**3. Protoporphyrin IX: a potential biomarker for cancer screening**

More recently, besides the analysis of porphyrin metabolites [39] and profiles for toxicological and pharmacological applications [40–42], PPIX has been investigated as tumor marker for bladder, colorectal and kidney cancer [10, 11, 32]. Tumor cells are able to produce porphyrins naturally or after administration of ALA, which is also reflected in elevated plasma fluorescence of cancer patients. The spectral characteristics of blood from normal control subjects differ significantly from those of cancer patients in renal cell carcinoma, prostate cancer and colorectal adenocarcinoma [8–11].

PPIX analysis is, however, not straightforward in a clinical setting. Factors such as unrelated diseases and medication may influence the measured porphyrin concentration [8]. Lualdi and co-workers [11], e.g., confirmed their findings of enhanced plasma fluorescence in colorectal adenocarcinoma patients by HPLC coupled to high-resolution MS and detected mainly PPIX and coproporphyrin I. Ota et al. [32] applied HPLC-FLD for the determination of PPIX in plasma of bladder cancer patients after ALA administration. The patients showed significantly higher plasma PPIX concentrations compared to healthy adults. It was extrapolated that the accumulation of PPIX in cancer cells is common to almost all types of cancer [8–10] and that the specific measurement of PPIX is advantageous for cancer screening [32].

A further application of PPIX is above-mentioned photodynamic diagnosis, where PPIX is applied as an intraoperative marker especially for brain tumors. Using ALA-induced PPIX-fluorescence in tissue during surgery of high-grade glioma, the resection is more complete and the patients have a higher 6-month progression-free survival compared to those without FGR [7]. Unfortunately, due to the infiltrative growth of these tumors, complete tumor resection is still impossible and tumors can recur. Clinically, diagnosis of high-grade glioma and glioblastoma multiforme (GBM) as well as their recurrence requires multidisciplinary strategies such as contrast enhancement magnetic resonance imaging, computer tomography and biopsy [6, 7, 46, 47]. Therefore, a sensitive and cost-effective method for tumor monitoring is highly desirable supporting early diagnosis and treatment of GBM as well as better prognosis for patients. So far, the survival prognosis for GBM patients is one of the lowest in modern day oncology [47]. As PPIX is an approved marker for GBM tissue in ALA-FGR, here, the hypothesis was tested if it could also be a blood biomarker for GBM screening and diagnosis.

### **4. PPIX quantification**

For the detection of PPIX in whole blood or serum, we developed an HPLC-MS method using an HP1100 HPLC (Agilent, Waldbronn, Germany) coupled to an Esquire 3000 ion trap mass spectrometer (Bruker Corp., Bremen, Germany).

**61**

*Protoporphyrin IX Analysis from Blood and Serum in the Context of Neurosurgery…*

Mesoporphyrin (MPIX) (**Figure 3**) was chosen as internal standard (IS), because it provided high structural similarity to PPIX and isotope labeled standards for PPIX were not available. Distinction of PPIX from ZnPPIX was possible during sample preparation. The method described below allowed the quantification of metal-free PPIX in whole blood, the determination of endogenous PPIX in serum and the measurement of endogenous ZnPPIX in whole blood (200 μl, respectively).

PPIX LLE extraction from serum and whole blood was achieved with only water and acetonitrile (ACN). Hemolysis with water was crucial for good recovery as observed by others working with pre-dilution [22]. It was followed by protein precipitation with ACN; concomitant porphyrins were extracted into the supernatant [48], which was further purified using anionic-exchange solid phase extraction (SPE) cartridges. The extracts of whole blood and serum had a pH 8–9 so that PPIX, ZnPPIX and MPIX had deprotonated propionic acid side chains and were negatively charged. No conversion of ZnPPIX into metal-free PPIX was observed before loading the extracts onto the SPE cartridge. All three porphyrins were retained on the cartridge presenting quaternary ammonium groups. MPIX and PPIX were eluted using ACN containing 2% formic acid (FA), ZnPPIX with increased FA content (20%). No elution or hydrolysis of ZnPPIX was detected at 2% FA; only metal-free PPIX was seen in the first eluate. The higher percentage of FA in the second step caused the acidic release of the Zn2+ ion. ZnPPIX was thus detected as metal-free PPIX.

As already demonstrated in the literature for other porphyrins [40], PPIX ion-

preferentially occurs on the side chains (**Figure 3**). The spectra in **Figure 4** illustrate the stepwise fragmentation of PPIX from preselected precursor ions in subsequent MS/MS and MS/MS/MS experiments in the ion trap. The abundant loss of an ethanoic acid substituent (-CH2COOH; 563-59 u) results in a fragment ion at *m*/*z* 504.3

chain losses (-CH3, 15/30 u; -COOH, 45 u; -CH2COOH, 59 u; -CH2CH2COOH, 73 u). For selected compounds, it was discussed that even-electron ions generated in the ESI source can produce radical cations with odd-electrons by hemolytic cleavage. The most common process in radical fragmentation is the elimination of a methyl group as proposed for flavonoids, antraquinones and terpenoids [49]. It was shown that the radical elimination of the methyl group is a low energy process in flavonoids. The loss of 59 u by radical cleavage was also already described for FePPIX in previous studies on metalloporphyrins and other compounds with

The fragmentation of MPIX is similar to PPIX. MPIX has two saturated ethyl side chains at positions 8 and 13 (**Figure 3**) so that precursor and fragment ions

ZnPPIX was measured in MS/MS mode as it showed lower ionization efficiency than MPIX and PPIX. The time-segmented method switched from MS/

This approach proved advantageous in comparison to continuous MS/MS and MS3 switching for porphyrins and significantly increased sensitivity. In comparison to the ion traces of matrix-free porphyrin standard solution, the background signal was about five times higher in whole blood extracts, but that did not hamper detec-

species (*m*/z 563.3) in ESI-MS. MS/MS fragmentation

fragmentation which then generates further side

mode for MPIX and PPIX measurement.

*DOI: http://dx.doi.org/10.5772/intechopen.95042*

**4.1 Sample preparation**

**4.2 Gas phase fragmentation**

izes as singly charged [M+H]+

that is used as precursor for MS3

extended π-electron systems [50–52].

MS mode for ZnPPIX detection to MS3

tion with the specific MS3

differ by four mass units in comparison to PPIX.

method.

Mesoporphyrin (MPIX) (**Figure 3**) was chosen as internal standard (IS), because it provided high structural similarity to PPIX and isotope labeled standards for PPIX were not available. Distinction of PPIX from ZnPPIX was possible during sample preparation. The method described below allowed the quantification of metal-free PPIX in whole blood, the determination of endogenous PPIX in serum and the measurement of endogenous ZnPPIX in whole blood (200 μl, respectively).
