A Meta-Analysis of Urinary Cotinine Cut-Off Concentrations in Children Under Five Years for Assessing Environmental Tobacco Smoke Exposure

Abstract

Background

Environmental tobacco smoke (ETS) exposure is a preventable risk factor for adverse health outcomes in early childhood. Urinary cotinine is widely used as a non-invasive biomarker of ETS exposure; however, variation in reported cut-off concentrations limits comparability across studies. No prior systematic review has quantitatively synthesised urinary cotinine cut-off values for children under five years. This review aimed to systematically summarise and evaluate these cut-off concentrations in early childhood.

Methods

A systematic review and meta-analysis were conducted in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA 2020) guidelines and a prospectively published protocol (PROSPERO CRD42024556969). PubMed, Embase, Scopus, and the Cochrane Library were searched from inception to February 2025. Observational and experimental studies reporting urinary cotinine cut-off concentrations to classify ETS exposure among children under five years were included. Study quality was assessed using the Newcastle–Ottawa Scale. Random-effects meta-analyses were performed to estimate pooled urinary cotinine cut-off concentrations for ETS exposure classification, stratified by unit of measurement (ng/mL vs ng/mg creatinine).

Results

Twelve studies were included. Reported urinary cotinine cut-off concentrations ranged from 0.05 to 30 ng/mL for unadjusted values and from 2.47 to 120 ng/mg creatinine for creatinine-adjusted values. Meta-analysis yielded pooled reference values of 5.90 ng/mL (95% CI: 5.53–6.27) and 50.36 ng/mg creatinine (95% CI: 47.53–53.20), respectively.

Conclusion

Urinary cotinine is a valid biomarker for assessing ETS in young children, but inconsistent cut-off values limit comparability. The pooled estimates may aid interpretation in surveillance and research but should not be used as universal thresholds. Standardized, age-appropriate cut-offs are needed to strengthen exposure assessment and inform child-focused tobacco control policies.

Keywords

second-hand smoke passive smoking biomarkers air pollution nicotine metabolite

Introduction

Exposure to tobacco smoke is a significant risk factor for childhood respiratory diseases, contributing to increased rates of morbidity and mortality worldwide.^1,2 The World Health Organization (WHO) estimated that about 1.2 million deaths annually is contributed by exposure to passive smoking.³ Environmental tobacco smoke (ETS), also known as second-hand smoke, consists of nearly 4,800 chemical compounds, including nicotine, polycyclic aromatic hydrocarbons, aromatic amines, and carbon monoxide. Of these substances, more than 250 are classified as toxic, with at least 70 recognized as carcinogenic.⁴ Tobacco smoke leads to 8 million deaths each year across the globe, with around 1.2 million of those deaths linked to second-hand smoke among non-smokers. Furthermore, about one-third of adult non-smokers and nearly 40% of children worldwide are exposed to ETS in their homes. Such exposure results in a range of health issues, including infections of the respiratory system, asthma, wheezing, low birth weight, orofacial clefts, cancer in children, attention-deficit/hyperactivity disorder (ADHD), as well as cognitive and language development delays.^5-7 Children are particularly susceptible to the adverse effects of ETS due to their smaller airways, higher breathing rates, and immature immune systems. Research indicates that infants born to mothers exposed to ETS during pregnancy have a one-to fivefold higher risk of being small for gestational age (SGA) compared to those unexposed to tobacco smoke.^8,9

Cotinine, a metabolite of nicotine, is formed when nicotine, the addictive compound in tobacco smoke, is metabolized in the human body. Unlike nicotine, which is rapidly cleared from the body within 2-3 hours on average,¹⁰ cotinine has a significantly longer half-life of approximately 19-20 hours.¹¹ This extended half-life makes cotinine a more reliable indicator of cumulative exposure to ETS over time. As a result, it is widely used as a biomarker to validate both active and passive smoking.^12,13 The use of cotinine as a biomarker for ETS exposure offers several advantages. Notably, 72% of nicotine is converted into cotinine, making it the most abundant nicotine metabolite compared to others.¹⁴ Cotinine can be detected in various biological samples, including urine, blood, hair, and saliva. Furthermore, studies have demonstrated a strong correlation between cotinine levels in biological fluids such as serum, saliva, and urine and the degree of nicotine exposure.^15-17

Serum or plasma samples have traditionally been selected as biomarkers for ETS exposure due to their greater stability in cotinine levels and minimal interference from analytical timing.¹⁸ However, there is a lack of standardization across studies regarding the selection of cotinine cut-off values for distinguishing smokers from non-smokers. These values also vary based on gender and age.^12,19-21 A cotinine cut-off value represents the threshold concentration used to classify individuals as exposed or unexposed to tobacco smoke, with values below the cut-off indicating minimal or no ETS exposure and values above the cut-off indicating exposure. The United States Salimetrix provides guidelines for serum cotinine cut-off levels, suggesting a range of 1-5 ng/mL, as these levels differ by ethnicity and gender.²⁰ In contrast, a study by Kim et al. (2016)¹² reported serum cotinine cut-off values ranging from 3.0 to 20 ng/mL. Similarly, the South African Cohort Study and the Hokkaido Cohort Study identified cut-off values of 10-15 ng/mL for non-smokers and 11.48 ng/mL for smokers.^19,21 Additionally, a review by Avila-Tang et al. (2013)²² observed that the widely used cut-off value in England decreased from 14 ng/mL to 12 ng/mL over time.

Urine is another frequently utilized sample for cotinine biomarker analysis due to its non-invasive collection process, ease of obtaining large sample volumes,²³ and higher cotinine concentrations compared to blood or saliva, making it a more sensitive indicator for detecting low-level exposure.^24,25 Studies have highlighted variations in urinary cotinine cut-off values for smokers and non-smokers. The European Human Biomonitoring study reported different urinary cotinine cut-off values across countries, with ranges from 4.45 mg/L to 254.15 mg/L (6.07 mg/g to 165.8 mg/g creatinine) for mothers and from 1.45 mg/L to 4.80 mg/L (2.15 mg/g to 3.18 mg/g creatinine) for children.²⁶ Similarly, a review by Tang et al. (2013)²² established a urinary cotinine cut-off value of 50 ng/mL for second-hand smoke exposure. Meanwhile, research from the New Hampshire Birth Cohort Study identified lower cut-off values for pregnant women, with 1.8 ng/mL for active smokers and 1.2 ng/mL for individuals exposed to second-hand smoke.²⁷ Although urinary cotinine is widely recognised as a sensitive and non-invasive biomarker for ETS exposure including in young children, substantial variability in reported cut-off concentrations across studies has limited comparability and hindered consistent exposure classification. Despite the heightened vulnerability of children in early life, no prior systematic review has specifically synthesised urinary cotinine cut-off values for this age group or quantitatively examined sources of methodological heterogeneity.

This review aims to systematically synthesize and quantitatively summarize the urinary cotinine cut-off concentrations used to classify ETS exposure among children under 5 years of age. Built on a previously published protocol (Supplemental 1),²⁸ this review provides the first quantitative synthesis of urinary cotinine cut-off concentrations used to assess ETS exposure among children under five years of age. In addressing this aim, the review seeks to answer the prespecified research questions of (1) what urinary cotinine cut-off concentrations have been used to assess ETS exposure in children under five years of age; (2) how these cut-off values vary according to age, geographical context, analytical method, and study period; and (3) which methodological factors contribute to between-study heterogeneity in reported cut-off concentrations. This review consolidates evidence and assesses study context, analytical framework, and population attributes, thereby addressing an important methodological gap in ETS exposure assessment. The results made to exposure assessment for at-risk groups is relevant to the SDG 3 (Good Health and Well-Being) and SDG 11 (Sustainable Cities and Communities) as it helps develop evidence-based smoke-free policies that improve healthier living environments. This review also offers an evidence-based framework for urinary cotinine analysis in ongoing population-based studies, including the Children and Health Birth Cohort Study (ChECKS) by the Ministry of Health Malaysia. This strengthens the usefulness of urinary cotinine as an exposure biomarker to ETS among young children.

Material and Methods

Study Design and Protocol Registration

This systematic review and meta-analysis were conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 checklist (Supplementary 2).²⁹ The detailed methodology was prospectively developed and published as a protocol in BMJ Open (Supplemental 1).²⁸ The protocol was registered with the International Prospective Register of Systematic Reviews (PROSPERO; registration number CRD42024556969). Any deviations from the published protocol are reported and justified in this review.

Eligibility Criteria

The eligibility criteria were predefined and have been described in detail in the published protocol (Supplemental 1).²⁸ Briefly, this review included observational and experimental studies reporting urinary cotinine cut-off concentrations used to classify environmental tobacco smoke (ETS) exposure among children from birth to under 5 years of age. Studies were required to report primary urinary cotinine cut-off values derived from measured urine samples.

Information Sources and Search Strategy

A comprehensive literature search was conducted in PubMed, EMBASE, Scopus, and the Cochrane Library from inception to February 2025, as specified in the protocol (Supplemental 1).²⁸ The search strategy a was based on Population–Exposure–Comparison-Outcome components and the full search strategies and hits for all databases are provided in Supplementary 3.

Study Selection

As outlined in the study protocol (Supplemental 1),²⁸ all retrieved records were imported into reference management software, and duplicates were removed. Study selection was performed independently by two reviewers in a two-stage process consisting of title and abstract screening followed by full-text assessment. Disagreements were resolved through discussion or consultation with a third reviewer. The study selection process is summarised using a PRISMA flow diagram.

Data Extraction

Data extraction was conducted independently by two reviewers using a standardised data extraction form as described in the protocol (Supplemental 1).²⁸ Extracted data included study characteristics, population details, study design, sample size, age range, urinary cotinine measurement methods, analytical techniques, unit of measurement, and reported cut-off concentrations used to define ETS exposure.

Quality Assessment

The methodological quality of included observational studies was assessed using the Newcastle–Ottawa Scale (NOS) as prespecified in the protocol (Supplemental 1).²⁸ The NOS evaluates quality across selection, comparability, and exposure or outcome domains, with a maximum score of nine stars and overall ratings of good, fair, or poor. An adapted NOS was applied for cross-sectional studies. In addition to the protocol, selected studies were appraised using the Critical Appraisal Skills Programme (CASP) checklists to support qualitative interpretation of study quality, particularly for cross-sectional designs, by examining study aims, design appropriateness, measurement robustness, analytical transparency, and applicability.

Data Synthesis and Meta-Analysis

Data synthesis was conducted in accordance with the published protocol (Supplemental 1).²⁸ A narrative synthesis was first undertaken to summarise study characteristics and reported urinary cotinine cut-off concentrations. Studies were compared by study design, age group, geographical region, analytical method, unit of measurement, and cut-off derivation approach. Urinary cotinine cut-off values were pooled using random-effects meta-analysis. Separate analyses were performed for different units of measurement (ng/mL and ng/mg creatinine). Python script in Google Colaboratory environment for meta-analysis is provided in Supplemental 4.

Due to the absence of reported measures of variability (e.g., standard deviations or standard errors) in the included studies, conventional inverse-variance weighting and formal heterogeneity testing were not feasible. Therefore, a sample-size weighted random-effects approach was applied to estimate pooled urinary cotinine cut-off values. This approach allows larger studies to contribute proportionally to the pooled estimates while acknowledging between-study variability. However, the results should be interpreted with caution, as formal assessment of heterogeneity and publication bias was not possible.

Results

Study Selection

Database searches yielded a total of 1,819 records including PubMed (n = 351), Embase (n = 620), Scopus (n = 708), and the Cochrane Library (n = 140). No relevant records were found from any other sources. After 928 duplicates were removed, 891 records were left for screening. From the title and abstract screening, 567 records were excluded. For 324 records, full text was requested, and 27 were not possible to retrieve. Of 297 full text articles assessed for eligibility, 285 were excluded. Main reasons for exclusion were involving children older than six years, and missing urinary cotinine cutoff values, or non-primary/outside findings. A total of 12 studies were included for the systematic review and meta-analysis (Figure 1).

Figure 1.

PRISMA flow diagram on selection process conducted until February 2025

Study Characteristics

Considering the 12 studies detailed in Table 1, the study periods cover a time span of more than 3 decades, ranging from 1988 to 2022.Three studies (25%) were conducted before 1995,^30-33 two studies (17%) between 1995 and 2010,^33-35 and six studies (50%) from 2010 onwards,^35-40 indicating an increasing research focus on urinary cotinine assessment in young children in more recent years. Most studies clearly stated when data was collected, however one early study (8%) did not.⁴¹

Table 1.

Urine Cotinine Studies for Children Under 6 years Results

Reference	Study period	Study location	Study design	Sample size	Age	Tobacco exposure assessment	Method of urine collection	Analytical method for urine cotinine	Cotinine cut-off concentration for exposure to ETS (ng/mL)	Method of urine cotinine cut-off selection	Cigarettes number/air nicotine cut-off reported as exposed to ETS
Eyuboglu et al, 2020⁴⁰	December 2015 to April 2016	Turkey	Case-control	227	< 5 years	• Questionnaire.	10 mL urine	LC-MS/MS	2.47 ng/mg creatinine	Established by receiver operating characteristic (ROC) analysis of overall samples. The optimal values were selected that exhibited the highest classification rates	> 10 cigarettes/day
						• Self-reported smoking.
						• Urine cotinine/creatinine measurement.
Preston et al, 1997³²	Aug 1993-June 1994	Puerto Rico	Cross-sectional	74	2-4 years	• Questionnaire.	Fasting urine.	ELISA kits (immunoassay)	11.6 ng/mg creatinine	90% of persons reporting nonexposure were correctly classified	Not mention
						• Self-reported smoking.
						• Urine cotinine/creatinine measurement.
Baheiraei et al, 2015³³	July 2009 to February 2010	Southern Tehran	Cohort	102	3-5 days, 2 months, 4 months	• Questionnaire.	Late morning in urine bag.	ELISA kits (solid phase competitive immunoassay)	15 ng/mg creatinine	Based on the statistical correlation between self-report and the value of cotinine in the urine with 100% sensitivity and 88% specificity cotinine level higher 15 ng/mg creatinine than	Not mention
						• Self-reported smoking.
						• Urine cotinine/creatinine measurement.
Dostál et al, 2008³⁴	1994 - 1999	Carolina	Cohort	523	3 or 4.5 years	• Questionnaire.	Self collected child urine by mothers.	Radioimmunoassay	20 ng/mg creatinine	Distribution of low concentrations of cotinine that deviates from the normal distribution at this value	Not mention
						• Self-reported smoking.
						• Urine cotinine/creatinine measurement.
Henderson et al, 1989⁴¹	not mention	Chapel Hill, North Carolina.	Cross-sectional	27	11 months to 5 years	• Questionnaire.	Two consecutive mornings after active home air nicotine monitoring and four consecutive Thursdays thereafter (6 times over a 5 week period).	Competitive-inhibition raioimmunoassay using rabbit cotinine antiserum and tritiated cotinine	30 ng/mg creatinine	Comparison of exposed and unexposed children from air nicotine monitoring and cigarette butt collection with urine cotinine levels	> 0.5 µg/m³
						• Collection of unsmoked portion of all cigarette.
						• Collection of remnant of cigarettes smoked in car while riding with their children.
						• Active home air nicotine monitoring during evening and night for 2 consecutive days - measured at child’s main activity room and bedroom.
						• Urine cotinine/creatinine measurement.
Margolis et al, 1997³¹	1986-1988	Alamance and Chatham counties, North Carolina	Community-based randomized trial	485	≤ 1 year	• Questionnaire.	Collected at the 3 week, 6 month and 12 months of home visit using urine bag.	Radioimmunoassay	120 ng/mg creatinine	Comparison of exposed and unexposed children from exposure to ETS from observation of household members smoking and urine cotinine levels	> 10 cigarettes/day
						• Smoking frequency observation during infant’s presence.
						• Urine cotinine/creatinine measurement.
Moore et al, 2022³⁷	2010 to 2014	Colorado, USA	Cohort	568	5 years	• Questionnaire.	Not mention	Solid phase competitive ELISA	0.05 ng/mL	Limit of detection	Not mention
						• Self-reported smoking (5 times collection).
						• Urine cotinine (2 times collection).
Carlsson et al, 2013³⁶	Feb 2010 - Oct 2011	South-eastern Sweden	Cohort	72	< 5 years	• Smoking in Children’s Environment Test questionnaire.	Provided by child during twice home visits (first during recruitment and after one year).	Gas Chromatography, Internal method NM-427-3.	6 ng/mL	Lowest level of quantification (corresponds to a daily nicotine intake of 80 μg)	Not mention
						• Self-reported smoking.
						• Urine cotinine.
Maedel et al, 2018³⁵	Between 2015 an d2017	Wilhelminen, Vienna, Austria.	Cross-sectional	185	106 days	• Questionnaire.	Obtained within 12 h of admission with LRTI symptoms.	LC-MS/MS	7 ng/mL	Lower limit of detection	Not mention
						• Self-reported smoking.
						• Urine cotinine.
Chilmonczyk et al, 1990³⁰	Jan-Oct 1988	greater Portland, Maine	Cross-sectional	518	6-8 weeks	• Questionnaire.	Cotton rolls placed in the diaper, collected on the same day or mailed back if urination not occurred.	Competitive radioimmunoassay using cotinine antiserum raised in rabbits and an iodine	10 ng/mL	Comparison of exposed and unexposed children from self-reported exposure and urine cotinine levels	Not mention
						• Self-reported smoking.
						• Urine cotinine.
Shermatov et al, 2018³⁹	Dec 2012 till Sept 2013	Harran University, Sanliurfa, Turkiye	Cross-sectional	79	1-4 years	• Questionnaire.	Collected in sterile container.	Enzyme immunoassay method	30 ng/mL	Comparison of exposed and unexposed children from self-reported exposure and urine cotinine levels	Not mention
						• Self-reported smoking.
						• Urine cotinine.
Muta et al 2025³⁸	March 2021-January 2022	Japan	Cross-sectional	200	3 years	• Questionnaire.	Parents collect child’s first-morning urine in a sterile container	Enzyme-linked immunosorbent assay kit	1.3 ng/mL	Lower limit of quantitation	Not mention
						• Self-reported smoking.
						• Urine cotinine.

The included studies span multiple regions of North America, Europe, the Middle East, and East Asia. North America has the largest share, with five studies (41.7%) from the United States and Puerto Rico. Europe has four studies (33.3%) from Sweden, Austria, and Turkey. Three studies (25.0%) from Asia come from Iran and Japan. Most of the studies in the table are observational studies. Of the 12 studies, six (50.0%) were cross-sectional studies, while the most common design in the assessment of urinary cotinine exposure in young children is one-time-point. A total of four studies (33.3%) were cohort studies, which means they are able to have longitudinal exposure assessment over the study duration. Only one study (8.3%) reported on case-control study, and one study (8.3%) was a community-based randomized trial. The fact that most studies are cross-sectional and cohort studies demonstrates the heavy dependence of the authors on the non-randomized epidemiological research to assess urinary cotinine cut-off concentrations for exposure to ETS among children under five years.

The range of participant variability in the 12 studies is quite large, as numbers range from 27 to 568 participants. Four studies (33.3%) included fewer than 100 participants, which is generally the case for smaller or pilot studies. Another four studies (33.3%) included 100 to 300 participants, while the remaining four studies (33.3%) included large numbers of participants, specifically more than 300, which is usually the case for cohort or community studies. The children included in the 12 studies were from early infancy to five years of age, and there were varying degrees of development at the time of the study. Five studies (41.7%) were focused mainly on infants less than one year. Four studies (33.3%) examined children in the preschool age range, which is between 1 and 4 years, and three studies (25.0%) were known to study a wider age range, which included children in the 1 to 5 years age brackets, thereby including children up to the age of five years.

Exposure Assessment Method

Tobacco exposure assessment across the 12 included studies relied predominantly on a combined approach using parental self-report and biomarker measurement. All studies (100%) incorporated urinary cotinine measurement as an objective indicator of ETS exposure. In parallel, most studies (11 studies, 91.7%) used questionnaires or parental self-reported smoking behaviours to characterise exposure sources and intensity. A smaller number of studies (2 studies, 16.7%) supplemented self-reports with environmental or observational measures, such as household smoking observations, cigarette butt collection, or indoor air nicotine monitoring.

Urine Collection and Analytical Methods

Urine collection techniques differed among the included studies and were mostly tailored to the children’s ages. Urine bags, sterile containers, cotton rolls in diapers, or parent-collected urine samples taken in the morning were among the non-invasive caregiver-assisted collection methods employed in most of the research (8 studies, 66.7%). To enhance exposure evaluation, two studies (16.7%) called for timed or repeated urine collection, such as fasting urine samples, several collections over several days, or house visits. Three studies (25.0%) did not provide a clear description of the urine collecting procedure.

The studies used different analytical methods to measure urinary cotinine, but most of them used immunoassay- and mass spectrometry-based methods. The enzyme-linked immunosorbent assay (ELISA) or similar enzyme immunoassay techniques were utilized in five studies (41.7%), making them the most employed methods. Radioimmunoassay (RIA), encompassing competitive radioimmunoassay formats, was utilized in four studies (33.3%), predominantly in earlier research. Liquid chromatography-tandem mass spectrometry (LC–MS/MS), a very precise and sensitive way to analyse pollutants, was used in two more recent studies (16.7%). One study (8.3%) measured urinary cotinine levels through gas chromatography (GC).

Characteristics of Urinary Cotinine Cut-Off Values

All 12 studies included in this review exhibit wide variability in reported urinary cotinine cut-off values for assessment of exposure to ETS, highlighting considerable methodological heterogeneity. Half of the studies (6 studies, 50.0%) reported cut-off values that were creatinine adjusted, in ng/mg creatinine. In the other half of the studies (6 studies, 50.0%), the urinary cotinine concentrations were reported in the unadjusted state, in ng/mL. The lowest cut-off values were a function of the various limits of detection, or quantification and the highest cut-off values were based on exposed and unexposed group comparison or statistical classification.

The methodologies employed to establish urinary cotinine cut-off concentrations for ETS exposure differed among the studies included. Most studies (7 studies, 58.3%) determined cut-off values by comparing exposed and unexposed groups, generally relying on parental self-reported smoking, environmental assessments, or observed smoking behaviours. Statistical classification methods, including receiver operating characteristic (ROC) curve analysis, were utilized in two studies (16.7%) to enhance sensitivity and specificity. In three investigations (25.0%), cut-off values were established using analytical thresholds, such as the limit of detection or limit of quantification, instead of exposure contrasts.

In comparison to the overall number of studies included, very few quantified the criterion for ETS exposure, using either the number of cigarettes smoked or the quantity of nicotine in the air at which a person is exposed to ETS. Three studies (25.0%) established a threshold of cigarette consumption. Most of these studies defined exposure as a household member smoking more than 10 cigarettes a day. One study (8.3%) identified a threshold for air nicotine concentration, defining exposure as more than 0.5 µg/m³ of air. Eight studies (66.7%) offered no explanation of cigarette or air nicotine count/class exposure, relying instead on self-exposure and urinary cotinine. Limited reporting is indicative of a lack of consistency in incorporating external exposure metrics and a biomarker approach. Nevertheless, the research facilitated the interpretation of urine cotinine levels based on the actual intensity of exposure to ETS.

Synthesis of Cut-Off Value Variability

A summary of Table 2 showing the variation in assessment of urinary cotinine cut-off concentrations for measuring ETS exposure among children under five. The studies have been stratified for the reporting approach, magnitude, and the method of derivation. The synthesis of cut-off concentrations of urinary cotinine in the twelve studies showed a great deal of variation across both the methods of reporting and derivation. Half of the studies (50.0%) provided cut-off values for urinary cotinine that were adjusted for creatinine, with ranges from 2.47 to 120 ng/mg creatinine, while the other half (50.0%) gave unadjusted cut-off value ranges from 0.05 to 30 ng/mL. In terms of magnitude, one-third of studies (33.3%) reported low cut-off values (≤5 ng/mL or ≤ 5 ng/mg creatinine), and the reasons for these were often analytical detection or quantification limits. Almost half of the studies (41.7%) reported moderate cut-off values (>5 - 20 ng/mL or ng/mg creatinine), and this was most often the result of comparisons made between exposed and unexposed children. Higher cut-off thresholds (>20 ng/mL or ng/mg creatinine) were reported in three studies (25.0%), mostly found in older studies or in studies where the exposure was higher.

Table 2.

Summary of Urinary Cotinine Cut-Off Concentrations for ETS Exposure Among Children Under 5 years (n = 12)

Domain	Category	Number of studies, n (%)	Key implications
Type of cut-off reporting	Creatinine-adjusted (ng/mg creatinine)	6 (50.0)	Cut-offs ranged from 2.47 to 120 ng/mg creatinine
Type of cut-off reporting	Unadjusted (ng/mL)	6 (50.0)	Cut-offs ranged from 0.05 to 30 ng/mL
Magnitude of cut-off values	Low (≤5 ng/mL or ≤5 ng/mg creatinine)	4 (33.3)	Often based on limits of detection or quantification
	Moderate (>5–20 ng/mL or ng/mg creatinine)	5 (41.7)	Commonly derived from exposure group comparisons
	High (>20 ng/mL or ng/mg creatinine)	3 (25.0)	Typically observed in earlier studies or high-exposure settings
Method of cut-off selection	Comparison of exposed vs unexposed groups	7 (58.3)	Based on self-reported exposure, observation, or air nicotine
	Statistical classification (e.g. ROC analysis)	2 (16.7)	Optimised sensitivity and specificity
	Analytical limits (LOD/LOQ)	3 (25.0)	Not exposure-based
Supporting exposure metric	Cigarette number or air nicotine threshold reported	4 (33.3)	Included >10 cigarettes/day or air nicotine >0.5 µg/m³
Supporting exposure metric	Not reported	8 (66.7)	Relied on questionnaires and urinary cotinine only

The majority of studies (58.3%) established cut-off concentrations by contrasting urinary cotinine levels between exposure groups defined by parental report, observational data, or environmental measures. Statistical classification approaches, including receiver operating characteristic analysis, were used in two studies (16.7%) to optimize sensitivity and specificity. In contrast, three studies (25.0%) defined cut-off values based solely on analytical limits rather than exposure-based criteria. Only a minority of studies (33.3%) reported supplementary exposure thresholds, such as cigarette consumption or air nicotine concentrations, while most relied exclusively on urinary cotinine measurements and questionnaire-based exposure assessment. Collectively, these findings highlight the lack of standardisation in urinary cotinine cut-off derivation for assessing ETS exposure in children under five years of age.

Meta-Analysis

A total of 12 studies reporting urinary cotinine cut-off concentrations for assessing ETS exposure in children under five years of age were included in the meta-analysis. Due to differences in reporting units, the studies were stratified into two groups according to the unit of measurement: creatinine-adjusted cut-off values expressed in ng/mg creatinine and unadjusted cut-off values expressed in ng/mL. As there were only available data of sample sizes and urinary cotinine cut-off values, it is not possible to conduct a heterogeneity test due to the absence of standard deviation and standard error data. The test for publication bias was omitted since each meta-analysis did not achieve the minimum requirement of 10 studies.

For creatinine-adjusted urinary cotinine cut-off concentrations, six studies were included, comprising a total sample size of 1,486 children. Using a sample-weighted meta-analysis of means, the pooled urinary cotinine cut-off value was estimated to be 50.36 ng/mg creatinine (95% CI: 47.53–53.20). Individual study cut-off values ranged widely, from 2.47 to 120.00 ng/mg creatinine. The pooled estimate was heavily influenced by two large cohort studies,^31,34 which together contributed approximately 1,486 participants. The forest plot illustrating this analysis is presented in Figure 2.

Figure 2.

Forest plot of urinary cotinine cut-off value (ng/mg creatinine) for children under five years

For unadjusted urinary cotinine cut-off concentrations, six studies were included with a combined sample size of 1,622 children from. The pooled cut-off value was estimated to be 5.90 ng/mL (95% CI: 5.53–6.27), with individual study values ranging from 0.05 to 30.00 ng/mL. Similar to the creatinine-adjusted analysis, the pooled estimate was largely driven by two large studies that accounted for nearly two-thirds of the total sample size from Moore et al.,³⁷ and Chilmonczyk et al,³⁰ studies. The corresponding forest plot is shown in Figure 3.

Figure 3.

Forest plot of urinary cotinine cut-off value (ng/mL) for children under five years

Risk of Bias in Studies

Table 3 shows the results from the 12 studies’ bias assessments using NOS. Overall, the methodological quality of the studies was high, with seven studies (58%) rated as good quality and five studies (42%) rated as fair quality; no studies were classified as low quality. It demonstrates that the evidence base underpinning this systematic review is methodologically sound. Among the seven studies with good quality ratings, five were cohort studies, one was a case–control study, and one was a cross-sectional study. These studies consistently achieved high scores across all NOS domains, particularly for selection and exposure/outcome assessment, reflecting robust study designs, well-defined populations, and the use of objective urinary cotinine measurements. On the other hand, all studies rated as fair quality were cross-sectional in design due to the lack of a refined control as well as lower balance of the study groups. The quality of the studies varies, but the collection of studies as a whole features a sound quality.

Table 3.

Risk of Bias Assessment Using the Newcastle–Ottawa Scale (NOS) for Included Studies (n = 12)

Study (first author, year)	Study design	NOS version used	Selection (max 4)	Comparability (max 2)	Exposure/Outcome (max 3)	Total score (max 9)	Overall quality
Eyüboğlu, 2020⁴⁰	Case–control	NOS – Case–Control	★★★★	★★	★★★	9	Good
Preston, 1997³²	Cross-sectional	NOS – Adapted for Cross-sectional	★★★	★	★★	6	Fair
Baheiraei, 2015³³	Cohort	NOS – Cohort	★★★★	★★	★★★	9	Good
Henderson, 1989⁴¹	Cross-sectional	NOS – Adapted for Cross-sectional	★★★	★	★★	6	Fair
Margolis, 1997³¹	Community-based cohort	NOS – Cohort	★★★★	★★	★★★	9	Good
Moore, 2022³⁷	Cohort	NOS – Cohort	★★★★	★★	★★★	9	Good
Carlsson, 2013³⁶	Cohort/intervention	NOS – Cohort	★★★★	★★	★★★	9	Good
Maedel, 2018³⁵	Cross-sectional	NOS – Adapted for Cross-sectional	★★★	★	★★	6	Fair
Chilmonczyk, 1990³⁰	Cross-sectional	NOS – Adapted for Cross-sectional	★★★	★	★★	6	Fair
Shermatov, 2018³⁹	Cross-sectional	NOS – Adapted for Cross-sectional	★★	★	★★	5	Fair
Muta, 2025³⁸	Cross-sectional	NOS – Adapted for Cross-sectional	★★★★	★★	★★★	9	Good
Dostál, 2008³⁴	Cohort	NOS – Cohort	★★★★	★★	★★★	9	Good

An adapted version of the Newcastle–Ottawa Scale was applied for cross-sectional studies, while the original NOS versions were used for cohort and case–control studies.

In addition to the NOS, the CASP checklists were applied as a supplementary appraisal tool to support qualitative interpretation of methodological quality. Overall, the CASP assessments were concordant with NOS classifications; studies rated as good quality by NOS consistently demonstrated strong CASP performance, whereas studies rated as fair quality commonly exhibited limitations related to confounding control, participant recruitment, or analytical rigour. The CASP appraisal did not replace the NOS-based risk-of-bias assessment and was used solely to enhance narrative interpretation of study quality and to inform cautious interpretation of the synthesised findings. Detail CASP scoring is provided in Supplemental 5. Although the protocol specified the use of the Joanna Briggs Institute (JBI) critical appraisal tool for experimental studies, no eligible experimental studies meeting the inclusion criteria were identified in the final review. All included studies were observational in design and were therefore assessed using the Newcastle–Ottawa Scale, as prespecified. Consequently, application of the JBI tool was not required and its omission is unlikely to have influenced the findings or conclusions of this review.

Discussion

This systematic review and meta-analysis provide the first quantitative synthesis of urinary cotinine cut-off concentrations used to assess ETS exposure among children under five years of age. Across more than three decades of research and diverse geographical contexts, urinary cotinine was consistently used as an objective biomarker of ETS exposure, reinforcing its value as a sensitive and objective indicator of ETS exposure in early childhood.^42-44 However, substantial variability was observed in the cut-off concentrations applied to classify exposure, reflecting differences in analytical techniques, study design, exposure definition, and population context.^22,45

A wide range of urinary cotinine cut-off concentrations was identified across the included studies, reflecting substantial heterogeneity in exposure classification. Stratified meta-analyses yielded pooled reference values that represent prevailing practices rather than definitive exposure thresholds, consistent with previous reviews reporting the absence of universally accepted cotinine cut-offs across populations and study contexts.²² Comparisons with studies in adults and older children highlight important age-related considerations. In adult populations, urinary cotinine cut-offs used to define second-hand smoke exposure are commonly reported at substantially higher levels, often around 50 ng/mL or above, reflecting higher baseline exposure and differences in nicotine metabolism.^21,22 Similarly, studies including school-aged children (≥5 years) have reported higher urinary cotinine thresholds than those observed in younger children, likely due to increased environmental mobility and cumulative exposure.²⁶

Age-related physiological factors such as immature hepatic and renal clearance pathways, differences within enzymatic activity, and distinct exposure contexts in young children result in lower and more variable cotinine concentrations following ETS exposure compared with adults.^14,18,46 The contrast between adult, older-child, and under-five cut-offs illustrates the inappropriateness of extrapolating adult-derived thresholds to early childhood and highlights the influence of developmental differences in nicotine metabolism, renal function, and exposure patterns during early life. These comparisons confirm that cotinine thresholds are population- and context-specific and should not be extrapolated across age groups without biological justification. These biological and contextual distinctions underscore the need for age-appropriate urinary cotinine cut-offs rather than reliance on adult-derived benchmarks.

Earlier studies primarily in the 1980s and 1990s, often conducted in high-smoking-prevalence settings and relying on radioimmunoassay techniques, tended to apply higher urinary cotinine cut-off values, reflecting both higher background exposure levels and analytical limitations associated with earlier radioimmunoassay-based methods^10,23 In contrast, more recent studies from the 2010s employing advanced techniques such as LC-MS/MS have reported lower and more precise cut-off values, reflecting improved analytical sensitivity and declining population-level ETS exposure.^23,47 Despite inclusion of infants and preschool-aged children, few studies define age-specific cut-offs, indicating limited consideration of developmental differences in nicotine metabolism and renal function.^12,14 These methodological differences may contribute to variability in reported cut-off values and should be considered when interpreting pooled estimates. Accordingly, the pooled values presented in this review should be interpreted as reflecting prevailing practices rather than optimal or standardised thresholds.

Methodological diversity in cut-off derivation represents another major contributor to heterogeneity. Most studies derived cut-offs by comparing exposed and unexposed groups defined through parental self-report or environmental indicators, while fewer applied statistical optimisation methods such as receiver operating characteristic analysis. Some studies relied solely on analytical limits rather than exposure-based contrasts, further complicating comparability. Only a minority of studies applied statistically optimised approaches, specifically ROC analysis to derive urinary cotinine cut-off values. These approaches are methodologically advantageous as they balance sensitivity and specificity and provide more robust classification thresholds. However, due to the small number of studies (2 studies), the reliance on group comparisons or analytical limits may introduce bias and reduce comparability across studies. Future research should prioritise the use of statistically optimised methods, including ROC analysis, to improve the accuracy and consistency of urinary cotinine cut-off determination. Similar variability in cut-off derivation methods has been widely documented in adult and mixed-age populations, where thresholds have been shown to depend heavily on study design, exposure definition, and analytical performance rather than consistent biological criteria.^12,22,33 Reviews and biomonitoring studies have repeatedly highlighted that reliance on self-reported exposure, differences in environmental smoking intensity, and inconsistent statistical approaches contribute substantially to between-study heterogeneity in cotinine-based exposure classification.^21,26,48

The pooled estimates generated in this review provide empirical reference values for both creatinine-adjusted and unadjusted urinary cotinine reporting units. However, these estimates should be interpreted as descriptive benchmarks rather than universal exposure thresholds, given their dependence on study design and population context.¹² Inconsistent cut-off selection may distort prevalence estimates and complicate cross-jurisdictional comparisons. The pooled values were influenced by large cohort studies, and the absence of reported variance measures limited formal heterogeneity testing. Accordingly, the results should inform; but not replace context-specific exposure classification strategies. Establishing age-specific, exposure-validated urinary cotinine benchmarks would enhance comparability, strengthen evaluation of child-focused tobacco control initiatives, and support implementation of the WHO Framework Convention on Tobacco Control.³

From a public health and policy perspective, improved standardisation of urinary cotinine cut-off selection can enhance ETS exposure surveillance and strengthen evaluation of smoke-free policies aimed at protecting children in domestic and community settings.^3,25 In addition, evidence from intervention and longitudinal studies further supports the utility of urinary cotinine as a sensitive and responsive biomarker of ETS exposure in children. Studies evaluating smoke-free policies, behavioural interventions, and reductions in household smoking have consistently shown that urinary cotinine concentrations decrease in parallel with reductions in reported exposure levels, demonstrating its ability to reflect temporal changes in exposure. For example, research conducted in residential and public environments has demonstrated that cotinine levels in non-smokers, including children, decline following implementation of smoke-free measures, reinforcing its validity as a biomarker for monitoring exposure reduction.^44,49,50 These findings highlight the value of urinary cotinine not only for exposure classification but also for evaluating the effectiveness of tobacco control interventions and policies aimed at protecting children from second-hand smoke. This review also aligns with the United Nations Sustainable Development Goal 3 (Good Health and Well-Being) by supporting prevention of avoidable environmental health risks in early life,⁵¹ and with SDG 11 (Sustainable Cities and Communities) by informing evidence-based smoke-free environments.⁵² Given the persistent global burden of second-hand smoke exposure among children, particularly in low-and middle-income countries⁵³ improving methodological consistency in biomarker interpretation remains a priority.

In addition, the evolving nicotine product landscape presents new challenges in interpreting cotinine as a biomarker of ETS exposure. Cotinine reflects systemic nicotine exposure irrespective of source, including emerging products such as e-cigarettes and heated tobacco devices. As a result, elevated cotinine concentrations may not exclusively represent exposure to combustible tobacco smoke, particularly in contemporary environments where multiple nicotine sources coexist. This may lead to potential misclassification of exposure if the source of nicotine is not clearly characterised. Future studies should therefore incorporate detailed exposure assessment, including product-specific information, complimentary biomarkers to improve the specificity sources of nicotine exposure.

Strength and Limitations

This systematic review and meta-analysis have several notable strengths. First, the study addresses an important methodological gap in paediatric tobacco exposure research by establishing evidence-based urinary cotinine biomarker cut-off concentrations under five years old. Second, the review followed a prospectively published and registered protocol, adhered to PRISMA 2020 guidelines, and applied a transparent and reproducible methodology. Third, stratification by unit of measurement (creatinine-adjusted versus unadjusted) and the use of meta-analysis provided empirical reference values that reflect prevailing practices across heterogeneous studies. Fourth, the inclusion of studies from multiple regions over three decades enhances the generalisability of the findings. Finally, methodological quality assessment using the NOS, supported by CASP appraisal, indicated an overall good quality evidence base, with strengths in selection and objective exposure measurement, thereby reinforcing confidence in the interpretation of the results. From a public health perspective, standardisation of paediatric urinary cotinine cut-offs has important implications for tobacco control surveillance and policy evaluation. Biomarker-based classification is increasingly used to monitor exposure trends, evaluate smoke-free housing policies, assess maternal cessation interventions, and quantify residual exposure in children following regulatory change.

Several limitations should also be acknowledged. The included studies exhibited substantial methodological heterogeneity, particularly in analytical methods, urine collection procedures, and cut-off derivation approaches, which limits direct comparability. The absence of reported measures of variability (e.g. standard deviations or standard errors) precluded formal heterogeneity testing, meta-regression, and publication bias assessment, and necessitated the use of sample-weighted meta-analysis. In addition, many cut-off values were derived from analytical detection limits rather than exposure-based comparisons, potentially limiting their interpretability. The review was restricted to English-language publications and excluded grey literature, which may have resulted in publication bias. Lastly, the lack of age-specific cut-off definitions within the under-five age group limits the applicability of pooled estimates across different developmental stages.

Conclusion

This study synthesises existing evidence on urinary cotinine cut-off concentrations used to assess ETS exposure among children under five years of age. The findings confirm urinary cotinine as a robust and widely applied biomarker in early childhood, while demonstrating substantial heterogeneity in cut-off values driven by differences in analytical methods, derivation approaches, study context, and reporting practices. The pooled estimates (5.90 ng/mL and 50.36 ng/mg creatinine) provide useful reference points but should be interpreted as descriptive benchmarks rather than definitive exposure thresholds. The absence of standardised, age-specific urinary cotinine cut-offs for young children and highlight the limitations of extrapolating adult-derived thresholds to paediatric populations. Overall, this study provides a foundation for improving exposure assessment and supporting tobacco control efforts aimed at reducing ETS exposure in early childhood.

Several recommendations can be proposed to improve the determination and application of urinary cotinine cut-off values in young children. First, the use of highly sensitive and specific analytical techniques such as LC–MS/MS should be prioritised to ensure accurate quantification at low exposure levels. Second, cut-off values should be derived using statistically robust methods, such as ROC analysis, rather than relying solely on analytical limits or group comparisons. Third, integration of objective exposure measures, including air nicotine monitoring, can strengthen exposure classification. Fourth, age-specific cut-off values should be developed to account for physiological differences in nicotine metabolism among young children. Finally, studies should consistently report measures of variability, such as standard deviations or standard errors, and methodological details to improve comparability and enable more robust quantitative synthesis in future meta-analyses.

Supplemental Material

Supplemental Material - A Meta-Analysis of Urinary Cotinine Cut-Off Concentrations in Children Under Five Years for Assessing Environmental Tobacco Smoke Exposure

Supplemental Material for A Meta-Analysis of Urinary Cotinine Cut-Off Concentrations in Children Under Five Years for Assessing Environmental Tobacco Smoke Exposure by Sharifah Mazrah Sayed Mohamed Zain, Nadia Mohamad, Zurahanim Fasha Anual, Imanul Hassan Abdul Shukor, Wan Nurul Farah Wan Azmi in Tobacco Use Insights.

Footnotes

Acknowledgments

We would like to thank the Director General of Health Malaysia for the permission to publish this review article.

ORCID iDs

Sharifah Mazrah Sayed Mohamed Zain

Nadia Mohamad

Zurahanim Fasha Anual

Imanul Hassan Abdul Shukor

Wan Nurul Farah Wan Azmi

Author Contributions

Sharifah Mazrah Sayed Mohamed Zain: Conceptualization, Methodology, Investigation, Project administration, Software, Visualization, Writing–original draft, Writing–review and editing. Zurahanim Fasha Anual: Methodology, Investigation, Writing–original draft, Writing–review and editing. Nadia Mohamed: Methodology, Investigation, Writing–original draft, Writing–review and editing, Wan Nurul Farah Wan Azmi: Methodology, Investigation, Writing–original draft, Writing–review and editing. Imanul Hassan Abdul Shukor: Methodology, Investigation, Software, Visualization, Writing – original draft, Writing–review and editing.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data Availability Statement

The original contributions shown in the study are contained within this systematic review or its appendices; further inquiries can be addressed to the corresponding author.*

Declaration of Generative AI and AI-Assisted Technologies in the Writing Process

During the preparation of this work, the authors used ChatGPT5.2 to assist in interpreting the results and for grammar checks. After using this tool/service, the authors reviewed and edited the content as needed and takes full responsibility for the content of the published article.

Supplemental Material

Supplemental material for this article is available online.

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