ANALYrICAL APPROACH
Rkhard H. Eckerlin, Joseph G. Ebei, Jack D. Henion
+.,
Equine Drug Testing and Toxicology Diagnostic Laboratory New York State College 01 Veterinary Medicine Cornell Unlversity 925 Warren Dr. Ithaca. NY 14850
Thomas R. Covey
Sciex. Inc. 55 Glencameron Rd., #202 Thornhill, Ontario. Canada L3T 1P2
Recently, accidental and malicious contamination of pharmaceuticals has been reported in the United States (I). Fortunately, through observations by alert medical professionals, the signs of poisoning have often led to a timely diagnosis and catastrophic consequences have been avoided. Unfortunately, there is much less control and monitoring of similar problems in veterinary medicine. Therefore adverse drug reactions in animals caused by contaminated pharmaceuticals and feeds may occur. In many instances these problems are not recognized. A general lack of control over an animal's environment clouds the clinical picture and does not provide clues to the possible source of the problem. We recently encountered a situation in which a grossly contaminated vial of injectable dexamethasone caused the death of four animals. The suspect dexamethasone was rigorously analyzed using standard chemical techniques, to no avail. Thus new analytical technology had to be applied to identify the toxic component in the tainted injectable dexamethasone formulation.
HUw A referring veterinarian contacted our laboratory staff because of a drugrelated incident. A bottle of generic injectable dexamethasone with an assumed formulation similar to that of a brand-name injectable dexamethasone was used in a clinically acceptable 0003-270018910381-053AI$O 1.5010 @ 1988 American Chemical Society
1
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manner. However, the loss of three animals and the near loss of a fourth animal (all of whom were injected with dexamethasone from the same vial) alerted the veterinarian to a potential toxicology problem. A normal dog acquired from a client for euthanasia was then injected with the suspect dexamethasone and died within 10 min. The suspect vial and a similar vial with the same lot number from the animal clinic were given to ow laboratory for chemical analysis.
Initial methodologies The two vials of suspect injectable dexamethasone were compared with a vial of control dexamethasone using extensive thin-layer chromatography (TLC) and gas chromatography/mass
spectrometry (GC/MS). However, no differences were found among the three vials. The samples were also analyzed for the presence of cyanide and fluoride as well as for gross contamination by inorganic materials via energydispersive X-ray fluorescence spectroscopy; the results showed no differences. Inductively coupled plasma emission spectroscopy also did not reveal any significant differences among the three vials.
Ailemate methodologies Because the TLC and GC/MS results were inconclusive, further biological testing was undertaken to verify whether the toxic component was still present and active in the suspect dexamethasone sample. The goal of these experiments was to reproduce the syndrome with a similar dosage in a clinically healthy dog as well as in another species (mice). If this effect could be demonstrated, we would have a biological means of monitoring the fate of the toxic component following efforts to isolate it by selective extraction from the aqueous medium. If the unknown toxic component could be separated in
ANALYTICAL CHEMISTRY, VOL. 61, NO. 1, JANUARY 1, 1989 * 5 3 A
ANALYTICAL APPROACH a more pure form from the dexamethasone sample, further chemical analyses might lead to successful identification. A female dog was acquired, examined, and found to he in good health. This animal was prepared for electrophysiological monitoring via electroencephalogram and electrocardiogram and given the suspect injectable dosage (5 mL) subcutaneously in the intrascapular region (between the shoulder blades). The subject was observed during the period following the injection. Marked physiological changes were observed that were consistent with an overdose of a general anesthetic; all of these clinical signs occurred within 20 min following injection of the drug. The dog subsequently recovered fully and was placed in a private home as a pet. Experimental mice were then procured for the study of the suspect material. Some of these animals were injected with 0.05 mL of the control injectable formulation and observed for 1h postinjection. No ill effects were noted. Other mice were similarly injected with the suspect formulation of dexamethasone. Each of the mice died within 2 min after receiving the injection. Dilution studies of the suspect material showed that the material diluted with physiological saline still retained ita mouse lethality a t dilutions up to 1:4. The suspect formulation was then heated a t 100 "C for 10 min. This material again proved to be lethal to mice at the same dosage. The control vial of dexamethasone was also heated a t 100 'C and injected subcutaneously (0.05 mL),but was not lethal. Liquid-liquid solvent extraction studies were then conducted using the mouse as an indicator for the presence of the lethal component in the solvent extract. A variety of organic solvents ranging from hexane to ethyl acetate were used in an effort to separate the toxic substance from the aqueous injectable mgdium. In all cases, biological experiments revealed that the toxic substance remained entirely within the aqueous phase, but this uhase became inactivated when its nH was raised to 10.0 by the addition of 6 M NaOH. Reversed-phase high-performance liquid chromatography (HPLC) with diode array detection was employed to detect differences between the suspect and control vial contents. These HPLC experiments indicated no chemical difference between the control and toxic aqueous extracts. Similarly, radioimmunoassay methods used for the detection of potent narcotics gave no positive results. The suspect and control vial contents were tested by an outside 54A
Analytical techniwes used for comparing control samples with tainted dexamethasone samples Test
procedure
Resuits
Acid and base TLC W / M S (neat and with bis(trimethylsilyl)trifiuoroacetamide. BSTFA) HPLC (diode array detector) Cyanide and fluoride analyses X-ray fluorescencespectroscopy inductively coupled plasma emission spectroscopy UV-vis spectroscopy Radioimmunoassay for narcotics
No differences No differences
Endotoxin assay Bioiogical testing (dog)
Negative Toxicity established Toxic principle in aqueous phase
Biological testing (fraction study)
No differences Negative
No differences No differences No differences
Negative
i
Figure 1. LC/MS total mn current profiles scanning from 100 to 1000 G a ~ ~ u w A. 2.1 mm X 7.5 cm amino-bonded column was used wlth a gradlent from 10090 methanol to 100% water over 15 mln at a flow rate of 200 pLlmin. q m emact 01 control &aammsone toTmulatlon BM (b) aqbsous emact Irom ioxlc dexamethasons tonnulalion. 181 A
ANALYTICAL CHEMISTRY. VOL. 61, NO. 1, JANUARY 1, 1989
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laboratory for the presence of endotoxins, and the results were negative. A summary of results from all the routine, conventional analytical experiments is shown in the box on p. 54 A.
LC/MS/MS Because the toxic component of the sample appeared to be a very polar, water-soluble species, we decided to use our ion spray liquid chromatography/mass spectrometry (LC/MS) interface to detect and identify it (2). This interface is well suited for introducing compounds that exist as ions in solution into an atmospheric pressure ization mass spectrometer system. When it is used to couple an HPLC system with a mass spectrometer, one has a means of providing on-line liquid chromatographic separation with m e a spectrometric detection. A Sciex TAGA 6000E tandem triple quadrupole system was used for these experiments. This system was used in an attempt to detect a chemical difference between the toxic dexamethasone sample and that of the control dexamethasone. The LC/MS experiments that follow were all conducted on the aqueous medium (layer) remaining after successive liquid-liquid extraction by solvents ranging from hexane to ethyl acetate. Preliminary experiments were conducted by injecting the aqueous extracts into a flowing stream of 50/50 CH3CN/0.010 M NHlOAc without an in-line HPLC column. Thus no chromatographic separation of the sample extract was provided in the absence of an HPLC column. The mass spectra obtained from these experiments were identical and revealed a predominance of polyethylene glycols (PEGS)with no difference observed between the control and the toxic extract. The presence of PEGS is not surprising because these compounds are known carriers in drug formulations.’Their high concentration in this sample masked the presence of other components, making the detection of the toxic component difficult. Dexamethasone was not detected in these or later LC/MS experiments because it had been removed in the liquid-liquid extraction sequence described above. From these results it was apparent that .HPLC separation would be required to separate the P E G from other possible compounds. The LCiMS total ion current profiles for the reversed-phase gradient HPLC separation from the LC/MS analysis of the control and the toxic dexamethasone sample extracts were also identical. The PEGS eluted in the column b6A
void, and, because of their high concentration, initially suppressed the ion current and then gradually bled from the HPLC system during the entire run. These experiments were performed using a C-18 HPLC column where the gradient went from 100%water to 100% methanol. The flow rate was maintained at 200 pL/min, and the mass spectrometer was operated in the full-scan ion spray L C M S mode scanning from m/z 100 to lo00 Daltons. No obvious difference in the total ion current profiles of the samples was observed even though biological testa on mice clearly indicated that the latter sample was toxic. Careful inspection of the mass spectra across the time scale gave no indication of differences between the two samples. After these discouraging results it was tempting to give up looking for analytical evidence of a toxic component, but the tragic effects of the tainted sample encouraged us to look more carefully for this elusive unknown component. Because the extraction results suggested a highly polar component that might not be retained on a C-18 reversed-phase HPLC column, a more polar amino column was installed in the ion spray LCiMS system. A gradient from 100% methanol to 100%water was used to obtain the total ion current profdes shown in Figure l. For the first time, a chemical difference was observed between the extracts of the
Flgun 2. Backgroundsubtractedmass sp Flgure lb.
control and toxic dexamethasone formulations. The high levels of PEGS known to be present in the samples again eluted unretained early, in the chromatogram and bled slowly during the course of the run.However, Figure l b clearly shows a chromatographic peak eluting at about 12.6 min that did not appear at the same retention time in Figure la. Inspection of the mass spectrum of this chromatographic peak was straightforward; full-scan mass spectral acquisition had taken place during the analysis. Figure 2 shows the positive ion mass spectrum for the 12.6-min component in Figure lb. The ion at m/z 145 suggests a molecular weight of 144 for the unknown substance if the usual gas-phase protonation processes were taking place. However, careful inspection of the mass spectrum indicates that the isotope has unusually low abundance relative to the 12C isotope (see insert in Figure 2). For carboncontaining compounds of this mass, one would expect a relative abundance between 7% and 12% of the 12C isotope. One explanation for this observation is that the unknown peak is attributed to either an inorganic material or an unusual organic compound composed primarily of elements that do not have higher mass isotopic contributions. Another explanation is that the molecular ion at rn/z 145is multiply charged, thus giving mass spectral peaks at these low-
me inwt dmhorm mt, mlr 140-150 region wlm the relmlve abu-
ANALYTICAL CI-EEMISTRY, VOL. 61. NO. 1. JANUARY 1, 1969
le 1 2 . h i n peak observed in arnplllledby 10.
jure
3. ion profile mass spectrum of
the region ObSeNed at mlr 145 in F i g ure 2. The mass spearurn was acquired Mder -6 gectmmu~cresolution mndnim wproxime. ly twice mOae used lor nwmal mit ro6olution op eration.
er mass-to-charge values. Both the ion spray (2) and electrospray (3) interfaces produce ions by the ion evaporation mechanism, and it has been observed that ionic compounds with more than one ionizable functional group will produce multiply charged gasphase ions whose number of charges correspondsto the number of functional groups. There are several ways to determine the presence of a multiply charged ion and the number of charges that reside on the molecule. Mathematical routines have been reported (4, 5 ) , and MS/MS mass spectra will show product ions higher in mlz than the precursor ion if the precursor ion is multiply charged (2). For this problem, the ion current profile mass spectrum of the ions in question was obtained and examined to determine the mass shift for the isotopes. Figure 3 shows that the maas shift between the centroids of the ion at mlz 145 and its 13C isotope is 0.5 maas-to-charge units. This supports the hypothesis of a multiply charged ion and identikres it as being doubly charged. Therefore, the mass of the unknown molecular ion is now determined to he 290 Daltons, with the 1% isotope properly spaced one Dalton higher at mlz 291.The doubly charged 291 mass would occur at mlz 145.5 (see Figure 3). The only question remaining is whether the doubly charged molecular ion was produced by the addition of two protons to the molecule in solution or if the molecule exists in solution as a permanently charged species irrespective of pH, as is the case with a molecule containing two tetraalkyl quaternary ammonium cations. If the former case were true, it would indicate a mo-
I
I
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Fbure 4. Full-scan product ion MSIMS mass spectra of (a) succinylcholine and (b) the suspect toxin hom the dexamethasone formulation. mls m858 spearurn was obtained with a wllision hnergy of 50 eV uslnp arwn 88 th8 oolllllion gas.
lecular weight of 288 whereas the latter would indicate a molecular weight of 290. Adjusting the pH of the mobile phase to more basic conditions did not affect the mass spectrum, which identified the molecule in question as being one with two quaternary ammonium cations with a mass of 290 Daltons. The ion spray LC/MS data represented the first clear evidence of a chemical difference between the toxic and control dexamethasone samples. From this information, the toxic compound could be described as a doubly charged positive ion in solution with a molecular weight of 290. The next step was to refer to the Ninth Merck Index Table of Molecular Weights Index (6) and determine which entries had a molecular weight of 290, had two quater-
nary ammonium cation functionalities, and fit the limited analytical and biological information available. Only one entry fit our data: succinylcholine, a neuromuscular blocking agent that is lethal in overdose situations. Authentic succinylcholine was subjected to the same LC/MS analysis protocol described above, but with the tandem triple quadrupole mass spectrometer operated in the MS/MS mode (7). This experiment was performed to gather additional structural support for the identification of the suspect compound. Thus the mlz 145 doubly charged ion was focused into the central collision cell via the first quadrup l e followed by collision-induced dissociation (CID) of this ion, which produced the full-scan product ion maw
ANALYTICAL CHEMISTRY, VOL. 61, NO. 1. JANUARY 1, I989
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ANALYTICAL CHEMISTRY. VOL. 61. NO. 1, JANUARY 1, 1989
rn-rn9Dbsa ~m,r-M8w
ANALYTICAL APPROACH spectrum shown in Figure 4a. Higher mass singly charged ions and lower mass fragment ions were observed for authentic succinylcholine. Identical LCIMSNS experimente performed on the toxic dexamethasone extract provided the full-scan product ion mass spectrum shown in Figure 4b. This was identical to the mass spectrum for authentic succinylcholine (Figure 4a.) We now had conclusive proof that the toxic dexamethasone extract contained succinylcholine, which was not present in the control dexamethasone extract. Furthermore, it was determined by L C N S quantitative analysis studies that the concentration of succinylcholine in the injectable dexamethasone was 8.8 mg/mL. A 15-kg dog treated with 5 mL of this solution would have received 44 mg or 2.93 mgkg of succinylcholine. The standard dosage of this drug is 0.3 mgkg. It is little wonder that the animals succumbed to the accidental treatment of succinylcholine described here. Postdlscovey analyses After the toxic analyte was identified, a renewed effort was made to detect succinylcholine by conventional methods. TLC was performed on silica gel plates using the original drug solutions rather than the extracts. The best solvent system found was a 1:l solution of chloroform and methanol. This removed the polyethylene glycols and parabens from the analyte of interest, which remained at the origin of the TLC plate. Spraying the plate with Dragendorfs reagent yielded a bright red color that turned black after overspraying with copper chloride and sodium nitrate. This was consistent with the authentic standard. Preparative TLC for capillary GC/ MS analysis consisted of scrapes from the origin (R,= 0.0) after development in the 1:l chloroform/methanol solution. The scrapes were eluted from the silica gel using acidified water, diluted with methanol, and concentrated to dryness under nitrogen in a hot water bath. The residue was redissolved in methanol and chloroform (L4)and analyzed by capillary GCNS. An injection port pyrolysis technique previously reported (6)was used to verify the presence of succinylcholine by standard GUMS. Instrumentation consisted of a Hewlett-Packard Model 5890 capillary gas chromatograph coupled to a Model 5970 mass selective detector. The column employed was an HP 100% methylsilicone fused-silica capillary column (0.2 mm id. X 12 m). The initial oven temperature (170"C) was held for 1min and was followed by
a 15 "Clmin temperature program to 280 "C. To pyrolyze the succinylcholine, the injection port had to be heated to 320 "C. The mass spectrum produced for the analyte matched the published spectrum for the pyrolysis product of succinylcholine (8). These results highlight the importance of having access to new analytical technologies, including those that provide the capability for on-line separation and detection of polar analytes by, for example, LC/MS/MS. The modem toxicology laboratory must have these capabilities to solve particularly difficult analytical problems. The increasing prevalence of very polar toxic analytes in medicine highlights the need for the ability to detect and identify these elusive substances. We thank W. P. Cadwalladerfor bringing thisease to our attantion and aaaiating us with the biologiealatudirsandC. E.Shart foraaaistingus with the biological teats at Cornell. We dm thank G.A.
Practices
Maylin. director of the laboratory, for his continued support of our work.
I
Re(erences (1) Wolnik,K.A.;Fricke,F.L.;Bonnin,E.; Gaston, C. M.; Satzger. R. D. Anal. Chem. 1984,56,467 A 4 7 4 A. (2) Bruins. A.P.; Covey, T.R.; Henion,
J. D. Anal. Chem. 1987.59,264246. (3) Whitehouse, C. M.; Dreyer, R. N.; Yamashita, M.; Fenn. J.B. Anal. Chem. 1985,57.675-79.
(4) Mann, M.; Men C K Fenn, J. B. Presented at the 3 6 8 AS& Conference on Mess Spectrometry and Allied Topics,
San Francisco, CA. June 5-10,1988.
( 5 ) Covey, T. R.; Thomson, B. A.; Shushan. B.; Bonner, R.; Henion, J. D. Rapid Com-
munication in Moss Spectrometry, in
press.
(6) Table of Molecular Weiahts, A Comparison Volume to the Me& Index, 9th ed.: Windholtz, M.; Budavsri. S.; Fenig, M. N.:Schonbern.C.A..Edr.:Merckand Co.. Inc.: Rahway. NJ, 1978. (7) Tandem Mass Spectrometry; MeLafferty. F. W., Ed.; John Wiley and Sons:
New York, 1983.
(8) Lukaszewski, T. J. Analytical Toxicology
1985,9.101-8.
R
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Joseph G. Ebel. Jr., (left) is an analytical toxicologist for the Diagnostic Laboratory a t the New York State College of Veterinary Medicine, Cornell Uniuersity, and the director of laboratory operations for the toxicology department. He received a B.S. degree from Cornell and has been with the Equine Drug Testing and Toxicology Program since 1974. His interests include developing new and unique methodologies and analytical techniques for identifying drug and pesticide residues in biological samples. Jack D. Henion (second from left). a n associate professor of toxicology for the Equine Drug Testing and Toxicology Program a t the New York State College of Veterinary Medicine, received a Ph.D. from the State University of New York a t Albany. His research interests include combining HPLC, supercritical fluid chromatography, and capillary zone electrophoresis with conventional and tandem MS. He frequently uses these techniques to solue real-world analytical problems. Thomas R. Couey (third from left). a research scientist for Sciex, Inc., received a Ph.D. in analytical toxicology from Cornell University and has held research positions a t Finnigan Corp. and Nermag Inc. His research interests center around the deuelopment of HPLC and electrophoretic inlet systems for MS and the biomedical applications of these systems. Richard H. Eckerlin (right) is a clinical toxicologist for the Diagnostic Laboratory at the New York State College of Veterinary Medicine and director of the toxicology laboratory. After receiving a D.V.M. degree from Cornell, he was in private veterinary practice for 13years. Eckerlin's interest in studying real-world toxicological problems led him to join the Cornell veterinary faculty.
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