Robert D. Voyksner

Determination of penicillin G, ampicillin, amoxicillin, cloxacillin and cephapirin by high-performance liquid chromatography—electrospray mass spectrometry

This report contributes to a preliminary investigation of high-performance liquid chromatographic (HPLC)-mass spectrometric (MS) methods for confirming beta-lactam antibiotic residues in bovine milk. Initial work for each antibiotic evaluated the collisional activated dissociation (CAD) spectra that could be generated between the capillary and skimmer in the electrospray (ESP) interface. The drugs show various characteristic fragmentation, mostly within the beta-lactam ring and the amide group. Response for a particular compound in a given solvent can vary drastically. Usually, the more organic component in the solvent, the higher the ESP response. In many cases use of acetonitrile also results in slightly better ion currents than for methanol when comparing equal percentages of either organic solvent in water. The ESP response of most of the tested antibiotics can be enhanced by the addition of formic acid or acetic acid to the mobile phase methanol-water (1:1). In general, the negative ion spectra are lower in intensity, exhibiting an [M-H]- ion and producing less fragmentation at higher CAD voltages as compared to positive ion spectra. An isocratic reversed-phase HPLC method for the separation of a mixture of five common beta-lactam antibiotics was developed using acetic acid as a mobile phase additive and optimized for detection with a new ESP HPLC-MS interface. A post-column split ratio of 70:1 for the eluent from a 150 x 2 mm I.D. column was chosen to provide the required lower flow-rate (approximately 4 microliters/min). The limit of detection for the simultaneous determination of these antibiotics was estimated to be 100 ppb. Electrospray HPLC-MS could be used to confirm these antibiotics for quantities down to about 100 pg entering the mass spectrometer. Multiresidue analysis with microbore HPLC-ESP-MS has the advantage that no post-column splitting of the eluent is required and all of the analyte (on-column injected) will be transferred into the ESP interface. Preliminary work showed good mass spectrometric sensitivity down to the level of regulatory interest, but chromatographic separation efficiency must be improved.

Investigating the use of an octupole ion guide for ion storage and high-pass mass filtering to improve the quantitative performance of electrospray ion trap mass spectrometry.

An octupole radio frequency (rf) ion guide was evaluated for storage and filtering of ions generated by electrospray prior to introduction into an ion trap mass spectrometer. The control of the rf potential on the ion guide enabled its operation as a high-pass mass filter, removing low-mass chemical noise that would normally fill the ion trap and result in reduced sensitivity, mass resolution, and dynamic range. Also, the ion guide can serve to store the high-pass filtered ions during the ion trap mass analysis, enabling nearly 100% duty cycle and reduction of the cycle time by a factor of two. The linearity, precision, and detection limits of the liquid chromatography (LC) electrospray - ion guide-ion trap MS/MS system were evaluated for the determination of ceftiofur in milk. A linear calibration (linear correlation coefficient of 0.986) from 2-200 ppb was obtained with a relative standard deviation for replicate analysis of less than 8%. A quantitation detection limit of 100 pg of ceftiofur on-column (2 ppb) was achieved from a milk extract. This detection limit is nearly a factor of 10 lower compared with the determination on the same electrospray ion trap system not using a rf ion guide for high-pass mass filtering and ion storage.

Analysis of selected pesticides by high-performance liquid chromatography—mass spectrometry

Abstract A Finnigan 4500 mass spectrometer was modified to perform direct liquid introduction high-performance liquid chromatography—mass spectrometry (DLI-HPLCue5f8MS). The HPLCue5f8MS analysis of some 25 pesticides, including carbamates, chlorinated carboxylic acids and methyl ureas is described. The qualitative appearance of the spectra as well as detection limits are presented for both positive and negative ion detection. In most cases DLI-HPLCue5f8MS, using an 1:100 split, provided molecular weight information with sub-microgram detection limits and greatly increased specificity over HPLC detection.

Analysis of some metabolites of t-2 toxin, diacetoxyscirpenol and deoxynivalenol by thermospray high-performance liquid chromatography—mass spectrometry

Detection and identification of mycotoxin metabolites is a very challenging task. In order to achieve adequate sensitivity and specificity an analytical technique must overcome serious matrix interferences. Gas chromatography-mass spectrometry (GC-MS) which has the sensitivity and specificity to detect and identify mycotoxin metabolites requires hydrolysis of conjugated metabolites as well as derivatization. Thermospray high-performance liquid chromatography-mass spectrometry (HPLC-MS) offers the sensitivity, specificity, and structural information to detect and identify some mycotoxin metabolites in fecal and urine samples without derivatization. The mycotoxins evaluated in this study include deoxynivalenol (DON), T-2 toxin, and diacetoxyscirpenol. The de-epoxy and hydroxy metabolites of each toxin and the glucuronide conjugate of DON were isolated, extracted, and analyzed to detect their occurrence in animals. The thermospray mass spectra of the toxins showed an [M + H]+ ion and numerous structurally significant fragment ions in the positive ion detection mode. Negative ion detection exhibited primarily [M + acetate]- cluster ions with less fragmentation than observed by positive ion detection. The operation of the interface in the filament-on mode greatly increased the sensitivity in both positive and negative ion detection mode. Detection limits of 50-500 pg injected on column are obtained for these toxins and their metabolites using multiple ion detection. The urine and fecal extracts from rats, hens, and cows did not interfere with the HPLC-MS analysis for the specific metabolites or the glucuronide conjugate.

Trace analysis of explosives by thermospray high-performance liquid chromatography-mass spectrometry

Abstract A series of explosives were analyzed by thermospray high-performance liquid chromatography—mass spectrometry (TSP-HPLC—MS). Both positive and negative ion spectra were acquired in thermospray and chemical ionization (filament-on) modes. The explosives proves most sensitive in the negative ion mode under chemical ionization conditions. The spectra for the explosives analyzed provided molecular weight information with few fragment ions. Detection limits from 200 pg for trinitrotoluene to 5 ng for ammonium picrate were obtained under full scan mass spectrometer conditions. TSP-HPLC—MS proved useful in separating and identifying components in commercial explosive mixtures without interferences from plasticizers as well as in detecting explosives from hand swabs with excellent sensitivity and specificity. The plasticizers primarily were detected in the positive ion mode while the explosives were most sensitive with negative ion detection.

Quantitative determination of ceftiofur in milk by liquid chromatography-electrospray mass spectrometry

A liquid chromatography-electrospray mass spectrometry (LC-ES-MS) was developed for the quantitation of ceftiofur in milk at the 50 ppb tolerance level set by the US Food and Drug Administration (FDA) for the drug. The method used ultrafiltration as a simple and rapid means to prepare the sample for analysis. A 100 microliters volume of ultrafiltrate containing ceftiofur was concentrated on-column for LC-MS analysis. The LC separation was accomplished using an acetonitrile gradient with the ion-pair reagent heptafluorobutyric acid (HFBA). Propionic acid was added after the LC column to minimize electrospray signal suppression, enhancing the response for ceftiofur by a factor of 10. The transmission ions from the electrospray interface to the MS was enhanced by a factor of 7 by using a Rf ion guide. The development method could detect ceftiofur to 10 ppb and quantitate the antibiotic from 25-200 ppb (linear correlation coefficient of 0.993). The analysis indicated that bovine milk collected 32 h after dosing with ceftiofur was above the FDA tolerance of 50 ppb, while milk collected 48 h after dosing was found to contain 24-31 ppb of ceftiofur.

Simultaneous determination of enrofloxacin and its primary metabolite ciprofloxacin in bovine milk and plasma by ion-pairing liquid chromatography

A simple and sensitive high-performance liquid chromatographic method has been developed for the simultaneous determination of enrofloxacin and ciprofloxacin in bovine milk and plasma. Sample preparation consisted of mixing equal volumes of milk or plasma with acetonitrile-0.1 M sodium hydroxide (1:1, v/v), followed by ultrafiltration through 3000 Da molecular mass cut-off filters. Separation of these two fluoroquinolones in milk or plasma ultrafiltrate was accomplished by ion-pairing liquid chromatography using a reversed-phase analytical column eluted with acetonitrile-methanol-water. Ultraviolet absorbance of the column effluent was monitored over the 230-350 nm range with a photodiode-array detector (lambda max 278 nm). Recoveries of enrofloxacin from bovine milk and plasma were 92-107% and 80-84%, respectively. Recoveries of ciprofloxacin from bovine milk and plasma were 92-105% and 73-75%, respectively. The limit of detection for the two compounds was 5 ng/ml. Enrofloxacin was administered intravenously to a lactating cow at a dose of 2.5 mg/kg. Enrofloxacin was detected in milk within 15 min after injection and the metabolite ciprofloxacin rapidly appeared in plasma and milk. Both enrofloxacin and ciprofloxacin were below the limit of detection (5 ng/ml) by 48 h after drug administration.

Thermospray, particle beam and electrospray liquid chromatography-mass spectrometry of azo dyes

Abstract A high-performance liquid chromatographic-mass spectrometric (HPLC-MS) method was used for the analysis and characterization of fourteen commercial azo and diazo dyes. Thermospray analysis of these dyes using the “filament-on” chemical ionization operation mode produced mass spectra consisting primarily of [M + H] + ions with very few fragments. Particle beam electron impact ionization mass spectrometric analysis resulted in molecular ion information and various fragment ions. Characterization of the azo dyes could be achieved by observing typical fragment ions formed by cleavage of the Cue5f8N bonds on either side of the azo linkage, and cleavage of the Nue5fbN double bond with transfer of one or two hydrogen atoms to form an imine or amine. Many commercial azo dyes contain precursors, by-products of the synthesis, degradation products or other impurities. Some of these components were tentatively identified by their mass spectra and by review of the chemistry of dye manufacture. Electrospray negative ion operation was suitable for the characterization of a disulfonated azo dye and showed singly and doubly molecular ions and fragment ions at higher capillary-skimmer potential differences. The base peak was attributed to a doubly charged naphthol cleavage product.

Displacement of the bidentate malonate ligand from (d,l-trans-1,2-diaminocyclohexane)malonatoplatinum(II)by physiologically important compounds in vitro

Previous studies of platinum(II) compounds with bidentate leaving ligands have emphasized the contrast between the stability of the bidentate leaving ligand in vitro (T1/2 greater than 11 days in water) and the apparent reactivity of these bidentate platinum compounds in vivo. However, none of these studies actually measured the stability of these compounds in tissue culture medium (or in any other reaction mixture resembling in vivo conditions). The experiments described in this paper were designed to measure the stability and fate of (d,l-trans-1,2-diaminocyclohexane)malonatoplatinum(II) [Pt(mal)(trans-dach)] in RPMI-1640 tissue culture medium. The T1/2 for displacement of the malonate ligand in this medium was 9.5 hr at 37 degrees. Of the inorganic anions present in the medium, chloride accounted for the greatest displacement of the malonate ligand. However, at the concentrations with which it is found in tissue culture medium and in blood, bicarbonate was nearly as effective as chloride at displacing the malonate ligand. This observation is of particular significance because the bicarbonatoplatinum complex is unstable and the bicarbonate displacement reaction appears to represent a major non-enzymatic pathway for the formation of the biologically active aquated platinum complexes. At the concentrations with which they occur inside the cell, phosphates may play a similar role. Of the amino acids present in the medium, glutathione and the sulfur-containing amino acids were 50- to 400-fold more effective at displacing the malonate ligand than the other amino acids in RPMI-1640 medium. In the case of methionine, the reaction with Pt(mal)(trans-dach) was shown to be a direct displacement (SN2) reaction at physiological methionine concentrations. When Pt(mal)(trans-dach) was incubated at 37 degrees for 24 hr in RPMI-1640 medium, the major transformation products formed were (d,l-trans-1,2-diaminocyclohexane)methionineplatinum(II) (38%), other amino acid-platinum complexes (19%), and (d,l,-trans-1,2-diaminocyclohexane)dichloroplatinum(II) (14%). Eleven percent of the Pt(mal)(trans-dach) remained intact. Mass spectrometry and 1H-NMR indicated that the (d,l-trans-1,2-diaminocyclohexane)methionineplatinum(II) complexes that formed in RPMI-1640 medium consisted of approximately 60% of the bidentate mono-methionine complex coordinated to platinum at the sulfur and alpha-amino positions and 40% of the bis-methionine complex, presumably coordinated at the sulfurs.(ABSTRACT TRUNCATED AT 400 WORDS)

Solvent degradation of cloxacillin in vitro: Tentative identification of degradation products using thermospray liquid chromatography-mass spectrometry

Abstract The determination of cloxacillin and the other penicillin antibiotics at trace levels in pharmaceutical samples and biological fluids and tissues is difficult. Degradation of penicillins occurs during exposure to chemicals and solvents used in sample extraction, storage of extracts and chemical analysis. One of the most important factors for the determination of cloxacillin in bovine milk and tissues is the choice of solvent used for extraction. Cloxacillin stored in different solutions underwent two types of degradation: hydrolysis with decarboxylation to yield cloxacillinpenilloic acids when water or aqueous solutions of acetonitrile or 2-propanol were used, and alcoholysis to form cloxacillinpenicilloic acid ester when methanol or ethanol was used. Cloxacillin stored in aqueous methanol or acetonitrile solution underwent faster degradation when stored in water or aqueous solutions of 2-propanol or ethanol. The solvent combinations that resulted in minimum sample degradation of cloxacillin were acetonitrile-ethanol-water (25:25:50) or ethanol-water (50:50). Degradation of cloxacillin was faster at the lower levels (500 and 50 ng/ml) of the antibiotic for all solvents tested. Nearly complete degradation of cloxacillin was observed at the 50 ng/ml level after 2 h at 20°C. Degradation was nearly non-existent at the 1 mg/ml level of the antibiotic in each solvent.