Journal of Pharmaceutical and Biomedical Analysis
The high performance liquid chromatography electrospray ionization mass spectrometry analysis of diverse basic pharmaceuticals on cyanopropyl and pentafluorophenylpropyl stationary phases
Introduction
Bonded C8 and C18 phases have been widely used for the HPLC analysis of basic drugs [1], [2]. However, buffer salts and various additives, such as pairing- or counter-ions, which include alkylsulphonates, alkylamines or quaternary ammonium compounds, are often needed with these hydrophobic phases to achieve good peak shape and good retention [3], [4]. In ESI-MS, volatile buffers and additives must be used; thus many conventional additives and ion-pairing agents must be avoided. Even when ion-pair agents, such as trifluoroacetic acid [5], or ion suppressing agents, such as triethylamine [6], are used the ESI-MS signal is decreased. To analyze drugs such as codeine or morphine by HPLC without the use of silanol suppressing agents is problematic because these basic molecules (pKa 8.2 and 9.9, respectively) interact with the silanols to produce tailing peaks [7]. In addition, drugs such as these are hydrophilic; thus, with reversed-phase columns (C8 or C18), low concentrations (<10%) of organic solvent or ion-pairing agents must be used to provide adequate retention of the solutes [8]. However, it has been reported that when the concentration of the aqueous solvent in the mobile phase is increased, the ESI-MS signal is decreased [9], [10], [11]. Therefore, because of the poor peak shape and inefficient desolvation in the ESI process due to high concentrations of aqueous solvent when C18 columns are used, the overall MS response is highly reduced.
Ion suppression in the ESI interface has recently received much attention [12], [13], [14], [15]. Ion suppression can occur when endogenous interferences are co-eluted with the analytes. These endogenous compounds can diminish the ionization of the analyte in the ESI interface. The final result is a reduced and imprecise MS signal. Improvements in the sample preparation [12] or the HPLC separation [14] or a combination of both can reduce ion suppression. It has been reported that capacity factors (k′) greater than 4 were necessary to separate analytes from endogenous interferences and thus significantly decrease ion suppression [16]. Our goal is, therefore, to find HPLC stationary phases that provide good peak shape and good retention (k′>4) for the HPLC/ESI/MS analysis of basic drugs using a mobile phase with high concentrations of organic solvent. We have investigated a number of stationary phases and found that cyanopropyl (CN) and pentafluorophenylpropyl (PFPP) stationary phases provided good peak shape and good retention for basic drugs with the use of 90% acetonitrile in the mobile phase [17], [18]. By retaining the drugs with a high concentration of acetonitrile in the mobile phase, the CN and PFPP stationary phases provided signal enhancements greater than a factor of 10 when compared with a C18 stationary phase. We found other hydrophobic phases such as C8 and C4 had retention characteristics similar to C18 phases. However, only two classes of basic drugs were tested and it was not known whether the analyses obtained could be universally applied to all basic drugs. Thus, we report on the results of the use of a CN and PFPP stationary phase for the HPLC/ESI/MS analysis of a variety of basic drugs. Fig. 1 shows the structures of the CN and PFPP stationary phases.
Section snippets
Reagents and standards
The drug, gepirone was obtained from Mr Bob Behme of Scientific Resources, Inc. (Evansville, IN). Sumatriptan, bufuralol and hydroxy-bufuralol were obtained from Ms Jessica Dunn of Pfizer Inc. (Groton, CT). All other compounds were obtained from Sigma Chemical (St Louis, MO). Standard stock solutions (1.0 mg/ml) were prepared by dissolving a weighed amount of the compounds in H2O/MeOH (90:10 v/v%). The solutions were sonicated in an Ultrasonicating Bath 3200 (Bransonic, Danbury, CT) for 10 min.
Results and discussion
Mass spectral, drug class and pKa information for the solutes are shown in Table 1. All the solutes formed predominant protonated molecules [M+H]+ in the ESI source.
To demonstrate the broad applicability of the CN and PFPP stationary phases for the HPLC/ESI/MS analysis of basic compounds, we chose basic drugs with a wide-range of characteristics. The basic drugs ranged in polarities, had a pKa range from 7.2 to 10.7 and ranged in molecular weight from 227.7 to 385.5 Daltons. In addition, we
Conclusions
The CN and PFPP phases offer significant advantages when compared with C18 or C8 phases for the HPLC/ESI/MS of basic drugs. By retaining polar, basic drugs with 90% acetonitrile in the mobile phase, the ESI-MS signal is enhanced by over a factor of 9 when compared with a C18 phase which required 6.0% acetonitrile for the analysis of a model solute, codeine. The improvement in signal is due to more efficient desolvation in the ESI source with the use of higher concentrations of acetonitrile.
Acknowledgements
The authors thank Mr Keith Duff of Restek Corporation for the synthesis of the stationary phases and his insightful discussions. The authors would also like to thank Mr Bob Behme of Scientific Resources, Inc. for his donation of the drug, gepirone and Ms Jessica Dunn of Pfizer Inc for the use of the standards of sumatriptan, bufuralol and hydroxy-bufuralol.
References (21)
- et al.
J. Chromatogr. A.
(1998) - et al.
J. Pharm. Biomed. Anal.
(1999) - et al.
J. Chromatogr. B Biomed. Sci. Appl.
(1998) - et al.
J. Am. Soc. Mass Spectrom.
(1995) - et al.
J. Chromatogr. B Biomed. Sci. Appl.
(1998) - et al.
J. Chromatogr. B Biomed. Sci. Appl.
(1999) - et al.
J. Chromatogr. A
(1998) - et al.
J. Am. Soc. Mass Spectrom.
(1996) - et al.
J. Chromatogr. B Biomed. Sci. Appl.
(1998) - et al.
J. Pharm. Biomed. Anal.
(1998)
Cited by (31)
Development of a rapid method for the simultaneous separation and determination of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and its N- and O-glucuronides in human urine by liquid chromatography-tandem mass spectrometry
2013, Analytica Chimica ActaCitation Excerpt :These endogenous compounds can diminish the ionization of the analytes in the ESI interface. Improvement in the sample preparation or the LC separation or a combination of both can reduce ion suppression [26]. Moreover, in the MS/MS mode, the major product ions of NNAL-N-Gluc and NNAL-O-Gluc were all obtained by the fragmentation of the [M+H]+ ion m/z 386.0.
Synthesis, characterisation and chromatographic evaluation of pentafluorophenyl and phenyl bonded silica phases prepared using supercritical carbon dioxide as a reaction solvent
2013, Journal of Chromatography ACitation Excerpt :Zhang [32] suggested that fluorinated phases could show different separation mechanisms depending on the type of analytes. “U shape” retention profile for polar and basic analytes with increased amount of organic modifier in the mobile phase [40,46,47] have been observed on PFPP. The “U-shape” behaviour has also been observed on non-fluorinated phases, e.g. polar-embedded stationary phases [48,49].
Optimization of a high-performance liquid chromatography method for the analysis of complex polyphenol mixtures and application for sainfoin extracts (Onobrychis viciifolia)
2010, Journal of Chromatography ACitation Excerpt :The combination of reversed- and normal-phase behavior forms a “U-shape” relationship between retention and organic modifier percentage and can be rationalized by the presence of hydrophobically assisted ion-exchange mechanism or additional independent interactions due to the presence of the pentafluorophenyl ligands [20–22]. Needham et al. found that the pentafluorophenylpropyl modified silica columns gave good retention of several kinds of basic drugs with a mobile phase containing 90% acetonitrile, whereas, to achieve good retention on C18 columns, 40% acetonitrile has to be used [23–25]. In a similar study from Marín and Barbas, both reversed-phase and normal-phase-like characteristics for certain analytes have been observed [17].
Determination of betaine metabolites and dimethylsulfoniopropionate in coral tissues using liquid chromatography-time-of-flight mass spectrometry and stable isotope-labeled internal standards
2010, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences