Urinary biomarkers of smokers’ exposure to tobacco smoke constituents in tobacco products assessment: a fit for purpose approach

Figures & data

Table 1. Smoke constituents and their biomarkers in urine: assay validation parameters summary.

Smoke constituent	Biomarker	Analytical technique*	Limit of detection [in matrix] (Ref)	Lower limit of quantification (Ref)	Storage studies in matrix temp. duration (Ref)	Precision‡ (Ref)	Accuracy (Ref)	Recovery (Ref)
NNK†	Total NNAL†	GC-TEA	0.04 pmol/ml [8.4 pg/ml] (Yuan et al., 2009) 0.1 pmol/ml [20.9 pg/ml] (Church et al., 2010a)		−20 °C 4 years (Yuan et al., 2009)	10.9% intra-day RSD (Yuan et al., 2009) 6.4% (Church et al., 2010a)	>95% (Church et al., 2010a)
		LC-MS/MS	2.0 pg/ml (Xia et al., 2005), (Kavvadias et al., 2009b)	5.0 pg/ml (Kavvadias et al., 2009b) 20.0 pg/ml (Bhat et al., 2011)	21 °C 24 h, 10 °C 6 days, −20 °C 8 months (Kavvadias et al., 2009b) Freeze–thaw 3 cycles (Kavvadias et al., 2009b)	6.0–11.9% (Kavvadias et al., 2009b)	94.1–103.0% (Bhat et al., 2011) 100.4–110.7% (Kavvadias et al., 2009b)	30.3–31.7% (Shah et al., 2009) 32.1–45.5% (Kavvadias et al., 2009b)
NNN†	NNN	GC-TEA	5.7 pg/ml (Stepanov & Hecht, 2005)
		LC-MS/MS	0.8 pg/ml (Kavvadias et al., 2009b)	2.0 pg/ml (Kavvadias et al., 2009b)	21 °C 24  h, 10 °C 6 days, −20 °C 8 months (Kavvadias et al., 2009b) Freeze–thaw 3 cycles (Kavvadias et al., 2009b)	1.9–4.4% (Kavvadias et al., 2009b)	90.3–91.1% (Kavvadias et al., 2009b)	37.9–56.8% (Kavvadias et al., 2009b)
NAT†	Total NAT	GC-TEA	2.65 pg/ml (Stepanov & Hecht, 2005)
		LC-MS/MS	0.7 pg/ml (Kavvadias et al., 2009b)	2.0 pg/ml (Kavvadias et al., 2009b)	21 °C 24 h, 10 °C 6 days, −20 °C 8 months (Kavvadias et al., 2009b) Freeze–thaw 3 cycles (Kavvadias et al., 2009b)	1.4–13.2% (Kavvadias et al., 2009b)	93.5–103.9% (Kavvadias et al., 2009b)	46.3–77.4% (Kavvadias et al., 2009b)
NAB†	Total NAB	GC-TEA	3.44 pg/ml (Stepanov & Hecht, 2005)
		LC-MS/MS	1.1 pg/ml (Kavvadias et al., 2009b)	5.0 pg/ml (Kavvadias et al., 2009b)	21 °C 24 h, 10 °C 6 days, − 20 °C 8 months (Kavvadias et al., 2009b) Freeze–thaw 3 cycles (Kavvadias et al., 2009b)	1.1–11.7% (Kavvadias et al., 2009b)	88.2–97.2% (Kavvadias et al., 2009b)	42.0–76.3% (Kavvadias et al., 2009b)
Pyrene	1-OHP†	HPLC-FD	5 pg/ml (Lafontaine et al., 2006) 0.05 pmol/ml (Carmella et al., 2004)		−20 °C >12 month (Feng et al., 2006)	4.6% (Feng et al., 2006) 4.2% 30 replicates RSD (Carmella et al., 2004)	88.6–91.7% (Feng et al., 2006) 90% (Scherer et al., 2007a)	50% (Carmella et al., 2004)
		GC-MS	2.0–3.3 pg/ml (Suwan-ampai et al., 2009)			1. 5–5.7% (n > 2500) (Suwan-ampai et al., 2009)
Benzo[a]pyrene	3-OHBaP†	HPLC	134 pg/ml (Jongeneelen et al., 1986)
		HPLC-FD	0.05 pg/ml (Lafontaine et al., 2006)
		GC-MS	10.5 pg/ml (Suwan-ampai et al., 2009)			2.4–7.9% Inter-assay CoV (n > 2500) (Suwan-ampai et al., 2009)
		LC-MS/MS		50 fg/ml (Sarkar et al., 2010)		3.6–10.9% Inter-day CoV (Sarkar et al., 2010)	96.4--100.2% (Sarkar et al., 2010)
Aromatic amines	1-AN	GC-MS	0.05 pg/ml (Grimmer et al., 2000) 150 pg/ml (Weiss & Angerer, 2002)		stable on >1 freeze-thaw cycles (Weiss & Angerer, 2002)	7.9% (Grimmer et al., 2000) 2.8–8.2% Within series RSD (Weiss & Angerer, 2002)	70--90% (Weiss & Angerer, 2002)	93.9% (Grimmer et al., 2000)
	2-AN	GC-MS	0.05 pg/ml (Grimmer et al., 2000) 75 pg/ml (Weiss & Angerer, 2002) 4 pg/ml (Riedel et al., 2006)		stable on >1 freeze-thaw cycles (Weiss & Angerer, 2002)	6.8% (Grimmer et al., 2000) 2.6–6.3% Within series RSD (Weiss & Angerer, 2002)	90% (Weiss & Angerer, 2002)	92.5% (Grimmer et al., 2000)
	4-ABP	GC-MS	0.05 pg/ml (Grimmer et al., 2000) 50 pg/ml (Weiss & Angerer, 2002) 4 pg/ml (Riedel et al., 2006)		stable on >1 freeze–thaw cycles (Weiss & Angerer, 2002)	3.3% (Grimmer et al., 2000) 2.7–5.9% Within series RSD (Weiss & Angerer, 2002)	100--108% (Weiss & Angerer, 2002)	96.8% (Grimmer et al., 2000)
		GC-MS/MS		0.9 pg/ml (Seyler & Bernert, 2011)
	o-tol	GC-MS	50 pg/ml (Weiss & Angerer, 2002) 1 pg/ml (Riedel et al., 2006)		stable on >1 freeze-thaw cycles (Weiss & Angerer, 2002)	7.0–7.3% Within series RSD (Weiss & Angerer, 2002)	95–106% (Weiss & Angerer, 2002)	90% (Weiss & Angerer, 2002)
1,3-Butadiene	DHBMA†	GC-MS/MS	5 ng/ml (van Sittert et al., 2000)
		LC-MS/MS	76 ng/ml (Urban et al., 2003)	10 ng/ml (Schettgen et al., 2009)	−24 °C 9 months (Urban et al., 2003)
		HPLC-MS/MS	12 pmol/ml (Carmella et al., 2009) 0.14 ng/ml (Ding et al., 2009)		10 Freeze–thaw cycles (Ding et al., 2009)	8.7% (Carmella et al., 2009) 3.5–5.3% (Ding et al., 2009)	109% (Carmella et al., 2009) 103% (Ding et al., 2009)
	MHBMA†	GC-MS/MS	0.1 ng/ml (van Sittert et al., 2000)
		LC-MS/MS	2.7 ng/ml (Urban et al., 2003)	2.0 ng/ml (Schettgen et al., 2009)	−24 °C 9 months (Urban et al., 2003)
		HPLC-MS/MS	3.2 pmol/ml (Carmella et al., 2009) 0.05 ng/ml (Ding et al., 2009)		10 Freeze–thaw cycles (Ding et al., 2009)	16.0% (Carmella et al., 2009) 1.7–2.5% (Ding et al., 2009)	58.9% (Carmella et al., 2009) 100–106% (Ding et al., 2009)
	1-MHBMA	UPLC-HILIC-MS/MS	0.05 ng/ml (Sterz et al., 2012)	0.15 ng/ml (Sterz et al., 2012)		2.7-6–1% (Sterz et al., 2012)	98–103% (Sterz et al., 2012)
	2-MHBMA	UPLC-HILIC-MS/MS	0.24 ng/ml (Sterz et al., 2012)	0.72 ng/ml (Sterz et al., 2012)		3.9–7.4%(Sterz et al., 2012)	85.3–107% (Sterz et al., 2012)
	THBMA†	HPLC-MS/MS	<0.1 ng/ml (Kotapati et al., 2011)	1.0 ng/ml (Kotapati et al., 2011)	3 Freeze–thaw cycles (Kotapati et al., 2011)	0.9–6.5% Intraday RSD (Kotapati et al., 2011)	96.9–113% (Kotapati et al., 2011)	54.5% (Kotapati et al., 2011)
Benzene	tt-MA†	GC-MS	3 ng/ml (Scherer et al., 2007a), (Ruppert et al., 1995)		−20 °C >12 month (Feng et al., 2006)	3% (Feng et al., 2006)	92% (Scherer et al., 2007a)	83–92% (Feng et al., 2006)
	SPMA†	GC-MS	0.03 pg/ml (Kim et al., 2006)
		HPLC-MS/MS	0.03 ng/ml (Scherer et al., 2007a) 0.013 pmol/ml (Carmella et al., 2009) 0.01 ng/ml (Feng et al., 2006) (Ding et al., 2009)		−20 °C >12 month (Feng et al., 2006) 10 Freeze–thaw cycles (Ding et al., 2009)	<5% (Scherer et al., 2007a) 7.6% (Carmella et al., 2009) 2.8–7.5% (Ding et al., 2009)	96.2% (Carmella et al., 2009) 101–103% (Ding et al., 2009)	97.6–100.4% (Feng et al., 2006)
Catechol	catechol	GC-MS	< matrix background (Kerzic et al., 2010), (Waidyanatha et al., 2004)	< matrix background (Kerzic et al., 2010), (Waidyanatha et al., 2004)		10.5% (urine) (Waidyanatha et al., 2004) 4.6 % (serum) (Kerzic et al., 2010)
Acrolein	3-HPMA†	LC-MS/MS	50 ng/ml (Mascher et al., 2001)	0.9 ng/ml (Carmella et al., 2007)		1.6% Six measurements RSD (Carmella et al., 2007)	125% (Carmella et al., 2007)	Circa 40% (Carmella et al., 2007)
		LC-MS/MS	6.0 ng/ml (Scherer et al., 2007a)			<2.5% (Scherer et al., 2007a)
		HPLC-MS/MS	2.3 pmol/ml (Carmella et al., 2009) 0.2 ng/ml (Ding et al., 2009)		10 Freeze–thaw cycles (Ding et al., 2009)	9.4% (Carmella et al., 2009) 2.6–3.0% (Ding et al., 2009)	100% (Carmella et al., 2009) 95–109% (Ding et al., 2009)
Crotonaldehyde	HMPMA†	LC-MS/MS	28 ng/ml (Scherer et al., 2007b)	92 ng/ml (Scherer et al., 2007b)		1.8–3.5% (Scherer et al., 2007b)	105–8% (Scherer et al., 2007b)
	CMEMA†	LC-MS/MS	32 ng/ml (Scherer et al., 2007b)	104 ng/ml (Scherer et al., 2007b)		4.6–7.3% (Scherer et al., 2007b)	101–2% (Scherer et al., 2007b)
Acetaldehyde	Mainly DNA adducts
Formaldehyde	Mainly DNA adducts
Ethylene Oxide	HEMA†	HPLC-MS/MS	0.024 pmol/ml (Carmella et al., 2009) 0.03 ng/ml (Ding et al., 2009)		10 Freeze–thaw cycles (Ding et al., 2009)	12.1% (Carmella et al., 2009) 3.1–3.6% (Ding et al., 2009)	93.4% (Carmella et al., 2009) 103% (Ding et al., 2009)
Acrylamide	Acrylamide	HPLC-MS		0.5 ng/ml (Fuhr et al., 2006)
	Glycidamide	HPLC-MS		2.5 ng/ml (Fuhr et al., 2006)
	AAMA†	HPLC-MS/MS	0.8 ng/ml (Urban et al., 2006)	5 ng/ml (Fuhr et al., 2006)		1.3–1.6% (Urban et al., 2006)	98.2–99.5% (Urban et al., 2006)	83.5% (Urban et al., 2006)
	GAMA†	HPLC-MS/MS	0.5 ng/ml (Urban et al., 2006)	5 ng/ml (Fuhr et al., 2006)		0.8–1.3% (Urban et al., 2006)	80.0–96.3% (Urban et al., 2006)	87.2% (Urban et al., 2006)
Acrylonitrile	CEMA†	LC-MS/MS	0.5 ng/ml (Schettgen et al., 2009) 0.06 ng/ml (Minet et al., 2011a)	1.0 ng/ml (Schettgen et al., 2009) 0.17 ng/ml (Minet et al., 2011a)		4.9% Intraday RSD (Schettgen et al., 2009) 2.0–2.6% (Minet et al., 2011a)	94–109% (Schettgen et al., 2009) 92–105% (Minet et al., 2011a)	110% (Schettgen et al., 2009)
Cadmium	Cadmium	ICP-MS	0.01 ng/ml (Huang & Yang, 1997)
		AAS	0.1 ng/ml (Paschal et al., 2000) 0.05 ng/ml (Hoffmann et al., 2000)			1–18% relative measurement error (Hoffmann et al., 2000)	100 ± 5% (Paschal et al., 2000) Between labs <10% difference (Paschal et al., 2000)

†Abbreviations: 1-AN, 1-aminonaphthalene; 1-OHP, 1-hydroxypyrene; 2-AN, 2-aminonaphthalene; 3-OHBaP, 3-hydroxy-benzo[a]pyrene; 3-HPMA, 3-hydroxypropylmercapturic acid; 4-ABP, 4-aminobiphenyl; AAMA, N-acetyl-S-(2-carbamoylethyl)-l-cysteine; CEMA, N-acetyl-S-(2-carboxyethyl)-l-cysteine; CMEMA, 2-carboxy-1-methylethylmercapturic acid; DHBMA, dihydroxybutyl mercapturic acid; GAMA, N-(R,S)-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-l-cysteine; HEMA, N-acetyl-S-(2-hydroxyethyl)-l-cysteine; HMPMA, 3-hydroxyl-methylpropylmercapturic acid; MHBMA, monohydroxybutenyl mercapturic acid; NAB, nitrosoanabasine; NAT, nitrosoanatabine; NNAL, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol; NNK, 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone; SPMA, S-phenylmercapturic acid; THBMA, trihydroxybutyl mercapturic acid; tt-MA, trans,trans-muconic acid; Total NAB, NAB + NAB-N-glucuronide measured after sample glucuronidase treatment; Total NAT, NAT + NAT-N-glucuronide, measured after sample glucuronidase treatment; Total NNAL, NNAL + NNAL-glucuronide measured after sample glucuronidase treatment.

*Techniques: AAS, atomic absorption spectroscopy; FD, fluorescence detection; GC, gas chromatography; HILIC, hydrophilic interaction chromatography; HPLC, high-performance liquid chromatography; ICP, inductively coupled plasma; LC, liquid chromatography; MS, mass spectrometry; MS/MS, tandem mass spectrometry; TEA, thermal energy analysis; UPLC, ultra-performance liquid chromatography.

‡Precision (intra-day coefficient of variation, unless stated) and Accuracy are as defined by the FDA (Food and Drug Administration, 2001) as the closeness of individual measures of an analyte when the procedure is applied repeatedly to multiple aliquots of a single homogeneous volume of biological matrix. Generally, precision was measured using pooled urine samples sometimes spiked and several replicates were measured to achieve this. Whereas accuracy was generally was measured in most papers using spiked samples at several levels. However, in some paper it is merely stated that these guidelines were followed and no information on how samples were prepared for accuracy and precision measures were described.

Table 2. Biomarker qualification summary.

Smoke constituent†	Biomarker†	Analytical technique†	Ranges in smokers (Ref)	Ranges in non-smokers (Ref)	Short-term repeatability‡ (Ref)	Long-term study (Ref)	Inter-laboratory proficiency test (Ref)
NNK	Total NNAL	GC-TEA	99.8–1420.8 ng/24 h urine (Hecht et al., 1999) 0.6–1.8 pmol NNAL/mg creatinine 0.5–6.7 pmol NNAL glucuronide/mg creatinine (Carmella et al., 1997)	0.0016–0.0094 ng/mg creatinine (Meger et al., 2000), (Anderson et al., 2003)	13.7% inter-day RSD (Yuan et al., 2009) 7.8% (Church et al., 2010a)
		LC-MS/MS	153 ± 132 pg/ml mean ± SD (Kavvadias et al., 2009b) 189.5 ± 161.1 ng/24 h mean ± SD (Lindner et al., 2011)	2.7 ± 3.1 pg/ml mean ± SD (Kavvadias et al., 2009b) 0–24.4 pg/ml (Bernert et al., 2010) 6.06 ng/24 h mean (Sarkar et al., 2008)	3.9–11.5% (Kavvadias et al., 2009b)	Statistically non-significant changes over 6 months in control group smokers (Sarkar et al., 2008)
NNN	Total NNN	GC-TEA	0.182 ± 0.12 pmol/mg creatinine mean ± SD (Stepanov & Hecht, 2005)
			8.51 pg NNN-glucuronide/mg creatinine mean (Stepanov & Hecht, 2005)
		LC-MS/MS	7.20 ± 6.2 pg/ml mean ± SD (Kavvadias et al., 2009b)	< LOD (Kavvadias et al., 2009b) 0.49 ng/24 h mean (Sarkar et al., 2008)	5.1–11.4% (Kavvadias et al., 2009b)
NAT	Total NAT	GC-TEA	0.187 ± 0.11 pmol/mg creatinine mean ± SD (Stepanov & Hecht, 2005)
		LC-MS/MS	161 ± 150.9 pg/ml mean ± SD (Kavvadias et al., 2009b)	< LOD (Kavvadias et al., 2009b) 0.04 ng/24 h mean (Sarkar et al., 2008)	2.5–8.8% (Kavvadias et al., 2009b)
NAB	Total NAB	GC-TEA	0.041 ± 0.04 pmol/mg creatinine Mean ± SD (5)
		LC-MS/MS	47.0 ± 47.5 pg/ml mean ± SD (Kavvadias et al., 2009b)	< LOD (4) 1.21 ng/24 h mean ± SD (Sarkar et al., 2008)	3.5–6.5% (Kavvadias et al., 2009b)
Pyrene	1-OHP	HPLC-FD	0.346 µg/24 h mean (Scherer et al., 2000) 0.196 ± 145 µg/24 h mean ± SD (Scherer et al., 2007a) 0.129--1.157 µg/24 h (Lafontaine et al., 2006) 1.36 ± 0.78 nmol/24 h mean ± SD (Carmella et al., 2009)	0.157 µg/24 h mean (Scherer et al., 2000) 0.101 ± 0.070 µg/24 h mean ± SD (Scherer et al., 2007a) 0.024--0.523 µg/24 h (Lafontaine et al., 2006) 1.09 ± 1.97 nmol/24 h^§ mean ± SD (Carmella et al., 2009)	4.6% (Scherer et al., 2007a) 23–130% 7-day variability RSDs (n = 13) (Carmella et al., 2004)	No seasonal effect detected (Carmella et al., 1997) 6-weeks individual RSDs 8.6–71% (n = 13) (Carmella et al., 2004)	G-EQUAS^++
		HPLC-MS/MS	291 ± 123 ng/24 h mean ± SD (Lindner et al., 2011) [LC-MS/MS]	123 ± 101 ng/24 h mean ± SD (Lindner et al., 2011) [LC-MS/MS]		Non-significant changes over 6 months in control group smokers (Sarkar et al., 2008) [LC-MS/MS]
		GC-MS	circa 150 ± 80 pg/ml mean ± SD (Suwan-ampai et al., 2009)	circa 50 ± 30 pg/ml mean ± SD (Suwan-ampai et al., 2009)	1.5–5.7% Inter-assay CoV (n > 2500) (Suwan-ampai et al., 2009)
Benzo[a] pyrene	3-OHBaP	HPLC-FD	< 40–367 pg/24 h urine (Lafontaine et al., 2006)	< 44–224 pg/24 h urine (Lafontaine et al., 2006)
		GC-MS	>50% sample <LOD (smokers & non-smokers) (Suwan-ampai et al., 2009)	>50% sample <LOD (smokers & non-smokers) (Suwan-ampai et al., 2009)	2.4–7.9% inter-assay CoV (n > 2500) (Suwan-ampai et al., 2009)
		LC-MS	193.1 ± 100.6 pg/24 h mean ± SD (Riedel et al., 2006)		3.6–10.9% (Scherer et al., 2010)
Aromatic amines	1-AN	GC-MS	506.7 ± 670.0 ng/24 h (Grimmer et al., 2000) mean ± SD	68.9 ± 105.4 ng/24 h (Grimmer et al., 2000) mean ± SD	12.6–18.6% Inter-day RSD (Weiss & Angerer, 2002)
	2-AN	GC-MS	84.5 ± 102.7 ng/24 h mean ± SD (Grimmer et al., 2000) 17.5 ± 30.9 ng/24h mean ± SD (Lindner et al., 2011) [LC-MS/MS]	120.8 ± 272.2 ng/24 h mean ± SD (Grimmer et al., 2000) 1.79 ± 3.6 ng/24 h mean ± SD (Lindner et al., 2011) [LC-MS/MS]	12.9–13.0% Inter-day RSD (Weiss & Angerer, 2002)
	4-ABP	GC-MS	78.6 ± 85.2 ng/24 h mean ± SD (Grimmer et al., 2000) 24.7 ± 75.4 ng/24 h mean ± SD (Lindner et al., 2011) [LC-MS/MS]	68.1 ± 91.5 ng/24 h mean ± SD (Grimmer et al., 2000) 2.77 ± 11.8 ng/24 h mean ± SD (Lindner et al., 2011) [LC-MS/MS]	9.8–15.1% Inter-day RSD (Weiss & Angerer, 2002)
		GC-MS/MS	8.69 (7.43–10.16) pg/mg creatinine geo mean (95% CI) (Seyler & Bernert, 2011)	1.64 (1.30--2.07) pg/mg creatinine geo mean (95% CI) (Seyler & Bernert, 2011)
	o-tol	GC-MS			10.6–14.1% Inter-day RSD (Weiss & Angerer, 2002)
		LC-MS/MS	179 ± 491 ng/24 h mean ± SD (Lindner et al., 2011)	63.5 ± 128 ng/24 h mean ± SD (Lindner et al., 2011)
1,3-Butadiene	DHBMA	LC-MS/MS	“not diff from non-smokers” (Urban et al., 2003)	“not different from smokers” (Urban et al., 2003)			G-EQUAS
		HPLC-MS/MS	1038 ± 514 nmol/24 h mean ± SD (Carmella et al., 2009)	662 ± 248 nmol/24 h^§ mean ± SD (Carmella et al., 2009)	8.7% (Carmella et al., 2009)
		LC-MS/MS	113–1830 ng/ml (Ding et al., 2009) 166–1092 μg/g creatinine (Ding et al., 2009)	ND-329 ng/ml (Ding et al., 2009) ND-582 μg/g creatinine (Ding et al., 2009)		7 month QC inter-day 12–13% (Ding et al., 2009)
	MHBMA	LC-MS/MS	ND-132 ng/ml (Ding et al., 2009) ND-59.7 μg/g creatinine (Ding et al., 2009) 3.27 ± 3.02 µg/24 h mean ± SD (Lindner et al., 2011)	ND-73.4 ng/ml (Ding et al., 2009) ND-122 μg/g creatinine (Ding et al., 2009) 0.30 ± 0.40 µg/24 h mean ± SD (Lindner et al., 2011)		7 month QC Inter-day 10–12% (Ding et al., 2009) NS changes over 6 months in control group smokers (Sarkar et al., 2008)	G-EQUAS
		HPLC-MS/MS	66.1 ± 69.4 nmol/24h mean ± SD (Carmella et al., 2009)	3.66 ± 2.41 nmol/24 h§ mean ± SD (Carmella et al., 2009)	16.0% (Carmella et al., 2009)
	THBMA	HPLC-MS/MS	21.6 ± 10.2 ng/mg creatinine mean ± SD (Kotapati et al., 2011)	13.7 ± 7.9 ng/mg creatinine mean ± SD (Kotapati et al., 2011)	1.6–7.9% Inter-day RSD (Kotapati et al., 2011)
Benzene	tt-MA	GC-MS	165 ± 126 μg/24 h mean ± SD (Scherer et al., 2007a)	113 ± 139 μg/24 h mean ± SD (Scherer et al., 2007a)	3% (Scherer et al., 2007a)		G-EQUAS
	SPMA	HPLC-MS/MS	3.20 ± 3.80 nmol/24h mean ± SD (Carmella et al., 2009) ND-37.7 ng/ml ND-18.4 μg/g creatinine (Ding et al., 2009) 4.13 ± 3.36 µg/24h mean ± SD (Lindner et al., 2011)	0.21 ± 0.21 nmol/24h§ mean ± SD (Carmella et al., 2009) ND-0.26 ng/ml ND-0.45 μg/g creatinine (Ding et al., 2009) 0.39 ± 0.27 µg/24h mean ± SD (Lindner et al., 2011)	< 5% (Scherer et al., 2007a) 7.6% (Carmella et al., 2009)	7 month QC Inter-day 8% (Ding et al., 2009) Non-significant changes over 6 months in control group smokers (Sarkar et al., 2008) [LC-MS/MS]	G-EQUAS
Catechol	catechol	GC-MS	2.07 µg/ml mean (Waidyanatha et al., 2004)	1.69 µg/ml mean (Waidyanatha et al., 2004) 1.27 (0.3–11.9) µg/ml median (range) (59) [All subjects inc smokers]
Acrolein	3-HPMA	LC-MS/MS	2.8 mg/24 h urine mean (Mascher et al., 2001) 1297 ± 1164 µg/24 h mean ±SD (Scherer et al., 2007a) 1.81 ± 1.26 mg/24 h mean ± SD (Lindner et al., 2011) 0.68, 1.13, 1.81 mg/24 h medians (1 mg, 4 mg, 10 mg products) (Minet et al., 2011a)	0.8 mg/24 h urine mean (Mascher et al., 2001) 337 ± 383 µg/24 h mean ± SD (Scherer et al., 2007a) 0.63 ± 0.80 mg/24 h mean ± SD (Lindner et al., 2011) 0.23 mg/24 h median (Minet et al., 2011a)	< 2.5% (Scherer et al., 2007a)	Non-significant changes over 6 months in control group smokers (Sarkar et al., 2008)	4 labs (Minet et al., 2011b)
			1.3 mg/24 h mean (Scherer et al., 2006)	0.3 mg/24 h urine mean (Scherer et al., 2006)
			80.9–4030 ng/ml (Ding et al., 2009) 75-3678 μg/g creatinine (Ding et al., 2009)	ND-128 ng/ml (Ding et al., 2009) ND-245 μg/g creatinine (Ding et al., 2009)		7-month QC Inter-day 8–12% (Ding et al., 2009)
		HPLC-MS/MS	10020 nmol/24 h (Carmella et al., 2009) 53.5–3795 ng/ml 104–4423 µg/g creatinine (Eckert et al., 2011)	1500 nmol/24 h^§ (Carmella et al., 2009) 32.6–2325 ng/ml 39.3–1562 µg/g creatinine (Eckert et al., 2011)	9.4% (Carmella et al., 2009) 6.7–8.5% (Eckert et al., 2011)
Croton-aldehyde	HMPMA	LC-MS/MS	2122 ± 1286 µg/24h urine mean ±SD (Scherer et al., 2007b)	752 ± 457 µg/24 h mean ± SD (Scherer et al., 2007b)	1.1–2.0% (Scherer et al., 2007b)
	CMEMA	LC-MS/MS	1970 ± 2500 µg/24h urine mean ± SD (Scherer et al., 2007b)	1071 ± 513 µg/24 h urine mean ± SD (Scherer et al., 2007b)	1.7–4.1% (Scherer et al., 2007b)
Ethylene Oxide	HEMA	HPLC-MS/MS	ND-20.8 ng/ml ND-16.0 μg/g creatinine (Ding et al., 2009) <LOD--49.4 ng/ml 1.1--67.7 µg/g creatinine (Eckert et al., 2011)	ND-1.4 ng/ml ND-1.1 μg/g creatinine (Ding et al., 2009) <LOD–5.0 ng/ml 0.6–8.1 µg/g creatinine (Eckert et al., 2011)	4.2–5.6% (Eckert et al., 2011)	7 month QC Inter-day 11–15% (Ding et al., 2009)	G-EQUAS
Acrylamide	Acrylamide	HPLC-MS
	Glycidamide,	HPLC-MS
	AAMA	HPLC-MS/MS	185.7 ± 107.8 µg/24 h mean ± SD (Urban et al., 2006)	73.1 ± 46.6 µg/24 h mean ± SD (Urban et al., 2006)	6.3–6.6% (Urban et al., 2006)		G-EQUAS
	GAMA	HPLC-MS/MS	27.6 ± 20.1 µg/24 h mean ± SD (Urban et al., 2006)	15.9 ± 13.0 µg/24 h mean ± SD (Urban et al., 2006)	2.7–5.9% (Urban et al., 2006)		G-EQUAS
Acrylonitrile	CEMA	LC-MS/MS	240 ng/ml median, 2.0–1382 ng/ml range (Schettgen et al., 2009) 75.4, 140, 187 µg/24 h medians (1 mg, 4 mg, 10 mg product smokers) (Minet et al., 2011a)	2.0 ng/ml median, <LOQ--21.3 ng/ml range (Schettgen et al., 2009) 1.1 µg/24 h median (Minet et al., 2011a)	6.6% Inter-day RSD (Schettgen et al., 2009) 2.9–5.6% (Minet et al., 2011a)		G-EQUAS
Metals	Cadmium	ICP-MS	0.59 µg/g creatinine mean (McElroy et al., 2007b)	0.30 µg/g creatinine mean (McElroy et al., 2007b)			G-EQUAS
		AAS	0.412 (0.392-0.434) ng/ml geometric mean (95% CI) (Hoffmann et al., 2000) Smokers > non-smokers at every age group >20 years (Paschal et al., 2000)	0.226 (0.216–0.236) ng/ml geometric mean (95% CI) (Hoffmann et al., 2000)	1–18% relative measurement error (Hoffmann et al., 2000)

†Abbreviations: as for Table 1.

‡This parameter is sometimes described as “inter-day precision” and we have captured many variations in its presentation. The inter-day coefficient of variation, unless stated.

§Value from Smokers 56 days after cessation (>100 half-lives).

++G-EQUAS, German External Quality Assessment Scheme (see www.g-equas.de).

Table 3. Other biomarker parameters.

Smoke constituent †	Biomarker†	Direct comparison of tobacco products or user groups? (Ref)	Half-life‡ (Ref)	Metabolic enzymes (Ref)
NNK	total NNAL	Switching study (Sarkar et al., 2008) Product yield study (Kavvadias et al., 2009a) Reduction on smoking cessation (Carmella et al., 2009)	40–45 d (Hecht et al., 1999, Hecht et al., 2004) 10–18 d (Goniewicz et al., 2009)	CYP1B1, CYP2A13, CYP1A2, HSD11D1, AKR1B10, AKR1C1, AKR1C2, AKR1C4, CBR1, DCXR, UGT1A9, UGT1A4, UGT2B7, UGT2B10, UGT2B17, MRP1, MRP2 (Leslie et al., 2001, Carmella et al., 2002, Breyer-Pfaff et al., 2004, Wiener et al., 2004, Bao et al., 2005, Lazarus et al., 2005, Martin et al., 2006, Church et al., 2010b, Chiang et al., 2011, Ter-Minassian et al., 2012)
NNN	NNN	Switching study (Sarkar et al., 2008) Product yield study (Kavvadias et al., 2009a)	45 min (Upadhyaya et al., 2002) patas monkey	CYP2A6, CYP2A13, CYP2E1, CYP3A4, UGT2B10 (Chen et al., 1980, Patten et al., 1997, Wong et al., 2005)
NAT	total NAT	Switching study (Sarkar et al., 2008) Product yield study (Kavvadias et al., 2009a)	90 min (Li et al., 2006) rabbit
NAB	total NAB	Switching study (Sarkar et al., 2008) Product yield study (Kavvadias et al., 2009a)	30 min (Li et al., 2006) rabbit
Pyrene	1-OHP	Reduction in EHC vs CC (Feng et al., 2006, Roethig et al., 2007, Frost-Pineda et al., 2008a) No statistically significant reduction on smoking cessation (Carmella et al., 2009)	6 h in 8 US smokers (St Helen et al., 2012)	CYP1A1, CYP1B1, GSTM1, GSTP1, UGT1A6, UGT1A7, UGT1A9 (Nerurkar et al., 2000, Pal et al., 2000, Luukkanen et al., 2001, Chuang & Chang, 2007)
Benzo[a]pyrene	3-OHBaP	Reduction on switching to snus versus CC and on smoking cessation (Sarkar et al., 2010)		CYP1A1, CYP1B1, CYP1A2, CYP3A4, UGT1A1, UGT1A7, UGT1A10, UGT1A8, UGT2A1, SULT1B1, EPHX1 (Kim et al., 1998, Shimada et al., 1999, Zheng et al., 2002, Lodovici et al., 2004, Shimada & Fujii-Kuriyama, 2004, Bushey et al., 2011)
Aromatic amines	1-AN			NAT1, NAT2, CYP1A2, SULTs, UGTs (Frederickson et al., 1992, Badawi et al., 1995, Kimura et al., 1999, Finel et al., 2005, Al-Zoughool & Talaska, 2006, Sarkar et al., 2006, Butler et al., 2011)
	2-AN	Reduction in EHC versus CC (Frost-Pineda et al., 2008a)
	4-ABP	Reduction on smoking restriction and on switching to smokeless product (Sarkar et al., 2010) and EHC (Frost-Pineda et al., 2008a)	15 h (Frederickson et al., 1992)
	o-tol	Reduction in EHC versus CC (Frost-Pineda et al., 2008a)
1,3-Butadiene	DHBMA	No statistically significant reduction on smoking cessation (Carmella et al., 2009)	5–9 h (van Welie et al., 1992)	CYP2E1, CYP3A4, CYP2A6, EPHX1, GSTT1, GSTM1, MPO (Seaton et al., 1995, Himmelstein et al., 1996, Nieusma et al., 1998, Fustinoni et al., 2002, Abdel-Rahman et al., 2005, Tan et al., 2010)
	MHBMA	Reduction in Test versus CC (Sarkar et al., 2008) Reduction on smoking cessation (Carmella et al., 2009)	5–9 h (van Welie et al., 1992)
	THBMA	25–50% reduction at 56 days following smoking cessation (Kotapati et al., 2011)
Benzene	tt-MA	Reduction in EHC versus CC and non-smoker versus smoker (Kim et al., 2006)	6–12 h (Ruppert et al., 1995, Kim et al., 2006)	CYP2E1, CYP2B1, CYP2F1, GSTT1, GSTM1, COMT, NQO1, EPHX1, AKR1C1, SULT1A1, UGT1A6, MPO (Rossi et al., 1999, Dougherty et al., 2008, Manini et al., 2010, Angelini et al., 2011, Mansi et al., 2012)
	SPMA	Reduction in EHC versus CC and non-smoker versus smoker (Feng et al., 2006, Roethig et al., 2007) Reduction on smoking cessation (Carmella et al., 2009)	6–12 h (Kim et al., 2006)
Catechol	Catechol		3–7 h Metabolite of benzene	[See benzene]
Acrolein	3-HPMA	Reduction in EHC versus CC and nonsmoker versus smoker (Roethig et al., 2007) Reduction on smoking cessation (Carmella et al., 2009)	5–9 h (Scherer et al., 2006)	ALDH, ALR, GSTM1, GSTP1, GSTA1, GGT1, GGT2, GGT3, NAT1, NAT2 (Berhane et al., 1994, Pal et al., 2000, Mascher et al., 2001)
Crotonaldehyde	HMPMA	Significantly reduced on cessation or switch to lower toxicant cigarette (Scherer, 2005, Scherer et al., 2007b) Reduction on smoking cessation (Carmella et al., 2009)	5–9 h (van Welie et al., 1992, Scherer et al., 2006)	GSTP1 (Pal et al., 2000)
	CMEMA	No significant change on cessation or lower toxicant cigarette (Scherer, 2005, Scherer et al., 2007b)	5–9 h (van Welie et al., 1992, Scherer et al., 2006)
Acetaldehyde				ALDH2, ALDH1B1 (Weiner & Wang, 1994, Stagos et al., 2010)
Formaldehyde				ALDH2, ADH5 (Hedberg et al., 2001, Wang et al., 2002b, Thompson et al., 2010)
Ethylene oxide	HEMA	Reduction on smoking cessation (Carmella et al., 2009) Significantly reduced on cessation or switch to lower toxicant cigarette (Scherer, 2005, Scherer et al., 2010)	<5 h (Haufroid et al., 2007)	GSTT1 (Muller et al., 1998, Fennell et al., 2000)
Acrylamide	Acrylamide		2.4 h (Fuhr et al., 2006)	CYP2E1, EPHX1, CYPs (Doroshyenko et al., 2009, Huang et al., 2011a, Huang et al., 2011b)
	Glycidamide,
	AAMA		17.4 h (Fuhr et al., 2006)
	GAMA		25.4 h (Fuhr et al., 2006)
Acrylonitrile	CEMA	Significantly reduced on cessation or switch to lower toxicant cigarette (Scherer, 2005, Scherer et al., 2010)		CYP2E1, GSTP1 (Kedderis et al., 1993, Thier et al., 2001, Thier et al., 2002, Wang et al., 2002a, Suhua et al., 2010)
Metals	Cadmium		Months (Huang & Yang, 1997, Hoffmann et al., 2000, Paschal et al., 2000, McElroy et al., 2007a, McElroy et al., 2007b)

†Abbreviations as in Table 1. CC, conventional cigarette; EHC, electrically heated cigarette.

‡Approximate half-life based on elimination from body fluid or measurement in urine.

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