ANALYTICAL APPROACH
The Case of the Tainted Richard H. Eckerlin, Joseph G. Ebel, Jr., Jack D. Henion Equine Drug Testing and Toxicology Diagnostic Laboratory New York State College of Veterinary Medicine Cornell University 925 Warren Dr. Ithaca, NY 14850
Dexamethasone
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 (1). 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. History
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 0003-2700/89/0361-053A/$01.50/0 © 1988 American Chemical Society
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 our 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. Alternate 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 · 53 A
ANALYTICAL· APPROACH a more pure form from the dexametha sone sample, further chemical analyses might lead to successful identification. A female dog was acquired, exam ined, and found to be in good health. This animal was prepared for electro physiological monitoring via electroen cephalogram and electrocardiogram and given the suspect injectable dosage (5 mL) subcutaneously in the intrascapular region (between the shoulder blades). The subject was observed dur ing the period following the injection. Marked physiological changes were ob served 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 pro cured for the study of the suspect ma terial. Some of these animals were in jected with 0.05 mL of the control in jectable formulation and observed for 1 h postinjection. No ill effects were not ed. Other mice were similarly injected with the suspect formulation of dexa methasone. Each of the mice died with in 2 min after receiving the injection. Dilution studies of the suspect material showed that the material diluted with physiological saline still retained its mouse lethality at dilutions up to 1:4. The suspect formulation was then heated at 100 °C for 10 min. This mate rial again proved to be lethal to mice at the same dosage. The control vial of dexamethasone was also heated at 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 medium. In all cases, bio logical experiments revealed that the toxic substance remained entirely within the aqueous phase, but this phase became inactivated when its pH 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 dif ference between the control and toxic aqueous extracts. Similarly, radioim munoassay methods used for the detec tion of potent narcotics gave no posi tive results. The suspect and control vial contents were tested by an outside
Analytical techniques used for comparing control samples with tainted dexamethasone samples Test procedure
Results
Acid and base TLC GC/MS (neat and with bis(trimethylsilyl)trifluoroacetamide, BSTFA) HPLC (diode array detector) Cyanide and fluoride analyses X-ray fluorescence spectroscopy Inductively coupled plasma emission spectroscopy UV-vis spectroscopy Radioimmunoassay for narcotics Endotoxin assay Biological testing (dog) Biological testing (fraction study)
No differences No differences No differences Negative No differences No differences No differences Negative Negative Toxicity established Toxic principle in aqueous phase
mm Figure 1. LC/MS total ion current profiles scanning from 100 to 1000 Daltons. A 2.1 mm X 7.5 cm amino-bonded column was used with a gradient from 100% methanol to 100% water over 15 min at a flow rate of 200 μί/ιτπη. (a) Aqueous extract of control dexamethasone formulation and (b) aqueous extract from toxic dexametha sone formulation.
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ANALYTICAL APPROACH
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 mass 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/O.OIO M NH4OAc 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 PEGs from other possible compounds. The LC/MS 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
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 ^L/min, and the mass spectrometer was operated in the full-scan ion spray LC/MS mode scanning from m/z 100 to 1000 Daltons. No obvious difference in the total ion current profiles of the samples was observed even though biological tests 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 LC/MS system. A gradient from 100% methanol to 100% water was used to obtain the total ion current profiles shown in Figure 1. For the first time, a chemical difference was observed between the extracts of the
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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 lb 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 13C 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 m/z 145 is multiply charged, thus giving mass spectral peaks at these low-
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m/z Figure 2. Background-subtracted mass spectrum of the 12.6-min peak observed in Figure 1b. The inset shows the m/z 140-150 region with the relative abundances amplified by 10.
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(Μ) 2+ 145
(a) 100η
C0 2 CH 2 CH 2 N(CH 3 ) 3 80
(ÇH 2 ) 2 59
C0 2 CH 2 CH 2 N(CH 3 ) 3
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40 Η 159
Figure 3. Ion profile mass spectrum of the region observed at m/z 145 in Fig ure 2. The mass spectrum was acquired under mass spectrometric resolution conditions approximate ly twice those used for normal unit resolution op eration.
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ibl.ii ..ι. 1 τ~-\—ι—Γ
