of phosgene, the chemical synthesis of d-MCF was carried out in a well-ventilated hood. The

procedure included two steps and had a total yield of 70%. 5.1 g of triphosgene was mixed with 10 ml of toluene and 40 mg of

tetrabutylammonium chloride in a round-bottom flask (Pasquato et al., 2000). The reaction flask was equipped with a condenser and

connected to a condensation flask with a PVC hose. This recovery flask was dipped in dry ice/acetone bath (-70°C) to liquefy the

phosgene gas and connected to atmosphere through a drying tube filled with anhydrous calcium chloride. The triphosgene/toluene

mixture was periodically heated to 40°C under constant stirring for 10-15 min. After 15 min the decomposition of triphosgene started

and was followed by the release of phosgene gas, which was collected in the condensation flask. If the phosgene formation stopped,

the reactor flask was heated gently. The reaction ended when all solid triphosgene had completely decomposed. The flask with liquid

phosgene was weighed. Methanol-d4 was added drop-wise over 10 min to the phosgene containing flask in a dry ice/acetone bath. The

reaction mixture was stirred for 1 hour then warmed to room temperature. The product was refluxed for 15 min to allow evaporation

of deuterochloric gas and excess phosgene. d-MCF was distilled and the fraction with boiling point 71-73°C was collected. The mass

of d-MCF was 2.3 g. The structure was confirmed by 13C NMR (δ 150.35, 77.0 (CHCl3), 56.72 (q, J 22.9Hz)). Figure S1 shows the

scheme for d-MCF synthesis.

S-2

S2. Detailed calculations for metabolite absolute concentration CH (using valine as example).

Input data:

Parameter Valine Alanine-d4 DBP

Abundance of protonated

metabolite (AbH)

870464 657344 3309056

Abundance of deuterated

metabolite (AbD)

387904 286208 -

Expected abundance of

alanine-d4

- 734978 -

RF 0.0937 - -

Y-intercept -0.0127 - -

Concentration in analysed

sample, mM

- - 0.118

Step 1. Calculation of the coefficient proportional to the matrix effect (ME) of a sample (Equation 1):

k ME=AbD

AbDBP

where k ME is a coefficient proportional to the sample matrix, AbD (the abundance of deuterated metabolite derivative) and AbDBP (the

abundance of DBP).

k ME=387904

3309056=0.117.

S-3

Step 2. The matrix effect corrected metabolite abundance AbHME has a value as calculated below (Equation 2):

AbHME=870464 × (1+0.117 )=972308.

Step 3. Loss of alanine-d4 can be determined according to Equation 3:

k SP=1−( 714199734978 )=0.028.

Step 4. Final correction step (Equation 4):

AbHME ,SP=972308× (1+0.028 )=999533.

Step 5. The absolute concentration of metabolite c Hcan be calculated (Equation 5):

c H=( 9995333309056

−(−0.0127 ))× 0.1180.0937

=¿0.4 mM.

Using these 5 steps, the absolute metabolite concentration is calculated for the chloroform fraction obtained after chemical

derivatization step comprising 400 µL of solvent. In this example only 40 µL of water-based solution sample was used, giving the

final valine concentration of 4.0 mM.

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S-5

Cl O O Cl

Cl

Cl

O Cl

Cl

ClBu4N+

toluene Cl Cl

O

3

Cl Cl

O+ CD3OD Cl O

CD3

O

DCl-

Figure S1. The two-step reaction scheme used to synthesize d-MCF. The phosgene gas was generated by decomposition of triphosgene with a catalytic amount of tetrabutylammonium chloride in toluene followed by liquefaction of the gas. The second step included mixing methanol-d4 with liquid phosgene, producing d-MCF

S-6

Figure S2. The slope (a) of a linear function built by plotting the ratio of metabolite concentration to concentration of DBP (internal standard and mass reference) against the ratio of corresponding abundances represents the response factor (RF) for each respective metabolite. Both RF and Y-intercept determined for each metabolite are further used for described quantitative approach

Table S1. Analytical parameters of metabolites found in our in-house MCF MS library based on 7-10 calibration points (n=3) and obtained on an Agilent GC-MS instrument. RF and Y-intercept were determined for linear part of calibration function

Metabolite RT, min

RF Y-intercept

Quantification range, mM

Metabolite RT, min

RF Y-intercept

Quantification range, mM

10-Heptadecenoic acid 22.753 0.1046 -0.0384 0.02-4 Glyceric acid 11.752 0.0102 -0.0183 0.01-110-Pentadecenoic acid 18.933 0.1001 -0.0402 0.02-4 Glycerol 10.266 0.0146 0.0187 0.01-111,14,17-Eicosatrienoic acid 26.878 0.0863 0.0238 0.01-2 Glycine 11.386 0.0652 -0.0428 0.05-1011,14-Eicosadienoic acid 26.597 0.0916 0.0288 0.01-2 Glyoxylic acid 11.085 0.0105 0.0269 0.01-113,16-Docosadienoic acid 29.680 0.0801 -0.0388 0.01-2 Gondoic acid 26.493 0.0690 0.0051 0.02-4

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1-Aminocyclopropane-1-carboxylic acid 13.347 0.0362 0.0232 0.01-1 Heneicosanoic acid 28.476 0.1167 -0.0253 0.01-22,4-Diaminobutyric acid 22.831 0.0422 -0.0374 0.02-4 Hexanoic acid 5.792 0.1169 -0.0164 0.02-42,6-Diaminopimelic acid 29.397 0.0464 -0.0413 0.05-10 Hippuric acid 21.216 0.0567 -0.0064 0.05-102-Aminoadipic acid 19.786 0.0327 -0.0261 0.05-10 Histidine 26.892 0.0106 0.0136 0.05-102-Aminophenylacetic acid 18.155 0.0504 -0.0424 0.02-4 Homocysteine 23.193 0.0133 0.0316 0.02-42-Hydroxybutyric acid 10.300 0.0428 0.0087 0.05-10 Indole-3-butyric acid 25.964 0.0280 -0.0258 0.01-12-Hydroxycinnamic acid 21.048 0.0560 -0.0196 0.05-10 Isocitric acid 20.605 0.0281 -0.0231 0.05-102-Hydroxyisobutyric acid 9.009 0.0595 -0.0189 0.02-4

Isocitric acid secondary peak 20.257 0.0296 0.0272 0.05-10

2-Isopropylmalic acid 12.711 0.0563 0.0009 0.05-10 Isoleucine 13.980 0.0626 0.0302 0.05-102-Methyloctadecanoic acid 24.269 0.0887 -0.0231 0.02-4 Itaconic acid 10.069 0.0376 -0.0216 0.05-102-Oxoadipic acid 14.193 0.0456 0.0237 0.01-2 Lactic acid 9.103 0.0272 0.0270 0.02-42-Oxobutyric acid 5.702 0.0443 -0.0380 0.05-10 Leucine 14.044 0.0827 -0.0154 0.05-102-Oxoglutaric acid 13.859 0.0246 0.0066 0.05-10 Levulinic acid 8.607 0.0762 -0.0312 0.05-102-Oxovaleric acid 7.167 0.0278 -0.0113 0.01-1.5 Lignoceric acid 33.455 0.0762 -0.0323 0.01-22-Phosphoenolpyruvic acid 14.474 0.0416 0.0033 0.05-1 Linoleic acid 23.848 0.1115 0.0161 0.02-42-Phosphoglyceric acid 16.882 0.0455 -0.0241 0.01-1 Lysine 26.097 0.0628 0.0034 0.05-103,5-Diiodo-L-tyrosine 11.249 0.0374 0.0139 0.01-1 Malic acid 11.271 0.0360 0.0029 0.05-103-Hydroxybenzoic acid 17.018 0.0345 0.0204 0.05-10 Malonic acid 7.362 0.0265 -0.0416 0.05-103-Hydroxydecanoic acid 15.206 0.0629 0.0054 0.05-10 Margaric acid 22.880 0.1426 0.0131 0.02-43-Hydroxyoctanoic acid 12.407 0.0964 0.0300 0.02-4 Methionine 18.087 0.0389 -0.0038 0.08-2.83-Hydroxypropionic acid 13.260 0.0686 -0.0431 0.05-10 Myristic acid 17.458 0.1179 -0.0077 0.02-43-Methyl-2-oxopentanoic acid 7.689 0.0799 0.0121 0.05-10 Myristoleic acid 17.397 0.0374 0.0037 0.01-23-Oxoadipic acid 14.025 0.0782 -0.0371 0.05-10 N-Acetylcysteine 21.083 0.0541 -0.0199 0.02-44-Aminobenzoic acid 23.169 0.0162 -0.0301 0.05-10 N-Acetylglutamic acid 18.961 0.0172 0.0074 0.02-44-Aminobutyric acid 14.394 0.1038 -0.0406 0.05-10 N-alpha-Acetyllysine 28.903 0.0214 -0.0262 0.01-14-Hydroxycinnamic acid 22.082 0.1137 -0.0378 0.02-4 Nicotinamide 6.026 0.0138 -0.0346 0.1-54-Hydroxyphenylacetic acid 18.827 0.1108 0.0022 0.05-10 Nicotinic acid 10.696 0.0343 -0.0250 0.01-14-Methyl-2-oxopentanoic acid 7.787 0.0831 -0.0079 0.02-4 Nonadecanoic acid 25.219 0.1143 -0.0397 0.02-45-Hydroxy-L-lysine 30.085 0.0952 -0.0367 0.05-10 Norvaline 13.336 0.0794 -0.0301 0.05-105-Methoxytryptophan 39.918 0.0178 -0.0267 0.05-10 O-Acetylserine 15.747 0.0850 -0.0344 0.01-25-Oxotetrahydrofuran-2-carboxylic acid 14.186 0.0998 -0.0146 0.01-2 Octanoic acid 9.150 0.1195 0.0264 0.02-49-Heptadecenoic acid 22.860 0.0858 -0.0264 0.02-4 Oleic acid 23.798 0.0816 -0.0024 0.02-4

S-8

Adipic acid 11.859 0.1165 0.0265 0.05-10 Ornithine 24.667 0.0339 0.0137 0.05-10Adrenic acid 29.362 0.0795 -0.0429 0.01-2 Oxalic acid 5.892 0.0462 -0.0210 0.05-10Alanine 10.975 0.0782 -0.0322 0.05-10 Oxaloacetic acid 9.722 0.0084 0.0251 0.01-1Anthranilic acid 14.380 0.1000 -0.0219 0.05-10 Palmitic acid 21.093 0.1079 -0.0096 0.02-4Arachidic acid 26.807 0.1044 -0.0099 0.01-2 Palmitoleic acid 20.744 0.0701 0.0108 0.02-4Arachidonic acid 26.206 0.0875 -0.0241 0.02-4 para-Toluic acid 11.454 0.0571 0.0251 0.05-10Asparagine 16.639 0.1117 -0.0094 0.01-1 Pentadecanoic acid 18.965 0.1217 -0.0249 0.02-4Aspartic acid 16.215 0.0935 -0.0114 0.05-10 Phenylalanine 19.800 0.0509 -0.0224 0.05-10Azelaic acid 16.363 0.0355 0.0134 0.05-10 Pimelic acid 13.714 0.0330 -0.0221 0.05-10Behenic acid 29.821 0.0958 -0.0119 0.01-2 Proline 14.890 0.0998 0.0299 0.05-10Benzoic acid 9.492 0.0248 0.0192 0.05-10 Putrescine 21.528 0.0168 -0.0343 0.05-10bishomo-γ-Linolenic acid 26.429 0.0914 0.0009 0.02-4 Pyroglutamic acid 16.427 0.0649 -0.0032 0.01-1cis-4-Hydroxyproline 19.733 0.0409 -0.0269 0.02-4 Pyruvic acid 6.041 0.0143 -0.0062 0.02-2cis-Aconitic acid 15.392 0.0212 -0.0303 0.05-10 Quinic acid 16.315 0.0082 -0.0077 0.05-10cis-Vaccenic acid 23.821 0.0959 0.0125 0.02-4 Salicylic acid 16.798 0.0334 0.0162 0.05-10Citraconic acid 10.131 0.0550 -0.0206 0.05-10 Sebacic acid 17.612 0.0713 0.0091 0.05-10Citramalic acid 10.578 0.1064 -0.0301 0.05-10 Serine 17.256 0.0152 -0.0271 0.05-10Citric acid 16.229 0.0578 -0.0040 0.05-10 Sinapic acid 29.619 0.0124 0.0163 0.05-10Citric acid secondary peak 15.831 0.0493 0.0290 0.05-10 Stearic acid 24.004 0.1191 -0.0394 0.02-4Creatinine 16.661 0.0956 0.0082 0.05-10 Suberic acid 15.074 0.0573 -0.0385 0.05-10Cystathionine 34.577 0.0090 -0.0121 0.1-3.5 Succinic acid 8.955 0.0599 0.0107 0.05-10Cysteine 19.873 0.0136 -0.0133 0.07-2.5 Syringic acid 23.555 0.0522 -0.0110 0.05-10Decanoic acid 12.199 0.1212 -0.0093 0.02-4 Tartaric acid 20.310 0.0439 0.0174 0.05-10DHA 29.436 0.0682 -0.0022 0.01-2 Threonine 15.554 0.0159 0.0255 0.05-10Dipicolinic acid 18.086 0.1158 -0.0291 0.05-10 trans-4-Hydroxyproline 17.788 0.0413 -0.0415 0.05-10Dodecanoic acid 14.935 0.1197 0.0161 0.01-2 trans-Cinnamic acid 14.277 0.0699 0.0044 0.05-10DPA 29.597 0.0602 -0.0307 0.01-2 Tricosanoic acid 31.431 0.0942 -0.0185 0.01-2EDTA 27.241 0.0274 -0.0240 0.05-10 Tridecanoic acid 16.190 0.1213 0.0022 0.01-2EPA 26.496 0.0889 0.0009 0.01-2 Tryptophan 33.123 0.0763 0.0190 0.05-10Erucic acid 29.589 0.0711 0.0308 0.01-2 Tyrosine 28.558 0.0348 0.0278 0.05-10Ferulic acid 25.469 0.0400 0.0031 0.05-10 Undecanoic acid 13.592 0.1839 -0.0393 0.01-2Fumaric acid 9.104 0.0604 -0.0031 0.05-10 Valine 12.690 0.0937 -0.0127 0.05-10Glutamic acid 17.995 0.0342 -0.0042 0.05-10 Vanillic acid 20.709 0.0151 0.0286 0.05-10Glutamine 25.427 0.0079 0.0090 0.5-7.5 α-Linolenic acid 23.723 0.1019 -0.0116 0.02-4Glutaric acid 9.928 0.0215 -0.0344 0.05-10 β-Alanine 12.652 0.0390 -0.0188 0.05-10Glutathione 19.566 0.0214 0.0286 0.05-1.5 γ-Linolenic acid 24.018 0.0961 0.0237 0.02-4

RT, retention time; RF, response factor

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Table S2. The list of calculated fatty acid response factors and their drifts (%RSD, n=3) within 6 month time

Fatty acidChain length

RF 1 RF 2 Drift, %RSD

Myristic acid 14:0 0.1125 0.1179 3.31Palmitic acid 16:0 0.1032 0.1079 3.15Palmitoleic acid 16:1n7 0.0672 0.0701 2.99Stearic acid 18:0 0.1137 0.1191 3.28Oleic acid 18:1n9 0.0765 0.0816 4.56Linoleic acid 18:2n6 0.1081 0.1115 2.19Bishomo-γ-Linolenic acid

20:3n6 0.0874 0.0914 3.16

Arachidonic acid 20:4n6 0.0937 0.0875 4.84Docosahexenoic acid 22:6n3 0.0722 0.0682 4.03RF: response factor; RSD: relative standard deviation

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Table S3. The list of calculated correction factors for problematic metabolites within 6 month time

Metabolite Correction factor 1 Correction factor 1Phosphoenolpyruvic acid

1.25 6.75

Glutamic acid 0.70 2.46Glutamine 1.43 naMethionine 1.33 1.39

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Table S4. List of the most common metabolites quantified in biological samples used for method validation (%RSD shown in brackets, n=3). Results were presented as a mean value for (A) rat plasma, (B) rat urine and (C) rat liver extract together with published literature values for these metabolite concentrations

A

MetabolitesConcentration,

mg/LConcentration reported (Milakofsky et al., 1985; Milakofsky et

al., 1986), mg/LAlanine 25.52 (1.28) 37.6, 42.6Asparagine 13.97 (4.15) 11.8, 12.7Aspartic acid 2.64 (4.85) 1.0, 1.1Glutamic acid 7.74 (1.20) 7.6, 9.0Glutamine 38.47 (12.76) 58.6, 61.2Glycine 21.07 (3.86) 20.3, 22.1Histidine 14.41 (2.74) 10.2, 11.3Isoleucine 8.31 (1.73) 15.2, 17.8Leucine 26.54 (2.13) 29.0, 33.2Lysine 42.78 (3.78) 59.1, 64.8Ornithine 7.95 (1.95) 7.7, 8.4Phenylalanine 10.87 (2.78) 12.3, 14.3Serine 15.84 (5.21) 20.4, 25.0Threonine 33.08 (11.26) 34.8, 40.6Tryptophan 6.50 (6.18) 15.4, 18.7Tyrosine 17.01 (10.46) 16.6, 17.2Valine 22.78 (6.78) 28.6, 35.5

B

MetabolitesConcentration,

mg/LConcentration reported (Wang et al., 2013; Tang et al., 2014),

mg/LAlanine 6.76 (3.27) 1.36, 50.78Asparagine 3.92 (0.79) 0.67Aspartic acid 2.01 (1.62) 0.22, 0.67 Citric acid 4.27 (4.12) 0.37, 5.76Glutamic acid 10.81 (1.17) 2.22, 125.06Glycine 3.51 (4.60) 2.28Hippuric acid 8.24 (3.05) 0.54, 14.33

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Histidine 7.13 (1.57) 2.27, 15.52Isoleucine 10.18 (5.91) 0.29, 9.18Leucine 2.04 (3.20) 0.40, 2.62Lysine 6.08 (3.63) 13.98, 38.01Ornithine 6.75 (2.36) 0.91, 6.61Phenylalanine nd 0.41, 1.65Proline 3.76 (4.58) 0.68, 10.36Serine nd 0.66, 12.61Threonine 12.40 (1.85) 5.96, 52.41Tryptophan 4.43 (0.25) 0.08, 1.02Tyrosine 5.25 (1.07) 5.91, 5.44Valine 1.10 (1.76) 3.14, 4.69

C

MetabolitesConcentration,

mg/g tissue

Amino acids

Alanine 40.85 (2.99)

Aspartic acid 7.31 (6.32)

Glutamic acid 28.23 (0.82)

Glycine 2.74 (3.58)

Histidine 83.24 (0.38)

Isoleucine 3.70 (3.95)

Leucine 9.41 (0.07)

Lysine 17.62 (5.12)

Ornithine 17.36 (0.67)

Proline 4.72 (0.97)

Serine 14.19 (4.27)

Threonine 25.99 (9.47)

Tryptophan 5.63 (1.61)

Tyrosine 22.02 (4.54)

Valine 5.15 (1.41)

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Fatty acids

Arachidonic acid 49.64 (7.21)

DHA 43.23 (1.79)

EPA 47.59 (7.09)

Linoleic acid 23.54 (7.52)

Myristic acid 1.56 (2.27)

Oleic acid 37.27 (3.14)

Palmitic acid 50.06 (1.64)

Palmitoleic acid 2.39 (8.02)

Stearic acid 51.08 (6.20)

nd, not detected

References

Milakofsky, L., Hare, T.A., Miller, J.M., & Vogel, W.H. (1985). Rat plasma levels of amino acids and related compounds during stress. Life Science , 36, 753-761.

Milakofsky, L., Miller, J.M., & Vogel, W.H. (1986). Effect of acute ethanol administration on rat plasma amino acids and related compounds. Biochemical Pharmacology , 35, 3885-3888.

Pasquato, L., Modena, G., Cotarca, L., Delogu, P., & Mantovani, S. (2000). Conversion of Bis(trichloromethyl) Carbonate to phosgene and reactivity of triphosgene, diphosgene, and phosgene with methanol. Journal of Organic Chemistry , 65, 8224-8228.

Tang, X., Gu, Y., Nie, J., Fan, S., & Wang, C. (2014). Quantification of amino acids in rat urine by solid-phase extraction and liquid chromatography/electrospray tandem mass spectrometry: application to radiation injury rat model. Journal of Liquid Chromatography and Related Technologies, 37, 951-973.

Wang, C., Zhu, H., Pi, Z., Song, F., Liu, Z., & Liu, S. (2013). Classification of type 2 diabetes rats based on urine amino acids metabolic profiling by liquid chromatography coupled with tandem mass spectrometry. Journal of Chromatography B , 935, 26-31.

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