netural molecule. The transition promoting the single electron from the half-filled orbital to the lowest empty orbital is the A transition, and is analogous to the lowest-energy transition in the neutral. T h e remaining transitions involve several types of excited state configuration, and are all classed as B transitions; they lie a t energies too high to he of concern here. The conjugated dienes are expected to exhibit two peaks, corresponding to the lone I transition (in the visible), and the A transition (in the uv) (21, 22). (Note that within the *-electron approximation methods, the radical anion and the radical cation of alternant hydrocarbons should have identical spectra by the well known "pairing theorem.") As has been discussed in detail ( 8 ) ,the observed spectrum of butadiene cation agrees excellently with calculation in the position of these two bands. It is apparent from Figures 3 and 4 that substituents (and possible cyclic constraints) have a substantial effect on the peak positions, and again these effects remain to be studied in detail. Calculation on the conjugated diene system suggests that the uv peak should he substantially more intense than the visible peak (21, 2 2 ) , and while this is not true for butadiene cation, it is obviously true for the substituted butadiene ions. While it has been seen that the intensities of photodissociation peaks need not correlate with absorption intensities this observation offers some slight encouragement in thinking that the observed intensities may be qualitatively meaningful. I t is known that saturated hydrocarbons with several carbons have a multitude of occupied molecular orbitals within a few eV of the highest occupied orbital (23):since all of these can give rise to I-type transitions in the cation, with no immediately apparent selection rules, the interpretation of spectra such as those in Figure 6 is likely to be a difficult task. The spectra are included here to illustrate the possibly surprising fact that the saturated hydrocarbon cations are quite strongly colored. T h e two peaks in the spectra of benzene cation derivatives arise again from the lowest I transition (visible) and the A transition (uv; this degenerate configuration splits to give several predicted peaks, of which only the lowest is observed in this work.) These spectra agree very well with calculations, as has been discussed in detail for toluene cation (8).
ACKNOWLEDGMENT The author is grateful to Peter Armentrout for obtaining several of the spectra shown.
LITERATURE CITED H. Lew and I . Heiber. J. Chem. Phys., 58, 1246 (1973). G. Herzberg. 0.Rev., Chem. SOC.,25, 201 (1971). R. C. Dunbar and E. Fu, J. Am. Chem. SOC.,95, 2716 (1973). R. C. Dunbar, J. Am. Chem. Soc., 95, 6191 (1973). P. P. Dymerski. E. Fu, and R. C. Dunbar. J.Am. Chem. SOC.,96, 4109 (1974). (6) R. C. Dunbar. in "Ion-Molecule Interactions", P. Ausloos, Ed., Plenum Press, New York, 1975. (7) M . T. Riggin and R. C. Dunbar, Chem. Phys. Lett., in press. (8) R. C. Dunbar, Chem. Phys., Lett., 32, 508 (1975). (9) E. Fu, P. P. Dymerski, and R. C. Dunbar, J. Am. Chem. SOC.,in press. (10) B. S. Freiser and J. L. Beauchamp, J. Am. Chem. SOC., 96, 6260 (1974). ( 11) M. Comisarow and A. G. Marshall, Chem. Phys. Lett., 25, 282 (1974). (12) R. H. Staley, R . R. Corderman, M. S. Foster and J. L. Beauchamp, J. Am. Chem. SOC.,96, 1260 (1974). (13) R. C. Dunbar, J. Am. Chem. SOC.,93, 4354 (1971). (14) J. D. Baldeschwieler and S. S. Woodgate, Acc. Chem. Res., 4, 114 (1971). (15) J. L. Beauchamp, Ann. Rev. Phys., Chem., 22, 527 (1971). (16) R. C. Dunbar, in "Chemical Reactivity and Reaction Paths", G. Klopman, Ed., Wiley-lnterscience, New York, 1974. (17) T. B. McMahon and J. L. Beauchamp, Rev. Sci. Instrum., 43, 509 (1972). (18) R. T. Mclver and R. C. Dunbar, "Pulsed Ion Cyclotron Double Resonance for the Study of Ion-Molecule Reactions", Int. J. Mass Spectrom. Ion Phys., 7, 471 (1971). (19) R. C. Dunbar, to be published. (20) K . B. Wiberg, "Physical Organic Chemistry", Wiley, New York, 1964, p 179 f f . (21) T. Shida and S. Iwata, J. Am. Chem. Soc., 95, 3473 (1973). (22) R. Zahradnik and P. Carsky, J. Phys., Chem., 74, 1235, 1240 (1970). Note that within the .rr-electron approximation methods, the radical anion and the radical cation of alternant hydrocarbons should have (1) (2) (3) (4) (5)
identical spectra by the well known "pairing theorem". (23) A . D. Baker, D. Betteridge, N. R . Kemp. and R. E. Kirby, J. Mol. Structure, 8, 75 (1971)
RECEIVED for review October 8, 1975. Accepted January 8, 1976. Acknowledgment is made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, and to the National Science Foundation for partial support of this research. T h e author is an Alfred P. Sloan Fellow, 1973-75.
Mass Fragmentographic Assay for 1I-Hydroxy-Agtetrahydrocannabinol from Plasma J. M. Rosenfeld" and V. Y. Taguchi Department of Pathology, McMaster University, Hamilton, Ontario, Canada
A mass spectrometric assay for 1l-hydroxy-A9-tetrahydrocannabinol is presented. It is sensitive to 3 ng/ml of plasma and has a relative standard deviation of 4 % . The technique relies on the derivatization of the phenol moiety by extractive alkylation. This reaction is novel to the cannabinoid series. Since all cannabinoids are phenols, this procedure may have wide utility. It was possible to monitor 11-OHA9-THC in dog plasma for 2 h after its administration. However, no 11-OH-A9-THC was detected in dog plasma after oral or intravenous administration of AS-THC.
The understanding of cannabis pharmacology is difficult because of the unresolved question of the existence and/or the nature of an active metabolite. A metabolite proposed to be active is ll-hydr0xy-1~-tetrahydrocannabinol(11726
ANALYTICAL CHEMISTRY, VOL. 48, NO. 4, APRIL 1976
OH-A9-THC)( I ) . The elucidation of the biochemical pharmacology of a drug has been predicated upon the availability of assays capable of measuring the drug in plasma and/ or serum (2, 3 ) . Assays based on mass fragmentography for Ag-tetrahydrocannabinol ( 19-THC) have been reported by Agurell et al. ( 4 ) and Rosenfeld e t al. ( 5 ) These were found to be sufficiently sensitive for use in pharmacokinetics in human studies. T o date, there has been no reported mass fragmentographic assay for 1l-OH-19-THC. The mass fragmentographic assays reported for A9-THC differed in the methodology of the incorporation of deuterium for use as an internal standard as well as in the extraction procedure. Agurell synthesized A9-THC labeled a t the l', 2' positions and used an LH-20 column to purify t h e sample, The McMaster group utilized I-0-perdeuteri0methyl-1~-tetrahydrocannabinol(1-OCDI-A9-THC) and derivatized their extract using N , N , N -trimethyl anilinium
hydroxide to convert 19-THC to its methyl ether. The extraction procedure was based on extractability of the phenolic I 9 - T H C into Claisen's Alkali from 5% isoamyl alcohollhexane. Thus, both the extraction and the derivatization of A9-THC relied on the chemistry of the phenol moiety. Since all cannabinoids and their metabolites contain the phenol group, it was felt that similar exploitation of the chemistry of this group would make available assays for all of the cannabinoids and their metabolites. However, the chemistry of 11-OH-A9-THC was more complex than that of 19-THC and the on-column methylation procedure could not be used. Nevertheless, the development of the assay still relied on the successful utilization of the chemistry of the phenolic moiety. T h e report describes the development of an assay for 11-OH-L9-THC based on a novel cannabinoid reaction.
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EXPERIMENTAL Assays were performed on a Varian CH-7 or a Varian CH-5 gas chromatograph-mass spectrometer unit (GC-MS) equipped with a n accelerating voltage alternator (AVA) for multiple ion detection. Ionization voltages and emission current were 70 eV and 300 M A ,respectively. T h e ion source was kept a t a temperature of 290 "C. Gas chromatography was performed on a coiled 6-ft column containing 1,5?(0SE-30 on Chromosorb W High Performance (H.P.), 80/100 mesh or 1.5O'a SE-52 on Chromosorb W (H.P.), 80/ 100 mesh. Injector port and interface to the mass spectrometer were kept a t 300 "C. There was no difference in the sensitivity or precision when t h e different columns or instruments were used. Reagents. Solvents were analytical reagent grade. Tetrahexylammonium hydroxide (THAH) was prepared by treating a 0.1 M solution of tetrahexylammonium iodide (THAI) in methanol (MeOH) with 1 M equiv of silver oxide and spinning for 2 h. T h e silver oxide was then removed by filtration through a short packed bed of Celite previously washed with MeOH. The 11-OH-19-THC was obtained from R. A. Graham. Chief of Scientific Services, Health Protection Branch, Department of Health and Welfare, Canada. Analysis by gas chromatography ( G C ) and ultraviolet (UV) spectrometry indicated that the material was pure. Procedure. Extractions were performed in glass tubes with Teflon-lined screw caps. A stock solution of 11-OH-A9-THC was prepared by weighing 36 mg of crystalline material and diluting to 25 ml with 9,5% ethanol (EtOH). This solution, when stored at 4 O C , was stable for extended periods. T o prepare the 1-0-ethyl-11-hydroxy-19-tetrahydrocannabinol ( 1 - O C ~ H ~ - 1 1 - O H - 1 9 - T H Cand ) l-0-perdeuterioethyl-ll-hy~roxy-lg-tetrahydrocannabinol (1OC2D~-11-OH-19-THC) standard solutions, 1 ml aliquots were concentrated under nitrogen (Nz), dissolved in 5 ml of 0.1 N sodium hydroxide (NaOH), and shaken vigorously for 5 min with 5 ml of a 0.5 M solution of either ethyl iodide (C2HjI) or perdeuterioethyl iodide (C2DiI) in methylene chloride ICH2CIp) and 100 p l of 0.1 M THAH/MeOH. T h e entire organic extract was transferred to a FlorisillCH2Clr column (140 mm X 6 mm i.d.1. The column was washed with CH2Cln (1 X 10 ml) and the product eluted with diethyl ether (Et201 (1 X 10 ml). The ether fraction was concentrated under NP and then transferred to weighing vessels with EtzO. T h e samples were weighed accurately to less than 1%on a Mettler M5 Microbalance. T h e yields of' 1-OC:IHi- and 1 -OCzDs-11-OH-A9-THC were 1.470 (9441)and 1.466 mg (93"/0),respectively. T h e stock solutions were prepared by dissolving the samples in 5 ml95% EtOH. A :3-ml aliquot of the stock solution was diluted to 10 ml with 95Oh EtOH to preparr the CY sample. The UV spectra of 1-OCZHj2760 nm (log E 3.34), and l - O C ~ D , ~ - l l - O H - l g - Tshowed H C , , ,A 2815 nm (log E ,'3.34) and, , ,A 2765 nm (log 3.331, 2815 nm (log E 3.33). respectively. Reference standards having concentrations of 20 ng/pl were prepared hy diluting 680 p1 of the stock solutions to 10 ml with hleOH. T h e 11-OH-19-THC stock solution was prepared by weighing 1.13; mg and diluting to 50 ml with MeOH. Standard solutions having concentrations of 660. 1340, 2000. 4000, 7960. and 12 100 ng/ml were prepared hy diluting aliquots of the stock solution to 10 ml with MeOH. The corresponding plasma standards having concentrations of 6.6, 13. 20, 40, 80. 121 ng/ml were prepared by diluting 100 pl of the methariolic solutions to 10 ml with plasma. One-ml aliquots of these plasma samples were used for the plasma standard curves and for quality control. Blanks were prepared by
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Figure 3. Plasma levels of 1 l-hydro~y-1~-tetrahydrocannabinol after intravenous dose of 1 l-hydro~y-1~-tetrahydrocannabinol to a dog
adding 10 pl MeOH to 1 ml of plasma. T h e plasma assay was initiated by adding 10 ml CH2C12 to 1 ml of plasma, shaking vigorously for 5 min, and then centrifuging for 15 min a t 2500 rpm. Three phases resulted: the bottom organic phase, the top aqueous phase, and a protein cake a t the interface. T h e tubes were removed carefully from the centrifuge to prevent rupture of the protein cake. The top layer was discarded. In order to coagulate the protein cake, a large spatula of sodium chloride (NaCI) was added and the tube was shaken for 5 s. After the precipitate settled, the CHzC12 was transferred to a new tube containing 20 ng of the deuterated internal standard solution. At this stage, the sample could be stored overnight in a refrigerator if necessary. Then, sufficient CpHsI (0.4 ml) to make the CH2Clz phase 0.5 M in CaHjI was dissolved in the extract. After the addition of 5 ml 0.1 N NaOH and 100 p1 0.1 M THAH/MeOH, the tube was shaken for 5 min. The Florisil columns were prepared and the chromatography of the organic phase was performed as before. The ether wash from the column. which could be stored overnight, was concentrated under Ne and then transferred to a silanized 5-ml "Reacti-Vial". T h e samples were evaporated to dryness under Nz and then stored under argon in a refrigerator until analysis. At that time, each sample was dissolved in 20 pl of a 90% BSTFA/lO% T M C S solution. At room temperature, the reaction was complete within 5 min. A 1-3 pl aliquot was injected for analysis. Ions moni- T H332 C for 1tored were 327 for ~ - O C ~ H S - ~ ~ - O T M S - A ~and O C ~ D S - ~ ~ - O T M S - A ~ (Figures - T H C 1 and 2). Spiked plasmas were analyzed simultaneously with all pharmacokinetic samples. ANALYTICAL CHEMISTRY, VOL. 48, NO. 4, APRIL 1976
727
mSULTS AND DISCUSSION I t was originally proposed t h a t t h e technique for AST H C reported by Rosenfeld e t al. ( 5 ) would be exploited to develop a n assay for 11-OH-A9-THC. However, when this molecule, or its 1-0-methyl ether were subjected to the oncolumn methylating conditions, two products were formed, one of which was the 1-0,1 1 - 0 , dimethyl ether as identified by its low resolution mass spectrum; the other compound remained unidentified. Furthermore, on repeated injection of either 11-OH-A9-THCor t h e 1-0-methyl ether, the ratio of these products was variable. T h e use of the oncolumn methylating procedure as a potential route to the analysis of 11-OH-A9-THC was therefore abandoned. Because of the apparent instability of 11-OH-hS-THC and its derivatives to the severe reaction conditions, a milder form of derivatization was investigated. Extractive alkylation has been reported for a wide variety of compounds (6-8). Given the acidity of the phenol group, this procedure was attempted for 11-OH-A9-THC. When tested on the preparative scale (1mg) and on t h e nanogram scale, 11-OH-A9-THC was converted to the 1-0-alkyl derivative in greater than 90% yield. Furthermore, only 1-0-alkylation occurred over a wide range of methyl iodide (CH3I) concentrations (0.5 t o 5 M) in CH2C12, NaOH concentrations (0.1 to 2 M), and shaking times (5 t o 30 min). In addition, we observed t h a t both CH3I and C2HBI reacted completely within 5 min. I t was also determined that, in the absence of THAH, no alkylation took place and only 11-OHA9-THC was recovered. This reaction was apparently well suited for derivatization of the 11-OH-A9-THC. It was mild and it only gave one product, thus avoiding the possibility of two functionalities (phenol, allylic hydroxyl) reacting a t different rates. T h e latter possibility might have given a complex reaction mixture. The use of T H A H resulted in the co-extraction of the corresponding iodide (THAI) in t h e CH2C12 phase (6).This material could interfere with t h e assay of drugs by virtue of the large excess of the product formed. The separation of l-OR-ll-OH-Ag-THC (R= C2H5 or C ~ D Sfrom ) t h e iodide was effected simply by passing t h e CHZCl2 phase through a short Florisil column, and eluting the product with EtzO. T h e Et20 wash contains l-OR-ll-OH-Ag-THC in quantitative yield while t h e THAI remained on the column. When the 1-OR-ll-OH-Ag-THCwas subjected to mass spectrometry, we observed t h a t the fragmentation pattern was not constant. Specifically the problem was that, while the same fragments always appeared, their relative intensity varied. This was attributed to pyrolysis in conjunction with, or previous to ionization. However, when the silyl derivative was prepared, the fragmentation pattern was highly reproducible (Figures 1 and 2). The silylation also enhanced the gas chromatographic properties allowing for analysis a t a lower temperature. T h e reaction conditions for silylation are sufficiently simple that this step adds no difficulty in running t h e assay. The choice of the ethyl group for derivatization was based on the work of Estevez who reported t h e presence of l-O-methyl-ll-hydroxy-A9-tetrahydrocannabinol (1-OCH311-OH-A9-THC) in t h e central nervous system of rats treated with Ag-THC (9). Given the possibility t h a t the 10-methyl ether may be present in plasma, the choice was made to use the ethyl derivatives for t h e reaction. There was no overriding chemical reason for using t h e methyl derivative, since either ethyl or methyl derivatization was equally simple. Because of this simplicity of reaction, it would also be possible to prepare 1-OCH3-11-OH-A9-THC in plasma. In addition, the nonselective nature of the extraction procedure described suggests t h a t it may be possi728
ANALYTICAL CHEMISTRY, VOL. 48, NO. 4, APRIL 1976
ble to co-extract the 1-OCH3-11-OH-A9-THC. The extractive alkylation-silylation technique was quantitative, giving a linear standard curve ranging from 6.6 to 120 ng/ml of plasma. T h e limiting factor of this assay appears to be a background of 3 ng/ml, which is attributed to column bleed. Because 5 deuteria are incorporated into the internal standard, it is unlikely t h a t isotopic impurities contribute significantly to the background. T h e standard curve was based on triplicate determinations of each point. Linear regression analysis gave the equation y = 0.003 0 . 0 0 2 5 7 ~with a relative standard deviation of 4% (n = 18). This procedure, to our knowledge, represents the first sensitive, nonradioactive assay for 11-OH-A9-THC in mammalian plasma. In order to test the viability of the assay in pharmacokinetic studies, two separate studies were performed. In the first instance, 10 mg of A9-THC as an oil in a capsule was given orally to a 27-kg dog and plasma taken for 5.5 h a t half-hour intervals. At no time was the 11-OH-A9-THC observed, whereas the spiked samples, run simultaneously, gave excellent results. I t was possible that A9-THC was not absorbed; accordingly, a second experiment relying on intravenous administration was performed. In the first stage, 1.75 mg of 11-OH-A9-THC in 1.75 ml EtOH was administered to a 15-kg dog and plasma was drawn. T h e variation of blood levels with time is shown in Figure 3. However, when 1.32 mg of Ag-THC in 1.4 ml of EtOH was administered to the same animal 24 h later, and blood sampled for 5 hr, there was still no detectable level of 11-OH-Ag-THC. However, the same dose (88 pg/kg) produced strong psychological effects when administered to humans via the smoking route. The lack of detection of the metabolite of A9-THC can have many explanations. Our data suggest t h a t ll-OH-AgT H C is not rapidly metabolized (Figure 3 ) , and, therefore, the rationalization that ll-OH-Ag-THC is rapidly metabolized may not be applicable. It is possible t h a t less than 3 ng/ml is formed and t h a t this is sufficient t o produce the psychological effect. Furthermore, although the dog has been used on a classical bioassay for Ag-THC (IO), this species may not metabolize the A9-THC to the 11-OH metabolite. Finally, consideration should be given to the possibility that Il-OH-A9-THC is not a major circulating metabolite in vivo. Final elucidation of these possibilities will involve pharmacokinetics of Ag-THC and 11-OH-A9-THC, studies using larger numbers of animals, various dosing schedules and different species including man. It may also be necessary to develop assays of higher sensitivity. This report describes a sensitive and precise analytical technique. It also raises some pharmacological questions which will require resolution. T h e assay and the chemistry described could prove to be useful in elucidating the complex pharmacology of A9-THC.
+
ACKNOWLEDGMENT We thank J. D. Fitzgerald, F. Anderson, and M. Robertson for their assistance and advice. LITERATURE CITED (1) G. G. Nahas, "Marihuana-Deceptive Weed", Raven Press, New York, 1973. (2) J. Koch-Weser, N. fngl.J. Med., 287, 227-231 (1972). (3) "Biological Effects of Drugs in Relation to Their Plasma Concentrations. A British Pharmacological Society Symposium", B. S. Davies and B. N. C. Prichard. Ed., University Press, 1973. (4) S. Agureil, B. Gustafsson, B. Holmstedt, K . Leander, J. Lindgren. I. Nilsson, F. Sandberg, and M. Asberg, J. Pharm. Pharmacol., 25, 554 (1973). (5)J. Rosenfeld, B. Bowins, J. Roberts, J. Perkins, and A. S. Macpherson. Anal. Chern., 46, 2232 (1974). (6) M. Ervik and K . Gustavil, Anal. Chern., 46, 39 (1974). (7) B. Lindstrom and M. Molander, J . Chromatop., 101, 219 (1974).
(8)A. Arbin, P. Edlund, Acfa Pharm. Suec., 12, 119 (1975). (9)V. S . Estevez. L. F. Englert, and B. T. Ho, Res. Commun. Chem. Patho/. Pharmacol.,6 , 821 (1973). (10) Y. Grunfeld and H. Edery, Psychopharmacologia, 14, 200 (1969).
RECEIVEDfor review November 10, 1975. Accepted December 29, 1975. This work was supported by Grant DA-18 from the Medical Research Council of Canada.
Determination of Hydrogen Sulfide and Methyl Mercaptan in Mouth Air at the Parts-per-Billion Level by Gas Chromatography A. R. Blanchette" and A. D. Cooper Analytical Chemistry Department, Vick Divisions Research and Development, 1 Bradford Road, Mount Vernon, N. Y. 10553
Gas chromatographic techniques were adapted for quantifying hydrogen sulfide and methyl mercaptan at the low parts-per-billion level in mouth air samples. Major innovations were calibration of the system with hydrogen sulflde and methyl mercaptan permeation tubes, use of a variable stream splitter to produce a wide range of vapor concentrations, and adaptation of the sampling equipment to handle discrete samples of limited volume. The limits of sensltlvlty were 7 ppb for hydrogen sulfide and 15 ppb for methyl mercaptan in a 10-ml mouth air sample.
The quantification of volatile sulfur compounds in mouth air presents an interesting and unusual problem in chemical analysis, a problem which in several ways has more demanding requirements than those of air pollution studies. Interest in the clinical applications of such quantification arises from the fact t h a t two of these compounds, hydrogen sulfide and methyl mercaptan, are important constituents of oral malodor (1, 2). Determination of the range of concentrations of these compounds in mouth air samples before and after various hygienic procedures presents the following requirements. 1) For ideal functioning the calibration system should be adapted specifically for hydrogen sulfide and methyl mercaptan. 2) Noncontinuous sampling must be used because of the limited volume of mouth air available (25 to 35 ml), instead of the continuous-flow sampling utilized for air pollution studies. 3) Measurements must be made a t the low ppb level (usually well below 1000 ppb), but in a range of concentrations due to the considerable natural variation between subjects and even in the values obtained for a single subject within a single day. 4) The method should be simple and quick, since it will be used for repeated measurements in a group of waiting subjects. T o meet these requirements, in the research described below, we made certain modifications or adaptations in existing instrumentation, as utilized for air pollution studies. Key contributions to the progress of the air pollution studies were the development of a flame photometric detector with excellent sensitivity and selectivity for sulfur-containing compounds (such as described in ( 3 ) ) ,use of permeation tubes to produce low concentrations of volatile compounds in gaseous mixtures, thereby providing a means for accurate calibration of the detector ( 4 ) ,and utilization of a gas chromatograph, equipped with such a detector calibrated with permeation tubes, for the detection and quantification of sulfur compounds in the ambient air ( 5 ) .
EXPERIMENTAL Figure 1 shows our adaptation of the equipment for analyzing
hydrogen sulfide and methyl mercaptan. Details of the system are as follows. Calibration System. Known levels in the ppb range of hydrogen sulfide and methyl mercaptan for calibration are produced as follows. The flow of nitrogen gas through the flow controller and the flowmeter (0 to 200 ml/min) is held constant a t 150 ml/min, although other flow rates can be used, depending on the desired range of concentrations. T h e nitrogen then passes through a copper tube (lk-in. 0.d. X 10 ft) immersed in a constant-temperature bath to equilibrate the gas a t 30 & 0.1 "C. T h e mixture of gases emerging after the equilibrated nitrogen has flowed through the 500-ml gas-washing bottle containing the permeation tubes is split a t the tee in a ratio adjustable by the needle valve. Unused gas is measured by a second flowmeter (0 to 200 ml/min). The smaller fraction of the split stream then passes through a glass capillary restrictor into a three-way valve where it may be diverted for measurement with a soap-film flowmeter or else may continue to the Teflon low-resistance mixing chamber (1.0-cm i.d. X 8 cm) for dilution by additional nitrogen to the desired concentration. A gas sample for calibration can then be drawn from this chamber through the transfer line, located 3.5 cm downstream from the entering mixture of diluted permeation gases. Sampling and Chromatographic Analysis. Either a calibration sample or a mouth air sample withdrawn via the disposable sampling probe (a 2- to 3-in. length of 0.12-cm i.d. Teflon tubing) is delivered through the selector valve to the Chromatronix sampling valve, from where nitrogen carrier gas (72 ml/min) sweeps it into the chromatograph for separation of the volatile sulfur compounds on the 30 ft X %-in. 0.d. FEP Teflon chromatographic column. The column is packed with 5% w/w polyphenyl ether and 0.05% w/w phosphoric acid on 30/60 mesh Teflon. T h e column temperature is either 58 or 72 "C. As the volatile sulfur compounds emerge from the column, they are detected by the flame photometric detector equipped with a 394-nm sulfur-specific optical filter. T h e detector functions a t a temperature of 120 "C, with a hydrogen flow of 100 ml/min, and with a n air flow of 70 ml/min. All parts of the system in contact with the sulfur compounds were constructed of Teflon, with the exception of the capillary restrictor, the splitting tee, and the gas-washing bottle, all of which were glass. Specific modifications made to meet the requirements listed in the introduction for mouth air studies were: 1) Permeation tubes of hydrogen sulfide and methyl mercaptan were used to produce ppb levels of these two compounds, equivalent to the range expected in mouth air samples, for specific calibration of the flame photometric detector. Previous clinical workers (1, 6 ) had extrapolated from gas chromatographic responses to t h e concentrations produced by a sulfur dioxide permeation tube; however, we wished to bypass the problem noted by other authors ( 7 ) of differing losses, largely by adsorption, in the system for the various sulfur compounds. 2) In air pollution studies a sampling pump is commonly used in conjunction with a 6- or &port gas sampling valve to fill the sample loop. Because of the small size (10 ml) of the discrete samples of mouth air, we used a 20-ml syringe instead of the pump, after preliminary tests had shown that this modification did not adversely affect the sensitivity or reproducibility of the system. Further preliminary tests were performed t o develop a standardized procedure for the actual sampling of mouth air. These tests demonstrated that the observed sulfur levels in mouth air ANALYTICAL CHEMISTRY, VOL. 48, NO. 4, APRIL 1976
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