Coupling mass spectrometry (MS) to high performance liquid chromatography (HPLC) methods has become commonplace over the last two decades as many take advantage of both the sensitivity and the identity aspects of such instrumentation. The specificity of tandem MS using parent–daughter combinations (multiple reaction monitoring [MRM], for example) allows for quantifying individual compounds in a complex mixture, even when such compounds are not fully resolved from other analytes in a mixture. However, some resolution between analytes and ion-suppressing impurities is required for good MS quantitation. Combining shorter HPLC columns with MS has been the solution for developing sensitive methods for quantitating several analytes in a single analysis.

Over the last five years, several instrument and column manufacturers have invested much in marketing and advertising about ultrahigh-pressure LC (UHPLC) and the advantages of switching to higher efficiency columns for analytical separations. Showing performance gains for HPLC applications using UV detection by switching to UHPLC columns and instruments has been a rather straightforward process. Sampling rates of newer UV detectors are such that a large number of data points per second can be acquired, allowing one to visualize and quantitate the improved chromatography that UHPLC columns deliver. With LC–MS methods, visualizing improvements in chromatography are less apparent. The scan rate for MS instrumentation can sometimes be limited to a few points per second. This rate can be even slower for methods that scan large molecular weight ranges or when numerous parent–daughter pairs are being acquired. In addition, extracolumn volume in the MS interface or detectors postcolumn can mask any separation gain from a UHPLC column. As a result, realized performance gains for LC–MS methods using UHPLC have been significantly less than standalone LC methods.

Recently, a new type of UHPLC column has been introduced using core-shell media. These media use unique particle geometry versus the standard fully porous silica media; a nonporous core particle is surrounded by a thin porous layer of functionally bonded silica. The improved porosity and mass transfer of such media are much higher than that of fully porous media of the same particle size, resulting in performance on par with fully porous media much smaller than the core-shell media. In the case of the major core-shell products on the market, the core-shell media are slightly larger than 2.5 µm in size with performance on par with the major sub-2-µm media on the market with back pressures more in line with 3-µm fully porous HPLC media. The greater implication of such core-shell media is that one could get UHPLC performance on their existing HPLC system without having to buy a new ultrahigh pressure system.

Efforts were undertaken to adapt various existing LC–MS methods using standard HPLC systems to using the new core-shell media. Examples of high-throughput applications for pharmaceutical and environmental analysis were developed using core shell media. Key in developing such methods include maintaining reasonable resolution of key analytes from ion suppressing impurities while balancing the limitations of the MS being used for the analysis.

Materials and Methods

Figure 1

Chromatography standards (amphetamine, aflatoxin, sulfa drugs, and antibiotic mixtures), urine, and plasma were purchased from Sigma Chemical (St. Louis, Missouri). Various buffers and solvents were purchased from EMD (San Diego, California). Gemini 5 µm C18 (150 mm × 3.0 mm) and Kinetex 2.6 µm C18 and PFP (both 50 mm × 2.1 mm) HPLC columns were obtained from Phenomenex (Torrance, California). StrataX, StrataX-C, and Strata Florisil solid-phase extraction (SPE) tubes (50 mg/3 mL tubes) also were used. Food matrices were obtained from local grocery sources. Test samples were spiked into separate sample matrices. For the pharmaceutical–toxicology sample, amphetamines were spiked into urine. For environmental analysis, aflatoxin was spiked into peanut butter, sulfa drugs in honey, and an antibiotic mixture was spiked into a meat sample. While every SPE procedure was unique to the application, all had several common features: sample is diluted then loaded on their specific SPE cartridge; cartridge is washed, then analyte is eluted with an organic mixture. Samples are then dried down and reconstituted in aqueous mobile phase before injection on HPLC.

Figure 2

All samples were injected on an HP1100 HPLC system with an autosampler, solvent degasser, and multiwavelength detector (all from Agilent Technologies, Santa Clara, California). HPLC-UV data were collected using Chemstation software (Agilent Technologies). The aqueous mobile phase used was either 0.1% formic acid in water or a mixture of formic acid with ammonium formate. For organic mobile phase, either acetonitrile or methanol was used. Various gradient methods were used based upon the analyte mixture and columns used. For LC–MS analysis, the HPLC instrument was linked postcolumn (excluding the UV detector) directly to the electrospray ionization (ESI) interface of an API3000 tandem mass spectrometer (Applied Biosystems, Foster City, California). While individual MS parameters differ based upon the application, in most cases, the MS system was run in positive ion mode using MS-MS multiple reaction monitoring, in which analytes were quantitated using specific parent– daughter ion transitions. In many cases, a multiply deuterated internal standard was used for quantitation.