Determination of arecoline (areca nut alkaloid) and nicotine in hair by high-performance liquid chromatography/electrospray quadrupole mass spectrometry

RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2005; 19: 3416–3418 Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.2183 RCM Letter to the Editor To the Editor-in-Chief Sir, Determination of arecoline (areca nut alkaloid) and nicotine in hair by highperformance liquid chromatography/ electrospray quadrupole mass spectrometry The areca nut (or betel nut), composed of the sliced nut (areca nut) of the areca palm (Areca catechu), the leaf of the betel pepper (Piper betle), cut tobacco and lime is the fourth most commonly used drug in the world after tobacco, alcohol and caffeine.1 It is commonly consumed as betel quid (folded leaf package) chewing or betel nut smoking by Asian populations (both men and women) and Asian communities living in Europe and North America.2,3 Different chemical compounds have been identified in the nut, but arecoline, the principal alkaloid from the areca nut, is thought to be responsible for a central cholinergic stimulation and monoamine transmission, which then activates both sympathetic and parasympathetic effects.4 Chewing areca nut on a habitual basis is known to be deleterious to human health, especially in relation to the risk of the development of oral cancer.5,6 Recently, we developed and validated a high-performance liquid chromatography method with mass spectrometric detection to determine arecoline in the meconium of newborns from betel chewer mothers to assess fetal exposure to this alkaloid and in the placenta, to be associated with studies on the morphology of placental tissue from consumer mothers.7 Two adverse birth outcomes were observed within the six newborns, whose meconium was positive to arecoline and focal inflammatory changes in the amniochorial membranes (focal acute chorioamnionitis) were observed in placentas originating from these two cases.8 Whereas meconium is a biological matrix for assessing chronic exposure to xenobiotics during the prenatal period, hair is used to reveal repetitive exposure not only in newborns, but also in children and adults.9 For this reason, we sought to develop a digestion and extraction procedure for arecoline and nicotine (the hair biomarker for consumption of tobacco, which is usually present in betel preparations) in keratin matrix combined with the analysis of the extracts by highperformance liquid chromatography/ electrospray quadrupole mass spectrometry (LC/ESI-MS) methodology already validated for meconium, cord serum and urine.7 Figure 1 shows the structures of analytes under investigation together with the internal standard (IS) used. Hair samples, collected from chronic betel nut consumers, were cut at the scalp in the vertex region and the entire strand was washed three times (2 min) with 3 mL dichloromethane in an ultrasonic waterbath and allowed to dry at room temperature. Washed hair specimens were then pulverized with a ball mill (Retsch, Haan, Germany) for 10 min at 90 amplitude units. The 50 mg of pulverized hair were added to 5 mL of IS working solution (10 mg/mL) and incubated with 2 mL 12 M NaOH at 408C for 18 h. After the incubation, the alkaline mixture was extracted with 5 mL chloroform/isopropanol (95:5, v/v) by vortex-mixing for 2 min and centrifuging at 2000 g for 5 min. The organic layer was transferred to another tube containing 2.5 mL 0.5 M HCl. The tube was vortex-mixed for 2 min, centrifuged at 2000 g for 5 min, and the organic layer discarded. The acidic layer was neutralized with 1 mL 1 M NaOH and made alkaline with 2 mL NH4Cl saturated solution adjusted to pH 9.5 with concentrated ammonia. Re-extraction with 5 mL of chloroform/ isopropanol (95:5, v/v) was finally conducted for 5 min. The organic phase was evaporated to dryness under a stream of nitrogen and the residue was dissolved in 100 mL of 10 mM ammonium acetate (pH 4.3) solution. A 20 mL volume was injected onto the LC column. When the concentrations of the analytes in hair were higher than those in the calibration curve range, samples were re-injected after appropriate dilution in mobile phase. Chromatographic separation was achieved by an LC system (Agilent 1100 series HPLC system; Agilent Technologies, Palo Alto, CA, USA) equipped with a C18 reversedphase column (Phenomenex Luna C18, 150
4.6 mm
3 mm; Chemtek Analytica, Anzola, Emilia, Italy), using a 10 mM ammonium acetate (pH 4.3)/acetonitrile (90:10, v/v) Figure 1. Molecular structures of arecoline, nicotine and the internal standard, pilocarpine. Copyright # 2005 John Wiley & Sons, Ltd. Letter to the Editor solution as a mobile phase at a flow rate of 0.5 mL/min. The mass spectrometer (Agilent 1100 series G1946D) was operated in positive ESI mode using selected ion monitoring (SIM) acquisition. The following ESI conditions were applied: drying gas (nitrogen) heated at 3508C at a flow rate of 10.0 L/h; nebulizer gas (nitrogen) pressure of 40 psi; capillary voltage 1500 V, fragmentor voltage (applied to the exit end of the capillary) 110 V, dwell time 139 ms, and mass peak width 0.10 min. Characteristic ions were m/z 156, 140, and 118 for arecoline, 163, 132 and 106 for nicotine, and m/z 209, 96 and 95 for pilocarpine. The protonated molecules at m/z 156 for arecoline, m/z 163 for nicotine, and m/z 209 for pilocarpine were selected for quantification. Chromatograms of an extract of drug-free hair sample spiked with IS only (A), an extract of drug-free hair sample spiked with 1 ng/mg arecoline and nicotine (B) and an extract of hair sample from betel nut consumer (C) are shown in Fig. 2. The method was tested in a 3-day validation protocol, following the accepted criteria for bioanalytical method validation.10 Selectivity, recov- ery, matrix effect, linearity, precision, accuracy, limits of detection and quantification were assessed. Calibration standards containing 0.3, 1, 2, 4 and 10 ng arecoline/mg hair and 0.8, 2, 4, 8 and 15 ng nicotine/mg hair were prepared daily for each analytical batch by adding suitable amounts of methanolic working solutions to 50 mg drug-free hair. Quality control (QC) samples of 8.0 ng/mg arecoline and 12.0 ng/mg nicotine (high level), 3.2 ng/mg arecoline and 6.8 ng/mg nicotine (medium level), and 0.5 ng/mg arecoline and 1.2 ng/mg nicotine (low level) were prepared in drug-free hair, aliquoted and stored at 208C. Over-curve samples containing 20 ng arecoline and nicotine/mg hair were prepared, to be tested for accuracy and precision once diluted 5 and 10 times. Peak area ratios between compounds and the IS were used for weighted (1/concentration) least-squares regression analysis (SPSS, version 9.0.2 for Windows). Absolute analytical recoveries were calculated by comparing the peak areas obtained when QC samples were analyzed by adding the analytical reference standards in the extract of drugfree hair prior to and after the extrac- 3417 tion procedure (four replicates at each concentration). For an evaluation of matrix effects, the peak areas of extracted blank samples spiked with standards at three QC concentration levels after the extraction procedure were compared with the peak areas of pure diluted substances. Five replicates at each of the QC concentrations added to blank hair and over-curve samples after appropriate dilution were analyzed for the determination of intra-assay precision and accuracy. The inter-assay precision and accuracy were determined for three independent experimental assays. Five replicates of blank hair samples were used to calculate the limits of detection and quantification. Standard deviation (SD) of the mean noise level over the retention time window of each analyte was used to determine the limit of detection (LOD ¼ 3 SD) and the limit of quantification (LOQ ¼ 10 SD). The LOQ value was tested for precision and accuracy variation to be better than 20%. The method exhibited good linearity along the calibration range studied. Mean calibration curves (n ¼ 3), presented the following parameters: slope Figure 2. SIM chromatograms of protonated molecules of (A) an extract of 50 mg drug-free hair sample spiked with internal standard only; (B) an extract of 50 mg drug-free hair sample spiked with 50 ng arecoline and nicotine; and (C) an extract of 50 mg hair sample from a betel nut consumer containing 1.10 ng/mg hair arecoline and 8.02 ng/mg hair nicotine. Copyright # 2005 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2005; 19: 3416–3418 3418 Letter to the Editor day) and four betel quid chewing women are shown in Table 2. Although these data are preliminary and the number of analyzed samples does not allow any definite conclusion (precise number of betel nut cigarettes was unknown in men, nor the time of quid chewing in women), it can be noted that the level of arecoline present in hair from women (mean  SD: 1.27  0.20 ng/mg hair) is significantly higher than that measured in hair from men (mean  SD: 0.61  0.52 ng/mg hair). Conversely, hair nicotine shows a trend toward higher values in men (mean  SD: 11. 73  11.86 ng/mg hair) than in the samples from women (mean  SD: 4.71  3.62 ng/mg hair). One plausible hypothesis is that betel quid, normally placed in the mouth and held against the mucosa of the buccal cheek and molar teeth to be episodically chewed to extract juice, releases more arecoline than that made available by smoking a betel nut cigarette in a few minutes. The reason for the higher content of hair nicotine in men is that the majority of them also smoke tobacco cigarettes while women from our study declared to be nonsmokers. In addition, betel quid and betel nut cigarettes probably contain 1.7614, intercept 0.2021, determination coefficient (r2) 0.9997 for arecoline; slope 4.8943, intercept 3.8387, determination coefficient (r2) 0.9937 for nicotine. Absolute analytical recoveries (mean  SD) ranged from 81.2  2.6% for arecoline to 79.2  2.1% for nicotine. With respect to the matrix effect, the comparison between peak areas of analytes spiked in extracted blank samples versus those for pure diluted standards showed less than 10% analytical signal suppression due to coeluting endogenous substances. The intra- and inter-assay precision and accuracy data are presented in Table 1. Over-curve samples, tested for accuracy and precision after diluting 5 and 10 times, gave values always better than 10% relative standard deviation (RSD) and Error %. The LODs were 0.09 and 0.24, and LOQs were 0.30 and 0.80 ng/mg hair for arecoline and nicotine, respectively. Coefficients of variation for precision and accuracy at the LOQ were always better than 20%. The method presented here has been applied to the analysis of hair samples from 11 chronic areca nut consumers (range: 2–35 years of consumption). Data from the seven men declaring betel nut smoking (more than once per Table 1. Intra- (n ¼ 5) and inter-assay (n ¼ 15) precision and accuracy obtained from analytes under investigation Intra-assay Compounds Inter-assay Concentration ng/mg hair Precision (RSD%) Accuracy (Error %) Precision (RSD%) Accuracy (Error %) 0.5 3.2 8.0 1.2 6.8 12.0 4.00 4.84 8.07 3.70 1.93 3.33 4.23 4.71 2.62 6.11 0.94 9.36 4.91 4.79 4.70 3.96 2.44 2.27 3.78 2.77 1.41 4.44 1.15 5.44 Arecoline Nicotine Table 2. Arecoline and nicotine content in hair samples from drug consumers Sample Sex Type of consumption 1 2 3 4 5 6 7 8 9 10 11 F M F M M M F M F M M Betel quid chewing Betel nut smoking Betel quid chewing Betel nut smoking Betel nut smoking Betel nut smoking Betel quid chewing Betel nut smoking Betel quid chewing Betel nut smoking Betel nut smoking Copyright # 2005 John Wiley & Sons, Ltd. Arecoline ng/mg hair Nicotine ng/mg hair 1.18 0.81 1.26 0.33 0.30 0.41 1.10 1.71 1.55 0.35 0.36 1.72 33.02 1.44 5.37 12.46 2.44 8.02 5.50 7.65 22.25 1.06 different amounts of areca nut and cut tobacco. These observations should be confirmed in a higher number of individuals consuming different preparations of areca nut and with data on the number of times per day that the subjects are consuming the drug. In conclusion, for the first time, arecoline, the major alkaloid of areca nut, has been determined in hair from consumers by a simple, reliable and validated LC/MS method after alkaline digestion of keratin matrix and liquid–liquid extraction of analytes. The results suggest that hair can be a non-invasive biological matrix for monitoring chronic use of areca nut preparations. Emilia Marchei1, Abhilasha Durgbanshi2, Silvia Rossi1, Óscar Garcia-Algar3, Piergiorgio Zuccaro1 and Simona Pichini2* 1 Department of Drug Research and Evaluation, Istituto Superiore di Sanitá, Rome, Italy 2 Department of Criminology and Forensic Sciences, Dr. H.S. Gour University, Sagar, India 3 Paediatric Service, URIE, Hospital del Mar, and Universitat Autònoma, Barcelona, Spain *Correspondence to: S. Pichini, Istituto Superiore di Sanità, V.le Regina Elena 299, 00161, Rome, Italy. E-mail: pichini@iss.it REFERENCES 1. Gupta PC, Warnakulasuriya S. Addict. Biol. 2002; 7: 77. 2. Winstock A. Addict. Biol. 2002; 7: 133. 3. Warnakulasuriya S. Addict. Biol. 2002; 7: 127. 4. Chu NS. Addict. Biol. 2002; 7: 111. 5. Chang YC, Yang SF, Tai KW, Chou MY, Hsieh YS. Oral Oncol. 2002; 38: 195. 6. Warnakulasuriya S, Trivedy C, Peters TJ. Br. Med. J. 2002; 324: 799. 7. Pichini S, Pellegrini M, Pacifici R, Marchei E, Murillo J, Puig C, Vall O, Garcia-Algar O. Rapid Commun. Mass Spectrom. 2003; 17: 1958. 8. Garcia-Algar O, Vall O, Alameda F, Puig C, Pellegrini M, Pacifici R, Pichini S. Arch. Dis. Child Fetal Neonatal Ed. 2005; 90: F276. 9. Villain M, Cirimele V, Kintz P. Clin. Chem. Lab. Med. 2004; 42: 1265. 10. Guidance for Industry, Bioanalytical Method Validation, US Department of Health and Human Services, Food and Drug Administration, May 2001. Available: http://www. fda.gov/cder/guidance/ 4252fnl.htm. Received 8 July 2005 Revised 4 September 2005 Accepted 4 September 2005 Rapid Commun. Mass Spectrom. 2005; 19: 3416–3418