Sulfur-Specific Detection of Impurities in Cimetidine Drug Substance

Anal. Chem. 2001, 73, 4722-4728

Sulfur-Specific Detection of Impurities in Cimetidine Drug Substance Using Liquid Chromatography Coupled to High Resolution Inductively Coupled Plasma Mass Spectrometry and Electrospray Mass Spectrometry E. Hywel Evans,*,† Jean-Claude Wolff,‡ and Christine Eckers‡

Plymouth Environmental Research Centre, Department of Environmental Sciences, University of Plymouth, Drake Circus, Plymouth, Devon PL4 8AA, U.K., and GlaxoSmithKline, New Frontiers Science Park North, Third Avenue, Harlow, Essex CM19 5AW, U.K.

The use of liquid chromatography coupled to sector field inductively coupled plasma mass spectrometry (SF-ICPMS) for the specific detection of sulfur-containing compounds is described. In the sulfur-containing drug substance cimetidine, structurally related impurities well below the 0.1% mass fraction level relative to the main drug substance could easily be detected. The structure of most of the impurities was confirmed by electrospray mass spectrometry (ESI-MS), and thus, the complementarity of the two techniques for drug analysis is shown. The limit of detection by SF-ICP-MS for cimetidine in solution was ∼4-20 ng‚g-1, but it was blank-limited. In the pharmaceutical industry, the detection and identification of impurities/metabolites structurally related to the drug substance are of utmost importance. Conventional methods of analysis to detect, track, quantify, and identify drug substances and related impurities use molecular mass spectrometry via atmospheric pressure ionization (API-MS)1,2 or, in the case of drug metabolism and pharmacokinetics, use radiolabeling.3,4,5 A significant number of drug substances contain heteroatoms that can be detected using element-specific detectors such as inductively coupled plasma mass spectrometry (ICP-MS) or chemical reaction interface mass spectrometry (CRIMS).6 Structurally related compounds can then be more easily identified. ICP-MS has been widely used as an element-selective detector for both gas and liquid chromatography.7 It is particularly suited for coupling with high performance liquid chromatography (HPLC), because it is compatible with typical HPLC flow rates, and it has extremely low limits of detection for most elements in the periodic table. * Phone/Fax: +44 (0)1752 233040. E-mail: [email protected]. † University of Plymouth. ‡ GlaxoSmithKline. (1) Eckers, C.; Haskins, N.; Langridge, J. Rapid Commun. Mass Spectrom. 1997, 11, 1916. (2) Xu, K.; Arora, V. K.; Chaudhary, A. K.; Cotton, R. B.; Blair, I. A. Biomed. Chromatogr. 1999, 13, 455. (3) Griffiths, R.; Lee, R. M.; Taylor, D. C. Int. Congr. Ser.-Excerpta Med. 1977, 416, 38. (4) Steffens, T. G.; Davis, R.; Holohan, P. D.; Sokol, P. P. Trends Biomembr. Bioenerg. 1990, 1, 93. (5) Braendle, E.; Greven, J. Arch. Int. Pharmacodyn. Ther. 1991, 314, 169. (6) Eckers, C.; Abramson, F. P.; Lecchi, P. Rapid Commun. Mass Spectrom. 2001, 15, 602. (7) Sutton, K.; Sutton, R. M. C.; Caruso, J. A. J. Chromatogr. 1997, 789, 85.

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Table 1. Operating Conditions for SF-ICP-MS ICP nebulizer gas flow/L‚min-1 auxiliary gas flow/L‚min-1 coolant gas flow/L‚min-1 nebulizer spray chamber torch interface sampling cone skimmer cone single ion monitoring resolution slit settings masses monitored dwell time/ms scanning resolution slit settings masses monitored dwell time/ms points

0.74 0.6 14 Meinhard concentric jacketed quartz cyclonic and single pass spray chambers in series, cooled to 5 °C quartz Fassel-type without shield nickel, 1 mm i.d. nickel, 0.7 mm i.d. 6000 source, 245; collector, 155 single ion monitoring for 32S+ 500 6000 source, 245; collector, 155 scanning between 31.94 and 32.11 m/e 40 40

Element-specific detection via quadrupole ICP-MS of chlorineand bromine-containing drugs has been successfully applied.8,9,10 In the same context, analysis of protein phosphorylation by monitoring for phosphorus has been used.11 The technique does suffer from several serious interferences due to the formation of polyatomic ions;12 however, most of these can be resolved using a high resolution magnetic sector mass spectrometer rather than the more commonly used quadrupole.13 The latter technique was used to identify protein fractions containing trace metal elements as well as sulfur and phospho(8) Nicholson, J. K.; Lindon, J. C.; Scarfe, G.; Wilson, I. D.; Abou-Shakra, F.; Castro-Perez, J.; Eaton, A.; Preece, S. Analyst 2000, 125, 235. (9) Corcoran, O.; Nicholson, J. K.; Lenz, E. M.; Abou-Shakra, F.; Castro-Perez, J.; Sage, A. B.; Wilson, I. D. Rapid Commun. Mass Spectrom. 2000, 14, 2377. (10) Nicholson, J. K.; Lindon, J. C.; Scarfe, G. B.; Wilson, I. D.; Abou-Shakra, F.; Sage, A. B.; Castro-Perez, J. Anal. Chem. 2001, 73, 1491. (11) Wind, M.; Edler, M.; Jakubowski, N.; Linscheid, M.; Wesch, H.; Lehmann, W. D. Anal. Chem. 2001, 73, 29. (12) Evans, E. H.; Giglio, J. J. J. Anal. At. Spectrom. 1991, 8, 1. (13) Stuewer, D.; Jakubowski, N. J. Mass Spectrom. 1998, 33, 579. 10.1021/ac0103017 CCC: $20.00

© 2001 American Chemical Society Published on Web 08/31/2001

Figure 1. Mass spectral scan on the SF-ICP-MS at a resolution of 6000 for DDW passed through the Novapak C18 column.

Figure 2. Sulfur-specific chromatogram obtained by SF-ICP-MS for a 100-µL injection of a 5.2 mg‚g-1 solution of cimetidine (batch 92/085) (a) ×1 scale; (b) ×10 scale expansion. See Table 3 for key.

Analytical Chemistry, Vol. 73, No. 19, October 1, 2001

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Figure 3. (a) UV chromatogram at 230 nm and (b) ESI-MS total ion chromatogram for a 10-µL injection of the cimetidine sample at 18.8 mg‚mL-1. See Table 2 for key.

rus.14 In the context of this work (i.e., the analysis of sulfurcontaining cimetidine drug substance) the main isotope of sulfur (32S, abundance 95.018%) suffers from a serious interference as a result of the polyatomic ion 16O16O+ at nominal m/e 32. Fortunately, the two species can be resolved at a resolution of ca. 2000, thereby allowing the sensitive and selective determination of sulfur.

(between 5 and 8 mg‚g-1). Dissolution was aided by the use of an ultrasonic bath.

EXPERIMENTAL SECTION Reagents and Compounds Investigated. The mobile phases were prepared using methanol (HPLC grade, Fisher, U.K.) and ammonium acetate (Fisher, U.K.) and deionized distilled water (DDW, 18 MΩ, Elga, U.K.). A stock solution of sulfur was prepared by dissolving 0.1762 g of cimetidine (N-cyano-N′-methyl-N′′-[2-[[(5-methyl-1H-imidazol4-yl)methyl]thio]ethyl]guanidine, C10H16N6S, (I), nominal mass 252 g‚mol-1) in 10.5998 g of DDW, and subsequently diluting it in 2% nitric acid to prepare calibration standards. The cimetidine used was a GSK factory batch (GlaxoSmithKline, U.K.). Samples of cimetidine that were to be analyzed by HPLC were dissolved in 0.05 mol‚L-1 ammonium acetate adjusted to pH 5 with acetic acid and methanol in a proportion of 70:30 volume fraction 4724

Analytical Chemistry, Vol. 73, No. 19, October 1, 2001

A sample of SKF-92408 (GlaxoSmithKline), N-methyl-N′-[2-[(4methyl-5-imidazolyl)methylthio]ethyl] guanidine, C9H17N5S, (II) was prepared in the same way as was cimetidine.

Mass Spectrometry. High-Resolution ICP-MS. All experiments were performed using a sector-field inductively coupled plasma

Figure 4. Sulfur-specific chromatogram obtained by SF-ICP-MS for a 100-µL injection of a 6.573 mg‚g-1 solution of SKF-92408: (a) unspiked, (b) spiked with 5 µg‚mL-1 of cimetidine (0.08% m/m).

mass spectrometer (SF-ICP-MS, Thermo Elemental Axiom, Winsford, U.K.), which was capable of mass resolution up to 10 000. Operating conditions are shown in Table 1. The HPLC column was coupled with the SF-ICP-MS simply by inserting the end of the tubing from the column into the sample uptake inlet of the nebulizer. ESI-MS. Positive electrospray MS experiments were carried out using a Micromass Q-ToF quadrupole orthogonal acceleration time-of-flight mass spectrometer (Micromass U.K. Ltd., Wythenshawe, U.K.) equipped with a Z-spray ion source. The spray voltage was 3 kV. The source and desolvation temperatures were set to 130 and 350 °C, respectively. The nitrogen desolvation and nebulizer gas flow rates were set to 400 L‚h-1 and 90 L‚h-1, respectively. The cone voltage was set to 25 V. Experimental data were aquired over a 100-1000 Da mass range at an acquisition rate of 1 spectrum/s. Chromatography. An Agilent 1100 chromatography system (Agilent Technologies, Stockport, Cheshire, U.K.) equipped with

a binary pump, autosampler, and diode array detector was used for the LC/MS experiments on the Q-ToF. The injection volume for the cimetidine sample was 10 µL. For the SF-ICP-MS measurements, a Dionex tertiary gradient pump (Dionex GP40, Sunnyvale, CA) was used. Samples were injected using a rotary injection valve (Rheodyne, Cotati, CA) equipped with a 100-µL loop. PEEK tubing of 0.01-in. i.d. was used throughout. The HPLC column was a Waters NovaPak C18 (3.9 × 150 mm, 4 µm, Waters Corp, Milford, MA). The flow rate was 1 mL‚min-1, and the reequilibration time was 10 min. The run started at 100% 0.05 mol‚L-1 ammonium acetate in DDW, held for 3 min, and then a gradient to 40% methanol after 30 min was applied. RESULTS AND DISCUSSION Preliminary Studies on SF-ICP-MS. A resolution setting of 1801 is theoretically necessary to separate 32S+ at m/e 31.97207 from its major interfering agent, the polyatomic ion 16O16O+ at Analytical Chemistry, Vol. 73, No. 19, October 1, 2001

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Table 2. Structures of Compounds Detected by ESI-MS

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Analytical Chemistry, Vol. 73, No. 19, October 1, 2001

Table 2 (Continued)

m/e 31.98982. However, in practice, a resolution of at least 3000 is necessary, because the interfering species is much more abundant than 32S+, and there is a distinct low-mass tail. This is illustrated in Figure 1, which shows a mass scan at a resolution of 6000 while introducing water as a mobile phase through the Novapak C18 column. As can be seen, the 32S+ blank is made up of two components: a sulfur blank in the reagents and the tail from the 16O16O+ peak. It is possible to reduce the 16O16O+ tail by desolvating the mobile phase;15 however, in this case, it is evident that the majority of the background was caused by the reagent and instrumental blank. To establish the figures of merit, a calibration series was made by diluting cimetidine stock solution and nebulizing the standards into the instrument (i.e., no chromatography was performed at this point). The limit of detection was typically between 4 and 20 ng‚g-1 cimetidine (i.e., 0.6-2 ng‚g-1 S), and the sensitivity of the instrument was ∼6000 cps/ng‚g-1 of cimetidine. Similar detection limits were reported in earlier work16 for the analysis of inorganic sulfates and amino acids by LC/ICP-MS. HPLC Separation of Cimetidine. A sulfur-specific chromatogram obtained by SF-ICP-MS of 100 µL of a 5.2 mg‚g-1 solution of cimetidine (batch 92/085) is shown in Figure 2, that is, 520 µg of cimetidine was injected on-column. Most of the peaks in the

Table 3. Sulfur-Containing Compounds Detected by SF-ICP-MS ret time, min

rel ret time, min

mol form, MW

E F

9.3 9.9 10.5 12.6 14.2 14.8 16.8 17.6 18.2 25 25.3 26.2

0.51 0.54 0.58 0.69 0.78 0.81 0.92 0.96 1 1.37 1.39 1.44

G

26.7

1.47

H I

27.5 28.4 29 30.2 32.7

1.51 1.56 1.59 1.66 1.80

unknown unknown unknown C10H16N6OS, 268 unknown unknown C11H18N6S, 266 C11H18N6OS, 282 C10H16N6S (cimetidine), 252 unknown C11H20N8S2, 328 C10H15N5S2, 269; C10H18N8S2, 314 C16H26N10S2, 422; C15H22N8S, 346 C12H22N8S2, 342 C16H24N8S2, 392 unknown unknown, 468, 454 unknown

peak

A B C D

J

chromatogram represent impurities, all of which were below 0.1%, based on peak area relative to the peak for cimetidine, hence, satisfying the objective of detecting impurities below this level. Analytical Chemistry, Vol. 73, No. 19, October 1, 2001

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The same cimetidine batch was analyzed by LC/ESI-MS, and a UV chromatogram at 230 nm and a total ion current chromatogram (TIC) are shown in Figure 3. The amount on-column was ∼1/3 the amount for the SF-ICP-MS experiment, that is, ca. 180 µg. Impurities identified in the sample are listed in Table 2 and are in accordance with previous work.1,17 The sulfur-specific chromatogram obtained by SF-ICP-MS matches both UV and TIC chromatograms. The tailing due to cimetidine is less prominent in SF-ICP-MS. The shift in retention times and even relative retention times is explained by the use of two different chromatography systems (e.g., the mixing of the eluents being slightly different could lead to a slightly different gradient elution). All sulfur-containing impurities detected by SF-ICP-MS are listed in Table 3, and the letters refer to those in Figure 3 and Table 2 showing the equivalence of compounds identified by both techniques. Several impurities detected by SF-ICP-MS, especially eluting prior to cimetidine, probably have ionization efficiencies that are too low to be detected using ESI-MS. Further investigation is necessary to elucidate the structure of those impurities. This shows that both of the techniques complement each other and that SF-ICP-MS has the potential of detecting drug-related traceto-ultratrace impurities by element-specific detection well below the 0.1% mass fraction level. (14) Wang, J.; Dreessen, D.; Wiederin, D. R.; Houk, R. S. Anal. Biochem. 2001, 288, 89. (15) Prohaska, T.; Latoczy, C.; Stingeder, G. J. Anal. At. Spectrom. 1999, 14, 1501. (16) Jiang, S. J.; Houk, R. S. Spectrochim. Acta, Part B 1988, 43B, 405. (17) Eckers, C.; Wolff, J.-C.; Haskins, N. J.; Sage, A. B.; Giles, K.; Bateman, R. Anal. Chem. 2000, 72, 3683.

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To confirm this, a solution of SKF-92408, an impurity compound that did not contain cimetidine, was spiked with cimetidine at a concentration of 5 µg‚mL-1 to give an equivalent mass fraction of 0.08%. Figure 4 shows two chromatograms of SKF-92048, one unspiked (Figure 4a) and one spiked (Figure 4b) with cimetidine. Evidently, cimetidine could be detected at a mass fraction well below 0.08% relative to the main component. It is important to note the changing baseline in the chromatograms shown in Figures 2 and 4. This was caused by the increasing concentration of methanol in the mobile phase affecting the sensitivity of the instrument, which changed by approximately a factor of 10 throughout the run. This can be avoided if desolvation of the mobile phase is performed, which should also allow the use of more volatile mobile phases, such as acetonitrile, that the instrument cannot tolerate without desolvation. It is also important to note that the detection limit was limited by the reagent/instrumental blank, hence, 2 orders of magnitude improvement in the limit of detection should be possible if the blank can be eliminated. CONCLUSION Reverse-phase HPLC has been successfully coupled with SFICP-MS for the determination of sulfur-containing impurities in cimetidine at mass fractions below 0.1% relative to cimetidine. Limits of detection were blank-limited. For most of the impurities that were detected, structures were determined by using ESI-MS. Received for review March 14, 2001. Accepted June 29, 2001. AC0103017