**4.1 Sample preparation**

*Mass Spectrometry in Life Sciences and Clinical Laboratory*

For the investigation of porphyrins in blood, predominantly plasma or red blood

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 adenocar-

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

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

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).

blood biomarker for GBM screening and diagnosis.

**4. PPIX quantification**

cells were used [11, 40, 43]. In plasma, MS was applied for the quantification of coproporphyrin isomers for monitoring of drug interactions [43], the elucidation of fluorescing compounds after detection of elevated fluorescence [11], and the qualitative analysis of porphyrin patterns facilitating the differential diagnosis of human porphyrias [40]. Despite all these efforts, no short and sensitive HPLC-MS/MS method for specific PPIX quantification from whole blood or serum was yet available although great data have been shown for less complex cell culture extracts [44, 45].

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

**60**

cinoma [8–11].

screening [32].

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.
