Protoporphyrin IX purification from blood and serum for mass analysis – Considerations with respect to neurosurgery

Protoporphyrin (PPIX) and its precursor 5-aminolevulinic acid (ALA) are routinely used in fluorescence-guided resection (FGR) in neurosurgery of glioblastoma. Orally administered prior to surgery, ALA diffuses preferentially into the tumour-bearing brain region and is there transformed into PPIX as part of the heme biosynthesis. During the resection of high-grade gliomas (HGG), the fluorescent properties of PPIX enable the differentiation of healthy and malignant brain tissue – resulting in a more complete tumour resection and, a better prognosis for patients. 1,2

glioma cells contained PPIX, 9 but none of these investigations used mass spectrometry (MS) as detector.
Whole blood contains the most PPIX, but it is not the best matrix for such studies, because of unspecific contributions from erythrocyte ZnPPIX. For serum or plasma, though, the published PPIX levels, obtained with traditional methods, are low, often below the limit of detection, and not reliable. 10,11 Taking advantage of the high sensitivity of MS, we have been aiming for a reproducible LC/MS workflow for the use of serum and tested our protocol in comparison to whole blood. 5 Thereby, the reliable purification, respective recovery, of PPIX from the biological matrix proved particularly difficult and we have therefore evaluated different preparation methods. Initial liquid-liquid extraction (LLE) with water and acetonitrile (ACN) was crucial for best sensitivity. 5,12 However, an additional purification step was required to minimize LC-column aging and clogging.
We first tried to separate the small molecular weight substances from macromolecules using syringe and centrifugal filters. It soon turned out, however, that of some of the membrane material we testedpolytetrafluoroethylene, polyvinylidene fluoride, modified polyether sulfone, nylon and cellulosein particular nylon (polyamide 6.6) and cellulosequantitatively adsorbed PPIX. This observation led to experiments using punched-out membrane pieces for the specific extraction of PPIX directly from the biological matrix, because PPIX could subsequently be easily removed from the membrane using dimethyl sulfoxide (DMSO). The results from crude preliminary experiments were indeed promising considering a recovery of about 60-70% for PPIX from a reference extract, but handling was difficult, and reproducibility was hampered by the need to produce and process small to tiny pieces of nylon. The ratio between the amount of porphyrin and the nylon surface area was crucial as well as the pH during sample loading and the elution volume. Moreover, the extraction of mesoporphyrin (MPIX), our internal standard, from spiked serum was not as efficient as that of PPIX when working with serum instead of standard solution ( Figure 1). At that point we switched to anionic-exchange solid-phase extraction (ae-SPE), but we did feel that the potential of nylon with respect to PPIX isolation should be kept in mind.
Ultimately, ae-SPE worked best for our purpose, but, interestingly, not all sorbent material performed with the same quality. We tested three products with different types of ae-sorbent.
All cartridge bed materials consisted of quaternary amine functionalized polymers with strong ae-properties; they differed only in the polymer and the particle size (Table 1). We evaluated a reference solution mimicking the LLE extract and spiked whole blood as well as native and spiked serum. The recovery on styrene (Sty)-based cartridges was the lowest for all matrices ( Figure 2) when using a typical SPE workflow including bed preparation, ion exchange, analyte washing and elution ( Figure 3) and thus the determination of native PPIX in serum was not attempted with this material. The other two cartridge types performed similarly with recoveries above 80% for spiked PPIX and MPIX (Figures 2A and 2B).
Fully water-wettable polymeric-based cartridges possess the advantage, compared with silica and partially water-wettable material, that an aqueous sample can be loaded directly without performing sorbent conditioning and equilibration steps ( with a surface area of 20 mm 2 that were manually punched-out with a metal hollow punch. The disks were washed using ACN/water F I G U R E 1 Nylon adsorption experiments. Recovery of PPIX and MPIX using membrane disks for purification from a reference solution and spiked serum T A B L E 1 Characteristics of ae-SPE cartridges used for purification of porphyrins according to information from the manufacturer. Further parameters were the pH-stability from 0-14, a bed volume of 60 mg and polyethylene frits with 20 μm pore width F I G U R E 2 Recovery rates for MPIX and PPIX purified using MMae, HLB and Sty-based cartridges with the protocol shown in Figure 3 (A,C). Bars show averaged recovery (blue/dotted for MPIX; red/dashed for PPIX), error bars indicate the deviation in two separate purifications and triplicate LC/MS measurements. For whole blood and serum there was a contribution of native PPIX beside spiked PPIX resulting in recovery rates greater than 100%. Peak area for native PPIX extracted from 500 μL serum (B,D)