Methods & Platforms

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 analytes.*

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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 CID	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 CID	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	0.5	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 CID	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	0.62^c	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.
^cXiaofei 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.

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 CID	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	0.12	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	0.5	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 CID	Reported conc.	LOD	CV Wtn	CV Btw	Matrix
5-Methyl-tetrahydrofolate	mTHF	Folate	135398561	>7.5	0.07	5-9	4-5	s, p
5-Formyl-tetrahydrofolate	fTHF	Folate	135403648	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 CID	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 CID	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

NAD metabolome.

Sample volume required: 50 µL
LC (HILIC)-MS/MS

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

Metabolites	Abbr	PubChem CID	Reported conc.	LOD	CV Wtn	CV Btw	Matrix
N1-methylnicotinamide	mNAM	457	0.1-3	0.01	0.9	1.5	w
Nicotinamide	NAM	936	5-25	0.025	3.5	5.6	w
Nicotinamide adenine dinucleotide (oxidized)	NAD	5893	10-50	0.25	2.6	2.4	w
Nicotinamide adenine dinucleotide (reduced)	NADH	439153	0.5-3	0.25	2.1	3.1	w
Nicotinamide adenine dinucleotide phosphate (oxidized)	NADP	5886	10-40	0.25	10.2	10.9	w
Nicotinamide adenine dinucleotide phosphate (reduced)	NADPH	5885	1-4	0.25	8.0	10.1	w
Nicotinamide mononucleotide	NMN	14180	2-10	0.2	2.9	5.4	w
Nicotinamide N-oxide	NAMO	72661	0.005-1.5	0.01	2.3	4.5	w
Nicotinamide riboside	NR	439924	0-9		5.6	2.9	w
Nicotinic acid	NA	938	0-0.6	0.025	1.5	2.5	w
Nicotinic acid adenine dinucleotide	NAAD	165491	0.005-1	0.015	8.3	11.4	w
Nicotinic acid riboside	NAR	161233	3-15	0.01	2.4	2.7	w
N-methyl-2-pyridone-5-carboxamide	m2PY	69698	0-20	0.075	1.7	2.7	w
N-methyl-4-pyridone-3-carboxamide	m4PY	440810		0.0025	1.8	2.8	w

Bile acids.

Sample volume required: 50 µL
LC-MS/MS

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

Metabolites	Abbr	PubChem CID	Matrix
7α-hydroxy-cholesten-3-one	C4	123743	s, p
Chenodeoxycholic acid	CDCA	10133	s, p
Cholic acid	CA	221493	s, p
Deoxycholic acid	DCA	222528	s, p
Glycochenodeoxycholic acid	GCDCA	12544	s, p
Glycocholic acid	GCA	10140	s, p
Glycodeoxycholic acid	GDCA	3035026	s, p
Glycohyocholic acid	GHCA	71361462	s, p
Glycohyodeoxycholic acid	GHDCA	114611	s, p
Glycolithocholic acid	GLCA	115245	s, p
Glycoursodeoxycholic acid	GUDCA	12310288	s, p
Hyocholic acid	HCA	131750324	s, p
Hyodeoxycholic acid	HDCA	5283820	s, p
Iso-litocholic acid	isoLCA	164853	s, p
Lithocholic acid	LCA	9903	s, p
Lithocholic acid sulfate	LCAsulf	451489	s, p
Tauro-alfa-muricholic acid	TaMCA	101657566	s, p
Taurochenodeoxycholic acid	TCDCA	387316	s, p
Taurocholic acid	TCA	6675	s, p
Taurodeoxycholic acid	TDCA	2733768	s, p
Taurohyocholic acid	THCA	11954195	s, p
Taurohyodeoxycholic acid	THDCA	119046	s, p
Taurolithocholic acid	TLCA	439763	s, p
Tauroursodeoxycholic acid	TUDCA	9848818	s, p
Ursodeoxycholic acid	UDCA	31401	s, p

Short-chain fatty acids.
Sample volume required: 50 µL
GC-MS/MS

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 CID	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 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 CID	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	0.5	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.
Sample volume required: 35 µL
LC-MS/MS

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 CID	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	0.62^c	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

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

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; 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 CID	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	0.12	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	0.5	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