Review
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Recent advances in MS methods for nicotine and metabolite analysis in human matrices: clinical perspectives
Tobacco smoking is a major global health issue and represents the leading cause of preventable death in the developed countries. Nicotine is a major alkaloid found in tobacco products and its detection with its metabolites in human matrices is generally used for assessing tobacco consumption and second hand exposure. Several analytical techniques have been developed for the detection of nicotine and its metabolites, and MS coupled with chromatography is considered the standard reference method because of its superior sensitivity and specificity. In this work, we reviewed nicotine metabolism, clinical MS and the latest (2009–2014) development of MSbased techniques for measurement of nicotine and metabolites in human matrices. Appropriate biomarker and matrix selection are also critically discussed.
Tobacco smoking and addiction is a major global public health issue with welldocumented health risks, including cancer, cardiovascular disease and chronic pulmonary obstructive disease [1] . Over the last two decades, awareness regarding the adverse health effects of tobacco use has been on the rise and the prevalence of cigarette smoking has been declining [2] . Nevertheless, smoking and exposure to second-hand smoke (SHS) remains the leading cause of preventable deaths in the USA and is responsible for one in five deaths in US adults [2,3] . As a result, smoking is associated with substantial healthcare costs in the USA (estimated at US$133 billion a year) [4,5] and worldwide [6–8] . This has led US legislators to take action to reduce the impact of smoking on healthcare and the economy. As a result, smoking in enclosed public spaces and commercial establishments is banned in most states to curb the effects of SHS [9] and insurance companies were required to cover costs associated with smoking cessation programs without any patient cost sharing as mandated by the Affordable Care Act of 2010 [10] . Similar policies have been implemented worldwide [11–13] . In addition, some employers (mainly hospitals) have taken a step further by imposing a ban on hiring smokers or any
10.4155/BIO.14.176 © 2014 Future Science Ltd
Joe M El-Khoury‡,1 & Sihe Wang*,1 1 Department of Clinical Pathology, Cleveland Clinic, Cleveland, OH 44195, USA *Author for correspondence: Tel.: +1 216 445 2634 Fax: +1 216 445 0212 [email protected] ‡ Current address: Department of Laboratory Medicine, Yale University, New Haven, CT 06511, USA
applicant whose urine tests positive for nicotine use [14] . Consequently, laboratory testing for nicotine and/or metabolites has surged worldwide owing to compliance monitoring for smoking cessation programs, pre-employment drug screening programs, and ongoing public health studies. Traditionally, nicotine and/or metabolites have been measured for the evaluation of subjects for organ transplantations (as donors or recipients) [15] , for acute nicotine poisoning [16] , and to assess exposure to environmental tobacco smoke, especially during prenatal phase and childhood [1] . In addition, our physicians have also begun to monitor tobacco consumption (smoking and smokeless) for a defined period of time before admitting them for lumbar spinal fusion surgery. This is because nicotine has been shown to inhibit lumbar spinal fusion and promote disc degeneration, and in a large prospective Swedish study (n = 4555) smokers had less improvement after surgery than non-smokers [17] . Hence, nicotine testing has significantly increased over the last few decades and as always laboratories must adapt and ensure that high-quality test results are provided in a timely manner. A plethora of methods have been developed for the measurement of nicotine and/or
Bioanalysis (2014) 6(16), 2171–2183
part of
ISSN 1757-6180
2171
Review El-Khoury & Wang
Key term ESI: A technique that generates ions at atmospheric pressure by spraying a sample solution through a capillary tube with a high electric voltage in the presence of a flow of warm inert gas to assist in desolvation.
metabolites in a variety of human matrices [18] . These methods include radioimmunoassay, ELISA, HPLC, GC–MS and LC–MS [18] . MS, especially when coupled with GC or LC separation techniques, is a very powerful analytical tool that provides high sensitivity and specificity for the analysis of clinically relevant analytes in biological fluids [19] , and is considered the standard reference method for nicotine and metabolites [18] . The aim of this work was to review the most recent advances in mass spectrometric method development for the analysis of nicotine and metabolites in human matrices and to provide recommendations for selection of suitable biomarkers, methods and cut-offs for distinguishing active smokers from passive smokers and nonsmokers. An overview of clinical MS applications and nicotine metabolism is provided, followed by a critical review of MS-based assays for nicotine and/or metabolites published in the last 5 years (March 2009 to February 2014). A systematic approach was used for article selection. A PubMed search using the logic ‘nicotine’ OR ‘cotinine’ OR ‘nornicotine’ OR ‘anabasine’ AND ‘mass spectrometry’ restricted to the last 5 years was performed on Friday 28 February 2014 and yielded 306 results. All 306 abstracts were reviewed and articles reporting development of new MS methods for the measurement of nicotine and/or metabolites in human matrices were selected. Limitations of our approach include being restricted to articles indexed in PubMed and published in English. Clinical MS applications MS is a powerful qualitative and quantitative analytical technique involving the separation and detection of gas-phase ions according to their mass-to-charge ratios. This technology has been around for over a century but its use was largely restricted to highly
technical research laboratories for the most part. Adoption of MS in clinical laboratories has been rapidly increasing in the last decade largely due to the development of soft ionization techniques, such as ESI and MALDI, which allow the analysis of a wide range of small and macro-molecules in biological fluids [20] . As a result, ESI/MS coupled to LC (LC–ESI/MS) has found applications in many areas, such as endocrinology, therapeutic drug monitoring, inborn errors of metabolism, clinical and forensic toxicology, and nutrition assessment, while MALDI coupled to MS has applications in microbiology, proteomics and lipidomics [21,22] . The main driving force behind the implementation of MS testing in all these different areas of laboratory medicine is the superior sensitivity and selectivity that this technique provides. This advantage, combined with the ability of simultaneous multianalyte quantification, and the versatility makes MS a valuable tool for clinical research and laboratory medicine. The strengths and limitations of MS are summarized in Table 1, and the reader is encouraged to review these excellent references for more information on MS and the role it plays in laboratory medicine [23–26] . MS has certainly proven its utility for clinical laboratories, however, there are still several key challenges it must overcome, including high instrument cost, appropriate field service support, interfacing with the laboratory information system, standardization, throughput, robustness and automation. Nicotine pharmacokinetics Nicotine (Figure 1) is the principal tobacco alkaloid and exists mainly in the levorotatory form (S)-nicotine, while (R)-nicotine accounts for only 0.1–1.2% of total nicotine [27] . It is carried proximally on tar droplets after distillation from burning tobacco then absorbed rapidly in the pulmonary system. Absorption is pH dependent and nicotine is more rapidly absorbed across biological membranes in its basic unionized form, while acidic conditions reduce its absorption [27] . This also explains why nicotine in its basic form is well
Table 1. Strengths and limitations of MS.
2172
Strengths
Limitations
• High sensitivity and specificity • Ability to measure a wide range of compounds • Ability to measure multiple analytes in a single run (profiling) • Low reagent cost per sample • Provides high flexibility and speed for new assay development
• High instrument cost • Relatively low throughput • High complexity requiring extensive training and highly skilled personnel • Lack of appropriate service support from manufacturers • Often involves labor-intensive sample preparation • Not fully adapted for clinical laboratories
Bioanalysis (2014) 6(16)
future science group
Recent advances in MS methods for nicotine & metabolite analysis in human matrices: clinical perspectives
N N
+
Nicotine-N-oxide glucuronide (Nic-Oxi-GIu) [0.4%, 0.2%, 0.1%]
N
HO2C
OH
N+
OH
N
OH Nicotine-N-b glucuronide (Nic-GIu) [1.8%, 1.3%, 2.1%]
N
OH
CO2H
CYPs 2A6 2A13
4-hydroxy-4-(3-pyridyl) butanoic acid (HyPyBut)
CH3
N+ O
Cotinine-N-oxide (Cot-Oxi) [2.8%, 3.3%, 3.6%]
OH
AO
CH3
Nornicotine (Nornic) [3.6%, 6.9%, 4.7%]
Nornicotineglucuronide (Nornic-GIu) [0.1%, 0.1%, 0.1%]
O
CH3
N
Trans-3’-hydroxycotinine (Hcot) [20.8%, 26.8%, 38.7%]
Cotinine (Cot) [10.4%, 8.6%, 9.3%] CYP 2A6
UGTs 2B7,1A9 N
N
N CYP 2A6
CH3
N
CYP 2A6
N H
O
N
CYP 2A6 2A13
[10.2%, 9.7%, 7.7%]
4-hydroxy-4-(3-pyridyl) butanoic acid-glucuronide (HyPyBut-GIu)
O
N
UGTs 2B1010,1A4
Nicotine (Nic) [22.8%, 7.7%, 2.2%]
N
[0.7%, 0.7%, 0.6%]
OH
H3C O
FMO-3
N
CH3
OH
Nicotine-N-oxide (Nic-Oxi) [10.2%, 15.4%, 10.4%] UGTs 2B10,1A4
O
Cotinine-N-b-glucuronide (Cot-GIu) [8.0%, 9.7%, 11.0%]
O HO2C
Cotinine-N-oxide glucuronide (Cot-Oxi-GIu) [0.1%, 0.3%, 0.4%]
O
OH
CH3
N+
Review
O
N
HO O
Norcotinine (Norcot) [1.3%, 2.0%, 1.5%]
N N
Norcotinineglucuronide (Norcot-GIu) [0.1%, 0.1%, 0.99) [32,33] , and as a result researchers have preferred saliva as the specimen of choice owing to its easy collection and observation. Reference intervals for nicotine and Cot in saliva, plasma and urine are summarized in Table 2. It is important to note that the ranges provided in Table 2 are general. While several studies have been recently published that provide optimal cotinine cut-offs for serum [34] , urine [35] and saliva [36] , we choose to refrain from providing a single cut-off value because cotinine concentrations of passive smokers will vary depending on the prevalence of smoking due to different cultures and laws. In addition, for subjects undergoing nicotine replacement therapies, measurement of urinary anabasine and/or anatabine are the recommended biomarkers to assess abstinence because they are present in tobacco but not in nicotine medications [37,38] . Another limitation for the measurement of Cot in saliva, plasma or urine is that it only reflects recent exposure to tobacco (3-4 days) because of its short half-life. As a result, the search for other markers or matrices that can predict long-term exposure to tobacco use have led to analysis of nicotine and/or metabolites in a variety of other human matrices, including hair, toenails, meconium and fetal brain (post-mortem). The latest development of MS methods covering all these matrices and others will be discussed in detail in a later section (‘MS-based assays’). In short, selection of the appropriate biomarkers and matrices to assess nicotine exposure depends on the intended use and the target population. Biomarkers for tobacco exposure
Despite its high specificity, nicotine’s relatively short half-life (2 h) and low concentrations in blood make it a poor marker and its measurement for tobacco intake or exposure is not recommended. Cotinine, which is also highly sensitive, has a much longer half-life (16 h) and circulates at concentrations five- to ten-fold higher than nicotine making it the best biomarker for monitoring tobacco use [27] . Limitations of measuring Cot or any single biomarker in blood, saliva and urine have led some groups to develop methods for the detection of multiple metabolites in urine. The molar sum of nicotine and its metabolites in urine has been proposed as a measure of total nicotine exposure and is referred to as nicotine equivalence (NE) [41] . It is believed that if a high percentage of the metabolites excreted in urine are covered, it would provide a useful tool
future science group
Recent advances in MS methods for nicotine & metabolite analysis in human matrices: clinical perspectives
Review
Table 2. Suggested reference intervals and cut-offs for nicotine and cotinine in saliva, plasma and urine to help distinguish non-smokers, passive smokers and active smokers. Smoke exposure Non-smokers
Saliva (ng/ml)
Plasma (ng/ml)
Urine (ng/ml)
Nic
Cot
Nic
Cot
Nic
Cot
N/A
For reprint orders, please contact [email protected]
Recent advances in MS methods for nicotine and metabolite analysis in human matrices: clinical perspectives
Tobacco smoking is a major global health issue and represents the leading cause of preventable death in the developed countries. Nicotine is a major alkaloid found in tobacco products and its detection with its metabolites in human matrices is generally used for assessing tobacco consumption and second hand exposure. Several analytical techniques have been developed for the detection of nicotine and its metabolites, and MS coupled with chromatography is considered the standard reference method because of its superior sensitivity and specificity. In this work, we reviewed nicotine metabolism, clinical MS and the latest (2009–2014) development of MSbased techniques for measurement of nicotine and metabolites in human matrices. Appropriate biomarker and matrix selection are also critically discussed.
Tobacco smoking and addiction is a major global public health issue with welldocumented health risks, including cancer, cardiovascular disease and chronic pulmonary obstructive disease [1] . Over the last two decades, awareness regarding the adverse health effects of tobacco use has been on the rise and the prevalence of cigarette smoking has been declining [2] . Nevertheless, smoking and exposure to second-hand smoke (SHS) remains the leading cause of preventable deaths in the USA and is responsible for one in five deaths in US adults [2,3] . As a result, smoking is associated with substantial healthcare costs in the USA (estimated at US$133 billion a year) [4,5] and worldwide [6–8] . This has led US legislators to take action to reduce the impact of smoking on healthcare and the economy. As a result, smoking in enclosed public spaces and commercial establishments is banned in most states to curb the effects of SHS [9] and insurance companies were required to cover costs associated with smoking cessation programs without any patient cost sharing as mandated by the Affordable Care Act of 2010 [10] . Similar policies have been implemented worldwide [11–13] . In addition, some employers (mainly hospitals) have taken a step further by imposing a ban on hiring smokers or any
10.4155/BIO.14.176 © 2014 Future Science Ltd
Joe M El-Khoury‡,1 & Sihe Wang*,1 1 Department of Clinical Pathology, Cleveland Clinic, Cleveland, OH 44195, USA *Author for correspondence: Tel.: +1 216 445 2634 Fax: +1 216 445 0212 [email protected] ‡ Current address: Department of Laboratory Medicine, Yale University, New Haven, CT 06511, USA
applicant whose urine tests positive for nicotine use [14] . Consequently, laboratory testing for nicotine and/or metabolites has surged worldwide owing to compliance monitoring for smoking cessation programs, pre-employment drug screening programs, and ongoing public health studies. Traditionally, nicotine and/or metabolites have been measured for the evaluation of subjects for organ transplantations (as donors or recipients) [15] , for acute nicotine poisoning [16] , and to assess exposure to environmental tobacco smoke, especially during prenatal phase and childhood [1] . In addition, our physicians have also begun to monitor tobacco consumption (smoking and smokeless) for a defined period of time before admitting them for lumbar spinal fusion surgery. This is because nicotine has been shown to inhibit lumbar spinal fusion and promote disc degeneration, and in a large prospective Swedish study (n = 4555) smokers had less improvement after surgery than non-smokers [17] . Hence, nicotine testing has significantly increased over the last few decades and as always laboratories must adapt and ensure that high-quality test results are provided in a timely manner. A plethora of methods have been developed for the measurement of nicotine and/or
Bioanalysis (2014) 6(16), 2171–2183
part of
ISSN 1757-6180
2171
Review El-Khoury & Wang
Key term ESI: A technique that generates ions at atmospheric pressure by spraying a sample solution through a capillary tube with a high electric voltage in the presence of a flow of warm inert gas to assist in desolvation.
metabolites in a variety of human matrices [18] . These methods include radioimmunoassay, ELISA, HPLC, GC–MS and LC–MS [18] . MS, especially when coupled with GC or LC separation techniques, is a very powerful analytical tool that provides high sensitivity and specificity for the analysis of clinically relevant analytes in biological fluids [19] , and is considered the standard reference method for nicotine and metabolites [18] . The aim of this work was to review the most recent advances in mass spectrometric method development for the analysis of nicotine and metabolites in human matrices and to provide recommendations for selection of suitable biomarkers, methods and cut-offs for distinguishing active smokers from passive smokers and nonsmokers. An overview of clinical MS applications and nicotine metabolism is provided, followed by a critical review of MS-based assays for nicotine and/or metabolites published in the last 5 years (March 2009 to February 2014). A systematic approach was used for article selection. A PubMed search using the logic ‘nicotine’ OR ‘cotinine’ OR ‘nornicotine’ OR ‘anabasine’ AND ‘mass spectrometry’ restricted to the last 5 years was performed on Friday 28 February 2014 and yielded 306 results. All 306 abstracts were reviewed and articles reporting development of new MS methods for the measurement of nicotine and/or metabolites in human matrices were selected. Limitations of our approach include being restricted to articles indexed in PubMed and published in English. Clinical MS applications MS is a powerful qualitative and quantitative analytical technique involving the separation and detection of gas-phase ions according to their mass-to-charge ratios. This technology has been around for over a century but its use was largely restricted to highly
technical research laboratories for the most part. Adoption of MS in clinical laboratories has been rapidly increasing in the last decade largely due to the development of soft ionization techniques, such as ESI and MALDI, which allow the analysis of a wide range of small and macro-molecules in biological fluids [20] . As a result, ESI/MS coupled to LC (LC–ESI/MS) has found applications in many areas, such as endocrinology, therapeutic drug monitoring, inborn errors of metabolism, clinical and forensic toxicology, and nutrition assessment, while MALDI coupled to MS has applications in microbiology, proteomics and lipidomics [21,22] . The main driving force behind the implementation of MS testing in all these different areas of laboratory medicine is the superior sensitivity and selectivity that this technique provides. This advantage, combined with the ability of simultaneous multianalyte quantification, and the versatility makes MS a valuable tool for clinical research and laboratory medicine. The strengths and limitations of MS are summarized in Table 1, and the reader is encouraged to review these excellent references for more information on MS and the role it plays in laboratory medicine [23–26] . MS has certainly proven its utility for clinical laboratories, however, there are still several key challenges it must overcome, including high instrument cost, appropriate field service support, interfacing with the laboratory information system, standardization, throughput, robustness and automation. Nicotine pharmacokinetics Nicotine (Figure 1) is the principal tobacco alkaloid and exists mainly in the levorotatory form (S)-nicotine, while (R)-nicotine accounts for only 0.1–1.2% of total nicotine [27] . It is carried proximally on tar droplets after distillation from burning tobacco then absorbed rapidly in the pulmonary system. Absorption is pH dependent and nicotine is more rapidly absorbed across biological membranes in its basic unionized form, while acidic conditions reduce its absorption [27] . This also explains why nicotine in its basic form is well
Table 1. Strengths and limitations of MS.
2172
Strengths
Limitations
• High sensitivity and specificity • Ability to measure a wide range of compounds • Ability to measure multiple analytes in a single run (profiling) • Low reagent cost per sample • Provides high flexibility and speed for new assay development
• High instrument cost • Relatively low throughput • High complexity requiring extensive training and highly skilled personnel • Lack of appropriate service support from manufacturers • Often involves labor-intensive sample preparation • Not fully adapted for clinical laboratories
Bioanalysis (2014) 6(16)
future science group
Recent advances in MS methods for nicotine & metabolite analysis in human matrices: clinical perspectives
N N
+
Nicotine-N-oxide glucuronide (Nic-Oxi-GIu) [0.4%, 0.2%, 0.1%]
N
HO2C
OH
N+
OH
N
OH Nicotine-N-b glucuronide (Nic-GIu) [1.8%, 1.3%, 2.1%]
N
OH
CO2H
CYPs 2A6 2A13
4-hydroxy-4-(3-pyridyl) butanoic acid (HyPyBut)
CH3
N+ O
Cotinine-N-oxide (Cot-Oxi) [2.8%, 3.3%, 3.6%]
OH
AO
CH3
Nornicotine (Nornic) [3.6%, 6.9%, 4.7%]
Nornicotineglucuronide (Nornic-GIu) [0.1%, 0.1%, 0.1%]
O
CH3
N
Trans-3’-hydroxycotinine (Hcot) [20.8%, 26.8%, 38.7%]
Cotinine (Cot) [10.4%, 8.6%, 9.3%] CYP 2A6
UGTs 2B7,1A9 N
N
N CYP 2A6
CH3
N
CYP 2A6
N H
O
N
CYP 2A6 2A13
[10.2%, 9.7%, 7.7%]
4-hydroxy-4-(3-pyridyl) butanoic acid-glucuronide (HyPyBut-GIu)
O
N
UGTs 2B1010,1A4
Nicotine (Nic) [22.8%, 7.7%, 2.2%]
N
[0.7%, 0.7%, 0.6%]
OH
H3C O
FMO-3
N
CH3
OH
Nicotine-N-oxide (Nic-Oxi) [10.2%, 15.4%, 10.4%] UGTs 2B10,1A4
O
Cotinine-N-b-glucuronide (Cot-GIu) [8.0%, 9.7%, 11.0%]
O HO2C
Cotinine-N-oxide glucuronide (Cot-Oxi-GIu) [0.1%, 0.3%, 0.4%]
O
OH
CH3
N+
Review
O
N
HO O
Norcotinine (Norcot) [1.3%, 2.0%, 1.5%]
N N
Norcotinineglucuronide (Norcot-GIu) [0.1%, 0.1%, 0.99) [32,33] , and as a result researchers have preferred saliva as the specimen of choice owing to its easy collection and observation. Reference intervals for nicotine and Cot in saliva, plasma and urine are summarized in Table 2. It is important to note that the ranges provided in Table 2 are general. While several studies have been recently published that provide optimal cotinine cut-offs for serum [34] , urine [35] and saliva [36] , we choose to refrain from providing a single cut-off value because cotinine concentrations of passive smokers will vary depending on the prevalence of smoking due to different cultures and laws. In addition, for subjects undergoing nicotine replacement therapies, measurement of urinary anabasine and/or anatabine are the recommended biomarkers to assess abstinence because they are present in tobacco but not in nicotine medications [37,38] . Another limitation for the measurement of Cot in saliva, plasma or urine is that it only reflects recent exposure to tobacco (3-4 days) because of its short half-life. As a result, the search for other markers or matrices that can predict long-term exposure to tobacco use have led to analysis of nicotine and/or metabolites in a variety of other human matrices, including hair, toenails, meconium and fetal brain (post-mortem). The latest development of MS methods covering all these matrices and others will be discussed in detail in a later section (‘MS-based assays’). In short, selection of the appropriate biomarkers and matrices to assess nicotine exposure depends on the intended use and the target population. Biomarkers for tobacco exposure
Despite its high specificity, nicotine’s relatively short half-life (2 h) and low concentrations in blood make it a poor marker and its measurement for tobacco intake or exposure is not recommended. Cotinine, which is also highly sensitive, has a much longer half-life (16 h) and circulates at concentrations five- to ten-fold higher than nicotine making it the best biomarker for monitoring tobacco use [27] . Limitations of measuring Cot or any single biomarker in blood, saliva and urine have led some groups to develop methods for the detection of multiple metabolites in urine. The molar sum of nicotine and its metabolites in urine has been proposed as a measure of total nicotine exposure and is referred to as nicotine equivalence (NE) [41] . It is believed that if a high percentage of the metabolites excreted in urine are covered, it would provide a useful tool
future science group
Recent advances in MS methods for nicotine & metabolite analysis in human matrices: clinical perspectives
Review
Table 2. Suggested reference intervals and cut-offs for nicotine and cotinine in saliva, plasma and urine to help distinguish non-smokers, passive smokers and active smokers. Smoke exposure Non-smokers
Saliva (ng/ml)
Plasma (ng/ml)
Urine (ng/ml)
Nic
Cot
Nic
Cot
Nic
Cot
N/A
