II SAMPLE PREPARATION Jfb
A. Water and Wastewater Samples
There are basically two methods used for the analysis of A^-nitrosamines in water samples. Those are liquid-liquid extraction using an organic solvent, and adsorption onto a sorbent material.
1. Liquid-Liquid Extraction
The most popular solvent is the dichloromethane (DCM) for two reasons: the volatile nitrosamines are highly soluble in this solvent, and the boiling point of the DCM is low, hence preventing the volatilization or the degradation of nitrosamines through a subsequent concentration step.
In this extraction method,15,60-63 a known volume of water is filtered and adjusted to pH 12 with a 50% solution of NaOH in water. NaCl (14 g/l) can be added to break up any emulsion.63 The sample is repeatedly extracted with (3 to 5 X 40) ml aliquots of DCM by shaking. The extracts are dried over anhydrous sodium sulfate or passed through a column containing anhydrous sodium sulfate to eliminate water. Then, they are concentrated on a Kuderna-Danish evaporative concentrator, which already contains a nitrosation inhibitor. Aliquots of the final concentrate are further analyzed by GC or high-performance liquid chromatography (HPLC). Separatory funnel extraction of wastewater samples was not practical due to the formation of emulsions during shaking. Therefore, wastewater samples are better extracted for 6 h61 or overnight62 with DCM by continuous liquid-liquid extraction.
Sen et al.15 described a method for the determination of NDMA in drinking water practically free of artifactual formation. Such formation is minimized by extracting the samples in the presence of sulfamic acid, an excellent inhibitor of A^-nitrosation. The water sample is acidified with the addition of H2SO4 and sulfamic acid and afterwards it is extracted with DCM. The extracts are washed with KOH solution.
2. Adsorption on Solid Supports
Several types of solid sorbents have been utilized for the removal of A^-nitrosamines from water samples. They include active carbon, carbonaceous adsorbents, XAD resins, and others.
In the case of active carbon, the water samples are adjusted to pH 7 with hydrochloric acid and then the samples are passed through a mini activated carbon column by a sweep pump at a fixed flow rate. The carbon is then partly dried by flushing the column with purified nitrogen to remove most of the residual water before the column is eluted with acetone or chloroform.64 The concentration of the organic extracts can be accomplished on a rotary evaporator under reduced pressure, at ambient conditions or slightly above ambient temperature. It is advisable to dry the organic extracts with anhydrous sodium sulfate prior to any final concentration step. This approach can be used only for the less volatile A^-nitrosamines unless care is taken during the concentration step to avoid losses of NDMA or NDEA. Queiroz et al.60 obtained better extraction efficiencies for the most volatile nitrosamines with two adsorbents coupled in series (C8 and active carbon) and using ethanol as eluent.
Carbonaceous adsorbents like Ambersorb 572 have been used to remove NDMA from water.24 The sample is added with 200 mg of carbonaceous adsorbent and extracted by shaking the solution for 1 h at 200 rpm. Ambersorb beads are vacuum filtered onto a glass fiber filter, dried in air for 30 min and then soaked with methylene chloride for 20 min before the analysis.
Kimoto et al.65 have used Ambersorb XE-340 to remove trace levels of volatile nitrosamines from tap water. In order to wet the adsorbent, it was first covered for 1 h with methanol and then washed with distilled water. Ambersorb XE-340 was packed in a 26 X 260 mm copper pipe equipped with a copper fitting on both ends. One fitting was connected to the faucet and the other to a valve which controls the flow rate. Water was sampled for 8.5 to 11.75 h. At the end of the water sampling, methanol was added to remove the water from the column. DCM (700 ml) was next passed through the column. The first 500 ml DCM contained more than 92% of the A^-nitrosamines. The extracts were dried over anhydrous Na2SO4 and concentrated in a Kuderna-Danish evaporator.
The XAD-type resins have been used to remove A^-nitrosamines from water samples and sewage effluent. The samples are filtered to remove suspended particles and then they are passed through a column at a flow rate between 11 and 15 ml/min. Acetone-DCM66 or DCM-diethyl ether (75:25)67 have been used to desorb the organic compounds retained by the XAD resin. After an appropriate concentration, the final solution can be analyzed directly by HPLC or GC. Alternatively, it may be necessary to perform a further clean-up of the extract prior to the analysis. This clean-up will depend largely on the nature of the water samples extracted and on the complexity of the organic materials present in the sample.
B. Soil Samples
Depending on the soil type, it may be possible to extract the A^-nitrosamines using different solvents. The mineral oil distillation procedure can be used to extract volatile A^-nitrosamines from soils which are not adequate to solvent extraction.
The mineral oil distillation procedure introduced by Fine et al.,68 and modified by many authors,69-74 is the most popular extraction method for volatile A^-nitroso compounds and it has been applied to a wide variety of samples. This method consists of a distillation of the sample at reduced pressure from an alkaline medium containing mineral oil. The mineral oil ensures an effective and uniform heat distribution within the distillation mixture. It also improves the efficiency of distillation by reducing the distillation time and increasing the recoveries of the volatile nitrosamines with higher boiling points.75 The first step is to homogenize the sample with a nitrosation inhibitor, such as ascorbic acid, sulfamic acid, or tocopherol. Afterwards, it is added to a round-bottom flask along with an equal volume of mineral oil. The mixture is then distilled under vacuum and the temperature is slowly increased up to 100°C over 40 to 50 min. The distillate is collected in a glass finger immersed in liquid nitrogen and quantitatively transferred to a separating funnel for further purification.
In determination of solid samples, Eisenbrand et al.75 have pointed out the importance of adding water to the flask before distillation. The water increases recovery of the less volatile nitrosamines and avoids exposing the sample to excessive temperatures.
A modification of the mineral oil distillation procedure has been proposed using a gas purge system to trap nitrosamines on a ThermoSorb/N cartridge.76 This procedure eliminates solvent extraction and evaporation steps, hence reducing the possibility of artifact formation. On the other hand, some methods have also been described for the vacuum distillation of alkaline suspensions without mineral oil.77 The distillation is carried out from a slightly basified sample with carbonate potassium or from a highly basified sample with potassium hydroxide.
The direct extraction using different solvents is a more rapid alternative than the distillation for determination of A^-nitrosamines in soils. The soil, altogether with suitable nitrosation inhibitors such as ascorbic acid and tocopherol, is homogenized with a suitable extraction solvent. The solvent is then filtered and purified prior to the analysis. You et al.78 extracted 100 g of soil with a 50 ml portion of DCM during 20 min. The samples were then filtered to remove insoluble particulates. Afterwards, the filtrate was treated with anhydrous sodium sulfate and concentrated under a stream of dry nitrogen at 30°C. The nitrosamines were analyzed by HPLC with a precolumn fluorescence derivatization. A similar approach has been used by Ross et al.79 obtaining a NDPA recovery of 80% at the 4 ppb level and of 45% at the 1 ppb level.
C. Air Samples
In order to determine the A^-nitrosamines in the atmosphere, it is necessary to pass about 200 l of air through a suitable trap at a flowrate of roughly 2 l/min. The contents of the trap are then extracted into an organic solvent or desorbed by heating. Several trapping techniques have been employed and some of them will be described below. The A^-nitrosamines precursors are generally present at levels from 100 to 1000 times greater than the A^-nitrosamines. Therefore, great care must be taken to ensure that the concentration step does not also concentrate the precursors. The concentration of the precursors would lead to an important artifact formation of nitrosamines.
ThermoSorb/N cartridges are commercially available and have been specifically designed for the quantitative collection of A^-nitrosamines in outdoor air.80'81 The cartridge contains two sorbent zones. The first zone selectively traps and removes amines from the incoming air, hence preventing the subsequent formation of nitrosamines by airborne nitrogen oxides. The second zone contains a nitrosating inhibitor system which prevents the formation of Af-nitrosamines followed by elution of the ThermoSorb/N cartridge. The cartridges have a relatively moderate sampling capacity (1500 ng/cartridge); however, they can be connected in series to increase the total capacity of the sampling system. A^-Nitrosamines are removed from the cartridge by reverse elution with 0.5 ml of a special eluting solvent. An aliquot of the eluate is introduced into the GC or HPLC for further analysis. The concentration of the eluting solvent is not required, thereby eliminating the possibility of artifact formation during this step. Marano et al.82 carried out determination of trace levels of nitrosamines in air using ThermoSorb/N cartridges. These cartridges were preeluted to remove interfering compounds prior to nitrosamine elution and selective ion monitoring MS detection. Enhanced sensitivity was achieved by using a commercial concentrator which allowed the introduction of 40 /a1 of eluent onto fused silica capillary and packed columns.
Sampling techniques using Tenax were developed by Pellizzari et al.83-85 A^-Nitrosamines and other organic vapors were collected from ambient air on a 1.5 X 6 bed of Tenax GC (35/60) in a glass cartridge. All cartridges were preconditioned by heating at 275°C prior to field sampling. They are desorbed by heating in a stream of helium and afterwards the contents are caught in a gold-lined trap held at —192°C. The gold trap is then flash heated, driving the contents directly into the capillary column of a GC-MS system. The main disadvantages of the method are: the Tenax may trap precursor amines, which could form A^-nitrosamines during desorption and heating steps; and the Tenax has a relatively small breakthrough volume for NDMA, and this A^-nitrosamine is often the one of maximum interest.
Rounbehler et al.86 have examined several types of sorbents for their ability to collect and retain quantitatively a variety of volatile nitrosamines under simulated air-sampling conditions. Also, the artifactual formation of nitrosamines from trapped amines and air containing nitrogen oxides were studied. The dry solid sorbents included activated charcoal, activated alumina, silica gel, Florisil, Tenax, and ThermoSorb/N cartridges. It was found that a ThermoSorb/N cartridge was the only sorbent which was both free of artifact formation and capable of retaining 100% of the preloaded nitrosamines.
D. Environmental Tobacco Smoke Samples
The most highly developed and validated method for sampling volatile A^-nitrosamines from tobacco smoke employs an aqueous buffered solution (pH 4.5 citrate-phosphate) with 2.10"2M ascorbic acid contained in several impingers connected in series.87-90 Ascorbic acid is added to the solution as a nitrosating inhibitor to prevent the formation of artifact A^-nitrosamines from the amines and NOX in tobacco smoke.89 An ascorbic acid-impregnated Cambridge filter is placed upstream of the impinger solution to remove particles. Buffer solutions and Cambridge filter are extracted with DCM. The extracts are concentrated and afterwards subjected to a clean-up on alumina. For elution 1% methanol in DCM is used.
For determining volatile nitrosamines in mainstream and sidestream, Kataoka et al.91 employed 5% hydrochloric acid as the tripping solution and diethyl ether containing 25% 2-propanol as the extractant. A similar approach was used by Cardenes et al.92 using DCM as the extractant.
These methods are limited by the air-flow rate (especially for midget impingers), and also the aqueous solutions are not a very convenient sampling medium, particularly for field experiments. Sample recovery problems are often encountered in liquid-liquid extraction due to the aqueous buffers or to the emulsion formation. Labor-intensive sample preparation procedures and the relatively fast degradation of the aqueous ascorbic acid solution are the two additional drawbacks when using this sampling method.
The use of the ThermoSorb/N cartridges, specifically designed for the quantitative collection of Af-nitrosamines in out air, eliminates the most common problems associated with the aqueous sampling medium. This approach has been evaluated by Mahanama et al.93 for A^-nitrosamines in a complex matrix like environmental tobacco smoke. Besides the TheromoSorb/N cartridges, ascorbic acid-impregnated Teflon filters were used to remove particles. The cartridges were back-flushed with 2 ml methanol. The extract was concentrated on a rotatory evaporator and loaded onto an alumina-B Sep-Pak, using 10% of chloroform in DCM to recover nitrosamines. This clean-up procedure eliminates polar interferences in the extracts, which could contaminate the capillary GC column and could also interfere in the later analysis. The procedure yields a 97.8 ± 2.8% recovery for the internal standard, NDEA, using nine cartridges loaded with environmental tobacco smoke samples.
Wu et al.94 used an ascorbic acid-impregnated Cambridge filter pad to measure five TSNA in the particulate phase of mainstream tobacco smoke. Each pad with trapped smoke particulate was first spiked with an internal standard and then extracted with DCM, back extracted into an aqueous solution and further purified by solid-phase extraction (Water Oasis HLB 60 mg), using 100% ethanol as eluent.
Among other A^-nitrosamines collection methods, the use of wet traps such as 1 N KOH, cold traps, and Tenax traps have been reported, each one with its own limitations.95'96
E. Additional /V-Nitrosamines Extraction Methods 1. Solid-Phase Microextraction
The newly developed solid-phase microextraction (SPME) technique, first reported by Pawliszyn97 in 1989, is increasingly used for the gas chromatographic determination of a wide variety of volatile and semivolatile organic compounds in water or aqueous extracts of different substrates. Basically, it involves the extraction of specific organic analytes directly from aqueous samples or from the headspace of these samples in closed vials. The extraction is achieved onto a fused-silica fiber coated with a polymeric liquid phase. After equilibration, the fiber containing the absorbed or adsorbed analyte is removed and thermally desorbed in the hot injector port of a gas chromatograph or in an appropriate interface of a liquid chromatograph.97-102
The technique is very simple, fast, and does not employ any organic solvents either for sample preparation or clean-up. This makes it highly desirable because, unlike other methods, it does not release environment-polluting organic solvents into atmosphere. Thus far, the technique has been successfully applied to the determination of a wide variety of organic compounds. However, the application of SPME to determine the nitrosamines could eliminate some problems like the widespread solvent use and the lengthy and time-consuming sample preparation steps (that are common in most of the published methods in this area).
Sen et al.103 have reported a SPME analytical method for the determination of Af-nitrosodibutylamina and A^-nitrosodibenzylamina. This method is based on the isolation of the compounds by steam distillation, followed by the SPME in the distillate headspace using a polyacrylate coated silica fiber, and the determination by GC-TEA. Recoveries ranged between 41% and 112%. Nitrosamines in environmental matrixes (air, tobacco, and seawater) were preconcentrated on polydimethylsiloxane/divinylbenzene and the recovery of different nitrosamines from different matrixes varied between 95% and 98%.104
An automated in-tube solid-phase microextraction HPLC method for NNK and several metabolites have been developed by Mullett et al.105 In-tube SPME is an on-line extraction technique where analytes are extracted and concentrated from the sample directly into a coated capillary by repeated draw-eject steps. A tailor-made polypyrrole-coated capillary was used to evaluate their extraction efficiencies for NNK and several metabolites in cell cultures. This automated extraction and analysis method simplified the determination of the tobacco-specific Af-nitrosamines, requiring a total sample analysis time of only 30 min.
2. Supercritical Fluid Extraction
Because of U.S. Environmental Protection Agency regulations,106 there is a strong incentive to reduce or replace organic solvents, particularly those containing halogens that are usually employed in residue analysis. These regulations are designed to reduce the use of solvents that are potentially harmful to the environment and to reduce the costs of solvent disposal. Supercritical fluid extraction (SFE) has the potential to achieve effectively the selective extraction in a single step and to concentrate the analyte as it is ready for instrumental analysis with a minimum amount of solvent.
SFE applications in environmental analysis, particularly for heterogeneous solid samples, are emerging as viable alternatives to more traditional methods employing polar liquids or mixtures as extractants. Most of these approaches utilize CO2 as the fluid with or without methanol as modifier.107
The current emphasis on methods that use less solvent makes SFE an attractive alternative for the analysis of nitrosamines. However, only limited studies have been carried out on SFE with nitrosamines. Prokopczyk et al.108,109 reported extraction efficiencies of 83% to 98% for the major nicotine-derived TSNAs in smokeless tobacco and snuff, with methanol-modified supercritical carbon dioxide. These compounds were extracted from cigarettes using SFE and purified by a sodium hydroxide wash of the ethyl acetate eluting solvent and solid-phase extraction.110
SFE has been demonstrated to be a good extraction technique for A^-nitrosamines in rubber products. In addition, SFE allows fast analysis with a reduction in solvent waste, time, and manipulation. Although recoveries are not too good, especially for the smaller A^-nitrosamines, SFE could be considered as a useful tool to determine these analytes, considering that through its selectivity it provides quite clean extracts in one step.111 Reche et al.112 determined A^-nitrosamines in latex products by combining supercritical fluids and chemical derivatization. The addition of a denitrosation reagent into the extractor combined with an adequate liquid trap allows elucidation of the presence of A^-nitrosamines as well as their potential precursors.
F. Derivatization Reactions for Gas Chromatographic Determination of N-Nitrosamines
Derivatization of A^-nitrosamines can be employed not only to convert nonvolatile nitrosamines into volatile materials suitable for GC analysis, but also to improve selectivity and sensitivity, and to reduce the analysis time. The commonly used derivatization reactions for GC analysis of nitrosamines are stated below.
A^-Nitroso compounds can be easily reduced to their hydrazines, which are further derivatized. The most satisfactory approach is the conversion of the hydrazine to its 3,5-dinitrobenzaldehyde hydrazone, which is then analyzed using GC with electron capture detection.113
In a similar way, the A^-nitrosamines can be converted into secondary amines. The produced secondary amines can be converted into appropriate derivatives for their gas chromatographic analysis. One of the most common derivatives is the heptafluorobutyryl amide (HFB-amide), formed by the reaction of the HFB-acid chloride and the amine. Afterwards, this derivative can be analyzed by GC-electron capture or GC-MS.114,115 Kataoka et al.91 reported the determination of seven nitrosamines by GC-flame photometric detection. The method is based on denitrosation with hydrobromic acid and the subsequent diethylthiophosphorylation of secondary amines.
The Af-nitroso compounds can be converted into their oxidation products, the nitramines, by several oxidation pathways. Thus, the peroxidation of nitrosamines with trifluoroperoxyacetic acid, prepared by the reaction of trifluoroacetic acid or anhydride with 30% to 90% of hydrogen peroxide, was used in the past for detecting nitrosamines. This is because the electron capture detector shows greater sensitivity and selectivity for nitramines than other detectors available for nitrosamines at that time. This peroxidation reaction has been applied for the detection of NDMA in ambient air and in cigarette smoke.96 The peroxidation reaction with pentafluoroxybenzoic acid, a stable solid peroxyacid, has been used to confirm the NDMA and NPYR. This reaction has the advantage of minimizing the repeated use of concentrated hydrogen peroxide.116 Cooper et al.117 reported the conversion of a series of A^-nitrosamines into their corresponding A^-nitramines analogs by pertrifluoroacetic acid oxidation. The nitramines were detected by GC-electron capture.
The formation of ether derivatives of hydroxynitrosamines converts these nonvolatile A^-nitroso derivatives into volatile compounds suitable for analysis by GC and their sensitive detection by TEA or MS.118-121 In this sense, the trimethylsilylation using A^-methyl trimethylsilyl trifluoroacetamide or teri-butyl dimethylchlorosilane/imidazole and trimethyldiclorosilane/hexa-
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