Experiments using inductively coupled plasma-mass spectrometry (ICP-MS) as a detection system for reversed-phase chromatographic methods have been conducted for the analyses of various pharmaceutical compounds. Interfacing the ICP-MS instrument to the high performance liquid chromatography (HPLC) system was straightforward. The eluent from the HPLC column was pumped through a photodiode array (PDA) and then directly into the ICP-MS nebulizer using standard sample introduction components at flow rates ranging from 0.5 to 1.5 mL/min. Two separate sets of experiments were carried out. Initial speciation experiments were conducted on the B supplements cyanocobalamin (vitamin B₁₂), biotin, and thiamine (vitamin B₁) using isocratic separations. ICP-MS chromatograms were generated by monitoring cobalt at m/z 59, phosphorus at m/z 31, and sulfur at m/z 34 simultaneously. Linear regression data were obtained for cyanocobalamin, thiamine hydrochloride, and biotin. Elemental detection was used to resolve coeluted thiamine and cyanocobalamin peaks in a rapid isocratic separation. The signal for biotin in the sulfur chromatogram was determined to be much larger than that of the corresponding UV chromatogram at 250 nm. In the second set of experiments, an active pharmaceutical ingredient (API) was hydrolyzed with base to monitor the parent compound and two degradation products: degradation product 1 (DP1) and degradation product 2 (DP2). The API molecule contains bromine, chlorine, and sulfur; DP1 contains bromine and sulfur; and DP2 contains chlorine. The ions monitored for analysis of API, DP1, and DP2 were m/z 79 for bromine, m/z 35 for chlorine, and m/z 34 for sulfur. The calculation of relative amounts of API, DP1, and DP2 from the bromine, chlorine, and sulfur chromatograms was performed. The data suggest that the same calculation performed from the UV chromatogram introduces a bias due to the difference in extinction coefficients for each compound. The capacity for monitoring UV absorbance and elemental ions sequentially was demonstrated to provide enhanced analytical specificity without requiring interpretation of complex mass spectra.

Applications were investigated for inductively coupled plasma–mass spectrometry (ICP-MS) detection with reversed-phase high performance liquid chromatography (HPLC) for the speciation of pharmaceutical compounds containing a transition metal (cobalt) or the nonmetallic heteroatoms sulfur, phosphorus, chlorine, and bromine. Multiple elements were monitored simultaneously by ICP-MS in order to fully utilize specificity of elemental detection. An objective of this investigation was to demonstrate the use of elemental detection by ICP-MS with minimal modification of typical packed-column HPLC isocratic and gradient methods. Background signals from polyatomic isobaric interferences at m/z 31, 34, 35, and 79 were attenuated by using a hexapole collision cell charged with a mixture of 1% ammonia–helium.

Proof of concept investigations of this technique were performed using the vitamin B supplements cyanocobalamin (vitamin B₁₂), which contains cobalt and phosphorus; thiamine (B₁), which contains sulfur; and biotin, which contains sulfur. These data were collected using isocratic HPLC methods. These compounds were chosen for method development because several HPLC-UV methods have been published to detect water-soluble B vitamins (1–5). Two methods have been reported to detect water-soluble B vitamins, including cyanocobalamin, thiamine, and biotin, using UV detection (6,7). ICP-MS detection has been used previously to detect vitamin B₁₂ (8) by monitoring cobalt at m/z 59. For these experiments, UV and elemental detection were used in a serial arrangement. Cyanocobalamin chromatograms were acquired while monitoring UV absorbance with a photodiode array, along with phosphorus (m/z 31) and cobalt (m/z 59) simultaneously using the ICP-MS.

Table I: Analyte formulae and structures

Experimental

Table II: Element ions and associated polyatomic interferences

HPLC System

A Waters 2695 HPLC system equipped with a Waters 996 PDA detector (Milford, Massachusetts) was used for these studies. An Agilent Zorbax Eclipse XDB-C18 column (150 mm × 4.6 mm, 3.5 μm; Agilent Technologies, Wilmington, Delaware) was used for the B-supplement experiments. An Agilent Zorbax Eclipse XDB-C8 column (150 mm × 4.6 mm, 3.5 μm) was used for the analysis of the API, DP1, and DP2. All of the connections were made with 0.007-in. i.d. PEEK tubing. The eluent from the PDA detector cell was plumbed directly into the ICP-MS sample introduction system.

Reagents, Standards, and Samples

Cyanocobalamin, thiamine hydrochloride, and biotin were obtained from Sigma-Aldrich (St. Louis, Missouri). API material was obtained from Eli Lilly research facilities (Indianapolis, Indiana). HPLC-grade methanol and acetonitrile were purchased from Mallinckrodt (Phillipsburg, New Jersey). Ammonium acetate, acetic acid, and monobasic potassium phosphate were purchased from Sigma-Aldrich. Phosphoric acid was purchased from Mallinckrodt. Sodium hydroxide (1.0 N) was purchased from Red Bird Services (Batesville, Indiana). Milli-Q water (18.3 Mohm-cm) was used throughout (Millipore, Bedford, Massachusetts).

Individual stock solutions of cyanocobalamin, thiamine hydrochloride, and biotin were prepared at a 1000-mg/mL concentration in water. Ammonium hydroxide was added drop-wise to the biotin stock until dissolution. Fresh calibration standard solutions were prepared daily by serially diluting the stock solutions in water. The standards contained a mixture of the three analytes, ranging in concentrations from 5 to 100 mg/mL.

A 1.0-mg/mL API solution was prepared in 0.1 N sodium hydroxide. This solution was thermally stressed at 80 °C for 72 h to form degradation products DP1 and DP2. This solution was injected directly. The 25 mM ammonium acetate buffer was prepared by dissolving ammonium acetate in water and adjusting the final pH to 4.0 with acetic acid. The 25 mM potassium phosphate buffer was prepared by dissolving monobasic potassium phosphate in water and adjusting the final pH to 2.5 with phosphoric acid.