New Picogram Detection System Based on a Mass Spectrometer with an External Ionization Source at Atmospheric Pressure E. C. Horning, M. G. Horning, D . I. Carroll, I. Dzidic, and R . N. Stillwell lnstitute for Lipid Research, Baylor College of Medicine, Houston, Texas 77025
A novel mass spectrometer with an external ionization source can be used to detect picogram quantities of compounds of biologic interest. The source contains a 63Ni foil, and is at atmospheric pressure. Samples are introduced in a flowing gas stream in selected common solvents. Positive ions are formed by a complex series of ion molecule reactions. The ionization reaction for the sample may involve either proton transfer or charge transfer. Negative ions are formed by either resonance or dissociative capture of thermal electrons, or by ion-molecule interactions. In a favorable case (very little adsorption on the reaction chamber walls), 5-10 picograms could be detected by single ion monitoring, and a scanned mass spectrum could be obtained with as little as 25 picograms. The potential uses include incorporation into LC-MS-COM and GC-MS-COM analytical systems.
Table I. Instrument Parameters 180-200 "C 200 "C 1.7 ml/sec 760 Torr
Sample inlet temperature Source temperature Gas flow Source pressure Mass spectrometer pressure Electric field in source
4 X
10-5Torr
None
reaction chamber with a flowing gas stream at atmospheric pressure; and to design GC-MS-COM and LC-MSCOM (liquid chromatograph-mass spectrometer-computer) systems based on the approach described in this paper, and incorporating recently developed thermostable glass capillary columns (I) for a GC-MS-COM system. The results of work related to the first two objectives are described here.
EXPERIMENTAL Analytical systems of the GC-MS-COM (gas chromatograph-mass spectrometer-computer) type provide the most powerful means of investigation now available for studies of drug metabolism, in toxicology and in many problems involving human metabolic pathways. These systems are still in an early stage of development, however, and their capabilities are restricted more by historical patterns of investigation than by technological limitations. Mass spectrometers in these systems are usually based on design considerations which no longer apply. Sample size was not an important matter earlier; now, high sensitivity in detection is often of paramount importance. The function of the system is to detect and quantify molecular entities in a flowing gas stream, but mass spectrometers are designed largely as high vacuum instruments with internal sources and multiple pump assemblies. We have initiated a program with three objectives: to define ionization reactions occurring at atmospheric pressure for compounds of biologic interest, initiated by electrons from 63Ni rather than from a'heated filament; to define the characteristics of an MS-COM system designed as a detector and operating with continuous sampling of a 936
ANALYTICAL C H E M I S T R Y , VOL. 45, NO. 6, M A Y 1973
Instrumentation. The apparatus used to obtain the experimental results reported here is shown in schematic form in Figure 1 and the instrument parameters are given in Table I. The reaction chamber is at atmospheric pressure and is separated from the mass spectrometer high vacuum region by a 25-p pinhole aperture. Primary ions are produced in the carrier gas by a 12.5-mCi nickel-63 (on gold foil) radiation source. The ion molecule reactions occurring in the chamber are described in detail in the discussion section. The ions from the atmospheric pressure source are entrained in the gas flow through the pinhole aperture, into the high vacuum mass spectrometer region. The mass spectrometer is pumped by a 6-inch high speed oil diffusion pump, with liquid nitrogen trap, which has an effective pumping speed of 900 l./sec. The measured pressure a t the pumping system inlet was 4 x Torr. The actual pressure in the ion lens and mass analyzer region was not measured but is believed to be an order of magnitude higher. Sampled ions are separated from neutral molecules in the high vacuum region by a series of ion lenses. A conventional electron impact source is incorporated into the ion lens assembly to permit spectra to be taken for calibration purposes. The ion lens and quadrupole bias voltages are adjustable and can be optimized for either positive or negative ion operation. The ion entrance energies are set between 10 and 15 eV. (1) A. L. German
and E. C. Horning, J. Chromatogr. Sci.. in press.
The quadrupole mass analyzer is a modified unit based on the Finnigan Model 1015 mass spectrometer. The modifications consist of providing a quadrupole rod-bias voltage (to permit operation with a ground potential source), and substitution of a Bendix Model 4039 Spiratron electron multiplier for the normally supplied dynode multiplier. The nanosecond low level pulse output of the multiplier is capacitively coupled to a Solid State Radiation Model 1120 Amplifier-Discrimator and Model 1105 Data Converter. The capacitive coupling of the output permits the multiplier anode to be operated a t the +5 kV potential required for negative ion operation. The sample inlet consists of an eighth-inch stainless steel Swagelok elbow located in the carrier gas inlet line and arranged for injection through a silicone septum. This inlet was heated by an aluminum block and a 100-watt cartridge heater. The reaction chamber and mass spectrometer housing were heated by means of "Briskheat" heating mantles and proportional temperature controllers (Therm0 Electric Model 400). Temperatures were measured using iron-constantan thermocouples attached to the inlet elbow, the reaction chamber, and mass spectrometer housing. The reaction chamber and inlet temperatures were maintained a t 200 "C. A PDP8/E computer (Digital Equipment Corp.) with laboratory interface and display scope was used for acquisition and analysis of data from the mass spectrometer. The digital output signal of the SSR counter is connected to a Schmitt trigger input of the interface. This signal is a 5-V pulse of approximately 0.5 psec duration a t every pulse from the Spiratron electron multiplier. The digital-to-analog converter (DAC) output which drives the X-axis of the display scope is also used as the input to an operational amplifier circuit to supply a signal of 0 to -10 V to the external scan plug of the mass analyzer circuit. The system is operated under control of a program (PCMS) which is based in part on the post-stimulus histogram program (PSTLAT) supplied as part of the computer software package. For acquisition of mass spectral data, the DAC is stepped through a specified range a t a specified rate while counts from the electron multiplier are accumulated in a buffer whose pointer is stepped in synchronization with the mass spectrometer scan. Acquisition continues either for a fixed number of scans or until the operator intervenes. The accumulated mass spectrum is displayed during the acquisition phase. At the end of acquisition, the program enters the display mode. The display can be scaled up or down, and plotted on an X-Y recorder. A cursor can be generated and positioned on the screen either by means of a potentiometer connected to an A-D channel (floating mode) or, for finer adjustment, by stepping one bit left or right on command from the teletype keyboard (fixed mode). The voltage corresponding to the cursor position is typed on command from the teletype, or all of the display except the cursor can be erased, allowing measurement with a digital voltmeter. Four 512word buffers are available and can be combined into larger buffers if necessary, so that more than one scan can be stored and recalled for display. Spectra can also be dumped on to paper tape and read back in a t a later time. Operation. High purity Matheson nitrogen was used as the carrier gas. The gas was cleaned of residual organic and water vapors prior to use by passage through a 2.5-liter stainless steel cylinder containing 4-8 mesh type 13X molecular sieve. Passage of the gas through the molecular sieve reduces the vapor pressure of water to Torr. Consistent initial background ion spectra were obtained by increasing the reaction chamber and inlet temperatures to 300 "C overnight with the carrier gas flowing. Occasional bakeouts a t 300 "C under vacuum of 10-7 Torr were required to remove particularly persistent samples. Solutions of reference compounds and mixtures of biologic origin were injected directly into the inlet with a Hamilton 7001 syringe. The usual volume was 1-2 pl. Two modes of operation were usually employed. In the scanning mode, signal averaging over 15 scans (approximately 2 sec for each scan over 0-750 amu), generally gave satisfactory spectra. Narrow range scanning was employed in the deuterium labeling experiments. The programming also permitted single ion monitoring (an example of the chart record in an experiment of this kind is in Figure 6). The usual carrier gas flow rate was 100 ml/min. The retention of compounds in the source was dependent on the temperature and gas flowrate, and also upon the extent to which adsorption occurred. Traces of pyridine and silylating reagents were difficult to remove from the reaction chamber. Calibration. The mass spectrometer was routinely calibrated using the electron impact source. Positive ion spectra were cali-
brated using the electron impact spectrum of sulfur hexafluoride. More extensive calibrations with perfluorotributylamine were made as required to further check resolution and mass calibration. Negative ion spectra were calibrated using the characteristic ions C1-, Br-, I-, Is-. SFs-, and SFs- generated in the reaction chamber by dissociative and resonance electron capture. The negative and positive ion mass calibration were the same within experimental limits of k0.5 amu and were very stable from day to day. The stability of the instrument was easily monitored on a daily basis by examining solvent or background ion spectra. Resolution. The mass spectrometer was initially calibrated and adjusted to unit resolution at mass 614 using perfluorotributylamine as a reference compound. A large mass discrimination effect was found. This was determined as follows: in the negative ion mode the ion current can be converted entirely to I- ions by injecting iodobenzene into the reaction chamber. Under these conditions the total ion current. a t the detector, was measured as 106 cps, and the resolved I- ion amplitude was adequate with a 1-second scan. The ion current was then converted entirely to Isions by injecting elemental iodine. Under these conditions the total ion current was again measured and found to be unchanged. At unit resolution, however, the 13- ion could not be detected. To attain high sensitivity, the resolution was decreased to 2-3 amu; for unit mass resolution, a narrow scanning range was used with reduced sensitivity in detection. These characteristics are determined by the nature of the mass analyzer. The limits of detection and the nature of the spectra are indicated in the Figures; the discussion section contains information about ion currents in the source and in the analyzer region. Solvents, Reference Compounds and Biologic Experiments. Solvents were Nanograde quality; all were examined to ensure the absence of contaminants. When chloroform was employed in studies of positive ion formation, the small amount of ethanol usually present provided the major ions. Typical solvent spectra are illustrated in the Figures. Stock solutions of reference compounds (of the highest available purity) were prepared and stored in all-glass containers under deep freeze conditions. Working samples obtained by dilution of reference solutions were prepared in glass vials with Teflon (Du Pont)-lined caps. These solutions, when exposed to light a t room temperature, usually showed evidence of decomposition or alteration of samples within a few days. For this reason, fresh working solutions were prepared as required from the refrigerated stock solutions. Phenobarbital (110 mg/kg sodium salt) was administered by intraperitoneal injection to a rat (200-gram Sprague-Dawley), and the urinary metabolites (24 and 24-48 hours) were isolated according to Harvey, Glazener, Stratton, Nowlin, Hill, and Horning (2). The metabolites from 8 ml of rat urine (24-48-hour period) were dissolved in 2.0 ml of methanol; derivatives were not prepared. A control urine sample was obtained prior to drug administration; this was treated in the same way.
RESULTS AND DISCUSSION Ion-Molecule Reactions. Positive Ions f r o m Nitrogen, W a t e r and Benzene. Ion-molecule reactions in irradiated pure nitrogen, and nitrogen containing traces of water, at pressures up to 4 Torr were studied byGood et al. (3). The following sequence of reactions (1 through 6) was established on the basis of kinetic data: N2
+
e -N2+
Nz++ 2N2-N,+ N4+ + H20-Hz0+ HzO+ H30+
+
+
H20
+
H20-H30+
+
+ 2e
(1)
+
(2)
N2
+ 2Nz + OH
N,-H+(Hz0)2
+
t N,
(3) (4) (5)
+ Nz (6) In reaction 1 the Drimarv ion N7+ is Droduced by electron impact. This the; reacts with additional nitrogen to give H+(H20),-1
H,O
N2-H+(Hz0),
(2) D. J. Harvey. L. Glazener, C. Stratton, J. Nowlin, R . M .Hill, and M . G . Horning, Res. Commun. Chem. Path. Pharm.. 3 , 557 (1972) (3) A. Good, D. A . Durden, and P. Kebarle, J. Chem. Phys.. 52. 212 (1970). A N A L Y T I C A L C H E M I S T R Y , VOL. 4 5 , N O . 6 , M A Y 1973
937
A'1
D20 SOLVENT
PREHEATED CARRIER GAS INLET 1 BETA SOURCE
I
CALI BRATION I O N SOURCE
QUADRUPOLE RODS
Figure 1. Schematic diagram of the apparatus. The reaction volume is about 1 cm in diameter and 1 cm in length
(reaction 2) which would be the final product in the absence of water vapor. In the presence of water vapor, however, charge transfer occurs (reaction 2) to give HzO+, which in a subsequent step (reaction 4) reacts with another molecule of water to give the observed ion H&+. Subsequently, association of H30+ with additional water molecules occurs to give the ion clusters H+(H20),. The distribution of clusters depends upon the water concentration and the temperature of the reaction chamber. H+(H20), clusters were also observed with a-particle irradiated gases at 200-Torr pressure (4), in corona discharge studies of nitrogen a t atmospheric pressure (5), and in this work at 1 atm with /3 particle irradiated nitrogen. Traces of water present on the walls of the reaction chamber which lead to Hr(H20), formation at high pressures cannot be easily removed, and these ions were always present in background spectra (before injection of solvent). When 1-2 pl of benzene was vaporized into the reaction chamber, two major ions, C6H6+ and C12H12+, were observed. The C&+ ion is probably produced by charge transfer (reaction 7), since the ionization potential of benzene is lower than that of water and the concentration is much greater than that of background water. The C12H12+ ion is formed by reaction 8; this was observed by Wexler (6) with 0.005- to 0.32-Torr pressure of benzene in the ion source. Nq+
N4+ -k C&3-C,H6+
-k 2N2
(7)
CsH6+ -k C&I,j-C&12+ (8) According to Wexler (6), the C12H12+ ions are apparently produced by "sticky" collisions of C & j + ions with benzene molecules, and these collision complexes do not require further de-exciting collisions for stabilization. Other ions of simple structure can be produced in the reaction chamber from other solvents. Methanol and ethanol, for example, gave spectra corresponding to protonation and proton-dimer ion formation. An advantage in using benzene (demonstrated in the course of the work) is that the C6&+ ion can participate in both proton transfer and charge transfer reactions. The ionization potential of benzene is reported to be between 9.5-10.5 eV (7), so that compounds with a higher ionization potential introduced in benzene solution would presumably not be detected. (4) P. Kebarle and A. M . Hogg, J . Chem. Phys., 42, 668 (1965), (5) J. M. Shahin, J . Chem. Phys., 45, 2600 (1966). (6) S.Wexler and R. P. Clow, J. Amer. Chem. SOC.,90,3940 (1968). (7) J. L. Franklin et a / . , "Ionization Potentials, Appearance Potentials and Heats of Formation of Gaseous Positive Ions," NSRDS-NB526, National Bureau of Standards, Washington, D.C.. 1969.
938
A N A L Y T I C A L C H E M I S T R Y , V O L . 45, NO. 6, V A Y 1973
Figure 2. Mass shift from MH' to M D + for 2,6-dimethyl-y-pyrone, using narrow scan range with unit resolution
The concentration of primary ions (from nitrogen] and secondary ions (from water or benzene) is dependent upon the reaction conditions, the concentration of neutral molecules, the proton affinities, and the ionization potentials of each molecular species. The water spectrum disappears, for example, when benzene is swept through the source. Chloroform can also be used as a solvent; in this instance (positive ion detection), the chief ions were those from ethanol present in the chloroform. Ionization of Sample Molecules by Proton or Charge Transfer. Small amounts of organic compounds introduced into the reaction chamber along with a large amount of benzene would be expected to undergo reaction 9 if the ionization potential of compound A is lower than that of benzene, or reaction 10 if the gas phase basicity of compound B is greater than that of the phenyl radical. C6H6' -k A-A+
+
+ C&6 + CsH5
(9)
C6H,+ B -BH+ (10) For example, the calculated heat of reaction for aniline, pyridine, or dimethylamine in reaction 10 is 12, 21 and 21 kcal/mole exothermic, respectively (8). It seems likely, therefore, that most amines would undergo reaction 10 (9). A few nonnitrogenous compounds, including some unsaturated ketones, are known to exhibit high gas phase basicity, and these would also be expected to ionize through proton transfer ( I O ) . This reaction was investigated for 2,6-dimethyl-y-pyrone; the product ion was formed through reaction 10. This was clearly demonstrated through use of deuterated benzene; Figure 2 shows the amu shift due to deuterium transfer. A similar effect was observed when water and deuterated water were introduced into the reaction chamber along with the pyrone; in this case, protonation is also the only reaction that would be expected to occur. Some scrambling of the label occurred, since MH+ was always present along with MD+ in the labeling experiments; this may have been due to background water. Charge transfer (reaction 9) would be expected to occur for nonbasic compounds with an ionization potential lower (8) The heat of reaction 10 was calculated by using the heats of formation of C6H6+, CsH5 and the neutral compounds from reference 7, and the proton affinities from reference 9. (9) J. P. Briggs, R . Yamdagni and P. Kebarle, J. Amer. Chem. SOC.. 9 4 , 5128 (1972). (10) I. Dzidic and J. A . McCloskey, Org. Mass Speclrom., 6, 939 (1972).
MASS SPECTRUM OF M I X T U R E
304 MH'
Ijj
Figure 3.
MW COCAINE 15ng 303 METHADONE 12ng 309 15 SCANS 0 - 7 5 0 A M U
SAMPLE M I X T U R E
Positive ion mass spectrum of a chloroform solution of cocaine and methadone fer reactions. A relatively high concentration of reactant ions is maintained in the chamber, and both drugs are presumably converted to ions a t approximately the same rate. A solution containing 20 ng of nicotine, 2 ng of cocaine, 30 ng of methadone, and 500 ng of caffeine in 1 pl of chloroform was injected. The purpose of this experiment was to determine if a relatively large concentration of a commonly occurring urinary constituent would interfere with ion formation for the relatively low concentrations of the other bases. All components gave M H + ions; no interference was detected. Reactant ions were present throughout the process. Negative Ions. The reaction conditions used in this work are not greatly different from those present in a GC electron capture detector. Negative ions are formed by attachment of thermal electrons to species possessing sufficient electron affinity either by resonance capture (reaction 11) or by dissociative capture (reaction 12). Negative ions can also be formed through an ion-molecule reaction (reaction 13).
than that of benzene. The charge transfer process was therefore investigated for testosterone and for the trimethylsilyl (TMS) derivative of Sa-androstane-3a,l7P-diol. A single ionic product was observed for testosterone, while the TMS derivative showed three product ions. The nature of the testosterone ion was established as M + through experiments similar to those carried out with 2,6-dimethvl-y-pyrone. No transfer of deuterium was observed when deuterated benzene was used as the solvent. A similar study of the ions from the T M S derivative indicated that M + and not M H + was formed, but further studies of steroids are needed to establish the patterns of ionization which occur under these conditions. It is probable that most T M S derivatives will yield M + ions, but methoximes (MO derivatives) may yield M H + ions (by analogy with results of chemical ionization studies at 1 Torr). Unsaturated ketones may show basicity in the gas phase, but evidently the energetically favored route for the ionization of testosterone is charge transfer. These results indicate that a complex sequence of ionmolecule reactions may be used to generate ions from small amounts of sample in a reaction chamber of the type described here. The primary ions, as far as is known, are those involving nitrogen. Charge transfer reactions with solvent molecules, introduced in relatively large amount, provide a second group of reactant ions which then react with the sample. The product ions (from the sample) are produced by either charge transfer or proton transfer when benzene is used as the solvent. As is true for chemical ionization reactions carried out a t 1-2 Torr in conventional mass spectrometers, the spectra of most samples contain one or only a few product ions (11). Basic Drugs. The ionization of a basic drug, introduced into the ionization chamber with secondary reactant ions arising from water, methanol, ethanol, or benzene, should occur by proton transfer. Several types of experiments were carried out to define suitable reaction conditions. In the first, a mixture of two basic drugs, cocaine and methadone, was injected in chloroform solution. As expected, the only product ions observed corresponded to M H + . The positive ion spectra given by chloroform and by the chloroform solution of the two drugs is shown in Figure 3. The ions due to the solvent are derived from ethanol present in about 0.5% concentration; chloroform apparently does not interfere with positive ion formation or with proton trans-
Reaction 13 represents the gas phase conversion of a Bronsted acid BIH into its conjugate base BI-. This reaction should occur if the difference in bond dissociation energies, D(B2-H) - D(B1-H) > ' electron affinity E.A.(B2) - E.A.(Bi). The lower chart of Figure 4 shows the negative ion spectrum of chl'oroform. The ions present include C1-, Cl-(CHC13), and Cl-(CHC13)2. The C1- ions are produced by dissociative electron capture and the cluster ions are subsequently formed through association of Cl- ion with chloroform (12). The relative concentration of cluster ions depends on the temperature of the chamber and the concentration of chloroform. The introduction of a trace amount of sample dissolved in chloroform will still result in negative ion formation if the sample undergoes reaction 13. Figure 4 (upper chart) shows the ions resulting from injection of a chloroform solution containing four barbitu-
(11) F. H. Field,AccountsChem. Res., 1,42 (1968).
(12) R. Yamdagni and P. Kebarle, J. Amer. Chem. SOC.,93,7139 (1971). A N A L Y T I C A L C H E M I S T R Y , V O L . 45, N O . 6, M A Y 1973
939
23 I
NEGATIVE ION MASS SPECTRUM OF MIXTURE rn/e
ng
211 8 225 8 231 IO 237 IO 20 SCANS 0-750 AMU
Butabarbifol Pentobarbital Phenobarbital Secobarbital
I
I
SAMPLE MIXTURE IN CHLOROFORM I
Figure 4.
Negative ion mass spectrum of a chloroform solution of four barbiturates
HYDROXY PHENOBARBITAL
NEGATIVE ION MASS SPECTRA OF UNDERIVATIZED RAT URINE EXTRACT
DIHYDROXYPHENOBARBITAL PH EN0 BARB ITA L RAT
Figure 5.
Negative ion mass spectrum of a biologic sample (in methanol) containing phenobarbital and two metabolites
rates. Each barbiturate, BzH, was converted to B1- by reaction 13. This mechanism for the production of barbiturate negative ions in the presence of C1- was suggested by Kebarle (13). Cluster ions derived from barbiturates were not observed. Negative ions were not observed when acidic hydrogens on the barbiturate ring were replaced by methyl groups, as would be expected. Biologic Samples. The experimental work with reference compounds indicated that two circumstances were particularly favorable for the detection of specific types of compounds without interference from solvents or from many naturally occurring substances. In the positive ion mode of operation, basic drugs should be detectable through MHf ion formation. Interference through generation of additional ions would be expected if charge transfer reactions occurred a t the same time for other substances in the reaction chamber; it is possible, however, that proton transfer would still be the principal route of reaction with an appropriate reactant ion. In the negative ion mode, less complex circumstances are present. A very low background is usually observed with samples of bio(13) P. Kebarle, personal communication, 1972.
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A N A L Y T I C A L C H E M I S T R Y , V O L . 45, NO. 6, M A Y 1973
logic origin. Compounds with high electron affinities (pesticides, for example) are detectable in an electron capture detector in low concentration with little interference from other materials. Since barbiturates can also be detected by negative ion formation, as indicated in the work with reference compounds, an analysis was carried out for a sample of urine obtained after administration of phenobarbital to a rat. During the first 24 hours, the metabolites are excreted in large quantity. Since the drug is metabolized relatively slowly, it is possible by GC-MS-COM analyses to find unchanged drug and drug metabolites several days after administration of the drug. In this work, a 24-48-hour urine specimen was used. Figure 5 shows the background negative ion spectrum for a urinary fraction in methanol, from a control experiment without drug, and the negative ion spectrum for a urinary fraction in methanol, after drug administration. Derivatives were not prepared. The drug spectrum (from 1 11 of methanol solution) shows (M - 1)- ions resulting from ion molecule reactions, corresponding to unchanged phenobarbital, an ion corresponding to a monohydroxyphenobarbital as the major metabolite (known to be p hydroxyphenobarbital), a dihydroxyphenobarbital [also
2,6- D I M E T H Y L - T - P Y R O N E
1000
t
S I M 125(MH+) PICOGRAM S A M P L E S
$i zl
50
Figure 6. Single ion monitoring response vs. time for picegram
samples of 2,6-dimethyl-y-pyrone known to be present in urine and believed to be 3,4dihydroxyphenobarbital (2)], and very small peaks of unidentified origin. The estimated concentration of barbiturates, from comparison with reference solutions, is about 10-20 pg/ml of methanol solution, or about 3-5 kg/ml for the original urine sample. Concentrations below 1ng/ml of urine could be detected by this method. Detection of Ions. Total Ion Current. The e3Ni foil source is electrically isolated; this allowed measurement of the total ion current within the chamber and the aperampere. ture plate. The standing current was 6.8 x The current entering the high vacuum region, measured on the first lens element under normal operating condiampere. Thus, only about one ion tions, was 5.8 x in lo4 produced in the reaction chamber enters the high vacuum region. The total ion current reaching the multiplier was measured by removing the direct current ramp voltage from the quadrupole rods. The observed pulse signal rate was about lo6 cps; the expected rate based on the ion lens current was 3.5 x IO6 cps, indicating about 30% efficiency of the ion lens and rod structure. The total ion current, measured a t the detector, was not affected when a draw out potential was applied within the reaction chamber. A field-free condition was used in all experiments reported here. An estimate of the ion current to be expected a t the ion lens can be calculated based on assumptions with respect to self absorption and ion recombination rate. The 63Ni source (12.5 mCi) emits 4.6 x lo8 electrons per second, each with a mean energy of 60 KeV. About 3 .x IO3 ion pairs should be produced for each electron. Assuming 2 T geometry and approximately 50% self absorption, the rate of formation of primary ions should be 3.4 x ion pairs cc-l sec-l. If the recombination rate is 10-6 cc sec-l, the steady state ion pair concentration would be about 6 x 10s cc-l. The conductance of the aperture is about 5 x 10-2 cc sec-1 or 3 x 107 ion sec-1, corresponding to a current of about 5 x ampere. The measured current, 5.8 x ampere, is less than calculated, but this may be due to a higher effective recombination rate than estimated, or to greater self absorption in the source. The ion current due to sample ions can also be calculated. The fraction of the total current converted to sample ion current is dependent upon the ratio of the average ion lifetime in the source to the half-life of the ion molecule reaction which converts neutral sample molecules to sample ions. The average ion lifetime in the source is inversely proportional to the product of the ion density and the ion-ion and/or ion-electron recombination rate constant. The calculated value of the ion lifetime for the source is 2 x second. For sample materials of interest, the reaction rate constant for conversion of neutral sample molecules to sample ions is typically between 10-9 and cc sec-l. One picogram of a sample of molecular weight
1
10
100 PICOGRAMS
1000
IO, 000
Figure 7. Integrated single ion response vs. sample size, showing linearity of response for 2,6-dimethyl-y-pyrone
124 in the source will give a sample molecule concentration of 5 x IO9 molecules cc-l. Under these conditions, the half-life of the reaction converting neutral molecules to sample ions is not more than two seconds. Thus, the ratio of the ion lifetime (2 x sec.) to the conversion reaction half-life (2 seconds) is 1 x Since the total ion signal is lo6 ion sec-1 this conversion ratio of 1 X will give a sample ion signal of 1 x lo3 cps. This is 100 times the ideal base-line noise level of 10 cps or 10 times the typical experimental noise level of 100 cps. These calculations indicate that a sample of one picogram should be detectable. In the following section, these calculations will be shown to be in good agreement with experimentally observed values. Limits of Sample Size. The mass analyzer-detector assembly was originally designed (Franklin GNO Corp.) to be used with a Plasma Chromatograph for the purpose of identifying ions present in the drift tube. A small computer (PDP8/E) with a laboratory interface was added so that computer-controlled ion monitoring could be carried out (for quantitative work) and signal averaging could be employed in scans. The use of pulse counting technology leads to greatly improved sensitivity in detection, and resolution was enhanced by signal averaging. 2,6-Dimethyl-y-pyrone does not show large adsorption effects, and was chosen for use in experimental tests of system behavior with small samples. Solutions in benzene were prepared so that sample sizes ranging from 5 picograms to 1 nanogram in 1 p1 of solvent could be injected. The chart record of ion monitoring for MH+, using the smallest samples, is shown in Figure 6. About 10-20 seconds were required to clear the reaction chamber for small amounts of the pyrone. The peak shape is due to the nature of the inlet-reaction chamber assembly; a chromatographic process was not involved. The “zero” value, obtained by solvent injection alone, is believed to represent an authentic response due to about 2 picograms of the pyrone adsorbed on walls of the assembly and removed by solvent flushing. The smallest sample employed was 5 picograms; this gave an easily detectable peak. The linearity of the response/mass relationship was examined. Figure 7 shows the integrated response (estimated by weighing peak shapes cut from a Xerox copy of the chart record) observed for sample sizes to 1 nanogram. The linearity is evident, and the mass intercept calculated from the regression equation (presumably indicating irreversible adsorption) was less than 0.5 picogram. These results indicate that the system, when used in an ion monitoring mode, can detect ions from as little as ANALYTICAL CHEMISTRY, VOL. 45, NO.
6 , M A Y 1973
941
78 C6H6'
I
I
156 C12H12*
1
'25.
MASS SPECTRUM OF 2 , 6 - D I M E T H Y L - F P Y R O N E IO SCANS 0-750AMU
A
BENZENE SOLVENT
m/e
Figure 8. Scanned spectrum over 0-750 amu range for 25 and 160 pg of 2,6-dimethyl-y-pyrone
c6r+ 78
MASS SPECTRUM OF T E S T O S T E R O N E 2 0 SCANS 0-750 AMU
BENZENE SOLVENT
--,
m /e Figure 9. Scanned spectrum over 0-750 amu range for 1 ng of testosterone
5-10 picograms of sample. The implications of this result are significant for applications where the biologic sample size is limited (blood samples from the newborn, for example) or where the concentration of the substances to be analyzed is low (catecholamines in tissue, for example). The limiting factor in reducing sample size may prove to be adsorption; if this is true, it will be necessary to find ways of decreasing the extent to which adsorption effects occur on columns and in ion-molecule reaction chambers. Another, and perhaps more easily attainable solution, lies in the use of I3C labeled compounds as both internal reference compounds and carriers, with isotope ratio measurements. An indication of the sample size required for scanning over 0-750 amu was obtained in another group of experiments. Figure 8 shows three scans observed for sample sizes of 25 and 160 picograms of 2,6-dimethyl-y-pyrone, and for a solvent (benzene) injection alone. Judging from the experimental observations made during the ion monitoring experiments, the solvent injection alone might have released 1-2 picograms of material from the inlet and reaction chamber walls, but no ion peak for the pyrone was observed. The peak for MH+ is clearly evident at 25 picograms and is very strong a t 160 picograms. The background was low; high mass ions were not detectable under the conditions of operation, but very little interference 942
A N A L Y T I C A L CHEMISTRY, V O L . 4 5 , NO. 6 , M A Y 1973
from solvent contamination or solvent desorption of previous samples was observed. Coupled with the results found earlier in ion-molecule reaction studies for mixtures, these data suggest that analyses of samples for one or more components could be carried out with component sample sizes of 25-50 picograms, provided that selective ionization can be accomplished in order to decrease the background. With the present prototype apparatus, the limit of detection would be higher for substances of greater mass; this effect is related to the nature of the mass analyzer. A representative example of a steroid mass spectrum is shown in Figure 9; this is the charge transfer spectrum for testosterone in benzene.. The steroid was not derivatized; a 1-ng sample was injected in 1 pl of solvent. In other experiments, it was determined that the ionization step leads to M + rather than to MHf, and that the limit of detection was about 50 picograms. Adsorption of the steroid on inlet and reaction chamber walls was probably responsible for the detection limit, but 1 nanogram was a sufficiently large sample to provide a scanned mass spectrum. Other examples of scanned mass spectra are in Figures 3-5. Effects of Large Samples. Solutions containing 1 microgram of component under study may represent unduly large samples. Amounts of 1-20 nanograms are conve-
nient. Very little interference is seen when both large (,WO-ng) and small (2-ng) amounts of samples are ionized a t the same time, but high concentrations of sample ions may lead to cluster ions if these are stable under conditions existing in the reaction chamber. For example, Figure 8 shows the presence of ions, in low concentration, which correspond to associative structures. This effect was not found in other examples.
CONCLUSION Ionization reactions, initiated by electrons from a 63Ni source, can be carried out in a flowing gas stream at atmospheric pressure for many organic compounds. It is possible to introduce solvent molecules into the stream which react through proton transfer or charge transfer processes with compounds of biologic interest to produce sample ions. Alternatively, negative ions can be generated from compounds with high electron affinity. These ions can be sampled continuously with a mass analyzer-detector-computer assembly. The lower limit of
sample size, established for single ion monitoring and for scanning, is much lower than that currently attained in conventional instruments operated with flowing gas streams for sample introduction. Three possible ways of using these results are in the analysis of samples which do not require chromatographic separation, in the development of LC-MS-COM systems (some liquid partition chromatography effluents might be directly vaporized through the reaction chamber) and in the development of GC-MS-COM systems, with thermostable glass capillary columns to provide high resolution (100,000 or more theoretical plates). Received for review November 24, 1972. Accepted January 22, 1973. This work was aided by Grants GM-13901 and GM-16216 of the National Institute of General Medical Sciences, Grant HE-05435 of the National Heart Institute, Grant Q-125 of the Robert A. Welch Foundation, and Contract 69-2161 of the National Institute of General Medical Sciences.
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