Ana/. Chem. 1983, 55, 549-553
ACKNOWLEDGMENT
(9) Herbst, E.; Patterson, T. A,; Lineberger, W. C. J . Chem. Phys. 16174, 61, 1300-1304. (10) Smith, G. P.; Lee, L. C.; Cosby, P. C. J . Chem. Phys. 1979, 7 1 , 4464-4470. (11) Zimmerman, A. H.; Brauman, J. I. J . Am. Chem. Soc. 1977, 99, 3565-3566. (12) Hotop, H.; Lineberger, W. C. J . Chem. Phys. 1973, 5 8 , 2379-2387. (13) Simon, R. K. Ph. C). Dissertation, Kansas State University, Manhattan, KS, 1980. (14) Barratt, R. S. Analyst (London) 1981, 706, 817-849. (15) McGee, P. R.; Cleveland, F. F.; Meister, A. G.; Decker, C. E.; Miller, S. I. J . Chem. Phys. 1953, 27,242-246. (16) Kreurer, L. B. Anal. Chem. 1978, 5 0 , 597A-606A. (17) Compton, R. N.; I3einhardt. P. W.; Cooper, C. D. J . Chem. Phys. 1978, 68, 4360-4367. (16) Jovicevlc, S.; Skenderi, S . ; Konjevic, N. Spectrosc. Left. 1981, 74, 415-422. (19) Ball, J. J.; Keller, F?. A. APCA J . 1975, 25, 631-633. (20) Andersen, E.; Simons, J. J . Chem. Phys. 1977, 6 6 , 2427-2430. (21) Balm, M. A.; HIII, ti. H. Anal. Chem. 1982, 5 4 , 38-43. (22) . . Bomse. D. S.; Beauchamp, J. L. J . A m . Chem. SOC. 1980, 702, 3967-3969. (23) Chantry, P. J. J . Chem. Phys. 1969, 57,3369-3379. (24) Phillips, M. P.; Sievers. R. E.; Goldan, P. D.;Kuster, W. C.; Fehsenfeld, F. C. Anal. Chem. 1979, 5 1 , 1819-1825.
This project was initiated by a suggestion from A. Tmkevich of the TJniversity of Chicago. Helpful discussions with D. Bomse and L. Babcoclr of Los Alamos are acknowledged. Furthermore, the following are acknowledged for the kind loan of equipment: M. Buchwald for the C 0 2 laser head, D. Casperson for the COz laser power supply, B. Stine for the gas chromatograph, all of Los Alamos, and J. Eng of Princeton University for the electron capture cell. Registry No. CF,Br, 75-63-8; NOz, 10102-44-0; 2-propanol, 67-63-0.
LITERATURE CITED (1) Hirschfeld, T. Anal. Chem. 1976, 4 8 , 16A-31A. (2) Knorr, F. J.; Thorsheimi, H. R.; Harrls, J. M. Anal. Chem. 1981, 53, 821-825. (3) Spence, D.; Schulz, G. J. J . Chem. Phys. 1973, 5 8 , 1800-1803. (4) Wentworth, W. E.; Chen, E. J . Gas Chromafogr. 1967, 5 , 170-1’79. (5) Pettitt, B. C.; Simmonds, P. G.; Zlatkis, A. J . Chromafogr. Sci. 1969, 7, 645-650. (6) O’Malley, T . F. Phys. fiev. 1966, 150, 14-29. (7) Henderson, W. R.; Fite, W. L.; Brackmann, R. T. Phys. Rev. 1969, 183, 157-166. (8) Chen, C. L.; Chantry, P. J. J . Chem. Phys. 1979, 7 1 , 3697-3907.
549
RECEIVED for review May 20, 1982. Accepted November 9, 1982. This material is based upon work supported by the U.S. Department of Energy.
Determination of Drugs in Blood Serum by IMass SpectrometryIMass Spectrometry Harry 0. Brotherton’ and Richard A. Yost” Department of Chemistry, University of Florida, Gainesville, Florida 326 7 7
Screenlng procedures for drugs and metabolites often involve lengthy sample preparatlon and derlvatlratlon procedures followed by chromatographic analysls. Conflrmatlon is usually performed by GCIMS. Here we describe a rapld, sensitive technlque based on tandem mass spectrometry (MSIMS) for both screenlng and conflrmatlon. This MS/MS technique replaces the chromatographlc separation wlth a mass spectrometric separation and greatly reduces the amount of sample preparation required. It allows the simultaneous screening for as many as 50 drugs; and metabolltes in less than 5 mln. The technlque Involves an Initial screening by monitoring 88lected parent ioddaughter Ion pairs for each targeted cornpound, or selected neutral losses characterlstlc of targeted classes of compounds,, Conflrmation Involves obtalnlng daughter spectra of the parent ions for all “posltlves” and matching them wlth library daughter spectra of the pure compounds. Detectlon limits for most drugs, wlth 1 pL of blood serum placed on1 the solids probe, are In the low parts-per-milllon (ng/pL) range. A simple extraction of the serum reduces these detectlon limits to the low parts-perbillion (pg/pL) range.
The detection, identification, and quantitation of drugs in blood serum are challenging analytical problems. Very small amounts of narcotics, stimulants, and other drugs can be used ‘Present address: Department of Chemistry, Northeast Louisiana University, Monroe, LA 7 1209.
to alter the performance of athletes (both human and anim,al) while larger doses of some drugs can cause convulsions, coma, and death. The detection of drugs, as well as studies of metabolic disorders and pathways, requires the ability to identify and quantitate trace amounts of drugs and metabolites in blood serum. This type of analysis has traditionally been performed by using a chromatographic screening step, often thin-layer chromatography (TI&), followed by a more selective confirmation step, usually gas chromatography/mass spectrometry (GC/MS) (1). While improved screening techniques, particularly gas chromatography and high-performance liquid chromatography, are being developed, the most common chromatographic screening technique today is still TLC. Chromatographic screening often necessitates extensive cleanup procedures and derivitization, greatly increasing the time required for the analysis. In cases where rapid analyses are needed, such as the identification of an unknown drug in the blood serum of an overdose victim or preevent screening of athletes for illegal drugs, the current methods are far from satisfactory. Tandem mass spectrometry (MS/MS) is a relatively new analytical technique which has proven to be very useful for the trace analysis of selected compounds in complex mixtures (2-4). Pioneering research on reversed-geometry double-focusing mass spectrometers in the laboratories of Cooks (5,6) and McLafferty (7) demonstrated the potential for mixture analysis by using mass separation in lieu of chromatographic separation, followed by a second step of mass spectral analysis. The subsequent development of the triple quadrupole mass spectrometer by Yost and Enke (8)has made MS/MS a viable
0003-2700/83/0355-0549$01.50/00 1983 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 3, MARCH 1983
Table I. Drugs Screened in This Study, and Their Therapeutic Categories and Suppliers drug apomorphine aspirin caffeine camphor chloropromazine cocaine diethylcarbamazine ephedrine fentanyl furosemide glycerol guaiacolate meperidine methadone methocarbamol methylphenidate morphine nikethamide p-aminobenzoic acid pemoline phenacetin phenobarbital phenylbutazone procaine propylparaben reserpine theobromine theophylline
therapeutic category
supplier
emetic, expectorant analgesic, antipyretic, antirheumatic, anticoagulant cardiac and respiratory stimulant, diuretic internally, stimulant and carmenative; externally, antipruretic, counterirritant, and antiseptic antiematic, tranquilizer, sedative, peripheral vasodilator local anesthetic, CNS stimulant anthelmintic, antimicrobilarial sympathomimetic, mydriatic, CNS stimulant analgesic, tranquilizer diuretic, antihypertensive expectorant narcotic, sedative, analgesic, anesthetic narcotic, analgesic skeletal muscle relaxant central stimulant narcotic analgesic, sedative, preanesthetic, gastric sedative respiratory stimulant treatment for eczema nasi in dogs central stimulant analgesic, antipyretic anticonvulsant, sedative analgesic, antiinflammatory local anesthetic pharmaceutical aid (antifungal preservative) hypotensive, tranquilizer diuretic, myocardial stimulant, vasodilator diuretic, cardiac stimulant, vasodilator
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Merck Sharp & Dohme, West Point, PA. Aldrich, Metuchen, NJ. Chem. Service, West Chester, PA. R. J. Perchalski, Gainesville V.A. Hospital, FL. e Applied Science, State College, PA,, f Sigma, St. Louis, MO. Janesen Pharmaceutical, New Brunswick, NJ. USV Pharmaceutical, Tuckahoe, NY. I Florida State Division of Parimutual Wagering, Miami, FL. I CIBA Pharmaceutical, Summit, NJ. Jean DeGraeve, Universite De Liege, Belgium. Matheson, Coleman and Bell, East Rutherford, NJ. rn Warner-Lambert, Ann Arbor, MI. a
’
analytical technique for routine analysis. The triple quadrupole mass spectrometer consists of, in series, a dual chemical ionization/electron impact ionization source, a quadrupole mass filter, a n RF-only quadrupole, a second mass filter, and an electron multiplier. Three operating modes of the tandem mass spectrometer (8)-neutral loss scan, daughter scan, and selected reaction monitoring (SRM)-are used in the current study. Collection of a neutral loss spectrum entails simultaneous scanning of quadrupole one and quadrupole three with a fixed mass difference which is characteristic of a given class of compounds or functional group. A daughter spectrum is collected by passing the parent ion of the compound of interest through the first quadrupole, fragmenting it by collisionally activated dissociation (CAD) with a gas in the second (RF-only) quadrupole, and then scanning the third quadrupole to determine the fragmentation of the parent. During selected reaction monitoring, only the most intense daughter ion from each parent ion is monitored with the third quadrupole. Here we describe a new MS/MS procedure for the rapid screening and confirmation of trace amounts of drugs and metabolites in blood serum. The procedure involves the use of SRM for preliminary screening t o indicate possible “positive” drugs and/or neutral loss screening t o indicate the presence of any member of a given class of drugs, followed by the acquisition of complete daughter spectra for confirmation. The screening is carried out either with whole blood serum or with acid-neutral and base extracts of the serum. Data are collected in intervals of 0.1 s or less over the single mass selected, enabling sequential screening for 25 t o 50 compounds or drug classes during the approximately 100 s that the solids probe is heated ballistically from 30 t o 325 “C. Compounds for which the selected reaction yields signal above background during the screening procedure are then confirmed
’
by collecting complete daughter spectra of the indicated compounds’ parent ions from a second sample, and matching them with a library of the daughter spectra of pure compounds. This procedure permits the screening of 12-14 serum samples/ h. EXPERIMENTAL SECTION Instrumentation. The instrument used in this study is a Finnigan MAT triple stage quadrupole mass spectrometer/data system (9). The mass spectrometer was operated in the positive chemical ionization mode using 99.9% pure methane (Matheson) at a pressure of 0.20 torr as reagent gas. All samples were placed in glass sample vials and inserted into the ion source on the tip of a heated solids probe. The Model 4500 ion source includes interchangeable ion volumes which can be rapidly (1 min) exchanged. Zero grade nitrogen (Matheson) was used as the collision gas a t a pressure of 1.8mtorr in the center quadrupole, with an ion energy of 18 eV. Chemical. Drugs used in this study are listed in Table I, together with their pharmacological uses and their sources. Equine blood serum was obtained from the University of Florida School of Veterinary Medicine. Procedure. Blood serum samples were analyzed either directly, after deproteination, or after extraction. Samples were deproteinated by adding an equal volume of acetone to the sample while vortexing. The mixture was then centrifuged and the aqueous layer decanted and analyzed. Acid-neutrals were extracted with 2 mL of ether from 1 mL of serum acidified with 0.1 M HC1 to a pH of 3. The ether was then evaporated to dryness and the residue taken up in 1 mL of chloroform. The pH of the serum was then adjusted to 10 with 0.1 M NaOH and the bases were extracted with 2 mL of chloroform. The volume was then reduced to 1 mL. RESULTS AND DISCUSSION The rapid screening of complex mixtures such as blood serum for several trace substances by tandem mass spec-
ANALYTICAL CHEMIISTRY, VOL. 55, NO. 3, MARCH 1983
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Figure I. Daughter spectra of the MH' ions of (a) procaine and (b) phenylbutazone.
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Figure 2. Screening for three drugs in 1 pL of chloroform, each at 10 ppm, by selected reaction monitoring. Also monitored was the MH' ion for the three of 181".
trometry is best accomplished by using chemical ionization in combination with selected reaction monitoring. The use of chemical ionization, a more efficient and more gentle ionization technique than electron impact, maximizes the conversion of the sample to parent ions and minimizes fragmentation. Although negative chemical ionization is even more sensitive than positive chemical ionization for many classes of compounds, positive chemical ionization is more sensitive for most of the drugs that have been studied. Furthermore, the structural stability of the negative ions decreases the efficiency of collisionally activated dissociation of the parent ions in the center quadrupole, decreasing the sensitivity of the screening technique. The daughter ions which are monitored during SRM are selected by first acquiring a complete daughter spectrum of the pure drug's molecular ion. The most intense daughter ion is monitored except in cases where two drugs have parent ions of the same mass which fragment to give the same daughter ion. The most intense unique daughter ion from each parent ion is then monitored. Figure 1 shows the daughter spectra of the molecular ions of two drugs, procaine (237") and phenylbutazone (309+),from which the loo+ and 188" ions are selected as4the daughter ions to be monitored with the third quadrupole. In order to demonstrate the potential of SRM as a rapid, selective screening technique, we examined a mixture of two isomers arid a third compound with the same nominal molecular weight of 180. The daughter ions 124', 138+,and 139' were monitored for the drugs theophylline, theobromine, and
50 TIME ( S E C )
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Figure 4. Mass chromatograms of 25 ppm procaine in (a) chloroform, (b) fresh blood serum, and (c) in serum which was frozen for 7 days after procaine addition.
propylparaben, respectively, while the 181' parent ions were passed through the first quadrupole. The resulting "mms chromatograms" (plots of ion current vs. time as the probe is heated) are shown in Figure 2. Each compound show!j a distinct peak which is slightly offset from the others, indicating some separation arising from thermal distillation of the mixture off the solids probe. Note that this separation is not required, however, siince the selected daughter ions are unique. The bottom trace shows the ion current produced by moiiitoring the 181' parent ion. The ability of tandem mass spectrometry to identify trace amounts of specified compounds in a complex mixture can be demonstrated by adding 30 ppm theobromine to equine blood serum. The thiermal decomposition of the blood serum a t about 300 "C pralduces numerous mass peaks, including 181+,effectively masking the existence of the trace component. If the analysis is carried out with selected reaction monitoring, however, the theobrolmine can be detected as shown in Figure 3. The theobromine fragmentation trace shows a peak shiftlad to higher temperature (70 s) than in the standard, as well as the beginning of a peak near 100 s. The latter peak, which occurs a t all monitored masses, is an increase in background due to the fragmentation of 181' ion from the thermally decomposed blood serum. The peak at 70 s shows the presence of theobromine: a parent mass of 181+which fragments to give a daughter ion of mass 138'. Possible interference from the thermal decomposition of blood serum components can be eliminated by use of the extraction procedure. The shift of the mass 138' peak from 25 s in chloroform to 70 s in blood serum is due to the binding of the theobromine to the proteins in the blood serum. Similar results are observed for 25 ppm procaine, as shown in Figure 4. In chlo-
ANALYTICAL CHEMISTRY, VOL. 55, NO. 3, MARCH 1983
552
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of a mixture containing five drugs and two internal standards (tribenzylamine for base extracts and 2-amino-5-chiorobenzophenone for acid extracts).
roform, procaine desorbs a t 25 s (Figure 4a); in fresh serum, it desorbs at 70 s, indicating that essentially all the procaine is protein bound (Figure 4b). If the serum is frozen for 7 days and the analysis repeated, however, much of the drug is now released from the protein. At high concentrations (100 ppm), procaine exists in both forms even in fresh serum. This indicates the potential for determining the ratio of free/bound drugs in serum. A series of procaine solutions of varying concentrations in CHC1, were prepared and a calibration plot (screening for the 237' 100' reaction) was constructed to evaluate quantitation with the tandem mass spectrometer system. Three determinations were made at each concentration. The plot is linear over 4 orders of magnitude, has a slope of 1.01, and gives a detection limit of 30 ppb (30 pg) for procaine. Two compounds have been selected as internal standards, to be added prior to the appropriate extraction step: 2-amino-5chlorobenzophenone for acid-neutral extracts, and tribenzylamine for base extracts of the serum. The parent ion of each of the internal standards fragments to give one major 154' for 2-amino-5-chlorobenzophenone daughter ion: 232' and 288' 91' for tribenzylamine. A plot of the ratio of the sample and internal standard signals vs. concentration for each of three drugs, theophylline, theobromine, and phenylbutazone, is linear from the low parts-per-million (ng) range to concentrations approaching 200 ppm using a 25 ppm concentration of internal standard. The compounds were mixed into equine serum, the mixture was extracted, and the extract volume brought to the original volume of the serum extracted. The recovery of the compounds from serum is approximately 80%. A mixture of five drugs (theobromine, theophylline, phenylbutazone, propylparaben, and procaine) in serum was extracted and analyzed by using the screening technique. The results of screening the acid-neutral extract are shown in Figure 5 . Each of the four drugs which extracted as acidneutrals, as well as the 2-amino-5-chlorobenzophenoneinternal standard, gives a distinct peak in its "mass chromatogram" indicating that the compound is present. The mass chromatograms of the bases show only low intensity background indicating they are not present in the acid extract. The screening of the base extract indicates the presence of procaine and the tribenzylamine internal standard. The presence of all compounds which are probably "positives" based on the screening procedure may be confirmed by library comparison. Screening for members of a selected class of drugs (those containing a terminal -N(C,H& group) was carried out on -+
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Figure 7. Mass chromatograms from the screening of canine serum extract for drugs: (a) diethylcarbamazine, (b) procaine, and (c) phenylbutazone.
a mixture of seven drugs. Included in the mixture were three members of this class (nikethamide, diethylcarbamazine, and procaine) as well as four other drugs (ephedrine, methylphenidate, meperidine, and methadone). A scan for neutral loss of mass 73 (diethylamine) from the molecular ion was collected as shown in Figure 6. Peaks at 106, 127, and 164 represent the (MH - 73)' peaks for nikethamide, diethylcarbamazine, and procaine, respectively. The drugs' identity may be confirmed by collecting a full daughter spectrum of the MH+ ion indicated by the neutral loss scan, and comparing it to a library of CAD spectra collected by using pure compounds. As an example of the analysis of real (not spiked) serum samples, consider the screening and confirmation of three different drugs in the serum of two greyhounds. The dogs were treated once daily with therapeutic dosages (oral) of diethylcarbamazine for 3 days, and with therapeutic dosages of diethylcarbamazine, procaine, and phenylbutazone on the fourth day. Blood serum samples were collected 2 h after administration of the fourth-day dose. Screening for the drugs was performed both on the whole serum and on extracts prepared as described above. Phenylbutazone, an acidic drug, was present in the serum at the low parts-per-million level and could easily be screened and confirmed in the whole serum. The two basic drugs, diethylcarbamazine and procaine, present at mid-parts-per-billion levels, could not be reliably confirmed in whole serum because of the thermal decomposition background. All three drugs are clearly identified when the serum extract is screened, however, as shown in Figure 7. For confirmation, daughter spectra were collected for each MH' ion in the extract and compared to a library containing daughter spectra of the drugs cited in Table I. The library matches for diethylcarbamazine and phenylbutazone show excellent agreement, as shown in Figure 8. The library match
ANALYTICAL CHEMISTRY, VOL. 55, NO. 3, MARCH 1983
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-
___-
Table 11. Detection Limits for Six Drugs in 1 pL Samples by Selected Reaction Monitoring CHCl, procaine phenylbutazone PABA theophylline theobromine morphine -____
30ppb 800 ppb 1 ppm
2ppm 1 0 ppm 7Dppm
blood serum whole extract 4ppm 2 ppm 30ppm 16ppm 20 ppm 90ppm
100ppb 1.2 pprn 3ppm 8ppm 14 ppm 75ppm
for procaine, also shown in Figure 8, is much less positive, since the drug is present in the serum at approximately 60 ppb, near the detection limit. The detection limits for several drugs obtained with these screening techniques are compared in Table 11. The detection
553
limits for most compounds are 2 to 40 times higher in whlole serum than in chloroform. This is due to the protein binding of the drugs and the background produced by the thermal decomposition of the serum. The addition of a simple acidbase extraction scheme to the screening procedure reduces the detection limits into the range of those obtained for the pure drugs in chloroform. The use of extractions as part of the analytical procedure is particularly important when screening for base extracts due to the low levels at which basic drugs appear in serum. Acidic drugs can usually be screened in the serum itself. A further advantage of the use of extracts for screening is the' increase in number of samples (from approximately 50 to 90) that can be analyzed before replacing the ion volume. If samples are screened daily (approximately 100 samples/day), the instrument must be disassembled once a week to clean the ion source and once a month to clean the first quadrupole. Work is currently under way to evaluate the use of these screening techniques on more actual serum and urine samples. The technique will be evaluated by comparing results from these samples with those obtained with current analytical procedures. The speed, selectivity, and sensitivity of this screening technique by using tandem mass spectrometry promise a much more rapid screening technique than is currently available and shows potential as a useful tool in clinical and forensic chemistry.
ACKNOWLEDGMENT The authors thank Ron Gronwall and Walter Stone of the University of Florida School of Veterinary Medicine for their assistance.
LITERATURE CITED Tobin, T. J. Equine Med. Surg. 1978, 2 , 518-524. McLafferty, F. W. Science 1981, 274, 280-286. Cooks, R. G.; Gllsh, G. L. Chem. Eng. News 1981, Nov 30, 40-4.2. McLafferty, F. W. Blomed. Mass Spectrom. 1981, 8 , 446-448. Kondrat, R. W.; Cooks, R. G. Anal. Chem. 1978, 50, 81A-92A. Gllsh, G. L.; Shaddock, V. M.; Harmon, K.; Cooks, R. G. Anal. Chern. 1980, 52, 165-167. (7) McLafferty, F. W.; Bockhoff, F. M. Anal. Chern. 1978, 50, 69-76. (8) Yost, R. A.; Enke, C. G. Anal. Chem. 1979, 57, 1251A-1264A. (9) Slayback, J. R. 6.; Story, M. S. Ind. Res. Dev. 1981, Feb, 128-133.
RECEIVED for review August 16, 1982. Accepted December 15, 1982. This work was supported in part by Grant CHE8106533 from the National Science Foundation.
