Elsevier

Journal of Chromatography B

Volume 928, 1 June 2013, Pages 139-145
Journal of Chromatography B

Simultaneous serum nicotine, cotinine, and trans-3′-hydroxycotinine quantitation with minimal sample volume for tobacco exposure status of solid organ transplant patients

https://doi.org/10.1016/j.jchromb.2013.03.032Get rights and content

Highlights

  • A rapid LC–MS/MS assay quantitating nicotine and its metabolites was validated.

  • We developed this assay using only one-hundred microliter of serum.

  • Quantitating both cotinine and 3-OH-Cot is essential to evaluate tobacco exposure

Abstract

Concentrations of nicotine and its metabolites in blood are indicative of patients’ current tobacco exposure, and their quantifications have been clinically applied to multiple assessments including demonstration of abstinence prior to heart–lung transplantation. For the purpose of transplant evaluation, the laboratory work up is extensive; thereby an assay with minimal sample volume is preferred. We developed and validated a rapid LC–MS/MS assay to simultaneously quantitate nicotine and its major metabolites, Cotinine and trans-3′-OH-cotinine (3-OH-Cot), in serum. 100 μL of serum was spiked with deuterated internal standards and extracted by Oasis HLB solid phase extraction cartridge. Nicotine and metabolites in the reconstituted serum extract were separated by Agilent Eclipse XDB-C8 3.5 μm 2.1 mm × 50 mm HPLC column within 4.7 min, and quantified by MS/MS with positive mode electrospray ionization and multiple reaction monitoring. Ion suppression was insignificant, and extraction efficiency was 79–110% at 50 ng/mL for all compounds. Limit of detection was 1.0 ng/mL for nicotine and 3-OH-Cot, and <0.5 ng/mL for Cotinine. Linearity ranges for nicotine, cotinine and 3-OH-Cot were 2–100, 2–1000, and 5–1000 ng/mL with recoveries of 86–115%. Within-day and twenty-day imprecision at nicotine/cotinine/3-OH-Cot levels of 22/150/90, 37/250/150, and 50/800/500 ng/mL were all 1.1–6.5%. The reconstituted serum extracts were stable for at least 7 days stored in the HPLC autosampler at 5 °C. Our method correlates well with alternative LC–MS/MS methods. We successfully developed and validated an LC–MS/MS assay to quantitate concentrations of nicotine and its metabolites in serum with minimal sample volume to assess tobacco exposure of heart–lung transplant patients.

Introduction

Tobacco use is one of the main preventable risk factors leading to major diseases, cancers, and death worldwide. It has also been found even more addictive than other substances of abuse, such as alcohol, cocaine, and marijuana. One of the chemical constituents in cigarette smoke causing the addiction is nicotine. It is most effectively absorbed by the lungs through inhalation of the tobacco smoke and rapidly delivered to the brain. Nicotine activates nicotinic acetylcholine receptors in both the central and sympathetic nervous systems and subsequently releasing several neurotransmitters, such as epinephrine and norepinephrine giving a stimulant effect. Other sedative, relaxant neurotransmitters like serotonin and β-endorphin are also released by the nicotinic acetylcholine receptor mechanism, and their effects are more apparent at a higher dose. In addition, some other substances present in cigarette smoke are monoamine oxidase inhibitors [1], [2]. They prohibit oxidation of the nicotine-releasing neurotransmitters, thereby enhance the nicotine effects. Such co-administration of substances through tobacco smoking ultimately causes the addiction behavior. Chronic tobacco smoking adversely impacts human organs and wound healing [3]. Abstinence from tobacco smoking is thus one of the crucial requirements for being a qualified solid organ transplant recipient.

Although nicotine effects on solid organ transplant outcomes are yet elusive, a number of studies have demonstrated the higher graft failure, increased risks of malignancy, and lower survival rates for organ recipients who was abstinent for less than five years prior to and relapsed smoking habits following the surgery [4], [5], [6]. Surprisingly, post-transplant active smokers are quite prevalent: 21% in heart transplants [5] and 25% in kidney transplants [7]; such data is not available amongst liver transplants, but one study indicated that 50% of liver transplant candidates who were active smokers continued to smoke at least within three months post-transplant [8]. Continuous monitoring of transplant patient's exposure to tobacco smoke and smoking cessation therapies are therefore essential practices in qualifying transplant candidates and transplant management.

The extent and length of exposure to tobacco smoke can be estimated by nicotine level in body fluids. Nicotine can reach 10 ng/mL in the blood after one cigarette is smoked, and plateau in average of 40 ng/mL when more cigarettes are consumed during the day [9]. Plasma elimination half-life is only 1–2 hrs [10], thus quantitating nicotine in plasma alone is not a sensitive enough measure of tobacco smoke exposure. About 2–35% of nicotine is excreted to the urine depending on urinary pH, and 70–80% is metabolized by the liver to cotinine involving the Cytochrome P450 2A6 enzyme. Cotinine is further metabolized to trans-3′-hydroxycotinine (3-OH-Cot), and both compounds have much longer half lives, 18 and 7 h, respectively [11]. Other metabolites include nicotine N′-oxide (4–7%), nicotine glucuronide (3–5%), nornicotine (1%), 2′-OH-nicotine (1%) which are readily excreted into urine. Simultaneously quantitating nicotine, cotinine, and 3-OH-cot gives more accurate interpretation about the recent nicotine intake. Quantitating the three compounds is valuable for the transplant management, as nicotine alone elevates risks for microvascular injuries, malignancies, and microbial infections that impede success of transplantation.

Transplant evaluation requires extensive blood tests, including immunocompatibility tests, serology tests for various infectious diseases, complete metabolic panel, cancer markers, and substance use [12]. A nicotine quantitation assay requiring minimal blood volume without sacrificing sensitivity is desired. The lower limit of quantitation (LLOQ) should be as low as 2 ng/mL for at least cotinine, so that physicians can identify patients inhaling second hand tobacco smoke, which can also impact transplant outcomes [9].

The sample volume, sample treatment and LLOQ from previously reported serum/plasma nicotine and metabolite HPLC assays are briefly described as the following. Bernert et al. [13] extracted 1 mL of serum with methylene chloride only quantitating cotinine as low as 0.6 ng/mL. Moyer et al. [9] used an SPE approach to extract 1 mL of plasma and reached lower limit of quantitation (LLOQ) of 2 ng/mL. Byrd et al. [14] extracted 100 μL serum with SPE cartridges and LLOQ were 1.8 and 7.7 ng/mL for nicotine and cotinine, respectively; 3-OH-Cot was not included in this assay. Beyer et al. [15] used an SPE approach to extract 1 mL plasma reaching LLOQ of 1 ng/mL for both nicotine and cotinine, but did not include 3-OH-Cot either. Massadeh et al. [16], extracted 500 μL plasma by liquid-liquid extraction reaching LLOQ of 1.0 and 0.9 ng/mL for nicotine and cotinine. Miller et al. [17] took 1 mL of plasma with a combination of two different sorbent-based SPE cartridges reaching LLOQ of 1 ng/mL for all nicotine, cotinine and 3-OH-Cot. Baumann et al. [18] used 500 μL serum extracted by an SPE approach and LLOQ was 2 and 5 ng/mL for nicotine and cotinine. Jacob et al. [19] treated 100 μL of plasma with protein precipitation followed by liquid-liquid extraction, LLOQ was 1 ng/mL for only cotinine and 3-OH-Cot.

Here we present an LC–MS/MS assay quantitating nicotine, cotinine and 3-OH-Cot as low as 2 ng/mL with only 100 μL of serum through a rapid sample preparation procedure.

Section snippets

Sample collection and handling

Patient blood was collected in a red-top tube. Following sufficient clotting and centrifugation, serum was aliquoted and sent out to referral laboratory for nicotine quantitation, and a separate serum aliquot was stored under −20 °C until in-house analysis. Only the serum samples reported with greater than 2 ng/mL of either nicotine, 3-OH-Cot, or cotinine by the referral laboratory were selected for further correlation studies herein. A total of 27 such serum samples were selected during a three

Ion suppression and extraction efficiency

The traces of all three compounds infused to HPLC eluent from reconstitution solution versus extracted serum were recorded and compared in both quantifying ion and confirmatory ion transitions (Fig. 2). The overlayed ion intensities in each ion transition channel do not show significant ion suppression by the extracted serum at the compound's retention time.

The effect of serum extract on each compound ionization was determined by the peak areas from A and B types of samples described in the

Discussion

Reference ranges for different extent and length of tobacco exposure established by Moyer et al. [9] have been widely used. Briefly, all three analytes of interest herein would be <2 ng/mL if one is free of tobacco exposure or considered abstinent for more than two weeks; second hand tobacco smoke would give 2–8 ng/mL cotinine while nicotine and 3-OH-Cot could still be <2 ng/mL. Active cigarette consumption would have ranges of 30–50 ng/mL, 200–800 ng/mL, and 100–500 ng/mL for nicotine, cotinine, and

Acknowledgements

We thank Drs. David Bernard and Wayne Chandler for providing insights to method validation, and Joanne Topper for collecting specimens to for method comparison.

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