Volatile IPRs

Liquid chromatography mass spectrometry (LC-MS) is now routinely used in analytical laboratories. Traditional IPRs are non-volatile salts that are not compatible with MS techniques because they play a major role in source pollution that is responsible for reduced signals. Moreover the final number of charged ions that reach the detector is impaired by ion-pair formation actually IPRs added to the mobile phase to improve analytes retention exert a profound effect on analyte ionization. Chromatographers who perform IPC-MS must optimize the eluent composition based on both chromatographic separation and compatibility with online detection requirements. 

Different strategies devised for effective IPC-MS methods are discussed in greater detail in Chapter 14. This chapter examines the advantages of using volatile 

IPRs that hold much promise in this field and also for easy IPC evaporative light scattering detector techniques. 

Small organic (e.g., formic and acetic) acids are effective volatile IPRs. They impact the retention behaviors of pH-sensitive compounds, changing their charge status and providing pairing anions that may easily interact with protonated solutes. Many chromatographic separations benefit in terms of retention, resolution, and peak shape under acidic conditions due to suppression of silanol activity. Furthermore, the acidity of these IPRs facilitates the formation of the protonated molecular ion [M + H] measured by mass spectrometry in the usual positive ion mode. 

The volatility of perfluorinated IPR was also beneficial when an evaporative light scattering detector was employed [5,17,24,50]. Perfluorinated carboxylic adds proved suitable for preparative chromatography [51]. Interestingly, since they prevent strong IPR build-up in the column, they are particularly useful when gradient elution is performed to reduce analysis time without compromising resolution [28,52-54]. 

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IPC-MS combined methods must be optimized with respect to separation and compatibility with online detection involving the constraints detailed in Table 12.2 regarding the composition and volatility of the mobile phase. The major concern of chromatographers who deal with this combined technique is the reduced signal caused by source pollution of non-volatile IPRs. Moreover, the efficiency of droplet development, which in turn affects the number of charged ions that ultimately reach... 

Since alterations in global DNA methylation are implicated in various pathobio-logical processes, a gradient IPC-ESI-MS/MS method with a volatile IPR was used to determine cytosine and 5-methylcytosine in DNA quantification relied on stable isotope dilution [58], Muscular dystrophies caused by various mutations in the dystrophin gene are amenable to easier prenatal diagnosis via a multiplex polymerase chain reaction (PCR)/IPC assay [59]. Some guidelines for the analysis of genomic DNA by IPC-ESl-MS can be found in Reference 60. 

IPC proved valnable for estimating peptide hydrophobicity [13]. Pharmaceutical science utilized IPC to monitor rat serum esterase activities [14] and also to analyze relationship between plasma concentrations at the end of infusion and toxicity profiles of fixed-dose-rate gemcitabine plus carboplatin [15]. An IPC trap was also used in an online desalting-mass spectrometry system. This system allows ionic compounds in a nonvolatile buffer to be introduced into a MS for strutural elucidation. The trap column was equilibrated with a volatile IPR, the target analyte and the nonvolatile buffer ions (phosphate and sodium ions) were transferred into the trap column, but only the target analyte that interacts with the volative IPR can be retained phospahte buffer ion were eluted from the trap column and the target analyte was eluted by oragnic solvent in a backflush mode and introduced into the MS. 

A soln of the diphenyl [l-(benzyloxycarbonylamino)alkyl]phosphonate 4 in 45% HBr/AcOH (150 mL per 0.1 mol) was kept at rt for 1 h. Removal of the solvent and volatile products in vacuo yielded the oily hydrobromide, which crystallized upon addition of anhyd Et20. The free amine was obtained by suspending the hydrobromide in CHC13 and shaking in a separatory funnel with an equal volume of 2M NaOH until all the solid dissolved. The CHC13 layer was separated, washed with brine, dried (MgS04), and concentrated to yield 19 in >90% yield mp (R1 = Me) 36-39°C mp (R = iPr) 60-63°C mp (R1 = iBu) 57-58 °C mp (R = Ph) 63-65 °C mp (R = Bzl) 65-67 °C. 

The addition of many classes of IPRs leads to a modification of the eluent pH. Volatile amines, greatly appreciated for their volatility in IPC-MS use, increase the eluent pH. Conversely, using perfluorinated acids of different chain length as IPRs for basic analytes involves a concomitant lowering of the eluent pH that in turn provides the protonation the basic analytes need under IPC conditions. The IPR counter ion is also important when dealing with eluent pH for example, the behavior of tetrabutylam-monium hydroxide would be very different from that of the corresponding chloride. 

Ionic modifiers in the IPC eluent can provide a charge for neutral molecules so that they become detectable via the ESI interface. Conversely, counter ions reduce the charge state of an oppositely charged analyte or even convert it to the opposite polarity. If they are polycharged, for example, ESP and ESP modes were comparatively evaluated for the detection of nucleotides that are negatively charged. It was straightforward to use the ESP mode that detects the [M - H]"ion (with low levels of sodium and potassium adducts present), but ESP was a viable alternative because the volatile N,N-dimethylhexylamine IPR yields ion-pairs with the nucleotides. The most abundant relevant ion was the adduct between the compound and... 

Bisphosphonates (bone resorption inhibitor drugs) were subject to many investigations. They can chelate to metal surfaces, producing chromatographic peak tailing. Different tetraalkylammonium salts, commonly selected as IPRs in separation of bisphosphonates [85], were replaced by volatile organic amines when ELSD was used [86]. 

An IPC-ESI-MS/MS method using volatile perfluorinated carboxylic adds as IPRs added directly to the sample solution (not incorporated into the mobile phase) was developed for the quantitative assay of methadone in human plasma. This cost-effective strategy enhanced the efficiency of separation and minimized ion suppression because no IPR was present in the eluent [103]. Table 13.1 lists other potential uses of IPC in the pharmaceutical and clinical analysis fields. 

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