The bottleneck of sample analysis has always been the sample preparation. Online SFE-SFC-MS/MS minimizes sample preparation by allowing solid samples to be completely analyzed without performing sample extraction and cleanup at the lab bench. The online method allows automatic solid sample extraction to be completed. This is subsequently followed by chromatography separation and ultimately detection by triple quadrupole mass spectrometry. The online extraction process is up to seven times faster than the current QuEChERS method for pesticide analysis in food. Other fast sample preparation applications include preserving labile compounds in dietary supplements, cleaning validation in pharmaceuticals, biomarkers for dried blood spots, and trace additives in polymers.

Supercritical fluids (SCF) have some properties similar to liquids and others similar to gases. For example, SCF density behaves more like a liquid due to its higher density and therefore higher solubility. With respect to viscosity and diffusivity, SCF behaves more like a gas. The higher diffusivity provides faster mixing and better extraction. The lower viscosity allows lower pressure, higher flow rates and faster analysis times, similar to a gas chromatograph (GC). SFC with added high pressure (9600 psi or 66MPa) can reduce analysis time by two-thirds that of HPLC methods.

The “Achilles Heel” of the SFC-MS/MS interface has been the back pressure regulator (BPR). The purpose of the BPR is to keep the SCF in that state through pressure. The problem is current technologies require splitting the sample 4:1 between the BPR and the MS to avoid peak broadening in the BPR. As a result, only 20 percent of the sample makes it to the MS. Overcoming the Achilles Heel of this technique is accomplished by the incorporation of a newly patented BPR splitless design. The new SFC-MS/MS technology enhances sensitivity by at least five times compared with current technologies for SFC-MS/MS, as shown below.

The first supercritical fluid chromatography (SFC) reference is credited to Klesper^[1] et al. in 1962. The authors described the technique as high-pressure gas chromatography (HPGC). In their work, the supercritical fluids used were dichlorodifluoromethane (Tc = 112°C, Pc = 1000 atm) and monochlorodifluoromethane (Tc = 96 °C, Pc = 1400 atm).

In the mid-1960s, Sie^[2-6] et al. published a series of papers using carbon dioxide (Tc = 31.3°C, Pc = 72.9 atm) as the mobile phase. Since carbon dioxide has a low supercritical temperature and pressure, it has been used extensively since the 1980s as an extraction method. CO2 offers a number of other advantages, including being inexpensive, non-toxic, non-flammable and relatively “green” compared to other solvents with respect to the environment.

The first coupling of SFE to SFC with a capillary column was by Hawthorne ^[7] and Wright ^[8] in 1987, while McNally^[9] and Engelhardt^[10] reported the coupling with a packed column in 1988. Packed column is the choice of methods with SFC today.

This article introduces the analysis of solid samples with little sample preparation in the areas of foods, dietary supplements, pharmaceutical, clinical, and polymers using the first online SFE-SFC-MS/MS.

Experimental

Shimadzu’s Nexera UC (Unified Chromatography) was used for all experiments. The online configuration was SFE-SFC-MS/MS. The Nexera UC system can also use other detectors such as UV-Vis, Photodiode Array (PDA), and single quadrupole mass spectrometers.

Results and Discussion

Sample preparation may involve many steps such as blending, shaking, filtering, partitioning, centrifuging, evaporating, concentrating, extraction, and cleanup as well as using lots of glassware and solvents. The ideal workflow would allow analysis of solid samples with minimal sample preparation and without human intervention. This ideal workflow is accomplished with online SFE-SFC-MS/MS or SFE-SFC-UV.

Food Analysis

QuEChERS methodology developed by Steven Lehotay and Michelangelo Anastassiades was a significant improvement in extraction and cleanup for analysis of pesticides in foods over traditional methods. This led to the development of AOAC (2007.01) and European (EN 15662) methods because they can use 95 percent less solvent and 95 percent less consumables, and offer a 90 percent reduction in preparation time. Shown in Figure 2 is a schematic of typical steps required for sample preparation, extraction and cleanup using QuEChERS for a spinach sample. Sample preparation time can be up to 35 minutes. 

 

 

 

 

Because of the increased selectivity of SFE, the sample preparation and extraction time can be reduced from 35 minutes to 5 minutes. A proprietary absorbent is used to absorb the water.

After the extraction, traditionally the next question is whether to use GCMS or LCMS for analysis. Generally, GCMS is used for low polarity and low molecular weight compounds as shown in Figure 4. For GCMS, ideally the compounds are volatile and thermally stable regardless of the MS detection design. For higher polarity and higher molecular weight compounds, the ideal choice is LCMS. ESI (electrospray ionization) is typically used for higher polarity and molecular weight compounds. Shimadzu’s DUIS ionization source includes both the ESI and APCI (atmospheric pressure chemical ionization) sources.

SFC covers a wider range of polarities compared to GCMS or LCMS. The molecular weight of SFC is not as high as that of LCMS, but many compounds such as pesticides are not high molecular weight compounds.

During the SFE-SFC, methanol is added to the mobile phase in order to solubilize more of the polar compounds since the supercritical fluid CO2 is non-polar. Thus, SFE-SFC-MS/MS can analyze non-polar and polar type compounds.

Dietary Supplement

Coenzyme Q10 is a liable compound which is oxidized during the traditional solvent extraction process. The analyst typically would assume the original content in the dietary supplement was in the reduced form. However, assumptions are not always correct. By performing an online SFE-SFC analysis, one can clearly see the extraction and chromatography results differ, thus illustrating the advantages of the online coupled technique.

Pharmaceutical Cleaning Validation

Two of the traditional methods for pharmaceutical cleaning validation are by total organic carbon (TOC) and HPLC. For TOC analysis, the sample must be soluble in water. It is also worth noting that the exacted compound(s) cannot be determined since the method only gives total organic carbon. Using the HPLC method requires solvent extraction followed by a concentrating method since the method is not very sensitive. Alternatively, SFE-SFC simply requires breaking the swab tip off and adding it to the sample extraction vessel.

Clinical Biomarkers from Dried Blood Spots

The conventional analysis of biomarkers in dried blood spots (DBS) requires a numbers of steps such as cutting off the DBS, solvent extraction, stirring/shaking, solvent soaking, filtration and evaporation before analysis in the LCMS. Online SFC-SFE-MS/MS only requires placing the sample in the sample vessel, placing the sample vessel inside the instrument and selecting the start button.

Polymers with Trace Additives

Analysis of a polymer by chromatography typically requires eight to 24 hours Soxhlet extraction followed by manually transferring the extraction to the HPLC. Alternatively, by cutting the polymer into small samples and placing them in the sample vessel, analysis can be completed in a few minutes. The sample contained Irganox 1010, which is an antioxidant and a highly effective, non-discoloring stabilizer used in polymers.

Conclusion

Online SFE-SFC-MS/MS provides several advantages over traditional sample preparation and analysis of solid samples. The advantages affect all areas of the analysis workflow, including sample preparation, chromatography and detection. Sample preparation can be up to seven times faster, chromatography up to three times faster, and sensitivity up to five times higher when compared to other techniques. In addition, it offers a wider range of sample polarities then GC-MS/MS or LC-MS/MS and protection of liable samples.