805
Anal. Chem. 1983, 55, 805-807
rod vaporization system (2) are also shown in Table 11. A comparison of the absolute detection limits expressed as analyte mass shows that in most cases a superior power of detection was obtained with the present method. The relative standard deviation was 2-4% for boron, lead, phosphorus, and tin, and 5-10% for germanium and zinc with a 1 pg/mL concentration. A rectilinear calibration curve was obtained from 0.001 pg/mL over 4 orders of magnitude for boron, from 0.01 pg/mL over 3 orders of magnitude for phosphorus, lead, tin, and zinc, and from 0.01 pg/mL over 2 orders of magnitude for germanium. The author acknowledges that the present study is limited to relatively volatile elements. It is not known whether the same advantages of this small chamber system would also occur with less volatile elements. This will be the subject of future investigation in this laboratory.
ACKNOWLEDGMENT The author is grateful to Atsushi Mizuike, Horishi Kawaguchi, Fumio Nagata, and Hisao Kojima for their helpful suggestions. Registry No. B, 7440-42-8; Ge, 7440-56-4; P, 7723-14-0;Pb, 7439-92-1; Sn, 7440-31-5;Zn, 7440-66-6. LITERATURE CITED (1) Nixon, D. E.; Fassel, V. A.; Kniseley, R. N. Anal. Chem. 1974, 46, 2 10-2 13. (2) Gunn, A. M.; Millard, D. L.; Kirkbright, G. F. Analyst (London) 1978, 103, 1066-1073. (3) Millard, D. L.; Shan, H. C.; Klrkbright, G. F. Analyst (London) 1980, 105, 502-508. (4) Kawaguchl, H.; Vallee, B. L. Anal. Chem. 1975, 4 7 , 1029-1034.
RECEIVED for review April 13, 1982. Accepted December 9, 1982.
Breath Analysis by Atmospheric Pressure Ionization Mass Spectrometry Frank M. Benoit” Environmental Health Dlrectorate, Health and Welfare Canada, Tunney’s Pasture, Ottawa, Ontario K I A OL2, Canada
W. R. Davldson, A. M. Lovett, Sabatlno Nacson, and Angle Ngo Sciex Inc., 55 Glencameron Road, Thornhill, Ontario L3T 7P2, Canada
The value of human breath analysis in medical, forensic, and environmental applications has long been recognized. Sampling techniques have consisted of the collection of large volumes of breath in Teflon ( I ) , Mylar (2-5), or Plastigas (6) bags, in glass tubes ( 4 , 5 , 7-9),or in cryogenic traps ( 1 0 , I I ) followed by concentration of the volatiles onto Tenax (1,111, charcoal (3), silica gel (6),or cryogenic (2) traps prior to analysis by infrared spectrometry, gas chromatography, or gas chromatography/mass spectrometry. A recent report (12) suggested that direct introduction of expired breath into the ion chamber of API/MS system would allow the extraction of the organics directly from the gaseous mixture by ionmolecule ionization. However, direct breath introduction into the ion source caused difficulties due to interferences from ammonia (12). In the present study a novel breath inlet system which allows the direct sampling of exhaled human breath for analysis by API/MS is described.
EXPERIMENTAL SECTION Breath Inlet. The breath inlet shown in schematic form in Figure 1 consisted of a mouthpiece, filter tube, manifold, and mixing chamber mounted on a metal frame. The disposable polyethylenemouthpiece (Inspiron,Rancho Cucamonga, CA) was separated from the glass fiiter tube (22 mm 0.d. X 270 mm, Sovirel, France) by a diaphragm one way valve (Inspiron, Rancho Cucamonga,CA). The filter tube could be filled with filter material contained between two loose fitting glass wool plugs, if required. The filter tube was fitted with a port (Swagelok fittings) though which carrier gas could be directed, if required, to simulate an exhalation for calibration purposes. The filter tube assembly was attached to the stainless steel manifold via a tee connection of which one arm, fitted with an adjustable valve, led to the atmosphere and the second arm, containing a replaceable glass capillary, led to a stainless steel manifold. The manifold was connected to a mixing chamber containing perforated macor disks to ensure mixing of the breath sample with the carrier gas prior to introduction into the ion chamber. The manifold and filter tube assembly were fitted with outlets for the pressure transducers such that the pressure differential across the capillary was measured by two manometers connected in parallel. One ma0003-2700/83/0355-0805$01.50/0
nometer (Magnehelic,range 0-2 in. of water, Dwyer Instruments, Michigan City, IN) provided a visual reading to the subject who was required to maintain a prescribed constant pressure differential across the capillary during exhalation. The second manometer (Robinson Halpern, range 0-10 in. of water, Plymouth Meeting, PA) controlled the scan of the TAGA ion analyzer and provided a reading of the pressure differential during each scan period. The carrier gas, zero grade air (Matheson) was passed through a filter (molecular sieves Linde 13X and Linde 4A) to remove impurities in the air prior to introduction into the manifold. The carrier gas flow was controlled by a Lee Jet orifice such that the flow could be set by adjusting the regulator pressure.
RESULTS AND DISCUSSION Atmospheric Pressure Ionization. In the positive mode of operation a series of protonated water clusters H+(H20), are generated from the moisture in the ambient air following a series of ion-molecule reactions (13)initiated by the ionization of molecular nitrogen in a point to plane corona discharge. Ionization of a sample molecule (T) produces protonated moleculewater clusters of varying size depending on n (reaction 1).
T + H+(H,O),
+
TH+(HZO),
Upon passage of a clustered target ion through a gas (N,) curtain located between the ion chamber and the mass analyzer (14) collisions with the neutral nitrogen molecules activate the ion sufficiently to strip the loosely held water molecules from the ion and a protonated molecule, (eq 2) is transmitted to the analyzer. One consequence of the “declustering” is that the distribution of ions collected at the detector does not necessarily correspond to the original distribution of ions generated in the ion source a t the moment of ionization. In the negative mode of operation the reagent ion 02-is generated by point to plane corona discharge ionization of oxygen in ambient air. Some of the observed reactions with 0 1983 American Chernlcal Soclety
806
ANALYTICAL CHEMISTRY, VOL. 55, NO. 4, APRIL 1983 200000
SPIKING PORT, I”
\
” ’
TETRACHLOROETYYLENE
r 9
8 ”
TAGA
J L
-
Flgure 2.
In1 GAS
j - \ O N EV A L WAY VE ‘MOUTHPIECE
Flgure 1. Schematic of breath Inlet. the sample molecule are charge transfer (reaction 3) and proton abstraction (reaction 4)depending upon the relative electron and proton affinities, respectively, of the sample molecule and the reagent ion.
(3)
+ HO2
Ion
100 200 CONCENTRATION Ipprnl
IO
abundance vs. concentration (ppm range)
relative to that of the reagent ion. During the residence time in the ion chamber the ionized species undergo numerous collisons with the neutral molecules from the air matrix. However, should there be present in the gaseous mixture species of higher proton affinity, a collision could be reactive and proton transfer between sample molecules (reaction 5)
-
T + 02--.* (T-H)-
0
(4)
One problem associated with the API ion source is ion signal saturation a t relatively low (low parts per million) sample molecule concentrations. In the positive mode the usual total abundance of protonated water clusters is ca lo6 counts/s. However, reagent ion clusters are not all equally reactive. As the size of the cluster increases, the proton affinity of the cluster increases and the reactivity of the protonated cluster by proton transfer decreases. Hence only a portion of the reagent ions may be reactive toward a particular sample molecule and the maximum concentration of sample molecule ions that may be generated is limited by the concentration of reagent ions clusters that have a proton affinity lower than the proton affinity of the sample molecule. A complicating factor is that a water cluster has a higher proton affinity than the water molecule alone and the initial sample ion formed is a water clustered sample ion (reaction 1);therefore the prediction of the reactivity of a sample molecule based on the proton affinities of the simple water and sample molecules may not be valid. However, the proton affinities of water clustered molecules are unavailable at present; hence the use of the proton affinities of the simple molecules represents the best compromise. In addition, when analyzing mixtures, the ion signal may be partitioned among the various components which in effect further reduces the concentration of reagent ions available for a particular reaction. Consequently the number of sample molecules entering the ion source may exceed the number of reagent ions available for reaction, in which case the target ion signal is no longer proportional to the target molecule concentration in the ion source. A plot of ion abundance vs. molecule concentration shows onset of curvature at the low parts per million (20 ppm) level (Figure 2) which is generally the upper concentration limit for quantitative work. It is essential therefore in quantitative work to establish the concentration range for linear response for the particular reaction under study. Fortunately for samples of high concentrations it is always possible to dilute the sample to lower concentrations that fall within the linear response range. A second problem associated with the API ion source is interference from extraneous compounds in a multicomponent gas mixture, in which individual ionization reactions will be dependent upon the proton affinities of the sample molecules
TIH++ T2
-
T2H++ T1
(5) deplete the ion signal for TIH+. In the limit, at “higher” T2 concentrations, TIH+ may “disappear”. Problems of interferences are generally eliminated by dilution of the sample although the limit of detection of components of lower concentration is raised when dilution of the sample is required. Breath Inlet System. The direct introduction of exhaled human breath into the ion chamber of the API/MS produced both the saturation and interference effects; although not the major component of breath, ammonia was present in sufficiently high concentrations in breath and had a higher proton affinity than the other breath components which resulted in a saturated signal at m/z 18, NH4+(12). In order to overcome these problems a novel inlet was designed with the aim of diluting and filtering (if desired) the sample of breath prior to introduction into the ion source. The breath inlet, which is described in the Experimental Section, achieved controlled dilution of the breath sample by bleeding a portion of the breath sample through a capillary of known bore and length into a stream of carrier gas (N,) of known flow prior to mixing and introduction into the ion source. The excess breath was dumped to the atmosphere via a variable valve. The volume of breath transmitted through the capillary was determined by the pressure differential across the capillary which was measured with a manometer in view of the subject. With a capillary of 0.50 mm i.d. and of 5 cm length, an exhalation pressure of ca. 0.2 in. of water and a carrier gas flow of 90 cm3/s, a dilution factor of ca. 1000 was obtained. Under these conditions the difficulties with saturation and interferences were eliminated. Performance of Breath Inlet System. To enhance the sensitivity of the API/MS the electron multiplier was operated in the ion counting mode in which the analyzer, under computer control, remained “open” at a mass window until either a selected number of ions (set by operator within narrow limits) had been detected or a preset time limit (from s to 1 s) had expired. Even with the fastest scan speed (‘/15 s per amu) a complete scan over 200 amu could not be obtained during one exhalation. In fact two to three exhalations were usually required. To obtain meaningful results, it was therefore necessary to suspend the scan between exhalations. This was achieved by initiating and maintaining the scan only when the inlet pressure exceeded a selected preset value (usually 0.2 in. of water). The spectra presented were then obtained by subtraction of background carrier gas spectra from the exhaled breath spectra. In previously reported ( I , 10) profiles of human breath, acetone was observed as the most abundant organic species. In Figure 3 a full scan (positive mode) of a breath sample using
Anal. Chem. 1983, 55, 807-808 vu
I
119
0
Figure 3.
100 m/z
50
150
I
200
Full scan (positive mode) of human breath.
0 0 Figure 4.
BREATH
10 20 30 SCAN N U M B E R
40
Acetone (MH', m l r 59) as a function of exhalations. 32
BREATH NEGATIVE
W W O
>z
MODE
60
< ;a 0 50J Z
u3
wm
16
4
50 0,
Figure 5.
1 .
'
76 '
I
Full scan (negative mode) of human breath.
the breath inlet system exhibited a prominent peak at mlz 59 corresponding to the protonated acetone molecule. The peaks at mlz 19 and mlz 37 corresponded to protonated water clusters H+(H20)and H+(H20)2,respectively. The peak at mlz 18 due to NH4+which dominated the spectrum of nondiluted breath despite its low concentration is no longer the most prominent peak when the breath sample is diluted via the breath inlet system prior to introduction into the ion source; thus the dilution technique successfully eliminated the interferences from ammonia and, despite the dilution factor of ca. 1000, the system retains sufficient sensitivity to detect volatile organic vapors on breath. Other minor peaks observed include mlz 33 and mlz 47, corresponding to protonated methanol and ethanol, respectively.
a07
In Figure 4 (M + H)+ of acetone, mlz 59 is shown as a function of time over several exhalations with the instrument operating in the selected ion mode. The observed response of the system to an exhalation is rapid as shown by the sharp onset when the exhalation is swept into the ion source. Further, at the end of an exhalation there is a sharp decrease in the ion signal to background levels. There is no evidence of contamination from one exhalation to another at least for the compound under study. In the negative ion mode the breath spectrum (Figure 5) contained large peaks at mlz 16, mlz 59, m / z 60, and mlz 61 in addition to the reagent ion peak at m/z 32. The negative ion spectrum of expired breath differed markedly from the positive ion spectrum. Compounds of high proton affinities (ionized in positive mode) generally do not have high electron affinities (ionized in negative mode) and vice versa. The largest peak, other than the reagent ion peak, corresponded to COS- m/z 60 which resulted from reaction of 02-with C02 in expired breath. The peak a t m / z 61 corresponding to HC03- likely originated from the interaction of COB-and moisture in the breath. Endogenous organics contributed to a few minor peaks at mlz 26 (CN-) and m/z 93 (C,H,O-). Registry No. Acetone, 67-64-1;methanol, 67-56-1;ethanol, 64-17-5; tetrachloroethylene, 127-18-4. LITERATURE CITED (1) Krotoszynskl, B.; Gabriel, G.; O'Nelll, H. J. Chromatogr. Scl. 1977, 15, 239. (2) Levey, S.; Balchum, 0.J.; Medrano, V.; Jung, R. J . Lab. Clln. Med. 1064, 63. 574. (3) Jansson, B. 0.; Larsson, B. T. J. Lab. Clin. Med. 1969, 74, 961. (4) Stewart, R. D. I n "Essays In Toxlcology"; Hayes, W., Ed.; Academic Press: New York, 1974; Vol. 5, Chapter 5. (5) Stewart, R. D.; Hake, C. L.; Wu, A. Scand. J. Work, Envlron. Health 1976, 2 , 57. (6) Gage, J. C.; Lagesson, V.; Tunek, A. Ann. Occup. Hyg. 1977, 20, 127. (7) Stewart, R. D.; Hake, C. L.; Peterson, J. E. Arch. Environ. Health 1974, 6 , 29. (8) Pasqulni, D.A. Am. Ind. Hyg. Assoc. J . 1978, 3 9 , 55. (9) Verberk, M. M.; Scheffers, T. M. L. Envlron. Res. 1980, 21, 432. ( I O ) Conkle, J. P.; Camp, B. J.; Welch, B. E. Arch. Environ. Health 1975, 30, 290. (11) Gearhart, H. L.; Pierce, S. K.; Payne-Bose, D. J. Chromatogr. Sci. 1977, 15, 480. (12) Lovett, A. M.; Reid, N. M.; Buckley, J. A.; French, J. B.; Cameron, D. M. Blomed. Mass Spectrom. 1979, 6 , 91. (13) Good, A.; Durden, D. I.; Kebarle, P. J. Chem. Phys. 1970, 52, 212. (14) French, J. B.; Reid, N. M.; Poon, C. C. Twenty-Fifth Annual Conference on Mass Spectrometry and Allied Topics. Washington, DC, 1977. (15) Carroll, D. 1.; Dzidic, I.; Stillwell, R. N.; Hornlng, M. G.; Hornlng, E. C. Anal. Chem. 1974, 46, 706.
RECEIVED for review October 8,1982. Accepted December 20, 1982. This work was carried out by Sciex, Inc., under contract with Health and Welfare Canada.
Modification of a Centrifugal Filtratlon Device for Direct Filtration into an Autosampler Vlal I. A. Elseman and L. A. Pachla" Warner-LambertIParke-Davls Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, Michigan 48 105
Samples for high-performance liquid chromatography are frequently derived directly from biological materials, pharmaceutical preparations, or TLC spots. Often the analytical sample requires filtration to remove suspended matter. Presently there is commercially available a centrifugal mi-
crofilter which is capable of filtering microliter volumes with greater than 95% recovery (Bioanalytical Systems, MF-1 centrifugal mirofilter). This filtration device has been successfully applied to the analysis of phenolics ( I ) . This device is an excellent tool when samples are manually injected into
0003-2700/83/0355-0607$01.50/0 0 1983 American Chemlcal Society
