Methods

Short-chain fatty acids.
Volume: 50 µL

All concentrations are reported as µmol/L; CVs (%) shown as within-day (Wtn-day) and between-day (Btw-day) variations; sample matrices: serum (s), plasma (p), cerebrospinal fluid (csf).

Amino- and carboxylic acids. TCA metabolites and intermediates. Volume: 50 µL

All concentrations are reported as µmol/L; CVs (%) shown as within-day (Wtn-day) and between-day (Btw-day) variations; sample matrices: serum (s), plasma (p), urine (u).

Metabolites	Abbr	Category	PubChem	Reported	LOD	CV Wtn	CV Btw	ICC	Matrix	Method
Methylmalonic acid	MMA	Carboxylic acid	487	<0.26	0.03	2.4	3	0.81	s, p
Total homocysteine	tHcy	Sulfur amino acid	91552	5-15	0.1	1.4	2.7	0.72	s, p
Total cysteine	tCys	Sulfur amino acid	5862	150-350	50	1	2	0.62	s, p
Methionine	Met	Essential amino acid	6137	18-50	1	2.2	4.1	0.33	s, p
Serine	Ser	Polar amino acid	5951	95-200	5	1.3	1.7	0.71	s, p
Glycine	Gly	Hydrophobic amino acid	750	150-350	3	1.3	1.7	0.81	s, p
Cystathionine	Cysta	Thioether	439258	0.07-0.55	0.02	2.3	2.9	0.63	s, p
Sarcosine*	Sarc	Methylated amino acid	1088	0.7-2.3	0.035	1.3	1.6	0.68	s, p
Histidine	His	Essential amino acid	6274	40-150	1	2	3	0.44a	s, p
Tryptophan	Trp	Essential aromatic amino acid	6305	40-90	0.1	1.6	2.6	0.51	s, p
Kynurenine	Kyn	Trp metabolite	161166	1.0-3.3	0.12	1.9	2.3	0.68	s, p
Ornithine	Orn	Charged amino acid	6262	30-90	1	2.2	2.7	0.55a	s, p
Aspartic acid	Asp	Charged amino acid	5960	0-26	0.5	1.8	4		s, p
Glutamic acid	Glu	Charged amino acid	33032	20-140	0.5	1.7	2.7		s, p
Lysine	Lys	Essential amino acid	5962	120-290	1.2	1.4	1.9	0.54a	s, p
Alanine	Ala	Hydrophobic amino acid	5950	230-510	1	1.2	1.4	0.43a	s, p
Phenylalanine	Phe	Essential aromatic amino acid	6140	26-85	0.5	1.5	3	0.44a	s, p
Isoleucine	Ile	Essential BCAA**	6306	40-140	0.5	1.5	1.8	0.53a	s, p
Leucine	Leu	Essential BCAA**	6106	70-170	0.5	3.4	4	0.52a	s, p
Proline	Pro	Hydrophobic amino acid	145742	110-360	0.5	2.8	3.7	0.71a	s, p
Valine	Val	Essential BCAA**	6287	150-350	0.5	2.2	2.7	0.38a	s, p
Asparagine	Asn	Polar amino acid	6267	20-130	0.6	3	3	0.40a	s, p
Glutamine	Gln	Polar amino acid	5961	390-700	10	2.1	2.7	0.42a	s, p
Threonine	Thr	Essential amino acid	6288	70-240	1.2	2.4	3.3	0.44a	s, p
Tyrosine	Tyr	Aromatic amino acid	6057	40-110	0.5	2.1	2.8	0.55a	s, p
α-Hydroxyglutaric acid	aHG	α-Hydroxy acid	43	0.15-2	0.10	4.2	7.0		s, p
3-Hydroxyisobutyrate	3HIB	Val metabolite	440873	15-25	3	2.4	2.9		s, p
2-Hydroxybutyrate	aHB	α-Hydroxy acid	11266	10-60	3	1.7	2.3		s, p
3-Hydroxybutyrate	bHB	Keton body	441	20-700	3	1.9	3.4		s, p
Acetoacetate	AcAc	Keton body	96	10-200	5	5	5		s, p
Carboxymethyllysine	CML	an AGE***	123800	0.05-0.3	0.03	4.3	4.5		s, p
Carboxyethyllysine	CEL	an AGE***	23400779	0.05-0.4	0.03	4.1	4.4		s, p
2-Aminoadipic acid	2AAA	Lys metabolite	469	0.3-2.0	0.2	4.8	6.9		s, p
β-Aminoisobutyrate	BAIBA	Val metabolite	64956	0.3-2.0	0.2	2.5	3.7		s, p
β-Alanine	bAla	β-Amino acid	239	0.5-10	0.3	3	4.3		s, p
Citrate	Cit	Tricarboxylic acid	311	90-200	4	3	3.1		s, p
Isocitrate	iCit	Tricarboxylic acid	1198	2-22	0.3	2.8	3		s, p, csf
α-Ketoglutarate	aKG	Keto acid	51	2-40	0.5	3.7	3.7		s, p, csf
Fumarate	Fum	Dicarboxylic acid	444972	0.5-4	0.2	1.7	5.2		s, p, csf
Malate	Mal	β-Hydroxy acid	525	2-21	0.4	2.6	2.6		s, p, csf
Pyruvate	Pyr	α-Keto acid	1060	80-160	5	6	6.5		s, p, csf
Lactate	Lac	α-Hydroxy acid	612	500-1600	10	4.6	4.8		s, p, csf

*Sarcosine cannot be measured in EDTA-tubes from some suppliers because of the presence of sarcosine in these tubes; **BCAA, Branched chain amino acid; ***AGE, Advanced glycation end product. 
^aXiaofei Yin, Orla Prendiville, Aoife E. McNamara, and Lorraine Brennan. Targeted Metabolomic Approach to Assess the Reproducibility of
Plasma Metabolites over a Four Month Period in a Free-Living Population. J. Proteome Res. 2022, 21, 683−690.

Choline derivatives. Charged, methylated or sulfur amino acids. Short- and medium-chain acylcarnitines.
Volume: 35 µL

All concentrations are reported as µmol/L; CVs (%) shown as within-day (Wtn-day) and between-day (Btw-day) variations; sample matrices: serum (s), plasma (p), urine (u).

Metabolites	Abbr	Category	PubChem	Reported	LOD	CV Wtn	CV Btw	ICC	Matrix	Method
Choline	Chol	Quarternary ammonium	305	5–15	0.5	2.8	3.2	0.36	s, p, u
Trimethylamine N-oxide	TMAO	Amine oxide	1145	0.5–60	0.1	4	4.1	<0.38	s, p, u
Betaine	Bet	Methylated amino acid	247	15–60	0.5	3.8	4.5	0.65	s, p, u
Dimethylglycine	DMG	Methylated amino acid	673	1.5–5	0.25	5.5	5.9	0.64	s, p, u
Total homocysteine	tHcy	Amino acid	91552	<15	0.1	1	2	0.72	s, p, u
Creatine	Crn	α-Amino acid	586	10–100	1	3.9	4		s, p, u
Creatinine	Creat	α-Amino acid	588	50–100	0.25	2.6	3.1	0.79	s, p, u
Methionine	Met	Essential amino acid	6137	18–50	1	4.1	4.5	0.33	s, p
Methionine sulfoxide	MetSo	Polar amino acid	158980	0.5–5	0.06	7.1	7.9	0.36	s, p
Total cysteine	tCys	Sulfur amino acid	5862	150–400	0.1	2	6	0.56	s, p
Histidine	His	Essential amino acid	6274	40–200	0.25	3.5	3.8		s, p
3-Methylhistidine	3-MH	Amino acid	92105	2.7–10	0.6	4.4	4.9		s, p, u
1-Methylhistidine	1-MH	Amino acid	64969	0.5–15	0.25	3.7	3.9		s, p, u
Citrulline	Ctn	Amino acid	9750	10–60	4	7-8	6-9		s, p
Arginine	Arg	Charged amino acid	6322	10–100	0.25	2.6	3	0.53	s, p
Asymmetric dimethylarginine	ADMA	Guanidinated amino acid	123831	0.4–1	0.08	5.2	5.5	0.53	s, p
Symmetric dimethylarginine	SDMA	Guanidinated amino acid	169148	0.3–0.9	0.08	4.8	5.2	0.62	s, p
Homoarginine	hArg	Guanidinated amino acid	9085	1–6	0.1	5.8	6	0.65	s, p
Trimethyllysine	TML	Methylated amino acid	440121	0.3–2	0.2	4	4.4	0.15	s, p
Butyrobetaine	BB	Quarternary ammonium	725	0.4–2	0.2				s, p
Carnitine	C0	Carnitine	288	20–80	0.5			0.69a	s, p
Carnitine, total**	tC0	Carnitines		30–90	0.5				s, p
Acetylcarnitine	C2	Acylcarnitine	7045767	3–15	0.1			0.63	s, p
Propionylcarnitine	C3	Acylcarnitine	107738	0.2–.5	0.05			0.62	s, p
Butyrylcarnitine	C4	Acylcarnitine	213144	0.1–0.5	0.012				s, p
Isovalerylcarnitine	iC5	Acylcarnitine	6426851	0.06–0.2	0.05			0.68	s, p
Glutarylcarnitine	C5DC	Acylcarnitine	71464488	<0.05	0.01				s, p
Hexanoylcarnitine	C6	Acylcarnitine	6426853	0.01–1.2	0.01				s, p
Octanoylcarnitine	C8	Acylcarnitine	123701	0.06–0.3	0.05			0.41	s, p
Decanoylcarnitine	C10	Acylcarnitine	10245190	0.2–1	0.02				s, p
Dodecanoylcarnitine	C12	Acylcarnitine	168381	<0.4	0.02				s, p

*Measured in a separate assay after sample treatment with phospholipase D that does not affect the other metabolites of platform C, which are obtainable; **Measured in a separate assay involving alkaline hydrolysis of acylcarnitines.

Kynurenine pathway and B vitamins (B1, B2, B3, B6).
Volume: 60 µL

All concentrations are reported as µmol/L or nmol/L. CVs (%) shown as within-day (Wtn-day) and between-day (Btw-day) variations; sample matrices: serum (s), plasma (p), urine (u), cerebrospinal fluid (csf).

Metabolites	Abbr	Category	PubChem	Reported	LOD	CV Wtn	CV Btw	ICC	Matrix	Method
Pyridoxal 5-phosphate	PLP	B6 vitamer	1051	15–150 nmol//L	0.2	3.5	5.7	0.7	s, p
	PLP			7–70 nmol					csf
Pyridoxal	PL	B6 vitamer	1050	5–150 nmol/L	0.2	3.5	5.1	0.62	s, p
	PL			10–50 nmol/L					csf
4-Pyridoxic acid	PA	B6 vitamer	6723	10–200 nmol/L	0.5	3.6	5.8	0.58	s, p
	PA			0–15 nmol/L					csf
Pyridoxine	PN	B6 vitamer	1054	Exogenous metabolite, reflecting B vitamin supplement use	1	4.5	5.2		s, p
Thiamine	Thi	B1 vitamer	1130	2–50 nmol/L	0.5	3.5	4.9		s, p
	Thi			5–40 nmol/L					csf
Thiamine monophosphate	TMP	B1 vitamer	1131	2–15 nmol/L	0.5	7.1	11.6		s, p
	TMP			25–75 µmol/L					csf
Riboflavin	Ribo	B2 vitamer	493570	5–100 nmol/L	0.2	6	7.1	0.79	s, p
	Ribo			5–25 nmol/L					csf
Flavin mononucleotide	FMN	B2 vitamer	643976	3–30 nmol/L	0.4	5.7	8.1	0.69	s, p
	FMN			1–2 nmol/L					csf
Cystathionine	Cysta	Thioether	439258	70–550 nmol/L	0.02	3.4	4.5	0.63	s, p
Neopterin	Neopt	Pteridine	4455	5–50 nmol/L	0.7	5.3	6.6	0.6	s, p
	Neopt			5–50 nmol/L					csf
Tryptophan	Trp	Essential aromatic amino acid	6305	40–90 µmol/L	4	3.1	3.9	0.51	s, p
	Trp			2–7 µmol/L					csf
Kynurenine	Kyn	Trp metabolite	161166	1.0–3.3 µmol/L	0.007	3.6	5.1	0.68	s, p
	Kyn			0.02–0.12 µmol/L					csf
Kynurenic acid	KA	Trp metabolite	3845	20–100 nmol/L	0.4	4.4	5.4	0.68	s, p
	KA			0.5–15 nmol/L					csf
Quinaldic acid	Qld	Trp (KA) metabolite	7124	0.2–100 nmol/L	0.5	6-7	6-8		s, p
	Qld			0.1–10 nmol/L					csf
Anthranilic acid	AA	Trp metabolite	227	7–30 nmol/L	2	7.4	9.5	0.64	s, p
	AA			3–14 nmol/L					csf
3-Hydroxykynurenine	HK	Trp metabolite	11811	25–80 nmol/L	2	3.2	4.5	0.62	s, p
	HK			0.1–25 nmol/L					csf
Xanthurenic acid	XA	Trp metabolite	5699	2–35 nmol/L	0.5	4.8	5.7	0.58	s, p
	XA			<0.5 nmol/L					csf
3-Hydroxyanthranilic acid	HAA	Trp metabolite	86	10–80 nmol/L	2	4.6	6	0.54	s, p
	HAA			0–7 nmol/L					csf
Picolinic acid	Pic	Trp metabolite	1018	20–100 nmol/L	8	6-7	5-8		s, p
	Pic			5–100 nmol/L					csf
Quinolinic acid	QA	Trp metabolite	1066	150–700 nmol/L	1.6	7-8	7-10		s, p
	QA			10–150 nmol/L					csf
Nicotinic acid	NA	B3 vitamer	938	<20 nmol/L	10	4	5.4		s, p
Nicotinamide	NAM	B3 vitamer	936	100–600 nmol/L	20	4.2	6.1		s, p
	NAM			5–200 nmol/L					csf
N1-methylnicotinamide	mNAM	B3 vitamer	457	20–250 nmol/L	5	4	5.5		s, p
	mNAM			5–100 nmol/L					csf
Cotinine	Cot	Nicotine metabolite	854019	Non-smokers < ~5 nmol/L; passive smokers 5 – 85 nmol/L; smokers > 85nmol/L; heavy smokers (>25 cigarettes/day) > 1700 nmol/L	1	3	4.2	0.92	s, p, u
Trans-3-hydroxycotinine	OHCot	Nicotine metabolite	107963	Concentrations relative to cotinine, with OHCot/cotininine ratio ranging from 0.02-0.90	2	3.6	4.5	0.85	s, p, u
Trigonelline	Trig	Alkaloid	5570	0.4–25 µmol/L	0.01	2.9	3.8	0.66	s, p
Trimethylamine N-oxide	TMAO	Amine oxide	1145	0.5–60 µmol/L	0.1	4.2	6.9	<0.38	s, p, u
	TMAO			0.2–12 µmol/L					csf
Succinate	Suc	Dicarboxylic acid	1110	2–12 µmol/L					s, p
Imidazole propionate	ImP	Histidine metabolite	70630	3–500 nmol/L	2				s, p
3-Indoxyl sulfate	3IS	Indole, Trp metabolite	10258	0.9–30 µmol/L	0.2				s, p
Indole-3-acetamide	IAM	Indole, Trp metabolite	397	0–10 nmol/L	1				s, p
Indole-3-acetate	IAA	Indole, Trp metabolite	802	0.3–23 µmol/L	0.05				s, p
Indole-3-aldehyde	IAld	Indole, Trp metabolite	10256	10–200 nmol/L	4				s, p
Indole-3-lactate	ILA	Indole, Trp metabolite	92904	0.08–5 µmol/L	0.025				s, p
Indole-3-propionate	IPA	Indole, Trp metabolite	3744	0.5–12 µmol/L	0.04				s, p

Folate species and catabolites.
LC-MS/MS: 60 µL vol.

All concentrations are reported as nmol/L. CVs (%) shown as within-day (Wtn-day) and between-day (Btw-day) variations; sample matrices: serum (s), plasma (p).

Metabolites	Abbr	Category	PubChem	Reported	LOD	CV Wtn	CV Btw	Matrix	Method
5-Methyl-tetrahydrofolate	mTHF	Folate	439234	>7.5	0.07	5-9	4-5	s, p
5-Formyl-tetrahydrofolate	fTHF	Folate	6006	0-2	0.52	8-9	6-11	s, p
Folic acid	FA	Folate	6037	0-2	0.27	7-8	6-12	s, p
Serum folate as pABG equivalents	S pABG	Folate		>7.5				s, p
RBC folate as pABG equivalents	E pABG	Folate		>340				e
Para-aminobenzoylglutamate	pABG	Folate catabolite	196473	0-0.5	0.08	6-9	3-10	s, p
Acetamidobenzoylglutamate	apABG	Folate catabolite	3080868	0.1-1	0.13	5-6	5-9	s, p
4-α-hydroxy-5-methyl-THF	hmTHF	Oxidized folate			0.2	6-10	6-7	s, p

Folate (B9) and cobalamin (B12).
Microbiological assay: 15-35 µL vol.

All concentrations are reported as nmol/L or pmol/L.

Metabolites	Abbr	Category	Reported	LOD	CV Wtn	CV Btw	ICC	Matrix	Method
Serum folate	s-Folate	B vitamin	>7.5 nmol/L	2	4	5	0.56	s, p
Erythrocyte folate	e-Folate	B vitamin	>340 nmol/L					e
Serum cobalamin	s-B12	B vitamin	>150 pmol/L	30	4	5	0.82	s, p

Protein biomarkers and variants.
MALDI-TOF MS: 30 µL vol.

All concentrations are reported as µmol/L or %.

Proteins	Abbr	Category	UniProt	Proteoforms	Reported	LOD	CV Wtn	CV Btw	ICC**	Matrix	Method
C-reactive protein	CRP	Protein	P02741	1	0-10 µg/mL***	0.1	4-5	7-9	0.5-0.7	s, p
Serum amyloid A	SAA	Protein	P0DJI8, P0DJI9	18	0-20 µg/mL	0.05	4-5	8-10	0.4-0.7	s, p
Calprotectin	S100A	Protein	P05109, P06702	6*	0-30 µg/mL	0.02	4-7	7-8	0.3-0.5	s, p, csf
Cystatin C	CnC	Protein	P01034	5*	0-8 µg/mL	0.01	4-5	4-5	0.7	s, p, csf
Glycated hemoglobin	HbA1c	Protein	P69905, F8W6P5		4-14 %		2-6			w

*Mass interference of S100A and CnC proteoforms; **ICCs of proteoforms can be found here.

Lipid-soluble vitamins and long-chain acylcarnitines.
LC-MS/MS: 50-100 µL vol.

All concentrations are reported as µmol/L or nmol/L.

Metabolites	Abbr	Category	PubChem	Reported	LOD	CV Wtn	CV Btw	ICC	Matrix	Method
All-trans retinol	Vit. A	A vitamer	445354	0,3-8 µmol/L	0.1	2.9	3.6	0.87	s, p
25-hydroxy vitamin D2	25OH-D2	D vitamer	5710148	0-60 nmol/L	5	6.6	8.1		s, p
25-hydroxy vitamin D3	25OH-D3	D vitamer	5283731	10-280 nmol/L	3.3	5.1	6.1	0.71	s, p
α-tocopherol	aToc	E vitamer	14985	11-106 µmol/L	2	4.3	5.8	0.86	s, p
γ-tocopherol	gToc	E vitamer	92729	0,5-11 µmol/L	0.1	3.7	5		s, p
Phylloquinone	Vit. K1	K vitamer	5284607	0,5-20 nmol/L	0.5	4.8	7.4	0.49	s, p
Myristoylcarnitine	C14	Acylcarnitine	6426854	10-216 nmol/L				0.38a	s, p
Palmitoylcarnitine	C16	Acylcarnitine	461	40-310 nmol/L				0.49a	s, p
Stearoylcarnitine	C18	Acylcarnitine	52922056	17-100 nmol/L				0.40a	s, p
Oleoylcarnitine	C18:1	Acylcarnitine	46907933	40-412 nmol/L				0.66b	s, p
Linoleylcarnitine	C18:2	Acylcarnitine	6450015	10-297 nmol/L					s, p

^aFloegel A, Drogan D, Wang-Sattler R, Prehn C, Illig T, et al. (2011) Reliability of Serum Metabolite Concentrations over a 4-Month Period Using a
Targeted Metabolomic Approach. PLoS ONE 6(6): e21103. doi:10.1371/journal.pone.0021103
^bXiaofei Yin, Orla Prendiville, Aoife E. McNamara, and Lorraine Brennan. Targeted Metabolomic Approach to Assess the Reproducibility of
Plasma Metabolites over a Four Month Period in a Free-Living Population. J. Proteome Res. 2022, 21, 683−690.

This platform is under development.
Short-, medium- and long-chain acylcarnitines.
LC (HILIC)-MS/MS.

All concentrations are reported as µmol/L.

^aFloegel A, Drogan D, Wang-Sattler R, Prehn C, Illig T, et al. (2011) Reliability of Serum Metabolite Concentrations over a 4-Month Period Using a
Targeted Metabolomic Approach. PLoS ONE 6(6): e21103. doi:10.1371/journal.pone.0021103
^bXiaofei Yin, Orla Prendiville, Aoife E. McNamara, and Lorraine Brennan. Targeted Metabolomic Approach to Assess the Reproducibility of
Plasma Metabolites over a Four Month Period in a Free-Living Population. J. Proteome Res. 2022, 21, 683−690.