TY - JOUR
AU - Joza,, Peter
AB - Abstract A new isotope dilution liquid chromatography/tandem mass spectrometric method was developed for the analysis of potential hydrazine present in tobacco smoke. The sample preparation was performed via an optimized derivatization method using an aqueous buffer:methanol solution of 2-nitrobenzaldehyde (10 g/L) used as a derivatizing agent. The mainstream smoke of cigarettes was passed through a glass fiber filter pad followed by a trapping solution containing an isotopically labeled 15N2-hydrazine used as internal standard. After smoking, the filter pad was extracted with the trapping solution and then incubated for 30 minutes at 35°C. An aliquot of the extract was centrifuged and the resultant hydrazone was quantified by liquid chromatography tandem mass spectrometry (LC-MS/MS). The isotope dilution standard calibration curve demonstrated good linearity (R2 > 0.999) from 0.079 to 248 ng/mL, with limits of quantification in mainstream smoke of 0.2 and 0.4 ng/cig for ISO and Canadian Intense smoking regimens, respectively. The method recovery was assessed using samples spiked with solutions of known amounts of hydrazine. The results showed good accuracy with recoveries ranging from 98 to 111%. Although there were no detectable levels of hydrazine in the reference cigarettes used in the validation (KR3R4F), the method precision was estimated to be ~10% based on the variability observed in the spiked samples. Trapping efficiencies were assessed using a hydrazine permeation tube providing a known amount of hydrazine vapor such that the distribution between the vapor phase and particulate phase of mainstream smoke could be determined. Introduction Hydrazine is a small, polar and strongly reducing agent primarily used as a chemical intermediate to produce agricultural chemicals and water-treatment chemical (1–2). It is widely used as a rocket propellant and has significant applications in the synthesis of polymers and pharmaceuticals (3–6). Carcinogenic potency of hydrazine in experimental animals has been reported by IARC in 1974, 1982, 1985, 1987 and 1999 (7–11) and US Agency for Toxic Substances and disease Registry(12). When administered by inhalation, hydrazine induced alveolarogenic carcinomas and lymphosarcomas in mice (13–16). In humans, a significant dose–response relationship between hydrazine exposure and lung-cancer incidence and mortality and a significant increase in colorectal-cancer incidence were reported for the first time in a study conducted among aerospace workers (17–18). Hydrazine is identified in Smoking and Tobacco Control Monograph 2 (19) and by IARC in 2007 (20) as a carcinogenic agent in smokeless tobacco products. The recent FDA publication of a list of harmful and potentially harmful constituents (commonly referred as HPHC list) recommends the reporting of hydrazine found in mainstream smoke (21). The presence of hydrazine in tobacco smoke was attributed to be mainly the result of the use of maleic hydrazide applied to tobacco prior to harvest as a means of reducing sucker growth (22). Several methods have been developed for measuring hydrazine levels in biological, environmental and pharmaceutical matrices. With respect to detection, there have been numerous techniques suggested for analysis of hydrazine in water and air samples. Hydrazine level in various matrices has been quantified by either spectrophotometric determination (23–25) voltammetry (26), infrared tunable diode laser spectroscopy (27–28), liquid chromatography (HPLC/UV) (29–31) or ion chromatography (32). Gas chromatography such as GC-FID/ECD or GC-MS (33–39) was also used. Due to its polar characteristic, a direct determination of hydrazine is quite cumbersome and requires a derivatization step that allows producing compounds that are more amenable to gas chromatographic separation. The conventional methodology is based on a Schiff base formation where the primary amine function reacts with a carbonyl group to generate the Schiff base (Scheme 1). In presence of excess of the reagent, the Schiff base can react with a second carbonyl group to produce the azine function (40, 41). Scheme 1 Open in new tabDownload slide Hydrazine reaction in presence of excess of reagent containing a formyl (CHO) group Scheme 1 Open in new tabDownload slide Hydrazine reaction in presence of excess of reagent containing a formyl (CHO) group With respect to analysis of hydrazine in tobacco smoke, only few studies have been reported. Due to hydrazine tendency to react with other compounds during extraction procedure, a direct, rapid and quantitative isolation procedure is required. The efficiency of the extraction from the smoke has a major role in preventing the loss of hydrazine caused by either its reaction with other freshly generated smoke constituents, such as aldehydes and ketones, in the traps or the poor stability of the derivatization products. Hoffmann et al. (33) isolated for the first time hydrazine in mainstream and sidestream smoke of a US blended cigarette without filter tip and reported mean levels of 31 ng/cig and 94 ng/cig, respectively. The authors extracted hydrazine from smoke by direct reaction with a methanolic phosphate buffer solution containing a highly reactive trapping agent (pentafluorobenzaldehyde) as derivatization agent. The resulting hydrazone derivative was extracted by liquid–liquid chromatography, enriched by a series of thin-layer chromatographic separations using silica gel and aluminum oxide plates and then quantified by gas chromatography with ECD detector. In another study published in 2002 (27), hydrazine in cigarette smoke was examined by directly measuring a flowing stream of a fresh smoke on a puff-by-puff basis. The hydrazine was trapped from tobacco smoke by reaction with pentafluorobenzaldehyde. The azine product was then enriched by absorption chromatography on alumina plates and then back extracted with diethylether. The analysis was done using an improved Infra-red diode laser that provided a hydrazine detection limit of 0.1 ng/cig. The authors did not detect hydrazine in tested cigarette smoke suggesting that hydrazine was most likely adsorbed on the cigarette filter. The lack of recent methodology published for the determination of hydrazine in cigarette smoke along with the long turn-around time due to multi-step procedures proposed in the literature, were the main reasons for us to look into an alternate analytical approach for analysis of hydrazine in cigarette smoke. The main objective of our experimental investigations focused on improving the efficiency of trapping via optimization of extraction/derivatization procedure. Therefore, the starting point consisted to select a suitable derivatization reagent allowing a quantitative isolation of hydrazine. To this purpose, we assessed the derivatization efficiencies of three aldehyde reagents: benzaldehyde, 2-nitrobenzaldehyde and pentafluorobenzaldehyde. The second point was to optimize the assay performance by introducing the use of an adequate surrogate standard that allows compensating for the potential effects of matrix interferences, performance variations of derivatization, extraction efficiency and instrument performance variations. The fittingness of two isotopically-labeled compounds was investigated: a deuterium-labelled dimethylhydrazine (DMH-d6) and an isotopically-labelled hydrazine (hydrazine-15N2). Both compounds have been used in the past as surrogate standard for quantification of 1,1-dimethylhydrazine in cigarette mainstream smoke (36) and hydrazine in drinking water (38). In an effort to increase assay sensitivity and specificity for hydrazine detection, we took advantage of the mass spectrometric capabilities (i.e. MS/MS combined with isotope dilution). Two approaches were investigated consisting of GC/MS and HPLC-MS/MS techniques. Finally, the trapping mechanism for isolation of hydrazine vapour from cigarette mainstream smoke was optimized by using a vapour of pure hydrazine allowing to determine the distribution of hydrazine between particulate and vapour phases and the volume of impinging solutions required for quantitative isolation of hydrazine from cigarette smoke. This paper describes and discusses the results of our investigative experiments during method development and proposes a rapid, sensitive and specific method for analysis of hydrazine in tobacco smoke. The assay sensitivity, accuracy, and precision were investigated during the method validation. The method performance and its application are also discussed. Experimental Reagents and chemicals Benzaldehyde (BA), 2-nitrobenzaldehyde (2-NBA), and 2,3,4,5,6-pentafluorobenzaldehyde (PFBA) were purchased from Sigma-Aldrich (Oakville, ON, Canada) and were labeled as 99%, 98% and 98% pure, respectively. The salts of hydrazine dihydrochloride (>99%, pure) and isotopically labeled hydrazine-15N2 dihydrochloride (99%) were also obtained from Sigma-Aldrich (Oakville, ON, Canada). The deuterium labeled 1,1-Dimethyl-d6-hydrazine HCl (DMH-d6) was purchased from CDN Isotopes (Pointe-Claire, Quebec) and labeled as ≥98% pure. The hydrazine permeation tube with certified permeation rate at 356 ng/minute at 50°C was supplied by KIN–TEK Laboratories (La Marque, TX, USA). Potassium phosphate monobasic, formic acid (>98%) and reagent grade 2,4-dinitrophenylhydrazine (2,4-DNPH) (97%) were purchased from Sigma-Aldrich (Oakville, ON, Canada). Perchloric acid (60% pure) was supplied by EMD Millipore (Billerica, MA, USA). Methanol and acetonitrile were distilled-in-glass grades (VWR International, Mississauga, ON, Canada). Type I reagent water was generated in-house as per American Society for Testing and Materials D1193 standard specification. The standard stock solutions of hydrazine dihydrochloride and all isotopically labeled compounds were prepared in water. All Internal standard spike solutions were prepared in methanol. Two sets of calibration standards were prepared in buffer: methanol and in methanol. All derivatization reagent stock solutions were prepared in methanol. An acidified solution of 2,4-dinitrophenylhydrazine (12 mM) was prepared by dissolving the chemical in a binary acetonitrile:water (50,50, v/v) mixture containing perchloric acid (26 mM). Analyses The analysis of derivatization products of hydrazine (i.e. hydrazone and azine) was investigated using both GC-MS and LC-MS techniques. The GC-MS system consisted of a Varian CP-3800 gas chromatograph system coupled with a 320MS triple quadrupole mass analyzer, equipped with a conventional EI/CI source. The chromatographic separation was investigated on an OV-1701 (14% cyanopropylphenyl methylpolysiloxane phase) capillary column (30 m × 0.25 mm × 0.25 μm film thickness) and a polar DB-Wax (polyethylene glycol phase) capillary column (30 m × 0.25 mm × 0.25 μm), both purchased from VWR International (Mississauga, ON, Canada). Various GC conditions were investigated to establish optimized chromatographic parameters. An optimized GC oven temperature program was established, where the oven temperature was initially held at 55°C for 2 minutes, ramped to 150°C at a rate of 15°C/min and held for 25 minutes, ramped to 240°C at rate of 10°C/min and held for 2 minutes. Helium was used as carrier gas at a constant flow of 1.4 mL/min. The GC injector was set to 260°C and 2-μL aliquots of the calibration standards were injected in a splitless mode. Both EI and positive-CI modes were explored for ionization of hydrazine derivatization products in the source. Ammonia was used as the CI reagent gas. The source temperature and the transfer line temperature were set to 170 and 240°C. The mass analyzer was operated under single-ion-monitoring mode with analyzer monitoring expected molecular species mass/charge ratios. The LC-MS setup comprised an Agilent 1200 LC system coupled with an API3000 triple quadrupole mass spectrometers (Applied Biosystems, ON, Canada), equipped with interchangeable interfaces allowing either for conventional electrospray and atmospheric pressure chemical ionization. The LC column employed for chromatographic separation was a Kinetex PFP, 100 × 4.6 mm, 2.6 μm thickness (Phenomenex, Torrance, CA, USA). A binary gradient of acidified type 1 water, and methanol was used to investigate HPLC separations. The optimized chromatographic separation was achieved with a solvent flow rate of 300 μL/min with a linear gradient of 50% methanol to 90% methanol in 8 min and then held for 6 min at 90% methanol. The column temperature was set to 40°C and the injection volume was 5 μL. The positive-ion ESI experiments, both MS and MS/MS (CID, daughter-ion) were conducted by direct infusion at a flow rate of 10-25 μL/min to the ESI source. The analytes were prepared in type 1 water. All instrument parameters (both source and compound dependents) were optimized to achieve maximum sensitivity for the expected protonated species of derivatization products. The optimized ion voltage for ES source was 5.5 kV and the de-clustering potential (orifice potential) was 21 V. The nebulizer and curtain gas were set to 9 and 12 mL/min, respectively. The temperature of the source block for desolvation was set to 400°C. Smoke generation and “direct” derivatization of hydrazine The reference cigarette 3R4F was purchased from the University of Kentucky (Lexington, KY, USA). Cigarettes were conditioned and smoked under the environmental conditions specified in ISO 3402 (42). Mainstream tobacco smoke was generated and collected on a 20-port rotary smoking machine (Borgwaldt, Hamburg, Germany). The smoking parameters and smoking machine specifications that were used are set out in the International Organization for Standardization standard ISO 3308, Fourth Edition 2000-04-15 (43). Mainstream yields were obtained under “ISO” conditions (puff volume, 35 mL; interval, 60 sec; duration, 2 sec; vent blocking, 0%). The smoke collection system included a 92-mm glass fiber filter pad and two Dreschel-type bottle containers mounted in series enclosing the derivatization reagent. The mainstream smoke was passed through the filter pad and then the impinging traps, allowing “direct” derivatization of hydrazine during sample generation. Evaluation of method efficiency The extraction of the hydrazine vapor from cigarette smoke requires a quantitative isolation procedure in order to minimize the loss of hydrazine via its oxidation with other reactive smoke constituents in mainstream smoke (such as carbonyls present at μg/cig levels). In order to accurately evaluate the efficiency of our smoke collection system, we needed to somehow introduce pure hydrazine vapor in the smoke collection. The hydrazine would then follow the same sample path as hydrazine in smoke. Due to its hygroscopic and its strong reducing nature, the commercially available hydrazine is mainly offered in the form of its water-soluble salt (i.e. sulphate or dihydrochloride). The permeation tube technology is the only way to generate a flow of pure-hydrazine vapor. A permeation tube is a small tubular device made of a selective membrane that has liquid analyte sealed inside the tube. Permeation tubes are generally used to dispense a very small and extremely stable flow of analyte vapor by molecular permeation through the membrane at constant temperature (44). In our experimental set up (Figure 1), the hydrazine vapor was generated by flowing nitrogen gas through a glass container holding a polymeric (Teflon®) membrane containing pure hydrazine (Trace Source™ Disposable Permeation Tube, catalogue no 2013, KIN-TEK Analytical Inc., TX, USA). The glass container was enclosed in a brass coil housing placed on a heating plate. The heated temperature inside the container was monitored using a digital thermocouple. The generated hydrazine/nitrogen vapour mixture was drawn through the pad holder and the two Dreschel-type bottles, mounted in series, under a negative pressure generated by using a constant flow air sampling device (Model P21661, SKC Inc., PA, USA). The concentration of hydrazine collected in the trapping solution is function of the membrane permeability and the operating temperature of the membrane (P), the flow rate (F) of the carrier gas flowing around the membrane, the volume of the trapping solution (V) and the sampling-time (t). The following formula (Formula 1) was used to calculate hydrazine amount (mass concentration) collected in each wash bottle: Figure 1 Open in new tabDownload slide Experimental set up: assessing the smoke collection efficiency; hydrazine flow goes from the left to the right. Figure 1 Open in new tabDownload slide Experimental set up: assessing the smoke collection efficiency; hydrazine flow goes from the left to the right. To verify the system suitability with respect to hydrazine simultaneous isolation and derivatization, a series of experiments were conducted in which the sampling-time was increased from 5 to 60 minutes. The trapping solution from each trap (Dreschel bottle) was analyzed at the end of each experiment and concentrations of the derivatization products were measured. The hydrazine equivalent-concentrations were then calculated and used to determine the linearity of the procedure. The least square value of correlation coefficient of the correlation curve (response of quantified hydrazine equivalent plotted against the hydrazine concentration) was satisfactory (R2 > 0.9, five data points) demonstrating the excellent trapping efficacy of our experimental set up for extracting the hydrazine from the vapor phase (Figure 2). Results and Discussion Our method development approach was based on a systematic scouting of four key components of the method that are the hydrazine derivatization step, the internal standard, the detection method and the hydrazine extraction from smoke. Derivatization reagent The suitable derivatization reagent shall provide a high and specific reactivity vis-à-vis hydrazine while meeting other criteria, such as stability in trapping solvent and inertness vis-à-vis other smoke constituents generated and trapped in the trapping solutions. Preliminary experiments aimed to compare the performance of three derivatives of benzaldehyde (BA, 2-NBA, and PFBA) as candidate reagents for the derivatization of hydrazine. To assess the derivatization efficiency of selected reagents, an aqueous solution of hydrazine HCl was combined with a large excess of each reagent at a constant molar ratio of 1:30 (hydrazine:reagent) in each reaction mixture. Hydrazine-reagent mixtures were then heated to 35°C and held at this temperature for 2 hours to allow the completion of the derivatization reaction. Aliquots from each mixture were taken at 1, 30, 60 and 120 minutes after mixing the reactants. The hydrazone products were analyzed by GC-MS. The kinetics of all three derivatization reactions were also evaluated by plotting over time the peak areas of hydrazone-products. All three benzaldehyde-hydrazones showed a plateau starting around 30 minutes indicating that the derivatization of the substrate was complete within 30 minutes at 35°C. Also, an excess of the reagent was detected at the end of the study time (2 hours), proved that the derivatization of the substrate was complete. Under these experimental conditions, the 2-NBA (with a product signal at least 30-fold higher than BA and PFBA-hydrazone products) was found to be the most reactive and efficient agent vis-à-vis hydrazine. Figure 2 Open in new tabDownload slide Performance evaluation of the experimental set up for trapping-derivatization of hydrazine vapor. Figure 2 Open in new tabDownload slide Performance evaluation of the experimental set up for trapping-derivatization of hydrazine vapor. Internal standard The performance of both DMH-d6 and hydrazine-15N2 as surrogate internal standards were evaluated by preparing two sets of hydrazine standards in a methanolic solution of 2-NBA spiked with either DMH-d6 or hydrazine-15N2. Aliquots of derivatized standards were analyzed in triplicates at various time intervals after derivatization of standards. The calibration standards containing DMH-d6 as internal standard were analyzed at 1, 16, 35 and 86 hours after the end of derivatization step (30 minutes at 35°C) and calibration curves were built. Figure 3 compares all four calibration curves recorded over the study time. The graph shows a drastic change in the instrument response represented by changes in the calibration curves regression fit from linear to quadratic. Further investigation allowed us to attribute the calibration curve behavior over time to either the differences between substrate reactivities vis-à-vis the reagent (caused by kinetic differences in “Shiff base” formation) or to the differences in the relative stability of their respective hydrazone products in solution. However, the use of DMH-d6 as internal standard was abandoned. Figure 3 Open in new tabDownload slide Calibration curves constructed using standards containing DMH-d6 as internal standard—curve linearity evolved over time from a linear to a quadratic regression fit. Figure 3 Open in new tabDownload slide Calibration curves constructed using standards containing DMH-d6 as internal standard—curve linearity evolved over time from a linear to a quadratic regression fit. In the case of hydrazine-15N2, calibration curves were recorded at 5, 28 and 74 hours after derivatization. All three calibration curves (not shown) exhibited excellent linear regressions that clearly indicates similar reactivity of both hydrazine and hydrazine-15N2 vis-à-vis the reagent (2-NBA). The 15N2-hydrazine was proved to be a suitable internal standard for the purpose of this study and chosen for the analysis. Detection The performance of both GC-MS and LC-MS platforms for the analysis of the derivatization products were investigated. We first investigated the GC-MS capability under both electron impact (EI) and positive-chemical ionization modes. Under EI ionization mode, no hydrazine derivatization products were detected. This is most likely due to high energy transfer under EI ionization mode causing substantial fragmentations of the expected molecular species in the source. As opposed to EI, CI/NH3 mode generated an abundant ionic specie corresponding to the protonated hydrazone (MH+, m/z 166 RCH=HN+-NH2) but the expected dinitrobenzalazine (R-CH=N-N=CH-R) product was not detected. Overall, the mass chromatograms exhibited a relatively high baseline and also low hydrazone responses. Furthermore, hydrazone peak eluted in the vicinity of an abundant peak showing a constant concentration independently to the target substrate (hydrazine) concentration. The full-scan mass spectrum of the unknown peak was dominated by an abundant ion at m/z 166. Further investigative MS/MS experiments were conducted on m/z 166 to elucidate its structure. The CID product-ion spectrum showed an abundant ion (m/z 134) formed by loss of 32 mass units (CH3OH). These results suggest an oxonium structure (R-CH = O+-CH3) for the precursor ion m/z 166. The latter is most likely formed after loss of a molecule of water from a protonated hemiacetal structure (R-CHOH-O-CH3) formed itself via an adverse reaction of 2-NBA (reagent) with methanol (solvent). Figure 4 Open in new tabDownload slide Solvent effect on derivatization—comparison of MRM traces of benzalazines and hydrazones with either methanol (in blue) or an aqueous citrate-phosphate buffer:methanol (in red) mixture used as derivatization solvents. Figure 4 Open in new tabDownload slide Solvent effect on derivatization—comparison of MRM traces of benzalazines and hydrazones with either methanol (in blue) or an aqueous citrate-phosphate buffer:methanol (in red) mixture used as derivatization solvents. The overall low sensitivity, the poor chromatographic elution profile and the relatively long analysis time were the major drawbacks to the adoption of GC-MS for the analysis and therefore, further investigation into a quantitative GC-MS approach was suspended. The LC-MS was also investigated for analysis of the single (hydrazone) and double-derivatization (azine) products of hydrazine. Electrospray ionization (ESI) experiments were conducted in the positive ion mode. The ESI produced abundant pseudo-molecular ions (MH+) for benzalazine and hydrazone at m/z 299 and 166, respectively. The identities of the molecular ions of each of the analytes were confirmed by recording product ion MS/MS spectra. The identity of the reaction products was further confirmed by analyzing labeled products formed via reaction between hydrazine-15N2 and the reagent (2-NBA). The acquired mass spectra of benzalazine-15N2 and hydrazone-15N2 showed peaks at m/z 301 and 168, respectively. As opposed to GC-MS results, both benzalazine and hydrazone were detected upon ESI. LC-MS assay provided superior specificity (by allowing detection of azine product) and sensitivity compared to GC-MS, and was thus proved to be the method of choice for the purpose of this study. Trapping solvent Another advantage of LC-MS technique is the capability to perform the analysis of analytes in aqueous solvents/matrices. This feature allowed us to study the efficiency of hydrazine derivatization in aqueous solvents. The main reason for this investigation was to find an alternative solvent to replace the use of methanol that had the disadvantage to react with the aldehyde reagent forming a large amount of hemiacetal. A citrate-phosphate buffer:methanol (55:45, v/v) solution of the reagent (2-NBA) was prepared and used for derivatization of hydrazine. The MRM mass spectra were recorded and analytical results were compared to those for hydrazine derivatization products produced in presence of a methanolic solution of the reagent. The typical MRM traces acquired from both systems are reproduced in Figure 4. The MRM traces for corresponding benzalazines show no significant differences in signal indicating that the azine formation rates are similar in both solvents. On the other hand, with the aqueous buffer:methanol as solvent, hydrazone was generated in the highest abundance compared to data obtained when pure methanol was used. In addition, the hemiacetal formation occurs to a much lower extent (almost 60%) when methanol amount was decreased by half. Based on these results, we concluded that the citrate-phosphate buffer:methanol solution of 2-NBA was a better choice for derivatization of hydrazine. The other main advantage of using an aqueous solvent over pure methanol is that the loss of solvent during the sampling process of the smoke is significantly reduced and there is no need to have a cooling system for the impinging solutions during sample generation (i.e. smoking). Table I A brief description of experimental parameters implemented for the trials Glass Fibre Filter Pad (ng/mL) Trap Solution A (ng/mL) Blank filter pad 546 (85%) 96 (1%) Smoke Condensate 621 (98%) 14 (2%) DNPH-treated filter pad 766 (120%) 6 (1%) Glass Fibre Filter Pad (ng/mL) Trap Solution A (ng/mL) Blank filter pad 546 (85%) 96 (1%) Smoke Condensate 621 (98%) 14 (2%) DNPH-treated filter pad 766 (120%) 6 (1%) Permeation tube temperature, 70°C; Trap solution volume, 2 × 40 mL (A and B); and sampling time, 15 minutes. Recovery values (%) calculated as per formula: |$\frac{{[\mathrm{Azine}]}_{\mathrm{measured}}}{{[\mathrm{Azine}]}_{\mathrm{ref}(\mathrm{trap})}}\times 100$| Open in new tab Table I A brief description of experimental parameters implemented for the trials Glass Fibre Filter Pad (ng/mL) Trap Solution A (ng/mL) Blank filter pad 546 (85%) 96 (1%) Smoke Condensate 621 (98%) 14 (2%) DNPH-treated filter pad 766 (120%) 6 (1%) Glass Fibre Filter Pad (ng/mL) Trap Solution A (ng/mL) Blank filter pad 546 (85%) 96 (1%) Smoke Condensate 621 (98%) 14 (2%) DNPH-treated filter pad 766 (120%) 6 (1%) Permeation tube temperature, 70°C; Trap solution volume, 2 × 40 mL (A and B); and sampling time, 15 minutes. Recovery values (%) calculated as per formula: |$\frac{{[\mathrm{Azine}]}_{\mathrm{measured}}}{{[\mathrm{Azine}]}_{\mathrm{ref}(\mathrm{trap})}}\times 100$| Open in new tab Method trapping efficiency The candidate analytical procedure was further evaluated by conducting four sets of trials focused on assessing the efficacy of hydrazine extraction/derivatization procedures. For these trials, a permeation tube was used as hydrazine source to generate a constant flow of hydrazine vapor. The experimental set-up was described earlier in Materials and Method section. Table 1 gives a brief description of experimental parameters implemented for our trials. A “reference trapped” value was determined by drawing the hydrazine vapor directly (no filter pad used) through two wash bottles, mounted in series, with each containing 40 mL of a citrate-phosphate buffer:methanol (55:45, v/v) solution of 2-NBA. In other three trials, different filter pads were used: an un-smoked (blank) filter pad; a cigarette smoke condensate filter pad (prepared by passing the mainstream smoke of 10 cigarettes through the filter pad); and a 2,4-DNPH treated filter pad. The use of the pre-treated filter pad was undertaken to evaluate the possibility of chemical stripping of carbonyls from the smoke by 2,4-DNPH, and therefore, minimizing the amount of carbonyl substrates reaching the trapping solution and potentially reacting with hydrazine. The benzalazine was solely detected in the first wash-trap when no filter pad was placed between the hydrazine source and wash-bottles. From blank filter trial results, it appeared that over 85% of hydrazine is absorbed on the filter pad and the rest reached the first impinging trap. In contrast, when the smoke condensate pad was used, 100% of the diffused hydrazine was trapped on the filter pad. Finally, we found an average percentage recovery value from the 2,4-DNPH-treated pad at 120% indicating additional formation of hydrazine in the acidic extract most likely generated from 2,4-DNPH itself. Method validation Based on the findings presented above, the following procedure was proposed: the mainstream smoke of cigarettes is drawn through a 92-mm glass fiber filter disk and into a single Dreschel-type bottle containing a citrate-phosphate buffer:methanol (55:45, v/v) solution and the internal standard (2-nitrobenzaldehyde, 10 g/L). Immediately after smoking, the filter pad is extracted with the trap solution, diluted with an additional 40 mL fresh buffer:methanol and then incubated for 30 minutes at 35°C. An aliquot of the extract is then centrifuged and the derivatization products are quantified by LC-MS/MS using the operation parameters described above. The analytical method was validated and results are summarized hereafter: The calibration curves showed excellent linear regression curves for a concentration range of 0.4–800 pg/cig. The method detection and quantification limits were established to be 116 and 400 pg per cigarette, respectively. The method accuracy and precision were determined by calculating the recovery percentage values of 16 replicate smoked pad samples fortified with known amounts of hydrazine. The method accuracy for validation study was between 85–110% for a hydrazine fortified range of 30–60 ng/mL. The quality control chart shown in Figure 5 reports the individual recovery percentage values calculated from measurement of over 50 fortified samples prepped over eight-month period. The chart demonstrates the excellent method accuracy and precision (6%) of the method over a large number of observations. Figure 5 Open in new tabDownload slide Method accuracy/precision: individual recovery percentage values for over 50 fortified matrix samples prepped over 8 month time interval Figure 5 Open in new tabDownload slide Method accuracy/precision: individual recovery percentage values for over 50 fortified matrix samples prepped over 8 month time interval The precision calculated based on the data variability of all recovery percentage values was 6%. The sample extracts were proved to be stable for up to 28 days. Conclusion The results of this study demonstrate several key points regarding the extraction and the analysis of trace level hydrazine in mainstream cigarette smoke. 2-nitrobenzaldehyde was proved the most suitable reagent for the derivatization of hydrazine. The reaction efficiency was optimized by electing an aqueous buffered:methanol binary solvent for simultaneous extraction/derivatization of hydrazine vapour. The analytical performance of both GC-MS and LC-MS techniques for quantification of derivatized products was investigated. The LC-MS approach proved to be a sensitive, fast and reliable method for the quantitative analysis of hydrazine in mainstream cigarette smoke. The isotopically-labelled 15N2-hydrazine was chosen as internal standard. The method was validated and successfully applied to various tobacco smoke matrices generated under “ISO” and “intense” smoking regimens. To the best of our knowledge, the proposed method is the first reported single procedure (simultaneous derivatization/trapping) allowing unambiguous quantification of hydrazine vapor in mainstream smoke by LC-MS/MS instrumentation. Acknowledgements The authors would like to express their gratitude to Professor William (Bill) Rickert, founder of Labstat International ULC. 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TI - A Sensitive and Quantitative Isotope-Dilution LC-MS/MS Method for Analysis of Hydrazine in Tobacco Smoke
JO - Journal of Chromatographic Science
DO - 10.1093/chromsci/bmz069
DA - 2020-01-23
UR - https://www.deepdyve.com/lp/oxford-university-press/a-sensitive-and-quantitative-isotope-dilution-lc-ms-ms-method-for-neXMxt5h3T
SP - 1
VL - Advance Article
IS - 
DP - DeepDyve
ER -