Selective extraction of tobacco-specific nitrosamines from urine

Introduction

Tobacco-specific nitrosamines, TSNAs, are carcinogens found in tobacco products, including cigarettes, e-cigarettes and smokeless tobacco. NNN (N-nitrosonornicotine), NNK (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone), and NNAL (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol) are the most analyzed TSNAs. NNK has been attributed to lung cancer in humans (1), NNAL is a metabolite of NNK, and NNN has been associated with the risk of causing esophageal cancer in smokers (2).

TSNAs can be difficult to accurately detect as, like nicotine, they may be subject to false positive results caused by second-hand and third-hand contamination. Second-hand contamination, or second-hand smoke, may cause non-smokers to test positive for TSNAs when highly sensitive analytical methods are used. Third-hand contamination can arise from residual material present on test tubes, extraction media, pipette tips, or other consumables used throughout the sample extraction and analysis process. In addition, urine is a complex biological matrix that can introduce matrix effects and interfere with sensitive LC-MS/MS measurements, making effective sample preparation critical for reliable quantification.

The strongest basic pKa values of the analytes are 4.79, 3.96, and 4.73 for NNN, NNK, and NNAL, respectively. The octanol-water partition coefficient (logP) values are 1.011, 0.579, and 0.492 for NNN, NNK, and NNAL, respectively.

This application note demonstrates an optimized sample preparation and extraction workflow for NNN, NNK, and NNAL in urine prior to UPLC-MS/MS analysis.

Analytes

N-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL).

Sample preparation procedure

Sample extraction experiments were conducted using non-smoker donor urine (pH 6.30) to evaluate the recovery of TSNAs. Synthetic urine (UriSub, pH 7.4) was used for calibration solutions to avoid potential interference from human urine that may contribute to the TSNA signal.

Format

EVOLUTE® EXPRESS CX 30 mg Fixed Well Plate, Part Number: 601-0030-PX01

Processing

Samples were processed using Biotage® PRESSURE+ 96 Positive Pressure Manifold, (P/N PPM-96). Alternatively, sample preparation can be automated using the Biotage® Extrahera™ automated sample preparation workstation.

Sample pre-treatment

Sample pre-treatment was performed by mixing 150 µL urine from non-smoker human donor or synthetic urine (UriSub, pH 7.4) with 850 µL 0.5 M ammonium formate and 20 µL formic acid. The pre-treated sample was fortified (pre-spike) with 200 pg/mL TSNAs (NNN, NNK, and NNAL) for extraction recovery performance experiments. Non-fortified pre-treated urine was used for blank, pre-spike, and post-spike experiments. Calibration solutions were prepared using pre-treated synthetic urine (UriSub) fortified with 1.0-1000.0 pg/mL of TSNAs.

Extraction procedure

Sample loading

Load 1.0 mL of pre-treated urine (blank/fortified) sample onto the plate and apply vacuum or positive pressure (1-2 psi) to load the sample.

Wash 1

Elute polar interferences with 10% methanol in water (v/v, 3x1000 µL)

Wash 2

Elute nonpolar interferences with 100% methanol (2x1000 µL)

Elution

Elute analytes with 500 µL 5% ammonium hydroxide in methanol (v/v)

Post-extraction

Dry the extract in a stream of air or nitrogen using a TurboVap® Dual at a gas and plate temperature of 50 °C, plate height 70 mm, and gas flow of 25 L/min for 20 minutes.
Reconstitute in 200 µL of the initial mobile phase composition (acetonitrile:2 mM ammonium formate, 15:85 v/v).

Analytical conditions

U/HPLC conditions

• Instrument: Shimadzu Nexera X2 (LC-30AD)

• Column: Restek Raptor ARC-18 (2.7 µm, 100 x 2.1 mm, p/n 9314A12)

• Mobile phase(s): acetonitrile/2 mM ammonium formate (15:85, v/v)

• Flow rate: 0.45 mL/min

• Gradient details: isocratic method, 5.10 min

• Column temperature: 50 ᵒC

• Injection volume: 5.00 µL

• Sample temp (cooler temperature): 8 ᵒC

MS/MS conditions

• Instrument (SCIEX, 5500 MS/MS)

• Polarity: Positive

• Source temp: 500 ᵒC

• Curtain gas: 20

• Collision Gas (CAD): 8

• IonSpray voltage (IS): 5500

• Source gas(s): 50, 50

• MRM parameters

Table 1. Analyte MRM transitions and ionization settings. 

Results

Using the method presented in this application note, a panel of three TSNA compounds were spiked into pre-treated human urine and extracted using EVOLUTE® EXPRESS CX 30 mg Fixed Well Plate. All analytes demonstrated excellent recovery (>85%) and satisfactory matrix effect (0.73-0.95) (Table 2, Figure 1). Extraction recovery for TSNAs were 88%, 90%, and 93%, for NNN, NNK, and NNAL respectively (Figure 1(A)), demonstrating the robustness of the extraction method. Matrix effects for TSNAs were 0.87, 0.95, and 0.73 for NNN, NNK, and NNAL respectively (Figure 1(B)) indicating relatively lower matrix suppression for the analytes representing clean sample extract.

Table 2. Analyte LOQ, linearity, recovery, and matrix effect.

Figure 1. (A) Extraction recovery and (B) matrix effect for TSNAs in human urine. NNN (n-nitrosonornicotine), NNK (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone), and NNAL (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol). %RSD shown as error bars (n=3). 

Linearity and LOQ

The concentration range for TSNAs calibration curves was 1.0, 10.0, 20.0, 50.0, 100.0, 150.0, 200.0, 500.0, and 1000.0 pg/mL. Calibration curves for TSNAs showed good linearity with R2 values of 0.9998, 0.9996, and 0.9997, for NNN, NNK, and NNAL, respectively (Figure 2(A-C)). Limits of quantification (LOQs) determined by linear regression analysis were 20, 13, and 36 pg/mL for NNN, NNK, and NNAL respectively (Figure 2(D)).

Figure 2. Calibration curves for (A) NNN, (B) NNK, (C) NNAL, and (D) limit of quantification for TSNAs in UriSub.

Summary and conclusions

This application note demonstrates an effective extraction procedure for the quantification of TSNA from human urine at low LOQ levels. The method enables TSNAs to be quantified within clinically relevant quantification ranges (3-5). Due to the potential for contamination of control urine samples, spiked water or synthetic urine should be run to determine LOQ/LODs along with water or synthetic urine that has not been spiked. This will help determine at what concentration the negative samples may contain TSNAs. Running several donor urine samples from both smokers and non-smokers could help to determine reasonable cutoff concentrations. Careful control of second-hand and third-hand TSNA contamination is important during method development and validation.

EVOLUTE® EXPRESS CX well plates and columns offer an efficient alternative to traditional and time-consuming liquid-liquid extraction (LLE) process for bioanalytical sample preparation (6). The CX sample clean-up procedure provides high analyte recoveries and can be fine-tuned to enrich target analyte for the detection and quantification at sub-pico gram levels (7). On the other hand, supported liquid extraction (SLE) protocol developed earlier offers a simple, more indiscriminate extraction method for the analysis of broader analyte panel at clinically relevant concentration levels.

Using EVOLUTE® EXPRESS CX  enables a rapid sample preparation workflow while generation cleaner extracts and maintaining strong analytical performance. 

Chemicals and reagents

• TSNA standards are NNK (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone) (AccuStandard, 99.9%), NNAL (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol) (LGC Standards, 99.94%), and NNN (n-nitrosonornicotine) (LGC Standards, 99%).

• The working standard solutions were prepared by diluting or dissolving in mobile phase through serial dilutions and stored in the refrigerator (<4 ᵒC).

• Synthetic urine (UriSub, pH 7.4) was used without further modification. Human urine was collected from a healthy non-smoker donor and used fresh without further modification

Standard statements

All extract cleanliness data shown in this application note was generated using real, intact matrix, obtained from human volunteers or other sources, as stated.

References:

1. Derby, K.S., Cuthrell, K., Caberto, C., Carmella, S., Murphy, S.E., Hecht, S.S. and Le Marchand, L. (2009), Exposure to the carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in smokers from 3 populations with different risks of lung cancer. Int. J. Cancer, 125: 2418-2424.
2. Yuan, J.M.; Knezevich, A.D.; Wang, R.; Gao, Y.T.; Hecht, S.S.; Stepanov, I. Urinary levels of the tobacco-specific carcinogen N'-nitrosonornicotine and its glucuronide are strongly associated with esophageal cancer risk in smokers. Carcinogenesis 2011, 32, 1366–1371.
3. Stephen S. Hecht, Irina Stepanov, and Steven G. Carmella. Exposure and Metabolic Activation Biomarkers of Carcinogenic Tobacco-Specific Nitrosamines. Acc. Chem. Res. 2016, 49, 1, 106–114.
4. Delshanee Kotandeniya, Steven G. Carmella, Xun Ming, Sharon E. Murphy, Stephen S. Hecht. Combined Analysis of the Tobacco Metabolites Cotinine and 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol in Human Urine. Anal. Chem. 2015, 87, 3, 1514–1517.
5. Nikam, S.S., Gurjar, M., Singhavi, H. et al. Simultaneous analysis of urinary total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol, N′-nitrosonornicotine, and cotinine by liquid chromatography-tandem mass-spectrometry. Sci Rep 11, 20007 (2021)
6. Sepehr, E., Woodling, K. A., & Bryant, M. S. (2018). Rapid quantitation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in rat urine using ultra-fast liquid chromatography mass spectrometry (UFLC/MS/MS). Journal of Liquid Chromatography & Related Technologies, 41(8), 422–429.
7. Xia, B., Xia, Y., Wong, J., Nicodemus, K.J., Xu, M., Lee, J., Guillot, T. and Li, J. (2014), Quantitative analysis of five tobacco-specific N-nitrosamines in urine by liquid chromatography–atmospheric pressure ionization tandem mass spectrometry†. Biomed. Chromatogr., 28: 375-384
   

Literature number: AN1034

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