Methods

Key data of our analytical platforms are described below and include PubChem numbers, reported biomarker concentrations, LODs, CVs, ICCs, sample matrix and method description for most of our biomarkers.*

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Analytical platforms

Short-chain fatty acids.

Sample volume required: 50 µL
GC-MS/MS

All concentrations are reported in µmol/L.

Metabolites	Abbr	Class	PubChem	Reported conc.	LOD	CV Wtn	CV Btw	Matrix
Acetate	Ace	Short-chain fatty acid	175-176	5-300	5.0	3-5	3-7	s, p
Butyrate	But	Short-chain fatty acid	264	1-30	0.2	3-6	3-6	s, p
Formate	Frm	Short-chain fatty acid	284	8-100	7.0	3-6	3-6	s, p, csf
Isobutyrate	iBut	Short-chain fatty acid	6590	0.7-6	0.05	3-6	3-8	s, p, csf
Isovalerate	iVlr	Short-chain fatty acid	10430	0.3-3	0.02	3-6	3-8	s, p
Propionate	Pro	Short-chain fatty acid	1042	1-13	0.06	2-6	3-6	s, p
Valerate	Vlr	Short-chain fatty acid	7991	0.2-1	0.05	3-6	4-8	s, p
α-Methylbutyrate	aMB	Short-chain fatty acid	8314	0.3-3	0.05	3-7	4-7	s, p
β-Hydroxy β-methylbutyrate	HMB	Leu metabolite	69362	0.1-10, 2-6 (csf)				s, p, csf

Amino- and carboxylic acids. TCA metabolites and intermediates.

Sample volume required: 50 µL
GC-MS/MS

All concentrations are reported in µmol/L.

Metabolites	Abbr	Class	PubChem	Reported conc.	LOD	CV Wtn	CV Btw	ICC	Matrix
2-Aminoadipic acid	2AAA	Lys metabolite	469	0.3-2.0	0.2	4.8	6.9		s, p
2-Hydroxybutyrate	aHB	α-Hydroxy acid	11266	10-60	1	1.7	2.3		s, p
3-Hydroxybutyrate	bHB	Ketone body	441	20-700	1	1.9	3.4		s, p
3-Hydroxyisobutyrate	3HIB	Val metabolite	440873	15-25	1	2.4	2.9		s, p
Acetoacetate	AcAc	Ketone body	96	10-200	5	5	5		s, p
Alanine	Ala	Hydrophobic amino acid	5950	230-510	1	1.2	1.4	0.43^a	s, p
Asparagine	Asn	Polar amino acid	6267	20-130	0.6	3	3	0.40^a	s, p
Aspartic acid	Asp	Charged amino acid	5960	0-26	0.5	1.8	4		s, p
Carboxyethyllysine	CEL	an AGE***	23400779	0.05-0.4	0.03	4.1	4.4		s, p
Carboxymethyllysine	CML	an AGE***	123800	0.05-0.3	0.03	4.3	4.5		s, p
Citrate	Cit	Tricarboxylic acid	311	90-200	4	3	3.1		s, p
Cystathionine	Cysta	Thioether	439258	0.07-0.55	0.02	2.3	2.9	0.63	s, p
Fumarate	Fum	Dicarboxylic acid	444972	0.5-4	0.2	1.7	5.2		s, p, csf
Glutamic acid	Glu	Charged amino acid	33032	20-140	0.5	1.7	2.7		s, p
Glutamine	Gln	Polar amino acid	5961	390-700	10	2.1	2.7	0.42^a	s, p
Glycine	Gly	Hydrophobic amino acid	750	150-350	3	1.3	1.7	0.81	s, p
Histidine	His	Essential amino acid	6274	40-150	1	2	3	0.44^a	s, p
Isocitrate	iCit	Tricarboxylic acid	1198	2-22	0.3	2.8	3		s, p, csf
Isoleucine	Ile	Essential BCAA**	6306	40-140	0.5	1.5	1.8	0.53^a	s, p
Kynurenine	Kyn	Trp metabolite	161166	1.0-3.3	0.12	1.9	2.3	0.68	s, p
Lactate	Lac	α-Hydroxy acid	612	500-1600	10	4.6	4.8		s, p, csf
Leucine	Leu	Essential BCAA**	6106	70-170	0.5	3.4	4	0.52^a	s, p
Lysine	Lys	Essential amino acid	5962	120-290	1.2	1.4	1.9	0.54^a	s, p
Malate	Mal	β-Hydroxy acid	525	2-21	0.4	2.6	2.6		s, p, csf
Methionine	Met	Essential amino acid	6137	18-50	1	2.2	4.1	0.33	s, p
Methylmalonic acid	MMA	Carboxylic acid	487	<0.26	0.03	2.4	3	0.81	s, p
Ornithine	Orn	Charged amino acid	6262	30-90	1	2.2	2.7	0.55^a	s, p
Phenylacetylglutamine	PAGln	n-acyl-alpha amino acid	92258	2-100	1	5-6	6-7		s, p
Phenylalanine	Phe	Essential aromatic amino acid	6140	26-85	0.5	1.5	3	0.44^a	s, p
Proline	Pro	Hydrophobic amino acid	145742	110-360	0.5	2.8	3.7	0.71^a	s, p
Pyruvate	Pyr	α-Keto acid	1060	80-160	5	6	6.5		s, p, csf
Sarcosine*	Sarc	Methylated amino acid	1088	0.7-2.3	0.035	1.3	1.6	0.68	s, p
Serine	Ser	Polar amino acid	5951	95-200	5	1.3	1.7	0.71	s, p
Threonine	Thr	Essential amino acid	6288	70-240	1.2	2.4	3.3	0.44^a	s, p
Total cysteine	tCys	Sulfur amino acid	5862	150-350	50	1	2	0.62	s, p
Total homocysteine	tHcy	Sulfur amino acid	91552	5-15	0.1	1.4	2.7	0.72	s, p
Tryptophan	Trp	Essential aromatic amino acid	6305	40-90	0.1	1.6	2.6	0.51	s, p
Tyrosine	Tyr	Aromatic amino acid	6057	40-110	0.5	2.1	2.8	0.55^a	s, p
Valine	Val	Essential BCAA**	6287	150-350	0.5	2.2	2.7	0.38^a	s, p
α-Hydroxyglutaric acid	aHG	α-Hydroxy acid	43	0.15-2	0.10	4.2	7.0		s, p
α-Ketoglutarate	aKG	Keto acid	51	2-40	0.5	3.7	3.7		s, p, csf
β-Alanine	bAla	β-Amino acid	239	0.5-10	0.3	3	4.3		s, p
β-Aminoisobutyrate	BAIBA	Val metabolite	64956	0.3-2.0	0.2	2.5	3.7		s, p

*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.

Sample volume required: 35 µL
LC-MS/MS

All concentrations are reported in µmol/L.

Metabolites	Abbr	Class	PubChem	Reported conc.	LOD	CV Wtn	CV Btw	ICC	Matrix
1-Methylhistidine	1-MH	Amino acid	64969	0.5–15	0.25	3.7	3.9		s, p, u
3-Methylhistidine	3-MH	Amino acid	92105	2.7–10	0.6	4.4	4.9		s, p, u
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
Betaine	Bet	Methylated amino acid	247	15–60	0.5	3.8	4.5	0.65	s, p, u
Carnitine	C0	Carnitine	288	20–80	0.5			0.69^a	s, p
Carnitine, total^b	tC0	Carnitines		30–90	0.5				s, p
Choline	Chol	Quarternary ammonium	305	5–15	0.5	2.8	3.2	0.36	s, p, u
Citrulline	Ctn	Amino acid	9750	10–60	4	7-8	6-9		s, p
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
Dimethylglycine	DMG	Methylated amino acid	673	1.5–5	0.25	5.5	5.9	0.64	s, p, u
Histidine	His	Essential amino acid	6274	40–200	0.25	3.5	3.8		s, p
Homoarginine	hArg	Guanidinated amino acid	9085	1–6	0.1	5.8	6	0.65	s, p
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
Symmetric dimethylarginine	SDMA	Guanidinated amino acid	169148	0.3–0.9	0.08	4.8	5.2	0.62	s, p
Total cysteine	tCys	Sulfur amino acid	5862	150–400	0.1	2	6	0.56	s, p
Total homocysteine	tHcy	Amino acid	91552	<15	0.1	1	2	0.72	s, p, u
Trimethylamine N-oxide	TMAO	Amine oxide	1145	0.5–60	0.1	4	4.1	<0.38	s, p, u
Trimethyllysine	TML	Methylated amino acid	440121	0.3–2	0.2	4	4.4	0.15	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
^bMeasured in a separate assay involving alkaline hydrolysis of acylcarnitines.

Kynurenine pathway and B vitamins (B1, B2, B3, B6), indoles, nicotin, coffee markers, and inflammation markers.

Sample volume required: 60 µL
LC-MS/MS

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

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

Folate species and catabolites.

Sample volume required: 60 µL
LC-MS/MS

All concentrations are reported in nmol/L.

Metabolites	Abbr	Class	PubChem	Reported conc.	LOD	CV Wtn	CV Btw	Matrix
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).

Sample volume required: 15-35 µL
Microbiological assay

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

Metabolites	Abbr	Class	Reported conc.	LOD	CV Wtn	CV Btw	ICC	Matrix
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.

Sample volume required: 30 µL
MALDI-TOF MS

All concentrations are reported in µg/ml or %.

Proteins	Abbr	Class	UniProt	Proteoforms	Reported conc.	LOD	CV Wtn	CV Btw	ICC**	Matrix
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 are reported here; *** high sensitive CRP (hs-CRP)

Lipid-soluble vitamins.

Sample volume required: 50-100 µL
LC-MS/MS

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

Metabolites	Abbr	Class	PubChem	Reported conc.	LOD	CV Wtn	CV Btw	ICC	Matrix
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

Short-, medium- and long-chain acylcarnitines.

Sample volume required: 60 µL
LC (HILIC)-MS/MS

All concentrations are reported in µmol/L. Read more about acylcarnitines here.

Metabolites	Abbr	Class	PubChem	Reported conc.	LOD	CV Wtn	CV Btw	ICC	Matrix
Butyrobetaine	BB	Quarternary ammonium	725	0.2-2	0.01	4.3	2.5		s, p
Carnitine	C0	Carnitine	288	7–90	0.50	2.7	2.4	0.69	s, p
Acetylcarnitine	C2	Acylcarnitine	7045767	1–35	0.06	3.0	2.1	0.63	s, p
Propionylcarnitine	C3	Acylcarnitine	107738	0.1-1.5	0.01	3.8	1.6	0.62	s, p
Malonylcarnitine	C3DC	Acylcarnitine	91825606	0-0.5	0.05	2.8	2.4		s, p
Butyrylcarnitine	C4	Acylcarnitine	213144	0.04-1.2	0.005	3.4	3.1		s, p
Hydroxybutyrylcarnitine	C4OH	Acylcarnitine	90659885	0.01-0.5	0.01	5.3	1.6		s, p
Succinylcarnitine / Methylmalonylcarnitine	C4DC	Acylcarnitine	131802075	0.01-1	0.01	8.4	12.7		s, p
Isovalerylcarnitine	iC5	Acylcarnitine	6426851	0.04-0.5	0.01	2.7	1.8	0.68	s, p
Glutarylcarnitine	C5DC	Acylcarnitine	71464488	0.01-0.3	0.01	8.0	8.2		s, p
Methylcrotonylcarnitine / Tiglylcarnitine	C5:1	Acylcarnitine	71750254	0.005-0.1	0.005	5.0	3.7		s, p
Hexanoylcarnitine	C6	Acylcarnitine	6426853	0.005-2.1	0.005	5.6	3.8		s, p
Octanoylcarnitine	C8	Acylcarnitine	123701	0.03-1.1	0.005	3.5	3.7	0.41	s, p
Decanoylcarnitine	C10	Acylcarnitine	10245190	0.03-2.1	0.005	3.2	1.7		s, p
Dodecanoylcarnitine	C12	Acylcarnitine	168381	0.01-0.5	0.01	2.3	1.4		s, p
Tetradecanoylcarnitine (Myristoylcarnitine)	C14	Acylcarnitine	6426854	0.015-0.3	0.015	5.4	2.7	0.38	s, p
Hydroxytetradecanoylcarnitine	C14OH	Acylcarnitine	71464541	0.005-0.08	0.005	7.6	4.1		s, p
Hexadecanoylcarnitine (Palmitoylcarnitine)	C16	Acylcarnitine	461	0.04-1.3	0.01	3.1	2.6	0.49	s, p
Hydroxyhexadecanoylcarnitine	C16OH	Acylcarnitine	126456228	0-0.05	0.005	5.2	4.1		s, p
Octadecanoylcarnitine (Stearoylcarnitine)	C18	Acylcarnitine	52922056	0.01-0.5	0.006	5.0	6.6	0.4	s, p
Hydroxyoctadecanoylcarnitine	C18OH	Acylcarnitine	71464560		0.01	9.9	4.6		s, p
Octadecenoylcarnitine (Oleylcarnitine)	C18:1	Acylcarnitine	46907933	0.02-1	0.01	3.4	2.2	0.66	s, p
Octadecadienylcarnitine (Linoleylcarnitine)	C18:2	Acylcarnitine	6450015	0.02-0.3	0.01	3.5	2.4		s, p

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).

Metabolites	Abbr	Class	PubChem	Reported conc.	LOD	CV Wtn	CV Btw	Matrix
Acetate	Ace	Short-chain fatty acid	175-176	5-300	5.0	3-5	3-7	s, p
Butyrate	But	Short-chain fatty acid	264	1-30	0.2	3-6	3-6	s, p
Formate	Frm	Short-chain fatty acid	284	8-100	7.0	3-6	3-6	s, p, csf
Isobutyrate	iBut	Short-chain fatty acid	6590	0.7-6	0.05	3-6	3-8	s, p, csf
Isovalerate	iVlr	Short-chain fatty acid	10430	0.3-3	0.02	3-6	3-8	s, p
Propionate	Pro	Short-chain fatty acid	1042	1-13	0.06	2-6	3-6	s, p
Valerate	Vlr	Short-chain fatty acid	7991	0.2-1	0.05	3-6	4-8	s, p
α-Methylbutyrate	aMB	Short-chain fatty acid	8314	0.3-3	0.05	3-7	4-7	s, p
β-Hydroxy β-methylbutyrate	HMB	Leu metabolite	69362	0.1-10, 2-6 (csf)				s, p, 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	Class	PubChem	Reported conc.	LOD	CV Wtn	CV Btw	ICC	Matrix
2-Aminoadipic acid	2AAA	Lys metabolite	469	0.3-2.0	0.2	4.8	6.9		s, p
2-Hydroxybutyrate	aHB	α-Hydroxy acid	11266	10-60	1	1.7	2.3		s, p
3-Hydroxybutyrate	bHB	Ketone body	441	20-700	1	1.9	3.4		s, p
3-Hydroxyisobutyrate	3HIB	Val metabolite	440873	15-25	1	2.4	2.9		s, p
Acetoacetate	AcAc	Ketone body	96	10-200	5	5	5		s, p
Alanine	Ala	Hydrophobic amino acid	5950	230-510	1	1.2	1.4	0.43^a	s, p
Asparagine	Asn	Polar amino acid	6267	20-130	0.6	3	3	0.40^a	s, p
Aspartic acid	Asp	Charged amino acid	5960	0-26	0.5	1.8	4		s, p
Carboxyethyllysine	CEL	an AGE***	23400779	0.05-0.4	0.03	4.1	4.4		s, p
Carboxymethyllysine	CML	an AGE***	123800	0.05-0.3	0.03	4.3	4.5		s, p
Citrate	Cit	Tricarboxylic acid	311	90-200	4	3	3.1		s, p
Cystathionine	Cysta	Thioether	439258	0.07-0.55	0.02	2.3	2.9	0.63	s, p
Fumarate	Fum	Dicarboxylic acid	444972	0.5-4	0.2	1.7	5.2		s, p, csf
Glutamic acid	Glu	Charged amino acid	33032	20-140	0.5	1.7	2.7		s, p
Glutamine	Gln	Polar amino acid	5961	390-700	10	2.1	2.7	0.42^a	s, p
Glycine	Gly	Hydrophobic amino acid	750	150-350	3	1.3	1.7	0.81	s, p
Histidine	His	Essential amino acid	6274	40-150	1	2	3	0.44^a	s, p
Isocitrate	iCit	Tricarboxylic acid	1198	2-22	0.3	2.8	3		s, p, csf
Isoleucine	Ile	Essential BCAA**	6306	40-140	0.5	1.5	1.8	0.53^a	s, p
Kynurenine	Kyn	Trp metabolite	161166	1.0-3.3	0.12	1.9	2.3	0.68	s, p
Lactate	Lac	α-Hydroxy acid	612	500-1600	10	4.6	4.8		s, p, csf
Leucine	Leu	Essential BCAA**	6106	70-170	0.5	3.4	4	0.52^a	s, p
Lysine	Lys	Essential amino acid	5962	120-290	1.2	1.4	1.9	0.54^a	s, p
Malate	Mal	β-Hydroxy acid	525	2-21	0.4	2.6	2.6		s, p, csf
Methionine	Met	Essential amino acid	6137	18-50	1	2.2	4.1	0.33	s, p
Methylmalonic acid	MMA	Carboxylic acid	487	<0.26	0.03	2.4	3	0.81	s, p
Ornithine	Orn	Charged amino acid	6262	30-90	1	2.2	2.7	0.55^a	s, p
Phenylacetylglutamine	PAGln	n-acyl-alpha amino acid	92258	2-100	1	5-6	6-7		s, p
Phenylalanine	Phe	Essential aromatic amino acid	6140	26-85	0.5	1.5	3	0.44^a	s, p
Proline	Pro	Hydrophobic amino acid	145742	110-360	0.5	2.8	3.7	0.71^a	s, p
Pyruvate	Pyr	α-Keto acid	1060	80-160	5	6	6.5		s, p, csf
Sarcosine*	Sarc	Methylated amino acid	1088	0.7-2.3	0.035	1.3	1.6	0.68	s, p
Serine	Ser	Polar amino acid	5951	95-200	5	1.3	1.7	0.71	s, p
Threonine	Thr	Essential amino acid	6288	70-240	1.2	2.4	3.3	0.44^a	s, p
Total cysteine	tCys	Sulfur amino acid	5862	150-350	50	1	2	0.62	s, p
Total homocysteine	tHcy	Sulfur amino acid	91552	5-15	0.1	1.4	2.7	0.72	s, p
Tryptophan	Trp	Essential aromatic amino acid	6305	40-90	0.1	1.6	2.6	0.51	s, p
Tyrosine	Tyr	Aromatic amino acid	6057	40-110	0.5	2.1	2.8	0.55^a	s, p
Valine	Val	Essential BCAA**	6287	150-350	0.5	2.2	2.7	0.38^a	s, p
α-Hydroxyglutaric acid	aHG	α-Hydroxy acid	43	0.15-2	0.10	4.2	7.0		s, p
α-Ketoglutarate	aKG	Keto acid	51	2-40	0.5	3.7	3.7		s, p, csf
β-Alanine	bAla	β-Amino acid	239	0.5-10	0.3	3	4.3		s, p
β-Aminoisobutyrate	BAIBA	Val metabolite	64956	0.3-2.0	0.2	2.5	3.7		s, p

Choline derivatives. Charged, methylated or sulfur amino acids.
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	Class	PubChem	Reported conc.	LOD	CV Wtn	CV Btw	ICC	Matrix
1-Methylhistidine	1-MH	Amino acid	64969	0.5–15	0.25	3.7	3.9		s, p, u
3-Methylhistidine	3-MH	Amino acid	92105	2.7–10	0.6	4.4	4.9		s, p, u
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
Betaine	Bet	Methylated amino acid	247	15–60	0.5	3.8	4.5	0.65	s, p, u
Carnitine	C0	Carnitine	288	20–80	0.5			0.69^a	s, p
Carnitine, total^b	tC0	Carnitines		30–90	0.5				s, p
Choline	Chol	Quarternary ammonium	305	5–15	0.5	2.8	3.2	0.36	s, p, u
Citrulline	Ctn	Amino acid	9750	10–60	4	7-8	6-9		s, p
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
Dimethylglycine	DMG	Methylated amino acid	673	1.5–5	0.25	5.5	5.9	0.64	s, p, u
Histidine	His	Essential amino acid	6274	40–200	0.25	3.5	3.8		s, p
Homoarginine	hArg	Guanidinated amino acid	9085	1–6	0.1	5.8	6	0.65	s, p
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
Symmetric dimethylarginine	SDMA	Guanidinated amino acid	169148	0.3–0.9	0.08	4.8	5.2	0.62	s, p
Total cysteine	tCys	Sulfur amino acid	5862	150–400	0.1	2	6	0.56	s, p
Total homocysteine	tHcy	Amino acid	91552	<15	0.1	1	2	0.72	s, p, u
Trimethylamine N-oxide	TMAO	Amine oxide	1145	0.5–60	0.1	4	4.1	<0.38	s, p, u
Trimethyllysine	TML	Methylated amino acid	440121	0.3–2	0.2	4	4.4	0.15	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

^bMeasured in a separate assay involving alkaline hydrolysis of acylcarnitines.

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

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

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	Class	PubChem	Reported conc.	LOD	CV Wtn	CV Btw	Matrix
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.

Metabolites	Abbr	Class	Reported conc.	LOD	CV Wtn	CV Btw	ICC	Matrix
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.

Proteins	Abbr	Class	UniProt	Proteoforms	Reported conc.	LOD	CV Wtn	CV Btw	ICC**	Matrix
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
*** high sensitive CRP (hs-CRP)

Lipid-soluble vitamins.
LC-MS/MS: 50-100 µL vol.

Metabolites	Abbr	Class	PubChem	Reported conc.	LOD	CV Wtn	CV Btw	ICC	Matrix
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

Short-, medium- and long-chain acylcarnitines.
LC (HILIC)-MS/MS.

All concentrations are reported as µmol/L.

Metabolites	Abbr	Class	PubChem	Reported conc.	LOD	CV Wtn	CV Btw	ICC	Matrix
Butyrobetaine	BB	Quarternary ammonium	725	0.2-2	0.01	4.3	2.5		s, p
Carnitine	C0	Carnitine	288	7–90	0.50	2.7	2.4	0.69	s, p
Acetylcarnitine	C2	Acylcarnitine	7045767	1–35	0.06	3.0	2.1	0.63	s, p
Propionylcarnitine	C3	Acylcarnitine	107738	0.1-1.5	0.01	3.8	1.6	0.62	s, p
Malonylcarnitine	C3DC	Acylcarnitine	91825606	0-0.5	0.05	2.8	2.4		s, p
Butyrylcarnitine	C4	Acylcarnitine	213144	0.04-1.2	0.005	3.4	3.1		s, p
Hydroxybutyrylcarnitine	C4OH	Acylcarnitine	90659885	0.01-0.5	0.01	5.3	1.6		s, p
Succinylcarnitine / Methylmalonylcarnitine	C4DC	Acylcarnitine	131802075	0.01-1	0.01	8.4	12.7		s, p
Isovalerylcarnitine	iC5	Acylcarnitine	6426851	0.04-0.5	0.01	2.7	1.8	0.68	s, p
Glutarylcarnitine	C5DC	Acylcarnitine	71464488	0.01-0.3	0.01	8.0	8.2		s, p
Methylcrotonylcarnitine / Tiglylcarnitine	C5:1	Acylcarnitine	71750254	0.005-0.1	0.005	5.0	3.7		s, p
Hexanoylcarnitine	C6	Acylcarnitine	6426853	0.005-2.1	0.005	5.6	3.8		s, p
Octanoylcarnitine	C8	Acylcarnitine	123701	0.03-1.1	0.005	3.5	3.7	0.41	s, p
Decanoylcarnitine	C10	Acylcarnitine	10245190	0.03-2.1	0.005	3.2	1.7		s, p
Dodecanoylcarnitine	C12	Acylcarnitine	168381	0.01-0.5	0.01	2.3	1.4		s, p
Tetradecanoylcarnitine (Myristoylcarnitine)	C14	Acylcarnitine	6426854	0.015-0.3	0.015	5.4	2.7	0.38	s, p
Hydroxytetradecanoylcarnitine	C14OH	Acylcarnitine	71464541	0.005-0.08	0.005	7.6	4.1		s, p
Hexadecanoylcarnitine (Palmitoylcarnitine)	C16	Acylcarnitine	461	0.04-1.3	0.01	3.1	2.6	0.49	s, p
Hydroxyhexadecanoylcarnitine	C16OH	Acylcarnitine	126456228	0-0.05	0.005	5.2	4.1		s, p
Octadecanoylcarnitine (Stearoylcarnitine)	C18	Acylcarnitine	52922056	0.01-0.5	0.006	5.0	6.6	0.4	s, p
Hydroxyoctadecanoylcarnitine	C18OH	Acylcarnitine	71464560		0.01	9.9	4.6		s, p
Octadecenoylcarnitine (Oleylcarnitine)	C18:1	Acylcarnitine	46907933	0.02-1	0.01	3.4	2.2	0.66	s, p
Octadecadienylcarnitine (Linoleylcarnitine)	C18:2	Acylcarnitine	6450015	0.02-0.3	0.01	3.5	2.4		s, p

*Multianalyte determination is carried out in a single aliquot. Volume requirement is independent of the number of metabolites. Reported concentrations are not strictly normal reference range, which depends on the population, and several factors, including age, gender, ethnicity and procedures for sample handling and analytical technology. Reported concentrations are values that we and others have observed or reported in various cohorts of healthy subjects. For some biomarkers the assay performance may be different from those given in published articles. This is due to continuous improvements of the methods. LODs, CVs, and ICCs are not available for all biomarkers.

October 30, 2024

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Methods

Analytical platforms

Short-chain fatty acids.

Amino- and carboxylic acids. TCA metabolites and intermediates.

Choline derivatives. Charged, methylated or sulfur amino acids.

Kynurenine pathway and B vitamins (B1, B2, B3, B6), indoles, nicotin, coffee markers, and inflammation markers.

Folate species and catabolites.

Folate (B9) and cobalamin (B12).

Protein biomarkers and variants.

Lipid-soluble vitamins.

Short-, medium- and long-chain acylcarnitines.

Short-chain fatty acids.
Volume: 50 µL

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

Choline derivatives. Charged, methylated or sulfur amino acids.
Volume: 35 µL

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

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

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

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

Lipid-soluble vitamins.
LC-MS/MS: 50-100 µL vol.

Short-, medium- and long-chain acylcarnitines.
LC (HILIC)-MS/MS.

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Methods

Analytical platforms

Short-chain fatty acids.

Amino- and carboxylic acids. TCA metabolites and intermediates.

Choline derivatives. Charged, methylated or sulfur amino acids.

Kynurenine pathway and B vitamins (B1, B2, B3, B6), indoles, nicotin, coffee markers, and inflammation markers.

Folate species and catabolites.

Folate (B9) and cobalamin (B12).

Protein biomarkers and variants.

Lipid-soluble vitamins.

Short-, medium- and long-chain acylcarnitines.

Short-chain fatty acids.Volume: 50 µL

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

Choline derivatives. Charged, methylated or sulfur amino acids.Volume: 35 µL

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

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

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

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

Lipid-soluble vitamins.LC-MS/MS: 50-100 µL vol.

Short-, medium- and long-chain acylcarnitines.LC (HILIC)-MS/MS.

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Short-chain fatty acids.
Volume: 50 µL

Choline derivatives. Charged, methylated or sulfur amino acids.
Volume: 35 µL

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

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

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

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

Lipid-soluble vitamins.
LC-MS/MS: 50-100 µL vol.

Short-, medium- and long-chain acylcarnitines.
LC (HILIC)-MS/MS.